VITAMIN D THIRD EDITION
http://www.elsevierdirect.com/companion.jsp?ISBN=9780123819789
Vitamin D David Feldman, Editor-in-Chief, J. Wesley Pike and John S. Adams, Associate Editors
VITAMIN D THIRD EDITION VOLUME I Editor-in-Chief
DAVID FELDMAN Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA Associate Editors
J. WESLEY PIKE Department of Biochemistry, University of Wisconsin, Madison, WI, USA
JOHN S. ADAMS UCLA-Orthopaedic Hospital Department of Orthopaedic Surgery, University of California, Los Angeles, CA, USA
AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier
Academic Press is an imprint of Elsevier 32 Jamestown Road, London NW1 7BY, UK 225 Wyman Street, Waltham, MA 02451, USA 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA First edition 1997 Second edition 2005 Third edition 2011 Copyright Ó 2011, 2005, 1997 Elsevier Inc. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+ 44) (0) 1865 843830; fax (+44) (0) 1865 853333; email:
[email protected]. Alternatively, visit the Science and Technology Books website at www.elsevierdirect.com/rights for further information Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Medicine is an ever-changing field. Standard safety precautions must be followed, but as new research and clinical experience broaden our knowledge, changes in treatment and drug therapy may become necessary or appropriate. Readers are advised to check the most current product information provided by the manufacturer of each drug to be administered to verify the recommended dose, the method and duration of administrations, and contraindications. It is the responsibility of the treating physician, relying on experience and knowledge of the patient, to determine dosages and the best treatment for each individual patient. Neither the publisher nor the authors assume any liability for any injury and/or damage to persons or property arising from this publication. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-381978-9 Two Volume Set ISBN: 978-0-12-387035-3 Volume 1 ISBN: 978-0-12-387034-6 Volume 2 For information on all Academic Press publications visit our website at www.elsevierdirect.com Typeset by TNQ Books and Journals Printed and bound in United States of America 11 12 13 14
10 9 8 7
6 5 4 3
2 1
Contents
11. Target Genes of Vitamin D: Spatio-temporal Interaction of
Preface to the 3rd Edition ix Preface to the 2nd Edition xi Preface to the 1st Edition xiii Contributors xv Introduction xxi Abbreviations xxiii Relevant Lab Values in Adults and Children xxix
12.
13.
VOLUME I
14.
I 15.
CHEMISTRY, METABOLISM, CIRCULATION 1. Historical Overview of Vitamin D 3
Chromatin, VDR, and Response Elements 211 Carsten Carlberg Epigenetic Modifications in Vitamin D Receptor-mediated Transrepression 227 Alexander Kouzmenko, Fumiaki Ohtake, Ryoji Fujiki, Shigeaki Kato Vitamin D and Wnt/b-Catenin Signaling 235 Jose´ Manuel Gonza´lez-Sancho, Marı´a Jesu´s Larriba, Alberto Mun˜oz Vitamin D Response Element-binding Protein 251 Thomas S. Lisse, Hong Chen, Mark S. Nanes, Martin Hewison, John S. Adams Vitamin D Sterol/VDR Conformational Dynamics and Nongenomic Actions 271 Mathew T. Mizwicki, Anthony W. Norman
Hector F. Deluca
2. Photobiology of Vitamin D 13
III
Michael F. Holick
3. The Activating Enzymes of Vitamin D Metabolism
(25- and 1a-Hydroxylases) 23 Glenville Jones, David E. Prosser 4. CYP24A1: Structure, Function, and Physiological Role Rene´ St. Arnaud 5. The Vitamin D Binding Protein DBP 57 Roger Bouillon 6. Industrial Aspects of Vitamin D 73 Arnold Lippert Hirsch
MINERAL AND BONE HOMEOSTASIS 16. Genetic and Epigenetic Control of the Regulatory Machinery
43
17. 18.
II
19.
MECHANISMS OF ACTION 7. The Vitamin D Receptor: Biochemical, Molecular,
20.
Biological, and Genomic Era Investigations 97 J. Wesley Pike, Mark B. Meyer, Seong Min Lee 8. Nuclear Vitamin D Receptor: Natural Ligands, Molecular StructureeFunction, and Transcriptional Control of Vital Genes 137 Mark R. Haussler, G. Kerr Whitfield, Carol A. Haussler, Jui-Cheng Hsieh, Peter W. Jurutka 9. Structural Basis for Ligand Activity in VDR 171 Natacha Rochel, Dino Moras 10. Coregulators of VDR-mediated Gene Expression 193 Diane R. Dowd, Paul N. MacDonald
21. 22. 23.
v
for Skeletal Development and Bone Formation: Contributions of Vitamin D3 301 Jane B. Lian, Gary S. Stein, Martin Montecino, Janet L. Stein, Andre J. van Wijnen Vitamin D Regulation of Osteoblast Function 321 Renny T. Franceschi, Yan Li Osteoclasts 335 F. Patrick Ross Molecular Mechanisms for Regulation of Intestinal Calcium and Phosphate Absorption by Vitamin D 349 James C. Fleet, Ryan D. Schoch The Calbindins: Calbindin-D28K and Calbindin-D9K and the Epithelial Calcium Channels TRPV5 and TRPV6 363 Sylvia Christakos, Leila J. Mady, Puneet Dhawan Mineralization 381 Eve Donnelly, Adele L. Boskey Vitamin D Regulation of Type I Collagen Expression in Bone 403 Barbara E. Kream, Alexander C. Lichtler Target Genes: Bone Proteins 411 Gerald J. Atkins, David M. Findlay, Paul H. Anderson, Howard A. Morris
vi
CONTENTS
24. Vitamin D and the Calcium-Sensing Receptor 425 Edward M. Brown 25. Effects of 1,25-Dihydroxyvitamin D3 on Voltage-Sensitive Calcium Channels in Osteoblast Differentiation and Morphology 457 William R. Thompson, Mary C. Farach-Carson
40. Vitamin D and the Renin-Angiotensin System 707 Yan Chun Li
41. Parathyroid Hormone, Parathyroid Hormone-Related Protein, 42. 43.
IV TARGETS 26. Vitamin D and the Kidney 471 27. 28. 29. 30. 31. 32.
33.
Peter Tebben, Rajiv Kumar Vitamin D and the Parathyroids 493 Justin Silver, Tally Naveh-Many Cartilage 507 Barbara D. Boyan, Maryam Doroudi, Zvi Schwartz Vitamin D and Oral Health 521 Ariane Berdal, Muriel Molla, Vianney Descroix The Role of Vitamin D and its Receptor in Skin and Hair Follicle Biology 533 Marie B. Demay Vitamin D and the Cardiovascular System 541 David G. Gardner, Songcang Chen, Denis J. Glenn, Wei Ni Vitamin D: A Neurosteroid Affecting Brain Development and Function; Implications for Neurological and Psychiatric Disorders 565 Darryl Eyles, Thomas Burne, John McGrath Contributions of Genetically Modified Mouse Models to Understanding the Physiology and Pathophysiology of the 25Hydroxyvitamin D-1-Alpha Hydroxylase Enzyme (1a(OH) ase) and the Vitamin D Receptor (VDR) 583 Geoffrey N. Hendy, Richard Kremer, David Goltzman
44. 45.
and Calcitonin 725 Elizabeth Holt, John J. Wysolmerski FGF23/Klotho New Regulators of Vitamin D Metabolism 747 Valentin David, L. Darryl Quarles The Role of the Vitamin D Receptor in Bile Acid Homeostasis 763 Daniel R. Schmidt, Steven A. Kliewer, David J. Mangelsdorf Vitamin D and Fat 769 Francisco J.A. de Paula, Clifford J. Rosen Extrarenal 1a-Hydroxylase 777 Martin Hewison, John S. Adams
VI DIAGNOSIS AND MANAGEMENT 46. Approach to the Patient with Metabolic Bone Disease 807 Michael P. Whyte
47. Detection of Vitamin D and Its Major Metabolites 823 Bruce W. Hollis
48. Bone Histomorphometry 845 Linda Skingle, Juliet Compston
49. Radiology of Rickets and Osteomalacia 861 Judith E. Adams
50. High-Resolution Imaging Techniques for Bone Quality Assessment 891 Andrew J. Burghardt, Roland Krug, Sharmila Majumdar 51. The Role of Vitamin D in Orthopedic Surgery 927 Aasis Unnanuntana, Brian J. Rebolledo, Joseph M. Lane
VII V HUMAN PHYSIOLOGY 34. Vitamin D: Role in the Calcium and Phosphorus 35. 36. 37. 38. 39.
Economies 607 Robert P. Heaney Fetus, Neonate and Infant 625 Christopher S. Kovacs Vitamin D Deficiency and Calcium Absorption during Childhood 647 Steven A. Abrams Adolescence and Acquisition of Peak Bone Mass 657 Connie Weaver, Richard Lewis, Emma Laing Vitamin D Metabolism in Pregnancy and Lactation 679 Natalie W. Thiex, Heidi J. Kalkwarf, Bonny L. Specker Vitamin D: Relevance in Reproductive Biology and Pathophysiological Implications in Reproductive Dysfunction 695 Lubna Pal, Hugh S. Taylor
NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY 52. Worldwide Vitamin D Status 947 Paul Lips, Natasja van Schoor
53. Sunlight, Vitamin D and Prostate Cancer Epidemiology 965 Gary G. Schwartz
54. Nutrition and Lifestyle Effects on Vitamin D Status 979 Susan J. Whiting, Mona S. Calvo
55. Bone Loss, Vitamin D and Bariatric Surgery: Nutrition and Obesity 1009 Lenore Arab, Ian Yip 56. Genetics of the Vitamin D Endocrine System 1025 Andre´ G. Uitterlinden 57. The Pharmacology of Vitamin D 1041 Reinhold Vieth 58. How to Define Optimal Vitamin D Status 1067 Roger Bouillon
Volume I Color Plate Section
vii
CONTENTS
76. Analogs of Calcitriol 1461
VOLUME II
VIII
77.
DISORDERS
78.
59. The Hypocalcemic Disorders: Differential Diagnosis and 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73.
Therapeutic Use of Vitamin D 1091 Thomas O. Carpenter, Karl L. Insogna Vitamin D Deficiency and Nutritional Rickets in Children 1107 John M. Pettifor Vitamin D and Osteoporosis 1129 Peter R. Ebeling, John A. Eisman Relevance of Vitamin D Deficiency in Adult Fracture and Fall Prevention 1145 Heike Bischoff-Ferrari, Bess Dawson-Hughes Clinical Disorders of Phosphate Homeostasis 1155 Karen E. Hansen, Marc K. Drezner Pseudo-vitamin D Deficiency 1187 Francis H. Glorieux, Thomas Edouard, Rene´ St-Arnaud Hereditary 1,25-Dihydroxyvitamin-D-Resistant Rickets 1197 Peter J. Malloy, Dov Tiosano, David Feldman Glucocorticoids and Vitamin D 1233 Philip Sambrook Drug and Hormone Effects on Vitamin D Metabolism Barrie M. Weinstein, Sol Epstein Vitamin D and Organ Transplantation 1291 Emily M. Stein, Elizabeth Shane Vitamin D and Bone Mineral Metabolism in Hepatogastrointestinal Diseases 1299 Daniel D. Bikle Vitamin D and Renal Disease 1325 Adriana S. Dusso, Eduardo Slatopolsky Idiopathic Hypercalciuria and Nephrolithiasis 1359 Murray J. Favus, Fredric L. Coe Hypercalcemia Due to Vitamin D Toxicity 1381 Natalie E. Cusano, Susan Thys-Jacobs, John P. Bilezikian Vitamin D: Cardiovascular Effects and Vascular Calcification 1403 Dwight A. Towler
79.
80. 81.
Lieve Verlinden, Guy Eelen, Roger Bouillon, Maurits Vandewalle, Pierre De Clercq, Annemieke Verstuyf Analogs for the Treatment of Osteoporosis 1489 Noboru Kubodera, Fumiaki Takahashi Non-secosteroidal Ligands and Modulators 1497 Keith R. Stayrook, Matthew W. Carson, Yanfei L. Ma, Jeffrey A. Dodge The Bile Acid Derivatives Lithocholic Acid Acetate and Lithocholic Acid Propionate are Functionally Selective Vitamin D Receptor Ligands 1509 Makoto Makishima, Sachiko Yamada CYP24A1 Regulation in Health and Disease 1525 Martin Petkovich, Christian Helvig, Tina Epps Calcitriol and Analogs in the Treatment of Chronic Kidney Disease 1555 Ishir Bhan, Ravi Thadhani
X CANCER 82. The Epidemiology of Vitamin D and Cancer Risk 1569 Edward Giovannucci
83. Vitamin D: Cancer and Differentiation 1591 1245
84. 85. 86. 87. 88. 89. 90.
Johannes P.T.M. van Leeuwen, Marjolein van Driel, David Feldman, Alberto Mun˜oz Vitamin D Effects on Differentiation and Cell Cycle 1625 George P. Studzinski, Elzbieta Gocek, Michael Danilenko Vitamin D Actions in Mammary Gland and Breast Cancer 1657 JoEllen Welsh Vitamin D and Prostate Cancer 1675 Aruna V. Krishnan, David Feldman The Vitamin D System and Colorectal Cancer Prevention 1711 Heide S. Cross Hematological Malignancy 1731 Ryoko Okamoto, H. Phillip Koeffler Vitamin D and Skin Cancer 1751 Jean Y. Tang, Ervin H. Epstein, Jr. The Anti-tumor Effects of Vitamin D in Other Cancers 1763 Donald L. Trump, Candace S. Johnson
IX ANALOGS 74. Alterations in 1,25-Dihydroxyvitamin D3
Structure that Produce Profound Changes in in Vivo Activity 1429 Hector F. DeLuca, Lori A. Plum 75. Mechanisms for the Selective Actions of Vitamin D Analogs 1437 Alex J. Brown
XI IMMUNITY, INFLAMMATION, AND DISEASE 91. Vitamin D and Innate Immunity 1777 John H. White
92. Control of Adaptive Immunity by Vitamin D Receptor Agonists 1789 Luciano Adorini
viii
CONTENTS
93. The Role of Vitamin D in Innate Immunity: Antimicrobial 94. 95. 96. 97.
Activity, Oxidative Stress and Barrier Function 1811 Philip T. Liu Vitamin D and Diabetes 1825 Conny Gysemans, Hannelie Korf, Chantal Mathieu Vitamin D and Multiple Sclerosis 1843 Colleen E. Hayes, Faye E. Nashold, Christopher G. Mayne, Justin A. Spanier, Corwin D. Nelson Vitamin D and Inflammatory Bowel Disease 1879 Danny Bruce, Margherita T. Cantorna Psoriasis and Other Skin Diseases 1891 Jo¨rg Reichrath, Michael F. Holick
99. Vitamin D Receptor Agonists in the Treatment of Benign 100. 101. 102. 103. 104.
XII THERAPEUTIC APPLICATIONS AND NEW ADVANCES 98. The Role of Vitamin D in Type 2 Diabetes and Hypertension 1907 Anastassios G. Pittas, Bess Dawson-Hughes
105.
Prostatic Hyperplasia 1931 Annamaria Morelli, Mario Maggi, Luciano Adorini Sunlight Protection by Vitamin D Compounds 1943 Rebecca S. Mason, Katie M. Dixon, Vanessa B. Sequeira, Clare Gordon-Thomson The Role of Vitamin D in Osteoarthritis and Rheumatic Disease 1955 M. Kyla Shea, Timothy E. McAlindon Vitamin D and Cardiovascular Disease 1973 Harald Sourij, Harald Dobnig Vitamin D, Childhood Wheezing, Asthma, and Chronic Obstructive Pulmonary Disease 1999 Carlos A. Camargo Jr., Adit A. Ginde, Jonathan M. Mansbach Vitamin D and Skeletal Muscle Function 2023 Lisa Ceglia, Robert U. Simpson The VITamin D and OmegA-3 TriaL (VITAL): Rationale and Design of a Large-Scale Randomized Controlled Trial 2043 Olivia I. Okereke, JoAnn E. Manson
Index 2057 Volume II Color Plate Section
Preface to the 3rd Edition The 3rd edition of Vitamin D was written at a time of great interest, exuberant hype, and even commotion in the public and lay press about vitamin D as a potential drug to treat and/or prevent multiple important and common diseases. Recent noteworthy events impacting the vitamin D field were the launching of the VITAL trial to discover whether vitamin D supplementation can reduce the risk of severe and life-threatening disease and the Institute of Medicine (IOM) report setting new dietary reference intakes (DRIs) for calcium and vitamin D. The IOM report expressed doubt on how well current data supported the beneficial actions of vitamin D on nonskeletal sites and called for more research to prove the hypothesis. This volume marshals the currently available data on basic mechanisms, normal physiology, and effects on disease and lays out for the reader up-todate and expert information on the role of vitamin D in health and many disorders. These and other current trends in vitamin D research are extensively covered in this new edition. The editors have continued our basic plan to constantly renew and remodel this book with each successive edition. To this end, we have added a new editor, Dr. John Adams, who has broad skill and knowledge in many areas of vitamin D research at both the basic science and clinical levels. John replaces Francis Glorieux who has undertaken to edit a separate book on pediatric bone disease. We thank Francis for his years of exemplary service to this book and wish him well in his new endeavors. John adds new energy and expertise to the editorial team. The 3rd edition has 105 chapters, making the book approximately the same size as the 2nd edition. However, the editors have worked very hard to revise and update this edition with new material and the presentation of fresh and different perspectives from respected authors. Some chapters covered in the 2nd edition have not been continued in this edition because relatively little new research was added in those areas. We thank the authors who are no longer contributing to this edition for their previous efforts. They may well be asked to write in the next edition as we continue our strategy of rotating authors. All chapters have been revised and updated and new references added. In our revitalization of the material in the book we
have added 32 new chapters to cover previously uncovered areas of research. In addition, we have changed the authorship of 20 additional chapters that are now written by different authors who have been charged with revising and updating previous chapters. These extensive modifications, with major updates and expansion of the content and the addition of totally new material in half of the chapters, has resulted in a substantially reorganized, modified, and modernized book compared to the 2nd edition. Finally, the expanded internet availability of the text and the figures will make access to the material easier and more flexible. Among the areas given new emphasis are nutrition, additional diseases that may be affected by vitamin D, and newly recognized biological pathways that regulate or are regulated by vitamin D. As we appreciate the full scope of vitamin D action, it has become clearer that the vitamin D endocrine system affects most if not all tissues in the body. We have tried to keep up with these advances in the state of knowledge about vitamin D by increasing our coverage of these newly recognized areas. We have enlisted the leading investigators in each area to provide truly expert opinion about each field. We would like to thank the excellent team at Elsevier/Academic Press for their outstanding support of our efforts to produce this new edition. We especially thank Mara Conner and Megan Wickline for their indispensable contributions to make this edition possible. We also want to extend our thanks and appreciation to the many authors who contributed to this volume. Without their hard work there of course would be no new edition. We therefore wish to express our gratitude for their willingness to offer their time and knowledge to make this book a success. Finally, we hope that this book will provide for our readers the authoritative information that they seek about the significance and importance of vitamin D in health and disease and serve as the means to keep their knowledge current about the continuing growth of the field of vitamin D biology. David Feldman J. Wesley Pike John S. Adams
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Preface to the 2nd Edition Those interested in the vitamin D field will not be surprised that this second edition is considerably larger than the first edition. A great deal of progress has been made since the first edition was published in 1997. However, our goal in planning this updated version remains the same. We have endeavored to provide investigators, clinicians, and students with a comprehensive, definitive, and up-to-date compendium of the diverse scientific and clinical aspects of vitamin D, each area covered by experts in the field. Our hope for the second edition is that this book will continue to serve as both a resource for current researchers, as well as a guide to assist those in related disciplines to enter the realm of vitamin D research. We hope that this book will illuminate the vitamin D field and help investigators identify areas where new research is needed as well as educate them about what is currently known. We believe that the first edition succeeded in stimulating interactions between researchers and clinicians from different disciplines and that it facilitated collaborations. As we move from basic science and physiology to the use of vitamin D and its analogs as pharmacological agents to treat various diseases, the need for crosscollaborations between researchers and clinicians from different disciplines will increase. We hope that this new volume will continue to be a valuable resource that plays a role in this advancement and stimulates and facilitates these interactions. Enormous progress in the study of vitamin D has been made in the approximately eight years since the first edition was written and we hope that this book has contributed in some way to this progress. The first edition proved to be highly valuable to its readers and the chapters have been cited frequently as authoritative reviews of the field. However, it became clear to us that the time was ripe to organize a second edition. Building on the original, we hope this second edition will
incorporate all of the progress made in the field since the first edition was published so that our objective of an up-to-date compendium containing everything you wanted to know about vitamin D will continue. The second edition is essentially a new and reinvigorated book. We have changed the symbol on the cover to reflect its updated content and the field’s continued evolution into the molecular world. This new edition now includes 104 chapters. In order to cover the growth of new information on vitamin D, we have had to publish this new edition in two volumes. In addition, the book has undergone some major remodeling. There are 33 completely new chapters and 18 other chapters have had major changes in authorship and are totally rewritten. While approximately half of the chapters have some of the same authors, all have had major updates and many have new co-authors with new perspectives. We have endeavored to attract the leading investigators in each field to author the chapter covering their area. We are especially pleased with the roster of authors who have written for the second edition. They really are the leaders in their respective fields. We wish to express our thanks to Tari Paschall, Judy Meyer, and Sarah Hajduk as well as the rest of the Elsevier/Academic Press staff for their expertise and indispensable contribution to bringing this revised edition to fruition. Most of all, we thank the authors for their contributions. We hope that our readers will find this updated volume useful and informative and that it will contribute to the burgeoning growth of the vitamin D field. David Feldman J. Wesley Pike Francis H. Glorieux
xi
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Preface to the 1st Edition Our reasons for deciding to publish an entire book devoted to vitamin D can be found in the rapid and extensive advances currently being made in this important field of research. Enormous progress in investigating many aspects of vitamin D, from basic science to clinical medicine, has been made in recent years. The ever-widening scope of vitamin D research has created new areas of inquiry so that even workers immersed in the field are not fully aware of the entire spectrum of current investigation. Our goal in planning this book was to bring the diverse scientific and clinical fields together in one definitive and up-to-date volume. It is our hope that this compendium on vitamin D will serve as both a resource for current researchers and a guide to stimulate and assist those in related disciplines to enter this field of research. In addition, we hope to provide clinicians and students with a comprehensive source of information for the varied and extensive material related to vitamin D. The explosion of information in the vitamin D sphere has led to new insights into many different areas, and in our treatment of each subject in this book we have tried to emphasize the recent advances as well as the established concepts. The classic view of vitamin D action, as a hormone limited to calcium metabolism and bone homeostasis, has undergone extensive revision and amplification in the past few years. We now know that the vitamin D receptor (VDR) is present in most tissues of the body and that vitamin D actions, in addition to the classic ones, include important effects on an extensive array of other target organs. To cover this large number of subjects, we have organized the book in the following manner: Section I, the enzymes involved in vitamin D metabolism and the activities of the various metabolites; Section II, the mechanism of action of vitamin D, including rapid, nongenomic actions and the role of the VDR in health and disease; Section III, the effects of vitamin D and its metabolites on the various elements that constitute bone and the expanded understanding of vitamin D actions in multiple target organs, both classic and nonclassic; Section IV, the role of vitamin D in the physiology and regulation of calcium and phosphate metabolism and the multiplicity of hormonal, environmental, and other factors influencing vitamin D metabolism and action; and Sections V and VI, the role of
vitamin D in the etiology and treatment of rickets, osteomalacia, and osteoporosis and the pathophysiological basis, diagnosis, and management of numerous clinical disorders involving vitamin D. The recent recognition of an expanded scope of vitamin D action and the new investigational approaches it has generated were part of the impetus for developing this volume on vitamin D. It has become clear that in addition to the classic vitamin D actions, a new spectrum of vitamin D activities that include important effects on cellular proliferation, differentiation, and the immune system has been identified. This new information has greatly expanded our understanding of the breadth of vitamin D action and has opened for investigation a large number of new avenues of research that are covered in Sections VII and VIII of this volume. Furthermore, these recently recognized nonclassic actions have led to a consideration of the potential application of vitamin D therapy to a range of diseases not previously envisioned. This therapeutic potential has spawned the search for vitamin D analogs that might have a more favorable therapeutic profile, one that is less active in causing hypercalcemia and hypercalciuria while more active in a desired application such as inducing antiproliferation, prodifferentiation, or immunosuppresion. Since 1a,25dihydroxyvitamin D [1,25(OH)2D] and its analogs are all presumed to act via a single VDR, a few years ago most of us probably would have thought that it was impossible to achieve a separation of these activities. Yet today, many analogs that exhibit different profiles of activity relative to 1,25(OH)2D have already been produced and extensively studied. The development of analogs with an improved therapeutic index has opened another large and complex area of vitamin D research. This work currently encompasses three domains: (1) the design and synthesis of vitamin D analogs exhibiting a separation of actions with less hypercalcemic and more antiproliferative or immunosuppressive activity, (2) the interesting biological question of the mechanism(s) by which these analogs achieve their differential activity, and (3) the investigation of the potential therapeutic applications of these analogs to treat various disease states. These new therapeutic applications, from psoriasis to cancer, from immunosuppression to neurodegenerative diseases, have drawn into the field an expanded
xiii
xiv
PREFACE TO THE 1ST EDITION
population of scientists and physicians interested in vitamin D. Our goal in editing this book was to create a comprehensive resource on vitamin D that would be of use to a mix of researchers in different disciplines. To achieve this goal, we sought authors who had contributed greatly to their respective fields of vitamin D research. The book has a large number of chapters to accommodate many contributors and provide expertise in multiple areas. Introductory chapters in each section of the book are designed to furnish an overview of that area of vitamin D research, with other chapters devoted to a narrowly focused subject. Adjacent and closely related subjects are often covered in separate chapters by other authors. While this intensive style may occasionally create some redundancy, it has the advantage of allowing the reader to view the diverse perspectives
of the different authors working in overlapping fields. In this regard, we have endeavored to provide many cross-references to guide the reader to related information in different chapters. We express our thanks to Jasna Markovac (Editor-inChief), for encouraging us to develop this book and guiding us through the process; to Tari Paschall (Acquisitions Editor) and the Academic Press staff for their diligence, expertise, and patience in helping us complete this work. Most of all, we thank the authors for their contributions that have made this book possible. David Feldman Francis H. Glorieux J. Wesley Pike
Contributors Nutrition
Mona S. Calvo US Food and Drug Administration, Laurel, MD, USA (979)
John S. Adams UCLA-Orthopaedic Hospital, Los Angeles, CA, USA (251, 777)
Carlos A. Camargo Jr. Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA (1999)
Judith E. Adams Manchester Royal Infirmary, Manchester, UK and Imaging Science and Biomedical Engineering, The University, Manchester, UK (861)
Margherita Cantorna The Pennsylvania State University, University Park, PA, USA (1879)
Steven A. Abrams USDA/ARS Children’s Research Center, Houston, TX, USA (647)
Luciano Adorini (1789, 1931)
Carsten Carlberg University of Luxembourg, Luxembourg and University of Eastern Finland, Kuopio, Finland (211)
Intercept Pharmaceuticals, Perugia, Italy
Thomas O. Carpenter Yale University School of Medicine, New Haven, CT, USA (1091)
Paul H. Anderson SA Pathology, Adelaide, South Australia, Australia and University of South Australia, Adelaide, South Australia, Australia (411)
Matthew W. Carson Lilly Indianapolis, IN, USA (1497)
Lenore Arab David Geffen School of Medicine at UCLA, Los Angeles, CA, USA (1009)
Lisa Ceglia
Research
Laboratories,
Tufts University, Boston, MA, USA (2023)
Hong Chen Emory University School of Medicine, Atlanta, GA, USA (251)
Gerald J. Atkins University of Adelaide, Adelaide, South Australia, Australia (411)
Songcang Chen University of California at San Francisco, San Francisco, CA, USA (541)
Ariane Berdal Universities Paris 5, Paris 6 and Paris 7, Paris, France and Rothchild Hospital, Assistance PubliqueHoˆpitaux de Paris, Paris, France (521)
Sylvia Christakos University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ, USA (363)
Ishir Bhan Massachusetts General Hospital, Boston, MA, USA (1555)
Fredric L. Coe The University of Chicago Pritzker School of Medicine, Chicago, IL, USA (1359)
Daniel Bikle Veterans Affairs Medical Center and University of California, San Francisco, CA, USA (1299)
Juliet Compston Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK (845)
John P. Bilezikian Columbia University College of Physicians and Surgeons, New York, NY, USA (1381)
Heide S. Cross Retired, Medical University of Vienna, Austria (1711)
Heike Bischoff-Ferrari University of Zurich, Switzerland and University Hospital Zurich, Switzerland (1145)
Natalie E. Cusano Columbia University College of Physicians and Surgeons, New York, NY, USA (1381)
Adele L. Boskey Hospital for Special Surgery, affiliated with Weil College of Cornell Medical School, New York, NY, USA (381)
Bess Dawson-Hughes USA (1145, 1907)
Roger Bouillon Laboratory of Experimental Medicine and Endocrinology, K.U. Leuven, Leuven, Belgium (57, 1067, 1461)
Tufts
University,
Boston,
MA,
Pierre De Clercq Universiteit Gent, Vakgroep Organische Chemie, Gent, Belgium (1461)
voor
Hector DeLuca University of Wisconsin-Madison, WI, USA (1429)
Barbara D. Boyan Georgia Institute of Technology, Atlanta, GA, USA (507)
Michael Danilenko Ben-Gurion University of the Negev, Beer-Sheva, Israel (1625)
Alex J. Brown Washington University School of Medicine, St. Louis, MO, USA (1437)
Valentin David University of Tennessee Health Science Center, Memphis, TN, USA (747)
Edward M. Brown Brigham and Women’s Hospital, Boston, MA, USA (425)
Hector F. Deluca University Madison, WI, USA (3)
Danny Bruce The Pennsylvania State University, University Park, PA, USA (1879)
of
Wisconsin-Madison,
San
Marie B. Demay Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA (533)
Thomas Burne The Park Centre for Mental Health, Wacol, Australia and University of Queensland, St. Lucia, Australia (565)
Francisco J.A. de Paula Maine Medical Center Research Institute and Department of Internal Medicine, School of Medicine of Ribeira˜o Preto, USP, Ribeira˜o Preto, SP, Brazil (769)
Andrew J. Burghardt University Francisco, CA, USA (891)
of
California,
xv
xvi
CONTRIBUTORS
Vianney Descroix Universities Paris 5, Paris 6 and Paris 7, Paris, France and Pitie´-Salpeˆtrie`re Hospital, Assistance Publique-Hoˆpitaux de Paris, Paris France (521) Puneet Dhawan University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ, USA (363)
David G. Gardner University of California at San Francisco, San Francisco, CA, USA (541) Adit A. Ginde University of Colorado Denver School of Medicine, Aurora, CO, USA (1999)
Katie M. Dixon University of Sydney, NSW, Australia (1943)
Edward Giovannucci Harvard School of Public Health, Boston, MA, USA and Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA (1569)
Harald Dobnig Medical University of Graz, Graz, Austria (1973)
Denis J. Glenn University of California at San Francisco, San Francisco, CA, USA (541)
Jeffrey A. Dodge Lilly Research Laboratories, Indianapolis, IN, USA (1497) Eve Donnelly Hospital for Special Surgery, affiliated with Weil College of Cornell Medical School, New York, NY, USA (381) Maryam Doroudi Georgia Institute of Technology, Atlanta, GA, USA (507) Diane R. Dowd Case Western Cleveland, OH, USA (193)
Reserve
University,
Marc K. Drezner William H. Middleton Veterans Administration Medical Center, Madison, WI, USA (1155) Adriana S. Dusso Experimental Nephrology Laboratory, Lleida, Spain (1325) Peter R. Ebeling Australia (1129)
University
of
Melbourne,
Victoria,
Thomas Edouard Shriners Hospital for Children, Montreal, Quebec, Canada (1187) Guy Eelen Katholieke Belgium (1461)
Universiteit
Leuven,
Leuven,
John A. Eisman Garvan Institute of Medical Research, Sydney, Australia (1129) Tina Epps (1525)
Cytochroma Inc., Markham, Ontario, Canada
Ervin H. Epstein Jr. Children’s Hospital Oakland Research Institute, Oakland, CA, USA (1751) Sol Epstein Mount Sinai School of Medicine, New York, NY, USA (1245) Darryl Eyles The Park Centre for Mental Health, Wacol, Australia and University of Queensland, St. Lucia, Australia (565) Mary C. Farach-Carson USA (457)
Rice University, Houston, TX,
Murray J. Favus The University of Chicago Pritzker School of Medicine, Chicago, IL, USA (1359) David Feldman Stanford University School of Medicine, Stanford, CA, USA (1197, 1591, 1675) David M. Findlay University of Adelaide, Adelaide, South Australia, Australia (411) James C. Fleet USA (349)
Purdue University, West Lafayette, IN,
Renny T. Franceschi MI, USA (321) Ryoji Fujiki
University of Michigan, Ann Arbor,
University of Tokyo, Tokyo, Japan
(227)
Francis H. Glorieux Shriners Hospital Montreal, Quebec, Canada (1187)
for
Children,
Elzbieta Gocek University of Wroclaw, Wroclaw, Poland (1625) David Goltzman McGill University and Royal Victoria Hospital of the McGill University Health Centre, Montreal, Quebec, Canada (583) Jose´ Manuel Gonza´lez-Sancho Universidad Auto´noma de Madrid, Madrid, Spain (235) Clare Gordon-Thomson Australia (1943)
University
of
Sydney,
NSW,
Conny Gysemans Katholieke Universiteit Leuven, Leuven, Belgium (1825) Karen E. Hansen USA (1155)
University of Wisconsin, Madison, WI,
Carol A. Haussler University of Arizona, Phoenix, AZ, USA (137) Mark R. Haussler USA (137)
University of Arizona, Phoenix, AZ,
Colleen E. Hayes University Madison, WI, USA (1843) Robert P. Heaney (607)
of
Wisconsin-Madison,
Creighton University, Omaha, NE, USA
Christian Helvig Cytochroma Inc., Markham, Ontario, Canada (1525) Geoffrey N. Hendy McGill University and Royal Victoria Hospital of the McGill University Health Centre, Montreal, Quebec, Canada (583) Martin Hewison UCLA-Orthopaedic Hospital, Los Angeles, CA, USA (251, 777) Arnold Lippert Hirsch AGD Nutrition LLC, Lewisville, Texas, USA (73) Michael F. Holick Boston Medical Center and Boston University School of Medicine, Boston, MA, USA (13, 1891) Bruce W. Hollis Medical University of South Carolina, Charleston, SC, USA (823) Elizabeth Holt Yale University School of Medicine, New Haven, CT, USA (725) Jui-Cheng Hsieh USA (137)
University of Arizona, Phoenix, AZ,
Karl L. Insogna Yale University School of Medicine, New Haven, CT, USA (1091)
xvii
CONTRIBUTORS
Candace Johnson Roswell Park Cancer Institute, Buffalo, NY, USA (1763)
Paul Lips VU University Medical Center, Amsterdam, The Netherlands (947)
Glenville Jones Queen’s University, Kingston, Ontario, Canada (23)
Philip T. Liu University of California at Los Angeles, CA, USA (1811)
Peter W. Jurutka Arizona State University at the West Campus, Glendale, AZ, USA (137)
Thomas S. Lisse UCLA-Orthopaedic Hospital, Los Angeles, CA, USA (251)
Heidi J. Kalkwarf Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA (679)
Yanfei L. Ma Lilly Research Laboratories, Indianapolis, IN, USA (1497)
Shigeaki Kato
Paul N. MacDonald Case Western Reserve University, Cleveland, OH, USA (193)
University of Tokyo, Tokyo, Japan
(227)
Steven A. Kliewer University of Texas Southwestern Medical Center, Dallas, TX, USA (763) H. Phillip Koeffler Division of Hematology and Oncology, Cedars-Sinai Medical Center, Los Angeles, CA, USA and National University of Singapore, Singapore (1731) Hannelie Korf Katholieke Universiteit Leuven, Leuven, Belgium (1825) Alexander Kouzmenko University of Tokyo, Tokyo, Japan and Alfaisal University, Riyadh, Kingdom of Saudi Arabia (227) Christopher S. Kovacs Memorial University of Newfoundland, Health Sciences Centre, St. John’s, Newfoundland, Canada (625) Barbara E. Kream University of Connecticut Health Center, Farmington, CT, USA (403) Richard Kremer McGill University and Royal Victoria Hospital of the McGill University Health Centre, Montreal, Quebec, Canada (583) Aruna V. Krishnan Stanford University School of Medicine, Stanford, CA, USA (1675) Roland Krug University of California, San Francisco, CA, USA (891) Noboru Kubodera Chugai Pharmaceutical Co., Ltd, Tokyo, Japan, present address: International Institute of Active Vitamin D Analogs (1489) Rajiv Kumar Mayo Clinic and Foundation, Rochester, MN, USA (471) Emma M. Laing University of Georgia, Athens, GA, USA (657) Joseph M. Lane USA (927)
Hospital for Special Surgery, New York, NY,
Marı´a Jesu´s Larriba Cientificas (235)
Consejo Superior de Investigacio nes
Seong Min Lee University of Wisconsin-Madison, Madison, WI, USA (97) Richard D. Lewis University of Georgia, Athens, GA, USA (657) Yan Li University of Michigan, Ann Arbor, MI, USA (321) Yan Chun Li (707)
The University of Chicago, Chicago, IL, USA
Jane B. Lian University of Massachusetts Medical School, Worcester, MA, USA (301) Alexander C. Lichtler University of Connecticut Health Center, Farmington, CT, USA (403)
Leila Mady University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ, USA (363) Mario Maggi University of Florence, Florence, Italy (1931) Sharmila Majumdar University of California, San Francisco, CA, USA (891) Makoto Makishima Nihon University School of Medicine, Tokyo, Japan (1509) Peter J. Malloy Stanford University School of Medicine, Stanford, CA, USA (1197) David J. Mangelsdorf University of Texas Southwestern Medical Center, Dallas, TX, USA (763) Jonathan M. Mansbach Children’s Hospital Boston, Harvard Medical School, Boston, MA, USA (1999) JoAnn E. Manson Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA (2043) Rebecca S. Mason University of Sydney, NSW, Australia (1943) Chantal Mathieu Katholieke Universiteit Leuven, Leuven, Belgium (1825) Christopher G. Mayne Medical College of Wisconsin, Milwaukee, WI, USA (1843) Timothy M. McAlindon Tufts Medical Center and Tufts University School of Medicine, Boston, Massachusetts (1955) John McGrath The Park Centre for Mental Health, Wacol, Australia and University of Queensland, St. Lucia, Australia (565) Mark B. Meyer University of Wisconsin-Madison, Madison, WI, USA (97) Mathew T. Mizwicki CA, USA (271)
University of California, Riverside,
Muriel Molla Universities Paris 5, Paris 6 and Paris 7, Paris, France and Rothchild Hospital, Assistance PubliqueHoˆpitaux de Paris, Paris, France (521) Martin Montecino Chile (301)
Universidad Andres Bello, Santiago,
Dino Moras Universite´ de Strasbourg, 67404 Illkirch, France (171) Annamaria Morelli University of Florence, Florence, Italy (1931) Howard A. Morris SA Pathology, Adelaide, South Australia, Australia and University of South Australia, Adelaide, South Australia, Australia (411)
xviii
CONTRIBUTORS
Alberto Mun˜oz Consejo Superior Cientificas, Madrid, Spain (1591)
Investigacio nes
Gary G. Schwartz Wake Forest University School of Medicine, Winston-Salem, NC, USA (965)
Mark S. Nanes Emory University School of Medicine, Atlanta, GA, USA (251)
Zvi Schwartz Georgia Institute of Technology, Atlanta, GA, USA (507)
Faye E. Nashold University Madison, WI, USA (1843)
Vanessa Sequeira University of Sydney, NSW, Australia (1943)
of
de
Wisconsin-Madison,
Tally Naveh-Many Hadassah Hebrew University Medical Center, Hadassah Hospital, Jerusalem, Israel (493)
Elizabeth Shane Columbia University College of Physicians & Surgeons, New York, NY, USA (1291)
Wei Ni University of California at San Francisco, San Francisco, CA, USA (541)
M. Kyla Shea Wake Forest University School of Medicine, Winston-Salem, NC, USA (1955)
Corwin D. Nelson University Madison, WI, USA (1843)
Wisconsin-Madison,
Justin Silver Hadassah Hebrew University Medical Center, Hadassah Hospital, Jerusalem, Israel (493)
University of California, Riverside,
Robert U. Simpson University of Michigan Medical School, Ann Arbor, MI, USA (2203)
Anthony W. Norman CA, USA (271) Fumiaki Ohtake
of
University of Tokyo, Tokyo, Japan
(227)
Ryoko Okamoto Division of Hematology and Oncology, Cedars-Sinai Medical Center, Los Angeles, CA, USA (1731)
Linda Skingle Cambridge University Hospitals Foundation Trust, Cambridge, UK (845)
NHS
Eduardo Slatopolsky Washington University School of Medicine, St. Louis, MO, USA (1325)
Olivia I. Okereke Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA (2043)
Harald Sourij (1973)
Medical University of Graz, Graz, Austria
Lubna Pal Yale University School of Medicine, New Haven, CT, USA (695)
Justin A. Spanier University Madison, WI, USA (1843)
Martin Petkovich Cytochroma Inc., Markham, Ontario, Canada and Queen’s University, Kingston, Ontario, Canada (1525)
Bonny L. Specker South Dakota State University, Brookings, SD, USA (679)
of
Wisconsin-Madison,
John M. Pettifor University of the Witwatersrand and Chris Hani Baragwanath Hospital, South Africa (1107)
Rene´ St-Arnaud Shriners Hospital for Children, Montreal, Quebec, Canada and McGill University, Montreal, Quebec Canada (43, 1187)
J. Wesley Pike University of Wisconsin-Madison, Madison, WI, USA (97)
Keith R. Stayrook Indiana University School of Medicine, Indianapolis, IN, USA (1497)
Anastassios G. Pittas Tufts Medical Center, Boston, MA, USA (1907)
Emily M. Stein Columbia University College of Physicians & Surgeons, New York, NY, USA (1291)
Lori A. Plum (1429)
Gary S. Stein University of Massachusetts Medical School, Worcester, MA, USA (301)
University of Wisconsin-Madison, WI, USA Queen’s University, Kingston, Ontario,
Janet L. Stein University of Massachusetts Medical School, Worcester, MA, USA (301)
L. Darryl Quarles University of Tennessee Health Science Center, Memphis, TN, USA (747)
George P. Studzinski UMD-New Jersey Medical School, Newark, NJ, USA (1625)
Brian J. Rebolledo Weill Cornell Medical College, New York, NY, USA (927)
Fumiaki Takahashi Japan (1489)
Jo¨rg Reichrath Universita¨tsklinikum Homburg, Germany (1891)
Jean Y. Tang Stanford University School of Medicine, Stanford, CA, USA (1751)
David E. Prosser Canada (23)
Natacha Rochel (171)
des
Saarlandes,
Universite´ de Strasbourg, Illkirch, France
Chugai Pharmaceutical Co., Ltd, Tokyo,
Hugh S. Taylor Yale University School of Medicine, New Haven, CT, USA (695)
Clifford J. Rosen School of Medicine of Ribeira˜o Preto, USP, Ribeira˜o Preto, SP, Brazil (769)
Peter Tebben Mayo Clinic and Foundation, Rochester, MN, USA (471)
F. Patrick Ross Washington University School of Medicine, St. Louis, MO, USA (335)
Ravi Thadhani Massachusetts General Hospital, Boston, MA, USA (1555)
Philip Sambrook University of Sydney, Sydney, NSW, Australia (1233)
Natalie W. Thiex South Dakota State University, Brookings, SD, USA (679)
Daniel R. Schmidt University of Texas Southwestern Medical Center, Dallas, TX, USA (763)
William R. Thompson USA (457)
Ryan D. Schoch USA (349)
Susan Thys-Jacobs Columbia University College of Physicians and Surgeons, New York, NY, USA (1381)
Purdue University, West Lafayette, IN,
University of Delaware, Newark, DE,
xix
CONTRIBUTORS
Dov Tiosano Meyer Children’s Hospital, Rambam Medical Center, Haifa, Israel (1197) Dwight A. Towler Washington University in St. Louis, St. Louis, MO, USA (1403) Donald Trump Roswell Park Cancer Institute, Buffalo, NY, USA (1763) Andre´ G. Uitterlinden Erasmus Medical Center, Rotterdam, The Netherlands (1025) Aasis Unnanuntana Hospital for Special Surgery, New York, NY, USA and Siriraj Hospital, Mahidol University, Bangkok, Thailand (927) Maurits Vandewalle Universiteit Gent, Vakgroep voor Organische Chemie, Gent, Belgium (1461) Marjolein van Driel Erasmus Medical Center, Rotterdam, The Netherlands (1591) Johannes P.T.M. van Leeuwen Erasmus Medical Center, Rotterdam, The Netherlands (1591) Andre J. van Wijnen University of Massachusetts Medical School, Worcester, MA, USA (301) Natasja van Schoor VU University Medical Center, Amsterdam, The Netherlands (947) Lieve Verlinden Katholieke Universiteit Leuven, Leuven, Belgium (1461) Annemieke Verstuyf Laboratorium voor Experimentele Geneeskunde en Endocrinologie, Leuven, Belgium (1461)
Reinhold Vieth University of Toronto, Toronto, Canada and Mount Sinai Hospital, Toronto, Canada (1041) Connie M. Weaver IN, USA (657)
Purdue University, West Lafayette,
Barrie M. Weinstein Mount Sinai School of Medicine, New York, NY, USA (1245) JoEllen Welsh University at Albany, Rensselaer, NY, USA (1657) John H. White (1777)
McGill University, Montreal, Canada
G. Kerr Whitfield USA (137)
University of Arizona, Phoenix, AZ,
Susan J. Whiting University of Saskatchewan, Saskatoon, Saskatchewan, Canada (979) Michael P. Whyte Shriners Hospital for Children and Washington University School of Medicine at BarnesJewish Hospital, St Louis, MI USA (807) John J. Wysolmerski Yale University School of Medicine, New Haven, CT, USA (725) Sachiko Yamada Nihon University School of Medicine, Tokyo, Japan (1509) Ian Yip David Geffen School of Medicine at UCLA, Los Angeles, CA, USA (1009)
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Introduction
On November 30, 2010, after nearly two years of deliberation, an Institute of Medicine (IOM)-appointed committee released their findings, “2011 Report on dietary reference intakes (DRIs) for calcium and vitamin D.” Among their many recommendations the important conclusions regarding vitamin D were: (1) most of the population was not currently vitamin-D-deficient: (2) that 600 IU/day for ages 1e70 and 800 IU/day if over age 70 was adequate to protect bones; and (3) that all of the other potential benefits of vitamin D, besides bone health, did not yet have compelling evidence to support advising higher doses. They concluded that higher doses of vitamin D should not be advised on a public health basis until further research was done. It should be noted that the European counterpart to this report concluded that 800 IU was the suggested daily intake. Although the IOM report was meant to provide the populations of Canada and the United States with Recommended Dietary Allowances (RDAs) and Tolerable Upper Intake Levels (ULs) for calcium and vitamin D, the committee also identified a large number of uncertainties surrounding the DRI values that they recommended. For instance, the committee expressed a need for more research into both the skeletal and nonskeletal actions of vitamin D. Despite the thousands of publications on vitamin D, the committee was clearly disappointed by the lack of rigorous randomized trials and convincing clinically applicable knowledge on the subject of vitamin D benefits beyond the skeleton. With this in mind, how does the IOM report impact what is written by the contributors to the third edition of “Vitamin D”? It is important for the readers of this book to know that the authors of each of the 105 chapters were asked to consider revising their chapters on the basis of the IOM report. For those authors contributing chapters in the book’s Sections III (Mineral and Bone Homeostasis), V (Human Physiology), VI (Diagnosis and Management), VII (Nutrition, Sunlight, Genetics and Vitamin D Deficiency), and VIII (Disorders) this
task was of particular importance, because, as mentioned above, the IOM determined there was insufficient causeand-effect evidence to support a role for vitamin D beyond its effects on bone health. That is not to say that vitamin D does not impact other human health conditions; the IOM committee simply stated that conclusive causal evidence was lacking in these areas and existing data were insufficient to support a public health statement for nonskeletal outcomes. As the authors in Sections IV (Targets), IX (Analogs), X (Cancer), XI (Immunity, Inflammation, and Disease) and XII (Therapeutic Applications and New Advances) remind us over and over again, definitive, randomized, clinical trial data supporting a role for vitamin D in the pathophysiology and/or treatment of nonskeletal human diseases are still wanting. However, as covered in essentially every chapter in the book, data highly suggestive of benefit in a multitude of diseases are so strong that many vitamin D researchers are persuaded that vitamin D will eventually be convincingly demonstrated to be efficacious in many disease states. Furthermore, many authors express the viewpoint that avoidance of vitamin D deficiency will be shown to prevent, delay, or reduce the development of numerous diseases. What is the reason convincing clinical studies are missing from the published literature? Most of the previous NIH-sponsored trials of vitamin D have focused on bone or musculoskeletal health. Moreover, there is a lack of pharmaceutical company interest in a nonpatentable small molecule like vitamin D as a therapeutic. Pharmaceutical companies are at work developing vitamin D analogs, but most of this work has not progressed beyond preclinical studies. Hopefully, much of the lack of interest in the use of vitamin D itself as a preventive or therapeutic agent for extraskeletal chronic diseases, including cardiovascular disease, cancer, diabetes, hypertension, cognitive decline, depression, lung disorders, infections, and autoimmune diseases, will be allayed by the recently initiated, NIH-funded, randomized, placebo-controlled VITamin
xxi
xxii
INTRODUCTION
D-OmegA-3 TriaL (VITAL); VITAL is reviewed by its principal investigator, Dr. JoAnn Manson, in Chapter 105 of this text. The central aim of VITAL is to determine whether the administration of 2000 IU daily with or without 1 g of marine omega-3 fatty acids (in a 2 2 factorial design) reduces the risk of developing heart disease, stroke, or cancer in those without a prior history of these illnesses. It is of note that all study participants will be allowed to take up to 800 IU of personal vitamin D supplements (a dose, when added to dietary sources, exceeds the IOM recommended daily intake). If shown to be efficacious alone or in combination with omega-3 fatty acids in preventing the leading causes of death of American men and women, then vitamin D supplementation at a daily dose higher than the IOM guidelines will be justified. However, even if a 2000 IU dose of vitamin D3 daily reduces the risk of one or more of these nonskeletal diseases, other controversies raised by the IOM report will no doubt persist or surface. For example, the IOM report claimed that most of the US population is not vitamin-D-deficient. This obviously raises the discussion of where the cut-points for deficiency should be placed. The IOM has chosen 20 ng/ml (50 nmol/L) as the cut-off, a concentration they felt was sufficient to maintain bone health. Some would argue this is not
high enough even for bone health, let alone the other potential diseases that vitamin D may benefit. There will be much continued discussion of this report in the literature and no doubt there will be spirited debate about some of its findings. It is not our intent to carry out a pro and con discussion of the report but to emphasize several points. Importantly, public health policy must be conservative and risk averse and the IOM concluded that it should await more convincing data before recommending higher vitamin D intakes. The IOM was also concerned that, on a public health level, advising millions of people to take higher doses of vitamin D for extended periods of time could raise safety issues not observed in much smaller and shorter studies. These are real concerns. Finally, the IOM called for continued research efforts to develop compelling data to demonstrate the benefits of vitamin D claimed by many researchers. Although there is disagreement about the potential risks of not instituting vigorous vitamin D supplementation now, the editors and authors agree that more and better research would be welcome. It is our hope that the compilation of evidence about vitamin D action in normal and disease states contained in this volume will help to clarify the state of the science and be of use in elucidating the role of vitamin D in health and disease.
Abbreviations
AA AC ACE ACF ACTH ADH ADHR ADP AHO AI AIDS Aj.AR ALP ANG II ANP APC APD APL AR ARC 5-ASA ATP ATRA AUC Bmax BARE bFGF BFU BGP BLM BMC BMD BMI BMP BMU bp BPH
BSA BUA [Ca2+]i
arachadonic acid adenyl cyclase angiotensin converting enzyme activation frequency adrenocorticotropin antidiuretic hormone (vasopressin) autosomal dominant hypophosphatemic rickets adenosine diphosphate Albright’s hereditary osteodystrophy adequate intake acquired immunodeficiency syndrome adjusted apposition rate alkaline phosphatase angiotensin II atrial natriuretic peptide antigen presenting cell aminohydroxypropylidene bisphosphonate atrichia with papular lesions androgen receptor activator recruited cofactor 5-aminosalicylic acid adenosine triphosphate all-trans-retinoic acid area under the curve maximum number of binding sites bile acid response element basic fibroblast growth factor burst-forming unit bone Gla protein (osteocalcin) basal lateral membrane bone mineral content bone mineral density body mass index bone morphogenetic protein basic multicellular unit base pairs benign prostatic hyperplasia
CaBP CAD CaM cAMP CaSR or CaR CAT CBG CBP CC CD CDCA CDK or Cdk cDNA CDP Cdx-2 CFU cGMP CGRP CHF CK-II CLIA cM Cm. Ln. CNS CPBA cpm CRE CREB CRF CsA CSF CT CTR
xxiii
bovine serum albumin bone ultrasound attentuation internal calcium ion molar concentration calcium-binding protein coronary artery disease calmodulin cyclic AMP calcium-sensing receptor chloramphenicol acetyltransferase corticosteroid-binding globulin competitive protein-binding assay chief complaint Crohn’s disease chenodeoxycholic acid cyclin-dependent kinase complementary DNA collagenase-digestible protein caudal-related homeodomain protein colony-forming unit cyclic GMP calcitonin gene-related peptide congestive heart failure casein kinase-II competitive chemiluminescence immunoassay centimorgans cement line central nervous system competitive protein-binding assays counts per minute cAMP response element cAMP response element binding protein chronic renal failure cyclosporin A colony-stimulating factor calcitonin or computerized tomography calcitonin receptor
xxiv CTX CVC CYP CYP24 DAG DBD DBP DBP DC DCA DCT DEXA or DXA 7-DHC DHEA DHT DIC DMSO DR DRIP DSP DSS E1 E2 EAE EBT EBV EC EC50 or ED50 ECaC ECF EDTA EGF ELISA EMSA EP1 ER ERE ERK Et
ABBREVIATIONS
cerebrotendinous xanthomatosis calcifying vascular cells cytochrome P450 cytochrome P450, 24-hydroxylase diacylglycerol DNA-binding domain diastolic blood pressure vitamin-D-binding protein dendritic cell deoxycholic acid distal convoluted tubule dual energy X-ray absorptiometry 7-dehydrocholesterol dehydroepiandrosterone dihydrotachysterol or dihydrotestosterone disseminated intravascular coagulation dimethyl sulfoxide direct repeat vitamin D receptor interacting protein dental sialoprotein dextran sodium sulfate estrone estradiol experimental autoimmune encephalitis electron-beam computed technology Epstein-Barr virus endothelial cells effective concentration (dose) to cause 50% effect epithelium calcium channel extracellular fluid ethylenediaminetetraacetic acid epidermal growth factor enzyme-linked immunosorbent assay electrophoretic mobility shift assay PG receptor-1 estrogen receptor or endoplasmic reticulum estrogen response element extracellular signal-regulated kinase endothelin
FACS FAD FCS FDA FFA FIT FMTC FP FRAP FS FSK FXR g g G0, G1, G2 GAG GC-MS G-CSF GDNF GFP GFR GH GHRH GIO GM-CSF GnRH GR GRE GRTH GWAS HAT HDAC HEK HHRH HIV HNF HPI HPLC HPV
fluorescence-activated cell sorting or sorter flavin adenine dinucleotide fetal calf serum US Food and Drug Administration free fatty acid Fracture Intervention Trial familial medullary thyroid carcinoma formation period fluorescence recovery after photobleaching Fanconi syndrome forskolin farnesoid X receptor gram acceleration due to gravity gap phases of the cell cycle glycosaminoglycan gas chromatography-mass spectrometry granulocyte colony-stimulating factor glial-cell-derived neurotrophic factor green fluorescent protein glomerular filtration rate growth hormone growth-hormone-releasing hormone glucocorticoid-induced osteoporosis granulocyte-macrophage colonystimulating factor gonadotropin-releasing hormone glucocorticoid receptor glucocorticoid response element generalized resistance to thyroid hormone genome-wide association study histone acetyltransferase histone deacetylase human embryonic kidney hereditary hypophosphatemic rickets with hypercalciuria human immunodeficiency virus hepatocyte nuclear factor history of present illness high-performance liquid chromatography human papilloma virus
ABBREVIATIONS
h HR HRE HSA Hsp HSV HVDRR HVO IBD IBMX IC50 ICA ICMA IDBP IDDM IDM IEL IFN Ig IGFBP IGF-I, -II IGF-IR IL i.m. IMCal iNKT i.p. IP3 IRMA IU IUPAC i.v. JG JNK Kd Km kb kbp kDa KO LBD LCA LDL
hour hairless hormone response element human serum albumin heat-shock protein herpes simplex virus hereditary vitamin-D-resistant rickets hypovitaminosis D osteopathy inflammatory bowel disease isobutylmethylxanthine concentration to inhibit 50% effect intestinal calcium absorption immunochemiluminometric assay intracellular vitamin-D-binding protein insulin-dependent diabetes mellitus infants of diabetic mothers intraepithelial cells interferon immunoglobulin IGF-binding protein insulin-like growth factor type I, II IGF-I receptor interleukin (e.g., IL-1, IL-1b, etc.) intramuscular intestinal membrane calciumbinding complex invariant NKT intraperitoneal inositol trisphosphate immunoradiometric assay international units International Union of Pure and Applied Chemists intravenous juxtaglomerular c-Jun NH2-terminal kinase dissociation constant Michaelis constant kilobases kilobase pairs kilodaltons knockout ligand-binding domain lithocholic acid low-density lipoprotein
Li. Ce. LIF LNH LOD LPS LT LXR M M MAPK Mab MAR MAR MARRS MCR M-CSF MEN2 MGP MHC min MIU MLR Mlt MR MRI mRNA MS MT MTC NADH NADPH NAF NBT NcAMP NCP NFkB NGF NHANES III NHL NIDDM NIH NK cell
xxv lining cell leukemia inhibitory factor late neonatal hypocalcemia logarithm of the odds lipopolysaccharide leukotriene liver X receptor mitosis phase of cell cycle molar mitogen-activated protein kinase monoclonal antibody matrix attachment region mineral apposition rate membrane-associated rapid response steroid metabolic clearance rate macrophage colony-stimulating factor multiple endocrine neoplasia type 2 matrix Gla protein major histocompatibility complex minute million international units mixed lymphocyte reaction mineralization lag time mineralocorticoid receptor magnetic resonance imaging messenger ribonucleic acid multiple sclerosis metric ton medullary thyroid carcinoma nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate nuclear accessory factor nitroblue tetrazolium nephrogenous cAMP noncollagen protein nuclear factor kappa B nerve growth factor National Health and Nutrition Examination Survey III Non-Hodgkin’s lymphoma non-insulin-dependent diabetes mellitus National Institutes of Health natural killer cell
xxvi NLS NMR NOD NPT NR Ob Oc OCIF OCT ODF 1a-(OH)D3 25(OH)D3 1,25(OH)2D3 24,25(OH)2D3 OHO Omt OPG OPN OSM OVX Pi PA2 PAD PAM PBL PBMC PBS PC PCNA PCR PCT PDDR PDGF PEIT PHEX
PG PHA PHP PIC PKA PKC
ABBREVIATIONS
nuclear localization signal nuclear magnetic resonance nod-like sodium/phosphate cotransporter nuclear receptor osteoblast osteocalcin or osteoclast osteoclastogenesis inhibitory factor (same as OPG) 22-oxacalcitriol osteoclast differentiation factor (same as RANKL) 1a-hydroxyvitamin D3 25-hydroxyvitamin D3 1a,25-dihydroxyvitamin D3 24,25-dihydroxyvitamin D3 oncogenic hypophosphatemic osteomalacia osteoid maturation time osteoprotegerin osteopontin oncostatin M ovariectomy inorganic phosphate phospholipase A2 peripheral arterial vascular disease pulmonary alveolar macrophage peripheral blood lymphocyte peripheral blood mononuclear cells phosphate-buffered saline phophatidyl choline proliferating cell nuclear antigen polymerase chain reaction proximal convoluted tubule pseudovitamin D deficiency rickets platelet-derived growth factor percutaneous ethanol injection therapy phosphate regulating gene with homologies to endopeptidases on the X chromosome prostaglandin phytohemagglutinin pseudohypoparathyroidism preinitiation complex protein kinase A protein kinase C
PKI PLA2 PLC PMA PMCA PMH p.o. poly(A) PPAR PR PRA PRL PRR PSA PSI PT PTH PTHrP PTX PUVA QCT QSAR 9-cis-RA RA RA Rag RANK RANKL RAP RAR RARE RAS RBP RCI RDA RFLP RIA RID RNase ROCs ROS RPA
protein kinase inhibitor phospholipase A2 phospholipase C phorbol 12-myristate 13-acetate plasma membrane calcium pump past medical history oral polyadenosine peroxisome proliferator-activated receptor progesterone receptor plasma renin activity prolactin pattern recognition receptors prostate-specific antigen psoriasis severity index parathyroid parathyroid hormone parathyroid hormone-related peptide parathyroidectomy psoralen-ultraviolet A quantitative computerized tomography quantitative structureeactivity relationship 9-cis-retinoic acid retinoic acid rheumatoid arthritis recombination activating gene receptor activator NF-kB receptor activator NF-kB ligand receptor-associated protein retinoic acid receptor retinoic acid response element renineangiotensin system retinol-binding protein relative competitive index recommended dietary allowance restriction fragment length polymorphism radioimmunoassay receptor interacting domain ribonuclease receptor-operated calcium channels reactive oxygen species ribonuclease protection assay
ABBREVIATIONS
RRA RT-PCR RXR RXRE SBP SD SDS SE SEM SH SHBG SLE SNP SNPs SOS Sp1 SPF SRC-1 SSCP SV40 SXA t1/2 T3 T4 TBG TBP TC TF TFIIB TG TGF TIO TK TLR TmP or TmPi TNBS TNF TPA
radioreceptor assay reverse transcriptase-polymerase chain reaction retinoid X receptor retinoid X receptor response element systolic blood pressure standard deviation sodium dodecyl sulfate standard error standard error of the mean social history sex-hormone-binding globulin systematic lupus erythematosus single nucleotide polymorphism single nucleotide polymorphisms speed of sound selective promoter factor 1 sun protection factor steroid receptor coactivator-1 single-strand conformational polymorphism simian virus 40 single energy X-ray absorptiometry half-time triiodothyronine thyroxine thyroid-binding globulin TATA-binding protein tumoral calcinosis tubular fluid general transcription factor IIB transgenic transforming growth factor tumor-induced osteomalacia thymidine kinase toll-like receptor tubular absorptive maximum for phosphorus trinitrobenzene sulfonic acid tumor necrosis factor 12-O-tetradecanoylphorbol13-acetate
TPN TPTX TR TRAP TRAP TRP TRE TRE TRH Trk TSH TSS UF US USDA UTR UV VDDR-I VDDR-II VDR VDRE VDRL VEGF VERT VICCs VSMC VSSCs WHI WRE WSTF WT XLH XRD ZEB
xxvii total parenteral nutrition thyroparathyroidectomized thyroid hormone receptor tartrate-resistant acid phosphatase thyroid hormone receptor associated proteins transient receptor potential thyroid hormone response element TPA response element thyrotropin-releasing hormone tyrosine kinase thyrotropin transcription start site ultrafiltrable fluid ultrasonography US Department of Agriculture untranslated region ultraviolet vitamin-D-dependent rickets type I (see PDDR) vitamin-D-dependent rickets type II (see HVDRR) vitamin D receptor vitamin D response element vitamin D receptor ligand vascular endothelial growth factor Vertebral Efficacy with Risedronate Therapy studies voltage-insensitive calcium channels vascular smooth muscle cell voltage-senstive calcium channels Women’s Health Initiative Wilms’ tumor gene, WT1, responsive element Williams syndrome transcription factor wild-type X-linked hypophosphatemic rickets X-ray diffraction zinc finger, E box-binding transcription factor
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Relevant Lab Values in Adults and Children
CRITERIA FOR VITAMIN D DEFICIENCY: 25(OH)D SERUM LEVELS Adult IOM recommendations
Deficient Normal Excessive
Conventional units
SI units
<20 ng/ml 20 ng/ml >50 ng ml
<50 nmol/L 50 nmol/L >125 nmol/L
Frequently used vitamin D cut-points by many laboratories
Deficient Insufficient Sufficient
Conventional units
SI units
< 20 ng/ml 20 to 29.9 ng/ml >30 ng/ml
<50 nmol/L 50e74.9 nmol/L >75 nmol/L
Pediatric (The IOM and the Pediatric Endocrine Society have agreed on these cut-points.)
Deficient Normal
Conventional units
SI units
<20 ng/ml 20 ng/ml
<50 nmol/L 50 nmol/L
xxix
xxx
RELEVANT LAB VALUES IN ADULTS AND CHILDREN
APPROXIMATE NORMAL RANGES FOR SERUM VALUES IN ADULTSa Measure
SI units
Conventional units
Conversion factorb
Ionized calcium Total calcium Phosphorous, inorganic 1,25(OH)2D
1.12e1.32 mmol/L 2.17e2.52 mmol/L 0.77e1.49 mol/L 60e108 pmol/L
4.5e5.3 mg/dl 8.7e10.1 mg/dl 2.4e4.6 mg/dl 25e45 pg/ml
0.2495 0.2495 0.3229 2.40
APPROXIMATE NORMAL RANGES FOR SERUM VALUES IN CHILDRENa Measure
SI units
Conventional units
Conversion factorb
Ionized calcium Total calcium Phosphorous, inorganic 1,25(OH)2D
1.19e1.29 mmol/L 2.25e2.63 mmol/L 1.23e1.62 mol/L 65e134 pmol/L
4.8e5.2 mg/dl 9.0e10.5 mg/dl 3.8e5.0 mg/dl 27e56 pg/ml
0.2495 0.2495 0.3229 2.40
USEFUL EQUIVALENCIES OF DIFFERENT UNITS Vitamin D Calcium Phosphorus
a
1 mg ¼ 40 IU 1 mmol ¼ 40 mg 1 mmol ¼ 30 mg
Normal ranges differ in various laboratories and these values are provided only as a general guide.
b
Conversion factor X conventional units ¼ SI units.
S E C T I O N
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C H A P T E R
1 Historical Overview of Vitamin D Hector F. Deluca Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA
present in natural foods was missing from the purified diets. Hopkins developed a growth test in which natural foods were found to support rapid growth of experimental animals whereas purified materials could not [3]. Funk had found similar results for the prevention of neuritis and reasoned that there were "vital amines" present in foods from natural sources and actually provided the basis for the term "vitamins" used later to describe essential micronutrients [5].
DISCOVERY OF THE VITAMINS Early Nutritional Views The field of nutrition was largely dominated in the nineteenth century by German chemists, led by Justus von Liebig [1]. They taught that adequacy of the diet could be described by an analysis of protein, carbohydrate, fat, and mineral. Thus, a diet containing 12% protein, 5% mineral, 10e30% fat, and the remainder as carbohydrate would be expected to support normal growth and reproduction. This view remained largely unchallenged until the very end of the nineteenth century and the beginning of the twentieth century [2e5]. However, evidence opposing this view began to appear. One of the first was the famous study of Eijkman who studied prisoners in the Dutch East Indies maintained on a diet of polished rice [6]. A high incidence of the neurological disorder beri-beri was recorded in these inmates. Eijkman found that either feeding whole rice or returning the hulls of the polished rice could eliminate beri-beri. Eijkman reasoned that polished rice contained a toxin that was somehow neutralized by the rice hulls. Later, a colleague, Grijns [7], revisited the question and correctly demonstrated that hulls contained an important and required nutrient that prevented beri-beri. Other reports revealed that microorganic nutrients might be present. The development of scurvy in mariners was a common problem. This disease was prevented by the consumption of limes on British ships (hence, the term “Limey” to describe British sailors) and sauerkraut and fruits on other ships. This led Holst and Frohlich to conclude that scurvy could be prevented by a nutrient present in these foods [8]. Experiments by Lunin, Magendie, Hopkins, and Funk showed that a diet of purified carbohydrate, protein, fat, and salt is unable to support growth and life of experimental animals [2e5]. This suggested that some unknown or vital factor
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10001-0
McCollum and Osborne and Mendel’s Discovery of Vitamin A and B Complex A key experiment demonstrating essential micronutrients was one carried out at the Wisconsin Agricultural Experiment Station, engineered by Stephen Moulton Babcock and carried out by E. B. Hart supported by McCollum and Steenbock [9]. Herds of dairy cows were maintained on a diet composed individually only of corn, oats, or wheat or were fed a mixture of all of these grains, all receiving the same amount of carbohydrate, protein, fat, and salts and all providing equal analysis according to the German chemists [1]. The animals on the corn diet did very well, produced milk in large amounts, and reproduced normally. Those on the wheat diet failed to thrive and soon were unable to reproduce or lactate. The oat group was found to be intermediate between the corn and wheat groups, and the mixture approximated the growth and reproduction found with corn. Yet all these diets had the same proximate analysis. The conclusion of the Wisconsin Experiment Station study was that there are unknown nutrients present in corn and not found in wheat that are essential for life and reproduction. This led E. B. Hart, Chairman of Biochemistry at Wisconsin, to conceive that a search for these nutrients must begin. Professor McCollum
3
Copyright Ó 2011 Elsevier Inc. All rights reserved.
4
1. HISTORICAL OVERVIEW OF VITAMIN D
was asked to search for these nutrients using small experimental animals. McCollum and Davis demonstrated there was present in butter fat a substance that prevented xerophthalmia and was also required for growth. They termed this "a lipin-soluble growth factor" [10]. McCollum later named this factor "vitamin A" [11]. This substance was absent from lard and other fats but was found in large amounts in cod liver oil. In constructing the diets, McCollum obtained the carbohydrates and salts from milk whey that, unknown to him, supplied the vitamin B complex group of micronutrients that permitted him to observe a vitamin A deficiency. McCollum at Wisconsin [11] and Osborne and Mendel [12] at the Connecticut Experiment Station carried out experiments in which cod liver oil was used as a source of fat in the diet but the minerals were supplied from pure chemicals mixed to approximate the mineral composition of milk. Starch or sugar was used as the carbohydrate. These animals developed a different group of symptoms, namely, neuritis, which could be cured by the provision of the milk components. McCollum and Osborne and Mendel correctly concluded that this activity was due to a different micronutrient called "vitamin B." This ushered in the concept of the organic micronutrients known as vitamins.
History of Rickets The disease rickets was very likely known in antiquity but was described in the fifteenth century as revealed by later writings. Whistler first provided a clear description of rickets in which the skeleton was poorly mineralized and deformed [13]. Rickets undoubtedly in ancient times appeared only on rare occasions and hence was not considered a problem. However, at the end of the nineteenth century, the Industrial Revolution had taken place: a highly agrarian population had become urbanized, and smoke from the industrial plants polluted the atmosphere. Thus, in low-sunlight countries such as England, rickets appeared in epidemic proportions. In fact, it was known as the English Disease [14]. Some reports of the beneficial action of cod liver oil had appeared. However, they were not given scientific credence. With the discovery of the vitamins, Sir Edward Mellanby in Great Britain began to reason that rickets might also be a disease caused by a dietary deficiency [15]. Mellanby fed dogs a diet composed primarily of oatmeal, which was the diet consumed where the incidence of rickets was the highest (i.e., Scotland). McCollum inadvertently maintained the dogs on oatmeal indoors and away from ultraviolet light. The dogs developed severe rickets. Learning from the experiments of McCollum, Mellanby provided cod liver oil to cure or prevent the disease. Mellanby could not decide whether
the healing of rickets was due to vitamin A known to be present in the cod liver oil or whether it was a new and unknown substance. Therefore, the activity of healing rickets was first attributed to vitamin A.
Discovery of Vitamin D McCollum, who had moved to Johns Hopkins from Wisconsin, continued his experiments on the fat-soluble materials. McCollum used aeration and heating of cod liver oil to destroy the vitamin A activity or the ability to support growth and to prevent xerophthalmia [16]. However, cod liver oil treated in this manner still retained the ability to cure rickets. McCollum correctly reasoned that the activity in healing rickets was due to a new and heretofore unknown vitamin that he termed "vitamin D." On the basis of the experiments of McCollum and of Mellanby, vitamin D became known as an essential nutrient.
DISCOVERY THAT VITAMIN D IS NOT A VITAMIN At the same time that Sir Edward Mellanby was carrying out the experiments in dogs, Huldshinsky [17] and Chick et al. [18] independently found that rickets in children could be prevented or cured by exposing them to sunlight or to artificially induced ultraviolet light. Thus, the curious findings were that sunlight and ultraviolet light somehow equaled cod liver oil. These strange and divergent results required resolution. Steenbock and Hart had noted the importance of sunlight in restoring positive calcium balance in goats [19]. At Wisconsin, with McCollum carrying out experiments in small experimental animals (i.e., rats), Steenbock was required to work with larger animals. Steenbock then began to study goats because they would consume less material and could serve as better experimental animals than cows. Steenbock began to study the calcium balance of lactating goats and found that those goats maintained outdoors in the sunlight were found to be in positive calcium balance, whereas those maintained indoors lost a great deal of their skeletal calcium to lactation [19]. Steenbock and Hart, therefore, noted the importance of sunlight on calcium balance. This work then undoubtedly led Steenbock to realize that the ultraviolet healing properties described by Huldschinky might be related to the calcium balance experiments in goats. By irradiating the animals and diets, Steenbock and Black found that vitamin D activity could be induced and rickets could be cured [20]. A similar finding was reported soon thereafter by Hess and Weinstock [21]. Steenbock then traced this to the
I. CHEMISTRY, METABOLISM, CIRCULATION
DISCOVERY OF THE PHYSIOLOGICAL FUNCTIONS OF VITAMIN D
nonsaponifiable fraction of the lipids in foods [22]. He found that ultraviolet light activated an inactive substance to become a vitamin-D-active material. Thus, ultraviolet light could be used to irradiate foods, induce vitamin D activity, and fortify foods to eliminate rickets as a major medical problem. This discovery also made available a source of vitamin D for isolation and identification.
ISOLATION AND IDENTIFICATION OF NUTRITIONAL FORMS OF VITAMIN D From irradiation of mixtures of plant sterols, Windaus and colleagues isolated a material that was active in healing rickets [23]. This substance was called "vitamin D1," but its structure was not determined. Vitamin D1 proved to be an adduct of tachysterol and vitamin D2, and thus vitamin D1 was actually an error in identification. The British group led by Askew was successful in isolating and determining the structure of the first vitamin D, vitamin D2 or ergocalciferol, from irradiation of plant sterols [24]. A similar identification by the Windaus group confirmed the structure of vitamin D2 [25]. Windaus and Bock also isolated the precursor of vitamin D3 from skin, namely, 7-dehydrocholesterol [26]. Furthermore, 7-dehydrocholesterol was synthesized [27] and converted to vitamin D3 (cholecalciferol) as identified by the Windaus group [28]. Thus, the structures of nutritional forms of vitamin D became known (Fig. 1.1). Windaus and Bock, having isolated 7-dehydrocholesterol from skin, provided the presumptive evidence that vitamin D3 is the form of vitamin D produced in skin, a discovery that was later confirmed by the chemical identification of vitamin D3 in skin by Esvelt et al. [29] and of a previtamin D3 in skin by Holick et al. [30]. Synthetic vitamin D as produced by the irradiation process replaced the irradiation of foods as a means of fortifying foods with vitamin D and was also rapidly applied to a variety of diseases including CH3
5
rickets and tetany and in the provision to domestic animals such as chickens, cows, and pigs. Windaus’ group provided chemical syntheses of the vitamin D compounds, confirming their structures and thus ending the era of the isolation and identification of nutritional forms of vitamin D and making them available for the treatment of disease. Although Windaus received the 1928 Nobel Prize in chemistry, it was for his general work on steroids.
DISCOVERY OF THE PHYSIOLOGICAL FUNCTIONS OF VITAMIN D Intestinal Calcium and Phosphorus Absorption Besides bone mineralization, the earliest discovered function of vitamin D is its important role in the absorption and utilization of calcium. The first report of this finding was in the early 1920s by Orr and colleagues [31]. Kletzien et al. [32] demonstrated that vitamin D plays an important role in the utilization of calcium from the diet, and a number of experiments were carried out on the utilization of calcium and phosphorus from cereal diets. Nicolaysen was responsible, however, for demonstrating unequivocally the role of vitamin D in the absorption of calcium and independently of phosphorus from the diet [33]. Nicolaysen also followed the early work of Kletzien et al. [32] in which animals adapted to a low-calcium diet were better able to utilize calcium than were animals on an adequate calcium diet. This work was confirmed by Nicolaysen, who postulated the existence of an "endogenous factor" that would inform the intestine of the skeletal needs for calcium [34]. This endogenous factor later proved to be largely the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) [35]. Strong support for this concept was provided by the studies of Ribovitch et al. [36] that showed animals maintained on a constant exogenous source of 1,25(OH)2D3 are unable to change intestinal calcium transport in response to changes in dietary calcium levels.
20
Mobilization of Calcium from Bone
25
8
7
3
HO
2
1
HO Cholecalciferol
FIGURE 1.1
Ergocalciferol
Nutritional forms of vitamin D.
For many years, investigators have attempted to show that vitamin D plays a role directly on the mineralization process of the skeleton. However, early work by Howland and Kramer [37], later work by Lamm and Neuman [38], and more recent work by Underwood and DeLuca [39] demonstrated very clearly that vitamin D does not play a significant role in the actual mineralization process of the skeleton but that the failure to mineralize the skeleton in vitamin D deficiency is due to inadequate levels of calcium and phosphorus in the
I. CHEMISTRY, METABOLISM, CIRCULATION
6
1. HISTORICAL OVERVIEW OF VITAMIN D
plasma. Thus, the action of vitamin D in mineralizing the skeleton and in preventing hypocalcemic tetany is the elevation of plasma calcium and phosphorus [40]. These discoveries laid to rest the concept of a role of vitamin D in mineralization. However, Carlsson [41] and Bauer et al. [42] were the first to realize that a major function of vitamin D is to induce the mobilization of calcium from bone when required. Thus, in animals on a low-calcium diet, the rise in serum calcium induced by vitamin D is the result of actual mobilization of calcium from bone [43]. This important function is known to be essential for the provision of calcium to meet soft tissue needs, especially those of nerves and muscle, on a minute-to-minute basis when it is in insufficient supply from the diet. It is likely that the function of vitamin D in mobilizing calcium from bone is an osteoclastic-mediated process [44]. It is clear, however, that both vitamin D and parathyroid hormone are required for this function [45]. Furthermore, it is clear that vitamin D plays an important role in osteoclasticmediated bone resorption [46], which is certainly the first and essential event in bone modeling [47].
Renal Reabsorption of Calcium and Phosphorus A final site of vitamin D action to elevate plasma calcium is in the distal renal tubule. Although experiments were suggestive of a role for vitamin D in increasing renal tubule reabsorption of calcium, a clear demonstration of this did not occur until the late 1980s at the hands of Yamamoto et al. [48]. The renal tubule reabsorbs 99% of the filtered calcium even in the absence of vitamin D. However, reabsorption of the last 1% of the filtered load requires both vitamin D and parathyroid hormone. Thus, these agents work in concert in the renal reabsorption of calcium as well as in the mobilization of calcium from bone. Both agents are required to carry out this function.
Discovery of New Functions of Vitamin D With discovery of the receptor for the vitamin D hormone (described below) came the surprising result that this receptor could be found in a variety of tissues not previously appreciated as targets of vitamin D action. It localizes in the distal renal tubule cells, enterocytes of the small intestine, bone lining cells, and osteoblasts in keeping with its known role in calcium metabolism [49,50]. However, its appearance in tissues such as parathyroid gland, islet cells of the pancreas, cells in bone marrow (i.e., promyelocytes), lymphocytes, and certain neural cells raised the question of whether the functions of vitamin D might be broader than previously anticipated [49,50]. As a result of those findings, new functions of vitamin D have been found. For
example, vitamin D plays a role in causing differentiation of promyelocytes to monocytes and the subsequent coalescing of the monocytes into multinuclear osteoclast precursors and ultimately into active osteoclasts [51,52]. The target of vitamin D in this function is the osteoblast and osteocyte. In response to the hormonal form of vitamin D (see below), RANK ligand is produced that signals osteoclastogenesis and osteoclastic activation [51,52]. Suppression of parathyroid cell growth and suppression of parathyroid hormone gene expression represent other new vitamin D actions [53,54]. In keratinocytes of skin, vitamin D appears to play a role in suppression of growth and in cellular differentiation [55]. Likely, discoveries of many new functions of 1,25(OH)2D3 will be made and are well on their way, as described in later chapters of this volume.
DISCOVERY OF THE HORMONAL FORM OF VITAMIN D Early Work of Kodicek The true pioneer of vitamin D metabolism was Egan Kodicek working at the Dunn Nutritional Laboratory in Cambridge. Kodicek used a bioassay at first to study the fate of the vitamin D molecule and found that much vitamin D was converted to biologically inactive products [56]. Clearly, however, this approach of assaying vitamin D activity following administration of known doses of vitamin D was of limited value in determining metabolism.
Radiolabeled Vitamin D Experiments Professor Kodicek then began to synthesize radiolabeled vitamin D2. Unfortunately, the degree of labeling was not sufficient to permit the administration of truly physiological doses of vitamin D. Nevertheless, Professor Kodicek continued investigations into this important area. At the conclusion of 10 years of work, he concluded that vitamin D was active without metabolic modification and that the metabolites that were found were biologically inactive [57]. This conclusion was reached even as late as 1967, when it was concluded that vitamin D3 itself was the active form of vitamin D in the intestine [58]. However, chemical synthesis of vitamin D3 of high specific activity in the laboratory of the author proved to be of key importance in the demonstration of biologically active metabolites [59]. By providing a truly physiological dose of vitamin D, it could be learned that the vitamin D itself disappeared and instead polar metabolites could be found in the target tissues before those tissues responded [60]. The polar metabolites proved to be more biologically active
I. CHEMISTRY, METABOLISM, CIRCULATION
DISCOVERY OF THE HORMONAL FORM OF VITAMIN D
and acted more rapidly than vitamin D itself [61]. Thus, presumptive evidence of conversion of vitamin D to active forms had been obtained as early as 1967.
Isolation and Identification of the Active Form of Vitamin D By 1968, the first active metabolite of vitamin D was isolated in pure form and chemically identified as 25hydroxyvitamin D3 (25(OH)D3) [62]. Its structure was confirmed by chemical synthesis [63] that provided it for study to the medical and scientific world. For a couple of years, 25(OH)D was visualized as the active form of vitamin D. However, when it was synthesized in radiolabeled form, it was rapidly metabolized to yet more polar metabolites [64]. By this time, the Kodicek laboratory reawakened their interest in metabolism of vitamin D and began to study the metabolism of latritium-labeled vitamin D [65]. Furthermore, polar metabolites of vitamin D were found by Haussler, Myrtle, and Norman [66]. The Wisconsin group labeled these metabolites as peak 5 [64], the Norman group called it peak 4B [66], and Lawson, Wilson, and Kodicek described it as peak P [65]. Kodicek et al. claimed that the metabolite of vitamin D found in intestine was deficient in tritium at the 1-position [65]. However, Myrtle et al. reported that peak 4B did not lose its tritium [67]. Thus, the suggestion of a modification at the 1-position could not be confirmed. The DeLuca group, however, isolated the active metabolite from intestines of 1600 chickens given radiolabeled vitamin D, and, by means of mass spectrometric techniques and specific chemical reactions, the structure of the active form of vitamin D in the intestine was unequivocally demonstrated as 1,25(OH)2D3 [68]. Of great importance was the finding of Fraser and Kodicek that the peak P metabolite could be produced by homogenates of chicken kidney and that anephric animals are unable to produce the peak P metabolite [69]. They correctly concluded that the site of synthesis of the active form of vitamin D is the kidney. The Wisconsin group then chemically synthesized both 1a,25(OH)2D3 [70] and 1b,25(OH)2D3 [71] and provided unequivocal proof that the active form is 1a,25(OH)2D3. Furthermore, this group was able to synthesize la(OH)D3, an important analog that assumed great importance as a therapeutic agent throughout the world [72].
7
mobilization, whereas animals receiving 25(OH)D3 at physiological doses did not [73e75]. Furthermore, the experiment of nature, namely, vitamin D-dependency rickets type I, an autosomal recessive disorder, provided final proof [76]. This disease could be corrected by physiological doses of synthetic 1,25(OH)2D3, whereas large amounts of vitamin D3 or 25(OH)D3 were needed to heal the rickets. The exact defect in this disease is now clearly known and is described elsewhere in this volume (see Chapter 64). 25(OH)D3 at pharmacological doses likely acts as an analog of the final vitamin D hormone, 1,25(OH)2D3 (Fig. 1.2).
Discovery of the Vitamin D Endocrine System Immediately after the identification of 1,25(OH)2D3 as the active form of vitamin D came studies in which it could be shown that animals on a low-calcium diet produce large quantities of 1,25(OH)2D3, whereas those on a high-calcium diet produce little or no 1,25(OH)2D3 [77]. A reciprocal arrangement was found for the metabolite 24R,25(OH)2D3. Thus, when calcium is needed, production of 1,25(OH)2D3 is markedly stimulated and the 24-hydroxylation degradation reaction is suppressed. When adequate calcium is present, production of 1,25(OH)2D3 is shut off and the 24-hydroxylation reaction is turned on. This discovery also satisfactorily provided evidence that 1,25(OH)2D3 is the likely endogenous factor originally described by Nicolaysen et al. [34]. The next important step was the demonstration that it is parathyroid hormone that activates 1a-hydroxylation of 25(OH)D3 in the kidney [78]. Thus, parathyroidectomy eliminates the hypocalcemic stimulation of 1ahydroxylation and suppression of 24-hydroxylation, whereas administration of parathyroid hormone restores that capability. Fraser and Kodicek also provided evidence that, in intact chickens, injection of parathyroid hormone stimulated the 1a-hydroxylation reaction [79]. Thus, the basic vitamin D endocrine system was largely discovered and reported in the early 1970s, being completed by 1974. Hypophosphatemia also stimulates 1a-hydroxylation [80,81] and 1,25(OH)2D3 increases absorption of phosphate [82,83]. Further, 1,25(OH)2D3 induces synthesis of the FGF23 that in turn causes phosphate elimination in the kidney (see Chapter 26) [84]. Thus, vitamin D is also a participant in the regulation of serum phosphate.
Proof that 1,25(OH)2D3 is the Active Form of Vitamin D
Other Metabolites of Vitamin D
Proof that 1,25(OH)2D3 and not 25(OH)D3 is the active form was provided by experiments in which anephric animals respond to 1,25(OH)2D3 by increasing intestinal absorption of calcium and bone calcium
During the 21,25(OH)2D3 25,26(OH)2D3 21,25(OH)2D3
course of identification of 1,25(OH)2D3, was reported as a metabolite, as was [85,86]. However, the identification of was in error and was corrected to
I. CHEMISTRY, METABOLISM, CIRCULATION
8
1. HISTORICAL OVERVIEW OF VITAMIN D
OH
HO
Liver
Kidney
Microsomes (Mitochondria)
Mitochondria
HO Vitamin D3
FIGURE 1.2
OH
HO 25-hydroxyvitamin D3
OH
3
Activation of the vitamin D3 molecule.
24,25(OH)2D3, with the correct stereochemistry as 24R,25(OH)2D3 [87]. Over the late 1970s and early 1980s, as many as 30 metabolites of vitamin D were identified [88]. These are covered in other chapters in this volume. Of great importance was the use of the fluoro derivatives of vitamin D to illustrate that the only activation pathway of vitamin D is 25-hydroxylation followed by 1-hydroxylation [89]. Thus, 24-difluoro-25 (OH)D3 supported all known functions of vitamin D for at least two generations of animals [90]. 24Difluoro-25(OH)D3 cannot be 24-hydroxylated. Furthermore, other fluoro derivatives such as 26,27-hexafluoro-25(OH)D3 [91] and 23-difluoro-25(OH)D3 [92] are all fully biologically active, illustrating that 26hydroxylation, 24-hydroxylation, and 23-hydroxylation are not essential to the function of vitamin D. The enzyme that carries out the 1a-hydroxylation is the CYP27B1 [93], while the enzyme that carries out the 23- and 24-hydroxylation is the CYP24A1 [94]. The 24R,25(OH)2D3 was at first believed to be another hormonal form of vitamin D, but these ideas were eliminated not only by the experiments with the 24,24-difluoro-25(OH)D3 but also by the CYP24A1null mice created by St. Arnaud and colleagues [95]. The 24-hydroxylase pathway is the degradative pathway of vitamin D metabolism and is induced by 1,25(OH)2D3 through the vitamin D receptor (VDR) and in VDR knockout mice, the lifetime of 1,25 (OH)2D3 is markedly increased [96]. This enzyme is responsible for the modifications resulting in calcitroic acid [97], the excretory product of vitamin D metabolism. The exact gene(s) and enzyme(s) responsible for the initial 25-hydroxylase step remain to be identified, although CYP2R1 is the most likely candidate [98].
Discovery of the Vitamin D Receptor Zull and colleagues provided evidence that the function of vitamin D is blocked by transcription and protein
synthesis inhibitors [99]. Thus, it became clear very early that a nuclear activity is required for vitamin D to carry out its functions. This work confirmed and extended the earlier work of Eisenstein and Passavoy [100]. With the discovery of the active forms of vitamin D came new attention to the idea that vitamin D may work through a nuclear mechanism. Thus, Haussler et al. reported vitamin D compounds to be associated with chromatin [66]. However, these experiments did not exclude the possibility that the vitamin D compounds might be bound to the nuclear membrane. The first clear demonstration of the existence of a VDR was at the hands of Brumbaugh and Haussler [101]. Furthermore, the experiments of Kream et al. [102] provided strong and unequivocal evidence of the existence of a nuclear receptor for 1,25(OH)2D3. Intense efforts toward purification of the receptor and its study appeared with the knowledge that it is an approximately 55,000 molecular weight receptor protein. In 1987, a partial cDNA sequence for the chicken VDR was determined [103]. This was followed by isolation of the full coding sequence for the human [104] and rat [105,106] receptors. Cloning of the cDNAs encoding the vitamin D receptor in human and mouse permitted the isolation of the gene encoding the vitamin D receptor [107e109]. The human gene was completely described and the mouse promoter was isolated and shown to be a TATA-less Sp1-driven promoter [109]. The human gene appears to have alternate promoters [107]. Two groups have prepared the receptor null mutant mice, permitting extensive experiments with vitamin D receptorless animals [110,111]. From a historical point of view, one of the most important discoveries was vitamin D-dependency rickets type II [112], which is now known to be due to a defect in the receptor gene [113,114] (discussed in Chapters 64 and 65). This discovery essentially provided receptor knockout experiments in humans, allowing unequivocal proof of the essentiality of the VDR for
I. CHEMISTRY, METABOLISM, CIRCULATION
REFERENCES
the functions of vitamin D. The nature of the receptor and how it functions are described in subsequent chapters along with current thinking on the molecular mechanism of action of 1,25(OH)2D3. However, the discoveries of vitamin D-responsive elements in the osteocalcin, osteopontin, preproparathyroid, and 24-OHase genes represent important historical developments [115e118]. This led to a consensus sequence and, most important, the development of the 3,4,5-rule of Umesono et al. [119]. It is now clear that vitamin D-responsive elements represent two imperfect repeat sequences separated by three nonspecified nucleotides. The VDR will bind to these response elements but it requires the presence of another nuclear factor which proved to be the retinoid-X receptor (RXR) [120e122]. It is quite clear that the vitamin D receptor forms a heterodimer on the vitamin D-responsive elements with the RXR protein on the 5’ arm of the responsive element and the VDR on the 3’ segment [123]. The work of Rosenfeld and Glass [123] has demonstrated that the RXR protein, when complexed with the VDR on the responsive elements, will not accept an RXR ligand. Details of what is known concerning the role of the VDR in transcription are described fully in subsequent chapters. That the VDR is regulated in kidney and parathyroid gland by 1,25(OH)2D3 itself and calcium is quite clear [124,125]. No VDRE(s) were found in the promoter of the VDR gene. However, elements were found elsewhere in the gene by Chip/Chip analysis, which has led to the idea that multiple enhancers distal from the promoter may be responsible not only for activation of this gene but many others as well [126,127]. Much remains to be discovered regarding the mechanisms through which 1,25(OH)2D3 regulates gene expression as described in several subsequent chapters.
Acknowledgments This work was supported by a fund from the Wisconsin Alumni Research Foundation.
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for binding of the 1,25-dihydroxyvitamin D3 receptor and 1,25dihydroxyvitamin D3 enhancement of mouse secreted phosphoprotein 1 (Spp1, osteopontin) gene expression, Proc. Natl. Acad. Sci. USA 87 (1990) 9995e9999. M.B. Demay, M.S. Kiernan, H.F. DeLuca, H.M. Kronenberg, Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D3, Proc. Natl. Acad. Sci. USA 89 (1992) 8097e8101. C. Zierold, H.M. Darwish, H.F. DeLuca, Two vitamin D response elements function in the rat 1,25-dihydroxyvitamin D 24-hydroxylase promoter, J. Biol. Chem. 270 (1995) 1675e1678. K. Umesono, K.K. Murakami, C.C. Thompson, R.M. Evans, Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors, Cell 65 (1991) 1255e1266. V.C. Yu, C. Delsert, B. Andersen, J.M. Holloway, O.V. Devary, A.M. Naar, et al., RXRb: a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements, Cell 67 (1991) 1251e1266. M. Munder, I.M. Herzberg, C. Zierold, V.E. Moss, K. Hanson, M. Clagett-Dame, et al., Identification of the porcine intestinal accessory factor that enables DNA sequence recognition by vitamin D receptor, Proc. Natl. Acad. Sci. USA 93 (1995) 2796e2799. C.H. Jin, J.W. Pike, Human vitamin D receptor-dependent transactivation in Saccharomyces cerevisiae requires retinoid X receptor, Mol. Endo. 10 (1996) 196e205. J. DiRenzo, M. Soderstrom, R. Kurokawa, M.-H. Ogliastro, M. Ricote, S. Ingrey, et al., Peroxisome proliferator-activated receptors and retinoic acid receptors differentially control the interactions of retinoid X receptor heterodimers with ligands, coactivators, and corepressors, Mol. Cell. Biol. 17 (1997) 2166e2176. K.D. Healy, J.B. Zella, J.M. Prahl, H.F. DeLuca, Regulation of the murine renal vitamin D receptor by 1,25-dihydroxyvitamin D3 and calcium, Proc. Natl. Acad. Sci. USA 100 (2003) 9733e9737. A.J. Brown, M. Zhong, J. Finch, C. Ritter, E. Slatopolsky, The roles of calcium and 1,25-dihydroxyvitamin D3 in the regulation of vitamin D receptor expression by rat parathyroid glands, Endocrinology 136 (1995) 1419e1425. L.A. Zella, S. Kim, N.K. Shevde, J.W. Pike, Enhancers located within two introns of the vitamin D receptor gene mediate transcriptional autoregulation by 1,25-dihydroxyvitamin D3, Mol. Endocrinol. 20 (2006) 1231e1247. S. Kim, M. Yamazaki, L.A. Zella, N.R. Shevde, J.W. Pike, Activation or receptor activator of NF-kappaB ligand gene expression by 1,25-dihydroxyvitamin D3 is mediated through multiple long-range enhancers, Mol. Cell. Biol. 26 (2006) 6469e6486.
I. CHEMISTRY, METABOLISM, CIRCULATION
C H A P T E R
2 Photobiology of Vitamin D Michael F. Holick Vitamin D, Skin, and Bone Research Laboratory, Department of Medicine, Endocrinology, Nutrition and Diabetes Section, Boston Medical Center and Boston University School of Medicine, Boston, Massachusetts
sunlight was a common denominator associated with the high incidence of rickets in children living in the inner cities when compared to children living in underdeveloped countries. Both Sniadecki [4] and Palm [5] encouraged systematic sunbathing as a means of preventing and curing rickets, but their recommendations went unheeded. Huldschinsky [6] was the first to clearly demonstrate that it was exposure of the skin to ultraviolet radiation that was responsible for the antirachitic activity of sunlight that had been postulated by Sniadecki [4] and Palm [5]. He exposed four rachitic children to radiation from a mercury arc lamp and reported dramatic reversal of rickets within 4 months. He further reasoned that the radiation responsible for producing the antirachitic factor was the same radiation that caused melanization of the skin. The intimate relationship between sunlight and its antirachitic properties in humans was finally demonstrated in 1921 when Hess and Unger [7] took rachitic children and exposed them to sunlight on the roof of a New York City hospital and demonstrated that within 4 months the rachitic lesions had healed. The concept that rickets was caused by a nutritional deficiency was supported by the observation in 1827 by Bretonneau, who treated a 15-month-old child with acute rickets with cod liver oil and noted the incredible speed at which the patient was cured [8]. His student Trousseau used liver oils from a variety of fish and aquatic mammals including herring, whales, and seals for the treatment of rickets and osteomalacia. In the early twentieth century, Mellanby [9] conducted classic studies where he demonstrated that rachitic beagles that were fed cod liver oil were cured of their bone-deforming disease. Originally, it was thought that the active antirachitic factor in cod liver oil was vitamin A. However, McCollum et al. [10] heated cod liver oil to destroy the
INTRODUCTION Vitamin D is neither a vitamin nor a hormone, but is created when adequate exposure to sunlight is available to promote the synthesis of vitamin D in the skin [1]. First produced in ocean-dwelling phytoplankton and zooplankton, vitamin D has been made by life forms on earth for almost 1 billion years [2]. Although the physiological function of vitamin D in these lower life forms is uncertain, it is well recognized that the photosynthesis of vitamin D became critically important for land-dwelling vertebrates that required a mechanism to increase the efficiency of absorption of dietary calcium. The major physiological function of vitamin D in vertebrates is to maintain extracellular fluid concentrations of calcium and phosphorus within a normal range. Vitamin D accomplishes this by increasing the efficiency of the small intestine to absorb dietary calcium and phosphorus, and by stimulating the mobilization of calcium and phosphorus stores from the bone [3].
HISTORICAL PERSPECTIVE It was the lack of appreciation of the beneficial effect of sunlight in preventing rickets that caused the widespread endemic outbreak of this bone-deforming disease in the seventeenth through nineteenth centuries. As early as 1822, Sniadecki [4] had suggested that the high incidence of rickets that occurred in Warsaw, Poland, in the early 1800s was likely caused by the lack of adequate exposure to sunlight. This was based on his observations that whereas children who lived in Warsaw had a very high incidence of rickets, children who lived in the rural areas outside of the city did not suffer the same malady. Seventy years later Palm [5] also recognized that lack of
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10002-2
13
Copyright Ó 2011 Elsevier Inc. All rights reserved.
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2. PHOTOBIOLOGY OF VITAMIN D
vitamin A activity and demonstrated that the preparation maintained its antirachitic activity. He, therefore, coined the term “vitamin D” for the antirachitic factor. The appreciation that exposure to sunlight could cure rickets in children prompted Steenbock and Black [11] and Hess and Weinstock [12] in 1924 to expose a variety of foods and other substances including vegetable oils, animal feed, lettuce, wheat, and grasses to ultraviolet radiation. They found that this process imparted antirachitic activity to all of the substances. These observations led Steenbock [13] to conclude that there would be great utility in irradiation of food substances for the prevention and cure of rickets in children. This concept initially led to the addition of provitamin D (ergosterol or 7dehydrocholesterol) to milk and its subsequent irradiation to impart antirachitic activity. Once vitamin D (D without a subscript refers to either D2 or D3) was inexpensive to produce, vitamin D was directly added to milk. This simple concept led to the eradication of rickets as a significant health problem in the United States and other countries that followed this practice. A more comprehensive summary of this historical perspective can be found in Chapter 1.
production of vitamin D3 in the skin due to chronic excessive exposure to sunlight, especially in people who live near the equator. Although it is likely that increased pigmentation was important for the prevention of skin cancer, there is little evidence that skin pigmentation evolved to prevent vitamin D intoxication. The reason for this is that once previtamin D3 is formed in the skin, it either can isomerize to vitamin D3 or can absorb solar ultraviolet B radiation and isomerize into biologically inactive photoisomers lumisterol and tachysterol [17] (Fig. 2.1). Furthermore, when vitamin D3 is made, it can either enter into the circulation or absorb solar ultraviolet photons and isomerize to at least three photoproducts including suprasterol I, suprasterol II, and 5,6-transvitamin D3 [18] (Fig. 2.1). Thus, sunlight itself can regulate the total output of vitamin D3 in the skin by causing the photodegradation of previtamin D3 and vitamin D3. This is the likely explanation for why there are no reported cases of vitamin-D-intoxication-induced hypercalcemia from chronic excessive exposure to sunlight. Melanin, on the other hand, provides the body with an effective natural sunscreen. Thus, increased melanin pigmentation will reduce the efficiency of the SUN
PHOTOBIOLOGY OF VITAMIN D Photosynthesis of Vitamin D3
SUN
SUN
When human skin is exposed to sunlight, it is the solar ultraviolet B photons with energies between 290 and 315 nm that are responsible for causing the photolysis of 7-dehydrocholesterol (provitamin D3; the immediate precursor in the cholesterol biosynthetic pathway) to previtamin D3 [1,14] (Fig. 2.1). This photochemical process occurs in the plasma membrane of skin cells; as a result, the thermodynamically unstable cis, cis isomer of previtamin D3 is rapidly transformed by a rearrangement of double bonds to form vitamin D3 [15] (Fig. 2.2). Approximately 50% of previtamin D3 is converted to vitamin D3 within 2 h. As vitamin D3 is formed in the membrane, its open flexible structure is likely jettisoned from the plasma membrane into the extracellular space. Once vitamin D3 enters the extracellular fluid space, it is attracted to the vitamin D binding protein (DBP) in the circulation, and thus enters the dermal capillary bed.
Regulation of Vitamin D Synthesis by Sunlight In the late 1960s, a theory was popularized that human skin pigmentation evolved to play a critically important role in regulating the cutaneous photosynthesis of vitamin D3. Loomis [16] speculated that melanin pigmentation in humans evolved for the purpose of preventing excessive
H HO 7-DEHYDROCHOLESTROL
SUN H
HO
LUMISTEROL3
SUN HO
PREVITAMIN D3 CH3
H BLOOD VESSEL
OH TACHYSTEROL3
DBP HO
SUN
VITAMIN D3
SUN
SUN
OH SUPRASTEROL I
HO SUPRASTEROL II OH 5,6-TRANSVITAMIN D
FIGURE 2.1 Photochemical events that lead to the production and regulation of vitamin D3 in the skin. Reprinted with permission from Holick [76]. DBP is the plasma vitamin D binding protein.
I. CHEMISTRY, METABOLISM, CIRCULATION
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PHOTOBIOLOGY OF VITAMIN D
OH
s-trans,s-cis-PRE-D3
SUN
C HO
A
D
B
19
HEXANE
PRO-D3
HO
3
C
D
T1/2
10 9
91 HRS
5 6 7
s-cis,s-cis-PRE-D3 25 C
CH3
SUN HO
VITAMIN D3
T1/2 8 HRS
SKIN
HO PRO-D3
HO s-cis,s-cis-PRE-D3
Photolysis of provitamin D3 (pro-D3) into previtamin D3 (pre-D3) and its thermal isomerization to vitamin D3 in hexane and in lizard skin. In hexane, pro-D3 is photolyzed to s-cis,s-cis-pre-D3. Once formed, this energetically unstable conformation undergoes a conformational change to the s-trans,s-cis-pre-D3. Only the s-cis, s-cis-pre-D3 can undergo thermal isomerization to vitamin D3. The s-cis,s-cis conformer of pre-D3 is stabilized in the phospholipid bilayer by hydrophilic interactions between the 3-hydroxyl group and the polar head of the lipids, as well as by van der Waals interactions between the steroid ring and side-chain structure and the hydrophobic tail of the lipids. These interactions significantly decrease the conversion of the s-cis,s-cis conformer to the s-trans,s-cis conformer, thereby facilitating the thermal isomerization of scis,s-cis-pre-D3 to vitamin D3. Reprinted with permission from Holick et al. [15].
FIGURE 2.2
Influence of Latitude, Season, and Time of Day on Vitamin D Synthesis It was recognized at the beginning of the twentieth century that the incidence of rickets was much higher during the winter and early spring months and that rickets was less prevalent during the summer and autumn [21]. An evaluation of the effect of season on the cutaneous production of vitamin D3 in Boston revealed that during the summer months of June and July, 7-dehydrocholesterol was most efficiently converted to previtamin D3 [22] (Fig. 2.3). There was a gradual decline in the production of previtamin D3 after August, and there was essentially no previtamin D3 formed in human skin after November (Fig. 2.3). Previtamin D3 photosynthesis commenced in the middle of March. To evaluate the influence of latitude on the cutaneous production of vitamin D3, similar studies were conducted
30
25
% Photoproducts from 7-DHC
sun-mediated photosynthesis of previtamin D3 [19]. This is particularly important in Blacks who live in northern latitudes and who ingest very little dietary vitamin D. It is the likely explanation for why Blacks have lower circulating concentrations of 25-hydroxyvitamin D3 (25(OH)D) and are more prone to developing vitamin D deficiency [20].
20
15
10
5
0
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
Month Photosynthesis of previtamin D3 after exposure of 7-dehydrocholesterol (7-DHC) to sunlight. Measurements were as follows: in Boston (42 N) after 1 h (V) and 3 h (,) and total photo products (previtamin D3, lumisterol, and tachysterol) after 3 h in Boston (C); in Edmonton, Canada (52 N), after 1 h (-); in Los Angeles (34 N) (:) and Puerto Rico (18 N) in January (B). Reprinted with permission from Webb et al. [22].
FIGURE 2.3
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2. PHOTOBIOLOGY OF VITAMIN D
Influence of season, time of day, and latitude on the synthesis of previtamin D3 in northern (A and C) and southern hemispheres (B and D). The hour indicated in C and D is the end of the 1-h exposure time. (Reproduced with permission from Chen TC 1998 The Photobiology of Vitamin D. In: Holick MF (ed) Vitamin D e Physiology, Molecular Biology and Clinical Applications. Humana Press, Totowa, NJ, pp. 17e37).
FIGURE 2.4
in Edmonton, Canada (52 N), Los Angeles (34 N), and San Juan, Puerto Rico (18 N). In Edmonton, the photosynthesis of previtamin D3 essentially ceased by midOctober and did not resume until mid-April. However, in Los Angeles and San Juan, the production of previtamin D3 in the skin occurred throughout the year (Fig. 2.3). The cutaneous production of vitamin D3 was also evaluated in Boston every hour from the time the sun rose to the time the sun set in the middle of the month on cloudless days for a full year. It was found that during the summer, sunlight was capable of producing vitamin D3 in the skin from 10:00 to 17:00 hr Eastern Standard Time (EST) (Fig. 2.4). However, as the zenith angle of the sun increased in the spring and autumn, previtamin D3 photosynthesis in the skin began at approximately 10:30 and ceased at approximately 16:00 EST [1].
Influence of Sunscreen Use, Melanin, Clothing, Glass, and Plastics on Vitamin D Synthesis Any substance such as melanin, clothing, or a sunscreen that absorbs ultraviolet B radiation will reduce the cutaneous production of vitamin D3
[1,23e25]. Sunscreen use is now widely accepted as an effective method for diminishing the damaging effects due to chronic excessive exposure to sunlight. Sunscreens are of great benefit because they can prevent sunburn, skin cancer, and skin damage associated with exposure to sunlight. The solar radiation that is most responsible for causing damage to the skin is the ultraviolet B (UVB) radiation. Because the major function of sunscreens is to absorb solar UVB radiation on the surface of the skin before it penetrates into the deeper viable layers, sunscreen use diminishes the total number of UVB photons that can reach the 7-dehydrocholesterol stores in the viable epidermis to form previtamin D3. Topical application of a sunscreen will substantially diminish or completely prevent the cutaneous production of previtamin D3 [25]. The topical application of a sunscreen with a sun protection factor (SPF) of 8, followed by whole-body exposure to one minimal erythemal dose of simulated sunlight, prevented any significant increase in the blood levels of vitamin D3 in healthy young adult volunteers. On the other hand, without the sunscreen, there was a marked 10e20-fold increase in circulating concentrations of vitamin
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ROLE OF SUNLIGHT AND DIETARY VITAMIN D IN BONE HEALTH, OVERALL HEALTH, AND WELL-BEING
60
Without Sunscreen
60 40
Serum 25-Hydroxyvitamin D (ng/ml)
Serum Vitamin D (nmol/L)
80
With Sunscreen
20 0
2
0
2
4
8 6 Days
10
12
14
16
FIGURE 2.5 Effect of sunscreen on vitamin D status. Circulating
concentrations of vitamin D were measured in young adults after application of a cream either with a sun protection factor of 8 or without sunscreen (topical placebo cream) following a single exposure to one minimal erythemal dose of simulated sunlight. Reprinted with permission from Matsuoka et al. [23].
D3 [23] (Fig. 2.5). The elderly are often very concerned about their health and appearance, and some will consistently apply a topical sunscreen on all sunexposed areas and/or wear clothing over most sunexposed areas before going outdoors. Since aging significantly decreases the capacity of the skin to produce vitamin D3 because of the marked decline in 7-dehydrocholesterol in the epidermis, chronic sunscreen use by the elderly can increase the risk of vitamin D deficiency [1,23] (Fig. 2.6). Similarly, since clothing absorbs ultraviolet B radiation, the wearing of clothing on body surfaces prevents the cutaneous production of vitamin D3 in those covered surfaces [24] (Fig. 2.7). Thus, in cultures that require the covering of almost the entire body with clothing, such as Bedouins living in the Negev Desert, despite the intense sunlight environment, the Bedouin women have an increased risk of vitamin D deficiency and osteomalacia
Serum Vitamin D3 (ng/ml)
80 60 40 20 0
None
Summer
Autumn
Summer and Sunscreen
FIGURE 2.6 Effect of clothing and sunscreen on vitamin D status. Circulating concentrations of vitamin D were measured in human subjects who wore either no clothing, summer-type clothing, autumntype clothing, or summer-type clothing and sunscreen 24 h after a whole-body exposure to one minimal erythemal dose of ultraviolet B radiation. Reprinted with permission from Matsuoka et al. [24].
50
PA IL
40
30
20
10
0 Without Sunscreen
With Sunscreen
Effect of chronic sunscreen use on vitamin D status. Circulating concentrations of 25-hydroxyvitamin D were measured in adults from Pennsylvania (PA) and Illinois (IL) who always wore a sunscreen or never wore a sunscreen. Reprinted with permission from Matsuoka et al. [25].
FIGURE 2.7
and their children are more prone to developing vitamin-D-deficiency rickets [26]. Because glass and Plexiglas efficiently absorb most if not all ultraviolet B photons, exposure of the skin to sunlight through glass or Plexiglas will not produce any vitamin D3 (Fig. 2.8).
ROLE OF SUNLIGHT AND DIETARY VITAMIN D IN BONE HEALTH, OVERALL HEALTH, AND WELL-BEING Vitamin D deficiency is finally being recognized as an epidemic for all age groups in most industrialized countries [3,27e35]. Ninety to 95% of our vitamin D requirement comes from exposure to sunlight [3,36]. Most experts agree that in the absence of sun exposure, at least 1000 IU and preferably 2000 IU of vitamin D/day is required to satisfy the adult’s vitamin D need [37,38,40]. Children need at least 400 IU of vitamin D/ day and 1000 IU/d may have additional health benefits for them [36] (Table 2.1). If children and adults received sensible exposure to sunlight during the spring, summer, and fall, they are able to store vitamin D in their body fat and call upon it during the winter when the sun is incapable of producing vitamin D in the skin. However, because of inadequate dietary intake of vitamin D and the increased concern about the risk of skin cancer from sun exposure and because both children and adults have fewer outdoor activities, there is a resurgence of vitamin D deficiency for the population
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2. PHOTOBIOLOGY OF VITAMIN D
ovarian, and esophageal cancers and non-Hodgkin’s and Hodgkin’s lymphomas [48e53] by some studies but not by all [77e80]. In addition, vitamin D deficiency may increase the risk of children developing type I diabetes, asthma, upper respiratory tract infections, influenza infection and also may put adults at increased risk of developing hypertension, heart disease, type II diabetes, multiple sclerosis, and rheumatoid arthritis [3,54e62]. The evidence for and against this possibility is discussed in many of the chapters in this volume.
% Previtamin D3 Synthesis
7 6
5.98%
5 4 3 2 1 0
No Shield
ND Glass
ND Plexiglas
ND Plastic
FIGURE 2.8 Prevention of previtamin D3 formation by UV radi-
ation by common light-shielding materials. The effects of glass, plastic, or Plexiglas (DuPont Chemical Company, Memphis, TN) placed between the simulated sunlight source and the provitamin D3 were measured. ND, Not detectable. Reprinted with permission from Holick [76].
at large. Nesby-O’Dell et al. [39] reported that 42% of African-American women aged 15e49 years were found to be vitamin D deficient throughout the United States at the end of the winter. Tangpricha et al. reported that 32% of healthy young men and women aged 18e29 years were vitamin D deficient at the end of the winter in the Boston area [31]. Sullivan et al. [35] reported that 48% of young girls aged 9e11 years were vitamin D deficient at the end of the winter and 17% were vitamin D deficient at the end of the summer in Maine. It has been estimated that 50% of children 1e5 years old and 70% of children 6e11 years old in the US are vitamin D deficient (25(OH)D < 20 ng/ml) or insufficient (25 (OH)D 21e29 ng/ml) [40,41]. For further discussion of the worldwide problem of vitamin D deficiency and its causes see Chapters 52, 53 and 54. Vitamin D deficiency has also become a major health concern for pregnant women and infants who receive their sole nutrition from breastfeeding [42]. Rickets, which was considered to be a disease of the nineteenth century, has become much more common, especially in children of color who are solely breastfed [3,42]. It has been estimated that to put enough vitamin D in her milk to satisfy her infant’s requirement a lactating woman requires 4000e6000 IU of vitamin D/day [42]. Vitamin D deficiency during pregnancy increases risk of preeclampsia [43] and increases the need for a cesarean section during delivery [44]. For preadolescent and adolescent children, vitamin D deficiency will prevent them from attaining their genetically programmed peak bone mass. For adults, it will precipitate and exacerbate osteoporosis, increase risk of fracture, and cause the painful bone disease osteomalacia [3,45e47]. It is now also recognized that insufficient exposure to sunlight and vitamin D deficiency increases risk of many common cancers, including colon, breast, prostate,
SUNLIGHT, VITAMIN D, AND SKIN CANCER There is no question that chronic excessive exposure to sunlight and sun-burning experiences, especially during childhood and young adulthood, increases risk of squamous and basal cell carcinoma and wrinkling [63,64] and possibly melanoma as discussed in detail in Chapter 89. There is, however, little evidence that sensible and limited exposure to sunlight to satisfy the body’s requirement for vitamin D will substantially increase risk of developing either basal or squamous cell carcinoma [3,36]. Furthermore, if basal or squamous cell carcinoma develops and is detected early, this type of cancer is easily curable. Melanoma, on the other hand, is often a deadly skin cancer. Although it has been suggested that melanoma is associated with sun exposure, it should be noted that some melanomas occur on the least sun-exposed areas, i.e., back of leg, abdomen, etc., and rarely occur on the face [64,65]. Furthermore, a recent study in Scandinavia suggested that melanoma is associated with increased number of nevi (moles), color of the hair, i.e., red and blonde, and number of sun-burning experiences [66]. They also reported that there may be an association with frequent exposure to a tanning bed. However, the data were not statistically significant and the authors concluded that there was only a suggestion of an association. Again, the data for melanoma risk are discussed in detail in Chapter 89. To put this into perspective, it is estimated that approximately 1200 people per year will die in the United States of squamous and basal cell carcinomas that are clearly associated with excessive sun exposure. However, if the cancerevitamin D deficiency causality link is proven, there are estimates that the lack of adequate sun exposure and/or adequate vitamin D nutrition puts at risk more than 150 000 lives for dying of many common cancers [67,68]. There has been no estimate of the toll that vitamin D deficiency potentially has on mortality rates due to cancers, heart disease, and complications of type I diabetes, type II diabetes, autoimmune diseases, and infectious diseases, which has now been linked but not yet proven to be caused by vitamin D
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SUNLIGHT, VITAMIN D, AND SKIN CANCER
TABLE 2.1 Recommended Adequate Intakes (AI), Estimated Average Requirement (EAR), Recommended Dietary Allowance (RDA) and Tolerable Upper Limit (UL) by the Institute of Medicine (IOM) and Dr. Holick’s recommendation for Daily Allowance and safe Upper Limit (UL) for vitamin D for children and adults who are not obtaining adequate vitamin D from sun exposure and who are at risk for vitamin D deficiency. Dr. Holick’s Recommendations for Patients at Risk for Vitamin D Deficiency
IOM Recommendations Life Stage Group Infants
AI
EAR
RDA
UL
Daily Allowance (IU/d)
0 to 6 mo
400 IU (10 mg)
e
e
1000 IU (25 mg)
400e1000
2000
6 to 12 mo
400 IU (10 mg)
e
e
1500 IU (38 mg)
400e1000
2000
1e3 y
e
400 IU (10 mg)
600 IU (15 mg)
2500 IU (63 mg)
600e1000
4000
4e8 y
e
400 IU (10 mg)
600 IU (15 mg)
3000 IU (75 mg)
600e1000
4000
9e13 y
e
400 IU (10 mg)
600 IU (15 mg)
4000 IU (100 mg)
1500e2000
4000
14e18 y
e
400 IU (10 mg)
600 IU (15 mg)
4000 IU (100 mg)
1500e2000
4000
19e30 y
e
400 IU (10 mg)
600 IU (15 mg)
4000 IU (100 mg)
1500e2000
10 000
31e50 y
e
400 IU (10 mg)
600 IU (15 mg)
4000 IU (100 mg)
1500e2000
10 000
51e70 y
e
400 IU (10 mg)
600 IU (15 mg)
4000 IU (100 mg)
1500e2000
10 000
> 70 y
e
400 IU (10 mg)
800 IU (20 mg)
4000 IU (100 mg)
1500e2000
10 000
9e13 y
e
400 IU (10 mg)
600 IU (15 mg)
4000 IU (100 mg)
1500e2000
4000
14e18 y
e
400 IU (10 mg)
600 IU (15 mg)
4000 IU (100 mg)
1500e2000
4000
19e30 y
e
400 IU (10 mg)
600 IU (15 mg)
4000 IU (100 mg)
1500e2000
10 000
31e50 y
e
400 IU (10 mg)
600 IU (15 mg)
4000 IU (100 mg)
1500e2000
10 000
51e70 y
e
400 IU (10 mg)
600 IU (15 mg)
4000 IU (100 mg)
1500e2000
10 000
> 70 y
e
400 IU (10 mg)
800 IU (20 mg)
4000 IU (100 mg)
1500e2000
10 000
14e18 y
e
400 IU (10 mg)
600 IU (15 mg)
4000 IU (100 mg)
1500e2000
10 000
19e30 y
e
400 IU (10 mg)
600 IU (15 mg)
4000 IU (100 mg)
1500e2000
10 000
31e50 y
e
400 IU (10 mg)
600 IU (15 mg)
4000 IU (100 mg)
1500e2000
10 000
14e18 y
e
400 IU (10 mg)
600 IU (15 mg)
4000 IU (100 mg)
1500e2000
10 000
19e30 y
e
400 IU (10 mg)
600 IU (15 mg)
4000 IU (100 mg)
1500e2000
10 000
31e50 y
e
400 IU (10 mg)
600 IU (15 mg)
4000 IU (100 mg)
1500e2000
10 000
UL (IU)
Children
Males
Females
Pregnancy
Lactation*
deficiency. Data to support or refute the vitamin De cancer link is carefully analyzed in Chapter 82. Some experts advocate the use of vitamin D supplements to reduce these risks rather than attempting to balance the amount of sunlight needed to supply adequate vitamin D. There is concern in many quarters about advocating tanning beds as a source of vitamin D
because of the risk of over-usage and over-exposure to harmful UV-A rays (see also Chapter 89). Additional opinions about the risks of vitamin D deficiency are discussed in many chapters in this volume. Discussion of the optimum target levels for serum 25(OH)D are also discussed in Chapters 57 and 58, where aggressive and conservative views are presented.
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2. PHOTOBIOLOGY OF VITAMIN D
CONCLUSION This chapter presents the opinions of this author and to obtain a balanced view of the subject, the reader is advised to also read the opinions expressed by other authors in various chapters throughout this volume. Of particular relevance will be different opinions on the subjects of: risks of vitamin D deficiency, safety of sunlight, the use of UV exposure vs supplements to achieve vitamin D sufficiency, and optimum target levels of serum 25(OH)D. The skin has a huge capacity to produce vitamin D [3,36,65]. Children and adults exposed to natural or artificial ultraviolet B radiation can satisfy their vitamin D requirement [3,65] (Fig 2.9). Exposure of a healthy adult in a bathing suit to one minimal erythemal dose (a light pinkness to the skin) of sunlight or tanning bed UV radiation is equivalent to taking between 10 000 IU and 20 000 IU of vitamin D [1,3] (Fig. 2.10). Because melanin is such an effective sunscreen, African Americans require 5e10 times the exposure that a white person requires to satisfy their body’s vitamin D requirement [19]. Although aging markedly diminishes the capacity of the skin to produce vitamin D3, because its capacity is so high, elders exposed to sunlight can still raise their blood levels of 25(OH)D into a satisfactory range [70e72]. Thus, there needs to be a reexamination of the message that any exposure to sunlight requires some type of sunscreen or sun protection. This extreme position has pervaded the psyche of the population at large. It unfortunately has put many people at risk for vitamin
Comparison of serum vitamin D3 levels after a whole-body exposure (in a bathing suit; bikini for women) to 1 MED (minimal erythemal dose) of simulated sunlight compared with a single oral dose of either 10 000 or 25 000 IU of vitamin D2. Reproduced with permission from Holick [76].
FIGURE 2.10
D deficiency and many of the serious chronic diseases that have been associated with inadequate sun exposure and vitamin D deficiency. Sensible and limited exposure to sunlight, typically no more than 5e15 minutes a day on arms and legs (depending on time of day, season, skin sensitivity, latitude) between 10 a.m. and 3 p.m. during seasons when vitamin D can be produced in the skin will satisfy most people’s vitamin D requirement [3,68e75]. Always protect the face. It is the most sun-exposed area on the body and only represents 5e9% of the body surface area. Taking a multi-vitamin containing 400 IU of vitamin D will satisfy approximately 20% of an adult’s requirement. Thus, there is a need for additional supplementation and/or foods that contain vitamin D to satisfy the 1000e2000 IU and 400e1000 IU of vitamin D that adults and children respectively require to raise their blood levels of 25 (OH)D above 30 ng/ml, which is considered to be a healthy level [36] (Table 2.1).
Acknowledgment This work was supported in part by the NIH CTSI Grant # UL1RR025771 and the UV Foundation.
References
FIGURE 2.9 The serum 25-hydroxyvitamin D levels in healthy adults with skin types II, III, and IV exposed to 0.75 MEDs of simulated sunlight in a bathing suit three times a week for 12 weeks compared to healthy adults receiving a daily dose of 1000 IU of vitamin D3 daily for 12 weeks. )p < 0.01. Copyright Holick 2010; reproduced with permission.
[1] M.F. Holick, 2004 Vitamin D: importance in the prevention of cancers, type 1 diabetes, heart disease, and osteoporosis. Robert H. Herman Memorial Award in Clinical Nutrition Lecture, Am. J. Clin. Nutr. 79 (2003) 362e371. [2] M.F. Holick, Phylogenetic and evolutionary aspects of vitamin D from phytoplankton to humans, in: P.K.T. Pang, M.P. Schreibman (Eds.), Vertebrate Endocrinology: Fundamentals and Biomedical Implications, vol. 3, Academic Press, Orlando, FL, 1989, pp. 7e43. [3] M.F. Holick, Vitamin D deficiency. New Engl, J. Med. 357 (2007) 266e281.
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[4] J. Sniadecki, cited by W Mozolowski, Jerdrzej Sniadecki (1768e1883) on the cure of rickets, Nature 143 (1939) 121. [5] T.A. Palm, The geographic distribution and etiology of rickets, Practitioner 45 (1980) 270e279. 421e442. [6] K. Huldschinsky, Curing rickets by artificial UV-radiation (Heilung von Rachitis durch kunstliche Hohensonne), Deut. Med. Wochenschr. 45 (1919) 712e713 (in German). [7] A.F. Hess, L.F. Unger, Cure of infantile rickets by sunlight, JAMA 77 (1921) 39. [8] J. Mayer, Armand Trousseau and the arrow of time, Nutr. Rev. 15 (1957) 321e323. [9] T. Mellanby, The part played by an accessory factor in the production of experimental rickets, J. Physiol. 52 (1918) 11e14. [10] E.F. McCollum, N. Simmonds, J.E. Becker, P.G. Shipley, Studies on experimental rickets; and experimental demonstration of the existence of a vitamin which promotes calcium deposition, J. Biol. Chem. 53 (1922) 293e312. [11] H. Steenbock, A. Black, The reduction of growth-promoting and calcifying properties in a ration by exposure to ultraviolet light, J. Biol. Chem. 61 (1924) 408e411. [12] A.F. Hess, M. Weinstock, Antirachitic properties imparted to inert fluids and green vegetables by ultraviolet irradiation, J. Biol. Chem. 62 (1924) 301e313. [13] H. Steenbock, The induction of growth-prompting and calcifying properties in a ration exposed to light, Science 60 (1924) 224e225. [14] J.A. MacLaughlin, R.R. Anderson, M.F. Holick, Spectral character of sunlight modulates photosynthesis of previtamin D3 and its photoisomers in human skin, Science 216 (1982) 1001e1003. [15] M.F. Holick, X.Q. Tian, M. Allen, Evolutionary importance for the membrane enhancement of the production of vitamin D3 in the skin of poikilothermic animals, Proc. Natl. Acad. Sci. USA 92 (1995) 3124e3126. [16] F. Loomis, Skin-pigment regulation of vitamin D biosynthesis in man, Science 157 (1967) 501e506. [17] M.F. Holick, J.A. MacLaughlin, S.H. Doppelt, Factors that influence the cutaneous photosynthesis of previtamin D3, Science 211 (1981) 590e593. [18] A.R. Webb, B.R. deCosta, M.F. Holick, Sunlight regulates the cutaneous production of vitamin D3 by causing its photodegradation, J. Clin. Endocrinol. Metab. 68 (1989) 882e887. [19] T.L. Clemens, J.S. Adams, S.L. Henderson, M.F. Holick, Increased skin pigment reduces the capacity of the skin to synthesize vitamin D, Lancet 1 (1982) 74e76. [20] N.H. Bell, A. Greene, S. Epstein, M.J. Oexmann, W. Shaw, J. Shary, Evidence for alteration of the vitamin D endocrine system in Blacks, J. Pediatr. 76 (1985) 470e473. [21] M. Kassowitz, Tetany and autointoxication in infants (Tetani and autointoxication in kindersalter), Wien. Med. Presse. 97 (1987) 139 (in Dutch). [22] A.R. Webb, L. Kline, M.F. Holick, Influence of season and latitude on the cutaneous synthesis of vitamin D3: exposure to winter sunlight in Boston and Edmonton will not promote vitamin D3 synthesis in human skin, J. Clin. Endocrinol. Metab. 67 (1988) 373e378. [23] L.Y. Matsuoka, L. Ide, J. Wortsman, J.A. MacLaughlin, M.F. Holick, Sunscreens suppress cutaneous vitamin D3 synthesis, J. Clin. Endocrinol. Metab. 64 (1987) 1165e1168. [24] L.Y. Matsuoka, J. Wortsman, M.J. Dannenberg, B.W. Hollis, Z. Lu, M.F. Holick, Clothing prevents ultraviolet-B radiationdependent photosynthesis of vitamin D3, J. Clin. Endocrinol. Metab. 75 (1992) 1099e1103. [25] L.Y. Matsuoka, J. Wortsman, N. Hanifan, M.F. Holick, Chronic sunscreen use decreases circulating concentrations of
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[45] M.F. Holick, Sunlight “D”ilemma: risk of skin cancer or bone disease and muscle weakness, Lancet 357 (2001) 4e6. [46] H. Glerup, K. Mikkelsen, L. Poulsen, E. Hass, S. Overbeck, H. Andersen, et al., Hypovitaminosis D myopathy without osteomalacic bone involvement, Calcif. Tissue Int. 66 (2000) 419e424. [47] A.O. Malabanan, A.K. Turner, M.F. Holick, Severe generalized bone pain and osteoporosis in a premenopausal black female: effect of vitamin D replacement, J. Clin. Densitometr. 1 (1998) 201e204. [48] C.F. Garland, F.C. Garland, E.K. Shaw, G.W. Comstock, K.J. Helsing, E.D. Gorham, Serum 25-hydroxyvitamin D and colon cancer: eight-year prospective study, Lancet 18 (1989) 1176e1178. [49] C.F. Garland, F.C. Garland, E.D. Gorham, J. Raffa, Sunlight, Vitamin D, and Mortality from Breast and Colorectal Cancer in Italy. Biologic Effects of Light, Walter de Gruyter, New York, 1992. 39e43. [50] C.L. Hanchette, G.G. Schwartz, Geographic patterns of prostate cancer mortality, Cancer 70 (1992) 2861e2869. [51] M.H. Ahonen, L. Tenkanen, L. Teppo, M. Hakama, P. Tuohimaa, Prostate cancer risk and prediagnostic serum 25-hydroxy-vitamin D levels (Finland), Cancer Causes Control 11 (2000) 847e852. [52] W.B. Grant, An ecologic study of dietary and solar ultra-violet-B links to breast carcinoma mortality rates, Am. Cancer Soc. 94 (2002) 272e281. [53] H.A. Bischoff-Ferrari, E. Giovannucci, W.C. Willett, T. Dietrich, B. Dawson-Hughes, Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health outcomes, Am. J. Clin. Nutr. 84 (2006) 18e28. [54] E. Hypponen, E. Laara, M.-R. Jarvelin, S.M. Virtanen, Intake of vitamin D and risk of type 1 diabetes: a birth-cohort study, Lancet 358 (2001) 1500e1503. [55] S.G. Rostand, Ultraviolet light may contribute to geographic and racial blood pressure differences, Hypertension 30 (1979) 150e156. [56] Y.C. Li, J. Kong, M. Wei, Z.F. Chen, S.Q. Liu, L.P. Cao, 1,25dihydroxyvitamin D3 is a negative endocrine regulator of the renineangiotensin system, J. Clin. Invest. 110 (2002) 229e238. [57] R. Scragg, R. Jackson, I.M. Holdaway, T. Lim, R. Beaglehole, Myocardial infarction is inversely associated with plasma 25hydroxyvitamin D3 levels: a community-based study, Int. J. Epidemiol. 19 (1990) 559e563. [58] A. Zitterman, S. Schulze Schleithoff, C. Tenderich, H. Berthold, R. Koefer, P. Stehle, Low vitamin D status: a contributing factor in the pathogenesis of congestive heart failure? J. Am. Coll. Cardiol. 41 (2003) 105e112. [59] J. Moan, A.C. Porojnicu, A. Dahlback, R.B. Setlow, Addressing the health benefits and risks, involving vitamin D or skin cancer, of increased sun exposure, Proc. Natl. Acad. Sci. USA 105 (2008) 668e673. [60] T.J. Wang, M.J. Pencina, S.L. Booth, P.F. Jacques, E. Ingelsson, K. Lanier, Vitamin D deficiency and risk of cardiovascular disease, Circulation 117 (2008) 503e511. [61] A.A. Ginde, J.M. Mansbach, C.A. Camargo, Association between serum 25-hydroxyvitamin D level and upper respiratory tract infection in the Third National Health and Nutrition Examination Survey, Arch. Intern. Med. 169 (2009) 384e390. [62] M. Urashima, T. Segawa, M. Okazaki, M. Kurihara, Y. Wada, H. Ida, Randomized trial of vitamin D supplementation to prevent seasonal influenza A in schoolchildren, Am. J. Clin. Nutr. 91 (2010) 1255e1260. [63] T.S. Housman, S.R. Feldman, P.M. Williford, A.B. Fleischer Jr., N.D. Goldman, J.M. Acostamadiedo, Skin cancer is among the
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I. CHEMISTRY, METABOLISM, CIRCULATION
C H A P T E R
3 The Activating Enzymes of Vitamin D Metabolism (25- and 1a-Hydroxylases) Glenville Jones 1, 2, David E. Prosser 1 1
Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada 2 Department of Medicine, Queen’s University, Kingston, Ontario, Canada
covered in the first and second editions of this book [9,10] and in reviews of the subject [11,12]. Also, while it is not the purview of this chapter to review knowledge of the vitamin-D-inactivating enzyme, 25(OH)D3-24hydroxylase, also known as CYP24A1 (covered in Chapter 4), the similarities of its structure to the activating enzymes make it inevitable that there will be occasional references to both activating and inactivating enzymes.
INTRODUCTION The activation of vitamin D3 is accomplished by sequential steps of 25-hydroxylation to produce the main circulating form, 25-hydroxyvitamin D [25(OH) D3] followed by 1a-hydroxylation to produce the hormonal form, 1a,25-dihydroxyvitamin D3 [1,25 (OH)2D3] [1] (Fig. 3.1). The initial step of 25-hydroxylation occurs in the liver [2], while the second step occurs both in the kidney and extra-renal sites [3,4]. While work performed over the past four decades in humans and a variety of animal species has revealed that several cytochrome P450 enzymes (CYPs): CYP2R1, CYP27A1, CYP3A4, CYP2D25, and perhaps others, are capable of 25-hydroxylation of vitamin D3 or related compounds and are thus referred to as vitamin D3-25-hydroxylase, it is CYP2R1 that is emerging as the physiologically relevant enzyme [5]. (The nomenclature of all cytochromes P450, including those involved in vitamin D metabolism, is the responsibility of an internationally acknowledged group headed by D. Nelson [8]. Individual CYP names are based upon sequence similarity, function, and other considerations.) On the other hand there is no ambiguity over the second step of 1a-hydroxylation or the 25(OH) D3-1a-hydroxylase enzyme responsible, which is carried out by a single cytochrome P450 named CYP27B1 [6,7]. This chapter assembles the most currently pertinent literature on the activating enzymes of vitamin D metabolism: protein structure and enzymatic properties, crystal structures, gene organization, mutational analysis and regulation. Due to space restrictions, this overview will not cover all of the rich history which went into the early enzymology or cloning of these cytochrome-P450-containing enzymes, much of which has been extensively
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10003-4
GENERAL INFORMATION REGARDING VITAMIN D HYDROXYLASES Table 3.1 summarizes pertinent information about all of the vitamin-D-metabolizing CYPs including both the activating and inactivating enzymes. CYPs are classified into two main subtypes based upon their subcellular location: microsomal or mitochondrial; vitamin D metabolism featuring both subtypes. Both microsomal and mitochondrial CYP subtypes do not function alone but are components of electron transport chains, microsomal CYPs (e.g., CYP2R1) requiring a single generalpurpose protein NADPH-cytochrome P450 reductase (Fig. 3.2A). As with all mitochondrial CYPs, the functional enzyme activity for mitochondrial vitaminD-related CYPs (e.g., CYP27A1, CYP27B1, CYP24A1) requires the assistance of two additional electron-transporting proteins consisting of a general purpose ferredoxin reductase, a general purpose ferredoxin and a highly specific CYP (Fig. 3.2B). All of the vitamin-Drelated CYPs catalyze single or multiple hydroxylation reactions on specific carbons of the vitamin D substrate using a transient Fe-O intermediate. The exact site of hydroxylation, termed regioselectivity, can be somewhat
23
Copyright Ó 2011 Elsevier Inc. All rights reserved.
24
3. THE ACTIVATING ENZYMES OF VITAMIN D METABOLISM (25- AND 1a-HYDROXYLASES)
FIGURE 3.1 Pathways of vitamin D activation and inactivation. Vitamin D3 absorbed from the diet or synthesized in the skin on exposure to
UVB is stored in the liver and fat. The first step in activation, 25-hydroxylation, occurs primarily in the liver and is mediated either by a lowcapacity, high-specificity microsomal 25-hydroxylase, CYP2R1, or a high-capacity, low-affinity mitochondrial hydroxylase, CYP27A1. The 25(OH)D3 is transported from the liver to the kidney bound to the plasma vitamin-D-binding protein (DBP, also known as Gc protein) where it is internalized by megalin-dependent cubilin-mediated endocytosis in the renal proximal tubule [125]. The second step in activation occurs primarily in the proximal tubule through the action of the 1a-hydroxylase, CYP27B1, to yield the active seco-steroid hormone, 1a,25(OH)2D3. The active hormone is transported to the kidney, intestine, bone, and vitamin D target tissues where it binds to the nuclear vitamin D receptor (VDR). Ligand-bound VDR heterodimerizes with retinoid-X receptor (RXR) and modulates the expression of 200e800 genes, including up-regulation of the 24-hydroxylase, CYP24A1, which degrades 1a,25(OH)2D3. The 24-hydroxylase and the 1a-hydroxylase are reciprocally expressed to regulate 1a,25(OH)2D3 levels. Expression of 24-hydroxylase activity produces 24,25(OH)2D3 from 25(OH)D3 which is putatively involved in bone fracture healing, but CYP24A1 predominantly inactivates 1a,25(OH)2D3 by a series of sequential hydroxylation and oxidation reactions to yield either calcitroic acid, which is excreted in the bile, or the 1a,25(OH)2D3-26,23-lactone, which itself is a weak VDR antagonist.
variable with vitamin-D-related CYPs, human CYP24A1 being documented to hydroxylate at C23, C24, or C26. From alignments of the vitamin-D-related CYPs (Fig. 3.3), it is immediately apparent that all CYP proteins possess around 500 amino acids and a size of 50e55 kDa, featuring abundant highly conserved residues which suggest a common secondary structure with multiple highly conserved helices (designated AeL) connected by loops and b-sheet structures. All
CYPs possess a cysteine residue and two other residues near to the C terminus which covalently bind and align the heme group, in addition to several other domains for interaction with the electron-transferring machinery, such as ferredoxin or NADPH-cytochrome P450 reductase. The N-terminus is thought to insert into the endoplasmic reticular membrane for microsomal CYPs or the inner mitochondrial membrane for mitochondrial CYPs. The substrate-binding pocket is formed by several
I. CHEMISTRY, METABOLISM, CIRCULATION
TABLE 3.1 Vitamin D Metabolizing CYPs Subcellular location
Size (aa)
Human gene locus
Mouse gene locus
Activity
Human >47 species
Liver
micro.
501
11p15.2 [23]
7-7E3 [23]
25-hydroxylation of D3 25-hydroxylation of D2
CYP27A1
Human >56 species
Liver Macrophage
mito.
531
2q33-qter [30]
25-hydroxylation of D3 24-hydroxylation of D2
CYP2C11
Rat
Liver (male)
micro.
500
25-hydroxylation of D3 25-hydroxylation of D2
[135]
CYP2D25
Pig
Liver
micro.
500
25-hydroxylation of D3
[136]
CYP2J2 CYP2J3
Human Rat
Liver
micro.
502
1p31.3-p31.2 [134]
25-hydroxylation of D2 25-hydroxylation of D3
[137]
CYP3A4
Human
Liver Intestine
micro.
503
7q22.1 [138]
25-hydroxylation of D2
[138] [43,44]
CYP27B1
Human >39 species
Kidney
mito.
508
12q13.1-q13.3 [65]
1a-hydroxylation of D3 1a-hydroxylation of D2
CYP24A1
Human >51 species
Target tissue
mito.
514
20q13.2-q13.3 [140]
23- & 24-hydroxylation of 25(OH)D/1,25 (OH)2D
Species
CYP2R1*
Disease state human or (mouse XO)
Function
Ref.
VDDR-1B (unknown)
Physiological 25-hydroxylase
[5] [132] [133] [134]
CTX
Pharmacological 25-hydroxylase
[27] [128]
VDDR-1A (Rickets)
1a-hydroxylase
[65] [75] [69] [139]
24-hydroxylase
[140e142]
GENERAL INFORMATION REGARDING VITAMIN D HYDROXYLASES
I. CHEMISTRY, METABOLISM, CIRCULATION
Tissue location
P450
CYP2R1 maps near PTH (11p15.3-p15.1), calcitonin and calcitonin-related polypeptide (11p15.2-p15.1), and insulin and insulin-like growth factor 2 (11p15.5).
25
26
3. THE ACTIVATING ENZYMES OF VITAMIN D METABOLISM (25- AND 1a-HYDROXYLASES)
(A) Microsomal NADP
+
NADPH
NADPH P450 Reductase
_ 2e
_ 2e e.g. CYP2R1 Fe
P450 Product R-OH + H2O
Endoplasmic Reticulum NADP
+
NADPH
3+
FPred
P450-Fe
P450 Reductase
Cytochrome P450
FPox
P450-Fe
2H
+
VITAMIN D3-25-HYDROXYLASES
2+
R-H + O 2 substrate
(B) Mitochondrial Ferredoxin Reductase
NADP
+
_ 1e
_ 2e
(twice)
_ 1e
Ferredoxin
NADPH
e.g. CYP27A1 CYP27B1 CYP24A1
Fe
P450 Product R-OH + H 2O
Inner Mitochondrial Membrane NADPH
NADP
+
FPox
crystallography-derived models of CYP2R1 [18] and CYP24A1 [19]. Mutational analyses to pinpoint residues involved in contact with the main functional groups (hydroxyls) or hydrophobic cis-triene of the vitamin D substrate, as well as define those amino acid residues which are closest to the hydroxylation-sensitive 1aposition in CYP27B1 or the side-chain C-23 to 27 carbons in the side-chain hydroxylases (CYP2R1, CYP24A1, and CYP27A1), are currently in progress in various laboratories.
2+
NHI-Fe
Reductase
Ferredoxin
FPred
NHI-Fe
3+
3+
P450-Fe
Cytochrome P450
2H
+
2+
P450-Fe
R-H + O 2 substrate
Electron transport chains and protein components of the vitamin D hydroxylases. (A) In the endoplasmic reticulum, electron equivalents from nicotinamide adenine dinucleotide phosphate (NADPH) are captured by the NADPH P450 reductase (also known as P450 oxidoreductase, POR). The two electrons from NADPH are transferred sequentially to the microsomal P450 (e.g., CYP2R1). (B) In mitochondria, NADPH is oxidized by the flavoprotein, ferredoxin reductase, which transfers single electrons through a pool of ferredoxin ironesulfur proteins to the mitochondrial P450s on the inner membrane. In both systems, the P450þ electron-transfer protein interaction is not well characterized, but is presumed to occur through close proximity of the heme with the redox center. The electron equivalents are used in the two-electron reduction of molecular oxygen at the heme iron atom, consuming two protons to liberate H2O and to generate a reactive ironeoxygen intermediate capable of regio- and stereo-selectively abstracting a hydrogen atom from a substrate ligand and replacing it with a hydroxyl group or, more generally, mediating P450-specific oxidation reactions.
FIGURE 3.2
secondary structures folded around the distal face of the heme group so that the substrate can be brought to ˚ of the iron atom for hydroxylation. within 3.2A Attempts to identify the key substrate-binding residues were originally guided by homology models [13e17] based upon 10e20 available crystal structures from unrelated soluble prokaryotic CYPs. Recently, the study of the active site of vitamin-D-related CYPs has been further advanced by the emergence of X-ray
As outlined above, there has been no shortage of CYPs proposed as candidates for the title of physiologically relevant vitamin D3-25-hydroxylase. Early work suggested that there were both mitochondrial and microsomal 25-hydroxylase enzyme activities [20,21], and experiments with the perfused rat liver suggested that these might be a low-affinity, high-capacity mitochondrial enzyme and a high-affinity, low-capacity microsomal enzyme [11,22]. More than three decades later we can use several criteria to decide the answer to the question: which CYP is the physiologically important 25-hydroxylase in vivo? These criteria include: a. substrate specificity towards D3 and D2 substrates b. Km and Vmax and enzymatic properties of the expressed enzyme c. tissue and subcellular location d. occurrence of natural mutations e. disease consequence of gene deletion or mutation in human and animal models. Currently, based upon available data for these criteria, we can conclude that the answer to the above question is still not fully resolved, since there is still insufficient evidence that deletion of any single CYP results in a rickets phenotype in the mouse or vitamin D deficiency/rickets in humans. Indeed, it is possible that in vivo several CYPs could contribute to 25-hydroxylation of vitamin D and its analogs under a broad substrate concentration range. However, all available evidence suggests that CYP2R1 is probably the physiologically relevant enzyme at normal vitamin D concentrations (nM) but that it is possibly backed up by others that operate when substrate concentrations rise into the pharmacological range (high nMelow mM). Consequently, we have reviewed relevant information firstly on CYP2R1 and then the other candidate CYPs.
CYP2R1 The discovery of CYP2R1 in 2003 by a research group headed by David Russell and David Mangelsdorf [23]
I. CHEMISTRY, METABOLISM, CIRCULATION
VITAMIN D3-25-HYDROXYLASES
27
Sequence alignments of structurally determined or predicted secondary structures for vitamin D hydroxylases. Residues conserved in both mitochondrial and microsomal P450s (shaded) are structurally or functionally important, although elements of substrate recognition, binding, and specificity are inherently less conserved. The locations of missense mutations leading to autosomal CYP27A1 deficiency (cerebrotendinous xanthomatosis, CTX) and CYP27B1 deficiency (vitamin-D-dependent rickets type IA) are indicated by the dark shading. Heme-binding residues and ERR-triad residues are also indicated.
FIGURE 3.3
I. CHEMISTRY, METABOLISM, CIRCULATION
28
3. THE ACTIVATING ENZYMES OF VITAMIN D METABOLISM (25- AND 1a-HYDROXYLASES)
arguably ended a three-decade-long search for the elusive physiologically relevant vitamin D3-25-hydroxylase. CYP2R1 satisfies most, if not all, of the criteria listed above to describe the location and properties of the enzyme activity first defined by Bhattacharrya and DeLuca in the early 1970s [20,21]. CYP2R1 is a liver microsomal cytochrome P450 that is 501 amino acids in size and was cloned from mouse and human and shown by real-time PCR to be primarily expressed in liver and testis [23]. The full amino acid sequence of hCYP2R1 is shown in Figure 3.3 and alignments of all known CYP2R1 isoforms (current databases hold 47 species) reveal that it is highly conserved in comparison to other CYP2 family members which are not highly conserved between species presumably because they are usually broad-specificity, xenobioticmetabolizing enzymes [24]. The initial Cheng et al. studies demonstrated that CYP2R1, unlike all other putative 25hydroxylases, would 25-hydroxylate both vitamin D2 and vitamin D3 equally well at physiologically relevant substrate concentrations [23]. Subsequent work [25] using nanomolar substrate concentrations of [3H]1a(OH)D2, a vitamin D2 analog, has reinforced the finding that transfected mouse and human CYP2R1 enzymes are able to synthesize the predominant in vivo metabolite 1,25(OH)2D2, and not 1,24(OH)2D2, the minor in vivo product of 1a(OH)D2 which is also the major in vitro product of 1a(OH)D2 incubated with CYP27A1. Recent work [18] using bacterially expressed human CYP2R1 protein in a solubilized system revealed enzyme kinetic properties consistent with both of the earlier studies. hCYP2R1 showed Km values of 4.4, 11.3, and 15.8 mM for vitamin D3, 1a-OHD2, and 1a-OH-D3 respectively, while Kcat values of 0.48, 0.45, and 1.17 mol/min/mol P450 were observed for the same three substrates. As defined in the associated LC analysis (Fig. 3.4), the regioselectivity of hCYP2R1 was clearly confined to the C-25 position with no peaks corresponding to 24- or 26-hydroxylated products, this being in sharp contrast to the findings with CYP27A1 [18, supplemental material]. Furthermore, CYP2R1 failed to metabolize 25(OH)D3, cholesterol, or 7-dehydrocholesterol thereby demonstrating a high specificity for the C-25 position on vitamin D and not other sterol substrates. Thus, the evidence suggests that CYP2R1 has the enzymatic properties needed for a vitamin D-25-hydroxylase capable of appropriately activating known vitamin D precursors in vivo. Strushkevich et al. [18] also solved the crystal structure of a functional form of CYP2R1 in complex with vitamin D3, this representing the first crystal structure of a vitamin-D-related CYP. The crystal structure generally confirmed the helical nature and binding pocket of CYP2R1 predicted from other CYPs using homology modeling [13]. Co-crystallized vitamin D3 in the CYP2R1 occupied a position with the side chain pointing
towards the heme group, but somewhat paradoxically, it was not optimally placed for hydroxylation, since the C˚ from the heme iron. It is unclear at 25 carbon was 6.5 A this point if the substrate was trapped in an ingress channel or if there is some other explanation for the data. Another piece of evidence that strengthens the case for CYP2R1 being the vitamin D3-25-hydroxylase is the finding of a human Leu99Pro mutation in a Nigerian family which results in vitamin-D-dependent rickets, type 1B [26]. This disease was postulated four decades ago [27] following the elucidation of vitamin D metabolism. Unfortunately, the genetic nature of the Leu99Pro mutation of CYP2R1 was determined by Cheng et al. [5], a decade after the initial identification of the Nigerian rachitic patient, making patient and family follow-up difficult. However, subsequent genetic analysis of exon 2 of CYP2R1 by Cheng et al. [5] in 50 Nigerian individuals revealed one heterozygote with the leu99pro mutation suggesting that there may be a founder gene effect in the Nigerian population, and where vitamin D deficiency is quite prevalent [28]. Though the Leu99 residue is not in a region of the CYP2R1 coding for substratebinding domain, it is involved in water-mediated hydrogen bonding to the Arg445 amide nitrogen located three residues from the heme coordinating Cys448, and thus a Leu99Pro mutation probably results in a misfolded protein with little or no enzyme activity. Numerous attempts to bacterially express both hCYP2R1 with a Leu99Pro mutation and wild-type hCYP2R1 simultaneously failed, leading Strushkevich et al. [18] to conclude that CYP2R1 with Leu99Pro is misfolded or shows poor protein stability. To date there is no animal model or mouse knockout with defective CYP2R1 reported in the literature to confirm that CYP2R1 is essential and thus the possibility that there is some redundancy in the liver vitamin D3-25-hydroxylase “family” of enzymes that can compensate for the deletion of CYP2R1 cannot be confirmed. Recently, a genome-wide association study of the genetic determinants of serum 25-hydroxyvitamin D concentrations [29] concluded that variants at the chromosomal locus for CYP2R1 (11p15) were the second strongest association of only four sites, DBP or Gc, CYP24A1 and 7-dehydrocholesterol reductase (DHR7) being the others. Notably, variants of the other 25-hydroxylases such as CYP27A1 were not identified to be associated with serum 25(OH) D concentrations, again arguing for the fact that they play no role in 25-hydroxylation of vitamin D at physiological substrate concentrations.
CYP27A1 This was the first cloned vitamin D-25-hydroxylase in the early 1990s again discovered by David Russell’s group. CYP27A1 is a liver mitochondrial cytochrome
I. CHEMISTRY, METABOLISM, CIRCULATION
29
VITAMIN D3-25-HYDROXYLASES
0.05
0.05 1 (OH)D3
1 (OH)D2
0.04
Absorbance (265nm)
0.04 1 ,25(OH)2D2
0.03
0.03
0.02
0.02
0.01
0.01
0.00
0.00
0
5
Comparison of the enzymatic properties of two vitamin D-25-hydroxylases: CYP2R1 and CYP27A1. The substrates used are the prodrugs, 1a(OH)D2 and 1a(OH)D3, to gauge the hydroxylation site and efficiency of the two vitamin D 25-hydroxylases towards D2 and D3 family members. Straight-phase chromatograms of metabolites from (A) in vitro reconstituted CYP2R1 enzyme [18] and (B) transiently transfected CYP27A1 in COS-1 cells [31]. The lack of CYP27A1-mediated 25-hydroxylation towards 1a(OH)D2 is evident, although the major product, 1a,24(OH)2D2, is detectable in serum of animals given large doses of vitamin D2 [33] and is a known VDR agonist [126].
FIGURE 3.4
(A) CYP2R1
10
15
20
1 ,25(OH)2D3
0
5
10
15
20
Absorbance (265nm)
(B) CYP27A1
Time (minutes)
Time (minutes)
P450 with a homolog in >56 species that is 531 amino acids in size in the human. It was originally cloned from rabbit but also from human [30,31]. Even the earliest claims, that CYP27A1 was a vitamin D25-hydroxylase, were controversial as the purified liver enzyme seemed to be a better cholesterol-26-hydroxylase than vitamin D-25-hydroxylase. Thus, it was proposed as a bifunctional enzyme involved in both bile acid and vitamin D metabolism [32]. Work with the recombinant protein demonstrated that CYP27A1 is a low-affinity, high-capacity vitamin D3-25-hydroxylase that also 25-hydroxylates 1a-OH-D3 but seems incapable of the 25-hydroxylation of vitamin D2 or 1a-OH-D2 catalyzing 24-hydroxylation to 24-OH-D2 or 1,24S-(OH)2D2 instead (Fig. 3.4) [31,33]. Figure 3.4 shows that while CYP27A1 exhibits the ability to 24- and 26(27)-hydroxylate 1a-
OH-D3, its primary site of hydroxylation is C25; whereas with 1a-OH-D2, this switches to C24-hydroxylation with a small amount of 26(27)-hydroxylation. The inability of CYP27A1 enzymatic properties to explain the formation of 25(OH)D2 in animals in vivo became the main impetus for the search for an alternative 25-hydroxylase that culminated in CYP2R1 [23]. Parallel enzymatic work with bile acid substrates clearly showed that CYP27A1 could 25- and 27-hydroxylate the side chain of cholesterol and play a role in the trimming of C-27 sterols without the secosteroid, open B-ring nucleus [34]. The same workers performed mutagenesis studies which established the important residues involved in ferredoxin interaction. Although there is currently no crystal structure of CYP27A1, numerous models have been proposed for the enzyme
I. CHEMISTRY, METABOLISM, CIRCULATION
30
3. THE ACTIVATING ENZYMES OF VITAMIN D METABOLISM (25- AND 1a-HYDROXYLASES)
predicted from other CYPs using homology modeling [13,35]. Until the recent emergence of crystal structures of CYP2R1 and CYP24A1, these models offered the best structural insights into the general structure and substrate-binding pockets of vitamin-D-related CYPs. Several pieces of biological or clinical information argue against CYP27A1 being the physiologically relevant vitamin D-25-hydroxylase. Firstly, the CYP27A1null mouse phenotype does not include rickets or any other bone lesion [36]. However, this animal model is complicated by the absence of any significant bile acid defect either. Secondly, though human CYP27A1 mutations have been documented in the literature, these result in a bile-acid-related condition known as cerebrotendinous xanthomatosis (CTX) rather than rickets [37]. Affected individuals usually have normal serum 25(OH) D, though some of these individuals can exhibit low serum 25(OH)D and a type of osteoporosis [38]. Current opinion is that CTX is a defect in bile acid metabolism and that the bone disease is the result of malabsorption of dietary vitamin D caused by bile acid insufficiency rather than an inadequate 25-hydroxylase enzyme activity [39]. Thirdly, the genome-wide association study of the determinants of serum 25-hydroxyvitamin D concentrations [29] concluded that variants at the locus for CYP2R1 (11p15) but not CYP27A1 are associated with serum 25(OH)D concentrations arguing for the fact that CYP27A1 plays no role in 25-hydroxylation of vitamin D at physiological substrate concentrations. A more likely possibility for an in vivo role for CYP27A1 in vitamin D metabolism is as a pharmacologically relevant 25-hydroxylase, in the activation of the prodrugs, 1a-OH-D3 and 1a-OH-D2. The 1a-hydroxylated vitamin D analogs are popular prodrugs in the treatment of osteoporosis and metabolic bone disease or the secondary hyperparathyroidism associated with chronic kidney disease (CKD) [40]. Thus establishing the activating enzyme needed to convert them into active 25-hydroxylated products has some clinical importance. It is worth noting that in vitro CYP27A1 synthesizes 1,25-(OH)2D3 and 1,24S-(OH)2D2, metabolites from 1a-OH-D3 and 1a-OH-D2 respectively, 24-hydroxylated compounds such as 1,24S-(OH)2D2 are also observed in vivo following administration of pharmacological amounts of vitamin D2 compounds [33,41,42]. Thus, CYP27A1 may contribute to the metabolism of vitamin D compounds, including the 1a-hydroxylated compounds when they are present at high concentrations, but seems unlikely to be involved in vitamin D metabolism at physiologically relevant concentrations.
Other CYPs Over the past three decades, there have been numerous reports that in addition to CYP2R1 and
CYP27A1, a number of other specific microsomal CYPs, partially purified from tissues or cells, or studied in bacterial or mammalian expression systems can 25-hydroxylate spectrum vitamin D substrates, but only at micromolar substrate concentrations. These include: CYP2D11, CYP2D25, CYP2J2&3, and CYP3A4 (see Table 3.1). Some of these are expressed in one mammalian species (e.g., pig or rat) and have no obvious human equivalent, show gender differences not observed for human vitamin D-25-hydroxylation in vivo or fail to 25-hydroxylate vitamin D2 or 1a-OH-D2. Again, as with CYP27A1, lack of regioselectivity for the C-25 position surfaces as an important distinguishing feature compared with CYP2R1, as many other microsomal CYPs (e.g., CYP3A4) catalyze the 24hydroxylation of vitamin D2 and D3 compounds [43e45]. Based upon the emergence of the strong case for CYP2R1 being the vitamin D-25-hydroxylase, the pursuit of these other nonspecific CYPs is becoming less urgent, but at least one of these CYP3A4 deserves special mention. A multifunctional nonspecific enzyme such as CYP3A4, which is estimated to metabolize up to 50% of known drugs, would probably not attract special interest here were it not for the fact that recently it been shown to be selectively induced by 1a,25(OH)2D3 in the intestine [45e47]. CYP3A4 has been shown to 24- and 25-hydroxylate vitamin D2 substrates more efficiently than vitamin D3 substrates [43,44], and also 23R- and 24S-hydroxylates the already 25-hydroxylated 1a,25-(OH)2D3 [45]. However, CYP3A4 is known to have Km values for vitamin D compounds in the micromolar range, a property that questions its physiological but not pharmacological relevance. Recent work [48,49] has demonstrated that both human intestinal microsomes and recombinant CYP3A4 breakdown 1a,25-(OH)2D2 at a significantly faster rate than 1a,25(OH)2D3 suggesting that this nonspecific cytochrome P450 might limit vitamin D2 action preferentially in selective target cells where it is expressed (e.g., intestine), particularly in the pharmacological dose range. Such an observation may also offer an explanation for the welldocumented lower toxicity of vitamin D2 compounds as compared to vitamin D3 compounds in vivo, the vitamin D2 compounds not causing such severe hypercalcemia by virtue of reduced effects on intestinal calcium absorption. The same type of mechanism involving differential induction of nonspecific CYPs, such as CYP3A4, may also underlie the occasional reports of drugedrug interactions involving vitamin D, where co-administered drug classes, e.g. anticonvulsants [50,51], causing accelerated degradation of vitamin D2 over vitamin D3. Thus, while CYP3A4 might be occasionally considered as a vitamin D-25-hydroxylase, its main relevance to vitamin D metabolism may lie in its
I. CHEMISTRY, METABOLISM, CIRCULATION
25-HYDROXYVITAMIN D-1a-HYDROXYLASE
involvement in inactivation of vitamin D compounds at high concentrations.
25-HYDROXYVITAMIN D-1a-HYDROXYLASE The 25-hydroxyvitamin D-1a-hydroxylase has been investigated virtually from the day that the hormone 1a,25-(OH)2D3 was discovered [3,52]. As soon as it became apparent that the 25-hydroxyvitamin D-1ahydroxylase was a central regulatory axis of the calcium and phosphate homeostatic systems, subject to up-regulation by PTH, low Ca2þ and low PO3e 4 levels [1,53,54], the need to define its activity in biochemical terms was apparent. It was quickly recognized that serum 1a,25(OH)2D3 was predominantly made in the kidney [3,55] with a PTH-regulated form located in the proximal convoluted tubule and a calcitonin-regulated form in the proximal straight tubule [56e59]. Biochemical investigations showed that the enzyme involved was a mixed-function oxidase with a cytochrome P450 component [60], but the exact molecular description of this enzyme took another 25 years to unravel. During this time, there have been emerging reports of so-called “extra-renal” 25-hydroxyvitamin D-1a-hydroxylase activity in several sites including placenta, bone, and macrophage [61e66], which evoked the question of whether there was more than one cytochrome P450 with 25-hydroxyvitamin D-1a-hydroxylase activity. Unlike with the liver vitamin D-25-hydroxylase, this does not appear to be the case and with the cloning of CYP27B1 as a single gene, this story has become much simpler. In 1997, several groups coincidentally cloned, sequenced, and characterized CYP27B1 from rat, mouse, and human species [67e69]. Though many of these groups used kidney libraries as the source of the enzyme, interestingly other groups reported finding the same CYP27B1mRNA in keratinocyte [70] and human colonic cell HT-29 [71] libraries, suggesting that the enzyme was identical in all locations. Subsequently, it has been confirmed that the CYP27B1 protein is identical in all locations [4,71,72], whether renal or extrarenal, though the regulation in these different tissue locations almost certainly involves different hormones and effectors. hCYP27B1 is a 507-amino-acid protein with a molecular mass of ~55 kDa. Best available information suggests that the enzyme 1a-hydroxylates 25(OH)D2 and 25(OH)D3 equally efficiently to give the active metabolite of each form of the vitamin. The genetic rachitic condition termed vitamin D dependency rickets (VDDR Type I), in which the 1a-hydroxylase enzyme was absent or defective, presumably due to mutation of CYP27B1, had been recognized in the early 1970s by
31
Fraser and colleagues [27,73]. These authors showed that patients had low or absent serum 1,25-(OH)2D and they could be successfully treated using small amounts of synthetic 1,25-(OH)2D3. VDDR Type IA involves a resistant-rickets phenotype, characterized by hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and undermineralized bone. It is essentially cured by physiological (microgram) amounts of 1,25-(OH)2D3 or pharmacological (milligram) amounts of 25(OH)D3 or vitamin D, which is consistent with a block in 1a-hydroxylation activity [27]. Subsequent work mapped the CYP27B1 gene to 12q13.1eq13.3, which is the same location established for the VDDR Type I disease [67]. Human CYP27B1 mutations occur throughout the gene (Fig. 3.5) resulting in defective and misfolded proteins with little or no activity [74e78]. At least two groups have created CYP27B1-null mice [79,80], which exhibit a lack of 1a-hydroxylated metabolites in the blood and tissues, revealing that CYP27B1 is the sole source of 1,25-(OH)2D in the body. The mouse phenotype mirrors human VDDR Type IA in terms of resistant rickets. The animals also show a reduction in CD4- and CD8-positive peripheral lymphocytes and female mice are infertile [79]. Detailed bone histomorphometric analyses of the CYP27B1 and CYP27B1/ PTH double knockout mice established that 1,25(OH)2D3 deficiency resulted in epiphyseal dysgenesis and only minor changes in trabecular bone volume [81]. Bikle and colleagues showed that CYP27B1 is also required for optimal epidermal differentiation and permeability barrier homeostasis in the skin of mice [82]. Administration of a normal diet supplemented with either small amounts of 1,25-(OH)2D3 or use of a high-calcium “rescue diet” largely corrects the mineral metabolism and bone defects seen in the CYP27B1-null mouse [83e85]. Tissue-specific knockout of the mouse CYP27B1 gene in chondrocytes has been achieved and suggests that local production of 1,25-(OH)2D3 plays a role in growth plate development [87,88]. The availability of specific CYP27B1mRNA and antiCYP27B1 protein antibodies has allowed for a more rigorous exploration of the extrarenal expression of the enzyme. Diaz et al. [89] used Northern analysis and RT-PCR to examine mRNA expression in human synctiotrophoblasts and concluded that there was CYP27B1 expression in human placenta. Using similar techniques, several groups reported low but detectable expression of CYP27B1 in a variety of cultured cell lines and freshly isolated cell explants, e.g. prostate and colonic cells [90e92]. Immunohistochemistry data from analysis of animal and human tissues has revealed the presence of the CYP27B1 protein in several tissues purported to express 1ahydroxylase activity, e.g. skin, colon, macrophage, prostate, breast [4,72]. Not all studies have supported the
I. CHEMISTRY, METABOLISM, CIRCULATION
32
3. THE ACTIVATING ENZYMES OF VITAMIN D METABOLISM (25- AND 1a-HYDROXYLASES)
L99P
CYP2R1 -helix
ER
A’
B
A
B’
C
D
E
F
G
H
I
J
A216P
K259R
ER
B
A
B’
C
D
E
F
G
H
I
J
G102E R107H P112L G125E P143L D164N E189GL
Q65H
-strand
B
A
B’
C
D
E
F
G
H
I
J
R
K
508
L 3a 4 3b
1
5
P382S R389CGH T409I R429P V478G R453C P497R
T321R S323Y R335P L343F
ER
A’
531
L 3a 4 3b
CYP27B1 -helix
R
K
1
-strand
5
P384L R395CHQS P401R G472A N403K P441S R474QW T339M D354G R405QT R479CGS
CYP27A1 A’
501
L 3a 4 3b
R127QW R137QW G145ER
-helix
K
1
-strand
R
5
Missense mutations identified in patients with 25-hydroxylase deficiency rickets (VDDR-Type IB, CYP2R1), the cholesterol and bile acid metabolism disorder, cerebrotendinous xanthomatosis (CTX, CYP27A1), and vitamin-D-dependent rickets type I (VDDR Type IA, CYP27B1). The relative positions of the conserved a-helices and b-strands are indicated. Heme-binding residues are denoted by the open diamonds. The positions of the ERR-triad residues in the K-helix (E R R) and meander region (E R R) form the core of a structurally rigid motif which may stabilize ferredoxin binding and electron transfer to the heme iron. Secondary structures positioning determinants of substrate orientation in P450 crystal structures include the b-1, A-helix, B’-helix, B’/C loop, F/G loop, I-helix, b-3a, b-3b, and b-5 structures. Figure adapted from Malloy and Feldman [78], previously tabulated mutations [113], and other literature [76,77,127e131].
FIGURE 3.5
conclusion that CYP27B1 is expressed outside of the kidney in normal, nonpregnant animals. Using a b-galactosidase reporter system, Vanhooke et al. [86] found no evidence for expression of CYP27B1 in murine skin or primary keratinocytes, although there was expression in kidney and placenta. It is possible that the lack of detection of low-abundance extrarenal CYP27B1 transcripts is due to some inherent insensitivity of the b-galactosidase reporter system, whereas it is sufficiently sensitive to detect abundant renal CYP27B1 transcripts. Despite the fact that the existence of the extrarenal 1ahydroxylase remains tentative, there has been much speculation about the role of this enzyme in health and disease [93e95]. It is now widely believed the enzyme exists in nonrenal tissues to boost local production of cellular 1,25-(OH)2D3 in a paracrine/intracrine system. Such a role would suggest that cellular 1,25-(OH)2D3 concentrations in extrarenal CYP27B1 tissues might be higher than in the tissues of the classical endocrine system which depend upon renally synthesized, blood-derived 1,25-(OH)2D3 at a concentration around
10e10 to 10e9M (e.g., intestine, bone, parathyroid gland). In turn, the genes regulated in extrarenal tissues (e.g., macrophage, colon, prostate) might be a less-sensitive cell differentiation and antiproliferative subset, known to be regulated in cancer cell lines at 1,25-(OH)2D3 concentrations of 10e8 to 10e7 M under cell culture conditions. A role for the extrarenal CYP27B1 is also consistent with the finding that serum 25(OH)D levels are associated with various health outcomes from bone health to cardiovascular health. In particular, low serum 25(OH)D levels are associated with increased mortality for colon, breast, and prostate cancer; increased autoimmune diseases and greater susceptibility to tuberculosis; increased cardiovascular diseases and hypertension. The presence of CYP27B1 in cells of the colon, breast, prostate, monocytes/macrophages, and vasculature could explain why serum 25(OH)D levels are so critical to the normal functioning of these tissues. Chronic kidney disease (CKD) with five stages defined by decreasing glomerular filtration rate (GFR) is well established to be accompanied by a gradual fall
I. CHEMISTRY, METABOLISM, CIRCULATION
25-HYDROXYVITAMIN D-1a-HYDROXYLASE
in serum 1,25-(OH)2D3 (normal range ¼ 20e60 pg/mL), widely assumed to be due to a gradual decline in CYP27B1 activity [96]. Whether this is in turn due to loss of the CYP27B1 protein mass caused by renal damage is debatable. It is possible that the fall in serum 1,25-(OH)2D3 to values below 20 pg/mL by the end of CKD Stage 2 could be due in part to increased FGF23 levels, a known down-regulator of CYP27B1 expression in normal kidney cells [97]. Recent reports of marked increases in FGF23 levels in CKD Stage 5 dialysis patients with phosphate retention are consistent with FGF23 playing a major role in vitamin D dysregulation and mortality in chronic kidney disease [98]. The regulation of CYP27B1 (summarized in Fig. 3.6) has been a major focus ever since the enzyme’s discovery in the early 1970s [1]. Ca2þ and PO3 4 ions, probably through the hormones, PTH, calcitonin, and FGF23, regulate CYP27B1 expression through complex signal transduction processes [59,99,100,101], while 1,25-(OH)2D3, the end-product of the enzyme, downregulates its own synthesis at the transcriptional level by VDR-mediated action at the level of the CYP27B1 gene promoter [100,102,103]. Evidence is also accumulating that CYP27B1 expression is down-regulated through DNA methylation and up-regulated through DNA demethylation [103,104]. While it is logical to isolate CYP27B1 from the rest of the calcium/phosphate homeostatic system, in practice there is a reciprocity between CYP27B1 and CYP24A1 that suggests that the factors up-regulating one enzyme, down-regulate the other. This is evident in the isolated perfused kidney from the rat fed a low-Ca vitamin-D-deficient diet, or low-PO4 vitamin-D-deficient diet which is in the 1ahydroxylation mode, and which over a 4-hour perfusion period after being exposed to its 25(OH)D3 substrate turns off CYP27B1 expression and 1a-hydroxylation and turns on CYP24A1 and 24-hydroxylation [105]. The vitamin D metabolic system seems ideally designed to avoid synthesis of excessive amounts of the hormone IFN TLR2/4 Low
Other inflammatory Cytokines: IL-1 , IL-15, IGF-I, EGF, TGF-
Ca2+
PTH
CYP27B1
Epigenetic
1 ,25(OH)2D3
Low PO43-
Calcitonin
FGF23, leptin
NFRegulation of CYP27B1. Factors identified which regulate positively or negatively the expression of renal or extrarenal CYP27B1.
FIGURE 3.6
33
and also to degrade the hormone, or even its substrate, by superinduction of catabolic processes including CYP24A1. In the VDR-null mouse, we see a complete breakdown of this autoregulation process because CYP27B1 is not suppressed by excessive 1,25-(OH)2D3 production and CYP24A1 is not actively stimulated, both steps requiring VDR-mediated events. The regulation of the extrarenal 1a-hydroxylase has also received attention over the last couple of decades. What is clear is that the renal and extrarenal enzymes are regulated by different factors: the kidney CYP27B1 by calcium and phosphate homeostatic hormones described above; while the extrarenal enzyme is regulated by tissue-specific factors, including cytokines. Adams et al. [106] have shown that macrophages in the granulomatous condition, sarcoidosis, are driven by proinflammatory cytokines, such as g-interferon, which also stimulate extrarenal CYP27B1 activity, that can cause excessive serum 1,25-(OH)2D3, which left unchecked results in hypercalciuria and hypercalcemia. The mechanism of g-interferon-mediated up-regulation of CYP27B1 appears to involve the Janus kinase-signal transducer and activator of transcription, MAPK, and nuclear factor-kappaB pathways, with a crucial role for the transcription factor CCAAT/enhancer binding protein beta [107,108]. Also, the usual CYP24A1 counter-regulatory mechanism seems to have been replaced in the monocyte/macrophage system by an inactive splice-variant of CYP24A1 allowing the CYP27B1 activity to go largely unchecked and cellular 1,25-(OH)2D3 to rise to super-normal levels [109]. The nature of the down-regulator(s) of the extrarenal CYP27B1 in these and other cells of the immune system remains largely unknown. Recently, the normal regulation of the monocyte/ macrophage CYP27B1 system was elucidated [95,110,111]. Toll-like receptors (TLRs) on the cell surface respond to the presence of bacteria (e.g., M. tuberculosis) with a signal transduction process which results in upregulation of VDR and CYP27B1. Uptake of 25(OH)D bound to its blood carrier DBP, allows the cells to then manufacture 1,25-(OH)2D3, which in turn stimulates VDR-mediated gene transcription of cathelicidin. Cathelicidin is an antimicrobial peptide, which specifically kills M. tuberculosis. Stubbs et al. [112] have demonstrated the existence of a high-VDR, CYP27B1expressing subpopulation of immune cells making cathelicidin that can be selected by cell-sorting techniques in CKD Stage 5 dialysis patients treated with high doses of cholecalciferol (40 000 IU twice per week). Since these patients are virtually devoid of circulating 1,25-(OH)2D3 because of their low renal CYP27B1 activity, the data suggest that the monocyte/macrophage extrarenal CYP27B1 survives during renal failure and is responsible for cathelicidin production.
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3. THE ACTIVATING ENZYMES OF VITAMIN D METABOLISM (25- AND 1a-HYDROXYLASES)
ADDITIONAL TOPICS Crystal Structures and Homology Models of Vitamin D-related CYPs Dating back to their first description as enzyme activities in the 1970s, biochemists have wondered about the structures of the family of vitamin-D-related hydroxylases. The cloning and characterization of these CYPs during the 1990e2003 period opened the door to defining the structures of these proteins, particularly the nature of their substrate-binding domains. Though the elucidation of these structures has been a significant challenge it has been aided by the fact that the vitaminD-related cytochrome P450s are part of a large group of highly conserved proteins across all phyla that metabolize both endogenous and exogenous small-molecule substrates. Consequently, structural information gleaned from the study of all cytochrome P450s has established that though the amino-acid sequences show wide variability, the secondary and tertiary structures have been highly conserved across phyla. Over the past 20 years, genome sequencing projects have yielded dozens of species orthologs for each of the various cytochrome P450s and the number of available crystal structures has increased impressively, starting with soluble bacterial CYPs but reaching over the past few years to membrane-bound mammalian CYP structures including some of the 59 human CYPs. These crystal structures together with the parallel advancement in protein homology modeling led to the emergence of a number of useful homology models for vitamin-D-related CYPs, first for CYP27A1 then for CYP24A1 [13e17,113]. In the last 2 years, the first crystal structures of vitamin-D-related CYPs in the form of the human microsomal CYP2R1 and the rat mitochondrial CYP24A1 have been released [18,19] and these have validated the homology modeling approach very well. In addition, two bacterial vitamin D hydroxylases capable of sequentially hydroxylating vitamin D3 to 1a,25 (OH)2D3 at production levels, CYP105A1 from Streptomyces griseolus (2zbz.pdb) [114] and P450 Vdh from Pseudonocardia autotrophica (3a4g.pdb) [115], have been determined. In general, the structures of all cytochrome P450s are similar and comprise a series of highly conserved helices (designated AeL) connected by loops and b-sheet structures (see Fig. 3.7, top). Though sequence homology is poorly conserved in the cytochrome P450 superfamily, the presence of various highly conserved heme-binding and structurally or functionally important amino acids and crystallographically determined secondary structure make it relatively easy to prepare useful sequence alignments for secondary structure prediction, model building, and site-directed mutagenesis studies. All
CYPs contain a cysteine residue near to the C-terminus (see Fig. 3.3) to which the heme group attaches. Heme binding is stabilized by an additional 4e5 amino acid sidechains which hydrogen bond with the two heme propionic acid groups. The heme-binding domain is further stabilized by a number of structurally implicit water molecules and protein backbone interactions which extend a large measure of structural stability into the surrounding secondary structures. Other structural motifs such as b-sheets, hydrophobic clusters, the ERR-triad, the ferredoxin, or NADPH-P450 reductase binding site, and remarkably well-conserved sidechain hydrogen bonds between secondary structures further maintain the folded protein and the characteristic structure of a cytochrome P450 [13]. The N-terminus of the mitochondrial CYPs contains an ~30-amino-acid targeting sequence which is cleaved during mitochondrial import. The N-terminus of the mature mitochondrial and the microsomal CYPs is thought to be membraneassociating. The substrate-binding pocket is formed by several secondary structures folded around the distal face of the heme group that precisely position the ˚ of the heme iron atom. An analsubstrate within ~3.2 A ysis of the heme-ligand geometry of 49 substrate-bound crystal structures revealed the hydroxylation target carbons actually adopt a spatially conserved orientation to the heme iron and this can be triangulated (as shown in Fig. 3.7, middle) for use in docking studies [16]. Somewhat paradoxically, none of the published crystal structures of the vitamin D hydroxylases have been very useful in understanding how a vitamin D substrate leaves the hydrophobic membrane, gains entry to the active site through a substrate access channel, and is precisely positioned in the active site. Given that the cytochrome P450 is associated with the membrane, the prevailing paradigm is that the lipid bilayer of the membrane flows into the substrate access channel and the substrate floats or diffuses in. Unfortunately, it has been difficult to ascertain exactly where the substrate access channel is located because it is usually closed in CYP crystal structures (and therefore not apparent) or it is situated in different positions in different CYP subfamilies [116]. In some cases, such as CYP3A4 and CYP24A1, the active site is so widely open that one could wonder how the substrate could spend any time in one place. Of the existing vitamin D hydroxylases, only two CYPs have been co-crystallized with vitamin D. In the bacterial CYP105A1 [114] the protein was cocrystallized with its product 1a,25(OH)2D3 in a transient, nonproductive orientation too distant from the heme iron. The vitamin D 3b-hydroxyl was hydrogen bonded to Ser236 (I-helix) and Arg193 (G-helix) which were bridged by a water molecule to Glu232 (I-helix). It was also bound to a water molecule networked to a B0 /C loop backbone carbonyl and a second water molecule
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35
ADDITIONAL TOPICS
Modeling, docking, and crystal structure studies of the vitamin D hydroxylases. The top panel depicts a stereographic view of a model of CYP27A1 with 1a(OH)D3 in the heme distal cavity active site. An analysis of hemeligand geometry in cytochrome P450s revealed the existence of a preferred binding orientation for the hydroxylation target carbon (49 crystal structures) and the position of azole nitrogen interacting with the heme iron (18 crystal structures). The atoms of interest were triangulated by calculating the distances to the heme methyl carbons CMA, CMB, and CMD and the averages used to dock 1a (OH)D3 in CYP27A1. A similar process was used to dock 1a,25(OH)2D3 [16] and ketoconazole in CYP24A1. The middle panel shows the triangulated distances to the average substrate target carbon and inhibitor azole nitrogen above the heme. The lower panel depicts 1a(OH)D2 binding in CYP2R1 crystal structure (3dl9.pdb) [18]. Several key hydrogen bonds in the substrate access channel of CYP2R1 are directed towards the A-ring hydroxyls to position the substrate in a hydrophobic active site. These hydrogen bonds are directed towards the backbone carbonyls of Ala250 (G-helix) and Ile301 (I-helix) and to a water molecule (marked with an *) coordinated by Asn217 (F-helix). The substrate access channel opening between the B’-, G-, and I-helices is extensively stabilized by a network of water molecules centered in the B’/C loop and stabilized by Glu306 (I-helix) and Asn126 (B’/C loop).
FIGURE 3.7
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36
3. THE ACTIVATING ENZYMES OF VITAMIN D METABOLISM (25- AND 1a-HYDROXYLASES)
hydrogen bonded to the 1a-OH. This is the only example, outside of the VDR, where a vitamin D Aring hydroxyl was hydrogen bonded to an amino acid side-chain. In CYP2R1 [18], the A-ring hydroxyls of bound vitamin D structures were hydrogen bonded to the backbone carbonyls at Ala250 (G-helix) and Ile301 (I-helix) (see Fig. 3.8, bottom). As shown in Figure 3.7, bottom and Figure 3.8, bottom, the 3b-OH was also hydrogen-bonded to a water molecule stabilized by Asn217 (F-helix). This emphasizes that the acidebase chemistry of the vitamin D hydroxyls appears to be directed towards networked water molecules in the active site and backbone amide bond carbonyls and nitrogen atoms. If this is true, it will be difficult to predict how vitamin D substrates are positioned in CYP active sites without high-resolution crystal structures showing the positions of structurally implicit water molecules. Even with such data, the CYP2R1 and CYP105A1 structures would not reveal mechanistically how the hydrophobic parts of vitamin D substrates interact with the predominantly hydrophobic residues lining the active site to position the hydroxylation target carbon so precisely for reaction. Hopefully, a clearer picture of substrate binding will be forthcoming in substrate-bound crystal structures of the mammalian vitamin D hydroxylases or the pending release of P450 Vdh mutants obtained by directed evolution co-crystallized with vitamin D3 (3a50.pdb) and 25(OH)D3 (3a51.pdb) [115]. In the meantime, continuing studies of vitamin D docking in CYP27A1, CYP27B1, and CYP24A1 will focus on naturally occurring autosomal mutations [e.g., 117] (Fig. 3.5) and on site-directed mutagenesis studies of hydrophobic contact residues in the active site. The most successful applications of the latter approach were the identification of an Ala-Gly polymorphism at Ala326 in CYP24A1 which affected C24 and C23 pathway catabolism of 1a,25(OH)2D3 [16], a Thr416Met mutation in ratCYP24A1 which had a similar (smaller) effect [14], CYP24A1 mutations at Ile131, Trp134, Leu148, Met246, and Gly499 [15], Phe249 mutations in rat CYP24A1 [118], and Phe248, Ile514, Val515, Leu516 mutations in CYP27A1 [13] among others. However, even with these data sets, a deterministic explanation of substrate binding will be difficult. What can be said for certain is that the hydrophobic core and active site residues are extensively conserved across 40e50 species orthologs in the mammalian CYPs (see Fig. 3.8) and that current research will identify hydrophobic substrate contact residues but will have difficulty in explaining how they work.
Inhibitors of Vitamin-D-related CYP Enzymes The structural analysis of the vitamin-D-related CYPs has been performed in parallel to a search for inhibitors
of these enzymes. Because of the timing of the cloning work and the perception of the relative importance of these vitamin-D-related CYPs most of this work has been directed towards CYP24A1 and to a lesser extent CYP27B1. CYP24A1 inhibitors offer the promise that they would raise intracellular 1,25-(OH)2D3 levels and might have utility as treatments for psoriasis, in cancer therapy, and in the treatment of secondary hyperparathyroidism in CKD. CYP27B1 inhibitors offered the promise that they would lower serum 1,25-(OH)2D3 levels and might have utility as agents to treat forms of vitamin-D-related hypercalcemia: possibly nephrolithiasis, idiopathic hypercalcemia. Sandoz/Novartis initiated a program to synthesize a family of CYP24A1/ CYP27B1 inhibitors based upon chemical modification of the common azole template used successfully for other CYP family members (e.g., general P450 inhibitor, ketoconazole or aromatase inhibitor, letrazole). Schuster et al. succeeded in developing both CYP27B1- and CYP24A1selective inhibitors [119e121] though these were never developed clinically. These inhibitors, most notably VID-400, have been used with some degree of success in animal research settings [122] to try to establish the relative importance of CYP27B1 and CYP24A1 in a target cell context. Another commercial company, Cytochroma Inc., has also developed inhibitors to vitamin-D-related CYPs based upon a vitamin D template and tuning out the VDR-binding properties [123]. Several of these molecules, including CTA-018, are under development as CYP24A1 inhibitors for potential clinical use in CKD, psoriasis, and cancer [124]. All of these vitamin-D-related CYP inhibitors are usually specific enough for use as single-purpose drugs and rarely inhibit other unrelated CYPs and occasionally trigger VDR-mediated gene transcription. Accordingly, their properties indicate that the vitamin-D-related CYPs must have a binding pocket that retains some specificity for its vitamin D substrate but also shares a degree of similarity to the ligandbinding pocket of the VDR and the substrate-binding pockets of nonspecific CYPs.
Future Directions In addition to the predictable improvement in the resolution of the structures of vitamin-D-related CYPs over the coming years, several other future developments can be speculated upon. Firstly, there will be a continued search for natural mutations of any of the known CYPs. Loss of function mutations have manifested themselves as VDDR Type 1A and 1B, but other variants can be predicted as the techniques for exploring the human genome become more powerful and more widely used. It is possible that some of these variants are associated with previously unconnected disease states. Secondly, one can predict that the concept of
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37
ADDITIONAL TOPICS
Crystal-structure-based analysis of the conservation of amino acid sequences in mitochondrial vitamin D hydroxylases and substrate binding in the crystal structure of CYP2R1 [18]. The top panel depicts the frequency of conserved residues in CYP27A1 (41 species, excluding nine fish and frog) mapped from sequence alignments onto the crystal structure of CYP24A1. The extensively open active site seen in the crystal structure of CYP24A1 is very likely conserved in CYP27A1 and CYP27B1 and provides a clear view through to the heme. The positions of two mutations causing autosomal CYP27A1 deficiency (cerebrotendinous xanthomatosis, CTX) are indicated. The first residue, Thr339, is highly conserved (with only minor exceptions) in the I-helix of cytochrome P450s and is thought to structurally distort a helical turn necessary for molecular oxygen staging during the redox cycle. The second residue, Arg405, forms a hydrogen bond with the heme A-ring propionate to structurally stabilize the active site. Many microsomal P450s, including CYP2R1, use histidine for the same purpose. In the middle panel, mutation of corresponding residues causes CYP27B1 deficiency (vitamin-D-dependent rickets type I, VDDR-I). The frequency of conserved residues in CYP27B1 (39 species including five fish and frog) appears to be marginally better than CYP27A1. Approximately 40% of the residues are >95% conserved (green) across species and these are mostly in the core of the enzyme and on the proximal heme ferredoxin-binding surface. The less conserved yellow (80e95%), orange (60e80%), and blue (<60%) residues are generally found on the surface of the enzyme where the selective pressure of structuree function relationships is less. The lower panel shows CYP2R1 co-crystallized with vitamin D3. In this structure (2ojd.pdb and also 3c6g.pdb), the 3b-OH group in the vitamin D3 A-ring mediates hydrogen bond contacts to the protein backbone at the carbonyl of Ala250 and a water molecule stabilized by Asn217. The position of the hydroxylation target carbon (C25) is slightly out of range of target carbons geometrically triangulated from 49 P450 crystal structures (pink spheres above heme) possibly due to the fact that the substrate exhibits a trans-triene and not the cis-triene characteristic of vitamin D. Please see color plate section.
FIGURE 3.8
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3. THE ACTIVATING ENZYMES OF VITAMIN D METABOLISM (25- AND 1a-HYDROXYLASES)
CYP2R1 being the physiologically relevant vitamin D25-hydroxylase will be more thoroughly tested with the development of a CYP2R1-null mouse model or other genetic approach. Thirdly, the role of the extrarenal CYP27B1 will be further explored in tissues outside of the immune system. Fourthly, we may see the further development of CYP inhibitors which might be used to partially block overproduction of 1,25-(OH)2D3, in conditions where mild hypercalciuria or hypercalcemia are found. Fifthly, no doubt there will be further development of vitamin D analogs based in part upon subtle details derived from new knowledge of the structures of vitamin-D-related CYPs rather than being based upon VDR-binding alone or upon building an “armor-plated” structure to block metabolism or a metabolism-sensitive molecule. Time will tell which, if any, of these future directions will become a reality.
References [1] H.F. DeLuca, Vitamin D: the vitamin and the hormone, Fed. Proc. 33 (1974) 2211e2219. [2] G. Ponchon, H.F. DeLuca, The role of the liver in the metabolism of vitamin D, J. Clin. Invest. 48 (1969) 1273e1279. [3] D.R. Fraser, E. Kodicek, Unique biosynthesis by kidney of a biological active vitamin D metabolite, Nature 228 (1970) 764e766. [4] M. Hewison, J. Adams, Chapter 79: Extra-renal 1a-hydroxylase activity and human disease, in: D. Feldman, W. Pike, F. Glorieux (Eds.), Vitamin D, second ed., Academic Press, San Diego CA, 2005, pp. 1379e1403. [5] J.B. Cheng, M.A. Levine, N.H. Bell, D.J. Mangelsdorf, D.W. Russell, Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase, Proc. Natl. Acad. Sci. USA 101 (2004) 7711e7715. [6] R. St-Arnaud, S. Messerlian, J.M. Moir, J.L. Omdahl, F.H. Glorieux, The 25-hydroxyvitamin D 1-a-hydroxylase gene maps to the pseudovitamin D-deficiency rickets (PDDR) disease locus, J. Bone Miner. Res. 12 (1997) 1552e1559. [7] K. Takeyama, S. Kitanaka, T. Sato, M. Kobori, J. Yanagisawa, S. Kato, 25-Hydroxyvitamin D3 1a-hydroxylase and vitamin D synthesis, Science 277 (1997) 1827e1830. [8] D.R. Nelson, The cytochrome p450 homepage, Hum. Genomics 4 (2009) 59e65. [9] M. Gascon-Barre, Chapter 4: The vitamin D 25-hydroxylase, in: D. Feldman, W. Pike, F. Glorieux (Eds.), Vitamin D, second ed., Academic Press, San Diego CA, 2005, pp. 47e69. [10] H.L. Henry, Chapter 5: The 25-Hydroxyvitamin D 1a-hydroxylase, in: D. Feldman, W. Pike, F. Glorieux (Eds.), Vitamin D, second ed., Academic Press, San Diego CA, 2005, pp. 69e85. [11] G. Jones, S. Strugnell, H.F. DeLuca, Current understanding of the molecular actions of vitamin D, Physiol. Rev. 78 (1998) 1193e1231. [12] G.K. Whitfield, P.W. Jurutka, C. Haussler, J.-C. Hsieh, T. Barthel, E.T. Jacobs, et al., Chapter 13: Nuclear Receptor: Structure-Function, Molecular Control of gene Transcription and Novel Bioactions, in: D. Feldman, J.W. Pike, F.H. Glorieux (Eds.), Vitamin D, second ed., Academic Press, San Diego CA, 2005, pp. 219e262. [13] D.E. Prosser, Y.-D. Guo, Z. Jia, G. Jones, Molecular modelling of CYP27A1 and site-directed mutational analyses affecting vitamin D hydroxylation, Biophys. J. 90 (2006) 3389e3409.
[14] H. Hamamoto, T. Kusudo, N. Urushino, H. Masuno, K. Yamamoto, S. Yamada, M. Kamakura, M. Ohta, K. Inouye, T. Sakaki, Structure-function analysis of vitamin D 24-hydroxylase (CYP24A1) by site-directed mutagenesis: amino acid residues responsible for species-based difference of CYP24A1 between humans and rats, Mol. Pharmacol. 70 (2006) 120e128. [15] S. Masuda, D.E. Prosser, Y.D. Guo, M. Kaufmann, G. Jones, Generation of a homology model for the human cytochrome P450, CYP24A1, and the testing of putative substrate binding residues by site-directed mutagenesis and enzyme activity studies, Arch. Biochem. Biophys. 460 (2007) 177e191. [16] D.E. Prosser, M. Kaufmann, B. O’Leary, V. Byford, G. Jones, Single A326G mutation converts hCYP24A1 from a 25(OH)D324-hydroxylase into -23-hydroxylase generating 1a,25(OH)2D3-26,23-lactone, Proc. Natl. Acad. Sci. USA 104 (2007) 12673e12678. [17] A.J. Annalora, E. Bobrovnikov-Marjon, R. Serda, A. Pastuszyn, S.E. Graham, C.B. Marcus, J.L. Omdahl, Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: insights into substrate specificity and membrane bound structure-function, Arch. Biochem. Biophys. 460 (2007) 262e273. [18] N. Strushkevich, S.A. Usanov, A.N. Plotnikov, G. Jones, H.W. Park, Structural analysis of CYP2R1 in complex with vitamin D3, J. Mol. Biol. 380 (2008) 95e106. [19] A.J. Annalora, D.B. Goodin, W.X. Hong, Q. Zhang, E.F. Johnson, C.D. Stout, Crystal structure of CYP24A1, a mitochondrial cytochrome P450 involved in vitamin D metabolism, J. Mol. Biol. 396 (2010) 441e451. [20] M.H. Bhattacharyya, H.F. DeLuca, The regulation of rat liver calciferol-25-hydroxylase, J. Biol. Chem. 248 (1973) 2969e2973. [21] M.H. Bhattacharyya, H.F. DeLuca, Subcellular location of rat liver calciferol-25-hydroxylase, Arch. Biochem. Biophys. 160 (1974) 58e62. [22] M. Fukushima, Y. Nishil, M. Suzuki, T. Suda, Comparative studies on the 25-hydroxylations of cholecalciferol and 1 alphahydroxycholecalfierol in perfused rat liver, Biochem. J. 170 (1978) 495e502. [23] J.B. Cheng, D.L. Motola, D.J. Mangelsdorf, D.W. Russell, Deorphanization of cytochrome P450 2R1: a microsomal vitamin D 25-hydroxylase, J. Biol. Chem. 278 (2003) 38084e38093. [24] D.R. Nelson, Comparison of P450s from human and fugus: 420 million years of vertebrate P450 evolution, Arch. Biochem. Biophys. 409 (2003) 18e24. [25] G. Jones, V. Byford, S. West, S. Masuda, G. Ibrahim, M. Kaufmann, J. Knutson, S. Strugnell, R. Mehta, Hepatic Activation & Inactivation of Clinically-Relevant Vitamin D Analogs and Prodrugs, Anticancer Res. 26 (2006) 2589e2596. [26] S.J. Casella, B.J. Reiner, T.C. Chen, M.F. Holick, H.E. Harrison, A possible genetic defect in 25-hydroxylation as a cause of rickets, J. Pediatr. 124 (1994) 929e932. [27] D. Fraser, S.W. Kooh, H.P. Kind, M.F. Holick, Y. Tanaka, H.F. DeLuca, Pathogenesis of hereditary vitamin-D-dependent rickets. An inborn error of vitamin D metabolism involving defective conversion of 25-hydroxyvitamin D to 1a,25-dihydroxyvitamin D, N. Engl. J. Med. 289 (1973) 817e822. [28] T.D. Thacher, P.R. Fischer, J.M. Pettifor, J.O. Lawson, C.O. Isichei, G.M. Chan, Case-control study of factors associated with nutritional rickets in Nigerian children, J. Pediatr. 137 (2000) 367e373. [29] T.J. Wang, F. Zhang, J.B. Richards, B. Kestenbaum, J.B. van Meurs, D. Berry, et al., Common genetic determinants of vitamin D insufficiency: a genome-wide association study, Lancet 376 (2010) 180e188.
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[135] M. Rahmaniyan, K.S. Patrick, N.H. Bell, Characterization of recombinant CYP2C11: a vitamin D 25-hydroxylase and 24-hydroxylase, Am. J. Physiol. Endocrinol. Metab. 288 (2005) E753eE760. [136] F. Hosseinpour, I. Ibranovic, W. Tang, K. Wikvall, 25-Hydroxylation of vitamin D3 in primary cultures of pig hepatocytes: evidence for a role of both CYP2D25 and CYP27A1, Biochem. Biophys. Res. Commun. 303 (2003) 877e883. [137] I. Aiba, T. Yamasaki, T. Shinki, S. Izumi, K. Yamamoto, S. Yamada, et al., Characterization of rat and human CYP2J enzymes as Vitamin D 25-hydroxylases, Steroids 71 (2006) 849e856. [138] K. Inoue, J. Inazawa, H. Nakagawa, T. Shimada, H. Yamazaki, F.P. Guengerich, et al., Assignment of the human cytochrome P-450 nifedipine oxidase gene (CYP3A4) to chromosome 7 at band q22.1 by fluorescence in situ hybridization, Jpn. J. Hum. Genet. 37 (1992) 133e138. [139] G.K. Fu, A.A. Portale, W.L. Miller, Complete structure of the human gene for the vitamin D 1a-hydroxylase, P450c1, DNA. Cell Biol. 16 (1997) 1499e1507. [140] M. Labuda, N. Lemieux, F. Tihy, C. Prinster, F.H. Glorieux, Human 25-hydroxyvitamin D 24-hydroxylase cytochrome P450 subunit maps to a different chromosomal location than that of pseudovitamin D-deficient rickets, J. Bone Miner. Res. 8 (1993) 1397e1406. [141] K.-S. Chen, J.M. Prahl, H.F. DeLuca, Isolation and expression of human 1,25-dihydroxyvitamin D3 24-hydroxylase cDNA, Proc. Natl. Acad. Sci. USA 90 (1993) 4543e4547. [142] Y. Ohyama, M. Noshiro, K. Okuda, Cloning and expression of cDNA encoding 25-hydroxyvitamin D3 24-hydroxylase, FEBS Lett. 278 (1991) 195e198.
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C H A P T E R
4 CYP24A1: Structure, Function, and Physiological Role Rene´ St-Arnaud Genetics Unit, Shriners Hospital for Children, Montreal (Quebec), Canada; Departments of Medicine, Surgery, and Human Genetics, McGill University, Montreal (Quebec), Canada
In the first case, the reaction leads to the formation of 24,25(OH)2D, a metabolite that circulates in the bloodstream. In the second instance, the initial, short-lived enzymatic product is 1,24,25(OH)3D. For CYP24A1dependent hydroxylation, electrons are derived from NADPH and transferred in sequence through NADPH-adrenodoxin reductase and adrenodoxin to the heme molecule in the enzyme’s active center where molecular oxygen is bound, then split into a reactive oxyferryl center that is directed to the targeted substrate and subsequently reduced to a hydroxyl group. The enzyme is termed a mixed-function oxidase because the other oxygen atom is reduced to H2O [1]. The specificity of this catalytic reaction is determined by substrate orientation within the enzyme’s active site, where the target carbon atom must be in close proximity to the hemeeoxyferryl center. Interestingly, the CYP24A1 enzyme is able to hydroxylate both the C23 or the C24 side-chain carbons of 25(OH)D or 1,25 (OH)2D [2]. The relative level of C23- and C24-hydroxylase activity appears species-specific and the structural basis of this altered specificity has recently been examined using sequence alignment and site-directed mutagenesis and is described below. C24 hydroxylation leads to side-chain cleavage and oxidation to a carboxylic acid (C24 oxidation pathway), while hydroxylation at carbon 23 results in side-chain lactone formation (C23 hydroxylation pathway).
OVERVIEW In a classic endocrine negative feedback loop, the vitamin D hormone, 1,25(OH)2D, induces in target tissues the expression of the main effector of its catabolic breakdown, the CYP24A1 enzyme. This VDR-mediated transcriptional response insures attenuation of the 1,25 (OH)2D biological signal inside target cells and helps regulate vitamin D homeostasis. Catabolism of 1,25(OH)2D occurs through CYP24A1mediated modification of the secosteroid’s aliphatic side-chain (Fig. 4.1). CYP24A1 is a mitochondrial innermembrane cytochrome P450 enzyme that utilizes the soluble ironesulfur protein adrenodoxin and the flavoenzyme adrenodoxin reductase for NADPH-derived electron transfer into its heme center. The enzyme exhibits multifunctionality and the pathways catalyzed by the protein have been mapped out. The physiological relevance of these pathways has been confirmed in mice deficient for the Cyp24a1 gene. Recent work has focused on structureefunction analysis and several homology models were described before the crystal structure of the rat CYP24A1 protein was reported. This chapter will review the enzymatic pathways, the structureefunction relationships, and the role of CYP24A1 in the catabolism of 1,25(OH)2D. In addition, the putative additional biological roles of the enzyme and one of its main catalytic products will be presented, together with the perspectives offered by pharmacological modulation of its activity.
C24 Oxidation Pathway In the mid- to late-1970s, the C24-hydroxylation of 1,25(OH)2D was shown to be induced by 1,25(OH)2D itself [3,4]. Since the product of that reaction, 1,24,25 (OH)3D, was ten times less active than the 1,25(OH)2D
CYP24A1-CATALYZED PATHWAYS The main substrates for the hydroxylation reactions catalyzed by CYP24A1 are 25(OH)D and 1,25(OH)2D.
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10004-6
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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4. CYP24A1: STRUCTURE, FUNCTION, AND PHYSIOLOGICAL ROLE
FIGURE 4.1 Enzymatic pathways catalyzed by CYP24A1. The C24-oxidation pathway products are shown on the left; the C23-hydroxylation products are depicted at the right. Only the products of 1,25(OH)2D are represented; refer to the text for the 25(OH)D products.
substrate [3], investigators began to reason that the C24hydroxylation reaction was perhaps the first step in an inactivation process. It was also discovered that the C24-hydroxylated metabolites, 24,25(OH)2D and 1,24,25(OH)3D, could be further converted to different metabolic products sporting C24-oxo and/or C23hydroxy groups [5e7]. Studies using perfused rat kidney then led to the identification of additional
metabolites: a C23-alcohol [8] and a C23-acid, calcitroic acid [9,10]. Those metabolites had not been previously identified in vitro. Calcitroic acid was shown to be the main biliary excretory form of 1,25(OH)2D [11]. Each metabolite was identified by a combination of high-performance liquid chromatography (HPLC), UV spectrophotometric, and mass spectrometric techniques.
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CYP24A1-CATALYZED PATHWAYS
With most metabolites identified, investigators deduced that C24-hydroxylation initiates the C24 oxidation pathway that leads to 1,25(OH)2D degradation [9,10]. This pathway comprises five enzymatic steps (Fig. 4.1): it begins with 24-hydroxylation of 1,25 (OH)2D to yield 1,24,25(OH)3D. This metabolite is ketonized to 24-oxo-1,25(OH)2D. Carbon 23 is then hydroxylated to generate 24-oxo-1,23,25(OH)3D, and this compound is metabolized by oxidative cleavage of the carbonecarbon bond between C23 and C24 to produce 24,25,26,27-tetranor-1,23(OH)2D. This C23 alcohol converts to calcitroic acid, the excretory product of 1,25 (OH)2D in bile. When the initial substrate is 25(OH)D, the metabolic intermediates are 24,25(OH)2D, 24-oxo-25(OH)D, 24oxo-23,25(OH)2D, 24,25,26,27-tetranor-23(OH)D, and finally calcitroic acid. The 1,25(OH)2D-inducible 24-hydroxylation and calcitroic acid production were observed in several cell lines from kidney, bone, intestine, skin, and breast [12e14], demonstrating that the C24 oxidation catabolic pathway can be induced in a number of vitamin D target cells.
C23 Hydroxylation Pathway The discovery of a C23 oxidative pathway for 25(OH)D emerged from the identification of 25(OH)D-26,23lactone, 1,25(OH)2D-26,23-lactone, and their respective metabolic precursors [15e20]. Using kidney mitochondria isolated from a variety of species, the 24-hydroxylase and 23-hydroxylase activities were found to copurify [21]. Interestingly, some species use both pathways, such as in humans [2,22e24], while others preferentially 23hydroxylate (guinea pig, opossum) [25,26] or primarily 24-hydroxylate (rat) [21]. Classical biochemical studies hinted that the two enzymatic activities might have different kinetic parameters [21], but for quite some time it was unclear whether the reactions were catalyzed by a single enzyme or by distinct proteins. The cloning and expression of recombinant CYP24A1 from different species resolved the discrepancy and demonstrated that a single polypeptide chain was capable of both C23- and C24-hydroxylation activities. The structural basis of the C23- vs. C24-hydroxylation regioselectivity has been examined using site-specific mutagenesis [27] and will be detailed below. The human CYP24A1 was expressed in bacteria and used in a reconstituted system consisting of the bacterial membrane fraction, adrenodoxin, and adrenodoxin reductase. This system was used to study the metabolism of 25(OH)D or 1,25(OH)2D by HPLC and mass spectrometric analysis [2]. All steps of the C23-hydroxylation pathway were catalyzed by the recombinant human CYP24A1 enzyme: from 25(OH)D to 23,25
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(OH)2D, then 23,25,26(OH)3D, followed by conversion to 25(OH)D-26,23-lactol and finally 25(OH)D-26,23lactone [2]. When the initial substrate is 1,25(OH)2D, the metabolic intermediates are 1,23,25(OH)3D, 1,23,25,26 (OH)4D, 1,25(OH)2D-26,23-lactol and finally 1,25 (OH)2D-26,23-lactone (Fig. 4.1). The biological activity of the C23-hydroxylation metabolites is unclear but there have been claims that the terminal 1,25(OH)2D-derived product, 1,25(OH)2D26,23-lactone, could act as a VDR antagonist [28,29]. It appears surprising that CYP24A1 would catalyze two independent pathways from the 25-hydroxylated forms of vitamin D. The finding that 1,25(OH)2D-26,23lactone acts as a VDR antagonist [28,29] suggests that the lactone pathway provides a redundant or fail-safe mechanism to more efficiently and rapidly dampen the vitamin D signal [27].
CYP24A1, A Multifunctional Enzyme The CYP24A1 enzyme has been purified to homogeneity from rat kidney mitochondria [30] and the purified protein was used to raise antibodies [31] that permitted cloning of the cDNA [32]. This then allowed production of the recombinant protein in parallel with the cloning of the gene from various species [33e35]. The recombinant CYP24A1 protein, when associated with its electrontransport cofactors, NADPH-adrenodoxin reductase and adrenodoxin, has been shown to perform multiple steps of the C24-oxidation pathway. This includes 23hydroxylation, dehydrogenation of the 24-hydroxyl group, and side-chain cleavage [22,23]. CYP24A1 is thus a bona fide multicatalytic enzyme. The recombinant human CYP24A1 protein exhibited release of the product at each step of the C23- and C24-hydroxylation pathways [2]. Thus, multiple intermediate metabolites, such as for example 24,25(OH)2D, can be released from the CYP24A1 substrate-binding pocket. When metabolism of 25(OH)D was studied using kidney homogenates from vitamin-D-treated or vitamin-D-deficient chicks, conversion from 25(OH)D to 25(OH)D-26,23-lactol to 25(OH)D-26,23-lactone was measured in samples from both D-replete and D-deficient animals [36]. Since Cyp24a1 is a 1,25(OH)2Dinduced gene, the conversion to 25(OH)D-26,23-lactone measured in vitamin-D-deficient chicks hints that it can be catalyzed even in the absence of CYP24A1, although it is clear that the recombinant CYP24A1 protein has the ability to perform this conversion. It has been suggested that an unknown aldehyde oxidase could catalyze this reaction in vitamin-D-deficient chicks [36]. Conversion rates from 1,25(OH)2D-26,23-lactol to 1,25(OH)2D-26,23-lactone in tissue homogenates from 1,25(OH)2D-deficient chicken range from 55e61% of
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4. CYP24A1: STRUCTURE, FUNCTION, AND PHYSIOLOGICAL ROLE
those measured in 1,25(OH)2D-supplemented chicken [37]. These measured differences may represent the relative contributions of the putative aldehyde oxidase and of CYP24A1 for the conversion to the lactone under physiological conditions.
findings suggest that the Cyp24a1-null survivors adapt to the impaired vitamin D catabolism not by using an alternative catabolic route, but by limiting the synthesis of the active compound [41].
STRUCTUREeFUNCTION RELATIONSHIPS
Biological Relevance of the C24 Oxidation Pathway The hypothesis that the main role of the C24 oxidation pathway is attenuation of the 1,25(OH)2D biological signal inside target cells was tested in vitro using cytochrome P450 inhibitors. Blocking P450 activity by treatment of cells with the antifungal imidazole derivative, ketoconazole, inhibits catabolism and results in 1,25 (OH)2D accumulation and extended hormone action [38]. This hypothesis was also tested and confirmed in vivo by engineering Cyp24a1-deficient mice to examine the role of the CYP24A1 enzyme in vitamin D homeostasis [39]. Fifty percent of Cyp24a1e/e mice die before 3 weeks of age [39,40]. Analysis of macrophage function ruled out impaired responses to infection as the cause for postnatal death. The perinatal lethality is most likely a consequence of hypercalcemia secondary to hypervitaminosis D, since the inactivation of the Cyp24a1 gene in mice impaired the ability of the animals to clear 1,25(OH)2D. Bolus and chronic 1,25(OH)2D administration resulted in a marked elevation in serum 1,25(OH)2D levels in the mutant animals [39]. Chronic 1,25(OH)2D administration in Cyp24a1e/e mutants resulted in histological changes consistent with hypervitaminosis D in the kidney: cortical tubular dilation, necrotic debris and mineralization (nephrocalcinosis). The inability to regulate 1,25(OH)2D and calcium homeostasis presumably leads to fatal hypercalcemia. Indeed, extremely high levels of circulating 1,25(OH)2D and calcium were measured in runted animals that died before weaning [39]. Since half of the mutant progeny appear unaffected by the Cyp24a1 deficiency, these surviving animals most likely use alternate means of regulating vitamin D homeostasis. The pharmacokinetics of labeled 1,25 (OH)2D were measured in Cyp24a1e/e survivors and heterozygote controls. These experiments have shown that Cyp24a1-null mice have impaired clearance of 1,25 (OH)2D. Surprisingly, Cyp24a1e/e mice demonstrate total absence of both 24-hydroxylated metabolites and 1,25(OH)2D-26,23-lactone [41]. Similar results were obtained in VDR-knockout mice [41,42], demonstrating that the VDR-dependent induction of Cyp24a1 expression is required for production of calcitroic acid and 1,25(OH)2D-26,23-lactone production in vivo. These data raise doubts about any physiological contribution of an unknown aldehyde oxidase for the conversion to the lactone [36], at least in mice. Furthermore, these
There has been significant interest in performing structureefunction analyses and generating homology models of the CYP24A1 structure while awaiting crystal structure resolution, which fortunately was recently reported [43]. Efforts stemmed from two different perspectives: on the one hand, investigators were intrigued by the species-specific bias for C23- or C24hydroxylation [2,21e24]. On the other hand, there has been steady pharmacological interest in the development of CYP24A1 inhibitors to prolong the bioactivity of 1,25(OH)2D or its relevant analogs [44e47]. Information on tertiary structure of the substrate-binding pocket was perceived as relevant to develop specific inhibitors. A seminal finding within the cytochrome P450 field was obtained when the crystal structure of class I P450s (bacterial/mitochondrial, receiving their electrons from a two-protein redox chain) was aligned with that of class II enzymes (microsomal, receiving electrons from a single reductase). This led to the observation that despite extensive differences in amino acid sequence, all P450 molecules possess similar tertiary structure [48]. This feature was used extensively to generate CYP24A1 homology models and test the role of multiple residues in substrate binding and catalysis using site-directed mutagenesis [27,49e53]. These modeling studies successfully predicted multiple residues in the CYP24A1 binding pocket as confirmed by the crystal structure. Recombinant CYP24A1 crystals were obtained following adrenodoxin-sepharose affinity chromatography purification of bacterially expressed rat Cyp24a1 sequence deleted of its mitochondrial import signal (D2e32) [49] and mutated at residue 57 (S57D) to stabilize the recombinant protein [43,54]. The CYP24A1 crystal structure displays the canonical P450 fold, including the 12 a-helices (AeL) and four b-sheet systems (b1eb4). Five additional short helices (A0 , B0 , G0 , K0 , and K00 ) were also identified [43]. The substrate-binding cavity is defined by the b1 and b4 sheets, the BeC loop, and helices E, F, G, I, and K surrounding the heme. Alignment of helices A0 and G0 with other mitochondrial P450 sequences identifies them as membrane insertion sequences (MIS) and computational modeling suggests that residues in the hydrophobic surfaces of helices A0 ˚ into the mitoand G0 can penetrate approximately 7 A chondrial inner membrane to serve as anchors flanking the substrate access channel (Fig. 4.2).
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STRUCTUREeFUNCTION RELATIONSHIPS
Structure of the rat CYP24A1 protein inserted in the inner mitochondrial membrane. The ribbon structure was obtained from the atomic coordinates deposited in the Protein Data Bank, code 3K9V (http://www.pdb.org). The arrow defines the substrate access channel and points to the heme. MIS, membrane insertion sequence. Please see color plate section.
FIGURE 4.2
The crystal structure identified 19 residues (from nine regions of the folded sequence) that surround the activesite cavity [43]. These are Leu129 and Ile131 (from the BeB0 region); Trp134 (within the B0 -helix); Met148 (B0 eC region); Met245, Met246, and Phe249 (from the F-helix); His271 and Trp275 (within the G-helix); Leu325, Ala326, Glu329, and Thr330 (four residues of the I-helix); Val391 (K-helix/b1-4 loop); Phe393, Thr394, and Thr395 (part of the b1e4 sheet); and Gly499 and Ile500 (from the b4-1/b4-2 turn). Of these 19 residues, 13 were correctly predicted as relevant for substrate binding and catalysis in homology modeling studies: Ile131, Trp134, Met148, Met245, Met246, Phe249, Ala326, Glu329, Thr330, Val391, Thr394, Gly499, and Ile500 [50,51,53]. The P450 structuralmotif-based method of 3D-homology modeling used by Masuda et al. [53] appears particularly effective as it allowed the authors to make strong predictions concerning the positioning and role of six residues (Ile131, Trp134, Met148, Met246, Ala326, and Gly499) within the CYP24A1 active site. These predictions were borne out by the solved crystal structure. As mentioned previously, CYP24A1 is a multifunctional enzyme that, amongst other catalyzed reactions, can either 23-hydroxylate or 24-hydroxylate the 1,25 (OH)2D substrate. The degree to which CYP24A1 performs each reaction is species-dependent [2,21e24] and it was reasoned that species-specific sequence differences could explain the behavior of the enzyme. A systematic site-directed mutagenesis effort to convert the putative substrate-binding residues of the rat sequence to those of the human sequence identified
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the residues at positions 416 (Thr in rat and Met in human) and 500 (Ile in rat and Thr in human) as important for C23-hydroxylation. However, the changes in C24-/C23-hydroxylation ratio measured in that study were quite modest [52]. Furthermore, the amino acid differences between the human and rat sequences at residues 416 and 500 are not consistently different in nonhuman species including opossum and guinea pig, with predominantly 23-hydroxylate vitamin D substrates. This observation hinted that it was unlikely that residues 416 and 500 were the primary determinants of the species-specific differences in regioselectivity. Alignment of the I-helix (residues 312e345) from 19 species identified residue 326 (Ala in human and Gly in opossum) as a potential key determinant [27]. When alanine 326 in the human CYP24A1 is changed to a glycine, as it is in opossum and guinea pig, the catalytic pattern is dramatically altered to favor 23-hydroxylation. No other mutation produced a comparable radical change in hydroxylation pattern [27]. The side chain of Ala326 appears as a major determinant of the depth of substrate penetration within the enzyme’s binding pocket and of the alignment of the hydroxylation site above the heme iron atom. Helix I was confirmed as a structural element defining the substrate-binding cavity in the crystal structure [43]. When substrate docking simulations with 1,25(OH)2D were performed on the open CYP24A1 crystal structure [43], the calculations yielded proper nanomolar affinity (Ki ¼ 2.65 nM) and a reproducible conformation in accord with predictions made by homology modeling [50,51,53]. In the crystal structure-based simulations, the 1,25 (OH)2D molecule was stabilized by two hydrogen bonds and multiple hydrophobic interactions among key conserved residues identified in the homology models: Ile131, Trp134, Met246, Phe249, Ala326, Val391, Thr394, Thr395, Gly499, and Ile500. The two hydrogen bonds were computed between the C3-hydroxyl group of the secosteroid A ring and the BeB0 loop at amino acid Leu129, and between the C25-hydroxyl group of the side chain and residue Leu325 within the I-helix [43]. In the calculated configuration using the open conformation of CYP24A1 [43], the C21-methyl group is positioned over the heme iron, and thus it is unlikely that the open structure computation represents the substrate’s terminal binding configuration relevant for catalysis (which requires the C23 or C24 carbons to be aligned with the heme). However, the ability of the 1,25(OH)2D substrate to bind deep within the active site and interact with the heme group should accommodate the cavity collapse to a closed state required to exclude solvent for catalysis and for proper substrate positioning. Improper substrates bound in a shallow conformation would not be able to stabilize the closed state, and this may contribute to enzymatic specificity [43].
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Site-directed mutagenesis of a related vitamin D metabolic enzyme, CYP27B1, had identified the invariant arginine pair within helix L (Arg465 and Arg466) as critical for protein folding and/or heme binding and more clearly linked Arg466 to the electron and oxygen transfer steps required for catalytic function [55]. The crystal structure of CYP24A1 reveals that these basic residues’ side chains protrude below the heme and that Arg466 is positioned to promote electron transfer between adrenodoxin and the heme iron. The side ˚ chains of Arg465 and Arg466 are also within 8e10 A of the conserved K-helix residues (Lys378 and Lys382) involved in adrenodoxin recognition and electron transfer in CYP27B1 [55]. Overall, the CYP24A1 crystal structure identified conserved residues from helices K, K00 , and L that face an adjacent lysine-rich loop to provide the interface for binding of the redox protein [43]. The CYP24A1 crystal structure thus provides a template for understanding membrane insertion, substrate binding, and electron chain partner interactions. This molecular understanding of the structuree function relationships of the CYP24A1 protein will greatly facilitate biorational drug development.
PUTATIVE CYP24A1 INVOLVEMENT IN NONCLASSICAL SYSTEMS The prevalent view implicates CYP24A1 mainly in the attenuation of the calcemic 1,25(OH)2D biological activity. But the enzyme is inducible in all 1,25(OH)2D target tissues and evidence has emerged that CYP24A1 expression might be relevant for vitamin D functions distinct from mineral homeostasis. Furthermore, intermediate metabolites can be released from the CYP24A1 substrate-binding pocket. One such metabolite, 24,25(OH)2D, is the most abundant dihydroxylated metabolite in the circulation and it has been proposed that it may exert biological effects.
Role of 24,25(OH)2D in Chondrocyte Maturation An extensive literature demonstrates that Cyp24a1 is expressed in growth plate chondrocytes and that cells from the growth plate respond to 24,25(OH)2D in a cell maturation-dependent manner (reviewed in [56,57]). Most of these studies were performed using the in vitro rat costochondral primary culture system. Dissection of the tissue allows isolation of cells from different regions of the growth plate. Each region represents a different maturation stage along the chondrocytic differentiation pathway. In this model system, the less differentiated cells of the resting zone, also called the reserve zone, respond to 24,25(OH)2D. The more mature cells of the
growth zone, including the prehypertrophic and hypertrophic compartments, respond primarily to 1,25 (OH)2D. The effects of 24,25(OH)2D were also recently observed in the well-characterized prechondrocytic cell line ATDC5 [58,59]. In resting zone cells, 24,25(OH)2D decreases cell proliferation but stimulates differentiation and maturation: the metabolite stimulates extracellular matrix production through stimulation of the synthesis of sulfated glycosaminoglycans [60]. It also causes the cells to produce extracellular matrix vesicles that contain neutral metalloproteinases [61] but reduces total matrix vesicle metalloproteinase activity [62,63]. 24,25(OH)2D was shown to act on resting zone chondrocytes via phospholipase D [64,65]. The proposed mechanism invokes that 24,25(OH)2D-mediated stimulation of phospholipase D promotes the conversion of phosphatidylcholine to phosphatidic acid, leading to lysophosphatidic acid (LPA) production. LPA in turn stimulates increases in alkaline phosphatase activity and sulfation, while protecting resting zone cells from apoptotic cell death [66]. Interestingly, treatment of resting zone chondrocytes with 24,25(OH)2D induces a change in maturation state, resulting in down-regulation of responsiveness to 24,25 (OH)2D and up-regulation of responsiveness to 1,25 (OH)2D [67]. These observations support the hypothesis that 24,25(OH)2D plays a role in cartilage development. It is worth mentioning that growth plates from Cyp24a1e/e mice do not show major defects [39,68]. These observations suggest that the absence of CYP24A1 activity does not affect growth plate development and that 24,25(OH)2D is not required for chondrocyte maturation in vivo [39]. It remains possible, however, that a redundant endocrine system is able to compensate for the function of 24,25(OH)2D in animals.
CYP24A1 as a Candidate Oncogene In addition to its effects on mineral homeostasis, the vitamin D hormone, 1,25(OH)2D, regulates many cell types with respect to their growth, differentiation, and apoptosis. These findings, coupled with seminal epidemiological results [69], have stimulated interest for exploring the use of calcitriol or suitable analogs as anticancer agents [70]. The cancer-related pathways targeted by 1,25(OH)2D are numerous and include inhibition of cell-cycle progression, induction of apoptosis, as well as reduction of angiogenesis [70]. The antiproliferative effects of 1,25(OH)2D are significantly reduced in tumors derived from VDR-deficient mice [71], indicating that the VDR mediates these antitumor effects. As detailed above, the bioactivity of 1,25(OH)2D is regulated by catabolic pathways in target tissues, and thus the antitumor effects of the vitamin D hormone may be restricted by the activity of CYP24A1. Indeed,
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PUTATIVE CYP24A1 INVOLVEMENT IN NONCLASSICAL SYSTEMS
copy number or expression of CYP24A1 has been found to be increased in human tumors. Assessment of the expression or possible amplification of CYP24A1 may thus be clinically relevant for cancer treatment. The human CYP24A1 gene maps to the 20q13.2e20q13.3 chromosomal region [72]. The 20q region is of particular significance in cancer research as it shows gains or amplifications in a number of adenocarcinoma types [73e80]. Using array comparative genomic hybridization (CGH), three studies have examined chromosome 20 aberrations in breast cancer, prostate cancer, and gastric adenocarcinomas to define the location of amplicon boundaries and amplification maxima [73,78,81]. The chromosome region where CYP24A1 is located was amplified in each type of cancer. The results revealed three breast tumors in which a relatively narrow region contained two peaks with the highest copy number [73]. These types of peaks in copy number are thought to include the “driver” gene responsible for progressive trimming of the amplified segment, a phenomenon that has been described in cultured cells under drug selection [82], and the “driver” gene is often an oncogene. CYP24A1 was located in the distal peak and was considered the candidate “driver” gene for it, leading the authors to propose its assignment as a putative oncogene [73]. While the oncogene label may be an exaggeration, it remains that CYP24A1 was shown to be overexpressed in breast tumors [73], esophageal cancers [83], skin cancers including squamous [84] and basal cell carcinomas [85], ovarian cancers [86], colon adenocarcinomas [86,87], and primary lung tumors [86,88]. The expression of the gene is also elevated in a number of human cancer cell lines from various tissue origins such as esophagus [83], colon [87], lung [88,89], and prostate [90,91]. In several of these model systems, the growth inhibition induced by 1,25(OH)2D was shown to be inversely proportional to the level of CYP24A1 expression or CYP24A1 activity [83,86,88,91,92], providing a mechanistic basis for the poor prognosis of CYP24A1-overexpressing cancers [83]. Preclinical model systems may also provide mechanistic clues. While there is no evidence that mice deficient for Cyp24a1 develop spontaneous cancers [39], the distal mouse chromosome 2, which is one of the largest regions of conserved synteny between mouse and human autosomes and corresponds to human chromosome 20q [93], is amplified in mouse islet carcinomas [94]. More interestingly, DNA methylation-based epigenetic changes at the Cyp24a1 gene promoter were shown to be involved in the differential response to 1,25(OH)2D of tumor-derived endothelial cells compared with endothelial cells of normal origin [95]. The observation that aberrant DNA hypermethylation targets the Cyp24a1 locus to affect endothelial cell responses suggests that
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endothelial cells may be a vitamin D target in the tumor microenvironment and may open up avenues for therapeutic intervention. There has also been recent interest in the putative association between polymorphisms in genes of the vitamin D pathway and cancer risk [96]. The 20.5-kblong human CYP24A1 gene contains 12 exons and several single nucleotide polymorphisms (SNPs) have been identified using HapMap or resequencing data [97e99]. As all of these are within noncoding regions, it is unclear whether they have any functional effect, except perhaps for the SNP characterized in the CYP24A1 promoter that results in lower expression of the CYP24A1 protein [99]. The HapMap-derived SNP genotypes were not associated with prostate cancer risk [98]. The CYP24A1 polymorphism from intervening sequence (IVS) 4 (intron 4), IVS4-66T > G, identified by resequencing of the gene in Caucasian samples, showed a statistically significant protective association with risk of colon cancer overall, and particularly for proximal colon cancer [97]. Three other CYP24A1 polymorphisms showed statistically significant association with risk of distal colon cancer: IVS4 þ 1653C > T (lower risk); IVS9 þ 198T > C (increased risk); and þ4125bp 30 of STPC > G (higher risk, in whites only) [97]. Of these, IVS9 þ 198T > C could be involved in the regulation of splicing of an alternate exon [97], a mechanism involved with different patterns of constitutive and inducible CYP24A1 activity [100e102]. The þ4125bp 30 of STPC > G variant, located in the 30 -untranslated region, could modulate mRNA stability [103]. Future studies will be required to confirm the risk associations and to determine the functional significance of the sequence variations.
CYP24A1 in Chronic Kidney Disease Chronic kidney disease (CKD) shows steadily increasing worldwide incidence due to an aging population and to augmenting obesity with its associated complications of hypertension and adult-onset diabetes [104]. Stages 3 and 4 CKD (moderate) are characterized by progressively decreasing kidney function as assessed by glomerular filtration rate. Severe CKD (stage 5) is associated with minimal or altogether absent kidney function and patients require regular dialysis or kidney transplant for survival [105]. With declining renal function, kidney failure patients experience declining 1,25 (OH)2D levels, which causes decreased systemic calcium levels and leads to the development of secondary hyperparathyroidism (SHPT), a disorder characterized by elevated serum levels of parathyroid hormone (PTH) [106e108]. Patients with SHPT of renal failure also develop disorders of bone termed uremic osteodystrophy, characterized by disorganized bone
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4. CYP24A1: STRUCTURE, FUNCTION, AND PHYSIOLOGICAL ROLE
remodelling and accompanied by loss of bone strength and integrity, with associated morbidity [109e111]. Untreated, SHPT of renal failure has also been associated with increased mortality through increased cardiovascular calcification and associated ischemic events [112e114]. Treatment with vitamin D analogs is advocated for the clinical care of renal patients [115,116]. These compounds act to lower PTH levels close to the normal range and help to maintain normocalcemia, bone health, and cardiovascular integrity [117e120]. Recent results suggest that in CKD, CYP24A1 expression can be aberrantly elevated [121,122]. This may be due in part to processes related to kidney damage, as well as to hormonal replacement therapeutic regimen with current vitamin D analogs. As CYP24A1 inactivates pro-hormonal, hormonal, and analog forms of vitamin D, the aberrant elevation in CYP24A1 expression may contribute to vitamin D insufficiency and exacerbate SHPT in CKD patients. These novel findings suggest that treatment of CKD patients might be another clinical management situation that could benefit from the availability of specific inhibitors of CYP24A1 [123].
24,25(OH)2D and Fracture Repair It has been proposed that 24,25(OH)2D, the enzymatic product of the CYP24A1 activity on the 25(OH)D substrate, might also play a role in fracture repair, but there is limited information available on this putative function of the metabolite. The healing of fractures is a unique postnatal biological repair process resulting in the restoration of injured skeletal tissue to a state of normal structure and function. Fracture repair involves a complex multistep process that involves response to injury, intramembranous bone formation, chondrogenesis, endochondral bone formation, and bone remodeling. Several studies have described a complex pattern of gene expression that occurs during the course of these events [124e127]. Taken together, results from gene expression monitoring during bone repair suggest that the molecular regulation of fracture healing is complex but recapitulates some aspects of embryonic skeletal formation [128,129]. A role for 24,25(OH)2D in fracture repair is supported by the observation that the circulating levels of 24,25 (OH)2D increase during fracture repair in chickens due to an increase in CYP24A1 activity [130] (Fig. 4.3, top panel). When the effect of various vitamin D metabolites on the mechanical properties of healed bones was tested, treatment with 1,25(OH)2D alone resulted in poor healing [131]. However, the strength of healed bones in animals fed 24,25(OH)2D in combination with 1,25 (OH)2D was equivalent to that measured in a control population fed 25(OH)D [131]. These results support
Cyp24a1 expression during fracture healing in chicks and mice. Top panel: the changes in CYP24A1 activity and circulating 24,25(OH)2D concentrations are listed besides the temporal sequence of fracture healing. [, increase; N.D., not determined. The putative expression of a receptor/binding protein for 24,25(OH)2D at day 10 post-fracture is expressed by a question mark. Based on the data described in references 130e133. Bottom panel: quantitative reversetranscription PCR (RT-qPCR) on mRNA extracted from the callus of the fractured right tibia (Fracture) and a diaphysial section of the left nonfractured tibia (Contralateral) of wild-type mice at 14 days postfracture. The expression of Cyp24a1 was significantly increased in the fractured bone, confirming the observations previously obtained in chicks. *, p<0.05.
FIGURE 4.3
a role for 24,25(OH)2D as being an essential vitamin D metabolite important for fracture repair. It is likely that 24,25(OH)2D would act through receptor-mediated signaling (as do other vitamin D metabolites and hormones), and circumstantial evidence suggests the presence of a nonnuclear membrane receptor for 24,25 (OH)2D in the chick tibial fracture-healing callus [132,133]. Cell fractionation to isolate a membrane fraction followed by ligand binding studies using hydroxyapatite to separate bound and free ligands described a receptor/binding protein for 24,25(OH)2D in the fracture-healing callus membrane fraction from vitamin-Ddepleted chicks [133]. These observations were never followed through and, to date, no molecular entity corresponding to this binding activity has ever been cloned or purified for complete characterization. The Cyp24a1-deficient mouse strain [39] represents an invaluable tool to examine the putative role of 24,25
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PERSPECTIVES
(OH)2D in mammalian fracture repair. Cyp24a1 mutant animals that survive past weaning appear to use an alternative pathway of 1,25(OH)2D catabolism to regulate circulating levels of the hormone [41] and are normocalcemic and normophosphatemic when fed regular rodent chow. This has allowed us to study bone healing in these animals. As a first step, the induction of the expression of the Cyp24a1 gene during fracture repair was confirmed in mice. Wild-type mice were subjected to a stabilized, transverse mid-diaphysial fracture of the tibia. To stabilize the fracture without disrupting the bone marrow microenvironment, we used a smallscale version of the Ilizarov distraction osteogenesis device used in orthopedic patients [134e136]. RNA was extracted from the fracture callus at 14 days postosteotomy, reverse-transcribed, and analyzed by RTqPCR. Cyp24a1 mRNA levels were significantly elevated in the fracture callus as compared to the undamaged contralateral bone (Fig. 4.3, bottom panel). Importantly, these results confirm the data reported previously for chicken [130] and support the hypothesis that the activity of the CYP24A1 enzyme is important for bone fracture repair. Fracture repair was then compared between Cyp24a1/ mice and wild-type controls. We have observed a delay in the mineralization of the cartilaginous matrix of the soft callus in Cyp24a1e/e mutant animals, accompanied by altered expression of differentiation marker genes (data not shown). The repair delay and the aberrant pattern of gene expression could be rescued by treatment with 24,25(OH)2D. We then used the Cyp24a1-deficient mice as a source of tissue to identify differentially expressed genes in the callus of Cyp24a1-deficient mice. This has led to the identification of a restricted set of genes which we are currently characterizing for their ability to bind 24,25(OH)2D. Taken together, our preliminary results strongly support a role for 24,25(OH)2D in mammalian fracture repair.
PERSPECTIVES 24-Hydroxylated Metabolite Supplementation for Fracture Repair Since it is a rather lengthy process, it can be argued that repair of long bones has not been optimized by evolution [137]. To survive a fracture and avoid being prey to predators, animals would have needed to restore long bone function within days. Mutations that could shorten healing time, even by half, would not have allowed survival, and thus evolutionary pressure towards optimized fracture repair must have remained slight. But because the process is not optimized, it should be quite possible to enhance it through
51
pharmacological intervention. Efforts to develop suitable preclinical models and beneficial treatments have been described [137]. The observation that mice deficient for Cyp24a1 exhibit a delay in bone fracture healing that can be corrected by exogenous administration of 24,25 (OH)2D suggests that treatment with vitamin D metabolites hydroxylated at position 24, such as 24,25(OH)2D, could be useful in the treatment of bone fractures subsequent to trauma or metabolic bone diseases. It can be argued that 24,25(OH)2D is an abundant circulating vitamin D metabolite and that it is present in sufficient amounts to efficiently promote bone healing without the need for additional supplementation. However, it is now recognized that a sizeable proportion of the population suffers from vitamin D insufficiency [138e140], which may have deleterious effects for optimized fracture repair. Thus fracture healing could benefit from supplementation with 24,25(OH)2D or a suitable analog.
CYP24A1 Inhibitors It has long been surmised that inhibition of CYP24A1 activity could be beneficial to enhance 1,25(OH)2D action. The rationale for such an inhibitory treatment was apparent when thinking of improving the efficacy of the vitamin D hormone and D analogs in cancer therapy and in the treatment of hyperproliferative diseases such as psoriasis. Novel findings suggest that CYP24A1 inhibitors could also help in the clinical management of chronic kidney disease. The first identified inhibitors were antifungal imidazole derivatives, such as ketoconazole and liarozole. They lack specificity since they inhibit steroidogenesis by interfering broadly with cytochrome P450 enzyme systems [141], although this feature could prove beneficial in the treatment of prostate cancer [90]. The prominent phytoestrogen in soy, genistein, was shown to inhibit CYP24A1 in cultured cells [142e146] as well as in mouse colon in vivo [147]. It appears to act through several mechanisms as it was shown to inhibit both CYP24A1 expression (at the transcriptional level) [144] and the activity of the CYP24A1 protein [143]. Phytoestrogens have a beneficial effect on cancers [148,149] and the discovery of their action on vitamin D metabolic pathways suggests that part of their antitumorigenic effects could be mediated by vitamin D [147]. It also raises the possibility that inhibition of CYP24A1 could be achieved in part via nutritional means [150,151]. As part of the effort to chemically synthesize vitamin D analogs with low calcemic activity, potent inhibitors of CYP24A1 based on the 1,25(OH)2D secosteroid structure were identified [123]. These include sulfone [152] and sulfoximine [44] derivatives of 1,25(OH)2D. The 16,23diene-25 sulfone analog (compound CTA018/MT2832)
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[123] is particularly interesting from a therapeutic standpoint as it exhibits a dual mechanism of action. It acts as a potent and low-calcemic inhibitor of CYP24A1, and in addition was shown to be a potent activator of VDRmediated transcription [123]. The compound was shown to exhibit adequate pharmacokinetic and pharmacodynamic profiles, and to effectively suppress elevated PTH without affecting calcemia or phosphatemia in a preclinical rodent model of CKD [123]. This new class of analogs with a dual mechanism of action may be able to achieve the desired therapeutic response of preventing secondary hyperparathyroidism in late-stage chronic kidney disease without leading to acquired resistance to vitamin D analog therapy caused by induction of CYP24A1. It is possible that yet further refinement of pharmacological CYP24A1 inhibitory strategies could be achieved through biorational drug development based on the solved crystal structure of the enzyme [43].
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C H A P T E R
5 The Vitamin D Binding Protein DBP Roger Bouillon Laboratory of Experimental Medicine and Endocrinology (Legendo), K.U.Leuven, Herestraat 49 ON1 bus 902, 3000 Leuven, Belgium
the fast-growing side of actin monomers is blocked completely through a perfect structural fit with DBP, demonstrating how DBP effectively interferes with actin-filament formation and this could have major implications for maintaining the microcirculation. Comparisons of the DBP structures with the structure of serum albumin, another family member, reveal a similar topology but also significant differences in local folding. These structural differences explain the unique vitamin-D3-binding and actin-binding properties of DBP. DBP can also bind to fatty acid at a binding site different from the cleft for vitamin D metabolites. Finally DBP can be enzymatically transformed in DBPmacrophage activating factor (DPB-MAF) and thereby influence the function of macrophages and osteoclasts. The different functions of DBP have been reviewed extensively in the previous edition of Vitamin D [10e12] and in several reviews dating back several years [13]. Therefore, this chapter will only summarize its main functions and specifically deal with recent advances over the last 5 years.
INTRODUCTION Group-specific component of serum (Gc globulin) was originally identified in 1959 by serum electrophoresis as a polymorphic protein [1]. At that time its function was not known, although it became useful in population genetics [2] and forensic medicine [3]. Its function as binding protein for all vitamin D metabolites in serum was first discovered by Daiger while looking for polymorphic proteins [4] and independently confirmed by several groups that isolated this binding protein [5,6]. The human serum vitamin-D-binding protein (DBP) has many physiologically important functions, ranging from transporting vitamin D3 metabolites, binding and sequestering globular actin and binding fatty acids to possible roles in inflammation and in the immune system. The functional implications of the polymorphic nature of DBP are still largely unknown. DBP is structurally related to albumin and a-fetoprotein, and the DBP gene is a member of the albumin and a-fetoprotein gene family. Despite large-scale human studies no cases of complete absence of DBP have yet been identified whereas this has been well documented for albumin. While this suggests that DBP may be essential for normal development and survival, DBP-null mice are viable and do not display an obvious phenotype when fed a normal diet [7]. The crystal structure of DBP in complex with the vitamin D3 metabolite 25hydroxyvitamin D3 (25(OH)D3) was solved, as well as the structure of DBP in complex with a vitamin D3 analog. Both structures reveal the vitamin-D-binding site in the N-terminal part of domain I [8]. DBP is also capable of binding monomeric actin and thereby facilitates the depolymerization of actin filaments [9]. The crystal structure of the DBPeactin complex was deter˚ resolution. This structure reveals that mined at 2.4 A
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10005-8
VITAMIN D BINDING PROTEIN: PROTEIN AND GENE STRUCTURE Gene The human DBP gene is located on chromosome 4q11-q13 as revealed by in situ hybridization to metaphase chromosome spreads [14]. This sublocalization overlaps with the known positions of albumin and a-fetoprotein [15]. These three genes have been assigned to chromosome 13 in the rat [16] and to chromosome 5 in the mouse [17]. The rodent chromosomes encoding DBP are syntenic with human chromosome 4, thus
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5. THE VITAMIN D BINDING PROTEIN DBP
demonstrating conservation of the DBPea-fetoproteinealbumin linkage in three species. A linkage between DBP and albumin has also been noted in horses and in chickens [18,19]. A fourth gene in the DBP/ albumin/a-fetoprotein family has been described by two groups and named afamin by one group [20] and a-albumin by the other [21]. a-Albumin is selectively expressed in the liver during the late stages of development, suggesting that it may be a phylogenetic intermediate between a-fetoprotein and albumin and may share some of the functions of these proteins [21,22]. Gene analysis indicates that DBP is the oldest member of the family whereas albumin and a-fetoprotein are more recent members. Indeed in zebrafish the only member of this family, based on genome analysis, is DBP and thus preceedes albumin and a-fetoprotein in vertebrate evolution [23]. Physical and meiotic mapping has determined that the organization of the gene family in humans is centromereeDBPealbuminea-fetoproteine a-albuminetelomere. Human DBP mRNA contains 1690 nucleotides and encodes a 458-amino-acid secreted protein. Rat and human DBP genes span 35 and 42 kb, respectively, and both contain 13 exons. The signal sequence of the protein is encoded on the first exon, whereas the last exon contains the entire noncoding 30 -untranslated region. Moreover, DBP cDNAs from the mouse [17], rabbit [24], chicken [25], a turtle, Trachemys scripta [26], and zebrafish [23] have been cloned and sequenced. There is a 16-amino-acid signal peptide, based upon alignment of the sequenced amino terminus of the mature protein [27,28] with the cDNA-predicted sequence of the primary translation product. A number of models have been proposed to account for the evolution of the DBP multigene family and for the shared, triplicated internal domain structure. In one model, the ancestral internal domain is encoded by four exons that subsequently triplicated, creating the present gene encoding a three-domain protein. This domain triplication likely predated vertebrate evolution [29,30]. The separation of a unique DBP gene from this precursor is estimated to have occurred 560e600 million years ago [31,32]. It has been postulated that albumin duplicated from the ancestral gene and began to diverge about 280 million years ago, just after the time of the amphibian/reptile divergence about 350 million years ago. The absence of a larval a-fetoprotein gene in amphibians and the existence of a-fetoprotein in chickens is consistent with this conclusion [31].
Protein Structure The predicted mature DBP proteins in rat, mouse, and rabbit are each 460 amino acids long, two amino acids longer than the human sequence, whereas that of T.
scripta is 466 amino acids in length. There is an N-linked glycosylation consensus sequence in human, rat, and mouse but not rabbit or T. scripta DBP. Based on the presence of sequence homology and nearly identical disulfide bridge pattern in DBP, human serum albumin (HSA), a-fetoprotein, and afamin, the overall folds of these proteins are believed to be homologous [13]. Serum DBP is a polymorphic, monomeric, serum aglobulin of approximately 58 kDa, its size being dependent upon its glycosylation state [6]. Periodic positioning of 28 cysteine residues predicts a characteristic secondary structure demarcated by internal disulfide bonds and defines the presence of three internally homologous domains [27]. The crystal structure of DBP has now been described by several groups [8,33e36]. The three domains of DBP consist entirely of a-helices, ten in domain 1, nine in domain 2, and four in domain 3. The tertiary structure of DBP can be based ˚ on the crystal structure of human DBP, solved to 2.3 A resolution [8]. This crystal consisted of two DBP molecules present in the asymmetric unit, one in complex with 25(OH)D3, and one in apoprotein form. As in human serum albumin (HSA) [37,38], DBP has an all a-helical structure and contains three structurally similar domains. DBP and the other members of its protein family (human serum albumin, afamin, and afetoprotein) are postulated to have evolved from a progenitor that arose from the triple repeat of a 192amino-acid sequence [39]. This three-domain structure has been preserved in DBP; however, the third repeat is largely truncated at the C terminus. The human albumin structure [37,38] indicates that each domain consists of ten a-helices. In the DBP structure, the first domain (residues 1e191) has this a-helical arrangement. However, the second domain (residues 192e378) has a similar topology but helix 7 is replaced by a coil folding, and the third domain (residues 379e458) contains only helices 1e4. The three domains of DBP do not pack in a spherical manner but adopt a rather peculiar shape with two large grooves (Fig. 5.1). Despite the sequence similarity, the only function DBP shares with the other family members is its fatty-acid-binding ability [40,41]. Superposition of the respective domains of the DBP and HSA structures shows similar topologies. Although the folding within each corresponding domain shows some parallels, the global orientation of the three domains in both molecules is strikingly different, resulting in two totally different structures. Residual electron density that can accommodate 25(OH)D3 is observed in DBP holoprotein, close to the biochemically identified vitamin-D-binding residues [10,11]. Residues belonging to helices 1e6 of domain I form the complete vitamin-Dbinding site (Fig. 5.1B). This binding site designation was further confirmed by the elucidation of the structure
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FIGURE 5.1 DBP-25(OH)D in three dimensions. (A) The DBP three-dimensional structure with its three structural domains. The numbering of the helices is the same as in human serum albumin. (B) The conformation of 25(OH)D in DBP. Helices 1e6 of the apoprotein (light blue) and the holoprotein (DBP-25(OH)D) are superimposed. 25(OH)D is shown in ball-and-stick representation. Two water molecules present in the binding site are indicated as red balls. (Reproduced from figures 2a and 4a of the manuscript of Verboven et al. [8].) Please see color plate section.
of DBP in complex with a vitamin D analog, 22-(mhydroxyphenyl)-23,24,25,26,27-pentanor vitamin D3 with known high affinity for DBP. The vitaminD-binding site is lined predominantly by hydrophobic residues, allowing favorable interactions with the hydrophobic vitamin D3 ligand. Binding of 25(OH)D3 is further improved by hydrogen bond formation of the 25-hydroxyl with Tyr 32 (OeH.O distance ˚ ), and the 3-hydroxyl with Ser 76 (O.HeO 2.85 A ˚ ) and Met 107 (OeH.S distance distance 2.81 A ˚ 3.01 A). Despite the low detail in the observed electron density, the proposed orientation of the molecule in the binding site is consistent with extensive biochemical and modeling data and, therefore, is assumed to be correct. Modeling of 1,25(OH)2D3 binding to DBP illustrates that this molecule can make the same hydrogen bonds with DBP as 25(OH)D3 but also shows that the axial 1-hydroxyl causes steric hindrance with Met 107. This may explain the lower DBP binding affinity observed for 1,25(OH)2D3. The vitamin-D-binding site is a cleft; consequently, certain parts of the vitamin D3 molecule have no interaction with DBP e for example, the b-side of the C- and D-rings of the vitamin D3 molecule. Analogs substituted in these parts of the molecule display affinities similar to the affinity between 1,25 (OH)2D3 and DBP. Furthermore, modeling of the analogs with large substituents on the C ring (11bphenyl-1a,25-dihydroxyvitamin D3, 11a-phenyl-1a,25dihydroxyvitamin D3) illustrates that large substituents, such as a vinyl group or a phenyl ring, can easily fit in the binding site. These observations confirm the proposed orientation of 25(OH)D3 in the vitamin-Dbinding site (Fig. 5.1). The binding site of DBP for vitamin D metabolites is quite different from that of the vitamin D receptor VDR. In VDR, the vitamin-D-binding site is a closed pocket formed in the inner structure of the receptor, whereas in DBP it is a cleft located at the surface of the molecule and partly in contact with the surrounding solvent. Moreover, no parallel for both of these
physiologically important binding sites is found in ligand conformation, hydrogen bonding, or hydrophobic interactions. In the VDR pocket, the A-ring of the vitamin D3 ligand has a B-chair conformation (3hydroxyl axial), whereas in the DBP cleft the A-ring has an A-chair conformation (3-hydroxyl equatorial). In both binding sites, the C5eC6eC7eC8 torsion angle of the ligand is non-planar. The angle is 149 in DBP and e149 in VDR. The torsion angles responsible for the side-chain orientation of the vitamin D3 molecule are different as well (C13eC17eC20eC22 ¼ e77 in DBP and 89 in VDR; C17eC20eC22eC23 ¼ e70 in DBP and e156 in VDR). DBP (or group-specific component, Gc) was originally characterized in humans by serum electrophoresis as the product of two autosomal, codominant alleles (Gc1 and Gc2). Isoelectric focusing allowed the further characterization of slow (Gc1S) and fast (Gc1F) subtypes of Gc1, resulting in six common phenotypes. The protein isoforms produced by the Gc1F and Gc1S alleles represent a mixed population containing or lacking a single N-acetylneuraminic acid on a threonine residue at position 420. In contrast, the Gc2 allele produces a single protein that contains a nonglycosylated lysine at position 420. The DBP gene contains an Alu middle repetitive element located at the end of DBP intron 8 resulting in four gene polymorphisms created by differences in the size of the Alu poly(A) tract. Each of these polymorphisms is equally distributed among the three common alleles. Although the major alleles can be distinguished at the DNA level by restriction digestions or PCR-single-strand conformation polymorphism analysis, protein electrophoretic techniques remain the most usual initial approach to assigning protein isoform phenotypes. Although commonly used for forensic purposes in the past [3], DBP phenotyping is now more frequently used to map population dynamics, whereas its link with disease susceptibility is still unclear. In addition to the three common alleles, there are also more than 124 rare variant alleles described worldwide.
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The geographic occurrences of these variants often correspond to patterns of human population migrations and thus are of anthropological interest [2,10]. The molecular bases for some of these rare variants have been determined by sequencing of exons amplified by the polymerase chain reaction. A number of genetic studies have suggested the presence of a DBP-null allele [42,43], but these have not been clearly distinguished from low-expressing “pseudo-silent” alleles [44]. The lack of a naturally occurring DBP-null homozygote in the human population suggested that such a genotype would be embryonically lethal. A homozygous, DBPdeficient mouse line has, however, been generated that is viable and fertile [7]. DBP polymorphisms have also been identified in non-human primates, rodents, a variety of ungulates, domestic cats, marsupials, and birds [10]. The high degree of polymorphic variation in the human population and its widespread occurrence among vertebrates make the DBP locus among the most polymorphic known.
Synthesis and Turnover of DBP Like albumin and other serum proteins, DBP is produced primarily in the liver with virtually no production elswhere. The half-life of DBP is in the order of a few days and therefore its daily production is high, in the order of 10 mg/kg/day of DBP in humans [45]. Clearance of DBP from the plasma in the rabbit, like that of albumin, is described by a multiexponential curve with an estimated t1/2 of 1.7 days. This is less than half that of albumin with a t1/2 of 5 days, but over five times faster than that of its major ligand, 25 (OH)D, suggesting that ligand recycling is likely [46]. DBP is primarily cleared by the kidneys as a result of its uptake by megalin in the proximal tubule epithelium [45,47e49]. The relatively low concentration of hepatic DBP mRNA suggests that the DBP transcript, like that of albumin, may be very stable and exhibit a long cytosolic half-life [50]. The circulating DBP concentration in adult mammals and birds is in the micromolar range [10]. DBP concentration increases in the postnatal period and is fairly stable thereafter. Most studies have failed to link vitamin D status or disturbances of mineral homeostasis with alterations to DBP concentration [51e54]. DBP concentration increases during pregnancy [55e57], and under the influence of female sex hormones in humans and birds [58e60]. In rodents, DBP concentration increases in response to testosterone and is higher in males than in females [61]. Malnutrition [62], liver failure [55,63], and pronounced proteinuria results in a decrease in circulating DBP concentration [64,65]. Marked tissue necrosis or damage releases intracellular actin, increases circulating actineDBP complexing and decreases DBP concentrations. DBPeactin
complexes are cleared up to three times more rapidly than unliganded DBP, primarily by hepatic filtration [66,67].
FUNCTIONS OF DBP Vitamin D Transport Lipophilic steroid hormones are largely bound to plasma proteins for their extracellular transport. For cortisol this is cortisol-binding globulin or CBG, and for sex steroids sex steroid binding b-globulin or SBBG. Also thyroid hormones use specific transport proteins predominantly thyroxine-binding globulin or TBG, whereas vitamin A or retinol mainly uses retinolbinding globulin for its transport. It is therefore no surprise that all vitamin D metabolites use a similar system of plasma transport. The major transport protein is DBP transporting more than 95% and probably even 99% of 25(OH)D, whereas albumin and lipoproteins have minor transport functions as they transport a small fraction of all vitamin D metabolites despite their much higher serum concentration in comparison with DBP. Based on competition experiments and on the crystal structure of the holoprotein (DBP plus 25(OH)D or analogs) it is clear that there is only a single binding site for all the vitamin D metabolites. The affinity of DBP for these metabolites is however quite different with the highest affinity for 25(OH)D lactones, followed by 25(OH)D and its catabolic metabolites such as 24,25and 25,26-dihydroxyvitamin D, whereas 1,25(OH)2D has about a 10- to 100-fold lower affinity for DBP than 25(OH)D. These differences in affinity can be reasonably explained by the structure of the cleft-like binding site on DBP. Vitamin D itself has still a much lower affinity. Vitamin D2 metabolites bind slightly less well to human DBP than vitamin D3 metabolites, whereas chick DBP has a much lower affinity for vitamin D2 metabolites for otherwise unexplained structural reasons. It is also well known that vitamin D2 has poor antirachitic properties in birds and it is attractive to link that to differences in DBP affinities, although direct proof is missing. The vitamin D receptor VDR has a much higher (about 100-fold) affinity for 1,25(OH)2D than for 25 (OH)D with little difference between D2 and D3 metabolites whether in humans, rodents, or birds. The binding proteins for hormones in general do not all belong to the same gene family as do the CYP P450 enzymes responsible for the metabolism of vitamin D, whereas also the receptors of these hormones all belong to the class of nuclear transcription factors. However all these binding proteins have the same consequences: due to the universal law of mass action and due to the relative high affinity of these binding proteins for their
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circulating ligands, most of the ligand is bound to its binding protein and only a small fraction is unbound or “free.” This resulted in the "free hormone" hypothesis [68]. Indeed, as the small lipophilic free ligands are able to cross the cell membrane and thereby gain access to the cytoplasmic or nuclear-binding proteins, whereas the protein-bound ligand cannot freely cross the cell membrane, the biologically active fraction is the free ligand. The protein-bound ligands or hormones are thereby largely excluded from direct cellular entry and thus play essentially a role as circulatory reservoir for local delivery of the free ligands. A slightly more subtle modification of this hypothesis also takes into account the total time needed for blood to cross a particular tissue so that part of the bound hormone can be released during tissue perfusion to replace the free hormone take up by cells during this tissue perfusion. This implies that the real available hormone is higher than the static free hormone concentration due to dissociation of free hormone during tissue perfusion. A large number of biochemical, cellular, and physiologic data strongly support this free hormone hypothesis. The serum transport of 25(OH)D and 1,25(OH)2D also likely behaves as the other steroid or thyroid hormones. Indeed the cellular entry of 25(OH)D into most tissues decreases in the presence of serum protein or DBP confirming the sequestering characteristics of DBP (Table 5.1). This has been clearly demonstrated in keratinocytes and monocytes. Indeed the activity of 1,25(OH)2D or 25 (OH)D in human monocytes can be evaluated by measuring the expression of cathelicidin. The biological activity was much higher when such monocytes were cultured in the presence of serum from DBP-null mice than in the presence of normal serum or DBP-enriched medium [69]. No megalin-mediated uptake of DBP could be detected in monocytes. The inhibition of the biological activity was two- to three-fold higher in the presence of Gc1F-1F compared to other DBP polymorphisms indicating that the biological activity of 1,25 (OH)2D or 25(OH)D may be different according to DBP polymorphism. This inhibitory effect has also been shown for cultured kidney cells as the presence of DBP decreases the production of 1,25(OH)2D. This phenomenon can be easily explained by the high affinity of 25(OH)D for extracellular DBP and a much lower affinity for the intracellular VDR, with the overall effect of slow entry of 25(OH)D into cells. This is confirmed by in vivo studies. Indeed, the distribution volume of 25 (OH)D as evaluated by radiotracer studies is very similar to the distribution volume of DBP and thus corresponds to the extracellular fluid compartment. This would be in line with the slow dissociation of 25 (OH)D bound to DBP and slow entry of 25(OH)D inside the intracellular compartment. The situation of 1,25 (OH)2D is more complex. Addition of serum proteins
TABLE 5.1
Characteristics of Human DBP
Features Isoelectric point
4.5e4.8
Electrophoretic migration
a-globulin
Size
58 kDa, single-chain glycoprotein
Plasma concentration
4e8 mM (232e464 mg/liter)
Plasma half-life
2.5e3.0 days
Daily production rate
~10 mg/kg
Altered plasma levels Increased
Estrogen, pregnancy
Decreased
Nephrotic syndrome, liver disease, malnutrition, acute critical illness, extensive tissue damage
Vitamin D sterol binding Plasma capacity
mol/mol (2.4 mg D sterol/liter)
Normal sterol occupancy
<5%
Affinity (KD) ~nanomolar
25(OH)D, 24,25(OH)2D
~micromolar
1,25(OH)2D, vitamin D
Clearance by
Renal proximal tubule epithelial cells
Actin binding Plasma capacity
mol/mol (270 mg/liter)
Affinity (KD)
Nanomolar
DBPeactin complexes
Seen with tissue injuries, inflammation
Clearance by
Hepatic phagocytic cells, sinusoidal endothelium
Binding of free (especially unsaturated) fatty acids with micromolar affinity Cell associations Proximal renal tubule (luminal) Cell “receptor”
Megalin, cubilin
Internalization
Yes
Physiology
Protein clearance, delivery of ligand
Other functions Cochemotactic properties together with complement C5a for neutrophils Precursor for DBPemaf after partial deglycosylation: DBP-maf has antiviral, proinflammatory and antitumoral activities
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or pure DBP to cell cultures clearly decreases the biological activity of 1,25(OH)2D as studied by gene transcription or cellular functions, again demonstrating the sequestering characteristics of DBP. However, the affinity of intracellular VDR for 1,25(OH)2D by far exceeds its affinity for DBP so that over time a favorable transport from extracellular to intracellular compartment is likely. This could then explain why the distribution volume of 1,25(OH)2D corresponds to the intracellular fluid volume (and thus much higher than the 25(OH)D distribution volume). Changes in serum DBP due to estrogens, pregnancy, or diseases have generally little effect on serum 25 (OH)D concentrations. This is in line with the concept of 25(OH)D being a true vitamin-D-like substance whose concentration is essentially dependent on supply and partially on catabolism but without a clear feedback system to maintain total or free 25(OH)D constant. This is in contrast with most hormones and 1,25(OH)2D where physiologic feedback mechanisms maintain the free hormone as close to normal when total DPB changes, so that the total hormone concentration fluctuates directly to changes in serum transport proteins. That explains why pregnant women or women on estrogen therapy first develop high concentrations of CBG, TBG, or DBP due to estrogen effect on liver protein synthesis. This is rapidly followed by increases in total concentrations of their hormonal ligands, cortisol, thyroxine or T3, and 1,25(OH)2D without clear biological signs of hormone excess as would be expected on the basis of their total hormone concentration. This is such a well-accepted hypothesis that in clinical practice free hormones are routinely measured or calculated to estimate the biological hormone concentration (especially for thyroid hormones and sex steroids and indirectly for cortisol). There is much less clinical experience with total or free 1,25(OH)2D concentrations but generally the free 1,25(OH)2D concentration corresponds better to biological activity than the total concentration. Another unusual argument for the free hormone hypothesis is found in the extremely high (more than 100-fold) concentration of total 1,25(OH)2D in serum of rabbits immunized against 1,25(OH)2Deprotein complex [70] without causing vitamin D toxicity. However, the free hormone hypothesis has been challenged by a number of observations that suggest it may not fully explain the relationship between sterols, their binding proteins, and target cells. Indeed the measurement of free 1,25(OH)2D is technically very difficult and also subject to technical errors due to impurity of the tracer, adherence of small concentrations to surfaces and the need to add so much labeled steroid that it is no longer a tracer amount but approaches the endogenous concentration and thus modifies the natural equilibrium situation. So most people rely on calculated free levels.
Secondly, in view of the high capacity/high affinity binding of DBP to 25(OH)D the free 25(OH)D concentration is serum is extremely low and one can question whether the free diffusion of such low 25(OH)D concentration is sufficient to produce the microgram amounts of 1,25(OH)2D produced in the kidney in normal or calcium-stress situations. Finally and most importantly, the presence of tissue-specific DBP-binding proteins in cell membranes opens the possibility that the holoprotein, DBP plus 25(OH)D, can gain direct access to some cells or tissues. Several mouse models with genetically modified genes however allow clarification of this hypothesis. DBP-Null Mice DPB mice are viable and develop normally without a clear phenotype related to either vitamin D, calcium, or bone homestasis nor display symptoms related to its other putative roles (see below). Indeed, DBP-null mice are normocalcemic without a clear bone phenotype, yet they have very low serum levels of 25(OH)D and 1,25(OH)2D [7]. This is, however, in line with the free hormone hypothesis as the free 1,25(OH)2D is biologically active and low due to DBP sequestering in normal mice (free levels are only about 1% of the total level) and low due to feedback regulation in DBP-null mice. The serum 1,25(OH)2D levels are so low that they fall below the detection limit of routine assays but even when the sensitivity is markedly improved, levels are still around the detection limit [71]. This is indeed in line with the free 1,25(OH)2D level calculated on the basis of DBP concentration and affinity [57]. It is therefore no surprise either that the major vitamin-D-dependent gene expression in the intestine (TRPV5 and 6, calbindin D9k and PMCA1b) is normal in DBP-null mice. Also tissue levels of 1,25(OH)2D are normal, all in line with the free hormone hypothesis [71]. The reasons for low 25(OH)D levels may be different from the reasons for low serum 1,25(OH)2D. Indeed the synthesis or production of 25(OH)D is thought to be substrate-dependent and not truly feedback-regulated. However the pool of circulating 25(OH)D is markedly lower (probably 100-fold lower) whereas the clearance of 25(OH)D in DBP-null mice is probably higher than normal. This can explain the more rapid development of vitamin D deficiency symptoms in DBP-null mice after receiving a vitamin-D-deficient diet [7] and also their greater resistance to vitamin D toxicity. A major yet unresolved question arises from the observation [71] that the kinetics of vitamin D metabolites or analogs did not fit with the predicted sequestering effect of DBP delaying the clearance of 25(OH)D in comparison with analogs with low DBP binding. Therefore this observation is not in line with the free hormone hypothesis. Despite a different and well-argued interpretation of
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FUNCTIONS OF DBP
the authors [71] their extensive and detailed studies rather confirm than contradict the free hormone hypothesis for 1,25(OH)2D, with still a number of unresolved questions. Megalin and Megalin-Related Proteins Null Mice Megalin is a scavenging receptor with broad ligand specificity that constitutes the main pathway for tubular reabsorption of filtered plasma proteins [72]. This receptor is abundantly expressed on the brush border (luminal) surface of the proximal tubular cells [73,74]. Megalin-null mice or mice with a kidney-selective deficiency of megalin develop low-molecular-weight proteinuria and excrete the receptor ligands [47,75e77]. Among other proteins, megalin-knockout mice lose DBP in the urine (Fig. 5.2). The identity of the tubular reuptake pathway for 25 (OH)D/DBP and its significance for vitamin D metabolism was discovered through studies on megalin, a multiple carrier endocytic receptor. Megalin-deficient mice excrete approximately 0.3 mg of DBP per day [47]. This is very close to the expected amount of DBP that passes through the glomerular filtrate in a normal kidney in 24 hours [46]. Massive urinary loss of DBP in megalin-knockout mice results in a concomitant loss of 25(OH)D bound to its binding protein. The total 25
FIGURE 5.2 The renal uptake of 25(OH)D by proximal tubular cells (PTC). DBP/25(OH)D is filtered in the glomerulus and taken up at the luminal side of the PTC at coated pits. DBP is degraded in lysosomes and 25(OH)D becomes available for further metabolization. This mechanism prevents urinary loss of 25(OH)D and provides ample substrate supply for the synthesis of 1,25(OH)2D. However, serosal upake of non-DBP-bound 25(OH)D is possible as demonstrated in DBP-null mice.
63
(OH)D loss amounts to 0.3 ng to 0.5 ng in 24 hours [47]. Therefore megalin-null mice easily develop nutritional vitamin D deficiency on a normal vitamin D intake and even more pronounced on a low vitamin intake. Such animals then develop the typical bone phenotype of rickets or osteomalacia (increase in osteoid surfaces and dramatic reduction in mineralizing surfaces and in total bone mineral content), and associated hypocalcemia and hyperparathyroidism. On a vitamin-D-enriched diet, the plasma levels of 25(OH) D and 1,25(OH)2D are still reduced by more than 70% in the total KO mouse and by 50% in the conditional knockout line (that retains some residual megalin activity). This dietary supplementation with vitamin D allows megalin-deficient mice to compensate the urinary loss of 25(OH)D and therefore serum calcium, phosphorus, and PTH levels are normal and rickets is prevented. Further support for the essential role of the megalin pathway in renal reuptake of 25(OH)D comes from studies in rats treated with megalin antagonists. Infusion of receptor antagonists in proximal tubular cells (PTC) of rats can largely block the conversion of 25(OH)D to 1,25(OH)2D. This suggest that the megalin receptor pathway provides PCT cells with the precursor to produce the active hormone 1,25(OH)2D [47]. Megalin or glycoprotein 330 or low-density lipoprotein (LDL) receptor-related protein-2 is a giant 600-kDa cell surface protein and was initially identified as the autoantigen in Heymann nephritis [78]. In this animal model, autoantibodies directed to kidney homogenates in fact react with megalin and create immune deposits in the glomerular wall, causing severe tissue damage and renal failure [78,79]. Megalin is genetically highly conserved and already expressed in nematodes. Megalin gene and protein are structurally closely related to members of the LDL receptor gene family, a group of multifunctional endocytic receptors [80e82]. The large protein has many structural domains involved in either endocytosis or pH-dependent release of ligands in endosomes. A 22-amino-acid transmembrane domain is responsible for its membrane attachment. The cytoplasmic tail contains three asparagineproline-X-tyrosine (NPXY) elements that represent signals for coated-pit internalization [83] and binding sites for cytosolic adapter proteins such as Disabled (Dab)-2 [84]. Megalin is expressed in many epithelia of embryonic (yolk sac and the neuroepithelium) and adult tissues, especially in the proximal convoluted kidney tubule (PCT) and the small intestine [72]. In these tissues, megalin expression is restricted to the apical cell surface (and not in the serosal site facing the blood circulation) and to endosomal compartments and this confirms its role in apical clearance pathways [74]. Megalin is a nonspecific bulk transporter; many ligands bind to
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5. THE VITAMIN D BINDING PROTEIN DBP
megalin in vitro and are subsequently taken up by the receptor into cells (whether in cell culture or in vivo). These ligands include: (1) proteases and protease/ inhibitor complexes; (2) lipoproteins; and (3) vitamins or hormones bound to carrier proteins (reviewed in [12,72,85]). Indeed, not only the holoprotein 25(OH) D/DBP complex but also retinol (vitamin A) with its retinol-binding protein [76] and vitamin B12 with the carrier transcobalamin [77] are reabsorbed in the PCT, indicating a role as a general retrieval receptor for filtered metabolites. The function of the receptor in other epithelia is less well characterized. Forebrain defects in global megalin-null mice suggest an important role of the protein in brain development [86]. DBP binds to megalin with a Kd of 108 nM regardless of the presence or absence of 25(OH)D. Megalin is also associated with cubulin, a protein that was initially identified as the intestinal receptor responsible for the uptake of intrinsic factor (IF)/vitamin B12 complexes and is therefore also known as the intrinsic factor/ cobalamin receptor [87]. Cubulin is a 460-kDa protein but lacks a membrane anchor. As it binds to megalin it is plausible that cubulin and megalin function as a coreceptor complex present at the cell membrane and cycling as a complex throughout endocytic uptake and recycling process. In mice genetically deficient for megalin, cubilin biosynthesis and intracellular transport are significantly impaired [88]. Cubilin and megalin colocalize in the apical part of epithelial cells, in particular in clathrincoated pits, in dense apical tubules, and in endosomes, all in line with a coreceptor system [89]. Patients with a natural mutation of cubulin suffer from hematologic problems especially megaloblastic anemia in line with its role for uptake of vitamin B12 [90,91]. Such patients also develop a low-molecular-weight proteinuria and excrete ligand/carrier complexes such as 25(OH)D/ DBP [48]. The megalinecubulin complex is internalized via an endocytic pathway starting in clathrin-coated pits and endocytic vesicles and lysosomes. To recycle through the endocytic compartments, scaffold or adaptor proteins are needed. Disabled (Dab)-2 is an adaptor protein required for proper function of megalin in PCT. Dab-2-knockout mice have indeed impaired tubular endocytosis and excrete DBP in the urine [92]. Other proteins are probably also involved as voltagegated chloride-channel-5-null mice display endocytic malfunction resulting in disruption of megalin-mediated uptake of 25(OH)D/DBP complexes, in urinary loss of these metabolites, and in substantial decrease in serum levels of 25(OH)D and 1,25(OH)2D [93]. ClC-5 is responsible for chloride conductance required for import of Hþ and acidification of the endosomes. This process is thus part of the overall endocytosis recycling pathway including the recycling pathway of megalin/
cubulin. The human equivalent of mouse ClC-5 deletion, Dent’s disease, also results in a severe bone and mineral phenotype [94]. The most likely scenario of the endocytic pathway of DBP in PTC is shown in Figure 5.2. Megalin and cubulin are internalized together with DBP and 25(OH)D or other vitamin D ligands and processed in lysosomes. DBP is degraded, 25(OH)D is released and becomes available as substrate for CYP P450 enzymes capable of activating or inactivating 25 (OH)D, and the coreceptors are recycled to the apical membrane. This megalin pathway helps to explain the frequently severe vitamin D deficiency seen in patients with nephrotic syndrome as first described in the Lancet [95] and later confirmed by others [96e98]. The high urinary loss of DBP causes urinary loss of 25(OH)D that exceeds the nutritional supply, thereby depleting the plasma vitamin D pool (all the vitamin D metabolites). This can be due to increased glomerular permeability so that albumin and smaller proteins (DBP) are massively lost and cause the nephrotic syndrome and deficiency of some small molecules such as 25(OH)D. In some patients, the glomerular structure is intact and the defect is a purely tubular one inactivating the megalin/cubulin-mediated reuptake of ligands of this coreceptor complex and causing small molecule deficiencies as in the megalin-null mice. The DBP-megalin-mediated access of 25(OH)D to the proximal renal tubule is well documented but the question arose at to whether other cells also use such or a similar mechanism for cellular entry of 25(OH)D. This would have major implications for understanding the physiology of vitamin D as such a mechanism would allow some cells to have access to the total concentration of serum 25(OH)D for intracellular (in)activation and thus would allow such cells to produce important amounts of 1,25(OH)2D in a paracrine or autocrine fashion. Similarly, such DBP-megalin-mediated uptake of 1,25(OH)2D would expose such cells and tissues to much higher concentrations of circulating 1,25(OH)2D than other cells that rely on free 1,25(OH)2D for cellular uptake. There are presently only few data on megalin or other receptor-mediated uptake of vitamin D metabolites. Hepatic Ku¨pner cells can take up DBP in vitro when appropriately stimulated [99] but this may be important for DBP or DBP/actin clearance rather than for vitamin D metabolism. Monocytes have received substantial attention and conflicting data have been published but the overall interpretation is that the presence of DBP does not facilitate but limits the cellular entry of vitamin D metabolites, making a receptor-mediated uptake unlikely or of limited physiologic importance [69]. Whereas there is little doubt for the existence of the renal filtering and reuptake process of DBP-25(OH)D there is some doubt whether this selective delivery of
I. CHEMISTRY, METABOLISM, CIRCULATION
FUNCTIONS OF DBP
25(OH)D via the apical membrane is absolutely essential for renal 1,25(OH)2D synthesis. Indeed, higher than normal vitamin D supplementation of megalin-null mice or patients with nephrotic syndrome can restore a normal calcium and bone homeostasis, indicating that serosal uptake of free 25(OH)D may be sufficient for normal 1,25(OH)2D synthesis. Also DBP-null mice on a vitamin-D-replete diet do not develop rickets or metabolic bone diseases indicating that the free serum 25(OH)D concentration is sufficient for delivering the substrate for the renal synthesis of 1,25(OH)2D in the absence of DBP-megalin import of 25(OH)D. It is therefore likely that a dual access mechanism is in place to ensure adequate synthesis of 1,25(OH)2D (Fig. 5.2).
Polymorphism of DBP Gc/DBP was initially discovered as a highly polymorphic protein and a large number of studies have tried to find associations between this polymorphism and human diseases. Some studies found small differences in bone size, mass, or density linked to DBP polymorphism [100,101] in otherwise healthy people. Many studies suggested an increased risk for autoimmune diseases for some DBP polymorphism but this may be disease- or population-specific and thus cast doubt on whether the DBP gene or protein itself is involved rather than a closely associated other gene [102]. No relation could be found between DBP polymorphism and the risk of multiple sclerosis in a large nested caseecontrol study [103]. Polymorphism of DBP has however an important impact on circulating 25(OH)D. Indeed, a large consortium study tried to identify genetic determinants of serum concentration of 25 (OH)D using data from more than 50 000 subjects [104]. This revealed that polymorphism of genes coding for metabolism of vitamin D such as 7-dehydrocholesterol-dehydrogenase and the CypP450 enzymes coding for a 25-hydroxylase (CYP2R1) and, to a lesser extent, also 24-hydroxylase activity had a significant effect on serum 25(OH)D concentration. However the major effect was found for the common polymorphism of the DBP gene so that the risk for being modestly to severely vitamin-D-deficient was dependent on DBP polymorphism. Whether such an effect is simply a reflection of minor differences in binding properties of DBP or is due to different production or catabolism of 25(OH)D or vitamin D metabolites is presently unclear [104,105]. The effect of DPB polymorphism on serum 25(OH)D levels was already observed in previous smaller studies [106e108]. This may also be linked to a difference in response to oral vitamin D intake as DBP polymorphism influenced the increase in serum 25(OH)D after supplementation with physiological doses of vitamin D [109].
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The DBPeActin Complex Role of DBP in the Actin-scavenger System Actin is a highly abundant intracellular protein present in all eukaryotic cells and has a pivotal role in muscle contraction as well as in cell movements. Actin also has an essential function in maintaining and controlling cell shape and architecture. It is the essential building block of the microfilament system, a cytoskeletal structure that complements two other cytoskeletal structures (the microtubules and the intermediate filaments). In low-salt buffers actin exists as a monomeric protein (globular actin, G-actin), but it polymerizes under physiological salt conditions into a double helical 10-nm-thick filament structure (filamentous actin, Factin). In vivo, the equilibrium between G-actin and Factin is controlled mainly by the action of several actin-binding proteins with distinct activities (e.g., gelsolin or profilin) [11]. Severe cell injury, such as by trauma, shock, sepsis, and fulminant hepatic necrosis, causes the release of large quantities of actin in the systemic circulation. The presence of actin filaments in blood, leading to an increase in blood viscosity, is dangerous and may be fatal [110]. In addition, actin filaments can promote clot formation by their ability to aggregate platelets [111]. Therefore, the presence of a protective system, preventing actin polymerization and ensuring actin’s fast elimination from serum, is essential. The vitamin-D-binding protein is an actin-binding protein [9,13] and acts as an actin-sequestering agent in extracellular space. DBP and gelsolin, the only other plasma protein that binds actin avidly, play a crucial role in the clearance of actin filaments from the circulation, a process known as the “actin-scavenger system” [110]. Gelsolin has three actin-binding properties: (i) it severs F-actin; (ii) it caps the end of F-actin; and (iii) it nucleates actin filament assemblies. Gelsolin will shift the equilibrium between actin assemblyedisassembly toward actin depolymerization, but will basically not prevent actin reassembly. DBP, by forming a tight complex with G-actin, prevents the (re-)formation of actin filaments. Therefore DBP and gelsolin have complementary functions: upon severing the actin filaments by gelsolin, the generated globular actin is locked in its monomeric state by DBP. The clearance of this DBPeactin complex is even substantially faster compared to the clearance of free DBP [66,67,112,113]. The complementary action of both proteins is further illustrated by the previous observation that both DBP and gelsolin are required to inhibit actinstimulated platelet aggregation [111]. General Features of the DBPeActin Structure The DBPeactin structure was solved by X-ray diffraction by Verboven et al. [36] as well as by others [33,34].
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5. THE VITAMIN D BINDING PROTEIN DBP
The structure of the DBPeactin complex reveals that Gactin binds in one of the DBP grooves, mainly formed by helix 10 of domain I, helix 6 of domain II, and helix 3 of domain III, allowing DBP and actin to fit as two pieces of a jigsaw puzzle. The surface area buried at the interface ˚ 2. Numerous interof this complex is as large as 3600 A molecular hydrogen bonds, hydrophobic contacts, and electrostatic interactions stabilize the complex. The actin monomer consists of two domains with each domain further subdivided in two subdomains. The DBPeactin structure demonstrates that actin residues of subdomains 1 and 3 constitute the DBP binding interface. Upon complexation with DBP, the changes in the actin structure are restricted to some small regions rather than affecting the general fold. According to limited proteolysis experiments performed with DBP [114] the actin-binding site is located in its C-terminal part, and more specifically residues 350e403 (of domains II and III) are involved in actin binding. However, our DBPeactin structure shows that all three DBP domains interact with actin. Only minor folding differences are observed between the DBP conformations in the presence or absence of actin. In agreement with previous biochemical observations [9,115], the DBPeactin structure proves that the bound actin and the observed folding differences in domain I do not hinder the binding of vitamin D3 compounds to the vitamin-Dbinding site of DBP. The DBP residues involved in actin binding are relatively well conserved between different species in all presently known DBP sequences. Together with the highly conserved amino acid sequence of actin throughout evolution (e.g., human and rabbit skeletal muscle actin are 100% identical) it explains why the actin-binding property of DBP is universally observed in vertebrates. In comparison with a-actin, a twofold decrease of DBP-binding affinity for the nonmuscle actin isoforms, b-actin and g-actin, was observed [115]. The presence of DBP on actin seems not to hinder the interaction with other actin molecules at the pointed end side. Filament growth is, however, impossible. In conditions where the molar G-actin concentration is less than or equal to that of DBP, virtually all G-actin subunits will be captured and blocked by DBP. Implications for the ActineScavenger System The observed perfect fit between actin and DBP provides the structural basis for the important role of DBP in the extracellular actinescavenger system. Furthermore, although DBP and gelsolin are both present in large concentrations (mM) in blood, their capacity to scavenge actin may still be overwhelmed during massive cell injury. Indeed, the saturation of the actinescavenger system leads to thrombi formation and microangiopathy [13,116,117], and excessive amounts of actin in the circulation may lead to
a condition resembling “multiple organ dysfunction syndrome.” Indeed, most patients with this syndrome have reduced serum DBP levels [118,119]. The DBP concentration has some value in predicting survival from hepatic failure [120,121] and may identify highrisk patients after multiple trauma [122]. Consequently, the DBPeactin structure, revealing the DBP residues essential for the interaction with actin and for the actin sequestering, is of major importance for the development of a drug that could be applied in the pathological conditions just mentioned. Despite intensive analysis no DBP-deficient individual has been identified, leading to the suggestion that certain functions of DBP might be essential for survival. However, viable DBP-knockout mice have been generated [7]. These mice were only used for analysis of DBP’s role in vitamin D metabolism and action. Although DBP-knockout mice are viable in normal laboratory conditions, it might well be that the role of DBP in the actinescavenger system is confined to cases of severe tissue damage in which DBP is essential for survival. Experimental evidence for this hypothesis in DBP-null mice is however still lacking. The structural observations and the perfect conservation of DBPeactin interaction during the evolution of vertebrates however suggest an evolutionary advantage.
The Role of DBP in Inflammatory Processes and in the Immune System DBP has been associated with the inflammatory process, including the prevention of thromboembolic events in the micro-vasculature by actin (see previous paragraph), the stimulation of chemotaxis by phagocytic neutrophils, and the activation and stimulation of phagocytic function by macrophages. Chemotaxis is the directional migration of cells in response to an extracellular cue, usually in the form of a chemical gradient. A wide variety of chemoattractants have been identified, with varying degrees of specificity. Complement 5a-stimulated chemotaxis of leukocytes is enhanced in the presence of serum and the responsible protein was subsequently identified to be DBP. DBP alone has no chemotactic activity. The mechanism(s) by which DBP enhances neutrophil response to C5a is unknown, but several studies document such an effect [123e127]. Preincubation of DBP with neutrophils is sufficient for enhanced C5a-stimulated chemotaxis. This is apparently mediated via DBPecell surface proteoglycan interaction [124,127,128] without further internalization [123]. A neutrophil-associated serine protease, elastase, is responsible for cleaving and regulating the number of available chondroitin-sulfate-binding sites necessary for the binding of DBP to neutrophil surfaces. In addition it seems that there is a second serum protein,
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CONCLUSIONS AND PERSPECTIVES
thrombospondin 1, derived from platelets, needed for this DBP cochemotactic activity [129]. Antigenic stimulants cause the activation of phagocytic function in leukocytes and other cells via several pathways. DBP can be converted to DBP-maf via the inducible, stepwise actions of B-lymphocytic b-galactosidase and T-lymphocytic sialidase [130e132], resulting in selective deglycosylation of DBP until only one GalNAc residue is left over. The exact mechanism by which DBP-maf is generated or becomes active is unknown; however, the complete deglycosylation of DBP-maf by the removal of the terminal alpha-N-acetylgalactosamine (GalNAc) moiety results in diminished macrophage-activating activity. DBP-maf activity is specific to Fc-mediated phagocytosis and does not appear to be involved in complement-primed phagocytic processes that can be stimulated by lipopolysaccharide [133]. DBP-maf-activated macrophages demonstrate significant tumoricidal activity [134e136]. The specific stimulants for DBP-maf generation and the particular phagocytic pathways that it activates suggest that the principal role of DBP-maf may be in the immune response to neoplasia. DBP-maf may also have direct antiangiogenic effects on endothelial cells and be able to inhibit the angiogenic actions of a number of tumorderived growth factors. Daily in vivo administration of DBP-maf to immune-compomised mice bearing a human pancreatic cancer inhibited tumor growth and at higher dose even caused tumor regression [137]. Histological examination revealed reduced microvessel density, increased number of infiltrating macrophages and increased apoptosis. This suggests that DBP-maf is an antiangiogenic molecule and stimulates macrophages to attack growing malignancy. Tumor-derived alpha-Nacetylgalactomanidase (nagalase) can deglycosylate DBP rendering it unable to be converted in DBP-maf and nagalase activity in serum can even be used as a marker of tumor burden. Treatment of a few patients with either metastatic colorectal or mammary carcinoma with DBP-maf (100 ng once weekly) during several months normalized serum nagalase activity and prevented tumor progression for 4e7 years’, follow-up [138,139]. These remarkable data certainly deserve confirmation in larger groups of patients treated with well-characterized DBP-maf. If confirmed this would represent a totally new view on the importance of DBP and its derivatives. Another groups also reported the synthesis of a 66-residue DBP-maf modeled on the structure of DBP-maf that mimics the activity of the natural product [140]. DBP-maf is also absent in HIV-infected patients due to total deglycosylation of DBP by nagalase secretion of HIV-infected cells. Nagalase activity is part of the gp120 viral envelope protein. DBP-maf treatment activates macrophages that prevent the infectivity of HIV-infected cells in vitro. In vivo treatment of
67
HIV-infected patients with DBP-maf for 16 weeks restored normal nagalase activity and reduced the infectivity of their blood lymphocytes and prevented disease progression for several years [141]. DBP-maf has also been reported to stimulate boneresorptive activities of osteoclasts in vitro [142]. It has been proposed that a defect in lymphocytic b-galactosidase and/or some other deficiency in DBP-maf generation may be involved in the pathogenesis of some osteopetrosis syndromes [143e145]. This activity is proposed to operate via the CD36 receptor, expressed on both macrophages and endothelial cells [137,146]. Several rodent strains of osteopetrosis (rat op and ia strain and mouse op and mi strain) have defects in the production of DBP-maf and in vivo treatment of these mice, but not of toothless osteopetrotic mice, can improve the bone phenotype [147]. The variety of functions of DBP or its metabolite in inflammatory, immune processes and its potential role in macrophage and osteoclast activation is in sharp contrast with the absence of a bone or immune phenotype in DBP-null mice [7]. Indeed, no differences in phagocytic cell recruitment in response to intraperitoneal treatment with thioglycollate medium [148] or to infection with Listeria monocytogenes or Toxoplasma gondii [10] could be attributed to the inactivation of DBP. DBPnull mice do not demonstrate osteopetrotic lesions [7], and this seriously cast doubt on the obligatory role of DBP-maf in osteoclastic function. The reasons for these apparent discrepancies between in vitro or ex vivo actions of DBP and observations in the DBP-null mouse are as yet unclear. Of course it is still possible that immune or inflammatory defects related to DBP may only become visible in DBP-null mice, when appropriately challenged.
CONCLUSIONS AND PERSPECTIVES DBP is a serum tranport protein for all vitamin D metabolites and evolved during evolution at the time of the other components of the vitamin D endocrine system (VDR and CYP450 enzymes involved in vitamin D metabolism) during early evolution of the vertebrates. The presence of DBP limits the ready access of 25(OH)D and 1,25(OH)2D to cells and prolongs the half-life of these metabolites in serum. The presence of a DBP receptor in renal tubular cells facilitates the renal access to 25(OH)D but the role of DBP in selective delivery of 25 (OH)D to other tissues is still unclear. Most but not all data however point towards the free ligand hypothesis whereby only DBP free 25(OH)D and 1,25(OH)2D have direct access to the nuclear receptor. DPB also binds to many other proteins, including actin, or cell membranes. Its role in the protection of the microcirculation,
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inflammation, infections, tumor progression, and osteoclast activation is still unclear, as such well-described in vitro effects are not reflected in the normal phenotype of DBP-null mice. Some biological properties of a partially deglycosylated DBP, called DBP-maf, are however very exciting because of its very potent antiviral and antitumoral activity.
[17]
[18] [19]
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C H A P T E R
6 Industrial Aspects of Vitamin D Arnold Lippert Hirsch AGD Nutrition LLC, Lewisville, Texas, USA
produced a substance equivalent to the fat-soluble vitamin when exposed to sunlight or UV-light. In 1923, Hess and Weinstock [7] showed that animal skin irradiated with ultraviolet light, when fed to rachitic rats, provided absolute protection against rickets and consequently the fat-soluble vitamin was not an essential dietary trace constituent. Merging these two cures for rickets, Steenbock and Black [8] in 1925 demonstrated that eating some foods that had been irradiated was an effective treatment for the prevention and cure of rickets. The irradiation of certain foods became standard practice in the 1920s for preventing rickets in the general population. The antirachitic factor was shown to be fat-soluble and became classified as a vitamin. The link between irradiation and plant materials led to the conclusion that ergosterol was an antirachitic substance. The UV spectrum of provitamin D changed with UV irradiation and produced antirachitic activity; leading to the conclusion that vitamin D was derived from the provitamin. In the 1930s, Adolph Windaus determined the formula for vitamin D2 to be C28H44O which was isomeric with the provitamin [9] (see also [1] for review of Windaus’ work). In 1937, he also isolated 7-dehydrocholesterol from pig skin, characterized the molecule, and produced vitamin D3 by UV irradiation of it. Thus began the commercial production of vitamin D. Rygh [10] showed that one rat unit of cod liver oil was 100 times more effective in chicks than one rat unit of vitamin D2. The fact that two forms of the vitamin were identified led to the commercialization of the production of both materials. The antirachitic component of cod liver oil was shown to be identical to the newly characterized vitamin D3 by Brockman in 1937 [11]. These results clearly established that the antirachitic substance vitamin D was a seco-steroid (a steroid in which the 9, 10 bond of the B ring has been broken, i.e. see (12)e(14) in Fig. 6.7).
HISTORY OF VITAMIN D LEADING TO COMMERCIALIZATION The late 1800s experienced the onset of the Industrial Revolution. With it, there was an increased incidence of the bone disease rickets in Europe and America. A concerted effort was made to find a way to prevent and/or cure this crippling disease of children. Scurvy and beriberi were known to be prevented by the addition of citrus fruits containing vitamin C and whole grain rice containing vitamin B1 to the diet, and researchers began to look for foods which would cure rickets in a similar fashion. The success of these investigations led to the commercial production of vitamin D products for medicine and nutrition in humans as well as animals. An excellent summary of this history is found in “The Discovery of Vitamin D: The Contribution of Adolf Windaus” by George Wolf [1]. A brief review of this history with relation to the commercialization of the manufacture and use of vitamin D is in order. In 1919, Sir Edward Mellanby [2], describing the results of his studies, said “The action of fats in rickets is due to a vitamin or accessory food factor which they contain, probably identical with the fat-soluble vitamin.” Huldschinski [3] realized in the same year that UV light cured rickets and impacted on its causation. He additionally identified cod liver oil as an effective antirachitic agent and he and other researchers discovered the fact that sunlight exposure was also effective in preventing and curing rickets. These efforts led towards the realization that rickets was somehow related to lack of exposure to sunlight and that there was also a food substance that could be added to the diet to prevent or cure rickets. In 1922, this was followed by investigations of Harriett Chick and her coworkers [4], which showed that rickets in children could be cured by whole milk or cod liver oil. In 1923, Goldblatt and Soames [5,6] identified a vitamin D precursor in the skin of animals which
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10006-X
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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6. INDUSTRIAL ASPECTS OF VITAMIN D
The fact that exposure of chickens to direct UV irradiation resulted in protection from the effects of rickets, while ergosterol irradiated with UV light to form vitamin D2 was not effective in chickens (although it was antirachitic in rats) led to the conclusion that the vitamin D3 effective in chicks and made from the provitamin in cholesterol was different from ergosterol. Although the focus on vitamin D was initially concerned with the material as an antirachitic agent in humans, it was soon realized that animals grown for commercial food were subject to the same ailment and in much greater numbers. The need to develop a material to add to animal feed for the prevention of rickets and maintenance of good health in growing livestock was recognized. As poultry and other animals began to be raised entirely or in part indoors and out of direct sunlight, it became critical to add vitamin D to the diet of these animals. Thus, vitamin D became one of the most important, if not the most important, micronutrients in the diet of livestock. Cod liver oil was used as the major source of the antirachitic ingredient in both human and animal nutrition until synthetic methods could be developed to meet the commercial requirements. Initially, cod liver oil became the standard against which all other materials were measured in comparing their antirachitic properties. By 1936, however, Eliot and coworkers [12] and others had shown that crystalline vitamin D3 added to milk at 400 USP units was more effective than cod liver oil or viosterol (irradiated ergosterol; vitamin D2). Companies involved in the manufacture of vitamin D products for use in milk fortification and nutritional aids as well as animal feed ingredients became interested in the synthesis of the vitamin D for commercial purposes. Fish liver oils are naturally high in vitamin D content, but as the requirements to refine the oil developed, the vitamin content was diminished and became variable, containing 50 to 45 000 IU/gram of vitamin D3 [13]. In the past several years, because the value has been variable and difficult to standardize for nutritional purposes, synthetic vitamin D sources are preferred. The major vitamin D products used in industry today are vitamin D3, vitamin D2, 25-hydroxyvitamin D3, and 1a-hydroxyvitamin D3. Other derivatives such as 1a,25-dihydroxyvitamin D3 and synthetic analogs are used primarily in pharmaceutical applications and are made in much smaller volumes. For example, Calipotriene (Dovenex), which is a derivative of vitamin D2, is used for psoriasis as are Doxercalciferol (Hectoral) and Paricalcital (Zemplar) for secondary hyperparathyroidism associated with chronic renal failure. Largevolume products, however, are used in human and animal health and nutrition, with the largest amount of material produced for animal nutrition. This is mainly
because of the large number of animals used for meat, milk, and egg production. Vitamin D is a critical component of the diet of animals, especially for those raised indoors without exposure to sunlight and no source of the vitamin. The vitamin is critical to the good health of the animal and to its efficient growth. Vitamin D3 has also become a critical nutritional supplement for humans as well, since large numbers of people have been found to have low vitamin D blood levels. This appears to be caused primarily by a lack of adequate exposure to sunlight (people are afraid of developing melanoma and therefore avoid sun exposure or use a sunscreen, limiting UV skin irradiation even when in sunlight). Research over the past 10 years has demonstrated that vitamin D appears to play a critical role in the maintenance of good health. The nutritional requirements of animals and the maintenance of good health in humans have placed an ever-increasing challenge to produce vitamin D and its derivatives in an efficient and economical fashion.
MANUFACTURE OF THE PROVITAMINS The manufacture of the vitamins and their derivatives involves the synthesis of the provitamin from cholesterol (1) (Fig. 6.1) in the case of vitamin D3 (2) or the isolation of the ergosterol (3) (Fig. 6.2) from plant sources such as yeast for the manufacture of vitamin D2 (4). The vitamin is then generated by UV irradiation of the provitamin. It should be noted that ergosterol (3) and 7dehydrocholesterol share similar structures with only two differences. Ergosterol possesses an additional double bond at position 22,23 and has an additional methyl group on position 24 of the steroid side chain. This fact accounts for the similar behavior of the two molecules with regard to activation. They both undergo the same photochemical transformations to form the same series of photoisomers and have very similar biological activities. The major difference is that vitamin D2 is not active in poultry.
7-Dehydrocholesterol The finding that vitamin D3 derived from the irradiation of cholesterol was a more efficient form of vitamin D than that found in plant sources (particularly when used in poultry feed) led to investigations to convert cholesterol into 7-dehydrocholesterol for the commercial production of vitamin D. Isolation of cholesterol from one of its natural sources is the first step in the commercial production of vitamin D3. Cholesterol occurs in almost all animals, and it can be extracted from the spinal cords and brains of large animals. The major commercial source is from sheep’s wool. Spinal cord and brain extractions
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FIGURE 6.1 Cholesterol (with numbering of carbon atoms) and vitamin D3.
FIGURE 6.2 Ergosterol (with numbering of carbon atoms) and vitamin D2.
were acceptable sources for many years and were used when cholesterol prices rose to levels that justified the more expensive processing costs over those for the isolation of cholesterol from wool. However, today this source of cholesterol is not acceptable because of the danger of coextracting prion protein which can give rise to transmissible spongiform encephalopathy. Cholesterol obtained from wool grease is therefore the primary acceptable source of cholesterol for the manufacture of vitamin D. Sheep’s wool must be cleaned after shearing and the wool grease that is obtained as a by-product from the washing contains about 15% cholesterol. The grease is a mixture of long-chain fatty-acid esters. These are saponified to give fatty acid soaps (usually isolated as the calcium salt) and an approximately equal weight of
wool grease alcohols (containing a mixture of cholesterol, lanosterol, D2, and other sterols and fatty alcohols). The acid soaps are separated by filtration and are used as heavy greases in industry. The cholesterol is separated from the other wool wax alcohols (usually by complexation with calcium or magnesium chloride). The solid complex is broken and the product is crystallized and purified with methanol. Cholesterol of the highest quality is necessary for the preparation of 7-dehydrocholesterol since the yield and quality of the provitamin is dramatically affected by the purity of the cholesterol used as starting raw material. Companies such as the National Oil Products Company (NOPCO), and Winthrop Chemical Company, as well as others, became involved in the vitamin business because of their interest in fish oil.
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FIGURE 6.3
6. INDUSTRIAL ASPECTS OF VITAMIN D
The Windaus procedure [10].
These were among the first companies to examine the synthetic manufacture of vitamin D3 as an alternative to their use of cod liver oil. Several methods were investigated for the conversion of cholesterol to the provitamin. Windaus and Schenk [14,15] filed patents in 1935 assigned to the Winthrop Chemical Company for the chemical production of 7-dehydrocholesterol and its irradiation to form vitamin D3. This procedure involved the oxidation of cholesterol (1) (with the 3-hydroxy group protected as an acetate ester (5)) to form the 7-keto cholesteryl acetate (6) which was then reduced to the 7-hydroxycholesterol (7) with aluminum isopropylate in isopropyl alcohol. The 3,7-dihydroxycholesterol is benzoylated followed by dehydration of the 3,5-dibenzoate (8) at elevated temperatures to give the 7-dehydrocholesterol benzoate (9) (see Fig. 6.3). This procedure gave relatively low yields of the product of only about 4% and other methods were sought to improve the conversion of cholesterol into 7dehydrocholesterol. The Windaus procedure has been
improved recently to a point where the procedure is now used currently for a majority of the 7-dehydrocholesterol production. The bromination-dehydrohalogenation process developed in the 1940s (see Fig. 6.4) was for many years the most generally used and most economical process for the production of 7-dehydrocholesterol for vitamin D3 production. It involved the Ziegler allylic bromination [16] of the 7-position of cholesterol (1). The 3bhydroxyl group is protected from oxidation by esterification, usually as the acetate or benzoate (5). The free radical bromine addition is accomplished by a number of agents, e.g., N-bromosuccinimide, N-bromophthalimide, or preferably 5,5-dimethyl-1,3-dibromohydantoin [17,18]. Bromine in carbon disulfide has also been employed using photo catalysis to generate the freeradical bromine [19]. 1,3-Dibromo,5,5-dimethylhydantoin (commonly called “brom 55”) has been, and is still, the most efficient reagent for the introduction of bromine into the 7-position of cholesterol. This
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MANUFACTURE OF THE PROVITAMINS
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FIGURE 6.4 Preparation of 7-dehydrocholesterol from cholesterol.
bromination is not stereo-specific and conditions must be carefully controlled to optimize the production of the 7-a-bromo derivative in favor of the 7-b isomer (10a and 10b). The elimination of the bromine atom from the 7-a position can occur by the dehydrohalogenation with the 8beta hydrogen to generate the desired 7-dehydro conjugated diene (9). However, the allylic 4-hydrogen is also prone to elimination to produce the undesired 4,6 diene (11) probably from the 7-b-bromo confirmer. The formation of the 4,6-diene isomer lowers the amount of 5,7isomer and also makes the purification of the 5,7-product very difficult. This can result in low yields. Dehydrohalogenation is generally accomplished through the use of various bases such as trimethyl phosphite or pyridine bases, but extreme care must be taken to avoid the generation of the unwanted 4,6-diene isomer. Holwerda [20] showed that the reaction of the 7a-bromocholesterol with 2,4,6-trimethylpyridine (collidine) followed firstorder kinetics with respect to the 7-bromo substrate
and zero-order kinetics with respect to the collidine. A 98% yield of the 4,6-diene was obtained in DMSO, while a 70% yield of the 5,7-diene product was observed in decalin or dioxane. This investigator then demonstrated that collidine was not acting in a normal E2 elimination. Rather, an ion-pair mechanism was proposed. The rate is dependent on the bromide ion concentration and the first-order kinetics with collidine is a result of the precipitation of collidine hydrobromide which results in a constant bromide ion concentration. Collidine is a unique base in this regard. Rappoldt and co-workers reported at the 5th Workshop on Vitamin D in 1982 [21] that the use of tetra-butylammonium fluoride was more efficient than the chloride or bromide in producing the 5,7-diene. They reported a 60e70% yield of 5,7-diene with purity of 90e100% when the 7-bromocholesterol was dissolved in THF, equilibrated with tetra butylammonium bromide to optimize the 7a-bromo isomer content and then dehydrobrominated with purified tetrabutylammonium fluoride.
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FIGURE 6.5
6. INDUSTRIAL ASPECTS OF VITAMIN D
An alternate preparation of 7-dehydrocholesterol.
In commercial practice, the dehydrobromination is generally best accomplished using purified 2,4,6-trimethylpyridine at 120 C [22]. The yield of this process is usually on the order of 60% of the 5,7-isomer and gives rise to overall yields of only 50e60% (by weight) at best of the 7-dehydrocholesterol (12) from cholesterol. The purity of the collidine is critical, as traces of the isomeric trimethylpyridines or other pyridines results in considerably diminished yield of the desired 7-dehydrocholesterol. This is a result of the higher amounts of the 4,6-isomer that are produced together with other isomers; i.e. cholesta-5,8-diene-3b-ol [23]. The purification of the isolated de-brominated product is accomplished by recrystallization of the mixture of isomers to isolate a product with a high 5,7diene content. This is critical to obtaining an efficient irradiation of the material to form vitamin D3. Mixtures of solvent such as toluene and acetone can be used in various proportions to crystallize material predominantly rich in the desired 5,7-isomer. Another method to produce 7-dehydrocholesterol involves the treatment of 7-a-bromo steroid with sodium phenyl selenolate [24] to form a 7-b-phenyl selenide (13). This compound can then be oxidized and the corresponding phenyl selenoxide eliminated to form the 7-dehydrocholesteryl ester (9) (see Fig. 6.5). This method has not replaced the direct bromination/dehydrohalogenation method because of economics but may be practical for use in the manufacture of vitamin D derivatives. Despite early modifications of the Windaus oxidation procedure [25] to improve yield, the direct bromination/ dehydrohalogenation procedure became the method of choice and continues to be used because of its economics. However, despite the many years of work
to obtain better yields, it continues to give relatively low conversion yields from cholesterol (about 50%) with a concomitant buildup of the 4,6-diene by-product. Procedures to recover this material by conversion to the 5,7-diene or recycling back to cholesterol have proven inefficient and expensive. In the last few years, modifications of the Windaus oxidation procedure have been developed and are being used for production of the majority of vitamin D3 products in the world today. This method oxidizes a cholesterol ester at the 7-carbon atom to a carbonyl which is converted to a hydrazone with a substituted hydrazine [26]. The hydrazone is then reacted with base under mild conditions to produce 7-dehydrocholesterol in high yield and good quality at an economical cost [27]. This method has the advantage that no halogen is used in the process. This improves on the bromination/dehydrohalogenation methods which may leave bromine-containing impurities in the 7-dehydrocholesterol. This can give rise to the release of HBr during the irradiation process for the production of the vitamin. This acid generation can result in significant loss in vitamin D product. This new methodology gives fewer side reactions, leads to higher yields and produces a purer 7-dehydrocholesterol than the brominationdehydrohalogenation methodology.
Ergosterol Commercial ergosterol (3) of 90e100% purity is isolated exclusively from plant sources (usually yeast fermentation) and often contains up to 5 wt% of 5,6dihydroergosterol. Many of the companies which began producing ergosterol early on were involved in yeast fermentation, such as breweries. The product
I. CHEMISTRY, METABOLISM, CIRCULATION
IRRADIATION OF 7-DEHYDROCHOLESTEROL AND ERGOSTEROL
was obtained through isolation of total sterol content from the fermentation product and the subsequent separation of the provitamin from the other sterols. This isolation of the sterol fraction involves extraction of the total fat component, saponification, and extraction of the unsaponifiable (sterol containing) portion, usually with an ether. Another method is the saponification of the total fermentation material, followed by isolation of the nonsaponifiable fraction. Separation of the sterols from the unsaponifiable fraction was done by crystallization using a suitable solvent, e.g., acetone or alcohol. The ergosterol was then recrystallized from ethylene dichloride, alone or mixed with methanol. Ergosterol is particularly difficult to remove from yeast by simple extraction, usually resulting in only ca. 25% recovery. Ergosterol procedures were developed in which digestion with hot alkalies or with amines was used [28e33]. Variations of the isolation procedure have been developed; after saponification, for example, the fatty acids may be precipitated as calcium salts, which absorb the sterols. The latter are then recovered from the dried precipitate by solvent extraction. More recently new methods have been developed in China. Most of the vitamin D2 made today comes from factories in South China where an abundant source of molasses from sugar cane or cassava is available for use in the fermentation media. In this medium, molasses represents the carbon source, yeast and urea are the nitrogen source and potassium phosphate is the phosphorus source. A variety of mineral salts including magnesium sulfate are also present. The 38hour fed batch process controls the substrate concentration by maintaining the sugar level at 0.3e0.5%. The yield is in the range of 42 grams/liter of yeast cells containing 3e4 grams of ergosterol/100 grams of yeast cells [34]. The ergosterol is isolated by saponification of the yeast fermentate at 130 C with hot alkalis or amines, and removal of the protein and nucleic acid fraction by methanol extraction. The solvent is concentrated and the product is crystallized and isolated by filtration. Ethanol, acetone, and dichloroethylene or mixtures thereof are used to recrystallize the product. Annual production in China is over 10 metric tons by this method. In North China where there is a preponderance of antibiotic fermentation facilities [35], ergosterol has also been produced using extraction from hyphae of penicillin production. Wet penicillin hypha contains 0.76% of ergosterol. Saponification is accomplished with 2.5 mol/L NaOH, 25% methanol solution at 100 C for 180 minutes. Extraction using ether or petroleum ether results in ergosterol with a yield of about 0.71% (4 g ergosterol/kg hyphae with a purity of 85e90%). Aspergillus niger for citric acid fermentation and Rhizopus for lactic acid fermentation are also
79
organisms that serve as sources for ergosterol manufacture.
IRRADIATION OF 7DEHYDROCHOLESTEROL AND ERGOSTEROL The photochemical conversion of 7-dehydrocholesterol (12) is used to manufacture most of the vitamin D produced in the world today as vitamin D3. Ergosterol (3) is irradiated to form vitamin D2 (4), although this form of the vitamin is not used as extensively as it once was. It offers no real price advantage and has been shown to be less active in the pig, chicken, cow, and horse [36]. These molecules contain a 5,7-diene moiety in the B ring which can be excited by UV light of 250e350 nm wavelength leading to a p to p* excitation. This results in the cleavage of the 9,10 bond of the B ring to form a secosteroid (2,4) (secosteroids are steroids in which two of the B-ring carbon atoms, C9 and C10, are not joined). In 1938, it was estimated that 7.5 1013 quanta of light were required to convert ergosterol to 1 USP unit of vitamin D2 [37]. The value was later determined to be 9.3 1013 quanta. The product of this reaction is pre vitamin D (14), commonly referred to as “pre.” Pre undergoes thermal equilibrium to form cis vitamin D (2), commonly called “cis.” Because these two substances form equilibrium mixtures with concentrations dependent on temperature and time, both are considered the active form of vitamin D. The equilibrium composition is normally around 80% vitamin D and 20% pre vitamin D in commercial production in which the resin is heated at 60e80 C for several hours. The reaction is an antarafacial hydride {1e7} sigmatopic shift which involves a rigid cyclic transition state [40]. At e20 C, the pre isomer equilibrates to less than 5% cis in a month and it takes 2204 days to form 80% cis. At 20 C, 80% cis is formed in 13 days while at 40 C, 80% cis is formed in hours. Conversely, 100% cis at 40 C forms 10% pre in 43 hours (see Fig. 6.6). Pre vitamin D can also undergo photoisomerization to undesired by-products. Irradiation with UV light, leads to (Z)e(E) photoisomerization to the 6,7-(E)isomer, tachysterol (15). The B-ring 9,10 bond can also reform to regenerate the starting provitamin (7-dehydrocholesterol), as well as its isomer, lumisterol (16) which results from the free rotation about the 6,7 bond giving rise to the 10a-methyl isomer. The quantum yields of these reversible reactions are shown in Figure 6.7. They form equilibrium mixtures which are
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6. INDUSTRIAL ASPECTS OF VITAMIN D
FIGURE 6.6 Thermal equilibrium of pre and cis vitamin D.
dependent on the wavelength of light used for the reaction as well as time and temperature. It has been shown by theoretical calculations and confirmed experimentally that the optimum wavelength of light for the conversion of the provitamin into pre vitamin D is 296 nm [41]. A combination of photocyclization and photoisomerization pathways results in the formation of a variety of isomers. For a review of the extensive work done on the photochemistry of vitamin D see Jacobs and Laarhoven [42] as well as Dauben, McInnis and Michno [43]. It is assumed that only cZc conformers undergo photocyclization and that the rest undergo only photoisomerization resulting in a complex mixture of isomers. Care must be exercised in the irradiation process to assure product with a normal composition of isomers. These isomers can give rise to many other by-products in the event of overirradiation or exposure to air, heat, and light (see Table 6.1 for a summary of normal products in D2 and D3 irradiation and Table 6.2 for a description of abnormal products which might form under various conditions which could result from improperly controlled irradiation and heating procedures). The irradiation of 7-dehydrocholesterol (7-DHC) or ergosterol with UV light from Hg lamps gives rise to linear formation of the corresponding pre vitamin D
with the concomitant reduction in provitamin concentration. As the pre levels build, irradiation of pre begins to generate tachysterol and lumisterol and these isomers build in concentration with continued irradiation. Pre also is in equilibrium with the 7-dehydrocholesterol. In addition to time of irradiation and frequency of the light, temperature, solvent, and concentration of substrate affect the ratio of isomers in the product (see Fig. 6.8). Toxisterols, suprasterols, and other unusual forms of the type shown below (Fig. 6.9) can also form upon prolonged heating and irradiation [45e49]. There is little evidence that these materials are toxic despite their nomenclature. These materials show little if any biological activity and are not found in vivo [11,49e52]. It is important to note that normal irradiation procedures for the production of vitamin D utilize sufficiently low temperatures and controlled irradiation so as to limit these products, and that the by-products of overirradiation and heating as shown in Table 6.3 do not form; they are not found in normal commercial samples of D3 products. The most common commercial process involves the use of mercury lamps as the light source; however, several papers and patents have also been published in which light sources generating UV light near the optimum for conversion of provitamin to pre vitamin D (about 292 nm) are described. Bromine eximer lamps [53] that emit 292 nm light have been proposed for use in vitamin D2 and D3 manufacture. Laser light formed by an eximer or exciplex emitter that emits quasi-monochromatically according to the corona discharge mechanism [54] can give very specific monochromatic light at 282 nm. 7-Dehydrocholesterol (7-DHC) conversion is limited and the unused 7-DHC is recovered and recycled. While pure pre vitamin is obtained, it is produced at a low rate of conversion and thus the cost/photon is not economical for large-scale commercial production. The maximum molecular extinction coefficients (at various wavelengths) of the four main components of the irradiation are 4500 at 254 nm and 1250 at 300 nm for 7-dehydrocholesterol, 725 at 254 nm, 930 at 300 nm and 105 at 330 nm for pre vitamin D3; 11 450 at 254 nm and 11 250 at 300 nm and 2940 at 330 nm for tachysterol; 4130 at 254 nm and 1320 at 300 nm for lumisterol [53]. It is clear that the absorption of light above 300 nm is favored by tachysterol with a quantum yield of 0.48 compared to 0.26 for the conversion of 7-DHC to pre. Based on this, another scheme to produce vitamin D involves the continued irradiation of provitamin to high conversion with light of low frequency (250e300 nm) that results in a mixture of pre and tachysterol. The product containing a high percentage of tachysterol is then subjected to a secondary
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FIGURE 6.7 UV irradiation of 7-dehydro steroids to make vitamin D.
irradiation with light using a laser at about 330e360 nm. For example, 350 nm light, which is generated with an yttrium aluminum garnet (YAG) laser, has been used. N2 laser at 337 nm and XeF at 350 nm have also been used [55e58]. The secondary irradiation is also accomplished by using a photosensitizer to give light in the area of 350 nm. This provides a high yield of vitamin D product low in provitamin, lumisterol, and tachysterol. Photosensitizers, such as eosin, erythrosin, dibromodinitrofluorescein, and others, can also be used to promote this photoconversion of tachysterol back to pre vitamin D by absorbing light of lower wavelengths from lamps
and emitting very specific wavelength light useful in this conversion. The photosensitizer can also be bound to a solid matrix to facilitate the removal of the potentially toxic material from the final product [59,60]. To date, the economics of these light sources have not proven to be practical in the large-scale manufacture of vitamin D3. The fact that they give high yields may justify their use primarily in the manufacture of more expensive vitamin D derivatives such as 25hydroxyvitamin D, 1a-hydroxyvitamin D and 1a,25dihydroxyvitamin D. In commercial practice, the irradiation is carried out in a facility having vessels and equipment adequate in
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TABLE 6.1 Normal Photochemical Vitamin D Products and Their CAS Registry Numbers [44e46] Photopyrocalciferol2 [41411-05-6]
Photopyrocalciferol3 [85320-70-3]
Ultraviolet overirradiation
Photoisopyrocalciferol2 Photoisopyrocalciferol3 Ultraviolet over[26241-65-6] [85354-28-5] irradiation 5,6-trans-vitamin D2 [14449-19-5]
5,6-trans-vitamin D3 [22350-41-0]
irradiation of calciferol in the presence of iodine [58,59]
Isocalciferol2 [469-05-6]
Isocalciferol3 [42607-12-5]
treatment of trans D with mineral or Lewis acids; also forms from trans D with heat
Isotachysterol2 [469-06-7]
Isotachysterol3 [22350-43-2]
from isocalciferol or vitamin D (via trans) upon treatment with acid
size to manufacture many kilograms per day of vitamin D. A flow diagram for a typical commercial production facility is show in Figure 6.10. Irradiation of 7-dehydrocholesterol or ergosterol is carried out by dissolving the steroid in an appropriate solvent, e.g., peroxide-free diethyl ether. Solvents such as ethanol, methanol, cyclohexane, and dioxane can also be used but should be free of dissolved oxygen. The cooled solution is pumped through UV-transparent quartz reactors which permit the light from a high-pressure mercury lamp to impinge upon the solution. A commercial-type lamp assembly is shown in Figure 6.11. Nitrogen gas is passed through the inner lamp well (section 9) to minimize ozone formation. Cooled water is passed through the outer well (section 7). The important feature here is the cooling jacket which controls the high temperature (ca. 800 C) of the mercury-vapor lamps and can be used as a light filter (see below). The irradiation of the 5,7-diene provitamin to make vitamin D must be performed under conditions that optimize the production of the pre vitamin while TABLE 6.2
avoiding the development of unwanted isomers. Among the light sources used for irradiation are carbon arcs, metal-corded carbon rod, magnesium arcs, and mercury-vapor lamps. The high-pressure mercury lamp is the most widely used, however. The solution is recycled until the desired degree of irradiation has been achieved. The resulting solution contains a mixture of unreacted 7-dehydrosterol, pre vitamin D, vitamin D, and irradiation by-products. Higher yields, with a more favorable isomer distribution, can be achieved if the frequency of light is kept at 275e300 nm. The optimum frequency for the irradiation is 295 nm. Water solutions for cooling may contain salts for screening frequencies of light to ensure more optimum frequencies of light. Light below 275 nm can be filtered by aromatic compounds. Inorganic salt solutions such as 5-wt% lead acetate can also be used to filter the unwanted low frequencies; glass filters can also be used as screens for frequencies which are outside the chemical filter ranges. Photosensitizer solutions can be used similarly to emit specific frequencies of UV light. When the desired amount of conversion of the provitamin to pre has been achieved, usually between 20e30%, the solution containing the vitamin D resin is stabilized against oxidation by the addition of 1 wt% butylated hydroxyanisole or butylated hydroxytoluene. The solution is then transferred to the isolation unit and the solvent is evaporated and recovered for reuse. The unconverted provitamin is recovered from an appropriate solvent, e.g., alcohol or methanol, by precipitation in a crystallization unit. The recovered sterol is reused in subsequent irradiations. The solution containing the vitamin D is then evaporated to recover the solvent and the residual oil is heated to isomerize the pre vitamin D to the cis isomer. The resulting vitamin D resin is a pale yellow-to-amber oil that flows freely when hot and becomes a brittle glass when cold. The activity of commercial resin is 20e30 106 IU/g. The resin is formulated without further purification for use in animal feeds.
Abnormal Photochemical Vitamin D Products and their CAS Registry Numbers [44e46]
Isopyrocalciferol2 [474-70-4]
Isopyrocalciferol3 [10346-44-8]
Photo induced cyclization of pre; conrotatory bond formation to give the 9b,10b-antiisomers
(9a,10a)-pyrocalciferol2 [128-27-8]
(9a,10a)-pyrocalciferol3 [10346-43-7]
Thermal cyclization at >100 C leads to 9,10-syn isomers by disrotatory bond formation mechanism [57]
Photopyrocalciferol2 [41411-05-6]
Photopyrocalciferol3 [85320-70-3]
Ultraviolet over-irradiation
Photoisopyrocalciferol2 [26241-65-6] Photoisopyrocalciferol3 [85354-28-5] Ultraviolet over-irradiation 5,6-trans-vitamin D2 [14449-19-5]
5,6-trans-vitamin D3 [22350-41-0]
irradiation of calciferol in the presence of iodine [58,59]
Isocalciferol2 [469-05-6]
Isocalciferol3 [42607-12-5]
treatment of trans D with mineral or Lewis acids; also forms from trans D with heat
Isotachysterol2 [469-06-7]
Isotachysterol3 [22350-43-2]
from isocalciferol or vitamin D (via trans) upon treatment with acid
I. CHEMISTRY, METABOLISM, CIRCULATION
83
METABOLITE MANUFACTURE
METABOLITE MANUFACTURE 1a-Hydroxyvitamin D3
FIGURE 6.8 [41].
Approximate course of irradiation of provitamin D
Vitamin D can be crystallized from a mixture of hydrocarbon solvent and aliphatic nitrile, e.g., benzene and acetonitrile, or from methyl formate to give the USP product [63,64]. Chemical complexation as well as column chromatography is also used for purification of the resin to obtain crystalline vitamin D for food and pharmaceutical usage. Vitamin D products are formulated in a variety of matrices to protect the vitamin from exposure to air, heat, light, and minerals which cause it to degrade. These formulations also allow for the dilution of the high-potency pure product into its final dosage form with adequate distribution so as to assure uniform dosage of the food, feed, or pharmaceutical preparation. Vitamin D2 is made from ergosterol using the same type of technology. The same isomer distribution occurs and the irradiation must be carried out with similar care as described above for vitamin D3.
CH3
1a-Hydroxyvitamin D3 is becoming an important supplement in poultry diets. It has the ability to reduce tibial dyscondroplasia and is additive with phytase in promoting phosphorus utilization [65,66]. The product was originally prepared by Barton in 1973 and many preparations have been reported in the literature since. The one used most frequently is a modification of the Barton method [67,68]. The commercial process [69] starts by treating vitamin D3 with SO2 to produce two cyclic adducts. The 3-OH group is protected with a silicon protecting group. The SO2 is removed with the formation of a derivative of a single isomer (5,6transvitamin D3) followed by allylic oxidation to introduce the 1a-hydroxy function. After de-protection and crystallization, the 1a-hydroxytrans vitamin D3 is photochemically isomerized to 1a-hydroxyvitamin D3. The 1a-hydroxyvitamin D3 in addition to its many applications for pharmaceutical uses by several companies is formulated in a starch matrix for use in animal feed products at a concentration of 0.04%. The major use of the product is in animal feeds and volume currently approximates 100 kg of crystalline product per year which will produce 20 000 000 metric tons of feed (5 mg of 1a-hydroxyvitamin D3/kg of finished feed). The fact that the process starts with crystalline D3 causes the product to be substantially more expensive than the vitamin D itself. The benefits of using the product to replace vitamin D must overcome this cost differential. It is, therefore, used as a supplement to vitamin D3 to achieve its additive benefits.
25-Hydroxyvitamin D3 25-Hydroxyvitamin D3 is also of commercial use, primarily in animal nutrition but also pharmaceutically for osteoporosis and other bone disease treatments. Its
CH3
CH3
CH3
CH3
O
HO (17) Suprasterol II
OH (18) Toxisterol-E
(19) Toxisterol E1
FIGURE 6.9 Unusual by-products of heat and prolonged irradiation of vitamin D3.
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84
6. INDUSTRIAL ASPECTS OF VITAMIN D
UV Absorbance Maxima of 7-Dehydrocholesterol, Pre- and Cis-Vitamin D [61]
TABLE 6.3
Absorbance 7-Dehydrocholesterol Pre vitamin D cis-Vitamin D @ (nm)
(E 1% 1 cm)
260
270
265
484
273
282
282
293
293
170
use in animal feeds is claimed to promote bone development, weight gain, and feed efficiency. It should be noted that when administered to an animal, vitamin D is hydroxylated in the liver soon after absorption to 25hydroxyvitamin D which then circulates in the blood. Feeding the metabolite bypasses this initial biological process. Therefore, one must evaluate the toxicity, cost, and benefits of this type of administration. The 1a-hydroxyvitamin D is not a naturally occurring material but upon absorption into the body undergoes 25-hydroxylation in a manner similar to (and at an equivalent rate to) the vitamin D. This process by-passes the kidney 1a-hydroxylation of the 25-hydroxyl metabolite, making the 1a,25-dihydroxyvitamin D3 hormone available at an accelerated rate. 25-Hydroxyvitamin D is made commercially primarily by a process which involves the fermentation
FIGURE 6.10
of a double mutant yeast to form 5,7,24-cholestatrienol [70e72]. The 25-hydroxyvitamin D3 is used at 62.5 mg/kg finished feed and current usage is estimated to be approximately 15 000 000 metric tons of feed per year. Thus, approximately 937.5 kg of 25-hydroxyvitamin D3 are used. The active hormonal form of vitamin D3, 1,25-dihydroxyvitamin D3 or calcitriol, can be made from the 25-hydroxyvitamin D [73]. 1,25-Dihydroxyvitamin D3 and its derivatives are used primarily in pharmaceutical preparations and are made by a variety of processes.
ANALYTICAL Vitamin D The early history of vitamin D led to the use of biological testing to determine the effectiveness of vitamin D products to reduce the effects of rickets. This ultimately led to standardized rat and chick tests in which laboratory animals are fed special diets devoid of vitamin D to produce rachitic conditions in the animal. The test animals are then dosed with the test substance and the bone growth at the proximal end of the tibia or distil end of the ulna are compared after staining with silver nitrate. Rats can be used to test vitamin D2 or D3, but the results can lead to false positives for use of the product in poultry, since chickens do not respond to
Vitamin D3 manufacturing flow diagram [61, p. 239].
I. CHEMISTRY, METABOLISM, CIRCULATION
85
ANALYTICAL
FIGURE 6.11 system [62].
vitamin D2. Therefore, chickens must be used to evaluate product that is to be used in poultry. This method is still approved by the AOAC (see AOAC 45.3.03 Chick Bioassay for Poultry Feed Supplement.932.16 [74], while material used for all other animals can use the AOAC Rat Bioassay 45.3.02 Rat Bioassay 936.14 [75]). These methods are slow, expensive, and lead to variable results. The elucidation of the chemical nature of the vitamins led to the use of chemical methods of analysis which were primarily dependent upon colorimetric procedures. These were approved by the major official governing organizations and used for many years as the official methods of analysis. The AOAC chemical method (Colorimetric method 975.42 45.1.17) [76] involves “saponification” of the sample (dry concentrate, premix, powder, capsule, tablet, or aqueous suspension) to release the vitamin from its matrix, with aqueous alcoholic KOH. The vitamin is then extracted using an appropriate solvent and the solvent containing the vitamin D is removed. Vitamin D is separated from extraneous ingredients by a chromatographic separation and the potency of the vitamin D is determined by a colorimetric determination with antimony trichloride in comparison with a solution of USP cholecalciferol reference standard. The procedure includes a step to treat unsaponifiable material
Commercial photochemical UV lamp
with maleic anhydride to remove any trans-isomer which may be present and lead to a falsely high result. The antimony trichloride colorimetric assay is performed on the trans-isomer-free material. This procedure cannot be used to distinguish isotachysterol and, if present, also gives rise to a falsely high result. A test must therefore be performed to check for the presence of isotachysterol. The USP XXXII [77] and AOAC 2010 (HPLC 979.24 [78]) both now recognize high-pressure liquid chromatography (HPLC) as the preferred method of analysis. HPLC allows the separation of the active pre- and cisisomers of vitamin D3 from other isomers and provides a means to analyze the active content by comparison with the chromatograph of a sample of pure reference cis-vitamin D3. Equilibration of a solution of the standard to a mixture of pre- and cis-isomers [12,13] is included in the procedure in order to evaluate the total active isomer content of the sample. The sensitivity of this method provides information on isomer distribution and allows for the accurate evaluation of the active pre- and cis-isomer content of a vitamin D sample. It is applicable to most forms of vitamin D, including the more dilute formulations, i.e., oils containing 100 000 IU cholecalciferol/g; resins 20 000 000 IU cholecalciferol/g; and powders and aqueous dispersions at
I. CHEMISTRY, METABOLISM, CIRCULATION
86
6. INDUSTRIAL ASPECTS OF VITAMIN D
25 000 IU cholecalciferol/g (AOAC Methods 979.24; 980.26; 981.17; 982.29; 985.27) [79e81]. The limiting factor in the assay of low-level formulations is the isolation of the vitamin material from interfering and extraneous components which may obscure the vitamin D HPLC peak. Vitamin D products formulated in a variety of matrices are then usually mixed with carriers to form premixes and final dosage compositions. The assay of the vitamin requires the matrix to be broken in such a way as to assure complete availability of the vitamin. Additionally, the ability to separate it from the formulated mixture must be efficient. The above methods all address this issue. A particularly useful extraction procedure involves the use of dimethyl sulfoxide [82]. The usual HPLC procedures utilize UV detection for quantification of the elution peaks. Recently HPLC has been coupled with mass spectrographic detectors to enable the assay of vitamin D to much more significant detection limits [83,84]. A number of methods have been developed for the paper, thin-layer, and column chromatographic separation of vitamin D and related substances but these are more tedious and difficult to perform on low-level samples. Gas chromatography requires derivatization and has been applied to metabolite analysis as well as assay of multivitamin tablets and vitamin D2 in milk and other formulations [85e90]. The USP [91] requires the following tests for the pure crystalline vitamin D: Identity by: (a) IR; A typical infrared spectrum of cholecalciferol will have the following parameters; the IR spectrum of the sample should be identical to the IR spectrum of a USP reference standard of cholecalciferol. Wavelength
Peak Ht.
Characteristic
3305 2934 1642 1458, 1438, 1375 1053
Strong Strong Weak Medium Strong
-OH stretch -CH stretch & bend -C¼C- stretch -CH stretch & bend -C-OH stretch
(b) UV; A typical UV curve for a 10 mg/ml solution of cholecalciferol has a molar extinction coefficient of 18 692 at lmax 264.8 (E1%max ¼ 484). (c) Chemical color (with acetic acid and sulfuric acid turns bright red changing to violet and then blue green).
Thin Layer Chromatography: USP <621> [77] (developed with SbCl3 in Acetyl Chloride which gives a yellow/orange color) with retention time compared to a USP reference standard. Specific Rotation; USP <781s> [92] aD ¼ between þ105 and þ112 . Assay: by High Pressure Liquid Chromatography (HPLC) USP <621> [77] against a USP reference standard. The international standard for vitamin D is an oil solution of activated 7-dehydrocholesterol. The International Unit (IU) is the biological activity of 0.025 mg of pure cholecalciferol. One gram of vitamin D3 is equivalent to 40 106 IU or USP units. Samples of reference standard may be purchased from US Pharmacopeial Convention [93]. Reference standards are also available from the World Health Organization (WHO) as well as the European (EP) and British Pharmacopeia (BP). USP also issues vitamin D3 capsules for AOAC determination in rats and an oil solution for the vitamin D3 AOAC determination in chicks. The various isomers of vitamin D exhibit characteristically different UV absorption curves. Cis vitamins D2 and D3 exhibit UV absorption maximum at 265 nm with an Emax (absorbance) of 450e490 at 1% concentration (Table 6.3). Mixtures of the isomers are difficult to distinguish but the pure substances and their concentrates can be assayed using their UV absorption. When chromatographically separated by HPLC, the vitamin D peaks can be identified by stop-flow techniques based on UV absorption scanning or by photodiode-array spectroscopy as well as mass spectroscopy. The combination of elution time and characteristic UV absorption curves can be used to identify the isomers present in a sample of vitamin D. Infrared and NMR spectroscopy have been used to help distinguish between vitamins D2 and D3 [94e96].
Provitamin Assay The molecular extinction coefficient of 7-dehydrocholesterol at 282 nm is 11 300 and is used as a measure of 7dehydro isomer content of the provitamin [97,98]. High pressure liquid chromatography can also be used to analyze the provitamins. There are a variety of chemicals that show characteristic colors when reacted with the provitamins. Some of these are listed below. • The Salkowski reaction (revised) treats the provitamin with CHCl3 and H2SO4 (conc.) to give a deep red color in CHCl3 layer and green fluorescence in the acid layer which differentiates from sterols lacking a conjugated diene.
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87
DIETARY REQUIREMENTS
• The Lieberman-Burchard reaction is run in CHCl3 with acetic acideH2SO4 added dropwise. A red color develops and changes to blue-violet to green. The test can be quantitative and acts similarly to the Salkowski reaction, but the red color lasts longer. • The Tortelli-Jaffe reaction is run in acetic acid with 2 wt% Br2 in CHCl3 which turns green with sterols having ditertiary double bonds including vitamin D and compounds that give similar bonds upon isomerization or reaction. • The Rosenheim reaction is run in CHCl3 with trichloroacetic acid in H2O. A red color develops and changes to light blue. When run with CHCl3 and lead tetraacetate in CH3COOH followed by the addition of trichloroacetic acid, the reaction gives a green fluorescence which is not given by esters of provitamin D and can be used to distinguish between provitamin and provitamin ester. The test is quantitative to 0.1 mg. • A mixture of crystalline provitamins and chloral hydrate heated slowly melts at 50 C and color develops and changes from red to green to deep blue while other sterols, e.g., cholesterol, do not react to give color. • The antimony trichloride reaction with CHCl3 and SbCl3 gives a red color. • The Chugaev reaction adds glacial acetic acid plus acetyl chloride and zinc chloride to the provitamin which is heated to boiling. An eosin-red greenish yellow fluorescence develops with a sensitivity of 1:80 000 [99].
Assay of 25(OH)D The extremely low levels of vitamin D and its metabolites in biological systems make it very difficult to assay the vitamin D products in these environs by traditional methods. The ability to assay these materials was initially developed by Haddad [100] with the use of a competitive protein-binding assay (CPBA) for 25-hydroxyvitamin D (25(OH)D). 25(OH)D is especially vulnerable to matrix effects in any protein-binding assay because of its lipophilic properties. These were overcome by the utilization of chromatographic sample purification prior to the complex formation with the calcium. A nonchromatographic radio immunoassay for circulating 25(OH)D was developed by Napoli and Hollis using an antigen that would generate an antibody that was cospecific for 25(OH)D2 and 25(OH)D3 [101]. The study of vitamin D and its metabolites and their effects in clinical disease over the past 30 years was made possible by the ability to assay these materials. The need to assay large numbers of samples to evaluate the
vitamin D blood levels of large populations requires an ability to perform these assays with a rapid, accurate, and valid method. In 2001, Nichols Diagnostics introduced the fully automated chemiluminescence CPBA ADVANTAGE 25(OH)D assay system [102] in which nonextracted serum or plasma is introduced directly into a mixture containing human D-binding protein (DBP), acridinium-ester-labeled anti-DBP, and 25(OH)D3-coated magnetic particles. Another chemiluminescence assay was developed in 2004 by the DiaSorin Corporation [103]. The assay, LIAISON 25(OH)D, is very similar to the ADVANTAGE assay but uses an antibody as a primary binding agent as opposed to the human DBP and is a radio immune assay (RIA) method. See Hollis [104] for a review of these methods and their application to the important assay of blood levels of 25(OH)D.
DIETARY REQUIREMENTS Humans Dietary Reference Intakes of vitamin D2 and vitamin D3 (DRIs) developed by the Food and Nutrition Board (FNB) at the Institute of Medicine of the National Academies (formerly National Academy of Sciences) were established in 1997 [105]. They recommended 200 IU/day from 1 month to 50 years of age and 400 IU/day from 51 years to 70 years and 600 IU/day after reaching the age of 71. In 2008, the American Academy of Pediatrics (AAP) issued recommendations for intakes for vitamin D that exceed those of FNB to 400 IU/day and the Surgeon General of the United States indicated that all citizens should make sure they took a minimum of 400 IU/g day to ensure good health. Many studies over the past 15 years have led to the suggestion that 1000e2000 units and as high as 5000 IU/day of vitamin D3 per day are necessary to provide enough vitamin D to maintain 25hydroxyvitamin D blood levels (37.5e50 nmole/L; 15e20 ng/ml) sufficiently high to provide all of the functions the vitamin and its metabolites serve to influence. The Food Nutrition Board established an expert committee in 2008 to review the DRIs for vitamin D (and calcium). The FNB issued its report, updating as appropriate the DRIs for vitamin D and calcium, in November 2010. The new recommendations are 600 IU/day from age 1 year to 70 years and 800 IU/day from age 70 and older. Upper level intake limits were set at 4000 IU/day for 9 year olds and older. As noted above, there is much evidence which suggests higher levels than this are needed and this problem will be one of ongoing concern [105a].
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88 TABLE 6.4
6. INDUSTRIAL ASPECTS OF VITAMIN D
Practical Feeding Levels of Vitamin D, 106 IU/ton [111]
Animal
Amount Animal
Amount
Poultry*
Dairy cattle
Chickens
Calf starter
1e2
Broilers
2e4
Calf milk replacer
2e4
Replacement birds
1e3
Replacement heifers
1e2
Layers
1e3
Dry cows
1e2
Breeding hens
2e4
Lactating cows
2e4
Bulls
2e4
Turkeys Starting
3e5
Growing
2e4
Beef cattle
Breeding
3e5
Calf starter
1e2
Replacement heifers
1e2
Ducks Market
1e3
Feedlots
2e3
Breeding
2e4
Dry pregnant cows
1e2
Lactating cows
1e2
Bulls
1e2
Swine Prestart (to 10 kg)
2e3
Starter (10e35 kg)
1e2
Sheep
Growing-finishing (35 kg to market)
1e2
Fattening lambs
2e3
Gestation
1e2
Breeding
1e2
Lactation
2e3
Boars
2e3
Other
ECONOMIC ASPECTS
Fish
Dogs
5e1
Cats
1e2
Horses
1e2
for various species: starting and growing chicks, 200 IU; laying and breeding hens, 500 IU; turkeys, 1100 IU; ducks, 200 IU; quail, 480e900 IU; geese, 200 IU; and swine 125e220 IU. Calves require 600 IU per 100 kg of body weight [107e109]. Higher levels are usually used in common practice in order to make sure the animals receive adequate dosage of the vitamin. Most species can safely tolerate four to ten times the NRC requirements during long-term feeding and short-term (<60 d) most species can tolerate 100 times their apparent dietary requirements [110]. Animals produce vitamin D3 when exposed to sunlight and do not require substantial dietary vitamin D. However, many animals are raised indoors with little exposure to sunlight and modern livestock management practices place an emphasis on high productivity. As a result, most feed manufacturers recommend vitamin D3 supplementation of diets. Recommendations for practical levels of vitamin D3 in feeds for various animals, as recommended by feed manufacturers, are listed in Table 6.4.
Trout
1e2
* Poultry cannot absorb vitamin D2.
Animals Rickets prevention or cure initially was the purpose for feed fortification with vitamin D3. Adequate levels were determined to be those sufficient to prevent rickets. In 1995, Edwards [106] found that, in the absence of UV light, different vitamin D3 levels result in the optimization of various effects of vitamin D3 in poultry. For example, 275 IU/kg is required for growth, 503 IU/kg for optimum bone ash, 552 IU/kg for proper blood plasma calcium, and 904 IU/kg for rickets prevention. The National Research Council recommends the following amounts of vitamin D per kilogram of feed
Vitamin D3 is available in a variety of forms. Cod liver oil and percomorph liver oil were good sources of vitamin D3 historically but crude cod liver oil processing involves alkali refining, bleaching, winterization, and deodorization. This vigorous treatment of the vitamincontaining oil substantially depletes the vitamin activity. Fully cleaned and deodorized cod liver oil is sold with synthetic vitamins added back. Most of the cod liver oils on the market fall into this category. This is the socalled high-vitamin cod liver oil, standardized at a maximum of 2500 IU vitamin A per gram (12 500 IU per teaspoon) and 250 IU vitamin D3 (1250 IU per teaspoon). Lower-potency oils are sold with the ratio of vitamin A to D3 of 10:1. In 2010, cod liver oil with a potency of 1700 IU vitamin A and 170 IU vitamin D3 sold for approximately $5.50 to $7.75 per kilogram. Most of the vitamin D sold is synthetic as opposed to natural. The term “natural” is often used to describe an “organic” product. It should be noted that vitamin D3 is the vitamin found in nature; it is made by all animals. Vitamin D2 and vitamin D3 (crystalline and resin materials) are Generally Recommended As Safe (GRAS) by the United States Food, Drug and Cosmetic Act for both human nutrition [112] and animal feeds [113]. Pharmaceutical formulations are made with USP crystalline vitamin D2 and. vitamin D3. The pure crystalline vitamin at 40 MIU/gram or resin (at 25e30 MIU/
I. CHEMISTRY, METABOLISM, CIRCULATION
ECONOMIC ASPECTS
gram) must be diluted in a carrier to a concentration that is practical for use in uniform dosing of the final product. Many supplement preparations such as tonics, drops, capsules, tablets, and oil-based injectables are also marketed. The primary regulations governing vitamin D fortification are 21 CFR 184.1950 and 184.1(b) (2). These regulations state that vitamin D may be added to a limited number of foods for the functional use of nutrient supplementation. The food categories included on the approved list include breakfast cereals, milk, milk products, grain products and pastas, infant formula, and margarine. Essentially all milk produced in the United States is fortified with vitamin D3. Preparations based on the use of vitamin D3 resin are less expensive than those using crystalline vitamin D3 and have been used for many years. Vitamin D2 as a concentrate or in microcrystalline forms is used in many pharmaceutical preparations, although vitamin D3 is preferred by many manufacturers and consumers because it is the form occurring naturally in animals. Vitamin D2 has been used as a feed supplement for cattle, swine, and dogs, but its use has declined in favor of vitamin D3. Vitamin D2 cannot be used in poultry and many feed producers would prefer not to use both D2 and D3 to avoid the possibility of a mix-up that would allow D2 to get into poultry feed which could result in catastrophic losses. Table 6.5 shows the metric tons of feed TABLE 6.5 Animal Use of Vitamin D by Species in the United States and Europe. In Metric Tons of 500 000 IU/ gram Equivalent US
1994%
2010%
Poultry
41
49
2010 MT D3 500
Layers/breeders
101
Broilers
306
Turkeys
73
Swine
13
11
Beef and dairy
44
32
109
Dairy
280
Beef
35
Pet foods
2
Total tons of 500 000 IU/gm
8
76 980
Europe
1994
Cattle
58
Swine
19
Poultry
20
Other
3
89
grade 500 000 IU/gram used by species in 2010. The usage levels shown for dairy and beef in 2010 are approximately 30% and 50% lower, respectively, than they have been recently due to current market fluctuations. Formulations combining vitamin A and D or A, D, and E (the fat-soluble vitamins) are used widely. These may be pre-emulsified to insure increased bioavailability. Vitamin D is not water-soluble. In order to make it compatible with aqueous systems, it is formulated by dissolving the vitamin in oil and using surfactants to enable the emulsification of the oil in an aqueous media [114]. In animal feeds, oil solutions of vitamin D3 or solutions of the vitamin in oil-on-dry carriers, e.g., corn or flour, can be used. Animal diets usually contain high levels of minerals, and the vitamin D3 in the feed is thus exposed to the minerals as well as to air, heat, light, and moisture when the feed is in use. Because of this, when used in most feeds, vitamin D3 is not stable unless it is protected. Several stable forms are patented and sold commercially; they include beadlets or powders of dry suspensions in gelatin, carbohydrates, wax, and cellulose derivatives. They can be spray dried or drum dried [115e119]. Animal feed formulations usually employ resin in these various stabilized forms and are sold predominantly at levels of 500 000 or 1 106 units of vitamin D3 per gram. Combination products containing vitamins A and D are also available, with 1 000 000 units of vitamin A and 200 000 units of vitamin D per gram of product being the most common dosage form. During the past year we have seen vitamin D3 in a price range of $10.00 to $100.00 USD per Million International Units (MIU). Raw material plays a large part in market pricing and with today’s cholesterol price, the intrinsic value of vitamin D3 is approximately $50.00/ MIU. The estimate of world usage of vitamin D3 as 500 000 IU/gram material in animal feed is estimated in Table 6.6. The total tonnage is approximately 77.5 metric tons of D3 as crystals equivalent in animal feed, 15.3 metric tons of crystals in pharmaceutical use and about 4.5 metric tons in human food, or 97.3 metric tons of D3 crystals worldwide. Eighty percent of the world market is made in China by four main producers. About 1000 MT is produced in Crenzach, Germany, which is mainly consumed by its manufacturer for internal use. This is supplemented in the market with 25-hydroxyvitamin D3 product with sales revenue in 2008 reported to be 35 million euros at an average price of V350/kg or approximately 100 000 kg of the metabolite. Another major supplier is planning production of vitamin D3 in India and is planning to be in operation by 2011.
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6. INDUSTRIAL ASPECTS OF VITAMIN D
TABLE 6.6 2010 World Usage of Vitamin D3 [111] Feed Grade
Food & Pharma
Food Grade
MT OF 40 MIU/GR
MT OF 100,000 IU/GR
MT D3 500 000 IU/GM -
MT OF 40 MIU/GR
Europe
1300
16.25
6.5
600
1.5
USA
1000
12.5
5.5
800
2
China
800
10
1.5
200
0.5
Brazil
300
Asia
1200
200
0.5
1800
4.5
Country
-
South America
500
MT OF 40 MIU/GR
MT OF 40 MIU/GR
-
3.75 15 6.25
Turkey
0.5
Iran
0.3
Japan
0.3
India
0.7
Australia Other
1100
13.75
TOTAL
6200
77.5
Approximately 200 kg/year of vitamin D3 formulations have been sold as rat poisons. The metabolites of vitamin D3 and synthetic derivatives are being used or developed for treatment of osteoporosis, skin psoriasis, and other diseases in humans. 1a-Hydroxyvitamin D3 is being used for milk fever in cows, and more importantly for the treatment of tibial dyschondroplasia and to facilitate phosphorus utilization. 25-Hydroxyvitamin D3 has been proposed for eggshell thickness in poultry and is being marketed as an animal dietary nutritional supplement. Additionally it is used for human pharmaceutical purposes. As mentioned earlier, vitamin D2 derivatives such as Calipotriene (Dovenex) a 24-hydroxy,25-cyclopropyl derivative is used for psoriasis, and 1-a-hydroxyvitamin D2 (Doxercalciferol; Hectoral) and 19-nor-1a,25 dihydroxyvitamin D2 (Paricalcital; Zemplar) are used for secondary hyperparathyroidism associated with chronic renal failure.
STORAGE AND SHIPPING Vitamin D is sensitive to air, heat, UV light, and mineral acids. These sensitivities are exaggerated by the presence of heavy-metal ions, e.g., iron. Therefore, care should be taken to store and ship vitamin D and its various product forms by methods that minimize exposure to these conditions.
15.3
97.3
Pharmaceutical grade vitamin D3 is packaged in the pure crystalline form. It usually is packaged in 100, 500, or 1000 g hermetically sealed pouches. The high potency of the product allows for smaller amounts of the material to be used in formulations and the smaller package size minimizes the need to store open packages of vitamin D. The crystalline vitamin D is used in vitamin D food and pharmaceutical applications as well as multivitamin preparations. Commercial sources of feed-grade vitamin D are usually a vitamin D3 resin stabilized by spray or by roll drying a starch or gelatin suspension of the vitamin. These products should be stored in a cool, dry area, preferably in opaque, hermetically sealed containers under an inert atmosphere such as nitrogen, CO2, or an inert gas. Shipping vitamin D in crystalline or resin form should be done in containers marked appropriately to indicate that the material is toxic by DOT standards. Its proper DOT labeling is DOT Hazard Class 6.1, poisonous (this is because of its high degree of potency per gram). Waste material should be placed in an appropriate landfill or preferably burned. The provitamins are similarly unstable to heat and light and should also be stored in a dark, cool, place with limited exposure to oxygen. The provitamin is more stable if stored and shipped with 10e15 wt% methanol rather than in a dry form.
I. CHEMISTRY, METABOLISM, CIRCULATION
REFERENCES
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[65] H.M. Edwards Jr., Studies on the efficacy of cholecalciferol derivatives for stimulating phytate utilization in broilers, Poultry Science 81 (2002) 1026e1031. [66] J.L. Snow, M.E. Persia, P.E. Biggs, D.H. Baker, C.M. Parsons, 1 a hydroxycholecalciferol has little effect on phytate phosphorus utilization in laying hen diets, Poult. Sci. 82 (2003) 1792e1795. [67] D.H.R. Barton, R.H. Hesse, M.M. Pashet, E. Rizzardo, A convenient synthesis of 1a-hydroxy-vitamin D3 [45], Journal of the American Chemical Society 95 (1973) 2748e2749. [68] D.R. Andrews, D.H.R. Barton, K.P. Cheng, J.P. Finet, R.H. Hesse, G. Johnson, et al., Synthesis of 25-hydroxy- and 1a,25-dihydroxy vitamin D3 from vitamin D2 (calciferol), J.Org. Chem. 51 (1986) 4819e4828. [69] World Patent Application WO/2011/002756 June 1, 2011, Vitamin D compounds and methods for preparing same, H.M. Edwards Jr., G. Majetich, R. Hill. [70] JP 2004141125, 2004, Metabolically engineering modified true fungi and method for producing sterol by using the same, Y. Hayashi, A. Nakajima. [71] JP 04089476 A 19920323, 1992, Preparation of 25-hydroxyprevitamin D derivatives, S. Nakagawa, H. Yanai, M. Shiono, T. Muramatsu, M. Mori, M. Amano, from Jpn. Kokai Tokkyo Koho. [72] Amoco BioProducts Corporation, The Evaluation of the Human Health Aspects of Using 25-Hydroxyvtiamin D3 as a Broiler Poultry Feed Ingredient, FASEB Life Sciences Research Office, Bethesda, Maryland, 1994. [73] CN 101607931, Dec. 23, 2009, Preparation method of calcitriol, M. Wang, L. Ren, G. Sun, Y. Chen, X. Liu. [74] W. Horwitz, G.W. Latimer Jr, Ed., “Official Methods of Analysis”; eighteenth ed, Revision 2 Association of Official Analytical Chemists International Gaithersburg, Md. 2007., Chapter 45 p. 79. [75] Ibid. Chapter 45 p. 75. [76] Ibid. Chapter 45 p. 25. [77] United States Pharmacopeia 32nd Revision/National Formulary 27th ed. US Pharmacopeial Convention, Rockville, Md. 2009 Vol. 1, 233 HPLC <621>. [78] W. Horwitz, G.W. Latimer Jr, Ed., “Official Methods of Analysis”, eighteenth ed, Revision 2 Association of Official Analytical Chemists International Gaithersburg, Md. 2007, Chapter 45 p. 29. [79] W. Horwitz, G.W. Latimer Jr, Ed., “Official Methods of Analysis”, eighteenth ed, Revision 2 Association of Official Analytical Chemists International Gaithersburg, Md. (HPLC Methods 979.24 45.1.18e45.1.22a), 2007 Chapter 45 p. 30e36. [80] H. Hofsass, N.J. Alicino, A.L. Hirsch, L. Amieka, L.D. Smith, Comparison of high pressure liquid chromatographic and chemical methods for vitamin D3 concentrates. II. Collaborative study, J. Assoc. Off. Anal. Chem. 61 (1978) 735e745. [81] E.J. De Vries, B. Borsje, Analysis of fat-soluble vitamins. XXIX. Liquid chromatographic determination of vitamin D in AD concentrates: Collaborative study, J. Assoc. Off. Anal. Chem. 68 (1985) 822e825. [82] R.J. Pastore, R.V. Dunnett, G.K. Webster, Dimethyl Sulfoxide Extraction Method for the Liquid Chromatographic Analysis of Microencapsulated Vitamin D3, J. Agric. Food Chem. 45 (5) (1997) 1784e1786. [83] W.C. Byrdwell, Comparison of analysis of vitamin D3 in foods using ultraviolet and mass spectrometric detection, Journal of Agricultural and Food Chemistry 57 (2009) 2135e2146. [84] D.R. Bunch, A.Y. Miller, S. Wang, Development and validation of a liquid chromatography-tandem mass spectrometry assay for serum 25-hydroxyvitamin D2/D3 using a turbulent flow online extraction technology, Clinical Chemistry and Laboratory Medicine 47 (2009) 1565e1572.
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[85] J.G. Bell, A.A. Christie, Gas-liquid chromatographic determination of vitamin D2 in fortified full-cream dried milk, Analyst 99 (1974) 385e396. [86] J.R. Evans, The gas chromatography of calciferol, dihydrotachysterol and cholesterol, Clin. Chim. Acta. 42 (1972) 167e174. [87] T.K. Murray, K.C. Day, E. Kodicek, The differentiation and assay of vitamins D2 and D3 by gas-liquid chromatography, Biochem. J. 98 (1966) 293e296. [88] L.V. Aviolo, S.W. Lee, Detection of nanogram quantities of vitamin D by gas-liquid chromatography, Anal. Biochem. 16 (1966) 193e199. [89] D. Sklan, P. Budowski, M. Katz, Determination of 25 hydroxycholecalciferol by combined thin layer and gas chromatography, Anal. Biochem. 56 (1973) 606e609. [90] D.O. Edlund, F.A. Filippini, J.K. Datson, Gas-liquid chromatographic determination of vitamin D2 in multiple vitamin tablets containing minerals and vitamin E acetate, J. Assoc. Anal. Chem. 57 (1974) 1089e1091. [91] United States Pharmacopeia 32nd Revision/National Formulary, twentyseventh ed., US Pharmacopeial Convention, Rockville, Md. 2009 Vol. 2, 1923. [92] United States Pharmacopeia 32nd Revision/National Formulary, twentyseventh ed., US Pharmacopeial Convention, Rockville, Md. 2009 Vol. 1, 304 <781s>. [93] U.S. Pharmacopeial Convention, Inc., Reference Standards Order Department, 12601, Twinbrook Parkway, Rockville, Maryland 20852. http://www.usp.org. Phone: 1e301e816e8129. [94] J. Carol, Application of infrared spectrophotometry to pharmaceutical analysis, J. Pharm. Sci. 50 (1961) 451e463. [95] W.W. Morris Jr., J.B. Wilkie, S.W. Jones, L. Friedman, Differentiation of vitamins D2 and D3 by infrared spectrophotometry, Anal. Chem. 34 (1962) 381e384. [96] R. Strohecker, H.M. Henning, Vitamin Assay-Tested Methods, CRC Press, Cleveland, Ohio, 1966, p. 281. [97] T.G. Hogness, A.E. Sidwell Jr., F.P. Zscheile Jr., Photoelectric spectrophotometry: An apparatus for the ultra-violet and visible spectral regions: Its construction, calibration, and application to chemical problems, J. Physical Chem. 41 (1937) 379e415. [98] W. Huber, G.W. Ewing, J. Kriger, The absorption spectra of the vitamins and provitamins D, J. Am. Chem. Soc. 67 (1945) 609e617. [99] Vitamins (Vitamin D) in Kirk Othmer Encyclopedia of Chemical Technology fourth ed., Exec. Editor, Jacqueline L. Koschwitz; editor, Mary Howe-Grant, Vol. 25, p. 217e256 by A. L. Hirsch, A.L. Laboratories, Inc., John Wiley and Sons, New York, New York (1998). Reprinted with permission of John Wiley & Sons, Inc., p. 237. [100] J.C. Haddad, K.J. Chyu, Competitive protein-binding radioassay for 25-hydroxycholecalciferol, J Clin Endocrinol Metab 33 (1971) 992e995. [101] B.W. Hollis, J.L. Napoli, Improved radioimmunoassay for vitamin D and its use in assessing vitamin D status, Clin Chem 31 (1985) 1815e1819.
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[102] Product insert, Nichols ADVANTAGE 25-hydroxyvitamin D assay, Nichols Institute Diagnostics, San Juan Capistrano, CA, 2001. [103] Product insert, LIAISON chemiluminescence 25-hydroxyvitamin D assay, DiaSorin Corporation, Stillwater, MN, 2004. [104] B.W. Hollis, Editorial: The determination of circulating 25hydroxyvitamin D: No easy task, J Clin Endo Met 89 (7) (2004) 3149e3151. [105] Dietary reference intakes for calcium, phosphorus, magnesium, vitamin D, and fluoride/Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, Food and Nutrition Board, Institute of Medicine 1997, National Academy of Sciences Press, Washington, D.C. p. 250. [105a] Dietary reference intakes for calcium and vitamin D. Committee to Review Dietary Reference Intakes for Vitamin D and Calcium, Food and Nutrition Board, Institute of Medicine, November 2010, Report Brief. [106] H.M. Edwards Jr, “Factors Influencing Leg Disorders in Broilers,” 1995 in (Proceedings of the Maryland Nutrition Conference, University of Maryland, College Park, Maryland) p.21. [107] Board of Agriculture, Nutrient Requirements of Poultry, nineth ed., National Academy of Science, National Research Council, Washington, D.C, 1994, p. 15. [108] Board of Agriculture, Nutrient Requirements of Swine, nineth ed., National Academy of Science, National Research Council, Washington, D.C, 1988, p. 35. [109] Board of Agriculture, Nutritional Requirements of Dairy Cattle, third ed., National Academy of Science, National Research Council, Washington, D.C, 1966, p. 1349. [110] Board of Agriculture, Vitamin Tolerances of Animals, National Research Council, National Academy Press, Washington, D.C, 1987, p. 10. [111] AGD Nutrition Feeding Level Recommendations G. Vannorsdel; President AGD Nutrition LLC, 190 Civic Circle, Suite 255 Lewisville, Texas 75067. [112] Title 21 Code of the Federal Register Chapter 184. 1950 Vitamin D. [113] Title 21 Code of the Federal Register Chapter 582.5953 Vitamin D3 and Chapter 582.5950 Vitamin D2. [114] U.S. Pat. 2,417,299, Fat soluble vitamin solutions, L. Freedman, E. Green (to U.S. Vitamin Corp.). [115] U.S. Pats. 2,777,797 Vitamin Products and 2,777,798 stable fat-soluble vitamin-containing composition Jan. 15, 1977, M. Hochberg, M.L. MacMillan (to Nopco Chemical Co.). [116] U.S. Pat. 2,702,262, Feb. 15, 1955, Vitamin composition, A. Bavley, E. Timrech (to Charles Pfizer and Co., Inc.). [117] U.S. Pat. 2,827,452 Mar. 18, 1978, stabilization of materials, H. Schlenk, D.M. Sand, J.A. Tillotson (to University of Minnesota). [118] U.S. Pat. 3,067,104 Dec. 4, 1962, stable fat-soluble vitamin compositions, M. Hochberg, C. Ely (to Nopco Chemical Co.). [119] U.S. Pat. 3,143,475, Aug. 4, 1964, vitamin-containing gelatin beedlets and the process of preparing them, A. Koff, R.F. Widmer (to Hoffmann-LaRoche, Inc.).
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7 The Vitamin D Receptor: Biochemical, Molecular, Biological, and Genomic Era Investigations J. Wesley Pike, Mark B. Meyer, Seong Min Lee Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA
The idea that vitamin D might function as a steroidlike hormone emerged in 1968 [7] and actually predated the documented discovery of 1,25(OH)2D3, the active hormonal form of vitamin D3 [8e10]. The lipophilic nature of the small vitamin D molecule and its capacity to localize in target tissues provided an initial basis for this hypothesis. This idea was also supported by the exquisite and highly complex nature of the regulation of 1,25(OH)2D3 synthesis by the renal enzyme 25hydroxyvitamin D3 1a-hydroxylase (CYP27B1) (see Chapter 3) [11]. Seminal to the concept, however, was the discovery of a “binding protein” in specific target tissues that appeared to mediate nuclear localization of what eventually was determined to be the active vitamin D hormone, 1,25(OH)2D3 [12]. Further characterization of this macromolecule over the years as well as its eventual cloning in 1987 [13] provided final confirmation that 1,25(OH)2D3 was indeed a steroid-like hormone. As indicated above, this molecular biologic milestone ushered in a new era of research aimed at understanding molecular features of the mechanism(s) central to the ability of 1,25(OH)2D3 to regulate gene expression. Importantly, however, this era of biochemical and molecular biologic research has given way recently to even more novel genomic approaches that promise to both revise and expand our understanding of gene transcription in general and to define the integral role of the VDR in many of these processes more specifically. In this chapter, we provide a historical overview as well as a contemporary summary of the VDR’s central role in mediating the actions of the vitamin D hormone, which is summarized in the cartoon in Figure 7.1. We
INTRODUCTION Our understanding of the mechanisms of action of steroid hormones has advanced significantly over the past several decades [1e6]. Indeed, it is now well known that the sex and adrenal steroids, thyroid hormone, retinoic acid (RA), and 1,25-dihydroxyvitamin D3 (1,25 (OH)2D3) as well as a number of metabolic intermediates produced within cells all bind and activate specific intracellular receptors. These transcription factors function within the nucleus, in turn, to regulated gene expression. Virtually all of the genes that encode nuclear receptors are now cloned and identified, revealing that each is part of a larger gene family with highly related structural similarity. Importantly, the cloning of the nuclear receptor family of genes has provided an important and unique entre´e into new avenues of research that have and will lead to not only important new insights into the structures of the proteins themselves, but also to a more complete understanding of how these receptors function to regulate gene expression. Perhaps as important, the discovery of new receptor entities resulted in delineation of entirely new biological systems, as exemplified by the unique tissue activities of receptors such as retinoid x receptor (RXR), peroxisome proliferator activator receptor (PPAR), liver x receptor (LXR) and farnesoid x receptor (FXR), to name just a few. Physiologically, the impact of the nuclear receptor family in general is wide ranging, although perhaps not unexpected given the ability of many of the members of this family to mediate in a systemic fashion diverse endocrine systems.
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10007-1
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7. THE VITAMIN D RECEPTOR: BIOCHEMICAL, MOLECULAR, BIOLOGICAL, AND GENOMIC ERA INVESTIGATIONS
Calcium /Phosphate Homeostasis
C N
Vit D3 Cyp2R1
Bile Acids
Bile Acids
RXR
1,25(OH)2D3
1,25(OH)2D3
VDR
25OHD3
Cyp27b1
R V
RNA
– –
+ Functional Responses
+ Cyp24a1
Bile Acid Metabolism
briefly describe the discovery of the receptor, summarize its biochemical characteristics and comment on several of the biological processes with which the receptor is involved. We also provide a summary of the VDR’s structural domains and describe how these components of the receptor integrate various processes through which gene expression is regulated. The results of recent analyses which demonstrate new principles of transcriptional control at both genome-wide and epigenomic levels are also considered. Finally, we describe the structural organization of the human VDR chromosomal gene and, using new genomic approaches just referred to, provide new insight into how the VDR gene is regulated at the molecular level. The reader is referred to the many additional chapters in various sections of this book for more depth in various aspects of VDR biology including the protein’s biochemistry, structure, and biological function.
VITAMIN D BIOLOGY The fundamental actions of 1,25(OH)2D3 are to maintain calcium and phosphorous homeostasis in vertebrate organisms [14]. This activity is achieved through direct and highly orchestrated actions of the hormone on the intestinal tract, kidney, and bone and through feedback inhibition of PTH production at the parathyroid glands and through the induction of FGF23 from bone cells to control blood levels of calcium and phosphate, respectively. In intestine and kidney, the transepithelial transport of calcium is currently known to involve the apical calcium ion channel proteins TRPV5 and TRPV6, soluble intracellular components such as the calbindins (D9K and D28K), and both the basolateral ATPase-driven calcium pump PMCA1b and the sodium calcium exchanger NCX1 (see also Chapters 19 and 20 and references therein). Various forms are differentially
FIGURE 7.1 Model for the molecular mechanism of action of the vitamin D hormone. 1,25(OH)2D3 is synthesized via renal CYP27B1, enters the cell and interacts with the vitamin D receptor (VDR). Activation by this ligand or by non-vitamin-D ligands such as bile acid metabolites leads to binding of the VDR (V) and its heterodimer partner RXR (R) to DNA sequences that enable the regulation of expression of linked genes. Linked genes include those involved in the regulation of the VDR activation pathway (Cyp24a1, Cyp27b1 and VDR), genes involved in calcium and phosphorus homeostasis and genes involved in the cell-specific regulation of proliferation, differentiation, and phenotype.
regulated in the intestine or kidney by the vitamin D hormone. Phosphate transport, on the other hand, is regulated by a series of transporters including NaPi2a-c. These isoforms are differentially expressed in the kidney but also in intestine and bone cells and can also be regulated by 1,25(OH)2D3. In bone cells, the actions of 1,25 (OH)2D3 are both extensive and complex (see Chapters 16, 17 and 21 and references therein). In osteoblasts, the vitamin D hormone is involved in both the formation of osteoid matrix and in mineralization. 1,25(OH)2D3 also regulates the production of RANKL and OPG from these cells (see Chapter 18) [15,16]. RANKL is a TNFa-like factor that couples bone formation to bone resorption by promoting the formation, activation, and survival of the bone-resorbing osteoclasts. OPG represents a soluble decoy receptor that inhibits RANKL activity. Via these and other activities, vitamin D thus plays a significant role in normal adult bone remodeling. Vitamin D also manifests actions in both embedded osteocytes and periosteal bone-lining cells. A primary target of vitamin D hormone activity in the osteocyte appears to be FGF23, one of the peptide factors involved in monitoring and maintaining phosphate levels in the blood [17]. Each of these biological topic areas is covered extensively in other chapters within this volume and therefore will not be discussed in detail here. However, the central feature of response to 1,25(OH)2D3 in each of these tissues is the presence of the VDR. 1,25(OH)2D3 is also a regulator of cellular proliferation and differentiation, activities not unlike those manifested by many of the steroid hormones [18]. These growth-regulating actions of 1,25(OH)2D3, considered in more detail in Chapter 84 and others, highlight both important physiologic activities of the hormone as well as potential therapeutic roles for both the hormone and for synthetic vitamin D analogs in the treatment of cancer, regulation of the immune system and autoimmune disease [19e22] and in cardiovascular disease [23].
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These cellular growth-regulating activities of 1,25 (OH)2D3 are also apparent in the skin [24]. Not surprisingly, therefore, both the antiproliferative and antiinflammatory effects of vitamin D are being exploited actively in the treatment of psoriasis [25]. The physiologic as well as potential therapeutic activities of the hormone in each of these areas are considered in much greater detail in individual chapters on these topics in this volume and the reader is referred to those chapters as well. As with tissues that control mineral homeostasis, the underlying key feature of vitamin D response in each of these “nontraditional” target tissues is also the presence of the VDR. Accordingly, the purpose of this chapter is to document what is known of the actions of the VDR and how the protein functions to regulate these numerous biological processes.
DISCOVERY OF THE VITAMIN D RECEPTOR While preliminary evidence suggested the possibility of a “receptor” for vitamin D [12], it was the studies by Brumbaugh and Haussler [26] and Lawson and Wilson [27] in 1974 which firmly established the presence of a protein macromolecule in the chromatin fraction of the chick intestine that was capable of mediating the preferential nuclear uptake of active vitamin D. Subsequent studies by Brumbaugh and Haussler in 1975 [28] demonstrated that this putative receptor was indeed capable of binding 1,25(OH)2D3 with high affinity and also associated with an intestinal chromatin fraction in a hormonesensitive fashion, both required characteristics of a legitimate nuclear receptor [26]. These findings established the presence of a receptor for vitamin D and provided the impetus for further exploration into the protein’s properties, distribution, structure, and function. We refer to research on the VDR that occurred prior to its cloning as belonging to the Biochemical Era; the Molecular Biological Era closely followed the cloning of the VDR structural gene. The Genomic Era, as will be discussed below, has recently emerged due largely to the sequencing of mouse, rat, and human genomes and to the development of unique techniques capable of defining VDR action on genome-wide scales. Clearly, the research conducted during these periods of time overlap.
VDR RESEARCH: THE BIOCHEMICAL ERA Tissue Distribution of the VDR The VDR was first discovered in extracts prepared from chicken intestine, perhaps as a result of the
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considerable role the chicken played as a model system in delineating the function of vitamin D in calcium and phosphorus homeostasis. Despite this, the known actions of vitamin D extended well beyond those in the intestine as indicated earlier, and were also not limited to the avian species. Thus, additional target tissues for vitamin D included kidney, bone, and the parathyroid glands cells and in both avian as well as mammalian species [29e34]. The discovery of receptors in these and other tissues was facilitated by tritium-labeled 1,25(OH)2D3, whose availability with increasing radioisotopic specific activity provided the enhanced sensitivity necessary to detect VDR levels in tissues where the protein was less abundant. High specific radioactivity 1,25(OH)2D3 was a major determinant in the discovery and subsequent characterization of the VDR. Surprisingly, this technique is rarely utilized currently in research, which now favors molecular techniques of RNA and western blot analysis. 1,25(OH)2D3 with very high specific radioactivity enabled detection of VDR in many unexpected target tissues as well as in cultured cell lines. The search for receptors in these tissue and cell sources was prompted by emerging evidence that the biological effects of 1,25(OH)2D3 extended beyond that of calcium and phosphorus homeostasis to include a role in cellular proliferation, differentiation and function as considered above [35,36]. Accordingly, VDRs were discovered in tissues such as pancreas [30,37], placenta [30], pituitary [30,38], ovary [39], testis [40], mammary gland [41e43], and heart [44]. The VDR was also discovered through tritiated ligand-binding assays in a number of cultured cell lines. Since the receptor could be up-regulated as a result of cell culture propagation, transformation, or other events, however, the presence of the VDR in these lines was not always predictive of their presence in the tissues from which they were derived. Thus, although receptors are clearly present in certain hematopoietic cells, a variety of epithelial cell types and cells of mesenchymal origin such as fibroblasts, myoblasts, chondrocytes, and osteoblasts, and have been identified in many of the tissues from which these cells are derived, the VDR has not been identified in the liver or muscle despite apparent biological activities in these latter tissues. It is assumed that the activities of vitamin D in these tissues are indirect. Of a more fundamental nature, receptors were found in a vast series of cell lines of tumorigenic origin [45e50]. A partial listing of vitamin D target tissues and cells is documented in Table 7.1. While the analysis of tissue extracts led to the discovery of many VDR-containing sites, it was the use of scintillant-enhanced autoradiography employed early on by Zile et al. [51] and Jones and Haussler [52]
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System
Tissue
Gastrointestinal
Esophagus, stomach, small intestine, large intestine, colon
Renal
Kidney, urethra
Cardiovascular
Cardiac muscle
Endocrine
Parathyroid gland, thyroid, adrenal, pituitary
Exocrine
Parotid gland, sebaceous gland
boundaries of both the mouse and human VDR genes, thereby enabling the development of large bacterial artificial chromosome (BAC) transgenes expressing uniquely tagged functional VDR proteins in a tissuespecific manner identical to that observed for the endogenous gene (discussed below) [59,60]. Linking VDR expression in these transgenes to novel reporters will permit confirmation of existing sites of VDR expression, define new sites of action, and enable expanded studies of how the VDR gene is regulated at the molecular level in vivo.
Reproductive
Testis, ovary, placenta, uterus, endometrium, yolk sac, avian chorioallantoic membrane, avian shell gland
Properties and Organization of the VDR
TABLE 7.1
Cellular and Tissue Distribution of the VDR
Immune
Thymus, bone marrow, B cells, T cells
Respiratory
Lung alveolar cells
Musculoskeletal
Bone osteoblasts and osteocytes, cartilage chondrocytes
Epidermis/appendage
Skin, breast, hair follicles
Central nervous system Brain neurons Connective tissue
Fibroblasts, stroma
and later by Stumpf et al. [53,54] that led to a more thorough appreciation of the wide cellular distribution of vitamin D target tissues. These studies revealed such targets as the duodenum, ileum, and jejunum of the rat intestine, and more novel target tissues such as stomach, kidney, skin, pituitary, specific neurons in the brain, and numerous additional sites as well; consistent with early ligand-binding studies, no activity was observed in skeletal muscle or in liver using this technique. It is important to note, however, that the detection of tritiated vitamin D ligands in cells does not prove that they are indeed direct targets, because this technique does not provide a direct measure of the VDR. Thus, it was the development of an antibody to the VDR some years later that provided definitive proof of the presence of VDR in many of these novel tissues [41,42,55e57]. The use of immunohistochemistry is utilized extensively by modern cell biologists, and when used with appropriate controls, constitutes a powerful method for identifying the presence of VDR in tissues and specific cell types. Currently, the most frequently used methods to assess the presence of the VDR are northern blot [13], in situ hybridization [58], and more routinely RT-PCR analyses, techniques that emerged following the cloning of the VDR in 1987 (discussed below). Frequently ignored with these methodologies, however, is the fact that the presence of an RNA transcript for a gene does not mean, a priori, that the protein is actually present in this tissue. Interestingly, recent studies have now defined the functional
The VDR is expressed in low concentrations in target tissues and cultured cells, a property consistent with its role as a potent transcription regulatory molecule [61,62]. The level of receptor expression ranges from a few copies of the VDR/cell to 25 000 copies/cell. It might be intuitive to suggest that receptor concentration represents the primary determinant of cellular response to 1,25(OH)2D3. A wide variety of additional factors also play a significant role, however, including posttranslational modifications of the VDR itself, the presence and activities of vitamin D metabolite-binding proteins such as vitamin-D-binding protein (DBP) (see Chapter 5), vitamin-D-degrading enzymes such as CYP24A1 (see Chapter 3), vitamin D response element (VDRE)-binding proteins (VDRE-BPs), and intracellular vitamin-D-binding proteins (IDBPs) (see Chapter 14), and finally, a variety of comodulators that act downstream of the VDR but which participate directly in the regulation of gene expression (see Chapter 10). These cell-specific contributors to the activity of 1,25(OH)2D3 in cells may be even more significant than that contributed by receptor concentration itself [63,64]. Numerous physical and functional properties of the VDR were defined following the protein’s discovery, several of which are documented in Table 7.2. The most important characteristic of the VDR, however, was its capacity to bind 1,25(OH)2D3 with both high affinity and selectivity [65e69]. Accordingly, the VDR displayed an equilibrium dissociation constant (KD) of approximately 10e10 M for the natural ligand 1,25(OH)2D3 and bound this ligand’s precursors as well as less active metabolites with substantially lower affinities [67,68]. The contribution of both the 25-hydroxyl and the 1a-hydroxyl groups on the 1,25 (OH)2D3 molecule to specific, high affinity binding to VDR has been studied extensively [68]. The recent structural definition of the VDR via crystallography of the protein’s ligand-binding domain (LBD) now provides fine detail into the role of individual functional groups on the ligand and its interaction with specific amino acids within the receptor (see below
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TABLE 7.2 Biochemical Properties of the VDR and its RNA Transcript VDR PROTEIN Molecular weight: 50 kDa (human) to 60 kDa (chicken) Sedimentation coefficient (S): 3.2 (human, rat) to 3.7 (chicken) Equilibrium dissociation constant: 1e2 10e10 M Isoelectric point: 6.2 Phosphoprotein: Serine phosphorylated at residue 208 (human) in response to 1,25(OH)2D3 Lability: proteolytically sensitive Tissue abundance: 10 to 1000 fmoles of VDR/mg protein hVDR MRNA 4.8 kb (human), 3.0 kb (chicken)
and additional chapters in this section on Mechanism of Action in this volume) [70e72]. Interestingly, estimates of the equilibrium dissociation constant between the VDR and its hormonal ligand, and both on and off rates of the ligand, have always been determined using soluble forms of the VDR. It is possible that these measurements are not reflective of the “true” interaction of both ligand and receptor under conditions where the VDR is bound directly to its sites of action on chromatin and has formed a complex with coregulatory proteins that mediate the receptor’s downstream actions. An important discovery in the late 1970s was the observation that the VDR could bind to DNA, an anticipated property of a protein that activates transcription [61]. While a more thorough understanding of VDR DNA binding emerged following the identification of specific DNA-binding sites (vitamin D response elements or VDREs) within gene promoters, the finding that the VDR bound to nonspecific DNA set the stage for ensuing studies aimed at understanding the structural organization and function of the VDR. Interestingly, while the VDR’s “affinity” for nonspecific DNA quantitatively increased following ligand binding, the receptor also exhibited an obvious “affinity” for DNA in the absence of 1,25(OH)2D3 as well [69,73]. This property suggests that the structure of the VDR or perhaps the composition of the active receptor was transformed in the presence of the hormonal ligand even at the level of nonspecific DNA. Interestingly, VDRs derived from patients with hereditary 1,25(OH)2D3 resistant rickets (HVDRR) (see Chapter 67) that contain DNA-binding domain mutations which prevent specific VDRE binding still manifest low affinity for nonspecific DNA [74]. This suggests that the ligand-independent binding to nonspecific DNA by the VDR noted in these
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early analyses was likely due to the cationic nature of the DNA itself (extensive phosphate residues) and not to an inherent DNA-binding property of the VDR. Recent X-ray diffraction studies to be described later in this chapter as well as in Chapters 8 and 9 define the nature of the effects of ligand on the VDR and its DNA-binding capabilities at the atomic level [70e72]. The DNA-binding ability of the VDR was exploited strongly in the protein’s purification from the chicken intestine [61,75] and later from porcine intestine [76], which led ultimately to the development of monoclonal antibodies capable of recognizing the VDR from a wide variety of species. These reagents proved essential in further immunological characterization of the VDR, provided alternative receptor isolation methods, and finally provided the means whereby the VDR gene was cloned. Using antibodies, western blot analysis revealed for the first time that the precise molecular mass of VDR varied from species to species: approximately 60 kDa in the chicken [77] and 50 kDa in humans [78]. These relative sizes were confirmed following the molecular cloning of their individual genes. Immunological analyses also revealed that the chicken VDR was comprised of A and B isoforms but that the mammalian forms were largely of a single species [77]. The two forms of the chicken VDR are now known to arise from the existence of alternative translational start sites in the mRNA, although the functional consequence of two such proteins with differing amino-terminal extensions remains unknown [79]. More recently, studies suggest that the human VDR may be comprised of as many as three forms, two of which are due to the presence or absence of an ATG to ACG conversion at the first codon, and the third due to the possible use of an upstream in frame ATG from an alternative gene promoter (see also Chapter 65) [80,81]. This latter species has yet to be adequately characterized.
VDR RESEARCH: THE MOLECULAR BIOLOGICAL ERA Cloning of the Structural Gene for the VDR Biochemical purification of steroid receptors in the early 1980s led to the development of immunological probes that facilitated not only further characterization of the proteins, but the cloning of their structural genes as well. As a result, receptor cDNAs for virtually all the known steroid, thyroid, and vitamin hormones were recovered during the latter half of the 1980s [2]. Importantly, sequence similarities within a specific region rapidly identified as the DNA-binding domain of this family of receptors suggested the possibility that related
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transcripts might be recovered through low-stringency hybridization screening [6]. Accordingly, numerous additional cDNAs for unknown nuclear receptors were rapidly identified as well. Extensive research during the past two decades has led not only to the discovery of novel ligands for many of these receptors but to entirely new biological systems with which they are involved. Examples include the discovery of RXR and its ligand 9-cis RA [82], PPARg, and one of its ligands prostaglandin J2 [83], FXR and its ligand farnesol [84], liver LXR, and its oxysterol ligands [85e87] and several others as well. Surprisingly, most of these ligands do not represent high-affinity endocrine ligands, but rather local, low-affinity, metabolic ligands of intracellular origin, leading to the concept that some nuclear receptors function to monitor and are activated by intracellular levels of key nutrient components [88]. The development of the anti-VDR monoclonal antibody 9A7 enabled McDonnell et al. to recover a cDNA segment of the chicken VDR DNA-binding domain [89]. This partial cDNA was used by Baker et al. [90] to recover a full-length VDR cDNA transcript from human cell sources. The rat intestinal VDR was cloned shortly thereafter by Burmester et al. [91]. Sequences of the VDR were subsequently reported from mouse [92], Japanese quail [79], Xenopus [93] and lamprey [94] and are now present in all relevant genomic databases [95,96]. The cloning of the VDRs from various sources and species represents a milestone in the vitamin D field. Indeed, it provided the first direct evidence of a structural relationship between the VDR and other bona fide members of the steroid receptor family of genes [97]. It also enabled entry into the Molecular Biologic Era wherein the use of recombinant DNA methodologies could be employed to facilitate studies of both VDR structure and function and to explore the underlying molecular basis for diseases such as HVDRR that were believed to be due to defects in the VDR itself. The VDR is highly conserved across tissue sources and species. Interestingly, as alluded to earlier, two of three VDR isoforms identified are present in the human population [98] arising from a frequent single base pair transition that occurs in the first initiation site reported by Baker et al. [90] (ATG to ACG). The result of this polymorphism in the human gene, which can be detected through the presence or absence of a FokI restriction site, is the production of a smaller VDR of 424 amino acids which occurs in homozygous form in approximately 37% of the human population and in heterozygous form (one allele containing both initiation sites and the second containing only the downstream site) in approximately 48% of the population [99]. Whether the two proteins exhibit different biochemical properties and/or different functions remains an open
question, although it does appear that the smaller form may be more active [99e101]. This has been suggested to occur via a tighter contact with the transcription factor TFIIB that control the formation and activity of the initiation complex (see Chapter 8) [99]. Association studies indicate that this polymorphism may be linked to bone mineral density [100] as well as to a wide variety of additional disease states, although none of these data are particularly robust [102e104] (Chapter 68).
VDR as a Member of the Intracellular Receptor Gene Family The cloning of glucocorticoid [105] and estrogen [106,107] receptors in 1985 and 1986 were the first in a series of successful efforts to clone the intracellular receptor genes. Forty-nine members of this intracellular receptor gene family [6] have been identified in mice and 48 in humans, making it one of the largest transcription factor families currently known. Interestingly, over 284 members of this family are found in C. elegans where it appears that they are expressed and active [96]. Only several ligands for these molecules have been identified presently, however [108,109]. It is noteworthy that the cloning of this family of genes was so successful that publication of the sequence of both the mouse and human genomes in 2002 did not reveal a single unidentified functional nuclear receptor gene [95,110]. The evolutionary success of this family of transcription factors is highlighted both by its abundance as well as by its involvement in an incredibly wide range of biological processes that include growth, development, differentiation, and adult homeostasis. The reader is referred to many other reviews on the steroid receptor family for details [3e5,111,112] as well as to more extensive summaries in Chapters 13e16.
Structural Domains of the VDR Overview The cloning of the ER, and the GR shortly thereafter, revealed that these proteins were comprised of distinct regions or domains, leading to their designation as A, B, C, D, E, and F [106]. As illustrated in Figure 7.2A, the highly conserved DNA-binding domain is designated the C domain and provides a central focus for the remainder of the molecule. Accordingly, the nonconserved A/B domain comprises all residues of the amino terminal to the DNA-binding domain and the D domain or hinge region comprises residues lying downstream of C. The carboxyterminal region containing the ligandbinding domain is termed the E or E/F domain. Three stretches of sequence similarity exist in the E domain for virtually all members of the nuclear receptor gene
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A
NR A/B 1 24
B
C
D 89
E/F 232
2
23
C
427
hVDR
E/F Domain
2 9 1 27 31 34
6 3 41 42
VDR
427 0
27
0 5 7 31 35 37
8 5 44 45
TRβ
456 4
1 6 8 27 31 33
8 5 40 41
21
1 8 0 76 80 83
7 4 90 91
49
9 5 7 38 43 45
8 5 53 54
23
462
RARα 7
934
PR 3
595
ER 40
22
6 aa
FIGURE 7.2 Functional domain structure of the nuclear receptor superfamily. (A) The nuclear receptors (NR) are separated into five regions designated A/B, C, D, and E/F. (B) Residue boundaries of corresponding regions within the human VDR. (C) Three regions of sequence similarity and residue boundaries within the E/F domain of several nuclear receptors. Shown in addition to VDR are thyroid receptor b (TRb), retinoid receptor a (RARa), progesterone receptor (PR), and estrogen receptor (ER). Functions associated with the domains include transactivation (A/B), DNA binding (C), flexible hinge (D), and dimerization, ligand binding, transactivation, and repression (E/F) (see text for details).
family. The F domain exhibits extensive variability, much like that of the A/B domain, and is not conserved. The domain structure of mammalian VDRs is depicted in Figure 7.2B. As can be seen, the A/B domain is very short relative to other members of the nuclear receptor family, representing one of the most abbreviated of the entire gene family. The C region comprises the highly conserved DNA binding domain of the VDR [113]. The D domain is a highly flexible region within the VDR and links both its DNA-binding and hormone-binding components [113]. This region is not conserved among VDRs from other species or among other members of the steroid receptor family [90,91] (see Fig. 7.2C). The majority of this region is, however, essential for intact hormone binding, a feature unlike that of most other steroid receptors. The hinge region may be more complex, however, as it contains an additional segment that corresponds to a specific exon in the chromosomal gene [114]. Interestingly, this additional segment can be removed without compromising 1,25(OH)2D3 binding or transcriptional function [70,71]. Finally, the E/F domain contains the 1,25 (OH)2D3 binding region of the VDR [113] and, equally
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important, serves as a highly complex proteineprotein interface for a series of additional interacting proteins that are integral to receptor activity [115e117]. This region has been termed activation function 2 or AF-2. The ability of the VDR to bind both DNA and additional transcription factors highlights its central role in nucleating the formation of transcriptionally active complexes at specific sites of gene regulation. Since the general role of all members of the receptor gene family is similar, it is not surprising that the E region is moderately conserved across the entire gene family and, as depicted in Figure 7.2C, contains several sub-domains that are even more highly conserved. The F domain is absent in the VDR. Surprisingly, the organization of key features of the steroid receptors in general and the VDR in particular were determined prior to their cloning. Indeed, it was Gustafsson and colleagues [118e122] who demonstrated that the GR was comprised of distinct ligand-binding and DNA-binding domains; similar studies were conducted with the ER. In the case of the VDR, initial insights into the structural organization of this receptor emerged using techniques such as limited proteolytic digestion together with radiolabeled 1,25(OH)2D3 and immunologic probes. Initial studies by Pike [123] and Allegretto and coworkers [124,125] suggested that the VDR was comprised of at least two functional domains (ligandbinding and DNA-binding domains) that were separated by a proteolytically sensitive “hinge.” Additional preliminary evidence indicated that the DNA-binding domain was located by the amino terminal to the hormonebinding domain [126]. As described in the next section, these rudimentary insights into the overall structural organization of the VDR were proven correct via the subsequent cloning of the receptor’s structural gene. DNA-Binding Domain The DNA-binding domain of the VDR is encoded in domain C. This domain consists of two similar modules, each consisting of a zinc-coordinated finger structure. The zinc atoms are individually coordinated in a tetrahedral fashion through four highly conserved cysteine residues that serve to stabilize the finger structure itself. Importantly, the finger modules are structurally unrelated to the zinc fingers found in TFIIIA wherein zinc atoms are coordinated through two cysteine and two histidine residues [127,128]. Surprisingly, although the two zinc modules of the VDR appear highly related to each other structurally, they are not equivalent topologically because the chirality of the residues that coordinate the zinc atoms in each module is different [128]. The function of each of these modules in DNA binding is also different. Whereas the amino-terminal module (first zinc finger) directs specific DNA binding in the major groove of the DNA-binding site, the carboxy-terminal
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module (second zinc finger) serves as a dimerization interface for interaction with partner proteins [129,130] (discussed below). It seems likely that the two exons that encode these modules (see below) evolved separately, although it is possible that each was derived through duplication from a common ancestral gene and then diverged later. The three-dimensional structure of the DNA-binding domain of several of the receptors has been determined through both nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. The salient features of the finger structures are the presence of carboxy-terminal a-helices essential to interaction with DNA. Thus, our understanding of the structural organization of these modules as well as the mechanisms by which they function to interact with DNA and other proteins is considerable [131e135]. Ligand-Binding Domain The E region of the VDR represents a multifunctional domain that exerts absolute regulatory control over the DNA-binding as well as transcription-modifying properties of the VDR. Historically, this region and comparable regions found in other receptors were explored for both common structural features as well as individual functional properties. As indicated in the overview in this section, several regions of homology were identified. Since cognate ligands differ substantially, it appeared likely that these regions of similarity represented motifs integral to the receptors’ role in transactivation. Closer scrutiny revealed the presence of multiple heptad repeats that were located in the ligand-binding domain (LBD) of most members of the gene family including that of the VDR [136]. Despite this and many other important biochemical and molecular biologic discoveries, it is clear that the greatest insight into LBD structure emerged as a result of X-ray crystallographic studies that were initiated by Moras and colleagues with the retinoid receptors RARg and RXRa [137,138]. As of this date, the three-dimensional structures of the LBDs of virtually all of the key members of the nuclear receptor family including VDR have been determined [70e72,139e143]. With the exception of RXRa, the structures of the LBDs from most of the receptors have been solved in the presence of their cognate ligands. Thus, the bulk of our information of LBD structure in the absence of hormone derives from the apo structure of RXRa. RXRs serve a rather unique role as unliganded heterodimer partners for many members of the nuclear receptor family [5,112]. Accordingly, it seems unlikely that the LBD of this receptor will faithfully mimic that of the other apo-receptor forms. Nevertheless, the structure has revealed the presence of 12 a-helices (H1eH12) arranged in an antiparallel a-helical sandwich. Perhaps most interesting is the location of H12, which projects
away from the body of the RXRa LBD [137]. As will be discussed below and in other chapters in more detail, this projection is in contrast to the location of H12 in virtually all the liganded or holo-receptor which shows that H12 is packed tightly against the body of the LBD [70e72,138e143]. This presumptive change in nuclear receptor structure in the AF-2 region in response to hormone, together with many other changes evident in the overall conformation of the receptor, appears integral to its ability to attract DNA-binding partners and to recruit protein complexes essential for transcriptional activation. It is important to note here, however, that the idea that ligand-induced significant changes in receptor structure emerged many years earlier from both receptor stability and proteolytic digestion studies [125,126 and references therein]. The latter analyses have been refined recently through the use of metabolically labeled nuclear receptors derived from in vitro translated receptor cDNAs [144] and is described in detail in Chapters 18 and 83. The reader is referred to the many primary publications that report the threedimensional structures of numerous members of the steroid receptor superfamily as documented above. Very recently, the crystal structure of the ligandbound full-length PPARg/RXRa heterodimer bound to its cognate DNA response element has been reported by Rastinejad and colleagues [145]. This complex structure provides a novel view of the important contacts that occur between a nuclear receptor and its RXR partner as well as how this dimer binds to specific DNA. Indeed, the structure reveals that in addition to the anticipated contacts that are made between the PPARg DNAbinding domain and its cognate DNA-binding sites, an unexpected contact between the PPARg ligand-binding domain and DNA sequences outside the canonical response element is also made. This structure provides the first glimpse of how an intact nuclear receptor dimer interacts on DNA; the success of this endeavor is likely to invigorate efforts to define the structural features of other nuclear receptors on DNA as well. The reader is referred to the above reference for additional fascinating detail. As with many of the steroid receptors, the threedimensional structure of the LBD of the VDR has also been determined [70e72]. As this is the subject of Chapter 9, only a brief summary of the structure of the VDR will be described here. Rochel et al. [70] first crystallized the human VDR LBD in complex with 1,25(OH)2D3 as well as other ligands [71,146]. A recent report by Vanhooke and colleagues has confirmed and extended this important work using the rat VDR [72]. Essential to the successful crystallography effort of both groups was the removal of a portion of the D region that prevented crystal formation. The removal of this segment did not appear to affect the transcriptional
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the ligand. Importantly, the recent elucidation of the wild-type VDR structure from the zebrafish has confirmed that the removal of the “insertion region” located in the hinge region is without consequence to the overall structure of the receptor [147,148]. It is important to note, however, that the reported absence of changes in both VDR structure and transcriptional activity as a result of the removal of this insertion region has been made using techniques that are unlikely to reflect subtleties in either receptor structure or activity. After all, the insertion in the human VDR contains what is suggested to be an important phosphorylation site (S208) that is known to impact VDR activity. Until the activity of this mutated receptor can be explored in vivo and its effects on multiple tissues examined in detail, a conclusion that the removal of the insertion has no biological effect seems premature.
activity of the VDR. The three-dimensional structure of the LBD of the rat VDR is depicted in Figure 7.3B and C. The human and rat VDR structures revealed the presence of 13 a-helices and three b-sheets that together form at its core a highly hydrophobic ligand-binding pocket. Surprisingly, the overall volume of this pocket is much greater than that seen for many of the other steroid receptors. As seen in Figure 7.3C, 1,25(OH)2D3 binds in this pocket in an extended configuration with the A ring in the b chair conformation and the 1a-OH group equatorial. The ligand is anchored by six hydrogen-bonding interactions, the 1a-OH group with Ser233 and Arg270, the 3b-OH group with Tyr143 and Ser274, and the 25-OH group with His301 and His393 (using the rat VDR sequence numbering). Several of the LBD helices, including H9 and H10, provide a binding surface for the VDR’s DNA-binding partner RXR (see below). Importantly, H12 is packed against the body of the LBD such that it seals the ligand-binding pocket and along with H3 and H4 creates the AF-2 hydrophobic cleft that serves as a docking site for transcriptional comodulators of VDR action (to be discussed below as well as in detail in Chapters 13e18). Several of the residues that comprise H12 make direct contact with
Activation Functions As indicated above, hormone binding leads to the creation of a functional AF-2. The core of this functional domain appears to be residues associated with H12 such as human residues 416e423 [149,150], although early mutagenesis studies indicated more extensive
DNA Binding
A
AF2
N
C 1
21
88
240
423
Hinge Region
B
Ligand Binding
C
1,25(OH)2D3
N-term H1
His 301
H9 Trp 282 H10 H4
H8 His 393
peptide
Ser 274
H5 H3
H7
Ser 233
H12 C-term
Tyr 143
H2
Arg 270
H6 H11
G218
M159
FIGURE 7.3 Structural organization of the rat VDR in complex with l,25(OH)2D3. (A) Schematic organization of the specific functional domains of the VDR. (B) Ribbon representation of the rat VDR LBD in complex with l,25(OH)2D3 and the DRIP205 coactivator peptide. (C) Key amino acid contacts identified between 1,25(OH)2D3 and the VDR as described by Vanhooke et al. [72].
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involvement of components located upstream in the E/F region [6]. Indeed, H3 and H4 are also involved. The hydrophobic cleft formed by these three helices provides an essential binding site for single proteins that mediate linkage to more complex sets of factors that include the p160 histone acetyltransferases (HAT) coactivator complexes [151] and the D-receptor interacting protein (DRIP205; MED-1) complex (Mediator-D complex) [152,153], both of which are discussed below and in detail in Chapter 10. Proteins such as the p160 coactivators SRC-1, SRC-2, and SRC-3 and MED-1 interact directly within the ligand-induced H12 cleft via a short helical LxxLL motif that is common to these comodulators [154]. Indeed, studies have confirmed that LxxLL-containing peptides that bind to either the VDR or RXR function as dominant negative regulators of VDR action [155]. Vanhooke et al. [72] also recently provided three-dimensional structural insights into the interaction between the rat VDR and an LxxLL motif through cocrystallization of the rat VDR LBD with an LxxLL peptide derived from the second coactivator motif of MED-1, the MED-D component that interacts with the VDR. This group observed that the LxxLL peptide folds into a short a-helix that binds to the surface groove formed by H3, H4, H5, and H12 (see Figure 7.3B). The LxxLL interaction buries some 500 square angstroms of VDR surface area and involves direct contact with residues of VDR H3, H4, and H12 including Pro412, Leu413, and Glu416 of H12. These interactions form a “charge clamp” that has been described previously for both PPARg and its coactivator SRC-1 [142]. The side chains of many of the LxxLL motifs are buried within the pocket and surrounded by hydrophobic residues. Nonspecific amino acids project directly into the solvent and away from the binding cavity. These findings support a strong structural basis for the interaction of the VDR with at least one coactivator, albeit a small segment of the native protein, and may lead to new insights with regard to VDR-coactivator interaction and stability. Ligand Binding and Differential Activation Since 1,25(OH)2D3 acts as a small molecular switch to activate the VDR, it should not be surprising that the binding of ligand would induce sweeping conformational changes in the VDR LBD. Changes in conformation are also likely to be sensitive to the overall structure of the ligand, supporting the hypothesis that synthetic analogs of natural hormonal ligands may be capable of inducing unique changes in receptors, thereby altering their biological function. Indeed, synthetic ligands of the ER as well as other nuclear receptors have been identified that produce both unique receptor conformations as identified by their crystal structures [140,156,157] and that manifest surprising
tissue selectivity [158,159]. Numerous vitamin D analogs have also been created. Interestingly, many of these compounds manifest unusual actions both in vivo and in vitro (see below) as compared to 1,25(OH)2D3 [71,72]. Perhaps the most interesting and therapeutically useful is the finding that some display a reduced capacity to induce hypercalciuria and hypercalcemia in vivo, a classic feature of 1,25(OH)2D3. The underlying mechanism for this lack of calcemic activity is unclear. It is possible, however, that these analogs are simply less active compounds since most of them also exhibit reduced efficacy on other biological processes as well (see Chapter 74). What is clear is that none of these analogs has demonstrated an ability to induce a strikingly different conformation within the VDR that might account for its unusual actions. Protein crystal structures are oftentimes not entirely reflective of protein structures in solution, however. Thus, a final assessment of both the overall dynamic structure of the VDR and the potentially differential effects of synthetic ligands on this structure will have to await a VDR structure as determined by NMR or other novel methodologies that have emerged in recent years. An alternative explanation, however, is that the unusual activities of many of these synthetic analogs are derived from their differential pharmacokinetic and/or pharmacodynamic properties. Indeed, there is considerable evidence that at least several of the analogs examined in vivo interact differentially with serum-binding proteins or are differentially metabolized in a tissue-selective manner.
VDR Function Osteocalcin Regulation as a Model for VDR Regulated Transcription Osteocalcin (OC) is a relatively abundant noncollagenous protein found in bone. Genetic ablation of the OC gene in mice suggests that this osteoblast-specific protein may contribute to the density and structural integrity of bone [160] (see also Chapter 16). More recent studies have suggested that the decarboxylated form of OC, perhaps released in this modified form from bone during osteoclast-mediated resorption, may function as a unique skeletal endocrine hormone that plays a role in insulin release from the pancreas and is therefore involved in energy metabolism [161,162]. Despite the uncertainty surrounding this and other functions of OC, its expression at the transcriptional level is regulated by a number of cytokines, growth factors, and systemic hormones, including 1,25(OH)2D3 (163). This fact, together with the cloning of the human OC gene and its promoter in 1986 [164], provided the raw materials for an initial investigation of 1,25(OH)2D3 action on gene expression at the molecular level.
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Initial efforts focused on determining if 1,25(OH)2D3 could stimulate the activity of the human OC gene promoter linked to a reporter gene when introduced into bone cells via transfection [113,165,166], a traditional method of exploring the molecular mechanism of gene activation. The results suggested that 1,25(OH)2D3 strongly induced this gene promoter, indicating that the effects were direct [167]. Kerner et al. [165] localized the cis-acting element within the promoter subsequently to a region approximately 500 bp upstream of the transcriptional start site (TSS). This study and an additional one by Ozono et al. [168] precisely mapped the first vitamin D response element (VDRE) to a directly repeated hexanucleotide sequence separated by three basepairs. Subsequent studies by others using the rat OC gene promoter led to a similar identification [169e171]. These studies provided initial insights into DNA sequences capable of mediating vitamin-Dinducible activity within genes. In the ensuing years following the above studies, many genes were explored for their sensitivity to 1,25 (OH)2D3. Gene targets investigated include the mouse osteopontin (SPP1) (OPN) [172], mouse calbindin-D28K (CALB1) [173], rat calbindin D9K (S100g) [174], rat [175e177], mouse and human [178] 25-hydroxyvitamin D3 24-hydroxylase (CYP24A1) (two VDREs), human p21 (CDKN1A) [179] and a host of others. These sequences revealed that a "typical" VDRE was comprised of two hexanucleotide repeats of AGGTCA separated by a 3 bp spacer. Although the sequence of the "spacer" is not generally conserved, recent studies have suggested that these basepairs may influence receptor binding. Thus, the presence of a G in the third position of the spacer of a VDRE (adjacent to the second hexanucleotide) [180] is preferred by the VDR. Indeed, the results of new approaches to VDR gene activation, as discussed below, confirm this preference at the genome-wide level. Additional “atypical” VDRE configurations (see Chapter 11) have been identified as well. The validity of these elements, however, will require further study in the context of the genes in which they are found. The efforts to define VDREs were part of a larger effort by numerous investigators to learn more about steroid receptor response elements in general, both with regard to specificity as well as receptor selectivity [130,159]. These overall efforts led to our current understanding that nuclear receptor-binding sites can be categorized into three groups: palindromic half-sites that interact with homodimeric receptors for the sex steroids, directly repeated half-sites with variable spacing that interact with heterodimeric receptors for 1,25(OH)2D3, retinoic acid, thyroid hormone, and a number of other ligands, and single half-sites that mediate the actions of monomeric receptors such as NGF-IB [6]. A primary determinant of nuclear receptor selectivity for 1,25
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(OH)2D3, retinoic acid and the like is the length of the spacer itself. Thus, Evan and colleagues demonstrated that while RAR preferred sites separated by 2 or 5 bp, TR preferred sites separated by 4 bp and the VDR preferred half-sites separated by 3 bp. This feature was termed the “3e4e5” rule. The reader is referred elsewhere for a complete review of the nature of these DNA-binding sites [6]. 1,25(OH)2D3 also induces gene repression, an effect observed at a variety of genes including PTH, PTHrp, and CYP27B1. Importantly, the mechanisms through which repression occurs are highly variable. On the one hand, “negative VDREs” have been described, particularly as they relate to PTH and PTHrp [181e183]. On the other hand, Kato and colleagues have recently described a detailed mechanism for the suppression of CYP27B1 by 1,25(OH)2D3 [184e189]. The latter involves the ability of the VDR to interact directly with a prebound transcriptional activator termed VDIR. The VDR functions to suppress VDIR-activated expression of CYP27B1. Interestingly, suppression also involves an active epigenetic mechanism as well. The reader is referred to Chapter 12 of this volume for further details. It is fair to say, however, that our understanding of gene repression by the VDR and other nuclear receptors is currently quite rudimentary. VDR Binding to Specific DNA Involves RXR High-affinity binding of the VDR to DNA in vitro requires two regions, the DNA-binding domain and the LBD. This conclusion is drawn from numerous mutagenesis experiments that revealed that amino acid alterations in either the zinc finger modules [114,190e196] or in the VDR LBD [113,116,149,197] were capable of blocking DNA bind. Interestingly, the molecular basis for altered DNA binding in the two regions is different. In the first case, changes in the VDR DNA-binding domain prevent interaction directly with DNA. In the second, however, changes in LBD residues block the ability of the receptor to form the protein dimers that are essential for high-affinity VDRE binding [116,197]. The discovery that the VDR must form a dimer to bind DNA was suggested by the structural nature of VDREs themselves which are composed of two repeated half-sites. Surprisingly, however, it was found that the VDR bound to DNA not as a homodimer like steroid receptors but rather as a heterodimer as illustrated in Figure 7.1. Liao et al. [198] and Sone et al. [199e201] both observed that while VDR derived from mammalian cell extracts was fully capable of binding to DNA in vitro, receptor produced either by in vitro transcription/translation or through recombinant means in nonmammalian cells failed to produce DNA-bindingcompetent VDR. The inactivity of the VDR could be rescued, however, through the addition of general
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mammalian cell extract, suggesting the presence of a VDR-DNA-binding facilitator. This factor(s) was discovered in a variety of tissue and cell sources [200] and termed nuclear accessory factor (NAF); its presence was subsequently confirmed by others [202,203]. NAFs were also discovered for other nuclear receptors including those for thyroid hormone and retinoic acid. In 1991 and 1992, Yu et al. [204], Leid et al. [205], Zhang et al. [206], and Kliewer et al. [207] all discovered that RXR, a previously cloned member of the nuclear receptor family, was the likely NAF protein partner. Three isotypes of RXR (a, b, and g) are known and may be present in a given cell type, although the criterion through which the VDR, for example, utilizes a specific isotype is unknown. Also unclear is whether the use of different RXR partners can affect target gene selectivity or result in a different functional outcome. Considerable effort has gone into defining the nature and function of RXR-mediated DNA binding, and the reader is referred to the many reviews that deal with this complex issue [6,111]. Nevertheless, it is now clear that both VDR and RXR subunits are necessary for not only high-affinity DNA binding, but also for the activation process itself (see Chapter 8). Although there has been some disagreement with regard to domain requirements for dimerization with other receptors [208,209], more recent crystallographic studies define the key interacting domains within both the dominant receptor and RXR and suggest that the interactive surfaces of both partners include those of helices H9 and H10. While elucidation of the three-dimensional structure of VDR-RXR LBD heterodimer will be required to define precisely the interaction between VDR and RXR, the reader is referred to Chapter 9 for more details regarding VDR structure. Is RXR required for VDR binding at all specific sites on DNA in living cells as suggested by the above studies? Or more broadly, can RXR be found at all sites of VDR action on the genome? Unfortunately, these questions could not be answered during the Molecular Biologic Era because all cells contain RXR and thus the effects of its absence could not be ascertained at either the single gene level or at the genome-wide level, a scale that would be necessary to answer the broader question posed above. These questions have been answered, however, using techniques that became available at the beginning of the Genomic Era and will be addressed in a subsequent section of this chapter. Polarity of Binding The asymmetric nature of natural VDREs (direct repeats) coupled with the heteromeric nature of the receptor activation unit (VDR-RXR) indicates that the two receptor subunits must bind to the VDRE with a defined polarity. Studies by Jin and Pike [115] and
Freedman and coworkers [192] addressed this question in detail. Through the use of chimeric receptors and chimeric response elements, it is now clear that RXR binds to the upstream 50 half-element and the VDR binds to the downstream 30 half-element of VDREs oriented on the DNA sense strand. This organization is consistent with the relative polarity noted for both RXR-TR [209] and RXR-RAR heterodimers [210,211] bound to their respective response elements. The functional impact of this polarity remains unclear, however, because many response elements exhibit an opposite orientation relative to the TSS of target genes. Under these circumstances, the heterodimer is oriented in the opposite direction relative to the TSS with the VDR likely bound to the upstream segment and RXR to the downstream segment. Transactivation by the VDR and RXR Involves Coregulators Studies by McDonnell et al. [113] first demonstrated that the responsiveness of a transfected human OC promoter to 1,25(OH)2D3 in VDR-negative cell lines such as CV-1 or COS7 required co-introduction of VDR. This biological assay enabled an extensive examination of the domains of the VDR that were required for transactivation and revealed the not surprising crucial nature of both the DNA-binding and heterodimerization functions on transactivation. These investigators also revealed the critical role of an intact AF-2 function in the context of normal DNA-binding and heterodimer function for subsequent transactivation. Interestingly, this assay also established the inactive nature of many of the mutant VDRs that was identified in the human syndrome of HVDRR, considered in more detail in Chapter 65 [114,195,196]. Activation of the VDR involves a l,25(OH)2D3dependent conformational change within the LBD which as indicated above creates a functional AF-2 domain. The protein components that interact within the AF-2 region of the nuclear receptors in general include (i) the SWI/SNF complex that functions to remodel chromatin in an ATP-dependent manner [212,213], (ii) the p300/CBP coactivator complex [214,215] that contains nuclear receptor-interacting proteins SRC-1, SRC-2, and/or SRC-3 [151] that function to acetylate histones [216,217], (iii) Mediator-D complex that contains the nuclear receptor-interacting protein MED-1 that functions to facilitate entry of RNA polymerase II [152,153,218] and (iv) likely other [219] complexes as well. Interestingly, recent studies suggest that MED-1 may also participate in DNA looping [220]. The potential interactions of the SRC-1/CBP and the MED-1 complexes with the VDR/RXR heterodimer are illustrated in Figure 7.4. The exact role of each of these enzyme-containing complexes during
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SWI / SNF (Chromatin Remodeling) BAF180
BAF60
Histone Acetylation
BAF155
BAF53
CBP P300
BRG/hBRM B
SRC1 p160
P PCAF HAT H
VDR-RXR
ATP
VD DRE DR VDRE
Nucleosomes N
A
8 kb
77.016
Exon
Tnfsf11
5
Enhancers
Log2 Ratio
Log2 Ratio
VDR
RXR
Log2 Ratio
Log2 Ratio
C
TFIIE TFIIH T IH H TFIIB RNA poll TFIIA A TF TFIID TFIIF CTD C General Transcription Complex
FIGURE 7.4 Contemporary model for VDRand RXR-mediated gene regulation. The VDR and RXR bind as a heterodimer in response to 1,25(OH)2D3 to the regulatory region (VDRE) of a target gene. Transcriptionally active complexes that include the SRC/CBP/p300 HAT coactivator complex, the DRIP205/Mediator complex, or the SWI/SNF remodeling complex are subsequently recruited to facilitate changes in chromatin architecture and to enhance RNA polymerase II entry. All of the complexes may be needed to modulate gene expression.
Chr 14
(Mb) 77.000
B
Mediator Complex 13 130 80 Med6 150 230 M M Med7 05 5 100 205 1
3.6
77.032
43
2 In2a
77.048
77.064
77.080
77.096
77.112
77.128
77.140
1 (TSS) TSS
D1
D2
D7
D3
D4
D5
D6
Veh vs IgG
0.0 -2.0 3.6
1,25 vs IgG
0.0 -2.0 3.6
Veh vs IgG
0.0 -2.0 3.6
1,25 vs IgG
0.0 -2.0
ChIP-chip analysis of VDR/RXR binding at the RANKL gene locus. Mouse ST2 cells were treated with either vehicle or 1,25 (OH)2D3 for 3 h and then subjected to ChIP-chip analysis using antibodies to VDR, RXR, or IgG. A schematic diagram of the mouse Tnfsf11 gene locus and its location on chromosome 14 is depicted at the top. The transcriptional start site is designated TSS and the previously identified enhancers are designated D1eD7 and In2a and marked through vertical shading. The VDR and RXR data tracks represent the log2 ratios of fluorescence obtained from vehicle- or 1,25(OH)2D3-treated samples precipitated with antibody to the VDR or RXR vs IgG control. Red peaks represent statistically valid regions of VDR or RXR binding (FDR <0.05).
FIGURE 7.5
transcription is unclear at this time as is the temporal order in which they are recruited to receptor-bound promoters during activation. The VDR has been shown to interact directly with specific components of each of these complexes in vitro [219,221e223], however, and
the addition of many of these factors to cells via transfection assays strongly enhances l,25(OH)2D3-induced transcription. A number of proteins also interact with the VDR in a ligand-independent manner in regions outside that
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of AF-2. These include the protein NCoA62, a coactivator that may play a role in mRNA transcript splicing [224], WINAC [225], Alien [226], hairless [227], SUG1, a 19S proteasomal component that may play a role in VDR degradation [228], E6-AP, an E3 ligase that may play a role in VDR processing [Pike et al., unpublished], and corepressors such as NCoR [229] and SMRT [230]. Little is known of how these proteins interact with the VDR or of the domain(s) within the VDR that mediates the interactions (see Chapter 10) with perhaps the exception of hairless (HR). Here, the ligand-independent interaction of the VDR with HR protein in the hair follicle drives the hair follicle cycle. The mechanism of this ligand-independent function of the VDR is not entirely clear but does highlight a unique function of the VDR as coregulator recruitment via the AF-2 domain of the protein is not required (see Chapters 8 and 30 for details). It is possible, however, that the VDR may be activated in this situation by a novel ligand of unknown structure, as several novel non-vitamin-D ligands for this receptor have been identified (see Chapter 8). The VDR also interacts with the core promoter transcription factor TFIIB [117,231], suggesting that the biochemical mechanisms by which the VDR contributes regulatory inputs into the basal transcriptional apparatus will be multifaceted and complex. The reader is referred to other chapters in the Mechanism of Action section of this volume for additional details regarding the interaction of the VDR with potential comodulators and the core transcription factor TFIIB. Direct Involvement of RXR in Transactivation One question that has emerged from many of the functional studies of VDR is whether RXR contributes directly to the recruitment of the coactivators described above. This is particularly relevant since RXR also contains a functional AF-2 which interacts in vitro with most of the coactivator complexes described above. That RXR contributes directly to VDR-mediated transactivation has been demonstrated through mutagenesis of the RXR H12 region by Thompson et al. [232]. The concept that RXR is not a “silent partner” but rather a transcriptionally active partner of the VDR has been reinforced also through recent studies by Pathrose et al. [155]; these investigators used RXR-selective LxxLL peptides to demonstrate that blockade of RXR recruitment of coactivators also leads to inhibition of transcription induced by l,25(OH)2D3. Finally, recent studies by Bettoun et al. [233] also support the contribution of RXR to VDR-induced transcription. Thus, it seems likely that both subunits of the VDR heterodimer complex are essential to the regulation of gene expression by 1,25(OH)2D3. An additional role of RXR as a prebound “marker” or pioneer factor for potentially active VDREs on specific target genes is discussed below.
General Conclusions An understanding of the structure and functional activity of the VDR has emerged as a direct result of biochemical and molecular biological investigation. Many of these features of the VDR have been both confirmed and extended through follow-on analysis of the three-dimensional structure of the LBD of both the VDR as well as other nuclear receptors. It is, in fact, a testimony to the effectiveness of both biochemical and molecular biological techniques that the crystallographic studies have confirmed most of the structuree function relationships established via the former two approaches. As will be seen below, however, while both biochemical and molecular biologic approaches have greatly enhanced our understanding of nuclear receptor structure and function, studies using genomic tools are now extending these insights even further, broadening, sharpening, and in many cases introducing entirely new concepts into the principles of gene regulation.
VDR RESEARCH: THE GENOMIC ERA Studies conducted and the techniques utilized during the Molecular Biologic Era resulted in the cloning of the VDR structural gene, over-expression, purification and physical analysis of the VDR protein from bacterial, yeast, and mammalian cell sources, dissection of the various structural domains of the VDR and finally, examination of VDR function. They also facilitated efforts to define the underlying mechanisms involved in the ability of the VDR to regulate the expression of target genes, including the identification of direct target genes, elucidation of the DNA sequence of VDR binding sites (VDREs) and delineation of proteins such as RXR and coregulatory factors that participate with the VDR in gene activation. Despite these advances, the nature of the molecular techniques utilized raised questions as to the nature of the results obtained, primarily because many of these assays required extensive cell modification, raised the levels of key cellular components through transfection to extremely high concentrations, and assessed the activities of DNA plasmids in artificial, nonchromatinized environments. Perhaps even more important, the cloning restrictions associated with creating artificial “reporter genes” generally limited the analyses of target genes during this era to a few thousand basepairs near the vicinity of the gene promoters themselves [234]. These issues as well as advances over the years in our understanding of the mechanisms of gene expression led to the following general questions: (1) what are the temporal and spatial dynamics of vitamin D
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activation at known target genes?; (2) are unidentified regulatory elements present in addition to those discovered near specific gene promoters and do they contribute to expression?; (3) how are genes modulated that do not appear to contain promoter proximal elements?; (4) how do control regions function to modulate gene expression and are they all operated similarly?; (5) are the principles that govern the regulation of previously described genes representative of all the genes that are regulated from an entire genome in a given cell type?; (6) how do epigenetic modifications to the genome impact gene expression?; and, finally, (7) what determines where and how these modifications are imposed on a genome, how they are “translated” into novel regulatory structures and how they are disassembled? In this section, we describe new approaches to the study of transcription in general and vitamin D in particular that have extended our current understanding of how the VDR modulates the expression of genes at not only single gene loci but across an entire cellular genomes as well. We also comment on how genomic sites of VDR and RXR binding intersect with those of additional transcription factors that are activated via vitamin-D-independent signaling pathways.
New Approaches to the Study of Transcription Although a number of techniques have emerged over the past few years that have contributed to our understanding of how genes are regulated, perhaps the most significant is chromatin immunoprecipitation analysis (ChIP) linked early on to direct PCR analysis (termed ChIP-PCR) but more recently to tiled DNA microarray hybridization analysis (ChIP-chip) and now almost exclusively to massively parallel DNA sequencing methodologies (ChIP-seq) [235e238]. In ChIP analysis, proteins bound either directly or indirectly to DNA are cross-linked to DNA using formaldehyde, the chromatin is fragmented to form short DNA segments of predetermined size (500e2000 bp), and the protein/ DNA complex is then precipitated using antibodies specific to the protein(s) or modification of interest. Harvested DNA is isolated and quantitated and the concentration of a fragment considered proportional to the concentration of the protein through which it was coprecipitated. Thus, ChIP-PCR analysis culminates in either a direct or a real time PCR measurement using primers designed to assess DNA abundance through a specific site on the genome. ChIP-chip analysis, however, permits an examination of the individual abundance of numerous precipitated DNA fragments across an extended region or regions of the genome (individual gene loci, a chromosome, or an entire genome) using thousands of oligonucleotides synthesized and sequentially tiled on glass microarrays.
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In the most current version, ChIP-derived DNA is simply amplified and then sequenced using “next generation” parallel sequencing techniques; the small sequenced DNA segments are mapped to the genome based upon their homology to the DNA sequence of the genome itself. These latter two applications of ChIP analysis can measure indirectly the presence and levels of endogenously synthesized proteins and/or modifications to proteins at specific sites on chromatin across the entire genome in unmodified cells or tissues. It is these latter techniques, coupled to additional methodologies that include RNA-seq [238], wherein expressed cellular RNAs (the cellular transcriptome) are evaluated not by classical DNA microarray analysis but rather through parallel DNA sequencing, as well as others to be discussed later that have truly transformed transcription research.
VDR DNA Binding and Dynamics at Known Target Genes The initial binding of the VDR to endogenous gene promoters in living cells using the aforementioned ChIP analysis was examined by several investigators including Sierra et al. [239], Zhang et al. [240], and Pike and colleagues [241,242]. Importantly, 1,25(OH)2D3 stimulated the appearance of the VDR at previously identified sites of action on the OC [239] as well as Cyp24a1 and OPN [242] genes. 1,25(OH)2D3 also induced the appearance of RXR at these sites as well, confirming early molecular biologic studies which suggested that RXR was indeed a heterodimer partner of the VDR. Interestingly, these findings also indicated that the VDR was not “prebound” to sites on vitamin D target genes, but rather was induced to bind as a result of 1,25(OH)2D3-mediated activation and RXR heterodimerization. Direct evidence also emerged using these techniques that the VDR recruited some of the many coregulatory factors that were described earlier. Zhang et al. [240] and Kim et al. [242] both demonstrated that l,25(OH)2D3 induced the recruitment of SRC-1 to the Cyp24a1 promoter. Kim et al. [242] also showed that SRC-2 and SRC-3 were similarly recruited, although the temporal pattern of recruitment differed among these p160 coactivators. This group also observed that the HAT containing integrators p300 and CBP as well as mediator component MED-1 were also recruited to the Cyp24a1 promoter in response to l,25(OH)2D3. Importantly, the consequence of this recruitment, perhaps due to the increased presence of MED-1, appeared to be an up-regulation of RNA polymerase II (RNA pol II) density. Increased acetylation of histone 4 was also observed, perhaps due to an increase in the HAT activity inherent to members of the p160 complex. These studies provided clear evidence that the VDR not
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only interacted with these coregulators on specific genes in living cells but that the recruitment of these complexes resulted in functional consequences with regard to gene promoter status and transcriptional activity. The results of these ChIP assays both confirmed and extended all previous in vitro knowledge of the actions of vitamin D and its receptor at the OC, Cyp24a1, and OPN gene promoters. Following these studies, the technique of ChIP analysis of the VDR became a mainstream methodology. Apart from the involvement of VDR and RXR and the numerous other coregulators in transcription, studies of this type also suggested that the process was highly dynamic as well. Indeed, general studies in cultured cells using chromatin immunoprecipitation and other assays have indicated that the binding of nuclear receptors to target gene promoters was cyclic in nature [243e246]. Accordingly, hormone-induced binding of the receptor to gene promoters exhibited cycles with a periodicity ranging from 30 to 40 minutes. The recruitment of coregulator complexes was also cyclic, suggesting the possibility that a spatial and temporal order to the transcriptional process might be necessary for gene regulation [247]. Whether this cycling process is fundamental to the mechanism whereby genes are activated and transcription is maintained is currently unclear. The binding of VDR and RXR to the Cyp24a1 and OPN promoters in response to l,25(OH)2D3 was also
cyclic [242]. This binding is accompanied in parallel fashion by the recruitment of coactivators such as the p160 family of genes, CBP and p300 and of MED-1. While current efforts are in place to determine the fundamental role of cycling in nuclear receptor activation, it is clear that this process may well enable precise real-time cellular monitoring of ambient hormone concentrations.
New Insights Into the Actions of 1,25(OH)2D3 as Assessed by ChIP-chip and ChIP-seq Analysis In this section, we examine the actions of 1,25(OH)2D3 in intestinal and bone cells using both ChIP-chip and ChIP-seq analyses, with the initial intent to reexamine the regulatory regions of genes that were previously characterized, and to explore genes for which regulatory regions had not been identified through traditional molecular biologic analyses. We use the term ChIPchip/ChIP-seq analysis to denote the use of one or both of these techniques together as the transition from ChIP-chip to ChIP-seq analysis has been exceedingly rapid, resulting in studies that have employed both techniques in an overlapping manner. In the next section, we explore the activity of 1,25(OH)2D3 across the entire genome and describe important overarching principles of VDR action.
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FIGURE 7.6 ChIP-chip analysis of VDR/RXR binding at the CYP24A1 gene locus. Human LS180 cells were treated with either vehicle or 1,25 (OH)2D3 for 3 h and then subjected to ChIP-chip analysis using antibodies to VDR or RXR. A schematic diagram of the human CYP24A1 gene locus and its location on chromosome 20 is depicted at the top. The transcriptional start site is designated TSS where previously identified enhancers at e158 and e256 bp are located and marked through vertical shading. The newly identified cluster of intergenic enhancers located downstream of the gene are marked at e41, e50, e60, e66 and e69 kb and highlighted through vertical shading. The VDR and RXR data tracks represent the log2 ratios of fluorescence obtained from vehicle- or 1,25(OH)2D3-treated samples precipitated with antibody to the VDR or RXR vs input controls. Red peaks represent statistically valid regions of VDR or RXR binding (FDR <0.05).
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ChIP-chip/ChIP-seq Analysis of VDR/RXR at Bone Cell Genes As indicated in earlier sections, the bone osteoblast is a significant biological target of 1,25(OH)2D3, participating with epithelial cells from both the intestine and kidney in the homeostasis of calcium (see also Chapter 16). Thus, this cell target has been explored extensively using traditional molecular biologic methods for the presence of vitamin-D-responsive genes; many have been identified as outlined in the above sections as well as in many chapters within this volume, including OC, OPN, Cyp24a1, IBSP (bone sialoprotein), and others. In many, but not all, of these cases, regulatory regions have been defined and VDR-binding sites have been characterized. ChIPchip/ChIP-seq analysis was therefore used to reexamine these genes to confirm the presence of previously described enhancers, to search for additional regulatory regions, and to identify regulatory regions in genes for which mechanisms were obscure. As an example, OPN was previously shown to contain an enhancer region located some 700 bp upstream of the gene’s transcriptional start site (TSS) [172]. ChIP-chip analysis provided confirmation of this site but also identified a second regulatory region containing a VDRE approximately 1.2 kb further upstream [248]. Interestingly, significant binding of the VDR and its partner RXR was observed at these sites in the absence of 1,25(OH)2D3, providing for the first time an example of a pre-bound VDR/RXR heterodimer. Whether this pre-bound VDR/RXR heterodimer manifests activity on the OPN gene is unknown. As will be seen, many examples of this type of binding occur at other genes as well as when examined on a genome-wide scale. ChIP-chip/ChIP-seq analysis of the prototypic vitamin-D-responsive gene Cyp24a1 was particularly illuminating. Accordingly, in addition to binding sites previously described in 1994 [175e177], an intergenic cluster of enhancers located between 35 and 45 kb downstream of the mouse Cyp24a1 TSS was observed [249]. In this case, however, VDR/RXR binding at these sites was fully dependent upon 1,25(OH)2D3 activation. Additional comments regarding the activation of the human gene will be discussed below. The identification and characteristics of both previously identified as well as novel regulatory sites in these and other known vitamin D targets by these new methodologies clearly highlight the technical shortcomings of the more traditional molecular biologic approaches. While known to be regulated by 1,25(OH)2D3 in bone cells, the mechanism of regulation of many genes by the vitamin D hormone has gone unresolved for many years. Genomic targets in this category are numerous, but include the VDR gene itself as well as the RANKL gene (termed TNFSF11), the molecule responsible for paracrine activation of osteoclast differentiation and
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function following 1,25(OH)2D3 stimulation [250e252]. While the regulation of the VDR gene will be described in detail in the next section, the regulation of RANKL by 1,25(OH)2D3 represents a striking model for the power of ChIP-chip/ChIP-seq analysis and also provides a paradigm for the increased complexity that is frequently associated with the regulation of gene expression by steroid hormones and other local and systemic activators. Preliminary studies of the mouse and human RANKL gene promoter regions failed to demonstrate significant and reproducible transcriptional activity in response to 1,25(OH)2D3; the reason for the absence of this activity was uncertain [253,254]. Following ChIPchip analysis of over 500 kb of DNA surrounding the RANKL locus, however, the answer was clear, as documented in Figure 7.5. Accordingly, 1,25(OH)2D3 was found to induce binding of the VDR and RXR to at least five sites within the mouse RANKL gene locus located e16, e23, e60, e69 and e75 kb upstream of the RANKL TSS [255e257]. As was previously discovered through traditional methods, no activity was observed at the RANKL proximal promoter. Further analysis revealed the presence of functional VDREs within at least three of these enhancers and functional binding sites were also identified in these regions for several additional transcription factors known to activate RANKL expression as well [256,258]. These data together with data derived from additional studies of the human RANKL gene (additional sites at IN2A and D7) suggest that this important gene is regulated through multiple enhancer elements located significant linear distances from this gene’s TSS. These results suggest an unusually complex mechanism of gene activation; currently, many genes are now known to be regulated through complex mechanisms analogous to that observed for RANKL. Further studies, as discussed below, provide additional strong evidence that these regions are indeed active in the regulation of RANKL gene expression. ChIP-chip/ChIP-seq Analysis of VDR/RXR at Intestinal/Colon Cell Genes The intestinal tract is also a primary target of 1,25 (OH)2D3 activity, functioning together with the skeleton and the kidney to maintain calcium and phosphorous homeostasis, but also to regulate the metabolic degradation of secondary bile acids such as lithocholic acid, to facilitate the transport and metabolism of xenobiotics and to modulate the proliferation and differentiation of epithelial cells. Many genes are known to be regulated by 1,25(OH)2D3 in the intestinal tract including CYP24A1, the calcium-binding protein S100G (calbindin D9K), TRPV6, PMCA1b (ATP2b1), paracellular calcium transporters such as CLDN 2 and CLDN12, the phosphate transporters SLC34A1-3 (NaPi2a-c), the xenobiotic
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metabolizing enzymes and transporters CYP3A4, CYP3A7, SULT2A1, ABCB1, and others (see Chapters 19 and 20 and references therein). In most cases, regulatory regions for these genes have been described. Recently, however, ChIP-chip/ChIP-seq analyses together with transcriptome analysis (gene expression analysis) have been conducted in both mouse intestinal tissue and in the human intestinal/colon cell lines Caco-2 and LS180. In the latter cells, 1,25(OH)2D3 induced a number of genes that include many of the vitamin D targets enumerated above. Interestingly, ChIP-chip/ChIP-seq analysis not only confirmed binding of the VDR and its heterodimer partner RXR to previously identified sites on these known target genes but also provided a wealth of new mechanistic information for other genes relative to regulation by 1,25(OH)2D3 [249,259]. CYP24A1, for example, was shown as documented in Figure 7.6 to be regulated by not only the two VDREs located near the gene’s promoter, but also through an intergenic cluster of five enhancers located between 41 and 66 kb downstream of the CYP24A1 promoter [249]. As noted earlier in bone cells, strong ligand-independent binding of the VDR and RXR to one enhancer (at 50 kb), but not at others, was observed. The features of this site that are responsible for this selective activity are unknown. VDREs located at these sites were all shown to be functionally active (see below). The ChIP-chip/ChIP-seq approach also confirmed upstream regulatory sites for VDR/RXR previously identified in the TRPV6 gene [260,261]. Interestingly, a novel VDR/RXR binding site was also observed within the 30 end of the TRPV5 gene as well; TRPV5 is located adjacent to TRPV6 on chromosome 7. TRPV5 is largely expressed in the kidney and only weakly regulated by 1,25(OH)2D3 in the intestine. A promoter proximal site has also been described for the S100g (calbindin D9K) gene [174]. ChIP-chip/ChIPseq analysis did not confirm this site, although VDR/ RXR binding was identified 27 kb upstream of the TSS. While previous studies have suggested that the CLDN2 and CLDN12 are regulated by 1,25(OH)2D3, an underlying mechanism was not defined for either gene [262]. ChIP-chip/ChIP-seq studies revealed several sites of VDR/RXR binding at CLDN2 but not CLDN12. A regulatory region associated with SLC34A3 was also observed. CYP3A4, CYP2B6 and the transporter ABCB1 were also induced by 1,25(OH)2D3 [263e267]. ChIP-chip/ChIP-seq studies both confirmed the regulatory regions previously described for these genes but also identified additional regions of regulation as well. Finally, we also observed novel regulatory regions in both the c-FOS and c-MYC genes (see the following section) [259]. The results of these studies highlight the power of ChIP-chip/ChIP-seq analysis in the elucidation of gene regulatory mechanisms.
ChIP-chip/ChIP-seq Analysis of VDR/RXR at Key Genes in a Colon Cancer Cell Line The Wnt-activated b-catenin pathway represents a primary driving force for not only the proliferation of intestinal/colonic crypt epithelial stem cells but also for colorectal tumor cell proliferation (see also Chapter 13). This activity is countered by 1,25(OH)2D3, which is known to exert both antiproliferative and prodifferentiative effects on these and other cell types. Early studies suggested that the mechanisms by which VDR and other members of the nuclear receptor family exert these effects are complex and focused at a variety of levels. These include the ability of the VDR to interfere with b-catenin activity at target genes and to induce TCF4, a repressor of b-catenin-activated genes, and Wnt antagonists such as DKK-1 and DKK-4 (see also Chapters 8 and 13). Recent ChIP-chip/ChIP-seq analyses, however, suggest that the c-MYC proto-oncogene, a direct target of b-catenin action, is also directly regulated by 1,25(OH)2D3 and its receptor [259]. These studies show that 1,25(OH)2D3 induces the binding of VDR/RXR directly to an enhancer located e335 kb upstream of the c-MYC gene. This enhancer binds both TCF-4/b-catenin and other transcription factors as well and is associated with increase risk of colorectal, prostate, and breast cancer [268e271]. Indeed, a single SNP located in this enhancer alters both TCF4/b-catenin binding and activity. Interestingly, ChIPchip/ChIP-seq analysis also revealed an additional cluster of upstream enhancers between e139 kb and e165 kb that bound the TCF-4/b-catenin activation complex and/or the VDR-RXR heterodimer. As seen in Figure 7.7, 1,25(OH)2D3 also strongly induced c-FOS gene transcription via a regulatory site located e24 kb
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FIGURE 7.7 ChIP-seq analysis of VDR/RXR binding at the c-FOS gene locus. LS180 cells were treated with either vehicle or 1,25(OH)2D3 for 3 h and then subjected to ChIP-seq analysis. A schematic diagram of the human c-FOS gene is shown at the bottom of the figure. Tag counts normalized to 107 reads are plotted across the c-FOS locus on chromosome 14, with the scale of peak tags under vehicle- and 1,25 (OH)2D3-treated conditions indicated (96 vs 403) as compared to normalized input control reads. The dotted line indicates statistical validity above 25 tag counts.
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upstream of the gene’s TSS. This study suggests that 1,25 (OH)2D3 likely exerts a direct transcriptional action on pro-proliferative genes to inhibit the Wnt-b-catenin signaling pathway in colon cancer cells and perhaps in other cancer cell types as well. Assessing Mechanistic Consequences of VDR/RXR Binding to Target Genes ChIP-chip/ChIP-seq analysis can also be used to explore the functional consequences of VDR/RXR binding at target gene loci. These measurements are exceedingly important because they provide evidence that the sites to which the VDR/RXR complex binds are capable of regulating gene expression. As discussed earlier, a primary function of the VDR/RXR heterodimer is to recruit coregulatory complexes that contain enzyme activities essential for modulating
A
events associated with gene output. Indeed, studies using direct ChIP analysis revealed that coregulators such as the p160 family of histone acetyltransferase coactivators, the acetyltransferase cointegrators p300 and CBP, as well as MED-1 could be recruited as a function of 1,25(OH)2D3 treatment and VDR/RXR enhancer binding [242]. Recent studies using ChIPchip/ChIP-seq analysis have demonstrated that these coregulators together with the two corepressors NCoR and SMRT are also recruited in a similar fashion to a number of genes that represent targets of VDR/ RXR action [248,249,259]. The recruitment of these factors is likely responsible for dramatic epigenetic changes in the level of histone H3 and H4 acetylation that are evident at VDR-activated enhancers as well, as exemplified by the up-regulation of H4 acetylation in the Rankl gene (Tnfsf11) seen in Figure 7.8. These
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FIGURE 7.8 ChIP-chip analyses of histone 4 acetylation and CTCF transcription factor binding at the Tnfsf11 (RANKL) gene locus. Mouse ST2 cells were treated with either vehicle or 1,25(OH)2D3 for 3 h and then subjected to ChIP-chip analysis using antibodies to tetra-acetylated H4, CTCF, or IgG. A schematic diagram of the mouse Tnfsf11 gene locus together with its upstream and downstream neighboring genes (Akap11, NM_177629) and its location on chromosome 14 is depicted at the top. The transcriptional start site (TSS) is designated with an arrow on the opposite DNA strand. Previously identified enhancer elements located upstream of the designated TSS are indicated (D1eD7). The data tracks represent the log2 ratios of fluorescence obtained from vehicle- or 1,25(OH)2D3-treated samples precipitated with antibody to the tetra-acetylated H4 or CTCF vs IgG controls. Key boundary CTCF sites are ringed. Red peaks represent statistically valid regions of VDR or RXR binding (FDR <0.05). Please see color plate section.
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modifications covalently mark existing enhancers within active genes across the genome; increased levels of H3 and H4 acetylation are indicative of changes in chromatin architecture that occur prior to, and therefore are functionally important to the enhanced expression of associated genes. Interestingly, analyses of regions containing these and other structural epigenetic marks can be used to identify and localize enhancers to the genes that they regulate [272]. ChIP-chip/ChIP-seq analysis can also be used to measure an increase in the recruitment of RNA pol II, the enzymatic component that is essential for the production of RNA transcripts from protein coding and other genes. In many cases, changes in RNA pol II density are observed either at the TSS or across the entire transcription unit of activated genes [273]. Surprisingly, RNA pol II is also recruited to VDR/ RXR-bound enhancers as well [255]. The appearance of RNA pol II at these regulatory regions has led to the hypothesis that enhancers might function as recruitment centers for the RNA pol II complex, particularly since many additional components of the general transcriptional apparatus are also recruited to these sites as well [274]. Recent studies, however, suggest that productive transcription also occurs at these sites and results in the synthesis of several types of noncoding RNA transcripts [273,275]. In many cases, these RNAs appear to serve a significant regulatory function, operating either in cis or in trans to facilitate gene output from neighboring genes [276]. This new role of RNA pol II in the production of novel regulatory RNA transcripts that encompass over 85% of the human and other mammalian genomes was initially discovered using tiled microarray analysis. Using RNA-seq analysis as an analytical tool, it is likely that this will represent an extremely fertile area of research for the future. Finally, ChIP-chip/ChIP-seq analysis has confirmed and extended the concept that regulatory regions often span several kilobases or more of DNA and frequently contain binding sites for multiple transcription factor species. Thus, enhancers that bind the VDR in bone cells also contain regulatory sites for such transcription factors as RUNX2, and C/EBP and AP-1 family members as well [248]. In intestinal cells, the VDR colocalizes with C/EBPb, AP-1, CDX2, and TCF-4 among others [259]. A current focus of study is to assess the role of several of these factors in cell lineage determination [277,278].
Genome-Scale Analysis Reveals Overarching Principles of Gene Regulation by 1,25(OH)2D3 While the above studies have focused upon delineating mechanisms responsible for the regulation of
specific genes by 1,25(OH)2D3, the power of ChIPchip/ChIP-seq analysis resides more impressively in its ability to define these mechanisms at the same level of resolution as single genes yet on a genome-wide scale. Thus, ChIP-chip/ChIP-seq analysis can be used to identify and quantitate the total number of binding sites for the VDR/RXR heterodimer in a given cell type (termed cistrome analysis) and, coupled with transcriptome analysis (genome-wide RNA measurement), assess all the likely functional gene targets in the cell as well. Analyses of this type permit delineation of the set of overarching principles that characterize the molecular actions of 1,25(OH)2D3 in a particular cell type. While several of these types of analyses have been conducted on other nuclear receptors [279e289] and several on the VDR [248,259,290], we focus on the results of several of our own studies in intestinal/colon and bone cells [248,259]. Genome-wide Analysis of VDR/RXR Binding in Intestinal/colon Cells ChIP-chip/ChIP-seq analysis using antibodies to the VDR and RXR were conducted in LS180 cells following treatment with 1,25(OH)2D3 [259]. Bioinformatic analysis, as summarized in Figure 7.9A, revealed 262 binding sites for the VDR in the absence and 2209 sites in the presence of 1,25(OH)2D3, indicating that the VDR was prebound to a limited number of control regions in the absence of ligand and largely dependent upon activation by 1,25(OH)2D3 for full localization at most DNA targets. The vast majority of these binding sites (71%) were located within intergenic regions of the genome with 27% located intronically and only a few percent located near regulated promoters (see Fig. 7.9B). Thus, the vast majority of regulatory enhancers for 1,25(OH)2D3 and its receptor are located distal to TSSs. As documented in Figure 7.9C, de novo motif finding analysis revealed that 27% of these sites contain a sequence comprised of AGGTCA ctg GGTTCA which is highly representative of a classic VDRE. These data also indicate that VDRE configurations other than the classic VDRE are likely present. ChIP-chip/ChIPseq analysis revealed some 742 RXR-binding sites in the absence of 1,25(OH)2D3, supporting the concept advanced earlier that RXR participates as a heterodimer partner with other nuclear receptors, and approximately 4200 RXR-binding sites in the presence of 1,25 (OH)2D3. Importantly, VDR and RXR colocalize to 1674 sites across the LS180 genome (see Fig. 7.9B). Interestingly, RXR binding in the absence of ligand premarks a number of sites to which the VDR binds when induced by ligand, suggesting that RXR may “mark” genomic sites for activation by VDR. Additional ChIP-chip/ChIP-seq studies which examined the effect of 1,25(OH)2D3 on coregulator recruitment
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revealed a preference for the SRC-1 coactivator and for the NCoR corepressor. The binding of these coregulators was correlated with VDR binding near genes that were modulated at the transcriptional level by 1,25 (OH)2D3. The formation of these complexes also led to an increase in histone H4 acetylation at many, although not all, enhancer sites, at target gene promoters and often across the transcription units themselves. These findings confirm the concept, at a genome-wide level, that VDR and RXR bind as partners at sequences that comprise VDREs and that a consequence of this binding is the recruitment of coregulators such as SRC-1 that play a role in the modification of chromatin that is necessary for altering gene output. De novo motif finding analysis also revealed the presence of potential binding sites for a series of unrelated transcription factors that could also associate with these VDR/RXR-regulated enhancers and thus participate in the regulation of these target genes. In LS180 cells, the most abundant was C/EBPb, ETS1, AP-1, and CDX2, the latter a cell-specific homeobox factor essential to the embryonic development of the intestine and to the function of mature intestinal epithelial cells. Direct ChIP-chip/ChIP-seq analysis of these factors confirmed their presence at many VDR/RXRbinding sites. Interestingly, VDR-binding activity was also linked through de novo motif analysis to TCF-4binding sites as well, an observation that was similarly confirmed through direct ChIP-chip/ChIP-seq analysis of the TCF-4 and b-catenin cistromes. While the overlap between VDR/RXR and TCF-4/b-catenin binding was small, two gene targets were highlighted in this analysis, c-FOS and c-MYC. The binding of VDR/RXR and
-1.3e+03
FIGURE 7.9 Genome-wide analysis of the VDR/RXR cistrome in colorectal cancer cells. LS180 cells were treated with either vehicle or 1,25(OH)2D3 for 3 h and then subjected to ChIP-seq analysis using antibodies to VDR or RXR. Immunoprecipitated samples were prepared, amplified and then sequenced (36 bp reads) on an Illumina GAIIx sequencer. Images were processed using the Illumina base-calling pipeline to obtain FASTQ formatted sequence data and then mapped to the human genome. Peaks were called at a cut-off setting of 0.01 (FDR) and tag density normalized of 107 tags. Additional information can be found in the reference. (A) Quantitation of the VDR and RXR peaks in the absence or presence of 1,25 (OH)2D3 and the common intersection between both VDR and RXR peaks in the data set. (B) Genomic location of VDR/RXR-binding sites relative to the most proximal annotated gene. (C) The most common sequence motif found de novo upon analysis of the binding site intersection between VDR and RXR.
TCF-4/b-catenin at these two genes was described in an earlier section on colorectal cancer cells. Thus, ChIP-chip/ChIP-seq analysis has revealed not only genome-wide sites of action of ligand-activated VDR in LS180 cells, but also confirmed a relationship between it and RXR. It also validated the predominant structural organization and sequence of typical VDREs (two hexameric half-sites separated by three basepairs), and identified important relationships between the VDR/RXR heterodimer and other transcription factors of significance to not only intestinal/colon cells but colorectal cancer cells as well. In so doing, two important target genes involved in cellular proliferation were uniquely identified. Genome-wide Analysis of VDR/RXR Binding in Mouse Osteoblasts A similar genome-wide ChIP-chip/ChIP-seq analysis was also conducted in the mouse osteoblastic bone cell line MC3T3-E1 [248]. The results were very similar with regard to VDR and RXR binding, the location of these binding sites and the prevalence of typical VDREs within these regions. Not surprisingly, while an overlap between VDR/RXR-binding sites and C/EBPb was identified, a predominant overlap was also observed between the VDR/RXR and RUNX2 sites, the latter an essential osteoblastdetermining transcription factor. This study suggests the existence of potential relationships between primary lineage-determining factors and secondary regulatory transcription factors such as the VDR. Regardless of the speculation, the use of ChIP-chip/ ChIP-seq analysis seems likely to reveal new insights into the overarching principles of gene regulation
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and to answer many of the questions posed in the introduction to this section on the Genomic Era.
Linking Enhancers to the Genes They Regulate Recent studies, including those above, suggest that transcriptional control regions that are capable of regulating the expression of genes are often located many kilobases upstream or downstream of these genes. Moreover, intronic enhancers can often regulate adjacent genes rather than the gene in which they are located. Finally, an enhancer and its specific target gene are often separated by one or more unregulated transcription units. These relative placements have made it difficult to link a regulatory enhancer to the gene it modulates. Several approaches have been taken, however, which permit such identification. Primary among them is the use of bacterial artificial chromosomes (BAC), DNA segments of 200e300 kb in size, which under most circumstances span the gene transcription unit of interest but also contain large flanking intergenic segments that often extend to the neighboring genes located upstream and downstream as well. If an IRESluciferase reporter (together with a neomycin selectable marker) cassette is inserted into the final noncoding 30 exon of the gene of interest using recombineering methods [291], the entire construct can be introduced into the genome of target cells, selected using the drug G418, and a collection of stable cell lines created and utilized to assess gene transcriptional activity [60,249,292]. The activity of this construct can then be contrasted with the activities of additional constructs that contain mutations that cause selective deletion of one or more of the enhancers that are hypothesized to contribute to the gene’s regulation. This approach was utilized to assess the role of the enhancer cluster located downstream of the mouse and human CYP24A1 genes [249]. The results provide direct evidence that the downstream enhancers that were discovered using ChIPchip/ChIP-seq contribute to the regulation of both mouse and human CYP24A1 genes by 1,25(OH)2D3. Similar studies were also conducted for the RANKL gene using an extended BAC clone that contained the mouse RANKL locus [292]. An advantage of these large DNA constructs is that they can also be utilized to prepare transgenic mice wherein the role of identified/ mutated enhancers can be determined not only with respect to gene regulation but also with regard to tissue-specific expression as well. An example of this type of analysis will be discussed in the next section on the VDR gene itself. Of course, an analysis of a suspected enhancer can also be conducted by deleting the specific segment from the mouse genome using contemporary Cre recombination methodologies. This approach may represent the only means to assess an enhancer/
target gene relationship if the distance from the enhancer to the putative gene target is extremely large. Deletion of a primary enhancer responsible, in part, for both 1,25(OH)2D3 and PTH induction of the mouse RANKL gene resulted in a blunting of RANKL response to both hormones in the altered mouse strain and in osteoblasts derived from these mice [293]. Additional methodologies such as chromosome conformation capture (3C) analysis and more advanced versions of this technology [294e300] have also been utilized, although these methods provide strictly correlative support for the relationship between an enhancer and its target gene.
VDR CHROMOSOMAL GENE: STRUCTURE AND MOLECULAR REGULATION Cellular VDR Expression and Abundance as Determinants of 1,25(OH)2D3 Response The presence of the VDR is an absolute requirement for cellular response to 1,25(OH)2D3, an observation that is supported by several major lines of evidence. First, cells and tissues that display direct response to 1,25 (OH)2D3 all contain the VDR. What determines the expression of the VDR in each of these tissues has not been determined and will be discussed in a subsequent section below. Second, the absence of the VDR in tissues such as liver and skeletal muscle and in a wide variety of additional cell types, as previously discussed, is correlated with an absence of direct response to the vitamin D hormone. This situation is reinforced by the observation that de novo appearance of the VDR, such as that which occurs in T cells following T cell receptor activation, leads to an immediate response to 1,25(OH)2D3. Third, inherited mutations in the VDR such as those found in HVDRR (see Chapter 65) that prevent expression or compromise its functional activity, result in partial or total resistance to the actions of 1,25(OH)2D3 despite the fact that the levels of the hormone are generally very high. Finally, mouse strains in which this species’ genome has been selectively altered to create homozygous VDR-null alleles also manifest phenotypes similar to those seen in HVDRR, including total resistance to the vitamin D hormone (see Chapter 33 and others in this volume). Intuitive is the idea that the abundance of the receptor in cells represents a primary determinant of the magnitude of cellular response to 1,25 (OH)2D3. This concept is much more complex, however, as cellular response is also influenced by a myriad of additional factors that affect the expression of genes and gene subsets, including (i) specific characteristics of the gene target itself, (ii) the participation and degree
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of activation of downstream coregulators that are involved in mediating VDR action, and (iii) the degree to which key signaling pathways and their transcription factor targets contribute to the expression of genes that are activated by 1,25(OH)2D3. Despite these caveats, examples of the strong correlation that exists between cellular VDR levels and biological response are present. One example is the observation that protein kinase A (PKA) activators, such as cAMP or forskolin which induce VDR gene expression, potentiate the upregulation of cellular response to 1,25(OH)2D3 treatment [301]. This finding highlights the role of one membranesignaling process in cellular sensitization to 1,25(OH)2D3 activation. A second classic example is the direct correlation between VDR supplementation via transfection of a VDR expression vector and the degree of responsiveness of a cotransfected VDRE-containing reporter gene to 1,25(OH)2D3 [113]. While the latter situation demonstrates that VDR levels correlate directly with response to 1,25(OH)2D3 under circumstances in which ancillary factors are likely to remain unchanged, this type of situation is highly artificial and may not mimic entirely the effects obtained in an intact and unaltered target cell. Nevertheless, the idea that the abundance of a nuclear receptor in target tissues directly influences cellular responsiveness to hormonal activation is a general tenet of modern hormone biology.
Transcriptional Regulation of the VDR Gene Studies over several decades have shown that VDR abundance is regulated transcriptionally by a variety of systemic factors, peptide hormones, and calcium and phosphorous, a process termed heterologous regulation [302]. Early studies focused upon identifying the regulatory agents themselves, the signaling pathways that were involved and the transcription factors that mediated these modulatory events. VDR abundance is also controlled by 1,25(OH)2D3 itself. This control of VDR levels has been termed homologous up-regulation and is much more complex than the heterologous version because it includes at least three distinct processes: transcriptional activation, post-translational stabilization and finally post-translational modification [302]. In the following sections, we briefly discuss the early studies that provided evidence for homologous and heterologous VDR gene regulation both in cells in vitro and in tissues in vivo. We then discuss how these regulatory processes occur at the molecular level by first focusing upon the structural organization of the VDR gene and then by defining important control regions within the gene itself that are responsible for mediating the actions of these regulatory agents. A portion of the results of these studies are derived through a recent examination of the properties of VDR gene BAC clone
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stable cell lines and transgenic mice. We conclude by discussing the ability of 1,25(OH)2D3 to induce VDR protein up-regulation independent of transcription (a post-translational process) and to regulate the receptor phosphorylation state. The mechanisms through which these latter processes occur are unknown. Heterologous Transcriptional Regulation VDR expression is regulated by a multitude of systemic, local, and intracellular factors. While the identification of factors that regulate this gene is incomplete at present, studies conducted during the past several decades have suggested that steroid hormones, peptide hormones, growth factors, and mineral constituents all contribute to the transcriptional control of the VDR gene, many in tissue/cell-specific ways. Early studies were frequently driven by biological observations that indicated either enhanced or reduced biological response to the vitamin D hormone in selected tissues. Others identified agents that participated with 1,25 (OH)2D3 in the regulation of mineral homeostasis. Oftentimes, the regulation of the VDR occurred during development in selected tissues or was associated with striking physiological changes such as those that occur during growth or reproduction. Clearly, these latter physiologic events are precipitated by changes in the levels of specific steroid hormone such as estrogen or the androgens and are accompanied by striking changes in calcium and phosphorous utilization. With respect to steroid hormones, the VDR is significantly regulated by glucocorticoids, estrogens, and retinoids such as retinoic acid [302]. This regulation often occurs in a cell-typespecific manner (e.g., retinoid activity in bone cells and glucocorticoid activity in the intestine). Interestingly, VDR expression can be both increased or decreased with these agents, sometimes in a species-specific manner. The VDR is also regulated by several peptide hormones and growth factors including bFGF, EGF, IGF, and insulin and by the calcitropic peptide hormone PTH and its cellular counterpart PTHrp. The latter two peptides induce the VDR via the PKA pathway whose downstream activity is mediated, at least in part, by the transcription factor cyclic AMP response element binding protein (CREB). Growth factors, on the other hand, operate via a number of membrane-signaling pathways including those involving specific receptor tyrosine kinase (RTK) and G-protein-coupled receptor (GPCR) pathways and their specific transcription factor targets. Many of these signaling pathways play significant roles in the maintenance of intracellular levels of calcium and phosphorous as well. Extracellular calcium levels also modulate VDR expression in specific tissues including the kidney, bone, and the parathyroid glands. Here, some experiments suggest that the calcium-sensing receptor (CaSR) may be involved,
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triggering activation of the stress-activated protein kinase p38 which promotes an up-regulation of the VDR in the kidney and parathyroid gland (see Chapter 24) [303e305]. VDR expression is also regulated by other signaling pathways that converge on AP-1 [306], a transcription factor complex comprised of the heterodimer c-Fos and c-Jun, and on C/EBPa and b [307]. The VDR gene is also regulated in cancer cells by transcription factors such as SNAI and SLUG [308e314]. Aside from transcriptional studies of C/EBP, SLUG, SNAI, AP1, CREB, and perhaps RAR, however, demonstration of direct actions of transcription factors on the VDR gene promoter has been largely unsuccessful. The reader is referred to a more complete discussion of VDR gene regulation at this exploratory level in chapters on VDR regulation found in Vitamin D [315] and Vitamin D 2nd Edition [316].
the absorption of calcium via the intestine during this time is independent of vitamin D status, but apparently dependent upon prolactin. This provides an explanation for why mice homozygous for the VDR-null allele are not hypocalcemic at birth, but rather become hypocalcemic as they transition to a solid diet. As indicated earlier, the VDR gene is similarly not expressed in naı¨ve immune T cells until these cells are activated through the TCR [20,321,322]; recent experiments show that this upregulation is due to the activation of c-Fos [Bishop and Pike, unpublished]. In most cases, however, the molecular mechanism through which the VDR gene is induced and the identity of the factors that participate remains to be discovered. Whether the VDR gene is induced in other tissues such as muscle, liver, or endothelial cells or during unique physiological or pathological states is unknown.
Homologous Transcriptional Regulation
Molecular Mechanisms of VDR Gene Regulation
As suggested above, the actions of 1,25(OH)2D3 on the VDR are complex, involving cell-specific transcriptional up-regulation (termed autoregulation), VDR protein stabilization, and covalent VDR modification. Prior to the cloning of the VDR gene cDNA, it was difficult to distinguish between autoregulation and posttranslational stabilization [317] (see also [302]). Thus, the discovery that 1,25(OH)2D3 was capable of upregulating its own receptor at the mRNA level was provided unequivocally by McDonnell et al. [89]. These authors demonstrated that 1,25(OH)2D3 induced an upregulation of VDR mRNA directly in both chicken tissues and in mammalian cells. 1,25(OH)2D3 also induced VDR gene expression in a variety of cell types in vitro. In normal mice, however, significant doses of 1,25(OH)2D3 were capable of autoregulating the VDR only in calvarial osteoblasts and not in kidney or the intestine [318,319]. Whether VDR mRNA is up-regulated by 1,25(OH)2D3 in other tissues is somewhat controversial. Interestingly, while the human and mouse VDR genes were cloned and their structural organization defined over a decade ago, studies focused at the level of the VDR gene promoter failed to define a mechanism whereby 1,25(OH)2D3 induced VDR gene transcription until very recently (see below). Tissue-specific Expression of the VDR As described earlier in this chapter, the VDR is selectively expressed in a variety of tissues in higher organisms including the kidney, the intestine, bone cells, the parathyroid chief cells, insulin-producing b cells of the pancreas, and in a myriad of others. Surprisingly, the underlying molecular basis for this selective expression and the identity of the factors involved remain unknown. Interestingly, VDR gene expression is absent in neonatal rodent intestine until weaning [320]. Thus,
Organization of the VDR Gene The cloning of the VDR structural gene in 1987 initiated the Molecular Biological Era of VDR research on the mechanism of action of vitamin D. Unlike many other members of the nuclear receptor family, however, the VDR appeared to be the product of a single gene. Indeed, sequence analysis of both the mouse and human genomes subsequently confirmed that only one VDR gene was present [95,96], the human homolog located on chromosome 12 [323]. The initial organization of the introneexon structure of human VDR chromosomal gene corresponding to the sequence of the VDR reported by Baker et al. [90] was determined in 1988 [114]. Additional efforts defined the complete structure of the gene a number of years later [324]. Restriction mapping of several lambda clones and a series of four recovered human cosmid clones coupled to nucleotide sequence analysis revealed a gene spanning over 75 kb of DNA. Eight exons comprised the coding sequence of the VDR protein. The first of these was exon 2, which contained the most proximal 3 bp of the 50 noncoding sequence, the translation start site, and nucleotide sequence that encoded the first zinc finger module. Located approximately 15 kb downstream, exon 3 encoded the second zinc finger module. Exons 4, 5, and 6 defined the D region or hinge. Exons 6, 7, 8, and 9 encoded a portion of the hinge and the carboxyterminal E/F region together with approximately 3200 nucleotides of 30 noncoding sequence. It is clear that the human chromosomal gene for the VDR is not unlike other steroid receptor genes in size and exon organization. Figure 7.10 documents the overall organization of both the mouse and human VDR genes relative to the VDR protein structure.
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24
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427 aa
N
C DBD
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LBD
ATG 1 1D-like
2 3
TGA 4
56 7
8910
mVDR ATG 1A 1F 1E 1D 1B
ATG 1C 2
TGA 3
45 6
78 9
hVDR FIGURE 7.10 Organization of the mouse and human VDR transcription units. Linear depiction of the structure of the mouse and human VDR gene where the arrows indicate exons containing either an initiation or a termination codon and vertical bars represent exons. Functional promoters are present upstream of exon 1 in the mouse and exons 1c and 1a in the human. A possible promoter may be present near exon 1f of the human gene. Dotted lines encompass exons 3e10 in the mouse gene and exons 2e9 in the human gene that encode the mouse or human VDR protein (shown at the top of the figure).
The 50 end of the human VDR gene is highly complex, featuring one major promoter and one or more minor promoters responsible for the production of a number of alternatively spliced RNAs (see Fig. 7.10). This is not the case for the mouse VDR gene, which appears to contain a single promoter that is likely responsible for VDR expression [325,326]. With respect to the human VDR gene, two short exons lie upstream of exon 2 and account for the known 50 noncoding sequence reported by Baker et al. [90]. These exons, termed 1a and 1c, are 77 and 81 bp in length. The primary promoter (P1) is located immediately upstream of exon 1a. A possible promoter is located immediately upstream of exon 1c and is characterized by its GC-rich nature and the absence of a TATA box. Interestingly, an exon of 121 bp that was not found in the originally reported sequence of Baker et al. [90] and termed exon 1b is located between exons la and 1c. Variable use of exons 1b and 1c leads to the production of alternatively spliced mRNAs, whose nature and function remain unknown. Additional exons upstream of exon 1a have also been identified, with a potential promoter (P2) located at exon 1f [47,81]. Although there is some indication that the use of these promoters is cell-type-specific, the relative abundance of most of these unusual VDR RNA transcripts is exceedingly low and thus their contribution to the overall expression of the VDR protein and to an unusual larger form of the receptor is unclear. Initial studies of the major promoters and regions nearby for both the human and the mouse vdr genes using transient transfection methods revealed that transcription factors
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such as AP-1, SP1, C/EBP, and CDX2 were involved in basal activity. These studies did not, however, identify the mechanisms responsible for transcriptional regulation by factors such as the glucocorticoids, estrogen, retinoic acid, or 1,25(OH)2D3. Defining Enhancers of the VDR Gene As indicated above, many of the organizational features of the VDR gene emerged during the decade of the 1990s using traditional molecular biologic techniques. What was not uncovered, however, was any significant understanding of how the VDR gene was regulated by either peptide or steroid hormones, including 1,25(OH)2D3, or why the VDR gene was selectively expressed only in a subset of cellular and tissues targets. Given the size and complexity of the VDR gene, however, it was easy to speculate that many of the key regulatory components for the gene’s expression might be located distal to its promoter. Since both the mouse and human VDR genes contained more than 40 kb of sequence upstream of their transcriptional start sites and their exons were separated by very large introns, the foremost question was: where were these regulatory elements? The answer to this question was delayed for almost a decade until technical means capable of searching the entire VDR gene locus emerged, as typified by ChIP-chip/ChIP-seq methodologies. Using ChIP-chip analysis, we focused first on identifying enhancers within the VDR gene locus that mediated the autoregulatory capabilities of 1,25(OH)2D3 and its receptor [59]. A working hypothesis was that if VDR-regulated enhancers could be identified, they might also facilitate identification of enhancers that mediate the actions of other regulatory agents as well. Mouse bone cells were treated with 1,25(OH)2D3, and a large genomic region extending from 100 kb downstream to 100 kb upstream of the VDR gene transcription unit, a span of approximately 260 kb, was scanned for VDR- and RXR-binding sites. As illustrated in Figure 7.11, strong VDR- and RXR-binding activity was discovered within two introns of the VDR gene some 29 and 20 kb downstream of the VDR TSS; these regions were termed the S1/S2 and the S3 enhancers, respectively. Two more minor VDR/RXR-binding sites were also observed one near the VDR gene promoter and one approximately 6 kb upstream of the TSS as well [60,327]. Additional analysis revealed that 1,25 (OH)2D3-induced VDR/RXR binding at these sites also resulted in the recruitment of coregulators such as SRC-1 and MED1. Increased RNA pol II levels were seen in response to 1,25(OH)2D3, but were largely restricted to the proximal promoter (see Fig. 7.11). Interestingly, as also seen in Figure 7.11, histone H4 acetylation levels were focally present across the VDR gene in
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FIGURE 7.11 1,25(OH)2D3 induces VDR/RXR binding, RNA pol II recruitment and increased histone H4 acetylation at the mouse VDR gene. ChIP-chip analysis of basal and 1,25(OH)2D3-induced VDR and RXR binding, RNA pol II recruitment and increased histone H4 acetylation to the mouse VDR gene. MC3T3-E1 cells were treated with either vehicle or 1,25(OH)2D3 for 3 h and then subjected to ChIP-chip analysis using antibodies to VDR, RXR, RNA pol II, or tetra-acetylated H4. A schematic diagram of the mouse VDR gene locus and its location on chromosome 15 is depicted at the top. Previously identified enhancers are designated S1/S2, S3, PP, and U1 and marked through vertical shading. VDR, RXR, RNA pol II, and acetylated H4 data tracks represent the log2 ratios of fluorescence obtained from vehicle- or 1,25(OH)2D3-treated samples precipitated with antibody to the VDR, RXR, RNA pol II, or acetylated H4 vs corresponding sample input DNAs. Red peaks represent statistically valid regions of VDR or RXR binding, RNA pol II recruitment, or levels of acetylated H4 (FDR <0.05).
the absence of 1,25(OH)2D3, suggesting that this gene was actively being transcribed. Indeed, expression levels of the VDR in this bone cell were substantial. Following 1,25(OH)2D3 treatment, however, H4 acetylation levels were increased. Interestingly, all of these 1,25 (OH)2D3-induced epigenetic changes occurred within a region extending from several kb downstream of the VDR gene’s final exon to 50 kb upstream of the VDR gene’s TSS. Active insulator CTCF-binding sites, which appeared to define the VDR gene locus with respect to changes in H4 acetylation, are located at the extreme ends. Studies of the human VDR gene revealed similar, although not identical, features [60]. Finally, further direct study of the S1/S2 enhancer revealed the presence of an active VDRE capable of mediating the autoregulatory actions of 1,25(OH)2D3 and its receptor in bone cells. These findings provided a significant entre´e into subsequent investigations aimed (i) at understanding how regulatory agents such as the retinoids, estrogens, the glucocorticoids, and PTH might
modulate VDR gene expression and (ii) in identifying the transcription factors that might be responsible for the strong basal expression of the VDR gene in bone and other cells. To determine how retinoic acid and the glucocorticoid hormones modulated VDR gene expression, bone cells were treated with each hormone and the cells then subjected to ChIP-chip analysis using antibodies to RAR and to the glucocorticoid (GR) receptor. Strong binding sites for RAR/RXR as well as for GR were found in a subset of the enhancers identified earlier for the VDR/RXR heterodimer [60]. RAR binding was most prevalent at the S3 enhancer, confirming a preliminary observation made for the human gene in 1997 [324]. These findings substantiated the initial hypothesis that the discovery of VDR/RXR enhancers might facilitate the identification of regulatory regions for other agents as well. ChIP-chip analysis also revealed that PKA activation by forskolin (a PTH surrogate) induced localization of CREB to several of these regulatory sites as
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VDR CHROMOSOMAL GENE: STRUCTURE AND MOLECULAR REGULATION
Exploring VDR Gene BAC Clone Activity in Bone Cells A previous section described the utility of large BAC clones for evaluating the individual contributions of enhancers to the expression of linked genes. Indeed, a mouse VDR BAC clone was used to evaluate the ability of 1,25(OH)2D3 and other inducing agents to up-regulate VDR expression and to assess the contribution of the VDRE located in the S1 enhancer [60]. Accordingly, a mouse VDR BAC clone, engineered to contain a luciferase reporter in the noncoding 30 portion of the VDR’s final exon, as illustrated in Figure 7.12A, was introduced into bone cells and a collection of lines stable for this DNA segment identified. Importantly, the stable cell lines all expressed recombinant VDR and synthesized basal levels of the reporter gene, as measured by luciferase activity, from this modified VDR BAC clone. Significantly, 1,25(OH)2D3, retinoic acid, glucocorticoids, and forskolin all stimulated the production of the reporter gene as well as the levels of the recombinant VDR, indicating that this extended gene locus was capable of recapitulating responses to steroids and other inducers that were observed with the endogenous VDR gene. Perhaps as important, deletion of the VDRE within the S1/S2 enhancer in the mouse VDR BAC clone reduced the level of induction of the luciferase reporter to 1,25(OH)2D3 by over 60% relative to the wild-type VDR BAC clone when stably transfected into the same host cells (see Fig. 7.12B). Additional studies are likely to reveal the importance of other regulatory regions within the VDR gene in modulating activity by other steroid and peptide hormones as well. Thus, recombineered BAC clones represent a promising approach to exploring the individual and collective contributions of enhancers in genes that contain more than one control unit both in cell lines as well as in transgenic mouse models.
(A)
23 kb
67 kb
3'
5' Hdac7a
Col2a1 mVDR VDR BAC2 mVDR VDR BAC3 mVDR VDR BAC4 ΔS1 VDRE = IRES/Luc/Tk/Neo cassette mVDR VDR BAC2
(B)
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mVDR VDR BAC4
5 Fold Induction
well [60]. CREB also localized to the VDR gene promoter, a finding consistent with previous studies in the human gene by Christakos and colleagues [328]. Finally, the transcription factors RUNX2 and C/EBPb were also found across a subset of the enhancers identified for the above hormonal activators [60]. The binding of these factors may provide clues as to the nature of several signaling pathways that are likely operable in bone cells to control basal VDR gene expression and could facilitate the discovery of additional basal factors responsible for VDR gene expression in other cell types as well. The results of these studies also highlight the characteristic of modularity that is typical of most enhancers. Thus most enhancers contain binding sites for multiple transcription factors which can act additively, but more frequently in synergy with or in opposition to each other to control the expression of an enhancer’s target gene.
4 3 2 1
0 1,25(OH)2D3 (10-7 M)
–
+
–
+
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Wild-type and mutant VDR gene BAC clone structures and activities. (A) Structure of the endogenous mouse VDR gene locus and several mVDR BAC clone constructs examined. All contain a recombineered IRES/Luciferase/TK/Neo activity/selection cassette. mVDR BAC3 contains a deletion of all intergenic sequence downstream of the final VDR exon. mVDR BAC4 contains an additional deletion of the 15 bp VDRE sequence in the S1 regulatory region (delta S1 VDRE). (B) Activity of mVDR BAC2e4 in MC3T3-E1 cells. MC3T3-E1 cells were used to prepare collections of mVDR BAC2, mVDR BAC3, and mVDR BAC4 stable cells (>30) using G418. The cell lines were treated with either vehicle (e) or 1,25(OH)2D3 (þ) (10e7 M) and then harvested for luciferase activity and total protein 24 h later. Fold induction is indicated.
FIGURE 7.12
BAC Clones as VDR Transgenes in Mice The expression properties and regulatory behavior of the mouse and human VDRs and their reporters in transfected cells in vitro suggested that these recombineered segments of DNA might also be useful in the preparation of unique mouse transgenic lines. To this end, mouse and human VDR BAC clone DNAs were utilized to create multiple transgenic mouse lines containing functional copies of each of the transgenes [329,330]. Preliminary investigation revealed that each VDR BAC clone directed the synthesis of levels of recombinant VDR protein that were similar to those expressed endogenously; they also expressed significant levels of the luciferase reporter. Perhaps most importantly, immunohistochemical analysis revealed that expression of BAC clone-derived VDR and its reporter recapitulated the tissue-specific expression of the endogenous gene in the intestinal tract, kidney proximal and
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distal tubules, osteoblasts, parathyroid cells, pancreatic insulin-producing b cells, keratinocytes, and hair follicle epithelial cells and in other cell types as well. No VDR or luciferase activity was observed in either muscle or liver, consistent with the idea that the effects of 1,25(OH)2D3 in these tissues are likely indirect. These mouse models of VDR gene expression as well as additional models that assess the consequences of introducing mutations into these VDR BAC clones will be useful in defining not only the regulatory components necessary for tissue-selective expression of the VDR gene, but will also be useful in exploring the functional consequences of mutations within the coding regions of the mouse and human VDR proteins in vivo. They may also be useful in defining the effects of RFLPs and SNPs that are often found within the human VDR gene locus (see Chapter 56).
Post-translational Stability and Covalent Post-translational Modifications of the VDR In addition to its ability to stimulate VDR gene transcription, as discussed at length in the foregoing sections, 1,25(OH)2D3 also strongly up-regulates VDR protein levels and promotes selective phosphorylation of the VDR. These latter two events appear independent of transcription as they occur in the presence of actinomycin D and correlate temporally with receptor ligand binding [317,331]. Early studies in cell extracts suggested that ligand binding desensitized the VDR to proteolysis. Subsequent studies have shown that 1,25 (OH)2D3 binding alters the half-life of the VDR protein and that the protein interacts with components of the 19S proteasome such as SUG1 and is degraded by it as well [228]. Whether altered sensitivity to proteasomal degradation represents the underlying basis for ligandinduced stabilization is unknown, as 1,25(OH)2D3 also induces translocation of the VDR to the nucleus where it binds to sites on DNA. Perhaps this change in cellular compartmentalization plays a role in the accumulation of VDR protein levels. The VDR is also phosphorylated at several distinct sites, including S51, S182, and S208 (see Chapter 8) [332e338]. The site at S208 is contained within a casein kinase II phosphorylation consensus sequence and its phosphorylation appears to enhance the VDR’s transcriptional capabilities, although only modestly. Additional studies suggest that phosphorylation at this site alters the ability of the VDR to interact with coregulators such as MED-1 [339]. Surprisingly, although evidence suggests that the mouse VDR can be phosphorylated in the presence of 1,25(OH)2D3, the S208 site is not conserved in VDRs from either the mouse or the rat. Are post-translational stabilization and covalent modification of the VDR directly linked? Experiments to address this question remain to be
conducted. It would not be surprising, however, as the stabilization of many, if not most, proteins frequently involves post-translational modification at one or more amino acid residues.
CONCLUDING COMMENTS The basic elements of the mechanism of action of vitamin D have been defined. The pace of exploration into the actions of vitamin D accelerated enormously during the mid 1980s and 1990s, largely as a result of the molecular cloning of the VDR but also as a result of the availability of cloned vitamin D target genes. Through studies conducted during this time, we have gained considerable insight into the structure of the VDR and its organization into definable functional domains. The now available crystal structure of the VDR LBD has placed much of the biochemical and molecular biologic investigation into perspective. We have yet to determine the structure of the VDR/RXR heterodimer bound to its cognate response element, however, or to determine the structure of the VDR using solution approaches such as NMR. The availability of recombinant clones has allowed investigation of the interaction of the VDR with vitamin-D-inducible gene promoters and the definition of VDREs. Further studies have revealed that the VDR requires a protein partner for DNA binding in the form of RXR, a central regulator of several nuclear receptors. The application of new methodologies such as that of ChIP-chip and ChIP-seq analyses, chromosome conformation capture analysis (3C analysis and beyond) and BAC clone analysis now confirm many, although not all, of the fundamental concepts that emerged during the Biochemical and Molecular Biological Eras. Thus, for example, while the relationship between the VDR and RXR was confirmed, the idea that regulatory regions are routinely located in linear terms near the promoters they regulate has been entirely disproved. Indeed, most genes are regulated by a number of enhancers often located 10s if not 100s of kilobases away from the promoters they regulate. Some may be located in trans on other chromosomes. Perhaps most important, these new techniques are now enabling a detailed view of the interaction of the VDR/RXR heterodimer with not only specific target gene loci but also at sites across entire genomes. It is also clear that the expression of genes by the vitamin D hormone is significantly impacted by complex layers of epigenetic modifications that function, in part, to establish the regulatory structures that are so crucial to the expression of genes. Surprisingly, despite new insights into how genes are regulated, the question of how vitamin D analogs, which are believed to promote unusual conformations within the VDR protein,
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REFERENCES
generate tissue-selective activities still remains an enigma. Indeed, many of the proposed mechanisms appear to be flawed. Hopefully, new mechanisms that account for these selective actions will be identified in the coming years so that vitamin D analogs can be developed as useful therapeutic agents for a broad range of vitamin D indications that include skin diseases, immunologic disorders, and cancer. The cloning of the VDR also enabled the recovery of its chromosomal gene. Characterization of this gene enabled subsequent investigations into the nature of the human syndrome of HVDRR and revealed that the cause was mutations that produce dysfunctional receptor protein. This discovery, together with the genetic ablation of the VDR gene in mice, confirmed the central role of the VDR in the regulation of mineral metabolism. Importantly, ChIP-chip/ChIP-seq methodologies have proven to be essential in understanding how the VDR gene is transcriptionally regulated by both 1,25(OH)2D3 as well as other steroid and peptide hormones. The construction and utilization of mouse and human VDR gene BAC clones both in vitro and in vivo in the mouse will enable in vivo confirmation of the studies conducted heretofore in vitro, to assess the molecular determinants of VDR gene expression in primary tissues such as the intestine, kidney, and bone and to study mechanisms associated with liganddependent changes in stability and covalent modification. In view of our current understanding of VDR action on target gene promoters, future studies are likely to focus on the mechanisms whereby enhancers function to modulate gene expression. These include the roles of coregulators, the role of RNA pol II, the role of numerous RNA transcripts that are produced at enhancers, and the role of nuclear subdomains that are likely to orchestrate the processes of transcription at the cellular level. Perhaps as important, future studies are likely to focus also on more fundamental details, such as the role(s) of epigenetic modifications in creating regulatory enhancers that are capable of selectively controlling the expression of transcriptomes. Indeed, it is not the expression of a gene that determines a cell’s phenotype, but rather the creation of enhancers that provides the means through which these genes can be regulated. These concepts are likely to be more fully developed in the coming decade. Elucidation of many of these processes will be essential to assessing how 1,25(OH)2D3 controls gene expression in different cell types and perhaps how vitamin D analogs can exploit these complex activities to achieve unique biological outcomes. Also likely to emerge from these studies will be a better understanding of the regulation of the VDR gene at both the transcriptional and post-translational levels and the contribution of this regulation to
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the mechanism of vitamin D action. Perhaps more important is the likelihood that we will determine how vitamin D controls directly as well as indirectly the expression of broad networks of genes, and integrates their activities in the regulation of proliferation, differentiation, and complex mature cell function. Thus, while significant progress has been made in the past few years there is still much to accomplish.
Acknowledgment The work of the authors cited in this article was supported by numerous NIH grants to JWP. The authors thank past and current members of the Pike Laboratory for their contributions to this work. We also acknowledge Laura Vanderploeg for her artistic contributions to this work.
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a residue crucial to its trans-activation function, Proc. Natl. Acad. Sci. USA 88 (1991) 9315e9319. J.C. Hsieh, P.W. Jurutka, S. Nakajima, M.A. Galligan, C.A. Haussler, Y. Shimizu, et al., Phosphorylation of the human vitamin D receptor by protein kinase C. Biochemical and functional evaluation of the serine 51 recognition site, J. Biol. Chem. 268 (1993) 15118e15126. J.C. Hsieh, H.T. Dang, M.A. Galligan, G.K. Whitfield, C.A. Haussler, P.W. Jurutka, et al., Phosphorylation of human vitamin D receptor serine-182 by PKA suppresses 1,25 (OH)2D3-dependent transactivation, Biochem. Biophys. Res. Commun. 324 (2004) 801e809. P.W. Jurutka, J.C. Hsieh, P.N. MacDonald, C.M. Terpening, C.A. Haussler, M.R. Haussler, et al., Phosphorylation of serine 208 in the human vitamin D receptor. The predominant amino acid phosphorylated by casein kinase II, in vitro, and identification as a significant phosphorylation site in intact cells, J. Biol. Chem. 268 (1993) 6791e6799. P.W. Jurutka, J.C. Hsieh, M.R. Haussler, Phosphorylation of the human 1,25-dihydroxyvitamin D3 receptor by cAMP-dependent protein kinase, in vitro, and in transfected COS-7 cells, Biochem. Biophys. Res. Commun. 191 (1993) 1089e1096. P.W. Jurutka, J.C. Hsieh, S. Nakajima, C.A. Haussler, G.K. Whitfield, M.R. Haussler, Human vitamin D receptor phosphorylation by casein kinase II at Ser-208 potentiates transcriptional activation, Proc. Natl. Acad. Sci. USA 93 (1996) 3519e3524. R. Cook, G.T. Hilliard, N. Weigel, J. Pike, 1,25-Dihydroxyvitamin D3 modulates phosphorylation of serine 205 in the human vitamin D receptor: site-directed mutagenesis of this residue promotes alternative phosphorylation, Biochemistry 33 (1994) 4300e4311. G. Arriagada, R. Paredes, J. Olate, A. van Wijnen, J.B. Lian, G.S. Stein, et al., Phosphorylation at serine 208 of the 1alpha,25dihydroxy vitamin D3 receptor modulates the interaction with transcriptional coactivators, J. Steroid Biochem. Mol. Biol. 103 (2007) 425e429.
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C H A P T E R
8 Nuclear Vitamin D Receptor: Natural Ligands, Molecular StructureeFunction, and Transcriptional Control of Vital Genes Mark R. Haussler 1, G. Kerr Whitfield 1, Carol A. Haussler 1, Jui-Cheng Hsieh 1, Peter W. Jurutka 2 1
2
Department of Basic Medical Sciences, College of Medicine-Phoenix, University of Arizona, Phoenix, USA Division of Mathematical and Natural Sciences, Arizona State University at the West Campus, Glendale, AZ, USA
LIGANDS, GENE TARGETS, AND BIOLOGICAL ACTIONS OF VDR Endocrine Regulation of Vitamin D Metabolism and Overview of 1,25(OH)2D3 Actions in Target Tissues The vitamin D receptor (VDR) is a chromosomal protein [1] that binds 1a,25-dihydroxyvitamin D3 (1,25 (OH)2D3), the active vitamin D metabolite produced mainly in the kidney [2]. VDR is a DNA-binding transcription factor which generates an active signal transduction complex consisting of a heterodimer of the 1,25(OH)2D3-liganded VDR and unoccupied retinoid X receptor (RXR). An overview of the formation of the VDR ligand, 1,25(OH)2D3, and its actions in vertebrates with a mineralized skeleton is presented in Figure 8.1. The hormonal precursor and parent compound, vitamin D3, either can be obtained in the diet or formed from 7dehydrocholesterol in skin (epidermis) via a nonenzymatic, UV-light-dependent reaction. Vitamin D3 is then transported to the liver, where it is hydroxylated at the C-25 position of the side chain to produce 25-hydroxyvitamin D3 (25(OH)D3), the major circulating form of vitamin D3. The final step in the production of the hormonal form occurs mainly, but not exclusively, in the kidney, via a tightly regulated 1a-hydroxylation reaction (Fig. 8.1). The cytochrome P450-containing (CYP) enzymes that catalyze 25- and 1a-hydroxylations
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10008-3
are microsomal CYP2R1 [3] and mitochondrial CYP27B1, respectively. These reactions are discussed in detail in Chapter 3 of this volume. As depicted in Figure 8.1, 1,25(OH)2D3 circulates, bound to plasma vitamin-D-binding protein (DBP), to various target tissues to exert its actions which are largely mediated by the nuclear VDR and its RXR heterodimeric partner. Many of the long-recognized functions of 1,25(OH)2D3 involve the regulation of calcium and phosphate metabolism, raising the blood levels of these ions to facilitate bone mineralization, as well as activating bone resorption as part of the remodeling cycle [4]. The parathyroid gland also expresses VDR, and when the receptor is liganded with 1,25(OH)2D3, parathyroid hormone (PTH) synthesis is suppressed by a direct action on gene transcription [5]. This negative feedback loop, which limits the stimulation of CYP27B1 by PTH under low-calcium conditions (Fig. 8.1), serves to limit the bone-resorbing effects of PTH in anticipation of 1,25(OH)2D3-mediated increases in both intestinal calcium absorption and bone resorption, thus preventing hypercalcemia. Since the 2nd edition of Vitamin D was published, new understanding of the homeostatic control of phosphate has emerged, emanating originally from characterization of unsolved familial hypo- or hyperphosphatemic disorders which we now know are caused by deranged levels of bone-derived FGF23 [6]. In short, FGF23 has materialized as a dramatic new phosphate regulator, and a second phosphaturic
137
Copyright Ó 2011 Elsevier Inc. All rights reserved.
138
Vitamin D acquisition, regulation of metabolic activation/catabolism, and receptor-mediated endocrine and intracrine actions of the 1,25(OH)2D3 hormone.
8. NUCLEAR VITAMIN D RECEPTOR: NATURAL LIGANDS, MOLECULAR STRUCTUREeFUNCTION, AND TRANSCRIPTIONAL CONTROL
II. MECHANISMS OF ACTION
FIGURE 8.1
LIGANDS, GENE TARGETS, AND BIOLOGICAL ACTIONS OF VDR
hormone after PTH. We [7] and others [8] proved that 1,25(OH)2D3 induces the release of FGF23 from bone, specifically from osteocytes of the osteoblastic lineage (Fig. 8.1), a process that is independently stimulated by high circulating phosphate levels (Fig. 8.1). Thus, in a striking and elegant example of biological symmetry, PTH is repressed by 1,25(OH)2D3 and calcium, whereas FGF23 is induced by 1,25(OH)2D3 and phosphate, protecting mammals against hypercalcemia and hyperphosphatemia, respectively, either of which can elicit ectopic calcification. In addition to effecting bone mineral homeostasis by functioning at the small intestine, kidney, parathyroid gland, and bone, 1,25(OH)2D3 also acts through its VDR mediator to influence a number of other cell types. These extraosseous actions of 1,25(OH)2D3-VDR include differentiation of certain cells in skin [9] and in the immune system [10] (Fig. 8.1). Interestingly, the skin and the immune system are now recognized as extrarenal sites of CYP27B1 action to produce 1,25(OH)2D3 locally for autocrine and paracrine effects [11,12], creating intracrine systems (Fig. 8.1) for extraosseous 1,25(OH)2D3-VDR functions distinct from the renal endocrine actions of 1,25(OH)2D3-VDR on the small intestine and skeleton. Apparently, higher circulating 25(OH)D3 levels are required for optimal intracrine actions of 1,25(OH)2D3 (Fig. 8.1). This insight stems from the importance of attaining adequate levels of circulating 25(OH)D3 revealed in a multitude of epidemiologic associations between low 25(OH)D3 levels and chronic disease, coupled with statistically significant protection against a host of pathologies by much higher circulating 25(OH)D3 [13]. Thus, as depicted schematically in Figure 8.1, locally produced 1,25 (OH)2D3 appears to be capable of benefiting the vasculature to reduce the risk of heart attack and stroke, controlling the adaptive immune system to lower the incidence of autoimmune disease while boosting the innate immune system to fight infection, effecting xenobiotic detoxification, and exerting anti-inflammatory and anticancer pressure on epithelial cells prone to fatal malignancies (these effects of 1,25(OH)2D3-VDR are discussed in other chapters in this volume). As illustrated in Figure 8.1, using the kidney as an example, an important mechanism by which the 1,25 (OH)2D3/VDR-mediated endocrine or intracrine signal is terminated in all target cells is the catalytic action of CYP24A1, an enzyme that initiates the process of 1,25 (OH)2D3 catabolism (see Chapter 4). The CYP24A1 gene is transcriptionally activated by 1,25(OH)2D3 [14, 15], as well as by FGF23 (Fig. 8.1). In addition, the 1a-hydroxylase (1a-OHase) CYP27B1 gene is repressed by FGF23 and 1,25(OH)2D3, with the latter regulation effected by epigenetic demethylation [16] in a short negative feedback loop to limit the production of 1,25(OH)2D3 [17].
139
Therefore, the vitamin D endocrine system is elegantly governed by feedback controls of vitamin D bioactivation which interpret bone mineral ion status, and via feedforward induction of 1,25(OH)2D3 catabolism to prevent the pathologies of hypervitaminosis D. The vitamin D intracrine system, in contrast, appears to be dependent more on the availability of ample 25(OH)D3 substrate to generate local 1,25(OH)2D3 to lower the risk of chronic diseases of the epithelial (skin, colon, etc.), immune, cardiovascular, and possibly nervous systems.
VDR: Structure Overview, Gene Targets/ VDREs, Novel Ligands and Actions Relevant to Disease An introductory overview of human VDR, the focus of the present chapter, is illustrated in Figure 8.2. Various domains of the 427-amino-acid VDR are highlighted on a linear schematic of the protein (Fig. 8.2), with the two major functional units being the N-terminal zinc finger DNA binding domain, and the C-terminal ligand binding domain. To date, the Protein Data Bank (PDB) database contains 36 X-ray crystal structures for the VDR ligand-binding domain (LBD) and four of the DNA-binding domain (DBD) bound as a homo- or heterodimer on vitamin-D-responsive element (VDRE) DNA sequences. The original X-ray crystallographic structure of the VDR LBD consisting of 12a-helices [18] has been updated (PDB #3A78) and now contain 15ahelices, including three new short segments of 3-4 amino acids each between residues 150 and 290 of the human receptor. To be consistent with the literature, in this chapter we will retain the original nomenclature based on 12a-helices. As such, the VDR LBD is a sandwich-like structure presenting VDR surfaces for heterodimerization with RXR (predominantly helices (H) 9 and 10 and the loop between helices 8 and 9) as well as for transactivation via interaction with coactivators (CoAct). Coactivator interfaces in VDR consist of portions of helices H3, H5, and H12 (the last constituting the AF-2 or activation function-2 domain), plus a region immediately N-terminal of the zinc fingers (residues 18e22). Human VDR also complexes with basal transcription factors such as TFIIB (near the N-terminus of VDR), as well as with transcriptional corepressors such as the hairless (Hr) gene product, which associates with the VDR hinge and H3/H5 (Fig. 8.2). The ligand-binding domain of human VDR has been co-crystallized with 1,25(OH)2D3 [18] as well as with many vitamin D analogs occupying the hydrophobic pocket, and lithocholic acid has been shown to associate compatibly with VDR as determined by molecular modeling [19] and a recent X-ray crystal structure [20]. As discussed in detail below, we have identified several additional nutritional lipids as candidate low-affinity VDR ligands
II. MECHANISMS OF ACTION
8. NUCLEAR VITAMIN D RECEPTOR: NATURAL LIGANDS, MOLECULAR STRUCTUREeFUNCTION, AND TRANSCRIPTIONAL CONTROL
DNA Binding Domain (DBD)
Ligand Binding Domain
Transactivation
TFIIB +
Zn
Heterodimerization with RXRs
Zn
Hinge
T A
H3 N
1 24
Co-Act
Hr 89
111
159
Gene Bioeffect SPP1 BGP Bone RANKL Metabolism LRP5 Intestinal Ca2+ TRPV6 Transport FGF23 Phosphate Npt2c Homeostasis PTHrP Mammalian SOSTDC1 Hair Cycle S100A8 p21 Cell Cycle Control CYP24A1 1,25D Detoxification CYP3A4 Xenobiotic Detoxification Klotho Longevity
loop
140
H3 H5
H9&10 AF-2
201
COO–
427 OH
COOH
HO HO
OH
1a,25-Dihydroxyvitamin D3
Lithocholic Acid
COOH
COOH 20
22
Docosahexaenoic Acid “w3”
Arachidonic Acid “w6”
H
O
HO O
CH3
O
CH3 O OH
HO O
CH3
CH3
g-Tocotrienol
Curcumin
Functional domains in human VDR. Highlighted at the left is the human VDR zinc finger DNA-binding domain which, in cooperation with the corresponding domain in the RXR heteropartner, mediates direct association with the target genes listed at the lower left, leading to the indicated physiological effects. The official gene symbol for BGP is BGLAP, for RANKL is TNFSF11, for Npt2c is SLC34A3, for PTHrP is PTHLH, and for klotho is KL. Below the ligand-binding domain (at the right) are illustrated selected VDR ligands, including several novel ligands discussed in the text.
FIGURE 8.2
which may function locally in high concentrations. Figure 8.2 reveals that these novel putative VDR ligands include u3- and u6-essential polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (DHA), and arachidonic acid, respectively, the vitamin E derivative g-tocotrienol, and curcumin [21] which is a turmericderived polyphenol found in curry. The liganding of VDR triggers tight association between VDR and its heterodimeric partner, RXR, and only this liganded VDR-RXR heterodimer is able to penetrate the deep groove of DNA and recognize VDREs in the DNA sequence of vitamin-D-regulated genes [22,23]. Figure 8.2 provides a partial list of VDR-RXR target genes recognized by the combined zinc fingers of the two receptors and their T-box and A-box C-terminal extensions. These VDR-RXR-controlled genes encode proteins which determine bone growth and remodeling, intestinal calcium absorption, phosphate homeostasis, the mammalian hair cycle, cell proliferation, lipid detoxification, and possibly longevity. Table 8.1 constitutes
a more comprehensive list of 50 VDREs residing in or near vitamin D target genes, specifically those that are known to be under primary control by VDR-RXR via characterized VDREs. In general, VDREs possess either a direct repeat of two hexanucleotide half-elements with a spacer of three nucleotides (DR3) or an everted repeat of two half-elements with a spacer of six nucleotides (ER6), with DR3 motifs being the most common. One notable exception to this rule is the reported (putative) DR4 VDRE at e7853 in the hMDR1 (P-glycoprotein) drug resistance gene (Table 8.1). In positive DR3 VDREs, VDR has been shown to occupy the 30 half-element, with RXR residing on the 50 half-site [24]. The “optimal” VDRE, which was experimentally determined via binding of randomized oligonucleotides to a VDR-RXR heterodimer [25,26], is in general agreement with the repertoire of natural VDREs (Table 8.1) and defines the optimal VDRE as a direct repeat of two six-base halfelements that resemble estrogen-responsive element (ERE) half-sites, i.e., AGGTCA, separated by a spacer of
II. MECHANISMS OF ACTION
LIGANDS, GENE TARGETS, AND BIOLOGICAL ACTIONS OF VDR
three nucleotides. The highest affinity 30 (VDR) half-site is PGTTCA, where P is a purine base, and the highest affinity 50 (RXR) half-site is PGGTCA. In addition, the random selection results suggest that the guanine at position 3 of the spacer (shown in lower case in Table 8.1) is important for VDR binding, an observation that is consistent with the finding [27,28] that this base is partially protected by RXR-VDR in methylation interference assays. In contrast to the human CYP3A4 ER6 VDRE, in which both half-elements coincide with the randomly selected sequences, the half-sites that exist in natural DR3 elements usually contain one to three bases that do not match the optimal VDRE. These variant bases occur in both half-sites, with the 30 half-site comprising the majority (60%) of the dissimilarities. The multiple sequence variations in natural VDREs (Table 8.1) may provide a spectrum of affinities for the VDR-RXR heterodimer, thus enabling these elements to respond to differing concentrations of the receptors (or their ligands) [29]. Another possibility, for which increasing evidence is accumulating, is that variant VDRE sequences induce unique conformations in the VDR-RXR complex, thereby promoting association of the heterodimer with distinct subsets of coactivators [30], or permitting differential actions in the context of diverse tissues [29]. Many VDREs occur as a single copy in the proximal promoter of vitamin-D-regulated genes, for instance those in mouse osteocalcin (BGP), rat RUNX2 (rRUNX2), chicken PTH (cPTH), human sodium phosphate cotransporter 2c (hNpt2c), human p21 (hp21), and rat PTHrP (rPTHrP). However, the rat and human CYP24A1 VDREs are at least bipartite, as is the human CYP3A4 VDRE, with the 50 DR3 located some 7.5 kb upstream of the proximal ER6 VDRE in the latter case. Studies of these vitamin-D-controlled CYP genes introduced the concepts of multiplicity and remoteness to VDREs, as confirmed more recently by ChIP or ChIP scanning [31e34] of genomic DNA surrounding the transient receptor potential vanilloid type 6 (TRPV6), LRP5 and receptor activator of nuclear factor kB ligand (RANKL) genes, uncovering novel VDREs at some distance from the transcription start site (Table 8.1). Genes possessing multiple VDREs require all VDR-RXR docking sites for maximal induction by 1,25(OH)2D3 and the individual VDREs appear to function synergistically in attracting coactivators and basal factors for transactivation. The most attractive hypothesis is that remote VDREs are juxtapositioned with more proximal VDREs via DNA looping in chromatin, creating a single platform that supports the transcription machine, and this conclusion has been verified by chromatin conformation capture studies from two independent groups [35,36]. The concept, from analyzing a number of VDREs, is that the docking sequences for VDR-RXR in DNA consist of clearly defined DR3 and ER6 motifs, often in multiple
141
copies dispersed up to 100 kb 50 or 30 of the transcription start site in vitamin-D-controlled genes. In fact, the only boundaries on functional VDREs may be CCCTC-binding factor sites known as “insulators” that demarcate individual and clusters of genes in the genome [37]. Regulation of the expression of many of these genes by 1,25 (OH)2D3 in traditional vitamin D target tissues such as kidney, bone, parathyroid, skin, and the hematopoietic system has been well established [38], and the combination of Figure 8.2 and Table 8.1 paints a picture of the biological breadth of VDR-mediated gene control. Obviously, the 1,25(OH)2D3-VDR/RXR complex modulates the expression of a multitude of genes, and microarray studies [39e43] suggest that the number of vitamin-D-regulated genes is much greater than the well-characterized bioresponses discussed herein. Nevertheless, the catalogue of VDRE-containing genes listed in Table 8.1 can be grouped into the major biological realms influenced by VDR as follows: (a) bone, (b) mineral, (c) detoxification, (d) cell life (proliferation, differentiation, migration, and death), (e) immune, and (f) metabolism (amino acid, lipid, and carbohydrate). In toto, it is clear that VDR affects some of the most fundamental processes in life. As described below, the use of VDR-null animals has both validated the obligatory role of VDR in 1,25 (OH)2D3 signaling, and revealed which VDR responses are the most pathophysiologically relevant.
Consequences of VDR Ablation in Rodents and Humans Important insight into the biological impact of VDR to mediate the myriad of gene regulatory effects of 1,25 (OH)2D3 discussed above can be gained by examining the phenotype of mice in which the VDR has been ablated (VDR knockouts), as well as the phenotype of human patients with hereditary hypocalcemic vitaminD-resistant rickets (HVDRR). HVDRR is a consequence of defects in the VDR gene on human chromosome 12 [44e47]. Clinically significant HVDRR is an autosomal recessive disorder resulting in severe bowing of the lower extremities, short stature, and often alopecia [46]. The serum chemistry in HVDRR reveals hypocalcemia, secondary hyperparathyroidism, elevated alkaline phosphatase, variable hypophosphatemia, and markedly increased 1,25(OH)2D3. Many of these sequelae are the expected manifestations of the loss of 1,25(OH)2D3 regulation in the target genes outlined above, such as PTH, RANKL, CYP24, etc. Most aspects of HVDRR, with the exception of alopecia, mimic those of classic vitamin-Ddeficiency rickets, suggesting that VDR not only mediates all of the bone mineral homeostatic actions of vitamin D, but may also participate in hair follicle function in a manner independent of vitamin D status. As detailed later in this chapter, individual natural VDR
II. MECHANISMS OF ACTION
142
8. NUCLEAR VITAMIN D RECEPTOR: NATURAL LIGANDS, MOLECULAR STRUCTUREeFUNCTION, AND TRANSCRIPTIONAL CONTROL
TABLE 8.1
VDREs in Genes Directly Modulated in their Expression by 1,25(OH)2D3 and Possibly Other VDR Ligands
Gene
Bioeffect
Type
Location
50 -Half
Spacer
30 -Half
REF
Group
rBGP
Bone metabolism
Positive
e456
GGGTGA
atg
AGGACA
[178]
Bone
mBGP
Bone metabolism
Negative
e444
GGGCAA
atg
AGGACA
[239]
Bone
hBGP
Bone metabolism
Positive
e485
GGGTGA
acg
GGGGCA
[240]
Bone
mSPP1
Bone metabolism
Positive
e757
GGTTCA
cga
GGTTCA
[241]
Bone
mSPP1
Bone metabolism
Positive
e2000
GGGTCA
tat
GGTTCA
[242]
Bone
mLRP5
Bone anabolism
Positive
þ656
GGGTCA
ctg
GGGTCA
[34]
Bone
mLRP5
Bone anabolism
Positive
þ19 kb
GGGTCA
tgc
AGGTTC
[32]
Bone
rRUNX2
Bone anabolism
Negative
e78
AGTACT
gtg
AGGTCA
[243]
Bone
mRANKL
Bone resorption
Positive
e22.7 kb
TGACCT
cctttg
GGGTCA
[90]
Bone
mRANKL
Bone resorption
Positive
e76 kb
GAGTCA
ccg
AGTTGT
[161]
Bone
mRANKL
Bone resorption
Positive
e76 kb
GGTTGC
ctg
AGTTCA
[161]
Bone
cIntegrin-beta3
Bone resorption, platelet aggregation
Positive
e756
GAGGCA
gaa
GGGAGA
[244]
Bone
cCarbonic anhydrase II
Bone resorption, brain function
Positive
e39
AGGGCA
tgg
AGTTCG
[245]
Bone
cPTH
Mineral homeostasis
Negative
e60
GGGTCA
gga
GGGTGT
[246]
Mineral
mVDR
Autoregulation of VDR
Positive
þ8467
GGGTTA
gag
AGGACA
[166]
Mineral
hTRPV6
2þ
Intestinal Ca transport
Positive
e1270
AGGTCA
ttt
AGTTCA
[31]
Mineral
hTRPV6
Intestinal Ca2þ transport
Positive
e2100
GGGTCA
gtg
GGTTCG
[31]
Mineral
hTRPV6
Intestinal Ca2þ transport
Positive
e2155
AGGTCT
tgg
GGTTCA
[31]
Mineral
hTRPV6
Intestinal Ca2þ transport
Positive
e4287
GGGGTA
gtg
AGGTCA
[31]
Mineral
hTRPV6
Intestinal Ca2þ transport
Positive
e4337
CAGTCA
ctg
GGTTCA
[31]
Mineral
hNpt2a
Renal phosphate reabsorbtion
Positive
e1963
GGGGCA
gca
AGGGCA
[247]
Mineral
hNpt2c
Renal phosphate reabsorbtion
Positive
e556
AGGTCA
gag
GGTTCA
[34]
Mineral
rCYP24A1
1,25D detoxification
Positive
e151
AGGTGA
gtg
AGGGCG
[14]
Detox
rCYP24A1
1,25D detoxification
Positive
e238
GGTTCA
gcg
GGTGCG
[15]
Detox
hCYP24A1
1,25D detoxification
Positive
e164
AGGTGA
gcg
AGGGCG
[140]
Detox
hCYP24A1
1,25D detoxification
Positive
e285
AGTTCA
ccg
GGTGTG
[140]
Detox
hCYP3A4
Xenobiotic detoxification
Positive
e169
TGAACT
caaagg
AGGTCA
[62,72]
Detox
hCYP3A4
Xenobiotic detoxification
Positive
e7.7 kb
GGGTCA
gca
AGTTCA
[71]
Detox
rCYP3A23
Xenobiotic detoxification
Positive
e120
AGTTCA
tga
AGTTCA
[62,248]
Detox
hMDR1
P-glycoprotein, drug resistance
Positive
e7863
AGTTCA
atg
AGGTAA
[81]
Detox
II. MECHANISMS OF ACTION
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LIGANDS, GENE TARGETS, AND BIOLOGICAL ACTIONS OF VDR
TABLE 8.1 VDREs in Genes Directly Modulated in their Expression by 1,25(OH)2D3 and Possibly Other VDR Ligandsdcont’d Gene
Bioeffect
Type
Location
50 -Half
Spacer
30 -Half
REF
Group
hMDR1
P-glycoprotein, drug resistance
Positive
e7853
AGGTCA
agtt
AGTTCA
[81]
Detox
hp21
Cell cycle control
Positive
e765
AGGGAG
att
GGTTCA
[63]
Cell life
hFOXO1
Cell cycle control
Positive
e2856
GGGTCA
cca
AGGTGA
[249]
Cell life
hIGFBP-3
Cell proliferation/ apoptosis
Positive
e3282
GGTTCA
ccg
GGTGCA
[250]
Cell life
hInvolucrin
Skin barrier function
Positive
e2083
GGCAGA
tct
GGCAGA
[251]
Cell life
hPLD1
Keratinocyte differentiation
Positive
e246
GGGTGA
tgc
GGTCGA
[252]
Cell life
hCCR10
Homing of T-cells to skin
Positive
e110
GGGTCT
acg
GGGTCA
[253]
Cell life
rPTHrP
Mammalian hair cycle
Negative
e805
AGGTTA
ctc
AGTGAA
[217]
Cell life
hSOSTDC1
Mammalian hair cycle
Negative
e6214
AGGACA
gca
GGGACA
[90]
Cell life
rVEGF
Angiogenesis
Positive
e2730
AGGTGA
ctc
AGGGCA
[254]
Cell life
hMIS
Mu¨llerian-inhibiting substance
Positive
e381
GGGTGA
gca
GGGACA
[255]
Cell life
hHLA-DRB1
Major histocompatibility complex
Positive
e1
GGGTGG
agg
GGTTCA
[256]
Immune
hCAMP
Antimicrobial peptide
Positive
e615
GGTTCA
atg
GGTTCA
[257]
Immune
hKSR-1
Monocytic differentiation
Positive
e8156
GGTGCA
tat
AGGTCA
[258]
Immune
hKSR-2
Monocytic differentiation
Positive
e2501
AGTTCA
gca
TGGTCA
[259]
Immune
hKSR-2
Monocytic differentiation
Positive
þ3185
GGTTCA
aac
AGTTCT
[259]
Immune
mInsig-2
Regulation of lipid synthesis
Positive
e2470
AGGGTA
acg
AGGGCA
[260]
Metabolism
hPCFT
Intestinal folate transporter
Positive
e1680
AGGTTA
ttc
AGTTCA
[261]
Metabolism
hCystathionine b synthase
Homocysteine clearance
Positive
þ2563
AGGGCA
gtg
AGGACA
[236]
Metabolism
hCystathionine b synthase
Homocysteine clearance
Positive
þ7849
GGGACA
gat
AGTTCA
[236]
Metabolism
mutations observed in HVDRR have been very instructive in the elucidation of structureefunction relationships within the receptor, especially with regard to DNA, ligand, and coactivator binding. Several strains of homozygous VDR knockout mice have been created [48e50] and their phenotype has been characterized under various conditions. Somewhat surprisingly, the VDRe/e phenotype, except for alopecia, could be reversed if blood calcium levels were normalized by a rescue diet [51,52]. The successful rescue of VDR-null mice, in relation to bone mineral homeostasis, suggests that the dominant biological
action of 1,25(OH)2D3-VDR is to promote intestinal calcium and phosphate absorption, especially under conditions of low calcium availability. With respect to alopecia, it was observed that lack of VDR prevented hair follicles from entering the anagen phase of the hair cycle [53], but transgenic VDR expression targeted to keratinocytes in the background of a VDR-null animal was successful in restoring hair cycling [54]. These results not only confirmed the essential role of VDR in maintaining hair (and skin) health, but also pinpointed keratinocytes as an important cellular target for VDR action in skin.
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Subsequent studies using VDRe/e animals fed the rescue diet to normalize calcium have revealed additional roles of VDR that are not simply secondary effects of maintaining serum calcium and phosphate levels. VDRe/e mice, when compared to normal mice: (a) exhibit uterine hypoplasia [49], probably linked to reduced expression of aromatase (CYP19) [55]; (b) display hyperplasia of the mammary glands [56]; (c) have blunted insulin synthesis in pancreatic beta cells [57]; (d) have reduced synthesis of macrophage IL-18 and STAT4 in T helper subset type 1 (Th1) lymphocytes, leading to an impaired Th1 response [58]; and (e) are markedly more sensitive to the skin carcinogen 7,12dimethylbenzanthracene [59], as well as to UV-induced skin tumorigenesis [60]. Importantly, because heart disease and ischemic stroke comprise the leading causes of death in the US population, VDR-null mice display cardiovascular risk factors including hypertension, left ventricular hypertrophy, and fibrosis [61]. VDR action is clearly immunomodulatory, and VDR likely mediates the reported anticancer effects of vitamin D. In the context of chemoprevention, 1,25(OH)2D3 is known to possess potent antiproliferative, prodifferentiation, proapoptotic, and/or cell cycle arrest activities in epithelial cells [62]. The molecular mechanisms underlying these effects may be related to the ability of liganded VDR to arrest cells at the G1 stage via induction of cell cycle regulatory proteins like p21 [63] (Table 8.1) and p27 [64], to control cell growth transcription factors such as c-myc [65] and c-fos [66], or to elicit apoptosis by repression of the Bcl-2 antiapoptotic factor [67]. However, an additional intriguing possibility is that VDR is chemopreventative by inducing detoxification enzymes, e.g., CYP3A4, since several of the genes encoding these CYPs in mammals possess VDREs (Table 8.1). Figure 8.3 summarizes and integrates the endocrine regulation and actions of 1,25(OH)2D3-VDR in exerting the bone mineral homeostatic, immune, cardiovascular, and anticancer effects. While the predominant action of 1,25(OH)2D3-VDR is promoting intestinal calcium and phosphate absorption to prevent osteopenia, the signal for this function is PTH reacting to low calcium, whereas the hormonal agent that feedback controls these events to preclude ectopic calcification is FGF23. In this fashion, bone resorption and mineralization remain coupled to protect the integrity of the mineralized skeleton. Beyond bone, renal 1,25(OH)2D3, and especially locally generated extrarenal 1,25(OH)2D3, benefit the cardiovascular system in which VDR is expressed in endothelial cells, smooth muscle cells, as well as cardiac myocytes (Fig. 8.3). Finally, kidney- and locally derived 1,25 (OH)2D3 also influences many cells in the immune system to modulate its functions, as well as to exert anticancer actions in virtually all epithelial cells.
VDR AS A MEMBER OF THE NUCLEAR RECEPTOR SUPERFAMILY A proper understanding of the molecular actions of VDR requires an appreciation of its genetic placement within the nuclear receptor superfamily. The human genome sequence encodes 48 nuclear receptors [68], and interrelationships between these receptors have been documented previously [22]. All human nuclear receptors, along with those from other species, have been placed into six groups according to a unified system of nomenclature [69]. As with previous analyses using a subset of human receptors [69], VDR groups with the TRs, RARs, the peroxisome proliferator-activated receptors (PPARs), the rev-erb orphan receptors, and the retinoic acid receptor-related orphan receptors (RORs) in a single clade that has been named Group 1 [69]. A significant feature shared by Group 1 receptors is that many of them form active heterodimeric complexes with the RXRs from Group 2 [70]. The closest relatives of VDR within Group 1 are PXR and the constitutive androstane receptor (CAR), which, with VDR, form subgroup 1I (Fig. 8.4) (full designations are NR1I1 for VDR, NR1I2 for PXR, and NR1I3 for CAR) [69]. The next closest group of receptors to VDR is subgroup 1H, including the liver X receptors (LXRs) and the farnesoid X receptor (FXR). Clearly, the common thread connecting the 1I receptors, VDR, PXR, and CAR, is the induction of CYP enzymes that participate in xenobiotic detoxification, as summarized in Figure 8.4. A major target for VDR and PXR in humans is CYP3A4 [62,71,72], for which the detoxification substrates include LCA [73]. Initial studies focused on VDR liganded to 1,25(OH)2D3 as a regulator of CYP3A4 [62,72], but more recent experiments have revealed that LCA is also capable of binding VDR to up-regulate expression of human CYP3A4, or its equivalent in rats (CYP3A23) or mice (CYP3A11) [71]. There is also evidence that other CYP enzymes may be VDR targets [74]. In fact, many of the traditional targets of VDR-mediated regulation of vitamin D metabolism, namely 1a- and 24-hydroxylases, are CYPs (CYP27B1 and CYP24A1, respectively, according to standard CYP nomenclature) [75]. These studies therefore demonstrate that, in addition to its well-known role in calcium homeostasis, VDR also participates in the regulation of intestinal CYP genes in a manner similar to its closest relative, PXR [62,72]. As summarized in Figure 8.4, VDR and PXR may regulate some of the same genes, since PXR also utilizes DR3 and ER6 responsive elements in DNA for transcriptional activation [76]. The differential transcriptional effects of VDR and PXR may reside not in the genes that are regulated, but rather in the overlapping, yet distinct, ligand
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FIGURE 8.3 Schematic representation of the tissue and cell interrelationships in the PTH-1,25(OH)2D3-FGF23-klotho endocrine axis that executes calcium and phosphate homeostasis to lower the risk of osteopenia/osteoporosis, hyperphosphatemia/ectopic calcification, and vascular disease, as well as prevent epithelial cell malignancies and modulate the immune system to fight infection while ameliorating autoimmune disorders.
profile (Fig. 8.4, second column); for example, PXR does not respond to 1,25(OH)2D3 [77]. It should be noted that CAR, the third human receptor in subgroup 1I, is also implicated in CYP regulation [78,79]. Whereas CAR normally binds DR4 elements (Fig. 8.4), this relatively uncharacterized receptor may exhibit some cross-over
binding to DR3 elements; conversely, PXR may crossover to DR4 elements [80]. There is some preliminary evidence that VDR may also cross-over to DR4 elements [74], as it was reported that VDR can activate transcription from at least three elements that strongly resemble degenerate DR4s [81e83].
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Comparative features of VDR and closely related nuclear receptors from subgroups 1I and 1H. Abbreviations for each receptor are defined in the text. Comparison of cognate ligands, DNA-responsive elements, and target gene function. Chenodeoxycholic acid ¼ CDCA, and other ligand abbreviations are defined in the text. DNA-responsive elements are classified as direct repeats with spacers of 3 or 4 nucleotides (DR3, DR4), elements in which the two half-site repeats are everted with a spacer of 6 or 8 nucleotides (ER6, ER8), or inverted repeats with a spacer of one nucleotide (IR1). Under target genes, CYP refers to cytochrome P450-containing enzymes that are involved in detoxification of xenobiotics, or in the case of the CYP24 gene, degradation of 1,25(OH)2D3 and 25(OH)D3.
FIGURE 8.4
One notable feature of the 1H and 1I receptors is the ability of these receptors to recognize multiple ligands [84]. The diversity of ligands for PXR is especially broad and includes not only endogenous steroids, but also an array of other lipophilic compounds such as the secondary bile acid lithocholic acid (LCA), the antibiotic rifampicin, as well as xenobiotics such as hyperforin, the active ingredient of St. John’s Wort [84]. It is now recognized that VDR binds several ligands beyond the 1,25 (OH)2D3 hormone. The first non-vitamin-D-related VDR ligands discovered were LCA and its derivative, 3-ketolithocholic acid (3-ketoLCA) [71]. This finding is of considerable interest to human medicine, since it is known that LCA is a secondary bile acid with significant carcinogenic potential [71,85,86], and its binding to VDR could trigger the detoxification of this ligand via the induction of CYP3A4 mediated by the VDREs in this gene (Table 8.1). Considering the structures for prototypical PXR, FXR, and LXR ligands (listed in Fig. 8.4), and comparing these compounds with the ligandbinding profile of VDR, expanded to include LCA and its 3-keto derivative, it is evident that VDR exhibits a ligand profile resembling that of the closest VDR relatives in the nuclear receptor superfamily, especially when it is noted that both PXR [87,88] and FXR [71,89] are also activated by LCA to some extent. It is now appreciated that most group 1I and 1H members, with the exception of CAR and the two LXRs, can recognize bile acid ligands. Therefore, based upon an analysis of its evolutionary position among the nuclear receptors and data with bile acids, it is apparent that VDR binds ligands beyond the vitamin D congeners that regulate bone mineral homeostasis, and we [21,90] have shown
that VDR associates at low affinity with several dietderived lipophilic ligands to possibly effect extraosseous bioresponses. For example, curcumin, which is found in curry and known to be anti-inflammatory to the degree that it reduces inflammatory bowel disease [91], was compared with LCA as a potential VDR ligand. Strikingly, we observed that curcumin is slightly more active than LCA in driving VDR-mediated transcription, and that it also binds to VDR with approximately the same affinity as LCA based upon ligand competition assays [21]. The mechanism of action of curcumin is not known, and we suggest that at least part of its beneficial functions are mediated by the nuclear VDR, perhaps even its ability to lower the risk of colon cancer [92]. Another class of nutritionally available lipids, which is critical in maintaining cell membrane fluidity and serves as precursors of the prostanoids and leukotrienes, is the essential PUFAs. DHA, for example, is an u-3 PUFA responsible for infant brain development, which also is a known ligand for RXR [93], the heterodimeric partner of VDR. u-3 PUFAs are also ligands for PPARa, through which they lower VLDL and eventually LDL cholesterol to lessen coronary artery disease as well as reduce the incidence of metabolic syndrome [94]. We demonstrated that u-3 PUFAs such as DHA and eicosapentaenoic acid (EPA), as well as u-6 PUFAs such as linoleic acid and arachidonic acid, compete with tritiated 1,25(OH)2D3 for binding to VDR with affinities for the receptor some four orders of magnitude lower than that of the 1,25(OH)2D3 hormonal ligand [90]. Nevertheless, we conclude that high local concentrations of PUFAs could occur in select cells or tissues
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and exert VDR-mediated antiproliferation/prodifferentiation effects that may partially explain the chemoprotective nature of diets rich in PUFAs, plus their cardioprotective and anti-inflammatory influences. The lack of specificity among the PUFAs for VDR binding and activation [95] is unusual for ligand receptor interactions. However, not all lipophilic compounds bind VDR with low affinity, as dexamethasone, the synthetic glucocorticoid, and a-tocopherol, the antioxidant vitamin E, do not compete with 1,25(OH)2D3 for occupation of VDR. Surprisingly, we have noted that the vitamin E metabolite, g-tocotrienol (Fig. 8.2), is a lowaffinity VDR ligand that is capable of activating the receptor [96]. This finding reveals that, similar to the concept that vitamin D and even 25(OH)D are ineffective ligands for VDR whereas metabolism to 1,25 (OH)2D3 generates a high-affinity hormone, the basic PUFA “core” structure could be metabolically activated to yield an array of higher-affinity ligands that function as cell-specific activators of VDR. The general conclusion based upon examination of the evolutionary position of VDR among the 48 nuclear receptors is that it is extremely closely related to PXR, both structurally and functionally. In one respect, VDR appears to have evolved as a “specialty” regulator of intestinal calcium absorption and hair growth in terrestrial animals, providing both a mineralized skeleton for locomotion in a calcium-scarce environment, and physical protection against the harmful UV radiation of the sun. Yet VDR also has retained its PXR-like ability to effect xenobiotic detoxification via CYP induction. VDR could complement PXR by serving as a guardian of epithelial cell integrity, especially at environmentally or xenobiotically exposed sites such as skin, intestine, and kidney.
MOLECULAR STRUCTUREeFUNCTION OF VDR Overview VDR possesses the classical (Cys2-Cys2)2 zinc finger motif for DNA binding, and a multifunctional Cterminal domain for ligand binding, RXR heterodimerization, and transcriptional coactivator binding, but lacks the long N-terminal extension typical of the traditional steroid hormone receptors [97]. A unique feature of VDR and PXR that is probably related to their ligand-binding profile is the fact that both of these receptors have especially lengthy amino acid segments between the DNA-binding domain (DBD) and the highly conserved regions of the LBD. The presence of additional residues following the DBD in VDR and PXR has been proposed to confer
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upon these two receptors more spacious binding pockets for accommodating a range of ligands, such as xenobiotics [18,98].
The Zinc Finger DNA-binding Domain of hVDR Throughout the nuclear receptor superfamily, the DBD is the region of highest sequence similarity [69]. The two zinc finger modules in hVDR are followed in the primary sequence by a C-terminal extension (CTE), which contains a long a-helix. The entire DBD, extending from positions 22e110 in the human VDR amino acid sequence, has been crystallized [99]. It is important to consider that the hVDR DBD fragments were crystallized by Shaffer and Gewirth as a homodimer on three different DR3 VDREs or as a heterodimer with RXR, but in a configuration (VDR on the 50 half-site and RXR on the 30 half-site) opposite to that observed on positive VDREs [99,100]. Nevertheless, it is reasonable to assume that the DNA contacts established by these VDR DBD fragments approximate those of at least the core zinc finger region of the VDR partner when it binds to a VDRE as part of the full-length VDR-RXR heterodimer with VDR on the 30 half-site. In the homodimer, the first zinc finger of hVDR possesses an a-helix on the C-terminal side containing amino acid residues (specifically E42, K45, R49, and R50) that contact VDRE 30 half-element bases in the major groove of DNA. An additional a-helix situated on the C-terminal side of the second zinc finger is positioned to interact with the DNA phosphate backbone, and there is extensive contact between residues at one end of this a-helix (specifically R73, R74m and R80) and DNA phosphates. The combined energy provided by the DNA recognition and phosphate backbone binding a-helices is apparently what is required for specific VDRE binding by VDR, and this conclusion is supported by the location of VDR point mutations found in patients with HVDRR [46]. In order to bind to DNA and regulate gene expression, transcription factors require nuclear translocation after biosynthesis on the polyribosomes, and nuclear localization is dependent upon various arrangements of basic amino acids in short stretches within the primary amino acid sequence [101]. Four such segments of positively charged amino acids that mediate hVDR nuclear translocation [102,103] exist in the DBD. These residues align on one surface of hVDR when the CTE is in the heterodimeric orientation, perhaps creating an interaction surface for nuclear import factors. Some of the same hVDR amino acids that contact DNA also participate in nuclear localization, namely the basic residue clusters at positions 49e50 [102] and 102e104 [103]. Two additional basic clusters are also involved in hVDR nuclear translocation, located at residues
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53e55 [102] and 82e83 [103]. Interestingly, Barsony and colleagues [104] have confirmed the essential role of hVDR residues 49e50 and 53e55 [102], and demonstrated that positionally equivalent basic residues in RXR are also important in nuclear localization, especially of the unliganded VDR-RXR heterodimer [104]. Thus, it appears that a quasi-stable, hormone-unoccupied VDR-RXR heterodimer is the species of receptor that localizes in the nucleus of vitamin D target cells, probably bound nonspecifically to DNA with relatively low affinity.
Ligand-binding/Heterodimerization/ Transactivation Domains of VDR and Comparison with PXR As discussed above, X-ray crystallographic structures of the VDR ligand-binding/heterodimerization domain bound to 1,25(OH)2D3 and to other vitamin D analogs have been obtained. The structure by Rochel et al. [18], shown schematically in Figure 8.5A, contains two discontinuous segments missing a 52-amino-acid sequence between helices 1 and 2, spliced together to form a peptide that can be purified and crystallized [18]. Despite the discontinuity, the structure of the VDR ligand-binding/heterodimerization domain displayed in Figure 8.5B bears a strong resemblance to the X-ray crystal structures of the corresponding regions of other nuclear receptors [105], consisting of what has been described as a “sandwich” of 12e15 a-helices with a centrally located hydrophobic ligand-binding pocket (see Chapter 9). Within the general structure of the updated a-helical sandwich, the ninth and tenth helices (Fig. 8.5B) have been shown in several hVDR X-ray crystal structures to contain heterodimer contacts [18]. The surface formed by these helices in VDR appears similar to that observed in crystals of the LBD of RARa, RXRa, PPARg, and ER [106e109]. Moreover, there is a human HVDRR patient harboring a VDR in which arginine 391 (helix 10) is mutated to cysteine (Fig. 8.5B), and in vitro experiments have confirmed that RXR heterodimerization by the R391C mutant hVDR is severely impaired [110]. The R391C HVDRR patient, like all those with DNAbinding mutations in hVDR, displayed an alopecic phenotype, indicating that RXR heterodimerization is functionally linked to VDRE binding, and that both of these actions of VDR are required for hair cycling. Independently supporting an obligatory role for the RXRa heteropartner in hair cycling is the observation that mice with an RXRa conditional knockout in skin exhibit alopecia indistinguishable from that in VDR-null animals [111]. Crystallographic studies with a number of nuclear receptors have revealed that helices corresponding to
3, 5, and 12 in hVDR combine to form a platform for transcriptional coactivator binding [112e116] (illustrated for hVDR in Fig. 8.5B). The involvement of helix 12 in VDR action is also supported by the identification of an HVDRR patient, in whom glutamate 420 is altered to lysine [117]. Further testing has demonstrated that natural mutant E420K hVDR [117], as well as its synthetic counterpart, E420A [118], have abrogated transactivation capacity caused by their inability to interact with the comodulators steroid receptor coactivator-1 (SRC-1) and vitamin D receptor interacting protein 205 (DRIP205). The third natural hVDR mutant highlighted in Fig. 8.5B is an arginine 274 to leucine alteration that leads to a loss in binding of the 1,25(OH)2D3 ligand [110]. Arginine 274 directly contacts the 1,25(OH)2D3 ligand via hydrogen bonding of the 1a-hydroxyl moiety [18]. Thus, the three natural point mutations in the hVDR LBD highlighted in Figure 8.5B represent loss of function alterations for each of the three major molecular actions of this domain, namely ligand binding, heterodimerization, and transactivation, with the respective disruption of contact by hVDR with 1,25 (OH)2D3, RXR, and coactivators. In summary, just as clinically significant inactivating point mutations in the hVDR DBD prove the obligatory role of DNA binding in VDR action, the three HVDRR-associated point mutations in the C-terminal domain of hVDR highlighted in Figure 8.5B illuminate the necessity for ligand binding, RXR heterodimerization, and coactivator contact in order for VDR to transduce the signal for the prevention of rickets. Although the crystal structure of the unoccupied LBD of hVDR has not been solved, it is generally accepted [105] that unliganded nuclear receptors are configured such that the helix 12/activation function 2 (AF2) is in the “open” orientation, directed away from the a-helical sandwich and allowing for entry of the ligand into a hydrophobic pocket (Fig. 8.6). Upon ligand binding with 1,25(OH)2D3, helix 12 of VDR, which contains two ligand contact residues (valine 418 and phenylalanine 422), is repositioned to the agonist-bound “closed” orientation and interacts with helices 3 and 5 [18] to create a coactivator platform (Fig. 8.5B). 1,25(OH)2D3 ligand binding also alters the conformation of the LBD such that the surface presented by helices 9 and 10 is more favorable for association with RXR (Fig. 8.5B). Facilitation by 1,25(OH)2D3 of strong heterodimerization of VDR with RXR has been demonstrated [110,119,120], and the resulting VDR-RXR heterocomplex is capable of high-affinity interaction with VDREs. Therefore, VDR liganding with the 1,25(OH)2D3 hormone generates both RXR heterodimerization for specific DNA binding and coactivator docking for transcriptional activation.
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FIGURE 8.5 Structureefunction relationships in the human VDR ligand binding/heterodimerization/transactivation domain. (A) A sche-
matic view of human VDR, in which the following subdomains are highlighted: the third, fifth, and twelth helices (H3, H5, and H12), which have been implicated in binding of coactivators and transactivation; the ninth and tenth helices (H9 and H10) and the loop between helices 8 and 9, which contain an interface for interaction with the RXR heterodimeric partner; and finally, two b-strands in the VDR crystal structure [18]. A section of the VDR ligand-binding/heterodimerization domain from positions 165e215 was deleted (a box with crossed lines) by Rochel and colleagues [18] in order to facilitate purification and crystallization. (B) The X-ray crystal structure of human VDR (residues 118e164 spliced to residues 216e425) [18] bound to its natural 1,25(OH)2D3 ligand, as viewed in Jmol [262]. Three examples of natural mutations that cause HVDRR are indicated with hVDR residue numbers (274, 391, 420) and are discussed in the text. The position of the deletion is indicated by the highlighting of residues 164 and 216, at the top of the structure. The conformation of this deleted region may resemble that in the related receptor, PXR (panel C), as suggested by the dotted outline at the upper left of the panel. (C) A view of the human PXR (or SXR, steroid, and xenobiotic receptor) ligand-binding/heterodimerization domain bound to the synthetic ligand, SR12813 [98], created in Jmol to approximate the same view of hVDR in panel B. A contiguous fragment of PXR was used for crystallization (residues 142e431), but the region from residue 177 to residue 198 was unstructured (shown as a dotted line). Four b-strands are shown, with the lower two strands (designated b1 and b10 in the original publication [98]) residing in a region corresponding to the deletion in the crystallized hVDR. This conformation of strands b1 and b10 was employed as a model for a hypothetical structure of hVDR in this region, as shown in B. Please see color plate section.
The availability of a PXR structure [98] permits interesting comparisons between the ligand-binding/heterodimerization domain of VDR and that of PXR, its closest relative in the nuclear receptor superfamily. The overall structures of the domains of these two receptors are nearly superimposable [18,98] (compare Figs. 8.5B and 8.5C). The PXR LBD has a particularly large ligand3) [121]; the ligand-binding binding pocket (1150 A pocket of VDR, even with the deletion of residues 3) compared with 165e215, is also large (approx. 700 A other nuclear receptors that have been crystallized 3 [18] but ranging as high as 800 A 3 for (approx. 400 A LXRb) [122]. This suggests that like PXR and LXR,
VDR may be able to accommodate a variety of lipophilic ligands beyond 1,25(OH)2D3, including LCA/3-ketoLCA. Of additional interest is the fact that the PXR crystal, which was created from a continuous stretch of sequence from positions 142e431 of overexpressed human PXR [98], contains residues that correspond to most of the 165e215 region (or its equivalent) that is absent in the VDR LBD crystals. Thus, the PXR X-ray crystallographic solution can serve as a model for the VDR structure in this region. Of particular note are the additional two b-strands in PXR, for which the corresponding hypothetical positions in VDR are suggested by the dotted outline in Fig. 8.5B. Indeed, inclusion of
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(A) Ligand-dependent Activation AF2
2
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ix 3
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1,25(OH)2D3 Ligand Protein Kinase Coactivator(s)
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GGTTCA 3nt GGTTCA
+ DR3 VDRE
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1,25(OH)2D3-stimulated Transactivation via RXR-VDR
(B) Ligand-dependent Repression 2
Helix 9,10
re
OH
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1,25(OH)2D3 Ligand Protein Phosphatase Corepressor(s)
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1,25(OH)2D3-mediated Transrepression via VDR-RXR
Proposed mechanisms of gene induction and repression by VDR. (A) Allosteric model of RXR-VDR activation after binding 1,25 (OH)2D3 and coactivator, phosphorylation, and docking on a high-affinity positive VDRE (mouse osteopontin). See text for explanation. (B) Allosteric model for VDR-RXR inactivation after binding 1,25(OH)2D3 and corepressor, dephosphorylation, and docking in reverse polarity on a high-affinity negative VDRE (chicken PTH). See text for explanation.
FIGURE 8.6
these two b-strands, plus associated turns, in this speculative hVDR LBD structure could allow for the further expansion of the ligand-binding pocket. The assertion has been made that this region (corresponding to the missing amino acids 165e215 in hVDR) may not be necessary for the transactivation function of VDR [123]. However, a mutation was reported in an HVDRR patient in which alteration of cysteine 190 to tryptophan results in loss of hormone binding by hVDR [124]. Although it is possible that the substitution of cysteine 190 by the more nonpolar and much bulkier tryptophan side chain somehow disrupts the general tertiary structure of the VDR LBD, this finding could be interpreted as an indication that the deleted region [165e215] does in fact serve some important function that is not detected
by limited in vitro testing of the mutated receptor performed with only the 1,25(OH)2D3 ligand at high (10e7 M) concentrations [123].
MECHANISMS OF VDR-MEDIATED CONTROL OF GENE EXPRESSION As described above in Figures 8.1e8.5 and Table 8.1, several lipophilic ligands and target gene DNA sequences have been defined for VDR, and the structures of the 1,25(OH)2D3 hormone-occupied LBD as well as the VDRE-docked DBD have been elucidated. Very recently, the structure of the hVDR DBD and LBD together in the same protein, heterodimerized
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with full-length RXRa, docked on a VDRE, and occupied with 1,25(OH)2D3 plus a single coactivator, has been determined in solution via Small Angle X-ray Scattering and Fluorescence Resonance Energy Transfer techniques [125], rendering it now possible to visualize how the DBD and the ligand-binding/ heterodimerization domains are arranged relative to one another. This advance, by Moras and colleagues, adds significantly to our understanding of the precise molecular mechanism whereby VDR ligands signal the control of gene transcription. This process is best understood for VDR mediation of 1,25(OH)2D3-stimulated transcription, where RXR heterodimerization constitutes an obligatory initial step in the VDR activation pathway. At physiologic receptor concentrations and ionic strength, VDR will bind to VDREs only in the presence of both 1,25(OH)2D3 and RXR [120], and in an in vitro transcription system containing native chromatin, 1,25(OH)2D3 will transactivate via VDR only when RXR is included [126]. Moreover, based on the requirement for coactivator in both crystallization and Small Angle X-ray Scattering and Fluorescence Resonance Energy Transfer structural studies of VDR, it is likely that a comodulator protein stabilizes liganded VDR-RXR on the VDRE. Figure 8.6 illustrates in hypothetical schematic fashion how the hormonal ligand could be influencing VDR to interact more efficaciously with its heterodimeric partner, with a VDRE, and with coactivators. From experiments with VDR [38,127,128], as well as insight from the mode of action of other nuclear receptors [129e131], the key event in the allosteric model presented in Figure 8.6 is the binding of a ligand, namely the 1,25(OH) 2D3 ligand for VDR. There are several steps that apparently are set in motion by the ligand-binding event. The presence of the 1,25(OH)2D3 ligand in the VDR-binding pocket results in a dramatic conformational change in the position of helix 12 at the C-terminus of VDR, bringing it to the “closed” position to serve in its AF2 role as part of a platform for coactivator binding [118,132,133]. The attraction of a coactivator to the helix-3, -5, and -12 platform of liganded VDR likely allosterically stabilizes the VDR-RXR heterodimer on the VDRE, and may even assist in triggering strong heterodimerization by inducing the VDR LBD to migrate to the 50 side of the RXR LBD, and in so doing rotate the RXR LBD 180 degrees employing the driving force of the ionic and hydrophobic interactions between helices 9 and 10 in hVDR and the corresponding helices in RXR (Fig. 8.6A). Therefore, ligand-intensified heterodimerization, VDRE docking, and coactivator recruitment by VDR appear to be functionally inseparable, yet experimentally dissociable, events that occur in concert to effect 1,25(OH)2D3-elicited gene transcription.
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The conformational changes allosterically elicited in VDR by the above-described interactions with ligand, RXR, and DNA have the added effect of converting VDR into a more efficient substrate for one or more serine protein kinases [28]. The most prominent phosphorylation appears to be catalyzed by casein kinase II (CK2) on hVDR serine 208 [134,135], an event that has been shown to potentiate the transcriptional activity of the VDR-RXR heterodimer [136], likely by enhancing interactions with coactivators such as DRIP205 (Fig. 8.6A) [137,138]. Finally, as depicted in Fig. 8.6A and supported experimentally [127,128,139], the liganding of VDR conformationally influences its RXR heteropartner, and appears to cause the AF2 region of RXR to pivot into the “closed” or active position. The RXR member of the heterodimer may now be endowed with the potential to bind an additional coactivator (not shown in Fig. 8.6), and the allosteric repositioning of the RXR AF2 appears to greatly reduce the affinity of RXR for its 9-cis RA ligand. With RXR serving as a subordinate partner, VDR is referred to as a nonpermissive primary receptor within the heterodimer, because RXR may not be able to bind 9-cis RA when heterodimerized to liganded VDR [127,128,139]. Alternatively, as depicted in Figure 8.6, we leave open the possibility that in specific cell contexts with certain promoters containing unique VDREs, the ligand-binding pocket of RXR in the heterodimer is available to occupation by 9-cis RA or other RXR ligands such as DHA [93], rendering the RXR heteropartner capable of ligand occupation and coactivator recruitment for the purpose of synergistic activation by 1,25 (OH)2D3 and retinoids/fatty acids of genes such as CYP24A1 [140]. As discussed above (Fig. 8.5), the coactivator platform in other nuclear receptors has been shown in crystallographic studies to consist of the AF2 in concert with the equivalent helices 3 and 5 in VDR [112e116], and it has been suggested that most nuclear receptors have similar interaction domains [105,141], with the possible exception of the progesterone receptor [109,142]. These crystallographic studies indicate that coactivators of the p160 class, such as SRC-1, utilize one or more LXXLL motifs for binding to nuclear receptors (where L ¼ leucine and X ¼ any residue). There is evidence from site-directed mutagenesis experiments that helix 12 and helix 3 of VDR are indeed involved in coactivator contact [118,143]; this has now been confirmed in X-ray diffraction data from VDR LBD-coactivator co-crystals [144e149]. Ligand-dependent repression of gene transcription by VDR-RXR likely shares some molecular features with induction, but no doubt is more complex mechanistically because it appears to occur via multiple routes. One theme of repression is probably the recruitment of
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nuclear receptor corepressor(s) to alter the architecture of chromatin in the vicinity of the target gene to that of heterochromatin. This restructuring of chromatin would be catalyzed by histone deacetylases and demethylases attracted to the receptor-tethered corepressor. The initial targeting of the repressed gene, as illustrated in Figure 8.6B, is hypothesized to be docking of liganded VDR-RXR on a negative VDRE. In the case of repression, liganded VDR is apparently conformed such that it binds corepressor rather than coactivator. We postulate that the information driving this allosteric transformation of VDR is intrinsic to the negative VDRE DNA sequence [22]. Further, because nonconsensus nucleotides in negative VDREs appear to occur in either or both half-elements as discussed previously [22], we contend that such basepair changes may be sufficient to drive RXR-VDR into reverse polarity on the negative VDRE (Fig. 8.6B), an event that is documented to switch liganded RAR-RXR into a repressor [150], albeit on a DR1 instead of its normal DR5 enhancer. Our assumption is that docking in reverse polarity on the negative VDRE conforms liganded VDR such that it favors the recruitment of corepressor over coactivator to their overlapping docking sites in helices 3e6. VDR may also be prone to protein phosphatase rather than protein kinase activity in this altered conformation, again favoring corepressor attraction. Evidence for this model is provided by the observation [28] that okadaic acid, a protein phosphatase inhibitor, potentiates transactivation by VDR. A key question is the role of the RXR heteropartner in gene repression by VDR. One possibility is that RXR is simply a “silent” partner in VDRE binding, with negative nucleotides alone in the 50 half-site allosterically conforming VDR to attract corepressor, but because the negative cPTH VDRE (Fig. 8.5B) does not possess nonconsensus variations in its 50 half-site and it can be converted to a positive VDRE by altering the 30 terminal bases from GT to CA [151], we favor a role for the RXR LBD in allosterically locking VDR into a corepressor docking motif (Fig. 8.6B). Thus, since nonconsensus nucleotides occur only in the 30 half-element of the negative chicken PTH VDRE, it is conceivable that the RXR partner receives information from the 30 half-site nucleotides on which it is docked, and transmits a signal to its VDR heteropartner that allosterically conforms VDR to attract corepressor. Future experiments will be required to elucidate the mechanism of negative regulation by VDR and test the hypothetical model presented in Figure 8.6B. An integrated picture of gene expression control by 1,25(OH)2D3 is that liganded VDR-RXR serves as a “nucleus” to recruit comodulators for signal transduction as presented in Figure 8.7 in the form of a sequential recruitment hypothesis for multiple comodulators. The key tenet of this sequential model is DNA looping to
facilitate contact between comodulators tethered to enhancer elements and the transcriptional start site. VDR can be considered a typical nuclear hormone receptor [38], a member of the thyroid hormone, retinoic acid, oxycholesterol, and xenobiotic subfamily of nuclear receptors that primarily heterodimerizes with RXR in response to ligand binding in order to recognize direct repeat responsive elements in the promoters of regulated genes (steps 1 and 2 in Fig. 8.7A) [97,152,153]. Previous research with VDR-activated genes indicates that many factors participate in transactivation [22]. These include the following (Fig. 8.7A): step 3, factors capable of histone acetylation (HATs), such as SRC-1 [154], CBP/p300, or pCAF, or factors involved in ATP-dependent chromatin remodeling, such as the mammalian homologs of SWI/SNF [155]; step 4, TATA-binding protein-associated factors (TAFs, especially TAFs 28, 55, and 135 [156,157]); step 5, basal transcription factors such as TFIIB [158]; step 6, Dreceptor interacting proteins (DRIPs, especially DRIP205, which is a subunit of the mediator complex that couples transactivators to the C-terminal tail of RNA polymerase II) [133], and step 7, NCoA-62, a factor reported to serve as a coactivator for VDR and related nuclear receptors that might also couple transcription to RNA splicing [159]. Step 8, interaction with TRIP1, the mammalian homolog of the yeast SUG factor, resulting in progressive ubiquitination of VDR and ultimately leading to its recognition and degradation by the proteasome [160]. Many of these factors interact with the same region of VDR, namely the C-terminal AF-2 motif [22]; thus, it is difficult to conceive of these factors all interacting with VDR to effect transactivation except in a sequential manner (Fig. 8.7A) or in a complex in which multiple VDR-RXR heterodimers are present. The RANKL gene promoter (Fig. 8.7B) is being utilized as a model system for studying the steps in transcriptional activation by the liganded VDR-RXR heterodimer using in silico analysis as well as ChIP, gel mobility shift, and transcription assays. Based on our studies [34] and those of J.W. Pike and associates [161], Figure 8.7C depicts a postulated chromatin looping model for the mouse RANKL gene. Instead of separate events in which various factors bind to a single VDRRXR heterodimer in a defined sequence (Fig. 8.7A) [22], we propose that in genes such as RANKL that possess multiple VDREs, the chromatin looping model (Fig. 8.7C) allows for simultaneous binding of multiple factors in a supercomplex at the promoter. Most evidence for a chromatin-looping model in transcriptional control is derived from studies of the globin genes [162] and the T-helper type 2 cytokine locus [163]. There is also direct evidence for such looping in transactivation by nuclear receptors. Multiple binding sites for the estrogen receptor have been reported
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Chromatin looping model of gene regulation by VDR. (A) Sequential model of gene activation of the mouse osteopontin gene (containing a single VDRE) by the 1,25(OH)2D3-bound VDR-RXR heterodimer. Numbers inside circles refer to discreet stages in the activation of a VDR target gene as discussed in the text. (B) The presence of several potential VDREs in the 50 flanking region of the mouse RANKL gene, as reported by Kim et al. [161] and verified by our experiments (Table 8.1). (C) Depiction of how these VDREs might cooperate in a chromatinlooping model. As discussed in the text, multiple VDR-RXR heterodimers may be capable of simultaneously recruiting coactivators and other factors (pictured are the factors from the numbered stages of panel A) to form a regulatory supercomplex at the promoter.
FIGURE 8.7
flanking single genes, some as far as 144 kb from the transcriptional start site (TSS) [164]. Chromosomal looping has also been postulated for androgen-dependent transcription [165]. Indeed, direct evidence for chromosomal looping in VDR-mediated transcriptional modulation has been obtained via chromosome conformation capture technology by Carlberg and associates [35]. Moreover, active VDREs are located anywhere from 76 kb upstream in the RANKL gene [161] (Fig. 8.7B) to 19e29 kb downstream (within the first two introns) in the mouse LRP5 [32,34] and VDR [166] genes, and 2e4 kb upstream of the TRPV6 gene [31,34]. It therefore seems likely that nuclear receptors, including VDR, also utilize chromosomal looping in their mechanism of transactivation, at least in some settings. DNA looping obviates “crowding” of comodulators that may occur when only a single VDRE is present (Fig. 8.7A), and multiple VDREs allow for the formation of a “cloverleaf” structure (Fig. 8.7C) which, by analogy to a highway interchange moving traffic, permits the functioning of multiple coactivators just upstream of the TSS. As depicted in Figure 8.7C, in effect multiple comodulators influence transcriptional initiation sequentially by attraction to a complex nucleus of several VDREs with docked VDR-RXR.
The existence of multiple enhancer elements, some of varying strengths, may also provide an explanation for hitherto unresolved phenomena. For example, the dramatically different patterns of 1,25(OH)2D3-dependent regulation for the RANKL and osteoprotegerin (OPG) genes observed in different cell lines can perhaps be attributed to differing abilities to form the chromatin looping complex suggested by Figure 8.7C. This is an especially attractive hypothesis if, for example, the complex requires factors other than VDR and RXR that are limiting in certain cell lines. The time that may be required to form such complexes might also explain why, for many hormones, there is a distinctly different regulatory result when the hormone is chronically present versus situations in which there is an acute spike in hormone concentration. There is additional evidence, in the case of VDR-mediated regulation of RANKL, that overexpressing VDR in osteoblasts can eliminate or even reverse the induction of RANKL by 1,25(OH)2D3 [167]. One could speculate that high concentrations of VDR might lead to formation of supercomplexes in which low-affinity VDREs might attract corepressors rather than coactivators. Further characterization of the multiple potential VDREs at the RANKL locus will be required to obtain a complete picture of the complex
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regulation of RANKL by 1,25(OH)2D3-bound VDR, and the current working model depicted in Figure 8.7C may serve as a paradigm for other 1,25(OH)2D3-VDR-regulated genes that contain multiple VDREs with differential VDR binding affinity.
VDR-MEDIATED CONTROL OF VITAL GENES IN HEALTHFUL AGING AND DISEASE PREVENTION Bone and Mineral Regulatory Gene Expression Control by 1,25(OH)2D3-VDR 1,25(OH)2D3-VDR controls the expression of at least 11 genes (SPP1, TRPV6, LRP5, BGP, RANKL, OPG, CYP24A1, PTH, FGF23, PHEX, and klotho) which encode bone and mineral homeostasis effectors that also facilitate healthful aging. The first, osteopontin or SPP1, is dramatically induced by 1,25(OH)2D3 in osteoblast-like cells [4]. Osteopontin was initially named for its positive actions in bone, which include inducing ossification, especially in response to mechano-stress/fracture, and remodeling [168], both of which strengthen the skeleton to promote longevity and reduce fracture risk. Moreover, SPP1 facilitates angiogenesis and osteoclast accumulation for the resorption of ectopic bone; in fact, SPP1 is an inducible inhibitor of vascular calcification and associated disease [169]. Other benefits of SPP1 are the effecting of immune homeostasis and the promotion of myelination [170]. Intestinal calcium absorption is mediated, in part, by 1,25(OH)2D3-VDR induction of TRPV6 [31,34]. TRPV6 represents a key calcium channel gene product that supplies dietary calcium via transport to build the mineralized skeleton (Fig. 8.3) and thereby delay the inevitable calcium leaching from bone in senile osteoporosis. Accordingly, TRPV6-null mice have 60% decreased intestinal calcium absorption, decreased bone mineral density (BMD) and, strikingly, 20% of animals exhibit alopecia and dermatitis [171] similar to VDR knockout mice [48]. Since the skin phenotype in VDR-null mice is not ameliorated by the high-calcium rescue diet [52], we speculate that TRPV6 may mediate calcium entry into keratinocytes to elicit differentiation and hair cycling. Because calcium is protective against colon cancer [172], while hair plus a full stratum corneum reduce UV-induced skin damage and cancer, VDR-induced TRPV6 could also function in colon and skin to lower the risk of neoplasia in these two epithelial cell types (Fig. 8.3). 1,25(OH)2D3 significantly induces LRP5 [32,34], a gene product that promotes osteoblastogenesis via enhanced canonical Wnt signaling, and is thereby anabolic to bone [173]. Interestingly, LRP5 is not only beneficial to bone through an anabolic action in osteoblasts, but we
speculate that induction of LRP5 by 1,25(OH)2D3-VDR in duodenal enterochromaffin cells suppresses gut serotonin, a neurotransmitter which Karsenty and colleagues [174] have recently demonstrated is deleterious to bone and must be kept in check via an LRP5-triggered silencing mechanism. This is in contrast to central serotonin, which is downstream of leptin signaling and indirectly preserves bone via inhibition of sympathetic nervous system tone and b-adrenergic agonist release [175]. Osteocalcin (BGP) is another gene classically induced by 1,25(OH)2D3 in osteoblasts, particularly in the rat [176e178] and the human [179]. Recently, utilizing BGPnull animals, it has been shown that normal osteocalcin expression is important for robust, fracture-resistant bones [180]. Extracellular BGP also is thought to bind calcified bone matrix where it functions as a chemotactic agent for osteoclasts, perhaps strengthening the skeleton via enhanced remodeling cycles. Finally, osteocalcin has been identified by Karsenty and coworkers [181] as a bone-secreted hormone that both improves insulin release from pancreatic b-cells and increases insulin metabolic responsiveness in target tissues. Thus, vitamin-D-induced bone osteocalcin, by supporting insulin release and action, could be considered an important adjunct in insuring glucose control to delay or lower the risk of advanced glycosylation end product formation (“AGEing”) characteristic of uncontrolled diabetes mellitus which elicits retinopathy, neuropathy, and vascular disease. RANKL constitutes one of the most dramatically 1,25 (OH)2D3-upregulated bone genes, the product of which effects 1,25(OH)2D3-VDR mediated bone resorption through osteoclastogenesis (Fig. 8.3). We have shown that RANKL is induced over 5000-fold by 1,25(OH)2D3 in mouse ST-2 stromal cells in culture [4]. OPG, the soluble decoy receptor for RANKL that tempers its activity, is simultaneously repressed by 86% [4] to amplify the bioeffect of displayed (or secreted) RANKL. Thus, like PTH, 1,25(OH)2D3 is a potent bone-resorbing, hypercalcemic hormone and, although chronic excess of either hormone elicits a severe osteopenic pathology, physiologic bone remodeling can be argued to strengthen the skeleton. In other words, like a wellmineralized bone, an appropriately remodeled bone is a healthy bone, less susceptible to fractures and the eventual ravages of senile osteoporosis.
Feedback Control of 1,25(OH)2D3 Calcemic, Phosphatemic, and Bone-resorbing Actions The 1,25(OH)2D3-modulated genes enumerated in the previous paragraphs are all calcemic, phosphatemic, or affect bone remodeling. To govern these 1,25(OH)2D3induced phenomena, there exists a separate class of feedback regulatory genes which curb the mineralotropic and osteotrophic actions of 1,25(OH)2D3. Control
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VDR-MEDIATED CONTROL OF VITAL GENES IN HEALTHFUL AGING AND DISEASE PREVENTION
of these genes by 1,25(OH)2D3-VDR delimits bone mineralization to the defined endoskeleton, prevents ectopic calcification elicited by excesses of either calcium or phosphate, reduces age-related vascular pathology and atherosclerosis, protects against muscle and skin atrophy as well as respiratory failure, and generally prevents premature aging and lengthens lifespan [182]. Many of these pathologies are also the consequence of hypervitaminosis D [182]. Thus, excess vitamin D and its actions actually reduce lifespan, meaning that the level of 1,25(OH)2D3, as well as the sequelae of its effects through VDR, must be “detoxified” and sustained in an optimal range to maintain healthful aging. The vitamin D 24-hydroxylase, CYP24A1, is one of the most strongly 1,25(OH)2D3-induced genes, and its catalytic gene product is present in all cells expressing VDR. The endocrine and intracrine effects of 1,25 (OH)2D3 are curtailed by CYP24A1-catalyzed catabolism of 1,25(OH)2D3, providing an “off” signal once the hormone has executed its physiologic modulation of gene expression (Fig. 8.1). Mice with ablation of the CYP24A1 gene die early because of 1,25(OH)2D3 toxicity, and those that survive by compensatory adaptation lack endochondral bone formation caused by excess 1,25 (OH)2D3 [183]. Therefore, as summarized schematically in Figure 8.1, 1,25(OH)2D3 induces CYP24A1 to feed back to attenuate its actions through catabolism to inactive vitamin D metabolites. PTH is the major tropic hormone that stimulates the renal 1a-OHase, CYP27B1, to produce the 1,25(OH)2D3 hormone, primarily under low-calcium conditions when the calcium-sensing receptor in the parathyroid glands recognizes hypocalcemia and signals the synthesis and release of PTH. In a short feedback loop pictured in Figures 8.1 and 8.3, 1,25(OH)2D3-VDR feedback represses PTH gene expression to close the endocrine loop; PTH repression by 1,25(OH)2D3 is well documented [184], and a negative VDRE in the chicken PTH gene (Table 8.1) has been identified [151]. Calcium, mobilized from bone via resorption, or derived from 1,25(OH)2D3enhanced intestinal absorption, is a second negative feedback regulator of PTH elaboration. Ultimately, the combination of 1,25(OH)2D3-VDR and calcium closes the endocrine loop by suppressing PTH and eliminating its potentiation of CYP27B1, thereby reducing 1,25(OH)2D3 production. Normally, through its phosphaturic action, PTH would eliminate any excess, unneeded phosphate mobilized by 1,25(OH)2D3-VDR in the process of correcting hypocalcemia. However, because PTH is so quickly and effectively suppressed in this situation, there is a need for a second phosphaturic hormone to elicit both acute and long-term control of phosphate. FGF23 is the newly recognized phosphaturic peptide hormone which functions initially in concert with PTH, and chronically when PTH is suppressed by calcium
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and 1,25(OH)2D3. In fact, FGF23 directly represses PTH (Fig. 8.3) [185] to eliminate the activation of CYP27B1 by PTH, while at the same time taking over from PTH the role of phosphate elimination. Like PTH, FGF23 inhibits renal Npt2a and Npt2c to elicit phosphaturia (Fig. 8.3). Yet oppositely to PTH, FGF23 is strikingly up-regulated by 1,25(OH)2D3 as first shown by our group [7] in the case of rat UMR-106, osteocytelike cells of the osteoblast lineage (Fig. 8.3). Notably, FGF23 regulation is complex and multifactorial. For instance, PHEX, the gene encoding an endoproteinase which is loss-of-function mutated in X-linked hypophosphatemic rickets (XLH), acts to repress FGF23 expression [186], and excess FGF23 causes the hypophosphatemic pathology in XLH, tumor-induced osteomalacia (ectopic FGF23 secretion) [187], and autosomal dominant hypophosphatemic rickets (ADHR) [187] wherein FGF23 is altered to preclude proteolytic inactivation. 1,25(OH)2D3 represses PHEX expression (Fig. 8.3) in UMR-106 osteocyte-like cells [188], which is in accordance with the induction of FGF23 in that the PHEX suppressor is removed to permit maximal induction of FGF23 by 1,25(OH)2D3. It is conceivable that the mechanism of FGF23 induction by 1,25 (OH)2D3 is, in part or entirely, a consequence of PHEX repression, yet the PHEX substrate which ultimately regulates FGF23 transcription is not known. Although dentin matrix acid phosphoprotein 1 (DMP1) is apparently not a PHEX substrate, loss-of-function mutations in DMP1 cause a phenotype identical to XLH, with excess FGF23 producing hypophosphatemia. Conversely, as illustrated in Figure 8.3, hyperphosphatemia enhances FGF23 independently of 1,25(OH)2D3, rendering FGF23 the perfect phosphaturic counter-1,25 (OH)2D3 hormone because it inhibits both renal phosphate reabsorption and 1,25(OH)2D3 biosynthesis (Fig. 8.3). FGF23 also induces CYP24A1 (Fig. 8.1) in kidney to further reduce ambient circulating 1,25 (OH)2D3 levels. Regardless of the mechanism whereby FGF23 is induced, this hormone allows osteocytes in bone to communicate with the kidney to govern vitamin D bioactivation and circulating phosphate, thereby preventing excess 1,25(OH)2D3 function and ectopic calcification due to hyperphosphatemia. The primary mission of FGF23 is phosphate excretion to protect against hyperphosphatemia and ectopic calcification, which not coincidentally are the two dominant characteristics of the FGF23 knockout mouse [189]. As predicted, FGF23-null mice also possess markedly elevated 1,25(OH)2D3 in blood, and their phenotype is characterized by a shortened lifespan, skin atrophy, arteriosclerosis, and chronic obstructive pulmonary disease, with complications from the latter usually having fatal consequences [189]. Thus, FGF23, a gene induced by 1,25(OH)2D3, could be considered a longevity gene.
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Interestingly, double knockouts of FGF23 with either VDR [190] or CYP27B1 [191] essentially rescue the FGF23-null phenotype in mice, indicating that the capability of FGF23 to function as a counter-regulatory hormone to 1,25(OH)2D3 is the key to its health and longevity benefits. Thus, the physiologic activities of 1,25(OH)2D3 provide for healthful aging and prolong life by lowering the risk of chronic disorders of old age, but in pharmacologic doses or pathological excess, 1,25(OH)2D3 generates the phenotype of the FGF23 ablated mouse, including ectopic calcification, skin atrophy, osteoporosis, vascular disease, and emphysema. This situation of 1,25(OH)2D3 toxicity is analogous to the actions of the only other known toxic fat-soluble vitamin, namely vitamin A and its active retinoic acid metabolite. In physiologic quantities, retinoids mediate epithelial cell differentiation and barrier formation, embryonic development, etc., yet pharmacologic excesses of vitamin A and retinoic acid yield epithelial cell pathologies such as gastroenteritis and exfoliation, and embryopathy, respectively. Whereas no feedback control exists in the vitamin A system, the endocrine “Rosetta Stone” that allows the body to keep vitamin D in check is FGF23 and its signaling. FGF23 signals via renal FGFR1 and klotho coreceptors to promulgate phosphaturia and repress CYP27B1/ induce CYP24A1 [192], functioning through phosphoERK and resulting in induction of the early response gene Egr-1 [193]. Klotho is a bona fide longevity gene expressed primarily in the distal renal tubule which, when inactivated in mice, generates a phenotype apparently identical to that of FGF23-null mice [194], including premature aging/decreased lifespan, elevated 1,25 (OH)2D3, hyperphosphatemia, ectopic calcification, skin and muscle atrophy, osteoporosis, and hearing loss. FGF23 down-regulates its klotho coreceptor [8] but we recently discovered that 1,25(OH)2D3 induces klotho mRNA in kidney cells [4]. Up-regulation of klotho by 1,25(OH)2D3 is modest, but statistically significant, and consistent with potentiation of FGF23 signaling in kidney and perhaps in other cell types (e.g., vascular) where a secreted form of klotho is considered a potential beneficial hormone [195]. In one particularly striking study, klotho introduction via a viral vector prevented progression of spontaneous hypertension and renal damage in the SHR rat model [196]. We propose that many of the health and longevity benefits of 1,25(OH)2D3 may be effected through VDR-mediated enhancement of klotho expression in kidney (Fig. 8.3) and perhaps other cell types.
Interaction of 1,25(OH)2D3 and/or VDR with Wnt Signaling/b-Catenin Wnts consist of approximately 20 members of a secreted peptide signaling molecule family that act by
binding to transmembrane Frizzled (Fz) and low-density lipoprotein-related protein (LRP) coreceptors releasing intracellular Disheveled (Dsh) to inhibit the b-catenin degradation complex. The result is an accumulation of b-catenin which translocates into the nucleus and induces the transcription of target genes encoding proteins that function to alter cell proliferation, differentiation, polarity, and tissue remodeling. b-Catenin also functions extranuclearly to affect cell adhesion and motility. In the colon, b-catenin constitutes a proto-oncogene, with either mutational activation of its function [197] or loss-of-function mutation in its counterregulatory protein partner(s) [198] causing colon cancer. Calcium and 1,25(OH)2D3 are known to lower the risk of colon cancer [172], a disease prevalent at higher latitudes where vitamin D production from sunlight exposure is limited. 1,25(OH)2D3 protects against colon cancer by detoxifying LCA, the carcinogenic secondary bile acid [71] ligand for VDR, and by controlling the expression of genes which influence cell growth and differentiation such as TGFbR1, EGFR, etc. We have shown that VDR, in a 1,25(OH)2D3 ligand-dependent manner, suppresses the transcriptional activity of b-catenin in HT-29 human colorectal carcinoma cells [199], likely via direct VDRb-catenin protein interaction to divert transcriptional activity away from b-catenin target genes that promote proliferation, and toward VDR target genes that encode factors stimulating cell differentiation and apoptosis. Palmer and coworkers [200] have independently reached this same conclusion. Moreover, the interaction between VDR and b-catenin is significantly attenuated when VDR is instead liganded with carcinogenic LCA [199]. These observations provide further evidence for a mechanism whereby 1,25(OH)2D3-VDR acts to prevent colon cancer while LCA may contribute to tumorigenesis. The relationship between 1,25(OH)2D3-VDR and bcatenin is interestingly different in bone. VDR potentiates b-catenin-directed transcription in human osteoblast-like TE-85 osteosarcoma cells, and this effect is ligand-independent [4]. By enhancing b-catenin signaling in osteoblasts, VDR is apparently anabolic to bone via enhanced Wnt signaling. This action is consistent with the osteoporotic phenotype that occurs in LRP5 knockout [201] mice, and also with the fact that the most promising new bone anabolic drug potentially to treat osteoporosis is a sclerosterin-1 (SOST-1)-immunoneutralizing monoclonal antibody [202] which promotes Wnt/b-catenin signaling by removing the soluble, extracellular SOST-1 Wnt antagonist. Similar to the situation in bone cells, VDR ligandindependently up-regulates b-catenin transcriptional signaling in keratinocytes [4]. b-Catenin is absolutely required in keratinocytes [203], as is VDR [204], to permit mammalian hair cycling, and the ligand-independent action of VDR to mediate the hair
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CONCLUSIONS AND PERSPECTIVES
cycle is thought to involve Wnt signaling in skin stem cells [204,205]. Thus, mammalian hair cycling, which does not require a VDR ligand, appears to be driven by unliganded VDR (or VDR liganded with a nonvitamin-D lipid) through modulation of canonical Wnt signaling, with b-catenin as a key player. All of the effects of VDR on b-catenin can be considered “Fountain of Youth” actions because a lowered risk of colon cancer, enhanced bone volume, and competent skin protected by hair all lead to more healthful aging. As illustrated schematically in Figure 8.8, the regulation of mammalian hair cycling is complex, consisting of the convergence of two signaling pathways, BMP and Wnt. Starting at the upper left of Figure 8.8, Noggin from the dermal papilla initially antagonizes BMP4 signaling in bulb (or bulge) keratinocytes, allowing for the accumulation of Lef1/TCF (❶ in Fig. 8.8), a transcriptional coactivator which targets genes via DNA-binding partners such as b-catenin. Cessation of Noggin signaling reinstates BMP signal transduction via SMADs (❷ in Fig. 8.8) provided that the Wnt modulator in surface ectoderm (Wise or SOSTDC1), which antagonizes both Wnt and BMP pathways [206], has also been repressed. Wnt ligand, e.g., Wnt 10b, signaling and resulting accumulation of b-catenin facilitates cooperation with Lef1/TCF to induce the genes encoding factors, such as sonic hedgehog (Shh), that trigger the hair cycle to transition from telogen (resting) to anagen (growth). VDR apparently promotes b-catenin-Lef1/ TCF function by serving either as a coactivator of the b-catenin transcription complex, or inducing a positive member of this protein complex. Moreover, we propose that VDR also functions in keratinocytes to drive the hair cycle by controlling gene expression through negative as well as positive VDREs. This proposal is based upon gene ablation studies which indicate that VDR, its RXRa [111] DNA-binding heteropartner, as well as the coactivator DRIP205 [207] and the corepressor, hairless (Hr) [208], all are required for normal hair cycling. A candidate induced gene is the calcium channel TRPV6, which not only is required for hair cycling [171], but also is induced by ligandedVDR. Notably, intracellular calcium, which can be enhanced by TRPV6 activity, is itself a trigger for keratinocyte differentiation [209]. However, the most important role of VDR in controlling the hair cycle appears to be in repression of key target genes. This conclusion is based upon the observation that knockout of Hr, a VDR corepressor which colocalizes with VDR in the outer root sheath of the hair follicle [210], produces a phenocopy of the VDR-null mouse with respect to alopecia, but does not affect bone and mineral metabolism [211]. At least one of the molecular functions of Hr is recruitment of histone deacetylases (HDACs) to reconform chromatin to a repressive heterochromatin
157
architecture [212]; another proposed function of Hr is to catalyze histone demethylation (HDMe) to further attenuate transcription [213]. What genes are targeted by the VDR-RXRa-Hr complex for repression? Thompson and coworkers [205] have defined Wise (SOSTDC1) as a gene overexpressed in keratinocytes from Hr-null mice, and Kato and colleagues [214] characterized S100A8 as a gene overexpressed in VDR-null keratinocytes. In determining the effect of activated VDR on the expression of SOSTDC1 and S100A8 mRNA levels in human keratinocytes [4], we observed that Wise/SOSTDC1 is strikingly repressed after 18 hours of 1,25(OH)2D3 treatment of keratinocytes. Suppression of SOSTDC1 mRNA by 1,25(OH)2D3 was verified utilizing cDNA microarray analysis of human cells [4]. Because Wise/SOSTDC1 not only antagonizes the Wnt pathway by binding to LRP, but also inhibits the BMP pathway through neutralization of BMP4 [206], repression of Wise by VDR could constitute a major event in initiating the mammalian hair cycle (Fig. 8.8). Similarly, we observed that 1,25 (OH)2D3 rapidly represses expression in human keratinocytes of S100A8 and its obligatory S100A9 heteropartner in calcium binding [4]. This inhibition of S100A8/A9 expression by 1,25(OH)2D3-VDR is in stark contrast to the induction of S100A8/A9 observed in HL-60 promyelocytic leukemia cells when differentiated by 1,25 (OH)2D3 along the macrophage lineage [215]. Thus, 1,25(OH)2D3 regulates S100A8/A9 expression differentially in a cell-selective fashion. The S100A8 protein is a general biomarker for inflammation and malignancy [216], but we propose that its role in keratinocytes is to dampen intracellular calcium oscillations required for skin differentiation and hair cycling (Fig. 8.8). Therefore, repressing S100A8/A9 could conceivably restore the cellular calcium gradient which appears to be the ultimate messenger that controls keratinocyte function. One additional gene repressed by 1,25(OH)2D3-VDR, namely PTHrP [217], is already known to encode a suppressor of the telogen to anagen transition in the hair follicle, as well as promote entry into catagen [218], providing yet another VDR-RXRa-Hr repressed gene target that participates in hair cycle control (Fig. 8.8). In summary, VDR appears to usher the regeneration of hair, an obvious shield that protects skin and facilitates healthful aging, via both proteineprotein and proteineDNA interactions that potentiate Wnt, BMP, and calcium signaling.
CONCLUSIONS AND PERSPECTIVES The vitamin D receptor is a unique member of the nuclear receptor superfamily in the realm of ligand binding, mediating both the endocrine control of bone
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VDR action in keratinocytes to sustain the mammalian hair cycle. Keratinocytes or their precursors in the bulge of the hair follicle are thought to receive signals that stimulate the follicle to exit a resting phase (telogen) and enter a phase of active growth (anagen). A number of factors [218,263,264] have been identified that influence this transition, and a simplified diagram is presented here. Abbreviations not defined in the text are: Wnt, ortholog of Drosophila wingless and mouse int-1; Lef1, lymphoid enhancer factor-1; TCF, T cell-specific factor; msx-1 and msx-2, orthologs of Drosophila muscle-specific homeobox protein; factors that are membrane receptors or transporters are boxed. Solid arrows indicate activation and dotted lines ending in a solid perpendicular line denote inhibition. The black circled 1 and 2 in the BMP/SMAD pathway represent sequential negative and positive signaling as described in the text. The precise point(s) at which VDR or the RXR-VDR-Hr complex act has not been fully characterized, and it is not known whether VDR functions unliganded or occupied by a novel, non-vitamin D ligand. See text for an explanation of the model.
FIGURE 8.8
mineral metabolism and the detoxification of endobiotics such as lithocholic acid. Liganded VDR functions as a heterodimer with unliganded RXR, with the binary protein complex required for recognition and highaffinity association with VDREs in the promoters of regulated genes, as well as for the recruitment of transcriptional comodulator proteins that govern the activity of RNA polymerase II. As a nuclear receptor that heterodimerizes with RXR, VDR therefore possesses the molecular character of both its evolutionarily closest endocrine relative, the thyroid hormone receptor, and of its closest xenobiotic detoxifying receptor, PXR. The biology of VDR signaling primarily involves: (i) stimulation of intestinal calcium and phosphate absorption to prevent rickets/osteomalacia, (ii) enhancement of bone remodeling via osteoblast-induced osteoclast maturation, (iii) differentiation of skin cells and maintenance of the hair cycle, (iv) repression/induction of CYP enzymes for 1,25(OH)2D3 hormone synthesis/degradation as well as for the promotion of secondary bile acid detoxification, (v) modulation of the immune system, and (vi) potential anticancer actions via the control of epithelial cell growth, differentiation, and apoptosis.
Neoclassical Functions for Liganded and Unliganded VDR Figure 8.9 chronicles the actions of vitamin D and VDR within an iceberg, with the traditional bone and
mineral (antirachitic/antiosteoporotic) effects, as well as newly recognized bone anabolic and counter-ectopic calcification functions, as the fraction of the iceberg above the water line. The bulk of VDR’s actions are novel extraosseous effects that are diagramed in the submerged portion of the iceberg. These actions are all mediated by VDR, consistent with the broad cellular distribution of VDR expression. Many of the extraosseous effects of VDR appear to be triggered by locally produced 1,25(OH)2D3. Numerous tissues besides the kidney express the 1a-OHase enzyme, including cells of the immune system (e.g., T-cells), the pancreas, skin, etc. This locally produced 1,25(OH)2D3 does not contribute significantly to circulating 1,25(OH)2D3, but rather is active in a cell- and tissue-specific manner. Examples of local 1,25(OH)2D3-VDR actions include repression of IL-2 in T-cells [219], induction of defensin and cathelicidin as local antimicrobial effectors [220], stimulation of involucrin synthesis in skin [221], CYP3A4 and p21 induction in epithelial cells e especially in the colon [62], and promotion of insulin secretion from the b-cells of the pancreas [222]. By locally stimulating the above-mentioned genes, the vitamin D/VDR system emerges, likely redundantly with other regulators, as an immunomodulator that stimulates the innate and suppresses the adaptive immune system to effect both antimicrobial and antiautoimmune actions, detoxifies xenobiotics to be chemoprotective, controls cell proliferation, and regulates apoptosis to reduce
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FIGURE 8.9 A summary of the classical bone mineral effects of 1,25(OH)2D3-VDR with its relevant gene targets as the “tip of the iceberg,”
and novel “extraosseous” effects of the VDR-RXR heterodimer with candidate regulated genes as the proverbial submerged portion of the iceberg. Also shown at the upper left are the calcemic and phosphaturic hormones that participate in feedback loops to maintain bone mineral homeostasis as discussed in the text; a normally mineralized human vertebral body with its trabeculations is illustrated at the upper right. Thus the upper portion of the figure depicts actions of 1,25(OH)2D3 liganded VDR to maintain bone health, including interactions with other hormones (PTH, CT, FGF23). The lower portion of the figure summarizes the many extraosseous effects of VDR in the 1,25(OH)2D3-bound, unliganded, and novel ligand-bound states, as discussed in the text. Repressive actions of VDR are depicted as dashed arrows. We hypothesize that VDR occupied by locally generated 1,25(OH)2D3 uses cell context specific coactivators, and that VDR occupied by a novel ligand (NL) may utilize ligand selective comodulators.
cancer, and moderates type II diabetes by promoting insulin release as well as possibly enhancing fatty acid b-oxidation via induction of FOXO1 (Table 8.1). A second possibility obviating the need to locally generate 1,25(OH)2D3 would be for VDR to function unliganded. As indicated above, VDR, but not vitamin D, is required to sustain the mammalian hair cycle. Thus, as depicted in Figure 8.9 (lower center), the Hr corepressor could function as a surrogate VDR “ligand” to suppress Wise or other genes that normally keep the hair cycle in check. Also, unlike the case of intestine, kidney, and bone, calbindin induction by VDR does not require vitamin D in brain [223]. VDR is widely expressed in the central nervous system, as is Hr, raising
the possibility that unliganded VDR, along with Hr, acts in select neurons. Notably, it has been reported that VDR-null mice exhibit behavioral abnormalities including anxiety, etc. [224]. However, the ability of VDR to function unliganded is difficult to justify physicochemically because the tertiary structure of VDR and its functionally interactive surfaces cannot be stabilized unless the hydrophobic binding pocket is occupied by a lipophilic ligand. We therefore suggest that VDR binds one or more naturally occurring non-vitamin-D ligands to effect its extraosseous actions. As discussed above, we have identified several potential examples of non-vitamin-D-related VDR ligands, including LCA, PUFAs, and curcumin.
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Because VDR is capable of binding these lipids, albeit with low affinity, the receptor may have retained its promiscuity for ligand binding that presumably originated with its primitive detoxification function. Also, the ligand-binding pocket of VDR is second only to PXR in volume among the crystallized nuclear receptor ligand-binding domains, suggesting (but not proving) that it can accommodate a broad array of lipids. The question remains whether VDR coevolved higheraffinity local ligands that would explain the broad health benefits of vitamin D and other lipid nutrients beyond bone. For example, 1,25(OH)2D3-VDR is anti-inflammatory and suppresses NFkB [225,226]. This action would be desirable for instance in preventing atherosclerosis. Is there perhaps a local novel VDR ligand in endothelial cells that could trigger the anti-inflammatory influence of VDR? Combined with the anticalcification effect of FGF23, VDR would then be able to exert a two-pronged attack in preventing arteriosclerosis. Only the future will reveal the actual mechanisms for the apparent cardiovascular benefits of vitamin D/VDR. Clearly, VDR will emerge as a versatile therapeutic and preventative target once we understand fully the pleiotropic extraosseous effects of vitamin D. No doubt the faces of vitamin D and VDR will again change to reflect new frontiers in health and disease.
VDR as a Longevity Nuclear Receptor In the present chapter we have highlighted numerous genes for which the expression is modulated by 1,25 (OH)2D3-VDR, and theorized that through physiological regulation of the transcription of these and other genes, the nuclear VDR can be characterized as an “age-well” receptor. These genes fall into four general classes. The first group is comprised of genes for which the products homeostatically control bone and mineral metabolism, as well as the integrity of the endoskeleton. By inducing TRPV6/TRPV5 and Npt2b/Npt2c, VDR signaling supplies respective calcium and phosphorus minerals via absorption/reabsorption to generate the fully mineralized endoskeleton. By inducing the calcium-sensing receptor (CaSR) [227] (Fig. 8.3), repressing PTH and PHEX, and inducing the FGF23/klotho system, plus up-regulating CYP24A1, VDR prevents the production of excess 1,25(OH)2D3 hormone and protects against ectopic calcification arising from hypercalcemia and/or hyperphosphatemia. Through regulation of BGP, SPP1, LRP5, RANKL, and OPG, VDR ensures the formation of high-volume, fractureresistant bone with connectivity that is modeled for strength via osteocyte mechanosensing endocrine cells in the skeleton. Klotho can be considered to be in a class (second group) by itself because it has been shown to represent
a longevity gene, with actions that include but are not limited to control of phosphate metabolism [195]. The third group is composed of genes encoding factors impacting Wnt signaling that effects organogenesis (especially skin and bone) and hair growth, including LRP5 (also in group 1), SOSTDC1, S100A8/S100A9, and PTHrP. Control of this set of genes by VDR guarantees the proper formation of epithelial cell barriers, especially the cornified epithelium of the epidermis, to guard against invasion by microorganisms and elicits hair growth and cycling to shield against the age-related damages from UV irradiation. The fourth group of VDR-regulated genes is unrelated to skin, bone, and mineral metabolism, but regulation of these genes by vitamin D is well documented to facilitate healthful aging by delaying or eliminating a host of diseases. For example, 1,25(OH)2D3-VDR induces cathelicidin [220] to activate the innate immune system to fight infection, and represses IL-17 [10] to temper the adaptive immune system and lower the risk of autoimmune disorders such as type I diabetes mellitus, multiple sclerosis, lupus, and rheumatoid arthritis. Liganded VDR also functions as a detoxification nuclear receptor by inducing CYP3A4 [71] and SULT2 [228] to eliminate toxic xenobiotics that might affect the gastrointestinal tract, skin, and other tissues. High vitamin D concentrations are associated with longer leukocyte telomere length, a parameter which decreases with each cell cycle and increased inflammation, highlighting the potential beneficial effects of 1,25(OH)2D3-VDR on aging and age-related diseases [229]. 1,25(OH)2D3-VDR is antiinflammatory by blunting NFkB [225,226] and COX2 [230] and, because inflammation is considered a common denominator in maladies such as heart disease and stroke, as well as cancer, the inflammation-reducing actions of VDR could be crucial in the healthy aging property of the vitamin D receptor. VDR likely also reduces risk for many cancers by inducing the p53 [231] and p21 [231] tumor suppressors, as well as DNA repair enzymes in skin [232]. Thus, it is no surprise that the VDR-null mouse is supersensitive to DMBA-induced skin cancer [59] as well as UV-lightinduced skin malignancy [60]. VDR knockout mice exhibit enhanced colonic proliferation [233] plus amplified mammary gland ductal extension, end buds, and density [56], indicating that the fundamental actions of VDR to promote cell differentiation and apoptosis [199] play an important role in reducing the risk of age-related epithelial cell cancers such as those of the colon and breast. In the realm of cardiovascular disease, and neurodegenerative disorders of aging such as Alzheimer’s disease, excess circulating homocysteine is considered a negative risk factor [234,235]. Amazingly, 1,25(OH)2D3-VDR has recently been shown by the group of Bouillon [236] to induce cystathionine b-synthase
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BGP TRPV6/5 LRP5 RANKL SPP1
FGF23/Klotho CYP3A4 CYP24A1 cathelicidin FOXO3 Npt2b/c
cystathionine β-synthase
CaSR
SULT2
p21
p53
FOXO1
PHEX
NFκB
COX-2
OPG
IL-2/17
SOSTDC1
S100A8/9
PTH/PTHrP
1,25(OH)2D3-VDR FIGURE 8.10 Stylistic representation of VDR regulation of vital gene expression to “feed the Fountain of Youth”. Genes listed on the upper
portion of the fountain are induced by liganded VDR, whereas those appearing in italics in the lower tier of the fountain are repressed. We assert that the collective control of these genes allows one to age well by delaying: fractures, ectopic calcification, oxidative damage, infection, inflammation, pain, and malignancy.
(Table 8.1), a major enzyme catalyzing the metabolic elimination of homocysteine. Finally, 1,25(OH)2D3VDR induces FOXO3 [237], an important molecular player in preventing oxidative damage, a leading candidate for the cause of aging [238]. Clearly, as depicted in schematic form in Figure 8.10, through control of key genes, VDR indeed feeds the “Fountain of Youth” and allows one to age well by delaying fractures, ectopic calcification, oxidative damage, infections, autoimmunity, inflammation, pain, cardiovascular disease, and malignancy.
Conclusions In conclusion, in the time elapsed since the last edition of this volume, we have witnessed the determination, in solution via Small Angle X-ray Scattering and Fluorescence Resonance Energy Transfer techniques, the structure of the hVDR DBD and LBD together in the full-length receptor, heterodimerized with full-length RXRa, docked on a VDRE, and occupied with 1,25(OH)2D3 plus a single coactivator [125], allowing us to begin to understand the interactions between
the DBD and the ligand-binding/heterodimerization domains of both VDR and its RXR heteropartner. A second major realm of accomplishment has been the identification of numerous additional VDR-regulated genes, including those mediating calcium and particularly phosphate homeostasis, bone metabolism, detoxification, cell proliferation, differentiation, migration and death, immunity and antimicrobial action, as well as carbohydrate, lipid, and amino acid metabolism. Equally exciting has been the discovery of additional natural VDR ligands, especially in the more-recently identified and studied VDR target tissues such as the hair follicle/skin and immune systems. The availability of this information will facilitate molecular investigations of transcriptional control by VDR in target-cellspecific environments in the presence of novel ligands and/or in the context of a myriad of promoters/genes. Such experiments will likely extend our understanding of the variety of conformations and coregulator associations that the VDR-RXR heterodimer is capable of achieving while performing its multitude of extraosseous effects (Fig. 8.9) to lower the risk of the chronic diseases of aging.
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Acknowledgment Supported by NIH grants to M.R.H. and P.W.J., and an Arizona Biomedical Research Commission grant to J.-C.H. The authors thank other members of our laboratories, Leonid Bartik, Jan B. Egan, Ryan E. Forster, Julie K. Furmick, Zachary Hernandez, Jana L. Kubicek, Christine L. Lowmiller, Eric W. Moffet, Janky Patel and Yifei Wu, for their contributions.
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[257] A.F. Gombart, N. Borregaard, H.P. Koeffler, Human catidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3, FASEB J. 19 (2005) 1067e1077. [258] X. Wang, T.T. Wang, J.H. White, G.P. Studzinski, Induction of kinase suppressor of RAS-1(KSR-1) gene by 1, alpha25-dihydroxyvitamin D3 in human leukemia HL60 cells through a vitamin D response element in the 50 -flanking region, Oncogene 25 (2006) 7078e7085. [259] X. Wang, T.T. Wang, J.H. White, G.P. Studzinski, Expression of human kinase suppressor of Ras 2 (hKSR-2) gene in HL60 leukemia cells is directly upregulated by 1,25-dihydroxyvitamin D(3) and is required for optimal cell differentiation, Exp. Cell Res. 313 (2007) 3034e3045. [260] S. Lee, D.K. Lee, E. Choi, J.W. Lee, Identification of a functional vitamin D response element in the murine Insig-2 promoter and its potential role in the differentiation of 3T3-L1 preadipocytes, Mol. Endocrinol. 19 (2005) 399e408. [261] J.J. Eloranta, Z.M. Zair, C. Hiller, S. Hausler, B. Stieger, G.A. Kullak-Ublick, Vitamin D3 and its nuclear receptor increase the expression and activity of the human protoncoupled folate transporter, Mol. Pharmacol. 76 (2009) 1062e1071. [262] Jmol: an open-source Java viewer for chemical structures in 3D. http://www.jmol.org/. [263] E. Fuchs, B.J. Merrill, C. Jamora, R. DasGupta, At the roots of a never-ending cycle, Dev. Cell 1 (2001) 13e25. [264] E.M. Peters, K. Foitzik, R. Paus, S. Ray, M.F. Holick, A new strategy for modulating chemotherapy-induced alopecia, using PTH/PTHrP receptor agonist and antagonist, J. Invest. Dermatol. 117 (2001) 173e178.
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C H A P T E R
9 Structural Basis for Ligand Activity in VDR Natacha Rochel, Dino Moras De´partement de Biologie et de Ge´nomique Structurales, Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Sante´ de la Recherche Me´dicale, Universite´ de Strasbourg, 1 rue Laurent Fries, 67404 Illkirch, France
INTRODUCTION All nuclear receptors (NRs) are modular proteins that harbor one DNA-binding domain and one ligandbinding domain (LBD) (Fig. 9.1) [1,2]. NRs act as ligand-induced factors that enhance or suppress transcription of their target genes. Some receptors can act as silencers of transcription in the absence of ligands or the presence of antagonists. Agonists induce a change in the structure of the NR that allows interaction with coactivators that can acetylate histones, which prepare target gene promoters through decondensation of the corresponding chromatin [3]. Following decondensation, a second complex, Med1, appears to establish linkage to the basal transcriptional machinery [4]. The ligand-binding domain of nuclear receptors harbors a highly structured ligand-dependent activation function or AF-2, a major interface for dimerization with RXR and an interface for coactivators as well as corepressors. Detailed molecular insights into the structureefunction of nuclear receptors have been gained A/ B
C
by the elucidation of the crystal structures of the LBD alone or in complexes with agonists, antagonists, and coregulator peptides. The first 3D structures reported for NR LBDs were those of the unliganded RXRa [5], the all-trans retinoic acid-bound RARg [6], and the agonist-bound thyroid receptor TRb [7]. Unliganded receptors are referred to as apo receptor forms while liganded referred to as holo forms. To date, the crystal structures of most human NR LBDs [reviews in 8,9] and of only one full-length heterodimer PPAR/RXR [10] have been reported. The general fold of nuclear receptors LBD consists of a three-layered a-helical sandwich. The helices have been designated H1eH12, according to the first crystal structures of RXR and RAR [11]. The different crystal structures of apo and holo forms of RXR LBDs as well as extensive NR mutagenesis have demonstrated that LBDs undergo major conformational changes upon ligand binding (Fig. 9.2). Ligand-induced conformational changes for NRs have been likened to the “mousetrap mechanism” described for RAR [6]. Upon ligand binding, helix H11 is D
E
F
AD AD
N
C
DNA binding DIMERIZATION
dimerization NLS
LIGAND BINDING AF-2 Activation Function Ligand-dependent Interaction with cofactors
AF-1 Activation Function Ligand independent
Structural and functional organization of nuclear receptors. NRs consist of six domains (AeF) based on regions of conserved sequence and function. Please see color plate section.
FIGURE 9.1
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10009-5
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FIGURE 9.2 Superimposition of unliganded (green and yellow) and liganded (blue and red) hRXRa LBD monomers. The main conformational differences affecting helices H3, H11, and H12 are colored in yellow and red. The arrows show the main structural changes upon ligand binding. The ligand is depicted in yellow. Adapted from Figure 4A of Egea PF, Mitschler A, Rochel N, Ruff M, Chambon P, Moras D 2000. Crystal structure of the human RXRalpha ligand-binding domain bound to its natural ligand: 9-cis retinoic acid. EMBO J 19:2592e2601 [77]. Please see color plate section.
repositioned in the continuity of helix H10, and helix H12 swings to seal the binding cavity while the u-loop flips over underneath helix H6 carrying along the Nterminal part of helix H3. Helix 12, also referred as the activating domain (AD) of the AF-2 function, stabilizes ligand binding by contributing to the hydrophobic environment, making in some cases additional contacts with the ligand. The structural data reveal that H12, when folded back onto the core of the LBD, forms a hydrophobic cleft together with other surface-exposed residues that accommodates the “NR box” of coactivators such as members of the SRC-1/TIF2 family [12e14]. VDRs have been characterized from mammals [15e17], birds [18], Xenopus laevis [19], Paralichtus olivaceus [20], zebrafish (GenBank accession number AAF21427), and recently from lampreys [21]. Sequence analysis of the VDR subfamily members reveals that VDR presents a large insertion domain at the N-terminal part of the LBD in the peptide connecting helices H1 to H3. The length of this connecting region varies between 72
and 81 residues compared to 15e25 residues for the other nuclear receptors. This insertion region, for which no biological function has been found, is poorly conserved between the VDR species with only 9% identity. One mutation associated with the genetic disease vitamin-Dresistant rickets type II has been found in this region (Cys190Trp of hVDR) with no effect on ligand binding [22]. A phosphorylation site has also been identified at Ser208 [23]. Secondary structure prediction reveals that this region is not structured. Among the NR LBD crystal structures solved, it has been reported frequently that the region connecting helices H1 to H3 is poorly ordered and shows little if any secondary structure. In hVDR, this region is sensitive to proteolysis with a trypsin cleavage site at C-terminal of Arg174 [24]. Structural characterization of VDReligand interactions has been investigated using either hVDR LBD, rVDR LBD mutants or zVDR LBD wild type [25e27]. In the hVDR and rVDR mutants, a deletion of 50 residues in the region connecting helices H1 to H3 has been engineered in order to stabilize the protein by lowering the number of conformations adopted by the insertion region. This hVDRD has been fully characterized in solution and compared to the wild-type hVDR [25,28] and has been used to solve the initial crystal structure of VDR LBD in complex with its natural ligand [25]. Since the first crystal structure of VDR LBD in 2000 [25], 33 crystal structures of VDR LBD complexes have been deposited in the protein data bank so far.
CRYSTAL STRUCTURE OF hVDRD BOUND TO 1,25(OH)2D3 Deletion of the hinge region insertion in hVDRD stabilizes the protein and allows the crystallization of the hVDR-1,25(OH)2D3 complex [25]. The crystal struc by a combination of molecular ture was solved at 1.8 A replacement using a homology model based on the retinoic acid receptor RARg [6] and isomorphous replacement with a mercurial derivative. A higher resolution data set was collected later [29]. The overall (1.5 A) topology of the hVDRD LBD (Fig. 9.3) is that of the canonical NR LBD with 13 a helices sandwiched in three layers and a three-stranded b sheet. Helices H1 and H3 are connected by two small helices H2 and H3n, H3n replacing the u loop of the RARg structure [6]. The truncation of the hVDRD construct is positioned just before H3n as shown in Figure 9.3. The VDR LBD structure is closely related to that of the holo hRARg LBD structure [6] with a root mean square deviation (rmsd) of the over 179 residues. The superimposed structure of 1.2 A connection between helices H1 and H3 follows a path between H3 and the tip of the b-sheet similar to that of the ERa structure [30]. The tip of the b sheet is
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FIGURE 9.3 Overall fold of hVDRD ligand-binding domain. The helices are represented as cylinders and b sheets as arrows. The whole structure is colored in gray except the helix H12 in purple. The ligand is depicted in yellow. The insertion region location is shown in green. The reconnected residues are indicated, together with their sequence numbering. Adapted from Figure 1 of Rochel N, Wurtz JM, Mitschler A, Klaholz B, Moras D 2000. The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol Cell 5:173e179 [25]. Please see color plate section.
FIGURE 9.4 Intramolecular interactions of helix H12 in VDR. The backbone of the protein is colored in gray except for helix H12 in purple. The side chain residues involved in polar stabilization of H12 are shown together with the hydrogen bonds (green dots). The ligand is depicted in yellow and red for the oxygen atoms. Adapted from Figure 3 of Rochel N, Wurtz JM, Mitschler A, Klaholz B, Moras D 2000. The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol Cell 5:173e179 [25]. Please see color plate section.
consequently shifted outward and enlarges the ligandbinding cavity. The b-sheet tip is stabilized by hydrogen bonds with residues of the H2eH3n loop. Helix 12 is in the agonist position and stabilized by two interactions with the ligand. Helix 12 is also stabilized by several hydrophobic contacts with residues of H3, H5, and H11 and two polar interactions with residues of H3 and H4 (Fig. 9.4). Some of these residues contact the ligand, thus indicating an additional indirect ligandcontrol of the position of helix H12. In the hVDRD structure, a strong crystalline contact is observed between helix H3n and helices H3, H4, and H12 of a symmetrically related molecule, with H3n mimicking the coactivator SRC-1 peptide contacts [12e14]. Active vitamin D resides in a chair B conformation with the 19-methylene “up” and the 1a-OH and 3b-OH groups in an equatorial and axial orientation, respectively. A chair A conformation of the A-ring would disrupt the hydrogen bonds formed by the hydroxyl and the protein. The conjugated triene system connecting
A-ring to C- and D-rings accommodates an almost trans conformation with the C6eC7 bond exhibiting a torsion angle of e149 that deviates significantly from the planar geometry which results in the curved shape of the ligand bound to the receptor. The deviation explains the lack of biological activity of the analogs with a trans or a cis conformation of the C6eC7 bond [31]. The a face of the C-ring is lined by Trp286 whereas the methyl C18 on the b face points toward Val234 (H3). The ligand-binding pocket is lined by hydrophobic residues (Fig. 9.5). The elongated ligand embraces helix H3 with its A-ring orientated toward the C-termini of helix H5 and the 25-OH close to helices H7 and H11. The 1-OH group forms two hydrogen bonds with Ser237 (H3) and Arg274 (H5), while the 3-OH group is hydrogen-bonded to Ser278 (H5) and Tyr143 (loop H1eH2). The 25-OH moiety forms two hydrogen bonds with His305 (loop H6-H7) and His397 (H11). The triene is tightly fitted in a hydrophobic channel sandwiched between Ser275 (loop H5-b) and Trp286 (b1) on one side and Leu233 (H3) on the other side. The
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FIGURE 9.5 Stereo view of 1a,25(OH)2D3 in the hVDR-binding pocket. The ligand molecule is shown in the experimental electron density omit map contoured at 1.0 standard deviation. The hydroxyl groups are depicted as red spheres. Water molecules are shown as purple spheres. Hydrogen bonds are shown as green dotted lines. Carbon atoms are colored in gray, and oxygen and nitrogen atoms are colored in red and blue, respectively. Adapted from Figure 3 of Rochel N, Wurtz JM, Mitschler A, Klaholz B, Moras D 2000. The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol Cell 5:173e179 [25]. Please see color plate section.
aliphatic chain at position 17 of the D-ring adopts an extended conformation parallel to the C13eC18 bond with the C13eC17eC20eC22 torsion angle close to 90 ; it is surrounded by hydrophobic residues. 3) The ligand-binding cavity of hVDRD is large (697 A with the ligand occupying only 56% of this volume. A channel of water molecules near position 2 of the Aring makes an additional space that can accommodate ligands with a methyl group at position 2. The fourfold increase in binding affinity of the 2a-methyl analog is in agreement with this observation [32]. Additional space around the aliphatic chain is also observed. In order to investigate structurally and functionally important amino acid interactions within the ligandbinding pocket of the wild-type hVDR in the presence of several synthetic vitamin D analogs, Vaisanen et al. [33] have combined side-directed alanine mutagenesis with limited proteolytic digestion, electrophoretic mobility shift assay, and reporter gene assay. They have shown that structurally different agonists have distinct ligandereceptor interactions and that residues H229, D232, E269, F279, and Y295 are critical for the
agonist conformation [33]. Using a two-dimensional alanine scanning mutational analysis, Choi et al. [34] have studied the interactions between VDR and various vitamin D ligands. Eighteeen alanine mutants of residues forming the ligand-binding pocket were studied. These residues form either hydrogen bonds with the ligands (Y143, S237, R274, S278, H305, H397) or hydrophobic interactions (D144, L233, V234, I238, I268, I271, S275, C288, W286, V300, Q400, Y401). The transactivation potencies of these mutants with the natural hormone and 11 other ligands were evaluated through functional studies with luciferase reporter assay and a mouse osteopontin response element. The comparison of six mutants of hVDRD and wild-type hVDR has shown that both LBPs are similar, thus providing additional evidence to support identical structures of VDR mutant and wild type. The importance of these residues in the interactions with specific ligands was evaluated in this study. Eight residues (Y143, D144, L233, I271, R274, W286, H397, Y401) have been shown to be essential for transactivation by vitamin D ligands and mutations to less bulky
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hydrophobic residues increase the potency of 20-epi ligands [34]. The rat VDR LBD has been crystallized as a mutant with a deleted insertion domain corresponding to that of the human VDRD and in presence of a peptide containing the second LXXLL motif of Drip205 [26]. The structure of rVDRD is similar to the hVDRD one with a root mean square deviation of the superimposed The LXXLL peptide binds in a structures of 0.53 A. groove formed by helices 3, 4, and 12. This interaction 2 of the receptor’s surface. The side buries about 507 A chain of Leu630, 633, and 634 of Drip205 are buried within the pocket and surrounded by hydrophobic residues, while Met631 and Asn632 are oriented outwards from the solvent. The ligand 1,25(OH)2D3 binds rVDRD as reported for the hVDRD in an extended conformation.
CRYSTAL STRUCTURE OF zVDR WILDTYPE LBD BOUND TO 1,25(OH)2D3 In order to validate the conclusions made on the hVDRD structure and to find another crystal packing, the VDR from zebrafish (Danio rerio) was also studied. The zVDR exhibits 100% identity in the LBP lining residues and its transactivation potency is 50% of that of hVDR [27]. This activity is consistent with that of Xenopus laevis VDR or lamprey VDR, which show 50% [19] and 25% [21], of the activity of the hVDR, respectively. The zVDR LBD exhibits 69% identity and 79% similarity in its sequence with that of the hVDR LBD, while the insertion region (191e252 of zVDR) exhibits only 34% identity and 47% similarity. The zVDR LBD in complex with 1,25(OH)2D3 was crystallized in presence of a coactivator peptide that contains the second NR box of SRC-1 [27,35]. The zVDR LBD (Fig. 9.6) adopts the canonical active conformation observed for agonist-bound receptors. The SRC-1 peptide (687e696 HKILHRLLQE) forms an amphipatic a-helix interacting with a hydrophobic cleft on the LBD surface. These interactions are similar to those described for other NRs [12e14]. In particular, Glu446 from H12 [Glu420] forms hydrogen bonds with the backbone amide nitrogen of Leu690 and Leu691. At the other end, Lys274 from H3 (Lys246) forms a hydrogen bond to the main chain oxygen of Leu694. The structures of zVDR LBD and hVDRD LBD complexes with 1,25(OH)2D3 are similar with a root over 236 main chain mean square deviation of 0.72 A atoms. The binding pocket is identical and the natural ligand adopts the same conformation and forms the same interactions with the protein in the two structures [35]. Notably the channel of water molecules at position 2 of the ligand, which was observed in the hVDR
Overall fold of zVDR ligand-binding domain. The helices (in red) are represented as cylinders and b sheets (in yellow) as arrows. The SRC-1 peptide is shown in blue. The ligand 1a,25(OH)2D3 is depicted in gray. The green stars indicate the VDR-specific insertion region not seen in the crystal structure. Please see color plate section.
FIGURE 9.6
structure, is also observed in the zVDR. The differences observed when superimposing the two structures are small and primarily involve the loops. The ligandbinding pocket is conserved both in sequence and structure. The VDR-specific insertion region between helices H2 and H3 present in the zVDR construct and deleted in the hVDR mutant construct is not visible in the electron density map. This probably reflects the mobility of this region resulting in a static disorder. In the crystal structure of a VDR subfamily member, PXR [36,37], which also contains an insertion region between helices H1 and H3, the loop is also partially unresolved. The structural similarities between zVDR wild type and hVDRD LBDs bound to 1,25(OH)2D3 validate the overall biological relevance of the mutant hVDR structure.
STRUCTURE OF VDR LBD COMPLEXED TO SUPERAGONIST LIGANDS Superagonist analogs are at least ten times more potent in transactivation assays and act as antiproliferative agents with a magnitude of several orders higher than 1,25(OH)2D3 in vitro. The elucidation of the crystal
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structures of some of these superagonist ligands in complexes with VDR help to understand their biological potency.
20-epi Analogs Among the several synthetic analogs of vitamin D, the 20-epi compounds [38], which exhibit an inverted stereochemistry at position 20 in the flexible aliphatic chain, have attracted much attention. They are potent growth inhibitors and inducers of cell differentiation, while showing an affinity similar to that of 1,25 (OH)2D3 for VDR [38,39]. KH1060 (1,25-dihydroxy20epi-22oxa-24,26,27-trihomovitamin D3 [38]) a member of this 20-epi family exhibits similar properties with decreased calcemic side effects. 20-epi compounds induce VDR-dependent transcription at concentrations at least 100-fold lower than the natural ligand and provoke antiproliferative activity several orders of magnitude higher than the natural ligand [38e41]. The differences in biological activity of 1,25(OH)2D3 and the 20-epi molecules in general, and KH1060 in particular, are known to be VDR-LBD-dependent. However, they are not yet understood. The ability of 20-epi analogs complexed to VDR to induce transcription appears to correlate with the ability of these compounds to promote coactivator interaction [40]. Differing proteolytic digestion patterns of VDR in the presence of the natural ligand and the 20-epi compounds has been interpreted to reflect large conformational changes in the receptor upon binding of the later class of molecules. In order to investigate the binding mode of the 20-epi analogs to the VDR-LBD, the crystal structures of the hVDRD in complex with MC1288 (20-epi-1,25(OH)2D3 [29]) and KH1060 were determined and compared to those structures that were obtained with the natural ligand [25] (Fig. 9.7). When compared to hVDRD-1,25(OH)2D3 complex, the atomic models show an rms deviation on Ca atoms and 0.14 A for hVDRD-MC1288 complex and of 0.08 A hVDRD-KH1060 complex, respectively. Variations involve only some side chains located at the surface of the protein. In opposition to the view that the 20-epi analogs induce a different agonist conformation, the overall conformation and especially the position of helix H12 are strictly maintained in all three complexes. Furthermore, the ligand-binding cavity is unique and conserved for all three complexes. The rms deviations of all atoms comprising the ligand pocket are 0.09 A for hVDRD-MC1288 complex and 0.12 A for the hVDRD-KH1060 complex. The sizes of the three ligands 3, 375 A 3, and 392 A 3 for 1,25(OH)2D3, MC1288, are 381 A and KH1060, while the volume of the ligand pocket 3). The remains unaltered in the three complexes (660 A ligands occupy only 57% of the volume of the pocket
for the VDR-1,25(OH)2D3 and VDR-MC1288 complexes and 59% for the VDR-KH1060 complex. The A, seco-B, and C/D rings form identical contacts as previously described for the 1,25(OH)2D3-VDR complex. The hydroxyl groups make the same hydrogen bonds, 1-OH with Ser237 and Arg274, 3-OH with Tyr143 and Ser278, and 25-OH with His305 and His397. The deletion of 1-OH or 25-OH leads to the largest changes with a significant decrease in binding (1/1000), while that of the 3-OH has a smaller effect (1/20) [42]. The specific interactions observed in the three ligandeprotein complexes involve the hydrophobic contacts of the 17b-aliphatic chains (Fig. 9.7B). When comparing the natural ligand and MC1288, the main difference is the positioning of the methyl group C21 which results in different contacts with Val300, Leu309, and His397. In MC1288, the C21 moiety is closer to His397. Other proteineligand contacts differ, but to a lesser extent they involve the methyl groups at positions 23 (His305), 24 (His397), and 27 (His397 and Val418). In these two complexes, the carbon C22 makes In the case of no contact at a distance closer than 4.2A. KH1060, the methyl group C21 is quite close to that of the corresponding atom in 1,25(OH)2D3. Note that the oxygen atom at position 22 of KH1060 forms a van der Waals contact with Val300. The major differences observed between KH1060 and the two other ligands are the tighter and more numerous ligandeprotein contacts. The methyl groups C26a and C27a, specific to KH1060, form additional contacts with H3, loop 6-7, H11, and H12. A weak density is observed for the C26a methyl group, suggesting a structural disorder, whereas the C27a methyl group is clearly defined. In the three complexes, the ligands adopt an elongated conformation (Fig. 9.7C) similar to that described in the hVDRD-1,25(OH)2D3 structure. In the 20-epi complexes, the aliphatic chain is less constrained, thus allowing alternative conformations of the 17b-chain. The distances between the 25-OH group and the 1-OH and 3-OH are similar for the three complexes. These represent the anchoring points that must be maintained in order to obtain an active conformation. The aliphatic side chain of 1,25(OH)2D3 adopts an extended conformation parallel to the C13eC18 bond with the C13eC17eC20eC22 torsion angle close to 90 . The two 20-epi analogs use different strategies to fit into the cavity. KH1060 accommodates its longer chain by adopting an eclipsed conformation around the O22eC23 bond whereas MC1288 adopts a gauche conformation and has to compensate for a chain that is too short to maintain the 25-OH interactions because of the C20 inverted stereochemistry. Our preliminary docking experiments of the 20-epi analogs correctly positioned the methyl C21 in the same cavity as 1,25
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FIGURE 9.7 Crystal structures of the hVDRD LBD complexed to 1a,25(OH)2D3, MC1288, and KH1060. (A) Experimental electron density
omit map contoured at 2.0 standard deviation of (a) 1a,25(OH)2D3, (b) MC1288, and (c) KH1060. (B) Close-up view of KH1060 in the ligandbinding pocket. Secondary structure features are represented in blue (a-helices) and green (b-strands). The ligand is colored in yellow with the oxygen atoms in red. The volume of the cavity is represented in gray. (C) Superposition of 1a,25(OH)2D3 (yellow), MC1288 (green), and KH1060 (blue) ligands after superimposed VDR complexes. Oxygen atoms are colored in red. Adapted from Figures 2 and 3 of Tocchini-Valentini G, Rochel N, Wurtz JM, Mitschler A, Moras D 2001. Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands. Proc Natl Acad Sci USA 98:5491e5496 [28]. Please see color plate section.
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(OH)2D3, but failed to give the correct geometry of the aliphatic chains. The crystallographic structures provide the exact information regarding these changes and the details of the interactions with the protein. Compared to the natural ligand, the aliphatic chains of the 20-epi analogs are lining the opposite side of the pocket. The different orientation of the chains is reflected by the different values of the torsion angles C16eC17eC20eC22 and C17eC20eC22(O22 for KH1060)eC23. Further down the chain, while 1,25(OH)2D3 adopts an extended conformation, the MC1288 and KH1060 exhibit gauche and eclipsed conformations respectively to properly orient the 25 hydroxyl group. The energetically unfavorable eclipsed conformation observed in KH1060 (C20eO22eC23eC24 of KH1060 equal to 16 ) is made possible by the strong interactions formed by and C18 (3.24 A). A methylene O22 with C12 (2.9 A) group at this position as in MC1301 would be shifted away from the D-ring. A crude modeling analysis suggests that the methylene group would move away from C12, adopting a gauche (þ) conformation instead of the eclipsed one observed for KH1060. As a result, additional contacts within the protein pockets are formed (Val300) which may contribute to the higher affinity of MC1301; other contributions like solvation cannot be excluded. Similarly, the naturally occurring hydroxylation of C24a in KH1060 and C23 in 1,25(OH)2D3 would form steric clashes with His305 affecting the hydrogen bond network with 25OH. Based on the crystal structures this activity would depend on the capacity of the pocket to accommodate these additional groups. Both conformations and additional interactions of the 20-epi analogs are predicted to afford higher stability and longer half-life of the active complexes. Indeed, within 3 hours, 60% of 1,25(OH)2D3 dissociates from the VDR complex, whereas only 5e20% of MC1288 is dissociated [39]. Furthermore, receptors lacking the C-terminal helix 12 exhibit different dissociation behavior for the 20-epi analogs that are still capable of interacting with a mutated receptor [39,40]. Limited proteolytic digestion studies show that the 20-epi analogsereceptor complexes are more resistant to digestion, suggesting that they are more stable. Additionally, an important role might be played by the rate of assimilation, i.e. how synthetic agonists are metabolized as compared to the natural ligand in different cells types [43,44].
2-Substituted 19-nor Analogs Three 2-substituted 19-norvitamin D3 analogs were crystallized in complexes with the rVDRD LBD and a coactivator peptide [26]. Among them, 2MD which presents a 19nor configuration, a methylene moiety on
C2, and a 20epi configuration on C20 is highly potent in HL60 promyelocyte differentiation [45e47]. The overall conformation of the ligand in the rVDR LBP is extended and forms the same hydrogen bonds as the natural ligand. Like for KH1060 and MC1288 superagonists, no conformational change is observed. The CD ring of 2MD is notably tilted. As a consequence and due to the 20epi configuration, the aliphatic side chain takes another pathway in the LBP with the same anchoring points. Despite the CD tilt, the interactions of the atoms of the rings are similar for 2MD and 1,25 (OH)2D3. Specific contacts involve the C21 of 2MD. The extra carbon on C2 does not perturb the solvent molecules forming a channel. The 2-methylene carbon interacts with neighbor residues, Tyr143, Tyr147, and Phe150.
14-Epi Analogs Two 14-epi-analogs TX527 and TX522 (Fig. 9.8A) show a strongly enhanced antiproliferative action (at least ten-fold) coupled to markedly lower calcemic effects (50e400 times less than 1,25(OH)2D3, respectively) [48]. No differences are observed between those ligands and 1,25(OH)2D3 at the level of binding to VDR nor at the level of the binding between the ligand-bound VDR and its preferred dimerization partner, the retinoid X receptor (RXR) [48]. To elucidate the role of coregulator molecules in the superagonistic action of the analogs, the interaction between the VDR complexed to TX522, TX527, or the natural ligand and different coactivator molecules, including SRC-1, TIF2, and the 205-kDa subunit of the Mediator complex (DRIP205/Med1) was characterized [49]. Cells treated with TX522 or TX527 reveal stronger interactions between VDR and each of the three coactivators than in cells treated with 1,25(OH)2D3. Selective inhibition of the 24-hydroxylase enzyme (CYP24), which is the main enzyme involved in the catabolism of 1,25 (OH)2D3, shows that these differences can only partially be accounted for by a difference in metabolic stability between 1,25(OH)2D3 and the two analogs. The crystal structure of the hVDRD LBD in complex with TX522 reveals modified contacts of C12 and C22 of the ligand with the ligand-binding pocket (LBP) [49] (Fig. 9.8B). As a consequence of the epi-configuration of C14, the The C12 shift induces CD rings are shifted by 0.5 A. a closer contact of this atom to Val300 (H6) in the VDR-TX522. Due to the rigidity of the side chain of TX522, another pathway of the chain is taken in the pocket and makes an additional contact with the CD1 atom of Ile268 (H5). This study suggests that the enhanced potency to induce VDRecoactivator interactions may be the basis for the superagonistic profile of TX522 and TX527.
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Crystal structures of the hVDRD LBD complexed to the 14-epi TX522 ligand. (A) Chemical structures of TX522, TX527, and 1a,25 (OH)2D3. (B) Close-up view of TX522 in the ligand-binding pocket. Superposition of the VDR-1,25-(OH)2D3 (yellow) and VDR-TX522 (blue) are shown. The two residues Val300 and Ile268 making different contacts with TX522 are highlighted complexes. Only residues closer than 4.0 A in red. The ligands 1,25-(OH)2D3 and TX522 are shown in stick representation, with carbon and oxygen atoms in gray and red, respectively. The hydrogen bonds are shown as red dashed line. Adapted from Figure 8 of Eelen G, Verlinden L, Rochel N, Claessens F, De Clercq P, Vandewalle M, Tocchini-Valentini G, Moras D, Bouillon R, Verstuyf A 2005. Superagonistic Action of 14-epi-Analogs of 1,25-Dihydroxyvitamin D Explained by Vitamin D ReceptoreCoactivator Interaction. Mol. Pharm 67:1566e1573 [49]. Please see color plate section.
FIGURE 9.8
STRUCTURES OF VDR COMPLEXED TO 2a-SUBSTITUTED ANALOGS The crystal structure of the VDR in complex with 1,25 (OH)2D3 revealed the presence of several water molecules near the A-ring linking the ligand C-2 position to the protein surface. Several crystal structures of the VDR ligand-binding domain bound to selected C-2a substituted analogs that fill this water channel have been described [26,50,51]. The crystal structures of hVDR in complexes with C-2a substituted analogs bearing methyl, propyl, propoxy, hydroxypropyl, and hydroxypropoxy groups on C-2a position were solved [50]. These specific replacements do not modify the
structure of the protein structure or the conformation of ligands from the aliphatic side chain to the A-ring protein, with the exception of the methyl substituent, all analogs affect the presence and/or the location of the above water molecules. The methyl substituent is small enough not to affect the water molecules network (Fig. 9.9), while providing additional van der Waals contacts that explain the higher binding affinity of this analog [50]. The propyl and propoxy groups replace W1 and shift W2 into a more unfavorable position with a consequent loss or weakening of H-bonds, consistent with the lower binding affinity. While the hydroxypropyl and hydroxypropoxy groups replace both W1 and W2, but their terminal hydroxyl groups maintain
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FIGURE 9.9 Close-up view of the water molecules network near C-2 position of ligands. VDR complexes with 1a,25(OH)2D3 (A), 2a-methyl1a,25-dihydroxyvitamin D3 (B), 2a-propyl-1a,25-dihydroxyvitamin D3 (C), 2a-propoxy-1a,25-dihydroxyvitamin D3 (D), 2a-(3-hydroxypropyl)1a,25-dihydroxyvitamin D3 (E), and 2a-(3-hydroxypropoxy)-1a,25-dihydroxyvitamin D3 (F). The water molecules are shown as red spheres. The missing water molecules in CeF complexes are shown as dotted lines to red spheres. Residue side chains contacting the water molecules through H-bonds are shown. The ligands are shown in stick representation with carbon and oxygen atom in gray and red, respectively. The hydrogen bonds formed by the ligands and those formed by the water molecules are shown as yellow dotted lines. Adapted from Figure 4 of Hourai S, Fujishima T, Kittaka A, Suhara Y, Takayama H, Rochel N, Moras D 2006. Probing a water channel near the A-ring of receptor-bound 1 alpha,25dihydroxyvitamin D3 with selected 2 alpha-substituted analogs. J Med Chem. 49:5199e5205 [50]. Please see color plate section.
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STRUCTURES OF VDR WITH NONSTEROIDAL LIGANDS
essential channel interactions. The affinities of the C-2 substituted analogs for VDR reflect the balance between the loss of water-mediated H-bonds, additional van der Waals contacts, and entropic effect. Analogs (20S and 20R) bearing 2b-hydroxyethoxy group together with other modifications (16ene-22thia-19nor and C26 and C27 methylation) have been crystallized in complex with rVDRD and Drip205 coactivator peptide [26]. The 20S isomer acts as VDR superagonist [51]. The 2bhydroxyethoxy group in the 20S or 20R complex shows further stabilizing interactions via hydrogen bonds between the terminal OH moiety of the 2-substituent and both Arg270 (hArg274) and a water molecule. These interactions may stabilize the active receptor conformation and explain its increased potency.
STRUCTURES OF VDR WITH NONSTEROIDAL LIGANDS Nonsteroidal analogs lack the rigid CD-ring [52]. Among them, the analogs CD578, WU515, and WY1113 in which the C-ring has been deleted, have a significantly more potent prodifferentiating action on human SW480-ADH colon cancer cells, a stronger transactivating potency and a stronger induction of the interaction between the VDR and some coactivators [53]. Another group of analogs is composed of nonsecosteroidal ligands that mimics the natural calcitriol but with no direct structural relationship to calcitriol [54,55]. Few ligands of this type that activate VDR have been reported up to now. Among them, the diphenylmethane LG190178 and its derivatives exhibit tissue-selective activity [54,56]. Some of these nonsecosteroidal ligands present less calcium mobilization and are attractive therapeutics against psoriasis, osteoporosis, and cancer [56e58]. Few crystal structures of VDR complexes with nonsteroidal and nonsecosteroidal analogs are reported.
zVDR LBD Structure with the Nonsteroidal CD578 Analog The zVDR LBD in complex with an LXXLL-motif containing SRC-1 peptide and the nonsteroidal analog CD578 was crystallized and its structure solved [52,59,60]. This analog lacks the six-membered C-ring and is fluorinated on C26 and C27 of the side chain, a modification that renders 1,25(OH)2D3-analogs more resistant to metabolic degradation by the 24-hydroxylase (CYP24) enzyme. The structure of the zVDR complex was refined at a resolution [53]. The zVDR LBD in complex with CD578 of 2.7 A analog adopts the canonical active conformation observed for agonist-bound VDR receptor. The same conformations of the residues which form the LBD pocket are observed. The absence of the C ring in CD578 induces
181
a loss of a contact between Leu258 (hLeu230) and the atom C11 of 1,25(OH)2D3 and decreases interactions with Trp314 (hTrp286). Furthermore, the indole group closer to the ligand of Trp314 (hTrp286) is shifted by 0.6 A to fill the space created by the absence of the C ring (Fig. 9.10). The C9 atom of CD578 makes a new contact Most interestingly, with Leu341 (hLeu313) at 3.3 A. CD578 interacts through its fluorine atoms with Val444 (hVal418) and Phe448 (hPhe422) of H12 and with Leu440 (hLeu414) of the loop H11eH12 at a distance of respectively. In contrast, in the 3.5, 3.6, and 3.3 A, zVDR-1,25(OH)2D3 complex, the ligand made no contacts with the activation helix H12 within a distance cutoff of (the closest distance is 4.1 A for Val444 (hVal418)). 4.0 A The canonical hydrogen bonds formed between the hydroxyl groups of the ligands and the LBD are conserved. As a consequence of the stronger contacts with and the consequent stabilization of H12 in the zVDR-CD578 complex, the coactivator SRC-1 peptide makes additional interactions (80 interactions with VDR in the zVDRCD578 complex compared to 68 in the zVDR-1,25 These struc(OH)2D3 complex at a distance cutoff of 4.0 A). tural data provide explanation for the increased prodifferentiating action of these analogs not only by protecting the analogs from metabolic degradation but also by increasing the stability of the VDR H12 [53].
Nonsecosteroidal YR301-rVDR LBD Complex YR301 is a derivative of the nonsecosteroidal diphenyl ligand LG190178 (Fig. 9.11A) and has been shown to exhibit potent transcriptional activity in vitro [56]. The crystal structure of VDR bound to this analog is the only nonsecosteroidal complex described in the literature [61]. The overall structure of the VDR LBDeYR301 complex is similar to that of the VDR LBDe1,25(OH)2D3 complex. The YR301 ligand is embedded in the LBP in the same position as the natural ligand (Fig. 9.11B). The diethylmethyl group of YR301 occupies a similar space to the C and D rings of 1,25(OH)2D3 and interacts with Trp282 (hTrp305). The interactions between the receptor and the ligand involve both hydrophobic and electrostatic contacts. Only four of the six hydrogen bonds made by the hydroxyl group of the natural ligand are maintained in the YR301 complex. The 20-OH group of YR301 forms hydrogen bonds to His301 (hHis305) and His393 (hHis397) of VDR LBD. While the 2-OH group of YR301 interacts with Ser233 (hSer237) and Arg270 (hArg274). The hydrogen bonds Tyr139 (hTyr143) and Ser274 (hSer278) observed in the structure of VDR with the natural ligand are missing in the YR301 complex. The terminal hydroxyl group of YR301 forms three direct hydrogen bonds to Arg270 (hArg274) and interacts indirectly with Tyr232 and Asp144 via mediated water molecules.
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(A)
CD578
WU515
WY1113
(B)
FIGURE 9.10 VDR structure with the nonsteroidal CD578 analog. (A) Chemical structures of 1a,25(OH)2D3, CD578, Wu515, and WY1113. (B) Stereo view of the conformation of the bound ligands. CD578 (green) and 1a,25(OH)2D3 (pink) are shown in stick representation after superimposed VDR complexes. Adapted from Figure 3 of Eelen G, Valle N, Sato Y, Rochel N, Verlinden L, De Clercq P, Moras D, Bouillon R, Mun˜oz A, Verstuyf A 2008. Superagonistic fluorinated vitamin D3 analogs stabilize helix 12 of the vitamin D receptor. Chem Biol. 15:1029e1034 [53]. Please see color plate section.
STRUCTURES OF VDR WITH ANALOGS THAT INDUCE STRUCTURAL REARRANGEMENTS zVDR LBDeGemini Crystal Structure Among more than 3000 synthetic analogs of 1,25 (OH)2D3 synthesized to increase the potency and specificity of the physiological effects of vitamin D, Gemini (1,25-dihydroxy-21-(3-hydroxy-3methylbutyl)vitamin D3) (Fig. 9.12A) is an interesting molecule with two identical side chains branching at carbon 20. While Gemini binds less efficiently to VDR (38% compared to 1,25 (OH)2D3 [63]), its transactivation potency is similar to that of 1,25(OH)2D3 in ROS cells [62] and tenfold higher in Hela and COS-7 cells [63]. It has been shown that in the presence of an excess of corepressor, the VDReGemini complex shifts from an agonist to an inverse agonist conformation through the recruitment of NCoR, and mediates repression [63,64]. Its stereochemistry and its 25% increase in volume when compared to the natural ligand create a major docking problem using the original LBP as a template. The packing constraints of the crystal form obtained for the hVDRD discriminate complexes with even small conformational changes near the ligand-binding pocket. The Gemini was crystallized in complex with zVDR LBD and a coactivator peptide [35]. and the structure solved at resolution 2.6 A At the backbone level, the overall structure of zVDRGemini is almost identical to that of zVDR-1,25(OH)2D3 over 249 main chain with a rms deviation of 0.37A
atoms. However, the binding of the ligand’s second side chain results in a significant adaptation of the binding pocket that would have been difficult to predict [65]. A new pocket is created by the combined effect of a backbone shift and a side-chain conformational reorientation (Fig. 9.12B). The position of helices H11 and H12 are unchanged and the peptide coactivator makes the same interactions with the protein, underlying the similar agonist character of both structures. This crystal structure reveals for the first time the plasticity of the VDR LBP. The most striking effect that emerges from this study is the “formation” of a new channel that extends the original pocket. This liganddependent pocket emphasizes the adaptability of the LBD and confirms the induced-fit mechanism of ligand binding. Despite large discrepancies in the shape and size of the ligand pocket, the overall structure of VDR and more importantly the position of H12 and of the coactivator peptide remain unaltered consistent with the agonist character of Gemini.
22-Butyl-1,24-Dihydroxyvitamin D3 Derivatives Another class of ligands, the 22-butyl-1a,24-dihydroxyvitamin D3 derivatives have been shown to induce a structure rearrangement of the LBP of VDR [66,67]. The biological activities of eight different derivatives, all having a butyl group as the branched alkyl side chain were characterized [67]. One analog, the 22S-butyl-20epi-25,26,27-trinorvitamin D derivative, is a potent
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STRUCTURES OF VDR WITH ANALOGS THAT INDUCE STRUCTURAL REARRANGEMENTS
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FIGURE 9.11 VDR structure with the nonsecosteroidal analog YR301. (A) Chemical structures of the nonsecosteroidal ligands LG190178 and YR301. (B) Close-up view of YR301 in the ligand-binding pocket of rVDRD LBP. Superposition of the VDR-1a,25-(OH)2D3 (red) and VDR-YR301 (blue) complexes. Adapted from Figure 4 of Kakuda S, Okada K, Eguchi H, Takenouchi K, Hakamata W, Kurihara M, Takimoto-Kamimura M 2008. Structure of the ligand-binding domain of rat VDR in complex with the nonsecosteroidal vitamin D3 analog YR301. Acta Crystallogr Sect F Struct Biol Cryst Commun. 64:970e973 [61]. Please see color plate section.
VDR agonist, whereas the corresponding compound with the natural configuration at C(20) is a potent VDR antagonist [67] (Fig. 9.13A). While analogs with the full vitamin D3 side chain are less potent agonists, and whether they were agonists or antagonists, depends on the 24-configuration. The crystal structures of rVDRD LBD in complexes with coactivator peptide and the potent agonist ligand, 22S-butyl-20-epi-25,26,27-trinorvitamin D or the antagonist one, 22S-butyl-25,26,27-trinorvitamin D, are similar to that of VDR complex with the natural ligand. The 22-butyl group of the agonist
ligand is oriented toward helix 12, and the 24-hydroxyl group is oriented toward helix 6 to form a hydrogen bond with the carbonyl oxygen of the main chain of Val296 (hVal300) on helix 6. Consequently, some adaptations of some side chains of the VDR LBP are observed, notably of residues on helix 6, the loop 6e7, and the loop 11e12 (Fig. 9.13B). Thus the pocket is slightly expanded in the terminal region of H11. In the antagonist bound VDR crystal structure, the 22butyl group of the ligand induces a side chain reorientation of Leu305 (hLeu309). A novel cavity is produced in
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(A)
Gemini (B)
FIGURE 9.12 Adaptability of the vitamin D nuclear receptor to the synthetic ligand Gemini. (A) Chemical structure of Gemini. (B) Superimposition of the Gemini (in orange) and 1a,25(OH)2D3 (in green) zVDR-bound LBP. This view emphasizes the large conformational change of Leu337 (hLeu309) side chain that opens the additional channel and the central role of His333 (hHis305) that anchors the hydroxyl groups of both ligand side chains. Please see color plate section.
the region surrounded by helix 6, the subsequent loop 6e7, the N-terminal of helix 7, and helix 11, similarly to that observed in the zVDR/GEMINI complex. However, unlike GEMINI, this compound showed antagonistic activity.
STRUCTURAL BASIS FOR VITAMIN D RECEPTOR ANTAGONISM Few vitamin D analogs that have antagonistic activity have been reported, they include compounds that contain a bulky alkoxycarbonyl group at the side-chain
terminus, such as ZK159222 [68] (Fig. 9.14A), or that have a methylene lactone [69] structure in the side chain, such as TEI9647. While numerous agonist complex structures are reported, no antagonist conformation of VDR complex is reported. However, some crystal structures and molecular modelling provide some clues for the VDR antagonism [70e74].
Switching of Calcipotriol to a Receptor Antagonist by Further Side Chain Modification The crystal structure of hVDRD LBD bound to calcipotriol, a ligand that is characterized by its side chain
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STRUCTURAL BASIS FOR VITAMIN D RECEPTOR ANTAGONISM
185
(A)
22Sbutyl-25,26,27-trinorvitamin D
22Sbutyl-20epi-25,26,27-trinorvitamin D
(B)
FIGURE 9.13 Adaptability of the vitamin D nuclear receptor to the 22-butyl-1a,24-dihydroxyvitamin D3. (A) Chemical structures of the antagonist 22Sbutyl-25,26,27-trinorvitamin D and the agonist 22Sbutyl-20epi-25,26,27-trinorvitamin D. (B) Superimposition of the ligands in the rVDR/ 1a,25(OH)2D3 (green), the antagonist rVDR/22Sbutyl-25,26,27-trinorvitamin D (cyan) and the agonist rVDR/22Sbutyl-1a,24R-dihydroxyvitamin D3 (pink) complexes. Adapted from Figure 1 of Inaba Y, Nakabayashi M, Itoh T, Yoshimoto N, Ikura T, Ito N, Shimizu M, Yamamoto K 2010. 22S-Butyl1alpha,24R-dihydroxyvitamin D(3): Recovery of vitamin D receptor agonistic activity. J Steroid Biochem Mol Biol [66]. Please see color plate section.
modifications, was solved [70]. The crystal structure shows an identical protein conformation with an adaptation of the ligand to the binding pocket. Despite the of His305 is sufficient shorter side chain, a shift of 0.4 A to maintain the hydrogen bond with the hydroxyl group. A remarkable feature of calcipotriol compared to the natural ligand is the absence of direct contact with H12. Additional interaction observed concerns atoms of the cyclopropyl group and residues of H3
(CA atom of Ala231 in H3). The ZK antagonist molecules share the MC903 skeleton but present extension at the position 25 (Fig. 9.14A). Depending on the length and the structure of this extension these ligands act either as partial agonist (ZK159222, Schering AG) or as full antagonist (ZK168281, Schering AG) [68]. Docking the two ZK compounds in the binding pocket by anchoring the 24-hydroxyl group reveals that the chain, extending from the position 25 of these ligands,
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(A)
MC903
ZK159222
ZK 168281
(B)
FIGURE 9.14 Structural basis for vitamin D receptor antagonism. (A) Chemical structures of the agonist MC903, the partial agonist ZK159222, and the full antagonist ZK168281. (B) Model of VDR-ZK159222 based on the VDR-calcipotriol complex crystal structure (blue). Superposition of calcipotriol in stick representation with carbon and oxygen atoms in gray and red, respectively, and of two conformers of ZK159222. One conformer (in cyan) with the cyclopropyl orientated as in the VDR-calcipotriol crystal structure presents a steric clash with H3. In the other conformer (in pink), the cyclopropyl is rotated and the last four carbon atoms lie between H3 and H12. The resulting steric hindrance is most likely responsible for the displacement of H12 and the resulting antagonist effect. Adapted from Figure 3 of Tocchini-Valentini G, Rochel N, Wurtz JM, Moras D 2004. Crystal structures of the vitamin D nuclear receptor liganded with the vitamin D side chain analogs calcipotriol and seocalcitol, receptor agonists of clinical importance. Insights into a structural basis for the switching of calcipotriol to a receptor antagonist by further side chain modification. J Med Chem. 47:1956e1961 [70]. Please see color plate section.
clashes with helix H3 (Leu230, Val234). In order to avoid this, a rotation around the bond between C24 and C25 of the ligand (from e67 in MC903 to e154 in the ZK compounds) keeps the cyclopropyl ring and the carboxyl moiety of ZK159222 in the ligand binding pocket, close to H3 but further away from Phe422 (Fig. 9.14B). In this orientation, the last four carbon atoms extend towards helices H3 and H12. The aliphatic chain, if too short, can snuggle in small cavities. By extending this carbon chain, steric contacts are observed with Ala231 (H3) and Val418 (H12) suggesting that most likely the activation helix will not be correctly positioned. With ZK159222, the displacement of H12 allows still an interaction with coactivators (20% of 1,25(OH)2D3). In contrast, ZK168281 exhibits a much larger and more rigid chain, due to the shifted carboxyl group and the introduction of a double bond between the cyclopropyl ring and the carboxyl group.
Crystal Structures of VDR with Partial Agonists, the Adamantyl Analogs The crystal structures of the rVDRD LBD in complexes with 19-norvitamin D compounds that contain bulky adamantyl substituent at the side-chain terminus (Fig. 9.15A) (ADTT, ADNY, and ADMI4) and a coactivator peptide derived from DRIP205 were reported by Nakabavaski et al. [74]. These compounds show a series of partial agonistic activities. In all of these complex structures, the VDR LBD adopts the canonical agonist conformation regardless of their biological activity. The bulky adamantyl side chain does not crowd helix 12 but protrudes into the gap formed by helix 11, loop 11e12, helix 3, and loop 6e7, thereby widening the ligand-binding pocket (Fig. 9.15B). The LBP is consequently expanded. These structural changes destabilize the active protein conformation. The coactivator peptide traps the minor active conformation that can be crystallized.
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STRUCTURE-BASED DESIGN OF NOVEL SUPERAGONIST ANALOGS
187
9.15 Crystal of VDR bound to adamantly vitamin D analogs. (A) Chemical structure of 24-AD-24hydroxyl derivative. (B) Close-up of the interactions made by the adamantly group of the 25AD-25-hydroxyl derivative (green) superimposed to 1a,25(OH)2D3. Adapted from Figure 3 of Nakabayashi M, Yamada S, Yoshimoto N, Tanaka T, Igarashi M, Ikura T, Ito N, Makishima M, Tokiwa H, DeLuca HF, Shimizu M 2008. Crystal structures of rat vitamin D receptor bound to adamantyl vitamin D analogs: structural basis for vitamin D receptor antagonism and partial agonism. J Med Chem. 51, 5320e5329. [74]. Please see color plate section. FIGURE
STRUCTURE-BASED DESIGN OF NOVEL SUPERAGONIST ANALOGS In the crystal structures of VDR complexed with several agonist ligands, the ligand is tightly bound to the receptor around the A-, Seco B-, C-, and D-rings. In contrast, the aliphatic side chain is less constrained, thus allowing alternative conformations of the side chain for the 1a,25(OH)2D3 and the 20-epi analogs as described previously. Superimposition of the side chain conformations of the VDReligand complexes KH1060 and 1a,25 (OH)2D3 shows that the two lateral side chains exactly coincide, except at the C17 and C23 positions, where the two directions form a virtual heterocycle. Based on this structural information, novel ligands were designed that incorporate a rigid ring system: an oxolane group, to minimize the entropic loss, and maximize the number of proteineligand contacts (Fig. 9.16A). Due to the chirality of the atom C23, two epimers ((20S,23S)- and (20S,23R)epoxymethano-1 alpha,25-dihydroxyvitamin D3) with opposite stereochemistry at C23 of the oxolane moiety were generated [75]. 2a-Methyl group were also incorporated in these analogs (2 alpha-Methyl-(20S,23S)- and 2 alpha-methyl-(20S,23R)-epoxymethano-1 alpha,25-dihydroxyvitamin D3) to provide additional van der Waals contacts compared to that obtained in the complex hVDR LBD with its natural ligand [76]. The crystal structures of these four complexes reveals that hVDRD LBD adopt the canonical conformation of all previously
reported agonist-bound nuclear receptor LBDs and the position and conformation of the activation helix H12 are strictly maintained [75,76]. The ligands have the same orientation in the pocket. An adaptation of their conformation is observed to maintain the hydrogen bonds forming the anchoring points. The interactions between the protein and the secosteroid rings are identical and the hydroxyl groups make the same hydrogen bonds (Fig. 9.16B and C). Some specific contacts of the aliphatic side chains are made with the VDR LBP with Val300, His305, His397, and Val418. A novel feature of the C23S epimers is the additional van der Waals contact of O21 with Val300, which is also present but weaker in the VDR LBD-C23R analog complexes (Fig. 9.16B). Due to the inverse configuration at C23, the stereoisomers adopt different side chain conformation. and 1.2 A away from their positions C23 and C24 are 0.6 A in the (20S,23S)-epoxymethano-1 alpha,25-dihydroxyvitamin D3 and 2alpha-methyl-(20S,23S)-epoxymethano-1 alpha,25-dihydroxyvitamin D3 complexes thereby affecting their respective contacts. Consequently, the positions of C25, C26, C27, and 25-OH are also different and the direct interactions of C23R compounds with activation helix-12 are then weaker. The modification at the A-ring for 2alpha-methyl-(20S,23S)-epoxymethano-1 alpha,25dihydroxyvitamin D3 and 2alpha-methyl-(20S,23R)-epoxymethano-1 alpha,25-dihydroxyvitamin D3 analogs does not affect the half-boat conformation of the ring for both ligands. The 2a-methyl group fills a small empty
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188
9. STRUCTURAL BASIS FOR LIGAND ACTIVITY IN VDR
Structure-based design of superagonists ligands. (A) Chemical structures of the 1a,25(OH)2D3 and the analogs (20S,23S)- and (20S,23R)-epoxymethano-1a,25-dihydroxyvitamin D3, 2a-methyl-(20S,23S)- and 2a-methyl-(20S,23R)-epoxymethano-1a,25-dihydroxyvitamin D3. (B) Detailed structural representation of the 2a-methyl-(20S,23S)-epoxymethano-1a,25-dihydroxyvitamin D3 (green) superimposed to 1a,25 (OH)2D3 (orange) bound to hVDR LBD around the aliphatic chain (B) and the 2a-methyl group (C) showing the characteristic residues involved in interactions. Hydrogen and van der Waals bonds are shown in red and black dotted lines, respectively. Secondary structure of VDR is shown in cartoon. H2, H3, H6, H7, and H11 indicate helices. Please see color plate section.
FIGURE 9.16
cavity of the pocket and makes additional van der Waals interactions with Phe150, Leu233, and Ser237 (Fig. 9.16C). Transcriptional assays show that (20S,23S)-epoxy methano-1 alpha,25-dihydroxyvitamin D3 is a VDR superagonist, whereas (20S,23R)-epoxymethano-1 alpha, 25-dihydroxyvitamin D3 behaves like the natural ligand [75,76]. This specific property is associated with a more potent ability to reduce HL60 cell proliferation. Methylation at the C2a of (20S,23S)-epoxymethano-1 alpha,25dihydroxyvitamin D3 analog synergistically increases its superagonistic character. In contrast, C2a methylation of the C23R analog does not improve its ability to further induce VDR-directed transcription compared to its parental analog or the natural ligand. The superagonistic activity of the C23S analogs can be explained by a combination of enthalpic effects (additional and tighter intermolecular contacts due to the higher fraction of LBP being occupied) and entropic effects
(energetically favorable preformed conformations), resulting in an overall gain both in binding energy and kinetics. All these factors contribute to a better specificity of the ligand for VDR. Now with all the knowledge on the structures of VDR complexes, more potent superagonists that may be used to combat various types of cancer can be designed.
CONCLUSION The crystal structures of VDR LBD explain most features of ligand binding, superagonism, adaptability of the ligands and of the pocket in some specific cases, and provide clues for the antagonism mechanism. In all experimentally determined structures, the protein conformation, notably H12, is conserved and is in agonist conformation. The available structural information does not
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REFERENCES
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[71] M. Pera¨kyla¨, F. Molna´r, C. Carlberg, A structural basis for the species-specific antagonism of 26,23-lactones on vitamin D signaling, Chem. Biol. 11 (2004) 1147e1156. [72] A. Toell, M.M. Gonzalez, D. Ruf, A. Steinmeyer, S. Ishizuka, C. Carlberg, Different molecular mechanisms of vitamin D (3) receptor antagonists, Mol. Pharmacol. 59 (2001) 1478e 1485. [73] M.T. Mizwicki, C.M. Bula, P. Mahinthichaichan, H.L. Henry, S. Ishizuka, A.W. Norman, On the mechanism underlying (23S)25-dehydro-1alpha(OH)-vitamin D3-26,23-lactone antagonism of hVDRwt gene activation and its switch to a superagonist, J. Biol. Chem. 284 (2009) 36292e36301. [74] M. Nakabayashi, S. Yamada, N. Yoshimoto, T. Tanaka, M. Igarashi, T. Ikura, et al., Crystal structures of rat vitamin D receptor bound to adamantyl vitamin D analogs: structural basis for vitamin D receptor antagonism and partial agonism, J. Med. Chem. 51 (2008) 5320e5329. [75] S. Hourai, L.C. Rodrigues, P. Antony, B. Reina-San-Martin, F. Ciesielski, B.C. Magnier, et al., Structure-based design of a superagonist ligand for the vitamin D nuclear receptor, Chem. Biol. 15 (2008) 383e392. [76] P. Antony, R. Sigu¨eiro, T. Huet, Y. Sato, N. Ramalanjaona, L.C. Rodrigues, et al., Structure-function relationships and crystal structures of the vitamin D receptor bound 2 alphamethyl-(20S,23S)- and 2 alpha-methyl-(20S,23R)-epoxymethano1 alpha,25-dihydroxyvitamin D3, J. Med. Chem. 53 (2010) 1159e1171. [77] P.F. Egea, A. Mitschler, N. Rochel, M. Ruff, P. Chambon, D. Moras, Crystal structure of the human RXRalpha ligandbinding domain bound to its natural ligand: 9-cis retinoic acid, EMBO J. 19 (2000) 2592e2601.
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C H A P T E R
10 Coregulators of VDR-mediated Gene Expression Diane R. Dowd, Paul N. MacDonald Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, USA
INTRODUCTION The main physiological role of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is to promote the intestinal absorption of dietary calcium and phosphate which ensures that the serum concentration of these ions is optimal for normal mineralization of the skeleton. 1,25(OH)2D3 directly affects bone remodeling by causing osteoblasts to terminally differentiate into osteocytes and deposit calcified matrix [1]. 1,25(OH)2D3 also promotes the differentiation of precursor cells into mature osteoclasts which function to resorb bone and maintain appropriate bone remodeling [2]. In addition to its traditional role in maintaining calcium and phosphate homeostasis, the vitamin D endocrine system is also involved in a number of other physiological processes including blood pressure regulation, immune function, mammary gland development, and hair follicle cycling (reviewed in [3,4]). 1,25(OH)2D3 is generated by two sequential hydroxylations of vitamin D3 (cholecalciferol), a secosteroid precursor that is obtained in the diet or produced in the skin upon exposure to UV light (see chapters in Section I of this volume). 1,25(OH)2D3 is transported in the serum bound to the serum vitamin-D-binding protein. Once inside a target cell, 1,25(OH)2D3 is bound selectively by the vitamin D receptor or VDR (see other chapters in Section II of this volume). The binding of 1,25(OH)2D3 to VDR enhances the association of VDR with vitamin D response elements (VDREs) in the promoters of target genes. VDREs are characterized by two direct hexameric repeats with a spacer of three nucleotides (DR-3 elements). Purified recombinant VDR interacts weakly with DR-3 elements. High-affinity binding requires VDR heterodimerization with retinoid X receptor, or RXR (Fig. 10.1). The association of 1,25(OH)2D3
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10010-1
with VDR promotes both the heterodimerization with RXR and high-affinity binding to DR-3 VDREs, with the VDR occupying the 30 half-site and the RXR occupying the 50 half-site [5,6]. In this manner, the interaction of the liganded VDR/RXR with a VDRE confers target gene selectivity and ultimately influences the rate of RNA polymerase II (RNA Pol II)-directed transcription. Research spanning the past two decades reveals that 1,25(OH)2D3-mediated transcription is more complex than the simple binding of the receptor to DNA and the recruitment of RNA Pol II to initiate transcription. A phenomenon termed “squelching” was documented in the early 1990s in which the ligand-binding domain (LBD) of one nuclear receptor (NR) interfered with the transcriptional activation mediated by a second NR [7,8]. The theory was that there are limiting quantities of accessory factors or adapter proteins that interact with the receptor’s ligand-binding domain (LBD) and are necessary for NR-mediated transcription. Thus, the LBD of one NR competes with the other intact liganded receptor for binding to these proteins. The prediction was that a small group of common NR coregulatory proteins exist that interact with NRs to modify NR activity and they are required for ligand-stimulated transcription. Presently, there are more than 250 published coregulators that are known to interact with nuclear receptors and modify their transactivation potential [9]. The complexities of 1,25(OH)2D3- and VDR-mediated transcription and the multifaceted roles of these coregulatory proteins are only now becoming more fully appreciated. The regulation of VDR-mediated transcription involves a series of macromolecular interactions that occur in a temporally coordinated fashion between the
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FIGURE 10.1 1,25(OH)2D3-induced heterodimerization between VDR and RXR. VDR binds to its ligand, 1,25(OH)2D3, heterodimerizes with RXR, and binds to a DR-3 type VDRE in the promoter of 1,25 (OH)2D3-responsive genes. VDRs occupy the proximal or 30 half-site while RXR occupies the distal or 50 half-site.
VDR/RXR heterodimer and other transcription factors (see also Chapters 7 and 8). Association between the liganded VDR/RXR heterodimer and other transcriptional components may be classified into two general categories: general transcription factors and the coregulatory proteins. The interaction of VDR with the first group results in direct contacts with the preinitiation complex (PIC) which may facilitate assembly or recruitment of the PIC and thereby stimulate transcription by RNA Pol II. TFIIB and TATA binding-protein-associated factors (TAFs) are important examples of this class of factors that interact with the VDR to modulate the activity of liganded VDR [10,11]. In addition to direct contacts with the general transcription machinery, the liganded VDR is also linked to the transcriptional PIC by the NR coregulatory factors. NR coregulators are proteins that interact
directly with NRs and modulate, either positively or negatively, the ability of the NR to regulate transcription. NR coregulators are classified either as coactivators or corepressors, and they aid in the induction or repression, respectively, of ligand/receptor-mediated transcription. Coactivator proteins function to augment transcription by one of three proposed mechanisms. First, coactivators may function as macromolecular bridges between the liganded receptor and the general transcriptional machinery. In this capacity, they may recruit components of the PIC, aid in the assembly of the PIC, or promote the stability of the complex. Second, some coactivators possess, or recruit secondary coactivators which possess chromatin modification or remodeling activities. Activities include histone acetyl transferase activity (HAT), protein methyl transferase activity, and ATP-dependent alterations in chromatin structure. A third possible mechanism of coactivator function is to increase the rate of coupling between RNA Pol II-directed transcription and more distal events such as transcription elongation and RNA processing. For example, several coactivator proteins express domains that are consistent with RNA-processing proteins, thus, pointing to a role in both activated transcription and in mRNA maturation. These multifunctional proteins interact with the nuclear receptor, general transcription factors, chromatin remodelers, and the RNA splicing machinery to couple transcription to RNA processing and influence the rate at which a gene product is expressed. Coactivators may also be considered to be primary or secondary. Primary coactivators interact directly with NRs, whereas secondary coactivators interact with primary coactivators to regulate transcription. In contrast, NR corepressors are generally defined as proteins that interact with unliganded receptors to repress basal expression of hormone-responsive genes. They are distinguished from other general transcriptional repressors that interfere with NRs through distinct mechanisms such as by disrupting NR binding of ligand or DNA response element interaction. In this chapter, NR corepressors are discussed as nuclear factors that interact with the unliganded NRs and directly modify histones or recruit modifying enzymes, such as histone deacetylases (HDACs), to maintain chromatin in a tightly packaged state and silence transcription from the promoter in an NR-dependent manner. They also associate with antagonist-bound NRs to inhibit NR-dependent, target gene expression. A simplified, yet integrated two-step model of coactivator/corepressor function (Fig. 10.2) proposes that type II NRs maintain a transcriptionally inactive state at a promoter by recruiting corepressors and their associated HDAC activity. Upon binding ligand, the corepressors either dissociate from the receptor or
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COACTIVATORS OF VDR The binding of 1,25(OH)2D3 to the VDR initiates an orchestrated cascade of protein assembly ultimately leading to transcriptional activation of select target genes. Ligand-induced coactivator recruitment to the VDR/RXR heterodimer is one of the early events in this transactivation process. These initial interactions between VDR and coactivators are the seed for the assembly of intricate multiprotein complexes that remodel the chromatin structure, recruit the core transcriptional machinery, and induce expression of 1,25 (OH)2D3-regulated genes. Thus, understanding the molecular details of 1,25(OH)2D3-induced assembly of VDR and various coactivator complexes is central to understanding the process of 1,25(OH)2D3/VDR-activated transcription.
SRC Family of Coactivators
FIGURE 10.2 Two-step model of coregulator activity on VDRmediated gene expression. The VDR/RXR heterodimer is loosely bound to a VDRE in the absence of ligand. In this state, it may interact with corepressors which function to keep gene transcription repressed, in part, by keeping histone proteins deacetylated. Upon binding ligand, the corepressor is released and replaced by coactivators. The coactivators then function to remodel the chromatin and aid in the recruitment of RNA Pol II and other key components of the preinitiation complex.
are displaced from the complex by the entrance of coactivator molecules. These coactivators then acetylate core histones while recruiting other coactivators and transcription factors, thereby creating a transcriptionally permissive environment at the promoter. It seems likely that other senarios are also possible. At later stages, coregulators may also promote the recruitment or stability of the RNA-processing machinery to enhance the rate at which mature RNAs are made and subsequently translated. The focus of this chapter is on the roles of the coregulatory proteins of vitaminD-dependent transcription in the induction and repression of 1,25(OH)2D3-regulated gene expression. The next two sections describe general aspects and specific examples of VDR coactivators and corepressors known to be significant for VDR-mediated transcription. The final section extends this somewhat oversimplified two-step model to incorporate dynamic, cyclical NRecoregulator interactions in the mechanism of VDR gene regulation.
The first nuclear receptor coactivator to be identified was steroid receptor coactivator-1 (SRC-1) [12]. SRC-1 was initially identified as a progesterone receptorinteracting protein that selectively enhanced hormonedependent transcription. Subsequent studies have shown that SRC-1 is important for the transactivation of nearly all nuclear receptors including VDR [13e15]. SRC-1, also known as NCoA1, constitutes the founding member of the p160 or SRC family, which also includes SRC-2 (also known as GRIP-1, TIF2, and NCoA2 [16,17]) and SRC-3 (also known as p/CIP, RAC3, ACTR, AIB-1, TRAM-1, and NCoA3 [18e22]). (For review, see [23].) SRCs are composed of several domains that are highly conserved among the coactivator family (Fig. 10.3). A basic helixeloopehelix domain and a PAS domain exist in the amino terminus of SRCs. The PAS domain is characteristic of the Per/Arnt/Sim family of transcription factors. This aminoterminal region exhibits intrinsic transcriptional activity when tethered to a heterologous DNA-binding domain and is thought to mediate proteineprotein interactions with other transcription factors, perhaps including other PAS proteins [24]. Three LXXLL-containing NR boxes are located in the mid-region of the SRCs and these mediate the ligand-dependent interaction with NRs [25]. Although these three NR boxes were first presumed to be functionally redundant, subsequent mutational studies showed that the individual LXXLL motifs and surrounding sequences confer some degree of receptor selectivity [26e28]. Indeed, liganded VDR binds SRC NR boxes with micromolar affinity and shows a marked preference for the second and third NR boxes of SRC1, SRC2, and SRC3 compared to the first [29]. In the case of SRC-3, NR box III appears to be most important for interaction with VDR [29,30].
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FIGURE 10.3 Schematic of the conserved SRC coactivator domains. bHLH, basic helixeloopehelix domain; PAS, region characteristic of the
Per/Arnt/Sim family of transcription factors; NR boxes, nuclear receptor interaction boxes containing the LXXLL motifs; CBP interaction, region which interacts with CREB-binding protein; Q-rich, region rich in glutamine residues.
The interaction of SRC coactivators with VDR has been studied extensively. VDR, like other NRs, exhibits a modular structure with three principal domains [31] (see Chapters 7 and 8). The amino terminus of most NRs contains a transactivation function, although for VDR, this region is small (approximately 20 amino acid residues) and its function is poorly understood. Adjacent to this domain is the DNA-binding domain, which contains nine strictly conserved cysteine residues forming two zinc-binding modules that mediate highaffinity and highly selective binding to DNA. The large carboxyl-terminal ligand-binding domain (LBD) of VDR is organized into 12 alpha helices [32]. The LBD not only mediates association of VDR with 1,25(OH)2D3 [31], but also with its heterodimeric partner RXR [33e35] and with coregulatory proteins [13]. Helix 12 of the VDR LBD contains the ligand-dependent activation function-2 (AF2), which is essential for transactivation mediated by the VDR [13,35,36] (see additional chapters in this section including Chapter 9). The molecular mechanisms involved in AF2-dependent transactivation have become increasingly clear based on structural analysis of VDR and other NRs. As 1,25(OH)2D3 nestles into the ligand-binding cavity of VDR, the AF2 helix undergoes a subtle yet important conformational change. Helix 12 folds over the LBD [37] and together with helices 3, 4, and 5, creates a hydrophobic crevice that selectively interacts with the complementary, hydrophobic LXXLL or NR-box domain of the SRC coactivators (Fig. 10.4) [25,38]. The NR box forms an amphipathic a-helix whose orientation in the hydrophobic crevice of VDR is probably stabilized by a “charge clamp” composed of two conserved charged residues, one in the AF2 helix 12 (E420) and the other in helix 3 of the VDR (K246) [39]. Besides the three highly conserved leucines, other residues in the NR box are
also important for high-affinity binding and selectivity of coactivators for individual nuclear receptors [26,40,41]. It is interesting to note that while many coactivators interact with NRs via LXXLL motifs, others do not. Those coactivators that lack LXXLL motifs appear to interact with VDR and other nuclear receptors in an AF2-independent manner. Thus, it is apparent that NRs have the capacity to simultaneously contact multiple coactivators through distinct interaction domains and motifs. The diversity of coregulators and multiple multicomponent complexes could help explain receptor, target gene, and cell-selective responses to ligands such as 1,25(OH)2D3. Once targeted to the appropriate promoter through interactions with NRs, SRCs are thought to remodel the chromatin creating a template that is more accessible to the transcriptional machinery. This remodeling occurs, in part, through the covalent addition of acetyl groups onto the carboxyl-terminal lysine residues of histones. This acetyl modification weakens the electrostatic interaction between the positively charged histone tails and the negatively charged phosphate backbone of the DNA, thus inducing decondensation of the chromatin (reviewed in [42]). Such HAT activity is intrinsic to SRCs, residing in the C-terminal regions of SRC1 and SRC3 [20,43]. SRCs also efficiently recruit other transcriptional proteins to the promoter that possess HAT activity such as CREB-binding protein (CBP)/p300 [44,45] and p300/CBP-associated factor (p/CAF) [46]. The carboxyl-termini of SRCs contain two transactivation domains, AD1 and AD2 [24]. A glutamine-rich region, common to many transcriptional activators, characterizes this large carboxyl-terminus. The AD1 region of SRC-1 mediates interactions with the transcription factors CBP/p300 [26,47] and p/CAF [48]. The recruitment of CBP or p300 by SRCs to the chromatin
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FIGURE 10.4 The vitamin D receptor undergoes a conformational change upon binding hormone. The VDR binds ligand and this induces a conformational change in Helix 12 of the AF2 domain. Helix 12 folds to trap the ligand in the binding pocket. This change also creates a hydrophobic cleft or surface on VDR composed of helices H3, H4, H5, and H12 that are used by the LXXLL motifs in coactivator proteins for docking to the VDR.
is essential for transcriptional activation. In addition, p300/CBP binds directly to nuclear receptors and, together with SRC coactivators, they synergistically stimulate transcription [49,50]. SRC recruitment to the promoter-bound liganded NRs also results in histone methylation, and presumably, a relaxation of chromatin structure [51]. This occurs through the interaction of the SRC carboxy-terminal AD2 domain with the histone methyltransferase proteins, coactivator-associated arginine methyltransferase 1 (CARM1) and protein arginine N-methyltransferase 1 (PRMT1) [51,52]. Thus, CARM1 and PRMT1 function as secondary coactivators via
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SRC recruitment and act synergistically with SRCs through a combination of histone acetylation and methylation leading to chromatin reorganization and enhanced transcription. These interactions involve additional LXXLL motifs. Because the cellular concentration of the SRCs is limiting and they appear to interact with many diverse proteins, post-translational modification provides an efficient means to control their activity [23]. SRCs are post-translationally modified by extracellular stimuli resulting in changes in phosphorylation, ubiquitinylation, acetylation, or methylation. These modifications have roles in determining the stability, activity, and subcellular localization of the SRCs. One prime example is the methylation of SRCs by CARM1 resulting in disassociation of the coactivator complex and degradation of the SRC coactivator itself [53,54]. Thus, post-translational modifications modulate coactivator complex stability, assembly, or disassembly, while different combinations of post-translational modifications may allow for the integration of multiple upstream signals and specificity of a response. Murine knockout models of each of the SRCs have been generated [55e57]. These models show a degree of functional redundancy in the SRC family, especially in the development and maintenance of the female reproductive system. However, the individual SRCs may also have distinct physiological functions perhaps related to nuclear hormone receptor selectivity. For example, deletion of SRC-2 results in aberrant spermatogenesis and testicular defects, whereas deletion of either SRC-1 or SRC-3 has no effect on the male reproductive tract [56]. Mutation of SRC-3 causes decreased estrogen-mediated protection against vascular damage [57]. Knockout of SRC3 in mice retards mammary gland growth [58] and suppresses mammary tumor initiation [59,60]. In comparison, knockout of SRC1 suppresses metastasis without affecting primary tumor formation, and the SRC1-KO mice also exhibit a partial resistance to steroid and thyroid hormones [55,61]. The impact of SRC genetic deletions on VDR/1,25 (OH)2D3-targeted systems and the importance of SRC interaction with VDR in vivo is under investigation. While studies addressing the in vivo significance of SRCs in VDR action in mammals are lacking, a number of cellular studies indicate an important functional role. In vitro evidence suggests that SRC-2 may be a favored coactivator for VDR since SRC-2 is expressed in 1,25 (OH)2D3 target cells such as osteoblasts and preferentially augments 1,25(OH)2D3-induced expression of the osteocalcin gene [62]. Other work demonstrated that SRC-3 is required for VDR-mediated expression of IGF1-binding protein 3, a protein involved in stabilizing circulating IGF1 in the serum [63]. More recently, both SRC-2 and SRC-3 were shown to be required for
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VDR-mediated keratinocyte differentiation as well as epidermis-specific sphingolipid production and barrier formation [64e66]. SRC-3 also mediates VDR activity in more differentiated keratinocyte cultures [65]. These studies highlight a requirement of the SRC family of coactivators in the mechanism of VDR- and 1,25 (OH)2D3-activated transcription as well as elude to a cell-selective role in keratinocyte differentiation.
Mediator-D Complex In addition to SRCs, a large multiprotein complex called DRIP (vitamin D receptor interacting proteins) is a coactivator for VDR and other nuclear receptors [67,68]. DRIP shares many components with other transcriptional coactivator complexes, including thyroid hormone receptor (TR) activating proteins (TRAP), activator recruited cofactor (ARF), and the mammalian Mediator complex (MED) [69e71]. In fact, these four complexes are most probably one and the same. These complexes interact with a wide variety of mammalian transcriptional activators including NRs. Thus, they likely have fundamental roles in activator-induced transcriptional processes well beyond the VDR and other NRs. Due to the considerable similarity between proteins comprising these various complexes, a unified nomenclature was proposed which uses the Mediator complex as the basis for naming the multiple complexes and subunits [72]. Using this nomenclature, DRIP is referred to as Mediator-D. Mediator-D is proposed to interact with the liganded VDR bound to its enhancer element and to recruit RNA Pol II, thereby acting as a bridge between VDR and the general transcriptional machinery to promote the formation and function of the preinitiation complex (PIC) [72,73]. Although mediator complexes may recruit RNA Pol II to the promoter, the polymerase is not tightly bound, thereby allowing for its release and the efficient initiation of transcription. Moreover, yeast mediator does not interact with the hyperphosphorylated form of RNA Pol II involved in transcriptional elongation [74]. These data suggest that mediator complexes are associated with RNA Pol II only during preinitiation and the transition to elongation. In vitro transcription studies indicate that following the release of RNA Pol II, mediator remains bound to the promoter to function in the reinitiation of a second round of transcription [75]. While the Mediator-D complex efficiently stimulates VDR-mediated transcription in vitro on chromatinized templates, Mediator-D, and other mammalian mediator complexes lack detectable HAT activity [68]. This raises the question of when and how the recruitment of chromatin remodeling complexes and mediator complexes takes place. Chromatin immunoprecipitation studies often reveal that the entrance of Mediator follows the
entrance of the NRs, HATs, and the acetylation of histones surrounding the transcriptional start site. This supports an ordered, sequential recruitment of coactivators into the promoter of target genes, a model that is described in more detail below. The Mediator-D complex is composed of at least ten different proteins (and probably up to 25e30 subunits) anchored by MED1/DRIP205/TRAP220, which interacts directly with ligand-activated VDR/RXR heterodimers through the second of two LXXLL motifs (Fig. 10.5) [76]. While MED1/DRIP205 promotes interaction with the VDR, another component of Mediator-D, MED130, may stimulate assembly of the PIC on the promoter [77]. The interaction between VDR and MED1 is enhanced by phosphorylation of VDR, and this correlates with an increase in 1,25(OH)2D3-mediated transcription [78]. Additional support for the significance of this interaction comes from studies using 20epi analogs of 1,25(OH)2D3 which showed a link between the increased transcriptional activity of these higher-potency 20-epi compounds and increased binding of VDR to MED1 [79]. This enhanced binding and transcriptional activity is also reflected in an increase in cellular differentiation and a decrease in proliferation in response to these analogs. No differences were observed between 1,25(OH)2D3 and the 20epi analogs when comparing the ability of these ligands to promote binding of VDR to SRC-2, thus highlighting the importance of mediator complexes in agonistinduced VDR activity. More recently, silencing of MED1 in keratinocytes resulted in profound differences in keratinocyte morphology and proliferation rate as well as a loss in 1,25(OH)2D3-stimulated gene expression [80]. Genetic ablation of MED1 in mice causes early embryonic lethality [81]. Heterozygous mutants display growth retardation, impaired transcription, and
FIGURE 10.5 Model of the Mediator-D complex interacting with
the liganded VDR. Med-220 uses an LXXLL NR box motif for docking to the VDR.
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hypothyroidism, underscoring the primary importance of this mediator complex in TR-regulated processes and other NR-mediated transcriptional events. Studies in mouse embryo fibroblasts derived from MED1KO animals showed that MED1 ablation led to attenuated NR-mediated transcriptional responses and that MED1 is essential for peroxisome proliferator-associated receptor g (PPARg)-mediated adipocyte differentiation [81e83]. While the extension to the vitamin D endocrine system and VDR-mediated transcription in vivo is a logical one, such studies have not been addressed and it remains an open question whether MED1 is required for the in vivo actions of 1,25(OH)2D3 and VDR. Cell programs, such as proliferation and differentiation of keratinocytes, involve the sequential regulation of gene expression, and the VDR interacts with both the Mediator D and the SRC/p160 families of coactivators to control these processes. These two coactivator complexes have temporally specific, noninterchangeable roles in the regulation of keratinocyte proliferation and differentiation [29,84,85]. In the proliferating keratinocyte, the Mediator D complex is the main complex interacting with VDR [65]. It was later shown that the MED1/DRIP205 subunit controls proliferation by regulating b-catenin pathways [29]. The maturation and differentiation of the keratinocyte requires both Mediator D and SRC3. Once the keratinoctye is fully differentiated, SRC2 and SRC3 play a major role in binding to VDR and in regulating the induction of more differentiated functions such as lipid synthesis, barrier formation, and activation of the immune response that is triggered by disruption of the barrier. Thus, by selectively interacting with various coactivators in a temporally controlled fashion, VDR/1,25(OH)2D3 can regulate a large number of genes in a sequential and differentiation-specific fashion.
NCoA62/SKIP NCoA62 is a VDR and NR coactivator that was isolated as a VDR-interacting protein by a yeast two-hybrid screen [86]. Expression of NCoA62 in mammalian cells enhances vitamin-D-, retinoic acid-, estrogen-, and glucocorticoid-activated transcription. NCoA62 is structurally and mechanistically distinct from SRCs and Mediator-D. However, it is highly related to Bx42, a Drosophila melanogaster nuclear protein putatively involved in ecdysone-stimulated transcription [87]. NCoA62 was independently identified as a factor that interacts with the Ski oncoprotein and was termed Skiinteracting protein, or SKIP [88]. It is referred to as NCoA62/SKIP throughout this chapter. NCoA62/SKIP interacts with a diverse array of transcription regulatory factors thus highlighting a potential role for NCoA62/ SKIP in general aspects of target gene regulation by different classes of transcription factors [89e94].
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Unlike the SRCs and Mediator-D, the interaction between NCoA62/SKIP and VDR is independent of the AF2 domain of VDR [86]. Moreover, ligand is not required for the interaction in vitro, although ligand does enhance the interaction. NCoA62/SKIP does not contain obvious LXXLL motifs and it exhibits a marked preference for binding to the VDR-RXR heterodimer relative to VDR alone [95]. Because NCoA62/SKIP and SRCs contact different domains within the VDR-RXR heterodimers, both coactivators can simultaneously interact with liganded VDR to form a ternary complex in vitro, which suggests that liganded VDR can act as a bridge to recruit both SRCs and NCoA62/SKIP to the promoter complex (Fig. 10.6) [95,96]. Coexpression of NCoA62/SKIP and SRCs leads to a synergistic induction of VDR-mediated transcription, and protein interference studies indicate that both coactivators are required for optimal VDR-mediated transcription [95]. Evidence also exists for NCoA62 involvement in cellselective activation and repression of VDR transactivation [97]. Chromatin immunoprecipitation (ChIP) approaches show that NCoA62/SKIP is physically recruited in a 1,25(OH)2D3-dependent manner to the 1,25(OH)2D3responsive regions of native VDR target genes in osteoblasts including the osteocalcin and 24-hydroxylase promoters [98]. Interestingly, NCoA62/SKIP is associated with the promoter region after the entry of both VDR and SRC, suggesting it may function at more distal steps of the transactivation process compared to the SRCs. As mentioned previously, the SRCs exhibit histone acetyltransferase (HAT) activity and recruit other proteins such as CBP/P300 that possess HAT activity [43,99]. No significant HAT activity has been detected for NCoA62/SKIP. Thus, one possible interpretation is that NCoA62/SKIP enters the promoter region after the chromatin-remodeling step. NCoA62/SKIP interacts with basal transcription factors such as TFIIB [96], suggesting that NCoA62/SKIP may also be involved in connecting VDR to the general transcription machinery. Regardless, NCoA62/SKIP and SRCs apparently function through different mechanisms to enhance
FIGURE 10.6 Hypothetical model for VDR-SRC-NCoA62/SKIP complexes in VDR-activated transcription.
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VDR-mediated transcription and the function of both distinct classes of coactivators is needed for appropriate vitamin-D-activated target gene expression.
Nuclear Receptor Coactivators: Potential Links to RNA Processing In addition to chromatin remodeling and recruiting the transcription machinery, NR coactivators are implicated in more distal steps of gene expression including transcription elongation and RNA processing. In this regard, NR coactivators are proposed to be important coupling factors linking transcription and RNA processing [100]. The coupling concept implies that RNA processing events (e.g., RNA capping, polyadenylation, and splicing) are physically connected to RNA Pol II and that RNA processing begins on the nascent RNA emerging from the polymerase complex (reviewed in [101,102]). Putative candidates that may aid in this coupling process are predicted to associate with and alter the functional properties of proteins involved in both transcription and RNA processing. In fact, many NR coactivator proteins contain domains or regions that are associated with RNA-processing proteins making some NR coactivators strong candidates as transcriptionsplicing couplers [9]. For example, progesterone receptor (PR)-activated transcription is known to influence splicing decisions of alternatively spliced transcripts in a PR and progesterone response element-dependent manner [100]. NR coactivators such as CoAA and PGC1 have been shown to alter RNA-processing events in NR-mediated transcription [100,103]. Thus, activated steroid receptors bind to target gene promoters may recruit factors that are involved in both transcription regulation and the RNA splicing machinery. This is thought to ensure efficient coordination between RNA synthesis and the final mature mRNA. NCoA62/SKIP may also play a central role in coupling transcription to mRNA splicing. Several observations support this hypothesis. First, endogenous NCoA62/SKIP is recruited to native VDR-responsive promoters in a 1,25-(OH)2D3-dependent manner in osteoblast-like target cells proving its physical association with the transcriptional regulatory machinery at the responsive promoter regions [98]. Second, studies characterizing protein components of the spliceosome and interchromatin granules, where many splicing factors are present, identified NCoA62/SKIP as a component that is associated with the splicing machinery in general and at discrete steps in the splicing mechanism [98,104e106]. Finally, a dominant-negative NCoA62/ SKIP mutant effectively interferes with appropriate splicing of transcripts derived from a VDR-activated mini-gene cassette [98]. These studies indicate that disrupting the function of endogenous NCoA62/SKIP
inhibits 1,25(OH)2D3-activated transcription, in part, by interfering with appropriate splicing of the mRNA transcripts. This supports the hypothesis that NCoA62/SKIP may couple VDR-mediated transcription to RNA splicing and ensure appropriate, efficient processing of vitamin-D-regulated mRNA transcripts.
Other Coactivators While the number of putative nuclear receptor coactivator proteins continues to climb into the hundreds, relatively few of these have been documented to interact with VDR and alter its transcriptional activity. Presently, SRCs, DRIPs, and NCoA62/SKIP represent the most studied of the nuclear receptor coactivators in 1,25 (OH)2D3/VDR-activated gene expression. Several other factors that have been shown to interact with VDR and augment its activity are discussed below. VDR contacts Smad3, a transcription factor activated by TGF-b signaling [107]. Smad3 interacts with VDR in a ligand-dependent manner and stimulates VDR-mediated transcription. Additionally, SRC-1 enhances the binding of Smad3 to VDR, suggesting cooperation between these two coactivators. Given its function in two diverse signaling networks, Smad3 may serve as a bridge between 1,25(OH)2D3 and TGF-b signal transduction pathways. An additional binding partner for VDR is the helixeloopehelix transcription factor, Ets-1 [108]. This interaction occurs between the DNA-binding domains of both proteins. Unlike other VDR coactivators, Ets-1 can stimulate VDR-mediated transcription in the absence of ligand on select promoters, such as the prolactin promoter. Ets-1 is also unique because activation of VDR by Ets-1 is independent of the AF2 domain. The authors suggest that Ets-1 induces a conformational change in the receptor that allows for interaction with other coactivators and subsequent transcriptional activation. A novel multifunctional ATP-dependent chromatin remodeling complex called WINAC was shown to interact directly with the vitamin D receptor [109]. WINAC, a member of the SWI/SNF subfamily, also contains subunits associated with DNA replication, transcript elongation, and the Williams syndrome transcription factor (WSTF). The WSTF gene is deleted in patients with Williams syndrome, a complex neurodevelopmental disorder characterized by congenital vascular and heart disease, dysmorphic facies, mental retardation, growth retardation, infantile abnormal vitamin D metabolism, and hypercalcemia [110], the latter two characteristics supporting a role for WINAC in vitamin D biology. The WINAC complex was shown to promote both the assembly and disruption of nucleosome arrays in an ATP-dependent manner, and in addition, it can also reconstitute chromatin upon newly replicated DNA.
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COREPRESSORS
Transient expression assays revealed that WINAC potentiates the ligand-induced function of VDR in gene activation and repression through its ATP-dependent chromatin remodeling activity [111,112]. In this regard, WINAC is required for the ligand-bound VDR-mediated transrepression of the 25(OH)D3 1a-hydroxylase (1a (OH)ase) gene. In that system, WINAC associates with chromatin through a physical interaction between the WSTF bromodomain and acetylated histones, and that interaction occurs prior to VDR association with the 1a (OH)ase promoter. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) is a transcriptional coactivator that functions in regulating mitochondrial biogenesis and energy metabolism and is known to interact with numerous nuclear receptors [113]. Savkur et al. [114] demonstrated that VDR interacts with PGC1 alpha in a 1,25(OH)2D3- and AF2-dependent manner and that this interaction augments ligand-dependent VDR transcription. These data, combined with the muscle and adiposity phenotypes of the VDRKO mouse [115,116], suggest potential roles of PGC-1 alpha in VDRdependent regulation of genes involved in muscle development and/or energy metabolism. Meningioma-1 (MN1) is a gene that was originally identified as a gene that is disrupted by a balanced translocation in meningioma [117]. Subsequently, it was shown to be expressed in osteoblasts and regulated by 1,25(OH)2D3. In addition, MN1 interacts with VDR and enhances VDR- and 1,25(OH)2D3-activated transcription [118]. Thus, MN-1 serves as a coactivator for VDR and its gene is also a target for VDR-activated transcription, implying a feed-forward mechanism in target gene regulation by VDR [118]. MN1 knockout mice exhibit a defect in intramembranous ossification [119] and a defect in the proper differentiation of calvarial osteoblasts [120]. Thus, MN1 may fulfill an important role in 1,25 (OH)2D3-/VDR-dependent gene expression ensuring proper differentiation and formation of the osteoblast. Global protein interaction approaches were recently used to identify osteoblast-specific proteins that interact in a ligand-dependent manner with the VDR [121]. CCAAT displacement protein (CDP) was identified as a putative osteoblast-selective coactivator of VDR. Interestingly, CDP expression dramatically affected 1,25 (OH)2D3-dependent osteoblast differentiation, suggesting that this putative VDR coactivator may function to control VDR-mediated differentiation of preosteoblasts in this system.
COREPRESSORS The role of the corepressor in regulating gene expression is generally the converse of that of the coactivator
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(reviewed in [122]). In general terms, corepressors interact with DNA-bound transcription factors and play essential roles in silencing or repressing transcription. In the case of NR-regulated gene expression, this often involves a ligand-dependent switch of corepressor interaction with the NR. Corepressors and their associated proteins interact with unliganded NRs and are displaced when agonistic ligands bind. Corepressors generally function to lower basal promoter activity in the presence of unliganded receptor or antagonistbound receptor. In addition to contacting unliganded NRs, corepressors are tightly associated with enzymes that modify histone tails. In this case, the modifications leading to gene repression include histone deacetylation and select examples of methylation and ubiquitinylation. Histone deacetylation, catalyzed by histone deacetylases (HDACs), is one of the most significant processes that mediates transcriptional repression, and indeed, hypoacetylated histone tails are associated with transcriptionally repressed genes. Removal of the acetyl groups induces chromatin compaction that interferes with gene transcription.
SMRT and NCoR Two of the best-characterized NR corepressors are the ubiquitously expressed proteins SMRT [123] and nuclear receptor corepressor (NCoR) [124]. SMRT and NCoR are highly homologous proteins that are present in multisubunit protein complexes that have similar compositions. HDAC3 is the predominant HDAC present in SMRT and NCoR complexes. Transducin blike 1 (TBL1) and G-protein-pathway suppressor 2 (GPS2) are also present in addition to various other factors. SMRT and NCoR corepressors repress basal promoters in systems regulated by many nuclear receptors including VDR, TR, and the retinoic acid receptor (RAR). SMRT and NCoR corepressor interaction is mediated through multiple, short interaction domains known as corepressor NR (CoRNR) boxes [125,126]. CoRNR boxes are generally composed of the sequence L-X-XH/I-I-X-X-X-I/L, where X is any amino acid. Mutation of these sequences abolishes interaction of the corepressors with unliganded NR [125]. Mutational analysis defined residues responsible for proteineprotein interaction between SMRT and VDR and these studies revealed that SMRT mutants defective in interaction with VDR were also unable to repress endogenous VDR-target genes [127]. Although VDR is in the same NR family as TR and RAR, the silencing effects of SMRT and NCoR on VDR-regulated templates are weaker than in the other NR systems [128]. Indeed, overexpression of VDR/RXR does not significantly repress the basal activity of VDR-responsive reporter plasmids nor decrease transcript levels of endogenous target
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genes such as CYP24a1 [129]. This is most probably due to a reduced binding of SMRT and NCoR to VDR relative to that of TR and RAR, although a more likely possibility it that the VDR does not interact well with most target VDREs in the absence of 1,25(OH)2D3. SMRT/NCoR corepressors interact with VDR and other NRs through the same hydrophobic cleft on the LBD as SRC coactivators, making binding of corepressors and coactivators mutually exclusive. However, in contrast to SRC coactivators, SMRT/NCoR interaction with the VDR LBD is surprisingly independent of the AF2 domain, an integral part of the common interaction cleft of the LBD [126,130]. In fact, deletion of the AF2 helix enhances corepressor binding [123]. Structural studies of antagonist-bound LBDs suggest that in the liganded state or antagonist-bound state, the AF2 helix does not adopt a stable, “active” conformation and the cleft formed by H3e5, H12 is incomplete [131]. This favors interaction of the larger CoRNR boxes of the corepressors with the H3e5 surface, an interaction that is somewhat independent of the H12 AF2 domain. Ligand-induced folding of the H12 AF2 domain reduces the area of the hydrophobic cleft so that the shorter LXXLL NR boxes of the SRC coactivators fit, but the corepressors CoRNR boxes are excluded. Thus, 1,25 (OH)2D3 binding to the VDR results in a conformational change in the AF2 domain that interferes with SMRT/ NCoR binding to VDR, causing release of the corepressor complexes. In contrast, nuclear receptor antagonists alter positioning of the AF2 helix (compared to that of agonist-bound receptors) such that corepressor interactions are favored over coactivator binding. Indeed, in the presence of VDR antagonists such as ZK168281 (a 25-carboxylic ester 1,25(OH)2D3 analog), the interaction between VDR and NCoR is dramatically enhanced [132]. Thus, rather subtle alterations in the positioning of the AF2 helix in response to various 1,25 (OH)2D3 analogs dramatically influence the overall activity of the VDR and provide structural insight into the dynamics of VDR-controlled gene expression. NCoR and SMRT are large modular proteins that serve as platforms for the assembly of multiprotein repression complexes containing histone deacetylases or HDACs. HDACs function to deacetylate core histone proteins in their vicinity to result in localized region of compact or tightly wound nucleosomal packaging ultimately leading to repressed basal expression of the promoter. Thus, unliganded or antagonist-bound VDR recruits NCoR/SMRT corepressor complexes containing HDACs leading to a compaction of chromatin near the transcriptional promoter. Gene activation involves the 1,25(OH)2D3induced release of NCoR/SMRT-mediated repression and subsequent ubiquitinylation and degradation of corepressors presumably mediated through the transducer beta-like 1, X-linked (TBL1X) exchange factor [133,134].
Hairless The VDR plays a role in mammalian hair follicle cycling and disruption of the VDR gene leads to total alopecia in humans and mice (for review see [4]) (see Chapters 8 and 30). Inactivation of RXRa in keratinocytes [135] and global knockout of the hairless gene (hr) [136] result in hair, follicle, and skin phenotypes similar to VDRKO, thus implicating Hr in VDR/RXR pathways in the skin and hair follicles [137]. Indeed, the Hr protein was shown to interact with VDR and to repress transcription mediated by VDR [138,139], thus providing a molecular basis for its role in the maintenance of hair growth [139e141]. Although Hr lacks homology with SMRT and NCoR, it functions in a similar manner to repress VDR-mediated transcription. Like these other corepressors, Hr binds to VDR in a region localized to the central portion of the ligand-binding domain and independent of AF2 (139), the docking site for some coactivators. This interaction requires two F-X-X-F-F hydrophobic motifs in Hr, Hr1, and Hr2 [139], with Hr1 mediating ligand-dependent repression of VDR [142]. While Hr does not exhibit histone deacetylase activity, it is present in complexes together with SMRT and multiple HDACs [140,143e145]. Whereas SMRT and NCoR are ubiquitously expressed, the expression of Hr is restricted primarily to brain and skin, thereby indicating a more specialized role compared to the other corepressors (reviewed in [146]).
Alien Dressel et al. described a novel corepressor, Alien, which is unrelated to the SMRT/NCoR family of corepressors [147]. In these early studies, Alien interacted with TR in the absence of ligand and repressed TR-mediated basal transcription. Alien does not interact with the glucocorticoid receptor, RXR, or RAR. In this system, the mechanism of Alien-mediated silencing appears to be through the recruitment of Sin3A and its associated histone deacetylase activity. Alien did not interact with SMRT or NCoR and thus represents an independent pathway to Sin3 recruitment. Polly et al. [148] extended these findings to demonstrate that Alien also interacts with VDR and represses basal transcription from a DR-3 VDRE, but not an atypical IP9-type VDRE suggesting a level of specificity for repression. The interaction of Alien with unliganded VDR is independent of the AF2 domain of VDR, and appears to use a different interaction surface than does NCoR. In contrast to the studies by Dressel, a deactylase inhibitor had only a partial effect on the ability of Alien to repress transcription. Later studies demonstrated that Alien increases the efficiency of nucleosome assembly thereby sustaining a compact nucleosomal structure and
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CONCLUSION e INTEGRATED MODEL OF COREGULATOR ACTIVITY
inhibiting gene expression [149]. Thus, Alien appears to use at least two molecular mechanisms for transcriptional corepression e recruiting histone-modifying activities and enhancing nucleosome activities.
CONCLUSION e INTEGRATED MODEL OF COREGULATOR ACTIVITY Gene expression is a complex process involving the coordinated repression and activation of transcription. According to a simplified two-step model of nuclear hormone signaling, unliganded VDR, like other NRs, are DNA-bound and complexed with corepressor molecules that keep chromatin condensed and the promoter inaccessible to the transcription machinery. Upon binding 1,25(OH)2D3, the corepressors are displaced by coactivators to begin the transcriptional activation process. Immunodepletion, dominant negative, knockdown, and knockout approaches indicate that SRCs, Mediator-D, and NCoA62/SKIP have different functions and all are required for robust VDR-mediated transcription. Promoter-specific and more global genomic chromatin immunoprecipitation (ChIP) strategies have provided a more detailed view into the temporal and spatial details of diverse NR coregulator assembly onto 1,25(OH)2D3-responsive promoters [150]. For example, early ChIP assays revealed that both nuclear receptors and their coactivators cycle on and off the promoter [98,151,152]. Upon ligand addition, VDR, or other nuclear receptors, enters the transcriptional complex first, followed by SRCs (Fig. 10.7) [98,152]. SRCs likely loosen the chromatin structure by bringing both intrinsic HAT activity and recruiting extrinsic HAT activity to the complex, through their interaction with p300/CBP. SRCs then dissociate, allowing for binding of the Mediator-D multimeric complex [152,153]. Mediator-D is thought to recruit the PIC and RNA Pol II holoenzyme to initiate transcription of target genes [73]. NCoA62/SKIP also enters the VDR complex following SRC-1 [98]. It is possible that SRCs help to recruit NCoA62/SKIP to the complex as suggested by the data that these two proteins can form a stable ternary complex with VDR [95]. Once NCoA62/SKIP is bound, it may target the spliceosome complex to the actively transcribed gene [98], allowing for efficient splicing of the nascent transcript. Taken together, these ChIP studies suggest a temporal model of coactivator action in which all three major classes of coactivators enter the complex at distinct times and provide three different functions: chromatin remodeling, recruitment of the core transcriptional machinery, and coupled transcriptional activation and RNA splicing. More recent work indicates yet another level of complexity to this evolving model of coregulator
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involvement in VDR-mediated transcription. These studies suggest that the switch between gene repression and gene activation cannot be simply defined by the alternative recruitment of two different regulatory complexes. Indeed, a cyclical recruitment of corepressors, HDACs, and nucleosomal-remodeling complexes is also observed on NR- and VDR-activated promoters
Model of the sequential occupation of the promoter site with coactivators, the PIC, and the spliceosome. First, ligandactivated VDR/RXR binds to VDREs in target genes and recruits coactivators such as SRCs and p300/CBP. The HAT activity of these coactivators loosens the chromatin structure, allowing for a more transcriptionally permissive environment. Second, SRCs and p300/ CBP dissociate, allowing for binding of the Mediator-D multimeric complex. This complex is thought to recruit RNA Pol II and the core transcriptional machinery to initiate active transcription of the target gene. Finally, NCoA62/SKIP binds to the VDR/RXR complex and tethers the splicing machinery to the activated promoter, allowing for immediate splicing of the nascent transcript.
FIGURE 10.7
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10. COREGULATORS OF VDR-MEDIATED GENE EXPRESSION
in addition to coactivators and in response to ligand addition [129,154,155]. Thus, a more realistic view is the continuous cycling or exchange of coactivator and corepressors complexes (on the order of approximately 60 minutes) is required for 1,25(OH)2D3-activated transcription (reviewed in [150]). The transcriptional cycling phenomenon is thought to exist in order to allow for better control of the overall process of gene activation. In addition, the switch from active promoter (coactivators) to repressed promoter (corepressors) can be achieved by a series of sequential events that are mediated by multiple enzymatic activities that can be factor, promoter, and cell-type specific (for review, see [156,157]). For example, the basic model has unliganded receptors preferentially interacting with corepressors, however some VDR corepressors, such as Alien and Hr, can interact with receptors in the presence of ligand and may compete with coactivators by displacing them. The switch between corepressor- and coactivator-associated nuclear receptor may also require an exchange complex in addition to ligand binding [158]. The model begins to get even more complex when one begins to consider the role of post-translational modifications in the regulation of receptors and their coregulators. In fact, the function and cycling of coregulators can be altered by modifications as diverse as phosphorylation, acetylation, methylation, sumoylation, and ubiquitinylation. Each modification can be expected to have a distinct functional outcome including cellular localization, enzymatic activity, protein stability, and altered affinity for the transcriptional complex. Importantly, one must be cautious not to oversimplify what is obviously an extremely complex process that requires a variety of macromolecular machines and coordination of many complex pathways.
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hormone-responsive promoters in vivo, Proc. Natl. Acad. Sci. USA 99 (2002) 7934e7939. [154] R. Metivier, G. Penot, M.R. Hubner, G. Reid, H. Brand, M. Kos, et al., Estrogen receptor-alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter, Cell 115 (2003) 751e763. [155] M. Malinen, A. Saramaki, A. Ropponen, T. Degenhardt, S. Vaisanen, C. Carlberg, Distinct HDACs regulate the transcriptional response of human cyclin-dependent kinase inhibitor genes to Trichostatin A and 1alpha,25-dihydroxyvitamin D3, Nucleic Acids Res. 36 (2008) 121e132.
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[156] D.M. Lonard, B.W. O’Malley, Nuclear receptor coregulators: judges, juries, and executioners of cellular regulation, Mol. Cell 27 (2007) 691e700. [157] V. Perissi, M.G. Rosenfeld, Controlling nuclear receptors: the circular logic of cofactor cycles, Nat. Rev. Mol. Cell Biol. 6 (2005) 542e554. [158] V. Perissi, A. Aggarwal, C.K. Glass, D.W. Rose, M.G. Rosenfeld, A corepressor/coactivator exchange complex required for transcriptional activation by nuclear receptors and other regulated transcription factors, Cell 116 (2004) 511e526.
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C H A P T E R
11 Target Genes of Vitamin D: Spatio-temporal Interaction of Chromatin, VDR, and Response Elements Carsten Carlberg Life Sciences Research Unit, University of Luxembourg, L-1511 Luxembourg, Luxembourg; Department of Biosciences, University of Eastern Finland, FIN-70211 Kuopio, Finland
INTRODUCTION Transcriptional Regulation in the Chromatin Context Transcriptional regulation is a central process for nearly all physiological actions in eukaryotes. Interestingly, gene transcription is in most cases repressed, since eukaryotes have in their nucleus a complex of genomic DNA and nucleosomes, referred to as chromatin, that occludes binding sites of DNA-binding proteins [1]. This is essential for fundamental decisions in development, such as terminal differentiation of cells, which are mediated by long-lasting programming of chromatin [2]. However, the epigenetic landscape can also be highly dynamic and lead to short-lived states, such as a response of chromatin to stress and other endocrine signals [3]. Epigenetic changes originate, in part, from reversible post-translational modification of histone proteins, such as acetylation and methylation, that are directed by histone acetyltransferases (HATs), histone deacetylases (HDACs), and histone methyltransferases (HMTs) associating with DNA-binding transcription factors [4]. The “histone code” consists of specific sets of histone modifications, which are associated with genes that are actively transcribed or with those that are repressed [5], i.e. histone post-translational modifications correlate with either positive or negative transcriptional states. In turn, negative-acting marks on histone tails and methylation of genomic DNA at CpG islands are laid down across genes during repression by DNA-bound
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10011-3
repressor recruitment or across heterochromatic regions of the genome. Plasticity of chromatin is induced by ATP-dependent remodeling complexes, which rearrange the organization of the nucleosomes [6]. Rapid changes in chromatin activation make these complexes permissive for interference with transcriptional regulation. Most of the dynamic nature of transcriptional regulation has been observed using nuclear receptors and their target genes as model systems [7e9]. This chapter focuses on the nuclear receptor VDR and its target genes, but the principles discussed here may apply to many other nuclear receptors and to eukaryotic transcription factors in general.
Nuclear Receptors Nuclear receptors are the best-characterized representatives of the approximately 3000 different mammalian proteins that are involved in transcriptional regulation in human tissues [10]. The receptors form a superfamily with 48 human members, most of which have the special property of being activated by small lipophilic ligands the size of cholesterol [11]. Nuclear receptors modulate genes that affect processes as diverse as reproduction, development, inflammation, and general metabolism. The subgroup of endocrine nuclear receptors bind their specific ligands, which are the steroid hormones estradiol, progesterone, testosterone, cortisol, and aldosterol, thyroid hormone or the biologically active forms of the fat-soluble vitamins A and D, all-trans retinoic acid
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and 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3) with a Kd of 1 nM or less [12]. In contrast, adopted orphan nuclear receptors, such as peroxisome proliferator-activated receptors (PPARs) and liver X receptors, bind to dietary lipids, such as fatty acids and oxysterols, and constitutive androstane receptor (CAR) and pregnane X receptor (PXR) binding to xenobiotics in the micro- to low millimolar concentration range [13]. Finally, orphan nuclear receptors have no natural ligand and behave like normal transcription factors. On the basis of mRNA expression of all nuclear receptor genes in 39 different tissues in two different mouse strains, nuclear receptors are divided into clades with distinct physiological roles. In this classification, for example, VDR is grouped with bile acid and xenobiotic metabolism based upon its high expression in gastroenteric tissues, whereas PPARs are linked to lipid metabolism and energy homeostasis [14].
Coregulators Most coregulators are not exclusive to nuclear receptors but are also used in a similar manner by numerous other transcription factors. Based on their mode of action, coregulators can be classified into two main groups [15]. The first group contains factors that covalently modify histones through acetylation/deacetylation and methylation/demethylation mechanisms, a process that follows the above-mentioned precise and combinatorial histone code [5]. The second group of coregulators includes previously mentioned ATP-dependent chromatin remodeling factors that modulate the accessibility of a gene to transcription factors and to the basal transcriptional machinery [16]. The complex network of coregulators defines a coregulator code characterized by distinct patterns of coregulator recruitment and by their regulated enzymatic activities. The histone code can therefore be considered as a consequence as well as a determinant of this coregulator code, as histones are crucial targets for the enzymatic activities of coregulators but also have a key role in specifying coregulator recruitment based upon a reading of the histone code. Nuclear receptors recruit positive and negative coregulatory proteins, referred to as coactivators (CoAs) [17] and corepressors (CoRs) [18], respectively. In a simplified view of nuclear receptor signaling, in the absence of ligand the receptor interacts with CoR proteins, which in turn associate with HDACs leading to a locally more compact chromatin packaging [19]. The binding of ligand induces the dissociation of CoRs and the association of CoAs to the receptor [20]. Some CoAs have HAT activity or are complexed with proteins harboring such activity and this results in the net effect of local chromatin relaxation [21]. In a subsequent
step, nuclear receptors interact with a member of the mediator (Med) complex, which builds a bridge to the basal transcriptional machinery [22]. In this way, ligand-activated nuclear receptors serve first as adaptors between gene regulatory regions and the chromatin modifying enzyme complexes and then as activators of RNA polymerase II. Cell- and time-specific patterns of relative protein expression levels of certain coregulators can distinctly modulate nuclear receptor transcriptional activity. This aspect may have some diagnostic and therapeutic value in different diseases, such as cancer [23]. Relative to skin cancer, it was postulated that the stoichiometric ratio between CoAs of the p160 family and Med1 might regulate a 1,25(OH)2D3-dependent balance between proliferation and differentiation of keratinocytes [24]. However, the switch between gene repression and activation is more complex than a simple alternative recruitment of two different regulatory complexes [25]. Most coregulators are coexpressed in the same cell type at relatively similar levels, which raises the possibility of their concomitant recruitment to a specific promoter. Therefore, repression and activation are more likely achieved through a series of sequential multiple enzymatic reactions that are promoter- and cell-type specific. Moreover, it is presently not clear, whether coregulators already mark in some cases active regulatory elements independent of DNA-binding transcription factors or if they routinely require active recruitment to target sites. Both aspects emphasize that the spatio-temporal context, i.e. the nuclear organization and the timing of the association of transcription factors and their coregulators, plays an important role in controlling gene transcription.
Transcriptional Dynamics Most models of transcriptional regulation tend to be static and place transcription factors associated with their binding sites at the center. However, these binding sites and the associated DNA-binding transcription factors provide a platform for highly dynamic events of rapid association and dissociation of coregulatory proteins. Kinetic descriptions of transcriptional regulation were initially demonstrated using members of the nuclear receptor superfamily as examples [26]. The main techniques used to evaluate the kinetics of transcriptional processes were chromatin immunoprecipitation (ChIP) assays, quantitative real-time PCR (qPCR), chromosome conformation capture (3C) assays, fluorescence recovery after photobleaching (FRAP) and real-time, single live-cell imaging of fluorescent-tagged transcription factors [7,27]. Note that the molecular biological methods of ChIP, qPCR, and 3C have a resolution of minutes, whereas the biophysical methods of FRAP
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and live-cell imaging resolve events in the subsecond range. Therefore, both methods provide different types of information about the duration of a transcription cycle, with ChIP, qPCR, and 3C suggesting 30e60 min cycling [28,29] and FRAP and live-cell imaging indicating more rapid changes [30,31]. The apparent discrepancy may be explained by the fact that FRAP experiments detect bulk, rapid and potentially transient binding of factors, whereas ChIP assays may detect productive associations of regulatory genomic regions with specific transcription factors. Using time-resolved ChIP, Shang et al. [32] demonstrated that several CoA proteins were recruited in a cyclical fashion to an estrogen-responsive chromatin region of the human trefoil factor 1 (TFF1, also called pS2) gene. However, the master example of timeresolved monitoring of recruitment and release of cohorts of coregulatory complexes on a single transcription factor-binding site was provided by Metivier and colleagues on the same chromatin region [28]. The sequential and ordered recruitment of estrogen receptor a, RNA polymerase II, and many chromatin factors, such as CoAs, CoRs, HATs, HDACs, and HMTs, defined the direction of cycling on this chromatin region. Similar observations were made with the androgen receptor on the human kallikrein 3 (KLK3, also called PSA) gene [33], with thyroid hormone receptor (TR) on the human thyroxine deiodinase type I (DIO1) gene [34] and with VDR on the human genes 24-hydroxylase (CYP24A1) [35,36], cyclin-dependent kinase inhibitor 1A (CDKN1A, also called p21) [29], insulin-like growth factor binding protein 3 (IGFBP3) [37] and MYC [38]. All these examples show cyclical association of coregulators and, in part, also of the respective nuclear receptor with a periodicity of 30e60 min. Interestingly, the more recently published reports on CDKN1A and IGFBP3 also demonstrate the cycling of mature mRNA. Interestingly, a periodic limitation of transcription is generated by clearing the chromatin region of transcription factors and by recruiting of HDACs and HMTs. Moreover, the DNA methylation pattern at CpG regions close to regulatory chromatin regions also revealed cyclical changes [39]. All of this induces a restrictive chromatin environment for a brief period of time, which after several minutes is reversed and a new cycle begins.
TRANSCRIPTIONAL REGULATION BY 1,25(OH)2D3 VDR VDR is a classical endocrine nuclear receptor and the only nuclear protein that binds with high affinity to the biologically most active vitamin D metabolite, 1,25
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(OH)2D3 [40]. Like most members of the nuclear receptor superfamily, VDR contains two zinc finger structures forming a characteristic DNA-binding domain (DBD) of 66 amino acids [41] and a carboxyterminal ligand-binding domain (LBD) of approximately 300 amino acids formed by 12 a-helices [42] (see also Chapters 7, 8, 9 and 10). Ligand binding causes a conformational change within the LBD, whereupon helix 12, the most carboxy-terminal a-helix, closes the ligand-binding pocket via a “mouse-trap like” intramolecular folding [43]. The LBD is also involved in a variety of interactions with nuclear proteins, such as other members of the nuclear receptor superfamily, CoA, and CoR proteins [21]. As already mentioned above, CoR proteins, such as NCoR1 and SMRT/NCoR2, may link nonliganded, DNA-bound VDR to enzymes with HDAC activity that cause chromatin condensation [44]. The conformational change within VDR’s LBD after binding of 1,25(OH)2D3 or one of its agonistic analogs results in replacement of CoR by a CoA protein of the p160-family [20].
VDREs Each eukaryotic gene is under the control of a large set of transcription factors that can bind up- and downstream of its transcription start site (TSS) [45]. An essential prerequisite for a direct modulation of transcription via 1,25(OH)2D3-triggered proteineprotein interactions is the location of activated VDR close to the basal transcriptional machinery. This is achieved through the specific binding of the VDR to a VDRE in the regulatory region of a primary 1,25(OH)2D3 responding gene [46]. In some cases VDREs can be in a distance of 100 kb and more, either up- or downstream of the TSS (Fig. 11.1). As will be discussed later in more detail, there is considerable evidence that most primary VDR target genes use multiple VDREs for full functionality [47]. These VDREs are typically arranged near binding sites for other transcription factors into collections of neighboring sites, so-called modules or enhancers. Modules of transcription factors that act on focused genomic regions have been shown to be far more effective than individual factors at isolated locations. The complete sequence of the human genome and also that of other mammalian species, such as chimp, dog, mouse, and rat, is now available, so that transcription factor modules can be identified by parallel and comparative analysis of their binding sites [48]. This applies also for putative VDREs, but they may not be used by the VDR, since the repressive chromatin environment in most cases denies their access [49]. Fortunately, new experimental techniques for genome-wide analyses of chromatin modifications and transcription
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Schematic structure of a chromatin unit. A chromatin unit is a region between two MARs that often contains only one gene. DNA looping should enable every DNA sequence within the same chromatin unit to be located near the basal transcriptional machinery.
FIGURE 11.1
factor binding, such as ChIP-chip and ChIP-Seq, are now available [50]. This is presently revolutionizing our understanding of the genome-wide effects of the VDR and of the diversity of its target genes as outlined later in this chapter.
DNA Binding of the VDR The DBD of the VDR contacts the major grove of a hexameric sequence, referred to as core binding motif, with the consensus sequence RGKTSA (R ¼ A or G, K ¼ G or T, S ¼ C or G). The affinity of monomeric VDR to a single core binding motif is not sufficient for the formation of a stable proteineDNA complex and thus VDR requires the formation of homo- and/or heterodimeric complexes with a partner nuclear receptor in order to allow efficient DNA binding [51]. The heterodimeric partner of VDR is the retinoid X receptor (RXR), which is another nuclear receptor family member. The proteineDNA complex of a VDR-RXR heterodimer binding to a VDRE therefore can be considered as a molecular switch for primary 1,25(OH)2D3 responding genes. Most nuclear receptors are able to dimerize in solution via their LBDs, but the DBDs dimerize only in the presence of DNA [52]. The DBD and the LBD of all nuclear receptors are linked by a hinge region of 35e50-amino-acid residues that form a long a-helical structure [53]. The loop between this a-helix and the second zinc finger of the DBD contains a short 6amino-acid residue region, referred to as T-box, which has been suggested to provide a dimerization interface for the interaction with the DBD of RXR [54]. Steric
constraints allow dimerization of DBDs only on response elements (REs) with properly spaced corebinding motifs. Modeling of the DBDs of VDR and RXR on DNA [53] suggested that an asymmetric arrangement, i.e. head-to-tail, as a direct repeat with three intervening nucleotides (DR3) provides the most efficient interface of the core DBDs. This fits with the 3-4-5 rule of Umesono et al. [55], in which VDR-RXR heterodimers show optimal binding to DR3-type REs, whereas other nuclear receptors prefer DR4-type REs (for example TR, CAR, and PXR) and DR5-type REs (for example RARs). On DR3-, DR4-, and DR5-type REs, different heterodimers bind with the same polarity, in which RXR always binds to the upstream hexamer and the partner receptor, for example VDR, to the downstream hexamer [56,57]. This specific and directed dimerization of the DBDs appears to be the major discriminative parameter for selective RE recognition.
GENOMIC VDR-BINDING SITES DR3-type VDREs Numerous studies (for example [51,58]) have confirmed Umesomo’s suggestion [55] that VDR binds well to DR3-type REs, the motif most widely accepted as the classical structure of a VDRE. Every transcriptionally responsive primary VDR target gene has to contain at least one VDRE in its promoter region and these VDREs are often located relatively close to the TSS of these genes. It is assumed that matrix attachment regions (MARs) subdivide genomic DNA into units of
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an average length of 100 kB containing the coding region of at least one gene [59]. DNA looping should be able to bring any DNA site within the same chromatin unit close to the basal transcriptional machinery that is assembled on the TSS (Fig. 11.1). This model suggests that distant sequences can also serve as VDREs and that sequences downstream of the TSS may act as functional VDR-binding sites. Since the pleiotropic physiological effects of 1,25 (OH)2D3 are based on the transcriptional regulation of primary 1,25(OH)2D3 responding genes, the activation of these genes can be explained through ligandactivated VDR-RXR heterodimers bound to DR3-type VDREs. There have been attempts to explain the various effects of 1,25(OH)2D3 through a multiplicity of 1,25 (OH)2D3 signaling pathways that are based on DR3type VDREs [60]. This raises the question of whether each DR3-type VDRE has an individual functionality that may explain the specific effects of 1,25(OH)2D3 or whether all DR3-type VDREs function in the same way. The affinity of the known natural DR3-type VDREs for VDR-RXR heterodimers in vitro (at a fixed proteineDNA ratio) seems to be their major discriminating parameter. VDR-RXR heterodimers appear to form identical complexes on all DR3-type VDREs, since distinguishable heterodimer conformations have not been observed at these elements [61]. Moreover, VDR-RXR heterodimers formed on the different DR3-type VDREs display no significant differences in their interaction with a given CoA or CoR protein [61]. This suggests that comparative in vitro data are not indicative of multiple DR3-type VDRE-mediated 1,25(OH)2D3 signaling pathways. Investigations of VDR-RXR heterodimers and their conformations have provided important new insights into the functionality of these molecular switches that may well explain their function in living cells [44,62].
Other VDRE Types The results from a variety of DR3-type VDREs appear unable to explain the pleiotropic physiological action of 1,25(OH)2D3 or explain the dissociated profiles exhibited by some synthetic 1,25(OH)2D3 analogs. Thus, other VDRE structures such as direct repeats with four and six intervening nucleotides (DR4 and DR6) or everted repeats with nine spacing nucleotides (ER9), might offer an alternative explanation. A comparison of the individual VDRE core sequences based upon affinity for VDR-RXR heterodimers suggests that the degree of deviation from the core binding motif consensus sequence RGKTSA [63] is proportional to the loss of in vitro functionality [61]. Interestingly, the DR4-type RE of the rat pit-1 (Pou1f1) gene [64], which contains two perfect core binding motifs, was found to
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be stronger than any known natural DR3-type VDRE [61]. Does this suggest that DR4-type REs are even better VDREs than DR3-type structures? One has to consider that a DR4-type REs is also recognized by the heterodimeric complexes of TR and other nuclear receptors with RXR [65,66], whereas the same complexes bind to DR3-type REs less tightly than VDR-RXR heterodimers. The competitive situation on DR4-type REs may therefore be the reason why in vivo VDR-RXR heterodimers still prefer DR3-type REs. Moreover, VDR-RXR heterodimers bind to DR4-type REs in the same conformation as to DR3-type REs [66], i.e. there seems to be no differential action of VDR on these elements due to a differential complex formation with RXR. Dimerization facilitates cooperative, high-affinity interaction of a nuclear receptor heterodimer, such as VDR and RXR with specific hexameric core-binding motifs. VDR-RXR heterodimers bind to DR-type VDREs in a nonsymmetrical head-to-tail tandem arrangement. In contrast, ER-type VDREs are per se symmetric, since the heterodimeric partner receptors bind in a tail-to-tail arrangement. However, natural ER9-type REs were found to be sufficiently asymmetric in their core-binding sequences, in order to allow a polarity-determined binding of heterodimers [57]. Thus far, ER9-type VDREs have been described within the promoter regions of the human calbindin D9k (S100G) gene [57], the mouse Fos gene [67] and the rat osteocalcin (Bglap) gene [57]. On DR3-type VDREs, both receptor DBDs are located at roughly the same side of the DNA (tilted by 51.4 ), whereas on ER9-type VDREs the DBDs are at nearly opposite sites of the DNA (tilted by 154.3 ) [54]. Moreover, the distance between the DBDs along the axis of the DNA is threefold greater on ER9-type VDREs than on DR3-type VDREs. Computer modeling and experimental studies have both shown that the T-box of VDR contributes to the dimerization interface of the extended VDR DBD with the RXR DBD, i.e. on a DR3-type VDRE the VDR DBD contacts directly the RXR DBD, whereas a direct contact of both DBDs is not possible on an ER9-type VDRE. The lack of DBD dimerization on an ER9-type RE suggests that DNA-driven cooperativity between the partner DBDs is unlikely. In fact, the spacing of the two core-binding motifs is less stringent than on DR-type REs, as ER7-, ER8-, and ER10-type structures are also able to bind dimeric VDR complexes [68,69]. However, the Kd-value for the binding of VDRRXR heterodimers to DR3- and ER9-type REs has been determined to be in a similar range of 0.5e1 nM [57].
RE Clusters In mammals, the most responsive known primary 1,25(OH)2D3 target gene is CYP24A1, whose gene product is the key enzyme involved in the catabolism
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of 1,25(OH)2D3. Activation of this gene provides a negative feedback loop mechanism controlling the level of the hormonal ligand. The reason for the strong responsiveness of the CYP24A1 gene is that it contains two DR3-type VDREs separated by less than 100 bp and located in close proximity to the TSS. These DR3-type VDRE clusters are evolutionarily conserved between man and rodents [70,71]. The sequences of both VDREs deviate slightly from consensus hexameric sites, but this appears to be more than compensated for by the fact that they are located in close vicinity to each other and to the TSS of the gene. In addition, binding sites for the transcription factor Ets-1 have been characterized within the CYP24A1 promoter and seem to interfere synergistically with the two VDREs, i.e. the cluster of VDR and Ets binding sites form a unique and complex VDRE. RE clusters are also found in other genes that are primary targets of other members of the nuclear receptor superfamily, such as VDR’s close relatives CAR and PXR. Investigation of natural CAR and PXR target genes, such as CYP2B6 or CYP3A4, respectively [72,73], indicate that a single RE is insufficient for mediating the regulatory role of the receptors and that at least two CAR or PXR REs in close proximity to each other are likely required. In the case of CAR, these multiple RE clusters are called phenobarbital response enhancer modules (PBREMs), because they mediate the responsiveness to the CAR activator phenobarbital. The CYP2B6 gene contains two DR4-type REs with an additional binding site for the transcription factor NF-1 [72], whereas the PBREM of the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene is formed by three CAR REs [74]. In contrast, the CYP3A4 gene contains an ER6-type RE proximal to the TSS and a more distal DR3/ER6 cluster [73]. Taken together, these assemblies represent two or more simple nuclear receptor REs together with binding sites for other types of transcription factors. Their overall function seems to follow the same rules, i.e. the greater the number of nuclear receptors and other transcription factors that bind to such promoter regions, the greater the chance is that they will induce histone acetylation and chromatin decondensation. Another interesting observation in relation to the RE clusters is that they are rather promiscuously bound by related members of the nuclear receptor superfamily, such as VDR, CAR, and PXR. VDR was shown to activate the CYP2B6, CYP3A4, and CYP2C9 genes by replacing CAR and PXR within the respective RE clusters [75]. In particular, the CYP3A4 gene, which contains a DR3-type RE within its RE cluster, has been shown to be stimulated effectively by 1,25(OH)2D3, and can be considered as a primary 1,25(OH)2D3 target gene. This suggests that 1,25(OH)2D3 and the VDR may have an impact on the metabolism of prescription drugs, of
which 60% are metabolized by the CYP3A4 enzyme [76]. Note that the VDR seems to be able to act also as an intestinal bile acid sensor, because certain bile acids have been identified as low-affinity VDR ligands [77]. In this context, the bile-acid-activated VDR can also induce the CYP3A4 gene. It is very likely that future investigations will reveal more of these 1,25(OH)2D3 target genes, thus enlarging the physiological importance of this hormone to an even greater extent.
The Concept of Multiple VDREs per Gene For a detailed analysis of the regulatory regions of primary nuclear receptor target genes and for the confirmation of the binding of a nuclear receptor to a given RE in living cells, ChIP analysis has become the gold standard. For example, for the VDR target genes CYP24A1 [36], 25-dihydroxyvitamin D3 1a-hydroxylase (CYP27B1) [78], cyclin C (CCNC) [79], and CDKN1A [29,80] some 7e10 kb of their promoter regions were individually investigated by using a set of 20e25 overlapping genomic regions. This approach identified four functional REs for both the CYP24A1 and the CCNC gene, three in the CDKN1A promoter and two in the CYP27B1 gene. Although the in vitro DNA-binding affinity of VDR-RXR heterodimers to the REs described for these genes differs (compare [36,79e81]), at the chromatin level all RE-containing promoter regions show comparable association strength with VDR and RXR. Each of the multiple 1,25(OH)2D3-responsive promoter regions is able to contact independently the basal transcriptional machinery. This suggests that the simultaneous communication of the individual promoter regions with the RNA polymerase II complex occurs through a discrete three-dimensional organization of the promoter. Such a model could resemble the traditional “DNA looping model“ discussed previously to explain the activity of upstream enhancer elements [82]. An alternative approach to the identification of primary nuclear receptor target genes has been conducted with the six members of the IGFBP gene family. Here, an in silico screen for VDR-RXR binding was performed first, and was then followed by the analysis of candidate 1,25(OH)2D3-responsive sequences in ChIP assays [81]. Induction of gene expression was confirmed independently using quantitative PCR. By using this approach, the genes IGFBP1, IGFBP3, and IGFBP5 were demonstrated to be primary VDR target genes. The in silico screening of the 174 kb of genomic sequence surrounding all six IGFBP genes identified 15 candidate REs, ten of which were shown to be functional in ChIP assays. This approach also confirmed the concept of multiple REs per nuclear receptor target gene. In a comparable study the human arachidonate 5lipoxygenase (ALOX5) gene was also analyzed and
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shown to be a primary 1a,25(OH)2D3 target gene. However, from 22 in silico derived REs identified in the whole ALOX5 gene sequence (e10 kb to þ74 kb), only two have been validated to be functional in vitro and in the living cells. One of these REs is located far downstream of the TSS (þ42 kb) [83], i.e. this study revealed candidate REs located more than 30 kb from their target gene’s TSS. Based on the present understanding of enhancers, DNA looping and chromatin units being flanked by insulators or MARs (Fig. 11.1) these long distances do not appear to be limiting [84]. Interestingly, the number of REs within a promoter does not correlate with the inducibility of a nuclear receptor target gene, since the average short-term transcriptional response of most primary nuclear receptor target genes is only twofold or less [85]. However, most of them are simultaneously under the control of other transcription factors, such as p53 in case of the CDKN1A gene [80], and therefore possess significant basal levels of transcription.
Negative VDREs Expression profiling using whole genome microarrays indicates that the number of genes that are downand up-regulated by 1,25(OH)2D3 are similar [86]. In general, the mechanisms of down-regulation by 1,25 (OH)2D3 is much less well understood, but also seem to require the binding of a VDR agonist. It is obvious that only genes, which show basal activity, can be down-regulated, i.e. these genes exhibit basal activity due to the activity of other transcription factors bound to their promoters. There are several different models that attempt to explain how 1,25(OH)2D3 and the VDR can mediate down-regulation of genes, but a common theme is that VDR counteracts the activity of specific transcription factors. For the physiologically important down-regulation of the CYP27B1 gene by 1,25(OH)2D3 a negative VDRE located at position e0.5 kb has been proposed, where VDR-RXR heterodimers do not bind directly but via the transcription factor VDR interacting repressor (VDIR, also called TCF3) [87]. In addition, two positive VDREs located e2.6 and e3.2 kb upstream of the TSS also modulate the cell-specific activity of a negative VDRE [78]. Association of VDR-RXR heterodimers to TCF3-binding sites may also occur through liganddependent chromatin looping from more distal regions that directly bind the VDR [78]. In situations where these activating transcription factors are other nuclear receptors or transcription factors that bind to composite nuclear receptor REs, VDR could simply compete for DNA-binding sites [88,89]. In a similar way, VDR could also compete for binding to partner proteins, such as RXR, or for common CoA proteins, such as SRC-1 or CBP [90]. In all these situations the down-regulating
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effects of the VDR may be of a general nature, i.e. the same mechanism may be relevant to other genes as well. So far, however, no general down-regulating effects of 1,25(OH)2D3 have been reported. The binding of 1,25(OH)2D3 to the VDR results in a ligand-dependent conformational change, which leads to an exchange of proteins that bind to the LBD of the VDR, i.e. altering proteineprotein interaction profiles but no changes in VDReDNA interaction properties. In most cases, however, 1,25(OH)2D3-dependent downregulation of a gene involves specific DNA-binding sites on a specific promoter, which are referred to as negative VDREs. Experiments suggest that DNA binding of the VDR to down-regulated genes such as the parathyroid (PTH) gene does not involve RXR [91,92], whereas other studies such as those for the atrial natriuretic factor (NPPA) gene [93] come to opposite conclusions. Investigation of the 1,25(OH)2D3-mediated repression of the parathyroid hormone-like hormone (PTHLH) gene also indicates that protein kinases may be involved in the downregulation of this gene [94]. The regulatory region of primary 1,25(OH)2D3 responding genes may contain both negative and positive VDREs as shown at the example of the human MYC gene [38]. The activities of the different VDREs are determined through promoter context and may not be active simultaneously. Some of the VDREs identified within genes that are down-regulated by 1,25(OH)2D3 are more likely to represent negative VDREs. The sequences are not revealing, however, because in the absence of a natural promoter context and normal chromatin organization, these VDREs behave like positive VDREs. This highlights again the fact that promoter context, the chromatin status, and the cluster of neighboring transcription factors are important for the function of a simple VDRE. Thus, simple VDREs cannot act as negative VDREs. In the case of the calcitonin (CALCA) gene, two separate promoter regions are suggested to be responsible for the down-regulation of gene transcription by 1,25(OH)2D3 [95]. Together, this information indicates that mechanisms governing 1,25(OH)2D3mediated down-regulation are complex.
In Silico Screening for VDREs The specificity of VDR for its DNA-binding sites allows a description of VDRE properties that can be used to predict potential binding sites in genomic sequences. For this the VDR binding preference, often expressed as position weight matrix, has to be described on the basis of experimental data, such as a series of gel shift assays with a large number of natural binding sites [68,96e98]. However, VDR-RXR heterodimers do not only recognize a pair of the nuclear receptor consensus binding motifs AGGTCA, but also a number of variations to it.
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Depending upon the individual position weight matrix description, this leads to a prediction of VDREs every 1e10 kb of genomic sequence. This is certain to contain many false-positive predictions, due to scoring methodology and limitations that are imposed by chromatin and supported by all available experimental data. Wang et al. combined microarray analysis and in silico genome-wide screens for DR3- and ER6-type VDREs [99]. This approach identified several novel VDREs and VDR target genes, but most of the VDREs await a confirmation by ChIP and 3C assays. In a position frequency matrix, the quantitative characteristics of a transcription factor, i.e. its relative binding strength to a number of different binding sites, is neglected, since the total number of observations of each nucleotide is simply recorded for each position. Moreover, in the past there was a positional bias of transcription-factor-binding sites upstream in close vicinity to the TSS. This is apparent from the collection of identified natural VDREs [46], but is in contrast with a multigenome comparison of nuclear-receptor-binding site distribution [100] and other reports on wide-range associations of distal regulatory sites [101]. Internet-based software tools, such as TRANSFAC [102], screen DNA sequences with databases of matrix models. The accuracy of such methods can be improved by taking the evolutionary conservation of the binding site and that of the flanking genomic region into account. Moreover, cooperative interactions between transcription factors, i.e. regulatory modules, can be taken into account by screening for binding site clusters. The combination of phylogenetic footprinting and position weight matrix searches applied to orthologous human and mouse gene sequences reduces the rate of false predictions by an order of magnitude, but leads to some reduction in sensitivity [103]. Recent studies suggest that a surprisingly large fraction of regulatory sites are not conserved yet are functional, which suggests that sequence conservation revealed by alignments does not capture many relevant regulatory regions [104]. Most putative transcription-factor-binding sites are covered by nucleosomes, so that they are not accessible to the transcription factor. This repressive environment is found in particular for those sequences that are either contained within interspersed sequences, are located outside of transcription factor modules, or lie outside of insulator sequences marking the border of chromatin loops [105]. This perspective strongly discourages the idea that isolated, simple VDREs may be functional in vivo.
Genome-wide View on VDREs Most of the well-characterized natural VDREs [61] have a DR3-type structure and, because they were
investigated in the pregenomic era when only limited promoter sequences were available, most of them were identified within the first 1 kb of promoter sequence upstream of the TSS. These VDREs suggest that the consensus VDR core-binding motif is RGKTSA, although some natural hexameric sequences show a significant deviation from this consensus sequence. Moreover, only a very few of these VDREs, such as that of the rat Bglap gene, are understood in their promoter context, i.e. in the context of chromatin organization and flanking binding sites for other transcription factors. Recently published ChIP-Seq studies reported genome-wide 5000e10 000 binding sites per nuclear receptor [106,107], while microarrays showed some tenfold fewer genes being primary target genes within the same cell type [107,108]. The first ChIP-Seq results for VDR confirmed this ratio by reporting 2776 genomic VDR-binding sites and 229 target genes [109]. Note that this study analyzed VDR binding and gene regulation 36 h after ligand stimulation, i.e. a number of the targets may be secondary. All ChIP-Seq studies agree that the majority of identified transcription-factor-binding sites are distal to promoters [110,111]. This also confirms the above-mentioned concept that the regulatory unit of a gene involves multiple transcription-factor-binding sites at various positions and that the spatial organization of the unit is important to bring via DNA looping at least one activated nuclear receptor protein close to the TSS of the respective primary nuclear receptor target gene.
VDR TARGET GENES Classical VDR Targets The most striking effect of severe vitamin D deficiency is rickets. Rickets can also result from mutations in the CYP27B1 or the VDR gene. 1,25(OH)2D3 is essential for adequate Ca2þ and Pi absorption from the intestine and hence for bone formation [112]. Liganded VDR has been shown to induce expression of the gene encoding for the major Ca2þ channel in intestinal epithelial cell, transient receptor potential cation channel, subfamily V, member 6 (TRPV6), by direct binding to a VDRE at 1.2, 2.1 and 4.3 kb from the TSS [113]. The sodium-phosphate transport protein 2B (SLC34A2) gene was also found to be induced by 1,25(OH)2D3 although no VDREs have yet been identified for this gene [114]. 1,25(OH)2D3 also down-regulates the expression of the PTH gene that opposes 1,25(OH)2D3 in regulation of serum Ca2þ and Pi levels, but up-regulates the fibroblast growth factor 23 (FGF23) gene, whose gene product, like PTH, lowers serum Pi levels [115]. The induction of the tumor necrosis factor ligand superfamily, member 11
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(TNFSF11, also called RANKL) gene by liganded VDR via multiple distant VDREs (up to 76 kb from the TSS) leads to stimulation of osteoclast precursors to fuse and form new osteoclasts, resulting in enhanced resorption of bone [116]. Thus, this study also confirms that functional VDREs can be a large distance from the target gene TSS.
VDR Targets in Cell Cycle Regulation The main antiproliferative effect of 1,25(OH)2D3 on cells is a cell cycle block at the G1 phase. This can be explained by changed expression of multiple cell cycle regulator genes. Among the first targets described, expression of cyclin-dependent kinase inhibitor (CDKI) genes CDKN1A (also called p21) and CDKN1B (also called p27) were found to be up-regulated by ligand treatment [117,118]. For the CDKN1A gene, a VDRE in the proximal promoter was characterized, thus establishing CDKN1A as a direct 1,25(OH)2D3 target gene [119]. More recently, it has been questioned whether CDKN1A actually represents a primary or a secondary target, the latter process via up-regulation of TGF-b or IGFBP3, and whether the described VDRE is truly functional [120,121]. Indeed, by screening 7 kb of the CDKN1A promoter with overlapping ChIP regions, three novel regions with 1,25(OH)2D3-induced VDR enrichment were identified, two of which also bound p53 as well [80]. The functionality of these characterized 1,25(OH)2D3-responsive regions relative to CDKN1A expression was illustrated through ChIP and 3C analyses [25,29]. Additional CDKIs, such as CDKN2B (p15), CDKN2A (p16), CDKN2C (p18), and CDKN2D (p19), also show transcriptional response to 1,25(OH)2D3, although the CDKN2A response appears to be secondary as it can be blocked by protein synthesis inhibitors [119,122]. In addition, the genes cyclin E1 (CCNE1), cyclin D1 (CCND1), and cyclin-dependent kinase 2 (CDK2) were also found to be down-regulated by 1,25 (OH)2D3 [120]. It remains to be elucidated whether these effects are primary and occur via functional VDREs on regulatory regions of these genes. Another interesting 1,25(OH)2D3 target gene is CCNC. The cyclin C-CDK8 complex was found to be associated with the RNA polymerase II basal transcriptional machinery [123] and is considered to be a functional part of mediator protein complexes that are involved in gene repression [124]. The fact that the CCNC gene, being located in chromosome 6q21, is deleted in a subset of acute lymphoblastic leukemias, suggests that it may be involved in tumorigenesis [125]. In addition, growth arrest and DNAdamage-inducible, alpha (GADD45A) and members of the IGFBP gene family also respond to 1,25(OH)2D3 [81,126]. GADD45A plays an essential role in DNA repair and GADD45 proteins displace cyclin B1 from Cdc2 and
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thus inhibit the formation of M phase-promoting factor that is essential for G2/M transition [127]. GADD45A has been shown to be a direct transcriptional target of 1,25 (OH)2D3 with a functional VDRE within the fourth exon of the gene [128]. IGFBPs modulate the activity of the circulating insulin-like growth factors (IGF) I and II. The IGFBP3 gene was first discovered to be up-regulated by 1,25(OH)2D3 and contains a functional VDRE [129]. As described above, IGFBP1 and IGFBP5 are also primary 1,25(OH)2D3 target genes [81]. Another interesting primary 1,25(OH)2D3 target is the PPARD gene, which contains a potent DR3-type VDRE in close proximity to its TSS [130]. PPARd and VDR proteins are widely expressed and in an apparent overlap in the physiological action of the two nuclear receptors, both are involved in the regulation of cellular growth, particularly neoplasms. High PPARD expression in tumor seems to be positive for the prognosis of associated cancers [131]. Overall, 1,25(OH)2D3 restricts cell cycle progression in several phases via multiple and partially redundant targets on parallel pathways that when combined provide a robust antiproliferative effect.
Relative Expression of VDR Target Genes The steady state mRNA expression levels of some VDR target genes, such as that of the CYP24A1 gene, are very low in the absence of ligand, but are induced up to 1000-fold by stimulation with 1,25(OH)2D3 [36]. Most other known primary 1,25(OH)2D3 target genes, such as CCNC and CDKN1A, often show an inducibility of twofold or less after short-term treatment with 1,25 (OH)2D3 [132,133]. These latter genes, however, exhibit 10 000e100 000-fold higher basal expression levels as compared to that of the CYP24A1 gene. Thus, when the relative levels are taken into account, two- to 20-fold more CCNC and CDKN1A than CYP24A1 mRNA molecules are produced after induction with 1a,25(OH)2D3.
VDR TARGET GENE ANALYSIS Transcriptome Analysis There are a number of modern methods for the identification and characterization of VDR target genes. The effect of 1,25(OH)2D3 on mRNA expression, i.e. 1,25 (OH)2D3-induced changes of the transcriptome, has been assayed by multiple microarray experiments in cellular models (either an established cell line or primary cells) or in in vivo models (mostly rodents). While the focus is on identification of primary VDR target genes, the stimulation times were short (2e6 h), but when the overall physiological effects are the center of the study, longer treatment times were used
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(24e72 h). For a limited number of putative VDR target genes, quantitative PCR can be applied, but for a wholegenome perspective on VDR signaling, microarrays or RNA-Seq have to be used. A few years ago, cDNA arrays with an incomplete number of genes were used and rather short lists of VDR target genes from colon [133], prostate [23,85,134,135], breast [132], and osteoblasts were developed [136,137]. In squamous cell carcinoma more than 900 genes respond to 1,25(OH)2D3 after 12 h treatment in the presence of the protein synthesis inhibitor cycloheximide [99]. However, the number of overlapping VDR target genes in these lists was low. Since the setups of these microarray analyses were different in treatment times and probe sets, this suggests that most VDR target genes respond to 1,25(OH)2D3 in a very tissue-specific fashion and may manifest only a rather transient response. On the basis of these results, however, the total number of convincing primary 1,25(OH)2D3 target genes is on the order of 250. Secondary 1,25(OH)2D3responding genes contribute to the physiological effects of 1,25(OH)2D3, but their induction may be delayed by a few hours or even days; these are probably mediated by primary 1,25(OH)2D3-responding gene products, such as transcription factors or coregulator proteins [133]. For a more detailed meta-analysis of VDR target genes, standardized microarray procedures performed on whole-genome chips from Affymetrix, Illumina or other commercial suppliers will be essential.
ChIP-chip and 3C Assays The combination of ChIP assays with hybridization of the resulting chromatin fragments on microarrays, the so-called ChIP-chip assays, provides an additional step for a larger-scale analysis of VDR target genes. The ChIP-chip technology has been applied to the analysis of the VDR gene [138], the CYP24A1 gene [139], the intestinal calcium ion channel gene TRPV6 [113], the Wnt signaling coregulator LRP5 [140], and the TNF receptor ligand gene RANKL that promotes the formation of calcium-resorbing osteoclasts [116]. For all those genes, a number of VDR-associated chromatin regions were identified, some of which were far upstream or downstream of the gene’s TSS. These studies confirmed that many, if not all, VDR target genes have multiple VDR-associated regions. Presently, 3C assays are the most appropriate method to confirm the physical contact between a distal transcription factor and the basal transcription machinery located on the TSS [141]. Therefore, 3C assays provide an important additional proof for the functionality of an RE. So far, 3C assays confirmed the functionality of VDREs in the human genes CYP27B1 [78], CDKN1A [25], and CYP24A1 [139,142].
TRANSCRIPTIONAL CYCLING Models As already mentioned above, VDR belongs to those transcription factors for which transcriptional cycling has been reported [29,35e38]. Generally, transcriptional cycling is a phenomenon that depends on stimulus availability, association and dissociation of the transcription factor and its coregulators, and finally their possible removal through proteasomal degradation. Metivier and colleagues postulate that transcriptionally productive cycles are rather slow, because the initiation of transcription requires specific sequences of events to occur, which are ordered, kinetic and directional and dependent on productive events that occur infrequently from many rapid, stochastic, transient, and unproductive associations of factors [7]. In fact, many nuclear proteins rapidly but nonproductively associate with regulatory chromatin regions before a deterministic event takes place. Such continuous scanning is essential for transcription and is mirrored in the high mobility seen by FRAP and live cell imaging [143]. A model based on stochastic modeling, in which it is assumed that at least 30 proteins and six irreversible (i.e., energy-consuming) steps participate in each transcription cycle [144], requires energy consumption [145]. In principle, the recruitment and assembly of these complexes could occur in a random fashion, in a partially random fashion (partially determined order) or in a uniquely defined sequential order. In addition, the complexes could be preformed already in the solution of the nucleoplasm or they assemble on the DNA. Based on physiologically relevant protein concentrations, on/ off rates and equilibrium constants, we found that only the models based on sequential or partially determined orders of transcription complex assembly produce outputs that are consistent with the kinetics of our experimental observations. Per transcription cycle, our model distinguishes three phases (Fig. 11.2): (i) an activation phase in which transcription factors and HATs are recruited to the regulatory regions in order to locally open chromatin, (ii) an initiation phase in which Med proteins loop to the RNA polymerase II binding at the transcription start site and mRNA transcription starts, and (iii) a deactivation phase where HDAC and CoR association lead to chromatin condensation. Transcriptional cycling at the mRNA level can be observed if a gene fulfills two essential conditions: it manifests bursts of transcription and produces a transcript with a short half-life. Thus, cycling at the mRNA level is restricted. A burst of transcription results, in most cases, through activation by an inducible dominant transcription factor such as a member of the nuclear receptor superfamily. However, bursty transcription is
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Model of transcriptional cycling. The model monitors the three phases of transcriptional cycling, of which only the initiation phase results in the synthesis of mRNA, while mRNA degradation occurs at all phases. If a gene shows bursty transcription and the half-life of the mRNA is short enough, this will result in cycling of mRNA levels. Note that only the core proteins of the respective complexes are shown, as we assume that each protein complex contains up to 30 components. Ac ¼ Acetylated histones, Me ¼ methylated DNA (dark gray) or histones (light brown), Pol II ¼ RNA polymerase II. Please see color plate section.
FIGURE 11.2
a general phenomenon involving a vast array of mechanisms, for example nuclear translocation and second messengers oscillations (Ca2þ, cAMP) [146,147]. The length of the initiation phase influences the size and duration of a transcription burst and is modulated by epigenetic changes of the involved chromatin regions. This suggests that transcription bursts are cell- and gene-specific. Transcription of mature mRNA can be observed only with those genes, whose half-life of the induced mRNA transcript is shorter than the periodicity of cyclical association of transcription factors and their cofactors, i.e. in average 60 min or less. Only under this condition is there within one transcription cycle enough mRNA degradation, in order to observe cycling of transcript levels (Fig. 11.2). This reduces the list of genes that show transcriptional cycling to those that encode short-lived regulatory proteins, such as transcription factors and kinases. Moreover, in order to see transcriptional cycling at the cell population level, cells must be synchronized. Stimulation with a nuclear receptor ligand has been shown to be sufficient for a population-level synchronization of cells [29,37], although in some studies [28,32] pretreatment with the RNA polymerase II inhibitor a-amanitin was employed.
What is the Impact of Transcriptional Cycling? The most obvious answer to this question is that transcriptional cycling allows for better control of gene transcription. A gene can be silenced far quicker, when it has to confirm every 60 min, if its transcription is still required, than without this control mechanism. As
discussed above, transcription is a dynamic process with high mobility of transcription factors and their coregulators [148]. These proteins contact each other and their specific chromatin-binding sites only for a relatively short time [30], which provides transcriptional regulation with a stochastic component. This is further extended by the rapidly changing epigenetic state of the involved chromatin regions, as shown for cycling CpG methylation in the regulatory region of the TFF1 gene [39]. Could therefore transcriptional cycling be the expression of noisy transcription [143,149]? Positive feedback processes are able to enhance noise, while negative feedback mechanisms in most cases reduce the effect of noise [150]. Regular oscillations are a widespread phenomenon in cell biology, including those in glycolysis [151], calcium signaling [152], and circadian rhythms [153]. They show remarkable fidelity and resistance to noise. Moreover, they are entrained by periodic exposure to signals, but are capable of “free running” without any external signals. By analogy, we assume that the phenomenon of transcriptional dynamics represents a transcriptional clock, which is entrained by a transcription factor stimulus as exemplified by the ligands for nuclear receptors.
Other Forms of Transcriptional Dynamics The action of reusable factors, such as transcription factors and their coregulators, and of the chromatin activation status is intrinsically cyclic, since they act as catalysts or scaffolds. Ensembles of such systems can subsequently display synchronized cycles depending on the stochastic distribution functions of their cycling
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time. For example, low-frequency stimulations of cells with tumor necrosis factor induce cycling of the abundance of the transcription factor nuclear factor kB in the nucleus [154]. Moreover, pulsative exposure of cells with ultradian release of cortisol stimulates transcriptional cycling of the nuclear receptor glucocorticoid receptor (GR) [27]. Interestingly, these transcriptional cycles of GR are not observed, when its synthetic ligand dexamethasone is used, which stabilizes the receptor for longer periods than the natural ligand cortisol [27]. We made similar observations when using in constant, i.e. nonpulsative, stimulation experiments the potent synthetic VDR agonist Gemini. Gemini failed to induce transcriptional cycling of the human IGFBP3 gene, while the natural ligand does [37]. These observations may have implications for the therapeutic application of synthetic nuclear receptor ligands and may explain some of their side effects.
CONCLUSION The sequencing of the complete human genome and the genomes of other species, i.e. the availability of all regulatory sequences, enable a more mature understanding of the diversity of 1,25(OH)2D3 target genes. Perhaps the idea of simple isolated VDREs should shift to the concept of complex VDREs, of which the simple VDRE represents the core. Depending on the temporal presence of cell-specific transcription factors, these complex REs may act positively or negatively in respect to 1,25(OH)2D3. The coordinated action of these different types of VDREs could explain the individual response of target genes to 1,25(OH)2D3. Methods incorporating both experimental- and informatics-derived evidence to arrive at a more reliable prediction of VDR targets and binding modules can bring all available data together with the ultimate aim to predict the outcome in a specific context. One can envision that in the future, the emphasis will shift from target genes to target regulatory modules to alter a physiological response and from individual genes to whole-genome response. Therefore, a much larger challenge lies ahead where we will be confronted with the higher order of regulated networks of genes, where the sum effect of ligand treatment may be defined. There is no doubt that transcriptional regulation is a dynamic process. However, the impact of the cyclical phenomena of transcription factor and coregulator association with regulatory chromatin regions discussed here are not fully explored relative to their impact on transcriptional regulation. In many cases, the cycling of nuclear proteins and chromatin activation stages will not translate into a cycling of mRNA or protein
levels, i.e. they may not have any direct impact on a physiological function of the cell.
Acknowledgments Grants from the University of Luxembourg, the Academy of Finland and the Juselius Foundation supported this research.
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12 Epigenetic Modifications in Vitamin D Receptor-mediated Transrepression Alexander Kouzmenko 1, 2, Fumiaki Ohtake 1, Ryoji Fujiki 1, Shigeaki Kato 1 1
Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan 2 College of Science, Alfaisal University, Riyadh 11533, Kingdom of Saudi Arabia
INTRODUCTION Vitamin D plays a pivotal role in the control of calcium homeostasis and bone mineral metabolism. Vitamin D has been also shown to play a critical role in the regulation of a wide range of fundamental physiological processes including cell growth and differentiation, embryonic development, inflammatory reaction, and immune response [1e4]. Vitamin D is a prohormone that is ultimately converted into the active calciotropic hormone 1,25(OH)2D3, also known as calcitriol, by the 1a-hydroxylase or Cyp27b1 in kidney proximal tubular cells. The expression of CYP27B1 is negatively controlled by 1,25(OH)2D3 through a mechanism involving negative feedback regulation [5]. The majority of the pleiotropic effects of 1,25(OH)2D3 are thought to be mediated through the action of vitamin D receptor (VDR). VDR is a ligand-inducible transcriptional factor and a member of the nuclear hormone receptor (NHR) superfamily [6]. The essential role of the VDR in mediating 1,25(OH)2D3 physiological actions has been demonstrated by clinical analysis of VDRdeficient subjects (see Chapter 65) and in experimental studies on VDR-null mice [7e10]. Activation by the ligand leads to dimerization of VDR with RXR, and the ability of VDR/RXR heterodimers to recognize and bind to vitamin D response element (VDRE) DNA sequences in the promoters of 1,25 (OH)2D3 target genes [11]. Upon binding to the VDRE, VDR recruits various coregulator complexes with various chromatin-remodeling and histone-modifying enzymatic activities that cause changes in chromatin conformation and rearrangement of nucleosomal arrays [12e14] (Fig. 12.1).
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10012-5
Recent experimental findings indicate that the VDR also acts as a transcriptional cofactor through its recruitment by other DNA-binding regulatory proteins to specific promoters without direct interaction with the DNA. Interestingly, VDR may act as a cofactor in both ligand-free and ligand-activated states [14,15]. This chapter presents recent progress in our studies on the mechanisms of genomic actions of the VDR that include various epigenetic modifications at the target genes.
WINAC: A NOVEL VDR-ASSOCIATING ATP-DEPENDENT CHROMATIN REMODELING COMPLEX Identification of novel factors that modulate VDR action is an important step in unveiling the mechanisms of vitamin D regulatory effects. In the search for chromatin proteins that may account for occupancy of VDR at the target promoters in the absence of ligand, the William syndrome transcription factor (WSTF) was found to interact with VDR and recruit the receptor to chromatin in a ligand-independent manner. Further biochemical analysis and protein purification have shown that WSTF links VDR to a large nuclear protein complex designated as WINAC (WSTF Including Nucleosome Assembly Complex) [14]. WINAC was found to consist of 13 subunits including ATPases (Brg1 and hBrm) and several BAF components of the known SWI/SNF type complexes [16] together with DNA replication and repair associated factors, such as TopoII, FACTp140, and CAF-1p150 that had not been known to associate with ATP-dependent chromatin remodeling complexes [17e19] (Fig. 12.2).
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Schematic presentation of 1,25(OH)2D3-induced VDR transactivation. Upon 1,25(OH)2D3 binding, VDR heterodimerizes with RXR. VDR/RXR dimers associate with VDREs and recruit various coregulator complexes at the target gene promoters. Epigenetic modifications and chromatin remodeling result in activation of the target gene expression.
FIGURE 12.1
WINAC displays a capacity to rearrange nucleosomal arrays in the vicinity of the VDR-bound DNA. Furthermore, while WINAC mediates the association of unliganded VDR at VDRE in the target gene promoters, the recruitment of VDR coregulators requires presence of the ligand. Such differential recruitment capacity clearly indicates that interaction of sequence-specific regulators with chromatin remodeling complexes may render nucleosomal arrays favorable for coregulator accessibility at specific genomic loci [14]. WINAC displays significant functional versatility and appears to be involved through the WSTF in both
VDR-mediated transactivation and transrepression, albeit by different mechanisms [14,20].
E-BOX MOTIF NEGATIVE VDRE MEDIATES 1,25(OH)2D3-INDUCED TRANSREPRESSION Liganded VDR/RXR heterodimers transactivate through direct binding to the DNA of target gene promoter VDREs composed of two hexameric sequences (A/G)G(G/T)TCA arranged as direct
WINAC complex components in comparison with other major known ATP-dependent chromatin remodeling complexes. ATPase subunits are indicated in darker shade ovals.
FIGURE 12.2
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WINAC MEDIATES LIGAND-INDUCED TRANSREPRESSION BY VDR
repeats with three intervening nonspecific nucleotides (otherwise known as a DR3 VDRE) [1]. Some VDRregulated genes have multiple VDREs spread within several thousands of nucleotides in promoter regions [21]. While ligand-dependent transcriptional activation by the VDR has been studied extensively and its underlying mechanisms seem to share significant similarity with gene activation by other members of the nuclear hormone receptor superfamily [1,2,9], liganddependent VDR transrepression is far less well understood and appears to involve diverse sets of mechanisms [15,22,23]. The first type of negative VDRE (nVDRE) was identified in the human and rat parathyroid hormone (PTH) and parathyroid-hormone-related peptide (PTHrP) gene promoters [23,24]. These VDREs contain a single copy (half-site) of the consensus DNA sequence of the positive DR3 VDRE and have been presumed to bind either ligand-activated VDR homodimers or VDR/ RXR heterodimers [23e25]. A novel and unrelated type of nVDRE was initially identified in the human CYP27B1 (1a-hydroxylase) gene and then designated as 1anVDRe [15]. Later, similar nVDREs have also been found in the promoters for the human PTH and PTHrP genes [26]. These nVDREs contain no VDRE consensus DNA-binding site and, consistently, do not bind VDR, but instead are composed of E-box (CANNTG)-like motifs. They are, therefore, referred to as E-box nVDREs (Fig. 12.3).
COREGULATOR SWITCHING AT E-BOX nVDREs IN LIGAND-DEPENDENT TRANSREPRESSION BY VDR The E-box nVDRE is recognized by a bHLH-type transcriptional factor VDIR (VDR-interacting repressor).
FIGURE 12.3 Two types of functional nVDRE DNA sequences identified in the indicated gene promoters.
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In the absence of 1,25(OH)2D3 or VDR, the VDIR effectively activates transcription via E-box nVDRE binding and recruitment of p300/CBP histone acetyltransferase (HAT) activator complexes. This activation may account for high levels of CYP27B1 expression in VDR-KO mice despite high levels of circulating 1,25 (OH)2D3 [7,9]. VDIR transactivation is enhanced by protein kinase A (PKA) signaling through the phosphorylation of serine residues in the VDIR transactivation domains that stimulate the recruitment of HAT coactivator complexes. Since PKA signaling is activated by the PTH plasma membrane receptors [27], it is conceivable that VDIR mediates induction of the CYP27B1 expression by PTH/PTrP [15]. In the absence of 1,25(OH)2D3, VDR displays a very weak affinity for VDIR [20] and does not affect its transcriptional activation function. In the presence of ligand, VDR binds to VDIR and induces dissociation of HAT coactivator complexes and the recruitment of corepressor complexes with histone deacetylase (HDAC) activity and containing Sin3A, HDAC2, N-CoR, and possibly other factors as well [15,26]. In contrast to the previously described mechanisms of repression of bHLH activators by other transcriptional factors that included inhibition of the DNA binding and/or coactivator complex recruitment [20,29], ligand-dependent transrepression by VDR involves replacement of HAT coactivator complexes with HDAC repressor complexes at the E-box nVDRE (Fig. 12.4). Thus, 1,25(OH)2D3-dependent VDR transrepression of the VDIR target genes represents a novel mechanism of ligand-induced repression by NHR of other activator proteins by coregulator switching, rather than preventing DNA binding or recruitment of coactivator complexes.
WINAC MEDIATES LIGAND-INDUCED TRANSREPRESSION BY VDR While WINAC enhances VDR transactivation at the positive VDREs, ablation of WSTF, the WINAC-VDR bridging factor, also significantly impaired the 1,25 (OH)2D3-dependent VDR transrepression of CYP27B1 promoter [20]. Using ChIP assay in kidney cells, the ligand-dependent recruitment of WSTF/VDR complexes to the VDIR has been shown at the E-box nVDRE in the genomic CYP27B1 loci [14,30]. These data suggest that WINAC plays an important role in both VDR transactivation and transrepression. Our studies on the involvement of WINAC in VDR transrepression of CYP27B1 show that WINAC potentiates an association of VDR with the E-box nVDRE irrespective of the presence of ligand only when the CYP27B1 promoter DNA is configured as nucleosomal
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FIGURE 12.4 Schematic illustration of coregulator switching model at the CYP27B1 E-box nVDRE. Phosphorylation of VDIR by PTHactivated PKA promotes recruitment of a HAT coactivator complex at the VDIR bound nVDRE and stimulates CYP27B1 expression. Increased serum concentrations of 1,25(OH)2D3 lead to activation of VDR. 1,25(OH)2D3-bound VDR associates with VDIR, leading to dissociation of the HAT coactivator complex and recruitment of an HDAC corepressor complex.
array. In contrast, naked promoter DNA associated exclusively with liganded VDR via its binding to VDIR. Consistently, WSTF knockdown decreased VDR occupancy on the endogenous CYP27B1 promoter [14,20]. WSTF deletion mutant analysis has revealed that the C-terminal WSTF bromodomain serves as a chromatin-targeting module that “escorts” unliganded VDR to the promoter via physical interaction with acetylated histones [20]. These results indicate that WINAC acts as a tether between VDR and promoter nucleosomal arrays and that in the absence of ligand, the WSTF bromodomain targets VDR/WINAC complexes to the acetylated nucleosomal arrays in the vicinity of the E-box nVDRE within the actively transcribed CYP27B1 loci. Unliganded VDR was reported to associate with HDAC corepressor NCoR [13] which was displaced upon ligand binding. In contrast, ligand binding significantly increased the stability of interaction between VDR/WINAC and HDAC complexes [20]. Thus, WINAC may assist in the 1,25(OH)2D3-dependent VDR transrepression through stabilization of liganded VDR association with HDAC corepressor complexes and by rearrangement of nucleosomal arrays to favor HAT complex dissociation and/or HDAC recruitment. This process results in a coregulator complex switch and in a transition from the transactivation state into the repression state at the VDR/VDIR bound chromatin (Fig. 12.5).
1,25(OH)2D3-DEPENDENT VDR TRANSREPRESSION INVOLVES CpG SITE METHYLATION IN THE CYP27B1 PROMOTER Studies on reporter and endogenous gene expression in human embryonic kidney HEK293F cells and in mouse cortical tubular (MCT) cells have revealed that HDAC inhibitors are unable to completely abrogate 1,25(OH)2D3-dependent CYP27B1 transrepression by VDR [26]. This suggested that other histone-modifying activities, such as histone methyltransferases or demethylases, or even different epigenetic mechanisms might also be involved in this transrepression. To address this question, extensive analysis of various protein complexes interacting with VDR/VDIR was undertaken. It has been found that two DNA methyltransferases, DNMT1 and DNMT3B, associate with VDR/VDIR in the presence of ligand. Furthermore, DNMT1 and DNMT3B act as corepressors in 1,25(OH)2D3-dependent VDR transrepression of the CYP27B1 promoter in HEK293F cells, whereas DNA methylation inhibitor 5azacytidine or sRNAi knockdown of these DNMTs significantly weakened the transrepression. Consistently, 1,25(OH)2D3-induced recruitment of DNMT1 and DNMT3B to the endogenous CYP27B1 promoter was demonstrated by ChIP analysis [31].
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FIGURE 12.5 Model demonstrating the role of WINAC in the ligand-induced transrepression function of VDR at the CYP27B1 gene promoter. p300 coactivator complex is recruited to VDIR and acetylates nucleosomal histones at the CYP27B1 gene promoter (transactivation stage). WINAC, along with VDR, sequentially targets VDIR through interaction between unliganded VDR and VDIR, and is retained on the acetylated promoter via the WSTF bromodomain. VDR becomes receptive to 1,25(OH)2D3 binding (transition stage). Upon 1,25(OH)2D3 binding, HDAC corepressor complexes are recruited to the ligand-bound VDR/VDIR complex. VDR/VDIR-recruited HDACs deacetylate the nucleosomes, whereas WINAC exerts its ATP-dependent chromatin-remodeling activity (transrepression stage).
DNMT3B together with related DNMT3A are thought to be primarily responsible for de novo 50 -cytosine methylation [32], whereas DNMT1 plays a significant role in the maintenance of DNA methylation by employing hemimethylated DNA as a substrate [33]. Both DNMT3 and DNMT1 molecules appear to act within the same protein complexes and have been known to cooperate in gene silencing [34e36]. Consistent with their known biological activity, 1,25 (OH)2D3-induced recruitment of DNMT1/DNMT3B to the CYP27B1 loci resulted in DNA methylation of the CpG sites in the promoter and coding regions without detectable occupancy of the heterochromatization marker HP1a [31]. Thus, mechanisms of the vitaminD-induced CYP27B1 transrepression involve DNA methylation (Fig. 12.6).
CELL-CYCLE-INDEPENDENT DNA DEMETHYLATION DURING CYP27B1 TRANSCRIPTIONAL DEREPRESSION DNA methylation on cytosines of genomic CpG dinucleotides results in gene silencing during reprogramming
of cell genomic activity and plays key roles in the mechanisms of differentiation and carcinogenesis. It was previously thought that once established, methylation patterns were maintained throughout the life span of the cell, whereas demethylation was believed to be cellcycle-dependent and linked to the DNA replication [36e39]. We found that treatment with PTH significantly reduced 1,25(OH)2D3-up-regulated levels of DNMT enzymatic activity in the VDIR immunocomplexes and CpG methylation in the CYP27B1 loci. Significantly, in cells pre-exposed to 1,25(OH)2D3, treatment with PTH led to gradual demethylation of 50 -methylcytosine in the CYP27B1 CpG islands. However, PTH did not stimulate detectable incorporation of bromodeoxyuridine (BrdU) in proximal renal tubule cells from normal or VDR knockout (VDRe/e) mice. Similarly, arrest of the cell cycle after serum depletion or by treatment with aphidicolin did not abolish the PTH-induced demethylation of CYP27B1 genomic DNA in HEK293F cells [31]. Thus, CYP27B1 DNA demethylation during its transcriptional derepression is apparently independent from the cell cycle progression or DNA replication (Fig. 12.6).
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FIGURE 12.6 1,25(OH)2D3-dependent VDR transrepression and PTH-dependent derepression involve reversible CpG site methylation/ demethylation in the CYP27B1 promoter. DNMT1 and DNMT3B act as corepressors in 1,25(OH)2D3-dependent VDR transrepression of the CYP27B1. Mechanisms of the vitamin-D-induced CYP27B1 transrepression involve DNA methylation by DNMT1 and DNMT3B enzymatic activities. In the presence of PTH, MBD4 appears to trigger CYP27B1 promoter demethylation through its DNA glycosylase activity and acts as a downstream factor through the PTH signaling pathway to regulate CYP27B1 expression.
Recently, transient cyclical DNA methylation and demethylation has been reported at transcriptionally active estrogen-responsive PS2 gene promoter and few other genes [40,41] that appeared to be synchronized with previously described estrogen-induced cyclical recruitment and dissociation of various coactivator complexes [42,43].
MBD4 MEDIATES ACTIVE DNA DEMETHYLATION MBD4 belongs to a nuclear protein family that shares the methyl-CpG-binding domain (MBD) and is, therefore, capable of recognizing and binding to methylated DNA [44]. Distinct from other members of the family, MBD4 contains thymine glycosylase activity and is involved in DNA repair through its ability to target sites of cytosine deamination and to remove T/G mismatches [45]. MBD4 has been also implicated in transcriptional silencing and shown to directly interact with Sin3A and HDAC1 and to recruit HDAC complexes to methylated promoter DNA [46]. Several lines of evidence suggested that MBD4 might be involved in the regulation of CYP27B1 expression [31]. Association of MBD4 with CYP27B1 promoter depends on the promoter DNA methylation and requires VDIR. MBD4 was coimmunoprecipitated with VDIR in 1,25 (OH)2D3-dependent manner and regardless of the
presence of PTH or VDIR/DNMTs interaction. MBD4 knockdown abrogated PTH-induced demethylation of CYP27B1 promoter, whereas knockdown of other MBD proteins had no such effect. ChIP assay on the CYP27B1 promoter showed that PHT-dependent DNA demethylation was coupled to histone acetylation and H3K4 methylation. However, these epigenetic histone modifications were abolished by depletion of MBD4. Furthermore, MBD4 mutant with deletion in the glycosylase catalytic domain failed to mediate PTH-induced DNA demethylation in MBD4e/e cells [31]. Thus, considering that DNA glycosylases are known to mediate active DNA demethylation [39,47], MBD4 appears to trigger CYP27B1 promoter demethylation through its DNA glycosylase activity (Fig. 12.6). Indeed, ChIP analyses showed that components of the base-excision DNA repair complex, including apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1), DNA ligase I and DNA polymerase b [48,49], were recruited to the CYP27B1 promoter simultaneously with MBD4 [31]. This suggests that after glycosylation by MBD4, DNA demethylation is completed through a base repair reaction.
MBD4 IS A DOWNSTREAM MEDIATOR IN PTH SIGNALING-INDUCED TRANSCRIPTIONAL DEREPRESSION Together with vitamin D, PTH plays a pivotal role in maintaining calcium homeostasis. PTH acts through
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REFERENCES
multiple signaling pathways that lead to subsequent activation of protein kinase A and protein kinase C (PKA and PKC, respectively) [50]. While 1,25(OH)2D3 represses CYP27B1 through the mechanisms of negative feedback regulation, PTH signaling stimulates CYP27B1 expression [5]. Experiments in vitro and in HEK293F cells have shown that activated PKC phosphorylates MBD4 at serine 165 and 262 residues and that PKC inhibitors attenuate the enzymatic activity of MBD4 [31]. In MBD4-deficient cells, MBD4 phosphorylation mutants consistently failed to trigger DNA demethylation in response to PTH. Finally, in MBD4e/e mice, PTH effects on the CYP27B1 expression, CYP27B1 promoter methylation and serum levels of 1a,25(OH)2D2 were significantly impaired [31]. Taken together, these data suggest that MBD4 acts as a downstream factor in the PTH signaling pathway. It is noteworthy that PTHinduced protein kinases appear to stimulate both MBD4-mediated derepression and VDIR-mediated enhancement of CYP27B1 expression (Fig. 12.6).
CONCLUSION Physiological effects of vitamin D are mediated by several mechanisms that may lead to transcriptional activation or repression of target genes. Mechanisms of the ligand-dependent activation have been intensively studied, and transcriptional induction by vitamin D/ VDR follows the well-established scheme consistent with other known nuclear hormone receptors. In contrast, the general view of the ligand-dependent repression by nuclear receptors still remains obscure and modes of transrepression by various receptors appear to be diverse. A growing number of identified transcriptional/epigenetic factors coregulate transcriptional functions of nuclear receptors and more novel coregulators, particularly those involved in transrepression, are expected to be found. In the present chapter, we have described a molecular basis of transrepression of CYP27B1 by vitamin-Dbound VDR that represents a novel mechanism of the ligand-dependent transrepression. An intriguing variety of regulatory actions of the vitamin D/VDR system suggests the possibility that additional mechanisms of transcriptional repression will be described, given the fact that new gene targets that are negatively regulated by vitamin D have emerged in recent years.
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C H A P T E R
13 Vitamin D and Wnt/b-Catenin Signaling Jose´ Manuel Gonza´lez-Sancho, Marı´a Jesu´s Larriba, Alberto Mun˜oz Instituto de Investigaciones Biome´dicas “Alberto Sols”, Consejo Superior de Investigaciones Cientı´ficas-Universidad Auto´noma de Madrid, Arturo Duperier 4, 28029 Madrid, Spain
INTRODUCTION The most active vitamin D3 metabolite 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3, calcitriol), is a pleiotropic hormone with cell-type-dependent effects on cell proliferation, survival, differentiation, and function. 1,25 (OH)2D3 exerts its actions via binding and modulation of the vitamin D receptor (VDR), a member of the nuclear receptor superfamily of transcriptional regulators. 1,25 (OH)2D3 triggers a complex network of gene regulatory (genomic) effects (see Chapters 7e12, 14). It also modulates in a transcription-independent manner the activity of several types of signaling enzymes and ion channels (non-genomic effects) (see Chapter 15). In addition, 1,25(OH)2D3/VDR regulates cell phenotype and function through crosstalk with other signal transduction pathways. Research in the last decade has revealed that an important mechanism of 1,25(OH)2D3/VDR action in certain tissues is related to its interaction with the Wnt/b-catenin pathway. In this chapter, we will review the evidence and comment on the significance of this interplay, which highlights the biological relevance of 1,25(OH)2D3 and opens new possibilities for the therapeutic use of vitamin D and its analogs.
WNT SIGNALING Wnt factors are a family of highly conserved secreted glycoproteins that regulate cell proliferation, differentiation, polarity, movement, and survival during development and in adult tissue homeostasis [1e3]. The 19 human Wnts are cystein-rich molecules with 350e400 amino acids and highly related structures [4]. In addition to bearing an N-terminal signal peptide, Wnts undergo N-glycosylation which is required for secretion. They are also modified by the addition of two
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10013-7
lipids, palmitic and palmitoleic acids, which may account for the hydrophobicity and poor solubility of Wnt proteins, and thus explain their low range distribution and predominantly autocrine-paracrine activity [5,6]. Lipid modification is also required for the signaling activity of the secreted protein [7]. Several membrane receptors and coreceptors for Wnt proteins have been described: Frizzled family members (ten in humans), low-density lipoprotein receptorrelated proteins (LRP) 5 and 6 (Arrow, in Drosophila), and RYK (Derailed, in Drosophila) and ROR2 families of single-pass transmembrane receptor tyrosine kinases. Upon binding to their receptors, Wnts trigger different signaling pathways: the so-called canonical or Wnt/bcatenin pathway and the b-catenin-independent, noncanonical pathways [3].
The Wnt/b-Catenin Pathway In normal epithelial cells, b-catenin protein is bound to the intracellular domain of the transmembrane E-cadherin protein in the intercellular adhesion structures adherens junctions. b-Catenin links E-cadherin to the actin cytoskeleton through its simultaneous binding to a-catenin, which is directly and indirectly bound to actin filaments. In the absence of Wnt factors b-catenin is mostly found at adherens junctions (Fig. 13.1A). Free b-catenin is phosphorylated by casein kinase 1 (CK1) and glycogen synthase kinase 3b (GSK3b) in a so-called destruction complex that also contains the scaffolding proteins Axin and APC. Phosphorylated b-catenin is ubiquitinated by the E3 ubiquitin ligase b-transducin repeat-containing protein (b-TrCP), and thus targeted for degradation by the proteasome [3] (Fig. 13.1A). Wnt binding to Frizzled and the subsequent formation of a ternary complex with LRP5/6 coreceptor induces b-catenin protein stabilization (Fig. 13.1B). Wnt binding
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13. VITAMIN D AND WNT/b-CATENIN SIGNALING
FIGURE 13.1 Wnt/b-catenin signaling pathway. (A) In the absence of Wnt factors, b-catenin (b-Cat) is located at plasma membrane adherens
junctions bound to E-cadherin. Free b-catenin is phosphorylated by GSK3b and CK1 in a destruction complex that includes also APC and Axin. This phosphorylation targets b-catenin for degradation by the proteasome. The transcription of Wnt/b-catenin target genes is inhibited by TCF/ LEF via the recruitment of corepressors such as Groucho(Gro)/TLE1. (B) Wnt factors promote the stabilization of cytosolic b-catenin through the inactivation of the destruction complex. b-Catenin enters the cell nucleus and associates with TCF/LEF proteins activating the transcription of Wnt/b-catenin target genes. Activated Wnt pathway reduces E-cadherin expression through the induction of transcriptional repressors of CDH1/E-cadherin gene and of proteases that degrade E-cadherin protein (for a review see [159]).
to Frizzled/LRP triggers coclustering of receptor complexes in signaling structures named LRP-signalosomes, which leads to phosphorylation of LRP by GSK3b and CK1g located in the vicinity of the plasma membrane [8,9]. Axin docking to the phosphorylated residues in LRP promotes the inactivation of the destruction complex and the accumulation of b-catenin (Fig. 13.1B). Then, a population of b-catenin molecules translocate into the cell nucleus, where they join members of the T-cell factor/lymphoid enhancer factor (TCF/LEF: TCF1, TCF2/LEF1, TCF3 and TCF4) family of transcription factors (Fig. 13.1B). TCF/LEFs bind to specific DNA sequences referred to as Wnt-responsive elements (WRE: CCTTTGA/TA/T). In the absence of Wnt signals, TCF/LEF proteins are mostly repressors, although in some cases they may activate transcription of their target genes. TCF/LEFs repress gene expression by interacting with corepressors such as Groucho/TLE1, which promote histone deacetylation and chromatin compaction (Fig. 13.1A). Binding of b-catenin to TCF/
LEFs displaces corepressors [10] and recruits a plethora of coactivators which trigger transcriptional activation of TCF/LEF-bound genes that were previously repressed (Fig. 13.1B). b-Catenin-associated coactivators include BCL9 and Pygo, Mediator complex, p300/CBP, and TRRAP/TIP60 histone acetyltransferases (HATs), MLL1/2 histone metyltransferases (HMTs), the SWI/ SNF family of ATPases for chromatin remodeling, telomerase, and the PAF1 complex for transcription elongation and histone modification [11e13]. Intriguingly, recent evidence suggests that during Wnt-induced transcription b-catenin and its coactivators cycle on and off the WRE with a 60 min periodicity and are replaced by Groucho/TLE1 [3,14,15]. Recently also, APC has been found to antagonize b-catenin/TCF-dependent transcription by promoting the exchange of coactivators with corepressors within the nucleus in a stepwise and oscillating manner [14]. Thus, both coactivators and corepressors appear to be active during b-catenin-mediated transcription.
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WNT SIGNALING
Numerous b-catenin/TCF or Wnt target genes have been identified in diverse biological systems and they are mostly cell- and context-specific [16]. Products of Wnt target genes have a large variety of biochemical functions including cell cycle kinase regulation, cell adhesion, hormone signaling, and transcription regulation. The plurality and diversity of these biochemical functions reflect the variety of different biological effects of the Wnt pathway, including activation of cell cycle progression and proliferation, inhibition of apoptosis, and regulation of embryonic development and cell differentiation, growth, and migration [16]. For a comprehensive, updated overview of b-catenin/TCF target genes, we recommend Roel Nusse’s Wnt homepage (http://www.stanford.edu/~rnusse/wntwindow. html). Deregulation or abnormal activation in adult life of the Wnt/b-catenin signaling pathway contributes to the emergence and progression of several types of human cancer. This is not surprising as the Wnt1 gene (Int1 was its first name) was discovered in mouse mammary carcinomas as a target of insertional activation by the mouse mammary tumor virus [17]. Cancer cells with mutationally activated Wnt pathway overexpress at least 20 target genes that activate proliferation including proto-oncogenes c-MYC, c-JUN, and CCND1/cyclin D1 [16].
Noncanonical Wnt Pathways Noncanonical Wnt pathways are very diverse and are still evolving into an increasing number of branches [18]. Among them, Wnt/planar cell polarity (PCP) and Wnt/calcium pathways are relatively better characterized. The Wnt/PCP pathway was described in Drosophila melanogaster. It regulates the polarity of epithelial cells within the plane of the epithelium, e.g. orienting Drosophila wing hairs or regulating the organization of the ommatidia in the fly eye [19]. In vertebrates, a similar pathway has been described regulating convergent extension movements during gastrulation or neurulation and migration of neural crest cells [20e22]. The core element of the Wnt/PCP pathway includes the activation of Rho GTPases such as RhoA, Rac, or Cdc42 that can activate more downstream mediators like Jun N-terminal kinase (JNK) or Rho kinase (ROCK). Our understanding of PCP signaling has increased dramatically in the past years and its role in diseases such as cancer is an area of intense research (reviewed in [23]). Some Wnts (e.g., Wnt5a or Wnt11) can induce a rapid release of intracellular calcium that depends on heterotrimeric G proteins [24] and the activation of the phosphatidylinositol cycle [25]. This increase in calcium
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concentration activates different intracellular calciumsensitive enzymes such as protein kinase C [26,27], calcium-calmodulin-dependent kinase II [28], and the phosphatase calcineurin, which subsequently activates the transcription factor NFAT [29,30]. This pathway is involved in dorsoventral patterning of early Xenopus laevis and zebrafish embryos [28,29,31], regulation of the Wnt/b-catenin pathway [32], tumor formation [33e35], and the regulation of epithelialemesenchymal transitions [36].
Wnt Inhibitors In line with its crucial roles in essential processes during development and adult life, Wnt action is antagonized or modulated by numerous molecules that act at different locations and by different mechanisms. Extracellular inhibitors are secreted molecules that either bind Wnt factors in solution, thus preventing their interaction with the plasma membrane receptors or inhibit Wnt signaling by binding to LRP5/6 (Fig. 13.2). The first group comprises secreted Frizzled receptor-related proteins (SFRPs), Wnt inhibitory factor-1 (WIF-1), Crescent, and Xenopus Cerberus [37]. Wnt inhibitors that bind LRP5/6 include the Dickkopf (Dkk) and WISE/SOST families [38e41] (Fig. 13.2). In addition, an increasing number of intracellular inhibitors of Wnt signaling are known. Some of them function in the cytosol such as Naked or Axin2/ Conductin [42e44], while others such as Chibby or ICAT block b-catenin action within the cell nucleus either by direct binding or by binding to b-catenin partners and the promotion of b-catenin nuclear export [45,46]. The Dickkopf gene family encodes secreted proteins of 255e350 amino acids and comprises four main members in vertebrates (Dkk-1 to -4) [41]. A distant Dkk family member, Dkk-L/sgy (Dickkopf-like protein 1 or soggy), has been described in vertebrates [47] and shows unique homology to Dkk-3. Dkk-1 and Dkk-4 proteins act as pure inhibitors of Wnt/b-catenin signaling. In contrast, Dkk-2 and Dkk-3 can activate or inhibit the pathway depending on the cellular context [41,48,49]. The inhibitory effect of Dkks may be induced by two mechanisms. First, Dkk binding to LRP5/6 can directly block the WntFrizzled-LRP interaction [50], and second, Dkks can form a ternary complex with LRP5/6 and another class of high-affinity Dkk receptors named Kremen (Krm1/2), which induces rapid endocytosis and removal of LRP5/ 6 from the plasma membrane, thereby blocking Wnt/bcatenin signaling [51,52]. Recent biochemical and genetic studies suggest that Dkk-1 disruption of the Wnt-induced Frizzled-LRP6 complex is a more likely mechanism [53], with Kremen playing a minor modulatory role in specific tissues [54].
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FIGURE 13.2 Extracellular inhibitors of Wnt signaling. WIF-1, SFRPs,
and Cerberus bind directly to Wnt factors and block their interaction with Frizzled receptors. DKK and WISE/SOST families bind to LRP coreceptors and prevent Wnt-Frizzled-LRP interaction and signaling. In addition, DKKs induce LRP endocytosis in the presence of Kremen proteins.
ANTAGONISM OF WNT/b-CATENIN PATHWAY BY 1,25(OH)2D3 IN COLON CANCER Wnt Signaling in Normal Colon and Colon Cancer Colon is lined with a specialized simple epithelium organized into crypts (deep invaginations) and a flat surface epithelium. The bottom half of the crypts hosts
highly proliferative progenitor cells named transit-amplifying cells which give rise to two different cell lineages: the absorptive enterocytes and the secretory cells (mucus-secreting goblet cells and hormone-secreting enteroendocrine cells). Maturation of progenitor cells coincides with upward migration. Upon reaching the surface epithelium, the differentiated cells undergo apoptosis and are shed into the lumen. The self-renewing capacity of the colon depends on the presence of stem cells at the crypt bottom [55,56]. The Wnt/b-catenin pathway is the main driving force behind the proliferation of epithelial cells in the colonic crypts and functional studies have confirmed that this pathway constitutes the master switch between proliferation and differentiation of the epithelial cells [57,58]. Thus, active Wnt signaling is essential for the maintenance of crypt progenitor compartments in the intestine. This is evidenced by mice lacking the Tcf4 transcription factor [59], by the conditional depletion of b-catenin from the intestinal epithelium [60,61], and by transgenic inhibition of extracellular Wnt signaling through Dkk-1 [62,63]. In all cases, a dramatic reduction of proliferative activity was observed. In the converse experiment, activating the Wnt pathway through transgenic expression of the Wnt agonist R-Spondin-1 resulted in a massive hyperproliferation of intestinal crypts [64]. Although Wnt signaling is essential to the normal physiology of the intestine, it was first characterized by its association with colorectal cancer, one of the most common cancers in industrialized countries. Fearon and Vogelstein [65] have proposed that colorectal cancer results from an ordered sequence of mutations in what is called the suppressor pathway. Invariably, the initiating mutation occurs in a gene (APC, CTNNB1/b-catenin, or AXIN2) that encodes for a protein involved in the Wnt/b-catenin pathway. Loss of the tumor suppressor APC is the signature of the great majority of human intestinal tumors, both in the hereditary familial adenomatous polyposis and in sporadic colorectal cancers [66e68]. In the rare cases where APC is not inactivated, human colon tumors arise from activating mutations in CTNNB1/b-catenin itself [69,70], or from loss-of-function mutations in AXIN2 [71]. As a common result, b-catenin accumulates in the nucleus, constitutively binds to TCF/LEF transcription factors and induces the expression of target genes mainly involved in cell proliferation, leading to the formation of benign yet long-lived adenomas. Subsequently, other mutations follow (e.g., in K-RAS, SMAD4, or TP53.), ultimately resulting in metastasizing carcinomas.
1,25(OH)2D3 Inhibits the Transcriptional Activity of b-Catenin/TCF Complexes Results from our group have demonstrated that 1,25(OH)2D3 and several analogs antagonize the
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ANTAGONISM OF WNT/b-CATENIN PATHWAY BY 1,25(OH)2D3 IN COLON CANCER
Wnt/b-catenin signaling pathway in human colorectal cancer cells. 1,25(OH)2D3 inhibits the transcriptional activity of b-catenin by two mechanisms. First, it rapidly increases the amount of VDR bound to b-catenin, thus reducing the interaction between b-catenin and TCF4 [72] (Fig. 13.3A). In this way, 1,25(OH)2D3 modulates TCF/LEF target genes in the opposite way to b-catenin: those induced by b-catenin/TCF4 such as c-MYC, TCF1, PPAR-d, or CD44 are repressed by 1,25(OH)2D3, while zonula occludens (ZO)-1, which is inhibited by b-catenin/TCF4, becomes activated by 1,25(OH)2D3 [72]. Second, 1,25(OH)2D3 induces b-catenin nuclear export and relocation to the plasma membrane adherens junctions. As b-catenin is no longer in the nucleus, transcription of TCF/LEF target genes is halted (Fig. 13.3B). This happens concomitantly to an increase in E-cadherin protein expression. 1,25(OH)2D3 induction of E-cadherin in SW480-ADH human colon cancer cells was observed soon after treatment and peaked at 48e72 h, and correlated with a change to a more differentiated phenotype [72]. Thus, 1,25(OH)2D3 may cause a reduction in nuclear b-catenin concentration by promoting its sequestration at the adherens junctions bound to the newly synthesized E-cadherin (Fig. 13.3B). Alternatively, 1,25(OH)2D3 might stimulate b-catenin nuclear export through yet-unknown mechanisms. Although APC has been proposed as a contributor to b-catenin nuclear export [73], the relocation of b-catenin takes place in SW480-ADH cells that express a truncated APC protein. We have shown that transient activation of the RhoA small GTPase is necessary for the induction by 1,25 (OH)2D3 of the expression of CDH1/E-cadherin and many other genes in several cell types [74]. Interestingly, RhoA activity is also required for the promotion of b-catenin nuclear export and for the inhibition of b-catenin/ TCF4 transcriptional activity and cell proliferation [74], suggesting a relationship between E-cadherin expression and nuclear export. Moreover, it is worth noticing that although the export of b-catenin out of the nucleus contributes to the inhibition of b-catenin/TCF4-mediated transcriptional activity in some cell lines, the global antagonism of 1,25(OH)2D3 on this signaling pathway must be independent of E-cadherin induction, as it takes place in LS-174T colon cancer cells that lack E-cadherin expression [72]. In addition to 1,25(OH)2D3, the vitamin D analogs EB1089, MC903, and KH1060 [72] and the superagonistic fluorinated CD578, WU515, and WY113 compounds [75] also inhibit b-catenin/TCF transcriptional activity in a strictly VDR-dependent fashion, as inhibition does not occur in SW480-R or SW620 cells that lack VDR expression. Moreover, the transcription factors SNAIL1 and SNAIL2 (also called SLUG), which repress VDR expression [76,77], abolish this effect in vitro and in vivo [77,78].
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These results indicate that 1,25(OH)2D3 down-regulates the Wnt/b-catenin signaling pathway, and may thus control the phenotype of colon epithelial cells. Upon b-catenin stabilization in colon cancer cells due to its own mutation or that of APC or AXIN2, binding to VDR may buffer its stimulatory action on TCF4 target genes, a protective effect which can be lost along with VDR expression during malignant progression linked to SNAIL1/SNAIL2 up-regulation. Additionally, we found that nuclear b-catenin transiently potentiates VDR transcriptional activity before b-catenin moves out of the nucleus and/or VDR is extinguished [72]. Shah et al. [79] confirmed and extended the finding of VDR/b-catenin interaction. These authors have characterized the interacting domains in VDR and b-catenin: the activator function-2 (AF-2) domain of VDR and the C-terminal region of b-catenin. Moreover, they showed that acetylation of b-catenin C-terminal region differentially regulates its ability to activate TCF/LEF or VDRregulated promoters. The mutation of a specific residue in the AF-2 domain, which renders a VDR that can bind hormone but is transcriptionally inactive in the context of classical coactivators, still allows interaction with b-catenin and ligand-dependent activation of VDREcontaining promoters. Interestingly, VDR antagonists, which block the recruitment by VDR of classical coactivators, do allow VDR to interact with b-catenin, suggesting that some ligands would permit those functions of VDR that involve b-catenin interaction [79]. In addition to human colon cancer cells, the inhibition of b-catenin/ TCF transcriptional activity by 1,25(OH)2D3 or its analogs QW and BTW has been found in rat Rama 37 mammary cells [80]. In these cells different b-catenin transcriptional complexes distinctly modulate the activation of the OPN/Osteopontin gene promoter by ligand-activated VDR: b-catenin/LEF1 enhances the activation while b-catenin/TCF4 diminishes it. Recently, Egan et al. [81] have reported that the inhibition of the transcriptional activity of b-catenin/TCF complexes by 1,25(OH)2D3 in colon cancer cells is enhanced by wildtype APC. In addition, these authors have shown that the VDR ligand lithocolic acid also inhibits b-catenin transcriptional activity but to a lesser extent than 1,25 (OH)2D3 and concordantly it is also less effective than 1,25(OH)2D3 in promoting VDR binding to b-catenin. Nuclear hormone receptors other than VDR have also been reported to regulate b-catenin transcriptional activity in several types of cells and tissues, reflecting a complex crosstalk between hormonal systems and Wnt signaling [82,83]. Notably, a novel mechanism of Wnt/b-catenin signaling pathway inhibition by 1,25(OH)2D3 has recently been reported by Kaler et al. [84]. These authors have shown that THP-1 macrophages activate the Wnt/ b-catenin signaling pathway in HCT116 and Hke-3 colon
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FIGURE 13.3 1,25(OH)2D3 inhibits Wnt/b-catenin signaling in colon cancer cells by several mechanisms. (A) Ligand-activated VDR binds to b-catenin and inhibits the formation of transcriptionally competent b-catenin/TCF4 complexes. (B) 1,25(OH)2D3 induces E-cadherin expression and promotes b-catenin nuclear export and relocation at plasma membrane adherens junctions bound to newly synthesized E-cadherin. (C) 1,25 (OH)2D3 induces the expression of the DKK-1 Wnt inhibitor. (D) 1,25(OH)2D3 inhibits IL-1b secretion by tumor-associated macrophages and thus blocks the IL-1b-dependent inhibition of GSK3b activity and subsequent b-catenin stabilization in cancer cells.
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carcinoma cells via the STAT-1-mediated production and secretion of interleukin (IL)-1b, which blocks GSK3b activity and hence increases b-catenin/TCF transcriptional activity and proliferation of carcinoma cells (Fig. 13.3D). This mechanism, which might contribute to the tumorigenic effect of tumor-associated macrophages in vivo, is repressed by 1,25(OH)2D3 through the inhibition of the constitutive activation of STAT-1 and the production of IL-1b in macrophages [84] (Fig. 13.3D).
1,25(OH)2D3 Regulates the Wnt Inhibitor DICKKOPF-1 We have reported that 1,25(OH)2D3 increases the level of DKK-1 RNA and protein in SW480-ADH human colon cancer cells and this effect depends on the presence of a transcription-competent VDR [85]. The slow kinetics of DKK-1 RNA accumulation and the lack of VDR binding to the promoter region that is activated by the hormone, together with the absence of effect on the half-life of DKK-1 RNA and the requirement of VDR transcriptional activity strongly suggest that 1,25 (OH)2D3 up-regulates the transcription of DKK-1 via intermediate proteins encoded by early 1,25(OH)2D3 target genes that remain uncharacterized. In addition, DKK-1 is up-regulated by ectopic E-cadherin in SW480-ADH cells, and a blocking antibody against Ecadherin inhibits 1,25(OH)2D3-mediated DKK-1 induction. These data indicate that the regulatory effect of 1,25(OH)2D3 is an indirect consequence of the induction of E-cadherin and the epithelial adhesive phenotype [85]. The induction of DKK-1 by 1,25(OH)2D3 constitutes yet another mechanism by which this hormone antagonizes the Wnt/b-catenin pathway (Fig. 13.3C). The existence of several mechanisms of Wnt/b-catenin signaling antagonism by 1,25(OH)2D3 reinforces its importance for the biology and the maintenance of the normal status of the colonic epithelium. Since most colorectal cancer cells have mutations in APC that render an active Wnt/b-catenin pathway, the relevance of DKK-1 induction by 1,25(OH)2D3 is uncertain. Interestingly, DKK-1 seems to have antitumoral effects independently of the antagonism of b-catenin/ TCF transcriptional activity in H28 and MS-1 mesothelioma, HeLa cervical, and JAR and JEG3 human placental choriocarcinoma cancer cells [86e88]. In line with this, we have shown that in DLD-1 colon cancer cells, which bear a truncated APC gene and so have a constitutively active Wnt/b-catenin pathway, transfection of DKK-1 decreases cell growth in vitro and tumor formation in immunodeficient mice [89]. Activation of the Jun N-terminal kinase (JNK) pathway is involved in some of these tumor suppressor effects [86,88]. Thus, DKK-1 may control signaling cascades
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independently of LRP5/6 and b-catenin [41,90]. Additionally, LRP5/6 may have b-catenin-independent effects under the control of DKK-1 [91]. These data indicate that DKK-1 can inhibit tumorigenesis by different mechanisms and that its induction might be of unforeseen importance for the anticancer action of 1,25 (OH)2D3. We and others have observed that, in addition to 1,25 (OH)2D3, the transcription of the DKK-1 gene is enhanced by b-catenin/TCF itself acting on several sites in the promoter region [92e94]. Our group also reported the down-regulation of DKK-1 in human colon cancer [93] indicating the loss of this negative feedback control of the Wnt/b-catenin pathway in this neoplasia. Reduced DKK-1 expression is due, at least in part, to promoter methylation, which is specifically found in 25% of advanced, less-differentiated tumors (Dukes’ stages C and D) [89]. Thus, the induction of DKK-1 expression by 1,25(OH)2D3 may restore DKK-1 antitumoral effects in those colon tumors in which DKK-1 down-regulation is not due to promoter methylation. The finding that DKK-1 expression is silenced in dedifferentiated colorectal tumors and the association of DKK-1 expression with the differentiated phenotype suggests that DKK-1 accumulation is not only concomitant with, but also plays an active role in the differentiation process. Accordingly, we have also demonstrated a significant correlation between the expression of VDR and DKK-1 in human colon cancer [85]. VDR is considered a marker of differentiation in this neoplasia [95,96] and its expression is lost during colon cancer progression together with that of E-cadherin, and presumably in parallel to the up-regulation of the transcription factors SNAIL1 and SNAIL2 that repress both genes [76,77,97].
1,25(OH)2D3 Represses DICKKOPF-4 and Induces TCF4 in Colon Cancer Cells DKK-4 protein has been described as an antagonist of Wnt/b-catenin signaling [47,52] and, like DKK-1, it has also been shown to be transcriptionally induced by this pathway [98,99]. DKK-4 is a weaker Wnt inhibitor than DKK-1, although its effect is increased if Kremen 2 is overexpressed ([52] and our unpublished data). In apparent contradiction, DKK-4 inhibits the Wnt/b-catenin pathway but is overexpressed in several pathological diseases including some types of cancer, inflammation, and schizophrenia [99e103]. We and others have found DKK-4 RNA expression in human colorectal tumors but not in adjacent normal tissue [99,102,104]. Moreover, DKK-4 RNA levels are already increased in patients with inflammatory bowel disease [105,106]. These results contrast with the common silencing of the DKK-4 gene in colon cancer cell lines
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that we and others [107] have found and that may be related to cell culture conditions. They also contrast with results from Baehs and colleagues [108] who have reported DKK-4 down-regulation in colorectal cancer. Notably, 1,25(OH)2D3 inhibits DKK-4 expression in human colorectal (SW480-ADH, Caco-2) and breast (MCF-7, MDA-MB-468, MDA-MB-453) cancer cell lines [81,99]. The mechanism of DKK-4 repression is unclear. While in SW480-ADH cells a direct transcriptional repression mediated by VDR binding to the promoter is found [99], in Caco-2 cells a mutant VDR deficient in DNA binding mediates similar repression of DKK-4 to wild-type VDR [81]. In SW480-ADH cells, 1,25(OH)2D3 promotes the binding of VDR and also of the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressor to a consensus sequence adjacent to the transcription initiation site and the abrogation of histone H4 acetylation. Interestingly, our group showed an inverse correlation between VDR and DKK-4 RNA levels in human colorectal tumors which suggests that the regulation of DKK-4 observed in cell lines also occurs in patients [99]. In order to characterize DKK-4 effects in colon cancer and thus the importance of its regulation by 1,25 (OH)2D3, we ectopically expressed DKK-4 in two human colon cancer cell lines: SW480-ADH, which expresses low levels of the endogenous gene, and DLD-1, in which its expression is not detected. Exogenous DKK-4 protein enhanced the migratory and invasive potential in vitro of both cell lines. Moreover, the migration and morphogenetic capacity of primary human microvascular endothelial cells (HMVEC) were robustly increased in the presence of conditioned medium from DKK-4-expressing cells or recombinant DKK-4 protein [99]. Thus, DKK-4 enhances the capacity of colon cancer cells to invade and to induce sustained angiogenesis, both essential for incipient neoplasias to grow and metastasize. These findings suggest that DKK-4 inhibition by 1,25(OH)2D3 could explain some of the antitumoral effects of the hormone in colon cancer. Although DKK-4 can act as a Wnt inhibitor, these data support new roles for this protein in human colon cancer, probably inducing b-catenin-independent actions during the progression of this neoplasia. In addition, they suggest that the tumorigenic actions of DKK-4 could overcome its weak inhibitory effect on the Wnt/bcatenin pathway [99]. The effects may be dose related: small amounts of DKK-4 may predominantly be inhibitory for Wnt signaling while higher levels may promote cell malignancy. Accordingly, Wnt antagonists other than DKK-4 are also up-regulated and may contribute to tumorigenesis in different systems [109e111]. Thus, up-regulation of DKK-4 and other Wnt inhibitors in some cancer cell types implicate them in roles other than the control of this signaling pathway.
Byers and colleagues [112] have recently reported a decreased expression of Tcf4 (product of the TCF7L2 gene in humans) in the mammary gland of Vdr-null mice. In addition, these authors found that 1,25 (OH)2D3 increases TCF4 RNA and protein levels in several human colon cancer cell lines by an indirect mechanism that requires de novo protein synthesis and is completely dependent on VDR. This induction is unique to TCF4, as other TCF/LEF family members are not up-regulated. Although it is generally assumed that binding of b-catenin to members of the TCF/LEF family is cancer-promoting, recent studies have indicated that TCF4 functions instead as a transcriptional repressor with growth inhibitory activity. Thus, RNAimediated disruption of TCF4 expression facilitates b-catenin activity and cell growth in both DLD-1 cells (APC mutation) and HCT116 cells (activating CTNNB1/b-catenin mutation) [113]. Also, recent data show that TCF4 expression is lost in human breast cancers but abundant in the surrounding normal tissue, indicating that TCF4 might be a tumor suppressor in this tissue [114]. Consequently, it is possible that the 1,25(OH)2D3/VDR-mediated increase in TCF4 may have a protective role in colon and breast cancer.
WNT AND 1,25(OH)2D3 IN THE BONE Wnt Signaling and Bone Homeostasis The observation that Wnt signaling is critical in bone biology has been a major development in the area over the past few years. Bone-marrow-derived mesenchymal stem cells (BMSCs) can potentially differentiate into adipocytes, chondrocytes, or osteoblasts. Although the precise orchestration of Wnt signaling during bone development is dependent on complex microenvironmental cues, data from several groups suggest that Wnt signaling is central to osteoblastogenesis while it represses differentiation of BMSCs to alternative cell types, such as adipocytes [115,116]. A number of different Wnt proteins play a role in bone formation. Wnt10be/e mice have decreased trabecular bone and serum osteocalcin [117], while transgenic mice that express Wnt10b in bone marrow show increased bone mass and strength [117]. The expression of Wnt10b in mesenchymal progenitors induced the expression of Runx2 and Osterix, two transcription factors associated with osteoblast differentiation, and stimulated osteoblastogenesis [117]. Likewise, Wnt3aþ/e and Wnt5aþ/e mice showed a decrease in bone mass [118] which associates with a reduced number of osteoblasts. Wnt5a does not activate Wnt/b-catenin signaling but a noncanonical pathway, and has been shown to induce osteoblastogenesis by inactivation of peroxisome
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proliferator-activated receptor-g (PPARg), a key adipogenic transcription factor, and activation of Runx2 [118]. Thus both canonical and noncanonical Wnt signaling pathways play a role in osteoblast differentiation and bone formation. The involvement of Wnt/b-catenin signaling in the control of bone biology is further supported by findings related to the LRP5 gene. Loss-of-function mutations in this gene were associated with low bone mass in the osteoporosis pseudoglioma syndrome (OPPG) [119], whereas a single amino-acid substitution (G171V) in the same gene was associated with a high bone mass state in two kindreds [120,121]. This mutation inhibits the ability of DKK-1 and potentially other proteins to bind to LRP5 and inhibit Wnt signaling. In line with this, mice with disruption of Lrp5 in all cells, similar to patients with OPPG, have a low bone mass phenotype, which is secondary to reduced osteoblast proliferation [122]. Recently, Yadav et al. [123] have demonstrated that Lrp5 has an important role in inhibiting serotonin biosynthesis in the gut. Serotonin had previously been implicated in the regulation of bone mass [124] and gut-specific deletion of Lrp5 was shown to result in a low bone mass phenotype [123]. In contrast, osteoblast-specific deletion of Lrp5 did not cause a similar defect. Thus, bone formation appears to be controlled by Lrp5-mediated serotonin inhibition in the intestine. It is possible that LRP6, rather than LRP5, is the critical coreceptor for Wnt signaling in bone. Consistent with this, the Lrp6 loss of function bone phenotype is much more severe than the Lrp5 loss of function phenotype. Although Lrp6e/e mice die at birth [125], Lrp6 heterozygous mice display reduced bone mass [126]. In this regard, a missense mutation in LRP6 that resulted in impaired Wnt signaling was reported in a family with autosomal dominant early coronary artery disease, metabolic risk factors, and osteoporosis [127]. Wnt extracellular inhibitors also have a relevant role in bone biology. Dkk-1 overexpression in transgenic mice resulted in severe osteopenia with a 49% reduction in the number of osteoblasts [128]. In contrast, mice engineered to lack Dkk-1 showed increased bone formation and bone mass [129]. In humans, elevated expression of DKK-1 in myeloma patients has been associated with bone disease [130]. SFRP1 has also been identified as a regulator of osteoblast and osteocyte survival, with effects on trabecular bone mass. Expression of SFRP1 increases with advancing osteoblast differentiation and peak expression of SFRP1 occurs at the preosteocyte stage [131]. Mechanistically, deletion of Sfrp1 in mice led to a decrease in osteoblast and osteocyte apoptosis with a resultant increase in osteocyte number in vivo [132].
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The Wnt inhibitor Sclerostin, product of the SOST (SclerOSTeosis) gene, also has a critical role in the control of bone mass. Sclerostin is produced exclusively by osteocytes and inhibits bone formation. Inactivating mutations in SOST result in sclerosteosis, a sclerosing bone dysplasia [133,134]. Targeted deletion of the Sost gene in mice results in increased bone formation and strength [135]; conversely transgenic mice overexpressing SOST have low bone mass [136]. In summary, activation of the Wnt/b-catenin pathway leads to increased bone mass while suppression results in bone loss.
1,25(OH)2D3 Promotes Wnt/b-Catenin Signaling in Osteoblasts The important role of 1,25(OH)2D3 in bone homeostasis is well known (see Chapters 16e18). The function of VDR in osteoblasts seems to be modulated as a function of the differentiation stage of the cells. Indeed, calvarial osteoblasts from Vdr-null mice displayed enhanced osteogenesis in vitro, suggesting that VDR activation in preosteoblasts suppresses bone formation [137], whereas the expression of a Vdr transgene in mature osteoblasts results in increased bone mass [138]. This differentiation-dependent effect may however be species-specific, as it is not observed in humans (Hans van Leeuwen, personal communication). Some of the effects of 1,25(OH)2D3 in the bone are reminiscent of those orchestrated by Wnt signaling suggesting a crosstalk between both pathways. Indeed, it has been shown that 1,25(OH)2D3 can induce binding of the VDR to a response element within the mouse Lrp5 gene in both primary osteoblasts and osteoblastic cell lines [139]. This interaction between 1,25(OH)2D3-activated VDR and the Lrp5 gene led to both a modification in chromatin structure within the mouse Lrp5 locus and the induction of Lrp5 mRNA transcripts in vivo as well as in vitro [139]. Thus, through the induction of Lrp5 expression, 1,25(OH)2D3 enhances Wnt signaling in mouse osteoblasts. Interestingly, whereas the regulatory region in the mouse Lrp5 gene is highly conserved in the human genome, the vitamin D response element is not [139], which argues against a conserved mechanism for 1,25(OH)2D3/Wnt signaling interaction in the bone. Studies using BMSCs derived from Vdr-null mice showed that ablation of Vdr did not alter osteoblastic differentiation [140]. However, when cultured under adipogenic conditions, these BMSCs expressed higher mRNA levels of PPARg and other markers of adipogenic differentiation, and also of mRNA encoding the Wnt inhibitors Dkk-1 and Sfrp2 [140]. This increase was, at least in part, due to ligand-dependent actions of the VDR, since 1,25(OH)2D3 suppressed Dkk-1 and Sfrp2 expression in wild-type cultures. Thus, it is
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FIGURE 13.4 The interplay between Wnt/b-catenin pathway and 1,25(OH)2D3/VDR depends on the cell or tissue type. In colon cancer cells, 1,25(OH)2D3/VDR inhibits b-catenin transcriptional activity and target genes through the inhibition of b-catenin/TCF interaction, the induction of b-catenin nuclear export, and the regulation of DKK-1, DKK-4, TCF4, and IL-1b. In mouse osteoblasts and bone marrow stem cells, 1,25 (OH)2D3/VDR up-regulates Wnt/b-catenin signaling through the induction of Lrp5 and the repression of Dkk-1 and Sfrp2. In human osteoblasts, 1,25(OH)2D3 potentiates SOST induction by bone morphogenetic proteins. Unliganded VDR mediates the activation of Wnt/b-catenin target genes in mouse keratinocytes promoting hair follicle differentiation and inhibiting the formation of infiltrative basal cell carcinomas. In human keratinocytes, 1,25(OH)2D3 represses WISE/SOSTDC1.
concluded that ligand-dependent actions of the VDR in mouse BMSCs promote canonical Wnt signaling by inhibiting the expression of Dkk-1 and Sfrp2 and inducing the expression of Lrp5, leading to a repression of adipogenic differentiation (Fig. 13.4). 1,25(OH)2D3 effects on Wnt signaling are, however, complex and cell- or tissue-dependent. 1,25(OH)2D3 enhances the induction of the negative regulator of bone formation and Wnt inhibitor SOST gene by bone morphogenetic protein in human osteoblasts [141] while it represses the SOST homolog WISE/SOSTDC1 gene in keratinocytes [142]. Similarly, Dkk-1 expression is inhibited by 1,25(OH)2D3 in BMSCs while it is induced by the hormone in colon cancer cells [85,140] (Fig. 13.4).
WNT AND 1,25(OH)2D3 IN THE SKIN Wnt Signaling in the Epidermis The Wnt/b-catenin signaling pathway controls stem cell differentiation in the skin [143,144]. b-Catenin
transcriptional activity promotes differentiation of the hair follicle lineages in embryonic and adult epidermis and, in certain circumstances, can expand the stem cell compartment [145,146]. In contrast, b-catenin inhibits sebaceous gland differentiation [143,147] and actively suppresses interfollicular epidermis differentiation in developing skin [145,146]. Sonic hedgehog and Jagged1, ligands of the Hedgehog and Notch signaling pathways, respectively, are b-catenin target genes in the epidermis and both pathways act downstream of b-catenin to induce stem cell expansion and follicle formation [148,149]. Consistent with the relation between aberrant Wnt signaling and cancer, deletion of b-catenin renders the epidermis resistant to chemically induced tumors [150]. Moreover, prolonged activation of b-catenin in transgenic mice is sufficient to induce benign hair follicular tumors (pilomatricomas and trichofolliculomas) [151], which regress when the pathway is no longer active [147]. Interestingly, human pilomatricomas have been found to harbor activating b-catenin mutations [152].
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REFERENCES
The Vitamin D Receptor Mediates Wnt/ b-Catenin Signaling in the Epidermis VDR is essential for adult epidermal homeostasis [153] and mutations in the VDR gene in humans result in familial 1,25(OH)2D3-resistant rickets, which can be associated with alopecia [154]. In vivo, the expression of a mutant Vdr that can bind b-catenin but not 1,25 (OH)2D3 rescues alopecia in Vdr-null mice, demonstrating ligand-independent functions of VDR in the skin [155]. Vdr-null mice fail to undergo the first postnatal hair cycle; instead, the hair follicles are converted to cysts of interfollicular epidermis. Two independent groups have shown that the absence of Vdr impairs Wnt/b-catenin signaling in keratinocytes and leads to alopecia [156,157]. Cianferotti and colleagues reported that Vdr ablation results in gradual depletion of the hair follicle stem cell pool which correlated with a failure of b-catenin to induce proliferation [156]. In contrast, Pa´lmer and colleagues [158] defend that the degeneration of Vdrnull follicles does not reflect a loss of follicle stem cells. Alternatively, they suggest that the failure to maintain the hair follicle may represent an inability of the stem cells to migrate along the follicle [158]. These authors also found that many genes that are up-regulated by active b-catenin contain vitamin D response elements and that several of them are induced independently of TCF/LEF. They conclude that unliganded VDR is a Wnt effector and that b-catenin acts as VDR coactivator in epidermal keratinocytes [157]. For these researchers, the primary role of the VDR/b-catenin interaction in the skin is to promote the transcription of genes associated with differentiation of the hair follicle lineages. Although these genes are activated by Wnt signals in the absence of 1,25(OH)2D3, the combined treatment has a synergic effect [157] (Fig. 13.4). Prolonged activation of b-catenin in the absence of VDR results in the development not of benign trichofolliculomas but of undifferentiated tumors resembling basal cell carcinomas [157]. Conversely, activation of bcatenin in the presence of VDR and the vitamin D analog EB1089 prevents b-catenin-induced formation of trichofolliculomas. Interestingly, human trichofolliculomas have cells with high levels of nuclear b-catenin and VDR, whereas infiltrative human basal cell carcinomas have high b-catenin levels and low VDR levels [157]. Thus, vitamin D analogs may be beneficial in the treatment of skin tumors in which the canonical Wnt pathway is activated inappropriately. A corollary to these results is that b-catenin can no longer be considered as chiefly an activator of TCF/LEF target genes. The interaction of b-catenin with VDR and possibly other transcription factors is likely to contribute to the pleiotropic effects of the Wnt pathway, which has different target genes in different cell types (Fig. 13.4).
CONCLUSIONS 1,25(OH)2D3 and VDR regulate the Wnt/b-catenin signaling pathway depending on the cell or tissue type: while 1,25(OH)2D3 and analogs inhibit b-catenin transcriptional activity and target genes in colon tumor cells, up-regulation of the pathway by either ligand-activated or unliganded VDR occurs in osteoblasts and keratinocytes. The mechanisms of Wnt signaling control by 1,25(OH)2D3 and VDR are diverse: direct VDR/b-catenin interaction, induction of b-catenin nuclear export, variable regulation of the expression of Wnt inhibitors such as DKK-1 and -4, WISE/SOSTDC1, SOST and Sfrp2, of the Wnt coreceptor Lrp5 (in mouse cells) or of the nuclear b-catenin partner TCF4, and repression of IL-1b production by stromal macrophages (Fig. 13.4).
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[136] G.G. Loots, M. Kneissel, H. Keller, M. Baptist, J. Chang, N.M. Collette, et al., Genomic deletion of a long-range bone enhancer misregulates sclerostin in Van Buchem disease, Genome Res. 15 (2005) 928e935. [137] K. Sooy, Y. Sabbagh, M.B. Demay, Osteoblasts lacking the vitamin D receptor display enhanced osteogenic potential in vitro, J. Cell Biochem. 94 (2005) 81e87. [138] E.M. Gardiner, P.A. Baldock, G.P. Thomas, N.A. Sims, N.K. Henderson, B. Hollis, et al., Increased formation and decreased resorption of bone in mice with elevated vitamin D receptor in mature cells of the osteoblastic lineage, Faseb J. 14 (2000) 1908e1916. [139] J.A. Fretz, L.A. Zella, S. Kim, N.K. Shevde, J.W. Pike, 1,25Dihydroxyvitamin D3 regulates the expression of lowdensity lipoprotein receptor-related protein 5 via deoxyribonucleic acid sequence elements located downstream of the start site of transcription, Mol. Endocrinol. 20 (2006) 2215e2230. [140] L. Cianferotti, M.B. Demay, VDR-mediated inhibition of DKK1 and SFRP2 suppresses adipogenic differentiation of murine bone marrow stromal cells, J. Cell Biochem. 101 (2007) 80e88. [141] M.K. Sutherland, J.C. Geoghegan, C. Yu, D.G. Winkler, J.A. Latham, Unique regulation of SOST, the sclerosteosis gene, by BMPs and steroid hormones in human osteoblasts, Bone 35 (2004) 448e454. [142] M.R. Haussler, C.A. Haussler, G.K. Whitfield, J.C. Hsieh, P.D. Thompson, T.K. Barthel, et al., The nuclear vitamin D receptor controls the expression of genes encoding factors which feed the “Fountain of Youth” to mediate healthful aging, J. Steroid. Biochem. Mol. Biol. 121 (2010) 88e97. [143] J. Huelsken, R. Vogel, B. Erdmann, G. Cotsarelis, W. Birchmeier, beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin, Cell 105 (2001) 533e545. [144] T. Andl, S.T. Reddy, T. Gaddapara, S.E. Millar, WNT signals are required for the initiation of hair follicle development, Dev. Cell 2 (2002) 643e653. [145] K. Narhi, E. Jarvinen, W. Birchmeier, M.M. Taketo, M.L. Mikkola, I. Thesleff, Sustained epithelial beta-catenin activity induces precocious hair development but disrupts hair follicle down-growth and hair shaft formation, Development 135 (2008) 1019e1028. [146] Y. Zhang, T. Andl, S.H. Yang, M. Teta, F. Liu, J.T. Seykora, et al., Activation of beta-catenin signaling programs embryonic epidermis to hair follicle fate, Development 135 (2008) 2161e2172. [147] C. Lo Celso, D.M. Prowse, F.M. Watt, Transient activation of beta-catenin signalling in adult mouse epidermis is sufficient to induce new hair follicles but continuous activation is required to maintain hair follicle tumours, Development 131 (2004) 1787e1799. [148] V. Silva-Vargas, C. Lo Celso, A. Giangreco, T. Ofstad, D.M. Prowse, K.M. Braun, et al., Beta-catenin and Hedgehog signal strength can specify number and location of hair follicles in adult epidermis without recruitment of bulge stem cells, Dev. Cell 9 (2005) 121e131. [149] S. Estrach, C.A. Ambler, C. Lo Celso, K. Hozumi, F.M. Watt, Jagged 1 is a beta-catenin target gene required for ectopic hair follicle formation in adult epidermis, Development 133 (2006) 4427e4438. [150] I. Malanchi, H. Peinado, D. Kassen, T. Hussenet, D. Metzger, P. Chambon, et al., Cutaneous cancer stem cell maintenance is dependent on beta-catenin signalling, Nature 452 (2008) 650e653.
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[151] U. Gat, R. DasGupta, L. Degenstein, E. Fuchs, De Novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin, Cell 95 (1998) 605e614. [152] E.F. Chan, U. Gat, J.M. McNiff, E. Fuchs, A common human skin tumour is caused by activating mutations in beta-catenin, Nat. Genet. 21 (1999) 410e413. [153] D.D. Bikle, Vitamin D and skin cancer, J. Nutr. 134 (2004) 3472Se3478S. [154] M.R. Hughes, P.J. Malloy, B.W. O’Malley, J.W. Pike, D. Feldman, Genetic defects of the 1,25-dihydroxyvitamin D3 receptor, J. Recept. Res. 11 (1991) 699e716. [155] K. Skorija, M. Cox, J.M. Sisk, D.R. Dowd, P.N. MacDonald, C.C. Thompson, et al., Ligand-independent actions of the vitamin D receptor maintain hair follicle homeostasis, Mol. Endocrinol. 19 (2005) 855e862.
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C H A P T E R
14 Vitamin D Response Element-binding Protein Thomas S. Lisse 1, Hong Chen 2, Mark S. Nanes 2, Martin Hewison 1, John S. Adams 1 1
UCLA-Orthopaedic Hospital Department of Orthopaedic Surgery, Orthopaedic Hospital Research Center and Molecular Biology Institute, David Geffen School of Medicine at UCLA, 615 Charles E. Young Drive South, Los Angeles, CA 90095, USA, 2 VA Medical Center and Division of Endocrinology, Metabolism, and Lipids, Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA
INTRODUCTION Since the publication of the last edition of this book, much has been learned regarding the cellular machinery for the intracellular trafficking, genomic action, and metabolism of vitamin D. This chapter will re-chronicle the discovery and expand what we know, and are learning about, a family of heterogeneous nuclear ribonucleoproteins (hnRNPs) and interacting heat shock proteins (hsps) that together constitute a recently recognized mode of control over the expression of vitamin-Dregulated genes. As so often happens in science and medicine, this discovery evolved from the molecular analysis of a successful “experiment of nature.” In the bone field, a recent fitting example was the identification by astute observers of extraordinarily dense bone in a young man that showed up for an X-ray in an emergency room following a car crash. This individual was discovered to have an activating mutation in the wnt coreceptor LRP5 [1], an event that has opened up a whole new vista on intracellular signaling pathways controlling bone formation [2]. The story to be told in this chapter opens with our investigation of such an “experiment of nature” in vitamin D homeostasis and a novel form of metabolic bone disease in several species of New World primates (NWP) resident at the Los Angeles Zoo in the mid1980s. In recent years it has continued with the discovery of (i) a previously unrecognized human form of vitamin-D-resistant rickets and the cytoplasmic and nuclear proteins that conspire to legislate a vitamin D receptor (VDR)-independent resistant state and (ii) a similar set of proteins that act to cause estrogen
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10014-9
resistance in adolescent NWPs. Most recently, the story has evolved to encompass the functions of these proteins in controlling gene expression, beyond the level of transcription, encompassing chromatin remodeling, splicing and microRNA (miRNA) handling as additional sites of action.
CURRENT VIEW OF STEROID/STEROL HORMONE ACTION The schema in Figure 14.1 encapsulates the current view of the points of control for the endocrine action of sterol/steroid hormones acting on steroid hormone receptors. In all three compartments, the general circulation, the plasma membrane and cytoplasm as well as the nucleus of the target cell, there are host proteins and
circulation hormone binding protein pharmacologic
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FIGURE 14.1 Proteins and small molecules (pharmacologics such as SERMs, left panel) control access of the sterol/steroid hormone to its receptor and modifying enzymes (middle panel) and subsequently legislate transcription of the hormone-controlled gene (right panel). Shown in yellow are the hsp-related co-facilitator proteins and the hnRNP-related comodulator proteins discussed in this chapter. Selective estrogen receptor modulators (SERMs). Please see color plate section.
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other small molecules that have the potential to alter hormone-directed gene expression. As far as the growing human skeleton is concerned, (i) the absolute amount of hormone in the blood, (ii) the amount and polymorphic form (i.e., affinity) of the circulating hormone-binding proteins (e.g., sex-hormone-binding globulin and vitamin-D-binding protein (DBP)) for the hormone and (iii) the presence of a circulating pharmacologics (i.e., selective estrogen receptor modulators) will collectively determine the robustness of the extracellular hormone signal to the target cell. At the target cell itself the (i) plasma membrane “acceptor” or docking proteins such as megalin, (ii) hormone receptors (e.g., estrogen receptor (ERa) and VDR), and (iii) hormone-activating or -modifying enzymes, like the enzyme aromatase for intracellular conversion of androgens to estrogens and the enzyme 24-hydroxylase (CYP24A1) for binding and catabolism of 25-hydroxylated vitamin D metabolites [3,4], can all regulate the amount of intracellular hormone available to its cognate receptor. In the nucleus, a balance between (i) allosteric changes in the ligand-binding domain, (ii) receptor coactivator and (iii) receptor corepressor turnover and recruitment all can act to legislate gene-specific expression in response to steroid hormones. The work described in this chapter focuses on two additional sets of regulatory proteins: (1) heterogeneous ribonucleoprotein (hnRNP)-related comodulators first discovered in vitamin-D- and estrogen-resistant NWPs (right panel Fig. 14.1) and heat-shock protein (hsp) intracellular hormone-binding proteins (co-facilitators: middle panel, Fig. 14.1) that act in concert with the hnRNP comodulators. Both comodulators and cofacilitators are now known to be present and active in normal human cells.
Coactivators and Corepressors In common with other nuclear receptors, the VDR is a ligand-dependent transcription factor that recognizes and binds to cis-acting vitamin D response elements (VDREs) in regulatory regions of target genes thereby modulating transcription [5]. However, besides the VDR, there are many other factors that act to “fine tune” 1,25(OH)2D3 responsiveness [6]. Indeed, work in the field of coactivators and corepressors has revolutionized the way we think about steroid-directed gene expression [7,8]. Based on changes in tertiary structure of receptor induced by ligand binding, receptor-interacting coactivators and corepressors serve as “master” recruiters of proteins in the transcriptome that integrate signals to turn up or turn down transcription of the target gene, respectively. Of particular interest to us is the discovery that many of these coregulators are promiscuous in their effect on other nuclear and even
cytoplasmic machines with which they physically interact and functionally influence. For example, because of their physical proximity to the chromatin remodeling and splicing machinery, it is not surprising to learn that coregulators can influence both events [9,10]. Because some coregulators traverse the nuclear membrane, it is also not surprising that they can also influence the translation of mRNAs. Such an example is the steroid hormone coregulator SRC-3. It has been recently shown [10] that SRC-3 can serve as a coactivator for the transfactor NF-kB and transcription of cytokine genes in the nucleus and then dampen translation of mRNAs for those same genes. This kind of multilevel control of gene expression will become a major theme in this chapter, except that we will be focusing on the multifunctionality imparted by proteins e the hnRNPs e that bind first in cis to nucleic acid. By virtue of their ability to be attracted to and interact with nucleic acid elements in both double strand (ds) and single strand (ss) format, we theorize that the hnRNP-directed events, influencing chromatin remodeling, transcription, splicing, and post-transcriptional mRNA handling, permit the same hnRNP to regulate expression of genes and gene products in serial fashion at geographically proximate sites in the cell and have termed this the “hop-scotch” model (see below).
Beyond Coregulators In the early-to-mid 1980s we investigated an outbreak of deforming rachitic bone disease in the emperor tamarin (Saguinas imperator) colony at the Los Angeles Zoo [11,12] (see below). This disease was most prevalent in females during their adolescent growth spurts often resulting in death from fracture, inanition, and/or infection prior to complete sexual maturity and procreation. One such proband is shown in the left panel of Figure 14.2. When surveyed, all genera of NWPs, excepting Aotus trivergatus or night monkey, demonstrated resistance to 1,25(OH)2D3 and 17b-estradiol (E2) with very high circulating levels of the two hormones [13,14]. Resolution of the vitamin-Dresistant state was accomplished by supplemental UVB radiation exposure leading to both increased serum 25hydroxyvitamin D (25D) and 1,25(OH)2D levels in afflicted primates [11]. The right panel of Figure 14.2 shows the same afflicted primate as a 20-year-old, UVBtreated adult great, great grandmother. Purification, identification, and characterization of the so-called VDRE-BP and estrogen response elementbinding protein (ERE-BP) as two distinct members of the hnRNP super-family, hnRNP C1/C2 and hnRNP Clike proteins, respectively, were accomplished during the formative years of our research program [15,16]. Subsequent molecular cloning and overexpression of these proteins and their human homologs showed
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FIGURE 14.2 An adolescent female tamarin monkey (left panel) with X-ray evidence of rachitic bone disease (middle left panel e metaphyseal cupping and fraying of the tibial plateau, and decreased bone opacity) compared to an age-matched normal control tamarin without rickets (middle right panel), and after 20 years of vitamin D supplementation (right panel). Please see color plate section.
them to be (1) dominant-negative inhibitors of 1,25 (OH)2D3-VDR- and E2-ERa-directed transcription [17] and to (2) interact with another category of intracellular chaperone proteins in the extended heat shock protein family that bind both ligands and other regulatory proteins to fine tune transcriptional control over 1,25(OH)2D3- and E2-driven genes in subhuman and human primates [18,19]. We have termed the latter duo of hsp-related, vitamin-D- and estrogen-binding proteins as the intracellular vitamin-D-binding protein (IDBP or constitutively expressed hsc70) and intracellular estrogen-binding protein (IEBP or hsp27), respectively. The following chapter describes in detail the observations that led to the discoveries of the IDBP and VDREBP, and the subsequent work that incorporated these novel proteins into the vitamin-D-signaling pathway.
NEW WORLD PRIMATES Early Primate Evolution In the Eocene period the great southern hemispheric landmass, Pangea, ruptured and its parts started moving away from one another. This tectonic event resulted in the separation of the American landmass and Madagascar from Africa. This continental drift occurred early in the process of primate evolution, trapping primordial primates in South America, Africa, and Madagascar, respectively. As a consequence, the three major primate infraorders, platyrrhines or New World primates (NWPs), catarrhines or Old World primates (OWPs) and lemurs, evolved independently of one another [20] (Fig. 14.3). Unlike Old World primates, including our own species, which have populated virtually every landmass on our planet, NWPs have remained confined to Central and South America for the last 50 million years. Compared to OWPs, especially some of the terrestrial species like gorilla, NWPs are smaller in stature, a trait well-suited to their lifestyle as plant-eating arboreal sunbathers residing in the canopy of the periequatorial rain forests of the Americas.
Simian Bone Disease The appearance of generalized metabolic bone disease in captive primates has been recognized for the last 170 years [21]. The disease, which has not been well-studied from a histopathological standpoint, carries the clinical and radiological stigmata of rickets and osteomalacia [22]. Compared to OWPs reared in captivity, NWPs are particularly susceptible to the disease. The disorder affects primarily young, growing female animals and results in muscle weakness, particularly of the muscles of mastication, skeletal fragility, and in many instances death of the affected individual from inanition and complications of fall-associated long bone fractures. Rachitic bone disease of this sort has long presented a problem to veterinarians caring for captive platyrrhines, particularly in North American and European zoos [23], because death of preadolescent and adolescent primates prior to sexual maturity severely limits on-site breeding programs. Because the disease was reported to be ameliorated by either the oral administration of vitamin D3 in large doses or by ultraviolet B irradiation of affected primates, it was presumed to be caused by vitamin D deficiency [23]. The frequent occurrence of rickets and osteomalacia in NWPs was also ascribed to the relative inability of NWPs to effectively employ vitamin D2 in their diet [24]; a similar observation had been made for chickens [25]. Using assay technology that does not discriminate between 25-hydroxylated vitamin D2 and vitamin D3 metabolites, investigators [26] determined that 25hydroxyvitamin D (25OHD) levels were 2e3-fold higher when platyrrhines were dosed with supplemental vitamin D3 than with vitamin D2. These data suggested that 25-hydroxylation of vitamin D substrate in NWPs was much more effective when vitamin D3 was employed as substrate. However, in the same study two species of OWPs demonstrated similar discrimination against vitamin D2, in favor of vitamin D3. In summary, these results seemed to indicate that all subhuman primates, whether Old or New World, were relatively resistant to vitamin D2 in terms of its ability to
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FIGURE 14.3 New World primate evolution. Shown in geographic terms is the independent evolution of the three primate infraorders, Platyrrhini, Catarrhini, and Lemuridae, in South America (the New World), Africa (the Old World), and Madagascar, respectively. The superorder Hominoidea represents the human precursors.
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engender an increase in serum levels of 25OHD. Although still debated, this discrepancy in the handling of vitamin D2 and vitamin D3 also seems to be operative in human primates [27]. Hay and colleagues [28] suggested that NWPs may transport 25OHD in the serum by means of proteins that are dissimilar from those encountered in Old World primate species. This hypothesis was disproved by Bouillon et al. in 1979 [29] who showed that the vitamin-D-binding protein was the major carrier of 25OHD in their serum of both New and Old World primates. The question of why NWPs were more susceptible to vitamin D deficiency than were catarrhines began to be answered with the detection of extraordinarily high circulating levels of the active vitamin D metabolite, 1,25-dihydroxyvitamin D in NWPs [11,30,31]. These data confirmed that NWPs were resistant to the vitamin D hormone.
An Outbreak of Rickets in New World Primates at the Los Angeles Zoo Twenty-five years ago an outbreak of rickets in adolescent, female NWPs residing at the Los Angeles Zoo was investigated. This “experiment of nature” and that of an adolescent human female with a similar phenotype [19] led to the discovery of a novel means for vitamin D and estrogen resistance in primates, including man. The index case in the original studies was a preadolescent NWP of the Emperor tamarin species (left-most panels, Fig. 14.2). When investigated radiographically, this tamarin and those like her displayed classical rickets complete with growth retardation, decreased bone opacity, and metaphyseal
cupping and fraying characteristic of rickets. In order to investigate this rachitic syndrome, we collected blood and urine from involved monkeys as well as from control, nonrachitic NWPs and OWPs. That comparison yielded a biochemical phenotype that was most remarkable with an elevated serum 1,25(OH)2D in rachitic NWPs [11]. In fact, with the exception of nocturnal primates in the genus Aotus, NWPs in all genera had vitamin D hormone levels ranging to 100-fold higher than that observed in OWPs including man [11,13,32]. In the initial analysis NWPs affected with rickets were those with the lowest 1,25(OH)2D levels, while their healthy counterparts were those with the highest serum 1,25(OH)2D. These data were interpreted to mean that most NWP genera were naturally resistant to the vitamin D hormone, and that the resistant state could be compensated by maintenance of high 1,25(OH)2D levels. If this was true, then an increase in the serum 1,25(OH)2D concentration in affected primates should result in biochemical compensation for the resistant state and resolution of their rachitic bone disease. Compensation of the vitamin-D-resistant state and a marked reduction in morbidity and mortality were accomplished by supplemental UVB radiation exposure for 6 months with a consequent further increase in both serum 25OHD and product 1,25(OH)2D levels in afflicted primates [33]. Estrogen resistance was eventually compensated by ovarian hypertrophy as the female primates matured to adulthood [Adams, unpublished]. In summary, NWPs are periequitorial sunbathers for a reason. NWPs require robust cutaneous vitamin D synthesis in order to push their 25OHD and 1,25(OH)2D levels high enough to effectively interact
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with the VDR. The question remained as to why these primates were resistant to all but the highest levels of the vitamin D hormone?
Investigation of the Biochemical Nature of Vitamin D Resistance in New World Primates In order to answer the question of resistance in NWPs, cultured fibroblasts and immortalized cell lines from both resistant and hormone-responsive New and Old World primates were used to track, step by step, the path taken by the vitamin D hormone from the serum DBP in the blood in route to the nucleus and transactivation of hormone-responsive genes [11,13,16,32e38]. It was shown that the movement of hormone from DBP, across the cell membrane and through the cytoplasm and nuclear membrane was indistinguishable from that observed in OWP cells. It was also determined that the ability of the NWP VDR to bind 1,25(OH)2D3 or 1,25(OH)2D2 and induce receptor dimerization with the retinoid X receptor (RXR) was normal. In fact, when removed from the intranuclear environment, and distinct to previous reports [31], the VDR in NWPs was similar to the Old World primate VDR in all biochemical and functional respects [38]. Dissimilar was the reduced ability of VDR-RXR complex to bind to its cognate cis element and transactivate genes. In order to elucidate nuclear receptor events in NWP cells, the nuclei were isolated and extracted, and then characterized. In addition to the VDR-RXR, it was determined that these extracts contained a second protein that was bound by the VDRE. This protein was coined the vitamin D response element-binding protein or VDRE-BP [16]. In electrophoretic mobility shift assays (EMSAs) using the consensus VDRE as probe, Old World primate cell extract contained only the VDRRXR bound to the VDRE probe, while the NWP extract contained two probe-reactive bands, one compatible with the VDR-RXR and a second, more pronounced VDRE-BP-VDRE band (Fig. 14.4A). This VDRE-BPVDRE binding reaction was specific, as the VDRE-BP was competed from VDRE probe by the addition of excess unlabeled VDRE. These data suggested that VDRE-BP might function as a dominant-negative inhibitor of receptor-response element binding by competing in trans with receptor. When recombinant human VDR and RXR were permitted to interact with increasing amounts of nuclear extract from vitamin-D-resistant cells containing a VDRE-BP or from normal vitamin-Dresponsive cells, the addition of more control extract only amplified the VDR-RXR-retarded probe on the gel [19]. By contrast, increasing amounts of the hormoneresistant extract competed away receptor-probe binding in favor of VDRE-BP-probe binding.
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It is by virtue of its ability to bind DNA that VDRE-BP can be distinguished from traditional corepressor proteins [39]. When overexpressed, VDRE-BP can effectively squelch VDR-directed transactivation. VDREdirected reporter activity in a subclone of wild-type Old World primate cells stably overexpressing the NWP VDRE-BP as well as in naturally hormone-resistant NWP cells was compared [40]. Stable overexpression of VDRE-BP substantially squelched luciferase activity compared to the untransfected, wild-type cell to levels observed in hormone-resistant NWP cells that naturally overexpress the protein. This is strong confirmatory evidence that when overexpressed in vivo, the VDRE-BP is the cause of vitamin D resistance in these monkeys.
NEW WORLD PRIMATE-LIKE VITAMIN D RESISTANCE IN MAN Studies of the vitamin D resistance state in NWPs have been achieved for over 15 years with the expectation that this would provide valuable insight into how steroid hormones regulate gene expression in human primates. Prior to this time it was unknown whether there existed a human phenocopy to the vitamin-Dresistant state in NWPs.
Index Case In 1993 a patient with the classical signs of type II hereditary vitamin-D-resistant rickets (HVDRRII), including alopecia, was reported [41] (see Chapter 65). The biochemical phenotype of this patient included: hypocalcemia (2.03 mmol/L corrected for albumin (normal range, 2.25e2.55 mmol/L)); raised serum alkaline phosphatase (1101 U/L (normal range, <300 U/L)); and raised circulating levels of 1,25(OH)2D (466e650 pmol/L (normal range, 48e156 pmol/L)). Despite this, the patient had normal VDR expression; sequence analyses indicated that the coding regions, as well as the 50 and 30 untranslated regions of the VDR gene were normal. Furthermore, when extracted from cells, VDR from the patient displayed normal binding capacity and affinity for 1,25(OH)2D3. Transfection of VDR cDNA from the patient into receptordeficient (human) CV-1 cells resulted in normal transactivation in response to 1,25(OH)2D3 [41]. However, in the subject’s own cells the VDR was incapable of stable nuclear localization and transactivation after exposure to hormone 1,25(OH)2D3. These data suggested that an “extra-VDR factor” was interfering with the nuclear translocation and transactivation of the VDR in cells from this patient. The patient was without evidence of any other form of steroid-thyroid-retinoid
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Panel A depicts an EMSA using consensus vitamin D response element as probe, showing the presence of a second transbinding protein, in addition to the VDR-RXR, in nuclear extracts of vitamin-D-resistant New World primate cells. Addition of excess unlabeled VDRE is shown in lanes 3 and 7. Addition of anti-RXR antibody (lane 4) supershifts, while addition of either anti-VDR antibody (lane 5) or New World primate nuclear extract containing the VDRE-BP (lane 6) competes away probe-VDR-RXR binding. Shown in panels B and C, respectively, is the ability of the VDRE-BP (arrow) and ERE-BP (double arrow) to bind to the osteopontin VDRE and consensus ERE (AGGTCACAGTGACCTG) in either a double-stranded (ds) or single-stranded (ss) format. Affinity-purified human VDRE-BP and ERE-BP (150 pg of protein) were as a source of response element-binding protein and a molar excess of response element-containing oligonucleotides, in either ds or ss format, as competitor DNA. Panel D shows the ability of nuclear extracts of B-lymphoblasts from a normal, vitamin-D-responsive host (control; lanes 1e3) and from a vitamin-D-resistant patient (HVDRR; lanes 4e7) to bind a consensus dsVDRE probe in EMSA. Control cell extracts display only a VDR-VDRE complex which is competed away by a molar excess of radioinert probe and supershifted by anti-VDR antibody (aby). HVDRR nuclear extracts exhibit the presence of both a VDRE-BP-VDRE and VDR-RXR-VDRE complex with the latter being competed away from dsVDRE probe with ssDNA bearing the AGGTCA repeat motif of a consensus RXRE and dsRNA or ssRNA bearing a similar motif (AGGUCA).
FIGURE 14.4
hormone resistance, and correction of rachitic bone disease was accomplished with high-dose 1,25(OH)2D3 treatment (12 mg/day) and calcium supplements (1e3 g/day), although the alopecia persisted. It was recently discovered [19,42] that the underlying cause of insensitivity to 1,25(OH)2D3 in this patient resulted from overexpression of VDRE-BP with similarity to hnRNP C1/C2 proteins that cause receptor-normal hormone resistance in NWPs.
Suppression of RXR-VDR-Mediated Transactivation and VDRE Binding in HVDRR Cell Lines To investigate the presence of such an “extra-VDR factor” in cells from this HVDRR patient, promoterreporter assays were carried out using normal and HVDRR fibroblasts as transfection recipients [19]. Data confirmed the suppression of VDRE-mediated transactivation in HVDRR cells in the presence or absence of wild-type VDR. Furthermore, in contrast to control cells, HVDRR fibroblasts showed no induction of luciferase activity following treatment with 10 nM 1,25(OH)2D3. Based on these data, it was hypothesized that cells
from the HVDRR patient constitutively overexpress a nuclear protein that competes with the VDR-RXR for binding to cis recognition sequences. To test this hypothesis subsequent assays were carried out using EBV-transformed HVDRR and control B-lymphocytes, which could be cultured in large volumes to facilitate maximal protein recovery from nuclear extracts [19]. In contrast to control lymphocytes, cells from the HVDRR patient did not demonstrate any significant antiproliferative response to increasing doses of 1,25(OH)2D3 following mitogen (PMA) activation. To investigate this further, EMSAs were carried out with recombinant human VDR and RXR and the 3-nucleotide-spaced direct repeat VDRE (VDRE-DR3; cis sequence AGGTCAcagAGGTCA) probe in the presence or absence of increasing amounts of nuclear extract from control or HVDRR cells. Although the consensus VDRE sequence was used in these experiments, the VDRE-BP is known to bind other nongeneric halfsites located in promoter regions of key vitamin D target genes [42]. Data showed that addition of the HVDRR nuclear extract competitively displaced RXR-VDR binding to the VDRE in a dose-dependent fashion. In contrast, nuclear extracts from control cells were without effect.
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These results were confirmed by densitometric analysis of EMSA band density, in which a stepwise diminishment of probe-VDR-RXR complex formation with increasing doses of HVDRR nuclear extract (4, 8, and 12 mg) was achieved compared to equivalent amounts of control extract. EMSAs were then carried out in the absence of exogenously added recombinant VDR and RXR [19], indicating that nuclear extracts from HVDRR cells contained a protein(s) that retarded the mobility of labeled VDRE-DR3. A similar observation was made when the highly homologous consensus RXRE was used as probe (AGGTCAgAGGTCA). The presence of this response element complex was specific for the nuclear extract of the HVDRR patient, as equimolar concentrations of the control nuclear extract did not retard the VDRE or RXRE. Likewise, the RXRE-binding complex was not observed in other vitamin-D-responsive cell lines such as OWP Rhesus monkey breast cells, Colobus lymphocytic cells, or MCF-7 human breast cancer cells. Neither the mobility nor the intensity of the retarded HVDRR complex was altered by antihuman RXR or VDR antibodies, and HVDRR cells showed wild-type levels of RXRa and RXRb expression [19]. Furthermore, preincubation of nuclear extracts with 100 nM 1,25(OH)2D3, retinoic acid (RAR ligand) or 9-cis retinoic acid (RXR ligand) had no effect on the mobility or intensity of the HVDRR complex [19]. These results confirmed that neither RXR, VDR, nor their respective ligands participated in retardation of the probe by HVDRR nuclear extracts. Furthermore, although the consensus response elements were utilized, interaction of the BPs with nongeneric response elements are likely to influence selectivity and hence gene transactivation.
Identification and Characterization of a Vitamin D Response Element-Binding Protein in Nuclear Extracts from HVDRR Cells The cis element specificity of the HVDRR EMSA complex was assessed by competition analyses. Data confirmed that RXRE and VDRE competed out the hormone response element-binding complex; however, consensus sequences for other types of cis elements such as the ERE showed no displacement of the response element-binding complex [19]. The peptide nature of the complex component(s) was confirmed using HVDRR nuclear extracts that had been digested by trypsin; unlike untreated HVDRR extracts, these preparations were unable to compete out the EMSA complex formed by recombinant VDR-RXR [19]. In these initial studies, the classification of the VDRE-BP was mistaken to be hnRNP A1 at the time. However in subsequent studies [42], VDRE probe binding by
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HVDRR nuclear extracts was competed away by the addition of anti-hnRNP C1/2 antibody, suggesting that, in a fashion similar to that in NWP cells, HVDRR cells expressed an hnRNP-related vitamin D response element in the hnRNP C family capable of interacting with the VDRE. Characterization of the VDRE-BP by Western blot analysis showed increased expression of hnRNP C1/2 immunoreactive protein in HVDRR cells. In Southwestern blots, nuclear extracts from HVDRR and control cells were subjected to SDS-PAGE and the separated proteins analyzed for their ability to bind radiolabeled VDRE probe and RXRE probe. A consensus VDRE-DR3 and RXRE-reactive protein was observed only in the nuclear extracts from the HVDRR cells. In view of the RNA-binding capacity of hnRNPs, further studies were carried out to assess VDRE-BP binding to single-stranded nucleotide sequences [19]. When HVDRR nuclear extracts were incubated with a singlestrand radiolabeled DNA probe consisting of the upper strand of the RXRE, the VDRE-BP complex could only be competed out using an excess of unlabeled single(ss) or double-stranded (ds) RXRE (Fig. 14.4D). Results presented above suggested that resistance to 1,25 (OH)2D3 in the HVDRR patient correlated with overexpression of a VDRE-BP. To confirm a functional link between the cis binding of VDRE-BP and VDR-mediated transactivation, further reporter studies were carried out using the VDR-positive HKC-8 human renal cell line cotransfected with expression constructs for human and the NWP dominant-negative-acting hnRNP C1/2, which show strong homology to one another [16]. In the presence of added exogenous 1,25(OH)2D3 both the New World and Old World (human) VDRE-BPs suppressed VDRE-directed transcription to a similar degree. The fact that the hypocalcemia and rachitic bone disease in the HVDRR patient were responsive to high-dose 1,25(OH)2D3 treatment [41] indicated that vitamin D resistance was not absolute, but instead was likely to be determined by the relative abundance of the hnRNP C1/2 VDRE-BP and competent (e.g., 1,25 (OH)2D3-liganded) RXR-VDR heterodimer present in the target cell. For example, if the balance in cis element binding favored VDRE-BP, either because of a relative abundance of this protein and/or relative lack of the competitive RXR-VDR, then one would predict a 1,25 (OH)2D3-reversible vitamin-D-resistant phenotype in vivo. In fact, this prediction was compatible with the patient’s response to treatment with high-dose 1,25 (OH)2D3 whereby hypocalcemia and rachitic bone disease were corrected as long as the serum 1,25 (OH)2D3 level remained high [41]. These data also suggest that it is the VDRE-DR3 cis element which legislates the antirachitic action of the hormone in bone. It is
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interesting to note that the HVDRR patient with elevated VDRE-BP also presented with alopecia. This is frequently observed in patients with VDR mutations leading to HVDRR and is also a hallmark in the VDR KO mouse [43]. However, studies have shown that this is due to ligand-independent effects of the VDR involving the hairless corepressor protein [44]. In contrast, failure of the patient’s alopecia to be improved with an increase in serum 1,25(OH)2D3 levels suggests that: (1) the VDRE-BP does participate in control of genes rendering the hairless phenotype within the keratinocyte component of the hair follicle in a ligandindependent manner [44e46]; (2) maternal levels of 1,25(OH)2D3 are not high enough for fetal hair development; (3) VDRE-BP affinity for cis elements is higher for “hair”-related genes; and/or (4) as suggested by previous data from HVDRR patients and VDR-ablated animals, the role of 1,25(OH)2D3-VDR in hair follicle development involves only a prenatal mechanism.
HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEINS (HNRNPS) Basic Biology of hnRNPs Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of ubiquitously expressed nuclear DNAand poly-U RNA-binding proteins that share a wide array of nucleic acid targets as well as cellular and molecular functions [47e49]. At least 20 of these proteins, designated A through U, are present in abundance in the nucleus and are found in association with proteins to form hnRNP particles. While all of the hnRNPs are present in the nucleus, some appear to shuttle between the nucleus and the cytoplasm with distinct functions. The hnRNP family members and isoforms serve to mediate pre-mRNA/mRNA processing, splicing, packaging, export and stability, and chromatin remodeling through locus control region-associated remodeling complexes, recombination, telomere lengthening, and protein translation [47]. Functionally, the hnRNP proteins are proposed to influence the clinical and biological outcomes of viral infections [50,51], spinal muscular atrophy [52], myotonic dystrophy [53], Alzheimer disease [54], tumorigenesis, and hormone resistance [19,42,47,55]. In vertebrates, hnRNP C belongs to a subfamily of hnRNPs as a major constituent of nucleated 40S hnRNP particles and pre-mRNA, with multiple transcript variants encoding at least two major isoforms (C1 and C2) acting as tetramers ((C1)3C2) [56,57]. C proteins are found in the nucleus during interphase, but during mitosis they are dispersed throughout the cell being involved in a complex network of regulatory
and signal transduction pathways (Fig. 14.5A). Newly synthesized pre-mRNA is delivered as ribonucleoprotein complexes termed premessenger ribonucleoparticles (pre-mRNPs), which is controlled by hnRNP C proteins. hnRNP C1/2 is also known to play pleiotropic roles affecting the early steps of spliceosome assembly and pre-mRNA splicing [58,59] and is capable of interacting with poly-U tracts in the 30 -UTR or 50 -UTR of mRNA to modulate the stability and level of translation of bound mRNA molecules to regulate cell proliferation, for example [60]. The activities of topoisomerase [61,62] and telomerase [63,64] are regulated by hnRNP C proteins, as is the cellular repair machinery after DNA damage [65]. hnRNP C1/2 is known to directly inhibit apoptosis by interacting with multiple proteins [66], although much of the antiapoptic mechanism still remains unknown. All of these hnRNP C protein functions are influenced by a collection of known and yet-to-bediscovered associating proteins that make up its interactome (Fig. 14.5B). One can imagine that any imbalance between hnRNP C1/C2 and interacting proteins can lead to clinical disorders, for example, myotonic dystrophy, known to be caused by aberrant nuclear retention of an mRNA species regulated by the C proteins, PTBP1, and U2AF1 [67]. Furthermore, hnRNP C1/2 protein interactions with (i) ATPdependent nuclear DNA/RNA helicases (e.g., DXP9; [61]), involved in the unwinding of double-stranded DNA and RNA in a 30 to 50 direction, or (ii) heat shock proteins with ATPase activity (e.g., HSP90AA1; [62]), may alter their secondary structure to influence other interactions with proteins and/or nucleic acids responsible for transcriptional or chromosomal separation effects. Interestingly, heat shock protein 70 (HSP70) is known to interact directly with the VDR [68], as well as hnRNP C1/2 associated HSP90AA1 [69], revealing a potential interaction with hsp70/90 organizing protein (HOP) another mode of intracellular regulation of vitamin D responses. The overlapping domains in Figure 14.5B suggest concurrence of functions related to hnRNP C in regulating the elongation of (capped) intron-containing transcripts and cotranscriptional mRNA splicing (e.g., NCBP1/2, HNRPD). An attempt to better understand the exact function of hnRNP C proteins was made by analyzing the targeted knockout of the gene in mice [70]. The authors made an important discovery in generating isogenic embryonic stem cell lines from the embryonic lethal animals. The findings indicate that the C1 and C2 hnRNPs are not necessary for any crucial step in mRNA biogenesis or cell viability, albeit the C proteins may influence the fidelity of the mRNA biogenesis process. Thus other RNA-binding proteins (see below) may substitute for the loss of hnRNP C.
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FIGURE 14.5 The regulatory/signaling pathway modules and protein interactome of hnRNP C1/C2. (A) BiblioSphere (http://www. genomatix.de) litminer pathway view of cocitations and biological/molecular relationships for HNRNPC. Legend and displayed pathway associations: open arrow represents regulation; bold line indicates transcription factor binding site match in target promoters; dot indicates expert (Genomatix) curation; M is a gene product part of a metabolic pathway; ST is a gene product part of a Genomatix curated signal transduction pathway; TF is a transcription factor. (B) Known and predicted protein interactions with hnRNP C1/C2 (STRING v8.3; [75]). Direct (physical) and indirect (functional) associations with hnRNP C1/C2 include proteins coupled to DNA repair/topoisomerase/telomerase, the spliceosome, and pre-mRNP/hnRNP complexes which regulate its major molecular functions. The relationships are derived from four source interaction data: genomic context (co-occurrence), high-throughput experiments, coexpression and data-mining from multiple organisms. DHX9 (DEAH box protein 9, ATP-dependent RNA helicase A), U2AF1 (splicing factor U2 auxiliary factor 35 kDa subunit), PTBP1 (polypyrimidine tract-binding protein 1), HNRPA1 (heterogeneous nuclear ribonucleoprotein A1), HNRPD (heterogeneous nuclear ribonucleoprotein D), SFRS9 (splicing factor, arginine/serine-rich 9), NCBP1/2 (nuclear cap-binding protein subunit 1/2), HSP90AA1 (heat shock protein HSP 90-alpha), PCBP1 (poly(rC)-binding protein 1). II. MECHANISMS OF ACTION
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FIGURE 14.6 Human hnRNP C family of nucleic-acid-binding proteins. Schematic representation of hnRNP C isoforms and homologous proteins. Each protein contains a single RNA recognition motif (RRM) which harbors two conserved octa- and hexa-peptide sequences denoted RNP1 and RNP-2, respectively. Certain proteins contain a delineated nuclear localization signal (NLS), a basic leucine zipper-like motif (bZLM), and an encompassing acidic auxiliary domain thought to be involved in proteineprotein interactions that serve to increase specificity of nucleotide binding. HnRNP C2 contains an additional 13 a.a. and is expressed at one-third the level of hnRNP C1. An oligomerization domain (the C1eC1 interaction domain (CID)) is also present in hnRNP C1/2. RALY contains the BKRF1 (EBNA-1 nuclear protein) epitope. Numbers represent a.a. position.
The hnRNP C proteins are divided into distinct domains: (1) an N-terminal RNA-binding domain (RBD), also called RNA-recognition motif (RRM), (2) a central variable domain, and (3) a C-terminal auxiliary domain (Fig. 14.6) [71]. It is possible that these individual regions on their own may have a unique structure and function. It is also possible that these regions may function cooperatively through interaction or physical linkage between individual regions. Alone, the RRM (90e100 amino acids (a.a.)) consists of four-stranded antiparallel b-sheets and two a-helices capable of binding poly-U RNA with equal affinity to that of the
intact protein [72]. Sequence alignment of the human RRM family of hnRNP C proteins reveals that the Nterminal RRM domains of related proteins are highly conserved (Fig. 14.6). This is probably related to the conserved function of this domain in different proteins, i.e. binding to hnRNA. In humans, the RRM domain is followed by a region that is highly variable in length. The major difference between hnRNP C2 and C1 isoforms in this region is the loss of a 13 a.a. peptide in C1. The presence of the additional 13 a.a. in hnRNP C2, due to alternative splicing, enhances the specificity between the RRM,
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components of the auxiliary domain, or the full-length protein and its ligand, allowing each to bind specifically to sequences with high affinity. An even larger peptide (28 residues) is absent in the human hnRNP C homolog RALY (see below); therefore, the middle part of the variable region may confer a protein-specific function. In addition, two alternatively spliced transcripts have been reported for HNRNPC, one of which is missing two exons encompassing part of the RRM and N-terminal portion of the variable region. The last isoform is void of the majority of the variable region in addition to partial deletion of the leucine zipper domain housing the nuclear localization signal (NLS). The biological significance of these remains to be resolved. The C1 nuclear retention signal (NRS) comprises 78 a.a. (residues 88e165) partially overlapping the auxiliary and RNA-binding domains. Furthermore, the C1/2 NRSs contain proline-rich regions, clusters of basic residues, and potential phosphorylation and glycosylation sites. The structure and function of the auxiliary domain (about 200 a.a.) are not well understood. However, the auxiliary domain contains the residues necessary for the binding of the RRM to specific ligands and a basic leucine zipper motif (bZLM) that may function as a novel RNA-binding motif. After the variable region, there is a section of 35e39 a.a. which consists of 11 basic residues (Arg/Lys) within the auxiliary domain of hnRNP C1/2. The conservation of basic residues in the hnRNP C1/2 and other homologous proteins (e.g., RALY) suggests a common function for these residues. One major difference is that the former, not the latter, has imperfectly related Lys-Ser-Gly sequences. Therefore, the basic region for C proteins is also named the KSG box, similar to the RGG box containing the Arg-Gly-Gly repeats found in hnRNP A1, G, K, and U. In addition, the KSG box of the C proteins may also bind RNA since in hnRNP U the RGG box is the sole RNA-binding element [47]. This basic region is followed by a leucine zipper motif (31 residues) which is characterized by the presence of hydrophobic residues at every seventh and fourth positions. The secondary structure is predicted to be an ahelix to form a coiled-coil structure. The leucine zipper may be involved in dimer formation since a deletion construct of hnRNP C1 terminating before the leucine zipper is monomeric [73]. Furthermore, the C-terminal segment of the auxiliary domain is rich in acidic residues and contains a putative NTP (nucleoside triphosphate)binding and phosphorylation sites. Lastly, the C-terminal 50 residues of the human hnRNP C proteins contain elements required for tetramer assembly, and possible cleavage sites by interleukin 1b-converting enzyme-like proteinase during apoptosis [74]. Database searches (NCBI and String v8.3; [75]) with human hnRNP C sequences identified many
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homologous proteins that contain similar RRM (Fig. 14.6). Among these are the hnRNP C-like 1, RALY, and RALY-like proteins. Human HNRPCL1 encodes the hnRNP C-like 1 protein, which is 92.8% similar to hnRNP C1 sequence, and currently only the C1 isoform equivalent has been reported. hnRNP Clike 1 may play a role in nucleosome assembly by neutralizing basic proteins such as A and B core hnRNPs based on similarity and potential interactions [76]. Human RNA-binding protein RALY (also called hnRNP C-like 2) has a similar overall structure to hnRNP C1, with a single RRM at the amino terminus that is 70% identical to C1 RRM, and an auxiliary domain which is 30% similar in structure. In heterokaryon assays, it was shown that the hRALY auxiliary domain contains nondelineated sequences which can retain the protein in the nucleus despite not having a well-defined classic NLS motif [77]. In addition, the RALY protein contains a 16-amino-acid in-frame insert in the variable region of the protein. As alluded to above, the C-terminal acidic region of hnRNP C is dissimilar to that of RALY, suggesting that this region has diverged to serve a different function for these two groups of proteins. One distinct feature of RALY is the presence of a series of glycines and serines. This stretch of the human RALY is an autoantigenic epitope and represents a newly identified class of evolutionarily conserved autoepitopes [78]. The human RALY was originally identified as an autoantigen in individuals infected with the Epstein-Barr virus [79]. An epitope recognized by B-cells, which cross-reacted with the BKRF1 protein (EBNA-1 nuclear protein) of Epstein-Barr virus was identified. Through mouse experiments, the Raly gene may play an important role in preimplantation development in the early embryo, and deletion of its coding sequence is responsible for embryonic lethality (or retarded growth) associated with the yellow (Ay) mutation at the mouse agouti locus [80]. Nevertheless, RALY is a probable RNA-binding protein and could be a heterogeneous nuclear ribonucleoprotein involved in pre-mRNA splicing. The human RALYL protein (also called hnRNP C-like 3) is roughly 70% similar to both the hnRNP C and RALY protein sequences, and is a predicted nucleotide-binding protein. It contains a consensus RRM domain and at least one coiled-coil dimerization tag. Furthermore, the coiled-coil sequence in the RALYL protein may represent a nucleotide-binding domain with an a-b plait structure, which consists of a ferredoxin-like (ba-b) fold found in RNA-binding domains of various ribonucleoproteins. Although information on the composition, function, and mechanisms of hnRNP C1/2 and homologous proteins have accumulated in literature, the proteine protein interactions that participate in their functional
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recruitment at their sites of action along genomic DNA or RNA are elusive. Emerging evidence shows that the RNA polymerase (RNAP) II transcription apparatus is involved in this process, particularly through the Cterminal domain (CTD) of RPB1, the largest subunit of RNAP II, which was shown to bind several regulatory complexes at various stages of transcription [81,82]. The CTD was found to interact with transcription elongation factors, chromatin-modifying complexes, premRNA maturation enzymes, and other proteins, providing a molecular basis for the coupling between transcription and RNA processing. The pre-mRNA processing events, including 50 end capping, splicing, and 30 end cleavage/polyadenylation, were shown to occur cotranscriptionally [83]. During the transcription cycle, CTD phosphorylation stimulates efficient recruitment of 50 and 30 end processing factors to the pre-mRNA. The splicing machinery is comprised of small-nuclear ribonucleoprotein particles (snRNPs), each composed of various proteins and a specific snRNA (U1, U2, U4, U5, and U6), which together form the spliceosome responsible for performing the catalytic events leading to exon excision and intron ligation [84]. Many proteins such as the hnRNPs and serine/arginine-rich (SR) proteins regulate splice site selection [85], and possibly the more upstream general transcriptional mechanism of similar genes. This in-series set of hnRNP functions was further clarified in accessing hnRNP C’s regulation of c-myc translation [86]. These studies showed that hnRNP C can modulate translation of c-myc mRNA in a cell cycle phase-dependent fashion; that is, the hnRNP C protein is able to relocalize from the nucleus during interphase to the cytoplasm at the G(2)/M phase to perform distinct functions in a “hop-scotch”-like fashion (see below). HNRNPS
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While work presented so far points to the role of hnRNPs in transcription, the work of others is beginning to show that these proteins subserve a number of functions both inside and out of the eukaryotic cell nucleus. There are at least four protein machines in the nucleus and another in the cytoplasm of the human cell dedicated to the process of making transcripts of genes and converting those transcripts to proteins: the nucleosome, transcription apparatus, spliceosome, and exosome in the nucleus, and the ribosome outside of the nuclear compartment. There is now growing evidence that the hnRNPs are involved in the operation of many of these [87]. The nucleosome is the basic structural element in chromatin [88]. It consists of 146 bp of DNA wound
tightly around a core of histones. The nucleosomes and their chromatin are differentially compacted into highly condensed heterochromatin and euchromatin, the latter of which is less densely packed and representative of the sections of the genome that are accessible to transcription activators. It is the insertion of ds and ss DNA-binding proteins, including members of the hnRNP family [89,90], that initiates decompaction and provides accessibility to genes destined for transcription mediated by acetylases and deacetylases. The transcription apparatus is classically viewed as the collection of proteins associated with RNA polymerase II (RNAPII). Included among these proteins are a number of transcription factors that interact with specific cis elements in the promoter regions of genes targeted for transcription. These transcription factors include the sterol/steroid hormone receptors as well as members of the hnRNP family of ss/ds DNA proteins [15,16,37], which can compete with the receptor proteins for the same cis element. DNA strand separation is a crucial element of the transcription process. Strand separation is initiated by RNAPII as it is tethered to the promoter by a bridge of coregulator proteins [91]. Once initiation is fixed, then the process of “elongation” ensues and the coregulator tether is broken or dislodged from its DNA anchor site in the promoter. The RNAPII is then free to make its way down the coding strand of the DNA template. This chain of events makes it possible for the human hnRNPs, which can occupy the VDRE in the promoter and squelch hormone-directed transactivation [40], to also interact with the CTD of RNAPII. By successfully competing in cis for the VDRE, the liganded VDR-RXR can dislodge the hnRNPC, permitting the coactivator tether to assemble and interact with RNAPII. Once initiation of transcription is fixed, it is possible for the VDRE-BP to reoccupy the VDRE releasing the VDRRXR-anchored tether and permitting elongation. On the other hand, if the VDRE-BP remains bound to the CTD of RNAPII, then by virtue of its ability to bind singlestrand RNA it has the potential to influence the processing, export, and translation of the transcript by binding to its 30 UTR (see below). The spliceosome is one of the largest of the nuclear machines. It functions to recognize the introneexon junctions in eukaryotic genes, remove the introns, and then variably re-“splice” the transcribed exons in a cellspecific manner. The spliceosome is comprised of 145 distinct proteins. At least 30 of these proteins, including 12 in the hnRNP family, have recognized functions outside of the realm of splicing [92]. The function of these many spliceosome-associated hnRNPs is unknown. It is almost certain that their ability to recognize both RNA and other proteins is key in demarcating the scission and resplice junctions in pre-mRNA as part of the socalled exoneexon junction complex or EJC [93]. There
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is now convincing evidence that the processes of transcription, splicing, 30 polyadenylation and 50 capping are functionally coupled and temporally coincident [94] with the CTD of RNAPII proposed to serve as a platform for the ordered assembly of the different families of premRNA processing machines [87]. An especially relevant example is the recent study of Abouef and coworkers [95]. They showed that the liganded glucocorticoid receptor (GR) exerted coincident control over RNAPII and alternative splicing of the transcript initiated under the influence of the ligand. It is possible that VDRE-reactive VDRE-BP and VDR-RXR coordinately alter transcription and splicing in a similar fashion. The exosome constitutes the protein machinery responsible for the export of mRNA from the nucleus [96]. These events do not occur in isolation. Rather they are functionally linked to upstream events in RNA splicing and processing and downstream processes like nonsense-mediated messenger decay [97]. The hnRNPs are proposed to be principal players in the exosome, acting as a connector between the 30 end of the mRNA and constituent proteins of the nuclear pore [98]. Hence, it is possible that the hnRNP C-related VDRE-BPs by virtue of their protein- and nucleic-acidbinding capacity, can be passed from promoter-to-RNAPII-to-30 end of mRNA-to-exosome and contribute to the delivery of that same gene’s mRNA to the cytoplasm, perhaps in a cell-cycle-dependent manner [86]. These hypotheses are yet to be confirmed but are under active investigation. The ribosome and the process of translation is the final stop for a mRNA. hnRNPs serve as ribosome recognition proteins [99]. They participate in parking transcripts with tRNAs anchored in the 30s subunit of the ribosome. Therefore, it is possible that the influence of the human VDRE-BP and the nonhuman primate homolog VDRE-BPs can extend all the way from the promoter of a gene to the translation of that gene’s transcript into a functional protein. It is now clear that the process of making and translating RNA is not the result of a series of independent processes, but the outcome of functionally linked simultaneous events. It is also clear, as demonstrated by the hnRNPs, that molecules thought once to be involved with specific single steps in the making, processing, and translating RNA are not limited to single steps in that chain of events. Finally, as exemplified by the 154 proteins of the spliceosome machinery of which only half have a clearly defined splicing function [92], it is also clear that there remains much to learn about how these various complex machines described above function in concert with one another. The fact that the hnRNPs associated with vitamin D and estrogen-mediated transcription can bind ssDNA (events 1 and 2, Fig. 14.7) as well as RNA (events 3, 4
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FIGURE 14.7 Depiction of DNA and RNA cis elements that are known to or proposed to be (miRNA) bound by hnRNPs (tear drop; see bottom right legend) during the process of mRNA generation. The numbered dots represent serial hnRNP-regulated events in the production and processing of a 1,25-dihydroxyvitamin D (1,25D)- or estradiol (E2)-induced mRNA. The insert at event 2 describes competition between the dominant negative-acting hnRNP (response element-binding protein, REBiP) and the dominant positive-acting liganded receptor for binding to the response element. The geographic proximity of these events within the cell leads us to propose a “hopscotch” model of hnRNPs moving rapidly from one cis site to another to exert dynamic control over hormone-regulated gene expression. Please see color plate section.
and 5, Fig. 14.7) with a 6 bp motif of AGGT/UCA in helical or extended format (Fig. 14.4) further indicates that the hnRNPenucleic acid interaction is specified by a “grooved patch” on the surface of the hnRNP [100]. The hnRNP proteins have also been shown to be able to bind to a DNA AGGTCA-like, 6-bp motif in both ds- and ss-strand format as well as to ssRNA with an AGGUCA-like cis motif [40]. In fact, our recent findings indicate that the VDRE-BP [42] and the ERE-BP [55], respectively, occupy the VDRE and ERE in vivo in advance of these cis elements binding the VDR and estrogen receptor (ER) and ER and VDR potentially attracting other elements of the chromatin-remodeling machinery. This has led us to theorize that interaction of these hnRNPs with a 6-bp AGGTCA-like motif exposed on the outside of the DNA double helix (10e12 bp/turn) in heterochromatin (chromatin condensed about histones) would be an efficient means of controlling the modification and opening of the chromatin structure for binding of transcriptionally active dimers, like the RXR-VDR and ERa-ERa, that require both sides of dsDNA in a helical format and at least a 12e15 bp cis element for secure binding. As such, we predict that hnRNPs have the potential to regulate conversion of heterochromatin at that site in the genome to a more exposed, unwound euchromatic state on which transcription can take place through the potential recruitment of epigenetic chromatin modifiers. That these VDRE-BP and ERE-BP hnRNPs can also reside on dsDNA and compete in trans with the
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receptor dimers for occupation of their cognate response elements (Fig. 14.4 and see insert, left panel, Fig. 14.7) suggests that the exact same protein that affects chromatin remodeling can inhibit hormoneinduced gene transactivation if the dsVDRE or dsERE to which it is binding is an enhancer of transcription. This would seem to be a highly energy-efficient mode of cocontrol of both chromatin modification and transcription, as the hnRNP need not move from its cis element through the process of chromatin remodeling and initial stages of transcription (events 1 and 2, right panel, Fig. 14.7). In similar fashion we predict that these same hnRNPs need only “hop-scotch” [86] a short distance from one cis site to another in the nuclear environment to control splicing of the pre-mRNAs of the same 1,25(OH)2D3and E2-regulated genes (event 3, right panel, Fig. 14.7). Preliminary data suggest that relative overexpression of the same hnRNP that regulates chromatin modification and transcription will bind to ssRNA and/or ssDNA (after strand separation) and alter splicing of the product of that transcriptional event (unpublished). Finally, we hypothesize that by virtue of their ability to bind RNA, hnRNPs can effectively bind to noncoding (nc) RNA sequences, including microRNAs (miRNAs) transcribed from either a UTR or an intron (event 4, right panel, Fig. 14.7). These potential actions of the hnRNPs could effectively (1) limit the processing of mRNAs in the nucleus (e.g., through interaction with decoy dsRNA) and/or (2) alter their hybridization with complimentary mRNA sequences in ss format in the nucleus or cell cytoplasm in advance of their translation. As a consequence, the hnRNP could take the ncRNA “out of play” and promote transcript translation to protein, conspiring with the effects rendered by the same hnRNP during chromatin remodeling, transcription, and splicing to formulate a composite functional response within the cell.
Conceptual and Functional Innovation Presented by the Response Element Binding Proteins The concept that a single trans-acting protein by proteineDNA binding (not proteineprotein interaction as would be the case with coactivators and corepressors) can influence the fate of a specific steroid hormone-regulated gene product in serial fashion at the level of chromatin modification, transcription, splicing as well as transcript handling in the nucleus and cytoplasm is an appealing prospect. This is especially so when one considers that humans are getting by with far fewer structural genes (20 000) than either a mouse (35 000) or a plant (75 000). The complexity of our genome at the level of expression must, therefore, be invested in
at least two powerful strategies: (1) the use of a single protein to function as a cog in more than a single cellular machine (Fig. 14.7) and (2) the ability of the human cell to balance multiple levels of control to legislate a finely tuned bioresponse for the host. We propose that one example of this kind of complexity is the human cell’s use of VDRE-BP and ERE-BP (see below), respectively, to legislate coordinated, 1,25(OH)2D3- and E2-directed gene expression in bone-forming cells by operating at multiple functional sites in that cell.
THE ESTROGEN RESPONSE ELEMENT BINDING PROTEIN (ERE-BP) AND THE INTRACELLULAR ESTROGEN BINDING PROTEIN (IEBP) Molecular Cloning and Expression in vitro and in vivo of the Comodulator ERE-BP Considering that NWPs exhibit resistance to other steroid hormones in addition to vitamin D, and consistent with the finding that cells from NWPs exhibit squelching of normal ER-ERE-directed transcription [37], analyses with nuclear estrogen signaling similar to those performed for characterization of the VDREBP were performed. In this case, concentrated nuclear extracts from estrogen-resistant NWP cells were applied to DNA affinity columns eluted with a linear gradient of KCl and fractions tested in EMSAs for their ability to bind to a consensus ERE. Subsequent Southwestern and Western blot analysis of a 0.5 M KCl fraction revealed a 40-kDa protein that was detectable by antiserum to hnRNP C-like protein [15]. Microsequencing of tryptic fragments from this protein enable us to clone and sequence a cDNA referred to as the NWP ERE-BP [15]. The corresponding human EREBP has yet to be cloned but we have shown that the NWP ERE-BP shares 98% homology with human HNRNPCL. The functionality of the ERE-BP has been demonstrated in studies both in vitro (Fig. 14.4C) and in vivo [17,101]. In the case of the latter, transgenic mouse lines with varying degrees of ERE-BP overexpression were generated and confirmed that tissue-specific overexpression of ERE-BP results in estrogen resistance in vivo [17]. Given the fundamental role of estrogen in reproduction, it was reasoned that global overexpression of an estrogen-resistance gene was likely to have lethal consequences. Therefore, the ERE-BP cDNA was expressed using a pKBA plasmid containing 2.6 kb of the whey acid protein (WAP) promoter, which is known to be expressed predominantly in mammary gland tissue. Overexpression did not appear to have any effect on expression of ERa and was specific for mammary
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gland tissue. All offspring born to wild-type and “low” ERE-BP-expressing mothers were viable, whereas onethird of all pups born to mothers with “intermediate” ERE-BP died before weaning. Significantly, 100% of offspring born to “high” ERE-BP mothers died by postnatal day 7 [17]. Further analysis showed that this mortality was due to starvation as a result of decreased ingestion of breast milk; a rebound increase in body weight was observed in pups born of high ERE-BPexpressing mothers if fostered to a wild-type mother. Histological analysis of mammary glands from either virgin or lactating female mice showed that ERE-BP overexpression resulted in decreased numbers of mammary gland ducts and branches. It was proposed that increased ERE-BP expression lead to E2 insensitivity and impaired breast development. Parallel studies performed in vitro indicated that overexpression of ERE-BP dramatically blunted responses to E2, although it was possible to induce significant ERaERE signaling in ERE-BP overexpressing cells if one used E2 in high amounts to drive breast development and lactation [17].
Estradiol and Tamoxifen Rescue of ERE-BPblunted Breast Development and Function Based on the above-described observation that E2 could rescue the ERE-BP-mediated suppression of ERa-driven gene expression in vitro, further studies using ERE-BP transgenic mice were performed in which animals were provided with slow-release subcutaneous E2 pellets to differentially elevate their serum E2 levels [55]. Whole-mount and histological analyses showed that exposure to higher supplemental doses of E2 “rescued” the impaired mammary gland development in ERE-BP-overexpressing mice. Rescue of mammary gland development in virgin nonlactating female mice as well as in lactating mothers was accomplished with E2, indicating that the dominant-negative effect of ERE-BP was active at different stages of breast function. Significantly and unexpectedly, these studies showed that tamoxifen was as effective as E2 in counteracting the inhibitory effects of ERE-BP, suggesting that the selective estrogen receptor modulator (SERM) is capable of exerting effects on estrogen action that are distinct from its effects on ERa or its accessory, coregulatory proteins. Further clarification of this observation was obtained in experiments done in vitro in which the ability of ERa and ERE-BP to interact with chromatin was assessed by ChIP analysis of wild-type and EREBP-overexpressing MCF-7 breast cells treated with E2 or tamoxifen [55]. As with the hnRNP C1/2 VDRE-BP [42], cells overexpressing ERE-BP showed dysregulation of the spatiotemporal patterns of ERE occupancy; both E2 and tamoxifen were able to correct this dysregulation
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and promote binding of ERa to a pS2 gene ERE. These findings confirm the reversible nature of the physical interaction between hnRNP C-like proteins and receptor cis regulatory elements.
COMPENSATION FOR THE DOMINANTNEGATIVE ACTING, RESPONSE ELEMENT-BINDING PROTEINS Intracellular Vitamin-D-Binding Proteins During the process of discovery of the VDRE-BPs in NWP cells, it was also observed that these cells were extraordinarily efficient at accumulating 25-hydroxylated vitamin D metabolites in the cytoplasmic space. Accumulation here was the result of expression of a second set of resistance-associated proteins. These intracellular vitamin-D-binding proteins [102,103], or IDBPs as they have come to be called, exhibit both high capacity and high affinity for 25-hydroxylated vitamin D metabolites. In fact, among all of the vitamin D metabolites that have been tested, the IDBP(s) purified from vitamin-D-resistant NWP cells binds 25OHD3 and 25-OHD2 most avidly [13,103]; in a competitive displacement assay using radioinert 25OHD3 as competitor and [3H]25-OHD3 as labeled ligand, the concentration of metabolite required to achieve half-maximal displacement of labeled hormone (EC50) was <1 nM. Although normally present in OWP cells, including human cells, these proteins can be overexpressed some 50-fold in NWP cells. These IDBPs are highly homologous to proteins in the heat shock protein-70 family [104]. Hsp-70 family of proteins are involved in signal transduction, cell cycle regulation, differentiation and programmed cell death, which could be partly a result of changes in intracellular VDR concentration known to be affected by hsp-70 [105]. The first four members of this family, IDBP1e4, have been cloned and characterized [16,40]. They bear a high degree of sequence identity with constitutively expressed human heat shock protein-70, endoplasmic reticulum-targeted glucose regulated protein 78 (grp78), mitochondrial-targeted grp 75, and heat shock inducible heat-shock protein-70. The general domain structure of the IDBPs has been described [104] and all contain an ATP-binding-ATPase domain ahead of a proteineprotein interaction domain. Some like IDBP-3 also harbor an N-terminal organelle-targeting domain. The association of the chaperone is sensitive to chaperone-specific substrates for activity. Preliminary studies indicate that the vitamin D ligand-binding domain resides in the middle of the molecule near the C-terminal aspect of the ATPase domain [18]. Adding to the dynamic regulation of vitamin D responses, it is
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known that hsp-70 is able to dispose of certain proteins via interaction with CHIP (carboxyl-terminus of hsp-70 interacting protein), which is an E3 ubiquitin ligase, to pass proteins to the cell’s proteolysis pathways [106]. Interesting, it is known that the hsp-70 family of proteins are known to bind directly to the VDR [105], suggesting that perhaps 25OHD3 binding prevents association with CHIP or the converse. These issues remain to be resolved. What are these IDBPs doing inside the hormone-resistant NWP cell? Two countervailing hypotheses were considered to explain the function of these proteins. One hypothesis held that these IDBPs were “sink” molecules that worked in cooperation with the VDRE-BP in the nucleus to exert vitamin D resistance by disallowing access of the hormone to the VDR and the nucleus of the cell. The opposing hypothesis held that these were “swim” molecules that actually promoted the delivery of ligand to the vitamin D receptor, improving the ability of the VDR to dimerize and bind to DNA, antagonizing the actions of the VDRE-BP that was overexpressed in NWP cells. In order to determine which of these hypotheses was correct, IDBP-1, the most abundant of these IDBPs, was stably overexpressed in vitamin-D-responsive (i.e., wild-type) OWP cells and demonstrated that IDBP-1 was protransactivating [18]; the endogenous transcriptional activity of three different 1,25(OH)2D3responsive genes, the vitamin D-24-hydroxylase, osteopontin and osteocalcin genes, in OWP wild-type cells was markedly enhanced. It was concluded from these studies, at least for the function of transactivation, that IDBP-1 was a “swim” molecule for the vitamin D hormone to promote delivery of ligand to the VDR; a clear role for IDBP-2 and IDBP-3 in this regard has not yet been determined. Considering the facts that NWPs are required to maintain very high serum levels of 1,25(OH)2D in order to avert rickets, it was hypothesized that the IDBPs, which are known to bind 25OHD more avidly than 1,25(OH)2D, will also promote the synthesis of the active vitamin D metabolite via promotion of the 25OHD-1hydroxylase. When human kidney cells expressing the 25OHD-1-hydroxylase gene were stably transfected with IDBP-1 and incubated with substrate 25OHD3, 1,25(OH)2D3 production went up 4e8-fold compared to untransfected, wild-type cells [107]. This increase in specific 25OHD-1-hydroxylase activity occurred independent of a change in expression of the CYP27B1hydroxylase gene [107]. In fact, current data (see below) now strongly indicate that this increase in hormone production is the result of the ability of IDBPs to promote the delivery of substrate 25OHD to the inner mitochondrial membrane where the 25OHD-1-hydroxylase is anchored.
A New Model for Intracellular Vitamin D Trafficking Dogma has held that sterol/steroid hormones like vitamin D, by nature of their lipid solubility, move through the plasma membrane of the target cell and are randomly maneuvered around the cell interior until they encounter another specific binding protein like the 25OHD-1-hydroxylase or the VDR with which to bind. Recent results, developed from a compendium of confocal imaging studies with fluorescently labeled IDBPs and vitamin D metabolites as well as with proteineprotein interaction experiments [40], indicate that translocation of the hormone from the extracellular fluid compartment to the specific intracellular destinations involves a series of proteineprotein interactions which involve the hsp70 family of IDBPs (Fig. 14.8). For example, we now know from the work of Willnow et al. [108,109] that vitamin D metabolites can enter some target cells such as renal proximal tubule cells via internalized vesicles. The vitamin D metabolites stay bound to DBP, which is in turn bound by megalin and cubulin, members of the LDL superfamily of proteins. Once inside the cell there is interaction between the C-terminal domain of megalin, which protrudes into the cytoplasm, and the N-terminal
“Extra-VDR” mechanisms associated with the regulation of target cell responses to 1,25(OH)2D3. (1) Serum transport of 1,25(OH)2D3 (1,25D3) and 25OHD3 (25D3) by vitamin-D-binding protein (DBP). (2) Cellular uptake of 1,25D3 and 25D3 either by diffusion of free molecules or via megalin-mediated receptor uptake (meg). (3) Intracellular transport of vitamin D metabolites by heatshock protein 70 (hsc70). (4) Expression of heterodimer partner, retinoid X receptor (RXR). (5) Expression of coactivator and corepressor proteins (Co.). (6) Chromatin reorganization via histone acetylation and deacetylation enzymes. (7) Occupancy of the vitamin D response element (VDRE) by vitamin D response element-binding protein (VDRE-BP). Vitamin-D-activating enzyme 1a-hydroxylase (CYP27B1). Please see color plate section.
FIGURE 14.8
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domain of IDBP-1 [40]; if one incubates IDBP-1 overexpressing cells with a fluorescently labeled 25-hydroxylated vitamin D metabolite, one will observe a significant increase in the accumulation of the labeled hormone in the cell cytoplasm.
The Heat Shock Protein-Related Cofacilitators and their Functional Relationship to the EREBP Comodulators The NWP gene product bearing homologous function to the IDBP for estrogen is the intracellular estrogen-binding protein or IEBP. Its human ortholog is heat-shock protein 27 (hsp27) and in recent work, coimmunoprecipitation, colocalization, yeast-twohybrid, and GST-pulldown analyses to compare and contrast the function of hsp27 IEBP with that of EREBP in E2-responsive human cells was performed [55]. Data indicate that hsp27 IEBP can reostatically regulate E2 signaling in the host cell by (1) acting as a protein chaperone for ERa and ERE-BP, (2) serving as a competitive, cytosolic decoy for E2 in the cytosol, and (3) binding to ERE-BP and ERa directly, modulating the temporal competitive occupancy of the ERE by ERa and ERE-BP. IEBP and ERE-BP were shown to interact to form a dynamic complex that cycles between the cytoplasm and nucleus during the course of normal estrogen signaling to the nucleus. For example, overexpression of either IEBP or ERE-BP results in disruption of the usual subcellular distribution of the IEBP-ERE-BP complex with concomitant dysregulation of occupancy of the ERE by ERa. It is reasoned that, when overexpressed, the IEBP and ERE-BP act in a concerted fashion to promote estrogen resistance, as they do in NWP species, but when expressed at normal (e.g., much lower) levels, as they are in OWPs including human cells, the IEBP and ERE-BP are crucial to normal control of genomic estrogen signaling; the latter of which appears to involve a physical association between the two proteins to form a complex which, in turn, is able to interact with both E2 and ERa in cytosolic and nuclear compartments of the target cell.
CONCLUSION As our knowledge base increases, the mechanisms underlying the control of vitamin D action and metabolism within target cells continues to expand, sometimes in unexpected directions. The discovery of the hnRNPrelated vitamin D response element-binding proteins in subhuman primates and in humans is one such unexpected direction. Although originally linked to conditions of vitamin D resistance, these nuclear proteins
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are now known to play a key role in normal VDR signaling. The potential that these multifunctional nucleic-acid-binding proteins can alter hormonedirected transcription of genes in addition to controlling the post-transcriptional fate of mRNAs transcribed from those genes indicates that the traditional views of the various nuclear machines (i.e., transcriptosome, spliceosome, etc.) working independently of one another is no longer tenable. Moreover, the ability of these multifunctional hnRNPs to recruit the expression of other classes of proteins, like the hsp70 family of IDBPs, not previously known to be important in the chaperoning of small molecules, is adding further complexity to the means by which cellular responsiveness to vitamin D is modulated. Similar mechanisms also appear to be central to signaling by other steroid hormones, providing further support for these hsp-related cofacilitators and hnRNP comodulators as key components of nuclear receptor signaling.
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C H A P T E R
15 Vitamin D Sterol/VDR Conformational Dynamics and Nongenomic Actions Mathew T. Mizwicki, Anthony W. Norman Department of Biochemistry and Division of Biomedical Sciences, University of California, Riverside, CA 92521, USA
1a,25(OH)2-VITAMIN D3 (1,25(OH)2D3) REGULATION OF GENOMIC VERSUS NONGENOMIC SIGNALING 1a,25(OH)2-vitamin D3 (1,25(OH)2D3, Fig. 15.1) functions to alter cell physiology and function via cis regulation of the genome and by regulating the activity of extranuclear signaling factors localized to the plasma membrane, the cytosol, and intracellular organelles (e.g., the endoplasmic reticulum) [1e3]. Thus the shortand long-term effects of 1,25(OH)2D3 in an in vitro, ex vivo, or in vivo environment involve dynamic, complex signaling mechanisms that include extranuclear (i.e., nongenomic) and nuclear (i.e., genomic) signaling, and their socialization with one another, referred to as cross-talk in the vitamin D field (i.e., feed-back, feedforward effects) [4,5]. The general objectives of this chapter are to provide an overview of 1,25(OH)2D3 nongenomic responses, define the molecular tools used to investigate them, and define the role the nuclear vitamin D receptor (VDR) plays in nongenomic, extranuclear signaling. The principal focus will be to describe the structural/molecular details associated with the ability of the VDR to send signals dynamically. In addition, we will speculate on how some of the 1,25(OH)2D3 extranuclear, nongenomic, rapid responses are initiated through binding the VDR.
a measurable response following the administration of 1,25(OH)2D3 [6]. It is generally accepted in the hormone/ nuclear receptor (NR) fields that hormone-dependent effects observed within seconds to minutes involve only extranuclear signaling cascades [1]. These rapid, cellular effects do not require transcription of new mRNA (i.e., 1,25(OH)2D3-VDR cis regulation of genes) [7,8]. The advancement of biophysical instrumentation, molecular biology reagents, and knockout/in animal models has ushered in additional methods used to differentiate genomic and nongenomic responses that are not strictly dependent on time. For example immunohistochemistry and confocal microscopy have allowed researchers to demonstrate that the VDR and other NRs can be found localized or trafficked to the plasma membrane and/or various cytoplasmic compartments in response to hormone (e.g., 1,25 (OH)2D3) treatment [9e13]. Importantly, knockdown or knockout technologies, in conjunction with phosphorspecific western analysis (e.g., activation of MAPK), whole-cell patch clamp, and/or PCR can now be used to verify the role VDR-dependent nongenomic signaling plays in a given cellular function. However the most telling, powerful and widely used technique to differentiate 1,25(OH)2D3 nongenomic and genomic signaling is synthetic chemistry [14e19].
Differentiating the Signals
VITAMIN D3 STEROL (VDS) CHEMISTRY
A number of different methods and postulates are used to differentiate the relative role of each type of 1,25 (OH)2D3-dependent response in a specific function being addressed. The first and most common criterion used to differentiate 1,25(OH)2D3 genomic and nongenomic signaling is the time required for one to observe
The chemistry of vitamin D3 sterols (VDS, e.g., 1,25 (OH)2D3, Fig. 15.1) is unique when compared to other nuclear receptor (NR) ligands that have been shown to regulate both genomic and nongenomic signaling through the same receptor molecule. The VDS chemistry allows for the molecule to rapidly sample many
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10015-0
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FIGURE 15.1 Biogenesis of the vitamin D3 seco-steroid. 7-Hydrocholesterol (7-DHC), also referred to as provitamin D3, in response to UVB irradiation, undergoes a photolytic ring opening to produce previtamin D3 (pre-D3). This process involves the scission of the carbon-10 (C10) and C-19 single bond. Next the vitamin D3 C10/C19 methylene and seco-steroid triene system are produced by a [1,7]-sigmatropic shift. Once hydroxylated at the 25-OH position, vitamin D3 becomes more bioavailable because 25-hydroxyvitamin D3 has a high affinity for the serum vitamin-D-binding protein (DBP) [180,181]. The DBP and the nuclear vitamin D receptor (VDR) are the two most well-defined binding proteins in the vitamin D endocrine system and have 1 nM affinity for 25(OH)D3 and 1,25(OH)2D3 respectively. Importantly, the conversion of 25(OH)D3 to 1,25(OH)2D3 reduces the affinity for DBP, while enhancing the affinity for the VDR [27].
different conformations and therefore 3D shapes (Figs 15.1 and 15.2) [15,20,21]. Thus unlike traditional steroid hormones changes in the chemistry of the vitamin D3 carbon scaffold significantly alter both the conformational dynamics and the physicochemical properties of the ligand. The major message conveyed in this section and article is that ultimately the chemical change to the VDS underpins the change in function of the ligand observed, whether that be in the setting of a nongenomic, genomic, and/or cross-talk assay platform.
The Conformational Dynamics of the VDS Seco-B-ring and A-rings Vitamin D3 (VD3) is a seco-steroid hormone derived from 7-dehydrocholesterol (7-DHC, Fig. 15.1) [22,23].
The term seco, refers to the fact that VD3 has a fractured B-ring. The seco-B-ring (i.e., pre-vitamin D3 (pre-D3, Fig. 15.1)) is generated in the skin in response to exposure of 7-dehydrochloesterol (7-DHC) to UVB irradiation (Fig. 15.1) [24]. Following the production of pre-D3, VD3 is formed by a thermal [1,7]-sigmatropic shift [25,26]. This shift allows for 360 rotation about the carbon-6,7 single bond (Fig. 15.2A), allowing the molecule to sample cisoid and transoid conformations (Fig. 15.1, compare pre-D3 and VD3). Said differently, the A-ring (Fig. 15.2B) is free to rotate above and below the CD-ring plain of the VD3 molecule [27] (Fig. 15.2A). Thus, the sun provides the UVB and the heat required for endogenous production of the vitamin D secoB-ring (i.e., triene system), thereby conferring upon the VD3 molecule increased entropy (i.e., disorder) when
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o
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o
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o o 163
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D H
H
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1,25D3 dot map (D) Population A (Pop. A)
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20-epi-1,25D3 (IE) dot map
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compared to its metabolic precursors 7-DHC and pre-D3 (Fig. 15.1 and Fig. 15.3). Opening of the B-ring also frees the A-ring of VD3 to equilibrate between the a-chair and b-chair conformations and the intermediates known to exist for cyclohexane in this process (e.g., twist boats and boats; Fig. 15.2B). The chair equilibrium has been shown by solution [28] and solid state [29] NMR and confirmed by computation [30] to be nearly identical for VD3, 25(OH)D3, and 1,25 (OH)2D3 (w50:50). The two chair conformations differ in the spatial orientation of the exo-cyclic methylene (C19) and the C1- and/or C3-OH groups (Fig. 15.2B). It is noted that the spatial orientation of the C19 atom in the 6-s-trans conformations is used to define the a-chair (below the CDring plain, referred to as the a-face, Fig. 15.2A) and the b-chair (above the CD-ring plain, referred to as the b-face, Fig. 15.2A) rotomers of VD3. A common synthetic modification used in vitamin D drug design is to remove C19 (i.e., the exo-cyclic methylene of VD3) [31]. The 19-nor modification makes it energetically easier for the molecule to transition between the 6-s-cis and 6-s-trans conformation (Fig. 15.2A) and the a-chair and b-chair A-ring chair flips (Fig. 15.2B) [32] (see next paragraph). The 19-nor synthetic modification also removes the most common site on the VD3 molecule known to react with reactive oxygen species (ROS) [33]. Perhaps these changes or those remaining unidentified account for the reduced calcemic effects of 19-nor, 1,25 (OH)2D3 analogs [34]. Thus the A-ring and seco-B-ring molecular dynamics provide potential useful and underinvestigated sites for future vitamin D therapeutic design when compared to the exhaustively modified and studied 1,25(OH)2D3 side-chain [14].
The VDS Side-chain Conformational Dynamics: Dot Maps The most conformationally dynamic region of the VDS carbon scaffold is the cholesterol-like side-chain
(Fig. 15.2C). Hydroxylation of the C25 atom of 25(OH) D3 by the cytochrome P-450 enzyme, commonly referred to as the 25-OHase [35], produces a more bioavailable form of vitamin D3, 25(OH)-vitamin D3 (25(OH)D3) (Figs 15.1 and 15.3). The most common method used to assess the molecular dynamics of this region of the VDS is a conformational search calculation, commonly referred to as a dot map calculation [36]. Early dot map calculations performed in the Okamura and Yamada laboratories indicated that the 25-OH group of 1,25(OH)2D3 samples a large steric space, where most of the dots (i.e., 3D position of the 25-OH group) were located to the right of the D-ring [37,38] (Fig. 15.2C). When C20 of 1,25(OH)2D3 is epimerized (20-epi-1,25 (OH)2D3; IE) it was observed in the dot map that the side-chain preferred occupying steric space above the CD-ring [36,39] (Fig. 15.2C), a relative steric position later shown by X-ray crystallography to be the location of the 25-OH group with respect to the CD-ring, when bound to the VDR ligand-binding pocket [40,41]. Thus the dot map and the X-ray side-chain orientation were consistent with the results provided by a number of laboratories demonstrating that IE was a superagonist VDR ligand, because its chemistry favors the “active” side-chain trajectory (i.e., C16-C17-C20-C22 dihedral; Fig. 15.2C). Superagonists are able to activate VDR transcription of reporter constructs and cell differentiation at a tenfold lower concentration with respect to 1,25 (OH)2D3 [42,43]. More recently we showed that the initial dot map parameters/conditions replicated the side-chain rotomers observed when bound to the serum vitamin-Dbinding protein (DBP, Pop. C; Fig. 15.2D) and the two overlapping VDR ligand-binding pockets (Pop. B and Pop. A; Fig. 15.2D) [37]. In the population A rotomers the 1,25(OH)2D3 side-chain (carbons C16,17,20,22) adopts a gaucheþ conformation. This rotomer is favored by the VDR-AP and is observed 73% of the time in the 1,25(OH)2D3 dot map calculation (262 total conformers
=
FIGURE 15.2 Illustration of the vitamin D sterol (VDS) conformational ensemble model. (A) The seco-B-ring: the diagram shows 360 rotation about the C6eC7 single bond of vitamin D3 (i.e., vitamin D sterol, VDS) triene. This is indicated by the curved arrow in the 1,25(OH)2D3 structure shown in the upper left of the figure. Specific C5-C6-C7-C8 dihedral angles are highlighted in the figure that have been observed in the DBP and VDR X-ray complexes and also in the in silico flexible docking complexes obtained when 1,25(OH)2D3 was docked to the VDR genomic pocket (VDR-GP) and the VDR alternative ligand-binding pocket (VDR-AP, see text for more details). These conformations were first generated by a conformational search calculation using a modified PC_Model v9.2 GMMX protocol (see [1,44,45]). Thus the “active” seco-B-ring conformations of 25(OH)D3 and 1,25(OH)2D3 and other vitamin D sterols, can be produced in silico [1,37,44]. (B) The A-ring: because of the [1,7]-sigmatropic shift, the A-ring of 1,25(OH)2D3 is capable of undergoing a chair flip reorienting the axial and equatorial position of the C1 and/or C3 hydroxyl groups and orientation of the C19 methylene, either above (b-chair) or below (a-chair) the plain of the CD-ring (see panel A). The equilibrium between the two chairs is roughly 50:50, but can be altered by natural C-3 or synthetic C-1 epimerization, see text. (C) The side-chain: the cholesterol-like side-chain contains only sp3-hydridized sigma bonds and is therefore highly flexible. This flexibility was first simulated by Midland and Okamura [36] and referred to as a dot map. The black lines in the dot map diagrams highlight the side-chain region that must be populated [182] in order to form hydrogen bonds with H305 and H397 of the VDR (see Fig. 15.9B). (D) Population distribution of 1,25(OH)2D3 side-chain rotomers observed in the dot map calculation; w95% of the side-chain conformers possessed C16-C17-C20-C22 dihedral angles that fall into three 20 dihedral angle windows [37]. Empirical (X-ray) and theoretical (computation) structureefunction analyses show that Population A (Pop. A) is the most abundantly observed side-chain conformation in the VDR alternative ligand-binding pocket (VDR-AP, see text), Pop. B the VDR genomic pocket (VDR-GP, see Fig. 15.9A) or X-ray ligand-binding pocket and Pop. C the serum vitamin-D-binding protein (DBP) [1,37].
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DIET H
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H
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24R(OH)VD3
hv
HO
7-DHC
OH
H
H
HO
HO H H
H
OH
H
8 -OH-3-oxo 9,10-seco 25D3 H
19-nor-10-oxoVD3 OH
O
5(E)25D3
H
OH H
H
OH
OH
H
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OH
OH
25D3 H
19-nor-10-oxo25D3
O
OH
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24R(OH)25D3 23S(OH)25D3
H
OH
OH H
OH
OH
H
H
O
HO
H
OH
10-oxo24R(OH)25D3
H
HO
HO
H H
OH OH
25,26(OH)225D3
1,25D3 HO
OH
H OH
23,24-dehydro25D3 25R(OH)26,23S-peroxyLactone25D3
O OH O
OH
O
O
HO
HO
OH
25R(OH)26,23S-lactol25D3
HO
OH
3-epi 1,25D3
OH
OH OH
23S,25R,26(OH)31,25D3
OH
HO
OH
O
HO
23-OH Tetranor25D3 HO
H
HO
OH
24(OH)Trinor25D3
23S(OH)1,25D3 OH
24-oxo1,25D3
OH
O
OH
OH
HO
25R(OH)26,23S-lactone25D3 HO
H OH
O
HO
O O
OH
OH
HO
OH
HO
OH
23S(OH)-24-oxo1,25D3
OH
OH OH
25R,26(OH)21,25D3
24(OH)1,25D3
23S,25R,26(OH)325D3
H
OH
OH
OH OH
HO
H
HO
OH
HO
HO
24R(OH)1,25D3
24-oxo25D3
24(OH)25D3 HO
HO
HO
H
O
OH
OH
23S,25R(OH)225D3 HO
H
OH
OH OH
OH
H OH
O OH
OH
H
OH
H
OH
HO
OH
23-oxo25D3
H
1 ,24R(OH)2VD3
H
HO
O
OH
OH
H
HO
O
O
OH
25R(OH)26,23S-lactolOH 1,25D3
HO OH
23(OH)Tetranor1,25D3
OH
HO
O
Calcitroic acid OH
II. MECHANISMS OF ACTION
HO
OH
OH
O
O
OH HO
OH OH
23S-OH-24-oxo1,25D3
OH
23-COOH Tetranor25D3
OH
HO
25R(OH)26,23S-lactoneOH 1,25D3
276
15. VITAMIN D STEROL/VDR CONFORMATIONAL DYNAMICS AND NONGENOMIC ACTIONS
in this particular calculation). In contrast, the side-chain conformers observed in the X-ray crystallographic analysis are low in population in the 1,25(OH)2D3 dot map [37] (Pop. B; Fig. 15.2D). It is noted that oxidation (i.e., metabolism; Fig. 15.3) of the 25(OH)D3 and 1,25 (OH)2D3 side-chain C23 or C24 atoms (Fig. 15.3) reduces the free conformational space (i.e., entropy) sampled by the side-chain [32]. Thus in vivo 25(OH)D3 and 1,25 (OH)2D3 are the most conformationally dynamic of the over 30 natural VDS isolated and characterized in vivo (Fig. 15.3) [14]. This hard fact is supportive of the concept that the molecular dynamics of 1,25(OH)2D3 underpins its ability to stimulate both genomic and nongenomic functions. It also strongly suggests that assuming the conformation of 1,25(OH)2D3 observed in the VDR X-ray structure is the only physiologically relevant shape of 1,25(OH)2D3 is incorrect.
The VDS Conformational Ensemble A limitation of the original dot map protocol was that only the transhydrindane CD-ring/side-chain portion of the VDS could be used in the conformational search calculation. Fortunately, since the dot map protocols were first published, advancements in computational hardware/software have allowed for inclusion of the entire VDS molecule in the same type of conformational search calculation [44,45]. This type of calculation allows for all combinations of A-ring, seco-B-ring, and sidechain rotomers to be generated that range from 6-s-cis planar-like to 6-s-trans bowl and planar-like shapes (Fig. 15.4). In fact, the 1,25(OH)2D3 conformational ensemble generated using PC_Model v9.2 was recently combined with a flexible docking simulation (Discovery Studio v2.0) to blindly replicating the 1,25(OH)2D3 pose, energetics in molecular dynamics observed in the 1,25 (OH)2D3-VDR (aa118-427; D165-215) X-ray co-crystal [44,45]. Thus, the combination of PC_Model and DS2.0 may be useful in future VDR drug design efforts. Thus calculations that consider the dynamics of the vitamin D sterol A-ring, seco-B-ring and side-chain, demonstrate that oxidation of vitamin D3 (i.e., the
production of 1,25(OH)2D3 (Fig. 15.3)) in the liver (25OHase) and the kidney (1-OHase) does not alter significantly the conformational dynamics of the vitamin D sterol molecule when compared to vitamin D3. As stated previously this is not true for photolysis of the seco-Bring. Importantly, the reactions required for conversion of 7-DHC to 1,25(OH)2D3 (Fig. 15.1) have been observed in other tissues, namely the skin [46], where biogenesis begins and can be continued to form hormone D (i.e., 1,25(OH)2D3). Metabolic epimerization of the C3-OH group of 1,25 (OH)2D3, by an as yet to be determined epimerase, forms 3-epi-1,25(OH)2D3 (Fig. 15.3) and alters the A-ring chairchair equilibrium (Fig. 15.2B) [47e49]. According to computational studies a 3a-OH group causes the 1,25 (OH)2D3 A-ring to favor the a-chair conformation, given the introduction of a syn-1,3-diol intramolecular hydrogen bond (see Fig. 15.2B) [50,51]. 3-Epi-1,25 (OH)2D3 can function as a VDR agonist ligand, is produced in vivo [49] and shows activity in multiple cell types [52,53]. Thus mimicking the natural C3-epimerization may prove to be a useful modification in future drug design efforts in the vitamin D field.
Nongenomic Agonist Structural Features and Molecular Dynamics The unique conformational heterogeneity of 1,25 (OH)2D3 has provided researchers in the vitamin D field a unique tool used to differentiate genomic and nongenomic signaling, because it has been discovered that different shapes of 1,25(OH)2D3 initiate genomic and nongenomic signaling [54]. This theory is based in large on the results from multiple independent researchers that the 6-s-cis locked analog of 1,25(OH)2D3, 1a,25 (OH)2-lumisterol D3 (JN; Fig. 15.5), functions as a potent nongenomic agonist and rather weak genomic agonist in vitro and ex vivo, when compared to 1,25(OH)2D3 [4,32,37]. The A-ring of the JN molecule is locked in the a-chair conformation, where the 1a-OH group is axial and the 3b-OH is equatorial (Fig. 15.2B). This is the 1,25(OH)2D3 chair conformation that was shown to
=
Metabolism of vitamin D3 sterols. Once the sun and heat produce vitamin D3 the VDR A-ring and side-chain are oxidized at the C1 and C25 positions [183] (see Fig. 15.1 and text). This figure shows the structures of over 30 different vitamin D metabolites that have been identified in mammalian tissues, primary cells, and/or transformed cell lines. The figure highlights the three major forms of vitamin D3, vitamin D3 (VD3), 25(OH)D3 (25D3), and 1,25(OH)2D3 (1,25D3; see Fig. 15.1) and their C1, C3, C8, C10, C23, C24, C25, and/or C26 metabolites. The label for each metabolite uses VD3, 25D3, and 1,25D3 as the root of the name and calls out the specific carbon number(s), stereochemistry and functionality of each of their side-chain (see Figs 15.2C and 15.2D) metabolites. Those labels that are bold highlight the metabolites that have been most extensively studied in our laboratory and by the work of others. In general, the 25D3 or 1,25(OH)2D3 can be further stereospecifically metabolized by the CYP24 side-chain hydroxylase at the C23, C24, and/or C26 positions [35]. The C23 pathway ends with production of the 25ROH-26,23-lactone of 25D3 or 1,25(OH)2D3. The end of the C24 pathway produces calcitroic acid. It is largely assumed that the side-chain metabolism of 1,25(OH)2D3 is catabolic [35]; however, there exists increasing evidence that metabolism of the side-chain directly alters VDR conformation and therefore signaling [32] and can temper 1,25(OH)2D3-induced hypercalcemia in vivo [184e186]. The metabolism of vitamin D sterols has been recently made more complex by the finding that C3 of 1,25(OH)2D3 can be epimerized to form 3-epi-1,25(OH)2D3 in many different cell types. 3-epi-1,25(OH)2D3 is discussed in more detail in the text.
FIGURE 15.3
II. MECHANISMS OF ACTION
277
1,25(OH)2D3 MEDIATED RAPID, NONGENOMIC RESPONSES
C25
C3
C20
C20
C1
C19
C25
C3
C19 C25
C20
C1 C3 C19
25D3 “J-shaped”
1,25D3 “bowl-like”
1,25D3 “planar”
FIGURE 15.4 Shapes of vitamin D sterols (VDS) produced by conformational search calculations and observed in bound DBP and VDR complexes. The three structures in the figure are rendered in ball and stick format, with hydrogen atoms removed. The structures represent different shapes of vitamin D sterols observed in 1,25(OH)2D3-VDR X-ray complex [40], in PC_Model conformational search calculations (see text) and in an in silico simulation of 1,25(OH)2D3 binding to the VDR alternative and genomic overlapping ligand binding pockets (i.e., the VD-AP and VDR-GP, see Fig. 15.9A) complexes [1]. Moving from left to right, they represent the 25-hydroxyvitamin D3 (25(OH)D3) J-shape, a conformation accepted by the DBP binding cleft [27], the 1a,25(OH)2-vitamin D3 (1,25(OH)2D3) planar-like shape, a conformation preferred by the VDR-AP [37] (see Fig. 15.9A and 15.9C) and the bowl-shape of 1,25(OH)2D3, preferred by the VDR-GP, that is a conformation proven by many to be strongly associated with 1,25(OH)2D3-VDR cis regulation of the genome. Carbon atoms referred to throughout the text are labeled in each panel for reference.
be a preferred conformation, capable of selectively binding to the VDR alternative ligand-binding pocket (VDR-AP) [30,37] and to the serum vitamin-D-binding protein [55] (Figs 15.2A, 15.2B and 15.4). It is noted that the VDR-GP is highly selective with regard to the A-ring and seco-B-ring geometry required for strong interaction with the VDR-GP, X-ray pocket, where a b-chair and 6-s-trans configuration are required (Figs 15.2A, 15.2B and 15.4). The PC_Model calculations show that the side-chain atoms of 1,25(OH)2D3 and JN sample a similar molecular space based on their similar Pop. A-C ensemble distributions. This indicates that the similarities and changes in JN and 1,25(OH)2D3 function are underpinned by their similar A-ring but different seco-B-ring chemistries.
Nongenomic Antagonist Structural Features and Molecular Dynamics In addition to ligand shape differentiating 1,25 (OH)2D3 genomic and nongenomic function, researchers have discovered that 1b,25(OH)2D3 (HL; Fig. 15.5) is a nongenomic specific antagonist that is equipotent to JN in its ability to stimulate an osteocalcin vitamin D DNA response element reporter plasmid in CV-1 and COS-1 cells cotransfected with human VDRwt [30,32,37,38]. Like 3-epi-1,25(OH)2D3 epimerization of C-1 changes the A-ring chair equilibrium. For HL the b-chair is favored and stabilized by the C-1 and C-3 diaxial intramolecular hydrogen bond [30] (see Fig. 15.2B). Importantly, in the preferred HL (b-chair) and JN (a-chair) chair conformations the 1-OH is axial; however, the steric space it occupies with respect to
the rest of the VDS differs dramatically (see Fig. 15.2B). As we will describe in more detail below, comparison of 25(OH)D3, 1,25(OH)2D3, JN and HL chemistries, conformational dynamics and structureefunction profiles show that at least for ion channels the 1-OH group is not a required feature for a ligand to be capable of functioning as a nongenomic, rapid response agonist. However, it is required for HL to block all known nongenomic responses shown to be activated by all of the following: 1,25(OH)2D3, JN and 25(OH)D3. Perhaps most importantly, this is the exact opposite of the A-ring hydroxyl requirements that are well defined for genomic agonists, where the 1a-OH group is essential, while the 3b-OH group is not [56,57]. The molecular details that underlie the importance of A-ring hydroxyl groups and their role in dictating VDR structuree function is the focus of a later section in this chapter.
1,25(OH)2D3 MEDIATED RAPID, NONGENOMIC RESPONSES 1,25(OH)2D3 has been shown to modulate a number of nongenomic, rapid (i.e., extranuclear) responses that occur outside the nucleus of the cell. The Norman laboratory’s contribution to the previous vitamin D volume presented and discussed three nongenomic case studies in detail. They were whole-cell patch clamp analysis of ROS17/2.8 cells, 1,25(OH)2D3 stimulation of insulin secretion, and activation of phosphatidylinositol 3-kinase by 1,25(OH)2D3 [54]. Regulation of extranuclear signaling factors, in a rapid fashion, results in cellular responses that include but are not limited to increasing
II. MECHANISMS OF ACTION
278
15. VITAMIN D STEROL/VDR CONFORMATIONAL DYNAMICS AND NONGENOMIC ACTIONS
Non-genomic Agonists OH H
H
H OH
H
HO
H
1,25D3
OH
H
25D3
HO
HO F
H
OL
OM
OH
HO HO
F
H
H
OH H
OH
OH
H
H
H
OH
QW
F
H
IE
H D D3C HO
BT
OH
D HF
HO
OCH3
HO
OH
OH
H
HO
JP HO
H
JN HO
H
H
OH
OH
H
H
H
HO
OH
JM
COOH
H
HO
H
LCA
HO HO
HO
HO
Non-genomic Antagonists
Non-genomic Agonists/ Genomic Antagonists
O O S O
S
C
OH
O
H
OH
OH H
OH
N N O N O
OH
O H
O
HO
N H
OCH3
N VER
N O
O
O
O
O
HM
ZK
OH
O O
H
H
HO
HO
S
OCH3
H3CO HO
C
O S ODIDS O
N
H3CO
HL
MK
N
-
H
HO H
H
N H
ISR NIF
Ion Channel Blockers
OH
O O H3CO HO
CM O
OH OCH3
HO
OH
BDC
H
OH
New Botanical Ligands of the VDR II. MECHANISMS OF ACTION
H
O
O OH
O
H WFA
OH
1,25(OH)2D3 MEDIATED RAPID, NONGENOMIC RESPONSES
intracellular calcium, exocytosis/ATP secretion, vitamin D3 side-chain metabolism, and UV protection (Table 15.1). Recent evidence suggests that like other sex steroid hormones (e.g., 17b-estradiol), 1,25(OH)2D3 nongenomic effects are modulated at least in part by the classic nuclear receptor functioning outside the nucleus of the cell [2]. In this section we describe and evaluate the different nongenomic, rapid responses and highlight those that have been shown to require a functional extranuclear VDR protein and present two case studies.
Potentiation of Voltage-Gated Ion Channels The Farack-Carson, Norman, and Zanello laboratories have demonstrated that in multiple transformed and primary cell lines, 1,25(OH)2D3 treatment activates a rapid opening of voltage-gated chloride and calcium ion channels [58e62]. Both the CLC-type outwardly rectifying chloride channel (ORCC) and the L-type calcium channel, have been shown to be activated within seconds to minutes following addition of 1.0 nM concentrations of 1,25(OH)2D3 or JN (Table 15.1). In addition, in these studies all VDR agonist ligands were blocked by coincubation with equimolar HL (Table 15.1). In our nongenomic ORCC structureefunction analysis we have demonstrated that 25(OH)D3 is as effective as 1,25(OH)2D3 in potentiating chloride currents in osteoblasts, and TM4 Sertoli cells [63]. Given the link between changes in serum 25(OH)D3 levels and multiple physiological disorders [64,65] it would not be surprising if 25(OH)D3 functions as a nongenomic or perhaps even genomic VDR agonist ligand [66,67]. All current evidence suggests that in both of these cell types the presence of an extranuclear VDR, localized to a caveolae lipid, raft microdomain underpins the regulation of channel activity by 1,25(OH)2D3, JN and 25(OH)D3. Ion channels are classified based on the type of current regulated, ionophore selectivity and the response to small-molecule channel activators and/or blockers [68e70]. Figure 15.5 and Table 15.1 highlight the ion channel blockers used to attenuate 1,25 (OH)2D3, JN and 25(OH)D3 potentiation of outwardly rectifying chloride and L-type calcium channels. Of these four blockers we recently showed that DIDS is a VDR ligand with approximately 10 mM affinity [45].
279
Whether DIDS blocks activation of 1,25(OH)2D3-sensitive ORCC currents through competitive inhibition remains to be determined. Nonetheless, it blocks 1,25 (OH)2D3 activation of the ORCC currents and exocytosis in TM4 cells [63] and suggests that the CLC channel being regulated by 1,25(OH)2D3 in a VDR-dependent manner is CLC-3 and/or CLC-5 [71]. Given the similarities in agonist/antagonist profiles in other systems (e.g., osteoblasts) it is likely VDR nongenomic regulation of CLC-3 is a common cellular pathway, linking 1,25 (OH)2D3-VDR nongenomic actions to cell processes like secretion [63,72] or phagocytosis, depending on the cell type.
Control of Phosphatase, Kinase, and Phospholipase Activity Two phosphatase signaling factors whose activity is impacted by 1,25(OH)2D3 in both colon cancer and leukemia cells are PP1c and PP2A. Both of these phosphatases are activated in a 1,25(OH)2D3-VDR dependent nongenomic manner and linked to cell differentiation/ antiproliferation (Table 15.1). In both colon cancer and leukemia cells the VDR is complexed to the catalytic subunits of PP1 and PP2A and is dissociated by addition of 1,25(OH)2D3, activating the phosphatase [73,74] (Fig. 15.6). Interestingly the effect of 1,25(OH)2D3 activation of these two phosphatases leads to different effects on the p70(S6K) Ser/Thr protein kinase [75,76]. It was observed in colon cancer cells that an activation of PP1c leads to inhibition of p70(SK6) and G1-arrest (i.e., antiproliferation) [73]. Alternatively, in leukemia cells, phosphatase activation leads to activation of p70(S6K) and cell differentiation [74]. In theory, 1,25(OH)2D3 activation of PP1c could also provide a link to the rapid (w5 seconds to minutes) increase in intracellular calcium in response to 1,25 (OH)2D3 in fibroblasts, osteoblasts, pancreatic b-cells, immune cells (Table 15.1 and references therein) and yet to be determined cell/tissue types (reviewed in [14]). The potential link is based on the evidence that PP1c dephosphorylates and activates IBRIT (Inositol 1,4,5-Triphosphate (IP3) Receptor-binding Protein) [77]. IBRIT is an endogenous repressor of the IP3R that blocks IP3 access to its binding site in the IP3R and thus efflux of calcium from ER stores [78] (Fig. 15.6).
=
Chemical structures of the VDR ligands and/or ion channel blockers used in vitamin D sterol structureefunction studies discussed in Table 15.1 and the text. All of the vitamin D sterols that are synthetic analogs are labeled with a two-letter code. All vitamin D sterols, with the exception of HL and HM (i.e., 1b-OH analogs), have been described to function as nongenomic agonists in at least one tissue/ cell type (Table 15.1). All of the vitamin D sterols, with the exception of MK and ZK (i.e., genomic antagonists), function as genomic agonist ligands; however, they dramatically differ in their effective concentrations (see Tables 15.2 and 15.3). The L-type calcium channel blockers (isradipine, ISR; nifedipine, NIF; and verapamil, VER) are depicted in the middle, right panel of the figure. DIDS is a chloride channel blocker discussed further in the text and has been shown to bind specifically to the VDR. The chemical structures of nonsteroidal VDR botanical ligands are shown in the bottom panel of the figure. They are curcumin (CM), bisdemethoxycurcumin (BDC), and withaferin A (WFA).
FIGURE 15.5
II. MECHANISMS OF ACTION
280 TABLE 15.1
15. VITAMIN D STEROL/VDR CONFORMATIONAL DYNAMICS AND NONGENOMIC ACTIONS
Summary of the scope of nongenomic signaling regulated in various tissues by 1,25(OH)2D3 (1,25D3) and/or its analogs. The column headings highlight the cell type, species (h ¼ human; h* ¼ human natural mutations; m ¼ monkey; r ¼ rodent; c ¼ chicken), brief overview of the signaling factor(s) and the cellular response studied, the small molecule agonist(s) or antagonist(s) (i.e., inhibitors) applied in each individual nongenomic case study. The data in the table are organized based on the left hand column (i.e., the cell type utilized in each published work). Bold entries are those studies that have been defined as VDR-dependent by the authors and/or experimental design. Italic entries highlight those studies that showed cross-talk between a nongenomic effect and VDR-RXR gene transactivation. The chemical structures for all of the agonist and antagonist ligands are provided in Figure 15.5, with the exception of TPA (12-O-tetradecanoylphorbol-13-acetate), H7 [1-(5-isoquinolinesulfonyl)-3-methylpiperazine] and Bay K-8644 (Bay-K). If none is entered in the inhibitor column the authors commonly used siRNA, gene-KO (e,e) and/or other commercial agents to attenuate the production and/or the function of proteins listed in the signaling factor(s) column. Unconventional abbreviations present in the Cellular Response column are as follows: MG ¼ monoacylglycerol; EAE ¼ experimental autoimmune encephalomyelitis; VSMC ¼ ventricular smooth muscle cells; and CPD ¼ cyclobutane pyrimidine dimers
Cell type
Species
Signaling factor(s) 2þ
Cellular response
Agonist ligand(s)
Inhibitor(s)
Ref
Adipocytes
R
[Ca ]I/FAS expression/GADH activity
Lipid metabolism
1,25D3, JN
HL
[152]
Aortic smooth muscle
R
PI3K
VSMC migration
1,25D3, JN
HL
[153]
Bladder contraction
EL, BK
Isa, Ver
[154]
2þ
Bladder
R
L-type Ca
Cardiac myocytes
R
Cav-3
Contractility
1,25D3
none
[112]
Cardiomyocytes
R
VDR/ion channels
Contractility
1,25D3
none
[155]
Colonic epithelial (Caco-2)
H
PP1c/PP2A/p70(S6K)
Anti-proliferation
1,25D3
none
[73]
Colon cancer
H
[Ca2D]I/RhoA-ROCK/ p38MAPK-MSK1
Anti-proliferation (YWnt)
1,25D3
none
[156]
Colonic epithelial (Caco-2)
H
ERK1/2-MED1 and ERK5-Ets-1
Anti-proliferation
1,25D3
none
[157]
Colonic epithelial (Caco-2)
H
PKCa/IP3
[Ca2þ]I
1,25D3
TPA, H7
[158]
Colonocytes
R
cSrc/PLCg
PI-hydrolysis
1,25D3
none
[159]
Enterocytes
C
Tyr-kinase/MAPK
Structureefunction study
1,25D3, JN
none
[117]
Fibroblasts
h*
L-type Ca2D channel/ MEK1/2/Cyp24
[Ca2D]I and vitamin D metabolism
1,25D3
Ver
[58]
Hela and OB-6 cells
H
PI3K, Src, JNK, ERK, MEK
Anti-apoptosis
1.25D3, JN
HL
[80]
HeLa/COS-7/ROS
h/m/r
CaMKV
VDR transactivation
1,25D3
none
[160]
EAE incidence
1,25D3
none
[161]
2D
channel
Immune
R
[Ca
Intestine
C
Calcium channels
Transcaltachia
1,25D3, JN, JM, JP
HL
[162,163]
Keratinocytes
H
Raf/MAPK/Shc/Grb2/ Ras
DNA synthesis
1,25D3
none
[164]
Psoratic keratinocytes
H
MEK-ERK/NFkB
Antimicrobial peptides (cAMP)
1,25D3, BT, ZK-series
none
[93]
Keratinocyte/ fibroblasts
H
CPD/p53
UV photoprotection
1,25D3, JN, JM, QW
HL
[165,166]
Keratinocytes (NHEK)
H
MEK-ERK
Cathelicidin expression
LCA
none
[167]
Leukemia (THP-1/ HL-60)
H
PP1c/PP2A/p70(S6K)
Cell differentiation
1,25D3
none
[74]
Leukemia (NB4)
H
Ser-Thr/Tyr kinases
Monocytic cell differentiation
1,25D3TPA, HF, JN, JM, JP
HL, HM
[58,168-171]
]I
(Continued)
II. MECHANISMS OF ACTION
281
1,25(OH)2D3 MEDIATED RAPID, NONGENOMIC RESPONSES
TABLE 15.1 Summary of the scope of nongenomic signaling regulated in various tissues by 1,25(OH)2D3 (1,25D3) and/or its analogs. The column headings highlight the cell type, species (h ¼ human; h* ¼ human natural mutations; m ¼ monkey; r ¼ rodent; c ¼ chicken), brief overview of the signaling factor(s) and the cellular response studied, the small molecule agonist(s) or antagonist(s) (i.e., inhibitors) applied in each individual nongenomic case study. The data in the table are organized based on the left hand column (i.e., the cell type utilized in each published work). Bold entries are those studies that have been defined as VDR-dependent by the authors and/or experimental design. Italic entries highlight those studies that showed cross-talk between a nongenomic effect and VDR-RXR gene transactivation. The chemical structures for all of the agonist and antagonist ligands are provided in Figure 15.5, with the exception of TPA (12-O-tetradecanoylphorbol-13-acetate), H7 [1-(5-isoquinolinesulfonyl)-3-methylpiperazine] and Bay K-8644 (Bay-K). If none is entered in the inhibitor column the authors commonly used siRNA, gene-KO (e,e) and/or other commercial agents to attenuate the production and/or the function of proteins listed in the signaling factor(s) column. Unconventional abbreviations present in the Cellular Response column are as follows: MG ¼ monoacylglycerol; EAE ¼ experimental autoimmune encephalomyelitis; VSMC ¼ ventricular smooth muscle cells; and CPD ¼ cyclobutane pyrimidine dimersdcont’d Cell type
Species
Signaling factor(s)
Cellular response
Agonist ligand(s)
Inhibitor(s)
Ref
Leukemia (HL60)
H
MAPK
Differentiation
1,25D3, JN
HL
[172]
Macrophages (P388D1)
H
TNFa/NFkB/IkBa
Anti-inflammation
1,25D3, 1,24RD3
none
[149]
2þ
Microsomes/ microcytes
C
cAMP
Ca -uptake/Protein phosphorylation
1,25D3
Nif
[173]
Myoblasts
C
PKC/PLA2
[3H]-AA secretion
1,25D3
Nif, H7
[174]
2D
2D
Osteoblasts
R
L-type Ca /CLC-3 Cl- channels
[Ca ]I/exocytosis/ ATP secretion
25D3, 1,25D3, JN, Bay K
HL
[60-62,175]
Osteoblasts/kidney (CV1)
m/r
Akt/PI3K
Anti-apoptosis
1,25D3
none
[81]
Pancreatic islets
R
L-type Ca2þ channel
Insulinotropic effect
1,25D3, JN
HL, Nif
[94]
2þ
2þ
Pancreatic b-cell
R
L-type Ca channel/ Ryandodine receptor
[Ca ]I/insulin secretion
1,25D3
Nif
[95]
Parathyroid
P
PLC
Biphasic [ in DAG, MG and IP3
1,25D3
none
[176]
Sertoli
R
cAMP/PKC/PKA/ CLC-3 Cl channel
Exocytosis
25D3, 1,25D3, JN, MK, IE
HL, DIDS
[45,63]
Skeletal muscle
R
c-Src/ERK1/2/p38MAPK/Cav-1
Structureefunction study
1,25D3, JN
HL
[177]
Skin
R
p53/NOS
UV/ROS/CPD formation
1,25D3
none
[100]
Squamous cell carcinoma
H
PI3K/ERK1/2
Apoptosis
1,25D3
none
[178]
Of the kinases that have been shown to be regulated by 1,25(OH)2D3, the receptor tyrosine kinase Src is of considerable interest because it is often mutated in cancer cells and it links 1,25(OH)2D3 to yet another classical second messenger system that includes ras, raf, ERK1/2, MEK1/2, and/or p38-MAPK (Table 15.1 and Fig. 15.6). Kinases that have been shown to be activated by both 1,25(OH)2D3 and JN include Ser-Thr/Tyr kinases, PI3K, and PKA (Table 15.1). Of the kinases mentioned, the ability of HL to block the effect of 1,25 (OH)2D3 has been demonstrated for Ser-Thr/Tyrkinases and MAPK (Table 1.1). For example PKC and PKA have been shown to be required for 1,25(OH)2D3 potentiation of CLC-3 in TM4 Sertoli cells [63] (Table 15.1). It is noted that the VDR is also a substrate for some of these kinases and further dissecting how the VDR socializes with these and other extranuclear
signaling factors will aid in further defining the role 1, 25D3 nongenomic signaling plays in the global biology of the whole organism. The phospholipases that are activated by 1,25(OH)2D3 are PLC (e.g., PLCg, Fig. 15.6) and PLA2 (Table 15.1). These two phospholipases control IP3 and arachidonic acid (AA) concentrations by hydrolyzing the membrane phosphatidylinositolphosphates, PIP2 and PIP3. It is well documented that inositol-1,4,5-triphosphate (IP3) triggers the opening of the IP3R [78], which in turn facilitates an increase in cytosolic calcium by mobilizing it from the endoplasmic recticulum (ER; Fig. 15.6). Alternately the liberation of AA from PIPs leads to the production of prostanoids and/or leukotrienes. These derivatives of AA have a wide range of functions that include, but are not limited to, inflammatory signaling cascades [79]. Interestingly of the two PIPs, PIP3 is not
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15. VITAMIN D STEROL/VDR CONFORMATIONAL DYNAMICS AND NONGENOMIC ACTIONS
P
Cytoplasm
P
PP1c
?
P
1,25D3
PKC ↑
MNAR
PLC
Regulation of genes
c-Src ↑ [Ca]c
P
P
PP1c
P
IRBIT
BCL-2 IP3R
Regulation of genes
IP3R
IP3R
ERK1/2 ↑
ER Lumen
Key: = 1,25D3-VDR
FIGURE 15.6 A simplified model of VDR regulation of selected nongenomic signaling factors: a phosphatase (i.e., PP1c), a phosphoesterhydrolase (i.e., PLC), a kinase (PKCa), the cSrc/ERK1/2 signaling axis and their regulation of intracellular calcium, and cross-talk. The diagram represents the VDR as two concentric ellipses. The larger one is the VDR and the smaller the vitamin D ligand-binding surface. In the absence of nM levels of 1,25(OH)2D3 the VDR forms a complex with catalytic subunit of pp1 (see text). This releases PP1c allowing it to dephosphorylate multiple substrates that include IRBIT, a protein that blocks binding of inositol-1,4,5-triphosphate (IP3) to the IP3R and mobilization of calcium from the ER lumen and into the cytoplasm ([Ca]c) [187]. The figure depicts how binding of 1,25(OH)2D3 to the VDR-AP could stimulate PLCg [14,188] and other hydrolysis reactions by exposing bound PIP2 or PIP3. An increase in intracellular calcium is well documented to stimulate the catalytic activity of PKCa [189]. The holo-1,25(OH)2D3-VDR complex has also been reported to bind to Src and the scaffolding proteins, caveolae, tuberin, and MNAR (modulator of nongenomic activity of the estrogen receptor) [190,191]. By stimulating Src, 1,25 (OH)2D3-VDR can activate downstream ERK1/2. Both PKCa and ERK1/2 can function to activate and/or repress the transcription of genes through regulating the activity of second messengers, other transcription factors, and/or the nuclear receptor itself, a process referred to in the literature and the text as “cross-talk.”
found in yeast [79] and the enzyme that converts PIP2 to PIP3, PI3K, is a classic oncogene that, like c-Src, is mutated in most cancers. Thus the known 1,25 (OH)2D3/JN/HL nongenomic functional effects on PI3K (Table 15.1) [80,81] may have coevolved by the socialization of the VDR with both vitamin D sterols and phosphatidylinositolphosphates (PIP3, see sections on IVD and VE). It is noted that arachidonic acid (AA) can also be obtained from diacylglycerol (DAG), the other product of PLC hydrolysis of PIP2. In closing our overview and brief description of the extranuclear signaling factors whose activity is regulated by 1,25(OH)2D3, PLCg was recently shown to be up-regulated by 1,25(OH)2D3-VDR in antigen inexperienced naive T-cells [82]. In the study by von Essen et al., 1,25 (OH)2D3-VDR expression of PLCg was observed following the induction of VDR expression in response to T-cell antigen receptor (TCR) stimulation [82].
Up-regulation of VDR mRNA by TCR is p38-MAPKdependent and PTEN, the enzyme that converts PIP3 to PIP2, is thought to be a VDR target gene [83e85]. Giventhe evidence that 1,25(OH)2D3 regulates PIP2/3 and p38MAPK signaling in a nongenomic fashion (Table 15.1), it is possible that the innate and cognitive immune responses recently discovered for 25(OH)D3 and 1,25 (OH)2D3 [86,87] require cross-talk between 1,25(OH)2D3VDR genomic and nongenomic signaling. Said differently, the VDR may function to regulate both the expression of signaling factors like PLC and PTEN in the nucleus as well as their extranuclear enzymatic function.
Cross-Talk There are two fundamental forms of cross-talk that exist between extranuclear and nuclear vitamin D sterol (VDS) signaling. The first type involves the cell-specific
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THE PLASMA MEMBRANE VITAMIN D RECEPTOR
effect 1,25(OH)2D3 nongenomic signaling has on the VDR phosphorylation state. This type of cross-talk where 1,25(OH)2D3 activation of an extranuclear signaling factor can serve to regulate both VDR location within the cell and its transactivation potential [88e91]. The second type of cross-talk involves 1,25(OH)2D3 regulation of cytoplasmic second messengers which include, but are not limited to, cAMP, p53, NFkB, IkB, calcium, and ERK1/2 (e.g., c-fos) (Table 15.1). When activated, these second messengers can regulate genes that either do or do not contain a nuclear vitamin D response element (VDRE). Perhaps the crucial importance of VDR-dependent nongenomic signaling and cross-talk in whole cell function is best embodied in the antiproliferative effect 1,25(OH)2D3 and JN have in human acute promyelocytic leukemia NB4 cells [4] (Table 15.1). In NB4 cells the prodifferentiative effect of 1,25 (OH)2D3 and JN is blocked by coincubation with equimolar HL (Table 15.1), but not by addition of a tenfold excess of the human VDR genomic antagonist, analog MK (Fig. 15.5) [44,92]. In addition, the VDR genomic antagonist (ZK-series; Fig. 15.5) stimulates MEK-ERK signaling in psoriatic keratinocytes [93] and MK stimulates CLC-3 opening in TM4 Sertoli cells [63]. Other cellular functions shown and/or perceived to require nongenomic signaling and cross-talk include, but are not limited to, exocytosis (e.g., insulin [94,95] and ATP [61]), apoptosis (e.g., caspase-3 [96,97]), phagocytosis (e.g., soluble amyloid beta [98]) and protection against UVB skin damage and CPD ([99,100] and Table 15.1). Future work in the cross-talk subfield of vitamin D research is required to better define the interplay between 1,25(OH)2D3 nongenomic and genomic signaling in maintenance of good health and in therapeutic design.
THE PLASMA MEMBRANE VITAMIN D RECEPTOR Immunohistochemistry and confocal microscopy have convincingly shown that the classic nuclear VDR associates with a variety of cellular membranes and is observed in the cytosolic and nuclear compartments [11,72]. The Norman laboratory first discovered that the VDR was observed to localize to the lipid-raft caveolae-enriched microdomain in many different tissues and cell types. In these caveolae-enriched membrane fractions 1,25(OH)2D3, JN, HL, and other VDS showed similar binding affinities when compared with tritiated 1,25(OH)2D3 competition assays performed with nuclear preps from the same cells [101]. Perhaps most importantly in vitamin-D-deficient chicks a significant reduction in the capacity of the caveolae fraction to bind physiological levels of tritiated-1,25(OH)2D3 was
283
observed when compared to vitamin-D3-replete chicks [2,11]. In the study by Huhtakangus et al. [11], it was also determined that [3H]-1,25(OH)2D3 localized to caveolae, as assessed by isolation of caveolae-enriched membrane fractions and HPLC. Finally, in VDR-KO mice there was a loss of [3H]-1,25(OH)2D3 binding to the caveolae-enriched membrane fraction and an associated loss in 1,25(OH)2D3-mediated rapid responses of opening of chloride channels and stimulation of exocytosis [60]. Another 1,25(OH)2D3 membrane receptor has been proposed to play an important role in regulating calcium and phosphorous transport, namely the Membrane-Associated Rapid Response, Steroid (1,25 (OH)2D3-MARRS)-binding protein [102]. It is known that 1,25(OH)2D3-MARRS is a protein disulfide isomerase [102e104] and that disulfide isomerases contribute, in some way, to the nongenomic effects of 1,25(OH)2D3 in chondrocytes [105], osteoblasts [106], and other cell types [107e110]. Most recently, 1,25 (OH)2D3-MARRS was shown to regulate 1,25(OH)2D3 membrane effects in osteoblasts [106]. We will not address this proposed 1,25(OH)2D3 membrane receptor in more detail herein, in large because no structureefunction studies have been carried out that demonstrate the known shape specificity of 1,25(OH)2D3 for nongenomic responses. Nonetheless, the collective effects of 1,25(OH)2D3 suggest that it is possible that both the VDR and 1,25(OH)2D3-MARRS are required for the regulation of some nongenomic signaling factors/functions. In this section we overview what is currently understood concerning how the VDR is localized to the plasma membrane caveolae microdomain and the signaling factors and extranuclear scaffolds the VDR has been shown to interact with. We also highlight how the VDR is unique when compared to other nuclear receptors known to serve as membrane receptors for their endogenous steroid hormones.
Structural/Scaffolding Proteins (e.g., Caveolins) The main structural component of the plasma membrane caveolae microdomain is the membrane proteins, termed caveolins (e.g., Cav-1 and/or Cav-3) [111]. The VDR has been shown to colocalize with each of these molecules in a ligand-dependent (osteoblasts) [72] with ligand-independent (cardiac myocytes) [112] fashion, respectively. The important domains or amino acids of the VDR/Cav proteins required for their direct interaction are currently unknown. It is also unknown what specific function the interaction between the VDR and Cavs contributes to known nongenomic signaling cascades regulated by 1,25(OH)2D3. Nevertheless, the caveolae microdomain is home to an enhanced
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concentration of membrane PIP molecules and cholesterol [111,113,114], and it is also a cell loci enriched with signaling factors known to be regulated by 1,25 (OH)2D3 (Table 15.1) [114e116].
Other Signaling Factor/Scaffolding Proteins (e.g., PP1c and Src) In addition to Cav-1 and Cav-3, the VDR has been reported to bind directly to c-Src [117] and PP1c [73,74]. Initial findings indicate that like the Cav-3eVDR interaction, the PP1ceVDR interaction is attenuated by 1,25(OH)2D3, demonstrating that in some cases the VDR itself can function as a negative inhibitor of enzyme catalytic function (Fig. 15.6). Boland’s group provided evidence that the VDR mediates rapid changes in muscle protein tyrosine phosphorylation induced by 1,25(OH)2D3 [118]. This leads to the activation of the Src/MAP kinase pathway [117] (Fig. 15.6), presumably via the VDR interacting with the SH2-domain of Src. The interaction of VDR with the SH2-domain of Src is believed to occur via phosphorylation of a Tyr-residue located in the hinge domain of the VDR (e.g., Y143 or Y147) [117]. Src tyrosine kinases, Ga-subunits, and H-Ras have also been shown to share a common membrane-anchored scaffolding protein, caveolin. According to the report by Li et al. [118], caveolin binding negatively regulates the autoactivation of Src tyrosine kinases. The VDR has also been reported to bind to the scaffolding protein tuberin [120]; however, like the VDR interaction with Src, the molecular details of the interaction remain largely unknown. The functional relevance of the VDR interaction with tuberin has yet to be elucidated; however, the VDR may regulate different nongenomic signaling cassette(s) [121e123] by physically interacting with the tuberin/hamartin signaling scaffold [124]. Thus it appears that the VDR can socialize with nongenomic signaling factors via its reversible interaction with intracellular protein scaffolds in both a 1,25(OH)2D3-dependent and -independent manner. Certainly future work in this area of vitamin D research is warranted and is of paramount importance in providing a molecular basis [3] for how 1,25(OH)2D3 nongenomic, extranuclear, rapid responses contribute to cell physiology and ultimately the therapeutic effects associated with vitamin D supplementation and/or analogs of VDR ligands (i.e., 1,25(OH)2D3).
Covalent Modification (e.g., Palmitoylation) Another way that the VDR could in theory localize to the plasma membrane is through palmitoylation or myristylation. This hypothesis is in large part based on the recent evidence published by the Levin laboratory indicating some nuclear receptors, like the estrogen (ER),
androgen (AR), and progesterone (PR) sex hormone nuclear receptors (NR), contain a consensus sequence, marking the NR for palmitoylation and subsequent membrane anchoring [125,126]. Evaluation of the VDR primary sequence produced no such consensus sequence in the VDR ligand-binding domain. Thus it would seem that the VDR either localizes to the various cellular loci via socializing with various protein scaffolds and/or the VDR could bind directly to membrane lipids, like the phosphatidylinositolphosphates (e.g., PIP2, Fig. 15.6).
Membrane Binding (e.g., PIP2/3) We and others have shown over the past 10 years that the VDR does not “selectively” bind just 1,25(OH)2D3. Rather it can sample a very broad set of ligands that are capable of competing off specifically bound, tritiated 1,25(OH)2D3 [32,63,98,127,128]. It has also been recently demonstrated that the orphan nuclear receptors SF-1 and LRH-1 (NR5 family members) are capable of binding to membrane lipids [129] (e.g., phosphatidylinositol-3,4,5-triphosphate, PIP3). Molecular mechanics calculations indicate that both PIP2 and PIP3 are capable of binding to the VDR such that the inositol phosphate polar head group of the PIP is bound to the A-ring domain, the glycerol moiety is located in the region of the VDR-AP towards the surface of the VDR-AP (see below) and the hydrophobic tails are left free to suspend in the hydrophobic membrane (Fig. 15.7). This VDR membrane model has not yet been experimentally studied, but it does provide a direct link between 1,25(OH)2D3 and PLC hydrolysis of PIP2 and the subsequent rapid (w5 s) increase in cytosolic calcium from ER stores observed following hormone addition to cells [130] (Fig. 15.6). According to the molecular models, PIP2 is envisioned to be displaced from the VDR by 1,25(OH)2D3 allowing for it to be exposed and subjected to hydrolysis by PLC (Fig. 15.6).
THE VDR CONFORMATIONAL ENSEMBLE MODEL The first model describing ligand activation of a nuclear receptor was presented by the Moras laboratory over 10 years ago [131e133]. The model, termed the “mouse-trap” was based on comparison of the apo- and holo-retinoic acid receptors and posited that, in the absence of the high-affinity hormone ligand, the C-terminus of helix-11 occupies the NR ligand-binding pocket (Fig. 15.8A, center left ribbon structure). Helix11 therefore physically blocks the ability of hormone to bind the classical and highly conserved X-ray ligand-binding pocket [134] and orients helix-12 away
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THE VDR CONFORMATIONAL ENSEMBLE MODEL
PIP2
Nuclear H7
Coregulator
RXR
Surface
Surface
H3/H4/ H5/H12
H10
H1 The speculative VDR-PIP membrane model. In the figure the theoretical (i.e., computed) complex formed between the VDR-AP (Fig. 15.9A) and PIP2 is shown. The PIP2 is rendered in space filling with hydrogen atoms included. In the complex, the phosphate at C4 of the inositol ring forms a strong electrostatic interaction with R274. The R274 residue is central in the figure and is shown in space filling with hydrogen atoms absent. In this theoretical complex, the VDR RXR heterodimerization surface (H7 and H10) and the nuclear coregulator surface (portions of H3/H4/H5 and H12) are oriented in such a manner that they are theoretically accessible when the VDR is anchored noncovalently and reversibly to the underside of the plasma membrane. These surfaces are shaded in the figure and labeled. Please see color plate section.
FIGURE 15.7
from the remainder of the NR ligand-binding domain. Based on the tenants of the mouse-trap model the ligand induces a conformational change in the NR by replacing the C-terminal end of helix-11 in the ligandbinding pocket and causing helix-12 to swing into a closed position (Fig. 15.8A steps 1 and 2). In this position, helix-12 can form extensive intramolecular contacts with the ligand-binding domain and contact the ligand as well; this is discussed in detail in below. Closure of helix-12 completes the NR surface, termed the nuclear coregulatory binding site (Fig. 15.9A). Thus closure constitutes a switch from the transcriptionally off-state to an on-state (Fig. 15.8A steps 1 and 2), presumably by displacing nuclear corepressor and recruiting nuclear coactivators to the coregulatory binding site [135]. In 2004 we proposed that the VDR molecule, like its native high-affinity hormone ligand (i.e., 1,25 (OH)2D3), is a flexible body whose conformation is not static in the in vitro apo-state (i.e., the VDR is
285
unliganded; Fig. 15.8A). This hypothesis was largely based on the inability to form an apo-VDR crystal and solve its structure. The concept of a mobile helix-12 has been verified in solution by limited proteolytic digest [32] (Fig. 15.8B) and most recently by VDR hydrogen/deuterium exchange mass spectrometry [136]. The results from all of these methods showed no favored apo-VDR conformation [2] exists that correlates with that which would be predicted by the mousetrap to be of high population. Recently we showed that the conformational distribution of helix-12 of the apoVDR could be significantly altered by both changes in ligand chemistry and mutation of the VDR [32,44] (Fig. 15.8B). The ensemble concept also provides a sound, rationale, structural explanation for how the VDR antagonist, MK (Fig. 15.5), can be converted to a superagonist by a single point mutation and why removal of both hydrogen bonds to the 25-OH group of 1,25(OH)2D3 does not attenuate the ability of 1,25 (OH)2D3 to transactivate [44]. Based on the evidence the VDR is a highly flexible molecule and the structureefunction results that indicate that the VDR is, without question, involved in 1,25(OH)2D3 extranuclear, nongenomic, rapid cellular responses, we proposed the VDR could function as both a membrane receptor and transcription factor modulating the nongenomic and genomic functions of VDS [1,37]. While attempts have been made, X-ray crystallography has not provided a structural explanation for the mechanistic nuances (i.e., shape specificity, see above) of VDR nongenomic specific ligands (Fig. 15.5). Also X-ray crystallography has been unable to provide a unique structural understanding induced by genomic superagonist ligands [40,41,137]. This link between 1,25 (OH)2D3 chemistry and superagonism was discussed in greater detail above. Alternately, through the use of molecular mechanics modeling techniques, we obtained evidence that ligands could in theory regulate the population of different VDR ensemble conformations and thereby genomic and nongenomic signaling, by their ability to bind to different, yet overlapping, regions of the VDR ligand-binding and hinge domains [1,37, 44,63] (Fig. 15.9A). Thus we posit that the innate VDR conformational flexibility and the presence of two overlapping ligand-binding pockets allow the VDR to send signals dynamically [138].
The Overlapping Two-Pocket Model The ability of the VDR to function as a membrane receptor for 1,25(OH)2D3, JN, and 25(OH)D3 initiated rapid responses was for the first time structurally understood by the discovery that a hydrogen bond donore acceptor flip-flop between His229 and Tyr295 opened additional steric space in the VDR molecule forming
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15. VITAMIN D STEROL/VDR CONFORMATIONAL DYNAMICS AND NONGENOMIC ACTIONS
(B) I
holo-VDR H12 ensemble
(A)
VDR-1,25D3*
KH BS MW
+
+
+?
c1 c3
c2
+VDR ligand
1
30
hVDRwt
c3
KH BS
NL
34 I268Y/V300Y
MW
I C
NL
I
C
NL
c2
30
H305F/ H397F
hVDRwt
?
c3
20
HO
H12 H11 apo-VDR (inferred from apo-RXR )
C
4
3
H10
I
46
2 c1
+1,25D3
C
H H
c2
23 24 26 H O 25
OH H
KH
OH
O
BS
c1 HO
OH
HO
OH
apo-VDR H12 ensemble FIGURE 15.8 Aspects of the VDR conformational ensemble model. (A) Molecular dynamics: the ribbon diagrams in the upper left portion of
the figure illustrate a putative thermodynamic cycle for the VDR molecule and more specifically helix-12. For example, different helix-12 VDR conformations predicted from X-ray crystallization of various apo- and holo-NR complexes are illustrated in the upper, middle panel by coloring different conformations of the C-terminal activation helix of the VDR, helix-12 (H12, c1, c2, and c3). The numbers 1 and 2 highlight a theoretical induced-fit, where an apo-VDR, of favored conformation (i.e., high population) binds to 1,25(OH)2D3 (1) producing a transitory complex (VDRVDS*). This complex relaxes to prefer a specific distribution of VDR conformations. For the process of VDR-RXR transactivation, the major body of structural evidence suggests that helix-12 must be closed in order to potently up-regulate gene products which contain a positive vitamin D response element. The VDR conformational ensemble (2) or mutually induced-fit model [192] (3 and 4 in the cycle) differs from the induced-fit, mouse-trap model in that the unliganded VDR is considered to be a continuum of conformational isomers with a number of different sampled and populated states. According to this model, the VDR ligand pool and cell signaling state modulate the population distribution of VDR ensemble members [1,32]. In theory the VDR ensemble can be perturbed in vivo by differential and dynamic socialization with ligand and coregulatory/scaffolding protein pools. For example, nutritional intake, pro- and hormone metabolism, post-translational modifications, intracellular pH, isoelectric environment, intracellular localization, mutagenesis, content, and concentration of coregulatory molecules that either form the scaffold of a quaternary complex containing the VDR or form direct intermolecular interactions with the VDR surface, all can influence the VDR ensemble state (i.e., distribution of conformers) and/or the presence/movement of the VDR to different cellular loci. (B) The bottom panels in the figure are representative SDS-PAGE gels obtained following a limited proteolytic (i.e., trypsin) digest of radio-labeled apo- or holoVDR coding for the full-length hVDRwt, I268Y/V300Y, and H305F/H397F constructs. These two mutations significantly reduce the free volume of the VDR-GP and introduce intramolecular hydrophobic interaction with H12 residues (see Fig. 15.9B). The ligands used at 10 micromolar concentration were 1,25(OH)2D3 (C, control), BS, and KH (structures shown and labeled). These vitamin D sterols stabilize unique distribution of three VDR solution state conformations (c1, c2, and c3). The detailed structureefunction studies carried out with BS, a natural, terminal metabolite of 1,25(OH)2D3 side-chain metabolism (Fig. 15.3) and KH, a synthetic two side-chain analog of 1,25(OH)2D3 are compared in detail in Mizwicki et al. [32], but not elaborated on extensively herein. In both the I268Y/V300Y and H305F/H397F, no ligand (NL ¼ no exogenous vitamin D sterol added) showed significant protection of the VDR against trypsin cleavage, demonstrating that the population distribution of VDR conformational isomers can be largely influenced by changes in the chemistry of the ligand-binding surface and not just ligand itself.
what is termed the VDR alternative ligand-binding pocket (VDR-AP; Fig. 15.9A) [37,38]. The VDR-AP shares a significant amount of steric (i.e., 3D) space, termed the A-ring domain (Fig. 15.9A, dashed circle), with the VDR ligand-binding pocket defined by X-ray crystallography, termed the VDR genomic pocket (VDR-GP) (Fig. 15.9A).
When bound to the VDR-GP, the 1,25(OH)2D3 molecule is bowl-like in shape (Figs 15.4 and 15.8B). According to many lines of structureefunction evidence, obtained both before [14] and after [44] the 1,25 (OH)2D3-VDR X-ray structure, when bound to the VDR-GP, the 1,25(OH)2D3 A-ring and seco-B-ring are rigidly held in a b-chair (Fig. 15.2B), 6-s-trans
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The VDR two pocket model. (A) The solvent exposed surface of the VDR (aa118e427; D165-215) is shown as a transparent clear surface. In the diagram two amino acids are rendered in space filling and labeled R402 and K413. These two cationic residues serve as the residues recognized by the trypsin active site in the production of the c3 and c2 limited trypsin digest (i.e., protease sensitivity assay, PSA) fragments (see Fig. 15.8 and [32]). The VDR-AP is represented by a colored sky blue solid surface and the VDR-GP by a faint yellow transparent surface. The region where H1/ H2, H3, H5, and the b-sheet meet forms the region where the VDR-AP and VDR-GP ligand-binding pockets share three-dimensional, steric space. This region is termed the VDR A-ring domain, highlighted in the figure by a dashed circle. (B) The 25-OH hydrogen bonds, the C26 and C27 hydrophobic, van der Waals interactions, and the seco-B-ring intermolecular stabilization when 1,25(OH)2D3 (ball and stick) is flexibly docked to the VDR-GP are illustrated. Note that the 1,25 (OH)2D3 pose shown here is in strong agreement with the VDR X-ray pose of 1,25(OH)2D3 [40]. That same pose was produced blindly in the PC_Model conformational search calculation (see Figs 15.2 and 15.4 and the text). The residues forming favorable contacts with C5, C6, C7, C8, and C19 are W286 and C288. Hydrophobic, van der Waals (vdW) contacts are made between the terminal side-chain methyl groups of 1,25(OH)2D3 (i.e., C26 and C27) and the hydrophobic crown residues L227, L414, and V418. These hydrophobic interactions form the buried foundation of the nuclear coregulatory surface (labeled, shaded regions of H3 and H12 in panel A and Fig. 15.7). H305 and H397 are required for 1,25(OH)2D3 for genomic transactivation. This is because the dynamics of the 1,25(OH)2D3 side-chain must be energetically augmented so as to allow for it to interact with the hydrophobic crown residues in a manner that shifts the Boltzmann distribution of VDR conformations to favor the agonist conformation, where H12 is protected from trypsin and therefore ordered when compared to e1,25 (OH)2D3 (Fig. 15.8) [32]. (C) The polar or unsaturated amino acid R-groups that form the VDR A-ring domain: Y143, S237, R274, S278, W286, and C288. The b-chair, 6-s-cis, Pop. A conformation of 1,25 (OH)2D3 observed following the 1,25(OH)2D3/VDR-AP flexible docking simulation [37] is shown in the panel as a ball and stick structure. The nearest-neighbor contacts made between polar or unsaturated carbon atoms with the A-ring domain residues are indicated by solid lines and the distances in a˚ngstroms are provided in the figure. Please see color plate section.
FIGURE 15.9
(A)
H1
H5 H12 H2 R402
H3 K413
(B)
V418
L414 H305 C288
L227
H397
W286
(C)
S237 C288
W286
Y143 S278 S274
(Fig. 15.2A) conformation. The rigidity results from the hydrogen bonds formed between the equatorial 1a-OH group and S237 and R274 and the axial 3b-OH group and Y143 and/or S278 (Fig. 15.9B) and the buried nature of the VDR-GP (Fig. 15.9A). In contrast, the side-chain atoms of 1,25(OH)2D3 show enhanced heterogeneity, but are somewhat conformationally restricted by hydrogen bonds formed with His305 and His397 (Fig. 15.9B and see pdb code: 1DB1 heavy atom B-factors). Finally, the H-bonds between the 25-OH group (Fig. 15.1) and H305/H397 are intermolecular interactions that allow the C26 and C27 terminal methyl groups of 1,25 (OH)2D3 to contact crucial hydrophobic residues in the VDR-GP (Fig. 15.9B) that are essential for potent 1,25 (OH)2D3-VDR genomic agonism [44,139]. When bound to the VDR-AP, the A-ring of the 1,25 (OH)2D3 molecule can exist in either the a- or b-chair conformation (Fig. 15.2B). According to the molecular models, the seco-B-ring can exist in the 6-s-cis or 6-s-trans
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288
15. VITAMIN D STEROL/VDR CONFORMATIONAL DYNAMICS AND NONGENOMIC ACTIONS
conformations (Fig. 15.2C) and the side-chain prefers the lowest energy and highest populated Pop. A configuration (Fig. 15.2C) [37]. All of these 1,25(OH)2D3 conformational rotomers that are accepted by the VDR-AP have a more planar-like molecular shape (Fig. 15.2A). In the VDR-AP, the 3b-OH group forms hydrogen bonds with S237 and/or R274 and when in the b-chair, 6-s-cis conformation the 1a-OH group forms H-bonds with Y143 and/or S278 (Fig. 15.8C). The triene atoms of 1,25 (OH)2D3 make a significant number of contacts with Cys288 and also make intermolecular contacts with S278 and W286 (Fig. 15.9C). Based on the computational results that show the VDR-AP accepts more conformational isomers of 1,25(OH)2D3 when compared to the VDR-GP [37,45] and the evidence that the potential interaction energy and Gibbs free energy of binding (DGbinding) are better for the VDR-GP for nearly all VDS (Table 15.2), the VDR-AP has been described as being kinetically favored while the VDR-GP is the thermodynamically favored ligand-binding pocket for 1,25 (OH)2D3, if not all 1-OH vitamin D sterols (Table 15.2 and Fig. 15.9A legend).
TABLE 15.2
Experimental Support for the VDR-AP In order to test the validity of the VDR-AP model we designed single and double S237A/R274A or Y142A/S278A point mutations and measured and compared the binding affinity and genomic EC50 of 1,25(OH)2D3, 25(OH)D3, and 3-deoxy-1,25(OH)2D3 (analog CF) [30,32,37]. Based on the modeling results it was predicted that either single or double alanine mutants against S237/R274 would enhance the VDR affinity and genomic agonist potential of 25(OH)D3. This was because the hydrogen bonds formed between the 3-OH group and these residues in the VDR-AP would be lost in the Ala mutants (Fig. 15.9C), but those hydrogen bonds formed between the 3-OH group of 25(OH)D3 and Y143/S278 in the VDR-GP would still exist (Fig. 15.9B). These changes in the intermolecular interactions between the vitamin D sterol and the VDR A-ring domain would then have the consequence of increasing the fractional occupancy of the VDR-GP in the mutants as compared to VDRwt. Alternatively the VDR affinity and genomic agonist
Relative Competitive Index (RCI), genomic EC50 values and simulated binding properties of 1,25(OH)2D3 (1,25D3), JN, HL, 25(OH)D3 (25D3), CF, MK, and IE in the VDR-GP and VDR-AP. The table summarizes the theoretical and empirical binding affinities for the VDR ligands highlighted in this article. The (*) next to 1,25(OH)2D3, JN, HL, and IE indicates that the molecular formula of the sterol is identical, but the atom connectivity or stereochemistry at C1, C9/C10, or C20 differs when compared to 1,25(OH)2D3 (see Fig. 15.5 for the chemical structures). The (**) next to 25D3 and CF indicates that these two compounds have the same molecular formulas, but differ in the position of the A-ring hydroxyl group (C1 for CF and C3 for 25D3, see Fig. 15.2B). So long as the molecular formulas (i.e., the number and type of atoms in the ligand are identical) direct comparisons in computational affinities (i.e., DGbinding) can be made. The theoretical affinities for the VDRGP and VDR-AP are listed in the table. These values were obtained using the Discovery Studio 2.0 software package and a flexible docking simulation protocol, where vitamin D sterol conformations were generated using a PC_Model conformational search calculation (see Figs 15.2 and 15.4 and text). The first values listed in the VDR-GP column represent the average of the top seven VDR-GP complexes for each ligand. The top seven were averaged because it was observed that the averaged value for 1,25(OH)2D3 was identical to that computed using the ligand pose extracted from the 1,25(OH)2D3-VDR X-ray complex in the Gibbs free energy calculation [44]. Consistent with our previous VDR-AP and VDR-GP molecular mechanics calculations [30,32,37,38] a mixture of 6-s-cis and 6-s-trans, a/b-chair poses was observed in the VDR-AP flexible docking studies. Alternatively, a 6-s-trans,b-chair conformation was the only seco-B-ring, A-ring rotomer observed in the top 7 1,25(OH)2D3-VDR-GP flexible docking results [45]. The value in parentheses represents the highest affinity complex for each vitamin D sterol. This value is presented because it correlates better to the empirical affinity of the VDS for the VDR and their function where IE 1,25(OH)2D3 > JN HL (see text). This is based on the relative competitive index value for each VDS measured by a steroid competition assay where increasing concentrations of the cold ligand are measured for their ability to compete off [3H]-1,25(OH)2D3 (0.2 pmoles) and the genomic effective concentration (EC50) measured for each VDS in CV1 cells transiently transfected with VDRwt and a SEAP-reporter construct [139]
VDR ligand
VDR-GP DGbinding (kcal/mol)
VDR-AP DGbinding (kcal/mol)
Relative competitive index (RCI), chick intestinal mucosa
1,25D3*
e47.3 (e63.5)
e37.0
100%
JN*
e37.3 (e59.2)
e34.4
1.8% 0.5
178 37
HL*
e45.6 (e57.6)
e41.7
1.0% 1.5
160 1.8
25D3**
e36.7 (e43.7)
e37.2
0.15% 0.020
280 73
CF**
e44.7 (e50.8)
e29.7
5.7% 0.73
5.4 1.3
MK
e37.1 (e55.2)
e36.2
10.6% 5.3
>1000
IE*
e41.8 (e62.0)
e38.6
147% 72
0.019 0.0019
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Genomic effective concentration (EC50, nM) 1.4 0.15
289
THE VDR CONFORMATIONAL ENSEMBLE MODEL
TABLE 15.3
1,25(OH)2D3 (1,25D3), 25(OH)D3 (25D3), and 3-deoxy-1,25D3 structureefunction results in the VDR A-ring domain mutant constructs in transfected COS and CV1 cells. The table summarizes the relative competitive index (RCI) values and effective concentrations (EC50) of 1,25D3, 25D3 (1deoxy-1,25D3) and 3-deoxy-1,25D3 in COS-1 cells transfected with hVDRwt, S278A, Y143F/ S278A, S237A, R274A, and S237A/R274A VDR constructs [30,37,38]. RCI values were obtained using the cell lysate from the transfected cells in a steroid competition assay [179]. (ND) indicates that the ability of [3H]-1,25(OH)2D3 to bind the construct was too weak for an RCI to be determined. The EC50 value was obtained by cotransfecting CV1 cells with a secreted alkaline phosphatase reporter construct driven by an osteocalcin vitamin D response element [32].
VDR construct
Analog code
Relative competitive index, %
VDRwt
1,25D3
100
VDRwt
3-deoxy-1,25D3
VDRwt
Genomic transactivation EC50 (nM) 1.4 0.15 (n ¼ 21)
13 5.3
5.4 1.3 (n ¼ 3)
25D3
0.08 0.03
280 73 (n ¼ 3)
S278A
1,25D3
100
0.88 0.29 (n ¼ 3)
S278A
3-deoxy-1,25D3
Y143F/S278A
1,25D3
100
94 34 (n ¼ 3)
Y143F/S278A
3-deoxy-1,25D3
130 41
1.3 0.30 (n ¼ 3)
S237A
1,25D3
100
93 24 (n ¼ 2)
S237A
25D3
11.0 4.5
R274A
1,25D3
ND
6700 3700 (n ¼ 3)
R274A
25D3
ND
860 374 (n ¼ 2)
S237A/R274A
1,25D3
ND
21 000 64 000 (n ¼ 3)
S237A/R274A
25D3
ND
510 7.6 (n ¼ 3)
20 2.4
potential of 3-deoxy-1,25(OH)2D3 were posited to be increased in the Y143F and/or S278A mutants, because the mutation would, in theory, reduce the energetic stability of 3-deoxy-1,25(OH)2D3 in the VDR-AP and thereby increase its fractional occupancy of the VDR-GP. The actual structureefunction results were quite consistent with the predicted changes in the measured VDR affinity and genomic EC50 (Table 15.3 and [30,32,37]).
Understanding the Nongenomic Agonist/ Antagonist Affinity/Function Conundrum The vitamin D sterols, JN, 25(OH)D3, and HL, have all been shown to modulate nongenomic rapid responses when 1.0 nM of each VDS was added alone (i.e., agonist ligands, JN, and 25(OH)D3) or in the presence of 1,25 (OH)2D3 (i.e., antagonist HL). However, JN, 25(OH)D3 and HL bind to the VDR with a w50e500-fold reduced affinity, in a steroid competition assay, when compared to 1,25(OH)2D3 (Table 15.2). Thus when rapid, nongenomic responses were first identified it was unclear how 1 nM JN could stimulate and HL block nongenomic, rapid responses. These VDR affinityefunction conundrums formed the basis for this laboratory’s original hypothesis that a distinct, novel, membrane VDR
1.9 0.30 (n ¼ 5)
230 54 (n ¼ 2)
for rapid responses must exist that differs from the classic nuclear VDR [140]. Our most recent 1,25(OH)2D3, JN, 25(OH)D3, and HL molecular mechanics simulations [45] demonstrate that based on the overlay of the bound ligand poses and the potential interaction energy and Gibbs free energy of binding (DGbinding) calculations (Table 15.2), 1,25 (OH)2D3, JN, and 25(OH)D3 share similar VDR-AP physicochemical binding characteristics [30,37]. Alternately, HL is observed to form hydrogen bonds with only R274 and when bound to the VDR-AP is positioned differently with respect to the agonist ligands [1]. Lastly, 1,25(OH)2D3 is a significantly more stable VDR-GP ligand when compared to JN, 25(OH)D3, and HL (Table 15.2). Thus the models suggest that the enhanced VDRAP selectivity of JN and 25(OH)D3 (Table 15.2), when compared to 1,25(OH)2D3 is not a result of a significant increase in their VDR-AP affinity, but rather a reduced capacity to interact favorably with the VDR-GP. This is consistent with the hypothesis that the VDR-AP is kinetically favored for all VDR ligands [1]. In closing, the enhanced VDR-AP affinity of HL when compared to 1,25(OH)2D3 (Table 15.2) and its stabilization of a unique VDR-AP geometry, supports it functioning as a competitive antagonist of 1,25(OH)2D3, JN, and 25(OH)D3 nongenomic responses (Table 15.1).
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290
15. VITAMIN D STEROL/VDR CONFORMATIONAL DYNAMICS AND NONGENOMIC ACTIONS
Case Study I: Voltage-Gated Chloride Channels
Case Study II: W286R and Calcium Signaling
From the perspective of small-molecule specificity and nongenomic, rapid-response structureefunction studies, the system studied in the greatest detail is VDS potentiation of outwardly rectifying chloride channels (ORCC) of the CLC type (see above). Most recently we have extended our screening of vitamin D sterols for their ability to potentiate ORCC currents in TM4 Sertoli cells. Specifically, we have shown that 1,25(OH)2D3, JN, MK, IE (Fig. 15.5), and 25(OH)D3 all function as agonist ligands in this system (Table 15.1). The side-chain analogs MK and IE are most well known as an antagonist and superagonist, of VDR gene transcription respectively [14,141,142]. When flexibly docked to the VDR-AP and VDR-GP, MK displayed a good affinity in the VDR-AP (Table 15.2). In the VDRGP MK shows increased conformational heterogeneity when compared to 1,25(OH)2D3 [44], a result that supports the inability to form a cocrystal of the human VDRwt bound to MK [143]. The same was observed for IE, with the exception that its VDR-GP affinity was more similar to 1,25(OH)2D3 (Table 15.2). This evidence in conjunction with the results showing that 1,25 (OH)2D3, JN and 25(OH)D3 currents are blocked by coincubation with HL, clearly demonstrates that the changes in 1,25(OH)2D3 chemistry known to alter VDR genomic function do not necessarily alter VDR-dependent nongenomic potentiation of ORCC. This may also be true for other nongenomic signaling factors.
Individuals with the W286R VDR mutation do not possess the ability to stimulate VDRwt genomic responses; however, they do respond rapidly to 1,25 (OH)2D3, as manifested by an increase in intracellular calcium. Thus the W286R mutation is a natural mutation that causes vitamin-D-resistant rickets, but unlike other natural mutations which inhibit binding of the VDR to DNA, the mutation does not cause alopecia, considered a VDR genomic effect [130,144]. The conundrum here lies in the fact that 1,25(OH)2D3 binds very poorly to the W286R mutant (Fig. 15.10) yet can stimulate an increase in intracellular calcium at subnanomolar concentrations [130,144]. Thus, the change in VDR function induced by the W286R mutation is somewhat similar to that induced by the absence of the 1a-OH group or fusion of the 1,25(OH)2D3 B-ring. These topics are addressed in detail in above. Flexible docking molecular mechanics simulations show 1,25(OH)2D3 is not a good VDR-GP ligand in the W286R mutant; however, in the W286R mutant, there is no observed change in the affinity of 1,25(OH)2D3 for the VDR-AP (Table 15.4). According to the VDRPIP membrane model, when bound to a PIP, all of the known surface regions of the VDR molecule shown to interact with coregulator proteins would remain solvent exposed (Fig. 15.7). This may allow nuclear VDR-PIP to form a repressive complex with DNA and provide a basis for why patients with the W286R mutant lack
TABLE 15.4
Theoretical 1,25(OH)2D3 (1,25D3) and phosphatidylinositolphosphate (PIP) VDRwt and W286R complex scores. The table summarizes the computational results obtained when 1,25(OH)2D3 and the phosphatidylinositolphosphates, PIP2 and PIP3, were docked to the VDR using molecular mechanics. For docking 1,25(OH)2D3, the published flexible docking protocol was used [44,45], wherein 1,25(OH)2D3 conformational isomers generated using a PC_Model conformational search 3 VDR-GP or VDR-AP site spheres (see [45]). For the PIP-VDR calculation (Figs 15.2 and 15.4) were docked to a 10.0 A complexes, the PIP2 and PIP3 inositol head groups (i.e., inositol-1,4,5-triphosphate and inositol-1,3,4,5-tetraphosphate) where flexibly docked to the VDR A-ring domain (Discovery Studio 2.0, DS2.0), the phosphotriglyceride was then manually built off the highest scored complex. The resulting PIP2 or PIP3 complex was energy optimized to a convergence derivative of 0.01. As in Table 15.2, the 1,25(OH)2D3 Gibbs free energy of binding (DGbinding) values represent the average of the top seven complexes ranked using the cDOCKER scoring function of DS2.0. Alternatively the PIP DGbinding value is calculated from one complex. Based on the theoretical results the W286R mutation hinders the binding of 1,25(OH)2D3 to the VDRGP and PIP3 to the VDR-AP as deduced from the more positive (i.e., weaker affinity) DGbinding values when compared to their VDRwt results.
Ligand
VDR construct/binding pocket
DGbinding (kcal/mole)
1,25D3
VDRwt/VDR-GP
47.3
1,25D3
VDRwt/VDR-AP
37.0
1,25D3
W286R/VDR-GP
8.3
1,25D3
W286R/VDR-AP
33.7
PIP2
VDRwt/VDR-AP
178
PIP3
VDRwt/VDR-AP
233
PIP2
W286R/VDR-AP
192
PIP3
W286R/VDR-AP
92
II. MECHANISMS OF ACTION
REFERENCES
291
made and pocket selectivity observed [30,32,37,38,45]. As a consequence, each ligand can in some way mimic and/or differ from 1,25(OH)2D3 in the VDR functions it is able to modulate [1,32]. For example, 1,25(OH)2D3 [149], curcumin [150] and withaferin A [151] all inhibit NFkB; however, 1,25(OH)2D3 stimulates MAPK activity, while curcumin blocks it.
References
[3H]-1,25(OH)2D3 binding to the VDR W286R natural mutant. The graph shows the percent (%) of [3H]-1,25(OH)2D3 bound to the W286R construct. The value was obtained following incubation of expressed hVDRwt and W286R with 0.04 nM and 1.0 nM of tritiated 1,25(OH)2D3, in COS-1 cell lysate (Table 15.3). The percentage for hVDRwt was set to 100% and the amount specifically bound to W286R reported.
FIGURE 15.10
alopecia [130,144]. Thus our empirical evidence that [3H]-1,25(OH)2D3 can bind weakly to the W286R mutant (Fig. 15.10) confirms the results of Nguyen et al. [130,144]. The structureefunction and molecular modeling results provide the first model explaining why W286 patients lack alopecia. Also they provide evidence that alopecia may still occur when the VDR is occupied with a ligand and therefore not strictly a ligand-independent effect of the VDR [145,146].
VDR LIGAND SPECIFICITY: DOES AN UNLIGANDED VDR EVER EXIST IN VIVO? In the past 2 years it has become clear that botanical ligands isolated from the roots of plants (e.g., curcuminoids and withanolides, Fig. 15.5) can bind to the VDR [45]. Surprisingly, even tocopherols (i.e., vitamin E) have been shown to bind to the VDR [128]. When these results are considered with those showing that nonsteroidal synthetic analogs [147], DIDS [45], LCA [148] and 25(OH)D3, and 1,25(OH)2D3 metabolites [32] (Fig. 15.3) all bind specifically to the VDR and the computational results that suggest the VDR binds to PIPs (Fig. 15.7), it is hard to envision that the VDR is ever in a situation where it is unliganded in vivo. Perhaps these data will incline more researchers to consider the fact that when flexibly docked to the VDR-AP, VDR-GP, and/or the overlapping A-ring domain, all of the small molecules highlighted in this work show differences in the intermolecular contacts
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C H A P T E R
16 Genetic and Epigenetic Control of the Regulatory Machinery for Skeletal Development and Bone Formation: Contributions of Vitamin D3 Jane B. Lian 1, Gary S. Stein 1*, Martin Montecino 2, Janet L. Stein 1, Andre J. van Wijnen 1 1 2
Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, MA 01655, USA, Centro de Investigaciones Biomedicas, Facultad de Ciencias Biologicas y Facultad de Medicina, Universidad Andres Bello, Avenida Republica 239, Santiago, Chile
PROGRAMS OF BONE FORMATION Overview The structural and metabolic functions of bone tissue arise from its unique properties as a mineralized connective tissue. Specialized cell populations support activities that are dependent on the calcitrophic hormone 1,25(OH)2D3. This chapter will begin with a brief review of bone architecture and the interactions of functionally distinct cells. This crosstalk mediates competency for bone to respond to the physiologic signals that include the calcitrophic hormone axis, developmental osteogenic factors, secreted cytokines, and bone-specific transcription factors. Within this context of understanding bone formation and the mechanisms that control the progression of osteoblast differentiation, a major focus of the chapter will be the recent concepts that have identified (i) functional relationships between gene expression and nuclear structure in organizing the regulatory information for gene expression, (ii) the impact of epigenetic modifications of genes that control cell fate and lineage-specific phenotypes through association of transcriptional regulators with target gene loci on mitotic chromosomes, and (iii) chromatin modifications in transcriptionally active and repressed genes that are selectively influenced by Runx2 and the VDR receptor
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10016-2
complex to regulate the stage-specific expression of genes that mediate progression of osteoblast proliferation and differentiation. The dynamic integration of developmental signaling pathways and the unique genetic and epigenetic transcriptional regulators of cell growth and differentiation that coordinate skeletogenesis and the control of bone remodeling provide new dimensions for improving diagnosis and treatment of skeletal diseases.
Development of the Skeleton Inductive events for skeletal formation and patterning are governed by developmental signals that begin in the mesoderm layer to form the initial structures that develop the axial, appendicular, and craniofacial skeleton. HOX and related homeodomain proteins, clock genes, and other key transcriptional regulators (T-box proteins, Gli3, HAND2) together with Shh signaling, pattern the skeleton through integrated regulatory networks [1e5]. Recently small noncoding RNA regulation of cellular protein levels has been shown to support post-transcriptional control of organ development. Multiple lines of evidence demonstrate essential roles for microRNAs in development, organogenesis, cell growth, differentiation, and apoptosis. MicroRNA
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16. GENETIC AND EPIGENETIC CONTROL OF THE REGULATORY MACHINERY
regulation of BMP2-induced bone formation and bone mass has recently been reported [6e8]. The complexity of the multiple signaling pathways that converge to form the skeleton is first appreciated by the formation of skeletal elements through two distinct developmental pathways, intramembranous and endochondral bone formation. Recent advances have been made in understanding cellular and molecular mechanisms regulating the progression of mesodermal stem cells to the differentiated cell phenotypes that occur in mature bone. Intramembranous bone formation results from initial condensation of mesenchyme with direct differentiation of the cells into osteoblasts and gives rise to flat bones as illustrated by formation of the cranium. Growth of this tissue continues from progenitor cells within the periosteum covering the bone surface or the cranial sutures or from differentiation of mesenchymal- or stromalderived stem cells in the surrounding marrow. The process of endochondral bone formation (EBF) for the development and growth of long bones and vertebrae, involves initial formation of a cartilage tissue from condensing mesenchyme. Perichondrium develops around a cartilage anlagen and provides a future source of progenitor cells for differentiation to chondrocytes. In the core of the anlagen, chondrocytes hypertrophy producing a calcified cartilage matrix which subsequently is resorbed and replaced by bone tissue. Vascular invasion in hypertrophic cartilage leads to formation of the growth plates with clearly defined zones of proliferating and maturing chondrocytes. The growth plates spatially define the growing ends of the bone. Synthesis of a cartilage extracellular matrix competent to calcify and become vascularized is a pivotal stage of endochondral long bone growth as the calcified cartilage matrix is requisite for resorption by chondroclasts/osteoclasts. Following apoptosis of hypertrophic chondrocytes osteogenic cells are recruited to the cores of calcified cartilage for formation of trabecular bone spicules, concomitant with the resorption of the calcified cartilage. The coordination of chondrocyte maturation events at the growth plate with bone formation is tightly regulated through multiple signaling pathways, hormonal regulators, and growth factor responses to subpopulations of cells in the growth plate. These signals are transduced to intracellular intermediators and transcription factors for lineage commitment and expression of the appropriate genes for the dynamic transition from cartilage being replaced by bone formation. Both selective expression of secreted factors in chondrogenic subpopulations and feedback loops control the proliferation and maturation of chondrocytes and the pace of endochondral ossification [9] (Fig. 16.1). Two essential regulatory proteins include Indian hedgehog (Ihh), a mammalian
Regulation of endochondral bone formation (EBF). (A) Developmental signals suggest BMP/TGFb and the Wnt pathways required for specification of different cell phenotypes. These signals are ultimately transduced to the tissue-specific transcription factor that commits a cell to the indicated lineages. In addition, an important regulatory control in cell determination is through posttranscriptional mechanisms where microRNAs block translation of proteins that are required for commitment of cells to a specific lineage. Recently, BMPs have been shown to down-regulate a group of “osteomiRs” that inhibit the BMP receptors and various components of osteogenic Wnt signaling [6]. (B) Coordination of cellular activities for regulating the progression and pace of chondrocyte proliferation and differentiation at the growth plate for placement by bone tissue. The signaling pathways operative at the different growth plate zones are indicated with gene markers used to identify the different subpopulations and the key transcriptional regulators for chondrocyte maturation. Please see color plate section.
FIGURE 16.1
homolog of the Drosphila hedgehog secreted factor and parathyroid hormone-related peptide (PTHrP), which is secreted by many tissues, but has specialized autocrine and paracrine activities for regulating endochondral bone formation. PTHrP functions through the PTH (PTHrP) G-protein-coupled receptor and is abundantly expressed in the periarticular perichondrium and diffuses towards the prehypertrophic zone. Human mutations in this receptor characterize several chondrodysplasias [10]. In mice deficient in PTHrP, chondrocyte maturation is accelerated while overexpression of PTHrP stimulates chondrocyte proliferation, thereby inhibiting cartilage differentiation and delaying bone formation [11]. Ihh secretion in the hypertrophic zones increases PTHrP expression and consequently chondrocyte proliferation. In a negative loop PTHrP at the growth plate, by inhibiting the differentiation of proliferating chondrocytes to hypertrophic chondrocytes, inhibits Ihh synthesis. This intricate network ensures a synchronous timing of long bone growth. In addition,
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PROGRAMS OF BONE FORMATION
Ihh also promotes osteoblast differentiation in the bone collar and thereby Ihh coordinates expansion of the proliferative zone with bone formation [12,13]. A key event for endochondral bone formation is vascularization of the hypertrophic zone. Several in vivo studies have shown osteogenesis and angiogenesis are tightly coupled for bone formation and for bone healing. The mechanism involves activation of HIF-1a by low oxygen levels that leads to induction of VEGF (reviewed in Wan et al. [14]). Not to be overlooked is the role of 1,25 (OH)2D3 in facilitating maturation of chondrocytes at the growth plate, given that chondrocytes can support local synthesis of the hormone [15] and 1,25(OH)2D3 promotes vascularization by increasing expression of VEGF and MMP9 [16]. Both these genes are also targets of Runx2 [17]. Chondrocytes also express RANKL to facilitate osteoclastogenesis for removal of calcified cartilage and the transition to forming the primary spongiosa bone matrix [18]. Thus several distinct regulators operate on essential genes required to drive chondrocyte maturation for bone growth. Skeletal development and progression of endochondral bone growth at the hypertrophic stage of cartilage maturation producing a calcified matrix involve coupled positive and negative transcriptional control of gene expression (Fig. 16.1). Key transcriptional regulators of cell phenotypes at the growth plate include the Gli 2 and Gli 3 [19,20], the family of Sox genes, Sox 5, Sox 6 [21], and the essential Sox 9 for induction of
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the chondrocyte phenotype, Nkx3.2 a strong repressor transcription factor and one of the earliest mediators of chondrocyte commitment which allows for activation of Sox 9, required for chondrocyte differentiation [22], and Runx2 the transcription factor essential for hypertrophic chondrocyte and osteoblast differentiation [23]. Both gain- and loss-of-function studies have established that the reappearance of Runx2 and expression of Runx3 in the hypertrophic zone regulates chondrocyte maturation and vascular invasion through activation of VEGF and osteogenesis. Notably, Runx2 is expressed in prechondrogenic mesenchymal tissue prior to bone formation. A novel epigenetic mechanism has been discovered for Runx2 bookmarking of genes for activation and suppression of osseous and hypertrophic chondrocyte differentiated cells ([24]; and see below). However, suppression of Runx2 is required for onset of the chondrocyte phenotype and here Nkx3.2 initiates a cascade of events for both suppressing osteogenesis and activating chondrogenesis in mesenchyme [25,26]. Once chondrogenesis is initiated, Sox9 sustains Runx2 repression [27]. TRPS1 is another key factor repressing Runx2 in chondrocytes through a proteineprotein interaction [28]. Thus endochondral bone formation at the growth plate is highly regulated at different levels of gene regulation for promoting chondrogenesis, then derepressing the essential regulatory factors for maturation of hypertrophic cells and bone formation as indicated in Figure 16.2.
FIGURE 16.2 Regulation of osteoblastogenesis. (A) Examples of developmental (BMP/Wnt), hormonal (parathyroid hormone (PTH)), glucocorticoid (GC) and 1,25(OH)2D3 (VD3) and growth factors (IGF-1) influencing differentiation are indicated. (B) The major differentiation stages of osteogenic lineage cells are illustrated in histochemical stained cells with examples of markers for each stage. Growth e toluidine blue; ECM e extracellular matrix maturation stage showing alkaline phosphatase activity; mineralization e von Kossa stained. Markers frequently used to characterize stages of maturation include collagen type I, alkaline phosphatase, bone sialoprotein, osteopontin and osteocalcin. (C) Examples of transcription factors regulating progression of osteoblast differentiation are indicated. Please see color plate section.
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Osteogenic Signals Regulating Embryonic and Postnatal Bone Formation and Osteoblast Differentiation Multiple developmental signaling centers during embryonic development have coordinated activities with respect to location and timing to form the skeleton. Here three classes of morphogens that contribute to cell fate decisions through their downstream effects on expression of cell-specific transcription factors will be briefly reviewed. Fibroblast Growth Factor Receptors and Ligands The FGF receptors, FGFR1, FGFR2, and FGFR3 are expressed in limb mesenchyme and throughout skeletal cell populations. Several FGF ligands (FGF 2, 4, and 8) are essential for the earlier stages of limb bud outgrowth and many FGF ligands are expressed during both intramembranous and endochondral bone formation [29,30]. FGFR3 is highly expressed in differentiated chondrocytes and contributes to the regulation of progenitor and differentiated cells. The ligand FGF18, which is produced in the perichondrium and binds to FGFR3, limits the proliferation of chondrocytes, but has the opposite effect on osteoblasts, promoting their proliferation. FGFR3 appears as chondrocytes differentiate in long bones and is an important negative regulator of endochondral bone formation. The FGFR3 and PTHrP signals coordinate cartilage and bone formation [31]. Human mutations in the FGF receptors result in severe dwarfism and a spectrum of craniofacial and growth plate disorders [32]. Null mutation in the mouse leads to embryonic skeletal overgrowth [33], but osteopenia in the adult mouse [34]. Thus pre- and postnatal bone formation, as well as distinct subpopulations, are differentially regulated by the FGF receptors and ligands. Bone Morphogenetic Protein (BMP) Signaling TGFb and BMPs, members of the TGFb superfamily, are multifunctional growth and differentiation factors required at an early stage of embryogenesis for formation of the AER and dorsaleventral patterning of the limb [35] and throughout life in promoting osteogenesis from mesenchymal stem cells [36]. BMPs function through BMP1A and BMP1B type I and BMP type II receptors that transduce the signal by phosphorylating intracellular BMP-specific receptor Smad proteins (RSmads). BMPs can also signal through MEK/ERK and p38 MAPK to phosphorylate Smads. This event is followed by formation of a complex with a DNA-binding common-mediator co-Smad (Smad4) that enters the nucleus and is competent to either target gene promoters or form multimeric complexes on gene promoters with other transcription factors. Regulation of the BMP pathway is through an inhibitory Smad6
and by multiple antagonists including noggin, Dan, chordin, and gremlin [37]. The osteogenic BMPs 2, 4, and 7 are potent inducers of transcriptional regulators of bone formation and immediate early targets of BMP2 that are essential for the normal program of bone formation. These include induction of Msx, Dlx, and Hoxa10 genes which can increase expression of Runx2 and Osterix, both essential for differentiation of osteoblasts to the mineralization stage. BMPs that induce bone formation are also important for the development of other cell types forming the limb, e.g., muscle, blood vessels, etc. A compelling question is how a BMP transduces a cell-phenotype-specific outcome from a mesenchymal progenitor cell. A significant finding relevant to the osteogenic effects of BMPs is the interaction of Smad complexes with Runx2 resulting in osteoblast specificity for activation of genes characteristic of bone formation [38,39]. Runx2 is induced within a few hours of BMP2 treatment and together have synergistic effects in promoting osteogenesis [40,41]. BMPs induce a spectrum of other transcription factors essential for differentiation of skeletal tissues, including Sox genes which are required for chondrogenesis and C/EBP that uses Schnurri as a docking protein to Smad4 for adipogenesis and osteogenesis [42,43]. Thus, the chondrogenic and the osteogenic activity of BMPs are related to induction of specialized transcriptional regulators of cell differentiation and physical proteineprotein interaction between a tissue-specific transcriptional regulator and the BMP Smad provides for tissue-specific effects of BMPs. Wnt Signaling and Skeletal Development The Wnt pathway has many activities including directing cell fate during vertebrate development and regulating bone mass in the postnatal skeleton [44,45]. During embryogenesis, the b-catenin mediator of canonical Wnt signaling has been identified as a key regulator of formation of the apical ectodermal ridge (AER). Wnt ligands have specific functions in the developing limb (reviewed in Yang et al. [46]). Wnt7a contributes to establishment of the dorsaleventral axis of the limb; Wnt3a is required for somite formation and Wnt14 functions in induction of joint interzones. Other ligands regulate endochondral bone formation. Wnt5a and Wnt5b coordinate the pace and transitions between chondrocyte zones. Wnt5b which is expressed in the prehypertrophic chondrocyte zone, as well as in joints and perichondrium, delays hypertrophy, while Wnt4 blocks initiation of chondrogenesis, but accelerates hypertrophy. Wnt10b is highly anabolic for trabecular bone, in part by inhibiting adipogenic differentiation of mesenchymal cells [47]. Therefore, the developing limb and endochondral bone formation which both rely on the regulation of the proliferation, maturation,
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and spatial organization of chondrocytes, are processes highly dependent on Wnt signals. Wnt proteins bind to and activate receptor complexes consisting of the Frizzled family of G-protein-coupled receptors and the low-density lipoprotein (LDL) receptor-related proteins (LPR5/6) [48]. Although Wnt signaling can be transduced through three pathways [49], it is the canonical pathway through intracellular b-catenin that has decision-switching activities driving bipotential chondro-osseous progenitor cells into osteogenesis and with potent anabolic effects on bone formation in the adult skeleton. Wnt ligands binding to the LRP5/6 complex result in stabilization of b-catenin by inhibiting its phosphorylation involving casein kinase 1 and glycogen synthase kinase 3 within a protein complex, which prevents the targeting of b-catenin for ubiquitination/proteasome degradation. Subsequently, b-catenin is translocated into the nucleus to form heterodimers with the TCF1 or LEF transcription factors for expression of Wnt-responsive genes. In the absence of nuclear b-catenin, TCF/LEF is associated with transcriptional corepressors and suppresses Wnt target genes. The Wnt signaling pathway is regulated by several antagonists. Mouse models have revealed varying degrees of anabolic effects on the skeleton by deletion of Wnt antagonists. DKK (Dickkopf) and its associated coreceptors Kremen-1 and Kremen-2 [50] interact with the LRP5/6 receptor complex preventing transduction of the Wnt signal, while a class of secreted frizzledrelated proteins (SFRPs) interacts with Wnt proteins sequestering them from interaction with frizzled receptors, as does WIF (Wnt inhibitory factor-1) and Cerberus [51,52]. Null mutation of the antagonist sFRP1 in mouse, stimulates Wnt signaling in osteoblasts and results in a high bone mass in the mouse adult skeleton with a delay in bone loss in ages 7e9 months [52]. Another key inhibitor of Wnt signaling, produced by the osteocyte, is sclerostin, encoded by the Sost gene [53]. Sclerostin inhibits BMP-induced osteoblast differentiation and prevents Wnt signaling by binding to LRP5. These findings underscore the significance of Wnt signaling in maintaining bone mass in the adult skeleton. The lineage direction of a multipotential mesenchymal stem cell to commit to chondrogenic or osseous lineage is determined by cellular b-catenin levels. By increasing Wnt signaling, e.g., by ectopic or by expressing a stabilized form of b-catenin, produces enhanced ossification and suppression of chondrogenesis (reviewed in Hartmann et al. [54]). Complete ablation of b-catenin results in severe loss of bone from inhibited osteoblast maturation and increases osteoclast differentiation. The potent anabolic effects of canonical Wnt signaling for bone formation were revealed by an activating mutation (gain-of-function) in the Wnt coreceptor LRP5 resulting in the high bone mass trait in humans.
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The LRP5 loss-of-function mutation leads to an osteopenia accompanied by fractures in humans causing osteoporosis pseudoglioma syndrome (OPPG). These phenotypes are reproduced in mouse models [45]. Crosstalk between Wnt and early developmental signals that include BMPs and FGFs are known [55,56] and beginning to be addressed in bone. The effects can be synergistic or antagonistic and the mutual inhibition of each pathway may be a mechanism for supporting proliferative expansion of a cell population, followed by its differentiation. Many physiological regulators interface with Wnt signaling. For example, R-Spondins are secreted proteins which amplify the Wnt signal with Wnt ligands and the LRP5/6 receptor, and antagonize DKK1 activity [57]. Glucocorticoids have multiple repressive effects on Wnt signaling [58,59], while 1,25 (OH)2D3 stimulates Wnt activity through transcriptional activation of LRP5 [60] or by inhibiting the Wnt antagonists, DKK1 and SFRP2 [61]. PTH exerts its anabolic effects on bone in part through inhibition of sclerostin [62]. Clearly there are many components of Wnt signaling contributing to bone anabolism. Thus, the potential for stimulating canonical Wnt signaling as a therapeutic approach for bone loss safely, without the risk of forming tumors, could be achieved. For a discussion of targets being considered see Wagner et al. [63].
Transcriptional Control of Osteoblast Differentiation The progression of osteoblast differentiation requires the sequential activation and suppression of genes that encode phenotypic proteins and regulatory factors. Signaling molecules (morphogens, growth factors) indirectly result in a cascade of gene expression through induction of transcription factors that directly engage in proteineDNA as well as proteineprotein interactions [64]. A number of key studies have defined master genes that direct a pluripotent cell to different lineages as illustrated in Figure 16.2. Master genes are retained during mitosis and may provide a mechanism for epigenetic regulation of genes that retain the phenotype lineage properties of dividing cells [65]. These have been the subject of many reviews that have emphasized the reciprocal regulation between adipogenesis and osteogenesis [66], as well as chondrogenesis and osteogenesis [27]. Following commitment to osteogenesis by Runx2, a number of transcriptional regulators are required to complete the differentiation program to the mature osteocyte, while others regulate the timing for expression of genes that characterize the major stages of osteoblast maturation (Fig. 16.2). Mechanisms that are operative at this level of gene regulation include the interaction of transcription factors with co-regulatory proteins that modify the chromatin organization of the gene ([67]
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and see chapters in Section II for detail); the switching of members of transcription factor families at cognate regulatory elements as occurs with AP-1 and homeobox proteins; and the interaction of transcription factors with intracellular intermediates in response to physiological regulators or interactions with other neighboring factors on a gene promoter. During stages of osteoblastogenesis, certain classes of transcription factors are restricted to specific subpopulation of bone cells. Negative regulators of bone formation are highly expressed in proliferating cells. The helixloop-helix (HLH) family of proteins (Twist, Inhibitor of differentiation (Id), Scleraxis, Snail) are examples of negative transcriptional regulators at early stages of osteoblast development, but necessary for osteoprogenitor cell expansion. They are expressed in mesoderm tissue of developing embryo, but not detected at the onset of ossification [68]. The restricted expression of these factors in proliferating osteoblasts in vitro is consistent with their required down-regulation for differentiation to proceed. Twist transiently inhibits Runx2-mediated transcription by interacting with the Runx2 DNA-binding domain [69]. Snail represses both Runx2 and the VDR [70]. Thus, in vivo deficiency of these proteins results in premature osteoblast differentiation [69]. Functioning at later stages is Osterix, a bone-restricted SP1 family member (SP7) [71]. ATF4, based on a null mutation in the mouse, contributes significantly to the mineralization stage, but largely through cooperation with Runx2 and SATB2 factors [72]. The transcription factor FOXO1 was recently identified as promoting bone formation through multiple mechanisms by indirectly influencing p53 effects on bone and insulin secretion [73]. However, more direct effects on osteoblast differentiation indicate that FOXO1 directly contributes to Runx2 transcription and also interacts with ATF4 [74,75]. A typical molecular mechanism for control of gene expression involves a change in heterodimerization proteins to form either transactivating or repression complexes to influence competency for transcription of the genes at specific stages of osteoblast maturation. AP-1 factors include fos (c-fos, fra1, fra2) and jun (c-jun, jun-D, jun-B) oncogene-encoded transcription factors. These proteins regulate cell cycle and differentiation-related genes in cartilage and bone by forming homo- or heterodimeric complexes with their family members, and with other transcription factors, that include ATF members [63]. Genetic studies have established that several AP-1 family members are essential for normal bone development and osteoblasts differentiation (reviewed in Wagner et al. [63]). The concept of a regulatory network of transcription factors for osteoblast differentiation is best illustrated by the Msx2, Dlx3, and Dlx5 homeodomain (HD) proteins which
selectively associate with gene promoters at different stages of maturation generating molecular switches for repression and activation [76,77]. Msx2, restricted to proliferating and apoptotic cells, is followed by up-regulation of Dlx3 and Dlx5 expression in postproliferative cells. The first switch is Msx2 bound to osteocalcin in proliferating cells, but when transcripts are expressed by Runx2 binding to the promoter, Dlx3 replaces Msx2 at the conserved “OC Box” HD element. A second switch in HD protein occupancy at the OC promoter occurs at the mineralization stage with replacement of Dlx3 by Dlx5 binding to the gene [76]. In conclusion, these studies have provided evidence for coordinated control of gene expression throughout bone formation by family members of a class of transcription factors that form regulatory networks on promoters to regulate the timing of expression. Examples of cooperative effects between different transcriptional regulators are the requirements of Runx2 for mediating hormone responsiveness. Runx2 and cfos interaction is necessary for PTH responsiveness on the MMP13 promoter [78]. Large multimeric complexes are formed at steroid hormone response elements with Runx2 and the vitamin D receptor (VDR) (detailed for the osteocalcin gene below), and also with the androgen receptor [79]. The steroid hormone receptors recruit chromatin-remodeling factors that can bridge with transcriptional complexes at neighboring elements. In this manner, physiologic control of gene expression is coordinated among distinct regulatory elements on promoters and in a tissuespecific manner.
NUCLEAR ORGANIZATION OF THE REGULATORY MACHINERY FOR BONE FORMATION Overview Bone formation during development and skeletal remodeling throughout life requires the complex and interdependent expression of cell growth and phenotypic genes as reviewed above. There is a requirement for responsiveness to a broad spectrum of regulatory cues that transduce physiological signals from the extracellular matrix to sites within the nucleus where genes that mediate skeletogenesis reside [80,81]. As our understanding of gene-regulatory mechanisms expands, it becomes increasingly evident that there are unique parameters of transcriptional control that support the transient activation and suppression of genes for skeletal development and bone homeostasis. This section will discuss the concept that key components of the basal transcription machinery and several tissue-specific
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transcription factor complexes are functionally compartmentalized as specialized subnuclear domains [82]. Such compartmentalization may, at least in part, accommodate biological constraints; for example, the low representation of promoter regulatory elements and cognate transcription factors necessitate a subnuclear organization of nucleic acids and regulatory proteins that supports threshold concentrations for the activation and repression of gene expression. Other mechanisms are invoked for long-term obligations to gene expression that sustain the specialized properties of bone cells. Here epigenetic regulation is important for control of genes that govern cell fate and lineage commitment [83,84]. From a historical perspective, compartmentalization of the regulatory machinery for ribosomal genes in nucleoli and the organization of chromosomes during mitosis provide paradigms for intranuclear localization of genes and regulatory complexes. To be presented are novel epigenetic components that control gene expression. These were first identified in osteoblasts and strikingly reflected by retention of lineage-specific transcription factors at target gene loci during mitosis, thereby sustaining competency for gene expression that is linked to phenotype [85,86]. The recognition that master transcription factors associate with chromosomes during mitosis represents another level of epigenetic control in regulating a cell phenotype and ribosomal genes [65,87]. Lastly, nucleic acids and cognate regulatory factors are organized in subnuclear domains in nondividing cells which facilitate functional interrelationships between nuclear structure and gene expression. This architecturally associated organization of genes and regulatory machinery is a mechanism contributing to the spatial distribution of transcription factors within the dynamic three-dimensional context of the nucleus. Nuclear architecture controls the sorting of regulatory information and the transcriptionally competent or repressed chromatin configuration of gene promoters. There is growing appreciation for the repertoire of factors that influence gene expression for commitment to the osteoblast lineage and differentiation to the mature osteoblast (see Fig. 16.2). It is well documented that sequentially expressed genes support progression of osteoblast differentiation through developmental transition points where responsiveness to phosphorylation-mediated regulatory cascades determine competency for establishing and maintaining the structural and functional properties of bone cells [88]. The catalogue of promoter elements and cognate regulatory proteins which control skeletal gene expression is extensive and expanding. Two of the most thoroughly studied gene promoters, Runx2 and Osteocalcin, serve as regulatory infrastructure by functioning as blueprints for responsiveness to the flow of cellular regulatory signals
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(Fig. 16.3). These transcriptional regulators offer essential but insufficient insight into mechanisms that are operative in intact cells. To access specific genetic information necessitates understanding transcriptional control of skeletal genes within the context of the focal subnuclear organization of nucleic acids and regulatory proteins. Explanations are required for (1) convergence of multiple regulatory signals at promoter sequences; (2) the integration of regulatory information at independent promoter domains; (3) selective utilization of redundant regulatory pathways; (4) thresholds for initiation or down-regulation of transcription with limited intranuclear representation of promoter elements and regulatory factors; (5) mechanisms that render the promoters of cell growth and phenotypic genes competent for proteineDNA and proteineprotein interactions in a physiologically responsive manner; (6) the composition, organization, and assembly of sites within the nucleus that support transcription; (7) the intranuclear trafficking of regulatory proteins to transcriptionally active foci; and culminating in (8) crosstalk within and between regulatory networks that mediate physiological responsiveness. Figure 16.4 illustrates the impact of Runx2 on responses of the osteoblasts by multiple physiological signals. Hence, a mechanism that involves the intricate nature of the nuclear scaffold would provide for integrating multiple cues on a gene promoter in an architectural context. Runx2 mediates these responses integrating bone with other organ systems as both a transcriptional regulator and a coregulatory partner with other factors. Vitamin D serves as a principal modulator of skeletal gene transcription necessitating an understanding of the activity of this steroid hormone with regulatory cascades that are functionally linked to the regulation of skeletal genes [89]. Vitamin D control of gene expression serves as a paradigm for experimentally addressing mechanisms that govern the intranuclear targeting of regulatory factors to nuclear domains where chromatin remodeling and transcription of developmental as well as tissue-specific genes occur. We will discuss the cellular, molecular, biochemical, and genetic evidence that provides insight into the trafficking of regulatory factors that mediate vitamin-D-controlled gene expression to transcriptionally active subnuclear sites. Examples will be presented that suggest modifications in the intranuclear targeting of transcription factors abrogate competency for vitamin D control of skeletal gene expression during development and fidelity of gene expression in tumor cells. Together these studies provide evidence for an obligatory relationship between sites within the nucleus where regulatory complexes assemble and reside with fidelity of transcriptional control to support gene expression.
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Bone-regulatory elements in osteoblast gene promoters. Two well-characterized genes, the bone-specific osteocalcin maximally expressed in mature osteoblasts and Runx2, the bone essential transcription factor which is expressed early in mesenchymal cell-osteoprogenitor lineage, but increases in differentiated osteoblasts, are shown. Top panel e osteocalcin has two DNase hypersensitive domains, the distal vitamin D response transcriptional domain and the proximal promoter regulated by developmental factors. Lower panel e in the Runx2 promoter, the majority of transcriptional activity resides in proximal promoter. Both genes have multiple Runx sites which mediate activation of osteocalcin expression in nonosseous cells. For Runx2 autoregulation, the Runx sites mediate enhancement of Runx2 levels in progenitor cells, but suppression of Runx2 in mature osteoblasts. One distinct difference between osteocalcin and Runx2 is the positive enhancement of osteocalcin expression by 1,25(OH)2D3 with up to a several-hundred-fold induction. In contrast, the hormone has a very modest affect on Runx2 and mediates down-regulation of the gene. The osteocalcin gene has one highly conserved homeodomain response element in the Hox site in close proximity to Runx2, while the Runx2 gene has multiple homeodomains and Hox regulatory elements (not all are indicated), reflecting the importance of this regulation for expression of Runx2 early in the osteoblast lineage.
FIGURE 16.3
FIGURE 16.4 Signaling pathways that are transduced to gene expression through Runx2. Examples are shown in which Runx2 is posttranslationally modified by phosphorylation which alters its transcriptional activity on target genes. A second mechanism by which Runx mediates physiologic signals is through complex formation with intracellular receptors including Runx2eSmad complexes, Runx2 with WW domain proteins, steroid receptors and other transcription factors including AP-1 family members. These modifications can lead to either activation or repression on target genes that are in part dependent on promoter context of the regulatory element. Suppression of Runx2 activity can also result from interactions with proteins that E3 ubiquitin ligase activity, for example, Smurf-1 in response to TGFb signaling that decreases Runx2 cellular levels through proteosomal degradation.
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EPIGENETIC MECHANISM FOR LINEAGE COMMITMENT A fundamental question is whether regulatory complexes are retained during mitosis or are synthesized and reorganized in progeny cells immediately following cell division. The necessity for skeletal proliferation during development and to support bone remodeling as well as fracture repair on a continuous basis requires proliferation of lineage-committed skeletal regenerative cells. This process is therefore essential for epigenetically sustaining the structural and functional integrity of regulatory machinery for bone-tissue-specific gene expression. The principal components of epigenetic regulation of genes, that is the signature of chromatin that contributes to “status without transcription,” include (1) DNA methylation for silencing genes, (2) histone modification that alters the arrangement of nucleosomes, and (3) RNA-mediated gene silencing through Xist [90e93]. However, there are now examples of the growing dimensions to epigenetic control that were discovered from the study of Runx and other tissue-specific transcription factors. Consistent with the requirement for epigenetically mediated persistence of skeletal gene expression, the Runx2 transcription factor that is a master regulator of the bone phenotype has been shown to be: (1) maintained during mitosis as focal regulatory complexes [85] and (2) associated with target gene loci on mitotic chromosomes [86]. Runx2 is also retained at focal nuclear microenvironments in postmitotic progeny cells as sites for the organization and assembly of regulatory machinery for bone-tissue-specific gene expression in a manner that reflects the intranuclear organization of RNA polymerase II regulatory machinery during the G2 phase of the cell cycle prior to mitosis [24] (Fig. 16.5). This mitotic retention figure visualizes the association of Runx2 with functional nuclear structures. There is growing support for epigenetic retention of lineage-committed transcription factors for control of genes transcribed by RNA polymerase II, as well as by RNA polymerase I (ribosomal genes), providing a mechanistic basis for coordinating control of cell growth and phenotype [65]. The functional activity of Runx2 when associated with chromosomes and nucleolar organizing regions (NORs) in mitosis is akin to a “bookmarking” function to facilitate immediate regulation of phenotypic genes and protein synthesis after completion of mitosis. This novel dimension to epigenetic control that supports retention of phenotype competency extends beyond bone-tissue-specific gene expression. Recent results indicate that a similar mitotic retention of transcription regulatory machinery at target gene loci of mitotic chromosomes is operative for myoD, CEBP,
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Runx1, and Runx3, illustrating an architectural epigenetic mechanism for sustaining the muscle, adipocyte, hematopoietic, neural, and GI phenotypes [65,94]. Retention of the AML/ETO transformationefusion protein at target gene loci of mitotic chromosomes in AML leukemia suggests transcription-factor-mediated epigenetic maintenance of the AML/leukemia phenotype [95]. The function of Runx2 association with mitotic chromosomes and ribosomal genes without effecting transcription represents a “book-marking” activity. When the cell exits mitosis, Runx2 is prepared to regulate phenotype genes and protein synthesis in the interphase nucleus. Recognizing that all transcription factors are not retained on target genes during mitosis suggests that transcription-factor-mediated epigenetic control is selective and that additional regulatory mechanisms contribute to retention of cell fate and lineage commitment during proliferation. It is also necessary to extend understanding of the extent to which coregulatory factors, both coactivators and corepressors, are retained as mitotic complexes. The initial indication is that while mitotic persistence of coregulatory factors occurs, the extent to which this component of control is operative remains to be determined.
GENE EXPRESSION WITHIN THE THREEDIMENSIONAL CONTEXT OF NUCLEAR ARCHITECTURE The primary level of nuclear organization, the representation and ordering of genes and promoter elements, provides many options for physiological control. The molecular organization of regulatory elements, the overlap of regulatory sequences within promoter domains, and the multipartite composition of regulatory complexes increase options for responsiveness. Chromatin structure and nucleosome organization reduce distances between regulatory sequences, facilitate crosstalk between promoter elements and render elements competent for interactions with positive and negative regulatory factors [96e98]. The components of higherorder nuclear architecture that includes nuclear pores, the nuclear matrix, and subnuclear domains contribute to the subnuclear distribution and activities of genes and regulatory factors (reviewed in [82,84]). Compartmentalization of regulatory complexes is illustrated by focal organization of PML bodies [99], Runx bodies [100,101], the nucleolus, and chromosomes [102], as well as by the punctate intranuclear distribution of sites for replication [103], DNA repair, transcription [104,105], and the processing of gene transcripts [106]. The biological relevance for the intranuclear distribution of regulatory complexes is directly reflected by
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FIGURE 16.5 Novel epigenetic mechanisms for maintenance of cell phenotypes. This figure illustrates the concepts for Runx2 epigenetic functions, but have been identified for other tissue-specific transcription factors including myoD (muscle) and C/EBPa (adipocytes) [65]. (A) In contrast to ubiquitous transcription factors which are degraded during mitosis, Runx2 is retained in dividing cells and during telophase can still be visualized as a punctate foci with both small and large associated with chromosomes in metaphase. Large foci are localized to the nuclear organizing region (NOR). Following mitosis, Runx2 is restored to the interphase nucleus. (B) Higher magnification of the interphase nucleus shows Runx is associated with genes in transcriptional complexes bound to the nuclear matrix scaffold (left panel). Runx2 larger foci are seen in the periphery of the nucleolus (right panel). Studies have shown that Runx2, myoD, and C/EBPa contribute the regulation of protein synthesis through binding to regulatory elements in ribosomal RNA genes. Indeed, over 40e50 Runx regulatory elements can be found in promoters of rRNA genes [65,85]. Identification of Runx2 in nucleoli involved in transcription is confirmed by colocalization of Runx2 with upstream binding factor (UBF), essential for regulating ribosomal genes. (C) Illustration of the regulatory events in mitosis and interphase involving cell-typespecific transcription factors. These factors are “bookmarking” genes during mitosis for retaining phenotype fidelity after cell division; in the interphase nucleus, these tissue-specific transcription factors are contributing to protein synthesis regulation. Please see color plate section.
aberrant nuclear structureegene expression interrelationships that are associated with perturbations in skeletal development [107] and leukemia [108,109]. Association of osteoblast, myeloid, and lymphoid Runx transcription factors that mediate tissue-specific transcription with the nuclear matrix has permitted direct examination of mechanisms for targeting regulatory proteins to transcriptionally active subnuclear
domains [88]. Multiple lines of evidence support the biological specificity of these observations and are consistent with the concept that the nuclear matrix is functionally involved in gene localization and in the concentration and subnuclear localization of regulatory factors. Localization of Runx was established by both biochemical fractionation and in situ immunofluorescence, as well as by green fluorescent protein tagged
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Runx proteins in living cells. It has been shown that Runx transcription factors exhibit a punctate nuclear distribution (visualized in Fig. 16.5) that is associated with the nuclear matrix in situ. Colocalization of Runx1, 2, and 3 at nuclear matrix-associated sites indicate a common intranuclear targeting mechanism may be operative for the family of Runx transcription factors [110]. Association of osteogenic and hematopoietic Runx proteins with the nuclear matrix is independent of DNA binding and requires a nuclear matrix targeting signal, a 31-amino-acid segment near the C-terminus that is distinct from nuclear localization signals. The nuclear matrix targeting signal (NMTS) functions autonomously and is necessary as well as sufficient to direct the transcriptionally active Runx transcription factors to nuclear matrix-associated sites where gene expression occurs [111]. Transcriptionally active Runx proteins associate with the nuclear matrix but inactive C-terminally truncated Runx proteins do not [112]. Mice homozygous for the deletion (Runx2DC) do not form bone due to perturbed maturation or arrest of osteoblasts [107]. Heterozygotes do not develop clavicles, but are otherwise normal. These phenotypes are indistinguishable from those of the Runx2 homozygous and heterozygous null mutants, indicating that the intranuclear targeting signal is a critical determinant for function. The expressed truncated Runx2DC protein enters the nucleus and retains normal DNA-binding activity in the N-terminal, but shows complete loss of intranuclear targeting. These results establish that the multifunctional N-terminal region of the Runx2 protein is not sufficient for biological activity. Our results demonstrate that subnuclear localization of Runx factors in specific foci together with associated regulatory functions is essential for control of Runx-dependent genes involved in tissue differentiation during embryonic development. In summary, there is emerging recognition that nuclear structure and function are causally interrelated. Steroid receptor transcriptional regulators also exhibit punctuate foci in nuclear matrix domains. The glucocorticoid receptor was well established to function in the nuclear matrix compartment [104,105], and recently, it has been also reported that the VDR exhibits a punctate distribution in the nucleus that is enhanced upon ligand stimulation [113,114], indicating that VDR is interacting with components of the nuclear architecture in osteoblastic cells. Moreover, we have found that VDR binds to the nuclear matrix rapidly after addition of the ligand and does not require a functional VDR DNA-binding domain. Also a significant fraction of the nuclear matrix-bound VDR molecules are found to colocalize with the transcriptional coactivator DRIP205. A mechanism for targeting the VDR to subnuclear compartments remains still undefined. In addition, it is necessary to establish whether specific signaling
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pathways, including those activated by vitamin D through nongenomic actions at the cell plasma membrane [115], also contribute to VDR subnuclear localization. These findings indicate mechanisms involved in the selective trafficking of proteins to specialized domains within the nucleus where they become components of functional regulatory complexes. At least two trafficking signals appear to be required for subnuclear targeting of Runx and other transcription factors; the first supports nuclear import (nuclear localization signal) and a second mediates association with the nuclear matrix (nuclear matrix targeting signal). Targeting of Runx transcription factors to the nuclear matrix is important for their function and transcription. However, components of the nuclear matrix that function as acceptor sites remain to be established. With mounting evidence for organization of nucleic acids and regulatory proteins into subnuclear domains that are associated with components of nuclear architecture, the perception of a dichotomy between nuclear architecture and control of gene expression is difficult to justify. Rather, it is necessary to design experiments to define mechanisms that direct genes and regulatory factors to sites within the nucleus where localization integrates regulatory parameters of gene expression.
CHROMATIN REMODELING FACILITATES BONE-SPECIFIC AND VITAMIN-D-MEDIATED PROMOTER ACCESSIBILITY AND INTEGRATION OF REGULATORY ACTIVITIES It is well recognized that genomic DNA is packaged as chromatin, which can be structurally remodeled to accommodate requirements for transcription, emphasizing the extent to which architectural organization of genes is causally related to functional activity. The identification and characterization of proteins that catalyze histone acetylation, deacetylation, methylation, and demethylation [96,116] as well as the SWI/SNFrelated proteins [117e119] that facilitate chromatin remodeling and potentially the accessibility of promoter sequences to regulatory and coregulatory factors, represent an important dimension in control of the structural and functional activities of genes and promoter regulatory elements [120e123]. Additional levels of specificity are provided by structural modifications of gene promoters that influence competency for factor interactions. Simply stated, changes in the architectural properties of promoter elements determine effectiveness of gene regulatory sequences as substrates for interactions with regulatory factors. The regulatory and regulated parameters of chromatin
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remodeling and the rate-limiting steps in the relevant signaling cascades that are being actively pursued will unquestionably provide insight into skeletal gene regulatory mechanisms from structural and functional perspectives [124]. A specific chromatin-remodeling process at the P1 promoter region of the Runx2 gene is associated with transcriptional activity in osteoblastic cells [125]. This alteration in chromatin structure is reflected by the presence of two DNase I hypersensitive sites that span key regulatory elements within the first 400 bp of the Runx2 P1 promoter. This process of chromatin reorganization involves increased levels of histone H3 and H4 acetylation and is independent of SWI/SNF-mediated chromatin remodeling activity [125] (see Fig. 16.3). Highly relevant to bone cell biology and physiology is that the relationships of regulatory signaling pathways to enhance activities that modulate gene, chromatin, and chromosome organization can now be directly investigated [121,125,126]. The modularly organized promoter of the bone-specific osteocalcin gene contains proximal and distal regulatory elements that support basal, tissue-specific as well as growth factor, homeodomain, signaling protein, and steroid-hormone-responsive transcriptional control (reviewed in Montecino et al. [89]) (see Fig. 16.6). Remodeling of osteocalcin chromatin requires physiologically responsive accessibility of these proximal and upstream promoter sequences to regulatory and coregulatory proteins as well as proteineprotein interactions that integrate independent promoter domains. The nuclear matrix-associated Runx transcription factors contribute to the control of skeletal gene expression by sequence-specific binding to promoter elements of target genes and serving as scaffolds for the assembly and organization of coregulatory proteins that mediate biochemical and architectural control of promoter activity. The chromatin organization of the osteocalcin gene illustrates dynamic remodeling of bone-related promoters to accommodate requirements for lineagespecific developmental and steroid-hormone-responsive
= FIGURE 16.6 Chromatin remodeling of the osteocalcin gene. Factors that support basal tissue-specific transcription are recruited to the OC gene promoter and are organized in proximal and distal promoter domains for OC gene transcription. A positioned nucleosome resides between the proximal basal and distal enhancer regions of the promoter. The nuclease hypersensitive sites reflect the modifications in chromatin structure that facilitated the assembly of the regulatory complexes mediating regulated levels of OC during progression of the osteoblast phenotype (upper panel). Recruitment of Runx2 to the gene promoter is essential for transcriptional activation.
Low levels in the post-proliferative osteoblast are due to corepressor complexes with Runx2; in the differentiated AlkP positive cell, Runx2 complexes with coactivators and together with the down-regulation of other transcription factors that suppress osteocalcin (e.g., Msx2) allow for increased expression. Middle panel e in response to 1,25(OH)2D3, chromatin remodeling renders the upstream VDRE competent for binding the VDR/RXR heterodimer with its cognate element for maximal expression of osteocalcin in mature osteoblasts. Higher-order chromatin organization permits crosstalk between basal transcription machinery and the vitamin D receptor complex that involves direct interactions of the vitamin D receptor, Runx2, and TFIIB. Lower panel e there is an exchange in VDR coregulatory factors for fine tuning 1,25 (OH)2D3 enhanced levels of osteocalcin (see text).
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activity. Reduced CpG methylation is associated with transcriptional activation of the bone-specific osteocalcin gene in osteoblasts [127]. Nuclease digestion and chromatin immunoprecipitation analyses as well as in vitro nucleosome reconstitution studies establish the placement of nucleosomes in the proximal basal/ tissue-specific domain and at the upstream vitamin-Dresponsive element of the osteocalcin gene, blocking accessibility of these promoter sequences to regulatory proteins when this gene is not expressed or in immature bone cells when this skeletal-restricted gene is suppressed ([125] and reviewed in Montecino et al. [89]). Opening of the chromatin for accessibility to transcriptional activation occurs in sequential stages (Fig. 16.6). In response to developmental cues and skeletal regulatory signals, the striking positioning of a nucleosome and modifications in chromatin structure render the promoter regions of the osteocalcin genes accessible to regulatory and coregulatory proteins that support basal tissue-specific transcriptional activity (refer to Fig. 16.3). The most compelling evidence for a functional involvement of chromatin organization in skeletal gene expression is the obligatory relationship of dynamic changes in the biochemical and structural properties of osteocalcin gene promoter organization with competency for bonetissue-restricted and -enhanced transcription in response to vitamin D. Vitamin-D-linked remodeling of osteocalcin gene promoter organization is accompanied by acetylation of histones in the proximal basal and upstream vitamin-D-responsive element domains [128,129]. This post-translational modification of histone proteins reduces the tenacity of histone DNA interactions in a manner that is conducive to an open chromatin organization with increased access to regulatory factors. Vitamin D enhancement of osteocalcin gene transcription is also associated with alterations in the chromatin organization at the upstream vitamin-D-responsive element that facilitate binding of the vitamin D receptorassociated regulatory complex that includes many coactivators [130]. Thus, interaction of the vitamin D receptor at the distal promoter region of the bonespecific osteocalcin gene requires nucleosomal remodeling [89,131]. The retention of a nucleosome between the proximal and upstream enhancer domain reduces distance between the basal and vitamin-D-responsive elements and supports a promoter configuration that is conducive to proteineprotein interactions between the vitamin D receptor and the basal transcriptional machinery. Demonstration of functional interactions between the VDR and flanking Runx proteins is consistent with linkage of VDRE organization and Runx regulatory elements in cooperating to modify chromatin for full transcriptional activity [132]. In vitro and in vivo genetic approaches have demonstrated that Runx2
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controls developmental and steroid hormoneresponsive chromatin reconfiguration of the osteocalcin gene promoter [133,134]. Site-directed mutagenesis of osteocalcin genes that are genetically integrated in stable cell lines have established that Runx elements flanking the proximal and upstream promoter sequences are responsible for developmental, growth factor, and hormone-regulated transcription-induced chromatin remodeling [133]. This functional association between VDR and Runx2 appears to be evolutionary conserved and tightly associated with the osteoblastogenesis process [135]. Thus, insight into control of skeletal gene expression can be obtained from the understanding of mechanisms that alter the chromatin organization of the Runx2 and osteocalcin genes under biological conditions. Yet, despite the cogent support for a central role of chromatin remodeling in transcriptional control of the osteocalcin and Runx2 bone-related genes, there are open-ended questions. It is not justifiable to extrapolate from these findings to conclude that all genes activated and suppressed during skeletogenesis employ identical mechanisms. From a broader biological perspective there are multiple levels of control that must be mechanistically characterized to explain physiologically responsive regulation of chromatin structure within restricted and global genomic contexts. The now-available genome-wide approaches for detecting chromatin modifications are providing new insights for long-range effects of vitamin-D-mediated transcriptional control [124].
SCAFFOLDING OF REGULATORY COMPONENTS FOR COMBINATORIAL CONTROL OF GENE EXPRESSION Functional interrelationships between nuclear structure and gene expression are strikingly reflected by dual recognition of regulatory proteins, such as Runx transcription factors, for interactions with coregulatory proteins that modulate the structural and functional properties of targeted genes at microenvironments within the nucleus. The placement of Runx proteins at strategic sites where they provide scaffolds for proteine protein interactions and sequence-specific interactions with promoter elements results in the organization of machinery for a broad spectrum of regulatory requirements. Coregulatory proteins include enzymes for histone modifications and chromatin remodeling that establish competency for transcription factor binding (Fig. 16.7); and genomic conformations that interface activities at proximal and upstream promoter domains. Interactions with several transcription factors result in the formation of multimolecular complexes that regulate expression of a broad spectrum of genes [136]. Other
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FIGURE 16.7 Runx2 coregulatory factor interactions. (A) Runx2 interacts with different classes of coregulatory proteins as indicated to mediate cell signaling, growth control, and modulate expression of target genes during osteoblast differentiation. (B) Runx protein domains are illustrated indicating the key coregulatory interactions contributing to Runx2 functional activity. The DNA-binding partner CBFb is essential for Runx2 activity. Null mutations of CBFb lead to skeletal defects (see review by Komori [23]). TGFb and BMP signaling that leads to interactions between Runx2 and Smads is essential for maximal bone-specific activity of Runx2. It is significant that the interacting domain for Smad2 overlaps the nuclear matrix targeting signal (NMTS). Runx recruits Smads from the nucleus to the nuclear matrix scaffold [38]. (C) Shown below are Runx2 foci associated with the nuclear matrix scaffold following removal of soluble chromatin (shown in the electron micrograph).
factors include the intracellular mediators of signaling pathways that activate or suppress gene expression in a physiologically responsive manner, thereby facilitating the integration of regulatory cues. As a consequence, the Runx proteins are post-translationally modified (e.g., phosphorylated) to further influence the extent to which they engage in regulatory activity within the nucleus. A key component of the Runx complex is the p300/ CBP coactivator which functions as a transcriptional adaptor and is a component of the nuclear complex in osteoblastic cells [137]. P300 contains a domain with intrinsic histone acetyltransferase (HAT) activity and interacts with additional proteins containing HAT activity that include P/CAF, SRC-1, and ACTR, thereby implicated in chromatin structure alterations associated with modulation of gene expression [138]. With Runx2, formation of large multiprotein complexes that contribute multiple HAT activities provides a basis for bone gene specificity. Furthermore, when recruited to the osteocalcin gene promoter by Runx2, p300 stimulates both basal and vitamin-D-enhanced osteocalcin promoter activity. Thus, interactions of Runx2 with p300 support assembly of multisubunit complexes with several HAT-containing proteins at a series of regulatory regions of the bone-specific osteocalcin gene promoter. In a parallel manner, Kitabayashi et al. [139] have shown that in myeloid cells Runx1, a homolog of
the bone-specific Runx2, interacts with p300 and together they up-regulate myeloid-specific genes. It was also determined that a C-terminal region of the Runt domain in both Runx1 and Runx2, is critical for their interactions with p300 [137,139]. Considering the high degree of homology between these two members of the Runx transcription factor family it is likely that the structural determinants for Runx interactions with p300 are conserved. p300 can also be recruited to gene promoters by the transcription factor C/EBP [140]. Interestingly, a C/EBP-responsive regulatory element has been identified in the proximal promoter region of the rat OC gene adjacent to the Runx2 site C [141]. C/EBPb physically interacts with Runx2 and synergistically activates the osteocalcin promoter, suggesting that both proteins form a complex with p300 and together up-regulate basal tissue-specific transcription. C/EBPb has additionally been shown to interact with ATP-dependent chromatin remodeling complexes of the SWI/SNF family [142], recruiting these complexes to promoter sequences and activating cell-specific expression. In addition to functioning as transcriptional activators, Runx proteins suppress gene expression (transcription). Repression requires the recruitment of transcriptional repressors and corepressors with histone deacetylase activity (HDACs) to promoter regulatory elements of genes that are down-regulated. Combinatorial control that dampens transcription is illustrated by interaction of Runx2 with the transcriptional corepressors TLE/Groucho through a conserved VWRPY domain located at the C-terminus of the protein, which represses the expression of the bone sialoprotein (BSP) gene in osteoblastic cells [110]. Another example of combinatorial control that results in transcriptional suppression by Runx2 is down-regulation of the p21CIP/WAF promoter in fibroblastic and osteoblastic cells. Here HDAC6 interacts with a second repression domain that also resides in the C-terminal region of Runx2 and is recruited to chromatin by Runx2. Indeed, multiple HDACs interact with Runx2 for suppression at specific stages of osteogenesis (reviewed in Jensen et al. [67]). These results are consistent with combinatorial control that is mediated by Runx-dependent recruitment of coactivator and corepressors proteins that are associated with and organized as multiprotein complexes to activate or repress target genes in a physiologically responsive manner. In summary, transcription factors, like Runx, that function as scaffolds for interaction with coregulatory proteins provide an architectural basis for accommodating the combinatorial requirements of biological control. Combinatorial control supports replication, transcription, and repair by two mechanisms. Contextdependent combinations and permutations of regulatory
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proteins are assembled into multipartite complexes that increase specificity for gene expression. Scaffold associated proteineDNA and proteineprotein interactions permit integration of regulatory activities. Nuclear microenvironments are thereby organized, with gene promoters as focal points, where threshold concentrations of regulatory macromolecules are attained. The complexity that is achieved by these architecturally organized oligomeric factors can maximize options for responsiveness to diverse regulatory requirements for transient and long-term biological control.
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Acknowledgments Results presented in this chapter were in part supported by National Institutes of Health grants R37DE012528, R37DE012528-S1, R01AR039588, PO1AR048818, R01AR049069, FONDECYT 1095075, and T32DK007302, as well as Core resources supported by the Diabetes Endocrinology Research Center grant DK32520 from the National Institute of Diabetes and Digestive and Kidney Diseases. Gary Stein, Janet Stein, and Jane Lian are members of the UMass DERC (DK32520). The contents of this chapter are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.
References CLOSING REMARKS For both embryonic development and postnatal bone formation, a highly complex network of signaling factors that are transduced to transcriptional regulators of gene expression to bring about a phenotypic change in osteoblast-lineage cells, demands mechanisms that can accommodate the integration of multiple events in an ordered mechanistic manner. It would be presumptuous to propose a single model to account for the specific pathways that direct transcription factors to sites within the nucleus that support transcription. However, findings suggest that parameters of nuclear architecture functionally interface with components of transcriptional control. The involvement of nuclear matrix-associated transcription factors with recruitment of regulatory components to modulate transcription remains to be defined. Working models that serve as frameworks for experimentally addressing components of transcriptional control within the context of nuclear architecture can be compatible with mechanisms that involve architecturally or activity-driven assembly of transcriptionally active intranuclear foci. The diversity of targeting signals must be established to evaluate the extent to which regulatory discrimination is mediated by encoded intranuclear trafficking signals. It will additionally be important to biochemically and mechanistically define the checkpoints, which are operative during subnuclear distribution of regulatory factors, and the editing steps, which are invoked to ensure the structural and functional fidelity of nuclear domains, where replication and expression of genes occur. There is emerging recognition that placement of regulatory components of gene expression must be temporally and spatially coordinated to optimally mediate biological control. It is realistic to anticipate that further understanding of mechanisms that position genes and regulatory factors for establishment and maintenance of the bone cell phenotype will clarify nuclear structuree function interrelationships that are operative during osteoblast differentiation and vitamin D modulation of regulatory activity.
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C H A P T E R
17 Vitamin D Regulation of Osteoblast Function Renny T. Franceschi 1, 2, 3, Yan Li 1 1
Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, MI, USA, 2 Department of Biological Chemistry, School of Medicine, University of Michigan, Ann Arbor, MI, USA, 3 Department of Biomedical Engineering-College of Engineering, University of Michigan, Ann Arbor, MI, USA
INTRODUCTION Chondrocytes, osteoblasts, and osteocytes all likely trace their lineage to a common mesenchymal stem cell (MSC) progenitor. As discussed in Chapter 16, the lineage commitment events necessary to form osteoblasts, while not completely understood, require at least three specific transcriptional activators, Runx2, Osterix (Osx), and ATF4. Runx2 functions to initially commit MSCs to the chondrocyte/osteoblast lineage while Osx is necessary for overt osteoblast differentiation. ATF4, while not essential for osteoblast formation, is able to modulate the expression of osteoblast genes in response to the metabolic demands on bone (for review, see [1]). Progression of the osteo/chondroprogenitor population down the chondrocyte lineage requires Sox transcription factors (Sox 5, 6, 9) at early stages and Runx2 for the hypertrophic stage [2]. Together, these factors induce chromatin changes necessary for activation of bonespecific genes and expression of the differentiated phenotype. While gene deletion/mutation studies have firmly established the roles of Runx2, Osx, ATF4, and the Sox factors in skeletal development, this approach does not provide information regarding how osteoblast differentiation and function is controlled to maintain skeletal homeostasis. Yet, it is the regulation of osteoblast function that is of greatest relevance to understanding how factors like vitamin D function in bone. For this reason, the focus of this chapter will shift to a discussion of factors, including vitamin D, that regulate mature osteoblasts and osteocytes. The chapter will begin with a brief description of the overall properties of osteoblasts and related osteocytes, a summary of the major functions of these cells and discussion of how vitamin-D-regulated signaling pathways can control
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10017-4
osteoblast and osteocyte function and differentiation in vitro and in vivo. The final section will discuss interactions between vitamin D and two major signal transduction pathways in osteoblasts.
PROPERTIES OF MATURE OSTEOBLASTS AND OSTEOCYTES Once MSCs are committed to the osteoblast lineage, they undergo an orderly progression from periosteal preosteoblast cells adjacent to bone to secretory osteoblasts that form a monolayer on the bone surface. Osteoblasts are polarized cells having a prominent endoplasmic reticulum and Golgi apparatus, consistent with their major function to secrete the extracellular matrix (ECM) of bone. It is within this matrix that the hydroxyapatite mineral is deposited to produce a mineraleprotein composite material that is able to provide the strength and elasticity of mature bone. The overall composition of bone by mass is approximately 65e70% mineral, 10% water, and 20e25% organic matrix. This matrix is predominantly composed of type 1 collagen (approx. 90%) with the balance being noncollagenous proteins and proteoglycans (for review, see [3]). Since separate chapters are devoted to collagen, noncollagenous proteins and the mineralization process, these will only be briefly discussed here. Type I collagen, the prototypical fibrillar collagen, exists as a triple helix containing two identical a1(I) chains and a structurally similar, but genetically distinct, a2(I) peptide. Individual collagen triple helixes are arranged into fibrils with a characteristic quarter-staggered arrangement that gives the fibril its ultrastructural appearance. Fibrils also contain inter- and intrachain crosslinks between adjacent
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lysine residues to provide further structural rigidity. In lamellar bone, hydroxyapatite mineral first appears in the gap zones separating adjacent collagen triple helixes within fibrils and then propagates throughout the rest of the bone matrix [4]. However, there is still some debate about the site of primary mineral nucleation, which may be associated with certain noncollagenous proteins in complex with vesicular structures (also called matrix vesicles) secreted by osteoblasts [5]. The noncollagenous proteins are a broad class of molecules whose function in bone remains largely undefined. Unlike collagen, which is widely distributed in connective tissues, several of the noncollagenous proteins are preferentially expressed in osteoblasts and/or osteocytes and are thought to give these cells the unique ability to produce a mineralized ECM. The most studied of these are osteocalcin (Bglap1,2), bone sialoprotein (Ibsp), dentin matrix protein 1 (DMP1), bone acidic glycoprotein 75 (BAG75), matrix extracellular phosphoglycoprotein (MEPE), and alkaline phosphatase (Tnap). Although the functions of these proteins are still not well established, roles for bone sialoprotein, DMP1, BAG75, MEPE, and alkaline phosphatase in the mineralization process have been proposed. Bone sialoprotein can nucleate mineral formation under controlled in vitro conditions and is postulated to have a similar role in vivo in that Bsp-null mice have altered bone mineralization [6,7]. DMP1 can also nucleate mineral in vitro and DMP1-null animals have severe defects in bone and tooth mineralization [8]. However, results with DMP1 are complicated by systemic effects related to high levels of osteocyte FGF23 in DMP1-null mice which can induce systemic hypophosphatemia and secondary loss of bone mineral [9]. BAG75 may exist as part of a vesicular complex that also contains Bsp; it is proposed that a proteolytic processing event is necessary to activate this complex and nucleate mineralization [5]. MEPE may be a negative regulator of mineralization as MEPE-null mice have increased bone density [10]. MEPE’s inhibitory function requires a proteolytic event catalyzed by PHEX protein (phosphate-regulating neutral endopeptidase on chromosome X) to generate the mineralization inhibitor, ASARM peptide (acidic serine- and aspartate-rich MEPE-associated motif) [10e12]. In contrast, the role of alkaline phosphatase in mineralization appears to be indirect via its ability to degrade pyrophosphate, which is, itself, a potent inhibitor of mineralization [13,14]. Osteocalcin’s function in bone, if it has one, is related to suppression of mineralization in that deletion of the Bglap genes results in a mild elevation of bone mass [15]. More recently, however, osteocalcin has been shown to have a systemic role in regulating energy metabolism and insulin sensitivity [16]. Regardless of their function, Bglap and Ibsp genes have been most
useful as tools for understanding osteoblast-specific gene expression and its regulation by growth factors and hormones including 1,25-dihydroxyvitamin D3. Secretory osteoblasts have a limited in vivo lifetime estimated to be approximately 3 months in humans and only 10e20 days in mice [17]. Once they stop secreting bone matrix, osteoblasts have three potential fates: (i) they can become embedded in bone as osteocytes; (ii) they undergo apoptosis; or (iii) they become inactive bone-lining cells. In human bone, it is estimated that only about 30% of osteoblasts become osteocytes with the remaining cells either undergoing apoptosis or reverting to bone-lining cells [18]. The progression from preosteoblast to osteoblast and then to osteocyte was initially established by in vivo pulse/chase labeling [19]. More recently, real-time visualization of this process was achieved through the use of transgenic marking of osteoblasts and osteocytes using green fluorescent proteins emitting at different wavelengths. Individual transgenic mouse lines were developed expressing either green fluorescent protein (GFP) cyan under the control of a 2.3-kb Col1A1 promoter to mark osteoblasts or GFP topaz under the control of an 8-kb DMP-1 promoter to mark osteocytes and preosteocytes. Using this approach, a clear transition of individual osteoblast cells into osteocytes could be visualized in calvaria [20]. Although clearly derived from osteoblasts, osteocytes have a distinct phenotype and biological functions (for review see [17,21]). In contrast to the plump, polygonal morphology of osteoblasts, osteocytes gradually assume a more elongated shape with a smaller cell body and extensive dendritic processes as they become incorporated into the ECM. At early times, they continue to secrete matrix and may play an active role in the mineralization process (osteoid osteocytes). Mature osteocytes reside in lacunae within the mineralized ECM. Osteocytes communicate with each other and with osteoblasts on the bone surface through an extensive network of dendritic processes that use gap junctions to transfer signals between cells. Although they share some markers with osteoblasts (e.g., osteocalcin), they also uniquely express several proteins including DMP-1, MEPE, E11/gp38, PHEX, sclerostin, and FGF23. Because osteoblasts only line the surface of growing bone while osteocytes occupy the entire volume of bone matrix, this latter cell type vastly outnumbers the osteoblasts. It is estimated that 90e95% of all bone cells are osteocytes while only 4e6% are osteoblasts and 1e2% are osteoclasts. In contrast to osteoblasts that have lifetimes of weeks to months, osteocytes can persist in the bone matrix for many years. In this sense, osteoblasts and osteoid osteocytes can be viewed as transitional cell types that secrete and mineralize bone ECM for only a few weeks while mature osteocytes are the terminal cell in this lineage. Their long half-life and the fact that
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osteocytes are present regardless of whether or not active bone formation is taking place makes them an ideal cell to sense changes in the bone microenvironment associated with fluctuations in circulating calcium and phosphate levels or altered mechanical load. These possible regulatory roles of osteocytes will be discussed below. When considering how osteoblasts/osteocytes might be regulated by systemic factors such as 1,25(OH)2D3, two levels should be considered: (i) effects of the factor on regulatory functions of major cells in the osteoblast lineage and/or (ii) ability of the factor to affect differentiation or lineage progression to the mature cell phenotype. The vitamin D endocrine system may affect both aspects of osteoblast/osteocyte biology.
MAJOR REGULATORY FUNCTIONS OF OSTEOBLASTS AND OSTEOCYTES AND CONTROL BY THE VITAMIN D ENDOCRINE SYSTEM Three regulatory roles of osteoblasts/osteocytes will be discussed in this section; bone remodeling, regulation
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of phosphate homeostasis, and the response of bone to mechanical loads.
Bone Remodeling Although net bone growth ceases with the onset of adulthood, bone is constantly renewed by the concerted action of osteoclasts that resorb bone and osteoblasts that replace bone lost by resorption. Osteoblasts and osteoclasts form a recognizable anatomical structure known as the basic multicellular unit (BMU). Cells of the osteoblast lineage control both the formation and breakdown of bone necessary for remodeling (Fig. 17.1). Bone formation/mineralization, as discussed above, is a primary function of the secretory osteoblast and related osteoid osteocyte. In contrast, participation of the osteoblast lineage in bone resorption is indirect via activation of osteoclasts. Osteoblasts respond to a variety of resorptive signals including 1,25(OH)2D3, parathyroid hormone (PTH), IL-1, IL-6, and sympathetic tone (b-adrenergic receptor activation) by secreting receptor activator of NF-kB ligand (RANKL). RANKL, which, based on gene deletion studies, is indispensable for osteoclastogenesis
FIGURE 17.1 Actions of 1,25(OH)2D3 on cells of the osteoblast lineage. The right of the figure shows the lineage of osteoblasts/osteocytes from pluripotent mesenchymal stem cells, the relationship between osteoprogenitors, osteoblasts, and osteocytes on the surface of bone and the major transcription factors controlling lineage commitment decisions in this cell population. Reported effects of 1,25(OH)2D3 on cells of each developmental stage are indicated with relevant literature references. Mineralized matrix and entombed osteocytes are shown in gray.
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[22,23], functions by binding to its receptor, RANK. RANK activation stimulates a number of signal transduction pathways including IKK/IKb/NF-kB, mitogen-activated protein kinase (MAPK), Src, and phosphatidylinositol 3-kinase (PI3K)/AKT, which activate c-fos, NF-kB, and NFATc1 transcription factors to induce osteoclast differentiation from hematopoietic progenitors (for reviews see [24]). Because RANKL plays such a central role in osteoclastogenesis, a considerable effort has been devoted to understanding how it is controlled by 1,25(OH)2D3 and PTH. The basis for this control was independently reported by two groups in 2006 [25,26]. Interestingly, major regulation of the Rankl gene was localized to a distal region 76-kb upstream from the transcription start site called the RANKL distal control region (RL-DCR). The RL-DCR contains a functional VDRE, several glucocorticoid receptor response elements, a cAMP response element, and a binding site for Runx2. Deletion analysis established that the RL-DCR is critical for RANKL induction by PTH (via induction of cAMP and cAMP response element binding protein; CREB) and 1,25(OH)2D3 and that responsiveness could be modulated by Runx2. Of further significance, deletion of the RL-DCR in mice reduced PTH and 1,25 (OH)2D3 stimulation of RANKL mRNA and osteoclastogenesis in marrow cultures as well as RANKL mRNA induction in vivo. Furthermore, RL-DCR-null mice exhibited a high bone mass phenotype characterized by increased bone strength, reduced osteoclast and osteoblast numbers, and low bone turnover [27]. Although RL-DCR deletion inhibited both 1,25(OH)2D3 and PTH responsiveness, residual 1,25(OH)2D3 induction of RANKL mRNA and osteoclastogenesis was still seen. This may be explained by other more proximal VDREs in the Rankl promoter that were also shown to bind VDReRXR complexes as measured by chromatin immunoprecipitation assays [26]. Regulation of RANKL expression by 1,25(OH)2D3 is absolutely dependent on the VDR in that this regulation is not seen in VDR-null osteoblasts. However, VDR-null cells can still respond to PTH indicating that these two factors can function independently to control Rankl expression [28]. It has been assumed for many years that RANKL is produced by osteoblasts or cells of the osteoblast lineage. Several lines of evidence support this notion: (i) osteoprogenitor cells (i.e., MSCs), osteoblasts, or osteocytes all produce RANKL and coculture of any of these cells with osteoclast precursors produces functional osteoclasts (for review, see [29]), (ii) the RL-DCR of the Rankl gene contains a functional Runx2 binding site and Runx2 present in cells of the osteoblast lineage can enhance cAMP induction of RANKL(25), (iii) Runx2 can physically interact with the VDR and stimulate its activity [25]. However, it is still not clear which of these
cells is the major RANKL-producing cell in bone capable of responding to 1,25(OH)2D3 and PTH. In this regard, ablation of mature osteoblasts by ganciclovir treatment of transgenic mice expressing thymidine kinase (tk) under the control of the osteocalcin promoter, which is expressed exclusively in mature osteoblasts, did not alter bone resorption or osteoclast number [30]. Similarly, ganciclovir depletion of osteoprogenitors and osteoblasts in mice expressing a 3.6-kb Col1a1 promoter-tk construct did not affect RANKL induction by PTH [31]. In the same study, cAMP induction of RANKL was also not reduced in Runx2-null cells and shRNA suppression of Runx2 in a stromal osteoblast cell line did not inhibit PTH responsiveness. Also, comparison of sorted Osx promoter-positive (more mature osteoblasts) and negative calvarial cells (less mature) did not reveal any differences in RANK mRNA induction by either cAMP or 1,25 (OH)2D3 although Osx-positive cells were somewhat more responsive to PTH, probably because this osteoblast-enriched cell population expressed higher levels of the PTHR1. Unfortunately, the 3.6 Col1a1-tk osteoblast ablation study did not specifically examine 1,25(OH)2D3 induction of RANKL so it is not possible to conclude that mature osteoblasts are also not required for responsiveness to this hormone. However, as noted above, no differences in RANKL induction by 1,25(OH)2D3 were seen when Osxþ (mature osteoblast) and Osxe calvarial cells were compared, indicating that 1,25(OH)2D3 can act on nonosteoblast stromal cells or early osteoprogenitors [31]. In summary, cell ablation studies suggest that the major PTH-responsive RANKL-secreting bone cell is probably not an osteoprogenitor or secretory osteoblast since both these cells would be expected to express 3.6 Col1a1-tk and be sensitive to ganciclovir. It could be an earlier cell in the osteoblast lineage that does not express the 3.6 Col1a1 promoter-tk, a stromal cell, or an osteocyte. Regarding the latter possibility, osteocyte-like cells do, in fact, express RANKL, PTHR1, and the VDR. In addition, their cell processes may extend to the bone surface, making contact with osteoclast precursors at least theoretically possible [32]. Also, selective ablation of osteocytes in bone prevents the normal activation of RANKL seen with skeletal unloading, although it also stimulates rather than blocks RANKL expression in mice subjected to normal skeletal loading indicating an alternate source for RANKL under these conditions [33]. An alternate interpretation is that negative results obtained from cell ablation studies may be explained by incomplete cell destruction leaving enough intact cells of a given osteoblast differentiation stage (osteoprogenitors, osteoblasts, osteocytes) to support a resorption response. Ultimately, resolution of this controversy may require detailed immunohistochemistry studies that identify which cells produce
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increased RANKL in response to PTH and 1,25(OH)2D3 treatment in vivo.
Regulation of Phosphate Homeostasis The vitamin D endocrine system controls both calcium and phosphate levels by stimulating the uptake of these two ions in the gut and, together with PTH, by inducing osteoclast-mediated bone resorption. With PTH, it also stimulates renal tubular calcium reabsorption and inhibits phosphate reabsorption. However, because PTH secretion by the parathyroids and, indirectly, formation of 1,25(OH)2D3 (via PTH stimulation of the 25-hydroxyvitamin D 1a-hydroxylase-CYP27B1) is controlled by serum calcium concentrations, but not phosphate levels, these two endocrine factors are not able to directly control phosphate homeostasis. For many years, researchers sought an additional hormone that could directly respond to circulating phosphate concentrations. This phosphate-regulating hormone or phosphatonin is now known to be FGF23, a factor almost exclusively produced by osteoblasts and osteocytes [32]. FGF23 is preferentially released from these cells when serum phosphate levels become elevated and in response to elevated 1,25(OH)2D3. FGF23 acts on several target tissues. Like PTH, it blocks renal tubular reabsorption of phosphate by inhibiting the renal phosphate transporters, Npt2a and Npt2c, and inhibits renal CYP27B1 expression to reduce 1,25 (OH)2D3 synthesis and intestinal phosphate (and calcium) absorption [34,35]. Unlike PTH, FGF23 does not respond to changes in serum calcium and, therefore, provides a route for dealing with excess phosphate concentration when PTH is suppressed by normalization of serum calcium. Consistent with these observations, FGF23-null mice have hyperphosphatemia, moderate hypercalcemia, reduced serum PTH, and elevated 1,25(OH)2D3 [36,37]. FGF23 functions by binding to the FGF receptor, FGFR1(IIIc), together with a second protein, Klotho, previously associated with premature ageing [38]. Klotho-null mice have a similar phenotype to FGF23-nulls, consistent with this factor being required for FGF23 signaling [39]. Interestingly, the premature aging associated with Klotho deletions has been related to soft tissue calcification and elevated 1,25(OH)2D3 levels as would be expected with FGF23 resistance. FGF23 secretion from osteoblasts/osteocytes is controlled by both serum phosphate and 1,25(OH)2D3 through mechanisms that are still not well understood. In the case of 1,25(OH)2D3, this regulation is indirect since it can be blocked by inhibition of protein synthesis. Furthermore, no VDREs have been detected in the FGF23 gene nor is VDR binding to FGF23 chromatin detected by ChIP analysis. It has been speculated that
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1,25(OH)2D3 partially or totally induces FGF23 by suppressing expression of PHEX, a potent inhibitor of FGF23 function [40]. FGF23 is also negatively regulated by DMP-1. Osteocytes from DMP-1-null mice express high levels of FGF23. These mice have a similar hypophosphatemic phenotype to mice harboring mutations in the Phex gene. Similarly, humans with loss-offunction mutations in either PHEX or DMP1 genes also have elevated FGF23 levels and hypophosphatemia (for reviews, see [32,41]). Taken together, these studies suggest that the osteocyte proteins, DMP-1 and PHEX, may be part of a common pathway to regulate FGF23 and serum phosphate although their relationship to the vitamin D endocrine system is still not well understood (also see Chapter 42).
Response of Bone to Mechanical Loads Bone has the unique ability to alter its mechanical properties in response to the physical forces it experiences. While a detailed discussion of bone biomechanics is beyond the scope of this chapter, a brief overview of the subject is included because the responsiveness of bone to loading can be altered by endocrine factors, possibly including 1,25(OH)2D3 (for a more in-depth review, see [42]). When subjected to a load (stress), bone deforms in proportion to the magnitude of the load. The amount of deformation is called strain. Under physiological loading, maximum strains are 2e 3000 mstrain (displacements of 0.2e0.3%). When bone bends, one side is subjected to compression and the other to tension. Bone is able to modify its structure to minimize strain when exposed to dynamic as opposed to static loads [43]. Strains exceeding a certain threshold will stimulate new bone formation to strengthen the bone and thereby reduce maximum strains to below the threshold. A corollary to this concept is that bone mass will be lost with disuse when bone is subject to minimal strain [44]. There are many common examples illustrating how bone mass alters in response to loading or its absence. For example, the serving arm of professional tennis players has more bone mass than the nonserving arm [45]. In contrast, skeletal unloading during bed rest is associated with severe bone losses of up to 2% per month while weight-bearing exercise increases bone mass [46,47]. There are two main ways bone cells might detect strain in response to mechanical loads: (i) direct detection of strain to individual cells as transmitted by cell contacts with the extracellular matrix or (ii) detection of fluid flow through bone canaliculi in response to strain. This later response is initiated by the compression and tension across bone during loading, which squeezes interstitial fluid through canaliculi and through the marrow cavity. These two pathways are not mutually
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exclusive, and both types of response have been detected. Although no definitive experiments have excluded one or the other of these possibilities, fluid flow is thought to be the predominant way bone cells sense loads. Marrow stromal cells, including mesenchymal stem cells, osteoblasts, and osteocytes can all respond to fluid flow shear stress (FFSS) in vitro. However, the positioning of osteocytes within canaliculi allows them to be exposed to the most consistent load-induced fluid flow changes. Furthermore, when osteocytes are selectively ablated in bone by targeted expression of diptheria toxin using the DMP-1 promoter, bones are resistant to unloading-induced bone loss, suggesting that osteocytes are required for mechanotransduction [33]. This approach, which killed 70e80% of osteocytes, left osteoblasts largely intact, yet prevented the induction of RANKL that normally accompanies unloading. The response of animals to mechanical stimulation was not reported. However, reloading of tailsuspended mice stimulated new bone formation regardless of whether or not osteocytes had previously been ablated. FFSS stimulates a number of primary and secondary responses in target cells including stimulation of L-type calcium channels [48], nitric oxide synthase [49,50], cyclooxygenase, and prostaglandin synthesis [51], Wnt protein secretion and down-regulation of the Wnt inhibitor, sclerostin [52,53]. In addition, FFSS stimulates integrin-mediated activation of focal adhesion kinase (FAK) [54] and stimulation of several signal transduction pathways including ERK, p38, and JNK MAPK pathways and the PI3K/AKT pathway [55,56]. The end result is to stimulate mesenchymal stem cell recruitment and osteoblast differentiation leading to new bone formation [57]. The responsiveness of bone to mechanical stimulation is modified by several humoral factors including PTH [58], estrogens [59,60], and insulin-like growth factors [61]. In many cases, effects of hormones on mechanoresponsiveness may be related to modulation of common pathways regulated by both signals. For example, modulation of mechanoresponsiveness by estrogens may be related to postulated nongenomic effects of estrogen receptors a and b (ERa, ERb) on the ERK/ MAP kinase pathway [62,63]. Both ERs are required for maximal activation of ERK by mechanical stimulation of osteoblasts or osteocytes [63]. Also, nuclear localization of receptors is not required in that ERK activation can be achieved with ERs lacking a DNA-binding domain. Rather, a subfraction of ER can function in association with caveolin-1 on the plasma membrane consistent with a non-nuclear function. Since the VDR can also affect ERK/MAPK and related pathways (see below), it may also be able to modulate mechanoresponsiveness in this way. In a preliminary report, transgenic mice
overexpressing the VDR in osteoblasts were reported to have greater lamellar and cortical bone responses to mechanical loading although the basis for this response was not examined [64]. In contrast, the in vitro induction of RANKL expression by 1,25(OH)2D3 was reported to be blunted by application of mechanical strain to marrow stromal cells [65].
EFFECTS OF 1,25(OH)2D3 ON OSTEOBLAST DIFFERENTIATION In vitro Effects of 1,25(OH)2D3 on Osteoblasts in Cell Culture Actions of 1,25(OH)2D3 on the differentiation of marrow stromal cells, osteoblasts, osteoblast-like osteosarcoma cells, and osteoblast cell lines in tissue culture have been extensively described over the past two decades (for review, see [66]). In fact, isolated osteoblasts and osteoblast-like osteosarcomas were the classic systems used for describing the genomic actions of the VDR. As described in more detail elsewhere in this volume (see Section II Mechanisms of Action), much of what we know about how the VDR controls transcription comes from studies using the osteocalcin gene (Bglap1/2), which is almost exclusively expressed in osteoblasts. Deletion studies on Bglap were first used to identify a functional VDRE in the rat gene promoter and to show that the VDR binds this enhancer sequence as a heterodimer with the retinoid X receptor (RXR) [67,68]. Subsequently, a number of osteoblast-related genes including osteopontin (Ibsp2) [69], bone sialoprotein (Ibsp) [70], RANKL [26], and Runx2 [71] were all shown to contain functional VDREs that could be regulated by 1,25-(OH)2D3 in isolated osteoblasts. As would be expected if the vitamin D endocrine system were able to modulate osteoblast differentiation, the VDR also physically and functionally interacts with Runx2 to regulate osteocalcin expression [72]. Nevertheless, while effects of 1,25-(OH)2D3 on osteoblast gene expression have been extensively documented, the magnitude and direction (stimulatory or inhibitory) of hormone effects are actually quite variable, being dependent on the stage of differentiation when 1,25-(OH)2D3 is added and the duration of treatment. For example, in early cultures of rat calvarial osteoblasts, acute 1,25-(OH)2D3 treatment inhibits proliferation and histone mRNA levels, has no effect on the normally low to undetectable levels of osteocalcin mRNA while strongly stimulating matrix Gla protein and osteopontin expression. In contrast, 1,25(OH)2D3 stimulates osteocalcin in differentiated cells. Also, sustained 1,25(OH)2D3 treatment of proliferating cells blocks collagen, osteocalcin and alkaline phosphatase expression and mineralized nodule
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formation while the same treatment of differentiated cultures stimulates osteocalcin and mineralization [73]. Other studies also support a model where 1,25(OH)2D3 stimulates bone markers in mature osteoblasts while inhibiting ECM synthesis and differentiation of osteoprogenitors [74,75]. However, hormone effects on the early stages of differentiation varied with species, maturational stage of cells, time and duration of hormone treatment [76e78]. Overall, the disparate results of in vitro studies prevent reaching firm conclusions regarding the physiological effects of 1,25(OH)2D3 on osteoblast differentiation.
Direct Actions of 1,25(OH)2D3 on Osteoblast/ Osteocyte Differentiation in Vivo Given that the classic functions of vitamin D are all related to bone and cartilage function and that several well-defined VDR target genes are expressed in osteoblasts/osteocytes, one might expect that the direct actions of 1,25-(OH)2D3 on osteoblasts in vivo would be well documented. Surprisingly, evidence for this is actually quite limited with results being largely explained by systemic effects of the vitamin D hormone on calcium homeostasis. During embryonic development, the VDR is first expressed coincident with the formation of mesenchymal condensations in bone primordia (at embryonic day 13 in rats) and persists throughout the osteoblast/ osteocyte and chondrocyte lineages [79]. Furthermore, this receptor is clearly functional in vivo since osteocalcin mRNA extracted from whole bone is induced when vitamin-D-deficient rats are treated with 1,25(OH)2D3 [67]. At issue has been whether normal bone formation requires direct actions of 1,25(OH)2D3 on osteoblasts or if it can occur in the absence of vitamin D signaling. Evidence for the latter possibility dates back many years to studies showing that stringently vitamin-D-deficient rat dams could produce offspring that were only slightly smaller than normal. Furthermore, these mice did not show signs of vitamin D deficiency until weaning [80]. Subsequent work showed that during nursing, pups acquired calcium through a non-vitamin-D-dependent mechanism facilitated by milk lactose and the availability of this calcium pool allowed normal bone development. In another early study, normalization of serum calcium and phosphate in vitamin-D-deficient rats by continuous infusion was shown to maintain normal bone density, rates of bone formation, and epiphyseal growth plate width [81]. These early investigators concluded that vitamin D and its metabolites are not necessary for reproduction and fetal development nor is the vitamin D endocrine system necessary to promote bone mineralization as long as adequate amounts of serum calcium and phosphate are provided.
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More recently, genetic approaches have been used to further address this issue by examining bone formation in animals lacking either the ability to make 1,25(OH)2D3 via knockout of the CYP27B1 gene encoding 25-hydroxyvitamin D 1a-hydroxylase or respond to 1,25(OH)2D3 by knockout of the VDR. Similar to early results obtained with vitamin-D-deficient animals, either CYP27B1 or VDRe/e animals or double knockout mice (CYP27B1e/e x VDRe/e) developed severe osteopenia and reduced bone size, hyperparathyroidism, and hypocalcemia. However, a rescue diet containing lactose and high calcium was able to completely normalize serum calcium, parathyroid hormone levels, and bone mineral, indicating that these processes directly require serum calcium rather than the vitamin D endocrine system [82]. Further evidence for this concept came from studies where VDR expression was selectively rescued in the intestines of VDRe/e animals, resulting in normalized serum calcium and bone mineralization. However, more detailed examination of knockout animals revealed subtle effects of 1,25(OH)2D3 and the VDR on bone formation and osteoblast differentiation even after serum calcium was normalized with the rescue diet [82]. For example, osteoblast number was reduced in bones from CYP27B1e/e, VDRe/e and CYP27B1e/eVDRe/e mice regardless of diet and this reduction was reflected by a decrease in total fibroblast colony forming units (CFU-F) and osteoblast colony forming units (CFU-Ob) in marrow stromal cells isolated from these animals. Consistent with the idea that 1,25(OH)2D3 signaling can stimulate osteoblast differentiation, transgenic overexpression of the VDR using an osteocalcin promoter also increased both cortical and trabecular bone [83], although in this case VDR expression would presumably be elevated only in mature osteoblasts. On the other hand, some studies suggest that vitamin D signaling actually inhibits bone formation by more mature cells. Thus, calvarial osteoblasts isolated from VDRe/e mice exhibited more rapid expression of osteoblast markers and mineralization than did cells from wild-type littermates. Also, VDRe/e bones formed more mineral than wild-type bones when implanted into wild-type recipient animals [84]. Lastly, in a preliminary report, bone mineral density was shown to increase in mice harboring an osteoblastspecific deletion of the VDR generated by crossing VDRf/f mice with animals harboring Cre recombinase driven by the a1(I) collagen promoter [85]. Taken together, studies with genetically modified mice suggest that 1,25(OH)2D3 has differentiation-stage-specific effects on osteoblasts and osteoblast precursors with stimulatory effects on the maturation of early mesenchymal progenitors and inhibitory actions on more mature osteoblasts. Also, it can clearly regulate the expression of osteoblast-associated genes such as Bglap in vivo. However, more detailed analysis of conditional
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VDR knockouts at various stages of osteoblast differentiation will be necessary to understand the full range of and significance of 1,25(OH)2D3 action in osteoblasts. The current availability of a broad range of promoters to drive Cre expression in cells at different points in the osteoblast lineage will greatly facilitate this analysis. Whether these studies in mice reflect that which occurs in humans remains to be fully established.
REGULATION OF INTRACELLULAR SIGNALING PATHWAYS BY 1,25(OH)2D3 How might 1,25(OH)2D3 affect osteoblast function? In some cases, hormone actions involve direct binding of the 1,25(OH)2D3eVDR complex to VDREs in regulatory regions of target genes as is the case for RANKL or osteocalcin regulation described above. 1,25(OH)2D3 can also alter the activity of several osteoblast-related signal transduction pathways either through classic actions of the VDR on transcription of pathway intermediates or by nongenomic effects on pathway activity. Two pathways will be discussed; mitogen-activated protein kinases and Wnt. These pathways are necessary for responsiveness to hormones and growth factors, extracellular matrix and mechanical cues, and in many cases can affect osteoblast differentiation or function.
MAPK Signaling Pathways Three related mitogen-activated protein kinase (MAPK) pathways have been described in mammals; ERK/MAPK, p38, and Janus kinase (JNK) pathways. All three are active in osteoblasts although the former two have been most carefully studied. The canonical ERK/MAPK pathway is activated by multiple signals encountered by osteoblasts/osteocytes including those initiated by growth factor receptors (e.g., receptors for insulin, IGF-1, fibroblast growth factor and BMPs [39]), extracellular matrix/integrin binding and focal adhesion kinase (FAK) activation [40], related biomechanical stimulation [43] and certain nongenomic actions of estrogens [41]. Regardless of the initiating stimulus, downstream signals involve activation of Raf kinases that phosphorylate and activate the dual specificity kinases, MEK1/MEK2. MEKs subsequently phosphorylate/activate ERK1/ERK2 to stimulate gene expression by phosphorylating specific transcription factors [50]. There is compelling in vivo evidence that ERK/MAPK signaling is critical for osteoblast differentiation. As shown by Ge et al. [86], sustained transgenic overexpression of constitutively active (Mek-sp) or dominant negative MEK1 (Mek-dn) in osteoblasts, respectively, increased or decreased skeletal maturation during mouse development [58]. Furthermore, crossing TgMek-sp mice
with Runx2þ/e animals was able to partially rescue the cleidocranial dyplasia (CCD) phenotype caused by Runx2 deficiency while TgMek-dn, Runx2þ/e mice died at birth with a more severe CCD, consistent with the concept that MAPK activates the limiting amounts of Runx2 in heterozygous animals. In addition, selective inactivation of ERK2 in mesenchymal cells (osteoblast and chondrocyte progenitors) of ERK1-null mice using a Prx1-Cre was shown to cause severe defects in endochondral and intramembranous bone formation, decreased osteoblast differentiation, and ectopic cartilage formation [87]. These studies provide conclusive proof that the ERK/MAPK pathway stimulates osteoblast differentiation and inhibits chondrogenesis in vivo. In the case of osteoblasts, a major ERK substrate is Runx2, an essential transcription factor for the osteoblast lineage [88]. ERK binds to Runx2 via a specific docking site in the runt domain and directly phosphorylates two critical residues at S301 and S319 (mouse sequence, type II Runx2 isoform) as measured by mass spectroscopy and biochemical assays. Serine to alanine mutation of these sites blocks the ability of Runx2 to stimulate osteoblast-specific gene expression and differentiation. Interestingly, the ERKeRunx2 interaction was shown to occur on the chromatin of target genes and is essential for subsequent stimulation of gene expression [89]. The ERK/MAPK pathway can also indirectly affect osteoblast differentiation by activating the ERK1/2 substrate, RSK2, which subsequently phosphorylates ATF4, another critical factor for osteoblast differentiation [90]. Mutations in RSK2 cause Coffin-Lowry syndrome, an X-linked disorder associated with mental retardation and skeletal anomalies. The stress and cytokine-activated MAPK, p38 MAPK, may also participate in Runx2 activation and osteoblast differentiation, likely via a mechanism similar to that described above for ERK/MAPK [91]. In addition to signaling through SMAD proteins, TGF-b, and BMP receptors activate TAK1 (TGF-b-activated kinase), which stimulates ERK, p38, and JNK MAPK pathways [92]. Osteoblast-selective deletion of TAK1 in mice causes severe defects in endochondral and intramembranous bone formation. Furthermore, deletion of the p38 pathway intermediates, Mkk3, Mkk6, p38a, or p38b, also leads to reduced bone mass and defective osteoblast differentiation, suggesting that at least some of the effects of TAK1 deletion can be attributed to defects in p38 signaling. Furthermore, p38a and b can phosphorylate Runx2. Mass spectroscopy revealed that one of the sites phosphorylated by ERK (S319) is also a possible p38 substrate as well as additional sites at S28 and S250. Combined mutation of S28, S250, and S319 significantly reduced the ability of p38 to stimulate a Runx2 reporter gene. Studies on ERK and p38 MAPK signaling in osteoblasts raise the intruiging possibility that Runx2,
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via its phosphorylation state, may be able to integrate diverse signals from hormone/growth factor receptors, TGF-b/BMP receptors, integrins, and other mechanoreceptors to control the overall level of osteoblast gene expression. Like other steroid hormones, 1,25(OH)2D3 can rapidly stimulate ERK/MAPK activity in osteoblast-like cell lines [93]. This hormonal response does not appear to involve VDR-mediated transcriptional activity and is attributed to nongenomic actions of 1,25(OH)2D3 (see Chapter 15). ERK/MAPK activity can either stimulate or inhibit VDR transcriptional activity depending on the cell type examined. For example, Raf activation of ERK1/2 increases VDR-dependent transcription in MG-63 osteoblast-like osteosarcoma cells while inhibiting VDR activity in MC3T3-E1 preosteoblasts. These responses are related to the isoforms of the VDR partner, RXR, expressed in each cell type; MG-63 cells express RXRb and RXRg, which are activated by ERK1/2dependent phosphorylation, while MC3T3-E1 cells express RXRa, which is inhibited by phosphorylation. 1,25(OH)2D3 can also activate p38 signaling via nongenomic stimulation of calcium influx and activation of RhoA-Rho-associated coiled kinase (ROCK) in colon cancer cells. This pathway is necessary for activation of CYP24 (25-hydroxyvitamin D-24-hydroxylase), inhibition of Wnt-b-catenin signaling and cell proliferation [94,95]. However, only one study examined cross-talk between vitamin D and p38 and ERK MAPK signaling in osteoblasts. In this case, in vitro induction of differentiation in human preosteoblasts by 1,25(OH)2D3 was blocked with the ERK/MAPK inhibitor, PD09059, while the p38 inhibitor, SB203580, had no effect [96].
The Wnt Pathway and Control of Osteoblast versus Adipocyte Lineages Bone marrow contains mesenchymal stem cells capable of differentiating to osteoblasts or adipocytes. These cells have been a focus of considerable interest because of the well-known property of marrow to accumulate fat with aging and the association of marrow adiposity with reduced bone mass and osteoporosis [97,98]. The Wnt pathway controls cell migration, proliferation, and differentiation during tissue development as well as in certain cancers and can preferentially stimulate the lineage of marrow MSCs toward osteoblasts at the expense of adipocytes (see Chapter 13). In the absence of Wnt ligands, target genes are quiescent because of b-catenin phosphorylation and degradation catalyzed by a complex containing glycogen synthase kinase 3b (GSK3b), adenomatous polyposis coli (APC), and axin. The pathway is activated by binding of Wnt ligands to frizzled receptors and lowdensity lipoprotein receptor-related protein 5 and 6
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(LRP5,6) coreceptors. This inactivates GSK3b via phosphorylation, leading to disruption of the degradation complex and release of b-catenin. Active b-catenin then translocates to the nucleus where it interacts with T cell factor/lymphoid enhancer binding factor (TCF/ LEF) to stimulate the transcription of target genes. Wnt signaling can also be modulated by the inhibitors, secreted frizzled-related protein (SFRP2) and Dickkopf proteins (DKKs), which inhibit WnteLRP receptor interactions [99,100]. In bone, the Wnt signaling pathway is essential for osteoblastogenesis and osteogenesis [101]. Many of the components of this pathway have been characterized by gain-of-function or loss-of-function mutations in mice. These studies established an important role for Wnt signaling in bone formation. For example, conditional deletion of b-catenin in early osteochondroprogenitors or in Runx2þ, Osxþ progenitors blocked formation of mature osteoblasts and diverted the cell lineage to chondrocytes. In contrast, ectopic bcatenin expression enhanced osteoblast differentiation [102,103]. Consistent with these findings, transgenic overexpression of Wnt10b in the adipogenic cells of bone marrow increased bone mass presumably by stimulating resident marrow MSCs to progress down the osteoblast lineage [104]. In contrast, DKK1 haploinsufficiency increased bone formation while DKK1 overexpression in calvarial osteoblasts stimulated adipocyte differentiation [105,106]. 1,25(OH)2 D3 can regulate Wnt signaling via two different routes; direct regulation of Lrp5 transcription and inhibition of the Wnt inhibitors, SFRP2 and DKK1 [107,108]. A ChIP-chip screen of the mouse genome was used to identify three VDR- and RXR-binding sites in the Lrp5 gene locus. Further functional analysis showed that 1,25(OH)2D3 can up-regulate LRP5 transcription in a time- and dose-dependent manner, thus confirming that vitamin-D-induced Lrp5 expression is modulated through the binding of VDR to specific regions of Lrp5 gene [107]. 1,25(OH)2D3 also strongly inhibited SFRP2 and DKK1 in wild-type murine bone marrow stromal cells (BMSCs) and inhibited adipogenesis while BMSCs from VDR-null mice exhibited increased expression of SFRP2, DKK1, and increased adipogenesis. In contrast, osteoblast differentiation appeared normal in VDR-null BMSC [108] (although see [82]). This is to be contrasted with more homogeneous calvarial osteoblast cultures where the absence of VDR led to increased osteoblast differentiation [109]. Overall, these studies suggest a possible stimulatory role for 1,25(OH)2D3 in Wnt signaling that can suppress adipogenesis while increasing osteogenesis in MSC cells. However, much like studies showing diverse effects of 1,25(OH)2D3 on osteoblast differentiation discussed above, discrepant results were also seen when effects of 1,25(OH)2D3 on adipogenic differentiation were
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examined in vivo. Specifically, VDR-null mice actually have a lean body mass, lower plasma triglycerides and cholesterol levels and are resistant to the obesityinducing effects of a high-fat diet [110]. One study suggested that the unliganded VDR is actually necessary for adipogenesis even though the VDR-1,25(OH)2D3 complex can inhibit adipocyte formation [111]. This would explain why VDR-null mice are lean although it is inconsistent with the finding that MSCs from VDRnull marrow exhibit enhanced adipogenic differentiation in vitro. Taken together, these studies further stress the need for careful use of genetic models to discriminate between physiological and nonphysiological effects of the 1,25(OH)2D3 on the MSC lineage.
SUMMARY AND CONCLUSIONS Osteoblasts are derived from mesenchymal stem cell (MSC) progenitors and are only transiently present during periods of active bone formation when their differentiation from MSCs is stimulated. The primary function of osteoblasts is to secrete and mineralize the type I collagen-containing extracellular matrix of bone. A fraction of osteoblasts become entrapped in the matrix they secrete as osteocytes, a long-lived component of bone having a lifespan measured in years. Osteocytes do not secrete large amounts of ECM proteins, but may participate in the mineralization process, which occurs in bone osteoid distal to the osteoblast layer. Cells of the osteoblast lineage also have a number of regulatory functions including control of bone remodeling, regulation of phosphate homeostasis, and response to mechanical loads. The vitamin D hormone, 1,25 (OH)2D3, affects osteoblast function at multiple levels. It controls remodeling via induction of receptor activator of NF-kB ligand (RANKL), regulates phosphate homeostasis by increasing fibroblast growth factor 23 (FGF23) and may enhance the response of bone to mechanical loads via stimulation of mitogen-activated protein kinase signaling. Lastly, 1,25(OH)2D3 stimulates MSC differentiation to the osteoblast lineage and suppresses adipocyte formation via stimulation of the Wnt pathway. Although all cells in the osteoblast lineage (MSC-derived osteoprogenitors, osteoblasts, and osteocytes) express VDRs, 1,25(OH)2D3 may target different cells for different regulatory actions. For example, it stimulates RANKL expression in all three cell types while preferentially stimulating FGF23 only in osteocytes. Although 1,25(OH)2D3 has clearly documented effects on the in vitro differentiation of osteoblasts, there is generally poor agreement between different in vitro and in vivo model systems. Surprisingly, classic vitamin D deficiency studies and gene ablation experiments of
the VDR and/or the 1a-hydroxylase gene generally show only subtle direct actions of 1,25(OH)2D3 on osteoblast differentiation and function with most responses to 1,25(OH)2D3 being attributed to systemic effects on calcium and phosphate homeostasis via stimulation of intestinal calcium and phosphate absorption. Further work, particularly carefully controlled genetic studies, will be required to discriminate between direct actions of 1,25(OH)2D3 on osteoblasts and systemic effects on mineral metabolism.
Acknowledgments Work from the authors’ laboratory cited in this article was supported by NIH Grants DE 11723, DE13386, a research fellowship from the University of Michigan Center for Organogenesis (YL) and the Michigan Diabetes Research and Training Center (NIDDK Grant DE020572).
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C H A P T E R
18 Osteoclasts F. Patrick Ross Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA
metastasis lose bone mass, invariably as a result of greater net degradation.
INTRODUCTION Bone Formation and Turnover Bone is a more complex organ than envisaged as little as 20 years ago [1e6]. The appendicular skeleton, a mixture of cortical and trabecular bone, the latter providing much of the mechanical strength, develops as a result on endochondral ossification, while the bones of the skull and jaw arise by intramembranous ossification. Three integral cell types contribute significantly to regulation of bone mass; osteoblasts, of mesenchymal origin, are transducers of numerous endocrine and paracrine signals. Separately they secrete and calcify the unique bone matrix, which is degraded by myeloidderived osteoclasts. Most recently discovered and studied are osteocytes [7], nondividing cells that are generated by encapsulation of mature osteoblasts within the calcifying tissue. These latter cells, by far the most numerous in mammalian bone, are principally mechanosensors that respond to force by secreting molecules that modulate both osteoblast and osteoclast function. Finally, the recently developed field of osteoimmunology has led to the realization that lymphoid cells are also major transducers of signals impacting the other three cells types, often in a complex manner involving signals to one cell type that are then transmitted to a second [8,9]. A longstanding cell-based model which has stood the test of time suggests that osteoblasts and osteoclasts are “coupled” such that effete bone is degraded and replaced. In young healthy adults this turnover results in no net bone loss. In contrast, starting in utero, and continuing for about 30 years, bone mass increases as a consequence of net greater formation and is stable in early midlife. Alternatively, older adults and people with a wide range of metabolic or endocrine abnormalities, or those suffering from the complications of tumor
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10018-6
Basic Osteoclast Biology The osteoclast, the only cell capable of bone degradation, is of hematopoietic origin. This fact was confirmed in man by the elegant experiment in which transplantation of male marrow to a female recipient suffering from malignant osteopetrosis resulted in resolution of the disease, accompanied by appearance of osteoclasts bearing only the Y chromosome [10]. Many experiments have affirmed the original finding, including many examples of bone marrow rescue replicative of the early work, plus studies in which mice lacking the early myeloid-specific gene PU.1 fail to develop osteoclasts [11]. While it is clear that hematopoietic stem cells (HSCs) are osteoclast precursors, the initial steps in lineage development have not been defined completely [12]. Thus, the earliest precursor that can be isolated and manipulated readily in vitro is the bone marrow macrophage (BMM) or its splenic counterpart, cells which arise from HSCs by incompletely understood signaling pathways. It is possible now to generate sufficient mature murine osteoclast-like cells to perform a wide range of cell biology studies by exposing BMMs to just two cytokines, M-CSF and RANKL, whose receptors are c-Fms and RANK, respectively [13]. Genetic deletion and/or inactivation of either cytokine or its respective receptor, as a result of natural mutations or following manipulation of the genome, results in profound osteopetrosis accompanied by complete absence of osteoclasts [13,14]. Interleukin (IL) 34, whose threedimensional structure mimics that of M-CSF without the two molecules exhibiting detectable amino acid homology, was cloned recently and shown to replace the cytokine functionally both in vitro and in vivo [15].
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This observation explains the longstanding conundrum that deletion of c-Fms leads to a more profound osteopetrosis than that seen in the mice lacking M-CSF [16]. Nevertheless, the fact that administration of M-CSF to the op/op mouse and rat which cannot produce M-CSF, rescues the bone phenotype [17,18] speaks to the major role played by this molecule in osteoclast biology. Of interest, IL-34 is secreted at high levels by the stromal, presumptively oncogenic, component of giant cell tumor of bone [19], helping to explain why patients exhibiting this condition have marked lytic capacity. Finally, while M-CSF and RANKL are the basal proteins necessary to generate and activate osteoclasts, a range of secondary stimulators also play an important role in the same process. Major contributors, in man and/or mouse, include tumor necrosis factor (TNF) and interleukins (IL)-1 [20e23], -6 [24,25] -11 [26], and -17 [27e29] while the data on transforming factor beta (TGF-b) [30,31] and bone morphogenetic proteins (BMPs) [32] require further validation. Many other cytokines impact osteoclast formation by inducing RANKL and M-CSF, while reducing OPG in the stromal and lymphoid compartments of bone [5].
Overview of Osteoclast Function The mechanism by which osteoclasts resorb bone is now understood in some detail (Fig. 18.1). The fully differentiated polykaryon adheres to bone, largely via the integrin avb3 [33], following which the cell polarizes in a c-src-dependent manner [34]. The heterodimeric integrin binds several proteins in bone that contain the motif Arginine-Glycine-Aspartic acid [35]. Numerous intracellular lysosomal-like vesicles enriched in the acidic collagenase cathepsin K traffic to the boneapposed surface [36], where their fusion requires the calcium-binding protein synaptotagmin VII [37]; synpatotagmins play central roles in vesicleemembrane fusion across many phyla [38,39]. Fusion creates the characteristic ruffled membrane, the unique osteoclast organelle. It is presumed but unconfirmed that the membrane of the same fusogenic vesicles rich in cathepsin K contain high levels of the vacuolar ATPase complex and its charge-coupled chloride channel that secrete protons and chloride ions respectively, resulting in a markedly acidic pH in the resorptive lacuna. Discharge of the vesicular contents releases cathepsin K and matrix-metalloprotease (MMP) 9 into the same space. The protein responsible for chloride transport is the product of the ClC7 gene [40]. Key members of the multimolecular ATPase complex include the a3 and d2 subunits [41,42] and Ac45, with the latter facilitating movement of vesicles to the cell surface [43]. The same role has been ascribed to osteoclast specific transmembrane protein 1 (OSTM1) [44], the product of the
HCO3/-Cl-
HCO3/-Clexchanger
H2O+CO2 Carbonic Anhydrase ClCath K Sy H+ HCO3tV II RGD
OC
αvβ3
HCl
Bone
Overview of osteoclast function. The osteoclast is a multinucleated cell that adheres to bone matrix primarily via interaction of arginine-glycine-aspartic acid (RGD) sequences in several proteins with the dominantly expressed integrin avb3, generating the tightly apposed sealing zone. This ligation triggers undefined signals that stimulate movement of intracellular vesicles rich in cathepsin K, that also express on their surface the multicomponent vacular ATPase and its charge-coupled chloride channel. Fusion of the vesicles with the plasma membrane generates the highly invaginated ruffled border, the unique resorptive organelle of the cell and results in secretion of cathepsin K into the bone-apposed compartment. Hydrolysis of ATP leads to secretion of protons and chloride ions, the latter via the charge-coupled chloride channel. Intracellular pH is maintained by passive chlorideebicarbonate exchange on the contralateral surface. Acid dissolves the inorganic components of bone and acidifies the resorptive space, enhancing the capacity of cathepsin K to degrade collagen, the major protein in bone. Vesicle transport and fusion is mediated in part by synaptotagmin VII, a member of the large protein family that performs an analogous function in many cell types.
FIGURE 18.1
gl gene, mutation of which results in severe infantile osteopetrosis [45]. The acidic environment below the ruffled border dissolves the inorganic bone matrix, while cathepsin K, with an acidic pH optimum [46], degrades type 1 collagen, the major protein in bone. MMPs act in an auxiliary manner. Intracellular pH balance is achieved by a contralateral chlorideebicarbonate exchanger [47e49]. Protons and bicarbonate ions are generated mainly by the activity of carbonic anhydrase II [50]. Finally, collagen fragments are released in part by being transported across the cell [36] and in part by diffusion from the site following osteoclast migration; their serum levels are the basic of the current most specific market of osteoclast activity in man and rodents, the CTx-1 assay, while osteoclast number can be assessed by measurement of serum-tartrate-resistant acid phosphatase (TRAP) 5b levels [51]. Deletion or mutation of numerous genes in mice leads to osteopetrosis arising from impaired osteoclast formation or function [52]. In contrast, it was believed until
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recently that human osteopetrosis is characterized solely by malfunction, involving failure to acidify [50]. However, rare mutations leading to juvenile osteopetrosis have been reported for osteoclast-specific transmembrane protein 1 (OSTM1) and pleckstrin homology domain-containing family M member 1 (PLEKHM1), two molecules involved in osteoclast polarization [53e55]. Other human genetic abnormalities for which there is considerable molecular understanding include Paget’s disease of bone, caused by mutations in a RANK, RANKL, and p62, also called sequestesome 1 (SQSTM1), a protein in the RANKL signaling pathway [56,57]. A more contentious hypothesis, but for which there is experimental support is that paramyxoviral infection contribute to the disease [56]. Rarer conditions include pycnodystosis as a result of decreased or absent expression of cathepsin K [58], juvenile expansile osteolysis (JEO), and its related mutations [59] and recent reports of inactivating mutations in both RANKL [60] and RANK [61].
aspects of their differentiation into osteoclasts and the activation and lifespan. Like other receptor tyrosine kinases, binding of its ligand to c-Fms results in homodimerization and autophosphorylation in trans on a number of tyrosine residues. These phosphotyrosine sites act as docking stations for a variety of proteins, which can act as adapters, recruiting multimacromolecular complexes that contain cytosolic kinases or phosphatases, with enhanced or suppressed dissemination of a wide range of signals. Alternatively, specific docking sites attract kinases or phosphatases directly and the net activities of these activated molecules provide a dynamic set of cues that drive cellular function [66] (Fig. 18.2).
RANKL/RANK Signaling RANKL and RANK are members of the TNF superfamily which share many general features structurally
Overview of Signaling from c-Fms and RANK M-CSF and RANKL activate many signaling pathways, some independent and others overlapping, together mediated by numerous downstream effectors. A single protein in particular, however, is the protooncogene c-Src, the prototype src family kinase [62,63] and a central regulator of the osteoclast. Use of genetically deleted mouse became possible as a result of groundbreaking work in several laboratories in Europe and the USA. One of the first genes deleted was that coding for c-Src; the null animal was expected to have major phenotypes manifesting in a number of tissues, including brain; the great surprise was that severe osteopetrosis was the sole manifestation of the genotype [64]. Thus, mice lacking c-Src have many more osteoclasts which cannot polarize and hence exhibit a total failure to degrade bone. Later work identified the osteoblast as a second target of c-Src function [65], but numerous reports have revealed its central role in the osteoclast. While a number of functionally important c-Src targets in osteoclasts have been identified including PI3K [66], DNAX activating protein of 12 kilodaltons (DAP12) and spleen tyrosine kinase (Syk) [67], dynamin [68], ADP ribosylation factor (Arf) 6 [69], and cortactin [70], the list is probably incomplete (see for support of this assertion [69]). As for other key intracellular kinases, c-Src is present in osteoclasts in several signaling complexes, including those anchored by c-Fms [71], avb3 [72], phospholipase gamma (PLCg)2 [73], and proline-rich tyrosine kinase (Pyk)2 [68]. Signaling downstream of M-CSF/IL-34, by activation of the receptor tyrosine kinase c-Fms, contributes to the proliferation and survival of early precursors, key
c-Fms DAP12 PM
Akt
c-Src P Y
PI3K
c-Src PY PY
ERKs P Y p27 D-cyclins
Bcl-2, Bim, Mcl-1 IAP/survivin
Vav3
c-Fos
PRb
Proliferation
SYK
Rac GDP
Survival
Differentiation
Rac GTP
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FIGURE 18.2 Summary of major pathways downstream of the MCSF receptor c-Fms. Binding of M-CSF to c-Fms dimerizes and activates the membrane tyrosine kinase, resulting in autophosphorylation in trans on a number of residues. Four major sets of signals emanate, controlling proliferation, differentiation and survival (left side of cartoon) and reorganization of the actin cytoskeleton (right side). Specific phophotyrosine-containing regions of the cytoplasmic tail recruit c-Src and the MAPK family members, p42/44 (ERKs). c-Src assembles a signalsome that includes c-Cbl (not shown for simplicity) and PI3K, which phosphorylates Akt, which in turn regulates expression of p27 and the cyclin D family, key upstream suppressors of Rb phosphorylation. Hypophosphorylated Rb enhances levels of a number of genes that are required for G1/S transition. Akt, together with ERKs, decreases apoptosis by modulating the relative amounts of pro- and antiapoptotic proteins. ERKs, by phosphorylating C-Fos, increase its stability and capacity to act as a transcription factor required for osteoclast differentiation (see Fig. 18.3). Finally, c-Src, as a part of a second signalsome, phosphorylates first DAP12, an immunoreceptor that contains an ITAM motif (two tyrosines with specific spatial separation) and then SYK, which is recruited to the activated ITAM domain. Complex downstream events finally recruit and activate the Rac-specific GEF Vav3; activated Rac, again via multiple intermediary processes, leads to assembly of a growing actin chain, the key process in remodeling of the cytoskeleton. See text for more details.
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and functionally [74]. Ligation of RANK initiates homotrimerization, with recruitment of a number of adapter proteins of which the most important is TNF receptorassociated factor 6 (TRAF6), which then recruits several signaling complexes that control a wide range of effectors [3,75]. Further discussion of RANKL/RANK signaling requires introducing the area of osteoimmunology, which studies the interactions between and commonalities of the fields of bone and immunology [76]. Many proteins and pathways that are important in T and B cell biology are also key players in bone. Prominent molecules that meet this definition include RANKL and RANK themselves, DAP12 and FcRg, whose cytoplasmic tails contain phosphotyrosine motifs that recruit the cytosolic kinase Syk, two further kinases, Bruton’s tyrosine kinase (BTK) and tyrosine kinase expressed in hepatocellular carcinoma (Tec), the adapters SH-2 domain containing leukocyte protein of 65 or 76 kilodaltons (SLP65/76), the phosphatase PLCg2, Vav3, an activator of small GTPases, and several SFK members. Several of these proteins also mediate events downstream of c-Fms. The role of each of these will be discussed in more detail in the appropriate context. While osteoclast generation and activity represent a continuum in signal transduction, it is convenient to divide the process into separate steps: proliferation, differentiation, survival/apoptosis (programmed cell death), and function, which includes adhesion, migration, and resorption. M-CSF and RANKL play varying roles in each event. The sections that follow will discuss each topic briefly.
Osteoclast Differentiation As noted some time ago [80] M-CSF also regulates osteoclast differentiation. At a molecular level, activation of c-Fms induces expression of RANK [14], the receptor for RANKL, and phosphorylation of transcription factors c-Fos and microphthalmia-related transcription factor (MITF), the latter in an ERK-dependent manner following activation of c-Fms [81]. c-Fos is a transcription factor belonging to the AP-1 homo-/heterodimer family, which collectively plays several roles in osteoclast differentiation and function [75]. Phosphorylated MITF, also a RANKL target, trans-activates many osteoclast genes [82]. Pioneering studies characterized the critical steps in RANKL-induced osteoclast formation (Fig. 18.3). Liganding of RANK leads to homotrimerization and binding of several adapter proteins of which the most important is TRAF6, followed by recruitment and/or activation of three signaling complexes all containing a kinase; the I kappa B kinase (IKK complex), Jun N
RANK Signaling Pathway
Intracellular TRAF6 Ca2+
Activation of c-Fms provides the signals that stimulate precursor division [66]. Earlier studies examining c-Fms signaling involved overexpression of the receptor in cells that never express the molecules, leading to uncertainty about the results obtained. More recently two groups [77,78] used primary macrophages as targets for expression of the membrane-associated kinase containing wild-type or mutated forms of the cytoplasmic tail. Both showed that only a subset of tyrosine residues mediate signaling for proliferation via downstream effectors c-Src, extracellular signaling-regulated kinase ERKs)1/2, the serine/threonine kinase Akt, and phospholipase C. A more detailed examination revealed that major mediators of cell division are levels of (a) p27, an inhibitor of cyclin-dependent kinases (CDKs), (b) D-type cyclins that increase CDK function, and (c) retinoblastoma protein (Rb), a CDK substrate. Since hyperphosphorylated Rb stimulates expression of genes responsible for initiation of the cell cycle [79] the net effect is increased proliferation.
IKK complex
CaMK
CN
Precursor Proliferation
RANK
Extracellular
NFATc1
NIK
NFkBs
JNK/p38 c-Fos
Kinases MITF
TFs
Target proteins
FIGURE 18.3 Summary of major pathways downstream of the RANKL receptor RANK. RANK monomer is trimerized by binding of RANKL and undergoes conformation rearrangement, resulting in recruitment of several adapter proteins, among which TRAF6 is the most important for osteoclast differentiation, a complex process involving five major pathways. Intracellular calcium fluxes arise as a result of coupling between RANK and the two immune-related receptors DAP12 and FcRg, which together activate phospholipase C2 (PLC2). Calcium binds calmodulin, stimulating calmodulin kinase and the serine threonine/phosphatase calcineurin. These molecules initiate transcription of c-Fos and dephosphorylation of the inactive transcription factor NFATc1, which translocates to the nucleus. Four other kinases are downstream of TRAF6, two targeting NFkB, one Jun, and one MITF. NFATc1 autoactivates its transcription and is required for that of c-Fos, MITF and p50, p52 and p65, the active components of NFkB. The combined function of the four transcription factors leads to expression of the multitude of gene products required for osteoclast function. Not shown in detail is the fact that NFATc1 also induces a number of negative regulators of osteoclastic gene transcription, this representing a negative feedback loop.
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terminal kinase (JNK), or mitogen-associated protein kinase of 38 kilodalton p38. In combination the kinases target three transcription factors, nuclear factor kappa B (NFkB), the c-Fos/Jun complex, and MITF, respectively. A separate pathway involves DAP12 and FcRg and their downstream effectors, the kinases Syk, BTK, and Tec and the adapters SLP65/76, converging on PLCg. The events surrounding calcium release and function are the subject of active research. PLCg hydrolyzes PIP3 to DAG and IP3; the latter releases Ca from the SR by opening the SERCA channel [83], a signal augmented by secondary influx from two plasma membrane calcium channels, transient receptor potential vanilloid (TRPV)4 and TRPV5. Attesting to the concept that intracellular signals are compartmentalized these two related proteins play distinct roles in the osteoclast; TRPV4 regulates terminal differentiation [84], while TRPV5 impacts bone resorption via an NFkB-dependent pathway [85], while its deletion in human osteoclasts increases their number but decreases function [86]. The well-characterized epithelial calcium receptor also controls osteoclast differentiation and apoptosis [87], including the cell death stimulated by the drug strontium ranelate [88]. Finally raised intracellular calcium binds to calmodulin, which activates two pathways, each of which enhances differentiation by separate effectors. Calmodulin kinase 4 phosphorylates the transcription factor CREB, thus increasing expression of c-Fos [89], which binds with a JUN family member as an AP-I complex to the regulatory region of nuclear factor of activated T cells (NFAT)c1, also called NFAT2. Separately, calmodulin activates the phosphatase calcineurin, resulting in dephosphorylation of cytosolic NFATc1, whose subsequent nuclear translocation releases its transcriptional function [2]. While genetic evidence confirms the role of NFkB, c-Fos/Jun, and MITF as important transcription factors, the overwhelming evidence indicates that a single protein, NFATc1 is likely a master controller of osteoclastogenesis [2]. There are confirmed binding sites for NFATc1 in several thousand genes [90,91] and its regulatory domain contains an NFATc1 site, resulting in autoup-regulation. In consequence genetic deletion of NFAT in osteoclasts results in profound osteopetrosis. It is beyond the scope of any review to list all the genes controlled via the RANKL/RANK/NFATc1 pathway; suffice it to say that the majority of the proteins documented to be central to the differentiation and function of osteoclasts are induced temporally. Recent findings have uncovered a novel mechanism by which osteoclastogenesis is regulated in the form of RANKL/NFATc1-inducible genes that act as transcription suppressors. To date interferon regulatory factor (IRF)8, B lymphocyte-induced maturation factor
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(BLIMP)1, and B cell lymphoma (BCL)6, all suppressors of B and/or T cell ontogeny [92e94] and musculoaponeurotic factor (Maf)B, a protein involved in lineage commitment [12], have been documented to act in this manner. All four proteins are regulated by NFATc1; MafB and BLIMP1 are decreased, while BCL6 and IRF8 are increased following exposure of osteoclast precursors to RANKL. The current model suggests that MafB, IRF8, and Bcl6 inhibit NFATc1 function by competing for binding on target genes, while BLIMP1 is an upstream negative regulator of BCL6 [91,95e97]. These observations begin to provide an explanation as to how RANKL signaling is subject to feedback control.
Osteoclast Apoptosis Apoptosis is a highly regulated, complex set of events in which a key step is activation of the highly conserved protease caspase 3, resulting in irreversible cell death [98,99]. Two major pathways are upstream of caspase 3; one designated as intrinsic, in which mitochondria release cytochrome c, leading to formation of the apoptosome and cleavage of full-length, inactive caspase 3 [100] and the second as extrinsic, where signals from the cell surface create active caspase 8, which itself degrades caspase 3 [101]. Bcl-2 family proteins are pro- or antiapoptotic [98] and their net balance controls extrusion of mitochondrial cytochrome c; separately, caspase function is blocked by several inhibitor of apoptosis (IAP)/survivin family members [102]. The fact that osteoclast lifespan is short (days) compared to that for osteoblasts (months) and osteocytes (years) suggests that factors that regulate this event will be important for its function, a hypothesis supported by many studies. In short, M-CSF and RANKL increase osteoclast survival by coordinated control of a combination of overlapping and parallel signals. In early studies M-CSF, TNF, and RANKL increased osteoclast lifespan in vitro by activating the mammalian target of rapamycin (mTOR) pathway; no downstream pathways were investigated [103]. Later work showed that M-CSF decreases expression of proapoptotic Bim, by targeting it for proteasomal degradation [104,105], while up-regulating the prosurvival BCLXL members and Mcl-1 [106,107]; the latter was also stabilized following RANKL treatment [108]. Finally, by activating its downstream kinase TGFb-activated kinase (TAK1), which is also stimulated by RANKL [109], TGFb increased levels of BCL-XL and Mcl-1 [107], while the same agonist increased survival of human osteoclasts; the data provided limited mechanistic insight, but suggested a role for down-regulation of Bim [110].
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Osteoclast Adhesion and Migration As discussed previously a prerequisite for bone degradation is close apposition of the polarized osteoclast with its target matrix, an event mediated by several surface-residing proteins, the best studied of which is the integrin avb3 [33]. Deletion of the expressionlimiting beta change of the heterodimer results in development of moderate osteopetrosis with age, suggesting that other adhesion receptors can compensate partially. Likely candidates include the integrin a2b1 [111], CD44 [112, 113], a protein present in various splice forms [114] or other unknown receptors. Finally, ligation of the integrin a9b1 by the disintegrin domain of a disintegrin and metalloprotease (ADAM)8 regulates spreading of osteoclasts in vivo and in vitro [115]. Osteoclasts and their precursors are motile cells, a feature that requires they rapidly reorganize their tubulin- and actin-based cytoskeletons. While almost nothing is known about how signal transduction pathways control the former, there is a rich literature as regards the latter. Histone deacetylase (HDAC) 6, known best for its role in controlling the accessibility of transcription factors to condensed chromatin [116], also deacetylates osteoclastic gamma tubulin, resulting in decreased bone resorption [117e119]. Compelling data indicate that Fms-dependent movement requires synergistic signals from the integrin avb3. The pathway regulating cytoskeletal remodeling and hence migration requires integration of signals from the integrin and c-Fms. Surprisingly, the intracellular proteins involved in these events include DAP12 and Syk [67,72], SLP65/76 [120], the kinases BTK and Tec [121] and Vav3, a guanosine exchange factor (GEF) for Rac [122]. Rac is one member of the small GTPases of the Rho subfamily, Rac, cdc42, and RhoA; these molecules are important broad regulators of actin remodeling. The biochemistry underlying generation of fibrous actin from its monomeric precursor is now well understood. In short, linear aggregation involves a role for formins [123,124], while the Arp 2/3 complex drives branching [125,126]. The net activity of these assembly processes is countermanded by disaggregation at the opposite end of the polymer, a well-described process [125]. Deletion of Wiskott-Aldrich syndrome protein (WASp), an upstream effector of actin-related protein (Arp)2/3, suppresses bone resorption, [127]; similarly, down-regulation of one component of the Arp2/3 complex yields the same outcome [128]. Signals more proximal to actin assembly/disassembly are numerous and relatively complex, but a focal point is activation of a group of small GTPases, part of the Rho GTPase family [129], itself one of the five members of the Ras superfamily [130]. As with all small GTPases, activation involves generation of the GTP-bound form, while
dephosphorylation yields an inactive molecule; the enzymes mediating these two events are GEFs and GTPase-activating proteins respectively [129]. Five Rho GTPases have been characterized and analyzed partly in osteoclasts: Rac1 and Rac2, RhoA, cdc42, and RhoG (also called Wrch-1) [130e133]. Many of the studies in osteoclasts used overexpression of constitutively active or dominant negative forms of individual proteins, an approach posing significant problems, since there is crosstalk between members. Recently Rac1 and Rac2, the isoforms in osteoclasts, were deleted and the resulting mice have osteopetrosis [134]. Similarly, genetic manipulation of mice to create animals lacking cdc42 in osteoclasts or having a basally hyperactive form of the same GTPase, yielded reciprocal osteopetrotic and osteopenic phenotypes respectively [135]. The same report found that RANKL treatment of pro-osteoclasts also activates cdc42.
Osteoclast Fusion The macrophage is only one of three mammalian cell types to undergo fusion, the others being sperm/egg and myoblasts. There are few studies that provide significant insight into the fusion process in the context of the osteoclast. Deletion of dendritic cell-specific transmembrane protein (DC-STAMP) abrogated formation of polykaryons [136]. Since molecular modeling of DC-STAMP indicates it is a member of the seventransmembrane family of G-protein-coupled receptors (GPCRs), its endogenous ligand remains to be identified [137].
Secretion of OB Activators While it is well established that osteoclasts release active TGFb from bone matrix, there is growing evidence that they generate and secrete factors that act in an autocrine/paracrine manner [138,139]. The molecular basis has evolved slowly but recent studies have revealed that RANKL induces the expression of sphingosine 1 kinase [140,141]. Moreover, osteoclasts secrete the enzyme’s product, the soluble lysosphospholipid sphingosine-1 phosphate (S1-P) [142]. This bioactive moiety, chiefly the product of erythrocytes, circulates in plasma at levels higher than in tissue [143]. S1-P also modulates osteoclast and osteoblast differentiation [144] and, in accordance with its known role in the lymphoid system, [145] regulates trafficking of osteoclast precursors through marrow [141].
Bone Metastasis and the Role of Chemokines in Regulating the Osteoclast Three major types of cancer, breast, prostate, and multiple myeloma, metastasize to bone, as a result of
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INTRODUCTION
which they are responsible for significant morbidity and mortality. There is considerable knowledge of the mechanisms by which mestastatic breast cancer augments its progression, involving the well-described vicious cycle in which the initial epithelial cells secrete factors that stimulate resorption, which release molecules that stimulate expansion of cancer cells into the newly formed bone cavities. Progression is accompanied by secretion from the invading tumor of proteins that stimulate bone formation [146]. It is clear now that chemokines are important in this form of high-turnover osteolysis, as well as for the pathogenesis of myeloma-derived bone loss [147,148]. The situation is more complex in the case of prostate cancer, where the net outcome is often increased bone mass, most likely resulting from the presence of an abundance of osteotrophic factors; once again likely culprits include chemokines. Chemokines are a large family of small proteins, characterized by the presence of one of several cysteinecontaining motifs that allow classification into subgroups [149]; it is beyond the scope of this review to detail the roles of chemokines in all aspects of bone biology. In general the targets of these small proteins include osteoclast precursors themselves, as well as accessory cells in the marrow environment; in the latter circumstance their impact is to stimulate secretion of a range of molecules that enhance expression of RANKL and ILs, among others. Earlier work indicated an important role for the CXCR4eCXCL12/SDF1 axis in osteoclast biology (reviewed in [150,151]) but there have been questions about whether this represents a suitable therapeutic target [152]. Recent reports have expanded our knowledge to include other chemokines and their receptors, including CXCL2 [153], CXCL11 [154], and CCR1 [155].
The Role of Other Cell Types, Hormones, and Receptors in Osteoclast Biology As indicated above, my comments have focused on recent advances as to how M-CSF and RANKL regulate osteoclast formation and function, by impacting a single cell of myeloid origin. It is abundantly clear, however, that many other molecules and several cell types contribute to the same two events. In addition to mesenchymal cells in the bone marrow environment, their precursors in the bone niche are likely responsive to paracrine and/or endocrine signals, including those stimulated by PTH, prostaglandins, a number of interleukins, and TNF [5]. At least in the murine model there is an in vivo hierarchy in that IL-1 and M-CSF are downstream of TNF [21,156,157]. Consistent with the model in which osteoclast formation follows angiogenesis, endothelium secretes and responds to chemokines, thereby increasing expression of osteoclastogenic factors [158]. In addition to
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the plethora of soluble factor controlling osteoclast formation and function, several interactions between moieties located in the plasma membrane of myeloid and mesenchymal cells contribute to the same outcomes. Thus, ligation of Notch receptors by their ligands is important for both osteoblast and osteoclast biology [159] and the same appears to be true for the Eph-Ephrin families [160]. For a more general overview of osteoblasteosteoclast coupling mechanisms see also [161]. There is now a strong body of evidence implicating T and B cells as important effectors of several hormones, including estrogen and PTH [162,163]. Moreover, B cells secrete OPG, the endogenous circulating RANKL inhibitor, in response to IL-7 [163]. Finally, while the hedgehog pathway is an established regulator of embryonic skeletogenesis, recent studies reveal that the same signals control osteoblasts and osteoclasts postnatally (reviewed in [164]). Circulating steroids impact the skeleton in a complex manner [165e167]. To summarize, while estrogen is the major regulator of bone mass in both men and women, androgens, also produced by women at low levels and the source of male estrogens as a result of aromatization, have an anabolic effect per se. Glucocorticoids were postulated to control bone mass mainly by regulating osteoblast apoptosis [168] but their role has expanded to include modulating secretion of nitric oxide and perhaps other factors by osteocytes [169,170], potentially regulating the OC and thus bone mass. Finally, the adrenal steroid and synthetic analogs suppress osteoclast formation directly by inhibiting actin reorganization, while increasing the lifespan of the cell, the net effect being decreased bone loss in a murine model of postmenopausal osteoporosis [171]. An area of recent interest concerns endocannabinoids, generated by hydrolysis of cell-surface phospholipids, their plant-derived or synthetic analogs, and related compounds such as lysophosphatidyl serine. These molecules bind with varying affinity to cannabinoid receptors CB1, CB2, or the putative homolog CRB55, members of the large GPCR family, to regulate osteoblast and/or osteoclast function differentially. As summarized in [172] mice lacking CB1 or CB2 exhibit osteoporosis with age. The former have malfunctioning osteoclasts initially but bone formation is also impaired, while in contrast CB2-null animals have high bone turnover accompanied by uncoupled bone formation and resorption. In contrast, GPR55 suppresses osteoclast function with no impact on the osteoblast and hence the deleted animals are osteosclerotic.
Strategies for Inhibition of Osteoclasts in Vivo Currently the major drug used to inhibit osteoclast function is one of a number of amino-containing
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bisphosphonates, whose mode of action is largely blockade of the enzyme that adds a large hydrophobic isoprenyl group to proteins in the Ras superfamily, thus inhibiting processes that suppress apoptosis and/ or cytoskeletal reorganization [173]. Given the detailed knowledge about the key role of RANKL and the acidic collagenase cathepsin K in osteoclast biology researchers developed a humanized antibody (denosumab) against the first and a small molecule inhibitor of the second (odanacatib). Denosumab rapidly, potently, and reversibly blocks bone resorption and increases bone mass in postmenopausal women [174] and odanacatib exhibits similar results in early studies [175]. Denosumab is now FDA-approved, while odanacatib is in phase three clinical trials.
Vitamin D and Bone Many studies have documented that the hormonally active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH2)D3) is an important regulator on bone mass, via mechanisms that target the organ itself, the kidney, and the intestine. Since its role with respect to the latter two is reviewed elsewhere in this volume, I will comment only briefly on the overall picture and then focus on novel advances relating to cells in bone; detailed reviews covering this field have been published recently [176,177]. Numerous reports show that the seco-steroid targets cells of the mesenchymal/osteoblast lineage, increasing expression of the osteoclastogenic cytokines M-CSF and RANKL, while decreasing those of the inhibitor OPG. Importantly, levels of RANKL decrease during osteoblast differentiation (reviewed in [178]). Novel observations include the fact that 1,25 (OH2)D3 treatment of the severely osteoporotic mice lacking OPG increased bone mass by suppressing RANKL-induced expression of the transcription factor c-Fos and hence osteoclast number and activity [179]. These data indicate that although the hormone is anabolic overall, its activities include a direct suppressive role on the osteoclast. Another mechanistic insight was reported by Carmeleit and colleagues who found that VDR genomic action in chondrocytes stimulated expression of RANKL. Tissue-specific deletion of the receptor resulted in mice with higher bone mass, attesting to the importance of this finding [180]. Finally, recent studies have suggested a para- or intracrine role for the seco-steroid hormone. Both osteoblasts and osteoclasts in vitro express cyp27b1, the enzyme involved in generation of the active hormone from its circulating precursor D. Moreover, osteoblasts express cubulin and/or megalin, surface receptors required for internalization of the circulating vitamin-D-binding protein (DBP)-prohormone complex. Consistent with these
observations, their exposure to the precursor results in generation of measurable levels of 1,25(OH2)D3 [181,182]. While these findings suggest a novel paracrine role for the hormone, in vivo relevance of these findings awaits targeted deletion of the receptor, analogous to those performed in the context of the chondrocyte.
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C H A P T E R
19 Molecular Mechanisms for Regulation of Intestinal Calcium and Phosphate Absorption by Vitamin D James C. Fleet, Ryan D. Schoch Department of Foods and Nutrition, Purdue University, West Lafayette, IN, USA
AN OVERVIEW OF INTESTINAL CALCIUM ABSORPTION Whole-body calcium metabolism is controlled by actions occurring within and between a number of tissues including the intestine, kidney, bone, fat mass, and brain in an effort to maintain serum calcium within a narrow range [1,2]. In this light, intestinal calcium absorption from the diet is an essential process maintaining calcium balance and bone health. Calcium absorption is moderately efficient in freeliving humans (35% of a typical dietary load is absorbed). Kinetic modeling based on the efficiency of absorption across a wide range of luminal calcium concentrations shows that the transfer of calcium across the intestinal barrier occurs through both saturable (presumably transcellular) and nonsaturable (presumably paracellular diffusion) pathways [3e6] (see Fig. 19.1). The existence of these two transport routes can be modeled mathematically using a MichaelisMenton-like equation modified to include a linear component (see equation in Fig. 19.1). The saturable component of calcium absorption is prevalent in the proximal small intestine (duodenum and jejunum) and is under nutritional and physiological regulation (see below). This is an energy-dependent pathway whereby calcium movement from the mucosal to serosal side of the intestinal barrier can occur across a concentration gradient [7]. The saturable pathway is absent in the ileum [8] but some animal studies have reported that saturable calcium transport may also be functional in the lower bowel [9e11]. In contrast, passive transport occurs throughout the length of the intestine and is
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10019-8
a nonsaturable, linear function of luminal calcium concentration (13% of luminal load per hour in humans [6]). The Km for the saturable component of calcium absorption from the small intestine of adults is 265 mg in a meal (calculated from data in [5,6]). For typical levels of calcium intake (400 mg per meal), the saturable
Kinetic modeling of intestinal mineral absorption demonstrates the existence of saturable and nonsaturable pathways. Examination of calcium or phosphate absorption under a range of luminal mineral levels has demonstrated that the total amount of the mineral transported across the intestinal barrier is described by a curvilinear function. Total transport is the sum of a linear, concentration-dependent, and nonsaturable transport process (defined mathematically by a simple straight line) and a saturable component that can be defined by the Michaelis-Menton equation. A ¼ total mineral absorption; [M] ¼ luminal concentration of the mineral; C ¼ the slope of the non-saturable linear component assuming the intercept equals zero; Vmax ¼ the maximum transport rate seen for the saturable transport component; Km ¼ the luminal concentration of the mineral at 1/2 the Vmax.
FIGURE 19.1
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component of calcium absorption may account for as much as 60% of total calcium absorption. However, as Figure 19.1 clearly shows, as the calcium level in a single meal is increased, the contribution of the saturable calcium transport pathway to the total amount of calcium absorbed is reduced. Under adequate-to-high calcium intakes, the proportion of calcium transported in any given segment is determined by the presence of the saturable and nonsaturable pathways, the sojourn time through the intestinal tract, and the solubility of calcium within the intestinal segment (e.g., in the ileum and lower bowel, where pH is neutral or basic, calcium solubility may be <20% that seen in the duodenum [12]). As a result, even though calcium solubility is low and the saturable pathway is absent or very low, the total amount of calcium absorption is greatest in the ileum since transit time through this segment is ten or more times longer in comparison to the more proximal intestinal segments [13,14] (Fig. 19.2).
Habitual Dietary Calcium Intake is a Major Physiologic Regulator of Intestinal Calcium Absorption Efficiency The amount of calcium absorbed from the diet is a function of the amount of calcium consumed and the efficiency of the calcium absorption process. It is this later process that is strongly regulated by physiological state (e.g., pregnancy and lactation, maturity/aging)
A schematic demonstrating the relationships between features proposed to be critical for intestinal calcium absorption. Net calcium absorption is determined by many factors and different mechanisms for calcium absorption may be present in different intestinal segments. This schematic summarizes these features. Refer to the text for details about the various parameters listed in the figure. Tx ¼ transport. The number of “þ” signs indicates the relative magnitude of the parameter across tissues. A “e“ sign indicates that the parameter is absent in a segment.
FIGURE 19.2
and by habitual dietary calcium intake [15]. The concept that low habitual intake of calcium would increase the efficiency of intestinal absorption was originally suggested from calcium balance studies in rats [16] and humans [17]. Later studies showed this more directly. Using in situ loops of intestine, Pansu et al. [4] found that compared to a normal diet with 0.44% calcium, feeding a calcium-restricted diet (0.17% calcium) to rats for 5 weeks increased the efficiency of duodenal calcium absorption by specifically increasing the saturable component of transport (Vmax increased 55%). Similarly, Norman et al. [18] found that in humans, 8 weeks of feeding a diet with 300 mg of calcium per day increased calcium absorption efficiency by 43% compared to subjects consuming a 1600 mg calcium/ day diet. We now understand that the increased efficiency of calcium absorption resulting from habitual low calcium intake is due to adaptation mediated by increased renal production of the active, hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D) [19].
Role of Vitamin D Status and Vitamin D Signaling in Intestinal Calcium Absorption For over 70 years we have known that calcium absorption in the gut is dependent upon adequate vitamin D status [20,21]. The efficiency of intestinal calcium absorption is dramatically lower in vitamin-Ddeficient animals (reduced by >75%) [8] and in dialysis patients with compromised renal function and low circulating 1,25(OH)2D levels [22]. Recently, Need et al. showed that Ca malabsorption is a very late effect of vitamin D deficiency in elderly adults; secondary hyperparathyroidism maintains serum 1,25(OH)2D (and Ca absorption) until the deficiency is so severe (<0 nmol/L) that serum 1,25(OH)2D falls due to lack of substrate for conversion [23]. Paradoxically, in children with vitamin-D-deficiency rickets serum 1,25(OH)2D levels can be in the normal range even though serum calcium and phosphate levels are low [24,25]. However, vitamin D supplementation in rachitic children increases serum 1,25(OH)2D to supraphysiologic levels indicating a strong compensation to correct the symptoms of vitamin D deficiency or a renewed ability to respond to the elevated PTH levels seen during vitamin D deficiency. In animal models the deficit in calcium absorption caused by vitamin D deficiency can be restored by either repletion of vitamin D status or with injections of 1,25(OH)2D. In rat duodenum [8] and in differentiated monolayers of the human intestinal cell line Caco-2 [26], the effect of 1,25(OH)2D on calcium absorption is limited to the saturable component of transport, leading to an increase in Vmax but not Km (i.e., the maximal capacity and suggesting the production of more
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transporters). However, there is some evidence that the nonsaturable portion of calcium absorption in the human ileum is also vitamin-D-sensitive; the slope of the nonsaturable transport pathway is reduced in chronic renal disease patients and it returns to normal after 1,25(OH)2D injection [6]. The cellular mechanisms for these effects are discussed below.
CRITICAL ROLE OF VDR IN CONTROL OF INTESTINAL CA ABSORPTION Since the 1970s, we have understood that many of the biological effects associated with 1,25(OH)2D are dependent upon transcriptional events that require binding of the hormone to the nuclear vitamin D receptor, VDR [27,28]. The physiologic importance of the VDR for whole-body calcium homeostasis was first evident from subjects with type II genetic rickets who have inactivating mutations in the VDR gene. The importance of the VDR for normal intestinal calcium absorption was subsequently confirmed experimentally using VDR knockout mice where deletion leads to a >70% reduction in calcium absorption efficiency that causes the major phenotypes associated with the VDR gene knockout, i.e. poor growth, low serum Ca, high serum PTH, and severe osteomalacia [29,30]. Two studies have used transgenic expression of VDR directed to the intestine to rescue the VDR knockout mouse. Initial studies using the adenosine deaminase promoter/ enhancer to drive transgenic VDR expression to the distal duodenum and jejunum showed that this is inadequate for recovery of the VDR KO mouse phenotype on a chow diet but that it can improve the phenotype of VDR knockout mice on a high-calcium rescue diet [31]. This suggests that although the proximal duodenum and jejunum are highly responsive to 1,25(OH)2D in normal mice, VDR function in additional segments is necessary to optimize intestinal calcium absorption and normalize calcium metabolism. Consistent with this hypothesis, transgenic expression of VDR throughout the intestinal epithelium using the villin promoter was sufficient to completely recover the VDR knockout mouse phenotype, i.e. normalization of duodenal calcium absorption, serum PTH, and calcium and bone mineral density [32]. This work supports the hypothesis that the primary role for VDR signaling relevant to calcium metabolism is the control of intestinal calcium absorption efficiency. It has been proposed that intestinal VDR level or function may be an important factor influencing intestinal calcium absorption under a variety of conditions. For example, the loss of basal and vitamin-D-responsive calcium absorption during aging [33,34] or after estrogen depletion [35,36] is associated with a reduction
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in intestinal VDR. Consistent with this hypothesis, low VDR levels have been seen in the intestine with aging in humans [37] and after ovariectomy in rats [38]. In addition, inducible overexpression of VDR in the intestinal cell line Caco-2 increases 1,25(OH)2D-regulated transcellular calcium transport [39] while reduced intestinal VDR levels seen in mice heterozygous for the VDR knockout allele blunts 1,25(OH)2D-regulated intestinal calcium absorption efficiency in mice [40]. Other studies have shown that the F allele of the Fok I gene polymorphism results in the translation of a VDR protein that is shortened by three amino acids at its N-terminus and which is more transcriptionally active [41]. Two studies have reported that individuals homozygous for the shorter, more transcriptionally active F allele have greater calcium absorption efficiency compared to individuals with the f allele [42,43]. Collectively these data support the hypothesis that variations in VDR level or function can influence vitamin-D-regulated intestinal calcium absorption.
CAN HIGH VITAMIN D STATUS INCREASE INTESTINAL CALCIUM ABSORPTION? It is well established that 1,25(OH)2D treatment can increase the efficiency of intestinal calcium absorption [8,44,45]. However, there is some controversy regarding whether intestinal calcium absorption is responsive only to increased serum levels of 1,25(OH)2D or whether it can also increase in response to improved serum 25 (OH)D levels, independent of renal conversion to 1,25 (OH)2D. The first suggestion of an independent effect of high serum 25(OH)D levels was seen by Heaney et al. [46] who showed that calcium absorption (measured by appearance of 45Ca in serum 5 h after a meal containing 300 mg calcium) was increased by 25% after 4 weeks of treatment with 50 mg 25(OH)D/d even though serum 1,25(OH)2D levels did not change. This same group later showed that as serum 25(OH)D increased from 50 to 86.5 nmol/L, there was a 65% increase in calcium absorption efficiency in postmenopausal women (increased area under the curve of serum calcium 5 h after an oral load of 500 mg calcium). These studies suggest that the enzyme necessary for conversion of 25(OH)D to 1,25(OH)2D (CYP27B1) in the kidney is also present in the intestine where it produces 1,25 (OH)2D for local use. This hypothesis is supported by several findings. First, CYP27B1 promoter activity is seen in the jejunum and ileum of transgenic mice expressing a 1.5-kb CYP27B1 promoter-luciferase reporter gene [47] and CYP27B1 mRNA (by RT-PCR) and protein (by IHC) has been observed in the human duodenum [48]. In addition, in a study of duodenal
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biopsies collected from human subjects, the mRNA level of a well-characterized vitamin D target gene, TRPV6, correlated well with CYP27B1 mRNA in human duodenum but not with serum 1,25(OH)2D [48]. In contrast to the data supporting a strong role for high vitamin D status as a regulator of intestinal calcium absorption, Abrams et al. [49] found that higher serum 25(OH)D levels were not consistently associated with higher calcium absorption efficiency in a cross-sectional study of 251 school-age children. Also, studies by Need et al. [50,51] found that the efficiency of intestinal calcium absorption was normal across a wide range of vitamin D status levels (20e90 nmol 25(OH)D/L, 45Ca in serum 1 h after a 20-mg oral dose of Ca). Increasing vitamin D status was only beneficial when subjects were so deficient that their serum 25(OH)D levels were inadequate to support renal production of 1,25(OH)2D. Finally, Hansen et al. [52] found that raising serum 25(OH)D from 55 to 160 nmol/L by supplementing women with 50 000 IU vitamin D2 per day for 15 days increased calcium absorption efficiency only modestly (13% above baseline as determined by a dual stable isotope technique). Thus, the hypothesis that improving vitamin D status beyond that necessary to permit adequate renal production of 1,25(OH)2D can regulate intestinal calcium absorption is not strongly supported by the literature. However, although a cross-sectional study by Aloia et al. [53] did not find an association between Ca absorption and serum 25(OH)D levels in 495 health black and white women, they found that the positive relationship between serum 1,25(OH)2D and Ca absorption was stronger in subjects with low serum 25(OH)D levels than in those with high levels. Confirmation of this observation and identifying a mechanism mediating this interaction requires further study.
Molecular Models of Ca Absorption At this point we have shown that the vitamin D metabolite 1,25(OH)2D uses the VDR to regulate the saturable component of calcium absorption leading to increased capacity of the intestine to absorb calcium. Four models have been proposed to explain vitamin-D-regulated intestinal calcium absorption: facilitated diffusion, vesicular trafficking, transcaltachia, and regulated paracellular transport. In the following section we will review each of these models with an emphasis towards relating them to the physiologic data defining calcium absorption. However, prior to describing these models, two studies deserve special attention. By using ion microscopy to follow the movement of 44Ca across the chick duodenum, Chandra et al. [54] showed that calcium flows from the apical side of the enterocyte and through the epithelial cell over the course of 20 minutes whereas in vitamin-D-deficient chicks, calcium was able to enter
the enterocyte but was trapped in the region just below the microvilli. This group later showed that treating vitamin-D-deficient chicks with 1,25(OH)2D caused a progressive redistribution of calcium from the brush border region into the cytoplasm and towards the basolateral membrane [55]. The 1,25(OH)2D effect was first seen 2e4 h after the treatment, consistent with the induction of gene expression mediated through the VDR. These data strongly support the hypothesis that the 1,25(OH)2D-regulated, saturable component of duodenal calcium absorption is a transcellular process. Facilitated Diffusion The evidence supporting this model was summarized in 1986 by Bronner et al. [56]. They critically reviewed transport data from isolated brush border membrane vesicles, from isolated basolateral membrane vesicles, and a variety of whole intestine transport methods conducted in rats and chicks (e.g., everted gut sacs, in situ transport, Ussing chambers) and concluded that while both calcium uptake and calcium extrusion were vitamin-D-regulated processes, it was the intracellular diffusion of calcium that was the rate-limiting step in the transcellular calcium transport process. From these data, Bronner et al. and others have built the model shown in Figure 19.3. In the facilitated diffusion model vitamin-D-dependent uptake of calcium across the brush border membrane into the enterocyte is thought to be mediated by the transient receptor potential cation channel vanilloid family member 6 (TRPV6 aka CaT1 or ECAC2), an apical membrane calcium channel [57]. TRPV6 mRNA level is reduced by more than 90% in the duodenum of VDR knockout mice and the TRPV6 gene is strongly regulated by 1,25(OH)2D in the duodenum of mice [45,58] and in Caco-2 cells [59,60]. In addition, induction of TRPV6 mRNA precedes the increase in duodenal calcium absorption that occurs following a single 1,25 (OH)2D injection [45]. However, in contrast to the implication by some that TRPV6 induction is the rate-limiting step in transcellular calcium transport, calcium uptake is increased by only 30% in brush border membrane vesicles isolated from vitamin-D-treated rats [61] and in vitamin-D-deficient chicks calcium can enter the enterocyte but is trapped in the region just below the microvilli [54]. In addition, in TRPV6 knockout mice, intestinal calcium absorption across everted gut sacs was not affected by 1,25(OH)2D injection [62,63] and the improved absorption resulting from feeding a lowcalcium diet was reduced by only 40% [63]. While there may be some compensation by other calcium channels for the loss of TRPV6 in the knockout mouse, these data do not support the simple hypothesis that TRPV6 is the sole means by which calcium can enter the enterocyte during calcium absorption.
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FIGURE 19.3 Models for intestinal calcium absorption. Several models have been proposed to explain how calcium transverses the intestinal barrier. The facilitated diffusion and vesicular transport models are both transcellular absorption pathways. In contrast, the claudin 12 and 2 have been proposed to provide selectivity for calcium to the tight junction complex. For details of how vitamin D regulates various aspects of these models refer to the text.
The central player in the facilitated diffusion model is the cytoplasmic calcium-binding protein calbindin D. There are two forms of calbindin D, a 9-kd form found in mammalian intestine and mouse kidney (calbindin D9k) and a 28-kd form found in the avian intestine and kidney and in the mammalian kidney (calbindin D28k). Calbindins are small EF-hand proteins that can bind either 2 (D9k) or 4 (D28k) moles of calcium per mole of protein [64]. Calbindin D protein levels positively correlate to calcium absorption over a wide range of biological conditions [56]. In vitamin-D-deficient animals and in VDR knockout mice, calbindin levels in the intestine are significantly reduced [29,65]. In addition, 1,25(OH)2D injections increase duodenal levels of calbindin significantly, suggesting the calbindin D9k and D28k genes might be vitamin D target genes [66]. Finally, disruption of calcium binding to calbindins with theophyline disrupts intestinal calcium absorption [67]. Collectively these data suggest that calbindins are proteins that participate in calcium transport by either acting as an intracellular buffer (limiting second messenger signaling by calcium during the transport process) or as a ferry that permits calcium to move away from the apical membrane to the basolateral membrane [54,68]. However, several studies refute the hypothesis that calbindins are necessary for intestinal calcium absorption. For example, Spencer et al. [69] showed that after injecting vitamin-D-deficient chicks with a single dose of 1,25(OH)2D, calbindin
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D28k protein remained elevated even after calcium absorption had returned to normal. This was later confirmed for calbindin D9k in mouse duodenum by Song et al. [30,45]. A calbindin D9k knockout mouse has been created and characterized but this has not completely settled the controversy regarding the role of calbindin D9k in intestinal calcium absorption [63,70e72]. The facilitated diffusion model predicts that calbindin D9k is the critical protein mediating vitamin-D-mediated calcium absorption and suggests that calcium absorption cannot occur in the absence of calbindin D. However, in two different lines of calbindin D9k-null mice neither basal nor 1,25 (OH)2D-induced calcium absorption (measured using the everted sac technique) are affected [63,71]. However, although both single calbindin D9k-null mice and single TRPV6-null mice have normal duodenal calcium absorption after 1,25(OH)2D treatment, the ability of double calbindin D9k/TRPV6 knockout mice to increase calcium absorption in response to 1,25(OH)2D is reduced by 60% compared to wild-type mice [63]. This suggests that TRPV6 and calbindin D9k proteins together may have a special role in calcium absorption and that their interaction is more complex than the current iteration of the facilitated diffusion model predicts. The final step in the facilitated diffusion model is the extrusion of calcium from the cell. Favus et al. [7] used Ussing chambers to show that calcium absorption is energy-dependent and found that the ATPase inhibitor trifluoroperizine (TFP) could reduce transcellular calcium transport in duodenal segments and block the increase in calcium transport induced by prior treatment with 1,25(OH)2D. This ATP-dependent process is localized to the basolateral membrane and is necessary to move calcium up the concentration gradient that exists between the enterocyte cytoplasm and the serum. Wasserman et al. [73,74] later identified the plasma membrane calcium ATPase 1b (PMCA1b) as a basolateral protein whose protein and mRNA level was expressed throughout the chick intestine, reduced by vitamin D deficiency, and increased by vitamin D repletion (2e3-fold) or by consumption of low-calcium diets. While some have suggested that the basolateral extrusion of calcium may also be mediated by a sodiumecalcium exchanger [75], Favus et al. [7] reported that sodiumepotassium pump inhibitors that disrupt the sodium gradient necessary for sodiumecalcium exchange had no impact on calcium transport across duodenal segments mounted in Ussing chambers. Vesicular Transport An alternative mechanism to the central role proposed for calbindin D as a calcium ferry/buffer during transcellular intestinal calcium absorption is
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the sequestration of calcium into vesicles within the cell (Fig. 19.3). There are a number of observations that support this hypothesis. First, several groups have reported that 1,25(OH)2D treatment increased the number of lysosomes in chick intestine [76] and increased the release of lysosomal enzymes from isolated rat enterocytes [77] suggesting there is a vitaminD-dependent increase in the activity and cycling of lysosomes. Warner and Coleman [78] previously observed that calcium accumulates in lysosome-like structures during calcium absorption. Nemere et al. [79] later showed that calcium is associated with lysosomes in the chick intestine and that the level of lysosomal calcium is increased 3.1-fold within 10 h of 1,25 (OH)2D treatment. Also, during the process of calcium absorption, calcium is initially associated with endosomes in the brush border membrane of intestinal epithelial cells prior to its appearance in lysosomes [80]. Consistent with an essential role for lysosomes in intestinal calcium absorption, disrupting lysosomal pH with agents like quinacrine and chloroquine does not stop calcium entry into chick enterocytes but it prevents lysosomal calcium accumulation and blocks calcium absorption [79]. In the rat intestine quinacrine can also block vitamin-D-regulated calcium absorption through a mechanism that is independent of ATP-mediated calcium extrusion [81]. Collectively these data suggest that vesicular movement may be a legitimate pathway for uptake and movement of calcium through the intestinal epithelial cells. It is not clear, however, what makes the vesicular transport pathway specific for calcium. In chick intestine, Nemere et al. [79] identified calbindin D28k in an endosome-like compartment and in lysosomes containing calcium after 1,25(OH)2D treatment. However, similar observations have not been made in mammalian intestinal epithelial cells and, given the fact that calcium absorption is normal in calbindin D9k knockout mice, a role for calbindin D as the factor defining calcium specificity to the vesicular transport system seems unlikely. Transcaltachia The two models for vitamin-D-mediated calcium absorption described above both require a prolonged period for the effects of 1,25(OH)2D to be realized (i.e., intestinal adaptation requires transcriptional events mediated through the VDR). In contrast, transcaltachia is a mode of calcium transport that occurs within minutes of exposing the basolateral side of enterocytes to 1,25(OH)2D. This is consistent with a body of literature on the existence of rapid 1,25(OH)2D actions that are initiated at the cell membrane and that are independent of new transcriptional events, such as activating protein kinase C, mitogen-activated protein kinases, and AKT signaling pathways, as well as the opening of
chloride channels [82]. Transcaltachia has been directly demonstrated in the perfused chick duodenum where exposure to a physiologic dose of 1,25(OH)2D increased calcium appearance in the serosal perfusate by 40% within 14 minutes of exposure [83]. This effect occurs only in response to serosal exposure to 1,25(OH)2D and it is lost in the intestine of vitamin-D-deficient chicks. This suggests that in absorptive epithelial cells in the intestine there is a membrane receptor on the basolateral surface of whose level, or whose downstream components, are dependent upon vitamin-Dregulated synthesis of new proteins. The exact mechanism that calcium follows through the cell during transcaltachia has not been determined with certainty but these observations are consistent with use of the vesicular transport model [84]. The existence of a specific basolateral membrane vitamin-D-binding protein was first shown by 1,25 (OH)2D-binding studies using chick duodenum [85]. Some data suggest that this represents a novel, nonnuclear role for the vitamin D receptor [86] while other data indicate that transcaltachia is mediated through a new vitamin-D-binding protein called the membraneassociated rapid response steroid-binding protein (MARRS) [87]. In support of the VDR hypothesis, mouse intestinal VDR was found associated with a caveolaerich membrane fraction. Caveolae are a membrane region known to accumulate a variety of cell surface signaling molecules [86]. Activation of the rapid, nontranscriptional roles mediated through the VDR is proposed to occur through a unique alternative ligandbinding pocket [88]; specific vitamin D analogs that are proposed to fit into this alternative binding pocket can stimulate rapid effects of 1,25(OH)2D including transcaltachia [89]. MARRS is a protein with multiple functions and names (e.g., ERp57, PLCa, PDIA3). Ribozyme-mediated knockdown of MARRS was shown to reduce phosphate uptake into chick enterocytes [87] and studies recently reported at a national meeting found that intestinespecific deletion of MARRS in mice reduces 1,25 (OH)2D binding and disrupts 1,25(OH)2D-regulated calcium uptake into enterocytes [90]. However, this abstract did not report the physiological impact of MARRS deletion on either intestinal calcium absorption or bone density. In addition, there are other aspects to the transcaltachia model that limit its acceptance as a physiologically important pathway. For example, the rapid fluxes in serum 1,25(OH)2D needed for transcaltachia have not been reported, particularly during the consumption of calcium-rich meals when transcaltachia would have to occur for the physiologic benefit of the process to be realized. Also, PTH treatment has also been shown to cause transcaltachia [91] but neither the PTH 1 receptor nor the PTH 2 receptor are well
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expressed in the small intestine. While serum PTH levels vary significantly throughout the course of a day (i.e., high in the morning, lower after consuming calcium-rich meals), calcium absorption efficiency has not been shown to have a similar daily rhythm. Thus, for transcaltachia to gain full acceptance as a physiologically important mode for regulating intestinal calcium absorption, more research is needed to place regulation through this mechanism into important physiologic contexts. In the meantime, an alternative view of this phenomenon is that it could reflect activation of a cellsignaling pathway that artificially resembles transcellular calcium transport because of the design of the chick intestinal perfusion system, i.e. a polarized cell with high levels of calcium at the apical surface that can only extrude calcium efficiently from the basolateral pole after the calcium has entered the cell. Regulated Paracellular Movement through Tight Junctions Although much of the research on intestinal calcium absorption has focused on explaining the vitamin-Dregulated changes in saturable calcium transport that is predominant in the proximal small intestine, several studies have shown that vitamin D signaling increases diffusional, presumably paracellular, fluxes across the intestine, particularly in the jejunum and ileum [6,92]. Tudpor et al. [93] used Ussing chambers to find that 1,25(OH)2D induced calcium absorption in the rat duodenum through a solvent drag mechanism that was sensitive to PI3K, PKC, and MEK inhibitors. Their finding that 1,25(OH)2D induced ion movement and transepithelial electrical resistance (TEER) without affecting manitol flux suggests that the effect was due to a change in the charge selectivity of the tight junction. The paradigm that tight junction selectivity may be relevant for transepithelial mineral transport was previously demonstrated in the kidney when mutations in the tight junction protein paracellin 1 (aka claudin 16) were found to account for magnesium and calcium wasting associated with the genetic disease familial hypomagnesemia with hypercalciuria and nephrocalcinosis [94]. Recently, Fujita et al. [95] proposed that 1,25 (OH)2D-mediated induction of the tight junction proteins claudin 2 and claudin 12 accounts for increased 1,25(OH)2D-induced movement of calcium across Caco-2 cell monolayers. They found that 1,25 (OH)2D treatment significantly increased claudin 2 and 12 mRNA levels in Caco-2 cells, that the mRNA levels for these proteins fall dramatically in the jejunum of VDR knockout mice, and that siRNA against these claudins reduces calcium permeability in Caco-2 cell monolayers. While this is an intriguing new way to view calcium absorption this model must be further tested prior to its acceptance. For example, it should
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be determined whether the loss of claudin 2 and 12 influences the saturable or nonsaturable component of calcium absorption using kinetic modeling. If the impact of deletion has a significant impact on the linear diffusional component of calcium transport, this may explain the findings from Sheikh et al. [6] who found that the nonsaturable component of ileal calcium absorption was reduced in chronic renal disease patients with low serum 1,25(OH)2D levels. This would also be consistent with the observation that claudin 2 and 12 expression is highest in the ileum [96] but essentially absent from the duodenum where 1,25(OH)2D regulates the saturable component of calcium absorption. It is also interesting to reflect on the fact that claudin 2 expression is highest in the undifferentiated crypt cells of the small intestine [97] whereas, similar to what is seen for the absorption of other minerals (e.g., iron [98]), the expression of proteins proposed to be important for transcellular calcium movement (PMCA1b, calbindin D9k) increase as cells differentiate and migrate up the villus [75,99]. Conclusions Regarding the Role of Vitamin D on Intestinal Calcium Absorption These data show clearly that vitamin D, particularly the metabolite 1,25(OH)2D, is necessary for adequate intestinal calcium absorption. The effect of 1,25(OH)2D is dependent upon signaling through the VDR, which leads to an increase in the maximum capacity of the calcium transport system. This is consistent with the existence of 1,25(OH)2D-mediated transcriptional regulation leading to increased levels of a transporter (or transporters) that mediate saturable, transcellular calcium transport. Given that serum 1,25(OH)2D levels are elevated by habitually low dietary calcium intake, and given that low dietary calcium intake is common in the general population, the saturable, vitamin-Dregulated component of intestinal calcium absorption should play a significant role in maintaining the efficiency of calcium absorption in most individuals. The exact mechanism that calcium travels to make it through the enterocyte is still in question. Each of the three models that we presented to explain transcellular calcium transport (i.e., facilitated diffusion, vesicular transport, transcaltachia) have weaknesses that must be resolved through additional research. A recently proposed model for regulated paracellular calcium movement through tight junctions is not consistent with other data showing that calcium movement is transcellular in the duodenum but may help explain data suggesting vitamin D signaling can control nonsaturable, presumably paracellular, movement across the ileum. Thus, vitamin D signaling may use different mechanisms to regulate intestinal calcium absorption depending upon the segment of intestine studied.
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AN OVERVIEW OF INTESTINAL PHOSPHATE ABSORPTION In contrast to calcium, dietary phosphate is abundant in the Western diet. Although whole-body phosphate metabolism is regulated predominantly through the control of renal phosphate reabsorption (leading to essentially phosphate-free urine during hypophosphatemia) a significant amount of research has been conducted on the mechanism of intestinal phosphate absorption as well. Phosphate absorption is highly efficient, with 60e70% of an intestinal load being absorbed from a typical diet. There are some species differences in the intestinal location of phosphate absorption; rats [100e102] and humans [103] efficiently absorb phosphate from the jejunum and duodenum while in mice the highest efficiency of absorption is in the ileum [104,105]. In rats and humans phosphate transport has been shown to occur through both saturable and nonsaturable pathways [103,106] (see Fig. 19.1). The Km for the saturable component for phosphate absorption from the jejunum of adults is between 1e2 mM (calculated from data in [103]) and this concentration is less than the expected value for the luminal contents in humans after a typical phosphate-rich meal (e.g., 4e10 mM). Thus, at dietary phosphate levels typical in the US diet, the saturable component of transport is saturated and the bulk of absorption of phosphate is likely through the nonsaturable, paracellular route.
testis, and liver, while NaPi IIa and IIc are found predominantly in the kidney. NaPi IIb is a glycoprotein whose Na/Pi-co-transport is electrogenic (3 Naþ:1PO4). The Km for Pi transport of NaPi IIb is approximately 100 mM [110]. There is evidence that phosphate uptake mediated by NaPi IIb can be regulated by endocytosis and that protein kinase C-mediated phosphorylation events lead to internalization of the transporter [112]. This redistribution would be expected to occur when phosphate status is high and the need to absorb phosphate from the diet was reduced (Fig. 19.4). In contrast to the apical membrane uptake of phosphate, the mechanisms mediating the intracellular movement and basolateral export of phosphate from intestinal cells are not known with confidence. The Major Physiologic Regulator of Intestinal Phosphate Absorption is Prior Dietary Phosphate Intake Many groups have shown that efficiency of intestinal phosphate transport is influenced by the habitual consumption of high- or low-phosphate diets. Using in situ loops of intestine [106], brush border membrane vesicles [106,108], and Ussing chambers [113] investigators have shown that dietary phosphate restriction can increase phosphate transport efficiency, e.g. in pig jejunum, phosphate transport is 90% higher in animals fed a 0.25% phosphorus diet compared to a 0.4% phosphorus diet. The strength of this adaptation may be limited to certain segments of the intestine. In the rat,
Intestinal Pi Absorption is Sodium-Dependent and is Mediated through the NaPi IIb Transporter Using Ussing chambers to measure intestinal phosphate transport in rat jejunum, Eto et al. [107] estimated that >50% is sodium-dependent. Many phosphate transporters are expressed in the rat jejunum: Pit1, Pit2, BNPi, and NaPi IIb [108], but recent studies from knockout mice show that 90% of active, and 50% of total, phosphate transport is mediated by the sodiumdependent phosphate transporter NaPi IIb (SLC34A2) [109]. Consistent with this estimate, phosphate transport into brush border membrane vesicles falls 70% as the jejunum of the rat matures from 2 to 12 weeks and this is accompanied by a parallel decline in NaPi IIb mRNA levels [110]. NaPi IIb is part of a family of sodium-dependent phosphate transporters that includes NaPi IIa and IIc. Each of the NaPi II proteins have eight membrane-spanning domains with long intracellular N-terminal and C-terminal domains and all are thought to function as monomers [111]. NaPi IIb is located on the apical brush border membrane of epithelial cells in the intestine, mammary gland, lung,
A model for transcellular phosphate absorption. Phosphate absorption is a sodium-dependent process that depends upon the apical membrane sodiumephosphate cotransporter NaPi IIb. Phosphate movement is driven by the electrochemical gradient generated by the sodiumepotassium ATPase. Transcellular phosphate transport is sensitive to habitual phosphate intake (e.g., low habitual intake leads to high efficiency of phosphate absorption). The localization of NaPi IIb may be sensitive to habitual phosphate intake as indicated in the model. The mechanism for phosphate movement through, and extrusion from, the cell is unknown. For details of how vitamin D regulates various aspects of these models refer to the text.
FIGURE 19.4
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dietary phosphate restriction from 0.6 to 0.1% for 7 days increased jejunal phosphate transport in brush border membrane vesicles twofold but had no effect on phosphate transport from brush border membrane vesicles prepared from the duodenum [105]. In adult mice dietary phosphate restriction from 1.1% to 0.1% increased brush border membrane vesicles phosphate transport twofold in the ileum but only 50% in the jejunum [102]. The change in intestinal phosphate transport with dietary adaptation is accompanied by changes in NaPi IIb mRNA and protein levels [102,105]. The mechanism accounting for dietary-induced regulation of phosphate transport and NaPi IIb regulation following changes in dietary phosphate is not clear. However, investigators have observed that diet phosphate restriction increases serum 1,25(OH)2D levels by increasing renal CYP27B1 expression and activity [114]. Thus, researchers have assumed that phosphate transport is regulated in a manner similar to the well-established regulation of intestinal calcium absorption by 1,25 (OH)2D (see above).
Role of Vitamin D Status and Vitamin D Signaling in Intestinal Phosphate Absorption It is well established that vitamin D deficiency reduces the efficiency of intestinal phosphate absorption. This was first demonstrated in rats by Bauer and Marble in 1932 [115] and correction of this defect with vitamin D injection was shown by Carlsson in 1954 [116]. Later depletionerepletion studies in rats showed that phosphate transport across everted sacs of rat duodenum and jejunum increased by 50% after a vitamin D3 treatment [100]. The intestinal location of improved phosphate absorption following vitamin D repletion (mid jejunum > duodenum) is different from the location of improved calcium absorption that occurs following vitamin D repletion (duodenum >> jejunum). However, similar to what others have shown for calcium absorption, the beneficial effect of vitamin D3 repletion on intestinal phosphate absorption can be achieved with 1,25(OH)2D treatment. When vitaminD-deficient rats are given large doses of 1,25(OH)2D (325e650 pmoles), serum phosphate levels and intestinal phosphate absorption are significantly increased [117,118]. Similarly, in vitamin-D-replete rats, the reduction in phosphate absorption caused by thyroidparathyroidectomy is restored within 6 h after a 0.8 pmol/g body weight dose of 1,25(OH)2D [106]. Similar to the findings from rats, Davis et al. [103] showed that 1,25(OH)2D increased jejunal phosphate absorption in subjects with chronic renal failure (and presumably low serum 1,25(OH)2D). In this study 1,25 (OH)2D treatment increased the Vmax of the saturable component of phosphate transport approximately
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twofold without having a significant impact on the rate of nonsaturable phosphate transport. Similar findings of a 1,25(OH)2D-induced increase in the Vmax for phosphate transport across brush border membrane vesicles prepared from the jejunum of vitamin-D-deficient rats were reported by others [108]. The effect was not seen until 12 h after 1,25(OH)2D treatment suggesting that the effect of 1,25(OH)2D was due to an increased production of phosphate transporters. Marks et al. [104] confirmed this by demonstrating that treating vitaminD-deficient mice with 1,25(OH)2D increased the amount of NaPi IIb protein in brush border membrane vesicles. Although these data suggest that 1,25(OH)2D is a direct regulator of NaPi IIb gene expression, and by extension, phosphate absorption, other data do not support such a straightforward interpretation. For example, although 1,25(OH)2D increased NaPi IIb mRNA level in cultured rat intestinal epithelial (RIE) cells by an actinomycinD-sensitive mechanism, investigators were not able to find a classical VDRE in the NaPi IIb promoter [110]. In vivo, the ability of 1,25(OH)2D to increase NaPi IIb mRNA is present in 2-week-old mice with immature intestines [110], but it is lost in 12-week-old mice [110]. However, the effect of 1,25(OH)2D on phosphate transport into brush border membrane vesicles is still seen in vitamin-D-replete adults suggesting that the effect of the hormone is mediated through either new protein production from existing message or through the redistribution of existing protein to the apical membrane. Finally, both Davis et al. [103] in humans and Lee et al. [119] in mice found that the benefit of 1,25(OH)2D treatment on phosphate absorption is modest in vitaminD-replete subjects. This suggests that other factors may limit the effects of 1,25(OH)2D when phosphate status is already adequate. The Role of VDR and Phosphatonins in Phosphate Absorption There are other studies that further demonstrate the complexity of the role vitamin D signaling plays in the control of phosphate absorption. For example, while intestinal phosphate transport in brush border membrane vesicles is reduced by 30e70% in VDR knockout mice, it is accompanied by a reduction in NaPi IIb protein but not mRNA [120,121]. This is consistent with a model of 1,25(OH)2D-mediated regulation that is dependent on the presence of the VDR but not new gene transcription mediated through the VDR. It is not known how signaling through the VDR regulates NaPi IIb translation or trafficking. Another interesting observation is that dietary phosphate restriction can increase intestinal phosphate absorption in VDR- and CYP27B1-null mice so that the level of absorption is equivalent to that seen in phosphate-restricted wildtype mice. The ability of VDR and CYP27B1 knockout
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mice to up-regulate phosphate absorption after dietary phosphate restriction is similar to what Lee et al. [119] previously observed in vitamin-D-deficient rats (40% increase in vitamin-D-deficient rats vs 90% increase in vitamin-D-replete rats). Collectively, these data demonstrate that vitamin D signaling through the VDR is important for normal levels of phosphate absorption but that dietary phosphate restriction regulates phosphate absorption through a vitamin-D-independent mechanism. Another mechanism mediating the effects of dietary phosphate on phosphate absorption is through phosphatonins like matrix extracellular phosphoglycoprotein (MEPE), secreted frizzled-related protein 4 (sFRP-4), dentin matrix protein 1 (DMP1), fibroblast growth factor 7 (FGF7), and the most studied phosphatonin, fibroblast growth factor 23 (FGF23) [122]. FGF23 and other phosphatonins inhibit renal phosphate reabsorption and their serum levels are directly related to dietary phosphate intake and serum phosphate levels [123,124]. In vivo injections of phosphatonins FGF23 and MEPE can inhibit intestinal phosphate absorption in mice [125,126]. FGF23 binds to the FGF receptors 1c, 3c, and 4 and requires the presence of the coreceptor, klotho, to signal through FGF receptors [127,128]. However, since the messages of the FGF receptors for FGF23 (R1c, R3c, R4) and the klotho coreceptor are expressed at very low levels in the intestine (determined from BioGPS (http://biogps.gnf.org/?referer¼symatlas #goto¼welcome)), this suggests that FGF23 works indirectly upon the intestine to alter phosphate absorption. This is consistent with the observation that FGF23 suppression of jejunal phosphate absorption is lost in VDR-null mice [129]. Renal CYP27B1 activity and serum 1,25(OH)2D levels are inversely associated with serum FGF23 levels in normal healthy men and in mice [123,124]. Implantation of cells producing FGF23 into nude mice reduces the renal expression of CYP27B1 mRNA [130] while in cultured mouse renal proximal tubule cells, FGF23 (R176Q) treatment suppresses CYP27B1 mRNA levels by a MAPK-dependent mechanism [131]. Thus, low dietary phosphate may improve intestinal phosphate transport in part through increased serum 1,25(OH)2D resulting from releasing the FGF23mediated inhibition of CYP27B1 expression.
Conclusion regarding the role of vitamin D on phosphate absorption It is clear that under normal conditions, the saturable portion of intestinal phosphate absorption is dependent upon adequate vitamin D status. However, the mechanism for that regulation is not clear. The central player in phosphate absorption is the apical membrane sodiumephosphate cotransporter, NaPi IIb. However,
while vitamin D changes the level of the NaPi IIb protein at the membrane, it does not strongly regulate the NaPi IIb gene and if it does so at all, it appears to be indirectly through an as yet unidentified factor. More importantly, even though both serum 1,25(OH)2D and intestinal phosphate absorption increase during dietary phosphate restriction, the body still has the ability to adapt to low-phosphate diets even in the absence of VDR or CYP27B1. This suggests that a major regulator of phosphate absorption is vitamin-D-independent and that there are critical regulators of intestinal phosphate absorption that have yet to be discovered. Finally, vitamin D may have a minor role in phosphate absorption in normal healthy men. This is because the saturable component of intestinal phosphate absorption that is regulated by vitamin D status may not be critical for human health under the high dietary phosphate loads of the typical Western diet.
Acknowledgments This work was supported by NIH award DK054111 to JCF.
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[63] B.S. Benn, D. Ajibade, A. Porta, P. Dhawan, M. Hediger, J.B. Peng, et al., Active intestinal calcium transport in the absence of transient receptor potential vanilloid type 6 and calbindin-D9k, Endocrinology 149 (2008) 3196e3205. [64] S. Christakos, R. Gill, S. Lee, H. Li, Molecular aspects of the calbindins, J. Nutr. 122 (1992) 678e682. [65] R.H. Wasserman, A.N. Taylor, Vitamin D3-induced calciumbinding proteins in chick intestinal mucosa, Science 252 (1966) 791e793. [66] F. Bronner, M. Buckley, The molecular nature of 1,25-(OH)2-D3induced calcium-binding protein biosynthesis in the rat, Adv. Exp. Med. Biol. 151 (1982) 355e360. [67] D. Pansu, C. Bellaton, C. Roche, F. Bronner, Theophylline inhibits active Ca transport in rat intestine by inhibiting Ca binding by CaBP, Prog. Clin. Biol. Res. 252 (1988) 115e120. [68] J.J. Feher, C.S. Fullmer, R.H. Wasserman, Role of facilitated diffusion of calcium by calbindin in intestinal calcium absorption, Am. J. Physiol. 262 (1992) C517eC526. [69] R. Spencer, M. Charman, P.W. Wilson, D.E.M. Lawson, The relationship between vitamin D-stimulated calcium transport and intestinal calcium-binding protein in the chicken, Biochem. J. 170 (1978) 93e101. [70] G.S. Lee, K.Y. Lee, K.C. Choi, Y.H. Ryu, S.G. Paik, G.T. Oh, et al., Phenotype of a calbindin-D9k gene knockout is compensated for by the induction of other calcium transporter genes in a mouse model, J. Bone Miner. Res. 22 (2007) 1968e1978. [71] S. Akhter, G.D. Kutuzova, S. Christakos, H.F. DeLuca, Calbindin D9k is not required for 1,25-dihydroxyvitamin D3mediated Ca2þ absorption in small intestine, Arch. Biochem. Biophys. 460 (2007) 227e232. [72] G.D. Kutuzova, S. Akhter, S. Christakos, J. Vanhooke, C. Kimmel-Jehan, H.F. DeLuca, Calbindin D(9k) knockout mice are indistinguishable from wild-type mice in phenotype and serum calcium level, Proc. Natl. Acad. Sci. USA 103 (2006) 12377e12381. [73] R.H. Wasserman, C.A. Smith, M.E. Brindak, N. Detalamoni, C.S. Fullmer, J.T. Penniston, et al., Vitamin-D and mineral deficiencies increase the plasma membrane calcium pump of chicken intestine, Gastroenterology 102 (1992) 886e894. [74] Q. Cai, J.S. Chandler, R.H. Wasserman, R. Kumar, J.T. Penniston, Vitamin D and adaptation to dietary calcium and phosphate deficiencies increase intestinal plasma membrane calcium pump gene expression, Proc. Natl. Acad. Sci. USA 90 (1993) 1345e1349. [75] E.J. van Corven, C. Roche, C.H. Van Os, Distribution of Ca2þATPase, ATP-dependent Ca2þ-transport, calmodulin and vitamin D-dependent Ca2þ-binding protein along the villuscrypt axis in rat duodenum, Biochim. Biophys. Acta 820 (1985) 274e282. [76] W.L. Davis, R.G. Jones, Lysosomal proliferation in rachitic avian intestinal absorptive cells following 1,25-dihydroxycholecalciferol, Tissue Cell 14 (1982) 585e595. [77] I. Nemere, C.M. Szego, Early actions of parathyroid hormone and 1,25-dihydroxycholecalciferol on isolated epithelial cells from rat intestine: 1. Limited lysosomal enzyme release and calcium uptake, Endocrinology 108 (1981) 1450e1462. [78] R.R. Warner, J.R. Coleman, Electron probe analysis of calcium transport by small intestine, J. Cell Biol. 64 (1975) 54e74. [79] I. Nemere, V. Leathers, A.W. Norman, 1, 25 dihydroxyvitamin D3-mediated intestinal calcium transport. Biochemical identification of lysozomes containing calcium and calcium-binding protein (calbindin-D28k), J. Biol. Chem. 261 (1986) 16106e16114.
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[113] K.L. Saddoris, J.C. Fleet, J.S. Radcliffe, Sodium-dependent phosphate uptake in the jejunum is post-transcriptionally regulated in pigs fed a low-phosphorus diet and is independent of dietary calcium concentration, J. Nutr. 140 (2010) 731e736. [114] T. Yoshida, N. Yoshida, T. Monkawa, M. Hayashi, T. Saruta, Dietary phosphorus deprivation induces 25-hydroxyvitamin D (3) 1alpha-hydroxylase gene expression, Endocrinology 142 (2001) 1720e1726. [115] W. Bauer, A. Marble, D. Claflin, Studies on the mode of action of irradiated ergosterol: I. Its effect on the calcium, phosphorus and nitrogen metabolism of normal individuals, J. Clin. Invest. 11 (1932) 1e19. [116] A. Carlsson, The effect of vitamin D on the absorption of inorganic phosphate, Acta Physiol. Scand. 31 (1954) 301e307. [117] Y. Tanaka, H.F. DeLuca, Role of 1,25-dihydroxyvitamin D3 in maintaining serum phosphorus and curing rickets, Proc. Natl. Acad. Sci. USA 71 (1974) 1040e1044. [118] T.C. Chen, L. Castillo, M. Korycka-Dahl, H.F. DeLuca, Role of vitamin D metabolites in phosphate transport of rat intestine, J. Nutr. 104 (1974) 1056e1060. [119] D.B. Lee, M.W. Walling, N. Brautbar, Intestinal phosphate absorption: influence of vitamin D and non-vitamin D factors, Am. J. Physiol. 250 (1986) G369eG373. [120] H. Segawa, I. Kaneko, S. Yamanaka, M. Ito, M. Kuwahata, Y. Inoue, et al., Intestinal Na-P(i) cotransporter adaptation to dietary P(i) content in vitamin D receptor null mice, Am. J. Physiol. Renal. Physiol. 287 (2004) F39eF47. [121] P. Capuano, T. Radanovic, C.A. Wagner, D. Bacic, S. Kato, Y. Uchiyama, et al., Intestinal and renal adaptation to a low-Pi diet of type II NaPi cotransporters in vitamin D receptor- and 1alphaOHase-deficient mice, Am. J. Physiol. Cell Physiol. 288 (2005) C429eC434. [122] J. Marks, E.S. Debnam, R.J. Unwin, Phosphate homeostasis and the renal-gastrointestinal axis, Am. J. Physiol. Renal. Physiol. 299 (2010) F285eF296.
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C H A P T E R
20 The Calbindins: Calbindin-D28K and Calbindin-D9K and the Epithelial Calcium Channels TRPV5 and TRPV6 Sylvia Christakos, Leila J. Mady, Puneet Dhawan Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey, USA
INTRODUCTION AND GENERAL CONSIDERATIONS, THE CALBINDINS In the two major target tissues of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) action, intestine and kidney, one of the most pronounced effects of 1,25(OH)2D3 known is the induction of the calcium-binding protein, calbindin, the first identified target of 1,25(OH)2D3 action. There are two major subclasses of calbindin: a protein of approximately 28 000 molecular weight (calbindin-D28K) and a protein of approximately 9000 molecular weight (calbindin-D9K). Calbindin-D28K is present in highest concentration in avian intestine and in avian and mammalian kidney, brain, and pancreas. Calbindin-D28K has four functional high-affinity calcium-binding sites and is highly conserved in evolution. Calbindin-D9K has two calcium-binding domains, is present in highest concentration in mammalian intestine, and, unlike calbindin-D28K, is not evolutionarily conserved and has been observed only in mammals. There is no amino acid sequence similarity between calbindin-D9K and calbindin-D28K. The discussion that follows reviews the chemistry, localization, proposed functional significance, and regulation of these calcium-binding proteins. In addition, this chapter provides insight into the information obtained by studying these proteins concerning the multiple actions of the vitamin D endocrine system and the basic molecular mechanism of 1,25(OH)2D3 action. Findings indicating that calbindins can be regulated by a number of different hormones and factors are also reviewed. The study of the molecular
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10020-4
interactions of several members of the steroid hormonee retinoic acid family as well as the role of signal transduction pathways in the regulation of calbindin-D may be applicable to the regulation of other targets of 1,25 (OH)2D3 action. Elucidation of multiple factors and interactions regulating 1,25(OH)2D3 target genes should result in novel insights related to tissue-specific molecular mechanisms involved in calcium homeostasis. One of the most important findings in the vitamin D field has been the discovery by Wasserman and Taylor in 1966 of a 28 000 Mr vitamin-D-dependent calciumbinding protein in avian intestine [1]. Although previously known as the vitamin-D-dependent calcium-binding protein (CaBP), in 1985 it became officially known as calbindin-D28K and calbindin-D9K for the 28 000 Mr and the 9000 Mr proteins, respectively [2]. Initially identified in avian intestine [1], calbindinD28K has since been reported in many other tissues including kidney and bone, and in tissues that are not primary regulators of serum calcium such as pancreas, testes, and brain and in a variety of species [3e9] (see Christakos et al. [9] for review). The importance of the discovery of calbindin-D28K is that key advances in our understanding of the diversity of the vitamin D endocrine system have been made through the study of its tissue distribution and its colocalization with the vitamin D receptor (VDR). In addition, the biosynthesis of calbindin has provided a model for studies that have resulted in an important basic understanding of the molecular mechanism of action of 1,25(OH)2D3 in major target tissues such as intestine and kidney.
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20. THE CALBINDINS: CALBINDIN-D28K AND CALBINDIN-D9K AND THE EPITHELIAL CALCIUM CHANNELS TRPV5 AND TRPV6
Chicken and mammalian calbindin-D28K proteins contain 261 amino acid residues, have a molecular weight of approximately 28 000 (30 000 based on amino acid sequence and 28 000 based on migration on sodium dodecyl sulfateepolyacrylamide gels), and are blocked at the amino terminus [9e12]. The mammalian calbindin-D28K sequences are 98% similar to one another and 79% similar to chicken calbindin-D28K [9,11,12]. Calbindin-D28K is highly conserved in evolution, suggesting an important, fundamental role for calbindin-D28K in mediating intracellular calcium-dependent processes [9]. Unlike calbindin-D28K, calbindin-D9K is observed only in mammals. It has no amino acid sequence homology to calbindn-D28K. It is most abundant in mammalian duodenum, placenta, and uterus [13e16]. It is also present in mammalian yolk sac, lung, bone, and mouse kidney [14,17e23]. Human calbindin-D9K has 79 amino acid residues and a calculated molecular weight of 9015 [24,25]. It is 89% similar to the bovine and porcine sequences and 78% and 77% similar to rat and mouse calbindin-D9K, respectively [24,25]. Calbindin-D28K and calbindin-D9K belong to a family of high-affinity calcium (Ca2þ)-binding proteins (Kd ¼ 10e8e10e6 M) that contains more than 200 members and is characterized by the EF-hand structural motif [26] (Fig. 20.1). The EF-hand domain is an octahedral structure consisting of two alpha helices separated by a 12-amino-acid loop that contains side chain oxygens necessary for orienting the divalent calcium cation [26]. Calbindin-D28K contains six EF hands (Fig. 20.2); however, only four of these actively bind Ca2þ [27,28].
FIGURE 20.1 EF-hand structural motif (helixeloopehelix). The helices are represented by the extended forefinger and thumb. The clenched middle finger represents the loop that contains the oxygen ligands of the calcium ion. The EF hand is a recurring motif in calbindin and other calcium binding proteins. Reprinted with permission from Stryer L 1995 Biochemistry. Freeman, San Francisco, p. 1064.
Calbindin-D9K contains two calcium-binding sites [29]. Other calcium-binding proteins belonging to this family include calmodulin, parvalbumin, troponin C, calretinin, calcineurin, calpain, Spec I, myosin light chains, S100, and recoverin [26,30]. Although the structure of calbindin-D28K has yet to be elucidated by X-ray crystallography, circular dichroism experiments have shown that calbindin-D28K contains approximately 30% a helix, 20.6% b sheet, and 51% random coil [31]. The threedimensional structure of calbindin-D9K has been elucidated [32]. Calbindin-D9K has been shown to undergo limited conformational change in the presence or absence of calcium [33]. Both calbindins are heat-stable protein and acidic, having a pI value of approximately 5 [34,35]. The calbindins bind other cations in addition to calcium with reduced affinity: Ca2þ > Cd2þ > Sr2þ > Mn2þ > Zn2þ > Ba2þ > Co2þ > Mg2þ [36].
LOCALIZATION AND PROPOSED FUNCTIONAL SIGNIFICANCE OF THE CALBINDINS Intestine One of the most pronounced effects of 1,25(OH)2D3 is increased synthesis of intestinal calbindin. CalbindinD9K in mammalian intestine and calbindin-D28K in avian intestine have been localized primarily in the cytoplasm of absorptive cells [37], which supports the proposed role of calbindin in intestinal calcium absorption [38e40]. Early studies in chicks established a strong correlation between the level of calbindin and an increase in intestinal Ca2þ transport [41e43]. In the intestine, 1,25(OH)2D3 effects the transfer of Ca2þ across the luminal brush-border membrane, the transfer of calcium through the cell interior, and active calcium extrusion from the basolateral membrane. Vitamin-D-inducible apical calcium channels have been identified in intestine and kidney, suggesting, for the first time, a mechanism of calcium entry [44e46]. It is thought that calbindin acts to facilitate the diffusion of calcium through the cell interior toward the basolateral membrane [40,42]. Supporting this hypothesis are findings observed in vitamin D receptor knockout mice. In these mice, the major defect that results in rickets is in intestinal calcium absorption [47e49]. The defect in intestinal calcium absorption is accompanied by a 50% reduction in intestinal calbindin-D9K mRNA [50]. The 1,25(OH)2D3 regulation of intestinal calbindin-D9K is also evident in 25-hydroxyvitamin-D3 1a-hydroxylase knockout mice. In these mice, characterized by hypocalcemia, hyperparathyroidism, and skeletal abnormalities characteristic of rickets, intestinal calbindin-D9K mRNA is absent [51].
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FIGURE 20.2 Position of intervening sequences within the structure of chicken calbindin-D28K. Locations of introns are indicated by circled numbers. Numbers above amino acids indicate codon positions. Invariant Glu/Leu and Gly amino acids are indicated by black circles. Calciumbinding domains are separated from the a-helix region by vertical lines. Reprinted with permission from Minghetti et al. [122].
Recent studies, however, using calbindin-D9k knockout (KO) mice have challenged the traditional model of vitamin-D-mediated transcellular calcium absorption in intestine. Calbindin-D9k KO mice show no difference in phenotype from wild-type mice and are able to maintain normal serum calcium levels regardless of age or sex [52]. In response to 1,25
(OH)2D3 treatment, calbindin-D9k KO mice are fully able to absorb calcium from the intestine, illustrating that calbindin-D9k is not required for 1,25(OH)2D3induced active intestinal calcium transport [53]. Furthermore, active intestinal calcium transport is similarly induced in both calbindin-D9k KO mice and wild-type mice in response to a low-calcium diet or 1,25(OH)2D3
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treatment [54]. These findings suggest that calbindinD9k may be compensated for by another factor or that calbindin-D9k may have another role in intestine, for example as a modulator of the activity of the vitamin-Dinducible epithelial calcium channel in the intestine and/or as a cytosolic buffer to prevent toxic levels of calcium from accumulating in the intestinal cell during vitamin-D-mediated translocation of calcium [40].
Kidney Immunocytochemical studies have reported the exclusive localization of calbindin-D28K in the distal nephron (distal convoluted tubule and connecting tubule) in a variety of species including mammals, chickens, and reptiles [3,9,55e57]. Renal calbindinD28K is localized in the cytosol and the nucleus and is not associated with membranes or filamentous elements. Both calbindin-D28K and calbindin-D9K are localized in mouse distal nephron and perinatal rat distal nephron [23]. Autoradiographic data indicated that the VDR is also predominantly localized in the distal nephron and both calbindins have been reported to be induced by 1,25(OH)2D3 in the kidney [58]. Although micropuncture data [59] as well as studies using a mouse distal convoluted tubule cell line [60] have indicated that vitamin D metabolites can enhance calcium transport in the distal nephron, little information is available concerning the exact role of vitamin-D-inducible renal calbindins in this process. Transcellular calcium transport in the distal convoluted tubule, similar to transcellular intestinal calcium absorption, involves calcium entry through the apical plasma membrane, diffusion of calcium across the cell, and active extrusion of calcium across the basolateral membrane mediated by a calcium-dependent ATPase [61]. The epithelial Ca2þ channel transient receptor potential vanilloid-subtype 5 (TRPV5) is coexpressed with calbindin-D28K and coregulated by 1,25(OH)2D3 and dietary Ca2þin the distal nephron and acts as a gatekeeper of apical Ca2þ entry during active reabsorption [62e64]. Calbindin-D28K directly interacts with TRPV5 at the apical membrane under conditions of low intracellular Ca2þ and has been shown to regulate the activity of the epithelial Ca2þ channel [65]. These findings suggest an additional role for renal calbindin-D28k as a modulator of calcium influx. It has also been suggested that calbindin-D28K may act to ferry calcium across the cell, as in the intestine, to buffer calcium, resulting in protection against calcium-mediated cell death [61,66]. Calbindin-D9K has been reported to have a different cellular action, i.e. to bind calcium and to stimulate ATP-dependent extrusion of calcium at the basolateral membrane [67]. The different functions of renal calbindin-D28K and
calbindin-D9K suggest different mechanisms that may be involved in the enhancement by 1,25(OH)2D3 of calcium transport in the distal nephron. Studies using immunosuppressant drugs (CsA and FK-506) that result in nephrotoxicity have noted decreases in calbindin-D28K in the rat coincident with increases in urinary calcium and intratubular calcification [68,69], providing additional evidence for a role of calbindin-D28K in distal tubular calcium reabsorption. In addition, calbindin-D28K KO mice fed a high-calcium diet were found to have significantly increased urinary calcium/creatinine ratio compared to wild-type controls [61,70]. The regulation of renal and intestinal calbindinD9K was found to be similar in wild-type and knockout mice, indicating that changes in calbindin-D9K were not compensating for the lack of calbindin-D28K and further suggesting different roles for these two vitamin-Ddependent calcium-binding proteins [61]. Serum calcium was not different in the wild-type and calbindin-D28K KO mice, suggesting compensatory changes in bone or in intestinal calcium absorption [61,70]. However, it should be noted that mechanisms within the kidney, independent of calbindin-D28K, are also associated with hypercalciuria [71]. To further elucidate the functional significance of mammalian calbindin-D28k, VDR/calbindin-D28K double KO mice were generated [72]. Mice deficient only in VDR display secondary hyperparathyroidism, rickets and osteomalacia, and a reduction of renal calbindin-D9K to 10% of typical levels (thus in these mice calbindin-D9k would not compensate for the loss of calbindin-D28k). VDR/calbindin-D28K double KO mice have increased urinary calcium excretion, more severe secondary hyperparathyroidism, lower bone mineral density, a more distorted growth plate and more osteoid formation in the trabecular region compared to VDR KO mice [72]. Although a high-calcium/lactose diet restored normal levels of serum ionized calcium in both VDR KO and double KO mice, the skeletal abnormalities were not completely corrected in the double KO mice [72]. These findings indicate the importance of renal calbindin-D28K in maintaining calcium homeostasis.
Bone Calbindin-D28K and calbindin-D9K are both present in chondrocytes of growth plate cartilage in rats and calbindin-D28K is present in the growth plate cartilage of chicks [22,73,74]. Although it is not clear whether calbindin is vitamin-D-dependent in chondrocytes, 1,25 (OH)2D3 receptors have been reported in developing chick bone, specifically in dividing chondrocytes [75]. It has been suggested that calbindin may be involved in the movement of calcium in the process of calcification in the chondrocyte [73]. Calbindin-D9K and
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calbindin-D28K have also been localized to osteoblasts and ameloblasts of rodent teeth, and it has been reported that calbindin-D9K and calbindin-D28K mRNAs are induced by 1,25(OH)2D3 in these cells [20,21]. It has also been suggested that elevated expression of calbindin may phenotypically characterize cells that are involved in calcium handling during mineralization [20]. In addition to an association with mineralization, more recent evidence has indicated that calbindin-D28K is able to protect against apoptosis of bone cells. Calbindin-D28K was found to protect osteoblastic cells against tumor necrosis factor (TNF)-induced apoptosis (Fig. 20.3) as well as to prevent glucocorticoid-induced apoptosis of osteoblastic and osteocytic cells [76,77]. The protection against both TNF- and glucocorticoidinduced cell death was found to be at least partially due to the ability of calbindin-D28K to inhibit endogenous caspase-3, a key mediator of apoptosis in response to multiple signals. Calbindin-D28K was found to inhibit caspase-3 but was not cleaved by the caspase. In addition, the inhibition of caspase-3 by calbindin-D28K was reported to be independent of its calcium-binding ability. As the only other known natural nononcogenic
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inhibitor of capsase-3 besides the inhibitor of apoptotic proteins (IAPs), calbindin-D28K may be an important target in the prevention of cellular degeneration in bone cells.
Pancreas The pancreas was the first nonclassic target tissue in which receptors for 1,25(OH)2D3 were identified [78]. Although 1,25(OH)2D3 has been reported to play a role in insulin secretion, the exact mechanisms remain unclear [79e80]. An early indication that the pancreas may be a target for 1,25(OH)2D3 was the immunocytochemical study of Morrissey et al. [3], which localized calbindin-D28K to the islet. In the chick, calbindin-D28K is detected exclusively in insulin-producing b cells [81] and is responsive to vitamin D [82]. In the rat, however, calbindin-D28K has been reported to be localized in a as well as b cells of the pancreas [83]. Because autoradiographic data have indicated that 1,25(OH)2D3 receptors are localized only in rat b cells [84], and because insulin but not glucagon secretion is affected in vitamin-Ddeficient animals [79], studies in the rat suggest that
FIGURE 20.3 Overexpression of calbindin-D28K suppresses nuclear fragmentation of osteoblastic cells induced by TNFa. Cells were
transfected with the expression vector pREP4 alone (empty vector) or containing the cDNA for calbindin-D28K (calbindin-D28K) together with an expression vector containing the coding sequence of green fluorescent protein with a nuclear localization sequence. Forty-eight hours after transfection, cells were exposed to 1 nM TNFa for 16 h. Cells were fixed, mounted, and examined with a Zeiss confocal laser scanning microscope. Note the presence of apoptotic nuclei in the TNFa-treated vector-transfected cells but not in the calbindin-transfected cells similarly treated. Please see color plate section.
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b-cell calbindin-D28K may be regulated by 1,25(OH)2D3 while non-b-cell calbindin may be independent of vitamin D. Calbindin-D28K has also been identified in human pancreatic islet cells [85]. Studies using pancreatic beta cell lines as well as calbindin-D28K knockout mice have suggested that calbindin-D28K, by regulating intracellular calcium, modulates depolarization-stimulated insulin release [86]. In rat pancreatic beta cells, the alpha1 subunit of the L-type channel can interact with calbindin-D28K and in the presence of calbindinD28K, L-type channels show enhanced sensitivity to Ca2þ-dependent inactivation [87]. In addition to modulating insulin release, more recent studies have indicated that calbindin-D28K, by buffering calcium, can protect against destruction of beta cells by cytokines by preventing calcium-mediated mitochondrial damage and the resultant generation of free radicals [88]. These findings have important therapeutic implications for type 1 diabetes and the prevention of autoimmune destruction of pancreatic b-cells. Together, these findings indicate the involvement of pancreatic calbindin-D28K in intracellular Ca2þ homeostasis and modulation of Ca2þ influx.
Reproductive Tissues Calbindin-D28K has been reported in both chick and rat testes [5,6]. In chick and rat, immunocytochemical studies have revealed that calbindin-D28K is present in spermatogonia and spermatocytes of the seminiferous tubules and some interstitial Leydig cells [5,6]. It has been reported that vitamin-D-deficient chicks have significantly (threefold) lower testicular calbindin levels than vitamin-D-replete chicks [89]. As calbindin-D28K as well as VDR (which is also present in seminiferous tubules) have been shown to correlate with testicular maturation [6,90], the involvement of calbindin-D28K and vitamin D in spermatogenesis and steroidogenesis has been suggested [89]. Calbindin-D9K is present in the placenta and yolk sac of rats and mice [14,17]. Calbindin-D9K is also present in rat endometrium and myometrium [16]. In pregnant rats calbindin-D9K is also expressed in the uterine epithelium [16]. In placenta and yolk sac calbindin-D9K increases at the end of gestation, when there is increased calcium need of the fetus, suggesting a role for calbindin-D9K in the transport of calcium to the fetus [14,17]. Calbindin-D9k KO mice reproduce similar to wild-type mice and calbindin-D9k KO pups are indistinguishable from wild-type pups [52], suggesting compensation for the absence of calbindin-D9k in placenta by another factor. Unlike calbindin-D9K, calbindin-D28K is not present in rat reproductive tissue. However, calbindinD28K has been localized in the tubular gland cells of the shell gland in the chick [91], which are involved in calcium secretion during egg-shell formation.
Calbindin-D28K is also found in the reproductive tissues of female mice (endometrium and glandular epithelium of mouse uterus, mouse oviduct epithelium, and in primary follicles of mouse ovary) [92]. 1,25(OH)2D3 has no effect on calbindin in these tissues. However, calbindin-D9K and calbindin-D28K in rat and chick uterus, respectively, are under the positive control of estradiol [16,93]. In the mouse, calbindin-D28K gene expression is down-regulated in the uterus but not in the ovaries and oviduct, suggesting tissue- and species-specific regulation of calbindin-D28K by estradiol [92]. It has been suggested that transcellular calcium transport in epithelial cells of the uterus and oviduct is facilitated by calbindin [92]. The presence of calbindin in the myometrium suggests the involvement of calbindin in the modulation of intracellular calcium that may alter the frequency and strength of uterine contractions.
Nervous Tissue Calbindin-D28K is widely distributed throughout the brain of mammals, avians, reptiles, amphibians, fish, and mollusks [9]. It is present in most neuronal cell groups and fiber tracts and is localized in neuronal elements and some ependymal cells [8,94,95]. In brain, calbindin-D28K is not vitamin-D-dependent [9]. Neurons containing calbindin-D28K are found in the cerebral cortex in layers 2e4, primarily in pyramidal neurons as well as in hypothalamus, amygdala, thalamus, hippocampus, and cerebellum [8,94,95]. In the hippocampus, both basket cells and pyramidal neurons in CA1 stain positively for calbindin, as do granule cells and fibers in the dentate gyrus [8,94,95]. Purkinje cells of the cerebellum stain most intensely for calbindin-D28K [8,94,95]. It is of interest that the phenotype of the calbindin-D28K knockout mouse is impaired motor coordination [96]. It has been suggested that this phenotype may be the result of abnormal cerebellar activity due to the alteration of synaptically evoked calcium transients in the Purkinje cells in the absence of calbindin [96]. Calbindin-D28K is also present in the suprachiasmatic nucleus (SCN) which is a fundamental regulator of circadian function and entrainment in mammals [97]. During development, calbindin-D28K is markedly expressed in the mouse SCN and is coexpressed in adult melanopsin producing retinal ganglion cells [98]. Significant abnormalities are observed in circadian locomotor rhythmicity and entrainment to light/dark cycles in calbindin-D28K knockout mice, suggesting a role for calbindin-D28K in circadian organization and light transduction [98,99]. In addition, specific neuronal sensory cells have been shown to contain calbindin-D28K [100]. These cell populations include cochlear and vestibular hair cells in the inner ear [100], avian basilar papilla [101], cone but not
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rod photoreceptor cells of the retina [102,103], and conelike, modified photoreceptor cells (pinealocytes) of pineal transducers [104]. The presence of calbindin in specific cells of the sensory pathway suggests the possible involvement of calbindin in mechanisms of signal transduction. In the nervous system it has been suggested that neuronal calbindin, by buffering calcium, can regulate intracellular calcium responses to physiological stimuli and can protect neurons against calcium-mediated neurotoxicity [105]. It has been demonstrated that introduction of exogenous calbindin into sensory neurons can modulate calcium signaling by decreasing the rate of rise of intracellular calcium and by changing the kinetics of decay of the calcium signal [106]. Using adenovirus as an expression vector, overexpression of calbindin-D28K in hippocampal neurons was reported to suppress post-tetanic potentiation, possibly by restricting and destabilizing the evoked calcium signal [107]. Calbindin-D28K was also reported to play a role in the control of hypothalamic neuroendocrine neuronal firing patterns [108]. The whole-cell patch-clamp method was used to introduce calbindin into rat supraoptic neurons. Calbindin-D28K suppressed Ca2þ-dependent depolarization afterpotentials and converted phasic into continuous firing [108]. As different firing patterns promote the release of different hypothalamic hormones, it was suggested that calbindin-D28K, by regulating firing patterns, may be involved in the control of hormone secretion from hypothalamic neuroendocrine neurons. These studies [106e108] indicated, directly by introduction of exogenous calbindin-D28K via the patch clamp method or by transfection and overexpression, that calbindin is an important and effective regulator of calcium-dependent aspects of neuronal function. Correlative evidence between decreases in neuronal calbindin-D28K and neurodegeneration in studies of ischemic injury [109], seizure activity [110], and chronic neurodegeneration (Alzheimer, Huntington, and Parkinson diseases) [111e113] have been reported. It has been suggested that decreased calbindin levels may lead to a loss of calcium buffering or intracellular calcium homeostasis, which leads to cytotoxic events associated with neuronal damage and cell death. Direct evidence of a protective role of neuronal calbindin-D28K against a variety of insults including exposure to hypoglycemia and IgG from amyotrophic lateral sclerosis patients has been shown in primary cultures of neuronal cells or in neuronal cell lines in which the calbindin-D28K gene has been transfected [114,115]. Expression of calbindin-D28K in neural cells was also found to suppress the proapoptotic actions of mutant presenilin 1 (PS-1), which is causally linked to about 50% of the cases of early-onset familial Alzheimer’s disease [116]. Mutant
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PS-1 has been reported to sensitize cells to apoptosis induced by amyloid b peptide (Ab), the major component of plaques in Alzheimer disease. Ab has been reported to damage neurons by a mechanism involving oxidative stress and disruption of calcium homeostasis. Calbindin-D28K protected against the proapoptotic action of mutant PS-1 by attenuating the increase in intracellular calcium and preventing the impairment of mitochondrial function [116]. In rats, pretreatment with a calbindin-D28K fusion protein (protein transduction domain (PTD)-calbindin-D28K) has been shown to attenuate neuronal insults induced by ischemia and reperfusion [117]. Protein transduction domains are small peptides that serve to deliver larger molecules into a variety of cells, including those of the bloodebrain barrier, without employing traditional endocytic mechanisms [118]. PTD-fused proteins have been shown to retain their biologic activity [119] and thus represent a potentially novel mode of neurobiological therapy. Since calbindin-D28K can prevent neuronal damage in neuropathies, including ischemic injury, these findings have important therapeutic implications.
Calbindin-D28K and Apoptosis Calcium is thought to play an important regulatory function in apoptosis, but the precise mechanism(s) by which calcium promotes cell death is unknown. The first study suggesting that calbindin-D28K plays a protective role in the process of apoptosis used subtraction analysis between the cDNA libraries of two human prostate cell lines [120]. One of the cell lines was androgen independent and the other one androgen dependent, and the results revealed that a hybrid calbindin-D28K gene was specifically expressed in the hormone-independent cell line. Apoptosis has been observed in prostate cells on androgen depletion. Androgen deprivation of prostate cells triggers an influx of calcium ions into the cells, leading to an increase in intracellular calcium. It was suggested that calbindin-D28K might buffer intracellular calcium and contribute to protection against apoptosis and thus androgen independence in the prostatic cell line. It has been reported that stable transfection and overexpression of calbindin-D28K in lymphocytes protect against apoptosis induced by calcium ionophore, cAMP, and glucocorticoid [121]. A similar protective role for calbindin-D28K has been observed in apoptosissusceptible cells in the central nervous system [114e116] as well as in human embryonic kidney cells (HEK 293), osteoblasts, and pancreatic b cells [66,76,77,88]. These findings indicate that calbindinD28K has a major role in different cell types in protecting against apoptotic cell death. A further understanding of the mechanisms involved will have important
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therapeutic implications for the prevention of a number of diseases including osteoporosis and diabetes.
REGULATION OF CALBINDIN GENE EXPRESSION Calbindin-D28K Genomic Organization of the Calbindin-D28K Gene The genomic organization of the chicken calbindinD28K gene has been elucidated [122,123] and a partial structure for the human gene was also reported [12,124]. The human gene (symbol: CALB1) is located on chromosome 8 [124,125] and is believed to consist of 11 exons, analogous to that demonstrated for the avian gene. Moreover, the total size of the gene is reported to be 18.5 kb in chicken and 24 kb in human. The protein coding region of the mouse gene shares 77% sequence similarity with the chicken gene [126]. However, no obvious sequence similarity exists between the mammalian and avian promoters except in the region of the TATA box [12,127]. Regulation by 1,25(OH)2D3 It is well known that calbindin-D28K in the avian intestine [1,128] and kidney [128] and in the mammalian kidney [129,130] is induced by 1,25(OH)2D3. In chicken, a putative calbindin-D28K vitamin D response element (VDRE) was suggested after computer analysis of the promoter sequence [131], but only a twofold response to 1,25(OH)2D3 was detected in primary kidney cells after transfection with a 2.1-kb segment of the 50 flanking region of the promoter [132]. A relatively inactive putative VDRE was also reported in the chicken calbindinD28K promoter by others [133,134] and the response of the mouse calbindin-D28K promoter to 1,25(OH)2D3 is modest (fivefold maximal induction in chloramphenicol acetyltransferase (CAT) activity) [127]. The modest response reflects previous in vivo findings that indicated a small transcriptional response to 1,25(OH)2D3 [125,130]. Similar findings were reported for the in vivo induction of the chick intestinal calbindin-D28K gene by 1,25(OH)2D3 [135]. In addition, in VDR knockout mice only a small reduction in basal levels of renal calbindin-D28K is observed. However, the response of renal calbindin-D28K in the VDR knockout mouse to 1,25(OH)2D3 is compromised [50]. Also in 25-hydroxyvitamin D3 1a-hydroxylase knockout mice the expression of renal calbindin-D28K is reduced [51]. There is now increasing evidence that the large induction of calbindin-D28K mRNA long after 1,25(OH)2D3 treatment may be due primarily to post-transcriptional mechanisms [125,130,135,136]. Exactly how this action is exerted is not known, but one report suggests that 1,25
(OH)2D3 may regulate the expression of an intermediate protein that may be involved in calbindin-D28K mRNA accumulation [136]. These studies suggest that the mechanism of action of 1,25(OH)2D3 on calbindin-D28K regulation is more complicated than the conventional hormone receptoretranscriptional activation model, and that this regulation may involve other factors and is mostly post-transcriptional. Regulation by other Steroids and Factors Further studies in intestine and kidney have provided evidence that the calbindin-D28K gene is not exclusively regulated by 1,25(OH)2D3 and that other factors can modulate gene expression. It has been reported that glucocorticoids can inhibit the levels of calbindin-D28K mRNA and protein in intestine of vitamin-D-treated chicks, resulting in a comparable decrease in intestinal calcium absorption [137,138]. These findings suggest the involvement of the inhibition of intestinal calbindin in the clinically important hypocalcemic action of glucocorticoids. Alterations in dietary Ca2þ and phosphorus have also been shown to modulate avian intestinal and renal calbindin-D28K gene expression, further suggesting that the regulation of calbindin is more complex than previously thought [139,140]. In other tissues where calbindin-D28K is present in significant amounts, for example, in parts of the brain, the regulation of calbindin-D28K appears to be very different from that in the intestine and kidney. In the central nervous system (CNS), 1,25(OH)2D3 has no apparent effect on the levels of calbindin-D28K [9]. Instead, a variety of different factors have been reported to be involved in regulating neuronal calbindin-D28K. Using rat hippocampal cultures, evidence has indicated that neurotropin 3 (NT-3) [141,142], brain-derived neurotropic factor (BDNF) [141,142], fibroblast growth factor (FGF) [141], and tumor necrosis factors (TNFs) [143,144] all can induce calbindin. It has been reported that neurotropic factors may protect against excitotoxic neuronal damage [145]. The induction of calbindin by those factors suggests a role for calbindin-D28K in the process of protection against cytotoxicity. In addition, corticosterone administration in vivo has been reported to increase calbindin-D28K expression in rat hippocampus [146,147]. The glucocorticoid response is specifically localized to the CA1 region [147]. Retinoic acid has also been reported to induce calbindin-D28K protein and mRNA in medulloblastoma cells, which express a neuronal phenotype [148]. Furthermore, the content of calbindin-D28K in cultured Purkinje cells can be increased by insulin-like growth factor I (IGF-I) [149]. Similarly IGF-I, as well as insulin, promoted the expression of calbindin-D28K protein in cultured rat embryonic neuronal cells [150]. Thus, neuronal calbindin-D28K can be regulated by steroids as well as by
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factors that affect signal transduction pathways. These different modes of activation may be important for cell-specific effects of calbindin-D28K. The molecular basis for tissue-specific calbindin-D28K regulation is still not understood, but in vivo experiments using transgenic mice suggest that tissue specificity of calbindin-D28K expression to some degree is controlled by separate elements on the promoter [151]. The transgenic mouse study demonstrated the importance of an in vivo system to investigate the role of sequence elements needed for tissue-specific gene expression and regulation [151]. Gender-specific hormones have also been suggested to modulate calbindin-D28K expression. As discussed previously, calbindin-D28K is also present in the avian egg-shell gland and in the reproductive tissues of female mice. In the avian egg-shell gland, estradiol-17b induces calbindin-D28K in in vivo experiments [93]. In the mouse, calbindin-D28K gene expression was found to be downregulated by estradiol in the uterus and oviduct [92,152] but up-regulated in the ovaries [152]. Multiple imperfect half-palindromic estrogen-responsive elements, which are likely to mediate the estrogen responsiveness of the calbindin-D28K gene by estradiol-17b, were present in two regions (e1075/e702 and e175/e78) of the promoter [152]. The contrasting responses to estradiol in chick and mouse suggest species-specific regulation of calbindin-D28K by estradiol. It has also been shown that differences in gender influence Ca2þ handling in the kidney as male mice display greater urinary Ca2þ loss than female mice [153]. Significantly decreased levels of TRPV5 and calbindin-D28K are also observed in male mice compared to female mice [154]. Furthermore, levels of renal TRPV5 and calbindin-D28K mRNA and protein are increased in androgen-deficient male mice and are unaccompanied by significant differences in serum estrogen, parathyroid hormone, or 1,25 (OH)2D3 levels [154]. Testosterone treatment suppresses the elevation of renal TRPV5 and calbindin-D28K in these mice [154]. Thus, it has been suggested that gender differences in renal Ca2þ reabsorption may, in part, be explained by the inhibitory actions of androgens on renal TRPV5 and calbindin-D28K. Though calbindin-D28K was one of the first identified targets of 1,25(OH)2D3 action, the complex regulation of calbindin-D28K illustrates that the utility of calciumbinding proteins extends beyond the traditional paradigm of the vitamin D endocrine system.
Calbindin-D9K Genomic Organization of the Calbindin-D9K Gene The size of the calbindin-D9K gene is 2.5 kb, and the gene consists of three exons and two introns [155]. The first exon contains the 50 untranslated region. The second
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exon codes for the first calcium-binding site. The third exon codes for the second calcium-binding site and the 30 untranslated region. It is related to the S100 family of calcium-binding proteins. The human calbindin-D9k gene (symbol: CALB3) spans 5.5 kb and consists of three exons. The chromosomal localization of the human calbindin-D9k gene is assigned to Xp22.2. [156]. Regulation of Calbindin-D9K by 1,25(OH)2D3 Similar to the regulation of avian intestinal calbindinD28K and mammalian renal calbindin-D28K, in vivo findings have indicated that intestinal calbindin-D9K is regulated by 1,25(OH)2D3 by a small, rapid transcriptional stimulation followed by a post-transcriptional effect accounting for a sustained accumulation of mRNA long after cessation of 1,25(OH)2D3 treatment [157]. Unlike renal calbindin-D28K, there is a marked decrease of basal as well as 1,25(OH)2D3 induced levels of intestinal calbindin-D9K mRNA in VDR knockout mice, suggesting that intestinal calbindin-D9K is more sensitive to control by VDR-mediated mechanisms than calbindin-D28K [50]. Studies using transgenic mice have shown that the proximal promoter of the calbindin-D9K gene from e117 to þ365 and distal element at e3731 to e2894 together but not separately confer the 1,25(OH)2D3-induced transcriptional response [158]. Since this region does not contain a classical VDRE, these findings suggest that the 1,25(OH)2D3 regulation of calbindin-D9K may involve a nonconventional activation pathway. Regulation of Calbindin-D9K by other Steroids and Factors In vitro footprinting and gel shift assays suggested that several transacting factors other than the VDR, including a ubiquitous factor (NF1), liver-enriched factors (HNF1, C/EBP alpha and beta, and HNF4), and the intestine-specific transcription factor caudal homeobox-2 (Cdx-2), may be important for intestinespecific calbindin-D9K gene expression [159]. Further studies using transgenic mice showed that a mutation in the distal Cdx2-binding site of calbindin-D9K promoter dramatically decreased intestinal expression of the calbindin-D9K gene, directly demonstrating the crucial role of Cdx2 for the transcription of this gene in the intestine [160]. With regard to other steroids, besides 1,25(OH)2D3, the expression of calbindin-D9K in the intestine is also regulated by glucocorticoids. Glucocorticoids have been reported to inhibit intestinal calbindin-D9K expression [161]. It has been suggested that this decrease may be involved in the reported decrease by glucocorticoids in intestinal calcium absorption. Whether the effect on intestinal calbindin-D9K expression is a primary or a secondary action of glucocorticoids is not yet known.
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In the uterus, calbindin-D9K is under the control of estrogen but is unaffected by 1,25(OH)2D3. An imperfect estrogen response element (ERE) that binds the estrogen receptor (ER) has been identified at the border of the first exon and the first intron [162,163]. In vivo experiments using transgenic mice suggest the functionality of this imperfect ERE [164].
EPITHELIAL CALCIUM CHANNELS Transient Receptor Potential Vanilloid 5 (TRPV5) General Considerations and Genomic Organization of the TRPV5 Gene Transient receptor potential vanilloid (TRPV) cation channels make up one of four main classes of the transient receptor potential (TRP) superfamily [165]. In 1999, TRPV5 (ECac/CaT2) was cloned from distal convoluted tubules in rabbit kidney using Xenopus oocytes [44]. Of all TRP channels, TRPV6 shares the highest sequence homology with TRPV5 (75%) [166]. The human TRPV5 gene has been mapped to chromosome 7q31.1-q31.2, just upstream of TRPV6, and contains 15 exons coding for a protein of 729 amino acids with a molecular mass of 83 kDa [167]. The TRPV5 protein consists of six transmembrane domains with a putative pore-forming region between domains 5 and 6 as well as cytosolic N- and C-terminal domains and exists functionally as a tetramer forming a single Ca2þ channel [167]. TRPV5 Ca2þ-binding properties are lost upon disruption of the aspartate-542 residue located within the pore formed between domains 5 and 6 [168]. Immunohistochemical studies have localized TRPV5 to the apical membrane of the distal convoluted tubule and connecting tubule of the kidney and have shown colocalization of renal TRPV5 with calbindin-D28K [169]. Regulation of TRPV5 by 1,25(OH)2D3 Putative vitamin D response elements have been identified in the TRPV5 promoter [170]. Studies in mice and rats have shown that renal TRPV5 mRNA is induced by 1,25(OH)2D3 [62e64]. However the expression of renal TRPV5 mRNA is unchanged in VDR KO mice (unlike intestinal TRPV6 mRNA which is markedly reduced in VDR KO mice compared to WT mice), suggesting that factors in addition to 1,25(OH)2D3 are involved in the regulation of TRPV5 in the kidney [171,172]. Proposed Functional Significance The generation of mice lacking TRPV5 has provided an understanding of the functional significance of this
epithelial calcium channel. Renal calcium reabsorption is compromised in TRPV5 KO mice, resulting in hypercalciuria and significant changes in bone morphology, including reduced trabecular and cortical bone thickness as well as impaired bone resorption [173,174]. Furthermore, these mice show significantly elevated levels of serum 1,25(OH)2D3 and a compensatory increase in intestinal TRPV6 mRNA, intestinal calbindin-D9K mRNA, and intestinal calcium absorption [173]. Micropuncture studies localize the site of defective calcium reabsorption to the distal convoluted tubule, the site of localization of TRPV5, thereby suggesting that hypercalciuria in the KO mice results primarily from the absence of TRPV5 [173]. In TRPV5/ 1a-hydroxylase double KO mice up-regulation of intestinal TRPV6 and calbindin-D9k mRNA is not observed, indicating that the elevated serum 1,25(OH)2D3 levels are responsible for the compensatory increase in intestinal calcium transporters and intestinal calcium absorption in the TRPV5 KO mice [175]. Together, these findings demonstrate a role for TRPV5 in active calcium reabsorption in the distal nephron. Studies have also been done comparing TRPV5 KO mice, calbindin-D28K KO mice, and TRPV5/calbindinD28K double KO mice [176]. Double KO mice display compensatory up-regulation of intestinal calcium absorption (similar to that described previously in TRPV5 KO mice), increased renal calbindin-D9K expression, and up-regulation of intestinal calbindin-D9K and TRPV6 [176]. Intestinal calcium absorption and expression of calbindin-D9K and TRPV6 remain unchanged in calbindin-D28K KO mice compared to WT mice [176]. The similarities between TRPV5 KO mice and TRPV5/ calbindin-D28K double KO mice suggest that loss of calbindin-D28K may be compensated by up-regulation of renal calbindin-D9K in the double KO mice and provide further evidence of the importance of TRPV5 in renal calcium reabsorption in the distal part of the nephron.
Transient Receptor Potential Vanilloid 6 (TRPV6) General Considerations and Genomic Organization of the TRPV6 Gene Transient receptor potential vanilloid (TRPV) cation channel 6 (TRPV6) was first cloned from rat duodenum [45]. TRPV6 is expressed in highest concentrations in duodenum and cecum and in lower concentrations in proximal jejunum [177,178]. Very low levels are also expressed in the colon [177,178]. TRPV6 is expresssed in villi tips and not in villi crypts [177,178]. TRPV6 is also expressed in placenta, acinar cells of the pancreas, ductal epithelial cells of mammary glands and in skin [177,178]. The human TRPV6 gene has been mapped to chromosome 7q33-q34 downstream of TRPV5 and
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CONCLUSION
TRPV6 (epithelial calcium channel cloned from rat duodenum): predicated membrane topology and domain structure. TRPV6 contains six transmembrane domains. The putative N-linked glycosylation site (branched chain) is marked. Reprinted with permission from Peng et al., 1999 [45].
FIGURE 20.4
consists of 15 exons coding for a protein of 725 amino acids [179]. Similar to TRPV5, TRPV6 contains six transmembrane (TM) segments and cytosolic N- and Cterminal domains [178] (see Fig. 20.4). TRPV6 as well as TRPV5 channels contain ankyrin repeats at their aminoterminal which are part of functional tetrameric channels [180]. Regulation of TRPV6 by 1,25(OH)2D3 and Functional Significance In the human TRPV6 promoter putative vitamin D response elements (VDREs) were identified at e1.2, e2.1, e3.5, e4.3, and e5.5 kb. The VDREs at e4.3 and e2.1 were found to mediate the 1,25(OH)2D3 response in intestinal cells [181]. TRPV6 is colocalized with calbindin-D9k in duodenum and jejunum. A similar regulation of intestinal TRPV6 and calbindin-D9k has been observed [64]. Both TRPV6 and calbindin-D9k are markedly induced at weaning, the time of onset of active intestinal calcium transport and intestinal responsiveness to 1,25(OH)2D3 and both are strongly induced under low calcium conditions and after 1,25(OH)2D3 injection of vitamin-D-deficient mice [64]. After a single injection of 1,25(OH)2D3 intestinal TRPV6 mRNA and calbindin-D9k mRNA are induced prior to the peak of induction of intestinal calcium absorption [64]. Since TRPV6 mRNA was found to be more markedly decreased in the intestine than calbindin-D9k in VDR KO mice, it was suggested that the expression of TRPV6 may be a rate-limiting step in the process of vitamin-D-dependent intestinal calcium absorption [172]. However, when fed a standard rodent chow diet TRPV6 KO mice were found to have serum calcium levels similar to those of WT mice [54,182,183]. Serum PTH levels have been reported to be significantly increased in TRPV6 KO mice, consistent with an observed 9.6% decrease in femoral bone density [182].
Thus, TRPV6 may have an indirect role in regulation of bone formation and/or mineralization. 1,25(OH)2D3 administration to vitamin-D-deficient TRPV6 KO mice results in a significant increase in active intestinal calcium transport, indicating that in the KO mice there is compensation by another channel or protein [54,183]. Recent studies have found that TRPV6 can interact with other proteins that modulate its function including calmodulin which facilitates rapid inactivation of TRPV6, Rab11a which has been shown to play a role in recycling of TRPV6 to the plasma membrane, and S100A10-annexin 2 protein complex which may be involved in the constitutive trafficking of TRPV6 to the plasma membrane [184e186]. These TRPV6-associated proteins may represent novel components of 1,25 (OH)2D3-regulated intestinal calcium transport that specifically influence calcium entry mechanisms.
CONCLUSION We once viewed calbindin-D28K and calbindin-D9K as exclusively vitamin-D-dependent proteins. It is now evident that the calbindins are not under the exclusive regulatory control of 1,25(OH)2D3. Calbindin-D28K and calbindin-D9K are present in many different tissues (see Table 20.1) and may serve many different functions. Accordingly, the regulation of these calcium-binding proteins is varied and quite complex. The calciumselective epithelial calcium channels, TRPV5 and TRPV6, are colocalized with calbindin in kidney and TABLE 20.1
Distribution of Calbindin
Calbindin-D9K
Calbindin-D28K
Mammalian intestine [13]
Avian intestine [3,34,37]
Mouse and neonatal rat kidney [23]
Avian, reptilian, amphibian, and mammalian kidney [3,55e57]
Rat and mouse yolk sac [14,17]
Hen egg-shell gland (uterus) [93]
Rat uterus [15,16]
Mouse reproductive tissues (uterus, oviduct, ovary) [92]
Rat and mouse placenta [14]
Avian and mammalian beta cells of the pancreas [81,85] Alpha cells of the rat pancreas [83]
Rat growth cartilage [22]
Rat and chick growth cartilage [22,73,74]
Ameloblasts and osteoblasts of rodent teeth [20,21]
Ameloblasts and osteoblasts of rodent teeth; mouse osteoblasts [20,21,76]
Rat lung [18]
Brain (avian, reptilian, amphibian, molluskan, fish, and mammalian brain) [9,94,95]
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intestine respectively. Studies in TRPV5 KO mice have indicated that TRPV5 is important for renal calcium reabsorption. Calbindin-D28k interacts with TRPV5 and regulates its activity, suggesting a role for calbindinD28k as a modulator of calcium influx. Studies using calbindin-D9k and TRPV6 KO mice suggest that in the KO mice there is compensation by another calcium channel or protein and that other factors involved in 1,25 (OH)2D3 mediated intestinal calcium absorption remain to be identified.
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[179] J.B. Peng, X.Z. Chen, U.V. Berger, S. Weremowicz, C.C. Morton, P.M. Vassilev, et al., Human calcium transport protein CaT1, Biochem. Biophys. Res. Commun. 278 (2000) 326e332. [180] I. Erler, D. Hirnet, U. Wissenbach, V. Flockerzi, B.A. Niemeyer, Ca2þ-selective transient receptor potential V channel architecture and function require a specific ankyrin repeat, J. Biol. Chem. 279 (2004) 34456e34463. [181] M.B. Meyer, M. Watanuki, S. Kim, N.K. Shevde, J.W. Pike, The human transient receptor potential vanilloid type 6 distal promoter contains multiple vitamin D receptor binding sites that mediate activation by 1,25-dihydroxyvitamin D3 in intestinal cells, Mol. Endocrinol. 20 (2006) 1447e1461. [182] S.D. Bianco, J.B. Peng, H. Takanaga, Y. Suzuki, A. Crescenzi, C.H. Kos, et al., Marked disturbance of calcium homeostasis in mice with targeted disruption of the Trpv6 calcium channel gene, J. Bone Miner. Res. 22 (2007) 274e285. [183] G.D. Kutuzova, F. Sundersingh, J. Vaughan, B.P. Tadi, S.E. Ansay, S. Christakos, et al., TRPV6 is not required for 1alpha,25-dihydroxyvitamin D3-induced intestinal calcium absorption in vivo, Proc. Natl. Acad. Sci. USA 105 (2008) 19655e19659. [184] I. Derler, M. Hofbauer, H. Kahr, R. Fritsch, M. Muik, K. Kepplinger, et al., Dynamic but not constitutive association of calmodulin with rat TRPV6 channels enables fine tuning of Ca2þ-dependent inactivation, J. Physiol. 577 (2006) 31e44. [185] S.F. van de Graaf, J.G. Hoenderop, D. Gkika, D. Lamers, J. Prenen, U. Rescher, et al., Functional expression of the epithelial Ca(2þ) channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex, EMBO J. 22 (2003) 1478e1487. [186] S.F. van de Graaf, Q. Chang, A.R. Mensenkamp, J.G. Hoenderop, R.J. Bindels, Direct interaction with Rab11a targets the epithelial Ca2þ channels TRPV5 and TRPV6 to the plasma membrane, Mol. Cell Biol. 26 (2006) 303e312.
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C H A P T E R
21 Mineralization Eve Donnelly, Adele L. Boskey Hospital for Special Surgery, 535 E 70th Street, New York, NY 10021; affiliated with Weil College of Cornell Medical School, New York, NY 10021, USA
INTRODUCTION Definitions “Biologic mineralization” is the physicochemical process leading to deposition of inorganic crystals (minerals) on an organic matrix within the cell or outside it. This term is more specific than “mineralization,” as biologic mineralization implies a relation between the cells, the organic matrix, and the mineral. The cell-mediated biomineralization process comprises oriented deposition of mineral within or adjacent to cells or upon an extracellular matrix. Examples of the former include iron oxides and sulfides in magnetotactic bacteria [1] and silicates in diatoms [2]; examples of the latter include calcium carbonates in shells [3] and exoskeletons [4] and calcium phosphates in bones and teeth [5]. The mineral in physiologically calcified vertebrate tissues is an analog of the geologic mineral hydroxyapatite (Fig. 21.1). The physiologic hydroxyapatite crystals are 10e60 nm in their largest dimension [6], much smaller than those found in geologic deposits, and have stoichiometries different from the predicted 10Ca:6P04:20H of the geologic mineral. For that reason biologic vertebrate mineral is often referred to as
FIGURE 21.1 The hydroxyapatite unit cell showing the major substituents occurring in biologic apatites and the ions for which they substitute.
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10021-6
“apatite” or “apatitic” meaning “like hydroxyapatite.” This chapter will focus on physiologic and dystrophic apatite formation in situ and in culture and on the effects of vitamin D on mineral formation. Crystalline deposits may form by several different mechanisms. De novo crystal deposition occurs when the solution supersaturation exceeds the solubility of the precipitating phase. Supersaturation refers to the ratio of the solution ion product to the solubility product of the phase in question. The process starts with “nucleation” in which several ions or ion clusters come together in solution with the orientation they will have in the final crystal. This first step requires a great deal of energy, and is facilitated by increasing the ion product or reducing the diffusion of ions in solution [5]. Most biomineralization is epitaxial or heterogeneous e occurring on the surfaces of pre-existing crystals or on protein and lipid templates, which resemble the surface of the crystal [7]. These processes use much less energy than de novo mineralization, and require less supersaturation. Crystal growth occurs as ions and ion clusters add on to the surface of the initial nuclei or other pre-existing crystals. Crystal growth requires less energy than nucleation, and is limited in the case of biologic mineralization by the template upon which the crystals are deposited. Crystals can grow in all dimensions by the addition of ions [7]; by agglomeration [8] in which crystals accumulate, not always in an oriented fashion; or by secondary nucleation. Secondary nucleation, as it is seen with hydroxyapatite crystals maturing in solutions in the presence of the dentin protein, phosphophoryn [9], is a branching process in which new nuclei form on the surfaces of existing crystals thus resulting in a new population of immature crystals.
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21. MINERALIZATION
Among the vertebrate mineralized tissues, enamel contains the largest apatite crystals, but these crystals do contain carbonate [10] and other environmental contaminants (e.g., strontium, fluoride, lead, etc. [11]). Bone and tendon-bone insertions contain the smallest apatite mineral crystals [12], 6e10% carbonate [13], as well as adsorbed and incorporated citrate, fluoride, and other trace impurities. Bone apatite crystals are also hydroxide-deficient [14], but are not totally devoid of hydroxyl groups [15,16]. Cementum and dentin mineral crystals have intermediate sizes and intermediate accumulation of foreign ions. In each of these tissues the crystals that are formed initially are smaller than those in the mature tissues due both to the growth of existing crystals and the removal of the smaller crystals during osteoclast remodeling [5]. In humans, mineral also deposits at abnormal (unexpected/pathologic) locations [17]. This pathologic mineral is frequently apatitic, but calcite (calcium carbonate) is found in pancreatic stones [18]; monosodium urate, sodium pyrophosphate, as well as apatite [17] are found in cartilage; and brushite, oxalates, and uric acid occur in kidney and salivary stones (Table 21.1). The mineral in pathologic calcifications may be formed by physiologic processes or may be associated with dying cells (dystrophic calcification). An example of dystrophic calcification induced by vitamin D toxicity is seen in arteries [19]. In some cases the tissue becomes bone-like, and genes associated with osteogenesis are activated [20e22]; however it is not clear whether the calcification or the osteogenic gene expression comes first [23]. Dystrophic calcifications are distinct from TABLE 21.1
Pathologic Mineral Deposits
Mineral phase
Found in
Affected by vitamin D
Apatite
Blood vessels Kidney and bladder stones Salivary stones Pulp stones Muscle Skin Hyaline and articular cartilage
þ þ þ þ þ þ þ
Calcium carbonate (aragonite) (calcite)
Pancreatic stones
0
Oxalates
Kidney and salivary stones Soft tissues
þ
Pyrophosphates
Hyaline and articular cartilage
0
Urates
Hyaline and articular cartilage Kidney stones
0 þ
Calcified intrauterine devices
þ Indicates that deposition in these tissues is accelerated in hypervitaminosis D.
physiologic mineralization as in the latter viable cells are required, while in the former cells die, releasing calcium and phosphate and degradative enzymes [24].
Direct and Indirect Effects of Vitamin D and Vitamin D Metabolites on Mineralization Physical Chemistry of Mineralization De novo apatite formation requires Caþ2, PO-3 4 , and OHe ions to come together in the correct orientation with sufficient energy and in sufficient numbers to form the first stable apatite crystal (nucleus). After this nucleus is formed, additional ions can add on to these small crystals (nuclei) causing crystal growth. As crystals become larger, new nuclei can branch off the surface (secondary nucleation), in a fashion analogous to glycogen formation. These new nuclei grow and exponentially form additional secondary nuclei. During cell-mediated biologic apatite formation, matrix molecules provide sites for accumulation of ions and templates to orient mineral growth. Some molecules may act as heterogeneous nucleators, facilitating the deposition of mineral. Others may bind to the crystals and regulate their shape and size [25]. Nucleation occurs at multiple sites along these templates, and crystal habit (shape) and size is regulated by the template and by other matrix proteins that bind to the surface of the apatite crystals. In cartilage, bone, dentin, tendon, and ligaments, mineral crystals form on a collagenous matrix. This collagen is the “template” [26] for initial mineral formation and crystal growth; mineralization of the collagen matrix gives it increased strength [27]. Differences in the distribution and post-translational modification [28] of extracellular matrix molecules associated with the fibrillar collagen influence the crystal size and the site of initial crystal deposition. In enamel, which does not have a collagen component, there is some debate as to which of the proteins initially present in the enamel matrix are the nucleators. Recent solution data showed that amelogenin, a large hydrophobic molecule that associates into nanospheres, can induce initial calcification [29,30], facilitate the ordering and agglomeration of crystals, and hence also regulate the size to which the crystals grow [30]. (There are several other proteins in enamel whose functions are not yet established, and it must be noted that results from solution studies do not always agree with cell culture or in vivo studies, and mice lacking amelogenin can form enamel [31].) One of the ways in which vitamin D is believed to influence mineralization is by stimulating the formation of these proteins and the enzymes responsible for their post-translational modification. The genes for many of these proteins, as well as many of the enzymes that
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INTRODUCTION
TABLE 21.2
Extracellular Matrix Proteins and Cellular Enzymes Regulated by Vitamin D and Associated with Mineralization: Solution Data
Protein
Effect on mineralization
Ref.
Regulated by vitamin D*
Ref.
Amelogenin
Regulator of crystal growth
a
þ
b
Aggrecan
Inhibitor
25
þ
c
Biglycan
Nucleator
25
?
133
Bone sialoprotein (BSP)
Nucleator
109
Expression inhibited
110, d
Collagen I
Template
25
þ
106,107
Dentin matrix protein-1 (DMP1)
Nucleator/inhibitor
e
þ
32
Dentin phosphophoryn
Nucleator
8
þ
b
Dentin sialoprotein
Weak nucleator/inhibitor
25
0
f
Enamelin
Regulator of crystal shape
30
þ
b
Fetuin
Inhibitor
g
þ
g
Matrix gla protein (MGP)
Inhibitor
25
þ
32
Matrix extracellular phosphoglycoprotein (MEPE)
Nucleator/Inhibitor
127,128
Osteopontin
Inhibitor
104,115
þ
115
Osteocalcin
Inhibitor
112
þ
111
Proteolipid
Nucleator
h
þ
117e119
ATPase
Hydrolyzes ATP, facilitates cellular Ca transport
i
þ
i
Matrix metallo-proteinases Stromolysin MMP3 Gelatinase MMP2
Degrade matrix molecules that inhibit mineralization
25
þ
32,j
EXTRACELLULAR MATRIX PROTEINS
32
ENZYMES
* Regulation by vitamin D depends on concentration, cell type and cell maturity ; hence + indicates that there is an effect and/or that the gene contains a vitamin D responsive element (VDRE). Readers are referred to other chapters to see precise effects. 0 ¼ No effect ? ¼ unknown. a. E. Beniash, J.P. Simmer, H.C. Margolis. The effect of recombinant mouse amelogenins on the formation and organization of hydroxyapatite crystals in vitro. J. Struct. Biol. 149 (2005) 182e190. b. P. Papagerakis, M. MacDougall, D. Hotton, I. Bailleul-Forestier, M. Oboeuf, A. Berdal. Expression of amelogenin in odontoblasts. Bone 32 (2003) 228e40. 156. A. Farzaneh-Far, P.L. Weissberg, D. Proudfoot, C.M. Shanahan. Transcriptional regulation of matrix gla protein. Z. Kardiol. 90 (2001) s38es42. c. D.J. Rickard, I. Kazhdan, P.S. Leboy Importance of 1,25-dihydroxyvitamin D3 and the nonadherent cells of marrow for osteoblast differentiation from rat marrow stromal cells. Bone 16 (1995) 671e678. d. J.J. Chen, H. Jin, D.M. Ranly, J. Sodek, B.D. Boyan. Altered expression of bone sialoproteins in vitamin D-deficient rBSP2.7Luc transgenic mice. J. Bone Miner. Res. 14 (1999) 221e229. e. A. Gericke, C. Qin, Y. Sun, R. Redfern, D. Redfern, Y. Fujimoto, et al. Different forms of DMP1 play distinct roles in mineralization. J. Dent. Res. 89 (2010) 355e359. f. H.H. Ritchie, H. Park, J. Liu, T.J. Bervoets, A.L. Bronckers. Effects of dexamethasone, vitamin A and vitamin D3 on DSP-PP mRNA expression in rat tooth organ culture. Biochim. Biophys. Acta. 1679 (2004) 263e271. g. T. Schinke, C. Amendt, A. Trindl, O. Poschke, W. Muller-Esterl, W. Jahnen-Dechent. The serum protein alpha2-HS glycoprotein/fetuin inhibits apatite formation in vitro and in mineralizing calvaria cells. A possible role in mineralization and calcium homeostasis. J. Biol. Chem. 271 (1996) 20789e20796. h. B.R. Genge, L.N. Wu, R.E. Wuthier. Mineralization of annexin-5-containing lipid-calcium-phosphate complexes: modulation by varying lipid composition and incubation with cartilage collagens. J. Biol. Chem. 283 (2008) 9737e48. i. W.E. Horton Jr., R. Balakir, P. Precht, C.T. Liang. 1,25-Dihydroxyvitamin D3 down-regulates aggrecan proteoglycan expression in immortalized rat chondrocytes through a post-transcriptional mechanism. J. Biol. Chem. 266 (1991) 24804e24808. j. Y. Nakano, W.N. Addison, M.T. Kaartinen. ATP-mediated mineralization of MC3T3-E1 osteoblast cultures. Bone 2007 41 (2007) 549e561.
post-translationally modify them have vitamin-Dresponsive elements (VDRE) [32]. Extracellular matrix proteins that in solution or in culture affect the formation of apatitic mineral are listed in Table 21.2.
Cells control the mineralization process, both by regulating local calcium and phosphate concentrations and pH [33,34] as well as by the production and post-translational modification of collagen and
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21. MINERALIZATION
noncollagenous proteins [28], which guide and direct mineral deposition. There is also a physicochemical process by which supersaturated solutions lead sometimes to mineral deposition in unwanted sites. One example of this physicochemical effect is the hypervitaminosis D syndrome [19], where injections of vitamin D into animals cause an elevation of circulating calcium and result in arterial and kidney calcification. The Nature of Vertebrate Mineral The mineral that forms in physiologically mineralized tissues e calcified cartilage, bone, dentin, and enamel, is an analog of the geologic mineral, hydroxyapatite. The chemical formula for this mineral is Ca10(PO4)6(OH)2. With the exception of enamel, physiologic mineral is deposited in an oriented fashion on a collagen template (Fig. 21.1). Unlike geologic hydroxyapatite crystals, which are quite large and visible to the naked eye, the physiologic mineral crystals are microscopic in size, <20 nm in their longest dimension in bone, dentin, and related collagen-based tissues, and ~1000 nm in the longest dimension in enamel. Because of the small crystal size (some crystals contain only a few unit cells) and the relatively large surface area, the physiologic mineral crystals contain a large number of surface impurities and vacancies. The presence of such imperfections in bone, cementum, calcified cartilage, and dentin crystals generally reduces the crystallinity (crystal size and perfection) of the physiologic mineral deposits and tends to make the crystals more soluble than geologic hydroxyapatite [33]. This solubility is essential both to enable remodeling, and to facilitate mineral homeostasis. The mechanisms of this homeostasis are highly dependent on vitamin D metabolites as reviewed in other chapters in this section. Mineralization Mechanisms in Bone, Cartilage, and Dentin In general, body fluids are supersaturated with respect to hydroxyapatite. In other words, the ion product [Ca]10 [PO4]6 [OH]2 exceeds the hydroxyapatite solubility product of 10e58 [33]. Yet biologic apatite only deposits physiologically in the so-called “mineralized tissues.” This is because the body fluids and tissues contain numerous mineralization inhibitors. These inhibitors prevent de novo precipitation, protect the cells from becoming engulfed in mineral, and also regulate the size and shape of the crystals that do form. Mineralization inhibitors include anionic molecules that can chelate calcium, e.g., citrate, ATP, pyrophosphate, glycosaminoglycans, and proteins that bind specifically to apatite crystals, e.g., osteonectin, osteopontin, matrixgla-protein, fetuin, and albumin [25].
The deposition of mineral in bones and teeth occurs at specific sites where the barriers to crystal deposition are diminished either by elevating CaxPO4 concentrations, removing the inhibitors, or exposing matrix molecules or structures that facilitate mineral deposition. Mineral deposition can be facilitated when mineral crystals are already present, in which case there is growth and proliferation of the initial crystals, when the surface structure of the matrix matches that of the precipitating phase, or when inhibitors of mineralization are removed [25]. There are several steps in the formation of physiologically mineralized tissues. These steps are common to all the tissues, but the details and the sequence vary across tissues. The first step is the differentiation of the precursor cells (marrow stromal cells, preosteoblasts, etc.) into a mature cell. That cell then secretes a matrix that can be mineralized. For bone, dentin, and cartilage that is predominantly a collagen matrix. For enamel it is mainly an amelogenin matrix. These matrices are then post-translationally modified so that mineral deposition can be favored at specific sites along the collagen fibers (or on the amelogenin nanospheres). There is no physiologic mineral deposition until the matrix is deposited and modified. Then mineralization begins simultaneously at multiple sites in a polarized direction. The next step is the nucleation of mineral crystals. The molecular factors responsible for nucleation and the sequence in which they act are more uncertain. In some slowly mineralizing tissues, extracellular matrix vesicles provide a site for accumulating mineral ions away from inhibitors. In other tissues, mineralization appears to start on the collagen fibrils. In physiologic mineralization mineral deposition always precedes matrix formation. Extracellular matrix vesicles [36], the site of initial mineral deposition in calcifying cartilage and mantle dentin (the first site of dentin mineral deposition), provide protected sites for the accumulation of calcium and phosphate ions and enzymes involved in mineral deposition [37,38]. Their membranes are also rich in enzymes required to disrupt mineralization inhibitors in the extracellular matrix, such as ATPase, pyrophosphatase, alkaline phosphatase, and matrix metalloproteinases. However, while the first few mineral crystals might be formed within the vesicle, the majority of physiologic mineral is believed not to be formed by a vesicle-mediated process. It is also not known whether vesicle-mediated calcification is a mandatory step in the calcification process, or just one that can facilitate the process. Most physiologic mineral forms at multiple discrete locations on type I collagen fibrils [39]. Collagen itself does not support de novo apatite formation; instead, specific anionic noncollagenous proteins that associate with collagen [40e43] can bind to and stabilize the
III. MINERAL AND BONE HOMEOSTASIS
INTRODUCTION
initially formed crystals and regulate the mineralization process [25]. In solutions and in cell culture, several of these proteins have been proven to be nucleators of apatite (Table 21.2). Their effects in the presence of fibrillar collagen are quite often different from the effects in its absence [41e43]. These in vitro observations have been validated in animals in which these proteins are ablated (knocked out) or overexpressed (Table 21.3). One of the challenges associated with deciphering the mechanism of biologic mineralization is determining which proteins are absolutely essential for initiation of mineralization, and then finding the order in which these proteins are expressed in different mineralizing tissues. Also important is the modification or removal of the inhibitors of mineralization [44] by enzymatic pathways [25]. Gene microarray and proteomics studies that report the temporal expression of cartilage, bone, and tooth matrix proteins should provide some insights into these challenges.
Methods for Quantifying Tissue Mineralization Several important questions must be addressed when examining a mineralized/mineralizing tissue: Where is the tissue mineralized? How much mineral is there? Is the mineral characteristic of physiologic mineral? (Is it a poorly crystalline apatite? Is it properly aligned with the collagen matrix? Is the composition of the mineral different from that in an age- and background-matched control tissue? What size are the crystals?) Multiple techniques can be used to address these questions. These techniques are illustrated by examples from vitamin-D-related studies. Where is the Tissue Mineralized? Radiographic methods, including plain films, dual energy X-ray absorptiometry (DEXA), quantitative computed tomography (pQCT), and micro-CT, show changes in X-ray attenuation when mineral is present. For example, plain films of rachitic (vitamin-D-deficient) growing animals show enlarged epiphyses and reduced tissue density. Plain X-ray films are routinely used to reveal the presence of decreased mineralization and bowing bones, characteristic of vitamin-D-deficient osteomalacia. Vitamin D toxicity is similarly recognized radiographically as prematurely closed epiphyses and denser bones than normal in animals and humans. Because the use of plain film radiographs and DEXA to assess reduced bone mineral content are well documented [45e47], here we focus on three-dimensional quantitative methods such as pQCT and micro-CT, which can provide more detail on bone mineral content and bone density. Quantitative computed tomography (QCT) and its recently developed high-resolution peripheral
385
counterpart HR-pQCT enable in vivo measurement of both cortical and trabecular density. QCT is capable of detecting bone loss in models of vitamin-D-stimulated remodeling [48]. Tibia of vitamin D- and calciumdeficient ovariectomized sheep characterized by QCT and mechanical testing showed reduced density and mechanical strength when contrasted with ovariectomized sheep given a diet rich in calcium and vitamin D [48]. Additionally, HR-pQCT scanners with isotropic resolution ~80 mm now allow in vivo imaging of 3D trabecular morphology at peripheral sites such as the distal radius [49,50]. The primary advantage of this technique over QCT is that trabecular bone can be resolved, and morphological parameters such as bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N) can be calculated. While there are not yet reports of HR-pQCT used to study vitamin D effects, increased availability of these systems will likely increase its application to in vivo investigations of metabolic bone diseases. At the highest resolution, microcomputed tomography (micro-CT) provides 3D ex vivo characterization of trabecular microarchitecture and tissue density with isotropic resolution as small as 6 mm. Micro-CT provides bone structural data including BV/TV, Tb.Th, Tb.Sp, Tb.N, trabecular connectivity, and tissue mineral density (TMD, mass mineral/volume bone tissue), in good, but not perfect agreement with parameters measured by 2D histomorphometry [51]. Whereas whole limbs or appendicular sites can be scanned with QCT, the maximum specimen size for micro-CT is ~14 mm diameter 36 mm length. Nevertheless, micro-CT is the gold standard for ex vivo quantification of 3D trabecular morphology and validation of morphological parameters from lower-resolution techniques such as HRpQCT [52]. This technique is capable of detecting changes in both cortical geometry and trabecular microarchitecture. For example, micro-CT analysis of hypophosphatemic (Hyp) mice revealed reduced cortical thickness and reduced bone volume fraction arising from reduced Tb.N and increased Tb.Sp with no change in Tb.Th relative to femora from wild-type controls [53]. Desktop in vivo micro-CT scanners now allow assessment of the geometry and microarchitecture of the bones of small rodents over time [54]. The ability to make measurements within living animals at multiple time points will enable longitudinal studies of skeletal development, adaptation, and treatment response. How Much Mineral is there? The standard in vitro method for quantifying the amount of mineral in a tissue is gravimetric measurement of the weight of the residue left after the tissue is dried (110 C) and the organic components removed by
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386 TABLE 21.3
21. MINERALIZATION
Proteins Associated with Mineralization and Regulated by Vitamin D: Mineral Properties in Knockout (KO), Overexpression (Tg), and Mutant (Mu) Animals
Protein
Model(s)
Phenotype
Ref.
Alkaline phosphatase (tissue nonspecific (Akp2))
KO
Decreased mineral content; no detected change in crystal size
a
Amelogenin
KO, Tg
Altered enamel density, Defective enamel mineralization
31, b
Biglycan
KO
Decreased bone mineral content, larger crystals
c
Bone sialoprotein (BSP)
KO Tg
Decreased mineral content at young age; decreased bone formation in older animals Increased remodeling
d,e f
Chondromodulin
KO
Impaired endochondral ossification
g
Collagen I
Osteogenesis imperfecta (mu (h) KO, Tg)
Brittle bones; Smaller crystals
h
Dentin Matrix Protein 1 (DMP1)
Mu (h) KO Tg
Decreased mineral content Decreased mineral content, increased crystal size No effect
i,j k
Dentin sialophosphoprotein (dspp)
Dentin dysplasias (mu/h) KO Tg
Impaired dentin formation Larger bone mineral crystals
len
Matrix extracellular phosphoglycoprotein (MEPE)
Tg
Increased mineralization, decreased turnover
o
Osteocalcin
KO
Increased bone mineral content, larger crystals
113
Osteonectin
KO
Increased collagen maturity, larger crystals
p
Osteopontin
KO
Increased bone mineral content, larger crystals
25
Phospholipase A2
KO
Accelerated age related bone mass
q
MMP-2
KO
Decreased early bone formation Decreased dentin mineralization
r
H ¼ human mutation, M ¼ mouse, if not indicated data are from mice. a. H.C. Anderson, J.B. Sipe, L. Hessle, R. Dhanyamraju, E. Atti, N.P. Camacho, et al., Impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice. Am. J. Pathol. 164 (2004) 841e847. b. M.K. Pugach, Y. Li , C. Suggs, J.T. Wright, M.A. Aragon, Z.A. Yuan, et al., The amelogenin C-terminus is required for enamel development. J. Dent. Res. 89 (2010) 165e169. c. T. Xu, P. Bianco, L.W. Fisher, G. Longenecker, E. Smith, S. Goldstein, et al., Targeted disruption of the biglycan gene leads to an osteoporosis-like phenotype in mice. Nat. Genet. 20 (1998) 78e82. c. L. Malaval, N.M. Wade-Gue´ye, M. Boudiffa, J. Fei, R. Zirngibl, F. Chen, et al., Bone sialoprotein plays a functional role in bone formation and osteoclastogenesis. J. Exp. Med. 205 (2008) 1145e1153. d. J.J. Chen, H. Jin, D.M. Ranly, J. Sodek, B.D. Boyan. Altered expression of bone sialoproteins in vitamin D-deficient rBSP2.7Luc transgenic mice. J. Bone Miner. Res. 14 (1999) 221e229. e. P. Valverde, Q. Tu , J. Chen. BSP and RANKL induce osteoclastogenesis and bone resorption synergistically. J. Bone Miner. Res. 20 (2005) 1669e1679. f. J. Zhang, Q. Tu, J. Chen. Applications of transgenics in studies of bone sialoprotein. J. Cell Physiol. 220 (2009) 30e34. g. K. Yukata, Y. Matsui, C. Shukunami, A. Takimoto, T. Goto ,Y. Nishizaki, et al., Altered fracture callus formation in chondromodulin-I deficient mice. Bone 43 (2008) 1047e1056. h. R.D. Blank, A.L. Boskey. Chapter 16: Genetic collagen diseases: influence of collagen mutations on structure and mechanical behavior. In: P. Fratzl (ed.) Collagen: Structure and Mechanics. Springer Science þ Business Media, LLC, 2008, pp. 447e474, 173. i. E.G. Farrow, S.I. Davis, L.M. Ward, L.J. Summers, J.S. Bubbear, R. Keen, et al., Molecular analysis of DMP1 mutants causing autosomal recessive hypophosphatemic rickets. Bone 44 (2009) 287e294. j. Y. Ling, H.F. Rios, E.R. Myers, Y. Lu, J.Q. Feng, A.L. Boskey. DMP1 depletion decreases bone mineralization in vivo: an FTIR imaging analysis. J. Bone Miner. Res. 20 (2005) 2169e2177. k. Y. Lu, C. Qin, Y. Xie, L.F. Bonewald, J.Q. Feng. Studies of the DMP1 57-kDa functional domain both in vivo and in vitro. Cells Tissues Organs 189 (2009) 175e185. l. A.D. McKnight, P.S. Hart, T.C. Hart, J.K. Hartsfield, A. Wilson, J.T. Wright, et al., A comprehensive analysis of normal variation and disease-causing mutations in the human DSPP gene, Hum. Mutat. 29 (2008) 1392e1404. m. K. Verdelis, Y. Ling, T. Sreenath, N. Haruyama, M. Macdougall, M.C. van der Meulen, et al., DSPP effects on in vivo bone mineralization, Bone 43 (2008) 983e990. n. S. Suzuki, T. Sreenath, N. Haruyama, C. Honeycutt, A. Terse, A. Cho, et al., Dentin sialoprotein and dentin phosphoprotein have distinct roles in dentin mineralization. Matrix Biol. 28 (2009) 221e229. o. V. David, A. Martin, A.M. Hedge, P.S. Rowe. Matrix extracellular phosphoglycoprotein (MEPE) is a new bone renal hormone and vascularization modulator. Endocrinology 150 (2009) 4012e4023.
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ashing at 600 C. The ash weight, or ash density, if expressed per volume, reveals the total amount of mineral in the tissue. Chemical analysis of Ca and PO4 content has also been used to calculate mineral content, but these values may be inaccurate due to the high organic phosphate content of these tissues. Bone mineral content can also be mapped in biopsied tissue with quantitative backscattered election imaging (qBEI) in a scanning electron microscope [55]. Quantitative BEI can be used to characterize the quantity and spatial distribution of mineral in a localized region of the specimen. Because the intensity of the backscattered signal is proportional to the atomic number of the specimen, the gray level intensity values in a BSE image can be related to tissue mineral density by comparing the intensity of each pixel in the image to that of osteoid and hydroxyapatite standards, from which calcium weight percent values are calculated. This technique has been used to describe the mineral distribution in the vitamin D receptor (VDR) transgenics discussed below [56]. Finally, micro-CT can also be used to quantify the mass and 3D spatial distribution of bone mineral in addition to tissue morphology. The mass of mineral within each bone voxel can be calculated from the X-ray attenuation using calibration phantoms. The tissue mineral density (TMD) is calculated as mass mineral/volume bone tissue (g/cm3) [57]. Is the Mineral Characteristic of Physiologic Mineral (is it a Poorly Crystalline Apatite)? The gold standard for determining the presence of bone mineral (or any mineral phase) is X-ray diffraction (XRD). The X-rays incident on a powdered sample are reflected at specific angles related to the spacing between lattice planes, which are characteristic of a particular mineral phase (Fig. 21.2A). While the small crystal size and numerous imperfections in the physiologic apatite crystals make their diffraction patterns quite broad, they are still easily recognizable as apatite. Additionally, the broadening of individual peaks due to the small size and imperfections in a particular dimension can be used to calculate an average crystallite size and perfection. Information on mineral particle thickness and alignment can be obtained by scanning smallangle X-ray scattering (scanning-SAXS). SAXS detects changes in particle sizes in the range of 2e25 nm,
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while XRD detects changes less than 1 nm. The data obtained agree with the crystal size predictions generated by infrared techniques [58]. As detailed below, scanning SAXS has been used to characterize the bone mineral in the transgenic mouse that overexpresses the vitamin D receptor [56]. Transmission electron microscopy (TEM) shows the size and shape of the mineral crystals and collagen fibers, and selected area electron diffraction, analogous to X-ray diffraction, yields a characteristic pattern that can be used to quantify crystallite size and composition. The advantage of TEM is that the mineral crystals can be observed along with the collagen fibrils, and physiologic mineral lies parallel to the collagen fibers, while dystrophic mineral may not always have that appearance [59]. Both XRD and electron diffraction have proven useful for characterizing mineral associated with matrices and matrix vesicles developed in culture [60]. The amount of mineral present can neither be quantified by X-ray diffraction nor electron diffraction. This can be achieved by quantitative analysis (chemical determination by microprobe (EDAX) or other chemical analysis) or by ashing. Energy-dispersive X-ray analysis (EDAX) can provide information on the chemical composition of the surface. Coupled to scanning electron microscopes, EDAX enables observation of the morphology of the mineral deposit and simultaneous determination of Ca:PO4 ratios of the mineral using appropriate chemical standards [61]. Atomic force microscopy (AFM) [62,63], enables examination of the surface structure of a variety of materials and can be used to image individual bone crystals as well as collagen fibers. This high-resolution technique depends on the use of a probe to visualize individual crystals. However, for in situ measurements of bone crystal size, the organic matrix must be etched from the tissue surface, a challenging process that might cause breakdown of some crystals. To date there have been no AFM analyses of any of the bones or soft tissues of animals with vitamin D abnormalities. Vibrational spectroscopic techniques can provide detailed information on the structure and chemical environment of the mineral and collagen phases in bone tissue. The vibrations of atoms in molecules are determined both by the bonds in which they are located, and by the environment surrounding these moieties. Vibrations that affect the dipole moment and
=p. A.L. Boskey, D.J. Moore, M. Amling, E. Canalis, A.M. Delany. Infrared analysis of the mineral and matrix in bones of osteonectin-null mice and their wildtype
controls. J. Bone Miner. Res. 18 (2003) 1005e1011. q. S. Ramanadham, K.E. Yarasheski, M.J. Silva, M. Wohltmann, D.V. Novack, B. Christiansen, et al., Age-related changes in bone morphology are accelerated in group VIA phospholipase A2 (iPLA2beta)-null mice. Am. J. Pathol. 172 (2008) 868e881. r. R.A. Mosig, O. Dowling, A. DiFeo, M.C. Ramirez, I.C. Parker, E. Abe, et al., Loss of MMP-2 disrupts skeletal and craniofacial development and results in decreased bone mineralization, joint erosion and defects in osteoblast and osteoclast growth. Hum. Mol. Genet. 16 (2007) 1113e1123.
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Wavenumber (cm-1) (A) X-ray diffraction pattern of a highly crystalline synthetic hydroxyapatite (dotted lines) and bone mineral (solid lines) showing the major peaks used for mineral analysis. The broadening of the 002 (c-axis) peak (arrow) is routinely used to estimate crystallite size and perfection. (B) Infrared spectrum of the same samples as shown in (A), showing the bands characteristic of mineral phosphate and protein.
FIGURE 21.2
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those which are symmetric, detected by infrared and Raman techniques, respectively, can be used for quantitative analysis of mineral composition and content. As in X-ray and electron diffraction, in which the poorly mineralized biologic apatite has broad diffraction peaks, the IR and Raman spectra characteristic of physiologic apatite are also broadened (Fig. 21.2B). Analysis of the relative phosphate to protein peak area ratios (v1/amide I) in Raman; v3/amide I in IR [64] is used to calculate mineral content. Curve fitting or chemometric analysis of specific subbands of the compound spectral features can be used to reveal mineral crystal size and perfection (crystallinity [57,65e70]), acid phosphate content [66,67], and carbonate substitution [13,71]. Infrared and Raman spectroscopic analysis of mineral to matrix ratio has been shown to correlate linearly with ash weight of synthetic mixtures [64,72]. Magnetic resonance imaging (MRI) and nuclear magnetic resonance (NMR) methods can also reveal the distribution of acid phosphate and features of the mineral crystal structure. Solid-state NMR spectroscopy has long been used for 1H, 13C, and 31P studies of bone mineral and calcium phosphates [73]. Magic angle spinning (MAS) and MAS coupled with cross polarization (CP-MAS) have been used to characterize initial mineral and matrix composition in developing bones and disease models [74e79]. While there are no published studies of mineral characteristics observed by these techniques in the presence and absence of vitamin D, the strong correlation between data obtained from them, with data obtained from X-ray diffraction, spectroscopic, and chemical analyses [74], indicates that NMR data would support the findings from XRD and IR described below. Methods for Studying in Vitro Mineralization Studies in cell and organ culture have been used extensively to assess the function of vitamin D metabolites and the vitamin D receptor in the mineralization process. While it is popular to use 10 mM beta-glycerophosphate as a substrate [e.g., 81e83], as will be discussed below, 5e10 mM organic phosphate can cause ectopic mineral deposition, even in the absence of cells or matrix, as long as alkaline phosphatase activity is present [59,83,84]. Mineral can be characterized in the culture system by measuring changes in solution calcium or phosphate concentrations (often by the use of radiolabeled ions) and the amount of mineral in the matrix (chemical analysis, diffraction, or spectroscopic methods). More recently it has become very popular to quantify calcium and phosphate accumulation in culture by the use of histochemical stains. Sometimes these values are quantified by extracting the stained culture. One of the
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problems with this histochemical approach is that these stains are often not specific for mineral. For example, von Kossa staining for phosphate [61,80] can equally label phosphate containing matrix molecules, especially cell membranes [85]. Alizarin red, used to stain for calcium [61,80] can show the presence of this ion in calcium phosphates, but also calcium bound to proteoglycans. Thus conclusive proof of the presence of mineral in a culture experiment comes from diffraction methods, electron microscopy, or spectroscopic techniques as discussed above.
Direct and Indirect Effects of Vitamin D on Mineralization Physicochemical Effects Vitamin D’s “direct” effect is to increase serum calcium levels by stimulating osteoclastic resorption, kidney calcium retention, and intestinal calcium absorption (see other chapters in this section). This in turn leads to an elevation in local calcium concentration, and often an elevation in phosphate. The increased circulating levels of calcium and phosphate, while not causing marked hypercalcemia or hyperphosphatemia (because of the counter effects of parathyroid hormone), are thought to be sufficient to raise the serum levels enough to promote dystrophic calcification, especially in blood vessels [86] and kidney [87]. Injection of vitamin D3 or 1,25(OH)2 D3 into animals can cause formation of apatite deposits in articular and growth cartilage as well as bone [88]. There is also a case report showing that milk accidentally fortified with excessive vitamin D caused dental pulp calcification in a child [89], showing that mineralizing tissues other than bone or cartilage can be affected. The vitamin-D-receptor knockout mouse has an impaired bone formation phenotype (see below), that can be rescued by calcium treatment [90]. Similarly, vitamin-D-deficient osteomalacia in animals can be cured by increasing serum calcium (by lactose and calcium infusion) without addition of vitamin D [91,92]. This suggests that the increased mineralization associated with vitamin D may simply reflect increased calcium or calcium and phosphate concentrations. However the mineral composition, crystal size, and crystal perfection have not been examined in any of these calcium-treated models. From the perspective of physical chemistry, to precipitate a basic calcium phosphate such as bone apatite, the solution needs to have an appropriate pH, and sufficient calcium and phosphate concentrations. In solution, at pH 7.4, when the CaxPO4 mM2 product exceeds 5.5 mM2, precipitation will occur even in the absence of a nucleator or a template for mineral deposition
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[93]. Thus, raising the local calcium or phosphate concentration, even slightly, can cause precipitation, all other factors being equivalent. This is extremely important when considering cell culture studies where ion concentrations are elevated beyond physiologic levels. Physiologic phosphate concentrations are 0.9e1.5 mM in most species [83]. Beta-glycerophosphate, often used in mineralizing cultures, and said to enhance osteoblastic differentiation, is a substrate for alkaline phosphatase. When 10 mM beta-glycerophosphate is used in culture, the phosphate concentrations of the media can be as high as 10 mM. With physiological calcium concentrations of 1.5 mM, the CaxPO4 concentration of the solution exceeds 5.5 mM2, and hence mineral will deposit in the absence of a matrix. As shown by Khouja [84], the occurrence of this dystrophic mineralization simply demonstrates the presence of alkaline phosphatase activity, not of physiologic mineralization. Physiologic calcium concentrations are ~2.1e2.5 mM. Raising the concentration to 2.9 or 4.2 mM [67] with phosphate at even 2 mM will give a CaxPO4 product of 5.8 mM2 or 8.4 mM2, greater than the value needed for de novo precipitation. While there is altered gene expression caused by the presence of these elevated calcium concentrations [81], it has not yet been demonstrated that this altered gene expression is due to the presence of the calcium, rather than to the presence of the dystrophic mineral that surrounds the cells. We know that gene expression is altered when mineral surrounds the osteocyte [94], and in this case the mineral surrounding the osteocyte is physiologic mineral. Thus caution must be exercised when looking at gene expression in such cases. In situ there are more controls on calcium concentrations: the elevations in calcium in tissues of animals treated with vitamin D may not directly cause precipitation, but may increase the ion product sufficiently that initial mineralization is facilitated, as is crystal growth. In vitamin-D-deficient animals (with rickets and/or osteomalacia) the mineral content of the bulk tissue, reflected by histochemical stains or ash weights, is decreased relative to controls (Fig. 21.3). The crystals present in the epiphysis and metaphysis, i.e., newly formed mineral crystals, tend to be larger than those in age- and gender-matched controls. It is not certain as to whether this is because osteoclast activity is increased or because existing crystals grow at the expense of new crystals being formed in the absence of an appropriate matrix. Increased osteoclast activity has been noted in some cases of human hypophosphatemic oncogenic osteomalacia [95,96] and in vitaminD-deficient animals [97,98]. However, the effects of vitamin D metabolites on extracellular matrix formation and composition suggest that impaired formation may be a contributing factor.
Effects of Vitamin D on Cells and Matrix Molecules INTRODUCTION
As reviewed elsewhere in this volume, vitamin D is a differentiation factor, facilitating the expression of mature chondrocyte and mature osteoblast activity, as well as osteoclastogenesis. Vitamin D alters the gene expression and protein synthesis of numerous matrix proteins that are regulators of biomineralization (Table 21.2). This regulation occurs through both genomic and nongenomic pathways and is modulated by calcium influx. The vitamin-D-regulated proteins include calcium-binding proteins and several enamel (e.g., enamelins, amelogenins), bone, and dentin matrix proteins (e.g., dentin sialoprotein (DSP) and dentin phosphoprotein (DPP)). Vitamin D also increases expression of membrane-bound enzymes such as alkaline phosphatase and the kinases and phosphatases [86] that regulate the phosphorylation and dephosphorylation of intracellular and extracellular matrix proteins. The effects of vitamin D metabolites on cell differentiation are reviewed elsewhere in this book. However, in terms of mineralization, it is important to note that in vitro 24,25-(OH)2 vitamin D stimulates chick chondrocyte [99] and rabbit osteoblast maturation [100], while 1,25-(OH)2D stimulates rat osteoblast differentiation in bone marrow stromal cell cultures [101,102]. Since it is the mature chondrocyte and osteoblast that synthesize the mineralizable matrix, and/or provide the enzymes necessary to modulate that matrix to facilitate mineralization, one indirect effect of vitamin D might be related to the differentiation and proliferation of the cells responsible for controlling mineralization. In cultured chick hypertrophic chondrocytes, for example, 1,25(OH)2D3 causes a concentration-dependent decrease in activity of alkaline phosphatase, while 24,25 (OH)2D3 stimulates this activity [103]. The effect of 1,25(OH)2D3 in stimulating kinase and phosphatase activity, and thereby regulating the extent of phosphorylation of osteopontin, is extremely interesting in light of data showing that the moderately phosphorylated bone osteopontin is an effective mineralization inhibitor [104]. In contrast, the highly phosphorylated milk osteopontin can induce hydroxyapatite formation in solution, and stabilize precursors of hydroxyapatite [105]. Several of the other matrix proteins’ activities depend on their state of phosphorylation, highlighting the importance of the phosphorylation process. THE INFLUENCE OF VITAMIN D ON BONE MATRIX PROTEINS
Vitamin D modulates the production of several molecules (Table 21.2) that are crucial for the synthesis and function of bone tissue, although there is no clear evidence that direct effects of vitamin D are required
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FIGURE 21.3 Mineral analyses in models of vitamin D deficiency. (A) Vitamin-D-deficient rats. Male Sprague-Dawley rats (21 days old) were maintained in the dark on a vitamin-D- and phosphate-deficient diet for 3 weeks. The first panel shows their serum Ca and PO4 levels. 1,25(OH)2vitamin D levels were not detectable in the vitamin-D-deficient animals. Tibias removed at sacrifice were used for analysis of ash weight and b002 (1/crystallinity). Reproduced with permission from Springer-Verlag [119]. (B) Female mice with hypophosphatemic rickets (Hyp) and their age- and sex-matched controls were 35 days old at sacrifice. Femora were used for analysis of ash weight, b002, and Ca/PO4 ratio of the ash. Reprinted with permission from Elsevier [117]. (C) Second-generation vitamin-D-deficient rats and their age-matched controls were 7 weeks old at sacrifice. Femora were used for analysis of ash weight and b002 and Ca/PO4 ratio of the ash. Reprinted with permission from the second edition of this book.
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for normal bone mineralization. The actions of these proteins have been described in detail elsewhere [25], hence only a few representative proteins are described in this section. Readers are referred to the references in Tables 21.2 and 21.3 for more detail. COLLAGEN The scaffold of bone matrix is made predominantly of type I collagen molecules. These molecules are produced early in the bone tissue formation, and their special morphology supports the mineral deposition in later stages. In addition, the collagen molecules provide the elasticity characteristic of the bone tissue, which is essential for the normal function in response to mechanical forces. The synthesis of collagen is a complex process, and it is regulated by several factors including vitamin D. The active metabolite 1,25(OH)2D3 down-regulates the transcription of a1(I) collagen by osteoblasts in a variety of cells [106,107]. This decrease in type I collagen expression appears to be cell-stage-dependent, occurring in early bone nodules formed in culture, but not in intermediate and mature ones [106]. Adding 1,25(OH)2D3 after the completion of extracellular matrix formation has been reported to inhibit further mineralization in chick osteoblast cultures [107], suggesting that 1,25(OH)2D3 affects the metabolic processes by which osteoblasts control mineralization independent of its effects on the formation of the collagenous matrix. However, Matsumoto showed that 1,25(OH)2D3
stimulated alkaline phosphatase activity in both early and late osteoblast cultures, and reported enhanced mineral deposition based on EM and 45Ca uptake when 10e10 M 1,25(OH)2D3 was added after the start of mineralization [108]. Similarly (Fig. 21.4), in differentiating chick limb-bud mesenchymal cell micro-mass cultures, 1,25(OH)2D3 added continuously to cultures after the matrix had been formed and mineralization was started (day 9), increased the rate of mineral accumulation compared to mineralizing cultures that did not receive exogenous vitamin D. This increase was in contrast to cultures to which vitamin D was added continuously while cells were differentiating (day 5) or when the cells were first beginning to deposit a cartilage matrix (day 7). Vitamin D also regulates the expression of the noncollagenous proteins in mineralized tissues (Table 21.2). Many of these proteins are believed to play direct roles in the regulation of biomineralization [5,25], and thus they account in part for the indirect effect of vitamin D on mineralization. NONCOLLAGENOUS PROTEINS The expression of bone sialoprotein (BSP), an in vitro apatite nucleator [109] and mineralization regulator [25], is suppressed by addition of 1,25(OH)2D3 to osteoblast cultures [82,107, 110]. In osteoblast cultures without exogenous vitamin D supplementation, BSP is expressed at its highest levels immediately prior to onset of extracellular matrix
0.50 C 0.40
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FIGURE 21.4 Effect of 10e8M 1,25(OH)2D3 on mineralization in differentiating chick limb bud mesenchymal cell micromass cultures. Data
are expressed as mg Ca/mg DNA. Continuous vitamin D treatment was started either at day 5 (when cartilage nodules formed), at day 7 (prior to the start of visible chondrocyte hypertrophy), or at day 9 (2 days before start of mineralization). Control cultures received a comparable volume (20 ml) of ethanol, the carrier for 1,25(OH)2D3. On days 12e21, Ca and DNA contents were determined in control and mineralizing cultures. The DNA contents of mineralizing and nonmineralizing cultures were not significantly different (not shown), and 1,25(OH)2D3 treatment did not significantly accelerate proliferation. There was a slight increase in DNA content of cultures treated on day 5, but neither this increase nor the gradual increase in DNA content with time was significant, indicating that the cultures had already achieved their plateau phase of proliferation. In contrast, cultures treated on day 9 showed an increase in Ca content relative to the other cultures.
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mineralization [110]. Thus its suppression in the presence of 10e8M 1,25(OH)2D3 may reflect a compensatory mechanism, preventing excessive initial mineralization. Osteocalcin (OCN) was one of the first matrix proteins whose expression was shown to be upregulated by vitamin D [111]. OCN is a member of a large family of hepatic and skeletal vitamin-K-dependent proteins which undergo post-translational modification and g-carboxylation at key glutamic acid residues (Gla), and have mineral-binding capacities. The mature 5.7 kDa OCN protein contains three Gla residues and accumulates in bone as a result of its high affinity for hydroxyapatite [25]. Initial osteoblastic expression of OCN occurs after the onset of extracellular matrix mineralization and increases with progressive mineralization and maturation of the osteoblast to a terminally differentiated state [82]. OCN can inhibit the formation of hydroxyapatite in vitro [112], a function that requires the presence of the Gla residues [113]. OCN also is important for osteoclast recruitment [25]. OCN-deficient mice have increased rates of bone formation despite a lack of detectable abnormalities in osteoclast numbers [25]. These mice also have increased mineral content and increased mineral crystallinity [113], supporting the role of OCN in regulating mineral turnover. Thus OCN has a role in the recruitment of osteoclasts to the surface of mineralized bone, contributing in this way to the regulation of both bone formation and resorption [110]. When 1,25(OH)2D3 is added to rat osteoblast cultures before the start of mineralization OCN biosynthesis is suppressed throughout the life span of the cultures, as a result of an arrested stage of cell differentiation. Once the cultures begin to mineralize and the cells are terminally differentiated, media OCN concentrations can be increased by continuous or acute application of 1,25(OH)2D3 [114]. Osteopontin (OPN), a glycoprotein produced in a number of tissues [25], is consistently up-regulated by 1,25(OH)2D3 in proliferating and differentiated mouse and rat osteoblasts [115]. In bone, OPN mediates autocrine and paracrine functions in the regulation of tissue formation. OPN is important for recruiting osteoclasts for bone remodeling [115] and it acts as a signaling protein in many tissues. OPN is an in vitro inhibitor of mineralization in muscle, cartilage, and bone [25,115]. Many of these effects may be mediated by the effects of vitamin D on phosphate transport, reviewed elsewhere [116].
Mineralization and Mineral Properties in Model Systems with Vitamin D Alterations The effects on mineralization of vitamin D, 1,25 (OH)2D3 and other vitamin D metabolites vary with dose and type, as well as with the system being studied. Even in similar species, the effects depend
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on animal age, gender, route of delivery, and method of analysis. In cell culture, results are also dependent on the maturity of cells or tissues, concentration of metabolite, and whether the metabolite is given acutely or continuously. It is well known that vitamin D deficiency, or even vitamin D insufficiency causes rickets (failure of growth plate cartilage to become mineralized) and osteomalacia (failure of osteoid to become mineralized) [116,117]; and that vitamin D toxicity (hypervitaminosis D) causes dystrophic calcification [118]. But even in rodents the effects of vitamin D deficiency (or insufficiency) and hypervitaminosis D can be variable. Moreover, in cell and organ culture the same vitamin D metabolites have been reported both to prevent mineralization and to enhance it. Understanding the reasons for this diversity can provide a good deal of insight into mineralization mechanisms.
Animal Models and Human Diseases with Mineral Alterations Vitamin D Deficiency Animal models of vitamin D deficiency may arise from either dietary intervention or genetic alteration. Examples of the former include the vitamin-Ddeficient rat [119] and the second-generation vitaminD-deficient rat, born to vitamin-D-deficient mothers [120], while the latter category includes the hypophosphatemic (HYP) mouse [53]. The animals in all of these models have negligible 1,25(OH)2D3 and reduced 25 (OH)D3 levels. Across all three models, the mineral content is decreased and the crystal size is increased relative to age-matched controls, but Ca/P ratios are quite variable (Fig. 21.3). The effect of first- vs. second-generation vitamin D deficiency is also inconsistent. In the second-generation vitamin-D-deficient rats, there are only small differences in bone ash weight, crystallinity, and Ca/P ratios when compared to age-matched controls [119]. These differences are greater in comparably aged, comparable gender and background, first-generation vitamin-D-deficient animals [120]. The initial electron microscopic studies of vitamin-Ddeficient bones [121] revealed that the total number of extracellular matrix vesicles was not different from that in the vitamin-D-sufficient rats, but the vesicles contained less mineral, and an extracellular matrix devoid of mineral. Healing of the rickets by treatment with vitamin D and phosphate was then associated with both matrix vesicle and extracellular matrix mineralization. In another classic study, EDAX was used to monitor changes in cellular and extracellular matrix ion concentrations in all zones of normal and rachitic growth plates [122]. Potassium levels were invariant, and calcium was
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found throughout the extracellular matrix in both normal and rachitic growth plates. The phosphate levels in the extracellular matrix did not increase until mineralization commenced, and hence was lower in the rachitic matrix, leading the authors to point out the importance of phosphate sufficiency for mineralization. Since vitamin D is known to regulate calcium and phosphate levels, these observations are no longer surprising, but are important in terms of the mineralization mechanisms discussed previously. In the hypophosphatemic mouse [53], in which failure to mineralize the growth plate and osteoid is due to a genetic abnormality in the endopeptidase known as PHEX (see Chapter 42), the Ca/P ratios are elevated probably due to abnormalities in renal phosphate transport. In addition, there is less bone mineral and the mineral crystals, which are present, tend to be larger and more perfect. A mechanism can be postulated to explain these mineralization abnormalities in hypophosphatemic animals’ bones: vitamin D may regulate mineralization by controlling PHEX-dependent [125] degradation of matrix proteins. This suggestion is based on the knowledge that a potential substrate for PHEX is matrix extracellular phosphoglycoprotein (MEPE) [123,124], and by analogy, the structurally related phosphorylated matrix glycoproteins, OPN, BSP, and DMP-1, and the finding that vitamin D causes down-regulation of PHEX expression [125]. Levels of FGF23, which regulate renal phosphate transport, are also regulated by PHEX [126]. In other words, vitamin D regulates the breakdown of multiple PHEX substrates, altering their effects on mineralization [127,128]. Although we know that many of these phosphoproteins can act both as nucleators and inhibitors [25], the detailed mechanism of the vitamin D PHEX effect is hypothetical and remains to be validated. The mineral properties of vitamin D insufficiency in human children have not been characterized. However mineral in adults with osteomalacia has been subject to more detailed analysis (see below). VDR Alterations The nuclear vitamin D receptor (VDR) binds 1,25 (OH)2D with high affinity and selectivity, as reviewed in Section II. Animals lacking VDR have normal circulating levels of vitamin D and reduced levels of 1,25 (OH)2D [129,130]. They have slight hypocalcemia and elevated PTH levels due to the inability of the vitamin D target organs to regulate calcium influx. The histologic appearance of the bone tissue of vitamin D receptor knockout (VDR-KO) animals resembles that of an animal with vitamin D deficiency [90]. The mice have a normal skeleton at birth but develop hypocalcemia and hyperparathyroidism shortly after weaning, comparable to human rickets type II [129,130]. While bone
volume is not decreased in these mice, the amount of unmineralized bone (osteoid) is increased 15-fold compared to the controls [129]. Femoral stiffness and strength are substantially reduced, by ~75% and 80%, respectively, in 10-week-old VDR-KO mice [131]. The rachitic malformation, growth retardation, and impaired structural performance in the bones of the VDR-KO animals can be rescued by dietary calcium supplementation or phosphorus restriction [131], or with high-dose vitamin 1,25(OH)2D3 [132]; however, while the vitamin D replenishment corrects the mineralization deficit, it does not alter PTH levels [132]. Prior to rescue, the VDR-KO animals have lower mineral content in their bones as compared to the wild-type and rescued KO [132], although their bone geometries are not altered, even during gestation and lactation. The VDR-null animals’ dentin is also undermineralized, while their enamel matures more rapidly relative to those of wildtype controls [133]. The VDR-KO animals have extended growth plates and an increased number of hypertrophic chondrocytes [129]. The hypertrophic chondrocytes express the correct relative amounts of phenotypic markers of chondrocyte maturity, type X collagen, vascular endothelial growth factor (VEGF), and osteopontin, but show decreased staining for annexin V-phosphatidyl serine, an early marker of apoptosis [134]. This suggests that impaired mineralization might be due to the failure of chondrocytes to undergo apoptosis, but another possibility is that the calcium and phosphate levels are not sufficient to mineralize the growth plates, with apoptosis following rather than preceding mineralization. Additionally, both annexin and phosphatidyl serine are membrane components of extracellular matrix vesicles; thus, decreased staining for these components might suggest that chondrocytes are not producing vesicles, or that vesicles, lacking these components, are not functional. Osteoclast numbers are normal in the VDR knockout [90] arguing against excessive remodeling as the origin of the mineralization defect. More is known about the mineral characteristics in the vitamin D receptor transgenics. Gardiner et al. [135] conditionally overexpressed the vitamin D receptor under control of the osteocalcin promoter. The long bones in these VDR transgenic mice have increased cross-sectional area and increased strength with twoand threefold elevations of osteoblast VDR levels [135]. Based on qBEI analysis [65], cortical bone peak mineral content (CaPeak) was enhanced in a dose-dependent manner with the mice with 3 overexpression having values higher than controls (p < 0.02), and the transgenic mice with 2 overexpression having intermediate values. In trabecular bone, the increase in CaPeak was significant for both two- and threefold overexpression. Calcified cartilage and primary spongiosa showed
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FIGURE 21.5 Infrared microspectroscopy of the effect of threefold VDR overexpression on the tibia of 4-month-old mice shows slight but not significant increase in mineral content (mineral:matrix ratio). Data were collected from cortical bone on the periosteal side (CP), in the center (CC) and on the endosteal side (CE) as well as within individual trabeculae (T). Reprinted with permission from the second edition of this book.
parallel trends. There was also increased homogeneity of mineralization in the transgenic mice. However, FTIR microspectroscopy, XRD, and scanning-SAXS failed to show any significant alterations in the mineral crystal properties of the transgenic bones, although there was a trend for the mineral content (mineral: matrix ratio) to be increased in the transgenic mice (Fig. 21.5) while crystallinity was identical in both groups [55]. Thus VDR overexpression did not markedly change the mineral properties and did not mimic effects of vitamin D toxicity. In humans, hereditary 1,25(OH)2D-resistant rickets (HVDRR), known as vitamin-D-dependent rickets type II, is a rare autosomal recessive disease that arises as a result of mutations in the gene encoding the VDR [136e138] (see Chapter 65). There have been no detailed mineral analyses in these patients but one would predict they would be similar to those changes in the VDR-KO mice. Vitamin D Carrier Protein Knockout Vitamin D is carried to its receptor (VDR) in the target tissues by the vitamin-D-binding protein (DBP). The mineral properties in the DBP knockout mice have not been reported, but they have an interesting bone phenotype [139]. The DBP knockout mice have low levels of serum vitamin D metabolites but otherwise appear normal and show none of the bone abnormalities seen in vitamin-D-deficiency rickets or osteomalacia. When stressed with excess exogenous vitamin D (1000 U/g body weight) to induce vitamin D toxicity, they had elevated serum calcium levels, but did not show the toxic effects found in wild-type
395
animals. Kidney mineral deposits were found in the wild-type but not in the DBP knockout mice. Additionally, when stressed with a diet low in vitamin D, histomorphometry showed increased thickening of the osteoid seams in the DBP knockout mice, consistent with impaired mineralization and “hypovitaminosis D osteopathy.” Recently, Zella et al. [140] demonstrated that while the concentration of 1,25(OH)2D in the blood of these animals was reduced, the concentration of this metabolite in their tissues was not significantly altered, and no significant alterations in targeted gene expression were detected. This important observation means that the individual tissues can maintain sufficient levels of 1,25(OH)2D3D for all key processes. However, since an analog of 1,25(OH)2D3 stimulates osteoblast activity in vitro and in ovariectomized rats [141], there is an argument for a direct effect of vitamin D on bone cells beyond the increase in calcium and phosphate levels discussed above. The stimulation in the rats occurs in periosteal bone, and significantly increases the strength of the bones relative to the controls [141]. The altered properties of the bones formed with this new analog appear to go beyond the stimulation of matrix protein synthesis, but the detailed properties of the mineral in animals treated with this drug are not known. The 1a-Hydroxylase Knockout Patients that lack the 1a-hydroxylase have secondary hyperparathyroidism, growth retardation, rickets, and osteomalacia [142]. Mice that lack the 1ahydroxylase also present with hypocalcemia, rickets, and osteomalacia [143], and mimic human pseudovitamin-D-deficiency rickets (PDDR). Although the phenotypes are similar to the VDR-KO animals, serum levels of vitamin D metabolites are different in the 1ahydroylase KO animals, which have undetectable 1,25 (OH)2D levels [144]. The mineral in the bones of the PDDR mice has not been characterized, but their mechanical properties are typical of rachitic animals with significantly reduced stiffness and reduced ultimate load [145]. Treatment of the 1a-hydroxylase-KO animals with high-calcium, -phosphate, and -lactose diets rescues the phenotype, but the bones do not grow as well as the VDR-KO mice given the same diet [145], implying that the 1,25(OH)2D may have properties beyond regulating serum calcium and phosphate levels. Similar to the VDR-KO mice, there are no differences in osteoclast number in the control and knockout mice given the normal or the enriched diet, suggesting that the reduced mineral content arises from decreased formation rather than from elevated resorption. Of interest, these 1a-hydroxylase-deficient mice can also be rescued by treatment with high-dose (10 or 20 the normal mouse requirement) vitamin D.
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Mice rescued with high-dose vitamin D had complete recovery of cortical bone as evidenced by micro-CT, but only partial recovery of trabecular bone. Such data indicate that high serum 25(OH)D can influence Ca and bone metabolism independent of its conversion to 1,25(OH)2D [146]. Osteoporosis and Osteomalacia in Humans Many individuals with osteoporosis are Cadeficient, demonstrate vitamin D insufficiency, and show histochemical evidence of osteomalacia [147,148]. Osteoporotic bones without evidence of osteomalacia have variably modified mineral content and consistently increased mineral crystallinity based on FTIR microspectroscopic analysis [149e152]. Since it is likely that some of these osteoporotic patients could have osteomalacia, we tested the hypothesis that the mineral in osteomalacic bone was different from that in normal bone. In tissue sections from female patients with osteomalacia (n ¼ 11, age 22e72), mineralization rates determined by histomorphometry were decreased relative to controls (n ¼ 7, age 36e57), while osteoid fraction was increased, revealing defective primary mineralization. In those sections, the degree of mineralization measured by qBEI in old bone (calcified tissue) was slightly higher than in four of the controls, reflecting the normal evolution of the secondary mineralization of bone tissue. FTIRI analysis of the biopsies [153] showed decreased mineral content in both cortical and trabecular bone of the osteomalacic patients and no differences in mineral crystallinity or collagen maturity (Fig. 21.6). These findings support the hypothesis that the reduced strength of osteomalacic bone originates in delayed primary mineralization, and reduced mineralized tissue volume.
FIGURE 21.6 Infrared imaging comparing the mean mineral
content (mineral:matrix ratio) and crystallinity in normal female patients to female patients with osteomalacia. Values are mean SD (n ¼ 7 and 4, respectively) as averaged from 3e5 fields from each biopsy. Reprinted with permission from the second edition of this book.
CONCLUSIONS Vitamin D affects mineralization predominantly by regulating local calcium and phosphate levels. Additionally, because vitamin D has genomic and nongenomic effects on the mineralizing tissues’ cells, vitamin D’s indirect actions on membrane properties, enzyme activity, and matrix protein expression and phosphorylation can affect the mineral that is formed in its presence or absence. Unfortunately there is a limited amount of information on the mineral properties in most of the new animal models in which mineralization is altered. Hopefully this chapter will continue to encourage analyses of those tissues.
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C H A P T E R
22 Vitamin D Regulation of Type I Collagen Expression in Bone Barbara E. Kream 1, Alexander C. Lichtler 2 1
Departments of Medicine, the University of Connecticut Health Center, Farmington, Connecticut 06030, USA, Reconstructive Sciences, the University of Connecticut Health Center, Farmington, Connecticut 06030, USA
2
INTRODUCTION Vitamin D has multiple functions in humans and animals [1e3]. It is probably best known as a nutrient required for adequate growth and mineralization of bone. Vitamin D, like parathyroid hormone (PTH), is an important calcium-regulating hormone. PTH is primarily responsible for the acute physiologic maintenance of serum calcium levels. In the presence of prolonged hypocalcemia, PTH increases the renal production of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D. 1,25(OH)2D3 together with PTH, normalizes serum calcium levels by increasing intestinal calcium transport, bone resorption, and renal calcium reabsorption. To carry out many of its biological actions in its target cells, 1,25(OH)2D3 binds with high affinity to a nuclear receptor, the vitamin D receptor (VDR), which in turn binds to DNA promoter elements and recruits coactivators to regulate gene transcription [4e6]. The mechanisms by which 1,25(OH)2D3 affects bone mineralization have been the subject of intense study. Some of the most well-recognized features of vitamin D deficiency are undermineralized bone in children (rickets) and adults (osteomalacia), a reduction in bone matrix formation [7] and mineralization [8] and an alteration in the pattern of collagen crosslinking [9]. Calcium deficiency also decreases bone formation and mineralization in rats [10]. Defective bone formation and mineralization in vitamin-D-deficient rats can be largely corrected by the administration of calcium and phosphate, suggesting that the trophic effect of vitamin D on the skeleton may be due to its ability to stimulate intestinal calcium absorption [11e13]. Accordingly,
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10022-8
preservation of mineral homeostasis in VDR-null mice reverses the abnormal skeletal phenotype (including excessive osteoid production) seen in these animals, suggesting that a key effect of vitamin D on bone mineralization may be due to its stimulation of intestinal calcium absorption [14]. The discovery of high-affinity 1,25(OH)2D3 receptors (VDRs) in cytosolic extracts of embryonic chick and fetal rat calvariae over three decades ago suggested that 1,25 (OH)2D3 also has direct effects on osteoblast function [15,16]. One notable effect of 1,25(OH)2D3 action in bone is its ability to stimulate osteoclastic bone resorption [17]. 1,25(OH)2D3 increases osteoclast formation and bone resorption by signaling in cells of the osteoblast lineage [18]. 1,25(OH)2D3 increases the expression of macrophage-colony-stimulating factor, which enhances proliferation and differentiation of osteoclast precursors, and receptor activator of nuclear factor-kappaB ligand (RANKL), which increases osteoclast differentiation and survival [18,19]. In addition to its effect on osteoclast formation and resorption, 1,25(OH)2D3 regulates the expression of matrix proteins in osteoblasts, including type I collagen [20], osteocalcin [21], and osteopontin [22]. Type I collagen is the most abundant protein in the body and comprises at least 90% of the organic component of the bone matrix. The biochemistry, molecular biology, and hormonal regulation of collagen genes have been extensively reviewed [23e28]. Type I collagen is produced at high levels by differentiated osteoblasts and is required for the formation of the mineralized bone matrix. Many other cell types synthesize and secrete type I collagen, although in lesser amounts. Each type I collagen molecule consists of three polypeptides; two a1(I) chains
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and one a2(I) chain. These polypeptides are encoded by separate genes (Col1a1 and Col1a2, respectively) expressed in a 2:1 ratio [29]. Collagen synthesis in bone is modulated by a variety of hormones, growth factors, and cytokines, some of which are produced locally by osteoblasts [28,30e32]. Insulin [33], insulin-like growth factor [34], and transforming growth factor-b [35] increase type I collagen synthesis whereas PTH [36], interleukin-1 [37], and tumor necrosis factor [37] are inhibitory. Glucocorticoids [38] and prostaglandins [39] can be either stimulatory or inhibitory in vitro depending on the model and culture conditions. Because of the critical role of type I collagen in maintaining the structure and function of the skeleton, it is important to understand the mechanisms by which 1,25(OH)2D3 regulates type I collagen expression. An overview of the effects of vitamin D on bone biology has been recently reviewed [3].
REGULATION OF BONE COLLAGEN SYNTHESIS Collagen synthesis in organ cultures of rodent calvariae and cell cultures has been assessed using several different assays [40,41]. In the most widely used assay, calvariae and cells are incubated with radiolabeled proline for several hours prior to the end of culture. The incorporation of radiolabeled proline into collagenase-digestible protein (CDP labeling) and noncollagen protein (NCP labeling) is measured in extracts of the cultures using highly purified bacterial collagenase [42]. The percent collagen synthesis is calculated from the CDP and NCP values after correcting for the greater abundance of proline in collagen relative to noncollagen proteins [43]. Collagen production can also be determined by measuring the hydroxyproline content of cell or organ cultures, since hydroxyproline is virtually unique to collagens. These methods do not distinguish between different types of fibrillar collagen. However, the collagen synthesized by bone organ cultures and most osteoblastic cell cultures is largely type I (>95%) so that the CDP labeling value usually reflects type I collagen synthesis. If desired, the production of different collagen types can be distinguished by ion-exchange chromatography and polyacrylamide gel electrophoresis of radiolabeled extracts of cell or organ cultures [44,45]. Type I collagen expression in human cell cultures has also been assessed by measuring secretion of the procollagen I C-terminal propeptide [46,47]. Finally, specific cDNA probes in Northern blotting and allele-specific primers in reverse transcriptase-polymerase chain reaction assays have been used to assess collagen mRNA expression in bone models. The measurement of the effect of 1,25(OH)2D3 on collagen synthesis and mRNA
levels has given comparable results using these different assays. 1,25(OH)2D3 inhibits collagen synthesis in organ cultures of 21-day fetal rat calvariae [20] and neonatal mouse calvariae [48] with little or no effect on noncollagen protein synthesis. Maximal inhibition of collagen synthesis by 1,25(OH)2D3 in rat calvariae (about 50%) occurs at 10 nM [20]. 1,24R,25-(OH)3D3 also inhibits collagen synthesis but is less potent than 1,25(OH)2D3 [20]. 25-(OH)D3 and 24R,25(OH)2D3 do not alter collagen synthesis below 100 nM [20,48]. Vitamin D metabolites inhibit collagen synthesis and stimulate resorption of fetal rat long bones with similar relative potencies that correlate with the affinity of the metabolites for the skeletal VDRs [49]. To determine the cell selectivity of the 1,25(OH)2D3 inhibition of collagen synthesis, organ cultures of fetal rat calvariae were treated with 1,25(OH)2D3 for 22 h and then incubated with tritiated proline for the final 2 h of culture. The central bone (mature osteoblasts) was dissected free of the periosteum (less mature osteoprogenitors and fibroblasts) and both compartments were analyzed separately for the incorporation of tritiated proline. 1,25 (OH)2D3 decreases collagen synthesis in the central bone but not the periosteum, indicating selectivity of the 1,25(OH)2D3 effect for mature osteoblasts [50,51]. Using an in vivo protocol in which neonatal rats were given multiple injections of tritiated proline to radiolabel newly synthesized bone matrix, 25 ng of 1,25 (OH)2D3 given on days 1, 3, and 5 inhibited bone matrix synthesis as assessed by histomorphometry of autoradiographs of tibia and calvariae [52]. 1,25(OH)2D3 also inhibits collagen production in rat osteoblastic osteosarcoma ROS 17/2.8 cells [53], primary rat [54,55] and mouse osteoblastic cells [56], and an immortalized murine osteoblast cell line (MMB-1) [57]. 1,25(OH)2D3 has a greater inhibitory effect on type I collagen synthesis during log phase growth of primary murine osteoblastic cells than at confluence, perhaps because proliferating cells contained more VDRs [58]. Likewise, 1,25(OH)2D3 inhibition of collagen synthesis is greater in sparse cultures of MMB-1 cells that have higher VDR levels than confluent MMB-1 cells [59]. 1,25(OH)2D3 inhibition of collagen synthesis is equivalent in sparse and confluent rat primary osteoblastic cells [54], but VDR number did not change during growth of the cells [60]. Taken together, these data show that the extent of inhibition of collagen synthesis by 1,25(OH)2D3 is largely determined by the cellular quantity of VDRs. 1,25(OH)2D3 inhibits collagen mRNA levels during the proliferative phase of longterm cultures of rat primary osteoblastic cells [61] and prevents the formation of mineralized bone nodules by these cultures [61,62]. These studies show that 1,25 (OH)2D3 inhibits the differentiation of osteoprogenitors
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MOLECULAR MECHANISMS OF REGULATION
that form mineralized nodules in primary rat osteoblastic cell cultures [62]. However, the inhibition of nodule formation by 1,25(OH)2D3 may be secondary to the suppression of type I collagen synthesis in the cultures. In contrast to the inhibitory effects described above, 1,25(OH)2D3 transiently stimulates collagen and noncollagen protein synthesis (about twofold), which peaks between 12 and 24 h, in the immortalized murine osteoblastic cell line MC3T3-E1 [63]. In this study, the percent collagen synthesized by the cultures (collagen relative to total protein synthesis) was not reported; as a result, it was not possible to determine the selectivity of the 1,25(OH)2D3 effect for collagen synthesis. 1,25(OH)2D3 also increases collagen expression in the human osteoblastic osteosarcoma cell line MG-63 [46,64,65] and primary cultures of human osteoblastic cells [66]. Interestingly, the increase in collagen synthesis by 1,25 (OH)2D3 in MG63 cells is blocked by insulin-like growth factor binding protein 5, which interacts directly to the VDR and prevents heterodimerization with the retinoid X receptor RXR [65]. However, in other studies, 1,25 (OH)2D3 has been shown to decrease the percent collagen synthesis in MC3T3-E1 cells [67,68]. MC3T3-E1 and MG-63 represent preosteoblastic cells that undergo in vitro osteogenic differentiation with ascorbic acid; 1,25(OH)2D3 inhibits cell growth and increases osteocalcin expression and alkaline phosphatase activity in both cell lines. MC3T3E1 cells, like most immortalized osteoblastic cell lines, display significant phenotypic variation [69]. Therefore some of these discrepant results may be due to variations in the cells used for the experiments. Collectively, these data suggest that 1,25(OH)2D3 may act as a differentiating hormone in early cells of the osteoblast lineage, which results in increased type I collagen expression. In contrast, 1,25(OH)2D3 inhibits type I collagen expression in mature osteoblasts.
MOLECULAR MECHANISMS OF REGULATION Some early studies showed that 1,25(OH)2D3 represses collagen synthesis in mature osteoblasts at a pretranslational level [50]. Measurements of procollagen mRNA activity by translation of total RNA in a reticulocyte lysate first showed that 1,25(OH)2D3 inhibited collagen mRNA in the osteoblast-rich central bone but not the periosteum of 21-day fetal rat calvariae [50]. 1,25(OH)2D3 at 10 nM inhibited procollagen mRNA activity at 6 h; maximal inhibition of about 50% occurred at 24 h [50]. A single subcutaneous injection of 1,25 (OH)2D3 (1.6 ng/g body weight) also decreased procollagen mRNA activity in calvariae [50]. Subsequently, specific cDNA probes were used to show that 1,25
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(OH)2D3 inhibited Col1a1 mRNA levels in ROS 17/2.8 cells [53], primary rat [55], and chick calvarial osteoblastic cells [70,71]. Nuclear run-on assays in ROS 17/2.8 cells showed that 1,25(OH)2D3 represses Col1a1 and Col1a2 mRNA levels by a transcriptional mechanism [72]. 1,25(OH)2D3 at 1 and 10 nM decreased the rate of Col1a1 and Col1a2 transcription by about 50%, similar to its effect on collagen synthesis and type I collagen mRNA levels, while actin and tubulin transcription were unaffected. 1,25(OH)2D3 repressed Col1a1 and Col1a2 transcription as early as 4 h with maximal inhibition at 24 h [72]. DNA motifs that mediate stimulatory effects of 1,25 (OH)2D3 on gene expression have been well characterized for several genes [6,73,74]. Vitamin-D-responsive elements (VDREs) that mediate 1,25(OH)2D3 induction of target genes such as human [75] and rat [76] osteocalcin, mouse osteopontin [77], rat 24-hydroxylase [78], and rat calbindin D-9K [79] contain two perfect or imperfect direct hexameric repeats of the consensus AGGTCA motif separated by three spacer nucleotides [6,73,74]. The consensus VDRE binds a heterodimer of the VDR and the retinoic acid X receptor (RXR) [80]. Negative promoter elements have also been identified. The negative VDRE in the avian PTH promoter is analogous to the consensus VDRE, since it contains two imperfect direct repeats separated by three spacer nucleotides and binds VDR and RXR [81]. In contrast, the negative VDRE in the human PTH gene contains a single AGGTTC motif, and binding of the VDR to this site does not require RXR [82,83]. The negative VDRE of the parathyroid-hormone-related protein (PTHrP) gene contains two potential VDREs, one similar to the negative VDRE in the human PTH gene and another identical to the stimulatory VDRE; both motifs bind the VDR [84]. To characterize the regions of the Col1a1 gene that are involved in its repression by 1,25(OH)2D3, we produced a chimeric gene containing a fragment of the rat Col1a1 gene extending from e3518 to þ116 bp fused to the chloramphenicol acetyl transferase (CAT) reporter gene termed ColCAT3.6 [85]. 1,25(OH)2D3 inhibited ColCAT3.6 activity in transiently transfected ROS 17/2.8 cells by 50%, similar to its effect on the endogenous Col1a1 gene [85]. We then generated a series of ColCAT constructs containing progressive 50 promoter deletions of the Col1a1 promoter to map 1,25(OH)2D3 response elements [86,87]. In stably transfected cells, 1,25 (OH)2D3 inhibited a Col1a1 promoter fragment deleted to e2295 bp (ColCAT2.3) but did not affect a promoter fragment deleted to e1670 bp [87]. These experiments localized an inhibitory 1,25(OH)2D3 element to a region of the Col1a1 promoter from e2295 to e1670 bp. Sequence analysis of the Col1a1 promoter revealed a site between e2240 and e2234 bp that had high homology to both the human and rat osteocalcin VDREs.
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We hypothesized that the VDR binding to this motif would inhibit Col1a1 transcription. Electrophoretic mobility shift assays using VDR expressed in COS cells or by an adenovirus vector demonstrated that the VDR bound to this sequence in vitro [87]. However, deletion of the sequence between e2256 and e2216 bp from the ColCAT3.6 or ColCAT2.3 constructs did not affect the inhibitory effect of 1,25(OH)2D3 on promoter activity [87]. Therefore, 1,25(OH)2D3 does not inhibit Col1a1 transcription in ROS 17/2.8 cells solely via the e2240/ e2234 bp site. To determine the effect of 1,25(OH)2D3 on Col1a1 promoter activity in vivo, we previously produced a series of transgenic mouse lines carrying ColCAT constructs [88,89]. 1,25(OH)2D3 inhibited ColCAT3.6 activity in organ cultures of 6e8-day-old transgenic mouse calvariae [90]. 1,25(OH)2D3 inhibited CAT mRNA as early as 3 h, and maximal inhibition of CAT mRNA (50%) was seen at 24 h. The inhibition of CAT mRNA by 1,25(OH)2D3 was not affected by cycloheximide, suggesting that new protein synthesis is not required for the effect. A series of Col1a1 promoter fragments deleted to e1719 bp were fully inhibited by 1,25(OH)2D3. However, a Col1a1 promoter construct deleted to e1670 could not be analyzed because it did not have detectable basal activity in transgenic calvariae [91]. Subsequently, we showed that the rat Col1a1 promoter contains a homeodomain protein motif immediately downstream from e1683 bp that is required for high levels of promoter expression in osteoblasts in vivo [89]. A similar element is also present in the rat Col1a1 promoter [92]. In organ cultures of transgenic mouse calvariae carrying ColCAT constructs, we showed that 1,25(OH)2D3 inhibited CAT activity when the promoter was further deleted to e1683 bp. Moveover, in a transgene having the e1719 bp promoter with a large internal deletion extending from e1284 to e318 bp, the inhibitory action of 1,25(OH)2D3 promoter activity was maintained (A. Ivkovic, A. C. Lichtler and B. E. Kream, unpublished). Taken together, studies in ROS 17/2.8 cells and transgenic calvariae suggest that down-regulation of the Col1a1 promoter by 1,25(OH)2D3 involves sites located between e1683/e1284 bp or in the proximal promoter downstream from e318 bp. There are no good matches to consensus VDREs within these regions, suggesting several possible mechanisms. For one, 1,25 (OH)2D3 repression of Col1a1 could involve binding of the VDR to a novel negative VDRE. Another possibility is that 1,25(OH)2D3 inhibition of Col1a1 expression involves displacement of a stimulatory transcription factor(s) from its cognate DNA-binding site, similar to the mechanism by which 1,25(OH)2D3 inhibits the interleukin-2 gene [93]. It is also possible that 1,25(OH)2D3 inhibition of Col1a1 involves interaction of the VDR with other transcription factors rather than binding of
the VDR to DNA. Such a mechanism has been described for the inhibition of collagenase expression by glucocorticoids [94,95]. Finally, 1,25(OH)2D3 repression of Col1a1 expression could be mediated by alternative signal transduction pathways. It has been suggested that some biological effects of 1,25(OH)2D3 may be mediated by the protein kinase C (PKC) signaling pathway [74]. We have shown that stimulation of PKC with phorbol myristate acetate inhibits collagen synthesis in fetal rat calvariae [96] and ColCAT3.6 expression in transgenic mouse calvariae [97]. Therefore, 1,25(OH)2D3 activation of the PKC pathway might inhibit Col1a1 expression. This could be mediated by a putative 1,25(OH)2D3 membrane receptor, which activates intracellular signal transduction pathways leading to alteration of gene transcription. Future experiments to identify 1,25(OH)2D3 response elements in the Col1a1 gene will involve the analysis of additional constructs having selected sitedirected mutations and internal promoter deletions in cultured osteoblastic cells and transgenic mice. The previous data provide evidence of a direct action of vitamin D on type I collagen expression in osteoblasts. In other systems, the effects of vitamin D may be indirect. For example, vitamin D blocks the fibrotic effects of TGFb in lung fibroblasts and epithelial cells, and although the precise mechanism is not clear, it may involve vitamin D inhibition of TGFb transcriptional activation [98]. Vitamin D has also been shown to inhibit 5-azacytodine induction of TGFb and type I collagen expression in C3H10T1/2 multipotent mesenchymal cells [99].
CONCLUSIONS AND PERSPECTIVES The effect of 1,25(OH)2D3 on collagen expression, either inhibitory or stimulatory, may depend in part on in vitro culture conditions such as cell density, the timing and concentration of 1,25(OH)2D3 addition, the presence of ascorbic acid, and the state of maturation of the model. A model has been proposed based on the premise that cells of the osteoblast lineage differ in their response to 1,25(OH)2D3 depending on their state of maturation [100]. 1,25(OH)2D3 stimulates osteoblast markers in immature osteoprogenitor cells (MC3T3-E1 and MG-63 cells) but inhibits these markers in mature osteoblasts such as rodent calvarial organ cultures, primary rodent osteoblastic cell cultures, and ROS 17/2.8 cells [100]. Such a model is consistent with the effects of 1,25(OH)2D3 on bone remodeling during periods of calcium and phosphate deficiency. When serum calcium and phosphate are low, PTH increases the synthesis of 1,25(OH)2D3. Both hormones increase bone resorption to increase the supply of calcium and phosphate for soft tissues. During periods of mineral
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REFERENCES
deficiency, it would be appropriate for 1,25(OH)2D3 to repress collagen synthesis and inhibit the differentiation of late osteoprogenitors as a means of temporarily limiting new bone formation. Such an effect would prevent calcium and phosphate from being redeposited at sites of new osteoid formation. At the same time, 1,25(OH)2D3 may stimulate the differentiation of early osteoprogenitors to differentiation into a new cohort of osteoblasts that would initiate the phase of coupled formation [100].
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C H A P T E R
23 Target Genes: Bone Proteins Gerald J. Atkins 1, David M. Findlay 1, Paul H. Anderson 2, Howard A. Morris 2 1
Bone Cell Biology Group, Discipline of Orthopaedics and Trauma, University of Adelaide, Adelaide, South Australia, Australia, 2 Chemical Pathology, SA Pathology, Adelaide, South Australia, Australia; School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia, Australia
VITAMIN D AND SKELETAL HOMEOSTASIS Genome-wide analyses indicate vitamin D, through its active metabolite 1,25-dihydroxyvitamin D3 (1,25 (OH)2D3) and the vitamin D receptor (VDR), has a potential to regulate the expression of some 3000 genes [1]. However, current evidence indicates that the strongest phenotype exhibited by vitamin-D-deficient humans or animals relates to impaired skeletal health. The first scientific reports in the early 1900s that rickets was due to vitamin D3 deficiency placed vitamin D as central to the regulation of calcium and phosphate homeostasis. In this context, it appears that the most critical and most widely studied endocrine role of vitamin D is its contribution to the maintenance of plasma calcium and phosphate at physiological levels through regulation of intestinal absorption of dietary calcium and phosphate. A central question, however, is whether vitamin D acts directly on bone tissue to modulate bone mineral homeostasis and bone strength. This question has been difficult to answer conclusively due in part to the direct actions of vitamin D on plasma calcium and phosphate levels, which indirectly affect bone mineralization and structure. One direct action that has been clearly demonstrated is the ability of plasma 1,25(OH)2D3, at least at supraphysiological levels, to stimulate bone resorption by the activation of osteoclasts [2]. The effects of vitamin D deficiency on bone in vivo can apparently be largely corrected by increasing dietary calcium and phosphate [3,4], suggesting that vitamin D is not an absolute requirement for optimal bone health. Indeed, the fact that the osteomalacic
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10023-X
phenotype observed in the vitamin D receptor gene knockout (VdrKO) mouse can be rescued by feeding a diet containing high levels of calcium and phosphate has led some to conclude that VDR-mediated activity in bone is essentially redundant [5,6]. Others have suggested that actions of VDR in bone may in fact impair mineralization [7,8]. These conclusions are, however, difficult to reconcile against an accumulating large body of evidence indicating that vitamin D activity in bone is critical for bone cell differentiation and optimal mineral status [9e12]. In this chapter, we examine the evidence for direct effects of vitamin D on the bone as a tissue, and its actions on the constituent cells of bone, in particular bone-matrix-forming osteoblasts, osteocytes, and bone-resorbing osteoclasts. There is now evidence that each of the major bone cells is capable of producing 1,25(OH)2D3 from the 25-hydroxyvitamin D3 (25(OH)D3) precursor, and that this activity is likely to account for the skeletal effects of circulating 25(OH)D3 (see Fig. 23.1). On the weight of this evidence, we have proposed that bone is an intracrine organ of vitamin D metabolism [13]. The actions of 1,25(OH)2D3 are mediated ultimately by direct effects on individual vitamin-D-responsive genes. However, the effects of vitamin D on bone tissue as a whole are not yet fully understood but are likely due to a combination of direct effects via VDREs, downstream effects of the induced gene expression and effects at specific stages of bone cell proliferation and differentiation. The effects of 1,25(OH)2D3 on important transcription factors, which in turn govern a downstream program of gene expression, will also be considered.
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23. TARGET GENES: BONE PROTEINS
UVb Skin
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25D Bone CYP27B1
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VD
R
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osteoblasts osteoclasts osteocytes -
proliferation differentiation mineralization development activity function
FIGURE 23.1 A major source of active 1,25(OH)2D3 to regulate
bone processes of turnover and mineral homeostasis is now considered to be the bone itself. The action of the 1a-hydroxylase enzyme (CYP27B1) in bone cells including osteoblasts, osteoclasts, and osteocytes, gives rise to the active form of vitamin D, 1,25(OH)2D3. 1,25 (OH)2D3 binds to the VDR in the nucleus of target cells whereby it can alter gene transcription of vitamin-D-responsive genes, including those involved in essential bone cell activities, such as bone formation and resorption.
The 1a-hydroxylation of 25(OH)D3 to 1,25(OH)2D3 in bone cells was reported almost three decades ago [23e25] and yet only recently has evidence arisen to suggest that locally produced 1,25(OH)2D3 in osteoblasts plays a role in osteoblast differentiation and mineralization and in the regulation of osteoclastogenesis and osteoclast activity [9e12,26,27]. We and others have characterized Cyp27b1 promoter activity and Cyp27b1 mRNA expression in bone tissue associated with trabecular bone and growth plate [13,16,28] (see Fig. 23.2). Cyp27b1 mRNA levels were significantly higher in rat fetal bone than in adult bone and were shown by in situ hybridization to be present largely in growth plate chondrocytes and osteoblasts [29]. We have demonstrated the age-related regulation of Cyp27b1 mRNA expression in rat bone, which is also increased in the presence of high dietary calcium levels and is positively associated with bone mineralization [20,30]. Similar findings of a relationship between circulating 25(OH)2D levels and osteoid thickness have been reported in two separate clinical studies [31,32]. Furthermore, the expression of bone Cyp27B1 mRNA is distinct from the regulation of renal Cyp27B1 mRNA [33]. The levels of Cyp24 mRNA in bone, a gene exquisitely regulated by 1,25(OH)2D3, are coupled with the levels of bone Cyp27b21 mRNA expression and independent of changes to circulating levels of 1,25(OH)2D3, suggesting that the local bone production of 1,25(OH)2D3 is responsible for regulating CYP24 activity [20,34].
EXOGENOUS AND ENDOGENOUS SOURCES OF 1,25(OH)2D3 Circulating 1,25(OH)2D3, under nonpathological conditions, is derived by the actions of the renal 25 hydroxyvitamin D 1a-hydroxylase (CYP27B1) enzyme, which is highly expressed under certain conditions in the proximal tubular epithelial cells of the kidney [14e16]. CYP27B1 is also expressed in a wide range of extrarenal tissues including bone. Except in the cases of sarcoidosis [17] and placentation [18], extrarenal production of 1,25(OH)2D3 does not appear to contribute significantly to circulating levels [19,20]. Thus, local or cell-specific production of 1,25(OH)2D3 in bone and other tissues has been generally postulated to act in an autocrine or paracrine manner to regulate parameters of cell growth and differentiation [21,22].
Bioluminescence grayscale heat-map showing regions of Cyp27b1 promoter activity in a femur (A) of the 1501 bp Cyp27b1 promoter-Luciferase reporter transgenic mouse. The darker intensities of gray (B) represent greater activity of the CYP27B1 promoter. The overlaid grayscale heat-map with the femur image (C) demonstrates greater Cyp27b1 promoter activity in proximal and distal regions of the bone consistent with growth plate and trabecular bone structures.
FIGURE 23.2
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EXOGENOUS AND ENDOGENOUS SOURCES OF 1,25(OH)2D3
In human osteoblasts, CYP27B1 mRNA expression and conversion of 25(OH)D3 into 1,25(OH)2D3 in transformed osteoblasts were demonstrated by Van Driel and colleagues. By using the pan inhibitor ketoconazole, they showed the reliance for this effect on cytochrome P450 activity [9]. We have also shown the expression of CYP27B1 mRNA in human primary osteoblasts isolated from adult femoral bone samples, and human osteosarcoma cell lines, and that 1,25 (OH)2D3 can be produced from 25(OH)D3 at physiological concentrations in these cells [12,35]. Using RNAi gene silencing, the synthesis of 1,25(OH)2D3 and the expression of osteocalcin, osteopontin, RANKL, and CYP24 mRNA in response to 25(OH)D3, were all dependent on CYP27B1 activity [12,35]. Thus, autocrine 1,25(OH)2D3 synthesis and activity is a common feature of human osteoblastic cells. We also showed that treatment with physiological levels of 25(OH)D3 inhibited cell proliferation and stimulated osteoblast differentiation, increasing the degree of matrix mineralization by human osteoblasts [12]. 25 (OH)D3 mainly circulates in vivo bound to the vitamin-D-binding protein, DBP, and this interaction is thought to exclude 25(OH)D3 from entering cells by passive diffusion [36]. The DBP-25(OH)D3 complex is reabsorbed specifically by renal tubules via the expression of two receptors for DBP, cubilin and megalin [37,38]. In further support of 25(OH)D3 metabolism representing a physiologically important pathway in osteoblasts, human osteoblastic cells have been shown to express both cubilin and megalin [9,35]. Many studies have shown an effect of 1,25(OH)2D3 on osteoclast formation, focusing on indirect effects via the osteoblast. Certainly, these effects are important, as indicated by studies using Vdr-null osteoblasts [39]. However, we have recently demonstrated that human peripheral blood mononuclear-cell-derived osteoclasts convert 25(OH)D3 into 1,25(OH)2D3 [27]. As will be discussed further below, conversion of 25 (OH)D3 to 1,25(OH)2D3 in osteoclasts dose-dependently inhibited the resorptive activity, with a maximal effect (~30% inhibition) seen at 25(OH)D3 levels >50 nmol/l. These data suggest that 25(OH)D3 metabolism in cells of the osteoclast lineage optimizes osteoclastogenesis and regulates the resorptive behavior of mature osteoclasts. Cells of the monocyte/macrophage lineage have been shown to express Cyp27b1 mRNA and convert 25(OH)D3 into 1,25(OH)2D3 [40,41]. Bone marrow macrophages also convert 25(OH)D3 into either 1,25 (OH)2D3 or 24,25(OH)2D3. Reichel and coworkers [42] demonstrated that, upon exposure to recombinant human interferon-gamma (IFN-g), bone-marrow-derived macrophages initially synthesized 1,25(OH)2D3 from 25(OH)D3.
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Growth plate chondrocytes also express Cyp27b1 mRNA and display 1a-hydroxylase activity [43,44]. Evidence suggests that 1,25(OH)2D3-VDR signaling is an important pathway for chondrocyte support of osteoclastogenesis during bone remodeling at the growth plate [45]. More recently, gene deletion and transgenic mouse models of CYP27B1 activity in chondrocytes demonstrated that locally produced 1,25(OH)2D3 in these cells regulated RANKL-mediated osteoclastogenesis, endochondral ossification, and chondrocyte development in vivo [46]. Intriguing preliminary data have been obtained suggesting that the locally produced 1,25(OH)2D3 in bone tissue exerts a differential response from that derived from the circulation. Thus in a rodent model, when circulating levels of 25(OH)D3 are below 80 nmol/L, bone loss is detectable as a result of increased bone resorption, increased osteoclastogenesis, and increased Rankl expression, driven by elevated circulating 1,25 (OH)2D3 levels [47]. On the other hand, when 25(OH) D3 levels are adequate and metabolized by bone cells to 1,25(OH)2D3 then Rankl expression is minimized. Furthermore there is clearly a differential response to induction of Cyp24 when osteoblastic cells are incubated with 1,25(OH)2D3 or 25(OH)D3 in vitro. Cells incubated with 25(OH)D3 at levels of approximately 100 nmol/L induce vitamin-D-responsive genes such as osteocalcin and osteopontin in a CYP27B1-dependent manner. Cyp24 is not significantly induced until levels of 25 (OH)D3 reach 400 nmol/L. However, when 1,25 (OH)2D3 is incubated with osteoblastic cells, Cyp24 is the first gene to be induced at the lowest levels of 1,25 (OH)2D3 [11]. The mechanism of such differential effects is unknown at this time; however it could be related to the ability of vitamin D, especially when locally metabolized, to induce osteoblast maturation. It has been well established that early or pre-osteoblast-like cells respond to vitamin D in a different manner from mature osteoblast or pre-osteocytic-like cells, as discussed in more detail later in this chapter. In summary, bone cells express the vitamin D metabolic enzyme CYP27B1 and have the capability of converting 25(OH)D3 into 1,25(OH)2D3, to elicit various effects central to bone remodeling, including bone resorption, bone formation, and mineralization. The potential and known effects of 25(OH)D3 metabolism during bone remodeling are depicted in Figure 23.3. Further innovative studies are required to identify and characterize these autocrine/paracrine networks of vitamin D metabolism and activity in the in vivo bone microenvironment to establish the relative roles of endogenous and exogenous sources of 1,25(OH)2D3. Such studies have the potential to establish a new paradigm for nutritional requirements for vitamin D for optimal skeletal health.
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OSTEOCLASTOGENESIS Stromal OB preOC
BONE FORMATION
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Immature OB Mature OB
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proliferation
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Time / Bone Remodeling FIGURE 23.3 The potential effects of 25(OH)D3 metabolism by bone cells. This cartoon depicts the sequence of cellular events in bone remodeling and the potential role of metabolism of 25(OH)D3 into 1,25(OH)2D3 in osteoblasts (OB) and osteocytes (OCy), as well as in osteoclast (OC) lineage cells. Stromal osteoblasts support osteoclast differentiation from immature OC precursors (preOC), which form bone-resorbing, mature OC. The resorbed site is then populated by immature OB, which proliferate and differentiate into mature OB. These synthesize an unmineralized bone matrix, termed osteoid. Certain OBs become entrapped in osteoid (osteoid osteocytes) and these differentiate further into mature OCy, a process concomitant with bone mineralization. The effects of 25(OH)D3 to 1,25(OH)2D3 conversion, both those reported in the text, and those inferred from the known effects of exogenous 1,25(OH)2D3, at each stage are listed below each target cell type.
DIRECT ACTIONS OF VITAMIN D IN BONE Osteoblast The osteoblast regulates bone matrix synthesis and contributes to the coordination of bone resorption during remodeling in response to a large number of regulatory signals of which 1,25(OH)2D3 appears to be an important and pleiotropic member. A primary function of the osteoblast is to secrete a specialized organic extracellular matrix, which consists mainly of type I collagen but also a number of noncollagenous proteins, such as osteocalcin, osteopontin, osteonectin, bone sialoprotein-1, proteoglycans such as versican, and the small chondroitin sulfate proteoglycans, decorin and biglycan. This organic matrix is ultimately mineralized at discrete sites by the incorporation of calcium and phosphate, to form a mature bone matrix (for an excellent review of this topic, see [48]). In vitro studies have shown that 1,25(OH)2D3 is capable of regulating osteoblast gene transcription, proliferation, differentiation, and mineralization [49e51]. The genes of matrix proteins, such as osteopontin and
osteocalcin, possess vitamin-D-responsive elements (VDRE) within their promoter regions, suggesting a direct action for 1,25(OH)2D3 on their expression. Other important bone-matrix-associated genes, such as type I collagen and osteonectin, may have nonclassical VDREs in their promoters or be indirectly regulated by 1,25(OH)2D3 [52]. As mentioned above, in vitro evidence suggests that 1,25(OH)2D3 exerts effects during both the resorptive and synthetic phases of bone remodeling. In association with other factors including PTH, 1,25(OH)2D3 can also indirectly induce osteoclastogenesis by stimulating the differentiation of bone-marrow-derived promyelocytes and monocytes to active osteoclasts [53,54]. The tumor necrosis factor (TNF) ligand member, RANKL, itself a 1,25(OH)2D3-inducible protein, has been shown to be a critical mediator of 1,25(OH)2D3, PTH or inflammatory cytokine-induced osteoclastogenesis [55,56]. Thus, a paradox exists in that 1,25(OH)2D3 can potentially induce a proresorptive expression pattern, by increasing expression of RANKL, or a pro-osteogenic pattern in osteoblast lineage cells. One possibility is that the support of osteoclastogenesis and osteogenesis are performed by different types of specialized
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osteoblasts. A second possibility is that these two diverse osteoblast functions are performed at different stages of osteoblast differentiation, as depicted in Figure 23.3. Our previous study [51] showed that in primary human osteoblasts, 1,25(OH)2D3 induced the expression of RANKL in phenotypically immature osteoblast precursors, identified by their expression of the marker STRO-1 [57]. However, in phenotypically mature osteoblasts, negative for STRO-1 expression, an osteocalcin response predominated [51]. This differential response was not related to levels of VDR expression, nor to the overall ability of the cells to respond to 1,25 (OH)2D3, evidenced by the expression of other “synthetic phase” 1,25(OH)2D3-responsive genes such as type I collagen and bone sialoprotein-1, which were found to be expressed independently of differentiation stage. Similar results were obtained in mineralizing cultures of primary mouse osteoblasts, where the 1,25 (OH)2D3 induction of RANKL expression decreased with increasing maturation of the osteoblast [58]. A third and combinatorial model is possible, with the sum of signals received by the osteoblast within a permissive window of its differentiation program determining either an osteoclastogenic or osteogenic response. The above studies imply that the particular cohort of genes expressed in response to 1,25(OH)2D3 in the osteoblast is regulated according to their stage of differentiation. Differential responses of osteoblasts to 1,25(OH)2D3 may result from different VDR signaling complexes. As detailed elsewhere in this volume, upon ligation of 1,25 (OH)2D3 with VDR and translocation to the nucleus, the complex forms a heterodimer with the retinoid X receptor (RXR). This induces a VDR conformation that is essential for effective binding to the VDRE. This association serves to recruit nuclear proteins as coactivators or corepressors, necessary for VDR-mediated transcriptional regulation. In short, the interaction of the 1,25 (OH)2D3-bound VDReRXR complex with nuclear proteins forms a so-called “pre-initiation complex,” which regulates the rate of transcription of the target gene [59]. The constitution, and therefore the promoter specificity, of this complex may change with differentiation stage of the cell. It remains to be seen whether other mechanisms of modifying the 1,25(OH)2D3 response, such as CpG methylation and inactivation of the VDRE, as has been shown for the RANKL promoter [60], occur for other bone protein genes. Osteoblast Proliferation and Matrix Synthesis In general, 1,25(OH)2D3 inhibits the proliferation of osteoblasts. This antiproliferative activity is associated with the ability of 1,25(OH)2D3 to induce osteoblast differentiation [61,62]. Numerous studies have shown the inhibition by 1,25(OH)2D3 of osteoblast proliferation in the human [51,63,64], rat [65,66], and mouse [67e69].
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The effects of 1,25(OH)2D3 on osteoblast proliferation are, however, dependent on species and maturity of the cell. For example, van den Bemd and coworkers [64] found that in human MG-63 cells, 1,25(OH)2D3 could suppress proliferation, whereas in rat osteosarcoma (ROS 17/2.8) cells, 1,25(OH)2D3 stimulated growth. Murray and coworkers [66] found that 1,25 (OH)2D3 inhibited proliferation of the rat osteoblast cell line, G2, and stimulated proliferation in rat osteoblast cell line, C12. The differences in the effects of 1,25 (OH)2D3 on osteoblast proliferation are unclear but may be due to the differentiation state of the osteoblast-like cell line tested. A further complication of the above studies is that immortalized cell lines, in general, proliferate in an uncontrolled fashion, making interpretation of the data difficult. Using carboxy-fluorescein succinimidyl ester (CFSE), a fluorescent dye that enables the number of cell divisions to be tracked with respect to other fluorescently tagged proteins [70], Atkins et al. [51] showed that while growth was inhibited overall in a heterogeneous population of human primary osteoblast-like cells in the presence of 1,25(OH)2D3, immature cells proliferated more than phenotypically mature cells. This implies that the degree of growth inhibition by 1,25 (OH)2D3 relates to the inherent growth potential of a particular cell type. Moreover, the effects of 1,25 (OH)2D3 on osteoblast proliferation appear to be dosedependent. For example, treatment of human osteoblasts with a low dose of 1,25(OH)2D3 (5 10e12 M) increased proliferation, whereas a pharmacological dose of 1,25(OH)2D3 (5 10e6 M) showed decreased proliferation [63]. Type I collagen is expressed in the proliferative stage of osteoblast development and is essential for the tensile strength of bone. Stein and coworkers [71] suggested that the inhibition of type I collagen gene expression prevents subsequent extracellular matrix development. The effect of 1,25(OH)2D3 on type I collagen expression during osteoblast proliferation, however, is dependent on the cell model of osteoblast studied. In human MG-63 osteosarcoma cells, 1,25(OH)2D3 stimulated the synthesis of type I collagen [64,72]. In rats and chickens, treatment of osteoblasts with 1,25(OH)2D3 reduced type I collagen mRNA transcription and protein synthesis [65,73e75]. The effects of 1,25(OH)2D3 on type I collagen synthesis in osteoblasts also appears to be conditional on the differentiation and proliferation state of the cells. In proliferating rat osteoblasts, acute 1,25(OH)2D3 treatment inhibited the high levels of type I collagen expression found at this stage of osteoblast development. During mineralization, however, low basal levels of type I collagen mRNA were stimulated by acute 1,25(OH)2D3 treatment and were unaltered by chronic 1,25(OH)2D3 treatment [65]. In the mouse, while 1,25 (OH)2D3 treatment was shown to promote type I
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collagen breakdown in calvarial osteoblasts [74], it has also been shown to stimulate type I collagen synthesis in early-phase MC3T3-E1 cells and have no effect in late-phase MC3T3-E1 cells [49]. Alkaline Phosphatase Alkaline phosphatase activity is important for the mineralization of bone and represents a useful biochemical marker of bone formation [76,77]. Osteoblasts express the bone- or tissue-non-specific isoform of alkaline phosphatase (TNAP), which is a glycosylphosphatidylinositol (GPI) anchored cell surface protein [78]. Treatment of rat osteoblast-like cells with 1,25(OH)2D3 promoted mineralization, which was associated with high alkaline phosphatase activity [79e81]. While the alkaline phosphatase gene promoter has no classical VDRE, 1,25(OH)2D3 was also shown to have a stimulatory effect on alkaline phosphatase mRNA levels, protein synthesis, and activity in human osteoblasts [82,83]. The stage of differentiation of osteoblasts has been shown to determine the response of alkaline phosphatase expression to 1,25(OH)2D3. During the proliferative period of osteoblast development, 1,25 (OH)2D3 inhibited the expression of alkaline phosphatase, whereas during mineralization, 1,25(OH)2D3 stimulated alkaline phosphatase mRNA expression [65]. In the mouse, however, 1,25(OH)2D3 stimulated alkaline phosphatase activity only in the early phase of osteoblast differentiation and not in the mineralization phase [49]. Matrix Gla Protein Matrix Gla protein (MGP), like osteocalcin, requires vitamin-K-dependent gamma-carboxylation for its function. MGP has been identified as a calcification inhibitor in cartilage and vasculature since MGP-null mice die soon after birth due to aberrant cartilage and arterial calcification [84]. In both rat UMR 106-01 and ROS17/ 2.8 cells, 1,25(OH)2D3 treatment markedly increased MGP mRNA and protein levels [85,86]. The stimulation of MGP mRNA by 1,25(OH)2D3 was shown to be less in the late stages of rat osteoblast differentiation [87] than in earlier stages of osteoblast growth [87].
Osteoid/Pre-osteocytes and Bone Mineralization Following cessation of proliferation and production of the collagenous matrix, certain osteoblasts become embedded in the matrix, signaling their differentiation into osteoid- or pre-osteocytes, sometimes referred to as mineralizing osteocytes. Matrix mineralization is an active process and evidence suggests that it is tightly regulated by protein products of the osteoid-osteocyte and mature osteocyte. It has been demonstrated that vitamin D augments matrix mineralization in vivo
and in vitro [11]. This may be due to effects of 1,25 (OH)2D3 on the expression of proteins known to be nucleators and regulators of mineralization, and possibly others, as will be discussed in the ensuing sections. Osteopontin Osteopontin (OPN), an extracellular glycosylated bone phosphoprotein, is one such gene that, in bone, is secreted by late-stage osteoblasts at the mineralization front [65,88]. In human bone marrow cultures, and in MG-63 cells, 1,25(OH)2D3 administration was associated with increased levels of OPN mRNA [89]. Cultured rat bone cells and ROS17/2.8 cells were both shown to increase OPN mRNA expression and protein secretion in response to 1,25(OH)2D3 administration [90]. Low basal levels of OPN mRNA were seen in rat calvarial cultures of intermediate maturity, which were markedly up-regulated by 1,25(OH)2D3 [91]. However, 1,25(OH)2D3-mediated stimulation of OPN mRNA in rat calvarial osteoblasts was shown to be far greater in premineralization cells than in mature mineralizing cells, where levels of OPN mRNA were already high [65]. OPN is a member of the small integrinbinding N-linked glycoprotein (SIBLING) family since it contains an ASARM (acidic serine- and aspartaterich motif) [92]. The OPN ASARM peptide with three phosphoserines can inhibit in vitro mineralization and is a substrate for PHEX which can rescue mineralization. It has been found that the helix-loop-helix-type transcription factor (HES-1) is expressed in osteoblastic cells and is suppressed by 1,25(OH)2D3. Overexpression of HES-1 in ROS17/2.8 cells suppressed the vitamin-Ddependent up-regulation of osteopontin gene expression in these cells [90]. TGF-b and PTH were also shown to abrogate 1,25(OH)2D3-mediated induction of OPN in ROS17/2.8 cells [93,94], suggesting that multiple transcription factors and hormones may be involved in regulating OPN activity. Bone Sialoprotein Bone sialoprotein (BSP) is largely specific for mineralized tissues and is highly expressed during the initial formation of bone and cementum [95]. The expression of BSP is suppressed by 1,25(OH)2D3 treatment in rat calvaria and ROS 17/2.8 cells [96]. A VDRE that is integrated with an inverted TATA box in the rat BSP promoter mediates the suppression of BSP transcription [97e99]. In human bone marrow stromal cells, 1,25 (OH)2D3 treatment alone did not significantly affect the expression of BSP mRNA [89]. However, data from our laboratory demonstrate a positive induction of BSP-1 mRNA by 1,25(OH)2D3 in normal human osteoblast-like cells (G.J. Atkins et al., unpublished
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data), suggesting further differences between human and rodent responses to 1,25(OH)2D3. Osteocalcin In the mature osteoblast, 1,25(OH)2D3 downregulates the expression of BSP and type I collagen [96,100] and increases the expression of OPN and osteocalcin (OCN) [101,102]. OCN has a high affinity for calcium ions of hydroxyapatite and is the most abundant noncollagenous protein in bone [103]. OCN is expressed in postproliferative osteoblasts as well as in osteocytes. Ablation of the OCN gene in mice increases both bone formation and bone mass, although the mechanism for this remains unclear [104]. While the precise function of this protein is not well defined, it has been shown to be a chemotactic factor for osteoclasts and their precursors [105]. OCN has been widely used as a marker of bone formation [77]. It has recently been reported to be released from the matrix in the intact form rather than as fragments during remodeling as a result of osteoclast activity [106]. A number of studies have reported the induction of both OCN mRNA and protein synthesis by 1,25 (OH)2D3 in human and rat bone cells [63,65,82,83, 91,107e111], although the pattern of 1,25(OH)2D3induced expression of the OCN gene seems to depend on the culture system and the stage of maturity of the cells. For example, in human MG-63 cells, 1,25(OH)2D3 induction of OCN was highest in subconfluent cultures and decreased in confluent cultures [112]. Similarly, OCN gene expression was found to have a decreased responsiveness to 1,25(OH)2D3 in mineralizing human osteoblasts, which was suggested to be due to an accumulation of OCN in the extracellular matrix [113]. There are, however, reports of 1,25(OH)2D3 down-regulating OCN expression both in chicken embryonic osteoblasts [75] and in mouse osteoblast cultures [114,115]. Recent studies in our laboratories (H.A. Morris & G.J. Atkins et al., unpublished data), using mouse primary osteoblasts derived from adult mouse cortical bone, indicate that 1,25(OH)2D3 may stimulate OCN expression in this species, in contrast to previously published findings, and the direction of OCN expression in response to 1,25 (OH)2D3 may depend on the differentiation stage of the cells in question. The characterization of the OCN gene has identified a number of factors that are potentially involved in the development of the osteoblast phenotype. Besides the identification of a VDRE in the distal promoter of the OCN gene, several other promoter sites have been shown to be critical in the expression of the OCN and in osteoblastic differentiation. For example, the requirement for the osteoblast transcription factor, core binding factor alpha (CBFA)-1/AML-3, is best demonstrated in the CBFA-1/AML-3-null mutant mouse. These mice
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died at birth and had major skeletal deformations characterized by disrupted mineralization of osteoblasts [116,117]. Mutations of the three CBFA-1 motifs identified on the osteocalcin promoter were found to lead to abrogation of responsiveness to 1,25(OH)2D3 [118]. Another important site identified in the promoter of the OCN gene is the AP-1 site juxtaposed to the VDRE of the OCN gene. While this AP-1 site is essential for 1,25(OH)2D3 induction of the OCN gene [119], the suppression of OCN gene expression at the onset of mineralization appears to be related to the interaction of specific transcription factors at this AP-1 site. In proliferating osteoblasts, the repression of OCN gene expression is partly due to the expression of c-fos/ c-jun, which binds as a heterodimer to the AP-1 site and blocks the binding of the VDR/RXR complex [120,121]. In contrast, in postproliferative osteoblasts approaching mineralization, the expression of fra-2 results in binding to the AP-1 site, which facilitates VDR/RXR binding to the VDRE and 1,25(OH)2D3mediated expression of the OCN gene [121,122].
Osteocytes Osteocytes are the most long-lived and numerous cell type in bone tissue and have emerged over recent years as key controllers of osteoblast behavior, bone mineralization, and potentially of osteoclast activity [123,124]. While it has not been studied in detail, it is likely that osteocytes are responsible for the majority of osteocalcin synthesis, which as discussed above is under the control of 1,25(OH)2D3. Other key osteocyte derived proteins appear also to respond to 1,25(OH)2D3. Fibroblast Growth Factor (FGF) 23 FGF23 is a bone-derived hormone with known endocrine activities in regulating the renal expression of CYP27B1, having a negative effect on this enzyme and thus inhibiting the renal synthesis of 1,25(OH)2D3. The osteocyte is a major source of FGF23 although its expression has also been linked to osteoblasts. Deletion of the DMP1 gene in mice has revealed a complex relationship with FGF23, with DMP1-null cells resembling immature osteocytes and expressing excessive levels of FGF23 [125]. It is not yet known whether immature osteocytes predominantly express FGF23 in wild-type mice or in human bone. Importantly, FGF23 has been demonstrated to be 1,25(OH)2D3-responsive [126,127]. Additionally, in a recent study, Tang and coworkers demonstrated that 25(OH)D3 metabolism in cultured neonatal rat calvarial osteoblasts also resulted in the up-regulation of FGF23 expression [127]. Interestingly, the action of FGF23 on kidney tubule cells is to decrease their expression of Cyp27b1 and inhibit their synthesis of 1,25(OH)2D3 [128]. However, FGF23 has been shown to
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increase Cyp27b1 expression in bovine parathyroid cells [129]. It remains to be seen whether FGF23 also regulates osteoblast/osteocyte synthesis of 1,25(OH)2D3. Overexpression of FGF23 has been linked to rickets in X-linked hypophosphatemia (XLH) and in mouse models of this disease, such as the Hyp mouse and DMP1 mutant mice [130e133]. The pathology in these cases appears due to the increase in renal phosphate wasting and the decrease in 1,25(OH)2D3 levels. Because of the reciprocal relationship between FGF23 and circulating 1,25 (OH)2D3 levels, it might seem attractive to treat patients with XLH with 1,25(OH)2D3. A recent study, however, showed that treatment of these patients with calcitriol and phosphate resulted in increased FGF23 levels indicating that such a treatment may be counter-productive [134]. Dentin Matrix Protein 1 (DMP1) DMP1 is an acidic phosphorylated extracellular matrix protein, and like OPN, is a member of the SIBLING family [135]. First described as a product of odontoblasts [136], it is now recognized to be highly expressed in osteocytes and important for the differentiation of these cells [125]. Roles for DMP1 and its proteolytic cleavage products, the N-terminal 37 kDa and C-terminal 57 kDa fragments, are not fully understood but include nucleation of mineralization, cell attachment, and possibly as a transcriptional regulator [137]. As discussed above, DMP1, FGF23, and the CYP27B1 have a complex interplay made more complex perhaps by the findings that DMP1 is itself 1,25(OH)2D3responsive [138]. The in vivo significance of this observation has yet to be determined.
Osteoclasts Osteoclasts are multinucleated cells of the monocyte/ macrophage lineage whose primary function is to resorb bone during bone remodeling. They accomplish this by attaching to the mineral surface forming a tight sealing zone, and creating a basolateral membrane termed the ruffled border, from which bone-matrix-degrading enzymes, such as cathepsin K, and protons are secreted via proton pumps such as the vacuolar ATP-ase complex (V-ATPase) and carbonic anhydrase II, to break down the collagenous matrix and release calcium and phosphate ions, respectively. Osteoclasts may also be involved in the active coupling of bone resorption to bone formation by inducing osteoblast proliferation. Osteoclasts are generated by the proliferation and fusion of mononuclear precursors. These complex processes include several key proteins whose genes are known to be 1,25(OH)2D3-responsive. More recent data indicate that osteoclasts, like macrophages, are a site of extrarenal 1,25(OH)2D3 synthesis by virtue of their expression
of CYP27B1, and that the prevailing level of blood 25 (OH)D3 may govern aspects of both osteoclast formation and their resulting activity. Effects of Exogenous Vitamin D Evidence suggests that 1,25(OH)2D3 has direct effects on osteoclast precursors, increasing the expression of the key adhesion molecule, aVb3 integrin, in both avian osteoclast precursor cells [139e141] and in the human myelomonocytic cell line, HL-60 [142], thus potentially promoting osteoclast adhesion and the formation of the sealing zone. 1,25(OH)2D3 has been shown to facilitate adhesion of osteoclast precursors to stromal osteoblasts by increasing the expression of the intercellular adhesion molecule, ICAM-1 [143]. 1,25(OH)2D3 has also been shown to increase the expression of the receptor for RANKL, RANK, in HL-60 cells [144]. We have recently demonstrated a direct effect of 1,25(OH)2D3 on RANKL-induced osteoclast formation from the mouse preosteoclast cell line, RAW 264.7, where 1,25(OH)2D3 in the copresence of RANKL, increased the resulting numbers of multinucleated TRAP-positive osteoclasts and significantly increased osteoclast multinucleation [145]. Effects of Endogenous Vitamin D It was recently described that, similar to other macrophage cell lines and primary cells, the RAW 264.7 cell line expresses CYP27B1 and also that CYP27B1 mRNA levels increased during their differentiation into osteoclast-like cells [9]. We recently confirmed that human PBMC-derived osteoclasts possess the molecular machinery to both respond to and metabolize 25(OH)D3, as they express cytoplasmic CYP27B1 and nuclear VDR proteins [26]. Furthermore, CYP27B1 mRNA expression increased in response to M-CSF/RANKL-induced differentiation of PBMC, suggesting that 25(OH)D3 metabolism plays a role in osteoclast differentiation. In a subsequent study, we confirmed that the capacity of osteoclasts to synthesize 1,25(OH)2D3 increases substantially with their differentiation [27]. Metabolism of 25 (OH)D3 into 1,25(OH)2D3 in human PBMC-derived osteoclasts resulted in the increased expression of a number of genes, principal among these being the osteoclast transcription factor NFATc1 [27]. This may or may not be responsible for the observed concomitant increase in expression of a number of osteoclastic genes, including calcitonin receptor, tartrate-resistant acid phosphatase (TRAcP), cathepsin K, carbonic anhydrase, and V-ATPase. Notably, 1,25(OH)2D3 treatment also up-regulated the expression of these genes but generally did so to a lesser extent. It remains to be determined if some or all of these genes are vitamin-D-responsive in the classical sense or whether their expression was as a consequence of cell
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REFERENCES
differentiation and the expression of transcription factors, such as NFATc1, that created a permissive environment for their expression. In the case of VATPase, which is a complex multisubunit enzyme [146], Lee et al. [147] have demonstrated that its activity is up-regulated by exogenous 1,25(OH)2D3 by an unidentified post-translational mechanism, suggesting that 1,25(OH)2D3 effects on osteoclast behavior may not be simply transcriptional. In terms of the overall effect of 1,25(OH)2D3 on the osteoclast, studies published to date [26,27] suggest that intracellular accumulation of 1,25(OH)2D3 promotes osteoclast formation but inhibits the resorptive capacity of these cells.
CONCLUDING REMARKS Overwhelming clinical evidence suggests that vitamin D is important for calcium/phosphate and skeletal homeostasis. Numerous direct and indirect effects of 1,25(OH)2D3 have been demonstrated on a range of critical bone proteins and 1,25(OH)2D3 appears to be involved in their regulation at all stages of osteoblast differentiation and, indeed, bone remodeling. Future studies will need to unravel the complexities surrounding the involvement of 1,25(OH)2D3 in the coordination of these processes. However, the evidence for a fundamental and nonredundant role for 1,25 (OH)2D3 in skeletal biology in vivo, namely, geneablated mouse models, is currently lacking. As suggested in earlier, this is in part due to an incomplete analysis of these potentially informative models, rather than a convincing lack of a biological effect. Additionally, some of the apparently contradictory data surrounding this question arise from the plethora of model systems (immortalized cell lines versus primary cells, the use of different mammalian and other vertebrate species) which have been utilized to investigate this issue, many of which may have significant and confounding limitations. Recent studies using 1,25(OH)2D3 analogs that show potent anabolic effects, for example, the compound 2-methylene-19-nor-(20S)-1-a,25dihydroxyvitamin D3 (2-MD) when administered to sham-operated or ovariectomized mice under otherwise normal dietary conditions [148], and other examples discussed elsewhere in this volume, must provide additional evidence for the bone-specific effects and importance of the natural hormone. The combination of clinical [149,150] and preclinical [47] studies provides strong evidence that adequate circulating 25(OH)D3 levels and not 1,25(OH)2D3 are required to optimize skeletal health. Such findings support the concepts described above that the local metabolism of vitamin D and the subsequent biological activity of autocrine/ paracrine-derived 1,25(OH)2D3 enhance the maturation
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and activities of each of the major bone cell types to optimize skeletal structure and strength.
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[130] ADHR Consortium Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat. Genet. 26 (2000) 345e348. [131] P.S. Rowe, The wrickkened pathways of FGF23, MEPE and PHEX, Crit. Rev. Oral. Biol. Med. 15 (2004) 264e281. [132] S. Liu, J. Zhou, W. Tang, X. Jiang, D.W. Rowe, L.D. Quarles, Pathogenic role of Fgf23 in Hyp mice, Am. J. Physiol. Endocrinol. Metab. 291 (2006) E38e49. [133] S. Liu, J. Zhou, W. Tang, R. Menard, J.Q. Feng, L.D. Quarles, Pathogenic role of Fgf23 in Dmp1-null mice, Am. J. Physiol. Endocrinol. Metab. 295 (2008) E254e261. [134] E.A. Imel, L.A. DiMeglio, S.L. Hui, T.O. Carpenter, M.J. Econs, Treatment of X-linked hypophosphatemia with calcitriol and phosphate increases circulating fibroblast growth factor 23 concentrations, J. Clin. Endocrinol. Metab. 95 (2010) 1846e1850. [135] L.W. Fisher, N.S. Fedarko, Six genes expressed in bones and teeth encode the current members of the SIBLING family of proteins, Connect. Tissue Res. 44 (Suppl. 1) (2003) 33e40. [136] A. George, B. Sabsay, P.A. Simonian, A. Veis, Characterization of a novel dentin matrix acidic phosphoprotein. Implications for induction of biomineralization, J. Biol. Chem. 268 (1993) 12624e12630. [137] C. Qin, R. D’Souza, J.Q. Feng, Dentin matrix protein 1 (DMP1): new and important roles for biomineralization and phosphate homeostasis, J. Dent. Res. 86 (2007) 1134e1141. [138] E.G. Farrow, S.I. Davis, L.M. Ward, L.J. Summers, J.S. Bubbear, R. Keen, et al., Molecular analysis of DMP1 mutants causing autosomal recessive hypophosphatemic rickets, Bone 44 (2009) 287e294. [139] H. Mimura, X. Cao, F.P. Ross, M. Chiba, S.L. Teitelbaum, 1,25Dihydroxyvitamin D3 transcriptionally activates the beta 3-integrin subunit gene in avian osteoclast precursors, Endocrinology 134 (1994) 1061e1066. [140] M.M. Medhora, S. Teitelbaum, J. Chappel, J. Alvarez, H. Mimura, F.P. Ross, et al., 1 Alpha,25-dihydroxyvitamin D3 up-regulates expression of the osteoclast integrin alpha v beta 3, J. Biol. Chem. 268 (1993) 1456e1461. [141] X. Cao, F.P. Ross, L. Zhang, P.N. MacDonald, J. Chappel, S.L. Teitelbaum, Cloning of the promoter for the avian integrin beta 3 subunit gene and its regulation by 1,25dihydroxyvitamin D3, J. Biol. Chem. 268 (1993) 27371e 27380. [142] G. Andersson, E.K. Johansson, Adhesion of human myelomonocytic (HL-60) cells induced by 1,25-dihydroxyvitamin D3 and phorbol myristate acetate is dependent on osteopontin synthesis and the alpha v beta 3 integrin, Connect. Tissue Res. 35 (1996) 163e171. [143] Y. Okada, I. Morimoto, K. Ura, K. Watanabe, S. Eto, M. Kumegawa, et al., Cell-to-cell adhesion via intercellular adhesion molecule-1 and leukocyte function-associated antigen-1 pathway is involved in 1alpha,25(OH)2D3, PTH and IL-1alpha-induced osteoclast differentiation and bone resorption, Endocr. J. 49 (2002) 483e495. [144] S. Kido, D. Inoue, K. Hiura, W. Javier, Y. Ito, T. Matsumoto, Expression of RANK is dependent upon differentiation into the macrophage/osteoclast lineage: induction by 1alpha,25dihydroxyvitamin D3 and TPA in a human myelomonocytic cell line, HL60, Bone 32 (2003) 621e629. [145] C. Vincent, M. Kogawa, D.M. Findlay, G.J. Atkins, The generation of osteoclasts from RAW 264.7 precursors in defined, serumfree conditions, J. Bone Miner. Metab. 27 (2009) 114e119. [146] H.C. Blair, M. Zaidi, Osteoclastic differentiation and function regulated by old and new pathways, Rev. Endocr. Metab. Disord. 7 (2006) 23e32.
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[147] B.S. Lee, L.S. Holliday, I. Krits, S.L. Gluck, Vacuolar Hþ-ATPase activity and expression in mouse bone marrow cultures, J. Bone Miner. Res. 14 (1999) 2127e2136. [148] H.Z. Ke, H. Qi, D.T. Crawford, H.A. Simmons, G. Xu, M. Li, et al., A new vitamin D analog, 2MD, restores trabecular and cortical bone mass and strength in ovariectomized rats with established osteopenia, J. Bone Miner. Res. 20 (2005) 1742e1755.
[149] H.A. Bischoff-Ferrari, E. Giovannucci, W.C. Willett, T. Dietrich, B. Dawson-Hughes, Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health outcomes, Am. J. Clin. Nutr. 84 (2006) 18e28. [150] H.A. Bischoff-Ferrari, W.C. Willett, J.B. Wong, E. Giovannucci, T. Dietrich, B. Dawson-Hughes, Fracture prevention with vitamin D supplementation: a meta-analysis of randomized controlled trials, JAMA 293 (2005) 2257e2264.
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C H A P T E R
24 Vitamin D and the Calcium-Sensing Receptor Edward M. Brown Division of Endocrinology, Diabetes and Hypertension, Brigham and Women’s Hospital, EBRC 223A, 221 Longwood Ave., Boston, MA 02115, USA
INTRODUCTION Calcium and vitamin D have been inextricably linked for decades by their combined roles in promoting mineral ion homeostasis and the growth and remodeling of the skeleton. For much of the history of vitamin D and the knowledge of its link to calcium metabolism, however, calcium was thought to be a largely passive partner. That is, stimulation of active intestinal calcium absorption required the receptor-mediated action of the active form of vitamin D (1,25(OH)2D3), but the resultant active transport of calcium across the intestinal epithelium did not imply any regulatory role for calcium ions per se. Similarly, during bone growth and remodeling, the role of calcium ions was primarily conceptualized as contributing to the maintenance of an adequate calciumephosphate product to enable mineralization of the bony matrix. Some early studies had shown that calcium could interact with vitamin D directly to modulate cellular functions, e.g., their coordinate up-regulation of the expression of calbindin D-28K in primary chick kidney cells [1]. It was the cloning of the extracellular calcium (Ca2þo)-sensing receptor (CaSR) in 1993 [2], however, that provided a molecular target by which Ca2þo could serve as an extracellular, first messenger. Since that time, there has been rapid progress in elucidating the mechanisms by which the CaSR exerts its regulatory roles, including its structure, intracellular signaling pathways, tissue distribution, and the range of cellular functions that it controls [3]. There has also been increasing appreciation of the wide range of tissues in which both calcium, acting via the CaSR, and 1,25(OH)2D3, acting through its nuclear vitamin D receptor (VDR), regulate the functions not only of those tissues involved in but also those uninvolved in mineral ion homeostasis in both health and
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10024-1
disease [4]. This chapter will briefly review the structure and functional properties of the CaSR and how it regulates, in concert with 1,25(OH)2D3, the various cells types expressing both the CaSR and VDR that participate in the maintenance of Ca2þo homeostasis as well as bone growth and development. It will then provide selected examples of the roles of the CaSR and VDR when they are coexpressed in selected cell types uninvolved in Ca2þo homeostasis. Since the VDR, its ligands, and its actions are reviewed in great detail in other chapters in this volume, the focus of this chapter will be to compare and contrast the actions of the CaSR and VDR and, if relevant, their interactions in a number of the cell types in which they are coexpressed.
WHAT IS THE CASR? To maintain near constancy of the blood Ca2þ level, a mechanism must exist that senses small changes in Ca2þo and responds in a manner that will normalize Ca2þo [5]. The CaSR is the major Ca2þo sensor involved in maintaining Ca2þo homeostasis. It is a G-proteincoupled receptor (GPCR), whose principal physiological ligand is Ca2þo. The isolation and characterization of the CaSR from bovine parathyroid gland by molecular cloning were reported in 1993 [2]. Soon afterward, the CaSR was cloned from human parathyroid gland [6] and, subsequently, from parathyroid and/or other tissues in a variety of species, including birds and fish. The CaSR is a member of family C of the GPCRs, which also comprises the metabotropic glutamate receptors (mGluRs), the GABAB receptors, whose ligand is gamma aminobutyric acid (GABA), and receptors for taste and pheromones. An additional aminoacid- and divalent-cation-sensing receptor, GPRC6A
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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[7], is also a member of the family C GPCRs. Whether the latter is principally a calcium-modulated amino acid receptor or an amino-acid-modulated calciumsensing receptor, or whether it plays a physiologically meaningful role in normal mineral ion and skeletal physiology remains controversial, and GPRC6A will not be discussed further here. The predicted protein structure of the human CaSR, which is very similar to the CaSRs from the other species studied to date, has a large, 612-amino-acid extracellular domain (ECD) [6], followed by a 250-amino-acid transmembrane domain (TMD) containing seven transmembrane helices, a signature of the GPCRs, and, lastly, a 216amino-acid carboxyterminal (C)-tail. During its biosynthesis, the CaSR is targeted to the endoplasmic reticulum by a hydrophobic signal peptide, where it dimerizes through intermolecular disulfide bonds involving cysteines 129 and 131 within each monomer [8,9]. The receptor is then extensively glycosylated in the Golgi apparatus before reaching the cell surface in its biologically active, dimeric form. The receptor-activity-modifying proteins (RAMPs), RAMP-1 and RAMP-3, facilitate translocation of the CaSR to the cell membrane in some cells [10]. The cell surface CaSR undergoes little desensitization upon repeated exposure to elevated levels of Ca2þo, at least
in parathyroid cells. Its resistance to desensitization results, in part, from its binding to the large, actinbinding scaffold protein, filamin-A [11], and is presumably important to ensure the CaSR’s persistent presence on the cell surface, thereby enabling it to continuously monitor Ca2þo. Other binding partners of the CaSR include the Kþ channels, Kir4.1 and Kir4.2, caveolin-1, and the E3 ubiquitin ligase, dorfin, which likely participates in regulating the proteasomal degradation of the receptor [12]. Molecular modeling, using the known three-dimensional structures of the extracellular domains of several mGluRs, strongly suggests that the CaSR’s ECD has a bilobed, venus flytrap (VFT)-like structure with a cleft between the two lobes [13] (Fig. 24.1). The CaSR responds over a much smaller range of Ca2þo (
disulfide bonds at C129, C131 Lobe 1
Lobe 1
Ca2+binding site
Ca2+binding site Lobe 2
Lobe 2
Calcimimetics
FIGURE 24.1 Model of the predicted structure of the human CaSR. Shown are two monomers of the receptor, each of which has two lobes, which are linked by disulfide bonds involving cysteines 129 and 131 on each monomer. The lower part of the figure shows the seven transmembrane domains that enable transmission by the activated receptor of the extracellular Ca2þo signal to the receptor’s G proteins and other intracellular effectors. Red segments of the receptor’s extracellular domain indicate alpha helices. The locations of a key calcium-binding site in the crevice between the two lobes of each monomer are shown, as are the separate sites for calcimimetics, which bind within the receptor’s transmembrane domains. From Huang et al. 2007 J Biol Chem 282:19000e19010, with permission. Please see color plate section.
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facilitates the conversion of the inactive form of the CaSR ECD to the active conformation. Subsequent conformational changes in the TMDs and intracellular domains are presumed to initiate signal transduction. Additional putative binding sites for Ca2þ have been identified by biophysical and biochemical studies that may contribute to positive cooperativity that generates the steep slope of the CaSR’s activation by Ca2þo [14]. Definitive identification of the Ca2þ-binding sites that participate in activation of the CaSR, however, awaits determination of the structure of its ECD by X-ray crystallography. Even this approach may not identify these sites definitively owing to the low affinity and presumably rapid off rates of the CaSR’s calcium-binding sites. In addition to the Ca2þ-binding sites in the receptor’s ECD, a binding site(s) likely resides within the receptor’s TMD, including extracellular loops between the transmembrane helices, since a headless receptor, e.g., lacking the entire ECD, still responds to Ca2þ under some conditions [13,16]. The biologically active, cell surface CaSR, upon binding Ca2þo, stimulates the G-proteins, Gq/11, Gi, and G12/13, which stimulate the activity of phospholipase C (PLC) (thereby increasing the formation of diacylglycerol and IP3), inhibit adenylate cyclase and activate Rho kinase, respectively [12]. In addition to directly inhibiting adenylate cyclase via Gi, the CaSR can also reduce cAMP levels indirectly by elevating Ca2þI, thereby decreasing the activity of Ca2þ-inhibitable adenylate cyclase and/ or stimulating phosphodiesterase [17]. In rare cases, the CaSR activates Gs, the G-protein-stimulating adenylate cyclase [18]. The receptor controls additional diverse intracellular signaling systems, including mitogenactivated protein kinases (MAPKs) (e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, and c-jun N-terminal kinase (JNK)), phospholipases A2 and D, and the epidermal growth factor (EGF) receptor, as reviewed recently [12]. These various intracellular signaling systems mediate cell-specific regulation by the CaSR. Several factors up-regulate expression of the CaSR gene, including Ca2þo [19] and calcimimetics [20] (drugs activating the receptor by an allosteric mechanism e see below), both of which do so by activating the CaSR, vitamin D (through vitamin-D-responsive elements (VDRE) in the two promoters of the CaSR gene) [21] and the cytokines interleukin-1b [22] and interleukin-6 [23]. Since the CaSR up-regulates the VDR gene [20,24], there is the possibility of synergistic interactions between the VDR and CaSR, as discussed in more detail later. In this scenario, activation of the CaSR increases its own expression and that of the VDR; the latter could potentiate vitamin D action via increased VDR occupancy, thereby further enhancing CaSR expression and action, and so forth.
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Ca2þo isn’t the only CaSR activator. Various di- (i.e., Mg2þ and Sr2þ) and trivalent (La3þ and Gd3þ) cations also activate the receptor, as do positively charged organic compounds, including polyamines (e.g., spermine), aminoglycoside antibiotics (i.e., neomycin), and polyarginine [25]. These polycationic agonists are termed type I agonists. They can activate the receptor without the need for extracellular Ca2þ to be present. Type II agonists, to the contrary, require that some Ca2þo be present, viz., in the millimolar range, in order to activate the CaSR [26]. Type II agonists comprise various L-amino acids, especially aromatic amino acids, and the low-molecular-weight, allosteric CaSR activators known as calcimimetics [26,27]. The physiological importance of the activation of the CaSR by amino acids is uncertain, but it takes place at physiologically relevant levels and may serve to coordinate protein/amino acid and calcium metabolism [27]. One calcimimetic, CinacalcetÒ (also called SensiparÒ), is employed clinically for suppressing severe secondary hyperparathyroidism in patients with end-stage renal disease receiving hemodialysis treatment [28]. As discussed later and in more detail elsewhere in this volume (see Chapter 70), calcimimetics are most commonly combined with vitamin D receptor activators (VDRAs), to achieve desired therapeutic endpoints in this patient population such as a serum intact PTH level of 150e300 pg/ml in dialysis patients. Calcimimetics interact with the CaSR’s transmembrane domain, including glu837, which binds the positively charged amino group that is present in the linker between the two hydrophobic ends of the molecule [13]. The latter interact with hydrophobic residues within the receptor’s TMD. Amino acids, in contrast, bind to the ECD, likely in close proximity to the binding site for Ca2þo that is present in the crevice between the two lobes of the VFT [29,30]. Calcilytics, which are allosteric inhibitors of the CaSR, have also been developed [31]. They interact with a site in the TMD that likely overlaps with that for calcimimetics. While calcimimetics appear to stabilize the active conformation of the CaSR, calcilytics likely do just the opposite, stabilizing the receptor’s inactive conformation.
OVERVIEW OF ROLES OF CASR AND VDR IN MAINTAINING CA2DO HOMEOSTASIS The Ca2þo homeostatic system has three key components: (1) the cells, tissues, and organs transporting Ca2þ out of or into the extracellular fluid (ECF) (kidney, bone, and intestine (and, in some stages of the life cycle, placenta and breast)); (2) hormones regulating these fluxes (principally parathyroid hormone (PTH), calcitonin (CT), and 1,25(OH)2D3); and (3) Ca2þo-sensors
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(primarily the CaSR) controlling the secretion/production of those hormones or the Ca2þ fluxes themselves [5] (see elsewhere in this volume). Of these three Ca2þo-regulating hormones, PTH is a Ca2þo-elevating hormone whose secretion is stimulated by low Ca2þo and inhibited by high Ca2þo. 1,25(OH)2D3 is also a Ca2þo-elevating hormone produced in the renal proximal tubule in response to PTH, hypocalcemia, and hypophosphatemia [32]. 1,25(OH)2D3 also feeds back to inhibit its own synthesis via the VDR. CT is a Ca2þolowering hormone secreted by the thyroidal C-cells in response to hypercalcemia [32]. A more recently discovered hormone regulating both calcium and phosphate homeostasis is fibroblast growth factor (FGF)-23 (see Chapter 42). It is a potent phosphaturic hormone released principally by osteocytes (osteoblasts encased in bone during bone formation) in response to 1,25 (OH)2D3 and hyperphosphatemia [33e35]. FGF-23 also inhibits both 1,25(OH)2D3 production and PTH secretion. The rapid developments regarding the roles of FGF-23 in phosphorus and calcium metabolism are detailed in recent reviews [33e35]. Hypocalcemia evokes PTH secretion by the parathyroid glands (Fig. 24.2). PTH, in turn, has three key
homeostatic actions on the kidney: stimulating (1) distal tubular calcium reabsorption, (2) phosphaturia, and (3) synthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) from its largely inactive precursor, 25-hydroxyvitamin D3. Hypocalcemia per se also has a direct stimulatory action on 1,25(OH)2D3 production in the proximal tubule that is likely CaSR-mediated (see discussion below on CaSR and VDR actions in the kidney) [36,37]. The renal calcium-retaining action of PTH occurs in both the cortical thick ascending limb of Henle’s loop (cTAL) as well as in the distal convoluted tubule (DCT) [32,38]. As a result there is, in effect, a “resetting” of calcium reabsorption by the kidney, shifting the curve relating serum calcium to urine calcium to the right so that more calcium is reabsorbed at a given level of extracellular calcium. Increased circulating levels of 1,25 (OH)2D3 have several physiological effects: (1) stimulating gastrointestinal absorption of calcium, (2) enhancing reabsorption of calcium in the DCT, (3) promoting release of skeletal calcium in conjunction with PTH, (4) directly inhibiting PTH production, and (5) inhibiting its own synthesis in the proximal tubule via the VDR as noted earlier [5,32]. The relative physiological importance of these various actions will be
FIGURE 24.2 Schematic diagram of the principal hormones and tissues participating in Ca2þo homeostasis. In response to hypocalcemia, the
parathyroid gland secretes more PTH, which acts on the kidney to increase phosphate excretion, enhance calcium reabsorption, and stimulate the synthesis of 1,25(OH)2D3. The latter acts on the intestine to increase absorption of calcium and phosphate. 1,25(OH)2D3 and PTH act together to increase net release of calcium from bone, although there are rapid changes in the fluxes of calcium into and out of bone that may participate in calcium homeostasis and take place largely independent of PTH and vitamin D status. Increased movement of calcium into the ECF from intestine and bone and reduced calcium excretion act to normalize Ca2þo. Shown with arrows are additional direct actions of Ca2þo and phosphate on various homeostatic processes. The actions of calcium are likely mediated by the CaSR FGF-23 (not shown) is a key phosphatelowering hormone and contributes to the integration of calcium and phosphate homeostasis by inhibiting both 1,25-dihydroxyvitamin D synthesis and PTH secretion (see text). From Brown et al. 1995 N Engl J Med 333:234e40, with permission.
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discussed later in the context of mouse models in which the CaSR and/or VDR have been inactivated (”knocked out”) by genetic means. Movement of calcium into the ECF from GI tract and bone, combined with greater renal tubular calcium reabsorption, will, except with severe calcium deficiency, normalize Ca2þo.
Defense Against Hypercalcemia The response of the Ca2þo homeostatic system to hypercalcemia is generally considered to be a sequence of events largely opposite to those observed with hypocalcemia. That is, high Ca2þo directly inhibits PTH secretion, directly and indirectly (e.g., via reduced circulating PTH levels) decreases 1,25(OH)2D3 synthesis, and stimulates CT secretion. The reduction in PTH and increase in CT diminish the formation and activity of boneresorbing osteoclasts [39], producing net movement of Ca2þ into bone. Concomitantly, the suppression of PTH secretion reduces renal tubular Ca2þ reabsorption in both cTAL [5] and DCT [40]. The decrease in 1,25 (OH)2D3 synthesis also suppresses Ca2þ reabsorption in the DCT [40], reduces bone resorption by diminishing the formation and activity of osteoclasts, and decreases intestinal Ca2þ absorption [5,40]. The resulting reduction in net Ca2þ release from bone, combined with reduced intestinal absorption and renal tubular reabsorption of Ca2þ, normalizes Ca2þo. Recent studies, however, suggest the need to update the scenario just described for the defense against hypo- and hypercalcemia. Mice that are hypoparathyroid owing to targeted inactivation of the PTH gene (PTHe/e) are hypocalcemic, to a degree equivalent to that present in mice lacking both the PTH gene and the CaSR (CaSRe/ePTHe/e) [41]. This result documents the importance of the CaSR-mediated regulation of PTH secretion by Ca2þo in the defense against hypocalcemia, effectively providing a “floor” for Ca2þo that serves as a robust protection against hypocalcemia. Moreover, even the loss of the full-length CaSR in the kidneys of the CaSRe/ePTHe/e mice, which reduces Ca2þ excretion substantially [41], is insufficient to prevent hypocalcemia in this setting. When high concentrations (e.g., 1e2%) of calcium are subsequently added to the drinking water of the mice, the serum calcium concentration in the PTHe/e mice increases to a high-normal level (10e11 mg/dl), equivalent to that in wild-type mice treated in the same way. In contrast, the serum calcium concentration in the CaSRe/ePTHe/e mice increases to a level (~14 mg/dl) dramatically higher than that in the other two genotypes [41]. Similarly, the double knock-out mice demonstrate exaggerated hypercalcemic responses to other calcium “loads” (e.g., 1,25 (OH)2D3 administration or PTH infusion) [42]. Therefore, the CaSR, independent of its regulation of PTH
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secretion, serves as an important “ceiling” for Ca2þo, acting, at least in part, through stimulation by high Ca2þo of renal calcium excretion and CT secretion (CT has a much more potent hypocalcemic action in rodents than in humans) [41]. Thus the various cells and tissues participating in Ca2þo homeostasis exhibit some degree of specialization in their contribution to the defenses against hypo- and hypercalcemia. To summarize, in the absence of PTH, enhanced renal tubular calcium reabsorption and decreased CT secretion cannot prevent hypocalcemia regardless of the presence or absence of the CaSR. CaSR-mediated stimulation of renal calcium excretion and CT secretion, in contrast, are key players in the defense against hypercalcemia, regardless of the presence or absence of PTH. Therefore, while the suppression of PTH secretion in normal animals no doubt contributes to preventing hypercalcemia, it is not essential.
Additional Mechanisms Maintaining Ca2Do Homeostasis In addition to the mechanisms just described, it is likely that there is additional homeostatic control of calcium fluxes into and out of bone. Mammals have a substantial capacity to “buffer” acute hypo- or hypercalcemic stresses over a timeframe of minutes to hours, even in the total absence of PTH. For example, administration of the calcium-chelating agent, EGTA, which effectively irreversibly sequesters calcium in the ECF, only transiently lowers the serum calcium concentration in rats studied 60 minutes after parathyroidectomy [43]. Thereafter, there is restoration, over 1e2 hours, of the serum calcium concentration present prior to the EGTA injection. The amount of calcium that must be mobilized to restore serum ionized calcium concentration to its former level, given the amount sequestered by EGTA, strongly supports bone as the source of the calcium. Similarly, administration of calcium intravenously in the same model induces only transient hypercalcemia (for < 30 minutes), followed by a return over 1e2 hours to the preinjection level [43]. A possible role for the CaSR in these observations was suggested by experiments showing that the increase in serum calcium concentration following the initial decrement caused by EGTA injection was blunted in animals that had received the calcimimetic, R-568 [43]. Ideally another control in these experiments would have been treatment of the animals with the inactive S-isomer of the calcimimetic, since both the R- and S-isomers can block calcium channels independent of the CaSR. Nevertheless, an action of the CaSR in bone, perhaps on the bone-lining cells that have been postulated to serve as a functional membrane-controlling calcium fluxes into and out of bone [44], could serve to acutely regulate movement of
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calcium into and out of bone in a PTH-independent manner. Vitamin D status also does not appear to impact the capacity to respond rapidly to induced hypocalcemia [45].
THE CASR AND VDR IN TISSUES PARTICIPATING IN CA2DO HOMEOSTASIS Several of the tissues participating in Ca2þo homeostasis express both the CaSR and VDR, and activation of these two receptors exerts additive/synergistic or, in some cases, antagonistic effects on the functions of these cell types. This section will delineate how the presence of the two receptors modulates the function of these cells and how this impacts their roles in maintaining Ca2þo homeostasis. This discussion will focus principally on the classical nuclear VDR. An abundant literature exists related to nongenomic actions of vitamin D [46], but this topic is beyond the scope of this discussion (see also Chapter 15).
The CaSR and VDR in the Parathyroid Both high Ca2þo and 1,25(OH)2D3 inhibit important parameters of parathyroid function that play key roles in Ca2þo homeostasis. High Ca2þo suppresses PTH secretion [26], PTH gene expression [47], and parathyroid cellular proliferation [5], and stimulates PTH degradation [48]. Like the CaSR, the VDR also suppresses PTH secretion [49], PTH gene expression [50], and parathyroid cellular proliferation [51e53]. The 1,25(OH)2D3induced suppression of PTH gene transcription likely contributes substantially to the 1,25(OH)2D3-evoked reduction in PTH secretion. Nevertheless, 1,25(OH)2D3 has rapid actions on the parathyroid cell, e.g., activating phosphoinositide turnover [54] and increasing the cytosolic calcium concentration [55], which are presumably mediated by a cell surface receptor and resemble those produced by the CaSR; accordingly, these might contribute to the inhibition of PTH secretion by 1,25 (OH)2D3. The effects of 1,25(OH)2D3 on parathyroid function in vivo are likely due not only to the actions of 1,25(OH)2D3 produced by the kidney, but also to the effects of local production of the hormone owing to the presence of the 1-hydroxylase in parathyroid cells. The latter converts 25-hydroxyvitamin D3 to 1,25 (OH)2D3, which can then act locally [56]. The mediatory role of the CaSR in the regulation of PTH secretion by Ca2þo has been proven by demonstrating that calcimimetic CaSR activators, such as NPS R-467 or R-568, acutely inhibit (within minutes) PTH secretion in vivo and in vitro [57]. These actions are too rapid to be accounted for by changes in the synthesis or degradation of PTH. The
molecular mechanisms by which the CaSR regulates PTH secretion remain elusive. Knocking out both of the G proteins, Gq and G11 [58], whose principal actions are to activate phospholipase C, in a mouse model results in a phenotype reminiscent of neonatal severe hyperparathyroidism (NSHPT), which in mice and humans results from homozygous knockout of the CaSR gene [59,60]. These two G proteins, therefore, must be key downstream effectors of the CaSR in the regulation of PTH secretion, presumably by activating phospholipase C. Production of arachidonic acid by phospholipase A2 has also been suggested to be a key downstream mediator of calciuminduced inhibition of PTH secretion [61], owing, at least in part, to conversion of arachidonic acid to products of the 12- and 15-lipoxygenase pathways [62]. A possible “distal” mechanism involved in the suppression of PTH secretion in response to CaSR-mediated changes in the relevant second-messenger pathway(s) is rearrangement of the cytoskeleton to a configuration that physically blocks the access of PTH-containing secretory vesicles to the plasma membrane [63]. A similar mechanism has been proposed to account for reduced expression of aquaporin-2 in the plasma membrane in the inner medullary collecting duct in response to hypercalciuria [64]. In addition to inhibiting PTH secretion per se, the CaSR promotes the degradation of full-length, biologically active PTH1-84 to PTH7-84 and smaller carboxyterminal fragments, thereby reducing the secretion of intact PTH still further during hypercalcemia and, conversely, increasing it during hypocalcemia [48]. In addition to their effects on PTH secretion, calcimimetics also decrease the levels of the mRNA encoding preproPTH [47,65], proving the CaSR’s role in regulating this process. The change in the level of PTH mRNA results from an alteration in preproPTH mRNA stability rather than in transcription of the PTH gene per se. Elegant studies by Naveh-Many and Silver have elucidated the molecular mechanisms underlying the control of the stability of preproPTH mRNA by calcium (Fig. 24.3). Exposure of parathyroid cells to elevated extracellular calcium concentrations activates the CaSR and, through a pathway involving stimulation of calmodulin (CaM) and protein phosphatase 2B, post-translationally modifies and decreases the binding of the preproPTH mRNA stabilizing factor, AU-rich factor (AUF-1), to an AU-rich element in the 30 untranslated region (UTR) of the preproPTH mRNA (Fig. 24.3) [66]. This enables binding of a second, destabilizing protein, K-homology splicing regulator protein (KSRP), to the same site. KSRP then interacts with and is activated by the peptidylprolyl isomerase, Pin-1, and, in turn, recruits the endoribonuclease, PMR1, part of the RNA-cleaving exosome, which degrades preproPTH mRNA by cleaving it internally (Fig. 24.3) [67,68] (see also Chapter 27).
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24.3 Mechanism by which Ca2þo, acting via the CaSR, decreases the expression of the mRNA for preproPTH. The stability of the mRNA is enhanced in the presence of low Ca2þo by the AU-rich binding factor as well as by the protein, upstream of N-Ras (Unr). A key destabilizer of the mRNA is KSRP, which in the presence of high Ca2þo binds to the same site in the 30 untranslated region of the PTH gene as AUF1 and displaces the latter. The petidylprolyl isomerase, Pin-1, then activates KSRP, and the latter recruits the endonuclease, PMR1, and the exosome, which cleaves the mRNA internally. See text for details. FIGURE
Unlike the CaSR, 1,25(OH)2D3 reduces PTH gene expression by a transcriptional rather than a posttranscriptional mechanism [69] (Fig. 24.4). The activated VDR acts on a negative responsive element in the 50 untranslated region of the gene [70]. Recent studies have elucidated in more detail the molecular mechanism underlying the negative regulation of the PTH gene by 1,25(OH)2D3 [71]. Transrepression of the human preproPTH gene is mediated by a heterodimer of the VDR and retinoic acid X receptor (RXR), but not through direct binding of the heterodimer to the negative vitamin-D-responsive element (nVDRE), which resides within nucleotides -87 to -60 in the 50 UTR of the human PTH gene. Instead, VDR/RXR binds to another protein, VDR interacting repressor (VDIR), and it is the latter that binds to the nVDRE, which has also been termed an Ebox-type element. VDIR recruits histone deacetylase-2 (HDAC-2) as a corepressor to mediate repression of the hPTH gene [71] (also see Chapter 27).
SP1, NF-Y, ?other = high PTH gene expression
Studies in humans with neonatal severe hyperparathyroidism (NSHPT) [60] owing to the presence of homozygous inactivating mutations in the CaSR or in mice homozygous for knockout of the CaSR [59] have established the importance of the CaSR in regulating parathyroid cellular proliferation. In both cases, there is marked parathyroid cellular proliferation and glandular enlargement despite severe hypercalcemia, documenting the CaSR’s essential role in tonically inhibiting proliferation of the parathyroid cell. Studies in uremic rat models, principally by Slatopolsky and coworkers, have shed light on the mechanisms by which calcium, acting via the CaSR, regulates parathyroid proliferation (Fig. 24.5). Induction of the cyclin-dependent kinase inhibitor, p21WAF1, and suppression of the growth factor, TGF-a, and of its receptor, the epidermal growth factor receptor (EGFR), which are both up-regulated in this setting, have been identified as key elements in the mechanism by which high dietary calcium induces arrest of
Control of parathyroid growth by CaSR
Transcriptional Start site
(+) (+) (+) SP1 NF-Y NF-Y
(A)
hPTH gene
Ca 2+o [1,25(OH)2D3-VDR-RXR]-VDIR- E-box (nVDRE) ( )
FIGURE 24.4 Mechanism by which 1,25(OH)2D3, acting via the nuclear VDR, inhibits PTH gene transcription. A constitutively high expression of the PTH gene is driven by factors such as the transcription factors, SP1 and NF-y. 1,25(OH)2D3 inhibits transcription by binding to the VDR-RXR heterodimer. The latter binds to the vitaminD-interacting protein, VDIR, and it is the VDIR that binds to a DNA element called an E-box and inhibits gene transcription. Distances between DNA elements are not drawn to scale. See text for details. VDIR is prebound to the gene and promotes activation. VDR binding/ tethering to DNA-bound VDIR down-regulates the latter’s activity and thus suppresses PTH expression.
=
Balance of cell cycle activators and inhibitors
TGF-
(-) EGFR
(+) CaSR PT growth
p21
(B) (2) Ca 2+o
(-) CaSR
ET-1
(+) ET-1R 2þ
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Mechanism by which high Ca o, acting via the CaSR, (A) inhibits or (B) stimulates parathyroid cellular proliferation. (A) High Ca2þo, like 1,25(OH)2D3, decreases the expression of TGF-a, an activator of the epidermal growth factor receptor, and reduces expression of the cyclin-dependent kinase inhibitor, p21. (B) In addition to producing effects opposite to those in (A), low Ca2þo increases the expression of endothelin-1, ET-1, which stimulates the endothelin receptor, ET-1R, activating cellular proliferation. See text for details.
FIGURE 24.5
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parathyroid growth in uremic rats [72] (Fig. 24.5A). Another mechanism that may participate in this setting is an endothelin-1-mediated proliferative pathway [73] (Fig. 24.5B). It is difficult to carry out similar studies in nonuremic animals because of their much slower rate of parathyroid cellular proliferation, but it seems likely that similar mechanisms come into play. In human parathyroid tumors, down-regulation of the cyclindependent kinase inhibitors, p21 and p27, may also contribute to dysregulation of parathyroid growth in both primary and secondary hyperparathyroidism [74]. The second messenger pathway(s) linking the CaSR to the control of parathyroid cellular proliferation have not yet been elucidated. Vitamin D has also been recognized for several decades as an inhibitor of parathyroid cell growth. It has been thought to play a particularly important role in the setting of renal insufficiency, where reduced synthesis of 1,25(OH)2D3 [75], combined with decreased expression of the VDR in the parathyroid (owing, at least in part, to the fact that 1,25(OH)2D3 up-regulates its own receptor), will diminish VDR signaling in the parathyroid glands. For this reason, administration of 1,25 (OH)2D3 or other vitamin D receptor activators (VDRAs) has been an important part of the therapy of secondary hyperparathyroidism in patients with chronic kidney disease [76] (see Chapter 70). What is the mechanism underlying the antiproliferative action of 1,25(OH)2D3 in the parathyroid? In a uremic rat model, administration of 1,25(OH)2D3 or its analogs inhibits parathyroid cell proliferation by inducing p21 expression [72] and reducing the content of TGF-a [77] (Fig. 24.6). Therefore, the CaSR and VDR utilize similar pathways in regulating parathyroid cellular proliferation, although there are presumably second messenger pathways between the CaSR and its control of proliferation that are distinct from the nuclear, VDR-mediated regulation of these processes. There are interesting interactions between the regulation of the CaSR and VDR in the parathyroid that may be of importance in understanding how these receptors Control of parathyroid growth by VDR
1,25(OH)2D3
= (+) VDR
Balance of cell cycle activators and inhibitors
TGFp21
PT growth
FIGURE 24.6 Mechanism by which 1,25(OH)2D3 inhibits parathyroid cellular proliferation. 1,25(OH)2D3, acting via the VDR, suppresses parathyroid cellular proliferation by decreasing the level of TGF-a, an activator of the epidermal growth factor receptor, and increasing expression of the cyclin-dependent kinase inhibitor, p21. See text for details.
participate in the regulation of mineral ion metabolism in health and/or disease. Activation of the CaSR by increases in serum calcium or calcimimetics up-regulates the CaSR in some [19,20] but not all studies [78]. Activating the CaSR also up-regulates the VDR [20,78]. Moreover, 1,25(OH)2D3 up-regulates not only its own receptor, but also the CaSR [21,78]. Therefore, for any functions of parathyroid cells (or other cells) that are regulated by the CaSR and VDR, there is the opportunity for signal amplification and synergy between the actions of Ca2þo and 1,25(OH)2D3 based on these interactions. For instance, up-regulation of the CaSR by the activated VDR could increase CaSR signaling without a change in the level of Ca2þo owing to a greater number of occupied receptors at any given level of Ca2þo as a result of the increase in CaSR expression. What is the relative importance of the CaSR and VDR in regulating the overall level of parathyroid function (i.e., taking into account the net effect of changes in PTH secretion, expression of the preproPTH gene, and parathyroid cell mass)? The CaSR can clearly regulate the secretion of PTH over a timeframe as short as seconds to minutes and is the dominant contributor to acute changes in secretory rate. Over a longer timeframe of 3 weeks, both vitamin D deficiency and hypocalcemia modulate mRNA levels for preproPTH in the rat, although hypocalcemia of ~6 mg/dl is a more powerful stimulus to increased expression of preproPTH than is vitamin D deficiency [79]. Most of the changes in preproPTH mRNA in this study were the result of alterations in gene expression rather than in parathyroid cell number. Administration of pharmacological doses of 1,25(OH)2D3, however, markedly suppressed preproPTH levels by >90% at 48 h after the dose [69]. Recent studies using mouse knockout models, however, have yielded surprising results with regard to the relative importance of the CaSR and VDR in regulating parathyroid gland function in vivo. As noted earlier, homozygous knockout of exon 5 of the CaSR in the mouse results in striking hyperparathyroidism with marked increases in both PTH levels and parathyroid gland size [59]. It should be noted that, if anything, this mouse model likely underestimates the impact of losing the CaSR on parathyroid function, because knockout of exon 5 of the CaSR results, in some tissues, in production of an alternatively spliced CaSR lacking exon 5 (which encodes part of the CaSR ECD) that can apparently still signal [80]. The VDR, therefore, seemingly has limited capacity to compensate for loss of the CaSR in this animal model. Parenthetically, while a clear phenotype as seen here establishes the importance of the gene that was knocked out, the lack of a phenotype does not mean that the gene of interest serves no function in vivo, but rather that its function is nonessential, and
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its absence can likely be compensated for by one or more other genes. In contrast, studies in mice deficient in 1,25(OH)2D3 [81] or in the VDR [82], or both, have shown that the CaSR can effectively compensate for loss of the nuclear vitamin D signaling pathway with respect to the control of parathyroid function. VDR (VDRe/e), 1a-hydroxylase [1a(OH)asee/e] and double knockout mice [VDRe/ e 1a(OH)asee/e] mice all develop markedly elevated levels of PTH and parathyroid enlargement [81]. Administration of a calcium-rich “rescue” diet, however, normalizes the serum calcium concentration and restores PTH levels to normal in these three genotypes [81,82]. Thus hypocalcemia rather than vitamin D deficiency per se was apparently the dominant contributor to the high PTH levels. Parathyroid gland size was markedly enlarged in all three mutant genotypes of mice when hypocalcemic. In the VDRe/e mice, the rescue diet, when administered beginning early in life, completely prevented the parathyroid enlargement, indicating that hypocalcemia rather than loss of vitamin D signaling was a key contributor to parathyroid cell growth in this setting. In the 1a(OH) asee/e mice, however, the rescue diet did not fully normalize parathyroid gland size, but administration of 1,25(OH)2D3 did so [81]. Thus in this latter model both 1,25(OH)2D3 and calcium contribute to inhibition of parathyroid cell growth in the context of an intact VDR and CaSR, confirming studies reviewed earlier that both the VDR and CaSR participate in the control of parathyroid cellular growth. The capacity of the CaSR alone to mediate nearly complete inhibition of parathyroid growth in the VDRe/e mice remains unexplained, but perhaps up-regulation of the CaSR or of CaSR signaling can compensate for loss of the VDR in this setting. In addition, it is possible, since 1,25 (OH)2D3 levels are frankly high in the VDRe/e mice, that 1,25(OH)2D3 can inhibit parathyroid cell growth in a VDR-independent manner [81]. An elegant study addressing this issue further was carried out recently by Meir et al., who deleted the VDR specifically in the parathyroid gland [83]. In this way, the effects of the VDR on the parathyroid could be isolated from systemic changes in mineral ion homeostasis, e.g., resulting from loss of the VDR in kidney and intestine. The mice with parathyroid-specific ablation of the VDR exhibited modest (~30%) increases in serum PTH but had no change in the number of proliferating parathyroid cells and had exhibited normal serum calcium concentrations. Therefore, although administration of 1,25(OH)2D3 can clearly inhibit PTH gene transcription and parathyroid cellular proliferation in vivo and in vitro, under normal physiological conditions it apparently has only a limited role in parathyroid physiology in vivo. These results do not, however, mean
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that 1,25(OH)2D3 does not have a useful role therapeutically, particularly in the secondary hyperparathyroidism of renal insufficiency (see Chapter 70). In addition, it would be of interest to assess CaSR signaling efficiency in mice with deficient signaling through the vitamin D pathway to determine to what extent compensatory change in the CaSR and its downstream signaling elements contribute to the observed phenotypes in the knockout mouse models just discussed.
The CaSR and VDR in the C-cell Studies in CaSR knockout mice have proven the receptor’s role in stimulating CT secretion by showing blunting of the high Ca2þ-induced increase in circulating CT levels in CaSRþ/e mice [84] and near total loss of Ca2þ-evoked CT secretion in CaSRe/ePTHe/e mice [41]. A plausible model for the mechanism underlying CaSR-stimulated CT secretion [85] involves CaSR-mediated activation of a nonselective cation channel, which depolarizes the cells, thereby activating voltage-sensitive calcium channels and producing the increase in cytosolic calcium concentration that activates exocytosis. In contrast to the parathyroid cell, in which the CaSR and VDR exert the same biological actions (e.g., inhibition of parathyroid cell proliferation), albeit by distinct mechanisms, the VDR in the C-cell inhibits rather than stimulates CT gene expression [86,87]. In the second of these two studies, 1,25(OH)2D3 produced about a 60% inhibition of cAMP-stimulated transcriptional activity via an nVDRE located within nucleotides e920 and e829 in the 50 flanking DNA of the CT gene. The physiological significance of this bidirectional control of CT secretion is unknown.
The CaSR and VDR in the Kidney The CaSR and Regulation of 1-Hydroxylation of 25-Hydroxyvitamin D in the Proximal Tubule Both the CaSR [88] and VDR [89] are widely expressed in the kidney. The three principal sites that will be discussed here in this regard are the proximal tubule, where the CaSR and VDR regulate 1-hydroxylation of 25-hydroxyvitamin D3, the cortical thick ascending limb of Henle’s loop (cTAL), a key site of regulated calcium reabsorption, and the distal convoluted tubule/connecting segment (DCT/CNT), where the CaSR and VDR regulate tubular reabsorption of calcium. As in the parathyroid, 1,25(OH)2D3 administration in the rat up-regulates expression of the CaSR in the kidney [21], although the site(s) where this upregulation takes place was not identified. Furthermore, high calcium concentration up-regulates the VDR in
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a proximal tubular cell line [90]. Therefore, similar to the situation in the parathyroid, there is the possibility of synergistic interactions between the CaSR and VDR, whereby activation of one receptor up-regulates the other. An important aspect of the regulation of the 1-hydroxylation of 25-vitamin D3 in the proximal tubule is the VDR-mediated inhibition of the gene that 1-hydroxylates 25-hydroxyvitamin D3, the 25-hydroxyvitamin D 1a-hydroxylase (CYP27B1). It is one of the five factors that physiologically regulate this gene in vivo: 1,25 (OH)2D3 itself, the serum calcium concentration (hypocalcemia stimulates and hypercalcemia inhibits), the serum phosphate concentration (hypophosphatemia stimulates and hyperphosphatemia inhibits), FGF-23, which inhibits, and PTH, which stimulates CYP27B1. Since hypocalcemia stimulates PTH release, it can upregulate CYPB27B1 indirectly through the associated changes in circulating PTH levels. The available evidence, however, indicates that there is also direct regulation of the 1-hydroxylase by Ca2þo and that this regulation is likely mediated by the CaSR. For some time, there has been both in vivo [36,37] and in vitro [91] evidence that changes in serum and medium calcium concentration, respectively, directly modulate the 1-hydroxylation of 25-hydroxyvitamin D3. To avoid the confounding effects of calcium-induced changes in circulating PTH levels in vivo, Treschel et al. and Weisinger et al. utilized thyroparathyroidectomized rats infused with PTH to “clamp” the level of circulating PTH. Alterations in circulating 1,25(OH)2D3 levels were then determined in response to changes in the serum calcium concentration. The steep inverse sigmoidal relationship between serum calcium and 1,25(OH)2D3 levels [37] was similar to that for the relationship of PTH to the serum ionized calcium concentration. Similar results were observed in vitro using an SV40-transformed human proximal tubule cell line, namely stimulation of 1,25(OH)2D3 production at low calcium concentration and inhibition at high calcium [91]. More recent evidence has implicated the CaSR as the mediator of the direct effects of extracellular calcium concentration on the 1-hydroxylation of 25-hydroxyvitamin D3. Maiti and Beckman used a proximal tubular cell line, HK-2G, in which they had shown that high extracellular calcium inhibits the expression of CYP27B1 [90], to demonstrate that high Ca2þo up-regulates the expression of the VDR [24]. This effect was mediated by a p38 mitogen-activated protein kinase (MAPK)dependent mechanism [92]. P38 is one of several MAPKs activated by the CaSR in various cell types. Since knocking down the CaSR with siRNA prevented the high Ca2þo-evoked increase in VDR expression, the latter was CaSR-mediated [92]. The use of siRNA to prove the CaSR’s role in the concomitant inhibition of
the expression of CYP27B1 was not reported [90]. It also remains to be determined whether the high Ca2þstimulated, CaSR-mediated increase in VDR expression in the proximal tubule is sufficient by itself to account for the accompanying suppression of the expression of CYP27B1. The CaSR and Regulation of Renal Tubular Reabsorption of Calcium Favus et al. have developed an interesting model, the genetically hypercalciuric rat, which was created by repeated inbreeding of Sprague-Dawley rats with the highest urinary calcium excretion in any given generation. The hypercalciuric rats have increased intestinal calcium absorption and normal serum 1,25 (OH)2D3 concentrations [93]. The level of the VDR in the intestine of the hypercalciuric rats is twice that in the control rats, and there is no difference in the affinity of the receptor in the two groups of rats. The level of the VDR is also elevated in the kidneys of the genetically hypercalciuric rats [94]. The increased expression of the intestinal VDR provides a likely explanation for the hyperabsorption of calcium in the intestine, e.g., via up-regulation of the vitamin-Dstimulated transcellular calcium transport system, but not necessarily for the hypercalciuria, unless the latter is simply the result of absorptive hypercalciuria. However, subsequent studies [95] revealed that the level of CaSR protein was increased fourfold in the kidneys of hypercalciuric rats compared to controls. Furthermore, the level of the CaSR protein increased further and to a higher level in the hypercalciuric rats after treatment with 1,25(OH)2D3, reflecting the known VDR-mediated up-regulation of the CaSR. While the site within the kidney where the increase in the level of CaSR in the hypercalciuric rats takes place was not identified, the highest level of expression of the CaSR in the kidney is in the cTAL, an important site where the VDR [89] and CaSR [88] are both localized and in which the CaSR inhibits the reabsorption of calcium by a CaSR-mediated mechanism when the level of calcium in the blood is elevated. However, despite the importance of this nephron segment in the renal handling of calcium by the CaSR (and PTH), there are little or no data on the functional significance of the VDR in the cTAL. The distal convoluted tubule (DCT) follows immediately after the cTAL, and the connecting segment (CNT) follows the DCT and precedes the cortical collecting duct; both DCT and CNT play important roles in regulating renal tubular reabsorption of calcium. As in the cTAL [96], PTH stimulates the reabsorption of Ca2þ in the DCT [97]. It was the cloning of the TRPV5 calciumpermeable ion channel [98], however, which is the key apical Ca2þ channel involved in the transcellular
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transport of calcium in the distal tubule, that clarified the biological relevance of 1,25(OH)2D3-stimulated reabsorption of calcium, as well as its molecular mechanism. Previously, it had been difficult to show convincingly a quantitatively important action of 1,25(OH)2D3 on calcium reabsorption in the DCT in vivo. Mice with knockout of TRPV5 (originally called ECAC for Epithelial Calcium Channel) [99], however, have marked hypercalciuria, along with a compensatory increase in circulating 1,25(OH)2D3 levels and intestinal hyperabsorption of calcium. 1,25(OH)2D3 acts in the DCT to increase active reabsorption of calcium by up-regulating the expression of the key molecules participating in transcellular calcium transport. These are the apical uptake channel, TRPV5, calbindins-D9K and D28K, which facilitate transcellular diffusion of calcium, and the sodiumecalcium exchanger, NCX1, and the plasma membrane calcium pump, PMCA1B. Both NCX1 and PMCA1B pump calcium across the basolateral plasma membrane. The role of vitamin D in regulating this pathway was shown unequivocally using 1a(OH)asee/e mice, which lack any endogenous 1,25(OH)2D3 [100]. Repleting these mice with 1,25(OH)2D3 increases the expression of TRPV5, calbindin-D28K, calbindin-D9K, NCX1, and PMCA1B. The effect of dietary calcium rescue on the expression of these same genes was also examined in this study. Administration of the calcium-enriched rescue diet to 1a(OH)asee/e mice normalized their serum calcium concentration in association with upregulation of TRPV5, calbindin D28K, NCX1, and PMCA1b. These effects of calcium supplementation were presumably related to direct actions of calcium on the same cell type(s) upon which 1,25(OH)2D3 acts in the distal tubule, but indirect effects were not formally ruled out. However, Clemens et al. had shown some years before [1] that 1,25(OH)2D3 and/or elevated concentrations of calcium in the medium up-regulate expression of calbindin-D28K in primary kidney cells from the chick. It seems highly likely that calcium and 1,25(OH)2D3 were acting on the same calbindin-D28Kexpressing cell type in this study, and, because it was carried out in vitro, this effectively rules out indirect effects of 1,25(OH)2D3 and calcium mediated by other cells inside or outside of the kidney. The rescue diet, however, did not normalize renal calcium handling in the VDRe/e mice, as urinary calcium excretion in the VDRe/e mice receiving the rescue diet was twice that in normal mice receiving the same diet [101]. Apparently, therefore, the dietary calcium-induced changes in the expression of the components of the transcellular absorption pathway for calcium could not completely substitute for loss of the VDR. A recent study supports the role of the CaSR in mediating the actions of calcium on the transcellular calcium
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transport system in the DCT [102]. In this study, the CaSR and TRPV5 were shown to be coexpressed in the same cells, and activation of the CaSR on the apical membrane stimulated the activity of TRPV5, with a resultant increase in the intracellular calcium concentration, by a pathway that involves a PKC-dependent phosphorylation of amino acid residues S299 and S654 in the channel protein. This activation of TRPV5 was inhibited by a dominant negative CaSR construct, documenting the receptor’s involvement. What is the purpose of a CaSR-dependent stimulation of calcium reabsorption in DCT? This contrasts with the CaSRmediated inhibition of the paracellular reabsorption of calcium in the CTAL [103], which plays an important role in the defense against hypercalcemia. Topala et al. [102] suggested that this represents a local feedback mechanism that adjusts the reabsorption of calcium in the DCT to the prevailing urinary calcium concentration, perhaps mitigating the risk of stones when the urine reaching the DCT has an excessively high calcium concentration.
The CaSR and VDR in Cartilage and Bone Our understanding of the functions of the CaSR in bone and cartilage has lagged behind that in parathyroid and kidney, owing, at least in part, to controversy about whether the receptor actually exists in skeletal cells, to say nothing about whether it exerts biological relevant effects on those cells. While some studies found clear evidence for the receptor’s presence in cartilage or chondrocytic cell lines [104,105] and/or bone [105] as well as in osteoblastic cell lines, and/or osteoclasts and related cell lines, others did not (for review, see [106]). The following discussion summarizes the current state of this field and compares and contrasts the relative roles of the CaSR and VDR in cartilage and bone cells. The CaSR in Cartilage The chondrocytic cell line, RCJ3.1C5.18, expresses readily detectable levels of the CaSR. When incubated with elevated levels of Ca2þo, it shows suppression of the early differentiation marker, aggrecan, and increased expression of the markers of terminal differentiation, osteopontin, osteonectin, and osteocalcin, as well as increased production of matrix mineral, indicating stimulation of differentiation [107]. Several of these effects of the receptor were potentiated by overexpression of the wild-type CaSR or inhibited by transfection of the cells with a signal-defective CaSR, consistent with a mediatory role for the CaSR. Growth plate cartilage also expresses the CaSR [105]. Initial studies of cartilage in mice with homozygous knockout of exon 5 of the CaSR demonstrated rickets [108], suggesting a functional
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role for the receptor in cartilage in vivo. However, when these severely hyperparathyroid mice were “rescued” by knocking out the PTH gene (CaSRe/ePTHe/e) [109] or the key transcription factor, Gcm-2, that is required for formation of the parathyroid glands (CaSRe/e Gcme/e) [110], there was no obvious cartilage phenotype. Other studies carried out at around the same time, however, had shown that keratinocytes from the CaSRe/e mice were capable of generating a variant CaSR in which exon 5 had been spliced out [111]. This observation raised the possibility that the “exon-5-less” CaSR could have biological activity and be capable of rescuing the CaSRe/ePTHe/e and CaSRe/eGcme/e mice from any skeletal defects that might otherwise result from loss of only the full-length CaSR. Indeed, when chondrocytes were isolated from CaSRe/e mice, they still exhibited the same responses to Ca2þo that were present in wild-type chondrocytes [80], consistent with the presence of a biologically active CaSR lacking exon 5. Regardless of this observation, however, it has not been possible as yet to directly demonstrate biological activity of the exon-5-less CaSR by expressing it in heterologous cell systems. To further study the biological roles of the CaSR in the skeleton in vivo using knockout models, Chang, Shoback and Bikle and coworkers developed mice in which exon 7 of the CaSR had been “floxed” by insertion of loxP sites at either end of this exon [112]. If these mice are then mated with a strain of mice expressing the Cre recombinase only in the tissue of interest, the recombinase excises the floxed exon, recombines the ends of the remaining gene, and transcription and translation of that gene will produce a protein in which exon 7 is missing. Thus, mating the mice with exon 7 of the CaSR floxed with mice expressing the Cre recombinase in chondrocytes was utilized to examine the consequences of knocking out the CaSR in cartilage. Exon 7 encodes the entire transmembrane domain of the CaSR and the CaSR gene lacking exon 7 can only generate a receptor comprising the CaSR ECD, which would be released into the extracellular fluid as a soluble protein and presumably be incapable of signaling [112]. Presently, there is no convincing evidence that this soluble ECD protein has any biological role(s). Chondrocytespecific deletion of the CaSR resulted in death of embryos by day 13 [112], a surprisingly severe phenotype given the multiple hormonal or other factors known to regulate chondrocyte development and function. By using an inducible Cre recombinase, it was possible to delete exon 7 of the CaSR on days 16e18 of embryonic life, i.e., after the time when the embryos died in the previous model, which resulted in viable embryos that nevertheless displayed delayed growth plate development. These data supported, therefore,
a critical, nonredundant role of the CaSR in growth plate function. VDR in Chondrocytes An early, classic observation in the vitamin D field was that rickets, with its disordered structure of the growth plate and associated impaired growth, could be cured by the stimulation of the endogenous production of vitamin D or its exogenous administration (see additional chapters in this section). It was widely assumed that this represented direct actions of vitamin D on the cartilaginous growth plate. Indeed, direct effects of 1,25(OH)2D3 on chondrocytes had been shown in vitro that could potentially occur in vivo, such as stimulation [113] or inhibition [114] of growth, and promotion of differentiation [115,116]. Some of these actions may be mediated by cell surface receptors for 1,25(OH)2D3 [114] (see Chapter 28). While there is some overlap in the functions of the VDR and CaSR in chondrocytes identified in these studies (i.e., in promoting cellular differentiation), there have been limited studies to date directly assessing functional interactions between the two receptors [117]. A seminal observation was made, however, in mice with targeted disruption of the VDR (reviewed in [118]). As noted earlier, VDRe/e mice developed hypocalcemia, hypophosphatemia, and high PTH levels along with severe rickets after weaning. When administered a rescue diet, however, which included highcalcium and high-phosphate contents and lactose, the levels of serum minerals normalized and rickets was totally prevented. These findings, therefore, indicated that a major mechanism by which vitamin D cures rickets is indirect, by ensuring normal circulating mineral levels, which, in turn, promote mineralization of bone independent of direct cellular effects mediated by the VDR in chondrocytes. Nevertheless, these findings do not rule out the possibility that signaling through the VDR participates in growth plate function in VDR intact animals. For instance, up-regulation of signaling though the CaSR or some other pathway in the VDRe/e mice might compensate for loss of the VDR. Moreover, 1-hydroxylase knockout mice, unlike the VDRe/e mice, do not totally normalize their growth plates when receiving a rescue diet [117]. Thus the total lack of 1,25(OH)2D3 in these mice may have a detrimental effect on growth plate function not fully explained by absent VDR signaling. Of note the VDRe/e mice have markedly elevated 1,25(OH)2D3 levels, as noted above. Perhaps the latter contributes to normalization of their growth plates on the rescue diet, potentially through a VDR-independent mechanism, such as a membrane receptor for 1,25(OH)2D3. In addition, recent studies have shown that specific inactivation of the VDR in chondrocytes results in
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reduced RANKL expression by these cells, which is accompanied by reduced osteoclastogenesis and decreased FGF-23 levels, the latter producing increased serum phosphate concentrations [119]. These studies directly demonstrate novel functions of the VDR in chondrocytes in vivo related to the regulation of bone and mineral ion homeostasis. The CaSR in Osteoblasts As noted earlier, some [105,120,121] but not all [122] studies demonstrated the presence of the CaSR in bone, primary cultured osteoblasts and osteoblastic cell lines (for review, see [106]). In osteoblastic cells expressing the CaSR, high Ca2þo, acting at least in part via the CaSR, exerts effects that would be expected to promote bone formation. These include stimulation of the proliferation of preosteoblasts [123], increased expression of the mRNAs encoding the osteoblast differentiation markers, CBFA-1, osteocalcin, osteopontin, and collagen 1, and enhanced mineralized nodule formation [124]. As was the case with studies of cartilage in mice with homozygous knockout of exon 5 of the CaSR, bone histology in these mice was dominated by severe hyperparathyroid bone disease owing to loss of the CaSR in the parathyroid, which precluded ready interpretation of the impact of loss of the CaSR on osteoblast function. Studies in the two “rescue” models described above, CaSRe/ePTHe/e [109] and CaSRe/eGcme/e [110], showed that there were little or no differences in their bone histology and histomorphometry from those of control mice with intact CaSR with or without PTH or Gcm, respectively, suggesting a minimal role for the CaSR in the formation and turnover of the skeleton. However, conditional knockout of exon 7 of the CaSR in osteoblasts using the Cre recombinase driven by the 2.3-kilobase promoter for type 1 collagen produced viable mice, which exhibited, however, poor postnatal growth and skeletal development, with small poorly mineralized skeletons [112]. Most mice developed long bone and rib fractures and died within 3 weeks of birth. The bones were osteopenic and poorly mineralized and exhibited decreased levels of both early and late markers of osteoblast differentiation, such as type 1 collagen, alkaline phosphatase, insulin-like growth factor-1 (IGF-1, a key osteoblast growth factor), and osteocalcin. There was also increased osteoblast apoptosis. These results suggest key roles for the CaSR in promoting proliferation, survival, and differentiation of osteoblasts as well as skeletal mineralization [112]. For unclear reasons, the control mice with exon 7 of the CaSR floxed, which had not been crossed with Cre mice, had mildly elevated serum calcium concentrations. The significance of this finding is unclear, and it would be important to compare their serum calcium concentrations to those
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of the strain of mice from which they were derived in order to rule out an impact of the insertion of the loxP sites on expression or function of the CaSR. The VDR and Osteoblasts There is an extensive literature addressing the roles of the VDR in osteoblasts (for review, see [125]), which will be summarized briefly here as this is covered in detail elsewhere (see additional chapters in this section). Fetal rat calvarial osteoblasts grown in culture undergo a characteristic cellular program of proliferation, followed by differentiation and then mineralization [126]. Type I collagen production is initially expressed at high levels during the proliferative phase, then decreases during differentiation, while the production of alkaline phosphatase, osteopontin, and osteocalcin increase in that order during the differentiation and mineralization phases. Several of these genes are vitamin-D-responsive, including type I collagen, osteocalcin, and osteopontin, to name just a few of the osteoblastic genes that have been shown to be regulated by vitamin D during osteoblastogenesis and subsequent mineralization of bone. In fact, the use of chromatin immunoprecipitation techniques (ChIP), which enables identification of genes associated with specific transcription factors, such as the VDR, bound to their regulatory regions, has identified several thousand binding sites for the VDR/RXR heterodimer in the DNA of the mouse osteoblastic cell line MC3T3-E1 [127]. The analysis of the phenotype of mice with knockout of the VDR [128] and/or the 1a(OH)ase [129] has had a major impact on our understanding of the role of 1,25(OH)2D3 and the VDR on osteoblast function, analogous to the impact it had on the roles of the VDR in parathyroid cell and chondrocyte biology (see Chapter 33). Knockout of the VDR alone, as noted above, revealed that a rescue diet normalizing serum calcium, phosphate, and PTH could heal the rickets of the VDRe/e mice with normalization of the growth plate in 70-dayold mice, as noted above. Histomorphometry of adjacent metaphysis (the flared bone connecting the epiphysis to the shaft of a long bone) was likewise normal, including osteoblast number and the ratio of osteoblast surface to total bone surface. In addition, femoral strength, providing an assessment of the integrity of cortical bone, was equivalent to that in control mice [128]. This study concluded that the principal function of the VDR in bone formation and modeling was indirect, e.g., ensuring adequate serum levels of calcium and phosphate, rather than by directly exerting essential actions of osteoblasts. A follow-up study by the same group, however, demonstrated that primary calvarial osteoblasts isolated from VDRe/e mice exhibited increased numbers of osteoblastic colony-forming units (a measure of the number of cells capable of
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differentiating to osteoblasts) [130]. Moreover, the osteoblastic cells in vitro manifested earlier onset and increased magnitude of alkaline phosphatase expression and earlier and sustained mineralized matrix formation. These results point out that even though loss of the VDR can clearly alter the function of osteoblastic cells in vitro, additional factors, including (and almost certainly not limited to) the CaSR when serum calcium is normalized can compensate to maintain normal or nearly normal bone formation in vivo. Panda et al. carried out a similarly designed study in older mice (120 days old), utilizing VDRe/e mice and/ or 1a(OH)asee/e mice [129]. When the mutant mice received the rescue diet, the length of the femurs of the mutants (which were markedly shorter than that in the wild-type if the rescue diet was not utilized), were nearly normalized, indicating near normal bone growth, reflecting, in part, much improved growth plate function. With regard to the properties of osteoblasts per se, the rescue diet reduced the number of osteoblasts in the three mutants below that in the wild-type mice, with an associated decrease in mineral apposition rate and bone volume [130]. Furthermore, in contrast to the observations of Amling et al., cultured osteoblastic cells from all three mutants after they had been maintained on the rescue diet, exhibited a reduced, rather than increased, capacity to form osteoblastic colonies. Finally, the expression of RANKL, the key osteoblastic protein needed to promote the osteoblast-dependent formation of osteoclasts was substantially below that in wild-type mice. These authors concluded that 1,25(OH)2D3 and the VDR serve an anabolic role in osteoblastic function in vivo [129]. The difference between these results and those of Amling et al. [128] were ascribed to differences in the ages of the mice (120 vs. 70 days, respectively). Thus, the impact of the loss of the VDR on bone in the two studies can be compensated partially [129] or nearly completely [128] in vivo by ensuring a normal milieu of mineral ions, presumably mediated by the crucial roles of the CaSR and perhaps other compensatory genes in chondrocytes and osteoblasts. The key role that VDR-mediated intestinal calcium absorption, rather than the VDR in bone cells per se, plays in maintaining normal development and growth of cartilage and bone was shown by the studies of Xue et al. [131], who showed that transgenic expression of the VDR on a VDR-null background normalized calcium transport in the intestine, serum calcium concentration, serum PTH, and somatic growth. There were changes in bone per se, however, as bone mineral density and bone volume were higher in the transgenic mice compared with the normal mice as a result of increased mineral apposition rate and osteoblast number. An increase in 1,25(OH)2D3 levels owing to loss of the VDR in kidney and a resultant decrease in degradation
by the 25-hydroxyvitamin D 24-hydroxylase and increase in synthesis of 1,25(OH)2D3 may have contributed to an anabolic effect of 1,25(OH)2D3 in bone postulated by others [129]. The CaSR in Osteoclasts While some studies have failed to detect the CaSR in osteoclasts [132], recent studies have provided strong evidence that the CaSR is expressed in cell lines thought to resemble osteoclast precursors (e.g., RAW 264.7) [133], in multinucleated osteoclasts differentiated from such precursors in vitro and in at least some mature osteoclasts in intact bone. The CaSR appears to serve a permissive role in osteoclastogenesis in vitro, but high Ca2þo concentrations also directly inhibit osteoclast activity and stimulate their apoptosis [133]. Thus assuming that the CaSR mediates similar actions in vivo, high Ca2þo, via the CaSR, inhibits bone resorption in a homeostatically appropriate manner, actions that at least theoretically could be mimicked by calcimimetics, although one in vitro study that failed to detect transcripts for the CaSR in osteoclast precursors or osteoclasts formed in vitro did not observe any functional effect of a calcimimetic on these cells [132]. The effects of extracellular calcium on osteoclast function have also been ascribed to an entirely different calcium-sensing mechanism (for review, see [134]). VDR in Osteoclasts In contrast to the extensive literature on the presence and actions of vitamin D and the VDR in chondrocytes and osteoblasts, that related to osteoclasts is much more limited. Instead, there has been a greater emphasis on indirect effects of 1,25(OH)2D3 on osteoclasts that are mediated by osteoblasts, such as up-regulation of RANKL, with subsequent stimulation of osteoclastogenesis and bone resorption by pre-existing osteoclasts [135] (for additional details see Chapters 17 and 18). Available data indicate that the VDR is expressed in osteoclasts and their precursors [136e138], but the functional implications of the VDR in these cells are not known. It should be pointed out, however, that studies on osteoclasts in intact bone carried out using mice with knockout of the VDR and/or 1-hydroxylase have provided some insights into the role of the VDR, at least in mice. As noted above, Amling et al. [128] studied 70-day-old VDRe/e mice when on a rescue diet and found little if any difference in osteoclast number or in the percent of the bone surface covered by osteoclasts. On the basis of these results, it was not possible to ascribe any function to the VDR in osteoclasts (or indirect effects of the VDR on osteoclasts mediated by osteoblasts) in the setting of the normocalcemic, normophosphatemic, and euparathyroid state. More recently, Panda et al. [129] performed similar studies in 120-day-old mice,
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utilizing VDRe/e, 1a(OH)asee/e mice and double knockout mice as before. While receiving the rescue diet, osteoclasts in all three of the mutant genotypes were normal in size and number. Of note, when hypocalcemic owing to lack of the rescue diet, all three genotypes of mutant mice did not show the expected increase in osteoclast number owing to hypocalcemia and secondary hyperparathyroidism [129]. This observation is most likely not due to an intrinsic osteoclast defect, but rather to the reduced RANKL expression by the osteoblasts of the mutant mice, as noted above. Neither of these studies, therefore, supports a defect in osteoclasts per se or their precursors in the absence of the VDR and/or 1,25(OH)2D3.
The CaSR and VDR in the Intestine While the CaSR is widely expressed in the gastrointestinal tract [17], many of its functions are not directly related to Ca2þo homeostasis (e.g., regulation of gastric acid secretion) and are not covered here. Some of these locations, e.g., the colon, provide useful examples, however, of how the CaSR and VDR can interact in ways unrelated to mineral ion homeostasis, such as in the development of colon cancer, and will be discussed later. With regard to Ca2þo homeostasis, the stimulation of transcellular calcium transport in the intestine, particularly the proximal small intestine, is one of the bestknown actions of vitamin D. Although there are rapid nongenomic effects of 1,25(OH)2D3 on intestinal calcium transport in vitro [139], the best-characterized intestinal actions of vitamin D mediated by the VDR involve upregulation of the transport machinery for transcellular transport of calcium. This includes the apical uptake channel, TRPV6 (which is closely related to TRPV5 in the kidney), calbindin-D9K, and PMCA1b. The expression of all of these components is up-regulated by 1,25 (OH)2D3 in 1a(OH)asee/e mice [140], thereby providing a molecular basis for the stimulation of intestinal Ca2þ absorption by 1,25(OH)2D3 through a genomic mechanism very similar to that in the DCT/CNT. The CaSR is expressed in the small intestine [141] (presumably in the same cells expressing the VDR but this has not been proven) as well as in cell lines derived from the intestine [142], but its functions in the intestine have not been well characterized in many cases. Nevertheless, available data suggest that it can act in concert with vitamin D or partially substitute for the actions of 1,25(OH)2D3 in certain circumstances. In organ cultures of fetal rat duodenum, for example, not only 1,25 (OH)2D3 but also increases in medium Ca2þ concentration increase the expression of the mRNA for calbindin-D9K [143]. More recently, van Abel et al. showed that administration of a rescue diet to 1a(OH)asee/e mice restored normocalcemia in association with
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statistically significant, >tenfold increases in the expression of the mRNAs for TRPV6 and calbindin-D9k [140] in the intestine, similar to what was observed in the kidney. Although not formally proven, it seems likely that these actions of calcium are mediated by the CaSR. What is the significance of these actions of dietary Ca2þ on intestinal calcium absorption? On the one hand, high concentrations of Ca2þ can apparently substitute, at least in part, for a lack of vitamin D in the maintenance of the level of expression of key elements of the transcellular calcium transport system in the intestine. In the presence of vitamin D, on the other hand, there might be the possibility of a physiologically undesirable feedforward mechanism, whereby intake of increasing amounts of calcium in the vitamin-D-sufficient state might foster excessive GI calcium absorption by the mechanism just described. However, as long as the source of vitamin D was not 1,25(OH)2D3 itself or one of its analogs, inhibitory feedback regulation on renal 1-hydroxylation via decreases in PTH or increases in serum calcium or phosphorus (e.g., by directly inhibiting the 1-hydroxylase) could prevent such a scenario.
The CaSR and VDR in the Placenta A key role that the placenta plays during intrauterine life is to provide adequate quantities of calcium for the developing fetal skeleton, primarily during the third trimester in humans. This is accomplished by “pumping” calcium transcellularly using the same machinery that is used in other Ca2þ-transporting epithelia, including TRPV6, calbindin D9K, and PMCA. In the mouse, much of this transport is thought to occur in the placental yolk sac, the site of expression of this transport machinery in this species. The most cogent evidence regarding the relative importance of the CaSR and VDR for placental Ca2þ transport comes from mouse knockout models. The role of the CaSR in regulating placental Ca2þ transport was explored by Kovacs et al. [144] utilizing mice heterozygous or homozygous for knockout of exon 5 of the CaSR. There were several phenotypes of fetal CaSRe/e mice relevant to calcium homeostasis. These include hypercalcemia, markedly elevated PTH levels owing to defective Ca2þ sensing by the fetal parathyroids, elevated levels of the bone resorption marker, deoxypyridinoline, and increased urinary calcium excretion, likely as a result of PTH-induced bone resorption. Thus, as is also observed postnatally in this knockout model, loss of the full-length CaSR leads to severe hyperfunction of the fetal parathyroid glands. In addition, however, placental transport in the CaSRe/e fetuses measured in vivo using 45Ca (each fetus has its own placenta) was significantly less than
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that of both the wild-type (CaSRþ/þ) and heterozygous (CaSRþ/e) fetuses [144]. This result implicates the CaSR in promoting normal placental transport of calcium. It does so, at least in part, through a PTHrPdependent pathway, since knocking out PTHrP in vivo results in a decrease in 45Ca transport to a level similar to that found in CaSRe/e fetuses [144], which no longer varies as a function of CaSR genotype. That is, the CaSR can only stimulate placental calcium transport in the presence of PTHrP, which, therefore, is presumably “downstream” of the CaSR. Despite the presence in the murine placenta of the vitamin-D-dependent calcium transport machinery similar to that present in intestine and kidney, cogent evidence obtained using VDR knockout mice indicates that the VDR is not required for fetal mineral ion homeostasis, skeletal mineralization, or for normal placental calcium transport, at least in mice [145]. When heterozygous, VDRþ/e mothers were mated, the offspring (VDRþ/þ, VDRþ/e, and VDRe/e fetuses) were similar with regard to placental 45Ca transport, serum calcium and phosphate, and the calcium content of their skeletons. Thus the CaSR, and not the VDR, functions in the placenta to regulate key placental functions vital to mineral ion and bone homeostasis.
The CaSR and VDR in Breast Analogous to the role of the placenta in maternoefetal calcium and bone homeostasis, the breast serves as the dominant source of calcium for the newborn and growing infant. Recent studies by van Houten and colleagues have identified the CaSR as a key player in this process [146]. Expression of the CaSR increases markedly during lactation in the mouse and then returns to baseline levels following the termination of breastfeeding. The receptor is expressed basolaterally in breast epithelial cells (i.e., on the opposite side of the cell from the apical surface facing the milk), particularly in the milk-producing alveoli. The CaSR in the lactating breast plays two key roles in the breast during lactation: (1) suppressing the secretion of PTHrP, and (2) stimulating the transport of calcium into breast milk. Any decrease in maternal serum calcium concentration stimulates PTHrP secretion, both into the milk as well as into the systemic circulation. The increase in circulating PTHrP level stimulates bone resorption, providing a mechanism for increasing the serum calcium concentration in the mother and thereby providing calcium for transport into the milk. Activation of the basolateral CaSR in the breast by the increase in maternal serum calcium concentration then stimulates the transport of calcium into the milk by a mechanism involving activation of the apical calcium pump, PMCA2 [147]. In a sense, therefore, the CaSR serves as
an “accessory parathyroid gland” during lactation, showing inverse regulation of a calcium-elevating hormone (PTHrP) that acts through the same mechanisms utilized by PTH, namely stimulating bone resorption and, presumably, renal tubular calcium reabsorption. Since the PMCA is stimulated by calmodulin (CaM) following activation of the latter by increases in the cytosolic calcium concentration [147], a CaSRstimulated increase in CaM activity could underlie the activation of PMCA, although the signal transduction pathways involved have not been studied in detail. Is there a role for the VDR during lactation? Relatively little work has been carried out on the role of the VDR and vitamin-D-responsive genes participating in calcium transport on calcium transport into milk during lactation. As assessed by binding of radioactive 1,25 (OH)2D3, epithelial cells of the ductal and alveolar cells of the breast express the vitamin D receptor, and the levels increase during pregnancy and lactation [148]. It is currently unknown, however, whether key elements of the epithelial calcium transport machinery, e.g., TRPV5/6, calbindin D9K and D28K, PMCA, etc., function in transcellular calcium transport in the lactating breast. In this regard it would be of interest to study the effect of 1,25(OH)2D3 on transport of calcium into milk in vitro and in vivo during lactation using the tools used to study the role of the CaSR in this process, including mice with global or conditional knockout of the VDR (and/or CaSR) in the breast. Recent studies have addressed the role of the VDR in regulating other aspects of breast structure and function. In mice with knockout of the VDR, there was accelerated lobuloalveolar development and premature expression of casein, a milk protein, as well as delayed postlactational involution compared with control mice [149]. Thus the VDR appears to regulate mammary cell turnover during the reproductive cycle. Comparable studies have not been carried out with regard to the CaSR, for example, using mice with conditional knockout of the CaSR in the breast to examine breast development and involution during lactation as well as at other times.
The Utility of CaSR-based Therapeutics Alone or in Combination with VDRAs for Treating Parathyroid Hyperfunction Nearly simultaneous with the isolation and characterization of the CaSR was the development of the calcimimetic CaSR activators, which provided a means of pharmacologically inhibiting hyperfunctioning parathyroid glands, particularly in the setting of chronic renal insufficiency. The discussion that follows provides an overview of this area, which is relevant to another clinically useful way of treating hyperparathyroidism in this setting, i.e., the use of vitamin D
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receptor activators (VDRAs) [150]. The latter share with calcimimetics the capacity to suppress PTH gene expression and parathyroid cellular proliferation, as detailed earlier. These shared actions plus the additional direct suppression of PTH secretion by the calcimimetics offer new opportunities for sequential or initial combined therapy with these drugs. Given the voluminous literature on VDR-based therapy for stage 5 CKD and a related discussion of this issue in this volume (see Chapter 70), the emphasis of the discussion that follows will be on CaSR-based therapy and our evolving understanding of its relationship to VDR-based therapy. Studies of the Utility of Calcimimetics in Animals with Uremic Hyperparathyroidism It has not yet been shown definitively that all of the actions of calcimimetics on rats with experimentally induced uremia are applicable to humans. However, these animal studies have provided proof-of-concept that administration of calcimimetics in vivo, as would have been expected, sensitize the CaSR in the parathyroid gland to Ca2þo, thereby producing the expected alterations in parathyroid function. Moreover, the results of these animal studies provide benchmarks for the subsequent and ongoing investigation of potentially beneficial actions that the drug might have in humans. Studies in normal rats demonstrated that oral administration of a single dose of the “second-generation” calcimimetic, cinacalcet (Fig. 24.7), lowered serum PTH, with a nadir at 1e2 hours [151]. Serum calcium concentration also declined with a maximal decrease at 1e4 hours, depending on the dose. Cinacalcet also increased circulating calcitonin levels, but the apparent in vivo EC50 values for suppressing PTH and elevating calcitonin were 0.5 and 16 mg/kg, respectively [151]. Therefore, doses of cinacalcet that suppress PTH release would have minimal, if any, effects on calcitonin secretion in vivo. An extensive body of evidence subsequently showed that the “first-generation” calcimimetic, NPS R-568, lowers serum PTH and calcium concentrations in rats with experimentally induced renal insufficiency, usually produced by subtotal (i.e., 5/6) nephrectomy [151]. A calcimimetic also decreased PTH mRNA [47], which likely
FIGURE 24.7 Chemical structure of the calcimimetic, CinacalcetÒ
Ca2þo (also known as SensiparÒ). From Nemeth et al. 2004 J Pharmacol 308:627e35, with permission.
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contributes to the reduction in PTH secretion, and upregulated both the CaSR and VDR in the parathyroid [152]. Administering calcimimetics to uremic rats also obviated the parathyroid hyperplasia that would otherwise take place in the setting of renal insufficiency [153], owing, in part, to up-regulation of the cyclin-dependent kinase inhibitor, p21 [154]. Some studies have also demonstrated regression of established parathyroid hyperplasia [155]. While there is little apoptosis of the long-lived parathyroid chief cells under normal circumstances, there is some evidence that calcimimetics induce apoptosis of this cell type, albeit at a high dose (10e4 M) [156]. Finally, treating uremic rats with a calcimimetic has reduced the hyperparathyroid bone disease, termed osteitis fibrosa cystica, that occurs in the setting of renal failure [157]. Further studies in rats revealed additional actions of calcimimetics in other tissues. Rats administered NPS R-568 manifested a reduction in rate of progression of renal failure that otherwise occurs in rats that have undergone subtotal nephrectomy [158]. The same study documented that the drug also decreased blood pressure and LDL cholesterol levels, which could have a favorable impact on the substantially increased risk of cardiovascular complications of uremic animals and humans [159]. Indeed, the hearts of the treated animals showed less interstitial fibrosis and had a reduction in arteriolar wall thickness compared to control animals. Subsequent studies have also shown decreases in the thickness and calcification of blood vessel walls outside of the heart, and the life span of uremic rats treated with calcimimetic was significantly longer than in controls [160]. The use of atherosclerosis-prone, apolipoproteinE-deficient mice made it feasible to demonstrate that NPS R-568 retarded uremia-induced vascular calcification and atherosclerosis in this animal model [161]. Finally, a recently developed calcimimetic, AMG 641, produced actual regression of pre-existent aortic and soft tissue calcification in uremic mice of this strain [162]. The relative contributions of alterations in PTH vs. serum mineral ions (i.e., Ca2þ, phosphate, and calciumephosphate product) vs. potentially direct actions of calcimimetics on the vasculature are topics of ongoing investigation. Active vitamin D compounds, such as 1,25(OH)2D3 and its less calcemic analog, paracalcitol, are an important component in the therapy of secondary hyperparathyroidism in humans with stage 5 CKD by virtue of their capacity to reduce PTH gene expression and parathyroid cellular proliferation [163]. Studies in uremic rats have, however, shown that vitamin D analogs, particularly 1,25(OH)2D3, while effective in lowering PTH, can decrease survival and cause extraosseous calcification and progression of renal failure [160]. These deleterious
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actions of the vitamin D analogs in rats can be significantly decreased by coadministering a calcimimetic [164]. Studies of the Utility of Calcimimetics in SHPT in Humans with Stage 5 CKD A substantial body of evidence in humans with stage 5 CKD has demonstrated actions of cinacalcet that are similar to those just described in animal models of uremia, albeit with some differences, as pointed out below (the rats are not being dialyzed for one). This field will be summarized relatively briefly, emphasizing gaps in our knowledge or areas of controversy, as this topic is covered in detail in recent reviews [165]. In randomized controlled studies, cinacalcet lowers serum PTH, calcium, phosphate, and the calciumephosphate product [28]. The decrease in serum phosphate was surprising at first. In a dialysis patient, there is little or no renal phosphate excretion, so the change in serum phosphate observed during therapy with cinacalcet must result from other, as yet poorly defined, mechanisms. The decreases in these indices of mineral metabolism have improved the attainment of targets developed for these parameters in patients with stage 5 CKD. The U.S. National Kidney Foundation (NKF) provides guidelines (so-called NKF KDOQI (kidney disease outcomes quality initiative) guidelines) for the levels of serum calcium, phosphate, PTH, and the calciumephosphate product that are to be achieved in patients with CKD. These guidelines have as their goal to reduce morbidity and mortality in this patient population (http://www.kidney.org/Professionals/Kdoqi/ guidelines_ckd/toc.htm). For stage 5 CKD, the goals for serum calcium, phosphorus, calciumephosphate product, and PTH are, respectively, 8.4e9.5 mg/dl, 3.5e5.5 mg/dl, <55 mg2/dl2, and 150e300 pg/ml. A more recently formed international committee (KDIGO, Kidney Disease Improving Global Outcomes) is currently developing its own guidelines (www.kdigo. org/clinical_practice_guidelines/index.php). The PTH levels referred to in these guidelines are generally those obtained with so-called “intact” assays. In point of fact, these assays actually recognize not only the fulllength PTH1-84 molecule but also N-terminally truncated species such as PTH7-84, which inhibits the activity of PTH on its receptor in kidney and bone and accumulates in the serum of patients with reduced renal function [166]. C-terminal fragments of PTH, including PTH7-84 and shorter C-terminal species, are also secreted in greater quantities, at the expense of reduced secretion of PTH1-84, in the setting of activation of the CaSR by hypercalcemia or calcimimetics, further decreasing the net secretion of bioactive PTH1-84 [167]. While there are newer, “whole” PTH assays that recognize only PTH1-84 [168], these assays do not afford a clearly superior assessment of the relative degree of parathyroid overactivity.
Cinacalcet increases the percentage of patients achieving KDOQI goals, but the frequency with which the individual targets or all four together are achieved remains suboptimal [28]. For instance, adding cinacalcet to standard therapy (mainly phosphate binders and vitamin D analogs) enhanced the percentage of patients who achieved the NKF guidelines for serum calcium (49% vs. 24% for controls), phosphate (46% vs. 3%), and PTH (56% vs. 10%), calciumephosphate product (65% vs. 33%), as well as for both calciumephosphate product and PTH simultaneously (41% vs. 6%) [169]. The actions of cinacalcet on serum mineral ions and PTH persist for at least 3 years, without a need for escalating the dose of the drug. The most frequent side effects of the drug are nausea and vomiting, which occur in ~30% of patients, although ~15% of the placebotreated controls experienced the same symptoms [28]. In addition to its actions on serum mineral ions and PTH, one investigation showed a decrease in the size of the hyperplastic parathyroid glands in dialysis patients treated with cinacalcet [170], particularly in those glands weighing less than 500 mg. Moreover, a small study found that cinacalcet reduced the degree of osteitis fibrosa cystica in dialysis patients [171]. There is some concern, however, that overly aggressive therapy with cinacalcet that reduces PTH below the recommended levels could lead to the development of so-called adynamic bone disease, which is characterized by reduced bone cell populations and decreased bone remodeling. Adynamic bone disease is thought to result from the presence of insufficient circulating levels of PTH to promote a normal rate of bone turnover [171]. Additional studies are needed to clarify the impact of therapy with calcimimetics on bone mineral density and bone histology in patients with stage 5 kidney disease. There has been considerable interest in establishing whether cinacalcet alone, vitamin D metabolites alone, or a combination of the two would be most effective in treating secondary hyperparathyroidism in stage 5 CKD [165]. While VDRAs and cinacalcet given individually reduce PTH to a similar extent, there are several differences between the two classes of drugs. (1) VDRAs, especially 1,25(OH)2D3, can produce both hypercalcemia and hyperphosphatemia at high doses, while cinacalcet lowers both modestly, as noted above. Symptomatic hypocalcemia does occur in a minority of patients receiving cinacalcet [165]. (2) There is a greater frequency of side effects with cinacalcet, mainly nausea and/or vomiting. (3) Observational studies (there have been no randomized control trials to date) have suggested that VDRAs prolong survival [172]. A recently published observational study suggests that cinacalcet may have a similar effect on mortality [173]. A growing body of evidence suggests that combining both classes of drugs may have advantages over either given
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individually: (a) The combination enables better control of SHPT than does a VDRA alone and (b) combined therapy permits the use of a lower dose of VDRA, which decreases the risk of VDRA-induced elevations in serum calcium and/or phosphate [165]. Will the use of cinacalcet (or another calcimimetic), by itself or in combination with a VDRA, decrease cardiovascular disease and associated mortality in prospective randomized control studies? One small meta analysis of four randomized, double-blind, placebo-controlled clinical trials utilizing cinacalcet or placebo in patients already receiving a VDRA and phosphate binders has assessed the impact of cinacalcet on end-points other than serum minerals and PTH [159]. This study showed that compared to patients who were not receiving cinacalcet and were on standard care, cinacalcet-treated patients had a >90% decrease in the rate of parathyroidectomy and about a 40% reduction in cardiovascular hospitalizations. The study was not sufficiently powered, however, to assess the effect of the drug on “hard” cardiovascular endpoints, such as myocardial infarction or death. Two large randomized controlled studies are currently under way to address this latter point, the ADVANCE (“A randomized study to evaluate the effects of cinacalcet plus low dose VDRA on vascular calcification in subjects with chronic kidney disease (CKD) receiving hemodialysis”) and EVOLVE studies (EValuation Of Cinacalcet HCl Therapy to Lower cardio Vascular Events) [174]. The latter has primary end-points of all-cause mortality and first nonfatal cardiovascular event in 3800 chronic dialysis patients who are being treated with a flexible regimen of traditional therapies and will additionally receive either cinacalcet or placebo [174]. In addition to the use of Cinacalcet for treating secondary hyperparathyroidism in stage 5 CKD, the drug has also been approved by the FDA for use in parathyroid cancer and the drug has been used off-label (i.e., without FDA approval) for other forms of primary hyperparathyroidism, lithium-induced hyperparathyroidism, secondary hyperparathyroidism in patients being treated for phosphate-wasting disorders, and others. The interested reader is referred to a recent review of this topic [175].
THE CASR AND VDR IN TISSUES UNINVOLVED IN CA2DO HOMEOSTASIS There has been increasing interest in the roles of calcium and dietary calcium intake, on the one hand, and vitamin D, on the other, in maintaining the normal function of various cells and tissues (e.g., keratinocytes) as well as in preventing or treating a variety of pathological conditions not directly related to Ca2þo homeostasis. These pathological states include cancer (e.g., colon,
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breast, prostate, and others), infectious disease, autoimmune diseases (viz., inflammatory bowel disease), hypertension, and other cardiovascular diseases, and obesity, metabolic syndrome, and type 2 diabetes [176,177]. In general, more data are available regarding the mechanisms by which 1,25(OH)2D3 and the VDR, as opposed to calcium and the CaSR, regulate normal and pathological tissues, owing to the relatively recent recognition that the CaSR is expressed in a multitude of tissues uninvolved in Ca2þo homeostasis [178]. Indeed, in only a minority of cases are the molecular mechanism(s) fully understood by which calcium exerts beneficial and, in some cases, detrimental effects on normal and neoplastic cells outside of Ca2þo homeostatic tissues. In only a few of these, in turn, is there convincing evidence that the CaSR serves as the molecular target for the effects of calcium, as opposed to some other mechanism, such as a calcium channel, for instance. Rather than trying to cover this rapidly growing field in detail, this discussion will focus on several examples where the CaSR and VDR both appear to contribute to modulating the function(s) of selected normal and pathological tissues outside of those participating in Ca2þo homeostasis.
CaSR, VDR, and Renin Secretion Renin plays a key role in sodium, volume, and blood pressure homeostasis: hypovolemia is sensed by the juxtaglomerular (JG) cells of the afferent arteriole of the kidney, which increase their release of renin. In turn, renin converts circulating angiotensinogen to angiotensin II, a potent endogenous pressor, which also stimulates the production of the sodium-retaining hormone, aldosterone, by the zona glomerulosa of the adrenal gland [179]. The combination of the direct elevation in blood pressure due to angiotensin II and the sodium retention caused by aldosterone will promote normalization of sodium and volume homeostasis. It has been known for several decades that raising the extracellular calcium concentration suppresses the release of renin by the JG cells [180]. Only more recently, however, has it been shown convincingly that the CaSR is expressed by JG cells [181] and that it mediates the inhibitory action of elevated calcium concentrations on renin release [182]. An unexpected observation in VDRe/e mice was that they exhibit increased production of renin and angiotensin, which leads to hypertension and cardiac hypertrophy [183,184]. The CaSR and the VDR, therefore, can modulate not only on mineral ion homeostasis but also on sodium, blood pressure, and volume homeostasis. These observations highlight unexpected roles of the CaSR and VDR that suggest previously unknown interactions between Ca2þo homeostasis, on the one hand, and sodium and volume homeostasis, on the
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other. What might the physiological significance of these observations be? In some studies, intravenous infusion of calcium produces an acute increase in blood pressure in normal individuals and especially in those with chronic renal insufficiency [185,186], who cannot efficiently excrete a sodium load. Viewed in this context, suppression of renin secretion and the resultant decrease in angiotensin II production could serve as a counterregulatory, blood-pressure-lowering mechanism. In some but not all studies, calcium administration can exert a beneficial effect on some forms of hypertension, e.g., preeclampsia [187], although the relevance of the CaSR in JG cells to this effect is unknown.
CaSR and VDR in the Growth and Differentiation of Normal and Neoplastic Tissues CaSR and VDR in Keratinocytes Both elevating Ca2þo and treatment with 1,25(OH)2D3 inhibit the proliferation and stimulate the differentiation of keratinocytes in culture in a manner that mirrors that occurring in normal skin. Keratinocytes cultured at concentrations of <0.1 mM Ca2þo actively proliferate [188]. Raising Ca2þo to 0.5e2 mM inhibits keratinocyte proliferation in association with up-regulation of the cell cycle inhibitors, p21waf1 and p27kip1, and downregulation of c-Myc. Differentiation ensues concomitant with up-regulation of differentiation markers, such as profilagrin, involucrin, and loricrin [189] and proceeds to terminal differentiation accompanied by formation of the cornified envelope. An important part of the terminal differentiation process is cellecell adhesion owing to the cell adhesion molecule, E-cadherin, which also contributes to intracellular signaling via phosphatidylinositol 3-kinase that is mediated by catenins bound to the cytoplasmic domain of E-cadherin [190]. The involvement of the CaSR in the keratinocyte “calcium switch” has been documented by the use of the exon 5 CaSRe/e mice. The epidermis of these mice shows reduced expression of loricrin and profilagrin [191] documenting the involvement of at least the full-length CaSR in epidermal differentiation. It is possible in this model that the exon-5-less CaSR present in skin partially rescues keratinocytes, a point that could be addressed using a keratinocyte-specific knockout of the CaSR using mice with exon 7 of the CaSR floxed. The CaSR has also recently been shown to mediate cellecell adhesion promoted by E-cadherin [190], which likely contributes to its promotion of differentiation. In addition, transgenic overexpression of the CaSR in the skin of mice results in accelerated cutaneous differentiation [192,193], further supporting the importance of the CaSR in cutaneous biology.
1,25(OH)2D3-induced inhibition of keratinocyte proliferation, which is observed at >1 nm 1,25(OH)2D3, is associated with reductions in the mRNA levels for the growth promoters, cyclin D1 and c-Myc, as well as increases in the cell cycle inhibitors, p21cip and p27kip (reviewed in [193]) (Fig. 24.8). In addition to 1,25 (OH)2D3 produced in the kidney, it is likely that 1,25 (OH)2D3 synthesized locally by CYP27B1 expressed by skin cells [194] also acts on the VDR present in the keratinocyte. 1,25(OH)2D3-induced changes in gene expression contributing to differentiation include increases in the expression of involucrin, loricrin, profilagrin, and transgluatminase, a key protein for the cross-linking the cornified envelope of the skin. VDRe/e mice have reduced cutaneous levels of involucrin and loricrin and loss of keratinohyalin granules, confirming the physiological importance of the VDR in the keratinocyte [118]. Thus, unlike the parathyroid gland, chondrocyte and osteoblast, where the CaSR can support normal or nearly normal cellular function in the absence of the VDR, both the CaSR and VDR appear to have similar, nonredundant roles in the keratinocyte. Therefore, there is substantial overlap in the mechanisms by which calcium and 1,25(OH)2D3, acting through two entirely different classes of receptors, produce growth arrest and differentiation of keratinocytes (Fig. 24.8). In fact, they synergistically up-regulate the expression of involucrin and transglutaminase [195], which results in part from up-regulation of the CaSR by 1,25(OH)2D3 [196]. The reciprocal experiment, examining the effects of elevated Ca2þo on the level of expression of the VDR, does not appear to have been carried out. It would be of interest to determine whether activation of the CaSR by high Ca2þo up-regulates this receptor, as in the parathyroid cell. Calcium and vitamin D also exert important biological actions on a variety of tumor cells which are potentially relevant to treatment of these tumors in vivo. This field is considerably more advanced with regard to the antiproliferative and other effects of vitamin D than with respect to similar effects mediated by the CaSR. It should also be noted that, even though there is experimental evidence, primarily in vitro, that the CaSR modulates the biology of tumor cells, it is far from certain that any effects of dietary calcium on cancer risk (for review, see [176]) are CaSR-mediated. CaSR and VDR in Colon Cancer Garland et al. carried out an early prospective study showing that reduced dietary intakes of calcium and vitamin D were associated with significantly increased risks of colon and/or rectal cancer [197]. A recent review reported that of 30 studies of colon cancer or adenomatous polyps [198], 20 studies found a significant benefit of vitamin D level or status (i.e., sunlight exposure) on
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FIGURE 24.8 Interactions between Ca2þo, acting via the CaSR, and 1,25(OH)2D3, acting via the VDR, in promoting keratinocyte differentiation. Activation of the CaSR stimulates phospholipase C in a G-protein-dependent manner, resulting in formation of diacylglycerol (DAG) and inositol tris-phosphate (IP3). The former activates protein kinase C bound to the receptor for activated PKC (RACK), while the latter releases calcium from intracellular stores in the endoplasmic reticulum (ER) and participates in activation of influx of extracellular calcium. The increase in cytosolic calcium and the active protein kinase C can activate the transcription factors (AP-1) and stimulate calcium-responsive DNA elements (CaRE). Increases in expression of keratinocyte genes, such as Involucrin and Loricrin, which are cross-linked through the activity of transglutaminase, enhance formation of the cornified envelope in the outer layer of skin. 1,25(OH)2D3 acts in concert with high Ca2þo by increasing expression of the CaSR as well as of that of phospholipase C and by increasing expression of the same keratinocyte genes. From Bikle et al. 2003 J Cell Biochem 88:290e295, with permission.
cancer risk or mortality or on the incidence of adenomatous polyps. Five other studies showed beneficial effects of borderline statistical significance and five showed no effect. Three recent, large studies in the US or Japan found statistically significant inverse relationship between calcium intake and risk of colorectal cancer with an average relative risk of about 0.7 in subjects with a higher intake of calcium [199e201]. Therefore, substantial epidemiological evidence supports the concept that increased intake or supplementation with calcium and/or vitamin D can substantially reduce the risk of colorectal cancer and the risk of recurrence of colonic polyps, thereby potentially substantially reducing the morbidity, mortality, and cost of medical care related to colorectal neoplasia in this country and elsewhere. What are the mechanisms by which calcium and vitamin D act to reduce the risk of colon cancer? Elevated levels of Ca2þo, acting via the CaSR, inhibit the proliferation of colon cancer cells. In Caco-2 cells,
high Ca2þo lowers c-Myc expression and up-regulates p21 [176]. Furthermore, in CBS colon cancer cells, in addition to increasing p21, high Ca2þo up-regulates E-cadherin expression and inhibits the nuclear transcription factor, TCF4, which influences signaling by the Wnt pathway [202,203]. Activation of the CaSR in colon cancer cells also down-regulates thymidylate synthase and survivin, which enhances cellular sensitivity to 5-FU. Kallay, Peterlik, and Cross have shown that vitamin D exerts antiproliferative actions in colon cancer cells by down-regulating cyclin D1 (176). 1,25(OH)2D3 also upregulates p21waf1 and p27 and down-regulates survivin, an inhibitor of apoptosis, and thymidylate synthase, which increases the sensitivity of colon cancer cells to the cytotoxic agent, 5-fluorouracil, as just noted. Of interest, these latter effects occurred in a CaSR-dependent manner [203]. As just detailed, a number of these actions are shared by high Ca2þo. Of note with regard to the in vitro effects of 1,25(OH)2D3, VDR knockout mice exhibit
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colonic hyperproliferation, supporting the role of 1,25 (OH)2D3 and the VDR in tonically suppressing proliferation of the colonic mucosa in vivo [204]. Therefore, high Ca2þo and 1,25(OH)2D3 each have a number of actions in colon cancer cells that would be expected to reduce proliferation and enhance differentiation (e.g., up-regulating E-cadherin, as in keratinocytes). Recent work indicates that these actions may not take place totally independently of one another, as noted above. There are also interesting and potentially important interactions between the CaSR and VDR in their actions on normal colon or colon cancer cells. Both high Ca2þo and 1,25(OH)2D3 up-regulate the CaSR [202], providing a feedforward mechanism by which high Ca2þo can amplify its own effects. In addition, high dietary calcium suppresses expression of the 24-hydroxylase in vivo, providing a potential mechanism by which elevating Ca2þo could increase the local level of 1,25(OH)2D3 by reducing its degradation, even though the colonic level of CYP27B1 did not change [176]. Future studies in this area could investigate in more detail the mechanisms, including signaling pathways and transcriptional control, by which Ca2þo exerts its actions on the colon and develop and utilize mice with conditional knockout of the CaSR in the intestine to further solidify the receptor’s role in the colon and its interaction with vitamin D and the VDR. CaSR and VDR in Prostate Cancer Thirteen of 26 studies in patients with prostate cancer found a statistically significantly reduced risk of prostate cancer with increasing vitamin D status, 11 reported no significant association and only one reported a significant inverse correlation, but the last of these only used latitude as an indirect measure of vitamin D status and prostate cancer risk [198]. Available data for the effect of calcium intake on prostate cancer risk, however, are even less clearcut than with vitamin D status. One study found a positive association with calcium intake and the risk of prostate cancer [205], while others found no such association [206]. However, a recent meta-analysis of 45 observational studies did not show a clearcut association between calcium intake and risk of prostate cancer [207]. Mechanisms by which vitamin D could exert beneficial effects on prostate cancer include cell cycle arrest through induction of p21, promotion of differentiation, and inhibition of tumor cell invasion and metastasis [208]. As with colon cancer and breast cancer cells, prostate cancer cells express the 1-hydroxylase enzyme, allowing for local production of 1,25(OH)2D3. The mixed effects of calcium intake on prostate cancer risk may relate to actions of the CaSR on prostate cancer cells that would enhance rather than reduce prostate cancer cell growth and/or metastasis, or it could exert a combination of stimulatory and inhibitory actions.
For example, the CaSR inhibits apoptosis of AT-3 prostate cancer cells [209] and activates the epidermal growth factor receptor in PC-3 prostate cancer cells, which would likely increase cellular proliferation and tumor growth [210]. The CaSR also promotes cellular proliferation and enhances metastasis in an in vivo mouse model in which the malignant and metastatic behavior of either control PC-3 cell or those with knockdown of the CaSR were compared [211]. The CaSRinduced stimulation of PTHrP production by prostate cancer cells [210], if it occurs in prostate cancer cells metastatic to bone in vivo, might aggravate malignant osteolysis owing to the stimulation of further bone resorption by the PTHrP secreted by cancer cells near sites of bone resorption (i.e., with a locally high level of Ca2þo), begetting further resorption, and so forth. Therefore, activation of the CaSR need not produce the same biological actions exerted by 1,25(OH)2D3 in cancer cells, and the effects of the CaSR and VDR in various forms of cancer must be compared on a caseby-case basis. Clearly, there is also no definitive evidence that the CaSR on prostate cancer cells serves as the molecular target for the potentially deleterious actions of calcium supplementation on the risk of prostate cancer.
POLYMORPHISMS OF THE CASR AND VDR AND CALCIUM AND BONE HOMEOSTASIS Identifying an association between polymorphisms in a gene (often single nucleotide polymorphisms (SNPs) and more recently the use of SNPs in genome-wide association studies (GWAS)) and disease states or alterations in physiological or other variables can provide clues into disease pathogenesis that can be pursued by experimental approaches that prove causality. There have been a number of studies examining the associations of CaSR polymorphisms with variables such as serum calcium concentrations in normal individuals, PTH level and severity of hyperparathyroidism, calcium excretion and renal stones, fractures and bone mineral density, and risk of colon cancer. In some studies, there have been concomitant investigations of association with polymorphisms of the VDR in order to look for interactions between polymorphisms in the CaSR and VDR genes. The following briefly describes some of these studies to provide a brief overview of the state of this field. There have been several studies of the associations between CaSR SNPs, particularly those producing changes in amino acids within the receptor’s C-tail (A986S, R990G, and Q1011E), and serum calcium concentration in normals. Most [212,213] but not all
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[214] found modest changes in serum calcium concentration associated with specific genotypes (i.e., higher serum calcium with A986S or with haplotypes such as SRQ/ARE (a haplotype is a series of SNPs or alleles on a single strand of DNA) [212]). A recent study utilized GWAS to show the association of a different polymorphism with serum calcium concentration [215]. The studies showing a positive association are consistent with the idea that SNPs that alter the function or the expression of the CaSR may contribute to small variations in serum calcium concentration that are observed within the normal population. Two studies found an association between the presence of homozygosity for arginine at residue 990 and the severity of hyperparathyroidism in dialysis patients [216] or in primary hyperparathyroidism [217]. Another study, however, found no relationship of the three polymorphisms in the CaSR’s C-tail to persistent hyperparathyroidism after kidney transplantation [218]. Only one [219] out of about eight studies (viz., [220,221]) found an association between CaSR polymorphisms and bone mineral density, and no studies have reported an association with fracture risk. Therefore, despite its importance in chondrocyte and osteoblast biology, at least in the mouse, genetic variation in the CaSR in the general population does not appear to impact bone health. Several studies have shown an association between certain polymorphisms in the CaSR and either urinary calcium excretion [222] or renal stone disease [223e225] in normocalcemic stone formers, as well as in patients with primary hyperparathyroidism [226,227], but some have not [228]. Therefore, the presence of these associations is consistent with the known role of the CaSR in the control of renal calcium excretion. In this regard, it is also possible that a CaSR antagonist could be developed that would be of clinical utility in patients in whom altered function of the CaSR appears to be contributing to hypercalciuria by reducing the receptor’s activity. Three [229e231] out of four studies have found a positive association between risk or recurrence of colon cancer and polymorphisms in the CaSR. For instance, the A986S polymorphism was associated with a fourfold greater risk of cancer in one study [230], while a different SNP (rs1801726) was associated with a lower risk of cancer in another [229]. In one of these three studies that yielded positive results, the association was only with cancer in the proximal colon [231]. These results are consistent with those from the experimental studies described earlier showing that the CaSR may participate in the control of cell growth in the colon, and suggest that it may be possible to identify genetic variants that could predict persons at increased risk for the disease. In no study to date out of about ten that have studied both VDR and CaSR polymorphisms at the same time
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has there been any interactions between polymorphisms in the two genes and parameters such as bone mineral density [221] or disease states, e.g., renal stone disease [228] and colon cancer [231]. Surprisingly, therefore, despite the many interrelationships and interactions between the two genes in experimental studies carried out either in vivo or in vitro that were detailed earlier, polymorphic variations in the two genes do not appear to interact with one another in the studies carried out to date.
SUMMARY AND PERSPECTIVES Research carried out over the past 20 years, largely as a result of ongoing improvements in molecular techniques and the development of mouse knockout models, has dramatically altered our understanding of how calcium and vitamin D interact in bone and mineral ion homeostasis. In this regard, the summary that follows will assume that mice and men are more similar than different in calcium and bone metabolism. An important advance in this area has been the identification and characterization of the CaSR [2]. As a result, it has become possible to understand the role of extracellular calcium ions as not just serving as a passive participant in processes such as bone development and renal calcium excretion, but also acting as an extracellular first messenger. As such, calcium can actively regulate the tissues that make up the systems governing mineral ion and bone metabolism. The following discussion highlights and summarizes key areas covered earlier in which there have been dramatic shifts in our understanding of how calcium and vitamin D act and interact through their respective receptors, the CaSR and VDR. All calcium destined to serve extra- or intracellular roles originally entered the body through the GI tract. Studies in mice with global knockout of the VDR in which this receptor was expressed transgenically in the intestine support VDR-stimulated absorption of calcium as a crucial function of the receptor in mineral ion and bone homeostasis [131]. However, the rescue diet utilized in the global VDR knockout animals can overcome the defect in calcium absorption resulting from loss of the VDR in the intestine simply by ensuring sufficient dietary mineral ions. Thus the main purpose of the VDR in the intestine is to increase the efficiency of calcium absorption to the point where adequate calcium is absorbed with physiologically relevant levels of calcium intake. Until recently, this dietary rescue might have been assumed to simply reflect massaction-driven, passive absorption of calcium through the paracellular pathway. Surprisingly, however, normalization of the serum calcium concentration in mice with knockout of the 1-hydroxylase is
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accompanied by up-regulation of several of the genes participating in the active transcellular pathway of intestinal calcium absorption [232,233]. The latter observation has not been formally proven to be CaSRmediated but appears to reflect the capacity of the CaSR to serve a backup role in calcium absorption in the presence of abundant calcium in the gut lumen. How about the other elements of the calcium homeostatic mechanism? Once adequate calcium is absorbed, a key function of the CaSR is to serve as a “calciostat” that ensures adequate, stable circulating levels of calcium in bodily fluids [5]. Experiments in which the VDR was knocked out only in the parathyroid suggest that the parathyroid VDR serves only a relatively minor, secondary role [83]. That is, the loss of the well-established capacity of vitamin D to inhibit PTH gene expression and parathyroid cellular proliferation can be largely substituted for by the CaSR, with maintenance of normal or nearly normal parathyroid function even without the VDR in the parathyroid. However, the capacity of the VDR and CaSR to up-regulate both their own expression as well as that of the other receptor noted earlier raises the possibility that the two can “tune” or adjust the gain of the other receptor in the parathyroid or other tissues, although the physiological relevance of such a mechanism has not been shown as yet. What is the relative importance of the two receptors in the kidney with regard to their roles in Ca2þo homeostasis? Knockout models have not yet explored the interplay between the VDR and CaSR in regulating the 1-hydroxylase in the proximal tubule. The VDR is clearly a key feedback inhibitor of the formation of 1,25(OH)2D3, but can the CaSR exert physiologically relevant inhibition of the 1-hydroxylase in VDRe/e mice? One scenario by which the CaSR has been postulated to inhibit the 1-hydroxylase is by up-regulating the VDR, i.e., acting by a VDR-dependent mechanism [92]. The development of a proximal tubule-selective knockout of the CaSR, VDR, or both would permit this issue to be addressed experimentally. A second key site where urinary calcium excretion is adjusted to suit the homeostatic needs of the organism is in the thick ascending limb of Henle’s loop [103]. Studies in mice with knockout of the CaSR and PTH genes [41] have shown a nonredundant role of the CaSR in cTAL in up-regulating renal calcium excretion, independent of the regulation of PTH secretion by the CaSR. Does the VDR have any role in Ca2þo homeostasis in this nephron segment? The data are sparse, but up-regulation of the CaSR by the VDR in CTAL may be a mechanism by which the VDR can enhance urinary calcium excretion, perhaps contributing to the body’s defense against hypercalcemia in this setting. It would be of interest to determine whether loss of the VDR in the thick ascending limb has any functional consequences.
Knockout models have also not explored the relative importance of the VDR and CaSR in the DCT/CNT. As in the intestine, the VDR is clearly a key regulator of the components of the transcellular pathway that stimulate calcium absorption in the DCT/CNT [233]. Again, however, as in the intestine, dietary calcium rescue in mice lacking the 1-hydroxylase is capable of up-regulating some of these components, suggesting a backup role for the CaSR not only in intestine but also in kidney if vitamin D is deficient but dietary calcium abundant. However, the dietary calcium rescue of VDRe/e mice is associated with substantial hypercalciuria [101], supporting an important role of the VDR, presumably in DCT/CNT, in renal calcium reabsorption. Thus, loss of the VDR in DCT/ CNT cannot be effectively compensated for by the CaSR. The relative contributions of PTH- and 1,25(OH)2D3mediated changes in bone resorption and formation to Ca2þo homeostasis vs. that of rapid alterations in calcium fluxes into or out of bone by a largely PTH- or 1,25(OH)2D3-independent mechanism(s) [43] is uncertain. Both the CaSR and VDR can promote bone formation [112,125], which could dispose of calcium in the setting of hypercalcemia, and yet both can support osteoclastogenesis [133,234]. High concentrations of calcium, in excess of those ever encountered in the blood, but relevant to the levels measured near actively resorbing osteoclasts, exert a homeostatically appropriate inhibition of osteoclast activity and promote osteoclast apoptosis [133]. These actions are mediated, at least in part, by the CaSR [133]. The relevance of these actions of the VDR and CaSR on osteoblast and osteoclast function to homeostatically significant changes in fluxes of calcium into or out of bone is not clearly defined. Some evidence also suggests a role for the CaSR in regulating rapid fluxes of calcium into or out of bone, presumably independent of changes in bone formation and/or resorption [43]. Although experimentally challenging, further studies of how the receptor exerts such an action would be illuminating in terms of providing further understanding of the homeostatic relevance of the regulated movement of calcium ions into or out of the skeleton over a wide range of timeframes. Our understanding of how the VDR and CaSR interact has perhaps been altered most dramatically with regard to their roles in the development and maintenance of the skeleton. The dramatic phenotypes of mice with conditional knockout of exon 7 of the CaSR in chondrocytes and osteoblasts [112] came as a surprise given earlier work with mice with knockout of exon 5, which showed little, if any, cartilage or bone phenotype, apparently due to the capacity of the exon-5-less CaSR to rescue the cartilage and bone phenotypes [109]. Subsequent studies using conditional knockouts have established unexpected, essential, and nonredundant roles of the CaSR in
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C H A P T E R
25 Effects of 1,25-Dihydroxyvitamin D3 on Voltage-Sensitive Calcium Channels in Osteoblast Differentiation and Morphology William R. Thompson 1, Mary C. Farach-Carson 2 1
Department of Physical Therapy, Program in Biomechanics and Movement Science; University of Delaware, Newark, Delaware, USA, 2 Department of Biochemistry and Cell Biology, Rice University, Houston, TX, USA
SYSTEMIC AND INTRACELLULAR CA2D HOMEOSTASIS Tight regulation of plasma and intracellular Ca2þ concentrations is essential to ensure proper cellular function and phenotype for essentially all cells in complex tissue. Mammalian Ca2þ homeostasis is hormonally regulated by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) at three major organ sites: the intestine, kidney, and bone. A decrease in serum Ca2þ concentration increases 1,25(OH)2D3 production, increasing intestinal Ca2þ absorption, decreasing excretion of Ca2þ into the urine, and increasing osteoclast activity ultimately to favor bone resorption. The first two processes occur quickly and are largely physiological responses, the latter is delayed and requires osteoclast expansion and activation. Approximately 99% of Ca2þ in the human body is held in the skeleton. During skeletal remodeling, Ca2þ released from mineralized tissue becomes freely exchangeable with the extracellular fluid, thus creating a buffer and reservoir system to help maintain circulating Ca2þ concentrations. A key function of bone-lining cells is to protect the mineral component of bone from being lost except at sites of bone remodeling. Intracellular Ca2þ levels remain in dynamic balance due to the activity of channels, pumps, and exchangers present in the plasma membrane and within organelles such as the endoplasmic reticulum, mitochondria, and nuclei. Organelles and intracellular Ca2þ binding proteins only transiently buffer cytosolic increases. The majority of intracellular Ca2þ regulation is accomplished
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10025-3
by extrusion of Ca2þ into the extracellular space by the concerted efforts of Ca2þ-ATPases and Naþ/Ca2þ exchangers [1,2]. Uptake of Ca2þ into the mitochondria and endoplasmic reticulum is an additional mode of buffering intracellular Ca2þ. Increases in cytosolic Ca2þ concentrations result from release of Ca2þ from internal stores in the endoplasmic reticulum through leak channels and by extracellular influx of Ca2þ through a variety of membrane channels that include voltage-sensitive calcium channels (VSCCs), voltage-insensitive calcium channels (VICCs), mechanosensitive divalent cation channels (MDCCs) and receptor-operated calcium channels (ROCs). Plasma membrane Ca2þ channel activity is modulated by various mechanisms including the influence of calcitropic hormones such as 1,25(OH)2D3, parathyroid hormone (PTH), and by mechanical stimuli. VSCCs are major regulators of Ca2þ permeability in osteoblasts and are the major Ca2þ channels expressed in the plasma membrane of these cells [3].
VOLTAGE-SENSITIVE CALCIUM CHANNELS VSCCs are ubiquitous, multimeric complexes that regulate Ca2þ influx in response to membrane depolarization. VSCCs regulate numerous intracellular processes including contraction, secretion, neurotransmission, and gene expression in excitable tissues and in most nonexcitable cell types. Several tissues involved in the vitamin D endocrine system express VSCCs,
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including the kidney [4], intestine [5], and bone [3,6]. Tight spatial and temporal regulation of Ca2þ influx [7,8] is achieved by the presence of multiple unique subtypes of VSCCs, each with differing pharmacological, kinetic, and electrophysiological properties. The highly regulated patterns of Ca2þ influx help balance the need for Ca2þ entry with the potential cytotoxic effects of high intracellular Ca2þ levels such as may occur around active sites of bone remodeling, when Ca2þ stores are being solubilized from the bone reservoir. VSCCs first were purified from skeletal muscle transverse tubules [9]. Solubilization and purification of the channel complex initially revealed three subunits, a1, b, and g with consensus sites for cAMP phosphorylation present within the a1 and b subunits [9]. Later analysis demonstrated the presence of covalently linked fourth and fifth subunits from a common precursor that associate together termed a2d [10]. A model was produced based on a detailed analysis of protein sequences, hydropathicity, and glycosylation sites of the five VSCC subunits that included a transmembrane a1 subunit associated in a complex with the intracellular b subunit, a transmembrane g subunit, and a disulfidelinked a2d dimer [11e13]. A diagram of this general structure is shown in Figure 25.1. The a1 subunit is the largest of the complex consisting of an approximate mass of 175 kDa and has been the focus of numerous functional studies because it incorporates the Ca2þ conduction pore and can generate a Ca2þ current in the absence of other subunits [14]. The a1 subunit is the putative binding site for three classes of organic Ca2þ channel blockers and is regulated by various second messengers and toxins [15,16]. Similar to the a subunit of Naþ channels, the a1 subunit of VSCCs contains four homologous domains (IeIV), with six transmembrane segments (S1eS6) in each. The fourth transmembrane segment of each domain serves as the voltage sensor and the pore loop between segments S5 and S6 determines ion conductance and selectivity. Interestingly, a change of only three amino acids in the pore loops of domains I, II, and IV converts a Naþ channel to Ca2þ selectivity [15]. Also similar to the topology of Naþ channels, the a1 subunit of VSCCs have both the short amino-terminal and long carboxyterminal segments positioned on the intracellular or cytoplasmic face where they are accessible for modification during signal transduction [17e20]. The a1 subunits of VSCCs are encoded by at least ten distinct genes [20,21] and historically there have been various names given to the corresponding gene products. These subunits first were named by a lettering system starting with a1S for the original skeletal muscle isoform and a1A through a1E for those discovered subsequently [22]. This arbitrary nomenclature frequently led to confusion; therefore, a more rational naming system
was adopted [23] based on the well-defined potassium channel nomenclature [24]. Under the new system, calcium channels were identified using the chemical symbol of the primary permeating ion (Ca) with the principal physiological regulator (voltage) indicated as a subscript (Cav). The gene subfamily of each a1 subunit is denoted with a number (currently 1 to 3) and the order of discovery of the a1 subunit within that subfamily (1 to n). Thus the VSCC subfamily responsible for mediating L-type Ca2þ currents is labeled Cav1.1eCav1.4, which includes channels formerly known as a1S, a1C, a1D, and a1F respectively. The Cav2 subfamily mediates P/Qtype, N-type, and R-type currents and are labeled Cav2.1eCav2.3 respectively. T-type Ca2þ currents are mediated by Cav3 subfamily channels (Cav3.1e3.3), which include those formerly known as a1G, a1H, and a1I, respectively [15]. Amino acid sequences of a1 subunits share greater than 70% similarity within a subfamily, but are less than 40% identical among the three subfamilies [15]. This variation on a common structure demonstrates how these Ca2þ permeating subunits may regulate a variety of specialized events in varied systems and yet also share a number of common features. The b subunit has an approximate molecular weight of 56 kDa and is highly and dynamically phosphorylated in vitro. The lack of a membrane-spanning segment suggests an intracellular orientation of this subunit and pulse chase analysis demonstrates the ability of b subunits to be palmitoylated, which aids in membrane association [25]. Additionally, b subunits generate a motif termed the beta interacting domain (BID), identified as the minimum sequence necessary to influence a1 subunit expression and binding to the a1 subunit [26,27]. Genes encoding four distinct b subunits have been identified in rabbit brain. The activation and inactivation kinetics of a1 subunits expressed in Xenopus oocytes, in the absence of b subunits, are significantly reduced compared to native cell preparations [16]. Normal channel activity is nearly fully restored following coexpression of recombinant b subunits, demonstrating the ability of these intracellular subunits to regulate channel kinetics, voltage-dependent gating properties, and channel density. The b subunit also plays a role in trafficking the a1 subunit to the membrane [28], reportedly by the masking of an ER retention sequence on the a1 subunit [29]. In osteoblasts, the b2 isoform is found in a complex with the L-type, Cav1.2 subunit, and with a large cytoplasmic protein ahnak. This association forms a stable complex that permits Ca2þ signaling independently of the cytoskeleton [30]. The ability of different b subunits to associate with the a1 subunit allows for calcium channels with diverse electrophysiological properties [16,25,31e34].
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VOLTAGE-SENSITIVE CALCIUM CHANNELS
459 FIGURE 25.1 General model of voltage-
gated calcium channel structure showing subunit organization. Note that the poreforming a1 subunit is complemented by the exteriorly disposed a2d subunit and the cytoplasmic b subunit. The g subunit is not always present. The a2d subunit is positioned such that it can interact with the ECM. The b subunit is intracellularly disposed such that it can be modified during signaling such as occurs following exposure to 1,25(OH)2D3 (see text). Figure modified with permission from Dooley et al. [109].
The a2d subunits are encoded as a single gene product, which is post-translationally cleaved and subsequently joined together by a disulfide bond [35]. The reduced form of this subunit yields a 143-kDa a2 and 27-kDa d polypeptide [13,36]. The a2 subunit is extensively glycosylated [13] and is positioned extracellularly, while the d subunit, with a single transmembrane domain, resides in the plasma membrane. There are four distinct genes that encode for the a2d subunit. Osteoblastic cells express the a2d1 and a2d3 isoforms [37]. The extracellular a2 subunit promotes assembly and trafficking [38] of the a1 subunit on the plasma membrane, and the ability of a2d to modulate a1-induced current is enhanced by coexpression with the b subunit [35,39e43]. These properties demonstrate that similar to the b subunit, the a2d subunit can regulate VSCC current amplitude [16,39,40]. Additionally, the a2d1 isoform facilitates spreading, migration, and attachment of myoblasts, suggesting the ability of this
subunit to form attachments to the extracellular matrix (ECM) [44]. Similar interactions of the external face of VSCCs, in particular the a2d subunit, with ECM components likely occur in other components of connective tissue, including bone. The g subunit is the last of the peptides that forms the native high-voltage activated (HVA) calcium channel complex. This highly glycosylated subunit is approximately 35 kDa and contains several hydrophobic regions consistent with membrane-bound proteins [45]. The function of this auxiliary subunit remains largely unknown and, although purification studies have not yet revealed a g subunit within cardiac tissue, a gsubunit-like cDNA has been isolated from cardiac mRNA by PCR cloning [2,16]. In bone, our earlier studies showed that osteoblastic cells did not have a classic g subunit [37], however we cannot exclude the presence of those other rarer g subunits discovered since the time that study was published.
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Calcium currents may be generated with only the a1 subunit; however, in the absence of auxiliary subunits, these channels display lower levels of membrane expression and altered kinetics and voltage dependence compared to native channels [14]. The presence of b subunits increases membrane trafficking while shifting the voltage dependence of activation and inactivation to more negative membrane potentials and increasing the rate of inactivation [11]. Coexpression of the a2d subunit enhances expression of the channel and confers normal gating properties [28]. The g subunit creates a negative shift of the voltage dependence of inactivation resulting in altered peak currents and a reduction in channel availability [46]. This is similar to that effect of 1,25(OH)2D3 on calcium currents in osteoblasts, discussed in the following section.
1,25-DIHYDROXYVITAMIN D3 ACTIONS ON VOLTAGE-SENSITIVE CA2D CHANNELS Calcium homeostasis is modulated by 1,25(OH)2D3, altering bone remodeling through a primary effect on regulation of osteoblast function. Resorptive signals generated by osteoblasts in response to 1,25(OH)2D3 alter osteoclast activity to ultimately favor bone resorption. Feedback between these two processes through paracrine regulation at sites of bone remodeling allows 1,25(OH)2D3 to function as a metabolic “switch” that maintains a balance of cellular activities and preserves bone mass in normal bone. Biological responses are generated by 1,25(OH)2D3 through two mechanisms, regulation of gene transcription and by rapid, membrane-associated events. Altered gene transcription occurs following long-term (hours to days) treatment with 1,25(OH)2D3 by binding the nuclear vitamin D receptor (nuclear VDR) [47e52]. Production of several noncollagenous matrix proteins, including osteopontin is stimulated by 1,25(OH)2D3 in mice [49]. Additionally, 1,25(OH)2D3 down-regulates osteocalcin (OCN) [50] and parathyroid hormone (PTH) production [51,52]. The most well-characterized cellular response to 1,25(OH)2D3 is the binding of 1,25(OH)2D3 to the nuclear VDR, translocation to the nucleus, interaction with coactivators, and modulation of gene expression, processes discussed in great detail in Chapters 7e13 of this volume. However, the rapidly initiated actions of 1,25(OH)2D3 have become more greatly appreciated and commonly accepted to occur in many cells and tissues. Membrane-initiated actions have been observed among other steroid hormones including estrogen [53,54], glucocorticoids [55], androgens [56,57], and progesterone [58], thus the role of 1,25(OH)2D3 in membraneinitiated events has warranted study in bone. Rapid
responses of 1,25(OH)2D3 have been associated with the induction of protein kinase C [59], phospholipase C [60], activation of the phosphatidylinositide-30 kinase/AKT (PI3K/AKT) pathway [61], exocytosis [62], and ATP secretion [63] in osteoblasts, phosphorylation of matrix proteins including osteopontin (OPN) [64], and modulation of intracellular Ca2þ levels [65]. Previous studies using microarray analysis demonstrated altered gene expression through nuclear VDRdependent and independent pathways in osteoblasts following treatment with 1,25(OH)2D3 [66]. Many genes lacking vitamin D response elements in their proximal promoters including stress response proteins, transcription factors, and various matrix proteins demonstrated changes in expression as early as 3 h following treatment with 1,25(OH)2D3. While it is possible that some of these responded owing to the presence of more distant response elements, these observations, along with 1,25(OH)2D3 responsiveness in nuclear VDR-free membranes, suggest the presence of separate nuclear and membrane receptors for 1,25(OH)2D3, both of which can alter gene transcription [66]. A potential membrane receptor for 1,25(OH)2D3 has been identified called 1,25D3-MARRS (membrane-associated rapid response to steroids) [67e70]. An antibody generated against the N-terminal sequence of a component of the membrane vitamin D response system in other tissues to functionally block this 64.5-kDa receptor, inhibited intracellular Ca2þ signaling mediated by 1,25(OH)2D3 [71e73]. Additionally, Wali and colleagues demonstrate that the nuclear VDR is not required for the rapid actions of 1,25(OH)2D3 in mouse osteoblasts [74]. These data indicate that 1,25D3-MARRS is one player that is involved in initiating rapid signals in response to 1,25(OH)2D3, perhaps by virtue of its ability to alter the activities of signaling molecules such as protein kinase C, which also modulate VSCC activities. Recently, Nemere and colleagues demonstrated that knockout (KO) mice lacking the 1,25D3-MARRS receptor exhibited blunted or completely absent Ca2þ uptake in response to 1,25(OH)2D3 in intestinal epithelial cells. Additionally, intestinal cells isolated from 1,25D3-MARRS KO mice show significantly diminished PKA activity in response to 1,25(OH)2D3 treatment [75]. Interestingly, Ca2þ responses following activation of the plasma membrane vitamin D receptor have been associated with altered gene expression, similar to that seen with prostaglandin treatment [59,66,76]. The rapid transcriptional effects of 1,25(OH)2D3 are related to Ca2þ influx rather than activation of the nuclear VDR, because changes in gene transcription are observed within 3 h following treatment with 1,25(OH)2D3 or with Ca2þ mobilizing vitamin D3 analogs [66]. Many of the rapid actions of 1,25(OH)2D3 are negated with the addition of Ca2þ channel blockers, including the ability to modify
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the phosphorylation state of OPN [64], demonstrating that the activity of plasma membrane Ca2þ channels is necessary for membrane-initiated actions of 1,25(OH)2D3. A rapid and transient increase in intracellular Ca2þ is observed in some cell types following addition of 1,25(OH)2D3 [77e79]. Intracellular Ca2þ elevation is dependent on the influx of Ca2þ through the plasma membrane and by release from internal stores [79]. A transient local elevation of intracellular Ca2þ can be elicited by low nanomolar concentrations of 1,25(OH)2D3; however, supraphysiological levels are necessary to promote release of Ca2þ from intracellular stores and yield measurable Ca2þ transients [77]. Several signaling pathways are activated upon binding of 1,25(OH)2D3 to the membrane vitamin D response system. In rat osteoblastic cells, 1,25(OH)2D3 stimulates VSCC activity resulting in activation of the PI3K/Akt pathway [62]. Electrophysiological studies have shown that the primary mechanism of Ca2þ influx in osteoblastic cells is through the L-type VSCC, which demonstrates a prolonged open time in response to 1,25(OH)2D3 treatment [3]. Addition of the L-type agonist Bay K 8644 or the 1,25(OH)2D3 analog AT (25-hydroxy-16-ene-23-yne-D3), which does not bind the nuclear VDR but does result in increases in Ca2þ influx [80], increases Ca2þ influx in osteoblasts and shifts the threshold of activation towards the resting potential, an event termed “left shift” [3,81]. A “left shift” of the resting membrane potential predicts that osteoblastic plasma membrane VSCCs are more susceptible to opening or are “primed” for the action of other hormones acting through ROCs or in response to small membrane depolarizations such as by activation of VICCs or MDCCs. This has been shown to be the case
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for interactions between 1,25(OH)2D3 and PTH in terms of generating calcium signals in osteoblastic cells and cultured bony calvaria [81]. If one considers the osteoblast to be a polarized cell as has been well demonstrated [82], then these hormonal interactions with VSCCs occur on the cellular face distal from the face attached to the mineralized matrix. This places the Ca2þ signaling face of the osteoblast near the blood supply, in an ideal location to sense physiological levels of circulating Ca2þ. The physiological outcomes in response to a “left shift” are apparent in the coordinated actions of 1,25(OH)2D3 and PTH. PTH stimulates Ca2þ influx in osteoblastic cells through gadolinium-sensitive Ca2þ channels and the resulting local depolarization can influence neighboring VSCCs [83]. Osteoblastic cell cultures pretreated with low nanomolar concentrations of 1,25(OH)2D3 for 10 min results in enhanced PTHinduced Ca2þ influx associated with increased bone resorption rates [81,84]. This indicates that 1,25(OH)2D3 serves a priming function to augment PTH-induced Ca2þ influx at the plasma membrane. Addition of the L-type VSCC inhibitor nitrendipine or removal of extracellular Ca2þ, inhibits the elevation of 1,25(OH)2D3 and PTH-induced Ca2þ influx [81], demonstrating that the enhanced PTH-induced Ca2þ influx is dependent on the presence and influx of Ca2þ through the L-type VSCC [84]. A diagram illustrating the concerted actions of calcitropic hormones in modifying the calcium permeability of the osteoblast plasma membrane is shown in Figure 25.2, where it is shown that the overall conductance of the channel is increased in the presence of 1,25(OH)2D3, which then allows for a greater degree of Ca2þ entry in the presence of a second calcitropic signal such as provided by PTH.
Calcitropic regulation of voltage-sensitive (VSCC) and voltage-insensitive (VICC) calcium channels in the plasma membrane of the osteoblast. The 1,25(OH)2D3 first serves to sensitize the VSCC channel by moving the threshold of activation toward the resting potential, a process referred to as “left shift.” Small amounts of Ca2þ enter the cell during this phase, insufficient to trigger Ca2þ release from stores. Subsequent activation of neighboring VICCs by PTH triggers a local depolarization that is sensed by the left-shifted VSCC to allow Ca2þ entry to occur. This Ca2þ entry (dark arrow) then is able to trigger release of Ca2þ from internal stores, resulting in full-fledged Ca2þ transient and physiological response. See text for details and references.
FIGURE 25.2
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1,25-DIHYDROXYVITAMIN D3REGULATED OSTEOBLAST DIFFERENTIATION Osteoblast differentiation is influenced by interactions with the ECM [85] and is regulated by mechanical [86] and hormonal signals [87]. Differentiation of mesenchymal precursors to osteoblasts is accompanied by increases in collagen type I, alkaline phosphatase (ALP) [88], and fibronectin (FN) [89,90]. Addition of 1,25(OH)2D3 to human osteoblastic cells increased expression of FN and enhanced cell-associated ALP activity, followed by altered morphology with cells containing more cytoplasmic processes [87], demonstrating the ability of 1,25(OH)2D3 to influence osteoblast differentiation. Previous studies have definitively determined that the L-type VSCC Cav1.2 is the primary subunit responsible for Ca2þ entry into the proliferating osteoblast [3,91] and that treatment with 1,25(OH)2D3 increases the mean open time of the L-type VSCC and enhances
permeability by shifting the threshold of activation toward the resting potential [3,84]. Blockage of the Ltype VSCC using benidipine promotes differentiation in murine preosteoblast cells [92], consistent with the idea that markers of osteoblast differentiation are enhanced following a loss of L-type membrane currents. Interestingly application of 1,25(OH)2D3 for 24e48 h to rat osteoblastic cells reduced L-type VSCC mRNA levels and increased markers of differentiation including OPN and OCN [93]. Previous studies also have demonstrated that terminally differentiated osteocytes express the T-type Cav3.2, and little or none of the L-type Cav1.2 VSCC subunit [94]. Figure 25.3 shows the distribution of expression of Cav1.2 and Cav3.2 in developing prenatal murine bone (Shao and Farach-Carson, unpublished). While Cav1.2 is present in rapidly growing cells present in both bone and cartilage at this stage of development, Cav3.2 is largely absent from cartilage, but is richly expressed by well-differentiated osteoblasts and by entrapped osteocytes [94]. Collagen X staining is strong in the hypertrophic zone of cartilage, separating FIGURE 25.3 Expression of calcium channels in developing bone. (A) Expression of L-type VSCC Cav1.2 in developing long bone (red). Note expression in cartilaginous growth plate, in bone marrow cells amongst trabecular bone spicules, and in both cortical and trabecular bone. (B) Expression of T-type VSCC Cav3.2 (red). Note more restricted expression with absence in the growth plate where only nuclei (green) can be seen. Also note absence in bone marrow. Staining (red) is seen at sites of osteocyte entrapment. (C) Staining of collagen X (red), specific to the hypertrophic zone separating the growth plate and the marrow cavity. Note that the yellow color results from overlap of the VSCC stain and the nuclea stain. Please see color plate section.
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the chondrocytic compartment of the growth plate from trabecular bone and bone marrow. Note down-regulation of staining for Cav1.2 in the hypertrophic zone where cells are not dividing. This is consistent for a role for Cav1.2 in growth and cell division, and for Cav3.2 in post-proliferative cells. Strong data suggest that 1,25(OH)2D3 plays a key role in the dynamic changes in VSCC expression during bone development [3,93]. Treatment of murine preosteoblasts for 24 hours with 1,25(OH)2D3 coordinately down-regulates L-type Cav1.2 and uniquely up-regulates T-type Cav3.2 VSCC expression at both the mRNA and protein levels [95]. Of the ten VSCC a1 subunits assayed, only the T-type Cav3.2 subunit was up-regulated. Application of 1,25(OH)2D3 also alters the Ca2þ permeability properties of the osteoblast membrane from a state of primarily L-current sensitivity to T-current sensitivity [95]. The physiological roles of T-type VSCCs are well characterized in neuronal [96] and cardiac cells [97], where T-type currents result in low-threshold Ca2þ spikes. These Ca2þ spikes are associated with burst firing and oscillatory behavior [98], leading to rapid neuronal depolarization and generation of pacemaker currents. Mice null for the T-type, Cav3.2 subunit are viable, but demonstrate focal coronary and arteriole defects as well as skeletal abnormalities [99] (Kronsberg A, Duncan RL, and Farach-Carson MC, in preparation). Up-regulation of the T-type VSCC Cav3.2 subunit following 1,25(OH)2D3 treatment suggests the need to compensate for the loss of L-type VSCC-mediated Ca2þ influx. If a subsequent up-regulation of another VSCC a1 subunit did not occur following the 1,25(OH)2D3-mediated down-regulation of the L-type Cav1.2, the ability of the cell to maintain Ca2þ-dependent cellular processes would be compromised. Alterations in Ca2þ permeability following 1,25(OH)2D3 application in osteoblasts demonstrate a drastic shift from L-type sensitivity to T-type sensitivity. One likely explanation for this change is that the high Ca2þ permeability in the preosteoblast supports cell proliferation and growth, whereas the depletion of Ca2þ permeability during 1,25(OH)2D3-stimulated differentiation attenuates Ca2þ-dependent proliferation signals and related gene expression. These alterations likely occur in parallel with the down-regulation of L-type Cav1.2 expression in terminally differentiated osteocytes [94]. Interestingly, treatment of human osteoblastic cells with 1,25(OH)2D3 results in a transition to a more stellate morphology with increased cytoplasmic processes [87], a phenotype characteristic of terminally differentiated osteocytes [100]. Additionally, the lipid growth factor lysophosphatidic acid (LPA) is a potent stimulator of osteocyte dendrite outgrowth and treatment of the osteocytic cell line MLO-Y4 results in
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a more stellate morphology [101]. The LPA receptor antagonist Kil6425 and pertussin toxin blocked the LPA-induced increase in dendrite formation [101]. Further studies demonstrated that 1,25(OH)2D3-induced osteoblast differentiation was inhibited in the presence of the Kil6425 and that 1,25(OH)2D3 and LPA act synergistically to generate mature osteoblasts, which was inhibited by pertussis toxin [102]. Increases in ALP in cells cotreated with LPA and 1,25(OH)2D3 were blocked with Y-27632, an inhibitor of Rho-associated coiled kinase (ROCK). Taken together, these data suggest that the alterations in osteoblast differentiation initiated by 1,25(OH)2D3 that alter VSCC expression may be augmented by cooperative actions of LPA [102].
INFLUENCE OF 1,25(OH)2D3 ON OSTEOBLAST CELL SURVIVAL Hormonal regulation of bone cell apoptosis has been implicated as a crucial mechanism to control osteoblast to osteoclast cell ratios, and thus the state of remodeling and mineralization of bone [61]. Several studies have demonstrated the ability of 1,25(OH)2D3 to promote cell survival in osteoblasts [61,103,104]. Various pathways have been shown to be influenced by application of 1,25(OH)2D3 to osteoblasts including activation of the PI3/Akt survival pathway [61] and inhibition of the proapoptotic Fas pathways [105]. Chronic elevations in intracellular Ca2þ are well known to participate in the initiation of apoptosis [106]. Application of 1,25(OH)2D3 for seconds to minutes increases the mean open time and Ca2þ permeability of the L-type VSCC [3,107]. Treatment of primary osteoblast cultures with 1,25(OH)2D3 produces an increase in Ca2þ entry that, along with subsequent release of Ca2þ from intracellular stores, elevates cytoplasmic Ca2þ levels [77,78]. Treatment of osteoblasts with 1,25(OH)2D3 results in down-regulation of the Cav1.2 VSCC subunit mRNA transcription and protein production [95]. Transcript levels of other L-type VSCC a1 subunits remained unchanged following application of 1,25(OH)2D3, indicating that the Cav1.2 subunit is the primary L-type pore-forming subunit whose transcriptional regulation is modulated by 1,25(OH)2D3 in osteoblasts. Prolonged exposure of osteoblasts to 1,25(OH)2D3 also revealed diminished Ca2þ entry through the L-type VSCC by radioactive 45Ca2þ influx assays, most likely because of decreased Cav1.2 expression [95]. We propose that a rational role for down-regulation of the Cav1.2 subunit in response to long-term 1,25(OH)2D3 treatment is to protect the cell from chronic increases in intracellular Ca2þ levels that could result in apoptosis. Hippocampal neurons react similarly in that neuronal vulnerability to excitotoxicity is mediated by Ca2þ influx
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through the L-type VSCC, and down-regulation of these channels following long-term exposure to 1,25(OH)2D3 enhances neuroprotection [108]. These studies demonstrate that short- and long-term exposure to 1,25(OH)2D3 have distinct and sometimes opposite effects. The initial application of 1,25(OH)2D3 elicits a rapid cellular response, including activation of various protein kinases, protein lipases, and cAMP, by increasing Ca2þ influx through the L-type Cav1.2 subunit. In contrast, long-term exposure down-regulates the Cav1.2 subunit resulting in diminished Ca2þ entry, preventing Ca2þ toxicity. The previously described shift from L-type VSSC currents to T-type currents during differentiation from osteoblasts to osteocytes also may have a role in protection of these terminally differentiated bone cells. The L-type channel has a conductance of ~25 pS, whereas the T-type VSCC has a much reduced conductance of ~8 pS. The ability of osteoblasts to simultaneously decrease L-type Cav3.2 levels and increase expression of the T-type Cav3.2 subunit in the presence of 1,25(OH)2D3 [95], along with the observation that osteocytes predominantly express the T-type Cav3.2 subunit [94], together suggest that the decreased current potential in osteocytes may protect these terminally differentiated cells from toxic levels of Ca2þ. Ongoing studies in our laboratory are designed to further test these ideas. Ultimately, we believe that a complete understanding of the responses of osteoblasts to 1,25(OH)2D3 will create new avenues to design pharmacological interventions that will maintain bone mass in the elderly, perhaps while also preserving function of other excitable tissues such as nerve and muscle that express high levels of calcium channels.
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Acknowledgments The authors would like to thank our long-term collaborator, Dr. Randall L. Duncan, for many helpful discussions and for sharing unpublished data of voltage-sensitive channel knockouts. We also thank Jason M. Koons for his assistance in the preparation of the figures. We especially thank Dr. Ying Shao for contributing her images for Figure 25.3. William Thompson was funded in part by a Florence Kendall Scholarship and a Promotion of Doctoral Studies II training fellowship from the Foundation for Physical Therapy and an Adopt-A-Doc Scholarship from the American Physical Therapy Association Section on Geriatrics.
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[56] A. Gill, M. Jamnongjit, S.R. Hammes, Androgens promote maturation and signaling in mouse oocytes independent of transcription: a release of inhibition model for mammalian oocyte meiosis, Mol. Endocrinol. 18 (1) (2004) 97e104. [57] D. Haas, S.N. White, L.B. Lutz, M. Rasar, S.R. Hammes, The modulator of nongenomic actions of the estrogen receptor (MNAR) regulates transcription-independent androgen receptor-mediated signaling: evidence that MNAR participates in G protein-regulated meiosis in Xenopus laevis oocytes, Mol. Endocrinol. 19 (8) (2005) 2035e2046. [58] V. Boonyaratanakornkit, M.P. Scott, V. Ribon, L. Sherman, S.M. Anderson, J.L. Maller, et al., Progesterone receptor contains a proline-rich motif that directly interacts with SH3 domains and activates c-Src family tyrosine kinases, Mol. Cell 8 (2) (2001) 269e280. [59] J.P. van Leeuwen, J.C. Birkenhager, G.J. van den Bemd, C.J. Buurman, A. Staal, M.P. Bos, et al., Evidence for the functional involvement of protein kinase C in the action of 1,25-dihydroxyvitamin D3 in bone, J. Biol. Chem. 267 (18) (1992) 12562e12569. [60] B. Grosse, A. Bourdeau, M. Lieberherr, Oscillations in inositol 1,4,5-trisphosphate and diacyglycerol induced by vitamin D3 metabolites in confluent mouse osteoblasts, J. Bone Miner. Res. 8 (9) (1993) 1059e1069. [61] X. Zhang, L.P. Zanello, Vitamin D receptor-dependent 1 alpha,25(OH)2 vitamin D3-induced anti-apoptotic PI3K/AKT signaling in osteoblasts, J. Bone Miner. Res. 23 (8) (2008) 1238e1248. [62] Z. Xiaoyu, B. Payal, O. Melissa, L.P. Zanello, 1Alpha,25(OH)2vitamin D3 membrane-initiated calcium signaling modulates exocytosis and cell survival, J. Steroid. Biochem. Mol. Biol. 103 (3-5) (2007) 457e461. [63] P. Biswas, L.P. Zanello, 1Alpha,25(OH)(2) vitamin D(3) induction of ATP secretion in osteoblasts, J. Bone Miner. Res. 24 (8) (2009) 1450e1460. [64] J.B. Safran, W.T. Butler, M.C. Farach-Carson, Modulation of osteopontin post-translational state by 1, 25-(OH)2-vitamin D3. Dependence on Ca2þ influx, J. Biol. Chem. 273 (45) (1998) 29935e29941. [65] G.J. Long, J.F. Rosen, Lead perturbs 1,25 dihydroxyvitamin D3 modulation of intracellular calcium metabolism in clonal rat osteoblastic (ROS 17/2.8) cells, Life Sci. 54 (19) (1994) 1395e1402. [66] M.C. Farach-Carson, Y. Xu, Microarray detection of gene expression changes induced by 1,25(OH)(2)D(3) and a Ca(2þ) influx-activating analog in osteoblastic ROS 17/2.8 cells, Steroids 67 (6) (2002) 467e470. [67] I. Nemere, M.C. Dormanen, M.W. Hammond, W.H. Okamura, A.W. Norman, Identification of a specific binding protein for 1 alpha,25-dihydroxyvitamin D3 in basal-lateral membranes of chick intestinal epithelium and relationship to transcaltachia, J. Biol. Chem. 269 (38) (1994) 23750e23756. [68] M.C. Farach-Carson, I. Nemere, Membrane receptors for vitamin D steroid hormones: potential new drug targets, Curr. Drug Targets 4 (1) (2003) 67e76. [69] A.W. Norman, X. Song, L. Zanello, C. Bula, W.H. Okamura, Rapid and genomic biological responses are mediated by different shapes of the agonist steroid hormone, 1alpha,25(OH) 2vitamin D3, Steroids 64 (1e2) (1999) 120e128. [70] M. Mesbah, I. Nemere, P. Papagerakis, J.R. Nefussi, S. OrestesCardoso, C. Nessmann, et al., Expression of a 1,25-dihydroxyvitamin D3 membrane-associated rapid-response steroid binding protein during human tooth and bone development and biomineralization, J. Bone Miner. Res. 17 (9) (2002) 1588e1596.
[71] Z. Jia, I. Nemere, Immunochemical studies on the putative plasmalemmal receptor for 1,25-dihydroxyvitamin D3 II. Chick kidney and brain, Steroids 64 (8) (1999) 541e550. [72] H.A. Pedrozo, Z. Schwartz, S. Rimes, V.L. Sylvia, I. Nemere, G.H. Posner, et al., Physiological importance of the 1,25(OH) 2D3 membrane receptor and evidence for a membrane receptor specific for 24,25(OH)2D3, J. Bone Miner. Res. 14 (6) (1999) 856e867. [73] I. Nemere, D. Larsson, K. Sundell, A specific binding moiety for 1,25-dihydroxyvitamin D(3) in basal lateral membranes of carp enterocytes, Am. J. Physiol. Endocrinol. Metab. 279 (3) (2000) E614eE621. [74] R.K. Wali, J. Kong, M.D. Sitrin, M. Bissonnette, Y.C. Li, Vitamin D receptor is not required for the rapid actions of 1,25dihydroxyvitamin D3 to increase intracellular calcium and activate protein kinase C in mouse osteoblasts, J. Cell Biochem. 88 (4) (2003) 794e801. [75] I. Nemere, N. Garbi, G.J. Hammerling, R.C. Khanal, Intestinal cell calcium uptake and the targeted knockout of the 1,25D3MARRS (membrane associated, rapid response steroid binding) receptor/PDIA3/Erp57. J. Biol. Chem. [76] A.M. Choi, R.W. Tucker, S.G. Carlson, G. Weigand, N.J. Holbrook, Calcium mediates expression of stressresponse genes in prostaglandin A2-induced growth arrest, FASEB J. 8 (13) (1994) 1048e1054. [77] R. Civitelli, Y.S. Kim, S.L. Gunsten, A. Fujimori, M. Huskey, L.V. Avioli, et al., Nongenomic activation of the calcium message system by vitamin D metabolites in osteoblast-like cells, Endocrinology 127 (5) (1990) 2253e2262. [78] M. Lieberherr, Effects of vitamin D3 metabolites on cytosolic free calcium in confluent mouse osteoblasts, J. Biol. Chem. 262 (27) (1987) 13168e13173. [79] G. Vazquez, J. Selles, A.R. de Boland, R. Boland, Rapid actions of calcitriol and its side chain analogues CB1093 and GS1500 on intracellular calcium levels in skeletal muscle cells: a comparative study, Br. J. Pharmacol. 126 (8) (1999) 1815e1823. [80] R. Khoury, A.L. Ridall, A.W. Norman, M.C. Farach-Carson, Target gene activation by 1,25-dihydroxyvitamin D3 in osteosarcoma cells is independent of calcium influx, Endocrinology 135 (6) (1994) 2446e2453. [81] W. Li, R.L. Duncan, N.J. Karin, M.C. Farach-Carson, 1,25 (OH) 2D3 enhances PTH-induced Ca2þ transients in preosteoblasts by activating L-type Ca2þ channels, Am. J. Physiol. 273 (3 Pt 1) (1997) E599eE605. [82] C.V. Gay, V.R. Gilman, T. Sugiyama, Perspectives on osteoblast and osteoclast function, Poult. Sci. 79 (7) (2000) 1005e1008. [83] R.L. Duncan, K.A. Hruska, S. Misler, Parathyroid hormone activation of stretch-activated cation channels in osteosarcoma cells (UMR-106.01), FEBS Lett. 307 (2) (1992) 219e223. [84] W. Li, M.C. Farach-Carson, Parathyroid hormone-stimulated resorption in calvaria cultured in serum-free medium is enhanced by the calcium-mobilizing activity of 1,25dihydroxyvitamin D(3), Bone 29 (3) (2001) 231e235. [85] A.M. Moursi, C.H. Damsky, J. Lull, D. Zimmerman, S.B. Doty, S. Aota, et al., Fibronectin regulates calvarial osteoblast differentiation, J. Cell Sci. 109 (Pt 6) (1996) 1369e1380. [86] S. Kapur, D.J. Baylink, K.H. Lau, Fluid flow shear stress stimulates human osteoblast proliferation and differentiation through multiple interacting and competing signal transduction pathways, Bone 32 (3) (2003) 241e251. [87] R.T. Franceschi, W.M. James, G. Zerlauth, 1 Alpha, 25dihydroxyvitamin D3 specific regulation of growth, morphology, and fibronectin in a human osteosarcoma cell line, J. Cell Physiol. 123 (3) (1985) 401e409.
III. MINERAL AND BONE HOMEOSTASIS
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[99] C.C. Chen, K.G. Lamping, D.W. Nuno, R. Barresi, S.J. Prouty, J.L. Lavoie, et al., Abnormal coronary function in mice deficient in alpha1H T-type Ca2þ channels, Science 302 (5649) (2003) 1416e1418. [100] L.F. Bonewald, Establishment and characterization of an osteocyte-like cell line, MLO-Y4, J. Bone Miner. Metab. 17 (1) (1999) 61e65. [101] S.A. Karagiosis, N.J. Karin, Lysophosphatidic acid induces osteocyte dendrite outgrowth, Biochem. Biophys. Res. Commun. 357 (1) (2007) 194e199. [102] J. Gidley, S. Openshaw, E.T. Pring, S. Sale, J.P. Mansell, Lysophosphatidic acid cooperates with 1alpha,25(OH)2D3 in stimulating human MG63 osteoblast maturation, Prostaglandins Other Lipid Mediat. 80 (1-2) (2006) 46e61. [103] A.M. Vertino, C.M. Bula, J.R. Chen, M. Almeida, L. Han, T. Bellido, et al., Nongenotropic, anti-apoptotic signaling of 1alpha,25(OH)2-vitamin D3 and analogs through the ligand binding domain of the vitamin D receptor in osteoblasts and osteocytes. Mediation by Src, phosphatidylinositol 3-, and JNK kinases, J. Biol. Chem. 280 (14) (2005) 14130e14137. [104] O. Morales, M.K. Samuelsson, U. Lindgren, L.A. Haldosen, Effects of 1alpha,25-dihydroxyvitamin D3 and growth hormone on apoptosis and proliferation in UMR 106 osteoblast-like cells, Endocrinology 145 (1) (2004) 87e94. [105] G. Duque, K. El Abdaimi, J.E. Henderson, A. Lomri, R. Kremer, Vitamin D inhibits Fas ligand-induced apoptosis in human osteoblasts by regulating components of both the mitochondrial and Fas-related pathways, Bone 35 (1) (2004) 57e64. [106] A. Verkhratsky, Calcium and cell death, Subcell Biochem. 45 (2007) 465e480. [107] X.T. Wang, S. Nagaba, Y. Nagaba, S.W. Leung, J. Wang, W. Qiu, et al., Cardiac L-type calcium channel alpha 1-subunit is increased by cyclic adenosine monophosphate: messenger RNA and protein expression in intact bone, J. Bone Miner Res. 15 (7) (2000) 1275e1285. [108] L.D. Brewer, V. Thibault, K.C. Chen, M.C. Langub, P.W. Landfield, N.M. Porter, Vitamin D hormone confers neuroprotection in parallel with downregulation of L-type calcium channel expression in hippocampal neurons, J. Neurosci. 21 (1) (2001) 98e108. [109] D.J. Dooley, C.P. Taylor, S. Donevan, D. Feltner, Ca2þ channel alpha2delta ligands: novel modulators of neurotransmission, Trends Pharmacol. Sci. 28 (2) (2007) 75e82.
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S E C T I O N I V
TARGETS
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C H A P T E R
26 Vitamin D and the Kidney Peter Tebben, Rajiv Kumar Departments of Medicine, Biochemistry and Molecular Biology, Divisions of Nephrology, and Endocrinology, Diabetes, Metabolism, and Nutrition, Mayo Clinic and Foundation, Rochester, Minnesota, 55905, USA
INTRODUCTION The kidney has a unique function in mineral homeostasis and plays a vital role in the control of plasma calcium and phosphorus. While examining how the kidney controls calcium and phosphate homeostasis it is worthwhile to keep the following facts in mind. 1. In humans, in a 24-hour period, about 8 grams of calcium are filtered at the glomerulus, and about 7.8 grams are reabsorbed in the proximal and distal tubules and the loop of Henle [1e5]. This is carried out in a manner such that, under normal circumstances, i.e. in states of neutral calcium balance, the amount of calcium in the urine closely approximates that absorbed in the intestine. The mechanisms by which calcium is reabsorbed in the kidney are complex and yield several insights into the cellular regulation of calcium transport. 2. The reabsorption of calcium in the kidney is controlled by several factors such as the filtered load of sodium, urine flow, and the activity of several hormones, most notably, parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25(OH)2D) and calcitonin, in addition to others [1e5]. 3. The kidney is the major site of synthesis of 1,25 (OH)2D, the active, hormonal form of vitamin D [6,7]. 4. The kidney expresses several 1,25(OH)2D-dependent proteins such as the plasma membrane calcium pump (PMCa), the epithelial calcium channel (ECaC), the sodium calcium exchanger, and the calbindins, some of which play a vital role in calcium transport. 5. The kidney expresses the vitamin D receptor (VDR). 6. The kidney expresses 25-hydroxyvitamin D3 1ahydroxylase (1a-hydroxylase or CYP27B1 gene) and 1,25-dihydroxyvitamin D3-24-hydroxylase (24-hydroxylase or CYP24A1 gene), and other
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10026-5
1,25-dihydroxyvitamin D3 and vitamin D analog metabolizing enzymes [6,7]. 7. Finally, the kidney has an equally important role in the control of plasma phosphate, and the filtration and reabsorption of phosphate. As in the case of calcium, the reabsorption and secretion of phosphate are under hormonal control and many of the same hormones and factors involved in calcium regulation, play a significant role in the regulation of phosphate reabsorption [8e13]. Given the complex role of the kidney in calcium and phosphate homeostasis, a brief overview of calcium and phosphate reabsorption in the kidney is in order.
Calcium Handling by the Kidney About 55% of total plasma calcium is ultrafilterable [14]. The ultrafilterable calcium concentration is about 1.35 mM (5.4 mg/dl) and closely approximates the concentrations of calcium present in glomerular fluid [15e17]. The total amount of calcium filtered at the glomerulus in a 24-hour period is about 8000 mg. Approximately 98% of the filtered load of calcium is reabsorbed in the tubules. Thus, the amount of calcium excreted in the urine in a 24-hour period is about 150e200 mg [1,14]. In the proximal tubule, about 50e60% of the filtered load of calcium is reabsorbed [2,3,18]. The reabsorption of calcium is thought to occur as a result of solvent drag by a paracellular route and is sodium-dependent. Volume expansion and a reduction of tubular sodium reabsorption inhibits calcium reabsorption, while volume contraction and an increase in sodium reabsorption enhance calcium reabsorption [2,19]. Inhibition of sodiumepotassium ATPase activity and sodium reabsorption by ouabain reduce the amount of calcium
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26. VITAMIN D AND THE KIDNEY
reabsorbed as does the substitution of sodium with lithium [19]. The concentration of calcium at the end of the proximal tubule is similar to that in the glomerular fluid. Importantly, calcium reabsorption in the proximal tubule is not influenced by thiazide diuretics, hormones such as PTH or 1,25(OH)2D3, or by hydrogen ions [2,3,18,19]. As we shall discuss later, some vitaminD-dependent proteins such as the calbindins, ECaC, and PMCa pump are either not expressed in the proximal tubule or are expressed in low amounts when compared with the amounts expressed in the distal tubule. Calcium reabsorption in the descending loop and the thin ascending limb of the loop of Henle is minimal. In the thick ascending limb of the loop of Henle about 20% of the filtered load of calcium is reabsorbed while another 10e15% is reabsorbed in the distal tubule with the remaining 5% being reabsorbed in the collecting ducts [2,3,18,19]. There are important distinctions between the factors influencing calcium reabsorption in the proximal tubule and the mechanisms of calcium reabsorption in the distal segments of the nephron. Firstly, the movement of calcium in the distal nephron occurs against a concentration gradient (lumen relative to extracellular fluid). Secondly, the lumen of the tubule is electronegative and becomes progressively more so towards the end of the distal tubule. Thirdly, calcium reabsorption can be dissociated from sodium reabsorption by thiazide diuretics which inhibit sodium reabsorption but enhance calcium reabsorption. Fourthly, hydrogen ions inhibit calcium reabsorption in the distal tubule whereas they have no effect on calcium reabsorption in the proximal tubule.
Phosphate Handling by the Kidney Virtually all inorganic phosphate in the serum is filtered by the glomerulus [8e10]. About 80% of filtered phosphorus is reabsorbed in the kidney, mostly in the proximal tubule. The amount of phosphorus reabsorbed in the proximal tubule is greatest in the first half of the proximal tubule and exceeds that of sodium. There is evidence for further phosphorus reabsorption in the pars recta. Little or no phosphorus reabsorption occurs in the loop of Henle or the distal tubule although there is some debate about whether there is phosphorus reabsorption in the distal tubule. The reabsorption of phosphate is sodium-dependent and is mediated by sodiumephosphate cotransporters (Na-Pi IIa,SLC34A1; Na-Pi IIc, SLC34A3, and Pit-2, SLC20A2) [20,21]. Na-Pi IIa activity is increased by a low-phosphate diet and decreased by PTH [22e25]. The recently described phosphatonins, fibroblast growth factor-23 (FGF-23) and secreted frizzled related protein-4 (sFRP-4) are also able to inhibit sodium-dependent phosphate transport [13,26]. In opossum kidney (OK) cells, NaPi II is
TABLE 26.1 Factors that Alter Renal Phosphate Excretion Increase
Decrease
1. High-phosphate diet 2. Parathyroid hormone 3. Calcitonin 4. Chronic vitamin D 5. Glucagon 6. Glucocorticoids 7. Volume expansion 8. Increased pCO2 9. Chronic acidosis 10. Starvation 11. Diuretics 12. “Phosphatonin” FGF-23 sFRP-4
1. 2. 3. 4. 5. 6. 7. 8.
Low-phosphate diet Thyro-parathyroidectomy Thyroxine Acute vitamin D Insulin Growth hormone Volume contraction Decreased pCO2
internalized from the cell membrane in response to FGF-23 and sFRP-4, similar to the effects of PTH [27,28]. Additional factors involved in phosphorus reabsorption are noted in Table 26.1.
ROLE OF THE KIDNEY IN THE METABOLISM OF 25(OH)D Formation of 1,25(OH)2D3 25-Hydroxyvitamin D3-1a-hydroxylase is a multicomponent, cytochrome P-450-containing enzyme in the mitochondria of renal proximal tubular cells [29e35]. The central role of the kidney in the formation of 1,25(OH)2D3 was first noted by Fraser and Kodicek, who demonstrated that nephrectomy abolished the formation of 1,25(OH)2D3 [36,37]. This was subsequently confirmed by others [38,39]. Nephrectomy greatly decreases circulating 1,25(OH)2D3 concentration in vivo except during pregnancy, granuloma-forming diseases, and lymphomas associated with the ectopic production of 1,25(OH)2D3 [40e43]. While the kidney is the major site of 1,25(OH)2D3 production, 25-hydroxyvitamin D3-1a-hydroxylase activity has been found in several other cell types throughout the body [44e49]. In vitro, chick renal epithelial cells in culture, mammalian nephron segments and homogenates derived from avian and mammalian (mostly rodent) renal cells all appear to metabolize 25(OH)D3 to 1,25(OH)2D3 [50e54]. For some time the proximal tubule was felt to be the only site of 1,25(OH)2D3 synthesis in the kidney. However, there is evidence that other tubular segments synthesize 1,25(OH)2D3 [55,56]. Using immunohistochemistry and in situ hybridization techniques, Zehnder et al. have demonstrated 1a-hydroxylase mRNA and
IV. TARGETS
EFFECTS OF VITAMIN D, 25(OH)D3 AND 1,25(OH)2D3 ON THE RENAL HANDLING OF CALCIUM AND PHOSPHORUS
protein in the distal convoluted tubule, cortical collecting duct, thick ascending limb of the loop of Henle, and Bowman’s capsule [56]. Recent experiments in which the 25-hydroxyvitamin D 1a-hydroxylase cytochrome P450 gene (CYP27B1) was deleted in mice point to the central role of this enzyme in vitamin D metabolism [57]. Table 26.2 summarizes some of the key factors known to regulate the activity of this enzyme in vivo and in vitro. The major regulators appear to be PTH, inorganic phosphorus, and 1,25(OH)2D3 itself. Regulators such as 1,25-dihydroxyvitamin D3 which alter the expression of the CYP27B1 gene have reciprocal effects on the expression of the CYP24A1 gene [6] that are mediated via vitamin-D-responsive elements in the promoters of the respective genes [58,59].
24-Hydroxylase Activity in the Kidney The 24-hydroxylase enzyme is a multicomponent cytochrome P450 enzyme expressed in the kidney as well as many other tissues [60e78]. In the kidney, 24-hydroxylase is expressed primarily in the proximal TABLE 26.2 Effect of Increased Level or Activity of Various Factors on 1,25(OH)2D3 Concentration or 1ahydroxylase Activity Factor
Animals
Humans
Ref.
Parathyroid hormone
[
[
[17,52,257e265]
Serum inorganic phosphorus
Y
Y
[255,266e268]
1,25(OH)2D3
Y
Y
[257,269]
Calcium (direct)
?
Y
[270,271]
Calcitonin
[,Y,0
[
[14,52,257,258,272,273]
Hydrogen ion
Y
0
[259,274,275]
Sex steroids
[
[
[254,276]
Prolactin
[
0
[277e279]
Growth hormone and insulin-like growth factor-1
[
[,Y,0
[184,271,280e285]
Glucocorticoids
Y,0
[,Y,0
[136,286e289]
Thyroid hormone
?
Y*
[290e292]
Fibroblast growth factor 23
Y
?
[293,294]
Frizzled related protein 4
Y
?
[13]
Pregnancy
[
[*
[295,296]
* Effects may be secondary to changes in calcium, phosphorus or parathyroid hormone. (With permission, modified from Kumar R [6].) [, Stimulation or increase; Y, suppression or decrease; 0, no effect; ?, effect not known.
TABLE 26.3
25(OH)D3
473
The Metabolism of 25(OH)D3 by the Kidney 24R,25(OH)2D3
24-keto-25(OH)D3
25S,26(OH)2D3
25(OH)D3-lactone
23S,25(OH)2D3
23-keto-25(OH)D3
tubule but is also present in more distal segments [79,80]. It is responsible for the conversion of 25hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 to 24,25-dihydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3, respectively. There is conflicting evidence whether 25(OH)D3 or 1,25(OH)2D3 is the preferred substrate for 24-hydroxylase [81]. Some have reported 1,25(OH)2D3 is the preferred substrate with a Km approximately tenfold lower than that for 25(OH)D3 [82,83] while others have found Km values substantially lower for 25(OH)D3 [84]. It has been suggested that these metabolites have certain unique properties and actions [85e88], however, others have not confirmed these observations [89e95]. The 24-hydroxylase enzyme activity and mRNA expression in the kidney is up-regulated by 1,25 (OH)2D3 [96e102]. The effect of 1,25(OH)2D3 is blunted in vitro and in vivo by parathyroid hormone [96e99]. The kidney is also capable of transforming 25(OH)D3 to several other compounds listed in Table 26.3 [103e123]. The specific physiological roles of these various metabolites are not known with certainty. Several polar metabolites of 1,25(OH)2D3 are formed in the liver, including calcitroic acid and glucuronide and sulfate conjugates of the hormone; these and small amounts of unchanged dihydroxylated and trihydroxylated metabolites of vitamin D are excreted in the urine [124e138]. Many of the tranformations that occur with 25(OH)D3 also occur in the case of 1,25(OH)2D3.
EFFECTS OF VITAMIN D, 25(OH)D3 AND 1,25(OH)2D3 ON THE RENAL HANDLING OF CALCIUM AND PHOSPHORUS Clinical studies have shown that vitamin D deficiency is associated with low urine calcium concentration whereas vitamin D excess or intoxication is associated with hypercalciuria [1]. The concentrations of calcium and phosphorus in the urine in these in vivo situations, however, reflect the decreased or increased calcium absorption in the intestine, the presence of hypo- or hypercalcemia, and the presence of diminished or elevated concentrations of circulating PTH. Puschett et al. examined the effect of vitamin D3 and 25(OH)D3
IV. TARGETS
474
26. VITAMIN D AND THE KIDNEY
on the renal transport of phosphate, sodium, and calcium in parathyroidectomized dogs [139e141]. They showed that short-term infusions of vitamin D3 and 25 (OH)D3 were associated with decreases in the clearances of phosphate, calcium, and sodium relative to the clearance of inulin. These studies were interpreted as showing that vitamin D3 and 25(OH)D3 enhance phosphate, calcium, and sodium reabsorption. A follow-up study by Puschett et al. showed that 1,25(OH)2D3 had similar effects on the excretion of phosphate, calcium, and sodium in thyroparathyroidectomized (TPTX) dogs [142]. We performed similar studies examining the effects of 25(OH)D3 on renal bicarbonate and phosphate reabsorption [143]. Unlike the studies of Puschett et al., we observed that phosphate and bicarbonate reabsorption increased only in intact animals and not in parathyroidectomized animals, suggesting the need for PTH. Our observations are similar to those of Popovtzer et al. [144]. Yamamoto et al. carried out perhaps the most comprehensive examination of the effects of 1,25 (OH)2D3 on the reabsorption of calcium [145]. VitaminD-deficient rats, vitamin-D-deficient rats supplemented with dietary calcium to normalize plasma calcium and PTH levels, and vitamin-D-replete rats were examined following TPTX and the infusion of graded amounts of calcium. Figure 26.1 shows the relationship between the amount of calcium excreted in the urine and serum calcium concentrations in the three groups of animals. Urinary calcium excretion was lower in vitamin-Dreplete rats than in vitamin-D-deficient rats, suggesting that vitamin D administration increased the efficiency of renal calcium reabsorption in the absence of PTH. In a second group of experiments, rats treated in the manner noted above were TPTX and infused with PTH. The results of this experiment show that a lower dose of PTH is needed to exhibit a comparable effect on renal calcium reabsorption in vitamin-D-replete rats when compared to vitamin-D-deficient rats (Fig. 26.2). This finding may be explained by in vitro studies of distal convoluted tubule cells in which 1,25(OH)2D3 increased PTH/PTHrp receptor mRNA levels [146]. Winaver et al. used micropuncture to examine the sites along the nephron at which 25(OH)D3 exerted its antiphosphaturic and hypocalciuric effects [147]. They observed that the effects of 25(OH)D3 were not mediated at the level of the superficial proximal tubule but were likely to have an effect on other segments of the nephron, most likely the distal tubule. These studies are similar to those of Sutton et al. and others who observed distal tubular effects of 25(OH)D3 in the dog [148]. These effects occurred shortly after the administration of 25(OH)D3 and were likely to have been independent of conversion to 1,25(OH)2D3 although this cannot be entirely ruled out since amounts of 25(OH) D3 and 1,25(OH)2D3 were not measured. Harris and
FIGURE 26.1 Relationships between urinary calcium excretion and serum calcium concentration in three groups of thyroparathyroidectomized (TPTX) rats. Serum concentration and urinary excretion of calcium were determined 16e19 h after continuous infusion of an electrolyte solution containing 0e30 mM of CaCl2. Each point represents the data pooled according to a continuous series of 0.25mM changes in serum calcium concentration. Horizontal bars indicate standard error of mean serum calcium concentration, and vertical bars indicate standard error of mean urinary calcium excretion. The lines were derived from the regression analysis of the linear portion of data. (:): Group A rats fed vitamin-D-deficient standard diet. (B): Group B rats fed vitamin-D-deficient diet containing high calcium and lactose. (•): Group C rats fed vitamin-D-replete standard diet. For any given serum calcium level, the urinary calcium excretion was significantly lower in vitamin-D-replete rats (group C) than in vitamin-Ddeficient rats (groups A and B). Thus, the apparent serum calcium threshold determined as an intercept of the regression line on the serum calcium axis was higher in vitamin-D-replete rats (~1.5 mM) than in vitamin-D-deficient rats (~1.0 mM). There was no significant difference in the calcium threshold between group A and group B. (With permission from Yamamoto M et al. [145].)
Seely examined the effects of 1,25(OH)2D3 on tubular calcium handling and concluded that the enhanced reabsorption of calcium occurred in distal tubular segments of the nephron [149]. All of these studies do not exclude an effect of the vitamin D analogs on a segment of the proximal tubule that is not accessible to micropuncture but are consistent with the localization of many vitamin-D-responsive proteins exclusively in the distal nephron. In vitro studies have shown that vitamin D deficiency is associated with decreased calcium uptake in membranes derived from the distal segments of the
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DISTRIBUTION AND REGULATION OF VITAMIN-D-DEPENDENT PROTEINS IN THE KIDNEY
475
FIGURE 26.2 The effects of PTH infusion on the renal handling of calcium among three groups of TPTX rats. The data are presented as described in the legend to Fig. 26.1. PTH was delivered at 2.5 U/h (•) on groups A and B rats, and 0.75 U/h (B) on group C rats. A, B, and C illustrate the results in group A, group B, and group C, respectively. The enhancement of calcium reabsorption by PTH is shown as the shifts of the lines to the right in each group. The striking difference exists between vitamin-D-deficient (groups A and B) and vitamin-D-replete (group C) rats in the doses of PTH required to induce a comparable shift in the calcium threshold. , data of TPTX rats in each group (from Fig. 26.1). (With permission from Yamamoto M et al. [145].)
nephron [150]. This occurs in membranes of both luminal and basolateral origin. In addition, Bindels et al. have demonstrated that cultured rabbit connecting tubule cells show an increase in the transport of calcium when exposed to 1,25(OH)2D3 [151]. Protein and mRNA expression of the recently described epithelial calcium channels (ECaC1 and ECaC2 or TRPV5 and TRPV6) are diminished in rats fed a diet deficient in vitamin D [152e154]. TRPV5 protein is present in the apical membrane of the distal convoluted tubule and is responsible for uptake of calcium from the tubule fluid into the cell [154,155]. TRPV6 is localized to the principal cells of the cortical and medullary collecting ducts and is also regulated by 1,25(OH)2D3 [153,154]. 1,25 (OH)2D3 increases mRNA and protein expression of the PMCa pump which is involved with active transport of calcium through the basolateral membrane of distal tubule cells [156]. A synthesis of the experimental results suggest that vitamin D3 metabolites have effects on the distal tubular reabsorption of calcium through several mechanisms. Expression of proteins responsible for distal tubule fluid uptake, intracellular trafficking, and basolateral transport of calcium are responsive to vitamin D3. Figure 26.3 depicts the transcellular transport of calcium through a distal tubule cell.
DISTRIBUTION AND REGULATION OF VITAMIN-D-DEPENDENT PROTEINS IN THE KIDNEY Several vitamin-D-dependent proteins are expressed in the kidney and many of these play a role in calcium
and phosphate transport. The VDR, calbindins, PMCa pump, and the ECaCs (TRPV5 and TRPV6) are all found in renal tubule cells and act coordinately in the regulation of calcium transport in the nephron [5,18,146,152,154,157e163]. We will discuss pertinent information regarding these vitamin-D-dependent proteins. Additional vitamin-D-responsive proteins involved in renal calcium and phosphate transport are listed in Table 26.4.
1,25-Dihydroxyvitamin D3 Receptor (VDR) in the Kidney The VDR mediates many, if not all, of the effects of 1,25(OH)2D3 in diverse organs [164e172]. The distribution of the VDR in the kidney has been assessed using a variety of techniques including ligand-binding assays with protein obtained from specific microdissected nephron segments, the localization of radiolabeled 1,25 (OH)2D3 by autoradiography, and by the use of various antibody techniques [79,173e177]. Using protein from microdissected nephron segments, the VDR was found in proximal and distal tubules [79,175]. With autoradiographic methods, following the administration of labeled ligand in vivo, silver grains were localized over distal tubule segments [79,174]. We have used sensitive polyclonal antibodies to localize the receptor in human and rat kidneys [161]. These antibodies were raised against highly purified antigen that was expressed in bacteria. The specificity of these antibodies was determined by absorption with purified antigen and by Western analysis which showed that they detected a protein band with a molecular mass of approximately 50 000 [161,178]. With these antibodies,
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26. VITAMIN D AND THE KIDNEY
FIGURE 26.3 Integrated model of active Ca2þ reabsorption in the distal part of the nephron. Apical entry of Ca2þ is facilitated by ECaC, Ca2þ
then binds to calbindin-D28K, and this complex diffuses through the cytosol to the basolateral membrane, where Ca2þ is extruded by an Naþ/Ca2þ exchanger and a plasma membrane Ca2þ-ATPase. The individually controlled steps in the activation process of the rate-limiting Ca2þ entry channel include 1,25(OH)2D3-mediated transcriptional and translational activation, shuttling to the apical membrane, and subsequent activation of apically located channels by ambient Ca2þ concentration, direct phosphorylation and/or accessory proteins. (With permission from Hoenderop et al. [250].)
we found that the VDR was present abundantly in the distal tubule and to a lesser extent in the proximal tubule (Fig. 26.4). Cells expressing calbindin-D28K also express the calcium pump and the epithelial calcium channel [152,179]. Interestingly, not all cells in the distal tubule expressed the receptor. Acid-secreting cells do not express the VDR in significant amounts. Taken together, the results are consistent with the notion that the VDR is present in significant amounts in the distal tubule where it regulates the amount and the activity of several vitamin-D-dependent proteins such as the plasma membrane calcium pump, epithelial calcium channel, and calbindin D28K. Although in lesser amounts, the
TABLE 26.4
Vitamin D Responsive Proteins in the Kidney
Vitamin D receptor 24-Hydroxylase Plasma membrane calcium pump Epithelial calcium channels, TRPV5 and TRPV6 Calbindin D28K Calbindin D9K Calcium sensing receptor PTH/PTHrp receptor Sodiumephosphate cotransporter type 2
proximal tubule also expresses the VDR where it regulates the activity of 1a-hydroxylase and 24-hydroxylase. We have shown that the VDR is present in cells of the developing rodent kidney [180] (Figs 26.5 and 26.6) and in the cultured metanephros (Fig. 26.7). The VDR was detected as early as day 15 post-coitum (p.c.) in the developing rat kidney in vivo. Significant amounts of the receptor were found in the metanephric mesenchyme as well as in the ureteric bud. As the kidney matured, the receptor was observed in the S-shaped and comma-shaped bodies and in the developing glomerulus, specifically in the parietal and visceral endothelial cells. The VDR staining in the latter cells persists in the adult kidney as well. Despite slightly different gestational periods, similar patterns of VDR were found in the developing mouse kidney in vivo. Of great interest is the observation that calbindin D28K appears in the distal tubule only around day 18 p.c. This is when urine flow begins in the kidney. The VDR is also present in mouse metanephric cultures in the same distribution pattern as is found in vivo. By day 3 of culture, the pattern of expression of the VDR was similar to that seen in the mouse kidney in vivo at day 15. Calbindin D28K does not appear in the cultured metanephros. These results showing that the VDR appears well before the appearance of calbindin D28K suggest that it may play a role in fetal renal development. Additional evidence for the regulation of calbindin D28K and calbindin D9K expression in the kidney can be found in
IV. TARGETS
DISTRIBUTION AND REGULATION OF VITAMIN-D-DEPENDENT PROTEINS IN THE KIDNEY
(A)
(D)
(G)
(B)
(E)
(H)
(C)
(F)
(I)
477
FIGURE 26.4 (AeC) Immunohistochemical detection of VDR in normal human kidney tissue with polyclonal anti-hVDR antibody 2-152 (A, 200; B, 400; C, 400). (DeF) Immunohistochemical detection of 25(OH)D3 24-hydroxylase cytochrome P-450 in human kidney (D 200; E, 400; F, 400). (G) Immunohistochemical detection of calbindin D28k in human kidney (G, 400). (H and I) Control panels showing kidney tissue stained with preimmune serum (H, 200; I, 400). (With permission from Kumar R et al. [161].) Please see color plate section.
VDR knockout mice where calbindin expression is reduced [181]. The VDR is regulated by several factors in diverse tissues [182]. Concentrations of the VDR in the kidney and parathyroid glands are mainly regulated by 1,25 (OH)2D3, PTH, and dietary calcium. Studies have shown that somewhat different results are obtained in vivo with respect to VDR abundance when 1,25(OH)2D3 concentrations are altered by dietary manipulations when compared to the effects of the intravenous administration of the hormone [183,184]. Exogenous administration of 1,25(OH)2D3 in rats increases VDR levels in duodenal and renal tissues [183,184]. When endogenous 1,25(OH)2D3 concentrations are increased by adapting an animal to a low-calcium diet (Table 26.5), VDR concentrations in the duodenum and kidney do not increase. The difference appears to be due to increases in the levels of PTH elicted by the low-calcium diet and decreased PTH following 1,25(OH)2D3 administration. Differences in CYP24A1 expression (decreased in the presence of a low-calcium diet, and increased following the administration of 1,25(OH)2D3) might also contribute to this difference in VDR expression. PTH has been shown to down-regulate VDR in osteosarcoma cells as well as block up-regulation of VDR in rats infused with both PTH and 1,25(OH)2D3 [185]. These
results demonstrate the opposing effects of PTH and 1,25(OH)2D3 on VDR expression in the kidney. The results obtained from in vivo studies are consistent with those obtained following the administration of 1,25(OH)2D3 to yeast cells transfected with a VDR construct [186]. There is evidence that in certain cells such as fibroblasts, 1,25(OH)2D3 may have its predominant effect to increase VDR abundance by ligandinduced stabilization of the protein [187]. Wiese et al. showed that the addition of 1,25(OH)2D3 to fibroblasts did not result in a significant increase in VDR mRNA concentrations but did result in increases in the amount of VDR protein suggesting that the predominant effect of 1,25(OH)2D3 in fibroblasts is the stabilization of preexisting VDR protein or the stabilization of preexisting vitamin D receptor mRNA. In addition to the VDR, several vitamin-D-dependent proteins are expressed in the kidney. The pattern of regulation of these proteins is of great interest, in as much as it casts light on the different mechanisms by which calcium is transported in the kidney. Those proteins involved in calcium transport include: calbindin D28K and calbindin D9K, the plasma membrane calcium pump, the epithelial calcium channel, and the calcium-sensing receptor. Additionally, although not involved in the transport of calcium, the 24-hydroxylase
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26. VITAMIN D AND THE KIDNEY
(A)
(A)
(B)
(B)
(A) Vitamin D receptor (VDR) immunostaining in metanephros of rat fetus on gestational day 15. (B) Higher-power view of boxed area in (A). Branching ureteric buds (arrows) and mesenchyme (M) are indicated. Bars ¼ 2.1 mm. (With permission from Johnson JA et al. [180].) Please see color plate section.
FIGURE 26.5
and the sodiumephosphate type 2 cotransporter (NaPi 2) are also expressed in the kidney. All of these proteins appear to be regulated by 1,25(OH)2D3.
Calbindins-D The calbindins-D are widely distributed in many tissues of the body and in a variety of species. Except in the brain, their synthesis is dependent on vitamin D but not calcium or phosphorus. There are two forms of calbindin-D, namely, calbindin-D28K and calbindinD9K that are variably distributed in different tissues of the body [188e205]. The apparent molecular weight of the larger protein is about 30 000 daltons whereas that of the smaller protein is 9000 daltons [188]. The proteins are classical EF hand proteins, the calbindinD28K having six EF hand structures and the calbindinD9K having two such motifs [206e225]. The proteins bind calcium with high affinity and in different molar amounts. Calbindin D28K binds 3e4 moles of calcium per mole of protein and calbindin D9K two moles of
FIGURE 26.6 VDR (A) and calbindin D28k (B) immunostaining in metanephros of rat fetus on gestational day 17. Parietal epithelial cells (small arrows), visceral epithelial cells (open arrows), and tubule portion of developing comma-shaped body (T) are indicated. Bars ¼ 2.1 mm. (With permission from Johnson JA et al. [180].) Please see color plate section.
calcium per mole of protein [188]. In the mouse kidney, both forms are present and are regulated by vitamin D. In other species, only calbindin-D28K is expressed in the kidney. Both proteins undergo conformational change upon binding to calcium. This phenomenon has been studied extensively by us in the case of calbindinD28K. We have shown that the protein undergoes a two-step change in conformation that is associated with binding to a high-affinity site in EF-hand 1 and a further change upon calcium binding to sites in EF hands -4 and -5 [215,217,226]. We recently solved the structure of Ca2þ-loaded calbindin-D28K [227]. The protein is comprised of a single, globular fold consisting of six distinct EF-hand subdomains, which coordinate Ca2þ in loops on EF1, EF3, EF4, and EF5. Ranbinding protein M, myo-inositol monophosphatase, and procaspase-3-derived peptides interact with the protein on a surface comprised of alpha5 (EF3), alpha8 (EF4), and the EF2-EF3 and EF4-EF5 loops. Fluorescence experiments reveal that calbindin-D28K adopts discrete hydrophobic states as it binds Ca2þ. The
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DISTRIBUTION AND REGULATION OF VITAMIN-D-DEPENDENT PROTEINS IN THE KIDNEY
(A)
(B)
FIGURE 26.7 VDR immunostaining in mouse metanephric organ culture explant following 120 h of incubation. Parietal epithelial cells (small arrow), visceral epithelial cells (large arrow), proximal tubule (P), and distal tubule (D) are indicated. Bars ¼ 2.1 mm. (With permission from Johnson JA et al. [180].) Please see color plate section.
TABLE 26.5
conformation change is probably what allows the protein to act as a modulator of the activity of Ranbinding protein M, myo-inositol monophosphatase, procaspase-3, the plasma membrane calcium pump, TRPV5, and TRPV6 channels (see below). Calbindin D28K and calbindin-D9K are regulated by 1,25(OH)2D3 in the kidney [219e225,228]. Both calbindins have lower expression in 1a-hydroxylase knockout mice [229]. Calbindin D28K and calbindinD9K expression was normalized by treatment with 1,25(OH)2D3, however, only calbindin D28K expression is increased when 1a-hydroxylase knockout mice were fed a high-calcium diet. In a mouse VDR-KO model, renal calbindin D9K expression is nearly abolished whereas calbindin D28K expression returns to control values as the animals age [230]. Cao et al. demonstrated that calbindin-D9K induction by 1,25 (OH)2D3 in vitro is absent in VDR-null cells [231]. Furthermore, calbindin D9K regulation by 1,25 (OH)2D3 was restored after transfection of the VDRnull cells with human VDR clearly showing the effect of 1,25(OH)2D3 is mediated through the VDR. In vitro, PTH has a synergistic effect with 1,25(OH)2D3 on calbindin D9K expression but has no effect alone [231]. In contrast, calbindin D28K expression is enhanced by PTH infusion without elevations in 1,25(OH)2D3 concentrations [232]. Using distal tubule membranes, preincubation with calbindin D28K increased calcium uptake in luminal membranes while calbindin D9K preincubation increased calcium uptake in basolateral membranes [233,234]. Calbindin D28K knockout mice fed a high-calcium (1%) diet have an elevated urinary
Effect of Dietary Calcium on Unoccupied VDR Content in Rat Duodenum and Kidney Day Diet
2
7
14
21
1% calcium
341 26
197 17
202 17
259 26
0.02% calcium
365 27
226 28
221 16
267 28
1% calcium
ND
163 11
165 9
124 8
0.02% calcium
ND
120 4*
131 10*
77 3*
1% calcium
9.98 0.18
9.82 0.08
9.16 0.16
9.52 0.41
0.02% calcium
9.45 0.12
9.15 0.17*
9.09 0.14
8.70 0.29*
1% calcium
8.40 0.17
8.88 0.22
8.73 0.17
7.92 0.29
0.02% calcium
8.15 0.22
8.65 0.13
8.37 0.28
8.41 0.23
1% calcium
153 11
113 13
139 9
160 32
0.02% calcium
180 20
392 45**
682 44**
829 59**
>
VDR
Duodenum
Kidney
>
Calcium (mg/dL)
>
Phosphorus (mg/dL)
1,25-(OH)2D> 3
(pg/mL)
>
Values are mean SEM. Unoccupied vitamin D receptor content expressed as fmols [3H]1,25(OH)2D3 bound per mg cytosol protein. * P < 0.05; ** P < 0.001. ND ¼ not done. Modified from Goff JP et al. [183].
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26. VITAMIN D AND THE KIDNEY
calcium/creatinine ratio despite no difference in serum calcium or PTH compared to wild-type littermates [235,236]. However, the elevated urinary calcium/ creatinine ratio in calbindin D28K knockout mice was not apparent after fasting [235]. This is consistent with our finding that fractional excretion of calcium is not altered in calbindin D28K knockout mice fed a normalcalcium diet [237]. The effects of calbindin D28K on renal calcium conservation in mice are modest, perhaps because of compensatory increases in calbindin D9K. The interactions between vitamin D, PTH, calcium, and the calbindins appears to be quite complex. The exact mechanisms and their interactions with other calcium transport proteins is not yet completely understood.
The Plasma Membrane Calcium Pump We raised monoclonal and polyclonal antibodies directed against the plasma membrane calcium pump and used them to examine the distribution of these proteins in the adult human kidney [18, 157e160]. We found that epitopes for the calciume magnesium ATPase (calcium pump) were expressed in the basolateral membrane of the distal tubular cells (Fig. 26.8). Similar patterns of expression were apparent in the rat [158] and rabbit [238] kidney. Interestingly, not all cells of the distal tubule stained positively for the calcium pump. Further investigation showed that cells expressing carbonic anhydrase and presumably involved in acid secretion did not express the plasma membrane calcium pump whereas the other cells of the distal tubule did so. We found that the calbindin D28K was present in the same cells of the distal tubule as the plasma membrane calcium pump. Our studies on the distribution of the plasma membrane calcium pump in the kidney and its localization, predominantly in the distal tubule of the kidney are supported by others who have shown that calcium ATPases are present mostly in the distal tubule [238]. In addition, it appears that the plasma membrane calcium pump is widely distributed in a large number of other calcium transporting tissues many of which display vitamin-D-dependent calcium transport [239e245]. Table 26.6 shows the distribution of the plasma membrane calcium pump in different tissues. In MDBK (bovine distal tubule) cells, 1,25(OH)2D3 increases PMCa pump mRNA and protein [156]. Bouthinay et al. have examined the effects of vitamin D deficiency on the activity of the plasma membrane calcium pump [233,234]. They observed that vitamin D deficiency was associated with a decrease in PMCa pump activity in the distal tubule of the kidney. These same authors also observed that calbindin D9K increased the
activity of the renal basolateral membrane calcium pump. In collaboration with Wasserman’s group at Cornell University, we have shown that the plasma membrane calcium pump is regulated in the intestine by vitamin D [240,246,247]. We showed by Western analysis using monoclonal antibodies directed against the pump that shortly after the administration of 1,25(OH)2D3 to vitamin-D-deficient chicks, there is an increase in the amount of immunoreactive plasma membrane calcium pump in the cells of the duodenum, jejunum, and ileum [240]. This is associated with an increase in the amount of mRNA for the pump in the same segments of the intestine [246]. The increase occurs within 3e6 hours following the administration of 1,25(OH)2D3 and the effect is dose-dependent. Furthermore, dietary calcium and phosphorus depletion are also associated with an increase in the amount of the pump expressed in the intestine. Thus, in the intestine, 1,25(OH)2D3 increases the synthesis of the plasma membrane calcium pump. In addition, 1,25(OH)2D3 increases the activity of the plasma membrane calcium pump in intestinal cell basolateral membranes. The mechanism by which up-regulation of the calcium pump occurs in the kidney and intestine is uncertain although several possibilities arise. It could be a direct effect of either 1,25(OH)2D3 or via stimulation of calbindin D9k or calbindin D28k [233,234]. There is also evidence that stimulation of the calcium-sensing receptor (CaSR) decreases calcium absorption by inhibiting PMCa pump activity [248].
The Epithelial Calcium Channel, VanilloidReceptor-Related Transient Receptor Potential Channels 5 and 6 Hoenderop et al. described epithelial calcium channels (ECaCs) which are expressed in the apical membrane of the distal tubule and principal cells of the collecting duct and are distinct from previously described calcium channels [153,155,179,249,250]. There are at least two members in this family of calcium channels, ECaC-1/TRPV5 and ECaC-2/TRPV6 [250]. ECaC1/TRPV5 expression is limited to the kidney while ECaC2/TRPV6 is expressed in several other tissues [155,250e252]. The ECaCs/TRPV channels have six putative transmembrane-spanning domains including a pore-forming hydrophobic region between transmembrane domains 5 and 6 [249]. Several putative vitaminD-response elements (VDRE) have been identified within the promoter region of the human TRPV5 channel. Hoenderop et al. also demonstrated that ECaC1/TRPV5 mRNA and protein levels are increased to near control levels after vitamin D rescue in rats fed a vitamin-D-deficient diet [152]. In a study using
IV. TARGETS
DISTRIBUTION AND REGULATION OF VITAMIN-D-DEPENDENT PROTEINS IN THE KIDNEY
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
481
Immunoperoxidase localization of Caþþ-Mgþþ ATPase within human kidney distal tubules and human spleen erythrocytes. Kidney: (a and b) Monoclonal antibody JA3. (c and d) Monoclonal antibody JA8. (e and f) Negative control. (g and h) Double stain, PAS, and JA3; (arrows) PASpositive proximal tubule brush border. Spleen: (i) Monoclonal antibody JA3. (j) Negative control. (a, c, e and g) 200. (b, d, f, h, i, and j) 640. (With permission from Borke et al. [157].) Please see color plate section.
FIGURE 26.8
1a-hydroxylase knockout mice, a greater than 50% reduction in ECaC-1/TRPV5 expression was found compared to control mice [229]. Additionally, renal ECaC-1/TRPV5 expression in 1a-hydroxylase KO mice is normalized after treatment with 1,25(OH)2D3 [229]. Similar findings were seen when examining calbindin D28K expression which colocalizes to the same distal tubule cells as ECaC-1/TRPV5 [179,229]. However, ECaC-1/TRPV5 is not regulated through vitamin D
effects on calbindin D28K. Calbindin D28K KO mice and cyclosporine A induced down-regulation of calbindin D28K has no effect on ECaC-1/TRPV5 expression [235]. Others have suggested that calcium also regulates ECaC-1 expression. Quantitative PRC techniques showed reduced expression of ECaC-1 in VDR KO mice compared to control mice. When fed high-calcium diets, VDR KO mice had normalization of ECaC-1 concentrations [252].
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482 TABLE 26.6
26. VITAMIN D AND THE KIDNEY
Distribution of Plasma Membrane Calcium Pump in Transporting Epithelia as Assessed by Immunohistochemistry
Tissue
Source
Cell type
Location in cell
Reference
Kidney
Rat, human
Distal convoluted tubule, principal cell
Basolateral
[18,157e160]
Intestine
Rat, chick
Absorptive cell
Basolateral
[239,240]
Trophoblast
Rat, human
Syncytiotrophoblast
Basal
[241]
Choroid plexus
Cat Human
Choroid plexus Secretory cell
Apical
[242]
Shell gland
Chick
Principal cell
Apical
[243]
Bone
Human
Osteoblast
Not vectorially oriented
[244]
Bone
Chick
Osteoclast
Not vectorially oriented
[245]
The 25-Hydroxyvitamin D3- and 1,25Dihydroxyvitamin D3-24-Hydroxylase The 24-hydroxylase enzyme is widely distributed in a number of renal and nonrenal tissues [70e78,87, 88,253]. Pioneering work by DeLuca’s laboratory showed that the renal 24-hydroxylase was regulated by calcium and phosphorus such that elevated or normal calcium levels induced the 24-hydroxylase whereas low calcium levels inhibited it [72e74,254]. Similarly, elevated serum phosphorus concentrations increased the synthesis of the 24-hydroxylase enzyme whereas low-phosphorus diets decreased the activity of the enzyme [255]. We have used antibodies against the 24-hydroxylase cytochrome P-450 to examine the distribution of the enzyme in the human kidney, and found exceptionally high concentrations of the cytochrome P-450 in distal tubular cells [161]. Lower amounts were found in the proximal tubule. Using enzymatic methods, several investigators have found 24-hydroxylase activity in kidney cells of the proximal tubule of the rat nephron. Iida et al. were unable to find 24-hydroxylase activity in microdissected distal tubule segment [256]. The reason for this apparent discrepancy between human and rat tissues is uncertain. Certainly, it would make biologic sense for the 24hydroxylase to be present in the distal tubule where other elements of the vitamin-D-responsive system are present. Other enzymes responsible for the transformation of 25-hydroxyvitamin D are also present in the kidney; they mediate the reactions shown in Table 26.3.
CONCLUSION The kidney plays a vital role in the conservation of calcium and phosphorus. Besides being the site of synthesis of 1,25(OH)2D3, the kidney responds to the hormone by increasing the efficiency of calcium and
phosphorus reabsorption. Elements of the calcium transport systems including calbindin D28K, calbindin D9K, the epithelial calcium channel, and the plasma membrane calcium pump all localize to the distal portion of the nephron and are regulated directly or indirectly by 1,25(OH)2D3.
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[267] T. Yoshida, N. Yoshida, T. Monkawa, M. Hayashi, T. Saruta, Dietary phosphorus deprivation induces 25-hydroxyvitamin D (3) 1alpha-hydroxylase gene expression, Endocrinology 142 (5) (2001) 1720e1726. [268] M.Y. Zhang, X. Wang, J.T. Wang, et al., Dietary phosphorus transcriptionally regulates 25-hydroxyvitamin D-1alphahydroxylase gene expression in the proximal renal tubule, Endocrinology 143 (2) (2002) 587e595. [269] H. Kawashima, S. Torikai, K. Kurokawa, Calcitonin selectively stimulates 25-hydroxyvitamin D3-1 alpha-hydroxylase in proximal straight tubule of rat kidney, Nature 291 (5813) (1981) 327e329. [270] R. Bland, E.A. Walker, S.V. Hughes, P.M. Stewart, M. Hewison, Constitutive expression of 25-hydroxyvitamin D3-1alphahydroxylase in a transformed human proximal tubule cell line: evidence for direct regulation of vitamin D metabolism by calcium, Endocrinology 140 (5) (1999) 2027e2034. [271] B. Lund, O.H. Sorensen, J.E. Bishop, A.W. Norman, Stimulation of 1,25-dihydroxyvitamin D production by parathyroid hormone and hypocalcemia in man, J. Clin. Endocrinol. Metab. 50 (3) (1980) 480e484. [272] N. Yoshida, T. Yoshida, A. Nakamura, T. Monkawa, M. Hayashi, T. Saruta, Calcitonin induces 25-hydroxyvitamin D3 1alpha-hydroxylase mRNA expression via protein kinase C pathway in LLC-PK1 cells, J. Am. Soc. Nephrol. 10 (12) (1999) 2474e2479. [273] N.D. Adams, R.W. Gray, J. Lemann Jr., The effects of oral CaCO3 loading and dietary calcium deprivation on plasma 1,25-dihydroxyvitamin D concentrations in healthy adults, J. Clin. Endocrinol. Metab. 48 (6) (1979) 1008e1016. [274] B. Sauveur, M. Garabedian, C. Fellot, P. Mongin, S. Balsan, The effect of induced metabolic acidosis on vitamin D3 metabolism in rachitic chicks, Calcif. Tissue Res. 23 (2) (1977) 121e124. [275] H.P. Weber, R.W. Gray, J.H. Dominguez, J. Lemann Jr., The lack of effect of chronic metabolic acidosis on 25-OH-vitamin D metabolism and serum parathyroid hormone in humans, J. Clin. Endocrinol. Metab. 43 (5) (1976) 1047e1055. [276] L. Castillo, Y. Tanaka, H.F. DeLuca, M.L. Sunde, The stimulation of 25-hydroxyvitamin D3-1 alpha-hydroxylase by estrogen, Arch. Biochem. Biophys. 179 (1) (1977) 211e217. [277] N.D. Adams, T.L. Garthwaite, R.W. Gray, T.C. Hagen, J. Lemann Jr., The interrelationships among prolactin, 1,25dihydroxyvitamin D, and parathyroid hormone in humans, J. Clin. Endocrinol. Metab. 49 (4) (1979) 628e630. [278] R. Kumar, W.R. Cohen, F.H. Epstein, Vitamin D and calcium hormones in pregnancy, New Engl. J. Med. 302 (20) (1980) 1143e1145. [279] E. Spanos, K.W. Colston, I.M. Evans, L.S. Galante, S.J. Macauley, I. Macintyre, Effect of prolactin on vitamin D metabolism, Mol. Cell Endocrinol. 5 (3-4) (1976) 163e167. [280] P.C. Eskildsen, B. Lund, O.H. Sorensen, J.E. Bishop, A.W. Norman, Acromegaly and vitamin D metabolism: effect of bromocriptine treatment, J. Clin. Endocrinol. Metab. 49 (3) (1979) 484e486. [281] J.M. Gertner, R.L. Horst, A.E. Broadus, H. Rasmussen, M. Genel, Parathyroid function and vitamin D metabolism during human growth hormone replacement, J. Clin. Endocrinol. Metab. 49 (2) (1979) 185e188. [282] C. Menaa, F. Vrtovsnik, G. Friedlander, M. Corvol, M. Garabedian, Insulin-like growth factor I, a unique calciumdependent stimulator of 1,25-dihydroxyvitamin D3 production. Studies in cultured mouse kidney cells, J. Biol. Chem. 270 (43) (1995) 25461e25467. [283] L. Condamine, C. Menaa, F. Vrtovsnik, F. Vztovsnik, G. Friedlander, M. Garabedian, Local action of phosphate
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C H A P T E R
27 Vitamin D and the Parathyroids Justin Silver, Tally Naveh-Many Hadassah Hebrew University Medical Center, Hadassah Hospital, Jerusalem 91120, Israel
INTRODUCTION The action of 1,25(OH)2D3 or its analogs to decrease PTH secretion is part of the management of all patients with chronic kidney disease in order to prevent or suppress their secondary hyperparathyroidism. There is ongoing academic and commercial activity in the development of drugs that may have more selective actions on the parathyroid whilst leading to less hypercalemia. These attempts are of great clinical and pharmaceutical interest but still remain to be proven by rigorous scientific testing and therefore despite the extensive literature on the subject, the final word on the analogs awaits prospective outcome studies [1,2] and are not discussed in detail in this chapter. However, there is discussion of the subject in Chapters 70 and 81. In this chapter we shall discuss the PTH gene and the regulation of PTH gene expression by 1,25(OH)2D3.
and cleavage occurs during protein synthesis, very little intact preproPTH is found within the parathyroid cell. Mature PTH has a molecular mass of approximately 9600 daltons and is the only form that is secreted from the parathyroid cell. The amino acid sequence has been determined in several species, and there exists a high degree of identity among species, particularly in the amino-terminal region of the molecule. The parathyroids synthesize another protein that is also secreted [5]. This protein, secretory protein I, is identical to chromogranin A isolated from the adrenal medulla, and it is present in other endocrine cells and neoplasms as well. Its function is not known, but it is stored and secreted with PTH despite its differential transcriptional regulation relative to PTH. A 26-kDa N-terminal fragment of chromogranin A (CgA) secreted by bovine parathyroid glands, when added to dispersed, parathyroid cells in primary culture, inhibited the low-calcium-stimulated secretion of both PTH and CgA, suggesting an autocrine or paracrine regulation of secretion [6].
PARATHYROID HORMONE BIOSYNTHESIS
THE PARATHYROID HORMONE GENE
Parathyroid hormone, a protein of 84 amino acids, is synthesized as a larger precursor, preproparathyroid hormone [3,4]. PreproPTH has a 25-residue “pre” or signal sequence, and a 6-residue “pro” sequence. The signal sequence, along with the short pro sequence, functions to direct the protein into the secretory pathway. Like other signal sequences, the pre sequence binds to a signal recognition particle during protein synthesis. The signal recognition particle then delivers the nascent peptide chain to the rough endoplasmic reticulum, where it is threaded through a protein-lined aqueous pore. During this transit, the signal sequence is cleaved off by a signal peptidase, and the pre sequence is rapidly degraded. Because the process of transport
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10027-7
The PTH Gene The human parathyroid hormone (PTH) gene is localized on the short arm of chromosome 11 at 11p15 [7,8]. The human and bovine genes have two functional TATA transcription start sites, and the rat only one. The two homologous TATA sequences flanking the human PTH gene direct the synthesis of two human PTH gene transcripts both in normal parathyroid glands and in parathyroid adenomas [9]. The PTH genes in all species that have been cloned, have two introns or intervening sequences and three exons [10]. Strikingly, even though fish do not have discrete parathyroid glands, they do synthesize PTH using two distinct genes that
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share the same exoneintron pattern found in tetrapod PTH genes [11,12]. The locations of the introns are identical in each case [13]. Intron A splits the 50 untranslated sequence of the mRNA five nucleotides before the initiator methionine codon. Intron B splits the fourth codon of the region that codes for the pro sequence of preProPTH. The three exons that result, thus, are roughly divided into three functional domains. Exon I contains the 50 untranslated region. Exon II codes for the pre sequence, or signal peptide and exon III codes for PTH as well as the 30 untranslated region. It is interesting that the human gene is considerably longer in both intron A and the 30 untranslated region of the cDNA compared to the bovine, rat, and mouse. The genes for PTH and PTHrP (PTH-related protein) are located in similar positions on sibling chromosomes 11 and 12. It is therefore likely that they arose from a common precursor by chromosomal duplication.
The PTH mRNA Complementary DNA encoding for human [14,15], bovine [16,17], rat [18], mouse [19], pig [18], chicken [20,21], dog [22], cat [23], horse [24], macaca [25], fugu fish [11], and zebrafish [12] PTH have all been cloned [13]. The PTH gene is a typical eukaryotic gene with consensus sequences for initiation of RNA synthesis, RNA splicing, and polyadenylation. The primary RNA transcript consists of RNA transcribed from both introns and exons, and then RNA sequences derived from the introns are spliced out. The product of this RNA processing, which represents the exons, is the mature PTH mRNA, which will then be translated into preproPTH. There is considerable identity among mammalian PTH genes, which is reflected in an 85% identity between human and bovine proteins and 75% identity between human and rat proteins. There is less identity in the 30 noncoding region. A more extensive review of the structure and sequences of the PTH gene has been published elsewhere [13] and in the book, Molecular Biology of the Parathyroid [26].
DEVELOPMENT OF THE PARATHYROID AND TISSUE-SPECIFIC EXPRESSION OF THE PTH GENE The thymus, thyroid, and parathyroid glands in vertebrates develop from the pharyngeal region, with contributions both from pharyngeal endoderm and from neural crest cells in the pharyngeal arches. Studies of gene knockout mice have shown that the hoxa3, pax 1, pax 9, and Eya1 transcription factors are needed to form parathyroid glands as well as many other pharyngeal pouch derivatives, such as the thymus. Glial cells
missing2 (Gcm-2), a mouse homolog of Drosophila Gcm, is a transcription factor whose expression is restricted to the parathyroid glands [27]. A human patient with a defective Gcm B gene, the human equivalent of Gcm-2, exhibited hypoparathyroidism and complete absence of PTH from the bloodstream [28]. The parathyroid gland of tetrapods and the gills of fish both express Gcm-2 and require this gene for their formation [29]. They also showed that the gill region expresses mRNA encoding the two PTH genes found in fish, as well as mRNA encoding the calcium-sensing receptor.
PROMOTER SEQUENCES Regions upstream of the transcribed structural gene often determine tissue specificity and contain many of the regulatory sequences for the gene. For PTH, analysis of this region has been hampered by the lack of a parathyroid cell line. It has been shown that the 5 kb of DNA upstream of the start site of the human PTH gene was able to direct parathyroid-gland-specific expression in transgenic mice [30]. Analysis of the human PTH promoter region identified a number of consensus sequences by computer analysis [31]. These included a sequence resembling the canonical cAMPresponsive element 50 -TGACGTCA-30 at position e81 with a single residue deviation. This element was fused to a reporter gene (CAT) and then transfected into different cell lines. Pharmacological agents that increase cAMP led to an increased expression of the CAT gene, suggesting a functional role for the cAMP responsive element (CRE). Specificity protein (Sp) and the nuclear factor-Y (NF-Y) complex are thought to be ubiquitously expressed transcription factors associated with basal expression of a host of gene products. Sp family members and NF-Y can cooperatively enhance transcription of a target gene. There is a highly conserved Sp1 DNA element present in mammalian PTH promoters [32]. Coexpression of Sp proteins and NF-Y complex leads to synergistic transactivation of the hPTH promoter, with alignment of the Sp1 DNA element essential for full activation [32]. The presence of a proximal NF-Y-binding site in the hPTH promoter highlights the potential for synergism between distal and proximal NF-Y DNA elements to strongly enhance transcription [33]. Several groups have identified DNA sequences that might mediate the negative regulation of PTH gene transcription by 1,25-dihydroxyvitamin D (1,25(OH)2D3). Demay et al. [34] identified DNA squences in the human PTH gene that bind the 1,25(OH)2D3 receptor (VDR). Nuclear extracts containing the VDR were examined for binding to sequences in the 50 -flanking region of the
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REGULATION OF PTH GENE EXPRESSION
hPTH gene. A 25-bp oligonucleotide containing sequences from e125 to e101 from the start of exon 1 bound nuclear proteins that were recognized by monoclonal antibodies against the VDR. The sequences in this region contained a single copy of a motif (AGGTTCA) that is homologous to the motifs repeated in the up-regulatory VDR response element (VDRE) of the osteocalcin gene. When placed up-stream to a heterologous viral promoter, the sequences contained in this 25-bp oligonucleotide mediated transcriptional repression in response to 1,25(OH)2D3 in GH4C1 cells but not in ROS 17/2.8 cells. Therefore, this down-regulatory element differs from up-regulatory elements both in sequence composition and in the requirement for particular cellular factors other than the VDR for repressing PTH transcription [34]. Russell et al. [35] have shown that there are two negative VDREs in the rat PTH gene. One is situated at e793 to e779 and bound a VDR/ RXR heterodimer with high affinity and the other at e760 to e746 bound the heterodimer with a lower affinity. Transfection studies with VDRE-CAT constructs showed that they had an additive effect. Liu et al. [36] have identified such sequences in the chicken PTH gene and demonstrated their functionality after transfection into the opossum kidney (OK) cell line. They converted the negative activity imparted by the PTH VDRE to a positive transcriptional response through selective mutations introduced into the element. They showed that there was a p160 protein that specifically interacted with a heterodimer complex bound to the wild-type VDRE, but was absent from complexes bound to response elements associated with positive transcriptional activity. Thus, the sequence of the individual VDRE appears to play an active role in dictating transcriptional responses that may be mediated by altering the ability of a VDR/RXR heterodimer to interact with accessory factor proteins. Further work is needed to demonstrate that any of these differing negative VDREs function in this fashion in parathyroid cells. The transrepression by 1,25(OH)2D3 has also been shown to be dependent upon another promoter element. Kato’s laboratory have identified an E-box (CANNTG)like motif as another class of nVDRE in the human 1a (OH)ase promoter [37,38]. In sharp contrast to the previously reported DR3-like motif in the hPTH gene promoter, a basic helix-loop-helix factor, designated VDR interacting repressor (VDIR), transactivates through direct binding to this E-box-type element (1anVDRE). However, the VDIR transactivation function is transrepressed through ligand-induced proteine protein interaction of VDIR with VDR/RXR. In the absence of 1,25(OH)2D3, VDIR appears to bind to 1anVDRE for transactivation through the histone acetylase (HAT) coactivator, p300/CBP. Binding of 1,25(OH)2D3 to VDR induces interaction with VDIR and dissociation
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of the HAT coactivator, resulting in recruitment of histone deacetylase (HDAC) corepressor for ligandinduced transrepression [37]. They have also characterized the functions of VDIR and E-box motifs in the human (h) PTH and hPTHrP gene promoters [39]. They identified E-box-type elements acting as nVDREs in both the hPTH promoter (hPTHnVDRE e87 to e60 bp) and in the hPTHrP promoter (hPTHrPnVDRE e850 to e600 bp e463 to e104 bp) in a mouse renal tubule cell line. The hPTHnVDRE alone was enough to direct ligand-induced transrepression mediated through VDR/retinoid X receptor and VDIR. Direct DNA binding of hPTHnVDRE to VDIR, but not VDR/retinoid X receptor, was observed and ligand-induced transrepression was coupled with recruitment of VDR and histone deacetylase 2 (HDAC2) to the hPTH promoter. They concluded that negative regulation of the hPTH gene by liganded VDR is mediated by VDIR directly binding to the E-box-type nVDRE at the promoter, together with recruitment of an HDAC corepressor for ligand-induced transrepression [39]. These studies were specific to a mouse proximal tubule cell line and await the development of a parathyroid cell line to confirm them in a homologous cell system.
REGULATION OF PTH GENE EXPRESSION 1,25-Dihydroxyvitamin D PTH regulates serum concentrations of calcium and phosphate, which, in turn, regulate the synthesis and secretion of PTH. 1,25(OH)2D3 has independent effects on calcium and phosphate levels and also participates in a well-defined feedback loop between 1,25(OH)2D3 and PTH [40]. 1,25(OH)2D3 potently decreases transcription of the PTH gene. This action was first demonstrated in vitro in bovine parathyroid cells in primary culture, where 1,25(OH)2D3 led to a marked decrease in PTH mRNA levels [41,42] and a consequent decrease in PTH secretion [43e46]. The physiological relevance of these findings was established by in vivo studies in rats [47]. The localization of VDR mRNA to parathyroids was demonstrated by in situ hybridization studies of the thyroparathyroid and duodenum. VDR mRNA was localized to the parathyroids in the same concentration as in the duodenum, the classic target organ of 1,25(OH)2D [48]. Rats injected with amounts of 1,25 (OH)2D3 that did not increase serum calcium had marked decreases in PTH mRNA levels, reaching <4% of control at 48 h. This effect was shown to be transcriptional both in in vivo studies in rats [47] and in in vitro studies with primary cultures of bovine parathyroid
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27. VITAMIN D AND THE PARATHYROIDS
cells [49]. When 684 bp of the 50 -flanking region of the human PTH gene was linked to a reporter gene and transfected into a rat pituitary cell line (GH4C1), gene expression was lowered by 1,25(OH)2D3 [50]. These studies suggest that 1,25(OH)2D3 decreases PTH transcription by acting on the 50 -flanking region of the PTH gene, probably at least partly through interactions with the VDR-binding sequences and/or the E box that binds VDIR noted earlier. The effect of 1,25(OH)2D3 may involve heterodimerization with the retinoid acid receptor RXR. This is because 9 cis-retinoic acid, which binds to the RXR, when added to bovine parathyroid cells in primary culture, led to a decrease in PTH mRNA levels [51]. Moreover, combined treatment with 1 10e6 M retinoic acid and 1 10e8 M 1,25(OH)2D3 decreased PTH secretion and preproPTH mRNA more effectively than either compound alone [51]. Alternatively, RXRs might synergize with VDRs through actions on distinct sequences in the PTH promoter. A further level at which 1,25(OH)2D3 might regulate the PTH gene would be at the level of the VDR. 1,25 (OH)2D3 acts on its target tissues by binding to the VDR, which regulates the transcription of genes with the appropriate recognition sequences. Concentration of the VDR in 1,25(OH)2D3 target sites likely allows a modulation of the 1,25(OH)2D3 effect, with an increase in receptor concentration leading to an amplification of its effect and a decrease in receptor concentration dampening the 1,25(OH)2D3 effect. Naveh-Many et al. [48] injected 1,25(OH)2D3 into rats and measured the levels of VDR mRNA and PTH mRNA in the parathyroid tissue. They showed that 1,25(OH)2D3 in physiologically relevant doses led to an increase in VDR mRNA levels in the parathyroid glands in contrast to the decrease in PTH mRNA levels. This VDR up-regulation in response to 1,25(OH)2D3 is a well-described effect, however it is complex in that it is tissue specific and various across species [52]. Weanling rats fed a diet deficient in calcium were markedly hypocalcemic at 3 weeks and had very high serum 1,25(OH)2D3 levels. Despite the chronically high serum 1,25(OH)2D3 levels, there was no increase in VDR mRNA levels. Furthermore, PTH mRNA levels did not fall and were increased markedly. The low calcium in the bloodstream may have prevented the increase in parathyroid VDR levels, which may partially explain PTH mRNA suppression. Calreticulin and the Action of 1,25(OH)2D3 on the PTH Gene Calreticulin is a calcium-binding protein present in the endoplasmic reticulum of cells that may also have a nuclear function. It regulates gene transcription via its ability to bind a protein motif in the DNA-binding domain of nuclear hormone receptors of sterol hormones.
Calreticulin has been shown to prevent VDR binding and action to regulate the level of osteocalcin expression in vitro [53]. Sela-Brown et al. [54] showed that calreticulin might also inhibit the action of vitamin D on the PTH gene. Both rat and chicken VDRE sequences of the PTH gene were incubated with recombinant VDR and RXR proteins in a gel retardation assay and the results showed a clear retarded band suggesting protein binding to the VDReRXR complex. Purified calreticulin inhibited binding of the VDReRXR complex to the VDREs in gel retardation assays. This inhibition was due to direct proteineprotein interactions between VDR and calreticulin. OK cells were transiently cotransfected with calreticulin expression vectors and either rat or chicken PTH gene promotereCAT constructs. The cells were then assayed for 1,25(OH)2D3-induced CAT gene expression. 1,25(OH)2D3 decreased PTH promotere CAT transcription. Cotransfection with sense calreticulin, which increases calreticulin protein levels, completely inhibited the effect of 1,25(OH)2D3 on the PTH promoters of both rat and chicken. Cotransfection with the antisense calreticulin construct did not interfere with the effect of vitamin D on PTH gene transcription. Calreticulin expression had no effect on basal CAT mRNA levels. In order to determine a physiological role for calreticulin in regulation of the PTH gene, levels of calreticulin protein were determined in the nuclear fraction of rat parathyroids. The rats were fed either a control diet or a low-calcium diet, which leads to increased PTH mRNA levels, despite high serum 1,25 (OH)2D3 levels that would be expected to inhibit PTH gene transcription [54]. It was postulated that high calreticulin levels in the nuclear fraction would prevent the effect of 1,25(OH)2D3 on the PTH gene. In fact, hypocalcemic rats had increased levels of calreticulin protein, as measured by Western blots, in their parathyroid nuclear faction. This may help explain why hypocalcemia leads to increased PTH gene expression, despite high serum 1,25(OH)2D3 levels, and may also be relevant to the refractoriness of the secondary hyperparathyroidism of many chronic renal failure patients to 1,25(OH)2D3 treatment. These studies, therefore, indicate a role for calreticulin in regulating the effect of vitamin D on the PTH gene and suggest a physiological relevance to these studies [54]. Russell et al. [55] studied the parathyroids of chicks with vitamin D deficiency and confirmed that 1,25(OH)2D3 regulates PTH and VDR gene expression in the avian parathyroid gland. Brown et al. [56] studied vitamin-D-deficient rats and confirmed that 1,25(OH)2D up-regulated parathyroid VDR mRNA. Rodriguez et al. [57] showed that administration of the calcimimetic R-568 resulted in increased VDR expression in parathyroid tissue. In vitro studies of the effect of R-568 on VDR mRNA and protein were conducted in
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REGULATION OF PTH GENE EXPRESSION
cultures of whole rat parathyroid glands. Incubation of rat parathyroid glands in vitro with R-568 resulted in a dose-dependent decrease in PTH secretion and an increase in VDR expression. Together with previous work on the effect of extracellular calcium to increase parathyroid VDR mRNA in vitro [58], they concluded that activation of the calcium-sensing receptor (CaR) up-regulates the parathyroid VDR mRNA. All these studies show that 1,25(OH)2D3, and calcium in certain circumstances, increases the expression of the VDR gene in the parathyroid gland, which would result in increased VDR protein synthesis and increased binding of 1,25(OH)2D3. This ligand-dependent receptor up-regulation would lead to an amplified effect of 1,25 (OH)2D3 on the PTH gene and might help explain the dramatic effect of 1,25(OH)2D3 to reduce PTH gene expression. 1,25(OH)2D3 may also amplify its effect on the parathyroids by increasing the activity of the CaR. Canaff et al. [59] showed that in fact there are VDREs in the human CaR’s promoter. The CaR is expressed in parathyroid chief cells, thyroid C-cells, and cells of the kidney tubule, and is essential for maintenance of calcium homeostasis. They showed that parathyroid, thyroid, and kidney CaR mRNA levels increased twofold at 15 h after intraperitoneal injection of 1,25 (OH)2D3 in rats. Functional VDREs have been identified in the CaR gene and probably provide the mechanism whereby 1,25(OH)2D3 up-regulates parathyroid, thyroid C-cell, and kidney CaR expression. The CaR is fully discussed in Chapter 24. The use of 1,25(OH)2D3 is limited by its hypercalcemic effect, and therefore a number of 1,25(OH)2D analogs have been synthesized that are biologically active but are less hypercalcemic than 1,25(OH)2D3 [60]. These analogs usually involve modifications of the 1,25(OH)2D side chain, such as 22-oxa-1,25(OH)2D3, which is the chemical modification in oxacacitriol [61], or a cyclopropyl group at the end of the side chain in calcipotriol [62,63]. Brown et al. [64] showed that oxacalcitriol in vitro decreased PTH secretion from primary cultures of bovine parathyroid cells with a similar dose response to that of 1,25(OH)2D. In vivo the injection of both vitamin D compounds led to a decrease in rat parathyroid PTH mRNA levels [64]. However, detailed in vivo doseeresponse studies showed that in vivo 1,25(OH)2D is the most effective analog for decreasing PTH mRNA levels, even at doses that do not cause hypercalcemia [65]. Oxacalcalcitriol and calcipotriol are less effective for decreasing PTH RNA levels but have a wider dose range at which they do not cause hypercalcemia. This property might be useful clinically. Paricalcitol was shown to be effective at reducing PTH concentrations without causing significant hypercalcemia or hyperphosphatemia as
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compared to placebo. Paricalcitol treatment was shown to reduce PTH concentrations more rapidly with fewer sustained episodes of hypercalcemia and increased Ca x P product compared to 1,25(OH)2D therapy [66]. The marked activity of 1,25(OH)2D analogs in vitro as compared to their modest hypercalcemic actions in vivo probably reflects their rapid clearance from the circulation [67]. Despite the great interest in the development and marketing of new 1,25(OH)2D analogs to decrease PTH gene expression and serum PTH levels without causing hypercalcemia, there have been few rigorous comparisons of their biological effects compared to those of 1,25(OH)2D itself [68,69]. There has been a lot of debate about the relative advantages of the so-called “nonhypercalcemic” vitamin D analogs over 1,25(OH)2D3. Drueke [2] has analyzed the clinical trials that have been performed and concluded that all clinical studies were retrospective in nature and suffered from the limitations of retrospective data analysis. The question is still open and is discussed more fully in Chapters 70 and 81. The ability of 1,25(OH)2D3 to decrease PTH gene transcription is used therapeutically in the management of patients with chronic renal failure. They are treated with 1,25(OH)2D, or its prodrug 1a(OH)-vitamin D3 in order to prevent or reduce the secondary hyperparathyroidism of chronic renal failure. The poor response in some patients who do not respond to treatment may well result from several factors: poor control of serum phosphate, decreased VDR concentration in the patients’ parathyroids [70], an inhibitory effect of a uremic toxin(s) on VDReVDRE binding [71], or tertiary hyperparathyroidism with monoclonal parathyroid tumors [72]. The development of calcimimetic drugs which act to directly activate the CaR has provided a significant advance in the treatment that we can offer patients with secondary hyperparathyroidism. In clinical practice these drugs are frequently used in combination with 1,25(OH)2D3 or its analogs. Studies on Mice with VDR Gene Deletion The VDR total knockout (VDRe/e) phenotype is characterized by high PTH levels, hypocalcemia, hypophosphatemia, bone malformations, rickets, and alopecia [73,74]. Most, but not all of the phenotype can be reversed by correcting the serum calcium concentration with a high-calcium-lactose diet [75] (see Chapter 33 for a discussion of the knockout mouse model). In order to investigate PTH regulation by the VDR as close as possible to the physiological conditions, we have generated parathyroid-specific VDR knockout mice (PTVDRe/e), by crossing PTH promoter-Cre mice with total body floxed-VDR mice [76] (Fig. 27.1). VDR expression was decreased specifically in the parathyroid glands of the PT-VDRe/e mice. In both the total knockout and the
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(A) Paraffin sections of thyroparathyroid tissue before (left panel) and after (right panel) removal of the parathyroid tissue by laser capture microscopy. (B) Schematic representation of the VDR mRNA (exons). The deleted exon 2 is shown as a box with a dashed line. (C) RT-PCR products of parathyroid RNA extracted from laser-captured tissue of VDRþ/þ, PT-VDRe/e and total body VDRe/e mice. Primers from exons 1 and 3 were used for detection of inclusion or deletion of exon 2 inVDR mRNA (left panel). Primers from exons 2 and 3 were used for detection of wt VDR mRNA (right panel). (D) Serum PTH in VDRþ/þ and PTVDRe/e mice demonstrating a significant increase in serum PTH in the PT-VDRe/e mice. (E) b-Terminal collagen crosslinks (CTX) levels in VDRþ/þ and PT-VDRe/e mice. CTX, a marker of bone resorption, was increased in the PTVDRe/e mice compared to VDRþ/þ mice. *, p < 0.05; n ¼ 7. Adapted from Meir et al. [76].
FIGURE 27.1
(A)
(B)
VDR mRNA ex 3
-/-
VD R
-/-
M
PT -
/+
M
VD R+
/-
VD R-
VD RPT -
VD R+
/+
/-
(C)
ex 2
VD R
ex 1
233 bp 85 bp
110 bp
ex.1-3 *
50 40 30 20 10 0
(E)
VDR+/+
PT-VDR -/-
8
* CTX (ng/ml)
PTH (pg/ml)
(D) 60
ex.2-3
6 4 2 0
VDR+/+
parathyroid specific knockout mice there was a decrease in parathyroid calcium receptor (CaR) levels (discussed in Chapter 24). In contrast the number of proliferating parathyroid cells was increased in the VDRe/e mice but not in the PT-VDRe/e mice. Serum PTH levels were moderately but significantly increased in the PTVDRe/e mice with normal serum calcium levels. The sensitivity of the parathyroid glands of the PT-VDRe/e mice to calcium was intact as measured by serum PTH levels after changes in serum calcium. This indicates that the reduced CaR in the PT-VDRe/e mice enables a physiologic response to serum calcium. Serum type I collagen C-telopeptides (CTX), a marker of bone resorption, was increased in the PT-VDRe/e mice with no change in the bone formation marker, serum osteocalcin, consistent with a resorptive effect due to the increased serum PTH levels in the PT-VDRe/e mice (Fig. 27.1). Therefore, deletion of the VDR specifically in the parathyroid decreases parathyroid CaR expression and only moderately increases basal PTH levels, suggesting that the VDR has a limited role in parathyroid physiology [76].
PT-VDR -/-
Calcium A remarkable characteristic of the parathyroid is its sensitivity to small changes in serum calcium, which leads to large changes in PTH secretion. This remarkable sensitivity of the parathyroid to increase hormone secretion after small decreases in serum calcium levels is unique to the parathyroid. All other endocrine glands increase hormone secretion after exposure to a high extracellular calcium. This calcium sensing is also expressed at the levels of PTH gene expression and parathyroid cell proliferation. Calcium and phosphate both have marked effects on the levels of PTH mRNA in vivo [77]. The major effect is for low calcium to increase PTH mRNA levels and low phosphate to decrease PTH mRNA levels. NavehMany et al. [78] studied rats in vivo. They showed that a small decrease in serum calcium from 2.6 to 2.1 mmol/liter led to large increases in PTH mRNA levels, reaching threefold that of controls at 1 and 6 h. A high serum calcium had no effect on PTH mRNA
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levels even at concentrations as high as 6.0 mmol/liter. Yamamoto et al. [79] also studied the in vivo effect of calcium on PTH mRNA levels in rats. They showed that hypocalcemia induced by a calcitonin infusion for 48 h led to a sevenfold increase in PTH mRNA levels. Rats made hypercalcemic (2.9e3.4 mM) for 48 h had the same PTH mRNA levels as controls that had received no infusion (2.5 mM). Therefore, hypercalcemia in vivo has a limited effect to decrease PTH mRNA levels. These results emphasize that the gland is geared to respond to hypocalcemia and not hypercalcemia. Mechanisms of Regulation of PTH mRNA by Calcium The mechanism whereby calcium regulates PTH gene expression is particularly interesting. Changes in extracellular calcium are sensed by a calcium sensor that then regulates PTH secretion [80,81]. Signal transduction from the CaR involves activation of phospholipase C, D, and A2 enzymes [82]. The response to changes in serum calcium involves the protein phosphatase type 2B, calcineurin [83]. In vivo and in vitro studies demonstrated that inhibition of calcineurin by genetic manipulation or pharmacologic agents affected the response of PTH mRNA levels to changes in extracellular calcium [83]. Moallem et al. [84] showed that the effects of calcium and phosphate are post-transcriptional and involve proteineRNA interactions at the 30 -untranslated region (UTR) of the PTH mRNA [84]. The mechanisms involved will be discussed after the independent effect of phosphate on the PT is considered.
Phosphate Regulates the Parathyroid Independently of Calcium and 1,25(OH)2D3 The demonstration of a direct effect of high phosphate on the parathyroid in vivo has been difficult. One of the reasons is that the various maneuvers used to increase or decrease serum phosphate invariably lead to a change in the ionized calcium concentration. A number of careful clinical and experimental studies suggested that the effect of phosphate on serum PTH levels was independent of changes in both serum calcium and 1,25(OH)2D3 levels. In dogs with experimental chronic renal failure, Lopez-Hilker et al. [85] have shown that phosphate restriction corrected their secondary hyperparathyroidism independent of changes in serum calcium and 1,25(OH)2D3 levels. They did this by placing the uremic dogs on diets deficient in both calcium and phosphate. This led to lower levels of serum phosphate and calcium, with no increase in the low levels of serum 1,25(OH)2D3. Despite this, there was a 70% decrease in PTH levels. This study suggested that, at least in chronic renal failure, phosphate affected the parathyroid cell by a mechanism
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independent of its effect on serum 1,25(OH)2D3 and calcium levels [85]. Therefore, phosphate plays a central role in the pathogenesis of secondary hyperparathyroidism, both by its effect on serum 1,25(OH)2D3 and calcium levels and possibly independently. A raised serum phosphate also stimulates the secretion of FGF23 which in turn decreases PTH gene expression and serum PTH levels [86,87]. This effect would act as a counter-balance to the stimulatory effect of phosphate on the parathyroid and is discussed separately in this chapter. Kilav et al. [88] bred second-generation vitamin-Ddeficient rats and then placed the weanling vitamin-Ddeficient rats on a diet with no vitamin D, low calcium, and low phosphate. After one night of this diet, serum phosphate had decreased markedly with no changes in serum calcium or 1,25(OH)2D3. These rats with isolated hypophosphatemia had marked decreases in PTH mRNA levels and serum PTH. However, the very low serum phosphates in these in vivo studies may have no direct relevance to possible direct effects of high phosphate in renal failure. It is necessary to separate nonspecific effects of very low phosphate from true physiologic regulation. To establish that the effect of serum phosphate on the parathyroid was indeed a direct effect, in vitro confirmation was needed, which was provided by three groups. Rodriguez was the first to show that increased phosphate levels increased PTH secretion from isolated parathyroid glands in vitro. The effect required maintainenance of tissue architecture [89e91]. The effect was found in whole glands or tissue slices but not in isolated cells. The requirement for intact tissue suggests either that the sensing mechanism for phosphate is damaged during the preparation of isolated cells or that the intact gland structure is important to the phosphate response. Parathyroid responds to changes in serum phosphate at the level of secretion, gene expression, and cell proliferation, although the mechanism of these effects is unknown. The effect of high phosphate to increase PTH secretion may be mediated by phospholipase-A2activated signal transduction. Bourdeau et al. [92,93] showed that arachidonic acid and its metabolites inhibit PTH secretion. Almaden [94] showed in vitro that a high phosphate medium increased PTH secretion, which was prevented by the addition of arachidonic acid.
ProteinePTH mRNA Interactions Determine the Regulation of PTH Gene Expression by Serum Calcium and Phosphate The clearest rat in vivo models for effects of calcium and phosphate on PTH gene expression are dietinduced hypocalcemia with a large increase in PTH mRNA levels and diet-induced hypophosphatemia
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with a large decrease in PTH mRNA levels. In both instances the effect was post-transcriptional, as shown by nuclear transcript run-on experiments [77]. Parathyroid cytosolic proteins were found to bind to in vitro transcribed PTH mRNA [84]. Interestingly, this binding was increased with parathyroid proteins from hypocalcemic rats (with increased PTH mRNA levels) and decreased with parathyroid proteins from hypophosphatemic rats (with decreased PTH mRNA levels). Proteins from other tissues bound to PTH mRNA, but this binding is regulated by calcium and phosphate only with parathyroid proteins. Intriguingly, binding requires the presence of the terminal 60 nucleotides of the PTH transcript. Naveh-Many and colleagues utilized an in vitro degradation assay to study the effects of hypocalcemic and hypophosphatemic parathyroid proteins on PTH mRNA stability [84]. This assay reproduced the differences in PTH mRNA levels observed in vivo. Moreover, the difference in RNA stability by the parathyroid extracts was totally dependent on an intact 30 -untranslated region (UTR) and, in particular, on the terminal 60 nucleotides. Proteins from other tissues in these rats were not regulated by calcium or phosphate. Therefore, calcium and phosphate change the properties of parathyroid cytosolic proteins, which bind specifically to the PTH mRNA 30 -UTR and determine its stability (Fig. 27.2). What are these proteins?
basal PTH mRNA
AUF1 P Unr
P An
KSRP
Pin1(active)
Pin1 (inactive) PTH mRNA low calcium or kidney failure
KSRP
P (inactive)
AUF1 (active)
Unr 5’-UTR
coding
3’-UTR ARE
An
FIGURE 27.2 Model for the regulation of PTH mRNA stability by PTH mRNA 30 -UTR binding proteins. Under basal conditions there is a balanced interaction of the PTH mRNA with its stabilizing proteins, AUF1 (AUF1 isoforms p37, p40, p42, and p45 are shown) and Unr, and the destabilizing protein, KSRP. In hypocalcemia or CKD, Pin1 is inactive resulting in KSRP phosphorylation and hence its inactivation. This would allow AUF1 and Unr to bind the PTH mRNA 30 -UTR ARE with a greater affinity leading to increased PTH mRNA stability. From [102] with permission of the American Society of Clinical Investigation.
Identification of the PTH mRNA 30 -UTR Binding Proteins that Determine PTH mRNA Stability AU-rich Binding Factor (AUF1) Sela-Brown et al. and Dinur et al. have utilized affinity chromatography using the PTH RNA 30 -UTR to isolate these RNA-binding proteins [95,96]. The proteins, which bind the PTH mRNA, are also present in other tissues, such as brain, but only in the parathyroid is their binding regulated by calcium and phosphate. A major band from the eluate of a PTH 30 -region RNA affinity chromatography was identical to AU-rich binding factor (AUF1) [95]. Recombinant AUF1 bound the full-length PTH mRNA and the 30 UTR. Added recombinant AUF1 also stabilized the PTH transcript in the in vitro degradation assay. These results showed that AUF1 is a protein that binds to the PTH mRNA 30 -UTR and stabilizes the PTH transcript (Fig. 27.2). The regulation of protein PTH mRNA binding involves post-translational modification of AUF1 [97]. AUF1 levels are not regulated in PT extracts from rats fed calcium- and phosphorus-depleted diets. However, two-dimensional gels showed post-translational modification of AUF1 that included phosphorylation [83]. There is no parathyroid cell line, but a PTH mRNA cis acting 63-nt element [98] is recognized in HEK 293 cells as an instability element. RNA interference for AUF1 decreased human PTH expression in cotransfection experiments [83]. Most patients with chronic kidney disease develop secondary hyperparathyroidism with disabling systemic complications (discussed in Chapter 70). Calcimimetic agents are effective tools in the management of secondary hyperparathyroidism, acting through allosteric modification of the calcium-sensing receptor (CaR) on the parathyroid gland to decrease PTH secretion and parathyroid cell proliferation. R-568 decreased both PTH mRNA and serum PTH levels in adenine-high phosphorus-induced chronic kidney disease [99]. The effect of the calcimimetic on PTH gene expression was post-transcriptional and correlated with differences in proteineRNA binding and post-translational modifications of the trans acting factor AUF1 in the parathyroid. The AUF1 modifications as a result of uremia were reversed to those of normal rats by treatment with R-568. Therefore, uremia and activation of the CaR mediated by calcimimetics modify AUF1 post-translationally. These modifications in AUF1 correlate with changes in proteinePTH mRNA binding and PTH mRNA levels [99]. UNR (UPSTREAM OF N-RAS)
A second parathyroid cytosolic protein which is part of the stabilizing PTH mRNA 30 -UTR binding complex was shown to be Unr by affinity chromatography [96].
IV. TARGETS
REGULATION OF PTH GENE EXPRESSION
Depletion of Unr by small interfering RNA decreased PTH mRNA levels in HEK293 cells transiently cotransfected with the human PTH gene. Overexpression of Unr increased the rat full-length PTH mRNA levels but not a PTH mRNA lacking the terminal 60-nucleotide cis-acting protein-binding region. Therefore, Unr binds to the PTH cis element and increases PTH mRNA levels, as does AUF1. Unr, together with the other proteins in the RNA-binding complex, determines PTH mRNA stability [96] (Fig. 27.2). Recent findings have identified an additional decay promoting protein, KSRP, that differentially interacts with PTH mRNA to recruit the degradatory machinery [100]. The balance between the stabilizing and destabilizing proteins determines PTH mRNA levels in response to physiological stimuli [101]. KSRP (K-HOMOLOGY SPLICING REGULATOR PROTEIN)
We have shown that mRNA decay promoting protein KSRP binds to PTH mRNA in intact parathyroid glands and in transfected cells [100]. This binding of KSRP is decreased in glands from calcium-depleted or experimental chronic kidney failure rats where PTH mRNA is more stable, compared to parathyroid glands from control and phosphorus-depleted rats where PTH mRNA is less stable. The differences in KSRPePTH mRNA binding counter those of AUF1. PTH mRNA decay depends on the KSRP-recruited exosome in parathyroid extracts. In transfected cells, KSRP overexpression and knockdown experiments show that KSRP decreases PTH mRNA stability and steady-state levels through the PTH mRNA ARE. Overexpression of isoform p45 of the PTH mRNA stabilizing protein AUF1 blocks KSRP-PTH mRNA binding and partially prevents the KSRP-mediated decrease in PTH mRNA levels. Therefore, calcium or phosphorus depletion, as well as chronic kidney failure, regulate the interaction of KSRP and AUF1 with PTH mRNA and its half-life. The balance between the stabilizing and destabilizing proteins determines PTH mRNA levels in response to physiological stimuli (Fig. 27.2). A Conserved Sequence in the PTH mRNA 30 -UTR Binds Parathyroid Cytosolic Proteins and Determines mRNA Stability in Response to Changes in Calcium and Phosphate We have identified the minimal sequence for protein binding in the PTH mRNA 30 -UTR and determined its functionality [98]. A minimum sequence of 26 nucleotides was sufficient for PTH RNAeprotein binding and competition (Fig. 27.2). Significantly, this sequence was preserved among species [13]. To study the functionality of the sequence in the context of another RNA, a 63-bp cDNA PTH sequence consisting of the 26 nucleotide and flanking regions was fused to growth
501
hormone (GH) cDNA. The conserved PTH RNA protein-binding region was necessary and sufficient for responsiveness to calcium and phosphate and determines PTH mRNA stability and levels [98]. The PTH mRNA 30 -UTR binding element is AU rich and is a type III AU-rich element (ARE). Sequence analysis of the PTH mRNA 30 -UTR of different species revealed a preservation of the 26-nt protein-binding element in rat, murine, human, macaque, feline, and canine 30 -UTRs [13]. In contrast to protein-coding sequences that are highly conserved, UTRs are less conserved. The conservation of the protein-binding element in the PTH mRNA 30 -UTR suggests that this element represents a functional unit that has been evolutionarily conserved. The cis-acting element is at the 30 distal end in all species where it is expressed. The Peptidyl-Prolyl Isomerase Pin1 Determines Parathyroid Hormone mRNA Levels and Stability in Secondary Hyperparathyroidism We have identified the peptidyl-prolyl isomerase Pin1 as a PTH mRNA destabilizing protein in rat parathyroids and transfected cells [102]. Accordingly, Pin1 activity is decreased in parathyroid protein extracts from hypocalcemic or CKD rats. Pharmacologic inhibition of Pin1 increases PTH mRNA levels posttranscriptionally in vivo in the parathyroid and in transfected cells. Pin1 regulates PTH mRNA stability and levels through a PTH mRNA 30 -untranslated region (UTR) cis-acting element. We showed that Pin1 interacts with the PTH mRNA destabilizing protein, KSRP, and leads to KSRP dephosphorylation and activation. In the parathyroid, Pin1 inhibition decreased the KSRPPTH mRNA interaction which contributes to the increased PTH gene expression. Furthermore, Pin1e/e mice had increased serum PTH and PTH mRNA levels. Therefore, Pin1 determines basal PTH expression in vivo and in vitro and decreased Pin1 activity correlates with increased PTH mRNA levels in CKD and hypocalcemic rats (Fig. 27.2). These results demonstrate that Pin1 is a key mediator of PTH mRNA stability and indicate a role for Pin1 in the pathogenesis of the secondary hyperparathyroidism of CKD [102].
Fibroblast Growth Factor 23 and the Parathyroid FGF23 Decreases PTH Expression Phosphate homeostasis is maintained by a counterbalance between efflux from the kidney and influx from intestine and bone. Fibroblast growth factor-23 (FGF23) is a bone-derived phosphaturic hormone that acts on the kidney to increase phosphate excretion and suppress biosynthesis of 1,25(OH)2vitamin D (discussed in detail in Chapter 26). FGF23 signals through fibroblast growth
IV. TARGETS
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27. VITAMIN D AND THE PARATHYROIDS
Parathyroids
Ca2+ R Ca
FGFR FGF23
Klotho
1,25(OH)2D VDR
PTH
Phosphate
factor receptors (FGFR) bound by the transmembrane protein Klotho [103] (see also Chapter 42). Since most tissues express FGFRs, expression of Klotho virtually determines FGF23 target organs. Takeshita et al. were the first to show that Klotho protein is expressed not only in the kidney but also in the parathyroid, pituitary, and sino-atrial node [104]. In addition, Urakawa et al. [105] injected rats with FGF23 and demonstrated increased Egr-1 (early growth response gene-1) mRNA levels in the parathyroid, suggesting that the parathyroid may be a further FGF23 target organ. Phosphate homeostasis is maintained by a counterbalance between efflux from the kidney and influx from intestine and bone. FGF23 is a bone-derived phosphaturic hormone that acts on the kidney to increase phosphate excretion and suppress biosynthesis of vitamin D. FGF23 signals with highest efficacy through several FGF receptors (FGFRs) bound by the transmembrane protein Klotho as a coreceptor. We have identified the parathyroid as a target organ for FGF23 in rats [106]. We showed that the parathyroid gland expressed Klotho and two FGFRs. The administration of recombinant FGF23 led to an increase in parathyroid Klotho levels. In addition, FGF23 activated the MAPK pathway in the parathyroid through ERK1/2 phosphorylation and increased Egr1 mRNA levels. Using both rats and in vitro rat parathyroid cultures, we showed that FGF23 suppressed both parathyroid hormone (PTH) secretion and PTH gene expression. The FGF23-induced decrease in PTH secretion was prevented by a MAPK inhibitor. These data indicate that FGF23 acts directly on the parathyroid through the MAPK pathway to decrease serum PTH (Fig. 27.3). This boneeparathyroid endocrine axis adds a new dimension to the understanding of mineral homeostasis. Krajisnik et al. [87] showed similar results using bovine parathyroid cells in primary culture. Interestingly, they also showed that FGF23 led to a dose-dependent increase in the expression of the 1ahydroxylase enzyme in the parathyroid. The increased
FIGURE 27.3 FGF23 acts on the parathyroid to decrease parathyroid hormone (PTH) synthesis and secretion e a novel bone-parathyroid endocrine axis. FGF23 is secreted by bone after the stimulus of a high Pi by acting on the Klotho-FGFR1c. In addition to FGF23’s action on the kidney to cause Pi excretion and decrease the synthesis of 1,25(OH)2 vitamin D, it is now shown to act on the parathyroid to decrease PTH synthesis and secretion. This new endocrine axis contributes to our understanding of how the metabolism of bone, Pi and Ca2þ are so tightly regulated. PTH is the major regulator of Ca2þ, FGF23 of Pi and together with vitamin D they contribute to normal mineral and bone metabolism. From [109] with permission.
1,25(OH)2D might then act in an autocrine manner to decrease PTH gene transcription. Resistance of the Parathyroid to FGF23 in CKD In chronic kidney disease (CKD) both serum FGF23 and PTH levels are increased. We and others have shown that a decrease in Klotho-FGFR1 expression and signal transduction may explain the resistance of the parathyroid to FGF23 in CKD [107,108]. In experimental CKD quantitative immunohistochemistry and RT-PCR using laser capture microscopy showed that Klotho and FGFR1 protein and mRNA levels were decreased in parathyroid sections of rats with adenine diet-induced advanced CKD. Similar results have been shown using the parathyroids from patients with advanced CKD. Moreover, in parathyroids of rats with advanced CKD, recombinant FGF23 failed to decrease serum PTH or activate the MAPK pathway. In rat parathyroid organ culture, FGF23 decreased secreted PTH and PTH mRNA levels in control or early CKD rats but not in advanced CKD. Therefore, in advanced experimental CKD, there is a decrease in parathyroid Klotho and FGFR1 mRNA and protein levels in rats and in patients with CKD. This decrease corresponds with the resistance of the parathyroid to FGF23 in vivo, which is sustained in parathyroid organ culture in vitro. The increased levels of FGF23 do not decrease PTH levels in established CKD because of a down-regulation of its receptor heterodimer complex Klotho-FGFR1c.
CONCLUSION 1,25(OH)2D acts on the PTH gene to decrease its transcription, and this action is used in the management of patients with CKD. The major effect of calcium on PTH gene expression in vivo is for hypocalcemia to increase PTH mRNA levels, and this is mainly posttranscriptional. Phosphate also regulates PTH gene
IV. TARGETS
REFERENCES
expression in vivo, and this effect appears to be independent of the effect of phosphate on serum calcium and 1,25(OH)2D3. The effect of phosphate is also posttranscriptional. Trans-acting parathyroid cytosolic proteins bind to a defined cis element in the PTH mRNA 30 -UTR. This binding determines the degradation of PTH mRNA by degrading enzymes and thereby PTH mRNA half-life. The post-transcriptional effects of calcium and phosphate are the result of changes in the balance of these stabilizing and degrading factors on PTH mRNA. These interactions also regulate PTH mRNA levels in experimental uremia. Pin1 is upstream to proteinePTH mRNA interactions leading to more rapid PTH mRNA decay (Fig. 27.2). In secondary hyperparathyroidism Pin1 is less active and is associated with an increase in PTH mRNA stability and levels and thus increased PTH secretion. FGF23 acts on its receptor, the Klotho-FGFR1c receptor, to decrease PTH mRNA levels and secretion (Fig. 27.3). In advanced CKD there is resistance of the parathyroid to the high FGF23 levels due to down-regulation of the FGF23 receptor. An understanding of how the parathyroid is regulated will help devise rational therapy for the management of secondary hyperparathyroidism.
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[59] L. Canaff, G.N. Hendy, Human calcium-sensing receptor gene. Vitamin D response elements in promoters P1 and P2 confer transcriptional responsiveness to 1,25-dihydroxyvitamin D, J. Biol. Chem. 277 (2002) 30337e30350. [60] A.J. Brown, Vitamin D analogs for the treatment of secondary hyperparathyroidsim in chronic renal failure, in: T. NavehMany (Ed.), Molecular Biology of the Parathyoid, Landes Bioscience and Kluwer Academic/Plenum Publishers, New York, 2005, pp. 95e112. [61] Y. Nishii, J. Abe, T. Mori, A.J. Brown, A.S. Dusso, J. Finch, et al., The noncalcemic analogue of vitamin D, 22-oxacalcitriol, suppresses parathyroid hormone synthesis and secretion, Contrib. Nephrol. 91 (1991) 123e128. [62] A.M. Kissmeyer, L. Binderup, Calcipotriol (MC 903): pharmacokinetics in rats and biological activities of metabolites. A comparative study with 1,25(OH)2D3, Biochem. Pharmacol. 41 (1991) 1601e1606. [63] D.B. Evans, M. Thavarajah, L. Binderup, J.A. Kanis, Actions of calcipotriol (MC 903), a novel vitamin D3 analog, on human bone-derived cells: comparison with 1,25-dihydroxyvitamin D3, J. Bone Miner. Res. 6 (1991) 1307e1315. [64] A.J. Brown, C.R. Ritter, J.L. Finch, J. Morrissey, K.J. Martin, E. Murayama, et al., The noncalcemic analogue of vitamin D, 22-oxacalcitriol, suppresses parathyroid hormone synthesis and secretion, J. Clin. Invest. 84 (1989) 728e732. [65] T. Naveh-Many, J. Silver, Effects of calcitriol, 22-oxacalcitriol and calcipotriol on serum calcium and parathyroid hormone gene expression, Endocrinology 133 (1993) 2724e2728. [66] S.M. Sprague, F. Llach, M. Amdahl, C. Taccetta, D. Batlle, Paricalcitol versus calcitriol in the treatment of secondary hyperparathyroidism, Kidney Int. 63 (2003) 1483e1490. [67] R. Bouillon, K. Allewaert, D.Z. Xiang, B.K. Tan, H. Van Baelen, Vitamin D analogs with low affinity for the vitamin D binding protein in vitro and decreased in vivo activity, J. Bone Min. Res. 6 (1991) 1051e1057. [68] A.J. Brown, Vitamin D analogues, Am. J. Kidney Dis. 32 (1998) S25eS39. [69] A. Verstuyf, S. Segaert, L. Verlinden, K. Casteels, R. Bouillon, C. Mathieu, Recent developments in the use of vitamin D analogues, Curr. Opin Nephrol. Hypertens 7 (1998) 397e403. [70] N. Fukuda, H. Tanaka, Y. Tominaga, M. Fukagawa, K. Kurokawa, Y. Seino, Decreased 1,25-dihydroxyvitamin D3 receptor density is associated with a more severe form of parathyroid hyperplasia in chronic uremic patients, J. Clin. Invest. 92 (1993) 1436e1443. [71] S. Patel, J.T. Rosenthal, Hypercalcemia in carcinoma of prostate. Its cure by orchiectomy, Urology 25 (1985) 627e629. [72] A. Arnold, M.F. Brown, P. Urena, R.D. Gaz, E. Sarfati, T.B. Drueke, Monoclonality of parathyroid tumors in chronic renal failure and in primary parathyroid hyperplasia, J. Clin. Invest. 95 (1995) 2047e2053. [73] Y.C. Li, A.E. Pirro, M. Amling, G. Delling, R. Baron, R. Bronson, et al., Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia, Proc. Natl. Acad. Sci. USA 94 (1997) 9831e9835. [74] S.J. Van Cromphaut, M. Dewerchin, J.G. Hoenderop, I. Stockmans, E. Van Herck, S. Kato, et al., Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects, Proc. Natl. Acad. Sci. USA 98 (2001) 13324e13329. [75] Y.C. Li, M. Amling, A.E. Pirro, M. Priemel, J. Meuse, R. Baron, et al., Normalization of mineral ion homeostasis by dietary means prevents hyperparathyroidism, rickets, and osteomalacia, but not alopecia in vitamin D receptor-ablated mice, Endocrinology 139 (1998) 4391e4396.
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[76] T. Meir, R. Levi, L. Lieben, S. Libutti, G. Carmeliet, R. Bouillon, et al., Deletion of the vitamin D receptor specifically in the parathyroid demonstrates a limited role for the VDR in parathyroid physiology, Am. J. Physiol. Renal. Physiol. (2009). [77] R. Kilav, J. Silver, T. Naveh-Many, Regulation of parathyroid hormone mRNA stability by calcium and phosphate, in: T. Naveh-Many (Ed.), Molecular Biology of the Parathyroid, Landes Bioscience and Kluwer Academic/Plenum Publishers, New York, 2005, pp. 57e67. [78] T. Naveh-Many, M.M. Friedlaender, H. Mayer, J. Silver, Calcium regulates parathyroid hormone messenger ribonucleic acid (mRNA), but not calcitonin mRNA in vivo in the rat. Dominant role of l,25-dihydroxyvitamin D, Endocrinology 125 (1989) 275e280. [79] M. Yamamoto, T. Igarashi, M. Muramatsu, M. Fukagawa, T. Motokura, E. Ogata, Hypocalcemia increases and hypercalcemia decreases the steady-state level of parathyroid hormone messenger RNA in the rat, J. Clin. Invest. 83 (1989) 1053e1056. [80] E.M. Brown, G. Gamba, D. Riccardi, M. Lombardi, R. Butters, O. Kifor, et al., Cloning and characterization of an extracellular Ca2þ-sensing receptor from bovine parathyroid, Nature 366 (1993) 575e580. [81] S. Yano, E.M. Brown, The calcium sensing receptor, in: T. Naveh-Many (Ed.), Molecular Biology of the Parathyroid, Landes Bioscience and Kluwer Academic/Plenum Publishers, New York, 2005, pp. 44e56. [82] O. Kifor, R. Diaz, R. Butters, E.M. Brown, The Ca2þ-sensing receptor (CaR) activates phospholipases C, A2, and D in bovine parathyroid and CaR-transfected, human embryonic kidney (HEK293) cells, J. Bone Miner Res. 12 (1997) 715e725. [83] O. Bell, E. Gaberman, R. Kilav, R. Levi, K.B. Cox, J.D. Molkentin, et al., The protein phosphatase calcineurin determines basal parathyroid hormone gene expression, Mol. Endocrinol. 19 (2005) 516e526. [84] E. Moallem, J. Silver, R. Kilav, T. Naveh-Many, RNA protein binding and post-transcriptional regulation of PTH gene expression by calcium and phosphate, J. Biol. Chem. 273 (1998) 5253e5259. [85] S. Lopez-Hilker, A.S. Dusso, N.S. Rapp, K.J. Martin, E. Slatopolsky, Phosphorus restriction reverses hyperparathyroidism in uremia independent of changes in calcium and calcitriol, Am. J. Physiol. 259 (1990) F432eF437. [86] I.Z. Ben Dov, H. Galitzer, V. Lavi-Moshayoff, R. Goetz, M. Kuro-o, M. Mohammadi, et al., The parathyroid is a target organ for FGF23 in rats, J. Clin. Invest. 117 (2007) 4003e4008. [87] T. Krajisnik, P. Bjorklund, R. Marsell, O. Ljunggren, G. Akerstrom, K.B. Jonsson, et al., Fibroblast growth factor-23 regulates parathyroid hormone and 1alpha-hydroxylase expression in cultured bovine parathyroid cells, J. Endocrinol. 195 (2007) 125e131. [88] R. Kilav, J. Silver, T. Naveh-Many, Parathyroid hormone gene expression in hypophosphatemic rats, J. Clin. Invest. 96 (1995) 327e333. [89] Y. Almaden, A. Canalejo, A. Hernandez, E. Ballesteros, S. Garcia-Navarro, A. Torres, et al., Direct effect of phosphorus on parathyroid hormone secretion from whole rat parathyroid glands in vitro, J. Bone Miner. Res. 11 (1996) 970e976. [90] E. Slatopolsky, J. Finch, M. Denda, C. Ritter, A. Zhong, A. Dusso, et al., Phosphate restriction prevents parathyroid cell growth in uremic rats. High phosphate directly stimulates PTH secretion in vitro, J. Clin. Invest. 97 (1996) 2534e2540.
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[91] P.K. Nielsen, U. Feldt-Rasmusen, K. Olgaard, A direct effect of phosphate on PTH release from bovine parathyroid tissue slices but not from dispersed parathyroid cells, Nephrol. Dialysis Transplant. 11 (1996) 1762e1768. [92] A. Bourdeau, J.-C. Souberbielle, P. Bonnet, P. Herviaux, C. Sachs, M. Lieberherr, Phospholipase-A2 action and arachidonic acid in calcium-mediated parathyroid hormone secretion, Endocrinology 130 (1992) 1339e1344. [93] A. Bourdeau, M. Moutahir, J.C. Souberbielle, P. Bonnet, P. Herviaux, C. Sachs, et al., Effects of lipoxygenase products of arachidonate metabolism on parathyroid hormone secretion, Endocrinology 135 (1994) 1109e1112. [94] Y. Almaden, A. Canalejo, E. Ballesteros, G. Anon, M. Rodriguez, Effect of high extracellular phosphate concentration on arachidonic acid production by parathyroid tissue in vitro, J. Am. Soc. Nephrol. 11 (2000) 1712e1718. [95] A. Sela-Brown, J. Silver, G. Brewer, T. Naveh-Many, Identification of AUF1 as a parathyroid hormone mRNA 30 untranslated region binding protein that determines parathyroid hormone mRNA stability, J. Biol. Chem. 275 (2000) 7424e7429. [96] M. Dinur, R. Kilav, A. Sela-Brown, H. Jacquemin-Sablon, T. Naveh-Many, In vitro evidence that upstream of N-ras participates in the regulation of parathyroid hormone messenger ribonucleic acid stability, Mol. Endocrinol. 20 (2006) 1652e1660. [97] G.M. Wilson, J. Lu, K. Sutphen, Y. Suarez, S. Sinha, B. Brewer, E.C. Villanueva-Feliciano, et al., Phosphorylation of p40AUF1 regulates binding to A þ U-rich mRNA-destabilizing elements and protein-induced changes in ribonucleoprotein structure, J. Biol. Chem. 278 (2003) 33039e33048. [98] R. Kilav, J. Silver, T. Naveh-Many, A conserved cis-acting element in the parathyroid hormone 30 -untranslated region is sufficient for regulation of RNA stability by calcium and phosphate, J. Biol. Chem. 276 (2001) 8727e8733. [99] R. Levi, I.Z. Ben Dov, V. Lavi-Moshayoff, M. Dinur, D. Martin, T. Naveh-Many, et al., Increased parathyroid hormone gene expression in secondary hyperparathyroidism of experimental
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uremia is reversed by calcimimetics: correlation with posttranslational modification of the trans acting factor AUF1, J. Am. Soc. Nephrol. 17 (2006) 107e112. M. Nechama, I.Z. Ben Dov, P. Briata, R. Gherzi, T. Naveh-Many, The mRNA decay promoting factor K-homology splicing regulator protein post-transcriptionally determines parathyroid hormone mRNA levels, FASEB J. 22 (2008) 3458e3468. T. Naveh-Many, M. Nechama, Regulation of parathyroid hormone mRNA stability by calcium, phosphate and uremia, Curr. Opin. Nephrol. Hypertens 16 (2007) 305e310. M. Nechama, T. Uchida, I.M. Yosef-Levi, J. Silver, T. NavehMany, The peptidyl-prolyl isomerase Pin1 determines parathyroid hormone mRNA levels and stability in rat models of secondary hyperparathyroidism, J. Clin. Invest. 119 (2009) 3102e3114. H. Kurosu, Y. Ogawa, M. Miyoshi, M. Yamamoto, A. Nandi, K.P. Rosenblatt, et al., Regulation of fibroblast growth factor-23 signaling by klotho, J. Biol. Chem. 281 (2006) 6120e6123. K. Takeshita, T. Fujimori, Y. Kurotaki, H. Honjo, H. Tsujikawa, K. Yasui, et al., Sinoatrial node dysfunction and early unexpected death of mice with a defect of klotho gene expression, Circulation 109 (2004) 1776e1782. I. Urakawa, Y. Yamazaki, T. Shimada, K. Iijima, H. Hasegawa, K. Okawa, et al., Klotho converts canonical FGF receptor into a specific receptor for FGF23, Nature 444 (2006) 770e774. I.Z. Ben Dov, H. Galitzer, V. Lavi-Moshayoff, R. Goetz, M. Kuro-o, M. Mohammadi, et al., The parathyroid is a target organ for FGF23 in rats, J. Clin. Invest. 117 (2007) 4003e4008. H. Galitzer, I.Z. Ben Dov, J. Silver, T. Naveh-Many, Parathyroid cell resistance to fibroblast growth factor 23 in secondary hyperparathyroidism of chronic kidney disease, Kidney Int. 77 (2010) 211e218. C. Kumata, M. Mizobuchi, H. Ogata, F. Koiwa, A. Nakazawa, F. Kondo, et al., Involvement of alpha-klotho and fibroblast growth factor receptor in the development of secondary hyperparathyroidism, Am. J. Nephrol. 31 (2010) 230e238. J. Silver, T. Naveh-Many, Phosphate and the parathyroid, Kidney Int. 75 (2009) 898e905.
C H A P T E R
28 Cartilage Barbara D. Boyan, Maryam Doroudi, Zvi Schwartz Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, School of Biology, and Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, USA
CHONDROGENESIS, ENDOCHONDRAL OSSIFICATION, AND VITAMIN D Properties of Cartilage Tissues Cartilage is a group of tissues produced by chondrocytes that is characterized by a relative lack of vascularity and consists of cells surrounded by a specialized extracellular matrix composed predominantly of type II collagen and proteoglycan, often in the form of proteoglycan aggregate. The glycosaminoglycan side chains on the proteoglycan core protein are highly sulfated in the mature tissue, resulting in a hydration state that can withstand compressive loads. Cartilage is generally thought of as a tissue at the ends of long bones, providing the articulating surface. Not all cartilages are articular, however. Cartilaginous tissues also include the ear, nose, trachea, xyphoid, and fracture callus, as well as the growth plates of the long bones, mandibular condyle, spheno-occipital synchondrosis, and costochondral junction. In this chapter, we focus on the growth plate since this tissue has been most extensively studied with respect to vitamin D. The growth plate represents a specialized situation in which the terminal differentiation of the chondrocyte occurs in a linear array so that chondrocytes appear as columns of cells traversing the lineage in clearly demarcated zones of maturation [1,2]. At one end of the growth plate is the resting zone, also called the reserve zone, in which the cells exhibit a hyaline cartilage-like phenotype, similar to articular cartilage found at the ends of long bones. The type II collagen extracellular matrix surrounding these cells is rich in large proteoglycan aggregates characterized by abundant sulfated glycosaminoglycan. Regulatory signals stimulate the cells to undergo a proliferative burst, after which they enter a prehypertrophic state. Hypertrophic
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10028-9
chondrocytes increase markedly in size and must make major changes in the composition of their extracellular matrix to accommodate this characteristic [3]. In addition, they must prepare their matrix for mineral deposition, and produce extracellular matrix vesicles that are enriched in alkaline phosphatase and serve as sites of initial calcium phosphate crystal formation as well as matrix metalloproteinases and enzymes to degrade the sulfated glysosaminoglycans. These changes to the extracellular matrix have also been reported for osteoarthritic cartilage. Many of the chondrocytes undergoing hypertrophy are also apoptotic [4], but how this contributes to the overall physiology of the growth plate is not clear. Once the matrix is calcified, it is resorbed by chondroclasts, vascular invasion occurs, and osteoprogenitor cells migrate onto the calcified cartilage scaffold and form bone.
Regulation by Vitamin D This process is regulated by vitamin D. In the absence of 1a,25-dihydroxyvitamin D3 (1a,25(OH)2D3) or in animals that lack the ability to respond to this vitamin D metabolite, the hypertrophic zone increases in size, resulting in a condition called rickets. This is because the cells at the base of the growth plate fail to mineralize their extracellular matrix and as a result, the tissue is not vascularized, although prehypertrophic cells continue to enter into the terminal differentiation pathway associated with hypertrophy [5]. Crystallographic studies suggest that the mineral crystals that do form in rachitic rats are less mature than those of normal controls [6]. The mechanical instability of the rachitic tissue leads to the pathology associated with rickets, including bowing of the epiphyses and shorter bones. This condition can be treated by restoring systemic levels of
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28. CARTILAGE
1a,25(OH)2D3 as well as by increasing the levels of circulating Caþþ sufficiently to initiate calcification in the hypertrophic cartilage [5,7e14], suggesting that the main function of this vitamin D metabolite is to ensure appropriate Caþþ availability. More recent data indicate that vitamin D and its metabolites regulate other growth plate functions that are not treatable simply by increasing Caþþ. These include regulation of tether formation [15] (Fig. 28.1); lipid metabolism [16e21]; regulation of matrix vesicle enzymes including alkaline phosphatase, matrix metalloproteinases (MMPs) [22e25], and carbonic anhydrase [26]; and protein kinase C (PKC) [27e29]. Many of these enzymes are
Zn-dependent and Zn metabolism is affected by 1a,25 (OH)2D3 [3]. The rachitic rat model has been of considerable value to investigators [3,21,30,31]. To maintain the rat in a rachitic state it is necessary to make it hypophosphatemic as well as vitamin-D-deficient. Rat growth plates remain open throughout life, making direct comparisons with the human growth plate more complex. Even so, the rat costochondral cartilage has been an exceptionally valuable model for understanding the role of maturational state on cellular response to vitamin D [32,33]. Investigators have turned more recently to the use of mouse models that lack responsiveness to
FIGURE 28.1 Requirement for a functional VDR in rachitic mice fed a high-lactose, high-calcium rescue diet. VDRe/e mice fail to form tethers, which are mineralized struts spanning the growth plate from epiphysis to metaphysis. Healing rickets by feeding the mice a high-lactose, high-calcium rescue diet heals the rickets, but does not restore tether formation to the levels seen in VDRþ/þ mice.
IV. TARGETS
SEPARATE ROLES FOR 24,25(OH)2D3 AND 1,25(OH)2D3 IN CARTILAGE
1a,25(OH)2D3, either due to a defect in the ligandbinding region of the vitamin D receptor (VDR) [34] or to a defect in the DNA-binding region [35,36]. In both instances the mice develop a rachitic phenotype. The importance of cartilage as a target tissue cannot be underestimated. When vitamin-D-replete rats are treated with radiolabeled 25(OH)D3, radiolabeled 1,25 (OH)2D3 and 24,25(OH)2D3 accumulate in this tissue [37]. This is also the case for chickens [38]. Although circulating levels of these metabolites are in the picomolar range, levels in the growth plate are in the nanomolar range [37], and 1,25(OH)2D3 and 24,25(OH)2D3 accumulate in fracture callus [39]. One explanation for the retention of these vitamin D metabolites in the cartilage is that they are bound by cartilage oligomeric matrix protein (COMP), which is a structural extracellular matrix protein present in both the growth plate and articular cartilage [40,41]. This suggests that vitamin D metabolites act as autocrine/paracrine regulators in cartilage. Growth plate chondrocytes possess both 1-hydroxylase (Cyp27B1) and 24-hydroxylase (Cyp24) enzymes and produce 1a,25(OH)2D3 and 24R,25(OH)2D3 in a regulated manner at nanomolar concentrations [42e44]. Recent studies using mice in which Cyp27B1 has either been knocked out or using transgenic mice in which Cyp27B1 is overexpressed show clearly that the local production of 1,25 (OH)2D3 in the hypertrophic cells of the growth plate is necessary for mineralization of the extracellular matrix and subsequent osteoclastic resorption of the calcified cartilage and new bone formation [45]. Receptors for 1a,25(OH)2D3 (VDR) are found throughout the growth plate [46], but in articular cartilage, these receptors appear to be present only in chondrocytes associated with osteoarthritic or rheumatic [28] lesions, suggesting that they mediate the actions of 1a,25(OH)2D3 on extracellular matrix degradation and calcification. This hypothesis is supported by the observation that VDRs are reduced or absent in tibial dyschondroplasia [47], a condition in which normal chondrocyte hypertrophy and extracellular matrix calcification fail to occur. It is clear that cartilage extracellular matrix is regulated by vitamin D. 1a,25(OH)2D3 regulates expression of MMP-3 (stromelysin-1) in articular cartilage cells [28,48,49], and activity of MMP-3 [36,50,51] in growth plate chondrocytes. Hyaluronic acid accumulates in the hypertrophic zone of growth plates of rachitic animals and this is reversed by treatment with 1a,25 (OH)2D3 [52]. Proteoglycan degradation is regulated by 1a,25(OH)2D3 [28], but the vitamin D metabolites also modulate growth plate maturation and calcification by regulating proteoglycan synthesis. In vitamin D deficiency in the chick, smaller aggregating proteoglycans are synthesized [53,54]. Restoration of calcium results
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in the production of larger proteoglycans [55]. In healing rickets in the rat, there is a substantial decrease in proteoglycan binding to hyaluronic acid [56]. This suggests that proteoglycan cleavage, which often occurs in the part of the molecule adjacent to the hyaluronicacid-binding region and likely mediated by stromelysin [57], is increased in the presence of vitamin D and retarded in rickets. Only a few studies on the effects of hypervitaminosis D on cartilage and bone in growing animals have been published. Wider epiphyses were reported with excess metaphyseal bone, causing “hypervitaminosis D rickets.” On the other hand, when pregnant rats were treated with high doses of vitamin D2 during days 10e21 of gestation, the fetuses had short bones, mainly due to short diaphyses. The cartilaginous epiphyses were of normal length, but they contained less calcium and atypical chondrocytes [58,59]. This same phenotype is observed in mice lacking 24-hydroxylase, due to high levels of circulating 1a,25(OH)2D3 [60,61].
SEPARATE ROLES FOR 24,25(OH)2D3 AND 1,25(OH)2D3 IN CARTILAGE Numerous studies have shown that 1a,25(OH)2D3 regulates the terminal differentiation of hypertrophic cartilage [3,59] and maintains the concentration of extracellular Caþþ so that calcification can occur [8,62]. However, in vivo studies of rachitic rats in which normal plasma levels of 1,25(OH)2D3 were retained during the first week on a rachitogenic diet showed that mild rachitic lesions occurred anyway [63], suggesting that vitamin D metabolites other than 1a,25(OH)2D3 might be involved, although studies of this type do not rule out the fact that low calcium and phosphate availability might be the critical factor. 24R,25(OH)2D3 may also play an important role, which is most evident in the resting zone, leading to the hypothesis that 24R,25(OH)2D3 is involved in promoting the differentiation of the resting zone chondrocyte into a more mature phenotype [64]. Local injection of 24R,25(OH)2D3 into the upper tibial growth plate [65] or systemic injection of 24R,25(OH)2D3 can heal rickets. It is unlikely that this is due to hydroxylation of 24,25(OH)2D3 to 1,24,25(OH)3D3 and subsequent actions of 1,24,25(OH)3D3 on Ca2þ release, as local injection of 1,25(OH)2D3 is relatively ineffective [65]. Rather, it is more likely that 24R,25(OH)2D3 acts directly on chondrocytes in the upper growth plate to promote their differentiation along the endochondral lineage. When 24R,25(OH)2D3 is injected with 1a,25(OH)2D3 into fracture callus, bone repair occurs more rapidly than when 1a,25(OH)2D3 is injected alone [66]. Receptors for 24R,25(OH)2D3 have been shown by autoradiographic
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28. CARTILAGE
studies in growth plate cartilage [38,46,60], further supporting the hypothesis that this vitamin D metabolite modulates these cells directly. Studies using fetal mouse long bone organ cultures have shown that maintenance of normal hydration, hypertrophy, columnar arrangement, and preservation of mineral in growth plate cartilage requires the presence of both 1a,25(OH)2D3 and 24R,25(OH)2D3 at physiological concentrations [67]. Similarly, both metabolites are required for maximal production of chondrocalcin (C-propeptide of type II collagen) and calcification of rat [68] and bovine [69] growth plate chondrocyte cultures. Cell culture models have made it possible to identify specific target cell populations for vitamin D metabolites. Among the most completely described cell models are the rat [25,32], rabbit [67,70e72], and mouse [73] costochondral cartilage systems. Studies using these models show that 24R,25(OH)2D3 and 1a,25(OH)2D3 exert independent effects on growth plate chondrocytes [70]. In vitro studies have demonstrated that growth plate chondrocytes exhibit differential responsiveness to 1,25(OH)2D3 and 24,25(OH)2D3. Using embryonic chick limb bud cells as a model, Boskey et al. [74] found that the less-mature cells are affected by 24R,25(OH)2D3, whereas the more mature cells are affected by 1a,25 (OH)2D3. When chick embryo cartilage cells were examined for evidence of VDR, the receptor was found only in growth cartilage cells and not in resting cartilage [71]. Increases in calcification of growth cartilage were paralleled by an increase in VDR levels. The differential response is seen in rats as well [75,76]. The response of rat growth plate chondrocytes to vitamin D3 metabolites depends on the zone of maturation from which the cells were originally derived [7] (Table 28.1). Rat costochondral resting zone chondrocytes respond primarily to 24R,25(OH)2D3, whereas growth zone chondrocytes respond primarily to 1a,25(OH)2D3. In addition, treatment of resting zone chondrocytes with 24R,25 (OH)2D3 causes a change in maturation state; cells become responsive to 1a,25(OH)2D3 and lose their responsiveness to 24R,25(OH)2D3 [64]. This indicates that this hormone has a very specific role in chondrocyte differentiation. The response of each cell to its metabolite includes differential regulation of plasma membrane and matrix vesicle enzyme activity [77,78] and fluidity [79], collagen and noncollagen protein synthesis and cell proliferation [80], calcium flux [81], phospholipid metabolism [81,82], and production of vitamin D metabolites [42]. The specific responses to 24R,25(OH)2D3 are also present in resting zone chondrocytes from VDRe/e mice [73], further supporting the conclusion that these cells are targets for this vitamin D metabolite. Primary functions of 1a,25(OH)2D3 in the growth plate are to inhibit proliferation, inducing terminal differentiation and apoptosis, to activate matrix-processing
TABLE 28.1
Differential Effects of 1a,25(OH)2D3 and 24R,25 (OH)2D3 on Rat Costochondral Resting Zone (RC) and Growth Zone (GC) Chondrocytes 1a,25(OH)2D3
24R,25(OH)2D3
Effect
RC
GC
RC
GC
Alkaline phosphatase activity
e
[
[
e
Proteoglycan sulfation
e
[
[
e
Phospholipase C (PLC) activity
e
[
e
e
Phospholipase D (PLD) activity
e
e
[
e
Protein kinase C (PKC) activity
e
[
[
e
Diacylglycerol (DAG) production
e
[
[
e
Inositol-trisphosphate (IP3) production
e
[
e
e
Mitogen-activated protein (MAP) kinase activity
e
[
[
e
Phospholipase A2 (PLA2) activity
e
[
Y
e
Arachidonic acid release
e
[
Y
e
Prostaglandin E2 (PGE2) production
e
[
Y
e
Protein kinase A (PKA) activity
e
[
Y
e
Membrane fluidity
e
[
[
Y
enzymes, and to prepare the matrix for calcium phosphate deposition by up-regulating production of appropriate matrix proteins and facilitating release of Caþþ ions into the extracellular environment. New evidence suggests that the primary functions of 24R,25(OH)2D3 are to ensure that the pool of chondrocytes in the resting zone of the growth plate is maintained, even in the presence of physiological apoptogens. 24R,25(OH)2D3 increases production of sulfated glycosaminoglycan-rich extracellular matrix, and reduces matrix metalloproteinase activity [47]. Recent evidence indicates that 24R,25(OH)2D3 acts to promote cell survival and maturation by mechanisms that involve both PKC activation and signaling through lysophosphatidic acid (LPA) [83], as described below. 24R,25(OH)2D3 increases PKC and LPA via activation of phospholipase D (PLD). LPA stimulates proliferation, maturation, inhibition of inorganic phosphate (Pi)induced apoptosis, and reduces p53 abundance [83]. Both 24R,25(OH)2D3 and LPA attenuate Pi-induced caspase-3 activity, requiring Gai, LPA receptor(s) 1 and/or 3, PLD, phospholipase C (PLC), and intracellular calcium, phosphoinositide 3-kinase (PI3K)
IV. TARGETS
RAPID ACTIONS OF VITAMIN D AND NONGENOMIC MECHANISMS
511
Schematic drawing of cells in the resting zone showing the interaction of 24,25(OH)2D3 and lysophosphatidic acid in regulating cell survival.
FIGURE 28.2
signaling, and nuclear export. 24R,25(OH)2D3 decreases both p53 abundance and p53-mediated transcription and inhibits Pi-induced cytochrome c translocation. Moreover, LPA induces increased mdm2 phosphorylation, a negative regulator of p53. Taken together, these observations show that 24R,25(OH)2D3 inhibits Piinduced apoptosis through Caþþ, PLD, and PLC signaling and through LPA-LPA1/3-Gai-PI3K-mdm2mediated p53 degradation, resulting in decreased cytochrome c translocation and caspase-3 activity (Fig. 28.2).
RAPID ACTIONS OF VITAMIN D AND NONGENOMIC MECHANISMS Definitions and Models for Studying Rapid Actions Some of the effects of vitamin D in cartilage occur via membrane-mediated mechanisms, some of which are nongenomic. Examples of nongenomic actions
include changes in membrane fluidity [79], rapid turnover of phospholipids [82,84], changes in calcium flux [81,85,86], and rapid activation of PKC [27]. In intact cells, however, many of these activities can ultimately lead to changes in gene expression. The rat costochondral chondrocyte culture model has been particularly useful for studying the membrane-mediated mechanisms of 1a,25(OH)2D3 and 24R,25(OH)2D3 action. One reason for this is that both resting zone and growth zone chondrocytes produce extracellular matrix vesicles in culture and these organelles can be separated from the cells that produce them by simple biochemical techniques that do not require homogenization or lysis. Because matrix vesicles are exterior to the cell, are isolated right side-out, and contain no DNA or RNA, any direct effect of the vitamin D metabolites on them is by definition nongenomic. The phospholipid composition [87] and enzyme activity [7,77] of these matrix vesicles depend on the cell of origin. Not only do matrix vesicles produced by growth zone chondrocytes differ from those produced by
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resting zone chondrocytes, but each type of matrix vesicle differs from the plasma membrane of the cell from which it was derived [79]. Thus, we can separate the genomic regulation of the cells resulting in gene transcription, protein synthesis, and new matrix vesicle production, from nongenomic regulation by comparing the effects of 24R,25(OH)2D3 and 1a,25 (OH)2D3 on cell cultures with those on isolated plasma membranes and isolated matrix vesicles [86].
Membrane Signaling by 1a,25(OH)2D3 The idea of a plasma membrane receptor for 1a,25 (OH)2D3 is an attractive one because it provides a new target for addressing conditions that are resistant to vitamin D therapy [88]. There are now considerable data to support the presence of such a receptor but there is also controversy as to what that receptor is. One candidate for an alternate receptor for 1a,25(OH)2D3 is Pdia3 (protein disulfide isomerase family A, member 3; aka 1,25-MARRS, ERp60, ERp57, and Grp58) [89e91]. We have shown that antibodies directed to the N-terminal, C-terminal, or mid-region of this protein, all inhibit rapid activation of PKC (PKC) by 1a,25(OH)2D3 in
plasma membranes isolated from growth plate chondrocytes and a number of osteoblast cell lines [92], in addition to blocking downstream responses to 1a,25(OH)2D3. Moreover, Pdia3 binds radiolabeled 1a,25(OH)2D3 [91] and experiments using ribozymes to ablate Pdia3 demonstrated a loss of 1,25(OH)2D3 binding, as well as loss of function in intestinal epithelial cells [93]. Mice with only one functional allele for Pdia3 (Pdia3þ/e) exhibit a rachitic phenotype characterized by an expanded hypertrophic cell zone and osteopenia [94]. Our studies using rat and mouse growth plate chondrocytes and MC3T3-E1 osteoblasts suggest that Pdia3 mediates the effects of 1a,25(OH)2D3 via a conserved mechanism in 1a,25(OH)2D3 responsive cells (Fig. 28.3) [95,96]. In growth plate chondrocytes, 1a,25(OH)2D3 causes a rapid increase in phospholipase A2 (PLA2) activity, catalyzing the release of arachidonic acid (AA), which is further metabolized via constitutive cyclooxygenase (COX-1), producing prostaglandin. PGE2 binds its EP1 receptor, generating Gaq [97]. In addition, lysophospholipid produced by the action of PLA2 causes activation of phosphatidylinositol (PI)specific phospholipase C (PLC), which then catalyzes the production of inositol-trisphosphate (IP3) and
FIGURE 28.3 Proposed mechanism for the rapid action of 1a,25(OH)2D3 on growth zone chondrocytes. In growth zone cells, 1a,25(OH)2D3, acting via a membrane receptor, increases phospholipase A2 (PLA2) activity in the membrane. This results in increased fatty acid turnover and changes in membrane fluidity. Changes in membrane fluidity can alter the activity of enzymes in the membrane and also calcium flux. 1a,25 (OH)2D3 also increases phospholipase C (PLC), which can act on phosphatidylinositol 4,5-bisphosphate (PIP2) to increase the production of inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 can stimulate the release of calcium from the endoplasmic reticulum (ER) and into the cell. DAG can activate protein kinase C (PKC). Stimulation of arachidonic acid release activates PKC. Increased production of arachidonic acid also increases the production of prostaglandins, such as prostaglandin E2 (PGE2), which are potent regulators of chondrocytes. PGE2 activates the G-protein pathway, stimulating adenylate cyclase (AC) activity and increasing protein kinase A (PKA) activity. Once PKC is activated, it can activate a signal transduction cascade, by phosphorylation of serine and threonine residues, leading to mitogen-activated protein kinase (MAPK) activation, phosphorylation of AP-1, and increased transcription from relevant gene promoters.
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diacylglycerol (DAG) [98]. IP3 stimulates release of intracellular Caþþ stores into the cytosol, and the Caþþ ions together with membrane-bound DAG, cause the translocation of PKCa to the PM and its activation. 1a,25(OH)2D3 activates phosphorylation of ERK1/2 MAP kinase [78], leading to new gene transcription. Among the new proteins that are regulated via this pathway is PKC-zeta (PKCz), which is incorporated into extracellular matrix vesicles produced by growth zone cartilage cells.
Membrane Signaling by 24R,25(OH)2D3 24R,25(OH)2D3 also activates PKCa and MAP kinase, but it does so only in costochondral resting zone cells and via a very different mechanism than is used by 1a,25(OH)2D3 in prehypertrophic and hypertrophic chondrocytes. It is unlikely that the effects of 24R,25(OH)2D3 are mediated by Pdia3 because antibodies to this protein do not block the activation of PKC. PLA2 plays a pivotal role in the differential response of resting zone cells and growth zone cells to 1a,25(OH)2D3 and 24R,25(OH)2D3. The mechanism of 24R,25(OH)2D3 action on resting zone cells is schematically shown in Figure 28.3. It is predicated on the observation that 24R,25(OH)2D3, acting via a hypothetical membrane receptor, inhibits PLA2 activity [77], an effect that will change fatty acid turnover, release of arachidonic acid, and production of PGE2 [82,99,100]. There is a resultant change in membrane fluidity [79] and Ca2þ flux. 24R,25(OH)2D3 increases DAG production [101], but does so through a two-step mechanism catalyzed by PLD2 [102]. DAG activates PKC, but PKC is not translocated to the plasma membrane [101]. Although resting zone cells possess PLCb1 and PLCb3, neither enzyme is activated by 24R,25(OH)2D3 and inhibition of PLC activity has no effect on PKC activity in response to 24R,25(OH)2D3 [98]. Interestingly, inhibition of PLA2 results in increased PKC activity [101], and PLA2 activation, or addition of lysophospholipids [98], exogenous arachidonic acid [103], or exogenous PGE2 [104], all inhibit PKC. The effect of PGE2 is via the EP2 receptor in these cells. These observations suggest that by inhibiting PLA2, even for a short period of time, the inhibitory effect of the enzyme and its products are reduced. Moreover, inhibition of PLA2 reduced levels of lysophospholipid, inhibiting PLC activity. As a result, the PLD-dependent mechanisms dominate in these cells. As noted above, PKC activation leads to an increase in the activity of ERK1/2 MAPK [78]. Whereas 1a,25(OH)2D3 increases PKCz in matrix vesicles produced by growth zone cells, 24R,25(OH)2D3 increases this PKC isoform in matrix vesicles produced by resting zone cells.
513
PHYSIOLOGIC RELEVANCE OF NONGENOMIC REGULATION OF MATRIX VESICLES Although the cell can down-regulate undesired nongenomic effects at the plasma membrane, this is more difficult in the matrix. To control events in the matrix, the cell may modulate the rate of production and the chemical composition of matrix vesicles [86,105,106]. Initially, matrix vesicles are produced under genomic control. 1a,25(OH)2D3 and 24R,25(OH)2D3 regulate the composition of matrix vesicles through new gene transcription, protein synthesis, and, finally, membrane synthesis. Once matrix vesicles are released into the extracellular matrix, the cells can regulate their maturation through secretion of vitamin D metabolites that act on the matrix vesicle through nongenomic mechanisms. Matrix vesicles may have multiple functions in the matrix. Those in the lower hypertrophic cell zone of cartilage are probably involved in matrix calcification [107]. In addition, matrix vesicles also appear to be involved in matrix maturation, as they contain matrixprocessing enzymes that degrade proteoglycans [108e111]. The activities of these enzymes are regulated in vitro and in vivo by 1a,25(OH)2D3 and 24R,25(OH)2D3 in a cell-specific manner [47,52]. 1a,25(OH)2D3 increases levels of acid metalloproteinases whereas 24R,25 (OH)2D3 decreases levels of these enzymes. In addition to controlling the levels of enzyme activity through genomic regulation, both metabolites can modulate matrix vesicle enzyme activity directly. 24R,25(OH)2D3 stabilizes the matrix vesicle, preventing the release of matrix-processing enzymes whereas 1a,25(OH)2D3 causes a loss of matrix vesicle membrane integrity leading to the release of these enzymes [112]. Matrix vesicles may also play an important role in activation of growth factors present in the extracellular matrix and this is regulated by the vitamin D metabolites [113]. Growth plate chondrocytes synthesize and store latent TGF-b1 as a macromolecular complex in the extracellular matrix. 24R,25(OH)2D3 modulates the expression of latent TGF-b binding protein-1 by resting zone chondrocytes whereas 1a,25(OH)2D3 modulates expression of the protein as well as deposition of TGFb1 together with its binding protein in the matrix [112]. At the same time, release of latent TGF-b1 and its activation are also controlled by 24R,25(OH)2D3 and 1a,25 (OH)2D3. 24R,25(OH)2D3-treatment of matrix vesicles produced by the growth plate cells stabilizes the matrix vesicle membrane, inhibiting the release of matrix vesicle enzymes. In contrast, 1a,25(OH)2D3-treatment of matrix vesicles produced by either resting zone cells or growth zone cells activates latent TGF-b1 [112,113]. Studies examining the direct regulation matrix vesicles
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produced by growth zone chondrocytes indicate that 1a,25(OH)2D3 causes the release of MMP-3 (stromelysin-1), which then catalyzes the release of latent TGFb1 from the extracellular matrix [112] and activates the latent growth factor. These observations suggest that nongenomic regulation of matrix vesicles can result in changes in local growth factor activation. This is a particularly attractive hypothesis in cartilage, where activation of latent growth factors by local decreases in pH (as occurs in osteoclasts) has not been reported. These observations led to the following hypothesis for nongenomic regulation of events in the extracellular matrix (Fig. 28.4). Chondrocytes produce matrix vesicles under hormonal and growth factor regulation. At the same time, vitamin D metabolites are synthesized by the cells and secreted in response to regulatory factors like 1,25(OH)2D3, 24,25(OH)2D3, TGF-b, or corticosteroids. These factors interact directly with the plasma membrane. In addition, they also interact with the matrix vesicle membrane, where their effects initiate a cascade of biochemical events that lead to maturation of the matrix vesicle, hydroxyapatite crystal formation, degeneration of the integrity of the matrix vesicle membrane,
and eventual release of active proteases. The proteases then degrade proteoglycan aggregates in the vicinity of the matrix vesicle, facilitating extracellular matrix calcification. In addition, they may activate latent growth factors that can then act on the cell in an autocrine manner or on adjacent cells via paracrine interactions. Further evidence that 1a,25(OH)2D3 and 24R,25 (OH)2D3 can directly affect proteoglycan degradation and matrix calcification via nongenomic effects on matrix vesicles is now available. When growth zone chondrocytes are treated with 1a,25(OH)2D3, there is an increase in matrix vesicle matrix metalloproteinase (MMP) activity [29]. Analysis of the direct effect of 1a,25(OH)2D3 on isolated membrane fractions indicates that plasma-membrane-associated PKC is increased, resulting in the PKCa-dependent phosphorylation of MMP-3 [28]. In this state, MMP-3 is then packaged into matrix vesicles and released into the matrix. However, no MMP activity is detectable in isolated matrix vesicles unless membrane integrity is lost [108,110]. When treated with 1,25(OH)2D3, isolated matrix vesicles contain increased PLA2, which destabilizes the matrix vesicle membrane, releasing the MMP into the matrix.
FIGURE 28.4 Mechanism for the rapid action of 24R,25(OH)2D3 on resting zone chondrocytes. In resting zone chondrocytes, 24R,25(OH)2D3,
acting via a hypothetical receptor, inhibits phospholipase A2 (PLA2) activity. This results in changes in fatty acid turnover, release of arachidonic acid, and production of prostaglandin E2 (PGE2). There is a resultant change in membrane fluidity and calcium flux. 24R,25(OH)2D3 increases diacylglycerol (DAG) production via phospholipase D (PLD), which activates protein kinase C (PKC). Inhibition of PLA2 results in increased PKC activity. Moreover, inhibition of PGE2 production increases PKC. In contrast, addition of PGE2 inhibits PKC through PKA. PKC may then change cell behavior through phosphorylation of different proteins.
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CONCLUSION
At the same time, alkaline phosphatase in the matrix vesicles is elevated in response to 1,25(OH)2D3. We hypothesize that alkaline phosphatase then dephosphorylates MMP-3, resulting in increased MMP activity. PKCz activity in the matrix vesicle is decreased by direct treatment with 1a,25(OH)2D3. Thus, this isoform of PKC has no effect on matrix vesicle MMP-3, leading to the hypothesis that the enzyme is then fully active in the matrix [28]. When isolated matrix vesicles are incubated in gelatin gels in the presence of proteoglycan, the
inhibition of crystal formation normally associated with proteoglycan is lost. Furthermore, treatment of the matrix vesicles with 1a,25(OH)2D3 causes an increase in the rate and extent of new crystal formation [111].
CONCLUSION This chapter has shown that cartilage, much like other tissues, is really a family of tissues spanning a broad
FIGURE 28.5 Proposed mechanism for the nongenomic regulation of matrix vesicles in the extracellular matrix. Chondrocytes produce matrix vesicles under hormonal and growth factor control. Systemic 1a,25(OH)2D3 or 24R,25(OH)2D3 interact with classic receptors or stimulate rapid membrane-mediated signal transduction pathways, resulting in new gene expression and, ultimately, new matrix vesicle production. Rapid membrane responses include release of arachidonic acid and prostaglandin E2 (PGE2) production as well as altered calcium flux. At the same time, the cells synthesize vitamin D metabolites. The vitamin D metabolites are secreted into the matrix and interact directly with the plasma membrane, causing PKCa-dependent phosphorylation of matrix metalloproteinase-3 (MMP-3). In addition, they also interact with the membrane of pre-existing matrix vesicles, where they initiate a cascade of events leading to matrix vesicle maturation, hydroxyapatite crystal formation, and, in matrix vesicles produced by growth zone chondrocytes, loss of matrix vesicle membrane integrity through stimulation of phospholipases. Once this occurs, active proteinases such as MMP-3 are released. The proteinases degrade proteoglycan aggregates, facilitating matrix calcification. In addition, they may activate latent growth factors. See the text for additional details.
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spectrum of cell maturation states. In growth plate, a subset of the cartilage phenotype, chondrocytes can be seen at distinct states of maturation in a linear array. Using a variety of in vivo and in vitro assays, investigators have been able to show that the growth plate is sensitive to vitamin D regulation, with 24R,25(OH)2D3 affecting less mature cells, particularly those of the resting zone, and 1a,25(OH)2D3 modulating activities in the growth zone (prehypertrophic and upper hypertrophic) cartilage. Both metabolites exert their effects through genomic mechanisms. However, some of the responses of the cell may involve nongenomic mechanisms (Fig. 28.5). Rapid cell-membrane-mediated events via Pdia3 and VDR may result in secondary genomic responses via protein phosphorylation cascades and MAP kinase; in matrix vesicles, rapid membrane effects may be termed nongenomic because no gene expression or protein synthesis is possible. This is relevant to in vivo regulation of endochondral ossification, since chondrocytes produce and secrete 1,25(OH)2D3 or 24,25 (OH)2D3, which then may interact with the matrix vesicles, resulting in modulation of the activity of this extracellular organelle. The consequences of this include activation of latent growth factors, degradation of matrix proteoglycans, and calcium phosphate deposition. Thus, 1a,25(OH)2D3 and 24R,25(OH)2D3 regulate chondrocyte proliferation, metabolism, differentiation, and maturation, as well as events in the extracellular matrix. The effects are cell maturation-dependent and organelle-specific, and may involve both VDR-dependent and VDR-independent genomic as well as nongenomic mechanisms.
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[78] Z. Schwartz, H. Ehland, V.L. Sylvia, et al., 1Alpha,25-dihydroxyvitamin D(3) and 24R,25-dihydroxyvitamin D(3) modulate growth plate chondrocyte physiology via protein kinase C-dependent phosphorylation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase, Endocrinology 143 (7) (2002) 2775e2786. [79] L.D. Swain, Z. Schwartz, K. Caulfield, et al., Nongenomic regulation of chondrocyte membrane fluidity by 1,25-(OH)2D3 and 24,25-(OH)2D3 is dependent on cell maturation, Bone 14 (4) (1993) 609e617. [80] Z. Schwartz, D.L. Schlader, V.M. Ramirez, et al., Effects of vitamin D metabolites on collagen production and cell proliferation of growth zone and resting zone cartilage cells in vitro, J. Bone Miner. Res. 4 (2) (1989) 199e207. [81] G.G. Langston, L.D. Swain, Z. Schwartz, et al., Effect of 1,25 (OH)2D3 and 24,25(OH)2D3 on calcium ion fluxes in costochondral chondrocyte cultures, Calcif. Tissue Int. 47 (4) (1990) 230e236. [82] Z. Schwartz, L.D. Swain, V. Ramirez, et al., Regulation of arachidonic acid turnover by 1,25-(OH)2D3 and 24,25-(OH)2D3 in growth zone and resting zone chondrocyte cultures, Biochim. Biophys. Acta 1027 (3) (1990) 278e286. [83] B.D. Boyan, J. Hurst-Kennedy, T.A. Denison, et al., 24R,25Dihydroxyvitamin D3 [24R,25(OH)2D3] controls growth plate development by inhibiting apoptosis in the reserve zone and stimulating response to 1alpha,25(OH)2D3 in hypertrophic cells, J. Steroid Biochem. Mol. Biol. 121 (1-2) (2010) 212e216. [84] H.K. Kimelberg, Alterations in phospholipid-dependent (Naþ þKþ)-ATPase activity due to lipid fluidity. Effects of cholesterol and Mg2þ, Biochim. Biophys. Acta 413 (1) (1975) 143e156. [85] Z. Schwartz, G.G. Langston, L.D. Swain, et al., Inhibition of 1,25-(OH)2D3- and 24,25-(OH)2D3-dependent stimulation of alkaline phosphatase activity by A23187 suggests a role for calcium in the mechanism of vitamin D regulation of chondrocyte cultures, J. Bone Miner. Res. 6 (7) (1991) 709e718. [86] M.C. Farach-Carson, J. Abe, Y. Nishii, et al., 22-Oxacalcitriol: dissection of 1,25(OH)2D3 receptor-mediated and Ca2þ entrystimulating pathways, Am. J. Physiol. 265 (5 Pt. 2) (1993) F705eF711. [87] H. Nakahara, K. Watanabe, S.P. Sugrue, et al., Temporal and spatial distribution of type XII collagen in high cell density culture of periosteal-derived cells, Dev. Biol. 142 (2) (1990) 481e485. [88] U.A. Liberman, Vitamin D-resistant diseases, J Bone Miner. Res. 22 (Suppl. 2) (2007) V105eV107. [89] I. Nemere, Z. Schwartz, H. Pedrozo, et al., Identification of a membrane receptor for 1,25-dihydroxyvitamin D3 which mediates rapid activation of protein kinase C, J. Bone Miner. Res. 13 (9) (1998) 1353e1359. [90] B.D. Boyan, V.L. Sylvia, D.D. Dean, et al., 1,25-(OH)2D3 modulates growth plate chondrocytes via membrane receptormediated protein kinase C by a mechanism that involves changes in phospholipid metabolism and the action of arachidonic acid and PGE2, Steroids 64 (1e2) (1999) 129e136. [91] I. Nemere, M.C. Dormanen, M.W. Hammond, et al., Identification of a specific binding protein for 1 alpha,25-dihydroxyvitamin D3 in basal-lateral membranes of chick intestinal epithelium and relationship to transcaltachia, J. Biol. Chem. 269 (38) (1994) 23750e23756. [92] B.D. Boyan, L.F. Bonewald, V.L. Sylvia, et al., Evidence for distinct membrane receptors for 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) in osteoblasts, Steroids 67 (3-4) (2002) 235e246. [93] I. Nemere, M.C. Farach-Carson, B. Rohe, et al., Ribozyme knockdown functionally links a 1,25(OH)2D3 membrane
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C H A P T E R
29 Vitamin D and Oral Health Ariane Berdal 1, 2, Muriel Molla 1, 2, Vianney Descroix 1, 3 1
Team “Molecular Oral Physiopathology”, Cordeliers Research Centre, INSERM UMRS 872, Universities Paris 5, Paris 6 and Paris 7, Escalier E, 75270 Paris Cedex 06, France 2 Reference Center “Oral Rare Dysmorphologies”, Rothchild Hospital, Garancie`re Dental Service, Assistance Publique-Hoˆpitaux de Paris, France 3 Oral Medicine Department, Pitie´-Salpeˆtrie`re Hospital, Assistance Publique-Hoˆpitaux de Paris, France
INTRODUCTION: THE DENTOMAXILLOFACIAL SKELETON The maxillofacial skeleton shows several unique features when compared to the axial and appendicular bone. Thus, vitamin-D-related physiopathology follows general but also additional site-specific pathways in the oral region. Vitamin D insufficiency plays a role in dental (altered formation) and oral bone pathologies (altered formation, periodontal disease and jaw osteonecrosis) the mechanisms of which rely on the specific behavior of oral cells. The oral region is composed of a variety of skeletal tissues. These include the hypermineralized enamel layer which covers the dental crown. Dental enamel is an exclusive example of epithelialderived mineralized tissue in the body. Mesenchymalderived tissues include dentine which constitutes the bulk of crown and dental root. Cementum attaches the teeth inside their bone sockets via a connective tissue, the periodontal ligament. Two anatomical compartments form the maxillary bones: alveolar bone is intimately associated with teeth during its formation, homeostasis, and physiopathology. The basal bone constitutes the structural basis of maxilla and mandible which anchors dental arches and their associated alveolar bone. The first original property of the oral skeleton is the presence of teeth. They are composed of unique epithelial (ameloblasts) and mesenchymal (odontoblasts and cementoblasts) cells which are more or less similar to osteoblasts. Consistently, dental cells are responsive to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) [1]. However, as established for bone, the complexity of dental vitamin-D-related physiopathology relies on the duality
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10029-0
of vitamin D action. Vitamin D metabolites may impact dental cells via genomic and nongenomic pathways. Additionally, vitamin D monitors calcium, phosphate, and PTH serum levels which secondarily influence dental cells and mineralization. This double-edge effect of vitamin D has been recently illustrated in teeth and associated alveolar bone by the differential phenotypes of vitamin D nuclear receptor ablated mice in hypocalcemic/hypophosphatemic and normocalcemic/normophosphatemic conditions [2,3]. It should be kept in mind, however, that tooth germs pertain to a second category of vitamin D target-organs, whose differentiation depends on epithelialemesenchymal cross-talk similar to that observed in hair follicles. Nutritional calcemia/phosphatemia rescue does not restore hair follicle phenotype in VDR-ablated mice. Similarly, but in a less evident manner, the molecular and tissue dental phenotype is only partially rescued by the nutritional normalization. The second specific property of the oral skeleton is a distinctive physiology and physiopathology of bone cells. The most exemplary and puzzling illustration of oral specificity is the occurrence of site-specific bone osteonecrosis in jaws under antiresorptive treatments that are not deleterious for the axial and appendicular bone [4]. This regional behavior of jaw bone depends on specific cellular and genetic circuitries of differentiation. Several transcription factors (Msx2) and signaling extracellular proteins (amelogenins) are emerging as major players [5] in the differential physiology of osteoblasts depending on their anatomical sites. These molecular determinants may interfere with vitamin D regulation. More studies are needed to decipher the regional identity of oral bone cells. Such knowledge
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could also contribute to an understanding of vitamin D impact on periodontal diseases, which is evidenced by recent epidemiological surveys (for review see [6]). Indeed, 1a-hydroxylase-null mutant mice show a reverse bone phenotype in the mandible and tibia, which may relate to differential sensitivity to PTH. In summary, oral health and diseases involve many different cells and signaling pathways whose analyses are rather complex. Dental defects constitute recognizable anatomical entities which are routinely used for the clinical diagnosis of rickets in pediatric medicine. In contrast, the different regulatory factors of vitamin D pathway have not yet been analyzed in depth either in vivo or in vitro. This is due to technical pitfalls in dental research (dental cell line availability, specific dentition of rodents). A cultural gap between pediatrics, endocrinology, and dental research constitutes another difficulty. This chapter is an attempt to provide an endocrinological overview of oral cells from basic knowledge to the emerging oral health concerns related to vitamin D.
DENTAL RARE DISEASES AND MOLECULAR DETERMINANTS OF DENTAL RICKETS Inherited disorders of odontogenesis have gained much attention in the last few years. Relative to these conditions, a number of dental genes whose expression is related to vitamin D pathways have been discovered and explored. Thus, the heterogeneous clinical forms of dental rickets constitute a panel of phenotypes which partially mimic nonsyndromic amelogenesis and dentinogenesis imperfecta [7,8].
Whatever its etiology, vitamin D bioinactivation during growth induces dental dysmorphologies along with bone rickets. Prenatal vitamin D deficiency is associated with an increased occurrence of enamel defects [9]. Vitamin D imbalances impact tooth development during childhood as dental tissues are formed until the adolescent age [10,11]. Transient vitamin D intoxication and hypercalcemia induce local alterations which record in a temporal fashion the systemic abnormalities within enamel structure (Fig. 29.1) [12]. The retrospective analysis of dental defects related to vitamin D insufficiency during growth is rather difficult to understand. The medical history is often difficult to define a posteriori. Indeed, other systemic factors induce enamel defects (fluoride, dioxin, antibiotics, etc. [8,10,11]). Genetic rare diseases of the vitamin D pathways are more informative. Vitamin-D-dependent rickets, types 1 and 2, and the most common hypophosphatemic vitamin-D-resistant rickets present different clinical oral features. The observed dental alterations show similarities with hereditary enamel [11] (Fig. 29.2) and dentine disorders [7]. Epithelial enamel is elaborated in two phases, the secretory phase where enamel matrix is deposited and the maturation phase where mineralization and matrix proteolysis are achieved. Amelogenesis imperfecta constitutes a heterogeneous group of clinical and genetic entities (Fig. 29.3). Partial or total enamel deficiencies (hypoplastic form) are related to specific mutations in genes encoding matrix proteins (X-linked OMIM #301200 for amelogenin, autosomal OMIM #104500 and 204650 for enamelin). Hypomature forms of amelogenesis imperfecta are associated with functional alterations of enamel enzymes. These are unable to correctly process extracellular proteins. The presence
FIGURE 29.1 Enamel biomineralization and calbindins. Ameloblast life-cycle follows the strict pattern of amelogenesis process. During the presecretion, ameloblasts polarize and differentiate. During the secretion, enamel proteins are produced and exported in the extracellular compartment where the biomineralization begins. During the postsecretion, ameloblasts undergo cyclical changes in morphology and function. Ruffled (approximately 20% time/length) and smooth (approximately 80% time/length) borders are observed by electron microscopy (white arrows). Calbindin-D9k and calbindin-D28k co-vary during these modifications, as shown here for calbindin-D28k by immunogold labeling (black dots). Ruffled-border ameloblasts harbor some common features with the principal cells of the distal convoluted kidney tubule: apical unfolding of the membrane and molecular organelles for ionic shuttling and buffering. These observations suggest that they may play a role in active calcium transport during postsecretion enamel hypermineralization.
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FIGURE 29.2 Phenotype and genotype record the hereditary nature of amelogenesis imperfecta and vitamin-D-deficiency diseases: (A) intraoral and radiographic illustration of vitamin-D-dependent rickets type II disease (OMIM 277440, Mutation of VDR gene); (B) intraoral and radiographic hypomineralization type of isolated amelogenesis imperfect; (C) recapitulation of genes implicated in amelogenesis imperfect and vitamin-D-deficiency diseases (bold print points out cases with dental analysis in human; * points out cases with tooth study in mouse model of gene inactivation). Please see color plate section.
of uncleaved enamel peptides interferes with hydroxyapatite crystal growth (OMIM #204700 for kalikrein 4 and OMIM # 612529 for MatrixMetalloProteinase MMP20). Such enamel defects are documented in vitamin-D-resistant and -dependent rickets. Interestingly, experimental data show decreased amelogenin and enamelin RNA and protein steady-state levels in vitamin-D-deficient and VDR-null mutant rodent teeth [2,13]. Hypomature enamel contains excessive amounts of unprocessed amelogenins in pigs harboring a mutation in the 1a-hydroxylase (CYP27B1) gene [14]. Therefore, it is possible that enamel hypoplasia and hypomineralization/maturation in vitamin D bioinactivation constitute partial phenocopies of inherited isolated enamel disorders. In the case of defective hydroxylase activity (1A e OMIM #26700 e see Fig. 29.2 and 1B e OMIM #600081), the timing of 1,25 (OH)2D3 treatment will be recorded within the enamel
structure. Hypoplastic/hypomature enamel will be formed before the systemic normalization of ionic homeostasis. Normal regions will define the time when vitamin D metabolites, calcium and phosphates are normal (unpublished data, Bailleul-Forestier, Berdal and Glorieux; [15]). When VDR functions are affected (OMIM #277440), enamel hypoplasia and hypomaturation are generalized and confer an abnormal shape to the permanent teeth (Fig. 29.3). This last clinical entity has not been systematically studied with respect to genotype/dental phenotype correlations. In contrast, the physiopathology of familial X-linked recessive hypophosphatemic rickets has been analyzed in detail, based on an important cohort of patients where PHEX gene mutations were identified [16]. The main target-tissue of this most common form of rickets (OMIM #307800) appears to be the dentin. In this pathology, the differential diagnosis is mandatory
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(A)
(B)
Compared phenotype of two temporal enamel defects: (A) dental phenotype of a clinical case of nutritional vitamin-D-deficiency rickets. Major enamel hypoplasia appears on central maxillar and mandibular incisors; (B) intraoral finding in MIH (hypomineralized molar incisor) showing brown coloration of hypomaturate enamel on the first permanent molar and permanent maxillary incisor. Please see color plate section.
FIGURE 29.3
with the different forms of dentinogenesis imperfecta and dentin dysplasia where the dentin sialophosphoprotein DSPP gene is mutated [7]. Patients show spontaneous abscesses that occur without any history of dental trauma or decay, due to important defects in dentin mineralization. PHEX impairment induces the cleavage of the matrix extracellular phosphoglycoprotein, resulting in the production of an inhibitor for mineralization, the C-terminal acidic serine- and aspartate-rich motif (ASARM) peptide [16]. 1,25(OH)2D3 decreased levels and hypophosphatemia may additionally play a part in the observed mysexpression of noncollagenous dentin proteins (osteocalcin and dentin matrix protein 1) by the odontoblasts [17]. Treatment with 1,25(OH)2D3 and phosphate normalizes dentin mineralization along with the decreased production of ASARM peptide in dentin [16]. As for bone deformities, the treatment corrects the dental phenotype provided that it takes place early in childhood and is carefully monitored until growth is achieved. This observation is in line with a classical concept of the indirect vitamin D action based on a second large cohort of rachitic children [18]. The systematic analysis of calcium and phosphate serum measurements led to the proposal that phosphate levels are the determinant in dentin mineralization. These studies support the major role played by DSPP gene products. Indeed, 50% of the encoded dentin phosphoprotein molecular weight is comprised of
phosphate. Dentin phosphoprotein is selectively secreted at the mineralization front, providing phosphates to the growing apatite crystals. In conclusion, the dental phenotypes reported in different inherited forms of rickets demonstrate a multifactorial impact of vitamin D on dental biomineralization depending on the nature of the involved partners and dental tissue-types. The present knowledge on the heterogeneity of hereditary rickets will need to be re-examined as these dental phenotypes were not systematically recorded along with gene mutation identifications. They have also not been analyzed as yet in available null mutant mice.
EXPERIMENTAL ANALYSIS OF DENTAL PATHWAY IN EXPERIMENTAL MODELS OF VITAMIN D BIOACTIVATION Dental cells harbor all the features of vitamin D target cells. They express both the VDR as well as 1,25(OH)2D3 membrane-associated rapid response steroid receptor during development and mineralization [1,19,20]. They convert 25(OH)D3 metabolites to 1,25(OH)2D3 as they express both CYP27B1 [21] and CYP24A1 [22]. As summarized for dental phenotype in rickets (see Chapter 3), animal models for vitamin D bioinactivation show dental defects. These include abnormal morphogenesis and cell differentiation as well as enamel and
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EXPERIMENTAL ANALYSIS OF DENTAL PATHWAY IN EXPERIMENTAL MODELS OF VITAMIN D BIOACTIVATION
dentin mineralization defects [2,3,23,24]. The expression of target-genes for 1,25(OH)2D3 varies according to vitamin D status in vivo and in vitro: this is also the case for calbindins [1,25], bone sialoprotein [26], amelogenin [13,14], enamelin, and ameloblastin [27] osteocalcin but not DSPP [28]. Vitamin D responses at various vitamin-D-target genes demonstrated in osteoblasts have been documented in dental cells in vitro. In dental mesenchyme, these include osteocalcin [29], osteopontin [22], alkaline phosphatase, and SPARC [30] but not DSPP [29]. Periodontal cells have been shown also to express receptor activator of nuclear factor kappa B ligand e RANK ligand in a vitamin-D-dependent fashion, thus behaving as the stromal cells in controlling osteoclast activity [31]. Vitamin D deficiency has been shown to affect enamel, dentin, and cementum and maxillary bone (for review see [32,33]). However, these data have been generated in diverse experimental models and analyzed through the window of confounding factors such as calcium, phosphate, and PTH levels as well as maternal environment. The most recent findings were obtained in
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null mutants for the VDR [2,3], Cyp27b1 [24], PTH, and calcium-sensing receptor (CaSR) [34]. Tooth defects were severe in hypocalcemic VDRe/e mice [3,35]. They exhibit (1) alterations in all dental tissues, particularly notably through predentine widening; (2) resorption of root tissues, including cementum, dentine, and predentine; and (3) alterations of dental cell differentiation, with, for instance, odontoblastic cell inclusions in the osteodentine, and ameloblast row disruption. The important interglobular dentin and predentin widening were normalized by nutritional rescue of calcium and phosphate with additional lactose (Fig. 29.4). Surprisingly, enamel biomineralization was not significantly affected. The temporal window during the secretion stage was reduced in the VDR-null mutant mice when compared to wild-type mice. Consistently, decreased expression of enamel proteins [2] and enamel prism size [3] were observed and persisted in hypocalcemic and normocalcemic VDRe/e mice. The documented smaller tooth volume of 1a-hydroxylase (Cyp27b1)-null mutants is consistent with an anabolic role of 1,25 (OH)2D3 in dental tissues [24]. In CaSR-deficient mice,
FIGURE 29.4 Root phenotype in VDR-ablated mice. VDR-ablated (VDRe/e) and control (VDRþ/þ) mouse litters are generated by mating VDRþ/e males and females [2,3]. Depending on the calcium, phosphate, and lactose regimen, VDRe/e show either normocalcemia/normophosphatemia (N) or hypocalcemia/hypophosphatemia (H). Microradiographs illustrate dentin (double arrow), cementum (CM), and mandibular bone. (B) Biomineralization defects in VDRe/e H were normalized in VDRe/e N and normal in VDRþ/þ. Morphological study reveals overactivity of osteoclasts in VDRe/e H which results in root resorption (arrow) involving cementum (CM), dentin (D), and predentin (PD). Resorption was absent in VDRe/e N mice. Tartrate-resistant acid phosphatase-positive cells were present and numerous within dental lacunae. These phenotypes illustrate the indirect action of vitamin D on oral mineralized tissues. Please see color plate section.
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Maternal and ionic microenvironment interference with the VDR pathway. A difficult task is to discriminate effects from maternal environment, the mutation of VDR in direct or indirect pathways (calcium, phosphate, and PTH). VDRe/e pups were generated either from VDRþ/e or VDRe/e N mothers and compared. VDRe/e pups were raised either on calcium/phosphate/lactose rescue or normal regimen. These different studies permit discrimination of the impact of VDR on enamel prism and dentine tubule histogenesis (red), of calcium and phosphate on mineralization (green) and of maternal environment on crown morphogenesis (blue). These confounding factors frequently compromise literature reports where first- and second-generation vitamin-D-deficient (either or not calcium- and phosphate-deficient) models have been used.
FIGURE 29.5
the respective role of calcium, phosphorus, and PTHrelated peptide (PTHrp) in the proper growth and mineralization of teeth were also apparent [34]. Finally, the precise role of the prenatal environment could be studied by comparing first and second generations of VDRe/e-null mutants as was apparent earlier with vitamin-D-deficient rats. Pathognomonic cusp features, previously described in the second-generation vitaminD-deficient pups [23], were documented only in VDRe/e pups born from VDRe/e mothers and not from VDRþ/e mothers [2]. Together, all of these data suggest complex vitamin-D-sensitive pathways in tooth cells (Fig. 29.5): 1. Major alterations of tooth morphogenesis imply the additional intervention of the maternal vitamin D environment. 2. Dental histogenesis (enamel prism size, tubule number) is directly dependent upon vitamin D, analogous to that shown for hair follicles [36]. 3. Dental biomineralization (more prominently dentin) is depend on the ambient concentration of calcium and phosphate. Therefore, the VDR pathways in dental rickets reflect the complexity of the tooth, which combines the properties of ectodermal appendages and bone.
SPECIFICITY OF ORAL BONE: POTENTIAL VITAMIN D IMPACT IN DEVELOPMENT, PERIODONTAL DISEASE, AND JAW OSTEONECROSIS The oral bone is composed of two distinct compartments: the basal region of the maxilla which anchors the alveolar bone intimately associated with the dental roots. The sensitivity of oral bone to vitamin D action has been analyzed in various transgenic mouse lines: VDR- [35], Cyp27b1 [24], PTH, and CaSR [34] null mutants. The data indicate that 1,25(OH)2D3 exerts an anabolic role on mandibular trabecular bone as shown in long bones. However, secondary hyperpathyroidism acts predominantly in long bones rather than mandibular bone [24]. Specific roles for calcemia, phosphatemia, and PTHrp are also evidenced in the specific compartment of alveolar bone. Hypocalcemia and hypophosphatemia in VDR-ablated mice are associated with a high resorption of alveolar bone which is normalized in rescued normocalcemic and normophosphatemic mice [35]. CaSR-null mutants show a decreased size of alveolar bone which is totally rescued by elimination of hypophosphatemia and increased levels of PTHrp in the adjoining tooth [34]. Therefore, vitamin D acts on maxillary and alveolar bone formation. Vitamin D
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SPECIFICITY OF ORAL BONE: POTENTIAL VITAMIN D IMPACT IN DEVELOPMENT, PERIODONTAL DISEASE, AND JAW OSTEONECROSIS
deficiency is also shown to affect maxillary bones in ovariectomized adult rats [37]. In aging populations, along with osteoporosis, the most prevalent bone illness is periodontal disease, resulting in tooth loss. Despite several decades of clinical research, there is no consensus regarding whether elderly people with systemic bone disease are more prone to periodontal bone loss or to dental implants failure. Periodontal disease (periodontitis) is a complex disease in which disease expression involves intricate interactions of the biofilm with the host immunoinflammatory response and subsequent alterations in bone and connective tissue homeostasis. The bacterial biofilm is deposited onto dental surfaces which stimulates periodontal inflammation, alveolar bone resorption, and tooth detachment [6]. The lost teeth are replaced by dental implants whose stability is dependent upon bone quality [38]. Proposed links between systemic disease of bone, osteoporosis and osteomalacia, and alveolar bone loss are based on the shared mechanisms of these two bone imbalances relative to aging [38]. More recent studies have focused on VDR polymorphism, dosage of calcium, phosphate, and vitamin D metabolites. These investigations suggest a strong relationship between the vitamin D endocrine system, periodontal inflammation, and bone loss [39]. Identified vitamin D targets in the disease are related to bone homeostasis (for review see [38]) as well as immune function, response to inflammation and bacterial infection such as cathelicidin LL-37 [40] and proinflammatory cytokines. The majority of clinical studies suggest that vitamin D and calcium supplementation reduced tooth loss and alveolar bone resorption [41]. Interestingly, analyses of the Third National Health and Nutrition Examination Survey (NHANES III) highlighted significant associations between vitamin D, calcium intake, and periodontal health [42]. All these clinical data support the view that lower dietary intake of vitamin D and calcium may contribute to reduced periodontal health. To date, no experimental study has been dedicated to studying the impact of vitamin D insufficiency on periodontal bone loss. The only explored facet relates to the therapeutic applications of vitamin D in dentistry. Vitamin D metabolites have been successfully used to control alveolar bone balance during orthodontic treatment [43]. Experiments on implant osseointegration revealed an important deleterious effect of vitamin D deficiency [44]. Periodontal disease analysis has progressed in the last few years. The emerging concept is that this entity is integrated within a more general framework of interrelated pathologies (metabolic imbalances, cardiovascular diseases, and cancer) due to disequilibrium in inflammation and immunity [39]. Interestingly, the number of these pathologies that are associated with
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vitamin D insufficiency and subclinical occurrence is frequent in the elderly. This suggests that the outcome of implants may be confounded by the increasing prevalence of vitamin D insufficiency in aging patients [38]. This new paradigm of oral diseases, as underlined by systemic disorders, leads to a final clinical question regarding vitamin D. An emerging oral pathology is bisphosphonate-induced jaws osteonecrosis (BONJ) (Fig. 29.6). Due to this alarming problem, a taskforce has been created within the American Society for Bone and Mineral Research (ASBMR). The American Association of Oral and Maxillofacial Surgeons (AAOMS) defines BONJ as exposure of portions of the jawbone in patients taking bisphosphonates (BP) that persists for more than 8 weeks in the absence of radiation therapy to the jaw [45]. This jawbone side-effect of the bisphosphonates is relatively uncommon and estimated to occur in 1e10% among patients treated with bisphosphonate intravenous infusion in oncology. These data are not applicable to the use of oral bisphosphonates for the treatment of osteoporosis. Whereas there have been reported cases of ONJ associated with oral bisphosphonate use [46], it is clear from the paucity of these reports that such events are significantly less common than in cancer patients treated with intravenous bisphosphonates. Clinical trial analysis has revealed three main types of risk factors for the development of ONJ: drug-related (potency and duration of therapy of bisphosphonate), local factors (include local anatomy, trauma due to dentoalveolar surgery, and concomitant oral disease), and demographic and systemic factors (like age, race, cancer diagnosis, and osteopenia/osteoporosis diagnosis). Studies have shown that the type of bisphosphonate and the duration of exposure play a role in the
FIGURE 29.6 Bisphosphonate-related osteonecrosis of the mandibular ridge. A 70-year-old man with a 5-year history of multiple myeloma, complicated by hypercalcemia, who was being treated with zoledronate, presented with a 1-month history of severe jaw pain. Please see color plate section.
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development of osteonecrosis. The nitrogen-containing bisphosphonates, pamidronate and zoledronate, have been described as the main culprits [45]. The mechanism for bisphosphonate-induced ONJ is unclear, although several possible pathogenic mechanisms need to be considered; for example, low bone turnover (osteoclast activity profoundly altered and decreased osteoblastic activity), bisphosphonate toxicity to bone and soft tissue or local infection [4]. Another interesting and complementary mechanism links osteonecrosis to local inflammatory processes (probably triggered by apoptotic osteocytes) and immune cell cytotoxicity with notably lymphocytes (IPP-induced gdT cells), monocytes, or macrophages [47,48]. The relationship between NBP and immunity represents the major adverse event of intravenously administered NBPs (incidence ranges between 10% and 50%) is the development of an acute-phase response (APR), which is a nonspecific physiologic immune-driven reaction to systemic challenge [49]. In the same way, the implications for vitamin D deficiency as a potential supplementary risk factor of ONJ have been evaluated. Experimental models combining bisphosphonate infusion and tooth extraction have been established, showing they are required but insufficient. Additional systemic actors are also necessary, either dexamethasone treatment or vitamin D deficiency [47] to obtain a significant amount of experimental osteonecrosis of the jaw in the rat. These preliminary data remain to be confirmed through clinical studies
FIGURE 29.7
on large size cohorts, but we can assume based upon the various actions of vitamin D on bone cells (osteoblast and osteoclast) and also on immunity and inflammation, that vitamin D deficiency is probably an additional risk factor for osteonecrosis of the jaw (Fig. 29.7). In conclusion, alveolar and maxillary bones harbor the general features of bones, as documented in the axial and appendicular skeleton. But, their physiological and physiopathological behavior diverge significantly from bone in the rest of the body. This specificity has been documented in oral pathologies related to vitamin D during growth (PTH refractoriness), periodontal and osteonecrosis diseases (dominant impact of regional factors). The specificity of oral bone may combine intrinsic (phenotype of bone cells) and extrinsic factors related to the presence of teeth (biomechanical constraints, signaling molecules produced by dental adjoining cells e see Fig. 29.8). One specificity of the oral skeleton is the embryonic origin of mesenchymal progenitor cells. Such is the case for odontoblasts forming the crown and root dentine, cementoblasts elaborating the cementum, as well as osteoblasts. Oral mesenchymal progenitors derive from cephalic neural crests while, in the axial and appendicular skeleton, mesenchymal bone cells are of mesodermal origin. Recent studies devoted to the study of neurectodermal mesenchymal cells have shown similar osteogenic properties of vitamin D metabolites [22] as widely established in mesoderm-derived bone cells. On the other hand, while skeletal patterning depends on the Hox homeobox gene clusters in the posterior part of the body, oral and
Hypothetical mechanism of additional role of vitamin D deficiency.
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FIGURE 29.8 Regional balance of periodontal bone homeostasis. Periodontal cells protect and connect the tooth and alveolar bone. Superficial periodontium, the mixed epithelialemesenchymal gingiva, is challenged by microorganisms and produces antimicrobial and immunomodulatory factors which control homeostasis [66]. Some of these events are vitamin-D-dependent [40]. The underlying periodontal ligament is comprised of a dense conjunctive tissue wherein collagen bundles are inserted within the two anchoring mineralized sides, the root cementum and alveolar bone. Some epithelial remnants, the Malassez rests, are also present along the radicular side which size and enamel protein production vary depending on the physiopathological situation [52]. Some transcription factors inhibit enamel protein production, notably amelogenin [5], while vitamin D is likely stimulatory for amelogenin expression in dental epithelial cells [2,27]. This signaling protein has been shown to up-regulate osteoblast differentiation and Runx 2 expression and repress the RANK activation pathway [67]. Therefore, the regional balance of alveolar bone is largely dependent on local regulators.
craniofacial patterning intervenes in an Hox homeobox gene-free territory. A set of divergent homeobox genes such as Muscle segment homeobox (Msx), Distal-less homeobox (Dlx), and Goosecoid (Gsc) genes drive oral and craniofacial morphogenesis. Interestingly, these developmental homeobox gene patterns are imprinted in postnatal mesenchymal bone cells and influence their fate and physiological behavior [50]. For instance, in the Hox-free mandible, Msx1 [51] and Msx2 [5,52] distinctly control the basal and alveolar bone morphodifferentiation during development, postnatal growth, and homeostasis. Msx and Dlx gene products provide a regional phenotype not only to cells devoted to tissue apposition but also to bone resorption [53]. Interestingly, vitamin D impacts Msx2 expression [54].
CONCLUSIONS: FACTS AND HYPOTHESIS The general understanding of dental physiopathology is driven by the advanced knowledge of vitamin D action on bone and calcium/phosphate homeostasis. The presence of vitamin D receptors and responsive genes in the odontoblasts and ameloblasts lead to the conclusion that they are classical target-cells [1]. The analysis of dental root cells is less advanced [20] yet promising as it will elucidate rootebone relationships underlying periodontal diseases where vitamin D is anticipated to be instrumental. On the other hand, the physiological framework of ionic transport during dental mineralization is complex, especially for enamel [55]. The dissection of passive and
active calcium transfer and investigations of calcium cell signaling has been impaired through a lack of appropriate experimental models relative to the situation in kidney or intestine. The numerous available mouse models have been evaluated, however, and have progressively delineated the specific impact of involved partners. Complementary cellular and molecular studies are now feasible, due to the availability of appropriate dental cell lines and dental gene knowledge. Human studies should scrutinize these geneedental phenotype relationships. But, dental defects in humans carrying inherited disorders of the vitamin D endocrine system are much more evident when compared to their corresponding mouse model phenotypes. One bias in endocrinological studies on teeth is the questionable legitimacy of the direct transposition from rodent species to humans. Monophyodont (one generation), rapidly growing (days), and continuously erupting rodent teeth diverge from human dentition. This latter shows diphyodonty (primary and permanent dentition), limited growth, and year-scale formation which occurs in diverse endocrinological contexts: pregnancy, childhood, and adolescent age. It may be that dental ionic homeostasis has been adapted due to the vital priority of tooth mineralization for rodent feeding and survival. Such adaptive strategies of the vitamin endocrine system have been evidenced in the vitamin-D-free subterranean naked mole-rat. In this species, vitamin D is devoted to a process of storing mineral specifically in teeth and maxilla and under extremely high pharmacological vitamin D doses [56]. All the molecular players of the vitamin D endocrine system are present, as specifically
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shown for ameloblast, odontoblast, and calbindins, but their relative impact may be circumstantial [55]. From a public health point of view, frequent periodontal disease and aggressive osteonecrosis require attention. They are part of the spectrum of pathologies relied to the subclinical vitamin D, calcium, and phosphate insufficiency described in modern aging populations. Basic knowledge on the nature of regional bone phenotypes and dental-bone signaling pathways will have to decipher the peculiar local tissue sensitivity to systemic hormones and drugs. Future studies will have to analyze in-depth oral physiopathology per se regarding the role of vitamin D and not to directly transpose knowledge form the bone field in general onto dental and periodontal cells. This might lead to future applications of vitamin D for oral disease treatment which have already proposed in orthodontic treatment.
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[29] H.H. Ritchie, H. Park, J. Liu, T.J. Bervoets, A.L. Bronckers, Effects of dexamethasone, vitamin A and vitamin D3 on DSP-PP mRNA expression in rat tooth organ culture, Biochim. Biophys. Acta 1679 (2004) 263e271. [30] P. Pavasant, T. Yongchaitrakul, K. Pattamapun, M. Arksornnukit, The synergistic effect of TGF-beta and 1,25dihydroxyvitamin D3 on SPARC synthesis and alkaline phosphatase activity in human pulp fibroblasts, Arch. Oral Biol. 48 (2003) 717e722. [31] D. Zhang, Y.Q. Yang, X.T. Li, M.K. Fu, The expression of osteoprotegerin and the receptor activator of nuclear factor kappa B ligand in human periodontal ligament cells cultured with and without 1alpha,25-dihydroxyvitamin D3, Arch. Oral Biol. 49 (2004) 71e76. [32] A. Berdal, Vitamin D action on tooth development and biomineralization, in: J.W.P. David Feldman, H. Francis, Glorieux (Eds.), Vitamin D, Academic Press, USA, 1997, pp. 1e1285. [33] A. Berdal, I. Bailleul-Forestier, J.L. Davideau, F. Le´zot, Dentoalveolar bone complex and vitamin D, in: J.W.P. David Feldman, H. Francis, Glorieux (Eds.), Vitamin D, second ed., Elsevier, USA, 2005, pp. 1e1791. [34] W. Sun, W. Sun, J. Liu, X. Zhou, Y. Xiao, A. Karaplis, et al., Alterations in phosphorus, calcium and PTHrP contribute to defects in dental and dental alveolar bone formation in calciumsensing receptor-deficient mice, Development 137 (2010) 985e992. [35] J.L. Davideau, F. Lezot, S. Kato, I. Bailleul-Forestier, A. Berdal, Dental alveolar bone defects related to Vitamin D and calcium status, J. Steroid Biochem. Mol. Biol. 89-90 (2004) 615e618. [36] Y.C. Li, M. Amling, A.E. Pirro, M. Priemel, J. Meuse, R. Baron, et al., Normalization of mineral ion homeostasis by dietary means prevents hyperparathyroidism, rickets, and osteomalacia, but not alopecia in vitamin D receptor-ablated mice, Endocrinology 139 (1998) 4391e4396. [37] F. Said, S. Ghoul-Mazgar, B. Ruhin, M. Abdellaoui, F. Chlaghmia, S. Safta, et al., Mandibular bone alterations of ovariectomized rats under vitamin D insufficiency, Histol. Histopathol. 23 (2008) 479e485. [38] E.K. Kaye, Bone health and oral health, J. Am. Dent Assoc. 138 (2007) 616e619. [39] W.B. Grant, Vitamin D, periodontal disease, tooth loss, and cancer risk, The Lancet Oncology 9 (2008) 612e613. [40] N. Mookherjee, L.M. Rehaume, R.E. Hancock, Cathelicidins and functional analogues as antisepsis molecules, Expert Opin. Ther. Targets 11 (2007) 993e1004. [41] E.A. Krall, C. Wehler, R.I. Garcia, S.S. Harris, B. DawsonHughes, Calcium and vitamin D supplements reduce tooth loss in the elderly, Am. J. Med. 111 (2001) 452e456. [42] T. Dietrich, K.J. Joshipura, B. Dawson-Hughes, H.A. BischoffFerrari, Association between serum concentrations of 25hydroxyvitamin D3 and periodontal disease in the US population, Am. J. Clin. Nutr. 80 (2004) 108e113. [43] M. Kawakami, T. Takano-Yamamoto, Local injection of 1,25dihydroxyvitamin D3 enhanced bone formation for tooth stabilization after experimental tooth movement in rats, J. Bone Miner. Metab. 22 (2004) 541e546. [44] J. Kelly, A. Lin, C.J. Wang, S. Park, I. Nishimura, Vitamin D and bone physiology: demonstration of vitamin D deficiency in an implant osseointegration rat model, J. Prosthodont. 18 (2009) 473e478. [45] S.L. Ruggiero, T.B. Dodson, L.A. Assael, R. Landesberg, R.E. Marx, B. Mehrotra, American Association of Oral and Maxillofacial Surgeons position paper on bisphosphonaterelated osteonecrosis of the jaws e 2009 update, J. Oral Maxillofac. Surg. 67 (2009) 2e12.
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[46] A. Barrier, G. Lescaille, A. Rigolet, V. Descroix, P. Goudot, B. Ruhin, [Jaw osteonecrosis induced by oral biphosphonates: 12 cases], Rev. Stomatol. Chir. Maxillofac. 111 (2010) 196e202. [47] A. Hokugo, R. Christensen, E.M. Chung, E.C. Sung, A.L. Felsenfeld, J.W. Sayre, et al., Increased prevalence of bisphosphonate-related osteonecrosis of the jaw with vitamin D deficiency in rats, J. Bone Miner. Res. 25 (2010) 1337e1349. [48] P. Lesclous, S. Abi Najm, J.P. Carrel, B. Baroukh, T. Lombardi, J.P. Willi, et al., Bisphosphonate-associated osteonecrosis of the jaw: a key role of inflammation? Bone 45 (2009) 843e852. [49] V. Kunzmann, E. Bauer, M. Wilhelm, Gamma/delta T-cell stimulation by pamidronate, N. Engl. J. Med. 340 (1999) 737e738. [50] K.C. Wang, J.A. Helms, H.Y. Chang, Regeneration, repair and remembering identity: the three Rs of Hox gene expression, Trends Cell Biol. 19 (2009) 268e275. [51] S.M. Orestes-Cardoso, J.R. Nefussi, D. Hotton, M. Mesbah, M.D. Orestes-Cardoso, B. Robert, et al., Postnatal Msx1 expression pattern in craniofacial, axial, and appendicular skeleton of transgenic mice from the first week until the second year, Dev. Dyn. 221 (2001) 1e13. [52] M. Aioub, F. Lezot, M. Molla, B. Castaneda, B. Robert, G. Goubin, et al., Msx2 / transgenic mice develop compound amelogenesis imperfecta, dentinogenesis imperfecta and periodental osteopetrosis, Bone 41 (2007) 851e859. [53] F. Lezot, B.L. Thomas, C. Blin-Wakkach, B. Castaneda, A. Bolanos, D. Hotton, et al., Dlx homeobox gene family expression in osteoclasts, J. Cell Physiol. 223 (2009) 779e787. [54] F. Lezot, V. Descroix, M. Mesbah, D. Hotton, C. Blin, P. Papagerakis, et al., Cross-talk between Msx/Dlx homeobox genes and vitamin D during tooth mineralization, Connect Tissue Res. 43 (2002) 509e514. [55] C.I. Turnbull, K. Looi, J.E. Mangum, M. Meyer, R.J. Sayer, M.J. Hubbard, Calbindin independence of calcium transport in developing teeth contradicts the calcium ferry dogma, J. Biol. Chem. 279 (2004) 55850e55854. [56] R. Buffenstein, M.T. Laundy, T. Pitcher, J.M. Pettifor, Vitamin D3 intoxication in naked mole-rats (Heterocephalus glaber) leads to hypercalcaemia and increased calcium deposition in teeth with evidence of abnormal skin calcification, Gen. Comp. Endocrinol. 99 (1995) 35e40. [57] K. Kikuchi, T. Okamoto, M. Nishino, E. Takeda, Y. Kuroda, M. Miyao, Vitamin D-dependent rickets type II: report of three cases, ASDC J. Dentist. Children 55 (1988) 465e468. [58] S.H. Al-Jundi, I.M. Dabous, G.A. Al-Jamal, Craniofacial morphology in patients with hypophosphataemic vitamin-Dresistant rickets: a cephalometric study, Journal of Oral Rehabilitation 36 (2009) 483e490. [59] P. Batra, Z. Tejani, M. Mars, X-linked hypophosphatemia: dental and histologic findings, Journal (Canadian Dental Association) 72 (2006) 69e72. [60] C. Chaussain-Miller, C. Sinding, M. Wolikow, J.J. Lasfargues, G. Godeau, M. Garabedian, Dental abnormalities in patients with familial hypophosphatemic vitamin D-resistant rickets: prevention by early treatment with 1-hydroxyvitamin D, J. Pediatr. 142 (2003) 324e331. [61] T. Murayama, R. Iwatsubo, S. Akiyama, A. Amano, I. Morisaki, Familial hypophosphatemic vitamin D-resistant rickets: dental findings and histologic study of teeth, Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics 90 (2000) 310e316. [62] C.M. Pereira, C.R. de Andrade, P.A. Vargas, R.D. Coletta, O.P. de Almeida, M.A. Lopes, Dental alterations associated with Xlinked hypophosphatemic rickets, J. Endodont. 30 (2004) 241e245.
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[63] D. Douyere, C. Joseph, C. Gaucher, C. Chaussain, F. Courson, Familial hypophosphatemic vitamin D-resistant rickets e prevention of spontaneous dental abscesses on primary teeth: a case report, Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics 107 (2009) 525e530. [64] J.R. Goodman, M.J. Gelbier, J.H. Bennett, G.B. Winter, Dental problems associated with hypophosphataemic vitamin D resistant rickets, Int. J. Paediat. Dentist./The British Paedodontic Society [and] the International Association of Dentistry for Children 8 (1998) 19e28. [65] G. Hillmann, W. Geurtsen, Pathohistology of undecalcified primary teeth in vitamin D-resistant rickets: review and report
of two cases, Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics 82 (1996) 218e224. [66] L. Yin, T. Chino, O.V. Horst, B.M. Hacker, E.A. Clark, B.A. Dale, W.O. Chung, Differential and coordinated expression of defensins and cytokines by gingival epithelial cells and dendritic cells in response to oral bacteria, BMC. Immunol. 11 (2010) 37. [67] J. Hatakeyama, T. Sreenath, Y. Hatakeyama, T. Thyagarajan, L. Shum, C.W. Gibson, et al., The receptor activator of nuclear factor-kappa B ligand-mediated osteoclastogenic pathway is elevated in amelogenin-null mice, J. Biol. Chem. 278 (2003) 35743e35748.
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C H A P T E R
30 The Role of Vitamin D and its Receptor in Skin and Hair Follicle Biology Marie B. Demay Endocrine Unit, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston MA 02114, USA
INTRODUCTION The skin is the only mammalian organ system capable of synthesizing vitamin D, of activating the pro-hormone by 25- and 1a-hydroxylation and responding to vitamin D receptor (VDR)eligand interactions by induction or suppression of a number of genes and signaling pathways. Thus, the skin is the only tissue identified to date that is capable of synthesizing all the critical factors required for vitamin D action and of responding to 1,25(OH)2D in a paracrine and autocrine fashion. While investigations into the role of 1,25 (OH)2D and the VDR in vivo have traditionally focused on the regulation of mineral ion homeostasis, the skin is an important target tissue. The observation that skin expresses the highest level of VDR mRNA in Xenopus [1], suggests that its role in this respect is evolutionarily conserved. Investigations into the molecular basis for the phenotype of mice with inactivating mutations in the 1a-hydroxylase and the VDR genes have demonstrated effects of this receptor that are dependent upon and independent of ligand binding [2e4].
production from keratins 5 and 14 to keratins 1 and 10. In addition to keratins 1 and 10, the cells in the spinous layer also synthesize involucrin. As cells mature, they transition to the granular layer, expressing profillagrin and loricrin. These proteins, along with the lipids synthesized by these cells, form the cornified layer which is an essential part of the epidermal barrier, preventing fluid loss and protecting the organism against invasion by pathogens. 1,25(OH)2D and calcium have been shown to attenuate epidermal keratinocyte proliferation and promote differentiation. These antiproliferative and prodifferentiation effects of 1,25(OH)2D have led to its clinical use
KERATINOCYTE DIFFERENTIATION The epidermis is composed of layers of keratinocyte that are characterized by their proliferative rate and expression of markers of differentiation. The basal layer lies on the basal lamina and contains epidermal stem cells that proliferate, giving rise to the keratinocytes that are found in the upper layers under normal conditions (Fig. 30.1). Differentiation of cells from the basal to the spinous layer is characterized by a shift in keratin
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10030-7
Schematic representation of keratinocyte differentiation. Cells in the basal layer rest upon the basal lamina, which separates the epidermis from the dermis. They are a source of epidermal keratinocyte stem cells that differentiate into cells expressing markers characteristic of the spinous layer, then the granular layer. The cornified layer is important for epidermal barrier function.
FIGURE 30.1
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in the treatment of psoriasis (discussed in Chapter 97). However, the effects of calcium and 1,25(OH)2D on keratinocyte proliferation and differentiation are largely redundant [5]. In vitro investigations in keratinocytes from mice lacking the VDR demonstrate that they are resistant to the effects of 1,25(OH)2D, but have normal proliferation and acquisition of markers of differentiation in response to calcium [6]. Similarly, in vivo studies in VDR-null mice demonstrate a decrease in markers of keratinocyte differentiation [7], a phenotype that is prevented by maintaining normal serum calcium levels [5]. Chapter 33 discusses the overall findings in the VDR and 1a-hydroxylase-null mice.
COACTIVATORS Like other nuclear receptors, the actions of the VDR are modulated by coactivators and corepressors (see Chapter 10 on coactivators). Studies in epidermal keratinocytes have demonstrated that different coactivator complexes predominate in proliferating versus differentiating cells [8]. The classic VDR interacting DRIP/Mediator complex is critical in proliferating keratinocytes. Knockdown of proteins present in this complex, including DRIP205/Med1 or Med21, result in increased proliferation and impaired differentiation, as well as impaired responsiveness to 1,25(OH)2D and calcium [9]. The expression of components of this complex decrease with differentiation, in association with an increase in Src3 [8]. This latter coactivator is important for the actions of 1,25(OH)2D on induction of differentiationspecific markers in keratinocytes and in the synthesis of lipid components of the epidermal barrier [10]. Of note, silencing of Src3 and VDR ablation have similar effects on the lipid barrier, suggesting that this coactivator is essential for the actions of the VDR on lipid barrier formation. Studies defining the use of specific VDR coactivators during epidermal differentiation have provided a paradigm demonstrating the molecular mechanism by which a single receptor/transcription factor modulates the expression of different subsets of genes during the course of differentiation in a specific cell type.
BARRIER FUNCTION Abnormalities in epidermal differentiation are often accompanied by impaired epidermal barrier formation. The importance of vitamin D metabolites in both formation of the cornified envelope of the skin and in host defense is well established. Similar to VDR-null mice, mice lacking CYP27B1, the enzyme responsible for 1ahydroxylation of 25(OH)D, demonstrate impaired
keratinocyte differentiation, associated with a decrease in the expression of the terminal differentiation markers involucrin, loricrin, and fillagrin [11]. While this is not accompanied by a gross epidermal defect, the CYP27B1-null mice exhibit an increase in transepidermal water loss and impaired recovery of barrier function following acute barrier disruption. 1,25(OH)2D also contributes to barrier function by regulating the synthesis of lipids that play a critical role in permeability barrier formation [10]. The impaired barrier function observed in the VDR-null mice is also associated with a decrease in the synthesis of glucosylceramides and altered barrier lipid composition in vivo. Thus, the effects of the VDR on the cornified envelope and lipid barrier are ligand-dependent. In addition to preventing water loss, the epidermis provides a barrier to invasion by infectious agents. Disruption of this barrier results in activation of the innate immune system. This leads to the synthesis of antimicrobial peptides, including cathelicidin, that play an important role in host defense to infectious agents. Activation of Toll-like receptors (TLRs) induces CYP27B1, leading to an increase in local production of 1,25(OH)2D, as well as induction of the VDR in monocytes and in keratinocytes. One of the downstream consequences of this activation of the VDR signaling pathway is an increase in the expression of cathelicidin and other antimicrobial peptides [12,13]. 1,25(OH)2D participates in a positive feedback loop in this system, since it has also been shown to induce the expression of TLRs [14]. This local activation of vitamin D, associated with induction of the VDR, identifies a critical in vivo role for the nonrenal CYP27B1 in host defense. The role of the VDR in host defense against infection and in barrier function is discussed in detail in elsewhere in this volume.
HAIR CYCLE While the actions of calcium and the VDR signaling pathways on epidermal keratinocyte differentiation are redundant, their effects on the hair follicle are not. Humans and mice with mutations in the gene required for 1a-hydroxylation of vitamin D do not exhibit a hair abnormality [2,15], whereas mice and several human kindreds with mutations in the VDR develop alopecia totalis (Fig. 30.2), in spite of having apparently normal hair development [16e19]. Chapter 65 discusses mutations in the human VDR leading to the syndrome of hereditary vitamin-D-resistant rickets (HVDRR) and alopecia. The skin of the VDR-null mice is characterized by large dermal cysts, dilated piliary canals, and expansion of the sebaceous compartment, features that are not normalized by maintaining normal mineral ion levels
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Phenotype of VDR-null mice. At 1 year of age, the VDR-null mice (foreground) are significantly smaller than their wildtype littermates and demonstrate severe alopecia. Please see color plate section.
FIGURE 30.2
(Fig. 30.3). Severely vitamin-D-deficient humans have not been reported to develop skin or hair abnormalities whereas those with VDR mutations do. Studies in mice rendered vitamin D deficient for several generations, resulting in the absence of detectable circulating levels of 25(OH)D and 1,25(OH)2D, confirm that the absence of ligand does not cause alopecia [3]. The observation that absence of ligand and absence of receptor function have different effects on the hair follicle led to the identification of the cellular compartment of the hair follicle where the actions of the VDR were required to prevent alopecia. During development, formation of the hair follicle is dependent upon reciprocal interactions between epidermal cells and a mesodermal condensate that subsequently gives rise to the dermal papilla. In mice, hair follicle morphogenesis ends the second week of postnatal life with the formation of a mature hair follicle. The bulge region of the hair follicle forms at this time, just below the sebaceous gland (Fig. 30.4). This bulge
(A)
contains keratinocyte stem cells required for the cyclic regeneration of the hair follicle that occurs postnatally. The hair cycle consists of three distinct phases: anagen, catagen, and telogen. The anagen phase of the hair cycle is characterized by proliferation of keratinocytes, leading to the formation of a mature hair follicle and subsequently, generation of a hair shaft. This is followed by catagen, which is characterized by apoptosis of the lower portion of the hair follicle below the bulge, resulting in approximation of these bulge cells to the mesodermal papilla component of the hair follicle. It is thought that the proximity of the dermal papilla and bulge stem cells, during the quiescent telogen phase of the hair cycle, allows paracrine signaling between these two populations of cells, which leads to the initiation of a new anagen phase. Hair reconstitution assays demonstrate that the actions of the VDR required for cyclic regeneration of the hair follicle are critical in the keratinocyte component of the hair follicle. These assays, which involve implantation of dermal papilla cells and neonatal keratinocytes into a cutaneous defect in nude mice, result in reconstitution of the epidermis and hair follicles. Studies which employ keratinocytes and dermal papilla cells differing in VDR status demonstrate that, while expression of the VDR was not required in either cell population for morphogenesis, VDR expression in the keratinocyte population, but not the mesodermal population, was required for hair cycling [3]. This was confirmed by in vivo studies in VDR knockout mice, expressing a VDR transgene specifically in keratinocytes [20]. These mice do not develop alopecia, nor do they have a defective response to anagen initiation. A similar rescue of the hair cycle defect in the VDRnull background is seen with transgenic overexpression of a VDR incapable of binding ligand or mediating ligand-dependent transactivation, confirming that the effects of the VDR on the hair cycle do not require 1,25 (OH)2D. Interestingly, deletion of the AF1 region and
(B)
(C)
Normalization of mineral ion homeostasis does not prevent skin changes in the VDR-null mice. (A) Skin section from a 70-dayold wild-type mouse; (B) from a 70-day-old VDR-null littermate with abnormal mineral ion levels demonstrating dermal cysts and dilatation of hair follicles; (C) from a 70-day-old VDR-null mouse with normal mineral ion homeostasis. Please see color plate section.
FIGURE 30.3
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Schematic representation of the anagen hair follicle. The keratinocyte stem cells that reside in the bulge provide a source of cells for cyclic regeneration of the hair follicle. They also are capable of differentiating into sebocytes which populate the sebaceous glands and of differentiating into epidermal keratinocytes during wound repair.
FIGURE 30.4
first zinc finger of the VDR results in expression of a truncated protein that contains domains required for RXR heterodimerization and ligand binding, as well as the AF2 domain [21]. These mice phenocopy mice with total absence of VDR protein, suggesting that sequences in the AF1 domain or DNA-binding domain of the VDR are essential for the prevention of alopecia. These regions of the VDR and their functions are discussed in detail in Chapter 7.
KERATINOCYTE STEM CELLS Identification of the keratinocyte as the cellular compartment in which expression of the VDR is required for maintenance of the hair cycle was somewhat paradoxical, based on studies demonstrating that calcium and 1,25(OH)2D play redundant roles in the regulation of keratinocyte proliferation and differentiation [5] and the observation that maintaining normal mineral ions in the VDR-null mice does not prevent alopecia [22]. Thus, these studies suggested that there was a specific population of keratinocytes in which the actions of the VDR were essential and where calcium could not play a compensatory role. The hair follicle bulge contains a unique population of keratinocyte stem cells that is responsible for cyclic regeneration of the hair follicle. These cells also give rise to sebocytes and participate in epidermal wound repair, contributing keratinocytes for epithelial regeneration. While formation of the bulge was not altered in the VDR-null mice, there was an age-dependent decrease in the CD34 immunoreactivity, characteristic of this stem cell niche, in the VDR-null mice [23]. Flow cytometry demonstrated that the number of bulge keratinocyte stem cells was normal at 4 weeks of age, a time when the VDR-null mice are unable to regenerate a hair follicle. However, there was a progressive decrease in the number of these keratinocyte stem cells with aging in the VDR-null mice [23,24], correlating with the
decrease in CD34 immunoreactivity. Targeting expression of a VDR transgene to the keratinocytes of the VDR-null mice prevented these abnormalities, demonstrating that the keratinocyte stem cell defect could be prevented by restoring VDR expression [23]. The decrease in the keratinocyte stem cell number that was observed with aging suggested a defect in stem cell self-renewal in the absence of the VDR. However, the inability of the VDR-null mice to regenerate a hair follicle at 4 weeks of age, a time when keratinocyte stem cell number was normal, suggested a functional defect in these cells. Further characterization of the skin defect demonstrated that the large dermal cysts observed in the VDR-null mice were filled with Oil red O staining lipid and their presence was accompanied by expansion of the sebaceous compartment of the hair follicle, suggesting a defect in the lineage progression of the bulge stem cells, favoring sebocytes over keratinocytes. This defect in lineage progression has also been proposed to include impaired ability of these cells to migrate along the follicle at the onset of anagen [24].
PATHWAYS IMPORTANT FOR KERATINOCYTE STEM CELL FUNCTION A number of signaling pathways and transcriptional regulators have been shown to play a role in hair follicle morphogenesis and in cyclic regeneration of the hair follicle postnatally. Notable in this respect, the skin phenotype of mouse models with mutations in three separate pathways resembles that of the VDR knockout mice. Mice with keratinocyte-specific ablation of RXRa, the major isoform of RXR expressed in these cells, develop alopecia and dermal cysts, accompanied by an inflammatory response, the latter of which is unique to the mice with RXRa ablation [25]. The actions of the VDR on activation of gene transcription involve heterodimer formation with RXR, thus targeted ablation of either the VDR or of RXR could prevent regulation of critical target genes by the VDR and result in the cutaneous phenotype observed. The more extensive abnormalities observed with ablation of RXRa could reflect impaired signaling by other nuclear receptors whose function is also dependent upon heterodimerization with RXR (discussed in Chapter 8). Mutation of the Hairless gene results in alopecia in both humans and mice [26,27]. This gene was first characterized as a thyroid hormone receptor corepressor [28,29] but was subsequently found to interact with additional nuclear receptors, including the VDR. Hairless binds the VDR and represses VDR-mediated transactivation in both the presence and absence of ligand [30,31]. Mice lacking Hairless develop alopecia the
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second week of life, accompanied by the development of large, lipid-laden dermal cysts and severe skin wrinkling. However, Hairless mRNA expression is not impaired in VDR-null keratinocytes [6] and is increased in the hair follicles of VDR-null mice [32], suggesting that Hairless is upstream of the VDR or acts on a parallel or unique pathway in the hair follicle. Notable in this respect, in addition to its interactions with the VDR, Hairless has been shown to suppress the expression of Soggy and WISE, two inhibitors of the canonical Wnt signaling pathway, a pathway that is critical for both hair follicle morphogenesis and regulation of postnatal hair cycling [33]. Thus, it is possible that dysregulation of the canonical Wnt signaling pathway in the absence of the VDR or of Hairless is the basis for the alopecia and skin phenotype observed with mutation of either of these genes. Like the VDR, canonical Wnt signaling is critical for keratinocyte stem cell lineage commitment and selfrenewal. Numerous genes regulated by b-catenin in keratinocytes have vitamin D response elements (VDREs) in addition to Wnt response elements [34]. The binding of Wnt ligands to cell surface receptors results in disruption of the axineAPC complex, impairing the phosphorylation of b-catenin by GSK3-b. This prevents proteosomal degradation of b-catenin, resulting in its translocation into the nucleus where it regulates target gene expression with TCF/Lef complexes (see Chapter 13 on the Wnt pathway). Expression of Wnt ligands by the dermal papilla is essential for the ability of these mesodermal cells to induce anagen [35]. Wnt signaling in the keratinocyte compartment of the hair follicle also regulates hair follicle formation and regeneration. Keratinocyte-specific ablation of b-catenin impairs hair follicle morphogenesis and postnatal hair cycling, whereas constitutive activation leads to de novo hair follicle formation and hair follicle tumors [36e38]. Mice lacking Lef1 are born with few hair follicles and develop alopecia by 12 days of age, whereas expression of a dominant negative Lef1 transgene in keratinocytes leads to alopecia and dermal cysts, findings similar to those observed in the VDR-null mice [23,39e41]. Investigations into the interaction of the VDR with the canonical Wnt signaling pathway have largely focused on the liganded VDR [42e44]. In colon cells, where canonical Wnt signaling plays a role in carcinogenesis, 1,25(OH)2D inhibits this pathway [41]. The presence of liganded VDR leads to translocation of b-catenin from the nucleus to the cell membrane where it participates in adherens junction formation with E-cadherin, whose expression is also induced by 1,25(OH)2D. However, the unliganded VDR has been shown to be required for synergistic activation of a Wnt reporter by b-catenin/ Lef1 in keratinocytes [23]. Studies of the physical interactions of these proteins have shown that the VDR
and b-catenin immunoprecipitate with Lef1 in COS-7 cells in the absence of ligand [23]. The VDR also immunoprecipitates with b-catenin in keratinocytes, but only in the presence of ligand and Wnt3a [34]. However, keratinocyte-specific expression of a constitutively active bcatenin transgene cannot prevent the hair cycle defect observed in the absence of a functional VDR [23]. The Hedgehog signaling pathway, which is regulated by the canonical Wnt signaling pathway, has also been shown to be essential for hair follicle development and cycling [45]. Hair follicle development is disrupted in the absence of Sonic hedgehog, whereas overexpression of Sonic hedgehog postnatally induces anagen [46]. Another member of the Hedgehog family, Indian hedgehog, is thought to promote sebocyte differentiation when canonical Wnt signaling is attenuated. Keratinocyte-specific expression of a dominant negative Lef1 transgene that impairs canonical Wnt signaling leads to increased sebaceous glands and sebaceous tumors that overexpress Indian hedgehog and its downstream mediator Gli1 [47]. In contrast, normal or excessive b-catenin signaling promotes Sonic hedgehog/Gli2 expression. Studies of genes expressed in the hair follicles of VDR-null mice demonstrate impaired expression of several effectors of this signaling pathway, including Sonic hedgehog, its receptor Patched, Gli1, and Gli2. Topical application of Sonic hedgehog to VDR knockout mice resulted in temporary induction of hair growth, but not to the extent observed in wild-type mice [32]. Thus, while the hedgehog pathway regulates the development and maturation of the hair follicle, activation of this pathway cannot compensate for the absence of a functional VDR.
CONCLUSIONS Investigations into the role of vitamin D and its receptor in skin homeostasis have dramatically enhanced our understanding of the mechanism of action and biological effects of the VDR. It remains unclear to what extent, if any, the skin contributes to the endocrine actions of 1,25(OH)2D. However, the skin is the only organ system known to date that is capable of synthesizing all the effectors of the vitamin D signaling system. While 1,25(OH)2D and calcium are thought to play redundant roles in regulating keratinocyte proliferation and differentiation, studies into the mechanism by which 1,25(OH)2D regulates this process have demonstrated a transition in the nuclear receptor comodulator complexes that associate with the VDR during differentiation, resulting in the expression of a different subset of genes during keratinocyte differentiation. Studies into the role of the VDR in hair follicle biology have identified a unique population of keratinocyte cells in which
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the actions of this receptor are ligand-independent. Characterization of the molecular partners of the VDR required to maintain cyclic regeneration of the hair cycle is expected to identify additional unique actions of the VDR in cutaneous homeostasis.
References [1] Y.C. Li, C. Bergwitz, H. Juppner, M.B. Demay, Cloning and characterization of the vitamin D receptor from Xenopus laevis, Endocrinology 138 (1997) 2347e2353. [2] O. Dardenne, J. Prud’homme, A. Arabian, F.H. Glorieux, R. StArnaud, Targeted inactivation of the 25-hydroxyvitamin D(3)-1 (alpha)-hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D-deficiency rickets, Endocrinology 142 (2001) 3135e3141. [3] Y. Sakai, J. Kishimoto, M. Demay, Metabolic and cellular analysis of alopecia in vitamin D receptor knockout mice, J. Clin. Invest. 107 (2001) 961e966. [4] K. Skorija, M. Cox, J.M. Sisk, D.R. Dowd, P.N. MacDonald, C.C. Thompson, et al., Ligand-independent actions of the vitamin D receptor maintain hair follicle homeostasis. Mol. Endocrinol. (Baltimore, Md.) 19 (2005) 855e862. [5] D.D. Bikle, Y. Oda, Z. Xie, Calcium and 1,25(OH)2D: interacting drivers of epidermal differentiation, J. Steroid Biochem. Mol. Biol. 89-90 (2004) 355e360. [6] Y. Sakai, M.B. Demay, Evaluation of keratinocyte proliferation and differentiation in vitamin D receptor knockout mice, Endocrinology 141 (2000) 2043e2049. [7] Z. Xie, L. Komuves, Q.C. Yu, H. Elalieh, D.C. Ng, C. Leary, et al., Lack of the vitamin D receptor is associated with reduced epidermal differentiation and hair follicle growth, J. Invest. Dermatol. 118 (2002) 11e16. [8] Y. Oda, C. Sihlbom, R.J. Chalkley, L. Huang, C. Rachez, C.P. Chang, et al., Two distinct coactivators, DRIP/mediator and SRC/p160, are differentially involved in vitamin D receptor transactivation during keratinocyte differentiation. Mol. Endocrinol. (Baltimore, Md.) 17 (2003) 2329e2339. [9] Y. Oda, R.J. Chalkley, A.L. Burlingame, D.D. Bikle, The transcriptional coactivator DRIP/mediator complex is involved in vitamin D receptor function and regulates keratinocyte proliferation and differentiation, J. Invest. Dermatol. Epub. (2010). [10] Y. Oda, Y. Uchida, S. Moradian, D. Crumrine, P.M. Elias, D.D. Bikle, Vitamin D receptor and coactivators SRC2 and 3 regulate epidermis-specific sphingolipid production and permeability barrier formation, J. Invest. Dermatol. 129 (2009) 1367e1378. [11] D.D. Bikle, S. Chang, D. Crumrine, H. Elalieh, M.Q. Man, E.H. Choi, et al., 25 Hydroxyvitamin D 1 alpha-hydroxylase is required for optimal epidermal differentiation and permeability barrier homeostasis, J. Invest. Dermatol. 122 (2004) 984e992. [12] G. Weber, J.D. Heilborn, C.I. Chamorro Jimenez, A. Hammarsjo, H. Torma, M. Stahle, Vitamin D induces the antimicrobial protein hCAP18 in human skin, J. Invest. Dermatol. 124 (2005) 1080e1082. [13] P.T. Liu, S. Stenger, H. Li, L. Wenzel, B.H. Tan, S.R. Krutzik, et al., Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response, Science (New York, NY) 311 (2006) 1770e1773. [14] J. Schauber, R.A. Dorschner, A.B. Coda, A.S. Buchau, P.T. Liu, D. Kiken, et al., Injury enhances TLR2 function and antimicrobial peptide expression through a vitamin D-dependent mechanism, J. Clin. Invest. 117 (2007) 803e811.
[15] F.H. Glorieux, A. Arabian, E.E. Delvin, Pseudo-vitamin D deficiency: absence of 25-hydroxyvitamin D 1a-hydroxylase activity in human placenta decidual cells, J. Clin. Endocrinol. Metab. 80 (1995) 2255e2258. [16] Y.C. Li, A.E. Pirro, M. Amling, G. Delling, R. Baron, R. Bronson, et al., Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia, Proc. Nat. Acad. Sci. USA 94 (1997) 9831e9835. [17] P.J. Malloy, Z. Hochberg, D. Tiosano, J.W. Pike, M.R. Hughes, D. Feldman, The molecular basis of hereditary 1,25-dihydroxyvitamin D3 resistant rickets in seven related families, J. Clin. Invest. 86 (1990) 2071e2079. [18] S.J. Van Cromphaut, M. Dewerchin, J.G. Hoenderop, I. Stockmans, E. Van Herck, S. Kato, et al., Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects, Proc. Nat. Acad. Sci. USA 98 (2001) 13324e13329. [19] T. Yoshizawa, Y. Handa, Y. Uematsu, S. Takeda, K. Sekine, Y. Yoshihara, et al., Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning, Nat. Genet. 16 (1997) 391e396. [20] C. Chen, Y. Sakai, M. Demay, Targeting expression of the human vitamin D receptor to the keratinocytes of vitamin D receptor null mice prevents alopecia, Endocrinology 142 (2001) 5386e5389. [21] R.G. Erben, D.W. Soegiarto, K. Weber, U. Zeitz, M. Lieberherr, R. Gniadecki, et al., Deletion of deoxyribonucleic acid binding domain of the vitamin D receptor abrogates genomic and nongenomic functions of vitamin D, Mol. Endocrinol. (Baltimore, Md.) 16 (2002) 1524e1537. [22] Y.C. Li, M. Amling, A.E. Pirro, M. Priemel, J. Meuse, R. Baron, et al., Normalization of mineral ion homeostasis by dietary means prevents hyperparathyroidism, rickets, and osteomalacia, but not alopecia in vitamin D receptor-ablated mice, Endocrinology 139 (1998) 4391e4396. [23] L. Cianferotti, M. Cox, K. Skorija, M.B. Demay, Vitamin D receptor is essential for normal keratinocyte stem cell function, Proc. Nat. Acad. Sci. USA 104 (2007) 9428e9433. [24] H.G. Palmer, D. Martinez, G. Carmeliet, F.M. Watt, The vitamin D receptor is required for mouse hair cycle progression but not for maintenance of the epidermal stem cell compartment, J. Invest. Dermat. 128 (2008) 2113e2117. [25] M. Li, H. Chiba, X. Warot, N. Messaddeq, C. Gerard, P. Chambon, et al., RXR alpha ablation in skin keratinocytes results in alopecia and epidermal alterations, Development (Cambridge, England) 128 (2001) 675e688. [26] W. Ahmad, Faiyaz, ul, M. Haque, V. Brancolini, H.C. Tsou, et al., Alopecia universalis associated with a mutation in the human hairless gene, Science New York, NY 1998 279:720e724. [27] S.J. Mann, Hair loss and cyst formation in hairless and rhino mutant mice, Anat. Rec. 170 (1971) 485e499. [28] G.B. Potter, G.M. Beaudoin 3rd, C.L. DeRenzo, J.M. Zarach, S.H. Chen, C.C. Thompson, The hairless gene mutated in congenital hair loss disorders encodes a novel nuclear receptor corepressor, Genes Develop. 15 (2001) 2687e2701. [29] G.B. Potter, J.M. Zarach, J.M. Sisk, C.C. Thompson, The thyroid hormone-regulated corepressor hairless associates with histone deacetylases in neonatal rat brain, Mol. Endocrinol. (Baltimore, Md.) 16 (2002) 2547e2560. [30] J.C. Hsieh, J.M. Sisk, P.W. Jurutka, C.A. Haussler, S.A. Slater, M.R. Haussler, et al., Physical and functional interaction between the vitamin D receptor and hairless corepressor, two proteins required for hair cycling, J. Biol. Chem. 278 (2003) 38665e38674.
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REFERENCES
[31] Z. Xie, S. Chang, Y. Oda, D.D. Bikle, Hairless suppresses vitamin D receptor transactivation in human keratinocytes, Endocrinology 147 (2006) 314e323. [32] A Teichert, H Elalieh, D Bikle, Disruption of the hedgehog signaling pathway contributes to the hair follicle cycling deficiency in Vdr knockout mice, J. Cell Physiol. (2010). In press. [33] G.M. Beaudoin 3rd, J.M. Sisk, P.A. Coulombe, C.C. Thompson, Hairless triggers reactivation of hair growth by promoting Wnt signaling, Proc. Nat. Acad. Sci. USA 102 (2005) 14653e14658. [34] H.G. Palmer, F. Anjos-Afonso, G. Carmeliet, H. Takeda, F.M. Watt, The vitamin D receptor is a Wnt effector that controls hair follicle differentiation and specifies tumor type in adult epidermis, PloS One 3 (2008) e1483. [35] J. Kishimoto, R.E. Burgeson, B.A. Morgan, Wnt signaling maintains the hair-inducing activity of the dermal papilla, Genes Develop. 14 (2000) 1181e1185. [36] U. Gat, R. DasGupta, L. Degenstein, E. Fuchs, De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin, Cell 95 (1998) 605e614. [37] J. Huelsken, R. Vogel, B. Erdmann, G. Cotsarelis, W. Birchmeier, beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin, Cell 105 (2001) 533e545. [38] D. Van Mater, F.T. Kolligs, A.A. Dlugosz, E.R. Fearon, Transient activation of beta-catenin signaling in cutaneous keratinocytes is sufficient to trigger the active growth phase of the hair cycle in mice, Genes Develop. 17 (2003) 1219e1224. [39] R. DasGupta, E. Fuchs, Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation, Development (Cambridge, England) 126 (1999) 4557e4568.
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[40] B.J. Merrill, U. Gat, R. DasGupta, E. Fuchs, Tcf3 and Lef1 regulate lineage differentiation of multipotent stem cells in skin, Genes develop. 15 (2001) 1688e1705. [41] C. van Genderen, R.M. Okamura, I. Farinas, R.G. Quo, T.G. Parslow, L. Bruhn, et al., Development of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient mice, Genes Develop. 8 (1994) 2691e2703. [42] H.G. Palmer, J.M. Gonzalez-Sancho, J. Espada, M.T. Berciano, I. Puig, J. Baulida, et al., Vitamin D(3) promotes the differentiation of colon carcinoma cells by the induction of E-cadherin and the inhibition of beta-catenin signaling, J. Cell biol. 154 (2001) 369e387. [43] S. Shah, A. Hecht, R. Pestell, S.W. Byers, Trans-repression of beta-catenin activity by nuclear receptors, J. Biol. Chem. 278 (2003) 48137e48145. [44] S. Shah, M.N. Islam, S. Dakshanamurthy, I. Rizvi, M. Rao, R. Herrell, et al., The molecular basis of vitamin D receptor and beta-catenin crossregulation, Mol. Cell 21 (2006) 799e809. [45] A.E. Oro, M.P. Scott, Splitting hairs: dissecting roles of signaling systems in epidermal development, Cell 95 (1998) 575e578. [46] N. Sato, P.L. Leopold, R.G. Crystal, Induction of the hair growth phase in postnatal mice by localized transient expression of Sonic hedgehog [see comments], J. Clin. Invest. 104 (1999) 855e864. [47] C. Niemann, A.B. Unden, S. Lyle, Ch. C. Zouboulis, R. Toftgard, F.M. Watt, Indian hedgehog and beta-catenin signaling: role in the sebaceous lineage of normal and neoplastic mammalian epidermis, Proc. Nat. Acad. Sci. USA 100 (Suppl. 1) (2003) 11873e11880.
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C H A P T E R
31 Vitamin D and the Cardiovascular System David G. Gardner, Songcang Chen, Denis J. Glenn, Wei Ni Diabetes Center and Department of Medicine, University of California at San Francisco, San Francisco, CA 94143-0540, USA
INTRODUCTION Vitamin D3, or cholecalciferol, is a secosteroid hormone that is either consumed in the diet or generated de novo through the interaction of ultraviolet light with precursor sterols in the basal layers of the epidermis. It is metabolized through two sequential hydroxylations at position 25 (largely occurring in the liver) and position 1 (occurs largely in the kidney but present in most tissues) [1] to generate the most polar and bioactive metabolite, 1,25-dihydroxyvitamin D (1,25(OH)2D3). 1,25(OH)2D3 exerts its biological activity by binding to and activating the vitamin D receptor (VDR), a single copy gene product and member of the extended nuclear receptor gene family [2]. The liganded VDR heterodimerizes with the retinoid X receptor (RXR), or occasionally the retinoic acid receptor (RAR), to form the core of the active transcription factor complex that associates with regulatory elements (REs) in or around target genes, thereby controlling transcriptional activity of those genes. The prototypical RE for the VDReRXR heterodimer is the DR3 sequence (AGGTCAnnnAGGTCA where “n” denotes any spacer nucleotide) (see Chapters 7e12). For many years it was assumed that the targets for 1,25(OH)2D3 were confined to the intestinal mucosa, bone and, to a lesser extent, kidney. More recently, it has become apparent that 1,25(OH)2D3 targets extend across multiple different organ systems, modulating functions ranging from epidermal development to immune function (see other chapters in this volume). Interestingly, one, until now, unanticipated target of 1,25(OH)2D3 appears to be the cardiovascular system. The VDR and synthetic machinery for production of 1,25(OH)2D3 are present throughout the heart and vasculature. This chapter will review (1) data identifying and
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10031-9
characterizing the 1,25(OH)2D3/VDR signaling system in the cardiovascular system, (2) the epidemiological evidence linking vitamin D deficiency to disorders affecting cardiovascular function, and (3) the animal and human interventional studies suggesting beneficial or palliative effects of vitamin D, or its metabolites, on experimental or clinical disorders affecting the cardiovascular system.
VITAMIN D AND THE VASCULATURE Identification of Components of Vitamin D System in Vascular Cells VDR VDR mediates most if not all biological functions of 1,25(OH)2D3 in target tissues. Historically VDR targets were thought to be concentrated in the small bowel and bone, where the calcitropic properties of the liganded receptor support normal skeletal mineralization. However, recent analyses indicate that vitamin D affects the expression of as much as 3% of the transcribed genome in target cells [1] (both direct and indirect targets), suggesting that the effects of this hormone extend well beyond the mineralization of bone. A combination of cell culture studies, clinical investigations, and studies leveraging the power of mouse genetics have demonstrated that the liganded VDR plays an important role in the maintenance of cardiovascular, renal, metabolic, immune, as well as skeletal function [3,4]. VDR is known to be present in endothelial cells. [3H]1,25(OH)2D3 bound specifically to a single class of macromolecules in nuclear extracts purified from bovine aortic endothelial cells (BAEC) [5]. The sedimentation
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velocity for this macromolecule was 3.2e3.5S, consistent with the sedimentation velocity of VDR reported previously [6]. [3H]-1,25(OH)2D3-labeled protein in these nuclear extracts was shown to associate with DNA cellulose and elute at 0.22 M KCl, similar to other nuclear hormone receptors [5]. More recently, the presence of VDR mRNA and protein has been demonstrated in cultured human coronary arterial endothelial cells by RT-PCR and Western blot analysis respectively [7]. Immunohistochemical staining carried out by two independent laboratories identified VDR in venular and capillary endothelial cells of human skin biopsies [5] and in endothelial cells lining the luminal surface of rat aorta [8], indicating a broad expression of VDR in endothelial cells from both small and large caliber blood vessels. Similarly, in vitro binding assays identified VDR in nuclear extracts from rabbit aorta [9] and cultured rat vascular smooth muscle cells (VSMC) [10]. Several laboratories have investigated the regulation of VDR gene expression and function in both endothelial and VSMC. Compared with confluent, density-arrested cells, rapidly proliferating BAEC express more VDR. In confluent BAEC, activation of protein kinase C increased VDR expression at both the transcriptional and translational level. These effects were prevented by sphingosine (PKC inhibitor), actinomycin D (transcription inhibitor), and cycloheximide (protein synthesis inhibitor) [5]. VDR expression is up-regulated by its ligand 1,25(OH)2D3 in cultured human umbilical vein endothelial cells (HUVEC) [11] and VSMC [12]. Changes in VDR expression have also been reported in animal models of disease. Endothelial staining for VDR was significantly lower in the aortic intima and intrarenal muscular arteries of 5/6 nephrectomized rats compared with sham-operated rats [13]. The suppressed endothelial expression of VDR was rescued by 1,25(OH)2D3 treatment. Interestingly, in this study there was no difference in VDR expression observed in aortic media between surgical groups or between 1,25(OH)2D3 treatment groups, suggesting differential regulation of VDR expression in endothelial cells vs. smooth muscle cells in the vascular wall [13]. However, an independent study provided support for vitamin-D-induced upregulation of VDR expression in VSMC. New Zealand rabbits were treated with excess vitamin D (10 000 IU, twice a week) for 1 month. The expression of VDR and 45 Ca uptake (an index of VDR activity) were enhanced in VSMC from vitamin-D-treated rabbits compared with the vehicle-treated group [14]. 1-a Hydroxylase (OHase) Using an antibody specific for the 1-a (OH)ase, Zehnder et al. localized this enzyme to endothelial cells from human renal artery and postcapillary venules from lymphoid tissue [15]. In situ hybridization analyses of
normal human renal tissue sections demonstrated the presence of 1-a (OH)ase mRNA in endothelial cells of both small and large caliber arteries and veins but not in other parts of the vessel wall [15]. In cultured HUVEC, expression of 1-a (OH)ase mRNA and protein has been confirmed by reverse transcription PCR and Western blot analysis respectively. 1-a (OH)ase enzymatic activity was documented based on conversion of radiolabeled substrate, [3H]-25(OH)D3 to [3H]-1,25 (OH)2D3 in HUVEC. A similar observation in BAEC demonstrated a substrate concentration-dependent conversion of [3H]-25(OH)D3 to [3H]-1,25(OH)2D3 with half-maximal conversion occurring between 10e9 and 10e8 M substrate [5], a concentration range comparable to that seen by the enzyme in vivo. Unlike the renal 1-a (OH)ase, the expression and function of which are down-regulated by its product 1,25(OH)2D3 [16], endothelial 1-a (OH)ase is not under negative feedback control by 1,25(OH)2D3. On the other hand, inflammatory cytokines have been shown to affect the production of 1,25(OH)2D3 in endothelial cells significantly. In cultured HUVEC, lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-a up-regulated 1,25 (OH)2D3 production by five- and threefold respectively, with no detectable change in the level of 1-a (OH)ase protein expression [15]. Cell-specific differences in the regulation of the 1-a (OH)ase gene may reflect differential regulation of alternatively spliced gene products [4] or intrinsic differences in the signaling modalities available to the expressing cell. The activity of the endothelial 1-a (OH)ase is affected by cell growth rate. In BAEC 1,25 (OH)2D3 production is substrate concentration-dependent in growing cells, but undetectable in confluent, growth-inhibited endothelial cells [5]. Although the existence of 1-a (OH)ase in VSMC was not supported in an earlier study [15], one recent report demonstrated the 1-a (OH)ase mRNA in cultured human VSMC using real-time PCR. Activity of the 1-a (OH)ase was confirmed by measurement of [3H]-1,25 (OH)2D3 production from radiolabeled substrate. Expression of the 1-a (OH)ase mRNA, as well as the production of 1,25(OH)2D3, has been shown to be upregulated by parathyroid hormone (PTH) and estrogenic compounds [17] in VSMC. The presence of functional 1-a (OH)ase in vascular endothelial and smooth muscle cells suggests that blood vessel cells have the capacity to synthesize 1,25(OH)2D3 from 25(OH)D3 precursor present in plasma. Thus, locally generated 1,25(OH)2D3 may well exceed the circulating concentration of this metabolite in plasma and, inferentially, provide the major portion of the 1,25 (OH)2D3-dependent activity in the vessel wall. In addition, if inflammatory cytokines truly change 1-a (OH) ase levels in endothelial cells, it is possible that in certain pathological conditions, such as atherosclerosis or other
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inflammatory vasculitides, the activity of the vascular vitamin D system could be up-regulated. In summary, there are numerous studies that document the presence of 1-a (OH)ase and VDR expression and activity in vascular endothelial and smooth muscle cells. Their presence in these cells raises the intriguing possibility of an autocrine/paracrine role for the vitamin D system in the vasculature. Understanding the role(s) which this system plays in regulating physiological and/or pathophysiological function in the vascular tree should serve to inform future use of VDR ligands (either 1,25(OH)2D3 or its less hypercalcemic analogs) in the management of cardiovascular disease.
by an eNOS inhibitor suggesting that paricalcitol affects nitric oxide production [18]. However, this result is not supported by another study using the same animal model [19], a discrepancy which could be related to different levels and duration of disease progression. In the study showing a positive effect of paricalcitol [16], animals were treated with the drug immediately following nephrectomy for a total of 6 weeks. In the negative study [17], paricalcitol treatment was started 15 weeks following surgery and lasted for another 12 weeks. These differences suggest that the VDR agonist is more effective when used early on in the disease process, well before chronic kidney disease is established.
The Function of VDR in the Vasculature: Studies in ex vivo Systems
Angiogenesis
Vascular Endothelium The vascular endothelium is composed of a monolayer of endothelial cells that line the luminal surface of blood vessels. The endothelium serves both as a barrier between circulating blood and the vascular wall and as a regulator of a variety of vascular functions (e.g., maintenance of vascular tone and control of hemostasis). Here, we will summarize the known effects of the vitamin D system in the control of endothelial physiology. Vasoactivity The endothelium has been shown to regulate blood vessel relaxation or contraction by producing and releasing endothelium-derived relaxing factor (EDRF) or endothelium-derived contracting factor (EDCF), respectively. Endothelial dysfunction is characterized by a disturbed EDRF vs. EDCF balance that typically favors higher levels of EDCF. The spontaneously hypertensive rat (SHR) displays endothelial dysfunction. Acetylcholine treatment of vessels from the SHR (vs. those from normotensive control Wistar-Kyoto (WKY) rats) caused more intense vasoconstriction that was both concentration- and endothelium-dependent. Interestingly, acute incubation of blood vessels with either 1,25(OH)2D3 or its analogs significantly reduced vascular tone by decreasing calcium influx into the endothelial cells, thereby decreasing the production of EDCF [8]. In addition to their inhibitory effect on EDCF, VDR activators have been shown to mitigate endothelial dysfunction by potentiating EDRF effects in animals with renal insufficiency [18]. Two weeks of paricalcitol (a 1,25(OH)2D3 analog) treatment dose-dependently restored acetylcholine-induced (endothelial EDRFdependent) relaxation in blood vessels from 5/6 nephrectomized rats. The improved relaxation was abolished
The inhibitory effect of 1,25(OH)2D3 on endothelial cell proliferation was noted soon after the identification of VDR in these cells. BAEC treated with 10e8 M 1,25 (OH)2D3 had significantly lower proliferation rates compared with control cells [5]. 1,25(OH)2D3 inhibited thymidine incorporation in BAEC in a dose-dependent manner, with a 70% reduction at a concentration of 10e8 M. 25(OH)D3 (10e8 M), the precursor for the active form of vitamin D, significantly inhibited thymidine incorporation with a magnitude comparable to 10e10 M 1,25(OH)2D3 [5]. Similarly, HUVEC proliferation was inhibited by both 1,25(OH)2D3 and 25(OH)D3 [15]. The effects of 25(OH)D3 in either system could be explained by the weak agonist activity of this ligand at the level of the receptor (it has reduced affinity for the VDR relative to 1,25(OH)2D3), or it could reflect the conversion of 25 (OH)D3 to the active form 1,25(OH)2D3 by the 1-a (OH)ase, which, as mentioned above, has been identified in BAEC [5,15]. It will be important to differentiate these two possibilities using a 1-a (OH)ase inhibitor or 1-a (OH)ase-deficient endothelial cells. Based on the observation above, the effect of VDR activation on blood vessel angiogenesis has been studied in different settings. In cultured BAEC [20] and retinal endothelial cells [21], experimental results from different investigators support the inhibitory role of VDR activation on endothelial cell sprouting, elongation, migration, proliferation, and capillary tube formation. In addition, 1,25(OH)2D3 and its less hypercalcemic analog, 1,25-dihydroxy-3-epi-vitamin D3, arrested HUVEC at the G0/G1 transition, induced cell apoptosis and reduced cell survival [22]. Furthermore, compared to normal endothelial cells (derived from implanted matrigel plugs), tumor-derived endothelial cells tended to be more sensitive to the antiangiogenic effects of 1,25 (OH)2D3 [23,24]. At the level of the whole animal, the effect of 1,25 (OH)2D3 on angiogenesis has been studied in two models. Tumor was induced by subcutaneous
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inoculation of MCF-7 breast carcinoma cells overexpressing vascular endothelial growth factor (VEGF) 121, plus MDA-435S breast carcinoma cells, into nude mice. Mice treated with 1,25(OH)2D3 had less vascularized tumors with smaller capillaries, although the tumor sizes were not different in the control and treatment groups [20]. On the other hand, tumor blood vessels in VDR knockout mice were enlarged with lower pericyte coverage compared to those of wild-type mice [25]. In a mouse model of oxygen-induced ischemic retinopathy, retinal neovascularization was evaluated by the whole mount collagen IV staining of retinal vasculature and the number of endothelial cell nuclei in hematoxylinand PAS-stained cross-sections. 1,25(OH)2D3 treatment did not change the expression of VEGF, which drives the growth of new blood vessels, in eyes from different groups. However, whole mount retinas from mice treated with 1,25(OH)2D3 revealed a significantly lower density of neovascular tufts. The highest dose of 1,25 (OH)2D3 in this study caused a 90% reduction in the number of endothelial cell nuclei [21]. In humans, there is an inverse correlation between serum vitamin D levels and the severity of diabetic retinopathy [26]. An association of a VDR polymorphism with diabetic retinopathy [27] has also been reported. Pathological angiogenesis contributes to the progression of many diseases, including cancer, diabetic retinopathy, psoriasis, and atherosclerosis. Vitamin D, by virtue of its antiproliferative and antiangiogenic effects, might be predicted to have efficacy in the management of these seemingly unrelated disorders. Vascular Inflammation As inflammation is triggered, cytokines are released that activate endothelial cells, which in turn release additional proinflammatory cytokines and present chemokines and adhesion molecules on their luminal surface. Several in vitro cell culture studies have shown that 1,25(OH)2D3 decreased lipopolysaccharide (LPS), a prototypical endotoxin, or advanced glycation endproduct-induced cytokine (e.g., IL-6) and chemokine (e.g., RANTES) expression and secretion from HUVEC [28,29] and from human microvascular endothelial cells (HMEC) [30,31]. A role for vitamin D in the regulation of vascular adhesion, leukocyte rolling, arrest and migration through endothelium has recently been explored [32]. E-selectin, an adhesion molecule, is expressed on inflamed endothelial cells. It interacts with its ligand on leukocytes to mediate leukocyte rolling. The reduction of TNFa-induced E-selectin expression by 1,25 (OH)2D3 treatment in cultured human coronary artery endothelial cells suggested that vitamin D may inhibit the initiation of the leukocyte adhesion cascade [7]. Intercellular adhesion molecule-1 (ICAM-1) and
vascular cell adhesion molecule-1 (VCAM-1), two proteins that play a critical role in the rolling phenomenon and mediate firm leukocyte adhesion are also regulated by 1,25(OH)2D3. Studies in cultured HUVEC showed that 1,25(OH)2D3 decreased ICAM-1 and VCAM-1 expression in unstimulated and LPS- or TNFa-stimulated cells [11,28]. These results are consistent with an earlier finding in rat pulmonary microvascular endothelial cells (PMVECs) that were stimulated with platelet activating factor (PAF). The reduction of adhesion molecule (E-selectin and ICAM-1) by 1,25 (OH)2D3 significantly decreased PAF-stimulated PMVEC-polymorphonuclear leukocyte (PMN) adherence [33]. A common downstream effector of proinflammatory cytokine signaling in endothelial cells is NFkB, which is sequestered in the cytoplasm in association with the inhibitory protein IkB while in the inactivated state. When activated, phosphorylated IkB loses inhibitory control of NFkB, leading to NFkB translocation to the nucleus and alteration of gene expression. Different assays of NFkB activation, including measurement of p65 (NFkB subunit) translocation [7], assay of p65 DNA-binding activity [28,29] and measurement of luciferase activity in an ELAM (E-selectin)-NFkB luciferase reporter system [30], have come to the same conclusion e that 1,25(OH)2D3 reduces NFkB activation in cultured proinflammatory stimulus-activated endothelial cells. This effect of 1,25(OH)2D3 could be due to an increase in the total IkB expression and/or reduced IkB phosphorylation [28,29]. Overall, vitamin D exerts multiple effects on endothelial cells to regulate blood vessel contractility, vascular angiogenesis, and production of local inflammatory cytokine and adhesion molecules. Further studies focusing on mechanisms that underlie these effects will be required if we are to bring this information to bear on the development of new therapies for cardiovascular disease.
Vascular Smooth Muscle Vasoactivity In endothelium-denuded aorta from WKY rats and SHR, phenylephrine induced comparable concentration-dependent contractions. No differences were observed in the presence or absence of 10e7 M 1,25 (OH)2D3 [8]. However, other studies have shown that incubation with 1,25(OH)2D3 in vitro increased the sensitivity of isolated mesenteric arteries from SHR, but not WKY rats, to norepinephrine [34]. Whether 1,25(OH)2D3 has a direct, acute effect on vascular smooth muscle to regulate contractility is still not clear. The effect of vitamin D on vasoactivity has also been studied in vitro using blood vessels from animals
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treated with vitamin D. In a mesenteric vascular bed perfusion system, perfusion pressure is varied inversely with arterial resistance to maintain flow at a constant rate. When the vascular bed is exposed to additives in the perfusate, an increase or decrease in the perfusion pressure is indicative of vasoconstriction or vasodilatation, respectively. The resting mesenteric vascular bed perfusion pressure is higher in the SHR compared to the WKY rats. Oral administration of cholecalciferol at a dose of 0.125 mg/kg body weight per day for 6 weeks completely normalized elevated resting mesenteric perfusion pressure in the SHR to the level seen in the WKY rats [35]. The same group subsequently demonstrated that this significant effect could be achieved with as little as 2 weeks’ treatment with cholecalciferol [36]. The restoration of resting perfusion pressure was accompanied by correction of the resting membrane potential of smooth muscle cells in SHR from a relatively higher (less negative) level to a normal level comparable to that measured in WKY rats [35,36]. Furthermore, after 6 weeks of cholecalciferol treatment, acetylcholine-induced vasorelaxation significantly improved in the mesenteric vascular bed and in isolated endothelium-intact mesenteric arterial rings from SHR [35]. One of the potential explanations for increased resistance in blood vessels from the SHR is that apamin-sensitive, Ca2þ-dependent Kþ channels are impaired, which might lead to a decreased responsiveness to hyperpolarizing factors released from the endothelium. Thus, cholecalciferol may protect blood vessels by the restoration of or synthesis of new apamin-sensitive Kþ channels. This is supported by the fact that apamin blocked acetylcholine-induced hyperpolarization in VSMC in endothelium-intact artery rings from WKY rats and cholecalciferol-treated SHR but not in those from untreated SHR [35]. The effect of vitamin D on Kþ channels is not limited to SHR. Similar findings have also been reported in 5/6-nephrectomized rats treated with paricalcitol, in this case leading to improved vasorelaxation in small mesenteric arteries [19]. In terms of the effects of vitamin D on arterial contractile properties, contradictory results have been reported. Vitamin D treatment reduced epinephrine-induced maximal contraction in aorta from SHR by recovering Kþ channels [36], a mechanism similar to that described above. 1a-OHD3 treatment significantly reduced maximal contraction induced by norepinephrine. This was not due to increased production of NO or other EDRF activity but due to 1a-OHD3-induced hypercalcemia and vascular calcification [37]. Paricalcitol reduced maximal contraction elicited by norepinephrine in mesenteric artery from rats with renal insufficiency. Similarly, this was attributed to calcium deposition in the vascular wall [19]. A negative correlation between
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maximal contraction and calcium content was also found in aorta from vitamin-D-treated rats fed with a normal or high-cholesterol diet [38]. On the other hand, some studies have shown that vitamin D treatment increases blood vessel response to vasoconstrictors. A 3-day 1,25(OH)2D3 treatment (35 ng/kg body weight) in normal Wistar rats increased both sensitivity and maximal contractile response of aorta to norepinephrine through enhanced Ca2þ mobilization, increases in myosin light chain phosphorylation and myofilament Ca2þ sensitivity [39]. A lower dose of 1,25 (OH)2D3 treatment (20 ng/kg body weight, which had no effect on serum Ca2þ) for 3e7 consecutive days increased vascular force generation in response to norepinephrine or arginine vasopressin. However, a single dose of 1,25(OH)2D3 had no effect [40]. In SHR, 1,25(OH)2D3 treatment increased the active stress response of resistance arteries to norepinephrine and arginine vasopressin, without changing sensitivity [41]. Moreover, the perfusion pressure induced by norepinephrine was significantly elevated in mesenteric vascular bed from 1,25(OH)2D3-treated, as well as nonhypercalcemic vitamin D analog (22-oxacalcitriol)treated rats compared to controls [42]. Thus, the effects of VDR activation on regulation of vascular contractility and their role in blood pressure control are complicated and, in some cases, the experimental data are conflicting. Further studies will be needed to clarify these issues. Cell Proliferation Published studies in this area have generated some complex and seemingly discrepant findings. An early study from Koh et al. showed that cultured rat aortic VSMC responded to 1,25(OH)2D3 (10e10 M) with increased proliferation rate [10]. At higher concentrations (10e6 M), even the precursor 25(OH)D3 promoted cell growth [10]. More recently, the promitogenic effect of 1,25(OH)2D3 (at a concentration of 10e8 M) was confirmed by Tukaj et al. [43], who used a procedure based on quantitative, sequential halving of stably incorporated fluorescent dye (carboxyfluorescein diacetate succinimidyl ester (CFSE)) to assess proliferative activity in neonatal rat aortic VSMC. 1,25(OH)2D3 has also been shown to regulate [3H]-thymidine incorporation in both quiescent and nonquiescent neonatal rat aortic VSMC [9]. When added simultaneously to quiescent VSMC, 1,25(OH)2D3 synergized with thrombin to promote cell growth and induce a 78-fold increase in [3H]-thymidine incorporation. In contrast, when nonquiescent cells were stimulated with thrombin, 1,25 (OH)2D3 inhibited cell growth by 50% [9]. The proproliferative effect of 1,25(OH)2D3 was also demonstrated in an in vivo study. Aortas from 5/6 nephrectomized mice, treated with 1,25(OH)2D3, were stained for Ki67
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(a marker for proliferating cells). More Ki67-positive cells were identified in the treated group compared to the control group [12]. The promitogenic effect of 1,25(OH)2D3 has been shown to be VEGF-dependent and is accompanied by shortening of the G1 phase of the cell cycle. Inhibition of VEGF activity using VEGF receptor antagonist (VGA1102) and VEGF-neutralizing antibody blunted 1,25(OH)2D3-induced adult rat aortic VSMC proliferation [12]. The interaction between 1,25(OH)2D3 and VEGF was further explored in A10 cells [44,45]. The A10 cell line was derived originally from the thoracic aorta of an embryonic rat and is commonly used to study VSMC biology in vitro [46]. 1,25(OH)2D3 alone, or in combination with TGF-b, induced VEGF release in A10 cells [45]. This effect was p38 MAPK-dependent [44,45]. Two vitamin D receptor response elements (VDREs) have been located in the promoter region of the rat VEGF gene. In transfected 293T cells, a VEGF promoter-linked reporter construct was activated following 1,25(OH)2D3 treatment [47]. This effect was lost with mutation of either VDRE. Thus, 1,25(OH)2D3 may positively regulate VSMC proliferation by directly regulating VEGF production. On the other hand, a variety of studies have demonstrated an inhibition of proliferative activity by 1,25(OH)2D3 and its analogs. 1,25(OH)2D3 inhibited [3H]-thymidine incorporation in VSMC from adult rat aorta [48] and human umbilical artery [17] in a concentration-dependent manner. In adult rat aortic VSMC, 1,25(OH)2D3 suppressed EGFinduced VSMC proliferation without altering epidermal growth factor binding to its receptor [48]. The effect of 1,25(OH)2D3 and its analog paricalcitol on cell proliferation has been compared in cultured human coronary artery smooth muscle cells [49]. Both drugs inhibited cell proliferation in a concentration-dependent manner with similar efficacy and potency. At 10e8 M, both 1,25 (OH)2D3 and paricalcitol inhibited thymidine incorporation by 46%. One recent study pursued the mechanism behind the inhibitory effect of 1,25(OH)2D3 on VSMC proliferation. In this study, 1,25(OH)2D3 and its less hypercalcemic analogs RO-25-6760 and RO-23-7553 inhibited endothelin (ET)-dependent DNA synthesis and cell proliferation. 1,25(OH)2D3 did not affect ETdependent induction of the MEK/ERK signal transduction cascade but significantly suppressed ET-induced activation of cyclin-dependent kinase 2 (CDK2), a key cell cycle kinase [50]. Therefore, evidence exists supporting 1,25(OH)2D3 as both a positive and negative modulator of VSMC proliferation. These divergent effects remain unexplained although cellular phenotype (e.g., neonatal vs. adult cells) and the presence of signaling modalities (e.g., VEGF) that may influence the direction of the growth regulatory effect are likely contributors.
Calcium Deposition As detailed in other chapters in this volume, 1,25 (OH)2D3 is well known to regulate calcium homeostasis. Its calcitropic properties have generated some concerns regarding its potential to promote vascular calcification e a property which might limit its acceptability as a therapeutic agent for the management of cardiovascular disease (Chapter 73). The 5/6 nephrectomized rat is an animal model that has been used to study uremic vascular calcification. 1,25(OH)2D3 treatment increased aortic wall thickness in both sham and 5/6 nephrectomized rats; however, more calcification and more proliferating cells were present in aortic walls of uremic rats with 1,25(OH)2D3 treatment [13]. Aortic rings from 1(OH)D2-treated but not paricalcitol-treated uremic rats showed elevated calcium levels in aorta and increased pulse wave velocity [51], an indicator of aortic stiffness. This is supported by a similar study showing that 1(OH)D2, but not paricalcitol, promoted vascular calcification and elevated 45Ca uptake ex vivo, even though paricalcitol had similar effects on serum calcium, phosphorus and calciumephosphate (Ca X P) product when compared to 1(OH)D2 [52]. The effect of 1,25(OH)2D3 on calcium deposition in VSMC is not due exclusively to its effects on calcium mobilization in VSMC. Recent studies have shown that in a VSMC and THP-1 (a monocytic cell line) coculture system, the combination of interferon gamma and 1,25 (OH)2D3 increased TNFa and oncostatin M production from the THP-1 cells. These two factors, in turn, increased alkaline phosphatase (an enzymatic marker of bone mineralization) production and activity in VSMC leading to a calcifying phenotype in VSMC [53]. In another study, 1,25(OH)2D3 or paricalcitol itself had limited effect on phosphate-induced calcification in cultured VSMC [54]. Coculture of VSMC with macrophage led to calcium deposition in VSMC [54]. Interestingly, when cocultured cells were treated with 1,25 (OH)2D3 or paricalcitol, calcification in VSMC was suppressed by osteopontin secreted from the macrophage. This protective effect of VDR agonists on vascular calcification has also been reported in human studies, especially in patients with chronic kidney diseases (CKDs) [55] (see Chapters 70 and 73). Blood Coagulation and Fibrinolysis The effect of 1,25(OH)2D3 on blood coagulation and fibrinolysis has only recently attracted attention. In human aortic VSMC [56,57] and coronary artery VSMC [58], 1,25(OH)2D3 and paricalcitol dose-dependently reduced plasminogen activator inhibitor-1 (PAI1) and thrombospondin 1 (THBS-1) mRNA and protein expression, while increasing thrombomodulin expression. In primary cultures of rat heart microvascular cells,
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The Function of VDR in the Vasculature: Observational Studies in Animal Models
diet [62]. However, a subsequent study from the same group reported that high-salt diet increased urinary 25 (OH)D3 binding activity by threefold and vitamin D3 metabolite content by approximately twofold in DSS rats compared to their low-salt-fed counterparts [63]. An inverse relationship was found between plasma 25 (OH)D3 concentration and the length of time that saltsensitive rats were fed a high-salt diet [64]. It has been hypothesized that the higher excretion of 25(OH)D3 in urine causes a low plasma 25(OH)D3 level in the DSS rats, thereby creating a state of vitamin D deficiency. Interestingly, plasma 1,25(OH)2D3 levels increased with high-salt diet in the DSS rats but not in the DSR rats [62]. Additionally, when compared to DSR rats, DSS rats had higher PTH, higher urinary calcium and proteinuria [62,65]. The hyperparathyroidism and hypercalciuria could result from a dysfunctional vitamin D system in these rats. Finally, plasma 25(OH) D3 levels were inversely correlated with blood pressure (r ¼ 0.76, P ¼ 0.047, n ¼ 7) in low-salt- and highsalt-fed DSS rats [63]. In similar fashion, an inverse relationship was found between plasma 25(OH)D3 concentrations and blood pressure across three group of DSS rats, including those on a low-salt diet, those on a high-salt diet, or those on a high-salt diet with exogenous 25(OH)D3 (r ¼ e0.99, P < 0.01) [66].
Dahl Salt-Sensitive Rats
Spontaneously Hypertensive Rats (SHR)
The Dahl salt-sensitive (DSS) rat and the Dahl saltresistant (DSR) rat, which represents the normotensive control, is one of the inbred rat models that has been widely used to study salt-sensitive hypertension. When the DSS and DSR animals are placed on a highsalt diet, DSR rats get only a minor increase in blood pressure, while DSS rats develop hypertension in 4e6 weeks. Several studies have shown that the DSS rats have lower levels of plasma 25(OH)D3. In addition, 24,25 (OH)2D3 concentrations in DSS rats were less than onethird of those found in DSR rats [62]. In that study plasma 25(OH)D3 levels were not changed significantly by low-salt (0.3% NaCl) vs. high-salt diet (2% NaCl) for 3 weeks in either group. However, in another study, feeding a high-salt (3% NaCl) diet to DSS rats for 28 days lowered plasma 25(OH)D3 levels by 36%, compared to the low-salt diet (0.3% NaCl) [63]. Compared to DSR rats, urinary 25(OH)D3 binding activity and urinary 25(OH)D3 levels were at least three times higher in DSS rats [62]. Significantly higher amounts of protein (predominantly albumin but including plasma vitamin-D-binding protein) were excreted in the urine of DSS rats compared with DSR rats [62]. In one study, high salt intake did not change urinary 25(OH)D3 or 25(OH)D3 binding activity in either salt-sensitive or salt-resistant rats compared to low-salt
The SHR is an inbred genetic model of experimental hypertension. Similar to human essential hypertension, multiple genes are involved in generating the blood pressure increase in the SHR. In contrast to DSS (lower plasma 25(OH)D3 inversely correlated with blood pressure), plasma 25(OH)D3 levels have been reported as either elevated [67] or unchanged [64,68] in the SHR when compared with age-matched WKY controls. In adult SHR (~12 weeks), the plasma concentration of 1,25(OH)2D3 was significantly lower than that in the WKY rats [67,68]. However, prehypertensive SHR (3.5 weeks) had higher serum 1,25(OH)2D3 levels compared to age-matched WKY controls [69].
1,25(OH)2D3 induced tissue plasminogen activator secretion, an effect that appeared to depend on both genomic and nongenomic effects of 1,25(OH)2D3 [59]. Direct evidence for involvement of the vitamin D system in blood coagulation and fibrinolysis comes from the vitamin D receptor knockout mouse. Platelet aggregation was enhanced in the VDR knockout compared to wild-type control mice [60]. The VDR knockout displayed an exacerbation in multiorgan thrombus formation after exogenous LPS injection. This phenotype was accompanied by a reduction of antithrombin in liver, reduced thrombomodulin in aorta, liver and kidney, and an elevation of tissue factor mRNA in liver and kidney in the VDR knockout vs. control mouse [60]. High doses of 1,25(OH)2D3 seem to reduce thrombosis in cancer patients. In a small clinical study, 250 cancer patients were randomized to receive placebo or high-dose 1,25(OH)2D3 (DN-101). Thirteen patients presented with thrombosis. Of these, 11 were in the placebo group and only two were in the 1,25(OH)2D3-treated group [61].
Deoxycorticosterone Acetate (DOCA)-Salt Rats DOCA-salt hypertensive rats represent a rodent model of mineralocorticoid excess-induced hypertension. This is a salt-dependent, low-renin model of hypertension that displays significantly higher serum 1,25 (OH)2D3 and PTH levels when compared to shamtreated rats on a normal-calcium diet [70]. Renal Mass Reduction, Salt-Induced Hypertension Model Hypertension in this model is induced by an 85% reduction in renal mass (often called 5/6 nephrectomized rats) and high-salt diet. These rats demonstrate
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lower plasma 1,25 (OH)2 D3, lower plasma 25(OH)D3, higher PTH, higher phosphate, and higher fibroblast growth factor 23 (FGF23) levels when compared to sham-operated controls [71].
The Function of VDR in the Vasculature: Interventional Studies in Animal Models Hypertension Daily injection of 1,25 (OH)2D3 (200 ng/kg) in normal 11-week Wistar rats for a period of 4 weeks raised blood pressure by 10 mmHg after 7 days of treatment. After 14 days of treatment, blood pressure gradually declined but plateaued at a level which was above that seen in vehicle-treated rats [40]. Seven days of treatment with the same dose of 1,25(OH)2D3 did not cause a further increase in blood pressure in 12-week-old SHR with established hypertension [40]. Subcutaneous infusion of 200 ng/kg/day 1,25 (OH)2D3, oxacalcitriol (OCT, a nonhypercalcemic analog of 1,25(OH)2D3) or 24,25(OH)2D3 for 14 days had no effect on blood pressure in normal Sprague-Dawley rats [42]. However, the pressor effect of norepinephrine or angiotensin II was potentiated in rats treated with 1,25(OH)2D3 or OCT but not in 24,25(OH)2D3-treated rats or calcium-chloride-infused rats. These results suggested that the enhanced pressor response to the vasoconstrictor agents was due to the direct effect of the VDR agonists on the vasculature rather than through its effect on calcium homeostasis. Although 1,25(OH)2D3 and its analogs have been reported to have beneficial effects in patients with endstage renal disease on dialysis [72], their effects in reducing blood pressure in rats with renal insufficiency are not as clearcut. 1,25(OH)2D3 treatment did not prevent the blood pressure increase in 5/6 nephrectomized rats [13]. 1,25(OH)2D3 or the calcimimetic (R-568) was given to 5/6 nephrectomized rats or sham-operated rats 4 days after surgery and continued daily for 12 weeks. 5/6 Nephrectomized rats had significantly higher systolic blood pressure (SBP) at week 12 (after surgery) compared to sham-operated rats. Neither 1,25 (OH)2D3 nor the calcimimetic reduced SBP in either sham-operated or 5/6 nephrectomized rats [13]. Several additional studies investigated whether 1,25(OH)2D3 or paricalcitol reduces blood pressure in renal-insufficiency-induced hypertension using different dosing regimens and durations of treatment. No blood pressure reductions were seen with treatment using either of these VDR agonists [18,71,73,74], although in some studies, improved heart [74] and kidney [73] function have been reported. In a study of DOCA-salt rats, DiPette et al. [70] found that a high-calcium diet reduced serum 1,25(OH)2D3
levels and blood pressure in these rats with no change in PTH levels. DSS rats develop hypertension on a high-salt diet. Exogenous administration of 25(OH)D3 through an osmotic mini pump did not prevent the elevation in blood pressure in DSS rats fed a high-salt diet with either normal or reduced levels of vitamin D [66]. The increase in plasma 25(OH)D3 was proportional to the concentration of 25(OH)D3 in the mini pump when DSS rats were fed a low-salt diet. A smaller increase was observed when these rats were fed a high-salt diet, supporting the hypothesis that the high-salt diet affects vitamin D clearance or metabolism [66]. Other studies tested the effect of cholecalciferol [63] or paricalcitol [75] on blood pressure reduction in DSS rats. Both reported negative results. However, treatment with paricalcitol resulted in cardio protection in the DSS rats with a 21% reduction in cardiomyocyte size compared to vehicle-treated controls [75]. An 8-week 6% NaCl diet induced an increase in SBP from 150 to 180 mmHg in WKY rats. This was accompanied by impaired endothelium-dependent (NO and EDHF) and endothelium-independent (NO donorinduced) relaxation in isolated mesenteric artery. Highcalcium diet (3%) prevented the blood pressure rise, with no change in total plasma calcium or body weight. It also restored the mesenteric artery relaxation response to acetylcholine (endothelium-dependent) and nitroprusside (endothelium-independent). 1a-OHD3 (1.2 mg/kg) partially reduced the blood pressure increase and elevated total plasma calcium. However, 1a-OHD3 treatment did not alter the impaired relaxation response in mesenteric artery. Blood vessels from 1aOHD3-treated rats had attenuated maximum contractions induced by norepinephrine and KCl, an effect that was postulated to be tied to vessel calcification [37]. Genetic Models Conflicting results have been reported regarding the effect(s) of vitamin D treatment on blood pressure in the SHR with individual studies reporting increased [41], reduced [35,36], or no effect [76,77]. Cholecalciferol (125 mg/kg body weight/day) for 6 weeks reduced blood pressure and reduced perfusion pressure of the mesenteric vascular bed in the SHR by releasing EDHF from endothelial cells and restoring the function of apamin and ATP-sensitive Kþ channels in the VSMC membrane [35,36]. The same dose of cholecalciferol did not alter blood pressure in normotensive WKY controls [35]. Short-term 1,25(OH)2D3 administration (50 ng/day over 3 days) increased serum 1,25 (OH)2D3 levels, ionized and total calcium levels, and phosphate levels with no reduction in blood pressure in the SHR [76]. 1,25(OH)2D3 treatment for 13 weeks protected cardiac function and prevented cardiac
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hypertrophy, but failed to reduce blood pressure in the SHRSP (heart failure-prone SHR) fed a high-salt diet [77]. 1,25(OH)2D3 may expedite hypertension progression in the SHR. In one study, blood pressure was tracked during 9 weeks of 1,25(OH)2D3 treatment in the SHR, beginning at 6 weeks of age. SHR received three doses of 1,25(OH)2D3 (200, 300, or 400 ng/kg body weight, i.p. daily) or vehicle. At week 5 of treatment, SHR in the 200 and 400 ng groups had higher blood pressure levels compared with other groups. After 6 weeks of treatment, blood pressure increases were similar in all groups including vehicle-treated SHR [41]. A mouse model of 1,25(OH)2D3 deficiency was developed through targeted ablation of the 1-a (OH)ase gene. Not surprisingly, with no active form of vitamin D these mice developed hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and rickets. Interestingly, these mice also had significantly higher SBP and cardiac abnormalities, suggesting an important role for the vitamin D system in maintaining cardiovascular function. Treatment of 1-a (OH)ase knockout mice with 1,25(OH)2D3 normalized the elevation in blood pressure by repressing the renin-angiotensin system, which is up-regulated in these mice at baseline [78]. Consistent with the findings in 1-a (OH)ase knockout mice, VDR knockout mice also had higher blood pressure, abnormal heart morphology and function, and increased renin gene expression [15]. However, a second, independent study reported that blood pressures were similar between the VDR knockout mice and wild-type mice at 3 and 6 months of age. The VDR knockout mice, in fact, had lower blood pressures relative to the wild-type controls at 9 months of age [79]. The reason for the significant discrepancy in blood pressure measurements between these two studies is not fully understood; however, the cardiac phenotype in these two studies is similar. Arteriosclerosis and Vascular Calcification Experimental injury in the rat or mouse artery (usually carotid artery) is typically generated using a balloon catheter to completely remove the intimal endothelium and create a mural injury through intraluminal distension. The operated vessel undergoes a reproducible remodeling response characterized by VSMC mitogenesis and migration (through phenotypic switching), VSMC apoptosis, partial vascular endothelial cell regeneration, enhanced matrix synthesis, and establishment of an invasive neointima [80]. Oral 1,25(OH)2D3 supplementation (0.25 mg/kg per day) for 4 weeks after balloon-induced injury exacerbated intimal hyperplasia compared with controls. Although 1,25(OH)2D3 has been shown to cause media degeneration in the vessel wall, no effect on vessel media was
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observed in either the sham-operated or injured group treated with 1,25(OH)2D3 in this study [81]. Vitamin D Excess and Arteriosclerosis Excess vitamin D intake induced arteriosclerosis in rat aorta e a finding that resembles Monckeberg’s medial calcific sclerosis in aged individuals or patients with diabetes [82]. The major characteristic of this condition is calcification and degeneration of the media in peripheral, medium-sized arteries. Studies have investigated the vascular response to vasoactive agents in aortas from animals with vitaminD-induced arteriosclerosis maintained on a normal vs. high-cholesterol diet [38]. A marked accumulation of calcium was found in the ergocalciferol (VD2) and VD2 plus cholesterol feeding groups, with higher calcium content in aortas from animals fed VD2 plus cholesterol. VD2 treatment potentiated the aortic cholesterol accumulation and lipid deposition [38,83]. There was increased sensitivity and reduced maximal contraction to vasoconstrictors (norepinephrine and 5-hydroxytryptamine) in aortas from VD2-induced arteriosclerotic rats [38]. Cholesterol feeding alone did not impair endothelial function. Acetylcholine-induced relaxation was attenuated in norepinephrine-precontracted aortas in both the VD2 and VD2 plus cholesterol feeding groups when compared to the control group. Acetylcholineinduced relaxation negatively correlated with calcium deposition in aorta [83]. In another study, VD2 (2000 IU/day) was given for 45 days following 45 days of high-cholesterol diet in sand rats [84]. The VD2 treatment amplified the lesions generated by the high-cholesterol diet. Pathology showed degeneration of the elastic lamina, smooth muscle cell proliferation, and lipidladen calcific plaques in the aorta [84]. High doses of vitamin D combined with nicotine (VDN) treatment of laboratory animals causes calcification of arteries and has been proposed as an animal model of arterial calcification associated with agerelated vascular pathology in humans [85]. VDN treatment induced a 15-fold increase in calcium content in the aortic wall, a reduction in desmosine and isodesmosine, two cross-linking amino acids of elastin, but caused no changes in internal diameter and media thickness of blood vessels. VDN-treated rats had reduced endothelium-dependent (carbachol-induced) relaxation of norepinephrine precontracted arteries [86]. In addition, these rats suffered from increased aortic stiffness and reduced diastolic pressure [85]. Allograft Arteriosclerosis Accelerated arteriosclerosis in heart transplant patients during chronic rejection has become the principal cause of allograft dysfunction and death [87]. This process is viewed primarily as an immune-mediated
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disease targeting the arterial intimal surface [88]. It features adventitial inflammation, reduction in smooth muscle cells in the media, and intimal thickening. In a rat model of aortic allograft, a vitamin D analog (MC1288) alone reduced the number of IL-2 receptorexpressing cells in aortic cross-section. MC1288 and cyclosporine, when used alone or in combination, decreased adventitial inflammation linked to acute rejection, reduced intimal thickening, and mitigated proliferation of the adventitial lymphoid cells. In this case the combination of both drugs was most effective. These effects of VDR agonists are probably due to its effect on immunomodulation rather than on vascular smooth muscle or endothelial cells [89]. Studies in Genetic Model for Arteriosclerosis and Vascular Calcification The low-density lipoprotein receptor gene knockout (LDLRe/e), high-fat-fed CKD (5/6 nephrectomy) model was generated by renal ablation in the atherosclerotic LDLRe/e mouse fed a high-fat Western diet (40% of calories from fat). Low-dose 1,25(OH)2D3 (20 ng/kg) or paricalcitol (up to 100 ng/kg) protected blood vessels from calcification in this model. At these doses, the VDR agonists showed protective effects, including the correction of secondary hyperparathyroidism; reduction in calcium content in the aorta and decreased osteoblastic gene expression in the aorta. These effects were accompanied by increased bone volume, increased osteoblast surfaces, increased osteoid volume, and reduced osteoclast surfaces. Whether these effects are caused by direct actions of the VDR agonists on aorta or result from indirect effects involving changes in bone or plasma calcium levels, or other, as yet undefined, systemic changes, remains to be determined. Consistent with studies in other animal models, high doses of VDR agonists led to vascular calcification in this study [90]. In summary, the liganded VDR seems to have vascular protective effects at least under certain conditions. However, higher doses of these ligands can cause toxicity, including deposition of calcium in the vascular wall.
this increase is mirrored in hypertrophied hearts in vivo [96]. Since it appears that the liganded VDR possesses antihypertrophic activity in the heart (see below), these findings suggest that this hormone-ligand system may operate in a negative feedback loop that regulates the magnitude and duration of the hypertrophic response. Additional studies have shown that ventricular tissue expresses the 1a(OH)ase implying that it has the capacity to produce 1,25(OH)2D3, the cognate ligand of the VDR [96], from circulating 25(OH)D3. Interestingly, it also expresses the 24 hydroxylase enzyme which is responsible for inactivating VDR ligands [96]. 1,25(OH)2D3 has been shown to up-regulate VDR expression in kidney cells [97,98]. To examine this in rat heart cells, we isolated neonatal rat cardiac myocytes and fibroblasts and prepared total RNA for real-time PCR analysis of VDR gene transcripts. VDR expression in the fibroblasts was several-fold higher than that in the myocytes. Interestingly, pretreatment with 1,25 (OH)2D3 increased VDR gene expression in cardiac myocytes but had no effect in cardiac fibroblasts (Fig. 31.1), raising the possibility that 1,25(OH)2D3 pretreatment may amplify liganded VDR signaling selectively in the cardiac myocytes.
Role of the VDR in Cardiac Development The VDR has been linked to cardiac atrial development [97,98]. Although the exact molecular mechanisms that control cardiomyocyte differentiation into either an atrial or ventricular phenotype remain undefined, the slow myosin heavy chain 3 gene (MyHC3), an atrial chamber-specific gene, has been used as a marker to investigate atrial and ventricular lineage specification
6
VDR mRNA expression Control VD3
(N=6) Fold Change
550
4
*
2
VITAMIN D AND THE HEART 0
Expression of VDR and 1a-Hydroxylase in Heart Studies have demonstrated the presence of VDR in the heart [91e96] and several of these have localized VDR specifically to the cardiac myocyte [91,96] or fibroblast [96]. Importantly, the expression of the VDR in cardiac myocytes and cardiac fibroblasts is up-regulated following exposure to hypertrophic stimuli in vitro and
Cardiac Myocyte
Cardiac Fibroblast
1,25(OH)2D3 (VD3) increases VDR gene expression in cardiac myocytes, but not fibroblasts. Neonatal rat cardiac myocytes and fibroblasts were placed in primary culture, then treated with 10e8 M 1,25(OH)2D3 or vehicle for 48 h. At that point cells were harvested, RNA was prepared and analyzed for VDR mRNA levels by real-time PCR. All mRNA measurements were normalized to glyceraldehyde dehydrogenase mRNA levels in the individual samples. * P < 0.05 relative to vehicle control. N ¼ 6/group.
FIGURE 31.1
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[97,99e101]. Slow MyHC3 is initially expressed throughout the early tubular heart. Atrial chamberrestricted expression is subsequently established by down-regulation of ventricular slow MyHC3 gene expression during chamber formation [97]. Wang et al. [97] reported that atrial expression of the slow MyHC3 gene is achieved through the positive effects of a GATA factor-binding element in the cardiac atria and by inhibition through a VDRE in the cardiac ventricle. Mutational analysis of the slow MyHC3 gene promoter has demonstrated that the VDRE alone is required for ventricular inhibition [97]. The VDRE binds a heterodimer of RXRa and VDR which suppresses slow MyHC3 expression within ventricular but not atrial cardiomyocytes [97,102]. Further study from the same group has documented that liganded VDR inhibits slow MyHC3 expression within ventricular cardiomyocytes through a process that involves an Iroquois family homeobox gene, Irx4. Expression of Irx4 is restricted to the ventricular myocyte throughout heart development [98]. Down-regulation of the slow MyHC3 gene by Irx4 requires the VDRE element. Interestingly, Irx4 does not bind directly to the VDRE. Instead it forms an inhibitory complex through interaction with the RXRa component of the VDR/RXRa heterodimer. Structureefunction analysis has identified the amino terminus of the Irx4 protein as being required for this inhibitory activity.
Relationship of the Liganded VDR to Hypertrophy of the Rodent Heart We have shown that the liganded VDR controls the development of hypertrophy in vitro, using the neonatal rat cardiac ventricular myocyte system. This cell culture model responds to a variety of agonists and mechanical stimuli with phenotypic changes that closely resemble those seen in the hypertrophied heart in vivo [103]. We have shown that 1,25(OH)2D3, as well as a number of less hypercalcemic analogs, act in both atrial [104] and ventricular [105] myocytes to inhibit the activation of phenotypic markers associated with hypertrophy. Endothelin-stimulated changes in fetal gene expression and promoter activity, cell size, and protein synthesis [105] are at least partially reversed by 1,25(OH)2D3 or its less hypercalcemic analogs (i.e., OCT). Similar findings have been reported by others using cultured cardiac HL-1 myocytes [91]. In the latter study, 1,25(OH)2D3 was found to decrease cell proliferation and reduce atrial natriuretic peptide (ANP) gene expression, a marker of the hypertrophy-sensitive fetal gene program. The structural requirements for VDR’s antihypertrophic activity in vitro have been characterized using human (h) ANP gene promoter activity as a surrogate marker of hypertrophy. Inhibition of this promoter requires an intact DNA-binding and ligand-binding
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domain of the VDR [102]. It also requires the capacity of the liganded VDR to heterodimerize with the RXR [106] and preservation of the activation domain of the receptor, particularly in the area surrounding the coactivator binding pocket [107]. Intriguingly, the same VDR residues that are critical for association with coactivator proteins and triggering an increase in target gene transcription, also play a role in mediating the inhibitory effect on hANP promoter activity [107]. More than 20 years ago, Simpson’s group using a vitamin-D-deficient rat model demonstrated a connection between vitamin D status and cardiovascular function [108]. They found that Sprague-Dawley rats administrated a low-vitamin-D diet for more than 2 weeks developed hypertension and significant hypocalcemia. However, the increase in blood pressure in the vitamin-D-deficient rats was transient. Although the rats were maintained on the same vitamin-D-deficient diet, by 8 weeks there was no difference in blood pressure between the vitamin-D-deficient group and control animals, and this persisted for up to 18 weeks [108]. The mechanism underlying this transient hypertension remains unknown. Using the same model, Weishaar et al. [109] found a significant increase in heart weight to body weight ratio, a marker of cardiac hypertrophy, in rats after 9 or 18 weeks of vitamin D deficiency. The cardiac hypertrophy was not accompanied by loss of soluble cardiac enzymes (e.g., creatine phosphokinase) or myocardial edema. The hypertrophic heart was not reversed by restoration of serum calcium to normal. Nor was it prevented by maintenance of serum calcium at normal levels during the period of vitamin D deficiency. Histological analysis of the ventricular sections demonstrated that myofibrils from the vitamin-D-deficient rat were smaller than those in vitamin-D-sufficient rats [109], and there was a significant increase in the amount of extracellular matrix protein [109]. Subsequent studies from the same group demonstrated that the cardiac hypertrophy is associated with myocyte hyperplasia and increased expression of the proto-oncogene c-myc [110], but the precise mechanism underlying cardiac hypertrophy in these vitamin-D-deficient rats remains unknown. Of note, Gezmish et al. recently reported that maternal vitamin D deficiency in Sprague-Dawley rats led to cardiac hypertrophy in offspring at 4 weeks of age. This was accompanied by an increase in cardiomyocyte number and size [111]. Both the VDR and the 1-a(OH)ase gene knockout mice are hypertensive with elevations in renin production in the kidneys [78,112] and in the case of the VDR knockout, the heart [113]. Both models also demonstrate elevated plasma angiotensin II levels. Increased plasma renin levels are independent of hypocalcemia and hyperparathyroidism in these mice [78,112]. Subsequent
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analyses have shown that CREB (Cyclic AMP Response Element Binding Protein), a transcription factor which binds to a cAMP response element (CRE) in the mouse renin gene promoter, is linked to the increase in renin gene transcription. Liganded VDR suppresses renin gene expression by directly interacting with CREB and preventing its association with the CRE in the mouse renin gene promoter [114]. These results suggest that the liganded VDR is a direct negative regulator of the renin gene. In addition to the elevations of blood pressure, both of these mouse models display significant cardiac hypertrophy with enlargement of individual myocytes. The VDR knockout mouse also displays elevations in plasma ANP levels and ventricular ANP transcript levels [113]. As mentioned above, renin activity is increased in both knockout mice, and treatment of these animals with angiotensin-converting enzyme inhibitors reverses hypertension and cardiac hypertrophy [78,112]. Renin expression has been shown to be increased in the hypertrophied heart itself in the VDR gene knockout mouse; however, the role of this cardiac vs. systemic renin activity in contributing to hypertrophy of the myocardium remains undefined. Subsequent studies from an independent group are only partially consistent with the results presented above. Simpson et al. reported that VDR knockout mice develop cardiac hypertrophy [79]. This is accompanied by significant hypertrophy of individual myocytes, as well as a marked increase in cardiac interstitial fibrosis. However, they found no difference in blood pressure between the wild-type and VDR knockout mice. They also found no evidence for elevated plasma renin activity, plasma angiotensin II, or plasma aldosterone levels in the VDR knockout mice, although the study included only a limited number of animals. The reasons behind these seemingly discrepant results [79,112] remain undefined. While all studies with the 1-a(OH)ase or VDR gene knockout mice consistently demonstrate cardiac hypertrophy that is seemingly independent of serum calcium and phosphorus levels, only limited data have been published dealing with cardiac function in these models. Zhou et al. [78] have shown impaired systolic function in 1-a(OH)ase gene knockout mice that normalized following 1,25(OH)2D3 administration. Paradoxically, cardiac myocytes isolated from 6-month-old VDR knockout mice have shown accelerated rates of contraction and relaxation as compared to age-matched wild type [94]. Interestingly, the same study showed that acute treatment of wild-type cardiac myocytes with vitamin D accelerates myocyte relaxation. Subsequent studies suggested that the vitamin D effect may be dependent upon an interaction between the VDR and caveolin-3 in the T-tubules and sarcolemmal membrane, operating through a rapid, nongenomic pathway [115].
Relationship of Liganded VDR to Pro-Fibrotic Activity in the Rodent Heart Myocardial extracellular matrix integrity is regulated by vitamin D through effects on the expression of matrix metalloproteinases (MMPs), as well as tissue inhibitors of metalloproteinases (TIMPs) [116]. Imbalance in the expression of MMPs and TIMPs in the myocardium is associated with the complex processes responsible for the initiation and progression of both diastolic and systolic dysfunction in the heart [117]. Our group has identified VDR expression in neonatal rat cardiac fibroblasts using real-time PCR analysis, Western blot analysis, and immunofluorescence [96]. This expression is up-regulated following exposure to the prohypertrophic/profibrotic agonist ET [96]. Interestingly, pretreatment with 1,25(OH)2D3 led to a reduction in preproendothelin gene expression (~20% decrease in endothelin mRNA levels relative to vehicle-treated controls) in the cultured cardiac fibroblasts (Fig. 31.2), implying the existence of a feedback loop in which activation of pathological hypertrophy/fibrosis, accompanied by an increase in preproendothelin gene expression, leads to an increase in VDR expression. VDR, in turn, feeds back to suppress endothelin gene expression and, inferentially, the prohypertrophic and profibrotic activity that it promotes (Fig. 31.3). Rats with vitamin D deficiency develop cardiac hypertrophy in association with expansion of the interstitial compartment [109]. VDR gene knockout mice display an increase in myocardial interstitial fibrosis [118] with increased expression of cardiac MMP-2 and MMP-9 and reduced expression of TIMP-1 and TIMP-3 implying that vitamin preproET mRNA expression 40
Fold change
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30
Control VD3 (N=5)
*
20 10 0
Cardiac Myocyte
Cardiac Fibroblast
FIGURE 31.2 1,25(OH)2D3 (VD3) inhibits preproendothelin gene expression in cardiac fibroblasts but not myocytes. Neonatal rat cardiac myocytes and fibroblasts were placed in primary culture, then treated with 10e8 M 1,25(OH)2D3 or vehicle for 48 h. Cells were harvested, RNA was prepared and preproendothelin mRNA was quantified using real-time PCR. All mRNA measurements were normalized to glyceraldehyde dehydrogenase mRNA levels in the individual samples. *P < 0.05 relative to vehicle control. N ¼ 5/group.
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FIGURE 31.3 Schematic diagram describing the effects of 1,25(OH)2D3-liganded VDR in cardiac myocytes and fibroblasts. 1,25(OH)2VD3 can be taken up from plasma or generated locally by 1a-hydroxylase present in myocytes and fibroblasts. Endothelin generated within the fibroblasts promotes myocyte hypertrophy, fibroblast proliferation, and extracellular matrix deposition. 1,25(OH)2VD3 blocks each of these events, at least in part by inhibiting ET gene expression. ETR, endothelin receptor; BP, binding protein; pro ET, proendothelin; ET, endothelin.
D plays an important role in suppressing fibroblastdependent remodeling activity in the myocardium. This is consistent with a study in humans, which showed an inverse correlation between MMP-9 levels and 25(OH)D3 concentrations [119]. Collectively, these findings suggest that the liganded VDR may play an important role in controlling remodeling activity in the ventricular myocardium, perhaps leading to preservation of ventricular function in the face of pathological stimuli like ischemia, hypertension, or myocardial inflammation.
Treatment with 1,25(OH)2D3 or its Analogs Suppresses Cardiac Hypertrophy Vitamin D deficiency in humans is associated with cardiomyopathy and congestive heart failure [120] which corrects, at least partially, with vitamin D repletion. The effect of vitamin D treatment on myocardial hypertrophy and function [75,78] has been studied in selected animal models. As noted above, mice with targeted deletion of the 25(OH)D 1-a(OH)ase which cannot produce endogenous active vitamin D, develop myocardial hypertrophy and cardiac dysfunction. The administration of 1,25(OH)2D3 not only normalizes serum calcium and phosphorus levels but also normalizes cardiac structure and function [78]. DSS rats display salt-sensitive hypertension and cardiac hypertrophy.
These rats have been shown to have low levels of plasma 25(OH)D3 [64] and they respond to treatment with pharmacological doses of a vitamin D analog (paricalcitol) with reversion of the cardiac hypertrophy [75]. SHR have demonstrated hypertension and cardiac hypertrophy with low levels of vitamin D [121]. Treatment of spontaneously hypertensiveeheart failure prone (SHR-HFP) rats fed a high-salt diet with 1,25(OH)2D3 resulted in reduced cardiomyocyte hypertrophy, left ventricular diameter, and stroke volume [77]. In uremic rats, which express low levels of the 1-a(OH)ase in kidney, treatment with paricalcitol suppressed left ventricular hypertrophy [122]. Collectively, these findings are consistent with the notion that 1,25(OH)2D3 or its less hypercalcemic analogs can reverse cardiac hypertrophy induced by vitamin D deficiency, although the mechanism underlying this antihypertrophic activity remains undefined at this point in time.
1,25(OH)2D3 and its Analogs Suppress Cardiac Fibrosis Cardiac fibrosis frequently accompanies cardiac hypertrophy, and it plays a major role in contributing to the dysfunctional state seen in advanced cardiomyopathy. 1,25(OH)2D3 has been shown to possess both antiinflammatory [30,123] and antifibrotic [124] activity in different experimental contexts. However, 1,25(OH)2D3
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and its analogs have demonstrated inconsistent results in the suppression of cardiac fibrosis. Koleganova et al. [74] have documented that treatment with subhypercalcemic doses of 1,25(OH)2D3 reduces interstitial fibrosis of the heart in subtotally nephrectomized rats. Similar results from another group have shown that paricalcitol ameliorates left ventricular hypertrophy as well as myocardial and perivascular fibrosis in uremic rats [122]. Interestingly, these findings have been associated with up-regulation of VDR gene expression, reduced myocardial proliferating cell nuclear antigen, labeling (an index of cellular proliferative activity), and reduced myocardial oxidative stress. In an acute myocarditisinduced, extensive fibrosis model, treatment of infected, susceptible mice with the vitamin D analog ZK 191784 led to decreased myocardial expression of osteopontin, metalloproteinase-3, TIMP-1, urinary plasminogen activator, and procollagen-1a [125]. These findings, in aggregate, have been associated with reduced fibrosis. In a contrasting study, Repo et al. [71] reported that paricalcitol aggravates cardiac perivascular fibrosis in rats with renal insufficiency, a model that has been assumed to be associated with low circulating 1,25(OH)2D3 levels. In another study, already alluded to above, while treatment with 1,25(OH)2D3 led to decreased cardiac hypertrophy in the SHR-HF rat, there was no significant reduction in myocardial collagen content [77].
CLINICAL EPIDEMIOLOGICAL CONSIDERATIONS OF VITAMIN D DEFICIENCY Hypertension Observational Studies Vitamin D deficiency has been associated with hypertension in several observational studies. Early studies linked living at higher latitudes, having deeply pigmented skin or reduced sun exposure to reduced vitamin D stores and increased risk of hypertension [126]. More recent observational studies have attempted to establish a more direct link between vitamin D deficiency and hypertension. A nested, case-controlled study, derived from both the Health Professional Follow Up Study and Nurse’s Health Study, demonstrated an increased risk of hypertension in those with baseline vitamin D deficiency [127]. In this study, the relative risk (RR) of hypertension was increased in those with vitamin D deficiency in both men (RR was 3.03, 95% CI: 0.94e9.76) and, to a lesser degree, in women (RR 1.42, 95% CI: 0.79e2.56). The same association was also demonstrated in a similar nested, case-controlled study derived from the Nurse’s Health Study [128], in which vitamin D deficiency at the time of enrollment
(<30 ng/ml) was associated with hypertension with an odds ratio of 1.47 (95% CI: 1.10e1.97). Of note several studies have failed to demonstrate a clear association between hypertension and reduced vitamin D stores [129,130], and a few have even suggested a direct relationship between 25(OH)D3 levels and blood pressure [131e133]. However, the majority of studies with sufficient sample size seem to support the inverse relationship between vitamin D levels and blood pressure [134]. In a recent systematic review, Pittas et al. [135] reported the results of a meta-analysis in which the RR for hypertension was found to be 1.76 (1.27e2.44) in those with lowest (vs. highest) 25(OH)D3 levels. Interestingly, several prospective studies of links between vitamin D status and risk for the development of future hypertension have not demonstrated a clear association. In a prospective study of vitamin D status and hypertension [136], 25(OH)D3 levels were inversely related to blood pressure at baseline but failed to predict hypertension over a 10-year follow-up. These results were largely confirmed in a Norwegian population [137] in which the baseline 25(OH)D3 levels did not predict the future development of hypertension. These studies would seem to suggest that vitamin D deficiency tracks with hypertension but may not be pathogenic in promoting the development of hypertension. A large prospective study of US women more than 45 years of age related dairy intake, calcium and vitamin D supplementation use to the incidence of hypertension [138]. The RR of hypertension was decreased across quintiles of low fat dairy intake, dietary calcium and dietary vitamin D, but did not change with supplemental vitamin D intake. It remains unclear why dietary but not supplemental vitamin D was associated with a decreased risk of hypertension. Interventional Studies Results of interventional studies evaluating the efficacy of vitamin D, vitamin D plus calcium supplements, or ultraviolet light exposure in treating or decreasing the incidence of hypertension have yielded mixed results. Several studies reported reduction in blood pressure while others failed to demonstrate a significant effect. In a randomized double blind trial of vitamin D (800 IU/d) and calcium (1200 mg/d) vs. calcium alone in women aged 70 or older, the vitamin D group demonstrated a significant reduction in systolic blood pressure [139]. The trial was relatively short term; however, similar effects were seen in diabetic patients with baseline 25(OH)D3 levels less than 50 ng/ml. In this patient population vitamin D supplementation was associated with a 9.3% reduction in SBP. In a study of ultraviolet B therapy, which increased 25(OH)D3 levels by 162%, systolic and diastolic blood pressure were reduced by 6 mmHg, while no change was seen in those receiving
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the control therapy with ultraviolet A light [140]. In the largest study to date, 36 282 women were enrolled in the Women’s Health Initiative and assigned to either vitamin D (400 IU/d) and calcium carbonate (1000 mg/d) or placebo [141]. In this study, vitamin D had no effect on self-reported incident hypertension or reduction in blood pressure after 7 years of follow-up. Surprisingly, supplementation appeared to increase the risk for incident hypertension among African-American participants (RR, 1.2 (95% CI: 1.0e1.4)). This result was largely unexpected given the results of smaller trials and cross-sectional studies. However, several methodological issues with this study may make interpretation difficult. Specifically, the failure of vitamin D to reduce blood pressure has been attributed to the possibility of inadequate dosing [142]. It should also be noted that this study was not designed to assess hypertension or cardiovascular events as the primary outcome. It is likely that precise definition of vitamin D’s role in the genesis of hypertension will require additional studies using higher doses of vitamin D.
Vascular Disease Alterations in vitamin D levels have been associated with vascular disease. Vascular pathology is typically classified anatomically, involving either the intimal layer (atherosclerotic disease) or the medial layer (Monckeberg’s calcification) of the vessel wall. In general, vitamin D deficiency has been linked to atherosclerotic disease, including coronary vessel disease (see below) and peripheral arterial disease (PAD), defined as obstructive disease of the vessels in the limbs. Conversely, vitamin D excess with hypercalcemia, most often in the setting of kidney disease, has been associated with medial layer calcification.
PAD Observational Studies The relationship between vitamin D status and PAD has been considered in several studies. PAD is quantified using the ratio of SBP at the ankle and brachial artery or ankle-brachial index. PAD, defined as an ankle-brachial index <0.9, was examined in 4839 participants of the National Health and Nutrition Examination Survey (NHANES) 2001e2004. After multivariable adjustment, the RR for PAD for the lowest compared to the highest 25(OH)D3 quartiles (<17.8 and 29.2 ng/ml, respectively) was 1.80 (95% CI: 1.19e2.74) [143]. Similar results were seen in a cross-sectional study of PAD and 25(OH)D3 status [144]. In that study the mean 25(OH)D3 level was 9.4 ng/ml in those with severe PAD vs. 20 ng/ml in a control group. Seventy-one per cent of patients
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with severe PAD had 25(OH)D3 levels that were <9 ng/ml. Of note, vitamin D levels were associated with subjective activity restriction suggesting that the low 25(OH)D3 levels could be associated with reduced sunlight exposure. The relationship between vitamin D status and PAD in African-Americans has received additional attention as several studies suggest that the increased risk for PAD among African-Americans may be related to vitamin D deficiency [143]. Unfortunately, randomized controlled interventional studies are lacking.
Coronary Atherosclerotic Disease Observational Studies Vitamin D status and coronary arterial disease (CAD) have been examined in both cross-sectional and prospective studies. A cross-sectional study of the NHANES III population revealed an association between vitamin D deficiency (25(OH)D3 <20 ng/ml) and CAD, defined as self-reported angina, myocardial infarction or stroke with an odds ratio of 1.20 (95% CI: 1.01e1.36) [145]. Similar results were seen in the Health Professionals Follow Up Study in which the RR for nonfatal MI or fatal cardiovascular disease was 2.42 (95% CI: 1.53e3.84) in those with vitamin D deficiency. In the Framingham Offspring Study the rate of cardiovascular events was also higher in those patients with 25(OH)D3 levels <15 ng/ml. Interestingly, the association was only seen in patients with hypertension, RR 2.13 (95% CI: 1.30e3.48). In contrast, a prospective study of postmenopausal women, which assessed dietary vitamin D intake and vitamin D supplement use, failed to demonstrate a reduction in cardiovascular disease with vitamin D supplement use [146]. Interventional Studies Randomized clinical trials designed to assess CAD as a primary outcome are not available; however, several studies have examined this relationship in secondary analyses. Overall, results fail to demonstrate a significant effect of vitamin D supplementation with or without calcium [135], but a trend towards benefit was seen in several studies. In a study of vitamin D supplementation over 4 months, a small but insignificant trend towards a reduction in cardiovascular death was observed [147]. Similar results were seen in a study designed to assess vitamin D status and fracture risk, in which a greater percentage of women receiving placebo (vs. vitamin D) had a cardiovascular event (2.0 vs. 1.3%), although this did not reach statistical significance [148]. Analysis of the results from the Women’s Health Initiative showed no significant difference in the rate of myocardial infarction, angina, or stroke
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[149] among groups with varying degrees of vitamin D deficiency. As discussed above, questions regarding the dosing regimen complicate the interpretation of this study.
Cardiomyopathy and Heart Failure Observational Studies Vitamin D deficiency is associated with heart failure and cardiomyopathy. Striking cases of heart failure have been reported in infants with rickets [150e152]. However, in this setting, profound hypocalcemia may contribute to the observed cardiac dysfunction. In cross-sectional studies, vitamin D deficiency has been linked to patients with heart failure [145,153]. In a study derived from the NHANEs III database, vitamin D deficiency, defined as 25(OH)D3 <30 ng/ml was associated with an OR of 1.7 (95% CI: 0.87e3.32) for heart failure and 3.52 (95% CI: 1.58e7.84) for cardiovascular disease and heart failure combined [154]. Similar results have been reported in a study of patients referred for coronary angiography. Vitamin D levels were negatively correlated with N-terminal pro-Btype natriuretic peptide, a marker of cardiac dysfunction and failure, and negatively correlated with NYHA classification and impaired LV function. After correction for cardiovascular risk factors, the hazard ratio for death due to heart failure was 2.84 (95% CI: 1.2e6.74) and 5.05 for sudden cardiac death (95% CI: 2.13e11.97) when vitamin-D-deficient patients with 25(OH)D3 levels <10 ng/ml were compared with replete patients with levels >30 ng/ml [155]. Interestingly, a recent report linked a functional polymorphism in the 1-a(OH)ase gene, the rate limiting step in the synthesis of active 1,25(OH)2D3, with increased risk for heart failure [156]. Interventional Studies The effect of vitamin D supplementation, in combination with other micronutrients, has been shown to improve left ventricular function [157]. It is important to note, however, that the intervention included several compounds thought to be beneficial in heart failure. Recently, a small randomized study compared the effect of vitamin D vs. placebo in patients with heart failure. During the 20-week study, vitamin D levels improved and BNP levels fell in the treatment arm. However there was no benefit in functional studies and perceived quality of life was reduced in those receiving vitamin D [158]. A similar small randomized study comparing calcium and vitamin D supplementation showed statistically significant reductions in TNFa and increases in the anti-inflammatory cytokine IL-10 in the vitamin D treatment group; however, no change in LV function was observed [159].
Vitamin D and Cardiovascular Disease in the Kidney Disease Population Alterations in vitamin D metabolism, assessed as either 25(OH)D3 or 1,25(OH)2D3 levels, are common in both CKD [160] and end-stage renal disease (ESRD) patients [161,162]. Vitamin D supplementation is recommended in kidney disease patients. Historically, supplementation was provided as a treatment for secondary hyperparathyroidism and the bone disease seen in these patients. However, more recently attention has been focused on the effect of vitamin D on survival and cardiovascular health. Observational Studies Since cardiovascular morbidity and mortality are high in the kidney disease population [163,164], the link to vitamin D deficiency has been examined in several observational cohort studies that included this patient population. The association has been supported by a historical cohort study comparing ESRD patients who received injectable 1,25(OH)2D3 with patients receiving no treatment [165]. In this study, patients who received vitamin D had improved 2-year survival with a hazard ratio of 0.8 (95% CI: 0.76e0.83) in the 1,25(OH)2D3-treated vs. untreated groups. In a separate study of incident dialysis patients, low vitamin D levels were associated with increased risk of mortality, with an OR of 1.9 (95% CI: 1.0e3.4) for those patients with 25(OH)D3 levels less than 10 ng/ml and 1.8 (95% CI: 1.2e2.9) for those with levels between 10e30 ng/ml [161]. In an observational cohort study from Shoji et al. [166] oral 1(OH)D3 (alfacalcidol) reduced risk of death from cardiovascular disease in patients on hemodialysis with a hazard ratio of 0.287 (95% CI: 0.127e0.649). Risk for death from noncardiovascular disease was not different between alfacalcidol users vs. nonusers. In a study of patients stratified by CKD stage, Mehrotra et al. demonstrated increased mortality in those patients with 25(OH)D3 levels <15 ng/ml. In this study greater than half of deaths were attributed to cardiovascular causes, but multivariate analysis failed to establish a statistically significant link between levels and cardiovascular death [167]. However, in a similar observational study in CKD patients, oral 1,25(OH)2D3 therapy was linked to improved survival with 26% reduced risk of death [168]. Patients with CKD not yet on dialysis who received 1,25(OH)2D3 therapy showed an incidence rate ratio for mortality of 0.35 (95% CI: 0.23e0.54) and for combined death and dialysis initiation of 0.46 (95% CI: 0.35e0.61) in fully adjusted models [169]. It is important to note that vitamin D supplementation can be associated with increased risk of hypercalcemia which is of particular concern in CKD and
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ESRD patients. In this setting, hypercalcemia and hyperphosphatemia (Ca X P product) may lead to increased vascular calcification [170,171]. These results have led some to conclude that vitamin D supplementation has a biphasic or U-shaped association with cardiovascular disease [172]. For this reason, there has been considerable interest in less hypercalcemic analogs of vitamin D. A cohort study comparing paricalcitol vs. 1,25 (OH)2D3 therapy [72] in patients with ESRD demonstrated an adjusted mortality rate 16% lower in the paricalcitol vs. calcitriol group, although the magnitude of this difference has been challenged by others [173]. Doxercalciferol, another ergocalciferol analog, was shown to be as effective as paricalcitol in reducing mortality rates in dialysis patients [173]. In summary, most, though not all, observational studies seem to indicate that vitamin D levels and, inferentially, 1,25(OH)2D3 are inversely correlated with cardiovascular mortality in the CKD and ESRD populations. Interventional Studies A number of interventional studies have examined the effect of vitamin D or vitamin D analogs on the biochemical profile of patients with CKD. In a systematic review and meta-analysis, Palmer et al. [174] found no clear mortality or cardiovascular benefit in randomized studies involving vitamin D or its analogs. In patients with ESRD on dialysis, interventional studies have focused on biochemical outcomes, such as serum calcium, phosphorus, and PTH levels; however, large randomized studies of vitamin D therapy looking at survival are needed to address this question in definitive fashion.
Insulin Resistance/Metabolic Syndrome/ Diabetes Mellitus Observational Studies Insulin resistance, metabolic syndrome, and type 2 diabetes mellitus are strongly associated with cardiovascular disease. Vitamin D deficiency has also been associated with type 2 diabetes. In a nested case-controlled study an association between measured 25(OH)D3 levels and diabetes mellitus incidence was demonstrated with an adjusted relative odds between the highest and lowest quartiles of 0.28 (95% CI: 0.10e0.81) in men and 1.14 (95% CI: 0.60e2.17) in women [175]. In other observational studies dietary intake of vitamin D and calcium has been shown to correlate inversely with the metabolic syndrome [176] and type 2 diabetes [177,178]. Interventional Studies In a recent randomized trial in New Zealand women, vitamin D supplementation was found to
improve insulin resistance. This study primarily involved women of South Asian descent who were randomized to receive vitamin D supplementation (4000 IU per daily) or placebo for 6 months. Insulin resistance was assessed by homeostatic model assessment (HOMA). A significant improvement in insulin sensitivity and fasting insulin levels were observed [179]. Of note, improvement was not seen before 6 months of therapy and not until vitamin D reached levels >80 nmol/L.
CONCLUSION The VDR and ligand-generating 1a(OH)ase are found throughout the cardiovascular tree and appear to be regulated by physiologically (and pathophysiologically) relevant stimuli. The liganded VDR promotes vasorelaxation through effects in the vascular endothelium. It also inhibits angiogenesis and suppresses vascular inflammation. Effects in vascular smooth muscle cells are less clearly defined. Low circulating 25(OH)D3 levels have been reported in numerous animal models of hypertension; however, treatment with a variety of VDR ligands has not demonstrated a convincing blood-pressure-lowering effect. The VDR plays an important role in maintaining the integrity of cardiac structure. Lack of VDR signaling leads to cardiac hypertrophy and fibrosis. Cardiac abnormalities induced by vitamin D deficiency can be reversed by treatment with 1,25(OH)2D3 and its less hypercalcemic analogs. 25(OH)D3 deficiency has been linked to a variety of cardiovascular (as well as noncardiovascular) diseases including hypertension, PAD, and heart failure. However, studies carried out to date have not demonstrated a clear beneficial role for vitamin D, its metabolites, or analogs in managing these disorders. Clinical trials designed to address these questions are planned or under way. The Vitamin D and Omega-3 Trial (VITAL), which plans to enroll 20 000 participants, will assess the effect of 2000 IU vitamin D and/or omega-3 fatty acid vs. placebo on the risk of cancer, heart disease, and stroke (www.vitalstudy.org). In addition, one arm of the Thiazolidinedione Intervention With Vitamin D Evaluation (TIDE) trial (clinical trial identifier #NCT00879970) will examine the effect of vitamin D supplementation vs. placebo on the risk of developing cancer or death over an ~10-year timeframe. Results of these and future studies should help to clarify the utility of vitamin D supplementation in the prevention and management of cardiovascular disease.
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Acknowledgments Supported by grants from NIH, American Heart Association, CeDAR, DAREF and the ExtenD Program of Abbott Laboratories. The authors are indebted to Mr. Christopher Law for assistance with figure preparation.
[16] [17]
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C H A P T E R
32 Vitamin D: A Neurosteroid Affecting Brain Development and Function; Implications for Neurological and Psychiatric Disorders Darryl Eyles 1, 2, Thomas Burne 1, 2, John McGrath 1, 2, 3 1
Queensland Centre for Mental Health Research, The Park Centre for Mental Health, Wacol, Q4076, Australia 2 Queensland Brain Institute, University of Queensland, St Lucia, Q4076, Australia 3 Department of Psychiatry, University of Queensland, St Lucia, Q4076, Australia
INTRODUCTION The last 10 years have been a fertile period for the investigation of vitamin D and its diverse functions in the brain [1]. For example, the distribution of the vitamin D receptor (VDR) and the enzyme associated with the synthesis of the active form of the hormone 1 alpha hydroxylase (CYP27B1) have been mapped in human brain [2]. These key components of the vitamin D system have discrete distributions within the brain, suggesting that vitamin D has paracrine and autocrine functions within this organ. In vitro studies in oligodendrocytes, astrocytes, and neurons (the major cell types in the brain) lend further weight to this proposal [3,4]. In this chapter we will provide a concise summary of the evidence showing that vitamin D affects not only the function of individual brain cells but the integrated function of the brain as a whole, i.e. behavior. The number of functions proposed for this vitamin in the brain over the past 10 years is staggering. Vitamin D can affect brain cell differentiation, neurotrophin expression, cytokine regulation, neurotransmitter synthesis, intracellular calcium signaling, antioxidant activity, and the expression of genes/proteins involved in neuronal structural and metabolism [5]. We will also present data from a number of studies that have manipulated vitamin D signaling through either dietary deficiency or by genetically ablating the receptor. Studies where the active form of the vitamin 1,25dihydroxyvitamin D3 (1,25(OH)2D3) is administered
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10032-0
will also be considered. The effects of perturbing vitamin D signaling in both the developing and adult brain will be discussed. We will also discuss possible mechanisms by which vitamin D is neuroprotective for the brain. Finally we will consider how changes in such a diverse array of cellular and molecular events could combine to affect brain function and how this may relate to certain disease states.
VITAMIN D SIGNALING IN THE BRAIN There is robust and convergent evidence showing vitamin D has a signaling role in the central nervous system (CNS). Evidence for the presence of the VDR in the brains of several experimental animals and humans has now been described. For example, this was initially shown in rats and hamsters using radiolabeled 1,25 (OH)2D3 and autoradiography [6e10]. Being a steroid, it was assumed that the brain availability of 25-hydroxyvitamin D3 (25(OH)D3) would be high. Early studies indicated that 25(OH)D3 penetration of the bloodebrain barrier is similar to retinoic acid but lower than that of the sex hormones [11]. Later when antibodies directed against the VDR became available, the immunohistochemical presence of the receptor was confirmed in the mouse, rat, and chick brains [12e15]. The VDR in the brain would appear functional in that it is able to specifically bind DNA response elements when ligated [16]. Recently, the VDR has been identified in the developing
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and adult brain of teleost fish, such as zebrafish [17]. Zebrafish have utility as the species for exploring developmental neurobiology given their transparency, ease of genetic manipulation, and high fecundity [18]. Early evidence that the VDR was expressed in human brain came from studies by Sutherland et al. [19] via in situ hybridization. Using radiolabeled cDNA probes, these researchers showed that VDR mRNA is expressed in the brains of patients with Alzheimer or Huntington disease. The VDR was also shown to be expressed in a human neuroblastoma cell line [20]. However the most convincing evidence that the VDR was present in adult human brain came from immunohistochemical studies [2].
Vitamin D Metabolites in the Brain The three major metabolites indicative of vitamin D turnover, 25(OH)D3, 1,25(OH)2D3, and 24,25(OH)2D3, have all been identified in the cerebrospinal fluid of humans [21]. Vitamin D metabolites are known to cross the bloodebrain barrier [11,22] in a similar fashion to other steroid hormones and small ligands that bind to nuclear receptors (e.g., vitamin A) [11]. However, the cytochrome P450 enzymes responsible for the formation of 1,25(OH)2D3 and 24,25(OH)2D3 (CYP27B1 and CYP24A1, respectively) are also present in the brain (see below). Therefore, it is plausible that the presence of metabolites in CSF may be the product of local synthesis in the CNS. The actual presence and regional distribution of these vitamin D species in the brain has not been described. An early study indicated that there were compounds that shared some chromatographic similarity with 25(OH)D3 and 1,25(OH)2D3 in chick brains when animals were dosed with cholecalciferol [23]. To date however no study has actually measured the concentrations of vitamin D metabolites in brain tissue. This situation possibly reflects incompatibilities with the bioassays that have been historically used to assess vitamin D metabolite levels. The new generation of chromatographic assays for vitamin D metabolites such as liquid chromatography coupled to tandem mass spectrometry (see Chapter 47) may help overcome these technical barriers.
Local Formation and Catabolism in Brain The enzyme which catalyzes conversion of 25(OH)D3 to 1,25(OH)2D3, CYP27B1, is classically expressed in the kidney but immunohistochemical techniques have also been used to describe the distribution of this enzyme in nonrenal human tissues [24]. In this study only two regions of the brain were examined but nevertheless strong expression of CYP27B1 was observed in the cerebellar Purkinje cells as well as within neuronal cells of
the cerebral cortex, suggesting local production of the active vitamin within human brain may be possible. CYP27B1 has also been detected in fetal human brain [25], as well as within glial cells in culture, which were also shown to be capable of the final hydroxylation of 25(OH)D3 to 1,25(OH)2D3 [26]. The distribution of CYP27B1 in the human brain was more fully described in 2005 [2]. In this study, confocal microscopy identified the enzyme in the cytosol of both neurons and glia. As with the VDR, the pattern of expression of this enzyme had regional and subregional specificity. The regions that stained the strongest were the supraoptic and paraventricular nuclei within the hypothalamus and the substantia nigra. The rich presence of both the VDR and CYP27B1 in the hypothalamus at least is consistent with that shown in rat [12]. One of the key enzymes involved in the catabolism of 1,25(OH)2D3 has also been identified in the brain cells. Hydroxylation of 25(OH)D3 at position 24 by the enzyme CYP24A1 (see Chapter 4) represents a major pathway for elimination of the active hormone. Although the activity of CYP24A1 has not been studied directly in brain, it has been investigated in both C6 glioma and rat primary glial cells in culture. The expression of CYP24A1 mRNA was not detected de novo in either cell system but CYP24A1 mRNA was induced in a dose-dependent manner upon the addition of 1,25 (OH)2D3 to the culture medium [27]. Therefore, not only can the active form of vitamin D be formed locally in the brain, it may also autoregulate its own elimination from cells within the brain.
Distribution of VDR in the Brain The VDR is broadly distributed throughout the adult mammalian brain. The regional organization of VDR is remarkably constant between rodents and humans. For example, VDR expression in both rat and human cerebellum is restricted to the granule cells and is completely absent from the Purkinje cells [2,13]. In the human hippocampus VDR immunoreactivity was strongest in CA1 and CA2 pyramidal cells with a marked reduction within CA3 [2], a finding previously reported in rats by two separate groups [13,15]. Within both the rat and human hypothalamus the most densely labeled nuclei were the supraoptic and paraventricular nuclei [2,12]. VDR immunoreactivity was completely absent in the large, presumably cholinergic neurons from the nucleus basalis in both rat and human [28], although VDR is present in the same cells of the nucleus basalis in hamster [9].
VDR and Non-neuronal Cells The VDR would appear to be present in most neurons and a subset of glia (the non-neuronal cells in the brain).
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Although human primary glioblastoma cells contain a large 220-kDa protein that is immunoreactive with VDR antibodies, no distinct VDR-like protein of known molecular weight could be detected by western blot in these cells [29]. In rodents, VDR immunoreactivity was shown to be present in oligodendrocytes within the white matter of the brain [30]. VDR content has also been examined in primary and secondary glial cultures. The VDR was shown to be present in glial fibrillary acidic protein (GFAP)-stained cells in primary rat hippocampal cultures indicating the VDR was present in astrocytes [16]. This was confirmed in secondary oligodendrocyte cultures and glial cell-lines [30]. The use of confocal microscopy has also established VDR nuclear staining in cells in human cortex that were colabeled for GFAP [2].
VDR in the Embryonic and Neonatal Brain The role of vitamin D as a potent differentiation agent in a variety of cell types has been extensively studied [31] (and see Chapter 84). Accumulating experimental evidence now indicates that vitamin D signaling is also involved in the orderly process of brain development (see below) [32,33]. CYP27B1 has also been detected in fetal human brain [25]. The immunohistochemical presence of VDR has also been described in the developing rodent brain. Studies in fetal rat dorsal root ganglion cells first suggested the VDR was present in developing brain [34]. Later the temporal nature of VDR expression in development was qualitatively mapped in both rat [35] and mouse brain [36]. The VDR emerges on embryonic day E12 in rats and E11.5 in mouse. Like the adult brain there is a broad distribution across a variety of brain regions. In the developing rat brain the VDR appeared to be preferentially localized in differentiating fields [35]. The suggestion that VDR signaling may be relevant to proliferation was further supported by Cui et al., who identified intense VDR immunohistochemical response in the ependymal surface of the lateral ventricles in neonatal rats [37]. This is a site that represents the richest source of cell division in the postnatal brain. The time course of VDR protein and mRNA expression in the rat brain between embryonic days 15 and 23 showed a distinct increase in expression over this critical window of development [38]. The timing of this increase in VDR expression was coincident with a decrease in cell proliferation and an increase in programmed cellular elimination. While a causal association cannot be directly established from these anatomical studies they strongly suggest that vitamin D via its receptor may either directly or indirectly mediate features of neuronal apoptosis and cell cycle.
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VITAMIN D DEFICIENCY, BRAIN DEVELOPMENT, AND FUNCTION Dietary Restriction The role of vitamin D in brain development has been directly explored in animal experiments. Our group has created a model of Developmental Vitamin D (DVD) deficiency in rodents. This is achieved via manipulating lighting and dietary conditions in female rodents prior to mating and during conception. The dam is returned to normal vitamin D status at birth [32,39]. The vitamin-D-deficient dams and DVD-deficient offspring have normal calcium and phosphate levels (i.e., neither the dams nor their offspring have the rickets-like phenotype that would result from more chronic vitamin D depletion) [40]. Offspring are vitamin-D-replete by 2 weeks of age. Consistent with the prodifferentiating and antiapoptotic properties of vitamin D, its absence during development was associated with a less-differentiated brain and proliferation was enhanced in the brain of DVDdeficient rats [32,33]. Moreover, it was shown that apoptosis in the developing brains of DVD-deficient rats followed a different trajectory compared to controls; at embryonic day 19 (E19) there was no difference in the number of apoptotic cells, however, at E21 and at birth DVD-deficient rats had fewer apoptotic cells than controls [33]. This study confirmed there were also corresponding changes in cell-cycle and apoptotic gene expression. Another way of examining cell proliferation in brain tissue is the use of neurosphere cultures [41]. When these cultures were prepared from the subventricular zone of DVD-deficient neonatal brains, the number of neurospheres was shown to be increased, further suggesting cell division is enhanced in these brains at birth [37]. This same study also revealed that the addition of 1,25(OH)2D3 decreased neurosphere number. Thus, both the addition and removal of vitamin D are capable of manipulating cellular proliferation in developing brain cells [37]. These findings in DVD-deficient embryonic brains are in accord with other in vitro studies in brain cells. When 1,25(OH)2D3 was added to primary hippocampal cells in culture it decreased the number of proliferating cells and increased neurite outgrowth in explant cultures [42]. More recently, in vitro studies using a hippocampal cell line have not only confirmed this but describe a limited time course for 1,25(OH)2D3 uptake within the nucleus. This period corresponded with decreased expression of genes related to cell proliferation, neurite formation, and the promotion of apoptosis [43]. When the anatomy of DVD-deficient embryonic brains was examined it was revealed that the newborn offspring of DVD-deficient rats had larger brains, increased volume of the lateral ventricles, and
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a smaller neocortical width when corrected for brain size [32]. An increase in brain size is consistent with increased cell proliferation and decreased cellular elimination.
The Impact of Developmental Vitamin D Deficiency on the Adult Brain The timing of the reintroduction of vitamin D appears to be important with respect to the persistence of some of these developmental changes into adulthood. The enlarged lateral ventricles seen in the DVD-deficient neonates [32] only persisted into adulthood if the introduction of a vitamin-D-replete diet was delayed until weaning. Thus, readdition of vitamin D to the diet from birth appeared to partially ameliorate the lateral ventricle changes observed in DVD-deficient rats [44]. The timing of the reintroduction of normal dietary vitamin D was also similarly important for the behavior of these adult animals (see below). Gene array and proteomics analysis have been used to explore gene expression in the whole brain and protein expression in the prefrontal cortex and hippocampus of adult DVD-deficient rats. DVD deficiency significantly altered expression of 74 genes and 36 proteins involved with such diverse functions as cytoskeleton maintenance, calcium homeostasis, synaptic plasticity and neurotransmission, oxidative phosphorylation, redox balance, protein transport, chaperoning, cell cycle control, and post-translational modifications [45,46]. A study of protein expression in the nucleus accumbens of DVD-deficient rats showed that although the degree of gene dysregulation was mild there were significant alterations in several proteins involved in either calcium binding (calbindin, calretinin, and hippocalcin), or mitochondrial function [47]. Although a number of studies have provided convincing data for enhanced proliferation in the brains of DVD-deficient rat embryos [32,33,37], one recent study of neurogenesis in DVD-deficient adults indicates that neurogenesis in adult hippocampus is decreased [48]. The VDR and CYP27B1 have a strong distribution in the dopamine-rich region of the human brain, the substantia nigra [2]. Curiously, dopamine is also able to induce VDR-mediated signaling in the absence of 1,25 (OH)2D3, suggesting a complex interaction between vitamin D and catecholamines [49]. Additionally the knowledge that other nuclear steroids such as estrogen could affect catecholamine levels in the brain led early investigators to explore the association between vitamin D and various neurotransmitter systems. Early studies reported that rat weanlings deprived of dietary vitamin D had increased catecholamine levels in cortical and hypothalamic areas [50], however these animals were also hypocalcemic. A later study also provided
some support for increased dopamine levels in striatum but again the influence of hypocalcemia could not be excluded [51]. This same study also suggested that the major inhibitory neurotransmitter in the brain, gammaaminobutyric acid (GABA), was increased in the brains of vitamin-D-deficient animals, but again this increase was abolished if calcium levels were normalized. In an effort to explore the effect of vitamin D on dopamine function in a normocalcemic environment, a recent study measured dopamine levels in normocalcemic developmentally vitamin-D-deficient neonatal forebrains and showed that although dopamine levels were normal, its metabolism was altered with increased ratios of 3,4-dihydroxyphenylacetic acid/homovanilic acid (DOPAC/HVA) (two major dopamine metabolites) [52]. This was accompanied by a reduction in catecholo-methyl transferase (COMT), the enzyme that converts DOPAC to HVA [52]. Therefore, it would appear that if calcium levels are normalized, vitamin D deficiency may not affect neurotransmitter levels in developing brains per se but may affect neurotransmitter turnover.
The Impact of Developmental Vitamin D Deficiency on Adult Behavior The earliest indication that vitamin D deficiency during development affects behavior comes from a study in which rats were deprived of vitamin D from weaning until 3 months of age [53]. In that study rats were assessed using a battery of behavioral tests examining motor performance, activity, perceptual learning, and memory functions. The study found a significant decrease in rearing in the open field and in the speed to complete each session in a test of working memory (8-arm radial maze) by the vitamin-D-deficient rats compared to controls, although musculoskeletal problems associated with hypocalcemia may have confounded the results. Our group has shown that preattentive mechanisms (prepulse inhibition of the acoustic startle response, PPI) are impaired in animals with combined prenatal and chronic postnatal depletion of vitamin D [54]. However, this result again may have been confounded by severe hypocalcemia, because in a separate study PPI was unaffected in that group of animals when calcium levels were normalized [55]. Comorbid hypocalcemia is avoided when behavior is examined in DVD-deficient animals. The behavioral phenotype of adult DVD-deficient rats is subtle and it has been established by the efforts of two collaborating laboratories using Sprague-Dawley rats in Brisbane, Australia [56] and Magdeburg, Germany [57]. Although there are several differences between laboratories, for example, maternal vitamin D depletion was obtained without calcium supplementation in the Brisbane protocol, whereas in the Magdeburg protocol
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the dams received 2 mM calcium in the drinking water, it should be noted that there were no effects on calcium levels at birth [40] or in the adult offspring [58]. Thus, DVD-deficient rats in either protocol do not have a rachitic phenotype (e.g., reduced body weight, size, hypocalcemia), thus allowing the examination of behavior without this confounding phenotype. When placed in a novel arena, DVD-deficient rats demonstrate spontaneous hyperlocomotion, with no effect on other behavioral domains, as measured on tests of anxiety or depression [39]. This novelty-induced hyperlocomotion is abolished if the animal is either briefly physically restrained (with or without injection) [55,59]. This is unlikely to be a stress-mediated mechanism because the animals have normal hypothalamic pituitary adrenal axis-mediated stress responses [60]. Locomotion in the open field was further assessed in these animals using the N-methyl-D-aspartic acid receptor (NMDA-R) antagonist, MK-801, an agent well known to induce hyperlocomotion. Adult male DVDdeficient rats had enhanced locomotor activity compared to controls [55]. The later period of gestation appeared to be most relevant for this behavior because rats experiencing a DVD deficiency during late gestation also showed this effect, whereas if the period of DVDdeficiency was restricted to early gestation this effect was not apparent [40]. DVD-deficient rats were also selectively sensitive to the locomotor-enhancing effects of amphetamine, a drug that induces presynaptic dopamine release [61]. Interestingly this effect was more pronounced in females who also were shown to have an increase in subcortical dopamine transporter affinity and density, and it is known that amphetamine induces dopamine release via this transporter [62]. A number of studies have also shown that DVD-deficient rats are selectively sensitive to postsynaptic dopamine blockade, in particular the dopamine 2 receptor blocker haloperidol (which is a widely used antipsychotic agent). The locomotorretarding effects of haloperidol appeared to be greater in DVD-deficient animals when hyperlocomotion had first been induced by MK-801 [55]. In a separate study haloperidol was shown to normalize an endogenous habituation deficit in DVD-deficient animals, whereas it resulted in habituation deficits if administered to control animals [63]. Using electrophysiological recordings from the hippocampus of freely moving rats, a subsequent study investigated long-term potentiation (LTP), which is a cellular correlate of learning and memory [64]. It was shown that DVD-deficient rats had enhanced LTP, and this was reversed by treatment with haloperidol. DVD-deficient rats also appeared to have normal PPI [55] and working memory but disrupted latent inhibition, which is a measure of attentional processing [57]. Although manipulating striatal dopamine
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release can affect all of these three behaviors this has not yet been investigated in vivo. The DVD-deficiency model has also been examined in two strains of mice (129/SvJ and C57BL/6J) [65]. Only one strain (129/SvJ) exhibited spontaneous hyperlocomotion in the open field arena, a finding in line with DVD-deficient rats. Both strains demonstrated increased frequency of head dips in a hole board arena, indicative of increased exploratory behavior [65]. This is in contrast to findings from DVD-deficient rats on the same test [63]. DVD-deficient mice were also assessed on a comprehensive screen of behavioral tests, including the elevated plus maze, forced swim test, prepulse inhibition, and social interaction test [65], however, there was no effect of maternal diet on performance in each of these four tests. Fernandes de Abreu et al. [66] trained DVD-deficient C57BL/6J mice on a hippocampaldependent memory task known as the olfactory tubing maze. A learning deficit was seen on the final day of training, with DVD-deficient mice showing a reduction in the number of correct responses when compared to controls. This was associated with a reduction in the size of the lateral ventricles of DVD-deficient mice [66]. Clearly the behavioral phenotype of the DVDdeficient rat is distinct from the mouse. Additionally the array of behaviors examined indicate a complicated mixture of pre- and postsynaptic alterations in the rat and mixed and subtle alterations in learning and memory in both species. However, based on these studies, there is no doubt that removal of this vitamin from the developing brain leads to persistent changes in brain function in the adult offspring [5].
Genetically Modifying Vitamin D Signaling An alternative approach to inducing vitamin D deficiency by dietary manipulation is genetically modifying various components of the vitamin D signaling pathway. One model has been developed in mice where production of the active hormone 1,25(OH)2D is blocked by ablating CYP27B1 [67,68]. Tissue-specific inactivation of CYP27B1 has also been demonstrated [69]. These homozygous mutants are likely to be less informative for subtle brain-related outcomes as they produce a confounding rickets-like phenotype. Interestingly the heterozygotes produce an intermediate phenotype as regards calcium-binding protein profile and effect on other enzymes responsible for vitamin D metabolism. The heterozygotes therefore may be less compromised by the hypocalcemia seen in the homozygous mutants. Unfortunately behavior in CYP27B1 heterozygous mice has not been comprehensively examined. There are also a number of transgenic and knockout mouse models examining vitamin D signaling through either complete ablation of the VDR or by expressing
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the human VDR in a mouse model [70]. Four different VDR knockout mouse lines have been generated by deletion of either exons 1e2 (Leuven strain) [71], exon 2 (Tokyo strain) [72], exons 3e5 (Boston strain) [73], or the first zinc finger of the DNA-binding domain of the VDR gene (Munich strain) [36]. In some strains the hormonebinding domain is intact [36], whereas others have no ligand-binding capacity [73]. However almost all behavioral studies have been performed on one strain of mice (the “Tokyo” strain [74]). Although these mice express a truncated form of VDR that still binds 1,25(OH)2D3, this binding is insufficient to activate gene transcription and the overall physiological phenotype of these mice is indistinguishable from the three other strains [75]. The two main groups that have examined behavior in this strain employ different calcium supplementation regimens. Kalueff’s group use the recognized so-called rescue diet containing 2% calcium, 1.25% phosphate, and 20% lactose. The group of Burne et al. supplement the drinking water with 2 mM calcium. However neither supplementation regimen fully normalizes serum calcium in homozygous mutants [76,77]. Whilst VDRnull mutant mice are born phenotypically normal they show growth retardation after weaning. Apart from hypocalcemia the phenotype of adult VDR knockout mice is characterized by alopecia, reduced body size and weight, impaired motor coordination, and impaired PPI [77]. These mice are able to swim but show an inability to float [76,78], as well as having post-exerciseinduced fatigue [78]. VDR knockout mice also show impairments on a range of behavioral domains, including anxiety [79], neophobia [80], altered nest building [81], as well as altered vestibular function [82], progressive hearing loss [83], and brain calcification [84]. To date there are no published studies using conditional or brain-specific VDR mutant mice to test the effects of transient or regional receptor disruption on brain development. Such studies would appear necessary because VDR knockout mice have a number of abnormalities outside the CNS which may confound interpretation of behavioral data, including hypertension and increased fluid intake [85], cardiac hypertrophy [86], altered heart function [87], impaired energy metabolism [88], and musculoskeletal changes [89]. Therefore although the VDR-null mouse may be useful in understanding certain limited aspects of brain function and a diverse array of physiological effects requiring genomic vitamin D activity, its application in understanding higher cognitive domains and more specifically how this may relate to serious psychiatric conditions would appear far more limited. For instance the fact that there is no association between VDR mutations and psychosis in a large Turkish pedigree [90] confirms that the global VDR knockout mouse may not be an appropriate model for psychosis in humans. The
VDR knockout mouse does however share some features with rats that have combined prenatal and postnatal vitamin D deficiency (e.g., reduced body weight, impaired PPI, and hypocalcemia) [54]. By contrast, as previously discussed transient prenatal vitamin D deficiency in mice produces no gross physiological abnormalities and a far more subtle behavioral phenotype [65,66]. Thus, until a way of targeting brain-specific vitamin D signaling can be developed the transient gestational DVD-deficient rodent would appear to be far more informative for modeling brain disorders with a developmental basis.
VITAMIN D AND NEUROLOGICAL DISORDERS Vitamin D and Models of Parkinson Disease In studies of Parkinson disease it is common to model the selective loss of substantia nigra neurons (a major dopaminergic nucleus in the brain) by either local addition of a dopaminergic toxin such as 6-hydroxy dopamine, or the local addition of an agent that will induce oxidative stress, i.e. iron. In such models, the addition of 1,25(OH)2D3 is associated with amelioration of toxicity by elevating the production of the ratelimiting synthetic enzyme for dopamine, tyrosine hydroxylase (TH) [91e93]. 1,25(OH)2D3 has also been shown to restore dopamine to control levels if given prior to other dopamine-depleting agents such as methamphetamine [94]. Indeed earlier studies have described how 1,25(OH)2D3 can increase TH expression in vitro [95]. However, 1,25(OH)2D3 does not appear to elevate TH in the brain under nonpathological conditions [91]. An alternate neuroprotective mechanism may be via the well-described actions of this vitamin on glial derived neurotrophic factor (GDNF) (see below). GDNF activates TH by phosphorylating this enzyme in dopamine-rich brain regions [96]. Another recent study has shown that in addition to iron, zinc can induce oxidative damage in the substantia nigra. Local delivery of zinc to the substantia nigra induces lipid peroxidation, apoptotic elimination of dopamine neurons, and reduces striatal dopamine content. If 1,25(OH)2D3 is systemically administered prior to exposure to this toxin, zinc-induced increases in lipid peroxidation and reductions in striatal dopamine are attenuated [97]. Therefore, taken together it would appear that the active metabolite of vitamin D is neuroprotective for dopamine neurons. Indeed several investigators have now linked either vitamin D insufficiency [98,99] or abnormalities in vitamin D signaling [100], with increased risk of Parkinson disease.
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Vitamin D and Models of Inflammation in the Brain There is now widespread agreement that vitamin D is a potent immunosuppressant (see Chapter 91). There is also strong epidemiological and experimental evidence to suggest that vitamin D may be a risk-modifying agent in multiple sclerosis (see Chapter 95). When autoimmune encephalitis (EAE) is induced as a model of acute demylination, animals exposed to foreign myeloid proteins reproduce an autoimmune and inflammatory cytokine response reflecting that seen in patients with multiple sclerosis. Since 1991 we have known that vitamin D can reduce inflammatory cytokine production and prevent this experimental phenotype in either rats or mice if given once clinical signs are evident [101e103]. The effect of vitamin D deprivation on inflammation of the brain is less clear. Studies in adult rodents show that predictably the absence of this putative protective factor enhances clinical symptoms [102]. However, somewhat paradoxically if the period of deficiency is restricted to early development (DVD deficiency) this would appear to be protective [104]. The authors of this latter study also found increased VDR expression in this model, which they suggest could contribute to this “imprinted” neuroprotection [104]. Given vitamin D’s well-described potential as an antiinflammatory agent (see Chapter 96) its use has been investigated in other models of brain inflammation. Progesterone is a well-known neuro-active steroid with anti-inflammatory potential [105]. Recent observations suggest that vitamin D might be synergistic with progesterone in treating ischemic brain injury. Accordingly when vitamin D deficiency was induced in aged rodents there was an increase in baseline levels of inflammatory cytokines in the brain [106]. When these animals were submitted to traumatic brain injury, progesterone treatment was ineffective. However, if progesterone was administered with 1,25(OH)2D3 brain inflammation was diminished. This has also been confirmed in vitro [107]. Local administration of lipopolysaccharides has also been used as a model of brain inflammation. The coadministration of 1,25(OH)2D3 with this inflammatory agent in rat hippocampus leads to a recruitment of macrophages and a reduction in nitric oxide synthesis [108]. These authors concluded that these actions were central to the reduction in cell elimination in this model.
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either direct administration of 1,25(OH)2D3 to the hippocampus or large doses of 25(OH)D3 intravenously increased the threshold for seizure activity in rats [109]. Subcutaneous treatment with 1,25(OH)2D3 was also shown to reduce the severity of pentylenetetrazole-induced convulsions in mice [110]. Prior administration of cholecalciferol in mice has also been shown to be anticonvulsant as it again increased electroconvulsive threshold in mice and enhanced the actions of the well-known anticonvulsants valproate and phenytoin [111]. Chemically induced seizures also occur more rapidly in VDR-null mutant mice [112]. In this particular study, use of the rescue diet rendered these homozygous VDR-null mutants technically normcalcemic even though mean serum calcium levels were still 16% lower than wild types. Therefore a brain-specific VDR-induced abnormality in calcium signaling in these animals can still not be ruled out [112]. In all the aforementioned studies the timeframe was too brief (5e40 minutes) to suspect these anticonvulsant actions were due to classic genomic regulation. A more plausible mechanism would appear to be a nongenomic direct regulation of calcium signaling in neurons via modulation of L-type voltage-sensitive calcium channels. Rapid effects of 1,25(OH)2D3 on L-type calcium channel activity have been observed in osteosarcoma cells [113] (see below). However, there are also longterm consequences for vitamin D’s actions in certain epilepsy models. For example, when seizures were induced in rats treated acutely with the potent muscarinic agonist pilocarpine, VDR mRNA was selectively up-regulated in the hippocampus up to 60 days post administration [114]. Clinically there are a number of case reports of severe vitamin D deficiency being associated with hypocalcemia-induced generalized seizures. In general seizures remit when the patient is treated with cholecalciferol to treat the hypocalcemia. There has also recently been a report of hypovitaminosis-D-induced hypocalcemia being related to increased seizures in infancy [115]. However, despite strong experimental evidence in animal models, the link between hypovitaminosis D in either pediatric or adult populations and increased seizure risk has not been well established. This picture is complicated somewhat by an apparent inverse relationship between antiepileptic drugs and 25(OH)D3 levels [116].
Vitamin D and Neurotransmitters
Vitamin D and Models of Epilepsy Vitamin D may have anticonvulsant activity in part due to the tight association between vitamin D and extracellular calcium levels. The early studies by Christakos and colleagues showed conclusively that
Dietary vitamin D levels have been linked with a number of serious psychiatric conditions (see below). The biological plausibility of this suggestion is enhanced by the knowledge that at least in vitro 1,25(OH)2D3 can alter cholinergic, dopaminergic, and noradrenergic
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neurotransmitter systems which are crucial to the major neuropsychiatric disorders such as Alzheimer disease, schizophrenia, and depression, respectively. Not surprisingly, early studies into the effects of 1,25 (OH)2D3 on brain function examined the hypothalamus and pituitary as not only were VDRs present in these nuclei but vitamin D was expected to behave as a “neurohormone” regulating homeostatic events. It was reported that 1,25(OH)2D3 increased choline acetyltransferase if given peripherally (but not if delivered centrally) [117]. Adult animals treated with high doses of 1,25(OH)2D3 long term have increased basal striatal dopamine levels and increases in evoked release of dopamine [118]. 1,25(OH)2D3 has also been administered to newborn rats and dopamine and norepinephrine measured in a variety of brain regions in these animals as adults. These authors showed that dopamine and norepinephrine were elevated mainly in the brainstem of these animals as adults [119].
POSSIBLE NEUROPROTECTIVE MECHANISMS OF VITAMIN D
Alternately vitamin D is also known to stimulate the expression of the intracellular-calcium binding proteins, parvalbumin and calbindin, which act to protect the cell by chelating intracellular calcium. However, the supportive evidence for this latter effect in brain cells is weak. One study has shown that 1,25 (OH)2D3 can up-regulate calcium-binding proteins in motor neurons [123]. Another study has shown chronic treatment with cholecalciferol had little effect on most calcium-binding proteins in CNS neurons except it induced a slight increase in parvalbumin expression [124]. Additionally the regulation of these proteins at a genomic level remains in doubt as baseline expression of these proteins in the brain is not affected by the genetic ablation of the VDR [36]. Therefore, on the balance of the available evidence it would seem that a direct down-regulation of neuronal L-type voltagesensitive calcium channels protecting the cell from the neurotoxic effects of excess calcium influx would appear to provide the most-likely functional neuroprotective mechanism [122].
Regulation of Reactive Oxygen Species
The evidence presented in the previous section indicating that vitamin D can protect the brain from certain toxins, inflammatory agents, or excess calcium release in vivo suggests that along with its many other functions vitamin D should be considered a neuroprotective neurosteroid. Although there are also likely to be a number of peripheral endocrine-like mechanisms at play including the regulation of extracellular calcium, inflammatory-mediated cytokines and stress-mediated agents such as the glucocorticoids there may also be a number of more direct vitamin-D-mediated actions in the brain. Here we review the evidence that vitamin D can regulate calcium signaling directly in the brain, modulate the production of brain-derived reactive oxygen species, stimulate the production of neurotrophic factors, and regulate at least certain aspects of stress hormone signaling in brain tissue.
Regulation of Calcium Unbuffered calcium is neurotoxic for brain cells. A role for vitamin D and calcium uptake in non-neuronal cells is of course well known. For example, physiologically relevant concentrations of 1,25(OH)2D3 are known to potently modulate calcium channel function in osteoblasts and osteosarcoma cells [113,120]. However, 1,25(OH)2D3 can also block the toxic effects of calcium influx in cultured mesencephalic neurons [121]. A highly plausible explanation for these findings comes from the discovery that 1,25(OH)2D3 can down-regulate L-type voltage-sensitive calcium channels in neurons [122].
As previously mentioned physiologically relevant concentrations of 1,25(OH)2D3 increase potent antioxidants in the brain such as glutathione [93,97]. It would appear to do this via inhibition of the synthetic enzyme for the formation of glutathione gamma-glutamyl transpeptiase [125]. At physiological concentrations 1,25(OH)2D3 also blocks the neuronal uptake of reactive oxygen species such as hydrogen peroxide. The exact mechanism for this was not clarified but it would appear that both transcriptional events and de novo protein synthesis were required for these protective effects suggesting some more traditional genomic regulation [121]. Although reactive oxygen species themselves induce nonspecific damage to lipid membranes there is some evidence to support a more direct and reversible interaction between such molecules and 1,25(OH)2D3. Nitric oxide, hydrogen peroxide, and peroxynitrite all directly inhibit nuclear vitamin D signaling by inhibiting complexation between the VDR and its coreceptor RXR in a dose-dependent manner [126]. This inhibition is largely reversible with the addition of 100 nM 1,25(OH)2D3. Thus, one potentially important mechanism of action of 1,25(OH)2D3 in the brain could be to inhibit the production of an important oxidant and signaling molecule, nitric oxide [3].
Regulation of Neurotrophic Factors Vitamin D has been shown to modulate a number of neurotrophic agents, most prominently nerve growth factor (NGF). NGF is essential for the growth and
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survival of many cells in the brain but in particular the cholinergic basal forebrain neurons which project to the hippocampus [127]. Because 1,25(OH)2D3 potently regulates NGF [128,129], vitamin D could modulate hippocampal development by increasing NGF production. For example, addition of 1,25(OH)2D3 increases neurite outgrowth in embryonic hippocampal explant cultures, an effect which was most likely due to its induction of NGF in vitro [42]. NGF induces at least some of its effects via the pan-neurotrophin receptor p75NTR [130]. The promoter region of this receptor contains a vitamin D response element, and indeed vitamin D has been shown to positively regulate the expression of the p75NTR receptor in glioma cells [131]. NGF and p75NTR are essential factors during programmed cell death in the brain [132]. Intracerebroventricular administration of 1,25(OH)2D3 is also capable of inducing NGF expression within the hippocampus of adult rats [133]. Therefore, vitamin D may directly modulate neuronal survival and differentiation during development. It is feasible that vitamin D may also indirectly influence neuronal development by altering neurotrophic factor production in non-neuronal cells. The addition of 1,25(OH)2D3 to rat primary glial cell cultures has been shown to increase the synthesis of NGF mRNA and protein, neurotrophin-3 (NT-3) mRNA and downregulate neurotrophin-4 (NT-4) mRNA [134]. The addition of 1,25(OH)2D3 can also increase the synthesis of GDNF mRNA in C6 glioma cells [135] but it does not appear to regulate GDNF production in primary glial cell cultures [136]. GDNF is integral to the development of the dopaminergic [137,138] and noradrenergic systems [139]. Therefore, vitamin D is capable of affecting cellular development in brain regions in which abnormal function is believed to be central to various psychiatric conditions.
Regulation of Glucocorticoids More speculatively, vitamin D may protect the brain from stress. The secretion of glucocorticoids is a classic endocrine response to stress and prolonged exposure to stress levels of this hormone induce neuronal atrophy and eventually cell death [140]. In general the cellular effects of 1,25(OH)2D3 and glucocorticoids are considered to be antagonistic. For instance early studies revealed cortisone altered VDR expression in rat or mouse osteoblasts [141,142] and up-regulated specific vitamin-D-related transport proteins [143]. Later it was shown that glucocorticoids could antagonize 1,25 (OH)2D3-mediated synthesis of NGF in skin fibroblast cells [144,145]. Although such cross-talk between glucocorticoids and vitamin D signaling has long been recognized (see Chapter 66) this has yet to be properly investigated in the brain. The best evidence to date
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that some interaction between these steroids may occur in brain tissue comes from a study by Obradovic and colleagues. In this study in hippocampal cells 1,25 (OH)2D3 completely antagonized the inhibitory effects of dexamethasone on cellular differentiation and directly interfered with glucocorticoid receptor function [146]. Whether any such interaction occurs in the brain, however, remains unknown.
VITAMIN D AND NEUROPSYCHIATRIC DISORDERS The links between vitamin D and multiple sclerosis have long been appreciated (see Chapter 95). Epidemiological studies provided the first clues linking vitamin D to this disorder (e.g., the pronounced latitude gradient in incidence). Over the last two decades epidemiological studies have suggested that vitamin D may also be implicated in a wider range of brain outcomes, including schizophrenia, depression, and cognitive impairment.
Schizophrenia Schizophrenia is largely considered a neurodevelopmental disorder. Many studies have shown that those born in winter and spring have a significantly increased risk of developing schizophrenia [147] and that those born at higher latitudes are also at increased risk [148], with both the incidence and prevalence of schizophrenia being significantly greater in sites from higher latitudes [149]. Furthermore, based on studies undertaken in the UK, the Netherlands, and Nordic countries, the incidence of schizophrenia is significantly higher in darkskinned migrants compared to the native-born [150]. Given that hypovitaminosis D is more common (a) during winter and spring, (b) at high latitudes, and (c) in dark-skinned individuals [151,152], low prenatal vitamin D “fits” these key environmental features. Epidemiological studies also provide some support for this association. For example, vitamin D supplements in the first year of life significantly reduced the risk of schizophrenia in males in a large Finnish Birth Cohort [153]. In addition, 25(OH)D3 serum levels in 26 mothers whose children developed schizophrenia were numerically (but not significantly) lower than those of 51 control mothers whose children did not develop the disease [154]. When these data were examined more closely a trend-level association between low maternal 25(OH)D3 levels and schizophrenia was revealed in a subgroup of children of African-American mothers. These mothers would be at greatest risk of vitamin D deficiency because of their increased skin pigmentation. Recently, a study based on Danish neonatal dried blood spots has provided more direct support for the
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association between neonatal vitamin D and risk of schizophrenia [155]. This study identified 424 individuals with a diagnosis of schizophrenia, and 424 sexand date-of-birth-matched controls and examined neonatal 25(OH)D3 status. Compared to neonates in the two upper quintiles (with 25(OH)D3 concentrations of greater than 40.5 nmol/L or 16 ng/mL), those in each of the lower two quintiles had a significantly increased risk of schizophrenia (twofold elevated risk). The nature of the relationship between neonatal 25 (OH)D3 and risk of schizophrenia was nonlinear (i.e., those with the highest concentrations of 25(OH)D3 also had a slightly increased risk), indicating that the nature of the relationship requires further investigation. The population-attributable fraction associated with neonatal vitamin D status in this sample was 44%. It is important to note that the DVD-deficient model in rodents continues to provide important information regarding the biological plausibility of this link. Like other animal models, the DVD-deficient model does not replicate every aspect of schizophrenia but it has several attractive features when compared to other animal models of this disease: (a) it is based on clues from epidemiology; (b) it reproduces the increase in lateral ventricle size (one of the most consistent neurobiological correlates of schizophrenia [156]; and (c) it reproduces two crucial behavioral endophenotypes associated with schizophrenia, i.e. behavioral sensitivity to NMDA antagonists and dopaminergic agonists [157] and disrupted latent inhibition [158]. Apart from risk of schizophrenia, there is preliminary evidence linking vitamin D intake with risk of isolated psychotic (subclinical) symptoms. A large populationbased study of Swedish women (n ¼ 33 623) reported a significant association between low vitamin D consumption and increased endorsement of psychotic-like symptoms [159]. This finding suggests that vitamin D status during adulthood may also influence risk of psychosis. Evidence from first-generation migrant studies (i.e., when migration occurs after birth) also suggests that postnatal exposure to low vitamin D may influence the subsequent risk of psychosis [160].
Depression Evidence linking hypovitaminosis D and depression is emerging. However, many of the positive associations must be interpreted with caution as many studies do not adjust for variables well known to affect 25(OH)D3 levels such as physical activity, latitude, season, or sunlight exposure. An initial link between vitamin D status and mood was made by Stumpf and colleagues, who noted the higher prevalence of seasonal affective disorder in high latitudes [161]. However to date the results of small trials of vitamin D supplements in
seasonal affective disorder have been inconclusive [162e164]. The evidence from observational studies exploring the association between 25(OH)D3 and depression is also mixed. Two studies have reported an association between low 25(OH)D3 levels and depressed mood [165,166]. However, neither of these studies included adjustments for physical activity, thus any apparent association between 25(OH)D3 and mood may simply reflect that depressed individuals are less likely to go outside and thus access ultraviolet radiation. Population-based studies have highlighted the importance of controlling for potential confounds such as place of residence and latitude. For example, a population-based study was undertaken in Beijing and Shanghai, China (n ¼ 3262, aged 50e70) [167]. In the crude analysis, the prevalence of depressive symptoms appeared lower in individuals with the highest tertile of 25(OH)D3 concentrations compared to the lowest. However, this association became nonsignificant when adjusted for various confounding factors (in particular, geographic location). Additionally when the analysis was stratified by location, no association was found. A community-based observational study from Japan [168] also reported a lack of association between 25 (OH)D3 and symptoms of depression. The influence of potential confounds has been carefully examined in a larger observational study from Europe [169]. Based on 1283 community-based elderly residents (65e95 years), it was reported that those with lower 25(OH)D3 levels were at significantly increased risk of both minor and major depression. The severity of symptoms was significantly associated with decreased serum 25(OH)D3 levels and increased serum PTH levels. These associations persisted when activity was controlled for. A 6-year prospective study of 954 adults aged 65 years and older examined the association between low vitamin D at baseline and subsequent (incident) depression [170]. Those with 25(OH)D3 less than 50 nM (or 20 ng/mL) at baseline (compared with those with higher levels) experienced significantly higher scores on measures of depression at 3- and 6-year follow-up. The study was able to adjust for a range of potential confounds, and examined the association between the variables of interest in a prospective fashion, thus lending weight to the hypothesis that low vitamin D may contribute causally to depression. The only way to resolve this issue is of course welldesigned adequately powered randomized controlled trials in the general community [171,172]. A randomized controlled trial of 800 IU vitamin D with calcium supplementation has been conducted that although originally designed to assess bone outcomes also assessed mental health [173]. In this large community-based sample of women (aged 70 years or more, n ¼ 1621) subjective psychological well-being was assessed, however there
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CONCLUSIONS
was no association between vitamin D supplementation and any mental health outcome. We still await studies with an adequate vitamin D dose.
Cognition and Dementia With respect to vitamin D and cognition, the results again are inconclusive. Several clinical studies have examined 25(OH)D3 levels and performance on neurocognitive measures using caseecontrol samples. Based on patients with secondary hyperparathyroidism (n ¼ 21) and matched controls (n ¼ 63), Jorde and colleagues reported no significant association between 25(OH)D3 levels and various cognitive measures [165]. Another study, based on a mixed sample of 80 elderly individuals (60 years or older; half with mild Alzheimer disease), reported no association between 25(OH)D3 levels and performance on a factor score derived from a large battery of neurocognitive tests [166]. A study based on a sample of 80 elderly individuals referred to a memory clinic, reported a significant positive correlation between 25(OH)D3 levels and performance on the Mini Mental State Examination [174]. In another US study in the elderly (n ¼ 60), an association between low 25 (OH)D3 and impaired scores on two cognitive screening scales (including the Mini Mental State Examination) was reported [175]. However, these studies lacked sufficient power to confidently detect small effect sizes, were not community-based, and were not able to address the important potential confound of reverse causality (i.e., those with impaired cognitive ability may be less likely to go outside, and thus may develop hypovitaminosis D as a consequence of impaired cognition). Some of these issues were addressed in a study based on the large population-based NHANES III survey. The study examined the association between 25(OH)D3 and several different neurocognitive measures (including measures of attention and memory) in an adolescent group (n ¼ 1676, age range 12e17 years), adult group (n ¼ 4747, 20e60 years), and elderly group (n ¼ 4809, 60e90 years). The study controlled for physical activity, as a proxy measure related to outdoor activity and possible ultraviolet light exposure. In the adolescent and adult groups, none of the psychometric measures were associated with 25(OH)D3 levels. However in the elderly group there was a significant difference between 25(OH)D3 quintiles and performance on a learning and memory task, however paradoxically those with the highest quintile of 25(OH)D3 were most impaired on the task, contrary to the original hypotheses. Lower 25 (OH)D3 levels were not associated with impaired performance on various psychometric measures. Other adequately powered studies have found some association between 25(OH)D3 levels and various cognitive outcomes. A population-based study from the UK
examined 1766 adults aged over 65 years. Cognitive impairment was assessed using the Abbreviated Mental Test Score. In the subgroup with cognitive impairment (n ¼ 212), there was a significant association between lower 25(OH)D3 and impairment on the cognitive task [176]. Buell and colleagues [177] examined the association between 25(OH)D3 and neurocognition in 1080 elderly individuals receiving home health services (age range from 65e99 years). The study was able to adjust for a wide range of potential confounds (including physical activity and body mass index). This study showed no association between 25(OH)D3 and memory tasks, but significant associations were found between lower 25 (OH)D3 levels and impaired performance on various tests of executive function and psychomotor speed (e.g., Trail Making task, digit symbol substitution). Curiously, based on this same sample, the authors found an association between 25(OH)D3 deficiency and (a) increased white matter hyperintensity volume, and (b) large vessel infarcts, as measured by magnetic resonance imaging [178]. Finally a large, populationbased multicenter study (n ¼ 3369 men, age 40e79 years) examined the correlation between 25(OH)D3 levels and performance on three cognitive measures (the Rey-Osterrieth Complex Figure test, the Camden Topographical Recognition Memory test, and the Digit Symbol Substitution Test). Importantly, this study was able to adjust for a range of variables including physical activity, functional performance, and mood/depression. When the model was adjusted for potential confounds, those with lower 25(OH)D3 did worse on only one of the measures, and this again was the Digit Symbol Substitution Test. Those with lower 25(OH)D3 also had slower psychomotor processing speed [179]. It would appear therefore that chronic hypovitaminosis D may exacerbate cognitive and other neuropsychiatric impairments. However longitudinal, prospective studies are required to confirm this hypothesis. While the animal experimental data support an association between low developmental vitamin D and altered brain development, studies on the impact of hypovitaminosis D during adulthood on brain function remain to be clarified. Recently, a detailed narrative review of the field reached similar conclusions [5]. More animal experimental work should help clarify the role of vitamin D on adult brain function, however more focused analytic epidemiological experiments are also required.
CONCLUSIONS Considering the preclinical with the direct and indirect clinical evidence, on balance it would appear that an adequate amount of vitamin D throughout one’s
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FIGURE 32.1 The impact of vitamin D deficiency on brain development, structure, and function, evidence from studies in rodents. Alterations have been reported at both a whole brain level and in select nuclei. The effects of vitamin D deficiency on embryonic brain have only been investigated in maternally vitamin-D-deficient animals. The effects of vitamin D deficiency on adult brain structure and behavior have been examined in models of dietary deficiency (during development or during adulthood) and in models where the vitamin D receptor has been genetically ablated. Outcomes in adults are presented separately based on calcium status (see text). Possible relevance to human disease is highlighted. VDR KO, vitamin D receptor knock-out; DOPAC/HVA, ratio of dihydroxyphenylacetic acid/homovanilic acid; COMT, catecholo-methyl transferase; DAT, dopamine transporter; GABA, gamma-amin butyric acid. ; decrease; : increase (see text for details) IV. TARGETS
REFERENCES
life is required for normal brain growth and function. Deficiency during development could well be related to the onset of childhood epilepsy. Developmental exposures may also increase one’s risk of developing serious psychopathologies as an adult such as schizophrenia. Finally it would appear that depression or degenerative conditions such as multiple sclerosis, Parkinson disease, and disorders of cognitive decline could also be associated with an individual’s 25(OH)D3 status. The current state of knowledge about the impact of vitamin D deficiency on brain development, structure, and function is summarized in Figure 32.1. The rapid accumulation of experimental evidence over the past 10 years indicating vitamin D could play some role in brain development and function is compelling [5]. The animal models, in particular the DVDdeficiency model, may provide important discoveries in aiding our understanding of the neurobiology of psychiatric diseases, in particular schizophrenia. The spectrum of neurological and psychiatric conditions linked to 25 (OH)D3 status continues to expand with a recent series of studies suggesting low levels of maternal or childhood vitamin D could also be implicated in autism [180e184]. Considering the epidemiological evidence linking vitamin D with a variety of adverse neurological, psychiatric, and cognitive outcomes [185,186], and the growing body of evidence implicating vitamin D in brain function [5], further well-designed observational studies, and, more importantly, randomized clinical trials of vitamin D supplementation are warranted in those with neurological and neuropsychiatric disorders. The attraction of using such a simple, safe, and inexpensive intervention to alleviate disease burden for many different adverse health outcomes is extremely attractive. There remain many unanswered questions regarding the dynamics of vitamin D status and its effects on brain structure and function. For instance what constitutes a minimum level of cholecalciferol supplementation or serum 25(OH)D3 level that would predispose an individual to various diseases remains a topic of intense debate [187]. Although vitamin D deficiency can be modeled in animals via dietary restriction or through ablating various factors responsible for vitamin D signaling there is insufficient research on chronic vitamin D insufficiency which is far more likely to be observed clinically. Whether the risk of adverse brainrelated outcomes is due to a concentration gradient or some unknown 25(OH)D3 threshold remains an important experimental variable to be addressed. Additionally we have little knowledge about whether an earlier deficiency/insufficiency can predispose an individual to later brain insults such as infection, autoimmunity, or oxidative damage. The plethora of data accumulated over these past 10 years has led to the tentative inclusion of vitamin D
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into the broader family of neuroactive steroids [4]. This group includes agents such as the sex steroids and glucocorticoids whose actions in shaping brain development and function have been well described. It would appear therefore that vitamin D has now finally “emerged from the shadows” and is no longer “The Neglected Neurosteroid” [1]. Given the alarming prevalence of hypovitaminosis D in both pregnant women [188,189] and in the general public, ensuring the diverse functional capacities of this neuroactive steroid in the developing and adult brain are preserved through either environmental or dietary interventions would appear to be a vital public health priority.
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[155] J.J. McGrath, D.W. Eyles, C.B. Pedersen, C. Anderson, P. Ko, T.H. Burne, et al., Neonatal vitamin D status and risk of schizophrenia: a population-based case-control study, Arch. Gen. Psychiatry (2010). In press. [156] P.J. Harrison, D.R. Weinberger, Schizophrenia genes, gene expression, and neuropathology: on the matter of their convergence, Mol. Psychiatry 10 (2005) 40e68, image 45. [157] M. Laruelle, W.G. Frankle, R. Narendran, L.S. Kegeles, A. Abi-Dargham, Mechanism of action of antipsychotic drugs: from dopamine D(2) receptor antagonism to glutamate NMDA facilitation, Clin. Ther. 27 (Suppl. A) (2005) S16eS24. [158] N. Kathmann, S. von Recum, C. Haag, R.R. Engel, Electrophysiological evidence for reduced latent inhibition in schizophrenic patients, Schizophr. Res. 45 (2000) 103e114. [159] M. Hedelin, M. Lof, M. Olsson, T. Lewander, B. Nilsson, C.M. Hultman, et al., Dietary intake of fish, omega-3, omega-6 polyunsaturated fatty acids and vitamin D and the prevalence of psychotic-like symptoms in a cohort of 33 000 women from the general population, BMC Psychiatry 10 (2010) 38. [160] J. McGrath, Is it time to trial vitamin D supplements for the prevention of schizophrenia? Acta Psychiatr. Scand. (2010). In press. [161] W.E. Stumpf, T.H. Privette, Light, vitamin D and psychiatry. Role of 1,25 dihydroxyvitamin D3 (soltriol) in etiology and therapy of seasonal affective disorder and other mental processes, Psychopharmacology (Berl.) 97 (1989) 285e294. [162] F.M. Gloth III, W. Alam, B. Hollis, Vitamin D vs broad spectrum phototherapy in the treatment of seasonal affective disorder, J. Nutr. Health Aging 3 (1999) 5e7. [163] A.T. Lansdowne, S.C. Provost, Vitamin D3 enhances mood in healthy subjects during winter, Psychopharmacology (Berl.) 135 (1998) 319e323. [164] T. Partonen, O. Vakkuri, C. Lamberg-Allardt, J. Lonnqvist, Effects of bright light on sleepiness, melatonin, and 25-hydroxyvitamin D(3) in winter seasonal affective disorder, Biol. Psychiatry 39 (1996) 865e872. [165] R. Jorde, K. Waterloo, F. Saleh, E. Haug, J. Svartberg, Neuropsychological function in relation to serum parathyroid hormone and serum 25-hydroxyvitamin D levels: the Tromso study, J. Neurol. 253 (2006) 464e470. [166] C.H. Wilkins, Y.I. Sheline, C.M. Roe, S.J. Birge, J.C. Morris, Vitamin D deficiency is associated with low mood and worse cognitive performance in older adults, Am. J. Geriatr. Psychiatry 14 (2006) 1032e1040. [167] A. Pan, L. Lu, O.H. Franco, Z. Yu, H. Li, X. Lin, Association between depressive symptoms and 25-hydroxyvitamin D in middle-aged and elderly Chinese, J. Affect. Disord. 118 (2009) 240e243. [168] A. Nanri, T. Mizoue, Y. Matsushita, K. Poudel-Tandukar, M. Sato, M. Ohta, et al., Association between serum 25-hydroxyvitamin D and depressive symptoms in Japanese: analysis by survey season, Eur. J. Clin. Nutr. 63 (2009) 1444e1447. [169] W.J. Hoogendijk, P. Lips, M.G. Dik, D.J. Deeg, A.T. Beekman, B.W. Penninx, Depression is associated with decreased 25-hydroxyvitamin D and increased parathyroid hormone levels in older adults, Arch. Gen. Psychiatry 65 (2008) 508e512. [170] Y. Milaneschi, M. Shardell, A.M. Corsi, R. Vazzana, S. Bandinelli, J.M. Guralnik, et al., Serum 25-hydroxyvitamin D and depressive symptoms in older women and men, J. Clin. Endocrinol. Metab. 95 (2010). [171] E.R. Bertone-Johnson, Vitamin D and the occurrence of depression: causal association or circumstantial evidence? Nutr. Rev. 67 (2009) 481e492.
[172] S.N. Young, Has the time come for clinical trials on the antidepressant effect of vitamin D? J. Psychiatry Neurosci. 34 (2009) 3. [173] J.C. Dumville, J.N. Miles, J. Porthouse, S. Cockayne, L. Saxon, C. King, Can vitamin D supplementation prevent winter-time blues? A randomised trial among older women, J. Nutr. Health Aging 10 (2006) 151e153. [174] R.J. Przybelski, N.C. Binkley, Is vitamin D important for preserving cognition? A positive correlation of serum 25-hydroxyvitamin D concentration with cognitive function, Arch. Biochem. Biophys. 460 (2007) 202e205. [175] C.H. Wilkins, S.J. Birge, Y.I. Sheline, J.C. Morris, Vitamin D deficiency is associated with worse cognitive performance and lower bone density in older African Americans, J. Nat. Med. Assoc. 101 (2009) 349e354. [176] D.J. Llewellyn, K.M. Langa, I.A. Lang, Serum 25hydroxyvitamin D concentration and cognitive impairment, J. Geri. Psychiatry Neurol. 22 (2009) 188e195. [177] J.S. Buell, T.M. Scott, B. Dawson-Hughes, G.E. Dallal, I.H. Rosenberg, M.F. Folstein, et al., Vitamin D is associated with cognitive function in elders receiving home health services. Js Gerontol. Series A, Biol. Sci. and Med. Sci. 64 (2009) 888e895. [178] J.S. Buell, B. Dawson-Hughes, T.M. Scott, D.E. Weiner, G.E. Dallal, W.Q. Qui, et al., 25-Hydroxyvitamin D, dementia, and cerebrovascular pathology in elders receiving home services, Neurology 74 (2010) 18e26. [179] D.M. Lee, A. Tajar, A. Ulubaev, N. Pendleton, T.W. O’Neill, D.B. O’Connor, et al., Association between 25-hydroxyvitamin D levels and cognitive performance in middle-aged and older European men, J. Neurol. Neurosurg. Psychiatry 80 (2009) 722e729. [180] J.J. Cannell, Autism and vitamin D, Med. Hypotheses 70 (2008) 750e759. [181] E. Fernell, M. Barnevik-Olsson, G. Bagenholm, C. Gillberg, S. Gustafsson, M. Saaf, Serum levels of 25-hydroxyvitamin D in mothers of Swedish and of Somali origin who have children with and without autism, Acta Paediatr. 99 (2010) 743e747. [182] D.W. Eyles, Vitamin D and autism: does skin colour modify risk? Acta Paediatr. 99 (2010) 645e647. [183] M.B. Humble, S. Gustafsson, S. Bejerot, Low serum levels of 25hydroxyvitamin D (25-OHD) among psychiatric out-patients in Sweden: relations with season, age, ethnic origin and psychiatric diagnosis, J. Steroid Biochem. Mol. Biol. (2010). [184] D.K. Kinney, D.H. Barch, B. Chayka, S. Napoleon, K.M. Munir, Environmental risk factors for autism: do they help cause de novo genetic mutations that contribute to the disorder? Med. Hypotheses 74 (2010) 102e106. [185] J.S. Buell, B. Dawson-Hughes, Vitamin D and neurocognitive dysfunction: preventing “D”ecline? Mol. Aspects Med. 29 (2008) 415e422. [186] W.B. Grant, Does vitamin D reduce the risk of dementia? J. Alzheimer’s Dis. 17 (2009) 151e159. [187] R. Vieth, H. Bischoff-Ferrari, B.J. Boucher, B. Dawson-Hughes, C.F. Garland, R.P. Heaney, et al., The urgent need to recommend an intake of vitamin D that is effective, Am. J. Clin. Nutr. 85 (2007) 649e650. [188] B.W. Hollis, C.L. Wagner, Vitamin D deficiency during pregnancy: an ongoing epidemic, Am. J. Clin. Nutr. 84 (2006) 273. [189] B.W. Hollis, C.L. Wagner, Nutritional vitamin D status during pregnancy: reasons for concern, Cmaj 174 (2006) 1287e1290.
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33 Contributions of Genetically Modified Mouse Models to Understanding the Physiology and Pathophysiology of the 25-Hydroxyvitamin D-1-Alpha Hydroxylase Enzyme (1a(OH)ase) and the Vitamin D Receptor (VDR) Geoffrey N. Hendy, Richard Kremer, David Goltzman Calcium Research Laboratory and Department of Medicine, McGill University and Royal Victoria Hospital of the McGill University Health Centre, Montreal, Quebec, Canada
INTRODUCTION The enzyme 25-hydroxyvitamin D-1a-hydroxylase (1a(OH)ase) catalyzes the 1a-hydroxylation of the vitamin D metabolite, 25-hydroxyvitamin D (25(OH)D) and is critical for the production of the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D) [1]. The 1a(OH)ase is a mitochondrial enzyme complex comprising a ferrodoxin, a ferrodoxin reductase and a cytochrome P-450 that provides the specificity for the 25(OH)D substrate. The renal enzyme is the major source of the circulating concentration of 1,25(OH)2D. Parathyroid hormone (PTH) acting via the PTH receptor (PTHR) by cyclic AMP signal transduction is a major regulator of the renal enzyme by enhancing expression of the CYP27B1 gene that encodes the cytochrome P-450 [2]. Independently, reductions in circulating calcium and phosphate up-regulate the CYP27B1 gene. The renal enzyme is product inhibited by 1,25(OH)2D which down-regulates transcription of the CYP27B1 gene. The osteoblast/osteocyte-derived phosphaturic fibroblast growth factor 23 (FGF23) is also a potent inhibitor of the renal 1a(OH)ase [3]. The 1a(OH)ase is expressed at extrarenal sites such as macrophages, placenta, and skin [4]. Extrarenal 1a(OH)ase has been implicated in the local generation of 1,25(OH)2D from
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10033-2
circulating 25(OH)D in many tissues and cells where the active metabolite acts in a paracrine/autocrine or intracrine fashion. The extrarenal 1a(OH)ases are regulated differently from the renal enzyme. After uptake into target cells 1,25(OH)2D binds the vitamin D receptor (VDR), a member of the nuclear receptor superfamily [5]. In the nucleus, the ligand-activated VDR heterodimerizes with the retinoid X receptor (RXR) and the dimer binds to vitamin D response elements (VDREs) on target genes [6]. Coregulators link the dimer to the basal transcriptional machinery and direct transcription in a gene-specific, tissuespecific, and differentiation stage-specific manner. A wide array of genes is affected, with individual genes being up-regulated or down-regulated in a gene-specific and cell-specific fashion. The widespread expression of the VDR is indicative that vitamin D actions extend beyond the calcium/skeletal homeostatic system. Rare genetic mutations in humans have confirmed the central importance of both 1,25(OH)2D and the VDR in calcium and skeletal homeostasis. Loss-offunction mutations in the CYP27B1 gene cause the autosomal recessive disorder vitamin-D-dependent rickets type 1 (VDDR1), also known as pseudo-vitamin-Ddeficiency rickets (MIM 264700) [7]. The abnormalities in the CYP27B1 gene, either homozygous or compound
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heterozygous mutations, lead to 1a(OH)ase deficiency and the disorder is characterized by low serum calcium and phosphate, secondary hyperparathyroidism, earlyonset rickets and low circulating levels of 1,25(OH)2D. Loss-of-function mutations in the VDR gene cause the autosomal recessive disorder vitamin-D-dependent rickets type 2 (VDDR2), also known as hereditary vitamin-D-resistant rickets (MIM 264700) [8]. Although presenting clinically with rachitic changes, this syndrome can be distinguished from VDDR1 by the elevated circulating levels of 1,25(OH)2D and lack of response to treatment with vitamin D. In these human disorders, characterized by either inability to synthesize the active form of vitamin D or inability of the ligand to act, skeletal and mineral abnormalities are the predominant phenotypic presentations underscoring the critical role of vitamin D in these functions.
MOUSE MODELS Mouse models null for either the 1a(OH)ase [9,10] or the VDR [11e14] have been generated. Use of these models has permitted a more controlled and extensive examination of the phenotypes than could be achieved by examination of the human VDDR1 and VDDR2 disorders. The mouse models have provided critical insights into both the skeletal and extraskeletal actions of the 1,25(OH)2D/VDR system.
1,25(OH)2D/VDR SYSTEM AND SKELETAL AND MINERAL HOMEOSTASIS We genetically engineered mice lacking the 1a(OH) ase by deleting the Cyp27b1 exons encoding the hormone-binding and heme-binding domains [9]. The null mutant mice were grossly normal from birth to weaning but thereafter displayed marked growth retardation. Expression of the mRNA for the important 1,25 (OH)2D target gene, Cyp24a1, encoding the 25-hydroxyvitamin D-24-hydroxylase enzyme (24(OH)ase), was almost completely ablated in the homozygous 1a(OH) ase-null mice. While circulating concentrations of 1,25 (OH)2D were undetectable, serum 25(OH)D levels were elevated reflecting its accumulation in the absence of the 1a- and 24-hydroxylating enzymes. Serum calcium and phosphate concentrations were reduced and serum PTH and urinary phosphate concentrations were markedly elevated. Typical histological features of rickets were present, including enlargement of the epiphyseal growth plates (mainly because of a widened and disorganized hypertrophic zone), inadequate mineralization of cartilage, primary spongiosa, and cortical bone, and an increase in osteoid in both
trabecular and cortical bone. The numbers of osteoblasts lining bone surfaces were increased and trabecular bone in the primary spongiosa was augmented. The phenotype of the 1a(OH)ase-null mice was compared with that of mice lacking the Vdr gene [12]. In mice lacking the VDR, expression of the renal 1a (OH)ase and 24(OH)ase mRNAs was elevated and suppressed, respectively. Thus, in the absence of the VDR, endogenous 1,25(OH)2D was unable to either suppress the 1a(OH)ase or stimulate the 24(OH)ase resulting in high circulating 1,25(OH)2D concentrations. We investigated the consequences of 1a(OH)ase deficiency on the VDR-null background by crossbreeding 1a(OH)asee/e and VDRe/e mice to obtain the compound 1a(OH) asee/e VDRe/e mutant mice. The three genetically diverse mutant strains were analyzed after exposure to different environments (diets to produce different calcium intakes and/or administration of exogenous 1,25(OH)2D) [15]. Thus, after weaning, mice received either a calcium intake on which they remained hypocalcemic, a high calcium intake with intraperitoneal injections of 1,25(OH)2D3 three times a week, or a so-called “rescue” diet containing high calcium, high phosphorus, and 20% lactose. This diet increases calcium transport in the rodent intestine independently of the 1,25(OH)2 D/VDR system and can normalize serum calcium levels in vitamin D deficiency by enhancing passive transport of calcium via a paracellular route.
1,25(OH)2D/VDR Action Regulates Calcium Transport in Intestine and Kidney Intestinal and renal calcium transport has a passive concentration gradient-dependent paracellular component and an active ATP-dependent transcellular component regulated by 1,25(OH)2D that is critical when calcium supply is low. In the kidney, passive calcium and sodium reabsorption takes place in the proximal nephron and the 1,25(OH)2D-regulated calcium reabsorption takes place in the distal part of the nephron. In the intestine, active 1,25(OH)2D-regulated calcium absorption occurs in the duodenum and passive absorption occurs in the jejunum. Epithelial cell apical calcium channels of the transient receptor potential vanilloid (TRPV) family (TRPV6 in intestine and TRPV5 in kidney) mediate the influx of calcium that is promoted by an electropotential gradient across the membrane. Cytosolic calcium-binding proteins, calbindins (CaBP-9k in intestine and both CaBP-9k and CaBP-28k in the kidney) transport calcium across the cell. Calcium is extruded across the basolateral membrane into extracellular fluid by an energy-requiring process. The plasma membrane calcium ATPase (PMCA1b) is expressed in the intestine and both Naþ/Ca2þ exchanger (NCX1) and PMCA1b are found in the kidney [16,17]. The 1,25(OH)2D/VDR
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system is known to enhance gene expression of several calcium transporters including Trpv6, Calbindin-D9k, Calbindin-D28k, and PMCA1b [13,18,19]. For greater detail with respect to these components and processes, see chapters in Section III of this volume. Intestinal Calcium Absorption Intestinal calcium absorption is markedly decreased (60%) in adult VDR-null mice [13,20]. The decrease correlates with a switch from predominance of passive calcium transport before weaning to a 1,25(OH)2D driven mechanism after [21,22]. Both 1a(OH)asee/e mice with intact VDR and VDRe/e mice with elevated endogenous 1,25(OH)2D were hypocalcemic after weaning onto the calcium intake. In the latter strain of mice, treatment with exogenous 1,25(OH)2D failed to normalize the serum calcium level whereas the same treatment normalized calcium concentrations in the 1a (OH)asee/e mice [15,23]. Hence intestinal calcium absorption requires both 1,25(OH)2D and the VDR. The expression of several duodenal calcium transport proteins is altered in VDRe/e and 1a(OH)asee/e mice. In VDR-null mice, TRPV6 and CaBP-9k levels are low [13,24] and the latter was also noted in 1a(OH)asee/e mice [9,10]. PMCA1b expression was not altered in VDR-null mice [13]. In both VDR-null and wild-type mice given the rescue diet, TRPV6 and CaBP-9k expression decreased to similar levels suggesting that when paracellular calcium transport is adequate to meet dietary needs, the genes responsible for active calcium transport are down-regulated [13,24,25]. In TRPV6-null mice both decreased and unaltered intestinal absorption have been observed despite the maintenance of normal serum calcium levels. On a low-calcium diet, TRPV6-null mice were able to increase calcium absorption but not to the extent of wild-type mice, resulting in hypocalcemia [26,27]. CaBP-9k-null mice had no defect in intestinal calcium absorption or serum calcium levels and its role is likely to be compensated for by other calcium transport proteins [28,29]. In double null TRPV6 and CaBP-9k mice, calcium transport was not altered when calcium intake was sufficient but exhibited a worsening of the effect of TRPV6 ablation alone on a low-calcium diet [27]. TRPV6 therefore appears to be redundant for intestinal calcium absorption when dietary calcium content is normal or high and passive diffusion likely contributes to maintain normal serum calcium levels in this situation. Claudin 2 and claudin 12, which are induced by 1,25(OH)2D, form paracellular calcium channels; their expression is reduced in VDR-null mice [30]. Thus, by regulating these epithelial cell junction proteins, 1,25 (OH)2D, may reroute calcium through the paracellular path. Other transport mechanisms, still to be largely identified, may also compensate for the loss of TRPV6,
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which might explain the rather mild phenotype of Trpv6-null mice. Renal Calcium Reabsorption The VDR is critical for proper calcium reabsorption in the kidney. VDR-null mice have inappropriately high urinary calcium excretion given the observed hypocalcemia suggesting impaired calcium reabsorption and renal calcium wasting. This was most marked in normocalcemic VDR-null mice on a rescue diet [14,31]. Expression of both renal CaBP-9k and CaBP-28k was decreased in VDR-null and 1a(OH)ase-null mice on a normal diet with reduced serum calcium levels. TRPV5 and NCX1 levels were normal in VDR-null mice but reduced in 1a(OH)ase-null mice; the expression of PMCA1b was unaltered in both strains [9,11,13,14,24,32]. Feeding wild-type, VDR-null, or 1a (OH)ase-null mice a rescue diet lowered CaBP-9k expression to comparable levels [13,20,32]. In 1a(OH) ase-null mice normalization of 1,25(OH)2D increased expression of each of the renal calcium transport proteins and normalized serum calcium [32]. TRPV5 is one of the major calcium transport proteins involved in calcium reabsorption in the kidney. TRPV5null mice have persistent hypercalciuria, and display increased 1,25(OH)2D levels and elevated intestinal calcium absorption [33] but are normocalcemic and do not develop rickets. Consequently TRPV5 appears to have little role in intestinal calcium absorption. However, TRPV5-null mice have reduced bone mass and cortical thickness [33]. Whether this is due to the high endogenous 1,25(OH)2D levels or to secondary hyperparathyroidism associated with the hypercalciuria, or both, remains to be determined. In contrast to the TRPV5-null mice, TRPV5/1a(OH)ase compound null mice do not have increased expression of calcium transporters and are very hypocalcemic [34], suggesting that 1,25(OH)2D through either an intestinal effect or a bone effect or both is important in maintaining normocalcemia in the TRPV5-null mice. CaBP-28k-null mice did not have disturbed calcium homeostasis, and manifested normal serum calcium and phosphate levels. In addition, urinary calcium excretion and any bone changes were subtle [35e38]. Furthermore, deletion of the CaBP-28k gene in TRPV5 mice did not worsen the phenotype suggesting that the role of CaBP-28k can be compensated for by CaBP-9k [39]. Studies of double null VDR and CaBP-28k mice also suggested that while CaBP-28k is involved in calcium homeostasis and skeletal mineralization, CaBP-9k largely compensates for this role [38]. However, the double null mice on the regular diet were more growthretarded than VDR-null mice and died prematurely. On the rescue diet the skeletal abnormalities were not completely corrected. Deletion of NCX1 or PMCA1b is
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embryonic lethal because of essential functions in maintaining intracellular calcium levels [40e43]. Thus, the consequence of deleting these components selectively in the intestine or kidney has not been examined in the context of the vitamin D system. Overall, these studies suggest that TRPV5, a 1,25 (OH)2D-inducible transport protein, is essential for renal calcium reabsorption whereas the 1,25(OH)2D-inducible proteins, CaBP-28k and CaBP-9k, may play overlapping roles in calcium homeostasis and skeletal mineralization. They also suggest that the key targets of 1,25 (OH)2D3 action in the intestine and kidney have not been identified.
Regulation of the 1a(OH)ase and the 24(OH)ase Enzymes Both 1,25(OH)2D and the VDR are required for downregulation and up-regulation of 1a(OH)ase and 24(OH) ase gene expression in vivo [1,44,45]. However, elimination of the hypocalcemia alone, using the rescue diet, normalized 24(OH)ase levels in the 1a(OH)asee/e mice and both 1a(OH)ase and 24(OH)ase levels in VDRe/e mice [15]. Thus, there is a calcium effect on gene expression of these enzymes that is independent of the 1,25 (OH)2D/VDR system. Whether this effect of calcium is entirely indirect through suppression of PTH levels [1,44,45], or is partly direct remains to be determined.
Regulation of Parathyroid Gland Function Extracellular calcium via the calcium-sensing receptor (CaSR) [46,47] inhibits PTH secretion, increases intracellular proteolysis of biologically active PTH to inactive fragments [48], and increases PTH mRNA degradation reducing PTH synthesis [49]. Calcium also inhibits parathyroid cell proliferation. 1,25(OH)2D inhibits PTH gene transcription [50] and parathyroid cell proliferation [51]. In the 1a(OH)asee/e [9,10] and the VDRe/e [12,15] mice on a normal or high-calcium diet, when hypocalcemia is present, enlarged parathyroid glands and increased circulating PTH concentrations occur. On the rescue diet that normalizes serum calcium concentrations, serum PTH levels fell in both mutant strains indicating that elevation of the ambient calcium alone normalizes PTH secretion. Parathyroid gland size was reduced somewhat but remained moderately enlarged in the 1a (OH)asee/e mice on the rescue diet. Treatment of these mice with exogenous 1,25(OH)2D normalized the serum calcium concentration and the parathyroid gland size. Consequently both calcium and 1,25(OH)2D act cooperatively to diminish PTH production and parathyroid gland size. A mouse strain in which the VDR was specifically deleted in the parathyroid was generated by crossing
mice with a floxed VDR gene with transgenic mice expressing the Cre recombinase under the control of the PTH promoter (PTH-Cre) [52]. In this study, the parathyroid gland VDR expression, although reduced, was not completely absent. The phenotype of these mice (PT-VDRe/e) was compared with that of wholebody VDR knockout mice (VDRe/e). While serum calcium levels were decreased in VDRe/e mice, they were normal in PT-VDRe/e mice. Serum PTH levels were markedly and moderately elevated in VDRe/e and PT-VDRe/e mice, respectively. In both strains there was a decrease in parathyroid CaSR levels. By the criteria of increased PTH levels in the face of normal calcium, the PT glands of the PT-VDRe/e mice would appear to have reduced sensitivity to extracellular calcium, although in this study the changes in serum PTH levels during experimentally induced alterations in serum calcium were taken as indicative of intact sensitivity of the PT gland to calcium. While numbers of proliferating PT cells were increased in VDRe/e mice they were normal in PT-VDRe/e mice indicating that reductions in serum calcium provide a major stimulus to proliferation whereas impaired vitamin D action is of lesser importance. The increased PTH levels in the PT-VDRe/e mice stimulated bone resorption without change in bone formation as indicated by increased serum C-terminal collagen cross links but no change in osteocalcin, respectively. Hence, from this study, the parathyroid gland VDR is suggested to have a limited role in parathyroid physiology. As VDR expression was not completely eliminated, additional studies are needed to explore this issue further.
Interaction of PTH and 1,25(OH)2D on Calcium and Phosphate Homeostasis Single PTH-null mice develop hypocalcemia and hyperphosphatemia, and low serum 1,25(OH)2D levels [53,54]. The PTH, 1a(OH)ase double null mice died at 3 weeks with tetany due to severe hypocalcemia suggesting the secondary hyperparathyroidsm of the 1a(OH)ase-null mice was controlling calcium homeostasis by acting on bone and kidney [55]. Treatment of the double null mice with PTH or PTHrP reduced the hypocalcemia by increased expression of renal calcium transporters and bone formation was enhanced [56]. In the double null mice treatment with 1,25(OH)2D3 also increased serum calcium levels and improved bone formation independently of PTH [57]. PTH suppresses apical Na/Pi-2a and Na/Pi-2b cotransporters that mediate phosphate uptake in proximal renal tubules and small intestine, respectively [58,59]. 1,25(OH)2D also up-regulates renal and intestinal phosphate transport [60,61]. However, feeding VDR or 1a(OH)ase-null mice a low-phosphate diet
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increased the renal and intestinal cotransporter protein levels indicating their expression can be regulated independently of the VDR [62,63].
Bone and Cartilage Remodeling In all hypocalcemic knockout models of the 1,25 (OH)2D/VDR system, osteoblast numbers, bone formation, and bone volume are markedly increased [9,10,12,15]. This is likely due to the anabolic effect of PTH that is markedly elevated in association with the severe secondary hyperparathyroidism in these animals. The increased bone volume is largely due to increased unmineralized osteoid. Although sustained elevation of PTH is usually associated with increased osteoclastic bone resorption as well as increased bone formation, the osteoclast number and resorbing surface were not elevated in these models. This suggests an uncoupling of bone turnover in the presence of a defective 1,25(OH)2D/VDR system that is required for an appropriate osteoclastic response to increased PTH. Because the production of osteoclasts and chondroclasts at the chondro-osseous junction may also be defective, removal of hypertrophic chondrocytes may be reduced in this region, leading to altered cartilage growth plate remodeling. The enlargement of the cartilaginous growth plate, particularly the hypertrophic zone, may be due in part to reduced activity of the 1,25(OH)2D/VDR system on chondroclast and osteoclast production [64].
Mineralization of Bone On a rescue diet, bone mineralization and osteoid accumulation normalized in 1a(OH)asee/e, VDRe/e, and compound 1a(OH)asee/e VDRe/e mutant strains [15,64,65]. Consequently, mineralization of bone is determined by ambient calcium and phosphate concentrations rather than through the direct participation of the 1,25(OH)2D/VDR system.
Effects of 1,25(OH)2D on Bone Volume 1,25(OH)2D is a potent stimulator of osteoclastogenesis in vitro, and administration of high doses of 1,25(OH)2D can exert an osteoclastogenic and boneresorbing effect in vivo [66]. However, in all three mutant strains at 4 months of age on the rescue diet to prevent hypocalcemia and secondary hyperparathyroidism, osteoblast numbers, mineral apposition rate and bone volume were suppressed below the levels of wild-type mice [15]. This suggests that the 1,25(OH)2D/VDR system may exert a skeletal anabolic effect that is necessary to sustain basal bone-forming activity and which is unmasked in the presence of a defective 1,25(OH)2D/VDR system and normal PTH
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levels. Evidence has been provided in other model systems of a bone anabolic effect of 1,25(OH)2D [67]. We examined skeletal development in 1a(OH)asee/e mice in the neonatal period in comparison with PTHe/e mice and compound 1a(OH)asee/e PTHe/e mice [55]. At 2 weeks of age all mutants showed reduced osteoblastic bone formation which was most pronounced in the compound mutants. The results showed that 1a (OH)asee/e mice are osteopenic as early as 2 weeks of age, suggesting that PTH plays a predominant role in appositional bone growth, whereas 1,25(OH)2D acts predominantly, although not exclusively, on endochondral bone formation. To identify 1,25(OH)2D-mediated skeletal actions independent of PTH, double-knockout mice, which are homozygous for both the 1a(OH)ase- and PTH-null alleles, were treated with 1,25(OH)2D from 4 to 14 days of age and compared with vehicle-treated mice [57]. Exogenous 1,25(OH)2D increased both trabecular and cortical bone, augmented both osteoblast number and type 1 collagen deposition in bone matrix, and up-regulated expression of the osteoblastic genes, alkaline phosphatase, type 1 collagen, and osteocalcin. The data indicate that administered 1,25(OH)2D can promote endochondral and appositional bone increases independently of endogenous PTH.
Direct vs Indirect Role of VDR or 1a(OH)ase Action in Chondrocytes VDR- and 1a(OH)ase-null pups are of normal length and morphology and mineral content of long bones and growth plates are normal. After weaning, long bone growth is impaired with features of rickets such as expansion of the epiphyseal growth plate. While resting and proliferating chondrocyte layers are normal, apoptosis of hypertrophic chondrocytes is impaired in VDR-null mice. Circulating phosphate levels are critical for hypertrophic chondrocyte apoptosis and in vitro studies suggest this occurs via the caspase-9-mediated mitochondrial pathway [68]. Normalization of circulating mineral levels by dietary intervention or 1,25(OH)2D administration rescued the rachitic phenotype in VDR- and 1a(OH) ase-null mice, respectively [23,24,64,65,69]. Therefore, VDR-dependent actions are apparently not required for growth plate development and maturation but altered mineral ion homeostasis underlies the rachitic changes. In support of this view, in one study, growth plate chondrocyte development was apparently not impaired in mice with chondrocyte-specific VDR deletion [70]. However, vascular endothelial growth factor (VEGF) and receptor activator of nuclear factor kB ligand (RANKL) expression was decreased leading to impaired vascular invasion and decreased osteoclast numbers in
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long bone metaphyses in young mice. Osteoblast FGF23 expression was decreased resulting in increased serum phosphate and 1,25(OH)2D levels. In another model, specific knockout of Cyp27b1 in chondrocytes led to decreased RANKL expression and reduced osteoclastogenesis, increased width of the embryonic growth plate hypertrophic zone, increased neonatal long bone volume, and increased expression of chondrocyte differentiation markers [71]. A delay in vascularization was suggested by reduced VEGF expression and decreased platelet/endothelial cell adhesion molecule-1 staining in the neonatal growth plate. Transgenic mice overexpressing the 1a(OH)ase in chondrocytes showed the mirror image of the knockout model, having reduction of the width of the embryonic growth plate hypertrophic zone, decreased neonatal long bone volume, and decreased expression of chondrocytic differentiation markers [71]. Together, the data supported an intracrine role for 1,25(OH)2D in endochondral ossification and chondrocyte development in vivo. In other studies, persistent abnormalities of the growth plate have been observed in 1a(OH)ase-null mice or double 1a(OH) ase/VDR-null mice on a rescue diet and in VDR/ RXRg double null mice [15,72]. The latter case suggests that 1,25(OH)2D may interact with an unknown nuclear receptor in chondrocytes that heterodimerize with RXRg.
Interaction of the 1,25(OH)2D/VDR System with FGF23 The circulating phosphaturic factor, FGF23, produced in osteoblastic/osteocytic cells stimulates renal phosphate wasting (see also chapters in Section V of this volume). Similar to PTH, FGF23 suppresses the expression of Na/Pi-2a and Na/Pi-2c cotransporters [73,74]. Also, FGF23 inhibits PTH expression and the 1a(OH) ase thereby decreasing 1,25(OH)2D levels [74,75]. Treatment of VDR-null mice with FGF23 decreased Na/Pi-2a and Na/Pi-2b cotransporter and 1a(OH)ase expression causing reduced renal and intestinal phosphate reabsorption and absorption, respectively [76e78]. FGF23-null mice are hyperphosphatemic, moderately hypercalcemic, have low PTH levels and high 1,25 (OH)2D levels e the latter being predominant over any FGF23 effect in modulating PTH synthesis and release. In the adult mice, the growth plate is disorganized and lacking hypertrophic chondrocytes, and mice have decreased mineralized bone mass with increased osteoid [74,79]. The mice have skeletal nodules and soft tissue calcification that are not present in FGF23/1a(OH)ase double null mice that also have hypophosphatemia rather than hyperphosphatemia likely due to the hyperparathyroidism and decreased activity of the Na/Pi-2a cotransporter [79,80].
Similar effects were observed in FGF23/VDR double null mice on the rescue diet [81]. Thus, mineral metabolism changes in the FGF23-null mice are mediated in large part by the 1,25(OH)2D/VDR pathway. FGF23 utilizes the transmembrane protein Klotho as a cofactor to enhance binding in FGF23eFGF receptor interactions [82]. The mineral and bone phenotypes of the Klotho- and FGF23-null mice are very similar [83]. Klotho-null mice have very elevated serum FGF23 levels, but hyperphosphatemia and hypervitaminosis D associated with increased expression of Na/Pi-2a cotransporter and 1a(OH)ase [80,84,85]. The phenotype was significantly rescued by feeding the mice a vitaminD-deficient diet [85]. Hyperphosphatemia was the most important factor in causing soft tissue calcifications. In contrast, Na/Pi-2a-null mice had hypophosphatemia with secondarily increased serum 1,25(OH)2D and calcium levels causing soft tissue calcifications, and this was rescued in Na/Pi-2a/1a(OH)ase double null mice [86]. Consequently 1,25(OH)2D, when contributing to either hyperphosphatemia or hypercalcemia may be associated with increased soft tissue calcifications. VDR-null mice have undetectable circulating FGF23 levels indicating a role for the 1,25(OH)2D/VDR system in regulating FGF23 production. However normalization of calcium and phosphate by dietary means in either the VDR- or 1a(OH)ase-null mice increases circulating FGF23 levels. Hence, there are 1,25(OH)2D/VDRdependent and -independent modes of regulation. These studies show the fundamental role of the 1,25 (OH)2D/VDR system on mineral and skeletal homeostasis and point to discrete and interacting functions of this system with calcium and phosphate in modulating mineral and skeletal homeostasis.
EXTRASKELETAL ACTIONS OF 1,25(OH)2D The VDR is widely distributed [87] and there is a broad spectrum of vitamin-D-dependent genes [88] pointing to the fact that vitamin D likely subserves important functions other than the regulation of mineral and skeletal homeostasis (Fig. 33.1). The fact that circulating 25(OH) D levels are a more valuable clinical biomarker than 1,25(OH)2D levels appears to be the result of the role of 25(OH)D as a substrate for the action of extrarenal 1a(OH)ases in producing locally active 1,25(OH)2D [89] which may then carry out extraskeletal actions of vitamin D (see also Chapter 47). Epidemiological studies provide support for the importance of extraskeletal actions of 1,25(OH)2D. One study reported a decrease in all-cause mortality (from 28.6% to 13.8% over 2 years) in hemodialysis patients supplemented with active vitamin D versus those not receiving
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Skeletal Actions
Mineral–Regulating Actions
Extra-Skeletal Actions
Parathyroid Kidney
VDR
PTH
Bone FGF23
25(OH)D Ca++,↑Pi
VDR
–
1,25(OH)2D VDR
Intestine
+
CV Immune Cell Cycle Skin +Hair CNS Muscle
Skeletal and extraskeletal actions of vitamin D. 1,25(OH)2D produced in the kidney from 25(OH)D can enhance calcium (Caþþ) and phosphate (Pi) absorption in the gut (solid arrow) through the action of the VDR and, at least in rodents, can also increase renal calcium reabsorption (solid arrow) resulting in increased Caþþ and Pi in the extracellular fluid. At high concentrations, 1,25(OH)2D can also increase bone resorption (þ) contributing to the increased extracellular Caþþ and Pi. The Caþþ and Pi are essential for mineralization of bone. Circulating and/or locally produced 1,25(OH)2D can inhibit PTH gene transcription, and inhibit parathyroid cell proliferation. Reduced PTH action can decrease entry of Caþþ and Pi from bone into the extracellular fluid, and increase urinary Caþþ excretion. 1,25(OH)2D may also have a direct anabolic action on bone () and can stimulate FGF23 release from osteoblasts/osteocytes. FGF23, through a phosphaturic effect, can decrease extracellular Pi and, by inhibiting 1(OH)ase, can reduce renal 1,25(OH)2D production which further limits increases in Caþþ and Pi. Increasing evidence also points to a role for circulating and/or locally synthesized 1,25(OH)2D in a variety of extraskeletal actions including effects on the cardiovascular system (CV), the immune system, the cell cycle and cancer development, skin and hair development, and central nervous system (CNS) and muscle function.
FIGURE 33.1
the vitamin D metabolite [90]. The study also reported a reduction in cardiovascular mortality, the major cause of death in hemodialysis patients, from 14.6% to 7.6% over 2 years. In end-stage chronic kidney disease, the deficiency of 1,25(OH)2D is extreme. However, a metaanalysis of all-cause mortality in 18 randomized controlled trials in which vitamin D treatment was used, generally for osteoporosis and fracture prevention (in nondialysis patients), also found a significant reduction in mortality of 7% [91] (see also Chapter 71). The generation of mouse models null for the 1a(OH) ase and the VDR genes has facilitated the examination of the consequences of vitamin D deficiency on several critical extraskeletal functions. These include immune function [92], the cell cycle and cancer development [93], glucose homeostasis [94], the cardiovascular system and blood pressure regulation [95], hair cycling and epidermal differentiation, [12] and muscle [96], brain [97] and reproductive systems [98]. (For additional insight, see specific chapters on these elsewhere in this volume.) We will review the evidence for only several of these functions.
The Vitamin D/VDR System and Cardiovascular Function Many cells in the cardiovascular system, including vascular endothelial cells, vascular smooth muscle cells,
cardiomyocytes, and monocytes/phagocytes express the VDR and respond to 1,25(OH)2D [99] (see Chapters 31 and 104). In addition, the juxtaglomerular cells of the nephron, which produce renin, are also 1,25(OH)2 D-sensitive. Renin is a protease that cleaves angiotensinogen to angiotensin I, which is subsequently converted to angiotensin II (see Chapter 40). Angiotensin II in turn activates specific receptors and regulates electrolyte, volume, and blood pressure homeostasis. In VDR-null mice, increased renal renin mRNA levels have been observed, leading to high plasma angiotensin II levels, systemic hypertension, and cardiac hypertrophy [100]. A VDRE has been identified in the renin gene promoter, and direct inhibition of renin expression by 1,25(OH)2D has been reported in vitro [100]. The effect of the absence of 1,25(OH)2D production on blood pressure regulation and cardiac structure and function have also been determined in the 1a(OH)asenull mouse and the consequences of repleting the mice with active vitamin D or treating with antihypertensives have been examined [101]. In 1a(OH)asee/e mice on a normal diet, systolic blood pressure, renal angiotensinogen and renin expression, circulating angiotensin II, renin and aldosterone were all increased. Myocardial structure was abnormal with heart-to-body ratio, myocyte diameter, interventricular mass relative to body weight, and relative wall thickness all being increased. Myocardial function was impaired with reduced percent
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fractional shortening and systolic ejection function. Any or all of the hypocalcemia, hypophosphatemia, and elevated serum PTH levels in these mice could potentially have an impact on the parameters measured and hence have a confounding effect on the interpretation. However, even when these were all normalized on the rescue diet, the elevated blood pressure, activation of the renin/angiotensin system, altered myocardial structure, and reduced function persisted. The 1a(OH) ase-null mice were treated with 1,25(OH)2D3, the angiotensin-converting enzyme inhibitor, captopril, or the angiotensin type 1 receptor antagonist, losartan. Each agent prevented the elevation in blood pressure, the development of myocardial hypertrophy, and the reduction in cardiac function. Cardiac and renal angiotensinogen and renin were elevated in the untreated mice and the 1a(OH)ase and VDR were present in the whole heart (although their compartmentalization was not evaluated in this study). Consequently while the circulating 1,25 (OH)2D can access and activate the VDR in tissues there is also the potential for a local system of 1,25(OH)2D production and action. In human studies, blood pressure in both normotensive [102,103] and hypertensive subjects [104] is inversely correlated with plasma 1,25(OH)2D and renin concentrations, and 1a-hydroxylated vitamin D has been reported to reduce blood pressure in hypertensive patients [105]. The regulation of the renin/angiotensin system by the 1,25(OH)2D/VDR system may also contribute to renoprotective effects of 1,25(OH)2D. Thus, after streptozotocin-induced diabetes, VDR-null mice exhibit an earlier onset and more severe diabetic nephropathy, characterized by increased proteinuria, higher renin, angiotensin and angiotensin receptor expression, more mesangial sclerosis, lower nephrin expression, and decreased podocyte number [106]. Renoprotective effects of the vitamin D system have also been reported in patients with chronic renal failure in whom vitamin D analogs were able to reduce proteinuria [107]. Even in the absence of hypertension, however, it has been reported that VDR-null mice develop cardiac hypertrophy and fibrosis, suggesting that 1,25(OH)2D may also have direct effects on prevention of cardiocyte hypertrophy [108] (see Chapters 31 and 104). Cardiac myocytes from VDR-null mice showed accelerated rates of contraction and relaxation, compared with WT mice, and 1,25 (OH)2D3 directly affected contractility in WT but not in VDR-null cardiac myocytes [109]. Deficient expression of tissue inhibitors of metalloproteases-1 and -3 leading to increased extracellular matrix production and fibrosis has also been described in VDR-null mice [110], and this could contribute to cardiac hypertrophy. There are several other beneficial effects of the 1,25 (OH)2D/VDR system on cells of the cardiovascular
system which may lead to abnormalities when vitamin D action is insufficient (Fig. 33.2). In VDRnull mice, platelet aggregation was increased and lipopolysaccharide (LPS)-induced, multiorgan thrombosis was enhanced, consistent with increased tissue factor expression in liver and kidney and decreased thrombomodulin expression in several tissues [111]. Based on in vitro effects of 1,25(OH)2D and in vivo effects in VDR-null mice, the overall effects of 1,25(OH)2D on cells of the vascular wall and on cardiomyocytes provide synergistic beneficial effects on cardiovascular function. Nevertheless, it is well known that vitamin D toxicity is associated with ectopic calcification, especially of the vascular wall (see also Chapter 73). This has been observed in rodents and patients exposed to high doses of exogenous vitamin D, as well as in mice with endogenous overproduction of 1,25(OH)2D due to targeted gene deletion of CYP24A1 [112], FGF23 [113], and Klotho [83]. These ectopic calcifications are also observed in the kidney and other soft tissues, leading to organ failure and early lethality. The ectopic calcifications are partially due to excess extracellular concentrations of calcium and phosphate, but in some tissues (for example, the vascular wall) a true cellular transformation of mesenchymal cells into osteoblast-like cells is induced by excess 1,25(OH)2D or excess serum phosphate and membrane phosphate transporter Pit 1 [114]. Thus, the therapeutic window for the efficacy of vitamin D on the cardiovascular system needs to be clarified and both deficiency and excess vitamin D need to be avoided.
The Vitamin D/VDR System and Cancer Vitamin D insufficiency has been implicated in the development of cancer [115] and several studies point to the importance of vitamin D in a variety of cancers including colon, ovarian, prostate, esophageal, nonHodgkin’s lymphoma, and breast cancer [116,117] (see also chapters in Sections VII and X of this volume). Epidemiological evidence links low levels of vitamin D to increased risk of cancer [116,118e120], for example, increased circulating concentrations of vitamin D and its metabolite 25(OH)D are associated with a lower risk of breast cancer whereas low concentrations are associated with faster progression of metastatic breast cancer [121]. The connection between cancer and vitamin D has also been made through analyses of the geographic distribution of cancer in comparison to sunlight exposure [118]. Vitamin D and 25(OH)D concentrations are inversely correlated with latitude and increase directly with increased sun exposure [122e124], and it has been documented that latitudes above 37oN and below 37oS of latitude, which result in
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EXTRASKELETAL ACTIONS OF 1,25(OH)2D
1,25(OH)2D
Insulin secretion, Insulitis, Insulin Resistance
Up-regulation of the renin/angiotensin system
Inflammation, atherogenesis, Protection against blood vessel injury
Diabetes Mellitus Cardiac fibrosis, Myocyte contractility
Atherosclerosis
HTN CH CVD FIGURE 33.2 Potential mechanisms for effects of the 1,25(OH)2D/VDR system on cardiovascular disease (CVD). Reduced action (Y) of the 1,25(OH)2D/VDR system can up-regulate the renin/angiotensin system contributing to hypertension (HTN) and cardiac hypertrophy (CH). The up-regulated renin/angiotensin system likely also contributes directly to CH. In addition, increased ([) cardiac fibrosis, enhanced myocyte contractility may contribute to CH. There is also evidence for reduced insulin secretion, inflammation of the islets (insulitis) and increased glucose intolerance (insulin resistance) in the absence of vitamin D that may predispose to diabetes mellitus and contribute to CVD. Finally, reduced vitamin D has been associated with increased inflammation and atherogenesis as well as increased vascular wall injury which may contribute to atherosclerosis and CVD.
the decreased synthesis of vitamin D3 [118], are associated with increased cases of cancer. Additionally, in various in vitro cancer cell models such as the keratinocyte HPK1 and HPK1A RAS cell lines, and the MCF-7 cell lines which mimic breast cancer, vitamin D has been shown to inhibit proliferation and promote differentiation [121,125e135] (see chapters in Section X of this volume). Furthermore, in vivo administration of 1,25(OH)2D3 or its analogs to animals shows reduced mammary cancer [127,128,136] and decreased malignancy-associated hypercalcemia [137]. As a result 1,25 (OH)2D has been seen as a potential treatment for cancer [129,138]. Several mechanisms have been implicated in the antineoplastic effects of vitamin D, including an effect on inhibiting cell proliferation and inducing differentiation (Fig. 33.3). Thus, in most cell types that express a functional VDR, 1,25(OH)2D produces an accumulation of cells in the G0/G1 phase of the cell cycle [139]. The exact sequence of events between VDR-mediated transactivation, or repression of target genes, and actual G0/G1 arrest is probably cell-type-specific and multiple pathways regulating the cell cycle have been implicated [140e142]. 1,25(OH)2D also induces apoptosis in a number of tumor models, including carcinomas of the breast, colon, and prostate, and again a variety of mechanisms have been invoked. In addition, 1,25 (OH)2D has been demonstrated to inhibit angiogenesis
in experimental models both in vitro and in vivo [143,144]. This antiangiogenic activity may be mediated by inhibition of proliferation of tumor-derived endothelial cells, and/or by repression of the release of angiogenic factors, such as vascular endothelial growth factor, TGF-alpha, and epidermal growth factor [145,146]. Finally 1,25(OH)2D may inhibit degradation of the extracellular matrix of tumor cells, a process required for invasion to secondary sites [147e149]. Regulation of production of 1,25(OH)2D at extrarenal sites including skin, prostate, colon, pancreas, brain, parathyroid glands, and breast [150e156] is substratedependent (see Chapter 45) and in vitro studies have demonstrated a linear increase in 1,25(OH)2D production with increasing availability of 25(OH)D [157]. This local production appears to be dependent on substrate availability from both skin production of vitamin D and oral vitamin D intake. Consequently, low exposure to vitamin D should result in lower tissue concentrations of both substrate 25(OH)D and its product 1,25(OH)2D, which may accelerate local cell growth and cancer susceptibility. Conversely, raising circulating 25(OH)D levels should deliver more substrate to these target organs, enhance local 1,25(OH)2D production and prevent tumor progression. 1,25(OH)2D tissue levels are also subject to regulation via degradation by the 24-hydroxylase pathway [158], thus protecting against excessive accumulation of 1,25(OH)2D in normal tissues
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Calcitroic acid
25(OH)D
1α(OH)ase [Cyp27B1]
24(OH)ase [Cyp24] vdr
1,25(OH)2D
Induce G0/G1 Arrest Stimulate differentiation or apoptosis inhibit angiogenesis Normal tissue development; Deletion of mutated cells
inhibit degradation of extracellular matrix Tumor prevention or regression
Potential mechanisms for effects of the 1,25(OH)2D/VDR system on cancer development and progression. Circulating 25(OH)D may act as a substrate for locally produced 1,25(OH)2D via the action of extrarenal 1a(OH)ase (Cyp27B1) in multiple cell types. The 1,25(OH)2D generated may act via the VDR in an intracrine and/or autocrine mode to modify gene transcription. The enzyme 24(OH)ase (Cyp24), induced by activation of the VDR may then metabolize 1,25(OH)2D, ultimately to calcitroic acid, and limit concentrations of active vitamin D within the cell. Activation of VDR can also alter genes contributing to cell cycle arrest, stimulation of differentiation, inhibition of apoptosis, inhibition of angiogenesis, and inhibition of matrix degradation. In contrast, reduced VDR signaling can result in enhanced proliferation, increased sensitivity to transformation, and increased tumor invasiveness.
FIGURE 33.3
(see Chapter 80). However, in cancer tissues this pathway may be overactive, resulting in lower 1,25 (OH)2D tissue levels and theoretically leading to enhanced local cell proliferation. Therefore, it has been proposed that 24-hydroxylase is a possible candidate oncogene [159]. Although there is strong epidemiological evidence that vitamin D insufficiency increases the risk and perhaps progression of several types of cancer, a direct causal relationship has been more challenging to establish. Immunodeficient mice transplanted with a human colon cancer cell line had enhanced tumor growth when raised on a vitamin-D-deficient diet [160]. Indirect evidence that vitamin D insufficiency leads to accelerated tumorigenesis also comes from studies using VDR knockout mice. Skin tumors were induced with DMBA in this model whereas wild-type animals only developed epidermal hyperplasia indicating that VDR signaling presumably through 1,25(OH)2D activation, protects against tumor development [161]. In the same VDR ablation model breast tumor development was also investigated by crossing these animals with a mouse mammary tumor virus (MMTV)-neu transgenic model [161]. In this model, loss of one copy of the VDR allele resulted in accelerated mammary tumor development. More recently, VDR knockout mice were crossed with
the LPB-Tag prostate cancer model. Prostate tumors progressed more rapidly in the VDR knockout animals compared to VDR wild-type animals [162]. These animal studies support a mechanism linking local substrate 25 (OH)D availability and tumor progression. Several studies using tissue-specific 1a-hydroxylase ablation to understand the role of local tumoral production of 1,25 (OH)2D have provided further evidence supporting its autocrine/intracrine function. Thus, in a mouse model of skin cancer, conditional ablation of 1a(OH)ase resulted in accelerated skin tumor growth in vivo [129], whereas administration of substrate 25(OH)D to animals transplanted with wild-type skin tumors significantly suppressed tumor growth. In a human melanoma metastatic model in vivo knockdown of 1a(OH) ase accelerated the development of bone metastatic lesions [163] demonstrating that local conversion of 25(OH)D to 1,25(OH)2D may be beneficial in preventing metastatic spread of melanoma to the skeleton. The postulated autocrine/paracrine role of 1a(OH) ase in breast cancer development and progression has also recently been investigated in the PyMT mouse model of breast tumor progression [164]. The PyMT mouse breast cancer model closely mimics many features of human breast cancer progression. Mouse mammary tumor virus (MMTV) is an archetypal
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B-type retrovirus whose presence is linked to the development of mammary adenocarcinomas [165]. The MMTV does not contain a conventional oncogene and therefore the induction of mammary adenocarcinomas by MMTV is closely tied to the activation of cellular proto-oncogenes such as Neu, Polyomavirus (PyV) large and middle T antigens by insertional mutagenic activation [166]. The long terminal repeats (LTR) of MMTV can activate nearby genes through steroid hormonedependent mechanisms. The tumors are restricted to the mammary epithelium because they are under the control of the mammary-gland-specific MMTV promoter/enhancer [167]. In the PyVMT transgenic model of breast cancer all female PyVMT rapidly develop multifocal mammary adenocarcinomas that are palpable as early as 5e7 weeks of age [168e170] and by 7e8 weeks of age mammary tumors develop in 100% of PyVMT mice. In addition, female PyMT mice also develop pulmonary metastases by about 11e13 weeks of age, with an extremely high penetrance (~90e100%) [165e167,169,171,172]. This mouse tumor model is characterized by the up-regulation of ErbB2/ Neu and cyclin D1 expression [167] and closely mimics the actual cascade of events that coincide with human breast cancer. In this model, administration of the inactive precursor 25(OH)D increased local production of 1,25(OH)2D in breast tumor cells and slowed the development and progression of breast cancer without inducing significant side effects such as hypercalcemia [173]. Subsequently, tissue-specific ablation of the 1a (OH)ase gene was achieved in these animals prior to the appearance of breast tumors using a Cre/loxP recombination system that disrupts 1a(OH)ase in the mammary epithelium [174]. The resulting homozygous mice (PyVMT-1a(OH)aseflox/flox; Creþ) therefore do not express 1a(OH)ase in the mammary epithelium, while the heterozygous mice (PyVMT-1a-hydroxylaseflox/þ; Creþ) present lowered levels of 1a(OH)ase expression. In animals fed a normal diet with normal vitamin D status, a reduction or elimination of 1a(OH)ase expression in the mammary epithelium resulted in a marked acceleration in tumor growth as compared to control mice. Tumor weight at sacrifice was almost double in homozygous animals as compared to controls. Overall these studies establish a plausible link between vitamin D insufficiency and breast cancer as a result of decreased availability of tumoral 1,25(OH)2D production favoring breast cancer development and progression. In summary the causal link between vitamin D insufficiency and cancer development suggested by epidemiological data seems to be supported by animal models that have been subjected to dietary or genetic manipulations. These studies point to the crucial importance of 1a (OH)ase expression in modulating tumor progression at the local tissue level independently of systemic 1,25
593
(OH)2D production. Nevertheless although 1,25(OH)2D potentiates the antitumor activities of multiple chemotherapeutics agents and of gamma irradiation in humans, well-designed clinical trials demonstrating a definitive role for vitamin D or its analogs in cancer treatment in humans remain to be performed [175].
The Vitamin D/VDR System and Skin The skin can synthesize vitamin D in response to UV radiation, metabolically activate vitamin D via 25(OH)D to 1,25(OH)2D, respond to the active metabolite via the VDR, and inactivate these metabolites via CYP24A1 (see also Chapters 3 and 30). Increases in calcium and 1,25(OH)2D promote differentiation of epidermal keratinocytes that express the VDR [176,177]. Epidermal keratinocyte differentiation is noticeably abnormal in VDR-null mice after 2 weeks of age, and this phenotype is rescued by normalization of the hypocalcemia [178]. In vitro, keratinocytes from VDR-null mice differentiate in response to calcium but not to 1,25(OH)2D [179]. VDR is Essential for Hair Cycling In the skin, in addition to the epidermal keratinocytes, the VDR is also expressed in the outer root sheath and hair follicle bulb and in sebaceous glands. Alopecia is a feature, in the majority of VDDR-II kindreds, of affected humans having VDR mutations [180] (see Chapter 65). Consistent with this, after 21 days of age VDR-null mice develop perioral and periorbital hair loss and eventually alopecia totalis, with large dermal cysts [11e14] (see Chapter 30). VDR-null mice fed a vitamin-D-deficient diet and UV restricted, and with undetectable 1,25(OH)2D concentrations, still develop alopecia whereas wild-type mice do not [24]. Hence the elevated levels of 1,25(OH)2D in the VDR-null mice are not implicated in the pathogenesis of the alopecia. Likewise, 1a(OH)ase-null mice and affected humans with homozygous 1a(OH)ase mutations in VDDRI kindreds do not develop alopecia [9,10,180] (see Chapter 64). Hence the effects of the VDR in the hair follicle do not require 1,25(OH)2D. The formation of the hair follicles has three stages. During anagen, a mature hair follicle that forms a hair shaft from keratinocyte stem cells is generated. Then, keratinocytes regress to the level of the hair follicle bulge during catagen followed by the quiescent phase, telogen. During telogen, the proximity of dermal papilla cells to the keratinocyte stem cells in the bulge enables signaling between them resulting in reinitiation of anagen. While proliferation of neonatal keratinocytes is normal in VDR-null mice, the subsequent response to anagen-initiating stimuli after hair follicle morphogenesis has finished is lost [179]. The VDR is critical for regulating postnatal hair cycles.
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Reconstitution assays using keratinocytes and dermal papilla cells from neonatal wild-type and VDR-null mice reimplanted subcutaneously into nude mice demonstrated that while dermal papilla cells from VDR-null mice functioned normally keratinocytes lacking the VDR did not [181]. Hence, while hair follicle morphogenesis is normal the postmorphogenic hair cycle is impaired. Crossing of transgenic mice expressing the VDR specifically in keratinocytes with the VDR-null mice, rescued the alopecia while the mineral and bone abnormalities remained unaltered [182,183]. Therefore the alopecia is due to impaired VDR action in the keratinocyte component of the hair follicle. Potentially, binding of the VDR to a novel endogenous ligand made in the skin might be responsible for normal hair growth and function in the absence of vitamin D and 1,25(OH)2D. Studies were conducted by crossing transgenic mice expressing mutant transgenes specifically in the keratinocyte with VDR-null mice. While a ligand-binding domain mutant unable to bind either 1,25(OH)2D or the alternative ligand, lithocholic acid, was fully capable of rescuing the alopecia defect, a VDR transgene with a mutation in the nuclear receptor coactivator binding (AF-2) domain retarded the onset while not entirely preventing the alopecia [184]. Thus, the epidermal VDR acts in a ligand-independent fashion but requires interactions with nuclear factors. The atrichia caused by VDR mutations is a phenocopy of the atrichia caused by the corepressor, Hairless [185]. Hairless binds the VDR and represses VDR-mediated gene transactivation [186,187]. However, neither of the mutants referred to above, with ligand-binding or AF-2 domain mutations, had altered VDReHairless interaction [184]. Canonical Wnt signaling plays a key part in hair follicle development and participates in postmorphogenic hair cycling (see also Chapter 13). VDR-null mice, Hairless-null mice, and mice in which a keratinocyte-specific transgene for the b-catenin-responsive Lef1 transcription factor that is mutated such that it cannot interact with b-catenin, all exhibit dermal cysts and increased sebaceous glands [188]. The VDR complexes with b-catenin and Lef1 and is essential for their activation of a Wnt response element containing promoter [189] (see Chapter 8 for molecular detail). Hairless also promotes Wnt signaling. Abnormal canonical Wnt signaling is a common pathway by which VDR and Hairless mutations cause cutaneous abnormalities. 1a(OH)ase is Required for Optimal Epidermal Differentiation Keratinocytes highly express the 1a(OH)ase. While there is no gross skin phenotype in 1a(OH)ase-null mice the expression of differentiation markers involucrin, profilaggrin, and loricrin is reduced [190]. 1a(OH)
ase-null mice have a reduced ability to recover normal barrier function after perturbation. Therefore, local synthesis of 1,25(OH)2D is important for normal epidermal differentiation. Inactivation of 1a(OH)ase alleles in a ras-transformed keratinocyte cell that produces squamous cell carcinoma in nude mice led to the tumors being unresponsive to growth inhibition by locally administered 25(OH)D, but responsive to the antiproliferative and prodifferentiating effects of 1,25 (OH)2D [129]. Thus, keratinocyte 1a(OH)ase activity is important for autocrine regulation of their growth and differentiation.
The Vitamin D/VDR System and the Immune System The 1a(OH)ase and the VDR are expressed in most cells of the immune system, including activated CD4þ and CD8þ T lymphocytes, B cells, and antigen-presenting cells (APCs) such as macrophages and dendritic cells (DCs) [191e193] (see chapters in Section XI). Regulation of expression and activity of the 1a(OH)ase in immune cells differs from that in the kidney. Thus the macrophage enzyme, originally discovered in association with granulomatous disease such as sarcoid, is primarily regulated by immune signals such as interferon (IFN)g which inhibits activity, and Toll-like receptor (TLR) which can stimulate it [194,195]. The enzyme is not feedback-inhibited by 1,25(OH)2D itself. Up-regulation of 1a(OH)ase, by DCs and other immune cells, typically occurs at later stages of immune activation, and the 1,25(OH)2D produced down-regulates many immune responses resulting in a negative feedback loop. The 24(OH)ase enzyme is also expressed in immune cells [196]. Undifferentiated monocytes are highly susceptible to 1,25(OH)2D-mediated 24(OH)ase induction, whereas differentiated/activated macrophages are resistant due to inhibition of 1,25(OH)2D production by IFNg. The 1,25(OH)2D/VDR complex in turn inhibits IFNg and granulocyte macrophage colony stimulating factor production and interferes with the signaling of transcription factors such as NFkB, nuclear factor of activated T cells (NFAT), and activating protein-1 (AP-1) that play a crucial role in regulating immunomodulatory genes [197] such as interleukin (IL)-8, IL-12, IL-2, and IL-4 [198]. Mechanistically, 1,25(OH)2D has many effects on the adaptive and innate immune systems. With respect to adaptive immunity, it inhibits the surface expression of MHC II-complexed antigen and of costimulatory molecules, as well as the production of the cytokine IL-12 in APCs, thereby shifting T cells from an (auto-) aggressive effector (Te) phenotype toward a protective or regulatory (Tr) phenotype. 1,25(OH)2D also exerts its immunomodulatory effects directly at the level of T cells. In
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EXTRASKELETAL ACTIONS OF 1,25(OH)2D
vitamin D deficiency the 1,25(OH)2D-mediated attenuation of pathological T helper 1 cell (Th1) immune responses is impaired. These immunomodulatory effects of 1,25(OH)2D on the adaptive immune system can lead to the protection of target tissues in autoimmune diseases whereas 1,25(OH)2D deficiency provides an increased risk for Th1-mediated autoimmune diseases such as inflammatory bowel disease [199], rheumatoid arthritis [200], systemic lupus erythematosus [201], multiple sclerosis [202], and type 1 diabetes [203] (see chapters in Section XI of this volume). Much evidence has been obtained from epidemiological data, which has linked vitamin D deficiency to increased prevalence of autoimmune diseases in humans. The consequences of absent VDR signaling on the immune system have been studied in VDR-null mice. In normocalcemic VDR-null mice, a defect in Th1related IFNg production was observed, presumably related to defective IL-18 production by macrophages and decreased expression of signal transducer and activator of transcription (STAT)4 in T cells per se [204]. Abnormalities in DCs from skin-draining lymph nodes were noted with a higher proportion of more mature DCs, and an accelerated maturation and increased responsiveness to IgE-mediated stimulation of mast cells, the main Th2-type effector cells that releases histamine [205]. As well, in VDR-null mice, the severity of different models of inflammatory bowel disease has been reported to be exacerbated [206], accompanied by increased expression of inflammatory and Th1-related cytokines and chemokines in the intestinal tract (such as IL-1, TNFa, IFNg, IL-12, and MIP1a). Vitamin D deficiency in animal models of autoimmune disease may also result in advanced presentation of the disease. On the other hand, normocalcemic VDR-null mice have been reported to be less susceptible to experimentally induced autoimmune encephalomyelitis, a model of multiple sclerosis [207]. In contrast, experimental autoimmune encephalomyelitis and inflammatory bowel disease are accelerated in vitamin-D-deficient animals [208] and vitamin D supplements have been reported to provide protection from disease [209]. Nonobese diabetic (NOD) mice, an animal model of type 1 diabetes that spontaneously develops this autoimmune disorder, have been reported to express a defect in up-regulation of 1a(OH)ase in response to immune stimuli, and vitamin D deficiency during early life, results in a more aggressive manifestation of type 1 diabetes in NOD mice [210]. Furthermore, 1,25(OH)2D and its analogs can prevent diabetes and insulitis in NOD mice when treatment is started before the onset of insulitis [211]. The positive effects of 1,25(OH)2D and analogs in this model appear to be a consequence of the restoration of defective suppressor cell activity; the enhanced
595
clearance of autoreactive T cells, by restoring sensitivity to apoptosis in immune system; and a shift from a Th1 to a Th2 cytokine expression profile locally in the pancreas and in the pancreas-draining lymph nodes. Analysis of the immune system of compound VDR-null/NOD revealed severe immune defects considered to be crucial for the development of diabetes, but the VDR-null/ NOD mice do not display increased susceptibility to diabetes. The apparent discrepancies between the phenotypes of deficiency of the vitamin D ligand and deficiency of the VDR in these models remain to be resolved. VDR-null mice are resistant to experimentally induced airway inflammation and asthma [212] (see chapters in Section XII of this volume). Altered T cell homing behavior could play a role not only in the resistance of VDR-null mice to allergy (as well as in their increased susceptibility to inflammatory bowel disease), but the resistance was proposed to be due to a failure of the lung environment to respond to inflammation and attract pathogenic immune cells (Th2 cells and eosinophils), and not to defects in the priming and lung homing of the immune cells themselves. VDR-null mice are more resistant to parasitic Leishmania major infection [213]. This differs markedly from the association between vitamin D deficiency and increased susceptibility for most nonparasitic infections, and may be related to the fact that parasitic infections may be more closely linked with Th2 defense mechanisms, whereas the innate immune system and Th1 arm of the adaptive immune system defend against other infections. In particular, the critical involvement of the innate immune system has been demonstrated in the role of myocbacterial TB infection in humans where TLR2 could increase 1,25(OH)2D production and, with 1,25(OH)2D, stimulate expression of the antibacterial protein cathelicidin [214], resulting in vitamin-D-promoted monocyte killing of M. tuberculosis [215]. Thus, mechanistically, 1,25(OH)2D can stimulate the innate immune system and strengthen the antimicrobial function of monocytes and macrophages, for example through enhanced expression of cathelicidin, eventually leading to better clearance of pathogenic microorganisms and thus to prevention of increased susceptibility to infections. Vitamin D can also act to promote tolerance as shown for example by studies of pancreatic islet transplantation in which lower rejection rates were observed in 1,25 (OH)2D3-treated mice [216]. Overall, therefore studies in mouse models with alterations in the vitamin D/VDR system and with dysregulated immune function have shed important light on the pathogenesis of immune disease, and provide evidence that prevention of vitamin D deficiency is important for a healthy immune system.
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33. CONTRIBUTIONS OF GENETICALLY MODIFIED MOUSE MODELS TO UNDERSTANDING THE PHYSIOLOGY
APC (egDC)
MHC-II
lL-12 (Th1 polarization factor) IL-23 (Th17 polarization factor) IL-10 (Treg promoting factor) Treg
CD83 CD 86
IL-10
CD 40 1,25(OH)2D
Th2
CD4+ cells
IL-4 IL-17
Th17
chemotaxis phagocytosis cathelicidin
B cell MФ
Th1
B cell proliferation Plasma cell differentiation
IL-21
IL-2 IFNγ
IgG, IgM production INNATE IMMUNE RESPONSE
ADAPTIVE IMMUNE RESPONSE
Effects of the 1,25(OH)2D/VDR system on the immune system. 1,25(OH)2D stimulates innate immune responses by enhancing the chemotactic and phagocytotic responses of macrophages (MV) as well as the production of antimicrobial proteins such as cathelicidin. 1,25 (OH)2D also modulates adaptive immunity. At the level of the APCs (such as DCs), 1,25(OH)2D inhibits the surface expression of MHC-IIcomplexed antigen and of costimulatory molecules such as CD83, CD86, and CD40 to reduce activation of CD4þ lymphocytes. In addition it inhibits the production of the cytokines IL-12 and IL-23, thereby indirectly shifting the polarization of T cells away from Th1 and TH17 phenotypes and towards a Th2 phenotype. 1,25(OH)2D also directly affects T cell responses, by inhibiting the production of Th1 cytokines (IL-2 and IFNg), Th17 cytokines (IL-17 and IL-21), and by stimulating Th2 cytokine production (IL-4). Furthermore, 1,25(OH)2D favors Treg cell development via modulation of DCs and by directly targeting T cells. Finally, 1,25(OH)2D blocks B-cell proliferation, plasma-cell differentiation, and IgG and IgM production.
FIGURE 33.4
In summary (Fig. 33.4), 1,25(OH)2D affects the adaptive immune system indirectly by decreasing monocyte-derived DC maturation, decreasing antigen presentation, and indirectly influencing the T cell repertoire. It also directly decreases Th1 cytokines and therefore cytotoxic T cell production, decreases Th17, and therefore reduces inflammation and autoimmunity, increases Th2 cytokines, and increases Treg and therefore increases suppression of helper T cells. 1,25(OH)2D also reduces B cell production and immunoglobulin production. 1,25(OH)2D affects innate immunity by increasing macrophage differentiation (for phagocytic acquisition and elimination of pathogens and cell debris) and by increasing bacterial killing (for example by increasing expression of cathelicidin). The clinical implications of vitamin-D-mediated immunity in humans now need to be explored in detail and the possible beneficial effects of supplementary vitamin D with respect to autoimmune and infectious diseases now needs to be proven [217].
CONCLUSION Genetically modified mouse models of the 1a(OH)ase and of the VDR have provided highly controlled conditions in which to study the actions of the 1,25(OH)2D/ VDR system. It has been possible to examine global actions of the system and tissue-specific actions, and to distinguish effects on skeletal and mineral homeostasis from extraskeletal actions. By controlling the environment in which these genetic models grow it has been possible to distinguish direct effects of the system and indirect effects mediated by calcium and phosphate. Genetic models may also facilitate assessment of the temporal actions of the vitamin D system. Careful comparison of the phenotypes of mice with targeted ablation of the 1a(OH)ase and those with targeted ablation of the VDR may provide insights into effects of 1,25 (OH)2D independent of the VDR and of ligand-independent effects of the VDR [218]. In vivo studies of the molecular physiology of the 1,25(OH)2D/VDR system have been expanded by ex vivo studies of tissues from
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REFERENCES
these models which have provide further mechanistic understanding of the actions of the system. Extrapolation of the insights from these models to the human condition is often more difficult to approach experimentally but results from the studies with genetically modified animals can set the stage for appropriate directions in order to define the role of vitamin D deficiency and vitamin D therapy in human disorders.
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enhances invasion in vitro and skeletal metastasis in vivo, J. Bone Miner. Res. 21s1 (2006) 1100. E.Y. Lin, J.G. Jones, P. Li, L. Zhu, K.D. Whitney, W.J. Muller, et al., Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases, Am. J. Pathol. 163 (2003) 2113e2126. S. Eash, K. Manley, M.L. Gasparovic, W. Querbes, W.J. Atwood, The human polyomaviruses, Cell Mol. Life Sci. 63 (2006) 865e876. K.A. Gottlieb, L.P. Villarreal, Natural biology of polyomavirus middle T antigen, Microbiol. Mol. Biol. Rev. 65 (2001) 288e318. M. Bocchetta, M. Carbone, SV40-Mediated oncogenesis. Malignant Mesothelioma Advances in Pathogenesis, Diagnosis, and Translational Therapies, Springer, New York, 2000, pp. 34e59. C.J. Dawe, R. Freund, G. Mandel, K. Ballmer-Hofer, D.A. Talmage, T.L. Benjamin, Variations in polyomavirus genotype in relation to tumor induction in mice, Am. J. Pathol. 127 (1987) 243e261. M. Berebbi, L. Dandolo, J. Hassoun, A.M. Bernard, D. Blangy, Specific tissue targeting of polyoma virus oncogenicity in athymic nude mice, Oncogene 2 (1988) 149e156. S.Z. Haslam, J.J. Wirth, L.J. Counterman, M.M. Fluck, Characterization of the mammary hyperplasia, dysplasia, and neoplasia induced in athymic female adult mice by polyomavirus, Oncogene 7 (1992) 1295e1303. P.M. Siegel, W.R. Hardy, W.J. Muller, Mammary gland neoplasia: insights from transgenic mouse models, Bioessays 22 (2000) 554e563. D.L. Dankort, W.J. Muller, Signal transduction in mammary tumorigenesis: a transgenic perspective, Oncogene 19 (2000) 1038e1044. L. Rossdeutscher, D. Huang, J. Li, T. Reinhardt, W. Muller, R. Kremer, Vitamin D delays breast cancer progression in the PyVMT transgenic mouse model: local conversion of the precursor 25(OH)D3 into 1,25(OH)2D3 is safer and more effective than systemic administration of 1, 25(OH)2D3. ASBMR 30th, Ann. Meet (2008). Montreal, September 12e16. J. Li, R. St-Arnaud, W. Muller, R. Kremer, Conditional ablation of the 25OHD3-1a-hydroxylase gene (CYP27B1) in mammary epithelial cells accelerates breast cancer development in vitamin D sufficient PyVMT transgenic mice, ASBMR 31st Ann. Meet. Denver (2009). Y. Ma, D.L. Trump, C.S. Johnson, Vitamin D in combination cancer treatment, J. Cancer 1 (2010) 101e107. D.D. Bikle, Y. Oda, Z. Xie, Calcium and 1,25(OH)2D: interacting drivers of epidermal differentiation, J. Steroid Biochem. Mol. Biol. 89e90 (2004) 355e360. D.D. Bikle, Vitamin D regulated keratinocyte differentiation, J. Cell Biochem. 92 (2004) 436e444. Z. Xie, L. Komuves, Q.C. Yu, H. Elalieh, D.C. Ng, C. Leary, et al., Lack of the vitamin D receptor is associated with reduced epidermal differentiation and hair follicle growth, J. Invest. Dermatol. 118 (2002) 11e16. Y. Sakai, M.B. Demay, Evaluation of keratinocyte proliferation and differentiation in vitamin D receptor knockout mice, Endocrinology 141 (2000) 2043e2049. P.J. Malloy, J.W. Pike, D. Feldman, The vitamin D receptor and the syndrome of hereditary 1,25-dihydroxyvitamin D-resistant rickets, Endocr. Rev. 20 (1999) 156e188. Y. Sakai, J. Kishimoto, M.B. Demay, Metabolic and cellular analysis of alopecia in vitamin D receptor knockout mice, J. Clin. Invest. 107 (2001) 961e966. C.H. Chen, Y. Sakai, M.B. Demay, Targeting expression of the human vitamin D receptor to the keratinocytes of vitamin D receptor null mice prevents alopecia, Endocrinology 142 (2001) 5386e5389.
[183] J. Kong, X.J. Li, D. Gavin, Y. Jiang, Y.C. Li, Targeted expression of human vitamin D receptor in the skin promotes the initiation of the postnatal hair follicle cycle and rescues the alopecia in vitamin D receptor null mice, J. Invest. Dermatol. 118 (2002) 631e638. [184] K. Skorija, M. Cox, J.M. Sisk, D.R. Dowd, P.N. MacDonald, C.C. Thompson, et al., Ligand-independent actions of the vitamin D receptor maintain hair follicle homeostasis, Mol. Endocrinol. 19 (2005) 855e862. [185] J. Miller, K. Djabali, T. Chen, Y. Liu, M. Ioffreda, S. Lyle, et al., Atrichia caused by mutations in the vitamin D receptor gene is a phenocopy of generalized atrichia caused by mutations in the hairless gene, J. Invest. Dermatol. 117 (2001) 612e617. [186] J.C. Hsieh, J.M. Sisk, P.W. Jurutka, C.A. Haussler, S.A. Slater, M.R. Haussler, et al., Physical and functional interaction between the vitamin D receptor and hairless corepressor, two proteins required for hair cycling, J. Biol. Chem. 278 (2003) 38665e38674. [187] Z. Xie, S. Chang, Y. Oda, D.D. Bikle, Hairless suppresses vitamin D receptor transactivation in human keratinocytes, Endocrinology 147 (2006) 314e323. [188] B.J. Merrill, U. Gat, R. DasGupta, E. Fuchs, Tcf3 and Lef1 regulate lineage differentiation of multipotential stem cells in skin, Genes Dev. 15 (2001) 1688e1705. [189] L. Cianferroti, M. Cox, K. Skorija, M.B. Demay, Vitamin D receptor is essential for normal keratinocyte stem cell function, Proc. Natl. Acad. Sci. USA 104 (2007) 9428e9433. [190] D.D. Bikle, S. Chang, D. Crumtine, H. Elalieh, M.Q. Man, E.H. Choi, et al., 25-Hydroxyvitamin D-1a-hydroxylase is required for optimal epidermal differentiation and permeability homeostasis, J. Invest. Dermatol. 122 (2004) 984e992. [191] M. Hewison, F. Burke, K.N. Evans, D.A. Lammas, D.M. Sansom, P. Liu, et al., Extra-renal 25-hydroxyvitamin D3-1-hydroxylase in human health and disease, J. Steroid Biochem. Mol. Biol. 103 (2007) 316e321. [192] C.M. Veldman, M.T. Cantorna, H.F. DeLuca, Expression of 1,25-dihydroxyvitamin D(3) receptor in the immune system, Arch. Biochem. Biophys. 374 (2000) 334e338. [193] J. Laureys, O. Rutgeerts, R. Saint-Arnaud, R. Bouillon, C. Mathieu, Identification and immune regulation of 25-hydroxyvitamin D-1-a-hydroxylase in murine macrophages, Clin. Exp. Immunol. 120 (2000) 139e146. [194] A.S. Dusso, S. Kamimura, M. Gallieni, M. Zhong, L. Negrea, S. Shapiro, et al., g-Interferon-induced resistance to 1,25-(OH) 2D3 in human monocytes and macrophages: a mechanism for the hypercalcemia of various granulomatoses, J. Clin. Endocrinol. Metab. 82 (1997) 2222e2232. [195] K. Stoffels, L. Overbergh, A. Giulietti, L. Verlinden, R. Bouillon, C. Mathieu, Immune regulation of 25-hydroxyvitamin-D3-1hydroxylase in human monocytes, J. Bone Miner. Res. 21 (2006) 37e47. [196] M. Vidal, C.V. Ramana, A.S. Dusso, Stat1-vitamin D receptor interactions antagonize 1,25-dihydroxyvitamin D transcriptional activity and enhance stat1-mediated transcription, Mol. Cell Biol. 22 (2002) 2777e2787. [197] X.P. Yu, T. Bellido, S.C. Manolagas, Down-regulation of NF-B protein levels in activated human lymphocytes by 1,25-dihydroxyvitamin D3, Proc. Natl. Acad. Sci. USA 92 (1995) 10990e10994. [198] R. Bouillon, G. Carmeliet, L. Verlinden, E. van Etten, A. Verstuyf, H.F. Luderer, et al., Vitamin D and human health: lessons from vitamin D receptor null mice, Endocr. Rev. 29 (2008) 726e776. [199] W.C. Lim, S.B. Hanauer, Y.C. Li, Mechanisms of disease: vitamin D and inflammatory bowel disease, Nat. Clin. Pract. Gastroenterol. Hepatol. 2 (2005) 308e315.
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REFERENCES
[200] M. Cutolo, K. Otsa, M. Uprus, S. Paolino, B. Seriolo, Vitamin D in rheumatoid arthritis, Autoimmun. Rev. 7 (2007) 59e64. [201] D.L. Kamen, G.S. Cooper, H. Bouali, S.R. Shaftman, B.W. Hollis, G.S. Gilkeson, Vitamin D deficiency in systemic lupus erythematosus, Autoimmun. Rev. 5 (2006) 114e117. [202] K.L. Munger, L.I. Levin, B.W. Hollis, N.S. Howard, A. Ascherio, Serum 25-hydroxyvitamin D levels and risk of multiple sclerosis, JAMA 296 (2006) 2832e2838. [203] E. Hypponen, E. Laara, A. Reunanen, M.R. Jarvelin, S.M. Virtanen, Intake of vitamin D and risk of type 1 diabetes: a birth-cohort study, Lancet 358 (2001) 1500e1503. [204] J. O’Kelly, J. Hisatake, Y. Hisatake, J. Bishop, A. Norman, H.P. Koeffler, Normal myelopoiesis but abnormal T lymphocyte responses in vitamin D receptor knockout mice, J. Clin. Invest. 109 (2002) 1091e1099. [205] E. Baroni, M. Biffi, F. Benigni, A. Monno, D. Carlucci, G. Carmeliet, et al., VDR-dependent regulation of mast cell maturation mediated by 1,25-dihydroxyvitamin D3, J. Leukoc. Biol. 81 (2007) 250e262. [206] M. Froicu, M.T. Cantorna, Vitamin D and the vitamin D receptor are critical for control of the innate immune response to colonic injury, BMC Immunol. 8 (2007) 5. [207] T.F. Meehan, H.F. DeLuca, The vitamin D receptor is necessary for 1,25-dihydroxyvitamin D(3) to suppress experimental autoimmune encephalomyelitis in mice, Arch. Biochem. Biophys. 408 (2002) 200e204. [208] M.T. Cantorna, C.E. Hayes, H.F. DeLuca, 1,25-Dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis, Proc. Natl. Acad. Sci. USA 93 (1996) 7861e7864. [209] K.M. Spach, C.E. Hayes, Vitamin D3 confers protection from autoimmune encephalomyelitis only in female mice, J. Immunol. 175 (2005) 4119e4126.
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[210] J.B. Zella, H.F. DeLuca, Vitamin D and autoimmune diabetes, J. Cell Biochem. 88 (2003) 216e222. [211] C. Mathieu, M. Waer, K. Casteels, J. Laureys, R. Bouillon, Prevention of type I diabetes in NOD mice by nonhypercalcemic doses of a new structural analog of 1,25-dihydroxyvitamin D3, KH1060, Endocrinology 136 (1995) 866e872. [212] A. Wittke, A. Chang, M. Froicu, O.F. Harandi, V. Weaver, A. August, et al., Vitamin D receptor expression by the lung micro-environment is required for maximal induction of lung inflammation, Arch. Biochem. Biophys. 460 (2007) 306e313. [213] J. Ehrchen, L. Helming, G. Varga, B. Pasche, K. Loser, M. Gunzer, et al., Vitamin D receptor signaling contributes to susceptibility to infection with Leishmania major, FASEB J. 21 (2007) 3208e3218. [214] A.F. Gombart, N. Borregaard, H.P. Koeffler, Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3, FASEB J. 19 (2005) 1067e1077. [215] P.T. Liu, S. Stenger, H. Li, L. Wenzel, B.H. Tan, S.R. Krutzik, et al., Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response, Science 311 (2006) 1770e1773. [216] S. Gregori, M. Casorati, S. Amuchastegui, S. Smiroldo, A.M. Davalli, L. Adorini, Regulatory T cells induced by 1 alpha,25-dihydroxyvitamin D3 and mycophenolate mofetil treatment mediate transplantation tolerance, J. Immunol. 167 (2001) 1945e1953. [217] M. Hewison, Vitamin D and the immune system: new perspectives on an old theme, Endocrinol. Metab. Clin. North. Am. 39 (2010) 365e379. [218] G.N. Hendy, D. Goltzman, Does calcitriol have actions independent from the vitamin D receptor in maintaining skeletal and mineral homeostasis? 14 (2005) 350e354.
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S E C T I O N V
HUMAN PHYSIOLOGY
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C H A P T E R
34 Vitamin D: Role in the Calcium and Phosphorus Economies Robert P. Heaney Creighton University, Omaha, Nebraska
INTRODUCTION
OVERVIEW OF THE CALCIUM ECONOMY
Vitamin D functions in many body systems, but perhaps the best attested of the nutrient’s actions e and certainly the one first associated with human disease e is its role in transferring calcium (and phosphorus) from ingested food into the body fluids. Calcium, like most divalent cations, is only partially absorbed from the chyme as it travels through the small intestine. This situation creates an opportunity for regulation of absorption, with room both to increase and to decrease calcium extraction efficiency in response to physiological controls. By contrast, phosphorus, mainly in the form of inorganic phosphate, is typically well absorbed in the intestine and its regulation in the body is predominantly by the kidney. Details of both the many cellular and tissue effects of vitamin D, and of the intestinal and renal absorptive processes, are covered in other chapters in this volume. Here I shall attempt to summarize mainly the meaning and importance of the vitamin-D-mediated transfer process from gut and tubular lumen to blood and to outline how these transfers fit into the maintenance of the calcium and phosphorus economies. My frame of reference will be the integrated functioning of the intact organism.
Body Calcium Compartments Body calcium in an adult human amounts to about 15e20 g (0.375e0.5 mol)/kg body weight. This calcium exists in three quite distinct divisions (or compartments). They are distinct because (1) movement of calcium atoms between them is both limited and regulated; and (2) they can and do vary in magnitude independently of one another. The first and most obvious compartment is the calcium in the bones and teeth. Here calcium exists as inorganic mineral crystals, arranged in an imperfect apatite lattice with variable stoichiometry, and embedded in a dense protein matrix. Although cells (osteocytes) ramify throughout bony tissue, the intercellular bony material itself lacks appreciable free water. As a result there is very limited exchange of calcium ions between the bone and the circulating body fluids. Isotopic exchange with tracers injected into the blood is confined to the surface layer of crystals in the bone situated along vascular channels and spaces, and to still incompletely mineralized new forming sites. Taken all together the exchangeable bone calcium moieties amount to only about 25 mmol (1000 mg), or ~0.1% of total skeletal calcium [1]. Moreover, the insolubility of bone mineral is such that, even when there is exchange, there is virtually never net transfer out of bone into the body fluids. Net transfer normally requires formation or resorption of bone tissue.1
1 One possible exception is the calcium carbonate of bone. Carbonate substitutes poorly for phosphate in the apatite lattice, and it is generally considered that, because of different valences and ionic radii for the two ions, carbonate is confined to crystal surfaces. Generally it is assumed that the carbonate is more or less uniformly distributed throughout the bony material. However, bone carbonate is substantially more labile than is bone calcium generally, and it is likely therefore that the carbonate is situated even more superficially than generally presumed, that is, primarily on anatomic bone surfaces, rather than diffusely on crystal surfaces generally. Calcium carbonate might, thus, be a kind of “icing” on the underlying mineralized matrix, sensitive to pH and pCO2 in the extracellular fluid. If this is the case, a limited amount of net movement of calcium into and out of bone would be possible without involving cell-mediated formation or resorption of bone tissue.
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10034-4
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A second, biologically critical compartment is intracellular calcium. Here calcium serves as a ubiquitous second messenger, linking signals from outside the cell to the mechanisms constituting the cell’s response. While free calcium ion concentrations in the cytosol are typically on the order of 1 10e5 mmol [2], total cell calcium is on the order of 0.5e2 mmol/kg tissue [1]. Most of this quantity (~99.99%) is bound to specialized calcium storage proteins (e.g., sequestrin, parvalbumin, calbindin) and located in storage vesicles, typically specialized units of the endoplasmic reticulum. Because the calcium ion is of just the right radius to fit neatly into folds of the peptide chain, and because calcium is capable of forming up to 12 (typically 6e8) coordination bonds with oxygen atoms in the side chains of amino acids projecting off the peptide chain, calcium stabilizes the tertiary structure of many catalytic molecules, thereby activating them. Cytosolic [Ca2þ] must be kept very low to prevent constant, uncontrolled activity of the many cell functions in which calcium plays a messenger or activation role. On the other hand, substantial intracellular stores of calcium are necessary because the binding avidity of cytosolic proteins for calcium is so high that, typically, the free path of a calcium ion in the cytosol is only a tiny fraction of a cell diameter [2]. If reservoirs were not diffusely distributed throughout the cytosol, and if extracellular calcium were the only source for this critical second
messenger function, activation would be limited to the zone immediately beneath the plasma membrane. Because the cell membrane is relatively impermeable to calcium ions, and because the cytosolic [Ca2þ] compartment is so tiny, tracer exchange between ECF calcium and cell stores of calcium is surprisingly slow e typically requiring hours or days to come into tracer equilibrium [1]. Further, cell calcium levels are typically unaffected by acute changes in calcium concentrations in the extracellular fluid (ECF). The third and smallest division of body calcium is the calcium present in the circulating blood and the ECF bathing all the body tissues. This compartment contains typically about 0.4 mmol calcium per kg body weight. Ionized calcium concentration in these fluids is ordinarily about 1.25 mmol/liter (5 mg/dl), a figure that is regulated across the higher vertebrate orders with the same exquisite precision as are, e.g., the concentrations of sodium and potassium. Departures from this level produce well-studied, significant effects on interneuronal signal transmission and on muscular excitability, among others. ECF [Ca2þ] is also important for extracellular protein activations such as those in the coagulation cascade. Into and out of this ECF compartment passes all the calcium entering and leaving the body from the outside, as well as entering and leaving bone. These fluxes are summarized schematically in Figure 34.1.
Bone
Diet Ca 20.0 Formation 9.0
Resorption 9.0 Skin
3.5 Extracellular Fluid
0.8
1.5
25 mmol 5.0 Filtered 200
Reabsorbed 197.5
Fecal Ca 17.7 Urine Ca 3.0 FIGURE 34.1 Schematic representation of the principal inputs and outputs of the extracellular fluid compartment. Units are in Ca mmol for extracellular fluid and mmol Ca/day for all other entries. (Copyright Robert P. Heaney, 2004. Reproduced with permission).
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Together they involve daily quantities amounting to 35e50% of the size of the entire compartment in healthy adults, and to several times that compartment size in infants. Without tight regulation, ECF [Ca2þ] would oscillate between possibly fatal extremes of hypo- and hypercalcemia as the organism goes from fasting to feeding.
Regulation of ECF [Ca2D] A detailed description of the processes involved in regulating ECF [Ca2þ] is beyond the purpose of this chapter. To situate vitamin D in this system it is necessary only to note that regulation occurs both by controlling the renal excretory threshold for calcium and by regulating calcium fluxes into and out of the ECF. Two of the transfers out of the ECF illustrated in Figure 34.1 drive the system and are essentially unregulated. The other transfers are at least partially responsive and constitute the basis of the regulatory control of ECF [Ca2þ]. The unregulated, driving transfers are (1) mineralization of newly deposited bone matrix at bone forming sites and (2) daily obligatory loss out of the body. Vitamin D and its metabolites play critical roles in the two regulated transfers into the ECF e from ingested food and from bone resorption. Driving Transfers MINERALIZATION OF BONE
Mineralization of bone constitutes an unregulated drain because it is a passive phenomenon, lagging several days, and even weeks, behind the osteoblastic cellular activity that initiates the process. The growing mineral deposits extract calcium and phosphorus from blood flowing past the mineralizing site at a decreasing exponential rate for up to 40 weeks after the matrix has been deposited. The only way, in theory, to stop the process is to shut down blood flow to bone. The magnitude of this mineralization drain varies with skeletal remodeling activity and with body size. It amounts to about 0.15 mmol/d/kg body weight in healthy mature adults. It is much higher during growth, of course, and rises once again after mid life [3,4]. OBLIGATORY LOSS
Obligatory loss consists of a combination of cutaneous loss and the fixed components of urinary and endogenous fecal calcium excretion. Cutaneous losses consist not just of sweat calcium but of the calcium contained in shed skin, hair, and nails. As has been noted above, all cells contain substantial amounts of calcium, and their loss from body surfaces inexorably takes calcium with them. Cutaneous losses have been hard to quantify but are estimated to be at least 0.4 mmol/day and more likely closer to 1.5 mmol/day
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[5,6]. Much higher losses have been reported with vigorous physical activity [7]. The fixed component of endogenous fecal and urinary calcium losses is somewhat more complicated [8]. On average, 3.5e4.0 mmol calcium enters the gut each day from endogenous sources, principally as a component of digestive secretions, but also as the calcium contained in shed mucosal cells (which turn over once every 4e5 days). The precise quantity varies directly with body size and with the amount of food consumed. For reasons not understood, the amount of calcium entering the gut in the digestive secretions varies directly also with phosphorus intake [8]. In any event, this endogenous calcium mixes with food calcium and much of it is subject to absorption, just as is the calcium of food. However, as noted earlier, calcium absorption is always incomplete. Gross absorption averages about 25e30% in healthy adults, and net absorption about 10e11%. Hence much of the secreted calcium is lost in the feces. Moreover, some of the endogenous calcium secretion enters the intestinal stream at a point so low in the gut as to be essentially unreclaimable (e.g., the calcium in colon mucus and in shed colon cells). On a normal diet this distal component has been estimated to be about 0.6 mmol [8]. Typical endogenous fecal calcium values average in the range of 3 mmol/ day. Since absorption efficiency in adults is essentially never above 60%, even on a very-low-calcium diet (see later discussion), there is an irreducible minimum loss of endogenous calcium through the gut averaging close to 2 mmol/day. If gross absorption is not greater than this figure, the gut becomes a net excretory organ for calcium. The third obligatory loss is through the kidney. It is generally held that renal calcium excretion is controllable, but this is only partly true. PTH certainly regulates tubular calcium reabsorption. However, there is a fixed limit to what that mechanism can accomplish. This limit is itself a function of other variables that are outside the regulatory loop. Best studied of these factors is the renal excretion of sodium, dietary intakes of protein and potassium, net endogenous acid production (NEAP), and absorbed dietary phosphorus. Sodium [9e11], protein [12,13], and NEAP [14] increase urinary calcium loss; phosphorus and potassium decrease it [15,16]. Their aggregate effect on renal calcium excretion constitutes obligatory (rather than regulated) loss because their input to the body is itself unregulated. Because sodium and calcium compete for the same reabsorption mechanism in the proximal tubule, the two ions influence one another’s excretion [17]. On average, urine calcium increases from 0.5 to 1.5 mmol for every 100 mmol of sodium excreted [9,10]. Similarly, urine calcium rises by about 0.25 mmol for every 10 g of
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protein ingested [12]. The result, for an adult woman ingesting the RDA for protein and the median sodium intake for North Americans, is a level of obligatory urinary loss amounting to about 2 mmol/day. Reducing sodium intake would certainly reduce this obligatory loss. Nevertheless such voluntary dietary change is clearly not a part of any physiological regulatory loop. And thus, to the extent that sodium intake influences obligatory calcium loss, it constitutes a demand to which the calcium homeostatic system must respond. Given typical adult diets in Europe and North America, the sum of these obligatory losses through skin, gut, and kidney is about 5 1 mmol/day, or about one-fifth of the total calcium in the ECF. To offset these losses (plus the demands of bone mineralization), the organism regulates countervailing inputs into the ECF from food and bone. It is in these transfers that vitamin D plays its role. The input from the first source is dependent both upon the presence of food in the upper GI tract and the presence of sufficient calcium in that food. Because neither condition can be guaranteed, the second source, bone, is the more reliable and constitutes, in fact, the first line of defense against hypocalcemia.2 The foregoing emphasis on gains and losses should not be interpreted as constituting the mechanism whereby ECF [Ca2þ] is maintained within such narrow limits. Instead, under conditions of health, it is the renal calcium threshold that is the primary determinant of ECF [Ca2þ]. The threshold is, conceptually, the level of ECF [Ca2þ] below which renal tubular reabsorption of filtered calcium is essentially quantitative, i.e., urine calcium is very low and ECF [Ca2þ] coming out of the kidney is about the same as that going in. The principal determinant of the threshold is PTH (which is why serum calcium rises in patients with hyperparathyroidism and falls in those with hypoparathyroidism. (The multiple effects of PTH are explored in the next section.) Serum calcitriol itself is calcemic, i.e., it raises ECF [Ca2þ] concentration. It is often considered that calcitriol does so by raising the renal calcium threshold, but the effect occurs even in anephric patients, and is probably not, in them, due even to increased intestinal absorption of calcium as the absorptive effect of calcitriol is severely blunted in patients with end-stage renal disease [19]. The only plausible remaining explanation is some alteration of the bloodebone interface. The role of bone in direct support of ECF calcium and phosphorus
concentrations has been an object of speculation and study for decades, but it is still very unclear how such a mechanism might work and, if it existed, how calcitriol might alter its setting. Response of the System to Demand PTH-MEDIATION OF RESPONSE
Briefly, a fall in ECF [Ca2þ] evokes a prompt rise in parathyroid hormone [PTH] release. PTH acts in a classical negative feedback loop to raise the ECF [Ca2þ], thereby closing the loop and reducing PTH release. The mechanisms by which PTH raises ECF [Ca2þ] illustrate well the complexities of the calcium economy. These mechanisms include: (1) increasing renal phosphate clearance, thereby lowering ECF phosphate levels; (2) increasing renal tubular reabsorption of calcium, thereby allowing system inputs to elevate ECF [Ca2þ]; (3) augmenting osteoclast work at existing resorption loci; (4) activating new bone resorption loci; and (5) increasing the activity of the renal 1-a-hydroxylase, thereby increasing serum levels of 1,25(OH)2D and augmenting absorption efficiency for ingested food calcium. These five effects reinforce one another in important ways. The earliest effects, occurring within minutes, are a decrease in renal tubular phosphate reabsorption and the resulting fall in serum phosphate. The latter immediately augments existing osteoclastic bone resorption [20] and increases activity of the renal 1-ahydroxylase [21]. The elevated production of 1,25 (OH)2D leads to increased intestinal absorption, elevating ECF [Ca2þ] and thereby closing the feedback loop. (1,25(OH)2D also suppresses parathyroid hormone release in its own right.) Finally, 1,25(OH)2D is necessary for efficient osteoclast work. In this last role, it is not known whether variations of 1,25(OH)2D in the physiologic range produce corresponding alterations in bone resorption, and it is a difficult question to study because of the tight regulation of the various components of the system. Nevertheless, it is well established that resorptive work is severely impaired in vitamin D deficiency states (see Fig. 34.2 and later discussion), and that 1,25 (OH)2D in supraphysiologic doses is capable of causing substantial increases in bone resorption. Finally, all of the components of the intestinal calcium transport system, including vitamin D receptors and calbindins, are also found in the distal tubule of the nephron [22]. In this way, 1,25(OH)2D may enhance recovery of
2
Because it is beyond the scope of this chapter, which is concerned mainly with calcium transfers, I shall not develop further what is actually an even more fundamental mechanism by which PTH regulates ECF [Ca2þ], that is, the control of the renal calcium threshold (which is covered in more detail elsewhere [18] Chapters 27 and 29). Suffice it to say here only that in the shift from a lower to higher PTH level, renal calcium losses are temporarily curtailed until calcium inputs from bone and gut succeed in elevating [Ca2þ]. Then, as the filtered load at the glomerulus rises, calcium excretion returns to its previous level (although renal calcium clearance is now reduced).
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OVERVIEW OF THE CALCIUM ECONOMY
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calcium content of the ingested foliage will be higher, and the “mineral debt” created by antler formation will be repaid from ingested greens. VITAMIN D DEFICIENCY
FIGURE 34.2 X-ray of the knee in a child with vitaminD-deficiency rickets. The alternating bands of high and low density reflect annual periods of greater and lesser vitamin D availability. (Reproduced from A Textbook of X-ray Diagnosis, Vol. 6, 4th edition, 1971, edited by S.C. Shanks, P. Kerley. H.K. Lewis & Co., Ltd, Toronto. Chapter XLIII, Metabolic and endocrine-induced bone disease by C.J. Hodson, p. 661, Fig. 774.)
filtered calcium and contribute to the PTH effect of elevating the renal calcium threshold. An example of the integrated response of the system to demand is afforded by what occurs during antler formation in deer in the spring [23]. The calcium demands of antler formation exceed the calcium available in the nutrient-poor late winter foliage. Other things being equal, this drain would lower ECF [Ca2þ], but the parathyroid glands, sensing minute reductions in ECF [Ca2þ], respond by increasing secretion of PTH, which in turn activates numerous remodeling loci throughout the deer’s skeleton. Since remodeling is asynchronous, with resorption preceding formation at each remodeling locus, the extra burst of newly activated remodeling sites provides a temporary surplus of calcium, which the animal promptly uses to mineralize matrix in the forming antler. A few weeks later, when the skeletal remodeling loci reach their own formation phase,
As already noted, vitamin D is essential for the mobilization of calcium from the skeletal calcium reserves. This is well illustrated in the course of nutritional rickets observed in the children of China and Northern Europe before the discovery of vitamin D, and now, unfortunately, occurring once again in exclusively breast-fed children not given vitamin D supplements [24] (see Chapter 60). Prior to routine prophylactic use of vitamin D in countries where rickets was endemic, summer sun exposure produced some vitamin D e enough to allow reasonably normal mineralization of the hypertrophic cartilage zone beneath the growth plate. Then, during the 8 or so months when solar vitamin D was lacking, the rachitic process resumed. The result, visible on X-ray, was a series of bands parallel with the growth plate, with densely mineralized bone tissue alternating with undermineralized layers (Fig. 34.2). One might have thought that, during the winter, PTH-mediated bone resorption would have attacked bone in the dense layers, making its calcium available to new bone-forming sites (as occurs with antler formation in deer). But in the absence of vitamin D, the bone resorptive process is severely impaired. As a result the rachitic child is unable adequately to mobilize its own calcium reserves. FEAST AND FAMINE
The diets of hominids were high in calcium [25], just as are the diets of contemporary deer and other higher mammals. Foods available to contemporary huntergatherers exhibit an annual mean calcium nutrient density of 1.75e2 mmol (70e80 mg)/100 kcal. For individuals of contemporary body size, doing the work of a hunter-gatherer, that value translates to calcium intakes in the range of 50e75 mmol (2000e3000 mg)/day. But, as noted, the environment could not be relied upon to supply calcium-rich food continuously. Periods of fasting, famine, or drought would undoubtedly have threatened hypocalcemia. This fact underscores the importance both of bone as a calcium reserve and of the vitamin-Deparathyroid hormone control system, with its ability to release calcium rapidly from bone.3 Contemporary humans in industrialized nations have the same paleolithic physiology as our hominid ancestors. We experience, however, a few crucial differences in external conditions. One is a generally lower exposure
3 Technically calcium is essentially never actually “released” from bone. (See, however, footnote 1.) Rather, a volume of actual bone tissue is resorbed. In the process its calcium is released into the ECF. Thus transfer of appreciable amounts of calcium out of bone always means some removal of bone tissue. Bone, however, is a very rich source of calcium. A single cubic centimeter of bone contains as much calcium as is contained in the entire circulating blood of an adult human.
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to sunlight. Rickets was endemic in Northern Europe in the nineteenth century, partly because of latitude, partly because of air pollution, and partly because of child labor. Routine use of vitamin D today has all but eliminated that problem in children, but adults, and particularly the elderly, are often vitamin-D-deficient. (The consequences of this low vitamin D exposure are explored in Chapter 52 and elsewhere in this volume.) Another difference is a much reduced nutrient density for calcium in our ingested food. The importance of this latter departure from primitive conditions lies in the fact that it limits the efficacy of vitamin D in augmenting input from the intestine. This limitation is not widely recognized and rarely is its quantitative impact adequately appreciated. This important component of the calcium economy is discussed in greater detail below.
Dawson-Hughes et al. [34] showed that blacks exhibited a greater response to dietary calcium reduction than did whites, with larger increments of both PTH and 1,25 (OH)2D, indicating a blackewhite difference in bony response (see Chapter 52). Clear evidence in this regard also comes from the calcium tracer studies of Abrams and his colleagues, showing higher calcium absorption and retention in adolescent black females than in whites at the same pubertal stage [32]. There seem also to be differences in vitamin D metabolism in the two ethnic groups, with blacks showing typically lower serum 25 (OH)D levels and higher 1,25(OH)2D levels than whites [33]. The relative importance of these two metabolites to the calcium economy, and of differences in their concentrations in ethnic groups, is still unclear (see later discussion).
Independence of PTH Effector Mechanisms The PTH regulatory system is unique in that the feedback loop operates through three independent effector mechanisms, already described (elevated renal calcium threshold, elevated intestinal calcium absorption, and elevated bone resorption). This seeming redundancy underscores the physiological importance of maintaining constant ECF [Ca2þ]. The independence of response of the three PTH effector mechanisms constitutes the substratum for still incompletely explored, but interesting, differences in the way the organism adapts to deficiency and surfeit of calcium. The feedback loop of calcium regulation is typically closed by some variable combination of all three effects. For example, when calcium intake falls, there is an obvious limit to what the absorptive mechanism can yield, forcing higher PTH levels and correspondingly greater renal calcium retention and enhanced net transfer out of bone. Relative differences in the intrinsic responsiveness of those effector organs have important consequences for bone mass [26]. Slightly lower intrinsic responsiveness of osteoclastic resorption to PTH, other things being equal, leads to a slightly higher PTH level, which, in turn, drives all three effector mechanisms slightly harder. The result is that the intestinal absorptive and renal recovery mechanisms for calcium operate at higher efficiency, and bone resorption, at lower efficiency. This leads to more bone and is probably a large part of the explanation for the higher bone mass in persons of black African ancestry. Although there has been some inconsistency in the data reported to date in this regard, the bulk of the evidence points to relative refractoriness to PTH-stimulated bone resorption in African-Americans [27e33]. For example,
OVERVIEW OF THE PHOSPHORUS ECONOMY Phosphorus is the sixth most abundant element in the body. However, unlike calcium, phosphorus is a trace element in the biosphere, with the bulk of phosphorus being tied up in biota or guano deposits. Hence the principal components of phosphorus homeostasis differ substantially from those regulating calcium homeostasis. For phosphorus, the regulatory apparatus is optimized to deal with environmental scarcity, while for calcium, the organism must deal with environmental surplus.
Body Phosphorus Compartments Phosphorus in nature is most commonly found in its pentavalent form, as phosphate, and it is as such that it functions in living organisms.4 Adult body phosphorus makes up about 1.0e1.4% of fat-free mass or ~12 g (0.4 mol) per kg. Eighty-five per cent of this total is in the mineral of bones and teeth, with 15% distributed through the soft tissues and blood. Thus a 70-kilogram adult with 25% fat mass would have a total body phosphorus of ~630 g (~21 mol). Phosphorus is an essential constituent of the fabric of all life. In addition to its role in the mineral component of the endoskeleton of vertebrates, phosphorus is involved in cell membrane structure as phospholipids, in information coding as DNA and RNA, in energy metabolism as ATP and GTP, and in enzymatic activation, by phosphorylation of catalytic proteins. Additionally, inorganic phosphate (Pi) concentration in extracellular fluid (ECF) plays a vital role in supporting
4
Nevertheless, when analyzed for in foods, tissues, or serum, the results are usually expressed as grams elemental phosphorus, not as phosphate (which has a mass approximately 3 that of elemental phosphorus). Expressing results in mmol (as in this chapter) obviates that possible confusion.
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both bone mineralization and tissue intermediary metabolism. ECF Pi makes up <0.1% of total body phosphorus, but it is into this virtual compartment that phosphorus is transferred from digestion of food phosphorus in the gut and resorption of bone in the process of bone remodeling; and it is out of the phosphorus of this compartment that urine phosphorus is derived and bone mineral is formed.
ECF [Pi] and its Regulation Bone Mineralization At physiological pH and pCO2, the calcium phosphate salt that would likely precipitate from the ECF is CaHPO4. Normally ECF is only about half saturated with respect to this product. However, in the presence of a hydroxyapatite crystal nucleus, ECF calcium and phosphorus concentrations are supersaturated. Hence normal ECF supports mineralization when and where the body chooses to mineralize (by creating a suitable crystal nucleus), but is indefinitely stable as it circulates and bathes the other tissues. The concentrations of ionized calcium and phosphorus in serum (and ECF) are typically 1.1e1.3 mmol/L for calcium and 0.9e1.4 mmol/L for phosphorus. These values produce a CaeP ion product y1.3 mmol2/L2. Since serum calcium is held much more constant than is serum phosphorus, most of the clinically encountered variation in the serum CaeP product is produced by variations in serum [Pi]. When ECF CaeP rises by a factor of about 2, the solubility constant for CaHPO4 is exceeded and spontaneous calcification of nonosseous tissues tends to develop. And when the ion product falls by a factor of about 0.5, bone mineralization effectively stops. Thus rickets or osteomalacia occurs when serum Pi falls below 0.5e0.6 mmol/L, and metastatic calcification occurs when Pi rises above 2.4e2.5 mmol/L. Even within the nominal normal range, the rate of bone mineralization is, to some extent, dependent upon the serum CaeP product. During infancy, rapid skeletal growth requires a high ECF CaeP, with serum Pi often as high as 2.0e2.4 mmol/L. It is not that a higher CaeP product is inherently more mineralizing, but rather that, as blood flow past a mineralizing site is pulsatile, the blood is more quickly depleted of its mineral at lower CaeP values, and mineralization slows until another pulse of blood comes by. Conversely, at higher CaeP values, there is simply more mineral in a pulse, facilitating greater transfer from blood to bone. Regulation of ECF [Pi] Concentration Like ECF [Ca2þ], ECF [Pi] is regulated mainly at the kidney and in healthy adults is little affected by diet phosphorus (intake or absorption). Approximately 200 mmol of phosphorus is filtered each day at the
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kidney, the vast majority of which is reabsorbed in the proximal tubule. This tubular reabsorption is mediated by sodium-dependent phosphate cotransporters in the brush border of the proximal tubular epithelium. This absorption has a limited capacity (known as the tubular maximum for phosphate (TmP)), which in turn is regulated up or down so as to adjust serum Pi concentration. The two factors most responsible for this regulation are parathyroid hormone (PTH) and several agents known collectively as phosphatonins, principally fibroblast growth factor-23 (FGF-23). (See also the Chapter 26.) FGF-23 is produced in bone by cells of the osteoblast lineage. Both FGF-23 and PTH function to lower the TmP and hence are said to increase renal phosphorus clearance, i.e., the virtual volume of serum effectively cleared of its Pi per unit time. When the TmP decreases (i.e., serum phosphorus clearance rises), serum [Pi] falls, and vice versa. PTH and FGF-23 also directly regulate renal synthesis of 1,25(OH)2D (calcitriol), but in opposite directions. PTH up-regulates and FGF-23 down-regulates the expression of the renal 1-a-hydroxylase. This discordance is a direct reflection of the differing control systems for which these hormones are the effector molecules. PTH is responsive to ECF [Ca2þ], and its up-regulation of calcitriol synthesis leads to increased calcium entry from the gut. FGF-23, on the other hand, is responsive to ECF [Pi] and adjusts renal clearance to keep [Pi] from fluctuating widely. Specifically, high-phosphorus diets elevate serum FGF-23, leading to increased renal phosphorus clearance; and low-phosphorus diets reduce serum FGF-23, thereby reducing renal phosphorus loss [35]. The effect of PTH on TmP, as noted in the section on calcium, above, lowers serum [Pi] and thereby enhances osteoclastic release of bone mineral at pre-existing resorption loci [20]. Serum Pi concentration itself is active in these systems, with low [Pi] leading to up-regulation of renal calcitriol synthesis. However, FGF-23, while lowering serum Pi concentration as a consequence of increasing renal phosphorus clearance, does not lead to elevated 1-a-hydroxylation of 25(OH)D, as would be predicted. This is because FGF-23 itself down-regulates the hydroxylase, thus preventing the increase in calcitriol synthesis that would otherwise have accompanied the drop in serum Pi concentration.
CALCIUM AND PHOSPHORUS ABSORPTIVE INPUTS Chapter 19 describes the mechanisms of calcium and phosphorus transfer from the intestinal lumen. Here I describe quantitative aspects of those transfers as a part of the integrated calcium and phosphorus
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economies, and will focus as well on the factors determining the magnitude of the input from the gut into the ECF.
Location and Timing of Calcium Absorption in the Gut As noted elsewhere in this volume (Chapters 7 and 19), there is a gradient of concentrations of vitamin D receptors and of calbindin in mucosa along the gut, with highest levels in the duodenum and lowest in the colon mucosa. Accordingly, the avidity (or rate) of active absorption is highest in the duodenum. It is sometimes said that absorption itself is highest there, but this is not correct. That conclusion is based on studies of isolated loops or gut sacs, where movement of the chyme along the intestine cannot occur. Absorbed quantity is the product of absorption rate and residence time; and residence time of the chyme in the duodenum is very brief. Only at very low calcium intakes (or test loads), and with maximal 1,25(OH)2D-stimulated active transport, will it be true that most of the calcium absorbed will be from the duodenum. At more usual intakes, the much longer residence time in the jejunum and ileum means that most of the quantity absorbed occurs from the lower small intestine. The importance of length of exposure to the absorptive surface is reflected in the finding that absorptive efficiency varies directly with mouth-to-cecum transit time [36]. Absorption does not occur from the healthy stomach, and thus the beginning of absorption is delayed until gastric emptying begins. This, in turn, is dependent upon the character of the ingested meal or other calcium source. Emptying tends to be most rapid with small
fluid ingestates and is slower with solid food and with fat. In healthy individuals ingesting light meals (such as would commonly be employed to test absorption efficiency), calcium absorption is nearly complete by 5 hours after ingestion [37]. Figure 34.3 presents data on the time course of absorption, using the ratio of the time-dependent apparent absorption fraction to its ultimate value in the individual being tested. As the figure shows, absorption has reached better than 80% of its ultimate value by 3 h after ingestion, and 96% by 7 h. There is then only a very gradual approach to completion over the next 20 h. This last component probably reflects a small amount of colonic absorption (or, alternatively, cecaleileal reflux, with delayed ileal absorption). It should be stressed that the percentage values in Figure 34.3 refer to the quantity absorbed, not the quantity ingested. Thus, with typically only 25e30% of a load absorbed (see later discussion), the 4e5% colonic component represents absorption of only about 1% of the ingested load.
Calcium Absorption as a Function of Intake It has long been recognized that absorption efficiency varies inversely with intake. Figure 34.4 illustrates this relationship with data obtained from healthy, middleaged women in whom unidirectional (i.e., gross) absorption fraction was measured under controlled metabolic ward conditions and plotted as a function of their ingested intakes [38]. The best fit regression line through the data shows the expected rise in gross absorption fraction at low calcium intakes. (Note, however, that even at the lowest intakes, predicted mean gross absorption efficiency is only ~45%.)
FIGURE 34.3 Time course of absorption (derived from Barger-Lux et al. [37]). The data plot the percentage completion of absorption (derived from expressing the double-isotope absorption fraction as a ratio of its value at any given time to its final value after 24 h). Completion of absorption is thus expressed as a value of 100%. As the curve shows, absorption is about 94% complete by 5 h after oral ingestion. The remaining 6% occurs more slowly and may be presumed to reflect absorption from the colon and/or from ileo-cecal reflux. (Copyright Robert P. Heaney, 1989. Reproduced with permission.)
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*
*
95
*
*
*
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Late phase absorption (colonic)
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End of early phase absorption (small intestine)
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FIGURE 34.4 Fractional absorption plotted as a function of usual calcium intake (in mmol/day) in 525 studies in healthy, middle-aged women [38]. The solid line is the least squares regression line derived from a log-log fit. (Copyright Robert P. Heaney, 1989. Reproduced with permission.)
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ABSORPTION FRACTION
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The higher efficiency at low intakes is traditionally attributed to adaptation, specifically to higher production of 1,25(OH)2D, with a corresponding increase in active calcium absorption. While that explanation is undoubtedly correct, it is also substantially incomplete. This is shown by the data in Figure 34.5, which plots nonadaptive absorption fraction as a function of a broad range of calcium load sizes. These studies were performed in women, assigned randomly on any given morning to intake loads spanning a 30-fold range, from 0.4 to 12.5 mmol [39]. Clearly, an inverse relationship is present, just as in the data of Figure 34.4. Equally clearly, it cannot be due to adaptation, since the test load was the first exposure these women had to the intake level concerned. Figure 34.6 plots these two sets of data together and shows that, while both exhibit an inverse relationship between absorption and intake, there is in fact a difference between them, with the adapted women absorbing more at low intakes than the nonadapted (as would be predicted). The zone between the two lines is a semiquantitative expression of the PTHevitamin-D-mediated adaptation to the lower intake.5 The most likely explanation for the inverse relationship observed under both sets of conditions is that calcium transfer, whether active or passive, is a slow, inefficient process, with only a limited number of carrier
40
50
0.9 0.8 ABSORPTION FRACTION
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0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0.1
1 CALCIUM LOAD (mmol)
10
Fractional absorption in nonadapted healthy women for loads ranging from 0.4 to 12.5 mmol Ca. Error bars are 2 SEM. The solid line is the least squares regression through the actual data (n ¼ 75), derived from Heaney et al. [39]. The units of the horizontal axis are natural logarithms of load size (in mmol Ca). (Copyright Robert P. Heaney, 1996. Reproduced with permission.)
FIGURE 34.5
molecules or pores available at any given instant. In the brief interval between exit of a bolus of food from the stomach and the time it reaches the colon, only so many calcium ions can use the available transport.
5 As Figure 34.6 shows, most of the difference occurs at intake loads below 500 mg (12.5 mmol). However, the two sets of observations were performed in different groups of women and even the non-adapted set must have had some basal level of 1,25(OH)2D-mediated adaptive absorption. Hence the true extent of adaptive absorption is undoubtedly somewhat greater than indicated solely by the difference between the two lines.
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ABSORPTION FRACTION
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Adapted women
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CALCIUM INTAKE (mmol)
FIGURE 34.6 Combination of the regression lines from Figures 34.4 and 34.5, showing the extent of difference produced by adaptation to the various intakes. (Copyright Robert P. Heaney, 1996. Reproduced with permission.)
If the number of ions reaching the absorptive site is small, then by numerical necessity the fraction absorbed will be larger than when the number of ions is large. Absorption fraction (or efficiency) is thus a potentially misleading measure (at least if we stop there). It is, however, a necessary starting point because it is the primary datum available from most studies of absorptive physiology. Figure 34.7 presents the regression line from Figure 34.4 and adds a second line representing the actual quantity of calcium absorbed in these same women (i.e., the product of absorption fraction and intake). This variable is obviously the nutritionally relevant one since, in offsetting obligatory losses (or special
demands such as antler building or fetal skeletal development), it is a quantity of calcium (not a fraction) that is needed to balance the drains created by calcium leaving the ECF. Figure 34.7 also illustrates another important aspect of this input to the calcium economy. At low intakes, absorption is quantitatively low, despite being relatively more efficient. A moment’s reflection suffices to show that a large fraction of a small number is, of necessity, a smaller number still. Thus, absorbing even a large fraction of a small intake cannot produce much calcium. The result is that, in the range of intakes commonly encountered among contemporary, industrialized humans, absorptive adaptation (via vitamin D) mitigates the problem created by a low intake, but it does not fully counterbalance it. A concrete example, employing realistic numbers, will help illustrate this point, and will show additionally how optimal operation of the vitamin D hormonal system is dependent upon e and in fact presumes e the kinds of high calcium intakes found among hunter-gatherer humans and high primates (in whom the system evolved). Contrast how two individuals are able to respond to the increased obligatory loss occasioned by regular daily ingestion of an additional 100 mmol sodium (approximately the sodium contained in a single fast-food chicken dinner). Assume that one individual is ingesting 5 mmol Ca (200 mg)/day (corresponding to the lower quintile of calcium intakes in US women [40]), and the other, 40 mmol (1600 mg) (approximately the NIH Consensus Conference recommendation [41] for estrogen-deprived, postmenopausal women). Using data from the curve in Figure 34.4, the individual with the lower intake absorbs at an efficiency of 44.5% prior
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Fractional absorption and mass absorption for the 525 studies of Figure 34.4. The left axis and solid line represent fractional absorption and the right axis and dashed line, mass absorption. (Copyright Robert P. Heaney, 1996. Reproduced with permission.)
FIGURE 34.7
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0.7
CALCIUM AND PHOSPHORUS ABSORPTIVE INPUTS
to the extra sodium load, and the individual with the higher intake, at 17.8%. (The first, therefore, has a gross absorbed quantity from the diet of 2.2 mmol/day, and the second, 7.1 mmol/day.) The increase in obligatory urinary loss occasioned by the increase in sodium intake will be about 1 mmol/day (see earlier discussion). To offset this loss, the first individual, with the low intake, would have to increase the absorbed quantity to 3.2 mmol/day, which means increasing the already high absorption efficiency by a factor of nearly 1.5 (from 44.5 to 64.5%). By contrast, the individual with the high intake needs to increase only from 17.8 to 20.3%. (These calculations are summarized in Figure 34.8.) The adjustment for the individual with a low calcium intake is substantially more than most adults can accomplish, while the second is easily accommodated. The first individual is forced, therefore, to get the needed additional calcium from bone, while the second easily gets it from her food. Thus, while vitamin D plays a critical role in increasing absorptive efficiency in response either to increased losses from the body or to decreased intake, it must be stressed that there is little room in which the PTHevitamin D endocrine system can operate when calcium intakes are already low. That does not mean that ECF [Ca2þ] regulation suffers. The bony calcium reserves are vast e effectively limitless. So long as vitamin D status is above rachitic levels, those reserves will readily be drawn upon to support ECF [Ca2þ], using the well-studied mechanisms already described. Naturally, if this drain continues, bone mass will inevitably decline. At high calcium intakes, such as prevailed during hominid evolution, the vitamin D 0.8
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FIGURE 34.8 Changes in absorption efficiency required to offset fully from diet an additional drain of 1 mmol Ca/d, at low and high regions of the curve of Figure 34.4. Numbers shown are the required increases in absorption fraction at the respective intakes. (Copyright Robert P. Heaney, 2003. Reproduced with permission.)
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hormonal system not only helps maintain ECF [Ca2þ], but total body calcium as well; at low intakes, only the ECF is protected. See Chapter 60 for a discussion of rickets caused by dietary deficiency of calcium.
Partition of Calcium Absorption between Active and Passive Mechanisms As described elsewhere in this volume (Chapter 19), absorption occurs both by vitamin-D-mediated active transport across the mucosal cells and by passive diffusion around the cells. Is it possible to partition absorption between the active, cellular process and the passive, paracellular process? To a limited extent, the answer is yes. As should be clear from the foregoing, absorption fraction (both passive and active) exhibits an inverse relation to intake or load. Absorption will be high by either mechanism, even approaching 100%, if the load is sufficiently small. But small loads are not nutritionally relevant, no matter how well they are (or are not) absorbed; so in this discussion I shall confine myself to consideration of loads or intakes in the range of (or above) currently recommended intakes. The right end of the regression line in Figure 34.4 represents an absorption fraction of approximately 0.15. In work published earlier from our laboratory [42], we extended intakes well above the 2 g (50 mmol) upper limit of Figure 34.4, to as high as 8.0 g (200 mmol) Ca/day. Absorption fraction in that study also averaged about 0.15 at these very high intakes, or approximately what we observed at 50 mmol in Figure 34.4. The essentially linear character of this absorption across such a broad range of intakes probably reflects the fact that, if passive absorption is due largely to solvent drag, the quantity of calcium absorbed will be a linear function of luminal calcium concentration. It is likely, at habitual intakes in the range of 40e50 mmol Ca/day, that active absorption would be minimal, and it is a virtual certainty that that would be so at supraphysiological intakes of 200 mmol. Studies in patients with end-stage renal disease, with limited ability to synthesize 1,25(OH)2D, also report absorption fractions in the range of 10e20% [43,44]. Taken together, these data indicate that unidirectional passive absorption is able to extract about 10e15% of the calcium in the ingested food at nutritionally relevant loads. Variation around that level will presumably relate to interindividual variations in mucosal mass and in intestinal transit time. What would an absorption efficiency of 15% mean for the calcium economy if passive absorption were the only means for extracting calcium from the diet, as, for example, must be the case with hereditary vitamin-Dresistant rickets (HVDRR) type II? (see Chapter 65).
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Assume an intake of 20 mmol/day. As noted earlier, digestive secretions add 3.5 mmol to the stream, 0.6 mmol of that total too low to be absorbed. Calculation from these data easily shows that, at a 15% absorption efficiency, the gut will be a net excretory organ (if only barely: approximately 0.1 mmol/day net loss). No calcium gain into the body could occur at such an intake. Hence a background, gross absorption of 15% without vitamin D seems entirely compatible with development of severe rickets, either as found in HVDRR or in typical nutritional vitamin D deficiency. Figure 34.9 systematizes those calculations for varying levels of active absorption and a broad range of calcium intakes, expressing the results in terms of net calcium absorption. It shows graphically, for example, that without active transport it takes an intake of about 26 mmol to ensure zero gut balance, and that an intake of 60 mmol would not suffice to offset extraintestinal losses of 5 mmol through the kidney and skin. By contrast, active absorption of ~16% is sufficient in mature adults to offset such losses at intakes in the range of current recommendations (25e30 mmol/d).
Phosphorus Absorption Because of the relative scarcity of phosphorus in the biosphere, most organisms get the phosphorus they need by consuming the tissues of other organisms (plant and animal), and they absorb that ingested phosphorus with high efficiency. In adult humans, for example, net phosphorus absorption typically ranges from 55 to 80%
of ingested intake, and in infants, from 65 to 90%. The most active site is the jejunum. Because phosphorus is intimately involved in virtually all of the functions and structures of living organisms, phosphorus content of most animal tissues varies little, ranging from 0.25 to 0.65 mmol per gram protein. The resulting ubiquitous distribution of phosphorus in all natural foods makes it all but impossible to construct, for patients on renal dialysis, a diet that is both nutritionally adequate and low in phosphorus. Most protoplasmic phosphorus in ingested foods is quickly hydrolyzed by intestinal phosphatases, and hence most absorbed phosphorus is in the form of inorganic phosphate. The principal exception is the phosphorus in phytic acid (inositol hexaphosphate), which is the storage form of phosphate in seed foods (e.g., wheat, soy, etc.). The human intestine cannot hydrolyze phytic acid; hence absorption of phytate phosphorus is low. However colonic bacteria possess phytase and some phytate phosphorus is thus absorbed from the distal bowel. With this exception, intrinsic bioavailability of most food phosphorus sources is high. Absorption of phosphorus, as for calcium, is considered to be by a combination of active transport and passive diffusion, with the former being the regulated component. It is widely held that active phosphorus absorption is influenced by vitamin D status. Indeed, it is almost an article of faith that the canonical function of vitamin D is to promote absorption of calcium and phosphorus. However, much of the evidence for this
35 48 ACTIVE ABSORPTION (percent)
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FIGURE 34.9 Relationship of vitaminD-mediated, active calcium absorption, calcium intake, and net calcium gain across the gut. Each of the contours represents a different level of active absorption above a baseline passive absorption of 12.5%. (The values along each contour represent the sum total of passive and variable active absorption.) The horizontal dashed lines indicate 0 and 5 mmol/d net absorption, respectively. The former is the value at which the gut switches from a net excretory to a net absorptive mode, and the latter is the value needed to offset typical urinary and cutaneous losses in mature adults. (Copyright Robert P. Heaney, 1999. Reproduced with permission.)
PHYSIOLOGICAL SOURCES OF VITAMIN D ACTIVITY
conclusion comes from animal experiments involving isolated gut loops or everted gut sacs, methods which, unfortunately, do not reproduce normal intestinal functioning. However, Ferrari et al. [35] showed in 29 normal humans that induced extreme changes in phosphorus intake induced not only the predicted changes in serum FGF-23 and urine phosphorus, but corresponding changes in serum 1,25(OH)2D. Briefly a large increase in phosphorus intake led to a decrease in serum 1,25 (OH)2D level, a change which, if associated with a reduction in phosphorus absorption, would suggest endocrine feedback regulation of phosphorus absorption. Ramirez et al. [45], using an intestinal wash-out method, showed an appreciable effect of large daily doses of calcitriol on meal phosphorus absorption in five patients on chronic hemodialysis, producing essentially normal phosphorus absorption efficiency, and suggesting that the loss of renal synthesis of calcitriol reduced phosphorus absorption in patients with end-stage renal disease. Nevertheless, as is well recognized clinically, dietary phosphorus absorption in ESRD patients is higher than the body can handle e which is the rationale for use of intestinal phosphate binders. It is also true that the molecular apparatus for vitamin-D-stimulated active absorption of phosphorus exists in the intestine (see Chapter 19). That being said, it is unclear whether vitamin D exerts any strong regulatory control over phosphorus absorption under normal circumstances. And, instead, such vitamin D effects on phosphorus absorption as can be found in intact humans may be more indirect than direct. Disorders of phosphate balance are discussed in Chapter 63. Heaney and Nordin [46] showed that, over a wide range of ingested calcium:phosphorus ratios, the principal determinant of fecal phosphorus (and therefore inversely of absorbed phosphorus) was fecal calcium, with phosphorus intake itself exerting a significant but weaker effect. Together, and altogether apart from vitamin D status, these two factors explain nearly three-fourths of the observed variance of phosphorus absorption in a large series of adult women. In their studies each 10 mmol of ingested calcium, by complexing phosphate in the intestinal lumen, blocked the absorption of ~4 mmol of diet phosphorus. (This phenomenon is, of course, the basis for the use of calcium salts as phosphate binders in patients with ESRD.) As calcium absorption rises in response to vitamin D, less calcium is left behind in the intestinal lumen to bind still unabsorbed phosphorus, and hence phosphorus absorption would predictably rise under conditions of high vitamin D status. But that would not necessarily mean that vitamin D directly stimulated phosphorus absorption. Thus experiments such as those of Brown et al. [47], in which calcitriol increased absorption of both calcium and phosphorus in rats, cannot be
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interpreted unambiguously. In brief, much of the actual effect of vitamin D on adult phosphorus absorption under usual conditions and in health remains unclear.
PHYSIOLOGICAL SOURCES OF VITAMIN D ACTIVITY Thus far I have spoken of vitamin D only generically. As discussed extensively elsewhere in this volume, native vitamin D (calciferol) has very little biological activity in its own right. It is converted in the body into a number of hydroxylated metabolites, the most important of which for the purposes of this chapter are 25(OH)D3 and 1,25(OH)2D3. It is well established that cholecalciferol is 25-hydroxylated in the liver. The reaction is loosely controlled by circulating levels of 1,25 (OH)2D as well as by limited end-product inhibition exerted by 25(OH)D itself. However, for the most part circulating 25(OH)D levels are driven mainly by the circulating levels of the precursor, either ergo- or cholecalciferol. Serum 25(OH)D3 rises by approximately 0.7e1.0 nmol/L for every one mg increment of daily oral cholecalciferol after 16e20 weeks of daily oral administration [48e50]. 1,25(OH)2D3 is produced in the kidney, as already described (Chapter 3), under control by serum PTH, serum Pi and FGF-23 concentrations. It is generally considered to be the biologically active form of the vitamin, responsible for its full spectrum of actions; it is also considered that the precursors (cholecalciferol and 25(OH)D3) exert activity in their own right only under conditions of intoxication. This conclusion is based mainly upon studies of both binding kinetics of the various metabolites with the vitamin D receptor and of consequent gene expression. If binding affinity for 1,25(OH)2D3 is taken as 1.0, reported values for 25 (OH)D3 are in the range of 1 10e3 to 1 10e4, and for native D, 1 10e6 or lower [51e53]. Accordingly, it is reasoned that normal serum levels of the precursor compounds are too low to exert significant action. However, since serum levels of 25(OH)D are typically three orders of magnitude higher than those of 1,25 (OH)2D, it is not clear, a priori, that 25(OH)D would be without effect under normal conditions. Protein binding in serum complicates interpretation of these relationships. It is generally presumed that total serum levels are physiologically less meaningful than free serum levels of a metabolite. As discussed elsewhere in this volume (Chapter 5), the vitamin D family of compounds is carried in serum complexed to a D-binding carrier protein (an alpha globulin designated DBP, with a gradient of affinities: highest for 25(OH)D, lower for native vitamin D3, and lower still for 1,25(OH)2D). However, binding to the carrier protein cannot be
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interpreted without reference to other factors, usually not readily determined in any given situation. These include the relative affinities of the binding protein and the receptor as well as the concentrations of both. Available data on binding at the intact organism level are not sufficient to resolve this issue of hormone dynamics. But such theoretical considerations aside, there is a substantial body of clinical data indicating that 25(OH)D exerts appreciable biological activity at normal physiological concentrations. Such activity may not be genomic, as would be the principal mechanism for the action of 1,25(OH)2D. It appears, for example, that both 25(OH)D and 1,25(OH)2D must be present for the canonical effect of vitamin D (promotion of active intestinal absorption) to be expressed. Thus, clinically, we note low calcium absorption in nutritional rickets and osteomalacia, despite normal to high serum values for 1,25(OH)2D. The same is true for patients with endstage renal disease, who do not show an appreciable absorptive response to 1,25(OH)2D [19]. In both situations, serum 25(OH)D is low. Several studies show a surprisingly strong correlation between serum 25(OH)D and intestinal calcium absorption efficiency in intact humans [36,54e58]. If 25 (OH)D were acting only as a precursor, one should have expected no correlation at all. Where 1,25(OH)2D levels were measured in these studies, the correlation of 1,25(OH)2D with absorption fraction was usually weaker than for 25(OH)D, or nonsignificant altogether. Colodro et al. [57], in a dose response study for both metabolites, using calcium absorption in healthy human adults as the endpoint, reported a molar potency for administered 25(OH)D relative to 1,25(OH)2D of 1:125, not the less than 1:2000e1:4000 figure predicted from in vitro receptor-binding studies. Barger-Lux et al. [59], in a similar study, found a nearly identical potency ratio (1:100). In this study the rise in absorption fraction produced by oral 25(OH)D occurred without a detectable change in 1,25(OH)2D level. Taken together these studies provide evidence that some fraction of circulating vitamin-D-like activity can be attributed to 25(OH)D.6 The basis for the interaction of the two vitamin D metabolites in intestinal calcium absorption is unclear. It may be that mucosal cell expression of the 1a-hydroxylase converts circulating 25(OH)D to 1,25(OH)2D within the cell, thereby augmenting the 1,25(OH)2D levels the receptor actually sees (see Chapter 45 for a full discussion). More likely, the two metabolites may act synergistically e 1,25(OH)2D in the canonical genomic manner, and 25(OH)D binding to membrane
receptors and opening calcium channels, a process termed “transcaltachia” [60,61] (see Chapter 15). In any event, there can be no question about the potency of 1,25 (OH)2D itself. This very active metabolite produces a strong enhancement of absorption efficiency when given to intact humans [57e59,62,63] with nondeficient levels of serum 25(OH)D. Transcaltachia, which is a rapid-response, nongenomic action of the D vitamin metabolites, requires occupancy of the nuclear receptor by 1,25(OH)2D [61]. This is shown, for example, in patients with HVDRR type II, who lack functional vitamin D receptors, and who are not able to absorb calcium efficiently despite normal to high circulating levels of both 1,25(OH)2D and 25(OH)D [64,65]. Thus vitamin-D-mediated absorption seems to require both a functioning receptor and some combination of 1,25 (OH)2D and 25(OH)D. Whatever the precise mechanism, the system operates as if there were a floor or background level of absorption in normal individuals that is determined in part by the long-half-life 25(OH)D, while 1,25(OH)2D produces a quick-acting fine tuning of the system.
OPTIMAL VITAMIN D STATUS Optimal vitamin D status can be defined as the daily intake or production of the vitamin (and/or the serum level of 25(OH)D) that is sufficient so that its availability does not limit any of the metabolic functions dependent upon the vitamin. This notion of limit is illustrated in Figure 34.10A. A large body of data indicates that vitamin D-mediated absorption follows a curve such as the one presented in Figure 34.10A, rising with intake at levels below the requirement, then flat through a range of sufficiency, then rising again at pharmacologic (or toxic) intakes. Figure 34.10B shows this behavior as related to actual serum 25(OH)D concentration, derived from two studies [66,67]. It is likely that the absorptive response, taken in isolation, would be a continuous smooth function of vitamin D dose, such as that indicated by the dashed line in Figure 34.10A. However, at the whole-organism level, once calcium absorption is optimal, other factors alter the response to vitamin D exposure. For example, at levels of calcium absorption higher than needed to offset daily losses, PTH levels would drop and 1,25(OH)2D synthesis would fall. Thus, despite a rising solar or oral dose of the precursor vitamin D, absorption plateaus. But when the dose becomes sufficiently high (as in vitamin D intoxication), system controls are saturated and
6
Some of the apparent action of 25(OH)D in oral dosing studies may be due to a direct effect of the metabolite on the mucosal cell (a first-pass effect). During absorption of 25(OH)D, mucosal exposure to the agent would be substantially higher than would occur from exposure to plasma 25(OH)D.
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OPTIMAL VITAMIN D STATUS
(A)
(B) ABSORPTION FRACTION
ABSORPTION FRACTION
0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.00 VITAMIN D INPUT
0
40
60 80 100 120 SERUM 25(OH)D (nmol/L)
140
160
FIGURE 34.10 (A) Schematic diagram of the likely relationship between absorptive performance and vitamin D input. The dashed line represents the curve in the absence of physiological controls (such as might be found in isolated gut loop studies), while the solid line represents the likely relationship in organisms with functioning control systems. (B) Paired sets of measured absorption values in groups studied at differing values for serum 25(OH)D. The lines connecting the points denote the pairs of values within each study [66,68]. (Copyright Robert P. Heaney, 1996, 2003. Reproduced with permission.)
bypassed, and absorption begins to rise once again.7 Intakes below the plateau are clearly insufficient, since they limit absorption anterior to any physiological controls. Intake levels beyond the plateau, by contrast, represent toxicity, i.e., the overriding of physiological controls. (The subject of vitamin D toxicity is discussed in Chapter 72.) Optimal status could thus be defined as an intake or production of the vitamin sufficient to get an individual onto the absorptive plateau. Optimal position on the plateau would depend upon population-level relative risks of toxicity and deficiency. Defining such a level can be approached operationally in several ways. One is by determining the level of vitamin D intake at which calcium absorption does not change further upon giving extra vitamin D at doses within the physiological range [66,68]. Since both PTH levels and PTH-mediated bone resorption are known to rise in the face of vitamin D insufficiency, a complementary approach would be to define the 25(OH)D level at which the parathyroid response evoked by inadequate absorbed calcium intake is minimized [69,70]. Using the absorptive response to supplemental D [53,54,66,68,70], such indices of vitamin D status as seasonal variation in serum iPTH and bone remodeling [72e77], or the point at which PTH concentration is minimized [69,70], available evidence converges on
a serum value of approximately 80 nmol/liter (32 ng/ml).8 This value is well above the bottom end of the range currently considered normal (~37.5 nmol/ liter (15 ng/ml)). (At the same time it is reassuring to note that a value of 80 nmol/liter is itself well below the mean for individuals with levels of sun exposure such as must have prevailed during hominid evolution [80].) Clinical confirmation of the approximate correctness of this level is found in a recent, large, randomized, controlled trial in which raising serum 25(OH)D from 53 nmol/liter to 76 nmol/liter resulted in a 33% decrease in typical osteoporotic fractures over 5 years of treatment [67]. Note that the untreated level (53 nmol/liter) was itself well within what had been considered the usual reference range and might therefore have been considered a “normal” value in the past. The fact that it permitted excess osteoporotic fractures shows clearly that the bottom end of the reference range can no longer be used to determine normality. Currently a “normal” value of approximately 80 nmoles/liter (32 ng/ml) is being adopted by many in the field. Since vitamin D is not found in appreciable concentrations in most of the items in the food supply (either primitive or modern), maintenance of optimal vitamin D status requires either sun exposure or, in high northern or southern latitudes, some degree of
7 This second rise in absorption has never been reported for purely solar sources of vitamin D; however, it is well recognized as a component of vitamin D intoxication. 8
The principal exception to this otherwise highly consistent body of data is found mainly in studies from the Netherlands, in which PTH is reported to be minimized below 50 nmol/L [78,79]. The reason for this discrepancy is unclear.
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supplementation or food fortification. Such a conclusion has often been uncongenial to traditional nutritionists, who have maintained that humans ought to be able to get all of the nutrients they need from a well-balanced diet. However, it must be stressed that vitamin D is an accidental nutrient, included with the other vitamins by mistake at an early stage of the development of nutritional science. Whatever the merit of the traditional nutritionists’ position, it cannot apply to this essential compound. Now that we understand the situation, we must see that it is certainly no more unnatural to sustain vitamin D status in high latitudes by supplementation than it is to sustain body temperature there by clothing or shelter. (See also chapters in Section VII of this volume.) Although more work needs to be done to define the bottom end of the acceptable normal range with precision, for now the prudent course would seem to be to aim for a vitamin D intake sufficient to produce a serum 25(OH)D level of at least 80 nmol/liter (32 ng/ml). As just noted, levels below that point result in calcium absorptive impairment [68] and carry a risk of bone loss and osteoporotic fractures [67]. The 80 nmol/liter level is certainly safe, since healthy college-age adults (with often generous sun exposure) commonly have levels twice that high. Oral cholecalciferol intakes required to produce a level of 80 nmol/liter will depend upon degree of cutaneous synthesis and pretreatment serum 25(OH)D concentration. As noted earlier, the most direct evidence for dose response in midlife individuals points to a rise of 0.7e1.0 nmol/liter for each microgram (40 IU) of daily oral cholecalciferol supplementation [48]. Less direct evidence, mostly obtained in the elderly, indicates that the rate of response from conditions of more extreme vitamin D depletion could be as much as 1.6e2.0 nmol/liter per mg of continuous daily oral supplementation. Whatever the actual response rate, it is unlikely to be higher than 2 or much lower than 0.6 mmol/liter/mg/d. Hence a patient with a baseline value of 40 nmol/liter can be estimated to require a daily dose ranging from 800 to 2600 IU to achieve the desired level. It is worth noting that the entirety of this range of required intakes is above the currently recommended intake.
SUMMARY AND CONCLUSIONS The best attested function of vitamin D is the facilitation of transfer of calcium (and phosphorus) into the extracellular fluid from ingested food and from bone. In this capacity, vitamin D functions as a part of a control system that operates to maintain constancy of the calcium ion concentrations in the extracellular fluid
against the demands of obligatory excretory losses and skeletal mineralization. In both transfers vitamin D works in concert with parathyroid hormone. Quantitative analysis of the inputs and drains of the calcium economy reveals that, at contemporary calcium intakes, D-mediated absorptive enhancement only partially mitigates the impact of low calcium intake or large calcium losses. However, at intakes closer to those prevailing during hominid evolution, minor shifts in vitamin Dmediated absorption are fully adequate to compensate for stresses on the calcium economy. While 1,25(OH)2D is clearly the most potent form of the vitamin, 25(OH) D exerts significant vitamin-D-like activity in its own right at physiological serum levels. Optimal vitamin D status is operationally defined as a level of D intake (or production) high enough to ensure that the D-mediated transfers are not limited by D availability. Available data point to a value for serum 25(OH)D of about 32 ng/ ml (80 nmol/liter) as the low end of the optimal range. Confirmation of this estimate comes from the demonstration that calcium absorption efficiency is suboptimal below ~80 nmol/liter and that risk of fragility fractures rises as serum 25(OH)D concentration falls below 80 nmol/liter.
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[52] P.F. Brumbaugh, M.R. Haussler, 1,25-Dihydroxyvitamin D3 receptor: competitive binding of vitamin D analogs, Life Sci. 13 (1973) 1737e1746. [53] H.F. DeLuca, The vitamin Decalcium axis, in: R.P. Rubin, G.B. Weiss, J.W. Putney Jr. (Eds.), Calcium in Biological Systems, Plenum Press, New York, 1983, pp. 491e511. [54] R.M. Francis, M. Peacock, J.H. Storer, A.E.J. Davies, W.B. Brown, B.E.C. Nordin, Calcium malabsorption in the elderly: the effect of treatment with oral 25-hydroxyvitamin D3, Eur. J. Clin. Invest 3 (1983) 391e396. [55] N.H. Bell, S. Epstein, J. Shary, V. Greene, M.J. Oexmann, S. Shaw, Evidence of probable role for 25-hydroxyvitamin D in the regulation of human calcium metabolism, J. Bone Miner. Res. 3 (1988) 489e495. [56] C.A. Reasner II, J.F. Dunn, D. Fetchick, Y. Liel, B.W. Hollis, S. Epstein, et al., Alteration of vitamin D metabolism in Mexican-Americans [Letter to the editor], J. Bone Miner. Res. 5 (1990) 13e17. [57] I.H. Colodro, A.S. Brickman, J.W. Coburn, T.W. Osborn, A.W. Norman, Effect of 25-hydroxyvitamin D3 on intestinal absorption of calcium in normal man and patients with renal failure, Metabolism 27 (1978) 745e753. [58] A. Devine, S.G. Wilson, I.M. Dick, R.L. Prince, Effects of vitamin D metabolites on intestinal calcium absorption and bone turnover in elderly women, Am. J. Clin. Nutr. 75 (2002) 283e288. [59] M.J. Barger-Lux, R.P. Heaney, S. Dowell, J. Bierman, Relative molar potency of 25-hydroxyvitamin D indicates a major role in calcium absorption, J. Bone Miner. Res. 11 (1996) S423. [60] A.W. Norman, Intestinal calcium absorption: a vitamin D-hormone-mediated adaptive response, Am. J. Clin. Nutr. 51 (1990) 290e300. [61] A.W. Norman, I. Nemere, L.-X. Zhou, J.E. Bishop, K.E. Lowe, A.C. Maiyar, et al., 1,25(OH)2-vitamin D3, a steroid hormone that produces biologic effects via both genomic and nongenomic pathways, Steroid Biochem. Molec. Biol. 41 (1992) 231e240. [62] J.C. Gallagher, C.M. Jerpbak, W.S.S. Jee, K.A. Johnson, H.F. DeLuca, B.L. Riggs, 1,25-Dihydroxyvitamin D3: short- and long-term effects on bone and calcium metabolism in patients with postmenopausal osteoporosis, Proc. Natl. Acad. Sci. 79 (1982) 3325e3329. [63] B. Dawson-Hughes, S.S. Harris, S. Finneran, H.M. Rasmussen, Calcium absorption responses to calcitriol in black and white premenopausal women, J. Clin. Endocrinol. Metab. 80 (1995) 3068e3072. [64] A. al-Aqeel, P. Ozand, S. Sobki, W. Sewairi, S. Marx, The combined use of intravenous and oral calcium for the treatment of vitamin D dependent rickets type II (VDDRII), Clin. Endocrinol. Oxf. 39 (1993) 229e237. [65] G. Simonin, B. Chabrol, E. Moulene, G. Bollini, S. Strouc, J.F. Mattei, et al., Vitamin D-resistant rickets type II: apropos of 2 cases, Pediatrie-Bucur. 47 (1992) 817e820. [66] M.J. Barger-Lux, R.P. Heaney, Effects of above average summer sun exposure on serum 25-hydroxyvitamin D and calcium absorption, J. Clin. Endocrinol. Metab 87 (11) (2010) 4952e4956.
[67] D.P. Trivedi, R. Doll, K.T. Khaw, Effect of four monthly oral vitamin D3 (cholecalciferol) supplementation on fractures and mortality in men and women living in the community: randomised double blind controlled trial, Br. Med. J. 326 (2003) 469e474. [68] R.P. Heaney, M.S. Dowell, C.A. Hale, A. Bendich, Calcium absorption varies within the reference range for serum 25-hydroxyvitamin D, J. Am. Coll. Nutr. 22 (2) (2003) 142e146. [69] M.-C. Chapuy, P. Preziosi, M. Maamer, S. Arnaud, P. Galan, S. Hercberg, et al., Prevalence of vitamin D insufficiency in an adult normal population, Osteoporos. Int. 7 (1997) 439e443. [70] M.K. Thomas, D.M. Lloyd-Jones, R.I. Thadhani, A.C. Shaw, D.J. Deraska, B.T. Kitch, et al., Hypovitaminosis D in medical inpatients, N. Engl. J. Med. 338 (1998) 777e783. [71] E.A. Krall, B. Dawson-Hughes, Relation of fractional 47Ca retention to season and rates of bone loss in healthy postmenopausal women, J. Bone Miner. Res. 6 (1991) 1323e1329. [72] J.M. McKenna, R. Freaney, A. Meade, F.P. Muldowney, Hypovitaminosis D and elevated serum alkaline phosphatase in elderly Irish people, Am. J. Clin. Nutr. 41 (1985) 101e109. [73] C.J. Rosen, A. Morrison, H. Zhou, D. Storm, S.J. Hunter, K. Musgrave, et al., Elderly women in northern New England exhibit seasonal changes in bone mineral density and calciotropic hormones, Bone Miner. 25 (1994) 83e92. [74] B. Dawson-Hughes, S.S. Harris, G.E. Dallal, Plasma calcidiol, season, and serum parathyroid hormone concentrations in healthy elderly men and women, Am. J. Clin. Nutr. 65 (1997) 67e71. [75] L.M. Salamone, G.E. Dallal, D. Zantos, F. Makrauer, B. Dawson-Hughes, Contributions of vitamin D intake and seasonal sunlight exposure to plasma 25-hydroxyvitamin D concentration in elderly women, Am. J. Clin. Nutr. 58 (1993) 80e86. [76] B. Dawson-Hughes, G.E. Dallal, E.A. Krall, S. Harris, L.J. Sokoll, G. Falconer, Effect of vitamin D supplementation on wintertime and overall bone loss in healthy postmenopausal women, Ann. Intern. Med. 115 (1991) 505e512. [77] E.A. Krall, N. Sahyoun, S. Tannenbaum, G.E. Dallal, B. DawsonHughes, Effect of vitamin D intake on seasonal variations in parathyroid hormone secretion in postmenopausal women, N. Engl. J. Med. 321 (1989) 1777e1783. [78] P. Lips, Vitamin D deficiency and secondary hyperparathyroidism in the elderly: consequences for bone loss and fractures and therapeutic implications, Endocr. Rev. 22 (2001) 477e501. [79] P. Lips, T. Duong, A. Oleksik, D. Black, S. Cummings, D. Cox, et al., A global study of vitamin D status and parathyroid function in postmenopausal women with osteoporosis: baseline data from the multiple outcomes of raloxifene evaluation clinical trial, J. Clin. Endocrinol. Metab. 86 (2001) 1212e1221. [80] L.Y. Matsuoka, J. Wortsman, B.W. Hollis, Suntanning and cutaneous synthesis of vitamin D3, J. Lab. Clin. Med. 116 (1990) 87e90.
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C H A P T E R
35 Fetus, Neonate and Infant Christopher S. Kovacs Memorial University of Newfoundland, Faculty of Medicine e Endocrinology, Health Sciences Centre, 300 Prince Philip Drive, St. John’s, Newfoundland A1B 3V6, Canada
OVERVIEW OF CALCIUM METABOLISM IN THE FETUS Adult calcium and bone homeostasis is largely regulated by the interactions of the classic calciotropic hormones: parathyroid hormone (PTH), 1,25dihydroxyvitamin D or calcitriol (1,25(OH)2D), calcitonin, FGF23, and the sex steroids. These hormones act to maintain a specific concentration of calcium (and other minerals) in the blood, a neutral calcium balance, and a fully mineralized skeleton. These hormones will also act to remove mineral from the skeleton when the demands for calcium outstrip the supply (lactation, renal insufficiency and other acidebase disturbances, hypercalciuria due to renal calcium leak, etc.). Deficiency of parathyroid hormone, vitamin D, or estradiol results in significant impairments in calcium and bone homeostasis. The blood calcium may be altered, skeletal mineralization becomes impaired, and a negative calcium balance results. Calcium and bone homeostasis is regulated differently during fetal development (reviewed in detail in [1,2]). The fetus has distinct goals from the adult, including that it must actively pump calcium from the maternal circulation against a concentration gradient, maintain a blood calcium higher than the maternal (adult) level, rapidly mineralize the skeleton during the final quarter of gestation, and achieve a positive calcium balance. Human babies accrete 80% of the required 30 g of calcium during the third trimester [3e5] while rats accrete 95% of the required 12.5 mg of calcium during the last 5 days of a 3-week gestation [6]. In achieving these goals fetuses do not appear to require vitamin D or the other calciotropic hormones listed above. There are limited data available from studies of human fetuses, and so human regulation of
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10035-6
fetal mineral homeostasis must be largely inferred from studies in animals. A caveat to keep in mind is that some of what has been discovered in other mammals may not apply to humans. Within the developing human, the serum calcium, ionized calcium, magnesium, and phosphorus are raised above the maternal values [1,7]. Whether these elevated values have physiological importance is uncertain but studies in fetal mice indicate that an elevated serum calcium and magnesium are needed for the skeleton to accrete the normal amount of these minerals by term [8e10]. Studies in rodents have shown that if the fetal blood calcium is reduced to the maternal level or below it, fetal survival to term is unaffected [8,11,12]. The high fetal blood calcium may protect against severe hypocalcemia at birth, when the onset of breathing raises the pH and causes an obligatory fall in the blood calcium (see neonatal section, below). The elevated serum calcium in fetuses is robustly maintained despite significant hypocalcemia due to various causes in the mothers; this observation holds true for animals and apparently for humans as well [1,7,9]. Among the calciotropic hormones, parathyroid hormone and 1,25(OH)2D are low in human babies and other mammals while calcitonin is elevated [1]. Recent human data indicate that intact FGF23 levels are low in cord blood but reach adult values by 5 days after birth [13]. Despite its low levels, parathyroid hormone is physiologically important for fetal development because in its absence fetal mice have reduced blood calcium, undermineralized skeletons, and altered placental expression of genes involved in cation transport [8,9,12]. The high serum calcium and phosphorus, and low parathyroid hormone, likely all contribute to suppression of the renal 1a-hydroxylase (CYP27b1) and maintenance of low levels of 1,25(OH)2D. Animal
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studies have demonstrated that 25(OH)D readily crosses the placenta while 1,25(OH)2D does not [14,15]. Human data are consistent with these animal studies since cord blood 25(OH)D levels are within 75e100% of the maternal value whereas cord blood 1,25(OH)2D levels are well below maternal levels [16e20]. Studies of perfused human placentas also indicate that 25(OH)D readily crosses the placenta [21]. The elevated calcitonin derives from fetal sources but the physiological importance of calcitonin for calcium homeostasis is unknown. Fetal mice lacking the gene that encodes calcitonin had normal serum calcium, placental calcium transfer, and calcium content of the skeleton at term whereas the serum magnesium and skeletal magnesium content were modestly reduced [10]. The role of FGF23 in fetal mineral and bone homeostasis has not been studied, although it has been noted that Fgf23-null mice have normal length, weight, appearance, and serum calcium and phosphorus at birth [22]. Another hormone, parathyroid-hormone-related protein (PTHrP), circulates at high levels in fetal rodents, sheep, and humans [1,12,23e25]. When full-length parathyroid hormone and PTHrP were measured in cord blood, PTHrP levels were 2e4 pmol/l and up to 15fold higher than simultaneous values for PTH (0.2e0.5 pmol/L) [23e25]. PTHrP plays a significant role in regulating fetal mineral homeostasis and skeletal mineralization. Fetal mice lacking PTHrP are hypocalcemic; display abnormal endochondral bone development with short-limbed dwarfism, rounded skull, and shortened mandibles with protruding tongues; and have reduced placental calcium transfer [11,26]. In humans the equivalent to the PTHrP-null state has not been described but null mutations in the PTH receptor cause a similar and lethal condition called Blomstrand chondrodysplasia [27,28]. Calcium and other minerals enter the adult organism via intestinal absorption but this route is trivial in the fetus because only the amniotic fluid is available to be swallowed and absorbed. At this stage of development intestinal calcium absorption occurs by passive and nonsaturable mechanisms. Fetal urine is the major source of fluid and solute in the amniotic fluid. The main route of entry for minerals into the fetus is via the placenta (Fig. 35.1). Calcium, magnesium, and phosphorus are actively transported across the placenta, and this is required in order to fully mineralize the fetal skeleton by term. Studies in fetal sheep, rats, and mice have shown that PTHrP is a major regulator of calcium and possibly magnesium transfer while parathyroid hormone may also play a role [1,9,11,29e31]. Active transfer of calcium becomes evident in the last 7 days of gestation in the rat and mouse, coinciding with marked up-regulation of calcium-binding proteins, TRPV6 (transient receptor potential vanilloid 6), and
Ca2þ-ATPase within trophoblasts, and rapid accretion of mineral within the fetal skeleton [32e37]. A complete cartilaginous skeleton with digits and intact joints is present by the eighth week of gestation in humans. Primary ossification centers form in the vertebrae and long bones between the eighth to twelfth weeks, but it is not until the third trimester that the bulk of mineralization occurs. At the 34rd week of gestation, secondary ossification centers form in the femurs, but otherwise most epiphyses are cartilaginous at birth, with secondary ossification centers appearing in other bones in the neonate and child [38]. As in the adult, the fetal skeleton participates in the regulation of mineral homeostasis with calcium being resorbed to help maintain the concentration of calcium in the blood. If human mothers are severely hypocalcemic due to hypoparathyroidism, fetuses will develop secondary
Calcium sources in fetal life. The main flux of calcium is across the placenta and into fetal bone. Some calcium returns to the maternal circulation (backflux); calcium filtered by the kidneys is partly reabsorbed into the circulation; calcium excreted by the kidneys into the urine and amniotic fluid may be swallowed and absorbed by the intestine; calcium is also resorbed from the developing skeleton to maintain the circulating calcium concentration. Calcitriol and the VDR do not appear to be major regulators of placental calcium transfer. Reproduced from [7] Ó2003 by Academic Press, used with permission.
FIGURE 35.1
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hyperparathyroidism, skeletal demineralization, and fractures in utero [1,39].
ANIMAL DATA RELEVANT TO VITAMIN D AND THE FETUS The fetal demand for calcium is largely met by upregulated intestinal calcium absorption in the mother beginning early in pregnancy. Several studies indicate that this maternal adaptation does not require vitamin D. Pregnancy in vitamin-D-deficient rats and in mice lacking the vitamin D receptor (Vdr-null) results in a marked up-regulation of intestinal calcium absorption to the same high rate achieved during pregnancy by normal rats and mice [40e42]. This up-regulated calcium absorption is physiologically significant because both vitamin-D-deficient rats and Vdr-null mice experienced a significant increase in skeletal mineral content or BMD during pregnancy [42e44]. Additional animal studies suggest that the Vdr-null mouse compensates by up-regulating intestinal expression of the calcium channel TRPV6 [42], and that prolactin and placental lactogen may stimulate intestinal calcium absorption independently of 1,25(OH)2D, possibly by stimulating TRPV6 [45,46]. Although the animal data are compelling that 1,25(OH)2D or its receptor are not required to up-regulate intestinal calcium absorption during pregnancy, as yet no clinical study has examined intestinal calcium absorption in vitamin-D-deficient versus vitamin-D-replete pregnant women. The impact of altered vitamin D physiology on systemic calcium homeostasis during pregnancy has been examined in severely vitamin-D-deficient rats [43,47e49], pigs with a null mutation of the 1a-hydroxylase [50], and Vdr-null mice [37,42]. In each model the nonpregnant adult is unwell with hypocalcemia, hypophosphatemia, and rickets/osteomalacia. (1a-Hydroxylase-null mice also have hypocalcemia and hypophosphatemia but are infertile [51,52].) Females with vitamin D deficiency or with these mutations conceive less often than normal, maintain low blood calcium and phosphorus levels, and bear smaller litters. Sudden, sporadic (presumably hypocalcemia-induced) maternal deaths occur late in pregnancy during the interval of rapid calcium transfer to the fetus in vitamin-D-deficient rats and Vdr-null mice, possibly indicating that the mother has difficulty maintaining her own blood calcium in the face of rapid, active transfer of calcium across the placenta to multiple fetuses [37,42,48,53]. In contrast to their mothers who are so unwell as a consequence of disrupted vitamin D physiology, the fetuses and newborns show normal blood calcium,
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magnesium, phosphorus, parathyroid hormone, weight, skeletal mineral content, and skeletal calcium content. This includes pups born of severely vitamin-D-deficient rats [48,49,53,54], 1a-hydroxylase-null pigs [50] and both Vdr heterozygous and Vdr-null mice [37]. 1a-Hydroxylase-null mice are reportedly normal at birth but extensive studies of fetal chemistries and skeletal mineral content have not been published [51,52]. The normal, heterozygous, and null fetuses born of Vdr heterozygous mice were indistinguishable in all parameters of mineral homeostasis, weight, and skeletal size, histomorphometry, morphology, gene expression, and mineral content [37] (Fig. 35.2). Heterozygous and null fetuses born of Vdr-null mothers were smaller, weighed less, but maintained normal blood calcium and normal mineral content for their proportionately smaller skeletons [37]. This reduction in fetal size and weight in pups born of Vdr-null mice was not observed in models of vitamin D deficiency, and may indicate that absence of the vitamin D receptor has effects on fetal growth which vitamin D deficiency does not. Placental calcium transfer from mother to fetus was normal to increased, as assayed indirectly in pups born of vitamin-D-deficient rats [55] and directly in fetuses from Vdr heterozygous and Vdr-null mice [37]. The placenta provides calcium to the fetus without relying on vitamin D metabolites, and the “vitamin D-dependent” factors calbindinD-9K and Ca2þ-ATPase e which are important for intestinal calcium absorption in the adult e are expressed at normal levels in vitamin-D-deficient and Vdr-null placentas [37,55e57]. On the other hand, expression of PTHrP and CaT1 (TRPV6) were both up-regulated in placentas of Vdr-null mice versus wt, consistent with the observed increase in placental calcium transfer in Vdr-nulls [37]. In all of the aforementioned animal models the adults have florid rickets, hypocalcemia, hypophosphatemia, and marked secondary hyperparathyroidism. However, as noted below in the neonatal section, none of these abnormalities develop until after weaning in vitaminD-deficient rats [53,54], 1a-hydroxylase-null pigs [50], 1a-hydroxylase-null mice [51,52], and Vdr-null mice [58,59]. These observations underscore that 1,25(OH)2D is not needed to regulate placental calcium transfer or skeletal mineralization in the fetus. Although these studies suggested no role for 1,25 (OH)2D in fetal mineral homeostasis, several other reports have suggested otherwise. Prior nephrectomy in fetal lambs reduced placental calcium transfer as assessed by perfusing the placenta in situ after the fetus had been removed, and treatment with 1,25(OH)2D partly restored the rate of calcium transfer across the isolated placenta [60]. Further, pharmacological doses of 1,25(OH)2D or 1a-OHD increased calcium transfer across in situ perfused placentas of lambs and rats [61,62]. All of
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FIGURE 35.2 Resilience of fetal blood calcium regulation despite absence of VDR and marked maternal hypocalcemia. Ionized calcium was measured on whole blood in fetuses obtained from Vdrþ/e mothers (A) and Vdr-null mothers (B) on embryonic days 17.5 and 18.5 (1 or 2 days before expected birth). No difference in ionized calcium level was noted among the fetal genotypes, and the mother’s genotype or ionized calcium level did not affect the fetal ionized calcium level. The mean maternal ionized calcium is indicated by the horizontal line in each graph, with SE values displayed on the right. Double-headed arrows emphasize the maternalefetal calcium gradient which was strikingly increased in fetuses of Vdr-null mothers as compared to fetuses of Vdrþ/e mothers (P < 0.001). The number of observations is indicated in parentheses. Reproduced from [37] Ó2005 by the American Physiological Society, used with permission.
these reports appear to have demonstrated pharmacological rather than physiological actions of 1,25(OH)2D. The studies of Vdr-null mice described above indicated that removal of the effective actions of 1,25(OH)2D caused an increase in the rate of placental calcium transfer (and placental expression of PTHrP and TRPV6) in intact mice, not a decrease as predicted by these studies in perfused placentas from lambs and rats. As noted above in the overview, maternal hypocalcemia due to hypoparathyroidism causes fetal hypocalcemia, secondary hyperparathyroidism, skeletal demineralization, and fractures in utero [1,39,63]. This outcome has not been reported with any animal model of disrupted vitamin D physiology. The explanation may be that hypoparathyroidism usually causes more severe maternal hypocalcemia which is also aggravated by the accompanying hyperphosphatemia. When hypocalcemia occurs due to disrupted vitamin D physiology (vitamin D deficiency, absence of 1,25(OH)2D, or absence of VDR), compensatory secondary hyperparathyroidism and consequent hypophosphatemia both serve to lessen the magnitude of maternal hypocalcemia. Collectively these findings indicate that fetal calcium homeostasis and skeletal development/mineralization are independent of vitamin D, 1,25(OH)2D, and its receptor. The animal studies predict that human babies born of vitamin-D-replete and vitamin-D-deficient mothers should have similar blood calcium, phosphorus, and skeletal mineral content at birth. As noted in the next section, low maternal 25(OH)D levels during late pregnancy have been associated in humans with increased risk of a variety of maternal outcomes (preeclampsia, bacterial vaginosis, elective C-section) and fetal/neonatal outcomes (asthma, type 1 diabetes, etc.). Most of these associations have not been tested in animal models. Vdr-null mice are not
known to be predisposed to any adverse pregnancyrelated outcomes except hypocalcemia and sudden (presumed hypocalcemic) deaths near term [37,42,64]. In the nonobese diabetic (NOD) mouse, which is genetically predisposed to develop insulitis followed by type 1 diabetes, there is evidence that superimposed vitamin D deficiency causes the diabetes to develop sooner [65]. However, treatment of NOD mice with 1000 IU of supplemental vitamin D daily (a supraphysiological dose for the mouse) beginning in utero and continuing up to 10 weeks of age with follow-up to 32 weeks did not reduce the incidence of type 1 diabetes [66]. Treatment with pharmacological doses of 1,25(OH)2D or analogs of it have been shown to significantly reduce insulitis and the occurrence of diabetes, presumably through immune-mediated mechanisms [67,68]. A more recent study crossed the NOD mouse with Vdrnull mice but loss of the VDR did not alter the rate of development of insulitis and diabetes [69]. Overall these studies indicate that severe vitamin D deficiency increases the risk of type 1 diabetes in a genetically predisposed animal whereas loss of the VDR does not, and that pharmacological (hypercalcemic) doses of 1,25 (OH)2D are needed to prevent the diabetes. The studies do not address whether vitamin D deficiency or insufficiency significantly increase the risk of type 1 diabetes in an animal that is not genetically predisposed to develop the condition.
HUMAN DATA RELEVANT TO VITAMIN D AND THE FETUS Unlike the animal studies, there have been no systematic and comprehensive studies comparing serum calcium, calciotropic hormones, or skeletal mineral
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content among babies born of vitamin-D-replete, vitaminD-insufficient, and vitamin-D-deficient mothers. In the only attempt at a systematic examination of the fetal skeleton [70], investigators in China examined the ash weight and mineral content of 15 fetuses that had died due to obstetrical accidents: seven of these were born of mothers known to have osteomalacia and the other eight were born of otherwise healthy mothers. The first three fetuses analyzed appeared to have a lower ash weight versus controls but the latter four fetuses had an ash weight that equalled or exceeded the controls. Overall there were no significant differences in ash weight or calcium, phosphorus, and magnesium content of the ash between the two groups. The authors also described that centers of ossification were normal and that there were no radiographic signs of rickets, although they did note in two of the seven fetuses “an apparent cupping of the end of the [ulna].which at once suggested the possibility that this was rickety in nature” [70]. The authors concluded that “there is no evidence of pre-natal rickets” but “there are definite changes in the skeletal system of the foetus born from mothers who are the subject of osteomalacia” [70]. The sample size was quite low in this old report and the osteomalacia in the mothers was confounded by malnutrition. A few rare, isolated case reports have unequivocally indicated that craniotabes and other suggestive skeletal changes can be detected at birth [71e74], whereas in other reports that described craniotabes or rickets being present “at birth” the diagnosis was actually made within the first or second week [74e79]. In one such case convincing radiographic findings were not present at day 2 after birth but had developed by day 16 [79]. In many cases of congenital rickets the cause was not isolated vitamin D deficiency. Instead, the mothers had significant malnutrition, malabsorption (e.g., celiac disease, pancreatic insufficiency), or very low intakes of both calcium and vitamin D [72,74,78]. More recent clinical experience is that vitamin-D-deficient rickets usually does not develop (or become recognized) until weeks to months after birth with a peak incidence between 6e18 months, even in regions where severe vitamin D deficiency during pregnancy is endemic [80e83]. Consistent with this, in a clinical trial that resulted in 25(OH)D levels of 138 nmol/L in fetuses of vitamin-D-treated mothers versus 25(OH)D levels in the rachitic range of 10 nmol/L in fetuses of placebotreated mothers, there was no difference in cord blood calcium or phosphorus between groups and no radiologic evidence of rickets [84] The clinical courses of children born with genetic disorders of vitamin D physiology have also been reported in assorted cases, series and reviews. Babies with 1a-hydroxylase deficiency (vitamin-D-dependent rickets type I; VDDR-I) and those lacking the vitamin
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D receptor (vitamin-D-dependent rickets type II or hereditary vitamin-D-resistant rickets; VDDR-II) have been described as normal at birth [85e90]. In both conditions the child inevitably develops hypocalcemia, hypophosphatemia, and rickets. VDDR-I presents as early as the neonatal period to within the first year of life; VDDRII can also present in infancy but more often is diagnosed during the second year of life [85e90]. Thus the limited data from humans are in agreement with the animal data that hypocalcemia and rickets are not present at birth but develop postnatally. The clinical course of pregnancy in women with VDDR-I or VDDR-II is uneventful when normocalcemia is maintained in the mother [91,92]. Overall the clinical findings in term infants with vitamin D deficiency and in all infants with VDDR types I and II indicate that hypocalcemia and skeletal changes of rickets are not present at birth but develop days to months afterward. But a few isolated case reports (described above) of vitamin-D-deficient babies born to osteomalacic mothers have shown that craniotabes and other early rachitic changes may be present at birth; the etiology in these cases may be maternal malnutrition, malabsorption, hypophosphatemia, or inadequate intake of both calcium and vitamin D, and not isolated vitamin D deficiency. In animal studies intestinal calcium absorption in the fetus is trivial and occurs through passive, nonsaturable mechanisms. Preterm human infants, the developmental equivalent of fetuses, also demonstrate passive, nonvitamin-D-dependent absorption of calcium [93,94], while normal term and preterm infants show a postnatal increase in the efficiency of intestinal calcium absorption similar to the animal studies described above [93,95,96]. This maturation of intestinal calcium absorption to a vitamin-D-dependent process parallels and explains the aforementioned clinical observation that vitamin-D-deficient rickets does not develop (or become recognized) until several weeks to typically 6e18 months after birth, even in regions where severe vitamin D deficiency during pregnancy is endemic [80e82]. Observational and clinical studies of human pregnancy described in the next several paragraphs have shown no relation of cord blood 25(OH)D levels with cord blood calcium or parathyroid hormone. This is consistent with the animal studies mentioned earlier in which vitamin D deficiency and absence of the vitamin D receptor did not alter the fetal blood calcium. Large, double-blind, randomized, placebo-controlled trials provide the highest level of evidence, but no such studies of vitamin D have been done during human pregnancy. Instead, small randomized and often unblinded clinical trials have been reported. These have shown that increased maternal intake of vitamin D supplements results in higher maternal and fetal
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(cord blood) 25(OH)D levels, but no clear proof of any additional benefits.
Intervention Studies In one of the earliest such studies [84], Brooke conducted a double-blind randomized trial of vitamin D supplementation in an Asian population living in England. The study involved 59 women treated with vitamin D and 67 controls. This population was quite vitamin-D-deficient (mean 25(OH)D level 20 nmol/L at baseline) and therefore was an ideal group to test whether correction of vitamin D deficiency resulted in any maternal or fetal benefits. Although the treatment group nominally received 1000 IU of vitamin D per day, they achieved a substantial increase in maternal 25(OH) D levels to a mean level of 168 nmol/L, indicating that the dose must have been 10 000 IU per day or more, or that the assay yielded unrealistic results. The mean cord blood 25(OH)D value was 138 nmol/L in babies born of the treated mothers versus 10 nmol/L in control babies. Treatment with vitamin D caused a modest increase in maternal serum calcium but cord blood calcium remained not significantly different between the vitamin-D-supplemented and control infants. The treatment did not affect birth weight, crowneheel length, forearm length, triceps skinfold thickness, and head circumference, whereas the fontanelle area was reduced from 6.1 0.7 to 4.1 0.4 cm2 (p < 0.05). In another intervention trial [97], Mallet administered 1000 IU/day of vitamin D2 during the last trimester of pregnancy, or a single dose of 200 000 IU of vitamin D2 at month 7, or no supplementation, to three groups of women (N ¼ 21, 27, and 29 respectively) living in France. The treatments were equally effective in increasing maternal and cord blood 25(OH)D levels versus controls. Maternal, cord blood, and neonatal calcium levels did not differ among the three groups, nor was there any effect on skeletal or anthropometric parameters in the infants. Another clinical trial randomized 40 women to 1000 IU vitamin D3 supplementation daily versus placebo beginning at 6 months of pregnancy and found no effect on skeletal parameters or on the cord blood calcium [98]. In a study of vitamin-D-deficient Asian women which generated two separate reports (randomization methodology was unclear), 800 000 IU of vitamin D2 administered in the 7th and 8th months of pregnancy to 20 women resulted in a small increase in cord calcium but no other benefit compared to 75 women who received no supplement and 25 women who received 1200 IU of vitamin D2 per day [99,100]. Yu conducted a small randomized but unblinded intervention trial in London to determine the effects of daily vs. single-dose vitamin D supplementation on
pregnancy outcomes [101]. The doses of vitamin D given were 800 IU/d of vitamin D2 or a single dose of 200 000 IU of vitamin D2, each beginning at gestational week 27, as compared to no treatment. Maternal serum 25(OH)D levels at delivery were significantly greater in both treatment groups compared to the control group (mean 42, 34, and 27 nmol/L, respectively). Similarly, mean cord 25(OH)D levels were significantly greater in the treatment groups compared to the controls (26, 25, and 17 nmol/L, respectively). However there was no significant difference between treatment and control groups for gestational age at delivery, birth weight, or incidence of low birth weight. More recently Wagner and Hollis have completed an NIH-sponsored trial in which 494 women were randomized at 12e16 weeks of gestation to receive 400, 2000, or 4000 IU of vitamin D3 per day for the duration of pregnancy. Only 350 (70.9%) of women completed the study. The primary outcomes and intention-to-treat analysis of this potentially illuminating study remain unknown at the time of writing; the results have not been peer-reviewed or published, although preliminary abstracts were seen. Wagner and Hollis also completed a smaller study of 257 women who were enrolled at 12e16 weeks of gestation to receive 2000 or 4000 IU of vitamin D3 per day. Only 160 (62.3%) of women completed this study and the results of it also remain unknown. The large dropout rates and expected low numbers of events (e.g., prematurity, preeclampsia) make it unlikely that these two studies would provide conclusive data about benefits and risks of such levels of vitamin D supplementation during pregnancy. However, the results are expected to be more compelling than those of the various small trials completed to date.
Associational Studies Associational and epidemiological studies are hypothesis-generating but do not prove causality. Such studies in humans have led to numerous associations being made between serum 25(OH)D in the mother and various outcomes of pregnancy, but the evidence is inconsistent and confounded. Among maternal outcomes of pregnancy that can adversely affect the fetus, preeclampsia has been associated with low maternal 25(OH)D levels [102], while use of vitamin D supplements has been associated with a lower risk of preeclampsia [103,104]. Observational studies also suggest that low 25(OH)D levels increase the risk of gestational diabetes [105,106]. However, these observations are significantly confounded by such factors as increased maternal weight, lower socioeconomic status, and poorer nutrition, all of which are associated with lower 25(OH)D levels. Overweight and obesity directly
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predispose to gestational diabetes but also reduce 25 (OH)D levels due to sequestration in fat and possibly other mechanisms. In support of this notion, weight reduction studies show that serum 25(OH)D levels rise when obese individuals lose body fat but maintain the same vitamin D intake [107e110]. A similar association of low 25(OH)D levels during pregnancy with increased risk of elective or primary cesarian section may also be confounded by maternal overweight and obesity [111]. There are no randomized, controlled trials that have tested whether any of these associations are causative. There are numerous studies testing associations of maternal or cord blood 25(OH)D levels on fetal outcomes. In an observational study assessing the effects of maternal vitamin D status on fetal bone health, Mahon compared 25(OH)D levels at 36 weeks’ gestation with radiologic evidence for bone development in the fetus [112]. The analysis of 424 mothereinfant pairs found no association between maternal vitamin D status and femur length. However there was an association between maternal serum 25(OH)D levels below 50 nmol/L and greater distal metaphyseal crosssectional area (RR ¼ e0.10 (95% CI: e0.02 to 0.00)), and with a novel index derived by the authors called the “femoral splaying index” (the metaphyseal crosssectional area divided by the femur length) (RR ¼ e0.011 (95% CI: e0.21 to e0.01)). A longitudinal study by Javaid in England evaluated the effect of serum 25(OH)D levels in late pregnancy on pregnancy outcomes at birth, 9 months, and 9 years of age [113]. No associations were found between maternal serum 25(OH)D and birth weight, birth length, placental weight, abdominal circumference, or head circumference. There was also no association found between vitamin D status in late pregnancy and cord blood calcium level. At the 9-month follow-up there was still no association of the maternal 25(OH)D status during pregnancy with skeletal and anthropometric parameters in the children. However, at 9 years of age the investigators were able to show a significant correlation between skeletal parameters and the estimated maternal 25(OH) D value in late pregnancy. A maternal serum 25(OH)D level below 27.5 nmol/L was associated with a modest but statistically significant decrease in bone mineral content (BMC) in offspring at 9 years of age compared to offspring of mothers whose 25(OH)D levels were 50 nmol/L or higher (1.04 0.16 vs. 1.16 0.17 g) (P ¼ 0.04). These findings are compatible with the theory that vitamin D sufficiency during fetal development may program childhood or peak bone mass and mineralization [114,115]. However, there are important confounders. Low 25(OH)D status in any adult correlates with obesity, poorer nutrition, lower socioeconomic status, etc., and so the association may not be casual. Moreover much time elapsed between birth (when no
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effect was seen) and 9 years of age (when reduced BMC was observed); the lower maternal 25(OH)D status in late pregnancy may indicate that such factors as lower socioeconomic status and poorer nutrition in the mother were shared with the child. A similar study by Morley examined 374 mothereinfant pairs and found no association of maternal 25 (OH)D level at 28e32 weeks of gestation with any infant measurement (body weight, skeletal lengths, skinfold thickness) [116]. An additional analysis stratified the women into those with 25(OH)D levels below 28 nmol/L compared to those with levels greater than 28 nmol/L. The infants born to mothers with 25(OH)D levels below 28 nmol/L were found to have a slightly shorter kneeeheel length but the difference was not statistically significant. No association was seen with crownerump length, weight, or other parameters. A more recent but much smaller study in Finland planned to measure maternal and cord blood 25(OH)D and pQT of the left newborn tibia in 125 mothereinfant pairs [20]. Although 125 women enrolled in the first trimester, only 90 women remained to be stratified into two groups based on 25(OH)D levels below or above the median of 42.6 nmol/L while only 72 infants had cord blood measurements that could be stratified by their mother’s 25(OH)D levels. Left unreported was the number of infants that had pQCT measurements done during the first several days after birth. With these limitations in mind, the authors reported that babies born to mothers above the median 25(OH)D level had a slightly higher tibial cross-sectional area (11.5 mm2) and tibial BMC (0.04 g/cm) while there was no difference in birth weight, length, or BMD of the tibia [20]. Vitamin D supplementation has also been proposed to increase birth weight, but the evidence for this is largely negative. In the controlled trials described above, there was no effect of vitamin D supplementation on birth weight. However, in two follow-up analyses of the small clinical trial by Brooke [84], severely vitaminD-deficient women treated with vitamin D gained more weight during pregnancy than controls, and although birth weight did not differ, their babies gained more weight postnatally than control infants [117,118]. Several associational studies mentioned above showed no effect on birth weight [112,113,116]. One prospective study of 2251 low-income, minority gravidae found that maternal intake of less than 200 IU vitamin D per day was associated with significantly reduced birth weight, but the association may have been the result of globally reduced nutrition rather than a specific effect of low vitamin D intake [119]. In a cohort of 279 pregnant women stratified by milk intake (> or <250 ml per day) there was no significant effect on birth weight, length, or head circumference; but in a multivariate analysis milk intake and estimated vitamin D intake were significant
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predictors of birth weight [120]. Lastly, in a prospective study of 123 Gambian mothereinfant pairs there was no association between maternal 25(OH)D level and birth weight, length, head circumference, BMD, or BMC of the infants even though maternal 25(OH)D levels ranged from 51e189 nmol/L at 20 and 36 weeks of pregnancy [121]. Associational studies among older adults have suggested that vitamin D deficiency and insufficiency may increase the risk of chronic diseases such as type 1 diabetes, asthma, multiple sclerosis, and others. For most of these associations there are no specific data relating the disease outcome to fetal or maternal 25(OH)D levels during pregnancy. However, there is some evidence with respect to risk of type 1 diabetes. A higher dietary intake of vitamin D (but not the use of vitamin D supplements) during pregnancy is associated with decreased prevalence of islet cell antibodies and diabetes in the offspring [122], and a history of vitamin D supplementation of pregnant women [123,124] or the infant directly [125e127] is associated with a lower childhood incidence of type 1 diabetes. There are no clinical trials and so this evidence is not definitive. The association requires further investigation with randomized trials before vitamin D can be recommended as a treatment proven to reduce the incidence of type 1 diabetes. Similarly while some studies have associated higher maternal intake of vitamin D during pregnancy with lower risk of childhood asthma and allergy in the offspring [128,129], other studies have found supplemental vitamin D use during pregnancy to increase the risk of childhood asthma and atopy [130e132]. In a follow-up analysis of the Javaid study cited above, the investigators examined whether exposure to maternal serum 25(OH)D levels above 75 nmol/L during pregnancy had any other effects on the health of offspring at 9 years of age [133]. No correlation was found between very high maternal vitamin D status and offspring growth, cognitive function, or cardiovascular function. However, the babies of women whose serum 25(OH)D levels were greater than 75 nmol/L during pregnancy were five times more likely to have asthma at 9 years of age compared to offspring of mothers with lower 25(OH)D levels during pregnancy. The bottom line is that current evidence indicates the human fetus may suffer no skeletal problems as a consequence of vitamin D deficiency and insufficiency, but after birth hypocalcemia and progressive rickets will develop in those with severe vitamin D deficiency. It remains unclear whether 25(OH)D levels in the fetus or pregnant mother have a direct influence on childhood bone mass, or on nonskeletal conditions such as type 1 diabetes and asthma. Furthermore, some of the existing associative studies suggest harm to the offspring from higher 25(OH)D levels or maternal intake of vitamin D
during pregnancy. Large randomized controlled trials of vitamin D supplementation during pregnancy are sorely needed.
OVERVIEW OF CALCIUM METABOLISM IN THE NEONATE AND INFANT Upon cutting the umbilical cord the placental calcium infusion and placental sources of PTHrP are lost. A rapid adjustment in the regulation of mineral homeostasis is forced to begin and be completed over the coming hours to days. The neonate becomes dependent upon intestinal calcium intake, skeletal calcium stores, and renal calcium reabsorption, in order to maintain a normal blood calcium at a time of continued skeletal growth and mineral accretion. A positive mineral balance must be maintained until peak bone mass is achieved in the young adult. Parathyroid hormone, 1,25(OH)2D, and FGF23 become more important for neonatal calcium homeostasis while PTHrP becomes less involved. After birth the total and ionized calcium concentrations fall, provoked by loss of the placental calcium pump and placental-derived PTHrP, and by a rise in pH that the onset of breathing causes. Studies in rodents show a fall in total and ionized calcium levels to about 60% of the fetal value by 6e12 hours after birth, and a subsequent rise to the normal adult value over the succeeding week [1]. Although data are less complete for humans, the progression in ionized and total calcium values appears to be similar. The ionized calcium in normal neonates falls from the umbilical cord level of 1.45 mmol/L to a mean of 1.20 mmol/L by 24 hours after birth [134]. Babies delivered by elective C-section were found to have lower blood calcium and higher parathyroid hormone levels at birth compared to babies delivered by spontaneous vaginal delivery [135], indicating that the mode of delivery can affect early neonatal mineral homeostasis. Phosphorus initially rises over the first 24 hours of postnatal life in humans and then gradually declines. Parathyroid hormone rises from the lower fetal levels to within or near the normal adult range by 24e48 hours after birth [1]. The increase in parathyroid hormone follows the early postnatal drop in the serum ionized calcium, and precedes the subsequent fall in phosphorus and rise in ionized calcium and 1,25(OH)2D. 1,25(OH)2D rises to adult levels over the first 48 hours of postnatal life, likely in response to the increasing levels of PTH. Serum calcitonin rises during the same time interval and then declines to adult levels. Based on one human study, serum intact FGF23 levels rise over the first 5 days post delivery to equal to adult values [13]. FGF23 appears to play an important role
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in phosphorus regulation after weaning, when Fgf23null mice develop progressive hyperphosphatemia, modest hypercalcemia, elevated 1,25(OH)2D, low PTH, skeletal abnormalities, and soft tissue and vascular calcifications [22,136,137]. PTHrP secretion from placenta, amnion, and umbilical cord is lost at birth. Secretion from the parathyroid glands (if ever present) is also apparently lost sometime after birth, since PTHrP circulates at low to undetectable levels during normal adult life in humans and animals. Animal studies suggest that PTHrP may persist in the neonatal circulation for some time, whether secreted by the parathyroids or due to absorption of PTHrP from milk (milk contains PTHrP at concentrations 10 000-fold higher than the level in the fetal circulation). Whether PTHrP present in milk contributes to the regulation of neonatal mineral homeostasis is unknown. Intestinal calcium absorption in newborns is largely passive, nonsaturable, and not dependent on 1,25 (OH)2D. The high lactose content of milk increases the efficiency of intestinal calcium absorption and net bioavailability of dietary calcium, through effects on paracellular diffusion in the distal small bowel [138e140]. As postnatal age increases, enterocytes express higher levels of the vitamin D receptor and the calcium-binding protein calbindin9KD. At the same time, vitamin-D-dependent active transport of calcium increases and passive transfer of calcium through the paracellular route declines. In weaned rodents active transport is the major route by which calcium enters the intestinal mucosa. Data from newborn humans are less complete, but the onset of 1,25(OH)2D-dependent active transport of calcium follows a similar postnatal course. The programmed postnatal maturation of the neonatal intestine limits the ability of preterm humans to absorb sufficient calcium to regulate the blood calcium and facilitate skeletal mineral accretion. Although data are limited, urinary calcium excretion rises in humans over the first 2 weeks after birth, consistent with increased efficiency of intestinal calcium absorption. The human neonatal skeleton continues to accrete calcium at 120e150 mg per day, similar to late gestation. Premature infants are prone to develop metabolic bone disease of prematurity, a form of rickets precipitated by loss of the placental calcium infusion at a time when the skeleton needs to accrete calcium at a peak rate. This form of rickets is not due to vitamin D deficiency per se but is the consequence of inadequate calcium and phosphate intake to meet the demands of the mineralizing neonatal skeleton. The limiting factor is the immaturity of the intestine which is not ready to express the vitamin D receptor and respond to 1,25 (OH)2D. Special oral or parenteral formulas that are high in calcium and phosphorus content will increase
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passive absorption and enable normal skeletal accretion of these minerals. In the neonatal period the consequences of maternal hyperparathyroidism during pregnancy may become apparent with prolonged (and sometimes permanent) suppression of the neonatal parathyroid function, hypocalcemia, tetany, and even death. The mechanism for this is uncertain but maternal hypercalcemia may cause increased flux of calcium across the placenta, activation of the calcium-sensing receptor and suppression of the fetal parathyroid glands in utero. This postnatal suppression has also been observed in infants of women with hypercalcemia due to familial hypocalciuric hypercalcemia. Maternal hypoparathyroidism with hypocalcemia during pregnancy can result in fetal parathyroid gland hyperplasia but is usually not recognized until the neonatal period [1]. Sparse data are available but in affected neonates parathyroid hormone is increased while the serum calcium may be normal or elevated. The skeletal findings generally resolve over the first several months after birth, but acute interventions may be required to raise or lower the blood calcium in the neonate. In severe cases neonatal hyperparathyroidism has caused persistent hypercalcemia with subtotal parathyroidectomy required to correct it. Neonatal hypocalcemia typically presents as seizures that onset between 4 to 28 days of age. The preterm infant is particularly prone to hypocalcemia due to inefficient intestinal calcium absorption. Other causes of neonatal hypocalcemia include congenital hypoparathyroidism, magnesium deficiency, maternal diabetes, vitamin D deficiency or resistance, and hyperphosphatemia.
ANIMAL DATA RELEVANT TO VITAMIN D AND THE NEONATE AND INFANT Data described earlier indicate that skeletal development and blood calcium, magnesium, and phosphorus are normal in offspring of mothers with severely altered vitamin D physiology, including vitamin-D-deficient rats [48,53,54], 1a-hydroxylase-null pigs [50], Vdr heterozygous and Vdr-null mice [37], and probably 1a-hydroxylase-null mice as well [51,52]. However, maternal hypocalcemia due to disrupted vitamin D physiology is not without consequences for the neonate since sudden (presumably hypocalcemia-induced) maternal deaths have occurred in the puerperium and during lactation for severely vitamin-D-deficient rats [48,53] and Vdr-null mice [37,42]. As reviewed in greater detail elsewhere [1,2,141,142], resorption of the maternal skeleton appears to be the main source of calcium contained in milk and provided
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The breast regulates skeletal demineralization and milk calcium supply during lactation. Suckling induces release of prolactin. Suckling and prolactin both inhibit the hypothalamic GnRH pulse center, which in turn suppresses the gonadotropins (LH, FSH), leading to low levels of the ovarian sex steroids (estradiol and progesterone). PTHrP production and release from the breast is controlled by several factors, including suckling, prolactin, and the calcium receptor. PTHrP enters the maternal bloodstream and combines with systemically low estradiol levels to markedly up-regulate bone resorption. Increased bone resorption releases calcium and phosphate into the bloodstream, which then reaches the breast ducts and is actively pumped into the breast milk. PTHrP also passes into the milk at high concentrations, but whether PTHrP plays a role in regulating calcium physiology of the neonate is unknown. Reproduced from [141] Ó2005 with kind permission from Springer Science and Business Medi B.V.
FIGURE 35.3
to the suckling neonate (Fig. 35.3). PTHrP (secreted from mammary tissue) and low estradiol during lactation combine to stimulate bone resorption and osteocytic osteolysis. Lactating rodents also maintain an increased rate of intestinal calcium absorption, approximately double the nonpregnant value [40,143], and this may be necessary in order to meet the proportionately greater calcium demands of multiple suckling pups. Vitamin D contributes to the normal regulation of calcium homeostasis in lactating mothers. 1,25(OH)2D levels are elevated in lactating rodents and increase further in response to a low-calcium diet or larger litter sizes [144,145]. This may indicate a compensatory mechanism that increases intestinal calcium absorption even further when extra demands are placed on the mother. However, studies in vitamin-D-deficient rats and Vdrnull mice indicate that sufficiency of vitamin D or responsiveness to 1,25(OH)2D are not required for lactation. Vitamin-D-deficient rats and Vdr-null mice lactated normally and resorbed the normal amount of bone
[42,43,146], although one study in vitamin-D-deficient rats found that more skeletal mineral content was lost than normal [147]. Intestinal calcium absorption remained up-regulated to twice normal in lactating vitamin-D-deficient rats, confirming that vitamin D is not required for this adaptation to take place [40,143]. As mentioned earlier, prolactin may stimulate intestinal calcium absorption independently of 1,25(OH)2D, possibly by stimulating TRPV6 [45,46]. At the end of lactation a rapid phase of bone formation occurs during which the maternal skeleton regains its prepregnancy bone mineral content. Full post-weaning recovery occurred in Vdr-null mice while two studies of vitamin-D-deficient rats noted some recovery of mineral content after lactation, with the final value exceeding the prepregnancy value in one study [42,43,146]. These findings suggest that 1,25(OH)2D and its receptor are not needed for skeletal recovery post-weaning. The role of FGF23 in skeletal recovery has not been studied. The fetal section above described how all models of disrupted vitamin D physiology have shown no evidence of rickets or hypocalcemia in utero or at term. But the adults will develop florid rickets, hypocalcemia, hypophosphatemia, and marked secondary hyperparathyroidism. None of these abnormalities are present until after weaning. This is true for vitamin-Ddeficient rats [53,54], 1a-hydroxylase-null pigs [50], 1ahydroxylase-null mice [51,52], and Vdr-null mice [58,59]. The most careful studies have been done in vitamin-D-deficient rats and Vdr-null mice, in which serum calcium and skeletal mineral content remain normal for the first 2e3 weeks after birth. Shortly after weaning the animals develop progressive hypocalcemia, hypophosphatemia, and histomorphometric evidence of rickets. This parallels the maturation of calcium absorption in the intestine, which changes from a nonsaturable, passive process in the newborn to an active, saturable, 1,25(OH)2D-dependent process [148e150]. These observations illustrate the importance of 1,25(OH)2D to regulate intestinal calcium absorption and facilitate skeletal mineralization in the adolescent and adult, but not in the neonate. Additional studies in both Vdr-null and 1a-hydroxylase-null mice have shown that if a high-calcium, high-phosphorus, lactose-enriched diet is initiated prior to weaning, then rickets is completely prevented [58,151e155]. The total alopecia remains in Vdr-null mice. The prevention of rickets by increasing the calcium content of the diet suggests that the main role of 1,25(OH)2D in calcium homeostasis is to stimulate active intestinal calcium absorption and that this role can be completely bypassed by manipulating the content of the diet. Overall, the animal data indicate that calcium is supplied to milk through up-regulated resorption of the maternal skeleton. Neither resorption of the
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maternal skeleton nor intestinal absorption of calcium by the mother appears to require vitamin D or 1,25 (OH)2D. In animals with disrupted vitamin D physiology that leads to rickets and hypocalcemia as adults, the newborn pups maintain normal serum calcium and phosphorus, calciotropic hormones, and skeletal mineral content until near the time of weaning. At that stage intestinal calcium absorption has become dependent upon 1,25(OH)2D. After weaning rickets will develop unless the impaired intestinal calcium absorption is bypassed by substantially increasing the calcium content of the diet.
HUMAN DATA RELEVANT TO VITAMIN D AND THE NEONATE AND INFANT The clinical course of VDDR-I and II in women during lactation has not been reported in the literature. Consequently there are no human data on what effect lack of 1,25(OH)2D or the vitamin D receptor might have on breast milk calcium content or maternal calcium and bone homeostasis during lactation. No clinical study has compared vitamin-D-deficient to vitamin-Dsufficient women during lactation, but numerous clinical trials and observational studies summarized below have examined the effect of vitamin D supplementation in women across a wide range of baseline 25(OH)D levels. As will be shown, even high-dose vitamin D supplementation has no effect on the calcium or phosphorus content of milk. Available clinical data from pediatric and older-age VDDR type II patients is consistent with the animal data, indicating that the deficiency in intestinal calcium absorption can be bypassed. Repeated calcium infusions or high oral dose calcium (3.5e9 g/m2 body surface area) will correct the biochemical abnormalities and prevent or heal underlying rickets or osteomalacia in VDDR type II patients [88,156,157]. In one such case series growth velocity increased, bone pain reduced, and several pediatric patients were able to walk for the first time [157]. Serum calcium, parathyroid hormone, phosphorus, and alkaline phosphatase values returned to normal within a year. Radiologic signs of healing occurred more rapidly in younger patients and when intravenous calcium was administered. Similar to Vdrnull mice, the total alopecia in VDDR type II subjects is not corrected by normalizing mineral homeostasis, indicating that the roles of 1,25(OH)2D and the VDR in hair follicle growth and development are independent of systemic calcium homeostasis. For VDDR type I the high-calcium approach has not been used because physiological doses of 1,25(OH)2D (0.25e1.0 mg daily) or 1a-OHD (0.5e3.0 mg daily) will normalize intestinal calcium absorption [87].
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In one attempt at a systematic examination of bone mineral content and vitamin D status in newborns, Congdon studied infants born to 45 Asian women, 19 additional Asian women who had received 1000 IU of vitamin D during the last trimester, and 12 Caucasian women who did not take supplements. Mean cord blood concentrations of 25(OH)D were 5.9, 15.2, and 33.4 nmol/L, respectively; there were no significant differences in serum calcium, phosphorus, and alkaline phosphatase. Bone mineral content of the forearm was assessed by SPA within 5 days of birth and did not differ among groups. Craniotabes was present in three babies from the unsupplemented Asians, four babies from Asians who received vitamin D, and in none of the Caucasians. The authors concluded that vitamin D deficiency did not impair skeletal mineralization at this early postpartum time point and that craniotabes is a nonspecific finding that should not be used to indicate the presence of rickets [158]. In a more recent systematic study, Weiler obtained cord blood 25(OH)D levels and used DXA to measure bone mineral content of the lumbar spine, femur, and whole body within 15 days after birth in 50 healthy term infants [159]. DXA has sufficient accuracy and precision to reliably measure BMD in anesthetized mice but movement artifacts limit its usefulness in human babies; the authors did not indicate whether the babies were sedated or anesthetized for the procedure. Caucasian infants were separated into sufficient and insufficient groups based on 25(OH)D levels in order to compare to Asian and First Nations babies who had insufficient levels of 25(OH)D. Mean cord blood 25(OH)D was 73 and 36 nmol/L in the sufficient and insufficient Caucasians, as compared to 44 nmol/L in Asians and 27 nmol/L in First Nations. There was no difference in bone mineral content of vitamin-D-sufficient versus -insufficient Caucasians. Lumbar spine bone mineral content of Asians was lower than Caucasian while that of First Nations babies was intermediate between the two. Whole body and femur bone mineral content were no different among the groups. The authors concluded that ethnic differences explained the variation in spine bone mineral content and that vitamin D sufficiency or insufficiency did not affect mineralization of the newborn skeleton [159]. In the days after birth vitamin D deficiency increases the likelihood of neonatal hypocalcemia. This coincides with maturation of intestinal calcium absorption from a passive, nonsaturable process facilitated by lactose to one that is saturable and dependent upon 1,25(OH)2D. Preterm infants demonstrate passive, nonvitamin-Ddependent absorption of calcium [93,94]. The lactose content of breast milk has also been shown to increase the efficiency of passive intestinal calcium absorption in human infants [160,161], as it does in rodents [138e140]. As normal term and preterm infants mature,
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1,25(OH)2D-mediated intestinal calcium absorption increases while passive absorption declines, similar to the animal studies described above [93,95,96]. This maturation of intestinal calcium absorption to a 1,25 (OH)2D-dependent process parallels and explains the clinical observation that vitamin-D-deficient rickets does not usually develop (or become recognized) until several weeks to typically 6e18 months after birth, even in regions where severe vitamin D deficiency during pregnancy is endemic [80e82]. Time will pass before reduced intestinal calcium absorption leads to impaired skeletal mineralization. Two recent case series found no radiological evidence of rickets or skeletal demineralization (by DXA) among infants presenting with hypocalcemia and low 25(OH)D levels [162,163]. This suggests that, as in the animal studies, hypocalcemia will precede the development of rickets. Vitamin D and 25(OH)D have low penetrance into breast milk, together comprising 40e50 IU/L of antirachitic activity, of which most of this is 25(OH)D [164e167]. A recent USDA survey found the vitamin D content of human milk to be 4.3 IU per 100 kcal, as compared to the 40e100 IU per kcal mandated for infant formulas [168]. This means that in exclusively breastfed infants who do not receive supplemental vitamin D or sufficient sunlight exposure, the serum 25(OH)D level will drop over several months in accordance with its half-life. The lower the 25(OH)D value at birth, the sooner the infant’s 25(OH)D will reach levels that predispose to hypocalcemia and rickets. This is why breastfed infants born of women with low vitamin D stores are at the highest risk of developing rickets during the first 18 months of life [169]. The higher the 25(OH)D level at birth, the more likely the infant will have adequate vitamin D stores until the time that solid (vitamin-D-supplemented) foods are established around 6 months of age. In infants receiving vitamin-D-fortified formula the 25(OH)D levels should not decline. In sufficiently sun-exposed infants the 25(OH)D level should not decline but sunlight exposure is normally avoided over concerns about actinic damage and risks of skin cancer. Specker studied the effects of sunlight exposure in exclusively breastfed infants and determined that 30 minutes per week wearing only a diaper, or 2 hours per week fully clothed but without a hat, would provide adequate 25(OH)D levels [170]. There are no data that have established what the optimal 25(OH)D level should be in the neonate or infant, although a systematic review of the available literature noted that a 25(OH)D level above 30 nmol/L is generally protective against the development of rickets [171]. Rickets has also developed at higher levels of 25(OH)D but especially in infants with extremely low calcium intakes or consumption of diets that are high in calcium-binding phytate. In such infants the rickets is
essentially due to pure calcium deficiency: since rickets is ultimately a disorder of impaired mineralization of osteoid which can be corrected by calcium infusion or high calcium intakes, no amount of vitamin D will prevent or treat rickets if there is little or no calcium in the diet [172]. Several observational studies have found that in such children calcium is very effective in treating rickets while vitamin D is ineffective [173,174]. This is supported by a study of nutritional rickets due to very low calcium intakes in which vitamin D supplementation had no effect on the fractional intestinal calcium absorption, despite raising the mean 25(OH)D level from a baseline of 50 nmol/L to 75 nmol/L [175]. In the days to weeks after birth in vitamin-D-deficient neonates, the serum calcium and phosphorus can be expected to decline while the parathyroid hormone level should rise. A low serum calcium may be detectable within a few days of birth, as noted in the randomized trial by Brooke [84], and in an observational study by Zeghoud and colleagues [176]. Zeghoud took blood samples from 80 neonates at 3e6 days after delivery and stratified them by 25(OH)D levels. The serum calcium was higher in neonates with 25(OH)D levels greater than 30 nmol/L as opposed to 16e30 nmol/L or less than 16 nmol/L (2.51 0.11 vs. 2.44 0.22 and 2.42 0.21 mmol/L, respectively) [176]. In the subsequent weeks to months after birth, vitamin D deficiency may present in diverse ways, including hypocalcemic seizures, growth failure, lethargy, irritability, and a predisposition to respiratory infections during infancy [177]. This presentation may be confounded by globally inadequate nutrition in the mother and infant which contributed to the vitamin D deficiency. A UK retrospective chart review identified 65 cases of vitamin D deficiency in children ranging from less than 1 year to 13 years of age and discovered two patterns of presentation. The first was hypocalcemia during periods of rapid growth (less than 3 years and more than 10 years of age) in which 17/29 subjects had no radiological evidence of rickets. Hypocalcemia presented as seizures, neuromuscular irritability, apnea, or stridor. The second group had no hypocalcemia but all had chronic skeletal effects of rickets and demineralization, including bow legs, pain, swollen joints, and other bone abnormalities. In both presentations all children had 25(OH)D levels below 15 nmol/L [178]. The prevalence of vitamin D deficiency depends upon the definition that is used as well as latitude, fortification practices, skin pigmentation, diet, and other factors. In a recent study among largely African-American and Latino infants and toddlers in Boston (latitude 42 N), the authors used the 25(OH)D level to estimate prevalence of vitamin D deficiency; 1.9% of infants and toddlers had a 25(OH)D level <20 nmol/L, 12.1% had a level <50 nmol/L, and 40.0% had a level
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<75 nmol/L [179]. The prevalence was quite similar among a predominantly Caucasian population of infants and toddlers much further north in the province of Newfoundland and Labrador, Canada (latitude range 46 to 61 N) during 2008; 3.6% had a 25(OH)D level <25 nmol/L, 12.7% had <50 nmol/L, and 54.5% had <75 nmol/L (unpublished data). The Boston survey identified that the main risk factor among infants for low 25(OH)D levels was being breastfed with no vitamin D supplement administered, whereas among toddlers the high-risk factors were low milk consumption and a higher body mass index [179]. The incidence of rickets was 2.9 per 100 000 in a recent 2-year survey of Canadian pediatricians, and the mean age at diagnosis was 1.4 years (range 2 weeks to 6.3 years) [169]; 94% of children with rickets had been breastfed. Additional risk factors included dark skin, living in the far north, born of mother who took no vitamin supplements, limited sun exposure, emigrated from a region where vitamin D deficiency is endemic, and delayed initiation of solid foods [169].
Intervention Studies With respect to maternal outcomes during the neonatal period, a number of observational studies [180e185] and clinical trials [186e193] have examined the effects of vitamin D supplementation in lactating women. These studies have generally shown that providing vitamin D to lactating mothers increased their 25(OH)D levels but had no significant effect on any other maternal or neonatal outcome. However, many studies measured no outcome other than the achieved 25(OH)D level in mothers and neonates and were not powered to look at outcomes such as hypocalcemia or clinical rickets [186,187,190e192]. The effect of maternal dietary calcium intake on the skeletal resorption that occurs during lactation has been carefully examined through randomized trials and in observational studies. The consistent finding is that calcium intakes ranging from very low to supplemented well above normal had no effect on the degree of skeletal demineralization that occurred during lactation although it did increase urinary calcium excretion [194e200]. In a randomized clinical intervention that studied the effect of consuming dietary calcium in excess of the recommended daily allowance (2.4 g daily), lactating women still lost 6.3% of bone mineral density at the lumbar spine and up to 8% from the radius and ulna, as determined by DXA [194]. In another RCT that included 97 lactating women randomized to 1 g calcium supplement versus placebo, there was no effect of calcium on the magnitude of BMD decline during lactation [195]. The calcium content of mother’s milk is an important lactation outcome relevant to the neonate and infant. A
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survey of all available studies indicated that at 30 days’ postpartum, the average calcium content of milk is 259 59 mg/L; assuming 0.78 L ingested per day, the average intake per baby is 202 46 mg per day [201]. Since both the 1a-hydroxylase and the VDR are expressed by human mammary tissue [202,203], a role for calcitriol in regulating the calcium content of milk seems plausible. However, breast milk calcium content is unaffected by maternal vitamin D status or any amount of vitamin D supplementation [191,192,204]. This includes a study by Hollis in which lactating women received 6400 IU daily and had a mean 25(OH)D level of 168 nmol/L [192]. Similarly maternal calcium intake does not alter the breast milk calcium content as demonstrated in several clinical trials. In one study of 97 women randomized to take 1 g calcium supplement versus placebo, breast milk calcium was unaffected [195]. In a study of 125 Gambian women with habitually low calcium intakes treated with 1500 mg supplemental calcium versus placebo, there was no effect on breast milk calcium content and infant weight, growth, or BMD during the first year of life [205]. Another study confirmed that the breast milk output predicts the maternal BMD decline during lactation, whereas calcium intake, breast milk calcium concentration, and vitamin D receptor genotype have no effect [197]. While one might expect a priori that low maternal vitamin D and calcium intake would accentuate skeletal losses in order to maintain breast milk calcium content, the majority of studies cited above suggest that this is not the case. These results are consistent with the notion that the calcium content of the milk largely supplied by resorption of the maternal skeleton, while the final calcium concentration of secreted milk is determined by local regulation within mammary epithelial cells (a process that does not require calcitriol or its receptor). The skeletal resorption is programmed by the obligatory rise in PTHrP and fall in estradiol, and not influenced by vitamin D status. Increasing calcium and vitamin D intake during lactation may simply increase the urinary calcium excretion and, thereby, the risk of kidney stones. Lactation is followed by an interval of several months during which the skeletal mineral content returns to the prepregnancy value. No clinical trial has examined the effect of vitamin D deficiency or insufficiency on the ability of the skeleton to recover from lactational losses. Assuming that it is desirable for the infant to have a 25(OH)D level over 75 nmol/L, several clinical trials have demonstrated that 300 or 400 IU of vitamin D given directly to the baby is more than sufficient to achieve this level of 25(OH)D, and that typical maternal doses of vitamin D supplements (400 or 1000 IU per day) have little or no effect on infant 25(OH)D levels
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[186e188,190,206,207]. In several pilot studies Hollis and colleagues examined the possibility that mothers could be given enough vitamin D so that their babies would achieve 25(OH)D levels greater than 75 nmol/L from breast milk alone [190e192]. A maternal dose of 4000 IU per day brought the mean infant 25(OH)D level to 75 nmol/L, whereas a maternal dose of 6400 IU per day brought all infants to greater than 75 nmol/L. Infants treated with 300 IU directly also achieved the same 25(OH)D level of greater than 75 nmol/L [190e192]. No adverse effects of 6400 IU per day were observed in the mothers or infants during these trials but the numbers of subjects involved were small. Treating the infant directly with 300 or 400 IU per day remains the preferred method but a maternal dose of 6400 IU per day is an alternative if it is desired to have all infant nutrition come from breast milk. Apart from the studies demonstrating the amount of vitamin D intake required to achieve a certain infant 25 (OH)D level, no other functional outcomes of significance have been found when comparing breastfed to formula-fed babies. One study randomized 51 breastfed infants to receive 400 IU vitamin D or no supplement, and an additional 40 formula-fed infants were followed as controls. The 25(OH)D level was 50 nmol/L at 2 weeks of age in the breastfed babies who did not receive a supplement, and the value did not change significantly over the following 6 months (mean 42.5 nmol/L at 2 months to 47.5 nmol/L at 6 months). The 25(OH)D level was higher in the formula-fed infants (peak 67.5 nmol/L) and intermediate in the breastfed infants who received a supplement (peak 57.5 nmol/ L). The differences among the three groups were statistically significant solely at the 2-month time point [208]. There were also no differences among groups at any time point with respect to weight, length, and skeletal mineral content (measured by SPA of the radius). Although small, this study of 91 infants suggested that there was no benefit achieved by raising the infant 25 (OH)D level above 50 nmol/L. Beyond preventing rickets, only a transient benefit on bone mass has been seen through the use of vitamin D supplements in infancy. In a study of 18 healthy term infants randomized to receive 400 IU or placebo at 3 weeks of age, 25(OH)D levels increased to 95 nmol/L versus 50 nmol/L and the BMC of radius and ulna (measured by SPA) was 23% higher than in the placebo group by 12 weeks of age (79.3 3.0 vs. 64 3.0 mg/cm, P < 0.001) [206,209]. The treatment continued but the BMC was identical between the two groups at the 6- and 12-month time points [206]. In a separate study of 46 healthy breastfed infants randomized to receive 400 IU vitamin D or placebo, the mean 25(OH)D level increased to 92.4 versus 47 nmol/L in the placebo group, but there were no differences between groups in BMC
(by SPA of radius) at 3 or 6 months of age [207]. Overall these studies indicate no benefit of a 25(OH)D level over 50 nmol/L. In the Brooke study cited earlier, in which 59 women received probably 10 000 IU of vitamin D3 daily and were compared to 67 unsupplemented, vitamin-D-deficient controls, there was no difference in cord blood calcium. However, at 3 and 6 days postnatal the plasma calcium levels of infants from the treated mothers were modestly but statistically significantly higher than in infants from control mothers (2.30 0.04 compared to 2.18 0.04 mmol/L, P < 0.05) [84]. The control infants had a 25(OH)D level of only 10.2 2.0 nmol/L as compared to a level of 137.9 10.8 nmol/L in the infants born of vitamin-D-supplemented mothers. Five of 67 infants in the placebo group had symptomatic hypocalcemia (manifest by irritability and serum calcium <1.8 mmol/L) whereas none of the 59 infants in the treatment group had symptomatic hypocalcemia. No infants showed radiologic signs of rickets. At this low level of 25(OH)D in the control infants, 1,25(OH)2Ddependent intestinal calcium absorption was likely submaximal. Data from older children indicate that intestinal calcium absorption is maximal with a 25 (OH)D level in the range of 30e50 nmol/L [210,211].
Associational Studies There have been numerous epidemiological studies that examined the effect of a history of lactation or recalled months of lactation on the risk of developing low bone mass, fractures, or osteoporosis later in life (reviewed in [1,212]). The vast majority of these have shown no effect, or a protective effect, of lactation on subsequent risk of these adverse outcomes. None of these studies were sufficiently powered to test whether vitamin D sufficiency or deficiency impacts on the effect that a history of lactation has on BMD or fracture risk. Neonatal hypocalcemia has been associated with low 25(OH)D levels in the days to weeks after birth. In one recent case series all hypocalcemic infants had 25(OH) D levels below 25 nmol/L (range 10e25 nmol/L; mean 20.3 9.8 nmol/L) [163]. In another series, all cases of neonatal hypocalcemia attributed to low vitamin D status had 25(OH)D values below 30 nmol/L (mean 20.5 9.0); the remaining cases of hypocalcemia had 25(OH)D levels greater than 50 nmol/L and were attributed to other causes (e.g., hypomagnesemia) [162]. In both case series, none of the infants who had both hypocalcemia and low 25(OH)D levels showed radiological evidence of rickets. In a third series of 42 cases of vitamin D deficiency presenting in the first 3 months of life, most presented with hypocalcemic seizures and all had 25(OH)D levels less than 27 nmol/L. No clinical signs of rickets were detected but subtle changes
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consistent with early rickets were noted on radiographs [213]. These observational studies suggest that hypocalcemia will develop before there are any significant skeletal changes. The possible effect of lower 25(OH)D levels during fetal development on childhood bone mass has been examined in an associational study by Javaid cited earlier in the fetal section [113]. Although no significant associations were observed at birth or 9 months of age, at 9 years of age there was a significant reduction in BMC in the children born of mothers who had a 25(O) D level below 27.5 nmol/L during pregnancy as compared to children born of mothers who had a 25 (OH)D level of 50 nmol/L or higher during pregnancy. This possibly confounded association needs to be tested in a clinical trial. 25(OH)D may have been a marker for poor nutrition and socioeconomic status during pregnancy and childhood. One group of investigators has done several followup analyses of a cohort of 696 Tasmanian women (out of an eligible 735 consecutive admissions) whose newborn children were considered to be at high risk of sudden infant death syndrome in 1988. Within a few days of childbirth, and again at 1 and 3 months postpartum, the women completed questionnaires that addressed such factors as nutritional intake during pregnancy and breastfeeding intake or practice. In the first follow-up analysis, the investigators measured BMD by DXA in 47% of the children at eight years of age. Compared to bottle-fed babies, breastfed babies weighed less at birth and had a higher proportion of preterm births. When term babies only were analyzed, breastfeeding 3 months or longer was associated with a slightly higher BMD (0.2e0.29 SD) at all sites by eight years of age as compared to bottle-fed babies. Breastfeeding for less than 3 months did not show this association and preterm babies showed no difference in BMD by bottle or breastfed status [214]. In a second report correlations between maternal nutritional intake during the third trimester and BMD at eight years of age were made in 173 infants (24% of the original cohort). Several statistically significant associations were found between maternal intake of magnesium, phosphorus, and potassium and BMD of spine, hip, or total body. However, maternal calcium intake during pregnancy did not correlate with childhood BMD, and vitamin D intake or status was not assessed [215]. In the most recent report, 216 adolescents (31% of the original cohort) had BMD done at age 16, and maternal intake of milk, fat and magnesium (but not calcium) during the third trimester of pregnancy were predictive of BMD at age 16 [216]. Overall these studies provide some support to the notion that in utero nutrient exposure may program childhood BMD or peak bone mass. However, the low follow-up rate (70e76% lost to follow-up or inadequate
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data) and abnormal status of the children at birth (at high risk for sudden infant death syndrome) make this data questionable. In another prospective cohort study, 70 preterm infants were randomized into four groups for a three month study: to receive a vitamin D dose of 500 or 1000 IU/day, and either calcium- and phosphorussupplemented or unsupplemented breast milk. These doses of vitamin D resulted in 25(OH)D levels that ranged from 100e400 nmol/L regardless of the dose. At 3 months of age, infants with a calcium-and-phosphorus supplemented diet had a 36% higher BMD of the left forearm shaft as measured by SPA. At 9e11 years of age only 50% of the cohort could be assessed. There was no effect of the prior history of calcium-andphosphorus supplementation on BMC (measured by DXA) of the lumbar spine, left forearm, or distal radius. At neither 3 months nor 9e11 years of age did the history of vitamin D supplementation affect the BMC result [217]. The sample size per group was quite small, ranging from 12 to 23; at the later follow-up the sample size was 8e11 per group. The study suggests no effect of vitamin D supplementation on childhood BMD. As mentioned briefly in the fetal section, epidemiological studies suggest that vitamin D supplementation of the infant is associated with a lower childhood incidence of type 1 diabetes [125e127]. However, the evidence is not definitive. A study in Finland asked mothers to recall if they had given their infants a 1000 IU vitamin D supplement during the first year of life (a national campaign at the time promoted the use of a 1000 IU vitamin D supplement for all babies). Over 30 years later, the cohort was assessed again to determine the prevalence of type 1 diabetes. A statistically significant increase was found in the incidence of type 1 diabetes in offspring who had never received vitamin D supplementation, but this analysis relied on only two cases of type 1 diabetes among infants whose mothers recalled not administering the vitamin D supplement. In a subanalysis that examined children who had reportedly received vitamin D during their first year, there was a statistically significant reduction in diabetes among the cohort who received the recommended dose of vitamin D versus those who had received a lower dose, but this also relied on just two cases of diabetes in children who had reportedly received a lower dose of vitamin D. This study is subject to recall bias especially at the one year time point when compliance with the national policy was considered evidence of being “a good mother.” A chance result cannot be excluded since in both the main analysis and the subanalysis, one instead of two cases of type 1 diabetes among those who did not receive vitamin D, or among those who received less than 1000 IU of vitamin D, would have eliminated any statistical significance [125]. In Norway
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a caseecontrol study compared 545 cases of childhoodonset type 1 diabetes with 1668 control subjects. Use of cod liver oil in the first year of life was associated with a significantly lower risk of type 1 diabetes (adjusted odds ratio: 0.74; 95% CI: 0.56e0.99). However, use of other vitamin D supplements during the first year of life had no protective effect [126]. Oddly, maternal use of cod liver oil or other vitamin D supplements during pregnancy were not associated with type 1 diabetes, in contrast to an earlier study by the same authors which reported that cod liver oil use during pregnancy reduced the incidence of type 1 diabetes [123,126]. Finally, a metaanalysis of observational studies concluded that vitamin D supplementation given to the infant reduces the risk of type 1 diabetes by 29% [127]. There are no prospective, randomized, controlled trials of the effect of vitamin D supplementation on the incidence of type 1 diabetes. The existing evidence is suggestive but not definitive; these findings need to be confirmed by randomized, controlled trials to rule out substantial known confounding effects of socioeconomic status, lifestyle, nutrition, etc., in those children whose mothers chose to administer a vitamin D supplement versus those who did not.
CONCLUSIONS Animal data are quite convincing that vitamin D, 1,25 (OH)2D, and the VDR are not required during fetal development for normal regulation of calcium homeostasis, skeletal development, and skeletal mineral accretion. It is only during the post-natal period that disrupted vitamin D physiology will lead to impaired calcium and phosphorus absorption, hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and rickets or osteomalacia. These postnatal outcomes can be avoided by providing the animal with a high calcium diet, especially one supplemented with lactose to facilitate passive absorption of calcium. Human data are sparse but appear consistent with the animal data, specifically that babies with vitamin D deficiency or VDDR types I or II will be born with normal blood calcium and skeletal mineral content, and that vitamin D supplementation during pregnancy will not alter the cord blood calcium. It is in the weeks to months after birth that impaired intestinal absorption of calcium and phosphorus leads to hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and rickets or osteomalacia. The risk is substantially higher in babies born of vitamin D deficient and especially malnourished mothers, and highest in breastfed babies because the vitamin D and 25(OH)D content of breast milk is normally very low. Babies who receive vitamin D-fortified
formulas are at lower risk of developing vitamin D deficiency. Breastfed infants need to receive a vitamin D supplement directly; an alternative approach is to give the mother 6400 IU per day in order to ensure that the baby achieves a 25(OH)D of 75 nmol/L or greater. However, the long-term safety of this approach in women has not been examined nor has it been demonstrated that the infant or neonate requires a 25(OH)D level of 75 nmol/L or higher. The available data do not show any convincing additional benefits in neonates or infants when the 25(OH)D level is greater than 50 nmol/L. Children with VDDR type II have demonstrated that calcium infusions or a high oral intake of calcium can prevent and heal rickets or osteomalacia, bypassing the need for vitamin D. The available human data do not exclude the possibility that subtler skeletal effects of vitamin D deficiency (or even of VDDR type I and II) will be discovered by systematic comparison of affected to unaffected babies at birth, or that a sufficiently powered clinical trial might demonstrate increased bone mass or skeletal mineral content in the infant or child as a result of vitamin D supplementation during pregnancy, the neonatal period, or childhood. But at present these concepts remain unproven. Numerous nonskeletal benefits of vitamin D have been proposed and the associations may be strongest for a possible role during fetal development and early childhood in preventing type 1 diabetes. However, the evidence is conflicting and the associational studies are confounded. Proper randomized, controlled trials are needed to determine whether any of the postulated extra-skeletal benefits of vitamin D are real. The foregoing data were used to support the committee’s conclusion that maternal vitamin D requirements are not increased during pregnancy and lactation, Consequently, 600 IU of vitamin D should be consumed daily by non-pregnant, pregnant, and lactating adolescents and adults. Infants require 400 IU of vitamin D daily and special attention should be paid to breastfed infants who will normally receive little or no vitamin D through breast milk.
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36 Vitamin D Deficiency and Calcium Absorption during Childhood Steven A. Abrams USDA/ARS Children’s Nutrition Research Center, 1100 Bates St., Houston, TX 77030, USA
This work is a publication of the U.S. Department of Agriculture (USDA)/Agricultural Research Service (ARS) Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children’s Hospital, Houston, TX. This project has been funded in part with federal funds from the USDA/ARS under Cooperative Agreement number 58-6250-6-001. Contents of this publication do not necessarily reflect the views or policies of the USDA, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
INTRODUCTION Although the importance of providing adequate calcium and vitamin D during childhood and adolescent growth is well known, there remain important gaps in our understanding regarding the process of calcium absorption and utilization in childhood. Certain time periods in development appear to be critical ones in which calcium or vitamin D deficiency can pose very high risks. The first such time period is in utero, or as commonly reflected in current medical care, the initial months of life of prematurely delivered infants. Preterm infants, especially those <1.5 kg at birth, are at very high risk for the development of clinical rickets or other manifestations of bone loss. This bone mineral deficiency is primarily related to difficulties in providing, via parenteral or enteral sources, adequate calcium and phosphorus for the extremely rapid bone growth that normally occurs via placental transfer to the fetus [1]. In healthy full-term infants, human milk is recommended as the sole nutritional source for the first 6 months of life [2]. The calcium content of human
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10036-8
milk appears adequate to meet the needs of infants during this time period. However, unless mothers have been supplemented with very high doses of vitamin D, the vitamin D content of human milk is very low and the photoconversion of vitamin D precursors to vitamin D is necessary to obtain adequate vitamin D levels [3]. Since this may not be possible in many infants, it is recommended that all infants receive supplemental vitamin D, either via infant drops or, for infant-formula-fed infants, as provided in the formula [4]. Although vitamin-D-deficient rickets is a welldescribed problem for older infants and toddlers, extremely little information is available regarding vitamin D requirements and the relationship between vitamin D status and calcium absorption in this age group. The optimal vitamin D intake or concentration to maximize calcium absorption, as well as the lowest intake of calcium and vitamin D needed to prevent severe bone loss or rickets, is not well described. Longterm consequences to variations in calcium absorption and bone mineralization in early life are not known. Despite the evidence that most infant formulas provide at least as much, if not more, absorbable calcium than human milk, some data suggest that, in later childhood, the bone mass of infants who are breastfed may be the same or greater than that of formula-fed infants [5,6]. An important issue then is to determine at what age it becomes critical to maximize mineral intake and absorption to lead to an optimal peak bone mass. Some controlled trials have indicated that supplementation of calcium before puberty may be important, whereas others have found benefit in pubertal children [7,8]. Interpreting such studies is difficult, however, as prestudy calcium intakes are often poorly assessed or controlled and
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supplemented intakes may exceed the absorptive threshold leading to little benefit. Because it is the time of most rapid mineralization of the skeleton, efforts to improve peak bone mass during childhood have focused on increasing the amount of absorbable calcium in the diet of adolescents. Efforts in this regard include advocacy campaigns to increase dairy product intake and considerable efforts to provide calcium-fortified foods and beverages to adolescents. Recent research efforts have looked at the effects of race, diet, gender, and other food components on maximizing calcium absorption [7,8]. As part of global efforts to develop nutritional planning, consideration has been given to the role of calcium and vitamin D supplements in food products designed for children in developing countries [9]. Although many of these are countries in which children are exposed to adequate sunshine, they may also have low calcium and vitamin D status due to social or dietary causes including the avoidance of dairy due to lactose intolerance or traditions of limited dairy intake. Extending research in calcium absorption to these populations is important not only to prevent bone loss, but also to understand and prevent an increase in osteoporosis in developing countries in the future.
PREMATURE INFANTS Human milk is recognized as the ideal food for virtually all infants [2]. The substantial health and developmental benefits of human milk feeding have been well documented even in the smallest of premature infants. However, for many decades, it has been known that one critical limitation is that the minerals, especially the calcium and phosphorus in human milk, do not meet the needs of rapidly growing premature infants. This and other factors place premature infants at high risk for nutritional rickets (Table 36.1). This can readily be seen in that the fetus accretes 100e120 mg/kg/d of calcium (about 50e60 mg/kg/d of phosphorus) during TABLE 36.1
High-Risk Criteria for Osteopenia in Premature Infants
Born at less than 27 weeks’ gestation Birth weight of less than 1000 g Long-term parenteral nutrition Severe bronchopulmonary dysplasia with use of diuretics and fluid restriction Long-term steroid use History of necrotizing enterocolitis High serum alkaline phosphatase activity (>900 IU/L) and low serum phosphorus (<5.2 mg/dl)
the third trimester. Since human milk contains about 25 mg/dl of calcium (13 mg/dl of phosphorus) and is usually fed at 150e200 mg/kg/d, even if all of the calcium and phosphorus were to be completely absorbed and retained, it cannot provide more than half of the minerals needed by extremely premature infants to meet the in utero accretion rate [1,10e12]. In reality, calcium absorption in unfortified human-milkfed infants is generally about 60%, leading to a net absorption of about 20e30 mg/kg/d or less than onethird of the in utero rate [13e15]. The addition of various forms of mineral salts and/or mineral fortifiers to human milk and the use of specialized preterm infant formulas with very high calcium content levels have been shown to enhance the amount of calcium and other minerals retained from the diet, to increase bone mineral content of the infants and to decrease the incidence of osteopenia and frank rickets in preterm infants [1,16,17]. Nonetheless, clinical rickets remains a problem in preterm infants, especially those who are less than 600e800 g body weight at birth [18]. The bioavailability of the calcium in these fortifiers may be a key aspect of their adequacy. Using a commercially available human milk fortifier, Schanler and Abrams [1] reported that net calcium retention was 104 36 mg/ kg/d in premature infants, a value approximating the in utero accretion rate during the third trimester. These retention values are well above those achieved using earlier human milk fortifiers [16] and those more recently reported using a human milk fortifier not used in the United States [19]. That fortifier led to minimally positive calcium retention and therefore little if any benefit to the baby. It is not clear why the bioavailability of the calcium in the fortified feeding was low, but it is likely related either to the form of the calcium or problems with the solubility of the calcium in the fortified milk. Of interest is that the calcium absorption percentages from both fortified human milk and from specialized preterm formula average 50e65% in many studies [14,15]. This percentage absorption is similar to that seen for unfortified human milk in both premature infants and full-term infants. However, in the case of preterm formulas or fortified milk, the calcium intake is about 220 mg/kg/d. This intake vastly exceeds the body-weight-adjusted calcium intake from human milk by full-term infants (10 mg/kg/d for a 10-kg infant) [5,12]. This constancy of absorptive fraction in premature infants suggests that some or most of the calcium absorption by premature infants and newborn full-term infants is not vitamin-D-dependent. In a review of over 100 balance studies, Bronner [14] showed that the calcium absorption fraction varied little with calcium intake in premature infants and thereby suggests that calcium absorption is partly or mostly vitamin-D-independent in these infants. Currently, no
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FULL-TERM INFANTS e CALCIUM
studies allow for a full understanding of when vitaminD-dependent absorption becomes predominant in preterm or full-term infants, or the level of vitamin D needed in infants for maximal calcium absorption. Multiple older studies have demonstrated that vitamin D intakes in premature infants of 400 IU/day (or 200 IU/kg up to 400 IU/d) leads to what are believed to be adequate vitamin D levels [20e22]. One study demonstrated adequate 25-hydroxyvitamin D concentrations and clinical outcomes with oral vitamin D intakes as low as 160 IU/d [23]. In addition, studies have generally failed to show any clinical benefit to increasing vitamin D intake above 400 IU/day in preterm infants. One study comparing 500 IU to 1000 IU found no short- or long-term benefit (up to 11 years) of higher amounts [24]. “There are no data to support the belief that preterm infants need a disproportionately high vitamin D dose in relation to their weight” [24]. Despite these findings, some advocate for modestly higher vitamin D intakes in preterm infants, generally up to 800 IU/d [25]. This may have particular benefits in older preterm infants and those who have been discharged from the hospital. The use of lower mineral-containing fortifiers and formulas, such as is done in Europe, may also be cause for considering higher doses of vitamin D in preterm infants. The effects of other formula components on mineral absorption have also been considered. A study using a triple lumen perfusion technique demonstrated that calcium absorption was greater using a solution that included a glucose polymer than one with lactose [26]. As glucose polymers are widely used in preterm formulas this effect may be clinically important. Altering the fat blend of infant formula to more closely resemble that of human milk may also enhance mineral absorption in premature infants [27e29].
FULL-TERM INFANTS e CALCIUM Infancy is a time of rapid body growth as infants may triple their birth weight in the first year of life. This rapid body growth is accompanied by comparably rapid bone growth [30]. Remarkably, however, the human-milk-fed baby readily mineralizes all of the calcium coming from mother’s milk in the first 6 months and most of it from human milk during the second 6 months of life [2]. This is possible due to the withdrawal of bone from the maternal skeleton to meet the infants’ needs and the high rates of calcium absorption from human milk. Dietary recommendations for calcium intake in infancy are based on the knowledge that calcium-deficiency rickets does not occur in healthy, vitamin-D-sufficient, breast-fed infants. Therefore, the calcium intake of the exclusively breast-fed infant, averaging 200 mg/day,
was set in 2011 by the National Academy of Sciences as the Adequate Intake (AI) for calcium in the first 6 months of life (Table 36.2) as part of the Dietary Reference Intake (DRI) process [31]. An AI was chosen as it was not possible to establish separately an Estimated Average Requirement (EAR) and a Recommended Dietary Allowance (RDA) based on a functional outcome, since it was accepted that the intake from breast milk was adequate for any functional outcome in infants. In the second 6 months of life, most calcium continues to come from human milk, but there is some from solid foods. The Adequate Intake for 7e12-month-old infants was therefore set at 260 mg/day, which is the sum of the usual intake of calcium from breast milk and solid foods. Infant formulas have a calcium concentration well above that usually found in human milk [31,32]. One justification for providing more calcium in infant formulas than human milk is the belief that calcium is more poorly absorbed from infant formula than from human milk. This perspective is primarily based on studies in which calcium concentrations were much greater in formula than in human milk [6]. These high concentrations may lead to lower fractional calcium absorption in the infants. This inverse relationship was demonstrated by Devizia [33] who reported decreasing fractional absorption in a very small group of infants as formula concentration of calcium increased. Several recent studies have suggested that in many cases, the fractional absorption of calcium from infant formulas [34e38] is similar to the value for human milk (Table 36.3) Studies of whole-body bone mineral content using DXA support these findings. Calculations from both Fomon and coworkers [6] and from earlier data using metacarpal morphometry [39] suggest a mean calcium accretion rate of approximately 80e100 mg/day during the first year of life [30]. Although there are few direct comparisons with formula-fed infants, recent studies suggest values of about 150 mg/day in the first 6 months of life [29,40] for formula-fed infants. This rate appears not to change substantially in the second 6 months of life after more solid foods are introduced [40,41]. More data are needed though, especially in the second 6 months of life to evaluate the interaction of calcium intake and vitamin D TABLE 36.2
Calcium and Vitamin D Recommendations for Infants and Children in the United States [31]
Calcium (mg/d)
Vitamin D
Infants 0e6 months
200
Infants 7e12 months
260
Children 1e3 years
700
Children 4e8 years
1000
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36. VITAMIN D DEFICIENCY AND CALCIUM ABSORPTION DURING CHILDHOOD
Calcium Absorption Fraction in Human-Milk- and Formula-Fed Infants Based on a Reference Intake of 780 ml/day
Ca Concentration Absorption Net Ca Absorption (mg/dl)
(%)
(mg/d)
Human milk [35]
25
61 23
97
Standard formula [36]
50
58 13
205
Partially hydrolyzed [34]
46
66 12
220
Human milk [6]
25
58 17
113
Cow-milk-based formula [38]
57
57 15
255
status in mineralizing bone. With these high absorption fractions, it appears that it is possible to markedly increase total calcium absorption and net calcium retention in infants above that of primarily human-milk-fed infants. However, caution should be raised about the benefits of targeting high levels of calcium absorption in infancy, either with high-mineral-containing formulas, or through the use of highly calcium-fortified solid foods for older infants. There are no data to support any long-term benefit in increasing peak bone mass or preventing osteoporosis to exceeding calcium absorption or bone mass in infancy. A study by Jones and coworkers [5] found a greater bone mineral density at the spine and whole body in 8-yearold children who had been breast-fed compared to those bottle fed as infants. This effect was only present for infants who had been breast-fed for at least 3 months. Studies in preterm infants have also failed to show any long-term benefit in adolescence to greater mineral intake during infancy [42]. This view is supported by animal data [43] in rabbits that do not show any benefit to increasing bone mineral content in early life and is consistent with similar classic data from Gershoff [44].
VITAMIN D IN FULL-TERM INFANTS Nutritional rickets in children is described in detail in Chapter 60. In this section, we will consider some specific issues related to the use of vitamin D supplements for infants, especially those in the United States. Human milk is a relatively small source of dietary vitamin D for most infants, usually providing an average of 10e20 IU/day [43,44]. Increased vitamin D in human milk is related to increasing maternal vitamin D status, but the level of supplementation required may be relatively high [3,4,45,46]. Infant formulas and cow’s milk are fortified by statute in the United States, although cow’s milk is not fortified with vitamin D in many other countries.
Therefore, those at greatest risk of vitamin D deficiency in the United States are infants who are breastfed without adequate sunshine exposure, or those who are weaned to diets containing little vitamin D. Infants who have dark skin pigmentation are at an additional risk, although rickets may occur in any infant. In addition to an increased frequency of breastfeeding of older infants and toddlers, social conditions (e.g., the requirement for occlusive dress) and the widespread vigorous use of sunblock has made it more common for infants to receive little sunlight exposure. Because of the resurgence of rickets in the US [47], it has become clear that policies of selective vitamin D supplementation of high-risk infants are not adequately protective. Therefore, the American Academy of Pediatrics [4] has recommended universal vitamin D supplementation for all infants. For breast-fed infants, the vitamin D is provided as part of a supplement drop, for formula-fed infants it is contained in the formula (Table 36.4). It is currently recommended by the AAP and the IOM to provide a total of 400 IU/day of vitamin D for all infants [4]. The choice of 400 IU/day is based on relatively little data [4,31]. Much more data are needed on this topic however, and given the high level of safety of vitamin D in this dose range, it is possible that higher doses may be beneficial, especially for some infants who are born with very low vitamin D status. The requirement for 400 IU/day of vitamin D in the United States can be met by an intake of one liter of infant formula daily. Many infants have a slightly lower formula intake and thus may not reach exactly this level. Complicating this issue is that infant formulas, like vitamin supplement drops, generally have a quantity of vitamin D at about 20% over the labeled amount. Thus, it remains uncertain whether formula-fed infants with an intake of slightly less that one liter/day should be given supplemental vitamin D. A further issue to be considered is the form of vitamin D provided for infants. In the United States, vitamin D3 TABLE 36.4
Common Supplemental Vitamin D Sources for Infants and Toddlers
Multivitamin dropsa
400 IU/ml
Vitamin D onlyb
8000 IU/ml
Vitamin D3
c
400 IU/ml
Infant formulas
400 IU/L
Whole milk/ fortified juices
100 IU/240 ml
a
Usually combined with vitamins A and C. Provides vitamin D3. Concentrated vitamin D2 source available in the United States. Not recommended for routine use. c Single source vitamin D3 (Mead-Johnson, Inc., Evansville, Indiana) available in the United States and Canada. b
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is provided in a liquid form, usually at 400 IU per ml of vitamin. A form of liquid vitamin D2 is also available, in a much more concentrated form of 8000 IU/ml. Although few data in infants are available to compare vitamin D2 to vitamin D3, it is likely that they are at least close to being comparably usable by infants and small children [48]. Extreme caution should be used when providing high concentration liquid forms of vitamin D to infants due to the risk of inadvertent intoxication with resultant hypercalcemia. Recent data indicate that high intakes of vitamin D given to lactating mothers can provide for enough vitamin D in the milk to meet the infant’s needs [3]. This suggests that an alternate approach to supplementing infants with vitamin D is to supplement the mothers. At this time, the relative benefits and potential safety issues with providing lactating women with over 5000 IU/day as is needed for this approach are poorly defined. Recommended vitamin D intakes for pregnancy and lactation were set in 2011 as the same as for nonpregnant or lactating women of the same age.
TODDLERS AND PREPUBERTAL CHILDREN There is a substantial gap in data regarding calcium absorption between infants and pubertal children. The 1997 calcium adequate intake (AI) of 500 mg/d for children from 12 to 48 months of age was developed based on extrapolation from desirable calcium retention for 4e8-year-olds [49]. We and others have shown that increasing calcium intake in small children leads to increased total calcium absorption [50e52]. Higher levels may significantly increase calcium absorption and retention without posing any risk to long-term bone development. Recently, stable isotope-based calcium absorption studies have been used to develop EAR and RDA in small children [53]. These and other data led to the establishment of both EAR and RDAvalues for calcium in children. For children age 1e3 years, the EAR was set at 500 mg/day and the RDA as 700 mg/day. For children 4e8 years of age, the EAR was set at 800 mg/day and the RDA at 1000 mg/day. Few data are available regarding calcium requirements in children prior to puberty. Most of these data ares based on balance studies conducted over 50 years ago on diets that are very different from those currently in place [51]. An increase in net calcium absorption when the intake of calcium in 3e5-year-old children was increased from 500 to 1200 mg/day has been reported [52]. The benefit was relatively modest however, and intermediate intake levels, as might more readily be achieved in preschool children, were not evaluated in this study. We compared measures of calcium
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metabolism in 7- and 8-year-old Mexican-American and non-Hispanic Caucasian girls living in southeastern Texas [54]. Fractional calcium absorption and total body bone mineral content in the girls was not significantly correlated to either PTH or vitamin D levels. We found lower 25(OH)D concentrations and higher PTH levels in the Mexican-American girls but these were not significantly inversely correlated to each other. Seasonal variability was seen for 25(OH)D concentrations in girls of both ethnic groups, but values in all of the girls were >12 ng/ml. Therefore, it appears that differences in 25 (OH)D and PTH concentrations between MexicanAmerican and Caucasian girls do not have a large effect on calcium absorption in vitamin-D-sufficient prepubertal children. Similar findings have been described for pubertal children as well [55,56]. The potential benefit to ultimate peak bone mass of increasing calcium intake has been studied in groups of prepubertal children. In one controlled calcium supplementation trial, an increase in bone mass was found when calcium supplements were given to children as young as 6 years of age [57]. However, relatively few children this young were studied, and the duration of effect of this supplementation and its impact on peak bone mass are uncertain. Further studies are needed to evaluate different levels of calcium intake in this age group. It is also not clear that benefits in bone mass achieved prepuberty will persist through puberty or once high intakes of calcium are stopped.
Effects of Puberty, Gender, Ethnicity, and Other Genetic Factors Population- and age-related variability in calcium absorption are largely related to factors that are not readily controlled such as pubertal status, ethnicity, and other genetic factors [58e63]. Identification of these factors and understanding their relative contribution to mineralization is an important area of ongoing research. A few recent studies have considered the effects of polymorphisms of the vitamin-D-receptor-related genes as well as other genes controlling estrogen and growth hormone [64,65] as described below. There are marked differences in bone mass and the incidence of osteoporosis between African-Americans and Caucasians. Several groups have found lower urinary calcium in African-American girls compared to Caucasians [60,62]. In addition, it appears that, at similar calcium intakes, African-American girls absorb more calcium than Caucasians [55,66]. There are no data available for this comparison among males. Numerous recent studies have focused on identifying the mechanisms of the relationship between genetics and osteoporosis by evaluating specific genetic markers
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and their relationship to bone mass. One of the unknown aspects of this important relationship is the relationship between these markers and calcium absorption and excretion. Because of the importance of puberty in determining peak bone mass, it is likely that an effect can be seen during pubertal development. We reported [64] a significant relationship between polymorphisms of the vitamin D receptor (VDR) Fok 1 genotype and calcium absorption. Children with the FF genotype absorbed on average 115 mg/d more calcium than those with the ff genotype. Furthermore, we found the FF genotype to be associated with greater bone mineral density in the study subjects. Recently we reexamined this relationship in a group of 99 young adolescents followed longitudinally over a year. We found that the ff genotype was associated with a significantly lower accrual of bone mineral during the year and lower calcium absorption in the adolescents [65]. These results confirmed the earlier report and suggest that further studies of the interaction of genes and dietary mineral intake may be of value in determining dietary requirements.
Effects of Inadequate Calcium and Vitamin D Intake Extremely low calcium intakes at all ages have been associated with fractures and rickets both in the United States and in other countries, especially Nigeria and South Africa. Much more common in Western countries are intakes of calcium by adolescents far below the RDA of 1300 mg/day but high enough to prevent overt clinical deficiency. For example, in girls 14e18 years old, the tenth percentile of usual intakes is 413 mg/day and the 25th centile is 541 mg/d. This means that nearly 25% of adolescent girls have a daily calcium intake of 40% or less of the recommended amount [31]. Clearly, there is a considerable ability to adapt to these low intakes by increasing fractional absorption. This has been shown in earlier studies by Matkovic [51] and in data from our group [63]. Net calcium retention remains far below that achieved on more appropriate intakes however. There are very few data on the health consequences or effects on calcium physiology of various levels of vitamin D intake or vitamin D status in children. One study from Finland indicated that there was markedly improved bone mineralization, especially at the lumbar spine in girls who had high vitamin D intakes. Those with the highest vitamin D levels showed the most increase longitudinally in bone mineral density at the lumbar spine [68]. This is consistent with findings that adolescent boys in France had decreased vitamin D status in winter associated with increased PTH levels [69]. Rickets has been reported in adolescents in Middle
Eastern countries who have little sunlight exposure for cultural reasons [70]. Goulding [71] found lower bone mass in girls with distal forearm fractures than in age-matched girls without fractures. A lower calcium intake was reported in the 11e15-year-old girls with fractures than in the controls. Wyshak [72] similarly reported that high calcium intakes decreased the risk of bone fractures in adolescents. Epidemiological evidence regarding the consequences of low calcium consumption in childhood and adolescence and ultimate adult bone mass has generally shown the expected benefits to higher intakes [73]. However, such data are not conclusive nor do they demonstrate a critical intake level of calcium at which long-term benefit or harm can be derived. Few longterm follow-up studies have been done to evaluate the effects of calcium intake in childhood and adolescence on adult bone mass.
Effects of Other Factors Including Soda Consumption Wyshak also reported a positive relationship between fractures and cola beverage intake [74]. However, this link does not prove a direct cause-and-effect relationship. Some have suggested that the high phosphorus content of the sodas leads to increased calcium losses and lower bone mass. Furthermore, there are no prospective data relating bone mass to carbonated beverage intake and no prospective studies of this relationship. It is likely that very high phosphorus intakes are needed to adversely affect calcium metabolism [75]. The amount of phosphorus in sodas is small relative to the total daily intake of most adolescents and therefore it is likely that reasonable soda or carbonated beverage consumptions is not a major cause of bone mass loss related to their phosphorus intake [76]. However, it remains of concern that excessive intake of some beverage products places such an adolescent at risk for calcium deficiency probably primarily due to lowered intake of healthier calcium-containing beverages. Further data on the relationship between dietary factors and fractures and bone loss are needed and at present this relationship remains highly controversial [77,78].
SPECIAL ISSUES Developing Countries In Mexican toddlers, Murphy [79] found relatively high levels of calcium intake (mean 735 199 mg/ day). This was far greater than the mean calcium intake
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SPECIAL ISSUES
of less than 220 mg/day in Egypt and Kenya. The higher intake in Mexico likely is due both to dairy products and lime-treated tortillas in parts of Latin America [80]. The bioavailability of the calcium in this diet, especially from the calcium-treated tortillas, may be poor however [81]. These calcium intakes may not occur among poorer populations of Latin America. Wyatt and Tejas [82] have reported large economic differences in calcium intakes in 4e6-year-old children in Southern Mexico. In children in the poorest areas, mean calcium intake was only 272 mg/day primarily coming from corn tortillas. This increased to more acceptable intake levels in mediumincome families of 625 mg/day due to the increased availability of dairy products in families of greater socioeconomic status. In Mexico City a mean calcium intake of 516 mg/day was reported for children 1e5 years of age [83]. In this group, 25% of the children had calcium intakes of less than 361 mg/day that was associated with higher blood lead levels (see below). This is in contrast to a 25th percentile value of 599 mg/day for 1e3-yearolds (and 649 mg/day for 4e8-year-olds) in the United States [31], suggesting a much greater prevalence of very low calcium intakes in Mexico compared to the United States.
Fortification of Foods for Children with Calcium and Vitamin D The potential for inadequate intake of calcium and vitamin D among children and adolescents throughout the world is considerable. Among the strategies for resolving this problem are (1) increasing the intake of foods which naturally contain calcium and vitamin D; (2) increasing the absorption of calcium from foods by additional food components or genetic modification (e.g., decreasing oxalate content) which might enhance calcium absorption; (3) universal pill or liquid vitamin and mineral supplementation; or (4) increased mandatory or optional fortification of food sources [84]. Each of these can and will be important in improving bone health in children and adults. Consideration to the use of prebiotics to enhance calcium absorption is provided below. Food fortification however, is likely to be a key component of any strategy. In considering food fortification with calcium or vitamin D, one must evaluate both the potential benefits of the fortification strategy and the risks related to excess intake from overly zealous fortification. To determine benefit, one must assure that fortification is necessary based on a low intake by at least one population subgroup, that the nutrient of importance has an important public health need, and that the nutrients being fortified are bioavailable. All of these criteria are easily met for both calcium and vitamin D fortification of food and beverage sources for children and adolescents.
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Good bioavailability of beverage calcium and vitamin D fortificants has been shown [85,86]. Available data indicate relatively good bioavailability of calcium added to bread and grain products [87] and fortified cereal [47]. From a safety perspective, it is worth noting that the upper limit for calcium intake was set in 2011 by the IOM as 2500 mg/day for ages 1e8 years and 3000 mg/ day for ages 9e18 years. These intakes are well above the 95th percentile of intakes for all population groups and above the 99th percentile of intake for all age and gender groups except adolescent males. Therefore, reasonable calcium fortification of grain products and juices is unlikely to pose a problem for children and adolescents. Traditionally in the US, relatively few foods and beverages have been vitamin D fortified except for milk. This situation is changing due in part to new regulations by the Food and Drug Administration and it is likely that an increasing number of commercial products will have added vitamin D. There is no apparent reason that products such as juices which are already fortified with calcium cannot also be fortified with vitamin D. The safety margin for this is also likely to be very favorable, as the upper limits for vitamin D intake in children over one year of age as set by the DRI of 2500e4000 IU/ day in children are likely well above the intakes of children who not taking high-dose supplements [31,88]. The combination of substantial public need and a high safety margin make calcium and vitamin D appropriate for food and beverage fortification efforts. However, continuing monitoring and education is important to ensure that excess fortification does not occur and that high-dose supplements are not used at the same time as fortificants. Consideration of the effects of calcium and vitamin D fortification on the status of other minerals, including magnesium and zinc, is also important.
Enhancers of Calcium Absorption in Children and Adolescents An alternative dietary strategy to enhancing net absorbed calcium is to identify dietary strategies which enhance the calcium bioavailability of the whole diet and which have other health benefits. For example, functional foods including prebiotics that alter intestinal flora such as nondigestible oligosaccharides (NDO) may be of benefit [89,90]. We recently completed several studies of the effects of an NDO composed of a mixture of long- and short-chain-length molecules on calcium absorption in young girls (aged 11e13.9 years) [91e93]. In each study, we found a significant increase in calcium absorption whilst consuming NDOs. In a longitudinal study [93], we further found that NDOs increased total body bone mineral content during puberty over a period of 1 year of consumption.
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The mechanism by which oligosaccharides might increase calcium absorption is not known. NDO resist digestion in the human gut, but are fermented to volatile fatty acids in the colon [90]. These fatty acids may have a local effect in the colon by reducing the pH and increasing solubility of mineral in the aqueous phase of the colonic contents permitting higher absorption of minerals in the colon, a site where little calcium absorption normally occurs. Alternatively the NDO, volatile fatty acids, or some other mediator may have a trophic effect on the gut [90], improving overall “gut health.” Such an effect could increase calcium absorption throughout the length of the gastrointestinal tract. Finally, NDOs may alter the composition of the colonic bacterial flora [89,90,94e96] and this might lead, directly or indirectly, to a change in mineral absorption of overall “gut health.” A recent kinetic study suggests that the effect is likely primarily in the colon [97].
References [1] R.J. Schanler, S.A. Abrams, Postnatal attainment of intrauterine macromineral accretion rates in low birth weight infants fed fortified human milk, J. Pediat. 126 (1995) 441e447. [2] Work Group on Breastfeeding, American Academy of Pediatrics, Breastfeeding and the use of human milk, Pediatrics 100 (1997) 1035e1039. [3] B.W. Hollis, C.L. Wagner, Vitamin D requirements during lactation: high-dose maternal supplementation as therapy to prevent hypovitaminosis D for both the mother and the nursing infant, Am. J. Clin. Nutr. 80 (Suppl. 6) (2004) 1752Se1758S. [4] C.L. Wagner, F.R. Greer, American Academy of Pediatrics Section on Breastfeeding; American Academy of Pediatrics Committee on Nutrition Prevention of rickets and vitamin D deficiency in infants, children, and adolescents, Pediatrics 122 (2008) 1142e1152. [5] G. Jones, M. Riley, T. Dwyer, Breastfeeding in early life and bone mass in prepubertal children: a longitudinal study, Osteoporos. Int. 11 (2000) 146e152. [6] S.J. Fomon, S.E. Nelson, Calcium, phosphorus, magnesium, and sulfur, in: S.J. Fomon (Ed.), Nutrition of Normal Infants, MosbyYear Book, St. Louis, 1993, pp. 192e218. [7] R.P. Heaney, S.A. Abrams, B. Dawson-Hughes, A. Looker, R. Marcus, V. Matkovic, et al., Peak bone mass, Osteoporos. Int. 11 (2000) 985e1009. [8] S.S. Baker, W.J. Cochran, C.A. Flores, M.K. Georgieff, M.S. Jacobson, T. Jaksic, et al., American Academy of Pediatrics. Committee on Nutrition. Calcium requirements of infants, children, and adolescents, Pediatrics 104 (1999) 1152e1157. [9] S.A. Abrams, S.A. Atkinson, Calcium, magnesium, phosphorus, and vitamin D fortification of weaning foods, J. Nutr. 133 (2003) 2994Se2999S. [10] R.J. Schanler, The use of human milk for premature infants, Pediatr. Clin. North Am. 48 (2001) 207e219. [11] F.R. Greer, A. McCormick, Improved bone mineralization and growth in premature infants fed fortified own mother’s milk, J. Pediatr. 112 (1988) 961e969. [12] A.J. Lyon, N. McIntosh, Calcium and phosphorus balance in extremely low birthweight infants in the first six weeks of life, Arch. Dis. Child. 59 (1984) 1145e1150.
[13] R.J. Schanler, W. Oh, Nitrogen and mineral balance in preterm infants fed human milks or formula, J. Pediatr. Gastroenterol. Nutr. 4 (1985) 214e219. [14] F. Bronner, B.L. Salle, G. Putet, J. Rigo, J. Senterre, Net calcium absorption in premature infants: results of 103 metabolic balance studies, Am. J. Clin. Nutr. 56 (1992) 1037e1044. [15] S.A. Abrams, N.V. Esteban, N.E. Vieira, A.L. Yergey, Dual tracer stable isotopic assessment of calcium absorption and endogenous fecal excretion in low birth weight infants, Pediatr. Res. 29 (1991) 615e618. [16] R.J. Schanler, The role of human milk fortification for premature infants, Clin. Perinatol. 25 (1998) 645e657. [17] R.J. Schanler, S.A. Abrams, C. Garza, Mineral balance studies in very low birth weight infants fed human milk, J. Pediatr. 113 (1988) 230e238. [18] S.M. Mitchell, S.P. Rogers, P.D. Hicks, K.M. Hawthorne, B.R. Parker, S.A. Abrams, High frequencies of elevated alkaline phosphatase activity and rickets exist in extremely low birth weight infants despite current nutritional support, BMC Pediatrics 9 (2009) 47. [19] A. Loui, A. Raab, M. Obladen, P. Bratter, Calcium, phosphorus and magnesium balance: FM 85 fortification of human milk does not meet mineral needs of extremely low birthweight infants, Eur. J. Clin. Nutr. 56 (2002) 228e235. [20] R. Cooke, B. Hollis, C. Conner, D. Watson, S. Werkman, R. Chesney, Vitamin D and mineral metabolism in the very low birth weight infant receiving 400 IU of vitamin D, J. Pediatr. 116 (1990) 423e428. [21] W.B. Pittard, K.M. Geddes, T.C. Hulsey, B.W. Hollis, How much vitamin D for neonates? Am. J. Dis. Child. 145 (1991) 1147e1149. [22] M.C. Backstrom, R. Maki, A.L. Kuusela, H. Sievanen, A.M. Koivisto, R.S. Ikonen, et al., Randomised controlled trial of vitamin D supplementation on bone density and biochemical indices in preterm infants, Arch. Dis. Child Fetal Neonatal 80 (1999) F161eF166. [23] W.W. Koo, S. Krug-Wispe, M. Neylan, P. Succop, A.E. Oestreich, R.C. Tsang, Effect of three levels of vitamin D intake in preterm infants receiving high mineral-containing milk, J. Pediatr. Gastroenterol. Nutr. 21 (1995) 182e189. [24] M.C. Backstrom, R. Maki, A.L. Kuusela, H. Sievanen, A.M. Koivisto, M. Koskinen, et al., The long-term effect of early mineral, vitamin D, and breast milk intake on bone mineral status in 9- to 11-year-old children born prematurely, J. Pediatr. Gastroenterol. Nutr. 29 (1999) 575e582. [25] C. Agostoni, G. Buonocore, V.P. Carnielli, M. De Curtis, D. Darmaun, T. Decsi, et al., ESPGHAN Committee on Nutrition Enteral nutrient supply for preterm infants: commentary from the European Society of Paediatric Gastroenterology, Hepatology and Nutrition, J. Pediatr. Gastroenterol. Nutr. 50 (2010) 85e91. [26] T.H. Stathos, R.J. Shulman, R.J. Schanler, S.A. Abrams, Effects of carbohydrates on calcium absorption in premature infants, Pediatr. Res. 39 (1996) 666e670. [27] V.P. Carnielli, I.H. Luijendijk, J.B. van Goudoever, E.J. Sulkers, A.A. Boerlage, H.J. Degenhart, et al., Feeding premature newborn infants palmitic acid in amounts and stereoisomeric position similar to that of human milk: effects on fat and mineral balance, Am. J. Clin. Nutr. 61 (1995) 1037e1042. [28] A. Lucas, P. Quinlan, S. Abrams, S. Ryan, P.J. Lucas, Randomised controlled trial of a synthetic triglyceride milk formula for preterm infants, Arch. Dis. Child. 77 (1997) F178eF184. [29] W.W. Koo, M. Hammami, D.P. Margeson, C. Nwaesei, M.B. Montalto, J.B. Lasekan, Reduced bone mineralization in infants fed palm olein-containing formula: a randomized, double-blinded, prospective trial, Pediatrics 111 (2003) 1017e1023.
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C H A P T E R
37 Adolescence and Acquisition of Peak Bone Mass Connie Weaver 1, Richard Lewis 2, Emma Laing 2 1
Purdue University, West Lafayette, IN, USA 2 University of Georgia, Athens, GA, USA
INTRODUCTION
PUBERTAL BONE ACQUISITION
Recent advances have been made in our understanding of the relationships between vitamin D and health and disease and defining optimal status in adults and the elderly. Unfortunately, similar progress toward filling the gaps in knowledge is lacking in adolescence and childhood. Adult criteria and cutoff levels for serum 25(OH)D defining deficiency, optimal status, and toxicity are often extrapolated to children. However, the rapid growth accompanying puberty reflects vastly different forces at play with growth regulators dominating homeostatic mechanisms and bone turnover imbalanced in favor of bone formation. Thus, the importance of vitamin D for musculoskeletal demands requires unique investigation distinct from the stable adult condition. Moreover, systems other than bone are also in flux during the pubertal growth spurt and may require vitamin D for optimal function. Though vitamin D is the focus of this book, many factors influence bone acquisition during puberty including genetics (which encompasses race), body size, timing of pubertal onset, physical activity, and diet. Thus, this chapter will consider the role of vitamin D in the context of these factors. Counterintuitive observations abound. Why do black adolescents have the greatest bone mass accretion in the face of relatively low vitamin D status? Why do Asian adolescents simultaneously have greater calcium absorption efficiency and lower bone mass? We have much to learn about the interplay of environment, including the role of vitamin D, and genetics during changes accompanying adolescence.
Almost half of adult skeletal bone mass is acquired during adolescence [1]. This reflects the high rate of skeletal accretion during puberty as shown from longitudinal whole-body bone mineral content (BMC) data collected in Canadian white boys and girls in Figure 37.1 [2]. The timing for peak BMC gain varies dependent on the skeletal site [3e5]. In females, the greater trochanter reaches its peak by age 14.2 years, femoral neck by age 18.5 years, total body by age 22 years, and spine by age 23 years. Another phenomenon illustrated in Figure 37.1 is that peak height gains occur prior to peak rates of bone mineralization as elongation of the long bones precedes consolidation. At the age of gains in peak height, adolescents have acquired 90% of their adult height, but only 60% of their adult total body BMC. This results in a period of relatively low bone mineral accrual and an increased vulnerability to fracture [6e8]. The incidence of childhood fracture in the US has increased dramatically in the past three decades, i.e. 56% in girls and 32% in boys [6], emphasizing the need to optimize bone strength. The large mineral acquisition during puberty results from bone modeling and remodeling processes whereby bone formation rates exceed bone resorption rates. Bone turnover is considerably higher during puberty compared to young adults and in young adulthood, bone remodeling is coupled such that bone formation and resorption are balanced. During growth, bone modeling leads to increased mineral accrual and expansion of the periosteal and endosteal diameters of bone resulting in changing size and shape, depending on the
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10037-X
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37. ADOLESCENCE AND ACQUISITION OF PEAK BONE MASS Peak BMC Gain in Boys
450
Total Body BMC Gain in g per year
400
Peak BMC Gain in Girls
350 300 250 200 150 100 50
Age at peak height gain in boys
0
Age at peak height gain in girls
9
10 11 12 13 14 15 16 17 18 19 Age in Years
FIGURE 37.1 and boys [2].
Age at peak BMC gain and height gain in white girls
stimulus [10]. Independent of bone mass, changes in bone size or shape can significantly impact bone strength [11]. Peak bone mineral acquisition as delineated in Figure 37.1 has been largely studied using dual energy X-ray absorptiometry (DXA) with BMC or areal bone mineral density (aBMD) as the primary outcome variables. This approach has been used to produce national and international multiethnic pediatric reference standards [12,13]. When using DXA to assess the impact of an intervention such as vitamin D on growing bone, BMC is the preferred outcome measure to aBMD [14]. Prentice et al. [15] caution that aBMD, which captures only two-dimensional space, does not account for differences in bone size. The effect on bone size is important during growth, which is adjusted away when BMC is divided by bone area to determine aBMD. Increasingly, peripheral quantitative computed tomography (pQCT) is being used to assess the three-dimensional aspects of bone including bone size and structure, volumetric BMD (vBMD), and to differentiate between cortical and trabecular bone. Childhood intervention studies that have utilized pQCT to assess the growing skeleton have gained important information that would have been lost with the use of DXA alone. For example, a calcium, weightbearing exercise, or combined calcium plus exercise intervention in young children showed the combined intervention improved leg BMC by DXA, but pQCT of the 20% tibia also showed increases in cross-sectional area (CSA) with exercise alone [16]. Moreover, the combined intervention resulted in both increased tibial
cortical CSA and cortical thickness. In another intervention study in 10e12-year-old girls, cheese was more effective in increasing cortical thickness than calcium carbonate [17]. This information gives a better understanding of an increase in bone strength than the BMC or aBMD from DXA. Interventions that influence bone mass, geometry, and/or size during growth can have a dramatic influence on bone strength and may affect the potential for future fractures [18]. The magnitude of peak BMC gains as well as the timing of onset of puberty, which sets in motion peak bone mass acquisition, varies with a variety of genetic and environmental factors. Timing is important because it influences the total period of skeletal growth [19]. Approximately 60e80% of peak bone mass is under genetic control [19,20]. The influence of sex differences is clearly shown in Figure 37.1 as the peak aBMD gain is higher and occurs later by about 1.5 years in boys compared to girls. Girls accumulate approximately 25% of their adult peak bone mass between the ages of 12e14 years, whereas, the sharp growth in boys occurs between ages 13e15 years. However, timing of peak bone mineral acquisition is more closely related to pubertal development than chronological age [2,19] and may be controlled in part by sex steroid hormones. For example, estrogen is more important than androgen in mineralization of the skeleton [21], and in adolescent boys, serum IGF-I concentration is a greater predictor of calcium retention than sex steroid hormones [22]. In that study [22], serum IGF-I was the most important predictor of calcium retention after calcium intake and explained 11.5% of the variation. Factors that are thought to influence timing of peak BMC gains are illustrated in Figure 37.2. A key genetic factor influencing the timing of peak BMC gain is race. For example, black and Asian girls on average have earlier menarche than white and Hispanic girls [23,24]. The difference between the age of menarche in black and white girls living in urban South Africa has been diminishing [25]. This secular trend and the trend towards earlier age at menarche were attributed to recent nutritional and socioeconomical changes demonstrating raceeenvironment interactions on bone. Plots of peak skeletal accretion rates in races other than whites in the study by Bailey et al. [2] in Figure 37.1 are very limiting. A low-resolution plot of cross-sectional means rather than averages of individualized plots for Chinese girls studied by Zhu et al. [26] compared to the white Canadian girls in the Bailey et al. study [27] is shown in Figure 37.3. These data show that the Chinese girls had an earlier peak BMC gain than the white Canadian girls. Lifestyle factors including exercise and diet account for 20e40% of peak bone mass [1,20], and these factors can also influence onset of menarche. In the prospectively studied cohort presented in Figure 37.1, participants who
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VITAMIN D IN CHILDREN AND ADOLESCENTS
450
Total Body BMC Gain in g per year
400 350 300 250
Black Male Poor diet Anorexia Excessive exercise
Calcium
200 150 100 50 0
9
10
11
12
13 14 15 16 Age in Years
17
18 19
Factors that shift timing of peak bone accretion from the baseline curve in white girls.
FIGURE 37.2
Total body BMC gain (g/y)
300 250
Chinese
White
200 150 100 50 0
9
10
11
12
13 Age (y)
14
15
16
17
FIGURE 37.3 Difference in bone accretion between Chinese and
white girls [28].
were more physically active during adolescence had greater BMC at the total hip and femoral neck through puberty and at adulthood [29]. Diets too low in energy or fat to produce sufficient concentrations of estrogen result in a delay of menarche or amenorrhea [30]. The condition anorexia nervosa can restrict bone gain or accelerate bone loss [31]. By the same token, energy expenditures in female athletes that exceed energy intakes can delay onset of menarche or alter menstrual function [32]. This can result in irreversible bone loss.
659
Moreover, specific nutrients such as dietary calcium have been shown to influence timing of menarche [33]. In a follow-up study of girls with a mean age 7.9 years that had been randomized to calcium-fortified foods for 1 year prepuberty, it was shown that those receiving extra calcium started menarche earlier by an average of 4.9 months compared to those receiving placebo foods. The extra time of estrogen exposure with an earlier menarche was presumed to be responsible for greater subsequent gains in bone mineral even after the calcium intervention was discontinued. It has been controversial whether effects of diet and exercise during adolescence can be sustained into adulthood. Diet- and exercise-induced BMC gains during growth have been shown to persist for several years or to be partially or totally lost following cessation of the intervention [34e36]. Evidence from animal studies clearly shows that diet during rapid growth influences bone strength in the mature skeleton. For example, rats fed a diet which provided calcium through fat dry milk had larger, stronger, and more dense bones than rats fed calcium as calcium carbonate [37]. When switched to a calcium-deficient diet at maturity, the rats raised on the dairy-based diet were largely protected from bone loss due to calcium deficiency compared to the rats fed calcium carbonate. In a randomized controlled trial in girls at Tanner Stage 2, calcium supplementation throughout puberty resulted in higher peak bone mass, but only in girls who achieved an adult height higher than average in the cohort [38]. Furthermore, increased calcium intake has been shown to suppress bone resorption in adolescents by an amount equivalent to the increase in absorbed calcium [39]. The roles of many nutrients in bone have been reviewed [40]. As the studies above indicate, calcium is the dominant mineral in bone mineral content and also suppresses bone resorption. Phosphorus and magnesium are other key elements that comprise bone mineral and proteins as connective tissue occupy half of bone volume. The high rates of bone mineral accrual during childhood and adolescence and the ability of bone to alter its shape or geometry provide a unique opportunity to intervene, improve bone strength and reduce the risk of fractures later in life. Because of the interdependency of calcium and vitamin D, more research is needed to understand the impact of their interactions on bone.
VITAMIN D IN CHILDREN AND ADOLESCENTS Circulating 25-hydroxyvitamin D (25(OH)D) concentrations assessed in children and adolescents worldwide vary depending on geographical location, season, race, age, sunscreen use, degree of urbanization, pubertal maturation stage, BMI, income, education, and genetic
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37. ADOLESCENCE AND ACQUISITION OF PEAK BONE MASS
variation factors [41e43]. Surprisingly, a higher than expected percentage of children are reported to have low concentrations of 25(OH)D and particularly in children sampled in the wintertime, those who are darkskinned, and those who reside at higher latitudes north or south of the equator. The relevance of these low levels is uncertain, as the 25(OH)D concentrations reflecting sufficient or optimal vitamin D status in children and adolescents are unknown. In adults, the vitamin D requirement is based not only on serum 25(OH)D responses to varying intakes of vitamin D, but also on responses to various functional indicators [44e47]. For example, a serum 25(OH)D concentration of 80 nmol/L is proposed as optimal in adults because this concentration is associated with maximal suppression of intact parathyroid hormone (iPTH), increased calcium absorption, attenuated bone loss, and reduced skeletal fractures. In children and adolescents functional outcome data in response to varying doses of vitamin
TABLE 37.1
D data are limited, but could include the absence of rickets, maximal suppression of iPTH, increased measures of bone mineral content, optimal calcium absorption, and/or decreased skeletal fractures [41]. Despite the fact that scientific data do not exist to support specific serum 25(OH)D cutoffs that define sufficiency, a significant number of children and adolescents have been classified as having either vitamin D deficiency or insufficiency. Though most investigators have employed a cutoff of >50 nmol/L to denote vitamin D sufficiency in children [42,48e54], values >60 to 80 nmol/L have also been used to define optimal vitamin D status based on limited iPTH data [42,54e56]. According to the Canadian Paediatric Society, vitamin D sufficiency for Canadian children has been defined as 75 nmol/L [57]. Table 37.1 summarizes the prevalence rates of vitamin D concentrations 50 nmol/L in children taking into account latitude, season, race, income, age, sex, and obesity. Using National Health and
Prevalence of Vitamin D Insufficiency and Deficiency in Children and Adolescents
Location
Population Sex1 Race2 Age
Ginty et al. [58] Switzerland 46e47 N
Design
I/D3,4 cutoffs5
Prevalence
M & F white (11e16) N ¼ 193
Serum 25(OH)D measured September through March
I < 50 I < 30
Overall, 76% <50 NoveMarch: 15% of boys and 17% of girls <30
Sullivan et al. [49] Bangor, Maine 44 N
F white (9e11) N ¼ 23
Serum 25(OH)D measured in September and in March for 3 years
I < 50
48% I 17% I in both September and March
Docio et al. [50] Cantrabria, Spain 43 N
M & F Spanish (6e10 y) N ¼ 94
Serum 25(OH)D measured in either winter (n ¼ 51, JaneApril) or in the summer (n ¼ 43, AugeOctober)
I < 50 D < 30
12% I (summer) 80% I (winter) 31% D (winter)
Gordon et al. [48] Boston 42 N
M & F 47% black, 16% white, and 26% Hispanic (11e18 y) N ¼ 307
Serum 25(OH)D measured year-round
I 50 D 37.5
42% I 24.1% Overall D 35.9% black D 17% Hispanic D 3% White D 4.6% 20
Jones et al. [51] Hobart, Tasmania 42 S
M & F (8 y) N ¼ 446
Serum 25(OH)D measured throughout the year
I < 50
10% I
Harkness et al. [52] Ohio 41 N
F white and black (12e18) N ¼ 307
Serum 25(OH)D measured throughout the year
I 50 D 37.5
17% Overall D 26% black D <1% white D 54% Overall I 71% black I 24% white I
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TABLE 37.1
Prevalence of Vitamin D Insufficiency and Deficiency in Children and Adolescentsdcont’d
Location
Population Sex1 Race2 Age
Jones et al. [53] Australia 41 S
Design
I/D3,4 cutoffs5
Prevalence
M white (16e18) N ¼ 136
Serum 25(OH)D measured in the winter (July through August)
I < 50 D 25
68% I 11% D
Rajakumar et al. [59] Pittsburgh, Pennsylvania 40.4 N
M & F black (6e10 y) N ¼ 41
Serum 25(OH)D during winter
I ¼ 25e50 D 25
49% I
Stein et al. [60] Athens, GA 34 N
F white and black (4e8 y) N ¼ 168
Serum 25(OH)D measured throughout the year
I < 50
2% I
El-Hajj Fuleihan et al. [61] Beirut, Lebanon 33.5 N
M & F Lebanese (10e16) N ¼ 169 (spring) N ¼ 177 (fall) N ¼ 83 (both)
Serum 25(OH)D measured in spring (MarcheApril) and fall (NovembereDec)
I ¼ 25e50 D < 25
65% I (winter) 40% I (summer) 42% (F) I (spring) 46% (F) I (fall) 46% (M) I (spring) 25% (M) I (fall) 32% (F) D (spring) 7.5% (F) D (fall) 9% (M) D (spring) 0% (M) D (fall)
Fares et al. [54] Beirut, Lebanon 33.5 N
M & F Lebanese (10e17) N ¼ 172
Serum 25(OH)D measured in fall (NovembereDec)
I < 50
40% I
Marwaha et al. [62] New Delhi, India 28 N
M & F Indian (10e18 y) N ¼ 760
Serum 25(OH)D
D ¼ 25e50, 12.5e25, <12.5
35.7% < 22.5 (overall) low SES group 11.2% < 12.5 39.5% 12.5e25 42.1% 25e50 upper SES group 4.9% < 12.5 25.5% 12.5e25 57.6% 25e50
M ¼ Male, F ¼ Female; NH ¼ Non-Hispanic; 3 I ¼ Insufficiency/Insufficient; 4 D ¼ Deficiency/Deficient; 5 Values are nmol/L. 1 2
Nutrition Examination Survey (NHANES) III data in US adolescents, 12e19 years of age, the national prevalence of vitamin D deficiency was estimated at 14% or 48% using cutoffs of 50 or 75 nmol/L, respectively [42]. In younger children aged 1e11 years, from NHANES 2001e2006 [63], the national prevalence of vitamin D deficiency was estimated to be 18% and 69% when using cutoffs of 50 or 75 nmol/L, respectively. Among females, 4e8 years of age, living in the southeast US (34 N), 2% and 35%, had serum 25(OH)D concentrations below 50 and 80 nmol/L, respectively [60].
Latitude and Season Mean circulating 25(OH)D concentrations are lower among children living at higher latitudes [48,49,52, 64,65]. For example, mean values for adolescents in Helsinki, Finland (latitude 60 N) are approximately 47 nmol/L [66], 62.0 nmol/L in the northern United States
(US; latitude 42 N) [48], and 94.0 nmol/L in the southern US (latitude 34 N) [60]. Though poor vitamin D status is observed primarily in children and adolescents living at higher, northern latitudes, low vitamin D concentrations are reported in southern regions. Data from NHANES III showed that up to 47% of 12e19-year-old males and females (all races combined) living in sunny locations (mean latitude 32 N) had wintertime 25(OH)D values <62.5 nmol/L [67]. Serum 25(OH)D concentrations also fluctuate with season whereby values are significantly lower in the wintertime months compared to the summer months (~20e30%) and this has been demonstrated for locations as far south as US latitude 34 N [48,49,52,60,66].
Race and Income Individuals with darker skin pigmentation synthesize less vitamin D for the same UVB exposure than lightskinned individuals [68]. This diminished synthesis is
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reflected in lower serum 25(OH)D levels, as black children have values approximately 20e40 nmol/L lower than white children [52,59,68e71]. When applying the cutoff used to define vitamin D sufficiency in adults (50 nmol/L) [72], 50e70% of black adolescents from NHANES had 25(OH)D concentrations <50 nmol/L and had ~20 times the risk of having serum 25(OH)D <50 nmol/L compared with white adolescents [42,67]. Mexican-American adolescents have also been shown to be at increased risk of vitamin D concentrations <50 nmol/L (odds ratio: 4.6) [42]. National prevalence data from NHANES 2001e2006 [63] showed that vitamin D concentrations in children aged 1e11 years <75 nmol/L was higher among black vs. white children (80 vs. 59%). Furthermore, adolescents studied in NHANES III with low-income status had three times the odds of deficiency (<50 nmol/L) compared to those with high-income status [42].
Age and Pubertal Maturation Older, more mature adolescents have lower serum 25 (OH)D concentrations than younger, prepubertal children and these observations have been shown prospectively in a sample of child and adolescent females [71] and from cross-sectional investigations [43,70]. The adjusted mean 25(OH)D concentrations for US children 12e19 years of age were lower than children 6e11 years of age (64 vs. 70 nmol/L, respectively) [70] and the estimated prevalence of 25(OH)D levels <75 nmol/L was higher among children aged 6e11 vs. 1e5 years (73 vs. 63%, respectively) [72]. Furthermore, late pubertal adolescents have significantly lower mean values than prepubertal children (75 vs. 95 nmol/L, respectively) [43]. The mechanism for the decreasing vitamin D levels with increasing age and maturation is unknown. In early pubertal girls increases in fatfree mass with growth are associated with decreasing plasma 25(OH)D concentrations [71]. The decreasing 25 (OH)D concentrations may be the result of plasma volume changes and a dilution effect with growth. It has been suggested that IGF-I, an important regulator of muscle growth, is associated with the conversion of 25(OH)D to 1,25(OH)2D [73], which could potentially lower 25(OH) D with age. The implications of decreasing 25(OH)D with growth is not clear, though children followed prospectively over seven years with lower 25(OH)D had greater gains in BMC [74]. Further investigation is needed into the interactions between 25(OH)D, 1,25(OH)2D, and IGF-I to better understand the role(s) of vitamin D in muscle and bone during growth.
Sex While the majority of pediatric vitamin D studies target females, data from cross-sectional [75,76] and
intervention [77] studies suggest that there are no significant sex differences in serum 25(OH)D concentrations. For example, mean standard deviation (SD) serum 25(OH)D values for 12-year-old boys and girls from Northern Ireland had median serum 25(OH)D values of 61 and 59 nmol/L, respectively [76]. Lebanese boys and girls ~13 years of age participating in a vitamin D supplementation trial had baseline serum 25(OH)D values of 37 17 and 40 15 nmol/L, respectively [77]. However, in adolescents from NHANES III, aged 12e19 years, means were lower for girls vs. boys in every racial category and black girls had the lowest 25 (OH)D values after adjusting for age, gender, body weight, education, income, degree of urbanization, and region (i.e., Northeast, Midwest, South, and West) [42]. Furthermore, the odds of having vitamin D concentrations <50 nmol/L was more than double for females compared with males [42]. In NHANES III data using a younger sample of 1e11-year-olds, the prevalence of 25(OH)D levels of <75 nmol/L was also higher among girls vs. boys (71 vs. 67%) [63]. The lower 25(OH)D concentrations in females compared to males from NHANES III could be associated with differences in pubertal maturation between the two sexes of the same chronological age [43].
Obesity Because vitamin D is fat-soluble, lower circulating levels may be exaggerated in those who are overweight/obese because of its suggested sequestration in body fat, which may decrease its bioavailability [76]. Using data from NHANES III in 12e19-year-old females [42], overweight (according to BMI) adolescents had 75% increased risk of 25(OH)D concentrations <50 nmol/L compared with normal-weight adolescents (a 1% increase in BMI for age percentile resulted in a 5% decrease in 25(OH)D concentrations). In a US study of 6e10-year-old black children, 25(OH) D concentrations <50 nmol/L were more pronounced in the obese vs. the nonobese group (57 vs. 40%, respectively) [59]. The implications of vitamin D deficiency or insufficiency in overweight adolescents may extend beyond skeletal functions and to other clinically relevant, cardiometabolic conditions, such as cardiovascular disease, metabolic syndrome, and impaired glucose tolerance.
VITAMIN D AND INTERMEDIATE ENDPOINTS OF VITAMIN D AND BONE METABOLISM Whether vitamin D supplementation during growth favorably alters functional outcomes related to vitamin
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VITAMIN D AND INTERMEDIATE ENDPOINTS OF VITAMIN D AND BONE METABOLISM
25(OH)D In adults, the oral vitamin D intake required to effect a change in serum 25(OH)D concentration has been estimated as approximately 0.7 nmol/L/mg/day by Heaney et al. [79]. Using this criterion, achieving serum 25(OH) D levels of 80 nmol/L would require an average intake of approximately 1000e1600 IU/day in adults living in the central US (latitude 40 to 42 N). Using the same equation from Heaney at al. [79] together with baseline levels of serum 25(OH)D in NHANES, inputs required to achieve 80 nmol/L were calculated for various subpopulations [44]. For those living in lower latitudes in the US during the winter season, it was estimated that up to 863 IU/day of additional vitamin D input was needed on average for males and females, aged 12e19 years, but it was highly racially dependent because of disparate starting levels of 25(OH)D for blacks. For example, black males and females aged 12e29 years were expected to require 1714 IU/day and 2154 IU/day, respectively, to achieve 25(OH)D levels of 80 nmol/L. White males and females of the corresponding age were expected to require far less vitamin D, up to 297 IU/day, to achieve 25(OH)D levels of 80 nmol/L. Vitamin D3 supplementation of 200 IU/day or 400 IU/day in healthy Finnish females, mean age 11.4 years, significantly increased serum 25(OH)D approximately 5 and 12 nmol/L, respectively, after 12 months [80]. In a study of 6e10-year-old black US children [81], vitamin D supplementation with 400 IU/day for 1 month significantly increased serum 25(OH)D almost 20 nmol/L, but only in those children who were vitamin-D-insufficient. In Lebanese females, 10e17 years of age, serum 25(OH) D was unaltered following 1 year without vitamin D supplements, though increased approximately 7.5 nmol/L with ~200 IU/day (not statistically significant) and 60 nmol/L with ~2000 IU/day [82]. In a companion study presenting data separately for males
of the same age [77], the authors found that the males responded similarly to supplementation., i.e., serum 25 (OH)D was unaltered following 1 year without vitamin D supplements, increased approximately 10 nmol/L with ~200 IU/day (not statistically significant) and 48 nmol/L with ~2000 IU/day. A study of Spanish boys and girls also found significant increases in 25 (OH)D with higher doses of vitamin D [50]. In this study, supplementation with 1600 IU/day for 1 week to children with low wintertime 25(OH)D concentrations (~31.5 nmol/L), increased 25(OH)D concentrations to approximately 80 nmol/L. In summary, it appears that higher doses of vitamin D may be required to significantly elevate serum 25(OH)D concentrations in children and adolescents and that basal levels are a key determinant of the effect, with greater responses in those with lower concentrations.
1,25(OH)2D Serum 1,25(OH)2D levels respond transiently to dietary calcium mediated by PTH release. Increases in serum 1,25(OH)2D occur when calcium intakes are low unless the precursor, 25(OH)D, is limiting. Serum 1,25 (OH)2D levels were higher in puberty (Tanner Stage 3) than in prepuberty or late puberty (Tanner Stage 4) in a cross-sectional study in 104 Norwegian boys aged 8.5e17.8 years and 87 girls aged 8.0e18 years, as shown in Figure 37.4 [83]. The authors of this study suggested that the rise in serum 1,25(OH)2D with puberty may be associated with the rise in iPTH, and the fall in late puberty may be associated with growth hormone or somatomedins. As previously discussed, serum IGF-I was the best predictor of calcium retention after calcium intake in adolescent boys [22].
60 1,25-(OH)2D,pg/ml ± SEM
D and bone metabolism is unclear. Contributing to this uncertainty is the fact that few vitamin D supplementation trials have been conducted in pediatric and adolescent populations. In order to accurately identify specific 25(OH)D cutoffs for adolescents defining sufficient or optimal status, the relationships between vitamin D inputs and change in 25(OH)D and other intermediate endpoints of vitamin D and bone metabolism need to be determined. Moreover, these relationships need to be examined using a wide range of vitamin D inputs and in adolescents representing the range of sex, race, and maturational stages. The published evidence regarding serum 25(OH)D, 1,25(OH)D2, iPTH, fractional calcium absorption, and bone biomarker responses to vitamin D supplementation in adolescents is limited and described below.
50
40
30
boys (n) 12 girls (n) <10.9
FIGURE 37.4 age [83].
V. HUMAN PHYSIOLOGY
14 18 11
13 11 12
26 13 13
10 8 14 Age
13 8 15
15 8 16
12 7 17
20 20-30
1,25-(OH)2D (mean þ SEM) relates to chronological
664
37. ADOLESCENCE AND ACQUISITION OF PEAK BONE MASS
The intervention trials to date that have investigated the relationship between vitamin D and 1,25(OH)2D concentrations have shown mixed results likely due in part to differences in baseline serum 25(OH)D levels, calcium intake, and genetic traits in the populations studied [50,71,80,82]. Vitamin D supplementation with 1600 IU/day [50] or ~2000 IU/day [82], but not with 600 IU/day [84] or ~200 IU/day [75,82] significantly increased serum 1,25(OH)2D in young males [50,85] and females [50,82,84] within the age range of 6e17 years. In the 1600 IU/day study [50], participants were supplemented for 7 days in both October and March and 25(OH)D, 1,25(OH)2D and iPTH were assessed before and after supplementation. In March, 1,25(OH)2D concentrations increased significantly after vitamin D supplementation and were accompanied by a significant reduction in iPTH. In October, vitamin D supplementation resulted in a much lower, though still significant, increase in 1,25(OH)2D serum levels; however, iPTH did not change. Lower baseline concentrations of 25(OH)D most likely contributed to the iPTH response seen in March, but not in October. Baseline concentrations of iPTH were not reported. In the 2000 IU/day study [82], 1,25(OH)2D concentrations increased significantly after 12 months of vitamin D supplementation; however, iPTH was not measured in this study. The participants in the studies discussed resided in Spain [50], Lebanon [75,82] and Denmark [84], and the effect of vitamin D supplementation on 1,25(OH)2D was observed mainly in participants with lower serum 25(OH)D concentrations at baseline. In the Maalouf et al. [77] study, the changes in 1,25 (OH)2D following 12 months of vitamin D supplementation (14 000 IU/week) were similar in boys and girls aged 10e17 years (pre- and post-pubertal maturation stages combined). The serum 1,25(OH)2D response to vitamin D supplementation in US adolescents of the same pubertal maturation is unknown as are the potential differences in the response among blacks and whites, since blacks have higher circulating concentrations of 1,25(OH)2D [73, 85].
25(OH)D and iPTH [48,55,66,69,79,89e94]. In the 2007 Agency for Healthcare Research and Quality review [95], it was concluded that there was fair evidence in children and adolescents for an inverse relationship between iPTH and serum 25(OH)D. Two cross-sectional comparisons, one in blacks aged 6e10 years living in the northeast US [80], and the other in males aged 13e16 years living in France [55], demonstrated nonlinear relationships between iPTH and 25(OH)D, which resulted in a plateau or inflection point at which iPTH declined minimally with increasing serum 25 (OH)D concentrations. In these studies, serum 25(OH) D concentrations of 75 and 83 nmol/L were identified as cutoffs defining 25(OH)D sufficiency. However, in a recent pooled analysis of data from three sites including Indiana, Boston, and Texas, no inflection point could be determined [96]. The case for an effect of vitamin D supplementation on iPTH is not as strong as the aforementioned crosssectional data have shown. Intervention trials in US black children [59,81], Finnish adolescent females [66], and male and female children from Denmark [84] showed no effect of vitamin D supplementation on iPTH. These studies used doses of vitamin D ranging from 200e600 IU per day. In contrast, supplementation with higher doses of vitamin D (~1600e1700 IU/day) in male and female children from Spain [50] and in male adolescents from France [65] significantly decreased iPTH. The suppression of iPTH, assessed at 7 days and 6 months after initiation of vitamin D replacement, respectively, was observed only in children with low initial serum 25(OH)D concentrations [50] or in those with low calcium intakes [65]. Based on the evidence presented above it seems as though higher doses of vitamin D are needed to elicit a meaningful iPTH response, although the response to supplementation in black adolescents remains unknown. Importantly, optimal levels of serum iPTH in children are also not known. Given that bone modeling is high in children to support growth, iPTH suppression may be counterproductive and overridden by growth hormones.
iPTH
Fractional Calcium Absorption
A third functional outcome measure for evaluating optimal vitamin D status is iPTH suppression [86]. Poor vitamin D status and low calcium intakes in adults lead to secondary hyperparathyroidism, which in turn increases bone remodeling and leads to increased fracture risk [45e47,87,88]. Serum 25(OH)D levels of approximately 80 nmol/L (range 75e110 nmol/L) would be optimal in adults as serum iPTH concentrations rise below this level and are unchanged above this level [45,88]. Several studies in youth have reported significant inverse linear relationships between serum
Calcium absorption occurs by an active, carrierdependent process and a passive, paracellular process. The active process is vitamin-D-dependent, but the passive process is not. When calcium intakes are low, 25(OH)D is converted to 1,25(OH)2D, which upregulates transcription of calcium transport proteins in the gut as described in Chapter 19. However, this homeostatic regulation mechanism is unable to correct for chronically low calcium intakes. If vitamin D stores are too low, conversion to 1,25(OH)2D is reduced. Vitamin D status would likely have to be very low,
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VITAMIN D AND INTERMEDIATE ENDPOINTS OF VITAMIN D AND BONE METABOLISM
i.e. <10 nmol/L [97] given the substrate pool of 25(OH) D is up to 1000-fold higher than 1,25(OH)2D. Because passive absorption is not vitamin-D-dependent, high calcium intakes can mitigate reduced calcium absorption efficiency in the face of low vitamin D status. Low vitamin D status therefore becomes a risk for bone health when calcium intakes are low. The gaps in knowledge in children and adolescents involve the calcium intake needed to overcome vitamin D insufficiency and the roles of vitamin D status, among other regulators associated with reduced calcium absorption efficiency, when calcium intakes are low. Calcium absorption efficiency in children is determined in only a few laboratories worldwide. The various approaches have been reviewed [98,99]. The best method for a double isotope tracer technique is where one calcium tracer is given by oral administration to determine absorption and adjusted for tracer dilution by a second intraveneously administered tracer. This method gives true fractional calcium absorption. Isotopic tracers are then measured in serum or urine samples. Recently, a single oral isotope tracer method has been validated in adolescents against the double isotope reference methods [100]. Another method is net calcium absorption determined by metabolic balance, which necessitates controlled diets and complete urine and fecal collections. This method does not correct for endogenous secretion, i.e., absorbed calcium that is re-excreted in the gut. Indirect methods of calcium absorption are changes in serum calcium or iPTH after a calcium challenge. Sequential blood draws following the calcium load allows calculation of area under the curve (AUC). The relationship between vitamin D and calcium absorption efficiency has not been studied extensively. Cross-sectional studies show no relationship between serum 25(OH)D and calcium absorption efficiency in adolescents with higher 25(OH)D concentrations [68,91], but in those with serum 25(OH)D concentrations below 50e62.5 nmol/L, the relationship appears negative [91,101] (Fig. 37.5). Conversely, when expressed as
total calcium absorbed, the relationship appears positive [43]. While the above data are meaningful, cross-sectional studies are unable to assign causal effects. Longer-term, supplementation studies provide greater insight with regard to the relationship between vitamin D and calcium absorption efficiency. For example, vitamin D supplementation at 1000 IU/d for 4 weeks improved vitamin D status, but resulted in decreased fractional calcium absorption despite no significant change in serum 1,25(OH)2D or PTH [102]. The unexpected decrease in calcium absorption with increased vitamin D status as shown in this study may occur if serum 25 (OH)D is a limiting substrate pool for forming 1,25 (OH)2D, such that at very low vitamin D status, the active hormone cannot be formed in sufficient quantities to stimulate calcium absorption. In adults, calcium absorption increases when vitamin D status falls below 10 nmol/L [97]. However, the mean presupplementation serum 25(OH)D level in the Park et al. [102] study was 48 nmol/L. Sufficiently high calcium intakes suppress iPTH release and therefore, conversion to 1,25(OH)2D. Calcium intake in the vitamin D supplementation study by Park et al. [102] was ~1000 mg/d, which may have been sufficient to suppress iPTH release, but does not explain the inverse relationship between serum 25(OH) D and calcium absorption. The inverse relationship between serum 25(OH)D and iPTH may explain the decreased calcium absorption at higher 25(OH)D concentrations through decreased conversion to 1,25(OH)2D. Indeed, serum 1,25(OH)2D has been shown to be positively correlated with calcium absorption efficiency in adolescent girls (r ¼ 0.35, P ¼ e0.001) [90]. Calcium absorption is influenced by an interaction of calcium intake and genetics, as well as diet. For example, the Fok1 polymorphism of the VDR gene was significantly related to calcium absorption (P ¼ 0.008), where those with the FF genotype had the greatest calcium absorption efficiency [103]. FGF-23 has an indirect role on calcium absorption since it down-regulates renal 1a-hydroxylase and decreases 1,25(OH)2D. Relation between vitamin D status and true fractional calcium absorption r ¼ e0.79, P < 0.01 [101].
FIGURE 37.5
100.0 80.0 TFCA (%)
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60.0 40.0 20.0 0.0 0.0
10.0
20.0
30.0 40.0 25-OHD (nmol/L)
50.0
60.0
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Calcium Metabolism in White Adolescent Girls and Young Women (mean SD) [9] Calcium, mg/d
Girls (n ¼ 14)
Bone formation Bone resorption rate Bone rate 1-84PTH, pg/ml 25(OH)D, ng/ml 1,25(OH)2, pg/ml Intake absorbed
Balance
29.97.4
282 269*
Young Women 25.2 10.6 (n ¼ 11)
27.6 8.5
37.5 7.4
1330
494 232*
1459 542*
1177 436*
33.0 7.9
36.1 6.8
1330
283 122
501 129
542 212
41 165
* P < 0.01.
Biochemical Markers of Bone Turnover The difference between bone formation and bone resorption rates determines bone balance as shown in Table 37.2 [9]. The impact of vitamin D status on bone turnover rates in puberty is understudied. No calcium kinetic studies have been undertaken in children to address the effect of vitamin D supplementation on true bone formation and bone resorption rates. In cross-sectional calcium kinetic studies in adolescents, vitamin D status has not predicted bone turnover rates [39,104,105]. However, the studies have been small and likely lacked statistical power to address the role of vitamin D on bone turnover [Table 37.2]. Biochemical markers of bone turnover are more commonly used to assess bone formation and bone resorption rates because assays are readily available in commercial kits. Although they are correlated with bone formation and bone resorption rates determined by calcium kinetic studies, these measures are highly variable [106]. Consequently, when diet perturbs bone turnover as measured by kinetics, biochemical markers of bone turnover in the same study may be unable to show a statistically significant effect [39]. In an evaluation of pooled data in 105 adolescent girls, urinary cross-liked N-telopeptides of type 1 collagen (NTx) were significant in a model for calcium retention along with race and calcium intake [69]. This finding reinforces the role of bone turnover as evaluated by biochemical markers in skeletal accretion. Furthermore, this required a larger sample size to find a small effect that was not apparent in the individual studies. A few studies have evaluated the relationship between vitamin D status and biochemical markers of bone turnover. In 301 Chinese adolescent girls, plasma bone alkaline phosphatase, a marker of bone formation, and urinary deoxypyridinoline:creatine ratio, a marker of bone resorption, were significantly lower in girls with serum 25(OH)D levels >50 nmol/L compared to those <50 nmol/L [107]. In other, smaller studies, results are mixed. For example, serum 25(OH)D was shown to be inversely correlated with pyridinoline (a bone resorption marker) in 136 adolescent males (r ¼ e0.23; P < 0.01) [53], but not to bone-specific alkaline phosphatase or
C-terminal telopeptide (CTX; a bone resporption marker) in 172 adolescent girls or boys from Lebanon [54]. Serum osteocalcin (a bone formation marker) concentrations in early pubertal girls were reported to be 17.6% lower (P < 0.001) in March compared to September [66], presumably due to lower 25(OH)D levels. No relationship between serum 25(OH)D levels and CTX or a marker of collagen formation (P1 NP) was observed in 196 Swiss boys and girls aged 11e16 years [58]. To date, few prospective trials have assessed bone turnover in relation to vitamin D status. In a 3-year longitudinal study in 9e15-year-old females, Lehtonen-Veromaa et al. [108] reported an inverse correlation between serum 25(OH) D and CTX (r ¼ e0.27; P < 0.001) suggesting that chronically low 25(OH)D levels could reduce bone formation and attenuate skeletal growth. The conflicting results may relate to use of different biochemical markers and their high variability, to confounding effects of pubertal status and sex, or to small sample sizes. To date, four intervention trials have assessed the impact of vitamin D supplementation on markers of bone turnover in children and adolescents [59,66,81,84]. Three of the trials found no significant changes in bone turnover markers following supplementation of 400e600 IU [59,81,84]. However, in the 12-month trial by Viljakaninen et al. [66], where 46 adolescent girls, mean age 11.4 years, were supplemented with either 200 or 400 IU per day supplementation significantly reduced deoxypyridinoline (compliance-based analysis only). In this study, supplementation had no effect on osteocalcin or pyridinoline. As with most cross-sectional studies, results from these supplementation trials were mixed. This may be due in part to large age ranges (and presumably the maturational stage ranges), relatively low doses of vitamin D used in most of the trials, and/or lack of sensitivity of the assays.
VITAMIN D AND OTHER PREDICTORS OF CALCIUM RETENTION Calcium retention is the balance between calcium intake and excretion. Over time, calcium retention determines bone acquisition since more than 99% of the
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Vitamin D status does not affect net calcium absorption (A) or calcium retention (B) (P > 0.05) in black and white adolescent girls, but black girls are more efficient at absorbing and retaining calcium (P ¼ 0.003); •black girls, Bwhite girls [69].
FIGURE 37.6
calcium in the body is within the skeleton. Short-term balance is measured by metabolic balance studies, whereas long-term balance is determined by total body bone mineral content. Studies evaluating the effect of vitamin D status and supplementation on bone mass acquisition will be discussed in the next section. Metabolic balance studies, by necessity due to their intensive nature, involve a small number of study subjects. This limits their ability to determine predictors of calcium retention other than the primary intervention, for which statistical power was determined. A few predictors that indicate pubertal growth have such a large effect during puberty that they are significant in models predicting calcium retention, even in small metabolic studies. For example, postmenarcheal age was the best predictor in one study (explaining 10% of the variation) of calcium retention after the dietary intervention, calcium intake (15%), in adolescent white girls [109]. In a pooled analysis of metabolic studies in white and black adolescent girls, calcium intake explained 12.3% and race explained 13.7% of calcium retention [93]. An additional 3.9% was explained by postmenarcheal age. In 31 white boys aged 13e15 years, calcium intake (the intervention over the range of 670e2003 mg/d) predicted 21.7% of calcium retention and serum IGF-I concentration explained an additional 11.5% of calcium retention [22]. In these studies, postmenarcheal age and serum IGF-I were markers of pubertal growth and testosterone could nearly replace serum IFG-1 in boys. A number of parameters associated with pubertal growth are often highly correlated, such as serum IGF-I and Tanner score (r ¼ 0.50, P < 0.001) [22].
Vitamin D status has not been a significant predictor of calcium retention in any metabolic study of calcium retention in adolescents conducted to date [22,93,103], nor in pooled data representing 158 observations (Fig. 37.6) [69]. In these studies, the range of vitamin D concentrations spanned 29.5e79.5 nmol/L. In a 4-week intervention of 1000 IU cholecalciferol/d in adolescent girls with baseline serum 25(OH)D levels between 23.5e44.3 nmol/L, calcium retention was likewise unchanged [102].
VITAMIN D AND BONE Appropriately designed intervention trials aimed at improving bone mineral accrual and bone strength in adolescents have focused primarily on calcium supplementation and exercise [1]. Much less is known regarding the vitamin D inputs required to optimize bone mass during this period [44]. This section will summarize key vitamin D and bone correlational studies, prospective observations, and intervention trials conducted to date. Significant relationships between serum 25(OH)D concentrations and bone indices are mixed, with some studies showing no relationship [91,92,110,111], some positive [70,80,91,107,112], and others negative [89,113]. There are several possible explanations for the discrepancies in these studies, including the range of serum 25(OH)D concentrations and the maturational status of the subjects, as well as differences in sample sizes and bone outcomes reported. For example,
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positive relationships were reported between 25(OH)D concentrations and spine, radius, and femoral neck bone mineral content (BMC) in early adolescent females with low mean baseline values of 35 nmol/L [80]. In contrast, no relationships were found between serum 25(OH)D values and total body and hip BMC, or bone structural parameters in late adolescents with higher mean 25(OH)D values of 75 nmol/L [111]. Using large sample sizes and wider ranges of 25(OH) D concentrations from the NHANES III dataset, Bischoff-Ferrari et al. [112] were able to detect significant associations between vitamin D status and aBMD in adults, but similar studies of this size are not available in children. Few studies have been conducted in adolescent populations examining vitamin D status and bone mineral accrual over time. In one such prospective study, female adolescents that had the greatest BMC gains at the spine, total hip and radius over a period of 1e7 years, were also in the lowest quartiles for plasma 25(OH)D [113]. Similarly, inverse relationships were reported between baseline serum 25(OH)D and 2-year changes in total body BMC [90] or 1-year changes in BMC of the spine (r ¼ e0.20), femoral neck (r ¼ e0.16), and radius (r ¼ e0.17) following supplementation [82]. These negative correlations indicate that subjects with low baseline 25(OH)D values respond to supplementation to a greater extent than those subjects with higher baseline 25(OH)D values. On the contrary, a 3-year prospective study in older, peripubertal Finnish girls, aged 9e15 years, showed that baseline (wintertime) serum 25(OH)D concentrations were significantly correlated with the 3-year change in lumbar spine and femoral neck aBMD (r ¼ 0.35, P < 0.001 and r ¼ 0.32, P < 0.001, respectively) [108]. For females in this study with advanced sexual maturation, the mean 3-year change from baseline lumbar spine aBMD was 26% higher in the highest tertile of serum 25(OH)D (at baseline), compared to the lowest tertile. Unfortunately, BMC, the most appropriate outcome variable for the assessment of growing bone [114], was not reported in this study. In both studies showing inverse relationships [80], mean calcium intakes were approximately 600e900 mg/day, lower than consumed by the Finnish adolescents who had calcium intakes greater than 1300 mg/day [10]. Though serum iPTH levels were not reported in these studies, knowledge of iPTH in the face of varying calcium intakes may provide greater insight with respect to the 25(OH)D and bone relationships in adolescents. As Stoffman and Gordon [115] indicated in a recent review, iPTH may be more important than vitamin D with respect to bone turnover and bone mineral accrual in adolescents. Findings from vitamin D intervention trials in adolescents are also inconsistent with respect to
skeletal outcomes [80,82,116]. For example, Cheng et al. [116] conducted a 2-year randomized, double-blind intervention trial in 10e12-year-old prepubertal females (Tanner stages 1 to 2) and randomized subjects into one of four groups, receiving daily: (1) 1000 mg calcium carbonate þ 200 IU vitamin D3, (2) 1000 mg calcium carbonate þ vitamin D3 placebo, (3) 1000 mg calcium from supplemented dairy products, or (4) calcium and vitamin D3 placebos. Having mean baseline 25(OH)D concentrations of approximately 46 nmol/L, subjects in the group supplemented with 1000 mg calcium from dairy products had greater increases in cortical thickness of the tibia measured by pQCT than the other three groups. In this study, vitamin-D3-calcium supplementation with 200 IU had no significant impact on bone mineral accrual. Viljakainen et al. [80] conducted a 12-month randomized, double-blind, placebo-controlled trial in Finnish girls, mean age 11.4 years and mean baseline 25(OH)D concentrations of 34 nmol/L, and found that vitamin D3 supplementation with either 200 or 400 IU improved femur BMC, and only the 400 IU dose produced an increase in spine BMC [80]. Since these positive bone responses to vitamin D supplementation were observed in the compliancebased analyses and not the intent-to-treat analyses, it is probable that the doses used in the above trials may have not been large enough to elicit positive BMC responses. Larger vitamin D doses than used in the aforementioned studies were administered to 10e17-year-old Lebanese females in a 1-year, double-blind, placebocontrolled vitamin D supplementation trial conducted by El-Hajj Fuleihan et al. [82]. The subjects in this study, having mean 25(OH)D concentrations of 35 nmol/L, were randomized into three groups: 1400 IU/week (~200 IU/day), 14 000 IU/week (~2000 IU/day), or placebo. For the entire study population, both vitamin D doses were associated with greater total hip BMC and fat-free mass gains compared to placebo in a dosedependent manner. For premenarcheal girls only, the 200 IU dose vs. placebo led to a significant increase in trochanter BMC and a marginally significant (P ¼ 0.08) increase in total hip BMC. For postmenarcheal girls, there were no significant group differences in changes in lean mass, aBMD, or BMC measures with any of the doses. Collectively, these intervention trials described above raise several issues with respect to skeletal responses to varying vitamin D inputs. Two of the trials showed positive responses with 200 IU, one basing their results from a complianceebased analysis, and both conducted among participants with low circulating baseline 25 (OH)D concentrations. Moreover, the osteogenic responses to such interventions appear to be more robust during the pre- vs. the late-pubertal years with
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respect to trochanter or total hip BMC [82], but greater at the lumbar spine in late-puberty [80]. Only one of the trials studied males aged 10e17 years, and no significant responses were observed [82]. Whether vitamin D supplementation improves bone mineral gains among adolescents without specific health-related conditions, in both males and females, and among other ethnic/ racial groups, is unknown. Furthermore, the dose(s) required among these groups to elicit changes in bone mineral is also unknown. Some of the inconsistencies observed in the aforementioned intervention trials may also be partially explained by geneeenvironment interactions [117e121]. Several investigators have reported significant associations between vitamin D receptor (VDR) polymorphisms and bone density [118,119,121], bone turnover [119], and calcium absorption [121]. To date, one vitamin D intervention trial has been conducted assessing the VDR genotype in adolescents [117]. In this study, it was reported that the VDR genotype influenced the skeletal response to 1 year of vitamin D supplementation in young females from Lebanon [117]. When participants from both vitamin D supplementation groups (1400 IU/week or 14 000 IU/week) were combined, the Bsm1 and Taq1 restriction enzymes were associated with greater BMC gains at most bone sites in the collective vitamin-D-supplemented groups, but not the placebo group, whereas the BB and tt genotypes demonstrated the least BMC gains. To help better ascertain the mechanisms of this functional effect, and in an effort to detect geneeenvironment interactions and potential interactions with other relevant gene polymorphisms, assessment of VDR polymorphisms should be included in future vitamin D intervention trials.
VITAMIN D AND MUSCLE The robust relationship between muscle strength/ size and bone strength in the growing skeleton has been well documented [122e125]. Emerging evidence from cross-sectional, prospective, and intervention trials in adolescents suggests a link between serum 25(OH)D concentrations and fat-free soft-tissue mass and muscle strength, leading to important ramifications with this line of inquiry. In addition to potentially impacting bone mineral accrual through the traditional calcium absorption mechanism, it may be that vitamin D directly affects muscle strength during pubertal growth, providing an alternative pathway by which vitamin D may strengthen bone. In the vitamin D supplementation study in Lebanese adolescent females using the equivalent of 200 and 2000 IU/day doses [82], a significant dose-dependent increase in fat-free soft-tissue mass was observed.
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When change in fat-free soft-tissue mass was used as a covariate in the regression model, the strength of the vitamin D effect on changes in BMC or on aBMD was reduced. Similar findings were reported by Willis et al. [71] who found that the age-related declines in serum 25(OH)D among US females were eliminated when correcting for changes in fat-free soft tissue. While the authors in the El-Hajj Fuleihan et al. [82] study did not observe improvements in hand-grip strength measures in adolescents supplemented with vitamin D, adolescent girls with adequate vitamin D status (>50 nmol/L) in the Foo et al. [107] study had greater forearm muscle strength and total body and forearm BMC, compared to those with inadequate serum concentrations of 25 (OH)D (<50 nmol/L). Further supporting these findings, Ward et al. [127] reported significant positive relationships between 25(OH)D concentrations and jump velocity, jump height, power, and force. Whereas, Gilsanz et al. [128] suggest that vitamin D insufficiency (72.5 nmol/L) may be associated with fatty infiltration of muscle in late adolescents, the possibility exists that fatty infiltration of muscle could lead to both reduced muscle strength and bone mass. The exact mechanisms by which vitamin D acts on muscle cells and influences muscle function are unknown, though the process is likely mediated through both genomic and nongenomic effects [129].
SEX AND RACIAL DIFFERENCES Calcium Metabolism Sex and race differences in calcium retention and metabolism during adolescence account for much of the differences in adult bone mass. Sex differences in skeletal calcium accretion during puberty illustrated in Figure 37.1 could either occur by higher calcium intakes and overall greater nutrient intakes, or by greater calcium utilization efficiency in boys compared to girls. When comparing boys and girls in metabolic balance studies using a range in calcium intakes of 700 to 2100 mg/d, boys were more efficient in skeletal calcium accretion than girls over the whole range of intakes by 17138 mg/d calcium retention [130]. In a similar study, black girls were shown to be more efficient than white girls in calcium retention across a wide range of calcium intakes [131]. Black vs. white girls on the same calcium intake, were shown to have increased calcium absorption, decreased urinary excretion, and greater bone formation rates [104]. Interestingly, Asian girls were shown to have even higher calcium absorption efficiency than black and white girls, especially at lower calcium intakes [105]. Does vitamin D status or supplementation play a role in modifying calcium metabolism
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during puberty beyond sex and racial differences? Race, but not vitamin D status, influenced net calcium absorption and retention in adolescent girls as shown in Figure 37.6. As shown in the figure, black girls were more efficient at absorbing and utilizing calcium than white girls despite having lower serum 25(OH)D levels (25.7 7.8 vs. 33.2 10.4 ng/ml). Even higher calcium absorption and utilization was observed in studies investigating Asian girls with even lower serum 25(OH)D levels (19.1 5.9 ng/ml) [102] or 13.7 4.4 ng/ml [101]. Doseeresponse vitamin D studies in adolescents of both sexes in various racial groups, with a range of vitamin D status to determine subpopulations that may be responsive to vitamin D interventions during the important period of pubertal growth. With current evidence, it would appear that vitamin D status in healthy adolescents plays little role in calcium metabolism.
Skeletal Accretion The prevalence of osteoporotic fractures differs by race and sex, where white and female groups characteristically experience more fractures than black and male groups, respectively [132]. Much of the research into sex and race differences in bone mass has focused on childhood and adolescence in an effort to verify if the divergence in bone masses among the race and sex groups occurs early in life and if these differences track into adulthood. This may be especially relevant considering the recent childhood bone study in 6e16-year-olds suggesting that a high percentage of individuals with either low or high BMC/aBMD Z-scores (at the total body, lumbar spine, total hip, and femoral neck), maintain their respective Z-score ranking 3 years later [133]. These data suggest that if the BMC/aBMD Z-score tracking persists into adulthood, without any interventions, those individuals with low Z-scores during adolescence may eventually become those that suffer skeletal fractures as adults. By and large, black adolescents have higher BMC or aBMD for most bone sites than their white counterparts [12,69,73,134]. Most of the racial differences in BMC velocity during growth are explained by changes in height [135]. Since two-dimensional DXA-derived measures of aBMD do not take into account differences in bone size and blacks have larger bones, the higher aBMD values of blacks vs. whites is misleading. That is, when aBMD is adjusted for differences in bone or body size (e.g., height), the higher aBMD values observed in blacks vs. whites are, for the most part, eliminated. If aBMD or BMC measures are likely not different between blacks and whites when corrected by bone or
body size as described above, differences in the threedimensional structural properties of bone among races and sexes more than likely contribute to the disparate fracture rates observed in adulthood. When examining the geometrical properties of bone with either CT or pQCT, and taking into account bone and body size variations, differences are indeed detected by race and sex. For example, in boys and girls aged 8e18 years, black children had higher vertebral cancellous bone density than white children, specifically by late maturity [136]. Femur cross-sectional area, but not cortical area, was also greater in these children, even when taking into consideration the significantly longer femurs in blacks vs. whites. Finally, Leonard et al. [136] showed that all tibia cortical bone measures, corrected for tibia length, including BMC, BMD, periosteal and endosteal circumferences, and section modulus, were greater in blacks vs. whites [137]. Unlike the Gilsanz et al. [136] study, the differences in vertebral cancellous BMD were less apparent by Tanner stage 5 in this study [137]. Wetzsteon et al. [138] showed in younger adolescents (9e12 years of age) that both blacks and Hispanics have greater radius and tibia vBMD, cortical area and bone strength than whites, after correcting for age, sex as well as bone and muscle size. Similarly, Pollock et al. [139] found in late adolescents that when correcting for muscle cross-sectional area and limb length, blacks vs. whites had greater trabecular and cortical bone strength at the tibia, but not the radius. From the existing data, it appears as though the greater bone strength in blacks vs. whites is apparent in the cortical bone measures by the prepubertal years, and later maturity for the axial skeleton, and this greater bone strength persists into adulthood. The mechanisms underlying the race differences in bone geometry are still unknown. During the pubertal years, males accrue more bone mineral than females, but like the skeletal differences observed between blacks and whites, the greater bone size in males accounts for the majority of the BMC differences. In early pubertal children (mean age ~11.5 years), Kontulainen et al. [140] observed a greater increase (10%) in total bone cross-sectional area and cortical area of the tibial midshaft over 20 months, in boys vs. girls, but the relative increase in cortical area was similar in both groups. Sex differences in bone are maturation-dependent. For example, in early puberty there are no sex differences in femoral shaft bone, yet by post-puberty, males have greater cortical dimensions, muscle area, and bone strength than females [141]. However, when correcting for muscle size in this study, females have greater cortical area and total bone cross-section than males. In sizematched late adolescent, military cadets, males had significantly greater BMC and aBMD of the femoral neck, trochanter, femoral shaft and total hip, but not
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the lumbar spine [142]. Cortical BMC and the cortical dimensions of the tibia are also greater in males than females, except cortical BMD which is higher in females, depending on Tanner stage [137]. These differences in cortical dimensions probably confer greater bone strength to males.
VITAMIN D AND DIABETES Evidence is accumulating on the possible role of vitamin D in the pathogenesis of type 2 diabetes in adolescents. Low circulating levels of vitamin D are associated with higher rates of type 2 diabetes in children, and this could pose a significant health risk worldwide, as an increasing number of adolescents are being diagnosed with type 2 diabetes or prediabetes. The majority of studies in this area have been cross-sectional with the focus on serum 25(OH)D, fasting glucose and insulin concentrations, and estimates of insulin sensitivity. Inverse associations have been reported between 25(OH)D levels and fasting plasma glucose (FPG) in adolescents, 12e19 years of age, from 2001e2004 NHANES [143] and in pediatric outpatients, 2e18 years of age (r ¼ e0.20, P < 0.001) [144]. In the NHANES study [143], adolescents in the lowest (<37.4 nmol/L) versus highest (>65 nmol/L) quartile of serum 25(OH)D had an adjusted odds ratio for fasting hyperglycemia of 2.54 (95% CI: 1.01e6.40), independent of adiposity. In another study [145], obese children and adolescents with 25(OH)D concentrations <75 nmol/L had higher BMI (P < 0.001), fat mass (p < 0.0001), and a lower quantitative insulin sensitivity check index (QUICKI) score (P < 0.005). Additionally, 25(OH)D was inversely correlated with HbA1c (r ¼ e0.23, P < 0.01) [145]. In obese African-American adolescent females, Ashraf et al. [146] found significantly lower markers of insulin sensitivity and significantly higher insulin area under the curve in participants with 25(OH)D concentrations 37 nmol/L, suggesting a potential threshold for negative effects of vitamin D insufficiency on insulin sensitivity. In order to determine if vitamin D indeed has a role in glucose homeostasis and the inputs needed to ameliorate hyperglycemia and insulin insensitivity, randomized, double-blind placebo controlled vitamin D intervention trials are needed in both type 2 and prediabetic adolescents.
VITAMIN D, ASTHMA, AND INFLUENZA Asthma is a disease characterized by airway hyperreactivity to stimuli, repeated episodes of reversible airways obstruction, and an inflammatory process; often mediated by an adaptive helper T cell type 2 (Th 2)
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immune response. The potent immunomodulatory properties of vitamin D have led to interest in a possible relationship between vitamin D status and asthma, though vitamin D has variable effects on human Th 2 response [147]. One study of 616 Costa Rican asthmatic children aged 6e14 years related serum 25(OH)D levels of deficient (<20 ng/ml, 3.4%), insufficient (20e30 ng/ ml, 28%), and sufficient (>30 ng/mL, 68.6%) to markers of allergy and asthma severity [148]. Higher concentration of serum 25(OH)D was associated with reduced odds of hospitalization and use of anti-inflammatory mediators, and increased metacholine airway responsiveness in the previous year. Lower vitamin D levels were associated with elevated total IgE and eosinophil counts. Similarly, in urban black children aged 6e20 years, 93 patients with asthma had lower serum 25 (OH)D levels than 21 control subjects without asthma (18.5 ng/ml vs. 40.4 ng/ml, P ¼ 0.002) [149]. Further research is needed to determine if vitamin D supplementation influences the incidence of asthma. Only one randomized, placebo-controlled trial has been conducted to date linking vitamin D to the incidence of influenza. In this study 1200 IU vitamin D3/d was administered to 334 school children aged 6e15 years with unmeasured baseline vitamin D status during the winter months in Japan [150]. Results of this study showed that only 10.8% of the children in the vitamin D supplementation group acquired influenza A compared with 18.6% in the control group, a significant (P ¼ 0.04) difference, but no difference in influenza B incidence was observed. The authors proposed that vitamin D supplementation enhanced innate immunity by up-regulating antimicrobial peptides or regulating cytokine release. Interestingly, the children with asthma did not respond to vitamin D3 supplementation with reduced incidence of influenza A. However, asthma attacks were reduced (P ¼ 0.006) in asthmatic children who were supplemented with vitamin D3.
VITAMIN D REQUIREMENTS IN ADOLESCENTS A few research groups have tried to set vitamin D requirements for adolescents. The World Health Organization recommends 200 IU/d for adolescents aged 10e18 years [151]. In 2008, the American Academy of Pediatrics raised their recommendations from 200 to 400 IU/d [152]. An EAR of 400 IU/d and RDA of 600 IU/d were set for adolescents in North America in 2010 [153]. Estimated mean vitamin D intakes of food plus dietary supplements for US adolescents aged 9e18 years are about 200 IU/d which do not meet the RDA [153].
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For vitamin D, the strategy for setting requirements is to determine the vitamin D status, i.e., the serum 25(OH) D levels associated with optimal health. Then, intakes to achieve these levels are determined for the individual or populations. The World Health Organization specifies that plasma 25(OH)D levels of 27 nmol/L are necessary to ensure normal bone health [151]. Serum 25(OH)D levels >50 nmol/L have been recommended as a cutoff for vitamin D sufficiency to ensure adequate calcium absorption and bone mineralization and to prevent increases in alkaline phosphatase [152]. The recommended intakes of vitamin D by the American Academy of Pediatrics and Canadian Pediatric Association increased to 400 IU to maintain levels >50 nmol/L were primarily based on adult data [155]. None of these current recommendations are based on true functional outcome measures associated with improved health in children. Limited data are available in children to determine the levels of vitamin D input to achieve a target serum 25(OH)D. From the four available vitamin D intervention studies in white adolescents that report changes in serum 25(OH)D over 1 month or more, the change in serum 25(OH)D with vitamin D supplementation can be predicted by the equation for a linear relationship (R2 ¼ 0.94, P < 0.001) as: DSerum 25ðOHÞD ðng=mlÞ ¼ 0:01130 x þ 1:13747 where x ¼ vitamin D (IU/d) supplementation. What is not known is if the relationship is similar for other races or for vitamin D input over 2000 IU/d. The equation predicts that vitamin D supplementation of 1000 IU/ d would lead to an increase in serum 25(OH)D of 12.4 ng/ml. It also predicts that for an individual to increase their serum 25(OH)D concentrations from 25 ng/ml (overt deficiency) to 50 ng/ml (borderline adequate), they should supplement their diet with at least 2000 IU/d. Public health recommendations for vitamin D assume no input from sunlight to cover the many individuals who do not receive sufficient ultraviolet-B (UVB) exposure or are unable to synthesize 1,25(OH)2D subcutaneously. The vitamin D recommendation for an individual largely depends on their vitamin D status at baseline. Generalized recommendations for the public are based on national survey data for serum 25(OH)D levels of various subpopulations [70]. Consideration of individuals at both ends of the range of 25(OH)D in the population for vitamin D recommendations is important to prevent insufficiencies at the low end and for safety purposes at the high end. Very little safety data exist for children, especially long term. The longest study at the highest dose was 14 000 IU/week (the equivalent of 2000 IU/d) in pubertal girls for 1 year [82], and this dose appeared to be well tolerated.
SUMMARY AND CONCLUSIONS Adolescence represents a unique stage of the lifecycle as evidenced by rapid bone growth, in terms of both size and structural properties. Race and sex differences in skeletal maturation have been identified, with skeletal size explaining much, but not all of the differences. Regulators of genetically programmed body size dominate skeletal expansion during this life stage. Modifiable lifestyle factors play important roles in determination of peak bone mass, with calcium intake being the strongest predictor of skeletal calcium accretion in healthy adolescents. Much less is known regarding the role of vitamin D with respect to bone mineral augmentation. Circulating levels of 25(OH)D vary depending on many factors including age, maturation, race, latitude, season, and body composition. The assumption that intermediate (i.e., biochemical) endpoints of vitamin D and bone metabolism respond to vitamin D supplementation similarly to that of adults is premature. Unlike adults, vitamin D status over a wide range of concentrations is not associated with calcium absorption or retention in adolescents. Moreover, an inverse relationship between serum 25(OH)D and iPTH has been reported, but suppressed iPTH may be undesirable during puberty when high rates of modeling are necessary to build and shape bone. Thus, criteria established for adults defining vitamin D deficiency, insufficiency, and sufficiency cannot be extrapolated to adolescents without further investigation. Few randomized controlled trials of vitamin D have been conducted in adolescents, though the trials published to date indicate that populations with low baseline serum 25(OH)D benefit most from vitamin D supplementation as evidenced by increased bone mineral accrual. Most of the evidence is from Caucasian females, with little known regarding males and other races. There is increasing interest in the possibility that vitamin D supplementation may impact adolescent bone by improving muscle strength. Dosee response studies are needed in adolescents to ascertain the benefits and safety of vitamin D with respect to skeletal and non-skeletal outcomes, such as immune function and risk of diabetes and obesity. Pubertal growth places adolescents into a unique category as growth dominates over the role of the skeleton in mineral homeostasis that is of primary concern in adults. Therefore, the appropriate outcome measures in adolescents likely vary from adults. Carefully planned and executed studies specifically in adolescents are necessary before serum 25(OH)D cutoff levels and vitamin D intake requirements can be appropriately determined.
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C H A P T E R
38 Vitamin D Metabolism in Pregnancy and Lactation Natalie W. Thiex 1, Heidi J. Kalkwarf 2, Bonny L. Specker 1 1
2
E.A. Martin Program in Human Nutrition, South Dakota State University, Brookings, SD, USA Division of General and Community Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
INTRODUCTION The primary role of vitamin D in pregnancy and lactation has historically been thought to be in the regulation of calcium metabolism of the mother and fetus. Significant changes in maternal calcium metabolism occur during pregnancy, lactation, and after weaning to provide the calcium needed for fetal bone mineral accretion, for the synthesis of breast milk, and for the restoration of the maternal skeleton. Multiple factors are involved in regulating these processes so that maternal blood calcium concentrations are maintained within a narrow range despite the large fluxes in calcium that occur. Primary strategies include changes in the efficiency of absorption of calcium from the intestinal tract, alterations in renal calcium reabsorption and thus urinary calcium excretion, and the flux of calcium in and out of maternal bone. Reproductive hormones have important effects on calcium homeostasis and bone metabolism, and during pregnancy and lactation their actions work in concert with the vitamin D endocrine system to ensure the calcium needs are met for fetal bone mineral accretion, for breast milk production, and to maintain circulating maternal calcium concentrations. Recently it has been suggested that low maternal vitamin D status during pregnancy may influence pregnancy outcomes and disease development and growth of the offspring later in childhood or adulthood.
ADAPTATIONS IN VITAMIN D AND CALCIUM METABOLISM Pregnancy Approximately 25e30 grams of calcium are transferred to the fetal skeleton by the end of pregnancy,
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10038-1
the majority of which is transferred during the last trimester. The fetus accumulates 2e3 mg/d during the first trimester, but 250 mg/d during the third trimester [1]. Maternal serum calcium concentrations decrease in the first half of pregnancy, reaching a nadir at mid-gestation due to plasma volume expansion and decreased albumin concentrations [2,3]. Serum concentrations of ionized calcium or calcium adjusted for albumin concentrations show less fluctuation and remain relatively stable throughout pregnancy [3,4]. Serum concentrations of intact parathyroid hormone (PTH) have been reported to decrease [3,5,6] or not change [2] over the course of pregnancy; whereas, serum parathyroidhormone-related peptide (PTHrP) concentrations increase during pregnancy [5]. Circulating concentrations of the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25-(OH)2D), are increased during pregnancy. By the second trimester serum concentrations of 1,25(OH)2D increase by 50e100% over prepregnant values, and in the third trimester they increase by 100% [2,7] (Fig. 38.1). The signal to increase 1,25-(OH)2D synthesis is not clear, as PTH concentrations are not elevated. Vitamin-D-binding protein concentrations increase in pregnancy possibly due to increased concentrations of estrogen [2,8]. Therefore, the increase in serum 1,25(OH)2D concentrations may be a response to the increase in vitamin-D-binding protein. However, the amount of free 1,25-(OH)2D is still elevated during pregnancy [2,8]. Most of the circulating 1,25-(OH)2D is thought to be of renal origin, but some may be of extrarenal origin as the placenta and decidua have been shown to synthesize 1,25-(OH)2D [9]. During pregnancy, high levels of CYP27B1 (1-a hydroxylase) and low levels of CYP24A1 (vitamin D 24-hydroxylase), gene expression are observed in the placenta [10e12]. Consistent with this, maternal 1,25-(OH)2D concentrations rapidly decrease
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38. VITAMIN D METABOLISM IN PREGNANCY AND LACTATION
180
intestinal calcium absorption
% Calcium Absorption
1,25-(OH)2D
65
140
50
100
35
60
20
1,25-(OH)2D (pmol/liter)
80
20 Prepregnant 1st trimester 2nd trimester 3rd trimester
FIGURE 38.1 Intestinal calcium absorption and serum 1,25-(OH)2D concentrations before and during pregnancy. Data from Ritchie et al. [2].
within a few days after delivery [13]. Fibroblast growth factor 23 (FGF-23) regulates renal expression of CYP27B1 and CYP24A1 [14]; however, no mention of FGF-23 effects on placental expression of these enzymes or of serum FGF-23 concentrations during pregnancy were found in the literature. The increase in 1,25-(OH)2D concentrations during pregnancy is accompanied by an increase in intestinal calcium absorption (Fig. 38.1). Fractional calcium absorption increases by 50e56% over prepregnant levels in the second trimester and by 54e62% in the third trimester [2,7]. Thus human data indicate increased maternal intestinal absorption of calcium is an important physiologic adaptation to secure sufficient amounts of this mineral for the fetus. However, increased intestinal calcium absorption in pregnant VDR-null mice suggests relative independence from vitamin D on intestinal calcium absorption during pregnancy [15,16]. Despite the increased need for calcium, urinary calcium excretion increases by 40e50% over the course of pregnancy. This is most likely due to the marked increase in glomerular filtration rate and increased absorptive load [2,3,17]. Several studies report increased concentrations of biochemical markers of bone turnover during pregnancy. Serum concentrations of biochemical markers of bone formation, namely bone specific alkaline phosphatase, and the pro-peptide of type 1 collagen (PICP), are elevated in the third trimester with a steep peak in the last month of pregnancy [3,6]. It is not clear whether there are changes in the first two trimesters of pregnancy as an increase, decrease, or no change in the concentration of these bone formation markers have been reported [3,6]. Osteocalcin concentrations have been found to decrease [6], decrease then increase [2], or not change during pregnancy [13]. Markers of bone resorption, namely the breakdown products of collagen such as pyridinoline, deoxypyridinoline, and cross-linked N-telopeptide of type I collagen (NTx), increase throughout pregnancy
reaching a peak at the end of pregnancy [2,3,6]. Although it appears that there is a dissociation of bone resorption and bone formation in the first two trimesters with elevated bone resorption predominating, it is difficult to predict whether there is a net loss of maternal bone during pregnancy using biochemical markers alone. Other factors confound the interpretation of biochemical markers of bone turnover during pregnancy such as whether they are of maternal, placental, or fetal origin, and the effects of pregnancy on the metabolic clearance of these proteins by the liver and kidney. It also has been suggested that some of the increase in bone turnover markers may be due to increased turnover of soft tissue collagen of the uterus and skin [6,18]. Increases in circulating concentrations of insulin-like growth factor-1 (IGF-1) and placental lactogen have been suggested as the possible mechanisms behind increased bone turnover during pregnancy [3,6,19]. Increases in IGF-1 concentrations precede the increase in bone formation markers, and IGF-1 concentrations correlated more strongly with markers of bone formation than bone resorption. Studies investigating changes in calcium-regulating hormones and osteoprotegerin (OPG) during pregnancy found that maternal serum OPG concentrations steadily increased with gestational age [13,20]. Receptor activator of nuclear factor-k B ligand (RANKL) is important in osteoclast differentiation [21], and OPG acts as a decoy receptor for RANKL thereby preventing the differentiation of osteoclast precursors into mature osteoclasts and decreasing bone resorption. Higher OPG concentrations during pregnancy, possibly of placental origin, might play a role in the control of bone metabolism throughout gestation. In summary, several adaptations in the maternal calcium economy occur in order to provide sufficient calcium for fetal bone mineral accretion during pregnancy (Fig. 38.2). The primary adaptive strategy is an increase in intestinal calcium absorption. Additional calcium may come from demineralization of maternal bone. The increased concentrations of 1,25-(OH)2D, estrogen, IGF-1, placental lactogen, and OPG interact to facilitate these changes. Despite the increased need for calcium during pregnancy, urinary calcium losses are increased due to increased glomerular filtration rate and an increased absorptive calcium load.
Lactation Lactating women secrete approximately 200e240 mg of calcium daily in breast milk [22]. Over 6 months of lactation this is equivalent to approximately 6% of the mother’s total skeletal calcium reserve. Despite this large transfer of calcium from the maternal circulation, maternal serum calcium concentrations are unchanged
V. HUMAN PHYSIOLOGY
ADAPTATIONS IN VITAMIN D AND CALCIUM METABOLISM
Dietary calcium
Intestinal calcium absorption
Fetus
Blood
Urinary calcium
Bone Adaptations in the calcium economy during pregnancy. Solid arrows indicate an increase with arrow thicknesses representing the magnitude of the fluxes.
FIGURE 38.2
[2,23] or slightly elevated [24,25]. There is no increase in PTH concentrations during lactation. In fact, serum PTH concentrations are lower in lactating as compared to nonlactating women in the first 3 months postpartum [23e26]. The lower PTH concentrations during lactation are likely to be a consequence of the rapid bone resorption that occurs especially early in lactation and the resultant increase in serum calcium concentrations. Two potential causes of bone resorption are hypoestrogenemia and elevated circulating concentrations of parathyroid-hormone-related peptide (PTHrP) [27e29]. Lactation results in prolonged postpartum amenorrhea and hypoestrogenemia due to suppression of the hypothalamicepituitaryegonadal axis. Hypoestrogenemia is known to result in bone resorption in a variety of clinical and experimental situations. PTHrP also stimulates bone resorption. PTHrP is made in the mammary gland and is present in very high concentrations in breast milk [30]. Presumably some of the PTHrP synthesized by the mammary gland may gain access to the maternal circulation. Circulating PTHrP has actions similar to PTH and acts through the PTH receptor [31]. PTHrP is a potent stimulator of bone resorption, and administration of PTHrP results in an immediate increase in serum calcium concentrations [32]. In lactating women, serum concentrations of calcium are more highly correlated with PTHrP than PTH [28], suggesting that the decrease in PTH may be secondary to elevated PTHrP concentrations and subsequently increased serum calcium concentrations. Whether there is a decrease in urinary calcium excretion during lactation is not clear. Some studies have found a 20e50% decrease in urinary calcium excretion in lactating women [2,25,33e36]. However, some of this decrease may be a postpartum phenomenon and not just a result of lactation. Studies that compared
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urinary calcium excretion in lactating women to that of nonlactating postpartum controls have not found urinary calcium excretion to be lower in lactating women [23,26,37,38]. Unlike during pregnancy, there is no increase in circulating concentrations of 1,25-(OH)2D in lactating compared to nonlactating postpartum women [23,24,37]. Commensurate with this finding is that there is no difference in intestinal calcium absorption in lactating compared to nonlactating women [2,35,39,40]. Intestinal calcium absorption is increased in lactating rats that are suckling multiple pups, but this does not occur in women nursing one infant. Greer et al. found that women nursing twins had elevated concentrations of PTH and 1,25-(OH)2D [41]. Urinary calcium excretion and intestinal calcium absorption were not measured, but presumably elevations in PTH and 1,25-(OH)2D concentrations resulted in changes in urinary calcium excretion and intestinal calcium absorption. Adolescents with a low calcium intake also may respond to lactation differently than lactating adult women with low calcium intakes. Bezerra et al. found that serum PTH was higher and urinary calcium was lower among both nonlactating and lactating adolescents compared to adults [42]. There was a significant increase in urinary deoxypyridinoline concentrations in lactating adult women, which was less pronounced in lactating adolescents, and the authors suggested that the effect of lactation on bone turnover is different in adults vs. adolescent women on low calcium intakes. Although bone demineralization, and not improved efficiency of intestinal calcium absorption, is the primary compensatory response to secure calcium in lactating women, it can be hypothesized that increased absorption efficiency may occur in situations of greater calcium demand such as women nursing multiple infants or in lactating adolescents, who may still be accreting bone. A recent study comparing lactating women to nonlactating postpartum women and healthy controls reported increases in a marker of bone resorption, serum C-telopeptide of type I collagen, and two bone formation markers (amino-terminal telopeptides of procollagen 1, osteocalcin, and bonespecific alkaline phosphatase). These results indicate that bone turnover remains coupled in lactation unlike other situations of rapid bone loss where marked uncoupling occurs [43]. In summary, the primary adaptive strategy to obtain calcium to support breast milk production is demineralization of maternal bone (Fig. 38.3; see below). Lactation also may result in urinary calcium conservation, although the results from studies are conflicting as to whether this is a lactation effect or a postpartum effect. These changes appear to be unrelated to vitamin D status.
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but the smaller sample size in that study may have limited their ability to detect a small increase [2].
Dietary calcium
Intestinal calcium absorption
CHANGES IN BONE MINERAL CONTENT AND DENSITY Pregnancy
Blood
Milk
Urinary calcium
Bone
FIGURE 38.3 Adaptations in the calcium economy during lactation. Solid arrows indicate an increase with arrow thicknesses representing the magnitude of the fluxes. Dashed arrows indicate a decrease.
Weaning Additional adjustments in calcium metabolism occur shortly after lactation has stopped as the maternal physiologic system switches from mobilizing calcium from the skeleton and secreting calcium into breast milk to restoring her calcium reserves (Fig. 38.4). Some studies have found an increase in serum concentrations of PTH shortly after weaning and decreased urinary calcium excretion, presumably in response to increased PTH concentrations [7,24,25]. However, these changes have not been found in all studies [23]. Kalkwarf et al. found that serum concentrations of 1,25-(OH)2D were higher in women shortly after weaning [39], this was accompanied by a higher intestinal calcium absorption (37% vs. 31%). Ritchie et al. did not find a significant increase in intestinal calcium absorption after weaning,
Dietary calcium
Intestinal calcium absorption
Blood
Urinary calcium
Bone Adaptations in the calcium economy after weaning. Solid arrows indicate an increase with arrow thicknesses representing the magnitude of the fluxes. Dashed arrows indicate a decrease.
FIGURE 38.4
Few studies have included bone mineral density measurements during pregnancy because of the potential risks to the fetus associated with radiation exposure. There is conflicting evidence as to whether there is a net change in bone density during pregnancy. Several different approaches have been used to evaluate the impact of pregnancy on maternal bone. Some longitudinal studies have measured bone density by dualenergy X-ray absorptiometry (DXA) before conception and shortly after delivery and have found no significant loss of bone density [2,7,44,45]. However, other studies report losses of 2e2.6% at the ultradistal radius [46,47], 2e4% at the spine [3,6,48], and 2.4e3.6% at the hip [3,49]. In some of these studies bone density was measured as far as 6 weeks’ postpartum, and significant losses of bone may occur within the immediate postpartum period thereby making it hard to interpret these results. A recent study measured women prepregnancy and 2 weeks’ postpartum and found significant decreases in size-adjusted BMC of the total body, spine, and hip [50]. Naylor and coworkers found that the changes in bone density during pregnancy varied according to skeletal site. Bone density at trabecular rich sites (pelvis and spine) decreased by 3e4% whereas bone density at cortical sites (arms and legs) increased by 2% [6]. A prospective study by Promislow et al. [51] obtained DXA measurements of the forearm at 16 and 36 weeks’ gestation and concluded that trabecular, but not cortical, bone was lost during pregnancy with 1.9% decrease in ultra-distal radius BMD. The loss was higher in women prescribed bedrest than those not. Many investigators have measured changes in bone density by use of ultrasound as it does not involve radiation exposure. Speed of sound (SOS) and bone ultrasound attenuation (BUA) are strongly correlated with bone density measured at the same skeletal site. Longitudinal studies over the course of pregnancy have documented a decrease in SOS and BUA measured at the os calcis or phalanges in the latter half of pregnancy, particularly in the third trimester [52e56]. These data are consistent with a loss of maternal bone mineral toward the end of pregnancy when the fetus is accreting bone mineral most rapidly. Longitudinal studies of bone density in women who do not lactate after delivery show that bone density at the spine and hip increases by about 2% in the first
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8 6 Percent Change
year postpartum [57e59]. It is possible that this increase in bone may compensate for bone lost during pregnancy. If so, this may explain why studies comparing bone density of women with different pregnancy histories have found no differences in bone density measured many years later [60e64]. Even grand multiparous women have not been found to have lower bone density later in life when compared to nulliparous women [64,65].
Nonlactating Groups
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Lactation
0.5
3
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Months Since Delivery
One of the primary changes in calcium homeostasis during lactation is the marked decrease in bone mineral content and density. Decreases of 3e9% at the lumbar spine and femoral neck have been reported [2,22,26,47,57,58,66e68]. The decreases in bone density of the spine and hip occur rapidly within the first 3e6 months of lactation, and bone density remains lower with continued lactation [69,70]. The rate of bone loss at these sites in the first 6 months of lactation is significant as it approaches 1% per month. In comparison, menopausal and early postmenopausal women lose bone at the rate of 1e2% per year [71]. The amount of bone lost during lactation is variable among women. Women who breastfeed longer, or who have a greater breast milk volume, have greater bone loss compared to women who breastfeed for shorter periods of time [22,47,69,72]. In addition, the length of postpartum amenorrhea is an important determinant of bone loss during lactation. Women who resume menses early have less bone loss than women who have longer periods of postpartum amenorrhea [47,58,70]. Although the length of postpartum amenorrhea and length of lactation are related, some women resume menses while still breastfeeding. Kalkwarf et al. found that the net change in bone density at the lumbar spine at 6 months’ postpartum was only e1.8% in women who had resumed menses whereas it was e4.4% in women who had not resumed menses despite the fact that both groups were breastfeeding five times a day [73]. Polatti et al. also found less of a deficit in bone density in lactating women who resumed menses by 5 months’ postpartum as compared to those who remained amenorrheaic (e3.0% vs. e5.8%) [58]. These findings underscore the importance of ovarian hormones in regulating bone loss during lactation. Dietary calcium intake does not appear to affect the amount of bone lost during lactation in women. Bone loss during lactation has been observed in women with high calcium intakes (>1500 mg/d) [26,47,69], and dietary calcium intake has not been shown to be a significant predictor of bone loss during lactation [22,37,69,72]. Furthermore, three randomized calcium supplementation trials have demonstrated that
Effects of calcium supplementation and lactation on the mean (SE) percent change in the bone mineral density of the lumbar spine during the first 6 months’ postpartum. Values are adjusted for baseline bone mineral density, height, weight, change in weight, dietary intake of calcium, and level of physical activity. P ¼ 0.01 for effect of calcium, P < 0.001 for the effect of lactation; and P ¼ 0.23 for the interaction between calcium supplementation and lactation. Reproduced from Kalkwarf et al. [57].
FIGURE 38.5
provision of supplemental calcium does not affect bone loss during lactation. Prentice et al. studied 60 lactating women in the Gambia who had a very low calcium intake (274 mg/d). Half of the women received an average of 714 mg of supplemental calcium per day, and half received a placebo. Overall there was a significant loss (1.1%) of bone mineral at the radial shaft by 13 weeks’ postpartum, but there was no difference in the amount of bone lost between supplemented and unsupplemented lactating women [33]. Kalkwarf et al. randomized 83 lactating women and 81 nonlactating postpartum women whose dietary calcium intake averaged 735 mg/d, to receive a calcium supplement (1 g/d) or placebo for 6 months. There was a small effect (þ1.2%) of calcium supplementation on bone density of the lumbar spine when considering all women [57]. However, bone loss did not differ between lactating women who received the calcium supplement and those who received placebo (4.2% vs. 4.9%) (Fig. 38.5). Similar results were observed in a supplementation trial conducted in 274 Italian women [58]. The primary adaptive strategy to obtain calcium to support breast milk production is demineralization of maternal bone (Fig. 38.3). This bone loss during lactation is related to postpartum amenorrhea and hypoestrogenemia and appears unrelated to vitamin D status. Changes that occur during lactation are mirrored during weaning.
Weaning Maternal bone density increases rapidly after weaning. Much of the bone density lost during lactation is recovered within the first 6 months after weaning.
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Laskey and coworkers demonstrated that the resumption of menses was as good a predictor of bone changes as was the length of lactation [70]. Increases in bone density after weaning occur earlier for the spine than for the femoral neck [47,67,69,70], which may be a consequence of the greater amount of trabecular bone at the spine. Although most studies show a complete recovery of bone density at the spine after weaning, it is not clear that the recovery of bone at the femoral neck is complete, as deficits in bone density at this site were still evident at the end of the study follow-up in most of these studies [47,67,70]. It is possible that a complete recovery of bone may have occurred with a longer follow-up period. Kalkwarf et al. conducted a calcium supplementation trial in 76 women who had lactated for 6 months and then weaned their infants, and 82 nonlactating postpartum controls to determine whether provision of supplemental calcium could enhance bone recovery after weaning [57]. By 6 months after the initiation of weaning (4 months after complete weaning), lactating women who had received 1 g/d of supplemental calcium had a significantly greater increase in spinal BMD compared to women who received the placebo (5.9% vs. 4.4%) (Fig. 38.6). The restoration of bone mass after lactation has ceased is important in maintaining maternal bone health. Bone mass and density increase in parallel with the return of menstruation and presumably normalization of circulating estrogen concentrations. The recovery of bone mass may be facilitated by an increase in intestinal calcium absorption efficiency and a decrease in urinary calcium excretion, but these alterations have Lactating Groups
8
Percent Change
6
Calcium
Weaning
4
Placebo
2
Calcium Placebo
0 Nonlactating Groups
-2 -4 -6
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9 Months Since Delivery
12
Effects of calcium supplementation and lactation on the mean (SE) percent change in the bone mineral density of the lumbar spine during the second 6 months’ postpartum. Values are adjusted for baseline bone mineral density, height, weight, change in weight, dietary intake of calcium, and level of physical activity. P < 0.001 for the effect of calcium; P < 0.001 for the effect of weaning and P ¼ 0.036 for the interaction between calcium supplementation and weaning. The lactating women were fully breast-feeding at baseline, and the arrow indicates the average time at which breastfeeding completely ended. Reproduced from Kalkwarf et al. [57].
FIGURE 38.6
not been found consistently across studies and whether vitamin D status of the mother is a significant determinant of the magnitude of the bone recovery is not known.
Long-term Effect on Maternal Bone Health Studies on the long-term effect of pregnancy and lactation on bone density are conflicting and may depend on calcium status, dietary calcium intake, or general nutrition of the population being studied. Some report a higher density later in life with increased parity and lactation [60,74e76], while other studies find a lower density [77,78], or no association [65,79]. Numerous studies have investigated whether parity or lactation are associated with hip fracture risk. The results are conflicting, but usually report either no association [80e83] or a reduced fracture risk with increasing parity [84e87]. In general, the larger caseecontrol and longitudinal studies find a protective effect, with hip fracture risk reduced by 9e10% per child [84,85]. The reduced fracture risk has been found to be independent of bone density [88], suggesting that some other factor other than density is responsible for the reduced risk. A study of 168 women with 0e16 children found an increase in periosteal diameter at the radius and femoral neck bone area with increasing parity, without any difference in areal or volumetric measures on bone density [64]. The larger bone size with higher parity could explain the decreased fracture risk that has been reported in grand multiparous women. The reason for the increased bone size is not known, but the authors speculated that the changes in weight that occur during pregnancy would place additional loads on the maternal skeleton, resulting in increased periosteal expansion. Another possible explanation for the larger bone size with greater parity could be the changes in estrogen concentrations with pregnancy and lactation. Estrogen has been postulated to have a negative effect on periosteal bone formation rates [89] and most of the women in this study breast-fed their infants and were therefore relatively estrogen-deficient while lactating. The combination of both increased bone loading and relatively low estrogen concentrations may explain the increase in bone area in these women.
LOW MATERNAL VITAMIN D DURING PREGNANCY Maternal vitamin D deficiency is more likely to occur in winter months, in countries that do not routinely fortify dairy or other food products with vitamin D, among members of ethnic groups who cover most of their skin, or among individuals with heavily
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Effects on the Mother Evidence is accumulating to suggest that maternal vitamin D deficiency during pregnancy also may be related to nonskeletal outcomes, in particular the development of preecalmpsia. Oken and coworkers did not find an association between dietary intake of vitamin D during pregnancy and preeclampsia in a small cohort of 1718 women who completed a food frequency questionnaire during the first trimester of pregnancy [90]. However, a larger study of 23 423 Norwegian women completed by Haugen and coworkers reported a reduced odds of preeclampsia among nulliparous pregnant women who had higher total vitamin D intake or vitamin D intake from supplements [91]. A study by Bodnar et al. [92] reported lower mean serum 25(OH) D concentrations before 22 weeks’ gestation in women who later developed preeclampsia compared to those who did not, even after adjusting for potential covariates. Mean serum concentrations of 1,25-(OH)2D in maternal and umbilical cord at the time of delivery have been found to be lower in preeclamptic women compared with normotensive women [93], suggesting a complex role between calcium and vitamin D metabolism and the risk of preeclampsia.
Effects on the Neonate Maternal vitamin D deficiency during pregnancy can affect neonatal calcium metabolism. Vitamin D deficiency is associated with secondary hyperparathyroidism and osteomalacia in the mother. Maternal hyperparathyroidism during pregnancy may lead to neonatal hypocalcemia or tetany [94,95]. In the early 1970s, Purvis and coworkers noted that the occurrence of neonatal tetany among 112 infants was inversely related to the amount of sunlight exposure the mothers had during the last trimester of pregnancy [96]. The authors speculated that the mothers developed hyperparathyroidism secondary to vitamin D deficiency leading to a transitory hypoparathyroidism and hypocalcemia in the neonate. Several investigators subsequently reported that infants of mothers with low
vitamin D intake during pregnancy had low serum calcium concentrations in cord blood or during the first week of life [97e99]. Several randomized trials of vitamin D supplementation during pregnancy were later reported [100e106]. Cockburn and coworkers randomized two obstetric wards, one with 506 women who received 400 IU vitamin D/d from the 12th week of gestation and another with 633 women who did not receive vitamin D [100]. They reported higher maternal, cord and infant 25-hydroxyvitamin D (25-OH-D) concentrations with vitamin D supplementation. They also found that the incidence of neonatal hypocalcemia was less with vitamin D supplementation, although this was modified by the infant’s feeding (hypocalcemia greater with formula feeding vs. breast-feeding). Incidence of hypocalcemia on day 6 in formula-fed and breast-fed infants is shown in Figure 38.7. Several randomized trials of vitamin D supplementation (1000 IU/d) during pregnancy subsequently found that infants of mothers receiving vitamin D had higher serum calcium concentrations within the first week of life than infants of mothers receiving placebo [105]. Brooke and coworkers conducted a randomized, double-blind trial of vitamin D supplementation (1000 IU/d from 28e32 weeks’ gestation) and found that infants of mothers receiving vitamin D had higher serum calcium on days 3 and 6 and a lower incidence of symptomatic hypocalcemia than infants of mothers receiving placebo [102,103]. These studies were completed in populations at increased risk for vitamin D deficiency, and show the importance of maternal vitamin D status on neonatal calcium homeostasis.
18 Incidence of Hypocalcemia (%)
pigmented skin. Few randomized nutritional vitamin D and calcium interventions have been conducted during pregnancy, and the importance of maternal vitamin D intake is best illustrated from observational studies of women with poor vitamin D and/or calcium intake. These observational studies, along with the results of the few clinical trials that have been conducted, indicate that maternal vitamin D and calcium status are important not only for the mother, but also in the neonatal handling of calcium, and possibly in fetal growth and bone maturation and mineralization.
16
Control Vitamin D
14 12 10 8 6 4 2 0 Human Milk
Formula
Incidence of hypocalcemia on day 6 by type of feeding (hatched bars are formula-fed; open bars are human-milk-fed) in infants whose mothers received either no vitamin D (control) or 400 IU vitamin D/d from 12th week of gestation (vitamin D). Data from Cockburn et al. [100].
FIGURE 38.7
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Maternal vitamin D deficiency during pregnancy also may lead to impaired fetal growth and bone development. The occurrence of vitamin D deficiency is high among Asians from the Indian subcontinent living in Great Britain. A trial of vitamin D supplementation (1000 IU/d) among pregnant Asian women found that a higher percent of the infants randomized to the placebo group (28.6%) were small-for-gestational-age (SGA) compared to infants in the supplemented group (15.3%) [105], but a later report found no difference in birth weight or length [107]. Some investigators [101,104], but not all [103,107], have reported lower birth weights of infants born to mothers with low vs. adequate vitamin D status. A recent study in the Netherlands among a large multiethnic cohort of 3730 women reported a higher SGA risk and lower birth weight in women with 25(OH)D concentrations less than 30 nmol/L during early pregnancy (median 13 weeks’ gestation) [108]. A study in the US reported an association with both low (<37.5 nmol/L) and high (>75 nmol/L) 25(OH)D concentrations prior to 22 weeks’ gestation and the incidence of small-for-gestational age (SGA) in a nested caseecontrol study of 77 white mothers [109] (Fig. 38.8). Although this relationship was not observed among 34 black mothers of SGA infants [109], the trend was similar. It also is important to note that the quartile definitions were defined within each race and cut-offs for serum 25(OH)D in the upper quartile among white women was almost twice that obtained for black women. Decreased skeletal mineralization in utero may be manifested as rickets or osteopenia in the newborn 4.5
Adjusted Odds Ratio for SGA
4.0
White Black
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
Quartile 1
Quartile 2
Quartile 3
Quartile 4
Serum 25-OHD at <22 Weeks Gestation
FIGURE 38.8 Odds for SGA (small-for-gestational-age) was
increased in both the low and high quartiles of serum 25(OH)D concentrations prior to week 22 of gestation. Although this was not significant for black women, the cut-offs for defining the quartiles were significantly lower in black women compared to white women. Quartiles 1 to 4 were defined as serum 25(OH)D concentrations of 21e58, 58.1e71.4, 71.5e90.6, and 90.7e245 nmol/L for white women and 13.8e30, 30.1e38.8, 40.4e49.3, and 49.4e137.2 nmol/L for black women. Data taken from Bodnar et al. [109].
infant. However, fetal or congenital rickets of the newborn are rare, although a recent report found greater splaying of the fetal distal tibia among women who were vitamin-D-deficient in the third trimester compared to women who were vitamin-D-sufficient [110]. Case reports of congenital rickets in newborn infants of mothers with severe nutritional osteomalacia associated with vitamin D or calcium deficiency have been reported [111e113]. Reif and coworkers, in a caseecontrol study, reported an association between craniotabes, or delayed ossification of the cranial vertex, and maternal and neonatal 25(OH)D concentrations [114]. However, these findings have not been replicated in other observational studies or trials [98,105]. Although Brooke and coworkers did not find an association between craniotabes and vitamin D status, they did find that infants of mothers who received placebo had larger fontanelles than infants of mothers supplemented with vitamin D, which is consistent with impaired skull ossification [105]. A study conducted in China also found possible evidence for a relationship between maternal vitamin D deficiency and impaired fetal bone ossification [115]. The presence of wrist ossification centers in neonates was associated with cord serum 25(OH)D concentrations. A higher rate of ossification centers in newborn infants of mothers with adequate vitamin D status was apparent when compared to infants of mothers with low vitamin D status. Few studies have investigated the role of maternal vitamin D status on infant bone mineralization. One of the original studies conducted investigating this relationship was one by Congdon et al., who measured forearm BMC using single-photon absorptiometry and found that BMC did not differ by history of vitamin D supplementation during pregnancy and was not correlated with cord serum 25(OH)D concentrations [98]. The majority of the vitamin D supplementation trials reported to date began supplements late in gestation. There are observational studies suggesting that maternal vitamin D status early in gestation may be important in fetal bone development. Seasonal differences in adult bone density have been reported by some [116,117], but not all investigators [118], and may be attributed to seasonal variations in vitamin D status. Studies that have examined seasonal differences in newborn BMC have had conflicting findings. Two studies conducted in the US found that infants born in the summer have lower BMC compared to infants born in the winter months [119,120]. However, Namgung et al. examined this association in infants born in Korea and found that summer-born infants had higher BMC than winter-born infants [121]. The authors speculated that one explanation for these contradictory findings is that many US women take prenatal vitamins containing vitamin D beginning in the second trimester of
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pregnancy. Thus the observed seasonal effects on infant BMC in the US may reflect vitamin D status in the first trimester of pregnancy. Because there is minimal fetal calcium accretion in the first trimester, these findings would indicate some other function of vitamin D on fetal bone development. More recent studies have supported the findings of Namgung et al. A study from Finland measured 25(OH)D concentrations in the 125 mothers during their first trimester and during the postpartum period, as well as measuring bone parameters in the newborn by both DXA and pQCT [122]. They found that mothers with mean 25(OH)D concentrations below 43 nmol/L had infants with lower total body bone mass and smaller cross-sectional area of the tibia than infants of mothers with mean 25(OH)D concentrations during pregnancy that were greater than 43 nmol/L. In summary, maternal vitamin D status during pregnancy has been shown to be associated with neonatal calcium homeostasis. There are conflicting reports indicating a possible role of maternal vitamin D status on fetal growth and bone development.
Effects on the Child Later in Life Childhood Growth and Bone Mass The study by Brooke et al., which did not find differences in birth weight or length in a vitamin D supplementation trial (1000 IU/d) among pregnant Asian-Indians in Great Britain, reported significantly greater weight and length during the first year of life among the infants of vitamin-D-supplemented mothers [107]. Weight was greater at 3, 6, 9, and 12 months of age, while length was greater at 9 and 12 months in infants of mothers who received the vitamin D supplement. The recent study of the Amsterdam cohort found that although birth weight was lower in mothers who were vitamin-D-deficient during the first trimester of pregnancy, these infants showed accelerated growth in both weight and length during the first year of life [108]. The effect of maternal vitamin D status during pregnancy on bone mass of the child later in life has been investigated in several longitudinal studies. Javaid et al. [123] reported the findings from a cohort study of 198 children aged 9 years of age whose mothers had serum 25(OH)D concentrations measured during the third trimester. Total body bone mass at 9 years was significantly correlated with maternal 25(OH)D concentrations during the third trimester. Milk intake and activity levels of the child were not associated with the child’s bone mass, but it is possible that other confounders could have explained the observed relationship: for example, a healthier lifestyle or greater outdoor activities could explain both a higher maternal 25(OH)D and a higher BMC in the child.
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Results from the Avon Longitudinal Study of Parents and Children (ALSPAC) also had interesting findings. The ALSPAC is a longitudinal study of 6995 children who were followed from pregnancy until 9 years of age. Some of the mothers (N ¼ 355) had 25(OH)D concentrations measured in the third trimester. The authors estimated UVB exposure of the mothers from meterological records and these UVB estimates were found to correlate with the mothers’ 25(OH)D concentrations. They then estimated UVB exposure during the third trimester for the population and found that it correlated with birth length, as well as height, weight, and bone and lean mass at 9 years of age. When potential confounders were included in the analysis the only significant finding was a correlation between estimated UVB exposure and bone area. When they examined bone mass in relation to maternal 25(OH)D concentrations in the subgroup with these measurements, no association was observed. Asthma Vitamin D has a number of immunological effects and may play a role in preventing autoimmune diseases (see Chapter 103). Observational studies on the relationship between maternal vitamin D during pregnancy and later risk of wheeze or asthma in the children are conflicting. The relationship between asthma risk later in childhood and maternal dietary vitamin D intake was observed in a study of 1669 children, but no relationship was observed if vitamin D from supplements was included [124]. Camargo et al. found an association between maternal vitamin D intake and asthma risk in 1194 children, which was not modified by maternal BMI [125]. Since mothers with high BMI should have lower serum 25(OH)D concentrations, it seems that the relationship between vitamin D intake and asthma risk would have been stronger in leaner mothers than more obese mothers, but this was not observed. A study of 1212 children found that maternal vitamin D intake during pregnancy was associated with self report of wheeze at 5 years of age in the offspring, but maternal vitamin D intake was not associated with asthma or spirometry [126]. To complicate these findings, a recent report by Gale et al. found that higher maternal 25(OH) D concentrations measured during pregnancy were associated with an increased risk of visible eczema and asthma in the children at 9 years of age [127]. Recall bias and confounding may be part of the explanation of these conflicting results that are likely to be answered only through randomized vitamin D supplementation trials. Diabetes Research in animals [128,129] and epidemiological evidence in humans suggests that low vitamin D levels
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may be related to development of type 1 diabetes (see Chapter 94). There is a geographical gradient of type 1 diabetes with greater incidence rates in regions with low UBV radiation (high latitudes, adjusted for cloud cover), while incidence rates approached zero in regions with high UVB exposure [130]. Furthermore, low serum concentrations of 25(OH)D are found in individuals at the time of type 1 diabetes diagnosis [131], and a metaanalysis of a small number of observational studies demonstrated that vitamin D supplementation during early childhood was associated with reduced risk of developing type 1 diabetes [132]. It also may be possible that maternal vitamin D status could influence the offspring’s risk of diabetes. Research detecting the presence of islet autoantibodies, precursors of clinical disease, in cord blood of children who later develop type 1 diabetes [133] suggests that autoimmunity predictive of development of clinical disease has origins in utero. In the USA, children born in the spring when maternal vitamin D levels are expected to be at their nadir have an increased risk of developing type 1 diabetes [134]. A caseecontrol study conducted by Stene et al. demonstrated that recall of cod liver oil intake during pregnancy, but not total vitamin D intake, was associated with a lower risk of type 1 diabetes in the offspring [135]. However, a follow-up study by the same research group with a larger cohort found no clear association between maternal intake of cod liver oil or other vitamin D supplements during pregnancy and diabetes in their offspring [136]. In the longitudinal DAISY (Diabetes Autoimmunity Study in the Young) study in the young cohort, in which children were recruited at birth, retrospective assessment of maternal dietary intake of vitamin D from food obtained by food frequency questionnaire during the third trimester of pregnancy reported an association between increased dietary vitamin D intake and decreased risk of islet autoimmunity in offspring [137]. In the ABIS (All Babies in Southeast Sweden) study, maternal vitamin D supplementation during pregnancy also was associated with reduced markers of diabetes-related autoimmunity (islet antigen-2 and glutamic acid decarboxylase autoantibodies) in offspring at 1 year of age (adjusted OR ¼ 0.7, P ¼ 0.03), but not at 2.5 years [138]. These results indicate that vitamin D status during pregnancy may play a role in autoimmunity of the child. Inconsistent findings among the epidemiological studies may be a result of the multifactorial nature of diabetes and possible geneeenvironment interactions. There are SNP mutations in genes associated with vitamin D metabolism (e.g., 9CYP27B1 in the vitamin D-1-hydroxylase gene) that are also implicated in the development of type 1 diabetes [139].
EFFECT OF LOW MATERNAL VITAMIN D DURING LACTATION ON THE INFANT Vitamin D deficiency leads to rickets in children. Infant formula is routinely fortified with vitamin D, but very low vitamin D concentrations have been found in human milk [140]. Infant serum 25(OH)D is correlated with maternal vitamin D status early in the neonatal period and is probably a result of placental vitamin D transfer and fetal stores. Beyond the neonatal period, the breast-fed infant serum 25(OH)D concentrations are neither correlated with breast milk vitamin D nor maternal serum 25(OH)D concentrations [141], and the infant is dependent upon endogenous synthesis or other dietary sources for vitamin D. Specker and coworkers found that exclusively breastfed infants residing in Cincinnati could maintain serum 25(OH)D concentrations above the lower limit of normal (11 ng/ml) with 2 hours of sunshine exposure per week if fully clothed except for the face [141]. The cutoff for defining low 25(OH)D was based on the concentration at which nutritional rickets had been observed at that point in time. Other factors such as latitude, season, weather conditions, and use of sunscreens may affect vitamin D status. Large seasonal differences in sunlight exposure and serum 25(OH)D concentrations over the first year of life have been observed in infants followed longitudinally [142]. These findings indicate that the infant’s sunlight exposure plays a more dominant role in determining his or her vitamin D status than the mother’s vitamin D status or milk vitamin D concentrations. Most reported cases of rickets have been of black infants, supporting the premise that individuals with dark skin have difficulty synthesizing adequate amounts of vitamin D due to the relative inability of sunlight to penetrate heavily pigmented skin [143]. In addition, the diet of mothers of rachitic infants appears to be low in vitamin D and the mothers may be vitamin-D-deficient themselves. Although some investigators have found breast milk vitamin D or 25(OH)D concentrations to be correlated with maternal intake of vitamin D, mothers who consume 600e700 IU vitamin D/d still have low concentrations of vitamin D in breast milk ranging from only 5 to 136 IU/L [144]. Investigators found that consumption of cod liver oil supplements among Icelandic women increased milk vitamin D concentrations, but the milk concentrations were still below the current Nordic recommendations [145]. In a series of vitamin D supplementation trials, Ala-Houhala and coworkers from Finland found that supplementing lactating mothers with up to 1000 IU vitamin D/d in northern latitudes during winter months increased maternal serum 25 (OH)D concentrations, but did not stabilize infant serum
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REFERENCES
FIGURE 38.9 Infant serum 25(OH)D concentrations after 20 weeks of either infant or maternal vitamin D supplementation. Data from Ala-Houhala et al. [146,147].
supplementation during pregnancy had adverse effects on the vasculature system compared to pups of mothers randomized to low vitamin D supplementation [152]; mean serum 25(OH)D concentrations in the high vitamin D group were 30 ng/ml, similar to what is observed in human populations. Few human studies have investigated cardiac outcomes of children in relation to the mother’s vitamin D status during pregnancy. A study by Gale and coworkers did not find an association between maternal 25(OH)D concentrations measured during the third trimester and measures of cardiac function in the offspring at age 9 years [127].
CONCLUSIONS 25(OH)D concentrations (Fig. 38.9) [146]. Maternal supplementation with 2000 IU/d, however, was found to normalize infant serum 25(OH)D concentrations [147]. There were no differences in infant serum calcium or alkaline phosphatase concentrations when mothers were supplemented with either 1000 or 2000 IU vitamin D/d or when infants were supplemented with 400 IU/d. A recent pilot study found that nine women who were supplemented with 6400 IU/d vitamin D3 had significantly greater increases in milk vitamin D activity than ten mothers who were supplemented with 400 IU/ d vitamin D3 [148]. The infants of women receiving 400 IU/d vitamin D3 were also receiving 300 IU/d of vitamin D3. Whether the women or infants who were supplemented with the higher dose of vitamin D have differences in functional outcomes compared to the women and infants receiving the lower dose are currently being investigated.
POTENTIAL RISK OF HIGH MATERNAL VITAMIN D DURING PREGNANCY During the 1960s large vessel calcification associated with infantile hypercalcemia was observed in the United Kingdom and was originally thought to be due to vitamin D fortification which had begun at this time [149,150]. It was later determined to be Williams syndrome and unrelated to the vitamin D fortification policy. In animal studies Norman et al. found that pups of rats supplemented with high vitamin D doses during pregnancy had a reduction in aortic elastin content, the number of elastic lamellae in the aorta, and force generation in aortic rings [151]. The humanequivalent dose of vitamin D in this study was much higher than what would be used in human populations, but a study by Toda et al. found that 6-week-old piglets of mothers randomized to high vitamin D
Multiple changes in the maternal calcium economy occur during pregnancy, lactation, and after weaning to protect maternal calcium concentrations while providing sufficient calcium for fetal bone mineral accretion, breast milk production, and maternal bone recovery. The strategies to secure calcium during these physiologic states differ. During pregnancy, the primary strategy to secure additional calcium is by increases in serum concentrations of 1,25-(OH)2D and intestinal calcium absorption. During lactation, maternal bone is demineralized, possibly due to the lactation-induced amenorrhea, in order to secure adequate availability of calcium for breast milk production. After weaning and the return of menses, maternal bone density increases. Lactation does not appear to have a long-term effect on maternal bone density nor does it increase osteoporotic fracture risk. The maternal calcium regulatory system is able to provide sufficient calcium to the fetus and for breast milk even when calcium intake is low. However, there is some evidence that neonatal calcium homeostasis and fetal bone mineral accretion may be compromised when maternal vitamin D status is low or calcium intake is below 600 mg/d. Whether vitamin D during pregnancy is associated with bone mass, asthma, or diabetes type 1 later in childhood requires further study.
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[143] P. Weisberg, K.S. Scanlon, R. Li, M.E. Cogswell, Nutritional rickets among children in the United States: review of cases reported between 1986 and 2003, Am. J. Clin. Nutr. 80 (2004) 1697Se1705S. [144] B.L. Specker, R.C. Tsang, B.W. Hollis, Effect of race and diet on human milk vitamin D and 25-hydroxyvitamin D, Am. J. Dis. Child 139 (1985) 1134e1137. [145] A.S. Olafsdottir, K.H. Wagner, I. Thorsdottir, I. Elmadfa, Fatsoluble vitamins in the maternal diet, influence of cod liver oil supplementation and impact of maternal diet on human milk composition, Ann. Nutr. Metab. 45 (2001) 265e272. [146] M. Ala-Houhala, 25-Hydroxyvitamin D levels during breastfeeding with or without maternal or infantile supplementation of vitamin D, J. Pediatr. Gastro. Nutr. 4 (1985) 220e226. [147] M. Ala-Houhala, T. Koskinen, A. Terho, T. Koivula, J. Visakorpi, Maternal compared with infant vitamin D supplementation, Arch. Dis. Child 61 (1986) 1159e1163.
[148] C. Wagner, T. Hulsey, D. Fanning, M. Ebeling, B. Hollis, High dose vitamin D3 supplementation in a cohort of breast-feeding mothers and their infants: a six-month follow-up pilot study, Breastfeeding Med. 1 (2006) 59e70. [149] A.U. Anita, H.E. Wiltse, R. Rowe, E.L. Pitt, S. Levin, O.E. Ottesen, et al., Pathogenesis of the supravalvular aortic stenosis syndrome, J. Pediatr. 71 (1967) 431e441. [150] H.B. Taussig, Possible injury to the cardiovascular system from vitamin D, Ann. Intern. Med. 65 (1966) 1195e1200. [151] P. Norman, I. Moss, M. Sian, M. Gosling, J. Powell, Maternal and postnatal vitamin D ingestion influences rat aortic structure, function and elastin content, Cardiovasc. Res. 55 (2002) 369e374. [152] T. Toda, Y. Toda, F.A. Kummerow, Coronary arterial lesions in piglets from sows fed moderate excesses of vitamin D, Tohoku. J. Exp. Med. 145 (1985) 303e310.
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C H A P T E R
39 Vitamin D: Relevance in Reproductive Biology and Pathophysiological Implications in Reproductive Dysfunction Lubna Pal, Hugh S. Taylor Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA
INTRODUCTION Resurgence in the appreciation of facilitatory roles for vitamin D and calcium in a myriad of biological processes has been witnessed in recent years [1 and references therein]. While amidst a burgeoning pandemic of vitamin D deficiency, beneficial roles for vitamin D are emerging in a spectrum of pathological processes ranging from autoimmunity, cardiovascular disease, and diabetes to carcinogenesis [1e8]. Indeed, critical roles for vitamin D and its active metabolites are being defined in processes regulating cellular growth and differentiation, and in the metabolic modulations specifically involving insulin action. Amongst the many physiological processes impacted upon, critical roles for the vitamin D hormone system in reproductive physiology have been previously suggested, albeit mostly in experimental and in vitro models [9e13]. Experiments investigating the significance of vitamin D for fertility and reproductive capacity, while demonstrating that the vitamin may not be essential for minimal female reproduction, do demonstrate compromised mating behavior, reduced fertility rates, decreased litter sizes, and impaired neonatal growth in 25(OH)D-deficient animals. Conversely, improved reproductive success with strategies involving vitamin D supplementation and/or calcium are also described, underscoring relevance of vitamin D and calcium homeostasis in reproductive biology [14e19]. Human data in this context, while sparse, similarly suggest relevance of vitamin D in reproductive biology. We provide an overview of our current understanding of the relevance of vitamin D to reproductive physiology. The first section provides a summary of
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10039-3
observations accrued in experimental models whereas the second section focuses on data accrued in humans. As will become apparent to the reader, the available data do implicate vitamin D as a key molecule in reproductive success across species and suggest pathophysiological mechanisms for reproductive compromise in the setting of vitamin D deficiency. This latter appreciation is especially relevant, given the pandemic of vitamin D inadequacy; aggressive repletion strategies may offer a cost-effective, safe, and easily accessible option that offers a potential for optimizing reproductive success.
PROCREATIVE RELEVANCE e ANIMAL MODELS The significance of vitamin D in reproduction is well studied in rodent models. While able to reproduce, vitamin D (25(OH)D)-deficient rats demonstrate diminished mating success and fertility capacity [11e13]. Reduced litter sizes, by as much as 40% are described in vitamin-D-deficient and hypocalcemic animals [12]. Impaired neonatal growth is additionally described when pups are nursed by a vitamin-D-deficient dam [13]. An overall reduction in fertility by as much as 75% is suggested in vitamin-D-deficient animals, attributed to a combination of decreased mating rates, diminished embryo implantation, and increased pregnancy complications. Conversely, treatment of the uterus with 1,25(OH)D induces decidualization and leads to improved reproductive success. Production of 1,25(OH)D and expression of vitamin D receptor (VDR) are well described in female reproductive
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39. VITAMIN D: RELEVANCE IN REPRODUCTIVE BIOLOGY AND PATHOPHYSIOLOGICAL IMPLICATIONS IN REPRODUCTIVE DYSFUNCTION
organs in the rat including uterus, oviduct, ovary, mammary gland, placenta, the pituitary gland, and hypothalamus [20] and in the hamster ovarian cell line [21]. Experimental studies with VDR-null mouse models demonstrate evidence of gonadal insufficiency, reduced aromatase gene expression, and reduced aromatase enzyme activity (both compromising the ovarian steroidogenic potential), impaired folliculogenesis and infertility. Further, features of hypergonadotropic hypogonadism [22] and end-organ sequelae of estrogen deficiency such as bone malformations and uterine hypoplasia are manifest as a result of impaired vitamin D signaling [23]. Similarly mice with a target mutation in the 25-hydroxyvitamin D-1a-hydroxylase (CYP27B1) gene also have small uteri and ovaries; these mice do not ovulate and are infertile due to uterine and ovarian defects [24]. Alterations in calciumephosphate metabolism are suggested to partly explain the reproductive sequelae of vitamin D deficiency [16e19]. In male rats, fertility is critically affected by calcium levels, independent of vitamin D and calcium has been shown to affect sperm maturation, capacitation and acrosome reaction [16]. In vitamin-D-deficient rats, normalization of reproductive capacity has been reported by feeding a high-calcium and -phosphate diet alone [19]. However others report disruptions in ovarian steroidogenesis, in uterine receptivity and in male reproductive physiology as direct sequelae of ineffective vitamin D signaling [14,15,22]. Consumption of a vitamin-D-deficient diet replete with calcium prior to and during pregnancy in rats still adversely affects fecundity rates [11]. Decreased aromatase activity and reduced aromatase gene expression in the ovary, testes, and epididymis of animals deficient in vitamin D are described, identifying its relevance in gonadal steroidogenesis [22]. Additionally, roles for 1,25(OH)D in placental steroidogenesis, calcium transport through the placenta, expression of placental lactogen, and decidualization of the endometrium are also suggested; vitamin D has been shown to promote calcium transport in the placenta, stimulate lactogen expression, and regulate decidualization of the endometrium [25e27]. The available data thus identify vitamin D as a key molecule in processes involved in reproductive success and the mechanistic roles for insufficient vitamin D stores in causing reproductive dysfunction appear well elucidated at least in rodent models.
VITAMIN D e RELEVANCE IN HUMAN REPRODUCTIVE PHYSIOLOGY As reflected by the finite number of studies discussed, it is apparent that the literature on this subject is in its
infancy; a need for future studies to better define the role of vitamin D in reproductive biology is clearly identified (Table 39.1).
Polycystic Ovary Syndrome Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age with estimated population prevalence of 3e11% ([28] and references therein). A spectrum of adverse health problems affect women with PCOS including: increased cardiovascular disease risk (CVD), type II diabetes (TII_DM), depression, menstrual irregularity, and sleep apnea among many others ([29e31] and references therein). Gestation-specific health concerns loom large for women with PCOS and include risks for hypertension, preeclampsia, gestational diabetes, and fetal growth abnormalities ([32] and references therein). The healthcare burden and the costs related to PCOS are thus excessive and underestimated. While hypothalamicepituitaryeovarian axis dysfunction has long been appreciated as contributing to the altered reproductive milieu of PCOS, insulin resistance, and the associated heightened systemic inflammation are now regarded as central to the pathophysiology of PCOS and its sequelae [33,34]. Indeed, facilitatory effects of insulin sensitizing interventions on reproductive, metabolic, and cardiovascular aberrations in women with PCOS underscore the importance of insulin resistance to the syndrome ([35,36] and references therein). This latter appreciation is especially relevant to any discussion on a potential role for vitamin D, as a large body of observational data suggest that vitamin D deficiency may be causative in insulin resistance and even contributory to a spectrum of disease processes that share insulin resistance as a common denominator, namely diabetes, cardiovascular disease, various autoimmune disorders (rheumatoid arthritis, lupus, IBD, multiple sclerosis), cancers, and obesity [1,3,5,6]. Of particular interest are observations that identify improved insulin sensitivity
TABLE 39.1
Evidence Supportive of Relevance of Vitamin D in Reproductive Biology
Parameter
Experimental models
Human studies
Folliculogenesis
þ
e
Spermatogenesis
þ
þ
Steroidogenesis
þ
e
Implantation
þ
þ
Relevance for pregnancy
þ
þ
Relevance for progeny
þ
þ
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and mitigation in metabolic burden with vitamin D supplementation [5,37]. Adequate dietary calcium and vitamin D intake has been shown to relate to reduced risk of subsequent diabetes and CVD [7,37]. Given the pivotal role of insulin resistance in the pathophysiology of PCOS, as currently understood, it is logical that one consider vitamin D insufficiency as an intermediary. Indeed, a relationship between vitamin D stores and PCOS has been assessed; vitamin D insufficiency is associated with many of the hormonal and metabolic features that are hallmarks of this entity. Table 39.2 provides a summary of the studies [38e47] that have explored vitamin D and calcium homeostasis in the context of PCOS. As is apparent as much from the brevity of this list, as by the sample size and spectrum of study designs, stringent and appropriately powered studies are much needed and will be critical in elucidating the relevance of sufficient vitamin D stores for optimal reproductive health in women with PCOS.
Premenstrual Syndrome Premenstrual syndrome (PMS) is a common gynecological entity that is characterized by cyclic occurrence of moderate to severe physical and affective symptoms that substantially interfere with normal life activities and interpersonal relationships. While as many as 85e90% of premenopausal women regularly experience some degree of symptoms before the onset of menses, approximately 8e20% experience symptoms of significant intensity and consistency to meet the clinical definition of PMS [48]. Cyclic modulations in ovarian hormones, specifically estrogen, are suggested to underlie the symptomatology of PMS and management strategies specifically target the menstrual-cycle-related hormonal undulations. Fluctuations in serum calcium, magnesium, and parathyroid levels are shown to accompany changes in estradiol across the different phases of the menstrual cycle. Both vitamin D and calcium have been studied in the context of PMS, which is recognized as a state of relative calcium and vitamin D deficit. In an epidemiological study, dietary vitamin D and calcium intake were observed to inversely relate to PMS; women in the highest quintile of total vitamin D intake (median, 706 IU/d) had a 41% risk reduction (relative risk 0.59, 95% confidence interval, 0.40e0.86) compared with those in the lowest quintile (median, 112 IU/d) (P ¼ 0.01 for trend), compared with women with a low calcium intake (median, 529 mg/d), participants with the highest intake (median, 1283 mg/d) had a relative risk of 0.70 (95% confidence interval, 0.50e0.97) (P ¼ 0.02 for trend). The intake of skim or low-fat milk was additionally associated with a lower risk of PMS (P ¼ 0.001) [49]. Protective effects of calcium
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on PMS have since been confirmed in prospective trials [50e52].
Endometriosis This is a common gynecological disorder, endometriosis causes subfertility, pelvic pain, and leads to an overall reduced quality of life in reproductive-age women [53]. Recognized as an estrogen-mediated disorder, endometriosis is characterized by an overgrowth of endometrial cells and stroma outside of the uterine cavity; the female pelvis is the most common site of affliction, although endometriotic foci are described in remote bodily areas including lungs, nasal mucosa, and even the brain! Altered immune surveillance is entertained as a mechanism whereby the focal bodily defenses are overwhelmed, allowing an aberrant overgrowth of endometrial tissue at ectopic sites. Indeed, immune-stimulating drugs have been shown to prevent and treat endometriosis in animal models [54] identifying the immune system as critical in the pathogenesis of the disease. Inflammation, both localized and systemic, is a recognized accompaniment to endometriosis, as reflected by elevated levels of proinflammatory chemokines within the peritoneal fluid and serum of patients diagnosed with endometriosis [55e57]. Given its immunomodulatory properties, 25(OH)D insufficiency can be theorized to impart a predisposition to endometriosis, a disease that may be characterized as a manifestation of immune surveillance failure. Hartwell et al. [58] explored the relevance of vitamin D and its metabolites in the context of endometriosis in humans. In a sample of 42 women with endometriosis, the authors observed higher serum 1,25(OH)2D but comparable 25(OH)D levels in patients compared to controls. Since 1a-hydroxylase enzyme activity, involved in the conversion of 25(OH)D to its active metabolite 1,25(OH)2D, is expressed in activated macrophages, increased serum levels of the active metabolite in the setting of endometriosis can be rationalized by the increased number of these cells in the peritoneal fluid of women with endometriosis. Somigliana et al. [59] however report opposing findings; significantly higher serum 25(OH)D levels were observed in severe cases of endometriosis compared to the controls; women with serum 25(OH)D >28.2 ng/ml were almost five times more likely to have laparoscopically proven endometriosis compared to those with lower serum levels (odds ratio 4.8, 95% confidence interval 1.7e13.4). In keeping with findings of the earlier study however, these authors did observe an increase in serum levels of 1,25(OH)2D with increasing severity of disease, although the differences were not of statistical significance. The authors proposed that higher levels of 25 (OH)D may allow availability of substrate for the
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Author
Design
Population
Results
Thys-Jacobs [38]
Uncontrolled observational trial
13 overweight women with PCOS by NIH criteria (mean age 31.6 7.9). Supplementation with ergocalciferol (D2) 50 000 IU once or twice a week 6 months plus elemental Ca 1500 mg/day
Resumption of menstrual cyclicity in 7/9 with history of oligomenorrhea within 2 months of supplementation. 2/11 achieved spontaneous pregnancy
Panidis et al. [39]
Observational caseecontrol study.
291 women with PCOS by NIH criteria and 109 healthy and regularly menstruating controls
Significant inverse correlations observed between 25(OH)D levels and BMI, insulin, and PTH. PCOS cases demonstrated significantly elevated levels of PTH compared to controls and this relationship was independent of BMI. Significant correlation between PTH, testosterone, and prolactin
Hahn et al. [40]
Prospective observational study
120 predominantly overweight/obese women with PCOS diagnosed by NIH criteria
Almost 2/3 of the population was deficient in vitamin D (25 (OH)D <20 ng/ml). Significant inverse correlations noted between 25(OH)D, BMI, body fat, insulin, leptin and FAI. Positive correlations noted between 25(OH)D and HDL. Inverse correlations noted between 25(OH)D and hirsuitism score
Kosta et al. [41]
Uncontrolled single-arm trial. Supplement with 1 mg/day alphacalcidol for 3 months
15 obese women with PCOS (by NIH criteria)
Significant improvement in serum 25(OH)D and PTH and in first-phase insulin secretion. Significant improvements in lipid profile following 3 months supplementation
Mahmoudi et al. [42]
Prospective observational study, caseecontrol design
85 women with PCOS (NIH criteria), ages 20e40 and 115 regularly cycling controls of proven fertility
Overweight/obese PCOS cases demonstrated significantly lower serum 1,25[OH]2D. Insulin and HOMA indices were significantly and positively correlated with PTH and inversely correlated with phosphate levels. PCOS cases demonstrated significantly higher serum 25(OH)D, PTH, phosphate, insulin, and HOMA indices
Rashidi et al. [43]
Randomized placebo controlled trial
60 infertile women (mean age 26.16 þ/e 3.95) with PCOS (Rotterdam criteria)
Improved menstrual cyclicity and improved ovarian follicular parameters noted in the combination group following 3 months of intervention
39. VITAMIN D: RELEVANCE IN REPRODUCTIVE BIOLOGY AND PATHOPHYSIOLOGICAL IMPLICATIONS IN REPRODUCTIVE DYSFUNCTION
TABLE 39.2 Summary of Published Literature Relating Relevance of Vitamin D and Calcium Metabolism to PCOS
Cross-sectional study
206 women diagnosed with PCOS (Rotterdam criteria)
73% of the population was vitamin D deficient. Significant inverse correlations observed between 25(OH)D and anthropometric indices of obesity (BMI, waist circumference), blood pressure, glucose and insulin (fasting and OGTT provoked), and with indices of insulin resistance. Significantly lower 25(OH)D levels seen in women meeting criteria for metabolic syndrome
Selimoglu et al. [45]
Uncontrolled, single-arm trial. Single mega dose of D3 followed by metabolic and hormonal assessments 3 weeks later
11 overweight/obese women (BMI > 25 kg/m2) with PCOS (Rotterdam criteria)
82% of the enrolled participants demonstrated serum 25(OH) D < 20 ng/ml. Significant increases in serum 25OHD levels followed intervention. Statistically significant improvements were seen in insulin resistance within 3 weeks of supplementation
Yildizhan et al. [46]
Cross-sectional study
100 women diagnosed with PCOS (Rotterdam criteria)
67% of the population demonstrated serum 25(OH) D < 20 ng/ml. Inverse relationship was observed between serum 25(OH)D and BMI, measures of insulin resistance, androgens, and cholesterol
Mahmoudi T [47]
Prospective observational study, caseeontrol design. Genetic polymorphism in the vitamin D receptor is assessed and risk of PCOS, as it relates to the receptor genotype FokI, BsmI, ApaI and TaqI assessed
162 women diagnosed with PCOS by NIH criteria (ages 19e42 years) and 162 controls (ages 18e54)
Significantly higher 25(OH)D levels observed in cases with bb or TT genotype compared to BB/Bb or Tt/tt variants. Aa genotype significantly reduced likelihood for PCOS; an aa genotype increased the risk of PCOS diagnosis. Increased insulin resistance noted in those with the FF genotype for FokI compared to Ff and ff polymorphisms
SHBG: sex hormone binding globulin; FAI: free androgen index ¼ Total testosterone/SHBG * 100; OGTT: oral glucose tolerance test; IVGTT: intravenous glucose tolerance test; PTH: parathyroid hormone; HOMA: homeostasis model assessment ¼ insulin (mU/ml) * glucose (mg/dl/405); QUICKI: Quantitative insulin sensitivity check index 1/log(fasting insulin mU/mL) þ log(fasting glucose mg/dl); vitamin D2 ¼ ergocalceferol; vitamin D3 ¼ cholecalciferol; alfacalcidol¼ 1a OH calceferol.
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Wehr E et al. [44]
699
700
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1a-hydroxylase enzyme that brings about 1,25(OH)2Ddirected suppression of the local adaptive immune response [1] within the pelvic peritoneum, thus allowing a “permissive environment” for establishment of endometriosis. Indeed, 1a-hydroxylase enzyme activity has been described in the human endometrium, eutopic and ectopic and increased endometrial expression of the enzyme has been shown in endometrial biopsy specimens from women with endometriosis when compared to samples from controls ([60] and references therein), thereby suggesting a pathophysiological role of vitamin D and its metabolites in the establishment of endometriotic lesions. These latter conjectures however merit substantiation in larger population samples. Vitamin-D-binding protein (VDBP) is a major carrier protein for vitamin D and its metabolites in plasma, VDBP has also been recognized as a component of the actin scavenger system [61,62]. While predominantly a protein of hepatic origin, expression of VDBP has also been described in the mammalian uterus, amongst other tissues [61]. Beyond its role as simply a carrier protein, more recently it has been shown that DBP may modulate inflammatory and immunoregulatory activities including complement-mediated chemotaxis and macrophage activation [62e64]. A mechanistic role for VDBP in endometriosis, by controlling tissue access to the active metabolites of the vitamin, as well as by influencing the local immune surveillance mechanisms, thus seems plausible. Serum levels of VDBP are influenced by the estrogenic concentration [62], suggesting that tissue access to free, i.e. unbound, vitamin D metabolites as well as to the immune regulatory VDBP may be regulated by reproductive hormones. A relevance of VDBP in endometriosis can thus be hypothesized. Lower peritoneal fluid, but not systemic, levels of VDBP were described in women with endometriosis compared to controls [65,66]. While the pathophysiological significance of these observations remains unclear, conversion of VDBP to macrophage activating factor (MAF) at the site of inflammation is described [67], suggesting that VDBP conversion to MAF, and increased MAF production may contribute to the higher number of activated macrophages present in the peritoneal cavity of patients with endometriosis. Conversely, the increased peritoneal fluid level of VDBP described in users of combined oral hormonal contraceptives (OC) [65] is in line with the concept that VDBP may be of pathophysiological relevance given that OCs are used as a first-line strategy in the management of endometriosis. The efficacy of OCs in the treatment of endometriosis may result from VDBP alterations along with the direct effect of OCs on endometrial cell differentiation. As described above, 1,25(OH)2D has also been shown to as necessary for endometrial differentiation (decidualization) in response to progesterone. Vitamin D
deficiency may therefore prevent adequate decidualization of ectopic endometrium in response to endogenous progesterone or in response to attempts at therapeutic administration of progesterone. Progesterone resistance is a hallmark of endometriosis and vitamin D deficiency likely contributes to this pathway. 1,25(OH)2D has myriad effects on the endometrium. Vitamin D supplementation has yet to be tested as a treatment for endometriosis, however it presents a promising new therapy.
Male Reproduction While the relevance of vitamin D and its metabolites is well studied in the animal models, data are relatively sparse for humans. VDR has been described in the testis, the prostate, and in the spermatozoa of humans [68]. More recently, vitamin-D-metabolizing enzymes have been described in the human testis, ejaculatory tract, and in mature spermatozoa, suggesting that 1,25 (OH)2D is important for spermatogenesis and maturation of the human spermatozoa [69]. Cytoplasmic coexpression of VDR and the metabolizing enzymes in the testicular Leydig cells was also evident suggesting that vitamin D might affect male reproductive hormone production. The authors suggest that the concomitant expression of VDR and the vitamin-D-metabolizing enzymes in male germ cells and reproductive tract indicates that local activation of VD may be important for spermatogenesis and in sperm maturation.
Fertility Beyond its role in reproductive steroidogenesis, a physiological role for vitamin D and metabolites in modulating the endometrial expression of HOXA-10, an endometrial implantation marker that is essential for embryo implantation and fertility, has been suggested [70,71]. HOXA10 is essential for endometrial differentiation and receptivity to the blastocyst. VDR mRNA and protein expression were detected in primary uterine stromal cells as well as in human endometrial stroma cell lines; treatment of primary endometrial stromal cells with 1,25(OH)2D3 increased HOXA10 mRNA and protein expression, demonstrating regulation of HOXA10 by 1,25(OH)2D3, findings that hold direct implications for reproductive success. Further, HOXA10 has a functional vitamin D response element (VDRE) in its 50 regulatory region. The HOXA10 and the classic VDRE from the osteopontin gene share 60% nucleotide sequence conservation. This significant difference may reflect a distinct regulatory role for the HOXA10 VDRE in a tissue where it is not primarily involved in calcium metabolism. While HOXA10 expression is regulated by sex steroids as well as 1,25 (OH)2D, it is clear that optimal endometrial receptivity
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depends on robust expression of this gene. Diminished fertility may result from lack of 1,25(OH)2D stimulation of HOXA10 expression. Indeed, existing data do imply that outcome of infertility treatments may be influenced by an individual’s vitamin D status. In a cross-sectional study of infertile women (varying etiologies) undergoing IVF treatment, ovarian follicular fluid levels of 25(OH)D related to the likelihood of clinical pregnancy following fresh embryo transfer [72]. Significantly higher follicular fluid 25(OH) D levels were observed in those achieving clinical pregnancy (Fig. 39.1) and significantly higher implantation and clinical pregnancy rates were observed across tertiles of follicular fluid 25(OH)D (Fig. 39.2). The ovarian response to controlled ovarian hyperstimulation was comparable between patients achieving clinical pregnancy and those with failed IVF cycles, suggesting that uterine endometrium may be preferentially modulated towards enhanced receptivity in the setting of replete vitamin D stores, a conjecture that ties in to the earlier discussed facilitatory effects of vitamin D metabolites on endometrial expression of HOXA10.
Vitamin D and Pregnancy Recent years have witnessed a blossoming of literature tying maternal vitamin D deficiency to common pregnancy-related morbidities that collectively account for the bulk of maternal and infant mortality, namely
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preeclampsia, gestational diabetes, intrauterine growth restriction, and preterm labor ([73,74] and references therein). It is thus especially concerning that as many as 50% of US gravidae may be deficient in vitamin D [74e76]. Darker skin tone is a recognized risk factor for vitamin D insufficiency [74,75] and notable is the parallel between prevalence and magnitude of maternal vitamin D insufficiency and gestation-specific disorders that preferentially afflict minority populations. Almost 10% of pregnancies in the United States may be affected by hypertensive disorders and preeclampsia is a major contributor to maternal and neonatal morbidity and death [77]. Seasonal patterns in preeclampsia have been observed with higher incidence in winter months compared to summer, suggesting a role for sunlight and vitamin D in disease predilection [78,79]. Epidemiological studies have related dietary vitamin D and calcium intake, supplementation strategies and higher serum 25(OH)D levels to reduced risk for preeclampsia [80e83]. Abnormal angiogenesis, trophoblast invasion of the maternal decidua, defective immune adaptive responses early in pregnancy, inflammation and aberrant activation of the renin-angiotensin system are recognized contributors to pathogenesis of preeclampsia, and vitamin D is noted to modulate each of these parameters [84e87]. Mechanistic role for maternal 25(OH)D status in the causality of preeclampsia is thus emerging, as demonstrated by reduced incident risk for preeclampsia with
Relationship between 25OH D levels and outcome of IVF cycles
20
40
60
80
Clinical Pregnancy
0
Follicular Fluid Level of 25-OH Vitamin D (ng/ml)
Not Pregnant
p=0.013*
FIGURE 39.1 Relationship between 25(OH)D levels and outcome of IVF cycles. Adapted from [71], reprinted, with permission from Elsevier.
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39. VITAMIN D: RELEVANCE IN REPRODUCTIVE BIOLOGY AND PATHOPHYSIOLOGICAL IMPLICATIONS IN REPRODUCTIVE DYSFUNCTION
FIGURE 39.2 25(OH)D levels (ng/ml) by tertiles (mean þ/e SD). Lowest 16.74 þ/e 3.38; middle 25.58 þ/e 3.17; highest 43.01 þ/e 10.65. Adapted from [71], reprinted, with permission from Elsevier.
supplementation strategies [88]. Maternal vitamin D deficiency has additionally been suggested as a mechanism that may partly explain predisposition of African-American gravidae to preeclampsia [89]. Gestational diabetes mellitus (GDM), a common gestational morbidity, has been linked to maternal vitamin D insufficiency [90]. Affecting up to 7% of pregnancies, the incidence is higher in minorities. Health implications of GDM are far-reaching and extend well beyond the affected pregnancy; women developing GDM are at an enhanced lifetime risk for developing type II diabetes and CVD ([90] and references therein). GDM results from a failure of pancreatic beta cell insulin reserves to overcome insulin resistance that results from secretion of human placental lactogen in pregnancy; GDM is a recognized risk for subsequent development of type II diabetes. Given its insulin-sensitizing effects as well as direct effects on the pancreatic beta cells to facilitate insulin secretion, the active metabolite of vitamin D appears involved in the pathophysiology of GDM. While a larger body of literature supports a role for vitamin D insufficiency in pathogenesis of type I and type II diabetes [91,92], evidence linking vitamin D to GDM is additionally emerging. Zhang et al. [93] explored this relationship in a nested caseecontrol study of predominantly white gravidae. Evidence of vitamin D deficiency at 16 weeks’ gestation was an independent predictor of enhanced risk for GDM later in pregnancy. A fairly robust body of literature has explored the relationship between maternal 25(OH)D status with infant size and growth parameters; while a number of studies relate maternal vitamin D inadequacy to compromised fetal growth and lower birth weights,
the data are far from equivocal [94]. The relationship between growth parameters and maternal vitamin D stores appears nonlinear ([95] and references therein) and heterogeneity in the offspring’s VDR genotype may modify the relationship between maternal serum 25(OH)D levels and the baby’s growth profile. Inflammation is a recognized mechanism in the cascade of events leading to preterm birth, and hence consideration of vitamin D’s role in preventing preterm labor is a logical quest. Data relating vitamin D insufficiency to risk of preterm delivery in humans are however lacking. Seasonality in preterm births, i.e. lower incidences in summer and fall, has been suggested to reflect fluctuations in vitamin D stores (highest in the summer and fall, relating to lowering incidence of preterm birth) [96]. Reduction in preterm birth with active vitamin D supplementation has been recently reported in preliminary analyses from a randomized controlled trial of vitamin D in pregnant women, final results of which are keenly awaited [97; also see reference 89]. Last but not least is the relationship between maternal and infant’s vitamin D stores. Maternal 25(OH)D diffuses across the placental barrier and 25(OH)D levels in fetal cord blood are reportedly lower than concentrations in maternal blood [98]. In the first few weeks of life, the newborn is dependent on its own vitamin D stores, a resource that was in turn dependent on the adequacy of maternal 25(OH)D levels in the last few weeks of gestation. Exclusively breast-fed infants are at particular risk for vitamin D insufficiency in the early months of life given the poor concentration of vitamin D in breast milk [99]. Vitamin D insufficiency in the neonatal period
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is related to a spectrum of disorders ranging from neonatal seizures, infectious morbidities, childhood asthma, to long-term skeletal sequelae [100]; transgenerational ramifications of maternal vitamin D status can thus not be minimized! To summarize, vitamin D is identified as having a key role in reproductive physiology and in procreative successes. The vitamin D hormone system influences ovarian and placental steroidogenesis, endometrial receptivity in females, and spermatogenesis in males. Emerging data identify transgenerational implications of maternal vitamin D status that impact the progeny’s health and wellbeing. Vitamin D inadequacy is emerging as a modifiable risk for common population morbidities; aggressive repletion strategies may offer a cost-effective, safe, and easily accessible option for improving reproductive health.
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[69] M. Blomberg Jensen, J.E. Nielsen, A. Jørgensen, E. Rajpert-De Meyts, D.M. Kristensen, N. Jørgensen, et al., Vitamin D receptor and vitamin D metabolizing enzymes are expressed in the human male reproductive tract, Hum. Reprod. 25 (5) (2010 May) 1303e1311. Epub 2010 Feb 18. [70] H. Du, G.S. Daftary, S.I. Lalwani, H.S. Taylor, Direct regulation of HOXA10 by 1,25-(OH)2D3 in human myelomonocytic cells and human endometrial stromal cells, Mol. Endocrinol. 19 (9) (2005) 2222e2233. [71] G.S. Daftary, H.S. Taylor, Endocrine regulation of HOX genes, Endocr. Rev. 27 (2006) 331e355. [72] S. Ozkan, S. Jindal, K. Greenseid, J. Shu, G. Zeitlian, C. Hickmon, et al., Replete vitamin D stores predict reproductive success following in vitro fertilization, Fertil. Steril. (2009 Jul 7). [Epub ahead of print]. [73] M.L. Mulligan, S.K. Felton, A.E. Riek, C. Bernal-Mizrachi, Implications of vitamin D deficiency in pregnancy and lactation, Am. J. Obstet. Gynecol. (2009 Oct 19). [Epub ahead of print]. [74] L.M. Bodnar, H.N. Simhan, R.W. Powers, et al., High prevalence of vitamin D insufficiency in black and white pregnant women residing in the northern United States and their neonates, J. Nutr. 137 (2007) 447e452. [75] L.M. Bodnar, J.M. Catov, K.L. Wisner, M.A. Klebanoff, Racial and seasonal differences in 25-hydroxyvitamin D detected in maternal sera frozen for over 40 years, Br. J. Nutr. 101 (2) (2009) 278e284. Epub 2008 Apr 23. [76] J.M. Lee, et al., Vitamin D deficiency in a healthy group of mothers and newborn infants, Clin. Pediatr. (Phila) 46 (2007) 42e44. [77] A.P. MacKay, C.J. Berg, H.K. Atrash, Pregnancy-related mortality from preeclampsia and eclampsia, Obstet. Gynecol. 97 (2001) 533e538. [78] P. Magnus, A. Eskild, Seasonal variation in the occurrence of pre-eclampsia, BJOG 108 (2001) 1116e1119. [79] J.K. Phillips, I.M. Bernstein, J.A. Mongeon, et al., Seasonal variation in preeclampsia based on timing of conception, Obstet. Gynecol. 104 (5 pt. 1) (2004) 1015e1020. [80] H.S. Ros, S. Cnattingius, L. Lipworth, Comparison of risk factors for preeclampsia and gestational hypertension in a population-based cohort study, Am. J. Epidemiol. 147 (1998) 1062e1070. [81] People’s League of Health, The nutrition of expectant and nursing mothers in relation to maternal and infant mortality and morbidity, J. Obstet. Gynaecol. Br. Emp. 53 (1946) 498e509. [82] L.M. Bodnar, J.M. Catov, H.N. Simhan, M.F. Holick, R.W. Powers, J.M. Roberts, Maternal vitamin D deficiency increases the risk of preeclampsia, J. Clin. Endocrinol. Metab. 92 (2007) 3517e3522. [83] S.F. Olsen, N.J. Secher, A possible preventive effect of low dose fish oil on early delivery and pre-eclampsia: indications from a 50-year-old controlled trial, Br. J. Nutr. 64 (1990) 599e609. [84] K.N. Evans, J.N. Bulmer, M.D. Kilby, M. Hewison, Vitamin D and placental-decidual function, J. Soc. Gynecol. Investig. 11 (2004) 263e271.
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[85] L. Dı´az, N. Noyola-Martı´nez, D. Barrera, G. Herna´ndez, E. Avila, A. Halhali, et al., Calcitriol inhibits TNF-alphainduced inflammatory cytokines in human trophoblasts, J. Reprod. Immunol. 81 (2009) 17e24. [86] Y.C. Li, J. Kong, M. Wei, Z.F. Chen, S.Q. Liu, L.P. Cao, 1,25Dihydroxyvitamin D(3) is a negative endocrine regulator of the renin-angiotensin system, J. Clin. Invest. 110 (2002) 229e238. [87] J.M. Roberts, H.S. Gammill, Preeclampsia: recent insights, Hypertension 46 (2005) 1243e1249. [88] R.K. Marya, S. Rathee, M. Manrow, Effect of calcium and vitamin D supplementation on toxaemia of pregnancy, Gynecol. Obstet. Invest. 24 (1987) 38e42. [89] L.M. Bodnar, H.N. Simhan, Vitamin D may be a link to blackwhite disparities in adverse birth outcomes, Obstet. Gynecol. Surv. 65 (4) (2010) 273e284. [90] M. Jennifer, J.M. Perkins, J.P. Dunn, J.M. Shubhada, Perspectives in gestational diabetes mellitus: a review of screening, diagnosis, and treatment, Clin. Diabetes 25 (April 2007) 57e62. [91] E. Liu, J.B. Meigs, A.G. Pittas, C.D. Economos, N.M. McKeown, S.L. Booth, P.F. Jacques, Predicted 25-hydroxyvitamin D score and incident type 2 diabetes in the Framingham Offspring Study, Am. J. Clin. Nutr. (2010 Apr 14). [Epub ahead of print]. [92] A.G. Pittas, B. Dawson-Hughes, Vitamin D and diabetes, J. Steroid. Biochem. Mol. Biol. (2010 Mar 18). [Epub ahead of print]. [93] C. Zhang, C. Qiu, F.B. Hu, et al., Maternal plasma 25-hydroxyvitamin D concentrations and the risk for gestational diabetes mellitus, PLoS One 3 (2008) e3753. [94] L.M. Bodnar, J.M. Catov, J.M. Zmuda, M.E. Cooper, M.S. Parrott, J.M. Roberts, et al., Maternal serum 25-hydroxyvitamin D concentrations are associated with small-forgestational age births in white women, J. Nutr. 140 (5) (2010 May) 999e1006. Epub 2010 Mar 3. [95] R. Morley, J.B. Carlin, J.A. Pasco, J.D. Wark, A.L. Ponsonby, Maternal 25-hydroxyvitamin D concentration and offspring birth size: effect modification by infant VDR genotype, Eur. J. Clin. Nutr. 63 (2009) 802e804. [96] L.M. Bodnar, H.N. Simhan, The prevalence of preterm birth and season of conception, Paediatr. Perinat. Epidemiol. 22 (2008) 538e545. [97. B. Hollis, Randomized controlled trials to determine the safety of vitamin D during pregnancy and lactation. 14th Workshop on Vitamin D, Scientific Program (update September 28, 2009)Brugge, Belgium, 4e8 October 2009. http://vitamind.ucr.edu/ 14thProgram.pdf (last accessed 4-30-10). [98] B.W. Hollis, W.B. Pittard III, Evaluation of the total fetomaternal vitamin D relationships at term: evidence for racial differences, J. Clin. Endocrinol. Metab. 59 (1984) 652e657. [99] J.A. Taylor, L.J. Geyer, K.W. Feldman, Use of supplemental vitamin D among infants breastfed for prolonged periods, Pediatrics 125 (1) (2010) 105e111. Epub 2009 Nov 30. [100] C.F. Casey, D.C. Slawson, L.R. Neal, Vitamin D supplementation in infants, children, and adolescents, Am. Fam. Physician 81 (6) (2010) 745e748. 15.
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C H A P T E R
40 Vitamin D and the Renin-Angiotensin System Yan Chun Li Department of Medicine, The University of Chicago, Chicago, IL 60637, USA
INTRODUCTION
THE RENIN-ANGIOTENSIN SYSTEM
The renin-angiotensin system (RAS) is a central regulator of renal and cardiovascular functions. Overactivation of the RAS leads to renal and cardiovascular disorders, such as hypertension and chronic kidney disease, the major risk factors for stroke, myocardial infarction, congestive heart failure, progressive atherosclerosis, and renal failure. Mounting epidemiological and clinical evidence has demonstrated an association of vitamin D deficiency or insufficiency with increased risks of renal and cardiovascular diseases, but the molecular basis remains poorly defined. The discovery of the vitamin D hormone as an endocrine repressor of the RAS provides a potential explanation for this association. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) down-regulates the expression of renin, the ratelimiting enzyme of the renin-angiotensin cascade, as well as angiotensinogen, the substrate of renin. Vitamin D deficiency leads to overexpression of renin and thus activation of the RAS, causing renal and cardiovascular injuries. It is speculated that the vitamin D hormone maintains the renal and cardiovascular homeostasis via suppressing the RAS. Pharmacologically vitamin D analogs can be used to target the RAS for treatment of renal and cardiovascular diseases. This chapter will focus on vitamin D regulation of the RAS and its physiological and therapeutic implications with regard to the renal and cardiovascular systems. Other relevant chapters in this book include vitamin D effects on renal disease discussed in Chapter 70 and in cardiovascular disease and risk in Chapters 31 and 102.
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10040-X
The Renin-Angiotensin Cascade and its Biological Functions The RAS is a systemic endocrine regulatory cascade consisting of multiple components (Fig. 40.1). The first and rate-limiting step of the RAS cascade is renin, an aspartyl protease that is primarily released from the juxtaglomerular (JG) cells in the JG apparatus of the kidney. The only known substrate for renin is angiotensinogen (AGT), produced predominantly in the liver. Renin cleaves AGT to angiotensin (Ang) I, an inactive 10amino-acid peptide; Ang I is then converted to Ang II, an 8-amino-acid peptide, by angiotensin-converting enzyme (ACE), which primarily resides in the endothelial cells in blood vessels. Further processing of Ang II by animopeptidase A and N produces Ang III and Ang IV [1]. ACE2, an ACE homolog, can convert Ang II to Ang (1e7) [2,3], and this enzyme is thought to play an essential role in heart functions [4]. Ang II is the central biological effector of the RAS. Systemic Ang II plays a central role in the regulation of blood pressure (Fig. 40.1). Ang II is the most potent vasoconstrictor. It acts on smooth muscle cells in the vasculature to increase vasoconstriction and thus enhances peripheral resistance. Ang II stimulates the synthesis and secretion of aldosterone from the adrenal cortex, a hormone that promotes sodium reabsorption in the renal tubular system. Ang II also stimulates the release of antidiuretic hormone (ADH, also called arginine vasopressin) from the hypothalamus/pituitary, which increases water retention from the kidney, leading
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Angiotensinogen Renin inhibitor
Renin
Prorenin
Renin gene
Angiotensin I (Pro)renin Receptor
ACE
ACEI
Angiotensin II ARB Angiotensin II Receptor
Thirst
ADH
H2O Intake
H2O Retention
Aldosterone
Na Retention
Extracellular Volume
FIGURE 40.1
Renin activity Renal & CV Injury
Vasocontriction
Blood Pressure
Fibrogenesis Atherogenesis Hypertrophy Proliferation Inflammation
The renin-angiotensin system. ACE, angiotensin-converting enzyme; ADH, antidiuretic hormone; CV, cardiovascular.
to expansion of extracellular volume. Finally, Ang II stimulates thirst sensation in the central nerve system and promotes water intake. Together, activation of the renin-angiotensin cascade ultimately causes volume expansion and enhances peripheral resistance. Because blood pressure is determined by the combination of cardiac output and total vascular resistance, overactivation of the systemic RAS results in the development of hypertension [5,6]. In fact, Ang II has diverse physiological and pathological activities. In addition to blood pressure control, it has been shown to promote fibrogenesis, inflammation, and cell hypertrophy and proliferation [7e9] (Fig. 40.1). Thus activation of the RAS usually poses detrimental effects. In addition to the systemic RAS, components of the RAS have been found inside many tissues including the brain, heart, vasculature, kidney, and reproductive system [10]. The tissue-specific RAS may function in a paracrine fashion and in some cases can cause tissue damages. The RAS within the brain is involved in the control of water drinking and blood pressure [11,12], the RAS within the heart may be involved in adaptive response to myocardial stress, and the RAS in the vasculature may be involved in vascular tone and endothelial functions [10]. The intrarenal RAS is known to play a key role in hyperglycemia-induced renal injury in diabetes mellitus [13]. The wide range of activities of Ang II is mediated by several G-protein-coupled receptors widely distributed in tissues [14]. Among these receptors, the type 1 receptor (AT1) mediates most of the activities involved in vasoconstriction, sodium retention, and hypertrophy, whereas the type 2 receptor (AT2) is involved in
vasodilation, natriuresis, and growth inhibition. Hypertension and hypertension-related organ damage resulting from excessive activation of the RAS are mostly mediated by the AT1 receptor [14].
Control of Renin Production and Secretion Renin, the central regulator of the renin-angiotensin cascade, is a highly specific aspartic peptidase with AGT as the sole known substrate. Renin is also species-specific in that human renin is not able to cleave murine AGT, and vice versa [15e17]. The structure of renin is composed of two b-sheet domains with the enzymatic active site residing in a cleft between these two domains [18,19]. Renin and its inactive precursor, prorenin, are synthesized and secreted from the JG cells, highly granulated smooth muscle cells located in the media of the afferent arteriole at the vascular pole of the glomerulus (Fig. 40.2). Renin is synthesized as a prepropolypeptide precursor during translation in the endoplasmic reticulum. The target signal sequence is cleaved during translocation in the endoplasmic reticulum, yielding the inactive prorenin. Prorenin is glycosylated and activated during intracellular transport through the Golgi apparatus, and eventually stored in secretory granules. The prosequence is removed during the activation process. Renin is secreted from these granules through exocytosis upon stimulation [20,21]. In the plasma the prorenin concentration is usually much higher (10e100 times) than the renin concentration. Prorenin is 43 amino acids longer than mature renin at the NH2-terminus, and this NH2-terminal prosegment is thought to block the enzymatic active site located in the cleft, thus preventing the interaction of
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FIGURE 40.2 Structure of the glomerulus. The tubular pole, which is where the proximal tubule leaves the corpuscle, is at the top, and the vascular pole, which is where the afferent arteriole enters and the efferent arteriole leaves the corpuscle, is at the bottom. The granulated reninproducing JG cells are located in the afferent arteriole at the vascular pole and are in close vicinity to the macula densa. (Adapted from Cormack (1984) Introduction to Histology, Fig. 15-5, p. 344, with permission.)
the active site with the substrate AGT. Although in vitro studies have shown that prorenin can be activated by endopeptidase such as trypsin and cathepsin B or by low pH, the mechanism involved in proteolytic prorenin activation in vivo and its physiological role remain poorly defined. High renin activity can result in inappropriate activation of RAS, leading to hypertension and end-organ damage. In fact, increased plasma renin activity is associated with hypertension [22], left ventricular hypertrophy [23], and renal dysfunction [24]. The (pro)renin receptor, a single transmembrane receptor initially identified in mesangial cells and vascular smooth muscle cells [25], binds to both renin and prorenin with high affinity. Renin bound to the receptor exhibits increased catalytic activity, and prorenin bound to this receptor exhibits full enzymatic activity comparable to that of mature renin. In addition,
binding of renin or prorenin to this receptor triggers intracellular signaling and phosphorylation of MAP kinase independent of Ang II generation. Stimulation of the (pro)renin receptor in mesangial cells with purified renin or prorenin promotes the synthesis of TGF-b [26], a profibrotic factor involved in the development of nephropathy. Thus, increased renin/prorenin can also cause tissue injury through the (pro)renin receptor independent of Ang II (Fig. 40.1). For instance, transgenic rats overexpressing prorenin in the liver with a high level of circulating prorenin developed severe vascular damage and diabetic renal complications in the absence of high blood pressure [27]. The exact physiological roles of the (pro)renin receptor, however, remain to be fully defined. Because of its central role in the renin-angiotensin cascade, the biosynthesis and secretion of renin is
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tightly regulated. The most common physiological factors that influence renin secretion include renal perfusion pressure, renal sympathetic nerve activity, and tubular sodium chloride load [20,28]. The perfusion pressure in the renal artery is the most profound parameter to influence renin secretion: when the renal perfusion pressure falls, renin secretion rises, and vice versa. This effect is mediated by a baroreceptor or stretch receptor mechanism in the JG cells [29]. The JG apparatus has rich sympathetic nerve endings, and stimulation of renin synthesis and release by sympathetic nerve activity is mediated by b-adrenergic receptors and intracellular cyclic AMP [30]. This pathway may exert a tonic stimulatory influence on renin production [31]. Renin secretion is also tightly regulated by the tubular sodium chloride load [32]. There is an inverse relationship between dietary sodium chloride intake and renin secretion. Tubular control of renin release is mediated by the macula densa, which is part of the distal tubule and anatomically in close association with the JG cells (Fig. 40.2). The macula densa senses the sodium chloride load and transduces the signal, possibly via p38 MAP kinase, PGE2, NO, and ATP/ adenosine, to the JG cells to influence renin production and secretion [28,33]. At the local level, renin synthesis and release are influenced by a variety of bioactive molecules. For instance, prostaglandins, nitric oxide, and adrenomedullin are known to stimulate renin secretion, whereas Ang II, endothelin, vasopressin, atrial natriuretic peptide, and adenosine are inhibitors of renin production [20,28]. Ang II is a potent negative feedback inhibitor of renin production and secretion that maintains the homeostasis of renin levels [34], and this inhibitory effect is mediated by the AT1 receptor [35,36].
Cyclic AMP is a major mediator for renin synthesis and secretion, and several cAMP response elements (CRE) have been identified in both murine and human renin gene promoters [21,28]. But both CREB-dependent and -independent mechanisms may be involved in the cAMP-PKA pathway in human renin promoter activation [40]. Transgenic studies have demonstrated that sequences required for the tissue-specific and development stage-specific expression of the renin, as well as for the response to a variety of physiological stimuli, are located within 5 kb of the 50 -flanking region of the murine renin gene [41e43]. In the 50 -flanking region of murine Ren-1c gene, a 223-bp minimal promoter (e117 to þ6) and a 242-bp enhancer (e2866 to e2625) have been found to be essential for high-level expression of the renin gene [44]. Genetic deletion of this enhancer region from the renin gene leads to reduction of renin expression in the JG cells and low blood pressure in mice [45]. Renin gene expression is regulated by a complex network of transcriptional factors [46]. In the renin gene promoter and enhancer regions, multiple transcription factor-binding sites have been identified, which are responsive to various signal transduction pathways including cAMP, retinoic acid, endothelin-1, and cytokines, to alter renin gene transcription. An array of transcriptional factors has been identified to be involved in the transcriptional regulation of renin gene expression. These factors include positive regulators such as LXRa, RAR/RXR, CREB/CREM, USF1/USF2, HOX genes, NFI, and SP1/SP3 [47e51], and negative regulators such as NF-Y and Ear-2 [52,53]. Thus, the production of renin is determined by a combined interplay of multiple transcriptional regulators available or activated under a specific physiological condition.
Transcriptional Regulation of Renin Gene Expression
Pharmacological Inhibition of the Renin-Angiotensin System
Renin is encoded by a single gene in humans. In mice, some strains (e.g., C57BL/6) have one renin gene (Ren-1c), whereas others (e.g., DBA/2, J129) contain two renin genes (Ren-1d and Ren-2), which are closely linked and probably result from a duplication of the 21 kb Ren-1c-like ancestral gene [37]. The human renin gene and the three mouse renin genes all share the same overall genomic organization (e.g., nine exons and eight introns) and encode highly homologous proteins. For instance, Ren-1 and Ren-2 share 97% amino acid identity [38]. It is believed that the Ren-1 protein is the major source of circulating renin and thus is the major systemic regulator of the renin-angiotensin cascade. Transgenic studies demonstrate that the Ren-1d and Ren-2 genes cooperate to preserve the homeostasis of the RAS [39].
The RAS has been a major therapeutic target for intervention of renal and cardiovascular disorders. Smallmolecule drugs that target the RAS include ACE inhibitors (ACEIs), Ang II type 1 receptor blockers (ARBs), and renin inhibitors. These drugs inhibit each major step of the renin-angiotenin cascade, respectively (Fig. 40.1). ACEIs and ARBs are probably the most successful and most widely prescribed antihypertensive drugs [54,55]. Aliskiren is the first FDA-approved renin inhibitor that specifically inhibits the enzymatic activity of renin and lowers blood pressure in hypertensive subjects [56,57]. There are a number of issues associated with the current ACEIs and ARBs that may compromise the efficacy of RAS blockade and cause undesired side effects. For example, Ang II conversion from Ang I can be
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catalyzed by other enzymes such as chymase [58], thus bypassing the ACE [59], and this reduces the efficacy of ACEIs. In addition to Ang I, ACE also recognizes other substrates such as bradykinin [60]; thus inhibition of ACE may also alter bradykinin metabolism and evoke undesirable side effects. Because Ang II has multiple receptors (e.g., AT1, AT2) with different functions [14], blocking the AT1 receptor with ABRs may increase the availability of Ang II to the AT2 receptor, leading to enhancement of unwanted AT2 activity. The increase in Ang II due to AT1 receptor blockade may lead to elevation of various Ang II metabolites, such as Ang (1e7), Ang III, and Ang IV [1], which are bioactive and may cause a variety of unwanted effects. A major problem associated with all current RAS inhibitors is the compensatory increase of renin concentration [61]. Patients receiving chronic dosing of ACEIs initially have lower plasma Ang II levels; however, Ang II, as well as aldosterone, often rises to the original baseline levels [62,63]. This phenomenon, often termed “ACE escape,” is caused by the disruption of the negative feedback loop in renin biosynthesis, leading to increased renin production. The negative feedback regulation is mediated by the AT1 receptor. The problem of plasma renin increase also exists in the case of ARBs and renin inhibitor aliskiren [64]. The huge increase in renin concentration and activity in the plasma and tissue interstitial space can stimulate the conversion of Ang I, which ultimately leads to the build-up of Ang I, Ang II, and other angiotensin metabolites in the body, through ACE-dependent and -independent pathways (see Fig. 40.5). Ang II accumulation limits the efficacy of RAS inhibition and may explain why the current RAS inhibitors are only suboptimal clinically. Therefore, it is speculated that agents that block the compensatory renin increase can enhance the efficacy of RAS inhibition.
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a complete vitamin-D-deficient model [68,69]. In 2002, we reported that VDR-null mice developed hyperreninemia due to dramatic up-regulation of renin expression in the kidney [67]. The up-regulation of renin was detected at both mRNA and protein levels, leading to marked increase in plasma renin activity and plasma Ang II levels (Fig. 40.3). The hepatic expression of AGT, the substrate of renin, was unchanged. Thus the increase in plasma Ang II production is mainly due to the increase in renin activity. As a consequence of aberrant RAS overstimulation, VDR knockout mice developed high blood pressure, cardiac hypertrophy, and an overdrinking behavior. Cardiac hypertrophy is reflected by an increase in the heart weight and in the size of left ventricular cardiomyocytes, as well as elevation of left
VITAMIN D REGULATION OF THE RENIN-ANGIOTENSIN SYSTEM Vitamin D Hormone as a Negative Endocrine Regulator of the Renin-Angiotensin System More than two decades ago, two articles reported an inverse relationship between circulating 1,25(OH)2D3 levels and plasma renin activity in hypertensive subjects [65,66]. The significance of these studies was hardly recognized until the discovery that 1,25(OH)2D3 is a negative endocrine regulator of renin gene expression [67]. This finding has enormous physiological, pathological, and pharmacological implications. The vitamin D receptor (VDR)-null mutant mouse, which lacks VDR-mediated vitamin D signaling, is
40.3 VDR-null mice develop hyperreninemia. (A) Northern blot showing marked up-regulation of renin mRNA expression in the kidney of VDR-null mice. (B) Quantitative results of the Northern blot data; ))), P < 0.001 vs. þ/þ mice. (C) Immunostaining of kidney cortex sections with renin antiserum. Arrows indicate the afferent glomerular arterioles in the juxtaglomerular region. Note the marked increase in renin staining in VDR-null kidney. (D) Plasma renin activity in wild-type and VDR-null mice. (E) Plasma Ang II concentrations in wild-type and VDR-null mice. )) P < 0.01; ))), P < 0.001 vs. þ/þ mice. þ/þ, wild-type; e/e, VDRnull; PRA, plasma renin activity; Ang II, angiotensin II. (From Li et al. (2002), with permission.) Please refer to color plate section. FIGURE
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ventricular ANP and plasma ANP levels, the surrogate marker of cardiac hypertrophy [70]. Urinary volume and urinary salt excretion are also increased, whereas plasma sodium and potassium concentrations remain normal in the mutant mice. All these abnormalities can be corrected by treatment with an ACEI or an ARB, confirming that RAS activation is responsible for these phenotypes [67,70]. As expected, plasma and urinary aldosterone levels are also markedly elevated in VDR knockout mice [71]. Moreover, renin expression in the brain is also up-regulated in VDR-null mice, leading to activation of the local RAS in the brain, which is mostly responsible for the overdrinking and polyuric phenotypes seen in the mutant mice [72]. Inactivation of VDR leads to development of hypocalcemia, secondary hyperparathyroidism, and alopecia [68], which may potentially influence renin production and secretion. Several lines of evidence demonstrate that vitamin D regulation of renin is independent of calcium, parathyroid hormone, or alopecia. First, normocalcemic neonatal VDR knockout mice have elevated renin expression, indicating that hyperreninemia develops prior to hypocalcemia; second, renin expression remains elevated in adult VDR-null mice whose serum calcium levels are maintained normal with a high-calcium diet; third, renin expression is normal in Gcm2-null mice that are as hypocalcemic as VDRnull mice [73,74]; and fourth, renin expression remains up-regulated in VDR knockout mice whose alopecia is rescued by targeted expression of human VDR in the skin [75]. Moreover, despite the high basal renin synthesis, the basic regulatory mechanisms that control renin production, including the Ang II negative feedback and salt- and volume-sensing mechanisms, remain intact in VDR-null mice [67,71], indicating that the vitamin D repression is independent of these mechanisms. The inhibitory role of vitamin D in renin biosynthesis has been confirmed by a transgenic approach. We recently produced transgenic mice that overexpress the human VDR in the JG cells. In these transgenic mice, renal renin mRNA levels and plasma renin activity were significantly suppressed while serum calcium and parathyroid hormone levels were normal. When the human VDR transgene was bred into VDR knockout mice to generate knockout mice that express VDR only in the JG cells, renin up-regulation was markedly reduced in these mice compared to VDR-null mice [76]. These data further prove that 1,25(OH)2D3 suppresses renin expression in vivo independent of parathyroid hormone and calcium. This conclusion is consistent with previous observations in humans that the inverse relationship between serum 1,25(OH)2D3 levels and plasma renin activity or blood pressure is independent of serum calcium levels [65,77].
The critical role of vitamin D in the regulation of the RAS in vivo has also been confirmed in another genetic mutant mouse model of vitamin D deficiency. These mutant mice lack Cyp27b1, the rate-limiting 1ahydroxylase required for the biosynthesis of 1,25 (OH)2D3 in the kidney. As seen in VDR knockout mice, Cyp27b1 knockout mice also developed hyperreninemia, hypertension, and cardiac hypertrophy as a result of renin up-regulation. Importantly, in this model these abnormalities were corrected not only by ACEI or ARB, but also by treating the mice with exogenous 1,25(OH)2D3 [78]. Consistently, in wild-type mice rendered vitamin-D-deficient by dietary strontium treatment, which inhibits 1,25(OH)2D3 biosynthesis [79], renin expression in the kidney was markedly upregulated. On the other hand, treatment of wild-type mice with 1,25(OH)2D3 significantly reduced renin expression [67]. Together these data firmly establish vitamin D hormone as a crucial negative endocrine regulator of the RAS.
Molecular Mechanism for Vitamin D Repression of Renin Expression As a ligand-activated transcription factor, VDR is involved in both positive and negative transcriptional regulations (see Chapters 7 and 8). While most positive regulations are mediated by vitamin D response elements (VDRE) in vitamin D target gene promoters, the mechanisms for negative regulation are diverse. Liganded VDR has been shown to physically interact with a variety of regulatory proteins including Smad3, b-catenin, and p65 NF-kB to down-regulate gene expression [80e83]. In the case of renin repression, 1,25 (OH)2D3 targets the cyclic AMP signaling pathway [84], a central stimulatory pathway involved in renin biosynthesis [28]. Cyclic AMP signals through cAMP response element (CRE), which interacts with members of the ATF/ CREB/CREM bZIP transcription factor family in homodimeric or heterodimeric forms. Intracellular cAMP is converted from ATP by adenylate cyclase. Cyclic AMP binds to the regulatory subunit of protein kinase A to free the catalytic subunit; the latter enters the nucleus and phosphorylates CREB at serine-133 or CREM at serine-117, resulting in the recruitment of ubiquitous co-activators CBP/p300 to promote gene transcription [85e87]. Through systematic deletion analyses of the mouse renin gene promoter, we demonstrated that the CRE at e2688 is necessary to mediate the suppression of renin gene transcription by 1,25(OH)2D3. This CRE is critical for the basal expression of renin [49]. Experimental data obtained from EMSA, ChIP assays, GST pull-down assays, and cell transfection experiments
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Schematic model of 1,25(OH)2D3-induced transrepression of renin gene transcription. PKA, protein kinase A; D, 1,25(OH)2D3; Pol II, RNA polymerase II.
FIGURE 40.4
demonstrated that 1,25(OH)2D3 disrupts the formation of the DNAeprotein complex on the CRE site. The complex contains CREB/CREM and CBP/p300. 1,25 (OH)2D3-activated VDR physically interacts with CREB, thus blocking CREB binding to this CRE. Consequently the DNAeprotein complex cannot be assembled on this CRE [84]. These data establish that 1,25 (OH)2D3 suppresses renin gene transcription, at least in part, by direct inhibition of CRE-mediated transcriptional activity. The proposed model is that in the basal state CREB, CREM, and CBP/p300 are recruited to the CRE to drive renin gene transcription; in the presence of 1,25(OH)2D3, liganded VDR binds to CREB, blocking CREB binding to the CRE and disrupting the formation of CRE-CREB-CBP/p300 complex. As a result, renin gene expression is inhibited (Fig. 40.4). Besides renin, it is possible that 1,25(OH)2D3 may also target the cAMP-CRE-CREB pathway in the regulation of other genes. This renin repression model has important physiological implications, because cAMP is the central intracellular signal that stimulates renin production and release in the JG cells. For instance, intracellular cAMP is critically involved in renin synthesis and release in response to sympathetic nerve stimulation (mediated by b-adrenergic receptor), as well as to stimulation by prostaglandins, dopamine, adrenomedullin, calcitonin gene-related peptide, and pituitary adenylyl cyclase activating polypeptide [28]. It is speculated that, by targeting the cAMP signaling pathway, 1,25(OH)2D3 may function as a general gatekeeper to counterbalance other renin-stimulating factors and prevent detrimental overproduction of renin.
Vitamin D Analogs as Renin Inhibitors A large number of vitamin D analogs have been synthesized with a wide range of pharmacological potency and calcemic index (see Section IX of this book). A few vitamin D analogs have been approved for clinical use [88,89]. Therefore, the concept of vitamin D analogs as therapeutic drugs is already sound. The notion that 1,25(OH)2D3 suppresses renin biosynthesis
provides a molecular basis to explore vitamin D analogs as renin synthesis inhibitors for therapeutic purposes. Low calcemic vitamin D analogs that have potent renin-inhibiting activity are particularly valuable. The vitamin D analog-based renin inhibitors, which inhibit renin gene expression, are different from another class of aliskiren-like renin inhibitors, which inhibit renin enzymatic activity. There are advantages to having two classes of renin inhibitors. For example, combination of these two classes of drugs simultaneously will inhibit renin at both the biosynthetic and enzymatic levels and thus may increase therapeutic efficacy. The renin-inhibiting activity of vitamin D analogs has been reported in a number of in vitro and in vivo models. Through cell culture screening, we have identified a group of vitamin D analog compounds that inhibit renin expression in vitro and in vivo without invoking severe hypercalcemia in mice, and some analogs are much more potent than 1,25(OH)2D3 in terms of renin repression [90]. Paricalcitol (19-nor-1,25-dihydroxyvitamin D2), an activated vitamin D analog, has been shown to suppress renin expression in mice with the same potency as 1,25(OH)2D3 but without induction of hypercalcemia [91]. Paricalcitol can also suppress renin expression in kidney mesangial cells [92]. In the rat model of chronic renal failure (5/6 nephrectomy) paricalcitol treatment was shown to significantly lower blood pressure and suppress the RAS in the remnant kidney [93]. Recently we showed that paricalcitol or doxercalciferol (1a-hydroxyvitamin D2) is able to suppress the RAS and effectively block cardiac hypertrophy in spontaneously hypertensive rats [94]. We also reported that patients with chronic kidney disease receiving vitamin D analog therapy have significantly lowered plasma renin activity, suggesting that vitamin D analogs can also suppress the RAS in humans [94]. Therefore down-regulation of the RAS appears to be a major therapeutic mechanism underlying the beneficial effects of vitamin D analogs in renal and cardiovascular diseases. As discussed above, the dramatic compensatory increase in plasma renin concentration that is associated with the current RAS inhibitors may compromise the
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efficacy of these drugs. One important application of vitamin D analogs is to block the compensatory renin increase at the transcriptional level when used together with the classic RAS inhibitors. The combination is expected to enhance the efficacy of RAS inhibition and achieve better therapeutic outcomes [95] (Fig. 40.5). We have tested the combination strategy (an ARB plus a vitamin D analog) in models of diabetic nephropathy and cardiac hypertrophy. We showed that combination of losartan and paricalcitol or doxercalciferol produced synergistic effects in prevention of albuminuria and glomerulosclerosis, the main pathogenic hallmarks of diabetic renal injury. The molecular basis underlying the therapeutic synergism is the blockade of the compensatory renin increase and intrarenal Ang II accumulation [96e98]. Similarly, in spontaneously hypertensive rats, the combination therapy markedly shrunk cardiac hypertrophy in a synergistic fashion, also as a result of inhibition of the compensatory rise of renin in the kidney and heart [94]. Clinical trials of vitamin D analogs in patients with chronic kidney disease who are already on ARB or ACEI therapy suggest that the concept of combination synergy also works in human patients [99]. Given the wide use of the RAS inhibitors, the combination strategy warrants more and larger clinical trials to test its therapeutic efficacy in humans.
Pathophysiological Implications The finding that 1,25(OH)2D3 regulates the RAS is consistent with the view that the vitamin D endocrine system plays multiple physiological roles. 1,25(OH)2D3 is a principal regulator to maintain calcium homeostasis. The notion of 1,25(OH)2D3 as a negative regulator of renin production implies that vitamin D deficiency or insufficiency can cause activation of the RAS. Adding to the complexity and controversy in defining vitamin D deficiency, the threshold of vitamin D status to induce hyperreninemia is unknown and might be different from that for hypocalcemia. Nevertheless, as described in the following sections, vitamin D deficiency is highly prevalent in patients with renal and cardiovascular problems. Given the broad pathological roles of the RAS in the development of renal and cardiovascular disorders [100,101], the notion of vitamin D affecting the RAS status suggests that vitamin D deficiency is not only associated with renal and cardiovascular diseases but may also promote them. This view has already gained support from clinical data [102]. Therefore, at least in part because of the RAS, long-term vitamin D deficiency may increase the risk of renal and cardiovascular diseases, whereas vitamin D supplementation and therapies with vitamin D and its analogs should be beneficial. In the following sections we will further discuss
Renin gene
Vitamin D & Analogs Angiotensinogen Renin
AT1 Receptor Renin Inhibitor
(Pro)renin Receptor
ARB Ang I Ang II
ACEI ACE
Compensatory increase of renin and its inhibition by vitamin D. Ang II feedback inhibition of renin expression is mediated by angiotensin type I (AT1) receptor. This negative feedback mechanism is required to maintain the homeostasis of the RAS. When the RAS is inhibited with renin enzymatic inhibitor, ACE inhibitor (ACEI) or AT1 receptor blocker (ARB), the feedback loop is disrupted, leading to overexpression of renin. Renin overproduction can increase Ang I conversion, which ultimately leads to Ang II increase, thus reducing the efficacy of RAS inhibition. Renin accumulation can also activate the (pro)renin receptor independent of Ang II to cause organ damage. When a vitamin D analog is used together with one of the three classes of RAS inhibitors, vitamin D analog will block the compensatory renin expression, thus enhancing the efficacy of RAS inhibition. That is the molecular basis for the synergism between vitamin D analogs and classic RAS inhibitors in combination therapy.
FIGURE 40.5
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VITAMIN D, BLOOD PRESSURE AND HEART DISEASE
the relationship between vitamin D status and renal and cardiovascular diseases in the human population and the potential of vitamin D and its analogs in the prevention and intervention of these diseases.
VITAMIN D, BLOOD PRESSURE AND HEART DISEASE Sunlight and Blood Pressure Hypertension, or high blood pressure, is one of the most prevalent health problems in the world. Hypertension increases the risk of heart attack, heart failure, stroke, atherosclerosis, and kidney disease. Data from the National Health and Nutrition Examination Surveys (NHANES) show that in the United States 29% adults had hypertension in 2005e2006. Despite intense prevention and intervention efforts, there was still no change in the prevalence of hypertension during 1999e2006 [103]. An increasing body of evidence has suggested a link between sunlight exposure, vitamin D, and blood pressure. As ultraviolet (UV) irradiation is essential for the cutaneous synthesis of vitamin D, circulating 25hydroxyvitamin D (25(OH)D) levels are influenced by geographic locations, seasonal changes, and skin pigmentations (see Chapter 2). UV irradiation decreases with the increase in latitude, and the data from the INTERSALT study show that the increase in latitude is correlated with the rise of blood pressure and the prevalence of hypertension in the general population around the globe [104]. An increasing gradient of hypertension prevalence and stroke incidents from south to north has also been reported in China [105]. Seasonal variations in blood pressure are seen in temperate climates, with blood pressure higher in the winter (low UV irradiation) than in the summer (high UV irradiation) [106,107]. Winter season is also associated with high incidence of myocardial infarction [108]. Dark skin pigmentation in the black population, which inhibits an efficient UV light penetration, is associated with higher blood pressure [109,110]. A small clinical study has shown that direct UVB irradiation is able to lower blood pressure in patients with mild essential hypertension [111].
Vitamin D Deficiency and High Blood Pressure Epidemiological studies have established a correlation between vitamin D deficiency and high blood pressure and cardiovascular risk factors. The NHANES III, a cross-sectional survey representative of the US civilian population from 1988 to 1994, shows an inverse relationship between serum 25(OH)D and blood pressure [112]. In this nonhypertensive population (n > 2500), systolic
715
and diastolic blood pressures are 3 and 1.6 mmHg higher, respectively, in the lowest quintile of serum 25 (OH)D compared to the highest quintile. This difference, although modest, has public health significance, as a 2e3 mmHg decrease in blood pressure could account for 10e15% decline in cardiovascular mortality on a population basis [113]. Analysis of the NHANES III database (n > 15 000 adults) revealed that serum 25(OH)D levels are also inversely associated with increased prevalence of cardiovascular risk factors including hypertension, diabetes, obesity, and hyperlipidemia [114]. Other epidemiological studies confirm the association between vitamin D deficiency and hypertension. Prospective cohorts from the Health Professionals’ Follow-Up Study (HPFS) (n ¼ 613) and the Nurses’ Health Study (NHS) (n ¼ 1198) showed that serum 25 (OH)D levels are inversely associated with the risk of incident hypertension during 4 years of follow-up [115]. A nested caseecontrol prospective study using the HPFS database (n > 18 000) also demonstrated an association of low serum 25(OH)D levels with higher risk of myocardial infarction, even after adjusting for factors known to be associated with coronary artery disease [116]. An examination of the Framingham Offspring Study participants without prior cardiovascular disease (n ¼ 1739) concluded that during the mean follow-up of 5.4 years low serum 25(OH)D (<10e15 ng/ml) is associated with incident cardiovascular disease after adjustment for C-reactive protein, physical activity, or vitamin use [117]. A similar inverse association is also found between serum levels of 1,25(OH)2D3 and blood pressure. A cross-sectional multivariate study with normotensive male industrial employees (n ¼ 100) showed an inverse and statistically significant association between serum 1,25(OH)2D3 levels and systolic blood pressure independent of serum parathyroid hormone and calcium levels [77]. In another population-based study with 34 middle-aged men serum levels of 1,25(OH)2D3 were also found to be inversely correlated to blood pressure [118].
Interventional Effects of Vitamin D on Blood Pressure Many clinical studies have reported cardiovascular benefits of vitamin D supplementation or therapy. In a double-blinded, placebo-controlled clinical trial with 39 hypertensive patients, blood pressure was significantly reduced after 4 months of 1a-hydroxyvitamin D3 treatment [119]. In a clinical trial involving 148 elderly women, 8-week supplement of vitamin D3 (800 IU) plus calcium (1200 mg) significantly reduced systolic blood pressure in these subjects [120]. A large prospective study with 28 886 middle-aged women in
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the US found that dietary intake of dairy products, calcium, and vitamin D are each inversely associated with risk of hypertension during 10 years of follow-up [121]. However, not all reported studies support a role of vitamin D in blood pressure control. In a clinical trial involving 189 elderly subjects, a single oral dose of 2.5 mg cholecalciferol (100 000 IU) in winter failed to change blood pressure [122]. A possible explanation is that a single dose of vitamin D supplement in the winter is not sufficient to raise the circulating vitamin D level to the blood-pressure-affecting threshold. Results from three large prospective cohort studies including NHS I and II and HPFS failed to find an association between high intake of vitamin D and low risk of incident hypertension [123]. These conflicting reports call for more rigorous and well-controlled investigations into the effects of vitamin D on blood pressure.
Vitamin D Status and the Renin-Angiotensin System Many studies have investigated the correlation between vitamin D status and the RAS in humans. Resnick et al. first demonstrated an inverse correlation between serum levels of 1,25(OH)2D3 and plasma renin activity in a study with 51 patients with essential hypertension [65] (Fig. 40.6). Subsequently, another smaller study confirmed the inverse correlation between the change in circulating 1,25(OH)2D3 and the change in plasma renin activity in subjects with high renin hypertension [66]. In the Ludwigshafen Risk and Cardiovascular Health (LURIC) study that includes more than 3000 patients referred for coronary angiography, both
serum 25(OH)D and 1,25(OH)2D3 levels were found to be independently and inversely associated with plasma renin concentration and Ang II levels [124]. Another study reported an inverse association between serum 25(OH)D levels and Ang II levels in normotensive individuals (n ¼ 184); it also showed an association of lower serum 25(OH)D levels with increased renal plasma flow in response to Ang II infusion [125], suggesting increased intrarenal RAS activity in individuals with lower serum 25(OH)D levels. Consistent with these observations, data from clinical studies also support a connection between vitamin D and renin. In a double-blinded, placebo-controlled clinical trial (32 subjects), 16 weeks of daily oral calcium supplementation, which suppresses plasma 1,25(OH)2D3 levels, resulted in a significant elevation of plasma renin activity, suggesting a suppressive role of vitamin D in renin regulation (although an effect of calcium independent of vitamin D is possible) [126]. In a clinical study involving 25 hypertensive patients with end-stage renal disease, 15 weeks of 1,25(OH)2D3 treatment reduced myocardial hypertrophy, with a concomitant reduction in plasma renin activity, Ang II and ANP levels [127]. 1,25(OH)2D3 treatment was also reported to reduce blood pressure and plasma renin activity in a patient with pseudohyperparathyroidism and high plasma renin activity [128]. Therefore, the beneficial effects of vitamin D on the cardiovascular system reported in the literature, including reduction of high blood pressure, likely include the down-regulation of the RAS [129,130]. Given the critical role of the RAS in the cardiovascular system, the relationship between vitamin D and plasma renin activity is likely part of the mechanism underlying the relationship between vitamin D and blood pressure. This notion is worth more translational and clinical investigations.
VITAMIN D AND CHRONIC KIDNEY DISEASE Increasing Prevalence of Chronic Kidney Disease
Inverse correlation between circulating 1,25(OH)2D3 levels and plasma renin activity in patients with essential hypertension. (Adapted from Resnick et al. (1986) Fig. 2, p. 652, with permission.)
FIGURE 40.6
Chronic kidney disease (CKD) affects more than 50 million people worldwide. The prevalence of CKD and kidney failure is continuously rising with the growing global epidemic of metabolic syndrome and diabetes. In the United States, the incidence and prevalence of end-stage renal disease have doubled in the past 10 years. The National Kidney Foundation-Kidney Disease Outcomes Quality Initiative estimates that CKD affects 11% of the US population. Diabetic nephropathy is the most common renal complication of diabetes. Diabetes is by far the leading cause of
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VITAMIN D AND CHRONIC KIDNEY DISEASE
CKD, accounting for 44% of new cases of end-stage renal disease in 2005. Current clinical care and management of kidney disease are costly with poor outcomes. Thus, new therapeutic strategies and methods for CKD treatment are urgently needed.
Vitamin D Deficiency in Chronic Kidney Disease Vitamin D deficiency has been increasingly recognized as a prominent feature of CKD. This is in part because the kidney is a key organ involved in vitamin D metabolism. The kidney not only provides the enzymatic system for the synthesis of 1,25(OH)2D3, but also is involved in the uptake of filtrated 25(OH)D from the urine, in the form of vitamin-D-binding protein (DBP)e25(OH)D complex, through megalin-mediated endocytosis [131]. Renal 1a-hydroxylase activity starts to decline even in the early stages of CKD. Continued progression of CKD leads to accumulation of phosphate in the serum, which suppresses 1a-hydroxylase activities. Proteinuria, a hallmark of renal disease, results in loss of DBPe25(OH)D from the urine. Therefore vitamin D deficiency, particularly 1,25(OH)2D3 deficiency, is very common in patients with CKD even at the early stages [132] (Fig. 40.7). Accumulating evidence has demonstrated a correlation between vitamin D deficiency and progression of CKD, and plasma vitamin D status is an independent inverse predictor of disease progression and death in patients with CKD [102]. Thus vitamin D deficiency may in fact accelerate the progression of kidney disease. In fact, a large number of retrospective observational studies have demonstrated multiple beneficial effects of calcitriol or vitamin
1,25(OH)2D (pg/mL)
iPTH (pg/mL)
D analog therapy in both hemodialysis and nondialysis CKD patients, leading to significant survival advantage for the patients receiving the therapy [133e137]. Because the risk of death is significantly lower in the treated patients with all levels of serum calcium, phosphorus, and PTH, the underlying protective mechanism of vitamin D likely extends beyond the impact on PTH and mineral metabolism. See Chapters 70 and 81 for further discussion of vitamin D therapy in renal disease patients.
Therapeutic Mechanisms of Vitamin D: Targeting the Local Renin-Angiotensin System It is well established that activation of the local, intrarenal RAS plays a key role in kidney damage [13]. Kidney cells have the capacity to synthesize all components of the RAS. Many pathological factors such as hyperglycemia, renal insufficiency, and vitamin D deficiency can activate the intrarenal RAS, leading to increased local production of Ang II that acts in a paracrine manner within the kidney. For example, in diabetes the intrarenal interstitial Ang II levels can reach as much as 1000 times higher than in the plasma. Ang II has a range of pathological activities that promote the progression of renal injury and renal failure [100,101]. Thus inhibition of the RAS has been the first line of treatment for kidney disease. ACEIs, ARBs, and the renin inhibitor aliskiren have been shown to reduce the progression of diabetic nephropathy in a number of large clinical trials [138e142]; however, the growing number of patients with CKD attests that these therapies are insufficient and ineffective to halt the epidemic of kidney disease.
25(OH)D (ng/mL) 150
50 45 †
100
30 25 †
20 15 10
†
5 0 80 n=61
50
n=1814 p<0.001
79–70 n=117
69–60 n=230
59–50 n=396
49–40 n=355
39–30 n=358
29–20 n=204
0 <20 n=93
eGFR Interval (mL/min/1.73 m2)
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iPTH level (pg/mL)
1,25(OH)2D (pg/mL) 25(OH)D (ng/mL)
40 35
717
FIGURE 40.7 Vitamin D status decreases along with progression of chronic kidney disease. Serum 25-hydrovitamin D (25(OH)D) and 1,25-dihydroxyvitamin D (1,25(OH)2D) levels begin to decline in CKD patients in early Stage 2. iPTH, intact parathyroid hormone. Adapted from Levin A et al. Kidney International (2007) 71:31e38, Figure 5.
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Proteinuria, glomerulosclerosis, and interstitial fibrosis are some of the key features of kidney disease. That vitamin D protects the kidney by suppressing the local RAS has been demonstrated in a number of kidney disease models. We have shown that in diabetic state or under unilateral ureteral obstruction (UUO) VDR-null mutant mice (with normal serum calcium) developed more severe renal injury compared with wild-type mice. Diabetic VDR-null mice showed earlier and more robust albuminuria and glomerulosclerosis, and VDR-null mice with UUO showed increased interstitial fibrosis in the obstructed kidney. In both models, the more severe kidney injury is largely due to the increased activation of the local RAS [92,143]. The subtotally nephrectomized rat model recapitulates renal insufficiency seen in advanced-stage CKD. The pathophysiology of this model is characterized by progressive glomerular and tubulointerstitial damage, glomerular hypertension and proteinuria, in which the activation of the RAS plays a pivotal role. Treatment with 1,25(OH)2D3 can reduce glomerulosclerosis and albuminuria and prevent podocyte injury in 5/6 nephrectomized rats [144,145]. Freundlich et al. reported that paricalcitol suppressed the activation of the local RAS in the kidney remnant, attenuated glomerular and tubulointerstitial damage and reduced high blood pressure and proteinuria in rats with 5/6 nephrectomy, confirming the importance of RAS blockade in prevention of renal function deterioration [93]. The mainstay treatment of diabetic nephropathy is to inhibit the RAS, but RAS inhibitors induce a compensatory rise of renin activity (see Fig. 40.5). We have demonstrated that blocking the compensatory increase of renin with vitamin D analogs markedly enhances the reno-protective efficacy in experimental models of both type 1 and type 2 diabetes mellitus [96e98]. In these studies, diabetic mice were treated with a combination of an ARB (losartan) and a vitamin D analog (paricalcitol or doxercalciferol) or with monotherapy. The combination strategy produced additive or synergistic therapeutic effects much better than the monotreatments, including inhibition of albuminuria and glomerulosclerosis and amelioration of glomerular filtration barrier damage. This is largely a result of blockade of renin induction and Ang II accumulation within the kidney. Similar combination therapies have also been used in the models of subtotal nephrectomy and UUO with significant better therapeutic outcomes compared to the monotherapy [146,147]. These preclinical investigations have important implications for the therapeutic treatment of kidney disease in humans.
Renoprotection of Vitamin D Therapy in Chronic Kidney Disease Albuminuria is a major risk factor for progressive renal function decline and is believed to be the initial step in an inevitable progression to proteinuria and renal failure in humans. Thus reduction of albuminuria is a major target for renoprotective therapy in CKD. A number of epidemiological and clinical studies have demonstrated potent antiproteinuric activity of vitamin D and vitamin D analogs. In a large cohort crosssectional analysis of data from the NHANES III, vitamin D insufficiency was found to be associated with increased prevalence of albuminuria [148], suggesting that vitamin D has an intrinsic antiproteinuric property. The therapeutic antiproteinuric activity of vitamin D analogs was first reported in a retrospective analysis of patients with CKD [149]. In that study the antiproteinuric effect of paricalcitol was seen even in subjects already treated with ACEI or ARB, indicating that the effects of vitamin D analogs are on top of those of ACEI and ARB. A recent randomized double-blinded pilot trial in patients with stage 2e3 CKD (n ¼ 24) showed that paricalcitol treatment for 1 month significantly reduced albuminuria and inflammation status in the drug-treated subjects, and these effects were independent of its effects on hemodynamics and PTH suppression [99]. Again, as the CKD patients in this study were already on ACEI or ARB treatment, the beneficial effects of vitamin D analogs are additive or synergistic to those of RAS inhibitors. These clinical data warrant larger and long-term randomized, controlled trials to confirm the therapeutic benefits of vitamin D and its analogs.
CONCLUSION Recent genetic, physiological, biochemical, and molecular studies have established 1,25(OH)2D3 as a negative endocrine regulator of the RAS. This discovery reveals an important physiological function of the vitamin D endocrine system. As such, longterm vitamin D deficiency can lead to overactivation of the RAS. Because of the broad involvement of the RAS in the development of renal and cardiovascular diseases, this finding has invaluable pathophysiological and therapeutic implications. It provides a mechanistic insight into the ever-increasing epidemiological and clinical evidence linking vitamin D deficiency to renal and cardiovascular problems in the general population. It also provides a molecular basis to explore the therapeutic potentials of vitamin D and its analogs in the prevention and intervention of these diseases. In
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REFERENCES
this regard, the rising prevalence of hypertension and chronic kidney disease around the world attests the urgent need for new and more effective therapeutic methods. Given the promising data obtained from recent translational and clinical studies, the future for vitamin D analogs to become renal and cardiovascular drugs appears to be bright.
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C H A P T E R
41 Parathyroid Hormone, Parathyroid HormoneRelated Protein, and Calcitonin Elizabeth Holt, John J. Wysolmerski Section of Endocrinology and Metabolism, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA
PARATHYROID HORMONE Introduction The parathyroid glands were first recognized as distinct from the thyroid in the late nineteenth century [1]. At the turn of the twentieth century it was noted that the absence of parathyroid tissue caused tetany and it was suggested that these glands were involved in the regulation of calcium metabolism. In 1925, Collip showed that acid extracts of the parathyroid gland could reverse tetany, documenting that the parathyroids made a soluble factor that supported circulating calcium levels [2]. However, it was not until 1959, when Aurbach and then Rasmussen and Craig isolated full-length parathyroid hormone (PTH) [3,4]. This led to the biochemical characterization of PTH and the ability to measure circulating levels. The PTH gene was characterized in the 1970s and 1980s and the PTH receptor gene was cloned and characterized in the early 1990s [1]. Thus, research on PTH has spanned almost 150 years. Although much progress has been made, there are still mysteries that remain unresolved. The reader is referred to Chapter 27 on the parathyroid gland for additional discussion of this topic.
PTH Gene Human PTH is encoded by a single gene located on chromosome 11 (11p15). The gene contains three exons; the first codes for the 50 untranslated portion of the mRNA, the second encompasses most of the preepro sequences, and the third encodes the actual protein sequence of the secreted hormone [1,5,6]. The PTH
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10041-1
gene is a member of a small gene family that also includes the parathyroid hormone-related protein (PTHrP) and the tubuloinfundibular peptide of 39 amino acids (TIP39) genes [6] (Fig. 41.1). Each of these genes has a similar exon/intron structure and all three bind related receptors with overlapping specificities (see below) [7]. The PTH and PTHrP genes may have arisen from a common ancestor. In mammals, PTHrP and TIP39 appear to act primarily as locally acting growth factors while PTH acts systemically as a classical peptide hormone [6]. The PTH gene is expressed almost exclusively by cells within the parathyroid gland. In humans, four parathyroid glands are derived from cells within the developing third and fourth branchial pouches. The formation of the parathyroid glands depends on the actions of several transcription factors, including hoxa3, Eya1, pax1, pax9, GATA3, SOX3, and Gcm2 [5]. Disruption of hoxa3, Eya1, pax1, and pax9 all result in complicated defects of several branchial pouch derivatives including the parathyroid glands [8e10]. However, disruption of the GATA3 SOX3 and Gcm2 genes appears to result in more specific defects in parathyroid development [11e13]. While these factors are necessary for parathyroid cell proliferation and/or differentiation, it is not clear if any of these factors individually or in combination are responsible for the tissue-specific expression of the PTH gene. In fact, transactivation of the PTH gene in a neuroendocrine tumor was found not to require the above transcription factors [14]. Thus, the molecular basis for parathyroid-specific expression of the PTH gene remains unclear. Three principal factors have been shown to modulate PTH mRNA levels within the parathyroid glands. These
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FIGURE 41.1 PTH, PTHrP, and TIP39 are related genes that comprise an evolutionarily conserved gene family. (A) Genomic structure of the hTIP39, hPTH, and hPTHrP genes highlighting the common organization of the three genes. White boxes represent exons encoding the pre-sequences, black boxes represent the pro-sequences, the gray boxes represent the coding section and the stipled boxes represent noncoding exons. Reproduced with permission from [189]. (B) The phylogentic analysis of the PTH, PTHrP, and TIP39 peptides. PTH and PTHrP split from a common ancestor related to TIP39 and GIP. Reproduced with permission from [190].
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are calcium, 1,25(OH)2 vitamin D (1,25(OH)2D), and phosphate, each of which will be discussed separately below [5]. Parathyroid cells are able to sense and respond to small changes in circulating calcium concentrations through the actions of the calcium-sensing receptor (CaSR), which is a G-protein-coupled receptor that binds and signals in response to ionized calcium and other cations (see Chapter 24) [15]. Changes in extracellular calcium lead to reciprocal changes in PTH secretion (see below) and gene expression [5,15]. That is, a drop in extracellular calcium increases PTH production and a rise in calcium suppresses PTH production. Studies in whole animals have demonstrated that PTH mRNA levels are more sensitive to hypocalcemia than hypercalcemia although high calcium levels do suppress PTH mRNA levels in parathyroid cells in culture [16]. The mechanisms through which CaSR signaling affects PTH gene expression are not well described. Although a negative calcium regulatory element has been identified in the PTH gene promoter region, it is currently thought that the primary effect of CaSR signaling is post-transcriptional [5]. PTH mRNA contains AU-rich elements in the 30 untranslated region (UTR), which regulate its stability. In the setting of low extracellular calcium concentrations, specific proteins bind to these elements and protect the mRNA from degradation. Activation of CaSR signaling displaces these factors and shortens the half-life of PTH mRNA [17]. The production of 1,25(OH)2D in the kidney is stimulated by PTH and, in turn, 1,25(OH)2D acts on parathyroid cells to inhibit PTH production, both by increasing circulating calcium levels and by inhibiting PTH gene transcription directly [5,6]. This effect is thought to be a result of reduced transcription of the PTH gene due to the binding of 1,25(OH)2D to the vitamin D receptor (VDR), which in turn binds to vitamin D response elements in the 50 flanking region of the PTH gene [18]. As in other cell types, the VDR is though to heterodimerize with the retinoic acid receptor and retinoic acid sensitizes parathyroid cells to the suppressive effects of vitamin D [19]. Vitamin D receptors are abundant in parathyroid tissue and their levels are modulated by calcium and 1,25(OH)2D itself [5]. Activation of the CaSR increases the expression of the VDR in parathyroid cells and 1,25(OH)2D increases the expression of the CaSR, mutually sensitizing the parathyroid glands to negative feedback in states of vitamin D or calcium excess. Interestingly, in chronic renal failure and in parathyroid adenomas, expression of both the VDR and the CaSR is reduced, likely contributing to increased PTH production [20e22]. Phosphate has been shown to stimulate PTH production and hyperphosphatemia is an important contributor to the development of secondary hyperparathyroidism in patients with chronic kidney disease
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[20,23]. Some of these effects are likely to be related to the drop in ionized calcium that attends any increase in circulating phosphate, but careful studies in vivo and in vitro have documented an independent effect of phosphate on PTH mRNA stability [23]. Similar to the effects of low calcium, elevations in serum phosphate lead to the binding of a protein complex to the 30 UTR of PTH mRNA that inhibits its degradation, thus increasing the steady-state levels of PTH message [17,23]. Changes in serum phosphate may also affect PTH indirectly via the actions of FGF-23, which is secreted from osteocytes in response to increases in 1,25(OH)2D or phosphate. In turn, FGF-23 interacts with a receptor complex composed of one of several FGF-receptors and a-klotho to inhibit the production of 1,25(OH)2D and to promote the renal excretion of phosphate [24]. The parathyroids express klotho and respond to FGF-23 but, somewhat paradoxically, FGF-23 has been described to reduce PTH gene expression and secretion [24e26]. The actions of FGF-23 on the parathyroid cells may counterbalance the direct effects of phosphate on PTH gene expression, but this is an evolving area and awaits further clarification. Further discussion of FGF23 and Klotho can be found in Chapter 42.
PTH Secretion PTH is initially synthesized as preepro-parathyroid peptide and undergoes post-translational modification to produce the active hormone. The 25-residue presequence is cleaved on entry of preepro-PTH into the endoplasmic reticulum. The shorter, six-residue prosequence is subsequently cleaved, yielding the mature, 84-amino-acid, full-length protein (PTH 1-84) [6]. PTH (1-84) is packaged for secretion into cytoplasmic granules that also contain several proteases. Full-length PTH is the biologically active form of the hormone. It has a very short half-life (minutes) in the circulation and is degraded by the liver and kidney. The process of degradation releases carboxy-terminal (C-terminal) fragments of PTH into the circulation. In response to hypercalcemia, the proteases found within parathyroid secretory granules inactivate full-length PTH by digesting its amino-terminal portion, resulting in the secretion of C-terminal fragments from the gland [27]. Therefore, the circulating concentration of C-terminal PTH species is a reflection of both parathyroid and peripheral metabolism of full-length PTH. These fragments are cleared by the kidney and may accumulate in the circulation in renal failure [28]. The minute-to-minute secretion of PTH is regulated by activation of the CaSR on the surface of parathyroid cells in response to changes in the extracellular ionized calcium concentration [29,30]. A steep inverse sigmoidal relationship exists between PTH secretion and calcium
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(A) The inverse sigmoidal relationship between increasing concentrations of extracellular calcium and PTH secretion from dispersed normal parathyroid cells in culture. (B) This relationship can be defined by the maximal rate of PTH secretion, the slope of the curve at the mid-point, the set-point (point at halfmaximal P secretion) and the minimal rate of PTH secretion. Reproduced with permission from [191].
example, mice with either global deletion or parathyroid-specific disruption of the CaSR gene have very high levels of circulating parathyroid hormone, which are not suppressed by severe hypercalcemia [31,32]. The same is true of humans with null mutations in the CaSR gene [33]. Furthermore, in both mice and humans, heterozygous disruption of the CaSR gene results in a milder form of hyperparathyroidism, demonstrating a dose effect between parathyroid CaSR levels and the sensitivity of PTH to calcium-mediated suppression [31e33]. Conversely, humans with activating mutations in the CaSR gene present with hypoparathyroidism characterized by a failure to secrete PTH in response to hypocalcemia [34]. The molecular mechanisms by which CaSR signaling inhibits PTH secretion are not well understood. Binding of calcium to the CaSR activates downstream signaling pathways (primarily induction of phospholipases and intracellular calcium transients) that, in turn, suppress PTH secretion [29,30]. The CaSR is a G-protein-coupled receptor (GPCR) known to activate both Gaq/11 and Gai. It is clear that activation of both Gaq and Ga11 is necessary for suppression of PTH secretion since parathyroid-specific disruption of the genes for both of these G proteins phenocopies the disruption of the CaSR itself [35]. However, it is not clear how their activation and the subsequent signaling events actually inhibit the secretion of PTH.
FIGURE 41.2
concentration and is defined by four parameters (Fig. 41.2). The first is the maximum secretory rate of the parathyroid glands. The second is the slope of the curve at the midpoint. The third is the parathyroid gland’s “set point,” or the extracellular ionized calcium concentration at which PTH secretion is half maximal. The final parameter is the basal, nonsuppressible rate of PTH secretion. The steep part of the curve represents the physiologic range for extracellular calcium, over which small changes in the concentration of ionized calcium elicit dramatic changes in the rate of PTH secretion [29]. Because of this steep slope, ionized calcium concentrations are maintained in a very narrow physiologic range. Parathyroid glands express high levels of CaSR and a great deal of data in both genetically altered mice and in humans with genetic disorders of calcium sensing have demonstrated the primary importance of the CaSR in controlling PTH secretion [29,30]. For
PTH Receptors PTH binds and activates a GPCR that is shared with PTHrP and is therefore named the type 1 PTH/PTHrP receptor (PTH1R) [7,36]. The human PTH1R is encoded by a single gene on chromosome 3. It is a member of a distinct subset of 15 GPCRs known as “class II” or “family B” receptors, which also includes the secretin and calcitonin receptors [7,37]. Two other PTH receptors are also included within the B family of GPCRs. The PTH2R is primarily a receptor for TIP39. PTH can bind weakly to this receptor and activate it, but it is not known if this represents a physiological interaction [7,37]. PTHrP cannot activate the PTH2R. There is also a third PTH receptor (PTH3R) documented in zebrafish, but it does not appear to be present within the human genome [7,37]. Thus, as with the PTH peptide family, it appears that evolution has reduced the diversity of PTH receptors. The amino-terminal portion (first 34 or fewer amino acids) of PTH is both necessary and sufficient for full activation of the PTH1R. The current model suggests that amino acids in the C-terminal portion of PTH (134) bind to the extracellular amino-terminal portion of the PTH1R and that the amino-terminus of PTH (1-34) binds to the juxtamembrane portion of the PTH1R in
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order to activate downstream signaling [37,38] (Fig. 41.3). The PTH1R has been shown to activate both the cAMP/protein kinase A (PKA) pathway and the phospholipase C/protein kinase C/calcium transient pathway. There is also evidence that the receptor activates phospholipase D and MAPK signaling [7,37]. MAPK activation can occur either through a PLC/ PKC-dependent pathway or via a G-protein-independent pathway that involves arrestins [39,40]. Many of the classic biological functions of PTH appear to be mediated to a great extent by the cAMP/PKA pathway, which requires the first two amino acids of PTH for activation [7,37]. Less is known about the biological role of the other signaling pathways. The PTH1R is widely expressed in many cells of the body outside of the skeleton and kidney, the classic target organs of PTH action [7,37]. The receptor at these sites may primarily serve as a PTHrP receptor. However, it must be kept in mind that circulating PTH could
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theoretically activate these receptors and may participate in physiological interactions outside the realm of calcium and bone metabolism, especially in the setting of hyperparathyroidism.
PTH Functions The classic functions of PTH involve the kidney and skeleton. Specific actions of PTH in these organs will be discussed below. Kidney PTH regulates the renal handling of calcium, phosphate, sodium, and hydrogen ions [41]. It also regulates the conversion of 25-hydroxyvitamin D (25(OH)D) to 1,25(OH)2D [41]. The PTH1R is expressed within several nephron segments including the glomerulus, proximal tubules, the cortical ascending limbs, and the distal
FIGURE 41.3 Model for binding of amino-terminal PTH to the PTHR1. The C-terminal end of PTH (1-34) (C) binds first to the extracellular
N-terminal portion of the receptor (B). Subsequently, the amino-terminal portion of PTH 1-34 (D) binds to the J-domain of the receptor. This results in a conformational change of the receptor such that it takes on a more closed shape, which increases its affinity for G-proteins and leads to their activation. Reproduced with permission from [1].
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convoluted tubules. Interestingly, in both the proximal and thick ascending limbs the receptor has been found on both the basolateral surface and the luminal surfaces of cells [42,43]. PTH was originally characterized as a phosphaturic substance [41]. Approximately 90% of plasma phosphate is filtered by the glomerulus and 80% is actively reabsorbed primarily by the proximal convoluted tubules. The rate-limiting step in phosphate reabsorption is entry of phosphate across the luminal membrane of the proximal tubule cells. Most phosphate entry occurs though two related sodiumephosphate cotransporters known as NPT2a and NPT2c, which are found in abundance on the brush border membranes of the proximal tubule [41,44]. PTH inhibits the entry of phosphate into these cells by leading to the withdrawal of NPT2a from the apical membrane into intracellular vesicles, which ultimately leads to its proteolytic degradation [41,45e48]. Interestingly, activation of both the luminal and basolateral PTH1R can lead to internalization of NPT2a although the different receptor populations appear to use different downstream signaling pathways to accomplish this task. Activation of basolateral receptors increases intracellular cAMP, which causes internalization of NPT2a. At the luminal surface, the PTH1R is associated with a scaffolding protein known as NHERF1, which favors the activation of the PKC pathway over the cAMP pathway [49,50]. Both pathways can mediate NPT2a internalization. NHERF1 has also been shown to bind to NPT2a and it has been suggested that dissociation of NHERF1 from NPT2a is necessary to allow internalization of NPT2a in response to PTH [41,51,52]. PTH increases calcium reabsorption by the kidney. This occurs through the stimulation of active transcellular calcium transport across epithelial cells in the cortical segment of the thick ascending loop of Henle and in the distal convoluted tubule [41]. Studies in distal tubule cells have suggested that stimulation of the PTH1R leads to the activation of cAMP/PKA signaling and phospholipase D, both of which are necessary to produce the full effects of PTH [53e58]. These signaling cascades lead to hyperpolarization of the membrane potential, which stimulates both the entry of calcium across the apical membrane as well as its extrusion via sodiumecalcium exchange across the basolateral membrane. PTH acts on the proximal tubule to augment the production of 1,25(OH)2D from 25(OH)D, thus increasing the active form of the hormone [6,7,41,59]. Through the actions of 1,25(OH)2D, PTH indirectly stimulates calcium absorption from the gut and activates bone cell activity. The increase in 1a-hydroxylase activity is the result of both up-regulation of the expression of the 1a-hydroxylase gene and the stimulation of 1a-hydroxylase enzymatic activity [41,60,61]. The
former appears to result primarily from activation of the cAMP pathway, while the latter effect may be the result of PKC signaling [41,62e65]. The effects of PTH on 1a-hydroxylase activity are inhibited by the CaSR and by 1,25(OH)2D itself [66e68]. PTH also inhibits the degradation of 1,25(OH)2D by decreasing 24-hydroxylase activity (the first step in vitamin D catabolism) in the proximal tubule [69,70]. PTH modulates sodiumehydrogen ion exchange in the proximal tubule, inhibiting acid secretion and leading to a mild compensated hyperchloremic acidosis [41]. This is the result of phosphorylation of the Naþ/ Hþ exchanger (NHE3) by PKA downstream of the PTH1R [48,71]. Although this action results in reduced bicarbonate and sodium reabsorption by the proximal tubule, the distal tubules compensate by increasing both sodium and bicarbonate absorption so that there is little change in serum bicarbonate concentrations or pH. In addition, sodium excretion is minimal. However, chronic elevations in PTH are often associated with mild hyperchloremia. The Skeleton PTH has complicated effects on the skeleton. Despite years of study, there are still many areas of uncertainty especially with regard to the effects of PTH on the osteoblast lineage [6,41,72,73]. There is no doubt that PTH increases bone turnover although it has long been established that the net effect on bone depends on the pattern of PTH administration. Continuous exposure to PTH causes net bone catabolism, which is seen in states of hyperparathyroidism. In contrast, intermittent exposure to PTH has an anabolic effect on the skeleton, which has been exploited pharmacologically in the treatment of osteoporosis. Below, we will discuss the actions of PTH on the three main cell types in bone, osteoclasts, osteoblasts, and osteocytes. PTH stimulates bone resorption due to an increase in the numbers and activity of osteoclasts [6,41,72,73]. Neither osteoclasts nor their precursors express the PTH1R, so this effect is the result of crosstalk between cells in the osteoblast lineage, which do bear the PTH1R, and osteoclasts. Osteoclast differentiation, activity, and survival depend on the actions of two cytokines, colony stimulating factor 1 (MCSF, CSF1) and receptor-activator of NFkB ligand (RANKL) [74,75]. CSF1 is produced by stromal/osteoblast cells and acts through its receptor c-fms to induce early monocyte/ macrophage precursors to enter the osteoclast lineage [74,75]. RANKL is also produced by cells in the stroma/osteoblast lineage and acts through its receptor, receptor-activator of NFkB (RANK) to promote the differentiation of osteoclast precursors into mature osteoclasts, to increase the bone-resorbing activity of mature osteoclasts and to increase the life-span of active
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osteoclasts [41,73e75]. RANKL can also be bound by a secreted decoy receptor known as osteoprotegerin (OPG), which inhibits osteoclast formation and activity and promotes osteoclast apoptosis [41,73e75]. Hence, in practice, the RANKL/OPG ratio is a dominant regulator of local osteoclastic activity (Fig. 41.4). PTH acts on cells in the stromal/bone-lining/osteoblast lineage to increase the secretion of both CSF1 and RANKL, thus increasing osteoclast numbers and activity [41,72,73,75,76]. Furthermore, PTH suppresses the secretion of OPG and raises the RANKL/OPG ratio, further boosting osteoclast numbers and bone resorption. These actions of PTH on bone are also discussed in the chapters on osteoblasts (Chapters 16 and 17) and osteoclasts (Chapter 18). PTH directly regulates the synthetic function of osteoblasts. The effects of PTH on these cells are complicated and vary depending on the duration of exposure to PTH as well as the state of differentiation of the cells. The reader is referred to more in-depth reviews for a detailed discussion of the specific effects of PTH on osteoblasts [41,72,73,76,77]; the following is an overall summary of these data. Both continuous (hyperparathyroidism) and intermittent (therapeutic) exposure to PTH lead to increased numbers of osteoblasts and to increased rates of bone formation in vivo [41,72,73,76,77]. With intermittent exposure to PTH, the increase in bone resorption is delayed relative to the increase in bone formation, resulting in an initial anabolic effect. The increase in osteoblasts does not appear to be the result of cell proliferation as most data suggest that either intermittent or
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continuous exposure to PTH inhibits the proliferation of committed osteoblast precursors and mature osteoblasts [73,78e80]. Intermittent exposure to PTH favors the commitment of mesenchymal precursors to the osteoblast lineage and stimulates osteoblast differentiation [41,73,81e84]. In addition, studies in vivo suggest that intermittent PTH administration can convert previously quiescent bone-lining cells into active osteoblasts [85]. In contrast, prolonged, continuous exposure to PTH appears to inhibit the latter stages of osteoblast differentiation [41,59,73]. Finally, several studies have suggested that intermittent PTH can inhibit osteoblast apoptosis [86e88]. However, there is some disagreement over this issue and there may be skeletal site-specific effects of PTH on osteoblast apoptosis [73]. The use of PTH to treat osteoporosis is discussed in Chapter 61. Osteocytes are also an important skeletal target for PTH action [89]. The PTH1R is expressed on these cells and amino-terminal fragments of PTH have been described to inhibit osteocyte apoptosis and to suppress the production of sclerostin, an important inhibitor of Wnt signaling in bone [77,90e92]. Recent experiments in mice have suggested that activation of the osteocyte PTH1R can increase bone mass and influence the activity of osteoblasts and osteoclasts on bone surfaces, demonstrating that the osteocyte may help to coordinate the hormonal regulation of bone turnover [92]. PTH may also modulate the ability of osteocytes to respond to mechanical stimuli [89]. In addition, C-terminal fragments of PTH affect osteocytes by binding to a specific C-terminal PTH receptor. It appears that activation of
FIGURE 41.4 Model for interactions between osteoblasts/stromal cells and osteoclasts as they differentiate. Osteoblasts secrete M-CSF (CSF-1) and RANKL, two critical cytokines necessary for the proliferation, fusion, and differentiation of osteoclast precursors to generate mature osteoclasts. Osteoblasts also secrete OPG, a soluble decoy receptor for RANKL, that inhibits the interaction of RANKL with its receptor, RANK. PTH as well as other hormones and local cytokines increases the production of M-CSF and RANKL by cells in the osteoblast lineage. PTH also inhibits the production of OPG by these same cells. This alters the local RANKL/OPG ratio, promoting the proliferation, differentiation, activity, and survival of osteoclasts, which, in turn, increases the rate of bone resorption. Reproduced with permission from [192].
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this receptor antagonizes PTH1R actions, at least with respect to osteocyte apoptosis [89].
Pathophysiology Disease may be associated either with excess PTH secretion or with lack of sufficient PTH secretion. A detailed discussion of hyperparathyroidism and hypoparathyroidism is beyond the scope of this chapter. We will simply outline the expected alterations in calcium metabolism in both instances. Excess production of PTH by abnormal parathyroid cells occurs in primary hyperparathyroidism. This disorder is most commonly caused by a benign clonal neoplasm of parathyroid cells known as a parathyroid adenoma, but can also result from multiple adenomas, parathyroid hyperplasia, parathyroid carcinoma, null mutations in the CaSR gene and, rarely, blocking autoantibodies to the CaSR [93e95]. Primary hyperparathyroidism is diagnosed by the simultaneous elevation of circulating calcium and PTH levels and should be distinguished from secondary hyperparathyroidism, in which the PTH level is elevated, but the calcium level is normal or low [93,94]. Secondary hyperparathyroidism most commonly occurs in patients with chronic kidney disease. Patients with primary hyperparathyroidism can suffer from kidney stones, osteoporosis, and excess fractures. The disorder has also been associated with neurocognitive deficits, insulin resistance, the metabolic syndrome, increased risk of cardiovascular disease and excess risk of cancer [93e95]. However, several of these latter associations are controversial and many patients with mild hyperparathyroidism appear to be asymptomatic [94,95]. The only cure is surgical removal of the offending parathyroid adenoma, but it is not clear if all asymptomatic patients require surgical cure of their hyperparathyroidism [96]. As a result, over the last
TABLE 41.1
20 years, a series of working groups have issued guidelines to aid clinicians in deciding on appropriate therapy for patients with asymptomatic disease [96] (Table 41.1). Medical therapy with cinacalcet, a CaSR agonist, will normalize serum calcium and reduce serum PTH. However, unlike parathyroidectomy, treatment with cinacalcet will not result in reduced bone turnover and restoration of a positive bone balance, so it is not considered to be a replacement for parathyroidectomy [97]. Hypoparathyroidism is defined as hypocalcemia and hyperphosphatemia in the setting of a low PTH level. Most commonly, this disorder is associated with genetic conditions leading to the agenesis of the parathyroid glands or with processes that destroy the parathyroid glands once they are formed [98]. Symptoms depend on the severity of hypocalcemia and include weakness, lassitude, muscle fasiculations, cramping, seizures, hypotension, and cardiovascular collapse. Treatment of hypoparathyroidism is aimed at restoring normal serum calcium. Absence of PTH in turn leads to reduced activity of renal 1-a hydroxylase and levels of serum 1,25(OH)2D that are inadequate, so supplementation in the form of oral calcitriol is provided, along with oral calcium supplements. Dosages of each are typically titrated to yield a serum calcium at the low end of the normal range and to minimize hypocalcemic symptoms. Careful monitoring is required, as excessive supplementation with calcitriol may lead to hypercalcemia and hyperphosphatemia. In the absence of the hypocalciuric effect of PTH, hypercalciuria may develop with resultant nephrolithiasis and nephrocalcinosis [98], hence the need to keep serum calcium at the low end of the normal range. Hypoparathyroidism also can be successfully treated with PTH itself [99,100]. However, given the short half-life of PTH, replacement therapy is limited by cost and inconvenience, so most patients are still treated with vitamin D analogs and calcium.
Comparison of New and Old Guidelines for Parathyroid Surgery in Asymptomatic PHPTa
Measurement
1990
2002
2008
Serium calcium (>upper limit of normal)
1e1.6 mg/dl (0.25e0.4 mmol/liter) 1.0 mg/dl (0.25 mmol/liter) 1.0 mg/dl (0.25 mmol/liter)
24-h urine for calcium
>400 mg/d (> 10 mmol/d)
>400 mg/d (>10 mmol/d) Not indicatedb
Creatinini clearance (calculated)
Reduced by 30%
Reduced by 30%
BMD
Z-score <2.0 in forearm
T-score <2.5 at any site
T-score <2.5 at any sitec and/ or previous fracture fragilityd
Age (yr)
<50
<50
<50
Reduced to < 60 ml/min c
a
Surgery is also indicated in patients for whom medical surveillance is neither desired nor possible. Some physicians still regard 24-h urinary calcium excretion >400 mg as an indication for surgery c Lumbar spine, total hip, femoral neck, or 33% radius (1/3 site). This recommendation is made recognizing that other skeletal features may contribute to fracture risk in PHPT and that the validity of this cut-point for any site vis-a`-vis fracture risk prediction has not been established in PHPT. d Consistent with the position established by the International Society for Clinical Densitometry, the use of Z-scores instead of T-scores is recommended in evaluating BMD in premenopausal women and men younger than 50 yr. Reproduced with permission from [96]. b
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Clinical Applications The observations that intermittent administration of PTH induces anabolic responses in the skeleton has led to the development of PTH analogs for the treatment of osteoporosis [1]. A landmark study published in 2001 demonstrated that once daily subcutaneous injections of PTH (1-34) increased bone density and decreased spine and hip fractures in postmenopausal women with osteoporosis [101]. Subsequent studies have demonstrated effectiveness in males with osteoporosis and in patients taking glucocorticoids [77,102]. Interestingly, some data suggest that PTH (1-34) may improve bone microarchitecture as well as bone mass [103]. Other analogs of PTH are also in development for the treatment of osteoporosis, including PTH (1-86) [77,102].
PARATHYROID-HORMONE-RELATED PROTEIN Introduction Fuller Albright first hypothesized in 1941 the existence of a parathyroid hormone (PTH)-like humor that caused hypercalcemia in some patients with cancer [104]. In the 1980s and 1990s the efforts of a number of investigators led to the biochemical characterization of humoral hypercalcemia of malignancy (HHM), the isolation of parathyroid-hormone-related protein (PTHrP) and the characterization of its gene [105e109]. We now know that PTHrP is related to PTH and that PTHrP shares with PTH the use of the classical PTH receptor (type I PTH/PTHrP receptor, PTH1R) [7,110,111]. PTHrP usually serves a local autocrine, paracrine, or intracrine role and normally does not circulate. However, in patients with HHM, PTHrP does reach the circulation and mimics the systemic actions of PTH. Not surprisingly, Fuller Albright was indeed prescient in his predictions.
The PTHrP Gene The human PTHrP gene is located on the short arm of chromosome 12. It encompasses eight exons and at least three promoters [106,109,110]. Alternative splicing at the 30 end of the gene gives rise to three distinct families of mRNA encoding proteins of 139, 141, or 173 amino acids. There is additional splicing of the 50 end of the gene so that each different 30 coding sequences can have a series of different 50 untranslated sequences. Although some data suggest that different cells may preferentially produce different 30 splice variants of PTHrP, the regulation of alternative splicing of PTHrP mRNA is not well understood. Moreover, the physiological significance of these different PTHrP transcripts
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remains unclear especially since in rodents and lower vertebrates, such as birds and fish, the gene has a much simpler structure [7,110,112]. The PTHrP and PTH genes share structural elements and sequence homology demonstrating that they are related, and that they likely arose through duplication of a common ancestor [106,108e110] (Fig. 41.1). There is high sequence homology at the amino-terminal portion of both genes such that the peptides share eight of the first 13 amino acids and a high degree of predicted secondary structure over the next 21 amino acids. Beyond this, however, the two genes diverge. PTHrP mRNA has been found in almost every organ at some time during its development or functioning. Many different hormones and growth factors regulate the transcription and/or stability of PTHrP mRNA. As with PTH, the CaSR has been found to regulate PTHrP gene expression in many cells [113,114]. Another common theme is the observation that mRNA levels are induced by mechanical forces [110]. 1,25(OH)2D has been shown to suppress PTHrP gene expression in several different cell types in vitro. It is not clear if this is important in vivo, although it is tempting to speculate that a local feedback loop involving PTHrP, 1,25(OH)2D and 1-a hydroxylase may exist in epithelial tissues. Locally produced PTHrP might up-regulate expression and activity of tissue 1-a hydroxylase (akin to the effects of PTH on the kidney), leading to increased local production of 1,25(OH)2D, which, in turn, would feedback to inhibit further PTHrP production. Dysregulation of such a local feedback loop may be important in the development or progression of epithelial malignancies.
PTHrP Protein Similar to the pro-opiomelanocortin (POMC) or calcitonin/CGRP genes, the primary translation product of PTHrP undergoes post-translational processing to generate a series of biologically active peptides [110]. The preepro sequence from amino acids e36 to 1, directs the nascent protein into the secretory pathway, after which these amino acids are removed. PTHrP’s primary sequence contains clusters of basic amino acids that direct processing enzymes to generate different peptides in a cell-type-specific manner. Although the details of PTHrP processing and the biological significance of the different PTHrP peptides are not entirely clear, several specific secreted forms of PTHrP have been defined. PTHrP 1-36 is secreted from a variety of cell types [110,115]. Longer forms of PTHrP containing the amino-terminus are also secreted from keratinocytes and mammary epithelial cells, and circulate in patients with cancer and during lactation [116e118]. The amino-terminus is necessary for interaction with the PTH1R. The secretion of mid-region peptides including
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amino acids 38-94, 38-95 and 38-101 has also been described [110,119]. The biology of these specific secretory forms is unclear, but the mid-region of PTHrP stimulates placental calcium transport and modulates renal bicarbonate handling, and this portion of the molecule contains nuclear localization signals (see below) [120e122]. Finally, C-terminal fragments consisting of amino acids 107-138 and 109-138 have been described. These peptides have been reported to inhibit osteoclast function and stimulate osteoblast proliferation [110,121].
lines, nuclear PTHrP has been implicated in the regulation of proliferation and/or apoptosis. Replacement of the endogenous mouse PTHrP gene with a mutant version encoding a protein that cannot enter the nucleus causes widespread cellular senescence, growth retardation, and early death [126]. This experiment suggests that nuclear PTHrP is of fundamental importance in many cell types.
PTHrP Functions PTHrP Receptors Like PTH, PTHrP binds to and activates the PTH1R. Most studies in vitro suggest that this receptor binds PTHrP and PTH with equal affinity and that both peptides trigger identical signaling events and biological effects. This is also true when amino-terminal fragments of PTH and PTHrP are infused into animals [7,110]. However, human subjects subjected to continuous infusion of the two peptides for 72 hours, were found to become hypercalcemic with lower doses of PTH 1-34 than PTHrP (1-36) [123]. In these same studies, PTHrP was also much weaker than PTH at stimulating renal 1,25(OH)2D production. This may be explained by physical differences in the binding of the two peptides to different conformational states of the receptor, so that the duration of cAMP production is shorter for PTHrP (1-36) than for PTH (1-34) [124]. Thus, the human PTH1R may respond differently to PTH and PTHrP, and this might help to explain differences in the biochemical profiles of HHM and hyperparathyroidism (see below).
Nuclear PTHrP Immunohistochemical studies have localized PTHrP to the nucleus of many different cell types [121,125]. Specific nuclear localization sequences (NLS) located between amino acids 84e93 allow PTHrP to shuttle into and out of the nucleus in a regulated fashion. This process requires binding to microtubules and a specific shuttle protein known as importin b1, which allows PTHrP to transit the nuclear pore [121,125]. Nuclear export is facilitated by a related shuttle protein known as CRM1 [121]. The nuclear trafficking of PTHrP is not completely understood but phosphorylation at Thr85 by the cell-cycle-regulated, cyclin-dependent kinase, p34cdc2 regulates nuclear import in a cell-cycle-dependent fashion [121]. The function(s) of nuclear PTHrP is unclear, but it can bind RNA and localizes to the nucleolus. This has led to the suggestion that PTHrP may be involved in regulating RNA trafficking, ribosomal dynamics, and/or protein translation [121,125]. In cell
Like many growth factors or cytokines, PTHrP has been suggested to have many functions. The reader is referred to more comprehensive reviews for a complete discussion of these findings [110,111]. What follows is a brief outline of areas where PTHrP has been rigorously documented to have physiological effects in intact organisms. The Skeleton Disruption of the PTHrP gene in mice alters chondrocyte differentiation in the growth plates of long bones and in costal cartilage and causes short-limbed dwarfism and a shield chest. Disrupting the PTH1R gene generates a similar phenotype and overexpressing PTHrP or a constitutively active PTH1R within growth plate chondrocytes in transgenic mice produces the opposite effect [127e129]. These and other animal models have documented that amino-terminal PTHrP acts through the PTH1R to coordinate the rate of chondrocyte differentiation in order to maintain the orderly growth of long bones during development [130]. PTHrP is secreted primarily by immature chondrocytes at the top of the growth plate in response to another molecule known as Indian Hedgehog (IHH) produced by differentiating hypertrophic chondrocytes. PTHrP, in turn, acts on its receptor located on proliferating and prehypertrophic cells to slow their rate of differentiation into hypertrophic cells. In this manner, IHH and PTHrP act in a local negative feedback loop to regulate the rate of chondrocyte differentiation (see Fig. 41.5) [130]. PTHrP is also produced in other cartilaginous sites such as the perichondrium that surrounds the costal cartilage and the subarticular chondrocyte population immediately subjacent to the hyaline cartilage lining the joint space [131,132]. In both of these sites, PTHrP appears to prevent hypertrophic differentiation of chondrocytes and the inappropriate encroachment of bone into these structures [131,132]. PTHrP also has important anabolic functions in bone. Heterozygous PTHrP-null mice are normal at birth, but develop osteopenia with increasing age [133]. In addition, selective deletion of the PTHrP gene from osteoblasts results in a decreased bone mass, reduced bone
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FIGURE 41.5 PTHrP and Indian hedgehog (Ihh) act as part of a negative feedback loop regulating chondrocyte proliferation and differentiation. The chondrocyte differentiation program proceeds from undifferentiated chondrocytes at the end of the bone, to proliferative chondrocytes within the columns and then to prehypertrophic and terminally differentiated hypertrophic chondrocytes nearest the primary spongiosum. PTHrP is made by undifferentiated and proliferating chondrocytes at the ends of long bones. It acts through the PTH1R on proliferating and prehypertrophic chondrocytes to delay their differentiation, maintain their proliferation and delay the production of Ihh, which is made by hypertrophic cells (1). Ihh, in turn, increases the rate of chondrocyte proliferation (2) and stimulates the production of PTHrP at the ends of the bone (3). Ihh also acts on perichondrial cells in order to generate osteoblasts of the bone collar (4). Reproduced with permission from [193].
formation, and mineral apposition, and a reduction in the formation and survival of osteoblasts [134]. These data suggest that PTHrP acts as an important local anabolic factor in the skeleton. Mammary Gland Not long after its discovery, PTHrP mRNA was found to be expressed in the lactating breast and PTHrP protein was measured in high concentrations in milk [135,136]. It is now known that PTHrP has important functions during breast development, is involved in regulating systemic calcium metabolism during lactation and contributes to the pathophysiology of breast cancer. The mammary gland forms as a bud-like invagination of epidermal cells that grow down into a developing fatty stroma as a branching tube that becomes the mammary duct system [137]. In mice, as soon as the mammary bud begins to form, epithelial cells produce PTHrP, which interacts with the PTH1R expressed on surrounding mesenchymal cells. This interaction is necessary for
proper differentiation of the dense mammary mesenchyme that surrounds the embryonic mammary bud so that these mesenchymal cells can maintain the mammary fate of the epithelial cells, initiate outgrowth of the duct system, and stimulate the formation of the specialized epidermis that comprises the nipple [138] (Fig. 41.6). The formation of the breast in human fetuses is similar to the formation of the fetal mammary gland in mice and PTHrP is necessary for the formation of breast epithelium in humans as well [139]. PTHrP is produced by breast epithelial cells during lactation and large quantities are secreted into milk [118,135]. The function of PTHrP in milk consumed by the suckling infant is unclear; ongoing studies are examining this issue. PTHrP is also secreted from the lactating breast into the maternal circulation, where it participates in the regulation of systemic calcium metabolism. The maternal skeleton is an important source of calcium for milk production and elevated rates of bone resorption and rapid bone loss are well documented in both nursing women and rodents [140]. Elevated levels of
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PTHrP regulates mesenchymal cell fate during embryonic mammary development. (A) During normal mammary development, PTHrP is secreted by epithelial cells within the forming mammary bud (circles) and interacts with the immature dermal mesenchyme (ovals) to induce formation of the dense mammary mesenchyme (light squares). These cells, in response to PTHrP, maintain the fate of the mammary epithelial cells, initiate branching morphogenesis and induce the formation of the specialized nipple skin (dark squares). (B) In PTHrP- or PTH1R-knockout embryos, the mammary bud forms, but the mammary mesenchyme does not. As a result, the mammary epithelial cells revert to an epidermal fate (ovals), morphogenesis fails and the nipple never forms. Adapted with permission from [194].
FIGURE 41.6
PTHrP correlate with bone loss during lactation in humans, and circulating levels of PTHrP correlate directly with rates of bone resorption and inversely with bone mass in mice [117,141]. In addition, mammary-specific disruption of the PTHrP gene during lactation reduces circulating PTHrP levels, lowers bone turnover, and preserves bone mass. These data demonstrate that the lactating breast secretes PTHrP into the circulation to increase bone resorption [118]. The lactating breast also expresses the CaSR, which signals to suppress PTHrP secretion in response to an increase in calcium delivery to the breast [114]. These interactions define a classical endocrine negative feedback loop, whereby mammary cells secrete PTHrP to mobilize calcium from the bone. Calcium, in turn, feeds back to inhibit further PTHrP secretion from the breast. Therefore, during lactation, the breast and bone communicate to regulate the mobilization of skeletal calcium stores in order to ensure a steady supply of calcium for milk production (see Fig. 41.7).
calcium transport [120,122]. Therefore this action of PTHrP cannot be mediated by the PTH1R.
Smooth Muscle and the Cardiovascular System Mechanical deformation increases the expression of PTHrP in many smooth muscle cell beds [110]. In turn, PTHrP acts in an autocrine or paracrine fashion through the PTH1R to relax the muscle that has been stretched [110,111,144e146]. In the stomach, bladder, or uterus, this feedback loop may be important in allowing for gradual filling. In the vasculature, PTHrP is induced by vasoconstrictive agents as well as stretch itself, and acts as a vasodilator to resistance vessels. Given these actions, PTHrP may act as a local modulator of blood flow [147].
Placenta During pregnancy, calcium must be actively transported across the placenta from mother to fetus. Furthermore, circulating calcium concentrations in the fetus are higher than in the mother, so that calcium must be transported against a gradient [140]. In PTHrPe/e mice, this gradient is abolished and PTHrP-deficient fetuses are relatively hypocalcemic, suggesting that fetal PTHrP is important in mediating placental calcium transport from the mother [122]. The source of the PTHrP is likely the placenta itself and placental production of PTHrP has been shown to be regulated by the CaSR [142,143]. Interestingly, experiments in sheep and mice have demonstrated that it is mid-region PTHrP, not the amino-terminal portion, that is responsible for placental
The breast and the skeleton communicate during lactation in order to provide a steady supply of calcium for milk production. The lactating breast secretes PTHrP into the systemic circulation during lactation. PTHrP interacts with the PTH1R in bone cells in order to increase the rate of bone resorption and liberate skeletal calcium stores. Mammary epithelial cells in the lactating breast express the CaSR and suppress PTHrP production in response to increased delivery of calcium, defining a classical endocrine negative feedback loop between breast and bone.
FIGURE 41.7
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PTHrP regulates the proliferation of vascular smooth muscle cells (VSMCs). Secreted amino-terminal PTHrP inhibits the proliferation of VSMCs by activating the PTH1R. However, mid-region and C-terminal portions of PTHrP act in the nucleus to stimulate the proliferation of these cells [148,149]. Furthermore, PTHrP expression is up-regulated by vascular damage following balloon angioplasty and in atherosclerotic lesions in rodents and in humans. Several studies have suggested that PTHrP plays an important role in the response of these VSMCs to injury and may contribute to the pathophysiologic development of a neo-intima following angioplasty [150,151]. Teeth Teeth develop within a cavity or crypt that is surrounded by alveolar bone. After they are formed, teeth must erupt through the roof of the dental crypt in order to emerge into the oral cavity. Tooth eruption relies on locally uncoupled bone turnover in which osteoclasts form over the crown of the tooth in order to resorb the overlying bone and bone formation at the base of the tooth propels it upward out of the crypt. Normally, just before the onset of eruption, PTHrP is produced by stellate reticulum cells and signals to dental follicle cells to drive the formation of osteoclasts above the crypt. In the absence of PTHrP, these osteoclasts do not appear, eruption fails to occur and teeth become impacted by the surrounding bone [152e154].
Pathophysiology HHM Humoral hypercalcemia of malignancy or HHM refers to a clinical syndrome in which patients with cancer develop hypercalcemia in the absence of significant tumor burden in the skeleton. Classically, HHM is associated most commonly with squamous cancers of the lung and head and neck, and with urothelial tumors such as transitional cell cancer of the bladder and renal cell carcinoma. However, many other tumor types have now been associated with the syndrome as have some benign tumors. The syndrome is defined by a typical biochemical signature, which includes an elevated serum calcium, a normal or low serum phosphate with a low TMP/GFR, low 1,25(OH)2D levels, suppressed PTH levels, and an elevated nephrogenous cAMP level. Many studies have demonstrated that this syndrome is caused by tumor cells secreting PTHrP into the circulation, and systemic levels of PTHrP are elevated when measured. Since PTHrP and PTH both activate the same PTH1R, when PTHrP is released into the circulation by tumors, it mimics the actions of PTH and produces a syndrome similar to primary
hyperparathyroidism (PHPT). The principal differences between HHM and PHPT include the low PTH levels in HHM and the lower 1,25(OH)2D levels in HHM. Recent studies have confirmed that PTHrP is less potent than PTH at stimulating 1a-hydroxylase activity and this appears to reflect differences in the ability of PTH and PTHrP to hold the PTH1R in a stably activated state. The most effective therapy for HHM is to treat the primary tumor. However, HHM is often a complication of advanced disease and surgical cure is often not possible. Therapy then focuses on resolution of hypercalcemia, which can be approached with a combination of hydration, forced saline diuresis, and treatment with bisphosphonates and/or denosumab in order to lower rates of bone resorption. Bone Metastases Lytic bone lesions are a feared complication of many malignancies including breast and prostate cancer and multiple myeloma. They are associated with hypercalcemia, bone pain, pathologic fractures, and increased mortality. Histological examination of lytic bone metastases typically demonstrates a halo of active osteoclasts between the tumor cells and the surrounding bone. It is known that the metastatic cells secrete soluble factors that enhance the local production and activity of otherwise normal osteoclasts to generate the osteolysis required for tumor expansion. It has also been demonstrated that bone resorption releases growth factors from the bone matrix that enhance the proliferation and survival of the tumor cells. This feed-forward loop or “vicious cycle” of interactions between bone and tumor cells enables the growth and lytic nature of the metastatic lesions [155]. A variety of growth factors, including PTHrP, have been shown to partake in establishing the vicious cycle of osteolysis. In particular, breast cancer cells have been shown to up-regulate PTHrP production in response to TGF-b, released from the bone microenvironment. In turn, PTHrP increases the local production of RANKL and enhances osteoclastic bone resorption leading to the release of more TGF-b.
CALCITONIN Introduction Calcitonin was first described in 1962 by Copp and Cheney who discovered its calcium-lowering actions in hypercalcemic dogs. It was thus named for its role in regulating calcium “tone.” Ultimately it was found to be produced by C-cells located in the thyroid gland [156,157]. Shortly after this, its ability to inhibit calcium release from bone was described [158]. Since then, its role in maintaining calcium homeostasis has been
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extensively studied although the physiological role of calcitonin in humans is still uncertain. Despite this, a variety of clinical uses for calcitonin have been defined.
Gene and Protein Calcitonin is produced by the C-cells of the thyroid gland in humans and by the ultimobranchial body in other animal species. The C-cells are derived embryologically from neural crest cells. During development, they migrate to the ultimobranchial body, a structure derived from the ventral portion of the fourth pharyngeal pouch. The ultimobranchial body later fuses with the developing thyroid gland allowing the C-cells to distribute to their final locations within the thyroid. In lower vertebrates, including reptiles, amphibians, fish, and birds, the ultimobranchial body remains a separate structure throughout life. Calcitonin is encoded by the CALC1 (also referred to as CALCA) gene, which is a member of a family of genes also encoding islet amyloid protein, calcitonin gene related peptide (CGRP), and the precursor of adrenomedullin. The CALC1 gene is located on chromosome 11 and consists of six exons, the first four of which encode calcitonin. In addition, alternative splicing of the CALC1 transcript to include exons 1e3, 5, and 6 yields a homologous peptide, calcitonin gene-related peptide (CGRP), which is produced in the nervous system [159] (Fig. 41.8). The calcitonin transcript directs translation of preprocalcitonin. The signal peptide is rapidly cleaved in the endoplasmic reticulum to yield procalcitonin and then subsequent proteolytic processing yields the mature, 32-amino-acid form of calcitonin [160].
Calcitonin Receptor The calcitonin receptor is a GPCR [161] in the same group B sub-family that also includes the receptors for vasoactive intestinal peptide, secretin, and PTH and
PTHrP [162]. Calcitonin receptor activation leads to a rise in cAMP/PKA and PKC activity. Calcitonin receptors have been described in many tissues throughout the body. The osteoclast is the primary target of calcitonin, but receptors also are present in the kidney, osteocytes, brain, testes, placenta, and lung [161]. Calcitonin receptor isoforms generated from alternative splicing have been described in a number of normal tissues and tumors. Some of these isoforms are nonfunctional and may have a regulatory effect by inhibiting calcitonin signaling [163]. The calcitonin receptor and other members of its family may complex to accessory proteins called receptor-activity-modifying proteins or RAMPs that change the cellular location or phenotype of the receptor. In the case of the calcitonin receptor, the association with a RAMP results in the creation of the receptor for amylin [162].
Regulation of Calcitonin Secretion Calcitonin is released in response to rising serum calcium levels within the normal physiologic range [164]. Like the parathyroid cells, the C-cells express the CaSR on their cell surface and thus are sensitive to small changes in extracellular fluid calcium concentrations. A rise in extracellular fluid calcium activates the CaSR on C-cells, which causes an influx of calcium through voltage-sensitive calcium channels. The resultant rise in intracellular calcium leads to the release of calcitonin. This signaling pathway in C-cells differs from that of parathyroid cells as it is release of calcium from intracellular stores in the parathyroid cell (rather than voltage-sensitive calcium channels) that is the primary modulator of PTH release [165]. Plasma calcitonin levels reflect primarily the rate of secretion from the C-cell, with degradation of the hormone occurring within minutes in the plasma and more significantly by the kidney [166]. In addition to calcium, a variety of gut hormones also control calcitonin release. Gastrin, a hormone produced by the parietal cells of the stomach and duodenum in
Both calcitonin and CGRP are the products of the same CALC1 gene. Alternative splicing of exons 4 and 6 leads to either calcitonin mRNA or CGRP mRNA. Reproduced with permission from [195].
FIGURE 41.8
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response to a meal or to hypercalcemia, acts on specific receptors on the C-cell to stimulate calcitonin release [167]. In contrast, somatostatin appears to down-regulate calcitonin production [168,169]. These relationships may activate calcitonin release in response to, or in preparation for, alterations in postprandial calcium absorption from the gut. Vitamin D also plays a role in calcitonin production, exerting an inhibitory effect on calcitonin gene transcription [170].
Calcitonin Functions In humans, calcitonin does not appear to be essential for maintenance of minute-to-minute calcium homeostasis, but it may have a role in the conservation of total body calcium, especially with aging and/or during periods of calcemic stress such as pregnancy or lactation [171]. The primary target of calcitonin in the skeleton is the osteoclast. Osteoclasts have abundant calcitonin receptors and exposure to calcitonin inhibits bone resorption rapidly [77,171]. Activation of the calcitonin receptor inhibits osteoclast motility and then causes retraction of the cell from the bone surface and disassembly of the ruffled border necessary for bone resorption [172e174]. These effects are the result of both an increase in cAMP as well as increases in intracellular calcium, and are associated with disruption of the cytoskeletal actin ring necessary for formation of a sealed resorption lacuna on the bone surface [171,172]. Activation of the calcitonin receptor also inhibits the production of tartrate-resistant acid phosphatase and carbonic anhydrase, two enzymes required for osteoclast function [171,172,175,176]. Calcitonin signaling causes a reduction in the number of calcitonin receptors expressed by osteoclasts and rapid development of calcitonin resistance, limiting its pharmacological use as an antiresorptive agent [177]. It has been difficult to determine if calcitonin contributes to the regulation of normal bone turnover or bone mass in any significant fashion in humans [171]. Reductions in circulating levels of calcitonin in patients with missing thyroid glands do not appear to affect mineral metabolism or bone mass [178,179]. Similarly, no defects in bone or mineral metabolism are evident in patients with elevated calcitonin levels due to medullary thyroid cancer [179]. Nonetheless, recent experiments in knockout mice have suggested that the disruption of calcitonin signaling results in an impaired ability to recover from hypercalcemia and excessive bone loss during lactation [180,181]. Most surprisingly, ablation of the calcitonin and calcitonin receptor genes has been associated with an increase in trabecular bone volume, suggesting that calcitonin may regulate bone formation [182,183]. A recent study has suggested that the effects of calcitonin
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on osteoblasts may be mediated by osteocytes. Gooi and colleagues demonstrated the presence of calcitonin receptor on osteocytes in vivo and noted that calcitonin treatment increased sclerostin in osteocytes, which would be expected to inhibit bone formation [184]. Thus, much remains to be resolved about calcitonin’s effects on the skeleton. Animal studies suggest that the role of calcitonin in lowering serum calcium may also be mediated through regulating levels of 1,25(OH)2D. Calcitonin down-regulates activity of the renal 1-a hydroxylase, thus reducing production of 1,25(OH)2D [68]. It also activates the renal 24-hydroxylase, an enzyme responsible for inactivating 1,25(OH)2D [185]. In addition to the skeleton, calcitonin receptors are expressed in many tissues and calcitonin may also be produced outside the thyroid gland [171]. This has led to many studies on the extraskeletal effects of calcitonin in organs such as the brain, lungs, uterus, placenta, mammary gland, and GI tract. In addition, the related CGRPs are widely expressed and knockout of the calcitonin receptor in mice leads to embryonic death [183]. Although we will not address the extraskeletal roles of calcitonin, these functions are clearly biologically significant.
Pathophysiology In humans it appears that calcitonin does not play a major role in regulating serum calcium levels. Evidence for this comes from individuals who have undergone thyroidectomy, where serum calcitonin levels are typically low or unmeasureable, yet serum calcium remains normal [178,179]. The few available studies of bone mineral density on patients post-thyroidectomy have shown variable effects of chronic lack of calcitonin, with some studies showing no effect on bone and others showing a detrimental effect [186,187]. A longer duration of follow-up may explain the finding of bone loss in the setting of low calcitonin in one study [187] but not the other [186]. High levels of serum calcitonin may be seen in patients with medullary thyroid cancer, a rare tumor derived from the C-cells. Individuals who have a considerable tumor burden and whose tumors produce high amounts of calcitonin may experience symptoms from the high calcitonin levels, including diarrhea and flushing. These individuals do not typically have hypocalcemia, however, perhaps due to the counter-effects of PTH [179].
Clinical Applications Calcitonin is a convenient serum tumor marker for medullary thyroid carcinoma, and can be used in longitudinal monitoring of patients for signs of recurrence or
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progression after surgery. There is considerable variability in the ability of individual medullary thyroid cancers to produce calcitonin, so baseline levels drawn prior to surgery are needed to interpret results [188]. The effects of calcitonin on the osteoclast have made it a useful agent in treatment of bone and calcium disorders [77,171]. Acute hypercalcemia in most cases results from increased osteoclast-mediated bone resorption, such as occurs in primary hyperparathyroidism, HHM, or local osteolytic hypercalcemia. In these conditions, subcutaneous calcitonin administration leads to prompt inhibition of osteoclast activity and a rapid reduction of serum calcium. Unfortunately, tachyphylaxis to the effects of calcitonin develops within a few days, so the benefits of calcitonin therapy in this setting are short-lived. Recombinant salmon calcitonin is used therapeutically as it is more potent than human calcitonin and has a longer duration of action. Intranasal calcitonin has been used in the treatment of osteoporosis, where its effects are modest in comparison with other currently available pharmaceuticals, such as the bisphosphonates. Subcutaneous calcitonin is also used in Pagets disease of bone, a disease characterized by increased and disordered osteoclast activity. Although calcitonin is effective in treating Pagets disease, it is now rarely used because of the superior efficacy and convenience of bisphosphonates.
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[133] N. Amizuka, A.C. Karaplis, J.E. Henderson, H. Warshawsky, M.L. Lipman, Y. Matsuki, et al., Haploinsufficiency of parathyroid hormone-related peptide (PTHrP) results in abnormal postnatal bone development, Dev. Biol. 175 (1996) 166e176. [134] D. Miao, B. He, Y. Jiang, T. Kobayashi, M.A. Soroceanu, J. Zhao, et al., Osteoblast-derived PTHrP is a potent endogenous bone anabolic agent that modifies the therapeutic efficacy of administered PTH 1-34, J. Clin. Invest. 115 (2005) 2402e2411. [135] A.A. Budayr, B.R. Halloran, J.C. King, D. Diep, R.A. Nissenson, G. Strewler, High levels of a parathyroid hormone-like protein in milk, Proc. Nat. Acad. Sci. USA 86 (1989) 7183e7185. [136] M.A. Thiede, G.A. Rodan, Expression of a calcium-mobilizing parathyroid hormone-like peptide in lactating mammary tissue, Science 242 (1988) 278e280. [137] G.W. Robinson, Cooperation of signalling pathways in embryonic mammary gland development, Nat. Rev. Genet. 8 (2007) 963e972. [138] J.R. Hens, J.J. Wysolmerski, Key stages of mammary gland development: molecular mechanisms involved in the formation of the embryonic mammary gland, Breast Cancer Res. 7 (2005) 220e224. [139] J.J. Wysolmerski, S. Cormier, W.M. Philbrick, P. Dann, J.P. Zhang, J. Roume, et al., Absence of functional type 1 parathyroid hormone (PTH)/PTH-related protein receptors in humans is associated with abnormal breast development and tooth impaction, J. Clin. Endocrinol. Metab. 86 (2001) 1788e1794. [140] C.S. Kovacs, Calcium and bone metabolism in pregnancy and lactation, J. Clin. Endocrinol. Metab. 86 (2001) 2344e2348. [141] J.N. VanHouten, J.J. Wysolmerski, Low estrogen and high parathyroid hormone-related peptide levels contribute to accelerated bone resorption and bone loss in lactating mice, Endocrinology 144 (2003) 5521e5529. [142] P. Hellman, P. Ridefelt, C. Juhlin, G. Akerstrom, J. Rastad, E. Gylfe, Parathyroid-like regulation of parathyroid-hormonerelated protein release and cytoplasmic calcium in cytotrophoblast cells of human placenta, Arch. Biochem. Biophys. 293 (1992) 174e180. [143] C.S. Kovacs, C. Ho, C.E. Seidman, J.G. Seidman, H.M. Kronenberg, Parathyroid calcium sensing receptor regulates fetal blood calcium and fetal-maternal calcium gradient independently of the maternal calcium levels, J. Bone Miner. Res. 22 (1996) S121. [144] M.A. Thiede, A.G. Daifotis, E.C. Weir, M.L. Brines, W.J. Burtis, K. Ikede, et al., Intrauterine occupancy controls expression of the parathyroid hormone-related peptide gene in pre-term rat myometrium. Proc. Nat. Acad. Sci. USA 87 (1990) 6969e6973 [145] M.A. Thiede, S.C. Harm, R.L. McKee, W.A. Grasser, L.T. Duong, R.M. Leach Jr., Expression of the parathyroid hormone-related protein gene in the avian oviduct: potential role as a local modulator of vascular smooth muscle tension and shell gland motility during the egg-laying cycle, Endocrinology 129 (1991) 1958e1966. [146] M. Yamamoto, S.C. Harm, W.A. Grasser, M.A. Thiede, Parathyroid hormone-related protein in the rat urinary bladder: a smooth muscle relaxant produced locally in response to mechanical stretch, Proc. Natl. Acad. Sci. USA 89 (1992) 5326e5330. [147] T. Massfelder, J.J. Helwig, Parathyroid hormone-related protein in cardiovascular development and blood pressure regulation, Endocrinology 140 (1999) 1507e1510. [148] N. Fiaschi-Taesch, B.M. Sicari, K. Ubriani, T. Bigatel, K.K. Takane, I. Cozar-Castellano, et al., Cellular mechanism through which parathyroid hormone-related protein induces proliferation in
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42 FGF23/Klotho New Regulators of Vitamin D Metabolism Valentin David, L. Darryl Quarles University of Tennessee Health Science Center, Memphis, TN, USA
INTRODUCTION Calcium and phosphorous are coordinately regulated to maintain circulating levels necessary for normal biological functions. The parathyroid glands, intestine, kidney, and bone are the primary organs involved in maintenance of calcium and phosphate homeostasis. Calcium and phosphate ingested in the diet are absorbed through the small intestine, excreted and reabsorbed by the kidney, and buffered by bone, which is used as a reservoir for rapid exchange of calcium and phosphate. Complex hormonal pathways have developed to control the levels of calcium and phosphate and prevent adverse consequences of calcium and phosphate excess and deficiencies. At present, at least five hormones have been identified, including 1,25-dihydroxyvitamin D (1,25(OH)2D), parathyroid hormone (PTH), calcitonin (CT), fibroblast growth factor-23 (FGF23), and Klotho. The vitamin D receptor (VDR), which mediates the transcriptional effects of 1,25(OH)2D, is ubiquitously expressed and is important in innate immune responses as well as mineral metabolism. The intestine and VDRdependent regulation of calcium absorption appear to be the key target organ and function of 1,25(OH)2D, since high calcium intake, or selective VDR replacement in the intestine rescues the VDR-deficient bone and mineral phenotypes of VDR-null mice [1,2]. The regulation and function of PTH, calcitonin, and 1,25(OH)2D are best understood, whereas the important physiological roles of FGF23 and Klotho are being elucidated. PTH and CT are secreted, respectively, by the chief cells in the parathyroid gland and C-cells in the thyroid. FGF23 is secreted by osteoblast/osteocytes in bone. 1,25(OH)2D is produced and secreted from the proximal
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10042-3
tubule in the kidney through the respective actions of Cyp27B1 to 1-a-hydroxylate 25-hydroxyvitamin D (25 (OH)D) to form 1,25(OH)2D and of Cyp24A1 to 24hydroxylate 25(OH)D leading to its degradation. The kidney also secretes a soluble form of Klotho, a poorly understood glucosidase and FGFR cofactor that affects calcium, phosphate, and energy homeostasis. We are beginning to understand how these hormones are organized into complex regulatory networks that involve integration of the function of the several tissues responsible for calcium and phosphate homeostasis. The regulation of 1,25(OH)2D production and degradation by these hormones is a central point for integrating and coordinating the functions of PTH, CT, FGF23, and 1,25(OH)2D itself.
REGULATION OF VITAMIN D METABOLISM The circulating concentration of 1,25(OH)2D depends on the relative rates of production and catabolism. Reductions in CYP27B1 expression and/or activity and enhanced CYP24A1, or both can lead to a decrease in biologically active 1,25(OH)2D3 levels, whereas the reciprocal changes in Cyp27B1 and Cyp24A1 lead to increased 1,25(OH)2D levels. CYP27B1 is predominantly expressed in the proximal tubule where it is responsible for the biosynthesis of 1,25(OH)2D in the circulation. There appear to be two distinct Cyp27B1 activities in the proximal convoluted and straight tubules that are differentially regulated. CYP24A1 is more widely expressed than is the CYP24A1 that catalyzes multiple hydrolyzing steps involving the side chains of 25(OH)D and 1,25(OH)D, mainly through
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42. FGF23/KLOTHO NEW REGULATORS OF VITAMIN D METABOLISM
a 24- or 23-hydroxylation pathways [3]. The major function of CYP24A1 is to tightly regulate the circulating 1,25 (OH)2D concentration by converting the 25(OH)D into 24,25(OH)D, thus depriving CYP27B1 of its substrate and by converting the 1,25(OH)2D into 1,24,25(OH)D, which is further metabolized into calcitroic acid (1-hydroxy-23-carboxy-vitamine D3) or 23,25(OH)D26,23-lactone, two inactive forms of vitamin D. The half-life of 1,25(OH)2D in the blood is approximately 2e3 hours [4] and reflects the activity of CYP24A1 [5,6]. PTH, hypophosphatemia, IGF1, insulin, and CT increase the expression of CYP27B1 [3,7]. PTH stimulates the activity of CYP27B1 promoter by inducing the phosphorylation of cAMP-dependent responseelement-binding protein (CREB) [8]. The ability of PTH to stimulate Cyp27B1 activity is enhanced by hypocalcemia and inhibited by hypercalcemia [9]. PTH and CT exert additive effects of CYP27B1 expression, indicating that these stimulatory effects on this enzyme are likely through separate signaling mechanisms to control CYP27B1. In addition, PTH targets the proximal convoluted tubule, whereas CT principally affects the proximal straight tubule. PTHstimulated CYP27B1 transcription and increased 1,25 (OH)2D synthesis is optimal in the setting of hypocalcemia; however, under normocalcemic conditions PTH has an attenuated effect to stimulate Cyp27b1 expression. Rather, CT, not PTH, is a major regulator of Cyp27b1 in the normocalcemic state [10]. Increments in calcium, phosphate, FGF23, and 1,25 (OH)2D reduce CYP27B1 expression and/or function. 1,25(OH)2D mediates down-regulation of CYP27B1 via VDR-dependent mechanisms [11]. FGF23 may decrease Cyp27b1 gene transcription and exert post-translational effects to decrease Cyp27B1 activity [12]. Loss of CYP27B1 function causes secondary hyperparathyroidism and rickets/osteomalacia. Inactivating mutations of CYP27B1 gene cause vitamin-D-resistant rickets type 1 [13]. 1,25-(OH)2D levels are undetectable in CYP27B1-null mice, whereas 25(OH)D levels are elevated and serum 24,25-(OH)2D levels are decreased [14]. Mice and children with inactivation of CYP27B1 have severe rickets unresponsive to normal vitamin D replacement but their phenotypic abnormalities can be largely corrected by supplementation of dietary calcium (in combination with high-lactose rescue diet) or 1,25(OH)2D treatment [14e18]. Inactivating mutations of VDR also leads to hypocalcemia and rickets/osteomalacia [19], but results in elevations of serum 1,25(OH)2D levels due to secondary hyperparathyroidism and stimulation of renal Cyp27b1 (1a-hydroxylase activity) and decreased 24-hydroxylase activity. 1,25(OH)2D, hyperphosphatemia, FGF23, and CT stimulate [20e23], whereas PTH suppresses CYP24A1 [24]. Calcitonin stimulates CYP24A1 transcription
through involvement of Ras-PKCzeta isoform pathway [21]. 1,25(OH)2D stimulates the transcription of CYP24A1 through its binding to the heterodimer VDRRXR which recognizes VDRE sites on the CYP24A1 gene promoter [22]. Another nuclear receptor, the pregnane X receptor (PXR), stimulates the expression of CYP24A1 through its interaction with the VDRE [25]. As the PXR is activated by antiepileptic or corticosteroids, administration of these drugs could lead to an increased catabolism of vitamin D analogs [25e28]. FGF23 reduces 1,25(OH)2D levels due to complex effects to inhibit CYP27B1 through either reduced expression and/or impaired translation and by stimulation of CYP24 to increase the catabolism of 1,25(OH)2D [29]. Studies using CYP24A1-knockout mice demonstrate increased 1,25(OH)2D levels and the total absence of calcitroic acid and 1a,25-(OH)2D3-26,23-lactone formation. The in vivo result of CYP24A1 ablation is decreased viability, with 50% of mice not surviving beyond weaning because of hypercalcemia and nephrocalcinosis. Altered CYP24A1 gene expression has been also associated with human diseases such as breast cancer and asthma [30e32], although no absolute human deficiency in CYP24A1 has been identified. The extrarenal production of 1,25(OH)2D is also regulated by CYP24A1. For example, an alternative splicing of CYP24A1 produces an inactive enzyme resulting in local increase in 1,25 (OH)2D [33]. The integrative physiology of PTH, 1,25(OH)2D, FGF23, and Klotho is providing new insights into the pathogenesis, diagnosis, and treatment of disordered mineral metabolism.
PTHeVITAMIN D AXIS: KEY REGULATOR OF CALCIUM HOMEOSTASIS The PTHevitamin D endocrine system is essential for calcium and bone homeostasis. The major function of this axis is to maintain normal serum calcium levels. Calcium is sensed by the calcium-sensing receptor, CASR, in the parathyroid chief cells in the parathyroid gland (PTG). Low calcium levels stimulate, whereas high calcium levels suppress, PTH secretion. The principal biological function of PTH is the prevention of hypocalcemia through its direct and indirect actions on several organs. Elevated levels of PTH target the PTH receptor in the kidney to increase the renal tubular absorption of calcium in the distal tubule and increase the synthesis of 1,25(OH)2D in the proximal tubule by activating the key anabolic enzyme 1-a-hydroxylase (Cyp27b1) in the kidney that converts 25(OH)D into the active state of 1,25(OH)2D. An elevated level of 1,25 (OH)2D increases intestinal absorption of calcium and
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FGF23 KLOTHO BONE KIDNEY AXIS: A PATHWAY COORDINATING PHOSPHATE AND VITAMIN D HOMEOSTASIS
phosphate. 1,25(OH)2D also acts on the VDR in osteoblasts to increase osteoclastogenesis and bone resorption leading to calcium and phosphate efflux and the resulting increase in serum calcium that acts on CASR in the PTG to reduce PTH. 1,25(OH)2D inhibits the synthesis and secretion of PTH [34] and prevents the proliferation of the parathyroid gland [34,35] cells. Additionally, 1,25 (OH)2D also up-regulates the calcium-sensing receptor [36]. Despite the fact that parathyroid cells contain a negative VDRE [34], ablation of VDR in the parathryoid glands has little effect on PTH secretion [37]. Finally, local 1,25(OH)2D production has been reported in the parathyroid gland [38], which may explain the relationship between PTH and 25(OH)D. PTH also targets PTH receptors in osteoblasts/osteocytes to modify calcium and phosphate efflux by modulating the bone-remodeling and mineralization processes. Although the resulting effect of a persistently elevated serum PTH level is increased bone turnover, intermittent PTH administration has a net anabolic effect due to increased bone formation greater than resorption. Continuous PTH administration and in most disease states of PTH excess, the net effect is a bone loss due to increased resorption greater than formation. PTH targets osteoblasts/osteocytes to down-regulate sclerostin [39,40], a secreted Wnt signaling inhibitor, thereby activating Wnt pathways leading to proliferation, differentiation, and activity of osteoblasts/osteocytes and enhanced osteogenesis [41]. PTH also modulates bone turnover through the OPG/ RANKL/RANK system. Osteoclasts express Receptor Activator of Nuclear Factor k B (RANK) a type I membrane protein, while osteoblasts express the ligand (RANKL) and also secrete a decoy receptor and physiological inhibitor of the RANKL/RANK interaction (for review see [42e43]), the osteoprotegerin (OPG). RANKLeRANK interaction stimulates osteoclastogenesis and the ratio of RANKL to OPG expressed by osteoblasts determines the level of osteoclast differentiation and activity. PTH up-regulates RANKL and inhibits OPG [44] thus increasing osteoclastogenesis. PTH also has phosphaturic effects that contribute to maintaining neutral phosphate balance in settings of excess phosphate or when acute elevations of serum phosphate lower ionized calcium. PTH targets PTH receptors in the proximal convoluted tubule to decrease the phosphate transporter protein content in the brush border membrane of the proximal tubule [45]. Thus, PTH and 1,25(OH)2D represent a coordinated and integrated cyclic feedback system to principally control serum calcium and secondarily to regulate phosphate. When exposure to calcium is in excess and suppression of PTH is not sufficient to prevent hypercalcemia, CT is secreted from the C-cells in the thyroid gland. Calcitonin is used therapeutically to lower calcium and
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inhibit bone resorption, but its physiological role appears to be the regulation of 1,25(OH)2D production and skeletal effects in pregnancy and lactation [46e47]. Patients with medullary thyroid carcinoma, a neoplasm of C-cells, have high calcitonin levels, high 1,25(OH)2D, but normal serum calcium [48], and the absence of calcitonin in animal models has no effect on serum calcium [49]. Moreover, calcitonin administration increases serum 1,25(OH)2D levels in both animal models [50] consistent with the direct effect of calcitonin to stimulate Cyp27b1 in the kidney. In patients with XLH, with low circulating 1,25(OH)2D levels, the response to PTH is blunted but the response to CT is preserved [51].
FGF23 KLOTHO BONE KIDNEY AXIS: A PATHWAY COORDINATING PHOSPHATE AND VITAMIN D HOMEOSTASIS WITH BONE MINERALIZATION Whereas the PTHevitamin D axis is principally designed to maintain neutral calcium and phosphate homeostasis and to stimulate 1,25(OH)2D production by the kidney, another hormone, FGF23, has been discovered that has similar effects to PTH to inhibit renal reabsorption of phosphate but opposite effects to decrease the circulating levels of 1,25(OH)2D levels. FGF23 is a circulating hormone produced by osteocytes/osteoblastic cells and to a lesser extent by endothelial cells in the central venous sinusoids, cells in the ventrolateral thalamic nucleus, and thymus. Whereas FGFs typically act as paracrine factors that activate FGF receptors in a process that requires heparin, FGF23 conserves an FGF N-terminal homology domain for binding to FGF receptors 1, 3, and 4 and a C-terminal domain that permits interaction with a-Klotho, a single-pass type 1 transmembrane protein required for FGFR23eFGFR binding [52]. In addition, the klotho gene encodes a truncated, secreted form derived from alternative RNA splicing [53]. Compared with the transmembrane form, this truncated gene product does not have the second internal repeat of the extracellular domain, the transmembrane domain, or the intracellular domain [53,54]. It only encodes the N-terminal half of klotho with its extracellular domain. The membrane-bound full-length Klotho and secreted extracellular domain of Klotho have distinct functions. Klotho is predominantly expressed in the kidney and the epithelium of the choroid plexus in the brain [55]. Besides these tissues, low expression of klotho is also reported in the pituitary gland, placenta, skeletal muscle, urinary bladder, aorta, pancreas, testis, ovary, and colon [56,57]. Knock-in of the lacZ gene downstream of the translational initiation codon of Kl shows
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expression of klotho in the parathyroid gland and sinoatrial cells of the heart [58,59]. The expression of membrane Klotho together with FGF-receptors imparts the tissue specificity to FGF23. Autosomal dominant hypophosphatemic rickets is caused by mutations in the RXXR domain of FGF23 that prevents its cleavage and inactivation by subtilisinlike proprotein convertases [60]. Comparative analysis of hereditary hypophosphatemic disorders, including autosomal dominant (ADHR), autosomal recessive (ARHR), and X-linked hypophosphatemic rickets (XLH) [61], plus the hyperphosphatemic disorder, autosomal recessive hyperphosphatemic familial tumoral calcinosis (HFTC) (reviewed in [62]) established the nonredundant role of FGF23 in regulating renal phosphate reabsorption and vitamin D metabolism. All of the hereditary hypophosphatemic disorders, as well as acquired disorders [62], characterized by excessive circulating levels of FGF23 have overlapping clinical characteristics, including renal phosphate wasting, inappropriately low 1,25(OH)2D levels, and rickets/ osteomalacia. Indeed, excess FGF23, both in response to acute administration of recombinant FGF23 protein and in disease states characterized by chronic elevations of FGF23, results in hypophosphatemia, aberrant vitamin D metabolism e to the degree of hypophosphatemia e and rickets/osteomalacia. The reductions in serum phosphate are due to FGF23-mediated decrements in proximal tubular reabsorption of phosphate due to the decreased expression of Na/Pi-2a and Na/ Pi-2c cotransporters [63e66] leading to renal phosphate wasting. In contrast, FGF23 deficiency results in hyperphosphatemia, moderate hypercalcemia, low PTH levels, markedly elevated levels of 1,25(OH)2D, and soft-tissue calcifications. FGF23 deficiency also results in a bone phenotype, characterized by a disorganized growth plate lacking hypertrophic chondrocytes and decreased mineralized bone mass with increased osteoid [67,68]. For example, hyperphosphatemic familial tumoral calcinosis caused by low bioactive FGF23 is characterized by hyperphosphatemia, elevated 1,25(OH)2D and soft-tissue calcifications and FGF23-null mice have a tumoral calcinosis phenotype [67]. Loss of 1,25(OH)2D actions from FGF23-null mice, by generating compound FGF23-null and either 1-alpha-hydroxylase or VDR-null mice, results in the disappearance of abnormal skeletal findings and soft-tissue calcifications, suggesting that at least some of the anomalies found in FGF23-null mice are mediated through increased 1,25(OH)2D activities [69]. Excess FGF23 in mice causes hypophosphatemia via inhibition of SLC34A1 (Npt2a) and SLC34A2 (Npt2c) sodium-dependent phosphate transport and suppresses 1,25(OH)2D via inhibition of CYP27B1 and stimulation of CYP24 in the proximal tubule of the
kidney [66,70e72]. Excess FGF23 also results in growth retardation and rickets/osteomalacia [66,72,73]. The importance of a-Klotho (a-Kl) in Fgf23 signaling is illustrated by both human and mouse genetic disorders where loss of Klotho results in end-organ resistance to Fgf23 and abnormalities resembling Fgf23 deficiency [57,74e76]. Hereditary hypophosphatemia and hyperparathyroidism (HHH) is caused by a promoter region translocation that increases a-Kl expression and its circulating levels [77]. Increased a-Kl might regulate PTH secretion through its maintenance of cell surface Naþ/Kþ-ATPase activity [74]. FGF23 decreases the expression of Kl and its receptors by the kidney, thereby creating complex feedback pathways for regulating phosphate and calcium metabolism [65,74]. At least two physiological functions of FGF23 have been identified. FGF23 is a counter-regulatory hormone for 1,25(OH)2D and functions to coordinate bone mineralization and turnover with renal handling of phosphate. Neither phosphate nor PTH appear to directly regulate FGF23 expression [78,79], but may indirectly regulate FGF23 through effects on bone remodeling (Fig. 42.1).
Counter-regulatory Hormone for 1,25(OH)2D The best-characterized physiological function of FGF23 is to act as a vitamin D counter-regulatory hormone. Prior to the discovery of FGF23, it was assumed that phosphate regulation occurred as a secondary action of PTH and 1,25(OH)2D. The administration of 1,25(OH)2D increases FGF23 levels, while the disruption of 1,25(OH)2D pathways reduces circulating FGF23 in mice [79,80]. Increased 1,25(OH)2D targets the gastrointestinal tract to increase calcium and phosphate absorption. Increments in calcium along with 1,25(OH)2D target the parathyroid gland to suppress PTH, which in turn targets the kidney to increase urinary calcium excretion to maintain neutral calcium balance. However, lowering of PTH levels decreases phosphate excretion and would potentially result in positive phosphate balance from vitamin-D-mediated increase in gastrointestinal phosphate absorption if not for compensatory elevations of FGF23, which also suppresses 1,25(OH)2D to counter the increase in vitamin D [79]. The expression of FGF23 is regulated by both VDRdependent and VDR-independent signaling. Stimulation of the 1,25(OH)2DeVDR pathway induces the expression of FGF23, as evidenced by increased FGF23 levels after 1,25(OH)2D administration. In line with these findings, VDR-null mice showed undetectable FGF23 levels [70,81]. In addition, normalization of serum calcium and phosphate levels by dietary means increased FGF23 levels in VDR-null mice, indicating
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Schematic representation of vitamin D function and regulation. Plain lines represent physiological functions for vitamin D (green), PTH (blue) and FGF23 (red). Please see color plate section.
FIGURE 42.1
that FGF23 expression is also regulated by a VDR-independent pathway [63e66]. FGF23 is not dependent on a functional 1,25(OH)2DeVDR system, as treatment of VDR-null mice with FGF23 further decreased hypophosphatemia due to reduced renal and intestinal phosphate absorption, accompanied by decreased Na/Pi-2a, Na/Pi-2b, and 1a-hydroxylase expression [70,82]. In hypophosphatemic disorders caused by FGF23 excess 1,25(OH)2D levels are suppressed for the degree of hypophosphatemia, which should increase 1,25 (OH)2D levels. FGF23 reduces 1,25(OH)2D levels due to complex effects on Cyp27b1 and Cyp24 to decrease production and increase the catabolism of 1,25(OH)2D [83]. Depending on the assay method, the chronicity of FGF23 exposure and other modifying factors, FGF23 increases Cyp24A1 [84] but has more variable effects to reduce Cyp27B1 expression [63,64,72]. FGF23 may also regulate the translation of the Cyp27b1 message to a functional protein [12,85]. Injection of recombinant FGF23 to wild-type mice results in a dose-dependent decrease in renal abundance of Cyp27b1 through a direct action on Cyp27b1 gene expression in human and mouse renal proximal tubule cells via an ERK1/2dependent mechanism [86] and leads to a rapid increase in Cyp24a1 message [63,83]. In contrast, in the Hyp mouse, homolog of XLH, which displays excess FGF23, defects in translation of Cyp27b1 message have been reported [87,88].
The importance of FGF23-mediated catabolism of 1,25(OH)2D and the interaction with PTH are revealed by the lethal phenotype induced by parathyroidectomy in mice with chronic FGF23 excess due to hypocalcemia [89]. The effect of PTH to stimulate 1,25(OH)2D is blunted whereas calcitonin stimulation of 1,25(OH)2D remains intact in the setting of excess FGF23 [51]. Excess FGF23 can promote secondary hyperparathyroidism by suppressing renal 25-hydroxyvitamin D (3)-1a-hydroxylase (Cyp27b1) activity while increasing that of renal 25-hydroxyvitamin D3 24-hydroxylase (Cyp24a1). Collectively these observations suggest that Cyp27b1 is the major target of PTH action, whereas FGF23 major effects are through increased catabolism of 1,25(OH)2D through increased expression of Cyp24 and impaired translation and/or post-translational modification of Cyp27b1. The effects of phosphate on FGF23 remain unclear, however, unlike calcium, where changes in serum calcium lead to predicable changes in PTH through the calcium-sensing receptor, it is clear that changes of secreted FGF23 are quite variable and modest when measured after high- or low-phosphate diets of long duration [90e92]. Indeed, serum FGF23 is regulated by dietary phosphate in some studies [93], whereas a subsequent study found that increased amount of dietary phosphate decreased FGF23 concentrations [94]. In rodents, high-phosphate diet increases circulating
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FGF23, but this seems to be a 1,25(OH)2DeVDRdependent mechanism, as dietary phosphate failed to increase FGF23 expression in absence of VDR [70]. However PTH-null mice [89] and patients with hypoparathyroidism [95] display increased FGF23 levels and phosphate levels and low 1,25(OH)2D, suggesting that low bone turnover may regulate FGF23 (see below). The regulatory loop of suppression of 1,25(OH)2D by FGF23 and stimulation of FGF23 by 1,25(OH)2D suggest that the principal function of FGF23 is to act as a counter-regulatory hormone for 1,25(OH)2D. If so, teleologically FGF23 may have evolved to prevent vitamin D toxicity [96].
Osteocyte Production of FGF23 Coordinates Renal Phosphate Handling with Extracellular Matrix Mineralization and Bone Turnover There is growing evidence that another physiological function of FGF23 is to respond to changes in bone mineralization and turnover to adjust renal phosphate handling to balance the phosphate flux from bone. Bone is a buffer for calcium and phosphate and can release calcium and phosphate into the circulation in high bone-remodeling states. Both low bone turnover and impaired mineralization would impair bone buffering capacity, leading to adaptive changes to excrete greater amounts of phosphate. Consistent with this possibility, there is an inverse relationship between FGF23 production by osteocytes and impaired mineralization in the XLH and ARHR caused by PHEX and DMP1 mutations [97,98]. The discovery that inactivating mutations of Phex leads to intrinsic abnormalities of mineralization and FGF23-dependent hypophosphatemia provided the initial insights into a bone kidney axis that coordinates bone mineralization and systemic phosphate homeostasis [99]. Phex, which colocalizes with FGF23 in osteocytes, is positioned to coordinate both FGF23 production and mineralization of extracellular matrix in bone. Indeed, inactivation of Phex leads to an intrinsic mineralization defect as well as increased FGF23 expression in osteocytes [100e102]. Conditional deletion of Phex in the osteoblast lineage in vivo is sufficient to reproduce the Hyp phenotype, confirming that an intrinsic defect in bone leads to increased FGF23 [103]. In the osteoblastic lineage Phex deficiency is sufficient, but not necessary to stimulate FGF23. The comparative analysis of the Hyp mouse homolog of XLH and dentin matrix acidic phosphoprotein 1 (Dmp1)-null mice established a link between defects in extracellular matrix mineralization and FGF23 expression. Ablation of Dmp1, an extracellular matrix SIBLING protein that regulates mineralization,
markedly stimulates the transcription of FGF23 by osteocytes in mice, leading to the discovery that ARHR is caused by inactivating mutations of DMP1 [104]. DMP1 contains an RGD domain for integrin binding, and an ASARM domain (for binding to PHEX). The ability of Dmp1 to bind to integrins through the RGD motif and Phex through ASARM motif provides a molecular basis for PhexeDmp1 interactions for regulating mineralization and FGF23 production. Primary defects in bone mineralization can also regulate FGF23 production. Inactivating mutations of Enpp1, which more typically cause hereditary generalized arterial calcification of infancy (GACI), can also cause a variant of autosomal recessive hypophosphatemic rickets, characterized by hypophosphatemia and elevated FGF23 levels in some patients [105,106]. Enpp1 generates inorganic pyrophosphate (PPi), an essential physiologic inhibitor of calcification, and substrate for alkaline phosphatase which converts it to Pi necessary for mineralization of bone. The inactivation of this Enpp1 reduces the ratio of PPi to Pi, leading to increased mineralization of soft tissues. Bone turnover also regulates FGF23 production. Lowturnover bone disease, such as adynamic bone, leads to decreased phosphate buffering by bone which could lead to increased production of FGF23. Impaired mineralization of extracellular matrix caused by inhibitors could also have a similar effect. More studies are needed to investigate the relationship between phosphate deposition in bone and FGF23 regulation; however, a phosphate-sensing mechanism linked to bone buffering capacity might reconcile the fact that in vitro phosphate does not regulate FGF23 expression nor FGF23 promoter transcriptional activities whereas in vivo phosphate loading can regulate FGF23 secretion [81] independently of 1,25(OH)2D. Consistent with this possibility, the antiresorptive agent osteoprotegerin produced a profound reduction in bone resorption and formation in male and oophorectomized female mice, accompanied by an increase in serum levels of FGF23 [107]. Theoretically, high rates of bone turnover would release calcium and phosphate from bone, leading to calcium-mediated suppression of PTH and elevated FGF23 to prevent hyperphosphatemia, but a role of primary increases in bone resorption leading to increased FGF23 is not supported by existing data, since OPG-null mice with high bone resorption have low FGF23 levels [108]. There is an association of increased FGF23 and plasma cell dyscrasias [109], but a formal assessment of the effect of increased osteoclastic-mediated bone resorption of FGF23 expression in bone has not been performed. Recent studies indicate that FGF23 is increased in callus during fracture healing, consistent with local matrixderived FGF23-stimulating factors [110]. It is also possible that the discrepancies between PTH lack of a direct effect on FGF23 promoter activity and the
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FIGURE 42.2 FGF23 regulation by Phex and Dmp1. A. Binding of extracellular matrix Dmp1 to membrane Phex, inhibits FGF23 transcription in osteocytes which in turn exerts a minimal action on phosphate excretion, maintaining the mineral balance. B. Absence of Phex-Dmp1 binding in pathological conditions results in increase in FGF23 transcription and consecutively to increased phosphate excretion, impaired mineral balance, osteoid accumulation and abnormal bone (re)modeling.
apparent ability of intermittent versus continuous administration of PTH to respectively inhibit and stimulate FGF23 may be due to primary effects of PTH to affect bone remodeling. Also, the recent observation that leptin stimulates FGF23 might also be mediated through effects on bone remodeling and also points to the complex interplay between nutrition, fat, bone, and energy metabolism [111] (Fig. 42.2).
RELATIONSHIP BEWEEN FGF23 AND PTH The parathyroid glands express FGF receptors and Kl, but whether FGF23 stimulates or inhibits PTH secretion is uncertain [55,57]. Elevated FGF23 levels are associated with hyperparathyroidism and the acute administration of recombinant FGF23 results in increments in Egr1 expression in parathyroid tissue in mice [52]. Conversely, FGF23 has recently been shown to inhibit expression of PTH mRNA and secretion of PTH from parathyroid cells [56,112]. A controversy is emerging regarding whether PTH directly regulates FGF23. Recent studies indicate that FGF23 suppresses PTH secretion and that PTH may stimulate FGF23, leading to another feedback loop (i.e., increased PTH / increased FGF23 / decreased PTH) [113]. PTH-FGF23 feedback challenges the simple
FGF23 counter-regulatory hormone for 1,25(OH)2D hypothesis. However, FGF23 is increased in primary hyperparathyroidism [114], in Jansens’ metaphyseal chondrodyplasia caused by activating PTH/PTHrp receptor mutations [115], and in some mouse models of excess PTH [114], and parathyroidectomy results in a decrease in FGF23 in CKD [116]. Indirect effects to stimulate FGF23 could be meditated through PTH-mediated increases in 1,25(OH)2D or the presence of cofactors modulating PTH effects. The apparent ability of PTH to increase or decrease FGF23 might reflect the differential anabolic and catabolic effects of PTH on bone remodeling. In this regard, intermittent administration of PTH leading to net increments in bone formation results in reduced FGF23 levels [107], consistent with the need to conserve phosphate. In contrast, continuous administration of PTH that leads to catabolic effects on bone might be predicted to stimulate FGF23, which would help eliminate the increased phosphate efflux from bone.
ROLE OF FGF23 IN PHOSPHATE HOMEOSTASIS While FGF23 regulates renal phosphate transport and loss of FGF23 leads to hyperphosphatemia, the relationship between changes in serum phosphate and FGF23 is less clear. Dietary phosphate loading can stimulate
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FGF23, but the magnitude of the phosphate regulation of FGF23 is small compared to the effects of 1,25(OH)2D and bone mineralization/remodeling. Unlike calcium, which has a calcium-sensing receptor (CaSR) that permits the sensing and tight control of calcium levels, a phosphate sensor has not been identified and the regulation of serum phosphate levels are not so tightly controlled. Phosphate homeostasis also is controlled by putative CNS- and gut-derived hormones, in addition to PTH and FGF23. Also the precise segments and the physiologically relevant receptor(s) that FGF23 targets in the kidney are not entirely clear. Although FGF23 can bind to FGFR3c, FGFR4, and FGFR1c in vitro, we have shown that neither deletion of FGFR3 nor FGFR4 impairs the phosphaturic actions of FGF23 in WT or Hyp mice [117], suggesting the remaining in vitro target, FGFR1, is the physiologically relevant receptor for FGF23 in the kidney. However, the highest levels of the FGFR1:Klotho complex are in the distal tubules, whereas the biological actions of FGF23 are in the proximal tubules [55]. Ex vivo studies of proximal tubular segments or cell lines have demonstrated variable effects of exogenously added FGF23 to inhibit sodium-dependent phosphate transport [71,118]. Interpretation of these findings are confounded by the use of nonphysiological amounts of FGF23 and the authenticity of the proximal tubular phenotype in cell culture models, which may be contaminated with distal tubular cells and/or undergo dedifferentiation [80,118e121]. Alternatively FGF23 actions on the proximal tubule may be indirect, possibly through FGF23 stimulation of the distal tubule and release of paracrine factors that regulate proximal tubule function (i.e., a “distal-to-proximal tubular feedback mechanism”), rendered possible by the close proximity of proximal and distal cells.
HORMONAL ACTIONS OF KLOTHO? As stated earlier, Klotho gene encodes both a membrane and a secreted form. Additionally, the transmembrane protein might be cleaved and secreted into the blood, cerebrospinal fluid (CSF), and urine. Indeed, the transmembrane Klotho is regulated by shedding (proteolytic cleavage and release) from the cell surface, which is a critical regulatory step in many normal and pathological processes. Klotho shedding is mediated mainly by ADAM10 and ADAM17, two members of the A Desintegrin and Metalloproteinase (ADAM) family [122], but other metalloproteinases could be involved. It is still unclear if the secreted form(s) of klotho is the consequence of shedding or alternative splicing, however, it is now clear that Kl may have dual actions
depending on its intracellular and secreted forms. In the kidney, Kl is exclusively coexpressed with calcium permeable Transient Receptor Potential V5 (TRPV5) channels, Na/Ca exchanger 1 (NCX1) and calbindinD28K (a vitamin-D-sensitive intracellular Ca2þ transporting protein) in a specialized region of the distal convoluted tubules where transepithelial Ca2þ reabsorption is actively regulated. This colocalization is important for the homeostatic control of Ca2þ by regulating Ca2þ reabsorption in the kidney. Indeed, mice lacking TRPV5 display diminished renal Ca2þ reabsorption, which causes severe hypercalciuria. The secreted form of klotho potentializes 1,25(OH)2D of renal reabsorption, as klotho stabilizes TRPV5 on the membrane by hydrolyzing the extracellular sugar moieties of TRPV5. A clinical presentation of hypophosphatemic rickets and hyperparathyroidism (HRH) is caused by mutation in Kl leading to increased circulating levels of alpha-klotho and FGF23 [61]. Plasma Klotho levels and betaglucuronidase activity are markedly increased in the affected patient; unexpectedly, the circulating FGF23 level is also markedly elevated. These findings suggest that the elevated Klotho level mimics aspects of the normal response to hyperphosphatemia and implicate Klotho in the selective regulation of phosphate levels and in the regulation of parathyroid mass and function; they also have implications for the pathogenesis and treatment of renal osteodystrophy in patients with kidney failure. The mineral and bone phenotype of Klotho-null mice is very comparable to the phenotype of FGF23-null mice [57]. a-Klotho mutant mice display a variety of accelerated aging-related disorders, including hypoactivity, sterility, skin thinning, decreased bone mineral density, vascular calcifications, ectopic calcification in various soft tissues (lung, kidney, stomach, heart, and skin), defective hearing, thymus atrophy, pulmonary emphysema, ataxia, and abnormality of pituitary gland, as well as hypoglycemia, hypercalcemia, and severe hyperphosphatemia in association with increased concentrations of 1,25(OH)2D. FGF23 serum levels are severely increased in Klotho-null mice, but these mice show hyperphosphatemia and hypervitaminosis D, associated with increased expression of Na/Pi-2a and 1a-hydroxylase [123,124]. A significant rescue of this phenotype was obtained when Klotho-null mice were fed a vitamin-D-deficient diet [125], which consecutively reduced abnormally high 1,25(OH)2D and circulating levels of calcium and phosphate. The altered mineral ion homeostasis displayed by the Klotho-null mouse, especially hyperphosphatemia, seemed to be the most important factor causing soft-tissue calcifications. Additional abnormalities are observed in association with FGF23 excess and deficiency, such as abnormalities in glucose homeostasis, growth retardation, abnormalities in thymic function, and aging phenotypes
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[67,69,97,126], consistent with a broader role for FGF23. The inter-relationships among FGF23, klotho, and vitamin D, and how these molecules coordinately regulate aging await further studies. Experiments have indicated that klotho is an antiaging factor; long-term studies will provide information on whether FGF23 might have similar antiaging effects.
HOW KNOWLEDGE OF FGF23 REGULATION AND FUNCTION CHANGES CONCEPTS, TREATMENTS, AND CLINICAL PRACTICE The discovery of FGF23 defines new physiological pathways and networks, and has led to reconsideration of the treatment of hereditary hypophosphatemic disorders, new insights into the pathogenesis of disordered mineral metabolism in chronic kidney disease, a new framework for understanding the mechanism of hyperphosphatemia caused by various drugs, and identification of a possible links between disordered mineral metabolism and cardiovascular mortality. The finding that FGF23 is a counter-regulatory hormone for 1,25 (OH)2D has challenged the current use of vitamin D analogs in combination with phosphate to treat XLH [127,128], as well as challenging the existing concept of the pathogenesis and treatment of secondary hyperparathyroidism in chronic kidney disease [129]. In addition, the FGF23 bone kidney axis offers new explanations for hyperphosphatemia that is observed with new cancer therapeutics that may disrupt FGF23 signaling pathways in the kidney as well as the effects of IV iron administration to cause hypophosphatemia through increased FGF23, possibly through inhibition of Phex [130]. Estrogen- and leptin-mediated stimulation of FGF23 provides further evidence of how the FGF23 boneekidney axis is more broadly integrated into estrogen and leptin biology [111]. The relationship between FGF23 and bone mineralization to regulate systemic phosphate and vitamin D homeostasis provides new insights into how bone mineralization and FGF23 may impact other systemic processes, such as vascular calcifications and cardiovascular abnormalities. The greatest impact of FGF23 may be to alter our view of the pathogensis of secondary hyperparathyroidism in chronic kidney disease. Prior to the discovery of the FGF23 boneekidney axis, secondary hyperparathyroidism in CKD was thought to be principally due to two factors: (1) a decline in 1a-hydroxylase-mediated production of 1,25(OH)2D by the kidney proximal tubule, and (2) hyperphosphatemia due to loss of nephron mass. In this “vitamin D-centric” view, CKD results in a functional vitamin-D-deficient state, with the corollary that the primary treatment should be active vitamin D
analogs with the principal goal of suppressing PTH and correcting the functional deficiency of active vitamin D. Cross-sectional studies indicate that FGF23 levels are also elevated early during the course of renal failure [131] in proportion to the decline in glomerular filtration rate (GFR). Circulating levels of FGF23 are also increased in end-stage renal failure and correlate with the degree of hyperphosphatemia [132,133]. The fact that FGF23 acts as a phosphaturic hormone that suppresses 1,25(OH)2D production suggests that the suppression of 1,25 (OH)2D in CKD, rather than representing a “functional deficient state” due to loss of renal mass, may represent a FGF23-mediated increased catabolism and suppression of 1,25(OH)2D production. If so, FGF23 actions may be the initial adaptive response that herald the subsequent decline in 1,25(OH)2D. Understanding if FGF23 is the initial biochemical abnormality of disordered mineral metabolism in CKD is important, because it may be a biomarker for earlier interventions before elevations in serum phosphate and PTH and may change the initial treatment approach to strategies that reduce FGF23, which would likely be phosphate binders and possibly other non-vitamin-D-based therapies. Indeed, FGF23 regulation of 1,25(OH)2D production by the kidney might be involved in mediating the known effect of dietary phosphate restriction to increase 1,25(OH)2D levels in chronic kidney disease [134]. Regardless, phosphate restriction very early in the course of CKD and consequent reduction of FGF23 might be expected to relieve FGF23-mediated suppression of 1a-hydroxylase activity in the kidney, thereby permitting a rise in 1,25(OH)2D levels and prevention of secondary hyperparathyroidism. On the other hand, administration of 1,25 (OH)2D or its active analogs would be expected to further elevate FGF23 levels in CKD, which, depending on the other actions of FGF23, could lead to untoward effects.
CONCLUSION Bone tissue is tightly regulated throughout life. Proper functioning of the bone organ, the basic unit of the vertebrate skeleton, relies on the intricate cooperation of many different tissues and regulatory systems, among which hormonal factors are best described. The vitamin D actions on the skeleton have been intensely described and well documented. However, the idea that bone is a passive buffer/reservoir organ for calcium and phosphate is long gone. Indeed, bone cells, and especially those of osteoblastic origin are recognized as being the source of hormones controlling energy homeostasis and mineral balance. Others have shown that osteocalcin [135e138], an osteoblast-secreted hormone, regulates insulin secretion. For years, our group has been demonstrating the complex role and regulation of
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Integrative physiology of vitamin D-FGF23-PTH multiorgan axis. Plain lines represent physiological functions for vitamin D (green), PTH (blue), FGF23 (red), leptin (pink), PO4 and Ca2+ (yellow) and bone remodeling (black). Please see color plate section.
FIGURE 42.3
FGF23, as a bone-secreted hormone [79,97e99], that plays a major role in phosphate homeostasis and vitamin D secretion. Thus, bone appears by all standards as a complex endocrine organ [62]. A more complex model of multiorgan interaction, in which bone plays a central role integrates both its endocrine functions in controlling energy and phosphate metabolism. Indeed, it has been known for years that low 25(OH)D levels were observed in association with obesity, either due to increased distribution to fat- of lipid-soluble vitamin D or preferential retention of vitamin D in fat. Moreover, serum 25(OH)D levels are increasing in obese patients after undergoing intestinal bypass surgery which results in rapid reduction of fat mass [139]. Increased serum concentrations of 1,25 (OH)2D are also associated with obesity as observed in leptin-deficient mice (ob/ob), as well as increased serum calcium and phosphate [140,141]. On the contrary, null mutations in the VDR or the CYP27B1 prevent abdominal fat mass accumulation and weight gain in mice [142,143]. Systemic 25(OH)D deficiency leads to
increased CYP27B1 directly to maintain 1,25(OH)2D or indirectly through secondary hyperparathyroidism [142,143]. Independently of the cause, elevated 1,25 (OH)2D may regulate energy metabolism and adipocyte function [143]. 25(OH)D deficiency is closely correlated to increased fat mass in humans [144,145] and mice [111,141], and leptin may provide a link between fat and vitamin D metabolism through regulation of FGF23. Interestingly, it has been shown in mice that administration of intraperitoneal leptin in ob/ob mice corrected abnormally elevated 1,25(OH)2D, calcium, and phosphate [140,141]. Recently, it has been shown that leptin stimulates FGF23 production in bone, which in turn is responsible for the reduction of 1,25(OH)2D and phosphate levels [111]. Furthermore, injection of FGF23 in leptin-deficient ob/ob mice corrected the overproduction of 1,25(OH)2D, whereas addition of leptin to renal tubular cells did not modify CYP27B1 activity. Leptin is secreted by white adipose tissue as a hormone that acts centrally on hypothalamus and peripherally to control energy intake and expenditure and bone
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REFERENCES
homeostasis. Leptin receptors are found in the hypothalamus, in bone marrow stromal cells, osteoblasts/osteocytes, chondrocytes, pancreas b cells, and kidney cells. Independently, leptin as a marker of lipid metabolism should be found elevated together with FGF23 in pathologies with increased cardiovascular risks. The discovery in a murine model that leptin uses FGF23 as a second messenger to counteract 1,25(OH)2D actions links both energy and mineral metabolism together placing bone at the center of regulatory loops involving brain, kidney, pancreas, adipose tissue, parathyroid gland, and cardiovascular system. This hypothesis is strengthened by the fact that a recent study in humans [146] FGF23 has been associated with fat mass and dyslipidemia. This opens a new field of research to study the actions of FGF23 and vitamin D. Despite all the recent discoveries, many questions remain unanswered about the regulation and function of FGF23, the integrative physiology of PTH, FGF23, and vitamin D and the pathological significances of these hormones in various disease states. The existence of biological actions of 1,25(OH)2D and klotho not related to mineral metabolism that include an antiproliferative, prodifferentiating effect on many cell types and immunoregulatory properties, and aging creates a broader role for these factors in health and disease. Similarly, increased levels of FGF23 are associated with increased morbidity and mortality even in the absence of renal failure [147e149]. A greater understanding of the regulation and function of FGF23 is needed. Understanding the integrative physiology of these hormones on the multiple organ systems that they target will help to better understand the pathological consequences of CKD or ESRD, conditions of vitamin D deficiency and excess and hyperand hypophosphatemic disorders (Fig. 42.3).
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C H A P T E R
43 The Role of the Vitamin D Receptor in Bile Acid Homeostasis Daniel R. Schmidt 1, Steven A. Kliewer 2, David J. Mangelsdorf 1, 3 1
Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, Texas 75390-9050, USA 2 Department of Molecular Biology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, Texas 75390-9050, USA 3 The Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, Texas 75390-9050, USA
INTRODUCTION Vitamin D is best known for its essential role in regulating calcium and phosphate homeostasis. Its active metabolite, 1a,25-dihydroxyvitamin D, functions as a hormone to regulate gene transcription by binding to and activating the nuclear vitamin D receptor (VDR). As a consequence of VDR activation, gene programs in the intestine, kidney, and bone are coordinated to increase calcium absorption and maintain serum calcium and phosphate levels. VDR is also activated by certain bile acids absorbed from the intestine and VDR has recently been shown to regulate bile acid biosynthesis. In this chapter we review the literature demonstrating that VDR is a bile acid sensor and plays an essential role in bile acid homeostasis. We also discuss how this new information regarding VDR provides insight into how a high-affinity hormone and its receptor may have evolved by co-opting an extant system with properties already designed to regulate nutrient absorption. Details of VDR target genes involved in mineral homeostasis, as well as their physiologic functions, are the topic of other chapters in this book.
BILE ACIDS IN PHYSIOLOGY AND DISEASE Bile acids are amphipathic sterols synthesized from cholesterol in the liver and secreted into the intestine
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10043-5
where they play an essential role in emulsifying dietary lipids. In the terminal small intestine most bile acids are reabsorbed and returned to the liver [1]. Approximately 5% of bile acids escape reabsorption and enter the large intestine where they are efficiently metabolized by colonic flora. The bacterial bile acid metabolites, termed secondary bile acids, are more hydrophobic than primary bile acids produced by the liver. The major secondary bile acids in humans are deoxycholic acid (DCA) and lithocholic acid (LCA). These hydrophobic bile acids cause direct damage to cell membranes and induce the generation of reactive oxygen species resulting in DNA damage, apoptosis, and necrosis (reviewed in [2] and [3]). Furthermore, secondary bile acids have been shown to promote intestinal and hepatic tumorigenesis in animal models, and their concentrations were reportedly higher in patients with colorectal cancer [4e7]. These studies have led to the idea that chronic exposure to elevated concentrations of secondary bile acids may contribute to the pathogenesis of cancer in gastrointestinal and hepatic tissues [8,9]. Fortunately, cells of the intestine, liver, and kidney are equipped with enzymes that detoxify bile acids through the addition of functional groups that decrease hydrophobicity and speed elimination from the body. In many cases expression of these enzymes is controlled by a subgroup of nuclear receptors that are activated by dietary lipids and xenobiotics. This topic is reviewed in detail elsewhere [10].
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FIGURE 43.1 Role of vitamin D in bile acid metabolism. Summary of known mechanisms by which vitamin D regulates bile acid metabolism. It is proposed that these mechanisms contribute to the chemopreventive functions of vitamin D in the colon. See text for details.
VDR AS A BILE ACID RECEPTOR The secondary bile acid, LCA, is one of the most toxic bile acids. Almost 10 years ago it was found that LCA and its bacterial metabolite, 3keto-LCA, could directly activate VDR [11]. Notably, VDR is a more sensitive receptor for LCA than the bile acid receptor/farnesoid X receptor (FXR). While LCA binds VDR with much lower affinity than 1a,25-dihydroxyvitamin D, studies in vitamin-D-deficient animals demonstrated that LCA could activate VDR in extraintestinal sites such as kidney and bone and was capable of inducing the same effects on calcium metabolism as vitamin D [12]. Within the intestine VDR induces expression of cytochrome P450 3A (CYP3A), which in turn detoxifies LCA [11,13e15]. Thus activation of VDR by LCA or vitamin D results in the induction of a feed-forward catabolic pathway for LCA in the intestine, implicating a paradigm for how the intestine protects itself from the toxic effects of LCA. It is noteworthy that vitamin D is associated with reduced risk of colorectal cancer (reviewed in [16,17]). One mechanism by which vitamin D may protect against colorectal cancer is through the induction of CYP3A and increased detoxification of LCA in intestine (Fig. 43.1). In addition, the recently discovered effects of vitamin D on bile acid biosynthesis (described in the following section) may also contribute to protection from colorectal cancer by reducing the overall concentration of bile acids to which the colon is exposed (Fig. 43.1).
REGULATION OF BILE ACID SYNTHESIS BY VDR Enzymatic conversion of cholesterol to bile acids in the liver is a multistep process that is tightly regulated to ensure that bile acid levels remain within a homeostatic range [18]. Feedback regulation occurs when end products of the pathway (bile acids) activate the farnesoid X
receptor (FXR) resulting in the induction of genes that suppress expression of CYP7A1, the rate-limiting enzyme for bile acid biosynthesis. In the postprandial state, this process begins with the induction of an intestinal hormone, fibroblast growth factor (FGF) 19 (also called FGF15 in rodents), which signals through a membrane receptor tyrosine kinase complex in hepatocytes to repress CYP7A1 transcription [19]. In addition, FXR activation in liver causes induction of the transcriptional repressor, short heterodimer partner (SHP), which binds to and suppresses the promoter of CYP7A1 (reviewed in [20]). In FXR-null mice, bile acid levels are increased due to low FGF15 levels and impaired bile acid feedback regulation [19]. Recently, it was found that VDR-null mice also have increased bile acid levels and decreased expression of Fgf15 [21]. Furthermore, it was shown that 1a,25-dihydroxyvitamin D suppressed bile acid synthesis through a mechanism that involved transcriptional regulation of Fgf15 by VDR [21]. This study demonstrated that both FXR and VDR are required to maintain Fgf15 expression and that VDR plays an essential role in the regulation of bile acid synthesis. It is noteworthy that a related mechanism involving transcriptional regulation of fibroblast growth factor 23 (FGF23) by VDR plays a role in renal phosphate metabolism (see Chapter 42). FGF23 is induced by vitamin D in bone and signals in a boneekidney axis to regulate phosphate reabsorption, while FGF15/19 is induced by vitamin D in intestine and signals in an intestineeliver axis to regulate bile acid biosynthesis [21,22]. These examples support a model in which endocrine FGFs function as downstream messengers to mediate the homeostatic effects of vitamin D and coordinate vitamin D signaling between organ systems (Fig. 43.2). Interestingly, another lipid-soluble vitamin, vitamin A, also suppresses bile acid synthesis [21]. The mechanism involves transcriptional regulation of both Fgf15 and Shp. Induction of Fgf15 by vitamin A appears to occur through activation of the retinoid X receptor (RXR)/FXR heterodimer, indicating that this complex
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EVOLUTION OF VDR: FROM BILE ACID METABOLISM TO MINERAL HOMEOSTASIS
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TABLE 43.1 Comparison of FXR and VDR Target Genes and their Functions
FIGURE 43.2 Endocrine FGFs as second messengers in the vitamin D signaling cascade. Model depicting vitamin-D-stimulated production of FGF15 in intestine and FGF23 in bone. These endocrine FGFs function as second messengers to mediate the homeostatic effects of vitamin D in the liver and kidney respectively.
functions as a sensor for both bile acids and dietary vitamin A. Given that bile acids promote absorption of lipid-soluble vitamins, it is possible that the mechanisms allowing vitamin A and D to control feedback repression of bile acid synthesis evolved to protect from exposure to potentially toxic levels of lipid-soluble vitamins in the diet or to allow increased absorption of lipid-soluble vitamins from a vitamin-deficient environment.
EVOLUTION OF VDR: FROM BILE ACID METABOLISM TO MINERAL HOMEOSTASIS Although, VDR has classically been regarded as a regulator of mineral homeostasis, the idea that VDR plays a role in intestinal detoxification is gaining momentum. In addition to its role in CYP3A regulation and detoxification of LCA, a recent study found that VDR may regulate a greater number of endobiotic/ xenobiotic detoxifying genes in the intestine than has previously been recognized [23]. Given these observations, we postulate that VDR may have evolved from an ancestral receptor with a role in bile acid or xenobiotic metabolism. One possibility is that gene duplication of an ancestral receptor occurred early in vertebrate evolution, perhaps coincident with the duplication of Hox genes, generating paralogs (e.g., FXR and VDR) that later evolved specialized roles in bile acid and calciumephosphate homeostasis. Although they have
Nuclear receptor Ligands
Target genes
Target gene function
ancestral bile acid receptor endobiotic? xenobiotic
? ? ?
transport detoxification endocrine hormone
FXR
Many bile acids
ABCB11 (BSEP) OSTa IBABP (FABP-6) SULT2A1 FGF15/19
bile acid transport bile acid transport bile acid binding bile acid detoxification bile acid homeostasis
VDR
Vitamin D lithocholic acid 3-keto lithocholic acid
TRPV5 CalbindinD9k CYP3A FGF15/19 FGF23
calcium transport calcium binding bile acid detoxification bile acid homeostasis phosphate homeostasis
taken on distinct roles, VDR and FXR retained the ability to regulate functionally related target genes; namely, membrane transporters, detoxifying enzymes, and endocrine hormones (Table 43.1). This idea is consistent with the appearance of VDR during the evolution of chordates into vertebrates. Bridging this gap are two well-studied invertebrate chordates, lancelets (“amphioxus”) and the sea squirt, and the most basal of vertebrates, the lamprey. Studies of nuclear receptor families in these species have provided a glimpse into early evolution of VDR and FXR. Although cephalochordates share a common ancestor with vertebrates they are likely the most basal organism of chordate lineage [24]. Like other protochordates, amphioxus has an intestinal tract and notochord but no vertebral column. Interestingly, amphioxus has no ortholog of VDR; however, gene duplication has led to multiple paralogs of FXR [25]. In contrast to amphioxus, Ciona intestinalis (“sea squirt”) represents another species of chordate invertebrates (subphylum urochordata), and is believed to be the closest extant relative of vertebrates. C. intestinalis contains a single VDR-like gene; however, this ortholog does not respond to vitamin D metabolites [26]. Lampreys, belonging to the subphylum vertebrata, superclass agnatha, represent the next branch in vertebrate evolution. These jawless fish have no bony skeleton or integumentary scales, yet they possess a functional ortholog of VDR that can be activated by vitamin D [27]. Taken together, these observations suggest that a functional receptor for vitamin D appeared early during vertebrate evolution, long before the evolution of a mineralized skeleton and the need for hormonal control of calcium absorption. Interestingly, larval lampreys are known to release bile acids in response to feeding [28]. Thus
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a bile-acid-like compound or endobiotic may have been the first endogenous ligand for VDR. It is worth noting that, as vertebrates evolved, the first site of ossification and calcification was in the skin, even while the skeleton remained cartilaginous [29]. In particular, placoderms, which are among the oldest known jawed fishes and preceded cartilaginous fishes, had well-developed bony dermal plates [30]. In addition to the intestine, VDR is also expressed in skin, particularly in mammals where it is required for normal hair growth [31]. Hair evolved both to provide warmth and protect against the damaging effects of UV light. Thus, it may not be merely coincidental that the skin also evolved to be the site of synthesis for the high-affinity hormonal ligand of VDR, which is essential for the maintenance of mineralized tissue. From a teleologic point of view, the evolution of the vitamin D endocrine system follows the evolution of vertebrates as they moved from an aquatic to a terrestrial environment. An important metabolic transition that occurred concurrently with this move was the need to develop an endocrine system that regulates calcium homeostasis. In aqueous environments calcium concentrations are not limiting, and fish receive virtually all of their calcium from the water. This is true even in teleost fish that have abundant calcium in their bones. Hence, in fish there is no need for an endocrine regulatory system to promote calcium absorption from the gut or its mobilization from bone. Thus, it is of interest that vitamin D, which is abundantly found in fish, has only a limited role in regulating intestinal calcium uptake or bone metabolism in fish. (Indeed, the role of vitamin D in fish has yet to be fully clarified. For an excellent review on the subject see Lock et al. [32].) Moreover, unlike terrestrial vertebrates that can make vitamin D through the action of sunlight in skin, the source of vitamin D in fish comes exclusively from their diet [32]. Again, these findings support the notion that VDR evolved originally as a sensor of a dietary lipid. Moving from water to land increased exposure of terrestrial vertebrates to UV light, which provided a convenient and dependable source for endogenous vitamin D production. As already discussed, the ancestral VDR was likely a sensor for bile acids and xenobiotics compounds and its original role may have been for protection against these compounds. A few mutations in the ligandbinding pocket of the receptor were likely all that was needed to permit it to become a high-affinity receptor for dietary vitamin D metabolites. The localization of VDR in the gut of primitive aquatic invertebrates and vertebrates furthered its adaptation to the regulation of intestinal calcium absorption. Since bone was already established as an organ rich in calcium and phosphate, it was adapted by terrestrial vertebrates to serve as the major storage site for these essential minerals. As early
aquatic species moved to land, the vitamin D endocrine system was established to permit communication between bone, intestine, kidney, and skin. Throughout the evolutionary process VDR appears to have retained its ability to bind to toxic bile acids, thereby maintaining its protective role in the colon. It is a fundamental principle of evolution to adapt an extant system and modify it through mutation to take on a new, advantageous role. The vitamin D receptor represents an excellent example of this evolutionary strategy, and its study with this concept in mind has continued to lead to new insights into the important role of this receptor and its ligands in biology.
References [1] A.F. Hofmann, The enterohepatic circulation of bile acids in mammals: form and functions, Front. Biosci. 14 (2009) 2584e2598. [2] H. Bernstein, C. Bernstein, C.M. Payne, K. Dvorak, Bile acids as endogenous etiologic agents in gastrointestinal cancer, World J. Gastroenterol. 15 (2009) 3329e3340. [3] M.J. Perez, O. Briz, Bile-acid-induced cell injury and protection, World J. Gastroenterol. 15 (2009) 1677e1689. [4] B.S. Reddy, K. Watanabe, J.H. Weisburger, E.L. Wynder, Promoting effect of bile acids in colon carcinogenesis in germfree and conventional F344 rats, Cancer Res. 37 (1977) 3238e3242. [5] B.S. Reddy, K. Watanabe, Effect of cholesterol metabolites and promoting effect of lithocholic acid in colon carcinogenesis in germ-free and conventional F344 rats, Cancer Res. 39 (1979) 1521e1524. [6] I. Kim, K. Morimura, Y. Shah, Q. Yang, J.M. Ward, F.J. Gonzalez, Spontaneous hepatocarcinogenesis in farnesoid X receptor-null mice, Carcinogenesis 28 (2007) 940e946. [7] F. Yang, X. Huang, T. Yi, Y. Yen, D.D. Moore, W. Huang, Spontaneous development of liver tumors in the absence of the bile acid receptor farnesoid X receptor, Cancer Res. 67 (2007) 863e867. [8] F.M. Nagengast, M.J. Grubben, I.P. van Munster, Role of bile acids in colorectal carcinogenesis, Eur. J. Cancer. 31A (1995) 1067e1070. [9] H. Bernstein, C. Bernstein, C.M. Payne, K. Dvorakova, H. Garewal, Bile acids as carcinogens in human gastrointestinal cancers, Mutat. Res. 589 (2005) 47e65. [10] D.R. Schmidt, D.J. Mangelsdorf, Nuclear receptors of the enteric tract: guarding the frontier, Nutr. Rev. 66 (2008) S88eS97. [11] M. Makishima, T.T. Lu, W. Xie, G.K. Whitfield, H. Domoto, R.M. Evans, et al., Vitamin D receptor as an intestinal bile acid sensor, Science 296 (2002) 1313e1316. [12] J.A. Nehring, C. Zierold, H.F. DeLuca, Lithocholic acid can carry out in vivo functions of vitamin D, Proc. Natl. Acad. Sci. USA 104 (2007) 10006e10009. [13] P. Schmiedlin-Ren, K.E. Thummel, J.M. Fisher, M.F. Paine, K.S. Lown, P.B. Watkins, Expression of enzymatically active CYP3A4 by Caco-2 cells grown on extracellular matrixcoated permeable supports in the presence of 1alpha,25dihydroxyvitamin D3, Mol. Pharmacol. 51 (1997) 741e754. [14] K.E. Thummel, C. Brimer, K. Yasuda, J. Thottassery, T. Senn, Y. Lin, et al., Transcriptional control of intestinal cytochrome P4503A by 1alpha,25-dihydroxy vitamin D3, Mol. Pharmacol. 60 (2001) 1399e1406. [15] W. Xie, A. Radominska-Pandya, Y. Shi, C.M. Simon, M.C. Nelson, E.S. Ong, et al., An essential role for nuclear
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receptors SXR/PXR in detoxification of cholestatic bile acids, Proc. Natl. Acad. Sci. USA 98 (2001) 3375e3380. S.A. Lamprecht, M. Lipkin, Chemoprevention of colon cancer by calcium, vitamin D and folate: molecular mechanisms, Nat. Rev. Cancer 3 (2003) 601e614. C.F. Garland, E.D. Gorham, S.B. Mohr, F.C. Garland, Vitamin D for cancer prevention: global perspective, Ann. Epidemiol. 19 (2009) 468e483. D.W. Russell, The enzymes, regulation, and genetics of bile acid synthesis, Annu. Rev. Biochem. 72 (2003) 137e174. T. Inagaki, M. Choi, A. Moschetta, L. Peng, C.L. Cummins, J.G. McDonald, et al., Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis, Cell Metab. 2 (2005) 217e225. J.Y. Chiang, Bile acids: regulation of synthesis, J. Lipid Res. (2009). D.R. Schmidt, S.R. Holmstrom, K. Fon Tacer, A.L. Bookout, S.A. Kliewer, D.J. Mangelsdorf, Regulation of bile acid synthesis by fat-soluble vitamins A and D, J. Biol. Chem. 285 (2010) 14486e14494. S. Liu, L.D. Quarles, How fibroblast growth factor 23 works, J. Am. Soc. Nephrol. 18 (2007) 1637e1647. G.D. Kutuzova, H.F. DeLuca, 1,25-Dihydroxyvitamin D3 regulates genes responsible for detoxification in intestine, Toxicol. Appl. Pharmacol. 218 (2007) 37e44. N.H. Putnam, T. Butts, D.E. Ferrier, R.F. Furlong, U. Hellsten, T. Kawashima, et al., The amphioxus genome and the evolution of the chordate karyotype, Nature 453 (2008) 1064e1071.
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[25] M. Schubert, F. Brunet, M. Paris, S. Bertrand, G. Benoit, V. Laudet, Nuclear hormone receptor signaling in amphioxus, Dev. Genes Evol. 218 (2008) 651e665. [26] E.J. Reschly, A.C. Bainy, J.J. Mattos, L.R. Hagey, N. Bahary, S.R. Mada, et al., Functional evolution of the vitamin D and pregnane X receptors, BMC Evol. Biol. 7 (2007) 222. [27] G.K. Whitfield, H.T. Dang, S.F. Schluter, R.M. Bernstein, T. Bunag, L.A. Manzon, et al., Cloning of a functional vitamin D receptor from the lamprey (Petromyzon marinus), an ancient vertebrate lacking a calcified skeleton and teeth, Endocrinology 144 (2003) 2704e2716. [28] W. Li, P.W. Sorensen, D.D. Gallaher, The olfactory system of migratory adult sea lamprey (Petromyzon marinus) is specifically and acutely sensitive to unique bile acids released by conspecific larvae, J. Gen. Physiol. 105 (1995) 569e587. [29] R.I.C. Spearman, The Integument: A Textbook of Skin Biology, Cambridge University Press, London, 1973. [30] G.C. Kent, L. Miller, Comparative Anatomy of the Vertebrates, The McGraw-Hill Companies, United States, 1997. [31] M.R. Haussler, C.A. Haussler, L. Bartik, G.K. Whitfield, J.C. Hsieh, S. Slater, et al., Vitamin D receptor: molecular signaling and actions of nutritional ligands in disease prevention, Nutr. Rev. 66 (2008) S98e112. [32] E.J. Lock, R. Waagb, S. Wendelaar Bonga, G. Flik, The significance of vitamin D for fish: a review, Aquacult. Nutr. 16 (2010) 100e116.
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C H A P T E R
44 Vitamin D and Fat Francisco J.A. de Paula 1, 2, Clifford J. Rosen 1 1
2
Maine Medical Center Research Institute Department of Internal Medicine, School of Medicine of Ribeira˜o Preto, USP
INTRODUCTION Vitamin D is one of the oldest hormones synthesized in the earliest life forms for over 750 million years [1]. However, only recently has it been thoroughly scrutinized; not surprisingly, the specialized antirickets hormone emerged as a multifunctional endocrine, paracrine/autocrine molecule with a broad spectrum of functions. Vitamin D seems to influence the expression of more than 200 genes, in addition to exerting its classical regulatory effects on mineral homeostasis, vitamin D seems to be integrated in the physiological control of cell proliferation [2,3], blood pressure [4,5], cardiac function [6,7], immunomodulation [8,9], muscle performance [10e12], and aging [13,14] and has a complex influence on the regulation of energetic metabolism [15,16]. Over the last few years, there has been great interest in delineating the physiological role of vitamin D in each of these functions and also its participation in the emergence of disorders such as cancer, hypertension, diabetes mellitus, and obesity. Knowledge of the intricate and independent regulation of the endocrine synthesis of the active form of vitamin D (1,25-dihydroxyvitamin D e 1,25(OH)2D) (involved in osteomineral control) and of its paracrine/autocrine synthesis (related to the other effects) is necessary to understand the discrepancies between the circulatory and tissue levels of this hormone [17]. The organism has a set of mechanisms involved in the maintenance of the renal synthesis of 1,25(OH)2D, the systemic source of hormonal 1,25(OH)2D, targeting the preservation of calcium homeostasis. Thus, usually individuals with vitamin D insufficiency show normal or even elevated serum levels of 1,25(OH)2D [18,19]. On the other hand, 1,25(OH)2D synthesis at other sites varies according to the supply of vitamin D or, more
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10044-7
precisely, depends on the availability of the prohormone, 25-hydroxyvitamin D (25(OH)D). It is still unknown if skeletal and mineral metabolism is more sensitive to the effects of mild vitamin D deficiency than the metabolism of other tissues. This especially applies to fat metabolism since adipose tissue is supposed to be the site for vitamin D storage [20]. This chapter was designed to discuss the most relevant aspects concerning the physiological relationship between vitamin D and fat metabolism and the consequences of vitamin D deficiency for energy metabolism.
VITAMIN D METABOLISM AND MECHANISM OF ACTION The secosteroid vitamin D3 is generated in the skin through a photochemical transformation of 7dehydrocholesterol after ultraviolet B radiation. Subsequently, vitamin D is processed to 25-hydroxyvitamin D (25(OH)D) by the liver in a constitutive metabolic step. 25(OH)D corresponds to the major circulating form of vitamin D and is widely used by clinicians as a surrogate marker of vitamin D status. Renal 1a-hydroxylation occurs in a closely controlled process to produce 1,25 (OH)2D. The renal synthesis of 1,25(OH)2D involves all the sophisticated steps of an endocrine pathway [19], considering that: (a) 1,25(OH)2D has distant target sites such as the intestine where it increases the absorption of calcium and phosphorus, (b) the production of 1,25 (OH)2D is inhibited by the hormone itself, by calcium and phosphorus and stimulated by parathyroid hormone (PTH), (c) 1,25(OH)2D also exerts a negative influence on the parathyroid synthesis of PTH, drawing a long negative loop of control, and (d) the action is orchestrated after the binding of 1,25(OH)2D to its high-affinity nuclear
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receptor (VDR), a member of the nuclear receptor superfamily. VDR is ubiquitously expressed in a variety of cells and tissues different from those involved in mineral homeostasis [21]. The endocrine production of 1,25(OH)2D is designed to be constantly maintained, independent of vitamin D status. Thus, circulatory 1,25(OH)2D neither rises with abundant sunlight exposure or vitamin D uptake nor usually falls with insufficient access to vitamin D sources [22]. In conditions of vitamin D sufficiency, serum levels of calcium, phosphorus, and PTH restrain the synthesis of 1,25(OH)2D to meet the physiological needs. Also, 1,25(OH)2D decreases its own synthesis through negative feedback and decreases the production and secretion of PTH by the principal cells in the parathyroid gland. In parallel, 1,25(OH)2D enhances the expression of 25-hydroxyvitamin D-24-hydroxylase to catabolize 1,25(OH)2D to an inactive product, calcitroic acid, 24,25(OH)2D which is cleared by bile excretion [22]. In contrast, in hypovitaminosis D low circulatory levels of calcium, phosphorus, and high PTH increase the metabolism of 25(OH)D to 1,25(OH)2D by the enzyme 25-hydroxyvitamin D-1a-hydroxylase (CYP27B1). These physiological adaptations allow the organism to maintain normal or even elevated serum levels of 1,25 (OH)2D in the presence of 25(OH)D insufficiency [22]. Ultimately, these mechanisms are part of a complex physiological network maintaining calcium homeostasis, in which 1,25(OH)2D has a critical role in the intestinal absorption of calcium and in the flux of calcium from bone to the blood circulation. Currently, there is a long and still expanding list of extrarenal tissues in which CYP27B activity was detected. In contrast to the pattern of tight endocrine control observed in the synthesis of 1,25(OH)2D in the kidney, extrarenal synthesis is largely dependent on 25 (OH)D levels, at least in disease-activated macrophages. Also, peripheral production most likely does not supply the systemic levels of 1,25(OH)2D [22]. Several lines of evidence suggest that local production of 1,25(OH)2D in the skin, breast, colon, and prostrate is important to restrain cell proliferation and to stimulate cell differentiation [23]. The possible antineoplastic paracrine/autocrine role of 1,25(OH)2D is currently being thoroughly investigated. Peripheral synthesis of 1,25(OH)2D seems to be also implicated in innate defense by macrophages and can also modulate the immune response of B and T lymphocytes [22,24]. The role of extrarenal synthesis in immunomodulation and in the control of cell proliferation is discussed elsewhere in this book. CYP27B1 activity has been identified in adipose tissue; however its role in fat homeostasis is still to be determined. The mechanism of action is another variable to be considered in the analysis of the physiological effects of vitamin D. 1,25(OH)2D exerts its effects through the
vitamin D receptor, classically, a nuclear receptor leading to gene expression, e.g., the calcium-binding protein. Alternatively, some cells and tissue (e.g., adipocytes) have a plasma membrane receptor (nongenomic receptor) and second messengers such as cyclic AMP, which transduces an intracellular Caþþ response to 1,25(OH)2D [25,26]. The latter response is very rapid and includes effects on the pancreas, vascular smooth muscle, and monocytes. Genomic and nongenomic actions of 1,25(OH)2D have been identified in adipose tissue. Intriguing results have revealed that suppression of 1,25(OH)2D by a high-calcium diet [25] and an animal model of vitamin D deficiency, with a relatively high 1,25(OH)2D, are associated with weight loss and a lean phenotype [27], respectively. At first glance, these conditions are in direct conflict with the common association between vitamin D deficiency and obesity as well as the putative relationship between vitamin D and diabetes mellitus.
VITAMIN D DEFICIENCY AND ADIPOSITY Obesity, the most common metabolic disease, reflects an imbalance between energy storage and expenditure. Besides the alterations in body composition, fat accumulation predisposes to diseases such as diabetes mellitus, cancer, and arterial hypertension, representing a permanent challenge for the determination of the hormonal, metabolic, and molecular mechanisms behind this disorder [28]. Traditionally, the impact of the diet on obesity has been investigated within the context of the consumption of major macronutrients such as fat, carbohydrate, and protein [29e31]. However, clinical and experimental investigations have documented that increased calcium ingestion can have a significant effect on overall weight maintenance due to its critical role in adipose tissue metabolism [32e34]. A hypercalcemic diet seems to act indirectly through the inhibition of 1,25(OH)2D synthesis (Fig. 44.1) [35]. Evidence indicates that 1,25(OH)2D plays a direct role in the modulation of adipocyte intracellular ionized calcium ([Caþþ]i) signaling, provoking both increased lipogenesis and decreased lipolysis [36]. The increase of [Caþþ]i within murine and human adipocytes stimulates the expression and activity of fatty acid synthase (FAS), a step-limiting enzyme in lipogenesis (Fig. 44.1) [37,38]. In addition to stimulating triacylglycerol synthesis, increasing intracellular calcium inhibits lipolytic activity, favoring fat storage. The antilipolytic effect of Caþþ is attributed to a direct activation of phosphodiesterase 3B, resulting in a decrease in cAMP, reducing the ability of agonists to stimulate the phosphorylation and activation of hormonesensitive lipase [39]. Therefore, a hypercalcemic diet can
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FIGURE 44.1 Dietary calcium regulation of adipocyte metabolism, involving 1,25-dihydroxyvitamin D (1,25(OH)2D) synthesis suppression.
be associated with a paradoxical decrease in [Caþþ]i due to the suppression of 1,25(OH)2D synthesis. As a consequence, the intracellular machinery of adipocytes is directed towards fatty acid catabolism. The hypothesis that 1,25(OH)2D positively affects triacylglycerol synthesis was further supported by the evaluation of the doseeresponse effect of 1,25(OH)2D on the cellular influx of Caþþ (Fig. 44.2). A steady-state, dose-dependent (1e50 nM) increase in Caþþ influx was observed in cultured human adipocytes [40]. A similar response was obtained with a specific agonist for the membrane VDR (mVDR), dihydroxylumisterol3 (1a,25(OH)2-lumisterol3), while this effect was completely prevented by 1b,25-dihydroxyvitamin D3, a specific antagonist of mVDR [36]. In addition, 1,25(OH)2D
1,25-Dihydroxyvitamin D (1,25(OH)2D) role on the control of adipocyte metabolism through genomic and nongenomic signaling.
FIGURE 44.2
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induces FAS expression and activation, as well as a significant increase in glycerol-3-phosphate-dehydrogenase (GPDH) and about 80% inhibition of isoproterenolstimulated lipolysis in adipose cell cultures. In the same study the authors observed that the specific mVDR agonist, 1a,25-(OH)2-lumisterol3, led to a more potent effect on adipocyte metabolism, provoking a 3.0-fold increase in FAS and GAPDH activity on the one hand, and being twice as potent in inhibiting basal lipolysis on the other. Taken together, these data indicate that 1,25(OH)2D regulates adipocyte Caþþ signaling and consequently exerts some control over both lipogenesis and lipolysis. The influence of calcium influx on adipocyte metabolism was also analyzed in another interesting experimental model, agouti mice, namely the overexpression of the murine agouti gene, which results in obesity, hyperinsulinemia, and insulin resistance [41,42]. Coincidentally, these mice have a high intracellular concentration of Caþþ in different tissues such as muscle [43] and the purified agouti gene product induces slow sustained increases in [Caþþ]i in smooth muscle cells and 3T3-L1 adipocytes [44]. It was observed that the agouti gene product enhances calcium flux into cultured and freshly isolated skeletal muscle myocytes from wild-type mice and it was hypothesized that the agouti peptide promotes insulin resistance and obesity in mutant mice through its ability to increase [Caþþ]i [44]. The human homolog of agouti is expressed primarily in human adipocytes, and it was shown that recombinant agouti protein increases adipocyte intracellular [Caþþ] i, thereby stimulating lipogenesis and inhibiting lipolysis in human adipocytes [45]. To determine the role of [Caþþ]i in the antilipolytic effect of agouti, an experiment was designed in which human adipocytes were treated with KCl or arginine vasopressin to stimulate voltage- and receptor-stimulated Caþþ influx, respectively. Both agents inhibited forskolin-induced lipolysis (P < 0.005). Furthermore, the antilipolytic effect of agouti was also blocked by the Caþþ channel blocker nitrendipine [45]. Concerning the effects of agouti on lipogenesis, it was demonstrated that agouti protein was correlated with FAS activity in adipose tissue from normal and overweight patients [46]. More recently, it was determined that a high-calcium diet, which suppresses 1,25(OH)2D synthesis, increases core temperature and white adipose tissue uncoupling protein 2 (UCP2) expression in aP2-agouti transgenic mice. Thereafter, the effect of 1,25(OH)2D in regulating human adipocyte UCP2 expression was evaluated. Human adipocytes treated with 1 nM 1,25(OH)2D for 2 days exhibited decreased UCP2 mRNA and protein levels and suppressed isoproterenol- or fatty-acid-stimulated UCP2 expression. In this case, 1a,25-dihydroxylumisterol, the specific mVDR agonist, was inefficient in
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inhibiting isoproterenol-stimulated, or fatty-acid-stimulated UCP2 expression. On the other hand, nuclear VDR disruption prevented the inhibitory effect of 1,25(OH)2D on adipocyte UCP2 expression and protein levels. These last results are indications that 1,25(OH)2D exerts an inhibitory effect on basal adipocyte function, isoproterenol- and fatty-acid-stimulated UCP2 expression; this effect is likely mediated via genomic signaling. Thus, 1,25(OH)2D has complex metabolic actions on adipocyte management of energy storage and fatty acid turnover, which may be mediated by a genomic pathway or, alternatively, by a membrane receptor pathway [47]. In addition to the functional association between a hypercalcemic diet and 1,25(OH)2D suppressionassociated fat loss, genetic models of severe vitamin D deficiency were recently used to show the role of 1,25 (OH)2D in body composition and to explore possible mechanisms involved in the metabolic effects of vitamin D (Table 44.1). Initially, it was observed that VDR knockout (VDRKO) mice exhibit atrophy of adipose tissue in mammary [48] and prostate glands [49]. Because bone marrow stromal cells from perinatal VDR-null mice did not exhibit defective adipogenesis in vitro, it was assumed that vitamin D signaling may become important in the control of adipogenesis during the aging process. In an elegant study, Narvaez et al. (2009) determined the age-related changes in adipose tissue mass, morphology, and gene expression in mice with ablation of either VDR or CYP27B1 [50]. The results showed that deficiency of VDR signaling resulted in age-related adipose atrophy and hypoleptinemia (Table 44.1). Decreased fat mass was not the result of diminished food intake since the mice presented a hyperphagic behavior. In contrast to wild-
TABLE 44.1
VDRKO
type mice, the white adipose tissue (WAT) from VDRKO mice presented smaller unilocular adipocytes and frequent areas of multilocular cell clusters reminiscent of brown adipose tissue (BAT) (Table 44.1). Both genetically modified mice, VDRKO and CYP27B1KO, exhibited a lean phenotype. Contrary to what was expected by the authors, higher FAS and PPARg gene expression was observed in adipose tissue from VDRKO mice than from WT mice. These challenges led to investigation by PCR screening array of 84 genes involved in insulin signaling in WAT of 6-month-old WT and VDRKO mice in order to clarify the factors underlying the lean phenotype of VDRKO. Of the genes implicated by the screening approach, ucp-1, which was 25-fold elevated in WAT of VDRKO mice compared with WT mice. UCP-1 is of particular interest because it functions as an uncoupler of mitochondrial ATP production, and thus its up-regulation in WAT could potentially explain the failure of VDRKO mice to maintain adipose stores. Furthermore, even though ucp-1 is a highly specific marker for brown adipocytes, the screening detected induction of UCP-1 in WAT of VDRKO mice. Western blotting of WAT lysate showed that ucp-1 was strongly detected in samples from VDRKO mice, but not in samples from WT mice. Further confirmation of UCP-1 protein expression in white adipocytes was obtained by immunohistochemistry, which detected large patches of UCP-1-positive cells in WAT of VDRKO mice but negligible staining in tissue from WT mice. Disruption of the VDR gene improved the effect of insulin on glucose and triacylglycerol metabolism. In parallel, another study carried out on VDRKO mice both complemented and supported the above results. In common the studies
Alterations Exhibited by Vitamin-D-receptor-disrupted Mice (VDRKO) in Body Composition, in Hormone and Metabolic Profile [49,50] Fat tissue (WAT) morphology
Fat tissue gene/protein
Hormones
Metabolism
Y weight
Smaller unilocular adipocyte
Yleptin mRNA (WAT, BAT)
Yleptin
[food ingestion*
Yglobal fat
Presence of multilocular cells clusters, reminiscent of BAT
[FASmRNA (WAT) e FASmRNA (BAT)
e T3
Yweight
Yvisceral fat
e PPARgmRNA e PPARg (WAT)
e T4
e glucose on high-fat diet
e BAT
[UCP1mRNA [UCP1 (WAT)
e TSH
e TG on high-fat diet
Body composition
[UCP1, 2 and 3 mRNA (BAT)
e insulin on high-fat diet
WAT: white adipose tissue; BAT: brown adipose tissue; FAS: fatty acid synthase; PPARg: peroxisome proliferator-activated receptor gamma; UCP: uncoupling protein.
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showed that VDRKO mice have less adipose tissue and lower plasma triglyceride, cholesterol, and leptin levels compared to WT mice [51]. Additionally, it was observed that the rate of fatty acid oxidation in white adipose tissue was higher, and the expression of UCP1, UCP2, and UCP3 was markedly up-regulated in VDRKO mice, suggesting higher energy expenditure in the mutant mice. UCPs are known to be under the direct regulation of b-adrenergic receptor 3 (Adrb3) [52,53]. Thus, these authors investigated and verified that Adrb3 expression was unchanged in the BAT of VDRe/e mice compared with WT mice, suggesting that, in this experimental model, it is not involved in VDR regulation of UCP expression. Experiments using primary brown fat culture confirmed that 1,25-dihydroxyvitamin D3 directly suppressed the expression of UCPs. Consistently, energy expenditure, oxygen consumption, and CO2 production were markedly higher in VDR-null mice than in WT mice. All the results obtained with VDRKO mice agree with those obtained with animals submitted to a highcalcium diet, indicating that 1,25(OH)2D inhibits UCP expression and thereby decreases energy expenditure by classical genomic signaling. Furthermore, it was recently shown that VDR heterozygoteþ/e mice, an experimental model of mild vitamin D deficiency, had a lower fat mass in comparison to WT type C57BL6 mice although bone mineral density was similar in both groups. Surprisingly, these results suggest a primordial involvement of vitamin D in the control of energy storage instead of bone development.
VITAMIN D AND OBESITY: CLINICAL IMPLICATION Clinical studies have directly linked vitamin D deficiency to obesity and insulin resistance, suggesting in general that vitamin D is a protective agent against these epidemic disorders. Vitamin D seems to act as a necessary cofactor for insulin secretion [54,55]. Vitamin D repletion improves insulin sensitivity and insulin secretion in animal studies [56]. An analysis suggests that these clinical studies conflict with experimental models showing that VDRKO and CYP27B1KO mice with severe vitamin D deficiency exhibit a lean phenotype and a metabolic advantage in handling a hypercaloric diet [50,51]. The last section of this chapter is dedicated to a discussion of these discrepancies, which are possibly related to existing limitations derived from the clinical diagnosis of vitamin D deficiency. Several studies have reported that serum 25(OH)D is negatively associated with obesity, suggesting that D hypovitaminosis leads to a higher risk of obesity [57,58]. In support of this hypothesis, 870 obese patients
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were assigned to three interventional groups (placebo/ placebo, 1500 mg/day calcium plus placebo and 1500 mg/day calcium plus 1100 IU/day vitamin D3) during a study period of 4 years [59]. The two groups on calcium interventional therapy, one of them associated with vitamin D, gained less trunk fat and maintained more trunk lean mass when compared to the placebo group. In this study, there was no group on single vitamin D therapy. In another study, obese children were evaluated in a cross-sectional and longitudinal study aiming to show the relationship between vitamin D, weight status, and insulin sensitivity in childhood. A total of 133 obese Caucasian children and 23 healthy Caucasian normal weight children participated in this investigation. Additionally, these parameters were determined in a subgroup of 67 obese children before and after their participation in a 1-year outpatient obesity intervention program. The results revealed that obese children had significantly higher PTH levels and lower 25(OH)D concentrations compared to nonobese children. PTH levels decreased and 25(OH)D concentrations increased significantly in obese children, who achieved a reduction of overweight in the course of 1 year in contrast to obese children who did not achieve a reduction of overweight. The authors interpreted this result to indicate that the alterations of PTH and 25 (OH)D are a consequence rather than a cause of overweight [59]. Furthermore, a relationship between PTH, vitamin D, and insulin sensitivity based on the HOMA index was not found in obese children [60]. Different results were obtained in another study that evaluated 127 obese children and adolescents (48 Caucasians, 39 Hispanics, and 39 African-Americans). Hypovitaminosis D was present in 74% of the cohort, but was more prevalent in the Hispanic (76.9%) and African-American (87.2%) groups than in the Caucasian group (59.1%). The vitamin-D-deficient group had higher body mass index, fat mass, and PTH, but had lower insulin sensitivity (quantitative insulin sensitivity check index (QUICKI)) than the vitamin-D-sufficient group. Whereas fat mass was negatively correlated with 25(OH)D, it was positively correlated with iPTH. Serum 25(OH)D was also positively correlated with QUICKI, but was inversely correlated with HbA1c. The authors suggested that obese children and adolescents with low 25(OH)D levels may be at increased risk of developing impaired glucose metabolism independent of body adiposity [61]. Independent of age and race, most studies have shown that vitamin D deficiency is associated with obesity and a higher risk of glucose intolerance. These data apparently are in conflict with: (a) experimental studies showing that severe vitamin D deficiency (VDRKO and Cyp27b1KO) in mice is associated with lean phenotype and better insulin sensitivity, (b) a hypercalcemic diet suppresses 1,25(OH)2D synthesis and
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thereby decreases the influence of [Caþþ]i, consequently provoking higher lipolysis and lipogenesis, and (c) 1,25 (OH)2D enhances intracellular calcium which promotes lipogenesis through the activation of fatty acid synthase and inhibits lipolysis via activation of phosphodiesterase 3B, which subsequently reduces catecholamineinduced lipolysis. However, the discrepancies disappear when we take into account the fact that the 25(OH)D metabolite is a surrogate parameter used for the diagnosis of vitamin D status in clinical investigation. As previously mentioned, due to secondary hyperparathyroidism, vitamin-D-insufficient patients often have elevated levels of 1,25(OH)2D despite low serum 25 (OH)D levels [62]. A recent study evaluated vitamin D status in 86 normal glucose-tolerant and 160 glucose-intolerant Thai individuals. Fasting blood samples were assayed for 25(OH)D, adiponectin, glucose, and insulin levels. Insulin resistance (HOMA-IR) and insulin secretion index (HOMA-B) were calculated by homeostasis model assessment. The prevalence of vitamin D deficiency defined by 25(OH)D levels of less than 20 ng/ ml and vitamin D inadequacy defined by 25(OH)D levels of less than 30 ng/ml was 44.3 and 91.9%, respectively. Abnormal glucose-tolerant subjects had slightly lower average 25(OH)D levels than the normal glucose tolerant group (21.0 þ/e 6.8 vs. 22.1 þ/ e 6.2 ng/ml). Serum 25(OH)D levels were positively associated with adiponectin levels and negatively associated with HOMA-IR and BMI only in abnormal glucose-tolerant subjects. The data suggested an association between insufficient vitamin D status and lower circulating adiponectin in subjects with abnormal glucose tolerance independently of adiposity, which may indicate the role of adiponectin as a link between vitamin D status and insulin resistance [63]. In addition to reaffirming the association of the pro-hormone 25(OH)D with obesity and insulin insensitivity, this study connects vitamin D to the control of the endocrine role of adipose tissue and another network may be proposed. Adiponectin, an insulin-sensitizing polypeptide originating in adipose tissue, also has osteometabolic activity, which still is to be fully delineated [64]. 1,25(OH)2D also stimulates osteoblast production of osteocalcin, a bone peptide which is now been thoroughly evaluated regarding its participation in the control of insulin synthesis and action [65e70]. Therefore, vitamin D seems to be part of an integrative physiological process involving bone and adipose tissue as well as mineral and energetic metabolism in reciprocal control. A full delineation of the role of vitamin D in this complex network will certainly help to understand the onset of chronic metabolic and osteoporotic disorders such as diabetes mellitus and osteoporosis.
References [1] M.F. Holick, Vitamin D: a millennium perspective, J. Cell Biochem. 88 (2) (2003) 296e307. [2] E.D. Gorham, C.F. Garland, F.C. Garland, W.B. Grant, S.B. Mohr, M. Lipkin, et al., Vitamin D and prevention of colorectal cancer, J. Steroid Biochem. Mol. Biol. 97 (1-2) (2005) 179e194. [3] A.V. Krishnan, D.L. Trump, C.S. Johnson, D. Feldman, The role of vitamin D in cancer prevention and treatment, Endocrinol. Metab. Clin. North Am. 39 (2) (2010) 401e418. [4] C.Y. Li, J. Kong, M. Wei, Z.F. Chen, Q.S. Liu, L.P. Cao, 1,25Dihydroxyvitamin D3 is a negative endocrine regulator of the rennin-angiotensin sytem, J. Clin. Invest. 110 (2) (2002) 229e238. [5] S.G. Rostand, Vitamin D, blood pressure, and African Americans: toward a unifying hypothesis, Clin. J. Am. Soc. Nephrol. 5 (9) (2010) 1697e1703. [6] G. Verhave, C.E. Siegert, Role of vitamin D in cardiovascular disease, Neth. J. Med. 68 (3) (2010) 113e118. [7] G. Zhao, R.U. Simpson, Interaction between vitamin D receptor with caveolin-3 and regulation by 1,25-dihydroxyvitamin D3 in adult rat cardiomyocytes, J. Steroid Biochem. Mol. Biol. 121 (1e2) (2010) 159e163. [8] D.A. Fernandes de Abreu, D. Eyles, F. Fe´ron, Vitamin D, a neuro-immunomodulator: implications for neurodegenerative and autoimmune diseases, Psychoneuroendocrinology 34 (Suppl. 1) (2009) S265eS277. [9] H.F. Deluca, M.T. Cantorna, Vitamin D: its role and uses in immunology, FASEB J. 15 (14) (2001) 2579e2585. [10] H.A. Bischoff-Ferrari, B. Dawson-Hughes, H.B. Staehelin, J.E. Orav, A.E. Stuck, R. Theiler, et al., Fall prevention with supplemental and active forms of vitamin D: a meta-analysis of randomized controlled trials, BMJ 339 (2009) b3692. [11] L.D. Moreira-Pfrimer, M.A. Pedrosa, L. Teixeira, M. LazarettiCastro, Treatment of vitamin D deficiency increases lower limb muscle strength in institutionalized older people independently of regular physical activity: a randomized double-blind controlled trial, Ann. Nutr. Metab. 54 (4) (2009) 291e300. [12] V. Gilsanz, A. Kremer, A.O. Mo, T.A. Wren, R. Kremer, Vitamin D status and its relation to muscle mass and muscle fat in young women, J. Clin. Endocrinol. Metab. 95 (4) (2010) 1595e1601. [13] T. Keisala, A. Minasyan, Y.R. Lou, J. Zou, A.V. Kalueff, I. Pyykko¨, P. Tuohimaa, Premature aging in vitamin D receptor mutant mice, J. Steroid Biochem. Mol. Biol. 115 (3e5) (2009) 91e97. [14] B. Lanske, M.S. Razzaque, Vitamin D and aging: old concepts and new insights, J. Nutr. Biochem. 18 (12) (2007) 771e777. [15] M.A. Beydoun, A. Boueiz, M.R. Shroff, H.A. Beydoun, Y. Wang, A.B. Zonderman, Associations among 25-hydroxyvitamin D, diet quality, and metabolic disturbance differ by adiposity in adults in the United States, J. Clin. Endocrinol. Metab. 95 (8) (2010) 3814e3827. [16] K. Brock, W.Y. Huang, D.R. Fraser, L. Ke, M. Tseng, R. Stolzenberg-Solomon, et al., Low vitamin D status is associated with physical inactivity, obesity and low vitamin D intake in a large US sample of healthy middle-aged men and women, J. Steroid Biochem. Mol. Biol. 121 (1-2) (2010) 462e466. [17] B.W. Hollis, Assessment and interpretation of circulating 25hydroxyvitamin D and 1,25-dihydroxyvitamin D in the clinical environment, Endocrinol. Metab. Clin. North Am. 39 (2) (2010) 271e286. [18] K.A. Kennel, M.T. Drake, D.L. Hurley, Vitamin D deficiency in adults: when to test and how to treat, Mayo Clin. Proc. 85 (8) (2010) 752e757.
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C H A P T E R
45 Extrarenal 1a-Hydroxylase Martin Hewison, John S. Adams Department of Orthopaedic Surgery and Molecular Biology Institute, David Geffen School of Medicine at UCLA, 615 Charles E. Young Drive South, Los Angeles, CA 90095, USA
INTRODUCTION The previous version of this chapter (2nd edition) was primarily defined by the rapid increase in our understanding of the basic molecular endocrinology of 25-hydroxyvitamin D-1a-hydroxylase (CYP27B1 or 1a-hydroxylase) e the vitamin-D-activating enzyme e that had taken place in the first 5 years of the new century. This latest version reflects the entirely new perspective on 1a-hydroxylase that has arisen since then in which studies have principally focused on the physiological impact of this enzyme in peripheral tissues. This fresh approach to extrarenal 1a-hydroxylase has been characterized by two key observations. The first stems from our revised view of what constitutes adequate vitamin D status. Until recently, the vitamin D status of an individual was defined simply by presence or absence of rickets (osteomalacia in adults), a relatively rare bone disease in modern society. However, as detailed in many chapters in this book, new parameters for vitamin D sufficiency suggest that many more people have subclinical vitamin D insufficiency. Inadequate vitamin D status may impact on several facets of vitamin D physiology but it is most likely to affect extrarenal 1a-hydroxylase activity, which is not regulated by the same endocrine factors associated with its renal counterpart. The second major new consideration for extrarenal 1a-hydroxylase relates to our increased understanding of the tissue distribution and regulation of this enzyme, notably within the immune system. We have incorporated these new developments into the framework of the original chapter alongside the seminal observations of extrarenal 1a-hydroxylase activity in diseases such as sarcoidosis. The fundamental structure of the chapter has been retained but additional sections have been added. After this brief introduction
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10045-9
(first section), the second section of the chapter will review the historical aspects of extrarenal synthesis of the active form of vitamin D 1,25-dihydroxyvitamin D (1,25(OH)2D) associated with inflammatory disease. The third will provide a comprehensive review of the various human diseases associated with the overproduction of active vitamin D metabolites from an extrarenal source. The fourth section of the chapter will describe the various studies that have documented expression and activity of 1a-hydroxylase in extrarenal tissues that are not associated with inflammatory or granulomatous disease. The fifth section will outline what we know about the mechanics and regulation of the vitamin-D-metabolizing enzymes present in an extrarenal setting. The sixth and final section of this chapter will address the clinical aspects of disordered extrarenal 1a-hydroxylase; this will include a discussion of the diagnosis, treatment, and prevention of hypercalcemia and hypercalciuria in the patient with endogenous vitamin D intoxication.
VITAMIN D AND GRANULOMAFORMING DISEASE: A HISTORICAL PERSPECTIVE Evidence of Endogenous Vitamin D Intoxication Associated with Sarcoidosis A pathophysiological relationship between vitamin D and sarcoidosis was first recognized 80 years ago by Harrell and Fisher [1]. Among the six hypercalcemic patients in their initial report, one was observed to experience a steep rise in serum calcium concentrations following ingestion of cod liver oil known to be enriched in vitamin D. Almost two decades passed before Henneman et al. [2] demonstrated that the hypercalcemic syndrome of sarcoidosis, characterized by
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45. EXTRARENAL 1a-HYDROXYLASE
increased intestinal calcium absorption and bone resorption, was remarkably similar to that of exogenous vitamin D intoxication and was treatable by the administration of glucocorticoids. In 1963, the first, large-scale seasonal evaluation of serum calcium levels in patients with sarcoidosis was performed [3]. They found that there was a significant increase in the mean serum calcium concentration in 345 patients with sarcoidosis from winter to summer but no such change in over 12 000 control subjects. This was the first evidence that there was an association between enhanced vitamin D synthesis, known to occur principally during the summer months, and the blood level of calcium in patients with sarcoidosis. This observation was prospectively confirmed by Dent [4] who was able to increase the serum calcium concentration in patients with active sarcoidosis upon whole-body exposure to ultraviolet (UV) light irradiation.
Evidence for Extrarenal Production of an Active Vitamin D Metabolite in Patients with Sarcoidosis The above-mentioned studies led Bell et al. [5] to propose that development of a clinical abnormality in calcium balance in patients with active sarcoidosis resulted from increased target organ responsiveness to vitamin D. This view persisted for more than decade. However, after the discovery of 1,25(OH)2D as the active vitamin D hormone [6e8] and the development of sensitive and specific assays for the hormone in blood [9e11], investigators were quick to determine that the hypercalcemia of sarcoidosis was the result of an increase in the circulating concentrations of a vitamin D metabolite that interacted with the vitamin D receptor (VDR) [12e15]. The fact that a vitamin D hormone was made outside the kidney in hypercalcemic patients with sarcoidosis was first discovered by Barbour and colleagues in 1981 [16]. These investigators reported high concentrations of a vitamin D metabolite detected as 1,25(OH)2D in the circulation of a hypercalcemic, anephric patient with active sarcoidosis. Two years later, Adams et al. [17] demonstrated that the macrophage was the extrarenal source of the active vitamin D metabolite. Unequivocal structural characterization of the metabolite as 1,25(OH)2D was later obtained by the same investigators [18,19].
Clinical Evidence for Dysregulated Extrarenal Production of 1,25(OH)2D As has been described in detail in earlier chapters, the synthesis of 1,25(OH)2D by the renal 1a-hydroxylase is normally strictly regulated with levels of the hormone product being some 1000-fold less plentiful in the
circulation than that of the principal substrate for the enzyme, 25-hydroxyvitamin D (25(OH)D). Hormone synthesis in the kidney is stimulated by increases in the serum parathyroid hormone (PTH) concentration, a decrease in the serum phosphate concentration, and a decrease in the activity of the competing vitamin D24-hydroxylase (24-hydroxylase) enzyme. Renal synthesis of 1,25(OH)2D is inhibited by decreased circulating levels of PTH, increased serum phosphate and associated fibroblast growth factor 23 (FGF23), and increased 24-hydroxylase activity. There are now several lines of clinical evidence to indicate that endogenous 1,25(OH)2D production in hypercalcemic/hypercalciuric patients with sarcoidosis is dysregulated and not bound by the same set of endocrine factors known to regulate 1,25(OH)2D synthesis in the kidney [20]. First, hypercalcemic patients with sarcoidosis possess a frankly high or inappropriately elevated serum 1,25 (OH)2D concentration, although their serum PTH level is suppressed and their serum phosphate concentration is relatively elevated [21,22]. If 1,25(OH)2D synthesis were under the regulation of PTH, phosphate, and 1,25 (OH)2D itself, then 1,25(OH)2D concentrations in such patients should be low. Second, the serum 1,25(OH)2D concentration in patients with active sarcoidosis is very sensitive to an increase in available substrate [14], while the serum 1,25(OH)2D level in normal individuals is not influenced by small of even moderate increments in the circulating 25(OH)D concentration. Clinically, this aspect of dysregulation is manifested by the long-recognized association of hypercalciuria and/or hypercalciuria in sarcoidosis patients in the summer months or following holidays to geographic locations at lower latitudes than those at which the patient normally resides [14,23]. This link between increased cutaneous vitamin D synthesis and the development of clinical abnormalities of calcium balance can be replicated by oral administration of vitamin D [14,22,24]. It can also be substantiated on a biochemical basis by demonstration of positive correlation between serum 25(OH)D and 1,25(OH)2D concentrations in patients with active sarcoidosis but not normal subjects [15]. Third, the rate of endogenous 1,25(OH)2D production, which is significantly increased in patients with sarcoidosis, is unusually sensitive to inhibition by factors (i.e., drugs) that do not influence the renal 1a-hydroxylase at the same doses [25]. Anti-inflammatory concentrations of glucocorticoids have long been recognized as effective combatants of sarcoidosis-associated hypercalcemia and have also been shown to dramatically lower elevated 1,25(OH)2D levels [15,24,26]. On the other hand, administration of the same glucocorticoid doses to patients without sarcoidosis is not associated with a clinically relevant reduction in the serum 1,25(OH)2D or calcium concentration. Chloroquine and its
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HUMAN DISEASES WITH EXTRARENAL OVERPRODUCTION OF ACTIVE VITAMIN D METABOLITES
hydroxylated analog, hydroxychloroquine, are other examples of pharmaceutical agents that appear to act preferentially on the extrarenal vitamin-De1a-hydroxylase reaction which is active in patients with sarcoidosis [27e30]. Fourth, the serum calcium and 1,25(OH)2D concentrations are positively correlated to indices of disease activity in patients with sarcoidosis [31e33]; patients with widespread disease and high angiotensin-converting enzyme activity are more likely to be hypercalciuric or frankly hypercalcemic. Investigators have now generated a substantial body of experimental data from cells, including inflammatory cells harvested directly from patients with sarcoidosis, to indicate that the dysregulated vitamin D hormone synthesis in sarcoidosis is not due to expression of a 1a-hydroxylase that is different from the renal enzyme, but rather to expression of the authentic 1a-hydroxylase (CYP27B1) in a macrophage, rather than a kidney cell [34]. In fact, each of the above-mentioned pieces of clinical evidence for dysregulated vitamin D hormone production in this disease can be borne out in vitro in cells from patients with this disease [18].
HUMAN DISEASES WITH EXTRARENAL OVERPRODUCTION OF ACTIVE VITAMIN D METABOLITES Over 30 conditions have been described in which elevated circulating levels of 1,25(OH)2D associated with extrarenal expression of 1a-hydroxylase are thought to be the cause of patient hypercalciuria or hypercalcemia (see Table 45.1). In some cases aberrant calcium homeostasis has been reported to be associated with only “high normal” concentrations of serum 1,25 (OH)2D [35]. Nevertheless, this would still represent an inappropriate elevation of 1,25(OH)2D given that suppression of PTH levels is common with most of the diseases listed in Table 45.1. The manifestation of raised serum 1,25(OH)2D and associated hypercalciuria/ hypercalcemia is likely to be highly dependent on the availability of substrate 25(OH)D, in other words the vitamin D status of the individual. This was illustrated clearly in recent studies of a patient with giant cell polymyositis (GCP) with low serum calcium levels who was prescribed vitamin D supplementation therapy and then went on to develop increased serum 1,25(OH)2D and hypercalemia [35]. Similar observations have also been made for other diseases associated with elevated extrarenal 1a-hydroxylase [36e38]. Most diseases listed in Table 45.1 are granulomatous in nature, ranging from inflammatory conditions and foreign body exposures to infections and neoplasms. The following section provides more comprehensive details for specific
TABLE 45.1 Human Diseases Associated with 1,25-Dihydroxyvitamin-D-mediated Hypercalcemia/Hypercalciuria INFECTIOUS Tuberculosis
[47,49,269]
Leprosy
[54,55,270]
Candidiasis
[36]
Cryptococcosis
[56,271,272]
Histoplasmosis
[273]
Coccidiodomycosis
[58]
AIDS-related pneumocystis
[59]
Cat-scratch fever
[274])
Mycobacterium avium infection
[275,276]
NON-INFECTIOUS Sarcoidosis
[13e16]
Silicone-induced granulomatosis
[60]
Langerhans cell histiocyosis
[61,63]
Wegener’s granulomatosis
[62,277,278]
Berylliosis
[279]
Lipoid pneumonia
[280,281]
Talc granulomatosis
[282]
Infantile fat necrosis
[66]
Slack skin disease
[283]
Giant cell polymyositis
[35]
Crohn’s disease
[67e69]
NEOPLASTIC Hodgkin’s disease
[75e77,80]
Non-Hodgkin’s lymphoma
[79e82]
Plasma cell granuloma
[284]
Dysgerminoma
[151,285]
Seminoma
[286]
human diseases that have been linked to overexuberant extrarenal 1a-hydroxylase.
Granuloma-Forming Diseases Sarcoidosis Sarcoidosis is the human disease most commonly complicated by endogenous vitamin D intoxication
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[1e5,13e17,19,39e41]. In the most expansive studies published on the topic, roughly 10% of patients with sarcoidosis develop hypercalcemia [41], and up to 50% suffer from hypercalciuria [41] at some time during the course of their disease. In their seminal, retrospective, worldwide review of serum calcium concentrations in 3676 patients with sarcoidosis, James et al. [41] recorded an 11% incidence of hypercalcemia (serum calcium 10.5 mg/dl). Studdy et al. [40] studied 547 patients with biopsy-proven sarcoidosis in Great Britain and found hypercalcemia to be 38% more frequent in men than women and more common among Caucasians than individuals of West Indian descent. Although not systematically studied, the frequency of hypercalcemia among patients with sarcoidosis tends to be consistently higher in North America than in Northern Europe [42]. This is perhaps due to the lower latitude and more direct sunlight exposure in the United States. Although fractional intestinal calcium absorption may be increased under the influence of 1,25(OH)2D and fractional urinary calcium excretion may be decreased in patients with renal insufficiency [32], the principal source of calcium which accumulates in the circulation of sarcoid patients is the skeleton. This fact is perhaps most clearly illustrated by the observations of Rizatto et al. [43] who documented in serial fashion a significant decrease in bone mineral density in a group of patients with chronic active sarcoidosis in whom antiinflammatory agents, including glucocorticoids, were not used in management compared to age- and sexmatched control subjects. This fact is confirmed by the long-standing observations that hypercalcemia persists in patients with active sarcoidosis in the absence of ingested calcium and may be contributed to by increased bone resorption [26]. The proximal cause of bone loss is increased osteoclast-mediated bone resorption and does not require the presence of extensive granulomata in the bone [44]. Tuberculosis Of the other human granuloma-forming diseases reported to be associated with vitamin D metabolitemediated hypercalcemia, tuberculosis is the most commonly reported. Hypercalcemia has been recognized as a complication of infection with Mycobacterium tuberculosis (M. tb) for over eight decades [45]. That this disturbance in calcium balance is caused by the extrarenal overproduction of an active vitamin D metabolite was confirmed by investigators in the mid-1980s [46,47]. As is the case with sarcoidosis, the circulating vitamin D metabolite causing hypercalcemia has the following properties: (1) appears to be 1,25(OH)2D [48,49]; (2) is synthesized by disease-activated macrophages [50,51]; (3) is abnormally responsive to small changes in the serum concentration of substrate 25
(OH)D [52]; and (4) is reducible under the influence of glucocorticoids in vivo [37,53]. The prevalence of hypercalcemia in patients may be as high as 26% [45] and may be even higher, particularly in the era of AIDS, because of frequent association of hypoalbuminemia (i.e., from malnutrition) in patients with tuberculosis. The source of 1,25(OH)2D in TB patients is, as it is in all of the other granuloma-forming diseases, extrarenal [46] most likely the macrophage [50]. Other Infectious Diseases Hypercalciuria or overt hypercalcemia has also been observed in a number infectious diseases, most characterized by widespread granuloma formation and macrophage proliferation in infected tissue. Included among these diseases are leprosy [54,55], disseminated candidiasis [36], crytococcosis [56], histoplasmosis [38,57], and coccidioidomycosis [58]. Hypercalcemia in most of these conditions has been documented to be associated with inappropriately elevated serum concentrations of 1,25(OH)2D. The true prevalence and incidence of hypercalcemia and hypercalciuria in patients with these diseases in unknown. However, it is likely that the dysregulated vitamin D metabolism and action associated with these diseases will increase in frequency as the number of immunocompromised patients, especially those with AIDS, increases worldwide. For example, hypercalcemia in association with elevated circulating levels of 1,25(OH)2D has been reported in an AIDS patient with pneumocystis [59]; both serum calcium and 1,25(OH)2D concentrations dropped in this patient with successful treatment of his opportunistic infection. Noninfectious Granuloma-forming Diseases The syndrome of extrarenal overproduction of 1,25 (OH)2D has also been documented in adult patients with widespread silicone-induced granulomata [60], eosinophilic granuloma [61], Wegener’s granulomatosis [62], Langerhans cell histiocytosis [63], and the systemic granulomatous disease GCP [35]. Although the active vitamin D metabolite was not measured, dysregulated calcium balance in the granuloma-forming pulmonary disease berylliosis is also attributed to the extrarenal production of 1,25(OH)2D [64]. In addition, 1,25(OH)2D-mediated hypercalcemia has been observed in newborn infants suffering from massive subcutaneous fat necrosis [65,66]; this is a transient disorder associated with birth trauma and characterized histopathologically by the proliferation of "foreign-body-type" giant cells around cholesterolshaped crystals in necrotizing, subcutaneous adipose tissue. Finally, there are also reports of elevated serum levels of 1,25(OH)2D and associated hypercalcemia in patients with Crohn’s disease, a form of inflammatory
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HUMAN DISEASES WITH EXTRARENAL OVERPRODUCTION OF ACTIVE VITAMIN D METABOLITES
bowel disease [67e69]. The possible impact of extrarenal 1a-hydroxylase in this clinical situation is an exciting new development in part because of the prevalence of Crohn’s disease particularly in developed countries [70], but also because of several recent reports which have documented expression of 1ahydroxylase along the gastrointestinal tract [71e74]. In the case of patients with Crohn’s disease presenting with elevated serum 1,25(OH)2D, it has been possible to use tissue biopsy material to show that the increased activation of 25(OH)D is due to enhanced expression of 1a-hydroxylase in areas of the colon with extensive granulomatous disease [68]. Vitamin D and inflammatory bowel disease is discussed in Chapter 96.
Malignant Lymphoproliferative Disorders By the 1980s accumulating data suggested that vitamin-D-mediated disturbances in calcium metabolism were not confined to patients with granulomaforming diseases and could also be observed in patients with lymphoproliferative neoplasms [75e78]. Subsequent reports indicated that the extrarenal overproduction of 1,25(OH)2D is the most common cause of hypercalciuria and hypercalcemia in patients with non-Hodgkin and Hodgkin lymphoma [79,80], especially in patients with B-cell neoplasms whether or not the tumor is associated with AIDS in the patient [78]. In fact, in the study by Seymour et al., 71% of normocalcemic patients with non-Hodgkin lymphoma had hypercalciuria (fractional urinary calcium excretion >0.15 mg/dl glomerular filtrate) and most of these had serum 1,25(OH)2D levels that were above the mid range of normal or frankly elevated [79]. As is the situation with hypercalciuric/calcemic patients with sarcoidosis or other granuloma-forming disease and elevated circulating 1,25(OH)2D levels, serum concentrations of PTH are suppressed and PTHrP normal (i.e., not elevated) in lymphoma patients, indicative of the state of dysregulated overproduction of 1,25 (OH)2D. Results of clinical studies of hypercalcemic patients with lymphoma pre- and post-successful antitumor therapy [78e81] are compatible with either the tumor being an immediate source of an active vitamin D metabolite or the source of a soluble factor which stimulates the production of 1,25(OH)2D in the kidney or other inflammatory cells. More recent data suggest that the latter is the case. Extensive analysis of a patient with hypercalcemia and raised circulating levels of 1,25 (OH)2D associated with a splenic B-cell lymphoma showed that the abnormalities in serum 1,25(OH)2D and calcium were corrected following resection of the spleen [82]. Subsequent immunonhistochemical
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analysis of this tissue revealed increased expression of 1a-hydroxylase in macrophages adjacent to the tumor but not in the tumor itself [82]. This raises further important questions: (i) what is the nature of the tumorderived factor that is able to stimulate macrophage 1ahydroxylase?; (ii) is macrophage-derived 1,25(OH)2D a contributing factor to the hypercalcemia associated with other types of tumors?; (iii) is overexpression of 1a-hydroxylase a feature of tumors in the absence of the macrophage-associated hypercalcemia associated with lymphoproliferative disorders? These questions will be addressed later in the chapter.
Other Inflammatory Diseases Reports describing circulating levels of 1,25(OH)2D in patients with inflammatory autoimmune diseases such as rheumatoid arthritis have been variable and include enhanced [83], decreased [84,85], or unchanged circulating levels [86] of the hormone. In other related diseases such as systemic lupus erythematosus (SLE) circulating levels of 1,25(OH)2D do not appear to be affected by disease status [86], even though SLE patients have been reported to exhibit low serum levels of 25 (OH)D [87e89]. Although patients with inflammatory disease may not present with the same elevated circulating levels of 1,25(OH)2D that are often characteristic of those with granulomatous disease, this does not detract from possible localized expression of extrarenal 1a-hydroxylase. Mawer and colleagues [170,171] demonstrated substrate-dependent accumulation of 1,25(OH)2D in the synovial fluid of patients with “inflammatory” arthritis including those with rheumatoid arthritis [90,91]. These observations prompted the suggestion that local induction of 1,25(OH)2D synthesis within the synovium may contribute to periarticular bone loss in such individuals. Peritonitis is another disease associated with membrane inflammation, in this case the serous membrane of the abdominal cavity. There are many potential causes of peritonitis including infectious and noninfectious forms. Amongst the former, peritonitis is frequently observed in chronic kidney disease (CKD) patients receiving peritoneal dialysis, particularly when afflicted with peritonitis. Peritoneal macrophages from such patients have been shown to metabolize 25(OH)D to 1,25(OH)2D in vitro [92e94]. The impact of this capacity for localized (peritoneal) synthesis of 1,25(OH)2D on circulating levels of the hormone is less easy to delineate than for other diseases such as sarcoidosis because CKD patients have underlying impairment of renal 1a-hydroxylase activity. Nevertheless, some reports have suggested that serum levels of 1,25(OH)2D in CKD patients receiving peritoneal dialysis are higher than those observed for patients receiving conventional hemodialysis [92].
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EXTRARENAL SYNTHESIS OF 1,25(OH)2D IN NORMAL PHYSIOLOGY AND CANCER The cloning of the gene for 1a-hydroxylase (CYP27B1) has enabled a much more comprehensive appraisal of the tissue distribution of this enzyme than was previously available. Indeed the human cDNA for CYP27B1 was cloned from keratinocytes, a well-established extrarenal source of 1,25(OH)2D [95]. As a result of these studies, the development of probes and antisera for 1a-hydroxylase have helped to define both the renal and extrarenal tissues that express this enzyme [71,96]. The following section will discuss some of the key extrarenal tissues that have been reported to express 1ahydroxylase in a “nondisease” setting (i.e., those not already listed as being associated with dysregulated serum levels of 1,25(OH)2D). However, it is important to recognize that some debate remains concerning the physiological impact of extrarenal 1,25(OH)2D, notably whether peripheral (nonrenal) expression of 1a-hydroxylase contributes to the circulating levels of this hormone in a nondisease setting, or whether locally generated 1,25(OH)2D remains local. Studies using cultured monocytes and dendritic cells (DCs) expressing 1a-hydroxylase have shown that treatment with 150 nM 25(OH)D is as effective as 100 nM 1,25(OH)2D in stimulating changes in monocyte or DC phenotype markers. This occurs despite the fact that the amount of intracrine 1,25(OH)2D generated by monocytes from 150 nM 25 (OH)D is only about 3 nM, 30 times lower than that obtained with exogenously added 100 nM 1,25(OH)2D [97]. It is therefore possible to conclude that, if expressed, the extrarenal 1a-hydroxylase system may provide a sensitive mechanism for mediating cellular responses to vitamin D. Nevertheless, it is important to recognize that, if present at high concentrations, 25 (OH)D itself may act as a ligand for the VDR and promote transcriptional responses independent of 1ahydroxylase activity. The affinity of VDR for 25(OH)D is approximately 500-fold lower than that observed with 1,25(OH)2D. Nevertheless it is increasingly common for studies of intracrine responses to 25(OH) D to employ 1a-hydroxylase inhibitors such as itraconazole to confirm this mechanism of action. The generation of knockout models for mouse Cyp27b1 [98,99] has attempted to address this question. In studies using a promoter-reporter cDNA to knock out the Cyp27b1 gene DeLuca and colleagues observed that active transcription of Cyp27b1 was only evident in two mouse tissues e the kidney and the placenta [99]. The overall conclusion was that in healthy mice expression of 1a-hydroxylase is very limited without an additional stimulus. Of course the situation in humans may be very different, but even in mice the role of
extrarenal 1a-hydroxylase may be more akin to a rapid response mechanism in which tissue-specific activators enhancing local synthesis of 1,25(OH)2D in response to a particular challenge. The following section will consider the various pieces of evidence that support a wider role for extrarenal 1a-hydroxylase in normal physiology by detailing specific examples of tissues where this has been extensively studied. In addition, Table 45.2 provides a more comprehensive list of tissues and cell types with reported extrarenal 1a-hydroxylase expression or activity. The number of tissues that do not appear to express 1a-hydroxylase appears to be quite small and includes the adrenal cortex, liver, and heart muscle. However, it is important to recognize that the techniques and reagents used to detect this enzyme are variable and this is documented in Table 45.2.
The Immune System As detailed above, description of extrarenal 1a-hydroxylase activity in macrophages from patients with sarcoidosis was one of the first pieces of evidence linking vitamin D with the immune system. Another important development stemmed from the ability of 1,25(OH)2D to stimulate differentiation of precursor monocytes to more mature phagocytic macrophages [100e103]. This response was shown to be associated with differential expression of VDR and 1a-hydroxylase during the differentiation of human monocytes/macrophages [104], indicating that normal human macrophages were also able to synthesize 1,25(OH)2D3 under the right conditions [105]. Localized activation of vitamin D, coupled with expression of endogenous VDR was consistent with an intracrine system for vitamin D action in normal monocytes/macrophages [106]. However, the functional relevance of such a mechanism was only defined much later in studies showing that VDR and CYP27b1 are specifically induced in monocytes by M. tb [107]. Subsequent experiments confirmed that precursor 25(OH)D was able to induce intracrine VDR responses in monocytes that had been treated with a toll-like receptor (TLR)-2 ligand that mimicks M. tb. In particular, combined treatment with a TLR2/1 ligand and 25(OH)D stimulated expression of the antibacterial protein cathelicidin, so that vitamin D was able to promote monocyte killing of M. tb [107]. Crucially this response was directly influenced by the 25 (OH)D status of the donor serum used for monocyte culture [107,108]. The importance of this mechanism in human innate immunity is detailed in Chapter 91, and the mechanisms that act to control extrarenal synthesis of 1,25(OH)2D by human monocytes are discussed later in this chapter. In addition to normal human monocytes, several other immune cell types have been shown to exhibit
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EXTRARENAL SYNTHESIS OF 1,25(OH)2D IN NORMAL PHYSIOLOGY AND CANCER
TABLE 45.2 Expression and Activity of 1a-hydroxylase in Renal and Extrarenal Tissues. Selected References Describing the Expression and Activity (in vivo or in vitro) of 1a-hydroxylase. * Includes Neoplastic Tissues or Cells Tissue/cell type
Detection
Proximal tubule
RNA, protein, enzyme activity, promoter activity, in vitro [96,99,213,287]
Distal nephron
RNA, protein, in vitro [96,288]
Placenta (decidua/trophoblast)
RNA, protein, enzyme activity, promoter activity, in vitro [99,115e117]
Immune system (various cells)
RNA, protein, enzyme activity, in vitro [17,105,110,113,114]
Epidermis (stratum basalis)
RNA, protein, in vitro [71,95,289]
Colon*
RNA, protein, enzyme activity, in vitro [71,170,290]
Parathyroid*
RNA, protein, in vitro [137e140]
Bone (osteoblasts)
RNA, protein, in vitro [179,180,183,291]
Growth plate (chondrocytes)
RNA, protein, enzyme activity, in vitro [184,185,188]
Vasculature (endothelial cells)
RNA, protein, enzyme activity, in vitro [292]
Breast*
RNA, protein, enzyme activity, in vitro [159e161]
Prostate*
RNA, protein, enzyme activity, in vitro [154,293,294]
Ovary*
RNA, protein, enzyme activity, in vitro [150,151]
Testis
RNA [295]
Brain
RNA, protein [71,296]
Muscle
RNA, enzyme activity, in vitro [297]
Pancreas (islets)
RNA, protein, activity, in vitro [143]
Thyroid*
RNA, protein [146]
Adrenal medulla
Protein [71]
Hair follicle
Protein [71]
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1a-hydroxylase expression and activity. For example, antigen-presenting dendritic cells (DCs) represent an important target for 1,25(OH)2D in mediating its actions on T- and B-cell function and subsequent effects on autoimmune activity and hostegraft rejection. In addition to the presence of VDR [109], DCs also express 1a-hydroxylase in a similar fashion to macrophages [110,111]. Monocyte-derived DCs show baseline 1ahydroxylase expression and activity that increases as they differentiate towards a mature phenotype [110]. Functional analyses have shown that DCs treated with 25(OH)D show suppressed maturation. This in turn leads to inhibition of T-cell proliferation, underlining the similarity of this intracrine pathway to that observed in macrophages [110]. Mature DCs express less VDR than immature DCs [110] and this may be advantageous in that mature antigen-presenting DCs will be less sensitive to the suppressive effects of 1,25(OH)2D, thereby allowing induction of an initial T-cell response. However, the high levels of 1,25(OH)2D synthesized by mature DCs can act on VDR-enriched immature DCs and thus prevent their further maturation [112]. In this way, the DC 1a-hydroxylase may act in a more paracrine rather than intracrine fashion to allow initial presentation of antigen to T cells whilst preventing continued maturation of DCs and overstimulation of T cells. Other reports of CYP27B1 expression in DCs have shown that this may play a role in mediating the effects of vitamin D on T-cell homing via interaction between chemokine ligand 27 and chemokine receptor 10 [113]. Interestingly, in this instance it was suggested that T cells may also act as a local source of 1,25(OH)2D by expressing CYP27B1. Similar expression of 1a-hydroxylase has also been reported for B cells [114], suggesting that some lymphocyte responses to vitamin D may be mediated via intracrine rather than paracrine metabolism of vitamin D.
Placenta The placenta was one of the first extrarenal sites shown to be capable of synthesizing 1,25(OH)2D from 25(OH)D [115,116]. Subsequent studies have characterized the spatiotemporal organization of placental 1ahydroxylase across gestation, showing that the enzyme is localized in both maternal decidua and fetal trophoblast, and is more abundant in 1st and 2nd trimester tissue [117,118]. Within maternal decidua, expression of 1a-hydroxylase is not restricted to decidualized stromal cells but is also detectable in decidual macrophages [117], consistent with studies of macrophages in many other tissues. It is also interesting to note that although CYP27B1 and VDR are strongly expressed in the placenta, there appears to be little or no parallel expression of the vitamin D catabolic enzyme 24hydroxylase. This is explained by recent reports
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describing extensive methylation of the CYP24A1 gene in placental tissue [119]. Based on this observation, it has been postulated that silencing of CYP24A1 provides a mechanism for maximal local generation of 1,25 (OH)2D3 from 25(OH)D3 in the placenta. Initially, the high level of placental 1a-hydroxylase activity was linked to the rise in maternal serum 1,25 (OH)2D that occurs at the end of the first trimester of pregnancy. However, studies of Cyp27b1-deficient animals and an anephric pregnant woman indicate that this is not likely to be the case [120]. Instead, the presence of VDR in the placenta suggests that vitamin D functions in an intracrine fashion at the fetalematernal interface [121]. One possible explanation is that 1,25(OH)2D acts as a regulator of placental calcium transport [121], but an immunomodulatory role has also been proposed [122]. The latter stems, in part, from studies highlighting immune activity within the heterogeneous cells that make up the placenta. Maternal and fetal cells are able to mediate innate [123e125] and adaptive [126,127] immune responses. Other groups have reported altered levels of 1a-hydroxylase in placentas from preeclampsia pregnancies [128,129]. Furthermore, in a recent nested caseecontrol study Bodnar and colleagues showed that vitamin D deficiency significantly increases the risk of preeclampsia [130]. This coupled with evidence of the prevalence of vitamin D insufficiency in pregnant mothers e particularly African-American mothers [131] e supports a role for vitamin D sufficiency in protecting against this prevalent complication of pregnancy [132].
Skin The human cDNA for CYP27B1 was cloned from fibroblasts and these cells have been used to demonstrate impaired synthesis of 1,25(OH)2D in patients with inherited mutations in CYP27B1 [95]. Studies in vitro have shown that expression and activity of 1ahydroxylase is dependent on the stage of keratinocyte development [133,134]. Proliferating keratinocytes characteristic of cells within the stratum basalis of the epidermis show relatively high levels of 1,25(OH)2D synthesis but this diminishes as the cells differentiate towards cornified envelope precursor cells. Down-regulation of keratinocyte 1a-hydroxylase activity is accompanied by increased 24-hydroxylase activity and decreased VDR expression [134]. These changes in vitamin D metabolism precede the induction of epidermal differentiation markers such as transglutaminase, suggesting an intracrine role for locally synthesized 1,25(OH)2D in the development of the normal epidermis. Studies using knockout mice for Cyp27b1 have confirmed that synthesis of 1,25(OH)2D is required for normal differentiation and function of epidermal
keratinocytes [135,136]. However, as yet, the specific relevance of epidermal 1a-hydroxylase versus the renal enzyme in mediating this process has still to be confirmed. The effect of vitamin D on keratinocytes is further discussed in Chapter 30.
Endocrine Glands and Reproductive Tissues One of the cornerstones of classical calcium endocrinology is the suppression of PTH secretion by 1,25 (OH)2D produced in the kidneys. However, it appears that the enzyme is also expressed in the parathyroid glands themselves. Analysis of mRNA expression showed that CYP27B1 was expressed in normal parathyroid glands, with transcript levels being increased in parathyroid adenomas and decreased in parathyroid carcinomas [137,138]. Paradoxically FGF23, which is known to suppress renal 1a-hydroxylase activity, acts to enhance expression of CYP27B1 in cultured bovine parathyroid cell [139]. Nevertheless, the presence of 1a-hydroxylase in the parathyroid glands raises the possibility that intracrine production of 1,25(OH)2D within this organ contributes to the regulation of PTH expression and secretion. This was addressed in studies using bovine parathyroid cells showing that 25(OH)D is able to suppress PTH secretion in a similar fashion to that classical observed with 1,25(OH)2D [140]. However, although the authors of this study demonstrated low rate conversion of 25(OH)D to 1,25(OH)2D in parathyroid cells, they were unable to suppress the effects of 25(OH)D using a 1a-hydroxylase inhibitor, and they concluded that the effects of 25(OH)D on PTH were direct actions on VDR [140]. It is therefore interesting to note more recent studies which suggest that parathyroid 1a-hydroxylase does indeed mediate the effects if 25(OH)D on these cells. In this report the authors used a parathyroid cell line to show that the PTH-suppressive effects of 25(OH)D could be blocked using the 1ahydroxylase inhibitor ketoconazole [141]. Although these observations support a role for extrarenal 1ahydroxylase in mediating the calcium/bone effects of low vitamin D (25(OH)D) status, a possible role for parathyroid 1a-hydroxylase as a factor in parathyroid tumor formation has been postulated. Specifically, it has been suggested that CYP27B1 may act as a parathyroid tumor suppressor gene by enhancing localized concentrations of antiproliferative 1,25(OH)2D [142]. Expression of CYP27B1 has also been reported for other endocrine glands. Protein for 1a-hydroxylase has been detected in human pancreas [71,143], as well as rat islets and a murine insulin-secreting cell line [143]. The VDR is also expressed in these different models and rat islets have been shown to be able to convert 25 (OH)D to 1,25(OH)2D, further supporting a possible intracrine mechanism for pancreatic response to vitamin
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EXTRARENAL SYNTHESIS OF 1,25(OH)2D IN NORMAL PHYSIOLOGY AND CANCER
D. Given the scope for potential effects of vitamin D on normal pancreatic physiology [144], and the link between vitamin D status and risk of diabetes [145], it seems likely that this will be a key target for future research. In a similar fashion to the parathyroid glands, normal thyroid tissue expresses mRNA for 1a-hydroxylase and transcript levels are similar for papillary thyroid carcinomas [146]. However, immunohistochemical analysis of 1a-hydroxylase suggests that the enzyme and VDR are more strongly expressed in the carcinoma tissue [146]. Other immunohistochemical studies have shown that 1a-hydroxylase is absent from the adrenal cortex, the location of many other CYP450 enzymes involved in steroidogenesis [71]. Instead 1a-hydroxylase protein was detectable in the adrenal medulla [71], suggesting a possible secretory function for localized synthesis of 1,25(OH)2D. Within male reproductive tissues, expression of mRNA for 1a-hydroxylase has been reported for testes in general [95,147], and more specifically in epididymis, seminal vesicle, prostate, and spermatozoa [147]. This suggests a possible role for vitamin D as a modulator of spermatogenesis, although effects of vitamin D on male and female gonadal estrogen synthesis have also been proposed [148]. Vitamin D regulation of estrogenic enzymes such as aromatase and 17b-hydroxysteroid dehydrogenase [149] has implicated vitamin D in the regulation of ovarian function. This has been underlined by expression studies describing the presence of 1ahydroxylase in normal ovaries [150,151], with expression being enhanced in ovarian carcinomas [152]. Intriguingly, in ovarian dysgerminomas 1a-hydroxylase expression and activity was also increased, with patients showing symptoms of elevated serum 1,25(OH)2D and hypercalcemia [151]. The similarity between this neoplasm and observations from granulomatous diseases was further underlined by immunohistochemistry showing that overexpression of 1a-hydroxylase in dysgerminomas was associated with infiltrating tumor macrophages [151]. The 1a-hydroxylase enzyme has also been detected in endometrial tissue, with expression being elevated in endometriosis [153], and cervix carcimonas [152].
Prostate Expression of 1a-hydroxylase has been reported in human prostate cells [154]. This has supported the hypothesis that locally synthesized 1,25(OH)2D may act in an intracrine fashion to regulate prostate cell proliferation, endorsing a possible role for vitamin D as an endogenous chemopreventative factor in cancer [155,156]. Subsequent studies using primary cultures and cell lines showed that 1a-hydroxylase activity was lower in prostate cancer cells relative to normal prostate cells [157,158]. This provides an explanation for the ability of
785
non-neoplastic prostate cells to show decreased cell proliferation when treated with either 1,25(OH)2D or 25 (OH)D, whereas some prostate cancer cells appear to be mostly responsive to 1,25(OH)2D [158].
Breast Analysis of paired normal and neoplastic biopsy tissue from a cohort of women with breast cancer revealed that although mRNA for 1a-hydroxylase was detectable in normal breast tissue, expression was much higher in breast tumors [159]. Similar observations have been made by other groups [74,160], whereas some studies have shown no change in 1a-hydroxylase mRNA levels [160]. Immunohistochemical analysis of 1a-hydroxylase protein have endorsed mRNA data showing increased expression of the enzyme in tumor, but have further indicated that expression of the enzyme is not restricted to tumor cells but is also a feature of the inflammatory infiltrate associated with breast tumors [159]. Thus, it is important to recognize that, in vivo, the expression and function of 1a-hydroxylase in tumors may be complicated by the heterogeneous composition of tumor tissue, and in particular the presence of cells from the immune system. Another observation that arose from studies of normal and breast tumor biopsies is that expression of the vitamin D catabolic enzyme 24-hydroxylase is elevated in a vitamin-D-independent fashion in breast tumors [159]. Tumor dysregulation of vitamin D metabolism such as this may play a key role in disrupting possible beneficial effects of locally synthesized 1,25(OH)2D. In contrast to studies of tumor tissue biopsies, expression of 1a-hydroxylase appears to be down-regulated in breast cancer cell lines relative to nonmalignant lines [159,161]. This may reflect the effects of the selection process by which malignant cells are isolated, with low expression of CYP27B1 and associated production of antiproliferative 1,25(OH)2D being diadvantageous to cell propagation. Alternatively this may simply be a reflection of the high 24-hydroxylase expression which is commonly observed with these cell lines. It is interesting to note that normal human breast epithelial cells showed decreased expression of VDR and 1a-hydroxylase when oncogenically transformed [162]. This resulted in decreased capacity for local generation of 1,25(OH)2D and impaired cell responses to 25(OH)D. Despite these complications, it has been shown that treatment with 25(OH)D can elucidate biological responses in breast cancer cells [163]. Notably this effect appears to involve megalin-mediated uptake of 25(OH)D bound to vitamin-D-binding protein (DBP), similar to that classically described for 1a-hydroxylation of 25(OH)D in proximal tubules of the kidneys [164]. Ex vivo and in vitro studies of breast 1a-hydroxylase are supported by analysis of mouse models which has demonstrated
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45. EXTRARENAL 1a-HYDROXYLASE
expression of Cyp27b1 in normal breast tissue [165], suggesting that locally synthesized 1,25(OH)2D can contribute to normal breast tissue development in these animals. Characterization of the VDR knockout mouse indicates that lack of the VDR is associated with abnormal ductal morphologic features, increased incidence of preneoplastic lesions, and accelerated mammary tumor development [166,167]. The actions of vitamin D in normal and malignant breast are discussed in Chapter 85.
extrarenal (i.e., colonic) activity of 1a-hydroxylase. Recent data have shown that vitamin D deficiency also increases the severity of experimental IBD in mice [175]. In this study although mice exhibited almost undetectable levels of serum 25(OH)D, their circulating levels of 1,25(OH)2D remained within the normal range, suggesting a greater role for localized e 25(OH)Ddependent e 1a-hydroxylase activity in protecting against IBD in these animals.
Bone
Gastrointestinal Tract Although the gastrointestinal tract is an important target for endocrine 1,25(OH)2D acting on gut VDR, there is increasing evidence that more localized metabolism of vitamin D may also be a feature of this tissue. Adenocarcinoma-derived Caco-2 and HT-29 human colonic cells express mRNA for 1a-hydroxylase and show some responses to 25(OH)D [168,169]. However, the expression of 1a-hydroxylase in colonic cell models appear to be highly dependent on the proliferation and differentiation status of specific cell clones [170]. In keeping with observations from colonic cell clones, analysis of normal colon tissue and tumors has shown that expression of 1ahydroxylase mRNA and protein is increased in moderate to high differentiated colon tumors but lost in highly undifferentiated tumors [73]. This suggests that the capacity for local colonic synthesis of 1,25(OH)2D is enhanced in early tumor initiation and may, at this stage, provide an intracrine target for 25(OH)D supplementation [171,172]. However, in established tumors this mechanism is completely corrupted [173]. Importantly, studies of 1a-hydroxylase expression in colonic tumors have been endorsed by HPLC analysis of vitamin D metabolites from freshly isolated tissues [73]. It is interesting to note that these latter studies reported that the level of 1,25(OH)2D in colonic tumors was inversely related to 24-hydroxylase activity [73], in a similar fashion to that described for breast tumor tissue [159]. In other reports immunohistochemistry has been used to demonstrate expression of 1a-hydroxylase protein in epithelial cells of human [71] and mouse colon [174]. In humans, the level of 1a-hydroxylase expression is increased in patients with Crohn’s disease, a form of inflammatory bowel disease (IBD), with the elevated levels of enzyme being primarily localized in diseaseaffected granulomatous tissue [68]. Similar results have also been demonstrated for mice with experimental forms of IBD where increased expression of 1ahydroxylase was noted in lymphomatous tissue within the proximal colon [174]. Like the VDR knockout model, mice lacking the Cyp27b1 gene are more susceptible to experimental forms of IBD [174], although it is still unclear to what extent this is due to loss of renal versus
Like the parathyroid glands, the skeleton is intimately associated with the endocrine actions of vitamin D. However, like the parathyroid glands there is now increasing evidence for the presence of 1a-hydroxylase within specific bone cells. In fact expression of the capacity for synthesis of 1,25(OH)2D from 25(OH)D was first reported for primary cultures of human and mouse osteoblasts more than 25 years ago [176e178]. More recent work has endorsed these observations by demonstrating expression of 1a-hydroxylase mRNA and protein in primary cultures of human osteoblasts and osteoblastic cell lines [179e181]. Significantly, these studies also showed that local conversion of 25(OH)D to 1,25(OH)2D by these cells promotes intracrine regulation of osteoblast differentiation and function, supporting the overall hypothesis that vitamin-D-mediated effects on the skeleton are not restricted to endocrine pathways [182,183]. Based on in vitro observations, a similar intracrine metabolism model has also been proposed for chondrocytes, with TGFb1 enhancing expression of 1ahydroxylase [184,185] in a similar fashion to that reported for keratinocytes [186]. These studies ex vivo and in vitro have been extended to include analysis of a chondrocyte-specific Cyp27b1 knockout mouse [187]. Detailed analysis of this animal model revealed a wide range of bone and growth plate abnormalities, with the opposite phenotype being observed in mice with transgenic overexpression of chondrocytic Cyp27b1 [188]. These data provide further evidence of a specific in vivo function for extrarenal 1a-hydroxylase activity in the regulation of normal physiological responses.
MECHANISMS FOR THE REGULATION OF EXTRARENAL 1a-HYDROXYLASE The original studies describing synthesis of 1,25 (OH)2D by activated macrophages and the potential consequences of this with respect to granulomatous diseases such as sarcoidosis has stimulated a much broader appreciation of extrarenal activation of vitamin D. Further investigation of macrophage 1a-hydroxylase activity also highlighted several crucial differences
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between the activity of the enzyme in these cells when compared to its classical renal counterpart. For example, early studies using tissue from sarcoidosis patients showed that the macrophage 1a-hydroxylase is not subject to the exquisite autoregulation characteristic of its kidney counterpart, raising the possibility that renal and extrarenal synthesis of 1,25(OH)2D is catalyzed by distinct enzymes. This and other mechanistic features of extrarenal 1a-hydroxylase are discussed below. The most significant contributing factor to our current understanding of extrarenal 1,25(OH)2D production has been the cloning of the CYP27B1 gene. After initial isolation of the mouse gene (Cyp27b1) from renal tissue [189] it is notable that the human homolog (CYP27B1) was cloned from keratinocytes, a well-established extrarenal site for 1,25(OH)2D production [95]. That this gene was identical to that in the kidney strongly supported the notion of a single but differentially regulated 1a-hydroxylase protein in renal and extrarenal tissues. Further support for this postulate was provided by Mawer and colleagues who showed that macrophages from patients harboring mutations in the CYP27B1 gene had impaired levels of 1,25(OH)2D production similar to that observed in the renal enzyme [34]. The availability of sequence information has also facilitated the development of specific antisera and probes for 1a-hydroxylase. This has further emphasized the widespread tissue distribution of 1a-hydroxylase but has also helped to confirm the identity between the renal and extrarenal enzymes [71,96]. Advances in our understanding of extrarenal 1a-hydroxylase have also led to its implication in both normal physiology as well as diseases beyond the original observation of abnormal synthesis of 1,25(OH)2D in some patients with sarcoidosis.
Subcellular Localization, Substrate Selectivity and Kinetics of the Macrophage 1a-Hydroxylase As is the case with the 1a-hydroxylase of renal origin, the macrophage enzyme is a mitochondrial mixed-
function oxidase with detectable cytochrome P450 activity [190] (Fig. 45.1). Like the renal 1a-hydroxylase reconstituted from mitochondrial extracts, the presence of a flavoprotein, ferredoxin reductase, an electron source, and molecular oxygen (O2) are all required for electron transfer to the cytochrome P450 and for the insertion of an oxygen atom in the substrate [190]. Also, like the renal 1a-hydroxylase, we now know the macrophage 1a-hydroxylase is inhibited by the naphthoquinones, molecules which compete with reductase for donated electrons, and by the imidazoles, compounds which compete with the enzyme for receipt of O2 [191]. Similar to the 1a-hydroxylase isolated from the mitochondria of proximal renal tubular epithelial cells, the macrophage enzyme requires a secosterol (vitamin D sterol molecule with an open B-ring) as substrate [192]. Also similar to the renal 1a-hydroxylase, the macrophage enzyme has a particular affinity for secosterols bearing a carbon-25 hydroxy group such as the two preferred substrates for this enzyme, 25(OH)D and 24,25-dihydroxyvitamin D (24,25(OH)2D) [192,193]; the calculated Km (affinity) of the 1a-hydroxylase in pulmonary alveolar macrophages derived directly from patients with active sarcoidosis is in the range of 50e100 nM for these two substrates [192,193]. The availability of cDNA sequences for 1a-hydroxylase expression studies has shed more light on the catalytic properties of the enzyme [95,189,194,195] but, as yet, has failed to provide a clear mechanism for the differential regulation of 1,25(OH)2D production in renal and extrarenal tissues. Some of the potential explanations for this are discussed in the following sections.
Macrophages Lack Responsiveness to PTH, Calcium, and Phosphate In vivo there appear to be four major regulators of the renal 1a-hydroxylase, the serum concentration of calcium, PTH, phosphate, and fibroblast growth factor 23 (FGF23) (Fig. 45.2). Hypocalcemia enhances the
mitochondrial membrane
NADPH
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+
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FIGURE 45.1 Mitochondrial electron transport associated with the vitamin-De1a-hydroxylase reaction. NADPH supplies the electron transport chain of accessory proteins associated with 1a-hydroxylase, consisting of a flavoprotein reductase (FP), a ferredoxin (Fdx), and the 1ahydroxylase cytochrome P450 (CYP1a). A stimulatory effect on the enzyme may also be mediated by relatively low intracellular NO levels. An electron (e-) generated from NO is donated to oxidized NADP, thus forming NADPH. On the other hand, the inhibitory effect on the enzyme which occurs at relatively high NO levels in the cell results from competition with O2 binding to the P450 heme group, inhibiting the enzyme.
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PTH PTHR
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1,24,25(OH)3D nucleus RXR VDR
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Model for the regulation of 1a-hydroxylase in the proximal renal tubular epithelial cells of the kidney. Under conditions of low serum calcium (Ca), enzymatic conversion of substrate 25-hydroxyvitamin D (25(OH)D) to produce 1,25-dihydroxyvitamin D (1,25(OH)2D) is enhanced by parathyroid hormone (PTH) which induced cyclic AMP (cAMP), phosphorylation of protein kinase A (PKA) and activation of a cAMP response element (CREB) site within the gene promoter for renal 1a-hydroxylase (CYP27B1). The resulting increase in circulating 1,25 (OH)2D acts in an endocrine fashion to enhance gastrointestinal uptake of Ca and phosphate (P), promote negative feedback regulation of PTH secretion by the parathyroid glands and increase serum P and fibroblast growth factor 23 (FGF23) levels. Elevated 1,25(OH)2D also acts in an intracrine and paracrine fashion by binding to the vitamin D receptor (VDR) and promoting transcriptional activity with VDR’s heterodimer partner RXR. This leads to induction of renal vitamin-De24-hydroxylase (CYP24A1) activity and the generation of either 24,25-dihydroxyvitamin D (24,25(OH)2D) or 1,24,25-trihydroxyvitamin D (1,24,25(OH)3D). FGF23, acting via a fibroblast growth factor receptor (FGFR)eklotho receptor complex, suppresses expression of CYP27B1 and decreases synthesis of 1,25(OH)2D by the kidney. Negative feedback regulation pathways are shown as dashed lines.
FIGURE 45.2
activity of the renal 1a-hydroxylase, but much of this stimulatory effect may be indirectly mediated through parathyroid PTH. Any decrease in the serum calcium concentration below normal is a stimulus for increased secretion of PTH [196,197] which, in turn, is a direct stimulator of the renal 1a-hydroxylase [198]. Promoterreporter analyses have shown that both PTH and calcitonin stimulate transactivation of 1a-hydroxylase [199,200], although other studies have suggested that PTH can also effect changes in 1,25(OH)2D production via transient alteration in the phosphorylation status of the ferredoxin which contributes electrons to 1a-hydroxylase [201,202]. Change in the serum phosphate concentration is another major regulator of renal 1,25(OH)2D production; in adult humans dietary phosphorus restriction causes an increase in circulating concentrations of 1,25(OH)2D to 80% above control values, an increase not due to accelerated metabolic clearance of this hormone [203].
Dietary phosphorous supplementation will have the opposite effect. Although the mechanism by which a drop in the serum phosphate level will increase renal 1,25(OH)2D production remains uncertain [204], there is no doubt that in humans there exists a concerted, cooperative attempt of the calciumephosphorousePTH axis to regulate the conversion of 25(OH)D to 1,25 (OH)2D in the kidney. For example, a drop in the serum calcium concentration will be immediately registered by the parathyroid cell calcium-sensing receptor which, in turn, relaxes its inhibition of PTH production and secretion. The resulting rise in circulating PTH directly stimulates the renal 1a-hydroxylase, while a PTH-mediated phosphaturic response and a subsequent decrement in the serum phosphate level indirectly promotes 1,25 (OH)2D production (see Fig. 45.2). The most recent addition to the collection of factors associated with endocrine regulation of renal 1a-hydroxylase is FGF23 [205]. In normal physiology FGF23 regulates urinary phosphate
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excretion to maintain systemic phosphate homeostasis [206]. However, FGF23 can also act on endocrine synthesis of 1,25(OH)2D by suppressing expression of renal 1a-hydroxylase; studies using FGF23 knockout mice showed that these animals had elevated serum levels of 1,25(OH)2D and increased kidney mRNA for 1a-hydroxylase [207]. Although 1,25(OH)2D is known to stimulate expression of FGF23 in bone cells [208], patients with chronic kidney disease commonly present with elevated circulating levels of FGF23, and this may be a key factor in the compromised renal 1a-hydroxylase activity that is characteristic of these patients [209]. The role of PTH and FGF23 as regulators of 1a-hydroxylase is presented in greater detail in Chapter 3 and Chapter 42. In contrast to its renal counterpart, the macrophage 1a-hydroxylase is unaffected by the stimulatory effects of PTH and phosphate [191,210] (see Fig. 45.3). The macrophage plasma membrane is not enriched with PTH receptors [211], and there is no evidence macrophages are responsive to PTH or PTHrP in terms of stimulating the protein kinase signaling pathways that are associated with induction of the renal 1a-hydroxylase. Similarly, the macrophage enzyme appears to be uninfluenced by changes in the extracellular phosphate
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concentration [191]. Moreover, exposure of activated macrophages expressing 1a-hydroxylase to a calcium ionophore stimulates the hydroxylation reaction [212], while increasing the extracellular calcium concentration has the opposite, inhibitory effect on the renal 1ahydroxylase [213]. Although effects of extracellular phosphate and serum FGF23 on macrophage 1a-hydroxylase activity have yet to be documented, the general conclusion is that the key extracellular signaling systems for renal 1a-hydroxylase activity are not heeded by the macrophage enzyme. This provides an explanation for why 1,25(OH)2D production by the macrophage in diseases such as sarcoidosis is not subject to the same negative feedback control that is mediated by a drop in the serum PTH concentration and an increase in the circulating calcium and phosphate level. Furthermore, with the possible exception of insulin-like growth factor-1 (IGF-1) [214], there is no evidence that the macrophage 1a-hydroxylation reaction is influenced by any of the other endocrine factors, including estrogen, prolactin, and growth hormone, purported to increase the renal production of 1,25(OH)2D [215e217]. By contrast, macrophage 1a-hydroxylase activity is potently
Model for the regulation of 1a-hydroxylase in monocytes/macrophages. Sensing of pathogen-associated molecular patterns for Mycobacterium tuberculosis (M. tb) by the toll-like receptor (TLR) 2/1 complex leads to transcriptional induction of the gene for 1a-hydroxylase (CYP27B1) and increased synthesis of 1,25-dihydroxyvitamin D (1,25(OH)2D) from 25-hydroxyvitamin D (25(OH)D). The resulting 1,25(OH)2D binds to the VDR and the liganded receptor acts as a heterodimer with the retinoid X receptor (RXR) to regulate transcription of antimicrobial proteins such as cathelicidin (LL37) and b-defensin 2 (DEFB4). Intracrine/paracrine 1,25(OH)2D also promotes negative feedback regulation by up-regulating expression of a 24-hydroxylase (CYP24A) splice variant (SV) that lacks mitochondrial targeting and is metabolically inactive. SV can act as cytosolic binding site for either 25(OH)D or 1,25(OH)2D, thereby preventing access to 1a-hydroxylase or the VDR. Intracrine macrophage 1,25(OH)2D also promotes feedback regulation by suppressing expression of TRL2 and TLR4. Negative feedback regulation pathways are shown as dashed lines.
FIGURE 45.3
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inhibited by anti-inflammatory agents such as glucocorticoids which have little or no effect on the renal enzyme. In vivo this is likely to be due in part to the effects of glucocorticoids on macrophage differentiation and apoptosis. However, studies in vitro suggest that there is also direct inhibition of macrophage 1ahydroxylase activity by glucocorticoids [18].
Macrophages Lack 1,25(OH)2D-directed 24-Hydroxylase Activity The other major contributor to the circulating 1,25 (OH)2D level is the competing activity of 24-hydroxylase. Like the 1a-hydroxylase, 24-hydroxylase is a heme-binding mitochondrial enzyme requiring NADPH, molecular oxygen, and magnesium ions [201,218,219]. The cDNA and gene sequences for human rat, and chicken 24-hydroxylase, now referred to as CYP24A1/Cyp24a1, were cloned several years prior to CYP27B1 and Cyp27b1 [220,221]. As depicted in Figure 45.2, expression of CYP24 is stimulated in kidney cells by 1,25(OH)2D, whereas PTH exerts an opposite, inhibitory effect on CYP24A1 gene transcription and 24,25(OH)2D synthesis [222]. There is dual impact of this mitochondrial, cytochrome P450-linked enzyme system on vitamin D and calcium balance in adult animals including man. Because 24-hydroxylase is coexpressed in the kidney along with the 1a-hydroxylase, the first point of regulation involves the bioavailability of substrate 25(OH)D to either enzyme. Like the 1ahydroxylase, the 24-hydroxylase exhibits a preference for 25-hydroxylated secosterol substrates [223]. Although the affinity of 24-hydroxylase for 25(OH)D is reported to be less than that of renal 1a-hydroxylase, its capacity for substrate is substantially greater [193]. Hence, when up-regulated under the influence of circulating or locally produced 1,25(OH)2D or diminished serum PTH levels, the 24-hydroxylase has the capacity to compete with 1a-hydroxylase for substrate 25(OH) D. Under physiological conditions, this state of competitive substrate deprivation for the 1a-hydroxylase will persist until the serum calcium and PTH concentration are normalized. The second point at which the vitamin D-24-hydroxylase can affect circulating concentrations of 1,25(OH)2D concentration is at the level of catabolism of 1,25(OH)2D itself. Although both 25(OH)D and 1,25 (OH)2D are metabolized by 24-hydroxylase [195], current data strongly suggest that the latter is the preferred substrate [219]. Considering the fact that the 24-hydroxylase is the initial step in the conversion of 1,25(OH)2D to nonbiologically active, water-soluble, excretable metabolites of the hormone, up-regulation of this enzyme will contribute to the lowering of 1,25 (OH)2D hormone levels.
Expression and activity of 24-hydroxylase appears to be common to all VDR-expressing cell types [224]. Studies using macrophage precursor monocytes [106] as well as bone marrow and pulmonary alveolar macrophages [192] have described very low levels of 24hydroxylase activity in basal cells, with only moderate elevations of the enzyme following treatment with added 1,25(OH)2D. Therefore, unlike renal tubular epithelial cells and indeed other epithelial cells [225], macrophages do not appear to have the same capacity for shunting substrate 25(OH)D, or the 1a-hydroxylase product 1,25(OH)2D, down the catabolic 24-hydroxylase pathway. This occurs despite the fact that expression of mRNA for CYP24A1 in monocytes and macrophages is readily induced by 1,25(OH)2D. A possible explanation for this is that monocytes/macrophages also abundantly express a splice variant form of the 24-hydroxylase protein [226]. In transfection studies this splice variant protein termed CYP24-SV was shown to be a more potent suppressor of macrophage 1,25(OH)2D3 production than CYP24A1 itself [226]. This occurs despite the fact that CYP24-SV is a truncated protein created as a consequence of alternative exon splicing, resulting in the introduction of a novel, in-frame, translation startsite within a pseudo exon 3. The absence of exons 1 and 2 from CYP24-SV means that it lacks a mitochondrial targeting sequence and its expression is therefore restricted to the cytoplasm. This eliminates the catalytic activity of CYP24A1 but because CYP24-SV retains its substrate-binding domain, it still has the potential to bind 25(OH)D or 1,25(OH)2D. Further discussion of the 24-hydroxylase can be found in Chapter 4. Molecular modeling of human CYP24A1 and CYP24SV based on the crystal structures of CYP3A4 and CYP2C8 respectively has shown that in addition to lack of mitochondrial targeting, CYP24-SV exhibits significant alterations in structure, particularly within helical regions adjacent to the heme-binding domain [227]. This alters the hydrophobicity of the substratebinding pocket immediately above the heme-binding domain leading to potential changes in the substrate preference of CYP24-SV versus CYP24A1. Based on these observations and the potent effects of CYP24-SV overexpression and knockdown on macrophage metabolism of 25(OH)D, it appears that the CYP24A1 splice variant functions to attenuate synthesis of 1,25(OH)2D. However, it is also possible that in cells such as macrophages that have a high capacity for generating 1,25 (OH)2D, CYP24-SV may also function as a decoybinding site for 1,25(OH)2D, thereby limiting access of the hormone to the VDR. In this way CYP24-SV may fulfill the same remit as CYP24A1 in limiting the cellular bioavailability of 1,25(OH)2D (see Fig. 45.3). Moreover, such a mechanism provides a potential explanation for unfettered accumulation and eventual systemic
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spillover of macrophage 1,25(OH)2D production in patients with sarcoidosis.
Macrophage 1a-Hydroxylase Exhibits Responsiveness to Immune Cell Regulators The lack of classical negative feedback control of macrophage 1a-hydroxylase described above does not adequately explain the fact that 1,25(OH)2D production rates in patients with inflammatory diseases such as sarcoidosis are increased well above normal in patients with these diseases at a time when the renal 1a-hydroxylase is inhibited. Several key questions remain: first, are there alternative, “non-classical” factors that stimulate the synthesis of 1,25(OH)2D by the macrophage but not by the kidney?; second, is there a specific mechanism involved in up-regulation of macrophage 1a-hydroxylase activity during normal immune responses?; third, is there a specific mechanism that triggers 1a-hydroxylase activity in diseases such as sarcoidosis?; and finally, why is macrophage 1a-hydroxylase activity pathologically elevated in patients with inflammatory diseases such as sarcoidosis? These issues are addressed in the following sections. Cytokines Clinical observations from sarcoidosis patients with diffuse, infiltrative pulmonary disease indicate that they are at greater risk of developing dysregulated vitamin D metabolism. Cultured pulmonary alveolar macrophages (PAM) from such patients are more likely to actively synthesize more 1,25(OH)2D in vitro on a per cell basis than PAM from a host with less intense or no alveolitis [228]. These results suggest that the specific activity of 1a-hydroxylase in macrophages from patients with active pulmonary sarcoidosis is regulated by endogenously synthesized factors which also modulated the intensity of the host immune response. Of the various bioactive cytokines concentrated in the alveolar space of patients with active sarcoidosis [77,78,229,230], IFNg was found to be the principal cytokine stimulator of the sarcoid macrophage 1a-hydroxylation reaction [77]; by itself at maximally effective concentrations in vitro, IFNg increased basal 1a-hydroxylase activity more than fourfold. However, it is now clear that other immunomodulators are also able to stimulate macrophage 1a-hydroxylase including other cytokines such as tumor necrosis factor a (TNFa) [79,43] and interleukin-2 (IL-2) [210], as well as pathogen-associated peptides such as bacterial lipopolysaccharide (LPS) [191,210]. As all of these factors have been implicated in the maturation of macrophage responsiveness within the innate immune system, it seems likely that up-regulated 1a-hydroxylase activity is a common feature of activated macrophages.
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The CYP27B1 promoter region includes putative AP1 and nuclear factor-kappa B (NF-kB) binding sites which are potential targets for cytokine-regulation of 1a-hydroxylase [199,200]. Thus several pathways may be involved in regulating transcription of CYP27B1 in an immune setting. IFNg signals via the janus kinase 1 (JAK1) and JAK2 pathways with subsequent phosphorylation of signal transducers and activators of transcription 1 alpha (STAT1a) and transregulation of target genes via cis-acting promoter elements [231]. However, the JAK/STAT pathway is essential for the effects of many cytokines and growth factors including some members of the interleukin family (e.g., IL-2 and IL-6) [232]. In macrophages, the JAK/STAT system may also interact with other signaling pathways including p38 mitogen-activated protein kinase (MAPK) and NF-kB [233,234]. Molecular analysis of the induction of monocyte CYP27B1 expression by IFNg and LPS, confirmed that multiple signal transduction pathways are involved [235]. In addition to JAK/STAT, p38 MAPK, and NF-kB, the authors also showed involvement of the p38 MAPK pathway in phosphorylation of C/EBPb. Binding of the latter to its recognition sites in the CYP27B1 promoter appears to be necessary for immune induction of the enzyme by IFNg and LPS [236]. Subsequent studies using mice with gene ablation of components in the IFNg signaling pathway have confirmed the importance of C/EBPb and STA1a as key regulators of 1a-hydroxylase activity in monocytes [237]. Signaling via cytokines such as IFNg may also lead to the activation of other, calcium-dependent pathways in the macrophage, specifically the protein kinase C (PKC) [238] and phospholipase A2 (PLA2) pathways [239,240]. Because the macrophage 1a-hydroxylase was not influenced by attempts to directly stimulate or inhibit the PKC, attention has focused on the PLA2 pathway and the endogenous arachidonic acid metabolic cascade as the signal transduction pathway of most influence over the macrophage enzyme. Further dissection of the intracellular arachidonate metabolic pathway in this cell demonstrated that signal transduction through the 5-lipoxygenase pathway, specifically with the generation of leukotriene C4 (LTC4), was most critical to an increase in 1,25(OH)2D synthesis [241]. These studies were extended to investigate another compound with potential actions in the PLA2-arachidonic acid pathway, the 4amino quinoline derivative chloroquine. Synthesis of 1,25(OH)2D by macrophages was completely inhibited by exposure to 10e6 M chloroquine in vitro [30]. Furthermore, this effect is independent of chloroquine’s apparent ability to alter the pH of intracellular organelles. When given orally to a hypercalcemic patient with sarcoidosis, chloroquine [27,30] or its analog hydroxychloroquine [28] can effectively reduce the serum 1,25(OH)2D and calcium concentration within 36 hours.
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Pathogen-associated Molecular Patterns (PAMPs) As outline above, the bacterial immunogen LPS is a potent inducer of macrophage 1a-hydroxylase expression and activity. Responses to LPS are mediated via a membrane receptor complex involving the monocyte marker cluster of differentiation 14 (CD14), lymphocyte antigen 96 (MD2), and the pathogen-recognizing receptor (PRR) TLR4 [242]. Binding of ligand LPS to the TLR4 complex activates MAPK, NF-kB and interferon regulatory factor (IRF), and all three of these appear to be involved in LPS regulation of 1a-hydroxylase [236]. However, there are now known to be 13 TLRs (TLR1eTLR13) in humans. These PRRs are expressed by many cell types and can respond to a variety of pathogenic stimuli [243]. Thus, the question arising from these observations is whether other TLRs or alternative PRRs can also promote extrarenal expression of 1a-hydroxylase? One of the most significant developments concerning the role of vitamin D and macrophage 1a-hydroxylase activity arose from studies aimed at identifying the mechanisms associated with innate immune responses to infection with Mycobacterium tuberculosis (M. tb). Using a toll-like receptor (TLR) 2 ligand to mimic M. tb Modlin and colleagues assessed the changes in monocyte gene expression that occur when the TLR2 ligand interacts with a complex of TLR2/1 [107]. Using DNA array analyses, they demonstrated induction of the genes for VDR and CYP27B1 following activation of TLR2/1 [107]. This suggested a potential intracrine system for vitamin D responses in monocytes but it also indicated that, like TLR4, TRL2/1 signaling was actively involved in promoting extrarenal 1a-hydroxylase. The precise mechanism by which this occurs has yet to be fully defined but is likely to involve similar intracellular signaling pathways to those described for LPS and TLR4 (see Fig. 45.3). However, it is interesting to note recent studies that have implicated the cytokine interleukin-15 (IL-15) as an intermediary in localized synthesis of 1,25(OH)2D [244]. Elevated expression of IL-15 is frequently associated with inflammatory diseases, notably sarcoidosis [245], and so IL-15-mediated induction of 1a-hydroxylase activity may provide a link between the regulation of 1a-hydroxylase in normal innate immunity, and the dysregulated 1,25 (OH)2D production associated with granulomatous disease. Chapter 93 provides further discussion of the role of TLRs, CYP27B1, and vitamin D in preventing and fighting infection. Nitric Oxide (NO) As outlined above, LPS and IFNg commonly activate different signal transduction pathways but, as outlined previously, there is potential for cross-talk between these pathways which may have a significant impact on
transactivation of CYP27B1. Notably, IFNg and LPS are also the two most effective stimulators of nitric oxide (NO) synthesis in macrophages, and this has supported the hypothesis that production of NO and 1,25(OH)2D in macrophage-like cells may be functionally linked [246e248]. The generation of NO in the macrophage is under the control of the enzyme inducible nitric oxide synthase (iNOS) [249]. In contrast to the more stringently regulated, constitutively expressed isoforms of the enzyme which are localized to endothelial cells and neurons, are regulated by calcium and are capable of producing only modest amounts of NO, the calcium-independent iNOS remains tonically active when “induced” and can generate large quantities of NO in and around the cell [250]. It is therefore interesting that two of the major stimulators of the human macrophage 1a-hydroxylase, IFNg and LPS, are also key transcriptional regulators of the iNOS gene [251,252], which is itself a cytochrome P450-linked oxidase [253]. These observations coupled with the fact that NO has established inhibitory effects on other cytochrome P450s [254,255] suggest a possible link with the enzymes involved in vitamin D metabolism. Data indicate that NO may act as an alternative to NADPH as a source of unpaired electrons for the 1a-hydroxylase reaction in macrophages [246,247] (see Fig. 45.1). However, as the amount of NO generated inside the macrophage continues to increase there is a corresponding decrease in 1,25(OH)2D production [248], suggesting that there is a built-in limit on the ability of cells to produce active vitamin D. This inhibitory effect of NO on the macrophage 1a-hydroxylase is almost certainly due to competition of NO with oxygen for binding to the heme center of the enzyme. A similar effect has been very recently demonstrated for a number of heme-containing enzymes [254,255], including those involved in steroid hormone metabolism [256]. Regulation of 1a-hydroxylase in Cells Other Than Macrophages Although most of our current knowledge of the factors involved in regulating extrarenal 1a-hydroxylase has stemmed from studies of the enzyme in monocytes and macrophages, it is important to recognize that other cells from the immune system may also actively synthesize 1,25(OH)2D. Transcripts for CYP27B1 have been reported in T cells [113] and B cells [114] but much work has also focused on the expression, regulation, and function of 1a-hydroxylase in DCs [110,111,113]. Expression and activity of 1a-hydroxylase is increased in human monocyte-derived DCs as they differentiate towards a mature antigen-presenting phenotype [110,111]. However, this can be further enhanced by several factors including ligation of CD40, or treatment with LPS, IFNg, TNFa, or polyinosinic:polycytidylic
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FIGURE 45.4 Model for the regulation of 1a-hydroxylase in keratinocytes. Epidermal wound-associated induction of transforming growth factor beta (TGFb) leads to increased Small Mothers Against Decapentaplegic (SMAD) induced transcription via Smad-binding DNA elements (SBE). TGFb-SMAD transcription increases expression of 1a-hydroxylase (CYP27B1) and enhanced synthesis of 1,25-dihydroxyvitamin D (1,25 (OH)2D) from 25-hydroxyvitamin D (25(OH)D). The resulting 1,25(OH)2D binds to the VDR and the liganded receptor acts as a heterodimer with the retinoid X receptor (RXR) to regulate transcription of toll-like receptors 2 and 4. Increased expression of TLRs leads to increased sensitivity to pathogen-associated molecular patterns and additional TLR-mediated transcriptional induction of CYP27B1. The resulting increased local concentration of 1,25(OH)2D leads to VDR-mediated transcriptional induction of antimicrobial proteins such as cathelicidin (LL37). Negative feedback regulation pathways are shown as dashed lines. Transcriptional pathways facilitated by TGFb1-induction of TLR expression are shown as double lines.
acid (poly I:C) [110]. The latter is a ligand for TLR3 and is structurally similar to double-stranded DNA. Other studies have confirmed the induction of DC 1a-hydroxylase by TLR3 and TLR ligands whilst showing that activation of TLR9 has no effect on the enzyme [257]. In this instance the induction of 1a-hydroxylase via TLR3 or 4 was associated with facilitation of DC lymphoid trafficking, with TLR9 activation failing to support this response. It would therefore appear that macrophage/ DC 1a-hydroxylase can be regulated by TLRs associated with Gram-positive (TLR2) and Gram-negative (TLR4) bacteria as well as viruses (TLR3). Other studies have shown that synthesis of 1,25(OH)2D by DCs is regulated by the TLR4-ligand monophosphoryl lipid A (MPLA) [258]. However, unlike LPS which utilizes the Myd88 adapter for TLR4 signaling [259], MPLA appears to signal via activation of the Toll-IL-1R domain-containing adapter-inducing IFN-b (TRIF) signaling pathway [260,261]. Given the vaccine adjuvant properties of MPLA, these data underline the potential importance of DC 1a-hydroxylase as a regulatory mechanism for antigen presentation. In addition they further illustrate the complexity of pathways involved in regulating expression of 1a-hydroxylase in immune cells.
With the exception of macrophages and the placenta, extrarenal 1a-hydroxylase has been most well studied in epidermal keratinocytes. Like monocytes, keratinocytes can respond to TLRs such as TLR2 but, because keratinocytes express low levels of TLRs, initial “priming” of these receptors is required to facilitate pathogen-sensing and induction of 1a-hydroxylase. Basal expression of keratinocyte 1a-hydroxylase is stimulated by transforming growth factor beta 1 (TGF-b1), and signaling via this growth factor appears to be sufficient for induction of localized synthesis of 1,25(OH)2D (see Fig. 45.4). This, in turn, leads to up-regulation of TLR2 expression which can then facilitate pathogen-sensing similar to that described for macrophages [186]. In human skin, expression of TGF-b1 is a feature of tissue injury and thus induction of epidermal 1a-hydroxylase activity may be part of a mechanism linking wound repair with enhanced 1,25 (OH)2D-induced innate immunity [186]. The precise overlap between mechanisms for vitamin D in the skin and similar systems in immune cells such as monocytes remains to be elucidated. In particular, it is interesting to contrast the 1,25(OH)2D-mediated induction of TLR2 in keratinocytes [186], with the suppression of TLR2 and TLR4 expression by 1,25(OH)2D in monocytes [262].
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In contrast to monocytes and keratinocytes, 1,25 (OH)2D appears to be constitutively synthesized by cells from the placenta [115,116]. Expression of 1a-hydroxylase and VDR is profoundly elevated in maternal decidua and fetal trophoblast early in the first trimester of pregnancy, remaining elevated until the third trimester [117]. In view of the fact that infection during pregnancy is a prevalent cause of preterm birth and fetal mortality, it is possible that constitutive elevation of 1ahydroxylase provides an optimal system for promoting synthesis of 1,25(OH)2D and antibacterial and antiinflammatory activity during pregnancy [118]. As yet, the precise mechanism for induction of 1,25(OH)2D production by decidua and trophoblasts remains unclear but the efficacy of this extrarenal 1a-hydroxylase is enhanced by suppression of the placental 24-hydroxylase following methylation of the CYP24A1 gene [119]. In an indirect fashion this inhibition of vitamin D catabolism will increase the localized concentration of 1,25(OH)2D within the placenta.
DIAGNOSIS, PREVENTION, AND TREATMENT OF THE PATIENT WITH ENDOGENOUS VITAMIN D INTOXICATION Diagnosis The diagnosis of so-called “endogenous” vitamin D intoxication is made when the following three criteria are met. First is the presence of hypercalciuria and/or hypercalcemia in a patient with an inappropriately elevated serum 1,25(OH)2D (i.e., the serum 1,25(OH)2D concentration is not suppressed below 20 pg/ml). Second is the presence in the serum of an appropriately suppressed PTH level if the patient’s free (ionized) serum calcium concentration is high; this is evidence that the calcium-sensing receptor in the plasma membrane of the host’s parathyroid cell is normally operative. This distinguishes the patient with primary hyperparathyroidism and elevated 1,25(OH)2D levels, in whom the calcium-sensing receptor signal transduction pathway to control of PTH synthesis and release is disrupted, and from the individual with endogenous vitamin D intoxication and elevated 1,25(OH)2D levels. The other major exception here is the patient with absorptive hypercalciuria who possesses, as a primary or secondary abnormality, an inappropriately elevated circulating 1,25(OH)2D concentration. Third is the exclusion of exogenous vitamin D intoxication arising from oral or parenteral administration of an active vitamin D metabolite or substrate for endogenous synthesis of an active vitamin D metabolite.
The most common cause of exogenous vitamin D intoxication occurs with the ingestion or injection of large doses of vitamin D2 (ergocalciferol) or vitamin D3 (cholecalciferol) and can usually be detected by measuring a frankly elevated serum 25(OH)D level. Exogenous vitamin D intoxication may occur in patients taking too much 1a-hydoxyvitamin D, dihydrotachysterol, or 1,25(OH)2D itself; the two former compounds undergo 25-hydroxylation in the host hepatocyte. In these instances the 1,25(OH)2D, not the 25(OH)D, concentration will be elevated, making a distinction between endogenous vitamin D intoxication from the extrarenal overproduction of 1,25(OH)2D impossible on strictly biochemical grounds. In these situations a complete knowledge of the medications to which the patient has access is critical for making the correct diagnosis. An example of such patients would be those receiving relatively large amounts of vitamin D, a vitamin D metabolite, or a vitamin D analog (i.e., patients with hypoparathyroidism, renal failure, and psoriasis, respectively). Most of the newer vitamin D analogs currently in clinical use will not be efficiently measured in the serum 1,25(OH)2D assay, so awareness of the use of these kinds of topically and orally administered agents is of particular importance to the diagnosing clinician. Chapter 72 also discusses the differential diagnosis and treatment of hypercalcemia.
Early Detection and Prevention of Hypercalciuria/Hypercalcemia Identifying Patients at Risk Considering the fact that the means of specifically inhibiting the production of active metabolites of vitamin D or of blocking the response of cells to active vitamin D derivatives is not yet available, the best way to treat vitamin-D-mediated abnormalities in calcium balance is to prevent their occurrence. The first step is to identify patients at risk. This encompasses primarily patients with granuloma-forming disease as well as patients with malignant lymphoproliferative disorders, especially B-cell and Hodgkin’s lymphoma. Disordered calcium balance in these groups of patients results from the endogenous and dysregulated overproduction of 1,25(OH)2D by inflammatory cells. Production of this vitamin D metabolite is, in turn, directly related to the amount of substrate 25(OH)D available to the macrophage 1a-hydroxylase as well as to the severity and activity of the underlying disease. In terms of sarcoidosis, for example, patients at risk would be those with: (1) widespread, active disease; (2) a previous history of hypercalciuria or hypercalcemia; (3) a diet enriched in vitamin D and/or calcium; (4) a recent history of sunlight exposure or treatment with vitamin D; and (5)
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an intercurrent condition, or medicinal treatment of an intercurrent condition, that increases bone resorption or decreases the glomerular filtration rate. Screening Patients at Risk Since hypercalciuria almost always precedes the development of overt hypercalcemia in this set of disorders, patients at risk should be checked for the presence of occult hypercalciuria. This is best accomplished by a fasting 2-hour urine collection for calcium and creatinine. If the calcium:creatinine ratio (g:g) is not abnormally high (<0.16), then a 24-hour urine collection for the fractional calcium excretion rate is necessary to establish hypercalciuria; presumably due to increased bone resorption, many patients with vitamin-D-mediated hypercalciuria will be hypercalciuric even in the absence of recent food (calcium) ingestion. If screening is to be done only on an annual basis, then the late summer or early autumn when 25(OH)D levels are usually at their peak is the best time. Prevention of Hypercalcemia Prevention of an overt disorder in calcium balance is obviously preferable to dealing with hypercalcemia. This is especially true for patients: (1) in whom one episode of hypercalcuria or hypercalcemia has already been documented; and (2) who are taking supplemental vitamin D and calcium preparations for another medical indication (i.e., osteoporosis). Prevention is best achieved by monitoring the serum calcium and urinary calcium excretion rate on a regular basis. If either is high, then the serum concentration of 25(OH)D and 1,25 (OH)2D level should be obtained; the former should be evaluated to rule out the existence or coexistence of exogenous vitamin D intoxication. For patients found to be at risk by the appropriate monitoring analyses, measures to prevent worsening hypercalciuria and frank hypercalcemia should be instituted. These measures should include: (1) the use of UVB-absorbing sunscreens on exposed body parts when they anticipate being out of doors for periods in excess of 20e30 minutes; (2) caution against ingestion of vitamin and food supplements containing 400 IU vitamin D; (3) education on the vitamin D and calcium content of foods, vitamin supplements, and medicinal agents like antacids; (4) caution against the regular ingestion of excess elemental calcium; and (5) education regarding the earliest signs of hypercalciuria (i.e., nocturia).
Treatment of Hypercalciuria and Hypercalcemia Because they reside on the same pathophysiological spectrum, the treatment aim of normalization of the
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urinary calcium excretion rate is the same whether the patient is hypercalciuric or frankly hypercalcemic. There are three general therapeutic goals. First is reduction in the serum concentration of the offending vitamin D metabolite or derivative. In patients with exogenous vitamin D intoxication this is usually accomplished by cessation or a reduction in the dose of the vitamin D preparation being used by that patient. Remembering that 25 (OH)D has a serum half-life of 2 weeks vitamin-D-intoxicated patients may require as much as a year off therapy to return to normal. In patients suffering from endogenous intoxication with 1,25(OH)2D made by inflammatory cells, reduction in the serum 1,25(OH)2D level can be most reliably achieved by treatment with anti-inflammatory doses of glucocorticoid (adult dose of 40 mg prednisone or equivalent per day). At these doses steroid therapy should have little effect on the renal 1a-hydroxylase so there is little concern for inducing hypocalcemia with glucocorticoid administration. In patients with extrarenal production of the hormone, steroid therapy should result in a drop in the serum 1,25(OH)2D concentration within a matter of 3e4 days, followed shortly thereafter by a decrease in the filtered load of calcium and urinary calcium excretion rate provided that the patient’s glomerular filtration rate is maintained. In patients who fail glucocorticoid therapy or in whom glucocorticoids are contraindicated, treatment with the 4-aminoquninolone class of drugs like chloroquine (250 mg twice daily) or hydroxy-chloroquine (up to 400 mg daily) may be effective [27,28,30]. A less desirable therapeutic alternative is the cytochrome P450 inhibitor ketoconazole [263]; it will effectively reduce the serum 1,25(OH)2D concentration [264,265], but the therapeutic margin of safety is narrow; doses of the drug that inhibit the macrophage P450 system are very close to those that will also inhibit endogenous glucocorticoid and sex steroid production, although these can be replaced. The second goal of therapy is to limit the actions of the vitamin D derivative at its target tissues, the gut and bone. A reduction in intestinal calcium absorption is best accomplished by elimination of as much calcium as possible from the diet. Such conservative measures are rarely effective in patients with active, widespread disease, so glucocorticoid administration may also be required to block vitamin-D-mediated calcium absorption and bone resorption [266]. Although not well studied, other skeletal antiresorptive agents, like calcitonin and the bisphosphonates, do not appear to be particularly effective in blocking active vitamin D metabolite-mediated bone resorption. The third goal of therapy is to enhance urinary calcium excretion to a point where the filtered load of calcium is insufficient to cause either hypercalcemia or hypercalciuria. This can be achieved by maintenance of the vascular volume (glomerular filtration rate) (therefore hydration is a mainstay of therapy)
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and urinary flow rate, and if needed, by the use of a “loop” diuretic like furosemide to inhibit calcium reabsorption from the urine (which would increase calciuria but decrease calcemia). The effects of successfully reducing the serum 1,25(OH)2D concentration and managing hypercalcemia/hypercalciuria on the patient’s skeleton long term is not known. There is preliminary evidence that successful treatment of exogenous vitamin D intoxication may result in a transient increase in bone mineral density [267].
CONCLUSIONS AND FUTURE PROSPECTS In the last 5 years there has been a quantum leap in studies of extrarenal 1a-hydroxylase expression, activity, and function. On one level in vitro and ex vivo studies have greatly increased the range of cells that appear to express mRNA or protein for the enzyme in nondisease settings. In addition, our understanding of the cell or tissue-specific induction of 1a-hydroxylase has been substantially increased so that it is possible to envisage situations in which tissue generation of 1,25 (OH)2D will only occur under conditions in which expression of CYP27B1 is markedly elevated due to localized recognition of PAMPs or other immune activators. Clearly extrapolation of the many pieces of in vitro evidence detailed in this chapter to unequivocal in vivo relevance still requires much work. This will include more studies aimed at tissue-specific knockout or overexpression of Cyp27b1 in mice. Other factors that will also need to be considered include the link between serum vitamin D status and tissue-specific 1a-hydroxylase activity. In recent studies exploring the induction of antibacterial proteins by vitamin D, we showed that a key determinant of this important innate immune response was the actual bioavailability of 25(OH)D to monocytes and macrophages [268]. For individuals with the same levels of circulating 25(OH)D, monocyte bioavailability of this metabolite was dictated by inherited variations in the serum vitamin-D-binding protein (DBP), with low-affinity forms of DBP increasing 25(OH)D bioavailability. This subject is discussed in Chapter 5. Thus, future studies to clarify the actual levels of substrate 25(OH)D and product 1,25(OH)2D within any given tissue are likely to be a pivotal feature of the continuing evolution of extrarenal 1ahydroxylase.
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[2] F. Albright, E.L. Carroll, E.F. Dempsey, P.H. Henneman, The cause of hypercalcuria in sarcoid and its treatment with cortisone and sodium phytate, J. Clin. Invest. 35 (1956) 1229e1242. [3] R.L. Taylor, H.J. Lynch Jr., W.G. Wysor Jr., Seasonal influence of sunlight on the hypercalcemia of sarcoidosis, Am. J. Med. 34 (1963) 221e227. [4] C.E. Dent, F.V. Flynn, J.D. Nabarro, Hypercalcaemia and impairment of renal function in generalized sarcoidosis, Br. Med. J. 2 (1953) 808e810. [5] N.H.G.J.J. Bell, F.C. Barter, Abnormal calcium absorption in sarcoidosis: evidence for increased sensitivity to vitamin D, Am. J. Med. 36 (1964) 500e513. [6] M.F. Holick, H.K. Schnoes, H.F. DeLuca, T. Suda, R.J. Cousins, Isolation and identification of 1,25-dihydroxycholecalciferol. A metabolite of vitamin D active in intestine, Biochemistry 10 (1971) 2799e2804. [7] D.E. Lawson, D.R. Fraser, E. Kodicek, H.R. Morris, D.H. Williams, Identification of 1,25-dihydroxycholecalciferol, a new kidney hormone controlling calcium metabolism, Nature 230 (1971) 228e230. [8] A.W. Norman, J.F. Myrtle, R.J. Midgett, H.G. Nowicki, V. Williams, G. Popjak, 1,25-Dihydroxycholecalciferol: identification of the proposed active form of vitamin D3 in the intestine, Science 173 (1971) 51e54. [9] M.R. Hughes, D.J. Baylink, P.G. Jones, M.R. Haussler, Radioligand receptor assay for 25-hydroxyvitamin D2/D3 and 1 alpha, 25-dihydroxyvitamin D2/D3, J. Clin. Invest. 58 (1976) 61e70. [10] T.L. Clemens, G.N. Hendy, R.F. Graham, E.G. Baggiolini, M.R. Uskokovic, J.L. O’Riordan, A radioimmunoassay for 1,25dihydroxycholecalciferol, Clin. Sci. Mol. Med. 54 (1978) 329e332. [11] R. Bouillon, P. De Moor, E.G. Baggiolini, M.R. Uskokovic, A radioimmunoassay for 1,25-dihydroxycholecalciferol, Clin. Chem. 26 (1980) 562e567. [12] M.F. Holick, The use and interpretation of assays for vitamin D and its metabolites, J. Nutr. 120 (Suppl. 11) (1990) 1464e1469. [13] N.H. Bell, P.H. Stern, E. Pantzer, T.K. Sinha, H.F. DeLuca, Evidence that increased circulating 1 alpha, 25-dihydroxyvitamin D is the probable cause for abnormal calcium metabolism in sarcoidosis, J. Clin. Invest. 64 (1979) 218e225. [14] S.E. Papapoulos, T.L. Clemens, L.J. Fraher, I.G. Lewin, L.M. Sandler, J.L. O’Riordan, 1, 25-Dihydroxycholecalciferol in the pathogenesis of the hypercalcaemia of sarcoidosis, Lancet 1 (1979) 627e630. [15] P.H. Stern, J. De Olazabal, N.H. Bell, Evidence for abnormal regulation of circulating 1 alpha,25-dihydroxyvitamin D in patients with sarcoidosis and normal calcium metabolism, J. Clin. Invest. 66 (1980) 852e855. [16] G.L. Barbour, J.W. Coburn, E. Slatopolsky, A.W. Norman, R.L. Horst, Hypercalcemia in an anephric patient with sarcoidosis: evidence for extrarenal generation of 1,25-dihydroxyvitamin D, N. Engl. J. Med. 305 (1981) 440e443. [17] J.S. Adams, O.P. Sharma, M.A. Gacad, F.R. Singer, Metabolism of 25-hydroxyvitamin D3 by cultured pulmonary alveolar macrophages in sarcoidosis, J. Clin. Invest. 72 (1983) 1856e1860. [18] J.S. Adams, M.A. Gacad, Characterization of 1 alpha-hydroxylation of vitamin D3 sterols by cultured alveolar macrophages from patients with sarcoidosis, J. Exp. Med. 161 (1985) 755e765. [19] J.S. Adams, F.R. Singer, M.A. Gacad, O.P. Sharma, M.J. Hayes, P. Vouros, et al., Isolation and structural identification of 1,25dihydroxyvitamin D3 produced by cultured alveolar
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[261] V. Mata-Haro, C. Cekic, M. Martin, P.M. Chilton, C.R. Casella, T.C. Mitchell, The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4, Science 316 (2007) 1628e1632. [262] K. Sadeghi, B. Wessner, U. Laggner, M. Ploder, D. Tamandl, J. Friedl, et al., Vitamin D3 down-regulates monocyte TLR expression and triggers hyporesponsiveness to pathogen-associated molecular patterns, Eur. J. Immunol. 36 (2006) 361e370. [263] D. Feldman, Ketoconazole and other imidazole derivatives as inhibitors of steroidogenesis, Endocr. Rev. 7 (1986) 409e420. [264] A.R. Glass, J.M. Cerletty, W. Elliott, J. Lemann Jr., R.W. Gray, C. Eil, Ketoconazole reduces elevated serum levels of 1,25dihydroxyvitamin D in hypercalcemic sarcoidosis, J. Endocrinol. Invest. 13 (1990) 407e413. [265] A.R. Glass, C. Eil, Ketoconazole-induced reduction in serum 1,25-dihydroxyvitamin D, J. Clin. Endocrinol. Metab. 63 (1986) 766e769. [266] A. Pont, P.L. Williams, D.S. Loose, D. Feldman, R.E. Reitz, C. Bochra, et al., Ketoconazole blocks adrenal steroid synthesis, Ann. Intern. Med. 97 (1982) 370e372. [267] J.S. Adams, G. Lee, Gains in bone mineral density with resolution of vitamin D intoxication, Ann. Intern. Med. 127 (1997) 203e206. [268] R.F. Chun, A.L. Lauridsen, L. Suon, L.A. Zella, J.W. Pike, R.L. Modlin, et al., Vitamin D-binding protein directs monocyte responses to 25-hydroxy- and 1,25-dihydroxyvitamin D. J. Clin. Endocrinol. Metab. 95:3368e3376. [269] R. Peces, A. Pobes, C. Diaz-Corte, E. Gago, Hypercalcemia, inappropriate calcitriol levels, and tuberculosis on hemodialysis, Scand. J. Urol. Nephrol. 34 (2000) 287e288. [270] C.E. Couri, N.T. Foss, C.S. Dos Santos, F.J. de Paula, Hypercalcemia secondary to leprosy, Am. J. Med. Sci. 328 (2004) 357e359. [271] I.K. Wang, T.Y. Shen, K.F. Lee, H.Y. Chang, C.L. Lin, F.R. Chuang, Hypercalcemia and elevated serum 1.25-dihydroxyvitamin D in an end-stage renal disease patient with pulmonary cryptococcosis, Ren. Fail. 26 (2004) 333e338. [272] M.Y. Ali, K.V. Gopal, L.A. Llerena, H.C. Taylor, Hypercalcemia associated with infection by Cryptococcus neoformans and Coccidioides immitis, Am. J. Med. Sci. 318 (1999) 419e423. [273] K.V. Liang, J.H. Ryu, E.L. Matteson, Histoplasmosis with tenosynovitis of the hand and hypercalcemia mimicking sarcoidosis, J. Clin. Rheumatol. 10 (2004) 138e142. [274] X. Bosch, Hypercalcemia due to endogenous overproduction of active vitamin D in identical twins with cat-scratch disease, Jama 279 (1998) 532e534. [275] E.G. Playford, A.S. Bansal, D.F. Looke, M. Whitby, P.G. Hogan, Hypercalcaemia and elevated 1,25(OH)(2)D(3) levels associated with disseminated Mycobacterium avium infection in AIDS, J. Infect. 42 (2001) 157e158. [276] J.W. Delahunt, K.E. Romeril, Hypercalcemia in a patient with the acquired immunodeficiency syndrome and Mycobacterium avium intracellulare infection, J. Acquir. Immune. Defic. Syndr. 7 (1994) 871e872. [277] J.L. Shaker, K.C. Redlin, G.V. Warren, J.W. Findling, Case report: hypercalcemia with inappropriate 1,25-dihydroxyvitamin D in Wegener’s granulomatosis, Am. J. Med. Sci. 308 (1994) 115e118. [278] X. Bosch, A. Lopez-Soto, A. Morello, A. Olmo, A. UrbanoMarquez, Vitamin D metabolite-mediated hypercalcemia in Wegener’s granulomatosis, Mayo. Clin. Proc. 72 (1997) 440e444. [279] O.P. Sharma, Hypercalcemia in granulomatous disorders: a clinical review, Curr. Opin. Pulm. Med. 6 (2000) 442e447. [280] T.M. Greenaway, I.D. Caterson, Hypercalcemia and lipoid pneumonia, Aust. N.Z. J. Med. 19 (1989) 713e715.
[281] A.R. Rolla, A. Granfone, K. Balogh, U. Khettry, B.L. Davis, Granuloma-related hypercalcemia in lipoid pneumonia, Am. J. Med. Sci. 292 (1986) 313e316. [282] A. Woywodt, W. Schneider, U. Goebel, F.C. Luft, Hypercalcemia due to talc granulomatosis, Chest 117 (2000) 1195e1196. [283] H. Karakelides, J.L. Geller, A.L. Schroeter, H. Chen, P.S. Behn, J.S. Adams, et al., Vitamin D-mediated hypercalcemia in slack skin disease: evidence for involvement of extrarenal 25hydroxyvitamin D 1alpha-hydroxylase, J. Bone Miner. Res. 21 (2006) 1496e1499. [284] M.A. Helikson, A.D. Havey, J.E. Zerwekh, N.A. Breslau, D.W. Gardner, Plasma-cell granuloma producing calcitriol and hypercalcemia, Ann. Intern. Med. 105 (1986) 379e381. [285] M. Hibi, F. Hara, H. Tomishige, Y. Nishida, T. Kato, N. Okumura, et al., 1,25-Dihydroxyvitamin D-mediated hypercalcemia in ovarian dysgerminoma, Pediatr. Hematol. Oncol. 25 (2008) 73e78. [286] T.H. Grote, J.D. Hainsworth, Hypercalcemia and elevated serum calcitriol in a patient with seminoma, Arch. Intern. Med. 147 (1987) 2212e2213. [287] M.G. Brunette, M. Chan, C. Ferriere, K.D. Roberts, Site of 1,25 (OH)2 vitamin D3 synthesis in the kidney, Nature 276 (1978) 287e289. [288] R. Bland, D. Zehnder, S.V. Hughes, P.M. Ronco, P.M. Stewart, M. Hewison, Regulation of vitamin D-1alpha-hydroxylase in a human cortical collecting duct cell line, Kidney Int. 60 (2001) 1277e1286. [289] D.D. Bikle, M.K. Nemanic, E. Gee, P. Elias, 1,25-Dihydroxyvitamin D3 production by human keratinocytes. Kinetics and regulation, J. Clin. Invest. 78 (1986) 557e566. [290] G. Bises, E. Kallay, T. Weiland, F. Wrba, E. Wenzl, E. Bonner, et al., 25-Hydroxyvitamin D3-1alpha-hydroxylase expression in normal and malignant human colon, J. Histochem. Cytochem. 52 (2004) 985e989. [291] F. Ichikawa, K. Sato, M. Nanjo, Y. Nishii, T. Shinki, N. Takahashi, et al., Mouse primary osteoblasts express vitamin D3 25-hydroxylase mRNA and convert 1 alphahydroxyvitamin D3 into 1 alpha,25-dihydroxyvitamin D3, Bone 16 (1995) 129e135. [292] D. Zehnder, R. Bland, R.S. Chana, D.C. Wheeler, A.J. Howie, M.C. Williams, et al., Synthesis of 1,25-dihydroxyvitamin D(3) by human endothelial cells is regulated by inflammatory cytokines: a novel autocrine determinant of vascular cell adhesion, J. Am. Soc. Nephrol. 13 (2002) 621e629. [293] J.F. Ma, L. Nonn, M.J. Campbell, M. Hewison, D. Feldman, D.M. Peehl, Mechanisms of decreased Vitamin D 1alphahydroxylase activity in prostate cancer cells, Mol. Cell Endocrinol. 221 (2004) 67e74. [294] T.C. Chen, L. Wang, L.W. Whitlatch, J.N. Flanagan, M.F. Holick, Prostatic 25-hydroxyvitamin D-1alpha-hydroxylase and its implication in prostate cancer, J. Cell Biochem. 88 (2003) 315e322. [295] I. Bieche, C. Narjoz, T. Asselah, S. Vacher, P. Marcellin, R. Lidereau, et al., Reverse transcriptase-PCR quantification of mRNA levels from cytochrome (CYP)1, CYP2 and CYP3 families in 22 different human tissues, Pharmacogenet. Genomics 17 (2007) 731e742. [296] D.W. Eyles, S. Smith, R. Kinobe, M. Hewison, J.J. McGrath, Distribution of the vitamin D receptor and 1 alpha-hydroxylase in human brain, J. Chem. Neuroanat. 29 (2005) 21e30. [297] D. Somjen, Y. Weisman, F. Kohen, B. Gayer, R. Limor, O. Sharon, et al., 25-Hydroxyvitamin D3-1alpha-hydroxylase is expressed in human vascular smooth muscle cells and is upregulated by parathyroid hormone and estrogenic compounds, Circulation 111 (2005) 1666e1671.
V. HUMAN PHYSIOLOGY
S E C T I O N V I
DIAGNOSIS AND MANAGEMENT
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C H A P T E R
46 Approach to the Patient with Metabolic Bone Disease Michael P. Whyte Center for Metabolic Bone Disease and Molecular Research, Shriners Hospital for Children; and Division of Bone and Mineral Diseases, Washington University School of Medicine at Barnes-Jewish Hospital; St Louis, Missouri, USA
INTRODUCTION Metabolic bone disease traditionally encompasses a considerable number and variety of conditions [1e3]. In fact, the list is rapidly growing as the molecular bases of inherited skeletal syndromes and dysplasias are elucidated using DNA technology [4,5]. Although these disorders are often rare, several are epidemic in various regions of the world (e.g., postmenopausal osteoporosis, vitamin-D-deficiency rickets). Some can be life-threatening (e.g., severe forms of osteopetrosis and osteogenesis imperfecta); others can be incidental findings (e.g., Paget bone disease and fibrous dysplasia). Patients reflect all ages. Cumulatively, the number of individuals with clinically important metabolic bone disease is significant [1e3]. Diagnosis and treatment of metabolic bone disease can be both intriguing and satisfying, but there are numerous challenges. We now have at our disposal a variety of techniques to image the skeleton and to measure bone mass, assays for many of the factors that condition mineral and skeletal homeostasis as well as biochemical markers of bone turnover, and qualitative and quantitative histopathological methods to directly examine osseous tissue [6]. Furthermore, molecular tests for genetic disorders of the skeleton are becoming increasingly available in commercial and research laboratories [4]. Initial and follow-up patient evaluation benefit greatly from skilled use of these tools. Nevertheless, clinical acumen is more important than ever. Judicious selection from among these advances in technology and circumspect interpretation of the information they provide comes from experience with patients. In fact, successful treatment
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10046-0
of metabolic bone disease often requires multidisciplinary medical approaches that may need to be especially broad-based when there is deformity or other structural problems of the skeleton. Significantly, a variety of potent hormones and drugs that regulate mineral homeostasis and alter bone remodeling are now available to clinicians [3]. This pharmaceutical armamentarium must be used knowledgeably for efficacy, yet safety. This chapter emphasizes a number of considerations for the approach to the patient with metabolic bone disease, particularly those with disturbances in vitamin D homeostasis. Other chapters in this and other sections of the book discuss in detail the radiology of rickets and osteomalacia (Chapter 49), bone histomorphometry (Chapter 48), measurement of the vitamin D metabolites (Chapter 47), and the pharmacology and therapeutic use of vitamin D preparations (Chapter 57). Other relevant chapters include new imaging techniques (Chapter 50), an orthopedic perspective (Chapter 51) and nutrition, diet, and lifestyle issues (Chapter 54).
DIAGNOSTIC EVALUATION Patients with metabolic bone disease are often challenging to physicians because many nutritional, environmental, genetic, pharmacological, and toxic factors can disturb mineral metabolism and impact the skeleton [2,3]. This book is testimony to the number and diversity of internal and external perturbations that can impair the biosynthesis, bioactivation, and actions of vitamin D.
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Patient age is yet another challenge for several reasons. In infants, children, and adolescents, complications of metabolic bone disease can be especially severe and complex because bone growth and modeling are occurring in addition to skeletal remodeling. All three physiological processes can be disturbed in pediatric patients, leading to impairment of growth and alterations in the shape of bones. The outcome is novel physical and radiological findings in children compared to adults. Patient age conditions the pathogenesis and clinical manifestations of these disorders, and also provides a guide to the etiology. However, diagnoses can be missed or delayed if the diagnostician is unaware that the reference ranges for some of the biochemical parameters of mineral homeostasis (e.g., serum inorganic phosphate, alkaline phosphatase) and all of the markers of skeletal turnover are different for infants and children compared to adults [2,3]. Elderly patients are also uniquely challenging, because they are especially likely to have metabolic bone disease with multifactorial etiology and pathogenesis [1e3,7]. Broad-based medical knowledge is necessary to fully understand the relationships between mineral and skeletal homeostasis and the consequences of specific or complex disturbances [8]. Clinicians who encounter patients with metabolic bone disease routinely need some of the skills of the endocrinologist, nutritionist, nephrologist, geneticist, and often the pediatrician or gerontologist. Familiarity with skeletal radiology and pathology is required. Furthermore, if there is significant bony deformity, the expertise of orthopedics, rheumatology, and rehabilitation medicine will be helpful. Metabolic bone disease is remarkable for the many subspecialties that can contribute to the comprehensive evaluation and effective care of patients. Diagnosis of metabolic bone disease must begin with the acquisition of all of the important information extractable from the medical history and obtainable from a thorough physical examination. This foundation should be supplemented with only the helpful and costeffective choices from the ever-expanding menu of biochemical tests, as well as the available radiological and histological studies. Most situations will require some biochemical and radiological investigation [2,3]. For the majority of these patients, however, histological assessment before therapy or during follow-up is not necessary. But, when it is, nondecalcified bone processing, staining, and interpretation following tetracycline labeling are often crucial. The importance of the medical history for evaluation of metabolic bone disease cannot be overemphasized. Foremost in diagnosing these disorders is this orderly accumulation of pertinent information directly from patients [9]. The revelations help to guide the physical examination and subsequent laboratory
studies and to disclose potentially important medical records. Examples are endless. For deficiency of vitamin D, historical details alone often explain how the patient has come to require supplementation. Prolonged breast feeding, lack of sunlight exposure together with failure to consume foods fortified with vitamin D, use of certain anticonvulsants, or a family history suggestive of a similar heritable disorder exemplify critical information that will be obtained by talking with the patient. For the physician skilled in medical history acquisition concerning metabolic bone disease, it is not unusual for the physical examination and laboratory testing to uphold a diagnosis. Physical examination of the patient with metabolic disease may show findings for or against the diagnosis suspected from the medical history, but the information is also important for revealing deformity or other structural problems of the skeleton requiring attention. Radiological investigation of metabolic bone disease may appropriately be minimal or extensive, but it should be directed by the complete medical history and physical examination. Not uncommonly, the diagnosis is established from characteristic radiographic findings (e.g., Paget bone disease, fibrous dysplasia) [10e12]. If not, X-ray studies often provide an important basis for differential diagnosis (e.g., osteopenia, osteosclerosis, rickets, etc.), or support the diagnostic impression that must instead be confirmed by additional tests (e.g., pseudohypoparathyroidism, mastocytosis, hyperparathyroidism). However, radiological studies are also useful because they may help to assess the severity and evolution of the disorder. In addition, they can reveal skeletal complications not detected by physical examination. Some changes (e.g., physeal widening in rickets, osteolytic lesions in Paget bone disease, etc.) can then be followed to precisely assess responses to medical treatment. Biochemical testing is crucial to characterize any disturbance of mineral homeostasis. Furthermore, biochemical testing provides quantitative information to help guide the intensity of medical treatment and to monitor the patient’s response. Proper selection of laboratory studies at the time of diagnosis is important to completely establish baseline data. Most metabolic bone diseases will be treated medically. If laboratory investigation is incomplete when pharmacological or nutritional intervention begins, an opportunity for diagnosis or for evaluating therapy may be lost. In fact, freezing away some patient serum and an aliquot of urine before medication is prescribed is sometimes worthwhile in case retrospective diagnosis may become necessary. Furthermore, this can help provide especially precise pre- and post-treatment comparisons. Additionally, in select circumstances where a therapeutic
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challenge is given, preserving pretreatment specimens can be a tactic for saving money. Expensive and lowyield testing can await results of a brief trial of therapy (e.g., suspected environmental vitamin-D-deficiency rickets). Among the biochemical tests for metabolic bone disease, there is now considerable redundancy (e.g., markers of skeletal turnover) [2,3,6]. Furthermore, some of these assays are useful primarily for research purposes where groups of patients are studied. The utility of a single measurement of some of the markers of bone turnover for an individual patient can be limited because of technical or physiological variability, leading to just modest correlation with pathological processes. Histological study of the skeleton is essential in relatively few clinical circumstances, although it often has considerable research importance. Here too, however, an opportunity for diagnosis and/or baseline assessment may be lost if pharmacologic therapy has begun [13].
Medical History The complete and accurate medical history and thorough physical examination is the cornerstone for diagnosis, effective and safe treatment, and sound clinical investigation of metabolic bone disease [9].* A detailed medical history helps assure that the many adverse external or heritable factors that can affect the patient with metabolic bone disease will be uncovered (Table 46.1). A questionnaire completed by the patient may serve as a beginning, but is hardly a satisfactory substitute. Only by talking with his/her patient will the physician sense how knowledgeable this individual might be, and therefore be able to judge the value of any historical information. Only by talking with the patient will the character of the major signs and symptoms be revealed. Subsequently, the medical history should be reported as a narrative description of the clinical problem. As discussed below, all of the principal elements of the medical history are potentially important for patients with metabolic bone disease. Chief Complaint The chief complaint (or CC) may readily lead to the diagnosis; for example, pain from hip fracture due to osteoporosis or increasing head size due to Paget bone disease. Often, however, the patient’s major concern is more subtle. The above conditions may manifest with chronic back discomfort or progressive leg deformity, respectively. Such less dramatic difficulties must be
TABLE 46.1
Some Potentially Adverse Influences on Mineral and Skeletal Homeostasis
Genetic Medical disorders Ethnic background Acromegaly Heritable disorders Anorexia nervosa/bulimia Lifestyle Celiac disease Alcohol abuse Cushing syndrome Inactivity (immobilization) Cystic fibrosis Smoking Early menopause Sunshine deficiency Fanconi syndrome Nutritional Fibrous dysplasia (polyostotic) Excessive tea drinking Gastrointestinal disease (fluorosis) Glycogen storage disease Low dietary calcium Hemochromatosis intake Hemolytic anemia High protein intake Hepatitis C infection Milk intolerance Hepatobiliary disease Vegetarian diet Homocystinuria Drugs Hyperparathyroidism Aromatase inhibitors Hypogonadism Anticonvulsants Hypophosphatasia Bisphosphonate excess Lymphoproliferative disease Chemotherapy Mastocytosis Cyclosporin A McCune-Albright syndrome Diuretics (producing Multiple myeloma calciuria) Oncogenic rickets/osteomalacia Fluoride Osteogenesis imperfecta Glucocorticoids Pancreatic insufficiency Gonadotropin-releasing Postmenopausal osteoporosis hormone (GnRH) Prolactinoma agonists or antagonists Renal failure (transplantation) Heparin Rheumatologic disorders Lithium Sarcoidosis Protease inhibitors Secondary amenorrhea Proton pump inhibitors Thyrotoxicosis Thyroid replacement Turner syndrome therapy Type I diabetes Vitamin A or D
recognized not only because they may be pointing to a diagnosis, but because they are objectives of treatment and can help monitor the efficacy of therapy. For example, when vitamin D deficiency causing weakness from myopathy or bone pain from osteomalacia is effectively treated, these difficulties should resolve. Furthermore, no matter how mild or severe the CC, this is the worry for which the patient with metabolic bone disease sought help. With disturbances in vitamin D homeostasis, there may be one or more major complaints that can be metabolic or skeletal in origin (Table 46.2). This aspect of the medical history is sometimes particularly challenging, because one problem can be emphasized from among this myriad collection of important symptoms or signs, or because complaints may seem vague or excessive.
*
The more resources we have, and the more complex they are, the greater are the demands upon our clinical skill. These resources are calls upon judgment and not substitutes for it. Do not, therefore, scorn clinical examination; learn it sufficiently to get from it all it holds, and gain in it the confidence it merits. Sir F.M. R Walshe (1881e1973) Canadian Medical Association Journal 67:395, 1952.
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810 TABLE 46.2
46. APPROACH TO THE PATIENT WITH METABOLIC BONE DISEASE
Vitamin D Deficiency: Age-dependent Signs and Symptoms
Metabolic
Skeletal
Lax ligaments
Bone tenderness
Limb deformity
Cranial sutures widened
Listlessness
Asthenia
Craniotabes (skull flattening or asymmetry)
Low back pain
Potbelly with lumbar lordosis
Dystocia
Pneumonia
Proximal myopathy
Flared wrists and ankles
Rachitic rosary
Waddling gait
Fracture
Rib deformity/respiratory compromise
Frontal bossing
Short stature
Caries
Harrison’s groove
Sternal indention or protrusion
Delayed eruption
Hypotonia
“String-of-pearls” deformity in hands
Enamel defects
Kyphosis
Hypocalcemia (see Table 46.4) Muscle
Dental
History of Present Illness Most metabolic bone diseases (including many caused by disturbances in vitamin D homeostasis) are chronic conditions. The detailed history of present illness (or HPI) may therefore be lengthy. Nevertheless, the time invested is crucial because this effort provides the most important historical information. Attention to the details also demonstrates to the patient that the physician understands and cares about his/her illness. Hence, this effort helps secure the patient’s confidence needed for effective treatment. From the HPI, the physician should gather an understanding of the temporal evolution of the disorder e likely essential for accurate and complete diagnosis and effective treatment. Clues predating the symptoms of vitamin D deficiency would be necessary to fully uncover the pathogenesis and etiology. Appreciation of past therapeutic attempts may reveal factors masking a diagnosis. Indeed, the outcome of previous therapy (successful or unsuccessful) may guide diagnosis and future treatment. I find it worthwhile at the outset (for the sake of time and effort, organization, and completeness) to alert the patient that information will be especially valuable if obtained in historical sequence. Patients often require some guidance as this effort begins, but most will then help present their medical history in this especially useful way. Tactfully diverting them from excessive or extraneous details usually becomes easy because they now appreciate that the physician is truly concerned and wishes to know, understand, and help them to organize this important information. Nutritional factors could be considered in the HPI. Both mineral metabolism and vitamin D homeostasis are influenced by diet in countries that fortify foods with vitamin D. Strict vegetarians (vegans), who avoid
all foods derived from animals, will not benefit from the safety net of vitamin D supplementation of milk products in the United States. Avoidance of dairy foods may also lead to a calcium-deficient diet (Table 46.3). Ovolactovegetarians, however, do drink milk. When the HPI is reported completely, not only will critical clues to disease etiology and pathogenesis emerge, but the physician may also gain important insight for treatment, and here may be a glimpse of the patient’s prognosis. Has this individual been compliant with regard to recommendations for diagnostic studies or medical care; if not, will pharmacologic therapy be safe? Have the manifestations of the disorder been lifelong (suggesting a congenital or genetic problem), or has there been the development of recent symptomatology that should prompt very different diagnostic TABLE 46.3 Vegetarians Vegans (strict vegetarians who do not consume dairy products or eggs) Ovolactovegetarians (vegetarians who will eat dairy products and eggs) Buddhists (some sects) Zen (Chinese) Ch’an (Japanese) Muslims (some Islamic sects) Ethnic groups of African, Hispanic, American-Indian, Jewish, or Oriental descent (who may have lactose intolerance) Yoga Seventh Day Adventists International Society for Krishna Consciousness Zen Macrobiotic Movement
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considerations and interventions? Have complications been substantial, and likely to remain so if medical therapy cannot effect a cure? Only by inquiring about the patient’s illness in detail is the physician likely to learn that previous medical records, radiographs, etc., are available to help avoid expensive duplication of effort, and perhaps to help address important diagnostic and prognostic issues. Finally, by carefully documenting this aspect of a metabolic bone disease, the physician is providing the basis for sound clinical research. Past Medical History A considerable number and variety of perturbations can cause metabolic bone disease, or can influence the outcome of medical treatment, by impacting on mineral and skeletal homeostasis (Table 46.1). The detailed past medical history (or PMH) will help to disclose these disturbances or factors that can influence or obscure a diagnosis. In the PMH, previous diagnostic studies may be revealed that could prove useful for assessing the metabolic bone disease. Radiographs (e.g., chest X-rays, intravenous pyelogram) taken years ago for other reasons could show whether osteopenia, osteosclerosis, or rachitic change is old or new. Routine biochemical testing reports might help to date the onset of vitamin D deficiency by documenting when serum alkaline phosphatase activity began to rise. Drug history must be carefully assessed here and, if relevant, perhaps detailed instead in the HPI. Many pharmaceuticals can adversely affect the skeleton and disturb mineral or vitamin D homeostasis (e.g., glucocorticoids, certain diuretics, anticonvulsants, etc.) (Chapter 67). A seemingly incidental problem like acne, if overlooked, might fail to reveal tetracycline or retinoid exposure. In fact, these concerns also apply to some over-the-counter preparations (e.g., vitamin A, calcium supplements, antacids). In large amounts, each of these nonprescription items can have important effects on mineral or skeletal homeostasis and cause illness. Nevertheless, they may inadvertently be dismissed during elicitation of a drug history (“Pardon me, doctor. You asked me what medications I was taking.”). Many patients will not consider vitamins, mineral supplements, or antacids to be in this category (Table 46.1). Furthermore, some medications confound interpretation of biochemical studies aimed at metabolic bone disease because they alter mineral homeostasis (e.g., diuretics that increase or decrease urine calcium levels), elevate serum alkaline phosphatase activity from the liver, etc. Fortunately, in the United States, pharmacological doses of vitamin D until recently required a prescription. Vitamin D intoxication has occurred from excesses inadvertently added to dairy
811
milk (and other errors) that would have gone unrecognized were it not for detailed medical histories. The PMH may also help to predict the nature and frequency of recurrent illness and therefore complications from pharmaceutical treatments. Depending on the patient’s prior medical problems and compliance, will use of vitamin D (with its prolonged biological effects) be safer or more risky than a shorter-acting, but more potent, vitamin D metabolite? Such information might help to prevent the abrupt development of hypocalcemia if therapy will be suddenly compromised, or prevent prolonged hypercalcemia if dosing might become excessive. Social History Patient compliance for medical treatment, especially for chronic disorders, is often imperfect. Recognition that an individual will be uncooperative or has health insurance problems or financial difficulties that could affect his/her ability or willingness to undergo diagnostic testing or remain compliant for therapy or follow-up may come in the social history (or SH). Such information can be necessary to formulate not only an effective but also a safe treatment plan, particularly when the disease is severe and/or requires potent medication. The various pharmaceutical preparations of vitamin D, such as 1,25-dihydroxyvitamin D3 (1,25 (OH)2D3), la-hydroxyvitamin D3 (1aOHD3), 25(OH)D3, and vitamin D, have very different potencies, biological half-lives, and price (Chapters 3 and 59). What will the issues of long-term treatment and drug cost mean for patient compliance, safety, and/or follow-up? The medical literature contains enough case reports of renal failure from vitamin D intoxication when patient monitoring was inadequate. Because vitamin D3 is produced naturally by exposure to ultraviolet light in sunshine, and because nutrition also importantly influences mineral homeostasis, appreciation of climate, clothing, diet, and skin pigmentation is important. Several ethnic, religious, and other groups have vegetarian members who will not consume dairy products for health, spiritual, or ethical reasons (Table 46.3). Recognition in the SH that the patient belongs to one of these populations may disclose a significant factor contributing to his/her metabolic bone disease. Physical factors (e.g., exercise and work activities) often impact patients with metabolic bone disease. Indeed, as regimens continue to improve for increasing low skeletal mass, much of what the clinician can do for a child or adult with osteoporosis still comes from cautioning them against potentially traumatic pursuits at play or work. Prevention of falls and proscription against heavy lifting for pediatric or adult forms of osteoporosis are important for minimizing fractures and spinal
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FIGURE 46.1 Considerable reconstitution of vertebrae (here, L3eL5) has occurred between ages 14 (left) and 16 (right) years in a boy with idiopathic osteoporosis. He was counseled against lifting and to avoid falls and stopped participating in traumatic exercises in physical education class. No pharmacological intervention had been attempted.
deformity. In fact, this advice, often guided by historytaking, can sometimes correct vertebral crush deformities in children with brittle bone disease who (unlike adults) can naturally reconstitute their spinal anatomy (Fig. 46.1). Family History The family history (or FH) is often revealing for metabolic bone disease, because many of these conditions are heritable [1e5]. In fact, a correct diagnosis may be disclosed by study of kindred members e familial benign (hypocalciuric) hypercalcemia or osteogenesis imperfecta are good examples. Inborn errors of vitamin D bioactivation or resistance are rare, but they may be uncovered in the FH. Furthermore, significant benefit may come from screening studies to identify, and then to treat or to counsel, other affected relatives who may also represent important clues to the patient’s future complications and prognosis. To report that the FH is “negative” without first establishing the value of this information is misleading. If the patient is adopted, he/she is unlikely to give relevant data. An understanding of the size of a family is essential before dismissing possible transmission of a heritable disorder. The patient who is the only child of only children, or from a disrupted family, is not as likely to disclose a heritable disorder as will be the patient from a large, cohesive kindred. This effort can facilitate a diagnosis. Medical records from similarly affected living or deceased family members may establish the diagnosis, and may be an important guide not only concerning prognosis, but also for treatment.
Review of Systems Metabolic bone diseases can cause a considerable variety of symptoms. This is especially true for disorders that disturb vitamin D homeostasis and lower extracellular calcium and phosphate levels (Table 46.2). Therefore, a careful “review of systems” may uncover a sufficient number of these problems so that a diagnosis becomes apparent, or a new or additional condition is suspected. Furthermore, this effort provides a baseline from which to judge the impact of subsequent medical therapy. Symptoms that persist after a course of otherwise effective treatment would need further investigation if they were expected to respond.
Physical Examination Many clinical signs as well as significant skeletal deformities can accompany or result from metabolic bone disease e especially in children. Furthermore, not all of these disorders manifest themselves with overt disturbances of hormone or mineral homeostasis (e.g., postmenopausal osteoporosis). Accordingly, physical examination is especially important. Discovery as well as comprehensive treatment of metabolic bone disease often depends on this skill. Occasionally, diagnosis of a metabolic bone disease starts with the identification of one physical finding; for example blue or gray sclerae (osteogenesis imperfecta), cafe-au-lait spots (McCune-Albright syndrome), tumor (oncogenic rickets/osteomalacia), premature loss of a deciduous tooth (hypophosphatasia), hallux
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valgus (fibrodysplasia ossificans progressiva), or brachydactyly (pseudohypoparathyroidism, type IA). For some metabolic bone diseases, a constellation of physical findings leads to the diagnosis. Paget disease of bone, when advanced, can feature an enlarging calvarium with bulging temporal arteries, deafness, asymmetrical bowing of the limbs, and localized areas of skeletal warmth. Postmenopausal osteoporosis causes loss of vertebral height with reduced stature leading to a “short-waisted” appearance, kyphosis or a gibbus (dowager’s hump), a protuberant abdomen (that the patient may mistakenly attribute to weight gain), ribs lowered toward (or in) the pelvic brim, paravertebral muscle spasm, and thin skin (McConkey’s sign) [7]. Unless such physical abnormalities individually or in combination are correctly identified, a diagnosis may be missed. Furthermore, these findings should focus attention on anatomical structures of concern, perhaps requiring treatment (e.g., vertebroplasty for osteoporosis, leg-bracing for rickets, etc.). With disturbances in vitamin D homeostasis, a plethora of physical findings can develop (Table 46.2). Patient age determines which are likely to be encountered. Low levels or ineffective action of vitamin D can be especially harmful for infants and for children. As discussed below, distinctive physical findings occur in the pediatric age group. Rickets disturbs the most actively growing bones. Because the skull is enlarging especially quickly at birth, craniotabes (flattened posterior skull) is characteristic of essentially congenital disease. A rachitic rosary (enlargement of the costochondral junctions) can appear during the first year of life, when the rib cage forms rapidly. During infancy or childhood, there may be flared wrists and ankles from metaphyseal widening, bony tenderness, and Harrison’s groove (rib cage ridging from diaphragmatic pull producing a horizontal depression along the lower border of the chest at costal insertions of the diaphragm). Although weight bearing typically bows the lower limbs, especially during the adolescent growth spurt a knock-knee deformity may occur instead. The patient can also have myopathy with reduced muscle strength and tone causing a waddling gait, lax ligaments, indentation of the sternum from forces exerted by the diaphragm and intercostal muscles, delayed eruption of permanent teeth, and enamel defects. In infants, floppiness and hypotonia are characteristic. Rachitic infants and young children commonly are listless and irritable. Bone pain can also occur from fracture and deformity. Hypocalcemia (Chapter 59) can also result from vitamin D deficiency (Chapters 52 and 60), pseudodeficiency (Chapter 64), or resistance (Chapter 65) [14]. Hence, it is important that symptoms of hypocalcemia are elicited during the medical history and physical signs
TABLE 46.4
Signs and Symptoms of Hypocalcemia
Nervous system Increased irritability with latent or overt tetany Mental status changes Seizures Basal ganglia calcification Mental retardation Cardiovascular Prolonged QT-interval with arrhythmia Cardiomyopathy with congestive heart failure Hypotension Other Papilledema Lenticular cataracts Intestinal malabsorption Dysplastic teeth Rickets/osteomalacia Integument changes Joint contractures Vertebral ligament calcification Reprinted with permission from MP Whyte 1993 Hypocalcemia. In: BEC Nordin, AG Need, HA Morris (eds) Metabolic Bone and Stone Disease, 3rd edn. Churchill Livingstone, Edinburgh pp. 147e162.
of latent or overt tetany are recognized during the physical examination (Table 46.4). Hypocalcemia enhances neuromuscular excitability. Consequently, there may be varying degrees (latent or overt) of tetany. Overt tetany usually presents with numbness and tingling around the mouth and in the fingertips, and can be followed by muscle spasm in the extremities, face, larynx (causing stridor), and elsewhere, and mental status changes including epileptic seizures. Symptoms and signs may be particularly striking when reductions in extracellular levels of ionized calcium are severe, or when hypocalcemia occurs rapidly. Typically, there is carpopedal spasm manifesting as adduction of the thumb, metacarpophalangeal joint flexion, and interphalangeal joint extension. Latent tetany can be unmasked by eliciting Chvostek’s sign or Trousseau’s sign. Chvostek’s sign is a spasm of the ipsilateral muscles of the face on tapping the facial nerve near its exit from the skull in the region of the parotid gland (just anterior to the ear lobe, below the zygomatic arch). A positive Chvostek’s sign ranges from twitching of the lip at the corner of the mouth, to spasm of all of the facial muscles on the stimulated side. However, slightly positive responses occur in as many as 10e15% of healthy adults. Trousseau’s sign is provoked when a sphygmomanometer is inflated on the arm to above the systolic blood pressure for up to 3 minutes [9]. Positive responses consist of carpal spasm with resolution occurring 5e10 seconds after the cuff is deflated (i.e., relaxation is not immediate). Both Chvostek’s and Trousseau’s signs can be absent, however, even in severe hypocalcemia. They seem to reflect the rapidity of change in serum calcium levels. Chronic hypocalcemia causes cataracts, dermopathy (Fig. 46.2),
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FIGURE 46.2 Hyperkeratotic dermatosis (shown here, posterior
neck) recurs in a 19-year-old black man with pseudohypoparathyroidism, who is poorly compliant with medical therapy. When he stops treatment with calcium and vitamin D, he becomes markedly hypocalcemic, and the hyperkeratotic lesions reappear.
contractures, and basal ganglia calcification [14]. Growth rate is an important parameter to follow in infants and children with metabolic bone disease, especially rickets. Improvement or correction of short stature can derive from effective treatment. With it should also come reduction or resolution of skeletal deformity if there is sufficient time for longitudinal growth before physes close at the end of puberty. Height or length is determined with a stadiometer or a tape measure with the patient in bare feet standing or supine, respectively. Weight should also be carefully assessed (and controlled, if excessive). Obesity or inordinate weight gain in girls during late childhood may transiently improve stature, not necessarily reflecting a favorable response to medical therapy. Instead, the physician can mistake the influence of excess weight on accelerating pubertal growth for efficacy of treatment. Here, the growth spurt has merely occurred early, but physes will fuse soon after menarche, negating any improvement in final adult stature. Skeletal deformation can cause much of the morbidity of metabolic bone disease. Bowing of the lower limbs predisposes to osteoarthritis especially in the knees. Prevention, control, or correction of deformities is an important goal of patient care. Without a complete physical examination, such important clinical problems may go unnoticed. Something as inexpensive as a shoe lift can be of considerable benefit, but the correct size and placement must come from accurate evaluation of leglength inequality if iatrogenic problems are to be avoided. For children with rickets, determination of height and arm span will help to quantitate skeletal deformity, as will measurements of the upper and lower segment lengths and calculation of their ratio. Simple measurements e including finger breadth separation of knees or ankles with bowed legs or knock knees, respectively
e are useful. With rickets (e.g., X-linked hypophosphatemia), some time may pass before the metabolic bone disease is controlled medically with active metabolites of vitamin D3 and phosphate supplementation. Accordingly, photography, gait analysis, and even videotaping of skeletal deformity may also help assess progression or document response to therapy. A “metabolic myopathy” is a prominent clinical feature of vitamin D deficiency, tumor-induced rickets or osteomalacia, and hypophosphatasia. Proximal muscle weakness of the limbs is suspected from a history of difficulty rising from a sitting position, negotiating stairs, or combing hair, and can be confirmed by physical examination. Gower ’s sign is a simple and excellent way to detect this problem in children who are observed getting up from a seated position on the floor e if they must push up with their hands on their thighs to achieve uptight posture, this is a positive test. Other routine assessments of muscle strength should be recorded [9]. In infants and children with rachitic disease, skull shape and calvarial growth should be followed e including recordings of head circumference using standard charts. Early closure of the cranial suture is not uncommon in these disorders [15]. Premature union of sagittal sutures commonly causes a dolichocephalic skull in X-linked hypophosphatemia, but this is usually a cosmetic concern only. In hypophosphatasia, however, there can be either functional or true premature fusion of multiple cranial sutures leading to a scafalocephalic skull, sometimes with raised intracranial pressure. Dystocia from too narrow a birth canal due to pelvic deformity from vitamin D deficiency during childhood was a major cause of puerperal mortality in mothers in the early twentieth century. This deformity should be searched for in pregnant women with a history of rickets. Alopecia is a distinctive clinical finding in some patients with hereditary vitamin-D-resistant rickets (vitamin-D-dependent rickets, type II) (Chapter 65). However, alopecia or hypotrichosis can also be a manifestation of far more prevalent vitamin-D deficiency rickets accompanying malnutrition. Additionally, hypotrichosis occurs in some forms of metaphyseal dysplasia that can be confused clinically and radiographically with rickets [5]. The benign tumors that cause oncogenic (tumorinduced) rickets or osteomalacia are often at least palpable if not visible, but they may be no more than pea-size and are sometimes hidden (Chapter 63). They are frequently found subcutaneously, but can occur anywhere on the body. Some have been discovered intravaginally or in the nasopharynx, and some lie within the skeleton. Because extirpation of these lesions is curative, especially thorough physical examination is essential when this disorder is suspected. If the physician cannot find the tumor on imaging studies
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(including bone, octreotide, or positron emission spectrophotometry scanning), patients should be instructed concerning periodic searches for subcutaneous masses or symptoms from tumor elsewhere. Physical examination yearly is warranted in hopes that a previously undetectable lesion has appeared.
Laboratory Testing The medical history and physical examination guide further assessment of the patient with metabolic bone disease by laboratory methods. Beginning the testing without this fundamental information, but instead with biochemical or radiological studies, is not appropriate. Of consternation for some physicians who only occasionally encounter patients with metabolic bone disease is the often bewildering array of expensive assays for factors that condition mineral or skeletal homeostasis, as well as the plethora of markers of skeletal apposition or resorption (Table 46.5). Although some biochemical testing is necessary for effective diagnosis or treatment of metabolic bone disease, especially disturbances of vitamin D homeostasis, a few studies are usually all that are needed. Radiological procedures used for skeletal disease are relatively limited, but often give critical information. However, it is wasteful to cover the diagnostic “waterfront” by ordering a “bone battery” of biochemical tests, or a series of low-yield radiological studies. Histopathological assessments are indicated in fewer situations, but may provide definitive or insightful findings.
Radiological Examination X-ray Images Radiographs of the skeleton chosen selectively are often crucial for diagnosis and follow-up of patients TABLE 46.5
Biochemical Markers of Bone Remodeling
Resorption (osteoclast products) Tartrate-resistant acid phosphatase (serum) Hydroxyproline (urine) Hydroxylysine glycosides (urine) Collagen cross-links (urine and serum) Total pyridinolines (Pyr and/or Dpy)* Free pyridinolines (Pyr and/or Dpy)* Cross-linked N- and C-telopeptides (NTX, CTX) Formation (osteoblast products) Propeptides of type I collagen (serum) C-propeptide N-propeptide Osteocalcin (serum) Alkaline phosphatase (serum) Total activity Bone-specific enzyme * Pyr, pyridinoiine; Dpy, deoxypyiidinoline.
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with metabolic bone disease (Chapter 49) [10e12,16]. However, the “skeletal survey” that examines all bones is an expensive, relatively insensitive, and laborious procedure, causing a not trivial exposure to X-irradiation. Visualization of the shape of the entire skeleton (but using films of just one upper and one lower extremity) is usually indicated to assess a bone dysplasia, but is rarely necessary for evaluation of metabolic bone disease. Instead, the “metabolic bone survey” generally gives the necessary radiographic information for diagnosis and follow-up. Here, one studies the appendicular, as well as the axial, skeleton, and therefore delineates both cortical and trabecular bone as well as “yellow” and “red” marrow spaces. The necessary films are a lateral view of the skull and thoracolumbar spine, posteroanterior view of a hand and wrist as well as the chest, and an anteroposterior view of the pelvis and a knee. Chapter 49, as well as several comprehensive texts [10e12,16], and other resources (London Dysmorphology Database, Oxford University Press and POSSUM e Pictures Of Standard Syndromes And Undiagnosed Malformations e The Murdoch Institute for Research Into Birth Defects, Melbourne, Australia), describe the radiographic findings of the metabolic bone diseases and help to distinguish the skeletal dysplasias. When there is rickets, posteroanterior radiographs of the hands and anteroposterior radiographs of the knees will precisely document the presence and degree of physeal and metaphyseal change. Long cassette films of the lower extremities, taken while the patient is standing, help quantitate bowing or knock-knee deformity. Radiographic studies can also provide clues to the particular etiology or pathogenesis of rickets. For those disorders that cause rickets by disturbing vitamin D homeostasis and result in secondary hyperparathyroidism, subperiosteal bone resorption and osteopenia may be noted in addition to growth plate widening and irregular metaphyses (Fig. 46.3). These findings contrast to most cases of X-linked hypophosphatemia where the skeleton typically has normal or sometimes increased radiodensity and evidence of hyperparathyroidism is generally absent. In hypophosphatasia, there are characteristically peculiar “tongues” of radiolucency that project from the physes into the metaphyses. Rachitic disease of recent onset will symmetrically widen growth plates, whereas longstanding rachitic disease with bony deformity alters the mechanical forces acting on the physes, which in turn become asymmetrically widened. Accordingly, chronic rachitic disease sometimes seems more difficult to diagnose with X-rays (Fig. 46.4). The rapidity of resolution of rachitic changes on Xray images also may be of diagnostic significance. With vitamin-D-deficiency rickets from lack of sunlight
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FIGURE 46.3 Characteristic changes of rickets (growth plate widening and frayed ends of metaphyses) occur in anteroposterior radiographs
of the knees of five boys each newly diagnosed with a different type of rachitic disease (AeE). However, as shown, additional information is apparent concerning the pathogenesis and/or etiology. (A) A 1-year-old boy with osteopenia due to secondary hyperparathyroidism from “nutritional” rickets. (B) Similar findings in a 5-year-old boy with osteopenia consistent with documented secondary hyperparathyroidism from anticonvulsant-induced rickets. (C) A 3-year-old boy has normal bone mass and no secondary hyperparathyroidism, consistent with X-linked hypophosphatemia (XLH). (D) A 10-year-old boy has symmetrical widening of the growth plates (seen best in the proximal tibia), but no long bone deformity. Together, these changes suggest recent-onset disease in keeping with suspected tumor-induced rickets. (E) A 10-year-old boy has “tongues” of radiolucency (arrows), that project from physes into metaphyses, characteristic of the childhood form of hypophosphatasia. (F) However, not all disorders that cause metaphyseal irregularity are forms of rickets. This 4-year-old boy, with unremarkable biochemical studies and iliac crest histology following “tetracycline labeling,” has metaphyseal dysplasia.
exposure, radiographic improvement can occur rapidly (within a few weeks) following a single pharmacological dose of vitamin D. Other forms of rickets, especially those due to renal phosphate wasting, generally take
longer (several months or more) to improve or correct with appropriate medical therapy. Sequential radiographs are essential to evaluate the response to therapy for rickets.
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FIGURE 46.4 A 9-year-old girl, who is poorly compliant for medical therapy for X-linked hypophosphatemia (XLH), has lower limb bowing that asymmetrically deforms the growth plates in her knees.
Bone Scintigraphy Bone scintigraphy is an excellent tool for uncovering a variety of abnormalities of the skeleton, but it does not establish a diagnosis [17]. Enhanced radioisotope uptake occurs in areas of increased blood flow to the skeleton, excess osteoid, and accelerated bone formation. Costeffective assessment starts with the bone scan followed by X-rays of the “hot spots” to guide further study. Bone scanning is generally unnecessary in children with rachitic disease, unless a search for a skeletal source of tumoral rickets is needed when physical examination fails to yield an obvious lesion. In adults, bone scanning can also disclose complications of osteomalacia, including “true” as well as “false” (pseudo) fractures. Bone Densitometry Bone mass quantitation is now widely available for clinicians [6]. Dual-energy X-ray absorptiometry (DEXA) and quantitative computed tomography (QCT) can give helpful assessments of skeletal mass and quality designated “bone mineral density” (BMD). However, DEXA or QCT densitometry does not provide a diagnosis. In fact, each of the principal categories of metabolic bone disease (see below) e namely, osteoporosis, osteomalacia, and osteitis fibrosa cystica e can manifest with low BMD. The presence of worrisome BMD revealed by these techniques is merely a point of departure for differential diagnosis. For those rare conditions associated with increased BMD, these tools are similarly helpful [18].
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Densitometry has some important technical caveats. When using DEXA for children, it is crucial to understand that the technique generates a so-called areal (two-dimensional) rather than volumetric (three-dimensional) assessment of BMD. BMD is measured as g/cm2 by DEXA, and as g/cm3 by QCT. Accordingly, values for BMD from DEXA are importantly dependent on body size. Children have lower BMD on DEXA compared to adults, but this is largely an artifact of their body size. Small-stature individuals, who have otherwise normal skeletons, will seem to have low BMD compared to taller subjects. Hence, the pediatric age group (or individuals who are small for reasons other than metabolic bone disease) may be incorrectly diagnosed with “osteopenia” or “osteoporosis” if this technical phenomenon with DEXA goes unrecognized or uncorrected. QCT can measure BMD with the advantage that it provides a volumetric measurement, and can focus on either cortical or trabecular bone, but with higher exposure to X-irradiation. QCT can also precisely evaluate the anatomy of the skeleton and readily detect extracellular calcification [17]. Quantitation of BMD in children uses Z-scores (standard deviations from mean values) matched for age and gender (and ideally for ethnicity), whereas T-scores (which refer to peak bone mass) are used to assess low bone mass in adults. Now, DEXAbased BMD reference ranges for various skeletal sites are available for children [18], but there are caveats (see above) for interpreting areal BMD values in children. Interpretation of high BMD values uses Z-scores for either children or adults [19]. Other Radiological Procedures Magnetic resonance imaging (MRI) is particularly useful for marrow space examination, including delineation of ischemic necrosis of bone. Fat-suppressed imaging showing bone edema may disclose a cause for unexplained bone pain. New applications of MRI include assessment of trabecular bone microanatomy [17]. Ultrasound studies of the skeleton may provide qualitative as well as quantitative information [6]. See Chapter 50 for a discussion of new imaging techniques.
Biochemical Investigation Understandably, circulating calcium levels are closely scrutinized in patients with metabolic bone disease, especially those suspected of having rickets or osteomalacia (Chapter 59). However, extracellular (serum) phosphate levels may be equally (perhaps more) important when there is defective skeletal mineralization (Chapter 63). Chronic hypophosphatemia occurs in a number of conditions associated with rickets or osteomalacia (Table 46.6). Accordingly, hypophosphatemia is an especially important finding. Clinicians must recognize that
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TABLE 46.6
Causes of Chronic Hypophosphatemia
Decreased intestinal absorption Alcohol abuse Antacid abuse Vitamin D deficiency Malabsorption Starvation Increased urinary losses Renal tubular defects X-linked hypophosphatemia (XLH) Oncogenic rickets or osteomalacia (tumor-induced osteomalacia) Abnormalities of vitamin D metabolism Vitamin D deficiency Vitamin-D-dependent rickets Hyperparathyroidism Renal transplantation Alcohol abuse Poorly controlled diabetes mellitus Metabolic or respiratory acidosis Drugs: calcitonin, diuretics, glucocorticoids, bicarbonate Respiratory alkalosis Extracellular fluid volume expansion Severe burns
the pediatric age group normally has higher fasting blood phosphate levels compared to adults. Serum phosphate levels should be assayed with fasting blood specimens, because food (depending on content) can acutely raise or lower the level. Assay of phosphate levels in a urine specimen can help to distinguish whether hypophosphatemia is due to renal phosphate wasting or to dietary deficiency. Detailed tests of renal phosphate handling using timed urine collections, e.g., tubular reabsorptive maximum for phosphorus/ glomerular filtration rate (TmP/GFR) ratios, provide a definitive assessment [2,3]. Disturbances in vitamin D stores or bioactivation commonly result in hypocalcemia, secondary hyperparathyroidism and, consequently, hypophosphatemia. Assay of the serum levels of the major active vitamin D metabolites e 25(OH)D and 1,25(OH)2D e is essential to detect disturbances in vitamin D stores or in vitamin D bioactivation, respectively (Chapter 47). The important effects of season on serum 25(OH)D levels should be considered. Furthermore, 25(OH)D is transported in the circulation bound to vitamin-D-binding protein (DBP) (Chapter 5). Accordingly, hypoproteinemia must be considered before interpreting a serum 25(OH)D concentration. Low levels of serum 25(OH)D usually indicate vitamin D deficiency, but this biochemical finding is merely a starting point for differential diagnosis. A considerable number of disorders cause rickets or osteomalacia where serum 25(OH)D levels are low (Table 46.7). Assay of serum 1,25(OH)2D concentration is
most helpful in exploring hypercalcemia, but is also necessary to characterize inborn errors of vitamin D bioactivation. Finding a low serum 1,25(OH)2D level also helps in the investigation of oncogenic osteomalacia. However, serum 1,25(OH)2D levels should be interpreted mindful that pediatric and adult reference ranges differ, and that low serum phosphate levels are expected to increase 1,25(OH)2D levels in healthy individuals. Hypophosphatemia without physiological elevation in 1,25(OH)2D levels points to a disturbance in renal 1,25 (OH)2D biosynthesis.
Histopathological Assessment Throughout the past century, clinicians diagnosed and dealt with three major types of metabolic bone disease based on the principal histopathological features and the mineral-to-matrix protein ratios e osteoporosis, osteomalacia, and hyperparathyroidism (osteitis fibrosis cystica). Biopsy of the iliac crest assesses cortical as well as trabecular bone and distinguishes especially well these types of metabolic bone disease [13]. Histological examination of the iliac crest must, however, follow in vivo “labeling” of the patient’s skeleton by ingestion of two 3-day courses of tetracycline [6,13] and nondecalcified sectioning and histomorphometric analysis of specimens. Which of these types of bone disease is present provides a starting point for differential diagnosis [2,3,13]. See Chapter 48 for discussion of histomorphometry. Bone biopsy is not routinely needed to diagnose rachitic disease, which is easily detected by radiographic studies of the physes of the wrist and knees together with biochemical testing. In adults with osteomalacia, however, growth plate changes on X-ray imaging have been “lost” as guideposts for diagnosis as well as for judging the efficacy of treatment. Thus, bone biopsy may be more helpful in adult patients.
TREATMENT Treatment of metabolic bone disease can range from simple (e.g., exposure to sunlight for environmental vitamin-D-deficiency rickets) to complex (e.g., bone marrow transplantation for the malignant form of osteopetrosis). When there is a disturbance in vitamin D homeostasis, pharmaceuticals are usually needed. However, additional treatment approaches may be required when there is skeletal deformity. Patients who require vitamin D sterols can benefit greatly; but medical care often must be especially skilled. Rickets or osteomalacia can occur from deficiency or impaired bioactivation of vitamin D. Either disturbance will decrease calcium absorption from the
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TABLE 46.7
Causes of Rickets or Osteomalacia
TABLE 46.7
Causes of Rickets or Osteomalaciadcont’d
b. Tumor-associated rickets and osteomalacia c. Neurofibromatosis d. McCune-Albright syndrome
I. Vitamin D deficiency A. Deficient endogenous synthesis 1. Inadequate sunshine 2. Other factors, e.g., genetic, aging, pigmentation B. Dietary 1. Classic “nutritional” 2. Fat-phobic II. Gastrointestinal A. Intestinal 1. Small-bowel diseases with malabsorption, e.g., celiac disease (gluten-sensitive enteropathy) B. Hepatobiliary 1. Cirrhosis 2. Biliary fistula 3. Biliary atresia C. Pancreatic 1. Chronic pancreatic insufficiency III. Disorders of vitamin D bioactivation A. Hereditary 1. Vitamin D dependency, type I (pseudovitamin D deficiency) 2. Vitamin D dependency, type II (hereditary vitamin D-resistant rickets) B. Acquired 1. Anticonvulsant therapy 2. Renal insufficiency (see below) IV. Acidosis A. Distal renal tubular acidosis (classic, type I) 1. Primary (specific etiology not determined) a. Sporadic b. Familial 2. Secondary a. Galactosemia b. Hereditary fructose intolerance with nephrocalcinosis c. Fabry’s disease 3. Hypergammaglobulinemic states 4. Medullary sponge kidney 5. Post renal transplantation B. Acquired 1. Ureterosigmoidostomy 2. Drug-induced a. Chronic acetazolamide use b. Chronic ammonium chloride use
VII. General renal tubular disorders (Fanconi syndrome) A. Primary renal 1. Idiopathic a. Sporadic b. Familial 2. Associated with systemic metabolic process a. Cystinosis b. Glycogenosis c. Lowe’s syndrome B. Systemic disorder with associated renal disease 1. Hereditary a. Inborn errors (i) Wilson’s disease (ii) Tyrosinemia 2. Acquired a. Multiple myeloma b. Nephrotic syndrome c. Transplanted kidney 3. Intoxications a. Cadmium b. Lead c. Outdated tetracycline VIII. Primary mineralization defects A. Hereditary 1. Hypophosphatasia B. Acquired 1. Bisphosphonate intoxication 2. Fluorosis IX. States of rapid bone formation with or without a relative defect in bone resorption A. Postoperative hypoparathyroidism with osteitis fibrosa cystica B. Osteopetrosis (“osteopetrorickets”) X. Defective matrix synthesis A. Fibrogenesis imperfecta ossium XI. Miscellaneous A. Magenesium-dependent B. Steroid-sensitive C. Axial osteomalacia
V. Chronic renal failure VI. Phosphate depletion A. Dietary 1. Low phosphate intake 2. Total parenteral nutrition 3. Aluminium hydroxide antacid abuse B. Impaired renal tubular (? intestinal) phosphate reabsorption 1. Hereditary a. X-linked, autosomal dominant, and autosomal recessive hypophosphatemic rickets b. Adult-onset vitamin D-resistant hypophosphatemic osteomalacia 2. Acquired a. Sporadic hypophosphatemic osteomalacia (phosphate diabetes) (Continued)
gastrointestinal tract, leading to variable degrees of hypocalcemia, secondary hyperparathyroidism, and hypophosphatemia. The hypocalcemia and hypophosphatemia together engender the defective skeletal mineralization (Chapter 21). Fortunately, it is possible to prevent, cure, or control most aberrations in vitamin D homeostasis. Vitamin D deficiency stemming from socioeconomic factors has relatively uniform etiology and pathogenesis, considerable prevalence, and a long history for mankind that make prevention or treatment well understood and theoretically straightforward. Inborn errors of vitamin
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D bioactivation can be relatively easy to control, e.g., merely by providing a “replacement” dose of 1,25 (OH)2D3 for pseudovitamin-D-deficiency rickets (vitamin-D-dependent rickets, type I), or extremely difficult to treat, as in some patients with hereditary vitaminD-resistant rickets (vitamin-D-dependent rickets, type II). Here, resistance to 1,25(OH)2D is so great in some cases that it must be “bypassed” therapeutically by providing calcium intravenously in order to heal the rickets. For other clinical situations (especially hepatobiliary, gastrointestinal, or pancreatic disease) leading to vitamin D deficiency, the precise regimen and duration of treatment will vary greatly from patient to patient and depend on measures to control the primary disorder. Hence, an accurate diagnosis, understanding of pathogenesis, and familiarity with the pharmacological armamentarium is essential for successful therapy that may require adjustment from time to time. Now, a considerable number of pharmaceuticals are available for the treatment of metabolic bone disease. In the United States, two major biological forms of vitamin D (vitamin D and 1,25(OH)2D3) can be prescribed. There are also several preparations that provide inorganic phosphate, and innumerable types of calcium supplementation. Hence, both repletion of vitamin D reserves as well as bypassing steps in vitamin D bioactivation are treatment options. Furthermore, direct (intravenous) administration of calcium is possible when there might be insufficient or delayed action of a vitamin D sterol on the gastrointestinal tract. Importantly, these drugs have different durations of fat and muscle storage and potency. Additionally, the lag times for onset of action and longevity of biological effects vary considerably for these preparations. Finally, the cost of these pharmaceuticals differs greatly (Chapter 59). An accurate diagnosis from among the many disorders that cause rickets or osteomalacia is essential for therapeutic efficacy, safety, and economy. An understanding of the pathophysiology of the aberration in vitamin D homeostasis is required for effective and safe therapy. Use of 1,25(OH)2D3, when there is depletion of body stores of vitamin D, merely circumvents but does not correct the basic problem. Because many disorders disturb vitamin D homeostasis and their severities can differ markedly, each patient and condition must be treated individually. From the above, it is apparent that follow-up of medical treatment for metabolic bone disease e especially rickets and osteomalacia e is essential for many reasons. Only some generalities can be discussed here. For infants, children, and adolescents with rickets, close monitoring of drug therapy is especially important because these individuals are growing. Not only will biochemical and skeletal dynamics change in response to treatment, but there is increasing body size to contend
with. Alterations in weight, a growth spurt, or healing of rachitic disease will all likely require changes in dosage. Greater weight means higher doses of vitamin D sterols and/or mineral supplements to control an otherwise static metabolic disturbance. A growth spurt can undo successful therapy if treatment does not increase to keep up with the greater skeletal demands. Conversely, healing of rickets or osteomalacia may require a reduction in dosage, because the skeletal “sump” is now satisfactorily mineralized. It should be clear that individualized follow-up, especially for pediatric patients, is crucial. When managing chronic forms of rickets, help from other medical and surgical (e.g., orthopedic) subspecialties is often necessary, and is optimal when there is significant interdisciplinary exchange. Children with rickets who do not respond to pharrnacologic therapy may benefit from bracing, epiphysiodesis (physeal stapling), or osteotorny to improve lower limb deformity. However, these procedures can reflect suboptimal medical therapy and are usually avoidable if dosing, compliance, and follow-up are satisfactory. For some patients, particularly children and adolescents, pill counts to assess compliance can provide important information. Cooperation and monitoring can be improved using pillboxes marked by days of the week. School nurses may help administer patient medications when family life is disrupted. If deformities are not obviously correcting, at least yearly orthopedic evaluation (perhaps biannually during the growth spurt) is advisable. Not all types of rickets or osteomalacia manifest with hypocalcemia or low serum levels of 25(OH)D or 1,25 (OH)2D. Hypophosphatemia, with or without hypocalcemia, can lead to defective skeletal mineralization. Nevertheless, many of these disorders respond to treatment with 1,25(OH)2D3 and mineral supplements [2,3]. Here again, the etiology and pathogenesis of the specific condition must be understood for successful medical management. The most common form of heritable rickets, X-linked hypophosphatemia (XLH), is transmitted as an X-linked dominant trait. The pathogenesis of XLH includes a renal tubular defect engendering loss of phosphate by the kidney [3]. Despite hypophosphatemia, serum 1,25 (OH)2D levels are paradoxically normal instead of elevated. More complex proximal renal tubular defects that also feature renal phosphate wasting (Fanconi syndrome) can reflect other inborn errors of metabolism, heritable disorders, or exposure to certain drugs or toxins including heavy metal poisoning (Table 46.6; and Chapter 63). Acquired hypophosphatemic rickets is also a common complication in McCune-Albright syndrome and is characteristic of oncogenic rickets. Treatment with 1,25 (OH)2D3 and phosphate is used for the rickets
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FIGURE 46.5 In X-linked hypophosphatemia (XLH), the extremes of lower limb deformity in children that can be corrected by pharmacological therapy alone are not widely appreciated. Accordingly, close cooperation between the medical and orthopedic disciplines is essential. At age 3 years, this girl with XLH has severe bowing deformity of the lower extremities that can be quantitated with standing, long-cassette radiographs. Compliance for 1,25(OH)2D3 and inorganic phosphate supplementation therapy was excellent, and her deformity is remarkably improved by age 10 years without osteotomy, epiphysiodesis, or lower extremity bracing.
complicating these disorders. Normalization of blood phosphate levels occurs only transiently with oral phosphate supplementation in conditions characterized by renal phosphate wasting. Nevertheless, clinical improvement can be substantial (Fig. 46.5), without
a likely hazardous push to correct phosphate levels measured with fasting blood specimens. Correction of hypophosphatemia should not be considered the objective of therapy, but rather correction of deformity and restoration of an adequate growth rate. Radiographic
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46. APPROACH TO THE PATIENT WITH METABOLIC BONE DISEASE
and other biochemical studies are, instead, useful for judging adequacy and safety or therapy.
CONCLUSION The complex interaction of the many exogenous and endogenous factors that impact vitamin D homeostasis, discussed throughout this book, explain why patients with disturbances in this endocrine process are especially challenging. Nevertheless, these individuals typically benefit greatly from the efforts of physicians who understand such disorders. Demonstration of concern for, and commitment to, the patient begins with a complete medical history and thorough physical examination that wins their confidence and trust. Such rapport will likely be essential for effective management of what will often prove to be a chronic disorder. Physical examination is crucial not only for diagnosis, but to uncover structural skeletal problems. Information gathered by the medical history and physical examination will be the guide to the myriad of biochemical, radiological, and other technologies that can assist to establish the etiology and pathogenesis and to set the stage for subsequent treatment and follow-up. Effective therapies are available for derangements of vitamin D homeostasis, but the pharmaceuticals vary significantly in potency, duration of effect, and cost. Once the proper clinical foundation is in place, the physician will usually be gratified by a patient that he/she has greatly helped.
Acknowledgments Supported by Shriners Hospitals for Children, The Clark and Mildred Cox Inherited Metabolic Bone Disease Research Fund, and the BarnesJewish Hospital Foundation.
References [1] L.V. Avioli, S.M. Krane, Metabolic Bone Disease and Clinically Related Disorders, Academic Press, San Diego, CA, 1998.
[2] F.L. Coe, M.J. Favus, Disorders of Bone and Mineral Metabolism, Lippincott, Williams & Wilkins, Philadelphia, PA, 2002. [3] C.J. Rosen, Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, seventh ed., American Society for Bone and Mineral Research, Washington, DC, 2008. [4] C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle, The Metabolic and Molecular Bases of Inherited Disease, McGraw-Hill, New York, NY, 2001. [5] McKusick V Online Mendelian Inheritance in Man, OMIM (TM) McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD) (http://www.ncbi.nlm.nih.gov/omim). [6] F.I. Tovey, T.C.B. Stamp, The Measurement of Metabolic Bone Disease, Parthenon Publishing Group, NewYork, NY, 1995. [7] R. Marcus, D. Feldman, D. Nelson, C.J. Rosen, Osteoporosis, third ed., Academic Press, San Diego, CA, 2007. [8] J.P. Bilezikian, L.G. Raisz, T.J. Martin, Principles of Bone Biology, third ed., Academic Press, San Diego, CA, 2008. [9] Degowen, Degowen Diagnostic Examination, McGraw-Hill Health Professionals Division, New York, NY, 1999. [10] J. Edeiken, M.K. Dalinka, D. Karasick, Edeiken’s Roentgen Diagnosis of Diseases of Bone, Williams & Wilkins, Baltimore, MD, 1990. [11] H. Taybi, R.S. Lachman, Radiology of Syndromes, Metabolic Disorders, and Skeletal Dysplasias, fifth ed., Mosby, St. Louis, MO, 2007. [12] P.A. Ravell, Pathology of Bone, Springer-Verlag, Berlin, Germany, 1985. [13] D. Resnick, Diagnosis of Bone and Joint Disorders, Saunders, Philadelphia, 2002. [14] M.P. Whyte, Hypocalcemia, in: B.E.C. Nordin, A.G. Need, H.A. Morris (Eds.), Metabolic Bone and Stone Disease, Churchill Livingstone, Edinburgh, UK, 1993, pp. 147e162. [15] B.J. Reilly, J.M. Leeming, D. Fraser, Craniosynostosis in the rachitic spectrum, J. Pediatr. 64 (1964) 396e405. [16] G.B. Greenfield, Radiology of Bone Diseases, Lippincott, Williams & Wilkins, Philadelphia, PA, 1990. [17] D. Resnick, Bone and Joint Imaging, Saunders, Philadelphia, 2005. [18] H.J. Kalkwarf, B.S. Zemel, V. Gilsanz, J.M. Lappe, M. Horlick, S. Oberfield, et al., The bone mineral density in childhood study: bone mineral content and density according to age, sex, and race, J. Clin. Endocrin. Metab. 92 (2007) 2087e2099. [19] M.P. Whyte, Misinterpretation of osteodensitometry with high bone density (BMD Z þ 2.5 is not normal, J. Clin. Densitom. 8 (2005) 1e6.
VI. DIAGNOSIS AND MANAGEMENT
C H A P T E R
47 Detection of Vitamin D and Its Major Metabolites Bruce W. Hollis Departments of Pediatrics, Biochemistry, and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, USA
In the interest of full disclosure, the author wishes to inform the readers that he has been a paid consultant to the DiaSorin Company.
INTRODUCTION Vitamin D is a 9,10-secosteroid and is treated as such in the numbering of its carbon skeleton (Fig. 47.1). Vitamin D occurs in two distinct forms: vitamin D2 and vitamin D3. As shown in Figure 47.1, vitamin D3 is a 27-carbon derivative of cholesterol; vitamin D2 is a 28-carbon molecule derived from the plant sterol ergosterol. Besides containing an extra methyl group, vitamin D2 differs from vitamin D3 in that it contains
28
CH3 22
21 18
CH3 20
12 11 13 9
8
14
17 16 15
24 23
26 25
CH3
27
7 6
4 3
5
2
CH2
CH2
10 1
HO
HO
Vitamin D3
Vitamin D2
FIGURE 47.1 Molecular structures of vitamins D2 and D3.
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10047-2
a double bond between carbons 22 and 23. The most important aspects of vitamin D chemistry center on its cis-triene structure. This unique cis-triene structure makes vitamin D and related metabolites susceptible to oxidation, ultraviolet (UV)-light-induced conformational changes, heat-induced conformational changes, and attack by free radicals. As a rule, the majority of these transformation products have lower biological activity than vitamin D. It is important to note that, in humans, vitamins D2 and D3 provide similar potency (although some controversy exists as discussed in Chapter 61), and in this chapter the term vitamin D refers to both compounds. Metabolic activation of vitamin D is achieved through hydroxylation reactions at both carbon 25 of the side chain and, subsequently, carbon 1 of the A ring. Metabolic inactivation of vitamin D takes place primarily through a series of oxidative reactions at carbons 23, 24, and 26 of the side chain of the molecule. These metabolic activations and inactivations are well characterized and result in a plethora of vitamin D metabolites (Fig. 47.2). Of the compounds shown in Figure 47.2, only four, vitamin D, 25-hydroxyvitamin D (25(OH)D), 24,25-dihydroxyvitamin D (24,25(OH)2D), and 1,25dihydroxyvitamin D (1,25(OH)2D) have been extensively quantitated, and to date only two of those, namely, 25(OH)D and 1,25(OH)2D, provide any clinically relevant information. However, the quantitation of vitamin D and 24,25(OH)2D can provide important information in a research environment. Thus, this chapter addresses the quantitation of these four important vitamin D compounds. Further, it is not the intent of this chapter to address the detailed history of vitamin D metabolite analysis, as this can be obtained from
823
Copyright Ó 2011 Elsevier Inc. All rights reserved.
824
47. DETECTION OF VITAMIN D AND ITS MAJOR METABOLITES
FIGURE 47.2 Summary of metabolic transformations of vitamin D3. From Bouillon R, Okamura WH, Norman AW 1995 Structure-function relationships in the vitamin D endocrine system. Endocrine Review 16 :200e257. Ó The Endocrine Society.
previous reviews [1e3]. Rather, the intent of this chapter is to describe how we currently measure vitamin D and its major metabolites in our laboratory, as well as to discuss the appropriate clinical judgments in the selection of a given compound for analysis. The first semiquantitative assay for vitamin D was a bioassay based on the rat-line test [4]. This assay was cumbersome, expensive, and relatively inaccurate. Real progress in vitamin D analysis was not achieved until the advent of high-specific-activity 3H-labeled vitamin
D3 compounds [5]. The introduction of these tracers led to the development of competitive protein-binding assays (CPBA) for vitamin D and 25(OH)D [6,7]. A short time later CPBA for 24,25(OH)2D and radioreceptor assays (RRA) for 1,25(OH)2D were introduced [8,9]. In the late 1970s, high-performance liquid chromatographic (HPLC) analytical procedures for vitamin D and 25(OH)D were described [10,11]. Subsequently, radioimmunoassay (RIA) techniques began to appear as a means to quantitate 25(OH)D and 1,25(OH)2D
VI. DIAGNOSIS AND MANAGEMENT
825
DETECTION OF VITAMIN D
[12,13]. Later advances in antirachitic sterol analysis have included RIA coupled with 125I-labeled and enzyme-linked tracers that require little or no chromatographic treatment of the sample [14,15] and the instrument automation for the direct detection of circulating 25(OH)D [16]. Finally, in the past 5 years the use of liquid chromatography coupled to mass spectroscopy (LC/MS) has emerged as a means to clinically assess circulating 25(OH)D [17]. The assays for vitamin D and its major metabolites that we currently utilize in our laboratory are described in this chapter. These assays are all standalone types of assays as opposed to the multiple-metabolite assays described in years past [18,19]. We chose to do this because seldom in a clinical situation does one require a battery of vitamin D metabolite values. Further, many assays, especially for 25(OH)D and 1,25(OH)2D, have been optimized as single metabolite procedures [14,15].
DETECTION OF VITAMIN D Background Vitamin D, the parent compound, is by far the most lipophilic of the antirachitic sterols, and for this reason it is the most difficult to quantitate (Table 47.1). The first serious attempt to quantitate vitamin D was performed in 1971 utilizing CPBA [7]. This initial study grossly overestimated the actual amount of circulating vitamin D because of insufficient sample prepurification prior to CPBA. It was later shown that vitamin D could be assessed by CPBA, but only following extensive chromatographic purification of the organic extract, including HPLC [20,21]. The first valid determination of circulating vitamin D was achieved in 1978 by utilizing direct UV detection following a two-step HPLC purification procedure [10]. A short TABLE 47.1
time later, valid CPBA were introduced for the quantitation of circulating vitamin D [20,21]. However, these procedures were cumbersome, as they required extensive sample prepurification prior to CPBA, including HPLC. Vitamin D is also difficult to quantitate because it is the only antirachitic sterol that cannot be extracted from aqueous media utilizing solid-phase extraction techniques [21]. Therefore, unlike its more polar metabolites, vitamin D must be extracted from serum, plasma, or tissue (e.g., fatty tissue where it is normally stored) using liquideliquid organic extraction techniques. Many of the initial studies used Bligh and Dyer-type total lipid extraction to extract vitamin D from serum samples [7,10,20]. However, these types of extractions remove an extraordinary amount of lipid from the plasma sample. We therefore utilized a more selective organic extraction procedure incorporating methanolhexane [23]. This extraction method coupled with open cartridge silica chromatography and direct UV quantitation of vitamins D2 and D3 following nonaqueous reversed-phase HPLC provides an accurate, convenient method to measure circulating vitamin D. This method is described here in detail.
Methodology Sample Extraction A 0.5e1-ml sample of serum or plasma is placed into a 13 100 mm borosilicate glass culture tube containing 1000 cpm of 3H-vitamin D3 in 25 ml of ethanol to monitor recovery of the endogenous compound through the extraction and chromatographic procedures. Following a 15-min incubation with the tracer, two plasma volumes of HPLC-grade methanol are added to each sample. The sample is then vortexmixed for 1 min followed by the addition of three plasma volumes of HPLC-grade hexane. Each tube is
Significant Methods for the Estimation of Vitamin D in Human Ssruma
Detection method
Extraction
Preliminary chromatography
Ref.
Detected circulating levelsb
CPBA
Methanol-chloroform
Silicic acid
Belsey et al. [7]
24e40 ng/ml
HPLC
Methanol-chloroform
Preparative HPLC
Jones [10]
2.2 1.1 ng/ml
CPBA
Ether-methylene chloride
Sephadex LH-20, preparative HPLC
Horst et al. [21]
e
CPBA
Methanol-methylene chloride
Lipidex-5000, preparative HPLC
Hollis et al. [20]
2.3 1.1 ng/ml
HPLC
Methanol-hexane
Silica cartridges, Preparation HPLC
Liel et al. [23]
9.1 1.0 ng/ml (normal), 1.3 0.1 ng/ml (obese)
a
As noted in the text, the Belsey method overestimated the circulating vitamin D levels because of insufficient prepurification prior to competitive protein-binding assay (CPBA). ng/ml 2.6 ¼ nmol/liter.
b
VI. DIAGNOSIS AND MANAGEMENT
826
47. DETECTION OF VITAMIN D AND ITS MAJOR METABOLITES
capped and vortex-mixed for an additional 1 min followed by centrifugation at 1000 g for 10 min. The hexane layer is removed into another 13 100 mm culture tube, and the aqueous layer is re-extracted in the same fashion. The hexane layers are combined and dried in a heated water bath, 55 C, under N2. The lipid residue is then resuspended in 1 ml of HPLC-grade methylene chloride and capped. Silica Cartridge Chromatography Silica Bond-Elut cartridges (500 mg) and a Vac-Elut cartridge rack were obtained from Varian Instruments (Harbor City, CA). The silica cartridges are washed in order with 5 ml HPLC-grade methanol, 5 ml HPLCgrade isopropanol, and 10 ml HPLC-grade methylene chloride. The sample, in 1 ml of methylene chloride, is then applied to the cartridge and eluted through the cartridge under vacuum into waste. This initial step is followed by 3 ml of 0.2% isopropanol in methylene chloride (discard) and 8 ml of the same solvent (vitamin D). The 8-ml fraction contains vitamins D2 and D3 and is subsequently dried in a heated water bath, 55 C, under N2. The elution profile of vitamin D from the silica cartridge is displayed in Figure 47.3. The silica
cartridges can be cleaned and regenerated by washing with methanol, isopropanol, and methylene chloride and reused many times. Preparative Normal-phase High-performance Liquid Chromatography High-performance liquid chromatography can be performed on any available HPLC system. This normal-phase HPLC step is performed in our laboratory using a 0.4 25 cm Zorbax-Sil column packed with 5 mm silica, but any equivalent column could be utilized. The mobile phase comprises hexaneemethylene chlorideeisopropanol (49.5 : 49.5 : 0.5, v/v) at a flow rate of 2 ml/min. The sample residue from the silica cartridge is dissolved in 150 ml of the mobile phase and injected onto the HPLC column that had been previously calibrated with 10 ng of standard vitamin D3. The elution of vitamins D2 and D3 (they coelute on this system) can be seen in Figure 47.4A. The vitamin D
(A) 0.004
Hexane: ISP
CH2Cl2:ISP
95:5
99.8:0.2
92:8
0.003
85:15
98.5:1.5
500
3H-1,25-(OH)
2-D3
0.002
300 100
Optical density 265 nm
0.001
500
Radioactivity (cpm)
D 2 + D3
3H-24,25-(OH) -D 2 3
300 100 500
3H-25-OH-D
0
D2 D3
(B) 0.004 0.003
3
300
0.002
100 500
3H-Vitamin
D3
0.001
300
0 100
2 0
4
8
12
16
20
24
28
32
36
Elution volume (ml)
Elution profiles of radioactive vitamin D3 and its metabolites chromatographed on a silica Bond-Elut (500 mg) cartridge.
FIGURE 47.3
4
6 8 10 12 Elution time (min)
14
High-performance liquid chromatographic profiles of standard vitamins D2 and D3 on normal-phase (A) and reversedphase (B) systems. Column calibration was achieved by injecting 10 ng of each compound and monitoring optical density at 265 nm. Column conditions are described in the text.
FIGURE 47.4
VI. DIAGNOSIS AND MANAGEMENT
827
DETECTION OF 25(OH)D
fraction is collected in a 12 75 mm glass culture tube and dried under N2 at 55 C. Quantitative Reversed-phase High-performance Liquid Chromatography The final quantitative step is performed using nonaqueous reversed-phase HPLC. The column is a Vydac TP-201-54, 5 mm, wide-pore, nonendcapped octadecylsilane (ODS) silica material, 0.4 25 cm. This particular column must be used for this procedure to work. The mobile phase comprises acetonitrileemethylene chloride (65 : 35, v/v) utilizing a flow rate of 1.2 ml/min. This system provides clear resolution of vitamins D2 and D3 (Fig. 47.4B). This system is calibrated with varying amounts of vitamin D2 and D3 (1e100 ng). The sample residue from normal-phase HPLC is dissolved in 15 ml methylene chloride followed by the addition of 135 ml of acetonitrile and injected onto the HPLC. After elution, final quantitation of vitamin D2 and D3 is by direct UV monitoring of 265 nm. The vitamin D3 portion is collected, dried under N2, and subjected to liquid scintillation counting in order to determine the final recovery of endogenous vitamin D3 from the sample. Calculations are then performed, and the results are reported in nanograms vitamin D2 and/or D3 per milliliter. A flow diagram of the entire procedure is displayed in Figure 47.5. 0.5 ml Sample or control + 3H-Vitamin D3 Incubate 15 min room temp Extract with methanol : hexane
Centrifuge and remove hexane layer Dry hexane under N2 and resuspend in methylene chloride Apply to silica cartridge and collect vitamin D-containing fraction Dry fraction under N2 and resuspend in normal-phase HPLC mobile phase Apply to normal-phase silica HPLC and collect vitamin D-containing fraction Dry fraction under N2 and resuspend in methylene chloride:acetonitrile Apply to nonaqueous reversed-phase Vydac ODS HPLC and quantitate vitamins D2 and D3 by direct UV absorption. Collect vitamin D3-containing fraction and monitor for 3H-content recovery correction
FIGURE 47.5 Flow diagram of the HPLC-UV assay for the quantitation of vitamins D2 and D3.
DETECTION OF 25(OH)D Background One of the major factors responsible for the explosion of knowledge related to vitamin D metabolism was the introduction of valid CPBA for 25(OH)D in the early 1970s [6,7] (Table 47.2). One of these assays in particular gained widespread use owing to its relative simplicity and, as a result, has been cited close to 1000 times [6]. The first assays utilized the vitamin-D-binding protein (DBP) from rat serum as a specific binding agent. These assays all contained some type of organic extraction coupled with sample prepurification by column chromatography, all used 3H-25(OH)D3 as a tracer, and all required individual sample recovery estimates to account for endogenous losses of 25(OH)D during the extraction and purification procedures. A nonchromatographic assay for circulating 25(OH)D was introduced in 1974 [24], but it was never widely accepted because of its nonspecificity and susceptibility to serum lipid interference. Various CPBAs for 25(OH)D dominated the literature until 1977 when the first valid direct UV quantitative HPLC assay was introduced [11]. 25(OH)D circulates in the nanogram per milliliter (nanomole/ liter) range and thus could be directly quantitated by UV detection following its separation by normal-phase HPLC. Also, HPLC detection provided the advantage of being able to individually quantitate 25(OH)D2 and 25(OH)D3. The disadvantages of HPLC quantitation methods are their requirements for expensive equipment and large sample size, cumbersome nature, and the technical expertise to perform this type of analysis. However, HPLC analysis for 25(OH)D is frequently used in research environments, including our own, and has provided a great deal of significant information. As the clinical demand for circulating 25(OH)D analysis increased, it was clear that simpler, rapid yet valid assay procedures would be required. To this point, all valid assays required liquideliquid organic extraction, some sort of chromatographic prepurification, and evaporation of the organic solvents, hardly practical for a clinical chemistry laboratory. Thus, in 1985, the first valid RIA for assessing circulating 25(OH)D was introduced [13]. This RIA eliminated the need for sample prepurification prior to assay and had no requirement for organic solvent evaporation. However, the method was still based on the use of 3H-25(OH)D3 as a tracer. This final shortcoming was solved in 1993 when an 125 I-labeled tracer was developed and incorporated into the RIA for 25(OH)D [14]. This assay has become the method of choice for assessing 25(OH)D status and has become the first test for vitamin D approved for
VI. DIAGNOSIS AND MANAGEMENT
828 TABLE 47.2
47. DETECTION OF VITAMIN D AND ITS MAJOR METABOLITES
Significant Methods for the Estimation of 25(OH)D in Human Serum
Detection method
Extraction
Preliminary chromatography
Ref.
Observed circulating levelsa
CPBA
Methanol-chloroform
Silicic acid
Belsey et al. [7]
18e36 ng/ml
CPBA
Ether
Silicic acid
Hadad and Chyu [6]
27.311.8 ng/ml
CPBA
Ethanol
None
Belsey et al. [24]
20e100 ng/ml
HPLC
Methanol-chloroform
Sephadex LH-20
Eisman et al. [11]
31.9 1.7 ng/ml
RIA
Acetonitrile
None
Hollis and Napoli [13]
25.5 11.8 ng/ml
RIA
Acetonitrile
None
Hollis et al. [14]
9.9e41.5 ng/ml
CLIA
None
None
DiaSorin Corp.[16]
9.5e52.0 ng/ml
LC/MS
Hexane
None
Manunsell et al. [26]
4.0e45.0 ng/ml
ng/ml 2.4 ¼ nmol/liter.
a
clinical diagnosis by the US Food and Drug Administration (FDA) and thus is considered the predicate device. Most recently an automated, nonextracted chemiluminescent immunoassay (CLIA) for the direct determination of circulating 25(OH)D has been introduced [16].
HPLC Methodology Sample Extraction A 0.5-ml sample of serum or plasma is placed into a 12 75 mm borosilicate glass culture tube containing 1000 cpm of 3H-25(OH)D3 in 25 ml of ethanol to monitor recovery of endogenous compound through the extraction and chromatographic procedures. Following a 15-min incubation with the tracer, 1 plasma volume of HPLC-grade acetonitrile is added to each sample. The sample is then vortex-mixed for 1 min followed by centrifugation at 1000 g for 10 min. The supernatant is removed into another 12 75 mm culture tube, and 1 plasma volume of 0.4 M K2 HPO4, pH 10.4, is added. Solid-phase Extraction Chromatography C18 silica Sep-Pak cartridges (500 mg) and a Sep-Pak rack were obtained from Waters Associates (Milford, MA). The C18 cartridges are washed in sequence with 5 ml HPLC-grade isopropanol and 5 ml HPLC-grade methanol. The sample is applied to the cartridge and eluted through the cartridge under vacuum into waste. This initial step is followed by 5 ml of 30% water in methanol (discard) and 3 ml of acetonitrile (25(OH)D). The acetonitrile fraction is dried in a heated water bath, 55 C, under N2. The lipid residue is then suspended in 1 ml of 1.5% isopropanol in hexane and capped. The C18 cartridges can be cleaned and regenerated by washing with 2 ml of methanol and reused many times.
Silica Cartridge Chromatography Silica Bond-Elut cartridges (500 mg) and a Vac-Elut cartridge rack were obtained from Varian Instruments. The silica cartridges are washed in order with 5 ml HPLC-grade methanol, 5 ml HPLC-grade isopropanol, and 5 ml HPLC-grade hexane. The sample, in 1 ml of 1.5% isopropanol in hexane, is then applied to the cartridge and eluted through the cartridge under vacuum into waste. This initial elution is followed by 4 ml of 1.5% isopropanol in hexane (discard) and 6 ml of 5% isopropanol in hexane (25(OH)D). The 6-ml fraction contains 25(OH)D2 and 25(OH)D3 and is subsequently dried in a heated water bath, 55 C, under N2. The elution profile of 25(OH)D3 from the silica cartridge is displayed in Figure 47.3. The silica cartridges can be cleaned and reused many times. Quantitative Normal-phase Liquid Chromatography
High-performance
The final quantitative step is performed using normal-phase HPLC with a 0.4 25 cm Zorbax-Sil column packed with 5 mm spherical silica. The mobile phase is composed of hexaneemethylene chloride eisopropanol (50 : 50 : 2.5, v/v) at a flow rate of 2 ml/ min. The sample residue from the silica cartridge is dissolved in 150 ml of mobile phase and injected onto the HPLC column previously calibrated with varying amounts of 25(OH)D2 and 25(OH)D3 (1e100 ng). This HPLC system provides clear resolution of 25(OH)D2 and 25(OH)D3 (Fig. 47.6A). After elution, final quantitation of 25(OH)D2 and 25(OH)D3 is by direct UV monitoring at 265 nm. The 25(OH)D3 portion is collected, dried under N2, and subjected to liquid scintillation counting in order to determine the final endogenous recovery of 25(OH)D2 and 25(OH)D3 from the sample. Calculations are then performed, and the results reported as nanograms 25(OH)D2 and/or 25(OH)D3
VI. DIAGNOSIS AND MANAGEMENT
829
DETECTION OF 25(OH)D
(A) 0.004
25OHD2
25OHD3
0.5 ml Sample or control + 3H-25OHD3
0.003
Incubate 15 min room temp 0.002
Solid-phase extract using C18 Sep-Pak 0.001
Dry acetonitrile under N2 and resuspend in methylene chloride
0
Optical density 265 nm
(B) 0.004 0.003
24,25(OH)2D2 24,25(OH)2D2
Apply to silica cartridge and collect 25OHD-containing fraction
0.002
Dry fraction under N2 and resuspend in normal-phase HPLC mobile phase
0.001
Apply to normal-phase silica HPLC and quantitate 25OHD2 and 25OHD3 by direct UV absorption. Collect 25OHD3 containing fraction and monitor for 3H-content for recovery correction
0
(C) 0.004 0.003
1,25(OH)2D2 1,25(OH)2D3
FIGURE 47.7 Flow diagram of the HPLC-UV assay for the quantitation of 25(OH)D2 and 25(OH)D3.
0.002
0.001
0 2
4
6 8 10 Elution time (min)
12
14
FIGURE 47.6 High-performance liquid chromatographic profiles of standard vitamin D metabolites. (A) 25(OH)D2 and 25(OH)D3; (B) 24,25(OH)2D2 and 24,25(OH)2D3; (C) 1,25(OH)2D2 and 1,25-(OH)2D3. The HPLC was performed on a normal-phase Zorbax-Sil column, and column calibration was achieved by injecting 10 ng of each compound and monitoring optical density at 265 nm. Column conditions are described in the text.
per milliliter. A flow diagram of the entire procedure is displayed in Figure 47.7.
RIA Methodology Preparation of Assay Calibrators One of the goals of the RIA procedure for 25(OH)D was to eliminate the need for individual sample recovery. Another goal was to obtain FDA approval for
clinical use of this procedure in the United States. Both of these goals place 25(OH)D3 in a human serum-based set of assay calibrators. To prepare these calibrators, human serum was “stripped” free of vitamin D metabolites by treatment with activated charcoal. Absence of endogenous 25(OH)D in the stripped sera was confirmed by direct UV detection of 25(OH)D in serum following HPLC. Subsequently, crystalline 25(OH)D3 dissolved in absolute ethanol was added to the stripped sera to yield calibrators at concentrations of 0, 5, 12, 40, 100 ng/ml. Sample and Calibrator Extraction To extract 25(OH)D from calibrators and samples, 0.5 ml of acetonitrile is placed into a 12 75 mm borosilicate glass tube after which 50 ml of sample or calibrator is dropped through the acetonitrile. After vortex-mixing, the tubes are centrifuged (1000 g, 4 C, 5 min) and 25 ml of supernatant transferred to 12 75 mm borosilicate glass tubes and placed on ice. Radioimmunoassay The assay tubes are 12 75 mm borosilicate glass tubes containing 25 ml of acetonitrile-extracted
VI. DIAGNOSIS AND MANAGEMENT
830
47. DETECTION OF VITAMIN D AND ITS MAJOR METABOLITES
calibrators or samples. To each tube add 125I-25(OH)D derivative (50 000 cpm in 50 ml 1 : 1 ethanole10 mM phosphate buffer, pH 7.4) that was synthesized as previously described [14]. Then added to each tube 1.0 ml of primary antibody diluted 1 : 15 000 in sodium phosphate buffer (50 mM, pH 7.4, containing 0.1% swine skin gelatin). Nonspecific binding is estimated using the above buffer minus the antibody. Vortex-mix the contents of the tubes and incubate them for 90 min at 20e25 C. Following this period, add 0.5 ml of a second antibody precipitating complex to each tube, vortex-mix, incubate at 20e25 C for 20 min, and centrifuge (20 C, 2000 g, 20 min). Discard the supernatant and bound the tubes in a gamma well counting system. 25(OH)D values are calculated directly from the standard curve by the counting system using a smooth-spline method of calculation. The entire 25(OH)D RIA procedure is displayed in Figure 47.8.
50 l Sample, standard or control
500 l Acetonitrile, 10 min spin
25 l Extract + 50 l tracer+ 1.0 ml primary antibody
Comments on the 25(OH)D RIA This 125I-based RIA is similar to an RIA we introduced several years ago that used 3H-25(OH)D3 as a tracer [13]. In both cases, antisera were raised against the synthetic vitamin D analog 23,24,25,26,27-pentanor vitamin D-C (22)-carboxylic acid. The syntheses of this analog and its 125I-labeled counterpart have been described in detail [13,14]. Coupling this compound to bovine serum albumin allowed us to generate antibodies that crossreacted equally with most vitamin D2 and D3 metabolites (Table 47.3). The structures for vitamins D2 and D3 differ only with respect to their side chains (Fig. 47.1). Because the analog retained the intact structure of vitamin D only up to carbon 22, the structural differences between vitamins D2 and D3 were not involved in the antibody recognition, and antibodies directed against this analog could not discriminate with respect to side chain metabolism of vitamin D. The antibody, however, was specific for the open B-ring cis-triene structure containing a 3b-hydroxyl group that is inherent in all vitamin D compounds. As of this publication, this antibody is the “only” totally cospecific antibody for detection of “total 25(OH)D,” 25(OH) D2 and 25(OH)D3. Many vitamin D metabolites other than 25(OH)D are present in the circulation; however, they contribute only a small percentage (6e7%) to the overall assessment of nutritional vitamin D status as compared with 25(OH) D [25]. This fact is supported by the comparison of the 25(OH)D described earlier on a variety of human serum samples (Fig. 47.9). TABLE 47.3 Cross-reactivity of Various Vitamin D Compounds with 25(OH)D Antiserum and 125I-labeled Vitamin D Derivativea
90 min Incubation at room temperature
+ 0.5 ml Precipitating complex
20 min Incubation at room temperature 20 min spin
Decant and count
Steroid
Cross-reactivity (%)b
Vitamin D2
0.8
Vitamin D3
0.8
DHT
<0.1
25(OH)D2
100
25(OH)D3
100
25(OH)D3-26,23-lactone
100
24,25(OH)2D2
100
24,25(OH)2D3
100
25,26(OH)2D2
100
25,26(OH)2D3
100
1,25(OH)2D2
2.5
1,25(OH)2D3
2.5
a
FIGURE 47.8 Flow diagram of the direct RIA or the quantitation of 25(OH)D.
From Hollis et al. Clin Chem 39:529e533. Ability to displace 50% of the 125I tracer from the 25(OH)D antiserum diluted 15 000fold.
b
VI. DIAGNOSIS AND MANAGEMENT
DETECTION OF 25(OH)D
RIA 25OHD determination (ng/ml)
200
RIA = 0.98(HPLC) + 0.01 r2 = 0.98 n = 63
150
100
50
0 0
50 100 150 HPLC 25OHD determination (ng/ml)
200
FIGURE 47.9 25(OH)D values obtained by the 25(OH)D RIA
(y axis) and by direct UV quantitation of 25(OH)D following HPLC (x axis).
Automated Instrumentation CLIA Methodology DiaSorin Corporation, Roche Diagnostics, and the now defunct Nichols Institute Diagnostics all introduced methods for the direct (no extraction) quantitative determination of 25(OH)D in serum or plasma using competitive protein assay chemiluminescence technology [16]. These assays appear quite similar on the surface but they are not. In 2001, Nichols Diagnostics introduced the fully automated chemiluminescence Advantage 25(OH)D assay system. In this assay system, nonextracted serum or plasma was added directly into a mixture containing human DBP, acridinium-ester labeled anti-DBP, and 25 (OH)D3-coated magnetic particles. Note that the primary binding agent was human DBP. Thus, this assay was a CPBA, much like the manual procedure introduced in 1974 by Belsey et al. [24]. The major difference between these procedures was that Belsey depotenized the sample with ethanol before assaying it. The calibrators for the Belsey assay were in ethanol. In the Advantage assay, the calibrators were in a serum-based matrix, and its developers assumed that this matrix would replicate the serum or plasma sample introduced directly into the assay system. In the end, the 1974 Belsey assay never worked and neither did the Advantage 25(OH)D assay. The company removed the assay from the market in 2006. In 2004, the DiaSorin Corporation introduced the fully automated chemiluminescence Liaison 25(OH)D Assay System [16]. This assay is very similar to the
831
late Advantage assay, with one major difference e the Liaison assay uses an antibody as a primary-binding agent as opposed to the human DBP in the Advantage system. Thus, the Liaison is a true RIA method. Details on this procedure are available elsewhere [16]. The Liaison 25(OH)D assay is cospecific for 25(OH)D2 and 25(OH)D3, so it reports a “total” 25(OH)D concentration. DiaSorin recently introduced a third-generation Liaison 25(OH)D assay. This new version has increased functional sensitivity and much improved assay precision. The Liaison 25(OH)D assay is the single most widely used 25(OH)D assay in the world for clinical diagnosis. The most recent addition to the automated 25(OH)D assay platforms is from Roche Diagnostics. Their test is an RIA called vitamin D3(25-OH) and it can be performed on their Elecsys and Cobas systems. Roche only released this assay in 2007, so very little information on it is available. However, the assay can only detect 25(OH)D3, so it will not be a viable product in countries in which vitamin D2 is used clinically, including the USA.
Liquid Chromatography-Mass Spectroscopy Researchers have recently revitalized LC/MS as a viable method to assess circulating 25(OH)D [17,26e28]. As with HPLC, LC/MS quantitates 25(OH) D2 and 25(OH)D3 separately. When performed properly, LC/MS is a very accurate testing method. However, the equipment is very expensive and its overall sample throughput cannot, when performed properly, match that of the automated instrumentation format. As a methodology, LC/MS can compare favorably with RIA techniques [26e28]. One unique problem with LC/MS is its relative inability to discriminate between 25(OH)D3 and its inactive isomer 3-epi-25(OH)D3. This problem has been especially noticeable in the circulation of newborn infants [17]. Next to the RIAs and ClIAs, LC/MS is the next most utilized procedure for the assessment of circulating 25(OH)D. However, it must be highlighted that while 25(OH)D RIAs and CLIAs are FDA-cleared devices, all LC/MS assays remain “home brew” methods in the United States. Because of this there are essentially no standards to establishing a 25(OH)D LC/MS assay. As a result, LC/MS assays for 25(OH)D can be as complex and rigorous as the one developed as a reference method by the National Institute of Standards and Technology (NIST) to the “crash and fire” methods used by reference labs that use LC/MS assays for clinical diagnostic purpose. The NIST method basically entails adding deuterated 25 (OH)D2 and 25(OH)D3 to a serum or plasma sample followed by equilibration, liquideliquid extraction of the sample with hexane ethyl acetate, drying the hexane,
VI. DIAGNOSIS AND MANAGEMENT
832
47. DETECTION OF VITAMIN D AND ITS MAJOR METABOLITES
drying the sample and resuspending in mobile phase followed by injecting into the LC/MS followed by a 30-minute run time to quantitate 25(OH)D2 and 25 (OH)D3 mass ions by MS [87]. This type of method is very accurate and analytically clean but will only allow about 80 samples in a day which is quite similar to a well-performed HPLC 25(OH)D assay. Compare this to a “crash and fire” method which basically is taking a serum or plasma sample, adding acetonitrile to “crash” the proteins and then injecting the supernate directly into the LC/MS which generates a “result” every 2e3 minutes. Before being acceptable as a clinical diagnostic tool, this type of procedure for 25(OH)D should fall under FDA regulation.
DETECTION OF 24,25(OH)2D Background Next to 25(OH)D, 24,25(OH)2D is quantitatively the most abundant circulating vitamin D metabolite, and, as a result, interest in its circulating levels has persisted. However, to this day, the biological function(s) of 24,25 (OH)2D3, if any, remains unresolved. The first assay for 24,25(OH)2D was first reported in 1977 and used CPBA in conjunction with sample prepurification on Sephadex LH-20 [8] (Table 47.4). However, it was soon discovered that more extensive sample prepurification was required prior to 24,25(OH)2D quantitation owing to substances that interfered in the 24,25(OH)2D CPBA [29]. To further complicate matters, the quantitation of 24,25(OH)2D is especially difficult when both the vitamin D2 and D3 forms are present in the circulation [18]. When both forms of 24,25(OH)2D are present, it is extremely hard to remove other vitamin D metabolites that coelute with 24,25(OH)2D2 and 24,25(OH)2D3 on HPLC separation [18]. Further, once 24,25(OH)2D2 and 24,25(OH)2D3 are adequately separated and ready for CPBA, varying affinities of the two metabolites for the TABLE 47.4
DBP require standard curves to be constructed for final quantitation [18]. A report published in 1994 questions the requirement of HPLC prepurification of the serum sample prior to CPBA [31]. However, we are firm believers that in order to perform a valid assay for 24,25(OH)2D, one has to incorporate HPLC prepurification into the assay protocol. We have also chosen to do the final quantitation of 24,25(OH)2D by RIA instead of CPBA. The RIA was chosen because the antibody used is cospecific for the vitamin D2 and D3 forms, and thus only one compound, 24,25(OH)2D3, is required to construct the standard curve (Table 47.3). This procedure is described here in detail.
Methodology Sample Extraction A 0.5-ml sample of serum or plasma is placed into a 12 75 mm borosilicate glass culture tube containing 1000 cpm of 3H-24,25(OH)2D3 in 25 ml of ethanol to monitor recovery of endogenous compound through the extraction and chromatographic procedures. Following a 15-min incubation with the tracer, 1 plasma volume of HPLC-grade acetonitrile is added to each sample. The sample is then vortex-mixed for 1 min followed by centrifugation at 1000 g for 10 min. The supernatant is removed into another 12 75 mm culture tube, and 1 plasma volume of distilled water is added. Solid-phase Extraction Chromatography C18 silica Bond-Elut cartridges (500 mg) and a VacElut cartridge rack were obtained from Varian Instruments. The C18 cartridges are washed in order with 5 ml HPLC-grade isopropanol and 5 ml HPLC-grade methanol. The sample is then applied to the cartridge and eluted through the cartridge under vacuum into waste. This initial step is followed by 5 ml of 40% water in methanol (discard), 5 ml of 1% methylene chloride in
Significant Methods for the Estimation of 24,25(OH)2D in Human Serum
Detection method
Extraction
Preliminary chromatography
Ref.
Normal circulating levelsa
CPBA
Methanol-methylene chloride
Sephadex LH-20
Haddad et al. [8]
3.7 0.2 ng/ml
CPBA
Methanol-methylene chloride
Sephadex LH-20, preparative HPLC
Shepard et al. [29]
3.5 1.4 ng/ml
HPLC
Methanol-methylene chloride
Sephadex LH-20, preparative HPLC
Dreyer and Goodman [30]
2.4 1.1 ng/ml
RIA
Methanol-methylene chloride
Sephadex LH-20, preparative HPLC
Hummer and Christiansen [31]
0.1e4.0 ng/ml
CPBA
Solid phase C18OH
Silica cartridges
Wei et al. [32]
3.1 0.7 ng/ml
ng/ml 2.4 ¼ nmol/liter.
a
VI. DIAGNOSIS AND MANAGEMENT
833
DETECTION OF 1,25(OH)2D
hexane (discard), and 5 ml of 5% isopropanol in hexane (24,25(OH)2D). The final fraction is dried in a heated water bath, 55 C, under N2. The lipid residue is then resuspended in 150 ml 5% isopropanol in hexane and capped. Preparative Normal-phase High-performance Liquid Chromatography The normal-phase HPLC step is performed using a 0.4 25 cm Zorbax-Sil column packed with 5 mm silica. The mobile phase is composed of hexaneemethylene chlorideeisopropanol (80 : 15 : 3.5, v/v) at a flow rate of 2 ml/min. The sample residue from the C18 silica cartridge is injected onto the HPLC column that had been previously calibrated with 10 ng of 24,25(OH)2D2 and 24,25(OH)2D3. The elution of these metabolites can be seen in Figure 47.6B. The fractions containing 24,25 (OH)2D2 and 24,25(OH)2D3 are collected individually in 12 75 mm glass tubes and dried under N2 at 55 C. The residue is then redissolved in 500 ml absolute ethanol and capped.
0.5 ml Sample or control +3H-24,25(OH)2D3 Incubate 15 min room temp Isolate 24,25(OH)2D by simultaneous extraction and purification using C18support in "phase-switching" mode Dry organics under N2 and resuspend in normalphase HPLC mobile phase Apply to normal-phase silica HPLC and individually collect 24,25(OH)2D3 and 24,25(OH)2D2 Remove a portion for recovery estimation
Quantitate 24,25(OH)2D2 and 24,25(OH)2D3 individually by RIA using antibody cospecific for each compound. 3H or 125I tracers are available for use
Radioimmunoassay The assay tubes are 12 75 mm borosilicate glass tubes containing 25 ml of the HPLC-purified extracts in ethanol. The standards for this RIA, which are 24,25 (OH)2D3 are placed in 12 75 mm tubes in 25 ml ethanol at concentrations between 0 and 200 pg/tube. To each tube add 125I-25(OH)D derivative (50 000 cpm in 50 ml 1 : 1 ethanole10 mM phosphate buffer, pH 7.4) or 3H25(OH)D3 (5000 cpm in 25 ml ethanol). Then add to each tube 1.0 ml of primary antibody diluted 1 : 15 000 in sodium phosphate buffer (50 mM, pH 7.4, containing 0.1% swine skin gelatin). Nonspecific binding is estimated using the above buffer minus the antibody. Vortex-mix the contents of the tubes and incubate them for 90 min at 20e25 C. Following this period, add 0.5 ml of a second antibody precipitating complex to each tube if 125I tracer was used, 0.2 ml of 0.1 M borate buffer containing 1.0% norit A charcoal and 0.1% dextran T-70 if 3H-25(OH)D3 was used, vortex-mix, incubate at 20e25 C for 20 min and centrifuge (20 C, 2000 g, 20 min). In the case of 125I tracer, discard the supernatant and count the tubes in a gamma well counting system. In the case of 3H-25(OH)D3, remove the supernatant into vials, add scintillation fluid, and monitor for radioactive content in a scintillation counter. 24,25(OH)2D values are calculated from the standard curve in picograms per tube. To convert this value to nanograms per milliliter, correct for dilution used as well as final recovery of 3H-24,25(OH)2D3 added at the beginning of the sample extraction procedure. The entire 24,25(OH)2D RIA procedure is displayed in Figure 47.10.
Dry organics under N2, and resuspend each fraction in ethanol
Flow diagram of the HPLC-RIA assay for the quantitation of 24,25(OH)2D2 and 24,25(OH)2D3.
FIGURE 47.10
DETECTION OF 1,25(OH)2D Background Of all the steroid hormones, 1,25(OH)2D represented the most difficult challenge to the analytical biochemist with respect to quantitation. 1,25(OH)2D circulates at low picogram per milliliter concentrations (too low for direct UV quantitation), is highly lipophilic, and is relatively unstable, and its precursor, 25(OH)D, circulates at concentrations in excess of 103 to 104 times that of 1,25 (OH)2D. The first RRA for 1,25(OH)2D was introduced in 1974 [9] (Table 47.5). Although this initial assay was extremely cumbersome, it did provide invaluable information with respect to vitamin D homeostasis. This initial RRA required a 20-ml serum sample, which was extracted using Bligh-Dyer organic extraction. The extract had to be purified by three successive laborious chromatographic systems (there was no HPLC at the time), and chickens had to be sacrificed and vitamin D receptor (VDR) harvested from their intestines at the time of the RRA. By 1976, the volume requirement for this RRA had been reduced to a 5-ml sample and sample prepurification had been modified to include HPLC [33].
VI. DIAGNOSIS AND MANAGEMENT
834 TABLE 47.5
47. DETECTION OF VITAMIN D AND ITS MAJOR METABOLITES
Significant Methods for the Estimation of 1,25(OH)2D in Human Serum
Detection method
Extraction
Preliminary chromatography
Ref.
Normal circulating levelsa
RRA
Methanol-chloroform
Silicic acid, Sephadex LH-20, celite
Brumbaugh et al. [9]
39.8 pg/ml
RRA
Methanol-methylene chloride
Sephadex LH-20, preparative HPLC
Eisman et al. [33]
29.2 pg/ml
RIA
Methanol-chloroform
Sephadex LH-20, preparative HPLC
Clemens et al. [12]
35 pg/ml
RRA
Solid phase C18OH
Silica cartridge
Reinhardt et al. [34]
37.4 2.2 pg/ml
RRA
Solid phase C18OH
None
Hollis [36]
28.2 11.3 pg/ml
RIA
Solid phase C18OH/silica
None
Hollis et al. [15]
32.2 8.5 pg/ml
pg/ml 2.4 ¼ pmol/liter.
a
However, the sample still had to be extracted using a modified Bligh-Dyer extraction, and then prepurified on Sephadex LH-20, and chicken intestinal VDR was still utilized as a binding agent. In 1978, the first RIA for 1,25(OH)2D was introduced [12]. Although it was an advantage not to have to isolate the intestinal VDR as a binding agent, this RIA was relatively nonspecific, so the cumbersome sample preparative steps were still required. Because of the extreme technical nature of these assays, and the cost of HPLC systems, few laboratories could afford to measure circulating 1,25 (OH)2D. Further, because these early techniques were so cumbersome, commercial laboratories did not offer 1,25(OH)2D determinations as a clinical service. This all changed in 1984 with the introduction of a radically new concept for the determination of circulating 1,25(OH)2D [34]. This new RRA utilized solidphase extraction of 1,25(OH)2D from serum along with silica cartridge purification of 1,25(OH)2D. As a result, the need for HPLC sample prepurification was eliminated. Also, this assay utilized VDR isolated from calf thymus, which proved to be quite stable and thus had to be prepared only periodically. Further, the volume requirement was reduced to 1 ml of serum or plasma. This assay opened the way for any laboratory to measure circulating 1,25(OH)2D. This procedure also resulted in the production of the first commercial kit for 1,25(OH)2D measurement. This RRA was further simplified in 1986 by decreasing the required chromatographic purification steps [35]. This purification procedure is still widely utilized today and recently has been identified as a citation classic [36]. Through the mid-1990s, no new advances were reported with respect to the quantitation of circulating 1,25(OH)2D. As good as the calf thymus RRA for 1,25(OH)2D was, it still possessed two serious shortcomings. First, VDR had to be isolated from thymus glands, which was still a difficult technique. Second, because the VDR is so specific for its ligand, only 3H-1,25(OH)2D3 could be
used as a tracer, eliminating the possibility of using a 125I-based tracer. This is a major handicap, especially for the commercial laboratory. As a result, we have developed and reported in 1996 the first significant advance in 1,25(OH)2D quantification in a decade [15]. This RIA incorporates a 125I-tracer, as well as standards in an equivalent serum matrix, so individual sample recoveries are no longer required. We describe this RIA for 1,25(OH)2D along with the standard RRA.
RRA Methodology Preparation of Calf Thymus VDR Frozen or fresh tissue is processed for VDR as follows (all steps are carried out at 4 C). Thymus tissue is minced with a meat grinder and homogenized (20% w/v) in a buffer containing 50 mM K2 HPO4, 5 mM dithiothreitol, 1 mM EDTA, and 400 mM KCl, pH 7.5. The tissue is homogenized using five, 30-s bursts of a Polytron PT-20 tissue disrupter at a maximum power setting. The homogenate is then centrifuged for 15 min at 20 000 g to remove large particles. The resulting supernatant is centrifuged at 100 000 g for 1 h, and the “cytosol” (actually a high-salt extract that includes nucleus VDR) is collected minus the floating lipid layer. The VDR is then precipitated by the slow addition of solid (NH4)2 SO4 to 35% saturation. The cytosole(NH4)2 SO4 mixture is stirred for 30 min while maintaining the temperature at 4 C. The mixture is then divided into 15-ml centrifuge tubes and centrifuged at 20 000 g for 20 min. The supernatant is discarded, and tubes are allowed to drain for 5 min. The precipitated VDR is lyophilized and stored under inert gas at e70 C. VDR prepared in this manner is stable for up to 60 h at room temperature. Sample Extraction A 1.0-ml sample of serum or plasma is placed into a 12 75 mm borosilicate glass culture tube containing
VI. DIAGNOSIS AND MANAGEMENT
835
DETECTION OF 1,25(OH)2D
700 cpm of 3H-1,25(OH)2D3 in 25 ml of ethanol to monitor recovery of endogenous compound through the extraction and chromatographic procedures. Following a 15min incubation with the tracer, 1 ml of HPLC-grade acetonitrile is added to each sample. The sample is then vortex-mixed for 1 min, followed by centrifugation at 1000 g for 10 min. The supernatant is removed into another 12 75 mm culture tube, and 1 volume of 0.4 M K2 HPO4, pH 10.4, is added followed by vortex-mixing. Solid-phase Extraction Chromatography
and
Purification
C18 OH silica Bond-Elut cartridges (500 mg) and a Vac-Elut cartridge rack were obtained from Varian Instruments. The C18 OH cartridges are washed in order with 5 ml HPLC-grade methylene chloride, 5 ml HPLCgrade isopropanol, and 5 ml HPLC-grade methanol. The sample is applied to the cartridge and eluted through the cartridge under vacuum into waste. This initial step is followed by 5 ml of 30% water in methanol (discard), 5 ml of 10% methylene chloride in hexane (discard), 5 ml of 1% isopropanol in hexane (discard), and 5 ml of 3% isopropanol in hexane (1,25(OH)2D) (Fig. 47.11). This final fraction is dried in a heated water bath, 55 C, under N2. The residue is then suspended in 200 ml absolute ethanol and capped. Radioreceptor Assay Prior to assay, the VDR-containing pellet is reconstituted to its original volume with assay buffer. The assay buffer contains 50 mM K2 HPO4, 5 mM dithiothreitol, 1.0 mM EDTA, and 150 mM KCl at pH 7.5. The receptor H2O
100
3H-25(OH)D
80 60 Percent of total radioactivity
CH3 OH:H2O
100%
70:30
pellet is dissolved by gentle stirring on ice using a magnetic stir bar. The receptor solution is allowed to mix for 20e30 min. Typically a small portion of the pellet resists solubilization and is removed by centrifugation at 3000 g for 10 min. The receptor solution is then diluted 1 : 3e1 : 9 with assay buffer and kept on ice until use. The correct dilution of receptor used in the assay is determined for each new batch of receptor. At the appropriate dilution for assay use, specific binding in the absence of unlabeled 1,25(OH)2D is 1600e2000 cpm; nonspecific binding is 200e300 cpm. These results assume a specific activity of 130 Ci/mmol for 3H-1,25 (OH)2D3 and a 40% counting efficiency for tritium. The assay tubes are 12 75 mm borosilicate glass tubes containing 50 ml of C18 OH-purified extracts in ethanol. The standards for the assay, 1,25(OH)2D3, are placed in 12 75 mm tubes in 50 ml ethanol at concentrations between 1 and 15 pg/tube. Nonspecific binding is estimated by adding 1 ng/tube of 1,25(OH)2D3. To each tube add 0.5 ml of reconstituted thymus cytosol, vortexmix, and incubate for 1 h at 15e20 C. Following this initial incubation, each tube receives 3H-1,25(OH)2D3 (5000 cpm in 50 ml ethanol) and the incubation proceeds for an additional 1 h at 15e20 C. Finally, place the assay tubes in an ice bath and add 0.2 ml of 0.1 M borate buffer containing 1.0% norit A charcoal and 0.1% dextran T-70, vortex-mix, incubate 20 min, and centrifuge (4 C, 2000 g, 10 min). Remove the supernatant into vials, add scintillation fluid, and monitor for radioactive content in a scintillation counter. 1,25(OH)2D values are calculated from the standard curve in picograms per tube. To convert this value to picograms per milliliter, correct for dilution Hexane: CH2CI2
Hexane: Isopropanol
90:10
99:1
97:3
3
40 20 100
3H-24,25(OH)
2D3
80 60 40 20 100
3H-1,25(OH)
2D3
2
4
80 60 40 20 0
FIGURE 47.11
6
8
10 12 14 Elution volume (ml)
16
18
20
22
24
Elution of 3H-vitamin D3 and its metabolites from a C18 OH Bond-Elut cartridge. From Hollis BW, Clin Chem 31:1815e1819.
VI. DIAGNOSIS AND MANAGEMENT
836
47. DETECTION OF VITAMIN D AND ITS MAJOR METABOLITES
used, as well as final recovery of 3H-1,25(OH)2D3 added at the beginning of the sample extraction procedure. The 1,25(OH)2D RRA procedure is displayed in Figure 47.12.
10 min. The supernatant is removed into another 12 75 mm culture tube, and 1 volume of 0.4 M K2 HPO4, pH 10.4, is added followed by vortex-mixing.
RIA Methodology
Solid-phase Extraction and Silica Purification Chromatography
Preparation of Assay Calibrators As was described for the 25(OH)D RIA, one of the goals of this RIA procedure was to eliminate the need for individual sample recovery. To prepare the assay calibrators, human serum was stripped free of vitamin D metabolites. The absence of endogenous 1,25(OH)2D in the stripped sera was confirmed by RRA for 1,25 (OH)2D as described above. Subsequently, crystalline 1,25(OH)2D3 dissolved in absolute ethanol was added to the stripped sera to yield calibrators at concentrations of 0, 5, 15, 30, 75, and 200 pg/ml. Sample and Calibrator Extraction and Pretreatment The 1,25(OH)2D is extracted from calibrators and samples as follows. First, 0.5 ml of serum of plasma is placed into a 12 75 mm borosilicate glass culture tube; 0.5 ml of HPLC-grade acetonitrile is added and vortexmixed for 1 min followed by centrifugation at 1000 g for 1.0 ml Sample or control + 3H-1,25(OH)2D3 1.0 ml Acetonitrile, 10 min spin Combine supernatant with 1 vol K2 HPO4, pH 10.4
Isolate 1,25(OH)2D by simultaneous extraction and purification Dry organics under N2 and resuspend in ethanol 50 l Extract + 500 l thymus receptor preparation 50 l for recovery estimation
1 hr Incubation at 15–20°C
+ 50 l 3H-1,25(OH)2D3 1 hr Incubation at 15–20°C + 200 l Dextran-charcoal solution
Decant and count
FIGURE 47.12
1,25(OH)2D.
20 min Incubation at room temp + 10 min spin
Flow diagram of the RRA for the quantitation of
C18 OH silica “Extra Clean” cartridges (500 mg) and a Vac-Elut cartridge rack were obtained from DiaSorin Corp and Varian Instruments, respectively. The C18 OHEC cartridges are washed in order with 5 ml HPCL-grade methylene chloride, 5 ml HPLC-grade isopropanol, and 5 ml HPLC-grade methanol. The sample is applied to the cartridge and eluted through the cartridge under vacuum into waste. This initial step is followed by 5 ml of 30% water in methanol (discard), 5 ml of 10% methylene chloride in hexane (discard), 3 ml of 1% isopropanol in hexane (discard), and 3 ml of 8% isopropanol in hexane (1,25(OH)2D). This final fraction is dried in a heated water bath, 55 C under N2 or in a rapid vacuum device such as a Labconco Rapid-Vap. The residue is first reconstituted with 50 ml of 95% ethanol with vortex-mixing. Each tube now receives 125 ml of I125-tracer solution with additional vortex-mixing. The sample may be capped and stored at e20 C or one may proceed to finish the assay at this point. Radioimmunoassay The assay tubes are 12 75 mm borosilicate glass tubes containing 75 ml of the ethanol/tracer-reconstituted extracted calibrators or samples. Then add to each tube 0.35 ml of primary antibody diluted 1 : 40 000 in sodium phosphate buffer (50 mM, pH 6.2, containing 0.1% swine-skin gelatin and 0.35% polyvinyl alcohol (Mr 13 000e23 000)). Nonspecific binding is estimated by using the above buffer without the antibody. Vortex-mix the contents of the tubes, incubate them for 2 h at 20e25 C, add 0.5 ml of second antibody precipitating complex, incubate at 20e25 C for 20 min, and then centrifuge (20 C, 2000 g, 20 min). Discard the supernatant and count the tubes in a gamma well counting system. 1,25 (OH)2D values are calculated directly from standard curve by the counting system using a smooth-spline method of calculation. The entire 1,25(OH)2D RIA procedure is displayed in Figure 47.13. Comments on the 1,25(OH)2D RIA Of the procedures developed for determining 1,25 (OH)2D status in humans, only a few RRA [34,35] have been able to quantify circulating 1,25(OH)2D without using HPLC for sample prepurification. Many RIA have been published and validated for the quantification of 1,25(OH)2D, but all have included HPLC steps for sample prepurification [12,37,38]. Development of an RIA for quantification of circulating 1,25(OH)2D has
VI. DIAGNOSIS AND MANAGEMENT
837
DETECTION OF 1,25(OH)2D
TABLE 47.6 Cross-reactivity of Various Vitamin D Compounds with 1,25(OH)2D Antiserum and 125I-labeled 1a-Hydroxylated Tracer
500 l Sample, standard or control 500 l Acetonitrile, 10 min spin
Combine supernatant with 1 vol K2PO4, pH 10.4
Steroid
Cross-reactivity (%)a
Vitamin D3
<0.001
25(OH)D3
0.002
24,25(OH)M2D3
0.012
25,26(OH)2D3
0.003
1,25(OH)2D2
100
1,25(OH)2D3
100
a
Ability to displace 50% of the 125I tracer from the 1,25(OH)2D antiserum diluted 1 : 40 000.
Isolate 1,25(OH)2D by simultaneous extraction and purification
Dry organics under N2 and resuspend in ethanol
75 l Extract/tracer + 300 l primary antibody
2 hr Incubation at room temp
Circulating 1,25(OH) 2D (pg/ml)
90 80
RIA RRA
70 60 50 40 30 20 10
+ 500 l Precipitating complex
0
20 min Incubation at room temp + 20 min spin
Normal subjects
Chronic renal failure
Hypoparathyroid
Biliary atresia
Pregnant subjects
Comparison of circulating 1,25(OH)2D measured by RIA and RRA. From Hollis et al. Clin Chem 42 :586e592.
FIGURE 47.14
Decant and count
FIGURE 47.13
1,25(OH)2D.
Flow diagram of the RIA for the quantitation of
been hampered from the beginning by the relatively poor specificity of the antibodies that have been generated. To date, the best antibodies toward 1,25(OH)2D have, at best, a cross-reactivity with the non-1-hydroxylated metabolites of vitamin D of approximately 1%. In comparison, the VDR used in the RRA has a crossreactivity of approximately 0.01% with these more abundant metabolites [34]. Given that the non-1-hydroxylated metabolites circulate at concentrations over 1000 times greater than that of 1,25(OH)2D, the magnitude of the problem becomes clear. However, the VDR is so specific that any attempt to introduce a radionuclide such as 125I into 1,25(OH)2D3 erodes the binding between this steroid hormone and the VDR. Therefore,
if one wishes to develop 125I-based assays to quantify 1,25(OH)2D, RIA is the only choice. Since the original paper on this assay was published [15], several improvements have been implemented. The major improvement involved the generation of a new antibody. This recent antibody has greater sensitivity, specificity as compared to the original antibody [15]. Further, the new antibody expresses 100% crossreactivity between 1,25(OH)2D2 and 1,25(OH)2D3 (Table 47.6). This new antibody has allowed the RIA to return to a single column format for sample purification along with the removal of a sample pretreatment step involving NaIO4. This RIA was the first 1,25(OH)2D assay to receive FDA approval for clinical diagnosis in humans and thus remains the FDA’s predicate device. The concentrations of 1,25(OH)2D as determined in serum from various groups of healthy and pathological subjects (Fig. 47.14) agree well with values reported in
VI. DIAGNOSIS AND MANAGEMENT
838
47. DETECTION OF VITAMIN D AND ITS MAJOR METABOLITES
400 Circulating 1,25(OH)2D (pg/ml)
previous studies [34,35]. It is very important to include pathological samples such as those from subjects with biliary atresia and vitamin D toxicity in any assay validation procedure for circulating 1,25(OH)2D. This importance was underlined in our previous report on an unpublished, unvalidated, but commercially available 125I-based RIA for 1,25(OH)2D that involves the immunoextraction of 1,25(OH)2D from serum samples and is marketed by IDS Ltd. (Tyne and Wear, UK) [39]. The basis of this kit is selective immunoextraction of 1,25(OH)2D from serum or plasma with a specific monoclonal antibody bound to a solid support. This antibody is directed toward the 1a-hydroxylated A ring of 1,25 (OH)2D [40]. We concluded that this immunoextraction procedure was highly specific for the la-hydroxylated forms of vitamin D [39]. However, there was a serious flaw in the assumptions made when this kit was designed: 1,25(OH)2D was the only significant lahydroxylated vitamin D metabolite that circulates. Many other la-hydroxylated metabolites exist in the circulation, including 1,24,25(OH)3D3, 1,25,26(OH)3D3, l,25(OH)2D3-26,23-lactone, 1,25(OH)2-24-oxo-D3, calcitroic acid, and probably various water-soluble, sidechain conjugates. Some of these compounds are bioactive, but most are not, and this assay cannot distinguish among them (Fig. 47.15). Further, compare how the IDS RIA for 1,25(OH)2D performs outside of normal or chronic renal failure human samples (Fig. 47.16). This assay appears to be inadequate when presented with selected pathological samples. We have specifically investigated the effects of l,25(OH)2D3-26,23-lactone on the “apparent” 1,25(OH)2D
200
100
Normal Chronic Hypo-para- Biliary Calcium Vitamin D subjects renal thyroid atresia deficient deficient failure rat rat
Circulating 1,25(OH)2D as determined by the RRA (squares) or IDS immunoextraction RIA (triangles) on a variety of clinical samples. The same samples were compared in each assay. Horizontal lines denote means.
FIGURE 47.16
levels using the immunoextraction technique and found it to interfere on an equal molar basis compared with 1,25(OH)2D3 (Fig. 47.17). We also know that 1,25 (OH)2D3-26,23-lactone is a significant in vivo metabolite in a variety of clinical samples, and we find its concentration to be 0e30% of the respective 1,25 (OH)2D concentration. Further, using the RIA based on immunoextraction, we have found “apparent” levels of 1,25(OH)2D to be grossly higher than the actual concentration in vitamin-D-intoxicated subjects, hypo-parathyroid subjects receiving vitamin D therapy, and biliary atresia patients (Fig. 47.16). We have also
120
Graphic description of the vitamin D metabolites assayed as “apparent” circulating 1,25(OH)2D by utilizing immunoextraction with a 1-hydroxy-specific monoclonal antibody (MAb) in conjunction with RIA.
1,25(OH)2D assayed (pg/ml)
Removed
1) 100% 1,25(OH)2D 1) All non-1-hydroxylated metabolites 2) 100% 1,24,25(OH)3D 3) 100% 1,25,26(OH)3D 4) 100% 1,23,25(OH)3D 5) 100% 1,25(OH)2D3-26,23-lactone 6) 100% Calcitroic acid 7) 100% Water-soluble conjugates of calcitroic acid
FIGURE 47.15
300
0
Immobilized 1-OH-specific MAb
Measured
RRA IDS-RIA
100 80 60 40 20 0
0
5 10 15 20 1,25(OH)2D3-26,23-lactone added (pg/ml)
50
Effect of exogenously added l,25(OH)2D-26,23lactone on the “apparent” serum concentration of 1,25(OH)2D. Concentrations were assessed by RRA (squares), RIA as described in the text (triangles), and IDS immunoextraction RIA (diamonds). From Hollis BW. Clin Chem 41:1313e1314.
FIGURE 47.17
VI. DIAGNOSIS AND MANAGEMENT
CLINICAL INTERPRETATION AND RELEVANCE OF ANTIRACHITIC STEROL MEASUREMENTS
observed some normal samples that displayed 100% elevation from the actual levels. What this assay is recognizing in these samples remains unknown, but it is undoubtedly some 1a-hydroxylated metabolite, probably a catabolic product. It is important to note that the RIA described in this chapter, which is based on classic separation procedures, appears to escape this problem of detecting inactive 1a-hydroxylated vitamin D metabolites (Fig. 47.14).
839
levels are greatly affected by short-term sun exposure and dietary intake of vitamin D [42,43]. Vitamin D has proved to be useful in assessing intestinal lipid absorptive capacity associated with fat malabsorption syndromes [44,45]. Recently, the vitamin D assay has been utilized to better understand the control of 25 (OH)D levels in human subjects [46,47]. However, this use is more of a research application as opposed to an application used in a clinical diagnosis. Table 47.7 lists a variety of clinical conditions for which the circulating levels of vitamin D have been defined.
CLINICAL INTERPRETATION AND RELEVANCE OF ANTIRACHITIC STEROL MEASUREMENTS
25(OH)D
Vitamin D
Determining and Defining a “Normal” Circulating 25(OH)D Level
The quantitation of circulating vitamin D is essentially of no clinical importance. The parent compound is a poor indicator of nutritional status because of its short circulating half-life. The circulating levels of vitamin D are also difficult to interpret because the
To define a “normal” circulating level of a given substance or nutrient one usually obtains blood samples from a diverse population, measures the substance in question, plots the data by Gaussian distribution and determines normality. This method works
TABLE 47.7
Relative Circulating Concentrations of Vitamin D, 25(OH)D, 24,25(OH)2, and 1,25(OH)2 in Various Disease States
Condition
Vitamin Da
25(OH)Db
24,25(OH)2Dc
1,25(OH)2Dd
Nutritional deficiency
Decreased
Decreased
Normal
Increased followed by decrease
Hypoparathyroidism
Normal
Normal
Normal
Decreased
Pseudohypoparathyroidism
Normal
Normal
Normal
Decreased
Hyperparathyroidism
Normal
Normal
Normal
Decreased
Tumor-induced osteomalacia
Normal
Normal
Normal
Decreased
Vitamin D-dependent rickets, type I
Normal
Normal
Normal
Decreased
Vitamin D-dependent rickets, type II
Normal
Normal
Normal
Increased
Sarcoidosis
Normal
Normal
Normal
Increased during hypercalcemia
Renal failure
Normal or decreased
Normal or decreased
Decreased
Decreased
Nephrotic syndrome
Decreased
Decreased
Decreased
Decreased
Hypervitaminosis D
Increased
Increased
Increased
Normal or decreased
Cirrhosis
Normal or decreased
Normal or decreased
Normal or decreased
Normal or decreased
Tuberculosis
Normal
Normal
Normal
Increased during hypercalcemia
Hodgkin’s disease
Normal
Normal
Normal
Increased during hypercalcemia
Lymphoma
Normal
Normal
Normal
Increased during hypercalcemia
Wegener’s granulomatosis
Normal
Normal
Normal
Increased during hypercalcemia
X-linked hypophosphatemic rickets
Normal
Normal
Normal
Decreased or normal
a
Observed range is 0e30 ng/ml (0e78 nmol/liter) and is extremely variable with respect to sunlight exposure and dietary intake. Normal range is 32e100 ng/ml (80e250 nmol/liter) and is related to season, latitude, diet and disease. c Observed range is 0.5e4 ng/ml (1.2e9.6 nmol/liter) and is directly related to circulating 25(OH)D. d Observed range is 20e60 pg/ml (48e144 pmol/liter). b
VI. DIAGNOSIS AND MANAGEMENT
840
47. DETECTION OF VITAMIN D AND ITS MAJOR METABOLITES
well for nutrients such as folate or vitamin E and was precisely how normative circulating levels of 25(OH)D were defined in humans beginning about 40 years ago by Haddad and Chyu [6]. They sampled a population of “normal” individuals whom were asymptomatic for disease, assessed circulating 25(OH)D and determined a mean value. In their study they also assessed 25(OH)D in a group of lifeguards and demonstrated their levels to be 2.5 times that of the “normal.” Countless similar studies performed over the ensuing decades reiterated the same conclusion. However, this author interpreted the original Haddad data differently; namely that 25(OH)D levels in the lifeguards were normal and the “normals” were actually vitamin-Ddeficient [48]. This interpretation has largely been validated by the current research. For all practical purposes, vitamin D does not naturally occur in foodstuffs that humans eat. There are exceptions such as oily fish and fish liver oil [48a]. The fact is, from an evolutionary standpoint, humans did not require vitamin D in their food supply because over millions of years humans evolved a photosynthetic mechanism in their skin to produce large amounts of vitamin D3. Thus, our skin is part of the vitamin D endocrine system, and vitamin D3 is really a preprohormone. The problem now is that humans avoid the sun, wear sunscreen and reside in latitudes that we are not programmed to live in. In light of this reality this author deems the dietary requirement for vitamin D in adults is 200 IU/d, as defined by the Adequate Intake (AI) by the Food and Nutrition Board as a gross underrepresentation of the real requirement [49]. As a result of these factors, we now define a “normal” circulating 25(OH)D range using various biomarkers of physiology or disease as opposed to a random population Gaussian distribution. The first use of biomarkers to define “normal” 25(OH) D levels, of course, started with parameters that affected skeletal integrity such as parathyroid hormone, bone mineral density, and intestinal calcium absorption [50e54]. These parameters demonstrated that a minimum circulating level of 25(OH)D should be a least 32 ng/ml (80 nmol) [48e55]. Presently, the “normal” circulating 25(OH)D level also relies on data based on the other diverse physiological function of 25 (OH)D including cancer prevention [56e64], infectious disease [65e68], cardiovascular health [69e73], diabetes [74e76], and autoimmune control [77]. Because of the diverse interaction of vitamin D with our genome this list is certain to grow [78]. For the present it is generally agreed that a normal level of circulating 25(OH)D is 32e100 ng/ml (80e250 nmol). Please take note that 32 ng/ml is not an “optimum” level but a minimum “normal” level. What constitutes an “optimum” level remains to be determined and may well be different for different physiological processes.
Clinical Reporting Concentrations
of
Circulating
25(OH)D
As highlighted earlier, only 25(OH)D assays are approved by the FDA for clinical utility. Currently, the diagnostic 25(OH)D tests sold by DiaSorin (Stillwater, Minnesota, USA) and IDS Diagnostics (Fountain Hills, Arizona, USA) are under strict FDA control and monitoring for assay performance and reliability. Regardless, many clinical reference laboratories are moving to replace these FDA-approved tests with “home-brew” LC/MS methods that are diverse and not under FDA scrutiny. The reasons for this switch in utilization are the “perceived” advantages of LC/MS technology being more accurate, precise, specific, cost-effective, and providing the separate determination of 25(OH) D2 and 25(OH)D3. With respect to accuracy and precision, the DiaSorin and IDS RIA methods perform at least as well as LC/MS methods according to the Vitamin D External Quality Assessment Scheme (DEQAS) operated out of London, UK. As far as specificity goes, the RIAs and CLIAs appear more specific than LC/MS methodology in that these assays do not detect the inactive 3-epimer of 25(OH)D3 [17]. Finally, LC/MS assays are marketed on their ability to separately measure 25(OH)D2 and 25(OH)D3 in a blood sample. Expert clinicians in this area concur that there is no advantage to this separate measurement claim [78a]. In fact, this separate reporting has been shown to confuse the clinician [79]. Further, 99% of all patient samples assayed will not contain any 25(OH)D2 (personal observation). The RIA has been used to generate all of the 25(OH) D data from the third National Health and Nutrition Examination Survey (NHANES III) [57e77,80]. Studies from the huge NIH-sponsored Women’s Health Initiative (WHI) used the DiaSorin LIAISON CLIA for 25 (OH)D for the first two major publications [64,81] with others to follow. The Harvard-based studies, the Health Professionals‘ Follow-up Study (HPFS) and the Nurses’ Health Study (NHS) have been used to establish much of the information in the last decade with regard to the relationship of circulating 25(OH) D levels and various disease states such as cancer, autoimmune, cardiovascular, and renal; all of these studies again utilized RIA-based assays [57e77,80,81].
24,25(OH)2D At the present time, there does not appear to be a compelling reason to measure circulating 24,25 (OH)2D in a clinical setting. Even in a research setting, the determination of 24,25(OH)2D is of questionable value as evidenced by the decreased usage of this assay in the literature since the early 1990s.
VI. DIAGNOSIS AND MANAGEMENT
ASSESSMENT OF ASSAY PERFORMANCE
1,25(OH)2D Circulating 1,25(OH)2D is diagnostic for several clinical conditions, including vitamin-D-dependent rickets types I and II, hypercalcemia associated with sarcoidosis, and other hypercalcemic disorders causing increased 1,25(OH)2D levels. These other disorders include tuberculosis, fungal infections, Hodgkin’s disease, lymphoma, and Wegener’s granulomatosis. In all other clinical conditions involving the vitamin D endocrine system, including hypoparathyroidism, hyperparathyroidism, and chronic renal failure, the assay of 1,25(OH)2D is a confirmatory test. It is also important to remember that circulating 1,25(OH)2D provides essentially no information with respect to the patient’s nutritional vitamin D status. Thus, circulating 1,25(OH)2D should not be used as an indicator for hypo- or hypervitaminosis D when nutritional factors are suspected (Table 47.7).
STABILITY OF 25(OH)D AND 1,25(OH)2D IN SERUM OR PLASMA Researchers have known for nearly 30 years that endogenous 25(OH)D and 1,25(OH)2D are extremely stable in serum or plasma [83] and showed that vitamin D metabolites in blood stored at 24oC for up to 72 h remain intact. Recent studies on the stability of 25(OH) D in plasma or serum that has undergone many freezeethaw cycles have reported the same stability [16,84]. We have used the same pooled human 25(OH)D and 1,25(OH)2 internal controls stored at e20oC for >10 years with no detectable degradation of either compound (personal observation). The Vitamin D External Quality Assessment Scheme (DEQAS), a major vitamin D quality assessment organization, ships its serum samples used by laboratories for quality assessment by ground post worldwide without affecting 25(OH)D and 1,25(OH)2D values. We have also performed experiments to try to destroy endogenous 25 (OH)D and 1,25(OH)2D in plasma to obtain a vitaminD-free human plasma to prepare various immunoassay procedure calibrators. When crystalline 25(OH)D3 or 1,25(OH)2D3 is placed in ethanol in an open glass Petri dish and exposed the dish to intense UV light, the UV light destroyed the compounds within a few minutes. However, when a similar experiment is conducted using serum or plasma, the 25(OH)D3 and 1,25(OH)2D3 levels did not change after 2 days of UV light exposure. Why are vitamin D and its metabolites so stable in serum or plasma when they are insulted with UV light, temperature shifts, or oxidation? One reason is that UV light penetrates aqueous media very poorly. However, the main reason is probably that in serum or plasma,
841
vitamin D and its metabolites are essentially bound completely to the serum DBP and this complex resists potential insults to the vitamin D molecule very effectively. In conclusion, 25(OH)D and 1,25(OH)2D are very stable in serum or plasma, so they require only minimal attention to storage conditions.
ASSESSMENT OF ASSAY PERFORMANCE DEQAS (Internet: www.deqas.org) was founded in 1989 to compare the performance of then-available 25(OH)D tests. DEQAS has since become the largest vitamin D quality assessment program in the world, with z700 participating laboratories worldwide. The organization’s major aim today is to assess the analytic reliability of 25(OH)D and 1,25(OH)2D assays. The organization achieves this goal by: • distributing serum pools at regular intervals; • conducting statistical analyses of submitted results; • appropriately manipulating pools to provide information on assay specificity and recovery; • assigning GC-MS target values to selected 25(OH)D pools; • helping participants and manufacturers evaluate methods by providing samples, technical support, and impartial advice; • offering advice and support to participants having difficulty achieving an acceptable level of assay performance; and • providing a forum for exchanging information on all aspects of vitamin D assay methodology. My laboratory has participated in both the DEQAS 25(OH)D and DEQAS 1,25(OH)2D survey since 1997, and the survey has been invaluable in maintaining the integrity of our assay procedures. The DEQAS survey has shown that (1) most current 25(OH)D assay protocols perform in a comparable fashion with respect to absolute values, assay linearity, and assay precision and (2) the only assays that quantitatively detect total 25(OH)D are HPLC methods, LC/MS methods and the RIAs/CLIAs. In fact, when DEQAS leaders question manufacturers about inconsistencies in their methods, most manufacturers attempt to address the issue identified. One example of the value DEQAS offers occurred when DEQAS informed Nichols Institute Diagnostics that its Advantage 25(OH)D automated assay was overestimating total 25(OH)D concentrations and that, contrary to the manufacturer’s claims, the method could not detect circulating 25(OH)D2 concentrations [85]. Nichols Institute Diagnostics chose not to respond to the concerns that DEQAS identified; as a consequence Advantage 25(OH)D assay is no longer on the market. As this example shows, DEQAS provides an invaluable
VI. DIAGNOSIS AND MANAGEMENT
842
47. DETECTION OF VITAMIN D AND ITS MAJOR METABOLITES
service to the vitamin D assay community. In the future, it is hoped that DEQAS can incorporate the new National Institute of Standards and Technology 25(OH)D calibrators into its survey in some fashion. Other quality control samples and organizations exist but have proven to be unreliable for their intended purpose. Current data have demonstrated that exogenous addition or 25(OH)D2 and/or 25(OH)D3 to human serum samples will not perform properly across all assay types [86]. The same situation exists in samples that are adulterated and freeze-dried [86]. Thus the calibrators currently available from both The National Institute of Standards and Technology (NIST) and The College of American Pathologists (CHP) are not reliable for assay comparisons or calibration especially with the CLIA tests [86].
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[13] B.W. Hollis, J.L. Napoli, Improved radioimmunoassay for vitamin D and its use in assessing vitamin D status, Clin. Chem. 31 (1985) 1815e1819. [14] B.W. Hollis, J.Q. Kamerud, S.R. Selvaag, J.D. Lorenz, J.L. Napoli, Determination of vitamin D status by radioimmunoassay with an 125I-labeled tracer, Clin. Chem. 39 (1993) 529e533. [15] B.W. Hollis, J.Q. Kamerud, A. Kurkowski, J. Beaulieu, J.L. Napoli, Quantification of circulating 1,25-dihydroxyvitamin D 10. by radioimmunoassay with an 125I-labeled tracer, Clin. Chem. 42 (1996) 586e592. [16] D.L. Ersfeld, D.S. Rao, J.J. Body, et al., Analytical and clinical validation of the 25 OH vitamin D assay for the LIAISON automated analyzer, Clin. Biochem. 37 (2004) 867e874. [17] R.J. Singh, R.L. Taylor, G.S. Reddy, S.K. Grebe, C-3 epimers can account for a significant proportion of total circulating 25(OH)D in infants, complicating accurate measurement and interpretation of vitamin D status, J. Clin. Endocrinol. Metab. 91 (2006) 3055e3061. [18] R.L. Horst, E.T. Littledike, J.L. Riley, J.L. Napoli, Quantitation of vitamin D and its metabolites and their plasma concentrations in five species of animals, Anal. Biochem. 116 (1981) 189e203. [19] P.W. Lambert, P.B. DeOreo, B.W. Hollis, I.Y. Fu, D.J. Ginsberg, B.A. Roos, Concurrent measurement of plasma levels of vitamin D3 and five of its metabolites in normal humans, chronic renal failure patients, and anephric subjects, J. Lab. Clin. Med. 98 (1981) 536e548. [20] B.W. Hollis, B.A. Roos, P.W. Lambert, Vitamin D in plasma: quantitation by a nonequilibrium ligand binding assay, Steroids 37 (1981) 609e613. [21] R.L. Horst, T.A. Reinhardt, D.C. Beitz, E.T. Littledike, A sensitive competitive protein binding assay for vitamin D in plasma, Steroids 37 (1981) 581e591. [22] C.J. Rhodes, P.A. Claridge, D.J.H. Traffold, K.L.J. Makin, An evaluation of the use of Sep-Pak Qg cartridges for the extraction of vitamin D3 and some of its metabolites from plasma and urine, J. Steroid Biochem. 19 (1983) 1349e1354. [23] Y. Liel, E. Ulmer, J. Shary, B.W. Hollis, N.H. Bell, Low circulating vitamin D in obesity, Calcif. Tissue Int. 43 (1988) 199e201. [24] R.E. Belsey, H.F. DeLuca, J.T. Potts, A rapid assay for 25-OHvitamin D3 without preparative chromatography, J. Clin. Endocrinol. Metab. 38 (1974) 1046e1051. [25] B.W. Hollis, W.B. Pittard, Evaluation of the total fetomaternal vitamin D relationships at term: evidence for racial differences, J. Clin. Endocrinol. Metab. 59 (1984) 652e657. [26] Z. Maunsell, D.J. Wright, S.J. Rainbow, Routine isotope-dilution liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of the 25-hydroxy metabolites of vitamins D2 and D3, Clin. Chem. 51 (2005) 1683e1690. [27] H. Chen, L.F. McCoy, R.L. Schleicher, C.M. Pfeiffer, Measurement of 25(OH)D2 and 25(OH)D3 in human serum using liquid chromatography-tandem mass spectrometry and its comparisons to a radioimmunoassay method, Clin. Chem. Acta 391 (2008) 6e12. [28] A.K. Saenger, T.J. Laha, D.E. Bremner, M.H. Sadzadeh, Quantification of serum 25-hydroxyvitamin D2 and D3 using HPLCtandem mass spectrometry and examination of reference intervals for diagnosis of vitamin D deficiency, Am. J. Clin. Pathol. 125 (2006) 914e920. [29] R.M. Shepard, R.L. Horst, A.J. Hamstra, H.F. DeLuca, Determination of vitamin D and its metabolites in plasma from normal and anephric man, Biochem. J. 182 (1979) 55e69. [30] B.E. Dreyer, D.B.P. Goodman, A simple direct spectrophotometric assay for 24,25-dihydroxyvitamin D3, Anal. Biochem. 114 (1981) 37e41.
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measurement of vitamin D3 in selected US foods, J. Food Comp. Analy. 21 (2008) 527e534. Institute of Medicine 1997, Dietary reference intakes: calcium, phosphorus, magnesium, vitamin D, fluoride, National Academy Press, Washington, DC, 1997, pp. 250e287. F.M. Gloth, C.M. Gundberg, B.W. Hollis, et al., Vitamin D deficiency in homebound elderly persons, JAMA 274 (1995) 1683e1686. R. Vieth, Y. Ladak, P.G. Walfish, Age-related changes in the 25(OH)D versus parathyroid hormone relationship suggest a different reason why older adults require more vitamin D, J. Clin. Endocrinol. Metab. 88 (2003) 185e191. R.P. Heaney, M.S. Dowell, C.A. Hale, A. Bendich, Calcium absorption varies within the reference range for serum 25hydroxyvitamin D, J. Amer. Coll. Nutr. 22 (2003) 142e146. A.C. Looker, M.E. Mussolino, Serum 25-hydroxyvitamin D and hip fracture risk in older white adults, J. Bone Min. Res. 23 (2008) 143e150. B. Dawson-Hughes, Calcium plus vitamin D and the risk of fractures, N. Engl. J. Med. 354 (2006) 2285e2287. B.W. Hollis, C.L. Wagner, Normal serum vitamin D levels, N. Engl. J. Med. 352 (2005) 515e516. S. Abbas, J. Linseisen, T. Slanger, et al., Serum 25-hydroxyvitamin D and risk of postmenopausal breast cancer e results of a large case-control study, Carcinogenesis 29 (2008) 93e99. E.R. Betone-Johnson, W.Y. Chen, M.F. Holick, et al., Plasma 25hydroxyvitamin D and 1,25-dihydroxyvitamin D and risk of breast cancer, Cancer Epidemiol. Biomarkers Prev. 14 (2005) 1991e1997. D. Feskanich, J. Ma, C.S. Fuchs, et al., Plasma vitamin D metabolites and risk of colorectal cancer in women, Cancer Epidemiol. Biomarkers Prev. 13 (2004) 1502e1508. E. Giovannucci, Y. Liu, E.B. Rimm, et al., Prospective study of predictors of vitamin D status and cancer incidence and mortality in men, J. Natl. Cancer Inst. 98 (2006) 451e459. W. Zhou, R.S. Heist, G. Liu, et al., Circulating 25-hydroxyvitamin D levels predict survival in early-state nonsmall-cell lung cancer patients, J. Clin. Oncol. 25 (2007) 474e485. S.S. Tworoger, I.M. Lee, J.E. Buring, et al., Plasma 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D and risk of incident ovarian cancer, Cancer Epidemiol. Biomarkers Prev. 16 (2007) 783e788. B. Mikhak, D.J. Hunter, D. Speigelman, et al., Vitamin D receptor (VDR) gene polymorphisms and haplotypes, interactions with plasma 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, and prostate cancer risk, Prostate 67 (2007) 911e923. K. Wu, D. Feskanich, C.S. Fuchs, et al., A nested case control study of plasma 25-hydroxyvitamin D concentrations and risk of colorectal cancer, J. Natl. Cancer Inst. 99 (2007) 1120e1129. J. Wactawski-Wende, J.M. Kotchen, G.L. Anderson, et al., Calcium plus vitamin D supplementation and the risk of colorectal cancer, N. Engl. J. Med. 354 (2006) 684e696. P.T. Liu, S. Sterdger, H. Li, et al., Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response, Science 311 (2006) 1770e1773. M. Zasloff, Fighting infections with vitamin D, Nat. Med. 12 (2006) 388e390. T. Dietrich, M. Nunn, B. Dawson-Hughes, H.A. Bischoff-Ferrari, Association between serum concentrations of 25hydroxyvitamin D and gingival inflammation, Am. J. Clin. Nutr. 82 (2005) 575e580. T. Dietrich, K.J. Josphipura, B. Dawson-Hughes, H.A. BishchoffFerrari, Association between serum concentrations of 25hydroxyvitamin D and periodontal disease in the US population, Am. J. Clin. Nutr. 80 (2004) 108e113.
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[69] J.P. Forman, E. Giovannucci, M.D. Holmes, et al., Plasma 25hydroxyvitamin D levels and risk of incident hypertension, Hypertension 49 (2007) 1063e1069. [70] D. Martins, M. Wolf, D. Pan, et al., Prevalence of cardiovascular risk factors and the serum levels of 25-hydroxyvitamin D in the United States, Arch. Intern. Med. 167 (2007) 1159e1165. [71] T.J. Wang, M.J. Pencina, S.L. Boothe, et al., Vitamin D deficiency and risk of cardiovascular disease, Circulation 117 (2008) 503e511. This study, from the Framingham Study population, provides a margin of circulating 25(OH)D levels that may decrease cardiovascular disease risk. [72] E. Giovannucci, Y. Liu, B.W. Hollis, E.B. Rimm, A prospective study of 25-hydroxy-vitamin D and risk of myocardial infarction in men, Arch. Inter. Med. 168 (2008) 1174e1180. This study, from the Physician’s Health Study, is a prospective study that defines a range of circulating 25(OH)D levels that may provide protection against myocardial infarction. [73] H. Dobnig, S. Pilz, H. Scharnagl, W. Renner, U. Seelhorst, B. Wellnitz, et al., Independent association of low 25(OH)D and 1,25(OH)2D levels with all-cause and cardiovascular mortality, Arch. Inter. Med. 23 (2008) 1850e1858. [74] K.C. Chiu, A. Chu, V. Go, M.F. Saad, Hypovitaminosis D is associated with insulin resistance and beta cell dysfunction, Amer. J. Clin. Nutr. 79 (2004) 820e825. [75] C.S. Zipitis, A.K. Abodeng, Vitamin D supplementation in early childhood and risk of type 1 diabetes: a systematic review and meta-analysis, Arch. Dis. Child 93 (2008) 512e517. [76] M. Chonchol, R. Scragg, 25-Hydroxyvitamin D, insulin resistance and kidney function in the third national health and nutrition examination survey, Kidney Internat. 71 (2007) 134e139. [77] K.L. Munger, B.W. Hollis, L.l. Levin, et al., Serum 25-hydroxyvitamin D levels and risk of multiple sclerosis, JAMA 20 (2006) 2832e2838. [78] L.E. Tavera-Mendoza, J.H. White, Cell defenses and the sunshine vitamin, Sci. Amer. 297 (2007) 62e72.
[78a] G.D. Corter, Accuracy of 25-hydroxyvitamin D assays: confronting the issues, Curr. Drug Targets 12 (2011) 19e28. [79] N. Binkley, M.K. Drezner, B.W. Hollis, Laboratory reporting of 25-hydroxyvitamin D results: potential for clinical misinterpretation, Clin. Chem. 52 (2006) 2124e2125. [80] S. Nesby-O’Dell, K.S. Scanlon, M.E. Cogswell, et al., Hypovitaminosis D prevalence and determinants among African American and white women of reproductive age: third national health and nutrition examination survey 76 (2002) 187e192. [81] R.D. Jackson, A.X. LaCroix, M. Gass, et al., Calcium plus vitamin D supplementation and the risk of fractures, N. Engl. J. Med. 354 (2006) 669e683. [82] A.J. Rovner, V.A. Stallings, J.l. Schall, et al., Vitamin D insufficiency in children, adolescents, and young adults with cystic fibrosis despite routine oral supplementation, Amer. J. Clin. Nutr. 86 (2007) 1694e1699. [83] D. Lissner, R.S. Mason, S. Posen, Stability of vitamin D metabolites in human blood serum and plasma, Clin. Chem. 27 (1981) 773e774. [84] O.M. Antoniucci, D.M. Black, D.E. Sellmeyer, Serum 25hydroxyvitamin D is unaffected by multiple freeze-thaw cycles, Clin. Chem. 51 (2004) 258e260. [85] G.D. Carter, R. Carter, J. Jones, J. Berry, How accurate are assays for 25-hydroxyvitamin D? Data from the International Vitamin D External Quality Assessment Scheme, Clin. Chem. 50 (2004) 2195e2197. [86] R.L. Horst, In vivo versus in vitro recovery of 25-hydroxyvitamins D2 and D3 in human samples using high performance liquid chromatography and the DiaSorin Liaison Total-D Assay, J. Steroid Biochem. Mol. Biol. (2010). In press. [87] S.C. Tai, M. Bedner, K.W. Phinney, Development of a candidate reference measurement procedure for the determination of 25(OH)D3 and 25(OH)D2 in human serum using isotope-dilution liquid chromatography-tandem mass spectrometry, Anal. Chem. 82 (2010) 1942e1948.
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C H A P T E R
48 Bone Histomorphometry Linda Skingle, Juliet Compston Dept of Medicine, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK
INTRODUCTION Bone histomorphometry describes the quantitative assessment of bone remodeling, modeling, and structure. It provides information that is not available from other investigative approaches, for example, bone densitometry and biochemical markers of bone turnover, and enables a more precise characterization of disease states and their response to treatment than can be obtained from qualitative examination of bone histology. Bone histomorphometry has been particularly valuable in determining the cellular pathophysiology of different forms of bone diseases and in defining the mechanisms by which drugs affect bone. In addition, it plays a central role in establishing the bone safety of drugs in humans. It can be applied either to bone histological sections or to high-resolution images produced by techniques such as microCT and microMR (discussed in Chapter 50).
BONE BIOPSY
supine position, and the specimen is obtained approximately 2 cm below and behind the anterior superior iliac spine. Most operators use a mild sedative, such as midazolam, and some also routinely administer an analgesic by mouth or injection before the procedure. However, if care is taken to ensure adequate local anesthesia during the biopsy, the latter measure is not generally necessary. The biopsy should be performed under sterile conditions. The area around the anterior superior iliac spine is infiltrated with local anesthetic, and the inner and outer periosteum are anesthetized. Using a small trocar and stilette which is driven with a weighted instrument until it lies just in the outer cortex, local anesthetic is infiltrated under the periosteum, and the trocar and stilette are advanced through the bone to the inner cortex, where the procedure is repeated. Alternatively, the outer and inner cortex can be anesthetized by introducing a needle through the skin from skin on either side. A small skin incision is made, and a hollow cannula with a serrated edge is introduced and placed firmly on the outer periosteum. A smaller, hollow cannula is then
Procedure The iliac crest is the preferred site for bone biopsy in patients with metabolic bone disease. Most investigators favor the transverse approach, in which a biopsy containing two cortices and intervening cancellous bone is obtained (Fig. 48.1), in contrast to vertical biopsies, which contain only one cortical plate. In growing individuals only the transverse approach should be considered, because of the presence of the growth plate along the top of the crest. A number of specially designed trephines are commercially available; ideally for bone histomorphometry, the internal diameter of the specimen should be at least 6 mm. In most cases, the biopsy is performed as an outpatient procedure. For a transiliac biopsy, the patient lies in the
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10048-4
Section of transiliac biopsy obtained with an 8-mm internal-diameter trephine. The biopsy contains inner and outer cortical plates and intervening cancellous bone. Please see color plate section.
FIGURE 48.1
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48. BONE HISTOMORPHOMETRY
inserted through the larger cannula, and the biopsy is obtained by advancing the serrated edge of this cannula through the iliac crest until it has reached the outer surface of the inner cortex. The cannula is withdrawn after rotation through 360 (to ensure that the core of bone has been freed from adjoining tissues), and the biopsy is removed from the cannula using a metal rod. The incision is sutured and dressed, and the patient is instructed to lie on the side of the biopsy to apply pressure to the site and reduce the risk of bruising. Ideally, this position should be maintained for around 2 h, and thereafter the patient should be advised to rest for 24 h.
Adverse Effects Bone biopsy is safe and generally well tolerated, although there is often some discomfort after the procedure, usually lasting between 24 and 48 h. The morbidity is low and mainly due to hematoma, which may occasionally be extensive; this is most likely to occur in obese subjects or in patients with bleeding diatheses. Other reported complications include infection, transient femoral nerve palsy, avulsion of the superior ramus of the iliac crest, fracture of the iliac crest, and osteomyelitis. It should be stressed, however, that these are extremely rare and, overall, the incidence of all complications is less than 1% [1]. In order to reduce the risk of hematoma, blood clotting should be routinely checked before the procedure and bone biopsies should not be performed in patients receiving anticoagulant therapy.
Indications for Bone Biopsy The majority of metabolic bone diseases can be diagnosed on the basis of clinical, radiological, and biochemical features and in clinical practice the role of bone biopsy is mostly confined to the diagnosis of osteomalacia and characterization of the different forms of renal osteodystrophy. In patients with suspected osteomalacia, the diagnosis may be evident from biochemical and/or radiological abnormalities, but in the absence of these, bone biopsy is required. Chronic renal failure is associated with several types of bone disease, as discussed in Chapter 70; accurate diagnosis is essential to establish the correct treatment. Bone biopsy may also be helpful in the diagnosis of some rarer forms of metabolic bone disease, for example, fibrogenesis imperfecta ossium and hypophosphatemic osteomalacia. For diagnostic purposes, qualitative assessment of bone by an experienced histopathologist is sufficient, and histomorphometry is not required. Although bone biopsy is a valuable research tool in osteoporosis, it is of little value for the diagnosis of
this condition, mainly because of the relatively weak correlations between bone mass in iliac crest biopsies and clinically relevant sites such as the spine and femoral neck.
HISTOMORPHOMETRY Theoretical Considerations Histological sections of bone provide a two-dimensional representation of a three-dimensional structure. Extrapolation from one to the other requires the application of stereological formulae that are based on the assumptions that sampling is random and unbiased and, for most applications, that the structure is isotropic (i.e., evenly dispersed and randomly orientated in space) [2]. Although these conditions are not strictly fulfilled in the case of bone, conversion of histomorphometric indices to three-dimensional units is used by some investigators, whereas others express their data as two-dimensional values. The absolute values generated by these different approaches clearly differ, but in practical terms either is acceptable provided that consistency is maintained. The requirement, for stereological purposes, for isotropy is clearly not fulfilled in bone in which the macroand microarchitecture are primarily determined by mechanical forces. However, random and unbiased sampling can be achieved using vertical sections, as first described by Baddeley et al. [3] and subsequently applied to bone by Vesterby et al. [4]; the vertical axis of the sections is kept parallel to the axis of the cycloid test system, in which the test lines are defined in relation to the axis (sine-weighted).
Methodology The use of manual techniques using grids and graticules has now been almost entirely replaced by interactive computerized systems. These are faster, more operator-friendly, and enable more complex measurements than can be achieved manually. A number of systems are now available, for example Bioquant and Osteometrics [5,6]. These systems enable the user to capture images via a camera attached to the microscope prior to histomorphometric analysis. The image capture requires a microscope with a camera attached, connected to a computer (this connection may be via a graphics card or Firewire (IEEE1394) interface). At the level of magnification required for sufficient resolution of the structures to be measured the section is often too large to be visible in one field of view and if required, individual images can be captured and aligned to provide a composite of the whole section. This can be
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HISTOMORPHOMETRY
achieved with a manual microscope stage but a motorized stage is quicker and more accurate. Images can also be imported from other sources. The image analysis is achieved by working through a series of protocols provided by the software. Measurements are carried out using a mouse or graphics tablet to highlight structures of interest (e.g., mineralized bone, osteoid, individual cells) or to outline lengths or perimeters (fluorescent label length, bone perimeter, wall width). Primary indices are measured in this way and from these the derived indices are calculated by the software and can then be exported to a spreadsheet.
Terminology In 1987 a committee of the American Society of Bone and Mineral Research (ASBMR) proposed a standard nomenclature for bone histomorphometric variables and this has subsequently become widely adopted [7]. It expresses all data in terms of the source (the structure on which the measurement is made), the measurement, and the referent. The recommended format (including punctuation) is source-measurement/referent, although because only one source is usually used in a particular study, it is unnecessary to specify this once it has been defined. Area or perimeter (two-dimensional nomenclature), or volume and surface (three-dimensional nomenclature) are used as referents for most measurements (Table 48.1). Histomorphometric data may be described in either two-dimensional or three-dimensional terms and the system used should be consistent within studies. Primary measurements are referred to as area, perimeter, and width (two-dimensional nomenclature) or volume, surface, and thickness (three-dimensional TABLE
48.1 Referents Commonly Histomorphometry
Used
in
Bone
Referent (3D/2D)
Abbreviation (3D/2D)
Bone surface/perimeter
BS/B.Pm
Bone volume/area
BV/B.Ar
Tissue volume/area
TV/T.Ar
Core volume/area
CV/C.Ar
Osteoid surface/perimeter
OS/O.Pm
Eroded surface/perimeter
ES/E.Pm
Mineralized surface/perimeter
Md.S/Md.Pm
Osteoblast surface/perimeter
Ob.S/Ob.Pm
Osteoclast surface/perimeter
Oc.S/Oc.Pm
3D, three dimensions; 2D, two dimensions. Adapted from Parfitt et al. [7] with permission from Wiley.
TABLE 48.2 Primary Histomorphometric Indices of Bone Remodeling Name
Abbreviation
Units
Bone area
B.Ar/T.Ar
%
Osteoid area
O.Ar/T.Ar or O.Ar/B.Ar
%
Osteoid perimeter
O.Pm/B.Pm
%
Osteoblast perimeter
Ob.Pm/B.Pm
%
Osteoid width
O.Wi
mm
Interstitial width
It.Wi
mm
Trabecular width
Tb.Wi
mm
Eroded perimeter
E.Pm/B.Pm
%
Osteoclast perimeter
Oc.Pm/B.Pm
%
Mineralizing surface
Md.Pm/B.Pm
%
Mineral apposition rate
MAR
mm/day
Wall width
W.Wi
mm
Erosion depth
E.De
mm
Erosion length
E.Le
mm
Erosion area
E.Ar
mm2
Cavity number
N.Cv./B.Pm or N.Cv./T.Ar
no./mm or no./mm2
Quiescent perimeter
Q.Pm
%
Reversal perimeter
Rv.Pm
%
Adapted from Parfitt et al. [7] with permission from Wiley.
nomenclature) (Table 48.2). It should be noted that absolute area and perimeter measured on conventional histological sections have no three-dimensional equivalent but, if three-dimensional nomenclature is adopted, these are referred to as volume and surface although the absolute values are identical. Cortical width and thickness are also numerically equal; however, in other situations, for example trabecular width, conversion to thickness requires division of width by 4/p (1.273) for isotropic structures or 1.2 for human iliac cancellous bone [2,8]. The units recommended for the revised nomenclature are micrometer and millimeter for length and day and year for time; surface/surface and volume/volume ratios are expressed as percentages whereas volume/ surface ratios are expressed in mm3/mm2. Some of the more commonly used derived histomorphometric indices are shown in Table 48.3. Most histomorphometric studies have been confined to cancellous bone, which because of its high surface to volume ratio exhibits greater remodeling activity than cortical bone. Although cortical bone predominates throughout the skeleton and is a major determinant of
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48. BONE HISTOMORPHOMETRY
TABLE 48.3 Derived Histomorphometric Indices of Bone Remodeling Name
Abbreviation
Units
Adjusted apposition rate
Aj.AR
mm/day
Bone formation rate
BFR/B.Pm BFR/B.Ar
mm2/mm/day %/year
Erosion rate
ER
mm/day
Mineralization lag time
Mlt
day(s)
Osteoid maturation time
Omt
day(s)
Formation period
FP
day(s)
Active formation period
FP(aþ)
day(s)
Erosion period
EP
day(s)
Reversal period
Rv.P
day(s)
Quiescent period
QP
day(s)
Remodeling period
Rm.P
day(s)
Total period
Tt.P
day(s)
Ac.f
/year
Tb.Sp
mm or mm
Tb.N
/mm
Activation frequency Trabecular separation Trabecular number
a
a
a
May also be measured directly. Adapted from Parfitt et al. [7] with permission from Wiley.
bone strength and fracture risk, it has until recently been largely ignored by histomorphometrists. The application of histomorphometric techniques to cortical bone was first described by Frost [9] in the rib and more recently a detailed analysis of the bone remodeling cycle in human cortical bone [10] and studies of cortical bone structure in the femoral neck and iliac crest [11e14] have been reported. Indices of remodeling balance and remodeling rate can be assessed in cortical bone as in cancellous bone.
Limitations of Bone Histomorphometry Certain limitations of bone histomorphometry should be recognized. Some of these are inherent in the restrictions imposed by a single biopsy site and disease heterogeneity, whereas others reflect imperfections in measurement techniques and difficulties in identification of some of the key processes in remodeling. A number of studies have documented the large measurement variance associated with bone histomorphometry, which arises from a variety of sources including intra- and interobserver variation, sampling variation, and methodological factors [15e17]. Intraand interobserver variation reflect the subjective approach to identification of many of the histological features assessed, for example, resorption cavities and
osteoid seams. Methodological factors include the criteria used for corticomedullary differentiation, the staining method used, and the magnification at which measurements are made. The technique used for quantitation may also affect the values obtained, as a result of different sampling procedures and variations in the number of sampling points utilized. Many of these sources of variance can be minimized by the standardization of staining, corticomedullary delineation, and magnification and the employment of criteria for identification of osteoid seams, resorption cavities, and newly formed bone structural units. In clinical practice the site of biopsy is generally restricted to the iliac crest, which may not always reflect changes occurring elsewhere in the skeleton. Some metabolic bone disorders, for example, osteomalacia and most forms of renal osteodystrophy, appear to affect the whole skeleton and in such cases an iliac crest biopsy is representative. In osteoporosis, however, there is clear evidence of disease heterogeneity and it is well documented that cancellous bone volume in iliac crest biopsies is a poor indicator of bone loss elsewhere in the skeleton [18]. There is also evidence for variations in bone turnover at different skeletal sites [19]. However, the demonstration of abnormalities of bone remodeling in iliac crest bone obtained from patients with osteoporosis indicates that changes responsible for the disease process are reflected, at least to some extent, in bone from this site. Similarly, changes in remodeling rate and balance have been shown in patients with treated osteoporosis and have generally been consistent with the observed changes in bone mass at sites such as the spine and hip. Finally, current histomorphometric techniques are limited by the lack of reliable markers for activation of remodeling and resorption. Dynamic indices related to these processes are at present calculated from bone formation rates, based on the assumptions that bone resorption and formation are coupled temporally and spatially and that bone remodeling is in a steady state; however, these assumptions are often unlikely to be tenable in untreated or treated osteoporosis [20].
ASSESSMENT OF MINERALIZATION In vivo Tetracycline Labeling The administration of two time-spaced oral doses of a tetracycline derivative prior to bone biopsy enables measurement and calculation of dynamic indices of bone formation and, by extrapolation, bone resorption [21]. Such information is not currently obtainable by other means, and tetracycline labeling should therefore be performed whenever possible. Various regimens
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ASSESSMENT OF MINERALIZATION
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have been described; most involve a 10e12-day gap between the two doses, bone biopsy being performed 3e5 days after the last dose. The regimen used by the author is as follows: Days 1 and 2 10 days Days 13 and 14
300 mg demeclocycline twice daily No demeclocycline 300 mg demeclocycline twice daily.
The bone biopsy is performed 3e5 days after day 14. Adverse effects of demeclocycline and related compounds are rare but include diarrhea and other gastrointestinal symptoms. Occasionally, skin rashes occur; these are sometimes severe and may exhibit photosensitivity. There is evidence that different tetracycline compounds differ with respect to their uptake by mineralizing bone. Parfitt et al. [22] reported that demeclocycline labeling resulted in a greater surface extent of fluorescence than oxytetracycline, significantly affecting values for dynamic indices of bone formation. These differences should be borne in mind when comparing data between centers and, in particular, when using control data obtained from other sources.
Measurement of Osteoid Osteoid may be distinguished from mineralized bone by several staining procedures, including von Kossa, toluidine blue, Goldner’s trichrome, and solochrome cyanin. Primary measurements of osteoid include area, perimeter, and seam width; assessment of dynamic indices of mineralization, for example, mineral apposition rate, mineralization lag time, and osteoid maturation rate, requires double tetracycline labeling prior to biopsy [7]. Calcification fronts along osteoid seams can be demonstrated by staining with toluidine blue (Fig. 48.2; see color insert), Sudan black B, or thionin, but the surface extent assessed in this way does not always correlate well with the mineralizing perimeter as measured using tetracycline uptake [23]. Osteoid seam width may be measured directly or calculated from osteoid area and perimeter. However, when relatively small amounts of osteoid are present, the latter approach is inaccurate because the value for osteoid area, expressed as a percentage of bone area, is influenced not only by the seam width but also by the mineralized bone area [24]. Measurements of osteoid, in particular its surface extent, are strongly influenced by the magnification used. At high magnification, it becomes difficult to distinguish osteoid seams from the thin endosteal membrane covering the quiescent bone surface, and
Toluidine blue-stained section of iliac crest cancellous bone showing mineralized bone (purple/blue) and osteoid (pale blue). The calcification front can be seen as a dark blue line at the interface of the osteoid and mineralized bone. Please see color plate section.
FIGURE 48.2
for this reason it is preferable to use defined criteria, for example, seams less than one lamella (3 mm) are excluded. Osteoid measurements may also be affected by the staining procedure used, and poor differentiation of osteoid from mineralized bone in images used for semiautomatic techniques may reduce the accuracy of measurements [16]. Finally, delineation of the corticomedullary junction may affect the values obtained for primary measurements of osteoid, as osteoid amount tends to be greater in the endosteal region than in pure cancellous bone.
Dynamic Indices of Mineralization The administration of time-spaced tetracycline labels prior to biopsy enables calculation of the dynamic indices of matrix formation and mineralization that are central to the histomorphometric diagnosis of osteomalacia. Tetracycline binds to calcium and becomes permanently incorporated into the mineralization fronts at sites of active formation (Fig. 48.3; see color insert). In this respect, the timing of the biopsy in relation to the labeling regime is critical, with a period of 3e5 days after the last label enabling deposition of a sufficiently thick layer of new mineral to retain the tetracycline label [25]. The primary histomorphometric indices of bone remodeling are listed in Table 48.2 and the derived indices in Table 48.3. Normative histomorphometric data from the author’s laboratory are shown for women (Table 48.4) and men (Table 48.5). Mineral apposition rate Mineral apposition rate (MAR) is calculated as the distance between two time-spaced tetracycline labels divided by the time between the administration of the
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48. BONE HISTOMORPHOMETRY
apposition rate should be used for the calculation of derived indices [27]. Adjusted apposition rate The adjusted apposition rate (Aj.AR) represents the mineral apposition rate averaged over the osteoid surface. In the absence of a mineralization defect the apposition of matrix and mineral, whilst not synchronous, can be assumed to occur at the same rate and under such circumstances the adjusted apposition rate is equivalent to the osteoid or matrix apposition rate. It is calculated as follows: Aj:AR ¼ MAR Md:Pm=O:Pm: FIGURE 48.3 Unstained section of iliac crest cancellous bone viewed by fluorescence microscopy. The tetracycline labels are seen as double yellow fluorescent bands. Please see color plate section.
labels. Measurements are made from the midpoint of each label at approximately equidistant points along the labeled surface, and the interlabel period is calculated as the number of days between the midpoints of the two labeling periods [26]. MAR is used in the calculation of many derived indices of bone formation, and accurate measurement is thus of key importance. In the absence of tetracycline uptake, mineral apposition rate and derived indices should be treated as missing data; in biopsies in which only single labels can be detected, the finite lower limit of 0.3 mm/day for mineral TABLE 48.4
From the above formula it is clear that Aj.AR is usually less than MAR and cannot exceed it. Mineralization lag time and osteoid maturation time The mineralization lag time (Mlt) is the interval between deposition and mineralization of a given amount of osteoid, averaged over the lifespan of the osteoid seam. It is calculated as Mlt ¼ O:Wi=Aj:AR: The osteoid maturation time (Omt) is the period between the deposition and onset of mineralization of a given amount of osteoid and results from processes
Normative Histomorphometric Data in Femalesa,b
Age range Parameter
19e30 years (n [ 5)
31e40 years (n [ 6)c
41e50 years (n [ 6)d
51e60 years (n [10)e
61e80 years (n [ 6)
BV/TV (%)
25.9 (3.1)
27.7 (5.5)
29.6 (2.1)
23.9 (4.5)
19.8 (3.9)
OV/BV (%)
2.4 (1.4)
2.7 (2.4)
2.3 (1.3)
3.1 (1.9)
4.7 (1.9)
OS/BS (%)
13.1 (5.9)
17.7 (11.5)
14.5 (7.8)
21.2 (11.3)
35.0 (12.1)
ES/BS (%)
2.15 (0.36)
1.84 (0.92)
1.78 (1.03)
Md.S/BS (%)
9.4 (1.8)
8.1 (3.4)
8.1 (4.2)
13.0 (6.7)
14.8 (8.1)
O.Th (mm)
5.4 (1.9)
3.9 (1.6)
5.8 (1.1)
5.8 (1.6)
5.8 (2.3)
W.Th (mm)
45.7 (4.9)
51.2 (6.6)
47.5 (4.9)
36.1 (2.9)
32.5 (3.6)
MAR (mm/day) Mlt (days) 3
0.59 (0.06) 12.2 (6.2)
2
BFR (mm /mm / day)
0.056 (0.013)
0.60 (0.12) 16.5 (11.8) 0.060 (0.022)
0.61 (0.09) 20.7 (8.7) 0.051 (0.031)
a
1.76 (0.83)
0.61 (0.10) 21.2 (19.6) 0.084 (0.045)
1.66 (0.66)
0.54 (0.07) 29.6 (13.5) 0.081 (0.043)
Results are expressed as means, with SD values in parentheses. Md.S/BS was calculated as the double plus half the single tetracycline-labeled surface. Data from Vedi S, Compston JE, Webb A, Tighe JR 1982 Histomorphometric analysis of bone biopsies from the iliac crest of normal British subjects. Metab Bone Dis Related Res 4:231e236 and Vedi S, Compston JE, Webb A, Tighe JR 1983 Histomorphometric analysis of dynamic parameters of trabecular bone formation in the iliac crest of normal British subjects. Metab Bone Dis Related Res 5:69e74 with permission from Elsevier. c n ¼ 4 for dynamic variables. d n ¼ 5 for dynamic variables. e n ¼ 9 for dynamic variables. b
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851
HISTOLOGICAL DIAGNOSIS OF OSTEOMALACIA
TABLE 48.5
Normative Histomorphometric Data in Malesa,b Age range
Parameter
19e30 years (n [ 3)
31e40 years (n [6)
41e50 years (n [ 3)
51e60 years (n [ 6)
61e80 years (n [ 6)
BV/TV (%)
31.3 (6.4)
22.2 (3.9)
26.9 (7.1)
23.0 (5.5)
21.4 (2.6)
OV/BV (%)
1.6 (0.8)
4.1 (1.6)
2.8 (0.9)
3.1 (0.9)
5.6 (3.6)
OS/BS (%)
10.0 (5.3)
28.3 (7.8)
26.3 (6.0)
20.0 (7.0)
34.8 (15.7)
ES/BS (%)
2.84 (1.27)
Md.S/BS (%)
9.9 (4.0)
O.Th (mm) W.Th (mm) MAR (mm/day) Mlt (days) 3
1.68 (0.32)
1.77 (0.68)
1.91 (0.42)
13.5 (8.4)
8.7 (7.0)
8.8 (1.5)
9.2 (5.1)
8.3 (3.0)
6.1 (1.3)
6.9 (2.9)
6.2 (2.0)
6.6 (2.8)
49.7 (9.6)
45.9 (4.4)
42.8 (4.0)
36.9 (2.0)
33.4 (3.3)
0.67 (0.07) 10.5 (1.9)
2
BFR (mm /mm / day)
0.066 (0.006)
1.69 (0.62)
0.60 (0.10) 28.5 (14.6) 0.079 (0.049)
0.55 (0.10) 36.1 (6.2) 0.047 (0.005)
0.57 (0.13) 34.7 (40.8) 0.051 (0.015)
0.53 (0.05) 49.4 (28.1) 0.045 (0.023)
a
Results are expressed as means with SD values in parentheses. Md.S/BS was calculated as the double plus half the single tetracycline-labeled surface. Data from Vedi S, Compston JE, Webb A, Tighe JR 1982 Histomorphometric analysis of bone biopsies from the iliac crest of normal British subjects. Metab Bone Dis Related Res 4:231e236 and Vedi S, Compston JE, Webb A, Tighe JR 1983 Histomorphometric analysis of dynamic parameters of trabecular bone formation in the iliac crest of normal British subjects. Metab Bone Dis Related Res 5:69e74 with permission from Elsevier. b
such as collagen cross-linking that are necessary before mineralization can proceed. In humans it is usually shorter than Mlt and never exceeds it. It is calculated as follows: Omt ¼ O:Wi=MAR:
HISTOLOGICAL DIAGNOSIS OF OSTEOMALACIA Generalized Osteomalacia Osteomalacia is essentially a histological diagnosis, although biochemical and radiological abnormalities may enable a clinical diagnosis to be made without the need for histological examination of bone. Nonetheless, osteomalacia may exist in the absence of biochemical and radiological abnormalities [28], and in such cases bone biopsy is the only certain means by which the diagnosis can be established. The cardinal feature of osteomalacia is defective mineralization, which results in accumulation of osteoid with an increase in the width of osteoid seams (Fig. 48.4; see color insert) and a reduction in the surface extent of osteoid showing tetracycline labeling. There is also often an increase in the width of individual tetracycline labels, and the distance between double labels is reduced or undetectable. In severe cases, tetracycline labeling may be absent. Increased surface extent of bone resorption, due to secondary hyperparathyroidism, is usually
present but becomes less apparent as the mineralized bone surface becomes covered with thick osteoid seams and, therefore, inaccessible to osteoclastic resorption. In such cases tunneling resorption may be apparent, and the irregular outline of mineralized bone beneath the thick osteoid seams provides evidence of previous resorption. Paratrabecular fibrosis is also seen in severe cases. In contrast, histological evidence of secondary hyperparathyroidism is absent in untreated hypophosphatemic osteomalacia. In histomorphometric terms, osteomalacia is defined as an increased osteoid seam width and a prolonged mineralization lag time [25]. The criteria for abnormality
Section of iliac crest stained by the von Kossa technique to show osteoid accumulation in a woman with severe privational osteomalacia. Please see color plate section.
FIGURE 48.4
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852
48. BONE HISTOMORPHOMETRY
in these indices depend on the source of the reference data, which may vary as a result of both geographical and methodological factors, but in most centers a mean osteoid seam width greater than 12.5 mm and a mineralization lag time in excess of 100 days would be regarded as abnormal. Although the mineral apposition rate is also reduced in osteomalacia, this is not a specific feature because low mineral apposition rates may also result from a reduction in matrix apposition rate as sometimes found in osteogenesis imperfecta and some forms of secondary osteoporosis. Similarly, the mineralization lag time will be increased in the presence of reduced matrix apposition rate and is therefore not, by itself, pathognomonic of osteomalacia. The distinguishing feature of osteomalacia is that the mineralization lag time is prolonged relative to the adjusted apposition rate, whereas in osteoporosis the reverse is true; this phenomenon accounts for the increase in osteoid seam width that is specific to osteomalacia.
Focal Osteomalacia Focal osteomalacia has been described in association with etidronate and pamidronate therapy and is characterized by the focal distribution of abnormally thick osteoid seams with impaired mineralization [29,30]. In such cases the total amount and surface extent of osteoid may be normal, and, because some osteoid seams are of normal width and exhibit normal mineralization, the abnormality may only be detected by examination of the distribution, within single biopsies, of values for osteoid seam width. The significance of these histological changes in terms of fracture risk has not been established but they do not appear to be associated with clinical symptoms or biochemical abnormalities.
ASSESSMENT OF BONE TURNOVER Bone turnover describes the amounts of bone removed and formed within a given volume of bone in a given time and is determined by the number of remodeling units and the focal balance within each remodeling unit. Histomorphometric indices commonly used as measures of bone turnover include mineralizing perimeter, bone formation rate, and activation frequency. These are described in more detail below.
Mineralizing Perimeter The extent of bone perimeter or surface that exhibits tetracycline fluorescence provides an estimate of the remodeling rate, determined by the number of recently formed remodeling units. When a double tetracycline
label has been administered, both double and single labels will be seen. Single labels reflect the labeling escape error, caused by initiation of mineralization before the first label or its termination between administration of the two labels [31], and probably also the switch from an active to resting state in a minority of osteoid seams (the so-called on/off phenomenon) [2]. Because of the former, the extent of double-labeled surface underestimates the actively mineralizing surface, and the double plus half the single label is generally used to estimate the mineralizing perimeter. In cases where only single labels can be detected, it has been suggested that the mineralizing perimeter should be expressed as half the single-labeled perimeter [27]. In the absence of tetracycline administration prior to biopsy, the surface extent of osteoid may provide some indication of remodeling rate. An increase in the surface extent of resorption cavities does not necessarily imply increased bone turnover since these do not always reflect active resorption but may rather indicate failure of formation to occur in previously resorbed cavities.
Bone Formation Rates Bone formation rates are usually expressed in terms of the bone perimeter or area. In the former case, either the osteoid perimeter may be used as the referent (adjusted apposition rate) or the total bone perimeter (tissue-based bone formation rate). They are calculated as follows: Adjusted apposition rate ¼ MAR Md:Pm=O:Pm; Bone formation rate ðtissue basedÞ ðBFR=BSÞ ¼ MAR Md:Pm=B:Pm: The bone formation rate/bone area (BFR/B.Ar) is calculated as BFR=B:Ar ¼ MAR Md:Pm ðB:Pm=B:ArÞ 100:
Bone Resorption Rates Because of the lack of markers of active resorption analogous to the use of tetracycline to identify actively forming bone, bone resorption rates can only be calculated indirectly from bone formation rates, based on the assumptions discussed earlier. Erosion rate (ER) is expressed in micrometers per day and calculated as follows: ER ¼ E:De=EP: Calculation of the erosion period (EP) is shown below.
VI. DIAGNOSIS AND MANAGEMENT
ASSESSMENT OF REMODELING BALANCE
Remodeling Periods The average duration of a single remodeling cycle is described as the remodeling period, which can be divided into quiescent, erosion, reversal, and formation remodeling periods [2]. The formation period (FP) can be further divided into the active (FP(aþ)) and inactive formation period (FP(ae)) [32]; the latter is a measure of the “off-time,” which accounts for the discrepancy between osteoid and mineralizing perimeter after correction for label escape [31]. Formation periods are calculated as follows: FP ¼ W:Wi=Aj:AR FPðaþÞ ¼ W:Wi=MAR; FPðaÞ ¼ FP FPðaþÞ; where W.Wi (wall width) is the mean width of completed bone structural units. Quiescent, erosion, and reversal periods are calculated as follows: QP ¼ Q:Pm=B:Pm FP; EP ¼ E:Pm=B:Pm FP; Rv:P ¼ Rv:Pm=B:Pm FP: where Rv.Pm ¼ E.Pm d osteoclastic perimeter; Q.Pm ¼ B.Pm. e (O.Pm) þ (E.Pm). The mean time between initiation of two successive remodeling cycles at the same site is defined as the total period and calculated as follows: Tt:P ¼ Rm:P þ QP:
Activation Frequency Activation frequency is defined as the probability that a new remodeling cycle will be initiated at any point on the bone surface [33]. It is defined as the reciprocal of the remodeling period (the average time between the initiation of two successive remodeling periods at the same site) and this definition translates into the equation used to calculate activation frequency, namely the bone formation rate at surface level divided by the wall width [2,7,34]:
853
remodeling units may be activated at sites that have not recently undergone bone remodeling, regardless of the total remodeling period at other sites. Thus if remodeling balance and rate are independent of each other, as suggested by a recent study [35], activation frequency as currently calculated may not accurately reflect remodeling rate and may be misleading when there is a dissociation between changes in BMU balance and remodeling rate.
ASSESSMENT OF REMODELING BALANCE Bone Formation Within individual remodeling units, the amount of bone formed is termed the wall width [36]. This is measured as the mean width of completed bone structural units that are identified under polarized light (Fig. 48.5; see color insert) or by stains such as toluidine blue or thionin [37], which demonstrate the cement line. Completed structural units are identified by the absence of resorption lacunae or osteoid. There is a large variation in reported values for wall width in both normal and osteoporotic subjects [20], reflecting differences in sampling procedures and difficulties in accurate identification of the cement line, which forms the base of the original resorption cavity. Investigation of the effect of a disease or its treatment on wall width (and calculated dynamic indices for which wall width is required) necessitates differentiation of those units formed during the period of observation from those formed prior to this time. This can only be achieved by identification of uncompleted bone structural units, which have a covering of osteoid and hence can be presumed to represent current or recent remodeling
Ac:f ¼ 1=Tt:P or ðBFR=B:PmÞ=W:Wi: The value generated therefore provides a measure of the frequency by which two successive remodeling cycles are initiated at the same site on the bone surface. Central to this definition is the assumption that remodeling rate is dependent on the duration of the remodeling cycle and the amount of bone formed at the BMU level. This in turn requires that remodeling rate and balance are coordinated. In reality, however, successive
Section of iliac crest biopsy viewed under polarized light to show a completed bone structural unit bounded by the cement line and mineralized bone surface. Please see color plate section.
FIGURE 48.5
VI. DIAGNOSIS AND MANAGEMENT
854
48. BONE HISTOMORPHOMETRY
activity. Reconstruction of these forming sites can then be achieved [38]. However, the number of such units that can be identified in any one biopsy is likely to be extremely small and variance correspondingly high.
Bone Resorption There are several problems associated with accurate assessment of the amount of bone resorbed during each remodeling cycle. The identification of resorption cavities is often difficult and always to some extent subjective; in addition, it is difficult to identify those cavities in which resorption has been completed. The use of polarized light microscopy to demonstrate cut-off lamellae at the edges of the cavity assists recognition [39] (Fig. 48.6; see color insert), as does the presence of osteoclast-like cells within the cavity. More precise identification of osteoclasts can be achieved by histochemical techniques that demonstrate the presence of tartrate-resistant acid phosphatase, although this is not specific to osteoclasts [40,41]. Finally, it is not usually possible to identify those cavities that have resulted in trabecular perforation.
FIGURE 48.6 (Top) Resorption cavity in cancellous iliac crest bone stained by toluidine blue. (Bottom) Same resorption cavity viewed under polarized light. Note the cut-off collagen lamellae at the edges of the cavity. Please see color plate section.
Indirect assessment of erosion depth was first reported by Courpron et al. [42], based on the postulate that the interstitial width (i.e., the distance between two bone structural units on opposite sides of a trabecula) is inversely proportional to erosion depth. In this model, interstitial width is calculated as the difference between the trabecular width and twice the wall width. However, the relationship between interstitial width and erosion depth is not a simple inverse one, as it is influenced by concomitant changes in wall width and trabecular width [43,44]. Another approach to the direct measurement of erosion depth was reported by Eriksen et al. [45]. In this method, the number of lamellae eroded beneath the bone surface is counted, and cavities are characterized according to the presence of osteoclasts, mononuclear cells, and preosteoblastic cells, these being specifically associated with increasing stages of completion of the resorptive phase. This approach depends critically upon the accurate identification, on morphological grounds, of these different cell types within resorption cavities; even in high-quality histological sections this can be difficult, and in the author’s hands 24% of resorption cavities were excluded from measurement because of failure to classify by cell type or inability to define and count the eroded lamellae. This method has not been widely adopted by other groups, although some have used a simplified approach in which the eroded lamellae are counted without subdivision of cavities by cell type [46]. The latter technique provides an estimate of the mean depth of cavities in all stages of completion and thus underestimates the final resorption depth. The computerized method developed by Garrahan et al. [47] involves reconstruction of the eroded bone surface by a curve-fitting technique (cubic spline) and provides measurements of mean and maximum erosion depth together with the area, surface extent, and number of cavities. Because all resorption cavities are included in the measurements, the mean values for mean and maximum depth and for area considerably underestimate the final resorption depth and area. This method may also be performed using interactive reconstruction of the eroded bone surface [48]. Another modification is to include for measurement only those cavities that contain a thin layer of osteoid, ensuring that the final resorption depth has been achieved; however, the number of such cavities that can be identified in a single biopsy is usually small. Interestingly, the values reported using this approach in normal human biopsies were approximately 17% lower than those obtained in the same biopsies by the technique of counting eroded lamellae and were more consistent with reported agerelated changes in trabecular width [49,50]. Further work is thus required to improve existing techniques for the measurement of erosion depth. The
VI. DIAGNOSIS AND MANAGEMENT
855
ASSESSMENT OF BONE STRUCTURE
approaches most widely used at present measure the depth of all resorption cavities and therefore underestimate the completed erosion depth, whereas measurements made using Eriksen’s method probably overestimate the true value [51]. Although these limitations preclude accurate assessment of remodeling balance at present, measurement of erosion depth has generated valuable information about the pathophysiology of bone loss in untreated and treated disease states.
ASSESSMENT OF BONE STRUCTURE Both cortical and cancellous bone structure are important determinants of bone strength. Structural indices can be derived from histological bone sections, as described below, or from images obtained using high-resolution techniques. The latter are almost exclusively confined to the peripheral skeleton at present and are described in more detail in Chapter 50.
Structural Determinants of Bone Strength Structural determinants of the mechanical strength of bone include cortical width and porosity and, in cancellous bone, trabecular size, shape, connectivity, and anisotropy. Early approaches to the quantitative assessment of cancellous bone structure were based on direct or indirect measurements of trabecular width, separation, and number [52e56]. Direct measurements of trabecular width can be made using an eyepiece
(A)
graticule or grid but nowadays are most commonly performed using computerized techniques [55,56]. These measurements provide information not only about the mean trabecular width but also about the distributions of trabecular width within individual biopsies. Calculation of trabecular width from area and perimeter measurements may also be performed [57], this approach being based on the assumption that the width of measured structures is small relative to their length. Calculation of trabecular separation and number from trabecular width and bone area is based on a parallel plate model [57] that may often be inappropriate in cancellous bone. Nevertheless, these approaches have generated useful information and have stimulated the development of more sophisticated techniques for analysis of bone structure. Because all of the structural determinants of cancellous bone strength are three-dimensional characteristics, their assessment on conventional histological sections provides only indirect information about these qualities, and three-dimensional images are required for direct measurement of connectivity, anisotropy, and trabecular size and shape [58]. Although further studies are required to examine the relationship between structural indices obtained using two- and three-dimensional approaches, there are several lines of evidence to support the contention that measurements from two-dimensional sections are representative of three-dimensional structure [59,60]. Structural indices of cortical bone include cortical width, cortical porosity, and Haversian canal number, density and area (Fig. 48.7; see color insert).
(B) H.Ca.Dm
Ps
Ct.Wi On.Dm
Ec
(A) Section of cortical bone viewed under polarized light showing the periosteal surface (Ps), endocortical surface (Ec), an osteon, and a Haversian canal (H.Ca). (B) Diagrammatic representation of cortical bone showing endocortical surface (Ec), periosteal surface (Ps), cortical width (Ct.Wi), Haversian canal diameter (H.Ca.Dm), and osteonal diameter (On.Dm). Adapted from Brockstedt et al. [71] with permission from Elsevier. Please see color plate section.
FIGURE 48.7
VI. DIAGNOSIS AND MANAGEMENT
856
48. BONE HISTOMORPHOMETRY
Two-dimensional Approaches Strut Analysis Strut analysis is based on the definition of nodes and termini and the topological classification of trabeculae and struts. Garrahan et al. [5] described a semi-automated procedure in which the binary image of a section is skeletonized and the different strut types are classified as shown in Figure 48.8 (see color insert). The total length of each strut type may be expressed as a percentage of the total strut length or in absolute terms. Node-to-node and node-to-loop strut lengths are positively related to connectivity, whereas node-to-terminus and terminus-to-terminus strut lengths are inversely related. Because of the edge effect, termini may be created artefactually, whereas node-to-node and nodeto-loop struts are true indices of connectedness, although their number may be underestimated. Termini may also result from sectioning through a trabecular window in a connected structure. Star Volume The star volume is defined as the mean volume of solid material or empty space that can be seen unobscured from a point of measurement chosen at random inside the material [61]. Its assessment in histological sections of bone was first reported by Vesterby [62] using the vertical section technique [3] and a cycloid test system. The method may be applied to measurement both of trabecular width (trabecular star volume) and trabecular separation (marrow space star volume) and theoretically provides an unbiased stereological approach to these indices. The method involves the
generation of intercepts from random sampling points, the cubed length of the intercepts being used in the calculation of star volume. The values generated by marrow star volume measurements are significantly influenced by biopsy size, particularly in poorly interconnected cancellous bone in which a large proportion of the measured intercepts may hit the boundary rather than bone, resulting in underestimation of star volume [63]. Trabecular Bone Pattern Factor This method is based on the concept that patterns or structures can be defined by the relationship between convex and concave surfaces [64], convexity indicating poor connectivity and concavity reflecting greater structural integrity. Using a computer-based system, convexity and concavity are assessed by measurement of the bone perimeter before and after computer-based dilatation of the trabecular surface; whereas thickening of convex structures increases their perimeter, the reverse applies to concave structures. The values obtained for trabecular bone pattern factor, which is calculated as the difference between perimeter measurements before and after dilatation divided by the corresponding difference in area, may be significantly influenced by the computer-based smoothing technique used, the degree of dilatation employed, and the magnification at which the measurements are performed. Fractal Analysis Fractal objects are characterized by scale invariance or self-similarity over a wide range of magnifications so that any one piece of a fractal, if magnified sufficiently, resembles the intact object [65]. Fractal analysis has been described in a number of biological systems including the bronchial tree and vascular networks; in bone, it has been applied to radiological images and histological sections of bone [66]. The value obtained for the fractal dimension is critically dependent on the magnification used for measurement, and the relationship between fractal dimension and connectivity in cancellous bone has not been established. Multidirectional fractal analysis can be used to assess structural anisotropy [67].
Three-dimensional Approaches FIGURE 48.8 Diagrammatic representation of different strut types in strut analysis. Cancellous bone is shown in gray. Lines represent the skeletonized axis of the original bone profile. Squares represent nodes (Nd) and termini (Tm) terminus-to-terminus (Tm.Tm), node-to-loop (Nd.Lp), and node-to-terminus (Nd.Tm) strut types are illustrated. Reprinted from Croucher et al. [63] with permission from Wiley. Please see color plate section.
A number of techniques have been used to generate three-dimensional images of bone. These include reconstruction of serial sections, scanning and stereomicroscopy, and techniques involving computed tomography and magnetic resonance imaging [68e70]. These are described in more detail in Chapter 50.
VI. DIAGNOSIS AND MANAGEMENT
REFERENCES
FUTURE DEVELOPMENTS An unmet need in the assessment of metabolic bone disease is the ability to assess bone remodeling activity at specific skeletal sites in vivo, for example the spine and femoral neck. An exciting development in this area is the use of (18)-fluoride positron emission tomography ((18)F-PET) which provides an assessment of regional bone blood perfusion and turnover and appears to reflect osteoblast number and activity. The technique is noninvasive, although expensive, and has the potential to provide unique site-specific information about untreated and treated bone disease [72e74]. As the resolution of in vivo imaging techniques improves, it will become possible to assess cortical and trabecular bone structure with increasing accuracy. Currently available techniques have limitations in terms both of resolution and the skeletal site to which they can be applied, but the use of high-resolution peripheral QCT has already provided some novel insights into age-related and drug-induced changes in cortical and trabecular bone in the appendicular skeleton. However, the structural changes observed using imaging techniques reflect the result of changes in bone remodeling caused by disease or its treatment but do not indicate the cause of those changes. Histomorphometric assessment of biopsy samples will therefore continue to play a major role in the evaluation of the safety and mechanisms of action of bone-active drugs [75e77], as well as providing unique information about the pathophysiology of metabolic bone diseases.
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[29]
[30]
[31]
[32]
[33] [34]
[35]
[36]
[37] [38]
[39]
[40] [41]
[42]
[43]
[44] [45]
[46]
48. BONE HISTOMORPHOMETRY
diagnosis of histological osteomalacia, J. Clin Pathol. 35 (1982) 625e630. B.F. Boyce, I. Fogelman, S. Ralston, E. Johnston, I.T. Boyle, Focal osteomalacia due to low-dose diphosphonate therapy in Paget’s disease, Lancet 1 (1984) 821e824. B.B. Adamson, S.J. Gallacher, J. Byars, S.H. Ralston, I.T. Boyle, B.F. Boyce, Mineralisation defects with pamidronate therapy for Paget’s disease, Lancet 342 (1993) 1459e1460. H.M. Frost, Bone histomorphometry: choice of marking agent and labelling schedule, in: R. Recker (Ed.), Bone Histomorphometry: Techniques and Interpretations, CRC Press, Boca Raton, Florida, 1983, pp. 37e51. M. Arlot, C. Edouard, P.J. Meunier, R.M. Neer, J. Reeve, Impaired osteoblast function in osteoporosis: comparison between calcium balance and dynamic histomorphometry, Br. Med. J. 289 (1984) 517e520. H.M. Frost, Dynamics of bone remodeling, in: H.M. Frost (Ed.), Bone Biodynamics, Little Brown & Co, Boston, 1964. E.F. Eriksen, Normal and pathological remodelling of human trabecular bone: three dimensional reconstruction of the remodelling sequence in normals and in metabolic bone disease, Endocr. Rev. 7 (1986) 378e408. J. Compston, S. Vedi, S. Kaptoge, E. Seeman, Bone remodelling rate and remodelling balance are not co-regulated in adulthood: implications for the use of activation frequency as an index of remodelling rate, J. Bone Miner. Res. 22 (2007) 1031e1036. P. Lips, P. Courpron, P.J. Meunier, Mean wall thickness of trabecular bone packets in the human iliac crest: changes with age, Calcif. Tissue Res. 26 (1978) 13e17. P. Derkz, D.H. Birkenhager-Frenkel, A thionin stain for visualizing bone cells, mineralizing fronts and cement lines in undecalcified bone sections, Biotech. Histochem. 70 (1995) 70e74. T. Steiniche, E.F. Eriksen, H. Kudsk, L. Mosekilde, F. Melsen, Reconstruction of the formative site in trabecular bone by a new, quick, and easy method, Bone 13 (1992) 147e152. S. Vedi, J.R. Tighe, J.E. Compston, Measurement of total resorption surface in iliac crest trabecular bone in man, Metab. Bone Dis. Related Res. 5 (1984) 275e280. M.S. Burstone, Histochemical demonstration of acid phosphatase activity in osteoclasts, J. Histochem. Cytochem. 7 (1959) 39e41. R.A. Evans, C.R. Dunstan, D.J. Baylink, Histochemical identification of osteoclasts in undecalcified sections of human bone, Miner. Electrolyte Metab. 2 (1979) 179e185. P. Courpron, P. Lepine, M. Arlot, P. Lips, P.J. Meunier, Mechanisms underlying the reduction with age of the mean wall thickness of the trabecular basic structure unit (BSU) in human iliac bone, in: W.S.S. Jee, A.M. Parfitt (Eds.), Bone histomorphometry, 3rd International Workshop, Armour Montagu, Paris, 1980, pp. 323e329. P.I. Croucher, R.W.E. Mellish, S. Vedi, N.J. Garrahan, J.E. Compston, The relationship between resorption depth and mean interstitial bone thickness: age-related changes in man, Calcif. Tissue Int. 45 (1989) 15e19. A.M. Parfitt, J. Foldes, The ambiguity of interstitial bone thickness: A new approach to the mechanism of trabecular thinning, Bone 12 (1991) 119e122. E.F. Eriksen, H.J.G. Gunderson, F. Melsen, L. Mosekilde, Reconstruction of the resorptive site in iliac trabecular bone; a kinetic model for bone resorption in 20 normal individuals, Metab. Bone Dis. Related Res. 5 (1984) 235e242. S. Palle, D. Chappard, L. Vico, G. Riffat, C. Alexandre, Evaluation of the osteoclastic population in iliac crest biopsies from 36 normal subjects: a histoenzymologic and histomorphometric study, J. Bone Miner. Res. 4 (1989) 501e506.
[47] N.J. Garrahan, P.I. Croucher, J.E. Compston, A computerised technique for the quantitative assessment of resorption cavities in trabecular bone, Bone 11 (1990) 241e246. [48] M.E. Cohen-Solal, M.-S. Shih, M.W. Lundy, A.M. Parfitt, A new method for measuring cancellous bone erosion depth: application to the cellular mechanisms of bone loss in postmenopausal osteoporosis, J. Bone Miner. Res. 6 (1991) 1331e1338. [49] R.S. Weinstein, M.S. Hutson, Decreased trabecular width and increased trabecular spacing contribute to bone loss with ageing, Bone 8 (1987) 137e142. [50] R.W.E. Mellish, N.J. Garrahan, J.E. Compston, Age-related changes in trabecular width and spacing in human iliac crest biopsies, Bone Miner. 6 (1989) 331e338. [51] A.M. Parfitt, Bone remodelling in type 1 osteoporosis (letter), J. Bone Miner. Res. 6 (1991) 95e97. [52] E. Wakamatsu, H.A. Sissons, The cancellous bone of the iliac crest, Calcif. Tissue Res. 4 (1969) 147e161. [53] W.J. Whitehouse, The quantitative morphology of anisotropic trabecular bone, J. Microsc. 101 (1974) 153e168. [54] J.E. Aaron, N.B. Makins, K. Sagreiya, The microanatomy of trabecular bone loss in normal aging men and women, Clin. Orthop. Rel. Res. 215 (1987) 260e271. [55] E.C.G.M. Clermonts, D.H. Birkenhager-Frenkel, Software for bone histomorphometry by means of a digitizer, Comput. Math Prog. Biomed. 21 (1985) 185e194. [56] N.J. Garrahan, R.W.E. Mellish, S. Vedi, J.E. Compston, Measurement of mean trabecular plate thickness by a new computerized method, Bone 8 (1987) 227e230. [57] A.M. Parfitt, C.H.E. Mathews, A.R. Villanueva, M. Kleerekoper, B. Frame, D.S. Rao, Relationship between surface, volume and thickness of iliac trabecular bone in aging and in osteoporosis: implications for the microanatomic and cellular mechanism of bone loss, J. Clin. Invest. 72 (1983) 1396e1409. [58] J.E. Compston, Connectivity of cancellous bone: assessment and mechanical implications, Bone 15 (1994) 463e466. [59] L.A. Feldkamp, S.A. Goldstein, A.M. Parfitt, G. Jesion, M. Kleerekoper, The direct examination of bone architecture in vitro by computed tomography, Bone 4 (1989) 3e11. [60] A. Odgaard, H.J.G. Gundersen, Quantification of connectivity in cancellous bone with special emphasis on 3-D reconstruction, Bone 14 (1993) 173e182. [61] J. Serra, Image Analysis and Mathematical Morphology, Academic Press, London, 1982. [62] A. Vesterby, Star volume of marrow space and trabeculae in iliac crest: sampling procedure and correlation to star volume of first lumbar vertebra, Bone 11 (1990) 149e155. [63] P.I. Croucher, N.J. Garrahan, J.E. Compston, Assessment of cancellous bone structure: comparison of strut analysis, trabecular bone pattern factor and marrow space star volume, J. Bone Miner. Res. 11 (1996) 955e961. [64] M. Hahn, M. Vogel, M. Pompesius-Kempa, G. Delling, Trabecular bone pattern factor d a new parameter for simple quantification of bone microarchitecture, Bone 13 (1992) 327e330. [65] B.B. Mandelbrot, Fractals: form, chance and dimension, Freeman, San Francisco, 1977. [66] R.S. Weinstein, S. Majumdar, H.K. Genant, Fractal geometry applied to the architecture of cancellous bone biopsy specimens, Bone 13 (1992) A38. [67] G. Jacquet, W.J. Ohley, M.A. Mont, R. Siffert, R. Schmukler, Measurement of bone structure by fractal dimension, Proc. Ann. Conf. IEEE/EMBS 12 (1990) 1402e1403. [68] S. Majumdar, Magnetic resonance imaging for osteoporosis, Skeletal. Radiol. 37 (2008) 95e97.
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[69] J.F. Griffith, H.K. Genant, Bone mass and architecture determination: state of the art, Best Prac. Res. Clin. Endocrinol. Metab. 22 (2008) 737e764. [70] H.K. Genant, K. Engelke, S. Prevrhal, Advanced CT imaging in osteoporosis, Rheumatol. 47 (suppl. 4) (2008) 9e16. [71] H. Brockstedt, M. Kassem, E.F. Eriksen, L. Mosekilde, F. Melsen, Age- and sex- related changes in iliac cortical bone mass and remodelling, Bone 16 (1993) 681e691. [72] M.L. Frost, G.M. Blake, G.J. Cook, P.K. Marsden, I. Fogelman, Differences in regional bone perfusion and turnover between lumbar spine and distal humerus: (18)F-fluoride PET study of treatment-naı¨ve and treated postmenopausal women, Bone 45 (2009) 942e948. [73] A.E. Moore, G.M. Blake, K.A. Taylor, A.E. Rana, M. Wong, P. Chen, et al., Assessment of regional changes in skeletal metabolism following 3 and 18 months of teriparatide treatment, J. Bone Miner. Res. 25 (2010) 960e967.
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[74] M.L. Frost, G.M. Blake, S.J. Park-Holohan, G.J. Cook, K.M. Curran, P.K. Marsden, et al., Long-term precision of 18Ffluoride PET skeletal kinetic studies in the assessment of bone metabolism, J. Nucl. Med. 49 (2008) 700e707. [75] R.R. Recker, P.D. Delmas, J. Halse, I.R. Reid, S. Boonen, P.A. Garcia-Hernandez, et al., Effects of intravenous zoledronic acid once yearly on bone remodeling and structure, J. Bone Miner. Res. 23 (2008) 6e16. [76] R.R. Recker, L.G. Ste-Marie, B. Langdahl, E. Czerwinski, B. Bonvoisin, D. Masanauskaite, et al., Effects of intermittent intravenous ibandronate injections on bone quality and microarchitecture in women with postmenopausal osteoporosis: the DIVA study, Bone 46 (2010) 660e665. [77] I. Reid, P. Miller, J. Brown, D. Kendler, A. Fahrleitner-Pammer, I. Valter, et al., on behalf of the Denosumab Phase 3 Bone Histology Study Group, Effects of denosumab on bone histomorphometry: The Freedom and Stand studies, J. Bone Miner. Res. 25 (2010) 2256e2265.
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C H A P T E R
49 Radiology of Rickets and Osteomalacia Judith E. Adams Manchester Royal Infirmary, Oxford Road, Manchester, UK and Imaging Science and Biomedical Engineering, The University, Stopford Building, Oxford Road, Manchester, UK
INTRODUCTION AND HISTORICAL ASPECTS Bone resorption is a one-stage process, with osteoclasts resorbing mineral and osteoid together. In contrast, bone formation occurs in two stages: osteoblasts lay down osteoid, which subsequently becomes mineralized. The mineralization of bone matrix depends on the presence of adequate supplies of not only vitamin D, in the form of its active metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D), but also calcium, phosphorus, and alkaline phosphatase, and on a normal pH prevailing in the body environment. If there is a deficiency of these substances for any reason, or if there is severe systemic acidosis, then mineralization of bone will be defective. There will be a qualitative abnormality of bone (in contrast to osteoporosis, which is predominantly a quantitative abnormality of bone), with reduction in the mineral/osteoid ratio, resulting in rickets in children and osteomalacia in adults. In the immature skeleton, the radiographic abnormalities predominate at the growing ends of the bones (metaphyses), where endochondral ossification is taking place, giving the classic appearance of rickets. When the skeleton reaches maturity and the process of endochondral ossification has ceased, the defective mineralization of osteoid is evident radiographically as Looser’s zones (pseudofractures, Milkman’s fractures), which are pathognomonic of osteomalacia. Rickets and osteomalacia are therefore synonymous and represent the same disease process, but are the manifestation in either the growing or the mature skeleton. A large number of different diseases can cause the same radiological abnormalities of rickets and osteomalacia [1e3]. Rickets appeared when people began to live in cities during the Industrial Revolution. The first descriptions
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10049-6
of the condition are attributed to Daniel Whistler, who in 1692, while a student at Merton College Oxford, wrote a thesis entitled, “Inaugural Medical Disputation in the Disease of English Children which is popularly termed Rickets” for his doctorate at the University of Leyden [4]. Glisson in 1650 wrote a treatise for the Royal College of Physicians on, “De Rachitide sive morbo puerili qui vulgo the Rickets dicitur” [5,6]. In 1666 a postmortem examination was reported by John Locke of a child with rickets who died of pneumonia [7]. The disease was well known in Europe at this time and was regarded as “the English disease.” The origin of the term “rickets” is still debated [8,9]. Fish oil was recognized as a popular cure for “chronic rheumatism” (osteomalacia) and limb deformities (rickets) in children [10]. Robert Darbey, a house surgeon and apothecary to the Manchester Infirmary, was quoted by Percival in 1783 [11] as saying that 50e60 gallons of cod liver oil were issued every year to patients, usually for the treatment of chronic rheumatism [6]. In 1890 Palm [12] published an essay on the distribution of rickets around the world and noted that it was common in cities where people were deprived of sunlight. He suggested that rickets could be treated with sunlight but did not proceed to perform the experiment. Others observed that the disease is more common in cities [13]. There was much confusion between conditions that had similar clinical features but different courses of progression and responses to therapies of the day [14,15]. It was the microscopic studies of Pommer in 1885 [16] which distinguished, for the first time, between rickets, achondroplasia, and osteogenesis imperfecta. In December1895 Roentgen discovered X-rays, and it then became possible to display the radiographic features of rickets and osteomalacia.
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The unraveling of the structure and function of vitamin D and its metabolites during the twentieth century has elucidated the causes for confusion which existed in the past as to the etiology of rickets and the variable response to treatment (see Chapter 1). Vitamin D deficiency may occur as a consequence of simple nutritional deficiency (diet, lack of sunlight; see Chapters 52, 53 and 54), due to malabsorption states, chronic liver disease which affects hydroxylation at the 25 position (see Chapter 69), and chronic renal disease in which the active metabolite 1,25(OH)2D is not produced (see Chapter 70). Consequently, a large variety of diseases may result in vitamin D deficiency [17e22] (see Chapter 60). The radiological features of all will be similar, being those of rickets and osteomalacia. This similarity of radiological features but variations in response to treatment contributed to some of the early confusion [2,20,21]. Rickets due to nutritional deprivation was cured by ultraviolet light or physiological doses of vitamin D (400 IU per day), but that associated with chronic renal disease was not, except if very large pharmacological doses (up to 300 000 IU per day) were used. These variations in response to treatment led to the terms “refractory rickets” and “vitamin-D-resistant rickets” being used for these conditions. In these groups were included the diseases that caused the clinical and radiological features of rickets but were related to phosphate, not vitamin D, deficiency, such as X-linked hypophosphatemia (see Chapter 63) and genetic diseases involving defects in 1a-hydroxylase (Chapter 64) and the vitamin D receptor (Chapter 65).
VITAMIN D DEFICIENCY The hormone 1,25(OH)2D plays an important role in calcium homeostasis by its actions principally on the bone and intestine, but also on the kidney and parathyroid glands. It promotes the intestinal absorption of calcium and phosphorus from the intestine. On the bone it has two actions; one is to mobilize calcium and phosphorus from the skeleton as required, and the other is to promote maturation and mineralization of the osteoid matrix. Deficiency of vitamin D results in rickets in children and osteomalacia in adults [19e23]. There are known to be seasonal variations in vitamin D status, with plasma levels being lower in the winter months [24]. The pathophysiology, clinical descriptions, and treatments are discussed elsewhere in this volume.
Rickets In the growing skeleton the effects of vitamin D deficiency and consequent defective mineralization of
osteoid are evident at the growing ends of the bones [25e27]. In the early phase there is widening of the growth plate, which is the radiolucent (unmineralized) gap between the mineralized metaphysis and epiphysis [28,29]. As the changes become more severe, there is “cupping” of the metaphysis with irregular and poor mineralization (Figs 49.1e49.3). There is some expansion in width of the metaphysis which results in the apparent soft tissue swelling around the ends of the affected long bones. This produces the expansion at the anterior ends of the ribs referred to as a “rachitic rosary” (Fig. 49.1C) [30]. There is often a thin “ghostlike” rim of mineralization at the periphery of the metaphysis, since this mineralization occurs by membranous ossification at the periosteum. The margin of outline of the epiphysis also appears indistinct as endochondral ossification at this site is also defective. A method for scoring the severity of rickets has been described [31]. The changes are most pronounced at the sites of bone which are growing most actively. These sites, in sequence, are around the knee, the wrist (particularly the ulna, Figs 49.2 and 49.3), the anterior ends of the middle ribs, the proximal femur, and the distal tibia. It is these anatomical sites that should be radiographed if rickets is suspected. As rachitic bone is soft and bends additional features that develop, once the child begins to walk, are bowing of the legs (genu valgum) or knock-knees (genu varum), deformity of the hips (coxa valga or, more usually, coxa vara), in-drawing of the ribs at the insertion of the diaphragm (Harrison’s sulcus), and protrusio acetabulae and tri-radiate deformity of the pelvis. The latter can result in problems with parturition at subsequent pregnancies. Involvement of the bones of the thorax and respiratory tract (larynx and trachea) can result in stridor and respiratory distress [32,33]. Paradoxically, in very severe rickets in which little growth is taking place (i.e., owing to nutritional deprivation or chronic ill health), the features of rickets may not be evident at the growth plate [34]. In mild vitamin D deficiency, the radiographic features of rickets may only become apparent during the growth spurt associated with puberty, and then the changes are most prominent at the knee (Fig. 49.2). The radiographic changes may be quite subtle and not involve the entire metaphysis (Fig. 49.2B). The causes, presentation and features of rickets may vary according to the age of onset [27]. In the rickets of prematurity there may be little abnormality at the metaphysis, as no skeletal growth is taking place in the premature neonate. However, the bones are osteopenic and prone to fracture. Periosteal reaction is probably due to the accumulation of unmineralized osteoid on the periosteal surface (Fig 49.4) [35]. The cause in preterm infants is an inadequate supply of phosphorus
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(A)
(C)
(B)
FIGURE 49.1 Nutritional vitamin-D-deficiency rickets in a young child. Radiograph of the wrist of a child of approximately 2 years (A) and 3.5 years (B) showing widening of the growth plate with cupping and expansion of the metaphysis, which is poorly mineralized. The expansion results in soft tissue swelling around the ends of the bones. The cortical “tunneling” (subcortical erosion) and hazy trabecular pattern indicate secondary hyperparathyroidism. (C) Chest radiograph in an infant showing the bulbous expansion of the anterior ends of the ribs (arrows) known as a “rachitic rosary.”
and calcium during periods in hospital, or those receiving only unsupplemented human milk. In young infants vitamin D levels are closely related to maternal vitamin D status [36]. Although there has been a decline in the incidence of rickets, with improved social and
(A)
environmental awareness, vitamin D deficiency remains a significant public health problem among children of Southeast Asian and Middle Eastern immigrants as a result of a number of factors. These include diminished cutaneous vitamin D synthesis due to migration and
(B)
(C)
FIGURE 49.2 Nutritional rickets in Asian adolescent (A) anteroposterior (AP) radiograph of the knee showing widening of the radiolucent growth plate due to poor mineralization and some splaying and cupping of the metaphysic. In other children in whom the vitamin D deficiency is mild, the features of rickets only become apparent clinically and radiographically during the growth spurt of puberty in the knee (B) and ankle (C). The changes might be quite subtle and not involve the entire metaphysic, as illustrated. The widened growth plate is evident only at the medial aspect of the metaphysis of the distal femur and proximal tibia (B), and in the lateral aspect of the distal tibia and in the fibula (C). There is not the cupping, splaying, and irregular and poor mineralization of the metaphyses, which is evident in more florid rickets.
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(A)
(B)
(C)
FIGURE 49.3 Rickets and effect of treatment. (A) Wrist before treatment. The radiolucent growth plate is increased in width, and there is some cupping of the metaphyses of the distal radius and ulna with irregular and deficient mineralization. Note that the ulna growth plate is more severely affected than that in the radius. This is a consequence of the ulna growing in length exclusively from its distal end, the proximal end forming the olecranon. This is the most sensitive site for assessing the radiographic presence of rickets. (B) Wrist 4 months after treatment with vitamin D. There is healing of the rickets, although the distal segments of the radius and ulna are still reduced in density. This indicates the stage of bone development at which the rickets occurred and the amount of growth that has taken place since then. With time and remodeling the appearances will become normal. (C) A series of radiographs of the wrist taken over a period of 4 months following treatment with vitamin D; left image: there are the characteristic features of rickets with widening of the growth plates of the radius and ulna and poor mineralization of the metaphysis. Middle image at approximately 6e8 weeks: the radiolucent growth plate is less wide and there is increased mineralization of the metaphyses, but minor abnormalities are still present. Right image: the abnormalities previously present have now disappeared almost completely with healing of the rachitic changes.
residency in more northern latitudes where the sun is lower in the sky and voluntary avoidance of sunshine due to religious and cultural practices in which the skin is covered and so not exposed to sunlight [25]. There are the additional factors of the rachitic role of vegetarian diets and prolonged breastfeeding without vitamin D supplementation [25e27]. In the newborn and young infant softening of skull bones may result in craniotabes [37] and frontal bossing. Depending on the age of onset, there will also be effects on the teeth (delay in dental eruption and enamel hypoplasia).
If the vitamin D deficiency is treated appropriately, then the radiographic abnormalities of rickets will heal over about 2e3 months (Fig. 49.3). The radiographic features of healing will lag behind the improvement in biochemical parameters (2 weeks) and clinical symptoms. With treatment the unmineralized osteoid of the growth plate of the metaphysis and epiphysis will mineralize. This section of abnormal bone may be visible for a period of time and gives some indication as to the age of onset and duration of the period of rickets (Fig. 49.3). Eventually, this zone becomes
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(i.e., malabsorption, chronic renal disease) than with simple nutritional vitamin D deficiency (J.E. Adams, personal observation). With vitamin D deficiency, there is associated hypocalcemia. To maintain calcium homeostasis, the parathyroid glands are stimulated to secrete parathyroid hormone (PTH). This results in another important feature of vitamin D deficiency rickets [40]. Evidence of secondary hyperparathyroidism, with increased osteoclastic resorption (erosions, bone cysts), is always evident histologically (see Chapter 48), although radiographically evident features are uncommon [41e43], and cystic lesions of bone (brown tumors) are rare [44]. In a child with rickets changes at the growth plates predominate; Looser’s zones may also, but rarely, be present (Fig. 49.4).
Osteomalacia
FIGURE 49.4 AP radiograph of the legs of a young child with severe rickets. The radiolucent growth plate is increased in width; the metaphyses are splayed and poorly mineralized. There are also Looser’s zones (pseudo fractures) evident as radiolucent lines through the distal fibula bilaterally (arrows). These have some radiodense callus formation indicating that vitamin D therapy has already commenced. There is also some periosteal reaction evident. This is thought to be caused by the accumulation of unmineralized osteoid on the periosteal surface of bone which lifts the periosteum, stimulating it to mineralize.
indistinguishable from the normal bone with time and remodeling. The zone of provisional calcification that was present at the onset of the disturbance to endochondral ossification may remain (Harris growth arrest line) (see Fig. 49.6) as a marker of the age of skeletal maturation at which the rickets occurred [38]. However, this is not specific for rickets and can result from any condition (i.e., a period of ill health, lead poisoning, intermittent intravenous bisphosphonate therapy [39]) that inhibits normal endochondral ossification. There is evidence of retarded growth and development in rickets, but this is more marked when the vitamin D deficiency is associated with chronic diseases that reduce calorie intake, general well-being, and activity
At skeletal maturity the epiphysis fuses to the metaphysis and longitudinal bone growth ceases. However, bone turnover continues throughout life to maintain the tensile integrity of the skeleton. Vitamin D deficiency in the adult skeleton results in osteomalacia, the pathognomonic feature of which is the Looser’s zone (pseudo-fracture, Milkman’s fracture) [45e50]. Looser’s zones are radiolucent areas in the bone that are composed of unmineralized osteoid. They appear as radiolucent lines in the bone that are perpendicular to the bone cortex, do not extend across the entire bone, and characteristically have a sclerotic margin (Fig. 49.5) [51]. They can occur in any bone, but typically are found in the medial portion of the femoral neck, the pubic rami, the lateral border of the scapula, and the ribs. They may involve the first and second ribs, in which traumatic fractures are uncommon and are usually associated with severe trauma. Other less common sites are the metatarsals and metacarpals, the base of the acromium, and the ilium (Fig. 49.5D). Traumatic fractures of the ilium are rare and also require severe trauma as a cause, so if there is what resembles a fracture in the ilium, but no history of trauma, then a Looser’s zone should be considered as an etiology. The etiology of why Looser’s zones occur in the anatomical sites that they do has been much debated [52,53]. At one time, it was thought that they were in the sites of vascular channels, but this theory has been discarded. Their site is most likely to be related to sites of stress in the skeleton. Looser’s zones must be differentiated from insufficiency fractures that occur in osteoporotic bone, particularly in the pubic rami, sacral ala, and calcaneus [54e56]. Insufficiency fractures consist of multiple microfractures and often have florid callus formation, which differentiates them from Looser’s
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(B)
(C)
(E)
(D)
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(G)
Osteomalacia. (A) Nutritional osteomalacia in an Asian. AP radiograph of the right hip. Looser’s zones are generally radiolucent lines that are perpendicular to the bone cortex, do not extend fully across the bone (unless a fracture has occurred through the site), and have sclerotic margins as illustrated. There is also some protrusio acetabulae due to softening of the bone. (B) Pelvis in untreated celiac disease. Bilateral protrusio acetabulae and triradiate deformity of the pelvis are apparent. There are Looser’s zones through both superior and inferior pubic rami (arrows), with complete fracture through that in the left superior pubic ramus. (C) Nutritional osteomalacia. Chest radiograph: in the left apical region there is a Looser’s zone through the entire width of the left first rib (arrow). Traumatic fractures through the first and second rib normally occur only with severe trauma. (D) Malabsorption osteomalacia. Pelvic radiograph: there is an extensive Looser’s zone through the left ilium and the right pubic ramus (arrows). There is evidence of pinning of a previous left hip fracture. Severe trauma is required to fracture the ilium. Malabsorption osteomalacia. (E) Chest radiograph: there are Looser’s zones in the lateral border of the right scapula and at the base of the acromium (arrows). (F) Forearm with Looser’s zones with sclerotic margins in both the radius and ulna. Nutritional vitamin D deficiency (G) hand radiograph: with Looser’s zones in the mid shaft of the third metacarpal.
FIGURE 49.5
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(B)
FIGURE 49.6 Stigmata of rickets in childhood. (A) Pelvis showing stigmata of past rickets with a triradiate deformity of the pelvis and varus deformity of the femoral necks. (B) Bowing of tibia and fibula with evidence of Harris growth arrest lines in the distal shaft of the tibia.
zones [57]. Incremental fractures occur in Paget’s disease of bone and resemble Looser’s zones in appearance (translucent zone, suggesting incomplete fracture, with sclerotic margin), but these tend to occur on the outer (convex) cortex of the bone involved. The typical features of Paget’s disease (sclerotic, disordered trabecular pattern, enlarged bone) serve as distinguishing radiographic features [58]. Complete fractures can occur through Looser’s zones. As in rickets, osteomalacic bone is soft and bends. This is evident by protrusio acetabulae, in which the femoral head deforms the acetabular margin so that the normal “teardrop” outline of the acetabulum is lost (Fig. 49.5A). There may be bowing of the long bones of the legs and triradiate deformity of the pelvis (Fig. 49.6), particularly if the cause of the vitamin D deficiency has persisted since childhood and has been inadequately treated or untreated. As in rickets, secondary hyperparathyroidism is present and can be manifested radiographically as subperiosteal erosions, particularly in the phalanges, but at other sites also (sacroiliac joints, symphysis pubis, proximal tibiae, outer ends of the clavicle, and “pepper-pot” skull), depending on the intensity of the hyperparathyroidism (Fig. 49.7). There can also be cortical tunneling
and a “hazy” trabecular pattern. There may be generalized osteopenia, and vertebral bodies may have concave end plates. This is due to softening of the osteomalacic bone, which is deformed by the cartilagenous intervertebral disc (“codfish” deformity) [59]. The etiology of this deformation is different from that which results in endplate irregularity in osteoporosis, in which microfractures occur owing to the bone being brittle rather than soft.
Secondary Hyperparathyroidism The most sensitive site for the radiographic features of hyperparathyroidism is the radial sides of the middle phalanges of the second and third fingers. There are characteristic erosions along the cortical surface of these bones (Fig. 49.7AeC). If there is florid hyperparathyroidism, then cortical erosions may be seen more widely and involve the distal phalanges (Fig. 49.7A), the outer ends of the clavicle (Fig. 49.7D), the symphysis pubis, the sacroiliac joints, the upper medial cortex of the tibiae, and the skull vault (“pepper-pot” skull) (Fig. 49.7E). The erosions of hyperparathyroidism in the sacroiliac joints tend to involve the iliac margin of the joint (Fig. 49.7F), in contrast to
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49. RADIOLOGY OF RICKETS AND OSTEOMALACIA
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(D)
(C)
(B)
(E)
(F)
(G)
FIGURE 49.7 Erosions of hyperparathyroidism. (A) Acrosteolysis and resorption of the distal phalanges resulting in pseudoclubbing. There
are also subperiosteal erosions in the lateral (radial) cortex of the middle phalanges of the second, third, and fourth fingers (azotemic osteodystrophy). (B) Intracortical tunneling of the cortex of the phalanges, with subperiosteal erosions in the radial cortex of the middle phalanx of the index finger. (C) Extensive subperiosteal erosions along the radial border of the middle phalanx of the third finger. There is adjacent metastatic vascular calcification in the digital artery indicating phosphate retention and azotemic osteodystrophy. (D) Erosions in the outer end of the clavicle. (E) Lateral skull showing erosions in skull vault giving pepper-pot appearance. (F) Erosions along the iliac margin of the left sacroiliac joint. (G) Erosions in the symphysis pubis (coronal tomogram).
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the involvement of both joint surfaces in inflammatory and erosive arthritides. The erosions of hyperparathyroidism in the skull vault (“pepper-pot” skull) must be differentiated from the “granularity” of the parietal region of the skull vault, which may be a variant of normal. In the former, the margins of the erosions are indistinct; in the latter they are distinct and corticated. Loss of the lamina dura around the teeth does occur but is not specific to hyperparathyroidism, occurring also in other conditions such as Paget’s disease of bone and dental infection. With intense hyperparathyroidism, there is an increase in cortical “tunneling” due to bone resorption and an indistinct, “hazy” trabecular pattern (Fig. 49.7B). Erosions may occur along the growth plate and result in displacement of the epiphysis from the metaphysis of the shaft of the bone. This is most likely to occur in association with chronic kidney disease (CKD) (see below), since the intensity of the hyperparathyroidism secondary to vitamin D deficiency is related to the duration and severity of the hypocalcemia which acts as the stimulant.
RENAL OSTEODYSTROPHY The bone disease that occurs in chronic renal impairment, namely, renal osteodystrophy or uremic (azotemic) osteodystrophy, is complex and multifactorial [60e63], and has changed in both clinical and imaging features over the past three decades (63,64). Previously there occurred a combination of vitamin D deficiency, which resulted in rickets and osteomalacia, and hypocalcemia [65,66], the latter inducing severe secondary hyperparathyroidism that stimulated osteoclastic resorption of bone [67e69]. This resulted radiographically in subperiosteal erosions, most frequently identified along the radial aspect of the middle phalanx of the second and third fingers (Fig. 49.7AeC). Because the stimulus to secondary hyperparathyroidism in CKD was intense and longstanding, the skeletal manifestations were often extensive and manifest radiographically not only as subperiosteal erosions in the hands, but other features also (e.g., “pepper-pot” skull, brown “tumors” causing bone cysts, osteosclerosis, and metastatic calcification). However, these features are now rarely evident on radiographs. This has occurred through the better understanding of vitamin D metabolism and improvements in therapeutic management of patients with chronic renal impairment (calcitriol, 1a-vitamin D, renal transplantation, and dialysis). Metastatic calcification in soft tissues and “adynamic” bone disease (related to aluminum) remain problematic, and new complications have developed as a consequence of treatments (dialysis and transplantation), including
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amyloid deposition, noninfective spondyloarthropathy, osteonecrosis, and osteopenia/osteoporosis, all of which may have characteristic imaging features (62,63,70e72).
Hyperparathyroidism Subperiosteal Erosions Subperiosteal erosions occur most commonly in the middle phalanges but are also present in the distal phalangeal tufts, causing acro-osteolysis and the clinical sign of “pseudo-clubbing” (Fig. 49.7A). Other sites of erosions include the outer end of the clavicle (Fig. 49.7D), the medial aspect of the proximal portion of the tibia, humerus, and femur, and the superior and inferior borders of the ribs [73]. Erosions may also occur adjacent to joints, and consequent damage to the articular subchondral bone can cause symptomatic arthritis [74]. These erosions may simulate those that occur in rheumatoid arthritis (RA) but tend to be a little further from the joint margin and are not generally associated with joint narrowing, periarticular osteopenia, or soft tissue swelling, which are early features of RA [75]. Joints that can be affected include the acromioclavicular, sternoclavicular, sacroiliac, and the symphysis pubis (Fig. 49.7G). In the hand, the distal interphalangeal joints and ulnar aspect of the metacarpophalangeal joints can be involved [76]. Subperiosteal erosions of the phalanges are diagnostic of hyperparathyroidism. In children, erosions can occur in the region of the growth plate, causing radiographic abnormalities which may be mistaken for rickets and which can result in slipped epiphysis and deformity [77,78] (Fig. 49.8). If the hyperparathyroidism is treated successfully erosions will fill in, and the cortex will revert to its normal appearance. However, if there has been severe resorption of the distal phalangeal tufts, these cannot reconstitute to their normal shape and may remain shortened and stubby (Fig. 49.7A). Bone resorption can occur in the regions of insertion of tendons and ligaments, particularly the trochanters, the ischial and humeral tuberosities, the inferior aspect of the calcaneus, and around the elbow. Excessive bone resorption in the skull vault causes the mottled texture of alternating areas of lucency and sclerosis referred to as a “salt and pepper” or pepper-pot skull (Fig. 49.7E). As the intensity of hyperparathyroidism is now much less intense and longstanding than previously, through the introduction of effective treatments (calcitriol, 1a-vitamin D, renal transplantation, and dialysis) there may be no features present radiographically, or only subperiosteal erosions in the phalanges. If there are no erosions in this site, then it is unlikely that they will be found in other sites; skeletal surveys previously
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49. RADIOLOGY OF RICKETS AND OSTEOMALACIA
(B)
(A)
Renal osteodystrophy in an adolescent in the past. (A) Pelvis showing rickets (widened growth plate at femoral metaphyses; varus deformity of femoral necks) and hyperparathryoidism (bone sclerosis and erosions in sacroiliac joints and along femoral metaphyses) resulting in slipped upper femoral epiphysis at right. (B) Anteroposterior view of ankle showing extensive erosions of the metaphyses due to severe secondary hyperparathyroidism. With improved treatment of patients with chronic kidney disease (calcitriol, 1a-vitamin D, renal transplantation, and dialysis), one should no longer see such cases of rickets and intense secondary hyperparathyroidism related to azotemic osteodystrophy.
FIGURE 49.8
performed in patients with chronic renal impairment are now inappropriate, particularly as parathyroid hormone levels can be measured directly; a hand radiograph would suffice.
Intracortical Bone Resorption Intracortical bone resorption is caused by osteoclastic resorption of Haversian canals within the cortex of the bones. Radiographically this causes linear translucencies within the cortex (Fig. 49.7B). This feature is not specific for hyperparathyroidism and can be found in other disorders in which bone turnover is increased (e.g., Paget’s disease of bone) [58]. Osteosclerosis Osteosclerosis occurs uncommonly in primary hyperparathyroidism but was a common feature of disease secondary to CKD. Radiographically the bones appeared increased in density (Fig. 49.9). This affected particularly the central (axial) skeleton. In the vertebral
bodies the endplates were preferentially involved, giving bands of dense bone adjacent to the end plates with a central band of lower, normal bone density. These alternating bands of normal and sclerotic bone gave a striped pattern described as a “rugger jersey” spine (Fig. 49.9A and B). The osteosclerosis may be more generalized. It may result from excessive accumulation of poorly mineralized osteoid, which would appear more dense radiographically than normal bone. It was also suggested that it results from an exaggerated osteoblastic response following bone resorption [79]. Brown Tumors Brown tumors represent cavities within the bone in which there has been excessive osteoclastic resorption. Histologically they are filled with fibrous tissue and osteoclasts, and may undergo necrosis and liquefaction. Radiographically they appear as radiolucent cysts within the bone. They can occur anywhere in the skeleton and may cause expansion of bones (Fig. 49.10). They constitute the osteitis fibrosa cystica of
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(A)
(C)
(B)
Renal osteodystrophy. (A) Bone sclerosis of the lateral spine radiographs showing endplate sclerosis, giving a “rugger jersey” appearance of alternating bands of dense stripes in the lumber (A) and thoracic (B) spine. (C) Hand radiograph showing Looser’s zone at the base of the first metacarpal, indicating osteomalacia, and generalized osteosclerosis due to secondary hyperparathyroidism. With improved management of chronic kidney disease the intensity of secondary hyperparathyroidism is less than in previous decades.
FIGURE 49.9
hyperparathyroidism first described by Von Recklinghausen. When appropriate treatment was given these bone cysts would fill with woven bone and increase in density (Fig. 49.10D). Osteoporosis With excessive bone resorption, combined with defective mineralization, the bones can appear osteopenic (reduced in radiographic density) in some patients [80]. Periosteal Reaction Periosteal new bone formation (radiodense line parallel to the periosteal cortex of a bone) used to be observed in up to 25% of patients with renal osteodystrophy. It occurred most frequently in those with severe bone disease and is thought to be a manifestation of intense hyperparathyroidism. It occurs in the metatarsals, femur, and pelvis and less commonly in the humerus, radius, ulna, tibia, metacarpals, and phalanges [81,82].
Metastatic Calcification With the reduced glomerular function of chronic renal failure there is phosphate retention [83]. This results in an increase in the calcium X phosphate product, and, as a consequence, amorphous calcium phosphate is precipitated in organs and soft tissues [84]. It is now recognized that fibroblastic growth factor-23 (FGF23) is elevated in CKD and is a powerful predictor of clinical outcomes [85e87]. FGF23 requires Klotho, a single-pass transmembrane protein expressed
in renal tubules, as an obligate coreceptor to bind and activate FGF receptors [88] (see Chapter 42). The metastatic calcification can occur in the eyes and skin, causing symptoms of “gritty,” sore eyes and itching, and, in severe cases, calciphylaxis (ischemic necrosis of soft tissue) [89,90]. Other organs in which calcium is deposited include the heart, stomach, kidneys, lung, and skeletal muscle, where it is rarely detected radiographically. However, radiographic evidence of metastatic calcification is seen in arteries and around the joints (Fig. 49.11). The periarticular calcification is more common around the large joints (hip, shoulder) but can also occur around small joints. The calcification can involve the joint capsule or tendons but more usually lies in the bursae adjacent to joints and bony protuberances (ischial tuberosity). These metastatic deposits of calcium will cause swelling and may be painful. They may increase rapidly in size and are sometimes erroneously diagnosed as “tumors” on initial clinical examination. Whereas rickets, osteomalacia, and secondary hyperparathyroidism have become much less evident radiographically in patients with CKD over the past three decades, the prevalence of metastatic calcification has perhaps become more frequent in recent years [91]. This may be due to a combination of excess phosphate not being removed effectively by dialysis, the increased use of calcium carbonate as a phosphate binder, and the occurrence of “adynamic” renal bone disease [92,93]. This may result in the skeleton being a less effective reservoir for calcium than normal, so that the calcium remains in the extracellular fluid.
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49. RADIOLOGY OF RICKETS AND OSTEOMALACIA
(A)
(B)
(C)
(D)
Brown “tumors” in renal osteodystrophy (osteiitis fibrosa cystica) in hyperparathyroidism. (A) Oblique radiograph of right lower ribs showing a lytic area expanding the rib (arrow). In renal osteodystrophy: right knee lateral (B) and anteroposterior (C) projections showing welldefined lytic areas in the distal femur and the proximal fibula. The lesions are causing expansion of the bone with cortical destruction and soft-tissue masses, radiological features of aggressive bone lesions. (D) Following parathryoidectomy and regression of the secondary hyperparathyroidism, the destructive lesions have filled in with woven bone and so have increased in density, and the cortex has reconstituted although there is still expansion of the fibula. Such florid features of hyperparathyroidism are now infrequently seen with improved management of CKD.
FIGURE 49.10
These calcific masses can regress with appropriate treatment, e.g. phosphate binders (Fig. 49.11B, C and E) [94]. Initially the masses may liquefy, and apparent fluid levels are seen on radiographs taken with the patient in the erect position. These fluid levels are the result of the interface between serum
and serum plus mineral. There can be complete regression of periarticular calcific masses with appropriate treatment; vascular calcification rarely regresses [95e97]. The metastatic calcification in end-stage CKD can involve the intimal layer of the coronary arteries. This
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(C)
(B) (A)
(D)
(E)
Metastatic calcification in renal osteodystrophy. (A) Extensive vascular calcification in vessels of the foot. (B) Extensive “tumor-like” calcification around the right shoulder. There is also bowing of the proximal humerus due to osteomalacic bone softeneing. (C) Following treatment with a phosphate binder (aluminum hydroxide or calcium carbonate), these calcified masses can liquefy and regress as illustrated. If the radiographs are taken with the patient in the erect position, there will be seen fluid/fluid mineral interface levels (arrows). (D) Pelvis showing extensive soft-tissue calcification around the ischia and left hip. Widening of the sacroiliac joints indicates erosions of hyperparathyroidism. (E) Fingers showing (left) extensive metastatic calcification around the distal phalanx and subperiosteal erosions along the radial cortex of the middle phalanx. (Right) Following therapy with oral phosphate binder there is reduction in the extent of the metastatic soft tissue calcification. Note the acro-osteolysis due to hyperparathyroidism. Although the features of osteomalacia and secondary hyperparathyroidism are now infrequently seen radiographically with improvement of CKD management, metastatic calcification is still prevalent. This may be due to a combination of ineffective removal of phosphate through dialysis, the use of calcium carbonate as a phosphate binder and “adynamic” bone disease.
FIGURE 49.11
is common, severe, and significantly associated with ischemic cardiovascular disease. The latter is the etiology of death in half the patients on dialysis. With recent developments in imaging (initially electronbeam computed tomography (EBT) and now spiral, multidetector computed tomography (MDCT)) quantitative assessment of the calcification in coronary arteries
is feasible [98e101]. The calcium in coronary arteries is highly correlated to myocardial infarction and angina in patients on dialysis. EBT and MDCT have the potential to identify those patients at highest risk of cardiovascular morbidity and mortality [99,101]. Abdominal aortic calcification (AAC) (Fig. 49.12) has also been found to be correlated to cardiovascular
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(B)
(A)
FIGURE 49.12 Quantitative assessment of the calcification: in coronary arteries, which is correlated to myocardial infarction and angina in patients on dialysis can be made with MDCT. Abdominal aortic calcification (AAC) has also been found to be correlated to cardiovascular morbidity and mortality. AAC can be assessed using lateral lumbar spine radiograph (A) and, more recently, on lateral spinal images (B) acquired on dual energy X-ray absorptiometry (DXA) scanners to assess for vertebral fracture (vertebral fracture assessment (VFA)) using a 24, or more simple 8, point scoring method.
morbidity and mortality. AAC can also be assessed using lateral lumbar spine radiographs (Fig. 49.12A) and, more recently, on lateral spinal images acquired on dual-energy X-ray absorptiometry (DXA) scanners used for bone densitometry to assess for vertebral fracture (vertebral fracture assessment (VFA)) (Fig. 49.12B) and on computed tomography [102e104], using a 24, or more simple 8, point scoring method [105,106].
Aluminum Toxicity Aluminum toxicity occurred in patients with chronic renal disease due to excessive ingestion of aluminum
hydroxide taken to reduce serum phosphate. It also occurs in those patients on hemodialysis in which the dialysate water contained excessive amounts of aluminum [107e109]. The control of aluminum content in dialysate water in recent years has reduced the prevalence of this disorder [110]. Aluminum accumulates at the bone/osteoid interface and inhibits mineralization. This results in rickets and osteomalacia, but the bone also becomes “adynamic,” with reduced osteoid production and turnover. Radiographically this results in reduced bone density and easy fracture [111]. These fractures can occur in unusual sites (second to fourth ribs, odontoid) or have an atypical appearance in
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long bones [111]. In patients with azotaemic osteodystrophy and aluminum toxicity, there is less osteosclerosis, fewer periosteal erosions and increase in the rate of osteonecrosis following transplantation, than occurs in those individuals with chronic renal failure but without excess aluminum [108]. Recent understanding of the role of the calciumsensing receptor and development of calcimimetics (e.g., cinacalcet) have shown that in CKD the latter will reduce parathyroid hormone, calcium (Ca), phosphate (P), and CaeP product levels, and so the risks of vascular calcification [112].
RENAL TUBULAR DEFECTS AND HYPOPHOSPHATEMIA Inorganic phosphate, glucose, and amino acids are absorbed in the proximal renal tubule; concentration and acidification of the urine in exchange for a fixed base occur at the distal renal tubule. Disorders of the renal tubules may involve either the proximal or distal tubule or both. They will result in a spectrum of biochemical disturbances that may result in loss of phosphate, glucose, or amino acids alone, or in combination, with additional defects in urine acidification and concentration. These defects may be inherited and present from birth (de Toni-Fanconi syndrome, cystinosis, X-linked hypophosphatemia or XLH) [113] or acquired later in life (e.g., Wilson’s disease, hereditary tyrosinemia, neurofibromatosis, mesenychymal tumors and cadmium poisoning; some drug-induced toxicity (ifosfamide)) [114e116] can interfere with tubular function and function can also be impaired by either crystal deposition (e.g., copper, in Wilson’s disease) or a humoral substance, such as is produced by tumors in tumor-induced osteomalacia (TIO), also known as “oncogenic” rickets [117]. It is the renal tubular disorders that cause phosphaturia which result in rickets and osteomalacia [118]. As the serum calcium is generally normal in these diseases, secondary hyperparathyroidism does not occur (see Chapter 63). Hypophosphatemic rickets has also been described in association with the epidermal naevus syndrome [119].
X-linked Hypophosphatemia (XLH) The genetic disorder XLH is transmitted as an X-linked dominant trait. Sporadic cases through spontaneous mutations also occur. An autosomal dominant variety with variable penetrance has also been described [120]. The incidence is approximately 1 in 25 000, and XLH is now the most common of genetically induced rickets [121e123]. The pathophysiology of XLH and its mode of treatment are discussed in detail in Chapter 63).
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The disease results through mutations in the PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome, previously known as PEX), which codes for a type 2 membrane glycoprotein that activates the putative phosphateregulating hormone known as “phosphatonin” [19,25, 124], now recognized to be FGF23 which is elevated [125e128]. The condition is characterized by lifelong phosphaturia, hypophosphatemia, and rickets and osteomalacia. Rickets becomes clinically evident around 12e18 months of age. The radiological features of XLH are characteristic. Although in the past affected patients were often short in stature with quite marked deformities (bow-legs), improvement in management (therapy with oral phosphate and 1,25(OH)2D3) have reduced these consequences of the disorder [129]. Rickets in XLH There is widening of the growth plate and defective mineralization of the metaphysis; however the metaphyseal margin tends to be less indistinct than in nutritional rickets, and there is less expansion in the width of the metaphysis (Fig. 49.13). Changes are most marked at the knee, wrist, ankle, and proximal femur. Healing does occur with appropriate treatment. The growth plates fuse normally at skeletal maturation. The bones are often short and undertubulated (shaft wide in relation to bone length), with bowing of the femur and tibia (Fig. 49.13D). Osteomalacia in XLH After skeletal maturation Looser’s zones persist in patients with XLH. These tend to occur in sites different from those in nutritional osteomalacia, and are often present in the outer cortex of the bowed femur (Fig. 49.13BeD), although they occur along the medial cortex of the shaft also [130]. Looser’s zones in the ribs and pelvis are rare. They may heal with appropriate treatment, but those that have been present for many years will persist and are presumably filled with fibrous tissue (see Fig. 49.15A and B). In the untreated patient there is no evidence of hyperparathyroidism as there is no hypocalcemia, in contrast to vitamin-D-deficiency osteomalacia. However, treatment with large doses of oral phosphate for long periods may induce secondary hyperparathyroidism, which may exceptionally be evident by subperiosteal erosions in the hand [131]. Osteosclerosis in XLH Although there is defective mineralization of osteoid in XLH, the bones are commonly increased in density, with a coarse and prominent trabecular pattern [132] (Fig. 49.13). This is a characteristic feature of the disease
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49. RADIOLOGY OF RICKETS AND OSTEOMALACIA
(A)
(B)
(D)
(C)
Familial X-linked hypophosphatemic rickets (XLH). (A) Anteroposterior view of ankle showing widened growth plate and Harris growth arrest lines. The appearance of the metaphysis is different from that in nutritional rickets (see Fig. 49.1). (B) Pelvis showing rachitic changes at the proximal femoral metaphyses, dense bones with a coarse trabecular pattern, and bowing of the femoral shafts with bilateral Looser’s zones (through medial cortex at right and outer cortex at left (arrows)). (C) Pelvis showing rickets at the proximal femoral metaphyses, dense bones with coarse trabecular and chronic Looser’s zones through the medial aspect of the proximal shafts of both femora. (D) Femora showing rickets at the proximal and distal femoral metaphyses, and the femoral are bowed and undertubulated with broad shafts. There are Looser’s zones through the medial cortex in the proximal shaft of each femur. The bones are also increased in density.
FIGURE 49.13
and is not related to treatment with vitamin D and phosphate supplements, as it can be present in those who have not received treatment. This bone sclerosis has been shown to involve the petrous bone and structures of the inner ear and may be responsible for the hydropic cochlear pattern of deafness that these patients can develop in later life [133]. Deformity and thickening of the skull and involvement of the skull base can rarely be associated with stenosis of the foramen magnum with a type 1 Chiari malformation and syringomyelia [134,135]. Abnormalities of Bone Modeling In XLH the bones are often short, with widening of the shaft (undertubulation). The ribs are broad and tend to slope downward more than normal, causing a bell-shaped chest (Fig. 49.14A). There can be
broadening of the distal end of the ulna (Fig. 49.14B), and often marked bowing of the femur and tibia [136]. Extraskeletal Ossification X-linked hypophosphatemia is characterized by an enthesopathy (inflammation in the junctional area between bone and tendon insertion) that heals by ossification of ligament and tendon insertions to bone in many affected patients [137,138]. This results in new bone formation around the pelvis and spine, with the changes resembling ankylosing spondylitis (Fig. 49.15). There can be complete ankylosis of the spine, which limits movements. As there is no inflammatory arthritis, the sacroiliac joints are normal, an important radiographic feature that serves to differentiate this condition from ankylosing spondylitis (Fig. 49.16A, B). Ossification can occur in the interosseous membrane of the
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(A)
(B)
Modeling deformities in familial X-linked hypophosphatemic rickets (XLH). (A) Chest radiograph showing that the ribs are broad and slope downward more than normal, giving a rather bell-shaped chest. (B) Wrist showing some bulbous expansion of the distal shaft of the ulna.
FIGURE 49.14
forearm, forming a synchondrosis between the radius and ulna, and in the leg between the tibia and fibula (Fig. 49.15D, E). Separate small ossicles can occur around the joints of the hands (Fig. 49.15C); there is also ossification at tendon insertions in the hands, causing “whiskering” of bone margins. Rarely, spinal cord compression may be caused by a combination of ossification of the ligamentum flavum, thickening of the laminae, and hyperostosis around the apophyseal joints [139] (Fig. 49.16CeF). It is the ossification of the ligamentum flavum that causes the most significant narrowing of the spinal canal [140,141]. This occurs most commonly in the thoracic spine and generally involves two or three adjacent vertebral segments. Patients may be asymptomatic, even with severe spinal canal narrowing, and acute cord compression can be precipitated by quite minor trauma. It is important to be aware of this rare, but recognized, complication of the disease since surgical decompression by laminectomy is curative. The extent of ossification cannot be predicted by the degree of paraspinal or extraskeletal ossification at other sites. Computed tomography (CT), with its cross-sectional depiction of anatomy, is a useful imaging technique for demonstrating the extent of intraspinal ossification (Fig. 49.16CeF) [140]. Extraskeletal ossification is uncommon in patients with XLH under 40 years of age. Osteoarthritis in XLH The deformity and bowing of the long bones in XLH cause altered stress through joints, predisposing to
degenerative joint disease. This is particularly prominent in the knee joint. The extent to which the radiographic abnormalities discussed above are present varies between affected individuals [142]. In some, all the features are present and so are diagnostic of XLH. In others, there may only be minor abnormalities, and the condition may be overlooked [143].
Tumor-Induced (“Oncogenic”) Rickets/Osteomalacia Tumor-induced osteomalcia (TIO) or “oncogenic” rickets and osteomalacia were first reported in 1947 [117,144]. This is a rare paraneoplastic syndrome occurring in patients between 7 and 73 years, although most patients are over 30 years of age [145e147] (see Chapter 63). The condition is associated with phosphaturia. Presentation is with gradual onset of muscle weakness and bone pain in the legs, hips, and back. Children may present with disturbance of gait, swollen joints and genu valgum or varum. The clinical and radiographic features of rickets or osteomalacia can precede identification of the causative tumor by long periods (1e19 years). Bilateral Looser’s zones can occur in characteristic sites, and have been described in the tibia, where they radiographically mimic stress fractures that might occur in athletes in this site [148,149]. There is hypophosphatemia due to excessive urinary phosphate loss, and serum concentrations of
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49. RADIOLOGY OF RICKETS AND OSTEOMALACIA
(A)
(B)
(C)
(D)
(E)
FIGURE 49.15 Enthesopathy and extraskeletal ossification in XLH. (A) Anteroposterior view of pelvis and left hip (B) showing a triradiate deformity of the pelvis indicative of rickets in childhood. There is new bone formation around the hip joints and at lesser trochanters. There is a chronic Looser’s zone in the outer cortex of the proximal shaft of the left femur. Note the normal sacroiliac joints which help in distinguishing the etiology of paravertebral ossification, which occurs in XLH, from that which occurs in ankylosing spondylitis. (C) Hands showing ossicles and bony outgrowths related to heads of the metacarpals and joints. (D) Forearm showing synostosis between the radius and ulna due to ossification of the interosseous ligament. (E) Lower leg showing deformity due to bowing and chronic Looser’s zones with ossification of the interosseous membrane. Such severe deformity reflects inadequate treatment during childhood.
1,25(OH)2D are inappropriately normal, low or undetectable. It is important to consider the diagnosis in cases of rickets and osteomalacia and specifically request measurement of the serum phosphate, as this may not be included in routine serum analysis. The syndrome is now known to be caused by production of FGF23 (or other phosphatonins) by the causative tumor [150e152]. The causative tumors are usually small, benign, and vascular in origin (hemangiopericytoma) [153,154], but some may be malignant [155] (Fig. 49.17). Approximately 50% of tumors are located in bone and 50% arise in soft tissues, and the most common anatomical sites are the legs and craniofacial regions [145,156,157]. The phosphaturic mesenchymal tumor (mixed connective tissue variant) (PMTMCT) is an extremely rare, distinctive tumor that is frequently associated with oncogenic
osteomalacia. Despite this association many PMTMCTs go unrecognized because they are erroneously diagnosed as other mesenchymal tumors [158]. Expression of FGF23 has been shown in a small number of mesenchymal tumors associated with oncogenic osteomalacia. Recognition of the syndrome and localization of the causative tumor is crucial as the levels of FGF23 fall and the rickets and osteomalacia will heal with surgical removal of the tumor [159e162]. Often the tumors are extremely small and elude detection for many years (Fig. 49.18). It is important that the patient is vigilant about self-examination and reports any small palpable lump or skin lesion. Modern imaging methods (CT, magnetic resonance imaging (MRI) and radionuclide scanning (ocreotide)) may be helpful in localizing more deep-seated lesions [145,163e171] (Fig. 49.19F). The condition is discussed
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RENAL TUBULAR DEFECTS AND HYPOPHOSPHATEMIA
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FIGURE 49.16 Changes in XLH. (A) Lateral lumbar spine showing ossification of the paraspinal ligaments and at apophyseal joints. The appearances resemble those of ankylosing spondylitis (AS) but can be differentiated from AS by the sacroiliac joints being normal and not eroded as would occur in the seronegative spondyloarthropathies. (B) Anteroposterior view of lumbar spine showing ossification of paraspinal ligaments resulting in ankylosis but normal sacroiliac joints (arrows). (CeF) Narrowing of the spinal canal may be caused by various factors. Computed tomography (CT) scan (C) through the thoracic spine showing thickening of the laminae and hypertrophy of the apophyseal joints causing narrowing and trefoil deformity of the spinal canal. (D) CT through the lower thoracic spine showing severe narrowing of the spinal canal by posterior ossification of the ligamentum flavum. (E) CT thoracic spine of a different patient showing narrowing of the spinal canal by new bone formation (arrows) lying anterior to the laminae severely narrowing the spinal canal. (F) Sagittal reformation of thin (3 mm) contiguous CT sections showing ossification of the ligamentum flavum (arrows), severely reducing the anteroposterior diameter of the spinal canal. C, E, and F reprinted from Adams and Davies [140] with permission. VI. DIAGNOSIS AND MANAGEMENT
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Deficiency in alkaline phosphatase will also result in rickets and osteomalacia. This occurs in hypophosphatasia, which is an inborn error of metabolism first reported in 1946 by Rathburn [180]. There is an accumulation of the enzyme substrates, including phosphoethanolomine, and of inorganic phosphate, which promotes the development of articular chondrocalcinosis. There are several forms of the disorder, which varies in its severity and age of onset [181].
VITAMIN D INTOXICATION
FIGURE 49.17 Tumor-induced or oncogenic osteomalacia in a 46-year-old woman who 18 months previously had presented with hypophosphatemic osteomalacia. There is a lytic lesion in the proximal fibula. This was a fibrosarcoma. The osteomalacia healed following surgical excision of the tumor.
in detail in Chapter 63). If no causal lesion comes to light with thorough imaging and spontaneous remission occurs the condition described as “pseudo-(tumorinduced) rickets” should be considered, to avoid prolonged medical treatment and futile searches for a neoplasm [172].
ACIDEMIA The mineralization of osteoid also requires a normal pH to prevail in the body environment. If there is a systemic acidosis then the features of rickets or osteomalacia may be evident radiographically. This may occur with ileal replacement of the ureters [173] and with chronic and excessive antacid ingestion [174,175].
In times gone by vitamin D was advocated in the treatment of a great variety of conditions, including tuberculosis (especially lupus vulgaris), sarcoidosis, rheumatoid arthritis, hay fever, chilblains, and asthma. This treatment had no beneficial effect in these conditions, and its use was eventually abandoned, but not before many cases of vitamin D intoxication had been described [182,183]. Although vitamin D intoxication has become less common with the advent of 1,25(OH)2D3 and other active metabolites, the new era of vitamin D usage to treat cancer, psoriasis, and immunological disease (see Section IX of this volume) may see a resurgence of interest in vitamin D intoxication. Clinically, the symptoms are fatigue, malaise, weakness, thirst and polyuria, anorexia, nausea, and vomiting due to hypercalcemia (see Chapter 72). The hypercalcemia results in hypercalciuria, nephrocalcinosis, renal impairment, and hypertension. Metastatic calcification and bone sclerosis also occur [184,185] (Fig. 49.20).
TECHNICAL ASPECTS OF IMAGING Radiographs
DIFFERENTIAL DIAGNOSES Other etiologies may cause radiographic abnormalities of the metaphyses that have to be distinguished from those of rickets. These are the syndromes associated with metaphyseal dysostoses (chondrodysplasias) (e.g., types of Jansen, Schmidt, etc.) (Fig. 49.19). The metaphyseal changes can vary in severity from mild, such as occur in the Shwachman-Diamond syndrome (Fig. 49.19A, B), in which there is associated neutropenia and pancreatic insufficiency, to more significant fragmentation of the metaphyses [176e178] (Fig. 49.19C, D). In contrast to rickets of vitamin D deficiency, although the metaphyses are fragmented and splayed, they do not exhibit the same cupping and widening of the radiolucent growth plate [179].
Despite tremendous developments and expansion in the imaging techniques available since the 1970s, radiographs remain the principal imaging method in the radiographic diagnosis of metabolic bone disorders, including those involving vitamin D deficiency, rickets, and osteomalacia. When radiographing the hands and feet, in the past image quality was optimized by using fine-grain, single-sided emulsion film and a fine X-ray focal spot (0.6 mm or less). Meticulous attention to detail of the radiographic technique used enhanced the diagnostic features present in the hand radiograph, such as the subperiosteal erosions and intracortical tunneling of hyperparathyroidism. Magnification techniques, either optical or radiographic, could further enhance identification of such diagnostic features of metabolic bone disease. High-resolution radiographs of the torso
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TECHNICAL ASPECTS OF IMAGING
(A)
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(B)
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FIGURE 49.18 Acquired hypophosphatemic osteomalacia. A 40-year-old man presented with low back and buttock pain. He was thought to have ankylosing spondylitis. (A) Pelvic radiograph, which was considered to be normal at the original hospital of referral. (B) Radionuclide scan of pelvis showing increased uptake in both femoral necks, which are sites of Looser’s zones. (C) Radionuclide scan of upper torso showing multiple hot spots in ribs. The distribution of the hot spots suggests Looser’s zones. A radionuclide scan may be more sensitive than a radiograph for identifying Looser’s zones. (D) The patient subsequently had a road traffic accident in which he suffered bilateral femoral neck fractures through the Looser’s zones, which in retrospect are subtly present in the original radiograph (A). The patient was found to have hypophosphatemia presumed to be tumor-induced, but no tumor was identified after 15 years, despite careful clinical examination and other imaging techniques. However, more recently he had a radionuclide ocreotide scan which showed a hot spot in the pelvis (E). The sagittal reformatted crude CT image (F) performed on the gamma camera at the end of the scan indicates the lesion lies anterior to the bladder. Modern PET CT scanners would give superior-quality CT images. (G) CT through the pelvis confirms a small sclerotic lesion in the left superior ramus, adjacent to the symphysis pubis. This lesion was treated by image-guided thermal ablation which successfully resulted in remission of the hypophosphatemia. Figures B, C, E and F courtesy of Dr Mary Prescott, Consultant in Nuclear Medicine, and Figure G courtesy of Dr Richard Whitehouse, both of the Manchester Royal Infirmary, UK. Figure E case report reference [199] (Moran and Paul Int Orthop 2002;26:61e62 with permission). VI. DIAGNOSIS AND MANAGEMENT
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49. RADIOLOGY OF RICKETS AND OSTEOMALACIA
(A)
(B)
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Differential diagnoses. Metaphyseal dysostosis (chondrodysplasia). The metaphyseal abnormalities may be mild and resemble rickets as evident in the knees (A) and proximal femora (B) in this patient with Shwachman-Diamond syndrome, in which there is associated neutropenia and pancreatic insufficiency. In more severe types there is greater fragmented mineralization of the metaphyses in the wrists (C) and the hips (D) which do not resemble the radiographic abnormalities of rickets. Hypophosphatasia, which is an inborn error of metabolism in which alkaline phosphatase levels are reduced, also results in rickets, as is evident in the knee (E) of this affected child, and osteomalacia in affected adults (F). As there is no specific therapeutic options Looser’s zones are chronic, as evident in the neck and shaft of the right femur, and require repeated orthopedic interventions, such as the intramedullary nailing, which has been undertaken in this case. Chondrocalcinosis may also be evident.
FIGURE 49.19
VI. DIAGNOSIS AND MANAGEMENT
TECHNICAL ASPECTS OF IMAGING
(A)
883
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Vitamin D intoxication with associated renal impairment. (A) Pelvis showing bone sclerosis, calcification of vessels in the pelvis, and periosteal reaction around the pelvic brim. There is also calcification of the iliolumbar ligaments (B) Lateral foot showing bone sclerosis and calcification of the ligaments in the foot. There was calcification of ligaments in other sites and of the falx and tentorium in the head.
FIGURE 49.20
regions of the body were generally precluded because of the high radiation doses required. Over the past decade computed radiography (CR) and digital radiography (DR) enable the acquisition of electronic images in picture archiving and communications systems (PACS) leading to the replacement of screen-film radiography by digital detectors. Although there has been some loss of spatial resolution the advantages of such digital imaging is a wide dynamic range and image enhancement and manipulation, which enable improved image presentation (e.g., magnification) and reduced rates of repeat exposures [186]. New imaging techniques are discussed in Chapter 50.
Nuclear Medicine In the imaging technique of the skeleton referred to as nuclear medicine, 99mTc-labeled phosphate compounds are administered intravenously [187,188]. They are incorporated into the skeleton, particularly in sites that have either increased vascularity or increased new bone formation. Such areas of increased uptake are evident as “hot spots” on the scan, which is performed, using a gamma camera, 2e4 h after administration of the radionuclide. This radionuclide scanning (RNS) technique is a good method to survey the entire skeleton which is very sensitive to disease in bone. However, RNS lacks specificity, in that numerous pathologies may cause hot spots, including infection, Paget’s disease of bone, metastases, and degenerative joint disease (hyperostosis). Radiographs of the relevant anatomical site will help to differentiate these various pathologies. The radionuclide bone scan is sensitive to detecting Looser’s zones that may not be evident radiographically [189e193] (Fig. 49.18). The areas of increased uptake of
radionuclide may be bilateral and symmetrical and be present in anatomical sites typical for Looser’s zones (femoral necks, ribs, pubic rami) [194]. If there is associated secondary hyperparathyroidism, there will be a generalized increase in uptake of the radionuclide by the skeleton (“super scan”), with elevation of the bone/soft tissue ratio. There will be poor renal uptake of the radionuclide if the cause of the osteomalacia and vitamin D deficiency is chronic renal disease. With appropriate treatment of the osteomalacia, the Looser’s zones will heal, and the hot spots will not be present on subsequent radionuclide scanning. Radionuclide scanning offers a method of monitoring response of osteomalacia to therapy [195,196]; however, clinical symptoms, biochemical parameters, and radiographs often suffice for this purpose. As mesenchymal tumors have somatostatin receptors, and may be associated with tumor-induced osteomalacia, Indium-111-labeled pentetreotide has become an important imaging technique to identify the presence and confirm the site of such tumors, which may otherwise prove elusive to clinical localization [197e199] (Fig. 49.18E). In the past if there was an abnormal area of uptake on the RNS then it was helpful to perform CT or MRI scanning targeted at the area of abnormality, since these imaging methods offer higher spatial resolution and superior anatomical detail of the site and size of the lesion (Fig. 49.19G). However, technical developments in scanners now combine the radionuclide positron emission tomography (PET) with CT through image coregistration [169,170]. Radionuclide scanning in children is of limited value in metabolic bone disorders, because there is high uptake in the normal metaphysis of the growing skeleton. In addition, the examination carries a significant ionizing radiation dose to the bone marrow.
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Other Imaging Techniques Ultrasonography (US), computerized tomography (CT), magnetic resonance imaging (MRI), and angiography generally play little part in the diagnosis and management of rickets and osteomalacia. The exception to this is their application to the identification and localization of tumors that cause oncogenic rickets. Such tumors are often very small and may be deep-seated, so they can be extremely difficult to identify. Wholebody MR scanning and CT have proved useful in such identification and localization [167,200].
Bone Mineral Densitometry Methods of bone densitometry play an important role in diagnosis of patients with osteoporosis and monitoring the efficacy of treatment. Integral (cortical and trabecular “areal” bone mineral density (BMDa; g/cm2) measurement using dual-energy X-ray absorptiometry (DXA) of the lumbar spine (L1e4) and hip (femoral neck and total hip) is the method most widely used in clinical practice [201e203]. Quantitative computed tomography (QCT) has some advantages (separate volumetric BMD measurement (mg/cm3) of cortical and trabecular bone in central and peripheral skeletal sites), but remains largely a research method [204e206]. In disease, and treatment, these techniques can provide complementary information because they measure different types of bone in different sites of the skeleton. In vitamin D deficiency and rickets or osteomalacia, there may be osteopenia, but it is important to remember that DXA will not differentiate if low bone calcium (reduced BMDa) is due to osteoporosis or osteomalacia. If there is secondary hyperparathyroidism the forearm cortical BMDa measurement might show the most marked reduction [207,208]. If the vitamin-D-deficiency osteomalacia is treated appropriately, there is very rapid increase (þ25% or more) in BMDa (2e4 weeks) on serial bone densitometry (see Chapter 60).
The pathognomonic feature of osteomalacia is the Looser’s zone (pseudofracture). Many different diseases that result in rickets and osteomalacia (vitamin D deficiency, calcium deficiency, hypophosphatemia, hypophosphatasia, and acidemia) may therefore have similar radiographic appearances. There may be distinguishing features, such as bone sclerosis and extraskeletal ossification in XLH. In conditions in which there is hypocalcemia (vitamin D deficiency), this acts as a stimulus to secondary hyperparathyroidism, features of which can be identified radiographically (bone erosions). The bone disease of CKD, known as renal osteodystrophy, is complex, being a combination of rickets and osteomalacia (1,25(OH)2D deficiency), secondary hyperparathyroidism (bone erosions, sclerosis), and metastatic calcification (due to phosphate retention and raised levels of FGF23). However, the pattern of bone disease in CKD has changed over the past 30 years with improved knowledge of vitamin D metabolism, treatments to prevent vitamin D deficiency (calcitriol, 1a-vitamin D, transplantation, and dialysis). It is therefore now rare in CKD to see florid radiographic evidence of rickets and osteomalacia with the associated features of severe and longstanding secondary hyperparathyroidism that was evident in the past. However, metastatic calcification in soft tissues still occurs and remains problematic. Radiographs remain the most important imaging technique for the diagnosis of metabolic bone disease; radionuclide scans may be more sensitive for identifying Looser’s zones. Radionuclide scanning (indium-111labeled ocreotide; PET CT) and other techniques (ultrasound, computed tomography, magnetic resonance imaging) have a role in localizing tumors that induce hypophosphatemic (“oncogenic”) osteomalacia. Multidetector computed tomography (MDCT) is particularly well suited to demonstrate the intraspinal ossification, which is a rare, but recognized, complication of the enthesopathy associated with XLH-linked osteomalacia.
References
CONCLUSIONS Mineralization of bone matrix depends on the presence of adequate supplies of not only vitamin D, in the form of its active metabolite 1,25(OH)2D, but also calcium and phosphorus and the presence of alkaline phosphatase and a normal pH. If there is deficiency of these substances for any reason, or if there is severe acidosis, then mineralization of bone will be defective. This results in rickets in childhood and osteomalacia in adults. Radiographically, rickets is evident by bone deformity caused by softening and metaphyseal abnormalities where endochondral ossification is defective.
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metabolic bone disease, Nucl. Med. Commun. 6 (1985) 141e148. B.D. Nguyen, E.A. Wang, Indium-111 pentetreotide scintigraphy of mesenchymal tumour with oncogenic osteomalacia, Clin. Nucl. Med. 24 (1999) 130e131. S.M. Jan de Beur, E.A. Streeten, A.C. Civelek, E.F. McCarthy, L. Uribe, S.J. Marx, et al., Localisation of mesenchymal tumours by somatostatin receptor imaging, Lancet 359 (2002) 761e763. M. Moran, A. Paul, Ocreotide scanning in the detection of a mesenchymal tumor in the pubic symphysis causing hypophosphatemic osteomalacia, Int. Orthop. 26 (2002) 61e62. S. Fukumoto, Y. Takeuchi, A. Nagano, T. Fujita, Diagnostic utility of magnetic resonance imaging skeletal survey in a patient with oncogenic osteomalacia, Bone 25 (1999) 375e377. J.E. Adams, Dual Energy X-ray Absorptiometry, in: S. Grampp (Ed.), Radiology of osteoporosis, second ed., Springer, Berlin Heidelberg, 2008, pp. 105e124. J.E. Adams, N. Bishop, Dual energy X-ray absorptiometry (DXA) in adults and children. In: American Society of Bone and Mineral Research (ASBMR) Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, seventh ed.; Editor: C. Rosen 2009 pp 152e158 G.M. Blake, I. Fogelman, Dual energy X-ray absorptiometry and its clinical applications, Semin. Musculoskel. Radiol. 6 (2002) 207e217. G. Guglielmi, T.F. Lang, Quantitative tomography, Semin. Musculoskel. Radiol. 6 (2002) 219e227. K. Engelke, J.E. Adams, G. Armbrecht, P. Augat, C.E. Bogado, M.L. Bouxsein, et al., Clinical Use of Quantitative Computed Tomography and Peripheral Quantitative Computed Tomography in the Management of Osteoporosis in Adults: The 2007 ISCD Official Positions, J. Clin. Densitom. 11 (2008) 123e162. J. Adams, Quantitative computed tomography, Europ. J. Radiol. 71 (2009) 415e424. J. Wishart, M. Horowitz, A. Need, B.E. Nordin, Relationship between forearm and vertebral mineral density in postmenopausal women with primary hyperparathyroidism, Arch. Intern. Med. 150 (1990) 1329e1331. P.L. Selby, M. Davies, J.E. Adams, E.B. Mawer, Bone loss in celiac disease is related to secondary hyperparathyroidism, J. Bone Miner. Res. 14 (1999) 652e657.
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C H A P T E R
50 High-Resolution Imaging Techniques for Bone Quality Assessment Andrew J. Burghardt, Roland Krug, Sharmila Majumdar Musculoskeletal Quantitative Imaging Research Group, Department of Radiology and Biomedical Imaging, University of California, San Francisco, CA, USA
INTRODUCTION The skeleton is composed of cortical and trabecular bone, both compartments contributing to bone strength and the resistance of bone to fracture. The strength of bone and risk of fracture are important outcomes in the study of growth and peak bone accrual, aging, postmenopausal bone loss, cancer-related bone loss, and conditions such as diabetes, osteogenesis imperfecta, osteoarthritis, rheumatoid arthritis, and others. Currently, determination of fracture risk is primarily based on assessment of bone mineral density (BMD) values obtained through areal or volumetric X-ray imaging techniques. While BMD has been shown to have utility in predicting bone strength [1], it does not entirely determine fracture risk [2,3] or adequately assess the full impact of therapeutic interventions [4,5]. For these reasons much interest currently exists in the investigation of other factors associated with bone mechanical competence and therapeutic response, including whole-bone geometry, cortical and trabecular microarchitecture, and tissue composition. Analysis of bone morphology e including the microarchitecture of trabecular bone, and the Haversian and lacunocanalicular ultrastructure of cortical bone (Fig. 50.1) e is critical in understanding bone mechanics, assessing fracture risk, and evaluating responses to disease, age, and therapy. Improved predictions of biomechanical properties have been found as a result of including measures of trabecular microarchitecture in statistical regressions [6,7]. Trabecular microarchitecture is also critical in the evaluation of therapeutic interventions, enabling researchers to explain a greater proportion of the effect of drugs on fracture risk than BMD alone [8,9]. Similarly, the ultrastructure of cortical
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10050-2
bone is an important determinant of bone strength [10,11], is critical in fracture initiation and propagation [12], and known to change with aging [13], disease [14], and therapy [15]. Therefore the development of technology that delivers quantitative assessment of these bone quality factors represents an important goal to advance the understanding of skeletal health. Quantitative imaging techniques to evaluate the threedimensional microarchitecture of trabecular and cortical bone have been developed using two primary modalities: X-ray computed tomography (CT) and magnetic resonance imaging (MRI). The hallmark of the high-resolution imaging techniques described in this chapter is that they allow independent assessment of cortical and trabecular compartments and have the ability to resolve the microstructural features of cortical and cancellous bone.
X-RAY COMPUTED TOMOGRAPHY (CT) Computed tomography (CT) is a 3D X-ray imaging technique, where the image intensity reflects the X-ray attenuation produced by the tissue in the threedimensional voxel. The X-ray attenuation depends on the atomic number of the tissue components within the volume and thus bone due to the relative electrondense inorganic component (calcium hydroxyapatite) of the matrix [16] produces a larger X-ray attenuation as compared to soft-tissue and fat. The attenuation is expressed in terms of Hounsfield units, which is 0 for water and the values increase with increasing bone density, while fat typically has a Hounsfield number that is negative. Quantitative calibration of X-ray attenuation to bone mineral density is accomplished by
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FIGURE 50.1 The primary morphologic features that contribute to bone strength are the macroscale bone geometry (A) and the spatial distribution of microscale features including the trabecular microarchitecture (B) and cortical ultrastructure (C).
imaging reference phantoms containing objects of known hydroxyapatite concentrations [17,18]. For an excellent resource on the image formation fundamentals and the technical aspects of CT imaging see Kalender [19]. In summary, the image formation process begins with the acquisition of sequential radiographic projections over a number of angular positions around the object of interest (Fig. 50.2) which are then reconstructed using established computational techniques based on Radon projection theory [19]. Computed tomography technology has become a critical tool for both preclinical and clinical research into disorders of the musculoskeletal system. This technology has been used for multiscalar assessment of bone, from the skeletal level, to the level of the trabecular microarchitecture, and as low as the cellular scale of individual osteocyte lacunae. Clinical whole-body CT, with modern systems featuring multiple detector rows
(MDCT), has been established for bone densitometric [20,21], geometric [22], and biomechanical analyses [23,24]. The use of these whole-body scanners to image trabecular bone in the axial and peripheral skeleton has been investigated at several research institutes [25e29]. Recently a standard MDCT gantry has been combined with 2D flat panel detector technology (fp-vCT) to provide rapid continuous acquisitions at high isotropic spatial resolution [30,31]. In the last 5 years a high-resolution, limited field of view CT device has become commercially available for dedicated imaging of bone structure in the peripheral skeleton [32e35].
Multidetector Computed Tomography (MDCT) Image Acquisition MDCT is a clinical CT technique, which is available in most diagnostic imaging departments using scanners
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pixel resolution that is desired. This is dependent on whether the data will be utilized for deriving densitometric, biomechanical, or trabecular structural measures. The ultimate trade-off for in vivo applications is radiation dose limits. Table 50.1 shows a comparison of different parameters, and radiation dose for CT. Image Analysis DENSITOMETRIC ANALYSIS
FIGURE 50.2 In this schematic, the generalized configuration for
computed tomography is illustrated. Photons emitted from an X-source (top) are directed towards a photon detector array (bottom). The object is situated within the X-ray beam and partially transmits X-rays according to its material composition. The X-ray source and detector rotate relative to the object, acquiring radiographic projections over a number of angular positions. The cross-sectional anatomy can be mathematically reconstructed from the stack of projections.
from a number of manufacturers. Therefore, a dedicated scanner is not required. Image acquisition in CT varies based on the scanner type, resolution, and the effective TABLE 50.1
With advances in CT technology, improved computational algorithms and quality assurance approaches [36], the use of CT as a measure of bone density, in addition to dual X-ray absorptiometry (DXA), has seen a recent resurgence. The prior issues related to poor reproducibility due to nonmatched regions being analyzed from single-slice acquisitions have been circumvented by volumetric acquisitions and computerized image registration and matching techniques [37,38]. The use of CT and MDCT provide separation of cortical and trabecular bone compartments, which given the potential differences in these two compartments to metabolic and therapeutic stimuli, makes CT a very attractive technique for assessing bone quantity, quality, and strength. The radiation dose in CT-based bone density assessments is higher than DXA (Table 50.1), depends on the resolution of the densitometric scan, and other parameters, however, it still remains lower than abdominal CT scans and some radiographs, thus making it a viable tool for skeletal assessments. Using CT, Black et al. showed therapeutic changes using QCT compared to dual X-ray DXA, as well as differences in response between trabecular and cortical bone [4,20,21,39]. Using QCT of the
Radiographic Imaging Techniques, Summary of Radiation Doses
Modality
References
Primary manufacturers
Radiograph
[56]
DXA MDCT/ fp-vCT
Effective dose
Skeletal sites
FOV size
Voxel size
Multiple
Lumbar spine: AP Chest
e
e
0.7 mSv 0.02 mSV
[56]
Hologic, Lunar
Hip Spine
e
e
9 mSv 13 mSv
[25,26,28,29,31, 43,47e49,56]
General Electric Phillips Siemens Toshiba
Specimens (ex vivo) forearm (in vivo) spine femur (in vivo)
100e250 mm
156e300 mm 300e500 mm thickness) 250e500 mm 300e700 mm thickness)
(in plane) (slice
e5 mSv 8 mSv
(in plane) (slice
mCT
[72e75]
Scanco Siemens Skyscan Xradia
specimens biopsies (ex vivo)
2e100 mm
0.3e100 mm (isotropic)
N/A
HR-pQCT
[32, 33, 282]
Scanco
Specimens (ex vivo) distal radius distal tibia (in vivo)
126 mm
41e123 mm (isotropic)
3e4 mSv
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radius Genant et al. have shown differences between placebo and Denosumab treated women over 24 months (Fig. 50.3) [40]. MORPHOMETRIC ANALYSIS
To characterize trabecular bone structure and cortical thickness, MDCT techniques have been extended into higher-resolution imaging modes where volumetric acquisitions achieve in-plane resolutions ranging from 150e300 mm, and a minimum slice thickness of 300 mm. These pixel sizes (not to be confused with actual resolution in CT) are greater than trabecular dimensions and resolving individual trabeculae is subject to considerable partial volume effects. The analysis of trabecular microarchitecture from MDCT and flat panel volumetric CT (fp-vCT) image data has primarily involved the application of traditional histomorphometry [26,28,29,41e43] where BV/TV, Tb.N, Tb.Th, and Tb.Sp are calculated in 2D using plate model assumptions [44]. Calibration studies have shown that, with the large width of intertrabecular spaces, trabecular bone parameters obtained with this technique correlate moderately well with those determined in contact radiographs from histological bone sections and mCT (r ¼ 0.53e0.70) [45,46] as well as mCT (r ¼ 0.44e0.99) [28,29,43,47e49]. Ito et al. used direct 3D measures of trabecular dimensions [50], connectivity [51], and SMI [52] in a MDCT study of the lumbar spine, finding a strong correlation (r ¼ 0.98) between BV/TV measured by mCT and MDCT. Other specialized measures of trabecular dimensions using the fuzzy distance transform (which does not require a threshold binarization process) were proposed by Krebs et al. [49]. BIOMECHANICAL ANALYSIS
Utilizing CT mineral density information and continuum assumptions from solid mechanics theory, mathematical finite element models have been constructed from CT image data to predict apparent bone mechanical properties [23]. In the last several years, investigators have applied these techniques to large clinical trial and normative databases of CT images to derive apparent biomechanical properties of the lumbar spine and proximal femur [53e55]. Applications The advantage of the MDCT technique is that central regions of the skeleton critically relevant to osteoporosis and fracture risk assessment, such as the spine [25,29,49] and proximal femur [26,28], can be visualized. However, to achieve adequate spatial resolution and image quality, the required radiation exposure is substantial, which offsets the technique’s applicability in clinical, routine, and scientific studies (Table 50.1). High-resolution CT protocols are typically associated with an effective dose of approximately 3 mSv (1.5 years of natural
background radiation) e several orders of magnitude greater than for standard DXA or HR-pQCT (4e13 mSv), and an order of magnitude higher than standard 2D QCT (0.06e0.3 mSv) [56]. To date in vivo human studies using MDCT to assess bone structure have been limited due to radiation dose concerns. Ito et al. demonstrated SMI and BV/TV measured from MDCT images of the lumbar spine provided superior fracture discrimination to aBMD by DXA [25]. Graeff et al. showed that teriparatide treatment effects were better monitored by architectural parameters of the spine obtained through MDCT than by BMD (Fig. 50.4) [27]. In a cross-sectional cohort study of adolescent girls with and without anorexia nervosa (AN), Bredella et al. observed significantly diminished trabecular microarchitecture at the distal radius in AN subjects compared to controls, despite no differences in lumbar aBMD. In a companion study, Lawson et al. found that the abnormal trabecular microarchitecture in these patients was predicted by IGF-1, leptin, and androgen levels [57].
Microcomputed Tomography (mCT) Microcomputed tomography (mCT) has become an important tool for investigating a wide range of aspects related to the biology of bone [58]. It has achieved widespread use in the laboratory for rapid, nondestructive, multiscale imaging of bone specimens [59e61] and noninvasive imaging in animal models [62,63]. The pervasive use of this technology has widely eclipsed traditional histomorphometry for quantitatively assessing trabecular microarchitecture and cortical ultrastructure. Additionally mCT image data can be used to characterize bone tissue composition [64e67] and to construct micromechanical models for computational virtual loading experiments [68e70]. Collectively, these technological advances have contributed to the accelerated pace of research on bone metabolism and skeletal disease. Image Acquisition CONVENTIONAL mCT
Laboratory mCT devices (Fig. 50.5) typically use a cone beam, polychromatic X-ray source, which produces photons spanning a broad range of energies [59]. While the first commercial mCT scanner consisted of a single-row 512-pixel detector [71], modern scanners employ areal CCD detectors up to 11 megapixels and capable of acquiring projection data for over 1000 slices simultaneously [72e75]. The nominal resolution (isotropic voxel size) of these imaging systems ranges from approximately 500 nm to 60 mm and covers a maximum field of view between 40 and 100 mm. The
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FIGURE 50.3 Dual-energy X-ray absorptiometry (DXA) and quantitative computed tomography (QCT) images showing the areas studied along the radius (A). The anatomic locations of the QCT slices used for this analysis are given relative to the corresponding DXA scan and illustrate the relative proportions of cortical and trabecular bone compartments. For placement comparison, a forearm DXA scan image is provided. The effects of denosumab on the total bone compartment at month 24 (B). Model-adjusted mean values are expressed as the percentage change from baseline with associated 95% CI. BMD, bone mineral density; BMC, bone mineral content; Circ, circumference; Q6M, every 6 months; Vol, volume. *P < 0.05; ***P < 0.001 vs. placebo. (Reprinted with permission, from [40].)
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actual spatial resolution (10% MTF) is typically two or three times the voxel size. Dedicated specimen ex vivo scanners are typically designed with the specimen oriented on a high-resolution motorized stage for translation and rotation (Fig. 50.5). In this scenario the source and detector remain in a fixed position during the scan, while the specimen rotates in the field of view.
(A)
SYNCHROTRON RADIATION mCT (SR-mCT)
The use of synchrotron radiation (SR) sources for high-resolution microtomographic imaging predates the commercial development of polychromatic mCT [60,76e80]. Access to SR beamlines is possible on a very limited basis at several particle accelerator facilities across the world (Fig. 50.6). Synchrotron radiation microtomography (SR-mCT) offers several important advantages over conventional mCT systems. Synchrotron accelerators yield high X-ray fluxes which allow for short acquisition times while maintaining a high signalto-noise ratio (SNR). A high flux also allows for the option of selectively passing a narrow energy, or monochromatic beam, thereby avoiding beam-hardening effects inherent to polychromatic mCT [66]. Furthermore, the parallel geometry of a synchrotron X-ray beam eliminates spatial artifacts seen with cone beam sources used in standard laboratory mCT systems. Collectively these features have made it possible to develop SR-mCT systems able to acquire high-contrast images with resolutions below the micron level [10,11,81]. In addition to the advantages related to image quality, a benefit of using a monochromatic X-ray beam is that image reconstruction yields true, energy-specific linear attenuation coefficients. Because the X-ray attenuation properties of calcium hydroxyapatite (HA) e the primary X-ray attenuating constituent of bone e are theoretically known, the linear attenuation values in the grayscale images are unambiguously related to tissue-level mineral density. The precision and accuracy of SR-mCT as a tool for quantifying tissue mineral density has been demonstrated in studies from several synchrotron facilities [64,66,82,83].
(B)
IN VIVO mCT
In contrast to ex vivo mCT, preclinical mCT systems designed for in vivo imaging of small animals utilize a fixed gantry with the X-ray source and detector ensemble rotating and translating about the field of view [62,63,84]. This modality is particularly advantageous because it allows for longitudinal monitoring of Rectangular “virtual biopsies” taken from T12 at baseline and after 6 and 12 months of teriparatide therapy (from left to right). Two orthogonal views of the rendered, segmented image data for each point in time are shown (A). The app. BV/TV values for this individual were 0.07, 0.10, and 0.14 at baseline, 6 months, and
FIGURE 50.4
12 months, respectively. Comparison of treatment-induced changes (B) in standardized units (per SD of baseline variability; mean SE). App. BV/TV increases are significantly larger compared with BMD and app. Tb.N increases (*P < 0.05; **P < 0.01). Reprinted with permission, from [27].)
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(A)
(B) FIGURE 50.5 Schematic diagram of the mCT imaging process. In this case and tube X-ray source (foreground), standard for laboratory mCT systems, emits X-ray photons in a cone-beam configuration. The photons are attenuated as they transmit through the object positioned in the field of view, casting a projected shadow on the 2D CCD detector (background). The object rotates in the field of view while the X-ray source and detector remain stationary in order to acquire projections over 180 . These projections are used to mathematically reconstruct a 3D image of X-ray attenuation.
bone quality within an individual animal, which reduces the effect of biological variability in a study and obviates the need for large numbers of individual animals for each time point of interest. Furthermore, with the aid of image registration to align baseline and follow-up images [85e87], the opportunity to monitor animal-specific microstructural changes in a spatially resolved fashion can provide unique insight into disease progression or the action of therapy [85,88,89]. For in vivo mCT studies, and longitudinal in vivo imaging experiments in particular, cumulative exposure to X-ray radiation may introduce adverse or confounding biological effects [90,91]. For detailed guidelines on experimental design and technical considerations for small animal mCT imaging we refer the reader to the excellent summary by Bouxsein et al. [92]. Image Analysis QUANTITATIVE mCT
Analogous to clinical QCT, the grayscale attenuation values of reconstructed mCT images can be converted to hydroxyapatite (HA) concentration. Various
FIGURE 50.6 Photograph of the experimental hutch from
a synchrotron radiation mCT beamline at the European Synchrotron Radiation Facility in Grenoble France (A) (Image courtesy of Dr. Galateia Kazakia, Ph.D., University of California, San Francisco). The specimen is seen mounted on a rotational/translational stage in the foreground, while the detector is immediately behind it, in the path of the parallel, monochromatic X-ray beam that enters the hutch from the right. This ensemble is capable of generating nanoscale CT images as seen in this reconstructed image (700 nm voxel size) of the spinal ossification center of an embryonic mouse at day 18 of gestation (B).
calibration procedures based on idealized phantoms or theoretical calculations have been established to derive relations between attenuation and bone mineral density [64,65,67,82,93,94]. Like clinical CT, the polychromatic X-ray source used in conventional mCT systems produces an X-ray beam with photons spanning a broad range of energies. Because X-ray attenuation in a material is an energy-dependent phenomenon, the energy spectrum of the X-ray beam is modified as it is transmitted: low-energy, or “soft” X-ray photons are
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(A)
(B)
Morphometric analysis of trabecular bone is performed in 3D from mCT images. Here a 5 mm 3 mm 3 mm block of trabecular bone from a cadaveric distal radius (18 mm voxel size) is visualized with the 3D distances mapped onto surfaces in pseudocolor demonstrating calculations for trabecular thickness, Tb.Th (A) and trabecular separation, Tb.Sp (B). Please see color plate section.
FIGURE 50.7
absorbed or scattered at a higher rate than high-energy photons, shifting the X-ray beam’s spectrum towards a higher mean energy e a process known as “beamhardening.” If unaccounted for, this phenomenon introduces spatially variant errors in the depiction of mineralization that additionally depend on object size and composition [95]. Therefore, minimization of beamhardening effects is required for reliable quantitative density calibration in conventional mCT. One common approach is to use filters of various compositions and widths to narrow the X-ray energy spectrum by blocking soft X-rays [94]. While all commercial scanners employ filtration at the aperture of the X-ray source, there is variability in the degree among manufacturers. The loss of X-ray intensity through filtration has a negative effect on noise performance and can be compensated for only by longer scan times. Another option is to derive empirical corrections for beam-hardening effects, which can be applied to the raw image data prior to reconstruction [65]. Using these procedures, apparent bone mineral density and tissue-level mineral density can be accurately measured [66]. MORPHOMETRIC ANALYSIS
Morphometric indices analogous to classical histomorphometry can be calculated from mCT images of trabecular and cortical bone. Comparison of structural parameters of specimens scanned with these systems, and mechanical testing suggest the amount of bone and the architecture of trabecular bone contribute to mechanical strength [96]. Advanced image-processing methodologies have been used to quantify trabecular bone microarchitecture beyond measures of bone volume fraction (BV/TV). Specifically, direct 3D measures of mean distances and measures of structural heterogeneity have been developed to characterize
trabeculae and marrow spaces (Fig. 50.7) [50,97]. The degree of anisotropy, a measure of the degree of structural orientation of the trabecular network, can be calculated from the principal structural directions calculated by the mean intercept length technique [98] and is highly related to the directional dependence of bone’s biomechanical properties [99]. A measure of the structural connectedness has also been adapted to bone, based on the Euler number [51]. The shape of the trabecular structure has been characterized using the structure model index (SMI) e an index of surface convexivity that estimates the degree to which the structure consists of rod-like or plate-like elements [52]. Furthermore, several groups have developed algorithms to decompose the trabecular structure in order to independently quantify the volume and scale of rod-like and platelike elements [100e102]. The ultrastructure of cortical bone is an important determinant of bone strength [10,11], is critical in fracture initiation and propagation [12], and known to change with aging [13], disease [14], and therapy [15]. Volumetric and morphologic characterization of the cortical ultrastructure has predominantly focused on the cannular features of cadaveric femoral neck specimens [103e107]. Resolution improvements heralding the evolution of nanocomputed tomography (CT with submicron resolution) have recently paved the way for a complete evaluation of cortical bone ultrastructure including the distribution of osteocyte lacunae [10,12]. BIOMECHANICAL ANALYSIS
While volumetric density and microarchitectural information provide improved fracture risk prediction and some explanation for treatment efficacy, more direct estimates of bone mechanical strength that inherently account for geometry, microarchitecture, and even
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composition are the ultimate goal for improving fracture risk prediction and management of osteoporosis. Computational modeling approaches have been introduced to take advantage of the detailed information in high-resolution images of bone. Finite element analysis (FEA) is a common computational tool in classical engineering fields critical to design and failure analysis. The basic concept is that the behavior of a complicated system under a simulated condition e in this case the biomechanical properties of bone e can be determined through subdivision into smaller constitutive elements for which the behavior is simple to determine. Applied to high-resolution images of bone, the apparent biomechanical properties (e.g., stiffness, elastic modulus, failure load, etc.) of a biologically complex microstructure are computed by decomposing the structure into small cubic elements (i.e., the voxels) with assumed mechanical properties (Fig. 50.8) [68,108]. In practice, voxel-based microfinite element (mFE) simulations require substantial computational resources, though the Mooresian advance of microprocessor technology [109] has led to concomitant decreases in the time required to solve a model. Linear simulations to calculate the elastic properties of a structure (apparent modulus, stiffness, etc.) can now be run on a modern multiprocessor workstation with shared-memory parallelization within minutes or hours, depending on the number of elements in the model [110]. For mFE modeling of nonlinear mechanical behavior (yield, failure, etc.) generally requires parallelized computing on high-performance computing (HPC) clusters [111,112]. Applications BASIC AND PRECLINICAL STUDIES
As a replacement for time-consuming serial-section histological approaches to bone histomorphometry, mCT has vastly accelerated the rate of basic research in the field of bone biology. In particular, mCT studies of small animal models of skeletal health and disease have contributed significant advances in the understanding of developmental [81,113,114], aging [115,116], environmental [84,117], genetic [118e120], and disease [121e123] effects on skeletal health. Furthermore, mCT is now a fixture in the drug discovery pipeline for pharmacologic agents to treat bone metabolic disorders, and in particular osteoporosis. In fact, the recent regulatory approval of denosumab marks the introduction of an antifracture compound for which mCT played an important role from the discovery of the OPG/RANKL pathway, and through the preclinical development phases of the candidate compound [124] that eventually paved the way for human trials [125,126].
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For microstructural finite element analysis (mFEA), the mCT or HR-pQCT image data are converted to a 3D model of cubic elements and simulated loads are applied according to chosen boundary conditions. Here a slab of bone is compressed uniaxially by 1% (A). The solution for the series of equations that describes the forceedisplacement behavior of each element results can be used to calculate the apparent mechanical properties of the whole bone, as well as visualize the distribution of stress at the microstructural level (B). Please see color plate section.
FIGURE 50.8
CLINICAL STUDIES
The application of mCT to clinical research is limited to examining small bone biopsy specimens ex vivo. There are numerous data over the previous 20 years establishing age, gender, and anatomic differences in the trabecular and cortical structure of cadaveric specimens of bone [97,104,127e131]. Anatomic sites most relevant to fracture risk, such as the spine and proximal femur, are generally precluded from clinical examination unless they are obtained via surgical intervention for indirect skeletal disorders such as osteoarthritis [132,133]. For routine assessment of bone quality, minimally invasive bone biopsies are typically acquired from the iliac crest [134]. Trabecular microarchitecture at the iliac crest has been shown to reflect vertebral
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fracture status [135,136] and to change following the onset of menopause [137]. Quantification of bone structure from iliac crest biopsies has also become an important endpoint in longitudinal drug-efficacy studies of PTH [138e141], strontium ranelate [142], and various bisphosphonates [15,143e148]. Borah et al. recently reported significant ultrastructural changes in cortical bone of the iliac crest following 5 years of treatment with risedronate [15]. Despite the image-quality advantages of in vitro imaging studies, the invasiveness of the procedure, inherent variability in specimen collection [149,150], and limited significance of the iliac crest as a fracture site are drawbacks to the clinical significance of mCT.
High-resolution Peripheral Quantitative Computed Tomography (HR-pQCT) The development of HR-pQCT represents the convergence of clinical CT with many of the technological features of desktop mCT. This imaging system, presently available from a single manufacturer (XtremeCT, Scanco Medical AG, Bru¨ttisellen, Switzerland), was designed specifically for in vivo quantitative evaluation of bone microarchitecture in the distal appendicular skeleton of human subjects. This device has the advantage of significantly higher SNR and spatial resolution (nominal isotropic voxel dimension of 82 mm) compared to clinical CT. Furthermore, the radiation dose is several orders of magnitude lower, and primarily does not involve critical, radiosensitive organs. There are also several disadvantages to this technology. Most notably it is limited to peripheral skeletal sites and therefore can provide no direct insight into bone quality in the lumbar spine or proximal femur e common sites for osteoporotic fragility fractures, which are associated with the most significant financial and quality-of-life burden for patients. This device was made available on a limited basis in the mid 2000s, with the first cross-sectional data published in early 2006 [32,33].
converts X-rays to visible light, which is coupled by a fiber optic taper to a 3072 256 CCD camera. The X-ray source and detector ensemble interface a highresolution motorized gantry that allows translation along the z-axis of the scanner and axial rotation across 180 for tomographic acquisitions. The readout electronics of the detector interface a computer workstation that provides storage and computational resources for image reconstruction and analysis. POSITIONING
A standard, manufacturer-recommended scan protocol has been used in the majority of the published literature to date. The techniques involved were primarily adapted from a previous-generation pQCT device [151]. The subject’s forearm or ankle is immobilized in a carbon fiber cast that is fixed within the gantry of the scanner (Fig. 50.9). A single dorsalepalmar projection image of the distal radius or tibia is acquired to define the tomographic scan region. This region spans 9.02 mm in length (110 slices) and is localized to a fixed offset proximal from the mid-jointline and extending
Image Acquisition HARDWARE
The HR-pQCT imaging system consists of a microfocus X-ray source with a 70-mm focal spot size that operates at a fixed voltage of 60 kVp and current of 900 mA. In order to minimize radiation dose to the patient and beam hardening effects, the X-ray beam is prefiltered with 0.3-mm Cu and 1-mm Al plates at the aperture. This shapes the output energy spectrum towards a higher effective energy by preferentially blocking “soft” low-energy X-rays. Analogous to mCT, the cone beam X-ray field is incident upon a 2D areal detector. The detector consists of a scintillator crystal (CsI) that
FIGURE 50.9 Image of the XtremeCT HR-pQCT imaging system manufactured by Scanco Medical AG, which can perform in vivo imaging of the distal extremities (radius, tibia) with an isotropic voxel resolution of 82 mm. (Image courtesy of Dr. Miki Sode, Ph.D. University of California, San Francisco.)
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Scout acquisition used to define the HR-pQCT scan region for the distal radius (A) and distal tibia (B). The solid region corresponds to the imaging location and consists of 110 slices spanning 9.02 mm longitudinally. In the radius the scan region is fixed 9.5 mm proximal from the mid-jointline, while in the tibia the scan region is 22.5 mm proximal from the tibial plafond.
FIGURE 50.10
proximally. In the radius the offset is 9.5 mm while in the tibia it is 22.5 mm (Fig. 50.10). It is important to note that this method does not account for differences in bone length and therefore positioning may be a confounding source of variability in cross-sectional studies [152]. In the radius, the default axial scan location has been found to partially overlap with the most common site for fracture and region where the cortical and trabecular structure are most strongly associated with whole bone strength [153,154]. There are several different protocol modifications that have been employed for developmental studies in children and adolescents to account for patient size and designed to avoid radiation exposure to the epiphyseal growth plate. In a cross-sectional study of age- and gender-related differences in the microstructure of the distal forearm of adolescent boys and girls Kirmani et al. used a fixed offset (1 mm) with respect to the proximal extent of the distal epiphyseal growth plate of the radius [155]. They reported that this resulted in no direct irradiation of the growth plate. Furthermore this location is consistent with the most frequent site of forearm fracture during adolescence [156]. In a similar population participating in a longitudinal study of bone development, Burrows et al. selected a region offset 8% of the total tibial length proximal to the tibial plafond. In their cohort this approach resulted in no overlap with the growth plate, allowed comparable localization for longitudinal measurements during growth, and did not require operator identification of the proximal extent of the growth plate e which can be highly variable and is therefore a potential source of operator-related error [157]. While there are a number of studies under way investigating other scan locations in adults e including more proximal sites dominated by cortical
bone e hardware constraints for this device preclude imaging true diaphyseal sites in the radius or tibia. TOMOGRAPHY
Prior to each tomographic acquisition, a precalibration procedure is performed to measure the dark bias signal in the detector (X-ray shutter closed) and the reference intensity of the X-ray source with an empty field of view (X-ray shutter open). For the actual tomographic acquisition, 750 projections are acquired over 180 with a 100-ms integration time at each angular position. The 12.6-cm field of view (FOV) is reconstructed across a 1536 1536 matrix using a modified Feldkamp algorithm, yielding 82-mm isotropic voxels (Fig. 50.11) [158]. The total scan time is 2.8 minutes and results in an effective dose to the subject of approximately 4.2 mSv e several orders of magnitude smaller than clinical CT and comparable to DXA and other planar radiographic modalities. In total, the raw projection data, reconstructed image data, and other derivative data require approximately 1 GB (1024 MB) of digital storage space per scan. Image Analysis The reconstructed images are analyzed using a standard protocol provided by the manufacturer. The operator initiates the segmentation process by drawing an approximate contour around the periosteal perimeter of the radius or tibia in the first slice of the dataset. This contour is then automatically adjusted using an edge detection process to precisely identify the periosteal boundary. The software iteratively proceeds through the remaining slices in the dataset while the operator visually verifies the accurate contouring of the periosteal surface, adjusting where necessary. The
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FIGURE 50.11 Typical HR-pQCT images from the distal radius (AeC) and distal tibia (DeF) of a healthy young adult. Images A and D correspond to the distal most slices of the scan, while images B and E correspond to the proximal most slices. Images C and F are 3D reconstructions of the extracted mineralized structure with the segmented cortical compartment highlighted in dark gray.
cortical bone compartment is segmented using a 3D Gaussian smoothing filter followed by a simple fixed threshold. The trabecular compartment is identified by digital subtraction of the cortical bone from the region enclosed by the periosteal contours. The trabecular bone structure is extracted using a laplace-hamming edge enhancement process followed by a second fixed threshold [159]. Based on this semiautomated contouring and segmentation process, the trabecular and cortical compartments are segmented (Fig. 50.11) automatically for subsequent densitometric, morphometric, and biomechanical analyses. For subjects with highly porous or very thin cortical bone this routine has been shown to poorly identify the cortical envelope [35,160]. Recently, more sophisticated techniques have been developed to robustly identify fine cortical structures [161,162]. In general reproducibility of densitometric measures is very high (CV < 1%), while biomechanical and morphometric measures typically have a coefficient of variation of 4e5% [32,33,35,163,164]. DENSITOMETRIC ANALYSIS
The grayscale attenuation values of the reconstructed HR-pQCT images are converted to hydroxyapatite (HA) concentration using a calibration procedure analogous to the process described for ex vivo microtomography [65]. The HR-pQCT calibration phantom (Scanco Medical AG, Bru¨ttisellen, Switzerland) is composed of five cylinders of HA-resin mixtures with a range of
mineral concentrations (0, 100, 200, 400, 800 mg HA/ cm3) where 0 mg HA/cm3 represents an organic, soft tissue equivalent material lacking mineralization. Based on this calibration volumetric BMD can be determined independently for cortical and trabecular bone compartments based on the segmentation process described above. HR-pQCT images have also been used to derive surrogate measures of areal BMD in the ultradistal radius [165]. This technique has shown a high level of agreement with multiple clinical DXA devices (R2 > 0.8). MORPHOMETRIC ANALYSIS
Morphometric indices analogous to classical histomorphometry are calculated from the binary image of the trabecular bone. Unlike MRI and MDCT, which have a large slice thickness relative to the in-plane resolution, the high isotropic resolution of HR-pQCT (82 mm) permits direct, 3D assessment of intertrabecular distances. These measures have been well validated against mCT gold standards in a number of studies [34,166,167]. From the binary image of the extracted trabecular structure, 3D distance transformation techniques are used to calculated trabecular number [50]. While the intertrabecular distances are large compared to the voxel dimension, the average trabecular thickness (100e150 mm) is on average only 1e2 voxels wide. Accordingly, direct measures of thickness and bone volume are complicated by significant partial volume effects. In the standard analysis protocol bone volume
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X-RAY COMPUTED TOMOGRAPHY (CT)
fraction (BV/TV) is derived from the trabecular volumetric BMD assuming a fixed mineralization of 1200 mg HA/cm3 for compact bone (BV/TV ¼ Tb.vBMD/1200). From the direct measure of Tb.N and the densitometrically derived BV/TV, trabecular thickness and separation (Tb.Th, Tb.Sp) are derived using standard stereological relations assuming a plate-model geometry [44,151]. There are several potential concerns with this approach. First, phantom studies by Sekhon et al. have documented significant errors in the measurement of trabecular vBMD related to biologically relevant variations in cortical thickness, as well as the magnitude of trabecular vBMD itself [168]. This is most likely related to X-ray scatter effects e the HR-pQCT does not have a collimated detector to block Compton scattered X-rays e and residual beam-hardening artifacts. These errors are primarily a concern for cross-sectional studies when cortical thickness and trabecular vBMD may span a broad range. It is less of a concern, and indeed may not be significant, in longitudinal studies where %-change is the primary endpoint as subject-specific age, pathology, or therapy-related changes in cortical thickness and trabecular vBMD are comparatively small. Second, the assumption of a fixed matrix mineralization is not consistent with the established action of many common antifracture therapeutic agents [169]. Changes in bone tissue mineral density would be expected to cause an increase in vBMD irrespective of bone volume changes and therefore result in an overestimation of BV/TV and propagate error to the derivative measures of trabecular thickness and separation, confounding any actual therapy-related effect on trabecular bone volume and structure. Several studies have investigated other measures of bone microarchitecture and topology from HR-pQCT images including connectivity, structure model index (a measure of the rod- or plate-like appearance of the structure), and anisotropy. However there is mixed evidence of their reliability at in vivo resolutions [34,166,167,170]. Recently, more sophisticated approaches to cortical bone segmentation have been proposed [161,162], which allow direct assessment of cortical thickness (Ct.Th) as well as quantification of cortical microarchitecture, including intracortical porosity [171,172]. BIOMECHANICAL ANALYSIS
HR-pQCT image data may be used to construct mFE models (Fig. 50.8) and has been validated against both higher-resolution models (mCT) as well as empirical measures of strength [164,167,173]. In most cases the binary bone image is converted to a model with homogeneous material properties and a uniaxial compressive load is prescribed to determine linear elastic apparent properties [173e175]. The feasibility of models with
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density-scaled material properties has been suggested by Macneil and Boyd, and may be important for longitudinal studies where tissue mineralization effects exist [173]. In addition to whole bone mechanics, mFEA can be used to determine the relative load distribution between cortical and trabecular compartments [176], and estimate mechanical implications of specific structural features such as intracortical porosity [171]. LONGITUDINAL ANALYSIS
For clinical investigations into longitudinal changes in HR-pQCT-derived measures of bone quality several important considerations must be addressed. First, because bone structure and geometry can change substantially along the axial direction [152] it is critical that baseline and follow-up scans be matched. To this end the scout scan and reference line position for all baseline measurements are recorded automatically. When a follow-up measurement is performed, the operator is automatically presented with an image of the baseline scout denoted with the original reference position as a visual aid for positioning the reference line for the follow-up acquisition. Nevertheless this is a subjective process subject to operator error. At our imaging center, repeat measurements are typically associated with a margin of error in the axial positioning of approximately 1 mm. Accordingly automated methods are needed to ensure comparable regions of interest are used for the image analysis. To that end the manufacturer provides software that automatically matches slices based on periosteal cross-sectional area and limits the analyzed region to the slices common to both baseline and follow-up [151]. Alternatively, MacNeil et al. have demonstrated that 3D image registration techniques can provide improved short- and mediumterm reproducibility compared to the default slicematching approach [163]. This approach may also be more appropriate in longitudinal studies where changes in cortical thickness would compromise registration based strictly on cross-sectional area. As discussed earlier, meaningful longitudinal comparisons in children or adolescents experiencing rapid growth is not trivial, and requires more careful consideration of standardization of scan positioning and analysis protocols [157]. Applications CROSS-SECTIONAL STUDIES
There is a growing body of literature featuring HRpQCT assessment of bone quality. The first crosssectional studies by Boutroy et al. and Khosla et al. reported gender-specific, age-related differences in trabecular bone microarchitecture [32,33]. Several centers have observed age-related differences in mFE
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Decade-wise trends for HR-pQCT-derived measures of total BMD (A), compressive stiffness determined by mFEA (B), cortical porosity (C), and the stiffness deficit attributed to cortical porosity determined by mFEA of models with and without the pore space assigned as bone (D). Bars represent mean with standard error indicated for female (black) and male (gray) subjects. Statistical significance between consecutive decades (Wilcoxon ranked sum with Bonferroni correction) is denoted by *P < 0.01 and **P < 0.001). (Adapted from [171].)
FIGURE 50.12
estimates of bone strength in normative cross-sectional cohorts [171,177,178]. Furthermore, Burghardt et al. [171] and Macdonald et al. [178] have demonstrated the ability of HR-pQCT to detect dramatic age-related differences in cortical porosity (Fig. 50.12) in females using new techniques for the analysis of cortical ultrastructure [162]. A microstructural basis for ethnicityrelated differences in bone strength between East Asian and Caucasian women was reported in two studies [179,180]. Sornay-Rendu et al. demonstrated cortical and trabecular morphology and provided additional fracture discrimination independent of aBMD in osteopenic women [181]. In the same OFELY cohort, Boutroy et al. showed that mFEA mechanical measures provided additional discriminatory power between osteopenic women with and without distal radius fractures [174]. While the initial focus has predominantly been related to fracture discrimination in postmenopausal osteopenia and osteoporosis [174,181e187], a number of studies have utilized HR-pQCT to investigate developmental changes in bone quality and fracture risk [155,188,189], as well as secondary causes of bone loss [190e194]. In a cross-sectional study of men and women with early chronic kidney disease (CKD), Bachetta et al.
reported modest early impairment of bone density in CKD patients compared to healthy controls [191]. Burghardt et al. have suggested that the welldocumented increased fracture risk in individuals with type 2 diabetes mellitus, despite normal or higher BMD, may be attributed to cortical bone ultrastructural deficits that can be detected by HR-pQCT (Fig. 50.13) [193]. LONGITUDINAL STUDIES
Most recently, data from the first HR-pQCT singleand multicenter longitudinal trials have been published. In a multicenter, head-to-head randomized placebocontrolled trial of denosumab (a RANKL inhibitor) and alendronate (a bisphosphonate), Seeman et al. reported more pronounced antiresorptive efficacy with denosumab compared to alendronate [126]. In particular cortical thickness was preserved or improved at the radius and tibia with either treatment, while cortical bone loss progressed in the control group. Burghardt et al. reported similar cortical bone changes and additionally showed a preservation of compressive bone strength by mFEA following 24 months of alendronate treatment [195]. Longitudinal microarchitectural
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FIGURE 50.13 HR-pQCT images of the distal tibia from a healthy control (top) patient with type 2 diabetes mellitus (T2DM) (middle), and
a T2DM patient with a history of fracture (bottom): distal-most slices (A, E, I); proximal-most slices (B, F, J); 3D visualization of the mineralized bone structure (C, G, K); 3D visualization of cortical bone (transparent light gray) and intracortical porosity (solid dark gray) (D, H, L). (Reprinted with permission from [193].)
changes have also been demonstrated for strontium ranelate [196] and teriparatide [197].
MAGNETIC RESONANCE IMAGING (MRI) Magnetic resonance imaging is an attractive modality for acquiring high-resolution images of cortical and trabecular bone in vivo. In contrast to X-ray imaging, which is based on the absorption of ionizing, electromagnetic radiation of high energy (20e150 keV), MRI uses the stimulation and subsequent emission of nonionizing electromagnetic radiation of low energy (10e8e10e6 eV) by biological tissue. Thus, MRI is a noninvasive imaging technique that does not require the use of ionizing radiation and is therefore well suited for assessing in vivo images in a clinical setting. Living tissue consists of approximately 60e80% water. Although other nuclei such as 23Na and 31P can also be used for MR imaging, the dominant nucleus used in MRI is the hydrogen nucleus (proton), as all other nuclei exist in vivo at much lower concentrations. Nuclei with an even mass number (protons and neutrons) and even
charge number have zero spin and cannot be used for magnetic resonance imaging. For MR imaging, three magnetic fields are required with different field strengths. The main magnetic field provides a strong, constant static magnetic field (B0) that can be up to several thousand times stronger than the earth’s magnetic field. The most common magnetic field strengths in a clinical setting are currently 1.5 Tesla and 3 Tesla. More recently, higher magnetic field strengths have also been explored. The B0 field is usually generated by a superconducting magnet, which is constantly operating. For this reason, safety precautions have to be taken before entering the MR scanner room. Another but smaller magnetic field gradient is superimposed to the main field generating different field strengths at different spatial locations in order to spatially encode the image. And finally, a radiofrequency (rf) pulse is required to excite the proton spins. The basic idea of MRI is based on the fact that nuclei with a net nuclear spin interact with the main magnetic field. The magnetic dipoles of the spins align with the applied field, in a similar manner to small bar magnets. They also spin or precess about the field just like
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a gyroscope precesses around the earth’s gravitational axis. The frequency of precession, known as the “Larmor frequency” uL, depends linearly upon the force of the magnetic field B0 and also on the specific nucleus used for imaging. For example, the water proton precesses with a frequency of approximately 64 MHz at 1.5 Tesla. This specific frequency has to be matched by the applied rf pulse. In biological tissue, the corresponding wavelength of the rf pulse is about 4.5 meters. Thus, it is clear that in MRI the spatial resolution does not depend on the wavelength of the electromagnetic pulse but only on the encoding magnetic field gradients. The MR signal is acquired by applying an rf pulse for a short time perpendicular to the static field. The pulse is produced by a “transmit” coil and rotates the precessing spins away from its alignment along the longitudinal direction of B0. This pulse synchronizes the phases of all spins to match that of the applied rf pulse and a new transversal magnetization is created which can be collected or “received” by the same coil. This rf signal is called the free induction decay. The duration and amplitude of this pulse are proportional to the angle of the spin vector relative to the main magnetic field. This is called the flip angle and a 90 flip angle would result in a maximum transversal magnetization maximizing the signal. Eventually, the so-excited protons flip back to their state of thermal equilibrium. The absorbed energy is thus released to the surroundings and this takes place along an exponential growth-course. The time point when exactly 63% of the maximum signal is reached is called the spin-lattice relaxation time or the T1 relaxation time. The T1 relaxation depends on the size of the molecules and their surroundings. The relatively small water-molecule has little possibility to release energy in the relaxed atom-fence of the liquid; the result is a long T1. Larger molecules will hand over their energy more quickly; the result is a short T1. The image contrast generated by this relaxation is generally referred to as T1-contrast. An additional process takes place in the transversal plane and thus overlaps the T1 effect. After the emission of an rf pulse, the originally synchronized spins gradually lose their phase coherence, resulting in attenuation and finally cancellation of the MR signal in the transversal plane. This loss of the phase coherence is called the spinespin relaxation or the T2 relaxation time, which denotes the point when only 37% of the maximal transversal magnetization is left. T2 is tissuespecific in a similar manner to T1. In the relatively dense atomic structure of a solid, the spins are continually exposed to locally fluctuating magnetic fields. Therefore, T2 is very short. In the less-packed molecular structure of liquids, T2 is relatively long. The so-generated contrast is called T2 contrast. These two processes, alongside proton density, render it possible to
distinguish between different tissue types and are thus the basic parameters for image contrast. In addition, MRI can distinguish a broader range of tissue properties such as motion (diffusion and perfusion), elasticity, susceptibility, and many more. In conventional MRI, mineralized bone is depicted by negative contrast relative to the high-background signal from bone marrow. The low signal from bone itself is due to the relatively low abundance of protons and an extremely short T2 relaxation time (<1 ms) typical of most solid-state tissues [198]. The high signal from bone marrow depends on the programmable acquisition procedure (“pulse sequence”) applied and the fat content of the marrow (fatty versus hematopoietic bone marrow). Thus, the aim of the applied MR pulse sequence is to maximize the marrow signal. The contrast is then primarily based on the proton density in the marrow. Other, more advanced pulse sequences can also provide signal from the bone itself. Some examples will be shown in the text. The following sections will focus on the assessment of trabecular and cortical bone structure using MRI in conjunction with advanced image analysis techniques. More recent topics like the imaging of bone and bone marrow fat quantification will also be discussed.
Trabecular Bone MRI Image Acquisition SIGNAL-TO-NOISE CONSIDERATIONS
The main challenge for the accurate depiction of the trabecular microstructure as needed for structural analysis lies in the requirement for very high spatial resolution achieved by the modality. As previously discussed, in MRI, the achievable spatial resolution is independent of the wavelength of the applied electromagnetic field but depends primarily on the strength of the applied field gradient. However, in modern MR scanners, the strength of these gradients (scanner hardware) is not the limiting factor and in theory very small voxel sizes are possible. The actual resolution limits are set by the detectable signal from the tissue in a voxel and therefore depend on the availability of sufficient signal-to-noise ratio (SNR) per voxel. In other words, very high spatial resolution is feasible but there would not be enough detectable signal in these small voxels. The obtained SNR is influenced by the detection sensitivity of the rf coils, the magnetic field strength of the scanner, the temperature, proton density of the tissue, and the imaging time. Current coil designs are largely optimized for high-resolution imaging [199] and the field strength is determined by the magnet. Thus, on clinical MR scanners, SNR can only be
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MAGNETIC RESONANCE IMAGING (MRI)
Shown is an in vivo magnetic resonance image of the proximal tibia (ankle). The bright signal from the bone marrow is clearly seen. The trabecular bone network is embedded in the fatty marrow and does not yield any MR signal in this case. The surrounding cortical shell can also be appreciated from this image.
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FIGURE 50.14
increased by optimized pulse sequences and/or increased imaging time. However, it is important to note that the voxel size scales with the square root of acquisition time. Thus, in order to double the spatial resolution in one dimension (e.g., halve the slice thickness), the imaging time needs to be quadrupled in order to maintain the same SNR per voxel. Nonpublished data in the authors’ laboratory suggest a minimum SNR of eight to ten for the accurate measurement of trabecular bone structure (see Fig. 50.14). In MRI, SNR is commonly measured by the mean signal intensity divided by the standard deviation of the background noise. Although there has been a lot of work on trabecular bone imaging of peripheral sites like the wrist, tibia, and calcaneus, very little work has been done on the proximal femur, a site of high fracture incidence. The reason for this is found in its deep location inside the body and the fact that the rf signal is attenuated in the body by tissues like fat and muscles. Only recently, through pulse sequence and coil optimization, the femur was made accessible for trabecular bone analysis [200]. Further optimization in the authors’ laboratory led to enhanced SNR efficiency and improved MR images of the proximal femur (Fig. 50.15). These in vivo images are currently acquired in 12 minutes with high spatial resolution (234 234 500 mm3). SPATIAL RESOLUTION
The spatial resolution currently achievable with MRI in a clinically feasible scan time is in the order of the trabecular thickness (100e150 mm) or above. However,
An in vivo image of the proximal femur (hip) is depicted. The featured spatial resolution is 234 234 500 mm3. The image was acquired in 12 minutes.
FIGURE 50.15
the Nyquist theorem in optical physics postulates a sampling rate twice the smallest structure present in the image for accurate depiction of the details. Thus, the only reason why the trabecular network can still be accurately depicted is because the spacing between the trabeculae is much larger than their size (600e890 mm). If this were not the case, the trabecular structure would not be resolvable due to partial volume effects. In addition to spatial resolution, the strength of the magnetic field has to be taken into account, which alters the image quality as well. Both, SNR and magnetic susceptibility effects increase linearly with field strength. The susceptibility effects are due to differences in the magnetic properties of bone and bone marrow (bone is more diamagnetic). They can severely change the homogeneity of the magnetic field and lead to signal cancellations near bone and bone marrow boundaries as will be further discussed below. Advanced image processing and analysis algorithms can be designed to overcome some of the resolution constraints. Information regarding structure, topology, and orientation of the trabecular bone network can be extracted from the images by applying these digital post-processing techniques. A large number of analysis parameters have been investigated in the past and related to osteoporotic fracture risk and response to treatment as outlined further below. Since spatial resolution and SNR are trade-off parameters in MRI, it is not clear whether enhancing resolution at the expense of SNR or the opposite way around increases or decreases detection sensitivity; and also
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representation of transverse trabeculae. The elastic moduli become increasingly underestimated with decreasing SNR. However, the high correlation between elastic parameters derived from mCT and simulated MR data suggested that there is potential for estimating mechanical moduli on the basis of in vivo MR images. This is a very important finding, since it could circumvent the need of deriving architectural parameters as surrogates of mechanical parameters. PULSE SEQUENCES The dramatic change of bone volume fraction (BV/TV) with decreasing signal-to-noise ratio (SNR) is depicted for three MR images. After image acquisition, additional noise was added, the image was thresholded and BV/TV was calculated. For SNR higher than 10, the calculated BV/TV values are constant.
FIGURE 50.16
what errors are incurred by imaging at anisotropic resolution with larger slice thickness than in-plane resolution. The impact of limited spatial resolution and noise on structural parameters and whether under these conditions, a small amount of bone loss and its associated structural manifestations can be detected, has been previously [201] investigated. To this end, 3D MRI models based on high-resolution CT images of human cadaveric bone cores were generated with different spatial resolution and noise. Systematic changes of the derived structural parameters were found with decreasing SNR (Fig. 50.16). However, these errors are correctable using simple linear transformations, thereby allowing the data to be normalized. In general, parameters obtained at a given SNR or spatial resolution are linearly related to those obtained under reference conditions, independent of the structural characteristics of the bone examined [201]. However, topological parameters inherently depend much more on spatial resolution and voxel size than structure parameters. For example, depending on voxel size, a narrow plate will either be classified as a curve or surface after skeletonization and thus changing the topological parameters. The implications of noise and spatial resolution on mechanical properties of trabecular bone have been previously estimated using image-based finiteelement analysis [202]. To this end various spatial resolutions and noise levels were simulated using mCT images at 21 mm isotropic voxel size from scores of 13 cadaveric human specimens. Elastic constants derived from actual MR images at 9.4 T and 50 mm isotropic voxel size were compared to those gained from highresolution mCT. The elastic moduli computed from simulated MR images were highly correlated with those obtained from mCT (R2 ¼ 0.99) and the data were relatively immune to noise. Thus, correlations between mCT and simulated MR-based elastic parameters improve at isotropic voxel sizes due to a more faithful
In MRI, a pulse sequence defines the contrast and to some extends the SNR of an image. It is a preselected set of defined rf and gradient pulses, usually repeated many times during a scan. Pulse sequences are computer programs that control all hardware aspects of the MRI measurement process. In principle, two main types of pulse sequences can be defined and are both used for bone imaging, namely, spin-echo-based pulse sequences and gradient-echo-based pulse sequences. With the term “echo,” a synchronization of spins is described. A gradient-echo refers to a balanced state of all applied magnetic field gradients where the sum of all gradients is zero. In a very homogeneous magnetic main field, all spins would then align and point in the same transversal direction to be detected. The resulting signal would then be the mean intensity of all spins. In a more realistic scenario, taking into account the field inhomogeneities, the spins would experience different precession frequencies and would not be synchronous at the time of the gradient-echo. However, by applying a second rf pulse a so-called spin-echo can be created. This method ensures that all spins are in-phase at the time of the gradient-echo no matter what field inhomogeneities they experience. Deviations from the main magnetic field are in particular strong near trabecular bone and bone marrow transitions. As bone is about 2.3 ppm [203] more diamagnetic than bone marrow, off-resonance frequencies of 100 Hz and more are expected [204] at 3 Tesla and more than 200 Hz at ultrahigh field strength of 7 Tesla and more [205]. The results are intravoxel spin dephasings, which can lead to significant signal attenuation and cancellations. In this context, it has to be noted that this effect can also be used to acquire information about the trabecular bone fraction in an indirect way by sampling the signal decay for each voxel [206e210]. The decay of the signal is expected to be faster with increasing off-resonance frequencies in a voxel depending on the number of bone and bone marrow transitions where the susceptibility effect occurs. The following characteristics can be used as guidelines for a suitable pulse sequence. Firstly, a threedimensional (3D) excitation usually provides more SNR than a 2D sequence. However, 2D acquisition techniques have also been used previously to image the
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MAGNETIC RESONANCE IMAGING (MRI)
trabecular structure of the calcaneus [211e213] albeit with lower spatial resolution (1 mm slice thickness). Secondly, the gradient-echo should be acquired immediately after the excitation pulse in order to avoid further spin dephasing and signal loss during the echo time. The readout bandwidth (rBW), which is inversely proportional to the readout time, should be as high as possible in order to reduce susceptibility induced spin dephasing during the readout time when the actual signal is acquired although this results in some SNR penalty. Finally, the imaging time has to be within a clinically feasible range. Image Processing Image analysis of trabecular bone images in MRI involves several post-processing steps. First, a region of interest containing the trabecular bone has to be chosen. Then, the signal sensitivity of the coil has to be corrected followed by bone/marrow segmentation, structural calculations and if needed serial image registration [214]. Before any analysis, the region of interest (ROI) containing the trabecular bone structure has to be defined. One method previously described [214] involves downsampling of the image by a factor of two to a spatial resolution of 0.312 mm in-plane and 1 mm slice thickness and cropping the image background outside the trabecular structure in order to increase processing speed and memory efficiency in the subsequent processing steps. A combination of gray-level erosion, dilation, and median filtering operations is then used to eliminate the trabecular structure while preserving the cortical shell. This results in a homogeneous marrow space surrounded by a darker cortical boundary. Initial regions for the radius and ulna are defined using intensity contouring and pattern matching. The initial regions are then projected and refined on successive slices in the SI direction using an adaptive intensity threshold level to outline the full 3D trabecular region. Finally, the automatically defined ROI is inspected by an experienced operator and manually adjusted if needed. COIL SENSITIVITY CORRECTION
Depending on the type of rf coil used, a correction of the sensitivity profile has to be applied in order to obtain an accurate depiction of the trabecular network. For quadrature or birdcage coils with satisfactory in-plane homogeneity, a simple phantom-based correction can be applied [214]. To this end, data from a uniform phantom are acquired in order to obtain a sensitivity profile of the coil. A third-order polynomial fit of the sensitivity profile can be used to define a longitudinal correction factor versus position function. The described coil sensitivity correction is completely automatic, using the position information from the image file headers. However, the method strongly depends on the accurate
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placement of the scanner landmark at the center of the coil. Another approach previously used for images acquired with surface coils [214] is based on a lowpass filter (LPF) correction scheme [215] where the image is LP filtered by convolution with a Gaussian kernel. In order to minimize edge effects, the LP image is generated from a masked image containing only the trabecular bone ROIs, with the background on each slice filled with the mean intensity of the ROI. The LP image reflects the coil sensitivity profile, which can be corrected for by dividing the original image by the coil profile. The LPF correction is fully automatic and does not require any operator interaction. Other methods estimate the local bias field by finding the intensity for which the discrete Laplacian is zero [216]. More recent approaches [217] use a fully automatic coil correction scheme based on a nonparametric nonuniform intensity normalization N3 approach [218] for coil-induced intensity inhomogeneities. The underlying assumption of this technique is that the bias field blurs the intensity distribution in the image and that this distribution can be restored by iterative deconvolution of the intensity distribution along with a smoothing of the bias field estimate using B-splines. The technique was evaluated ex vivo on proximal femur specimens scanned with both a surface coil and a volume coil with a more homogeneous sensitivity profile. Trabecular bone parameter values were compared with values obtained from high-resolution peripheral computed tomography (HR-pQCT). Also, in vivo reproducibility was evaluated. The interscan variability was found to be highly enhanced from CV ¼ 8.9e12.8% using LPF-based to CV ¼ 3.6e8.4% using N3 coil correction. Thus, N3 coil correction preserves image information while accurately correcting for coil-induced intensity inhomogeneities, which makes it very suitable for quantitative analysis of trabecular bone. IMAGE SEGMENTATION
A very basic approach to extract structural information of trabecular bone from MR images is based on binarized images where bone appears dark (no signal) and the fatty bone marrow yields high signal. From these images, fundamental structural parameters like the bone fraction BV/TV (bone volume to total volume fraction) can be derived by counting the bone pixels in a region of interest. This number can be easily extracted from ultrahigh-resolution images where the intensity histogram is bimodal by setting a single threshold. However, in the presence of partial volume effects, this becomes much more difficult and the two peaks of the intensity histogram blur into one single peak. Different approaches have been used to solve this problem, e.g. by measuring the bone’s signal in the image and making
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FIGURE 50.17 The image from Figure 50.1 is shown after thresholding resulting in only two phases of bone and bone marrow. The corresponding grayvalue histogram on the right side reveals the distribution of pixel values and emphasizes the challenges related to finding the appropriate threshold.
some basic assumption about the signal histogram [219] as shown in Figure 50.17. The drawback of this method is a significant loss of image information through binarization due to partial volume effects, and lack of spatial variability in intensities due to differences in marrow composition and coil shading. Thus, other methods seek to circumvent the binarization step and to process the original grayscale image. In theory, the grayscale value at each voxel should represent the percentage of bone (no signal) vs. bone marrow (bright signal) content. In practice, a number of technical considerations make this a challenging process. One approach [220] seeks to remove the noise by deconvolution. For each voxel a ROI is expanded until 50 voxels have an intensity 60% of the maximum intensity, and a local bias field estimate is determined as the most frequently occurring intensity greater than the mean intensity in the ROI. From the resulting noiseless image, a bone volume fraction map is generated. And from the voxel intensities the marrow volume fraction is determined. Although the method accounts for partial volume effects, noise, and intensity inhomogeneities, the assumption is made that the histogram is bimodal, which is typically not the case for resolutions achievable in vivo. Another method is based on local thresholding [216] and estimates the local marrow intensities within a nearest neighbor framework. Image Analysis MORPHOMETRIC ANALYSIS
For the assessment of structural information, three classes of parameters can be defined including scale, topology, and orientation [203,221]. Scale describes mainly the amount of bone in an ROI and the thickness of the trabeculae or the spacing between the trabeculae. Investigating the plate- or rod-like structure of the network assesses the bone topology. And finally the anisotropic character of the structure defines its orientation. Early assessments of trabecular bone applying the
principles of stereology are based on scale [222]. In MRI, trabecular thickness can be obtained from the mean intercept length (MIL) of parallel test lines across the ROI averaged over multiple angles [223,224]. From MIL and BV/TV measurements, trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp), and trabecular number (Tb.N) can be obtained [219]. These measures are usually called “apparent” parameters because of the limited spatial in vivo resolution of MRI. These measurements are usually conducted on a slice-by-slice basis. But also 3D approaches have been proposed such as 3D wavelets analysis [225] to compute a trabecular bone thickness map without the need for image binarization. Apparent BV/TV can also be determined from these maps by counting the number of pixels with thickness values different from zero and by dividing this number by the total number of pixels in the corresponding ROI. Another approach is based on fuzzy distance transform for trabecular bone analysis where the Tb.Th is obtained by computing the fuzzy distance along the medial axis of the trabeculae [226]. The method obviated the need for image binarization and is very robust to noise. Another method uses fuzzy clustering for trabecular bone segmentation and to evaluate BV/TV measurements (Fig. 50.18) [227]. A partial membership is assigned to each voxel based on the distance from the cluster center accounting for partial volume effects. The pixel intensity is the only feature used for clustering. This makes the method sensitive to noise and intensity inhomogeneities. An extended approach incorporates a local bone enhancement feature at multiple scales (BE-FMC) [228]. The local second-order structure is computed within a scale-space framework. This allows noise suppression while enhancing local relative intensity anisotropy and thus accounting for partial volume effects, noise, and signal intensity inhomogeneities. The new method proved to perform better then previously used fuzzy clustering using intensity as the only feature and a conventional dual-thresholding approach. BE-FCM could also significantly differentiate between
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MAGNETIC RESONANCE IMAGING (MRI)
(A)
(B)
(C)
(D)
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FIGURE 50.18 Analysis ROI defined in the femoral head (A); the ROI zoomed in (B). Trabecular bone segmentation of the ROI segmented using BE-FCM (C) which results in a significantly improved structure extraction in the presence of variable signal due to the presence of red and yellow marrow, compared to the established dual thresholding technique (D). (Image courtesy of Dr. Jenny Folkesson, Ph.D. University of California, San Francisco.)
control and fracture groups of 30 calcaneus specimens (from 17 subjects with and 13 without vertebral fracture). Also, the apparent BV/TV measurements based on BE-FCM segmentation had a lower mean value compared to the thresholding method (0.34 vs. 0.42) and were more consistent between different imaging sites. Furthermore, it was found that BE-FCM captures more bone structure and is less affected by partial volume effects, noise, and intensity inhomogeneities. Another class of structural bone analysis is bone topology. It has been emphasized in the literature that osteoporotic bone loss is very strongly related to a change in the bone’s topology which results in a fenestration of trabecular plates and finally a conversion from plate to a more rod-like bone structure [222,229e231]. For example, the measurement of bone’s connectivity has been conducted by computing a 3D Euler number [59]. Large negative Euler numbers represent a well-connected network, which is stronger than a broken
network. A more complex approach is digital topological analysis (DTA) [232], which was previously applied to trabecular bone measurements [233]. With this method, each voxel is classified into three categories it belongs to: curve, surface, or junction. The disadvantage of this approach is the requirement for binarized images since the trabecular network has to be first skeletonized. As previously discussed, the determination of an appropriate threshold is challenging and has an impact on the result. For topological analysis, an empirical threshold of BVF ¼ 0.25 was previously used [233]. Other 3D skeletonization techniques are based on topological invariant, and use a sequential thinning algorithm [234]. With this method, a 3D skeleton graph analysis is applied to count and to isolate all the vertices and branches of the skeleton graph. The technique was successfully applied to trabecular bone topology [235,236] and enhanced the prediction of mechanical properties and bone strength [237]. A more recent
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technique provides a complete assessment of scale, topology, and anisotropy using geodesic topological analysis (GTA) [238]. GTA quantifies the trabecular bone network in terms of its junctions, which play a central role in connectivity, and geodesic distances, defined as the shortest path between two points. By using the concept of geodesic distance, GTA assigns each bone voxel to its closest connected junction assuming that the relationship of a particular bone element is stronger to its closest connected junction than to any other junction in the network. Then, the trabecular bone architecture is divided into groups of voxels belonging to different junctions. This enables the quantification of individual junctions, junctions with their group of assigned voxels, as well as the interrelationship between junctions. A total of seven new apparent trabecular bone parameters that quantify the spatial distribution of trabecular bone in terms of its volume, spacing, and orientation were then computed based on the identified junctions and their assigned bone voxels. Two parameters quantify the trabecular bone volume, two the trabecular bone spacing, and three the trabecular bone orientation. It was found that GTA parameters yielded an average reproducibility of 4.8%, and the individual areas under the curve (AUC) of the receiver operating characteristic curve analysis for fracture discrimination performed better at 3.0 Tesla than at 1.5 Tesla reaching values of up to 0.78 (P < 0.001). Fracture discrimination was improved by combining GTA parameters as shown by logistic regression analysis. It was further demonstrated that GTA combined with BMD allowed for better discrimination than BMD alone (AUC ¼ 0.95; P < 0.001). In order to maximize bone strength, the trabecular orientation has to follow major stress lines [99]. This concept has been applied very early to trabecular bone structure using MIL [239] in order to investigate its relation to bone strength and bone loss [240,241]. The importance of connecting rods perpendicular to the major loading axis in the context of femoral bone strength has been previously emphasized [242]. More recent approaches include topology-based orientation analysis of trabecular bone networks [233] and 3D spatial autocorrelation function (ACF) [243,244]. ACF is defined as the convolution of the image with its complex conjugated mirrored image. The operation of autocorrelation does not require binarization but only returns the power information of the image and is thus nonlinear and irreversible. Compared to MIL, it was found that ACF is faster and considerably more robust to noise [244]. However, when comparing the structural anisotropy and principal direction of the computed fabric tensor for ACF with MIL in ten healthy postmenopausal women, good agreement was determined between the two methods. But ACF analysis yielded greater
anisotropy than MIL for both trabecular thickness and spacing. IMAGE REGISTRATION
Another important tool in the context of osteoporosis is the ability to compare images acquired at different time points. In particular, for treatment monitoring it is very important to be able to analyze the same trabecular sections at baseline and follow-up in order to detect small changes in the fine trabecular structure. To this end, two different registration approaches can be distinguished. The images can be either aligned after acquisition (retrospective registration) [214,245] or the acquisition process of the follow-up scan is adapted to exactly match the field of view of the baseline scan (prospective registration). The latter technique has only recently been applied to trabecular bone imaging [246e248]. Another possible method seeks to only align the ROIs and not to register the images. For example a coronal scout image on which the HR scan is prescribed can provide a longitudinal anatomic reference point for comparing results in similar regions between subjects [214]. In addition, for follow-up scans on the same subject, retrospective registration can be done using rigid body translations and rotations. The images can then be aligned using nearest-neighbor interpolation. But also more complex retrospective registration techniques have been previously used. For example, one algorithm uses local pattern of the trabecular structure near 100 randomly selected points to enable true 3D registration [245]. Prospective registration for trabecular bone MRI has some advantages as it does not require image interpolation since the images are already perfectly aligned after the acquisition [246]. This is a significant advantage for high-resolution MRI with isotropic voxels where the slice thickness is usually larger than the in-plane resolution and partial volume effects are always present. As a result, some image information is always lost during the interpolation process. Another advantage of the prospective registration technique is that it ensures that the ROI is placed on the same slice for both the baseline and follow-up. The method works as follows. First, the baseline images of the subject are acquired including a 3D low-resolution scan followed by the actual high-resolution scan. For the follow-up scan, one low spatial resolution image is again acquired for registration purposes. This scan takes only a few minutes. The low-resolution axial baseline and follow-up scans are then registered. Different registration algorithms such as mutual information [249] or cross-correlation maximization are commonly used for this purpose. Mutual information is computed from the joint probability distribution of the gray-level values. When two images are perfectly aligned, they should provide maximal information about each other and the
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MAGNETIC RESONANCE IMAGING (MRI)
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Cortical Bone MRI
a quantification of the amount of cortical porosity that contains bone marrow. Thus, the percentage, the number, and the size of each cortical pore containing marrow can be assessed. In a recent study, images of the distal radius and the distal tibia of 49 postmenopausal osteopenic women (age 56 3.7) were acquired with both HR-pQCT and MRI [253]. It was found that the amount of cortical porosity did not vary greatly between subjects but the type of cortical pore containing marrow versus not containing marrow, varied widely between subjects. Additionally, the number of cortical pores containing marrow did not depend on the amount of porosity and there was no relationship between cortical pore size and the presence of bone marrow. The data suggest that cortical pore spaces contain different components and that there may be more than one mechanism for the development of cortical porosity and more than one type of bone fluid present in cortical pores. However, this approach only captures relatively large cortical pores, which can be visualized within the resolution limits of MRI.
Image Acquisition
BONE WATER QUANTIFICATION
IMAGE CONTRAST
Using advanced MRI methods with ultrashort echo times (UTE), the bone water content in the microscopic pores of the haversian and the lacunocanalicular systems of cortical bone can be quantified. They contain approximately 20% water by volume [254,255]. A smaller water fraction is also bound to collagen and the matrix substrate and imbedded in the crystal structure of the mineral [256]. These micropores have usually a very small size in the order of a few micrometers and are thus difficult to visualize but the quantification of bone water using MRI could potentially provide a surrogate measure of bone porosity without resolving these individual small pores. The hydrated state of bone is essential in conferring bone material its unique viscoelastic properties [257]. However, the pore water protons commonly possess a very short T2 relaxation time (T2 < 500 ms) and ultrashort TE times have to be employed in order to capture the decaying signal immediately after rf excitation. Bone water quantification measurements have been previously conducted in sheep and human cadaveric specimen and the method’s sensitivity to distinguish subjects of different age and disease state has been evaluated [258]. The data were compared with areal and volumetric bone mineral density (BMD) from dual X-ray absorptiometry (DXA) and peripheral quantitative CT respectively. The bone water content was calibrated with the aid of an external reference (10% H2O in D2O doped with 27 mmol/L MnCl2) which was attached anteriorly to the subject’s tibial midshaft. Excellent agreement (R2 ¼ 0.99) was found in the specimen between the water displaced by using D2O exchange and water measured with respect to the reference sample.
joint probability distribution would yield a high mutual information value [250]. The image transformation usually involves a rotation matrix, characterized by three Euler angles, and a translation vector composed of three translation parameters. The rotation matrix and the translation vector define the movement of a point from the follow-up to the baseline image. The registration is performed while the patient remains in the scanner and usually takes less than 1 minute, including the time to upload the baseline and followup volumes. The output of this registration process is six registration parameters (three translations and three rotations). Thus, the pulse sequence of the follow-up scan has to be modified to allow for the input of these parameters. The registration error found was ~0.2 for rotations and ~1.1 mm for translations. The coefficient of variance was within a 2e4.5% range [246] thus allowing good reproducibility of the two scans.
Another important area of MR bone imaging is the cortical bone surrounding the trabecular bone compartment. Especially for the proximal femur, the ability of MRI to align the image plane perpendicular to the femoral neck is a great advantage and enables acquisition of the cortical architecture more accurately [251]. In contrast, HR-pQCT can image the cortical shell in much shorter scan time and with an isotropic voxel size [35]. However, HR-pQCT is restricted to peripheral sites like the distal radius and the distal tibia. Another challenge in MRI is the contrast between the cortical bone and other tissue. Whereas the fatty bone marrow usually appears bright, the contrast of the outer cortical shell to the surrounding dark muscle (long T1) and dark connective tissues like tendons and ligaments (short T2) is usually less pronounced. Furthermore, partial volume effects and chemical shift artifacts due to different resonance frequencies of water and fat hamper the segmentation process. Using higher readout bandwidths can mitigate the latter effect. In a previous study, investigating the cortical thickness of 41 postmenopausal osteopenic women and 22 postmenopausal osteoporotic women with spine fractures, significant changes in the cortical thickness between the two groups were found underlying the importance of morphologic measurements of the cortical bone structure [252]. CORTICAL POROSITY
Another more recent topic of interest is intracortical porosity. In contrast to HR-pQCT, MRI allows the visualization of soft tissues such as bone marrow and thus
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Measurements in vivo revealed that the bone water content was increased 65% in the postmenopausal group compared to the premenopausal group [258]. Patients with renal osteodystrophy had 135% higher bone water content than the premenopausal group whereas conventional BMD measurements showed an opposite behavior, with much smaller group differences. Further optimizations of 3D-UTE enabled increased spatial resolution with an isotropic voxel size of 0.4 0.4 0.4 mm3 as seen in Figure 50.19, allowing for instant image reformation [259]. PULSE SEQUENCES
The depiction of cortical bone with MRI has somewhat different requirements than trabecular bone imaging. In both cases, the bone itself appears dark and the signal contrast stems solely from the surrounding tissue. This is basically only bone marrow in the case of trabecular bone imaging. Subsequently, the used MR imaging techniques are tailored to maximize the signal of the bone marrow. In contrast, for the segmentation of the cortical bone, there is other soft tissue such as muscle and ligaments. Muscle has a relatively long T1 of more than 1 ms but short T2 and therefore yields usually very low signal. Thus, very long repetition times are preferred to increase SNR. Although 3D pulse sequences deliver higher SNR, the advantage of a 2D pulse sequence is the possibility of multiple
An ex vivo MR image of the human femoral shaft is shown acquired using 3D-UTE techniques. With this technique, the bone itself can be visualized in MR. Residual bone marrow shows up as a bright signal around the cortical bone. Two calibration phantoms with doped water are also depicted.
interleaved acquisitions allowing for much longer repetition times without compromising the clinically feasible scan time. Both techniques have been previously applied. For example, MR images of the distal radius have been acquired at 1.5 Tesla using a 3D fast gradient-echo pulse sequence with an in-plane resolution of 0.16 0.16 mm2 and a 2-mm slice thickness. The same protocol has also been used at 3 Tesla for both the distal radius and the distal tibia [35]. A 2D fast spin-echo (FSE) sequence with a voxel size of 0.47 0.47 2 mm3 was used to image the tibial cortex [251]. A very important site for cortical bone measurements is the neck of the proximal femur, which might be a site responsible for intracapsular hip fractures [260]. Cortical analysis of the hip is complicated by its deep location within the pelvis and the double-oblique angle between the neck and the body’s orthogonal axes. Thus, these measurements are performed on oblique slices perpendicular to the femoral axes. Previously used sequences for the proximal femur applied 2D gradient-echo pulse sequences [251]. An example image from the authors’ laboratory is shown in Figure 50.20. For measurements of cortical porosity [253], a balanced gradient-echo-type sequence was used to maximize spatial image resolution in order to resolve the small cortical pores. Two- and three-dimensional pulse sequences with a radial readout are usually applied for UTE MRI. The radial acquisition allows for very short echo times in the order of a few tens of microseconds [198,261e263]. Whereas for 3D UTE a nonslice-selective hardpulse can be applied, the excitation is more complicated for 2D UTE where usually special rf half pulses have to be implemented [264]. Echo times as short as TE ¼ 64 ms have been used for 3D UTE of cortical bone [259]. Similar values can also be achieved with 2D UTE pulse sequences for cortical bone measurements [258,265] on clinical MR scanners.
FIGURE 50.19
FIGURE 50.20 In vivo MR image of the proximal femur. The white box indicates the region of interest of the femoral shaft where an oblique image of the cortical bone is acquired (right).
VI. DIAGNOSIS AND MANAGEMENT
MAGNETIC RESONANCE IMAGING (MRI)
Image Analysis Image analysis techniques to segment both the inner and outer cortical boundary from their surroundings are commonly semiautomatic. An algorithm previously presented [252] featured a deformable contour (snake) to conform to the strongest gradient edges in the neighborhood of the manually placed ROI. The accuracy of the method was tested by comparing MR images (0.16 0.16 2 mm3) at 1.5 Tesla to high-resolution CT images (spatial resolution 0.07 0.07 0.8 mm3) of ex vivo porcine femora specimens. The in vivo feasibility was tested in the distal radius of a cohort of human subjects. The cortical area was calculated as the area enclosed by the concentric contours. The mean thickness was calculated as area divided by mean contour length. Using this method, very good in vivo reproducibility for cortical volume (CV ¼ 2.19%) and for cortical thickness (CV ¼ 1.96%) was found. Another method used is the distance transform method [50]. The measurements of cortical thickness are further improved by fitting a sphere into the segmented cortical shell. Comparing thickness measurements from MR data with HR-pQCT significant correlations were found but also higher values for the MRI data [35]. One limitation of MRI is the segmentation close to the endplate, where the cortex becomes thin and flares from the narrow diaphysis to the wider epiphysis. This geometry limits the visualization of the cortex in the axial MR images and therefore renders an accurate segmentation more difficult. Another semiautomatic segmentation technique has been presented for the proximal femur [251]. After a rough manual outline of the cortical contour, radial profiles perpendicular to the cortex were normalized to the marrow signal and further processed by morphological operators before computing the cross-sectional area and thickness. A similar CV of around 2% was found for the reproducibility of this technique. Applications The technological developments over the past few years have made quantitative MRI of bone clinically practical [211,212,236,258,266e273]. A substantial improvement in fracture discrimination by including structural information in addition to BMD has been well established [267e269]. The effect of testosterone replacement on trabecular architecture in hypogonadal men was investigated in the distal tibial metaphysis of ten severely testosterone-deficient hypogonadal men [273]. Dramatic topological changes in the bone were found, suggesting that antiresorptive treatment results in improved structural integrity. The effect of salmon calcitonin on bone structure was investigated at the distal radius and calcaneous of 91 postmenopausal women during a period of 2 years [274]. The treatment
915
group showed improved trabecular structure compared to the placebo group but no significant change in BMD was detected. Topological changes of the trabecular bone network after menopause and the protective effect of estradiol were recently reported [275]. Estrogen depletion after menopause is accompanied by bone loss and architectural deterioration of trabecular bone. A short-term temporal change in trabecular architecture after menopause was observed as well as a protective effect of estradiol ensuring the maintenance of a more plate-like TB architecture. This was shown in 65 early postmenopausal women of whom 32 were on estrogen. The temporal changes in trabecular and cortical architecture and density were assessed with MRI, DXA, and pQCT at baseline and after 12 and 24 months. MRI was performed at the distal radius and tibia. For comparison, pQCT of the same peripheral locations was conducted, and trabecular density and cortical structural parameters were measured. Areal BMD of the lumbar vertebrae and hip was also measured. In the distal tibia, substantial changes in trabecular architecture in particular related to topology were identified after 12 months for the control group. In contrast, none of the parameters measured in the estradiol group were significantly different after 12 months. The observed temporal alterations in architecture are consistent with remodeling changes that involve gradual conversion of plate-like to rod-like trabecular bone along with disconnection of trabecular elements. Thus, MRIbased in vivo micromorphometry of trabecular bone has great promise as a tool for monitoring osteoporosis treatment. The clinically prevalent field strength is currently 1.5 Tesla and consequently most of the previous studies were done at this field strength. However, as high-field (3 Tesla) and ultrahigh-field (7 Tesla and higher) modalities become more and more available at clinical sites, there is an increasing trend to use these scanners for bone imaging. Comparing both field strengths with mCT as the standard of reference [276], high correlations of trabecular bone structure were found. When comparing both field strengths in differentiating donors with spinal fractures from those without spinal fractures, MR imaging at 3 T provided a better depiction of the trabecular bone structure than did MR imaging at 1.5 T and a slightly better differentiation between fracture and nonfracture donors [277]. Different pulse sequences for trabecular bone imaging in vivo were previously compared at 1.5 T and 3 T by scanning eight healthy subjects at the proximal femur, calcaneus, and the distal tibia [204]. There was a significant increase in SNR at the higher field strength but also increased susceptibility differences between the more diamagnetic bone and bone marrow. Currently, there is a trend to higher field strengths beyond 3 Tesla. Magnets with 7 Tesla and higher are already installed at many
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sides. The feasibility of bone imaging at these higher field strengths and its impact on imaging quality have been previously investigated [205,278e280]. A substantial increase in SNR was found at 7 T and also an alteration in structural bone parameters as expected from susceptibility induced off-resonances [278]. In a study of ten untreated severely hypogonadal and ten eugonadal men, it was found that testosterone replacement in hypogonadal men improves the mechanical properties of their bone [281]. The hypogonadal men were treated with a testosterone gel for 24 months to maintain their serum testosterone concentrations within the normal range. Each subject was assessed before and after 6, 12, and 24 months of testosterone treatment using MRI of the distal tibia. A subvolume of each MR image was converted to a finite element model. No significant changes in estimated elastic moduli and morphological parameters were detected in the eugonadal group over 24 months but a significant increase in four estimated elastic moduli was found in hypogonadal men. These increases were accompanied by significant increases in trabecular plate thickness. The results of the study suggested that 24 months of testosterone treatment of hypogonadal men improves estimated elastic moduli of tibial trabecular bone by increased trabecular plate thickness.
References [1] P. Ammann, R. Rizzoli, Bone strength and its determinants, Osteoporos. Int. 14 (Suppl. 3) (2003) S13e18. [2] T.J. Beck, A.C. Looker, C.B. Ruff, H. Sievanen, H.W. Wahner, Structural trends in the aging femoral neck and proximal shaft: analysis of the Third National Health and Nutrition Examination Survey dual-energy X-ray absorptiometry data, J. Bone Miner. Res. 15 (2000) 2297e2304. [3] K.L. Stone, D.G. Seeley, L.Y. Lui, J.A. Cauley, K. Ensrud, W.S. Browner, et al., BMD at multiple sites and risk of fracture of multiple types: long-term results from the Study of Osteoporotic Fractures, J. Bone Miner. Res. 18 (2003) 1947e1954. [4] D.M. Black, S.R. Cummings, D.B. Karpf, J.A. Cauley, D.E. Thompson, M.C. Nevitt, et al., Randomised trial of effect of alendronate on risk of fracture in women with existing vertebral fractures. Fracture Intervention Trial Research Group, Lancet. 348 (1996) 1535e1541. [5] P.D. Delmas, E. Seeman, Changes in bone mineral density explain little of the reduction in vertebral or nonvertebral fracture risk with anti-resorptive therapy, Bone 34 (2004) 599e604. [6] C.L. Gordon, T.F. Lang, P. Augat, H.K. Genant, Image-based assessment of spinal trabecular bone structure from highresolution CT images, Osteoporos. Int. 8 (1998) 317e325. [7] T.M. Link, V. Vieth, R. Langenberg, N. Meier, A. Lotter, D. Newitt, et al., Structure analysis of high resolution magnetic resonance imaging of the proximal femur: in vitro correlation with biomechanical strength and BMD, Calcif. Tissue Int. 72 (2003) 156e165. [8] C.H. Chesnut 3rd, C.J. Rosen, Reconsidering the effects of antiresorptive therapies in reducing osteoporotic fracture, J. Bone Miner. Res. 16 (2001) 2163e2172.
[9] B.L. Riggs, L.J. Melton 3rd, Bone turnover matters: the raloxifene treatment paradox of dramatic decreases in vertebral fractures without commensurate increases in bone density, J. Bone Miner. Res. 17 (2002) 11e14. [10] P. Schneider, M. Stauber, R. Voide, M. Stampanoni, L.R. Donahue, R. Muller, Ultrastructural properties in cortical bone vary greatly in two inbred strains of mice as assessed by synchrotron light based micro- and nano-CT, J. Bone Miner. Res. 22 (2007) 1557e1570. [11] V. Sansalone, S. Naili, V. Bousson, C. Bergot, F. Peyrin, J. Zarka, et al., Determination of the heterogeneous anisotropic elastic properties of human femoral bone: from nanoscopic to organ scale, J. Biomech. 43 (2010) 1857e1863. [12] R. Voide, P. Schneider, M. Stauber, P. Wyss, M. Stampanoni, U. Sennhauser, et al., Time-lapsed assessment of microcrack initiation and propagation in murine cortical bone at submicrometer resolution, Bone 45 (2009) 164e173. [13] D.M. Cooper, C.D. Thomas, J.G. Clement, A.L. Turinsky, C.W. Sensen, B. Hallgrimsson, Age-dependent change in the 3D structure of cortical porosity at the human femoral midshaft, Bone 40 (2007) 957e965. [14] K.L. Bell, N. Loveridge, J. Power, N. Rushton, J. Reeve, Intracapsular hip fracture: increased cortical remodeling in the thinned and porous anterior region of the femoral neck, Osteoporos. Int. 10 (1999) 248e257. [15] B. Borah, T. Dufresne, J. Nurre, R. Phipps, P. Chmielewski, L. Wagner, et al., Risedronate reduces intracortical porosity in women with osteoporosis, J. Bone Miner. Res. 25 (2010) 41e47. [16] M.J. Berger, J.H. Hubbell, S.M. Seltzer, J. Chang, J.S. Coursey, R. Sukumar, et al., XCOM: Photon Cross Sections Database, National Institute of Standards and Technology, 1990. [17] W.A. Kalender, D. Felsenberg, H.K. Genant, M. Fischer, J. Dequeker, J. Reeve, The European Spine Phantom e a tool for standardization and quality control in spinal bone mineral measurements by DXA and QCT, Eur. J. Radiol. 20 (1995) 83e92. [18] J.E. Adams, Quantitative computed tomography, Eur. J. Radiol. 71 (2009) 415e424. [19] W. Kalender, Computed Tomography: Fundamentals, System Technology, Image Quality and Applications, Publicis MSD, Verlag, 2000. [20] D.M. Black, D.E. Thompson, The effect of alendronate therapy on osteoporotic fracture in the vertebral fracture arm of the Fracture Intervention Trial, Int. J. Clin. Pract. Suppl. 101 (1999) 46e50. [21] D.M. Black, J.P. Bilezikian, K.E. Ensrud, S.L. Greenspan, L. Palermo, T. Hue, et al., One year of alendronate after one year of parathyroid hormone (1-84) for osteoporosis, N. Engl. J. Med. 353 (2005) 555e565. [22] T. Lang, A. LeBlanc, H. Evans, Y. Lu, H. Genant, A. Yu, Cortical and trabecular bone mineral loss from the spine and hip in long-duration spaceflight, J. Bone Miner. Res. 19 (2004) 1006e1012. [23] K.G. Faulkner, C.E. Cann, B.H. Hasegawa, Effect of bone distribution on vertebral strength: assessment with patientspecific nonlinear finite element analysis, Radiology 179 (1991) 669e674. [24] T.M. Keaveny, P.F. Hoffmann, M. Singh, L. Palermo, J.P. Bilezikian, S.L. Greenspan, et al., Femoral bone strength and its relation to cortical and trabecular changes after treatment with PTH, alendronate, and their combination as assessed by finite element analysis of quantitative CT scans, J. Bone Miner. Res. 23 (2008) 1974e1982. [25] M. Ito, K. Ikeda, M. Nishiguchi, H. Shindo, M. Uetani, T. Hosoi, et al., Multi-detector row CT imaging of vertebral
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longitudinal studies, J. Magn. Reson. Imaging 29 (2009) 118e126. J. Blumenfeld, J. Carballido-Gamio, R. Krug, D.J. Blezek, I. Hancu, S. Majumdar, Automatic prospective registration of high-resolution trabecular bone images of the tibia, Ann. Biomed. Eng. 35 (2007) 1924e1931. J. Blumenfeld, C. Studholme, J. Carballido-Gamio, D. Carpenter, T.M. Link, S. Majumdar, Three-dimensional image registration of MR proximal femur images for the analysis of trabecular bone parameters, Med. Phys. 35 (2008) 4630e4639. C.S. Rajapakse, J.F. Magland, F.W. Wehrli, Fast prospective registration of in vivo MR images of trabecular bone microstructure in longitudinal studies, Magn. Reson. Med. 59 (2008) 1120e1126. I. Hancu, D.J. Blezek, M.C. Dumoulin, Automatic repositioning of single voxels in longitudinal 1H MRS studies, NMR Biomed. 18 (2005) 352e361. J.P. Pluim, J.B. Maintz, M.A. Viergever, Mutual-informationbased registration of medical images: a survey, IEEE. Trans. Med. Imaging 22 (2003) 986e1004. B.R. Gomberg, P.K. Saha, F.W. Wehrli, Method for cortical bone structural analysis from magnetic resonance images, Acad. Radiol. 12 (2005) 1320e1332. B. Hyun, D.C. Newitt, S. Majumdar 2005 Assessment of Cortical Bone Structure Using High-Resolution Magnetic Resonance Imaging Proceedings 13th Scientific Meeting, International Society for Magnetic Resonance in Medicine, Miami, 2005 J. Goldenstein, G. Kazakia, S. Majumdar, In vivo evaluation of the presence of bone marrow in cortical porosity in postmenopausal osteopenic women, Ann. Biomed. Eng. 38 (2010) 235e246. S.R. Elliott, R.A. Robinson, The water content of bone. I. The mass of water, inorganic crystals, organic matrix, and CO2 space components in a unit volume of the dog bone, J. Bone Joint Surg. Am. 39-A (1957) 167e188. K.H. Mueller, A. Trias, R.D. Ray, Bone density and compostiton. Age-related and pathological changes in water and mineral content, J. Bone Joint Surg. Am. 48 (1966) 140e148. P.A. Timmins, J.C. Wall, Bone water, Calcif. Tissue Res. 23 (1977) 1e5. E. Garner, R. Lakes, T. Lee, C. Swan, R. Brand, Viscoelastic dissipation in compact bone: implications for stress-induced fluid flow in bone, J. Biomech. Eng. 122 (2000) 166e172. A. Techawiboonwong, H.K. Song, M.B. Leonard, F.W. Wehrli, Cortical bone water: in vivo quantification with ultrashort echo-time MR imaging, Radiology 248 (2008) 824e833. A. Issever, P. Larson, S. Majumdar, D. Vigneron, R. Krug, W. Chunsheng, et al., 2009 High-Resolution 3D UTE Imaging Of Cortical Bone. Proceedings 17th Scientific Meeting, International Society for Magnetic Resonance in Medicine, Honolulu, 2009, p 3948 N. Crabtree, N. Loveridge, M. Parker, N. Rushton, J. Power, K.L. Bell, et al., Intracapsular hip fracture and the regionspecific loss of cortical bone: analysis by peripheral quantitative computed tomography, J. Bone Miner. Res. 16 (2001) 1318e1328. M.D. Robson, P.D. Gatehouse, M. Bydder, G.M. Bydder, Magnetic resonance: an introduction to ultrashort TE (UTE) imaging, J. Comput. Assist Tomogr. 27 (2003) 825e846. I.L. Reichert, M.D. Robson, P.D. Gatehouse, T. He, K.E. Chappell, J. Holmes, et al., Magnetic resonance imaging of cortical bone with ultrashort TE pulse sequences, Magn. Reson. Imaging 23 (2005) 611e618.
[263] D.J. Tyler, M.D. Robson, R.M. Henkelman, I.R. Young, G.M. Bydder, Magnetic resonance imaging with ultrashort TE (UTE) PULSE sequences: technical considerations, J. Magn. Reson. Imaging 25 (2007) 279e289. [264] C.J. Bergin, J.M. Pauly, A. Macovski, Lung parenchyma: projection reconstruction MR imaging, Radiology 179 (1991) 777e781. [265] A. Techawiboonwong, H.K. Song, F.W. Wehrli, In vivo MRI of submillisecond T(2) species with two-dimensional and threedimensional radial sequences and applications to the measurement of cortical bone water, NMR Biomed. 21 (2008) 59e70. [266] T.M. Link, S. Majumdar, P. Augat, J.C. Lin, D. Newitt, Y. Lu, et al., In vivo high resolution MRI of the calcaneus: differences in trabecular structure in osteoporosis patients, J. Bone Miner. Res. 13 (1998) 1175e1182. [267] F.W. Wehrli, S.N. Hwang, J. Ma, H.K. Song, J.C. Ford, J.G. Haddad, Cancellous bone volume and structure in the forearm: noninvasive assessment with MR microimaging and image processing, Radiology 206 (1998) 347e357. [268] S. Majumdar, T.M. Link, P. Augat, J.C. Lin, D. Newitt, N.E. Lane, et al., Trabecular bone architecture in the distal radius using magnetic resonance imaging in subjects with fractures of the proximal femur. Magnetic Resonance Science Center and Osteoporosis and Arthritis Research Group, Osteoporos. Int. 10 (1999) 231e239. [269] F.W. Wehrli, B.R. Gomberg, P.K. Saha, H.K. Song, S.N. Hwang, P.J. Snyder, Digital topological analysis of in vivo magnetic resonance microimages of trabecular bone reveals structural implications of osteoporosis, J. Bone Miner. Res. 16 (2001) 1520e1531. [270] F.W. Wehrli, P.K. Saha, B.R. Gomberg, H.K. Song, P.J. Snyder, M. Benito, et al., Role of magnetic resonance for assessing structure and function of trabecular bone, Top. Magn. Reson. Imaging 13 (2002) 335e355. [271] M. Benito, B. Gomberg, F.W. Wehrli, R.H. Weening, B. Zemel, A.C. Wright, et al., Deterioration of trabecular architecture in hypogonadal men, J. Clin. Endocrinol. Metab. 88 (2003) 1497e1502. [272] F.W. Wehrli, M.B. Leonard, P.K. Saha, B.R. Gomberg, Quantitative high-resolution magnetic resonance imaging reveals structural implications of renal osteodystrophy on trabecular and cortical bone, J. Magn. Reson. Imaging 20 (2004) 83e89. [273] M. Benito, B. Vasilic, F.W. Wehrli, B. Bunker, M. Wald, B. Gomberg, et al., Effect of testosterone replacement on trabecular architecture in hypogonadal men, J. Bone Miner. Res. 20 (2005) 1785e1791. [274] C.H. Chesnut 3rd, S. Majumdar, D.C. Newitt, A. Shields, J. Van Pelt, E. Laschansky, et al., Effects of salmon calcitonin on trabecular microarchitecture as determined by magnetic resonance imaging: results from the QUEST study, J. Bone Miner. Res. 20 (2005) 1548e1561. [275] F.W. Wehrli, G.A. Ladinsky, C. Jones, M. Benito, J. Magland, B. Vasilic, et al., In vivo magnetic resonance detects rapid remodeling changes in the topology of the trabecular bone network after menopause and the protective effect of estradiol, J. Bone Miner. Res. 23 (2008) 730e740. [276] C.A. Sell, J.N. Masi, A. Burghardt, D. Newitt, T.M. Link, S. Majumdar, Quantification of trabecular bone structure using magnetic resonance imaging at 3 Tesla e calibration studies using microcomputed tomography as a standard of reference, Calcif. Tissue Int. 76 (2005) 355e364. [277] C.M. Phan, M. Matsuura, J.S. Bauer, T.C. Dunn, D. Newitt, E.M. Lochmueller, et al., Trabecular bone structure of the calcaneus: comparison of MR imaging at 3.0 and 1.5 T with
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C H A P T E R
51 The Role of Vitamin D in Orthopedic Surgery Aasis Unnanuntana 1, 2, Brian J. Rebolledo 3, Joseph M. Lane 1 1
Department of Orthopaedic Surgery, Hospital for Special Surgery, 535 70th Street, New York, NY, 10021, USA 2 Department of Orthopaedic Surgery, Siriraj Hospital, Mahidol University, 2 Prannok Street, Bangkok Noi district, Bangkok, Thailand 3 Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, NY, 10021, USA
INTRODUCTION Vitamin D is necessary for efficient calcium and phosphate absorption from the intestine; decreased levels lead to hypocalcemia, resulting in an alteration of bone mineralization process and thus osteomalacia. Vitamin D deficiency is associated with a reduction in bone strength and increased fracture risk. There is a high prevalence of vitamin D deficiency in osteoporotic patients [1]. Unlike osteoporosis, patients with osteomalacia can present with nonspecific musculoskeletal symptoms such as fatigue and myalgias. In addition, fibromyalgia-like symptoms are common in patients with vitamin D deficiency [2]. In this chapter, we define normal vitamin D status as serum 25(OH)D level 32 ng/ml. Low serum vitamin D levels can be subcategorized to insufficiency and deficiency based on 25(OH) D levels: insufficiency (25(OH)D ¼ 20e31 ng/ml) and deficiency (25(OH)D < 20 ng/ml) [3]. Vitamin D status is a critical factor for maintaining musculoskeletal health, and remains an important consideration in patients undergoing orthopedic surgery. While vitamin D deficiency has emerged as a global health issue [4], the prevalence of vitamin D deficiency is also significant in the orthopedic population. A retrospective review of 723 patients who were scheduled to undergo orthopedic procedures at Hospital for Special Surgery, New York, NY, revealed that among six orthopedic services (Trauma, Sports Medicine, Arthroplasty, Foot and Ankle, Hand, and Metabolic Bone Diseases), 43% of patients had low serum vitamin D levels (defined as serum 25(OH)D levels <32 ng/ml), of these 40% were vitamin-Ddeficient (defined as 25(OH)D levels <20 ng/ml). Inadequate vitamin D levels were found to be most common
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10051-4
in the trauma (66%) and sports medicine (52%) services [4a]. Vitamin D deficiency has been linked to the pathogenesis of osteoporosis and fragility fractures [5e8], and has also been suggested to affect functional outcomes after orthopedic procedures [9,10]. Given the high prevalence of vitamin D deficiency in orthopedic surgery patients, the importance of evaluation and subsequent treatment will be necessary to prevent complications that arise from low vitamin D levels. This chapter will address the consequences of a low serum vitamin D level in common orthopedic conditions; not only how it contributes to the disease process but also how it affects the course of treatment. Subjects for discussion include fragility fractures, fracture healing, fracture fixation, and nonunion. Specific to pediatric orthopedics, the chapter will address rickets and slipped capital femoral epiphysis (SCFE). Additionally, the influence of vitamin D on the progression of osteoarthritis and the longevity of total joint implants will be discussed. Lastly, muscle strength and its function are dependent on vitamin D status. Since vitamin D inadequacy can affect various musculoskeletal disorders, orthopedic surgeons should be increasingly aware of the patient’s vitamin D status, and become familiar with the strategies for prevention and treatment of vitamin D insufficiency or deficiency.
IMPACT ON ORTHOPEDIC TRAUMA Fragility Fracture Vitamin D is critically important in bone metabolism. The major function of active vitamin D (1,25(OH)2D) in bone mineralization is to maintain the calciume
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phosphorus product in a supersaturated state while in circulation, thereby resulting in the passive mineralization of the collagen matrix (osteoid) laid down by osteoblasts [11]. In addition, 1,25(OH)2D has several effects on osteoblasts by increasing the expression of a number of key functional proteins, bone specific alkaline phosphatase, osteocalcin, osteonectin, and osteoprotegerin as well as a variety of cytokines [12,13]. Osteocalcin, osteonectin, and osteoprotegerin are noncollagenous proteins that make up the organic components of the bone matrix [14]. Since bone matrix material is one of the major determinants of bone quality, defects in this process lead to poor bone quality resulting in increased risk of fragility fracture. Fragility fracture, defined as low-energy fracture including a fall from no greater than a standing height, is increasingly common and related to osteoporosis or osteomalacia. More than 1.5 million osteoporotic fractures occur annually in the United States, the majority of which occur in the spine, hip, or wrist [15]. Women are predominantly affected by fragility fractures; it is estimated that as many as one in two women who are older than 50 years old will suffer an osteoporotic fracture [16]. These fractures can result in marked morbidity and mortality. For example, a single vertebral compression fracture in women is associated with a 1.2-fold increased age-adjusted mortality rate, and the presence of five fractures per individual increases that risk to 2.3-fold [17]. In addition, many patients who have had a fragility fracture will have another fragility fracture later in life. A history of previous fracture is associated with a twofold increase risk of fragility fracture in women [18]. Thus, orthopedic surgeons must be increasingly suspicious of the conditions that result in bone fragility and become familiar with the current strategies for diagnosis, prevention, and treatment of osteoporosis. The effect of vitamin D supplementation on the rate of fracture has been examined in several randomized controlled trials [19e25]. The reduced risk of fracture is a result of the combined effects of vitamin D on both muscle and bone metabolism [26e29]. A meta-analysis of randomized controlled trials revealed that supplementation with vitamin D lowers the risk of hip fracture by 26% and any nonvertebral fracture by 23% [5]. Diamond et al. reported that the prevalence of vitamin D deficiency (<20 ng/ml) among 41 men over 60 years of age who had sustained osteoporotic hip fractures was higher than the prevalence of these abnormalities among age-matched controls (63% versus 25% in the fracture and control groups, respectively). Furthermore, a multiple regression analysis of this study showed that vitamin D deficiency was the strongest predictor of hip fracture [6]. Although serum 25(OH)D level has been shown to be inversely related to fracture risk, some studies found no positive effect of vitamin D on fracture
rates [7,30]. However, in these studies, serum 25(OH)D levels were in the range of vitamin D insufficiency (serum 25(OH)D level between 20e32 ng/ml). In addition, compliance was a critical factor in these trials because only 50e60% of the participants were still taking the supplements at the indicated follow-up period. Nevertheless, based on the available evidence, a serum 25(OH)D level of approximately 32 ng/ml (80 nmol/L) or above is needed to lower the risk of fracture [31e33].
Fracture Healing Osteoporotic fracture healing is an arduous process. Numerous measures have been applied to enhance fracture healing in the setting of osteoporosis [34]. There has been extensive research to address this problem using various methods that include medications (calcium and vitamin D, 1-hydroxylated vitamin D metabolites and analogs, bisphosphonates, parathyroid hormone (PTH), estrogen, and calcitonin) and physical therapy. Vitamin D administration itself has been widely used for the prevention and treatment of osteoporosis [35]. Some reports have shown that administration of active vitamin D metabolites to a host inhibits bone resorption independent of its calcemic effects in vivo [36]. In addition, animal studies have shown increased mechanical strength of the callus and other beneficial effects with vitamin D and vitamin D metabolite treatment after a fracture [37e40]. A recent study to evaluate the effects of 1,25(OH)2D3 on fracture healing in an osteoporotic fracture rat model showed that the biomechanical testing data at 6 and 16 weeks’ postfracture in the 1,25 (OH)2D3 group were greater than those in the control group. Both micro-CT-based histomorphometric data and histology demonstrated that the fracture callus in the 1,25(OH)2D3 group showed better remodeling when compared to the control group [41]. Evidence shows that receptor for 1,25(OH)2D, the vitamin D receptor (VDR), are present in osteoblasts and chondrocytes [42e44]. Kato et al. described the VDR in the chick callus membrane [45]. In addition, Jingushi et al. demonstrated that the plasma concentration of 1,25(OH)2D rapidly decreased on day 3 after fracture and continued to decrease at 10 days [46]. The authors implied that the rapid disappearance of 1,25 (OH)2D in the early stages after fracture could be due to an increased uptake of 1,25(OH)2D by the fracture callus. This study also suggested that 1,25(OH)2D enhanced osteoporotic fracture healing by the accumulation of the hormone at the fracture site and acting on the VDR in local cells. Although there is much evidence to support the effect of vitamin D on fracture healing within animal studies, the data from clinical trials have been limited. Doetsch
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FIGURE 51.1 Radiographs of a patient with osteoporosis who sustained an intertrochanteric fracture of the right proximal femur and was treated with a long intramedullary nail. (A) Preoperative anteroposterior radiograph. (B) Postoperative anteroposterior radiograph 6 weeks after surgery. (C) Postoperative lateral radiograph of the femur 6 weeks after surgery.
et al. reported the role of vitamin D and calcium supplementation in treating patients with acute fracture of the proximal humerus. Thirty women with osteopenia or osteoporosis were randomized into two groups: placebo and treatment (supplement with calcium 1000 mg and vitamin D3 800 IU/day) groups. At 6 weeks, the bone mineral density (BMD) levels around the fracture site were higher in the treatment group compared with the placebo group; however, this benefit was not sustained at 12 weeks. The authors propose favorable changes in BMD being attributed to increased serum vitamin D and calcium levels in the treatment group [47]. In addition, several studies have demonstrated that fracture healing is affected by a number of endocrine and metabolic abnormalities; including vitamin D deficiency, which can result in nonunion as discussed later in this chapter [48e50].
Fracture Fixation The surgical indications for most fragility fractures are well defined. This includes consideration of the patient’s functional demands and expectations, as well as fracture characteristics, such as a fracture with multiple fragments (comminution), articular impaction, and fracture displacement. Load-sharing implants such as intramedullary devices are generally preferred to load-bearing devices such as plate and screws construct. When fixing fractures with poor bone quality, as encountered in the setting of osteomalacic or osteoporotic fractures, devices offering relative stability are preferable. These include bridge plates, buttress plates, and intramedullary nails. It is also important that the entire bone should be protected to avoid future fracture
(Fig. 51.1) [51]. Because of the decreased BMD and bone strength in this particular group of patients, synthetic bone grafts or augmentation with materials such as polymethylmethacrylate or calcium phosphate cements should be considered [52,53]. Alternatively, arthroplasty may be a better option for periarticular fractures such as those around the hip, knee, and shoulder. The decision between open reduction and internal fixation, and arthroplasty depends on many factors including the patient’s functional demands, life expectancy, and the preference and skills of the surgeon [54]. In some circumstances, immediate arthroplasty allows for rapid mobilization and decreases the risk of fixation failure and reoperation that exits when any form of internal fixation is attempted [55].
Nonunion Fracture nonunion is a multifactorial phenomenon that poses challenging problems for the orthopedic surgeon. Approximately 5e10% of fractured patients will develop complications with fracture healing, including either delayed union or nonunion (Fig. 51.2) [56,57]. The etiology of nonunion can be classified as biologic failure, fracture instability (mechanical failure), or a combination of both. From a biologic perspective, it is known that there are decreased numbers of osteoprogenitor stem cells in elderly patients and remaining cells demonstrate a decreased proliferative response to normal stimuli [54]. Although fracture healing proceeds through normal mechanisms in the elderly, the process is much slower than in younger adults [58e60]. A variety of other contributing factors for nonunion include cigarette smoking, certain medications,
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Sequential radiographs of a 50-year-old man treated with intramedullary nail and angle blade plate who developed nonunion of the distal femoral shaft. (A) Anteroposterior radiograph demonstrating a radiolucent line (arrow) at the distal femur extending from proximal medial cortex to distal lateral cortex. (B) Preoperative anteroposterior radiograph showing fracture of the angle blade plate at the level of nonunion (arrow). (C) Postoperative anteroposterior radiograph 3 months after revision surgery with distal femoral locking plate and injection with bone marrow and demineralized bone matrix. Note bridging callus at the proximal medial cortex (arrow) that represents fracture healing.
FIGURE 51.2
malnutrition, endocrine and metabolic abnormalities [61e72]. The response to fracture involves many metabolic and endocrine factors, including biochemical interactions of growth factors, bone morphogenetic proteins, vitamins, minerals, and hormones. Impairment of any of these factors could potentially affect the fracture-healing process. Many endocrine and metabolic disorders that affect these factors have been shown to be associated with alterations in bony metabolism [28,29,48,61,64e66]. Vitamin D deficiency results in a reduction in the efficiency of intestinal calcium absorption and a decrease in the serum calcium level, leading to the elevation of serum PTH levels [11]. All of these factors have been shown to be associated with impaired fracture healing [57]. Long exposure to elevated PTH levels increases bone resorption, as observed in secondary hyperparathyroidism from chronic renal failure [48]. As noted, intestinal calcium absorption is less effective with vitamin D deficiency, thus depriving the fracture site of the calcium necessary for mineralization. Therefore, any of these correlates of vitamin D deficiency can adversely affect fracture healing and contribute to the development of nonunion. Several studies have described adverse effects on bone healing and bone structure among patients with
metabolic abnormalities. Lancourt and Hochberg reported four patients for whom hyperparathyroidism was identified as a causal factor for fracture nonunion [49]. A caseecontrol study of 107 patients showed that patients with growth hormone deficiency have three times greater risk of fracture, suggesting that this hormone is an important determinant for bone strength [73]. Brinker et al. investigated a consecutive series of 683 patients with nonunion during 1998 to 2005 [50]. Among this group of nonunion patients, 37 patients met the following screening criteria: (1) nonunion without obvious technical error or identifiable etiology; (2) history of multiple low-energy fractures with at least one fracture progressing to a nonunion; (3) a nonunion of a nondisplaced pubic rami or sacral ala fracture. Eighty-four percent of the patients that met these criteria had one or more new diagnoses of endocrine or metabolic abnormalities. The most common newly diagnosed abnormality was vitamin D deficiency (68%) [50]. Although this study does not prove a causal link between metabolic and endocrine abnormalities and the development of nonunion, the authors concluded that nonunion patients who met their screening criteria should be referred to a specialist or endocrinologist for further evaluation.
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IMPACT ON PEDIATRIC ORTHOPEDICS
Stress and Insufficiency Fractures Stress or fatigue fractures are due to abnormal muscular stress applied to normal bone, whereas insufficiency fractures occur when normal muscular activity causes a fracture in bone that is already weakened [74]. The incidence of stress fractures in the overall population is unknown, but in certain groups of people, these fractures are relatively common. The majority of stress fractures occur in the lower extremity with tibia, metatarsals, and fibula being the most commonly reported in the literature [75]. Although the specific etiology of stress fractures is not well delineated, multiple factors, both intrinsic and extrinsic, play a major role in the evolution of stress injuries [76]. Once diagnosed with a stress fracture, nutritional assessment and metabolic work-up including serum vitamin D level should be performed to address the possible factors contributing to the development of the stress fractures. Ruohola et al. reported an association between low serum 25(OH)D levels and bone stress injuries in 756 military recruits with a mean age of 19 years. After 90 days of military training, 22 recruits (2.9%) had stress fractures with the incidence of 11.6 per 100 person-years. The authors found that low serum 25(OH)D level was a significant risk factor for stress fractures [77]. Conversely, insufficiency fractures are most commonly seen in elderly women [78]. There are multiple risk factors for insufficiency fractures, the most common being osteoporosis, osteomalacia, rheumatoid arthritis, prolonged corticosteroid treatment, and pelvic irradiation [79]. Pelvic and sacral insufficiency fractures are not uncommon and usually underdiagnosed. Sacral insufficiency fractures are often bilateral and occur mostly in the sacral alae [80]. The clinical manifestation is variable, although it usually presents with severe low back pain, exacerbated by movement and radiating to the leg or groin, with no history of trauma [79]. Computed tomography is an accurate, efficient and specific investigation for this particular fracture [78,80,81]. The diagnosis of insufficiency fracture mandates the need for further laboratory investigations to elucidate the cause of osteopenia, including measuring serum calcium, phosphate, bone-specific alkaline phosphatase, and 25(OH)D levels. The treatment for insufficiency fractures is generally conservative with limited weightbearing activities, appropriate pain control, and adequate calcium and vitamin D supplementation. Sacral fractures heal relatively fast, but the pubic fractures often have a protracted course [82]. Recently, there is a concern of the possible association between long-term treatment of bisphosphonates and the occurrence of insufficiency femoral fractures. Although the pathogenesis of this insufficiency fracture is not fully understood, it has been postulated that there
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is a profound osteoclast inhibition and the resulting suppression of bone turnover and bone remodeling, leading to skeletal fragility [83,84]. However, most of the literature on this subject is case reports, case series, or caseecontrol studies [85e87]. It is important to recognize that associations in these studies can be due to chance, biases, confounding factors, or may be truly indicative of causality. Therefore, concern regarding the association between bisphosphonates and insufficiency femoral fractures should not preclude the use of these agents in the treatment of osteoporosis. Nevertheless, these drugs should be used with caution and closely monitored by the clinicians. Patients with thigh pain who are currently being treated with bisphosphonates should undergo imaging studies such as plain radiographs, bone scan, or magnetic resonance imaging (MRI). For those patients with insufficiency fractures that are taking bisphosphonates, serum calcium, and vitamin D levels should be corrected, bisphosphonates should be stopped, and the use of an anabolic agent should be considered. Prophylactic internal fixation may be employed if there is no sign of radiologic union and the patient remains symptomatic [87a].
IMPACT ON PEDIATRIC ORTHOPEDICS Rickets Vitamin D deficiency in the pediatric population results in rickets. Rickets affects not only the bone structure, but also the epiphyseal cartilage. Structural changes in the epiphyseal plates are quite remarkable and are virtually diagnostic for rickets in growing children. The resting and proliferative zones are relatively normal in appearance, but there is a marked increase in the size and number of cells in the intermediate zone, with profusion of cell organization and structure. The zone of provisional calcification contains very little calcified material, and the zone of primary spongiosa has small irregular trabeculae with little bone and wide osteoid seams [88]. The axial height of the epiphyseal cartilage is much greater than normal and may extend far into the metaphysis of the underlying bone. These changes in the epiphyseal plate are dependent on the rate of growth, evidenced by findings in the distal femur, proximal tibia, proximal femur, and distal radius. Therefore, bowing or knock-knee deformities are commonly found in children with rickets. Surgical treatment to correct these deformities in patients with rickets is feasible but requires careful surgical planning and preoperative metabolic stabilization [89]. Since numerous types of rickets share similar histologic changes as well as clinical and radiographic characteristics, it is crucial to identify the underlying cause of
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rickets by analyzing biochemical laboratory profiles including serum calcium, phosphate, bone-specific alkaline phosphatase, PTH, 25(OH)D, and 1,25(OH)2D; determination of the fractional urinary excretion rate of phosphate is also usually required to confirm the diagnosis of hypophosphatemic rickets (see below), usually owing to the overproduction of a “phosphatonin” [90]. X-linked hereditary hypophosphatemic rickets (X-linked HPR) is the most common inherited etiology for rickets, marked by renal phosphate wasting, and abnormal mineralization of bone [91]. Further studies to define the genetic and molecular basis of the disease have led to the identification of phosphate-regulating genes on the X-chromosome (PHEX), which are known to be responsible for X-linked HPR [92e95]. It is also believed that X-linked HPR is caused by abnormalities in fibroblast growth factor-23 (FGF-23). FGF-23 inhibits tubular phosphate reabsorption, inhibits 1,25(OH)2D production, and stimulates 24,25(OH)2D production [90]. The clinical manifestations of X-linked HPR include disproportionate short stature and angular limb deformities such as genu varum (bowed legs) or genu valgum (knock-knees), which become apparent after the age of 1 or 2 years [96]. The objectives of management in children with HPR are to control hypophosphatemia, prevent deformities of long bones, enhance bone growth, and reduce bony lesions [97]. Treatment for this disease consists of oral phosphate and active vitamin D metabolite supplementation [98,99]. Pharmacologic treatment frequently improves the rate of growth, controls the progression of the deformity, and reverses radiographic signs of active rickets [100e102]. In addition to pharmacological treatment, bracing should be considered to slow or halt progression of the angular deformity until adolescence, when correction of the deformity can be performed with the minimum risk of deformity recurrence [9,98]. As children approach adulthood the requirement for medical intervention subsides due to the presence of lower bone turnover and closure of the epiphyseal plates. Therefore, an adult patient with X-linked HPR may be treated with only low-dose calcitriol or phosphorous, or a combination of both. It is unknown which patients will require long-term medical treatment and which patients are safe to discontinue the therapy [103]. Despite adequate medical treatment, some patients remain unresponsive and the deformities do not resolve, resulting in malalignment of the lower extremities that is severe enough to disturb gait and compromise adjacent joint mechanics [97,104,105]. In this setting, corrective osteotomy may be judiciously indicated even during the growing period (Fig. 51.3). Petje et al. reviewed 33 patients with HPR treated between 1978 and 2005. Ten of these patients with
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FIGURE 51.3 Radiographs of a 14-year-old girl with X-linked hypophosphatemic rickets. (A) Preoperative standing anteroposterior radiograph showing a 23.6 varus deformity of the left lower extremity. (B) Postoperative standing anteroposterior radiograph after surgical correction with angle blade plate at the distal femur and external fixation device at the proximal tibia. The postoperative alignment was substantially improved. (Radiographs courtesy of Dr. Roger F. Widmann, Hospital for Special Surgery, New York, NY.)
X-linked HPR had been treated with surgical treatment from early childhood to maturity. After a minimum of 10 years’ follow-up, the authors found that 90% of the deformities recurred after the first corrective procedure, while 60% recurred after a second procedure [104]. Similarly, Evans et al. reported a series of ten patients with HPR in which seven patients had been treated surgically. Five out of seven patients (71%) required more than one operation over a minimum of 9 years followup [98]. Furthermore, Choi et al. reported the results of deformities correction using Ilizarov method in 14
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IMPACT ON PEDIATRIC ORTHOPEDICS
patients with an average age of 13 years 9 months. The deformity recurred in one patient who was 3 years old. The authors also found that there was a significant negative correlation between the healing index and the serum phosphate level: those who had a serum phosphate level higher than 2.5 mg/dl showed relatively rapid bone healing compared with patients with less than 2.5 mg/dl [97]. Based on the available evidence, it is important to recognize X-linked HPR early in life. Although early diagnosis leads to appropriate medical management, some patients may require major operative procedures to correct residual deformities. However, recurrence of deformities in the lower limb is common and patients should be counseled to anticipate two or three corrective procedures during their growth development [104]. While nutritional rickets and osteomalacia are widely recognized forms of the hypocalcemic disorder, orthopedic surgeons should also be aware of the uncommon forms, especially those that relate to orthopedic conditions. Two of these rare forms are associated with genetic disorders. This includes resistant rickets in patients with multiple sites of fibrous dysplasia [106e108]; and a second type of resistant rickets in patients with type I neurofibromatosis [109e111]. The etiology of both disorders remains unknown. In addition, tumor-induced HPR (oncogenic rickets/ostemalacia) is another rare form that has been seen in the setting of both benign and malignant bone and softtissue tumors [112e116]. Most of these tumors secrete FGF-23, which leads to phosphaturia and hypophosphatemia, resulting in the development of rickets or osteomalacia [114,115,117]. The degree of osteopenia is usually more severe than seen in the other form of rickets, and generally intractable to traditional treatment with vitamin D supplementation. Once the diagnosis is made, removal of the tumor is essential to treat HPR [112,116,118]. Recurrence of disease is perhaps the best available marker for recurrent tumors or metastases [119]. The clinical presentation of tumor-induced HPR may be identical to familial or sporadic X-linked hereditary HPR. Therefore, in new HPR cases, a careful evaluation of other family members should be made for hypophosphatemia and osteomalacia or rickets [120]. Nonfamilial-related HPR should alert clinicians to carefully investigate for an associated tumor, which may be amenable to a surgical cure.
Slipped Capital Femoral Epiphysis (SCFE) SCFE is another orthopedic condition that occurs in the pediatric population and should raise concerns of underlying metabolic bone diseases. SCFE represents a skeletal deformity characterized by the femoral epiphysis becoming displaced from the metaphysis
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through the growth-plate cartilage [121]. Histologic examination of the growth-plate cartilage in patients with SCFE has shown abnormalities in the matrix of the hypertrophic cartilage zone where matrix mineralization occurs [122,123]. While the etiology of SCFE remains unclear, it is believed that mechanical and endocrine factors play major roles in the slippage [124e126]. Vitamin D, specifically 1,25(OH)2D, and PTH are important in matrix mineralization during growth spurts [88]. In addition, a number of in vitro studies showed that PTH induces proliferation of chondrocytes during chondrogenesis [127,128]. Therefore, vitamin D deficiency during these periods of rapid growth could significantly affect the epiphyseal region, leading to SCFE. Jinguishi et al. investigated 13 patients with SCFE who had measurements of serum 25(OH)D, 1,25(OH)2D, and three immunoreactive forms of PTH: the whole peptide (1-84)PTH, the carboxy-terminal portion of PTH and midportion of PTH. The authors found that serum levels of 1,25(OH)2D and midportion of PTH were significantly lower in SCFE patients than those of controls; however, the levels of these two markers returned to normal within a year after the onset of the disease [129]. Several studies reported a seasonal effect to the onset of symptoms of SCFE [130e132]. Loder et al. investigated 1630 children with 1993 SCFE from six continents and 33 orthopedic centers. The authors found a seasonal peak in the onset of symptoms related to SCFE in late June in North America and in late July in Europe. However, this seasonal variation was only present in children who lived above a latitude of 40 North [133]. Brown examined seasonal variation of hospital admissions for SCFE using a national database (National Inpatient Sample) and compared seasonal variation between the northern and southern United States, between genders, and between black and white children. All subgroups in the north and in the south had the greatest number of cases in September and during the 3-month period between August and October. Significant seasonal variation was found in the children who lived in the northern part. Some seasonal variation was present in the south; however, it did not reach the level of statistical significance [134]. Possible explanations for this peak incidence include seasonal changes in activity; seasonal patterns of growth and weight gain in adolescence; and vitamin D deficiency caused by its decreased synthesis during the winter [133,134]. Although these studies demonstrated the effect of seasonal variation to the occurrence of SCFE, they lack the ability to detect a direct relationship between vitamin D deficiency and SCFE. Nevertheless, we encourage orthopedic surgeons to incorporate metabolic evaluation as part of their routine surgical care. All patients diagnosed with SCFE should be instructed to supplement with adequate calcium and vitamin D.
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IMPACT ON TOTAL JOINT ARTHROPLASTY Osteoarthritis Osteoarthritis (OA) is a degenerative joint disease that is associated with changes in both cartilage and bone tissue. It is characterized by radiographic evidence of osteosclerosis in subchondral bone and osteophytes in the affected joint. In contrast, osteoporosis is characterized by a loss of BMD as measured from dual-energy X-ray absorptiometry. The relationship between these two musculoskeletal diseases is of special relevance to the aging population because both have increasing incidence and prevalence with age. There is a general belief that OA protects a patient from generalized primary osteoporosis. Foss and Byers reported the absence of osteoarthritic changes on the radiographs of fractured hips in osteoporotic subjects [135]. Subsequent studies also showed that the incidence of OA is inversely associated with the incidence of osteoporosis [136e139]. It is not uncommon to encounter postmenopausal patients with fragile cancellous bone both in the proximal femur and in the acetabulum during total hip arthroplasty (THA). In addition, some recent studies found coincidentally low BMD and vitamin D insufficiency in a substantial portion of patients undergoing THA [140,141]. The prevalence of vitamin D deficiency in patients undergoing total joint replacement ranges from 22% to 81%, depending on the cut-off reference value used in the analysis [140e142]. Makinen et al. investigated the incidence of osteopenia and osteoporosis in postmenopausal women who were scheduled for cementless THA. The authors found that the majority (73%) of the OA patients had BMD in the range of osteopenia (45%) or osteoporosis (28%), while the prevalence of vitamin D deficiency (defined as serum 25(OH)D levels 50 nmol/L or 20 ng/ml) was 36%. Interestingly, secondary osteoporosis was found in 9% of the cases in this study, and 15% of the patients needed preoperative consultation with an endocrinologist and occasionally rescheduling of the planned surgery [141]. Glowacki et al. performed a similar analysis of 68 postmenopausal women prior to THA, showing occult osteoporosis in 25% of the patients and vitamin D deficiency (defined as serum 25(OH)D3 levels 37.5 nmol/L or 15 ng/ml) in 22% of the patients [140]. Several investigators have assessed the possible relationship between the presence and progression of OA and vitamin D deficiency; however, the results remain controversial [143e145]. Three longitudinal epidemiologic studies showed that low vitamin D levels worsen the course of OA [143,146,147]. By using data from the original Framingham Study cohort, McAlindon et al. revealed that patients with pre-existing OA of the knee
who had vitamin D levels in the lowest and middle tertiles had a threefold increased risk of radiographic worsening of the OA [147]. Similarly, Lane et al. reported that participants with vitamin D levels in the lowest and middle tertiles were associated with incident hip OA, defined as development of joint space loss [146]. Furthermore, Bergink et al. investigated 1248 subjects from the Rotterdam Study over 6.5 years. The authors found that progressive radiographic OA of the knee occurred in 5.1% of the participants in the highest tertile of vitamin D intake against 12.6% in the lowest tertile, resulting in an adjusted odds ratio of 7.7 [143]. Conversely, Felson et al. investigated a study of 715 subjects recruited from the Framingham Offspring cohort and 277 subjects with established knee OA, recruited from the Boston Osteoarthritis of the Knee Study (BOKS). Progression of the OA was assessed by radiographs and/or MRI of the knee joint. In the Framingham Offspring cohort, 20.3% of the knees showed signs of worsening OA from the radiographs over a period of 9 years, while 23.6% of the knees from the BOKS showed signs of radiographic worsening over a 30-month follow-up period. There was no association between baseline 25(OH)D levels and radiographic worsening in either cohort. The authors concluded that vitamin D status is unrelated to the risk of joint space or cartilage loss in knee OA [148]. The clinical importance of conditions that compromised bone strength such as in vitamin D deficiency or osteoporosis to total joint replacement is still elusive. Osteoporotic bone has distinct morphologic characteristics. The diaphysis undergoes both endosteal cortical resorption and medullary expansion. The result is a thinning of the cortex and an overall increase in the diameter of the bone. These changes make it difficult to achieve stable prosthetic fixation. In the multivariate survival analysis, Kobayashi et al. identified that a medullary femoral canal with an unfavorable geometry (a stovepipe canal) was the only risk factor for femoral fixation failure. The 20-year survival rates were 92% and 99% for cemented femoral prostheses in stovepipe canals and nonstovepipe canals, respectively [149]. In addition, Nixon et al. reviewed 127 patients with cemented THA at an average follow-up of 7.7 years. The authors matched 78 patients with a loose prosthesis by age, gender, race, prosthesis, and time from surgery with 49 patients with a well-fixed stable hip replacement. Patients with loose components were more likely to have a history of fragility fracture, narrow femoral cortices, and lower BMD at the lumbar spine or periprosthetic area compared to the group with stable fixation. Low serum vitamin D level was common in both groups (41% had vitamin D deficiency, defined as serum 25(OH)D3 levels <50 mmol/L or 20 ng/ml); however, there was no significant difference between the two
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IMPACT ON MUSCLE STRENGTH AND REHABILITATION
groups [142]. Although there are concerns regarding long-term survival of total joint arthroplasty in the elderly population, on the basis of the currently available evidence, there are no definitive data on the clinical significance of decreased BMD or osteomalacic bone on the long-term survival of total joint replacement.
Periprosthetic Fracture Periprosthetic fracture is a devastating complication after total joint arthroplasty and is associated with a high rate of postoperative complications with often a poor clinical result (Fig. 51.4) [150,151]. According to the Mayo Clinic Joint Registry, the incidence of intraoperative fractures was 0.3% of patients with cemented prostheses and 5.4% of those with cementless prostheses after primary arthroplasty. During revision arthroplasty, 3.6% of patients with cemented and 20.9% of those with cementless prostheses experienced intraoperative fracture. For postoperative fractures, the incidence is estimated to be 0.1e2.5% of patients over the life of the implant [152]. Osteoporosis is a well-known risk factor for periprosthetic fracture [153e155]. However, only few studies have systematically investigated the effect of the patient’s bone quality on subsequent periprosthetic fracture risk [156,157]. Beals and Tower showed that 33 of 86 patients (38%) had either radiographic osteopenia or prior history of vertebral or metaphyseal fractures suggesting osteoporosis. The authors indicated
(A)
(B)
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that osteopenia/osteoporosis is a common finding in patients with periprosthetic fractures [157]. Wu et al. reported 16 postoperative fractures in a series of 454 consecutive cementless THA [156]. By using Singh’s index of osteoporosis and canal flare index of the proximal femur, preoperative osteoporosis was a predictive indicator of periprosthetic femoral fracture. Although there are no studies supporting the direct association between vitamin D deficiency and periprosthetic fractures, given the high coincidence between osteoporosis and osteomalacia and the fact that vitamin D influences bone quality, all total joint arthroplasty patients with compromised bone stock should be supplemented with adequate calcium and vitamin D. If possible, vitamin-D-deficient/insufficient states should be resolved in advance of elective surgical procedures.
Thigh Pain One of the most common complications following THA is thigh pain; defined as mid-thigh pain at the anterolateral aspect of the limb, grossly at the tip of the prosthetic stem [158]. The pain is absent at rest, reaches its peak in the start-up phase, and diminishes over the movement. The prevalence of thigh pain ranges from <1% to 40% [159e161]. Although many hypotheses have been proposed about the etiology of thigh pain, most can be narrowed down to one of the following mechanisms: tip micromotion or rigidity mismatch [158,162]. In the latter mechanism, there is an overload of stress around the stem tip, resulting in excessive bone strain and endosteal irritation. The hypothesis of rigidity mismatch is consistent with several risk factors for thigh pain already brought out by clinical observation: high modulus of elasticity of the stem, large and solid implant, and soft bone such as in osteoporosis or osteomalacia [163e166]. In the setting of osteoporosis, the femoral rigidity is reduced and the medullary canal is widened. This allows an implantation of a large femoral stem. Engh et al. reported that the rate of thigh pain was 11% among patients with good preoperative bone quality, while it was 26% in those with poor preoperative bone quality [166].
IMPACT ON MUSCLE STRENGTH AND REHABILITATION Radiographs of a patient with osteoporosis who sustained a periprosthetic fracture of the distal femur. (A) Anteroposterior radiograph demonstrating well-fixed femoral component with displaced distal femoral fracture. (B) Lateral radiograph showing fracture of the distal femur and superior pole of the patella. Note the tibial component was loosened and collapsed into the medial metaphysis of the proximal tibia.
FIGURE 51.4
Muscle strength is critically important in orthopedic surgery, particularly during the postoperative rehabilitation phase. Aging is accompanied by a reduction in muscle mass and muscle strength [167e169]. The agerelated change in hormone concentrations has been hypothesized to play a role in the loss of muscle mass
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and muscle strength with aging, the so-called condition known as sarcopenia [167e169]. The gradual loss of muscle strength results in functional impairment [170,171], the need for assistance in the performance of daily activities [172] and an increased risk of falling and nonvertebral fractures [173]. In male and female participants of the Longitudinal Aging Study Amsterdam, aged 65 years and older, grip strength (1008 participants), and appendicular muscle mass (331 participants) were measured during 1995e1996 and after a 3-year follow-up. After adjustment for physical activity level, season of data collection, serum creatinine concentration, chronic disease, smoking, and body mass index, persons with low baseline 25(OH)D levels (<25 nmol/L or 10 ng/ml) and high PTH levels (>4 pmol/L or 38.1 pg/ml) were associated with an increased risk of sarcopenia [174]. A number of studies demonstrated the important role of vitamin D to the muscle function [175e178]. The VDR has been reported to be found in skeletal muscle [179e181] and in the nerves through which muscle contraction and relaxation will be controlled by influx and efflux of calcium and in addition to the muscle protein synthesis [182]. A study in VDR-null mice showed that the absence of VDR on a global, whole-organism scale caused a reduction in skeletal muscle fiber size, based on an increased expression of myogenic regulation factors (Myf5, Myogenin, and E2A) through which the strict regulated differentiation and maturation of muscle cells will be disturbed [183]. This finding suggests that there may be an important role of the VDR in muscle development, although a muscle-specific knockout of the VDR has yet to recapitulate the phenotype of the global knockout. A recent study investigating the role of vitamin D for fatty degeneration and muscle function in the rotator cuff muscle showed that a lower serum vitamin D was related to higher fatty degeneration in the rotator cuff muscles. A subgroup analysis of 228 patients in this study who had full-thickness rotator cuff tear revealed that serum vitamin D level was an independent variable for fatty degeneration of the supraspinatus and infraspinatus muscles [10]. Active vitamin D metabolites (1,25(OH)2D3) induce rapid, nongenomic changes in calcium flux in the muscle cell [184]. Evidence indicates that 1,25(OH)2D3 binds a VDR in the muscle membrane, activating several interacting second-messenger pathways and resulting in enhanced calcium uptake into the cell [185,186]. Although 1,25(OH)2D3 is considered to be the active metabolite affecting target sites including muscle, clinical studies have reported a relationship between serum 25(OH)D and muscle strength and functional ability [187e189]. There are at least two possible mechanisms that might explain these findings. Firstly, serum 25
(OH)D concentrations are approximately 1000 times that of serum 1,25(OH)2D; as such, there is the potential for 25(OH)D to interact with and activate the VDR in that cell [190,191]. Secondly, peripheral tissues were found to express the CYP27b-1a-hydroxylase [192]; therefore, it is possible that local conversion of 25(OH) D to 1,25(OH)2D within a target tissues may be involved in regionally controlled cell function [193].
Vitamin D and Falls Falls are one of the most common geriatric problems threatening the independence of older people. Falls increase the risk of fracture by tenfold in people with osteopenia or osteoporosis [194]. Fear of falling is one of the most important factors influencing functional recovery after hip surgery in older people [195]; 16% of people with a tendency to fall limit their activity due to fear of falling and 30% reduce their participation in social activities [196,197]. As this is a critically important issue, several strategies should be implemented to lower the risk of fall including education to the highrisk groups, combined exercise program to improve muscle strength, flexibility, coordination and balance, and adequate nutrition [198,199]. Vitamin D deficiency has been shown to predominantly affect the proximal muscle groups and is clinically manifested by a feeling of heaviness in the legs, fatigue, and difficulty in climbing stairs and rising from a chair [200e202]. The majority of these muscle groups are weight-bearing antigravity muscles of the lower limb necessary for postural balance and walking [203]. A significant inverse correlation between 25(OH) D level and the occurrence of falls in the elderly population has been reported in many studies [187,189]. In a randomized, double-blind, placebo-controlled study, Dukas et al. showed that 1.0 mg of the active vitamin D metabolite alfacalcidol once a day significantly reduced the number of patients who fell in an elderly community-dwelling population [204]. Furthermore, another randomized, double-blind, placebo-controlled trial found an association between daily supplementation of vitamin D (cholecalciferol 700 IU) with calcium (calcium citrate 500 mg) and the reduction of falls in ambulatory elderly patients. During this 3-year study, vitamin Decalcium supplementation reduced falls by 46% in ambulatory women and 65% in less active women; nevertheless, supplementation had a neutral effect in men independent of their physical activity level. The explanation for these findings is less clear; however, it should be noted that 25(OH)D levels were similar at baseline and 3-year follow-up in the group of less active women when compared to other treatment subgroups that also received vitamin D and calcium supplementation [205].
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PREVENTION AND TREATMENT IN ORTHOPEDIC PATIENTS
Although available evidence indicates that vitamin D supplementation preserves muscle strength, reduces risk of falls and maintains functional ability, the question regarding the effect of vitamin D in muscle function, particularly after orthopaedic surgery that involves extensive muscle dissection during the procedure, and the risk of falls in a healthy individual remains unknown. Additional research, preferably by means of controlled randomized trials in this field, is therefore warranted.
PREVENTION AND TREATMENT IN ORTHOPEDIC PATIENTS The role of vitamin D in orthopedic surgery involves bone metabolism and muscle function. Vitamin D sufficiency enhances bone strength, improves muscle strength, and decreases the risk of falling. Normal 25 (OH)D levels also minimize circulating PTH levels and thus decrease osteoclastogenesis [206]. Therefore, every orthopedic patient, especially those with the diagnosis of osteoporosis, should have daily calcium supplementation and restoration of 25(OH)D levels to normal (see below). For optimal treatment, adequate calcium intake of 1000e1500 mg/day is required [1]. The current recommended dosages of vitamin D from the Institute of Medicine are 400e800 IU/day, depending on the age of the subject and the presence or absence of underlying bone disease [207]. However, many experts consider these recommendations to be too low [208]. In addition, several clinical trials that used at least 800 IU of supplemental vitamin D daily support the strong recommendation that adequate calcium and vitamin D intake is essential to decrease the risk of osteoporosis and fractures [7,8,209]. Thus, we and others propose that the minimum adult intake should be 1000e2000 IU/day of vitamin D [1,210,211]. The appropriate amount of vitamin D intake should be evaluated by monitoring serum 25(OH)D and PTH levels. The goal is to prevent an elevation of serum PTH; therefore, serum 25(OH)D level should be kept above 32 ng/ml [32,33]. Patients with fragility fractures should be screened for osteoporosis and metabolic bone diseases such as vitamin D insufficiency/deficiency. Low 25(OH)D levels should be rapidly corrected with pharmacological doses of vitamin D, after which adequate vitamin D should be given to sustain 25(OH)D levels above 32 ng/ml. In the United States, the only pharmaceutically available form of vitamin D is vitamin D2 (ergocalciferol) in a 50 000 IU dosage formulation. Malabanan et al. found that giving ergocalciferol 50 000 IU once a week for 8 weeks was effective to correct vitamin D deficiency and increase 25(OH)D levels by more than 100% [212]. After 8 weeks, serum calcium, vitamin D, and PTH levels should be
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monitored again. To prevent recurrent vitamin D deficiency and to maintain adequate levels in patients who are vitamin-D-sufficient, continuing vitamin D supplementation is necessary. This can be done by giving either vitamin D2 50 000 IU twice a month or the equivalent of 1000e2000 IU of vitamin D3 daily [210e213]. In fact, vitamin D3 given at 2000 IU/day is considered to be a conservative recommendation by many [214]. In the absence of an active granuloma-forming disease (e.g., sarcoidosis), toxicity is rare even if a daily dosage of 10 000 IU vitamin D3 is given for up to 4 months [215]. To improve medical management of fractured patients, a clinical pathway should be established in each institution. The clinical pathway includes developing a multidisciplinary team; promoting appropriate use of diagnostic tools and therapeutic approaches without compromising the quality of care; and educating patients and their relatives about the management of their disease (physical therapy, lifestyle modifications, and nutrition). Several clinical pathways have been developed to support orthopedic surgeons and the program was found to improve awareness of fragility fractures, rates of post-fracture follow-up, and management of fractures [216,217]. Recently, the American Orthopaedic Association (AOA) has developed a program known as “Own the Bone,” which is a webbased model for post-fracture osteoporosis management to prevent secondary fractures. The 10-month pilot study on this project on 635 participants over 14 sites found that the intervention produced significant improvements in patient counseling on calcium and vitamin D supplementation, exercise, fall prevention, and communication with primary care providers and the patients [218]. Therefore, the “Own the Bone” initiative offers tools to improve the prevention of secondary fracture and a structure to monitor patient compliance. The majority of orthopedic procedures are performed in the elderly population with the highest risk of osteoporosis [219,220]. Predominance of many studies that investigated the prevalence of vitamin D deficiency among the elderly supports that vitamin D deficiency is very common in patients with osteoporosis [221e224]. In addition, middle-aged and elderly adults are at high risk for vitamin D deficiency because of poor dietary intake, inadequate sun exposure, and an age-related decrease in vitamin D synthesis [3,225,226]. Multiple cross-sectional studies in community-dwelling older adults have found a direct association between serum vitamin D level and parameters of physical performance, especially when serum 25(OH)D levels are 30 ng/ml [227e231]. Although there is much evidence to support normalization of 25(OH)D levels in the treatment of osteoporosis, the role of vitamin D supplementation in the healthy younger population or during the rehabilitation period after an orthopedic
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procedure is still lacking. Future research in this area is therefore warranted.
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[224] S. Boonen, H.A. Bischoff-Ferrari, C. Cooper, P. Lips, O. Ljunggren, P.J. Meunier, et al., Addressing the musculoskeletal components of fracture risk with calcium and vitamin D: a review of the evidence, Calcif. Tissue Int. 78 (2006) 257e270. [225] S. Bakhtiyarova, O. Lesnyak, N. Kyznesova, M.A. Blankenstein, P. Lips, Vitamin D status among patients with hip fracture and elderly control subjects in Yekaterinburg, Russia, Osteoporos. Int. 17 (2006) 441e446. [226] M.J. McKenna, Differences in vitamin D status between countries in young adults and the elderly, Am. J. Med. 93 (1992) 69e77. [227] D. Shahar, M. Levi, I. Kurtz, S. Shany, I. Zvili, E. Mualleme, et al., Nutritional status in relation to balance and falls in the elderly: a preliminary look at serum folate, Ann. Nutr. Metab. 54 (2009) 59e66. [228] H.A. Bischoff-Ferrari, T. Dietrich, E.J. Orav, F.B. Hu, Y. Zhang, E.W. Karlson, et al., Higher 25-hydroxyvitamin D concentrations are associated with better lower-extremity function in both active and inactive persons aged > or ¼60 y, Am. J. Clin. Nutr. 80 (2004) 752e758. [229] L. Ceglia, Vitamin D and its role in skeletal muscle, Curr. Opin. Clin. Nutr. Metab. Care 12 (2009) 628e633. [230] P. Gerdhem, K.A. Ringsberg, K.J. Obrant, K. Akesson, Association between 25-hydroxy vitamin D levels, physical activity, muscle strength and fractures in the prospective populationbased OPRA Study of Elderly Women, Osteoporos. Int. 16 (2005) 1425e1431. [231] N.O. Kuchuk, S.M. Pluijm, N.M. van Schoor, C.W. Looman, J.H. Smit, P. Lips, Relationships of serum 25-hydroxyvitamin D to bone mineral density and serum parathyroid hormone and markers of bone turnover in older persons, J. Clin. Endocrinol. Metab. 94 (2009) 1244e1250.
VI. DIAGNOSIS AND MANAGEMENT
S E C T I O N V I I
NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
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C H A P T E R
52 Worldwide Vitamin D Status Paul Lips 1, Natasja van Schoor 2 1
Department of Internal Medicine, Endocrine Section, VU University Medical Center, Amsterdam, The Netherlands 2 EMGO Institute for Health and Care Research, VU University Medical Center, Amsterdam, The Netherlands
INTRODUCTION Vitamin D status has been determined in numerous studies covering all continents and many countries. The measurement of 25-hydroxyvitamin D (25(OH)D) has been used to assess vitamin D status, to diagnose vitamin D deficiency, and to determine the effect of vitamin D supplementation. There is no agreement on the required serum 25(OH)D concentration for good health. However, most clinicians agree that clinical vitamin D deficiency only occurs when serum 25(OH) D is lower than 25 nmol/L (10 ng/ml) [1,2]. Opinions differ on whether the optimal serum 25(OH)D for optimal bone mineral density, bone turnover, muscle strength, and nonclassical effects should be 50 nmol/L, 75 nmol/L or higher [3,4] (differing opinions are discussed in Chapters 57 and 58). Another related problem, when comparing vitamin D status between countries, results from assay differences between various studies [5]. There is some standardization between assays but differences in measured levels of serum 25(OH)D with different assays may still be as large as 30%. The vitamin D status depends on the available amount of ultraviolet light in the sunlight which varies with latitude and season, on actual sunlight exposure and on skin pigmentation, and the use of sunscreen and clothing. This also varies depending on cultural and religious background. The lower the amount of sunlight available, the more nutrition becomes important, especially the consumption of fatty fish, vitamin D-fortified foods, and vitamin D supplements. In the following paragraphs, vitamin D status and the occurrence of vitamin D deficiency will be discussed in different continents, North America, South America, Europe, the Middle East, Asia, Africa, and Oceania. Subsequently studies on vitamin D status in one or more continents
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10052-6
performed in a central laboratory will be discussed. In these studies interlaboratory variation in serum 25 (OH)D assays does not play a role. In the next sections, ethnic issues and nutrition will be discussed as far as these have consequences for vitamin D status in different countries and continents. In addition, risk groups will be discussed. In the final section, the consequences, i.e. the part of the population being vitamin-Ddeficient or vitamin-D-insufficient, will be discussed and finally conclusions will be drawn.
VITAMIN D STATUS IN NORTH AMERICA (INCLUDING CANADA AND MEXICO) Several large studies examined the vitamin D status in North America. Data of a selection of these studies are shown in Table 52.1. The table includes data on country, latitude, study population, age, mean levels of serum 25(OH)D, and percentage of the population with serum 25(OH)D below 25 nmol/L and below 50 nmol/L if available. The latitudes in the different studies ranged from 33o to 42o. The largest study is the National Health and Nutrition Examination Survey (NHANES). This US study comprised a nationally representative sample in which the following population groups were studied: pregnant (n ¼ 928) and nonpregnant women (n ¼ 5173), children (n ¼ 1799), and older people (n ¼ 18 883 in 1988e1994; n ¼ 13 369 in 2001e2004) [6e8]. Serum 25(OH)D was below 50 nmol/L in 33% of pregnant women, 42% of nonpregnant women, 18% of children, 22e36% of older people. Interestingly, the prevalence of low serum 25(OH)D increased between 1988e1994 and 2001e2004 in all age ranges, both sexes, and different ethnicities (Fig. 52.1).
947
Copyright Ó 2011 Elsevier Inc. All rights reserved.
948
TABLE 52.1 Vitamin D Status, Vitamin D Intake, and Prevalence of Vitamin D Deficiency in North America (Including Canada and Mexico) 25(OH)D References
Country
Study population
N
Representative sample of Canada (Atlantic provinces, Que´bec, Ontario, the Prairies, British Columbia)
Community-dwelling
Barake´ 2010 [32]
Que´bec, Canada
Healthy, independently 405 living elderly: Men
5306
6e79 6e11 12e19 20e39 40e59 60e79
Mean SD (nmol/L) a
67.7 (65.3e70.1) 75.0 (70.3e79.7)a 68.1 (63.8e72.4)a 65.0 (61.0e69.0)a 66.5 (63.8e69.2)a 72.0 (69.4e74.5)a
68e82
<50 nmol/L (%)
Comments
4.1 (<27.5) 10.6 (<37.5)
(<37.5) Winter: 12.6 Summer: 5.7 Winter: 8.7 Summer: 1.9
Women boys: 42.7e51.5 girls: 41.3e48.6
1.5e12.6 (<¼27.5) 7.8e37.8 (<¼37.5) latitude 45e48 N 1.5e10.1 (<¼27.5) 13.0e34.5 (<¼37.5) Jan-May
52.1 68.6
6.6 (deficient) 1.7 (deficient)
89 (insuff) 64 (insuff)
<19e>60
68.3 29.0
3.4
16.8 (<40)
928 5173
13e44
65 (61e68)a 59 (57e61)a
7 10
33 42
Children
1799
1e11
age 1e11: 68 (66e70)a age 1e5: 70 (68e73)a age 6e11: 66 (64e68)a
1
18
Nationally representative sample (NHANES)
NHANES III 1988e1994 NHANES 2001e2004
18 883 12e>¼60
75 (72.5e75)a
2
22
13 369
60 (57.5e62.5)a
6
36
Alabama, Minnesota, California, Pennsylvania, Oregon
Older men from general community
1606
62.8 19.8
2.9
25.7
Mark 2008 [33]
Que´bec, Canada
Youth
1753
Sloka 2009 [34]
Newfoundland & Labrador, Canada
Pregnant women: end of winter end of summer
304 289
Genuis 2009 [35]
Edmonton, Alberta, Canada
Clinical practices
1433
Ginde 2010 [6]
Nationally representative sample (NHANES)
Pregnant Non-pregnant
Mansbach 2009 [7]
Nationally representative sample (NHANES)
Ginde 2009 [8]
Orwoll 2009 [36]
<25 nmol/L (%)
9, 13, 16
73.8 5.9
latitude 53 N
52. WORLDWIDE VITAMIN D STATUS
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
Langlois 2010 [9]
Age (years)
Egan 2008 [10]
Cole 2010 [37]
Atlanta, Georgia
Pittsburgh, Pennsylvania
African-American: Men Women White: Men Women Low-income minority children: Hispanic Non-Hispanic black Pregnant: White
Black
99 99
50.3 (8.3) 51.6 (9.7)
42.5 (31.5e60.3)b 35.5 (22.3e50.5)b
5 (<20) 15 (<20)
99 98
53.8 (9.2) 54.7 (10.0)
69.5 (51.3e90.8)b 64.8 (43.3e79.8)b
2 (<20) 1 (<20)
141 149
2.7 1.2 2.3 1.1
64.7 14.8 66.3 22.3
200
<20 e >¼30
200
Cord blood: White Black
18.1 26.3
4e21w: 73.1 (69.4e76.9)a 37e42w: 80.4 (76.0e85.1)a 4e21w: 40.2 (37.9e42.7)a 37e42w: 49.4 (46.1e52.9)a 67.4 (63.8e71.3)a 39.0 (36.3e41.8)a
2.0 (<37.5)
Latitude 33 N
Latitude 40 N
5.0 (<37.5) 44.9 (<37.5) 29.2 (<37.5) 9.7 (<37.5) 45.6 (<37.5)
Lappe 2006 [38]
Eastern Nebraska
Rural postmenopausal white women
1179
66.7 7.3
71.8 20.3 AprileOct: 71.1 20.0
4 (<37.5)
14.4
Latitude 41 N
Merewood 2010 [12]
Boston, Massachusetts
Newborns Mothers
376 433
<20e43
43 (40e47)a 62 (58e64.5)a
38.0 (<37.5) 23.1 (<37.5)
58 35.8
Latitude 42 N Primarily low-income black and Hispanic
Araujo 2009 [39]
Boston, Massachusetts
Elizondo-Montemayor 2009 [40] a
Monterrey, Mexico
Latitude 42 N
Hispanic men: Puerto Rican Dominican Central American South American
121 82 82 73
50.4 52.6 46.3 46.6
Children: Obese Nonobese
99 99
9.0 2.0 8.9 2.0
11.6 12.3 10.8 11.4
82.5 40.8 91.8 59 87.5 34.3 91 47.8
26.1 21.1 10.8 8.5
57.8 13.5 66 15.3
27.3 13.1
Summer (June)
VITAMIN D STATUS IN NORTH AMERICA (INCLUDING CANADA AND MEXICO)
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
Bodnar 2007 [11]
Southeast USA
95% confidence interval; inter quartile range.
b
949
950
52. WORLDWIDE VITAMIN D STATUS
Serum 25(OH)D in the National Health and Nutrition Examination Survey 1988e1994 [III] and 2001e2004 [8]. A downward secular trend is visible in all age groups, both sexes, and different ethnicities. Reproduced with permission of the American Medical Association.
FIGURE 52.1
Several other prevalence studies were published using NHANES data. These are not reported here because of overlapping age ranges. Another large study was performed in Canada [9]. Data were collected from individuals aged 6e79 years from five regions of Canada (n ¼ 5306). Of these, 4.1% had a serum 25(OH)D level below 27.5 nmol/L, and 10.6% were below 37.5 nmol/L. When looking at other US studies, the prevalence of vitamin D deficiency and insufficiency was especially high in African-American men and women from the Southeast [10], black pregnant women from Pittsburgh and the cord blood of their children [11], and newborns in Boston [12].
VITAMIN D STATUS IN SOUTH AMERICA Only few studies have assessed the vitamin D status in South American countries (Table 52.2). Most of these studies were relatively small and limited to a few countries, especially Brazil, Chile, and Argentina. Latitudes range from 23o to 55o. In the majority of studies, a high prevalence of persons having serum 25(OH)D below 50 nmol/L was observed in adolescents, resident physicians, and older people (especially institutionalized older people). In a study in Argentina, higher mean 25
(OH)D values were observed at lower latitudes (closer to the Equator) [13].
VITAMIN D STATUS IN EUROPE Vitamin D status has been studied extensively in many European countries in different age groups (Table 52.3). Eastern Europe is less well represented. A general trend in these data is that vitamin D status is usually better in Nordic countries than around the Mediterranean despite higher latitude. However, this does not apply to Finland, where vitamin D status is not as good as in Norway and Sweden. This difference may be caused by the traditionally high intake of cod, cod liver, and cod liver oil in Norway and Sweden [14]. In studies using a central lab facility [15,16] a similar trend is visible, showing a positive correlation between serum 25(OH)D and latitude. The expected southenorth gradient with a decreasing serum 25(OH)D from south to north was visible in the French SUVIMAX study, where mean serum 25(OH)D decreased from 94 nmol/ L in the south-west to about 43 nmol/L in the most northern regions of France [17]. Representative data have been obtained in the United Kingdom in the National Dietary and Nutrition Survey [18] (Fig. 52.2). As expected, serum 25(OH)D was lower in older
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
TABLE 52.2 Vitamin D Status, Vitamin D Intake, and Prevalence of Vitamin D Deficiency in South America
References
City, state
Study population
Peters 2009 [41]
Sao Paulo, Brazil
Healthy adolescent students: Boys Girls
Saraiva 2005 [42]
Sao Paulo, Brazil
Independently living elderly
Premaor 2008 [43]
Porto Alegre, Brazil
Resident physicians 73
26.4 1.9
44.8 20
Gonzalez 2007 [44] Santiago, Chile
Healthy women: Premenopausal Postmenopausal
32.6 7.4 63.7 9.7
61.3 19.5 48.8 24.8
Oliveri 2004 [13]
Independently living elderly: North Mid South
Seven cities, Argentina
N
Age (years) Mean SD (nmol/L) <25 nmol/L (%) <50 nmol/L (%) Comments Latitude 23 S
64 72
18.0 0.1 18.3 1.0
71.8 21.3 74.0 22.8
0 0
63.9 (<75) 60.6 (<75)
Autumn (April & May) Rural
214 79.1 5.9
49.5 28.4
15.4
57.3
Latitude 23 S
57.4
Latitude 30 S
27 60
Latitude 33 S Half: winter (JuneeSept) Half: summer (DeceMarch)
30 60
0 (<22.5) 12 (<22.5)
339 70.0 4.9 72.0 5.5 70.6 4.9
51.8 18.5 44.8 20.5 35.5 14.0
2 11 14
52 64 87
32
86
Latitudes North 26e27 ; Mid 33e34 ; South 41e55 S End of winterebeginning of spring (AugeOct)
VITAMIN D STATUS IN EUROPE
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
25(OH)D
Latitude 34 S End of summer
Portela 2010 [45]
Buenos Aires, Institutionalized Argentina women
48
81.3 7.9
34.0 15.3
Tau 2007 [46]
Ushuaia, Argentinia
Children
18
7.3 4.4
Before treatment: 73.3 14.8
Latitude 55 S
Oliveri 1993 [47]
Ushuaia, Argentina
Children
42
8.5 1.8
Summer: 46.0 18.3 Winter: 24.5 9.5
Latitude 55 S End of winter þ end of summer
951
952
TABLE 52.3
Vitamin D Studies in Different European Studies 25(OH)D Country
Study population
N
Meyer 2004 [19]
Norway
Norwegian Pakistani
869 177
Brustad 2004 [14]
Norway
Volunteers
32
Snellman 2009 [48]
Norway
Twins
204
Toss 1980 [49]
Sweden
Home for elderly
47
Melhus 2010 [50]
Sweden
Older men
Gerdhem 2005 [51]
Sweden
Kauppi 2009 [52]
Age (years)
Mean SD (nmol/L)
<25 nmol/L (%)
<50 nmol/L (%)
Comments
74.8 23.7 25.0 13.6
0.2 59
13 91
Latitude 60
0
15
Latitude 70
84.8 27.4
0
8
Latitude 60
84
25
50
1194
71
68.7 19.1
0.8
17
Latitude 58
OPRA women
986
75 (75e76)
95 30
0
4.4
Latitude 56
Finland
Men Women
2736 3299
51 (30e97) 53 (30e94)
45.1 (5e132) 45.2 (7e134)
Latitude 60e68
Viljakainen 2010 [53]
Finland
Mothers Newborns
98 98
30.5 4
45 12 29.2
Latitude 60e68
Andersen 2005 [54]
Finland
Girls Older women
60 60
12.8 0.4 71.8 1.4
29.2 45.2 (14.9-90)
37 10
97 57
Latitude 60e68
Pekkarinen 2010 [55]
Finland
Older women
1604
62e79
45 (spring) 53 (autumn)
8.6
60.3
Latitude 60e68
Roddam 2007 [56]
UK
Pat. with fractures Controls
730 1445
52 52
82 40 81 38
21.7 20.9
Latitude 51e58
Khaw 1992 [57]
UK
General practice
138
45e65
28.9 11.6
Prentice 2008 [18]
UK
Nat Diet Nutr. Survey
16e80þ
see Figure 52.2
5e20
20e60
Latitude 51e58
Andersen 2005 [54]
Ireland
Girls Older women
19 43
12.2 0.8 72.3 1.5
41.3 (18e59) 43.7 (17e89)
26 14
89 60
Latitude 52e54
McKenna 1985 [58]
Ireland
Independent Nursing home
30 51
69 5 79 8
21 9
50 84
Latitude 52e54
Lips 1987 [59]
Netherlands
Independent Hip fracture
74 125
76 4 76 11
33 14 19 11
16 (<20) 62 (<20)
Latitude 52
Van Schoor 2008 [30]
Netherlands
LASA
1311
75.5 6.6
53.5 24.2
11.3%
48.4
Van Dam 2007 [60]
Netherlands
Hoorn cohort: Men Women
271 267
69.4 6.3 69.8 6.7
Summer: 1.7 Winter: 6.6
33.7 50.9
Ooms 1995 [61]
Netherlands
Home for elderly
348
80 6
28 13
Latitude 56
Latitude 51e58
34 (<20)
Latitude 52 Latitude 52
Latitude 52
52. WORLDWIDE VITAMIN D STATUS
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
References
Netherlands
Adult women and men
613: Dutch Turkish Moroccan Surinam Asian Surinam Creole African
18e65
Boonen 1997 [62]
Belgium
Hip fracture controls
40
Bouillon 1987 [63]
Belgium
Geriatric pat.
Woitge 1998 [64]
Germany
Chapuy 1987 [65]
67 27 30 24 27 33
6 41 37 51 45 19
73 6 72 5
26 22 47 18
62 (<30) 18 (<30)
240
78 7
29 21
15 (<12.5)
Pop. based
580
50e81
50 27
24 (in winter)
France
Older persons
193: 89 independent 104 geriatric
74 50 83 7
43 18 22 12
16 60
Chapuy 1996 [66]
France
EPIDOS women
440
80 3
43 25
39 (<30)
Latitude 43e49
Chapuy 1997 [17]
France
SUVIMAX
1569
50 6
61 30
14 (<30)
Latitude 43 , north 43 21 Latitude 49 SW 94 38
Burnand 1992 [67]
Switzerland
MONICA
3276
25e74
46 (median)
6 (<20)
Krieg 1998 [68]
Nursing home
Women 246 Men 103
85 7 81 8
23 18 26 21
65 48
Latitude 47
Theiler 1999 [69]
Non-institut. elderly
193
80 9
18 18
90
Latitude 47
Latitude 51 50
Small seasonal variation Latitude 48e52 Latitude 45
>50
Latitude 47
Quesada 1989 [70]
Spain
Elderly institut.
21 31
80 79
37 10
0 75
Latitude 38
Bettica 1999 [71]
Italy
Postmenop. women
570
59 8
45 20
28
Latitude 38e45
Isaia 2003 [72]
Italy
Multicenter
700
60e80
76
Latitude 38e45
Challa 2005 [73]
Greece
Breastfed children mothers
Lapatsanis 2005 [74]
Greece
Adolescents
Andersen 2005 [54]
Poland
Girls Older women
Kocjan 2006 [75]
Slovenia
Laktasic 2010 [76]
Croatia
Postmenop. women
Latitude 38
25 30
Latitude 38
47 61 65
12.6 0.5 71.6 1.4
448
17e89
120
61.1 8.8
30.6 (15.2e62.4) 32.5 (8.2e57.1)
46.9 16.8
VITAMIN D STATUS IN EUROPE
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
Van der Meer [20]
33 25
87 92
Latitude 51
30.5
66.4
Latitude 46
14.2 (<30)
63.3
Latitude 45
953
954
52. WORLDWIDE VITAMIN D STATUS
FIGURE 52.2 Percentage of the population in the UK with serum 25-hydroxyvitamin D lower than 25 nmol/L (vitamin D deficiency) or lower than 50 nmol/L (vitamin D deficiency and insufficiency). Data from the National Dietary and Nutrition Survey [18]. The prevalence of low serum 25(OH)D levels is remarkably high in adolescents and young adults. Reproduced with permission of Wiley-Blackwell Inc.
Serum 25-hydroxy vitamin D < 25 nmol/I 60
51,4
50
41,3
45,3
36,5
40
29,1
% 30
19,3
20 10
5,9
0
O =5 5 )
e
an
ric 7)
)
07
=1
=5
5)
(N
=7
(N
)
02
=1
n
(N
ia
(N
As
6)
Af
ol
n
re
ra
C
h
=9
ut
(N
So
n
ha
(N
Sa
er
b-
th
su
am
ca
am
rin
rin
Su
Su
oc )
ch
ut
21
D
=1
us
(N
no
h
is
ge
rk
or
M
Tu
di
in
FIGURE 52.3
from [20].
Prevalence of vitamin D deficiency (serum 25-hydroxyvitamin D <25 nmol/L) in different ethnicities in the Netherlands. Data
persons than in adults. Unexpectedly, serum 25(OH)D was very low in adolescents between 16e25 years. Older persons were extensively studied in the Netherlands and Belgium showing a low serum 25(OH)D. Vitamin D status was very poor in patients with hip fracture and the institutionalized. Similarly very low serum 25 (OH)D levels were observed in noninstitutionalized elderly in Switzerland. Also in Italy and Greece very low serum 25(OH)D levels were observed while sunshine is abundant in these countries. This may be caused by a more pigmented skin and by sun-avoidance behavior, especially in summer because of the high temperatures. Vitamin D status usually is very poor in immigrants from non-Western countries [19,20], compared with native people (Fig. 52.3). This is even worse in pregnant non-Western immigrants [21], in
which serum 25(OH)D often is lower than 25 nmol/L or undetectable.
VITAMIN D STATUS IN THE MIDDLE EAST Serum 25(OH)D is lower in these countries than should be expected based on the abundance of sunshine (Table 52.4). In Turkey and Jordan serum 25(OH)D was lower in women than in men. In women, vitamin D status depended on clothing style being lower in traditionally clothed women than in women with Westernstyle clothing. A very low serum 25(OH)D was observed in Saudi Arabia even with the very sunny climate. This may be explained by the completely covered skin in this
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
955
VITAMIN D STATUS IN AFRICA
TABLE 52.4 Vitamin D Status, Vitamin D Intake, and Prevalence of Vitamin D Deficiency in Middle East Countries According to Different Studies 25(OH)D References
Study Country population
N
Vitamin D Mean SD <25 nmol/L <50 nmol/L Age (years) intake (IU/day) (nmol/L) (%) (%) Comments
Alagol 2000 [77]
Turkey
Women
48
14e44
56 41 32 24 96
Atli 2005 [78]
Turkey
Institutionalized women and 138 men 87
75 7 76 7
60 75 95 73
Gannage-yared 2000 [79]
Lebanon Women Men
217 99
30e50 30e50
100 68
24 17
Goldray 1989 [80]
Israel
Geriatric patients
338
78 8
<100
33 22
Mishal 2001 [81] Jordan
Women Women Women Men
13 31 12 11
18e45
37 6 28 5 24 6 44 5
30 55 83 18
Western clothing Hijab Niqab Latitude 32
Sedrani 1983 [82] Saudi Arabia
Elderly
24
62 13
93
90
Completely covered Latitude 23
Meddeb 2005 [83]
Tunisia
Women Women
78 183
Adult
35 43
29 29
Veiled Nonveiled Latitude 35
Hashemipour 2004 [84]
Iran
Men and women population study
1210 20e69
20.6
67
25(OH)D increased with age in women Latitude 35
Moussavi 2004 [85]
Iran
Girls Boys
165 153
42 21 92 46
20 2
Latitude 35
76
14e18
Western clothing Traditional completely veiled Latitude 40 Latitude 40
84 48
Deficiency in veiled women Latitude 34 Latitude 32
country. Similar trends were visible in Tunisia and Iran. Another issue to explain the low levels of 25(OH)D in this part of the world is the extreme heat leading people to avoid being outdoors during the sunny parts of the day.
skin, skin covering, and sun-avoidance behavior. Vitamin D status was better in south-eastern Asian countries such as Malaysia and Japan.
VITAMIN D STATUS IN ASIA
The number of studies on vitamin D status in Africa is small. The literature was recently reviewed [23]. Studies from Central and South Africa are summarized in Table 52.6. The vitamin D status in central Africa was quite good in these studies. Even in patients with tuberculosis, the vitamin D status was very good. However, in southern Africa, in a study of nurses in the country of South Africa, serum 25(OH)D was similar in pre- and postmenopausal women but lower in black than in white women. More than 50% of the black women had a serum 25(OH)D lower than 50 nmol/L.
Vitamin D status was poor in patients with hip fracture in Yekaterinburg, Russia, [22] and in older control subjects (Table 52.5). A low vitamin D status was also observed in Mongolian children and pregnant women. Rickets is very common in Mongolia [18]. Similarly, teenage girls in China had a very low serum 25(OH)D in winter. Contrary to expectation, vitamin D status was poor to moderate in India situated at a latitude between 13 and 27 . This may be due to pigmented
VITAMIN D STATUS IN AFRICA
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
956
TABLE 52.5
Vitamin D Status, Vitamin D Intake, and Prevalence of Vitamin D Deficiency in Asian Countries According to Different Studies 25(OH)D N
Age (years)
Asian Russia
Hip fracture Controls
64 97
69 10 70 8
Fraser 2004 [86]
China
Girls
1277
12e14
Fraser 2004 [86]
Mongolia
Rachitic children Healthy children Pregnant women
40 22 57
Marwaha 2005 [87]
Northern India
Children
Sachan 2005 [88]
Northern India
Pregnant women
207
24 4
Arya 2004 [89]
Northern India
Hospital staff
92
Harinarayan 2005 [90]
Southern India
Postmenopausal women
Islam 2006 [91]
Bangladesh
Women
Rahman 2004 [92]
Malaysia
Chinese postmenop. women Malaysian postmenop. women
Country
Bakhtiyarova 2006 [22]
Vitamin D intake (IU/day)
40
Mean SD (nmol/L)
<25 nmol/L (%)
22 11 28 10
65 47
Latitude 57
12e13 winter 25e30 summer
45 (<12.5) 6 (<12.5)
Latitude 40
<50 nmol/L (%)
Comments
Latitude 42e50 Rickets very common
71 41 3 26 2 26 (lower class) 34 (upper class)
50 30
Latitude 28
35 23
42
Latitude 27
34 6
30 26
48
Latitude 27
164
54 8
36 17
30
Latitude 13
121
18e60
35
Latitude 24
173
50e65
69 16
2
Latitude 3
103
50e65
44 11
2 42 10
10e18 16 8
Nakamura 2001 [93]
Japan
Women
38 39
19e29 30e66
34 11 50 14
Ono 2005 [94]
Japan
Adults (winter)
197
43 13
38 18
Nakamura 2006 [95]
Japan
Women
151
66 7
60 7
Latitude 35 Latitude 35 (winter)
5
Winter, positive relation with fish
52. WORLDWIDE VITAMIN D STATUS
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
Study population
References
957
ETHNICITY/MIGRATION
TABLE 52.6
Vitamin D Status, Vitamin D Intake, and Prevalence of Vitamin D Deficiency in Africa According to Different Studies 25(OH)D Mean SD Age (years) (nmol/L)
References
Country
Study population
N
Aspray 2005 [96]
Gambia
Rural women
48 55e64
91 2.4
M’Buyamba 1987 [97]
Zaire
Men
33 31
65 39
Mehta 2009 [98]
Tanzania
HIV-infected women: Low D Adequate D
347 24.6 5 537 24.6 5
60.5 15 107.8 22.5
Friis 2008 [99]
Tanzania
Tuberculosis pa.
Glew 2010 [100]
Nigeria
Fulani men Fulani women
Wejse 2009 [101]
Guinea-Bissau Tuberculosis pa.
365 37 14
78.3 22.8
Haarburger 2009 [102]
South Africa
Unselected
216 All ages
48.3 (5.5e106)
37 (<45)
Nurses Premenopausal Black Premenopausal White Postmenopausal Black Postmenopausal White
74 105 65 50
48.2 (17e113) 65.7 (34e145) 47.5 (16e81) 64.5 (26e140)
>50
Daniels 1997 [103] South Africa
0
22 47.6 8.3 29 55.5 13.5
VITAMIN D STATUS IN OCEANIA A selection of studies in Oceania, i.e. Australia, New Zealand, and Pacific Islands, is reported in Table 52.7. Latitudes in these studies range from 27e46o. Although Oceania has a very sunny climate, vitamin D deficiency was very prevalent with the highest prevalence in East African immigrant children and adolescents living in Melbourne, Australia (87% having serum 25(OH)D <50 nmol/L), [24] and refugees living in Auckland, New Zealand (54% having serum 25(OH)D <50 nmol/ L) [25]. Also in Australia, higher mean 25(OH)D values were observed at lower latitudes [26].
MULTICENTER AND GLOBAL STUDIES USING A CENTRAL LABORATORY FACILITY Some studies have involved many countries or even several continents using one central laboratory facility. The advantage of these studies is that different assays for serum 25(OH)D and different laboratories are excluded as a source of variation. This is a great advantage as noted earlier, interlaboratory variation may be 30% or more [5]. The Euronut Seneca study was done in older persons in European countries from the Mediterranean to Northern Europe [16]. In this study, there was a positive correlation between serum 25(OH)D and
2.5 (<43)
Latitude 13 Latitude 5 Ne10 S
86.6 32.9
34.5 35 55 54
<25 nmol/L (%) <50 nmol/L (%) Comments
Latitude 2e10
41.2 (<75)
Latitude 2e10
45 (<75) 83 (<75)
Latitude 6 Latitude 10 Latitude 22e34 Latitude 22e34
>50
latitude, contrary to expectation, i.e. higher values in northern countries. This was confirmed by the baseline data of the MORE study, a study on the effect of raloxifene vs placebo in postmenopausal women with osteoporosis [15]. The MORE study and the baseline data of the bazedoxifene study [27] showed a positive correlation between serum 25(OH)D and latitude in Europe, the inverse of what was expected by sunlight exposure. In the latter (bazedoxifene) study the correlation between serum 25(OH)D and latitude in other continents was negative as should be expected. The baseline data of the bazedoxifene study showed also a relationship between serum 25(OH)D and affluence with lower 25(OH)D levels in Eastern Europe than in Western and Northern Europe. The MORE study, the bazedoxifene study and another global study [15,27,28] were all done in postmenopausal women with osteoporosis. Vitamin D status in these studies usually was better than in other studies because women participating in clinical trials usually are more concerned about their health. These three studies show a very poor vitamin D status in Middle Eastern countries confirming the data of national studies.
ETHNICITY/MIGRATION Ethnic differences in vitamin D status were clearly shown in a nationally representative sample (NHANES) in North America (Fig. 52.1) [8]. Lowest mean serum 25
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
Vitamin D Status, Vitamin D Intake, and Prevalence of Vitamin D Deficiency in Australia and New Zealand According to Different Studies 25(OH)D City, State
Study population N
Age (years)
van der Mei 2007 [26]
Three regions, Australia (Southeast Queensland, SQ; Geelong region, G; Tasmania, T)
Population based: SQ: Men Women G: Women T: Men Women
<60
Flicker 2003 [104]
Three states, Australia (Western Australia, New South Wales, Victoria)
Women in residential care: Low-level care High-level care
McGillivray 2007 [24]
Melbourne, Australia
Bowyer 2009 [105]
Sydney, Australia
Rockell 2005 [106] New Zealand Grant 2009 [107]
211 167
72.2 67.0
561
75.5
298 432
55.2 51.1
G: 7.9
G: 37.4
T: 13.0
T: 67.3
Latitudes SQ 27; G 38; T 43 S Three different studies
83.7 8.7 83.7 9.1
East African immigrant children
232
8.9 4.4
44
87
Latitude 37 S
Pregnant women Neonates
971 901
29.8 (23.2e37.1)b 52 (range: 17e174) 60 (range: 17e245)
15 11
48 40
Latitude 34 S
Children
1585
5e14
50 (45e54)c
4 (<17.5) 31 (<37.5)
Latitude 35e46 S
353
6e11 mo 12e17 mo 18e23 mo
62 (42e78)b 58 (44e76)b 49 (39e61)b
10 (<27.5)
Latitude 36 S
Auckland, New Zealand Urban children
Wishart 2007 [25]
869
17 (9e27)b
Bolland 2006 [109] Auckland, New Zealand Communitydwelling men
378
57 11
Lucas 2005 [110]
Auckland, New Zealand Healthy postmenopausal women
1606
73.7 4.3
Rockell 2008 [111]
Invercargill and Dunedin, New Zealand
Volunteers
342
Heere 2010 [112]
Pacific Islands
Fijian women: Indigenous Fijian Indian Fijian
511 306 205
Auckland, New Zealand Refugees
95% confidence interval; inter quartile range; c 99% confidence interval.
Winter/spring SQ: 40.5
667 952
21 987 >18
b
Winter/spring SQ: 7.1
Latitude 32e38 S
Bolland 2008 [108] Auckland, New Zealand Adults
a
Mean SD (nmol/L) <25 nmol/L (%) <50 nmol/L (%) Comments
39.7 20.3 31.4 19.7
22 45
48
Latitude 36 S
17
54
Latitude 36 S
85 31
Summer 0 Winter 0e2
Summer 0e17 Winter 0e20
Latitude 37 S
51.2 19.4
Summer 0e3 Winter 6e16
Summer 28e58 Latitude 36 S Winter 56e74 Latitude 45e46 S
Late summer: 79 Early spring: 51 15e44
76 (73e78)a 80 (76e84)a 70 (66e74)a
11
Mean 25(OH)D higher in rural than urban women
52. WORLDWIDE VITAMIN D STATUS
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
References
958
TABLE 52.7
959
CONCLUSIONS
(OH)D values were observed in non-Hispanic blacks, highest mean values in non-Hispanic whites. In all four ethnicities, lower mean serum 25(OH)D values were observed in 2001e2004 as compared with 1988e1994. The prevalence of vitamin D deficiency (serum 25(OH)D <25 nmol/L) increased in all four groups, but especially in non-Hispanic blacks. Vitamin D status in immigrants from non-Western countries was poor in Norway, the Netherlands and Australia [19e21,24]. A review on this subject concluded that serum 25(OH)D in non-Western immigrants in the Netherlands was much lower than in those born in the Netherlands and was also lower than in people in their country of origin [29].
NUTRITION In Europe, a northesouth gradient was observed for serum 25(OH)D with higher levels in Scandinavia and lower levels in Southern and Eastern European countries [15,16]. This indicates that other determinants than sunshine are of importance, e.g. nutrition, food fortification, and supplement use. Fortification of dairy products is practiced in the USA where vitamin D 400 IU is added per quart of milk. Fortification of milk is now also practiced in Sweden and Ireland.
RISK GROUPS Vitamin D deficiency is very common in certain risk groups, such as children with low birth weight (premature and small-for-gestational-age), pregnant women, older people, and non-Western immigrants. The poor vitamin D status in adolescents in the UK is rather unexpected [18]. Pregnant women, especially non-Western pregnant women and their children, are at high risk of vitamin D deficiency [21]. Older people have decreased dermal synthesis, and especially older nursing home residents who do not go outside frequently are at high risk. Non-Western immigrants migrating to countries at higher latitudes with limited UV-B irradiation are at high risk because of more pigmented skin, the habit to stay out of the sun, the wearing of well-covering clothes, and a diet low in dairy products [19e21,24,29].
IMPLICATIONS Vitamin D deficiency has been associated with many different outcomes, such as muscular weakness, decreased physical performance, falls, and fractures [4,30]. In recent years, vitamin D deficiency has also been associated with nonclassical outcomes, such as
cardiovascular disease, diabetes mellitus, multiple sclerosis, tuberculosis, respiratory infections, and several types of cancer [31]. However, for all of these outcomes, causality has not been established. The magnitude of the negative health effects attributed to vitamin D deficiency also depends on the percentage of the population having a low vitamin D status. Roughly 50% of the Western European population has a serum 25(OH)D level below 50 nmol/L at least in winter. This percentage is lower in North America, and appears higher in South America, although South American studies were limited to only three countries. The prevalence of vitamin D deficiency was more than 50% in South Africa, and around 50% in Oceania. From the Middle East and Asia, only prevalence rates were reported for serum 25(H)D <25 nmol/ L, revealing severe vitamin D deficiency in the Middle East, China, Mongolia, and India. It is important to do more research in these countries, especially in Asia, where a relatively large part of the world population lives. When the required serum 25(OH)D is set at 75 nmol/L as is advocated by many researchers, the prevalence of insufficiency might be in the order of 70e95% in many countries. In summary, to be able to estimate the burden of vitamin D deficiency, more prevalence studies are needed in parts of South America, the Middle East, Asia, and Africa. Prevention requires moderate sunlight exposure, consumption of fish, fortification of foods, and the use of vitamin D supplements. A supplement of vitamin D3 400 IU/d can be recommended for children and for adults who do not go outside or have dark skin. Pregnant and lactating women may require 400e800 IU per day. Older persons also require a supplement of 400e800 IU per day, the higher dose with insufficient sun exposure or dark skin. Patients with osteoporosis and older persons in rest or nursing homes require 800 IU per day. It will require an enormous effort to bring up serum 25(OH)D levels to more than 50 nmol/ L in all continents all year long.
CONCLUSIONS Vitamin D deficiency (serum 25(OH)D <25 nmol/L) and insufficiency (serum 25(OH)D 25e50 nmol/L) are very common in most countries around the world. Severe vitamin D deficiency is common in the Middle East, China, Mongolia, and India. Risk groups are children, especially those with low birth weight, adolescents, pregnant women, older persons, and nonWestern immigrants. Less than 50% of the world population has an adequate vitamin D status (serum 25(OH) D >50 nmol/L). Prevention requires moderate sunlight exposure, consumption of fish, fortification of foods, and the use of vitamin D supplements.
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
960
52. WORLDWIDE VITAMIN D STATUS
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[81] A.A. Mishal, Effects of different dress styles on vitamin D levels in healthy young Jordanian women, Osteoporos. Int. 12 (2001) 931e935. [82] S.H. Sedrani, A.W. Elidrissy, K.M. El Arabi, Sunlight and vitamin D status in normal Saudi subjects, Am. J. Clin. Nutr. 38 (1983) 129e132. [83] N. Meddeb, H. Sahli, M. Chahed, J. Abdelmoula, M. Feki, H. Salah, et al., Vitamin D deficiency in Tunisia, Osteoporos. Int. 16 (2005) 180e183. [84] S. Hashemipour, B. Larijani, H. Adibi, E. Javadi, M. Sedaghat, M. Pajouhi, et al., Vitamin D deficiency and causative factors in the population of Tehran, BMC Public Health 4 (2004) 38. [85] M. Moussavi, R. Heidarpour, A. Aminorroaya, Z. Pournaghshband, M. Amini, Prevalence of vitamin D deficiency in Isfahani high school students in 2004, Horm. Res. 64 (2005) 144e148. [86] D.R. Fraser, Vitamin D-deficiency in Asia, J. Steroid Biochem. Mol. Biol. 89-90 (2004) 491e495. [87] R.K. Marwaha, N. Tandon, D.R. Reddy, R. Aggarwal, R. Singh, R.C. Sawhney, et al., Vitamin D and bone mineral density status of healthy schoolchildren in northern India, Am. J. Clin. Nutr. 82 (2005) 477e482. [88] A. Sachan, R. Gupta, V. Das, A. Agarwal, P.K. Awasthi, V. Bhatia, High prevalence of vitamin D deficiency among pregnant women and their newborns in northern India, Am. J. Clin. Nutr. 81 (2005) 1060e1064. [89] V. Arya, R. Bhambri, M.M. Godbole, A. Mithal, Vitamin D status and its relationship with bone mineral density in healthy Asian Indians, Osteoporos. Int. 15 (2004) 56e61. [90] C.V. Harinarayan, Prevalence of vitamin D insufficiency in postmenopausal south Indian women, Osteoporos. Int. 16 (2005) 397e402. [91] M.Z. Islam, M. Akhtaruzzaman, C. Lamberg-Allardt, Hypovitaminosis D is common in both veiled and nonveiled Bangladeshi women, Asia. Pac. J. Clin. Nutr. 15 (2006) 81e87. [92] S.A. Rahman, W.S. Chee, Z. Yassin, S.P. Chan, Vitamin D status among postmenopausal Malaysian women, Asia. Pac. J. Clin. Nutr. 13 (2004) 255e260. [93] K. Nakamura, M. Nashimoto, S. Matsuyama, M. Yamamoto, Low serum concentrations of 25-hydroxyvitamin D in young adult Japanese women: a cross sectional study, Nutrition 17 (2001) 921e925. [94] Y. Ono, A. Suzuki, M. Kotake, X. Zhang, K. Nishiwaki-Yasuda, Y. Ishiwata, et al., Seasonal changes of serum 25hydroxyvitamin D and intact parathyroid hormone levels in a normal Japanese population, J. Bone Miner. Metab. 23 (2005) 147e151. [95] K. Nakamura, Vitamin D insufficiency in Japanese populations: from the viewpoint of the prevention of osteoporosis, J. Bone Miner. Metab. 24 (2006) 1e6. [96] T.J. Aspray, L. Yan, A. Prentice, Parathyroid hormone and rates of bone formation are raised in perimenopausal rural Gambian women, Bone 36 (2005) 710e720. [97] J.R. M’Buyamba-Kabangu, R. Fagard, P. Lijnen, R. Bouillon, W. Lissens, A. Amery, Calcium, vitamin D-endocrine system, and parathyroid hormone in black and white males, Calcif. Tissue Int. 41 (1987) 70e74. [98] S. Mehta, D.J. Hunter, F.M. Mugusi, D. Spiegelman, K.P. Manji, E.L. Giovannucci, et al., Perinatal outcomes, including motherto-child transmission of HIV, and child mortality and their association with maternal vitamin D status in Tanzania, J. Infect. Dis. 200 (2009) 1022e1030. [99] H. Friis, N. Range, M.L. Pedersen, C. Molgaard, J. Changalucha, H. Krarup, et al., Hypovitaminosis D is common among pulmonary tuberculosis patients in Tanzania
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C H A P T E R
53 Sunlight, Vitamin D and Prostate Cancer Epidemiology Gary G. Schwartz Wake Forest University, Winston-Salem, NC, USA
INTRODUCTION The hypothesis that vitamin D deficiency increases the risk for prostate cancer was first published in 1990 [1]. There was no literature on the effects of vitamin D on the prostate at that time and the hypothesis met with considerable skepticism. Today, we understand that the vitamin D hormone exerts pleiotropic anticancer effects on prostate cells. Studies of vitamin D feature prominently in research on prostate cancer epidemiology, biology, and therapy [2e4]. The roles of vitamin D in prostate cancer biology and therapy are described in Chapter 86. This chapter reviews the status of vitamin D in prostate cancer epidemiology. Interpretation of this literature requires an appreciation of methodological issues in epidemiologic studies as well as issues pertinent to the natural history of prostate cancer. Chapter 82 discusses the broad field of epidemiology of vitamin D and cancer.
TYPES OF EPIDEMIOLOGIC STUDIES Most epidemiologic studies are observational studies that compare the risk of disease, e.g. prostate cancer, among persons with different levels of exposure, e.g. serum vitamin D levels or levels of sunlight exposure. The simplest type of study is the ecologic study. In an ecologic study (or correlation study), data on disease and on exposure are obtained at the level of a group, frequently a country or state, and the correlation between these two variables is obtained. Ecologic studies can be fruitful tools for suggesting hypotheses about disease. However, because the data pertain to groups, these studies offer no evidence for an association
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10053-8
between exposures and disease at the level of the individual. In fact, correlations reported for groups often are not confirmed by studies conducted in individuals, a problem known as the “ecologic fallacy” [5,6]. Observational epidemiologic studies conducted among individuals are of two basic types, cohort and caseecontrol studies. The defining characteristic of a cohort study is that the population is free from the disease at the study’s outset. One group is exposed to the factor of interest, e.g. high levels of ultraviolet radiation, and another is not. The two groups are followed over time and the proportion of individuals from the exposed and unexposed groups which develop the disease is compared. The ratio of these proportions, the relative risk (RR), describes the direction and magnitude of the association between exposure and disease. RRs greater than 1.0 indicate that the exposure is associated with an increased risk of disease; RRs less than 1.0 indicate that the exposure is associated with a reduced risk. In contrast, caseecontrol studies are retrospective studies which begin with persons with the disease and a comparison (control) group without the disease. The odds of exposure in the two groups are determined and the ratio of these odds, the odds ratio, quantifies how much more or less likely exposure is among the diseased. The chief interpretive difficulty of caseecontrol studies is the problem of temporality. Since the disease has already occurred at the outset of the study, if the exposure and disease are associated, it is often impossible to determine whether the exposure caused the disease or the reverse. Another major difficulty of caseecontrol studies of chronic disease like prostate cancer is that the disease process often began many years previously. Patient recall of critical events may be inaccurate, or worse, may be distorted by the existence of
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disease such that patients mis-recall events that may be associated with disease (recall or “rumination” bias). A related study design is a caseecontrol study nested within a cohort. In the nested caseecontrol study, cases of disease that occur within a well-defined cohort are identified. A specified number of controls that have not developed the disease by the time the disease has occurred in the cases are identified for each case. An advantage of nested caseecontrol studies is that, when applied to samples such as stored blood, the samples are not contaminated by the effects of clinical disease. Thus, the nested caseecontrol design is particularly suited to study biologic precursors of disease [7].
SUBCLINICAL PROSTATE CANCER IS PREVALENT AND MAY DILUTE EFFECTS IN EPIDEMIOLOGIC STUDIES Prostate cancer is the commonest (non-skin) cancer among men in the Western world. Worldwide, ageadjusted mortality rates for prostate cancer vary more than 20-fold and are highest among African-Americans and northern Europeans [8]. Unlike mortality rates, subclinical prostate cancer (a.k.a. “latent” or “autopsy” prostate cancer) is highly prevalent among older men regardless of their race or geographic location [9]. For example, autopsies performed on men who died from causes other than prostate cancer show that approximately 27% of men in their 40s and more than 30% of men in their 50s have histological prostate cancer [10]. The prevalence of these subclinical cancers reaches 60% in men over the age of 80 and continues to increase with age [11]. The rarity of clinical tumors relative to the prevalence of subclinical tumors suggests that the occurrence of clinical prostate cancer depends upon factors that govern the growth of subclinical prostate tumors. The widespread use of prostate-specific antigen (PSA) as a screening test has markedly increased the detection of subclinical prostate tumors but has not been demonstrated to reduce prostate cancer mortality [12,13]. Most prostate cancers detected by PSA will not become fatal if untreated. This form of “detection bias” has implications for epidemiologic studies that involve incident (PSA-detected) cancer because the clinical behavior of incident cancers will resemble that of “autopsy” prostate cancers. In general, the effect of including incident prostate cancers in epidemiologic studies will be to weaken the estimates of the association between exposure and disease. The long duration of preclinical prostate cancer, estimated to be approximately 10 years, may influence the risk estimate even in prospective studies [14]. Thus, depending upon the length of follow-up, nested caseecontrol studies and cohort studies which aim to study the effects of serum
vitamin D on the risk of prostate cancer may also include effects of the cancer on serum vitamin D levels.
PROSTATE CANCER AND THE VITAMIN D HYPOTHESIS In 1990, we noted that the characteristic features of the descriptive epidemiology of prostate cancer, the increased risks associated with older age, Black race, and residence at northern latitudes, all are associated with a decreased synthesis of vitamin D. We proposed that vitamin D maintained the normal phenotype of prostatic cells and that vitamin D deficiency promoted the development of clinical prostate cancer from its preclinical precursors (Table 53.1) [1]. This hypothesis was supported by ecologic data at the level of the state that showed a negative correlation between ultraviolet light and state-wide mortality rates. Furthermore, in 1992 we showed that US mortality rates for prostate cancer among Caucasian men at the level of the county were inversely correlated with the availability of ultraviolet radiation, the major source of vitamin D [15,16]. The same year, Miller and colleagues demonstrated that prostate cancer cells possessed high-affinity receptors for the hormonal form of vitamin D, 1,25(OH)2D (vitamin D receptors, VDR) [17]. In 1993, Skowronski and colleagues demonstrated VDR in multiple prostate cancer cell lines and showed that 1,25(OH)2D exerted antiproliferative effects on these cells [18]. Pleiotropic anticancer effects of 1,25(OH)2D on normal and cancerous prostate cells later were described by numerous laboratories (see Chapter 86). The mechanisms for these effects in the prostate are not completely characterized but include inhibition of: cell proliferation (via cell cycle arrest) [19]; invasion TABLE 53.1 Risk Factors for Prostate Cancer and their Interpretation by the Vitamin D Deficiency Hypothesis Risk factor Age
Explanation by vitamin D deficiency hypothesis The prevalence of vitamin D deficiency increases with age
Race Black
Melanin inhibits synthesis of vitamin D
Asian
Traditional diet high in vitamin D (fish oil) protects against clinical cancer. Protection wanes as migrants adopt a Western diet
Geography
U.S. mortality rates from prostate cancer are inversely correlated with ultraviolet radiation.
Adapted from Schwartz GG, Hulka BS. Is vitamin D deficiency a risk factor for prostate cancer? (Hypothesis) Anticancer Research 10: 1307e1311, 1990.
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1,25(OH)2D IS SYNTHESIZED BY NORMAL PROSTATE EPITHELIAL CELLS AND ITS SYNTHESIS IS REDUCED IN PROSTATE CANCER CELLS
through the basement membrane (via inhibition of matrix metalloproteinases) [20]; inhibition of migration [21]; metastasis [22,23]; and angiogenesis [24]. In general, the inhibitory roles of vitamin D in prostate carcinogenesis occur in the various stages of cancer promotion/progression, rather than in tumor initiation (see chapters in Section X). This fact is important with regard to epidemiologic studies because it suggests that the effects of vitamin D should be greater on the risk of advanced or fatal cancer (i.e., cancers that have progressed) than on incident prostate cancer.
1,25(OH)2D IS SYNTHESIZED BY NORMAL PROSTATE EPITHELIAL CELLS AND ITS SYNTHESIS IS REDUCED IN PROSTATE CANCER CELLS As detailed in earlier chapters of this book, the synthesis of 1,25(OH)2D begins with the production of vitamin D3 (cholecalciferol) after 7-dehydrocholesterol in the skin is exposed to ultraviolet-B (UV-B) radiation or after vitamin D is ingested from the diet. Approximately 90% of vitamin D is sunlight derived [25]. Vitamin D is hydroxylated first in the liver at the 25th carbon, forming the prohormone, 25-hydroxyvitamin D (25(OH)D), and again at the 1-a position, forming 1,25(OH)2D, the active, hormonal form of vitamin D [26]. In the 1980s, most endocrine textbooks were emphatic that, with the exception of rare disorders like sarcoidosis and atypical organs like the placenta, the sole organ to hydroxylate the vitamin D prohormone into the active hormone was the kidney. This view was based on the observations that circulating levels of 1,25(OH)2D are basically undetectable in anephric individuals. We now recognize that the reasoning behind this view was in error; many organs have the capacity to synthesize 1,25(OH)2D locally but they do not usually release the hormone into the circulation. The importance of the local 1a-hydroxylase paracrine system is discussed in Chapter 45. The clue to the discovery of the autocrine synthesis of 1,25(OH)2D by prostate cells came from the descriptive features of prostate cancer epidemiology. The northesouth gradient in prostate cancer mortality and the higher mortality rates among US Blacks suggested a deficiency in 25(OH)D, whose serum levels are known to be lower at higher latitudes and among persons with dark pigmentation. However, the active vitamin D hormone is 1,25(OH)2D, not 25(OH)D. The conundrum was that serum levels of 1,25(OH)2D are tightly regulated, are not lower in Blacks than Whites, and (in normal individuals) are not correlated with serum levels of 25(OH)D [27]. Thus, it was difficult to understand how the northesouth gradient in prostate cancer mortality rates and the racial difference could
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be related to a deficiency of 25(OH)D. We reasoned that this paradox would be solved if prostate cells synthesized 1,25(OH)2D intraprostatically from circulating serum levels of 25(OH)D. In 1998, using high-pressure liquid chromatography (HPLC), we demonstrated that normal human prostate cells possess 25-hydroxyvitamin D3-1a-hydroxylase (1a-hydroxylase) and indeed synthesize 1,25(OH)2D from 25(OH)D [28]. Moreover, we and others showed that 25(OH)D inhibits the proliferation of prostate cells that possess 1a-hydroxylase [29]. Thus, the autocrine synthesis of 1,25(OH)2D in normal prostate cells provides a biochemical mechanism by which exposure to sunlight or vitamin D might prevent prostate cancer [30] (Fig. 53.1). Although normal prostate cancer cells express high levels of 1a-hydroxylase, 1a-hydroxylase expression appears to be diminished (but not absent) in prostate cancer cells. Hsu and colleagues compared 1a-hydroxylase activity in samples from normal prostate epithelial cells, cancer-derived prostate epithelial cells, prostate cancer cells lines, and samples of benign prostatic hyperplasia (BPH). Expression of 1a-hydroxylase was significantly reduced in BPH cells and was reduced further in the cancer-derived cells and cell lines [31]. Decreased expression of 1a-hydroxylase was correlated with a decrease in growth inhibition in response to 25(OH)D3. A loss of 1a-hydroxylase expression in prostate cancers versus benign and non-cancerous prostates was also shown by Whitlatch et al., who demonstrated that transfection of the cDNA for 1a-hydroxylase into prostate cancer cells that did not express the enzyme (LNCaP cells) and were not growth inhibited by 25(OH)D3, conferred growth inhibition by 25(OH)D3 in these cells [32,33]. These findings have implications for the interpretation of studies of prediagnostic serum levels of vitamin D. They suggest that, if higher serum levels of 25(OH)D are preventive because they are converted to 1,25(OH)2D intraprostatically, serum levels may be more effective before significant 1a-hydroxylase activity is lost in prostate cells (Fig. 53.2). The biological activity of 1,25(OH)2D in tissues requires the presence of VDR, a ligand-dependent transcription factor that is a member of the steroid nuclear receptor super-family [34e36]. The existence of VDR in human prostate cancer cells identified by Miller et al. in 1992 has been confirmed repeatedly by numerous investigators using cell lines and clinical samples [37e39]. For example, Krill et al. examined the expression patterns of VDR using immunohistochemistry in 27 clinical samples of normal human prostates that were free from adenocarcinoma or suspected carcinoma. They showed that VDR are widely expressed in human prostate cells and were expressed more abundantly in the peripheral zone of the prostate (the site of origin of most prostate cancers) than in the central zone.
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FIGURE 53.1 Endocrine and autocrine role of 1,25(OH)2D on prostate cells. In addition to the kidney (endocrine system), the prostate synthesizes its own 1,25(OH)2D (autocrine system) that controls local growth and proliferation (from [40]).
Laboratory investigations have provided strong experimental support for broad, anti-cancer effects of vitamin D on prostate cells. These studies have stimulated epidemiologic investigations of vitamin D including ecologic studies, studies of VDR and other polymorphisms, and studies of serum vitamin D concentrations and of exposure to sunlight [40]. In theory, prostate cancer risk might also be inversely related to diets high in vitamin D. The role of dietary vitamin D has been examined in several epidemiologic
studies, and these have not shown effects for dietary exposure [41e43]. However, until very recently, the forms of vitamin D generally available commercially, of 200e400 IU, are not adequate to alter serum levels of vitamin D by more than 2e4 nmol/L. Thus, unless conducted among populations with high levels of vitamin D consumption (e.g., Scandinavian countries where fish oils consumption is prevalent), dietary studies of vitamin D are unlikely to include biologically effective doses of vitamin D.
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FIGURE 53.2 1a-Hydroxylase activity in primary cultures of normal, BPH, and prostate cancer (CaP), and in human prostate cancer cell lines, DU 145, PC-3, and LNCaP cells. Bars are standard deviations of three determinations (reprinted from [32] with permission).
ECOLOGIC STUDIES OF SUNLIGHT AND PROSTATE CANCER The inverse relationship between geographic latitude or UV radiation and prostate cancer mortality in the USA that we showed in 1990 and 1992 has been replicated by numerous different authors, using different analytic techniques and over different periods of time [44e48]. In the most comprehensive analysis to date, we examined prostate cancer mortality rates among Caucasians in the USA at the level of the county and State Economic Area (groups of similar counties) over a 45-year period (1950e1994). Significant inverse correlations between UV radiation and prostate cancer mortality were observed at all 15 5-year intervals over this 45-year period (p < 0.0001). These correlations were significantly more pronounced at locations north of 40 N latitude, locations where vitamin D synthesis is limited to the non-winter months [49]. The consistency in these studies indicates that UV radiation, or something strongly associated with it, is related to an increased risk of prostate cancer mortality (Figs 53(a) and (b)).
STUDIES OF PROSTATE CANCER AND VDR POLYMORPHISMS The identification of the VDR in prostate cancer cells stimulated interest in the possible role of polymorphisms in the VDR and prostate cancer risk. The VDR gene is located on the long arm of chromosome 12 and spans over 100 kb [50] (see Chapter 7). Polymorphisms exist in exons 2, 8, and 9 of the VDR gene and involve
FIGURE 53.3 (a) First order trend surface map of annual UV radiation (from [16]). (b) First order trend surface maps of prostate cancer mortality by county, white males, 1950e1969 and 1970e1994 (from [16]).
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the FokI, Bsm1, and Taq1 restriction fragment length polymorphisms (RFLPs), respectively. The Bsm1 and Taq1 RFLPS do not alter the coding sequence, whereas the FokI polymorphism produces a VDR that starts at a methionine three amino acids distal to the usual start site yielding a VDR protein that is shorter by three amino acids at the N terminus. To date, more than 30 papers have evaluated prostate cancer risk in association with these and related polymorphisms (see [51e54] for reviews). Two early studies found three- to four-fold increased risks of prostate cancer associated with polymorphisms in the 30 end of the VDR gene [55,56]. However, a recent meta-analysis of 17 studies that assessed the 30 polymorphisms, TaqI, BsmI, and poly-A repeat polymorphisms as well as the FokI polymorphism concluded that none of these variants in isolation was a major determinant of prostate cancer risk [57]. This conclusion is not surprising since it is likely that the effect of functional VDR polymorphisms would be observed only in the presence of low levels of vitamin D (i.e., VDR appear to be risk modifiers, not main effects). Conversely, numerous studies of genetic polymorphisms in vitamin-D-related genes have reported significant effects when these polymorphisms were evaluated in the presence of low levels of vitamin D. Thus, John et al. found reduced risks associated with both TaqI tt and BglI BB genotypes but only in the presence of high sun exposure [58]. Likewise, Bodiwala et al. found a decreased risk for men with the GG polymorphism in the Cdx-2 gene, but only when considering men with high vitamin D exposure from sunlight [59]. Ma et al. [60] reported reduced risk associated with the TaqI tt genotype but only among men with low serum levels of 25(OH)D. Similar findings were reported by Li et al. who identified 1066 men with incident prostate cancer from 14 916 men initially free of cancer from the Physician’s Health Study. They reported that, compared to men with plasma 25(OH)D levels above the median and with the FokI FF or Ff genotype, men with 25(OH)D levels below the median and the less functional Fok I ff genotype had increased risks of total (OR 1.9; 95% CI 1.1e3.3) and aggressive prostate cancer (OR 2.5; CI 1.1e5.8; aggressive was defined as stage C or D, Gleason grade 7e10, and fatal prostate cancer) [61]. Conversely, in a nested caseecontrol study in the Health Professionals Follow-up Study, Mikhak et al. reported a reduced risk of total and poorly differentiated prostate cancer (Gleason sum 7) for carriers of the variant Cdx2-A allele who were deficient in plasma 25(OH)D (15 ng/ml) compared to non-carriers with higher levels of 25(OH)D [62]. Recently, in the most comprehensive analysis reported to date, Ahn et al. investigated the association of 48 SNPS in vitamin-D-metabolizing genes (CYP27A1, GC, CYP27B1, and CYP24A1) with serum
25(OH)D and 1,25(OH)2D levels as well as the association of these SNPS with 164 SNPS in downstream mediators of vitamin D signaling, including the VDR. The subjects were 749 incident prostate cancer cases and 781 controls in the Prostate Lung Colorectal and Ovarian Cancer (PLCO) Screening Trial. None of the SNPS overall were associated with cancer risk. However, among men in the lowest tertile of serum 25(OH)D, an increased risk was observed related to tag SNPS in or near the 30 untranslated region of the VDR. The strongest association was observed for rs11574143, with an odds ratio of 2.49 (95% CI 1.41e4.11, p ¼ 0.0007) for risk allele carriers versus wild type. Because of the effects of detection bias, polymorphisms in the VDR may be more strongly associated with advanced than with local disease. Thus, Hamasaki et al. genotyped 110 prostate cancer patients, 83 patients with benign prostatic hypertrophy and 90 age-matched controls for the Taq1 restriction site. Although the frequency of the TT genotype was not significantly different in the controls compared to the prostate cancer or BPH patients, it was significantly higher among prostate cancer patients with a moderately high Gleason grade (grade 5) compared to controls (p ¼ 0.0001) and in BPH patients with a high prostate volume (>50 cm3) compared to controls (p ¼ 0.001) [63]. Studies of vitamin D pathway gene variants have also been applied to the topic of prostate cancer prognosis. Holt et al. studied disease recurrence/progression in a cohort of 1294 Caucasian men who were followed for an average of 8 years. Significantly altered risk for disease recurrence was reported among for three VDR tag SNPS and five CYP24A1 tag SNPS [64]. The broad subject of VDR polymorphisms and risk of various diseases is discussed in Chapter 56.
SEROLOGICAL STUDIES OF THE VITAMIN D DEFICIENCY HYPOTHESIS Prediagnostic serum levels of vitamin D have been assessed in several prospective studies and are the subject of recent meta-analyses [65,66]. The first serological study of the vitamin D hypothesis was reported by Corder et al. who analyzed stored sera from members of the Kaiser Permanente Plan in northern California [67]. Among 181 men who subsequently developed prostate cancer, serum levels of 1,25(OH)2D were slightly but significantly lower among cases. The mean difference between cases and controls was 1.8 pg/ml. The effect was greatest in older men and in men with low serum levels of 25(OH)D. This finding has not been confirmed by subsequent studies. Several studies have reported evidence of a protective effect of serum 25(OH)D. Based on a 13-year
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follow-up of 19 000 men who participated in the Helsinki Heart Study, Ahonen et al. reported a three-fold increased risk of prostate cancer in men with low 25(OH)D (<40 nmol/l (<16 ng/ml)) [68]. As noted above, two studies conducted within the Physician’s Health Study, Ma et al. and Li et al., found increased risk with low levels of 25(OH)D in conjunction with polymorphisms in the VDR. Conversely, the majority of studies, including those conducted in Hawaii [69], Maryland [70], the Southeast [71], among physicians [72] and health professionals [73], among a cohort of elderly men [74], and among a multiethnic cohort in California and Hawaii [75], did not observe an association with serum 25(OH)D levels. However, the cutpoints for low 25(OH)D in these studies ranged from <21.4 ng/ml to <24.1 ng/ml and <34 ng/ml, i.e. approximately twice those in the Helsinki Heart Study. Thus, it is possible that increased risks are observed only when the low-exposure category reflects vitamin D deficiency (i.e., below 20 ng/ml (50 nmol/l)). Although the multiethnic cohort evaluated a cutpoint of 20 ng/ml, only 16% of these subjects were vitamin D deficient, far lower than the prevalence of vitamin D deficiency among the general US male population 70 years, 26.6% [76]. In their recent meta-analysis of 11 studies of prostate cancer, Gandini et al. reported a summary relative risk of 0.99 for prostate cancer (based on 11 studies). Ten of the 11 studies reviewed concerned incident disease. In a second Finnish study reported by Tuohimaa et al., in addition to an increase in risk associated with low 25(OH)D, these investigators reported that the risk was (paradoxically) elevated in men with high 25(OH)D levels, resulting in a “U-shaped” relationship between 25(OH)D and prostate cancer [77]. In a caseecontrol study nested within the Prostate, Lung, Colon and Ovarian Cancer Screening Trial, which patients were diagnosed from 1 to 8 years after blood draw, Ahn et al. found no effect for season-adjusted 25(OH)D and overall prostate cancer risk. However, they reported that serum concentrations greater than the lowest quintile were associated with an increased risk of aggressive cancer (defined as Gleason sum 7 or clinical stage III or IV disease) [78]. In contrast, in the largest serological study reported to date, Travis et al. compared serum 25(OH)D levels in 652 prostate cancer cases matched to 752 controls from seven European countries. The median follow-up time for a diagnosis of prostate cancer was 4.1 years. They observed no significant association between 25(OH)D levels and risk of prostate cancer (highest versus lowest quintile: odds ratio (OR) 1.28; 95% CI 0.88e1.88). Unlike Ahn, and consistent with most other studies, there was no association between vitamin D levels by histologic stage or grade of disease [79].
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Tretli et al. [82] recently reported on the effects of serum levels of 25(OH)D on prostate cancer survival using patients in the JANUS serum bank, a population-based cohort in Norway. Vitamin D was measured at the time of hospitalization. When 160 patients with prostate cancer were categorized by serum 25(OH)D level as low (<50 nmol/l), medium (50e80 nmol/l) or high (>80 nmol/l), men with medium or high 25(OH)D had a significantly longer survival compared to men with low 25(OH)D (RR 0.33; 95% CI 0.14e0.77; RR 0.16; 95% CI 0.05e0.43). The mean follow-up period from observation to death was 44.0 months. There was no evidence of a doseeresponse; that is, high levels of 25(OH)D were not better than medium levels. This finding is consistent with the hypothesis that it is vitamin D sufficiency (25(OH)D 50 nmol/l) that is associated with improved outcome, as discussed below. It is possible that the patients’ disease affected the serum 25(OH)D levels in this study, as patients who were ill would be expected to spend less time outdoors and/or might ingest less vitamin D or digest or metabolize it less efficiently. However, there was little evidence for this type of “reverse causality” in this study as patients with metastatic disease did not differ appreciably in their serum levels of 25(OH)D than patients with nonadvanced disease (median values of 62 versus 73 nmol/l) [80]. In summary, the overall serologic evidence in favor of the vitamin D hypothesis is weak, at least when serum levels of 25(OH)D are considered in isolation from genetic studies. The evidence is stronger for studies conducted at high latitudes, e.g. Scandinavia, where the prevalence of vitamin D deficiency and insufficiency is higher than in the USA and for studies that included polymorphisms in the VDR. In the original Finnish study, half the men had 25(OH)D levels below 40 nmol/l (16 ng/ml), which is below a common clinical cutpoint indicating vitamin D deficiency (<20 ng/ml). In the US studies, the proportions of men who had deficient 25(OH)D levels was much smaller, ranging from 5 to 13.3%. In the Hawaiian study, none of the men had 25(OH)D levels below 21 ng/ml. Thus, many of these studies do not address the hypothesis of vitamin D deficiency, but ask whether different degrees of vitamin D sufficiency are associated with an altered risk of prostate cancer. Paradoxically, data from one Finnish study and the US PLCO trial raise the possibility that high levels of 25(OH)D might be associated with increased prostate cancer risk. The conflicting reports highlight an important conceptual issue with critical implications for vitamin D supplementation programs. Whether vitamin D sufficiency decreases the risk for prostate cancer relative to vitamin D deficiency is a separate question from whether supra-sufficiency of vitamin D decreases the
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risk for prostate cancer relative to vitamin D sufficiency. That is, it is possible that vitamin D deficiency increases prostate cancer risk whereas vitamin D at doses higher than the threshold for sufficiency may not further alter the risk; or, if the findings of Tuohimaa et al. are valid, might even increase it. In this regard it is noteworthy that the “U-shaped” relationship of increased prostate cancer with both low and high levels of 25(OH)D associated with increased risk of prostate cancer has not been replicated by subsequent epidemiologic studies, including the large, pan-European study recently reported by Travis et al. [81]. Moreover, it is difficult to reconcile a “U-shaped” relationship with the geography of prostate cancer mortality. That is, if both low and high levels of 25(OH)D were associated with increased risk, one would expect that within the USA the lowest mortality rates for prostate cancer would be found in the middle latitudes. Conversely, geographic studies consistently demonstrate significantly lower mortality rates for prostate cancer in the southern regions of the USA. Our attempts to demonstrate a trough in mortality rates at middle latitudes, using trend surface analyses of prostate cancer mortality data from 1950e1994 at 15 5year intervals, failed to do so at any time period. Moreover, the proposed “U-shaped” relationship is not supported by laboratory studies which consistently show dose- and time-dependent inhibition of growth and invasiveness of prostate cancer cells with increasing doses of vitamin D (Chapter 86).
EPIDEMIOLOGIC STUDIES OF SUN EXPOSURE AND PROSTATE CANCER At least ten epidemiologic studies have examined the risk of prostate cancer in relation to exposure to UV radiation. The results of these studies are generally consistent with a protective effect of sunlight exposure. Luscombe et al. compared 210 men with prostate cancer in the United Kingdom to 155 men with benign prostatic hypertrophy (non-cancer controls) on several measures of lifetime sunlight exposure. They found that a high sunbathing score was significantly protective (OR 0.83; 95% CI 0.76e0.89). Multiple sunburns during childhood were significantly inversely associated with prostate cancer risk (OR 0.18; 95% CI 0.08e0.38). Among cases, men with low sunlight exposure were diagnosed at significantly younger ages than men with high exposure [81]. These authors also demonstrated that alleles linked to skin pigmentation (tyrosinase and melanocortin-1) were significantly associated with prostate cancer risk [82]. Similarly, Rukin and colleagues studied UV exposures, as determined by questionnaire, on prostate cancer stage and survival among 553 men with prostate cancer. They reported that UV exposure 10, 20, and 30
years before diagnosis was significantly inversely related to stage and to prostate cancer survival [83]. A positive relationship between vitamin D status and survival is consistent with the serological findings later reported by Tretli et al. An important issue in retrospective studies like that of Rukin et al. is recall bias. Due to the popularization of the sunlight/prostate cancer story in the popular press (e.g., [84]), some men with prostate cancer may underestimate their actual exposures to UV radiation, leading to a bias in support of the hypothesis. Four studies that circumvent this problem are those of Friedman et al., Weinrich et al., and two studies by John et al. Freedman et al. conducted a death-certificate-based caseecontrol study of mortality from prostate cancer in association with exposure to sunlight [85]. Cases were deaths from cancer between 1984 and 1995 in 24 states. Controls were deaths from causes other than cancer and other diseases thought to involve sunlight exposure. Residential exposure to sunlight was classified by state of residence at birth and at death. High residential exposure to sunlight was associated with a significantly decreased risk of fatal prostate cancer (OR 0.90; 95% CI 0.87e0.93). Because exposure to sunlight was classified from information on the death certificate, this study is unlikely to be influenced by recall bias. Weinrich et al. measured the association between self-reported solar exposure and an abnormal serum PSA among men attending a prostate cancer screening program. After adjustment for age, education, and income, frequent sun exposure (three or more times per week) was associated with a 55% reduction in the odds of an abnormal PSA (RR 0.45; 95% CI 0.21e0.97) [86]. John et al. analyzed data from the first National Health and Nutrition Examination Survey (NHANES I) Epidemiologic Follow-up Study in order to test the hypothesis that sunlight exposure reduces the risk of developing prostate cancer [87]. One hundred and fifty-three men with incident prostate cancer were identified from a cohort of 3414 white men who completed the dermatologic examination. State of longest residence in the South (RR 0.58, CI 0.38e0.88, p < 0.01), and high solar radiation in the state of birth (RR 0.48, CI 0.30e0.76, p < 0.01) were associated with substantial and significant reductions in the risk of prostate cancer. The data were adjusted for the confounding effects of education, income, body mass index (BMI), height, alcohol consumption, smoking, physical activity, energy intake, and intake of fat and calcium. The prospective design of this study precludes the possibility of recall bias. In a subsequent study, John et al. (2005) conducted a population-based caseecontrol study of advanced prostate cancer among men from the San Francisco Bay area. Interview data were collected on lifetime sun
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exposure for 450 Caucasian cases and 455 Caucasian controls. Using a reflectometer, skin pigmentation was measured at two sites: the upper underarm (a measure of sun-unexposed skin) and the forehead (a measure of sun-exposed skin). A halving of the risk of prostate cancer was seen for men with high sun exposure as determined by reflectometry (OR 0.51; 95% CI 0.33e0.80). Reductions in risk were also observed among men with high occupational outdoor activity. The use of skin reflectometry adds objective evidence for the effects of solar radiation that cannot be due to reporting bias. Using estimates of vitamin D status derived from the Health Professionals Follow-up Study, Giovannucci et al. reported inverse, non-significant protective effects of vitamin D status on prostate cancer incidence and mortality [88]. Using cancer registry data from Norway, a country with a very large variation in seasonal levels of 25(OH)D, Robsahm et al. reported a significantly longer cancer survival from prostate cancer for cases diagnosed in the summer and autumn, when 25(OH)D levels are at their highest [89]. However, this was an ecologic study and individual data on 25(OH)D were not obtained. de Vries and colleagues addressed the role of UV radiation in prostate cancer risk using non-melanoma skin cancer as a surrogate measure for chronic sunlight exposure. Among Caucasians, non-melanoma skin cancers, particularly those occurring on the head and neck, are believed to reflect chronic sunlight exposure (see Chapter 89). de Vries et al. examined the rate of subsequent prostate cancer among elderly men with non-melanoma skin cancer who were part of a population-based cancer registry in the Netherlands. Men with non-melanoma skin cancer subsequently experienced a significantly decreased incidence of prostate cancer (standardized incidence ratio 0.73; 95% CI 0.56e0.94) [90]. These findings are in agreement with those of Luscombe et al. and Rukin et al., and are not susceptible to recall bias. The decreased risk of prostate cancer among men with non-melanoma skin cancer is difficult to explain on the basis of detection bias since patients with a diagnosis of cancer tend to be examined more intensely for the occurrence of subsequent tumors. Conversely, this finding was not replicated by Levi et al. using incidence data on prostate cancer in the datasets of the Swiss Cancer Registries of Vaud and Neuchaˆtel [91]. Gilbert and colleagues recently reported on life course sun exposure in a large caseecontrol study nested within the Prostate Testing for Cancer and Treatment (ProtecT) study in the United Kingdom, a study based on 1020 PSA-detected cases and 5044 population controls [92]. They observed significantly increased risks among men with olive/brown skin (OR 1.47; 95% CI
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1.00e2.17) and among men with the lowest level of intense sunlight exposure in the 2 years prior to diagnosis (OR 1.24; 95% CI 1.03e1.50). This latter finding is consistent with the increased odds of an abnormal PSA among men with low sunlight exposure reported by Weinrich et al. Conversely, these authors reported that among men with prostate cancer, spending less time outside was associated with a reduced risk of advanced cancer and a high Gleason grade (OR 0.49; CI 0.27e0.89; OR 0.62; CI 0.43e0.91, respectively). Because the outdoor exposure in these men occurred predominantly in England and Scotland, the effective dose of UV radiation in this study may be relatively modest. In summary, in contrast to the inconsistent results of studies of serum levels of 25(OH)D, epidemiologic studies of sunlight exposure generally show protective effects of sunlight exposure on prostate cancer risk. What accounts for the differences between the two types of studies? It is important to note that the serologic studies have important limitations. The most important of these concerns the problem of the timing of the exposure. Although 25(OH)D is the best serological measure of vitamin D status, its half-life in blood is only about 3 weeks. Additionally, because we do not know during life when vitamin D exposures are most important, it is possible that, for example, studies of serum of middle-aged men a few years prior to diagnosis may have missed the “window of opportunity” for vitamin D to exert a preventive effect [93]. This interpretation is supported by the study by John et al. which found that early life exposure is more important than exposure later in life. In this regard, studies of habitual sunlight exposure, including those with objective measurements of skin pigmentation and diagnoses of skin cancer (related to sun exposure) are likely to more accurately reflect long-term vitamin D status than is a single serum measurement that is obtained at a time that may not be relevant to the natural history of prostate cancer. Studies of serum vitamin D on prostate cancer survival are less vulnerable to the problem of timing of exposure because they address the question whether vitamin D affects the course of disease rather than its occurrence. In summary, because the potentially relevant time periods for which vitamin D may be protective are not defined, studies of serum vitamin D are vulnerable to significant misclassification of exposure. Due to imprecision in defining biologically effective doses of solar radiation, which depend on skin type and on the intensity of sunlight exposure, studies of sunlight exposure also are vulnerable to misclassification. The studies that are the least vulnerable to the biases that traditionally affect epidemiologic studies are the studies of genetic polymorphisms.
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EPIDEMIOLOGIC STUDIES OF CALCIUM AND PROSTATE CANCER The relationship of calcium to prostate cancer is closely intertwined with the relationship of prostate cancer to vitamin D. Like the relationship of prostate cancer to sunlight, the relationship of prostate cancer to dairy products had its origin with ecologic studies showing a correlation between prostate cancer mortality rates and per capita dairy consumption [94]. Many of these studies identified calcium as the factor associated with increased risk. The subject of dietary calcium and prostate cancer risk has been the subject of several reviews and meta-analyses (e.g., [95,96]). In their review, the World Cancer Research Fund/American Institute for Cancer Research concluded that foods containing calcium are probably a cause of prostate cancer. Similarly, the Agency for Health Care Research and Quality recently noted that three of the four cohort studies rated A for methodological quality show significantly increased risk for prostate cancer with diets high in calcium [97]. For example, in the Health Professionals Study, Giovannucci and colleagues reported a significantly increased risk of advanced and fatal cancer at calcium intakes of 1500e1999 mg/day and was greatest at intakes >2000 mg/day (the present recommended allowance for men aged 51 and older is 1200 mg/day) [98]. However, other studies, especially those involving more moderate calcium intake, have not found evidence of increased risk, e.g. [99]. Giovannucci et al. initially speculated that the risk elevation is caused by calcium’s inhibition of the renal hydroxylation of 25(OH)D to 1,25(OH)2D [100]. However, recent studies have not confirmed previous findings of an inverse association between calcium intake and serum 1,25(OH)2D levels [101]. Thus, other potential mechanisms for the association between dietary calcium and prostate cancer risk must be sought. An increased risk of cancer in association with higher levels of calcium in serum was reported in 2008 by Skinner and Schwartz [102]. In the first NHANES and in the NHANES Epidemiologic Follow-up Study, 85 incident cases of prostate cancer and 25 prostate cancer deaths occurred over 46 188 person-years of follow-up. Serum calcium was measured an average of 9.9 years prior to the diagnosis of prostate cancer. When men in the top tertile of total serum calcium were compared to men in the bottom tertile, the multivariable adjusted relative hazard for fatal prostate cancers was 2.68 (95% CI 1.02e6.99; ptrend ¼ 0.04). For incident prostate cancer, the relative risk for the same comparison was not significantly elevated, 1.31 (95% CI 0.77e2.20). These results suggest that high serum calcium increases risk for fatal prostate cancer. In order to confirm this association, we
examined prostate cancer mortality in NHANES III. In addition to total serum calcium, NHANES III included measurements of ionized serum calcium, the biologically active fraction of total serum calcium. Twenty-five prostate cancer deaths occurred over 56 625 person-years of follow-up. Serum calcium was measured an average of 5.3 years prior to death from prostate cancer. Compared to men in the lowest tertile of total serum calcium, the multivariate-adjusted relative risk for death from prostate cancer for men in the highest tertile was 2.07 (95% CI 1.06e4.04). For the ionized fraction of total serum calcium, men in the highest tertile had a relative risk of 3.18 (95% CI 1.09e9.28). These results confirm the prospective association between serum calcium and prostate cancer mortality [103]. Because these are observational studies, it is important to consider possible non-causal explanations for these associations. These studies were prospective and men with prevalent cancer were excluded. Although it is possible that some men may have had occult, preexisting prostate cancer, reverse causality is an unlikely explanation for the findings because advanced prostate cancer is typically associated with normo- or hypocalcemia (i.e., the opposite of our findings) caused by the diversion of calcium from serum into bony metastases [104e106] (see [107] for a review). If these associations are valid, what is the underlying biology? There are several possibilities. Prostate cancer cells express the calcium-sensing receptor, a G-protein-coupled receptor that is activated by extracellular calcium [108] and calcium-dependent potassium channels that regulate prostate cancer cell proliferation via the control of calcium entry into the cells [109]. Increases in extracellular serum calcium cause a decrease in apoptosis and an increase in proliferation and migration of metastatic prostate cancer cells [110]. Thus, high levels of calcium in serum may promote the growth of potentially fatal cancers [111]. These findings can help explain the increases in risk for advanced or fatal prostate cancer that have been reported in association with diets high in calcium. Calcium levels in the diet over a large range of calcium intakes generally are not correlated with calcium levels in serum [112]. However, diets that are high in calcium, such as those associated with the use of calcium supplements, can override normal homeostatic controls and cause elevations in serum calcium, including frank hypercalcemia [113e115]. It is noteworthy that numerous epidemiologic studies have shown that within the normal reference range for 25(OH)D, serum calcium levels are positively correlated with serum levels of 25(OH)D (e.g., [101,116, 117]). Thus, an inverse relationship between serum levels of 25(OH)D and risk of prostate cancer, if one exists, may be confounded by a positive association between serum calcium and
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REFERENCES
prostate cancer. This may account for some of the inconsistent results observed in serological studies of 25(OH) D and prostate cancer. In this regard it is important to note that the role of serum calcium in prostate cancer risk is an emerging research area and the results of the two NHANES analyses await further confirmation.
CONCLUSIONS The vitamin D hypothesis has stimulated extensive investigations in prostate cancer, ranging from the laboratory to the clinic and to healthy populations. As discussed in other chapters, evidence that vitamin D exerts large and diverse anticancer effects on prostate cells and in animal models is unambiguous. Epidemiologic evidence that vitamin D and sunlight exert protective effects on the risk of prostate cancer in populations is less clear. A northesouth gradient in prostate cancer mortality in the USA is well established. However, studies of serum 25(OH)D on the risk of prostate cancer in individuals do not support a protective role for higher levels of 25(OH)D. However, an increased risk in the presence of vitamin D deficiency, particularly in conjunction with polymorphisms in the VDR, is supported by several studies. The evidence for a protective role of vitamin D in prostate cancer is stronger for epidemiologic studies of sunlight exposure, many of which demonstrate that lifetime solar exposure is protective. The differences in the results of these studies may be explicable, at least in part, by methodological differences in study design and by uncertainty about the time period when exposure to vitamin D is most important for protection. Prospective studies implicate both dietary and serum calcium with increased risk of advanced or fatal prostate cancer. We speculate that this reflects a direct effect of serum calcium on prostate cells. A positive effect of serum calcium on prostate cancer risk may confound the results of studies of 25(OH)D and prostate cancer risk.
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[92] R. Gilbert, C. Metcalfe, S.E. Oliver, D.G. Whiteman, C. Barin, A. Ness, et al., Life course sun exposure and risk of prostate cancer: population-based nested case-control study and metaanalysis, Int. J. Cancer 15 (2009) 1414e1423. [93] G.G. Schwartz, The “Cocaine Blues” and other problems in epidemiologic studies of vitamin D and cancer, Nutrition Reviews 65 (2007) S75eS76. [94] D.P. Rose, A.P. Boyar, E.L. Wynder, International comparisons of mortality rates for cancer of the breast, ovary, prostate, and colon, and per capita food consumption, Cancer 58 (1986) 2363e2371. [95] P.W. Parodi, Dairy product consumption and the risk of prostate cancer, Int. Dairy J. 19 (2009) 551e565. [96] World Cancer Research Fund/American Institute for Cancer Research, Food, nutrition, physical activity, and the prevention of cancer: A global perspective, AICR, Washington, DC, 2007. [97] Agency for Health Care Research and Quality, Vitamin D and calcium: a systematic review of health outcomes, AHHRQ Publication No. 09eE015, 2009.http://www.ahrq.gov/clinic/ tp/vitadcaltp.htm, 2009. [98] D.S. Straub, Calcium supplementation in clinical practice: a review of forms, doses, and indications, Nutr. Clinical Pract. 22 (2007) 286e296. [99] S.I. Berndt, H.B. Carter, P.K. Landis, M.L. Tucker, L.J. Hsieh, E.J. Metter, et al., Baltimore Longitudinal Study of Aging, Calcium intake and prostate cancer risk in a long-term aging study: the Baltimore Longitudinal Study of Aging, Urology 60 (2002) 1118e1123. [100] E. Giovannucci, Y. Lui, M.J. Stampfer, W.C. Willett, A prospective study of calcium intake and incidence of fatal prostate cancer, Cancer Epidemiol. Biomarkers Prev. 15 (2006) 203e210. [101] M. Tseng, V. Giri, D. Watkins-Bruner, E. Giovannucci, Dairy intake and 1,25-dihydroxyvitamin D levels in men at high risk for prostate cancer, Cancer Causes Control 20 (2009) 1947e1954. [102] H.G. Skinner, G.G. Schwartz, Serum calcium and incident and fatal prostate cancer in the National Health and Nutrition Examination Survey, Cancer Epidemiol. Biomarkers Prev. 17 (2008) 2302e2305. [103] H.G. Skinner, G.G. Schwartz, A prospective study of total and ionized serum calcium and fatal prostate cancer, Cancer Epidemiol. Biomarkers Prev. 18 (2009) 575e578. [104] N. Buchs, J.-P. Bonjour, R. Rizzoli, Renal tubular absorption of phosphate is positively related to the extent of bone metastatic load in patients with prostate cancer, J. Clin. Endocrinol. Metab. 83 (1998) 1535e1541.
[105] R.M.L. Murray, V. Grill, N. Crinis, P.W.M. Ho, J. Davison, P. Pitt, Hypocalcemic and normocalcemic hyperparathyroidism in patients with advanced prostatic cancer, J. Clin Endocrinol. Metab. 86 (2001) 511e516. [106] S.Q. Flores, M. Varsavsky, F. Valle Dı´az De La Guardia, ´ lvarez, et al., HiperparatirJ.L.M. Ortiz, M.M. Torres, E.R. A oidismo secundario en el cancer de pro´stata avanzado, Endocrinologı´a y Nutricio´n 57 (2010) 100e104. [107] G.G. Schwartz, Prostate cancer, serum parathyroid hormone and the progression of skeletal metastases, Cancer Epidemiol. Biomarkers Prev. 17 (2008) 478e483. [108] K.I. Lin, N. Chattopadhyay, M. Bai, R. Alvarez, C.V. Dang, J.M. Baraban, et al., Elevated extracellular calcium can prevent apoptosis via the calcium-sensing receptor, Biochem. Biophys. Res. Commun. 249 (1998) 325e310. [109] H. Lallet-Daher, M. Roudbaraki, A. Bavencoffee, P. Mariot, F. Gackie`re, G. Bidaux, et al., Intermediate-conductance Ca2þactivated Kþ channels (IKca1) regulate human prostate cancer cell proliferation through a close control of calcium entry, Oncogene. online publication 9 March (2009). doi:10.1038/ onc.2009.25. [110] J. Liao, A. Schneider, N.S. Datta, L.K. McKauley, Extracellular calcium as a candidate mediator of prostate cancer skeletal metastasis, Cancer Res. 77 (2006) 9065e9073. [111] G.G. Schwartz, Is serum calcium a biomarker of fatal prostate cancer? Future Oncology 5 (2009) 577e580. [112] R. Jorde, J. Sundsfjord, K.H. Bønaa, Determinants of serum calcium in men and women. The Tromsø Study, Eur. J. Epidemiol. 17 (2001) 1117e1123. [113] A.M. Patel, S. Goldfarb, Got calcium? Welcome to the CalciumAlkali Syndrome, J. Am. Soc. Nephrol. (2010). doi: 10.1681/ ASN.2010030255. [114] Y. Kato, K. Sato, A. Sata, K. Omori, K. Nakahima, K. Tokinaga, et al., Hypercalcemia induced by excessive intake of calcium supplements, presenting similar findings of primary hyperparathyroidism, Endocrine J. 51 (2004) 557e562. [115] W.P. Muldowney, S.A. Mazbar, Rolaids-yogurt syndrome: a 1990s version of milk-alkali syndrome, Am. J. Kidney Dis. 27 (1996) 270e272. [116] H.G. Skinner, G.G. Schwartz, The relation of serum parathyroid hormone and serum calcium to serum levels of prostate specific-antigen: a population-based study, Cancer Epidemiol. Biomarkers Prev. 11 (2009) 2869e2873. [117] P. Engel, G. Fagherazzi, A. Boutten, T. Dupre´, S. Mesrine, M.C. Bouton-Ruault, et al., Serum 25(OH)D and risk of breast cancer: a nested case-control study from the French E3N cohort, Cancer Epidemiol. Biomarkers Prev. 19 (2010) 2341e2350.
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C H A P T E R
54 Nutrition and Lifestyle Effects on Vitamin D Status Susan J. Whiting 1, Mona S. Calvo 2 1
University of Saskatchewan, Saskatoon, Saskatchewan, Canada 2 US Food and Drug Administration, Laurel, MD, USA
Disclaimer: The findings and conclusions presented in this chapter are those of the authors and do not necessarily represent the views or opinions of the US Food and Drug Administration. Mention of trade names, product labels or food manufacturers does not constitute endorsement or recommendation for use by the US Food and Drug Administration.
INTRODUCTION Vitamin D deficiency is a public health problem worldwide, even in countries with enough sunshine year round to promote adequate skin synthesis [1]. The circulating level of the transport metabolite 25hydroxyvitamin D (25(OH)D) is the most commonly used measure of vitamin D nutritional status. It is the intermediary metabolite made in the liver from vitamin D3 synthesized in skin during sun exposure or from dietary intake of vitamin D2 or D3 contained in foods [2,3]. The public health importance of 25(OH)D levels is critical as this metabolite acts as the only substrate for the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), a molecule with actions that extend far beyond its classic function in calcium regulation and bone health. Maintaining adequate circulating 25(OH)D concentrations is now considered critical to overall health maintenance and the function of the immune, integumentary, nervous, reproductive, and musculoskeletal systems of men and women of all ages [4,5]. To achieve the benefits of vitamin D, one requires sufficient skin synthesis and/or intake to maintain adequate plasma levels of 25(OH)D. This metabolite subsequently undergoes hydroxylation to form the active metabolite
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10054-X
1,25-dihydroxyvitamin D (1,25(OH)2D). The term vitamin D represents both the molecular precursors of 25(OH)D as well as all compounds having or potentially having the activity that we associate with the active metabolite of vitamin D. To avoid confusion, in this chapter the term vitamin D is used only in reference to the parent molecules cholecalciferol and ergocalciferol, vitamin D status refers to levels of 25(OH)D (and other biomarkers if appropriate), while vitamin D activity represents the ultimate action(s) of the 1,25(OH)2D molecule. The parent molecules cholecalciferol, made in the skin of mammals, and ergocalciferol, made after irradiation of yeast or fungi-derived molecules, are more commonly referred to as vitamin D3 and vitamin D2, respectively. Unless a specific difference is noted between vitamin D2 and D3, then “D” with no subscript is used. As is described later in this chapter, differences in metabolism between vitamin D2 and D3 do exist [6]; nevertheless, it should be emphasized that while they may not be of equal potency both D2 and D3 are effective in raising 25(OH)D levels when used appropriately for supplementation and food fortification [7]. The intermediary transport form of vitamin D, 25(OH)D, is delivered to tissues throughout the body which contain the mitochondrial enzymes needed to further hydroxylate it to the active form, 1,25(OH)2D. These tissues also contain the nuclear receptors and translational factors needed for the active or hormonal form to function in the regulation of expression of many genes and thus determine the transcription levels of a variety of cellular proteins. The metabolite 1,25(OH)2D regulates the transcription of proteins involved in many aspects of cellular activity including apoptosis, differentiation and synthesis of proteins involved in immune response,
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metabolism, and endocrine function [8]. The importance of vitamin D in the maintenance of overall health relates to the fact that adequate levels of 25(OH)D are needed to supply tissues with this critical hormonal precursor. A growing number of studies show significant association of higher serum 25(OH)D levels and lower risk of chronic and infectious diseases [9e11], mortality from all causes [12] as well as reduction in fractures and falls [13,14]. Analysis of chronic disease risk indicates that optimizing vitamin D status will significantly reduce the burden of health care costs [15]. To relate vitamin D intake to status, i.e. serum levels of 25(OH)D, it is important to understand how terminology is currently defined. There is consensus that deficiency represents levels not sufficient to provide protection against rickets, osteomalacia, osteoporosis, and falls. And most researchers and clinicians define this at 50 nmol/l (20 ng/ml). However, a level that is thought to provide protection against risk of chronic diseases beyond bone health is called optimal and is defined as being at or above 75 nmol/l (30 ng/ml) [16]. This chapter uses this latter value as the preferred level to attain through appropriate sun exposure and/or dietary intake of vitamin D. One hurdle in determining maintenance and repletion needs to achieve optimal vitamin D status lies in the significant differences in measurement of serum 25(OH)D that occur with the various assays currently in use. There are several methods used for measurement of 25(OH)D, and this situation has created some confusion regarding comparability between studies [17,18]; however, discussion of assay differences is beyond the scope of this chapter and is discussed in Chapter 47. It is important to emphasize that assay variability and between-laboratory imprecision together may be responsible for a 20% deviation between a laboratory result and the true measure [17]. A further cause of variability is within-individual (biological) variation, which also may be as high as 20%, although much of this measure may be reflecting seasonal changes in sun exposure [17]. Finally, there are aspects of the production of 25(OH)D that affect its circulating level. If the substrate, i.e. vitamin D3 or vitamin D2, is low, then this limits the rate of synthesis of 25(OH)D [19] so that at lower intakes or sun exposures, the amount of vitamin D3 or vitamin D2 limits synthesis of 25(OH)D. Over time, an increase in 25(OH)D produces an increase in 1,25(OH)2D that increases the rate of catabolism, by inducing 25(OH)D-24-hydroxylase (CYP24 which is also referred to as 24-hydroxylase). Thus, as the precursor molecule is made more available, there is increased formation of 1,25(OH)2D and then accelerated catabolism of vitamin D metabolites [20] such that levels of all the metabolites tend to plateau. The chapter objectives are to describe the determinants of vitamin D status, sun exposure, and dietary
intakes, and the many factors that can affect these two sources of vitamin D. There will be an emphasis on lifestyle choices and environmental factors that either positively or negatively affect these determinants, thus affecting vitamin D status. Strategies to improve vitamin D status through safe sun exposure and appropriate dietary intakes are discussed. Vitamin D has many functions beyond its role in calcium and bone health, and with this new knowledge has come the recognition that vitamin D insufficiency and deficiency is at epidemic proportions worldwide. In temperate countries more than half of the population is at risk and worldwide, even in tropical countries, vitamin D deficiency is a very serious concern due to changes in living and working conditions [1,21]. As an urgent public health concern, vitamin D deficiency has the potential to increase morbidity and mortality [15]. Thus worldwide, each country faces the task of determining a level of vitamin D, whether from sun exposure, dietary intake including food fortification and supplements, or combinations of these, which will be effective at maintaining what scientists believe to be the appropriate level of vitamin D activity in the body. For several reasons many of the examples in this chapter focus on activities in the USA and Canada. Both Canadians and Americans experience all possible permutations to sun exposure, have fortification practices in place that allow for evaluation of effectiveness, and have national survey tools generating data to help explain how factors affect vitamin D status.
DETERMINANTS OF VITAMIN D STATUS Setting recommendations of sun exposure and dietary intake for vitamin D must take both environmental and physiologic factors into account. The two major environmental determinants of poor vitamin D status are lack of sun exposure and poor dietary intake. It has been difficult to appreciate the unique contribution of each of these, as most studies have not controlled one factor when investigating the other. For example, it has often been stated that dietary intake of vitamin D plays no role in determining status. The main evidence cited in support of this statement is the third National Health and Nutrition Examination Survey (NHANES III), a study of over 16 000 Americans where diet influence on vitamin D status was reported to be minimal [22]. Surveys conducted in the winter often reveal a significant correlation between dietary intake of vitamin D and levels of 25(OH)D [23]. NHANES III was conducted in the North during the summer and in the South during the winter which may have masked the influence of dietary intake on vitamin D [24]. Thus it is important always to indicate whether sun exposure
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DETERMINANTS OF VITAMIN D STATUS
is a possible confounder in dietary studies, and whether the nature of the sun exposure could influence vitamin D synthesis, i.e. accounting for latitude, smog, sunscreen use, clothing, and time of day and season. Further, these lifestyle determinants are affected by where people live, what activities they are engaged in, such as occupation, leisure, and what foods are available. Cultural and social factors are important, and in this chapter we attempt to show how these factors are related to vitamin D status.
Overview of Vitamin D Metabolism Skin Synthesis A detailed discussion of the photobiology of vitamin D is given in Chapter 2 but a brief review is useful at this point in our discussion. In the skin, 7-dehydrocholesterol in the epidermis and dermis changes conformation when ultraviolet B (UVB) radiation in the wavelength range of 280e315 nm passes through these skin layers to form previtamin D3. Previtamin D3 forms rapidly; however, skin pigmentation (melanin) competes with 7-dehydrocholesterol for the UVB photons, and therefore reduces the amount of UVB that can act on 7-dehydrocholesterol to form previtamin D3. With prolonged exposure to UVB, inactive compounds are formed instead of previtamin D3. Over a prolonged period of time, the previtamin D3 that is formed is converted by thermal isomerization to vitamin D3. While the reaction to form previtamin D3 takes minutes, the reaction converting it to vitamin D3 takes hours to occur [25] and is a rate-limiting step. Should more UVB photons reach the epidermis and dermis, previtamin D3 is converted irreversibly to inactive compounds (tachysterol and luminsterol) having no vitamin D activity. Thus excess exposure to UVB does not result in excess vitamin D production [26]. Besides the skin pigment melanin, other factors reduce skin synthesis of vitamin D3. These include: clothing, although some loosely woven clothing does permit UVB to pass through; window glass; sunscreens formulated to block UVB; being indoors; cloudy days; smog and light-blocking air pollution; and wintertime, defined as the season when the sun does not rise far enough above the horizon to allow sufficient UVB irradiation to reach the ground. The term vitamin D winter refers to the time of year when UVB radiation is not sufficient for vitamin D3 synthesis in the skin [2,11]. Exposure to the UVB rays of sunlight can be quantified in erythemal doses, i.e. the appearance of reddening of the skin. A minimal erythemal dose (1 MED) causes reddening, and further exposure results in more severe sun burning. Tanning, the induction of melanin synthesis in the skin, also occurs but takes longer to manifest and occurs with UVA as well as UVB exposure [27]. Studies have shown that one full body (i.e., almost completely
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naked) minimal erythemal dose will synthesize as much as 500 mg (20 000 IU) of vitamin D3 [28]. This intensity of sun exposure is not recommended due to concerns about skin phototoxicity; an exposure less than 1 MED will provide sufficient vitamin D3 production depending on extent of body surface area exposed [27,29]. Accordingly, one can calculate, approximately, how much sun exposure at a given time of day and latitude for a reasonable amount of skin surface area is sufficient for vitamin D3 synthesis [29]. This is further described below. Metabolism of Vitamin D The full description of vitamin D metabolism is provided elsewhere in this book (Chapter 3), but a brief summary follows. The cholecalciferol made from skin synthesis is released from the epidermis into the blood, where it is bound to vitamin-D-binding protein (DBP). Dietary cholecalciferol and ergocalciferol are absorbed and carried to the liver in chylomicrons. Intestinal absorption is not a limiting factor except when there is fat malabsorption (e.g., cystic fibrosis, Crohn’s disease). Generally fat-soluble vitamins are better absorbed with dietary fat, but vitamin D3 added to orange juice is well absorbed [30]. Vitamin D (i.e., cholecalciferol and ergocalciferol) circulate for only one to two days. This quick turnover is due to hepatic conversion and uptake by fat and muscle cells [2,19,31]. The two steps leading to the active form of vitamin D, 1,25(OH)2D, are first, conversion to the major circulating form 25(OH)D. This step may involve four different hepatic cytochrome P-450 enzymes that can hydroxylate vitamin D at carbon 25 [2]. The resulting metabolites, 25(OH)D3 and 25(OH)D2 are released into the circulation. Hollis et al. [31] determined whether provision of the substrate cholecalciferol for synthesis of 25(OH)D was rate-limiting, through obtaining a wide range of substrate levels by giving subjects high doses of vitamin D3 or by investigating subjects living in a sunny near-equatorial region. They found that high doses of cholecalciferol from supplements (as much as 160 mg (6400 IU)) or sun exposure in Hawaii affected serum 25(OH)D similarly, and that the relationship is not linear (indicating a controlled, saturable reaction). Thus unless vitamin D is provided in sufficient amounts, the production of 25(OH)D is limited by its substrate. The second step for conversion of 25(OH)D to the active form, 1,25(OH)2D, occurs in many tissues. Two types of conversion occur: endocrine, in which the conversion takes place in one organ (in this case the kidney) with the product, 1,25(OH)2D, acting like a hormone and traveling through blood to its sites of action. This pathway is thus labeled the “endocrine pathway” [2]. The second pathway occurs in many cells of the body, where the enzymes needed for local 1,25(OH)2D production may exist, and under normal
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conditions, the product does not leave the cell or its environs. This pathway is thus labeled the “autocrine/paracrine pathway” [2] and is discussed in Chapter 45. In the endocrine pathway, occurring in proximal renal epithelial cells, 1,25(OH)2D is synthesized when the enzyme 1a-hydroxylase (CYP27B1) is stimulated, a major stimulus being parathyroid hormone regulated predominantly by serum calcium levels [2]. This is a tightly controlled endocrine system and considered to be the major contributor to the circulating levels of the active metabolite of vitamin D.
Overview of Determinants of Vitamin D Status Determining the need for vitamin D is difficult as factors such as sunlight exposure with appropriate UVB, age-related changes in cutaneous vitamin D formation in the skin, and availability of foods containing vitamin D all contribute to our difficulty to provide a single amount as a recommendation for intake. This chapter focuses on the two major determinants of vitamin D status, sun exposure providing UVB and dietary intake. The complexity of determining the mechanisms contributing to poor vitamin D status are evident especially when sun exposure is adequate and dietary vitamin D is available. A multitude of factors related to both skin synthesis and availability of dietary sources affect vitamin D status throughout the world. There are many influences on an individual’s ability to obtain adequate sun exposure. Seasonal and latitudinal differences to provision of UVB radiation can lead to variable rates of cutaneous synthesis of vitamin D [29,32]. Algorithms have been developed for calculating potential vitamin D synthesis from sun exposure by latitude and season (shown below). Several physiological factors, including skin pigmentation and old age, significantly influence the ability to make vitamin D with casual sun exposure. Major environmental considerations are season and latitude. The extent of UVB penetration of skin is further impeded by other factors including: occupation (work indoors or outdoors), rural or urban dwelling (e.g., presence of urban canyons where buildings block sunlight), extent of usual clothing practices, dominant local weather conditions, and altitude [33]. Other factors include beliefs about sun exposure, such as its contribution to skin cancer as well as cosmetic concerns regarding a desire to, or fear of, skin becoming darker due to tanning. Many factors also influence dietary intake of vitamin D. Foods containing natural sources of vitamin D are not widely found in usual diets or food supplies [21,34e35]. Lack of fortification of staple foods in many countries is a contributing factor for low vitamin D intakes [34e35]. Supplements are a possible choice for improving vitamin D status [36] but these are not easily
available in some countries. Even if any or all of these dietary sources are available, lack of education or low socioeconomic status may limit access.
Estimation of Amounts of Vitamin D Needed For a comprehensive discussion of the optimal target of 25(OH)D levels for good health, the reader is referred to Chapters 57 and 58 for two different viewpoints. In this discussion, we take the position that 75 nmol/l (30 ng/ml) is a reasonable target level for serum 25 (OH)D. Among people whose exposure to sunlight may be limited for whatever reason, dietary vitamin D becomes the major determinant of serum 25(OH)D concentrations [2,5,16]. However, with constant sunlight exposure that is available in tropical countries, dietary vitamin D should not be needed. Thus, to state a dietary vitamin D recommendation, the estimate must be made in the context of knowing the extent of sun exposure year round. Similarly, if sun exposure is the major or only determinant of vitamin D status, the amount of time exposed, the timing during the day of the exposure, and the amount of skin receiving the exposure should be quantified. In the middle of these extremes lies a large proportion of the world today, many of whom are involved with indoor work and indoor leisure. Along with usual attire that results in limited exposure to adequate sunlight year round, it is not difficult to see why there now is more reliance on dietary sources of vitamin D to maintain adequate vitamin D status. In Canada and the USA, naturally occurring rich sources of vitamin D are limited to specific foods such as oily fish that are not commonly consumed on a frequent basis by the general population due to expense, limited distribution, or aversion for different reasons. In this chapter, we present three contrasting scenarios as examples to illustrate the varying range of dietary needs experienced worldwide. In the first scenario, needed vitamin D originates only from sun exposure. In the second scenario, needed vitamin D comes from foods and supplements when only moderate amounts (or intermittent amounts) of sun exposure are possible. In the third scenario, needed vitamin D comes only from the diet. Results from a recent study provide estimates of dietary vitamin D requirements for these scenarios in young adults (age 19 to 39 years) living in the USA (latitude 38.5 N) [37]. As shown in Table 54.1, scenario 1 is represented by European-American subjects who achieved high sun exposure (i.e., 90 minutes per day with 35% body surface area exposed) in summer and fall in California; no dietary intake is needed to maintain 25(OH)D at a level of at least 75 nmol/l (30 ng/ml). Scenario 2 is illustrated by
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TABLE 54.1 Predicted Amount of Total Daily Dietary Vitamin D to Achieve a 25(OH)D Level of 75 nmol/L for Young Adults of African or European Descent Living in California, by Sun Exposure Lifestyle African-American
European-American
Season at latitude 38.5 N
Low sun exposure lifestylea
High sun exposure lifestyleb
Low sun exposure lifestyle
High sun exposure lifestyle
Fall
62.5 mg (2500 IU)
50 mg (1250 IU)
41.3 mg (1650 IU)
0
Winter
77.5 mg (3100 IU)
56.3 mg (2250 IU)
63.8 mg (2550 IU)
32.5 mg (1300 IU)
Spring
72.5 mg (2900 IU)
35 mg (1400 IU)
56.3 mg (2250 IU)
1.25 mg (50 IU)
Summer
52.5 mg (2100 IU)
25 mg (1000 IU)
25 mg (1000 IU)
0
a
Exposure to sun ~20 minutes per day with 18% body surface area exposed. b Exposure to sun ~90 minutes per day with 35% body surface area exposed. Modified from [37]. These authors assumed it would take 0.7 mg vitamin D for each 1 nmol/L rise in 25(OH)D.
European-American subjects, where a moderate dietary intake of 32.5 mg (1300 IU) is necessary to achieve a 25 (OH)D level of 75 nmol/l in subjects who had high sun exposure except in winter. Scenario 2 is also seen in those who have little sun exposure in the summer, such as European-American subjects who have low sun exposure (i.e., 20 minutes with only 18% of body surface area exposed), or African-American subjects in summer with high sun exposure; the predicted daily intake requirements are both 25 mg (1000 IU). The third scenario is represented by two groups: the EuropeanAmerican subjects in winter who have low sun exposure have similar high dietary needs, 63.8 mg (2550 IU) as do the African-American subjects with low sun exposure any time of year, who are predicted to need from 62.5 mg (2100 IU) to 77.5 mg (3100 IU) in order to achieve a 25(OH)D level of 75 nmol/l. Other studies provide agreement with these predictions. Scenario 3 is also seen in the data of Aloia et al. [38] who tested how much supplemental vitamin D3 was needed to raise 25(OH)D levels to over 75 nmol/l in almost all subjects studied in wintertime. In those whose baseline 25(OH)D was at least 50 nmol/l (i.e., not severely deficient), 95 mg (3800 IU) was needed. This amount is somewhat higher than the amount estimated by Hall et al. [37] as the former is an individual value while the latter is an average for the group. Scenario 2 is also seen in two studies of Caucasian adults who had summer sun exposure that resulted in an average 25(OH)D level close to 75 nmol/l. During winter, 12.5 mg (500 IU) of supplemental vitamin D3 maintained 75 nmol/l [39,40]. Scenario 1 is also seen in measurements of 25(OH)D in rural Africans residing near or at the equator and having no dietary sources of vitamin D; they have average 25(OH)D levels, for example in older women, of 92 26 nmol/l [41]. Thus for situations with little or no sun exposure, average dietary vitamin D requirements range from 50 mg (2000 IU) to 100 mg (4000 IU) [37,38]. When people
have previously obtained enough sun exposure to provide for storage of vitamin D, average dietary vitamin D requirements range from 12.5 mg (500 IU) to 32.5 mg (1300 IU) [37,39,40]. These estimates assume that intake and/or exposure amounts would be the amount of vitamin D necessary to maintain the stores of an already healthy person, which is the Dietary Reference Intake (DRI) estimate of Estimated Average Requirement (EAR) [42], the value that is then used to find the Recommended Dietary Allowance (RDA) which meets the needs of 97.5% of the population. There is also the consideration that in the deficient person, there must be enough vitamin D to replete stores. For the most part, supplemental sources of vitamin D are commonly used for repletion [43] although tanning beds that provide UVB are also used, especially in patients with malabsorption syndromes [44]. Table 54.2 shows a selection of recommendations for dietary vitamin D that have been set for individuals. In most cases, these recommendations are intended to provide enough vitamin D to maintain a level of 25(OH)D at 75 nmol/l; however, few of the recommendations are evidence-based in this regard. All of these recommendations for vitamin D appear to assume scenario 2 as none alone would maintain 25(OH)D without some sun exposure. This point is not stated explicitly in any recommendation. Table 54.2 includes the revised recommendations by the Institute of Medicine (IOM) Dietary Reference Intake (DRI) values which are used by Canada and the USA. These are discussed later in this chapter.
FACTORS INFLUENCING SKIN SYNTHESIS SUPPLYING VITAMIN D Physiological Factors There are three main physiologic modifiers of sun exposure: skin pigmentation, body size, and aging. To
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TABLE 54.2 A Selection of Recent Dietary Recommendations and Expert Guidelines for Health Professionals for Vitamin D Intake by Adults Organization and date
Age group
Recommendation
Comment from organization
Institute of Medicine (IOM), 2010 www.iom.edu
1e50 y 51e70 y 71þ y
15 mg (6 IU) 15 mg (6 IU) 15 mg (6 IU)
The Recommended Dietary Allowance (RDA) meets the needs of 97.5% of the population, and was set to maintain a 25(OH)D level of 50 nmol/l with minimal sun exposure
Osteoporosis Canada 2010 www.osteoporosis.ca
19e50 y 51þ y
10e25 mg (400e1000 IU) 20e50 mg (800e2000 IU)
A serum level of 25(OH)D above 75 nmol/L consistently improves clinical outcomes such as fracture risk
Dietary Guidelines for Americans, 2005, 2010 www.health.gov/ dietaryguidelines
Men and women at risk for low sun exposure or >50 y
25 mg (1000 IU)
In 2010: supplement those at risk with 25 mg (1000 IU) In 2010: “When necessary, individuals may consider vitamin D supplementation if they are having difficulty meeting the AI through vitamin-D rich foods”
International Osteoporosis Foundation 2010 www.iof.org
Adults
20e25 mg (800e1000 IU)
Achieve 25(OH)D level of 75 nmol/L
National Osteoporosis Foundation 2008 www.nof.org
<50 y 50þ y
10e20 mg 20e25 mg (800e1000 IU)
“Advice on adequate amounts . of vitamin D for individuals at risk of insufficiency”
Health Canada Canada Food Guide 2007 www.hc-sc.gc.ca/fn-an/ food-guide-aliment/context/ index-eng.php
19þ y
5e15 mg (200e600 IU)
“Have 500 mL (2 cups) of [fortified] milk every day for adequate vitamin D.” and, for “Men and women over 50 the need for vitamin D increases after the age of 50. In addition to following Canada’s Food Guide, everyone over the age of 50 should take a daily vitamin D supplement of 10 mg (400 IU)
Canadian Cancer Society 2007 www.cancer.ca
19 þ y
25 mg (1000 IU)
“Due to our northern latitude.we recommend that Canadian adults consider taking . 1000 international units (IU) a day during fall and winter months. “ Also, elderly; those with a dark skin; stay indoors or wear clothing covering most of your skin should consult a physician about supplement use all year round
National Osteoporosis Foundation 2008 www.nof.org
50þ y
20e25 mg (800e1000 IU)
Patients need advice on adequate amounts of vitamin D
Taken from IOM (Institute of Medicine) (2011) Dietary Reference Intakes for Calcium and Vitamin D. Washington, DC: The National Acadamies Press [144].
cover these in detail is beyond the scope of this chapter but Chapter 2 discusses these issues further; however, it is important to acknowledge their contributions. Thus a brief explanation of each factor follows. In the remainder of this chapter, considerations for vitamin D requirement are made for non-obese adults (excluding the very old) but skin pigmentation is specified when necessary. Other physiologic factors such as pregnancy and lactation are beyond the scope of this chapter. Skin Pigmentation Several studies have revealed large differences in vitamin D status among people in the same country with differing skin pigmentation, indicating that despite
similar environmental factors (as described in the following section), people experience varying levels of UVB penetration of skin to make vitamin D [24,45e47]. Generally these effects are due to differences in race and ethnicity (often characterized by skin pigmentation) in countries where sun exposure does not provide vitamin D synthesis year round. In most surveys, race and ethnicity are surrogates for skin pigmentation measures. Some studies that do provide both measures of skin pigmentation and self-reported ethnicity [23,48] provide validation of using ethnicity/race as determining factors for vitamin D status. Skin pigmentation is the result of the accumulation of the brown pigment melanin in the epidermis of the skin
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[49]. Melanocytes release both melanin and acetylcholine, the latter important in regulating body temperature. Melanin acts as a sunscreen to absorb UV radiation thus blocking it from reaching other parts of the skin [50]. Melanin’s production with sun exposure may also have been selected to more effectively camouflage the body [49]. Anthropologists speculate that the hairless human body required skin pigmentation to protect against harmful overexposure to ultraviolet radiation [50], and as humans migrated away from the equator, selection pressure resulted in loss of pigment. Skin color today, however, does not strictly predict latitude as evidenced in more recent migrations but evolutionary changes would take considerable time to occur. Light-skinned Europeans who migrated to Australia centuries ago present an example of skin color poorly adapted to the extremes of sun exposure encountered in central Australia [50]. Skin type can be characterized and used to estimate the resulting amount of melanin that would be produced in response to UV exposure. The Fitzpatrick skin type was originally developed in the USA in 1975 to facilitate UV dosage for psoriasis photochemotherapy in subjects with “white” skin, and characterized for skin types (I through IV); it was later expanded to categories V and VI, as shown in Table 54.3 [51]. These skin types vary in their ability to burn and tan. The time to burn, a minimal erythemal dose (1 MED) reflects the amount of melanin, and is an approximate indicator of the relative dose of UVB needed to synthesize an equivalent amount of previtamin D3. Skin type I needs only 40% of the time for 1 MED compared to skin type III, and skin type VI needs four times the exposure time of skin type III.
TABLE 54.3 Fitzpatrick skin typea
Studies have determined the amount of vitamin D formed in skin upon irradiation with UVB. Estimates vary from 250 mg (10 000 IU) under commercial tanning bed conditions to 500 mg (25 000 IU) when a near-naked individual is exposed to UBV light to 1 MED [52,53], with many experts using 20 000 IU [28]. These values are important as they indicate three important considerations about skin synthesis of vitamin D: (1) the body can safely handle this amount of vitamin D per day; (2) no further synthesis of vitamin D occurs with prolonged UVB exposure, thus indicating toxicity does not arise from sun exposure; and (3) the more surface area exposed, the more vitamin D made. There is a popular algorithm sometimes called Holick’s rule [54] which provides the time (in minutes) for sun exposure under ideal conditions (e.g., no clouds) using the finding of 500 mg (20 000 IU) for 100% body surface area. In the algorithm, the times for 1 MED for each skin type have been reduced to 25% MED (to prevent skin damage), and the amount of surface area exposed is assumed to be 25%. Calculating 25% surface area is shown in Table 54.4. A surface area of 25% would be obtained when a person is sleeveless (18%) and wears shorts or a short skirt (8%). To achieve a 35% body surface area as described from “high sun exposure” described above (scenarios 1e3), one would need to dress with minimal clothing (torso or full length of legs exposed). In Fig. 54.1, the times given for ¼ MED are estimates based on exposure at 42 N (i.e., Boston), at noon, in summer, and may be used to calculate the exposure time to reach a vitamin D dose of 25 mg (1000 IU) [29]. It can be seen from this figure that for some groups, times for sun exposure,
Categorization of Skin Type using a Traditional Dermatological System (Fitzpatrick skin type) and Association of these Categories with Vitamin D Synthetic Capacity of Skin Skin color
Common geographic origins
Skin response to sun exposureb
Relative MEDc
I
White (blue eyes, freckled, albino)
Northern European (Celtic)
Always burn, never tan
0.38
II
White (blond hair, blue or green eyes)
Northern European
Burn slightly, then tan slightly
0.75
III
White (brown eyes, darker complexion)
Southern European, Middle East Caucasian
Rarely burn, tan moderately
1.0
IV
White (“Mediterranean”)
Mediterranean countries
Never burn, tan darkly
1.3
V
Brown
Asian, Native American, Pacific Islander, South American
Never burn, tan darkly; Oriental or Hispanic skin
2.0
VI
Black
African
Never burn, tan darkly; black skin
3.8
a
Characteristics of previously unexposed skin after 30 minute direct exposure to sun. Relative minimal erythemal (MED) dose of UVB exposure is 2.5 SED, the standard erythemal dose, where 1 SED ¼ 100 J/m2 dose of UVB exposure. c Narrow-band reflectance corrected for hemoglobin (red) to estimate melanin content (higher number indicates greater melanin content. Modified from [136]. Additional information from [137]. b
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TABLE 54.4 Estimates of Percent of Body Surface Area (BSA) Exposed to Sun Based on the “Rule of Nines” and Clothing Worn1 Body region2
Clothing type
Head
Nothing
4
Baseball cap
2
Cowboy hat
1
Torso/Arms
Legs
Feet
% BSA exposed
Nothing
47
Bikini top
42
Tank top
18
Quarter length shirt
3
Long sleeves
0
Bikini bottom
38
Knee-length shorts/skirt
8
Full-length pants
0
Nothing
2
1
Modified from [37]. Hands contributed 4% of exposed BSA (0% if gloves were worn) and the neck 2% (0% if a scarf was worn).
2
especially at body surface area exposures of 25%, are not feasible for indoor workers who have a skin type at or above type V. Other factors related to timing, such as older age whereby skin synthesis is limited [55], have not been considered. One caveat to these calculations is that values provided by Webb and Engelsen [29] overestimate the time for exposure required by roughly a third, and underestimates exposure for persons with greater skin pigmentation, as discussed by Dowdy et al. [54].
Aging The main determinant of vitamin D status in aging is lack of vitamin D synthesis, even when there is sufficient UVB to induce adequate vitamin D synthesis in younger adults [55]. Since the early 1990s, it has been recognized that the elderly, especially those who are homebound, are at very high risk for vitamin D deficiency, particularly institutionalized elderly [56], and this concern continues today [57]. Recommendations for vitamin D in the previous 1997 Dietary Reference Intakes (DRIs) set for the USA and Canada reflected this age effect by having a higher recommendation for persons over 50 years compared to younger adults [42]. In the recently published DRIs, the elderly (>70 y) have a higher RDA of 800 IU compared to those 1 through 70 y (600 IU); however the EAR value of 400 IU is similar for all ages > 1 year [144] (Table 54.2). As one ages, there is a lessened ability to make vitamin D3 in skin due to lack of the precursor 7-dehydrocholesterol [55]. However, skin synthesis may not be the only determinant to status. Other factors such as reduced absorption of dietary sources of vitamin D may also be involved [43]. Once synthesized, dietary vitamin D binds to vitamin-D-binding protein (DBP), responsible for transporting this parent compound in the blood. As transport is dependent on the synthesis of DBP, this process may be limited in the elderly due to the lower rate of hepatic protein synthesis with increasing age [58]. Thus, it is important to acknowledge poor skin synthesis as well as decreased absorption and transport of vitamin D in the elderly. Furthermore, recommendations used in this chapter do not apply to older adults unless explicitly stated as such. Body Size Body size is also a factor for setting vitamin D requirements, but there is still controversy about the meaning of FIGURE 54.1 Time (minutes) of unobstructed sun exposure at 42.5 N first day of summer, in order to make 1000 IU vitamin D when 25% of skin surface area is exposed, by skin type (see Table 54.2 for detail of Fitzpatrick scale IeVI). Data derived from [29].
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this relationship. For example, using large American surveys such as Framingham [59] or the National Health and Nutrition Examination Survey (NHANES), adipose tissue levels or the body mass index (BMI) category were associated with 25(OH)D levels [59]. This relationship is not always seen in both men and women [60], nor is it stable through all seasons [61]. Some of the confounders include: lack of physical activity, which itself is associated with vitamin D status [62]; lack of sun bathing habit compared to leaner individuals [63]; and sequestering of vitamin D in adipose cells [64]. That vitamin D requirements might be a function of body size has not yet been put in practice. The level long recognized for infants, 10 mg (400 IU), is a much higher dose per kg body weight than levels currently recommended for adults (Table 54.2).
Environmental Factors Sunlight contributes UVB and UVA radiation, but only UVB promotes vitamin D3 synthesis. As previously described, exposure to sunlight can be quantified in erythemal doses, i.e. the appearance of reddening of the skin. A minimal erythemal dose (1 MED) causes reddening, and further exposure results in more severe sun burning [27]. Tanning (the induction of melanin synthesis in the skin) also occurs but takes longer to manifest and occurs with UVA as well as UVB exposure. Many factors can prevent or promote a person’s exposure to UVB, and thus vitamin D synthesis may not occur even with ideal conditions. For example, as shown in Table 54.4, wearing clothing that covers most or all of the body surface area can prevent conversion of 7-dehydrocholesterol to previtamin D3 [37]. Thus it is important to understand factors affecting vitamin D3 synthesis as these appear to be key to understanding the growing epidemic of vitamin D deficiency that exists worldwide. Latitude Latitude is the major determining factor for intensity of UVB irradiation e whether in the southern or northern hemisphere. The effect is not entirely linear as altitude, wind currents, smog, and other factors can disturb how much UVB reaches the ground. At the equator, vitamin D can be made year-round even in darker-pigmented skin. For those living above 37 N or below 37 S , there are approximately 4 months of vitamin D winter; at latitude 42 there are 5 months, and close to the poles there would be no time during the year when vitamin D synthesis in the skin could occur [29,32]. Yet, not all analyses of vitamin D status and latitude show the expected relationship [65]. In one large meta-analysis, only Caucasians showed a significant latitude effect of reducing 25(OH)D by 0.69 nmol/l per degree latitude north or
987
south away from the equator [66]. There is now considerable vitamin D deficiency and insufficiency in people living near the equator, in countries of the Middle East, southern Europe, and in India which will be discussed below [1,21,67,68]. Season is linked to latitude as during winter, the earth tilts away from the sun; subsequently the angle of the sun reaching the ground is too low in the sky for UVB radiance to be effective. Vitamin D winter can be defined as a UV Index of 0.5 or less [29]. The UV Index was developed by the United States National Weather Service to quantify the strength of solar UV radiation on a scale from 1 (low) to 11þ (extremely high) (http://www.epa. gov/sunwise/uvindex.html). Factors contributing to a low UV Index include: a high ozone level, a low solar zenith angle at noon, greater cloud cover, low altitude. Further details of these factors are provided in Table 54.5. However, little vitamin D is made between index values of 0.5 and 3, although the public is encouraged to take advantage of times when the UV Index is less than 3 to get sun exposure for vitamin D synthesis [69]. The effect of season can be demonstrated in Caucasians who are more likely to receive benefit from summer sun exposure. The data presented in Table 54.1 indicate that European-American subjects with either high or low sun exposure lifestyles would display seasonal variation in 25(OH)D levels. A seasonal effect is also seen in African-American data if there is high sun
TABLE 54.5 Environmental Factors that Affect Vitamin D Synthesis from Sun1 Factor
Effect on vitamin D synthesis
Solar zenith angle
At low angles, UVB photons are more likely absorbed by ozone or reflected back in space, thus attenuating UVB reaching the ground Rule of thumb: a UV index less than 3 provides little or no vitamin D synthesis
Clouds
More likely there is attenuation of UVB, by as much as 99% if completely overcast; broken clouds may enhance UVB radiation
Ozone
Absorption of UVB occurs
Surface reflection
UVB radiation can be reflected, especially by snow
Altitude
UVB radiation increases by ~7% per km in altitude if cloudless and more if in or above clouds
Windows
Windowpane glass absorbs ultraviolet B radiation
Sunscreen
Sunscreens block most UVB (SPF 8 or greater) Sun protection factor (SPF) indicates amount of UV blocked: SPF blocks at 1/SPF, e.g. SPF of 8 would allow 1/8 (~12%) of UV to penetrate
Information from [33], [71] and [138].
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54. NUTRITION AND LIFESTYLE EFFECTS ON VITAMIN D STATUS
exposure. However, the African-American data indicate a lack of a significant seasonal effect for those with low sun exposure. When vitamin D status is measured at a high latitude or when subjects have traveled to sunny destinations or when a sun tanning bed is used are excluded from analysis, there is a measurable decline in subjects of all ethnicities between early fall and midwinter [70], as shown in Fig. 54.2. Thus, studies that fail to show a seasonal or latitudinal effect are ones in which other lifestyle or environmental factors have intervened in terms of the expected amount of sun (or UVB) exposure subjects receive during the seasons. Other Environmental Factors Other factors that prevent UVB from reaching the skin are described in Table 54.5. The angle of the sun relates to latitude and seasonality, as described above. Weather influences vitamin D synthesis by blocking UVB penetration, although scattered clouds may have the opposite effect. The assumptions (Fig. 54.1) for the calculation of exposure time to provide vitamin D synthesis assume a cloudless day [29]. The ozone layer is an important protective layer that can filter out much of the UVB and all of the ultraviolet C (UVC) radiation entering the atmosphere. The assumption of the calculation of ¼ MED to provide vitamin D synthesis assumes a cloudless day [29]. Weather can also keep East Asian European South Asian Total Sample
80
Serum 25(OH)D (nmol/L)
70
60
50
40
30
20 Fall
Winter Season
FIGURE 54.2 Effect of season on mean serum 25(OH)D concentrations for the total sample and for each of three ancestry groups comprising young adults (age 18e35 years) living in Toronto Canada (43 N). From [70].
people indoors even if the weather can promote vitamin D synthesis. Very high temperatures and humidity in the summer months may keep people indoors or covered up much of the day [21]. In attempts to prevent overexposure to sun, people may apply sunscreen which, if properly applied, can block almost all vitamin D synthesis [71].
Social and Cultural Factors Living and Workplace Environment Where one lives and works may be a factor in degree of sun exposure an individual may obtain. One way to illustrate how this difference in exposure can impact on vitamin D status is through comparing urban versus rural living, or by comparing the nature of occupation (outdoors or indoors). In India there are marked differences in vitamin D status of Indian farmers versus factory workers [21]. Figure 54.3 illustrates that those in rural areas of India have higher 25(OH)D levels than urban dwellers, and the effect is most noticeable in men, presumably who are outdoor workers. Studies in tropical areas have measured urban workers while others have measured rural dwellers. The former, such as garment factory women in Bangladesh, have 25(OH)D levels averaging only 35 nmol/l (14 ng/ml), despite year-round sunshine [72]. It is estimated that in Bangladesh over 1.5 million work in that industry, often including 14-hour workdays. The latter, such as women farmers in the Gambia, have 25(OH)D levels well above 75 nmol/l (30 ng/ml) [41]. There are many reasons for the urbanerural differences beyond the obvious time spent in the sun. For urban dwellers, there may be a high degree of pollution. This was examined in a study of young children living in the Mori Gate district of Delhi, a high pollution area, who had 25(OH)D concentrations less than half that of an age-matched control group living in a lowpollution area [73]. Office workers have few opportunities for sun exposure due to long hours working indoors [74]. High temperatures and humidity in the summer months and avoidance of skin tanning for cosmetic reasons may also keep people indoors or covered up much of the day. In temperate Western countries, lifestyle differences in sun exposure are seen in leisure activities. There is a correlation between physical activity and vitamin D status [75]. In a study of older adults (>60 years), it was found that for every extra hour of outdoor activity there was an increase in 25(OH)D of 1.8 nmol/l [76]. Being outside in the sun for longer periods and wearing sports/casual clothing, such as the high sun exposure characteristics shown in Table 54.1, are factors that promote a better status. However, there are many
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989
Plasma 25(OH)D (nmol/L) in populations from India showing the distribution between rural and urban centers [21]. Line shows cut-off where rickets/osteomalacia occurs.
FIGURE 54.3
confounders; hence it may not be possible to make broad conclusions about outdoor work without full information on clothing, sunscreen use, and possibly even sun avoidance behaviors such as wearing protective clothing and headwear. Clothing Clothing can prevent UVB penetration, which can influence the vitamin D status of people in almost any country. When concealing clothing is worn most of the time, as illustrated in Table 54.1, a considerable amount of dietary vitamin D is needed to prevent deficiency. In many tropical countries it had been assumed that vitamin D is easily made in the skin, yet recent studies now show that commonly worn concealing clothing has a huge effect on vitamin D status [77]. In particular veiling, in which Muslim women wear modest, concealing clothing, is associated with lower vitamin D status (Table 54.6) in any season measured. Veiling is a plausible explanation for low vitamin D status in India where rickets has been found to be three to four times more common in the children of Muslims than Hindus, the latter group wearing less concealing veils than the former [77]. Not all studies show a difference in vitamin D status with veiling; among Bangladeshi women living in Dhaka, no difference in 25(OH)D concentrations was reported between veiled and nonveiled women (Table 54.6). In addition, there are behaviors such as intentional avoidance of sun such as staying indoors or in the shade on very hot days, which are reasons why people in tropical countries may have poor vitamin D status. Cultural Practices and Sun Exposure Preferences Two cultural practices that can lead to vitamin D deficiency are avoiding sun exposure to prevent skin from tanning, and avoiding sun exposure for prevention of skin cancer. In some cultures, many people avoid sun exposure likely due to the association of a tanned skin with performing outdoor chores or
having to work at outdoor occupations, either of which would be indicative of low social standing. Most studies in Asia show levels of 25(OH)D that are surprisingly low given the year-round ability to make vitamin D in skin [77]. In a study of older residents of Hong Kong (>50 years), 62% responded that they did not like going in the sun, and almost half used a parasol to shade themselves from the sun [78]. The authors were unable to determine the reasons for sunlight avoidance, but it can be speculated that the current cultural trend (prevalent among many Asian female populations) to have paler skin is a contributing factor for reducing sunlight exposure among the younger age groups in the survey. In a study of South Asian women living in New Zealand who were not getting sufficient sun exposure to synthesize adequate levels of vitamin D, subjects’ concerns about the strength of the New Zealand sun and the risk of skin cancer were the most prevalent reasons for avoiding sun. Although 29% of respondents said that they would not spend more time in the sun, 49% indicated they would spend more time in sun if they were not afraid of skin cancer [74]. The relationship between sun exposure and skin cancer is complex. Sun exposure, including UVB, induces formation of nonmelanoma cancers as well as skin aging [27]. As for melanoma, the relationship is less clear cut. Recent studies indicate the use of some sunscreens that absorb UVB but allow UVA to penetrate the skin. Sunscreens have included chemicals that absorb UVA only since 1989 [79]. As shown in Table 54.5, sunscreens effectively block vitamin D synthesis in the skin and so their use dictates the necessity of providing dietary vitamin D. In recognition that their sun avoidance message could be causing vitamin D deficiency in Canadians, the Canadian Cancer Society issued a recommendation in 2007 for vitamin D supplement use as shown in Table 54.2. The subject of sunlight and skin cancer is discussed in Chapter 89 and sunlight protection is discussed in Chapter 100.
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TABLE 54.6
Studies Showing Levels of 25(OH)D of Adults Where Skin Exposure was Varied due to Clothing Practices 25-Hydroxyvitamin D
Location and latitude
Sample characteristics
Season
Women in Western clothing
Spring
Mean (nmol/L)
MIDDLE EAST Ataturk 41 N
Women with only face and hands unclothed
Adana 37 N
Women in Western-style clothing
15 Summer
Women with only eyes exposed
Ankara 40 N
Winter
Women with only face and hands unclothed Amman Jordan 32 N
135 83
Women in Western clothing
56
Women wearing Western-style clothing
64 34
Summer
37
Women with face and hands exposed
28
Women fully covered
24
SOUTH ASIA Dhaka Bangladesh
Non-veiled women
Equivalent to summer
23 N
Veiled women
Kuala Lumpur Malaysia
Malay women e face and hands exposed
2 N
Chinese women wearing Western-style clothing
Khon Kaen Thailand
Older urban women
16 N
Older suburban women
30
32 Equivalent to summer
43
58 Equivalent to summer
79
90
Modified from [77].
Assessing Vitamin D Synthesis from Sun Exposure There are several ways to measure sun exposure and then make extrapolations to vitamin D synthesis. These include measures of skin reflectance as a quantitative method for measuring skin pigmentation, which can be inherited (constitutive) or UV-induced synthesis of melanin (facultative). Assumptions would then be made about potential for vitamin D synthesis. One can measure UVB exposure over time by using polysulfone dosimeter film badges placed on subjects. These objective measurements can also be used to validate subjective questionnaires assessing sun exposure. The following sections provide details of these techniques. Objective Measures of Sun Exposure to Estimate Cutaneous Synthesis of Vitamin D Exposure to UVB can be assessed using polysulfone dosimeter film badges. They consist of a polysulfone film layer mounted in a holder with a central aperture
measuring 1 cm2 [37]. In a recent study, subjects wore badges during daylight hours on their right wrist. The badge measurements were then adjusted for body surface area exposed to the sun as estimated in Table 54.4 [37]. UVB exposure is found after calibrating to the source spectrum, which is determined by placing badges in full sun the same day as measurements are taken. By multiplying the badge measurement (J/m2) by the mean area (m2) of exposed skin during time in the sun, investigators obtained an estimate of individual dose of sun exposure (J) received on that day [37]. An MED is equivalent to 250 J/m2. The error in the usage of polysulfone film as UV dosimeters is of the order of 10% [65]. One limitation of these films is that UVB light outside the range required for previtamin D3 formation is included. Therefore, polysulfone film is more useful for obtaining relative UVB exposures for each subject on a given day than an absolute objective measure of effective UVB for previtamin D3 synthesis [80]. A second measurement to estimate sun exposure is measuring skin pigmentation using reflectance
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spectrophotometry. Low reflectance represents low melanin content while high reflectance represents high melanin. A spectrophotometer assigns L and B values which represent the relative brightness of color (ranging from black to white) and degree of pigmentation, respectively. Skin pigmentation is best described by the Individual Typology Angle (ITA ): ITA ¼ Arc Tangent [(L 50)/B)] 180/p. The lower the ITA the darker the skin color [81]. When two body sites with differing sun exposure are measured, it is also possible to estimate the degree of tanning, i.e. melanin production in response to UV radiation. For example, Hall et al. [37] measured the middle upper inner right arm, the dorsum of the right hand between the thumb and index finger, and the middle of the forehead of subjects. Constitutive skin pigmentation is what is naturally present (i.e., due to genetics) while facultative skin color is that which results from sun exposure. When two sites with differing sun exposure (i.e., no exposure would be inner upper arm and high exposure the forehead or wrist) are measured, one can determine the extent of tanning. Different authors have reported one or the other skin color measurement is related to 25(OH)D levels. Gozdzik et al. [23] found constitutive skin pigmentation to be related to vitamin D status in measurements completed during winter. Rockell et al. [48] found facultative skin color to be a better indicator of vitamin D status during summer. Use of Questionnaires to Estimate Sun Synthesis of Vitamin D In 2008, McCarty [82] reviewed the question of whether vitamin D status could be assessed using sunlight questionnaires. Correlations between selfreported sun exposure and dosimeter data were generally low in the literature, although they did reach statistical significance. Few studies were available to validate questionnaires with measures of 25(OH)D, and her conclusion was that these questionnaires were too imprecise to be of value. Precision of questionnaires can be improved by carefully noting the time of day sun exposure occurred. In a study of Caucasian girls [80] activity diaries that included time spent outdoors were also adjusted for the relative strength of the UVB radiation. The study investigators found that in Maine (44.5 N) in the summer, sun exposure was not strong enough for the maximum conversion of 7-dehydrocholesterol to previtamin D3 [80]. A simple sun exposure questionnaire for Caucasian subjects at 40 N was shown to be valid in a recent publication [83]. In that study, a recall questionnaire assessed time in the sun during short time periods (<5 min, 5e30 min, >30 min) and also the amount of skin exposure (face/hands; face/hands and arms; face/hands and legs; and “bathing suit”) during a
991
1-week interval. A Sun Exposure Score was calculated and compared to 25(OH)D levels in both summer and winter. As expected, summer but not winter Sun Exposure Scores were significantly correlated with vitamin D status. While time in the sun was more important than the amount of skin exposure reported, both were necessary to accurately reflect potential for vitamin D synthesis. There is no single answer to how much sun exposure is needed to achieve and maintain an adequate vitamin D status. Some dermatologists have advocated for no sun exposure [27]. Nevertheless, the World Health Organization (WHO) Report [84] on solar UV radiation indicates that it is not appropriate to strive for zero sun exposure as this would create a huge burden of skeletal disease from vitamin D deficiency. It is important, however, to avoid excess exposure since this has been linked with skin cancers and skin photoaging. A rational scheme to achieve skin synthesis of vitamin D without significant risk of overexposure has been published [85] as shown in Fig. 54.1. While this subject remains a source of considerable controversy, the public now has access to information that will allow weighing of personal risk and benefits of sun exposure.
NUTRIENT INTAKE AS DETERMINANT OF VITAMIN D STATUS There is evidence which confirms that increasing dietary vitamin D intake will safely increase circulating 25(OH)D, the main status indicator. This was recently established in a systematic evidence-based review that specifically focused on the relation of vitamin D to bone and muscle health [86]. In one aspect of this meta-analysis, the authors examined the effect of vitamin D supplementation and food fortification on 25(OH)D concentrations, reporting a significant association between intake (either vitamin D2 or vitamin D3) and 25(OH)D concentration, as shown in Table 54.7. They found good evidence of a positive effect of vitamin-D-fortified food which varied in magnitude from 15 to 40 nmol/l; however, they could not determine if the positive effect varied by age, body mass index, or race/ethnicity. They determined a 1e2 nmol/l increase in 25(OH)D for each additional 100 IU of vitamin D which provides important evidence of the efficacy and safety of using diet to increase 25(OH)D [86].
Assessing Vitamin D Intake Relevant to understanding dietary recommendations is determining whether current dietary vitamin D intakes from food meet or exceed a country’s
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992 TABLE 54.7
54. NUTRITION AND LIFESTYLE EFFECTS ON VITAMIN D STATUS
Impact of Food Fortification on Vitamin D Status
Study group
Food fortified
Additional vitamin D intake with fortification mg/d (IU/d)
Absolute mean change in 25(OH) D relative to controls nmol/L
Elderly adults mean age 78 y
Milk
5 (200)
15.5
Young adults 17e54 y
Milk powder
3.4 (136)
16.0
Postmenopausal women mean age 59 y
Skim milk
10 (400)
14.5
Ambulatory men >49 y
Milk
20 (800)
18.6
Elderly adults mean age 79 y
Fruit/dairy products
10 (400)
30.0
Women 25e45 y
Bread
10 (400)
16.6
Adults 19e60 y
Orange juice
25 (1000)
34.5
Modified from [105].
recommendations. Relatively few studies take on the task of assessing vitamin D intake and when they do, almost all of them are largely inaccurate in their estimates of true vitamin D intake. This fact is attributed to several factors: the inaccuracies inherent in the methods used to determine food intake (e.g., Food Frequency Questionnaire, 24-hour recall, multiple-day food record) [87]; the large variability and incomplete nature of nutrient composition databases used to quantify vitamin D intake [88,89]; the variability in content of natural food sources due to seasonality [87]; the variation in dietary supplement use [34,35]; or the inconsistency in food fortification practices [34,35]. In national surveys, a 24-hour recall of all foods and beverages is the usual method for dietary assessment. This has the advantage of allowing for all sources of vitamin D to be included, as it is open-ended with respect to gathering food intake information. However, a single 24-hour recall does not provide the habitual intake of an individual. Studies where a more precise estimate of dietary intake is required must increase the number of days of collection, e.g. for 1 week. Many epidemiological studies employ food frequency questionnaires (FFQs) as these tools, compared to other methods for assessing food consumption of individuals, have lower respondent burden and greater ease of data analyses. An FFQ designed for assessment of vitamin D intake of a multiethnic group of healthy young Canadian adults was validated during the winter in order to utilize the biomarker of vitamin D status, 25(OH)D [90]. Additionally, 7-day food diary records provided open-ended estimations of total vitamin D intake. Because the study was completed in winter, it was consistently shown that dietary intake was significantly associated with serum 25(OH)D levels. While the FFQ itself provided valid estimates of intake such an instrument would need to be designed and validated for use
in different countries due to variability in fortification practices.
Dietary Recommendations for Vitamin D Since the early 1940s, the USA and Canada have set dietary intake recommendations for nutrients. In the mid 1990s, Canada and the USA worked jointly on the Dietary Reference Intakes (DRI) process, setting nutrient recommendations together for the first time [42]. The 1997 recommendations for vitamin D were set as Adequate Intake (AI) values, denoting the lack of scientific evidence needed to set Recommended Daily Allowances (RDAs) in the DRI process at that time [42]. Those 1997 AIs were based on maintenance of serum 25(OH)D levels in the absence of sunlight (i.e., through the winter) at or above 27.5 nmol/l for most age groups [91]. It was acknowledged that a dietary intake should maintain serum 25(OH)D above the concentration below which vitamin D deficiency rickets or osteomalacia occurs in the absence of sun exposure. A serum level of 25(OH)D that indicates optimal status is between 75 and 110 nmol/l (30 and 44 ng/ml) is based on analysis of recent studies including randomized trials [16]. Previously the threshold value used in establishing dietary guidelines by the USA and Canada of 27.5 nmol/l (11 ng/ml) and the UK of 25 nmol/l (10 ng/ml) was related only to bone health and is just above the range indicative of frank vitamin D deficiency observed in rickets or osteomalacia [42]. The threshold value of 25(OH)D required for optimal overall health is the most critical information needed [7]. Thus it is necessary to set a recommended nutrient value for vitamin D to reflect an amount that will bring 25(OH) D to the desired level. The Institute of Medicine has been charged by American and Canadian governments to develop
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NUTRIENT INTAKE AS DETERMINANT OF VITAMIN D STATUS
new Dietary Reference Intake values in 2010 for vitamin D with consideration of these objectives: (1) review evidence on indicators of adequacy and on indicators of adverse effects from excess intake relevant to the general US and Canadian population, including for groups whose needs for or sensitivity to the nutrient may be affected by levels of skin pigmentation, and by particular conditions which are widespread in the population such as obesity or age-related chronic diseases but excluding special groups under medical care whose needs or sensitivities are affected by rare genetic disorders or diseases and their treatments; (2) consider systematic evidenced-based reviews including those made available by the sponsors as well as others and carefully document the approach used by the committee to carry out any of its own literature reviews; (3) regarding selection of indicators upon which to base DRI values for adequate intake, give priority to selecting indicators of adequacy for the various age, gender, and lifestage groups that will allow for the determination of an Estimated Average Requirement (a value that meets the needs of 50% of the population); (4) regarding selection of indicators upon which to base DRI values for upper levels of intake, give priority to examining whether a critical adverse effect can be selected that will allow for the determination of a benchmark intake; (5) update DRI values, as appropriate, using a risk assessment approach that includes (a) identification of indicators of adequacy and hazard, (b)
TABLE 54.8
selection of the indicators of adequacy and the critical adverse effect, (c) intake-response assessment, (d) dietary intake assessment, and (e) risk characterization; and (6) identify research gaps to address the uncertainties identified in the process of deriving the reference values and evaluating their public health implications [92]. The 2010 DRI recommendations are shown in Table 54.2.
Intakes, Food Patterns and Dietary Sources of Vitamin D National Intakes Vitamin D intakes from foods recently estimated from the nationally representative survey NHANES conducted in the USA vary with gender and age (Table 54.8) [36]. There are higher intakes in men than women when food sources alone are considered and an increase in vitamin D intake with increasing age when supplements use is common. Much of the intake of vitamin D from foods in the USA is from fortified foods [93]. This explains why intake from foods is higher in the USA, as much as twice the intake in the UK, where few fortified foods are in the marketplace. Supplement use contributed to vitamin D intake in the USA, adding 1 to 2 mg (40e80 IU) in each group except for women and men over 50 years who showed an increase of approximately 3e6 mg (120e240 IU) [36]. In contrast, total vitamin D intakes in the UK rose only 0.1e0.7 mg (4e28 IU) [94].
Vitamin D Intake of Adults by Age and Sex According to Intake from Foods or Foods and Supplements (all sources) in Three Scenarios: a Country with Fortification Permitted and Supplement Available (USA) or these Sources Limited (UK)
Mean daily vitamin D intake, mg/da Young Country women
Young men
Mid-adult women
Mid-adult men
Older women
Older men
Many foods fortified and supplement use available without prescription 19e30 y
USAb
31e50 y
51e70 y
Food Sources
3.6 (0.3)
5.1 (0.3)
4.4 (0.3)
5.4 (0.3)
3.9 (0.4)
5.1 (0.3)
All Sources
5.8 (0.3)
6.6 (0.4)
7.7 (0.5)
7.9(0.3)
10.1 (1.0)
8.8 (0.4)
Some (limited) food fortification and supplement use available without prescription 19e24 y
UKc
35e49 y
50e64 y
Food Sources
2.3 (1.6)
2.9 (1.5)
2.8 (2.1)
3.7 (2.3)
3.5 (2.4)
4.2 (2.4)
All Sources
2.9 (2.5)
3.0 (1.6)
3.5 (2.9)
4.2 (3.1)
5.1 (4.1)
4.9 (3.2)
a
Values are from nationally representative surveys and presented as mean (SEM) except Ireland where values are mean (SD). US values from the 2003e2006 National Health and Nutrition Examination Survey [139]. c UK values from the 2000/01 National Diet and Nutrition Survey: adults aged 19 to 64 years [140]. b
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54. NUTRITION AND LIFESTYLE EFFECTS ON VITAMIN D STATUS
TABLE 54.9 Lawful Additions of Vitamin D to Foods in the USA. Foods are Separated According to Addition of Vitamin D2 and/or D3 Category of food
21 CFR citation
Maximum level allowedamg vitamin D2 or D3 (IU vitamin D2 or D3)
Enriched Farina
137.305
8.7 5 mg (350 IU)/100 g
Ready-to-eat breakfast cereals
137.305
8.75 mg (350IU)/100 g
Enriched rice
137.350
2.25 mg (90 IU)/100 g
Enriched cornmeal products
137.260
2.25 mg (90 IU)/100 g
Enriched noodle products
139.155
2.25 mg (90 IU)/100 g
Enriched macaroni products
139.115
2.25 mg (90 IU)/100 g
Fluid milk
131.110
1.05 mg (42 IU)/100 g
Acidified milk
131.11
1.05 mg (42 IU)/100 g
Cultured milk
131.112
1.05 mg (42 IU)/100 g
Concentrate milk
131.115
1.05 mg (42 IU)/100 g
Non-fat dry milk, A & D fortified
131.127
1.05 mg (42 IU)/100 g
Evaporated milk, fortified
131.130
1.05 mg (42 IU)/100 g
Dry whole milk
131.147
1.05 mg (42 IU)/100 g
Yogurt
131.200
2.22 mg (89 IU)/100 g
Low-fat yogurt
131.203
2.22 mg (89 IU)/100 g
Non-fat yogurt
131.206
2.22 mg (89 IU)/100 g
Cheese
133.165, 133.183
2.22 mg (89 IU)/100 g
Margarine
166.110
8.3 mg (331 IU)/100 g
Maximum level allowed b mg vitamin D3, (IU vitamin D3) except for soy foods where amount is vitamin D2 Calcium-fortified 100% fruit juicec
172.380
2.5 mg (100 IU)/240 mL
172.380
2.5 mg (100 IU)/240 mL
Soy-protein meal replacement beverage
172.380
3.5 mg (140 IU)/240 mL
Meal-replacement and other type bars
172.380
2.5 mg (100 IU)/40 g
172.380
2.02 mg (81 IU)/30 g
Calcium-fortified fruit juice drinks
d e
f
g
Cheese and cheese products
Maximum level allowed as mg vitamin D2 (IU vitamin D2)h Soy beverages
172.379
1.25 mg (50 IU)/100 g
Soy beverage products
172.379
2.23 mg (89 IU)/100 g
Soy-based butter substitute spreads
172.379
8.25 mg (330 IU)/100 g
Soy-based cheese substitutes and products
172.379
6.25 mg (270 IU)/100 g
a
Maximal level of vitamin D that can be added in accordance with 21 CFR 184.1(b)(2) for the category of food. b Vitamin D3 may be used safely in foods as a nutrient supplement in accordance with 21 CFR 172.380 at levels not to exceed those specified above. c Vitamin D3 may be added, at levels not to exceed 100 IU per 240 mL serving, to fruit juice drinks that are fortified with >10% of the RDI for calcium. Excludes fruit juice drinks that are specially formulated or processed for infants. d Vitamin D3 may be added at levels not to exceed 100 IU per 240 mL serving, to fruit juice drinks that are fortified with >10% of the RDI for calcium. Excludes fruit juice drinks that are specially formulated or processed for infants. e Vitamin D3 may be added at levels not to exceed 140 IU per 240 mL of soy protein-based meal replacement beverage (powder or liquid) used for special dietary purposes, weight reduction or maintenance at levels not to exceed 100 IU per 40 grams. f Vitamin D3 may be added to meal replacement and other bars used for special dietary purposes, weight reduction or maintenance at levels not to exceed 100 IU per 40 grams. g Vitamin D3 may be added to cheese and cheese products, excluding cottage cheese, ricotta cheese, and hard grinding cheese such as Parmesan and Romano, at levels not to exceed 81 IU per 30 grams. h Vitamin D2 safe use as a nutrient supplement in soy-based food products pertains only to the use of crystalline vitamin D2 and not the resin form of the vitamin.
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NUTRIENT INTAKE AS DETERMINANT OF VITAMIN D STATUS
TABLE 54.10
Differences in Vitamin D Status, Intakes, Food Sources and Supplements use from the Third National Health and Nutrition Examination Survey, 1988e1994, by Race White adults (n [ 6456)
Black adults (n [ 4316)
79.0 0.95
48.2 1.05*
All sources, mg/d
7.92 0.15
6.20 0.13*
Supplements only, mg/d
2.84 0.14
2.0 0.11*
Fluid milk, milk-based drinks, mg/d
1.99 0.06
1.01 0.44*
Plain fluid milk, mg/d
1.86 0.06
0.92 0.04*
Ready-to-eat breakfast cereals, mg/d
0.39 0.02
0.23 0.04*
Serum 25(OH)D, nmol/La Vitamin D intake:
Weighted mean SEM; adapted from [34]. Includes samples taken at all latitudes during both summer and winter, thus not controlled for sun exposure. * Significantly lower in black adults; p < 0.0001.
a
When considering intakes from food only [36], vitamin D intakes of those 50 years and younger met the previous Adequate Intake (AI) of 5 mg (200 IU) for 21e72% of the group; for those older than 50 years having AIs of 10 mg (400 IU)e15 mg (600 IU), less than 10% met the AI. When supplement intake was considered, intakes of those 50 years and younger met the current AI for 32e80% of the group; for those older than 50 years, 22e44% met the AI. Thus it is clear the food supply in the USA, despite having fortification permitted in many foods (Table 54.9), is not adequate to provide much more than 10 mg (400 IU) per day. As vitamin D supplement use is practiced by approximately only 40% of the population, this amount of fortification is not effectively reaching everyone [36]. Significant racial and ethnic differences in vitamin D nutritional status and intake can occur in a population. In Table 54.10, it can be seen that black men and women in the USA have significantly lower serum 25(OH)D than whites, and consume significantly lower vitamin D from milk, ready-to-eat cereals, and dietary TABLE 54.11 Ethnic group
supplements. Those in greatest need of dietary sources of vitamin D due to aging and/or darker skin have the lowest intakes of vitamin D, thus further contributing to their low circulating 25(OH)D. Table 54.11 shows that supplement use is much less in non-white Americans, and that this likely contributes to the poor vitamin D status. Black and Hispanic adults also have reduced potential for sun exposure, and as shown in Table 54.1 would need more dietary intake than white Americans. Similar low intakes and poor vitamin D status are observed in Canadian Aboriginal populations, especially among urban dwellers who no longer consume traditional diets, and among darker-skinned Canadians of Asian ancestry who tend to consume traditional vegetarian or vegan diets [23,95]. Food Composition Data The completeness of the food composition database used to estimate intake significantly influences national estimates of vitamin D intake. The USA has recently updated food composition data for vitamin D content in the US Department of Agriculture (USDA) database Standard Reference (SR) which is the authoritative source of food composition data for the USA [96]. The USDA National Nutrient Database was recently updated to incorporate the vitamin D content of nearly 3000 food entries, including mushrooms. Release 22 of the USDA SR (SR22) (http://afrsweb.usda.gov/ SP2UserFiles/Place/12354500/Data/SR22/sr22_doc. pdf) contains vitamin D values for approximately 3000 foods of the total 7520 food items listed. Canada also revised their nutrient file and has included items such as beef, lamb, veal, pork, and poultry products (Health Canada, personal communications). Applications of new analytical methods reveal measurable levels of vitamin D and its metabolite 25(OH)D in various meats shown in Table 54.12 [97]. Updated nutrient database and newly analyzed vitamin D food content values for natural and fortified foods are shown in Table 54.12. Given the differences in foods analyzed using very different nutrient content databases, it is difficult to accurately compare Canadian and American intake
Racial/Ethnic Differences in Prevalence of Vitamin D Deficiency by Supplement Usea No supplement use Mean 25(OH)D nmol/L
Prevalence % below 70 nmol/L
Supplement users Mean 25(OH)D nmol/L
Prevalence, % below 70 nmol/L
Total USA
74.4
48
79.5
39
White
78.8
42
83.3
33
Black
48.0
86
54.6
77
Hispanic
62.7
66
66.2
60
a
Table modified from [141]. Mean values presented.
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
996 TABLE 54.12
54. NUTRITION AND LIFESTYLE EFFECTS ON VITAMIN D STATUS
Updated Nutrient Databases and Newly Analyzed Vitamin D Food Content Values from Natural and Fortified Sources
Trout, raw
1 mg D3 22 mg 25(OH)D
1.05 mg D3
Chicken
0.25 mg 25(OH)D
0.59 mg D3 0.74 mg 25(OH)D* 4.29 mg total D activity*
Lean pork
<0.15 mg 25(OH)D
Margarine (fortified)
7.2 mg D3
Food
Canada
Margarine (fortified)
13.25 mg D3
Soy milk (fortified)
1.05 mg D2
Milk (fortified) Pork
USA
Butter (non-fortified)
1.2 mg D3
Full-fat milk (non-fortified)
0.15 mg D3
Reduced-fat milk (non-fortified)
0
0.96 mg D3 0.38 mg 25(OH)D* 2.86 mg total D activity*
Mackerel, smoked
8.0 mg D3
Salmon, microwaved
8.7 mg D3
Eel
20 mg D3
Herring
17 mg D3
Salmon
15 mg D3
Sardine
11 mg D3
Mackerel
10 mg D3
Trout
8 mg D3
Oysters
8 mg D3
Anchovy
7 mg D3
Tuna
5 mg D3
Halibut
4.3 mg D3
Blue fish
7.0 mg D3
Cod
2.6 mg D3
Gray sole
1.4 mg D3
Farmed salmon
6.18 mg D3
Wild-caught salmon
24.7 mg D3
Farmed trout
9.7 mg D3
Ahi-yt tuna
10.1 mg D3
Orange juice, calcium and vitamin D fortified
1.00 mg D3
Portabella mushrooms (raw, not UV light exposed)
0.3 mg D2
Portabella mushrooms (raw, UV light exposed)
11.5 mg D2
Shiitake mushrooms, raw
0.5 mg D2
Shiitake mushrooms, sundried
4.3 mg D2
Low-fat spread
2.20 mg D3
70% fat spread
1.98 mg D3
Cod fish, raw
1.49 mg D3
Premium ham
1.05 mg D3
France
* Assumes 5 activity factor. Reference sources: Canada [142], USA [30,34,103] and USDA nutrient values SR 22. Ireland [97,143], Netherlands [76], France [102].
estimates for vitamin D. For example, the recent Canadian Community Health Survey, 2004, showed 2.3 mg out of a total intake of 6.72 mg for males 9 years and older and 1.74 mg out of a daily mean total of 5.42 mg for females were contributed by meat and meat alternatives. In Canada meat contribution to vitamin D intake was second only to the contributions from fortified milk products [98]. Natural Food Sources
Chicken breast, breaded, 1.11 mg D3 fried Turkey slices
Netherlands
0.80 mg D3 <0.02 mg 25(OH)D* <0.90 mg total D activity*
Yogurt, selected fortified 2.5 mg D3 brands
Ireland
Vitamin D content (mg/100 g) 4 mg D3 11 mg 25(OH)D
Vitamin D content (mg/100 g)
Eggs, large
Food Salmon, raw
Country of origin
Rainbow trout
Country of origin
2.17 mg D3 (Continued)
Traditionally less attention has been given to improving the quantity or content of vitamin D in foods naturally rich in vitamin D2 or D3. In Canada and the USA, such foods are limited and not frequently consumed. Fatty fish represent the richest natural source
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of vitamin D commonly consumed in North America. Evidence from Japan and Norway illustrates how frequent consumption can be an effective way to maintain healthy 25(OH)D levels. Fish consumption contributes 1.5 to 1.8 mg/d to Norwegian daily intakes [99], 3.2 mg/d to Belgian adolescents [100], 6.4 mg/d to older Japanese women [101], and provides 25% of the French RDA for vitamin D [102]. Table 54.12 illustrates how rich a vitamin D source fish are. The vitamin D content of fish varies with season as to be expected, but also varies with environmental conditions and diet. Lu et al. [103] reported that vitamin D content of fish species may vary more widely. For example, they dispute the high value for vitamin D of mackerel that we report in Table 54.12 (10 mg/100 g), finding in their analysis only 0.6 mg/ 100 g. Further, they show that various cooking methods such as frying in oil can reduce vitamin D content by as much as half. Nevertheless, given that wild fish are generally a good source of vitamin D, this information would be helpful if displayed on the label. In the USA and Canada, food labels are not required to list the vitamin D content of foods naturally rich in vitamin D. Only foods that have been fortified with vitamin D are required to list it on the label. In both countries the vitamin D content is listed as a percent of the Daily Value (%DV). The DVs were developed by countries to help consumers compare the nutrient content of a product within the context of a normal diet, e.g. a 2000-calorie diet. Canada uses the value for the DV of 5 mg (200 IU), while a value of 10 mg (400 IU) is used on American labels. One possible solution to this source of confusion would be to have all countries adopt the method used by some countries where the vitamin D content per serving size is displayed in absolute amounts. Fortification Canadians and Americans are largely dependent on fortified foods and dietary supplements to meet their vitamin D needs because foods that are naturally rich in vitamin D are less frequently consumed. While Canada and the USA use the same dietary guidelines (DRI) for vitamin D and calcium and the same upper limits of safe intake for these nutrients, they have very different regulatory approaches to the lawful addition of vitamin D and calcium to foods [34]. Both countries recognize the potential for toxicity if vitamin D is consumed at very high doses and therefore both countries carefully regulate vitamin D addition to food. The Canadian approach is that of mandatory fortification of food staples through the Canadian Food and Drug Regulations [34]. Fortification of staple foods ensures nutritional benefit to all Canadians. Milk and milk alternatives and margarine are required to be
997
fortified in Canada. Fluid milk in Canada is labeled as providing 44% of the recommended daily intake (10 mg or 400 IU) per 250 ml serving [34]. Other milk products that require vitamin D fortification include: evaporated milk, powdered milk, goat’s milk, and milks of plant origin (soy, rice, and other grains), which also must be fortified with calcium [34]. All margarines in Canada are fortified with vitamin D at the level of 13.25 mg (530 IU) per 100 g [34]. Other foods for which vitamin D addition is permitted are meal replacements, nutritional supplements, and formulated liquid diets. Addition of vitamin D to such foods is optional but may be no less than 2.5 mg (100 IU) and no more than 10 mg (400 IU) per 1000 kcal, as long as the intended total energy intake is <2500 kcal [34]. Fortification of some egg products is also permitted at this time, but industrial milk used in baked goods and cheeses does not need to be fortified [34]. The approach to vitamin D fortification of foods in the USA is also very carefully regulated, but is largely optional for a number of food categories and is required only for fortified fluid milk and fortified evaporated milk (Table 54.9). The addition of vitamin D to foods as a nutrient supplement is in accordance with the US Code of Federal Regulations (CFR): 21 CFR 184.1(b)(2) which imposes strict limitations with respect to the categories of foods, functional use, and level of use [34] as shown in Table 54.9. Such regulatory limitations provide a control mechanism that limits over-fortification with vitamin D. In accordance with 21 CFR 184.1(b)(2), any addition of vitamin D to foods not in compliance with each of these established limitations requires a food additive regulation [34]. Recently, vitamin D3 may be added safely to foods as a nutrient supplement in accordance with 21 CFR 172.380 for those food uses presented in Table 54.9. The most recent fortification regulations concern the addition of vitamin D2 to soy and grainbased milks, and other products largely consumed by vegans and vegetarians are also shown in Table 54.9. In contrast to Canada, where fortification with vitamin D is mandatory for designated food staples, the lawful addition of vitamin D to eligible foods in the USA is voluntary in most cases, with the exception of fluid milk. Vitamin D fortification is required when the label declares that the milk is fortified. Although many food categories are eligible for controlled levels of vitamin D fortification in the USA, there is a large discrepancy between the number of eligible foods and the number and variety of vitamin-D-fortified foods currently in the US marketplace [34]. Fluid milk and ready-to-eat cereals are the major contributors to vitamin D intake in the USA, while milk and margarine are the major fortified foods contributing to Canadian vitamin D intake [35]. Significant racial/ethnic
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54. NUTRITION AND LIFESTYLE EFFECTS ON VITAMIN D STATUS
differences in vitamin D intake attributed to differences in the consumption of these fortified staples are shown in Table 54.10. There are marked differences in these fortified food consumption patterns between whites and blacks in the USA which are attributed to higher prevalence of lactose intolerance and low milk consumption in black men and women. Selective fortification of only a few staple foods that are not commonly consumed by individuals at greatest risk of poor vitamin D status is a major barrier to optimizing vitamin D intake. By encouraging US manufacturers to utilize these fortification options, the vitamin D intake of groups at risk could be significantly improved. Consideration could also be given to shifting optional fortification to mandatory for those eligible food staples commonly consumed by the entire US population. Cereal grain products (pasta, bread, and other baked goods) are frequently consumed by the general population and would serve as a good candidate for mandatory fortification with vitamin D [21,104]. Fortification of milk with vitamin D can result in a product with variable vitamin D content. As discussed by Holden et al. [96], several reports in the 1990s showed that fortified milk in the USA often fell below desirable fortification standards, many samples having very low levels, much less than the desired 400 IU per quart (0.95 l). In a 2001 survey of milk obtained throughout the country, two-thirds of samples of skim (no fat), and 1 and 2% (low fat) milks had levels of vitamin D between 300 and 600 IU per quart. Two samples were above 600 IU and two samples were below 300 IU [96]. While most vitamin D levels in these milks fell between 300 and 600 IU and showed no samples with little or no fortification (as was reported in the early 1990s), the data do indicate that fortification can result in variable amounts of vitamin D, a situation that needs to be considered when interpreting dietary assessment data. The effectiveness of fortifying foods other than milk and milk products with vitamin D must be demonstrated as the fat content of these foods may be low and thus be suspected of not allowing for vitamin D absorption. As shown in Table 54.7, there is good evidence that vitamin D is bioavailable from fortified foods [105]. These include: calcium-fortified orange juice [30], bread [106], cheese [107], and calcium-fortified milk [108,109]. There is also good evidence that vitamin D is bioavailable from natural food sources such as wild mushrooms or light-exposed mushrooms [110]. Discussion of vitamin D additions to infant formulas in countries such as Canada and the USA is beyond the scope of this chapter. Tangpricha et al. [30] performed two studies to determine the bioavailability of vitamin D in two non-fat beverages, skim milk and orange juice. The first study
compared the bioavailability of 625 mg (25 000 IU) vitamin D2 in whole milk, skim milk, and corn oil on toast. Each subject visited the research clinic on three different occasions, at least 2 weeks apart, and the sequence in which each subject received the three fortified foods was randomized. Serum was obtained at 0, 2, 4, 8, 12, 48, and 72 hours after ingestion of the fortified food to measure serum levels of vitamin D. The results found no significant difference in serum vitamin D concentrations between the three groups which suggests that fat is not necessary for vitamin D to be absorbed. The investigators then studied the feasibility of vitamin D fortification of orange juice. Two groups were included in the orange juice study: one group received 240 ml orange juice fortified with 350 mg calcium; and the other group received 240 ml orange juice fortified with 350 mg calcium plus 25 mg (1000 IU) vitamin D3. Subjects consumed one serving of the orange juice per day for 12 weeks. The group that received the orange juice containing vitamin D3 had significantly higher serum 25(OH)D levels than at baseline compared to the control group. In a fortification study using bread as the delivery medium, Natri et al. [106] provided 10 mg (400 IU) of vitamin D3 from fortified wheat or rye breads daily for 3 weeks to 41 healthy, middleaged women. The change in 25(OH)D in the two fortified-bread groups were compared with a group receiving unfortified wheat bread, and a group receiving unfortified wheat bread plus a 10 mg vitamin D supplement. The increase in 25(OH)D by subjects in the bread fortification groups was not significantly different from the group that received the same amount of vitamin D as a supplement. As is shown in Table 54.7, many foods may be considered candidates for fortification, thus allowing for cultural and religious preferences. Biofortification Biofortification is a term that normally refers to producing crops with higher levels of nutrients in foods through changes in plant breeding, thus modifying nutrient content pre-harvest rather than post-harvest or during food processing (the latter being traditional food fortification). In terms of vitamin D, one may expand this concept to consider those foods, whether plant, animal, or fungal/yeast, in which vitamin D levels are increased during growth or post-harvest. Two examples currently under development are light-exposed mushrooms and baker’s yeast and the addition of vitamin D (or its metabolite 25(OH)D) to animal feed. We offer a cautionary note with respect to vitamin D and changing agricultural practices which can work in the opposite direction of biofortification. Loss of vitamin D content in a food may occur with modern agriculture practices such as salmon farming. Farm-raised salmon
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
NUTRIENT INTAKE AS DETERMINANT OF VITAMIN D STATUS
have significantly lower vitamin D content than wildcaught salmon as shown in Table 54.12 [103]. IRRADIATION OF MUSHROOMS AND YEAST
Post-harvest light exposure techniques have been developed to significantly enhance the vitamin D content of fresh edible mushrooms [111]. A serving of portabella mushrooms without light exposure contains very little natural vitamin D2, approximately 0.3 mg (12 IU) in 100 g of raw mushroom (Table 54.13). A serving of UV-light-exposed portabella mushrooms can supply a fixed amount of vitamin D2, for example 11.5 mg (460 IU) of vitamin D2 in a serving (Table 54.12) by manipulating the length of time of UV exposure. This should represent an important natural food source of vitamin D2 for vegetarians and vegans. The concept of irradiating ergosterol from yeast or fungi with light is not new. This was the major source of vitamin D used to fortify milk and to treat rickets spearheading the public health campaign that successfully eradicated rickets in North America by the 1930s [32]. It is also possible to irradiate yeast with UV light, as was done originally to provide vitamin D for fortification purposes in the early 20th century. The company Lallemande (www.lallemande.com) has a patent pending process to convert ergosterol in the yeast to ergocalciferol (vitamin D2) while allowing the baker’s yeast to maintain leavening and flavor properties. Many different kinds of baked goods leavened with yeasts irradiated in this process may be produced in compliance with the regulatory levels of vitamin D allowed in grain products as shown in Table 54.9. FORTIFICATION OF ANIMAL FEEDS
In Canada meat contribution to vitamin D intake was second only to the contributions from fortified milk products [98]. Strategies are being developed for the natural enhancement of vitamin D content of animal foods through changes in diet composition [112] and the pharmacologic supplementation (vitamin D3 or 25(OH)D) of poultry and livestock feed to enhance meat and egg vitamin D content [113e115]. Eggs are considered as a dietary source of vitamin D, despite the fact that the amount of vitamin D in eggs is low (<1 mg per 100 g, see Table 54.12) and considering that this represents a daily two-egg serving which is generally not recommended or consumed. In the production of enriched eggs, vitamin D is added to hens’ feed, with studies showing an increase in vitamin D content of eggs up to seven times higher than regular eggs [116]. Hens fed up to 12 000 IU/kg food for 168 days showed no harmful effect on histopathological analyses or on the quality of eggs (e.g., emulsion capacity, gel-forming capacity, foaming properties, and eggshell strength) [117]. In this study, the final
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vitamin D content of the egg yolk was 22 mg/100 g or 880 IU/100 g. Further vitamin D activity is obtained when the level of 25(OH)D of the meat and eggs is taken into consideration. This metabolite is naturally present, as shown in Table 54.12. Although the bioequivalence of ingested 25(OH)D (calcidiol) is not known it may be as high as five-fold [97]. In human studies of calcidiol administration repletion of vitamin D deficiency proceeds more quickly [118]. Naturally occurring 25(OH)D levels and their biological equivalence to vitamin D are given in Table 54.12. In addition to naturally occurring 25(OH)D in some foods, calcidiol may be added to meat as a means of tenderizing. This addition to food must be evaluated in the final food products and also taken into consideration in estimating vitamin D intakes. FISH FARMING
Wild-caught salmon provide as much as 25 mg vitamin D3 per 100 grams (Table 54.12) and thus are often cited as an excellent source. Fatty ocean fish such as salmon achieve high levels of vitamin D through consumption of smaller fish that get their vitamin D ultimately from surface zooplankton. As salmon and other fish have no innate ability to synthesize vitamin D, the composition of the diet fed to them is important. In contrast to wild salmon, farmed salmon have lower amounts of vitamin D (24.7 mg/100 g and 9.7 mg/100 g, respectively, Table 54.12). As the aquaculture industry must seek plant sources of protein in order to reduce costs of production, farmed fish need to have vitamin D levels tested regularly as levels may drop even further. A solution to this problem could be enrichment of the diets fed to farmed fish so that levels are maintained or even enhanced. Vitamin D Supplements Dietary supplement use is another option for improving vitamin D intake and status. Table 54.8, showing intakes from all sources (foods and supplements), illustrates how supplement use can significantly increase vitamin D intake in countries having no fortification (e.g., UK) as well as countries with fortification, such as the USA. Supplement use by those in the UK increased average population intakes by only 0.1e1.6 mg (4e64 IU) but had a greater impact in the USA, increasing intakes by 1.5e6.2 mg (60e248 IU). Analyses of the NHANES III data demonstrate that the benefits gained from vitamin-D-containing supplement use are not uniform across all age, race/ethnic, and gender groups (Table 54.11). The benefits of supplement use were generally seen in people whose intakes were already above the median intake. Fewer individuals with intakes below the median intake used supplements [34]. Significant racial/ethnic differences have been
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Complete List of Foods Containing Vitamin D2 in the United States Department of Agriculture Standard Release (SR) 22
TABLE 54.13
Food name and types
SR 22 number
mg per 100 g
IU per 100 g
Mushroom shiitake, raw
11 238
0.4
16
shiitake, stir-fried
11 267
0.5
20
shiitake, dried
11 268
3.9
156
shiitake, boiled, no salt
11 269
0.7
28
shiitake, boiled with salt
11 798
0.7
28
chanterelle, raw
11 239
5.3
212
morel, raw
11 240
5.1
204
brown (Italian or Crimini), raw
11 266
0.1
4
white, raw
11 260
0.2
8
white, boiled with salt
11 797
0.2
8
white, boiled, no salt
11 261
0.2
8
white, stir-fried
11 263
0.2
8
white, microwaved
11 992
0.3
12
canned (drained)
11 264
0.2
8
portabella, raw
11 265
0.3
12
portabella, grilled
11 243
0.3
12
portabella, exposed to UV light, raw
11 998
11.2
448
portabella, exposed to UV light, grilled
11 939
13.1
524
enoki, raw
11 950
0.1
4
oyster, raw
11 987
0.7
28
maitake
11 993
28.1
1124
chocolate, with added (þ) calcium (Ca), vitamins A and D
16168
1.0
40
original and vanilla, þ Ca, vitamins A and D
16 139
1.1
44
all flavors, unsweetened, þ Ca, vitamins A and D
16 222
1.2
48
all flavors, enhanced
16 223
1.2
48
original and vanilla, light, þ Ca, vitamins A and D
16 225
1.2
48
chocolate and other flavors, light, þ Ca, vitamins A and D
16 227
1.2
48
original and vanilla, light, unsweetened, þ Ca, vitamins A and D
16 228
1.0
40
all flavors, low fat, þ Ca, vitamins A and D
16 229
1.0
40
all flavors, non-fat, þ Ca, vitamins A and D
16 230
1.0
40
chocolate, all flavors, non-fat, þ Ca, vitamins A and D
16 231
1.0
40
rice drink, unsweetened
14 639
1.0
40
milk, imitation, non-soy
43 543
1.1
44
Soymilk
Other plant-based “milk”
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NUTRIENT INTAKE AS DETERMINANT OF VITAMIN D STATUS
observed for the effects of daily intake of supplements containing >10 mg (400 IU), with African-Americans having lower use than whites [46]. The low use of supplements is problematic for African-Americans. African-Americans need more dietary sources of vitamin D than do European-Americans, as shown in Table 54.1 [37]. This situation is an example of the Inverse Care Hypothesis whereby those who need treatments, in this case vitamin D supplements, are the least likely to receive them. These findings underscore the need for race- or age-specific dietary supplements with a vitamin D content that would meet the specific needs of the target population [35]. Until recently non-prescription dietary supplements containing vitamin D were only available in multivitamin or single nutrient products containing 5e10 mg (200e400 IU) per tablet. New products have been introduced to the marketplace that contain 25e125 mg (1000e5000 IU) usually as cholecalciferol. The Dietary Supplements Labels Database of the US National Library of Medicine (http:// dietarysupplements.nlm.nih.gov/dietary/) lists over 400 brands of dietary supplements containing cholecalciferol (vitamin D3) or ergocalciferol (vitamin D2). The database presents the amount of vitamin D in IU per manufacturer’s recommended daily dose and the percent of the daily value (DV), the value used in labeling that assists consumers in choosing a product in the USA. In the USA, the DV for vitamin D is 10 mg (400 IU). The Dietary Supplements Labels Database does not specify whether the product contains vitamin D as ergocalciferol or cholecalciferol. For the brands listed, the vitamin D content ranges from 17 IU (4% DV) in a multiple vitamin and mineral product to 2000 IU (500% DV) found in a single nutrient supplement. Vitamin-D-containing supplements currently for sale in the USA are as high as 5000 IU (125 mg) per dose in the marketplace. As supplements may vary by as much as 20% from the stated value, label amounts are not an accurate reflection of content.
Issues Related to Dietary Vitamin D Use of Vitamin D2 The biological equivalency of vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) in humans is controversial [6,8,119]. These studies have called into question whether or not vitamin D2 has the same potency as vitamin D3 in raising circulating levels of 25(OH)D. This controversy stems from the faster clearance of vitamin D2 when given as a single large dose [119]. When used on a daily basis or slightly less frequently, vitamin D2 has been shown to effectively raise 25(OH)D levels in older women [120,121], adults
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[122], and infants and toddlers [123]. Consideration should be given to the fact that vitamin D2 is a veganfriendly form of dietary supplement or fortificant, and is a potentially safer form of the vitamin to use in situations where very high doses given frequently over time are required in order to achieve rapid repletion in severe deficiency as is frequently the case with renal dialysis patients. Foods that are enhanced with or have fortification of vitamin D2 are of particular interest to vegetarians. A complete list of foods with vitamin D2 content that are currently listed in the USDA SR22 as an indication of availability in the USA is given in Table 54.13. Use of Bolus Doses of Vitamin D There are other ways to replete vitamin-D-deficient young and older adults than daily oral or bolus loading of supplementary vitamin D. An example of using bolus vitamin D3 is seen in a study that compared different regimens [124]. In this study, elderly subjects were either low in vitamin D, having 25(OH)D levels less than 40 nmol/l, or only moderately low in vitamin D, having 25(OH)D levels close to 75 nmol/l. The treatments tested were: a large loading dose of vitamin D3 of 12 500 mg (500 000 IU) alone, the loading dose plus monthly doses of 1250 mg (50 000 IU), or monthly doses alone. As shown in Fig. 54.4, this protocol resulted in quick repletion of 25(OH)D after only 4 weeks in subjects who had initial levels below 40 nmol/l, and the continual monthly doses kept mean 25(OH)D levels close to 90 nmol/l. The monthly maintenance dose of 1250 mg (50 000 IU) is equivalent to 33 mg per day (1330 IU/d), an amount not much more than some public health recommendations. In contrast, in the same study, providing only the monthly doses (1250 mg (50 000 IU)) without first loading took 9 months to reach the maintenance level of the loading þ monthly dose group. Also shown in Fig. 54.4, if subjects begin repletion at a higher level of 25(OH)D, by 9 months all groups were at similar levels of 25(OH)D [124]. We cannot rule out some sun exposure as subjects in this study, who were an average age of 82 years, lived in the north island of New Zealand. A review of vitamin D2 protocols in an Atlanta, GA, hospital indicated that when ergocalciferol in 1250 mg (50 000 IU) daily doses was given three times weekly for 6 weeks, 82% of study subjects achieved circulating levels of 75 nmol/l [125]. As there are few head-to-head comparisons of these two forms of precursor or parent molecules, and given the differences in location, assays and other factors, it is not possible to conclude whether this regimen is as effective as studies using vitamin D3 [15]. Another factor to consider is whether annual doses are effective, as they tend to be a higher dose than monthly or weekly ones. In a recent study of falls, participants who received three to five annual doses of
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54. NUTRITION AND LIFESTYLE EFFECTS ON VITAMIN D STATUS
FIGURE 54.4 Effects of three regimens of oral vitamin D3 supplementation to elderly (>65 years) subjects. The loading dose was 12 500 mg (500 000 IU); the monthly dose was 1250 mg (50 000 IU). From [124].
12 500 mg (500 000 IU) had a higher incidence of falls and fractures [126]. While blood levels of 25(OH)D appeared reasonable after the first month of dosing (120 nmol/l), harmful effects might have occurred acutely. Thus there may be unintended risks associated with bolus dosing. A recent study involving annual dosing of vitamin D to older women had higher rates of falls and fractures [126] despite there being strong evidence of protection against falls and fractures by daily doses of vitamin D [13,14]. Availability of highdose vitamin D varies from country to country; for example, in the USA the only high dose currently available is 50 000 IU of vitamin D2. Safety concerns of high doses are addressed elsewhere in this volume. Excess Vitamin A A dietary source of vitamin D that often tops the list of the highest content per serving is cod liver oil. In the most recent USDA nutrient database (SR 22), a 15 ml serving (one tablespoon) is reported to contain 1360 IU of vitamin D and 13 600 IU of preformed (retinol) vitamin A. This amount of vitamin A is over the upper tolerable intake level (UL) of 10 000 IU (3000 mg).
Moreover, products often vary and most modern fish liver oils contain sub-physiological amounts of vitamin D, sometimes only 400 IU, but the vitamin A content of a suggested serving of fish liver oil can equal the UL of 10 000 IU (3000 mg), as is the case for halibut liver oil for sale in Canada. The reason for the alteration from traditional fish oils is unknown, but during processing, molecular distillation is used to remove the vitamin D which is then replaced but at a lower dose than those found in the fish liver itself [8]. Although the active metabolite of vitamin D requires binding to the retinoic acid receptor in order to function in gene transcription, high doses of vitamin A antagonize the action of vitamin D, probably at the site of the vitamin D receptor [127]. In humans, even vitamin A in a single serving of liver impairs vitamin D’s rapid intestinal calcium response [128]. In a recent dietary intake study, Oh et al. found high retinol intake ablated vitamin D’s otherwise protective effect on distal colorectal adenoma [129], and in that study they found a clear relationship between vitamin D and vitamin A intakes: the women in the highest quintile of vitamin D intake also ingested around 10 000 IU of retinol/day. The consumption of preformed retinol, even in amounts consumed by many Americans in fortified low-fat dairy products, eggs, fortified breakfast cereals, multivitamins, and fish liver oil appears to be causing low-grade, but widespread, bone toxicity [130]. Further, a review of 67 randomized controlled trials involving 230 000 participants found vitamin A significantly increased total mortality [131], with antagonism to vitamin D a potential mechanism.
LIFESTYLE STRATEGIES TO IMPROVE VITAMIN D STATUS In this chapter, we presented three contrasting scenarios to illustrate how vitamin D status could be maintained under different sun exposures: Scenario 1, vitamin D only from sun exposure; Scenario 2, vitamin D needed from foods and supplements when only moderate amounts (or intermittent amounts) of sun exposure are possible, and Scenario 3, where all sources of vitamin D sources are from diet. This final scenario best estimates the total amount of vitamin D needed daily. This total amount of vitamin D, irrespective of source, is estimated by several studies to be about 75 mg (3000 IU). One can conclude this amount is needed daily in all situations, whether it is from sun exposure or from dietary sources, or both. Scenario 1 occurs in tropical and semitropical countries where outdoor work predominates and clothing use allows for 80% of skin exposed. No dietary vitamin D is required. Scenario 2
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
LIFESTYLE STRATEGIES TO IMPROVE VITAMIN D STATUS
is seen in those with some sun exposure, but dietary vitamin D, estimated at 25 mg (1000 IU) is needed, possibly only seasonally. Strategies to improve vitamin D status, therefore, may be made for increasing effective safe sun exposure and for increasing dietary sources.
Strategies to Improve Vitamin D Status by Improving Sun Exposure In countries such as Canada, the USA, and Australia and New Zealand, extensive public campaigns have been conducted by cancer agencies to discourage the population from excess sun exposure. For example, in the USA, the message is “Be safe in the sun” (www. cancer.org). In Canada, the message is to “Protect yourself from UV light e it’s the best way to reduce your risk of skin cancer” (www.cancer.ca). In Australia and New Zealand, there is a caution against sun exposure when the UV index is 3 or greater [69]. Therefore, it is very difficult to promote effective sun exposure for vitamin D synthesis in the face of these health campaigns. For example, the best time for vitamin D synthesis is noon, when UVB is maximized thus requiring less time for synthesis and resulting in less time exposed to UVA [132]. To answer the question of whether sun exposure advice was a viable option, in a randomized control trial, Wicherts et al. compared sun exposure advice with supplementation [133]. Subjects were immigrants to the Netherlands and the timing of the study coincided with summer. One group of subjects was advised to have 30 minutes of sun exposure everyday, and they kept a diary of exposure times for 6 months. The supplement group received daily 800 IU over 3 months. A second supplement group received a monthly dose of 25 000 IU over a 3-month period (resulting in 100 000 IU, total). There was no control group. At baseline, 25(OH)D levels averaged 22 nmol/l. By 6 months, the 800 IU supplement group had improved 25(OH)D levels (89% >25 nmol/l) while the sun advice group had only modest changes (51% >25 nmol/l). Thus sun exposure advice is not a reliable method for improving vitamin D status, in part because compliance was poor and the amount of skin exposed not sufficient for adequate vitamin D synthesis. Further, as discussed by Webb and Engelsen [85], who provide ultraviolet exposure scenarios for 400 IU, 1000 IU, and 4000 IU vitamin D, migrant populations in higher latitudes would not achieve much more than 400 IU without risk of erythema.
Strategies to Improve Vitamin D Status by Improving Dietary Intake Dietary intake of vitamin D may be achieved through natural food sources, fortified (or enhanced) foods, and
1003
through supplements. Currently the food supply of natural sources and some fortification is not sufficient to achieve 25(OH)D levels of 75 nmol/l or greater [16]. However, fortification does work, as illustrated by the differences in dietary intakes of American and UK residents (Table 54.8). The problem to be solved is to expand the choices and amounts of vitamin D added to foods yet keep the food supply at a safe level. Supplementation is a well-established method of improving vitamin D status (e.g., [133]), but suffers from being unavailable to low-income groups [134]. Norman and Bouillon [5] describe the dilemma that is before us in terms of improving vitamin D status worldwide. They outline four options for changing the vitamin D status of countries, and what the consequences would be: (1) no change in present situation will promote an increase in rickets and osteomalacia; (2) strict implementation of the 1997 DRI values (i.e., intakes of 200 to 600 IU) would eliminate the number of individuals at risk for rickets and osteomalacia; (3) implementation of an intermediate approach whereby the target level of 25(OH)D would be 50 nmol/l (20 ng/ml), which would mean 400 IU for already adequate peoples and 800 IU for those with deficiency, with the results of having improved bone health in the elderly and some benefit to chronic disease rates; and (4) implementation of an interventionist policy, whereby intakes would be increased to 2000 IU (i.e., a level with the possibility of achieving over 75 nmol/l), so that there would be improvement in chronic disease outcomes for most populations. However, they express some concern about the fourth option as being untested for longer than 6 months. Further discussion of higher-dose supplementation is provided in Chapter 58. Canada illustrates option 3, the intermediate approach. There is mandatory fortification of some staple foods, namely milk and margarine, and promotion of moderate supplement use, of 400 IU for persons over 50 years in the Canada Food Guide and of 1000 IU by the Canadian Cancer Society for adults during winter (Table 54.2). These initiatives have resulted in 25(OH)D levels for Canadians of European descent (for whom sun exposure in the summer ensures vitamin D synthesis) of 71.2 nmol/l [47]. However, these initiatives are not enough for non-white residents, for whom dairy intake is not a preferred food staple [47], and for whom summer sun exposure is not adequate [70]. Governments should be encouraged to improve vitamin D status as economic analyses indicate significant reductions in health care costs if vitamin D status were raised to levels over 100 nmol/l [15,135]. In addition to personal initiatives to improve vitamin D status, there are many public health policies that can assist in improving the vitamin D status of a nation. Sun safety messages need to relay more accurate
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information about safe sun exposure, and set times that are appropriate for the population with respect to skin color. National fortification of staple foods can make a significant contribution to improving vitamin D intakes and nutritional status, an approach that was recently initiated in Jordan with the supplementation of bread flour [21]. Nutritional supplement use, especially in the USA, may be widespread and effective for many individuals, but more must be done for at-risk populations. Dietary guidelines need to identify and emphasize consumption of food staples naturally rich in or fortified with vitamin D, and look for ways to improve intakes of these foods by dark-skinned individuals and older men and women. Food labels should have vitamin D listed to identify good sources. Countries need to select the most appropriate ways to promote optimal vitamin D status, preferably a mixture of strategies that suits the cultural and social needs of the population.
[14]
[15]
[16]
[17] [18]
[19]
[20]
References [1] A. Mithal, D.A. Wahl, J.P. Bonjour, et al., Global vitamin D status and determinants of hypovitaminosis D, Osteoporos. Int. 20 (2009) 1807e1820. [2] M.F. Holick, Vitamin D deficiency, New Eng. J. Med. 357 (2007) 266e281. [3] S.J. Whiting, T.J. Green, M.S. Calvo, Vitamin D intakes in North America and Asia-Pacific countries are not sufficient to prevent vitamin D insufficiency, J. Steroid Biochem. Mol. Biol. 103 (2007) 626e630. [4] M.F. Holick, T.C. Chen, Vitamin D deficiency: a worldwide problem with health consequences. [review] [98 refs], Am. J. Clin. Nutr. 87 (2008) 1080Se1086S. [5] A.W. Norman, R. Bouillon, Vitamin D nutritional policy needs a vision for the future, Exp. Biol. Med. (Maywood) 235 (9) (2010) 1034e1045. [6] L.A. Houghton, R. Vieth, The case against ergocalciferol (vitamin D2) as a vitamin supplement, Am. J. Clin. Nutr. 84 (2006) 694e697. [7] M.S. Calvo, S.J. Whiting, Determinants of vitamin D intake, in: M.F. Holick (Ed.), Vitamin D. Physiology, Molecular Biology, and Clinical Applications, Springer, New York, 2010. [8] J.J. Cannell, R. Vieth, W. Willett, et al., Cod liver oil, vitamin A toxicity, frequent respiratory infections, and the vitamin D deficiency epidemic, Ann. Otol. Rhinol. Laryngol. 117 (2008) 864e870. [9] A. Gombart, The vitamin D-antimicrobial peptide pathway and its role in protection against infection, Future Microbiol. 4 (2009) 1151e1165. [10] M. Hewison, Vitamin D and the immune system: new perspectives on an old theme, Endocrinol. Metab. Clin. North Am. 39 (2010) 365e379. [11] J.H. White, Vitamin D signalling, infectious diseases, and reduction of innate immunity, Infect. Immun. 76 (2009) 3837e3843. [12] M.L. Melamed, E.D. Michos, W. Post, B. Astor, 25-hydroxyvitamin D levels and the risk of mortality in the general population, Arch. Intern. Med. 168 (2008) 1629e1637. [13] H.A. Bischoff-Ferrari, W.C. Willett, J.B. Wong, et al., Prevention of nonvertebral fractures with oral vitamin D and dose
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C H A P T E R
55 Bone Loss, Vitamin D and Bariatric Surgery: Nutrition and Obesity Lenore Arab, Ian Yip David Geffen School of Medicine, UCLA, Los Angeles, CA, USA
BACKGROUND Etiology/Pathophysiology of Obesity Obesity is becoming a major public health issue in the USA and around the globe. Obesity is a chronic, relapsing disease characterized by an excessive accumulation of body fat. The exact etiology of obesity is unknown, although genetic, environmental, metabolic, and behavioral factors are all likely to play important parts in its development. Obesity can be viewed as a disease of energy imbalance. When energy stored exceeds energy expended, body mass accrues in the form of both fat and non-fat tissues. Current societal pressures expose individuals to high-calorie foods, while improvements in technology promote sedentary behavior. Thus, westernized society fosters the development of obesity. Above a BMI of 25 (kg/m2) (Table 55.1) morbidity for a number of health conditions increases. Higher morbidity in association with a BMI greater than 25 has been observed for hypertension, type 2 diabetes, coronary heart disease, stroke, gallbladder disease, osteoarthritis, obstructive sleep apnea, and specific cancers (ovarian, TABLE 55.1
Assessing Obesity by BMI in Adults Obesity class
BMI (kg/m2)
Underweight
<18.5
Normal
18.5e24.9
Overweight
25.0e29.9
Obesity
I
30.0e34.9
Severe obesity
II
35.0e39.9
Morbid obesity
III
40.0e49.9
Super-morbid obesity
III
>50.0
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10055-1
endometrial, breast, prostate, colon) [1]. Obesity is associated with complications of pregnancy, menstrual irregularities, hirsutism, stress, urinary incontinence, and psychological disorders such as depression.
Epidemiology of Obesity The USA is experiencing an epidemic of obesity, defined as a body mass index (BMI) of 30 kg/m2, among both adults and children. The increasing prevalence of obesity makes it one of our most pervasive public health problems. According to the National Health and Examination Survey, the prevalence of obesity has increased from 15% in 1976e1980 to 35% in 2005e2006 [2]. Additionally, 67% of individuals 20e74 years of age were considered overweight (BMI of 25 kg/m2) in 2005e2006. The annual economic burden of obesity in the USA has reached $147 billion [3]. It is estimated that 15 million of our population are considered severely or morbidly obese (defined as BMI >40 kg/m2). Unfortunately, medical management with diet, exercise, and drugs has proven to be ineffective at achieving sustainable weight loss in these patients. The success of bariatric (weight loss) surgery in reliably achieving significant weight loss and reducing associated comorbidities in these patients has made it the preferred option for treating morbid obesity in appropriate patients.
EPIDEMIOLOGY OF BARIATRIC SURGERY Between 2003 and 2008, the number of bariatric surgical procedures performed worldwide more than doubled. The USA mirrors the global trend, with a dramatic increase in bariatric surgeries from 70 000
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55. BONE LOSS, VITAMIN D AND BARIATRIC SURGERY: NUTRITION AND OBESITY
cases in 2001 to 140 000 cases in 2004 and an estimated 220 000 cases in 2010 [4]. The absence of an effective, non-surgical alternative, reduced surgical morbidity and mortality with laparoscopic techniques, improved medical outcomes, increased insurance coverage for bariatric procedures, and rampant direct-to-consumer advertising, all suggest that these increases in the number of bariatric surgeries in the United States are likely to continue [5]. Based on responses to an email survey from 36 nations participating in the International Federation for the Surgery of Obesity and Metabolic Disorders as well as three non-participating countries (Denmark, Sweden, and Norway), 344 221 bariatric surgeries were performed in 2008 by 4680 bariatric surgeons. The absolute numbers of procedures by country for the top ten countries can be seen in Table 55.2. In 2008, of 344 000 procedures performed, 42.3% of all gastric banding procedures worldwide were laparoscopic, and 39.7% were laparoscopic standard Roux-en-Y gastric bypasses. Open sleeve gastrectomies represented 5.1% of the world total. Over 90% of those procedures were performed laparoscopically. The USA and Canada lead the countries by performing 65% of all the procedures conducted in the world, and surpassing the next closest runner-up country (Brazil) with a nine-fold greater number of surgeries. However, the number of surgeons is disproportionate to the number of surgeries. With only 1635 reported bariatric surgeons in the USA, the average number of surgeries per surgeon per year is 135, which is 30e35 more surgeries per surgeon than in the UK, Belgium, Luxembourg, Australia, and New Zealand, and 100 to 120 more surgeries per surgeon than reported in Brazil or Spain, which reports 15 surgeries per surgeon (Table 55.2). The worldwide average is 71 surgeries per surgeon worldwide. While the dramatic rise in procedure volume is shared, there are distinct regional differences in the TABLE 55.2
types of procedures favored in the USA and Canada as compared with Europe. Adjustable gastric banding increased dramatically from 9 to 44% of procedures in the USA, but decreased from 64 to 43% in Europe. In contrast, RYGB increased from 11 to 39% of procedures in Europe but dropped from 85 to 51% in the USA in the same time period. Although still an insubstantial percentage of total operations, sleeve gastrectomies have increased dramatically from 0 to 7% of all procedures in Europe and a bit more slowly in the USA, from 0 to 4% of all procedures. These numbers amount to 4667 sleeve gastrectomies in Europe and 8800 in the USA and Canada in the year 2008 (Fig. 55.1). While the cost of these procedures and their related medical treatment is substantial, the relative benefits of sustained reductions in medical morbidity distinguish bariatric surgery from unreliable medical alternatives. In a typical response, 6235 bariatric surgery patients reduced their medication use for diabetes, hypertension, and hyperlipidemia by 76%, 51%, and 59%, respectively [6]. The disadvantages of bariatric surgery include high rates of postoperative complications within the first year after the procedure, one-third of which are nutrition related, such as rapid bone loss [7]. Under the current payer system in North America, surgeons manage the initial postoperative period, but long-term post-bariatric follow-up is performed by primary care physicians, who must be vigilant to recognize and treat such sequelae.
INDICATIONS FOR BARIATRIC SURGERY Clinical guidelines developed by the National Heart, Lung, and Blood Institute Expert Panel on the identification, evaluation, and treatment of obesity for adults recommend that bariatric surgery be an option for
The Top 10 Countries Ranked by Numbers of Bariatric Procedures Conducted in 2008 [5]
Country
Number of bariatric surgery operations
% of total reported across all 39 countries
Number of surgeons by country
Number of surgeries per bariatric surgeon
USA/Canada
220 000
65.82
1635
135
Brazil
25 000
7.48
700
35
France
13 722
4.11
310
44
Mexico
13 500
4.04
150
90
Australia/New Zealand
11 914
3.56
118
101
Belgium/Luxembourg
8700
2.60
82
106
Spain
6000
1.80
400
15
United Kingdom
6000
1.80
60
100
344 221
100
4680
71
Totals reported worldwide
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FIGURE 55.1 Trends in various types of bariatric surgerical procedures worldwide. Reprinted with permission from Elsevier Ó 2010. All Rights Reserved.
carefully selected patients with clinically severe obesity (BMI >40 or >35 with comorbid conditions) when less invasive methods of weight loss have failed and the patient is at high risk for obesity-associated morbidity and mortality. The NIH statement “Gastrointestinal Surgery for Severe Obesity” concluded that the benefits outweigh the risks and that surgical treatment is reasonable in those who strongly desire substantial weight loss and have life-threatening comorbid conditions [8].
CLASSIFICATION OF BARIATRIC SURGERY There are two distinctive forms of bariatric surgery: restrictive and malabsorptive. In restrictive surgery such as vertical banded gastroplasty or lap-band, the stomach is stapled or restricted so that consumption of large quantities of food is not mechanically feasible, potentially leading to nausea or vomiting. However, the entire surface area for digestion and absorption of nutrients in the gastrointestinal tract is functionally intact. On the other hand, in malabsorptive bariatric surgery e jejunoileal bypass, Roux-en-Y gastric bypass, or biliopancreatic diversion e varying lengths of the intestines (duodenum and jejunum) are excluded from contact with nutrients by physical rearrangement. Therefore, micronutrient malabsorption after malabsorptive bariatric surgery is expected. Both restrictive and malabsorptive methods may be performed simultaneously, creating a third category of combination restrictiveemalabsorptive bariatric surgery. For the purpose of discussion, the authors of this chapter choose to combine both pure malabsorptive surgery and combined restrictive and malabsorptive surgery into the malabsorptive surgery category. All the techniques discussed can be performed laparoscopically or through an open incision.
Malabsorptive Bariatric Surgery The human small intestine is composed of duodenum, jejunum and ileum, measuring approximately 20 feet. The surface of the small intestine is where absorption of major macronutrients and micronutrients occurs. A surgical procedure where a portion of the intestine is bypassed creates malabsorption of nutrients and calories. Thus, malabsorptive surgery favors the energy balance equation and assists in weight reduction, but carries the risk of reduced absorption of important micronutrients. Jejunoileal Bypass (Fig. 55.2(a)) The oldest malabsorptive surgery is jejunoileal (J-I) bypass, where the first part of the jejunum is attached directly into the terminal ileum. Therefore, less than 2 feet of the small intestine are left for absorption of nutrients leaving more than 90% of the entire small intestine without contact with ingested food. The J-I was popular in the 1960s and 1970s until it was discovered that many patients developed severe malnutrition and unacceptable risks of many other medical complications such as bacterial overgrowth, inflammatory arthritis, calcium oxalate kidney stones, kidney failure, cirrhosis, and liver failure. Since then, J-I has been abandoned as a primary surgical tool for weight reduction. Biliopancreatic Diversion (BPD) (Fig. 55.2(b)) The biliopancreatic diversion was first performed by Scopinaro, who designed the surgery to be a safer alternative to the J-I bypass. The BPD eliminates the “blind loop” that exists with the jejunoileal bypass surgery. Bacterial overgrowth inside the blind loop was thought to be the origin of inflammatory reactions that cause reactive arthritis and liver failure. In this rearrangement, all digestive enzymes and bile will meet
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Various types of bariatric surgery. (a) Jejunoileal bypass (JIB); (b) biliopancreatic diversion (BPD); (c) biliopancreatic diversion with duodenal switch (BPDDS); (d) vertical-banded gastroplasty (VBG); (e) adjustable silicone gastric banding (lap-band); (f) Roux-en-Y gastric bypass (RYGB). Reprinted with permission from Elsevier Ó 2010. All Rights Reserved.
FIGURE 55.2
downstream in the ileum at about 50 to 100 cm from the large intestine, and thus reduce the amount of nutrients that can be digested and absorbed before they reach the colon. In BPD, patients usually can ingest more food without significant nausea and vomiting which is typical of restrictive procedure. Restrictive procedure creates a restriction in volume to less than 30 cc in the stomach. By definition, the BPD is a malabsorptive procedure without any restrictive component. Because BPD bypasses a large portion of the small intestine, less food is absorbed by the body. Most patients will experience frequent, foul-smelling bowel movement because of fat malabsorption. Other side-effects include flatulence, bloating, and body odor. In addition, there may be higher incidence of nutritional and vitamin deficiency, including vitamin D deficiency, after BPD. One
percent of all bariatric surgeries performed in the USA are BPD. Biliopancreatic Diversion with Duodenal Switch (BPDDS) (Fig. 55.2(c)) Biliopancreatic diversion with duodenal switch is a very complex bariatric operation that principally includes removing a large portion of stomach to promote smaller meal sizes and rerouting nutrients and digestive enzymes away from much of small intestine in order to promote malabsorption. The BPDDS procedure is most commonly used for patients who have a body mass index of over 50 kg/m2 (super-obese). The stomach is divided into left and right sections by stapling. A gastric sleeve is formed and the left side of the stomach along the greater curvature is excised. This partition is distinctively different from the original biliopancreatic
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silicone gastric banding that is easier to perform and less likely to develop staple line breakdown. Adjustable Silicone Gastric Banding (Fig. 55.2(e))
FIGURE 55.3 Sleeve gastrectomy.
diversion where the stomach is divided into top and bottom sections. The advantage of this partition involves preservation of the pyloric valve while allowing food to pass from the stomach to the duodenum without significant “dumping syndrome” commonly in patients who have Roux-en-Y gastric bypass surgery. BPDDS is not widely performed in the USA but is gaining attention for patients who are super-obese with BMI >50 kg/m2. Because of the concern about surgical morbidity for patients who are super-obese, BPDDS is often performed in two stages. The first operation involves the creation of the gastric sleeve alone as illustrated in Figure 55.3. When obese patients lose over 100 lb, the second stage of biliopancreatic diversion with duodenal switch will be done at a later date.
Restrictive Bariatric Surgery Vertical Banded Gastroplasty (VBG) (Fig. 55.2(d)) The VBG operation involves the creation of a 30 cc proximal gastric pouch with a vertical staple line measuring about 1 cm, punched out using a circular stapler. In addition, a plastic band is used as a collar to tighten and maintain an outlet of about 1 cm. The VBG is a successful restrictive procedure that is still performed by surgeons, but has been largely replaced by adjustable
This operation can be performed laparoscopically and is commonly referred to as the “lap-band.” Adjustable silicone gastric banding became popular first in Europe and was approved by the Food and Drug Administration in 2001 (http://www.accessdata.fda. gov/cdrh_docs/pdf/p000008a.pdf). A circular silicone band is used to encircle the proximal stomach and creates a 10e20 ml proximal pouch above the silicone band. The band has an accessible port implanted subcutaneously beneath the anterior abdominal wall. The circumference of the band can be adjusted by filling the port with normal saline in the ambulatory setting. The number of adjustments needed varies based on patient’s discomfort with nausea and vomiting, caloric intake, and amount of weight loss. Potential long-term complications include stomal obstruction, esophageal and gastric pouch dilatation, gastric erosion and necrosis, and access port problems. Laparoscopic adjustable gastric banding can be completely reversed. For patients who fail to lose weight after lap-band, revision procedures are usually performed to remove the band apparatus and convert the patient to a malabsorptive surgery, such as Roux-en-Y gastric bypass.
Combined Restrictive and Malabsorptive Bariatric Surgery: Roux-en-Y Gastric Bypass (Fig. 55.2(f)) Roux-en-Y gastric bypass (RYGB) is the current gold standard for bariatric surgery. It is estimated that approximately 60% of all bariatric surgeries performed nowadays are RYGB. Originally performed as an antiulcer operation before the availability of effective medical management, surgeons have been performing RYBG for over 50 years. RYBG is considered a combined restrictive and malabsorptive operation. The restrictive component involves the creation of a proximal pouch (<30 ml). The malabsorptive component is achieved through the creation of a “roux limb” using a variable length of jejunum (75 cm ¼ standard, 150 cm ¼ long limb). The surgery begins with dividing the stomach into a small pouch from the upper part with surgical staples. Next, the small intestine is divided from 15 cm from the ligament of Treitz, and the distal portion of the divided intestine is brought up as the roux limb and anastomosed to the gastric pouch. The proximal end of the divided intestine is anastomosed in a sideto-side fashion to the roux limb to create a jejunostomy anastomosis. In RYGB, the majority of the stomach and
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the duodenum are bypassed, resulting in a combination of restrictive and malabsorptive techniques.
Experimental Bariatric Surgery: Sleeve Gastrectomy (Fig. 55.3) Sleeve gastrectomy was first performed in the highrisk super-obese (BMI >50 kg/m2) patients to increase efficacy of weight loss surgery. In 2005, Mognol et al. reported good outcomes with sleeve gastrectomy and suggested that it can be utilized as a standalone bariatric procedure [9]. The sleeve gastrectomy has been gaining attention in the surgical community in the past 5 years. The surgery involves removal of a portion of the stomach and leaving the stomach in a sleeve form. This is a purely restrictive procedure which reduces the size and diameter of the stomach and allows for a sense of fullness with smaller amounts of food. In addition, it removes the fundus of the stomach where most of the cells that produce Ghrelin are located. Ghrelin is felt to be important for regulation of hunger and meal time [10]. In a systemic review of literature in 2009 by Brethauer et al., sleeve gastrectomy had resulted in weight loss and comorbidity reduction that is comparable to that of other bariatric procedures [11]. In addition, the postoperative major complication rates and mortality have been acceptably low [11].
SURGICAL COMPLICATIONS OF BARIATRIC SURGERY Surgical complications of bariatric surgery can be divided into intraoperative, early postoperative (within 30 days of surgery) and late (>30 days). According to an observational study published in the 2009 New England Journal of Medicine of over 4000 patients undergoing bariatric surgery at ten clinical sites in the USA between 2005 and 2007, the 30-day rate of death among patients who underwent Roux-en-Y gastric bypass or laparoscopic adjustable gastric banding was 0.3%. A total of 4.3% of patients had at least one major adverse outcome including deep venous thromboembolism, operative reintervention, and failure to be discharged from the hospital [9]. It is believed that the operative risk will continue to improve with the advancement of surgical techniques. The steady rise in the number of bariatric surgery operations has mandated the development of benchmarks for quality and patient safety. The American Society for Metabolic and Bariatric Surgery (ASMBS) founded the Surgical Review Corporation (SRC) in 2003 to advance the safety and efficacy of bariatric surgical centers. To achieve this objective, SRC established the Bariatric Center of Excellence around the globe [10].
Intraoperative and Early Postoperative Complications Venous thromboembolism is considered one of the major causes of morbidity and mortality in patients requiring bariatric surgery. While pulmonary embolism is the leading cause of perioperative death in bariatric patients, the incidence of pulmonary embolism is reported to be less than 1%. Despite the body of evidence guiding appropriate perioperative thromboprophylaxis in the general population, there is no consensus strategy to prevent deep venous thrombosis and pulmonary embolism in the morbidly obese patients after bariatric surgery. According to a statement published by the American Society of Metabolic and Bariatric Surgery, choice of anticoagulation, dosage regimen, duration of prophylaxis (including prolonged post-discharge administration), and the possible role of inferior vena cava filter placement have been controversial and recommendations regarding these issues have not been established. The incidences of gastrointestinal anastomotic leaks after gastric bypass surgery are the second most common cause of mortality after pulmonary embolism. While leak incidence has been reported to be as high as 5% in the past, improved operative techniques including appropriate staple size, staple line reinforcement, handsewnotomy closure, and intraoperative leak testing may lead to lower incidence of leaks in the future. Early detection may limit both mortality and morbidity. Fever, tachycardia, and abdominal tenderness are the most frequent signs and symptoms of gastrointestinal anastomosis; however, anastomosis leaks can be difficult to recognize and require a high index of suspicion. Gastrointestinal (GI) bleeding is another potential perioperative complication after bariatric surgery. The incidence of gastrointestinal bleeding ranges from 1 to 4%. Patients diagnosed with early GI bleeding (within 48 hours of surgery) are symptomatic with hematemesis, bright red blood in bowel movements, and/or hypotension. The majority of early GI bleeding results from staple line hemorrhage, and initial management consists of fluid resuscitation and blood transfusion. Reoperative intervention may be needed for persistently bleeding patients. GI bleeding that occurs beyond 48 hours postop can have a variety of etiologies including marginal ulcers, ulcers in the bypassed stomach, or duodenum ulcers.
Late Surgical Complications In the past, incisional ventral hernia represented the most common late complication after open bariatric surgery with an incidence rate of up to 20%. However, with the advancement of laparoscopic techniques, the incidence of incisional and ventral hernia has reduced
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dramatically. Small bowel obstruction (SBO) can occur in both the early and late postoperative period in up to 5% of patients after bariatric surgery. Patients are usually symptomatic with persistent nausea and vomiting. Abdominal CT and X-ray can usually provide the diagnosis. Adhesions, ventral hernias, and jejunojejunal anastomotic structures are among the most commonly observed etiologies. Other less common etiologies of SBO include jejunojejunal intussusceptions, kinking of small bowel and internal hernia. Initial management should consist of endoscopic nasogastric decompression performed by a gastroenterologist. Blind insertion of a nasal gastric tube is discouraged to avoid suture line and anastomotic rupture. Symptomatic gallstone disease commonly occurs in obese patients after bariatric surgery. It is thought to be secondary to decreased bile flow and biliary sludge formation that occurs in the setting of rapid weight loss. The incidence of sludge in the gallbladder is up to 50% postoperatively and gallstones approximately 32%. Prophylactic treatment with urosodiol for 6 months after surgery has been shown to reduce the incidence of gallstones to 2% [11]. One must bear in mind that after Rouxen-Y gastric bypass, access to the biliary tree via endoscopic retrograde cholangiopancreatography (ERCP) can be technically difficult, making the diagnosis of choledocholithiasis difficult to make. Prophylactic removal of the gallbladder during bariatric surgery is controversial among various surgeons. Some surgeons routinely performed prophylactic cholecystectomy whereas others believe it will increase postoperative complications. Adjustable gastric banding has been associated with band erosion, erosive esophagitis, and herniation of the stomach upward inside the band. Stomal ulcer occurs in patients after Roux-en-Y gastric bypass surgery. Stoma ulcer is a mucosal ulcer in the jejunum mucosa that occurs near the opening between the stomach and jejunum. The cause of stomal ulceration is unclear but has been postulated to be either leakage of acid through the staple line into the pouch or subclinical breakdown of the staple line. Stomal ulcers usually occur within the first 3 months after gastric bypass surgery. Most patients with these ulcers are seen for severe dyspepsia and vomiting and can be diagnosed by endoscopy. H. pylori must be either ruled out or treated as an etiologic factor.
HEALTH BENEFITS OF BARIATRIC SURGERY The many health benefits of bariatric surgery are directly or indirectly associated with the weight loss achieved. Increasing evidence has shown reduction in the risk factors associated with obesity such as type 2
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diabetes mellitus, obstructive sleep apnea, hypertension, hypercholesterolemia, non-alcoholic fatty liver disease, and arthritis as a result of the surgery. The Swedish Obesity Study (SOS) has demonstrated a significant long-term survival benefit for a patient who has bariatric surgery [12,13]. The majority of patients in the SOS study have undergone restrictive bariatric surgery. It is also reported that the life expectancy of gastric bypass patients is 2.4 years greater than patients who used conservative medical management to lose weight [14]. For patients with type 2 diabetes, 75% of patients who underwent laparoscopic adjustable gastric banding surgery had a complete remission of their diabetes compared to 13% of patients who were managed medically [15]. In addition, it has been shown that gastric bypass surgery can often produce a quick improvement and/or resolution of their diabetes. This phenomenon occurs within days of Roux-en-Y gastric bypass surgery irrespective of the little amount of weight loss [16]. Many attributed this observation to the alteration of neural and hormone regulations immediately after the rearrangement of the intestine after gastric bypass. Bariatric surgery has provided an excellent opportunity to study the role of various gastrointestinal, appetite, and satiety factors (leptin, ghrelin, CCK, NPY, Orexin, etc.) in controlling obesity.
MEDICAL COMPLICATIONS OF BARIATRIC SURGERY One of the most common nutritionally relevant complications of bariatric surgery is dumping, which occurs in 25e50% of patients. These patients often experience dizziness, palpitation, sweating, nausea, and vomiting soon after eating particularly sweets and hyperosmolar food. The exact etiology of dumping syndrome is unknown although it most likely involves multiple factors such as autonomic reflexes, and release of endocrine hormones from the gastrointestinal tract. Dietary modification that avoids eating such food items which illicit the dumping syndrome and smaller portions will often be sufficient to alleviate the symptoms. Nutrient deficiencies such as iron deficiency anemia are common in patients who have malabsorptive procedures and therefore iron supplementation is required for many patients. In addition, because of the complicated system involved in absorption of vitamin B12, vitamin B12 supplementation is critical in order to prevent pernicious anemia in these patients. Various forms of vitamin B12 supplement include monthly intramuscular injection or sublingual and nasal delivery system. Recommendations for daily supplementation of vitamins in bariatric patients run from 3 to 100 times the recommended daily allowances of healthy adults for
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55. BONE LOSS, VITAMIN D AND BARIATRIC SURGERY: NUTRITION AND OBESITY
vitamins A, B1, B12, C, D, E, K and folic acid [17]. The recommended level differs by procedure, with highest recommendations in biliopancreatic diversion, lower in RYGB and lowest in banding procedures [18].
BONE LOSS AFTER GASTRIC BYPASS One of the poorly understood medical consequences of bariatric surgery is rapid loss of bone density. This is of concern due to the immediate increase in risk of fracture as well as the long-term increases in the need for hip replacement. Each decrease in standard deviation for femoral neck bone density as measured by DXA increases the age-adjusted risk of hip fracture 2.6-fold (95% CL 1.9e3.6) [19,20]. Fractures cause greater morbidity, chronic pain and disability, increased dependence and potentially institutionalization, and are also associated with excess mortality [21]. The magnitude of increase in medical costs from future osteoporosis treatment and hip and joint replacement in these patients has yet to be estimated but is bound to be substantial. The publications on bone loss in this population (Table 55.3) report significant femoral neck and hip bone loss, measured 9 to 12 months after bariatric surgery using dual-energy X-ray absorptiometry (DXA) [22e25]. The studies show a total bone density reduction of 3e5% with greater site-specific reductions and an impact on cortical bone rather than trabecular bone. In the two studies that examined BMD reduction at the femoral neck, 9% reductions were seen [22,25]. All four studies showed a statistically significant and substantial reduction in hip bone mineral density (BMD), ranging from 8 to 11%. The lowest impact, at a 3e7% reduction, appears to be in the lumbar spine. It deserves note that the study of bone density changes in bariatric patients confronts two challenges. One is the problem that dual-energy Xray absorptiometry (DXA) scanners were not designed to accommodate people greater than 300 pounds in weight. This has resulted in spotty and incomplete measurement data that are limited either to lighter patients or measurements of the forearm. As seen in Table 55.1, some studies have lost as many as 90% of the subjects from bone density measures because they have TABLE 55.3
been too large for their DXA scanner. The second challenge has been concern about the validity of comparisons of bone density DXA scans due to the X-ray absorption changes related to weight loss. This latter concern has been addressed in a study of bias in bone measurement due to weight loss which was examined using DXA readings from scans performed in 34 obese subjects before and after weight loss. Errors in bone density measurement as a result of changes in soft tissue with weight loss were estimated to account for an inaccurate decrease of 1e2% in spinal BMD by DXA [26]. Considering this magnitude of potential measurement error, it appears that, despite imperfect measurements, massive and rapid bone loss, especially at the hip and femoral neck, is an expected sequelae of many common types of bariatric surgery.
Alternative Explanations of Bone Loss Subsequent to Bariatric Surgery As the underlying cause of the rapid post-surgical bone loss remains unknown, speculation on the cause of this dramatic bone loss includes one or more of the following: (1) loss of bone mineral density and increased bone turnover as a physiologic adaptation to weight loss and the altered mechanical loading of the skeleton; (2) rapid loss of adipose tissue and a corresponding change in adipokines that regulate bone turnover; (3) reduction in bioavailable vitamin D due to reduced absorption, reduced intake or a combination of both; or (4) a reduction in calcium bioavailability due to reduced intake, reduced uptake or lowered bioavailability resulting in secondary hyperparathyroidism.
Bone LosseWeight Loss Relationships in Bariatric Surgery and with Dieting Evidence suggests a close relationship between the amount and speed of weight loss and bone loss. In postoperative patients, correlations as great as 0.90 with femoral neck and 0.65 with hip were observed with the postsurgical weight loss over the course of 1 year [24]. This is consistent with the observation of greater bone mineral
Overview of Studies of Bone Loss After Bariatric (Roux-en-Y) Surgery
Author
Total subjects
N with DXA measurements
Total body % change
Femoral neck % change
Hip % change
Lumbar spine % change
Weight loss (kg)
Carrasco [22]
42
42
3.00
nm
10.90
7.40
31.8
Fleischer [24]
23
13
nm
9.20
8.00
<2%, ns
44.8
Johnson [25]
226
22
nm
nm
9.30
4.50
np
Coates [23]
15
12
5.10
9.3
7.80
3.30
36.0
nm ¼ not measured, np ¼ not presented in the report, ns ¼ not significant.
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density among the obese, particularly at weight-bearing sites such as hip and spine and that greater body weight is positively associated with total body bone mineral density across various racial groups [27,28]. Whether bone loss after bariatric surgery can be attributed entirely to weight loss remains controversial, despite recent evidence that weight loss drives the bone loss [24,29]. The fact that comparable bone losses are not seen in successful dietary weight loss regimens, as evidence of bone density reduction with diet-induced weight loss relate BMD reductions of 1.5 to 1.8% to weight losses ranging from 3.5 to 18 kg after 12 months, suggests that weight alone may not explain the bone loss completely [30,31]. Bone loss in diet trials is not proportional to the weight loss in these studies and hovers under 1.8%. These relatively small reductions in BMD may be simply within the range of the expected DXA measurement error in observed bone density with weight loss cited earlier [22].
The FateBoneeVitamin D Axis; Adipokines, Osteokines, and Bone-Regulating Hormones Bone homeostasis is partially regulated by adipokines, which is not surprising as adipocytes and osteoblasts share a common mesenchymal cell origin. Adipose tissue is an extremely active endocrine organ, producing leptin and adiponectin, TNF-alpha and IL-6, all of which are involved in the regulation of bone physiology. Adipokines interact with the “osteokines” _osteocalcin, osteopontin, osteonectin [32], and osteoprotegerin (OPG), secreted proteins that participate in bone remodeling [33]. Conversely, the bone-derived factors osteocalcin and osteopontin can act as endocrine factors to affect body weight control and glucose homeostasis. A number of these adipokines and osteokines e including osteocalcin, osteopontin, osteonectin and osteoprotegrin e are regulated by 1,25(OH)D [32,34e38]. Although this active boneeadipose axis is fairly well understood in weight-stable individuals, to date it remains poorly understood in bariatric patients. Leptin is also produced in adipocytes in proportion to adipocyte stores. Its receptors are expressed throughout the body, including in bone marrow stem cells, osteoblasts and osteoclasts. Leptin exerts a bone anabolic effect: inhibiting osteoclastogenesis by increasing osteoprotegerin (OPG) and decreasing levels of receptor activator of nuclear factor kappa B ligand (RANKL). It also induces osteoblast differentiation. Plasma leptin levels are seen to be independently and significantly correlated with BMD in epidemiologic studies of postmenopausal women and even among the non-obese. Leptin is believed to exert a protective effect on bone by limiting postmenopausal bone
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resorption [39,40]. The leptin pathway has recently been identified as a fat target for skeletal health [41]. Reduction in weight with the consequence of a reduction in leptin might drive the bone loss, but the few reports in bariatric surgery are contradictory. Sustained reductions in leptin post-RYGBP have been reported [42]. Furthermore, reduced leptin is a significant predictor of increased N-telopeptide of type 1 collagen (NTX) at both 6 and 18 months after RYGBP [43]. After lapband surgery, however, leptin initially drops and then rebounds after 6 months to a higher level [44]. At least one study has demonstrated that supplementation in humans with vitamin D in high doses (300 000 IU/m) results in a doubling of serum leptin [45]. Incidentally, both this study and the study by Arunabh et al. showed significant increases in BMI with therapeutic doses of vitamin D supplementation [45,46]. Other serum markers of bone turnover altered by bariatric surgery include osetocalcin, bone alkaline phosphatase, adiponectin, osteopontin, and NTX, all of which increase concomitantly to weight loss in the post-surgical period. NTX is inversely associated with (declining) leptin levels and positively associated with serum 25(OH)D in Roux-en-Y patients [43]. In biliopancreatic diversion, osteocalcin and b-CTx (serum C-terminal telopeptides) show increases that are related to the amount of body mass lost [47]. Adiponectin, which demonstrates antiosteogenic activity, likely through its role as ligand for NFkb, appears to increase, in vivo, post-surgery by about 48% at 6 months and twice that amount at 12 months [22]. Adiponectin receptors are expressed in primary human osteoblasts from femur and tibia and therefore adiponectin may provide an important signal that links fat and body weight to bone mineral density [48]. However, evidence suggests that it acts on bone mineral density independently from the body weight change impact on BMD (body mass density) [22]. Osteopontin is a proinflammatory matrix glycoprotein secreted from the osteoblast. Osteopontin has been implicated in a number of physiological events and is required for efficient bone resorption [49]. A key event in bone resorption is the binding of osteoclasts to the mineral matrix of bone surfaces. Osteopontin is highly enriched at regions of bone surface where osteoclasts are anchored. Therefore, osteopontin serves to initiate the process by which osteoclasts begin their bone resorption [50]. The serum osteopontin level decreases with diet-induced weight loss but increases after bariatric surgery weight loss in both gastric bypass surgery and gastric banding [51]. This increase appears to be greater and more consistent in RYGB patients [52,53]. This unexpected finding of a post-surgical increase is not related to weight loss or reduced insulin resistance in bariatric surgery patients. Ghrelin, a hormone produced in the fundus of the stomach, is reported to be dramatically reduced
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immediately after RYGB and to a lesser extent with other bariatric surgery procedures, remaining low for the post-surgical year [54]. However, a recent review questions the uniformity of this response and reports postsurgical elevations in some studies, including RYGP, may also play a role in bone [55,56].
VITAMIN D STATUS Low 25(OH)D levels are widespread in the population in the USA. Recently, a dramatic reduction in the mean serum 25(OH)D level from 24 to 30 ng/ml was reported in the population of the USA in general, as observed in various waves of NHANES studies over the last decade [57]. The prevalence of 25(OH)D levels 10 ng/ml tripled and 25(OH)D levels of 30 ng/ml or more decreased from 45 to 23%. Among non-Hispanic blacks, very low levels (10 ng/ml) more than tripled from 9% during NHANES III (1991e1994) to 29% during NHANES 2001e2004, and the prevalence of levels 30 ng/ml dropped from 12 to 3%. Lower circulating 25(OH)D levels have been associated with a 70% greater risk of fracture, and high levels appear to be related to lower fracture rates through both better bone and muscle health in elderly populations [58,59]. In addition to skeletal disorders, low 25(OH)D levels are associated with an increase in the risk of malignancies, of chronic inflammatory and autoimmune disease, of metabolic disorders (metabolic syndrome and hypertension), as well as peripheral vascular disease [60,61].
Vitamin D Status in Patients Seeking Bariatric Surgery Numerous studies from Europe and the USA have consistently demonstrated that obese individuals, both adults and children, are at even greater risk of vitamin D deficiency in general [46,62e68]. In multiple studies of morbidly obese patients seeking gastric bypass surgery, vitamin D deficiency, defined as 25(OH)D level <50 nmol/l or <20 ng/ml, was found in more than half of all patients studied [69e71]. The prevalence is double that among Hispanic and African-American patients as compared with Caucasians (78% vs. 36%) [69,70]. In Spain 80% of morbidly obese patients waiting for bariatric surgery have vitamin D deficiency [72]. Thus, a large proportion of patients who are seeking bariatric surgery have vitamin D deficiency before their operation [73]. This presurgical vulnerability to vitamin D deficiency is enhanced depending upon the type of bariatric surgery [74]. Restrictive bariatric surgery affects only the upper portion of the stomach. The entire gastrointestinal tract
remains intact and available for nutrient processing and absorption and thus severe malnutrition is not expected to occur in these patients. On the other hand, malabsorptive bariatric surgery alters the digestive tract. In Roux-en-Y gastric bypass, the majority of stomach and duodenum are bypassed and a moderate degree of vitamin D malabsorption is expected. In addition, as the purpose of the surgery is to limit food consumption, changes in eating behavior also increase risk of deficiencies. In biliopancreatic diversion with or without duodenal switch, the length of common channel left for absorption of nutrients is between 50 and 100 cm, and therefore significant malabsorption of macronutrients and micronutrients is expected. Bypassing the stomach, reduction in absorption sites in the jejunum and ileum, decreased dietary intakes overall, and low intakes of calcium and vitamin-D-rich foods can all play additive roles in post-surgical vitamin D deficiency.
Vitamin D Status in Patients after Bariatric Surgery Vitamin D deficiency with secondary hyperparathyroidism is common after bariatric surgery. The reported incidence varies widely in the literature. A recent review of vitamin D status in bariatric patients identified 14 studies involving a total of 1500 patients, 33 to 80% of which were considered vitamin-D-deficient. After surgery with supplementation, vitamin D levels increased in general but not enough to remediate the deficiencies noted pre- and postoperatively in most groups, and not enough to eliminate hyperparathyroidism in these patients and its accompanying bone resorption [75]. Several explanations might account for this varying incidence observed among different studies. First, most studies did not attempt to correct the vitamin D deficiency in obese individuals prior to bariatric surgery. Second, the time since surgery may be important, as an initial rise of 25(OH)D levels within the first few months of bariatric surgery related to the increase in supplementation or a yet to be proven possible release of vitamin D storage from fat cells during the acute weight loss phase. Third, the difference in bariatric surgeries needs to be taken into account. Restrictive surgery should not alter vitamin D homeostasis. One study reported significant changes in several gut hormones including ghrelin in patients who have RYGB when compared with those without the surgery [54]. Another study reported more favorable responses of peptide YY(3e36) and glucagon-like peptide-1 to glucose challenges in patients after gastric bypass as compared to patients with equivalent weight losses after gastric banding but no significant change in Ghrelin levels after either surgery [76]. Gastric or intestinal bypass surgery with the creation
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CALCIUM MALABSORPTION AFTER GASTRIC BYPASS SURGERY
of a Roux limb anastomosis may cause malabsorption of fat-soluble vitamins like vitamin D. In general, patients with biliopancreatic diversion had lower vitamin D levels and higher parathyroid hormone levels than those patients who had gastric bypass. The length of the bypass limb may also be important. Patients with a long Roux limb (>100 cm) have lower vitamin D levels and higher PTH levels 5 years after surgery than those patients with a short Roux limb (<100 cm) [25]. A significant incidence of secondary hyperparathyroidism is seen even among patients who had 25(OH)D levels >75 nmol/l (30 ng/ ml). This suggested that calcium malabsorption, which is inherent to gastric bypass surgery, may also play a role in the development of secondary hyperparathyroidism.
Vitamin D Sequestration in Adipose Tissue Inverse correlations between 25(OH)D level and BMI have been consistently reported in various ethnic groups [77,78]. In the Framingham Heart Study population, higher adiposity volumes in both subcutaneous and visceral areas were found to correlate with lower 25 (OH)D levels across different categories of body mass index. The prevalence of vitamin D deficiency was three-fold higher in individuals with high abdominal obesity [79]. The association between low plasma vitamin D levels and high adiposity can also be found in Hispanic and African-Americans [68,80]. Six hypotheses have been advanced to explain the negative association between circulating levels of 25(OH)D and elevated body mass index. They are: (1) decreased nutrient ingestion, including vitamin D, among overweight and obese as they tend to select nutritionally deficient diets [81]; (2) decreased production of vitamin D due to reduction in skin exposure to UV radiation in the middle of the day; (3) reduced hepatic synthesis of 25(OH)D possibly due to steatosis of the liver [75]; (4) increased clearance or sequestration into adipose tissue to protect the organism against toxicity; (5) reduced bioavailability with obesity [82]; and finally (6) increased clearance of 25(OH)D due to inflammation secondary to obesity and the large number of proinflammatory adipose cells. Vitamin D absorption at the intestinal level in patients with Roux-en-Y gastric bypass is unknown. Some authors suggested that there is bacterial overgrowth in both small intestine and the residual stomach which could affect bile salt delivery. Fat malabsorption is therefore common after RYGB with resulting diarrhea. Since fats trap fat-soluble vitamins A, D, E, and F, less vitamin D will be available for absorption. However, since it is known that 25(OH) D does not undergo enterohepatic circulation, malabsorption from bile is not a concern [83,84].
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The sequestration theory is supported by evidence from NHANES that in adults with BMI levels above 20 kg/m2, plasma 25(OH)D is linearly negatively associated with body mass index [85]. An inverse relationship between BMI and circulating vitamin D levels was also noted in an experiment designed to raise vitamin D levels among obese and lean individuals exposed to either whole-body ultraviolet irradiation or a 50 000 IU oral dose of vitamin D2. Despite similar precursor levels, the serum levels at 24 hours among the obese were 57% of the level achieved in the normal body weight, healthy, age-matched Caucasians. The authors also noted correlations of 0.56 between body weight and peak serum vitamin D2 from the oral dose and 0.55 between body weight and serum vitamin D3 after the wholebody irradiation. These findings are attributed to sequestration of the vitamin in adipose tissue [82]. Studies in Wistar rats under fasting and energy balance conditions support this hypothesis. Under high supplementation doses of vitamin D, cholecalciferol is accumulated rapidly in the adipose tissue of the rats. This reservoir is accessible during fasting when fat is combusted but, under conditions of energy balance, it is very slowly released [86]. Both of these studies suggest that in conditions of positive energy balance, vitamin D is sequestered in adipose tissue. They also find the absolute amount of adipose tissue to be inversely correlated with circulating vitamin D levels.
CALCIUM MALABSORPTION AFTER GASTRIC BYPASS SURGERY The current recommended daily allowances for calcium intake in the USA are 1000 mg for adult men and women and 1200 mg for menopausal women. Calcium content in food is generally higher in dairy products. In the USA, dietary calcium intake as nondairy components is approximately 200 to 300 mg per day. Therefore, depending on their dairy intake, women often require oral calcium supplementation to meet the RDA and to prevent osteoporosis. In humans, the bulk of body calcium is in the skeleton and teeth. The endoskeleton composed of crystalline hydroxyapatite Ca10(PO4)6(OH)2, which provides mechanical support, acts as a reservoir for this sparingly soluble mineral. Human bone is a dynamic tissue that undergoes remodeling throughout life. Osteoclasts begin the remodeling process by dissolving and resorbing bone while osteoblasts synthesize new bone to replace the bone that was resorbed. Less than 2% of our body’s calcium is in the plasma and extracellular fluid. The concentration of calcium in the extracellular fluid is tightly regulated in the range of between 2.2 and 2.6 mmol/l (8.8 to 10.3 mg/dl). This range is of
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importance for many vital functions of the body. The extracellular calcium concentration is maintained by a concerted effect of vitamin D and parathyroid hormone that involves the gastrointestinal tract, bone, and kidney. Calcium is primarily absorbed through the duodenum and jejunum by an active transport process which is facilitated by vitamin D in an acid environment [87]. Only a small amount of dietary calcium is absorbed through passive diffusion. Evidence suggests that both intake and absorption of calcium are reduced in patients who have undergone gastric bypass surgery [88]. In the lowacid environment of the gastric bypass, absorption of calcium carbonate is poor and calcium citrate is recommended [89]. Studies using food frequency questionnaires reviewed that most individuals did not have adequate calcium intake after gastric bypass surgery [90]. This may be due to the fact that after gastric bypass surgery, dairy products are often not tolerable and may provoke dumping syndromes. Therefore, patients may develop an aversion to consume dairy products which are a major source of calcium in the US diet. The current recommendations are approximately 1500 mg/d of calcium citrate for postoperative postmenopausal women [91]. Calcium malabsorption can result in Roux-en-Y gastric bypass as a consequence of bypassing the duodenum. Earlier studies using the stable calcium radioisotope technique in jejunoileal bypass patients have shown that calcium absorption decreased by up to 50% after surgery [92]. After gastric bypass surgery, the duodenum and proximal jejunum epithelium is excluded from contact with food and therefore calcium absorption is compromised. In a study by Riedt et al., the true fractional calcium absorption using the stable isotope technique was decreased by 33% after Rouxen-Y gastric bypass surgery [88]. However, after gastric bypass surgery, hypocalcemia does not generally occur except in severely malnourished patients. The body may compensate the decrease in calcium absorption by increasing parathyroid hormone production which in turn will mobilize calcium from bone in order to maintain calcium hemostosis. The intractability of secondary hyperparathyroidism, as seen in 40% of gastric bypass patients even after supplementation with 50 000 IU vitamin D per week, lends urgency to the discovery of the levels of calcium and vitamin D that can overcome this disturbance [93]. As active vitamin D metabolites are required in the active, transcellular transport of calcium in the intestine, lower levels of 25(OH)D in postoperative patients can further compound the severity of calcium malabsorption following RYGBP surgery [94]. Reduction in intestinal absorption of the lipid-soluble pre-/pro-hormone vitamin D leads to a decrease in the circulating concentrations of the vitamin D pro-hormone, 25(OH)D. This event, in turn, diminishes the efficiency of intestinal
calcium absorption, leading to a drop in the serum ionized calcium level with subsequent stimulation of parathyroid hormone (PTH) synthesis and release. Acting on the kidney, PTH stimulates expression of the CYP27B1-hydroxylase gene that converts 25(OH)D to the active vitamin D hormone, 1,25(OH)2D. A persistent rise in PTH stimulates bone turnover such that bone resorption outstrips formation. Increased resorption allows for (1) mobilization of calcium from bone to restore the serum calcium to normal, (2) release of the skeletal matrix protein osteocalcin into the general circulation which acts in fat to increase adiponectin and limit the release of leptin into the serum [95] and (3) release of FGF-23 from the bone into the general circulation which acts in negative feedback fashion to limit PTH-driven CYP27Bi-hydroxylase expression [96]. Therefore, adequate calcium intake and maintaining vitamin D sufficiency in the body are equally important for preserving bone mass in patients who have undergone gastric bypass surgery. It has also been suggested that magnesium deficiency, which is prevalent in the population at large and a greater risk after bariatric surgery because of the tendency of magnesium to bind with fatty acids, and be lost during fat malabsorption, is critical for passive calcium resorption. An extracellular calcium/ magnesium-sensing receptor has been reported as responsible for parathyroid sensing of serum calcium concentrations [97].
EVIDENCE OF EFFICACY OF SUPPLEMENTAL VITAMIN D Although recommendations continue to be vague, and in some places range from 600 to 50 000 IU/day, the standard recommended level of vitamin D supplementation in bariatric patients is currently between 800 and 2000 IU/day [91]. Supplementation among RYGBP patients using three dosages of vitamin D3 (i.e., 800, 2000, and 5000 IU/day) over a 12-month testing period, resulted in a small increase in serum levels of the major circulating metabolite of vitamin D, 25(OH)D [98]. In general, both 2000 and 5000 IU/day resulted in higher circulating levels of 25(OH)D, but neither was adequate to raise all subjects into the desired range (>30 ng/ml or 75 nmol/l). The 25(OH)D responses to 5000 IU per day were noted in nine of ten subjects. Even at this dose, three of ten patients remained under a serum 25(OH)D level of 75 nmol/l. In this trial, serum calcium did not change significantly and there were no cases of hypercalcemia or sustained hypercalciuria. The only other published trial of vitamin D supplementation in RYGBP patients was at a level of 50 000 IU/week among 60 patients selected for vitamin D deficiency (<20 ng/ml). In these patients vitamin D
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levels almost doubled, increasing from 19.7 to 37.8 ng/ ml after 12 months. No severe adverse events were seen. Bone loss at the hip, in those women in which they were able to measure it, was significantly retarded by 33% as compared to placebo; however, half the women did not fit in the DXA instrument used. This dose did not prevent secondary hyperparathyroidism and PTH levels remained unchanged at 40% of the studied population 1 year postop, suggesting that the dose of vitamin D was partially effective in preventing bone loss but not adequate to stem it. Thus, vitamin D replacement at greater than 5000 IU/day may be necessary in many patients to treat vitamin D deficiency following Roux-en-Y gastric bypass [99].
The Threshold Theory of Vitamin D and Calcium Absorption Efficiency Heaney suggests a threshold behavior of vitamin D such that a level of sufficiency is required for calcium absorption efficiency. He provides evidence to support his thesis that when 25(OH)D levels fall below 80 nmol/l or 32 ng/ml, calcium absorption drops and PTH rises and conversely when 25(OH)D levels average 86.5 nmol/l, calcium absorption efficiencies are 45e65% greater than at 25(OH)D levels for 50 nmol/l [100]. It is important to note that the mean 25(OH)D levels below this threshold are reported in all RYGBP studies, whether the period is 1 year post-surgery or 10 years after gastric bypass surgery. Consistent with this is the finding of significant reductions in fractional calcium absorption after surgery [88]. Heaney’s argument for a serum threshold to achieve normal calcium absorption is also supported by the persistent residual hyperparathyroidism noted in patients that are initially vitamin D deficient and do not rise above mean 25(OH)D levels of 38 ng/ml even following supplementation at 50 000 IU/week [93].
CONCLUSION Bariatric surgery is becoming commonplace in western countries and the rates continue to increase. RYGB, restrictive procedures, and sleeve gastrectomy are all increasingly employed worldwide. These diverse surgical procedures each have different success rates, different metabolic sequelae, and different influences upon nutrient adequacy. Rapid and severe loss of bone density is, however, seen with rapid weight loss by bariatric surgery across procedures, a problem that has not yet been successfully ameliorated through vitamin D supplementation at levels up to 5000 IU/day. Concomitant hyperparathyroidism remains prevalent.
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Post-surgical bone density change is a confirmed, disconcerting consequence of RYGBP and of unknown magnitude in sleeve gastrectomy patients. This may be modulated by leptin concentration changes due to depletion of adipose tissue [101]. The regulatory role of adipose tissue on bone, through the influence of adipokines on osteoblast and osteoclast number, has recently been established. Research on adipokineeosteokine interactions as well as the role of gastric hormones after various types of bariatric surgery is needed to disentangle the role of weight loss, fat loss, malabsorption, and preexisting micronutrient (including vitamin D) deficiencies in this patient population. The focus of such research should be on RYGB sleeve gastrectomy and restrictive procedures due to their popularity. Whether an adequate supply of vitamin D to meet some yet to be determined circulating threshold level may overcome these weight-loss-related changes and stem the PTH-driven bone loss remains to be demonstrated. Previous trials at levels up to 7000 IU vitamin D show a modest reduction of bone loss is possible, but in line with the threshold hypothesis higher doses appear necessary to reverse the concomitant hyperparathyroidism and prevent bone loss. Regardless, more research on approaches to prevent metabolic bone disease, including strategies involving preoperative loading with vitamin D, is needed.
Acknowledgment We are grateful for the assistance of Jasmine Y. Chen in the careful editing and formatting of this chapter.
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C H A P T E R
56 Genetics of the Vitamin D Endocrine System Andre´ G. Uitterlinden Erasmus Medical Centre, Rotterdam, The Netherlands
INTRODUCTION The secosteroid hormone vitamin D, its receptor (VDR), and the metabolizing enzymes involved in the formation of the biologically active form of the hormone, acting together, are major players in the vitamin D endocrine system. This system plays an important role in skeletal metabolism, including intestinal calcium absorption, but has also been shown to play an important role in other metabolic pathways, such as those involved in the immune response and cancer. In the immune system, for example, vitamin D promotes monocyte differentiation and inhibits lymphocyte proliferation and secretion of cytokines, such as IL2, interferon-g, and IL12. In several different types of cancer cells, vitamin D has been shown to have antiproliferative effects. These aspects of the vitamin D endocrine system are extensively discussed in other sections of this volume. At the same time it is also widely known that large inter-individual differences exist, e.g. in circulating serum vitamin D levels, but also in the risk of diseases in which vitamin D plays a role. One approach to understand the underlying molecular causes of such interindividual differences in the vitamin D endocrine system is to study the influence on phenotypes of variations in the DNA sequence of genes encoding important proteins of the vitamin D endocrine system. For example, very rare and deleterious mutations in the VDR gene cause 1,25-dihydroxyvitamin-D-resistant rickets, a rare monogenetic disease (see Chapter 65). Functional analysis of these rare DNA variants has taught us much about function of the VDR and its relation with disease. Yet, these variations are rare in the population and occur only at a few positions of the VDR gene. We now know there is a multitude of DNA sequence variations across the human genome
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10056-3
sequence, and such more subtle and commonly occurring sequence variations are called “polymorphisms” (more or less arbitrarily when they occur at a frequency of at least 1% in the population). We currently estimate there to be some 30 million positions across the human genome where variation in the population exists, on average 1 out of every 100 base pairs (bp). With a typical human gene being 100 000 bp, this high frequency results in a variance between individuals at every gene sequence in the human genome. And this includes of course the genes encoding proteins that are important in the vitamin D endocrine system, such as the VDR gene and the DBP gene. Therefore, the study of these polymorphisms may also be valuable in understanding more about the mechanism and downstream consequences of vitamin D action. However, while many of these polymorphisms have now been identified in large-scale sequencing projects such as dbSNP [1] (www.ncbi.nlm.nih.gov/snp), their effects on protein function are poorly understood. For example, the influence on the vitamin D endocrine system of VDR variants in particular has been under scrutiny in relation to a number of so-called complex diseases and traits, such as osteoporosis. Until recently these studies in the genetic dissection of complex traits (such as osteoporosis, immune disorders, or vitamin D levels, etc.) were mostly done in the framework of so-called “candidate gene approaches.” Yet, because of developments in genotyping technologies, more recently these studies have shifted to the so-called hypothesis-free approach using genome-wide association studies or “GWAS.” The interpretation of polymorphic variations in candidate genes of the vitamin D endocrine system (such as the VDR gene or the DBP gene) is severely hindered by the fact that, until recently, only few polymorphisms in these genes were studied. Initially, most of these were anonymous restriction fragment length
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polymorphisms (RFLPs), while later studies employed single nucleotide polymorphisms (SNPs) detected with a range of methods. One expects such variations to act as markers that are linked to truly functional polymorphisms elsewhere in the candidate gene (or in nearby gene(s)) which can then explain the associations observed. Thus, to understand the mechanisms underlying the associations one has to analyze the genomic organization of the loci, to identify which genes are present in the chromosomal area, to categorize all relevant polymorphisms, to determine the linked alleles (or haplotypes) across the gene, to determine their relationship with the markers used so far, and, finally, to perform association analyses with relevant phenotypic endpoints such as disease. While this used to be cumbersome, slowly progressing, and, above all, lead to controversial results, this situation has dramatically changed in the past 5e10 years. This is mainly due to the advent of the human genome project and its sequelae, such as the dbSNP database [1] (www.ncbi.nlm.nih.gov/snp) and the HapMap project [2] (hapmap.ncbi.nlm.nih. gov), as well as with the development of technology permitting a determination of the genotype for hundreds of thousands of SNPs in a single DNA sample in parallel using array technology. Application of such SNP arrays on large well-characterized sample collections has led to the introduction of hypothesis-free genome-wide searches for association of genetic variation to phenotypes of interest: the GWAS. Because so many polymorphisms are genotyped that are spread across the genome, GWAS can be seen as assessing the complete human genome across many samples for variation(s) that show association with a phenotype of interest. In addition, the standard design of the GWAS approach is a substantial improvement on the disadvantages of candidate gene studies in the past (such as small sample size and lack of replication in initial studies, leading to many irreproducible results). GWAS has been very successful in the past few years (see [3] www.genome.gov/GWAstudies for an overview of results) and has also been applied more recently to phenotypes that are relevant for this chapter on the genetics of the vitamin D endocrine system. Thus, below we will discuss which approaches genetic studies can take, and how genetic studies have evolved over the past decade, and then focus on some candidate genes and some GWAS on relevant phenotypes within the vitamin D endocrine system, and discuss the future perspectives.
GENETIC STUDIES Most common diseases such as diabetes, osteoporosis, and cardiovascular diseases as well as many
disease-related so-called intermediate traits or endophenotypes such as cholesterol levels, glucose levels, vitamin D levels, and bone mineral density (BMD) have strong genetic influences, as has been shown in many twin studies. Yet, the identification of genetic factors underlying these disorders and traits and clarifying their genetic architecture has been very problematic, given the complex nature of the phenotypes and the limited molecular tools available at the time to identify the underlying genetic factors. Several approaches have been used in the past decades to identify the genes responsible for complex traits and diseases and these are briefly discussed below (also reviewed in [4]).
Linkage Analysis Linkage analysis has been the most widely used approach for gene discovery in monogenic Mendelian human disease. It is a genome-wide, hypothesis-free, and top-down approach in which the (human or animal) genome is scanned for chromosomal loci showing cosegregation with the phenotype of interest. We can distinguish two subtypes of linkage analysis, parametric and non-parametric linkage analysis. Parametric linkage analysis involves specifying a model of inheritance for the disease within a family (such as dominant or recessive) and looking for evidence of segregation of the disease within a family according to that model. Linkage studies are usually carried out on a genome-wide basis which involves genotyping between 400 and 800 DNA markers (micro-satellites) spread at regular intervals across the genome. In recent years the microsatellite markers have been replaced by arrays with 10e50 000 SNPs. Non-parametric linkage has been more widely used for analysis of complex traits and diseases, and specify no model of inheritance but instead assume sharing of inherited alleles in relation to sharing of the disease phenotype. By observing a high correlation of phenotypic variance with genetic variance between relatives for a given DNA marker, we assume that the DNA marker is tightly linked to the disease-causing mutation, thus identifying a chromosomal position for the disease locus. The results of linkage studies are expressed as lodscores which are defined as the logarithm of the odds that the disease locus and marker locus are linked on the chromosome. In the case of parametric analysis, linkage is significant when the lodscore is >þ3.3, and “suggestive” when >þ1.9, while for non-parametric analysis these values are >þ3.6 and þ2.2. The weakness of these approaches is that they rely on the presence of a single mutation of very strong effect causing the disease (highly penetrant variants), and that they require large numbers of such related individuals especially in the case of mapping complex traits and diseases. Although linkage analysis has been very
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successful in identifying gene mutations underlying rare monogenic diseases, it has largely failed to identify genes involved in common complex diseases and traits as seen in the general population.
Linkage Studies in Animals Linkage studies in animal models provide another possible way of identifying genes which regulate complex phenotypes. These approaches rely on the assumption that at least some of the genes that regulate these phenotypes in animals will be the same as those in humans. Such animal studies have mostly involved crossing inbred laboratory strains of mice with low and high values of the phenotype of interest. By interbreeding offspring from the first generation (F1), a second generation (F2) of mice can be established with varying levels of the phenotype, because of segregation of the alleles which regulate the phenotype in the F2 offspring. A genome-wide search is then performed in the F2 generation and inheritance of alleles related to variance of the phenotype in the offspring. There are several advantages of these studies; the environment can be carefully controlled (thus minimizing the influence of confounding factors) and large numbers of progeny can be generated, giving excellent statistical power for linkage analysis. However, fine mapping of loci identified is challenging but can sometimes be achieved by backcrossing mice which inherit a locus for regulation of the phenotype of interest into the background strain, and selecting offspring which retain the phenotype. However, this is non-trivial and a timeconsuming process since the loci identified by linkage studies in inbred strains of mice are large (20e40 cM) and many generations of backcrossing need to be performed to narrow the critical interval to manageable proportions. In order to circumvent this problem, other strategies have been proposed such as performing genetic mapping in outbred mice, taking advantage of the smaller linkage disequilibrium in outbred strains to obtain a narrow region of interest. The abovementioned approach has been successful for some quantitative traits, but is too laborious for widespread application in complex genetics also given the necessary assumption of phenotypic equality between humans and mice.
Association Studies We now know that complex traits and diseases are typically (so not always) influenced by many genetic variants each with modest effect size while the variability in expression of the disease phenotype is most likely also influenced by environmental factors in interaction with the genetic factors. Two approaches were
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most commonly used in the past two decades to identify genetic susceptibility factors for such complex diseases: the (top-down) genome-wide linkage approaches and the (bottom-up) candidate gene approach building upon biologically mechanistic insights of relationships between certain genes and certain phenotypes. Currently, we know that linkage approaches in related subjects are unsuccessful in identifying genetic factors in complex traits and diseases, due to the low power to detect the subtle effects. In addition, the approach has low “genetic resolution,” i.e. very large chromosomal areas are (potentially) identified containing many possible candidate genes. Yet, while association studies have potentially bigger power, they have frequently suffered from irreproducible results mostly due to limited samples size and lack of standardization in phenotyping and genotyping. Several lessons have been learned from these initial attempts and have led to the formation of large consortia of collaborating investigators to address these problems. In addition, due to technological developments the association study design has further expanded its area of applications by now applying it on a genome-wide scale with an unprecedented density of genetic markers. This has led to the genome-wide association study design or GWAS. This renaissance has mostly been driven by the discovery of millions of single nucleotide polymorphisms or SNPs throughout the human genome and the development of so-called micro-array technology to type such SNPs accurately on a massive parallel scale.
Candidate Gene Association Studies Candidate gene association studies have been widely used in the genetics of complex traits and diseases over the past decades. They involve analyzing polymorphic variants in candidate genes with a known role in biology and relating carriage of a specific allele (or haplotype) to a quantitative trait or disease of interest. While initially this was limited to a single candidate gene with strong support for its involvement in the biology of the phenotype of interest, this can also be expanded to study all genes involved in a certain pathway or simply all candidate genes known. Caseecontrol study designs are used for categorical traits such as absence or presence of disease (e.g., diabetes, fracture, dementia, etc.) where allele frequencies are compared in the two groups. For quantitative traits such as glucose level, vitamin D level, or BMD, the mean values are calculated according to genotype or allele at the chosen polymorphic site and differences are assessed by analysis of variance, usually with inclusion of confounding factors in the statistical model (such as age, time of measurement (daytime, season), body weight, etc.). Association studies are straightforward in design, relatively easy to perform,
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and can be powered by expanding the sample size to detect even small effects of alleles. However, when executed carelessly they are prone to give spurious results, due to factors such as small sample size, lack of standardized phenotyping (comparing apples with pears) and genotyping (using studies with different genotyping methodologies in a single analysis), and population stratification when insufficient care has been paid to matching cases and controls. Another drawback of the candidate gene association studies performed so far has been the fact that only a very limited number of variants have been assessed across a gene of interest. However, we now know (see [1,2]) that most genes contain hundreds of common polymorphisms as well as many rare DNA variants. Since it is unknown a priori which of these is most likely to be involved in the phenotype of interest, it is important that analysis of candidate genes should be as comprehensive as possible. Until recently this was challenging but the prospects for comprehensive coverage of candidate genes have improved with advances in genotyping techniques. The transmission disequilibrium test (TDT) is a special type of association study performed in related individuals, which is less susceptible to confounding than a standard association study. Prior to the introduction of GWAS, this technique was widely used to reassess the results obtained from population-based candidate gene association studies. The TDT tests the hypothesis that a particular allele of a given polymorphism contributes to a trait or disease, by analyzing the frequency with which affected individuals inherit the allele from a heterozygous parent. If the allele contributes to the trait or disease of interest, then the probability that an affected person has inherited the allele from a heterozygous parent should vary from the expected Mendelian ratio of 50:50. Because the transmitted allele acts as the “case” and the non-transmitted allele acts as the “control,” the TDT is unaffected by confounding variables due to population stratification. Although TDT is a valuable technique, one important disadvantage is that only heterozygous individuals are informative (like in linkage analysis), which can reduce the effective sample size available for study and limit statistical power. In addition, the power is already limited to start with by the requirement to collect related individuals (trios of two parents and one offspring). It has therefore never been widely applied in candidate gene testing. Most of the problems of candidate gene association studies can be circumvented by careful study design, including the assembly of cohorts of adequate sample size and statistical correction for confounding factors. Many of these issues are being addressed by the creation of large consortia to address the genetic contribution to various complex diseases. For example, within the
osteoporosis field, the GENOMOS ([5] www.genomos. eu) and GEFOS ([6] www.gefos.org) consortia have been established to address the role of common genetic variants in the pathogenesis of osteoporosis. The GENOMOS consortium has focused on testing known candidate gene polymorphisms in a large-scale setting involving approximately 45 000 subjects, whereas the GEFOS consortium focuses on performing metaanalysis of GWAS datasets from about 20 000 subjects. Particularly in the field of vitamin D biology we have recently seen the formation of the SUNLIGHT consortium (Study of UNderLyIng Genetic determinants of vitamin d and Highly related Traits) which will be discussed below.
Genome-wide Association Studies Advances in genotyping technologies have now made it possible to perform association studies on a genome-wide basis by genotyping large numbers (100 000 to 2 000 000) of single nucleotide polymorphisms (SNP) spread at close intervals across the genome, rather than focusing on a specific candidate gene. Genome-wide association studies or GWAS have been successfully applied to the study of >150 complex traits and diseases and have identified >1000 loci across many different diseases and traits in less than 4 years (see [3] www.genome.gov/GWAstudies for an overview and [7] for a review). GWAS consists of screening the genome of many hundreds to thousands of subjects in a caseecontrol study or population base cohort study, with >500 000 single nucleotide polymorphisms (SNPs), followed by a simple association analysis between a phenotype and all the genetic markers. Such a GWAS then identifies genetic markers associated to the phenotype of interest with a certain statistical significance. This then assumes the genetic marker to be directly causal for the phenotype of interest, or, more likely, the marker to be linked to the causal variant elsewhere in the region identified. The size of the region is dictated by the linkage relationships among the markers used and usually encompasses 5000e50 000 bp. These are the so-called linkage blocks in the human genome which have originated through evolution as a result of recombination and mutation events across many generations. From data in the HapMap project we currently estimate there to be ~1 million of such linkage blocks across the human genome. Because of the multiplicity of testing with so many markers in a GWAS certain thresholds have been considered to declare an association “genome-wide significant” (gws). For example, when analyzing 500 000 markers this threshold is 1.107 (0.05/500 000) but given more recent imputation approaches to exploit the linkage relationships between the millions of SNPs in
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the HapMap database, 5.108 is now a more widely used gws level in GWAS. This latter threshold uses the presence of the estimated ~1 million linkage blocks and arrives at 5.108 as 0.05/1 million. Naturally, the chance of seeing such gws associations depends on factors such as the effect size of the markers on the phenotype, and the size of the study sample. Usually, a typical GWAS consists of a discovery sample with GWA data and a replication sample with GWA data. From the discovery GWAS the “top hits” (for example, all genetic markers that reach a significance of 1.104 or less) will be analyzed in the replication GWA dataset to see which markers will reach the gws level after combining the two datasets. Following such a GWAS, the remaining top hits (for example, any marker that has p < 1.105) will be analyzed in subsequent replication cohorts which do not necessarily have GWA data but which can be genotyped for the particular genetic markers identified. Thus, we have experienced a plethora of these GWAS that have produced dozens of common variants that confer modest risk for a variety of common disorders and traits. Yet, the current round of GWAS tend to focus on this low hanging fruit while we know that there are many more such common and less common variants to be discovered with less impressive p-values in the discovery phase due to even smaller effect size and/or even smaller population frequency. Identifying such small effects is possible but will require even larger sample sizes to detect them in a statistically robust and convincing way. While all GWAS currently focus on the common variants (say, >5% population frequency) we also suspect that there are less common variants (0.5e5% population frequency) and even rare variants (<0.5% population frequency) that will contribute to explain risk of disease. Examples highlighting the existence of such less common variants were demonstrated for sequence variations in cholesterol-related genes, in relation to cholesterol levels [8,9]. The challenge for the future will therefore be to identify also such more rare variants through deep sequencing approaches of the many genes identified through GWAS. Combinations of such rare and the more common genetic variants in these particular genes can then be scrutinized for their diagnostic potential in large well-phenotyped cohorts, also in relation to the more classical risk factors. Such combinations of genetic risk factors are expected to explain more of the genetic risk for particular common diseases than just the common variants or just the (very) rare variants. The explained genetic variance in complex genetic traits and diseases by the recently uncovered common variants in risk genes by GWAS, e.g. for diabetes, height, or bone mineral density, is still limited to, at most, 10%. Yet, we must realize these are still early days in complex
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genetics because we have just uncovered the “low hanging fruit” in these first rounds of GWAS. The complex genetics research community has now organized itself into still growing consortia of research groups who join (GWAS) datasets and are providing excellent forums for harmonizing phenotype definitions, such as GEFOS [6] (www.gefos.org), CHARGE [10] (web.chargeconsortium.org), and ENGAGE [11] (www.euengage.org). Such consortia will provide a very good testing ground to uncover more genetic variants in various disease areas. A major advantage of GWAS over candidate gene studies is that they offer the possibility of ranking the various association signals according to strength of the association signal across the genome, and of identifying novel pathways that contribute to the trait which is being studied. Disadvantages include the fact that currently available marker sets are designed to identify common alleles and are not well suited to study the effects of rare polymorphisms (<1e5% population frequency) within a gene of interest. The resulting dataset also allows one to assess in a comprehensive way, common variants across particular candidate gene(s). The statistical thresholds for significance in GWAS are stringent (p < ~1 107 or <5.10e8 when using imputed data) due to the large number of tests performed. In view of this, many polymorphisms that truly contribute to a trait but with a (very) small effect size will be missed by individual GWAS (of limited sample size of the discovery dataset) if one were to adhere strictly with formal thresholds for significance. In order to circumvent this problem researchers are starting to further increase the sample size by combining different GWAS (see below under “Meta-analysis”).
Genome-wide Sequencing Sequencing technology is now capable of generating a catalog of almost all variants present within a given DNA sequence, rather than having to rely on markers and patterns of linkage disequilibrium. These sequencing techniques are, for example, being used for analysis of selected areas such as candidate loci which have emerged from GWAS, or for analyzing the complete coding part of the genome, called the exome. These techniques will also provide complete human genome sequences in large collections of samples such as the “1000 genomes” project [12] (www. 1000genomes.org). This project aims to fully sequence the genome of 1000 individuals and to use this information as the basis for inferring (imputing) genetic variants in subjects who have been genotyped for a less dense set of markers (such as by GWAS). This will result in a second surge of genetic association studies generating
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comprehensive collections of sequence variations, common, less common, and (very) rare, including de novo events in individuals.
Meta-analysis The technique of meta-analysis is now common practice in the field of complex genetics because it provides the necessary statistical power which is required to detect the subtle effects of common genetic variants. Meta-analysis can be done retrospectively (based on published studies) and prospectively (with new and unpublished data). Retrospective meta-analysis combines data from different published studies to enhance sample size and obtain a more accurate estimate of the effect size of individual genetic variants than can be achieved by analysis of single studies. It is applicable to many study designs, from family-based linkage studies and population-based association studies to genome-wide linkage scans and GWAS. Prospective meta-analysis seeks the same increase in power by combining datasets, but uses unpublished datasets in which de novo genotyping has been performed. This approach is much more robust than retrospective meta-analysis since it will encompass standardization of phenotyping and genotyping, and it circumvents the problem of publication bias which can inflate the estimates derived from retrospective metaanalysis. A limitation of meta-analysis is that there is a selection for those effects and directions of a given genetic variant that are the same in all groups included in the meta-analysis. When such groups come from global efforts (such as in most if not all current consortia) meta-analysis thereby selects those variants that have universal effects across different human populations. Since geneeenvironment interactions are usually population-specific, meta-analysis of groups coming from the same genetic, geographical, ethnic, and cultural background is required to detect effects of common variants in such populations. Given the success of GWAS this underlines the necessity of generating GWAS data for as many epidemiological datasets as possible, especially within a country.
Functional Studies When an allelic association has been identified and replicated, the next step is to try to define the mechanism that underlies the association. For Mendelian diseases, functional analyses are usually straightforward since the causal mutation(s) can easily be identified given that they segregate with the disease in families and usually have a major effect on the protein-coding region of the gene. The effects of the mutation on function of the target protein can then be defined by in vitro studies of
the abnormal protein or by generating an animal model in which the disease-causing mutation has been knocked into the germ line of a model organism. It is much more difficult to define functional mechanisms for alleles of small effect, partly because the causal variant is difficult to identify among the many variants in a region, and because the effect size is small. Alleles associated with complex traits usually cluster together with a large number of other variants in the chromosomal area which are in linkage disequilibrium (LD) and which also could be responsible for the effects observed. In addition, such genetic associations can be identified in a chromosomal area without any annotation regarding function of that area (e.g., no gene is located there). While this can highlight existing new biology (such as presence of long-range regulatory regions containing transcription factor binding sites), it will be challenging and time consuming to characterize the exact underlying mechanisms. Finally, the relatively subtle effects detected on the phenotype of interest also require meticulous functional experiments with correctly chosen intermediate read-outs in the test systems of sufficient sample size to overcome the inevitable noise in many experimental systems. However, it is possible to gain insights into the mechanisms by which alleles of small effect regulate phenotype by performing a deletion of the gene in question, e.g. in an animal model. This was highly successful in the case of the FTO gene which was initially identified as susceptibility gene for type 2 diabetes which regulated body weight [13]. At the time of its original discovery, the function of FTO was poorly known but targeted deletion of the gene in mice demonstrated that it protected against obesity by affecting energy homeostasis [14]. In a similar way, one locus (i.e., the SORT1 locus) out of the 95(!) loci discovered in a very large meta-analysis of GWAS data (n ¼ ~100 000 subjects) on cholesterol levels, was functionally characterized by a range of experiments [15,16]. Thus, similar experiments can be contemplated for the novel genes coming from the many GWAS for complex traits and diseases including those relevant for the vitamin D endocrine system. Yet, because there are several complicating factors progress in this area is expected to be much slower than progress in identifying the responsible genetic factors.
CANDIDATE GENES As examples of two candidate genes that have been more or less extensively studied in the vitamin D endocrine system, we here discuss the vitamin D receptor gene and the vitamin D binding protein, mainly in relation to bone-related phenotypes such as osteoporosis.
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FIGURE 56.1 Vitamin D receptor chromosomal location, gene structure and polymorphisms.
The Vitamin D Receptor Gene The active metabolites of vitamin D play an important role in regulating bone cell function and maintenance of serum calcium homeostasis by binding to the vitamin D receptor (VDR) which regulates expression of various response genes. This has led to the VDR gene being extensively studied as a candidate gene mainly to osteoporosis but also to other diseases. We will here limit discussion to the available evidence for a possible involvement in osteoporosis. In Figure 56.1 the chromosomal structure and location of polymorphisms in the VDR gene discussed is shown. The study of Morrison that initiated the field of genetics of osteoporosis found a relatively strong association by current standards between polymorphisms affecting the 30 region of VDR and circulating osteocalcin levels [17]. In a subsequent study, the same group reported a significant association between a BsmI polymorphism in intron 8 of VDR and BMD in a twin study and a population-based study [18], but this association was later found to be much weaker than originally reported due to genotyping errors [19]. Many association studies of VDR alleles in relation to BMD and/or fracture followed, but with conflicting results, mainly because, as we now know through much larger studies, the effect of VDR polymorphisms on BMD and fracture risk is minimal if present at all. Thus, none of the studies was adequately powered and only looked at a very small number of VDR SNPs. A large-scale meta-analysis of a selected set of VDR alleles (Cdx2, FolkI, BsmI, ApaI, TaqI) in relation to BMD and fracture performed by the GENOMOS consortium involving 26 000 subjects failed to demonstrate any association between the BsmI, ApaI, and TaqI 30 polymorphisms in relation to BMD or fracture [20]. In addition,
the candidate gene meta-analysis of the GEFOS (GWAS) dataset from 19 000 subjects (somewhat smaller in size than the GENOMOS analysis and, thus, somewhat less powered) but now analyzing hundreds of SNPs across the VDR gene locus showed no significant association between any common VDR allele on BMD or fracture [21]. A common polymorphism in exon 2 of the VDR gene is a TeC transition within exon 2, which is a nucleotide polymorphism that is recognized by the FokI restriction enzyme in RFLP genotyping tests [22,23]. This transition introduces an alternative translational start codon that results in a shorter isoform of the VDR gene [22]. Association studies between this polymorphism, BMD and fracture have yielded conflicting results and in the GENOMOS study of 26 000 subjects no evidence for an association between this SNP and either BMD or fracture was found [20]. Another common G/A polymorphism affecting a binding site for the transcription factor Cdx2 in the VDR 50 promoter was originally found to be associated with BMD in a cohort of 261 Japanese women, with lower bone mass in carriers of the “A” allele [24]. This observation was confirmed in a much larger analysis of the Dutch Caucasian Rotterdam Study, and showed a protective effect on fracture risk which was in line with the association with BMD reported in that same study by Fang and colleagues [25]. An association between the VDR Cdx2 SNP and BMD and vertebral fracture was also found in the GENOMOS study, although the effect size was modest [20]. A comprehensive single study (before the Human Genome Project dbSNP project was fully matured and before the GWAS era) of genetic variation across the VDR gene in relation to osteoporosis-related phenotypes
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was conducted by Fang and colleagues who performed a large-scale study of haplotype tagging SNPs of the VDR gene in 6418 participants of the Rotterdam Study [26]. While some effects on BMD and fracture risk were detected, this was mainly driven by subgroup analysis and the effect sizes observed were modest. In hindsight we now conclude that this study, although comprehensive in terms of genetic variation covered across the VDR gene, has not documented any convincing genetic associations, again because of lack of power due to small sample size. We can hypothesize that the relation between VDR polymorphisms and BMD may be modified by environmental factors such as dietary calcium intake [27,28] and vitamin D status [29], but this has not been investigated in properly powered studies. Intestinal calcium absorption has been associated with the BsmI VDR polymorphism in some small studies [30,31] but this has not been widely replicated. The mechanism by which this occurs is unclear while, for example, no association was found between genotype and mucosal VDR density [32]. A positive association between the FokI polymorphism and intestinal calcium absorption was reported in one small study [33], but two other small studies yielded negative results [34,35]. The largest study of VDR alleles in relation to nutrient intake was that of Macdonald and colleagues who analyzed 3000 British Caucasian women, but they found no associations between VDR alleles and BMD, or any evidence for interaction between VDR alleles and dietary calcium intake, or serum 25(OH)D levels [36]. Several investigators have conducted functional analysis of individual VDR polymorphisms and haplotypes. Reporter gene constructs prepared from the 30 region of the VDR gene in different individuals have shown evidence of haplotype-specific differences in gene transcription, raising the possibility that polymorphisms in this region may be involved in regulating mRNA stability [18]. In support of this view, cell lines which were heterozygous for the TaqI polymorphism showed differences in allele-specific transcription of the VDR gene [37]. In this study, however, transcripts from the “t” allele were 30% more abundant than the “T”, which is the opposite of the result expected on the basis of Morrison’s results [18]. In another study, evidence of differences in allele-specific transcription were observed in relation to 30 VDR haplotypes in bone samples from male subjects in the MrOS study [38]. Specifically, carriage of haplotype 1 (baT) was associated with increased VDR mRNA abundance, and in that study this haplotype was shown to be also associated with an increased risk of fracture in men. In a comprehensive analysis of several cell lines, Fang et al. demonstrated that the baT (haplotype 1) variants were associated with decreased VDR mRNA level [26]. Other in vitro
studies have shown no differences in allele-specific transcription, mRNA stability or ligand binding in relation to the BsmI polymorphism [39e41]. Studies in vitro have shown that different VDR FokI alleles differ in their ability to drive reporter gene expression [22,42] and the polymorphic variant lacking three amino acids (“F”) has also been found to interact with human basal transcription factor IIB more efficiently than the longer isoform (“f”). Finally, peripheral blood mononuclear cells (PBMC) from “FF” individuals were also found to be more sensitive to the growthinhibitory effects of calcitriol than PBMC from “Ff” and “ff” individuals [43]. Contrasting with these results, however, Gross and colleagues found no evidence of functional differences between FokI alleles in terms of ligand binding, DNA binding, or transactivation activity. There is good evidence that the Cdx2 polymorphism within the promoter of the VDR gene is functional. Arai and colleagues noted that the G allele had reduced affinity for CDx2 protein binding and also had a 70% reduced ability to drive reporter gene expression compared with the A allele [24]. While this could be functionally in line with the genetic association studies on BMD and fracture, the effect size observed there is probably very small and therefore not detected in the relevant GWAS studies (such as in GEFOS). In summary, the studies which have been performed to date do not support the hypothesis that allelic variation at the VDR locus plays a major role in regulating bone mass or osteoporotic fracture. There is evidence that some of the polymorphisms described have functional effects, such as the Cdx2 polymorphism, at least in vitro, while for this variant there is also evidence to suggest that there may be an association with vertebral fracture risk, albeit modest.
The Vitamin D Binding Protein An important candidate gene in explaining interindividual variations in the vitamin D endocrine system is the vitamin D binding protein (DBP). In Figure 56.2 the chromosomal structure and location of polymorphisms in the DBP gene are shown. DBP was initially named group-specific component (Gc) and is a serum protein with different known functions. Three functions of DBP involve skeletal metabolism by binding to vitamin D. DBP: (1) binds to vitamin D metabolites (e.g., 25-hydroxyvitamin D3 (25(OH)D3), the major circulating metabolite, and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active form of vitamin D) at the sterol binding domain (domain I in Fig. 56.2(a)) [44], (2) transports vitamin D to liver, kidney, bone, and other target tissues, and (3) stores and prolongs the half-life of the circulating vitamin D metabolites [45]. Vitamin D metabolites are strongly
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FIGURE 56.2 Vitamin D binding protein chromosomal location, linkage blocks and polymorphisms.
and positively correlated to DBP levels in serum [46,47]. Besides its role in vitamin D metabolism, serum DBP can also be converted to a DBP-Macrophage Activating Factor (DBP-MAF) by the deglycosylation of DBP at the non-sterol binding domain (domain III in Fig. 56.2(a)), which involves the osteoclast activating domain [48,49]. DBP-MAF plays a role in osteoclast differentiation [50] and mediates bone resorption by directly activating osteoclasts [48]. DBP-MAF-treated osteopetrosis rats have increased number and activity of osteoclasts and decreased bone mass [51]. Hence, the contribution of DBP to bone metabolism is not only through assisting the vitamin D endocrine system but also directly influencing bone resorption. The three most commonly studied protein variations are identified by two polymorphisms in exon 11: a G/A substitution at codon 416 (E416D; rs7041), leading to a Glu/Asp amino acid change, and a C/A substitution at codon 420 (T420K; rs4588), leading to a Thr/Lys amino acid change [52]. Haplotypes of the nucleotide variants result in the protein isoforms Gc1s, Gc2, and Gc1f (Fig. 56.2(b)). By analysis of two DBP SNPs (Glu416Asp and Thr420Lys) in 6181 elderly Caucasians [53] it was shown that domains I and III (the III domain containing the two SNPs) are in different LD blocks (see Fig. 56.2). This indicates that all observed associations and interactions with the most widely studied Glu416Asp and Thr420Lys polymorphisms are independent of polymorphisms in the region corresponding to domain I. The protein and genomic structures also reveal at least two separate functions of the DBP in bone remodeling. The vitamin D binding region of DBP is located between residues 35 and 49 at the N-terminal end
(domain I in Fig. 56.2) [9]. Domains II and III are responsible for the non-sterol binding activities of DBP, while the macrophage/osteoclast activating activity is also related to domain III. The glycosylation of DBP in domain III is important for macrophage and osteoclast activation, while it has been shown that binding of vitamin D does not influence this activity [54]. This indicates that domain III plays an independent role from domain I (vitamin D binding domain) in the function of stimulating osteoclast activity. The two most widely studied SNPs are located in the C-terminal end (domain III, from residue 375 to the end) with a single glycosylation site nearby. In a subgroup of 1312 subjects, Fang et al. showed that DBP haplotype 1 (constructed by haplotypes of Glu416Asp and Thr420Lys and corresponding to the Gc1s protein isoform) was positively correlated with both 25(OH)D3 and 1,25(OH)2D3, while haplotype 2 (Gc2) was negatively correlated. Lauridsen et al. [55] demonstrated that the Gc1 isoform (consisting of 80% haplotype 1/Gc1s and 20% haplotype 3/Gc1f in the LD analysis) was associated with 8.5 nmol/l increased plasma 25(OH)D3 and DBP level in Danish Caucasian postmenopausal women, while DBP the Gc2 protein isoform was negatively correlated to plasma vitamin D and DBP level. Thus, the relationship between DBP haplotype 1 (Gc1) and vitamin D level is consistent among different reports in Caucasians [47]. Several studies demonstrated a relationship between a DBP haplotype 1 (or protein isoform, Gc1) with decreased BMD and increased fracture risk, namely one study in Japanese (56) and two in Caucasian populations [53,57]. There is evidence to suggest an
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interaction of DBP with calcium intake in relation to bone phenotypes. The number of osteoclasts significantly increases when rats are in a calcium deficiency condition, and the osteoclast number returned to a similar level as control groups after calcium or phosphorus replenishment [58,59]. Thus, both DBP-MAF and low calcium intake can influence osteoclast number and activity. It can therefore be hypothesized that DBP and the vitamin DeVDR system might influence bone resorption (via osteoclasts) and bone formation (via osteoblasts), respectively, which could result in osteoporosis. In the study of Fang et al. [53] among >6000 elderly Caucasians the DBP genotype was not significantly associated with fracture risk in the entire study population, but they observed in a subgroup with dietary calcium intake lower than the median (<1.09 g/day) that the DBP haplotype1-homozygotes (Gc1) had increased risk for fracture. Yet, while this interaction was not replicated in that study, interestingly, Lauridsen et al. [57] showed a similar relationship between DBPGc1 and fracture risk in a population of Danish Caucasians with a dietary calcium intake of 0.8 g/day level [55], while another study, in a Japanese population [56], found the Glu allele at codon 416 (corresponding to DBP haplotype 1, and protein isoform Gc1) to be associated with decreased radial BMD. The average dietary calcium intake of Japanese populations is considered to be much lower (0.6 g/day) [60] than the Dutch and the Danish study populations. So, also for DBP haplotype 1 (Gc1) and low calcium intake the studies seem to be in line with each other, but formal meta-analysis has to demonstrate this. This relationship cannot be explained by the effect of DBP on the vitamin D endocrine system since increased total vitamin D is not associated with increased fracture risk. Only free vitamin D hormone, which is only 12e15% of the total vitamin D level, has biological activity, rather than bound hormone [61]. Thus, the observed relationship between DBP and fracture risk could result from the function of DBPMAF in bone resorption. Thus, DBP haplotype 1 (Gc1) is associated with increased serum DBP level and might also be associated with increased DBP-MAF level relative to non-Gc1carriers. Since DBP-MAF is an activator of osteoclasts, this could result in increasing bone resorption and eventually osteoporosis.
GENOME-WIDE ASSOCIATION STUDIES Below we present some examples of GWAS for traits related to the vitamin D endocrine pathway, as examples of the results that GWAS will yield in the vast field of vitamin-D-related phenotypes and diseases. For BMD
this has already progressed substantially and large meta-analyses are ongoing by the time of writing. For vitamin D serum levels this has just begun and only the first meta-analysis is now known.
Bone Mineral Density The very first attempt to identify BMD loci through GWAS is presented by investigators from the Framingham study using the 100K Affymetrix platform and a limited sample size of n ¼ 1141 men and women [62]. This effort did not result in BMD loci that reached the so-called genome-wide significance (gws) and made it clear that larger sample sizes were to be used and also genotyping platforms with a higher genome coverage such as the Illumina 317K or 550K platforms. Subsequently, two GWAS on BMD were published that identified several loci contributing to BMD that all reached genome-wide significance. One study came from analysis of the TwinsUK cohort and the Rotterdam Study looking at Caucasian women from the UK and the Netherlands, respectively [21], and the other study was from deCODE and based on Icelandic subjects [63] (see Table 56.1 for an overview of some GWAS studies on BMD). One locus was overlapping between the two efforts, i.e. the OPG locus on chromosome 8, while the TwinsUK/Rotterdam Study effort reported on additional locus, i.e. LRP5, and the deCODE GWAS reported four additional loci. LRP5 was interesting because this gene and indeed this particular SNP (the exon 13 one) was just 1 month earlier reported by the GENOMOS consortium to be associated with very high confidence to BMD in the study by van Meurs et al. [64]. The explained variance of the genetic factors for BMD that was reached by both studies was low (1e3%). This indicates that, very similar to the earliest GWAS results for height [65], BMD is a truly complex trait with many loci (hundreds?) of small effect. This also indicates that even larger samples sizes than the one used by the deCODE study (n ¼ 13 000) are necessary to identify these additional common factors. Taken together, this now puts the GEFOS effort in the spotlight because this consortium is able to eventually accumulate >40 000 samples with GWAS data and therefore is well powered to identify the second wave of BMD loci in combination with the expanded GENOMOS consortium to include >100 000 replication samples. Initially, the GWAS in osteoporosis is focusing on BMD as a normally distributed quantitative trait as risk parameter for osteoporosis. Yet, in addition, it will be possible across the several GWAS to identify risk alleles for fracture risk (all types of fracture, hip fracture, vertebral fracture, etc.) and other phenotypes in osteoporosis such as
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TABLE 56.1
Characteristics of Genome-wide Association Studies for Osteoporosis and Vitamin D Level
Study
FOS*
TwinsUK
deCODE 1
GEFOS
SUNLIGHT
Reference
[62]
[21]
[63]
[66]
[68]
Discovery sample
Caucasians USA (n ¼ 1141)
Caucasian twins UK (n ¼ 2094)
Caucasian Icelanders (n ¼ 5861)
5 Caucasian cohorts (n ¼ 19 125)
5 Caucasian cohorts (n ¼ 16 125)
Replication cohorts
e
1 “in silico” (n ¼ 4081) 2 “de novo” (n ¼ 2410)
1 “in silico” (n ¼ 4165) 2 “de novo” (n ¼ 3760)
e
5 “in silico” (n ¼ 9367) 5 “de novo” (n ¼ 8504)
Total sample size
N ¼ 1.141
N ¼ 8585
N ¼ 3786
N ¼ 19 125
N ¼ 33 996
Genome-wide significant loci
0
2
5
20
3
Explained variance
e
0.6e1.0%
3%
2e3%
1e4%
* FOS ¼ Framingham Osteoporosis Study.
bone geometry parameters and bone structure phenotypes derived from ChromoTomography and Quantitative UltraSound (QUS) parameters. Yet, bone strength is only one of the risk factors for osteoporotic fracture. Together with loci coming from GWAS on cognitive function, muscle mass, and obesity/fat distribution the bone strength loci will contribute to explain the variance in fracture risk, that is to say, for the common risk alleles involved in osteoporosis. By the time of writing six individual GWAS have been carried out, in addition to the first meta-analysis of GWAS on BMD by the GEFOS consortium. Details of some of these and the GEFOS meta-analysis [66] are summarized in Table 56.1. The genes and loci which have been identified as being significantly associated with osteoporosis with p-values exceeding the threshold for genome-wide significance for BMD (p ¼ 5.10e8) are summarized in Table 56.2, including some summary statistics. From comparing the results of the individual GWAS with the meta-analysis, it shows that the metaanalysis is not just adding up the individual studies. By combing the data across studies several new loci are identified which were not seen by the individual studies. From these results it appears that more than half (58%) of the BMD loci identified by the GEFOS meta-analysis are from novel biological pathways, which were hitherto unknown to play a role in bone biology. In addition, bone-site-specific analysis showed that roughly one-third of the BMD loci showed genome-wide significant effects on both femoral neck BMD and lumbar spine BMD, suggesting more systemic effects of these BMD loci. Finally, for almost half of the loci an effect on fracture risk was observed in secondary analyses of these loci in one cohort (the Rotterdam Study) with fracture data. In summary, the experience with BMD, as with height as a phenotype, shows that adding an increasing number of GWAS datasets will lead to the identification of an increasing number of
genetic loci for the phenotype of interest. This principle is now being exploited for many phenotypes and diseases in complex genetics, leading to an impressive number of loci identified as being involved in many such traits and diseases (see [3] for an overview).
Candidate Gene Association Analysis in GWAS Data A particular use of GWAS data is to “look up” certain candidate genes in the GWAS dataset across different cohorts. Not only are the candidate genes well covered in terms of genetic markers (with usually several hundred SNPs being analyzed per gene), but also the dataset is relatively large, allowing robust statistics to be applied. Over the past decade approximately 150 candidate genes have been investigated in relation to osteoporosis in at least one study for their relationship with BMD or fractures in human population studies. Most of these genes have been investigated in less than five studies and most were underpowered. Accordingly the results of the vast majority of the candidate gene studies performed to date must be treated with great caution, given what we now know about the true effect size of common variants on phenotypes like BMD and fracture. In view of the lack of consistency between candidate gene studies in the osteoporosis field, Richards and colleagues [67] examined the candidacy of 150 genes previously implicated in the pathogenesis of osteoporosis in a cohort of about 19 000 individuals where GWAS data were available within the framework of the GEFOS consortium [6] (www.gefos.org). Here, SNPs within the gene of interest and in the 200 Kb of flanking sequence on either side were analyzed in relation to BMD and fracture. Only nine of the 150 genes analyzed were found to be significantly associated with BMD or fracture. These included SNP within or
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
1036 TABLE 56.2
56. GENETICS OF THE VITAMIN D ENDOCRINE SYSTEM
Genes and Loci by Chromosomal Position with Genome-wide Significant Evidence for Association with BMD or Vitamin D Level Locus
Novela
ZBTB40
1p36
Yes
GPR177
1p31.3
Yes
SPTBN1
2p16
Yes
CTNNB1
3p22
No
MEPE/IBSP/OPN
4q21.1
No
MEF2C
5q14
No
MHC
6p21
Yes
ESR1
6q25
No
STARD3NL
7p14
Yes
FLJ42280
7q21.3
Yes
TNFRS11B
8q24
No
SOX6
11p15
Yes
DCDC1 / DCDC5
11p14.1
Yes
LRP4/ARHGAP1/F2
11p11.2
Yes
LRP5
11q13.4
No
SP7
12q13
No
TNFSF11
13q14
No
MARK3
14q32
Yes
ADAMTS18
16q23.1
Yes
FOXL1
16q24
Yes
CRHR1
17q21
Yes
HDAC5
17q21
Yes
SOST
17q21
No
TNFRS11A
18q21
No
Total
N [ 24 loci
14/24 (58%)
GC
4p12
No
CYP2R1
11p15
No
DHCR7
11q12
No
CYP24A1
20q13
No
Gene(s) BMD
VITAMIN D LEVEL
a
Not previously known to play a role in phenotype.
close to the ITGA1, LRP5, SOST, SPP1, TNFRSF11A, TNFRSF11B, and TNFSF11 genes. Note that for most candidate genes there were no significant associations. The effect size for SNPs that were associated with BMD was small, ranging from 0.04 to 0.18 SD change in BMD per allele. Variants within or close to the
LRP5, SOST, OPN, and TNFRSF11A genes were also significantly associated with fracture risk, with odds ratios ranging between 1.13 and 1.43 per allele. The association with fracture remained significant after correction for BMD for the OPN and SOST loci. This indicates that susceptibility to fracture for these genes might be mediated by effects on bone quality or other BMD-independent predictors of fracture.
Serum Vitamin D Levels Determinants of circulating 25(OH)D concentrations include sun exposure and diet, but high heritability as derived from twin studies of up to 53% suggests that genetic factors could also play a part. The SUNLIGHT consortium (Study of Underlying Genetic Determinants of Vitamin D and Highly Related Traits) was formed in 2008. It represents a collaboration of cohorts from the UK, USA, Canada, the Netherlands, Sweden, and Finland. In their first study Wang et al. [68] aimed at identifying common genetic variants affecting vitamin D concentrations and risk of insufficiency through applying GWAS (summarized in Table 56.1). A GWAS was performed of 25(OH)D concentrations in 33 996 individuals of European descent from 15 cohorts. Five epidemiological cohorts were designated as discovery cohorts (n ¼ 16 125), five served as in silico replication cohorts (n ¼ 9367), and five served as de novo replication cohorts (n ¼ 8504). 25(OH)D concentrations were measured by radioimmunoassay, chemi-luminescent assay, ELISA, or mass spectrometry. Results of GWAS data across cohorts were combined using Z-scores and these were weighted in the meta-analysis. In total four loci were identified that associated with inter-individual variation in vitamin D levels (Table 56.2). Variants at three loci immediately reached genome-wide significance (p < 5.10e8) in the discovery cohorts for association with 25(OH)D concentrations, and were confirmed in replication cohorts. These were in decreasing order of effect size: 4p12 (rs2282679 in the GC or vitamin D binding protein DBP gene and including the previously described rs7041 which is Glu416Asp substitution and approximated the Gc1 protein isoform); 11q12 (rs12785878, near the DHCR7/NADSYN1 gene which is the 7-dehydrocholesterol reductase/NAD synthetase 1 gene); and 11p15 (rs10741657, near the CYP2R1 gene). Variants at an additional locus (20q13, CYP24A1) were genome-wide significant in the pooled sample (rs6013897) so combining discovery with replication cohorts. In a candidate gene analysis they noted that variants at the VDR, CYP27B1, or CYP27A1 were not (genome-wide) significant or could not be replicated. This GWAS showed that new biology in the vitamin D endocrine system can be uncovered by this approach.
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
1037
REFERENCES
DHCR7/NADSYN1 is a novel locus for association with vitamin D status, but has biological plausibility. DHCR7 encodes the enzyme 7-dehydrocholesterol (7-DHC) reductase, which converts 7-DHC to cholesterol, thereby removing the substrate from the synthetic pathway of vitamin D3, a precursor of 25(OH)D3. Rare mutations in DHCR7 lead to Smith-Lemli-Opitz syndrome, which is characterized by reduced activity of 7-DHC reductase, accumulation of 7-DHC, low cholesterol, and many congenital abnormalities [69]. The finding that common variants at DHCR7 are associated with circulating 25(OH)D concentrations suggests that this enzyme has a role in regulation of vitamin D status. The CYP2R1 gene encodes a hepatic microsomal enzyme and could be the enzyme underlying 25-hydroxylation of vitamin D in the liver, but this is still controversial. The GWAS finding that common variants at the CYP2R1 locus are associated with circulating 25(OH)D concentrations is thus strong evidence that CYP2R1 is indeed the enzyme underlying the crucial first step in vitamin D metabolism. The third gene, GC, encodes DBP or the vitamin D binding protein (see previous section on DBP). The current GWAS data thus confirm the association of rs7041 with circulating 25(OH)D. In the discovery cohorts, SNPs at the three confirmed loci (GC, DHCR7/NADSYN1, and CYP2R1) had expected subtle effects and accounted for 1e4% of the variation in 25(OH)D concentrations. On the other hand, when they compared mean concentrations of 25(OH)D by genotype category at each of the three loci in the two largest cohorts (combined n ¼ 12 208), they noted that differences in mean 25(OH)D concentrations between minor and major homozygotes for the strongest genetic variants were between 4 and 18 nM/L. This is similar to those seen with supplementation in these cohorts (9 nM/L), and close to differences recorded for a one season change (14e18 nM/L). The authors went on to define risk for vitamin D insufficiency which in this study was defined as concentrations lower than 75 nmol/L or 50 nmol/L. Genotype scores for having vitamin D deficiency were constructed for the three confirmed variants. This analysis showed that participants with a genotype score in the highest quartile were at 2.5 times increased risk of having 25(OH)D concentrations lower than 75 nmol/L, or at 2.0 increased risk for having concentrations lower than 50 nmol/L, compared with those in the lowest quartile of the genotype risk score. This first GWAS on circulating 25(OH)D levels showed that variants near genes involved in cholesterol synthesis, hydroxylation, and vitamin D transport affect vitamin D status in Caucasians. Already a combination of this genetic variation at these loci identifies individuals who have substantially raised risk of vitamin D insufficiency, and this suggests some clinical utility for such risk scores.
FUTURE DEVELOPMENTS In the coming years, we can expect a plethora of genetic variants that are identified to be associated to many different complex diseases and traits. This holds true for many of the phenotypes and diseases related to the vitamin D endocrine system, such as shown here for BMD and vitamin D level, but there are of course many more. Currently, all of such variants are coming from the many GWAS that are being performed worldwide, while in the near future we can expect the new high-throughput sequencing techniques to deliver even more variants, especially more rare ones, that are related to traits and diseases. In each and every case, firm epidemiological and population genetic evidence is required to establish the contribution of such genetic variants to explain risk for disease or variance of the trait. The combination of such rare and common genetic variants is likely to shed further light on the genetic architecture of the complex traits and diseases in a more comprehensive way than is now possible. Such improved genetic understanding together with improved biological understanding of the underlying mechanisms will bring the potential use of such markers in a susceptibility diagnostic or pharmaco-genetic setting closer to clinical practice. Such developments might be especially relevant for supplementation with vitamin D in health and disease.
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C H A P T E R
57 The Pharmacology of Vitamin D Reinhold Vieth University of Toronto, Toronto, Canada and Mount Sinai Hospital, Toronto, Canada
4. What, if any, are the biological markers to monitor toxicity, and what are our criteria for determining therapeutic effectiveness? What is the “therapeutic index,” the ratio between toxic versus beneficial dose levels?
INTRODUCTION This chapter focuses on vitamin D, the nutrient (cholecalciferol and to a lesser degree, ergocalciferol), as if it were a drug. While the distinction between a drug and a dietary supplement is largely semantic, the pharmacological perspective is important, because it imposes a discipline that has been overlooked too often by those who prescribe vitamin D. Vitamin D3 is the natural starting point for anyone wishing to administer vitamin D. I will address issues underlying various dosing strategies, and characterize the safety of 25(OH)D. In the USA, vitamin D3 is regarded as a dietary supplement, and as such it is subject to minimal regulatory oversight, while vitamin D2 in doses of 50 000 IU is a prescription medication. In most of the world, doses of vitamin D beyond 400 to 1000 IU daily are considered a prescription drug. If it is considered as a pharmaceutical, then in many ways, vitamin D possesses many of the attributes of an ideal drug (Table 57.1). Before allowing approval of any new drug, government regulators expect to see the answers to a number of standard questions. If vitamin D were a new drug, these questions should include the following: 1a. What are the established disease indications for the drug? 1b. What other clinical or health effects should we be looking for, based on preclinical animal and laboratory research? 2a. What are the most useful approaches to increasing vitamin D status? 2b. What is the appropriate dosage, route of administration, and interval between doses? 3. What is the desirable target for the plasma concentration, what dose would be needed to attain or ensure this?
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10057-5
At the end of this chapter I will return to these questions. As an introductory overview to the topic, Table 57.2 summarizes the basic pharmacology of vitamin D and its major metabolites. Vitamin D pharmacology is complex, because unlike a drug, it is rarely totally absent from the body because it is produced by exposure of TABLE 57.1
Features of Vitamin D in the Context of an Ideal Drug
DESIRABLE FEATURES Long half-life in the body. Hence, it is a “forgiving drug” that permits catch-up doses if doses are missed in the recent past Wide therapeutic index/wide margin of safety Pleiotropic benefits Amenable to monitoring Minimal interactions with other drugs Inexpensive Stable, long shelf-life of the medication NOT DESIRABLE Not specific to any one target tissue Slow development of evidence of response to therapy Correction of overdose/toxicity is not trivial and requires months Difficult to target specific serum levels because of variable background input due to environment, skin photoprotection, clothing, culture, and genetics Disease, particularly malabsorption, liver or renal disease, may diminish efficacy
1041
Copyright Ó 2011 Elsevier Inc. All rights reserved.
1042 TABLE 57.2
57. THE PHARMACOLOGY OF VITAMIN D
Pharmacokinetic Features of Vitamin D and its Key Metabolites Vitamin D3 (cholecalciferol)
Vitamin D2 (ergocalciferol)
25-Hydroxyvitamin D3
1,25-Dihydroxyvitamin D3
Volume of distribution
Larger than body size
Larger than body size
Larger than plasma volume
Plasma compartment
Tissue distribution for longer term
Adipose and muscle
Adipose and muscle
Blood, adipose, and muscle
Blood and tissues
Circulating half-life
2 days
2 days
2 weeks
12 hours
Functional half-life
2e3 months
2 months or less
2e3 months if generation from vitamin D3 stores is considered
12 hours
Physiologic dose rate per day
5e250 mg/day
N/A
5e60 mg/day
1e2 mg/day
Pharmacologic dose rate per day
>250 mg/day
>250 mg/day
>60 mg/day
>2 mg/day
Minimal toxic dose per day
>1000 mg/day
>1000 mg/day
Not tested, likely 400 mg/day
>2 mg/day
1 mg ¼ 40 IU of vitamin D3 or vitamin D2; dosages for other metabolites are properly considered in mass units.
skin to UVB light, and it is present in the diet. Therefore, classic pharmacokinetic studies are virtually impossible for vitamin D. Moreover, vitamin D is readily converted to a subsequent metabolite, 25(OH)D, that is the index of nutritional status and therapeutic response. While a typical micronutrient functions as a metabolic cofactor whose level might simply need to be maintained above a minimum threshold, vitamin D metabolites serve complex regulatory roles throughout the body, and doseeresponse curves probably vary, depending on the actions of vitamin D being characterized. Vitamin D differs from other nutrients because we have never had dietary intakes of vitamin D as a reasonable reference point for deciding on how much of this nutrient people should be consuming. Compared to the 250 mg (10 000 IU) of vitamin D that adults can obtain by exposing their full skin surface to the sunshine [1,2], foods generally contain such small amounts of vitamin D that it is difficult to acquire even 400 IU/day (10 mg/ day). For this reason, in Canada, the Food Guide advises supplements for those over age 50 years. The process of evolution and natural selection has effectively optimized the biology of primates, including humans, for life in equatorial Africa. Since those rare foods that do contain meaningful amounts of vitamin D (see Chapter 54) were not readily available to early humans. Diet during our evolution could not have played a role in determining human vitamin D requirements. Requirements for vitamin D were satisfied by the life of the naked ape that became our species, Homo sapiens, in our natural, tropical equatorial environment. The living conditions of modern humans differ from the tropical environment that affected our evolution e we avoid exposure of skin to the vitamin-D-forming UVB rays of sunshine. Even if we do spend time outdoors, the temperate latitudes do not simply provide less UVB than in the
equatorial regions, the annual amplitude of UVB fluctuates in the extreme, often with months of deprivation [3]. I contend that we modern humans might benefit if we could compensate for the biological consequences of modern life. The consequences in terms of vitamin D are not simply the endemic lack of vitamin D, but also the exaggerated annual cycles of rising and falling vitamin D supplies that can be corrected by appropriate supplementation.
OVERVIEW OF THE SYSTEM OF VITAMIN D METABOLISM AND ITS REGULATION Administration of vitamin D is unusual in the context of pharmacology or in endocrinology, because this molecule is two metabolic steps away from the biologically active agent 1,25(OH)2D. The laboratory test normally used to monitor the dose of vitamin D is the concentration of 25(OH)D, because this is the most abundant metabolite, and its levels change gradually over time. Measurement of vitamin D itself in the circulation is not helpful, because after sun exposure or oral consumption, its concentration rises sharply within 1e2 days and declines within 3e4 days. Figure 57.1 illustrates the metabolite “compartments” occupied by vitamin D after ingestion or exposure to sunshine. The two panels of the figure illustrate the metabolic adaptations that take place so that the vitamin D endocrine system can accommodate to a wide range in the substrate concentration. The vitamin D system is optimized to maintain plasma 1,25(OH)2D levels according to the requirements of calcium homeostasis. As vitamin D supplies decline progressively, the first factor limiting health is probably a diminished capacity of non-renal tissues to produce 1,25(OH)2D. The kidney possesses megalin, a protein which facilitates substrate access
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
OVERVIEW OF THE SYSTEM OF VITAMIN D METABOLISM AND ITS REGULATION
(A)
Vitamin D3
25(OH)D
1,25(OH)2D
24,25(OH)2D
Catabolism Excretion
(B)
METABOLITE “COMPARTMENT” Vitamin D3 Normally Plasma=0-5 ng/mL
(Context: 400 IU/quart milk = 10ng/mL)
25(OH)D 2-100 ng/mL
Paracrine signaling within tissues
1,25(OH)2D 15-80 pg/mL 24,25(OH)2D
(A) Vitamin D metabolism under conditions of low vitamin D supply. The mitochondrial vit D-25- vessels represent metabolic compartments, 1 Liver hydroxylase stages in the metabolism of vitamin D. The height of the shaded portion of each vessel Liver microsomal vit D-252 hydroxylase represents the relative concentration of each metabolite indicated in the figure. This figure illustrates the concept that vitamin D metaboRenal 25(OH)D-1lism in vivo functions below its Km, i.e. the hydroxylase (CYP27B1) 2 3 1 system behaves according to the first-order Tissue (non-renal) reaction kinetics. This is analogous to the flow 25(OH)D-1-hydroxylase 4 of water out of a vessel, where the height of 4 5 water in a vessel, together with the valve setting 3 determines the outflow. Likewise, the rates of 5 Renal mitochondrial 25(OH)D-24-hydroxylase metabolism through the vitamin D system are directly related to the concentration of precursor Non-renal 1,25(OH)2D-246 hydroxylase (CYP24) at each step, in conjunction with the corresponding adjustments at the enzymes. When vitamin D supplies are low, the flow of 25(OH)D An “unregulated” step in to the non-endocrine, paracrine pathways is the flow of metabolism compromised to preserve the plasma 1,25(OH) A regulated step in the flow 2D level that is the priority to sustain calcium 6 of metabolism homeostasis and life. (B) Metabolism of vitamin 7 Catabolism and excretion D under conditions of adequate vitamin D 7 supply. When there is enough vitamin D, then utilization of 25(OH)D by peripheral tissues for paracrine regulation is no longer compromised. Unlimited Vitamin D3 Higher 25(OH)D concentration makes possible Storage • Volume of distribution > body mass Capacity routes to service paracrine regulation in tissues • Half-life : in blood 2 days in other than the kidney. Furthermore, a higher in vivo >2 months PLASMA Muscle supply of vitamin D leads to an upregulation of • Weaker binding to DBP and 24-hydroxylase and the catabolic pathways Adipose To Bile associated with it, this accelerates rate of metabolic clearance and metabolite turnover in each 25(OH)D compartment. • Volume of distribution = PLASMA body mass? • Half-life 2 weeks • Tightest binding to DBP, • Plasma DBP capacity , 4300 nmol/L
Legend
METABOLITE “COMPARTMENT”
1043
FIGURE 57.1
Endocrine signaling via circulation
Within Tissues Possessing 1-OHase
1,25(OH)2 D
PLASMA
• Volume of distribution =
approximately plasma vol
• Half-life 0.5 day • Weaker binding
to DBP
Catabolism Excretion
[4e6] (see also Chapter 14). However, few non-renal tissues possess megalin, and hence their activity of existing 1-hydroxylase depends primarily on the diffusion from the circulating 25(OH)D concentration [7e10]. Reliance on free diffusion severely compromises the vitamin D system at non-renal tissues when 25(OH)D levels are low. This impaired access is represented in Figure 57.1 by the greater height of one of the valves (number 4 in the figure) representing nonrenal 1-hydroxylase on the vessel that represents the 25(OH)D compartment.
If one looks at the system of vitamin D metabolism in Figure 57.1 from the perspective of a system designed to control something, it becomes clear that this is a system better designed to cope with an abundance of supply, but poorly capable of adapting to a diminished supply. The only natural way to correct for deficiency of vitamin D is to redirect utilization of 25(OH)D toward renal 1,25(OH)2D production. The pathway toward 1,25(OH)2D is fundamental to life-sustaining calcium homeostasis. Although 1,25(OH)D has functions beyond calcium homeostasis, those functions are the
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
1044
57. THE PHARMACOLOGY OF VITAMIN D
consequence of local paracrine metabolism of 25(OH)D outside the kidney. If peripheral tissues such as the immune system, breast, brain, or prostate were reliant on circulating 1,25(OH)2D, instead of its local production, then there would be no opportunity for local paracrine control, because calcium deprivation is the key factor determining the renal secretion of 1,25(OH)2D and which would affect many tissues. That sort of control by renal 1,25(OH)2D production would be driven by calcium intakes, and it is not a plausible way to regulate the non-calcemic roles attributed to the vitamin D system. The serum and plasma levels of 25(OH)D play a far more profound role than 1,25(OH)2D when it comes to the non-calcemic actions of vitamin D. The system of vitamin D metabolism is effectively designed as one able to adapt to higher inputs of vitamin D, but with a poor capacity to adapt to lower inputs. The system possesses the equivalent of a brake pedal, in the form of CYP24 (24-hydroxylase), to increase the catabolism rate of 25(OH)D and its metabolites when there is abundance. But there is no efficient mechanism to adapt to low supplies of vitamin D other than the pathologic adjustments that occur once there is hypocalcemia. The impaired bone mineralization and hyperparathyroidism associated with vitamin D depletion are evidence of a system function beyond its design parameters. Human vitamin D metabolism was effectively designed through evolution and natural selection for people living at equatorial latitudes, without clothing and with a relative abundance of sun-derived vitamin D. In contrast, most modern humans cover 95% of their vitamin-D-forming skin surface with clothing and they tend to avoid exposure to direct sunshine. The vitamin D system entails endocrine, paracrine, and autocrine functions that could have never been optimized to cope with the lack of vitamin D created by our modern culture of clothing and sun avoidance. Despite our ability to survive under these conditions, the vitamin D nutritional status is neither natural nor optimal for humans, the only primate species that inhabits winter latitudes. Inadequate supplies of vitamin D limit the local, paracrine control that many tissues need so that they can function properly. The question is now whether the modern, “normal” prevalence of conditions such as those listed in Table 57.3 might not be reduced substantially if populations were to sustain higher serum 25(OH)D levels. The early inhabitants of the north overcame a lack of sunshine by dietary means. Early inhabitants of the arctic inhabited costal regions. Whether they were Inuit or Scandinavians, they consumed diets high in the vitamin-D-containing fish that can provide thousands of IU/day if consumed on a pound-per-day basis. Vitamin D truly is a vitamin because “insufficient
amounts in the diet may cause deficiency diseases” [11,12]. The original “vital amine” was the nutrient “antirachitic A” [11]. Lack of the antirachitic nutrient e i.e. what we now call “vitamin D” e was the very first thing that firmly established the existence of what we now refer to as a “vitamin” [11]. The concentrations of vitamin D and its metabolites are so low that some basic paradigms for control of it are very different from the regulation of other steroid hormones. For the metabolism of conventional steroid hormones, the concentration of substrate (cholesterol) is far higher than the substrate in the vitamin D system. Figure 57.2 illustrates the effective, in vivo Km of 1hydroxylase, in relation to the physiological concentration range of its substrate. In contrast to a typical human circulating cholesterol concentration in the order of 5 million nmol/liter (this is simply another way of saying 200 mg/dl); the 25(OH)D typically circulates at less than 200 nmol/liter. Cholesterol concentration is so high that it is not a rate-limiting aspect of the body’s capacity to generate steroid hormones; however, 25(OH)D concentration can be absolutely rate limiting for 1,25(OH)2D production. Circulating 1,25(OH)2D can remain stable through a fairly large range of 25(OH)D concentrations because megalin facilitates renal access to 25(OH)D, as discussed above, but the paracrine production of 1,25(OH)2D beyond the kidneys, and which underlies the non-calcemic effects of vitamin D suffers from limited access to 25(OH)D. In the acute situation, before adjustments can be made to 24-hydroxylase (CYP24) and catabolic pathways (before the “valves” in Fig. 57.1 can be adjusted), the in vivo production of 1,25(OH)2D is directly proportional to the circulating 25(OH)D concentration. In rats, the acute injection of 25(OH)D into the circulation produces a rapid, transient increase in 1,25(OH)2D, directly proportional to the percentage increase in 25(OH)D [10,13,14]. Since concentrations of 25(OH)D usually change slowly in vivo, over many months, this first-order relationship between 25(OH)D and 1,25(OH)2D is not normally evident in adults because there is a gradual adaptation of the synthesis and catabolic enzymes, respectively, CYP27B1 and CYP24 [15]. However, in situations where 1-hydroxylase is chronically stimulated, either because of primary hyperparathyroidism [16] or in granulomatous disease [17,18], modest increases in vitamin D supply will raise plasma 1,25(OH)2D concentration and aggravate hypercalcemia.
Hypothesis that Large Fluctuations in 25(OH) D Concentrations are Adverse The model of regulation represented by Figure 57.1 may help to explain the unusual U-shaped risk curves
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
TABLE 57.3
Clinical trial
Clinical Trials of Vitamin D3 at Doses >800 IU/day (>20 mg/day) and Effects of the Attained 25(OH)D Levels1
Study subjects
Treatment dose Primary and duration outcome
Mean (SD) or median Type of control (interquartile range) (double-blind unless stated Control 25(OH)D Treatment otherwise) nmol/L 25(OH)D nmol/L
Primary outcome improved with vitamin D
Reported adverse A secondary effect outcome attributable improved to treatment
Double placebo, 66 [24] without calcium or vitamin D
112 [30] [active D, placebo Ca group]
No
No
No
162 [74e226]
No
Yes
No
179 [76], at 12 months (varied through study)
NA since Phase 1e2 clinical trial
Yes
No
70 [23]
Yes
Yes
No
Aloia Men and women 4000 IU/day for 2010 [56] ages 20e80 4 months with or without 1200 mg calcium
Characterize interaction of calcium and vitamin D on bone-turnover markers
No effect of vitamin D on PTH or bone turnover markers. Calcium alone lowered these
Amir Women with 2010 [57] metastatic breast cancer
10 000 IU/day for 4 months
Overall pain scores
No effect on Single-arm trial overall pain or bone turnover markers, but significantly fewer pain sites
Burton Patients with 2010 [54] multiple sclerosis
Average 14 000 Safety and IU/day over 12 exploratory months (doses outcomes ranged up to 40 000 IU/ day)
Lower risk of progression of disability score
El-Haj Pre-menarcheal Fuleihan girls 2006 [52]
2000 IU/day for Bone density 12 months
Greater gains in Placebo hip and vertebral bone density, and lean body mass
Hitz 2007 Hip-fracture [58] patients
1400 IU/day plus 1200 mg calcium for 12 months
Bone density
Improved lumbar spine BMD
200 IU vitamin D 53 [17] only
82 [19]
Yes
No
No
Hitz 2007 Patients with 1400 IU/day [59] lower-extremity plus 1200 mg fracture calcium for 12 months
Bone density
Improved lumbar spine BMD
200 IU vitamin D 77 [18] only
90 [24]
Yes
No
No
88 [51e162] and 112 [47e193]
NA
Yes
No
Jorde Adults with BMI Weekly 20 000 IU Weight loss 2008 [50] >28 or 40 000 IU
70 [19e169]
Open label 83 [27] untreated group (many took up to 4000 IU/day vitamin D anyway)
Lower Placebo group (improved) Beck Depression Index
27[15]
50.0 [20e100]
1045
(Continued)
OVERVIEW OF THE SYSTEM OF VITAMIN D METABOLISM AND ITS REGULATION
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
Treatment effect (a secondary outcome if different from the primary one)
1046
TABLE 57.3
Study subjects
Treatment dose Primary and duration outcome
Lappe Adults over age 1100 IU/day, 2007 [55] 65 plus 1000 mg/ day calcium, 4 yrs
Bone density
Treatment effect (a secondary outcome if different from the primary one)
Mean (SD) or median Type of control (interquartile range) (double-blind unless stated Control 25(OH)D Treatment otherwise) nmol/L 25(OH)D nmol/L
Primary outcome improved with vitamin D
Reported adverse A secondary effect outcome attributable improved to treatment
Lower rates of new cancer diagnosis
Placebo
96 [21]
NA
Yes
No
125 [104e150]
Yes
No
No
71 [23]
NA
NA
No
Not measured
Yes
Yes
No
112 [41]
Yes
No
No
75 [50e83]
Yes
No
No
71 [20]
Mocanu Nursing-home 2009 [60] residents
5000 IU/day Bone density for 12 months, single-arm study
Smith Men and women 2009 [61] through the South Pole winter
2000 IU/day or 1000 IU/day for 5 months
Urashima School children 2010 [53] 6e15 yrs
1200 IU/day for Incidence of 4 months influenza A through winter
Lower risk of Placebo influenza A and lower risk of asthma attacks
Vieth Well thyroid 2004 [49] clinic outpatients
4000 IU/day for Well-being and 3 months and mood in 15 months February
Well-being Lower dose 79 [30] scores improved group, 600 IU/d into winter
Effect on serum NA 25(OH)D
von Hurst Insulin-resistant 4000 IU/day vs Insulin 2009 [51] Asian women 600 IU/day responsiveness for 6 months 1
Increase in bone Baseline of same 29 [20e36] density at both group in hip and vertebral spine
Improved insulin responsiveness vs placebo
Group receiving 57 [18] 400 IU/day
Placebo group
For reasons explained in the text, not included is the Saunders clinical trial, with annual 500 000 IU vitamin D3.
Not measured
21 [11e40]
57. THE PHARMACOLOGY OF VITAMIN D
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
Clinical trial
Clinical Trials of Vitamin D3 at Doses >800 IU/day (>20 mg/day) and Effects of the Attained 25(OH)D Levels1dcont’d
OVERVIEW OF THE SYSTEM OF VITAMIN D METABOLISM AND ITS REGULATION
The difference in enzyme kinetics between the vitamin D endocrine system and the substrate supply for conventional steroid hormone systems based on cholesterol. The (1) more 25(OH)D [ =D nutrition] or (2) more enzyme (Vmax) purpose of this figure is to emphasize that the range of physiologic concentration of 25(OH)D in Vmax mammals is less than the Michaelis-Menten constant (Km) of 1-hydroxylase that has been characterized in vitro [13], and in vivo [10]. There are two ways to improve capacity for 1,25(OH)2D (2) production: provide more substrate, or increase more 1-hydroxylase content of the tissue. This is a very Physiological enzyme different paradigm from every other part of the Km range of endocrine system. No other signaling molecule is ( Vmax ) 25(OH)D so strongly dependent on the arbitrary, external supply of its structural raw material. The concept of a masseaction relationship for 1,25(OH)2D Concentration production is the basis of the argument that operation of paracrine control systems that are 0 300 nmol/L 5 000 000 dependent on serum 25(OH)D for their substrate (1) More (For other hormones, the function more effectively with improved vitamin substrate substrate, e.g. cholesterol, D nutrition. is so abundant, its Mass action availability is not relevant to hormone regulation)
TWO WAYS TO INCREASE PRODUCTION OF HORMONE (1,25(OH)2D versus steroid hormones)
Rate of 1,25(OH)2D synthesis
1047
versus 25(OH)D that have been reported in recent years for prostate cancer [19], pancreatic cancer [20,21], and mortality [22]. While it remains possible that U-shaped risk curves are extremes of the statistical noise observable for anything being studied intensely, the observations do show a pattern, in that they have only been reported for northerly populations, but not in the south. Not only is there less UVB in the north, but also the seasonal amplitude in UVB is much greater in the north [23,24]. While the phenomenon may be attributed to consumption of too much vitamin A in the oily fish in the diet of Scandinavians, that is not likely to explain the similar latitudinal effect in the USA [21]. In 2004, I proposed the hypothesis that the mechanism for the paradoxical evidence of increased risk with higher 25(OH)D is attributable to the greater amplitudes of the seasonal cycles in 25(OH)D with higher latitude [25], and subsequent reports have been consistent with the prediction. The annual downward phase in the seasonal cycles of serum 25(OH)D concentration almost certainly creates a non-steadystate situation for paracrine production of 1,25(OH)2D. So long as serum 25(OH)D declines, tissues such as the prostate and pancreas will have difficulty in sustaining 1,25(OH)2D at its long-term set point for autocrine regulation [14]. High 25(OH)D concentrations may not be problematic per se, but it is well known that in temperate regions, the higher the summertime 25(OH)D levels the bigger the decline in 25(OH)D during winters [26,27]. Falling 25(OH)D concentrations are adverse. This hypothesis has predicted that if higher serum 25(OH)D relates to greater risk for
FIGURE 57.2
a cancer, then the relationship should be specific to high latitudes where winters produce prolonged, gradual declines in 25(OH)D levels [28]. Subsequent data have confirmed the hypothesis, in that risk for pancreatic cancer was reported as higher with higher serum 25(OH)D concentrations, specifically in the north, but not in the south [21]. While metabolism and regulation of the vitamin D system may appear complex, the practical implications are straightforward. The system functions optimally over the range of 25(OH)D concentrations for which our human biology was designed through evolution. Anthropologic considerations point to “biologically normal” 25(OH)D levels that exceed 75 nmol/l (30 ng/ ml), ranging to 225 nmol/l (90 ng/ml). Furthermore, because the enzymes of the vitamin D system function according to first-order reaction kinetics, if serum 25(OH)D concentrations are fluctuating, then the system cannot be in full equilibrium. Optimal serum 25(OH)D levels are those that are both high in the physiologic range, and stable throughout the year.
Role of Vitamin D Binding Protein (DBP) Differences between steroid hormones and the vitamin D system are amplified further by the large differences in concentrations of their respective plasma carrier proteins. Sex-steroid-binding globulin and glucocorticoid-binding globulin each circulate at concentrations of about 150 nmol/l, in the same order of magnitude as their ligands [29]. In contrast, the concentration of vitamin D binding protein is 4700 nmol/l [30];
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
1048
57. THE PHARMACOLOGY OF VITAMIN D
this represents a 50-fold excess over its vitamin-Dderived ligands. The dynamics of 25(OH)D in tissues are remarkable. Its carrier protein, DBP, is cleared from plasma with a half-life of 1.7 days, which is shorter than the 5-day half-life of albumin [31]. Within 1 hour after injection of radiolabeled DBP, the radiolabel is present in a greater concentration than in plasma within the following tissues: kidney, liver, skeletal muscle, heart, lung, intestine, testis, and bone [31]. In contrast to DBP, its ligand, 25(OH)D, is cleared slowly from the body, with a halflife of about 10 days in both rabbit [31] and human [32]. The binding of 25(OH)D to DBP does not affect the turnover or the tissue uptake of DBP [31]. As a short summary of the preceding, the DBP and/ or DBPe25(OH)D complex is removed from plasma by a variety of tissues. The DBP is degraded during this process, and most 25(OH)D released within those tissues is recycled. The molar excess of DBP to 25(OH)D in plasma and the relatively rapid turnover of DBP indicate that a high-capacity, high-affinity, and dynamic transport mechanism for vitamin D sterols exists in plasma. Although 25(OH)D released into cells because of the metabolic clearance of DBP is recycled, the clearance of DBP provides ready access of the liver and kidney to vitamin D, 25(OH)D, and its metabolites. The liver and kidney have preferential access to the vitamin D metabolites transported on DBP because liver and kidney are the two organs most involved in the clearance of DBP. The facilitated uptake of DBP by the liver and the kidney plays a dramatic and dynamic role in the metabolism of vitamin D. The reported molar replacement rate of DBP is more than 1000-fold higher than that of 25(OH)D, indicating that molecules of 25(OH)D are recycled many times during their residence time in the body [31]. An understanding of the megalin/cubulin system has shed light on the mechanisms of DBPetissue interactions, and tissue-specific uptake of DBP is discussed in Chapter 5 [33]. Megalin and cubulin are cell-surface, endocytic receptors, members of the low-density lipoprotein receptor gene family. These proteins help to regulate the concentration of vitamin D ligands in the extracellular fluids and deliver metabolites to cells in need of these metabolites [34]. Differences in tissue distribution of these cell-surface proteins will affect the accessibility of different tissues to circulating 25(OH)D, and this makes plausible the concept that tissues other than liver and kidney can have sufficient access to 25(OH)D so that it can be used as a substrate for the paracrine production of 1,25(OH)2D. Paracrine production of 1,25(OH)2D is the most widely accepted mechanism to explain the various putative effects of vitamin D status beyond bone and mineral metabolism.
PLACEBO-CONTROLLED CLINICALTRIAL JUSTIFICATION FOR AN OPTIMAL SERUM 25(OH)D LEVEL Bone and mineral-related diseases are the only conditions formally acknowledged by recent governmentaccepted reviews as benefited by vitamin D [35,36]. Other chapters in this book address the role of vitamin D in the treatment and prevention of osteoporosis (Chapter 61). The clinical trials into osteoporosis were generally designed to incorporate a placebo group versus a treatment that combined calcium plus 800 IU vitamin D3. Chapuy et al. were the first to publish antifracture efficacy of vitamin D with calcium, and they selected the dose arbitrarily, since aside from some suppression of PTH, they offered no rationale for the dose [37]. Chapuy et al. set the pattern for the 800 IU (20 mg) vitamin D dosage used in the successful clinical trials of osteoporosis fracture prevention [38], but no attempt has been made to find out whether there might be greater benefit with higher doses of vitamin D, or whether 1200 mg/day calcium is required together with the vitamin D. For vitamin D and calcium, the dosages still need to be defined better. The role of vitamin D in the prevention or treatment of cancers is a particularly controversial area. A recent review under the auspices of the World Health Organization concludes “.no compound should be recommended for cancer chemoprevention if its efficacy and side effects have not been evaluated in large, randomized trials. Ideally, these trials should be double-blind and placebo controlled” [39]. It is clear from this that what policymakers are led to demand are the same traditional criteria for evidence of benefit that are required to support the approval of proprietary pharmaceuticals. Those demands are unrealistic for a nutrient such as vitamin D. Figure 57.3 illustrates and summarizes the ways in which the traditional pharmaceutical model to demonstrate efficacy of a drug is different from what is realistically applicable to a nutrient such as vitamin D. The clinical research into the utility of simple vitamin D3 has been remarkably slow to develop, except for rickets, osteomalacia, and osteoporosis. There are three reasons for this. First, the financial incentive lies with the proprietary analogs of vitamin D, whose research is driven by private funding, and that diverts the focus of investigators who are able to do those studies without the need for the onerous effort of applying for the research grants to validate simple vitamin D3. Second, an optimized dose of vitamin D has never been established for adults, because research ethics committees discourage research beyond the dietary UL, even though the UL was never intended to be such a restriction [40]. Because of underdosing, “plain” vitamin D sometimes
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
PLACEBO-CONTROLLED CLINICAL-TRIAL JUSTIFICATION FOR AN OPTIMAL SERUM 25(OH)D LEVEL
(A) • Persons at high Response Outcome
risk or with existing condition
• Treat existing
Potential
condition
Effect for
• High likelihood
DRUG
to show effect in an individual
RCT “Evidence Based Medicine”
PLACEBO
TREATMENT
Relative Dose Difference
(B) • Healthy persons at low risk
Response Outcome
• Prevent future
Potential for Non-Index Nutrition RCT
condition
• Low likelihood of an effect in an individual
X
Y
Relative Dose Difference
RDA
TREATMENT
Relative Dose Difference The traditional randomized-clinical-trial pharmaceutical model to demonstrate efficacy of a drug, compared with the analogous situation for a nutrient such as vitamin D. (A) For a pharmaceutical clinical trial, subjects are affected by disease, or they are at unusually high risk of disease outcomes, the treatment includes a placebo, and there is a potentially large margin to detect the outcome response. (B) Doseeresponse curve illustrating an RDA for an accepted effect of a nutrient (solid line, X), and for a putative non-traditional effect of a nutrient (dashed line, Y). To study a nutrient to prevent disease, subjects are generally healthy, at low risk of disease, there can be no zero-dose group, and there is a very limited margin to detect the outcome response. This demands a far larger sample size with a nutrient, longer follow-up, and beyond that, healthy study subjects tend to be less motivated to adhere to the intervention for a long follow-up. These things explain why there are so few randomized interventional clinical trials into the use of vitamin D in adults younger than age 50 years. Efficient experimental designs aim to maximize the event rates needed for statistical power. Therefore, clinical trials focus on older adults, because they exhibit more of the key outcomes such as fractures, myocardial infarctions, and death. However, it is logical that the prevention of disease should start before age 50, but for younger age groups, there is no sign of disease-prevention clinical trials in the foreseeable future.
FIGURE 57.3
1049
compares poorly with 1,25(OH)2D and its analogs whose dose is more thoroughly optimized [41], and whose dose is usually designed to be very close to the point where it could cause hypercalcemia. Optimal doses of vitamin D probably vary, depending on the indication, so that one dose may not always be optimal. Third, the official misrepresentation that vitamin D2 and vitamin D3 are equal has resulted in efficacy studies at higher doses that usually involve vitamin D2 because high-dose commercial preparations of vitamin D are comprised of this metabolite. Clinical trials involving vitamin D2 alone have been conducted, but efficacy in those has generally been negative or inconclusive [42e45]. According to the United States Food and Drug Administration (FDA) as well as Health Canada, and other national drug regulatory agencies, the uses of prescription vitamin D are indicated “for use in the treatment of hypoparathyroidism, refractory rickets, also known as vitamin D resistant rickets, and familial hypophosphatemia.” To my knowledge, no prescription form of vitamin D is approved anywhere as a means to increase serum 25(OH)D. It should be noted that a prescription for a patient to use vitamin D to increase serum 25(OH)D is considered an “off-label use.” Overthe-counter vitamin D preparations of vitamin D are generally labeled as, “not intended to diagnose, treat or prevent any disease.” There is a disconnect between government regulations and the reasons why people take or prescribe vitamin D.
Non-bone Effects of Vitamin D Vitamin D nutrition almost certainly affects health beyond just bone. The mechanisms involved in mediating the non-classic (i.e., non-bone) effects of vitamin D are probably through 1,25(OH)2D produced locally, using circulating 25(OH)D as the substrate. Many tissues possess 25(OH)D-1a-hydroxylase (CYP27B1), including the skin (basal keratinocytes and hair follicles), lymph nodes (granulomata), pancreas (islets), adrenal medulla, brain, pancreas, colon, breast, prostate, and others [46] (see also Chapter 45). An even wider range of tissues possess receptors for 1,25(OH)2D (VDR) [47] (see also Chapter 7). All of this reveals a system for autocrine or paracrine regulation of tissue processes that involves the local production of 1,25(OH)2D [48] in addition to the renal-systemic production. Sufficient vitamin D nutrition, and hence appropriate 25(OH)D concentration, is essential to this local, paracrine role of 1,25 (OH)2D that is not generally reflected in the circulating level of 1,25(OH)2D. The paracrine components of the vitamin D endocrine/paracrine systems no doubt account for many of the effects of vitamin D nutrition and/or UVB light on health and disease prevention.
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
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57. THE PHARMACOLOGY OF VITAMIN D
As of late 2010, there are at least ten published, randomized clinical trials of doses of vitamin D higher than the osteoporosis dose of 800 IU/day (Table 57.3). What makes the summary analysis of this work complex is the variety of statistically significant beneficial outcomes. Moreover, none of the 13 trials of higherdose vitamin D reported a significant adverse event attributable to the vitamin D. With vitamin D3 at an average daily dose rate of approximately 4000 IU, the benefits for adults included general well-being [49], lower depression scores [50], and improved insulin responsiveness [51]. It is important to note that the 25(OH)D levels associated with benefit in Table 57.3 are those that exceed 75 nmol/l. The desirable target of 75 nmol/l is based primarily on placebo-controlled clinical trials. For adolescents, daily average vitamin D intakes of 2000 IU (14 000 IU weekly) improved the gain in bone density and muscle mass [52]. In another randomized trial during winter, supplementation with 1200 IU daily lowered the risk of influenza A and the incidence of asthmatic attacks [53]. Burton et al. reported an RCT in patients with multiple sclerosis showing that up to 40 000 IU of vitamin D are safe, and showing evidence of benefit in lowering relapse rates and severity scores of multiple sclerosis [54]. The Burton RCT was intended as a series of loading doses to accelerate attainment of super-physiological levels of serum 25(OH)D to test the safety of those levels; the RCT was not an assessment of therapeutic maintenance doses. The clinical trial of Lappe et al. demonstrated a reduction in cancer diagnosis in the group of women for whom serum 25(OH)D had been raised from an initial controlgroup mean of 71 nmol/l, to a mean of 96 nmol/l [55] (Table 57.3). Even though cancer was not the primary, fracture-preventive outcome of the trial, the Lappe publication represents a higher level of evidence than epidemiological data. Since the serum 25(OH)D in the control group was already at 71 nmol/l e essentially the same level defined as optimal in the meta-analysis of Bischoff-Ferrari et al. [38] e the Lappe clinical trial stands as a strong justification that desirable 25(OH)D levels are those that exceed 71 nmol/l. In another chapter, Bouillon concludes that a lower desirable level e 50 nmol (20 ng) per ml for 25(OH)D e remains justified unless and until there is more evidence from placebo-controlled trials to support the efficacy and safety of widespread use of doses beyond 800 IU/day (Chapter 5). The problem with the conservative approach to vitamin D recommendations is that it fails to acknowledge the existing evidence of safety and efficacy from randomized trials. It also begs the question of exactly how much evidence will be expected before the need for more vitamin D could be accepted.
A major problem with a conservative approach is that it ignores the consequence of failing to implement a beneficial public health measure. The evidence summarized in Table 57.3 is real, that clinical trials using higher doses of vitamin D improved bone density, insulin responsiveness, and well-being. None of the clinical trials with vitamin D showed any difference from placebo in terms of adverse events. The scientific objectivity of the vitamin D conservatives, can be tested with a thought experiment by considering the question, “If the clinical trials involving vitamin D had shown an opposite effect, i.e. if the higher dose of vitamin D were to be related to higher depression, cancer or worse tolerance to insulin, or lower bone density, would that evidence then not be accepted readily as reason for caution with vitamin D?” If evidence of harm would be accepted so readily, then why would one ignore the reality that there is evidence of net benefit with higher intakes of vitamin D?
DOSAGE CONSIDERATIONS Infants Cholecalciferol, or vitamin D3, given in the form of cod liver oil, has been a folk remedy in northern Europe since the 1700s [62]. Empirically, a teaspoon-full daily was found to help infants thrive. The 375 IU (9 mg) of vitamin D3 contained in that teaspoon [63] was confirmed relatively recently as appropriate for infants [64e66]. A French study utilizing vitamin D2 concluded that neonates might need somewhat more, 1000 IU [67]. If safety of vitamin D during infancy is a concern, it should be kept in mind that until the late 1960s, the recommended amount of vitamin D for infants in Finland was 2000 IU/day (50 mg/day). A large epidemiological study suggests that this higher dose lowered risk of juvenile diabetes before age 30 years by 85% compared to people not receiving vitamin D as infants [68]. In contrast to the adult, vitamin D nutrition in the infant and child has been well characterized, and it is the focus of Chapter 60. There are excellent reviews of the field and a wide consensus that a daily average vitamin D intake of 10 mg (400 IU) is appropriate for infants starting from birth [66,69]. Chapters 35, 36 and 37 discuss vitamin D issues in the perinatal, infancy and adolescence periods. The rest of this chapter focuses on the pharmacology of vitamin D e primarily cholecalciferol e in the adult.
Adults Until it became clear that vitamin D was important to the health of adults, there was very little thought
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DOSAGE CONSIDERATIONS
Vitamin D intake IU/day
1000
40000
4000
400
Serum or plasma 25(OH)D nmol/L
directed at how much vitamin D adults might require. At the time of writing, the UK official policy still presumes that in sunshine there is enough to sustain the health of adults who are not at high risk of vitamin D deficiency. Those who might require vitamin D include immigrant populations, and the sick or housebound [70]. Into the 1960s, the only criterion for adequate vitamin D nutrition was the absence of overt rickets or osteomalacia [71]. By that same criterion of bone deformity, anthropologists consider vitamin D nutrition to have been a relatively minor problem for ancient populations. That lack of evidence of bone deformity in ancient populations is, of course, explained by the recognition that the lack of vitamin D was the driving force for the natural selection favoring lighter skin color to prevent rickets and osteomalacia within defined environments [2]. Rickets and osteomalacia produce a misshapen pelvis that makes natural childbirth impossible. Women able to produce enough vitamin D to prevent rickets, osteomalacia, and a misshapen pelvis were, of course, the vast majority in any region. Survival depended on adequacy of vitamin D nutrition, and at latitudes away from the equator, the natural selection favoring lighter skin color helped to ensure adequacy for the quality of pelvis needed for vaginal birth. The seed for the recommended vitamin D intake of 200 IU (5 mg) daily in much of the world originated from the 1960s, when an expert committee on vitamin D could provide only anecdotal support for “the hypothesis of a small requirement” for vitamin D in adults. They thenceforth recommended one-half the infant dose e 200 IU daily e to ensure that adults obtain at least some from the diet [71]. Despite subsequent knowledge, new dietary vitamin D recommendations, which are imminent at the time of this writing, require an exceptional level of evidence to overcome prior government mandated recommendations that were never evidence based to the degree now demanded. The vitamin D recommendations of 1997 were referred to as an “adequate intake” (AI), because there was no clear sense of what the total supply of vitamin D from sunshine and diet might be for adults [72,73]. It remains to be seen whether anything changes in the guidelines issued in 2010. The objective measure of vitamin D nutritional status is the 25-hydroxyvitamin D (25(OH)D) concentration in serum or plasma [72]. This makes it possible for researchers to focus on a measurable target when it comes to vitamin D nutrition. Figure 57.4 is a dosee response curve showing the final average 25(OH)D concentrations attained in various studies reported in the literature [1,74]. Table 57.4 summarizes incremental responses to different published treatment strategies to
400000
Study Group Mean Data O Vit D2-Treated Group Mean Data x Individuals, Vit D Hypercalcemia
100
O
10
O O O
100 1000 Vitamin D Intake µg/day
1E4
Doseeresponse relationship between daily vitamin D intake and mean 25(OH)D concentration, based on data published in the literature. The solid points show mean results for groups of adults consuming the indicated doses of vitamin D. Results for groups of adults unambiguously consuming vitamin D2 are shown by the circled points. That vitamin D3 is more potent than vitamin D2 is evidenced here by the solid black circles that show that data for subjects consuming vitamin D2 lie below the trend-line based on vitamin D3. Both axes are log scale. The results represented by Xs are for individuals showing the classic hypercalcemia response to toxic levels of prolonged vitamin D consumption. The data used to generate this graph were compiled and published previously [1,74].
FIGURE 57.4
raise 25(OH)D to steady-state concentrations. Responsiveness to vitamin D administration, as measured by the nmol per liter increase per mg consumption per day, increases with: (1) lower vitamin D dosage, (2) lower initial 25(OH)D concentration; and (3) longer duration of supplementation, suggesting a long halflife and time to plateau. Table 57.5 summarizes the descriptions commonly applied to specific serum 25(OH)D concentrations, and it suggests long-term vitamin D3 dosage rates that can reasonably be expected to ensure levels above the target. The conventional approach to improving vitamin D nutritional status has been to give either vitamin D3 or vitamin D2 (ergocalciferol). Availability of 25(OH)D was an option some years ago (supply of this product has been discontinued). The discontinuation of 25(OH)D may have made sense at the time, because the objective of increasing plasma 25(OH)D concentrations can be almost as easily achieved by providing enough vitamin D3. Nonetheless, useful perspectives can be gained from the historic experience with 25(OH)D. Since the increase in the plateau plasma 25(OH)D concentration per mg dose is at least four times higher for 25(OH)D administration than for vitamin D3 administration [78,82], we can conclude
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
1052 TABLE 57.4
57. THE PHARMACOLOGY OF VITAMIN D
Strategies to Increase Circulating 25(OH)D Concentration in Adults: Effects of Compound, Dose, and Duration1
Compound
25(OH)D nmol/L increase per mg/day
25(OH)D3
4.1
50
4
206.4
[75]
25(OH)D3
4.0
10
4
40
[75]
25(OH)D3
3.8
20
4
76.1
[75]
Cholecalciferol
1.5
15
52
22
[76]
Cholecalciferol
1.4
20
8
27
[77]
Cholecalciferol
1.1
25
8
28.6
[75]
Cholecalciferol
1.1
21
20
23.4
[78]
Cholecalciferol
0.8
100
52
81
[76]
Cholecalciferol
0.8
25
20
19
[79]
Cholecalciferol
0.7
138
20
102.7
[78]
Cholecalciferol
0.6
275
20
169.8
[78]
Cholecalciferol
0.6
250
8
146
[75]
Cholecalciferol
0.5
100
20
51.8
[79]
Cholecalciferol
0.5
1250
8
643
[75]
Cholecalciferol
0.6
100
27
55.7
[80]
Ergocalciferol
0.3
36
104
11
[44]
Ergocalciferol
0.8
25
12
20
[81]
Cholecalciferol
1.5
25
12
37
Dose mg/day
Duration of dose wks
Absolute increase in 25(OH)D nmol/L
Reference
1
The results in this table represent recent work not included in Fig. 57.4. These data were assembled to permit comparison of efficacy dose of different strategies for increasing 25(OH)D concentration.
that only about 25% of vitamin D molecules taken orally will become 25(OH)D. One potential clinical advantage of 25(OH)D is that the half-life of its molecules is approximately 2 weeks [83], and this can offer an advantage if there is a concern about giving vitamin D to potentially hypersensitive individuals, such as those with hyperparathyroidism or granulomatous conditions. A shorter half-life with 25(OH)D treatment translates to a faster recovery from an overdose than with vitamin D. I am not aware that this potential advantage of more rapid clearance has been put to use. Besides, 25(OH)D is not readily available for clinical use.
The Case against Ergocalciferol, Vitamin D2 Vitamin D is available in two forms for nutritional supplementation, ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3). Vitamin D2 is manufactured by exposing a fat extract of yeast to UV light under tightly controlled conditions. Normally, plants contain little vitamin D, because light also degrades the vitamin D.
Normally, no metabolite of vitamin D2 is detectable in the blood of humans or primates [84,85]. I contend that vitamin D2 should be regarded as an artificial compound, a drug, and not a physiological nutrient. The present discussion focuses on vitamin D3, cholecalciferol, and the form of vitamin D naturally present in mammals. Vitamin D3 is the more potent form of vitamin D in all primate species and in humans [84,85]. Direct comparisons between the two versions of vitamin D [85], and a simple meta-analysis of effects on 25(OH)D (Fig. 57.4), indicate that vitamin D3 is about four times as potent as vitamin D2, i.e. 1 mg of D3 ¼ approximately 4 mg of D2. Nonetheless, vitamin D2 continues to be used clinically as if it is equivalent, since official guidelines [72] and pharmacopeias respond slowly to new evidence. Recently, Holick et al. published that the 25(OH)D response to vitamin D2 supplementation is not statistically significantly different from that of vitamin D3 [86]. However, the reasons for the lack of statistical significance are likely attributable to their low statistical power to detect a difference given the lower effect size
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DOSAGE CONSIDERATIONS
TABLE 57.5 The Clinical Interpretation of Serum 25(OH)D Levels and the Estimated Average Daily Intakes of Vitamin D Needed to Ensure these Levels (1 mg ¼ 40 IU)
Sufficiency (subjective, and under debate)
Desirable or “optimal” (subjective, and under debate)
Therapeutic/ potential excess (might increase urine and serum calcium)
25e40
50e75
>75
>225
10e16
16e40
30e64
>88
15e20 mg (600e800 IU)
No further benefit
100 mg (4000 IU)
25e100 mg (1000 e4000 IU)
100e250 mg (4000e10 000 IU)
>1000 mg (>40 000 IU)
Deficiency (rickets and osteomalacia)
Insufficiency (higher PTH and risk of osteoporosis)
Serum 25(OH)D nmol/l
0e25
Serum 25(OH)D ng/ml
0e10
Daily intake of vitamin D3 needed to reach the 25(OH)D above: ROAa From the literature reviewedb
0e5 mg (200 IU)
10e15 (400e600 IU)
a
Implications drawn from current National Academy of Sciences nutritional guidelines that the stated intake will deliver the level of adequacy, i.e. 25(OH)D concentration indicated (Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, 1997 NUTRITION1997 /id). The “adequate intake” recommendations for vitamin D vary according to age: adults <50, 5 mg/day; 50e70 years, 10 mg/day; >70 years, 15 mg/day. There is no RDA for vitamin D. b Based on literature [1,38,74,78].
achievable with their lower doses, 1000 IU/day, and fewer study subjects than the trials that did show a difference. Furthermore, all their vitamin-D2-treated groups showed lower (albeit not statistically lower) average final 25(OH)D levels than the comparable vitamin-D3-treated groups [86]. Probably most disconcerting in the context of vitamin D2 is the report by Armas et al. which showed that adults receiving an acute dose of vitamin D2 actually exhibited lower 25(OH)D concentrations after 1 month than at baseline, while those receiving vitamin D3 sustained elevated 25(OH)D levels throughout [87]. As the dose of vitamin D increases, the incremental 25(OH)D response to vitamin D2 diminishes more severely than with vitamin D3. The presumption that vitamin D2 and vitamin D3 are equivalent originated with what are now 70-year-old studies of rickets prevention in infants e even in that era, the evidence was acknowledged as weak [63,88]. The older rat data suggesting that vitamin D2 and vitamin D3 were equivalent lose their meaning when it is noted that the rat-line tests last done over half a century ago were bioassays to establish the very “units” to quantify the vitamin D not readily measured in any other way [89]. For a bioassay yielding “units,” equivalence is not the same thing as equivalence per milligram or per mole. Furthermore, all species tested show differences between the vitamin D2 and vitamin D3 [90,91], so why should humans be any different? Despite these obvious problems about units, the very conservative approach used by those who frame official statements about nutrients has remained unchanged, that one international unit of vitamin D is equivalent
to 25 nanograms of either vitamin D2 or vitamin D3 [72,89]. The differences between vitamin D2 and vitamin D3 are summarized in Table 57.6. Based on the many major differences between the two, it should be clear that unless there is a well-characterized reason to favor vitamin D2 (I am not aware of any, other than it is often the only prescription form available), all use of vitamin D for nutritional purposes should specify cholecalciferol, vitamin D3. In some countries, it is only vitamin D2 that is available as a prescription.
UVB Light Skin as a Dose of Vitamin D In the absence of clear knowledge about a dosee response curve for vitamin D, any discussion of vitamin D dosage needs to be in the context of what can be acquired from natural exposure of human skin to sunshine. As described elsewhere in this book (Chapter 2), the synthesis of vitamin D is a self-limiting reaction, reaching an equilibrium after 20e25 min of summer UVB exposure for people with white skin, and producing no net increase in vitamin D production after that [99]. Darker skin requires longer exposure but the potential yield of vitamin D is the same. Exposure of full skin surface to UVB light, in an amount less than an erythemal dose, is equivalent to a vitamin D consumption of about 250 mg (10 000 IU)/day [100e103]. Lifeguards in the USA and in Israel, as well as farmers in the Caribbean, all exhibit serum 25 (OH)D concentrations greater than 100 nmol/l [104e106]. Furthermore, even regular short periods in
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
1054 TABLE 57.6
57. THE PHARMACOLOGY OF VITAMIN D
The Case Against Vitamin D2, Compared to Vitamin D3
Vitamin D2
Vitamin D3
Not detectable in humans or primates unless administered from an external source
The natural metabolite generated within skin and the oils of fur
After a single 100 000 IU loading dose, serum 25(OH)D2 is actually lower a month later than at baseline
After a single 100 000 IU loading dose, serum 25(OH)D3 is sustained higher than baseline for at least a month
[87]
[92]
Generates metabolites for which there is no vitamin D3 equivalent Microsomal 25hydroxylase does not act on it
Ref.
Substrate for both microsomal and mitochondrial 25hydroxylases
Per mole of dose, the 25(OH)D increases by less than with vitamin D3
[93,94]
[85]
The 25(OH)D response to vitamin D2 is less in the elderly than in younger adults
25(OH)D response to vitamin D3 is the same for young vs older adults
[76,77,95]
All known cases of iatrogenic toxicity with vitamin D involved the vitamin D2 form (albeit, formulations >25 mg (1000 IU) have usually been vitamin D2)
All known adult cases of toxicity with vitamin D3 have been unintentional, "industrial" accidents
[1,96e98]
Vitamin D2 preparations are less stable
[71,85]
suntan parlors consistently raise serum 25(OH)D well beyond 80 nmol/l [107]. The highest 25(OH)D concentrations in the groups of adults acquiring vitamin D physiologically (via UV exposure) range up to 235 nmol/l [104,108], and none of these studies imply that such 25(OH)D levels have caused hypercalcemia. Since humans evolved as naked apes, whose native habitat was within 30 degrees latitude of the equator, our genome was selected under conditions of abundant vitamin D supply [1,2]. As such, it is reasonable to ask whether the substantially lower levels of 25(OH)D in modern societies have been accompanied by biological compromises. Barger-Lux and Heaney studied the effect of sunshine on healthy outdoor workers in the US Midwest, relating it to the vitamin D intakes needed to bring about the 25(OH)D levels observed [27]. They concluded that for
these men, the summertime supply of vitamin D from sunshine was approximately 70 mg (2800 IU)/day. This supply during summer, did not ensure sufficiency through the winter, when 25(OH)D fell to less than 50 nmol/l in three of 26 subjects and less than 75 nmol/l in 15 of 26 subjects. Similarly, Aloia et al. estimate that to exceed the target 25(OH)D level of 75 nmol/l, adults need from 107 mg/d (4280 IU) to 120 mg/d (4800 IU) [80].
PHARMACOKINETIC PRINCIPLES, VOLUME OF DISTRIBUTION, TURNOVER AND HALF-LIFE AS IT PERTAINS TO VITAMIN D A complete understanding of the pharmacokinetics of the vitamin D system has eluded researchers. This is because of the technical issue of measuring the nanomolar quantities of vitamin D potentially embedded within tissues or excreted in catabolized forms. It is extremely difficult to detect or to measure vitamin D and its metabolites when they exist among great excesses of other lipids. Perhaps the most careful study into the fate of physiological amounts of cholecalciferol was reported by Lawson et al. [109,110]. They exposed rats with shaved skin to ultraviolet light (UVB), and measured vitamin D and 25(OH)D in tissues at various times afterwards. Although adipose tissue concentration of vitamin D was never greater than the plasma concentration, it contained the largest exchangeable pool of vitamin D. Recovery of vitamin D3 in adipose was less than 5% of the amount produced within the skin [109], and this low recovery was attributed to vitamin D excretion into the bile. Lawson et al. estimated that the volume of distribution of unmetabolized vitamin D3 was approximately 4 liters per kg (based on concentration decay curves from plasma and total amounts recovered from tissues). Vitamin D is not detectable in the adipose tissue of normal rats [110,111], but with administration of pharmacological doses [112], or shaving of fur to increase yield five-fold [109], vitamin D is detectable. Brouwer et al. estimate the half-life of vitamin D in rat adipose tissue to be 96 days, which is plausible because it compares with the functional half-life of 25(OH)D in humans [1]. In contrast, Lawson et al. [109] estimated the vitamin D in rat adipose tissue to have a half-life of 13.8 days. The more rapid half-life reported by Lawson et al. was likely due to the younger age of the rats. What complicates the pharmacology even more is that unlike a drug, vitamin D is present in the body naturally. It is impossible to start with completely deprived individuals who would be ideal for the appropriate studies of pharmacokinetics. Furthermore the component of nutritional interest is 25(OH)D,
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
PHARMACOKINETIC PRINCIPLES, VOLUME OF DISTRIBUTION, TURNOVER AND HALF-LIFE AS IT PERTAINS TO VITAMIN D
a metabolite of either vitamin D2 or vitamin D3. Studies using isotopic techniques show that in humans, molecules of 25(OH)D have a plasma half-life of about 10e14 days [32,113]. However, a more practical measure of the half-life of 25(OH)D is reflected in the rate at which 25(OH)D concentrations decline upon the sudden elimination of sources of vitamin D (acute deprivation of ultraviolet light). Two studies show that when sailors embark upon 2-month-long missions in submarines, the 25(OH)D concentration decreases by approximately 50% [1,114,115]. Follow-up of 25(OH)D concentrations in adults who have been intoxicated with vitamin D3 suggests that the functional in vivo half-life is of the order of several months [96,98,116e118]. During summer, we can accumulate and store vitamin D well enough so that supplies of vitamin D do not deplete during the winter months. Within 3 days of a dose of vitamin D, very little of the original vitamin D is detectable in the plasma compartment of rats [119] or humans [120]. Most vitamin D entering the circulation appears to be excreted into the bile. The highest total concentrations of vitamin D and its metabolites occur in plasma. However, since plasma represents only 2.5% of body mass, larger pools of vitamin D and 25(OH)D exist in fat and muscle [121e123]. When there is a continuous supply of vitamin D, as is the situation with regular exposure of skin to tropical sunshine, the stores of vitamin D in the body are in an equilibrium state, maintaining a balance between vitamin D storage and removal. Under these natural, sun-derived circumstances, the 25(OH)D concentrations in plasma can sustain levels of more than 200 nmol/l [1]. At these levels of vitamin D nutrition, there has never been a concern raised that sudden loss of adipose tissue would either raise 25(OH)D or predispose to vitamin D toxicity. Likewise, despite 70 years of experience with the oral use of vitamin D in amounts that exceed by several fold the amounts that can be obtained through sun exposure, there has never been a report of an eventual, sudden excess of vitamin D caused by release from adipose stores because of weight loss.
Body Storage of Vitamin D and Inefficient Conversion to 25(OH)D There are many reports that obese individuals exhibit lower serum 25(OH)D concentrations than reference subjects with a lower body mass index [124e129]. The explanation that this is because greater amounts of adipose tissue sequester more fat-soluble vitamin D, thereby increasing requirements for the nutrient simply due to greater adiposity, is not convincing. One obvious problem with the explanation is that women, who average 50% higher adiposity per unit body weight
1055
than men, do not seem to suffer inordinately from low 25(OH)D levels, and they do not require higher intakes of vitamin D per unit body mass than men. More plausible is that lower 25(OH)D levels with obesity simply reflect a larger volume of distribution e greater body mass e and not adiposity per se. Furthermore, all the obesity studies cited about this are cross-sectional [124e127], and they fail to account for outdoor activity, and the proportion of skin subjects exposed to sunshine, both of which are more plausible explanations of low 25(OH)D concentrations in obese persons. The effects of vitamin D supplementation on 25(OH)D levels are only moderately less for people with higher percent body fat [125,127]. At physiological doses, cholecalciferol (unmetabolized vitamin D3) distributes widely into adipose tissue, skeletal muscle, and organs [109,121,123]. The turnover of vitamin D exhibits a long biological half-life of about 2e3 months [1,117,121,128]. As stored vitamin D gradually returns into the circulation, it is converted into 25(OH)D. The result is that when vitamin D treatment is discontinued, the level of serum 25(OH)D is sustained by the returning phase of the equilibrium between vitamin D in its various body compartments back into the circulation. When supplies of vitamin D are stopped, the serum 25(OH)D declines with a biological half-life of about 2 months [117,118,121]. Even though the half-life of molecules of 25(OH)D per se exhibit a half-life of only 2 weeks [32], the functional half-life of 25(OH)D is 2e3 months. Amounts of vitamin D recoverable from tissue stores account for only a fraction of the dose administered [109]. Animal data obtained at steady state after prolonged dosing with either vitamin D or 25(OH)D indicate a substantial difference in efficacy in raising the serum 25(OH)D concentration, leading to the conclusion that more than three-quarters of the molecules of vitamin D taken orally fail to become 25(OH)D [129]. There is no evidence that one additional hydroxyl group on a steroid-like molecule can alter intestinal absorption; moreover, unlike its parent vitamin D, if 25(OH)D is taken orally it can be metabolized pre-systemically [130]. Available evidence indicates that if there were a difference in availability from the intestine into circulation, 25(OH)D would be the lesser absorbed. The human data also support the greater potency of 25(OH)D and the concept that only a small proportion of vitamin D molecules ends up as 25(OH)D. In humans, when vitamin D or 25(OH)D is given over the long term, to achieve the same equilibrium concentration of serum 25(OH)D, it takes about four times as much vitamin D as 25(OH)D [75]. By definition, at that plateau in 25(OH)D, the exchange of vitamin D in its various body compartments is at equilibrium, where the release of stored vitamin D equals the storage of new vitamin D. When
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1056
57. THE PHARMACOLOGY OF VITAMIN D
comparing effects of doses of 25(OH)D and vitamin D, a four-fold difference exists in efficacy at sustaining 25(OH)D (Table 57.4). The difference in efficacy between 25(OH)D and vitamin D at sustaining plasma 25(OH)D concentrations cannot be explained by deposition of vitamin D into storage sites because the data summarized are at timepoints close to the plateau, equilibrium concentrations in the circulation. At equilibrium, the difference in efficacy between oral vitamin D and 25(OH)D at sustaining serum 25(OH)D concentrations can only be explained by loss of vitamin D to fates other than 25-hydroxylation or storage.
VITAMIN D TOXICITY AND SAFETY ISSUES Science is normally approached with a focus on one specific outcome. In contrast, the question of toxicity is an open-ended one. Safety considerations are precautionary and conclusions relating to safety can be based on levels of evidence that would never be acceptable in any other scientific context. The acknowledged, traditional criteria for vitamin D toxicity are objective measures: hypercalcemia, and to a lesser degree hypercalciuria [72,131]. However, the large amount of attention given to vitamin D in relation to disease prevention and treatment has brought with it some contra-intuitive findings suggestive of higher risk of cancer of the prostate and pancreas [19e21] and even mortality [22]. Although desirable trends with higher serum 25(OH)D levels are far more common, the rare exceptions have drawn particular attention in recent years. It has been pointed out above that the U-shaped risk curves have been specific to northerly regions, particularly Scandinavian countries [19,20,22]. Similar relationships have not been reported in studies of populations living further south, despite what should be generally higher levels of serum 25(OH)D. The basis for the theory to explain the paradoxically adverse phenomena relates to the relatively larger seasonal fluctuations in serum 25(OH)D at high latitude. The higher the summertime 25(OH)D levels, the bigger the decline through winter. Vitamin D supplementation will moderate the effect of seasonal 25(OH)D instability [14]. It is important to note that none of the reports suggestive of U-shaped risk involved an active intervention to increase intake of vitamin D. Therefore, there is little upon which to base a meaningful discussion about dosage or pharmacology in the context of U-shaped risk. What we do know from the clinical trials involving doses of vitamin D high enough to produce 25(OH)D in the high end of the putative U-shaped risk is that those trials have produced either desirable outcomes or no significant effect one way or the other (Table 57.3). It is also
important to note that across the wide range of doses used in Table 57.3, none of the vitamin-D-alone treatments produced a significant increase in serum calcium. It is generally presumed that vitamin D intake will eventually raise serum calcium, but there has been no evidence of that from the clinical trials, because the dosages used were not high enough. The rest of this section focuses on the traditional, objective, calciumrelated aspects of safety of vitamin D. Vitamin D, like everything that has an effect on living things, can be harmful if taken to an extreme. The reason vitamin D was perceived as toxic in the past was probably because the industrial synthesis of vitamin D2 in the first half of the 20th century made it possible for people to consume products providing milligram doses (over 40 000 IU), and now we know that these doses exceed by several fold the maximum physiological rate at which vitamin D could be acquired via exposure of skin to UVB light. Toxicity in normal adults requires intake of more than 1 mg (1000 mg, or 40 000 IU) per day on a long-term basis [1,132]. Milligram amounts of other nutrients are generally benign, so in comparison, vitamin D might be seen as toxic. In 1990, I proposed the theory that the mechanism of vitamin D toxicity involves saturation of circulating vitamin-D-binding protein (DBP) to the point that the percent of vitamin D and its metabolites that are free and accessible to target tissues increases inappropriately [133]. The theory was subsequently confirmed through laboratory testing of serum from patients intoxicated with vitamin D [1,134]. At toxic doses, the freely circulating vitamin D, along with its metabolites, accumulates in both adipose [112] and muscle [123]. The average capacity of human plasma DBP to bind vitamin D and its metabolites is 4700 nmol/l [30], and this exceeds by 20 times the physiologic total concentration of its vitamin-D-derived ligands. The 100 mg (4000 IU)/ day of vitamin D that is starting to be used in adult clinical trials [49e51,60] is physiologic and far below what would be needed to change the free fraction of vitamin D or its circulating metabolites [30]. The vast majority of cases of vitamin D intoxication have involved vitamin D2 [1]. The situations involving vitamin D3, to date, have been industrial accidents [97,98,136] or poisonings from an unknown source [96]. In our case, we assayed blood levels of vitamin D and its metabolites by chromatography and found that despite record-high 25(OH)D concentrations in humans (2400 nmol/l), they were still small in comparison to a large excess of vitamin D3 (17 000 nmol/l) suggesting that the capacity of liver to hydroxylate vitamin D is limited [96,135]. There are now many clinical trials of vitamin D3 supplementation in adults, involving the daily consumption of 100 mg (4000 IU) [79,80,132]. None of
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those trials has elicited the hypercalcemia in normal adults that was reported by Narang et al. [136], and which was used by the Food and Nutrition Board to establish the 50 mg/d (2000 IU/day) upper level for vitamin D intake. My colleagues and I have probably been the most active in attempting to characterize the point at which vitamin D supplementation starts to increase serum calcium. Based on clearly published data on calcium through various protocols, ranging from 4 months at 10 000 IU daily, to 15 months at 4000 IU daily to a year of an average intake of 14 000 [54,57,79,137,138], I can conclude on a firm basis of data that vitamin D in doses relevant to nutrition and therapeutics does not raise serum calcium e not even incrementally. That said, we have reported on a patient with multiple sclerosis who was motivated to maximize his serum 25(OH)D; he raised his calcium upon prolonged consumption of 80 000 IU (2000 mg) daily [118], a dose well beyond the realm of this discussion. By now, it should be obvious that the hypercalcemia elicited by Narang et al. (138) and which was the basis of the 1997 dietary upper level for vitamin D was essentially a bioassay showing that the vitamin D doses they actually administered were far higher than the 3800 IU daily they thought they had given. In the early 1990s, a dairy that delivered milk to 10 000 households made prolonged, gross errors in fortifying milk with several milligrams of vitamin D3 per quart. A rigorous epidemiological follow-up showed that the situation contributed to two deaths of susceptible elderly [97]. While hypercalcemia did occur, it was not widespread. By far the most susceptible group to the excess vitamin D was women aged over 65 years, suggesting that diminished renal function may have played a role, in the opinion of those authors. The average serum 25(OH)D concentration among the confirmed cases of vitamin D toxicity was 900 nmol/l (214 ng/ml) [97]; in comparison, physiologically attained 25(OH)D concentrations, obtained through sunshine exposure, can reach 235 nmol/l (90 ng/ml) safely, without hypercalcemia or hypercalciuria. When physiologically higher vitamin D nutrition is associated with hypercalcemia, this reflects aberrant control of 25(OH)D-1-hydroxylase. This would reflect either primary hyperparathyroidism, where PTH continuously stimulates the enzyme in the kidney [16,117], or granulomatous disease, where peripheral tissue loses its ability to regulate the 1-hydroxylase that normally serves a paracrine role [1,139]. Inappropriate production of 1,25(OH)2D is an unusual and special situation, and it can cause hypercalcemia. In people with abundant sun exposure, the serum 25(OH)D can exceed 150 nmol/l, and such a pre-supplement supply of vitamin D would be equivalent to at least 100 mg (4000 IU)/day [27]. If these individuals were to
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take an additional dosage by mouth of 100 mg/day of vitamin D, this would still be less than the dose of vitamin D shown to be safe in recent studies [54,78,138]. To date, only two clinical trials have been published whose purpose it was to define a limit of tolerability for vitamin D consumption and a serum 25(OH)D that would not result in hypercalcemia or hypercalciuria [54,140]. In a sense, both failed to achieve their purpose, because of the 49 combined subjects undergoing a doseescalation protocol that included a month of 40 000 IU per day (given as once-weekly 280 000 IU), neither elicited hypercalcemia or hypercalciuria. And while there was a modest rise in urinary calcium as should be expected, there was no change in serum calcium [54,140]. Hathcock et al. [1,132] have summarized case reports of vitamin D toxicity in reviews by Vieth. Based on the dose-escalation clinical trials that included a month of 40 000 IU/daily [54,140], and on the case reports where hypercalcemia was demonstrated along with elevated serum 25(OH)D [1,132], we know that long-term vitamin D consumption of at least 40 000 IU (1000 mg) per day would be needed to cause toxicity. By the conventional calcemic criteria, there is a wide margin of safety with 100 mg (4000 IU)/day e a dose that has been used in several clinical trials (Table 57.3), and which warrants further study. One concern sometimes expressed is that if adipose tissue were to break down, a sudden influx of vitamin D from adipose might be toxic [141]. In both rats and cattle, high doses of vitamin D are needed before vitamin D ends up as detectable in adipose tissue [112,123]. Despite being present in “significant” amounts in tissues, storage in tissues in not efficient. As a proportion of what enters the body via the skin or the diet, the amounts of vitamin D stored in adipose are a fraction of the total. In normal humans, adipose tissue content of vitamin D has been reported to be as high as 116 ng/g (approximately 5 IU/g adipose) [110]. In cattle intoxicated with 7.5 million IU vitamin D (to cause hypercalcemia, in an experimental process to activate proteases to make beef more tender after slaughter), muscle levels of vitamin D reached 91 ng/g tissue (4 IU/g). The highest tissue level reported in those animals was in the liver, which contained vitamin D at 979 ng/g (39 IU/g) [123]. The point is that while there is “meaningful” storage of vitamin D in tissues, all the evidence indicates that only a fraction of any vitamin D dose ends up in tissues to be withdrawn at later times. If there were a sudden breakdown of 1 kg of adipose tissue, or liver, this would release as much as 979 mg (39 000 IU) of vitamin D into the body. A toxic excess of vitamin D would require the break down daily of 1 kg of adipose tissue that had been primed by prior vitamin D intoxication, with daily adipose catabolism
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to continue for several weeks. When toxic doses of vitamin D are administered, the effect will be manifest during the period of administration. There is no evidence that enough residual vitamin D can be stored in adipose tissue that vitamin D toxicity could possibly arise at some later time, because of weight loss. Officially the safety limit for vitamin D, average, longterm intake without supervision by a physician is referred to as the “tolerable upper level” (UL). This is the cumulative average daily amount of vitamin D that adults can take without physician supervision on a long-term basis with no anticipation of harm. Guidelines in both North America [72] and Europe [131] have established the UL as 50 mg or 2000 IU/day. It should be clear that since the functional half-life of vitamin D approximates 2e3 months [1,96,144], it is perfectly appropriate to take vitamin D less often than daily. Different preparations of vitamin D, designed for daily, weekly or monthly use, have tended to produce differing effects on serum 25(OH)D, probably due to differences in bioavailability [145,146]. However, it is now well accepted that if the preparation is identical, with only the dosage adjusted to contain the same cumulative amount of vitamin D, then there is no difference between daily, weekly or monthly vitamin D [147]. The point is, the UL refers to cumulative average vitamin D consumption, and it does not pertain to an individual dose.
THE CONCEPT OF A LOADING DOSE There have been many attempts to improve vitamin D nutritional status quickly with convenient annual doses. The older literature referred to this as “stosstherapy” [1]. From my perspective, the search for a suitable loading dose for vitamin D has been an arbitrary hitand-miss process, and has never been approached from basic principles of pharmacology. Reports have been empirical, with bolus doses of vitamin D administered, followed by measures of serum 25(OH)D levels and calcium parameters usually done months later, and to my knowledge nobody has actually tried to match the loading dose to the desired maintenance dose [144,148e150]. Any statement about a loading dose is meaningless unless it is in the context of the subsequent maintenance dose. In the field of pharmacology, a loading dose is an amount of drug designed to fill the central volume of distribution for a drug to a concentration that matches the final plateau concentration achieved with the maintenance dose. The purpose is to achieve this final plateau sooner than the four half-lives required if the drug is simply administered at the maintenance dose rate [151]. Traditionally, a loading dose for a drug is based
upon the estimated volume of distribution (a theoretical number), and the dose is calculated to fill that volume to the desired plasma concentration. For vitamin D, I am not aware that the volume of distribution has ever been estimated meaningfully; all we know is that the apparent volume of distribution is very large. Furthermore, the half-life for vitamin D in the body is a long one, compared to other pharmaceuticals. And even beyond those complicating issues, about three-quarters of the vitamin D that is consumed orally and stored in tissues never does get metabolized to the 25(OH)D whose serum concentration is the target of interest. The situation is not hopeless, and there is a relatively simple solution. The half-life of a drug in the body provides an alternative means with which to estimate a required loading dose. At a steady-state plasma concentration, the half-life of drug molecules (if they were labeled with an isotope) in the body reflects their dilution and displacement by an equal number of drug molecules newly introduced during the half-life. In essence, we do not need to know the volume of distribution if we know the half-life of a drug. For vitamin D3, we know that the functional half-life within the body is in the range of 2 to 3 months [1,121,144]. (Note that since the half-life for vitamin D2 is not as well known, this discussion of loading dose is specifically referring to vitamin D3.) Given the preceding explanation of a pharmaceutical half-life at the steady state, then a suitable loading dose that is equivalent to the amount in the volume of distribution can be calculated without actually knowing the volume of distribution. The loading dose can be calculated as the cumulative maintenance dose that is planned to be given through one functional half-life of vitamin D in the body, we can calculate that loading dose as follows: Loading Dose ¼ ðDaily Maintenance DoseÞ ð60 to 90 days; depending on half-lifeÞ For example, if it is thought that a patient should need an additional 1000 IU/day of vitamin D3, and there is a lack of patience to wait the 8 months required to attain a final plateau in the serum 25(OH)D concentration, then a suitable loading dose should be 60 000 to 90 000 IU (1.5e2.25 mg) of vitamin D3. From the time when that loading dose is given onwards, the average daily intake should be sustained at 1000 IU/day. Such a protocol should permit attainment of the stable 25(OH)D outcome within a week or two. The effects of various loading doses are illustrated in Fig. 57.5, along with the effects of over- or underestimating the loading dose. To my knowledge the closest published approximation to the preceding protocol is the clinical trial of Bacon
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THE CONCEPT OF A LOADING DOSE
from the loading dose, the matching maintenance dose would have been equivalent to 500 000 IU divided by a 90-day half-life, or equivalent to 5560 IU per day. This scenario is presented to illustrate the relationship between the loading dose and the maintenance dose, and is not intended as a recommendation of any sort. The preceding strategy for estimating a loading dose to reach the maintenance plateau quickly is based on what is known about vitamin D and about pharmacokinetics [151], but to my knowledge this has not yet been put to use clinically or reported in the literature.
Serum 25(OH)D (arbitrary scale)
D
B
C
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A
The Problem with Weekly 50 000 IU Vitamin D2 as a Loading Dose –2
0 2 4 6 8 12 Months from start of treatment
14
FIGURE 57.5 Effect of the loading dose in relation to its corre-
sponding maintenance dose of vitamin D3. By definition, a loading dose must be selected in the context of the maintenance dose expected to produce the final, desired serum 25(OH)D concentration. (A) The lower solid-line curve represents the gradual rise in serum 25(OH)D concentrations in response to a daily maintenance dose alone, assuming a half-life rising time of 2 months. (B) The response to a perfect matching of the loading dose to the maintenance dose; this can be based on the approximation that loading dose ¼ cumulative intake of maintenance dose through one half-life. The consequence of underestimating the loading dose is indicated by the dashed line (C), which may still be useful because it shortens the time to the final plateau. The consequence of overestimating the loading dose is indicated by line (D), which tapers downward to the final maintenance dose plateau. Careful selection of maintenance and loading doses can minimize or eliminate the need for follow-up monitoring of serum 25(OH)D levels.
et al. who administered a loading dose of 500 000 IU, followed by monthly maintenance doses of 50 000 IU (equivalent to 1670 IU daily) [144]. In their case, the serum 25(OH)D concentration peaked at a higher level and tapered downwards with the time on the maintenance dose. This shows that the loading dose was too high in relation to the maintenance dose. What is also unfortunate with the administration of 500 000 IU all at once is that the loading dose can be so large that it can cause an acute initial phase of vitamin D toxicity, as explained in the next section of this chapter. A refinement to the loading dose protocol to make it safer would be not to take vitamin D much beyond 100 000 IU at one time. If a higher loading dose is desired, then provide it in weekly increments of 100 000 IU to allow vitamin D to clear from the circulation between each increment of the loading dose. A safer way to provide a very large loading dose like 500 000 IU would have been to provide it at a rate of 100 000 IU weekly for 5 consecutive weeks, and to then immediately begin the regular maintenance dose. Working
Patients prefer to have vitamin D by prescription if it is covered by health insurance. In the USA, vitamin D3 is not available by prescription, and so it has become a routine practice to prescribe 50 000 IU vitamin D2 weekly for 8 weeks, followed by a maintenance dose to be empirically determined based on follow-up testing of serum 25(OH)D [152]. This protocol has never been validated. The citations offered by Holick to justify it only reiterate versions of statements describing of the protocol, but provide no actual data to support it [153]. The protocol of weekly 50 000 IU vitamin D2 carries with it a risk that the prescription product may actually be used according to the package label. At least one published report exists of a patient prescribed vitamin D2, and who took it daily, causing prolonged toxicity manifest as hypercalcemia [117]. Vitamin D2 suffers from many disadvantages, as listed earlier in this chapter; it does behave differently from vitamin D3 [87], and to my knowledge no fracture prevention trial has been published for vitamin D2. Therefore, it is not pharmacologically rational to load with one form of a drug, with the intent to eventually maintain the patient on a different form of the drug that exhibits different behavior. If the purpose is to prevent fractures, then patients should be made aware that a prescription for vitamin D2 to raise serum 25(OH)D is an “off-label use” of a drug for which no clinical trial has shown efficacy in preventing fractures.
At What Point is a Loading Dose Toxic? It is not appropriate to regard a loading dose as a daily dose averaged over time. There does come a point when an acute dose is so large that it is toxic. The administration of annual bolus doses has recently been proven to cause adverse outcomes [45,150]. One remarkable feature of the Sanders clinical trial of annual vitamin D consistent with cycles of acute toxicity is that the temporal pattern of the falls and fracture events showed
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increases specifically during the first 3 months after each annual dose [150]. In contrast, a previous clinical trial using a comparable cumulative dose, but given in smaller individual doses, once every 4 months, did result in the desired lower rates of bone fracture [154]. It has been suggested that the practice of using “high vitamin D loading doses” might need to be reconsidered [155]. Although this is largely correct, it is important to note that Sanders et al. used 500 000 IU as an annual maintenance dose and not as a well-considered, onetime loading dose. Problems likely arose because of the magnitude of the acute dose, which exceeded by several-fold the buffering capacity of DBP, and secondarily, because the dosing interval was in any pharmacological context, a drug “washout” period. The paper of Sanders et al. should not be taken as an indication that vitamin D is toxic at a modest dose of 1400 IU/day (the annual dose used by Sanders et al. was on average equivalent to 1400 IU/day) and it should not be concluded that the peak 25(OH)D concentration they observed, 125 nmol/l (50 ng/ml), is harmful. Cipriani et al. published in greater detail the effects of a bolus, 600 000 IU dose of vitamin D [156]. Three days after the dose, there was a modest transient rise in serum calcium, and a doubling in the serum 1,25(OH)2D concentration, which subsided by 3 months to its baseline concentration e that transient in 1,25(OH)2D coincides with the timeframe in which Sanders observed higher rates of falls and fractures. Meanwhile, the serum 25(OH)D level declined in both the Sanders and the Cipriani studies at a rate consistent with a 2-month half-life [152]. A drug-free interval of four half-lives is normally regarded as a drug “washout period,” and outside the realm of vitamin D; such a long dosing interval in the context of the half-life is virtually unheard of as a treatment strategy. While an average vitamin D3 dose of 1369 IU per day may seem modest, and while larger but less frequent doses are appropriate, there does come a point when a one-time dose becomes toxic. We now know from the Saunders trial that a form of vitamin D toxicity occurs when a 1369 IU/day dose is given as 500 000 IU all at once [150,156]. A bolus dose of that magnitude overwhelms the capacity of vitamin-Dbinding protein (DBP) in the circulation and is toxic. The toxicity manifests as increased falls and fractures in the following 3 months [152]. As described earlier in this chapter, a key mechanism of vitamin D toxicity is that once vitamin D and its metabolites exceed the high but finite capacity of DBP, the biologically available “free” level of 1,25(OH)2D goes up inappropriately. Excessive 1,25(OH)2D at its target tissues induces the catabolic enzyme 25(OH)D24-hydroxylase (CYPP24) that eliminates 25(OH)D and 1,25(OH)2D at peripheral tissues. Since DBP normally circulates at 4300 nmol/l [30], the capacity of all the
DBP in the circulatory system to bind vitamin D and its metabolites is only about 12 900 nmol (assumes 3 l plasma volume), or equivalent to about 200 000 IU of vitamin D. Since a one-time dose of vitamin D of 500 000 IU (12 500 mg) is well in excess of the capacity of human plasma to bind vitamin D, it should not come as a surprise that there were adverse consequences. The conclusion to be drawn from the papers of Sanders et al. and Cipriani et al. is that a 500 000 IU bolus of vitamin D causes a transient rise in calcium not detectable a month later, and a 2-month period of higher circulating 1,25(OH)2D levels. That period of higher serum 1,25(OH)2D should be regarded as a mild, acute form of vitamin D toxicity that coincides with a 3-month period of higher risk of falls and osteoporotic fracture [150,156].
SUMMARY AND CONCLUSIONS This chapter has focused on vitamin D as if it were a drug. Pharmacology imposes a discipline about the administration of vitamin D that has been too often ignored, with the consequence of sometimes causing patients more harm than good. The irony with vitamin D is that, as with anything that actually works to a benefit, there is a risk of harm if it is taken in the extreme. Potential for risk is no reason to deny the benefits that are clearly evident for vitamin D. The biology of all primates, including humans, is optimized for tropical, UVB-rich environments that consistently provide vitamin D in equivalent daily doses well in excess of the 800 IU (20 mg) used in osteoporosis clinical trials in the elderly, and which are physiological and safe, up to 10 000 IU (250 mg)/day. I do not make this statement about safety to advocate that high a dose, but rather to reemphasize the safety of the doses shown in Table 57.3, which summarizes many clinical trials with desirable outcomes using vitamin D doses in excess of 1000 IU/day, and without harm, and across segments of the population ranging from young to old. Based on Table 57.3, a desirable serum 25(OH)D concentration is one that is at least 70 nmol/l (28 ng/ml). To deny the existing evidence from clinical trials begs the question, “How much evidence do you need?” As outlined in Figure 57.2, it is probably not realistic for anyone to expect that placebo-controlled, primary-disease-prevention clinical trials will ever be undertaken for all population groups. Nonetheless, there are those who contend that no dose of vitamin D should be advocated for any group beyond the 800 IU/day that was used in the osteoporosis clinical trials (see Chapter 5). In every other context, promotion of health and prevention of disease begins as early in life as possible, so why do we direct vitamin D advice primarily to older adults? The greatest
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REFERENCES
public health risk in the context of vitamin D stems from the lack of it and not its excess. In closing, I return to the pharmacological questions posed at the start of this chapter, and based on material covered in this chapter, address the questions here in the context of vitamin D: 1a. What are the established indications for use? Prevention of rickets and osteomalacia. Prevention of falls and fractures. Co-therapy with calcium to improve efficacy of drug treatments for osteoporosis. 1b. What other clinical or health effects should we be looking for, based on preclinical animal and laboratory research? Disease prevention: cancer, autoimmune conditions, including diabetes and multiple sclerosis, cardiovascular health, fibromyalgia, muscle strength, mood/depression, insulin responsiveness. 2a. What are the most useful approaches to increasing vitamin D status? Encourage exposure of a large percent of skin surface to summertime sunshine, 10 min daily for white skin, up to six times longer for very dark skin. Fortification of foods to physiologically meaningful levels of vitamin D, consumption of supplement preparations containing vitamin D. Since these approaches are often neither practical nor adequate, supplementation may be required. 2b. What is the best dosage and route of administration? Dosage depends on the target concentration of 25(OH)D desired. We can assume a rule of thumb, that a dose of 1 mg/day vitamin D increases 25(OH)D by 1 nmol/l, after 8 months of use (see Table 57.4). In non-SI units, this is the equivalent to saying that 100 IU/day increases 25(OH)D by 1 ng/ml. Oral vitamin D is probably more effective than injection. What is a suitable interval between doses? Since the halflife of serum 25(OH)D levels is effectively 2 months, doses of vitamin D could be given at monthly intervals (we use weekly in our studies). Less frequent dosing is possible, but the dose given at one time needs to balance the risk of acute toxicity (related to capacity of DBP). Furthermore, some health risks may result from large fluctuations in serum 25(OH)D, because the enzymes involved in the regulation of 25(OH)D metabolism function in a first-order relationship with substrate. 3. What is the desirable target for the plasma concentration, what dose would be needed to attain or ensure this? It was not the purpose of this chapter to lay out the evidence for the desirable minimum serum 25(OH)D level. Some contend the desirable minimum for 25(OH)D levels should be 50 nmol/l (20 ng/ml); but even if this is to be the minimum, then the average serum 25(OH)D would need to approximate 75 nmol/l. The level more commonly specified as
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desirable is one higher than 75 nmol/l (30 ng/ml) [38], which approximates an average serum 25(OH)D level for adults taking 25 mg (1000 IU)/day vitamin D, and who are getting sunshine. To ensure that 75 nmol/l is the minimum for a normal population requires a population average 25(OH)D concentration of about 120 nmol/l (48 ng/ml). This objective requires an intake of 100 mg (4000 IU)/day for all adults [49e51,60]. (This is by no means an official recommendation, but rather a scientific opinion offered by the author for research purposes. The dosage of vitamin D required for humans continues to be a subject of controversy.) 4. What, if any, are the biological markers to monitor toxicity, and what are our criteria for determining therapeutic effectiveness? What is the “therapeutic index”, the ratio between toxic versus beneficial dose levels? Hypercalcemia is the classic criterion for toxicity of vitamin D, its metabolites, and their analogs. “Non-calcemic” doses are those that do not cause hypercalcemia, and are considered “safe” by conventional criteria. However, the most sensitive clinical index of safety is urine calcium. This is easily monitored by measuring a morning urine calcium/ creatinine ratio (normal for this would be mmol/ mmol <1; or in non-SI units, mg/mg <0.35) [79,54]. Of some concern for the long-term use of vitamin D are the suggestions from epidemiology that pancreatic and prostate cancer risk may relate to higher serum 25(OH)D concentrations. However, these appear to be specific to regions with large seasonal fluctuations in environmental ultraviolet light and 25(OH)D [14]. Likewise, isolated 500 000 IU doses of vitamin D cause large fluctuations in serum 25(OH)D concentrations, and should be regarded as toxic because they have been shown to increase rates of fracture [152].
References [1] R. Vieth, Vitamin D supplementation, 25-hydroxyvitamin D concentrations, and safety, Am. J. Clin. Nutr. 69 (5) (1999) 842e856. [2] N.G. Jablonski, G. Chaplin, The evolution of human skin coloration, J. Hum. Evol. 39 (1) (2000) 57e106. [3] M.G. Kimlin, N.J. Downs, A.V. Parisi, Comparison of human facial UV exposure at high and low latitudes and the potential impact on dermal vitamin D production, Photochem. Photobiol. Sci. 2 (4) (2003) 370e375. [4] S.K. Moestrup, P.J. Verroust, Megalin- and cubilin-mediated endocytosis of protein-bound vitamins, lipids, and hormones in polarized epithelia, Annu. Rev. Nutr. 21 (2001) 407e428. [5] T.E. Willnow, A. Nykjaer, Pathways for kidney-specific uptake of the steroid hormone 25-hydroxyvitamin D3, Curr. Opin. Lipidol. 13 (3) (2002) 255e260. [6] T.E. Willnow, A. Nykjaer, Cellular uptake of steroid carrier proteins e mechanisms and implications, Mol. Cell. Endocrinol. 316 (1) (2010) 93e102.
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[135] R.P. Heaney, L.A. Armas, J.R. Shary, N.H. Bell, N. Binkley, B.W. Hollis, 25-Hydroxylation of vitamin D3: relation to circulating vitamin D3 under various input conditions, Am. J. Clin. Nutr. 87 (6) (2008) 1738e1742. [136] N.K. Narang, R.C. Gupta, M.K. Jain, K. Aaronson, Role of vitamin D in pulmonary tuberculosis, J. Assoc. Phys. India 32 (2) (1984) 185e186. [137] R. Vieth, S. Kimball, A. Hu, P.G. Walfish, Randomized comparison of the effects of the vitamin D3 adequate intake versus 100 mcg (4000 IU) per day on biochemical responses and the wellbeing of patients, Nutr. J. 3 (1) (2004) 8. [138] S.M. Kimball, M.R. Ursell, P. O’Connor, R. Vieth, Safety of vitamin D3 in adults with multiple sclerosis, Am. J. Clin. Nutr. 86 (3) (2007) 645e651. [139] N.H. Bell, Renal and nonrenal 25-hydroxyvitamin D-1alphahydroxylases and their clinical significance, J. Bone Miner. Res. 13 (3) (1998) 350e353. [140] S.M. Kimball, R. Vieth, A comparison of automated methods for the quantitation of serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, Clin. Biochem. 40 (2007) 1305e1310. [141] F.A. Muskiet, D.J. Dijck-Brouwer, D. Van, V, A. Schaafsma, . Do we really need >/¼100 mcg vitamin D/d, and is it safe for all of us? Am. J. Clin. Nutr. 74 (6) (2001) 862e863. [142] I. Munro, Derivation of tolerable upper intake levels of nutrients, Am. J. Clin. Nutr. 74 (6) (2001) 865e867. [143] A.A. Yates, Process and development of dietary reference intakes: basis, need, and application of recommended dietary allowances, Nutr. Rev. 56 (4 Pt 2) (1998) S5eS9. [144] C.J. Bacon, G.D. Gamble, A.M. Horne, M.A. Scott, I.R. Reid, High-dose oral vitamin D3 supplementation in the elderly, Osteoporos. Int. 20 (8) (2009) 1407e1415. [145] V. Chel, H.A. Wijnhoven, J.H. Smit, M. Ooms, P. Lips, Efficacy of different doses and time intervals of oral vitamin D supplementation with or without calcium in elderly nursing home residents: reply to comment by Vieth, Osteoporos. Int. 19 (5) (2008) 723. [146] R. Vieth, Comment on Chel et al., efficacy of different doses and time intervals of oral vitamin D supplementation with or without calcium in elderly nursing home residents, Osteoporos. Int. 19 (5) (2008) 721e722. [147] S. Ish-Shalom, E. Segal, T. Salganik, B. Raz, I.L. Bromberg, R. Vieth, Comparison of daily, weekly, and monthly vitamin D3 in ethanol dosing protocols for two months in elderly hip fracture patients, J. Clin. Endocrinol. Metab. 93 (9) (2008) 3430e3435. [148] M.O. Premaor, R. Scalco, M.J. Da Silva, P.E. Froehlich, T.W. Furlanetto, The effect of a single dose versus a daily dose of cholecalciferol on the serum 25-hydroxycholecalciferol and parathyroid hormone levels in the elderly with secondary hyperparathyroidism living in a low-income housing unit, J. Bone Miner. Metab. 26 (6) (2008) 603e608. [149] G.L. Van, S. Opdenoordt, S.A. Van, D. Telting, A. Giesen, B.H. De, Cholecalciferol loading dose guideline for vitamin D-deficient adults, Eur. J. Endocrinol. 162 (4) (2010) 805e811. [150] K.M. Sanders, A.L. Stuart, E.J. Williamson, et al., Annual highdose oral vitamin D and falls and fractures in older women: a randomized controlled trial, JAMA 303 (18) (2010) 1815e1822. [151] T.P. Kenakin, Pharmacokinetics. A Pharmacology Primer: Theory, Applications, And Methods, Elsevier, Oxford, UK, 2009; 179e214. [152] M.F. Holick, Vitamin D deficiency, N. Engl. J. Med. 357 (3) (2007) 266e281.
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[153] C.N. Holick, J.L. Stanford, E.M. Kwon, E.A. Ostrander, S. Nejentsev, U. Peters, Comprehensive association analysis of the vitamin D pathway genes, VDR, CYP27B1, and CYP24A1, in prostate cancer, Cancer Epidemiol. Biomarkers Prev. 16 (10) (2007) 1990e1999. [154] D.P. Trivedi, K.T. Khaw, Bone mineral density at the hip predicts mortality in elderly men, Osteoporos. Int. 12 (4) (2001) 259e265.
[155] B. Dawson-Hughes, S.S. Harris, High-dose vitamin D supplementation: too much of a good thing? JAMA 303 (18) (2010) 1861e1862. [156] C. Cipriani, E. Romagnoli, A. Scillitani, et al., Effect of a single oral dose of 600,000 IU of cholecalciferol on serum calciotropic hormones in young subjects with vitamin D deficiency: a prospective intervention study, J. Clin. Endocrinol. Metab. 95 (10) (2010) 4771e4777.
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C H A P T E R
58 How to Define Optimal Vitamin D Status Roger Bouillon Katholieke Universiteit Leuven, Leuven, Belgium
INTRODUCTION This chapter will examine the role of vitamin D in the prevention of disease by surveying its actions in bone and various extra-skeletal sites. Our goal is to characterize the best evidence for efficacy and safety in disease prevention and/or treatment and thereby to identify a target level of serum 25(OH)D that would improve human health on a global scale. It is our opinion that this target level should be 20 ng/ml of 25(OH)D. This is a conservative target but in our opinion it is safe and effective for the proven benefits that vitamin D can provide. Higher concentrations of 25(OH)D may have additional benefits but these are not yet proven by RCTs. It is our opinion that these benefits of vitamin D can therefore be achieved by supplementing the population with 800 or possibly 1000 IU of vitamin D3 per day. In contrast, Dr Vieth argues for a higher target and a higher level of supplementation in the preceding chapter. The vitamin D molecule appeared as an early biological event during evolution of life on earth as it was already an end-product of photochemical transformation of 7-dehydrocholesterol or 7-dehydroergosterol into vitamin D3 or D2 in phytoplankton and zooplankton after exposure to UV-B light. This photochemical reaction is thought to be beneficial for the stability of DNA by working as a sunscreen and this could have been the successful evolutionary advantage conferred by this reaction as UV-B was so prominent, more than today, during early evolution on earth. Vitamin D, however, is considered to have been a biologically inert product until the evolution of cartilaginous fish into bony fish more than 400 million years ago. At that time, the VDR receptor, the P450 enzymes needed for the conversion of parent vitamin D into 1,25(OH)2D, and the plasma protein needed for the transport of vitamin D (DBP or Gc protein), originated in fish and
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10058-7
is conserved in all later vertebrates. These proteins were generated from gene duplication of other genes: from other nuclear receptors for VDR, from other P450 enzymes for vitamin-D-metabolizing enzymes and from the common precursor of albumin and DBP for plasma DBP. This happened at a time of a major switch of life forms from a calcium-rich ocean environment to a calcium-poor fresh water or land area environment. It is therefore tempting to link these phenomena as an adaptation to calcium-poor environments by introducing a novel calciotropic hormone (the vitamin D hormone) that facilitates calcium absorption in the gills or gut or reabsorption in the kidney, instead of calcitonin, a major hormone for fish that can decrease serum calcium and enhances calcium excretion [e3]. During the whole of further vertebrate evolution, vitamin D originated mainly from photosynthesis as only a few species had access to vitamin-D-rich food from the ocean. Indeed the high vitamin D content of fatty fish is the pure consequence of the high vitamin D content in their food supply originally as inert products in phytoplankton and zooplankton, described above. Foods from terrestrial origins are vitamin-D-poor and thus not a major or reliable source of vitamin D. Vitamin D can also be synthesized endogenously using the energy of the sun and thus it is not a true vitamin for the human species. During the very recent evolution of human emigration from sunny Africa to less sunny regions of the world, easy and plentiful vitamin D synthesis became less available and there are indirect arguments that depigmentation of the human species was an adaptation to reduced sun exposure of humans during their northward migration [4,5]. So, vitamin D supplementation did not happen during the known history of humankind until very recent times and rickets seems to have been infrequent (although not absolutely absent as shown by disease description by Soranus of Ephese or Galenus [6] during the known history of
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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humans up to the 17th century). The first and excellent brief description of the clinical picture of rickets took place at the University of Lugdunum Batavorum (Leiden, the Netherlands) in 1645 by Daniel Whistler, followed a few years later by an extensive description in London by Francis Glisson and colleagues [7,8]. From their first edition in 1650 until the “editio postrema” in 1682, several versions must have been published indicating the popularity of the topic at that time (maybe matching the present situation!). There are also a number of historic records indicating that rickets was a frequent cause of mortality in children living in large cities in the UK and Europe, where smog and general living circumstances made access to sun exposure and its UV-B light problematic [9]. A similar situation happened years later in rich families in India and some European regions, where rickets became a clinical problem due to lack of exposure to sunlight for social or cultural reasons designed to maintain the skin as white as possible. This lack of exposure to UV-B light is also the major cause of vitamin D deficiency in most Muslim countries that practice the near total veiling of women and avoid exposure to sun for their children because of cultural and religious reasons. These subjects are discussed more fully in Chapters 52, 53 and 54.
Vitamin D Supplementation to Prevent Rickets Shortly after the discovery of the dual origin of vitamin D, both from dietary sources and from photosynthesis in the skin, widespread if not systematic vitamin D supplementation and to a much lesser extent better exposure to sunlight or artificial UV-B light virtually eliminated simple vitamin D deficiency rickets from the Western world. Indeed, although the large majority of clinical rickets could be prevented by such supplementation a few cases were “resistant” and this was later discovered to be due to other diseases such as hypophosphatemic rickets, calcium deficiency rickets or rare inborn errors of vitamin D metabolism (CYP27B1 or CYP2R1 or VDR mutations) [10]. However, simple vitamin D deficiency rickets is still problematic in major areas of the world despite the obvious well-known strategy to prevent it [11]. The amount of vitamin D needed to prevent rickets was of course defined at a time when even the concept of randomized controlled trials was yet to be invented [12]. However, careful observations published by clever clinicians, especially in the UK, French and German literature, led to the empirical practice that one teaspoon of fish liver oil was sufficient to prevent rickets and this eventually evolved into the practice of administering the equivalent of 400 IU of vitamin D3. There was extensive discussion about whether or not
vitamin D2 was equivalent to D3 with the general feeling that 400 IU of vitamin D2 was also sufficient in infants to prevent rickets. Lower dosages of vitamin D3, in the order of 100 to 200 IU/d were equally able to largely prevent rickets but this may not have applied to vitamin D2. Extensive clinical experience over many decades clearly showed that 200 IU of vitamin D3 per day is able to prevent rickets but 400 IU/d is equally safe and may correct compliance problems and may have additional advantages for extra-skeletal effects of vitamin D. Indeed, because of a potential link between perinatal vitamin D status and many other diseases later in life (see below) several countries now recommend a daily dose of 400 IU/d for infants and children [13]. There is probably only one randomized controlled trial in children living in an area with high prevalence of rickets whereby 400 IU of vitamin D3 per day totally eliminated new cases of rickets compared with a 4% incidence in the control group [14]. There are no good prospective data about levels of 25(OH)D needed to prevent rickets but from case reports and clinical experience serum 25(OH)D is usually well below 10 ng/ml and even 5 ng/ml in simple vitamin-D-deficiency rickets. The picture can of course be more complex when nutritional calcium deficiency is the predominant cause of rickets as seen in some African countries [10] or in children fed a calcium-poor soya milk equivalent instead of humanized milk in Western countries [15]. The subject of rickets is discussed in more detail in Chapter 60 and replacement in infants and children in Chapters 35 and 36. During the whole of human evolution, vitamin D supplementation was unknown and is only a very recent phenomenon. Therefore one might question whether such supplements are really needed and could not be replaced by simple exposure to more sunlight. In fact, the majority of infants and children around the world do not receive supplements and rely on UV-B exposure to effectively prevent rickets. Several reasons, however, clearly argue for systematic vitamin D supplementation of infants and children. First, UV-B is a photo-carcinogen especially for people with a fair skin phenotype [15a] and the long-term risk for skin cancer and photo-aging of the skin is greatest when UV-B exposure occurs early in life. The American Academy of Pediatrics as well as the Canadian Dermatology Society recommend that children below 6 or 12 months, respectively, should avoid direct sunlight so that their vitamin D requirement can only be met by supplementation. Moreover, most experts are convinced that the photo-damage caused by UV-B is cumulative over life so that sun exposure should be limited over a whole lifetime. This is further strengthened by the long life expectancy of the present human population
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whereas UV-B-related risks may have been negligible during earlier human evolution because the long lag time between UV-B exposure and clinical skin events exceeded the life expectancy of most people. Second, the climatologic conditions for a major part of the world population imply that natural sunlight does not provide sufficient UV-B for effective vitamin D synthesis during several months of the year because the earth’s atmosphere removes the shortest UV-B waves due to the low inclination of the sun. Third, social, cultural or religious reasons make sun exposure in practice too minimal for efficient vitamin D synthesis in many areas of the world. Additionally, there is a mismatch between skin pigmentation and chances for sun exposure for a large number of people due to recent historic migration waves to different geographical regions. Therefore, the practice of vitamin D supplementation of infants and children is undoubtedly necessary and efficient and should be continued and even introduced in areas where rickets is still endemic due to poor vitamin D status [11]. This is especially so because the well-established immediate benefits (prevention of rickets and improvement in bone health) far exceed the relatively cheap costs of systematic supplementation. The potential long-term benefits of improved vitamin D status during early life on other health aspects (see below) are then just an added value. In most countries the recommended daily dose is 200 IU or 400 IU, preferably D3 over D2. Much higher dosages with possibly beneficial extra-skeletal effects have still to be considered as experimental clinical research especially in view of a (not so strong) link between high vitamin D supplementation during pregnancy or early life and congenital malformations or infantile hypercalcemia [16]. The recent Institute of Medicine guidelines for dietary reference intake (DRI) of vitamin D also called for a daily intake of 400 IU.
VITAMIN D STATUS AND BONE HEALTH IN ADULTS AND THE ELDERLY In the first decades after the discovery of vitamin D, studies of its effects were mainly concentrated on its causal link with preventing rickets and the adult equivalent, osteomalacia. More recently, the vitamin D status in older age has been studied in relationship with ageassociated bone loss or osteoporosis. This was initially started following the epidemiologic observations of decreasing 25(OH)D levels, the best marker of the nutritional vitamin D status, with age [1]. In addition, the link to vitamin D status was strengthened by the simultaneous findings of increased serum parathyroid hormone [17] and the lower 25(OH)D levels observed in patients with a recent bone (especially hip) fracture compared
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to age-matched controls [18]. This led to the hypothesis that senile osteoporosis is due to, or at least accelerated by, increased bone resorption due to high PTH levels which are largely due to poor vitamin D status [19]. By now we realize that osteoporosis, whether postmenopausal or occurring later in life, has a much more complex origin (sex hormone deficit, osteoblast dysfunction, oxidative stress, PTH excess, decreasing muscle mass and associated lower mobility and bone strain, etc.) [20]. The formal proof for a role of vitamin D on bone health at older age has grown gradually over time based on many observations, initially mainly observational cross-sectional or prospective studies, and more recently also by prospective randomized trials. However, most of the studies were performed in elderly subjects (above 65 years of age) with very few in younger adults and just a few studies in adolescents. As the weight of evidence based on randomized controlled trials far exceeds the value of non-intervention studies I will here review only data on the importance of vitamin D nutritional status on surrogate and hard endpoints based on randomized controlled trials (or, if missing, on prospective long-term observational studies). Indeed, as vitamin D is clearly a precursor for other metabolites and especially first 25(OH)D and then the hormone 1,25(OH)2D3, one can expect there to exist a threshold level of vitamin D access that allows its full further metabolism into the active hormone, whether supplied by the renal production and systemic supply or whether locally produced as an autocrine or paracrine factor. Once such a threshold is reached, additional vitamin D or 25(OH)D will no longer increase the end-product but will allow only a better future supply in case access to new vitamin D becomes subcritical. This is very similar to the situation of iodine supply to produce the active thyroid hormones [1]. There are several surrogate markers for bone health related to vitamin D and the most important ones are summarized here. What is the optimal 25(OH)D level to normalize the serum concentration of 1,25(OH)2D3 and parathyroid hormone? What is the optimal or minimal level of 25(OH)D to optimize intestinal calcium absorption or bone turnover or bone mass? Of course the ultimate question for bone health is what level of 25(OH)D is needed for minimizing the risk of bone fragility fractures or to support fracture repair. For each of these surrogate or hard endpoints there are numerous cross-sectional studies with frequently contradicting data so that no clear picture or conclusion can be drawn. This led in fact to variable and sometime lively debates and “evangelical” attitudes of believers or disbelievers of “more is better” for every aspect of vitamin D action. See Chapter 57 for an alternative view of optimal vitamin D level.
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Vitamin D Status and Serum 1,25-Dihydroxyvitamin D3 Cross-sectional data on serum 25(OH)D and 1,25(OH)2D provided variable results as some studies did not reveal a positive correlation between the two metabolites. That is usually the case when the vitamin D status is sufficient in view of the precursor/threshold status of 25(OH)D so that the kidney, the only tissue that under normal conditions produces 1,25(OH)2D for export to serum, can synthesize the optimal amount of hormone, based on other input stimuli such as PTH, FGF23, and calcitonin [21]. However, other studies demonstrate a clear positive correlation and such studies are usually indicative of a vitamin D insufficiency status as this indicates that the precursor, 25(OH)D, is limiting the renal hormone production. Still other studies revealed a non-linear correlation with a positive relation between precursor and hormone at low 25(OH)D levels and a plateau at higher levels. In addition this relation is further complicated by variable renal function. This is especially so in the case of chronic renal failure as there are several studies showing that higher precursor levels of 25(OH)D can help the limited renal 1a-hydroxylase activity to overcome the limited 1,25(OH)2D production due to impaired kidney function [22,23]. So the optimal 25(OH)D status may be higher in case of chronic renal failure than in otherwise healthy subjects. Other factors may also be confounders such as habitual calcium intake: indeed a low calcium intake accelerates vitamin D metabolism and thus tends to lower serum 25(OH)D but also stimulates directly or indirectly the renal CYP27B1 production and thus may elevate serum 1,25(OH)2D concentrations. The same is frequently observed in cases of primary hyperparathyroidism. Therefore, intervention studies with vitamin D supplementation are a better strategy to define the minimal 25(OH)D level to optimize serum 1,25(OH)2D. The caveat here is that such intervention studies should not be short term as it may take several days or weeks for the whole vitamin D endocrine system to adapt to the new situation especially the resetting of the production of the various hormones responsible for 1,25(OH)2D homeostasis such as PTH, FGF23, and calcitonin. In addition, the dose of vitamin D should be in the physiologic range so as to avoid overwhelming the renal 1a-hyrdoxylase system. Our study on the vitamin D status of elderly subjects in Belgium [24] revealed that chronic vitamin D deficiency with low 25 (OH)D and low hormone 1,25(OH)2D concentrations (much lower than in healthy younger adults) was common. Two weeks after vitamin D supplementation of these initially deficient subjects (mean baseline 25 (OH)D levels around 5 ng/ml), serum 1,25(OH)2D increased to the level observed in young adults whereas mean serum 25(OH)D had only risen to about 15 ng/ml.
The amount of vitamin D supplementation needed to reach such a 25(OH)D concentration is relatively low and will be discussed below. This precursore end-product relationship indicates that the renal supply of just 15 ng/ml of 25(OH)D is sufficient for the production of a normal concentration of the end-product. In the case of chronic renal failure (CRF) stage III to IV, higher intake of vitamin D and concomitant higher levels of 25(OH)D to 30 ng/ml or higher are capable of increasing the initially low levels of 1,25(OH)2D as the renal capacity is now the rate-limiting step [22,23].
Vitamin D Status and Parathyroid Function As parathyroid hormone (PTH) is the primary regulator of the renal 1a-hydroxylase and is feedback controlled either indirectly by serum calcium/calciumsensing receptor or directly by serum 1,25(OH)2D concentrations, levels of PTH may provide a very good estimation of vitamin D status. Indeed, in several animal and human studies secondary hyperparathyroidism rapidly occurs in the situation of decreasing vitamin D status. Mice with genetically engineered selective VDR deficiency in the parathyroid gland develop mild hyperparathyroidism and increased bone turnover [25]. In addition, there is a clear expression of the CYP27B1 enzyme in parathyroid cells so that these cells can synthesize 1,25(OH)2D as an autocrine/paracrine factor. Cross-sectional studies are, however, disturbingly complex as nearly all studies revealed an inverse correlation between 25(OH)D and PTH levels if subjects with poor vitamin D status are included. However, the inclination point of rising PTH calculated by computer software programs or estimated by visual inspection of data plots can vary from as low as 12 or as high as 40 ng/ml of 25(OH)D. In addition, approximately 50% of the subjects below this 25(OH)D threshold do not display secondary hyperparathyroidism even when 25(OH)D falls to very low levels. When kidney function or habitual calcium intake and age are taken into account, further corrections occur. There is a shift towards a lower 25(OH)D concentration leading to the PTH inclination point when only subjects with normal renal function and normal calcium intake are selected. These studies are not only important to define the optimal vitamin D status but also have major implications for defining normal PTH levels and thus defining threshold levels for classifying primary hyperparathyroidism [26]. Based on such surrogate PTH data, variable definitions of optimal vitamin D status have been proposed but should be replaced by data based on intervention studies only. Two major studies have looked at PTH levels before and after vitamin D supplementation. Holick’s group [27] indeed found decreasing PTH levels
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Intervention Studies Vitamin D and PTH
• 35 patients with 25(OH)D <25 ng/ml • 50000 IU D2 once weekly x 8 weeks • 1000–1500 mg calcium/day • PTH and 25(OH)D before and after Rx • In patients with 25(OH)D ≥20 ng/ml, 25(OH)D levels increased by 66% but PTH levels did not change significantly
• Authors concluded that the risk of second hyperparathyroidism is minimized when 25(OH)D is ≥20 ng/ml
Intervention studies with vitamin D supplementation to define the optimal vitamin D nutritional status to normalize serum parathyroid hormone.
FIGURE 58.1A
after oral vitamin D3 supplementation of subjects with baseline 25(OH)D levels below 25 ng/ml but preplanned subgroup analysis revealed that serum PTH only decreased in subjects with a baseline 25(OH)D level below 20 ng/ml (Fig. 58.1(A)) and the decrease in PTH was more pronounced when baseline 25(OH) D values were lower. In the MORE trial, baseline 25 (OH)D was measured before and after long-term Changes in 25(OH)D & PTH Vitamin D3 400–600 IU + Calcium 500 mg daily Changes after 6 months Basal 25(OH)D ng/ml
25(OH)D ng/ml
PTH pmol/l
<10
↑ 23±13
↓ 0.8 ±1.8
10-20
↑ 16±10
↓ 0.5 ±1.5
>20
↑ 5±12
= 0.2±1.2
p<0.001
p<0.001
FIGURE 58.1B Changes in 25(OH)D and PTH vitamin D3 400e600 IU þ calcium 500 mg daily.
vitamin D replacement (Fig. 58.1(B)) and Lips’ group [28] found a significant decrease in serum PTH when baseline 25(OH)D levels were below 20 ng/ml. These data provide a clear indication that the normal parathyroid gland has a set point of PTH secretion that is no longer vitamin D dependent when serum 25(OH)D exceeds 20 ng/ml; or simply rephrased: the normal parathyroid gland is happy with serum 25(OH)D levels above 20 ng/ml.
Vitamin D Status and Intestinal Calcium Absorption The active intestinal calcium absorption is severely impaired in vitamin-D-deficient animals and humans and this has been amply confirmed in global VDR null mice [29]. The intestinal calcium absorption is in fact considered as the main primary and essential action of the vitamin D endocrine system and the cause of the calcium and bone phenotype of vitamin D or CYP27B1 deficient or VDR null animals and humans. Indeed, a very high calcium intake (oral or intravenous) can restore a (near) normal bone phenotype in vitamin-Ddeficient rats, and cyp27b1 or VDR null mice [30e33]. However, the role of intestinal VDR may well be more
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complex than just regulation of active calcium absorption as several genes involved in paracellular intestinal absorption are also vitamin-D-dependent so that both active calcium uptake and “passive” calcium diffusion may be at least partially vitamin-D-dependent [30,34e36]. In addition, the bone phenotype of selective intestinal VDR null mice is much more severe than that of global VDR null mice or mice with near zero calcium intake, so that other functions of VDR in the intestine or elsewhere may be operational and essential for global calcium/bone homeostasis [37]. However, these restrictions do not impede the essential and non-redundant role of the vitamin D endocrine system on intestinal calcium absorption. There are several techniques to measure active intestinal calcium absorption in vivo such as the measurement (multiple sampling) of a calcium (radioactive or non-radioactive) isotope in serum after its oral intake with a low and physiologic amount of normal calcium. Ideally this should be combined with a similar study of the behavior of another calcium isotope given intravenously so that a full calcium absorption and excretion study is possible and the net active calcium absorption can be calculated. A simplified version only measures the serum concentration of a calcium isotope at a single time point after its oral intake and for group evaluation this may be a valid alternative to the more complex dual isotope study or a full balance study of calcium intake with additional measurements of renal and fecal calcium excretion during at least 1 week. Some studies used measurement of serum calcium after ingestion of
a large oral dose of calcium but such changes in serum calcium are very small and the oral dose is above the normal physiologic meal size calcium intake so that such results are flawed. Nordin has performed numerous studies on intestinal calcium absorption in healthy and postmenopausal women using a single serum sample measurement of isotopically labeled calcium. In most of these studies he could not find a direct correlation between serum 25(OH)D and intestinal calcium absorption whereas such a positive correlation with serum 1,25(OH)2D was significant [38]. However, others, and especially Heaney’s group, have found a significant correlation between serum 25(OH)D and intestinal calcium absorption estimated from changes in serum calcium after oral calcium loading [39]. From a compilation of his numerous studies Heaney et al. found that intestinal calcium absorption increases with increasing serum 25(OH)D until a plateau is reached at higher than 32 ng/ml. This conclusion should be interpreted with caution in view of pooling of many studies with slightly different techniques that all are handicapped by non-optimal methodology. There are only few intervention studies in adults that used appropriate methods before and after vitamin D supplementation. In the Australian study a daily supplement of 1000 IU of vitamin D2 increased mean 25(OH)D levels from 18 to 24 ng/ml without any change in intestinal calcium absorption [40] (Fig. 58.2). In the US study a very large vitamin D2 supplement of 50 000 IU/d increased serum 25(OH)D from a baseline of 22 ng/ml to 60 ng/ml and increased the active
Calcium absorption and vitamin D status
Subjects: 302 elderly women (77yr) Therapy: calcium 1000 mg/d ± 1000 IU D2/d
Results control
after R/ D2
25(OH)D3 (ng/ml)
18 ± 5
24 ± 6
Fractional calcium absorption (1 h after 45Ca intake)
± 31%
± 31%
Fractional calcium absorption (%)
30
25
20 n = 18 p = 0.04
15
10
5
0
22 ± 4
64 ± 21
25OHD (ng/mL)
FIGURE 58.2 Intervention studies with vitamin D supplementation to define the vitamin D nutritional status to optimize intestinal calcium absorption [41].
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calcium absorption as measured by a gold standard method, only minimally although just significantly [41]. This observed small increase after such a huge increase in serum 25(OH)D would have little effect on net calcium absorption as the between person variation in calcium intake by far exceeds the small increase by a much higher vitamin D intake. Taken together, these data indicate that calcium absorption, measured with a reliable method, does not markedly increase by additional vitamin D intake once the baseline 25(OH)D is about 20 ng/ml, at least in healthy adults. Few data exist in children and adolescents but cross-sectional studies using a gold standard double isotope (non-radioactive) method could detect no relation between calcium absorption and 25(OH)D levels [42] but the correlation with serum 1,25(OH)2D was clearly present [43]. So it seems that intestinal calcium absorption is “happy” with baseline 25(OH)D levels of 20 ng/ml and is better correlated with serum 1,25(OH)2D than with serum 25(OH)D, thereby questioning a possible major contribution of locally (intestinal cell) produced 1,25(OH)2D. The reader is referred to Chapter 19 on calcium absorption and Chapter 34 on calcium homeostasis for additional discussion.
Vitamin D Status and Bone Mineral Density (BMD/BMC) There are numerous small- and even large-scale studies that compared serum 25(OH)D levels with several aspects of bone mineral density or bone mass. The results are quite different as could be expected since serum 25(OH)D levels were usually only measured once and the effects of vitamin D on bone turnover and structure may have a long lag time. Moreover, many other factors influence bone mass and it is extremely difficult to correct for these confounders. In the Amsterdam study, BMD was non-linearly related to serum 25(OH)D levels with a small decrease when 25(OH)D levels fell below 12 ng/ml [44]. According to an analysis of NHANES III data, Bischoff-Ferrari reported a significant increase in BMD with increasing serum 25(OH)D levels with the greatest slope (increase in BMD) when 25(OH)D was below 20 ng/ml but still further improvement of BMD with increasing 25(OH)D levels up to about 40 ng/ml in Caucasians [45] but with a non-significant decrease in BMD at still higher levels. A large longitudinal study in more than 1200 aging men (MrOS study) showed that BMD decreased more rapidly over a 4-year follow-up time period in subjects with baseline 25(OH)D levels below 20 ng/ml [46]. Numerous other studies, however, did not provide such clear evidence for an effect of vitamin D nutritional status on BMD. There are very few prospective studies on the effect
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of vitamin D supplementation on the evolution of BMD. Peacock could not detect an increase in BMD in more than 400 men and women above the age of 60 years treated with a calcium supplement and 15 mg of 25(OH)D per day for 4 years. The baseline 25(OH)D level increased from 24 to 48 ng/ml [47]. Some of the RCT data on vitamin D supplementation and fracture risk also measured BMD changes and modest increases (þ1 to 6%) have been observed, especially in institutionalized patients with poor vitamin D status at baseline [48].
Vitamin D Status and Fracture Risks Patients with a recent fracture frequently have lower levels of 25(OH)D than non-fracture controls [28,49] and this has especially been observed in patients with a recent hip fracture. Again, as the vitamin D status has effects only on bone, whether turnover, mineralization, structure or mass, after a long time lapse, it is difficult to define a causal link between vitamin D status and fractures and it is even more difficult to define an optimal threshold or level of 25(OH)D above which there is no further protective role. A nested caseecontrol study based on the WHI trial data revealed that a baseline 25(OH)D level below 19 ng/ml conferred a nearly two-fold increased risk for subsequent hip fracture than observed in women with a higher baseline level [50]. Of course intervention trials are needed to formally prove causality and fortunately there are quite a number of such intervention studies. Moreover a strikingly large number of meta-analyses have carefully evaluated these studies so that in fact there are less than 20 wellperformed RCTs on vitamin D supplementation (with or without calcium supplements) and fractures and about 12 meta-analyses of these data (Table 58.2). These primary studies and their combined analysis do not always come to the same conclusion. The Cochrane meta-analysis [51] could not detect a significant effect of vitamin D in monotherapy on fracture incidence but the calciumevitamin D combination had significant effects. This meta-analysis also included many more studies (n ¼ 45) than most other meta-analyses that were more critical and selected only well-designed and published RCTs. From the overall analysis of all these meta-analyses and their primary studies, there is a clear trend that vitamin D supplements reduce the risk of non-vertebral (including hip) fractures by about 20% in the target population (usually elderly subjects above age 65 years, mostly Caucasians). There is some disagreement regarding the minimal dosage of vitamin D needed but the trend is certainly that 400 IU/d is not as effective as higher doses so that a consensus is growing that 800 IU of vitamin D3 per day seems to be the optimal amount based on the present set of data
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(Table 58.2). Whether still higher doses might be more beneficial cannot be defined yet as there are no longterm RCTs using doses higher than 1200 IU/d. There is more controversy about the efficacy of vitamin D in monotherapy versus in combination with oral calcium supplements. This can partly be explained by the fact that most studies actually used a combination of both drugs and that there are few studies comparing the effect of vitamin D with the calciumevitamin D combination. Several meta-analyses (Table 58.2) therefore concluded that only the combination is effective in reducing fractures but an extensive in-depth analysis found that vitamin D alone was also effective if used at higher than 400 IU/d. Vitamin D2 was found to be less active than D3 in this analysis [52]. Finally, the best results on efficacy were found in institutionalized subjects with lower efficacy observed in elderly ambulatory subjects. Calcium supplementation alone does not look to be efficient in hip fracture prevention and may even increase this fracture frequency [53] and even increase cardiovascular events [54]. Another analysis found a small beneficial effect of calcium supplementation with or without vitamin D [55]. There are no prospective studies on the preventive action of vitamin D supplementation based on baseline 25(OH)D levels. So one cannot make strong statements regarding what baseline 25(OH)D concentration will generate the best results or which post-treatment 25(OH)D levels are needed for the preventive action of vitamin D. However, several post hoc analyses have been performed to try to identify the minimal 25(OH)D level needed for efficacy and 24 ng/ml has been put forward as a result of such an exercise [52]. The major problem with this analysis is, however, that 25(OH)D was not measured in the majority of subjects in the best RCTs and/or that in some older studies the techniques used for 25(OH)D measurements were notoriously inaccurate. Therefore there is no overall consensus possible about the target level of 25(OH)D to generate the beneficial effects of vitamin D supplementation.
Vitamin D Status and Bone Health: Conclusions The optimal vitamin D status for elderly subjects can be reasonably estimated by a series of RCTs using a variety of surrogate endpoints (Table 58.1). Numerous cross-sectional or prospective studies did not come to a reasonable consensus with regard to the minimal vitamin D status as defined by 25(OH)D serum concentrations as the results of different studies have generated very different threshold levels. Therefore, I concentrated the focus of this review on RCTs to define the minimal 25(OH)D levels needed to optimize bone health either using surrogate endpoints or using hard endpoints of
TABLE 58.1 Vitamin D Status and Bone Health: Surrogate and Hard Endpoints Endpoint Surrogate
Hard
Relationship 1,25(OH)2D PTH
25(OH)D threshold 15 ng/ml 25(OH)D threshold 20 ng/ml
Calcium absorption
Minimal or no change if baseline 25 (OH)D 20 ng/ml
BMD
Longitudinal and intervention studies suggest that if 25(OH)D is >20 ng/ml, vitamin D therapy does not further increase BMD
Falls
Recent meta-analysis found benefit only if 25(OH)D <20 ng/ml
Fractures
No fracture trials recruited on basis of initial 25(OH)D level EBM supports vitamin D 800 IU þ calcium to decrease fractures: this will increase 25(OH)D to 20 ng/ml in most subjects
EBM ¼ evidence-based medicine.
fracture incidence (Tables 58.1 and 58.2). The surrogate endpoints all suggest that when 25(OH)D levels are or exceed 20 ng/ml then the serum concentrations of 1,25(OH)2D or PTH or intestinal calcium absorption or bone mass and density reach a normal plateau level whereas lower 25(OH)D levels are responsible for abnormal surrogate endpoints. This level of vitamin D status is remarkable since it is also the threshold obtained in several RCTs for the reduction of non-vertebral fractures. This is based not on direct estimations of 25(OH)D in these RCTs because of technical (assay methodology) reasons. Fortunately, there is, however, much better indirect evidence for optimal 25(OH)D levels by extrapolating the most likely levels obtained during active treatment in all these studies as: (1) the dose of vitamin D3 in the trials has been correctly described (sometimes even with compliance rate); (2) there are reasonably good data on both the (measured or expected) baseline 25(OH)D levels in the target populations; and (3) finally one can reasonably calculate the expected increase in mean 25(OH)D levels after known extra intake of vitamin D3. The dosage of vitamin D supplementation that was most effective was about 800 IU/d and such dosage most likely will increase serum 25(OH)D levels by about 8 ng/ml taking into account that the treated populations were living with relatively low baseline 25(OH)D levels usually not exceeding mean 25(OH)D levels above 20 ng/ml. Such an analysis implies that the final 25(OH)D level was probably above 20 ng/ml in most of the treated subjects with mean levels in the high 20s to low 30s ng/ml. As this 25(OH)D level is closely approaching the threshold
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TABLE 58.2
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Meta-analysis of RCTs on Vitamin D Supplementation and Fractures
Meta-analysis
No. of studies evaluated
1. Avenell et al., 2005 [144]
n ¼ 45
Combined calcium and vitamin D but not vitamin D alone reduces hip fracture risk especially in frail older people
2. Bischoff-Ferrari et al., 2005 [145]
n ¼ 12
Vitamin D supplementation (~800 IU/d) decreases (hip and non-vertebral) fracture risks whereas 400 IU/d is not protective
3. Bischoff-Ferrari et al., 2007 [146]
n ¼ 17
Calcium supplements do not reduce the risk of hip or vertebral fractures and may even increase hip fracture risk
4. Boonen et al., 2007 [147]
n¼9
Combined calcium and vitamin D supplements are needed to reduce fracture risk (hip; non-vertebral, vertebral)
5. Jackson et al., 2007 [148]
n¼9
Vitamin D monotherapy reduces the incidence of falls and there is a trend for reduction of fractures
6. Tang et al., 2007 [55]
n ¼ 17
Calcium or calcium þ vitamin D supplementation reduces all type fracture risk. No additional benefits of vitamin D
7. Cranney et al., 2007 [51]
n ¼ 15
Combined calcium (500e1200 mg) plus vitamin D3 700e800 IU/d reduces fractures primarily in elderly institutionalized women
8. O’Donnell et al., 2008 [149]
n ¼ 16
1a-hydroxyvitamin D reduces vertebral and non-vertebral fracture risk but risk of hypercalcemia is also increased
9. Reid et al., 2008 [150]
n ¼ 17
Combined calcium and vitamin D supplements reduce hip fracture risk but calcium supplements only may increase this fracture risk
10. Bischoff-Ferrari et al., 2009 [53]
n ¼ 20
Non-vertebral (including hip) fracture prevention by vitamin D is dose-dependent and higher (z800 IU/d) dose ¼ 20% reduction for 65
11. Chung et al., 2009 [151]
n ¼ 15 þ 3
Inconsistent results on fractures risks
12. Dipart Group, 2010 [152]
n¼7
Calcium with vitamin D but not vitamin D alone reduces fracture risk (8% all fractures; 16% for hip)
13. Bergman et al., 2010 [153]
n¼8
Calcium with vitamin D reduces non-vertebral (RR ¼ 0.77) and hip fractures (RR ¼ 0.70)
Conclusions
level of 25(OH)D needed to normalize surrogate endpoints such as serum 1,25(OH)2D, PTH, intestinal calcium absorption or BMD, one can reasonably assume that the target population (elderly subjects as in the RCTs) should be treated with the effective dose (approximately 800 IU vitamin D3 per day). When such subjects are dealt with as individual patients and monitored for serum 25(OH)D levels, then the target level of 25(OH)D should be at least 20 ng/ml and this can guide the treating physician to adjust, if needed, the oral vitamin D supplementation. The choice between vitamin D3 and D2 is relatively easy. The natural product in people is D3, most of the RCTs have used vitamin D3 and the biological activity of vitamin D3 seems to exceed that of vitamin D2 in most but not all studies. Moreover the measurement of total 25(OH)D status is technically easier or more accurate when only D3 is used. Therefore I see no reason to prescribe D2 and see many practical advantages of using D3. Compliance with daily intake over many years may be problematic and vitamin D therapy is needed for many years if not over the rest of a person’s lifetime.
Therefore strategies that can improve compliance are essential and fortunately several options may work. Daily vitamin D3 supplements are virtually equivalent to weekly or monthly doses (of course with total similar cumulative amounts) to maintain similar 25(OH)D levels and this may also apply to dosages that cover supply for several months. Once yearly very high doses of vitamin D3 appear to be less optimal as initial values may necessarily be too high to maintain long-term 25(OH)D levels at target. This is especially so after the recent findings that a single yearly dose of 500 000 IU D3 given for 3 to 5 years yielded increased instead of the expected decreased incidence of fractures [56,57]. This recent study should not raise questions about the importance of vitamin D supplementation for bone health in general, as a post hoc analysis of these data indicated that the increased fracture risk coincided with high 25(OH)D levels (>>40 ng/ml) during the first 3 months after the yearly high oral vitamin D3 dose. However, the findings should caution against yearly high-dose treatment regimens and supraphysiologic 25(OH)D levels, until further studies create better guidelines. The effect of vitamin D on osteoporosis
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and fractures is further discussed in Chapters 62 and 61.
VITAMIN D AND EXTRA-SKELETAL HEALTH There are many reasons to accept the idea that the vitamin D endocrine system has a much wider spectrum of action than just regulation of extracellular calcium and bone homeostasis. Indeed, the vitamin D receptor is expressed in most cells although frequently at low level; the activating enzyme, CYP27B1, is expressed in many tissues so that the circulating precursor can be activated or regulated on a local basis for autocrine or paracrine functions, and most importantly, a very large number of genes are under direct or indirect control of the vitamin D hormone, 1,25(OH)2D. Indeed many microarray and related studies have identified a large number of genes to be up- or downregulated early or late after exposure to physiologic or slightly supraphysiologic concentrations of 1,25(OH)2D [58e60]. These studies identified moreover that the 1,25(OH)2D responsive genes belong to functional clusters involved in many coherent physiologic actions such as cell cycle progression, DNA repair, immune function, endothelium function and metabolism. Apart from purely gene expression studies many cellular studies involving both normal, diseased or malignant cells have identified again coherent reactions to exposure to 1,25(OH)2D with defined biological responses such as inhibition of cell cycle progression and inhibition of cell proliferation (normal and malignant cells), cell differentiation (e.g., immune cells), or other physiologic responses so that the gene expression patterns are in line with cellular biological events in vitro. These data are highly reproducible and have been confirmed in so many laboratories around the world using a wide variety of techniques that we can easily conclude that indeed the vitamin D endocrine system has a wide spectrum of activities (reviewed in [30]). In addition many preclinical studies in rodents or other animals e whether simply vitaminD-deficient or genetically modified animals with systemic or local knockout or overexpression of critical steps in vitamin D activation or actions e have confirmed and extended this concept as such animals are more prone to some diseases of cell proliferation (cancer predisposition or cancer evolution), immune diseases (such as autoimmune diseases), or show skin dysfunction or metabolic and cardiovascular abnormalities, etc. (for review see [30]). This wide spectrum of activities should not be a total surprise as this is the rule rather than the exception for all ligands of nuclear receptors such as androgens and estrogens, glucocorticoids, PPAR ligands, etc. Historically all of these
hormones were found to have a core group of specific target tissues that were discovered first and initially identified as the specific function of these hormones and their nuclear receptor, whereas their broader action was identified subsequently based on in vitro and in vivo studies especially by using transgenic animals. It is now generally accepted that the vitamin D endocrine system has a much wider set of activities than just calcium/ bone homeostasis. The more fundamental question that remains to be answered is: what is the contribution of the vitamin D endocrine system to all the physiologic systems identified by these genetic, cellular, and preclinical studies? And, of course, as a follow-up to that question: what are the implications for the origin or evolution of human diseases and thus global health? And if vitamin D plays a role: what is the level of vitamin D deficiency or excess that has an impact on human health? Indeed, essentially all of the genetic, cellular, or preclinical studies have been performed when either the vitamin D activation or action has been totally eliminated or when cells or animals were exposed to supra-physiologic concentrations of the vitamin D ligands or analogs. Of course, such extreme situations are rare in humans whereas the vitamin D nutritional or hormonal status often shows a wide variety of states of deficiency or sometimes excess. How sure are we that vitamin D status affects different various extra-skeletal systems in humans and what is the level of evidence to define the threshold vitamin status that prevents or minimizes disease risks in humans?
Skin Diseases VDR null mice and children with defective VDR (HVDRR) develop a special type of total alopecia as they are born with normal hair but cannot renew hair growth after its first normal or accelerated loss. However, vitamin-D-deficient people or animals do not display the same skin phenotype, nor do cyp27b1deficient mice so that this phenotype is directly related to VDR expression and function without involvement of the ligand [61]. Therefore, the vitamin D nutritional status is probably not relevant for this phenotype. Keratinocytes, and in fact all cells of the skin, are, however, targets of vitamin D action whereby especially keratinocyte proliferation and differentiation are vitamin-D-dependent. The expression of this action in experimental animals is, however, relatively small and only visible after previous skin damage as the repair of the barrier function of the skin is delayed in vitamin-D-deficient (cyp27b1 null) mice [62]. No clinical observations in humans, however, have yet revealed a major link between vitamin D status and clinical skin diseases. The human disease psoriasis is, however,
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characterized by increased proliferation and decreased final differentiation of keratinocytes, but inflammation and autoimmune reactions also contribute to its clinical expression. Topical application of 1,25(OH)2D or analogs on psoriatic plaques can markedly reduce the severity of these lesions so that such therapy has become the baseline treatment of psoriasis lesions [63] with or without combination with topical glucocorticoids. Vitamin D action on skin and hair is discussed in Chapter 30, studies in VDR null mice in Chapter 33, and in humans Chapter 65. Psoriasis is covered in Chapter 97.
Cancer Gene profiling after exposure to 1,25(OH)2D or its synthetic analogs has clearly confirmed that probably several hundred genes, directly or indirectly related to cell cycle progression, DNA repair, and cell proliferation in general, are 1,25(OH)2D-dependent. Of course a large number of these genes are not a direct target of 1,25(OH)2D but depend on “master switches” such as E2F transcription factors that subsequently regulate many other genes [30,58,64]. These data fit well with the nearly universal observation that 1,25(OH)2D decreases the progression of the cell cycle at the level of G0/G1 level with some secondary effects at the G2 level of cell cycle. This occurs in most normal non-transformed cells but also in a large number of cancer cells and cancer cell lines. However, the cell cycle inhibition is by far not a total block and does not lead to massive cell death as some chemotherapeutic agents do. Some cancer cells, however, become resistant to 1,25(OH)2D’s action for a series of variable reasons (Table 58.3). This development of resistance is not surprising as such and similar phenomena are fairly frequent and probably even necessary for cancer cells to escape normal inhibitory mechanisms and allow them to grow indefinitely. The mechanisms involved are quite logical as they involve escape mechanisms to TABLE 58.3 Mechanisms of Resistance of Cancer Cells to Inhibitory Effects of 1,25(OH)2D3 1. Loss of VDR expression (e.g., by enhanced SNAIL expression in colon cancer) 2. Altered intracellular metabolism: (a) Decreased CYP27B1 and decreased local production of 1,25(OH)2D3 (b) Increased CYP24 and vitamin D catabolism 3. Decreased VDR signaling: (a) RXR serine 260 phosphorylation (b) Increased repressor expression (SMRT in prostate cancer) (c) Decreased expression of cell adhesion molecules (e.g., E-cadherin)
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reduce the VDR expression or silence its action, or involve mechanisms to reduce the local production of the active hormone or induce its rapid degradation by massive upregulation of the degrading enzyme CYP24A1, or finally by developing post-receptor resistance by escape mechanisms in the distal signaling pathways by which 1,25(OH)2D regulates its inhibitory effects on cell proliferation. Apart from its effects on cell cycle genes, 1,25(OH)2D can also inhibit cancer cell migration and invasion and thus the process of cancer metastasis [65]. Also angiogenesis can be impaired by 1,25(OH)2D and again this could coherently add to its anti-cancer actions. Finally 1,25(OH)2D regulates different aspects of prostaglandin activation, inactivation and action with an overall anti-inflammatory and anti-cell proliferative effect [65]. The chapters in Section X of this volume discuss many basic and clinical aspects of vitamin D and cancer. If 1,25(OH)2D is acting as an inhibitory cell cycle control mechanism then it could be expected that vitamin-D-resistant or -deficient animals would be prone to cancer. However, VDR null mice do not develop more full-blown cancers during the first year of life (with very few data beyond this limit); however, they do show hyperplasia of the intestinal mucosa, breast, or prostate tissues [66,67]. In addition such VDR null mice are more prone to develop cancer after exposure to either chemocarcinogens or genetically induced oncogenes [68]. As 1,25(OH)2D may be too hypercalcemic to be used at high doses in the prevention or treatment of cancer, several synthetic agonistic analogs have been synthesized and tested for enhanced anti-proliferative and lower calcemic effects. Some of these analogs were found to be able to reduce cancer growth in vivo with minimal side effects [69]. Their use in human cancer prevention or treatment is, however, still limited to phase 1 and 2 studies with positive perspectives [70]. The chapters in Section IX of this volume discuss the various analogs under development. The human data on the relation between vitamin D status and cancer is much more complex than in animals as extreme vitamin D resistance or deficiency as studied in vitro or in animal models are exceptional in humans. By contrast there is a very extensive literature, probably more than a couple of hundred papers, on vitamin D intake or nutritional status in general and human cancer prevalence or incidence. With hindsight one could say that the initial studies started with the observation that there is a gradient of cancer incidence in humans in different countries according to latitude [71,72]. The Garland brothers subsequently linked these observations to UV-Bproduced vitamin D status and hypothesized that poorer exposure to sunlight caused lower vitamin D status and was so ultimately related to higher cancer
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incidence [73]. This hypothesis is, however, not fully supported by hard data as potential exposure to UV-B rays is not tightly linked with vitamin D status or 25(OH)D levels as based on NHANES data in the USA or epidemiologic surveys in Europe. In fact, in Europe the best vitamin D status is found in Scandinavian countries and the poorest vitamin D status in Mediterranean countries as the lifestyle attitudes (searching for or avoiding sun exposure) and nutrition (fatty fish) may be more important for 25(OH)D levels than the physical UV-B irradiation predicted by the lattitude of the region [74]. The sunlight hypothesis is explored in Chapter 53. Many but not all of the epidemiologic studies have generated results that link a lower vitamin D status, as defined by serum 25(OH)D levels, to a higher risk of cancer involving nearly all major human cancers such as cancer of the breast, prostate, colon, and even melanoma [65,75,76]. A few studies suggest that a poor vitamin D status also worsens the prognosis of existing cancers. However, there are also a number of negative studies (no relation between vitamin D status and cancer) that even suggest that higher levels of 25(OH)D are associated with higher risk of cancer, especially pancreas and prostate, or with more aggressive types of cancer [77,78]. The WHO ordered a critical analysis of all data on vitamin D status and cancer and this extensive report concluded that there are insufficient data to confirm a strong link but that the best data available suggested a possible link between vitamin D status and colon cancer [79]. This is in line with a subsequently published report on 25(OH)D status in several European countries and the incidence of colon cancer [80]. Others, however, look differently at the existing data and consider that the majority of studies support the idea of an association between 25(OH)D levels (either measured or estimated from several variables) with the major cancers such as breast, colon, and maybe prostate cancer [81,82]. There are very few intervention studies with vitamin D that directly prove the causality of such a relationship. The WHI trial with 400 IU of vitamin D3 per day could not demonstrate an effect on the risk of colon cancer during a 7-year follow-up period [83] but the compliance to the protocol cannot be described as perfect and the actual dose of vitamin D supplementation was, with hindsight, far too low to be considered effective to raise 25(OH)D above the most likely threshold for cancer prevention. A post-hoc analysis of the WHI trial including only women not assigned to the estrogen arm (estrogen by itself reduced the risk of colon cancer) revealed that even such low vitamin D supplementation was beneficial for the prevention of colon cancer [84]. A much smaller study by Lappe et al. [85] used a daily dose of 1100 IU of vitamin D3 per day for about 4 years and found a significant effect
on cancer-free survival. However, the total number of cancers observed was small and the effect of calcium supplementation alone was nearly as important as the effect of combined calcium and vitamin D supplementation [85]. As the relationship between cancer and vitamin D status cannot yet be considered as fully validated by intervention studies, the question about what level of 25(OH)D provides the best protection is of course even more difficult and will depend on future RCTs. From the available cross-sectional and prospective observational studies it is clear that the greatest cancer risks are found in subjects with 25(OH)D levels below 20 ng/ml compared to subjects with higher levels but there is also a trend for still lower risk at 25(OH)D levels higher than 40 or 50 ng/ml. The conclusion of the WHO analysis and a number of other experts is, however, that at present the data are not sufficiently validated to prescribe vitamin D supplementation solely on the basis of its potential anti-cancer effects and that no effective and safe 25(OH)D target concentration is yet possible for this indication.
Immune System and Vitamin D Status A very large number of genes involved in the immune system, from antigen-presenting cells to B cells and all subclasses of Tcells express the VDR at some stage of their development. Monocytes and macrophages also express (extra-renal) CYP27B1 when appropriately stimulated by several immune signals [86,87] so that they can locally produce the 1,25(OH)2D hormone now functioning as an autocrine/paracrine immune regulator. Indeed, the immune cells are clear targets of 1,25(OH)2D action with a different action on the native and acquired immune system. The native immune system and more specifically monocytes/macrophages are stimulated by 1,25(OH)2D and display enhanced antibacterial activities such as phagocytosis and bacterial killing. This was already observed many decades ago [88,89] but has more recently been better understood by the 1,25 (OH)2D effects on the expression of bactericidal natural defensins [90,91] (Table 58.4). By contrast 1,25(OH)2D downregulates the activation and action of T helper-1 cells and thus decreases autoimmune reactions. This is further demonstrated by similar inhibitory effects on Th17 cells and stimulatory effects on Treg cells which by nature suppress T helper cells. The overall picture is thus one of tapering down the acquired immune system and thus the risk of autoimmune diseases. In several experimental settings this has been confirmed either by using 1,25(OH)2D itself or even better by using less calcemic agonists. The effects include reduction of autoimmune diabetes, experimental allergic encephalitis (a model for multiple sclerosis), inflammatory bowel
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TABLE 58.4
Vitamin D and the Immune System Infections
Association rickets and (respiratory) infections: Historic records Recent studies in Middle East/Africa Association 25(OH)D levels and infections: Tuberculosis Respiratory infections: Finnish soldiers: þ63%; if 25(OH)D <16 ng/ml [154] NHANES III: OR of 1.36 if 25(OH)D <10 versus >30 ng/ml [155] especially in patients with asthma or COPD No association of serum 25(OH)D and mortality due to infections [156] No published RCT but several ongoing studies
disease, autoimmune nephritis models, and autoimmune prostatitis [30,86,92]. From these preclinical studies it is clear that a link between the vitamin D endocrine system and the immune system and immune disorders is highly likely. Indeed a poor vitamin D status is frequently observed in patients with active tuberculosis but also in patients with other infections such as otitis media, upper respiratory infections, or COPD [93,94]. Intervention studies are, however, yet equivocal as one study in Indian tuberculosis patients revealed an accelerated sputum clearance by vitamin D supplementation [95] whereas a US study could not demonstrate a beneficial effect of vitamin D supplementation on the evolution of tuberculosis [96]. Several clinical trials are, however, registered on the NIH clinical trial register so that one can expect a better understanding of the role of vitamin D for the innate immune system within the foreseeable future. Many patients with autoimmune diseases have a lower vitamin D status than their healthy controls (e.g., type 1 diabetic patients, patients with multiple sclerosis or inflammatory bowel disease, etc.). The largest nested control study in US army recruits demonstrated that a low vitamin D status (defined by 25(OH)D levels below 20 ng/ml) at the time of recruitment to the US army conveyed a nearly two-fold risk of later onset of multiple sclerosis compared to Caucasians with a better vitamin D baseline level [97] and the lowest risk in subjects with 25(OH)D levels above 40 ng/ml. Studies in the Netherlands also suggest that a poor vitamin D status predisposes to more frequent or severe relapses of multiple sclerosis [98,99]. For type 1 diabetes there are several retrospective studies indicating that vitamin D supplementation during early life reduces the later risk of developing this disease later in life [100] but as these studies were not designed for that purpose the quality of the data are not optimal. The overall reduction in incidence of type 1 diabetes by such intervention was about 30%. The dose needed for
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this effect has not been clearly defined but in one study up to 2000 IU per day during the first year of life was most effective and this dose is markedly higher than the presently accepted or recommended dose for infants (200e400 IU/d). The retrospective human observations were clearly reproduced in diabetes-prone NOD mice as vitamin D deficiency early in life, but normal intake later on substantially and highly significantly increased the subsequent risk of autoimmune diabetes [101,102]. Of course prospective human RCTs are needed to confirm this observation and to define the optimal safe dosage and such studies are now ongoing but will require a long time to provide an answer in view of the long lag time before diabetes onset is clinically visible or before surrogate markers for disease onset are detectable. For other autoimmune disorders no reliable intervention trials are yet available but the preclinical observations on inflammatory bowel disease are promising for the human situation so that such trials deserve a high priority score. The relationship of vitamin D to the immune system is extensively discussed in chapters in Section XI and some of the diseases mentioned above are discussed in chapters in Section XII of this volume.
Cardiovascular and Metabolic Risks/Diseases and Vitamin D Status Several genes that play an important role in the cardiovascular homeostasis are targets of the vitamin D endocrine system, such as renin, thrombospondin, thrombomodulin, tissue factor, PAI-1 and BNP, and other genes related to endothelial function. In vitro studies with endothelial or cardiac smooth muscle cells also demonstrate that 1,25(OH)2D is an important regulator with an overall beneficial profile [103]. VDR null mice develop high renin hypertension and cardiac hypertrophy and this has been confirmed in mice with inborn deficiency of the 1a-hydroxylase gene. This is in line with a negative regulation of the renin gene expression in the kidney [30,104]. In fact, the cardiac effect has probably a dual origin: due to systemic high renin hypertension but also due to direct effects on cardiac muscle as cardiac muscle-specific VDR null mice also develop cardiac hypertrophy and accelerated stress-induced hypertrophic response [105]. See Chapters 31 and 40 for further discussion of these subjects. There are numerous observational studies in men regarding vitamin D status and blood pressure. Indeed, in normotensive and hypertensive Caucasians, blood pressure is frequently correlated with 25(OH)D or 1,25-(OH)2D [106,107]. This has been confirmed in a large multicenter study dealing with Hispanic and AfricanAmericans [108]. Unfortunately, the link between
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vitamin D status and hypertension is complicated by a very strong (negative) association between increased BMI, a very well-known risk factor for hypertension, and decreased 25(OH)D. Prospective randomized intervention trials are therefore needed to prove causality and, if so, to define the optimal dosage for each target population. A meta-analysis of eight randomized trials examining the effects of vitamin D supplementation on blood pressure in hypertensive (140/90 mm Hg) men and women showed a small but significant reduction in diastolic blood pressure (3.1 mmHg) and a nonsignificant reduction in systolic blood pressure (3.6 mmHg) [109]. The link between vitamin D status and risk of cardiovascular disease may also involve a much broader impact beyond its association with hypertension. Indeed, VDR null mice are more prone to increased thrombogenesis and display decreased fibrinolysis [1,30]. The equivalent in humans is an association between the vitamin D status and a large number of cardiovascular risk factors or events as revealed by several cross-sectional or prospective studies. Vitamin D and cardiovascular risks are also extensively discussed in Chapter 102. In the Framingham Offspring Study, a low 25(OH)D level (<15 ng/ml) was associated with an increased risk for a first cardiovascular event during a 5-year follow-up period compared with events in subjects with 25(OH)D values 15 ng/ml (hazard ratio 1.62) [110]. In the National Health and Nutrition Examination Study 2001 to 2004, the prevalence of coronary heart disease (angina, myocardial infarction) was more common in adults with 25(OH)D levels <20 ng/ml compared with 30 ng/ml (odds ratio adjusted for age, race, and gender 1.49) [111] but this association lost its significance after adjusting for other risk factors (body mass index, chronic kidney disease, hypertension, diabetes mellitus, smoking, use of vitamin D supplements). The prevalence of heart failure and peripheral arterial diseases was also higher in those with 25(OH)D values <20 ng/ml (OR 2.10 and 1.82, respectively) with similar attenuation after adjustment for other risk factors. Vitamin D deficiency during early pregnancy may also be a risk factor for preeclampsia [112]. An extensive systematic review [113] summarized a large number of observational and intervention studies dealing with vitamin D and cardiovascular risks. The authors confirmed that five out of seven cohort studies revealed an inverse relation between 25(OH)D and a set of cardiovascular risks factors. Nevertheless the authors concluded that this association is still uncertain. A meta-analysis of all interventional RCTs did not reveal a significant reduction of cardiovascular risks [112] and a new study [114] also did not detect beneficial effects on cardiovascular or metabolic risks after
increasing baseline 25(OH)D levels from 24 ng/ml to well above 40 ng/ml. It may well be that the effects will only be found in subjects with a low vitamin D status at baseline and that above such a threshold no further beneficial effects will occur. Excess vitamin D may also have major negative effects on the cardiovascular system with ectopic calcification, organ failure and death as a consequence. So it seems that too little but also frank excess of vitamin D may both be deleterious for cardiovascular events [30]. Both sides of the debate on possible deleterious effects of increased vitamin D on vascular calcification are discussed in Chapter 73.
Diabetes and Metabolic Syndrome and Vitamin D Status Body mass index is linked with serum 25(OH)D concentration in most of the studies around the world [115]. Whether increased fat mass comes first and then lower vitamin D status or the other way around is not known. It could be that the fat-soluble vitamin D is just more easily stored into fat cells before being available for further metabolism. There is, however, also a possible closed feedback loop between fat cells and vitamin D metabolism. Indeed leptin-deficient or leptinresistant mice have higher serum 1,25(OH)2D levels and higher serum calcium and phosphate levels due to increased renal 1a-hydroxylase expression [116]. This can be explained by leptin’s stimulatory effect on osteoblastic FGF23 production which then negatively feeds back on CYP27B1 [117]. Thus, since leptin stimulates fgf23 and thus suppresses cyp27b1, leptin deficiency would increase 1,25(OH)2D, but its effect on serum 25(OH)D is unexplored. Moreover fat tissue is also a target for vitamin D action. VDR null mice as well as cyp27b1 null mice are leaner than controls and more resistant to diet-induced obesity by actions not completely understood. Whether this feedback loop is also operational in humans is yet unknown [21]. Also see Chapter 44 for a discussion of new concepts regarding vitamin D and adipose tissue. The contribution of a poor vitamin D status on the health consequences of a high BMI or obesity is not known but many consequences of both situations are overlapping. It is therefore no surprise that type 2 diabetes and in fact all conditions known to be part of the metabolic syndrome are associated with a poor vitamin D status [118e123]; see also Chapter 98. This applies to both adults and US adolescents [124]. A good example of this observation [125] demonstrated that low serum 25(OH)D levels (below 21 ng/ml) are associated with a nearly two-fold increased risk of fasting hyperglycemia or diabetes and a 1.5-fold increased risk of hypertension or hypertriglyceridemia. Such low
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VITAMIN D AND EXTRA-SKELETAL HEALTH
25(OH)D levels are also linked to a two-fold increase in the overall prevalence of the metabolic syndrome [126]. In patients with chronic renal failure accelerated cardiovascular diseases are well known and are the most important factor for their increased mortality. Such patients are indeed also frequently deficient in both 25(OH)D and 1,25(OH)2D, whereas treatment with vitamin D analogs, albeit in non-randomized trials, has been found to result in lower cardiovascular and overall mortality [127]; see also Chapter 81. There are a number of reasons to link type 1 and type 2 diabetes with vitamin D status [100]. For type 1 diabetes, the link is largely mediated by the effects of vitamin D on the autoimmune system. For type 2 diabetes, the potential mechanisms include the beneficial effects of 1,25(OH)2D on b-cell secretion of insulin as well as insulin sensitivity [100]. Intervention studies usually not primarily designed to answer this question are, however, mostly negative as reported in a recent meta-analysis [119]. However, a good RCT in severely vitamin-D-deficient Asians living in New Zealand revealed a modest improvement of their insulin sensitivity after 6 months of vitamin D supplementation [128]. The overall conclusion or interpretation of all this data is that poor vitamin D status is frequently linked with the metabolic syndrome but the existing intervention studies are either negative or showed only limited beneficial effects so that large-scale studies are needed before its potential benefit can be translated into clinical large-scale recommendations. While awaiting such results it is difficult or even impossible to define a 25(OH)D threshold for the prevention of cardiovascular or metabolic events but for both group of complications, epidemiologic data suggest that the highest risks are found in subjects with 25(OH)D levels below 20 ng/ml and a trend for further risk reduction with still higher 25(OH)D levels.
Muscle Function and Vitamin D Status Adult striated muscle has a low VDR content but its concentration may be more prominent in earlier stages of muscle development. Vitamin D and muscle function is discussed in Chapter 104. Muscle from VDR null mice shows clear developmental abnormalities as immature muscle genes and proteins survive in adult muscle [30]. Early clinical observations in patients with very severe vitamin D deficiency (such as in rickets, in chronic renal failure, or children with pseudovitamin D deficiency rickets due to genetic CYP27B1 deficiency) clearly demonstrated that vitamin D or 1,25(OH)2D therapy can rapidly improve their severe and incapacitating proximal muscle weakness [30]. Observational human studies have also associated muscle weakness
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with poor vitamin D status in children and elderly subjects [129e132]. The poorest vitamin D status (<<20 ng/ml and especially <10 ng/ml) was associated in most studies with the poorest muscle function and higher levels of 25(OH)D were associated with muscle improvement in some studies [131]. A critical analysis of all published data on vitamin D supplementation versus placebo could only demonstrate a clear benefit on muscle strength if baseline 25(OH)D levels were below either 10 or 20 ng/ml; whereas there was no overall benefit in muscle strength in elderly subjects with baseline 25(OH)D levels above 20 ng/ml. However, few studies used proximal muscle strength (the most affected muscle function in clinical vitamin D deficiency or pseudovitamin D deficiency rickets) as a true endpoint and no studies used higher than 2000 IU of vitamin D3 per day. Of course, the real endpoint is not muscle strength but falls. A more recent meta-analysis published by the Cochrane Database Systemic reviews concluded that vitamin D supplementation in older people living at home reduced the risk of falls only in people with low baseline 25(OH)D levels (below 20 ng/ml) [133], but its effects were more pronounced in older people residing in nursing care facilities and hospitals (RR 0.85, five trials in 1925 participants) [134]. An extensive meta-analysis by Bischoff-Ferrari et al. [52] of a selection of eight double-blind RCTs in older individuals (65 years) revealed that a dose of 700e1000 IU vitamin D3/d reduced the risk of falls by 19% (RR 0.81) and as much as 23% if final mean 25(OH)D levels exceeded 24 ng/ml (see Chapter 62).
Vitamin D in Pregnancy and Perinatal Period Nutritional status in general may affect prenatal growth and development and may also have longterm effects on health. Several observational studies in humans have linked a poor vitamin D status during gestation (poor vitamin D status of the mother) or infants to a wide variety of diseases, such as lower bone mass at age 9, higher risk of schizophrenia, type 1 diabetes, multiple sclerosis or atopic diseases [135e137]. Also fetal bone development may be affected by maternal vitamin D deficiency (25(OH)D levels below 20 ng/ml) as revealed by a greater metaphyseal cross-sectional area and femoral splaying at 19 weeks of gestation [138]. Intervention trials, however, are virtually absent except for a small study revealing better postnatal growth when Asian mothers received 1000 IU vitamin D2 during pregnancy [139]. More recently a major NIH-sponsored intervention study with 4000 IU vitamin D3/d during pregnancy improved the pregnancy outcome (especially by decreasing premature birth rate) [140]. Chapter 35 discusses vitamin D in the perinatal period.
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58. HOW TO DEFINE OPTIMAL VITAMIN D STATUS
Vitamin D and Non-skeletal Targets
1. In vitro data (genetic, molecular, and cellular data): nearly all cells respond to the vitamin D hormone by decreased cell proliferation, increased cell differentiation, and improved function 2. Animal data (at very low vitamin D or high analog doses) clearly demonstrate vitamin D-VDR effects on all cells of the immune system proliferation of cancer cells cardiovascular system muscle, . 3. Observational studies link poor vitamin D status to nearly all major human diseases such as cancer infections/autoimmune diseases CV risks and events diabetes and metabolic syndrome muscle dysfunction and falls
Conclusions: Extra-skeletal Effects of Vitamin D There is thus little doubt that vitamin D may have many extra-skeletal effects. This conclusion is based on strong genetic, molecular, cellular and preclinical data as well as by a series of very large observational studies in humans, either cross-sectional or prospective (Table 58.5). However, there are serious limitations in the human data as there are very few RCTs with genuine extra-skeletal hard endpoints, except for muscle function and falls in the elderly. Therefore one can only conclude that vitamin D supplementation with >400 to 1000 IU of vitamin D3 per day modestly reduces the risk of falls in the elderly, mostly vitamin-D-deficient elderly subjects and that this dosage will probably increase circulating 25(OH)D levels above 20 ng/ml in most of the target population. For the other potential extra-skeletal effects, epidemiologic data suggest that the greatest risk is found in subjects with 25(OH)D levels below 20 ng/ml with a trend for still lower risk with higher 25(OH)D levels (above 30 or 40 ng/ml). Neither the causal link with vitamin D nor the dosage of supplementation nor the target 25(OH)D levels to achieve benefit have yet been clearly demonstrated (except for falls).
VITAMIN D AND HUMAN HEALTH: WHAT IS THE OPTIMAL VITAMIN D STATUS The causal link between vitamin D status and bone health is beyond doubt as vitamin D deficiency is clearly the cause of most cases of rickets and osteomalacia. Vitamin D is also the causal link in the pathogenesis
and clinical expression of bone disease in patients with chronic renal failure and osteoporosis of the elderly. Numerous cross-sectional or observational studies have in general not uniformly identified the optimal threshold for vitamin D status based on vitamin D intake or 25(OH)D levels. A number of RCTs, however, revealed that both surrogate calcemic endpoints such as serum concentrations of 1,25(OH)2D, or PTH, intestinal calcium absorption or BMD are impaired when serum 25(OH)D falls below 20 ng/ml, with little evidence for much further improvement with still higher 25(OH)D concentrations. For the best endpoint, fracture risks, several RCTs have shown that vitamin D supplementation together with good calcium intake (>1 g/d) can modestly reduce non-vertebral and hip fractures by about 20% when given to elderly subjects, with the greatest effects seen in institutionalized and/ or severely vitamin-D-deficient subjects. The daily dose of vitamin D proven to be efficient was >400e1000 IU/d and vitamin D3 was most frequently used. There are less convincing data on vitamin D2, and once-yearly intermittent very high doses of vitamin D should be avoided as it may transiently even increase the fracture risk. The causal link between vitamin D status and extraskeletal health is not yet formally proven although much data have accumulated suggesting a widespread action of the vitamin D endocrine system. Based on very convincing preclinical data the hypothesis of major effects of poor vitamin D status on overall health is very plausible. These potential clinical targets for vitamin D include: the immune system (as vitamin D deficiency is associated with increased risk of infections and of autoimmune diseases); cancer prevalence or evolution or progression; cardiovascular and metabolic diseases (including hypertension and metabolic syndrome); neurologic diseases; and in fact nearly all major diseases of humankind. Due to a lack of RCTs focusing on these clinical endpoints, it is yet not possible to firmly conclude that supplementation is beneficial or should be introduced for the general public. Nor is it possible to define a clear threshold for serum 25(OH)D levels associated with the best global health situation. Based on RCTs aiming at fracture and fall prevention in the elderly, a daily (or equivalent weekly or monthly) supplement of 800 IU of vitamin D3 per day can be highly recommended as safe and moderately efficient for the elderly Caucasian population, whereas there is less evidence in other groups (non-Caucasians, younger adults, etc.). Such vitamin D supplements will probably increase serum 25(OH)D levels above 20 ng/ml in most otherwise healthy subjects. This 25(OH)D level is also the minimal target level that should be reached to optimize surrogate bone/calcemic endpoints. Moreover levels below 25(OH)D are associated or linked with
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REFERENCES
the greatest risks for all major diseases associated with vitamin D nutritional status. This target is still far away from reality in most human populations as the mean 25(OH)D level in otherwise healthy adults hardly exceeds 20 ng/ml as based on the overall analysis of all published data [141]. A mean level of 20 ng/ml is not even reached in a number of countries (e.g., Germany and many Muslim countries) or in some subgroups (Afro-Americans in the USA or immigrants with dark skin in western Europe). Therefore it is already a formidable task to increase 25(OH)D to 20 ng/ml in all adults as the necessary supplementation must reliably reach billions of people so that a well-planned strategy is needed to implement this action for all and especially for the subgroups that are now at greatest risk of severe deficiency. Therefore there is a clear need for a global vision for vitamin D nutritional policy [142,143]. First, the minimal action required is the general introduction of vitamin D supplementation for infants in areas around the world or in niche groups where vitamin D deficiency rickets is still prevalent. This easy and highly effective strategy should be a top priority for health care worldwide. Second, the elderly Western population needs to achieve a better vitamin D and calcium status and therefore vitamin D supplementation and optimal calcium intake (>1 g/d) should be implemented as a routine care for all elderly subjects. This is already a strong recommendation by several societies dealing with osteoporosis or geriatrics and has also been supported by the European Standing Committee of Medical Doctors (website http://www.cpme.be). The US Institute of Medicine has recently published new guidelines for DRIs for vitamin D and recommend a daily intake of 600 IU for adults, increasing to 800 IU above the age of 70 years. The level of vitamin D supplementation to be recommended should be based on the best available evidence and thus on RCT findings and therefore approximately 800 IU of vitamin D3 per day is the best option. This will probably increase serum 25(OH)D levels above 20 ng/ml in most subjects. This strategy is proven to be safe even when implemented on a very large scale. Above and beyond these target groups there are no hard data to make valid general recommendations but it seems logical, while waiting for further evidence, to try to obtain this minimal adequate vitamin D status for the whole population. Dependent on their background nutritional status it would require 400e1000 IU of vitamin D3 equivalent per day. Many epidemiologic studies suggest that still higher 25(OH)D levels might have additional beneficial effects on global health but reaching these higher levels (targeting mean levels of >30 or 40 ng/ml for the total population) would require a totally different strategy. This could indeed be obtained by substantially higher exposure to UV-B rays, but this
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strategy cannot be recommended because of the known risk for skin cancer and photoaging. The other alternative for such a strategy would be to substantially increase the total vitamin D intake to well above 2000 IU/d. However, this strategy has not been tested in large-scale RCTs, or in patients, or in healthy subjects, so that neither efficacy nor safety has been demonstrated. The VITAL trial is just under way testing the safety and efficacy of 2000 IU in a large RCT (see Chapter 105). Previous situations, where association studies suggested large-scale health benefits from what appeared at first sight to be safe interventions have more frequently been found not to generate health benefits and even to cause harm (e.g., hormone replacement therapy for postmenopausal women, anti-oxidants, vitamin B supplements, etc.). The best solution therefore is to recommend that the required RCTs be carried out to test the vitamin D and global health hypothesis. In the meantime, the conservative implementation of an 800e1000 IU/d strategy should generate a marked benefit for millions of people around the world.
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58. HOW TO DEFINE OPTIMAL VITAMIN D STATUS
receiving calcium supplementation: randomised controlled trial, BMJ 336 (2008) 262e266. [151] M. Chung, E.M. Balk, M. Brendel, S. Ip, J. Lau, J. Lee, et al., The relationships of vitamin D and calcium intakes to nutrient status indicators and health outcomes. Evidence Report No. XXX (prepared by the Tufts Evidence-based Practice Center under Contract No. HHSA 290-2007-10055-I). AHRQ Publication No. 0X-E0XX, Agency for Healtcare Research and Quality, Rockville, MD, 2009. [152] Patient level pooled analysis of 68 500 patients from seven major vitamin D fracture trials in US and Europe, BMJ 340 (2010) B5463. [153] G.J. Bergman, T. Fan, J.T. Mcfetridge, S.S. Sen, Efficacy of vitamin D3 supplementation in preventing fractures in elderly
women: a meta-analysis, Curr. Med. Res. Opin. 26 (2010) 1193e1201. [154] I. Laaksi, J.P. Ruohola, P. Tuohimaa, A. Auvinen, T. Ylikomi, An association of serum vitamin D concentrations <40 Nmol/L with acute respiratory tract infection in young Finnish men, Am. J. Clin. Nutr. 86 (2007) 714e717. [155] A.A. Ginde, J.M. Mansbach, C.A. Camargo Jr., Association between serum 25-hydroxyvitamin D level and upper respiratory tract infection in the Third National Health and Nutrition Examination Survey, Arch. Intern. Med. 169 (2009) 1443. [156] M.L. Melamed, E.D. Michos, W. Post, B. Astor, 25-Hydroxyvitamin D levels and the risk of mortality in the general population, Arch. Intern. Med. 169 (2008) 1075e1076.
VII. NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY
Color Plates Crystal-structure-based analysis of the conservation of amino acid sequences in mitochondrial vitamin D hydroxylases and substrate binding in the crystal structure of CYP2R1 [18]. The top panel depicts the frequency of conserved residues in CYP27A1 (41 species, excluding nine fish and frog) mapped from sequence alignments onto the crystal structure of CYP24A1. The extensively open active site seen in the crystal structure of CYP24A1 is very likely conserved in CYP27A1 and CYP27B1 and provides a clear view through to the heme. The positions of two mutations causing autosomal CYP27A1 deficiency (cerebrotendinous xanthomatosis, CTX) are indicated. The first residue, Thr339, is highly conserved (with only minor exceptions) in the I-helix of cytochrome P450s and is thought to structurally distort a helical turn necessary for molecular oxygen staging during the redox cycle. The second residue, Arg405, forms a hydrogen bond with the heme A-ring propionate to structurally stabilize the active site. Many microsomal P450s, including CYP2R1, use histidine for the same purpose. In the middle panel, mutation of corresponding residues causes CYP27B1 deficiency (vitamin-D-dependent rickets type I, VDDR-I). The frequency of conserved residues in CYP27B1 (39 species including five fish and frog) appears to be marginally better than CYP27A1. Approximately 40% of the residues are >95% conserved (green) across species and these are mostly in the core of the enzyme and on the proximal heme ferredoxin-binding surface. The less conserved yellow (80e95%), orange (60e80%), and blue (<60%) residues are generally found on the surface of the enzyme where the selective pressure of structuree function relationships is less. The lower panel shows CYP2R1 co-crystallized with vitamin D3. In this structure (2ojd.pdb and also 3c6g.pdb), the 3b-OH group in the vitamin D3 A-ring mediates hydrogen bond contacts to the protein backbone at the carbonyl of Ala250 and a water molecule stabilized by Asn217. The position of the hydroxylation target carbon (C25) is slightly out of range of target carbons geometrically triangulated from 49 P450 crystal structures (pink spheres above heme) possibly due to the fact that the substrate exhibits a trans-triene and not the cis-triene characteristic of vitamin D.
FIGURE 3.8
Structure of the rat CYP24A1 protein inserted in the inner mitochondrial membrane. The ribbon structure was obtained from the atomic coordinates deposited in the Protein Data Bank, code 3K9V (http://www.pdb.org). The arrow defines the substrate access channel and points to the heme. MIS, membrane insertion sequence.
FIGURE 4.2
FIGURE 5.1 DBP-25(OH)D in three dimensions. (A) The DBP three-dimensional structure with its three structural domains. The numbering of the helices is the same as in human serum albumin. (B) The conformation of 25(OH)D in DBP. Helices 1e6 of the apoprotein (light blue) and the holoprotein (DBP-25(OH)D) are superimposed. 25(OH)D is shown in ball-and-stick representation. Two water molecules present in the binding site are indicated as red balls. (Reproduced from figures 2a and 4a of the manuscript of Verboven et al. [8].)
A
40 kb
Chr 14 (Mb)
76.77
76.81
76.85
76.89
76.93
76.97
77.01
77.05
CTCF1 Tnfsf11
NM_177629
B
Log2 Ratio
6.5
Veh vs IgG
D1 D2
77.09 D7
77.13
D3 D4 D5 D6
77.17
77.21
77.25
CTCF2 Akap11
H4ac
Log2 Ratio
0.0 -2.0 6.5
1,25 vs IgG
0.0 -2.0
C Log2 Ratio
4.0
Veh vs IgG
CTCF
0.0
Log2 Ratio
-2.0 4.0
1,25 vs IgG
0.0
-2.0
FIGURE 7.8 ChIP-chip analyses of histone 4 acetylation and CTCF transcription factor binding at the Tnfsf11 (RANKL) gene locus. Mouse ST2 cells were treated with either vehicle or 1,25(OH)2D3 for 3 h and then subjected to ChIP-chip analysis using antibodies to tetra-acetylated H4, CTCF, or IgG. A schematic diagram of the mouse Tnfsf11 gene locus together with its upstream and downstream neighboring genes (Akap11, NM_177629) and its location on chromosome 14 is depicted at the top. The transcriptional start site (TSS) is designated with an arrow on the opposite DNA strand. Previously identified enhancer elements located upstream of the designated TSS are indicated (D1eD7). The data tracks represent the log2 ratios of fluorescence obtained from vehicle- or 1,25(OH)2D3-treated samples precipitated with antibody to the tetra-acetylated H4 or CTCF vs IgG controls. Key boundary CTCF sites are ringed. Red peaks represent statistically valid regions of VDR or RXR binding (FDR <0.05).
FIGURE 8.5 Structureefunction relationships in the human VDR ligand binding/heterodimerization/transactivation domain. (A) A sche-
matic view of human VDR, in which the following subdomains are highlighted: the third, fifth, and twelfth helices (H3, H5, and H12), which have been implicated in binding of coactivators and transactivation; the ninth and tenth helices (H9 and H10) and the loop between helices 8 and 9, which contain an interface for interaction with the RXR heterodimeric partner; and finally, two b-strands in the VDR crystal structure [18]. A section of the VDR ligand-binding/heterodimerization domain from positions 165e215 was deleted (a box with crossed lines) by Rochel and colleagues [18] in order to facilitate purification and crystallization. (B) The X-ray crystal structure of human VDR (residues 118e164 spliced to residues 216e425) [18] bound to its natural 1,25(OH)2D3 ligand, as viewed in Jmol [262]. Three examples of natural mutations that cause HVDRR are indicated with hVDR residue numbers (274, 391, 420) and are discussed in the text. The position of the deletion is indicated by the highlighting of residues 164 and 216, at the top of the structure. The conformation of this deleted region may resemble that in the related receptor, PXR (panel C), as suggested by the dotted outline at the upper left of the panel. (C) A view of the human PXR (or SXR, steroid, and xenobiotic receptor) ligand-binding/heterodimerization domain bound to the synthetic ligand, SR12813 [98], created in Jmol to approximate the same view of hVDR in panel B. A contiguous fragment of PXR was used for crystallization (residues 142e431), but the region from residue 177 to residue 198 was unstructured (shown as a dotted line). Four b-strands are shown, with the lower two strands (designated b1 and b10 in the original publication [98]) residing in a region corresponding to the deletion in the crystallized hVDR. This conformation of strands b1 and b10 was employed as a model for a hypothetical structure of hVDR in this region, as shown in B.
A/ B
C
D
E
F
AD AD
N
C
DNA binding DIMERIZATION
dimerization NLS
LIGAND BINDING AF-1 Activation Function Ligand independent
AF-2 Activation Function Ligand-dependent Interaction with cofactors
FIGURE 9.1 Structural and functional organization of nuclear receptors. NRs consist of six domains (AeF) based on regions of conserved sequence and function.
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FIGURE 9.2 Superimposition of unliganded (green and yellow) and liganded (blue and red) hRXRa LBD monomers. The main conformational differences affecting helices H3, H11, and H12 are colored in yellow and red. The arrows show the main structural changes upon ligand binding. The ligand is depicted in yellow. Adapted from Figure 4A of Egea PF, Mitschler A, Rochel N, Ruff M, Chambon P, Moras D 2000. Crystal structure of the human RXRalpha ligand-binding domain bound to its natural ligand: 9-cis retinoic acid. EMBO J 19:2592e2601 [77].
Overall fold of hVDRD ligand-binding domain. The helices are represented as cylinders and b sheets as arrows. The whole structure is colored in gray except the helix H12 in purple. The ligand is depicted in yellow. The insertion region location is shown in green. The reconnected residues are indicated, together with their sequence numbering. Adapted from Figure 1 of Rochel N, Wurtz JM, Mitschler A, Klaholz B, Moras D 2000. The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol Cell 5:173e179 [25].
FIGURE 9.3
Intramolecular interactions of helix H12 in VDR. The backbone of the protein is colored in gray except for helix H12 in purple. The side chain residues involved in polar stabilization of H12 are shown together with the hydrogen bonds (green dots). The ligand is depicted in yellow and red for the oxygen atoms. Adapted from Figure 3 of Rochel N, Wurtz JM, Mitschler A, Klaholz B, Moras D 2000. The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol Cell 5:173e179 [25].
FIGURE 9.4
FIGURE 9.5 Stereo view of 1a,25(OH)2D3 in the hVDR-binding pocket. The ligand molecule is shown in the experimental electron density omit map contoured at 1.0 standard deviation. The hydroxyl groups are depicted as red spheres. Water molecules are shown as purple spheres. Hydrogen bonds are shown as green dotted lines. Carbon atoms are colored in gray, and oxygen and nitrogen atoms are colored in red and blue, respectively. Adapted from Figure 3 of Rochel N, Wurtz JM, Mitschler A, Klaholz B, Moras D 2000. The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol Cell 5:173e179 [25].
Overall fold of zVDR ligand-binding domain. The helices (in red) are represented as cylinders and b sheets (in yellow) as arrows. The SRC-1 peptide is shown in blue. The ligand 1a,25(OH)2D3 is depicted in gray. The green stars indicate the VDR-specific insertion region not seen in the crystal structure.
FIGURE 9.6
FIGURE 9.7 Crystal structures of the hVDRD LBD complexed to 1a,25(OH)2D3, MC1288, and KH1060. (A) Experimental electron density
omit map contoured at 2.0 standard deviation of (a) 1a,25(OH)2D3, (b) MC1288, and (c) KH1060. (B) Close-up view of KH1060 in the ligandbinding pocket. Secondary structure features are represented in blue (a-helices) and green (b-strands). The ligand is colored in yellow with the oxygen atoms in red. The volume of the cavity is represented in gray. (C) Superposition of 1a,25(OH)2D3 (yellow), MC1288 (green), and KH1060 (blue) ligands after superimposed VDR complexes. Oxygen atoms are colored in red. Adapted from Figures 2 and 3 of Tocchini-Valentini G, Rochel N, Wurtz JM, Mitschler A, Moras D 2001. Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands. Proc Natl Acad Sci USA 98:5491e5496 [28].
Crystal structures of the hVDRD LBD complexed to the 14-epi TX522 ligand. (A) Chemical structures of TX522, TX527, and 1a,25 (OH)2D3. (B) Close-up view of TX522 in the ligand-binding pocket. Superposition of the VDR-1,25-(OH)2D3 (yellow) and VDR-TX522 (blue) are shown. The two residues Val300 and Ile268 making different contacts with TX522 are highlighted complexes. Only residues closer than 4.0 A in red. The ligands 1,25-(OH)2D3 and TX522 are shown in stick representation, with carbon and oxygen atoms in gray and red, respectively. The hydrogen bonds are shown as red dashed line. Adapted from Figure 8 of Eelen G, Verlinden L, Rochel N, Claessens F, De Clercq P, Vandewalle M, Tocchini-Valentini G, Moras D, Bouillon R, Verstuyf A 2005. Superagonistic Action of 14-epi-Analogs of 1,25-Dihydroxyvitamin D Explained by Vitamin D Receptor-Coactivator Interaction. Mol. Pharm 67:1566e1573 [49].
FIGURE 9.8
Close-up view of the water molecules network near C-2 position of ligands. VDR complexes with 1a,25(OH)2D3 (A), 2a-methyl1a,25-dihydroxyvitamin D3 (B), 2a-propyl-1a,25-dihydroxyvitamin D3 (C), 2a-propoxy-1a,25-dihydroxyvitamin D3 (D), 2a-(3-hydroxypropyl)1a,25-dihydroxyvitamin D3 (E), and 2a-(3-hydroxypropoxy)-1a,25-dihydroxyvitamin D3 (F). The water molecules are shown as red spheres. The missing water molecules in CeF complexes are shown as dotted lines to red spheres. Residue side chains contacting the water molecules through H-bonds are shown. The ligands are shown in stick representation with carbon and oxygen atom in gray and red, respectively. The hydrogen bonds formed by the ligands and those formed by the water molecules are shown as yellow dotted lines. Adapted from Figure 4 of Hourai S, Fujishima T, Kittaka A, Suhara Y, Takayama H, Rochel N, Moras D 2006. Probing a water channel near the A-ring of receptor-bound 1 alpha,25dihydroxyvitamin D3 with selected 2 alpha-substituted analogs. J Med Chem. 49:5199e5205 [50].
FIGURE 9.9
(A)
CD578
WU515
WY1113
(B)
VDR structure with the nonsteroidal CD578 analog. (A) Chemical structures of 1a,25(OH)2D3, CD578, Wu515, and WY1113. (B) Stereo view of the conformation of the bound ligands. CD578 (green) and 1a,25(OH)2D3 (pink) are shown in stick representation after superimposed VDR complexes. Adapted from Figure 3 of Eelen G, Valle N, Sato Y, Rochel N, Verlinden L, De Clercq P, Moras D, Bouillon R, Mun˜oz A, Verstuyf A 2008. Superagonistic fluorinated vitamin D3 analogs stabilize helix 12 of the vitamin D receptor. Chem Biol. 15:1029e1034 [53].
FIGURE 9.10
FIGURE 9.11 VDR structure with the nonsecosteroidal analog YR301. (A) Chemical structures of the nonsecosteroidal ligands LG190178 and YR301. (B) Close-up view of YR301 in the ligand-binding pocket of rVDRD LBP. Superposition of the VDR-1a,25-(OH)2D3 (red) and VDR-YR301 (blue) complexes. Adapted from Figure 4 of Kakuda S, Okada K, Eguchi H, Takenouchi K, Hakamata W, Kurihara M, Takimoto-Kamimura M 2008. Structure of the ligand-binding domain of rat VDR in complex with the nonsecosteroidal vitamin D3 analog YR301. Acta Crystallogr Sect F Struct Biol Cryst Commun. 64:970e973 [61].
(A)
Gemini (B)
Adaptability of the vitamin D nuclear receptor to the synthetic ligand Gemini. (A) Chemical structure of Gemini. (B) Superimposition of the Gemini (in orange) and 1a,25(OH)2D3 (in green) zVDR-bound LBP. This view emphasizes the large conformational change of Leu337 (hLeu309) side chain that opens the additional channel and the central role of His333 (hHis305) that anchors the hydroxyl groups of both ligand side chains.
FIGURE 9.12
(A)
22Sbutyl-25,26,27-trinorvitamin D
22Sbutyl-20epi-25,26,27-trinorvitamin D
(B)
FIGURE 9.13 Adaptability of the vitamin D nuclear receptor to the 22-butyl-1a,24-dihydroxyvitamin D3. (A) Chemical structures of the antagonist 22Sbutyl-25,26,27-trinorvitamin D and the agonist 22Sbutyl-20epi-25,26,27-trinorvitamin D. (B) Superimposition of the ligands in the rVDR/1a,25(OH)2D3 (green), the antagonist rVDR/22Sbutyl-25,26,27-trinorvitamin D (cyan) and the agonist rVDR/22Sbutyl-1a,24R-dihydroxyvitamin D3 (pink) complexes. Adapted from Figure 1 of Inaba Y, Nakabayashi M, Itoh T, Yoshimoto N, Ikura T, Ito N, Shimizu M, Yamamoto K 2010. 22S-Butyl-1alpha,24R-dihydroxyvitamin D(3): Recovery of vitamin D receptor agonistic activity. J Steroid Biochem Mol Biol [66].
(A)
MC903
ZK159222
ZK 168281
(B)
FIGURE 9.14 Structural basis for vitamin D receptor antagonism. (A) Chemical structures of the agonist MC903, the partial agonist ZK159222, and the full antagonist ZK168281. (B) Model of VDR-ZK159222 based on the VDR-calcipotriol complex crystal structure (blue). Superposition of calcipotriol in stick representation with carbon and oxygen atoms in gray and red, respectively, and of two conformers of ZK159222. One conformer (in cyan) with the cyclopropyl orientated as in the VDR-calcipotriol crystal structure presents a steric clash with H3. In the other conformer (in pink), the cyclopropyl is rotated and the last four carbon atoms lie between H3 and H12. The resulting steric hindrance is most likely responsible for the displacement of H12 and the resulting antagonist effect. Adapted from Figure 3 of Tocchini-Valentini G, Rochel N, Wurtz JM, Moras D 2004. Crystal structures of the vitamin D nuclear receptor liganded with the vitamin D side chain analogs calcipotriol and seocalcitol, receptor agonists of clinical importance. Insights into a structural basis for the switching of calcipotriol to a receptor antagonist by further side chain modification. J Med Chem. 47:1956e1961 [70].
9.15 Crystal of VDR bound to adamantly vitamin D analogs. (A) Chemical structure of 24-AD-24hydroxyl derivative. (B) Close-up of the interactions made by the adamantly group of the 25AD-25-hydroxyl derivative (green) superimposed to 1a,25(OH)2D3. Adapted from Figure 3 of Nakabayashi M, Yamada S, Yoshimoto N, Tanaka T, Igarashi M, Ikura T, Ito N, Makishima M, Tokiwa H, DeLuca HF, Shimizu M 2008. Crystal structures of rat vitamin D receptor bound to adamantyl vitamin D analogs: structural basis for vitamin D receptor antagonism and partial agonism. J Med Chem. 51, 5320e5329. [74]. FIGURE
Structure-based design of superagonists ligands. (A) Chemical structures of the 1a,25(OH)2D3 and the analogs (20S,23S)- and (20S,23R)-epoxymethano-1a,25-dihydroxyvitamin D3, 2a-methyl-(20S,23S)- and 2a-methyl-(20S,23R)-epoxymethano-1a,25-dihydroxyvitamin D3. (B) Detailed structural representation of the 2a-methyl-(20S,23S)-epoxymethano-1a,25-dihydroxyvitamin D3 (green) superimposed to 1a,25 (OH)2D3 (orange) bound to hVDR LBD around the aliphatic chain (B) and the 2a-methyl group (C) showing the characteristic residues involved in interactions. Hydrogen and van der Waals bonds are shown in red and black dotted lines, respectively. Secondary structure of VDR is shown in cartoon. H2, H3, H6, H7, and H11 indicate helices.
FIGURE 9.16
Model of transcriptional cycling. The model monitors the three phases of transcriptional cycling, of which only the initiation phase results in the synthesis of mRNA, while mRNA degradation occurs at all phases. If a gene shows bursty transcription and the half-life of the mRNA is short enough, this will result in cycling of mRNA levels. Note that only the core proteins of the respective complexes are shown, as we assume that each protein complex contains up to 30 components. Ac ¼ Acetylated histones, Me ¼ methylated DNA (dark gray) or histones (light brown), Pol II ¼ RNA polymerase II.
FIGURE 11.2
circulation hormone binding protein pharmacologic
plasma membrane cytoplasm
nucleus
acceptor receptor enzyme coco- facilitator
receptor coco- activator coco- repressor coco- modulator
FIGURE 14.1 Proteins and small molecules (pharmacologics such as SERMs, left panel) control access of the sterol/steroid hormone to its receptor and modifying enzymes (middle panel) and subsequently legislate transcription of the hormone-controlled gene (right panel). Shown in yellow are the hsp-related co-facilitator proteins and the hnRNP-related comodulator proteins discussed in this chapter. Selective estrogen receptor modulators (SERMs).
FIGURE 14.2 An adolescent female tamarin monkey (left panel) with X-ray evidence of rachitic bone disease (middle left panel e metaphyseal cupping and fraying of the tibial plateau, and decreased bone opacity) compared to an age-matched normal control tamarin without rickets, (middle right panel) before and after 20 years of vitamin D supplementation (right panel).
FIGURE 14.7 Depiction of DNA and RNA cis elements that are known to or proposed to be (miRNA) bound by hnRNPs (tear drop; see bottom right legend) during the process of mRNA generation. The numbered dots represent serial hnRNP-regulated events in the production and processing of a 1,25-dihydroxyvitamin D (1,25D)- or estradiol (E2)-induced mRNA. The insert at event 2 describes competition between the dominant negative-acting hnRNP (response element-binding protein, REBiP) and the dominant positive-acting liganded receptor for binding to the response element. The geographic proximity of these events within the cell leads us to propose a “hopscotch” model of hnRNPs moving rapidly from one cis site to another to exert dynamic control over hormone-regulated gene expression.
FIGURE 14.8 “Extra-VDR” mechanisms associated with the regulation of target cell responses to 1,25(OH)2D3. (1) Serum transport of 1,25(OH)2D3 (1,25D3) and 25OHD3 (25D3) by vitamin-D-binding protein (DBP). (2) Cellular uptake of 1,25D3 and 25D3 either by diffusion of free molecules or via megalin-mediated receptor uptake (meg). (3) Intracellular transport of vitamin D metabolites by heatshock protein 70 (hsc70). (4) Expression of heterodimer partner, retinoid X receptor (RXR). (5) Expression of coactivator and corepressor proteins (Co.). (6) Chromatin reorganization via histone acetylation and deacetylation enzymes. (7) Occupancy of the vitamin D response element (VDRE) by vitamin D response element-binding protein (VDRE-BP). Vitamin-D-activating enzyme 1a-hydroxylase (CYP27B1).
PIP2
Nuclear H7
Coregulator
RXR
Surface
Surface
H3/H4/ H5/H12
H10
H1 The speculative VDR-PIP membrane model. In the figure the theoretical (i.e., computed) complex formed between the VDRAP (Fig. 15.9A) and PIP2 is shown. The PIP2 is rendered in space filling with hydrogen atoms included. In the complex, the phosphate at C4 of the inositol ring forms a strong electrostatic interaction with R274. The R274 residue is central in the figure and is shown in space filling with hydrogen atoms absent. In this theoretical complex, the VDR RXR heterodimerization surface (H7 and H10) and the nuclear coregulator surface (portions of H3/H4/H5 and H12) are oriented in such a manner that they are theoretically accessible when the VDR is anchored noncovalently and reversibly to the underside of the plasma membrane. These surfaces are shaded in the figure and labeled.
FIGURE 15.7
The VDR two pocket model. (A) The solvent exposed surface of the VDR (aa118e427; D165-215) is shown as a transparent clear surface. In the diagram two amino acids are rendered in space filling and labeled R402 and K413. These two cationic residues serve as the residues recognized by the trypsin active site in the production of the c3 and c2 limited trypsin digest (i.e., protease sensitivity assay, PSA) fragments (see Fig. 15.8 and [32]). The VDR-AP is represented by a colored sky blue solid surface and the VDR-GP by a faint yellow transparent surface. The region where H1/ H2, H3, H5, and the b-sheet meet forms the region where the VDR-AP and VDR-GP ligand-binding pockets share three-dimensional, steric space. This region is termed the VDR A-ring domain, highlighted in the figure by a dashed circle. (B) The 25-OH hydrogen bonds, the C26 and C27 hydrophobic, van der Waals interactions, and the seco-B-ring intermolecular stabilization when 1,25(OH)2D3 (ball and stick) is flexibly docked to the VDR-GP are illustrated. Note that the 1,25 (OH)2D3 pose shown here is in strong agreement with the VDR X-ray pose of 1,25(OH)2D3 [40]. That same pose was produced blindly in the PC_Model conformational search calculation (see Figs 15.2 and 15.4 and the text). The residues forming favorable contacts with C5, C6, C7, C8, and C19 are W286 and C288. Hydrophobic, van der Waals (vdW) contacts are made between the terminal side-chain methyl groups of 1,25(OH)2D3 (i.e., C26 and C27) and the hydrophobic crown residues L227, L414, and V418. These hydrophobic interactions form the buried foundation of the nuclear coregulatory surface (labeled, shaded regions of H3 and H12 in panel A and Fig. 15.7). H305 and H397 are required for 1,25(OH)2D3 for genomic transactivation. This is because the dynamics of the 1,25(OH)2D3 side-chain must be energetically augmented so as to allow for it to interact with the hydrophobic crown residues in a manner that shifts the Boltzmann distribution of VDR conformations to favor the agonist conformation, where H12 is protected from trypsin and therefore ordered when compared to e1,25 (OH)2D3 (Fig. 15.8) [32]. (C) The polar or unsaturated amino acid Rgroups that form the VDR A-ring domain: Y143, S237, R274, S278, W286, and C288. The b-chair, 6-s-cis, Pop. A conformation of 1,25 (OH)2D3 observed following the 1,25(OH)2D3/VDR-AP flexible docking simulation [37] is shown in the panel as a ball and stick structure. The nearest-neighbor contacts made between polar or unsaturated carbon atoms with the A-ring domain residues are indicated by solid lines and the distances in a˚ngstroms are provided in the figure.
FIGURE 15.9
(A)
H1
H5 H12 H2 R402
H3 K413
(B)
V418
L414 H305 C288
L227
H397
W286
(C)
S237 C288
W286
Y143 S278 S274
Regulation of endochondral bone formation (EBF). (A) Developmental signals suggest BMP/TGFb and the Wnt pathways required for specification of different cell phenotypes. These signals are ultimately transduced to the tissue-specific transcription factor that commits a cell to the indicated lineages. In addition, an important regulatory control in cell determination is through post-transcriptional mechanisms where microRNAs block translation of proteins that are required for commitment of cells to a specific lineage. Recently, BMPs have been shown to down-regulate a group of “osteo-miRs” that inhibit the BMP receptors and various components of osteogenic Wnt signaling [6]. (B) Coordination of cellular activities for regulating the progression and pace of chondrocyte proliferation and differentiation at the growth plate for placement by bone tissue. The signaling pathways operative at the different growth plate zones are indicated with gene markers used to identify the different subpopulations and the key transcriptional regulators for chondrocyte maturation.
FIGURE 16.1
FIGURE 16.2 Regulation of osteoblastogenesis. (A) Examples of developmental (BMP/Wnt), hormonal (parathyroid hormone (PTH)), glucocorticoid (GC) and 1,25(OH)2D3 (VD3) and growth factors (IGF-1) influencing differentiation are indicated. (B) The major differentiation stages of osteogenic lineage cells are illustrated in histochemical stained cells with examples of markers for each stage. Growth e toluidine blue; ECM e extracellular matrix maturation stage showing alkaline phosphatase activity; mineralization e von Kossa stained. Markers frequently used to characterize stages of maturation include collagen type I, alkaline phosphatase, bone sialoprotein, osteopontin and osteocalcin. (C) Examples of transcription factors regulating progression of osteoblast differentiation are indicated.
FIGURE 16.5 Novel epigenetic mechanisms for maintenance of cell phenotypes. This figure illustrates the concepts for Runx2 epigenetic functions, but have been identified for other tissue-specific transcription factors including myoD (muscle) and C/EBPa (adipocytes) [65]. (A) In contrast to ubiquitous transcription factors which are degraded during mitosis, Runx2 is retained in dividing cells and during telophase can still be visualized as a punctate foci with both small and large associated with chromosomes in metaphase. Large foci are localized to the nuclear organizing region (NOR). Following mitosis, Runx2 is restored to the interphase nucleus. (B) Higher magnification of the interphase nucleus shows Runx is associated with genes in transcriptional complexes bound to the nuclear matrix scaffold (left panel). Runx2 larger foci are seen in the periphery of the nucleolus (right panel). Studies have shown that Runx2, myoD, and C/EBPa contribute the regulation of protein synthesis through binding to regulatory elements in ribosomal RNA genes. Indeed, over 40e50 Runx regulatory elements can be found in promoters of rRNA genes [65,85]. Identification of Runx2 in nucleoli involved in transcription is confirmed by colocalization of Runx2 with upstream binding factor (UBF), essential for regulating ribosomal genes. (C) Illustration of the regulatory events in mitosis and interphase involving cell-typespecific transcription factors. These factors are “bookmarking” genes during mitosis for retaining phenotype fidelity after cell division; in the interphase nucleus, these tissue-specific transcription factors are contributing to protein synthesis regulation.
FIGURE 20.3 Overexpression of calbindin-D28K suppresses nuclear fragmentation of osteoblastic cells induced by TNFa. Cells were
transfected with the expression vector pREP4 alone (empty vector) or containing the cDNA for calbindin-D28K (calbindin-D28K) together with an expression vector containing the coding sequence of green fluorescent protein with a nuclear localization sequence. Forty-eight hours after transfection, cells were exposed to 1 nM TNFa for 16 h. Cells were fixed, mounted, and examined with a Zeiss confocal laser scanning microscope. Note the presence of apoptotic nuclei in the TNFa-treated vector-transfected cells but not in the calbindin-transfected cells similarly treated.
disulfide bonds at C129, C131 Lobe 1
Lobe 1
Ca2+binding site
Ca2+binding site Lobe 2
Lobe 2
Calcimimetics
Model of the predicted structure of the human CaSR. Shown are two monomers of the receptor, each of which has two lobes, which are linked by disulfide bonds involving cysteines 129 and 131 on each monomer. The lower part of the figure shows the seven transmembrane domains that enable transmission by the activated receptor of the extracellular Ca2þo signal to the receptor’s G proteins and other intracellular effectors. Red segments of the receptor’s extracellular domain indicate alpha helices. The locations of a key calcium-binding site in the crevice between the two lobes of each monomer are shown, as are the separate sites for calcimimetics, which bind within the receptor’s transmembrane domains. From Huang et al. 2007 J Biol Chem 282:19000e19010, with permission.
FIGURE 24.1
FIGURE 25.3 Expression of calcium channels in developing bone. (A) Expression of L-type VSCC Cav1.2 in developing long bone (red). Note expression in cartilaginous growth plate, in bone marrow cells amongst trabecular bone spicules, and in both cortical and trabecular bone. (B) Expression of T-type VSCC Cav3.2 (red). Note more restricted expression with absence in the growth plate where only nuclei (green) can be seen. Also note absence in bone marrow. Staining (red) is seen at sites of osteocyte entrapment. (C) Staining of collagen X (red), specific to the hypertrophic zone separating the growth plate and the marrow cavity. Note that the yellow color results from overlap of the VSCC stain and the nuclea stain.
(A)
(D)
(G)
(B)
(E)
(H)
(C)
(F)
(I)
FIGURE 26.4 (AeC) Immunohistochemical detection of VDR in normal human kidney tissue with polyclonal anti-hVDR antibody 2-152 (A, 200; B, 400; C, 400). (DeF) Immunohistochemical detection of 25(OH)D3 24-hydroxylase cytochrome P-450 in human kidney (D 200; E, 400; F, 400). (G) Immunohistochemical detection of calbindin D28k in human kidney (G, 400). (H and I) Control panels showing kidney tissue stained with preimmune serum (H, 200; I, 400). (With permission from Kumar R et al. [161].)
(A)
(A)
(B)
(B)
(A) Vitamin D receptor (VDR) immunostaining in metanephros of rat fetus on gestational day 15. (B) Higher-power view of boxed area in (A). Branching ureteric buds (arrows) and mesenchyme (M) are indicated. Bars ¼ 2.1 mm. (With permission from Johnson JA et al. [180].)
FIGURE 26.5
VDR (A) and calbindin D28k (B) immunostaining in metanephros of rat fetus on gestational day 17. Parietal epithelial cells (small arrows), visceral epithelial cells (open arrows), and tubule portion of developing comma-shaped body (T) are indicated. Bars ¼ 2.1 mm. (With permission from Johnson JA et al. [180].)
FIGURE 26.6
(A)
(B)
FIGURE 26.7 VDR immunostaining in mouse metanephric organ culture explant following 120 h of incubation. Parietal epithelial cells (small arrow), visceral epithelial cells (large arrow), proximal tubule (P), and distal tubule (D) are indicated. Bars ¼ 2.1 mm. (With permission from Johnson JA et al. [180].)
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
Immunoperoxidase localization of Caþþ-Mgþþ ATPase within human kidney distal tubules and human spleen erythrocytes. Kidney: (a and b) Monoclonal antibody JA3. (c and d) Monoclonal antibody JA8. (e and f) Negative control. (g and h) Double stain, PAS, and JA3; (arrows) PASpositive proximal tubule brush border. Spleen: (i) Monoclonal antibody JA3. (j) Negative control. (a, c, e and g) 200. (b, d, f, h, i, and j) 640. (With permission from Borke et al. [157].)
FIGURE 26.8
FIGURE 29.2 Phenotype and genotype record the hereditary nature of amelogenesis imperfecta and vitamin-D-deficiency diseases: (A) intraoral and radiographic illustration of vitamin-D-dependent rickets type II disease (OMIM 277440, Mutation of VDR gene); (B) intraoral and radiographic hypomineralization type of isolated amelogenesis imperfect; (C) recapitulation of genes implicated in amelogenesis imperfect and vitamin-D-deficiency diseases (bold print points out cases with dental analysis in human; * points out cases with tooth study in mouse model of gene inactivation).
(A)
(B)
Compared phenotype of two temporal enamel defects: (A) dental phenotype of a clinical case of nutritional vitaminD-deficiency rickets. Major enamel hypoplasia appears on central maxillar and mandibular incisors; (B) intraoral finding in MIH (hypomineralized molar incisor) showing brown coloration of hypomaturate enamel on the first permanent molar and permanent maxillary incisor.
FIGURE 29.3
Root phenotype in VDR-ablated mice. VDR-ablated (VDRe/e) and control (VDRþ/þ) mouse litters are generated by mating VDR males and females [2,3]. Depending on the calcium, phosphate, and lactose regimen, VDRe/e show either normocalcemia/normophosphatemia (N) or hypocalcemia/hypophosphatemia (H). Microradiographs illustrate dentin (double arrow), cementum (CM), and mandibular bone. (B) Biomineralization defects in VDRe/e H were normalized in VDRe/e N and normal in VDRþ/þ. Morphological study reveals overactivity of osteoclasts in VDRe/e H which results in root resorption (arrow) involving cementum (CM), dentin (D), and predentin (PD). Resorption was absent in VDRe/e N mice. Tartrate-resistant acid phosphatase-positive cells were present and numerous within dental lacunae. These phenotypes illustrate the indirect action of vitamin D on oral mineralized tissues.
FIGURE 29.4 þ/e
FIGURE 29.6 Bisphosphonate-related osteonecrosis of the mandibular ridge. A 70-year-old man with a 5-year history of multiple myeloma, complicated by hypercalcemia, who was being treated with zoledronate, presented with a 1-month history of severe jaw pain.
FIGURE 30.2 Phenotype of VDR-null mice. At 1 year of age, the VDR-null mice (foreground) are significantly smaller than their wild-type littermates and demonstrate severe alopecia.
(A)
(B)
(C)
FIGURE 30.3 Normalization of mineral ion homeostasis does not prevent skin changes in the VDR-null mice. (A) Skin section from a 70-dayold wild-type mouse; (B) from a 70-day-old VDR-null littermate with abnormal mineral ion levels demonstrating dermal cysts and dilatation of hair follicles; (C) from a 70-day-old VDR-null mouse with normal mineral ion homeostasis.
VDR-null mice develop hyperreninemia. (A) Northern blot showing marked up-regulation of renin mRNA expression in the kidney of VDR-null mice. (B) Quantitative results of the Northern blot data; ))), P < 0.001 vs. þ/þ mice. (C) Immunostaining of kidney cortex sections with renin antiserum. Arrows indicate the afferent glomerular arterioles in the juxtaglomerular region. Note the marked increase in renin staining in VDR-null kidney. (D) Plasma renin activity in wild-type and VDR-null mice. (E) Plasma Ang II concentrations in wild-type and VDR-null mice. )) P < 0.01; ))), P < 0.001 vs. þ/þ mice. þ/þ, wild-type; e/e, VDR-null; PRA, plasma renin activity; Ang II, angiotensin II. (From Li et al. (2002), with permission.)
FIGURE 40.3
Schematic representation of vitamin D function and regulation. Plain lines represent physiological functions for vitamin D (green), PTH (blue) and FGF23 (red).
FIGURE 42.1
FIGURE 42.3 Integrative physiology of vitamin D-FGF23-PTH multiorgan axis. Plain lines represent physiological functions for vitamin D (green), PTH (blue), FGF23 (red), leptin (pink), PO4 and Ca2+ (yellow) and bone remodeling (black).
Section of transiliac biopsy obtained with an 8-mm internal-diameter trephine. The biopsy contains inner and outer cortical plates and intervening cancellous bone.
FIGURE 48.1
Toluidine blue-stained section of iliac crest cancellous bone showing mineralized bone (purple/blue) and osteoid (pale blue). The calcification front can be seen as a dark blue line at the interface of the osteoid and mineralized bone.
FIGURE 48.2
FIGURE 48.3 Unstained section of iliac crest cancellous bone viewed by fluorescence microscopy. The tetracycline labels are seen as double yellow fluorescent bands.
FIGURE 48.4 Section of iliac crest stained by the von Kossa technique to show osteoid accumulation in a woman with severe privational osteomalacia.
(Top) Resorption cavity in cancellous iliac crest bone stained by toluidine blue. (Bottom) Same resorption cavity viewed under polarized light. Note the cut-off collagen lamellae at the edges of the cavity.
FIGURE 48.6
FIGURE 48.5 Section of iliac crest biopsy viewed under polarized light to show a completed bone structural unit bounded by the cement line and mineralized bone surface.
(A)
(B) H.Ca.Dm
Ps
Ct.Wi On.Dm
Ec
(A) Section of cortical bone viewed under polarized light showing the periosteal surface (Ps), endocortical surface (Ec), an osteon, and a Haversian canal (H.Ca). (B) Diagrammatic representation of cortical bone showing endocortical surface (Ec), periosteal surface (Ps), cortical width (Ct.Wi), Haversian canal diameter (H.Ca.Dm), and osteonal diameter (On.Dm). Adapted from Brockstedt et al. [71] with permission from Elsevier.
FIGURE 48.7
FIGURE 48.8 Diagrammatic representation of different strut types in strut analysis. Cancellous bone is shown in gray. Lines represent the skeletonized axis of the original bone profile. Squares represent nodes (Nd) and termini (Tm) terminus-to-terminus (Tm.Tm), node-to-loop (Nd.Lp), and node-to-terminus (Nd.Tm) strut types are illustrated. Reprinted from Croucher et al. [63] with permission from Wiley.
(A)
(B)
Morphometric analysis of trabecular bone is performed in 3D from mCT images. Here a 5 mm 3 mm 3 mm block of trabecular bone from a cadaveric distal radius (18 mm voxel size) is visualized with the 3D distances mapped onto surfaces in pseudocolor demonstrating calculations for trabecular thickness, Tb.Th (A) and trabecular separation, Tb.Sp (B).
FIGURE 50.7
(A)
(B)
FIGURE 50.8 For microstructural finite element analysis (mFEA), the mCT or HR-pQCT image data are converted to a 3D model of cubic
elements and simulated loads are applied according to chosen boundary conditions. Here a slab of bone is compressed uniaxially by 1% (A). The solution for the series of equations that describes the forceedisplacement behavior of each element results can be used to calculate the apparent mechanical properties of the whole bone, as well as visualize the distribution of stress at the microstructural level (B).
VITAMIN D THIRD EDITION
http://www.elsevierdirect.com/companion.jsp?ISBN=9780123819789
Vitamin D David Feldman, Editor-in-Chief, J. Wesley Pike and John S. Adams, Associate Editors
VITAMIN D THIRD EDITION VOLUME II Editor-in-Chief
DAVID FELDMAN Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA Associate Editors
J. WESLEY PIKE Department of Biochemistry, University of Wisconsin, Madison, WI, USA JOHN S. ADAMS UCLA-Orthopaedic Hospital Department of Orthopaedic Surgery, University of California, Los Angeles, CA, USA
AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier
Academic Press is an imprint of Elsevier 32 Jamestown Road, London NW1 7BY, UK 225 Wyman Street, Waltham, MA 02451, USA 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA First edition 1997 Second edition 2005 Third edition 2011 Copyright Ó 2011, 2005, 1997 Elsevier Inc. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+ 44) (0) 1865 843830; fax (+44) (0) 1865 853333; email:
[email protected]. Alternatively, visit the Science and Technology Books website at www.elsevierdirect.com/rights for further information Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Medicine is an ever-changing field. Standard safety precautions must be followed, but as new research and clinical experience broaden our knowledge, changes in treatment and drug therapy may become necessary or appropriate. Readers are advised to check the most current product information provided by the manufacturer of each drug to be administered to verify the recommended dose, the method and duration of administrations, and contraindications. It is the responsibility of the treating physician, relying on experience and knowledge of the patient, to determine dosages and the best treatment for each individual patient. Neither the publisher nor the authors assume any liability for any injury and/or damage to persons or property arising from this publication. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-381978-9 Two Volume Set ISBN: 978-0-12-387035-3 Volume 1 ISBN: 978-0-12-387034-6 Volume 2 For information on all Academic Press publications visit our website at www.elsevierdirect.com Typeset by TNQ Books and Journals Printed and bound in United States of America 11 12 13 14
10 9 8 7
6 5 4 3
2 1
Contents
11. Target Genes of Vitamin D: Spatio-temporal Interaction of
Preface to the 3rd Edition ix Preface to the 2nd Edition xi Preface to the 1st Edition xiii Contributors xv Introduction xxi Abbreviations xxiii Relevant Lab Values in Adults and Children xxix
12.
13.
VOLUME I
14.
I 15.
CHEMISTRY, METABOLISM, CIRCULATION 1. Historical Overview of Vitamin D 3
Chromatin, VDR, and Response Elements 211 Carsten Carlberg Epigenetic Modifications in Vitamin D Receptor-mediated Transrepression 227 Alexander Kouzmenko, Fumiaki Ohtake, Ryoji Fujiki, Shigeaki Kato Vitamin D and Wnt/b-Catenin Signaling 235 Jose´ Manuel Gonza´lez-Sancho, Marı´a Jesu´s Larriba, Alberto Mun˜oz Vitamin D Response Element-binding Protein 251 Thomas S. Lisse, Hong Chen, Mark S. Nanes, Martin Hewison, John S. Adams Vitamin D Sterol/VDR Conformational Dynamics and Nongenomic Actions 271 Mathew T. Mizwicki, Anthony W. Norman
Hector F. Deluca
2. Photobiology of Vitamin D 13
III
Michael F. Holick
3. The Activating Enzymes of Vitamin D Metabolism
(25- and 1a-Hydroxylases) 23 Glenville Jones, David E. Prosser 4. CYP24A1: Structure, Function, and Physiological Role Rene´ St. Arnaud 5. The Vitamin D Binding Protein DBP 57 Roger Bouillon 6. Industrial Aspects of Vitamin D 73 Arnold Lippert Hirsch
MINERAL AND BONE HOMEOSTASIS 16. Genetic and Epigenetic Control of the Regulatory Machinery
43
17. 18.
II
19.
MECHANISMS OF ACTION 7. The Vitamin D Receptor: Biochemical, Molecular,
20.
Biological, and Genomic Era Investigations 97 J. Wesley Pike, Mark B. Meyer, Seong Min Lee 8. Nuclear Vitamin D Receptor: Natural Ligands, Molecular StructureeFunction, and Transcriptional Control of Vital Genes 137 Mark R. Haussler, G. Kerr Whitfield, Carol A. Haussler, Jui-Cheng Hsieh, Peter W. Jurutka 9. Structural Basis for Ligand Activity in VDR 171 Natacha Rochel, Dino Moras 10. Coregulators of VDR-mediated Gene Expression 193 Diane R. Dowd, Paul N. MacDonald
21. 22. 23.
v
for Skeletal Development and Bone Formation: Contributions of Vitamin D3 301 Jane B. Lian, Gary S. Stein, Martin Montecino, Janet L. Stein, Andre J. van Wijnen Vitamin D Regulation of Osteoblast Function 321 Renny T. Franceschi, Yan Li Osteoclasts 335 F. Patrick Ross Molecular Mechanisms for Regulation of Intestinal Calcium and Phosphate Absorption by Vitamin D 349 James C. Fleet, Ryan D. Schoch The Calbindins: Calbindin-D28K and Calbindin-D9K and the Epithelial Calcium Channels TRPV5 and TRPV6 363 Sylvia Christakos, Leila J. Mady, Puneet Dhawan Mineralization 381 Eve Donnelly, Adele L. Boskey Vitamin D Regulation of Type I Collagen Expression in Bone 403 Barbara E. Kream, Alexander C. Lichtler Target Genes: Bone Proteins 411 Gerald J. Atkins, David M. Findlay, Paul H. Anderson, Howard A. Morris
vi
CONTENTS
24. Vitamin D and the Calcium-Sensing Receptor 425 Edward M. Brown 25. Effects of 1,25-Dihydroxyvitamin D3 on Voltage-Sensitive Calcium Channels in Osteoblast Differentiation and Morphology 457 William R. Thompson, Mary C. Farach-Carson
40. Vitamin D and the Renin-Angiotensin System 707 Yan Chun Li
41. Parathyroid Hormone, Parathyroid Hormone-Related Protein, 42. 43.
IV TARGETS 26. Vitamin D and the Kidney 471 27. 28. 29. 30. 31. 32.
33.
Peter Tebben, Rajiv Kumar Vitamin D and the Parathyroids 493 Justin Silver, Tally Naveh-Many Cartilage 507 Barbara D. Boyan, Maryam Doroudi, Zvi Schwartz Vitamin D and Oral Health 521 Ariane Berdal, Muriel Molla, Vianney Descroix The Role of Vitamin D and its Receptor in Skin and Hair Follicle Biology 533 Marie B. Demay Vitamin D and the Cardiovascular System 541 David G. Gardner, Songcang Chen, Denis J. Glenn, Wei Ni Vitamin D: A Neurosteroid Affecting Brain Development and Function; Implications for Neurological and Psychiatric Disorders 565 Darryl Eyles, Thomas Burne, John McGrath Contributions of Genetically Modified Mouse Models to Understanding the Physiology and Pathophysiology of the 25Hydroxyvitamin D-1-Alpha Hydroxylase Enzyme (1a(OH) ase) and the Vitamin D Receptor (VDR) 583 Geoffrey N. Hendy, Richard Kremer, David Goltzman
44. 45.
and Calcitonin 725 Elizabeth Holt, John J. Wysolmerski FGF23/Klotho New Regulators of Vitamin D Metabolism 747 Valentin David, L. Darryl Quarles The Role of the Vitamin D Receptor in Bile Acid Homeostasis 763 Daniel R. Schmidt, Steven A. Kliewer, David J. Mangelsdorf Vitamin D and Fat 769 Francisco J.A. de Paula, Clifford J. Rosen Extrarenal 1a-Hydroxylase 777 Martin Hewison, John S. Adams
VI DIAGNOSIS AND MANAGEMENT 46. Approach to the Patient with Metabolic Bone Disease 807 Michael P. Whyte
47. Detection of Vitamin D and Its Major Metabolites 823 Bruce W. Hollis
48. Bone Histomorphometry 845 Linda Skingle, Juliet Compston
49. Radiology of Rickets and Osteomalacia 861 Judith E. Adams
50. High-Resolution Imaging Techniques for Bone Quality Assessment 891 Andrew J. Burghardt, Roland Krug, Sharmila Majumdar 51. The Role of Vitamin D in Orthopedic Surgery 927 Aasis Unnanuntana, Brian J. Rebolledo, Joseph M. Lane
VII V HUMAN PHYSIOLOGY 34. Vitamin D: Role in the Calcium and Phosphorus 35. 36. 37. 38. 39.
Economies 607 Robert P. Heaney Fetus, Neonate and Infant 625 Christopher S. Kovacs Vitamin D Deficiency and Calcium Absorption during Childhood 647 Steven A. Abrams Adolescence and Acquisition of Peak Bone Mass 657 Connie Weaver, Richard Lewis, Emma Laing Vitamin D Metabolism in Pregnancy and Lactation 679 Natalie W. Thiex, Heidi J. Kalkwarf, Bonny L. Specker Vitamin D: Relevance in Reproductive Biology and Pathophysiological Implications in Reproductive Dysfunction 695 Lubna Pal, Hugh S. Taylor
NUTRITION, SUNLIGHT, GENETICS AND VITAMIN D DEFICIENCY 52. Worldwide Vitamin D Status 947 Paul Lips, Natasja van Schoor
53. Sunlight, Vitamin D and Prostate Cancer Epidemiology 965 Gary G. Schwartz
54. Nutrition and Lifestyle Effects on Vitamin D Status 979 Susan J. Whiting, Mona S. Calvo
55. Bone Loss, Vitamin D and Bariatric Surgery: Nutrition and Obesity 1009 Lenore Arab, Ian Yip 56. Genetics of the Vitamin D Endocrine System 1025 Andre´ G. Uitterlinden 57. The Pharmacology of Vitamin D 1041 Reinhold Vieth 58. How to Define Optimal Vitamin D Status 1067 Roger Bouillon
Volume I Color Plate Section
vii
CONTENTS
76. Analogs of Calcitriol 1461
VOLUME II
VIII
77.
DISORDERS
78.
59. The Hypocalcemic Disorders: Differential Diagnosis and 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73.
Therapeutic Use of Vitamin D 1091 Thomas O. Carpenter, Karl L. Insogna Vitamin D Deficiency and Nutritional Rickets in Children 1107 John M. Pettifor Vitamin D and Osteoporosis 1129 Peter R. Ebeling, John A. Eisman Relevance of Vitamin D Deficiency in Adult Fracture and Fall Prevention 1145 Heike Bischoff-Ferrari, Bess Dawson-Hughes Clinical Disorders of Phosphate Homeostasis 1155 Karen E. Hansen, Marc K. Drezner Pseudo-vitamin D Deficiency 1187 Francis H. Glorieux, Thomas Edouard, Rene´ St-Arnaud Hereditary 1,25-Dihydroxyvitamin-D-Resistant Rickets 1197 Peter J. Malloy, Dov Tiosano, David Feldman Glucocorticoids and Vitamin D 1233 Philip Sambrook Drug and Hormone Effects on Vitamin D Metabolism Barrie M. Weinstein, Sol Epstein Vitamin D and Organ Transplantation 1291 Emily M. Stein, Elizabeth Shane Vitamin D and Bone Mineral Metabolism in Hepatogastrointestinal Diseases 1299 Daniel D. Bikle Vitamin D and Renal Disease 1325 Adriana S. Dusso, Eduardo Slatopolsky Idiopathic Hypercalciuria and Nephrolithiasis 1359 Murray J. Favus, Fredric L. Coe Hypercalcemia Due to Vitamin D Toxicity 1381 Natalie E. Cusano, Susan Thys-Jacobs, John P. Bilezikian Vitamin D: Cardiovascular Effects and Vascular Calcification 1403 Dwight A. Towler
79.
80. 81.
Lieve Verlinden, Guy Eelen, Roger Bouillon, Maurits Vandewalle, Pierre De Clercq, Annemieke Verstuyf Analogs for the Treatment of Osteoporosis 1489 Noboru Kubodera, Fumiaki Takahashi Non-secosteroidal Ligands and Modulators 1497 Keith R. Stayrook, Matthew W. Carson, Yanfei L. Ma, Jeffrey A. Dodge The Bile Acid Derivatives Lithocholic Acid Acetate and Lithocholic Acid Propionate are Functionally Selective Vitamin D Receptor Ligands 1509 Makoto Makishima, Sachiko Yamada CYP24A1 Regulation in Health and Disease 1525 Martin Petkovich, Christian Helvig, Tina Epps Calcitriol and Analogs in the Treatment of Chronic Kidney Disease 1555 Ishir Bhan, Ravi Thadhani
X CANCER 82. The Epidemiology of Vitamin D and Cancer Risk 1569 Edward Giovannucci
83. Vitamin D: Cancer and Differentiation 1591 1245
84. 85. 86. 87. 88. 89. 90.
Johannes P.T.M. van Leeuwen, Marjolein van Driel, David Feldman, Alberto Mun˜oz Vitamin D Effects on Differentiation and Cell Cycle 1625 George P. Studzinski, Elzbieta Gocek, Michael Danilenko Vitamin D Actions in Mammary Gland and Breast Cancer 1657 JoEllen Welsh Vitamin D and Prostate Cancer 1675 Aruna V. Krishnan, David Feldman The Vitamin D System and Colorectal Cancer Prevention 1711 Heide S. Cross Hematological Malignancy 1731 Ryoko Okamoto, H. Phillip Koeffler Vitamin D and Skin Cancer 1751 Jean Y. Tang, Ervin H. Epstein, Jr. The Anti-tumor Effects of Vitamin D in Other Cancers 1763 Donald L. Trump, Candace S. Johnson
IX ANALOGS 74. Alterations in 1,25-Dihydroxyvitamin D3
Structure that Produce Profound Changes in in Vivo Activity 1429 Hector F. DeLuca, Lori A. Plum 75. Mechanisms for the Selective Actions of Vitamin D Analogs 1437 Alex J. Brown
XI IMMUNITY, INFLAMMATION, AND DISEASE 91. Vitamin D and Innate Immunity 1777 John H. White
92. Control of Adaptive Immunity by Vitamin D Receptor Agonists 1789 Luciano Adorini
viii
CONTENTS
93. The Role of Vitamin D in Innate Immunity: Antimicrobial 94. 95. 96. 97.
Activity, Oxidative Stress and Barrier Function 1811 Philip T. Liu Vitamin D and Diabetes 1825 Conny Gysemans, Hannelie Korf, Chantal Mathieu Vitamin D and Multiple Sclerosis 1843 Colleen E. Hayes, Faye E. Nashold, Christopher G. Mayne, Justin A. Spanier, Corwin D. Nelson Vitamin D and Inflammatory Bowel Disease 1879 Danny Bruce, Margherita T. Cantorna Psoriasis and Other Skin Diseases 1891 Jo¨rg Reichrath, Michael F. Holick
99. Vitamin D Receptor Agonists in the Treatment of Benign 100. 101. 102. 103. 104.
XII THERAPEUTIC APPLICATIONS AND NEW ADVANCES 98. The Role of Vitamin D in Type 2 Diabetes and Hypertension 1907 Anastassios G. Pittas, Bess Dawson-Hughes
105.
Prostatic Hyperplasia 1931 Annamaria Morelli, Mario Maggi, Luciano Adorini Sunlight Protection by Vitamin D Compounds 1943 Rebecca S. Mason, Katie M. Dixon, Vanessa B. Sequeira, Clare Gordon-Thomson The Role of Vitamin D in Osteoarthritis and Rheumatic Disease 1955 M. Kyla Shea, Timothy E. McAlindon Vitamin D and Cardiovascular Disease 1973 Harald Sourij, Harald Dobnig Vitamin D, Childhood Wheezing, Asthma, and Chronic Obstructive Pulmonary Disease 1999 Carlos A. Camargo Jr., Adit A. Ginde, Jonathan M. Mansbach Vitamin D and Skeletal Muscle Function 2023 Lisa Ceglia, Robert U. Simpson The VITamin D and OmegA-3 TriaL (VITAL): Rationale and Design of a Large-Scale Randomized Controlled Trial 2043 Olivia I. Okereke, JoAnn E. Manson
Index 2057 Volume II Color Plate Section
Preface to the 3rd Edition The 3rd edition of Vitamin D was written at a time of great interest, exuberant hype, and even commotion in the public and lay press about vitamin D as a potential drug to treat and/or prevent multiple important and common diseases. Recent noteworthy events impacting the vitamin D field were the launching of the VITAL trial to discover whether vitamin D supplementation can reduce the risk of severe and life-threatening disease and the Institute of Medicine (IOM) report setting new dietary reference intakes (DRIs) for calcium and vitamin D. The IOM report expressed doubt on how well current data supported the beneficial actions of vitamin D on nonskeletal sites and called for more research to prove the hypothesis. This volume marshals the currently available data on basic mechanisms, normal physiology, and effects on disease and lays out for the reader up-todate and expert information on the role of vitamin D in health and many disorders. These and other current trends in vitamin D research are extensively covered in this new edition. The editors have continued our basic plan to constantly renew and remodel this book with each successive edition. To this end, we have added a new editor, Dr. John Adams, who has broad skill and knowledge in many areas of vitamin D research at both the basic science and clinical levels. John replaces Francis Glorieux who has undertaken to edit a separate book on pediatric bone disease. We thank Francis for his years of exemplary service to this book and wish him well in his new endeavors. John adds new energy and expertise to the editorial team. The 3rd edition has 105 chapters, making the book approximately the same size as the 2nd edition. However, the editors have worked very hard to revise and update this edition with new material and the presentation of fresh and different perspectives from respected authors. Some chapters covered in the 2nd edition have not been continued in this edition because relatively little new research was added in those areas. We thank the authors who are no longer contributing to this edition for their previous efforts. They may well be asked to write in the next edition as we continue our strategy of rotating authors. All chapters have been revised and updated and new references added. In our revitalization of the material in the book we
have added 32 new chapters to cover previously uncovered areas of research. In addition, we have changed the authorship of 20 additional chapters that are now written by different authors who have been charged with revising and updating previous chapters. These extensive modifications, with major updates and expansion of the content and the addition of totally new material in half of the chapters, has resulted in a substantially reorganized, modified, and modernized book compared to the 2nd edition. Finally, the expanded internet availability of the text and the figures will make access to the material easier and more flexible. Among the areas given new emphasis are nutrition, additional diseases that may be affected by vitamin D, and newly recognized biological pathways that regulate or are regulated by vitamin D. As we appreciate the full scope of vitamin D action, it has become clearer that the vitamin D endocrine system affects most if not all tissues in the body. We have tried to keep up with these advances in the state of knowledge about vitamin D by increasing our coverage of these newly recognized areas. We have enlisted the leading investigators in each area to provide truly expert opinion about each field. We would like to thank the excellent team at Elsevier/Academic Press for their outstanding support of our efforts to produce this new edition. We especially thank Mara Conner and Megan Wickline for their indispensable contributions to make this edition possible. We also want to extend our thanks and appreciation to the many authors who contributed to this volume. Without their hard work there of course would be no new edition. We therefore wish to express our gratitude for their willingness to offer their time and knowledge to make this book a success. Finally, we hope that this book will provide for our readers the authoritative information that they seek about the significance and importance of vitamin D in health and disease and serve as the means to keep their knowledge current about the continuing growth of the field of vitamin D biology. David Feldman J. Wesley Pike John S. Adams
ix
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Preface to the 2nd Edition Those interested in the vitamin D field will not be surprised that this second edition is considerably larger than the first edition. A great deal of progress has been made since the first edition was published in 1997. However, our goal in planning this updated version remains the same. We have endeavored to provide investigators, clinicians, and students with a comprehensive, definitive, and up-to-date compendium of the diverse scientific and clinical aspects of vitamin D, each area covered by experts in the field. Our hope for the second edition is that this book will continue to serve as both a resource for current researchers, as well as a guide to assist those in related disciplines to enter the realm of vitamin D research. We hope that this book will illuminate the vitamin D field and help investigators identify areas where new research is needed as well as educate them about what is currently known. We believe that the first edition succeeded in stimulating interactions between researchers and clinicians from different disciplines and that it facilitated collaborations. As we move from basic science and physiology to the use of vitamin D and its analogs as pharmacological agents to treat various diseases, the need for crosscollaborations between researchers and clinicians from different disciplines will increase. We hope that this new volume will continue to be a valuable resource that plays a role in this advancement and stimulates and facilitates these interactions. Enormous progress in the study of vitamin D has been made in the approximately eight years since the first edition was written and we hope that this book has contributed in some way to this progress. The first edition proved to be highly valuable to its readers and the chapters have been cited frequently as authoritative reviews of the field. However, it became clear to us that the time was ripe to organize a second edition. Building on the original, we hope this second edition will
incorporate all of the progress made in the field since the first edition was published so that our objective of an up-to-date compendium containing everything you wanted to know about vitamin D will continue. The second edition is essentially a new and reinvigorated book. We have changed the symbol on the cover to reflect its updated content and the field’s continued evolution into the molecular world. This new edition now includes 104 chapters. In order to cover the growth of new information on vitamin D, we have had to publish this new edition in two volumes. In addition, the book has undergone some major remodeling. There are 33 completely new chapters and 18 other chapters have had major changes in authorship and are totally rewritten. While approximately half of the chapters have some of the same authors, all have had major updates and many have new co-authors with new perspectives. We have endeavored to attract the leading investigators in each field to author the chapter covering their area. We are especially pleased with the roster of authors who have written for the second edition. They really are the leaders in their respective fields. We wish to express our thanks to Tari Paschall, Judy Meyer, and Sarah Hajduk as well as the rest of the Elsevier/Academic Press staff for their expertise and indispensable contribution to bringing this revised edition to fruition. Most of all, we thank the authors for their contributions. We hope that our readers will find this updated volume useful and informative and that it will contribute to the burgeoning growth of the vitamin D field. David Feldman J. Wesley Pike Francis H. Glorieux
xi
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Preface to the 1st Edition Our reasons for deciding to publish an entire book devoted to vitamin D can be found in the rapid and extensive advances currently being made in this important field of research. Enormous progress in investigating many aspects of vitamin D, from basic science to clinical medicine, has been made in recent years. The ever-widening scope of vitamin D research has created new areas of inquiry so that even workers immersed in the field are not fully aware of the entire spectrum of current investigation. Our goal in planning this book was to bring the diverse scientific and clinical fields together in one definitive and up-to-date volume. It is our hope that this compendium on vitamin D will serve as both a resource for current researchers and a guide to stimulate and assist those in related disciplines to enter this field of research. In addition, we hope to provide clinicians and students with a comprehensive source of information for the varied and extensive material related to vitamin D. The explosion of information in the vitamin D sphere has led to new insights into many different areas, and in our treatment of each subject in this book we have tried to emphasize the recent advances as well as the established concepts. The classic view of vitamin D action, as a hormone limited to calcium metabolism and bone homeostasis, has undergone extensive revision and amplification in the past few years. We now know that the vitamin D receptor (VDR) is present in most tissues of the body and that vitamin D actions, in addition to the classic ones, include important effects on an extensive array of other target organs. To cover this large number of subjects, we have organized the book in the following manner: Section I, the enzymes involved in vitamin D metabolism and the activities of the various metabolites; Section II, the mechanism of action of vitamin D, including rapid, nongenomic actions and the role of the VDR in health and disease; Section III, the effects of vitamin D and its metabolites on the various elements that constitute bone and the expanded understanding of vitamin D actions in multiple target organs, both classic and nonclassic; Section IV, the role of vitamin D in the physiology and regulation of calcium and phosphate metabolism and the multiplicity of hormonal, environmental, and other factors influencing vitamin D metabolism and action; and Sections V and VI, the role of
vitamin D in the etiology and treatment of rickets, osteomalacia, and osteoporosis and the pathophysiological basis, diagnosis, and management of numerous clinical disorders involving vitamin D. The recent recognition of an expanded scope of vitamin D action and the new investigational approaches it has generated were part of the impetus for developing this volume on vitamin D. It has become clear that in addition to the classic vitamin D actions, a new spectrum of vitamin D activities that include important effects on cellular proliferation, differentiation, and the immune system has been identified. This new information has greatly expanded our understanding of the breadth of vitamin D action and has opened for investigation a large number of new avenues of research that are covered in Sections VII and VIII of this volume. Furthermore, these recently recognized nonclassic actions have led to a consideration of the potential application of vitamin D therapy to a range of diseases not previously envisioned. This therapeutic potential has spawned the search for vitamin D analogs that might have a more favorable therapeutic profile, one that is less active in causing hypercalcemia and hypercalciuria while more active in a desired application such as inducing antiproliferation, prodifferentiation, or immunosuppresion. Since 1a,25dihydroxyvitamin D [1,25(OH)2D] and its analogs are all presumed to act via a single VDR, a few years ago most of us probably would have thought that it was impossible to achieve a separation of these activities. Yet today, many analogs that exhibit different profiles of activity relative to 1,25(OH)2D have already been produced and extensively studied. The development of analogs with an improved therapeutic index has opened another large and complex area of vitamin D research. This work currently encompasses three domains: (1) the design and synthesis of vitamin D analogs exhibiting a separation of actions with less hypercalcemic and more antiproliferative or immunosuppressive activity, (2) the interesting biological question of the mechanism(s) by which these analogs achieve their differential activity, and (3) the investigation of the potential therapeutic applications of these analogs to treat various disease states. These new therapeutic applications, from psoriasis to cancer, from immunosuppression to neurodegenerative diseases, have drawn into the field an expanded
xiii
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PREFACE TO THE 1ST EDITION
population of scientists and physicians interested in vitamin D. Our goal in editing this book was to create a comprehensive resource on vitamin D that would be of use to a mix of researchers in different disciplines. To achieve this goal, we sought authors who had contributed greatly to their respective fields of vitamin D research. The book has a large number of chapters to accommodate many contributors and provide expertise in multiple areas. Introductory chapters in each section of the book are designed to furnish an overview of that area of vitamin D research, with other chapters devoted to a narrowly focused subject. Adjacent and closely related subjects are often covered in separate chapters by other authors. While this intensive style may occasionally create some redundancy, it has the advantage of allowing the reader to view the diverse perspectives
of the different authors working in overlapping fields. In this regard, we have endeavored to provide many cross-references to guide the reader to related information in different chapters. We express our thanks to Jasna Markovac (Editor-inChief), for encouraging us to develop this book and guiding us through the process; to Tari Paschall (Acquisitions Editor) and the Academic Press staff for their diligence, expertise, and patience in helping us complete this work. Most of all, we thank the authors for their contributions that have made this book possible. David Feldman Francis H. Glorieux J. Wesley Pike
Contributors Nutrition
Mona S. Calvo US Food and Drug Administration, Laurel, MD, USA (979)
John S. Adams UCLA-Orthopaedic Hospital, Los Angeles, CA, USA (251, 777)
Carlos A. Camargo Jr. Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA (1999)
Judith E. Adams Manchester Royal Infirmary, Manchester, UK and Imaging Science and Biomedical Engineering, The University, Manchester, UK (861)
Margherita Cantorna The Pennsylvania State University, University Park, PA, USA (1879)
Steven A. Abrams USDA/ARS Children’s Research Center, Houston, TX, USA (647)
Luciano Adorini (1789, 1931)
Carsten Carlberg University of Luxembourg, Luxembourg and University of Eastern Finland, Kuopio, Finland (211)
Intercept Pharmaceuticals, Perugia, Italy
Thomas O. Carpenter Yale University School of Medicine, New Haven, CT, USA (1091)
Paul H. Anderson SA Pathology, Adelaide, South Australia, Australia and University of South Australia, Adelaide, South Australia, Australia (411)
Matthew W. Carson Lilly Indianapolis, IN, USA (1497)
Lenore Arab David Geffen School of Medicine at UCLA, Los Angeles, CA, USA (1009)
Lisa Ceglia
Research
Laboratories,
Tufts University, Boston, MA, USA (2023)
Hong Chen Emory University School of Medicine, Atlanta, GA, USA (251)
Gerald J. Atkins University of Adelaide, Adelaide, South Australia, Australia (411)
Songcang Chen University of California at San Francisco, San Francisco, CA, USA (541)
Ariane Berdal Universities Paris 5, Paris 6 and Paris 7, Paris, France and Rothchild Hospital, Assistance PubliqueHoˆpitaux de Paris, Paris, France (521)
Sylvia Christakos University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ, USA (363)
Ishir Bhan Massachusetts General Hospital, Boston, MA, USA (1555)
Fredric L. Coe The University of Chicago Pritzker School of Medicine, Chicago, IL, USA (1359)
Daniel Bikle Veterans Affairs Medical Center and University of California, San Francisco, CA, USA (1299)
Juliet Compston Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK (845)
John P. Bilezikian Columbia University College of Physicians and Surgeons, New York, NY, USA (1381)
Heide S. Cross Retired, Medical University of Vienna, Austria (1711)
Heike Bischoff-Ferrari University of Zurich, Switzerland and University Hospital Zurich, Switzerland (1145)
Natalie E. Cusano Columbia University College of Physicians and Surgeons, New York, NY, USA (1381)
Adele L. Boskey Hospital for Special Surgery, affiliated with Weil College of Cornell Medical School, New York, NY, USA (381)
Bess Dawson-Hughes USA (1145, 1907)
Roger Bouillon Laboratory of Experimental Medicine and Endocrinology, K.U. Leuven, Leuven, Belgium (57, 1067, 1461)
Tufts
University,
Boston,
MA,
Pierre De Clercq Universiteit Gent, Vakgroep Organische Chemie, Gent, Belgium (1461)
voor
Hector DeLuca University of Wisconsin-Madison, WI, USA (1429)
Barbara D. Boyan Georgia Institute of Technology, Atlanta, GA, USA (507)
Michael Danilenko Ben-Gurion University of the Negev, Beer-Sheva, Israel (1625)
Alex J. Brown Washington University School of Medicine, St. Louis, MO, USA (1437)
Valentin David University of Tennessee Health Science Center, Memphis, TN, USA (747)
Edward M. Brown Brigham and Women’s Hospital, Boston, MA, USA (425)
Hector F. Deluca University Madison, WI, USA (3)
Danny Bruce The Pennsylvania State University, University Park, PA, USA (1879)
of
Wisconsin-Madison,
San
Marie B. Demay Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA (533)
Thomas Burne The Park Centre for Mental Health, Wacol, Australia and University of Queensland, St. Lucia, Australia (565)
Francisco J.A. de Paula Maine Medical Center Research Institute and Department of Internal Medicine, School of Medicine of Ribeira˜o Preto, USP, Ribeira˜o Preto, SP, Brazil (769)
Andrew J. Burghardt University Francisco, CA, USA (891)
of
California,
xv
xvi
CONTRIBUTORS
Vianney Descroix Universities Paris 5, Paris 6 and Paris 7, Paris, France and Pitie´-Salpeˆtrie`re Hospital, Assistance Publique-Hoˆpitaux de Paris, Paris France (521) Puneet Dhawan University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ, USA (363)
David G. Gardner University of California at San Francisco, San Francisco, CA, USA (541) Adit A. Ginde University of Colorado Denver School of Medicine, Aurora, CO, USA (1999)
Katie M. Dixon University of Sydney, NSW, Australia (1943)
Edward Giovannucci Harvard School of Public Health, Boston, MA, USA and Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA (1569)
Harald Dobnig Medical University of Graz, Graz, Austria (1973)
Denis J. Glenn University of California at San Francisco, San Francisco, CA, USA (541)
Jeffrey A. Dodge Lilly Research Laboratories, Indianapolis, IN, USA (1497) Eve Donnelly Hospital for Special Surgery, affiliated with Weil College of Cornell Medical School, New York, NY, USA (381) Maryam Doroudi Georgia Institute of Technology, Atlanta, GA, USA (507) Diane R. Dowd Case Western Cleveland, OH, USA (193)
Reserve
University,
Marc K. Drezner William H. Middleton Veterans Administration Medical Center, Madison, WI, USA (1155) Adriana S. Dusso Experimental Nephrology Laboratory, Lleida, Spain (1325) Peter R. Ebeling Australia (1129)
University
of
Melbourne,
Victoria,
Thomas Edouard Shriners Hospital for Children, Montreal, Quebec, Canada (1187) Guy Eelen Katholieke Belgium (1461)
Universiteit
Leuven,
Leuven,
John A. Eisman Garvan Institute of Medical Research, Sydney, Australia (1129) Tina Epps (1525)
Cytochroma Inc., Markham, Ontario, Canada
Ervin H. Epstein Jr. Children’s Hospital Oakland Research Institute, Oakland, CA, USA (1751) Sol Epstein Mount Sinai School of Medicine, New York, NY, USA (1245) Darryl Eyles The Park Centre for Mental Health, Wacol, Australia and University of Queensland, St. Lucia, Australia (565) Mary C. Farach-Carson USA (457)
Rice University, Houston, TX,
Murray J. Favus The University of Chicago Pritzker School of Medicine, Chicago, IL, USA (1359) David Feldman Stanford University School of Medicine, Stanford, CA, USA (1197, 1591, 1675) David M. Findlay University of Adelaide, Adelaide, South Australia, Australia (411) James C. Fleet USA (349)
Purdue University, West Lafayette, IN,
Renny T. Franceschi MI, USA (321) Ryoji Fujiki
University of Michigan, Ann Arbor,
University of Tokyo, Tokyo, Japan
(227)
Francis H. Glorieux Shriners Hospital Montreal, Quebec, Canada (1187)
for
Children,
Elzbieta Gocek University of Wroclaw, Wroclaw, Poland (1625) David Goltzman McGill University and Royal Victoria Hospital of the McGill University Health Centre, Montreal, Quebec, Canada (583) Jose´ Manuel Gonza´lez-Sancho Universidad Auto´noma de Madrid, Madrid, Spain (235) Clare Gordon-Thomson Australia (1943)
University
of
Sydney,
NSW,
Conny Gysemans Katholieke Universiteit Leuven, Leuven, Belgium (1825) Karen E. Hansen USA (1155)
University of Wisconsin, Madison, WI,
Carol A. Haussler University of Arizona, Phoenix, AZ, USA (137) Mark R. Haussler USA (137)
University of Arizona, Phoenix, AZ,
Colleen E. Hayes University Madison, WI, USA (1843) Robert P. Heaney (607)
of
Wisconsin-Madison,
Creighton University, Omaha, NE, USA
Christian Helvig Cytochroma Inc., Markham, Ontario, Canada (1525) Geoffrey N. Hendy McGill University and Royal Victoria Hospital of the McGill University Health Centre, Montreal, Quebec, Canada (583) Martin Hewison UCLA-Orthopaedic Hospital, Los Angeles, CA, USA (251, 777) Arnold Lippert Hirsch AGD Nutrition LLC, Lewisville, Texas, USA (73) Michael F. Holick Boston Medical Center and Boston University School of Medicine, Boston, MA, USA (13, 1891) Bruce W. Hollis Medical University of South Carolina, Charleston, SC, USA (823) Elizabeth Holt Yale University School of Medicine, New Haven, CT, USA (725) Jui-Cheng Hsieh USA (137)
University of Arizona, Phoenix, AZ,
Karl L. Insogna Yale University School of Medicine, New Haven, CT, USA (1091)
xvii
CONTRIBUTORS
Candace Johnson Roswell Park Cancer Institute, Buffalo, NY, USA (1763)
Paul Lips VU University Medical Center, Amsterdam, The Netherlands (947)
Glenville Jones Queen’s University, Kingston, Ontario, Canada (23)
Philip T. Liu University of California at Los Angeles, CA, USA (1811)
Peter W. Jurutka Arizona State University at the West Campus, Glendale, AZ, USA (137)
Thomas S. Lisse UCLA-Orthopaedic Hospital, Los Angeles, CA, USA (251)
Heidi J. Kalkwarf Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA (679)
Yanfei L. Ma Lilly Research Laboratories, Indianapolis, IN, USA (1497)
Shigeaki Kato
Paul N. MacDonald Case Western Reserve University, Cleveland, OH, USA (193)
University of Tokyo, Tokyo, Japan
(227)
Steven A. Kliewer University of Texas Southwestern Medical Center, Dallas, TX, USA (763) H. Phillip Koeffler Division of Hematology and Oncology, Cedars-Sinai Medical Center, Los Angeles, CA, USA and National University of Singapore, Singapore (1731) Hannelie Korf Katholieke Universiteit Leuven, Leuven, Belgium (1825) Alexander Kouzmenko University of Tokyo, Tokyo, Japan and Alfaisal University, Riyadh, Kingdom of Saudi Arabia (227) Christopher S. Kovacs Memorial University of Newfoundland, Health Sciences Centre, St. John’s, Newfoundland, Canada (625) Barbara E. Kream University of Connecticut Health Center, Farmington, CT, USA (403) Richard Kremer McGill University and Royal Victoria Hospital of the McGill University Health Centre, Montreal, Quebec, Canada (583) Aruna V. Krishnan Stanford University School of Medicine, Stanford, CA, USA (1675) Roland Krug University of California, San Francisco, CA, USA (891) Noboru Kubodera Chugai Pharmaceutical Co., Ltd, Tokyo, Japan, present address: International Institute of Active Vitamin D Analogs (1489) Rajiv Kumar Mayo Clinic and Foundation, Rochester, MN, USA (471) Emma M. Laing University of Georgia, Athens, GA, USA (657) Joseph M. Lane USA (927)
Hospital for Special Surgery, New York, NY,
Marı´a Jesu´s Larriba Cientificas (235)
Consejo Superior de Investigacio nes
Seong Min Lee University of Wisconsin-Madison, Madison, WI, USA (97) Richard D. Lewis University of Georgia, Athens, GA, USA (657) Yan Li University of Michigan, Ann Arbor, MI, USA (321) Yan Chun Li (707)
The University of Chicago, Chicago, IL, USA
Jane B. Lian University of Massachusetts Medical School, Worcester, MA, USA (301) Alexander C. Lichtler University of Connecticut Health Center, Farmington, CT, USA (403)
Leila Mady University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ, USA (363) Mario Maggi University of Florence, Florence, Italy (1931) Sharmila Majumdar University of California, San Francisco, CA, USA (891) Makoto Makishima Nihon University School of Medicine, Tokyo, Japan (1509) Peter J. Malloy Stanford University School of Medicine, Stanford, CA, USA (1197) David J. Mangelsdorf University of Texas Southwestern Medical Center, Dallas, TX, USA (763) Jonathan M. Mansbach Children’s Hospital Boston, Harvard Medical School, Boston, MA, USA (1999) JoAnn E. Manson Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA (2043) Rebecca S. Mason University of Sydney, NSW, Australia (1943) Chantal Mathieu Katholieke Universiteit Leuven, Leuven, Belgium (1825) Christopher G. Mayne Medical College of Wisconsin, Milwaukee, WI, USA (1843) Timothy M. McAlindon Tufts Medical Center and Tufts University School of Medicine, Boston, Massachusetts (1955) John McGrath The Park Centre for Mental Health, Wacol, Australia and University of Queensland, St. Lucia, Australia (565) Mark B. Meyer University of Wisconsin-Madison, Madison, WI, USA (97) Mathew T. Mizwicki CA, USA (271)
University of California, Riverside,
Muriel Molla Universities Paris 5, Paris 6 and Paris 7, Paris, France and Rothchild Hospital, Assistance PubliqueHoˆpitaux de Paris, Paris, France (521) Martin Montecino Chile (301)
Universidad Andres Bello, Santiago,
Dino Moras Universite´ de Strasbourg, 67404 Illkirch, France (171) Annamaria Morelli University of Florence, Florence, Italy (1931) Howard A. Morris SA Pathology, Adelaide, South Australia, Australia and University of South Australia, Adelaide, South Australia, Australia (411)
xviii
CONTRIBUTORS
Alberto Mun˜oz Consejo Superior Cientificas, Madrid, Spain (1591)
Investigacio nes
Gary G. Schwartz Wake Forest University School of Medicine, Winston-Salem, NC, USA (965)
Mark S. Nanes Emory University School of Medicine, Atlanta, GA, USA (251)
Zvi Schwartz Georgia Institute of Technology, Atlanta, GA, USA (507)
Faye E. Nashold University Madison, WI, USA (1843)
Vanessa Sequeira University of Sydney, NSW, Australia (1943)
of
de
Wisconsin-Madison,
Tally Naveh-Many Hadassah Hebrew University Medical Center, Hadassah Hospital, Jerusalem, Israel (493)
Elizabeth Shane Columbia University College of Physicians & Surgeons, New York, NY, USA (1291)
Wei Ni University of California at San Francisco, San Francisco, CA, USA (541)
M. Kyla Shea Wake Forest University School of Medicine, Winston-Salem, NC, USA (1955)
Corwin D. Nelson University Madison, WI, USA (1843)
Wisconsin-Madison,
Justin Silver Hadassah Hebrew University Medical Center, Hadassah Hospital, Jerusalem, Israel (493)
University of California, Riverside,
Robert U. Simpson University of Michigan Medical School, Ann Arbor, MI, USA (2203)
Anthony W. Norman CA, USA (271) Fumiaki Ohtake
of
University of Tokyo, Tokyo, Japan
(227)
Ryoko Okamoto Division of Hematology and Oncology, Cedars-Sinai Medical Center, Los Angeles, CA, USA (1731)
Linda Skingle Cambridge University Hospitals Foundation Trust, Cambridge, UK (845)
NHS
Eduardo Slatopolsky Washington University School of Medicine, St. Louis, MO, USA (1325)
Olivia I. Okereke Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA (2043)
Harald Sourij (1973)
Medical University of Graz, Graz, Austria
Lubna Pal Yale University School of Medicine, New Haven, CT, USA (695)
Justin A. Spanier University Madison, WI, USA (1843)
Martin Petkovich Cytochroma Inc., Markham, Ontario, Canada and Queen’s University, Kingston, Ontario, Canada (1525)
Bonny L. Specker South Dakota State University, Brookings, SD, USA (679)
of
Wisconsin-Madison,
John M. Pettifor University of the Witwatersrand and Chris Hani Baragwanath Hospital, South Africa (1107)
Rene´ St-Arnaud Shriners Hospital for Children, Montreal, Quebec, Canada and McGill University, Montreal, Quebec Canada (43, 1187)
J. Wesley Pike University of Wisconsin-Madison, Madison, WI, USA (97)
Keith R. Stayrook Indiana University School of Medicine, Indianapolis, IN, USA (1497)
Anastassios G. Pittas Tufts Medical Center, Boston, MA, USA (1907)
Emily M. Stein Columbia University College of Physicians & Surgeons, New York, NY, USA (1291)
Lori A. Plum (1429)
Gary S. Stein University of Massachusetts Medical School, Worcester, MA, USA (301)
University of Wisconsin-Madison, WI, USA Queen’s University, Kingston, Ontario,
Janet L. Stein University of Massachusetts Medical School, Worcester, MA, USA (301)
L. Darryl Quarles University of Tennessee Health Science Center, Memphis, TN, USA (747)
George P. Studzinski UMD-New Jersey Medical School, Newark, NJ, USA (1625)
Brian J. Rebolledo Weill Cornell Medical College, New York, NY, USA (927)
Fumiaki Takahashi Japan (1489)
Jo¨rg Reichrath Universita¨tsklinikum Homburg, Germany (1891)
Jean Y. Tang Stanford University School of Medicine, Stanford, CA, USA (1751)
David E. Prosser Canada (23)
Natacha Rochel (171)
des
Saarlandes,
Universite´ de Strasbourg, Illkirch, France
Chugai Pharmaceutical Co., Ltd, Tokyo,
Hugh S. Taylor Yale University School of Medicine, New Haven, CT, USA (695)
Clifford J. Rosen School of Medicine of Ribeira˜o Preto, USP, Ribeira˜o Preto, SP, Brazil (769)
Peter Tebben Mayo Clinic and Foundation, Rochester, MN, USA (471)
F. Patrick Ross Washington University School of Medicine, St. Louis, MO, USA (335)
Ravi Thadhani Massachusetts General Hospital, Boston, MA, USA (1555)
Philip Sambrook University of Sydney, Sydney, NSW, Australia (1233)
Natalie W. Thiex South Dakota State University, Brookings, SD, USA (679)
Daniel R. Schmidt University of Texas Southwestern Medical Center, Dallas, TX, USA (763)
William R. Thompson USA (457)
Ryan D. Schoch USA (349)
Susan Thys-Jacobs Columbia University College of Physicians and Surgeons, New York, NY, USA (1381)
Purdue University, West Lafayette, IN,
University of Delaware, Newark, DE,
xix
CONTRIBUTORS
Dov Tiosano Meyer Children’s Hospital, Rambam Medical Center, Haifa, Israel (1197) Dwight A. Towler Washington University in St. Louis, St. Louis, MO, USA (1403) Donald Trump Roswell Park Cancer Institute, Buffalo, NY, USA (1763) Andre´ G. Uitterlinden Erasmus Medical Center, Rotterdam, The Netherlands (1025) Aasis Unnanuntana Hospital for Special Surgery, New York, NY, USA and Siriraj Hospital, Mahidol University, Bangkok, Thailand (927) Maurits Vandewalle Universiteit Gent, Vakgroep voor Organische Chemie, Gent, Belgium (1461) Marjolein van Driel Erasmus Medical Center, Rotterdam, The Netherlands (1591) Johannes P.T.M. van Leeuwen Erasmus Medical Center, Rotterdam, The Netherlands (1591) Andre J. van Wijnen University of Massachusetts Medical School, Worcester, MA, USA (301) Natasja van Schoor VU University Medical Center, Amsterdam, The Netherlands (947) Lieve Verlinden Katholieke Universiteit Leuven, Leuven, Belgium (1461) Annemieke Verstuyf Laboratorium voor Experimentele Geneeskunde en Endocrinologie, Leuven, Belgium (1461)
Reinhold Vieth University of Toronto, Toronto, Canada and Mount Sinai Hospital, Toronto, Canada (1041) Connie M. Weaver IN, USA (657)
Purdue University, West Lafayette,
Barrie M. Weinstein Mount Sinai School of Medicine, New York, NY, USA (1245) JoEllen Welsh University at Albany, Rensselaer, NY, USA (1657) John H. White (1777)
McGill University, Montreal, Canada
G. Kerr Whitfield USA (137)
University of Arizona, Phoenix, AZ,
Susan J. Whiting University of Saskatchewan, Saskatoon, Saskatchewan, Canada (979) Michael P. Whyte Shriners Hospital for Children and Washington University School of Medicine at BarnesJewish Hospital, St Louis, MI USA (807) John J. Wysolmerski Yale University School of Medicine, New Haven, CT, USA (725) Sachiko Yamada Nihon University School of Medicine, Tokyo, Japan (1509) Ian Yip David Geffen School of Medicine at UCLA, Los Angeles, CA, USA (1009)
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Introduction
On November 30, 2010, after nearly two years of deliberation, an Institute of Medicine (IOM)-appointed committee released their findings, “2011 Report on dietary reference intakes (DRIs) for calcium and vitamin D.” Among their many recommendations the important conclusions regarding vitamin D were: (1) most of the population was not currently vitamin-D-deficient: (2) that 600 IU/day for ages 1e70 and 800 IU/day if over age 70 was adequate to protect bones; and (3) that all of the other potential benefits of vitamin D, besides bone health, did not yet have compelling evidence to support advising higher doses. They concluded that higher doses of vitamin D should not be advised on a public health basis until further research was done. It should be noted that the European counterpart to this report concluded that 800 IU was the suggested daily intake. Although the IOM report was meant to provide the populations of Canada and the United States with Recommended Dietary Allowances (RDAs) and Tolerable Upper Intake Levels (ULs) for calcium and vitamin D, the committee also identified a large number of uncertainties surrounding the DRI values that they recommended. For instance, the committee expressed a need for more research into both the skeletal and nonskeletal actions of vitamin D. Despite the thousands of publications on vitamin D, the committee was clearly disappointed by the lack of rigorous randomized trials and convincing clinically applicable knowledge on the subject of vitamin D benefits beyond the skeleton. With this in mind, how does the IOM report impact what is written by the contributors to the third edition of “Vitamin D”? It is important for the readers of this book to know that the authors of each of the 105 chapters were asked to consider revising their chapters on the basis of the IOM report. For those authors contributing chapters in the book’s Sections III (Mineral and Bone Homeostasis), V (Human Physiology), VI (Diagnosis and Management), VII (Nutrition, Sunlight, Genetics and Vitamin D Deficiency), and VIII (Disorders) this
task was of particular importance, because, as mentioned above, the IOM determined there was insufficient causeand-effect evidence to support a role for vitamin D beyond its effects on bone health. That is not to say that vitamin D does not impact other human health conditions; the IOM committee simply stated that conclusive causal evidence was lacking in these areas and existing data were insufficient to support a public health statement for nonskeletal outcomes. As the authors in Sections IV (Targets), IX (Analogs), X (Cancer), XI (Immunity, Inflammation, and Disease) and XII (Therapeutic Applications and New Advances) remind us over and over again, definitive, randomized, clinical trial data supporting a role for vitamin D in the pathophysiology and/or treatment of nonskeletal human diseases are still wanting. However, as covered in essentially every chapter in the book, data highly suggestive of benefit in a multitude of diseases are so strong that many vitamin D researchers are persuaded that vitamin D will eventually be convincingly demonstrated to be efficacious in many disease states. Furthermore, many authors express the viewpoint that avoidance of vitamin D deficiency will be shown to prevent, delay, or reduce the development of numerous diseases. What is the reason convincing clinical studies are missing from the published literature? Most of the previous NIH-sponsored trials of vitamin D have focused on bone or musculoskeletal health. Moreover, there is a lack of pharmaceutical company interest in a nonpatentable small molecule like vitamin D as a therapeutic. Pharmaceutical companies are at work developing vitamin D analogs, but most of this work has not progressed beyond preclinical studies. Hopefully, much of the lack of interest in the use of vitamin D itself as a preventive or therapeutic agent for extraskeletal chronic diseases, including cardiovascular disease, cancer, diabetes, hypertension, cognitive decline, depression, lung disorders, infections, and autoimmune diseases, will be allayed by the recently initiated, NIH-funded, randomized, placebo-controlled VITamin
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xxii
INTRODUCTION
D-OmegA-3 TriaL (VITAL); VITAL is reviewed by its principal investigator, Dr. JoAnn Manson, in Chapter 105 of this text. The central aim of VITAL is to determine whether the administration of 2000 IU daily with or without 1 g of marine omega-3 fatty acids (in a 2 2 factorial design) reduces the risk of developing heart disease, stroke, or cancer in those without a prior history of these illnesses. It is of note that all study participants will be allowed to take up to 800 IU of personal vitamin D supplements (a dose, when added to dietary sources, exceeds the IOM recommended daily intake). If shown to be efficacious alone or in combination with omega-3 fatty acids in preventing the leading causes of death of American men and women, then vitamin D supplementation at a daily dose higher than the IOM guidelines will be justified. However, even if a 2000 IU dose of vitamin D3 daily reduces the risk of one or more of these nonskeletal diseases, other controversies raised by the IOM report will no doubt persist or surface. For example, the IOM report claimed that most of the US population is not vitamin-D-deficient. This obviously raises the discussion of where the cut-points for deficiency should be placed. The IOM has chosen 20 ng/ml (50 nmol/L) as the cut-off, a concentration they felt was sufficient to maintain bone health. Some would argue this is not
high enough even for bone health, let alone the other potential diseases that vitamin D may benefit. There will be much continued discussion of this report in the literature and no doubt there will be spirited debate about some of its findings. It is not our intent to carry out a pro and con discussion of the report but to emphasize several points. Importantly, public health policy must be conservative and risk averse and the IOM concluded that it should await more convincing data before recommending higher vitamin D intakes. The IOM was also concerned that, on a public health level, advising millions of people to take higher doses of vitamin D for extended periods of time could raise safety issues not observed in much smaller and shorter studies. These are real concerns. Finally, the IOM called for continued research efforts to develop compelling data to demonstrate the benefits of vitamin D claimed by many researchers. Although there is disagreement about the potential risks of not instituting vigorous vitamin D supplementation now, the editors and authors agree that more and better research would be welcome. It is our hope that the compilation of evidence about vitamin D action in normal and disease states contained in this volume will help to clarify the state of the science and be of use in elucidating the role of vitamin D in health and disease.
Abbreviations
AA AC ACE ACF ACTH ADH ADHR ADP AHO AI AIDS Aj.AR ALP ANG II ANP APC APD APL AR ARC 5-ASA ATP ATRA AUC Bmax BARE bFGF BFU BGP BLM BMC BMD BMI BMP BMU bp BPH
BSA BUA [Ca2+]i
arachadonic acid adenyl cyclase angiotensin converting enzyme activation frequency adrenocorticotropin antidiuretic hormone (vasopressin) autosomal dominant hypophosphatemic rickets adenosine diphosphate Albright’s hereditary osteodystrophy adequate intake acquired immunodeficiency syndrome adjusted apposition rate alkaline phosphatase angiotensin II atrial natriuretic peptide antigen presenting cell aminohydroxypropylidene bisphosphonate atrichia with papular lesions androgen receptor activator recruited cofactor 5-aminosalicylic acid adenosine triphosphate all-trans-retinoic acid area under the curve maximum number of binding sites bile acid response element basic fibroblast growth factor burst-forming unit bone Gla protein (osteocalcin) basal lateral membrane bone mineral content bone mineral density body mass index bone morphogenetic protein basic multicellular unit base pairs benign prostatic hyperplasia
CaBP CAD CaM cAMP CaSR or CaR CAT CBG CBP CC CD CDCA CDK or Cdk cDNA CDP Cdx-2 CFU cGMP CGRP CHF CK-II CLIA cM Cm. Ln. CNS CPBA cpm CRE CREB CRF CsA CSF CT CTR
xxiii
bovine serum albumin bone ultrasound attentuation internal calcium ion molar concentration calcium-binding protein coronary artery disease calmodulin cyclic AMP calcium-sensing receptor chloramphenicol acetyltransferase corticosteroid-binding globulin competitive protein-binding assay chief complaint Crohn’s disease chenodeoxycholic acid cyclin-dependent kinase complementary DNA collagenase-digestible protein caudal-related homeodomain protein colony-forming unit cyclic GMP calcitonin gene-related peptide congestive heart failure casein kinase-II competitive chemiluminescence immunoassay centimorgans cement line central nervous system competitive protein-binding assays counts per minute cAMP response element cAMP response element binding protein chronic renal failure cyclosporin A colony-stimulating factor calcitonin or computerized tomography calcitonin receptor
xxiv CTX CVC CYP CYP24 DAG DBD DBP DBP DC DCA DCT DEXA or DXA 7-DHC DHEA DHT DIC DMSO DR DRIP DSP DSS E1 E2 EAE EBT EBV EC EC50 or ED50 ECaC ECF EDTA EGF ELISA EMSA EP1 ER ERE ERK Et
ABBREVIATIONS
cerebrotendinous xanthomatosis calcifying vascular cells cytochrome P450 cytochrome P450, 24-hydroxylase diacylglycerol DNA-binding domain diastolic blood pressure vitamin-D-binding protein dendritic cell deoxycholic acid distal convoluted tubule dual energy X-ray absorptiometry 7-dehydrocholesterol dehydroepiandrosterone dihydrotachysterol or dihydrotestosterone disseminated intravascular coagulation dimethyl sulfoxide direct repeat vitamin D receptor interacting protein dental sialoprotein dextran sodium sulfate estrone estradiol experimental autoimmune encephalitis electron-beam computed technology Epstein-Barr virus endothelial cells effective concentration (dose) to cause 50% effect epithelium calcium channel extracellular fluid ethylenediaminetetraacetic acid epidermal growth factor enzyme-linked immunosorbent assay electrophoretic mobility shift assay PG receptor-1 estrogen receptor or endoplasmic reticulum estrogen response element extracellular signal-regulated kinase endothelin
FACS FAD FCS FDA FFA FIT FMTC FP FRAP FS FSK FXR g g G0, G1, G2 GAG GC-MS G-CSF GDNF GFP GFR GH GHRH GIO GM-CSF GnRH GR GRE GRTH GWAS HAT HDAC HEK HHRH HIV HNF HPI HPLC HPV
fluorescence-activated cell sorting or sorter flavin adenine dinucleotide fetal calf serum US Food and Drug Administration free fatty acid Fracture Intervention Trial familial medullary thyroid carcinoma formation period fluorescence recovery after photobleaching Fanconi syndrome forskolin farnesoid X receptor gram acceleration due to gravity gap phases of the cell cycle glycosaminoglycan gas chromatography-mass spectrometry granulocyte colony-stimulating factor glial-cell-derived neurotrophic factor green fluorescent protein glomerular filtration rate growth hormone growth-hormone-releasing hormone glucocorticoid-induced osteoporosis granulocyte-macrophage colonystimulating factor gonadotropin-releasing hormone glucocorticoid receptor glucocorticoid response element generalized resistance to thyroid hormone genome-wide association study histone acetyltransferase histone deacetylase human embryonic kidney hereditary hypophosphatemic rickets with hypercalciuria human immunodeficiency virus hepatocyte nuclear factor history of present illness high-performance liquid chromatography human papilloma virus
ABBREVIATIONS
h HR HRE HSA Hsp HSV HVDRR HVO IBD IBMX IC50 ICA ICMA IDBP IDDM IDM IEL IFN Ig IGFBP IGF-I, -II IGF-IR IL i.m. IMCal iNKT i.p. IP3 IRMA IU IUPAC i.v. JG JNK Kd Km kb kbp kDa KO LBD LCA LDL
hour hairless hormone response element human serum albumin heat-shock protein herpes simplex virus hereditary vitamin-D-resistant rickets hypovitaminosis D osteopathy inflammatory bowel disease isobutylmethylxanthine concentration to inhibit 50% effect intestinal calcium absorption immunochemiluminometric assay intracellular vitamin-D-binding protein insulin-dependent diabetes mellitus infants of diabetic mothers intraepithelial cells interferon immunoglobulin IGF-binding protein insulin-like growth factor type I, II IGF-I receptor interleukin (e.g., IL-1, IL-1b, etc.) intramuscular intestinal membrane calciumbinding complex invariant NKT intraperitoneal inositol trisphosphate immunoradiometric assay international units International Union of Pure and Applied Chemists intravenous juxtaglomerular c-Jun NH2-terminal kinase dissociation constant Michaelis constant kilobases kilobase pairs kilodaltons knockout ligand-binding domain lithocholic acid low-density lipoprotein
Li. Ce. LIF LNH LOD LPS LT LXR M M MAPK Mab MAR MAR MARRS MCR M-CSF MEN2 MGP MHC min MIU MLR Mlt MR MRI mRNA MS MT MTC NADH NADPH NAF NBT NcAMP NCP NFkB NGF NHANES III NHL NIDDM NIH NK cell
xxv lining cell leukemia inhibitory factor late neonatal hypocalcemia logarithm of the odds lipopolysaccharide leukotriene liver X receptor mitosis phase of cell cycle molar mitogen-activated protein kinase monoclonal antibody matrix attachment region mineral apposition rate membrane-associated rapid response steroid metabolic clearance rate macrophage colony-stimulating factor multiple endocrine neoplasia type 2 matrix Gla protein major histocompatibility complex minute million international units mixed lymphocyte reaction mineralization lag time mineralocorticoid receptor magnetic resonance imaging messenger ribonucleic acid multiple sclerosis metric ton medullary thyroid carcinoma nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate nuclear accessory factor nitroblue tetrazolium nephrogenous cAMP noncollagen protein nuclear factor kappa B nerve growth factor National Health and Nutrition Examination Survey III Non-Hodgkin’s lymphoma non-insulin-dependent diabetes mellitus National Institutes of Health natural killer cell
xxvi NLS NMR NOD NPT NR Ob Oc OCIF OCT ODF 1a-(OH)D3 25(OH)D3 1,25(OH)2D3 24,25(OH)2D3 OHO Omt OPG OPN OSM OVX Pi PA2 PAD PAM PBL PBMC PBS PC PCNA PCR PCT PDDR PDGF PEIT PHEX
PG PHA PHP PIC PKA PKC
ABBREVIATIONS
nuclear localization signal nuclear magnetic resonance nod-like sodium/phosphate cotransporter nuclear receptor osteoblast osteocalcin or osteoclast osteoclastogenesis inhibitory factor (same as OPG) 22-oxacalcitriol osteoclast differentiation factor (same as RANKL) 1a-hydroxyvitamin D3 25-hydroxyvitamin D3 1a,25-dihydroxyvitamin D3 24,25-dihydroxyvitamin D3 oncogenic hypophosphatemic osteomalacia osteoid maturation time osteoprotegerin osteopontin oncostatin M ovariectomy inorganic phosphate phospholipase A2 peripheral arterial vascular disease pulmonary alveolar macrophage peripheral blood lymphocyte peripheral blood mononuclear cells phosphate-buffered saline phophatidyl choline proliferating cell nuclear antigen polymerase chain reaction proximal convoluted tubule pseudovitamin D deficiency rickets platelet-derived growth factor percutaneous ethanol injection therapy phosphate regulating gene with homologies to endopeptidases on the X chromosome prostaglandin phytohemagglutinin pseudohypoparathyroidism preinitiation complex protein kinase A protein kinase C
PKI PLA2 PLC PMA PMCA PMH p.o. poly(A) PPAR PR PRA PRL PRR PSA PSI PT PTH PTHrP PTX PUVA QCT QSAR 9-cis-RA RA RA Rag RANK RANKL RAP RAR RARE RAS RBP RCI RDA RFLP RIA RID RNase ROCs ROS RPA
protein kinase inhibitor phospholipase A2 phospholipase C phorbol 12-myristate 13-acetate plasma membrane calcium pump past medical history oral polyadenosine peroxisome proliferator-activated receptor progesterone receptor plasma renin activity prolactin pattern recognition receptors prostate-specific antigen psoriasis severity index parathyroid parathyroid hormone parathyroid hormone-related peptide parathyroidectomy psoralen-ultraviolet A quantitative computerized tomography quantitative structureeactivity relationship 9-cis-retinoic acid retinoic acid rheumatoid arthritis recombination activating gene receptor activator NF-kB receptor activator NF-kB ligand receptor-associated protein retinoic acid receptor retinoic acid response element renineangiotensin system retinol-binding protein relative competitive index recommended dietary allowance restriction fragment length polymorphism radioimmunoassay receptor interacting domain ribonuclease receptor-operated calcium channels reactive oxygen species ribonuclease protection assay
ABBREVIATIONS
RRA RT-PCR RXR RXRE SBP SD SDS SE SEM SH SHBG SLE SNP SNPs SOS Sp1 SPF SRC-1 SSCP SV40 SXA t1/2 T3 T4 TBG TBP TC TF TFIIB TG TGF TIO TK TLR TmP or TmPi TNBS TNF TPA
radioreceptor assay reverse transcriptase-polymerase chain reaction retinoid X receptor retinoid X receptor response element systolic blood pressure standard deviation sodium dodecyl sulfate standard error standard error of the mean social history sex-hormone-binding globulin systematic lupus erythematosus single nucleotide polymorphism single nucleotide polymorphisms speed of sound selective promoter factor 1 sun protection factor steroid receptor coactivator-1 single-strand conformational polymorphism simian virus 40 single energy X-ray absorptiometry half-time triiodothyronine thyroxine thyroid-binding globulin TATA-binding protein tumoral calcinosis tubular fluid general transcription factor IIB transgenic transforming growth factor tumor-induced osteomalacia thymidine kinase toll-like receptor tubular absorptive maximum for phosphorus trinitrobenzene sulfonic acid tumor necrosis factor 12-O-tetradecanoylphorbol13-acetate
TPN TPTX TR TRAP TRAP TRP TRE TRE TRH Trk TSH TSS UF US USDA UTR UV VDDR-I VDDR-II VDR VDRE VDRL VEGF VERT VICCs VSMC VSSCs WHI WRE WSTF WT XLH XRD ZEB
xxvii total parenteral nutrition thyroparathyroidectomized thyroid hormone receptor tartrate-resistant acid phosphatase thyroid hormone receptor associated proteins transient receptor potential thyroid hormone response element TPA response element thyrotropin-releasing hormone tyrosine kinase thyrotropin transcription start site ultrafiltrable fluid ultrasonography US Department of Agriculture untranslated region ultraviolet vitamin-D-dependent rickets type I (see PDDR) vitamin-D-dependent rickets type II (see HVDRR) vitamin D receptor vitamin D response element vitamin D receptor ligand vascular endothelial growth factor Vertebral Efficacy with Risedronate Therapy studies voltage-insensitive calcium channels vascular smooth muscle cell voltage-senstive calcium channels Women’s Health Initiative Wilms’ tumor gene, WT1, responsive element Williams syndrome transcription factor wild-type X-linked hypophosphatemic rickets X-ray diffraction zinc finger, E box-binding transcription factor
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Relevant Lab Values in Adults and Children
CRITERIA FOR VITAMIN D DEFICIENCY: 25(OH)D SERUM LEVELS Adult IOM recommendations
Deficient Normal Excessive
Conventional units
SI units
<20 ng/ml 20 ng/ml >50 ng ml
<50 nmol/L 50 nmol/L >125 nmol/L
Frequently used vitamin D cut-points by many laboratories
Deficient Insufficient Sufficient
Conventional units
SI units
< 20 ng/ml 20 to 29.9 ng/ml >30 ng/ml
<50 nmol/L 50e74.9 nmol/L >75 nmol/L
Pediatric (The IOM and the Pediatric Endocrine Society have agreed on these cut-points.)
Deficient Normal
Conventional units
SI units
<20 ng/ml 20 ng/ml
<50 nmol/L 50 nmol/L
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xxx
RELEVANT LAB VALUES IN ADULTS AND CHILDREN
APPROXIMATE NORMAL RANGES FOR SERUM VALUES IN ADULTSa Measure
SI units
Conventional units
Conversion factorb
Ionized calcium Total calcium Phosphorous, inorganic 1,25(OH)2D
1.12e1.32 mmol/L 2.17e2.52 mmol/L 0.77e1.49 mol/L 60e108 pmol/L
4.5e5.3 mg/dl 8.7e10.1 mg/dl 2.4e4.6 mg/dl 25e45 pg/ml
0.2495 0.2495 0.3229 2.40
APPROXIMATE NORMAL RANGES FOR SERUM VALUES IN CHILDRENa Measure
SI units
Conventional units
Conversion factorb
Ionized calcium Total calcium Phosphorous, inorganic 1,25(OH)2D
1.19e1.29 mmol/L 2.25e2.63 mmol/L 1.23e1.62 mol/L 65e134 pmol/L
4.8e5.2 mg/dl 9.0e10.5 mg/dl 3.8e5.0 mg/dl 27e56 pg/ml
0.2495 0.2495 0.3229 2.40
USEFUL EQUIVALENCIES OF DIFFERENT UNITS Vitamin D Calcium Phosphorus
a
1 mg ¼ 40 IU 1 mmol ¼ 40 mg 1 mmol ¼ 30 mg
Normal ranges differ in various laboratories and these values are provided only as a general guide.
b
Conversion factor X conventional units ¼ SI units.
S E C T I O N
V I I I
DISORDERS
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C H A P T E R
59 The Hypocalcemic Disorders: Differential Diagnosis and Therapeutic Use of Vitamin D Thomas O. Carpenter, Karl L. Insogna Yale University School of Medicine, New Haven, CT, USA
PHYSIOLOGY Hypocalcemia and Its Manifestations Hypocalcemia refers to an abnormally low concentration of ionized calcium in extracellular fluid, almost invariably sampled from the bloodstream. Manifestations of hypocalcemia are related to increased neuromuscular irritability [1]. Tetany is the classic sign of hypocalcemia, yet it is variable in presentation. Paresthesias often occur first around the mouth or in the fingertips and may progress to overt spasm of the muscles of the face and extremities, the latter typified by carpopedal spasm. More subtle presentations have included complaints of writer’s cramp or generalized stiffness. Children with tetanic laryngospasm due to hypocalcemia have been mistakenly diagnosed with croup [2]. Infants are more likely than adults to present with jitteriness or twitching, which can progress to overt toniceclonic seizures. Lethargy and cyanosis have also been described in this age group. The term latent tetany refers to signs elicitable with provocative stimuli such as ischemia (Trousseau test) or percussion (e.g., of the facial nerve to elicit Chvostek’s sign). Neither the degree of hypocalcemia nor the rapidity with which it develops necessarily correlate with clinical manifestations. Indeed many individuals with chronic, mild hypocalcemia may be entirely asymptomatic as can be seen in congenital hypoparathyroidism. Hypomagnesemia or hyperkalemia may present with findings similar to those caused by hypocalcemia, which can be exacerbated in the setting of hypocalcemia. Conversely, hypermagnesemia or hypokalemia can mask symptoms in a hypocalcemic individual. Abnormalities of cardiac repolarization can occur with
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10059-9
hypocalcemia resulting in a prolonged Q-T interval on the electrocardiogram (EKG). The Q-T interval corrected for heart rate (Q-TC, which equals Q-T/(R-R interval)1/2), is normally less than 0.40 0.04 s. This abnormality is not always present during hypocalcemia, and it may also be seen in hypokalemia. Cardiac failure may rarely occur in the setting of hypocalcemia [3]. Papilledema has also been reported [4]. Chronic hypocalcemia caused by deficient calcium intake during periods of significant skeletal growth may result in rickets and osteomalacia (see Chapter 60). Severe osteoporosis and dental abnormalities have also been reported in long-standing untreated hypoparathyroidism [5,6]. A mineralization defect, distinct from rickets, has been described in hypoparathyroidism; however, these skeletal consequences appear to be more prevalent in normal individuals living in conditions of endemic calcium deficiency, where secondary hyperparathyroidism develops. Basal ganglia calcifications are typical findings in longstanding hypoparathyroidism as well [7]. Abnormalities in the integument including dry skin, coarse hair, and a form of psoriasis that responds to normalization of the serum calcium concentration [8] have all been described in states of long-standing hypocalcemia. Regulatory mechanisms maintain the concentration of ionized calcium within a remarkably narrow range of 4.48 to 5.28 mg/dl in whole blood [9]. The ionized fraction of total serum calcium is generally estimated to be approximately 50%, with the remainder of the total serum calcium being bound to serum proteins, most notably albumin, and to a lesser extent complexed with anions, such as citrate or sulfate. Only the ionized fraction of total serum calcium is physiologically important, and it is this component that is regulated on a minute-to-minute basis.
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Copyright Ó 2008 Elsevier Inc. All rights reserved.
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59. THE HYPOCALCEMIC DISORDERS: DIFFERENTIAL DIAGNOSIS AND THERAPEUTIC USE OF VITAMIN D
Although it is possible to measure ionized calcium in large clinical laboratories, the specimen must be obtained anaerobically and analyzed promptly. Therefore, total serum calcium is often used as an indirect assessment of the ionized calcium fraction. A decrease in serum protein concentrations (particularly albumin) often results in reduced total serum calcium concentrations, but does not affect the ionized calcium concentration. Patients in whom this occurs are asymptomatic, displaying none of the signs or symptoms of hypocalcemia. These findings are often present in patients with nephrotic syndrome, chronic illness, malnutrition, cirrhosis, and volume overexpansion. Occasionally ionized calcium levels may transiently fall when protein losses are substantial (e.g., in severe nephrotic syndrome and protein-losing enteropathies). This phenomenon is presumably due to massive losses of both ionized and protein-bound calcium. A number of clinical guidelines have been suggested that correct for the effect of a reduced serum albumin on total serum calcium concentration. One commonly cited rule of thumb is to add 0.8 mg/dl to the total serum calcium for every 1 g/dl decline in serum albumin below 4.0 g/dl. However, these estimates have been shown to be somewhat inaccurate under many circumstances, and it is preferable to directly determine the ionized calcium concentration in the setting where the total serum calcium measure is thought to not accurately reflect the concentration of ionized calcium [10].
Role of Parathyroid Hormone in the Acute Defense of Ionized Serum Calcium Concentration Parathyroid hormone (PTH) is secreted by the parathyroid glands in response to a fall in ionized serum calcium concentration [9]. The relationship between decrements in ionized calcium within the physiological range and increments in PTH secretion is quite steep, permitting rapid and substantial changes in PTH secretion in response to minor fluctuations in ionized calcium [9]. This response is mediated by the seven transmembrane-domain, G-protein-coupled calcium sensor, which is expressed in the parathyroid glands as well as in a variety of other tissues [11]. A rise in ionized calcium suppresses PTH secretion by activating this receptor [11]. Parathyroid hormone acts to regulate ionized calcium through its effects in three principal target tissues, bone, kidney, and intestine. The cellular actions of PTH are mediated by the PTH receptor, also a seven transmembrane-domain, G-protein-coupled receptor [12]. Downstream signaling from the PTH receptor involves activation of both protein kinase A- and protein kinase C-dependent pathways [13e15]. Parathyroid hormone acts to increase bone resorption, liberating calcium from the mineralized matrix of
bone and thereby increasing the ionized calcium concentration of the extracellular fluid [9]. Details of the cellular mechanism by which this occurs are incompletely understood. The principal target cell for PTH in bone appears to be the osteoblast or osteoblast-like stromal cell, rather than the resorbing cell itself, the osteoclast [16,17]. In response to PTH, osteoblasts or osteoblastlike stromal cells release locally active cytokines that appear to increase the number and activity of osteoclasts [18]. As the resorptive response to PTH is quite rapid, the early effects of the hormone are likely mediated by activation of existing osteoclasts. A miscible pool of incompletely mineralized calcium at the endosteal surface of bone may be the most readily accessible source of calcium liberated in response to this action. It has been speculated that osteocytes may mediate release of calcium from this pool [19]. These effects are evident in 6e12 hours [20]. The renal effects of PTH to defend serum calcium occur within minutes. PTH increases calcium reabsorption in the distal tubule. This effect is greatest in the distal convoluted tubule, where a sodium/calcium exchanger is regulated by PTH [21]. More recently the TRPV5 calcium channel, another mediator of calcium reabsorption in the distal tubule, has been shown to be regulated by PTH in different ways. First, PTH increases the calcium inward flux of TRPV5, a PKA-mediated function [22]. Second, PTH decreases endocytosis of TRPV5, a PKC-mediated function [23]. PTH also serves to increase expression of the TRPV5 [24]. Channel function of TRPV5 is also enhanced by the klotho protein, which serves to preserve its integration in the membrane due to exposure of glycosaminoglycan binding sites [25]. In the proximal renal tubule, PTH acts via a cAMPdependent mechanism to decrease phosphate reabsorption, resulting in increased phosphaturia. PTH effects this change by prompting the removal of sodium/phosphate cotransporters from the renal tubular apical membrane [26]. These two effects both serve to acutely increase serum calcium; one by causing less calcium to be excreted by the kidney, the other by lowering circulating concentrations of phosphate which favors an increase in ionized calcium. The third site of action of PTH in the defense of serum calcium is the intestine. This is an indirect effect, described in detail below, and is a consequence of the ability of PTH to stimulate the renal production of 1,25-dihydroxyvitamin D (1,25(OH)2D).
Vitamin D in the Long-term Maintenance of Eucalcemia Long-term eucalcemia is maintained, in large part, via the vitamin D endocrine system. This system operates in the classic manner of a steroid hormone, resulting
VIII. DISORDERS
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PHYSIOLOGY
in de novo protein synthesis directed by vitamin-Dresponsive genes [27] as discussed in detail in Section II of this volume. As noted above, acute changes in serum ionized calcium levels are sensed by G-proteincoupled calcium-sensing receptors located within the parathyroid cell membrane [11]; see also Chapter 24. PTH acts rapidly to correct a fall in serum calcium, and a sustained increase in PTH also stimulates production of 1,25(OH)2D, which enhances intestinal calcium absorption. PTH mediates this change by promoting the increased expression of CYP27B1, which encodes the catalytic component of the renal 25-hydroxyvitamin D (25(OH)D) 1-hydroxylase enzyme complex, located in the inner mitochondrial membrane of renal tubular cells. A number of physiological studies have demonstrated increased production of 1,25(OH)2D in animals that were administered PTH [28,29] and decreased production following parathyroidectomy [30]. The mechanism is discussed in Chapter 3. PTH also acutely regulates the 1a-hydroxylase enzyme complex by altering the phosphorylation state of the associated ferredoxin molecule, which serves to donate electrons to the catalytic complex [31]. A low extracellular calcium concentration can directly stimulate 1a-hydroxylase activity in the absence of PTH as described in parathyroidectomized rats [32,33] and in hypoparathyroid humans [5]. Increased synthesis of 1,25(OH)2D results in greater circulating levels of the metabolite, which gain access to specific vitamin D receptors (VDR). The hormoneereceptor complex then binds to vitamin D response elements (VDRE) in the regulatory regions of target genes (Chapters 7 and 8). Long-term control of calcium homeostasis has been thought to be classically mediated by 1,25(OH)2D induction of the calcium channel TRPV6 and the intestinal 9 kDa calcium binding protein, calbindin-D9k (Chapter 20), which is thought to play a role in vitamin-D-mediated increases in calcium absorption in the jejunum and duodenum [34] (see also Chapters 19 and 34). These proteins, however, may not be absolutely necessary for calcium absorption, since mice in which both these proteins have been deleted still demonstrate substantial active intestinal calcium transport [35,36]. Rapid, nongenomic actions of 1,25(OH)2D mediating calcium transport across intestinal mucosa have also been described [37]. During vitamin D deprivation, the initial decline in ionized serum calcium results in secondary hyperparathyroidism, which maximizes 1,25(OH)2D production and initially allows for maintenance of eucalcemia. Eventually, this compensatory mechanism fails, and intestinal calcium absorption is sufficiently compromised such that frank hypocalcemia develops. This may be compounded by an induced resistance to PTH
seen in severe hypocalcemic or vitamin-D-deficient states [1]. In children with hereditary resistance to vitamin D (HVDRR; see Chapter 65) due to loss-of-function mutations in the vitamin D receptor (VDR), the compensatory changes described above are disrupted because of nonfunctional VDRs that result in the inability of 1,25 (OH)2D to signal to the nucleus [38]. Untreated patients with HVDRR can have severe hypocalcemia leading to convulsions, coma, and death, as this homeostatic system is effectively absent. The vitamin D system has further complexities that are currently not well understood. For example, some children with vitamin D deficiency (defined by low 25(OH)D levels) may manifest hypocalcemia even when circulating 1,25(OH)2D levels are actually elevated. Possible explanations for this phenomenon include decrements in expression of calbindin during hypocalcemia, or a requirement for other circulating vitamin D metabolites which have yet to be identified. This situation is discussed further in Chapter 24. Whether changes in the concentration of VDR levels play a role in this “resistant” state is not clear, as receptor levels have been reported to increase, decrease, or not change with manipulation of the ambient calcium concentration [39e41] (see Chapter 7).
Biochemical Changes Induced by Hypocalcemia As noted above, the most rapid response to hypocalcemia is secretion of PTH. In addition to increasing serum calcium levels, PTH stimulates renal phosphate (Pi) excretion. The fall in serum phosphate may, however, be compensated by sufficient mobilization of phosphate (as well as calcium) from bone, so that circulating phosphate remains largely unchanged. The principle of mass action is thought to maintain the stability of the Ca X Pi ion product in the blood. As a consequence, local concentrations of the two major mineral components of hydroxyapatite (Ca and Pi) are able to influence the rate of movement in and out of the mineral phase of bone: ½Ca þ ½Pi 4 ½HA Thus, a fall in ionized Ca would favor an increase in serum phosphate concentration. In the aggregate, however, sustained hypocalcemia usually results in a biochemical picture of secondary hyperparathyroidism, elevated 1,25(OH)2D levels, and variable changes in serum phosphate. If hypocalcemia develops in the setting of diminished or absent PTH function, serum phosphate is usually elevated, owing to increased renal phosphate reabsorption. In this instance, treatment with 1,25(OH)2D would also increase serum phosphate,
VIII. DISORDERS
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59. THE HYPOCALCEMIC DISORDERS: DIFFERENTIAL DIAGNOSIS AND THERAPEUTIC USE OF VITAMIN D
MATERNAL D DEFICIENCY UV Exposure
25-OHD DIET
1,25-(OH)2D 1,25(OH)2 D
Ca ABSORPTION LOW Ca INTAKE
[Ca] i
peptide (PTHrP) receptor or postreceptor defects, or (3) in the setting of normal or increased PTH activity and normal PTH receptor function. Thus, although many of the etiologies relate to abnormalities in PTH, they are very relevant to this book because vitamin D metabolism is always involved and vitamin D is the cornerstone of therapy. Chapter 41 on PTH deals with some of these issues as well. Hypocalcemia due to Abnormalities of PTH Availability
PTH [PO4 ]
BONE MINERALIZATION
Mechanisms of the development of osteomalacia and rickets. Deficiency of vitamin D intake and/or limited ultraviolet light exposure lead to limited vitamin D stores as reflected by a decreased circulating 25(OH)D level. Reduced availability of this substrate is presumed to limit 1,25(OH)2D production, resulting in impaired intestinal calcium absorption. Calcium availability for skeletal mineralization is subsequently compromised, and secondary hyperparathyroidism, with concomitant hypophosphatemia, occurs. Restricted dietary calcium intake can also result in a similar pathophysiology. Increased turnover of vitamin D in the calcium-deficient state may result in a greater risk of vitamin D insufficiency. The paradox of elevated levels of 1,25(OH)2D in these disorders is well recognized.
FIGURE 59.1
since this metabolite enhances intestinal phosphate absorption. The effect of hypocalcemia on circulating vitamin D metabolites is complex. An increase in the biosynthesis of 1,25(OH)2D occurs, as reviewed above. This is largely secondary to the induced increase in circulating PTH but can be a direct consequence of the fall in calcium ion concentration. It has been determined that calcium deprivation results in a general increase in turnover of the parent vitamin D metabolite, 25(OH)D, such that vitamin D stores are depleted at a more rapid rate than normal [42]. The clinical implication of this finding is that susceptibility to vitamin D deficiency may be greater in the setting of concomitant calcium deprivation (see Fig. 59.1).
DIFFERENTIAL DIAGNOSIS OF HYPOCALCEMIA Classification A rapid increase in PTH serves as the major defense against acute hypocalcemia. We therefore classify these disorders as those which manifest hypocalcemia: (1) due to abnormalities of PTH availability, (2) due to PTH resistance as a consequence of PTH/PTH-related
A variety of congenital or acquired disorders can lead to developmental failure of the parathyroid glands, failure of functional hormone production, or destruction of the glands. These all present as hypocalcemia, usually with attendant hyperphosphatemia and undetectable or inappropriately low levels of circulating PTH. FAILURE OF ORGANOGENESIS: DIGEORGE SEQUENCE
DiGeorge sequence is an uncommon developmental disorder that affects the third and fourth branchial clefts and results in dysgenesis of the thymus and the parathyroid glands [43]. Tetany and seizures are common features of the early course of infants with DiGeorge sequence. However, abnormalities in T-cell function with subsequent increased risk for infection often become a major feature of this disorder later in life [44]. Deletions, translocations, and rearrangements of the chromosome 22q11.2 region occur in this disorder, and more recent evidence implicates TBX1, a member of the t-box family of transcription factors, as the gene responsible for its characteristic craniofacial, parathyroid, and cardiac anomalies [45e47]. It is believed that contiguous genes, as well as a variety of other genetic modifiers are responsible for the broad spectrum of findings that exist in patients with DiGeorge sequence. The prevalence of the disorder is approximately 1/3000; hypocalcemia is reported as a feature in up to 60% of cases [48]. Several other genes have been identified as critical to parathyroid development, and loss-of-function mutations of these factors have been identified as the genetic basis for some human hypoparathyroid syndromes. These include loss of function mutations in the parathyroid-specific transcription factor, GCMB (glial cells missing B), or GCM2, usually manifesting as familial, isolated hypoparathyroidism [49]. The combination of familial Hypoparathyroidism, sensorineural Deafness, and Renal dysplasia (referred to as the HDR syndrome) is an autosomal dominant disorder due to mutations in the GATA3 transcription factor [50]. An autosomal recessive condition of hypoparathyroidism, mental retardation, and dysmorphic features (Sanjad-Sakati syndrome) is mediated by loss-of-function mutations in the tubulin chaperone E (TBCE) gene [51]. Mutations
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in this gene are also associated with the Kenny-Caffey syndrome in which hypoparathyroidism, dwarfism, tubular deformities of the long bones, and eye defects occur [52]. Mitochondrial gene defects can also result in hypoparathyroidism [53]. X-linked hypoparathyroidism has been observed in patients with mutations in the SOX3 transcription factor [54]. Indeed many causes of so-called “idiopathic hypoparathyroidism” are likely related to the above described disorders. MOLECULAR ABNORMALITIES IN THE PTH GENE
PTH is secreted and synthesized by a classic secretory pathway. The initial translation product is a prepropeptide, which requires cleavage of the amino-terminal pre and pro sequences before secretion. A family with autosomal dominant inherence of hypoparathyroidism has been reported in which a missense mutation (Cys18 / Arg) results in an abnormal signal sequence and diminished uptake of preproPTH into the endoplasmic reticulum [55]. Another family with recessively inherited hypoparathyroidism has been reported in which the prepro sequence is deleted by a splicing mutation [56]. MOLECULAR ABNORMALITIES IN THE CALCIUMSENSING RECEPTOR GENE
The gene for the calcium-sensing receptor (CaSR) has been mapped to chromosome 3 [57]. As mentioned in “Role of parathyroid hormone in the acute defense of ionized serum calcium concentration,” above, ionized calcium is a ligand for this receptor, and receptor occupancy suppresses PTH secretion. Numerous individuals and families with a variety of activating mutations of CaSR have been reported; the associated condition is referred to as autosomal dominant hypocalcemia [58]. The renal expression of the mutant CaSR results in hypercalciuria, despite the hypocalcemia, and an increased risk for nephrolithiasis has been reported in individuals harboring one of these mutations. Progression to chronic kidney disease in this setting is a concern, particularly if urinary calcium excretion is not carefully monitored [59]. The CaSR is more fully discussed in Chapter 24. AUTOIMMUNE POLYGLANDULAR SYNDROME TYPE 1
An autoimmune disorder termed autoimmune polyglandular syndrome type 1 is characterized by early development of hypoparathyroidism in association with Addison’s disease and mucocutaneous candidiasis. The majority of affected individuals will manifest hypocalcemia by the age of 10 [60]. In addition to Addison’s disease, one-third of the patients will develop other endocrine disorders such as diabetes mellitus, pernicious anemia, or premature ovarian failure [60]. This disorder is now known to be associated with mutations
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in a gene (AIRE) encoding an autoimmune regulatory protein containing a zinc finger motif, and is a candidate transcription factor [61,62]. POSTSURGERY REDUCTION IN PTH
Given the close anatomic relationship of the parathyroid glands to the thyroid, complete or near complete extirpation of the thyroid gland as part of the management of either Graves’ disease or thyroid cancer can be complicated by destruction or vascular compromise of parathyroid tissue and varying degrees of hypoparathyroidism. With increasing specialization of surgery practices, this should be a rare complication of thyroid surgery, and with experienced thyroid surgeons occurs with a frequency of less than 10%. Even when destruction of the parathyroid glands does not occur following neck surgery, so-called “stunned” parathyroids with transient declines of approximately 1 mg/dl in total serum calcium are often observed in the first 24 to 48 hours postoperatively. This is presumably due to transient vascular or physical damage to the glands. Some surgeons treat with a brief course of calcitriol or calcium or both to protect against acute hypocalcemia immediately following surgery. Considerable variability in the degree of hypoparathyroidism following neck surgery occurs, ranging from asymptomatic reduction in parathyroid reserve to frank tetany, requiring chronic therapy with vitamin D and calcium. INFILTRATIVE DISEASES AND DEPOSITION OF HEAVY METALS
Although uncommon, malignant metastasis to the parathyroid glands with hypoparathyroidism has been reported, usually with breast cancer [63]. It has been postulated that granulomatous involvement of the parathyroids in sarcoidosis can lead to hypoparathyroidism [64]. Patients with transfusion-dependent thalassemia can develop hypoparathyroidism due to hemochromatosis secondary to deposition of iron in the glands [65]. In Wilson’s disease hypoparathyroidism can occur, presumably because of copper deposition [66]. RADIATION
Although the parathyroid glands are quite resistant to radiation, hypoparathyroidism following radioactive iodine treatment for hyperthyroidism has been described [67]. FUNCTIONAL DEFECTS IN PTH SECRETION
Severe hypomagnesemia is associated with suppressed parathyroid secretion [68]. This is discussed in further detail under “Hypomagnesemia,” below. Transient hypocalcemia in neonates has been reported to be associated with maternal hyperparathyroidism. Finally,
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impaired parathyroid reserve has been reported in diabetic patients with uremia [69]. Hypocalcemia Due to Resistance to the Actions of PTH Several disorders of PTH action have hypocalcemia as their principal manifestation. PSEUDOHYPOPARATHYROIDISM
Peripheral tissue insensitivity or resistance to PTH is classically termed pseudohypoparathyroidism (PHP) [70]. The characteristic biochemical manifestations of PHP are hypocalcemia and hyperphosphatemia, as in classic hypoparathyroidism; however, circulating levels of PTH are elevated, rather than low or undetectable. The renal tubule is the primary site of PTH resistance, although skeletal resistance may occur to variable degrees, and in some cases varies with treatment status [71]. However, if the skeletal response to PTH is normal, lesions characteristic of hyperparathyroidism, including osteitis fibrosa cystica, can develop. Normally, PTH stimulates renal cAMP production, and levels of cAMP increase in the urine following administration of the hormone. A direct correlation has been demonstrated between the degree of PTH resistance (as assessed by the failure of urine cAMP excretion or phosphate to change) and the ambient circulating PTH level [72]. This renal cAMP response provides the basis for a diagnostic test that allows partial classification of this heterogeneous group of disorders. Individuals with PHP that demonstrate a blunted urinary cAMP response have PHP type I. Those that generate a normal cAMP response have type II PHP. PHP type I has been further characterized into types Ia, Ib, and Ic. Type Ia describes those individuals with the Albright’s hereditary osteodystrophy (AHO) phenotype, which includes short stature and large frame, broad faces, and shortened fourth metacarpals. Soft tissue calcifications and multiple endocrine abnormalities are often present. These individuals often have a mutation in GNAS1, the a subunit of the stimulatory guanine nucleotide binding regulatory protein, Gs [73]. This regulatory protein couples membrane receptors to adenylate cyclase, thereby regulating receptor-dependent cAMP production. The presence of Gs in various cell types accounts for the generalized hormone resistance that may occur. For example, affected patients often have elevated thyrotropin (TSH) levels with a compensated euthyroid state. Variable degrees of gonadotropin, antidiuretic hormone (ADH), adrenocorticotropin (ACTH), and glucagon resistance have been described. Type Ib PHP refers to individuals with isolated PTH resistance, without the AHO phenotype, or resistance to other hormones. However, subsequent
detailed investigations have shown that this phenotype has some of the features of the classical disorder, in particular elevation in TSH levels. In some familial cases of type Ib PHP, mutations in STX16, a gene upstream of GNAS1 has been found to correlate with the presence of heritable PTH resistance; it has been speculated that the phenotype may result from mutations in STX16, which causes reduced Gsa expression only in the renal tubule [74]. Other mutations in GNAS1 have been identified in sporadic type Ib PHP, which may result in tissuespecific splice variants that alter the function of the protein in different tissues [75]. Maternal imprinting of GNAS1 and adjacent related genes also contributes to the clinical heterogeneity of type I PHP. Thus individuals within an affected family who have inherited the mutant GNAS allele from their father may have no clinical or biochemical evidence of hormone resistance, but demonstrate the AHO phenotype, referred to as pseudopseudohypoparathyroidism. The GNAS locus contains three transcripts that encode expressed proteins and two non-coding transcripts that are regulated by a complex interaction of alternative splicing and imprinting [76]. Patients without an identified mutation in GNAS1, but with associated hormonal resistance and/or AHO have been classified as having type Ic PHP. The catalytic subunit of adenylate cyclase is a possible site for the defect in this condition; however, no genetic basis for this variant of PHP has been identified. In sum, it appears that mutations in GNAS1 or in neighboring regions that may affect its imprinting can cause a spectrum of defects seen in type I PHP, and an overlap between the subcategories of Ia, Ib, and Ic is likely to exist. In contrast to type I PHP, type II PHP is characterized by isolated resistance to the phosphaturic effects of PTH while cAMP generation in response to PTH administration is normal. It has been suggested that this is due to variable defects distal to cAMP generation in the cascade of hormone action. AHO is absent, and no distinct skeletal phenotype is evident; various autoimmune findings have been described in some patients. This entity is quite rare, and some have questioned whether it exists as a distinct entity. Finally, others have suggested that a circulating PTH inhibitor may play a role in the pathogenesis of PHP and have suggested that this inhibitor may be generated by parathyroid tissue itself [77]. Resistance to PTH has also been described in hypomagnesemia, as described below. HYPOMAGNESEMIA
Magnesium is an important cofactor for parathyroid hormone secretion, apparently required for release of the stored hormone from secretory granules. Thus magnesium deficiency can interfere with parathyroid
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secretion and function [68]. Serum magnesium levels are usually moderately to severely depressed (below the range of 1.0e1.4 mg/dl) before this occurs. Despite hypocalcemia, PTH levels may be inappropriately low or only modestly elevated, and tetany refractory to calcium supplementation can ensue. Hypomagnesemia, per se, may cause tetanic symptoms, although concomitant hypocalcemia is more common. Insufficient PTH secretion is the most widely accepted cause of refractory hypocalcemia in magnesium deficiency [68], although it has been suggested that resistance to the calcemic actions of PTH and vitamin D may also play a role [78]. Impairment of vitamin D synthesis may also contribute [79]. As PTH stimulates conversion of 25(OH)D to 1,25(OH)2D, the functional hypoparathyroidism seen with severe hypomagnesemia may result in low circulating 1,25(OH)2D levels, further compromising the body’s defense against hypocalcemia [80]. Whether target tissue resistance to infused PTH occurs in magnesium deficiency remains controversial, and it has been suggested that this apparent resistance may simply reflect differences in the basal levels of circulating PTH [81]. Hypomagnesemia can be seen in the settings of chronic gastrointestinal disease or nutritional deficiency especially in alcoholics. To further complicate matters, vitamin D deficiency is often present in hypomagnesemic patients [82]. Resistance to the action of PTH at the level of bone and kidney may also contribute to the hypocalcemia seen in the setting of magnesium deficiency. Replenishment of magnesium stores promptly restores parathyroid function to normal. Hypomagnesemia may result from inherited disorders of magnesium excretion and/or absorption [83]. It can also be induced by the renal tubular effects of several drugs, including amphotericin B, aminoglycoside antibiotics, chemotherapeutic agents (particularly cis-platinum), diuretics, and cyclosporin. Finally, primary disorders of magnesium wasting, due to mutations in the TRPM6 ion channel, claudin family members CLDN16 (paracellin), CLDN19, and in the Na-CL cotransporter SLC12A3, may have associated defects in renal tubular handling of calcium [84]. Thus factors other than the effect of hypomagnesemia on PTH secretion/action may play a role in the hypocalcemia often accompanying this group of disorders. Hypocalcemia in the Setting of Normal or Increased PTH Activity and Normal PTH Receptor Function Despite normal PTH function and downstream signaling from its receptor, hypocalcemia can still occur due to disturbances in skeletal homeostasis, vitamin D metabolism, and a variety of medical illnesses.
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NEONATAL HYPOCALCEMIA
The newborn infant undergoes an acute transition to independently regulated mineral homeostasis at parturition (see also Chapter 35). When the maternal source of calcium is eliminated, the infant’s circulating calcium level transiently decreases, with recovery occurring by the third day postpartum. Infants of diabetic or preeclamptic mothers and infants who suffer perinatal asphyxia or other fetal complications may experience an exaggerated fall in serum calcium with a delayed recovery phase. Management with intravenous calcium supplementation is required for symptomatic hypocalcemia or when severely low serum calcium levels are detected. This condition is referred to as “early neonatal hypocalcemia” and is usually transient. It may be associated with transient hypomagnesemia. Hypocalcemia presenting at 5e10 days of life is referred to as “late neonatal hypocalcemia.” This presentation is more typical of term infants after enteral feeding has been established. Infants of hyperparathyroid mothers may present with symptomatic hypocalcemia within this period but have also been reported to present as late as 1 year of life. Familial forms of hypoparathyroidism may present as either “early” or “late” hypocalcemia. Mild to moderate neonatal hypocalcemia commonly occurs in patients with congenital heart disease (apart from those defects common in the DiGeorge sequence) [85] and in some cases can be attributed to transient impairment of parathyroid function. HYPOCALCEMIA DUE TO VITAMIN D MALNUTRITION (SEE FIG. 59.1)
Vitamin D synthesis in the skin requires adequate exposure to ultraviolet light. Thus vitamin D deficiency is less common in settings where sunlight exposure is abundant. In extremes of latitude (e.g., northern climates in North America), and where industrial pollution can interfere with transmission of UV light, normal vitamin D status is dependent on adequate dietary vitamin D intake. Supplementation of milk products with vitamin D has significantly reduced the incidence of vitamin D deficiency in North America. Despite these measures, certain populations are at significant risk for development of vitamin D deficiency, and severe hypocalcemia may be a presenting manifestation of the disorder (see also Chapter 52). A convergence of several risk factors for vitamin D deficiency occurs in breast-fed infants during the first 18 months of life. Breast-fed infants present with vitamin D deficiency most commonly during the winter or early spring in northern US cities. The limited direct sunlight exposure during the winter season is a major factor. Black children are at greater risk due to the greater quanta of UV light required to penetrate the pigmented
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dermis and induce previtamin D formation [86]. Breast milk contains only small amounts of vitamin D, even when the mother is receiving pharmacological doses of the vitamin. Others have pointed out that dietary practices, including vegetarianism and high grain intake, may place infants at greater risk for the development of this condition [87]. Another group at high risk for development of vitamin D deficiency is found at the other extreme of life, the elderly, because of general nutritional compromise and limited sunlight exposure (see Chapter 54). Biochemical findings in these conditions vary with the severity or duration of deficiency. The circulating 25(OH)D level is generally used to assess total body vitamin D status; however, the definition of vitamin D deficiency is highly controversial. Serum calcium levels in moderate vitamin D deficiency are often normal, compensated by secondary elevations in PTH [88]. In severe vitamin D deficiency, however, overt hypocalcemia can occur despite elevated circulating PTH. Serum phosphate levels tend to be slightly low or normal. Circulating alkaline phosphatase activity of bone origin is usually markedly elevated in children and can be elevated in adults. Bone symptoms (pain, leg bowing in children) may persist after periods when 25(OH)D levels have been low, even while blood levels of 25(OH)D have normalized. In children, radiographs of rachitic extremities imaged after therapy has begun can reveal several distinctive features including hyperdense lines of remineralization at the physes, consistent with recent exposure to vitamin D, despite the continuing presence of physical findings of rickets. Circulating 1,25(OH)2D levels may be low, normal, or elevated during vitamin D deficiency. This may appear paradoxical, but it should be recognized that 1,25(OH)2D circulates in nearly 1000-fold lower concentrations than 25(OH)D. Furthermore, in the setting of vitamin D deficiency, production of 1,25(OH)2D is maximized. Thus, efficient conversion of small amounts of newly ingested or synthesized 25(OH)D may markedly increase the circulating 1,25(OH)2D concentration. Perhaps a more intriguing paradox in this setting is the continued malabsorption of calcium despite normal concentrations of 1,25(OH)2D. The skeletal consequence of isolated severe vitamin D deficiency in children is rickets, a disorder of the epiphyseal growth plate, characterized by the expansion of the hypertrophic zone of epiphyseal chondrocytes with subsequent disorganization and expansion of the growth plate cartilage matrix [89]. The defective mineralization processes ultimately result in malalignment deformities of the long bones. In adult bone, vitamin D deficiency causes osteomalacia, which is characterized histomorphometrically by excess undermineralized osteoid and a markedly delayed mineralization rate.
Adults with osteomalacia may suffer painful pseudofractures, particularly in weight-bearing long bones. HYPOCALCEMIA DUE TO VITAMIN D MALABSORPTION
Because vitamin D is a fat-soluble vitamin, generalized fat malabsorption may result in vitamin D deficiency. Gastrointestinal diseases such as Crohn’s disease, celiac sprue, and pancreatic insufficiency can be accompanied by hypocalcemia due to vitamin D malabsorption [90] (see also Chapter 69). We have also encountered children presenting with vitamin-Ddeficiency rickets who have ultimately been diagnosed with cystic fibrosis with accompanying fat malabsorption. In addition, interruption of the enterohepatic circulation of both 25(OH)D and 1,25(OH)2D may lower body vitamin D stores. The possibility that the diseased bowel may not be able to respond to 1,25(OH)2D may also contribute to calcium malabsorption in these settings. Mild hypocalcemia and secondary hyperparathyroidism are also seen in cholestatic liver diseases such as primary biliary cirrhosis [90]. Circulating levels of 25(OH)D are reduced in this setting due to impaired hydroxylation of vitamin D in the liver and compromised enterohepatic circulation. HYPOCALCEMIA DUE TO 1a-HYDROXYLASE DEFICIENCY
Impaired metabolism of 25(OH)D to 1,25(OH)2D is characterized by hypocalcemia and severe rickets [91] see also Chapter 64. The disorder (also termed pseudovitamin-D-deficiency rickets (PDDR) or vitamin-Ddependent rickets, type 1) is inherited in an autosomal recessive manner and is characterized by biochemical features similar to those of vitamin-D-deficiency rickets, with the exceptions that circulating 25(OH)D levels are normal and circulating 1,25(OH)2D levels are low. Loss-of-function mutations in the gene encoding the ferrodoxin-binding component of the mitochondrial P450 enzyme, 25-hydroxyvitamin D 1a-hydroxylase (CYP27B1) have been shown to cause this condition [92]. Restoration of eucalcemia and correction of rickets is attainable with physiological doses of 1,25(OH)2D3. HYPOCALCEMIA DUE TO HEREDITARY RESISTANCE TO 1,25(OH)2D
A defect in target tissue responsiveness to 1,25(OH)2D was clinically described shortly after the capacity to measure circulating 1,25(OH)2D became available [38]. In this book and currently in the literature the disease is often referred to as hereditary vitamin D resistant rickets (HVDRR) and it is discussed in detail in Chapter 65. Patients with hypocalcemia caused by hereditary resistance to 1,25(OH)2D have severe
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manifestations of vitamin-D-deficiency rickets despite normal serum 25(OH)D concentrations and elevated serum levels of 1,25(OH)2D. This disorder is inherited in an autosomal recessive manner. Additional features in many patients include alopecia totalis and oligodontia. The disease is variably responsive to large doses of 1,25(OH)2D3 and oral calcium therapy. In the most resistant cases, long-term parenteral calcium infusions can normalize serum chemistries and cure the skeletal lesions [93]. The positive therapeutic response to parenteral calcium suggests that mediation of calcium absorption at the intestine is the critical systemic action for 1,25(OH)2D. Several defects in the coding region of the vitamin D receptor (VDR) that impair or prevent either hormone or DNA binding have been described in these patients. Reduced levels of the VDR have also been described, usually due to premature stop mutations. This condition is quite rare but serves as an interesting experiment of nature in which the receptor-mediated function of 1,25(OH)2D3 is specifically ablated. An animal model of the disease, where the VDR is null, is described in Chapter 33. HYPOCALCEMIA DUE TO DIETARY CALCIUM DEFICIENCY
Although uncommon, extremely low calcium intakes have been reported to be associated with mild hypocalcemia. Nigerian and South African children with calcium intakes of 150 mg/day or less were found to have reduced serum calcium values, secondary hyperparathyroidism, and rickets [94e96]. The children were not vitamin D deficient, and their biochemical abnormalities and bone disease responded to treatment with calcium alone. Studies using stable calcium isotopes have demonstrated that intestinal calcium absorption in this group is not impaired [97]. Similar findings have been observed in certain US populations. This phenomenon appears to occur after children have been weaned to diets with little to no dairy product content, with fluids consisting mostly of juices and soft drinks [98]. Thus, nutritional rickets may reflect calcium or vitamin D deficiency, and often some combination of the two, given the common dairy source of these nutrients. HYPOCALCEMIA INDUCED BY HYPERPHOSPHATEMIA
Since the 1930s, it has been appreciated that oral or parenteral phosphate can induce a decline in serum calcium concentrations. Herbert et al. have demonstrated that phosphate infusions lower serum calcium in both the presence and absence of parathyroid glands [99]. Moreover, they reported that the changes in peak urinary calcium excretion during phosphate administration are not sufficient to account for the fall in the serum
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calcium. The theory they advanced remains the best explanation available for this phenomenon and centers on the hypothesis that the calcium X phosphate molar product, when exceeded, leads to spontaneous precipitation of calcium salts in soft tissues. The Ca X P product, when estimated from total serum ion concentrations (as mg/dl), is normally taken to be <60 in adults or <80 in small children. Hyperphosphatemia sufficient to cause hypocalcemia is usually abrupt in onset and severe in magnitude. Typical clinical settings include (1) excessive enteral or parenteral phosphate administration, (2) the tumor lysis syndrome, and (3) rhabdomyolysis-induced acute renal failure. Hypocalcemia induced by either oral or parenteral phosphate administration is often associated with soft tissue calcification. Such ectopic calcification has been observed during the treatment of hypophosphatemia due to either diabetic ketoacidosis or acute alcoholism. Adults receiving phosphate-containing enemas and infants fed “humanized” cow milk rich in phosphate may also become hypocalcemic [100,101]. Under most circumstances discontinuation of exogenous phosphate intake leads to prompt return of the serum calcium level to normal. Hypocalcemia in the setting of massive tumor lysis results from the release of intracellular phosphate as a consequence of chemotherapy-induced cell death, usually during the treatment of rapidly proliferating neoplasms [102]. The hypocalcemia may continue beyond the period of hyperphosphatemia and appears to be aggravated by suppressed 1,25(OH)2D levels [103]. The use of phosphate-binding antacids, oral calcium, and, in severe cases, 1,25(OH)2D3, may help to correct the serum calcium level. Rhabdomyolysis-induced acute renal failure occurs with trauma and drug or alcohol abuse. Marked hypocalcemia can occur in the early oliguric phase, and moderate to severe hypercalcemia in the subsequent polyuric phase. Llach et al. have described hyperphosphatemia and suppressed serum 1,25(OH)2D levels during the initial hypocalcemic phase, suggesting a mechanism similar to that seen in the tumor lysis syndrome [104]. The appearance of hypercalcemia and high serum 1,25(OH)2D levels during the diuretic phase may result from rapid development of secondary hyperparathyroidism during the initial hypocalcemic period. Treatment includes restriction of phosphate intake and efforts to prevent hypocalcemia during the early stages of the disease. HYPOCALCEMIA DUE TO ACCELERATED SKELETAL MINERALIZATION
Bone remodeling is a controlled process of tissue renewal which, in healthy individuals, results in closely matched rates of bone resorption and formation. If
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skeletal mineralization exceeds the rate of bone resorption, hypocalcemia can occur. One setting in which this can be observed is following surgical correction of primary or tertiary hyperparathyroidism. The abrupt cessation of PTH-mediated osteoclastic bone resorption with concomitant rapid remineralization of an undermineralized skeleton can lead to “hungry bone syndrome” with severe, even life-threatening hypocalcemia [105]. Postoperative treatment should be instituted when the serum calcium level falls below 8.0 mg/dl, using oral or parenteral calcium supplements and if necessary 1,25(OH)2D3. In general, this condition resolves over the course of several days, although distinguishing it from permanent postoperative hypoparathyroidism can be difficult and requires gradual discontinuation of supportive therapy with careful monitoring. Patients with primary hyperparathyroidism who are taking cinacalcet should have their drug stopped 48e72 hours before parathyroid surgery to reduce the risk of postoperative hypocalcemia. Hypocalcemia also may occur in patients with bony metastases that induce bone formation, as with prostatic and breast cancer [106]. Finally, institution of therapy for vitamin-D-deficiency osteomalacia or rickets can sometimes lead to a fall in serum calcium associated with rapid mineralization of previously unmineralized osteoid [107]. This is self-limited and can usually be prevented with supplemental calcium.
that parathyroid gland reserve is subnormal in patients with AIDS although hypocalcemia is not a prominent feature of that disorder [113]. MEDICATIONS
A variety of medications have been reported to decrease serum ionized calcium concentration. Many of these drugs are used to treat hypercalcemia and/or excessive bone resorption, and hypocalcemia results from their overzealous use. Thus, mithramycin, calcitonin, and the bisphosphonates can all cause hypocalcemia. In susceptible individuals, prolonged therapy with diphenylhydantoin or phenobarbital can lead to hypocalcemia, owing in part to enhanced catabolism of vitamin D metabolites [114] (Chapter 67). Citrated blood products, particularly when used for large-volume transfusions or plasma plasmapheresis, can cause hypocalcemia [115]. Radiocontrast agents that contain EDTA (ethylenediaminetetraacetic acid) can also induce falls in serum ionized calcium levels [116]. Finally, foscarnet (trisodium phosphonoformate), used in the treatment of cytomegalovirus ocular infections and acyclovir-resistant herpes, has been reported to cause a decline in ionized serum calcium, perhaps through complexing extracellular calcium [117].
THERAPY FOR HYPOCALCEMIA Acute Management
MEDICAL ILLNESS
Hypocalcemia in the setting of renal failure results from hyperphosphatemia due to reduced renal phosphate clearance by the failing kidney and is complicated by impaired biosynthesis of 1,25(OH)2D [108]. Hypocalcemia and tetany were first reported in patients with pancreatitis in the early 1940s [109]. Pancreatic lipase released from the damaged gland is believed to liberate free fatty acids that chelate calcium, thereby removing it from the extracellular fluid [110]. Hypomagnesemia resulting from poor oral intake, alcohol use, or vomiting may contribute to the hypocalcemia. Hypocalcemia in the setting of pancreatitis often suggests a poor clinical course. Treatment consists of parenteral calcium and magnesium when indicated. Hypocalcemia may also occur in patients with acute sepsis. In one series, 20% of such patients evidenced reductions in ionized serum calcium [111]. Hypocalcemia in this series was associated with a poor prognosis (50% mortality, compared to 30% in eucalcemic patients). This phenomenon is most often reported with Gram-negative sepsis but has occurred in toxic shock syndrome caused by staphylococcal infection [112]. The pathophysiology of hypocalcemia in these two settings is unknown. Finally, it has been suggested
Newborns It may be necessary to treat early neonatal hypocalcemia. We generally provide supplementation when the circulating concentration of total serum is less than 5e6 mg/dl in premature infants, and less than 6e7 mg/dl in term infants. Appropriate emergency therapy of acute symptomatic hypocalcemia consists of a slow intravenous infusion (<1 ml/min) of calcium gluconate in a 10% (w/v) solution. The calcium gluconate salt consists of 9% elemental calcium. A wellfunctioning indwelling intravascular catheter should be used, to avoid extravasation. Calcium should never be administered intramuscularly because of local tissue toxicity. It is important to perform cardiac monitoring and careful observation during acute infusions. A total infusion of 1e3 ml will usually arrest convulsions, and no more than 2 mg of elemental calcium per kilogram body weight should be given as a single dose. Such bolus infusions may be repeated up to four times in a 24-hour period. If severe hypocalcemia persists, however, it is generally more effective to use a longterm calcium gluconate infusion, such that 20e50 mg of elemental calcium per kilogram body weight is infused over an entire 24-hour period. Calcium chloride
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THERAPY FOR HYPOCALCEMIA
is more irritating than calcium gluconate and is not the preferred salt for infusion. Neither bicarbonate nor phosphate should be coinfused with calcium in order to prevent precipitation of their respective calcium salts, either in the infusion line or in the vein. Adults In adults, emergency management consists of 10e20 ml of 10% calcium gluconate infused over a 10 to 15 min period. In the longer term one can dilute 100 ml of 10% calcium gluconate (representing 900 mg of elemental calcium) in 1 liter of 5% dextrose and, beginning at a rate of 50 ml/h, titrate the rate to maintain the serum calcium in the low normal range. It is important to monitor the patient’s EKG during calcium infusions to monitor for adverse cardiac effects such as excessive shortening of the corrected QT interval or bradyarrythmias. Finally, in the setting of acute exacerbations of calcium malabsorption, as may typically occur in patients with autoimmune hypoparathyroidism with associated gastrointestinal disorders, nocturnal nasogastric supplementation with calcium carbonate or calcium glubionate has been employed, providing up to 20 mg of elemental calcium per kilogram body weight per 8 hours, as necessary, until the underlying intestinal disturbance has resolved. Role of Magnesium Supplementation In the setting of hypomagnesemia, magnesium therapy may be required to restore PTH secretion and sensitivity of target tissues to the hormone. Prior to administration of magnesium salts, assessment of renal function and urinary output should be performed. Magnesium treatment in infancy consists of 5e10 mg of elemental magnesium per kilogram body weight. Although magnesium may be given intramuscularly, the intravenous route is preferred. Magnesium sulfate septahydrate (MgSO4 • 7H2O) is available as a 50% solution, containing 48 mg/ml of elemental magnesium. These small volumes may be further diluted, but they should be infused slowly; the dose may be repeated every 12e24 hours. In older individuals, up to 2.4 mg of elemental Mg per kilogram body weight can be given over a 10 min period (to a maximum of 180 mg). Others prefer a continuous infusion of 576 mg of elemental magnesium over 24 hours. The length of therapy must be individualized, and maintenance with oral magnesium salts should be implemented in cases where ongoing hypomagnesemia is anticipated. Magnesium levels should be monitored to avoid toxicity. Deep tendon reflexes can be examined, and therapy should be halted if they diminish. As with calcium therapy, cardiac monitoring should be performed and therapy stopped if EKG changes occur. Intravenous calcium gluconate is a useful antidote for
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magnesium intoxication, and should be available at the bedside.
Long-term Treatment Many of the causes of hypocalcemia discussed above are corrected by treating the underlying disorder (e.g., vitamin D deficiency, tumor lysis syndrome). Relatively few of these disorders require maintenance therapy for hypocalcemia, and of those the most important are hypoparathyroidism and pseudohypoparathyroidism. Vitamin-D-resistant states, although rare, comprise a third group of patients that require long-term treatment. Hypoparathyroidism, whether primary or secondary to trauma or surgery, is the most frequently encountered condition that requires chronic therapy to maintain eucalcemia. In these individuals, the goal is to maintain serum calcium in the low normal range (8.5 to 9.0 mg/ dl). This will reduce the likelihood of symptoms such as circumoral tingling, signs such as carpopedal spasm, as well as more long-term complications such as nephrocalcinosis, or potential vascular calcification. Although hormone replacement with PTH has been successful in studies of up to 3 years in duration it is not yet approved for this indication [118,119]. There is no single best way to achieve stable eucalcemia, although the combination of a vitamin D metabolite with calcium supplements is generally preferred. A wide variety of preparations of both are available (see Tables 59.1 and 59.2). Because of the prolonged toxicity that occurs with excessive ingestion of either ergocalciferol or 25(OH)D, we generally prefer to use rapid-acting preparations of vitamin D for the treatment of this disorder. Toxicity, when it occurs with these latter preparations, corrects more rapidly with discontinuation of the drug. Calcitriol (1,25(OH)2D3) is the usual preparation used for this purpose. Dihydrotachysterol (DHT) or 1a-(OH)D (alfacalcidol) are also available as alternatives to 1,25(OH)2D3. In general, it is best to aim for a stable dose of one of these agents and to further regulate the serum calcium by adjusting the intake of supplemental calcium, rather than by making repeated changes in vitamin D metabolite therapy. We use calcitriol in the majority of our patients. The dose of calcitriol can range from as little as 0.25 up to 2.0 mg/day [120,121]. We have estimated the biological half-life of the drug as 12e14 hours. Hypercalcemia, when it develops during therapy with calcitriol, usually resolves within 3e4 days after discontinuing the drug, although we have had patients in whom it has taken over 1 week for serum calcium to normalize. In addition to a highcalcium diet, calcium supplements are important for the treatment of hypoparathyroidism. Doses of 1000e2500 mg/day of calcium may be necessary. Preparations including the carbonate, citrate, lactate,
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59. THE HYPOCALCEMIC DISORDERS: DIFFERENTIAL DIAGNOSIS AND THERAPEUTIC USE OF VITAMIN D
TABLE 59.1
Calcium Preparations Dosage form
Elemental calcium (mg/tablet)
Cost per 1000 mg of elemental calcium*
Calcium carbonate (available in generic brands)
Various formulations, including capsule, gum, suspension, tablets, chewable tablets (depending on manufacturer)
100 mg/ml (susp), Caps: 500 mg/cap Tabs: 160 to 600 mg/tab (depends on manufacturer) Gum: 200 mg/piece
Cost will vary by manufacturer (generally lower than name brands)
Os-Cal 500þ DÒ
Tablet
500 mg
$0.16
Caltrate 600Ò
Tablet
600 mg
$0.14
TumsÒ (Regular, EX, Ultra)
Chewable tablet
200 mg, 300 mg, 400 mg
$0.15, $0.15, $0.14
Alka-MintsÒ
Chewable tablet
340 mg
$0.8
ViactivÒ
Chewable
500 mg
$0.3
Various formulations including tablets, capsules, effervescent tablets, oral suspension (depending on manufacturer)
180 mge760 mg (granules e depends on manufacturer)
Cost will vary by manufacturer (generally lower than name brands)
Syrup
115 mg/5 ml
$1.64
Drug CALCIUM CARBONATE
CALCIUM CITRATE Calcium citrate (available in generic brands)
CALCIUM GLUBIONATE Neo-CalgluconÒ CalcionateÒ CalciquidÒ
* Retail cost will vary between retail pharmacies
TABLE 59.2
Vitamin D and Related Agents
Name
Formulation
Typical dose
Solution: 8000 IU/ml
2000 IU/day
Vitamin D (calciferol) DrisdolÒ
Other solutions (including 400 IU/drop, 1000 IU/drop) Tablet 400 IU
Capsule: 25 000 and 50 000 IU
1 capsule/day
1,25 dihydroxyvitamin D (calcitriol) RocaltrolÒ
0.25 mg/capsule
0.5 mg/day
0.50 mg/capsule
0.5 mg/day
1.0 mg/ml (oral solution)
0.5 mg/day
Ò
Calcijex solution: ampules for IV use containing solutions with 1 mg/ml of drug VecticalÒ topical ointment 3 mg/gm (used for psoriasis) 1 mg vitamin D ¼ 40 IU
gluconate, and glucobionate salts are suitable for this purpose. We prefer calcium carbonate because it is inexpensive, well tolerated, and easily acquired. In some cases of hypoparathyroidism, a thiazide diuretic may be useful in augmenting serum calcium levels and reducing the hypercalciuria that can occur with the institution of treatment. Several vitamin D analogs
have been developed for the treatment of secondary hyperparathyroidism in the setting of renal failure (see Chapter 81) [122]. These analogs are relatively more selective inhibitors of parathyroid proliferation than calcitriol. The reduced calcemic activity of these compounds renders them less useful in the management of hypocalcemia.
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REFERENCES
Magnesium deficiency can occur in patients with hypoparathyroidism, most often secondary to steatorrhea which is seen in the autoimmune forms of this disorder [123,124]. This may render a patient relatively resistant to therapy, and therefore magnesium deficiency should be considered in individuals whose therapeutic requirements unexpectedly increase. In pseudohypoparathyroidism, the therapeutic approach is similar to that in primary hypoparathyroidism, the principal difference being that hypercalciuria is less of an issue and it is generally easier to maintain eucalcemia in these individuals. Untreated patients with pseudohypoparathyroidism may have varying degrees of osteomalacia and initially may require high-dose therapy to achieve eucalcemia as their bones remineralize. Requirements will drop as the bone lesion heals, often heralded by a fall in serum alkaline phosphatase and a rise in serum calcium levels. Individuals with 1a-hydroxylase deficiency (PDDR) have a defect in the ability to generate 1,25(OH)2D from the precursor metabolite 25(OH)D. In this disorder, eucalcemia can be achieved by supplying 1,25(OH)2D3 in physiological dosages [91]. In contrast, patients with hereditary resistance to 1,25(OH)2D (HVDRR) can present with a spectrum of resistance to calcitriol therapy, with some individuals responding to doses of calcitriol in the usual therapeutic range and others resistant to even massive doses of the drug [38]. As noted above, chronic therapy with parenteral infusions of calcium has resulted in improvement of the rickets and normalization of all serum biochemical parameters [93]. Although as discussed above, recently developed analogs of vitamin D have not been generally recommended for therapy of hypocalcemia, two novel analogs (20-Epi-1,25(OH)2D3 and JK-1626-2) have been found to be somewhat more efficacious than calcitriol in the treatment of cases of hereditary resistance to vitamin D (HVDRR) caused by mutations in the ligand-binding domain of VDR [125]. A variety of stresses such as trauma, infection, and pregnancy can increase the therapeutic requirements of patients with chronic hypocalcemia, and the clinician should be alert to this possibility.
Acknowledgments We greatly appreciate the assistance of Teri Tuma in the preparation of this manuscript, and Osama Abdelghany in providing availability and pricing of the various calcium preparations.
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[91] S. Balsan, Hereditary pseudo-deficiency rickets or vitamin Ddependency type I, in: F.H. Glorieux (Ed.), Rickets (Nestle Nutrition Workshop Series, vol. 21), Raven, New York, 1991, pp. 55e165. [92] X. Wang, M.Y. Zhang, W.L. Miller, A.A. Portale, Novel gene mutations in patients with 1alpha"-hydroxylase deficiency that confer partial enzyme activity in vitro, J. Clin. Endocrinol. Metab. 87 (2002) 2424e2430. [93] S. Balsan, M. Garabedian, M. Larchet, A. Gorski, G. Coumot, C. Tau, et al., Long-term nocturnal calcium infusions can cure rickets and promote normal mineralization in hereditary resistance to 1,25-dihydroxyvitamin D, J. Clin. Invest. 77 (1986) 1661e1667. [94] J.M. Pettifor, F.P. Ross, R. Travers, F.H. Glorieux, H.F. DeLuca, Dietary calcium deficiency: a syndrome associated with bone deformities and elevated serum 1,25-dihydroxyvitamin D concentration, Metab. Bone Dis. Related Res. 2 (1981) 301e305. [95] J.M. Pettifor, P. Ross, J. Wang, G. Moodley, J. Couper-Smith, Rickets in children of rural origin in South Africa: is low dietary calcium a factor? J. Pediatr. 92 (1978) 320e324. [96] P.J. Marie, J.M. Pettifor, F.P. Ross, F.H. Glorieux, Histological osteomalacia due to dietary calcium deficiency in children, N. Engl. J. Med. 307 (1982) 584e588. [97] G.E. Oramasionwu, T.D. Thacher, S.D. Pam, J.M. Pettifor, S.A. Abrams, Adaptation of calcium absorption during treatment of nutritional rickets in Nigerian children, Br. J. Nutr. 100 (2008) 387e392. [98] M.C. DeLucia, M.E. Mitnick, T.O. Carpenter, Nutritional rickets with normal circulating 25-hydroxyvitamin D: a call for reexamining the role of dietary calcium intake in North American children, J. Clin. Endocrinol. Metab. 88 (2003) 3539e3545. [99] L. Herbert, J. Lemann, J. Petersen, E. Lennon, Studies of the mechanism by which phosphate infusion lowers serum calcium concentration, J. Clin. Invest. 45 (1966) 1886e1894. [100] B. Chernow, T. Rainey, L. Georges, J. O’Brian, Iatrogenic hyperphosphatemia: a metabolic consideration in critical care medicine, Crit. Care Med. 9 (1981) 772e774. [101] P. Venkaraman, R. Tsang, F. Greer, A. Nogucbi, P. Laskarzewski, J. Steichen, Late infantile tetany and secondary hyperparathyroidism in infants fed humanized cow milk formula, Am. J. Dis. Children 139 (1985) 664e668. [102] K. Arrambide, R. Toto, Tumor lysis syndrome, Semin. Nephrol. 13 (1993) 273e280. [103] R. Dunlay, M. Camp, M. Allon, P. Fanti, H. Malluche, F. Llach, Calcitriol in prolonged hypocalcemia due to the tumor lysis syndrome, Ann. Intern. Med. 110 (1989) 162e164. [104] F. Llach, A. Felsenfeld, M. Haussler, The pathophysiology of altered calcium metabolism in rhabdomyolysis-induced acute renal failure, N. Engl. J. Med. 305 (1981) 117e123. [105] A. Brasier, S. Nussbaum, Hungry bone syndrome: clinical and biochemical predictors of its occurrence after parathyroid surgery, Am. J. Med. 84 (1988) 654e660. [106] E.C. Abramson, H. Gajardo, S.C. Kukreja, Hypocalcemia in cancer, Bone Miner. 10 (1990) 161e169. [107] C. Anast, T. Carpenter, L. Key, Metabolic bone disorders in children, in: L. Avioli, S. Krane (Eds.), Metabolic Bone Disease and Related Research, Saunders, Philadelphia, Pennsylvania, 1990, pp. 850e887. [108] F. Llach, J. Bover, Renal osteodystrophy, in: B. Brenner (Ed.), The Kidney, Fifth ed., Saunders, Philadelphia, Pennsylvania, 1996, pp. 2187e2273. [109] H. Edmondson, C. Berne, Calcium changes in acute pancreatic necrosis, Surg. Gynecol. Obstet. 79 (1944) 240e244.
[110] A.F. Stewart, W. Longo, D. Kreutter, R. Jacob, W.J. Burtis, Hypocalcemia due to calcium soap formation in a patient with a pancreatic fistula, N. Engl. J. Med. 315 (1986) 496e498. [111] G.P. Zaloga, B. Chernow, The multifactorial basis for hypocalcemia during sepsis. Studies of the parathyroid hormonevitamin D axis, Ann. Intern. Med. 107 (1987) 36e41. [112] R.W. Chesney, D.M. McCarron, J.G. Haddad, C.D. Hawker, F.P. DiBella, P.J. Chesney, et al., Pathogenic mechanisms of the hypocalcemia of the staphylococcal toxic-shock syndrome, J. Lab. Clin. Med. 101 (1983) 576e585. [113] P. Jaeger, S. Otto, R.F. Speck, L. Villiger, F.F. Horber, J.P. Casez, et al., Altered parathyroid gland function in severely immunocompromised patients infected with human immunodeficiency virus, J. Clin. Endocrinol. Metab. 79 (1994) 1701e1705. [114] R. Weinstein, G. Bryce, L. Sappington, K. King, B. Gallagher, Decreased serum ionized calcium and normal vitamin D metabolite levels with anticonvulsant drug treatment, J. Clin. Endocrinol. Metab. 58 (1984) 1003e1009. [115] J. Tofalletti, R.A. Nissenson, D. Endres, E. McGarry, G. Mogollon, Influence of continuous infusion of citrate on responses of immunoreactive PTH, calcium, magnesium components, and other electrolytes in normal adults during plasmapheresis, J. Clin. Endocrinol. Metab. 60 (1985) 874e879. [116] L.E. Mallette, L.S. Gomez, Systemic hypocalcemia after clinical injection of radiographic contrast media: amelioration by omission of calcium chelating agents, Radiology 147 (1982) 677e679. [117] M.A. Jacobson, J.G. Gambertoglio, F.T. Aweeka, D.M. Causey, A.A. Portale, Foscarnet-induced hypocalcemia and effects of foscarnet on calcium metabolism, J. Clin. Endocrinol. Metab. 72 (1991) 1130e1135. [118] K.K. Winer, J.A. Yanovski, G.B. Cutler Jr., Synthetic human parathyroid hormone 1-34 vs calcitriol and calcium in the treatment of hypo-parathyroidism, JAMA 276 (1996) 631e636. [119] K.K. Winer, N. Sinaii, J. Reynolds, D. Peterson, K. Dowdy, G.B. Cutler Jr., Long-term treatment of 12 children with chronic hypoparathyroidism: a randomized trial comparing synthetic human parathyroid hormone 1-34 versus calcitriol and calcium, J. Clin. Endocrinol. Metab. 95 (2010) 2680e2688. [120] R.G. Russell, R. Smith, R.J. Walton, C. Preston, R. Basson, R.G. Henderson, et al., 1,25-dihydroxycholecalciferol and 1alpha-hydroxycholecalciferol in hypoparathyroidism, Lancet 2 (1976) 14e17. [121] R.M. Neer, M.F. Holick, H.F. DeLuca, J.T. Potts Jr., Effects of 1alpha-hydroxy-vitamin D3 and 1,25-dihydroxy-vitamin D3 on calcium and phosphorus metabolism in hypoparathyroidism, Metabolism 24 (1975) 1403e1413. [122] E. Slatopolsky, A. Dusso, A.J. Brown, Control of uremic bone disease: role of vitamin D analogs, Kidney Int. Supp. 80 (2002) 143e148. [123] A. Rosier, D. Rabinowitz, Magnesium-induced reversal of vitamin D-resistance in hypoparathyroidism, Lancet 1 (1973) 803e804. [124] P. Ahonen, S. Mylla¨rniemi, I. Sipila¨, J. Perheentupa, Clinical variation of autoimmune polyendocrinopathy-candidiasisectodermal dystrophy (APECED) in a series of 68 patients, N. Engl. J. Med. 322 (1990) 1829e1836. [125] S.A. Gardezi, C. Nguyen, P.J. Malloy, G.H. Posner, D. Feldman, S. Peleg, A rationale for treatment of hereditary vitamin Dresistant rickets with analogs of 1 alpha,25-dihydroxyvitamin D(3), J. Biol. Chem. 276 (2001) 29148e29156.
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60 Vitamin D Deficiency and Nutritional Rickets in Children John M. Pettifor University of the Witwatersrand and Chris Hani Baragwanath Hospital, South Africa
INTRODUCTION Rickets is a clinical syndrome that occurs in children as a result of a failure of or delay in mineralization of the growth plate of growing bones. There are numerous causes, the majority of which can be grouped into three major categories: those which primarily result in a failure to maintain normal calcium homeostasis; those which primarily affect phosphate homeostasis; and those which directly inhibit the mineralization process. Globally, rickets due to nutritional causes (which fall into the calciopenic group) remains the most frequent form of the disease seen. However, in a number of industrialized countries, such as the USA, the genetic forms of hypophosphatemic rickets are now probably more prevalent than the nutritional causes outside the neonatal and infant periods, as a result of the fortification of foods with vitamin D and the use of vitamin D supplements in at-risk groups. Nevertheless the last 30 or more years have seen a resurgence of nutritional rickets in minority communities in a number of developed countries.
HISTORICAL PERSPECTIVE Although nutritional rickets is often considered to be a disease of industrialization, descriptions of rickets have been attributed to both Homer (900 BC) and Soranus Ephesius (130 AD). More recently, attention was drawn to rickets by Daniel Whistler in 1645 and five years later Francis Glisson (1650) provided a classic description of the disease. It was described as a disease which occurred in young children, produced severe deformities and was often fatal. The condition, which was known in Europe as “the English disease,” was
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10060-5
more common in the cities than in rural areas. Prior to the industrial revolution, it was associated with affluence, as the children of well-to-do families were often completely covered by clothing and were kept indoors. With the migration of large numbers of people from rural to urban areas at the time of the industrial revolution, the disease became associated with poverty and overcrowding in the developing urban slums. A number of studies in the late 19th and early 20th centuries documented the almost universal prevalence of rickets in young children in cities in northern Europe (for example, in Glasgow [1] and Vienna [2]). However, with the realization of the importance of ultraviolet light in preventing nutritional rickets and the discovery and isolation of vitamin D in the first quarter of the 20th century [3] (cf. Chapter 1), programs were introduced to prevent vitamin D deficiency. In the United Kingdom, a number of foods were fortified with vitamin D during the Second World War. This led to a rapid reduction in the number of children diagnosed with rickets, but in the following years the incidence of idiopathic hypercalcemia rose in infants which at the time was thought to be due to uncontrolled fortification of various foods (especially milk and cereals) leading to daily intakes of 100 mg (4000 IU) or more [4]. As a result, the fortification of foods and the use of vitamin D supplements fell into disrepute and the prevalence of vitamin D deficiency and nutritional rickets has increased, particularly among the immigrant Asian (Indian and Pakistani) population. In the USA, the universal fortification of milk with vitamin D at 400 IU/quart from the 1930s onwards had almost eradicated nutritional rickets except in families who exclude milk from their diets [5]. However, as had possibly occurred in the UK, a few cases of vitamin
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D toxicity have been reported as a result of the lack of monitoring of the fortification process [6]. In central Europe, rickets has been effectively prevented in infants and young children by the intermittent administration (every 3 to 5 months for the first 2 years of life) of high doses of vitamin D (“stosstherapie”) [7].
THE EPIDEMIOLOGY OF VITAMIN D DEFICIENCY AND NUTRITIONAL RICKETS Over the past 20 years, there has been much discussion and debate concerning the definition of vitamin D deficiency and the best methods for determining vitamin D status. Since the descriptions of the first assays to measure serum concentrations of vitamin D and/or its metabolites in the early 1970s, 25(OH)D has been the metabolite that has been used to define vitamin D status, as its serum concentrations are relatively stable (half-life 2e3 weeks), are not regulated to any great extent except by substrate concentration, and reflect vitamin D intake through the diet and synthesis by the skin [8]. In the pediatric literature, vitamin D deficiency has in the past been defined as that range of 25(OH)D concentrations associated with the development of rickets and osteomalacia, which have generally been accepted to occur at serum concentrations of <10e12 ng/ml or <25e30 nmol/l [9]. However, there is now considerable interest in the less obvious effects of vitamin D on bone mass and parathyroid hormone secretion and in the non-classical actions of vitamin D, such as its role in immune regulation, and in the prevention of autoimmune diseases, diabetes, and certain cancers [10]. This has complicated the determination of optimal vitamin D status, as there are few if any randomized controlled trials designed to assess optimal 25(OH)D concentrations in children taking into consideration these extra-skeletal functions. The consensus among some researchers, particularly in North America, is that in adults optimal concentrations of 25(OH)D should be greater than 75 nmol/l [11], although the recently released report from the Institute of Medicine did not find convincing evidence for those levels to be above 50 nmol/l. Information for children is more scarce e a conservative recommendation would be that vitamin D deficiency equates to 25(OH)D <25 nmol/l, vitamin D insufficiency to levels between 25 and 50 nmol/l, and vitamin D sufficiency to concentrations 50 nmol/l [12,13]. The Institute of Medicine has highlighted that the risk of vitamin D deficiency rickets increases as 25(OH)D concentrations fall below 30 nmol/l. In its report it did not find any evidence of additional beneficial effects of 25(OH)D concentrations above 50 nmol/l, and suggests that 40 nmol/l be
considered the median level to be aimed for in a population. Vitamin D deficiency is a prerequisite for the development of nutritional rickets in the majority of children. Thus the disease is typically associated with a lack of ultraviolet light exposure or dietary vitamin D. As commonly ingested foods are generally deficient in vitamin D (the exceptions being oily fishes or fortified foods), the normal diet contributes little to the vitamin D status of an individual, thus adequate skin exposure to ultraviolet radiation is essential for the prevention of rickets in most situations [14] (cf. Chapter 2). Consequently, rickets occurs most frequently in infants before they are able to walk and get out of doors, in children living in countries at the extremes of latitude, or in communities in which social or religious customs prevent adequate sunlight exposure through excessive skin coverage by clothing or through the practice of purdah. Vitamin D deficiency rickets is most prevalent in children under 2 years of age, with a peak incidence between 3 and 18 months [15,16]. The disease is uncommon in infants under 3 months of age, because 25(OH)D readily crosses the placenta [17,18], thus providing the newborn infant with some protection against vitamin D deficiency [19] (cf. Chapter 38). Because 25(OH)D is not the major storage form of vitamin D and has a half-life of only 3 to 4 weeks, serum levels fall rapidly after birth unless additional sources of vitamin D are obtained by the young infant [20]. Neonatal or congenital rickets has been described in infants born to mothers who are themselves vitamin-D-deficient [21e26], and hypocalcemia is a common finding in neonates born to vitamin-D-deficient mothers [27]. Over the last two decades, the role of maternal vitamin D status during both pregnancy and lactation in predisposing infants to vitamin D deficiency has become clear [28]. Furthermore, it is apparent that many mothers worldwide are frankly vitamin D deficient (figures of up to 80% in some communities) [29], despite some being given supplements during pregnancy [30e32]. There is a good association between maternal vitamin D deficiency and vitamin D deficiency in their infants. In a study from the Middle East, vitamin D deficiency was identified in nearly 100% of mothers who had infants with rickets, compared to just over 50% of the mothers whose infants did not have rickets [33]. There are several reasons for this association: infants born to vitamin-D-deficient mothers have no vitamin D stores at birth; the breast milk of mothers typically contains negligible amounts of vitamin D; and finally the social and environmental factors that produce vitamin D deficiency in the mother are similar for the infant. In a number of studies vitamin D deficiency rickets has been noted to occur more commonly in boys than
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in girls [34e36]; however, the mechanism for this remains unclear. It has been suggested that vitamin D deficiency rickets might be a hereditary disease, which manifests itself only under adverse circumstances [35,37]. In a study of infants with rickets and their parents [37], urinary excretion of a-amino acids was increased in one-third of the infants a long time after the rickets had healed, many of the parents had increased amino acid and phosphorus excretion, and a good correlation was found between the excretion of individual amino acids by an infant and its parents. The authors suggested that these findings indicate a genetic factor playing a role in predisposing a child to rickets; however, the mode of inheritance is unclear. Several recent publications have suggested that polymorphisms of the vitamin D receptor (VDR) gene might play a role in predisposing infants and children to rickets. (Specific polymorphisms are described in Chapter 56.) In a study of Turkish and Egyptian infants with rickets, the prevalence of the F allele was greater in children with rickets than controls [38], which is similar to the findings in a Nigerian study of children with dietary calcium deficiency rickets [39]. Furthermore, 1,25 (OH)2D concentrations in Egyptian children were lower in those who were FF homozygotes. In the same group of children, it was suggested that the B allele might predispose an individual to vitamin D deficiency. However, in Indian and Mongolian studies no relationships between rickets and various VDR polymorphisms were found [40,41]. In the early literature, breast-feeding was reported to be protective against rickets [42]. However, since then, it has been described as a risk factor for the development of rickets [5,43e45]. In the last 50 years, specifically designed breast milk substitutes have replaced natural cow’s milk as the major source of nutrients for the non-breast-fed infant. This alteration in feeding patterns may account for the apparent change in risk associated with breast-feeding for several reasons; first, breast milk substitutes are fortified with vitamin D at 400 IU/ l while natural cow’s milk (unless fortified) contains little or no vitamin D [46]; second, the calcium:phosphorus ratio in breast milk substitutes (ratio ~2:1) is more appropriate than that in cow’s milk (ratio ~1:1) for optimizing intestinal calcium absorption; and third, although breast milk usually contains only small quantities of vitamin D or its metabolites (between 20 and 65 IU/liter), the content is dependent on the vitamin D status of the mother [46,47]. However, there is evidence that vitamin D metabolites may cross into breast milk from the mother in sufficient quantities to maintain normal serum concentrations of 25(OH)D in the suckling infant if the mother receives vitamin D supplements in high doses (~2000 IU/day) [20,48]. More recently a randomized controlled trial has been conducted
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supplementing lactating mothers with vitamin D3 at 6400 IU/day. Preliminary results suggest that the suckling infants’ vitamin D status was maintained through the increased breast milk vitamin D content at levels similar to those obtained by supplementing the infant directly with 300 IU/day [49]. The available evidence suggests that it is the parent vitamin D that crosses into breast milk and not 25(OH)D or other metabolites to any significant degree [50,51]. In the breast-fed infant not receiving vitamin D supplements, the maintenance of an adequate vitamin D status is dependent mainly on the infant’s exposure to ultraviolet light [52e54]. Specker and coworkers [52,53] have shown a marked seasonal variation in serum 25(OH)D concentrations in breast-fed infants, which is dependent on the time spent outdoors and on the extent of skin exposed to sunlight. They have estimated that an infant in Cincinnati (latitude 39 09’ N) requires to be outdoors for either 20 minutes a week in a diaper only or for 2 hours a week fully clothed but without a hat to maintain circulating concentrations of 25(OH)D above 11 ng/ml (27.5 nmol/l), which might by today’s criteria not be considered optimal levels [52]. Seasonal variations in serum 25(OH)D concentrations have also been documented in a number of countries in older children and adults [55e57], and these variations appear to correlate with the amount of ultraviolet light reaching the earth [58]. These observations highlight the importance of the photo-biosynthesis of vitamin D3 in the skin to prevent vitamin D deficiency and thus rickets in many populations in the world. In a number of countries such as Turkey [59], Saudi Arabia [45], India [60], Algeria [57], Iran [15], Kuwait [61], Nigeria [62], Ethiopia [63], and in others in the tropics and subtropics [64e66] rickets remains a problem despite generally good daily hours of sunshine. A number of factors contribute to the persistence of the problem in these areas; these include overcrowding and poverty, atmospheric pollution [67], purdah, lack of access to sunlight, a lack of vitamin-D-fortified foods or regular vitamin D supplements, and diets which are low in calcium and high in inhibitors of calcium absorption. In the USA, despite the almost complete eradication of vitamin D deficiency among Caucasian children, several studies have highlighted the resurgence of the problem in specific groups [5,43,44,68e72], namely vegans and children on macrobiotic diets, children who are breast-fed for prolonged periods, and AfricanAmerican children [73]. It is suggested that the combination of decreased vitamin D3 formation in the dark skin, extensive skin coverage by clothing, low dietary vitamin D intakes because of the lack of dairy products, and the generally low dietary calcium intakes associated with vegetarian diets contribute to an increased risk for vitamin D deficiency in these groups.
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A similar pattern has also been documented in darker-skinned immigrant populations in a number of European countries [74e78], and in Australia and New Zealand [79e81]. Perhaps the most intensively investigated community has been the Asian population in Great Britain because of the high prevalence of vitamin D deficiency and “Asian rickets” in children of all ages [82] and adults [83,84]. The age distribution of Asian children with rickets is described as being biphasic, with one peak in the classical age group of vitamin D deficiency (9e36 months) and the other related to the pubertal growth spurt [1,85]. Since the initial descriptions of the resurgence of rickets in the UK in the early 1960s, numerous studies have been undertaken to determine why Asians are predisposed to the problem when other immigrants such as Afro-Caribbeans are thought to be less at risk [86]. Among the hypotheses put forward are simple vitamin D deficiency due to the dark skin and lack of skin surface exposed to sunlight [87,88], low calcium diets associated with vegetarianism [89], and impaired intestinal calcium absorption associated with high phytate diets [90]. A unifying hypothesis, proposed by Clements [74], suggests that in a situation of relative vitamin D insufficiency, the low dietary calcium and high phytate content of the typical Asian vegetarian diet leads to mild secondary hyperparathyroidism and a resultant increase in the catabolism of vitamin D due to the stimulation of 24-hydroxylase activity by elevated PTH concentrations. The progressive decline in vitamin D status culminates in the development of rickets (cf. “Dietary calcium deficiency,” below).
majority who later present with rickets pass through this phase without developing symptomatic hypocalcemia. Pseudotumor cerebri [94] and cataracts, probably due to hypocalcemia, have been reported in a young infant with rickets [95]. It has been suggested that symptomatic hypocalcemia in infants with vitamin D deficiency might be precipitated by an acute illness [96], in which there is a release of intracellular phosphate [97]. Why young infants are particularly predisposed to presenting in stage I is not clear, but it may relate to a delayed response by the parathyroid glands to hypocalcemia. As the deficiency progresses, the classical features of rickets become apparent. Typically the infant or young child presents with a delay in motor milestones, hypotonia, and progressive deformities of the long bones. The deformities are most noticeable at the distal forearm with enlargement of the wrist and bowing of the distal radius and ulna, and in the legs with progressive lateral bowing of the femur and tibia. The sites and types of deformity are dependent on the age of the child and the weight-bearing patterns in the limbs. Thus in the small infant deformities of the forearms and anterior bowing of the distal tibias are more common, while in the toddler who has started to walk an exaggeration of the normal physiological bowing of the legs (genu varum) is characteristic. In the older child valgus deformities of the legs or a windswept deformity (valgus deformity of one leg and varus deformity of the other)
CLINICAL PRESENTATION The majority of clinical signs in children with rickets results from the effects of vitamin D deficiency on the mineralization process at the growth plate or on calcium homeostasis. Fraser and coworkers [36] have described three stages in the progression of vitamin D deficiency. Stage I is characterized by hypocalcemia with clinical signs related to the presence of hypocalcemia, in stage II the clinical features of impaired bone mineralization become apparent, and in stage III signs of both hypocalcemia and severe rickets are present. This division of the progression of vitamin D deficiency rickets is conceptually useful, but there is considerable clinical overlap between the various stages. The early clinical manifestations of vitamin D deficiency (stage I) are related to hypocalcemia and are more commonly seen in young infants (less than 6 months of age). They may present with convulsions [91,92], apnoeic episodes [93] or tetany with no clinical signs of rickets. Few children present clinically in stage I as the
A young infant with vitamin D deficiency rickets, presenting with respiratory distress. The child shows the characteristic deformities of the chest associated with severe rickets. The lateral diameter of the chest is reduced and bilateral Harrison’s sulci are present. The abdomen has a protuberant appearance.
FIGURE 60.1
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may be apparent. The characteristic feature in the ribs is enlargement of the costochondral junctions leading to visible beading along the antero-lateral aspects of the chest (the rachitic rosary). In the infant or young child with severe rickets, the muscular pull of the diaphragmatic attachments to the lower ribs results in the development of Harrison’s sulcus (Fig. 60.1). The negative intrapleural pressure associated with breathing may result in narrowing of the lateral diameter of the chest (the violin case deformity) with consequent severe respiratory embarrassment. Increased sweating has also been described in young infants and probably relates to the increased work of breathing due to the decreased compliance associated with the excessively malleable ribs. In premature infants with rickets (which may also be due to dietary phosphorus deficiency), fractures of the ribs may be the first clinical sign to draw attention to the problem [98]. Other skeletal abnormalities include a delay in the closure of the fontanelles, parietal and frontal bossing (hot-cross bun appearance) and the presence of craniotabes [99]. Craniotabes is often considered to be highly suggestive of rickets if other causes such as hydrocephalus and osteogenesis imperfecta have been excluded; however, a number of studies have suggested it might be a normal finding in healthy young infants [100e 102]. A recent article refutes this latter contention; in a large study conducted in Japan, the prevalence of craniotabes was found to be affected by season of birth and at 1 month of age, those infants with craniotabes had a higher prevalence of elevated alkaline phosphatase concentrations, hyperparathyroidism and low 25(OH)D levels [103]. Craniosynostosis, involving the coronal or multiple sutures, has been described in approximately 25% of patients, who were followed up after having suffered from vitamin D deficiency rickets [104]. The development of craniosynostosis appears to be related to the degree of severity of the rickets and thus to the severity of the mineralization defect, and inversely to the age of onset of the rickets. A delay in tooth eruption is a feature of rickets in the young child and enamel hypoplasia of teeth may occur if rickets develops prior to the completion of enamel deposition. The latter has been reported in the primary dentition of infants born to mothers who are vitamin D deficient [105], and is seen in the secondary dentition of children who have suffered from rickets during early childhood. Hypotonia, decreased activity and a protuberant abdomen are characteristic features of advanced vitamin D deficiency rickets in the infant and young child. These signs are probably analogous to the proximal muscle weakness described in vitamin-D-deficient adolescents and adults [106]. In this situation deep tendon reflexes are retained and may be brisk. The pathogenesis of the
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myopathy is thought to be due primarily to vitamin D deficiency, rather than hypophosphatemia [107]). Dilated cardiomyopathy and cardiac failure [108e110] have also been described in young infants with vitamin D deficiency. In one series, of the 18 infants diagnosed with vitamin D deficiency and cardiomyopathy, eight were ventilated and three died, indicating the severity of this complication in young infants [110]. The mechanism is thought to be due to the effect of hypocalcemia on cardiac muscle function, rather than a direct effect of hypovitaminosis D [111]. Infants and young children with rickets are prone to an increased number and severity of infections [59,112]. Although the increase in respiratory infections may be explained on the thoracic cage abnormalities (softening of the ribs, the enlarged costochondral junctions and the decreased thoracic movement due to muscle weakness), other reasons for the increase in diarrheal disease must be sought. The now welldocumented role of 1,25(OH)2D in modulating immune function [113,114] may contribute to the observed increase in infections (cf. Section XI of this book e Immunity, Inflammation, and Disease). Impaired phagocytosis [115] and neutrophil motility [116] have been described in children with vitamin D deficiency rickets and the lack of production of cathelicidin following the activation of Toll-like receptors may play a role in predisposing vitamin-D-deficient subjects to M. tuberculosis infection [117]. A possibly associated abnormality is anemia, thrombocytopenia, leucocytosis, myelocytosis, erythroblastosis, myelofibrosis [118], myeloid metaplasia and hepatosplenomegaly (von Jacksch-Luzet syndrome) [119], which has been described in infants with rickets [120,121]. Although the exact pathogenetic mechanisms for this syndrome are unclear, vitamin D deficiency has been implicated based on the clinical observation that vitamin D therapy cures the condition and on experimental evidence showing that 1,25(OH)2D has antiproliferative activity on myeloid leukemia cell lines [122].
BIOCHEMICAL ABNORMALITIES The hallmark of vitamin D deficiency is a low circulating level of 25(OH)D. In healthy children, a range of approximately 12 to 50 ng/ml (30e125 nmol/l) has been found in the majority of studies [52,123,124] conducted in communities in which vitamin D deficiency rickets is uncommon. However, the normal range is dependent not only on the vitamin D and calcium contents of the diet and on the ultraviolet light exposure of the skin, but also on the definition of vitamin D deficiency (see “The epidemiology of vitamin D deficiency and nutritional rickets,” above). In a number of studies
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a marked seasonal variation in levels has been recorded [53,55,125], reflecting in part the seasonal changes in the amount of ultraviolet light reaching the earth. During the winter months, at latitudes above 40 N or S insufficient UV radiation reaches the earth to allow any cutaneous synthesis of vitamin D [126]. In countries at high latitude where foods are not vitamin D fortified, serum 25(OH)D concentrations in some “normal” children may be in the range documented in symptomatic children with vitamin D deficiency [55,82]. Thus the development of symptoms depends on the duration and severity of low 25(OH)D concentrations and on the ability of the kidney to achieve adequate 1,25(OH)2D concentrations in the face of decreased substrate for the gastrointestinal tract to maintain calcium absorption at a level appropriate to meet the demands of the growing child. In children with 25 (OH)D concentrations within the normal reference range, there is no correlation between serum 25(OH)D and 1,25(OH)2D concentrations. However, once 25(OH) D levels fall below ~12 ng/ml (30 nmol/l) 1,25(OH)2D concentrations correlate with those of 25(OH)D [127,128]. In the majority of studies in which 25(OH)D values have been measured in children with vitamin D deficiency rickets, concentrations have been found to be less than 4e5 ng/ml (10e12.5 nmol/l) in most patients [82,129,130], although other researchers have found higher values [131e133]. The classical biochemical changes in vitamin-D-deficient children who have radiological changes of rickets are a combination of hypocalcemia, hypophosphatemia and elevated alkaline phosphatase and parathyroid hormone concentrations. In the early phase of vitamin D deficiency before the development of radiological signs (stage I), hypocalcemia may be the only biochemical abnormality [36]. Acute illness may precipitate hypocalcemia in the vitamin-D-depleted infant through the sudden increase in serum phosphorus concentrations [97]. The biochemical picture in stage I rickets may be confused with that of hypoparathyroidism or pseudohypoparathyroidism [134], as serum hypocalcemia, hyperphosphatemia and normal alkaline phosphatase may be found. As the disease progresses, secondary hyperparathyroidism in response to the hypocalcemia induces a partial correction of the low serum calcium concentration, which may return to levels within the normal range, and increases phosphate excretion by the kidney resulting in hypophosphatemia (stage II) [135]. At this stage serum alkaline phosphatase concentrations are usually elevated and other renal manifestations of secondary hyperparathyroidism, such as increased cyclic AMP excretion, generalized aminoaciduria, impaired acid excretion, and decreased urinary calcium excretion, are found [136]. In stage III of the disease, the radiological features are more severe,
hypocalcemia once again becomes apparent and alkaline phosphatase concentrations rise further [36]. The elegant studies conducted by Fraser and coworkers [36] before the availability of immunoassays for the measurement of serum parathyroid hormone (PTH) concentrations, suggested that in stage I vitamin D deficiency serum concentrations of PTH are normal as serum phosphorus values and urinary amino acid excretion are within the normal range. Their patients only had radiologic evidence of calvarial demineralization without other bone changes of rickets. Other data support this conclusion as normal PTH concentrations have been reported in the early hypocalcemic phase of symptomatic vitamin D deficiency [91]. However, Kruse [137] found elevated PTH values and increased urinary cyclic AMP excretion in children with stage I rickets. This discrepancy can possibly be explained by the fact that the patients in the latter study might represent a slightly later stage of vitamin D deficiency than those in the other studies as the children were selected on the presence of radiologic changes. Evidence of end-organ resistance to PTH has been found in the young children with both mild and more severe radiological rickets [137,138]. In the study by Kruse [137] the children with mild rickets remained normophosphatemic and had normal renal handling of phosphate (TmP/GFR) despite elevated PTH concentrations and increased urinary cyclic AMP excretion. Similar indirect evidence of PTH resistance (hypocalcemia, normophosphatemia and a decrease in the phosphate excretion index) was noted by Taitz [138] in infants with more severe radiologic rickets. Resistance to PTH has also been described in hypocalcemic adolescents with mild rickets [139]. Usually, however, as the severity of the rickets increases (stages II and III), so PTH values rise further and renal hyporesponsiveness is overcome [137]. Thus hypophosphatemia and a decrease in TmP/GFR become hallmarks of the disease. Markers of bone turnover are typically elevated in nutritional rickets in response to the development of secondary hyperparathyroidism. Urinary hydroxyproline excretion may be within the normal range in stage I rickets, but is elevated in patients with radiologic rickets [137], and an increase in serum concentrations of bone resorption markers has been reported in children with untreated rickets [140,141]. Similarly serum alkaline phosphatase values may be normal in stage I of vitamin D deficiency, but rise with the degree of severity of the radiologic changes. Bone turnover markers (especially those of bone resorption) rise in the first 2e3 weeks of treatment, and then fall progressively to normal values over a period of 4e6 weeks [141]. Of all the readily available biochemical tests which might be deranged in nutritional rickets, alkaline phosphatase has been used most frequently as a screening
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test. However, although it is elevated in the vast majority of children with radiological changes, it lacks specificity [64,101,142]. Further, the degree of elevation of serum concentrations does not necessarily correlate with the radiological severity of the bone disease [64]. Whether or not the measurement of bone-specific alkaline phosphatase in patients with suspected rickets will be of greater sensitivity and specificity is unclear at present [143]. In a small study of rachitic subjects, the authors suggested that the measurement of deoxypyridinoline in a first morning void urine sample might be a useful indicator of rickets, values being significantly higher in patients than in age-matched controls [144]. Osteocalcin is a non-collagenous bone matrix protein that binds to hydroxyapatite and is secreted by osteoblasts during mineralization [145]. Serum concentrations are higher in children than adults and peak during the pubertal growth spurt [146]. In the few children with untreated vitamin D deficiency rickets, in whom serum osteocalcin concentrations have been measured, values have been reported to be low [141] or normal [147], and may rise rapidly on therapy to supranormal concentrations [148]. One Nigerian study [140] of 12 rachitic children found slightly elevated serum osteocalcin concentrations compared to values in age-matched controls, while another found levels to be similar or slightly lower in rachitic children than controls [149]. The children in both these studies had low dietary calcium intakes. In patients with vitamin D deficiency, serum 1,25(OH)2D concentrations have been reported to be low, normal, or even elevated [127,130,133,137,150,151], while 24,25(OH)2D values are low or undetectable [127,130,133,151,152]. Kruse [137] found that 1,25(OH)2D values were higher in children with stage II rickets than in those with either stage I or stage III rickets. The finding of normal or elevated levels of 1,25(OH)2D in vitamin D deficiency rickets has led some researchers to conclude that other vitamin D metabolites, such as 24,25(OH)2D, are necessary for the maintenance of normal calcium homeostasis [150,153,154]. Others have suggested that although concentrations are within the normal range, they are inappropriately low for the degree of hyperparathyroidism [137,151]. As discussed later, the latter hypothesis is more likely. A possible pathophysiological progression of vitamin D deficiency rickets in children may be described as follows [137,155]. As the child becomes progressively vitamin D depleted, a stage is reached when the serum 25(OH)D concentration falls below that required to maintain a serum 1,25(OH)2D level necessary to ensure the required intestinal calcium absorption for normal calcium homeostasis. The resultant hypocalcemia (stage I rickets) leads to secondary hyperparathyroidism, which through the stimulation
of 1a-hydroxlase, increases 1,25(OH)2D production despite falling 25(OH)D concentrations. In concert with PTH, 1,25(OH)2D increases bone resorption and intestinal calcium absorption thus returning serum calcium concentrations towards normal (stage II rickets). The presence of hypophosphatemia at this stage is probably responsible for the mineralization defect and the development of radiologic rickets. It is during stage II that serum 1,25(OH)2D concentrations may be elevated [137]. A possible explanation for the failure of the elevated 1,25(OH)2D levels to reduce the hyperparathyroidism and heal the bone disease at this stage is that the concentrations are not high enough to meet the increased calcium requirements associated with the generalized mineralization defect and increased bone turnover. Support for this hypothesis comes from data which show that 1,25(OH)2D concentrations rise to considerably higher levels (3e5 times normal) during the healing process even when only small doses of vitamin D are provided [127,137] and that intestinal calcium absorption may reach ~80% of dietary calcium intake during this phase [127]. As 25(OH)D concentrations fall further, 1,25(OH)2D levels once again fall, despite persistent hyperparathyroidism, because of the lack of substrate. Hypocalcemia again becomes apparent as intestinal calcium absorption falls and calcium mobilization from bone decreases due to the lack of 1,25(OH)2D, which has a permissive action on bone resorption by PTH [156,157]. The combination of both hypocalcemia and hypophosphatemia increases the severity of the bone disease (stage III).
RADIOLOGIC CHANGES The typical radiologic changes associated with vitamin D deficiency rickets have been well described and are discussed in Chapter 49. Stage I rickets characteristically shows few radiologic signs, although demineralization of the calvarium and loss of definition of the skull sutures have been described [36], but these signs are difficult to quantify. The changes of rickets are best visualized at the growth plate of rapidly growing bones. Thus in the upper limbs, the distal ulna is the site which may show best the early signs of impaired mineralization. In the older child, the metaphyses round the knees become more useful. The early signs of rickets include widening of the epiphyseal plates and a loss of definition of the provisional zone of calcification at the metaphyses [158]. As the disease progresses, the disorganization of the growth plate becomes more apparent with cupping, splaying, spur formation and stippling [97,159] (Fig. 60.2). The appearance of epiphyses may be delayed or they appear small, osteopenic, and ill-defined.
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FIGURE 60.2 The radiographic features of vitamin D deficiency rickets at the wrist. Left panel: untreated vitamin D deficiency showing underdevelopment of the epiphyses, widening of the epiphyseal plates, splaying and irregularity of the metaphyses and loss of the provisional zones of calcification. The shafts show coarsening of the trabecular pattern and loss of the normal cortical definition. Middle panel: response after 3 months of vitamin D therapy. The metaphyses show clear signs of healing with dense bands of calcification at the distal ends of the metaphyses, narrowing of the epiphyseal plates, and more clearly defined epiphyses. The trabecular pattern still appears coarse but shows improvement. Right panel: 6 months after starting vitamin D therapy. The radiographic changes of rickets have disappeared. The epiphyses, epiphyseal plates, metaphyses, and trabecular structure are normal. (Reproduced with permission [159].)
The shafts of the long bones show features of both hyperparathyroidism and osteomalacia. Osteopenia is a characteristic feature which in the so-called “atrophic” form of the disease may be very severe [64]. The cortices become thin and may show periosteal new bone formation, although this is more frequently seen during healing. The trabecular pattern is reduced and appears coarse. Deformities of the shafts of the long bones are typically present and in severe rickets, pathological fractures and Looser’s zones may be noted. In vitamin D deficiency rickets, features of hyperparathyroidism, such as subperiosteal erosions, are uncommon. However, loss of the lamina dura around the teeth is frequently seen. Enlargement and splaying of the costochondral junctions on the lateral radiographs of the chest have been used as a sign of rickets; however, in one study mild changes were found to be unreliable as their presence did not correlate with serum 25(OH)D concentrations or with other features of rickets at the distal radius and ulna [160]. Rickets during adolescence may be difficult to detect using the conventional radiographic sites of the wrist and knees as the epiphyseal plates narrow and epiphyses fuse. A radiograph of the pelvis may be useful in this situation as the secondary iliac and ischial ossification centers may be abnormally wide [161]. These centers appear at puberty and normally unite with the rest of the bone between the 15th and 25th years of age. The sign of early healing of rickets is described as the appearance of broadened bands of increased density replacing the normal sharp metaphyseal lines (Fig. 60.2). The demarcation of the broad bands on the diaphyseal side of the shaft may be poorly defined
[158]. Healing in more severe cases of rickets may first appear as bands of mineralization occurring distal to and separated from the irregular and frayed metaphyses. There is then gradual filling in of the demineralized area on the diaphyseal side of the initial band of mineralization with remodeling and the development of a normal trabecular pattern. Periosteal new bone formation may be seen which gradually becomes incorporated into the cortices of the long bones.
THE GROWTH PLATE IN RICKETS The characteristic feature in all forms of rickets is the abnormality that occurs at the growth plate. The cartilaginous plate consists of resting, proliferating and hypertrophic zones of chondrocytes, and in rickets it is the hypertrophic zone that is affected through widening and loss of organization of this zone. The widening is caused by marked impairment of apoptosis of the hypertrophied cells through an inhibition of the caspase-9-mediated mitochondrial pathway [162]. It appears that in all forms of rickets, the inhibition of apoptosis is as a consequence of hypophosphatemia, rather than through a direct effect of vitamin D or one of its metabolites [163,164]. Studies suggest that the sodium-dependent phosphate cotransporter (NaPi-IIc) is intimately involved as a phosphate transporter in hypertrophied chondrocytes [164], and thus as a regulator of apoptosis. The widening of the hypertrophic zone of the growth plate due to impaired apoptosis can be reversed or prevented by correcting the hypophosphatemia without altering the basic defect causing
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the rickets [162] (e.g., without altering PHEX or FGF23 in X-linked hypophosphatemia, or without correcting the VDR abnormalities in hereditary vitamin-D-resistant rickets). However, correction of the hypophosphatemia does not correct the subgrowth plate metaphyseal changes of rickets which manifest with impaired vascular invasion and reduced osteoclast numbers.
TREATMENT AND PREVENTION Treatment Vitamin D deficiency rickets can be effectively treated by the oral administration of small doses of either vitamin D2 or D3, provided there is no evidence of gastrointestinal malabsorption. Stanbury et al. [127] showed that an oral vitamin D dose of between 200 to 450 IU/day produced a rise in serum 1,25(OH)2D concentrations to normal values within 1e3 days. The latter climbed to reach a peak some five times the normal mean after 1 to 3 weeks, despite serum 25(OH)D values remaining less than 10 ng/ml (25 nmol/l). Similarly, spontaneous improvement in the biochemical features of rickets has been reported to occur in children with biochemical abnormalities during the summer months, associated with a rise in serum 25(OH)D values due presumably to increased ultraviolet light exposure [129]. More generally, however, doses of vitamin D between 5000 and 15 000 IU/day for 3 to 4 weeks are used in the management of rickets. Normalization of serum calcium and phosphorus concentrations occurs within 1 and 3 weeks [137], although serum alkaline phosphatase concentrations and urinary hydroxyproline excretion remain elevated for several months. Despite the return to normal of serum PTH, calcium and phosphorus values within 3 weeks, serum 1,25(OH)2D concentrations may remain elevated for up to 10 weeks [133,137]. Serum 24,24(OH)2D values, which are often undetectable in the untreated patient, rise with the progressive increase in serum 25(OH)D concentrations during treatment [133]. Lower doses of vitamin D (1000e2000 IU/day) do produce healing but the response is less rapid. A recent randomized clinical trial found that daily vitamin D2 or D3 (2000 IU) was as effective as weekly vitamin D2 (50 000 IU) over a 6-week period in correcting hypovitaminosis D in young children [165]. The authors concluded that the option of daily or weekly treatment may help to individualize treatment to suit parental needs and thus reduce the likelihood of noncompliance which has been reported to be a common problem especially when vitamin D supplementation is used long term for the prevention of hypovitaminosis D. The study also clearly showed the equivalence of
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vitamin D2 and D3 in the treatment of hypovitaminosis D, a finding confirmed by Holick and coworkers, who measured serum 25(OH)D concentrations and found similar values in the D2- and D3-treated groups in adult subjects treated with either vitamin D2 or D3 at daily doses of 500 IU or 1000 IU over an 11-week period [166]. In Central Europe, a single dose of 600 000 IU vitamin D (either orally or intramuscularly) has been found to be effective in treating vitamin D deficiency rickets, resulting in a rapid improvement in biochemical abnormalities within a few days and radiologic evidence of healing within 2 weeks [97,167]. A sustained drop in serum alkaline phosphatase is seen within 6 to 12 weeks [167]. Single-dose therapy has an advantage over smaller daily doses as it avoids the problem of compliance, which was thought to be responsible for the lack of response in 40% of children with vitamin D deficiency rickets in a study conducted in Kuwait [168]. Concern has been raised about the use of 600 000 IU vitamin D in the treatment of rickets as hypercalcemia was reported in a small number of infants a month after having received the vitamin D dose [169]. These authors suggest that a dose of 150 000 IU is equally effective as the larger dose in the management of the disease without running the risk of hypercalcemia. In a recent study conducted in Pakistan, the authors compared the efficacy of using either intramuscular or oral vitamin D3 (200 000 IU as a single dose) for the treatment of active rickets in young children. No difference in response rate, which was very good, was noted between the two groups; however, the parents preferred the intramuscular treatment [170]. A single intramuscular dose of vitamin D3 (10 000 IU/kg body weight) has also been reported to be effective and safe in treating rickets in children [171]. 25(OH)D concentrations were above 20 ng/ml (50 nmol/l) in all but 12.5% of the subjects 3 months after the injection. Besides ensuring an adequate vitamin D intake, the calcium content of the diet should be optimized (between 600 and 1000 mg/day) during the initial stages of management. This is particularly true for children who are on vegetarian or low-calcium-containing diets [172] and for those who are severely hypocalcemic. In symptomatic patients, a single dose of calcium gluconate (1e2 ml/kg of a 10% solution) may be given slowly intravenously and the diet supplemented with 10% calcium gluconate (5 ml/kg/day in divided doses).
Prevention As discussed in “The epidemiology of vitamin D deficiency and nutritional rickets,” above, vitamin D deficiency rickets remains a problem in a number of at-risk groups despite readily available methods of preventing the disease. A number of studies in several
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countries have been conducted prospectively in breastfed infants to assess vitamin D status. Several have shown a fall in serum 25(OH)D concentrations in those infants who were not vitamin D supplemented, to levels in the vitamin-D-deficient range [20,173,174], although this is not a universal finding [175,176]. Further, a number of studies have highlighted the high prevalence of vitamin D deficiency in mothers during pregnancy and lactation, which exacerbates the severity and onset of vitamin D deficiency in their offspring [19,84,177,178]. In a number of countries, a further group that probably does not receive enough attention is the adolescent, as the prevalence of vitamin D deficiency tends to rise in this age group [179,180]. In the USA, adolescent vitamin D deficiency is particularly prevalent in African-American teenagers, in the overweight, and in females [181]. Thus, preventive strategies should be directed not only at breast-fed infants but also at pregnant and breast-feeding women and adolescents where appropriate [182]. Both North America [183] and the United Kingdom [184] recommend dietary intakes of vitamin D of between 200 and 400 IU/day for pregnant and lactating women to ensure adequate circulating 25(OH)D levels. Yet, a number of studies have shown that these recommended intakes are not adequate to maintain vitamin D concentrations in the sufficiency range [31,50,51,185], and researchers are suggesting that intakes of 1000e2000 IU/day are more appropriate. Although at normal circulating maternal 25(OH)D concentrations, the vitamin D content of breast milk is limited (cf. “The epidemiology of vitamin D deficiency and nutritional rickets,” above), there is evidence that maternal supplementation with vitamin D at 2000 IU/ day may increase breast milk vitamin D concentrations sufficiently to maintain the infant’s 25(OH)D within the normal range. North America and the UK recommend dietary intakes of between 350 and 400 IU/d vitamin D for the breast-fed infant [183,184]. The American Academy of Pediatrics in its latest recommendations suggest that infants less than 6 months of age should be kept out of direct sunlight, that children’s activities should minimize sunlight exposure and that sunscreens should be used, because of the indirect evidence that early exposure to sunlight might determine the risk of skin cancer in later life. These recommendations make it imperative that if the above guidelines are followed supplemental vitamin D (400 IU/day) should be provided to all breast-fed and weaned infants ingesting less than 500 ml of infant milk formula/day [186e189]. In a prospective study conducted in China on infants from birth to 6 months of age, it was concluded that supplemental vitamin D at a dose of 400 IU/day produced more normal circulating 25(OH)D concentrations
than did either 100 or 200 IU/day [190]; however, in a small number of infants even 400 IU/day did not maintain 25(OH)D levels above 11 ng/ml (27.5 nmol/ l). Nevertheless, no radiologic evidence of rickets was found in any of the infants in the three groups at 6 months of age. The use of 400 IU vitamin D daily to prevent vitamin D deficiency in at-risk infants is supported by a study from Turkey, in which it was found that no rickets occurred in infants receiving the supplement compared to a prevalence of 3.8% in those that did not [59]. Although 400 IU/day of vitamin D prevents rickets and maintains a normal vitamin D status in the majority of infants [191], there is evidence to suggest that higher intakes may be required to reduce the prevalence of type 1 diabetes in children [192]. Infants fed milk formulas or cow’s milk fortified with vitamin D do not require vitamin D supplements, as their intake of milk generally provides sufficient vitamin D to prevent deficiency [175]. However, this recommendation is based on infants drinking more than 500 ml/ day. As discussed earlier, high single-dose therapy (stosstherapie) has been used with success in the treatment of vitamin D deficiency rickets in a number of countries. A similar dose has also been used on a regular intermittent basis of every 3 to 5 months for the first 18 months of life as a means of prevention of vitamin D deficiency. Little data are available on the efficacy of such prophylaxis. However, in a study to assess the effect of these high doses of vitamin D (600 000 IU) on calcium and vitamin D metabolism in infants [7], it was found that serum 25(OH)D concentrations reached very high levels 2 weeks after each administration, but that these had returned to normal prior to the next dose. 1,25(OH)2D generally remained within the normal range; however, 34% of infants were hypercalcemic at some stage during the study. These results led the authors to conclude that the dosage regimen as used during the study was excessive and unsafe [7]. However, a recent study, using the same dose (600 000 IU intramuscularly) annually in adults for the management of vitamin D deficiency, reported that at the end of 12 months, serum 25(OH)D concentrations were still in the optimal range and that the treatment appeared safe, although further studies were required to examine urine calcium excretion and the risk of hypercalcemia in a larger group of subjects [193]. Following on these results, a study to assess the efficacy of a single dose of vitamin D (600 000 IU or 15 mg) at 15 days of life, compared to 200 000 IU (5 mg) at birth or 100 000 IU (2.5 mg) at birth and three monthly for 9 months was undertaken [194]. Two weeks after the initial administration, 28 of 30 infants in the 15 mg group had serum 25(OH)D concentrations above the upper limit of normal (mean SD for the group;
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307 160 nmol/l) compared to 58% (150 55 nmol/l) in the 5 mg group and 23% (92 42 nmol/l) in the 2.5 mg group. At 6 months of age, 50% of the infants who had received 15 mg at birth still had elevated 25(OH)D concentrations (defined in this study as being >120 nmol/l), while in the 5 mg group none had elevated levels. In the group receiving 2.5 mg three monthly, serum 25(OH)D values were in the normal range on each occasion prior to receiving the next dose. Although hypercalcemia was not detected in any of the infants, serum calcium concentrations were higher in the 15 mg group 2 weeks after receiving the dose than in the other two groups. The authors concluded that intermittent doses of 15 mg vitamin D during the first year of life are excessive, and that 5 mg every 6 months or even better 2.5 mg every 3 months are more suitable for the prevention of vitamin D deficiency in at-risk infants. The same group of researchers has also studied the use of intermittent vitamin D supplementation to prevent vitamin D deficiency during the winter months in adolescents in northern France [195]. They recommend a dose of 100 000 IU in early autumn, mid winter and again at the beginning of spring to maintain 25(OH)D concentrations above 20 ng/ml. On this regimen, the usual winter rise in PTH and fall in vitamin D status were prevented. Vitamin D supplementation should be considered for all breast-fed infants living in temperate climates until they are ambulatory and are able to play outside [196]. Even in countries closer to the equator, where sunlight exposure should not be a problem, social customs may place the mother and infant at risk from vitamin D deficiency. In such situations (e.g., the Middle East and in Muslim communities in North Africa) vitamin D supplementation may also be necessary to reduce the high prevalence of vitamin D deficiency [178,197]. In a number of countries, vitamin D deficiency is not just a disease of breast-fed infants and their mothers. Rickets has been described in adolescents of Indian and Pakistani descent in the UK and in the Middle East [198,199], while hypovitaminosis D has been reported in adolescents in a number of European countries [200e203], India [204,205], and China [206]. In order to address this problem, Fuleihan and her coworkers have studied the use of weekly therapy over a year in adolescent schoolchildren [207]. A dose of 14 000 IU vitamin D3 weekly produced mean serum 25(OH)D concentrations in the mid thirties (ng/ml), while a dose 10 times smaller (1400 IU weekly) did not raise the mean above 20 ng/ml (50 nmol/l) in girl subjects. They concluded that a vitamin D dose of 14 000 IU/week (equivalent of 2000 IU/day) was safe and effective in ensuring vitamin D sufficiency in an adolescent population at risk of vitamin D deficiency.
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Besides the high prevalence of hypovitaminosis D among groups of adolescents, there is an increasing awareness of the high prevalence of vitamin D insufficiency in many elderly subjects in Europe and North America [208]. With the widespread nature of vitamin D deficiency in many countries, vitamin D supplementation is unlikely to be an effective means of combating the disease on a community basis, thus food fortification should be considered a possible solution. Although the untargeted fortification of foods other than milk and infant milk formulas has been used in the past as a means of addressing the high prevalence of vitamin D deficiency in countries where the risk of vitamin D deficiency is high, the problems experienced in the UK after the Second World War have led to it falling into disfavor (cf. “Historical perspective,” above). More recently, the use of targeted food fortification has been studied in the Asian community in Great Britain as a means of reducing the high prevalence of vitamin D deficiency in both adults and children in that community [209]. In a small pilot study, it was found that the fortification of chapatti flour at a level of 6000 IU/kg produced a sustained and significant rise in serum 25(OH)D concentrations to values within the normal range over a 6-month period comparable to that achieved by a weekly dose of 3000 IU vitamin D. Over the 6-month period serum calcium and phosphorus values rose and the number of subjects with biochemical abnormalities suggestive of rickets fell. The authors concluded that fortification of chapatti flour is a cheap and effective method of preventing vitamin D deficiency in the Asian community in Britain, and has the advantage over daily or intermittent vitamin D supplementation as the compliance utilizing the latter form of prevention is often poor. Nevertheless, food fortification remains an emotional public issue. In the USA not only have there been isolated reports of vitamin D toxicity related to inadequate monitoring of the fortification process, but under-fortification is also a problem. Holick [14] reports that in his study less than 30% of milk samples from all sections of the USA and in British Columbia contained the specified amount, and that 14% to 21% of skim milk samples contained no detectable vitamin D.
DIETARY CALCIUM DEFICIENCY Conventional wisdom would have it that nutritional rickets is primarily due to vitamin D deficiency, although dietary calcium intake modulates the severity and rapidity of onset of the disease [210,211]. However, over the last three decades evidence has been accumulating which implicates low dietary calcium intakes as a cause of rickets in the face of serum 25(OH)D
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60. VITAMIN D DEFICIENCY AND NUTRITIONAL RICKETS IN CHILDREN
concentrations above values generally associated with vitamin D deficiency rickets (>25 nmol/l). Isolated case reports of rickets developing in infants and toddlers, who were placed on very low calcium diets, have been published [212e214]. Their clinical and biochemical presentations were very similar to those of infants with vitamin D deficiency; however, in three of the five infants serum 25(OH)D and 1,25(OH)2D values were reported to be greater than 9 ng/ml (22.5 nmol/l) and 118 pg/ml (295 pmol/l), respectively. In none of the five infants was a therapeutic trial of calcium supplementation of the diet alone tried; however, the clinical and biochemical presentation suggested to the authors that dietary calcium deficiency was the primary factor responsible for the development of rickets. More convincing evidence of dietary calcium deficiency as a cause for rickets in children comes from studies in South Africa [215,216], Nigeria [217e220], India [204] and possibly Bangladesh [221], where the staple diets of children are characteristically low in calcium because of the lack of readily available dairy products and the low calcium content of the cereals (maize (corn), cassava, yam, rice, and plantain) [61,222e224]. In the South African children, dietary calcium intakes have been estimated to be between 90 and 300 mg/day in those children suffering from rickets compared to between 200 and 500 mg/day in agematched controls [222], while in the Nigerian children both patients and controls had similar but very low calcium intakes (200 mg/day) [225]. In South Africa, the children typically come from rural areas and present with signs and symptoms of rickets between the ages of 4 and 15 years [224], while in Nigeria they present younger, at a mean age of approximately 4 years [61]. In the South African series, half the children presented with knock-knees, while the others presented with either bow-legs or windswept deformities (Fig. 60.3). Bow-legs were more common in the Nigerian children, probably reflecting their earlier age of presentation [225]. Unlike vitamin D deficiency, symptoms of muscle weakness are characteristically absent in older children with dietary calcium deficiency. Radiologically, the features are typical of calciopenic rickets with osteopenia and features of hyperparathyroidism being frequent findings (Fig. 60.4). The severity of the metaphyseal changes is variable. Older children (teenagers) may have no radiologic changes of rickets despite features of osteomalacia on the iliac crest bone biopsy [226]. Younger children may show evidence of only minor degrees of impaired endochondral calcification, while in others the metaphyseal changes may be quite marked. In a Nigerian study, the degree of severity of radiologic rickets was correlated with serum alkaline phosphatase values [227].
FIGURE 60.3 The clinical presentation of children with dietary calcium deficiency. The deformities are typically more severe in the legs with a predominance of knock-knees or windswept deformities. Upper limb deformities are usually mild if present at all. (Reproduced with permission [224].)
The biochemical features are similar to those of other causes of calciopenic rickets. Hypocalcemia, low urinary calcium excretion and elevated serum PTH and alkaline phosphatase concentrations are characteristic, while serum phosphorus values are variable and often within the reference range for age [61,223,224]. Serum 25(OH)D values are above values generally considered to be associated with vitamin D deficiency (mean 16.4 ng/ml and 14.4 ng/ml in the South African and Nigerian children, respectively) and 1,25(OH)2D concentrations are
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The radiographic features of dietary calcium deficiency rickets in the lower limbs of a child. The long bones are osteopenic with deformities characteristic of long-standing rickets. The metaphyses show evidence of impaired mineralization and growth arrest lines.
FIGURE 60.4
markedly elevated [218,219,228]. In a Nigerian study [61] and in the South African children [147], serum osteocalcin levels were similar to those of non-rachitic controls in the majority of patients, although another report from Nigeria found slightly higher levels in rachitic patients than controls [140]. The finding of normophosphatemia and normal renal handling of phosphorus (TmP/GFR) in some children with active disease suggests that a peripheral resistance to PTH might be prevalent in this form of rickets. Iliac crest bone biopsies reveal evidence of osteomalacia and hyperparathyroidism in those children who have radiologic features of rickets [216], while in the teenagers without radiologic changes but lower limb deformities, the histologic picture varies from that of decreased bone volume, through features of hyperparathyroidism, to frank osteomalacia associated with hyperparathyroidism [226]. In both the Nigerian and South African studies, clinical, biochemical and radiologic healing has been achieved through increasing the calcium intake of the children to between 800 and 1500 mg/day without the administration of vitamin D supplements [61,215]. More recently, a study using a calcium supplement of only 350 mg/day reported complete healing within 6 months [220]. In a randomized controlled trial, calcium supplements alone or calcium and vitamin D together were equally effective in healing the bone disease and were significantly better than vitamin D therapy alone
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[218]. In the majority of the South African children, orthopedic corrective surgery has been necessary to correct the deformities of the legs once biochemical and radiologic healing has occurred. This has not been the pattern in the younger Nigerian children with rickets, who have shown remarkable remodeling and straightening of deformities without orthopedic surgical intervention [220]. Similarly, studies from Bangladesh suggest that early medical treatment reduces the number of children requiring corrective surgery [229]. The data available from epidemiologic studies conducted in a rural area in South Africa in which a number of the affected children live suggest that asymptomatic dietary calcium deficiency is prevalent in school children living in the area. Some 13% of children between the ages of 7 and 12 years were hypocalcemic, 41.5% had elevated alkaline phosphatase concentrations and 76% had low urinary calcium excretion [230]. It is unclear whether these children have long-term sequelae as a result of the poor calcium intakes. However, studies do indicate that asymptomatic children with biochemical abnormalities living in the rural community have lower appendicular bone mass than those with normal biochemistry [222] and that children in the community as a whole have lower appendicular bone mass than their urban peers [231]. Although dietary calcium intakes in children with biochemical changes suggestive of dietary calcium deficiency are very low, it is unclear what role the high phytate or oxalate contents of the diet play in aggravating the symptoms. Nevertheless, biochemical improvement can be achieved by supplementing the children with 500 mg calcium daily [232]. The finding of similarly low dietary calcium intakes in patients with rickets and age-matched controls in Nigeria is intriguing [225], as it suggests that factors other than low dietary calcium intakes might influence the development of rickets in affected children. Such factors might include differing amounts of inhibitors of calcium absorption in the diet, differing growth rates and therefore calcium requirements in the children, or genetic differences which make the rachitic children less able to adapt to low dietary calcium intakes than control subjects. A number of these factors are currently under investigation. A small study has found that there are significant differences in the frequency of vitamin D receptor polymorphisms between affected and control children with the FF genotype being more common in children with rickets, but the significance of these findings are unclear at present [39]. One of the hallmarks of dietary calcium deficiency which differentiates it from vitamin D deficiency is the markedly elevated serum concentrations of 1,25(OH)2D in untreated subjects. These values are even higher than those found in age-matched controls, who had dietary calcium intakes similar to those who
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developed rickets [225]. In a situation of low dietary calcium intake, it would be expected that these high 1,25(OH)2D concentrations would maximize intestinal calcium absorption. Several studies using stable isotopes of calcium have confirmed this hypothesis in children with active rickets in Nigeria [233,234]. The mean intestinal fractional calcium absorption of just over 60% in these children is much greater than that reported in subjects with vitamin D deficiency (10e15%) [13], and is in keeping with the primary pathogenetic mechanism being dietary calcium lack rather than vitamin D deficiency. It is possible that relative vitamin D insufficiency might play a role, as 25(OH)D concentrations are lower in children with active rickets than in their age-matched controls [225]; however, the addition of vitamin D to the calcium supplements did not significantly improve the response of rachitic children to treatment [218]. Nevertheless, bolus administration of vitamin D (50 000 IU) to untreated rachitic children results in a rapid and marked rise in 1,25(OH)2D concentrations, which peak on day 3 and then decline over the following 10 days [235]. These findings suggest that there might be relative substrate (25(OH)D) deficiency which is corrected by the provision of the bolus of vitamin D.
THE PATHOGENETIC SPECTRUM OF NUTRITIONAL RICKETS Nutritional rickets has been viewed for some time as being due to an inadequate supply of vitamin D through either an inadequate dietary intake or insufficient skin exposure to ultraviolet radiation, or more recently due to low dietary calcium intake in the face of a normal vitamin D status. However, these pathogenetic concepts are too simplistic. Early studies by Mellanby [236] had shown the effect of cereals in exacerbating the clinical development of vitamin D deficiency rickets in dogs. Subsequent studies in baboons have confirmed these findings [213]. The resurgence of rickets and osteomalacia in the Asian community in Great Britain provided the impetus for detailed studies into the pathogenesis of vitamin D deficiency and bone disease in that community. Although vitamin D deficiency as assessed by circulating 25(OH)D concentrations is the hallmark of the disease in Asians [82,237,238], the mechanisms for the low vitamin D status and the high prevalence of rickets were unclear. It is apparent that the majority of Asians in Britain do not spend less time outdoors than their Caucasian counterparts [239]. Further, although they have darker skins than Caucasians, which might reduce the amount of vitamin D formed in response to sunlight exposure, Afro-Caribbean immigrants living in Britain have even darker skins, yet very
few cases of rickets have been described in this ethnic group [74]. Within the Asian community, studies have highlighted that risk factors for the disease include living at high latitude, Hindu religion, immigration from East Africa, vegetarianism, high-fiber diets, and the consumption of chapattis [83,89,90]. The association with vegetarianism, high-fiber diets, and cereals of high extraction suggests that dietary factors play a role. Support for this comes from two studies which have documented healing of rickets on removing chapattis from the diet [240,241], although this is not a universal finding [88]. Over the past 25 years, research has shown that both high-fiber diets and intestinal malabsorption reduce the serum half-life of 25(OH)D by approximately one-third [242,243]. Further, experiments in rats have demonstrated that an elevation in serum 1,25(OH)2D concentrations, either by exogenous administration or endogenously through a low-calcium diet, increases the metabolic clearance rate of 25(OH)D without altering its rate of production [244e246]. The fall in serum 25(OH)D levels could be accounted for by an increase in polar metabolites appearing in the feces. Similar findings have been reported from studies in humans [247,248]. Conversely, increasing the calcium content of the diet has been shown to increase serum 25(OH)D and decrease serum 1,25(OH)2D concentrations [249]. Thus these studies convincingly show that dietary calcium and phytate content influences the catabolism of 25(OH)D through altering serum 1,25(OH)2D concentrations. In the light of the above studies, Clements [74] has proposed that the low dietary calcium and high-phytate diet of the Asian population in Britain increases vitamin D catabolism and thus vitamin D requirements. In the face of a marginal vitamin D status due to living at high latitude and the low dietary vitamin D content of the diet, the increased catabolism is sufficient to precipitate vitamin D deficiency and clinical rickets and osteomalacia. Thus nutritional rickets has a spectrum of pathogenetic mechanisms ranging from pure vitamin D deficiency associated with adequate calcium intakes, as might occur in the breast-fed infant, at one end of the spectrum, to pure dietary calcium deficiency with an adequate vitamin D status, as documented in Nigerian and South African rural children, at the other end [250]. In between these two extremes lies the situation exemplified by the Asian community in Britain, where both poor calcium intakes or absorption and marginal vitamin D status combine to lead to frank vitamin D deficiency and rickets (Fig. 60.5). It is likely that the high prevalence of rickets in vegetarian or immigrant children reported from the USA [43,44], Norway [76], Holland [77,78], and a number of tropical and
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Lack of UV Light Inadequate dietary vitamin D ↓
Low 25(OH)D
25(OH)D catabolism
Low 1,25(OH)2D
↑ 1,25(OH)2D
Impaired calcium absorption High dietary phytate/ low dietary calcium
Inadequate calcium absorption for requirement of growing child ↓
Dietary calcium deficiency
↓ Serum ionized calcium
PTH
Serum phosphate
↓
Impaired mineralization
RICKETS FIGURE 60.5 The spectrum of nutritional rickets. At either ends of the pathogenetic spectrum are vitamin D deficiency and dietary calcium deficiency. In between lie combinations in varying degrees of relative vitamin insufficiency and decreased dietary calcium content or bioavailability.
subtropical countries [64] might be due to a mechanism similar to that in the Asian community, while osteomalacia in Bedouin adults in the Middle East reflects mainly dietary calcium deficiency [251]. A number of recent studies have highlighted the complex interaction between vitamin D and calcium intakes in the pathogenesis of nutritional rickets in children. A review of 43 patients diagnosed as having nutritional rickets in New Haven, Connecticut, found low 25(OH)D levels in only 22%, and the majority of infants had been weaned onto diets with minimal dairy content [70]. The authors concluded that low dietary calcium intakes probably played a major role in the pathogenesis of the disease. Similar findings are reported from India, where it is suggested that low dietary calcium intakes were responsible for rickets in young children while vitamin D deficiency played a major role in adolescents [204].
CONCLUSIONS Despite readily accessible and effective means to eradicate rickets globally, the disease remains a major public health problem in many countries, not only in temperate regions of the world but also in tropical and
subtropical countries. In many developed countries, the promotion of exclusive breast-feeding during the first 6 months of life and the concerns about the longterm effect of sunlight exposure during this period have exacerbated the risks of vitamin D deficiency in the young infant. In some subtropical countries, social customs play an important role in preventing adequate vitamin D status not only in the young infant but also in the pregnant and lactating mother. In a number of developing countries, low dietary calcium intakes appear to play a major role in the pathogenesis of rickets in older children. Recent studies have helped to provide an all-embracing concept of the interaction of vitamin D and calcium intakes in the pathogenesis of rickets. There remains a need for international agencies to place the eradication of vitamin D deficiency among young children in many parts of the world as a priority. Nutritional rickets not only leads to an increased infant mortality but also has long-term sequelae.
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deficiency in the etiology of rickets in young children vs. adolescents in northern India, J. Trop. Pediatr. 49 (2003) 201e206. J. Rajeswari, K. Balasubramanian, V. Bhatia, V.P. Sharma, A.K. Agarwal, Aetiology and clinical profile of osteomalacia in adolescent girls in northern India, Natl. Med. J. India 16 (2003) 139e142. X. Du, H. Greenfield, D.R. Fraser, K. Ge, A. Trube, Y. Wang, Vitamin D deficiency and associated factors in adolescent girls in Beijing, Am. J. Clin. Nutr. 74 (2001) 494e500. J. Maalouf, M. Nabulsi, R. Vieth, S. Kimball, R. El-Rassi, Z. Mahfoud, G. El-Hajj Fuleihan, Short- and long-term safety of weekly high-dose vitamin D3 supplementation in school children, J. Clin. Endocrinol. Metab. 93 (2008) 2693e2701. M.S. Calvo, S.J. Whiting, Prevalence of vitamin D insufficiency in Canada and the United States: importance to health status and efficacy of current food fortification and dietary supplement use, Nutr. Rev. 61 (2003) 107e113. J. Pietrek, M.A. Preece, J. Windo, J.L.H. O’Riordan, M.G. Dunnigan, W.B. Mcintosh, et al., Prevention of vitamin-D deficiency in Asians, Lancet I (1976) 1145e1148. A.R.P. Walker, Does a low intake of calcium cause or promote the development of rickets? Am. J. Clin. Nutr. 3 (1953) 114e120. M.I. Irwin, E.W. Kienholz, A conspectus of research on calcium requirements of man, J. Nutr. 103 (1973) 1020e1095. H.E. Maltz, M.B. Fish, M.A. Holliday, Calcium deficiency rickets and the renal response to calcium infusion, Pediatrics 46 (1970) 865e870. M.R. Sly, W.H. Van Der Walt, D. Du Bruyn, J.M. Pettifor, P.J. Marie, Exacerbation of rickets and osteomalacia by maize: a study of bone histomorphometry and composition in young baboons, Calcif. Tissue Int. 36 (1984) 370e379. W. Proesman, E. Legius, E. Eggermont, 1988 Rickets due to calcium deficiency. Proceedings of the Symposium on Clinical Disorders of Bone and Mineral Metabolism, New York, p 15. J.M. Pettifor, P. Ross, J. Wang, G. Moodley, J. Couper-Smith, Rickets in children of rural origin in South Africa: is low dietary calcium a factor? J. Pediatr. 92 (1978) 320e324. P.J. Marie, J.M. Pettifor, F.P. Ross, F.H. Glorieux, Histological osteomalacia due to dietary calcium deficiency in children, N. Engl. J. Med. 307 (1982) 584e588. T.D. Thacher, S.I. Ighogboja, P.R. Fischer, Rickets without vitamin D deficiency in Nigerian children, Ambulatory Child Health 3 (1997) 56e64. T.D. Thacher, P.R. Fischer, J.M. Pettifor, J.O. Lawson, C.O. Isichei, J.C. Reading, et al., A comparison of calcium, vitamin D, or both for nutritional rickets in Nigerian children, N. Engl. J. Med. 341 (1999) 563e568. L.M. Oginni, M. Worsfold, O.A. Oyelami, C.A. Sharp, D.E. Powell, M.W. Davie, Etiology of rickets in Nigerian children, J. Pediatr. 128 (1996) 692e694. L.M. Oginni, C.A. Sharp, O.S. Badru, J. Risteli, M.W. Davie, M. Worsfold, Radiological and biochemical resolution of nutritional rickets with calcium, Arch. Dis. Child. 88 (2003) 812e817. P.R. Fischer, A. Rahman, J.P. Cimma, T.O. Kyaw-Myint, A.R. Kabir, K. Talukder, et al., Nutritional rickets without vitamin D deficiency in Bangladesh, J. Trop. Pediatr. 45 (1999) 291e293. C. Eyberg, J.M. Pettifor, G. Moodley, Dietary calcium intake in rural black South African children. The relationship between calcium intake and calcium nutritional status, Hum. Nutr. Clin. Nutr. 40C (1986) 69e74.
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[223] R. Bhimma, J.M. Pettifor, H.M. Coovadia, M. Moodley, M. Adhikari, Rickets in black children beyond infancy in Natal, S. Afr. Med. J. 85 (1995) 668e672. [224] J.M. Pettifor, Dietary calcium deficiency, in: F.H. Glorieux (Ed.), Rickets, Nestec, Vevey; Raven Press, New York, 1991, pp. 123e143. [225] T.D. Thacher, P.R. Fischer, J.M. Pettifor, J.O. Lawson, C. Isichei, G.M. Chan, Case-control study of factors assocaited with nutritional rickets in Nigerian children, J. Pediatr. 137 (2000) 367e373. [226] C.M. Schnitzler, J.M. Pettifor, D. Patel, J.M. Mesquita, G.P. Moodley, D. Zachen, Metabolic bone disease in black teenagers with genu valgum or varum without radiologic rickets: a bone histomorphometric study, J. Bone Miner. Res. 9 (1994) 479e486. [227] T.D. Thacher, P.R. Fischer, J.M. Pettifor, J.O. Lawson, B.J. Manaster, J.C. Reading, Radiographic scoring method for the assessment of the severity of nutritional rickets, J. Trop. Pediatr. 46 (2000) 132e139. [228] J.M. Pettifor, F.P. Ross, R. Travers, F.H. Glorieux, H.F. Deluca, Dietary calcium deficiency: a syndrome associated with bone deformities and elevated serum 1,25-dihydroxyvitamin D concentrations, Metab. Bone Rel. Res. 2 (1981) 301e305. [229] T. Craviari, J.M. Pettifor, T.D. Thacher, C. Meisner, J. Arnaud, P.R. Fischer, Rickets: an overview and future directions, with special reference to Bangladesh. A summary of the Rickets Convergence Group Meeting, Dhaka, 26e27 January 2006, J. Health Popul. Nutr. 26 (2008) 112e121. [230] J.M. Pettifor, F.P. Ross, G.P. Moodley, E. Shuenyane, Calcium deficiency in rural black children in South Africa e a comparison between rural and urban communities, Am. J. Clin. Nutr. 32 (1979) 2477e2483. [231] J.M. Pettifor, G.P. Moodley, Appendicular bone mass in children with a high prevalence of low dietary calcium intakes, J. Bone Miner. Res. 12 (1997) 1824e1832. [232] J.M. Pettifor, F.P. Ross, G.P. Moodley, E. Shuenyane, The effect of dietary calcium supplementation on serum calcium, phosphorus and alkaline phosphatase concentrations in a rural black population, Am. J. Clin. Nutr. 34 (1981) 2187e2191. [233] M. Graff, T.D. Thacher, P.R. Fischer, D. Stadler, S.D. Pam, J.M. Pettifor, et al., Calcium absorption in Nigerian children with rickets, Am. J. Clin. Nutr. 80 (2004) 1415e1421. [234] G.E. Oramasionwu, T.D. Thacher, S.D. Pam, J.M. Pettifor, S.A. Abrams, Adaptation of calcium absorption during treatment of nutritional rickets in Nigerian children, Br. J. Nutr. 100 (2008) 387e392. [235] T.D. Thacher, P.R. Fischer, C.O. Isichei, J.M. Pettifor, Early response to vitamin D(2) in children with calcium deficiency rickets, J. Pediatr. 149 (2006) 840e844. [236] E. Mellanby, An experimental investigation on rickets, Lancet I (1919) 407e412. [237] S.J. Iqbal, I. Kaddam, W. Wassif, F. Nichol, J. Walls, Continuing clinically severe vitamin D deficiency in Asians in the UK (Leicester), Postgrad. Med. J. 70 (1994) 708e714. [238] M.A. Preece, J.A. Ford, W.B. Mcintosh, M.G. Dunnigan, S. Tomlinson, J.L.H. O’Riordan, Vitamin-D deficiency among Asian immigrants to Britain, Lancet I (1973) 907e910. [239] M.G. Dunnigan, W.B. Mcintosh, J.A. Ford, Rickets in Asian immigrants, Lancet I (1976) 1346. [240] M.R. Wills, R.C. Day, J.B. Phillips, E.C. Bateman, Phytic acid and nutritional rickets in immigrants, Lancet I (1972) 771e773. [241] J.A. Ford, E.M. Colhoun, W.B. Mcintosh, M.G. Dunnigan, Biochemical response of late rickets and osteomalacia to a chupatty-free diet, Br. Med. J. 3 (1972) 446e447.
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[242] A.J. Batchelor, J.E. Compston, Reduced plasma half-life of radio-labelled 25-hydroxyvitamin D3 in subjects receiving a high-fibre diet, Br. J. Nutr. 49 (1983) 213e216. [243] A.J. Batchelor, G. Watson, J.E. Compston, Changes in plasma half-life and clearance of 3H-25-hydroxyvitamin D3 in patients with intestinal malabsorption, Gut 23 (1982) 1068e1071. [244] M.R. Clements, L. Johnson, D.R. Fraser, A new mechanism for induced vitamin D deficiency in calcium deprivation, Nature 325 (1987) 62e65. [245] B.P. Halloran, M.E. Castro, Vitamin D kinetics in vivo: effect of 1,25-dihydroxyvitamin D administration, Am. J. Physiol. 256 (1989) E686eE691. [246] B.P. Halloran, D.D. Bikle, M.J. Levens, M.E. Castro, R.K. Globus, E. Holton, Chronic 1,25-dihydroxyvitamin D3 administration in the rat reduces serum concentration of 25-
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hydroxyvitamin D by increasing metabolic clearance rate, J. Clin. Invest. 78 (1986) 622e628. M.R. Clements, M. Davies, D.R. Fraser, G.A. Lumb, B. Mawer, P.H. Adams, Metabolic inactivation of vitamin D is enhanced in primary hyperparathyroidism, Clin. Sci. 73 (1987) 659e664. M.R. Clements, M. Davies, M.E. Hayes, C.D. Hickey, G.A. Lumb, E.B. Mawer, et al., The role of 1,25-dihydroxyvitamin D in the mechanism of acquired vitamin D deficiency, Clin. Endocrinol. 37 (1992) 17e27. T. Berlin, I. Bjorkhem, Effect of calcium intake on serum levels of 25-hydroxyvitamin D3, Eur. J. Clin. Invest. 18 (1988) 52e55. J.M. Pettifor, Privational rickets: a modern perspective, J. Roy. Soc. Med. 87 (1994) 723e725. S. Shany, J. Hirsh, G.M. Berlyne, 25-Hydroxycholecalciferol levels in bedouins in the Negev, Am. J. Clin. Nutr. 29 (1976) 1104e1107.
C H A P T E R
61 Vitamin D and Osteoporosis Peter R. Ebeling 1, John A. Eisman 2 1
The University of Melbourne, Western Hospital, Victoria, Australia 2 Garvan Institute of Medical Research, Sydney, NSW, Australia
EFFECTS OF VITAMIN D ON THE SKELETON The major effects of vitamin D on the skeleton are recognized in deficiency states as the failure of normal mineralization of bone. This presents in childhood as rickets and in the mature skeleton as osteomalacia. The consistent finding between these two states is inadequate mineralization of the formed matrix, such that the bones are softened and deform. There has been considerable discussion as to whether this is entirely due to the major role of active vitamin D metabolites in stimulating gut calcium (and phosphate) absorption or whether there are additional specific effects on bone. In the former concept, it is proposed that there is failure of the provision of adequate calcium and phosphate that is required for mineralization to proceed. In the latter, it is proposed that there are additional roles of the active vitamin D metabolites in regulation of both bone formation and bone resorption. It is clear that parathyroid hormone (PTH), which rises in response to low serum calcium, stimulates renal production of 1,25-dihydroxyvitamin D (1,25(OH)2D), which in turn stimulates gut calcium absorption. When there is active absorption of intestinal calcium into the circulation, the rise in serum calcium decreases renal 1a-hydroxylase (CYP27B1 gene product) activity both directly and indirectly, through decreasing parathyroid hormone and increasing FGF-23 levels. This classic negative feedback loop, which has been well characterized, does not address the direct effect of vitamin D on the skeleton. The presence of the vitamin D receptor (VDR) in bone cells suggests direct effects on bone. These receptors are expressed in osteoblasts and in immature cells of the osteoclast precursor lineage. It was proposed some
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10061-7
time ago that the effect of the active vitamin D metabolites on osteoclasts was indirect via osteoblasts. The clearest potential to discriminate between these direct and indirect vitamin D effects on bone came through the VDR knockout models [1e8]. In these there was good evidence that the osteomalacic phenotype could be largely rescued by a high-calcium, high-lactose diet, which overcame the poor gut calcium absorption defect. However, these findings do not preclude other roles of vitamin D in modulating bone cell function. In these studies, a specific role is supported for the VDR in chondrocytes [9,10]. One set of findings that suggests a specific role of vitamin D in bone cells was the overexpression of the VDR in mature cells of osteoblastic lineage [11,12]. These studies reported increased bone mass in mice and stronger bones characterized by increased calcium content of the mineral phase. However in other studies, VDR knockout was associated with increased osteogenic activity [7]. The diametric difference between these studies is yet to be determined. In relation to the specific VDR overexpression model, there were both in vivo and in vitro studies, demonstrating decreased osteoclastic activity. In the in vitro model this effect appeared to be driven by the osteoblasts with higher intrinsic VDR levels [13]. Other preclinical models have explored the effects of vitamin D on osteoclastogenesis. Bone volume was positively associated with circulating 25(OH)D levels, while osteoclast surface levels were positively associated with receptor activator of nuclear factor-kappa B ligand (RANKL):osteoprotegerin (OPG) mRNA ratio, which were, in turn, higher in groups with lower serum 25(OH)D levels, but independent of serum 1,25(OH)2D levels. Serum 25D levels <32 ng/mL (or 80 nmol/L) resulted in osteopenia due to increased osteoclastogenesis, consistent with human data [14].
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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The skeleton also appears to be responsive to serum levels of the 1,25(OH)2D precursor, 25(OH)D, in terms of bone mineralization [15]. Human osteoclast cultures incubated with 25(OH)D produce measurable quantities of 1,25(OH)2D. The expression of osteoclast transcription factor, osteoclast marker genes, and an osteoblast coupling factor, ephrin-b2, is also increased in the presence of 25(OH)D in vitro. Levels of CYP27B1 and nuclear factor of activated T cells-1 mRNA correlated during osteoclastogenesis and also in a cohort of human bone samples. 25(OH)D dependently reduced the resorptive capacity of osteoclasts while, conversely, osteoclasts formed from VDR-null mouse splenocytes have increased resorptive activity. Thus, it is likely 25(OH)D metabolism is an important intrinsic mechanism for optimizing osteoclast differentiation and reducing osteoclast activity. In rodents, the major determinants of serum 25(OH)D are dietary vitamin D and dietary calcium. Compared with animals fed a diet deficient in calcium, animals fed 0.1% calcium had higher renal CYP27B1 mRNA expression and 12e18-fold increased levels of serum 1,25(OH)2D [16]. Thus, the reported effects of lowcalcium diets on bone loss may be, in part, due to the adverse effects on 25(OH)D metabolism leading to a reduction in vitamin D status. This potential interaction between dietary calcium and vitamin D metabolism has important implications, given the recent evidence for local synthesis of active vitamin D in bone tissue, and may explain why the combination of calcium and vitamin D is more effective than vitamin D alone in preventing fractures. Overall these findings suggest two distinct levels of function of the active vitamin D metabolites on bone: a general effect through changes in gut calcium absorption and a fine tuning effect on the specific activity of the osteoblasts and osteoclasts in bone.
THE ROLE OF VITAMIN D GENETIC FACTORS IN OSTEOPOROSIS AND POSSIBLE INTERACTIONS WITH VITAMIN D THERAPY There are powerful inherited contributions to osteoporosis with up to 75% of variance in bone phenotypes, such as bone density, size and geometry, attributable to genetic factors in healthy individuals. A variety of risk prediction models have examined family history and shown that a positive family history contributes to risk. Typically this increased risk has odds ratios of 1.1 to 1.5, but values of 3 or more have been reported in some subgroups and fracture types. Family history is used in the World Health Organization-supported FRAXÒ fracture risk calculator. The data, used to
estimate the effect size drawn from several international osteoporosis epidemiology cohorts including the Dubbo Osteoporosis Epidemiology Study, have been treated as the crude categorical yes/no criterion. Fracture history is often limited to an individual’s parents and often to hip fractures, which are considered to be more reliably recorded. The Centers for Disease Control and Prevention (CDC) now formally recommend a more complete family history for many major chronic diseases including any “negative” fracture history. The recognition of heritability in osteoporosis led initially to studies of many candidate genes and more recently to large-scale genome-wide studies. The initial studies, at a time when genome-wide scans were not feasible, took the candidate gene approach based on known roles of specific genes in bone and calcium homeostasis and bone structure, i.e. the vitamin D receptor (VDR) and collagen Ia1 genes. The initial demonstration of the relationship of the VDR to bone turnover and bone density [17e19] was followed by many candidate gene approaches [20e28]. Replication of these findings has been “variable” in different ethnicity and age samples, even for the two most studied loci, the VDR and collagen Ia1 genes, which are possibly influenced by geneeenvironment interactions [22,24,28e34]. The gene discovery approach of genome-wide analysis of extreme phenotypes (mutations) in small families led to discovery of the lowdensity lipoprotein receptor-related protein 5 (LRP5) [35,36]. This work opened understanding of new and previously unsuspected bone anabolic pathways [37e40]. Large-scale, population-based, genome-wide association studies in osteoporosis and fracture risk [41e43] have identified mainly known bone-related genes but also some other potential genetic targets. The osteoprotegerin, RANK and RANK-ligand, sclerostin and estrogen receptor genes were among those to be detected, but several other novel loci are yet to be investigated [42e44]. Another approach has been to perform genome-wide scans in subsamples of high and low bone density individuals selected from normal populations [45]. The primary phenotype in most such studies has been lumbar spine and proximal femur bone mineral density by dual-energy X-ray absorptiometry. This has been extended to a variety of geometric and mechanical parameters, including estimated femoral neck volumetric BMD, width, cross-sectional area, endosteal diameter, mean cortical thickness, cross-sectional moment of inertia, buckling ratio, and section modulus [46e50]. Rate of change of bone density and even fracture incidence have also been considered a phenotype. More recent studies have reported a strong association between serum 25(OH)D levels and polymorphisms in
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the vitamin-D-binding protein gene as well as genes involved in vitamin D metabolism, particularly one of the 25-hydroxylase genes [51e53]. One of the major challenges has been the relatively limited reproducibility of associations between studies. However, this may relate to environmental differences and to ethnic differences in other gene variants. This possibility is supported by recent studies reporting interactions between the variants of the VDR, vitamin-Dbinding protein and calcium-sensing receptor genes in determining response to therapy including to vitamin D [54e56]. A recent study provided new genetic data that might explain why there is considerable inter-individual variability of serum 25(OH)D levels [57]. As only 25% of this variability is accounted for by season, latitude or vitamin D intake, behavioral and genetic factors have been thought to contribute to this variability; however, previous data on the genetic determinants of vitamin D status have been very limited. This large, multicenter, genome-wide association study of 15 cohorts in Europe, Canada, and the USA, comprising about 30 000 white people from European descent, found that polymorphisms at three different loci involved in vitamin D metabolism affect serum 25(OH)D levels and the risk of vitamin D deficiency and insufficiency. After accounting for age, sex, body mass index (BMI), and season, polymorphisms in at least three, and perhaps four, loci influenced serum 25(OH)D levels. The first three genes below were identified in the discovery sample and confirmed in the replication sample while the fourth candidate gene was identified in pooled analyses of the discovery and replication samples: (1) 4p12 polymorphisms near or within the GC gene, which encodes vitamin-D-binding protein, the main transporter of vitamin D metabolites in the blood; (2) 11q12 polymorphisms near DHCR7/NADSYN1, encoding the enzyme 7-dehydrocholesterol (7DHC) reductase, which converts 7DHC into cholesterol in the skin thereby removing the substrate for production of vitamin D3; (3) 11p15 polymorphisms near CYP2R1, which encodes an enzyme that may be responsible for 25-hydroxylation of vitamin D in the liver; (4) CYP24A1 encoding 24-hydroxylase, which initiates degradation of 25(OH)D and 1,25(OH)2D. Participants with a genotype score (combining the three main variants) in the top quartile had twice the risk of having vitamin D insufficiency (<50 or 75 nmol/L) than those in the lowest quartile. It was also associated with a 1.5-fold risk of severe vitamin D deficiency (<20 nmol/L). These findings are important, as they confirm that common genetic variants may contribute to the inter-individual variability of serum 25(OH)D levels and may predispose to (or protect against) vitamin D deficiency and insufficiency. This study also opened up new questions regarding the genetic
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regulation of serum 25(OH)D: (1) Will the same genetic variants be found in other ethnic groups? (2) Will these genetic variants explain the high variability that is clinically apparent in response to either UVB exposure or to vitamin D supplementation? (3) Are there other less common genetic variants that also affect serum 25(OH)D levels?
Optimal Serum Levels of 25(OH)D for Musculoskeletal Health Many studies have demonstrated seasonal variations in serum 25(OH)D with a decline during the winter months [58e64]. More importantly, studies from both the northern and southern hemispheres have shown seasonal variations in serum 25(OH)D concentrations, accompanied by responsive changes in serum PTH concentrations, later increases in bone resorption markers and bone formation markers and decreases in bone mineral density (BMD). In 43 German subjects followed for one year, calciotropic hormones, markers of bone turnover, and BMD varied by season. During the winter months, bone turnover was significantly accelerated, and lumbar spine and femoral BMD declined by 0.3e0.9%. Supplementation with oral 500 mg calcium and 500 IU cholecalciferol per day for 1 year either reversed or abolished seasonal changes in calciotropic hormones and markers of bone turnover in the intervention group, while these changes remained in a control group. In the subjects receiving oral cholecalciferol and calcium, lumbar and femoral BMD increased significantly, whereas controls continued to lose bone [65]. Rates of hip fracture also vary annually, with a winter peak in both northern and southern hemispheres [66,67]. Inadequate vitamin D levels have been demonstrated in patients with osteoporosis [68], including in hip fracture patients in many countries, although low levels may be influenced by the fracture itself [69e71]. It is also important to note that vitamin D deficiency increases with aging and that this contributes independently to secondary hyperparathyroidism [72]. In addition, serum androgen levels in men are associated with serum 25(OH)D levels and both hormones have a concordant annual periodicity being lowest in late winter [73], suggesting important, and as yet unidentified, links between the vitamin D and sex steroid axes. Similarly in southeastern Australia (latitude 38e39 S), in 287 women drawn from an observational, cross-sectional, population-based study [74], annual periodicities of ultraviolet radiation, serum 25(OH)D, serum PTH, a bone resorption marker, serum C-telopeptide (CTx), BMD, falls and fractures were measured. Cyclic variations in serum 25(OH)D lagged 1 month behind ultraviolet radiation, peaking in summer and dipping in winter. The periodicity of serum
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FIGURE 61.1 Sine curves showing the periodicity of UV, serum 25 (OH)D, PTH, CTx, and hip and wrist fractures in the southern hemisphere. Amplitude set a maximum of þ1 and a minimum of 1 [74].
PTH was the inverse of serum 25(OH)D, with a phase shift delay of 1 month. Peak serum CTx lagged peak serum PTH by 1e2 months. In late winter, a greater proportion of falls resulted in fracture. Seasonal periodicity in 439 hip and 307 wrist fractures also followed a simple harmonic model, peaking 1.5e3 months after the trough in 25(OH)D (Fig. 61.1). Thus, a fall in serum 25(OH)D in winter is accompanied by increases in serum PTH levels, bone resorption, and the proportion of falls resulting in fracture, and more hip and wrist fractures [74]. Only one English study did not show such a seasonal variation in bone turnover markers [75]. Season appears to be more important than latitude in determining serum 25(OH)D in many countries. However, in Australia, both season and latitude accounted for less than 20% of the variation in serum 25(OH)D levels, highlighting the importance of behavioral factors, including sun avoidance [76]. Such seasonal declines in vitamin D metabolites and the associated increases in fracture rates make interventions with vitamin D a logical intervention to prevent fractures.
Determining Optimal Serum 25(OH)D Concentrations for Musculoskeletal Health Previous attempts to define an optimal serum 25(OH)D for musculoskeletal health have used indirect measures such as the relationships between serum 25(OH)D and PTH concentrations in normal adult populations with a plateau of serum PTH above 31 ng/ml (78 nmol/l) [77]. A major role for active vitamin D metabolites is in stimulating gut calcium (and phosphate) absorption. This effect may not be uniform across all age groups and in humans there is evidence of an age-related
intestinal resistance to 1,25(OH)2D that may be secondary to reduced levels of intestinal VDR [78]. Despite an age-related increase in serum PTH and in serum 1,25(OH)2D concentration, intestinal VDR concentration decreased with age while active calcium absorption did not change with age. These data are most consistent with impaired intestinal responsiveness to 1,25(OH)2 action. This gut defect could lead to compensatory increases in PTH secretion and 1,25 (OH)2D3 production, which maintain calcium absorption and serum calcium, but at the expense of increased bone loss. Serum 25(OH)D concentrations are also related to active calcium absorption [79], albeit not as strongly, and one study showed a plateau in active intestinal calcium absorption at serum 25(OH)D concentrations 32 ng/ml (80 nmol/l). Others have related serum 25(OH)D concentrations to fracture risk. In a nested, caseecontrol study from the Women’s Health Initiative, 400 hip fracture patients and 400 controls were matched on the basis of age, race, or ethnicity, and date of blood draw were followed for a median of 7.1 years to assess fractures. Lower serum 25(OH)D concentrations were associated with increased hip fracture risk (adjusted OR for each 25 nmol/l decrease, 1.33). Women with the lowest 25(OH)D concentrations (47.5 nmol/l) had a higher hip fracture risk than did those with the highest concentrations (70.7 nmol/l) (adjusted OR, 1.71), and the risk increased statistically significantly across quartiles of serum 25(OH)D concentrations. Importantly, this association was independent of number of falls, physical function, frailty, renal function, and sex-steroid hormone levels and was, in part, mediated by increased bone resorption. Thus, serum 25(OH)D concentrations 20 ng/ml (50 nmol/l) are associated with a higher risk for hip fracture [80]. In another study of 1311 community-dwelling older Dutch men and women followed for 6 years, a low serum 25(OH)D level (<12 ng/ml or 30 nmol/l) increased the risk of fracture in those individuals aged 65e75 years (HR ¼ 3.1; 95% CI 1.4e6.9), but not in the older group of individuals (75e89 years) [81]. The above inferences about optimal serum 25(OH)D levels are drawn from association studies. Stronger evidence relates to the effects of vitamin D supplementation on musculoskeletal endpoints, particularly in a reduction in falls and fractures and the serum 25(OH)D threshold concentrations needed to achieve these outcomes. The anti-fracture efficacy of oral vitamin D supplementation in women and men 65 years old has been assessed in a meta-analysis of 12 double-blind randomized controlled trials (RCTs) for non-vertebral fractures (n ¼ 42 279) and eight RCTs for hip fractures (n ¼ 40 886) [82]. The pooled relative risks (RR) were 0.86 (95% CI; 0.77e0.96) and 0.91 (95% CI;
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0.78e1.05) for the prevention of non-vertebral fractures or hip fractures, respectively. However, there was significant heterogeneity for both endpoints. Factors explaining the heterogeneity were the daily dose and achieved serum 25(OH)D concentrations. When examining the trials with a high (482e770 IU/day) received dose (i.e., dose adjusted for adherence) of vitamin D, nonvertebral fractures were reduced by 20% and hip fractures by 18%, whereas doses <400 IU/day did not show any effect. Furthermore, vitamin D has been shown to improve muscle strength and performance and to reduce the risk of falling in community-dwelling, as well as in the institutionalized elderly. A meta-analysis including eight double-blind RCTs (n ¼ 2376) demonstrated that falling was significantly reduced by 13% in vitamin D supplemented individuals compared with those receiving either calcium or placebo. Again, a significant heterogeneity by daily dose and achieved 25(OH)D levels was observed. Higher doses of vitamin D supplements (700e1000 IU/day) reduced the relative risk of falls by 19% [83e84]. Collectively, this translates to an achieved serum 25(OH)D level of at least 24 ng/ml (60 nmol/l) for anti-fall efficacy and at least 30 ng/ml (or 75 nmol/l) for anti-fracture efficacy [85e86]. Thus, most clinicians would regard a target range for serum 25(OH)D between 20 and 30 ng/ml (50e75 nmol/l) as being required for optimal musculoskeletal health. However, a recent Institute of Medicine report recommended at least 20 ng/ml (50 nmol/l) [86a], which appears overly conservative and does not take season into account.
Effects of Vitamin D Alone or Calcium and Vitamin D on Bone Mineral Density (BMD) A systematic review of 17 randomized controlled trials evaluated the effect of supplemental vitamin D2 or D3 on BMD, predominantly in populations of late menopausal women [87]. Most trials had small sample sizes, were 2 to 3 years in duration and used vitamin D doses of 800 IU per day. In addition, most trials used vitamin D3 and also included calcium 500 mg as a co-intervention. Combined results of trials of vitamin D3 plus calcium versus placebo were consistent with small positive effects on lumbar spine, femoral neck and total body BMD. The WHI trial found a significant benefit of vitamin D3 400 IU plus 1000 mg of calcium on total hip BMD [88]. Although there were no effects on spine BMD, this large study had several potential cofounders including 30% of participants already taking a calcium supplement, 30% having a high dietary calcium intake (>1200 mg/day), 52% receiving current HRT and a large proportion of patients
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taking a bisphosphonate at study end and poor adherence with calcium and vitamin D supplements. However, in combined trials of vitamin D3 plus calcium versus calcium, a significant increase in BMD was not observed, suggesting vitamin D3 may be of less benefit in calcium-replete postmenopausal women. Vitamin D3 alone versus placebo did not show significant increases in BMD, except in one trial that noted an increase in femoral neck BMD [89]. Only a few trials reported the impact of baseline serum 25(OH)D concentrations on BMD and in all of these trials, a lower baseline serum 25(OH)D was not associated with a greater BMD increase. Overall, there is good evidence that vitamin D3 plus calcium results in small increases in BMD of the spine, total body, femoral neck, and total hip. Based on this systematic review, however, it is less certain if vitamin D3 alone has any significant effect on BMD. A further meta-analysis examined effects of calcium or calcium combined with vitamin D on BMD and fractures in men and women aged >50 years [90]. Of the 23 trials (n ¼ 41 419) reporting BMD as an outcome, calcium and calcium in combination with vitamin D were associated with a reduced bone loss of 0.54% at the hip, and 1.19% at the spine. A positive treatment effect on BMD was evident in most studies; however, the treatment effect of vitamin D alone was not assessed. Calcium and vitamin D supplementation during winter results in increases in spinal and femoral neck BMD, compared with decreases in those older men and women not receiving supplements [65]. One recent trial examined the effect of the combination of calcium and vitamin D on bone structure and bone turnover compared with calcium alone [91]. Three hundred and two elderly women with baseline serum 25(OH)D concentrations <24 ng/ml (60 nmol/l) participated in a 1-year randomized, double-blind, placebo-controlled trial of 1000 mg calcium per day with either 1000 IU ergocalciferol (vitamin D2) or identical placebo. Baseline calcium intake was 1100 mg/d, and serum 25(OH)D was 18 ng/ml (44 nmol/l); this increased only in the vitamin D group to 24 ng/ml (60 nmol/l) after 1 year. Total hip and total body BMD increased significantly, and procollagen type I intact N-terminal propeptide (PINP) decreased by approximately 4% during the study, with no difference between the treatment groups. In addition, the increase in serum 25(OH)D achieved with vitamin D supplementation had no extra effect on active fractional intestinal calcium absorption, which fell equally in both groups by 15e17%. Thus, ergocalciferol (vitamin D2) 1000 IU for 1 year had no extra beneficial effect on bone structure, bone formation markers, or intestinal calcium absorption over an additional 1000 mg of calcium. In the same cohort, falls risk was measured every 6 months. Ergocalciferol (vitamin D2) 1000 IU combined
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with calcium reduced the risk of having at least one fall over 1 year by 39% compared with calcium supplementation alone [92]. When those who fell were grouped by either the season of first fall or the number of falls, ergocalciferol treatment reduced the risk of having the first fall in winter and spring (ergocalciferol group, 25.2%; control group, 35.8%; OR 0.55), but not in summer and autumn, and reduced the risk of having one fall by 50%, but not the risk of multiple falls. Thus, patients with a history of falling and vitamin D insufficiency benefited from vitamin D supplementation in addition to calcium, which was associated with an overall 19% reduction in the relative risk of falling, mostly in winter. A 5-year randomized, controlled, double-blind trial of 120 community-dwelling women aged 70e80 years compared 1200 mg/day calcium with placebo vitamin D (Ca group) or with 1000 IU/d ergocalciferol (vitamin D2) (CaD group), or double placebo (control) [93]. Hip BMD was preserved in both intervention groups, but not in controls at year 1 and maintained in the CaD group only over the long term at years 3 and 5. At year 1, compared with controls, the Ca and CaD groups had lower bone turnover markers. At 5 years, the suppression of bone formation and bone resorption markers was maintained only in the CaD group, and this decrease was also associated with reductions in PTH at 3 and 5 years compared with controls. Thus, although the combination of calcium with vitamin D had no additional effect over calcium alone in the short term (at 1 year), continuing skeletal benefits appear to be greater with this combination over the long term (up to 5 years). Another study examined effects of calcium and vitamin-D-fortified milk compared with normal diet over 24 months in older men [94]. Men received milk containing 1000 mg of calcium plus 800 IU of vitamin D3 or no additional milk. After 2 years, the mean percent change in BMD was 0.9e1.6% less in the milk supplementation compared with control group at the femoral neck, total hip, and radius. There was a greater increase in lumbar spine BMD in the milk supplementation group after 12 and 18 months, but not after 2 years. Serum 25(OH)D increased and PTH decreased in the milk supplementation relative to control group after both the first and second years of the study. After a further 18 months of follow-up, during which neither group received calcium- and vitamin-D3-fortified milk, the net beneficial effects of fortified milk on femoral neck and radius BMD at the end of the intervention (24 months) were sustained [95]. Non-significant between-group differences at the total hip also persisted at follow-up, but there were no lasting benefits at the lumbar spine, suggesting the possibility of sustained skeletal benefits after withdrawal.
Conclusion Treatment with the combination of calcium and vitamin D prevents bone loss and results in small increases in BMD at most sites. For long-term maintenance of BMD up to 5 years, the combination of calcium and vitamin D appears to be better than calcium alone. These skeletal benefits of calcium and vitamin D may be maintained at some, but not all, skeletal sites after withdrawal. However, there are no data to suggest that vitamin D alone is effective in maintaining or in increasing BMD. The addition of vitamin D to calcium is also likely to reduce the risk of falling, particularly in winter, in patients with a history of falling and vitamin D insufficiency. This may result in a reduction of falls-related fractures.
PRIMARY FRACTURE PREVENTION WITH VITAMIN D OR CALCIUM AND VITAMIN D Primary fracture prevention is the most important potential role for vitamin D supplementation. However, most studies have been performed using a combination of calcium and vitamin D supplementation rather than vitamin D alone, of which there are only a few studies. The quality of many studies has also been reduced first by poor long-term compliance with study medications and rates have mostly ranged from 55 to 75%, and second a high degree of variability of dosing regimens.
Effect of Vitamin D Alone on Fractures The study showing the most benefit of vitamin D supplementation alone on fracture reductions was by Trivedi et al. [96]. They studied 2037 men and 649 women aged 65e85 years, predominantly doctors, living in the general community in a randomized double-blind controlled trial of 100 000 IU oral vitamin D3 (cholecalciferol) or matching placebo every 4 months over 5 years. After 5 years 268 men and women had incident fractures, of whom 147 had fractures in common osteoporotic sites (hip, wrist or forearm, or vertebrae). Relative risks in the vitamin D group compared with the placebo group were 0.78 for any fracture (Fig. 61.2) and 0.67 for first hip, wrist or forearm, or vertebral fracture. Four hundred and seventy-one participants died; however, the relative risk for total mortality in the vitamin D group compared with the placebo group was not significantly reduced (RR ¼ 0.88). Meta-analyses, however, show no overall effect of vitamin D alone on fracture risk. A Cochrane review of 38 lower-quality randomized controlled trials in postmenopausal women or men over 65 years compared
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FIGURE 61.2 Cumulative probability of any first fracture with 100 000 IU cholecalciferol every 4 months versus placebo over 5 years; p ¼ 0.04 [96]. Reproduced from D.P. Trivedi, R. Doll, K.T. Khaw, Effect of four monthly oral vitamin D3 (cholecalciferol) supplementation on fractures and mortality in men and women living in the community: randomised double blind controlled trial, BMJ 326 (2003) 469, with permission from BMJ publishing group.
vitamin D supplements to placebo, no intervention, or calcium supplements [97]. Medication regimens varied from calcium 1000 mg to 1200 mg and vitamin D3 700 to 800 IU daily. Results from seven trials (10 376 participants) showed a significant reduction in incidence of new hip fracture (ES 0.81; 95% CI 0.68e 0.96) and nonvertebral fractures (RR 0.87; 95% CI 0.78e0.97) for vitamin D combined with calcium; however, results for institutionalized older adults may have influenced the overall analysis, as no significant effect was found for community-dwelling individuals. There was no evidence for the effectiveness of vitamin D alone for prevention of fractures. A good-quality systematic review (nine randomized controlled trials, n ¼ 53 260) investigated the need for calcium supplementation (500 to 1200 mg daily) in postmenopausal women and men receiving vitamin D (cholecalciferol 700 to 800 IU daily (six randomized controlled trials) or 400 IU daily (three randomized controlled trials)) for prevention of fractures [98]. Mean therapy duration was 20 to 84 months. Pooled results showed vitamin D alone was not associated with a reduction in risk of hip fracture (RR 1.10; 95% CI 0.89e1.36, p ¼ 0.38) or a reduction in risk of non-vertebral fractures (RR 0.98; 95% CI 0.83e1.16; p ¼ 0.79) compared with placebo. An indirect comparison of trials investigating vitamin D with calcium compared to those investigating vitamin D alone showed an RR of 0.75 (95% CI 0.58e0.96, p ¼ 0.021) for hip fracture. Another meta-analysis addressed the effect of vitamin D supplementation on all fractures in postmenopausal women and men aged 50 years or older [87].
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The pooled results for all fractures included ten double-blind randomized controlled trials and three open-study-design trials (n ¼ 58 712) and did not show a significant reduction of fractures with vitamin D alone (pooled odds ratio, 0.90; 95% CI 0.81e1.02). In this report, the benefit of vitamin D depended on additional calcium and was also primarily seen in institutionalized individuals. Such negative results might be explained by heterogeneity between studies in doses of vitamin D. In a further meta-analysis of 12 double-blind randomized controlled trials among individuals aged 65 years or older, the antifracture efficacy of supplemental vitamin D increased significantly with either a higher received dose or a higher achieved 25(OH)D level for any non-vertebral fractures and for hip fractures [82]. No fracture reduction was observed for a received daily dose of 400 IU, whereas a higher received daily dose of 482 to 770 IU of supplemental vitamin D reduced nonvertebral fractures by 20% and hip fractures by 18%. Subgroup analyses for the prevention of non-vertebral fractures with the higher received dose suggested possibly better fracture reduction with cholecalciferol compared with ergocalciferol, whereas additional calcium did not further improve anti-fracture efficacy. Non-vertebral fracture reduction with the higher received dose was significant among all subgroups by age and dwelling, including younger individuals aged 65 to 74 years and those living in the community. Using individual patient data from seven major randomized trials of vitamin D with calcium or vitamin D alone, yielding a total of 68 517 participants with a mean age of 70 years (14.7% men), the results indicated that vitamin D given alone in daily doses of 10e20 mg (400e800 IU) was not effective in preventing fractures [99]. However, trials using vitamin D with calcium showed a reduced overall risk of fracture (hazard ratio 0.92; 95% confidence interval 0.86e0.99, p ¼ 0.025) and hip fracture (all studies: 0.84; 0.70e1.01, p ¼ 0.07; studies using 10 mg (400 IU) of vitamin D given with calcium: 0.74; 0.60e0.91, p ¼ 0.005). No interaction was found between fracture history and treatment response, nor was there any interaction with age, sex, or hormone replacement therapy.
Conclusion Overall evidence from several meta-analyses shows no effect of vitamin D alone on fracture risk. Although the effects of vitamin D on fracture risk may be masked by heterogeneity of the received daily doses between studies, the evidence for vitamin D reducing the risk of non-vertebral and hip fractures is most compelling with the use of additional calcium.
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FIGURE 61.3 Increase in falls and fractures after an annual dose of 500 000 IU cholecalciferol [102]. Reprinted from K.M. Sanders, A.L. Stuart, E.J. Williamson, et al., Annual high-dose oral vitamin D and falls and fractures in older women: a randomized controlled trial, JAMA 303 (2010) 1815e1822, with permission from the American Medical Association.
Single Annual Doses of Vitamin D The first study investigating single large annual doses of vitamin D utilized an annual intramuscular injection of 150 000e300 000 IU vitamin D2 (ergocalciferol) in 479 Finnish men and women aged >85 years, who were living in their own home and 320 subjects aged 75e84 years living in a home for aged people [100]. Although treatment allocation was not adequately randomized or blinded, the group of subjects administered intramuscular vitamin D had a significantly lower rate of all fractures. This was particularly marked for fractures of the upper limb, but was not apparent for lower limb sites. The second study was a randomized, double-blind, placebo-controlled trial of 300 000 IU intramuscular vitamin D2 (ergocalciferol) injection or matching placebo every autumn over 3 years. Nine thousand four hundred and forty people (4354 men and 5086 women) aged 75 years were recruited from general practices in England [101]. Five hundred and eighty-five subjects had incident non-spine fractures (hip 110, wrist 116, ankle 37). Hazard ratios (HRs) for fracture in the vitamin D group were: 1.09 (95% confidence interval (CI) 0.93e1.28) for any first fracture, 1.49 (95% CI 1.02e2.18) for hip and 1.22 (95% CI 0.85e1.76) for wrist. There was no effect on falls: HR 0.98 (0.93e1.04). When the analyses were confined to fractures at the wrist, hip or both of these sites, fracture incidence tended to be higher in the vitamin-D-treated arm when compared with placebo. However, these differences were not statistically significant. There was, however, a significant interaction between gender and treatment group on the risk of any non-vertebral fracture, such that a detrimental effect of vitamin D was seen among women, but not among men (p < 0.03). This implies annual intramuscular vitamin D2 injections may have increased the risk of non-vertebral fractures in women in this study.
These data were confirmed in a recent double-blind, randomized controlled trial of annual doses of oral cholecalciferol 500 000 IU, given in autumn or winter, versus placebo performed by Sanders et al. [102]. Two thousand two hundred and fifty-six communitydwelling women, aged 70 years, were randomly assigned to receive cholecalciferol or placebo each autumn to winter for 3 to 5 years. Falls and fractures were ascertained using monthly calendars; details were confirmed by telephone interview, while fractures were radiologically confirmed. Women in the cholecalciferol (vitamin D) group had 171 fractures versus 135 in the placebo group; the fall rate was 83.4 per 100 person-years in the vitamin D group, and 72.7 per 100 person-years in the placebo group; incidence rate ratio (RR), 1.15, p ¼ 0.03. The incidence RR for fracture in the vitamin D group was 1.26 (95% CI 1.00e1.59, p ¼ 0.047) versus the placebo group (rates per 100 person-years, 4.9 vitamin D versus 3.9 placebo) (Fig. 61.3). A temporal pattern was observed in a post-hoc analysis of falls. The incidence RR of falling in the vitamin D group versus the placebo group was 1.31 in the first 3 months after dosing and 1.13 during the following 9 months (test for homogeneity; p ¼ 0.02). Although not significant, the incidence RR for fractures was also increased in the vitamin D group versus the placebo group in the first 3 months compared with the following 9 months after dosing (RR 1.53 versus 1.18). The median baseline serum 25 (OH)D was 49 nmol/l in this community-based study. In the vitamin D group, serum 25(OH)D levels increased at 1 month after dosing to approximately 120 nmol/l, and were approximately 90 nmol/l at 3 months. The cause of the increased rate of falls or fractures after dosing is unknown, but might relate to rapid fluxes in vitamin D metabolites or vitamin-D-regulated genes or, perhaps, even to increased levels of physical activity.
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Conclusion Large annual doses of vitamin D are not recommended either to treat vitamin D deficiency or to prevent fractures. In addition, the safety of high-dose vitamin D supplementation warrants further study, as the levels of 25(OH)D seen in this study (48 ng/ml or 120 nmol/l) may have had detrimental effects on falls and fractures in older women. In this regard, studies using either monthly doses of 50 000 IU cholecalciferol [103] or a loading dose of ten daily doses of 50 000 IU cholecalciferol [104] achieved more modest increases in serum 25(OH)D to just above the optimal target range (75 nmol/l) at 3 months.
Primary Fracture Prevention with Calcium and Vitamin D The effects of calcium and vitamin D on fractures have been studied more extensively than vitamin D alone. The pivotal study from which many meta-analyses derive their strength was performed in 3270 ambulatory elderly women (mean age 84 years) residing in institutional care (nursing homes or apartments) [105]. Supplementation with 1.2 g calcium and 800 IU cholecalciferol reduced hip fractures and non-vertebral fractures by 43% and 32%, respectively, and resulted in increases in hip BMD over 18 months (Fig. 61.4). The mean baseline serum 25(OH)D concentration was 16 ng/ml (40 nmol/l), meaning most women were vitamin D insufficient or deficient. The number needed to treat to prevent a hip or non-vertebral fracture was 41 and 29, respectively. These data were confirmed in a later study performed by the same authors [106]. Calcium and vitamin D also reduce fractures in community-dwelling healthy men and women. In 176 men and 213 women with a mean age of 70e72 years and having mean calcium intakes of 673e798 mg per day, 500 mg calcium and 700 IU
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cholecalciferol reduced non-vertebral fractures by 50% over 3 years and resulted in increases in hip, spine, and total body BMD [107]. The number needed to treat to prevent a non-vertebral fracture was 15. The largest study of calcium and vitamin D used daily doses of cholecalciferol (400 IU) that would now be considered to be suboptimal and the mean baseline calcium intake in this study was also high (1150 mg/d) [88]. Thirty-six thousand, two hundred and eighty-two postmenopausal women, aged 50 to 79 years, were randomly assigned to receive 1000 mg calcium with 400 IU cholecalciferol per day or placebo. Participants receiving calcium plus vitamin D supplementation had a non-significant decrease in hip fracture risk (HR 0.88 (95% CI 0.72e1.08)), HR 0.90 for clinical spine fracture (0.74e1.10), and HR 0.96 for total fractures (0.91e1.02). The risk of renal calculi increased with calcium plus vitamin D (HR 1.17; 1.02e1.34). Censoring data from women when they ceased to adhere to the study medication indicated a reduction in hip fracture risk by 29% (HR 0.71; 0.52e0.97). The most recent study of 3432 community-dwelling northern Finnish women aged 65e71 years assessed incident fractures in women randomly assigned to receive 1000 mg calcium with 800 IU of cholecalciferol per day or placebo over 3 years [108]. The risk of any fracture decreased in the vitamin D and calcium group by 17% (95% CI 0.61e1.12), and the risk of any non-vertebral fracture decreased by 13% (0.63e1.19). The risk of distal forearm fractures decreased by 30% (0.41e1.20), and the risk of any upper extremity fractures decreased by 25% (0.49e1.16), whereas the risk of lower-extremity fractures did not change. However, none of these effects reached statistical significance. This study provides further evidence that vitamin D and calcium supplementation does not prevent fractures in older community-dwelling postmenopausal women. Effects of calcium 1200 mg and cholecalciferol 800 IU per day on hip and non-vertebral fractures in ambulatory elderly women resident in nursing homes or apartments [105].
FIGURE 61.4
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Meta-analysis of fracture risk reduction with calcium or calcium and vitamin D [90]. Reprinted from B.M. Tang, G.D. Eslick, C. Nowson, C. Smith, A. Bensoussan, Use of calcium or calcium in combination with vitamin D supplementation to prevent fractures and bone loss in people aged 50 years and older: a meta-analysis, The Lancet 370 (2007), with permission from Elsevier.
FIGURE 61.5
Meta-analyses Meta-analyses indicate mixed evidence for the impact of oral calcium and vitamin D supplementation on reduction of fractures outside institutionalized settings. One good-quality systematic review (29 studies, 63 867 individuals, 92% female) reported on the effect of calcium supplementation (alone or in combination with vitamin D) in doses of 1000 to 1200 mg in adults aged over 50 years [90]. Overall, calcium supplementation was associated with a 12% reduction in risk of any fracture (RR 0.88; 95% CI 0.83e0.95), with similar reduction in risk of fractures in trials using calcium supplements alone (10% risk reduction) and those where calcium was administered in combination with vitamin D (13% risk reduction) (Fig. 61.5). The estimated NNT to prevent one fracture over 3.5 years was 63 in the overall population. For individuals who were elderly, lived in institutions, had a low body weight, had a low calcium intake (less than 700 mg per day), or were at a higher baseline risk of fracture, the NNT to prevent one fracture over 3.5 years was 30. The fracture risk reduction was significantly greater (24%) in trials in which the compliance rate was high (p < 0.0001). The treatment effect was better with daily calcium doses 1200 mg than with doses <1200 mg (0.80 versus 0.94; p ¼ 0.006), and with daily vitamin D doses of 800 IU than with doses <800 IU (0.84 versus 0.87; p ¼ 0.03). People with low vitamin D serum 25(OH)D concentrations
(<25 nmol/l) had a greater risk reduction compared with those whose serum 25(OH)D was normal (RR 0.86 versus 0.94); however, this result was not significant (p ¼ 0.06). A good-quality systematic review (nine randomized control trials, n ¼ 53 260) investigated the need for calcium supplementation (500 to 1200 mg per day in postmenopausal women and men receiving vitamin D (either cholecalciferol 700 to 800 IU per day (six randomized control trials) or 400 IU daily (three randomized control trials)) for prevention of fractures [98]. Mean therapy duration was 20 to 84 months. Results (six randomized control trials, n ¼ 45 509) of vitamin D in conjunction with calcium supplements compared with placebo or no treatment showed a significant reduction in risk of both hip fracture (RR 0.82; 95% CI 0.71e0.94) and non-vertebral fracture (0.88; 0.78e0.99). The NNT to prevent one hip fracture over 24 to 84 months was 276 and NNT to prevent one non-vertebral fracture was 72. An indirect comparison of trials investigating vitamin D with calcium compared to those investigating vitamin D alone showed a significant risk reduction in hip fracture (RR 0.75; 0.58e0.96). A Cochrane review of 38 lower-quality randomized control trials in postmenopausal women or men over 65 years compared vitamin D supplements to placebo, no intervention, or calcium supplements [97]. Medication regimens varied from calcium 1000 mg to 1200 mg and vitamin D3 700 to 800 IU daily. Results from seven
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trials (10 376 participants) showed a significant reduction in incidence of new hip fracture (ES 0.81; 95% CI 0.68e0.96) and non-vertebral fractures (RR 0.87; 95% CI 0.78e0.97) for vitamin D combined with calcium; however, results for institutionalized older adults may have influenced the overall analysis, as no significant effect was found for community-dwelling individuals. Conclusion There is mixed evidence on the effectiveness of vitamin D supplementation for the prevention of minimal trauma fractures in postmenopausal women and older men. There may be some benefit on primary fracture prevention for those who have inadequate serum levels of 25(OH)D, particularly in institutionalized patients, and when combined with calcium supplements. In women and men aged >50 years, the combination of vitamin D with calcium, but not vitamin D alone, has a modest effect in preventing fractures, particularly in those with long-term compliance rates 80%. The daily dose of vitamin D should be at least 800 IU (20 mg) with larger monthly doses being an alternative. Large single annual doses of vitamin D may increase fractures and falls and are not recommended.
Safety Vitamin D Vitamin D alone is safe at daily doses of 2000 IU per day and is likely to be safe up to 4000 IU per day. However, the recent study of Sanders et al. showing an increased risk for falls and, possibly, fractures in the first 3 months following an annual injection of 500 000 IU means the safety of high-dose vitamin D supplementation warrants further study (see above). Calcium and Vitamin D The safety of the combination of calcium and vitamin D has not been comprehensively assessed; however, concern has recently been raised regarding the safety of calcium supplements. One randomized control trial (n ¼ 1471) [109] and one meta-analysis using either individual patient or trial data [110] have reported on cardiovascular (CV) adverse events associated with calcium supplements compared to placebo or calcium and vitamin D in elderly, postmenopausal women. In the trial, there was no significant difference between groups in the risk of any CV event (angina, chest pain, myocardial infarction (MI), or sudden death), risk of stroke or risk of sudden death. The risk of MI was not significant between groups for number of validated events (RR 1.49; 95% CI 0.86e2.57). For the primary endpoint (risk of MI, stroke, or sudden death) there was no significant difference in number of validated events (RR 1.21;
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0.84e1.74); however, the rate ratio showed a significant increase associated with calcium (RR 1.43; 1.01e2.04, p ¼ 0.043). In the meta-analysis of individual patient data, the risk of myocardial infarction was increased (HR 1.31; 95% CI 1.02e1.67, p ¼ 0.035) [109]. The meta-analysis of trial level data showed similar results, with an increased incidence of MI in those allocated to calcium (RR 1.27; 1.01e1.59, p ¼ 0.038) [109]. However, it should be noted that all trials were designed to assess the effect of calcium on BMD and the power to detect a clinical effect for cardiovascular outcome measures is not reported. In addition, it remains unclear whether the addition of vitamin D might attenuate the proposed adverse cardiovascular effects of calcium supplements.
SECONDARY FRACTURE PREVENTION WITH VITAMIN D OR CALCIUM AND VITAMIN D Either vitamin D alone or the combination of calcium and vitamin D are ineffective in reducing fractures in patients who have already had a minimal trauma fracture. A large study of 5292 women and men (15%) aged >70 years with prevalent minimal trauma fractures examined secondary fracture prevention using 800 IU cholecalciferol (vitamin D3) per day versus 1000 mg calcium per day, or the combination of calcium and vitamin D3, or placebo over 2e5.2 years [111]. Thirteen percent of participants had a new minimal trauma fracture, 183 (26%) of which were hip fracture. The incidence of new, minimal trauma fractures did not differ significantly between participants allocated calcium and those who were not (HR 0.94; 95% CI 0.81e1.09); between participants allocated vitamin D3 and those who were not (1.02; 0.88e1.19); or between those allocated combination treatment and those assigned placebo (HR for interaction term 1.01; 0.75e1.36). The groups did not differ in the incidence of all-new fractures, fractures confirmed by radiography, hip fractures, death, number of falls, or quality of life. This trial was notable for low adherence (54.5%) with tablets at 2 years and the ability of patients to start other anti-osteoporotic therapy during the study. Compliance with tablets containing calcium was also significantly lower (difference: 9.4% 6.6e12.2), partly because of gastrointestinal symptoms. In a primary care, nurse-led study of 3314 women aged 70 and over with one or more risk factors for hip fracture, including the presence of a previous fracture, daily oral supplementation with 1000 mg calcium and 800 IU cholecalciferol was given for 2 years in an open randomized trial [112]. Adherence with therapy at 2 years was low (55%). After a median follow-up of 25 months (range 18 to 42 months), clinical fracture rates
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were lower than expected in both groups, but did not significantly differ for all clinical fractures (HR 1.01; 95% CI 0.71e1.43). The odds ratio for hip fracture was 0.75 (0.31e1.78). The odds of a woman having a fall at 6 and 12 months also did not differ between groups.
Conclusion Despite the limitation of poor adherence in both studies, there is no evidence that calcium or vitamin D, either alone or in combination, is effective in reducing fractures in older women and men with pre-existing minimal trauma fractures. In these individuals, antiosteoporotic drugs should be used instead. Interestingly, in women treated with commonly used anti-resorptive drugs, treatment responses are improved in those with optimal serum 25(OH)D levels. In an Italian study, 1515 women with postmenopausal osteoporosis treated with anti-resorptive drugs (alendronate, risedronate, raloxifene) for 13.1 months with an adherence rate >75% were classified as vitamin D deficient (<20 ng/ ml or 50 nmol/l) (n ¼ 453) or replete [113]. Vitamin-Ddeficient or -replete subjects differed significantly in annualized spine and hip bone mineral density (BMD) changes adjusted for confounding factors. One hundred and fifty-one patients suffered from a new incident clinical fracture. The adjusted odds ratio for incident fractures in vitamin-D-deficient compared with vitaminD-replete women was 1.77 (1.20e2.59, p ¼ 0.004). Thus, a target serum level 20 ng/ml or 50 nmol/l should be aimed for in women and men on anti-resorptive drugs to optimize skeletal responses. Most patients will require 800e2000 IU cholecalciferol per day to achieve these levels [114]. In addition, such levels will minimize the risk of hypocalcemia following bisphosphonate therapy [115e116] and may reduce the severity of the acute-phase reaction commonly seen after the first intravenous infusion of zoledronic acid [117].
EFFECTS OF ACTIVE VITAMIN D ANALOGS ON FRACTURES There is no evidence that related vitamin D compounds (analogs) have advantages in terms of effectiveness or reduced incidence of adverse effects compared with vitamin D. Four small trials of alfacalcidol, of which three were in Japan by the same author in patients with neurological diseases [118e121], suggest that alfacalcidol was effective in reducing the incidence of hip fractures in older people with and without preexisting osteoporotic fractures (four trials, 371 participants, RR 0.18; 95% CI 0.05e0.67). Positive results from these small studies need to be confirmed by other investigators in larger studies. Other small studies that
examined effects on fractures at other skeletal sites, and compared alfacalcidol with calcium (with or without vitamin D), or alfacalcidol and calcium with calcium, were inconclusive [122]. Two trials compared calcitriol with calcium [123e124]. Overall, there was no statistically significant effect on the incidence of non-vertebral fractures (two trials, n ¼ 663 participants, RR 1.19; 95% CI 0.09e15.77) or vertebral deformities (two trials, n ¼ 556 participants, RR 1.69; 95% CI 0.25e11.28). In Tilyard et al. [124] the duration of treatment was critical. At the end of 1 year, no effect could be shown. Fewer vertebral deformities occurred in the calcitriol group during the second year (RR 0.47; 95% CI 0.26e0.87), and also during the third year (RR 0.28; 95% CI 0.15e0.52). Thus, the effect of calcitriol in fracture prevention is unclear, with the best evidence for effectiveness coming from the trial where vertebral deformities were significantly reduced only in the second and third years. However, the use of calcitriol was associated with a statistically significant increase in the risk of hypercalcemia, so it is not recommended as a first-line drug to treat osteoporosis.
The Anabolic Vitamin D Analog, 2MD Most anti-osteoporosis drugs, including active vitamin D analogs, act by inhibiting bone resorption. 2MD (2-methylene-19-nor-(20S)-1a,25-dihydroxyvitamin D3) is a novel vitamin D analog that is a potent bone formation stimulating drug in vitro and also in vivo in an ovariectomized rat model [125]. However, in a randomized, double-blind, placebo-controlled study of osteopenic women, although daily oral treatment with 2MD caused a marked increase in markers of bone formation, it did not significantly increase BMD [126]. In this study 2MD also caused a marked increase in bone resorption, so 2MD stimulated both bone formation and bone resorption, thereby increasing bone remodeling. This critical difference between the preclinical and clinical studies could reflect underlying differences in bone metabolism, including continuing skeletal modeling in the rat.
FUTURE DIRECTIONS FOR VITAMIN D IN OSTEOPOROSIS There is mixed evidence on the effectiveness of vitamin D supplementation for the prevention of bone loss and minimal trauma fractures in postmenopausal women and older men. There may be some benefit on primary fracture prevention for those who have inadequate serum levels of 25(OH)D, particularly in institutionalized patients, and when combined with calcium supplements. However, there is little evidence that
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vitamin D alone can prevent fractures, and no evidence that the combination of calcium and vitamin D, or either alone, can prevent fractures in patients with pre-existing minimal trauma fractures. The mechanisms occurring in muscle and bone whereby a large single dose of vitamin D may result in the unexpected finding of falls and fractures, respectively, need to be determined. In addition, the role of recently identified genetic variants in explaining the high variability of clinical responses of serum 25(OH)D concentrations to either UVB exposure or to vitamin D supplementation needs to be elucidated. Other less common genetic variants that may also affect serum 25(OH)D levels need to be identified in order to determine individualized doses of vitamin D supplements to attain optimal target levels of serum 25(OH)D. Finally, the links between the vitamin D and sex steroid axes in regulating age-related bone loss in both sexes need to be elucidated.
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[95] R.M. Daly, N. Petrass, S. Bass, C.A. Nowson, The skeletal benefits of calcium- and vitamin D3-fortified milk are sustained in older men after withdrawal of supplementation: an 18-mo follow-up study, Am. J. Clin. Nutr. 87 (2008) 771e777. [96] D.P. Trivedi, R. Doll, K.T. Khaw, Effect of four monthly oral vitamin D3 (cholecalciferol) supplementation on fractures and mortality in men and women living in the community: randomised double blind controlled trial, BMJ 326 (2003) 469. [97] A. Avenell, W.J. Gillespie, L.D. Gillespie, D.L. O’Connell, Vitamin D and vitamin D analogues for preventing fractures associated with involutional and post-menopausal osteoporosis, Cochrane Database Syst. Rev. (2006) CD000227. [98] S. Boonen, P. Lips, R. Bouillon, H.A. Bischoff-Ferrari, D. Vanderschueren, P. Haentjens, Need for additional calcium to reduce the risk of hip fracture with vitamin D supplementation: evidence from a comparative metaanalysis of randomized controlled trials, J. Clin. Endocrinol. Metab. 92 (2007) 1415e1423. [99] B. Abrahamsen, T. Masud, A. Avenell, F. Anderson, H.E. Meyer, C. Cooper, et al., Patient level pooled analysis of 68 500 patients from seven major vitamin D fracture trials in US and Europe, BMJ 340 (2010) b5463. [100] R.J. Heikinheimo, J.A. Inkovaara, E.J. Harju, M.V. Haavisto, R.H. Kaarela, J.M. Kataja, et al., Annual injection of vitamin D and fractures of aged bones, Calcif. Tissue Int. 51 (1992) 105e110. [101] H. Smith, F. Anderson, H. Raphael, P. Maslin, S. Crozier, C. Cooper, Effect of annual intramuscular vitamin D on fracture risk in elderly men and women e a population-based, randomized, double-blind, placebo-controlled trial, Rheumatology (Oxford) 46 (2007) 1852e1857. [102] K.M. Sanders, A.L. Stuart, E.J. Williamson, J.A. Simpson, M.A. Kotowicz, D. Young, et al., Annual high-dose oral vitamin D and falls and fractures in older women: a randomized controlled trial, JAMA 303 (2010) 1815e1822. [103] C.J. Bacon, G.D. Gamble, A.M. Horne, M.A. Scott, I.R. Reid, High-dose oral vitamin D3 supplementation in the elderly, Osteoporos Int. 20 (2009) 1407e1415. [104] K.L. Hackman, C. Gagnon, R.K. Briscoe, S. Lam, M. Anpalahan, P.R. Ebeling, Efficacy and safety of oral continuous low-dose versus short-term high-dose vitamin D: a prospective randomised trial conducted in a clinical setting. Med. J. Aust. 192 (2010) 686e689. [105] M.C. Chapuy, M.E. Arlot, F. Duboeuf, J. Brun, B. Crouzet, S. Arnaud, et al., Vitamin D3 and calcium to prevent hip fractures in the elderly women, N. Engl. J. Med. 327 (1992) 1637e1642. [106] M.C. Chapuy, R. Pamphile, E. Paris, C. Kempf, M. Schlichting, S. Arnaud, et al., Combined calcium and vitamin D3 supplementation in elderly women: confirmation of reversal of secondary hyperparathyroidism and hip fracture risk: the Decalyos II study, Osteoporos. Int. 13 (2002) 257e264. [107] B. Dawson-Hughes, S.S. Harris, E.A. Krall, G.E. Dallal, Effect of calcium and vitamin D supplementation on bone density in men and women 65 years of age or older, N. Engl. J. Med. 337 (1997) 670e676. [108] K. Salovaara, M. Tuppurainen, M. Karkkainen, T. Rikkonen, L. Sandini, J. Sirola, et al., Effect of vitamin D(3) and calcium on fracture risk in 65- to 71-year-old women: a population-based 3-year randomized, controlled trialethe OSTPRE-FPS, J. Bone Miner. Res. 25 (2010) 1487e1495. [109] M. Bolland, B. Mason, A. Horne, R. Ames, G.D. Gamble, A. Grey, et al., Calcium supplementation improves lipid prpofile but does not decrease the incidence of cardiovascular events in postmenopausal women, BMJ (2008) 262e266. [110] M.J. Bolland, A. Avenell, J.A. Baron, A. Grey, G.S. MacLennan, G.D. Gamble, et al., Effect of calcium supplements on risk of
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62 Relevance of Vitamin D Deficiency in Adult Fracture and Fall Prevention Heike Bischoff-Ferrari 1, Bess Dawson-Hughes 2 1
University of Zurich and University Hospital Zurich, Zurich, Switzerland 2 Tufts University, Boston, MA, USA
MUSCLE EFFECTS OF VITAMIN D Four lines of evidence support a role of vitamin D in muscle health. First, proximal muscle weakness is a prominent feature of the clinical syndrome of vitamin D deficiency [1]. Clinical findings in vitamin D deficiency myopathy include proximal muscle weakness, diffuse muscle pain, and gait impairments such as waddling [2]. Second, the vitamin D receptor (VDR) is expressed in human muscle tissue [3,4], and VDR activation may promote de novo protein synthesis in muscle [5]. Mice lacking the VDR show a skeletal muscle phenotype with smaller and variable muscle fibers and persistence of immature muscle gene expression during adult life [6,7]. These abnormalities persist after correction of systemic calcium metabolism by a rescue diet [7]. Third, several observational studies suggest a positive association between 25-hydroxyvitamin D (25(OH)D) and muscle strength or lower-extremity function in older persons [8,9]. Fourth, vitamin D supplementation increases muscle strength and balance [10,11], and reduces the risk of falling in community-dwelling individuals [11e13], as well as in institutionalized individuals [10,14] in several double-blind randomized-controlled trials (RCTs) summarized in a 2009 meta-analysis discussed below [15]. See Chapter 104 for additional discussion of vitamin D effects on skeletal muscle. Regarding data from clinical trials, one uncontrolled biopsy trial in postmenopausal women with osteoporosis documented a relative increase in the diameter and number of type II muscle fibers after a 3-month treatment with 1-alpha-calcidiol (1a-(OH)2D3) [5]. These findings were supported by three recent double-blind RCTs with 800 IU vitamin D3 resulting in a 4e11% gain
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10062-9
in lower-extremity strength or function [10,11], and an up to 28% improvement in body sway [11,13] in older adults aged 65þ after between 2 and 12 months of treatment.
VITAMIN D MODULATES FRACTURE RISK IN TWO WAYS: BY INCREASING BONE DENSITY AND DECREASING FALLS Vitamin D is essential for bone growth [16,17] and preservation [18], and higher 25(OH)D levels are associated with higher bone density in younger and older adults [19]. Also, in double-blind RCTs, vitamin D supplementation increased bone density and reduced bone loss [20,21]. The effects of vitamin D on bone, bone density, and fracture are discussed in detail in Chapter 61. Severe vitamin D deficiency (serum concentrations below 25 nmol/l 25(OH)D) in the senior population causes secondary hyperparathyroidism, osteoporosis, and osteomalacia [22]. Histologic osteomalacia, characterized by accumulation of unmineralized matrix or osteoid in the skeleton, has been found to be common in several but not all hip fracture case studies (12e44%) [23e28]. At the same time it is well recognized that severe vitamin D deficiency is prevalent in about 50% of hip fracture patients [29,30] and that vitamin D supplementation reduces the risk of hip fracture [31]. Thus, it is conceivable that a significant fraction of hip fractures occurring in seniors are explained by osteomalacic changes that soften bone. Additionally, as a primary clinical sign of osteomalacia, muscle weakness may contribute to the risk of fracture [32].
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In fact, the direct effect of vitamin D on bone may not be the only explanation for its protective effect against fractures. Vitamin D deficiency may cause muscular impairment even before adverse effects on bone occur [33]. Further supported by the presence of the VDR in human muscle tissue [3] and an early (within 2 to 5 months) [10,14] and sustained [12,34e36] effect of vitamin D on falls [15], the observed fracture reduction with vitamin D may be modulated in part by its benefit on muscle. Moreover, the early effect of vitamin D supplementation on fall prevention [15] may explain a fracture reduction that was apparent within 6 months of treatment in the Boston STOP-IT [37] and the Decalyos I studies [38]. The early fracture risk reduction cannot be explained by bone density changes alone. This dual-benefit vitamin D on bone and muscle is especially attractive among seniors who have a high incidence of non-skeletal risk factors for fracture [39]. Mechanistically, fractures at later age are closely linked to muscle weakness [40] and falling [41,42]. Over 90% of fractures occur after a fall and fall rates increase with age [43] and poor muscle strength or function [43]. While the circumstances [39] and the direction [44] of a fall determine the type of fracture, bone density and factors that attenuate a fall, such as better strength or better padding, critically determine whether a fracture will take place when the faller lands on a certain bone [45]. Additionally, fear of falling may adversely affect bone density through self-restriction of physical activity [46,47]. After their first fall, about 30% develop a fear of falling [46], resulting in decreased mobility and quality of life [46]. Notably, anti-resorptive therapy alone is not adequate treatment in elders with muscle weakness and other risk factors for falls [48].
ANTI-FALL AND FRACTURE EFFICACY OF VITAMIN D Several recent meta-analyses of randomized, controlled trials have addressed the benefit of vitamin D on fracture risk reduction with conflicting findings. Different findings and conclusions may be explained by differences in the selection of trials included in the meta-analysis, with respect to design (e.g., double-blind only versus doubleblind and open trials), to vitamin D formulation (parent vitamin D only or inactive and active metabolites), and to route of vitamin D administration (oral only or oral and intra-muscular vitamin D).
Efficacy from Double-blind RCTs Two 2009 meta-analyses of double-blind, randomized controlled trials came to the conclusion that oral vitamin D supplementation reduces the risk of falls by 19% [15],
the risk of hip fracture by 18% [31] and the risk of any non-vertebral fracture by 20% [31]. However, for fall and fracture prevention, this benefit was dose-dependent (see Fig. 62.1(AeC)). Fall prevention was observed only in trials with a treatment dose of 700 to 1000 IU vitamin D per day (Fig. 62.1(C)), and fracture prevention required a received dose (treatment dose * adherence) of more than 480 IU vitamin D per day (Fig. 62.1(A,B)). The primary use of received dose (dose * adherence) as opposed to treatment dose from double-blind RCTs allowed for the assessment of anti-fracture efficacy by a dose that accounts for the low adherence in several recent large trials [49,50]. Any lower dose did not reduce fracture or fall risk. Importantly, the benefit of fall prevention and fracture prevention was present in all subgroups of the senior population at the higher dose of vitamin D [15,31]. At the higher dose of 700 to 1000 IU vitamin D, there was a 38% reduction in the risk of falling with a treatment duration of 2 to 5 months and a sustained significant effect of 17% fall reduction with treatment duration of 12 to 36 months, and the benefit was independent of type of dwelling and age. Thus, benefits of 700 to 1000 IU vitamin D per day on fall prevention are immediate and sustained and include all subgroups of the senior population [15]. Similarly, at the higher received dose, the prevention of non-vertebral fractures was present in all subgroups of the older population independent of age and type of dwelling, and additional calcium supplementation did not further improve antifracture efficacy [31]. There was a suggestion that vitamin D3 was superior to vitamin D2 for both fall and fracture prevention with vitamin D [15,31].
Results from Meta-analyses that Included also Open-design Trials In August 2007, a review and meta-analysis commissioned by the US Department of Health and Human Services (HHS) addressed the effect of vitamin D supplementation on all fractures in postmenopausal women and men aged 50 and older [51]. The pooled results for all fractures included 10 double-blinded and three open-design trials (n ¼ 58 712) and did not support a significant reduction of fractures with vitamin D (pooled odds ratio ¼ 0.90; 95% CI 0.81e1.02). The report suggested that the benefit of vitamin D at any dose (range 400 to 800 IU per day) may depend on additional calcium and may be primarily seen in institutionalized individuals, which is consistent with the meta-analysis of Boonen et al. [52]. One 2010 patient-based meta-analysis included seven large trials of vitamin D with 68 500 individuals aged 47 and older [53]. The authors defined criteria that permitted the inclusion of two open-design trials
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Fracture and fall prevention by dose of oral vitamin D in the treatment groups: (A) non-vertebral fracture prevention by received dose (dose * adherence) in the treatment group; (B) hip fracture prevention by received dose (dose * adherence) in the treatment group; (C) fall prevention by treatment dose in the treatment group. (A) and (B) are adapted from [31], all rights reserved. (C) is adapted from [15], all rights reserved. Triangles indicate trials with D3, circles trials with D2. Line ¼ Trendline through relative risks from different trials. (A) depicts the relative risk of non-vertebral fracture from 12 double-blind RCTs (n ¼ 42 279 individuals) plotted against the received dose (dose * adherence) of vitamin D in the different trials. For any non-vertebral fractures, anti-fracture efficacy increased significantly with higher received dose (metaregression: Beta ¼ 0.0007; p ¼ 0.003). The pooled RR was 0.80 (95% CI 0.72e0.89) for a received dose of 482 to 770 IU supplemental vitamin D per day, while the pooled RR was 1.02 (95% CI 0.92e1.15) for received doses of less than 482 IU per day. (B) depicts the relative risk of hip fracture from 8 double-blind RCTs (n ¼ 42 279 individuals) plotted against the received dose (dose * adherence) of vitamin D in the different trials. For hip fracture, anti-fracture efficacy increased significantly with higher received dose (meta-regression: Beta ¼ 0.0009; p ¼ 0.07). The pooled RR was 0.82 (95% CI 0.69e0.97) for a received dose of 482 to 770 IU supplemental vitamin D per day, while the pooled RR was 1.09 (95% CI 0.90e1.32) for received doses of less than 482 IU per day. (C) depicts the relative risk of falling from 8 double-blind RCTs (n ¼ 2426 individuals) plotted against the treatment dose of vitamin D in the different trials. By visual inspection of (C), anti-fall benefits of vitamin D started at a dose of 700 IU per day. A meta-regression indicated a significant inverse relationship between higher dose in the treatment group and the risk of sustaining at least one fall (Beta-estimate for dose: 700 IU or higher compared to less ¼ 0.337; p ¼ 0.02). The pooled RR was 0.81 (95% CI 0.71e0.92) for doses of 700 to 1000 IU vitamin D per day, while the pooled RR was 1.10 (95% CI 0.89e1.35) for doses of 200 to 600 IU per day.
FIGURE 62.1
[54,55], one trial with intra-muscular vitamin D [56], and four of the 12 double-blind RCTs included in the 2009 meta-analysis described above (one RCT using intermittent vitamin D2 without calcium [57], one RCT with 400 IU vitamin D3 without calcium [58], one trial with 800 IU vitamin D3 per day with and without calcium and less than 50% adherence [50], and one trial with 400 IU vitamin D with calcium [49]). Based on these criteria, their findings showed a reduced overall risk of fracture (hazard ratio ¼ 0.92; 95% CI 0.86e0.99) and a non-significant reduction of hip fractures (hazard ratio ¼ 0.84; 95% CI 0.70e1.01) for trials that used vitamin D plus calcium. Vitamin D alone, irrespective of dose, did not reduce fracture risk. The authors concluded that vitamin D, even in a dose of 400 IU
vitamin D per day reduces the risk of fracture if combined with calcium. Notably, this regimen was tested in 36 282 postmenopausal women in the Women’s Health Initiative Trial over a treatment period of 7 years and did not reduce the risk of fracture [49]. Discussion of the Recent Meta-analyses In all three reports reviewed under this section, heterogeneity by dose may have been missed due to the inclusion of open-design trials plus a dose evaluation that did not incorporate adherence. Biologically, the exclusion of heterogeneity by dose seems implausible even if a formal test of heterogeneity is not statistically significant. A doseeresponse relationship between vitamin D and fracture reduction as documented for the two 2009
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meta-analyses of double-blind RCTs [15,31], is supported by epidemiologic data showing a significant positive trend between serum 25(OH)D concentrations and hip bone density [59] and lower-extremity strength [8,9]. Factors that may obscure a benefit of vitamin D are low adherence to treatment [60], low dose of vitamin D, or the use of less potent D2 [61,62]. Furthermore, open-design trials [63] may bias results towards the null because participants who know that they have been randomized to the control group may purchase vitamin D themselves as it is available without prescription. Heterogeneity by trial quality is supported by the sensitivity analyses performed for the two 2009 metaanalyses, where heterogeneity was introduced by open-design trials for any dose of vitamin D, and lower and higher dose of vitamin D [15,31].
DESIRABLE 25(OH)D STATUS FOR OPTIMAL MUSCULOSKELETAL HEALTH A threshold for optimal serum 25(OH)D concentration and fracture and fall prevention has been addressed in a recent benefiterisk analysis [64] and is illustrated in Figure 62.2. Based on these data, 75 or better 100 nmol/l (30 or better 40 ng/ml) are suggested as an optimal threshold of 25(OH)D for fall and fracture prevention. The threshold of 75 nmol/l, 25-dihydroxyvitamin D (1,25(OH)2D) as desirable for optimal musculoskeletal health is supported by epidemiologic data for hip bone
Desirable serum 25-hydroxyvitamin D concentration for fall and fracture prevention. Data points show the relative risk of falls and the relative risk of sustaining any non-vertebral fracture from double-blind RCTs, by achieved 25-hydroxyvitamin D levels in the treatment groups. Data were extracted from two 2009 meta-analyses [15,31] and summarized in a recent benefiterisk analysis of vitamin D [64]. Based on these data, 75 or better 100 nmol/l (30 or better 40 ng/ml) are suggested as an optimal threshold of 25(OH)D for fall and fracture prevention.
FIGURE 62.2
density in younger and older adults [19], as well as lower-extremity function for older adults [8,65]. A threshold for optimal 25(OH)D and hip bone density has been addressed among 13 432 individuals of NHANES III (the Third National Health and Nutrition Examination Survey) including both younger (20e49 years) and older (50þ years) individuals with different ethnic racial background [59]. Compared to the lowest quintile of 25(OH)D, the highest quintile had higher mean bone density by 4.1% in younger whites (test for trend; p < 0.0001), by 4.8% in older whites (p < 0.0001), by 1.8% in younger Mexican-Americans (p ¼ 0.004), by 3.6% in older Mexican-Americans (p ¼ 0.01), by 1.2% in younger blacks (p ¼ 0.08) and by 2.5% in older blacks (p ¼ 0.03). In the regression plots higher serum 25(OH)D levels were associated with higher BMD throughout the reference range of 22.5 to 94 nmol/l in all subgroups (Fig. 62.3(A,B)). In younger whites and younger Mexican-Americans, higher 25(OH)D was associated with higher BMD even beyond 100 nmol/l. The threshold of at least 75 nmol/l for serum 25(OH)D concentrations is supported by the recent IOF position statement on vitamin D [66].
DOSING INTERVAL OF VITAMIN D AND MUSCULOSKELETAL HEALTH In 2010, a large double-blind RCT by Sanders et al., including 2256 community-dwelling women aged 70 years and older, tested the benefit of 500 000 IU vitamin D3 given orally once a year, on fall and fracture prevention [67]. In those women, mean age 76, considered to be at risk of fracture, 500 000 IU vitamin D once a year did not reduce but instead increased the risk of falls by 15% and the risk of fractures by 26% compared to placebo, with the greatest increase in falls occurring during the first 3 months after dosing. These findings are consistent with another trial that tested 300 000 IU vitamin D2 as an intra-muscular injection once a year [56]. Whether the large dose of vitamin D tested in the Sanders trial was too much of a good thing or not enough to provide a sufficient supply of vitamin D over 12 months is speculative [68]. The temporal pattern of events suggests that the high dose of vitamin D may have induced a “protective” reaction resulting in an acute decrease in 1,25(OH)2D [69]. Alternatively, the undocumented potential effect of vitamin D on muscle strength [65] and overall health (i.e., fewer infections and hospital admissions [70]) in the Sanders trial may have improved mobility and decreased “down time,” ironically leading to an increased opportunity to fall and fracture. As a result of the Sanders trial and given the half-life of vitamin D is 3 to 6 weeks, a daily, weekly,
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FIGURE 62.3 Desirable serum 25-hydroxyvitamin D concentration for hip bone mineral density in younger and older adults: (A) younger adults (age 20e49); (B) Older adults (age 49þ). (A) and (B) are adapted from [59], all rights reserved. Regression plot of difference in bone mineral density by 25(OH)D in younger (20 to 49 years (A)) and older adults (50þ years (B)). Symbols represent different ethnicities: circles are Caucasians, squares are Mexican-Americans and triangles are African-American individuals. The intercept was set to “0” for all race/ethnicity groups to focus on the difference in BMD by 25(OH)D levels, as opposed to differences in BMD by race/ethnicity. The reference range of the 25(OH)D assay (22.5e94 nmol/l) is marked as vertical lines. The reference range of the Diasorin assay has been provided by the company and was established using 98 samples from apparently healthy normal volunteers collected in the south-western USA (high latitude) in late autumn (www.fda.gov/cdrh/pdf3/k032844.pdf). Regression plots adjust for gender, age, body mass index, smoking, calcium intake, estrogen use, month, and poverty income ratio. Weighting accounts for NHANES III sampling weights, stratification and clustering.
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or monthly dosing interval may be most advantageous and safe [64,71].
HOW TO ACHIEVE A SERUM CONCENTRATION OF AT LEAST 75 NMOL/L FOR MUSCULOSKELETAL HEALTH Studies suggest that 700 to 1000 IU of vitamin D per day may bring 50% of younger and older adults up to 75e100 nmol/l [72e74]. Thus, to bring most older adults to the desirable range of 75e100 nmol/l, vitamin D doses higher than 700e1000 IU would be needed. According to a recent benefiterisk analysis on vitamin D, mean levels of 75e110 nmol/l were reached in most RCTs with 1800e4000 IU vitamin D/d without risk [64]. In a recent trial among acute hip fracture patients, 70% reached the 75 nmol/l threshold with 800 IU vitamin D3 per day, and 93% with 2000 IU vitamin D3 per day, at 12-month follow-up and with over 90% adherence [75]. Consistently, Heaney and colleagues, in a study of healthy men, estimated that 1000 IU cholecalciferol per day are needed during winter months in Nebraska to maintain a late summer starting level of 70 nmol/l, while baseline levels between 20 and 40 nmol/l may require a daily dose of 2200 IU vitamin D to reach and maintain 80 nmol/l [76,77]. These results indicate that individuals with a lower starting level may need a higher dose of vitamin D to achieve desirable levels, while relatively lower doses may be sufficient in individuals who start at higher baseline levels. Due to seasonal fluctuations of 25(OH)D levels [78], some individuals may be in the desirable range during summer months. However, these levels will not sustain during the winter months even in sunny latitudes [79,80]. Thus winter supplementation with vitamin D is needed even after a sunny summer. Furthermore, several studies suggest that many older persons will not achieve optimal serum 25(OH)D levels during summer months suggesting that vitamin D supplementation should be independent of season in older persons [80e82]. Even among younger persons, the use of sunscreen or sun-protective clothing may prevent a significant increase in 25(OH)D levels [82]. The most vulnerable to low vitamin D levels are older individuals [80,83] living in northern latitudes with prolonged winters [78,84], obese individuals [85], and individuals of all ages with dark skin pigmentation living in northern latitudes [59,86,87]. Naturally high 25(OH)D levels observed in healthy outdoor workers are 135 nmol/l [88] in farmers
and 163 nmol/l [89] in lifeguards. As a first sign of toxicity, only serum 25(OH)D levels of above 220 nmol/l have been associated with hypercalcemia [90,91].
SUMMARY Based on evidence from RCTs, oral vitamin D supplementation reduces both falls and non-vertebral fractures, including those at the hip. However, this benefit is dose-dependent and a dose of 700e1000 IU vitamin D per day is required to assure both fall and fracture prevention. For optimal fall and fracture reduction a serum 25(OH)D concentration of at least 75 nmol/l is required. This threshold may be reached with 800 to 1000 IU vitamin D in 50% of adults, whereas higher doses of vitamin D would be required to shift all adults to this threshold.
Discussion on the IOM report While the IOM recommendation of an increase in vitamin D intake is supported by the available data from double-blind RCTs of fracture risk, a threshold of 50 nmol/l for its 25(OH)D blood level is not. In two 2009 meta-analyses of double-blind RCTs, a threshold of 50 nmol/l was insufficient for fracture or fall reduction based on achieved 25(OH)D levels in the treatment groups [92,93] (see Figure 62.2). Also, in the very large population-based NHANES analysis, bone density increased with higher 25(OH)D levels far beyond 50 nmol/l in younger and older adults suggesting that the IOM threshold recommendation is too low for optional bone health in adults [94] (see Fig. 62.3). In contrast to the IOM report, the IOF recommended in their 2010 position paper on vitamin D a threshold of 75 nmol/l for optimal fall and fracture reduction and recommended 800 to 1000 IU vitamin D per day for seniors age 60 years and older [95]. The IOM synopsis is that the evidence of vitamin D on fall prevention is inconsistent, which is in contrast to the 2011 assessment of the Agency for Healthcare Research and Quality (AHRQ) for the US Preventive Services Task Force [96], the 2010 American Geriatric Society/British Geriatric Society Clinical Practice Guideline [97], and to the 2010 assessment by the IOF [95], all three of which identified vitamin D as an effective intervention to prevent falling in older adults.
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[36] M. Pfeifer, B. Begerow, H.W. Minne, K. Suppan, A. FahrleitnerPammer, H. Dobnig, Effects of a long-term vitamin D and calcium supplementation on falls and parameters of muscle function in community-dwelling older individuals, Osteoporos. Int. 20 (2009) 315e322. [37] B. Dawson-Hughes, S.S. Harris, E.A. Krall, G.E. Dallal, Effect of calcium and vitamin D supplementation on bone density in men and women 65 years of age or older, N. Engl. J. Med. 337 (1997) 670e676. [38] M.C. Chapuy, M.E. Arlot, F. Duboeuf, J. Brun, B. Crouzet, S. Arnaud, et al., Vitamin D3 and calcium to prevent hip fractures in the elderly women, N. Engl. J. Med. 327 (1992) 1637e1642. [39] S.R. Cummings, M.C. Nevitt, Non-skeletal determinants of fractures: the potential importance of the mechanics of falls. Study of Osteoporotic Fractures Research Group, Osteoporos. Int. 4 (Suppl. 1) (1994) 67e70. [40] S.R. Cummings, M.C. Nevitt, W.S. Browner, K. Stone, K.M. Fox, K.E. Ensrud, et al., Risk factors for hip fracture in white women. Study of Osteoporotic Fractures Research Group, N. Engl. J. Med. 332 (1995) 767e773. [41] Centers for Disease Control and Prevention, Fatalities and injuries from falls among older adults e United States, 19932003 and 2001-2005, MMWR Morb. Mortal Wkly. Rep. 55 (2006) 1221e1224. [42] A.V. Schwartz, M.C. Nevitt, B.W. Brown Jr., J.L. Kelsey, Increased falling as a risk factor for fracture among older women: the study of osteoporotic fractures, Am. J. Epidemiol. 161 (2005) 180e185. [43] M.E. Tinetti, Risk factors for falls among elderly persons living in the community, N. Engl. J. Med. 319 (1988) 1701e1707. [44] N.D. Nguyen, S.A. Frost, J.R. Center, J.A. Eisman, T.V. Nguyen, Development of a nomogram for individualizing hip fracture risk in men and women, Osteoporos. Int. 17 (2007) 17. [45] M.C. Nevitt, S.R. Cummings, Type of fall and risk of hip and wrist fractures: the study of osteoporotic fractures. The Study of Osteoporotic Fractures Research Group, J. Am. Geriatr. Soc. 41 (1993) 1226e1234. [46] B.J. Vellas, S.J. Wayne, L.J. Romero, R.N. Baumgartner, P.J. Garry, Fear of falling and restriction of mobility in elderly fallers, Age Ageing 26 (1997) 189e193. [47] C.L. Arfken, H.W. Lach, S.J. Birge, J.P. Miller, The prevalence and correlates of fear of falling in elderly persons living in the community, Am. J. Public Health 84 (1994) 565e570. [48] M.R. McClung, P. Geusens, P.D. Miller, H. Zippel, W.G. Bensen, C. Roux, et al., Effect of risedronate on the risk of hip fracture in elderly women. Hip Intervention Program Study Group, N. Engl. J. Med. 344 (2001) 333e340. [49] R.D. Jackson, A.Z. LaCroix, M. Gass, R.B. Wallace, J. Robbins, C.E. Lewis, et al., Calcium plus vitamin D supplementation and the risk of fractures, N. Engl. J. Med. 354 (2006) 669e683. [50] A.M. Grant, A. Avenell, M.K. Campbell, A.M. McDonald, G.S. MacLennan, G.C. McPherson, et al., Oral vitamin D3 and calcium for secondary prevention of low-trauma fractures in elderly people (Randomised Evaluation of Calcium Or vitamin D, RECORD): a randomised placebo-controlled trial, Lancet 365 (2005) 1621e1628. [51] A. Cranny, T. Horsley, S. O’Donnell, H. Weiler, L. Puil, D. Ooi, et al., Effectiveness and safety of vitamin D in relation to bone health (2007)
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[53] DIPART (Vitamin D Individual Patient Analysis of Randomized Trials) Group, Patient level pooled analysis of 68 500 patients from seven major vitamin D fracture trials in US and Europe. BMJ 340 (2010) b5463. [54] J. Porthouse, S. Cockayne, C. King, L. Saxon, E. Steele, T. Aspray, et al., Randomised controlled trial of calcium and supplementation with cholecalciferol (vitamin D3) for prevention of fractures in primary care, BMJ 330 (2005) 1003. [55] E.R. Larsen, L. Mosekilde, A. Foldspang, Vitamin D and calcium supplementation prevents severe falls in elderly communitydwelling women: a pragmatic population-based 3-year intervention study, Aging Clin. Exp. Res. 17 (2005) 125e132. [56] H. Smith, F. Anderson, H. Raphael, P. Maslin, S. Crozier, C. Cooper, Effect of annual intramuscular vitamin D on fracture risk in elderly men and women e a population-based, randomized, double-blind, placebo-controlled trial, Rheumatology (Oxford) 46 (2007) 1852e1857. [57] R.A. Lyons, A. Johansen, S. Brophy, R.G. Newcombe, C.J. Phillips, B. Lervy, et al., Preventing fractures among older people living in institutional care: a pragmatic randomised double blind placebo controlled trial of vitamin D supplementation, Osteoporos Int. 18 (2007) 811e818. [58] H.E. Meyer, G.B. Smedshaug, E. Kvaavik, J.A. Falch, A. Tverdal, J.I. Pedersen, Can vitamin D supplementation reduce the risk of fracture in the elderly? A randomized controlled trial, J. Bone Miner. Res. 17 (2002) 709e715. [59] H.A. Bischoff-Ferrari, T. Dietrich, E.J. Orav, B. Dawson-Hughes, Positive association between 25-hydroxy vitamin d levels and bone mineral density: a population-based study of younger and older adults, Am. J. Med. 116 (2004) 634e639. [60] A.M. Grant, A. Avenell, M.K. Campbell, A.M. McDonald, G.S. MacLennan, G.C. McPherson, et al., Oral vitamin D3 and calcium for secondary prevention of low-trauma fractures in elderly people (Randomised Evaluation of Calcium Or vitamin D, RECORD): a randomised placebo-controlled trial, Lancet 365 (2005) 1621e1628. [61] L.A. Armas, B.W. Hollis, R.P. Heaney, Vitamin D2 is much less effective than vitamin D3 in humans, J. Clin. Endocrinol. Metab. 89 (2004) 5387e5391. [62] L.A. Houghton, R. Vieth, The case against ergocalciferol (vitamin D2) as a vitamin supplement, Am. J. Clin. Nutr. 84 (2006) 694e697. [63] J. Porthouse, S. Cockayne, C. King, L. Saxon, E. Steele, T. Aspray, et al., Randomised controlled trial of calcium and supplementation with cholecalciferol (vitamin D3) for prevention of fractures in primary care, BMJ 330 (2005) 1003. [64] H.A. Bischoff-Ferrari, A. Shao, B. Dawson-Hughes, J. Hathcock, E. Giovannucci, W.C. Willett, Benefit-risk assessment of vitamin D supplementation, Osteoporos. Int. 21 (2009) 1121e1132. [65] H.A. Bischoff-Ferrari, T. Dietrich, E.J. Orav, F.B. Hu, Y. Zhang, E.W. Karlson, et al., Higher 25-hydroxyvitamin D concentrations are associated with better lower-extremity function in both active and inactive persons aged > or ¼ 60 y, Am. J. Clin. Nutr. 80 (2004) 752e758. [66] B. Dawson-Hughes, A. Mithal, J.P. Bonjour, S. Boonen, P. Burckhardt, et al., IOF position statement: vitamin D recommendations for older adults. Osteoporos Int. 21 (2010) 1151e1154. [67] K.M. Sanders, A.L. Stuart, E.J. Williamson, J.A. Simpson, M.A. Kotowicz, D. Young, et al., Annual high-dose oral vitamin D and falls and fractures in older women: a randomized controlled trial. JAMA 303, 1815e1822. [68] B. Dawson-Hughes, S.S. Harris, High-dose vitamin D supplementation: too much of a good thing? JAMA 303, 1861e1862.
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[69] M.J. Beckman, J.A. Johnson, J.P. Goff, T.A. Reinhardt, D.C. Beitz, R.L. Horst, The role of dietary calcium in the physiology of vitamin D toxicity: excess dietary vitamin D3 blunts parathyroid hormone induction of kidney 1-hydroxylase, Arch. Biochem. Biophys. 319 (1995) 535e539. [70] H.A. Bischoff-Ferrari, B. Dawson-Hughes, A. Platz, E.J. Orav, H.B. Stahelin, W.C. Willett, et al., Theiler Effect of high-dosage cholecalciferol and extended physiotherapy on complications after hip fracture: a randomized controlled trial. Arch. Intern. Med. 170, 813e820. [71] V. Chel, H.A. Wijnhoven, J.H. Smit, M. Ooms, P. Lips, Efficacy of different doses and time intervals of oral vitamin D supplementation with or without calcium in elderly nursing home residents, Osteoporos. Int. 19 (2008) 663e671. [72] V. Tangpricha, E.N. Pearce, T.C. Chen, M.F. Holick, Vitamin D insufficiency among free-living healthy young adults, Am. J. Med. 112 (2002) 659e662. [73] M.J. Barger-Lux, R.P. Heaney, S. Dowell, T.C. Chen, M.F. Holick, Vitamin D and its major metabolites: serum levels after graded oral dosing in healthy men, Osteoporos. Int. 8 (1998) 222e230. [74] B. Dawson-Hughes, Impact of vitamin D and calcium on bone and mineral metabolism in older adults. Biologic Effects of Light 2001, in: M.F. Holick (Ed.), Kluwer Academic Publishers, Boston, MA, 2002, pp. 175e183. [75] H.B. Bischoff-Ferrari, B. Dawson-Hughes, A. Platz, J.E. Orav, H.B. Staehelin, W.C. Willett, et al., Effect of high-dosage vitamin D3 cholecalciferol and extended physiotherapy on complications after hip fracture: a randomized controlled trial, Archives of Internal Medicine (2010). in press. [76] R.P. Heaney, K.M. Davies, T.C. Chen, M.F. Holick, M.J. BargerLux, Human serum 25-hydroxycholecalciferol response to extended oral dosing with cholecalciferol, Am. J. Clin. Nutr. 77 (2003) 204e210. [77] R.P. Heaney, The Vitamin D requirement in health and disease, J. Steroid Biochem. Mol. Biol. 15 (2005) 15. [78] B. Dawson-Hughes, S.S. Harris, G.E. Dallal, Plasma calcidiol, season, and serum parathyroid hormone concentrations in healthy elderly men and women, Am. J. Clin. Nutr. 65 (1997) 67e71. [79] W.B. Grant, M.F. Holick, Benefits and requirements of vitamin D for optimal health: a review, Altern. Med. Rev. 10 (2005) 94e111. [80] M.J. McKenna, Differences in vitamin D status between countries in young adults and the elderly, Am. J. Med. 93 (1992) 69e77. [81] R. Theiler, H.B. Stahelin, M. Kranzlin, G. Somorjai, L. SingerLindpaintner, M. Conzelmann, et al., Influence of physical mobility and season on 25-hydroxyvitamin D-parathyroid hormone interaction and bone remodelling in the elderly, Eur. J. Endocrinol. 143 (2000) 673e679. [82] M.F. Holick, Environmental factors that influence the cutaneous production of vitamin D, Am. J. Clin. Nutr. 61 (suppl) (1995) 638Se645S. [83] R. Theiler, H.B. Stahelin, A. Tyndall, K. Binder, G. Somorjai, H.A. Bischoff, Calcidiol, calcitriol and parathyroid hormone
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C H A P T E R
63 Clinical Disorders of Phosphate Homeostasis Karen E. Hansen 1, Marc K. Drezner 2 1
2
University of Wisconsin, Madison, WI, USA William H. Middleton Veterans Administration Medical Center, Madison, WI, USA
INTRODUCTION Extensive studies over the past several decades have established that phosphate homeostasis and vitamin D metabolism are reciprocally regulated. As discussed in Chapters 19 and 34 calcitriol promotes phosphate absorption from the intestine, mobilization from bone, and reabsorption in the renal tubule [1]. In turn, phosphate depletion or hypophosphatemia stimulates renal production of 1,25(OH)2D (calcitriol), while phosphate overload or hyperphosphatemia inhibits renal 25(OH) D-1a-hydroxylase (1a-hydroxylase) activity [2]. Since phosphorus is one of the most abundant constituents of all tissues, disturbances in phosphate homeostasis can affect almost any organ system [3]. Indeed, a deficiency or excess of this mineral can have profound effects on a variety of tissues. Consequences of hypophosphatemia include osteomalacia, rickets, red cell dysfunction, rhabdomyolysis, metabolic acidosis, and cardiomyopathy. By contrast, hyperphosphatemia may lead to soft tissue calcification, hypocalcemia, tetany, and secondary hyperparathyroidism. In many cases, the interrelationship between phosphate homeostasis and vitamin D metabolism precludes establishing whether the consequences of hypo- or hyperphosphatemia are singularly related to this abnormality or are modified by changes in calcitriol production. Occasionally, however, discrimination between these possibilities has been achieved by evaluation of the therapeutic response to phosphate supplementation or depletion. Such studies indicate that few of the phosphate homeostatic disorders respond adequately to therapeutically induced alterations in phosphate alone, but do regress upon coincident modification of the vitamin D status. The detailed control mechanisms that regulate phosphate homeostasis and vitamin D metabolism in
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10063-0
intestine and kidney are reviewed in Chapters 19 and 26 and overall physiology in Chapter 34. The following is a summary of important elements of the phosphate homeostatic schema and the regulation of vitamin D metabolism that pertain to an understanding of the diseases described in the remainder of this chapter and in Chapter 42.
Regulation of Phosphate Homeostasis Most phosphate (85%) resides in bone, where it associates with calcium; 14% of phosphate is in cells, as a component of lipids, proteins, nucleic acids, and small molecules of metabolic and signaling pathways. Phosphate in serum and extracellular fluids accounts only for 1% of total-body phosphate, but the serum phosphate concentration correlates, in most circumstances, with total-body phosphate content. The kidney is the major arbiter of extracellular phosphate (Pi) homeostasis and plays a key role in bone mineralization and growth. Most of the filtered phosphorus is reabsorbed in the proximal tubule, with approximately 60% of the filtered load reclaimed in the proximal convoluted tubule and 15e20% in the proximal straight tubule [4]. In addition, a small but variable portion (<10%) of filtered phosphorus is reabsorbed in more distal segments of the nephron. The reabsorption of phosphate filtered by the glomerulus is an active, hormonally regulated process and the amount of phosphate reabsorbed is a key determinant of the serum phosphorus concentration [5]. Transepithelial phosphorus transport is effectively unidirectional and includes uptake at the brush border membrane of the renal tubule cell, translocation across the cell and efflux at the basolateral membrane [6]. Apical sodium-dependent phosphate (Na/Pi)
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63. CLINICAL DISORDERS OF PHOSPHATE HOMEOSTASIS
cotransport across the luminal (brush border) membrane is rate limiting and the target for physiological/pathophysiological alterations. The uptake at this site is mediated by Naþ-dependent phosphate transporters that reside in the brush border membrane and depend on the Naþ, Kþ-ATPase to maintain the Naþ gradient that drives the transport system [7]. In contrast, basolateral Pi-transport systems are not well defined. Efflux of Pi across the basolateral membrane may involve an anion exchange mechanism and/or a “Pi leak” to complete transcellular reabsorptive flux, and an Naþ-dependent Pi uptake mechanism to guarantee Pi uptake from the interstitium if apical influx is insufficient to maintain cellular metabolism. There are three types of sodium-phosphate cotransporters in the renal proximal tubule cells, all of which have the capacity to induce an increase in Na-dependent Pi uptake in heterologous expression systems [8]. The type 1 cotransporter (NPT1) is an anion carrier that does not specifically mediate phosphate transport and does not contribute significantly to regulation of proximal tubular Pi flux [9,10]. Indeed, heterologous expression studies indicate that the type-I-mediated Na/Pi cotransport does not have the characteristics and regulatory features of brush border membrane Na/Pi cotransport. Rather, these studies suggest that the type I cotransporter has a channel function, mediating the flux of chloride and organic anions [8,10,11]. The type 2 cotransporter family includes three carriers: NPT2a (encoded by the SLC34A1 gene), NPT2b (encoded by the SLC34A2 gene), and NPT2c (encoded by the SLC34A3 gene). Type 3 consists of two transporters: sodium-dependent phosphate transporter 1 (PiT1, encoded by the SLC20A1 gene) and sodium-dependent phosphate transporter 2 (PiT2, encoded by the SLC20A2 gene). NPT2a, which plays a key role in renal Pi reabsorption and phosphate homeostasis, is located almost exclusively in the apical membrane of renal proximal tubular cells [8]. Indeed, studies in adult animals support the vital function of the type 2a receptor in several ways: [1] loss of 70e80% of brush border membrane Na/Pi cotransport upon disruption of the gene encoding the NPT2a [12]; (2) correlation of NPT2a protein abundance in brush border membranes with Na/Pi cotransport activity under a variety of physiological/pathophysiological conditions [8]; and (3) disruption of the NPT2a gene in mice increases urinary phosphate excretion and causes marked hypophosphatemia. The NPT2b transporter is expressed predominantly in the lungs and the small intestine and only weakly in the kidney [13]. After birth, disruption of the gene leads to decreased gastrointestinal phosphate absorption. However, the serum phosphorus concentration remains normal due to a compensatory increase in renal NPT2a.
NPT2c, located at the renal brush border of the proximal tubule, when mutated in humans results in hypophosphatemia, rickets, and hypercalcuria, the HHRH syndrome. In contrast, mice null for Npt2c exhibit no renal phosphate wasting when fed a normal phosphorus diet, suggesting that NPT2a is the major player in renal phosphate transport. However, Segawa et al. [14] demonstrated that mice null for both Npt2a and Npt2c had more severe phosphate wasting and subsequent rickets than mice null for Npt2a or Npt2c alone, indicating a synergistic role of both transporters in phosphate balance. The type 3 Na/Pi cotransporters are cell-surface receptors, first identified as receptors for retroviruses [15], and appear to exhibit ubiquitous renal and extrarenal expression. Although type 3 mRNA expression is detected in all nephron segments [7], studies to localize the type 3 protein in apical or basolateral cell membranes are lacking. However, the type 3 Na/Pi cotransporters may be responsible for basolateral Pi influx in all tubule cells to maintain cell metabolism, as well as in proximal tubule cells under conditions of limited apical influx. Proximal tubular Na/Pi cotransport is regulated by a variety of hormones/metabolic factors, which elicit an increase or decrease in Pi reabsorption through alteration of the NPT2a and NPT2c protein availability. Parathyroid hormone (PTH)-dependent inhibition of Naþ-Pi cotransport depends upon internalization of the cell surface NPT2a protein [16]. In contrast, FGF23 alters renal phosphate reabsorption by altering expression of NPT2a and NPT2c. FGF23 binds predominantly to the renal FGFR1 receptor through a process that requires membrane-bound protein, klotho, located in the distal tubule. Interaction of FGF23 with the heteromeric FGFR1/Klotho receptor elicits MAPK signaling in the distal convoluted tubule, which likely downregulates NPT2a and NPT2c expression in the proximal convoluted tubules by an ill-defined paracine process [17]. Acute renal adaptation to phosphate restriction is associated with an increase in NPT2a protein, which is rapidly reversed by a high-Pi diet [18]. Not surprisingly, NPT2a protein availability is also central to modulation of the abnormal renal phosphate transport underlying a large number of phosphate homeostatic disorders. These changes in Na/Pi cotransport and NPT2a protein expression occur predominantly in the absence of variations in NPT2a mRNA levels. Thus, rapid increments or decrements in Na/Pi cotransport follow either membrane insertion of NPT2a protein, which may be preceded by de novo synthesis of the protein, or membrane retrieval of NPT2a protein, followed by lysosomal degradation [8]. Although such membrane trafficking of NPT2a protein is an unequivocally central element in the regulation of Na/Pi cotransport, changes
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INTRODUCTION
Phosphate-dependent Modulation of Vitamin D Metabolism The production of 1,25(OH)2D, the active metabolite of vitamin D, is under stringent control (see Chapter 3). Indeed, 1a-hydroxylation represents the most important regulatory mechanism in the metabolism of vitamin D [24]. In normal adults, serum 1,25(OH)2D concentrations change little in response to repeated dosing with vitamin D, and remain normal, or even decline, in vitamin D intoxication. Phosphorus is one of the three major factors that regulate the activity of the 1a-hydroxylase enzyme [25]. Phosphate depletion and resultant hypophosphatemia stimulate 1a-hydroxylase activity and increase the serum 1,25(OH)2D concentration, whereas phosphate loading and consequent hyperphosphatemia inhibit formation of this metabolite. The mechanism whereby phosphorus modulates this adaptive effect remains unknown. Regardless of the mechanism,
the potential role of phosphorus in regulating vitamin D metabolism is central to understanding and appropriately treating many of the clinical disorders of phosphate homeostasis. As will become evident in the remainder of this chapter, however, the aberrant regulation of vitamin D metabolism encountered in many of these diseases is considered paradoxical. Thus, in virtually all disorders secondary to a primary abnormality in renal phosphate transport, hypophosphatemia or hyperphosphatemia is perplexingly associated with decreased or increased serum 1,25(OH)2D levels, respectively. These observations suggest that the effect of phosphorus on 1a-hydroxylase activity may, in fact, occur indirectly and secondary to alterations in renal phosphate transport systems. In this regard, phosphate depletion and hypophosphatemia and phosphate loading and hyperphosphatemia are associated with compensatory changes in renal phosphate transport that may mediate changes in 1,25(OH)2D production. In accord, the prevailing serum calcitriol levels in normal patients and in patients with disorders of renal phosphate transport, X-linked hypophosphatemia (XLH), tumor-induced osteomalacia (TIO), and tumoral calcinosis (TC) display a highly significant positive correlation with the renal tubular maximum phosphate reabsorption per liter of glomerular filtrate (TmP/GFR) [26], supporting the possibility that renal tubular reabsorption of phosphate may be a major determinant of renal 1,25(OH)2D production (Fig. 63.1). Of course, these data do not
84 72 Plasma 1.25(OH)2D (pg/ml)
in NPT2a mRNA levels do occur after prolonged treatment with 1,25(OH)2D, PTH, FGF23 or thyroid hormone, following chronic changes in dietary Pi and in disorders of Pi homeostasis, such as X-linked hypophosphatemia (XLH). Thus, a secondary mechanism operates under select conditions to regulate Na/Pi cotransport. The intracellular signaling mechanisms involved in insertion/retrieval of NPT2a protein remain incompletely understood. However, several studies have identified numerous signals for NPT2a internalization. PTH effects on NPT2a protein are initiated by binding to receptors that activate protein kinase C on apical membranes and protein kinase A and C on basolateral membranes [19]. In contrast, atrial natriuretic proteininduced internalization of NPT2a protein is mediated by protein kinase G activation [20]. Additional investigations suggest that these different signaling pathways converge on the extracellular signal-regulated kinase/ mitogen-activated protein kinase (ERK/MAPK) pathway to internalize the NPT2a protein [21]. In any case, the signaling process regulating internalization/ retrieval of NPT2a is insufficient to explain the integrated regulation of Pi homeostasis at the kidney. Rather it appears that protein/protein interactions may also participate in regulation of Pi reabsorption. In this regard, NaPi-Cap 1 and NHERF-1, brush border membrane proteins, may interact with NPT2a via one of multiple PDZ-domains, resulting in release of the protein from an apical scaffold, thereby permitting internalization [22,23]. Clearly, further studies are necessary to determine the interrelated mechanisms regulating the many processes that affect the abundance of NPT2a in brush border membranes.
60 XLH TIO Normals Tumoral calcinosis
48 36
r = 0.85 p<0.001
24 12
0
1
2
3
4 7 5 6 TmP/GFR (mg/dl)
8
9
10
Correlation between renal TmP/GFR and plasma 1,25(OH)2D levels in normals and patients with XLH, TIO, and tumoral calcinosis. Significant linear correlation is evident, suggesting a potential relationship between renal phosphate transport and the prevailing plasma levels of active vitamin D. TIO, tumor-induced osteomalacia. From [29].
FIGURE 63.1
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10 8 mg/dl
*
*
6 4 2 0
Control Dietary Control Renal –P –P
Renal 25(OH)D-1-hydroxylase activity
*
14 (fmoles/mg kidney/min)
Serum phosphorus concentration
(A)
1,25-Dihydroxyvitamin D produced
63. CLINICAL DISORDERS OF PHOSPHATE HOMEOSTASIS
12 10 8 6 4 2 0
Control Dietary Control Renal –P –P
*Significantly different from control at p,0.05
Renal 25(OH)D-1-hydroxylase activity
Serum phosphorus concentration
(B) 10
4 2
1,25 OH2D produced
**
(fmoles/mg kidney/min)
mg/dl
** 6
0 Dietary- P PFA- P
**
12
8
FIGURE 63.2 (A) While hypophosphatemia resulting from phosphate restriction increases renal 25(OH)D-1a-hydroxylase activity, hypophosphatemia of similar magnitude secondary to phosphonoformic acid (PFA)-mediated renal phosphate wasting has no effect on enzyme function. These data suggest that hypophosphatemia is not the proximal trigger mechanism for 1,25(OH)2D production. (B) In similar experiments the effects of phosphate depletion and consequent hypophosphatemia on renal 25(OH)D-1a-hydroxylase activity are blocked by the coincident administration of phosphonoformic acid, which prevents the expected compensatory change in renal phosphate flux attendant upon the depletion. These observations reaffirm the potentially important role of renal phosphate transport in modulating the effects of phosphate for 1,25(OH)2D production.
10 8 6 4 2
0 0
0
**Significantly different from respective control (0,0) at p<0.01
0 Dietary- P PFA- P
0 0
0
PFA, Phosphonofomic acid -P, Phosphate depletion
establish that alterations in 1a-hydroxylase activity, in response to phosphate depletion or loading, are dependent upon renal phosphate transport. However, the observations that phosphate depletion does not increase 1,25(OH)2D production in mice subjected to treatment with phosphonoformic acid, which precludes a compensatory alteration in TmP/GFR, suggests an important role for the renal phosphate transport system in modulating phosphorus-mediated effects on 1a-hydroxylase activity under all conditions (Fig. 63.2). Additionally, preliminary studies have documented that mice with targeted disruption of the NHERF-1 gene, which singularly promotes internalization of proximal tubule Npt2a, manifest ~50% decreased apical membrane localized Npt2a and consequent renal P wasting and hypophosphatemia [22], and paradoxically exhibit inappropriately normal serum 1,25(OH)2D levels, similar to Hyp mice (the murine homolog of XLH) with comparable Npt2a deficiency. In contrast, Tenenhouse et al. [27] concluded from studies in the Npt2ae/e mouse that
Npt2a and renal P transport do not influence 25(OH)D-1a-hydroxylase activity. However, these data may have limited applicability to understanding the role of renal phosphate transport on vitamin D metabolism, since it is well known that compensatory developmental changes often confound studies in knockout mice with complete absence of a protein function. In this regard, several investigators [28,29] reported that the sodium-dependent P cotransporter, Npt2c, normally expressed in murine kidneys of young animals, has sustained activity in adult Npt2ae/e mice, which is likely responsible for the “inappropriate” residual renal P transport in these mutants and the resultant limited hypophosphatemia. The absence of similar Npt2c expression in the kidneys of adult normal and NHERF knockout mice [30] substantiates that enhanced Npt2c expression in adult Npt2ae/e mice may be a unique compensatory mechanism, which limits the applicability of studies in this model to conclusions regarding regulation of vitamin D metabolism.
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DISORDERS OF PHOSPHATE HOMEOSTASIS
While these data indicate that the renal production of 1,25(OH)2D and the renal tubular reabsorption of phosphate are linked, the precise mechanism(s) underlying this association remain uncertain. However, it is clear that understanding the pathophysiology of, and defining appropriate treatment regimens for, many clinical disorders of phosphate homeostasis require investigation of the vitamin D regulatory system.
TABLE 63.1
Disorders of Phosphate Homeostasis
ABNORMAL RENAL PHOSPHATE TRANSPORT Hypophosphatemic syndromes Genetic diseases X-Linked hypophosphatemia (XLH) Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) Tumor-induced osteomalacia (TIO) Autosomal dominant hypophosphatemic rickets (ADHR)
DISORDERS OF PHOSPHATE HOMEOSTASIS
Autosomal recessive hypophosphatemic rickets (ARHR) McCune Albright syndrome (MAS)
The variety of diseases, therapeutic agents, and physiological states that affect phosphate homeostasis are numerous and reflect a diverse pathophysiology. Indeed, rational choice of an appropriate treatment for many of these disorders depends on determining the precise cause for the abnormality. In general, defects in phosphate homeostasis result from impaired renal tubular phosphate reabsorption and a consequent change in the TmP/GFR, an altered phosphate load due to varied intake, or abnormal gastrointestinal absorption or translocation of phosphorus between the extracellular fluid and tissues (Table 63.1). The disorders of renal phosphate transport are the most common of these diseases and have been intensively studied. Indeed, investigation of these conditions has provided new insight into the regulation of renal phosphate transport and the reciprocal regulation of phosphate homeostasis and vitamin D metabolism. In the remainder of this chapter, several clinical states that represent disorders of phosphate homeostasis will be discussed and the potential role of the vitamin D endocrine system in their pathogenesis and phenotypic presentation highlighted.
Fanconi syndrome, type I (FS I) Familial idiopathic Cystinosis (Lignac-Fanconi disease) Oculocerebrorenal (Lowe) syndrome Glycogen storage disease Wilson disease Galactosemia Tyrosinemia Hereditary fructose intolerance Neurofibromatosis Linear nevus sebaceous syndrome Fanconi syndrome, type II (FS II) Acquired disorders Tumor-induced osteomalacia Mesenchymal, epidermal and endodermal tumors Light chain nephropathy Renal transplantation Multiple myeloma Cadmium intoxication
Disorders of Renal Phosphate Transport: Hypophosphatemic Diseases
Lead intoxication Tetracycline (outdated) administration
The disorders of renal phosphate transport, which lead to phosphate wasting and hypophosphatemia, are by far the most common disturbances of phosphate homeostasis. However, the pathophysiological basis of these disorders has remained elusive, largely because the hormonal/metabolic control of phosphate homeostasis at the kidneys and in bone is not completely understood. In this regard, the function of the PTHevitamin D axis is not sufficient to explain the physiological complexity of systemic phosphate homeostasis. However, researchers have identified a class of circulating factors that promote renal phosphorus wasting in patients with TIO, XLH, autosomal recessive hypophosphatemic rickets (ARHR), and autosomal
Hyperphosphatemic syndromes Tumoral calcinosis ALTERED PHOSPHATE LOAD Hypophosphatemic syndromes Decreased phosphate availability Phosphate deprivation Gastrointestinal malabsorption Transcellular shift of phosphate Alkalosis Glucose administration
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(Continued)
1160 TABLE 63.1
63. CLINICAL DISORDERS OF PHOSPHATE HOMEOSTASIS
Disorders of Phosphate Homeostasisdcont’d
Combined mechanisms Alcoholism Burns Nutritional recovery syndrome Diabetic ketoacidosis Hyperphosphatemic syndromes Vitamin D intoxication Rhabdomyolysis Cytotoxic therapy Malignant hyperthermia
dominant hypophosphatemic rickets (ADHR). These phosphatonin(s) regulate renal phosphate transport and bone mineralization and participate in the pathophysiological cascade of events underlying many of the renal phosphate wasting disorders. In fact, current theories have emerged, which suggest that a common metabolic pathway underlies many of these hypophosphatemic diseases. The Common Pathway Underlying the Pathogenesis of TIO, ADHR, ARHR, and XLH As detailed below, XLH, ADHR, and ARHR are genetic disorders characterized by hypophosphatemia, due to impaired renal tubular reabsorption of phosphate, inappropriately normal or decreased serum 1,25(OH)2D levels, and defective cartilage and bone mineralization. In contrast TIO is an acquired hypophosphatemic disorder caused by production of a factor(s) by tumors, which leads to phenotypic features similar to those of the hereditary phosphate wasting diseases. Based on the shared phenotype of these disorders, several groups have postulated the existence of a unique circulating protein(s), common to each of these diseases, which inhibits sodiumdependent phosphate reabsorption by the renal proximal tubule through PTH distinct mechanisms, impairs bone and cartilage mineralization, and deters hypophosphatemia-mediated increments in the renal production of 1,25(OH)2D. Initial evidence lending credence to the possibility that there is such a common metabolic pathway underlying these diseases derived from studies of patients with TIO. By culturing a tumor associated with this disease, Cai et al. [31] demonstrated that supernatants of such tumor cells, maintained in culture, contained a factor(s) that inhibited sodium-dependent phosphate reabsorption in renal cultured epithelia. The effects of this factor(s) were specific, cyclic AMP independent,
and distinct from those of PTH. These data, as well as subsequent reports from other groups [32e34], indicated the existence of a novel substance(s), putatively named phosphatonin(s) [35], that alters phosphate reabsorption in the renal tubule. Further investigation, utilizing serial analysis of gene expression and array profiling, resulted in identification of at least 10 genes consistently overexpressed in tumors from patients with TIO, relative to patients with mesenchymal tumors but without TIO [36]. These overexpressed genes included those that encode fibroblast growth factor (FGF) 23, secreted frizzle-related protein (sFRP)-4, matrix extracellular phosphoglycoprotein (MEPE), dentine matrix protein 1 (DMP1), and PHEX [37e41]. Although, in the vast majority of patients with surgically confirmed TIO, hormone overproduction by the tumor results in secretion of FGF23, as evidenced by the elevated serum concentration of this hormone in 92% of such patients [42], the independent role of this phosphatonin in the genesis of the syndrome has not been established. The possibility that these observations linked the pathogenesis of TIO to that of ADHR was suggested by the seminal discovery that FGF23 has a pathogenetic role in patients with ADHR [43,44]. Patients with this syndrome have missense mutations in the FGF23 gene at the subtilisin-like proprotein convertase (SPC) cleavage site, 176-RXXR-179 (R176Q, R179W, and R179Q), rendering the FGF23 protein resistant to cleavage/hydrolysis and inactivation by SPC proteolytic enzymes [43,45]. The resultant increase in circulating levels of cleavage-resistant FGF23 was presumed responsible for phosphaturia and abnormal bone mineralization due to direct actions of this hormone on the renal proximal tubule and the osteoblasts. Since tumors in patients with TIO overexpress and hypersecrete FGF23 and mutations in the FGF23 gene underlie ADHR, the possibility that FGF23 is the presumed phosphatonin operative in these diseases seemed plausible. Subsequent studies established the biological activity of FGF23, an approximately 26 kDA circulating protein, consisting of an N-terminal FGF homology domain and a novel 71-amino-acid C-terminus of uncertain function. Shimada et al. [40] reported that administration of biosynthetically prepared full-length FGF23 to mice resulted in phosphaturia, but did not lead to upregulation of 1,25(OH)2D production. Bai et al. [46] also discovered that intact FGF23 has enhanced in vivo biological potency and tumors derived from cells that overexpressed FGF23 caused a more severe osteomalacia and rickets in nude mice than observed due to hypophosphatemia alone, suggesting a direct effect of FGF23 on cartilage and bone. In concert, Bowe et al. [38] found that FGF23 inhibited sodium-dependent phosphate
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DISORDERS OF PHOSPHATE HOMEOSTASIS
transport in cultured renal epithelia by decreasing the expression of the NPT2a mRNA and protein. Conversely, FGF23-null mice exhibited elevated serum phosphorus levels and increased 1,25(OH)2D production, confirming an essential and non-redundant role of FGF23 in regulation of phosphate homeostasis [47], while FGF23 transgenic mice exhibited a phenotype consistent with a severe form of ADHR [48]. In addition, several groups have found that circulating FGF23 levels are elevated in patients with ADHR, who manifest hypophosphatemia and other elements of the disorder, as in patients with TIO [49e51]. Moreover, further studies have documented that the well-known variability of symptoms and disease severity in patients with ADHR fluctuate with the serum FGF23 concentration [52]. Absent from these studies, however, is documentation that FGF23 has an independent role in the genesis of the rickets/osteomalacia characteristic of ADHR or if the rickets/osteomalacia in patients with ADHR is phosphate-responsive or -resistant. More recent studies have determined that the phenotype of autosomal recessive hypophosphatemic rickets (ARHR) bears a striking resemblance to that of both TIO and ADHR. ARHR, characterized by renal phosphate wasting, aberrant regulation of 25(OH)D-1ahydroxylase activity and rickets/osteomalacia, is caused by inactivating mutations of dentin matrix protein 1 (DMP1). A relationship between ARHR, TIO, and ADHR was assured by documentation of elevated circulating levels of FGF23 in patients with ARHR and its Dmp1-null mouse homolog [53e56]. The common pathophysiological basis for these diseases was thereafter confirmed by the observation that Dmp1 deficiency in the mouse homolog of ARHR also results in a selective increase in osteocyte production of FGF23. In addition, the recognition that ablation of Fgf23 in Dmp1-null mice results in increased serum phosphorus and 1,25(OH)2D levels in the Dmp1-null mice indicates an independent role for FGF23 in regulation of phosphate and 1,25(OH)2D levels in ARHR. These observations provide unprecedented evidence that links the pathophysiological cascade underlying TIO, ADHR, and ARHR. Nevertheless, the absence of studies addressing the mechanisms whereby Dmp1 deficiency stimulates transcription of FGF23 in osteocytes and inhibits bone mineralization preclude determining that FGF23 alone underlies the bone phenotype (osteomalacia/rickets) in this disorder. Indeed, hypophosphatemia and/or the presence of an alternative factor (similar to that possibly present in TIO) may complement the effects of FGF23 in the genesis of the ARHR phenotype. However, the recent observation that the ARHR phenotype is rescued by overexpression of the Dmp1 57 kDa C-terminal fragment either supports that FGF23 alone underlies the phenotype or suggests that
1161
Dmp1 deficiency alters another factor(s) that regulates bone mineralization [57]. Studies of XLH have further reinforced that FGF23 is the phosphatonin, providing a common link to the pathogenesis of the phosphate wasting diseases and provided insight to the enigma surrounding the role that FGF23 plays in regulation of bone and cartilage mineralization. Early studies by Meyer et al. [58], testing parabiosis between Hyp and normal mice, suggested the presence of a circulating factor that causes hypophosphatemia in the mouse model of XLH. In concert, Lajeunesse et al. [59] discovered the presence of a serum factor in Hyp mice that inhibits phosphate transport in renal epithelia, and others reported that a similar factor is elaborated by Hyp mouse osteoblasts, which not only inhibits renal phosphate transport but impairs osteoblast mineralization [60,61]. Further, renal cross-transplantation studies confirmed the presence of a phosphate transport inhibitory factor in Hyp mice and excluded the possibility that XLH is due to an intrinsic defect in the renal phosphate co-transport system [62]. Upon discovery that mutations of the PHEX gene underlie XLH (see below), a series of observations established the interrelated events linking the HYP phenotype to FGF23. Most notably, the absence of Phex in the kidneys of Hyp mice [63e69] indicated that the gene mutation must indirectly regulate the expression of NPT2a/Npt2a in renal tubular cells. In accord, several groups [70e72] documented that inactivation of Phex in the osteoblasts or osteocytes of Hyp mice leads to decreased Fgf23 proteolysis and to upregulation of Fgf23 mRNA expression, which collectively increase circulating Fgf23 levels not only in Hyp mice [72], but in the vast majority of patients with XLH [49,50]. Interestingly, in Hyp mice and patients with XLH, the mechanisms underlying the elevated circulating FGF23 levels, limited FGF23 degradation and increased FGF23 mRNA production, are the same that occur in patients with ADHR and TIO/ARHR, respectively. The elevated FGF23 levels in XLH promote phosphaturia, and subsequent renal phosphate wasting may contribute to unmineralized cartilage/bone, as noted in both Hyp mice and XLH. The observed amelioration of hypophosphatemia and rickets in Hyp mice, secondary to antibody-mediated neutralization of FGF23, supports this role for FGF23 [72]. Information regarding the downstream effects by which inactivating mutations of PHEX decrease FGF23 proteolysis and enhance FGF23 mRNA production, thereby increasing serum FGF23 levels, remains incomplete. Indeed, despite extensive characterization of the PHEX/Phex gene and PHEX/Phex protein in humans and mice, repeated studies over the past 10 years have
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63. CLINICAL DISORDERS OF PHOSPHATE HOMEOSTASIS
failed to definitively identify a substrate for the protein [38,73e80] or the downstream effects of PHEX/Phex, which result in the classical renal and bone phenotype in XLH [48,76,81,82]. In this regard, several studies have definitively excluded FGF23/Fgf23 as a substrate for PHEX/Phex [38,73,74]. Moreover, investigations to establish that changes in serum P mediate alterations in serum FGF23/Fgf23, and thereby hormone degradation or production, are variable and inconclusive [83e87]. Thus, the ability to alter FGF-23 and positively influence human diseases, through modification of PHEX interaction with FGF-23 or an alternative substrate, has not been possible. More recently, however, the downstream Phex-dependent effects that modulate Fgf-23 production and degradation have been elucidated. These studies revealed not only that Fgf-23 degradation and production occur primarily in the osteoblast/osteocyte but are seemingly regulated by 7B2 and 7B2•SPC2 activity, a subtilisin-like proprotein convertase. In these investigations Yuan et al. (88) documented that a decrease in the 7B2 chaperone protein mRNA in Hyp mouse osteoblasts, and consequent diminished 7B2•SPC2 enzyme activity, limits Fgf23 degradation and enhances Fgf23 mRNA production. Moreover, the effects on Fgf23 production are mediated by a downstream effect of decreased 7B2•SPC2 enzyme activity, impaired Dmp1 degradation and resultant deficiency of the 57 kDa C-terminal proteolytic Dmp1 fragment, which is a mechanism identical to that by which FGF23 is increased in ARHR (Fig. 63.3). Collectively, the aforementioned observations form the basis of the model for a common pathogenesis of XLH, TIO, ADHR, and ARHR. According to this model, FGF23 is the phosphatonin that inhibits sodium-dependent phosphate uptake in the renal proximal tubule and contributes, by an unknown mechanism, to impaired bone mineralization. This model presumes that only fulllength FGF23 is phosphaturic and impacts mineralization. Further, the model predicts that FGF23 is increased: (1) in ADHR because mutations in FGF23 render it resistant to subtilisin-dependent proprotein convertasedependent cleavage; (2) in TIO and ARHR because overproduction of FGF23 overwhelms the inactivation capacity of degradative mechanisms; and (3) in XLH because downstream effects of PHEX limit FGF23 degradation and stimulate FGF23 mRNA production. Consistent with this model, multiple studies have linked the biological activities of FGF23 to the phenotype of the hypophosphatemic diseases. However, presumed restoration of normal FGF23 activity in Hyp mice by a variety of techniques fails to normalize renal phosphate transport and hypophosphatemia [72,89e91], albeit flawed experimental design and subsequent data from studies evaluating mice with knockout of the Phex gene negate the message of these investigations.
Nevertheless, there are a number of additional inconsistencies and unexplained observations that raise concern about whether this simple FGF23 hypothesis is correct. First and foremost, the existence of other phosphatonins raises the plausible possibility that proteins other than FGF23 may be operative in TIO, ARHR, and XLH. Second, the known biologic activities of FGF23 do not explain the panorama of phenotypic abnormalities common to the hypophosphatemic disease. Third, the downstream effects of PHEX that regulate FGF23 degradation and production are not unequivocally established. These issues are considered in the remainder of this section. The most compelling of the observations, challenging the FGF23 hypothesis, is the existence of additional phosphatonins. Thus, while it is tempting to speculate that TIO is due solely to excessive production of FGF23, this is not necessarily the case since tumors overexpress the mRNA of other molecules, including sFRP-4 and MEPE [41], that exhibit the characteristics of a phosphatonin. In this regard, Berndt et al. [92] provided compelling evidence that sFRP-4 has unmistakable characteristics of a phosphatonin. Thus, recombinant sFRP-4 inhibits sodium-dependent phosphate transport in cultured opossum renal epithelial cells, indicating a possible direct action of sFRP-4 on proximal tubular phosphate transport. Further, the systemic administration of recombinant sFRP-4 caused phosphaturia (and hypophosphatemia) in normal rats without stimulating renal 25-hydroxyvitamin D-1a-hydroxylase activity. Moreover, the effects on renal epithelia occurred through a mechanism that involved antagonism of Wnt-dependent b-catenin pathways. And, finally, Berndt et al. [92] extended these observations by showing that sFRP-4 is present in normal human serum and in the serum of patients with TIO. Similar experiments have established MEPE as a phosphatonin. In this regard, extensive experiments have documented that MEPE is exclusively expressed in osteoblasts and osteocytes in rodents [89,93e97] and in abundance in human bone, as well as in human brain, albeit in lesser amounts [93,95e97]. Moreover, MEPE expression occurs in all tumors from patients with TIO and is notably absent in non-phosphaturic tumors [37,40,93]. Further, Rowe et al. [98] have demonstrated that MEPE inhibits phosphate transport in renal epithelia in vitro and when administered in vivo to rats results in significant dose-dependent phosphaturia and hypophosphatemia. However, a report regarding the MEPE null-mutant mouse phenotype showed that the MEPE-deficient mice did not have abnormalities of serum phosphorus, as might be expected if MEPE played an important role in phosphate homeostasis [97]. Moreover, Liu et al. [99] reported that transfer of MEPE deficiency onto the Hyp mouse background failed
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DISORDERS OF PHOSPHATE HOMEOSTASIS
(A)
PHEX/Phex
7B2 mRNA
2 DMP1 (104kd)
ProBMP1
Normal Osteoblasts
SPC2•7B2
SPC2 mRNA
BMP1 DMP1 + DMP1 (57kd) (37kd)
1 Normal Degradation
C-term Fgf23 + N-term Fgf23
Fgf23
Suppressed Production
(B)
PHEX/Phex
Hyp Osteoblasts
Fgf-23 mRNA SOST mRNA
7B2 mRNA ProBMP1
SPC2 mRNA
SPC2•7B2
DMP1 (104kd)
BMP1 Decreased Degradation Increased Fgf-23
Fgf23
C-term Fgf23 + N-term Fgf23
Decreased Increased Production
Increased
DMP1 + (57kd)
DMP1 (37kd)
Fgf-23 mRNA SOST mRNA
(A) In the normal osteoblast a series of reactions occur that regulate the degradation of Fgf23 (box 1) and the production of Fgf23 and Sclerostin (box 2). In ADHR a genetic mutation of Fgf-23 alters the cleavage site, precluding normal degradation of the full-length bioactive protein and resulting in an accumulation of the Ffg23. The attendant increased serum Fgf23 concentration results in the characteristic biochemical phenotype in ADHR. In ARHR an inactivating mutation of DMP1 limits the production of the 57kd C-terminal fragment of DMP1, which normally suppresses production of Fgf23 and SOST mRNA and thereby the coded proteins Fgf23 and Sclerostin. The resultant increased serum Fgf23 concentration causes renal phosphate wasting and the characteristic biochemical phenotype, whereas the increased serum Fgf23 and Sclerostin are apparently responsible for the bone mineralization abnormality in ARHR. (B) In the osteoblast from the Hyp-mouse, an inactive mutation of PHEX/Phex causes a downstream effect, decreased 7B2 mRNA. The resultant decreased 7B2 protein decreases the SPC2•7B2 enzyme activity. The diminished enzyme activity retards Fgf23 degradation and increases the concentration of full-length bioactive Ffg23. The decreased enzyme activity also diminishes conversion of ProBMP1 to BMP1, which limits the degradation of DMP1 to its 57kd Cterminal fragment. The decreased carboxy-terminal fragment lifts suppression of Fgf23 and SOST mRNA, resulting in increased transcription and respective protein concentrations. The increased Fgf23 causes renal phosphate wasting and the characteristic biochemical phenotype, whereas the increased serum Fgf23 and Sclerostin are apparently responsible for the bone mineralization abnormality in XLH. Thus, in XLH abnormalities of the processes in box 1 and box 2 (A) occur. The box 1 abnormality, in essence, recapitulates the nature of the abnormality in ADHR, while that in box 2 is similar to that in ARHR. It is important to note that in other renal phosphate wasting diseases in humans, most notably TIO, the pathophysiology simply involves increased production of Fgf23 and possibly Sclerostin, giving rise to a similar phenotype but not requiring the genetic mutations that result in the increased mRNA production.
FIGURE 63.3
to rescue the hypophosphatemia in the mutants, suggesting that MEPE may not cause phosphaturia in the setting of inactivating Phex mutations. Thus, it is possible that MEPE may influence only bone mineralization and not phosphate homeostasis, thereby requiring the tumor secretion of a complementary phosphatonin to create the complete TIO phenotype. The potential effects of MEPE on bone mineralization have been supported in ancillary studies. First, MEPE-deficient mice exhibit increased bone formation and mineralization, consistent with the possibility that MEPE, under physiological conditions, plays an inhibitory role in bone mineralization and formation. Second, Rowe et al. [98] documented that MEPE dose-dependently inhibited BMP2-mediated mineralization of a murine osteoblast cell line (2T3) in vitro. Thus, the ever-present MEPE
overproduction in tumors of patients with TIO precludes establishing with certainty an independent role of FGF23 in genesis of the syndrome. Similar uncertainty about the independent role of FGF23 in the genesis of the HYP phenotype exists. Indeed, multiple studies have identified that the production rate and/or the circulating level of the phosphatonins, FGF23, MEPE, and sFRP-4, are increased in patients with XLH and/or Hyp mice [100]. However, Yuan et al. [100] found that while Hyp mice and mice with global knockout of Phex (Cre-PhexDflox/y) exhibited increased production and serum levels of FGF-23, MEPE, and sFRP-4, mice with a targeted knockout of Phex in osteoblasts (OC-Cre- PhexDflox/y), despite manifesting the characteristic HYP phenotype, had only an increased osseous production rate and serum level of
VIII. DISORDERS
1164 TABLE 63.2
63. CLINICAL DISORDERS OF PHOSPHATE HOMEOSTASIS
Activity of Candidate Phosphatonin/Minhibin Proteins FGF23
sFRP4
MEPE
In vitro
?
?
þ
In vivo
þ
?
?
In vitro
þ
þ
þ
In vivo
þ
þ
þ
In vitro
þ
þ
e
In vivo
þ
þ
e
In vitro
Z
e
e
In vivo
Z
e
\
In vitro
?
?
?
In vivo
?
?
?
Bone Inhibits mineralization
Kidney Inhibits Naþ-Pi transport
Decreases Npt2a transcription
Vitamin D metabolism Alters 25(OH)D-1a-hydroxylase mRNA
Alters 25(OH)D-1a-hydroxylase protein
Fgf23. These data suggest that increased bone production and serum levels of MEPE and sFRP-4 are not critical for development of the classical HYP phenotype, whereas increased osseous production and serum Fgf23 concentration appear requisite for this biological function. Indeed, the observations that a combined Fgf23-deficient and Phex-deficient mouse model displayed no evidence of abnormal phosphate homeostasis supports the conclusion that FGF23 is the sole phosphatonin that contributes to the genesis of the HYP phenotype [71]. However, these data do not establish that FGF23 is solely responsible for the bone mineralization abnormality in these diseases. Indeed, available studies do not preclude that a non-phosphatonin protein may complement the effects of FGF23 to create the bone phenotype in the hypophosphatemic diseases. Alternatively, in some of the hypophosphatemic diseases the bone abnormality may be only a mild form of phosphate-dependent rickets/osteomalacia. The inadequate evidence supporting FGF23 as the inhibitor of bone mineralization is best summarized in Table 63.2. As illustrated, studies do not yet exist that demonstrate the in vitro inhibitory activity of FGF23 on osteoblast mineralization. Yet, studies in Hyp mice
and/or transgenic Fgf23 mice clearly indicate that the mineralization defect in the hypophosphatemic disorders exceeds that expected from hypophosphatemia alone and does not normalize with phosphate therapy. Hence, an effect of FGF23 on osteoblast mineralization function superficially seems certain. However, Yuan et al. [101] have presented data from comparative studies of Hyp mice and mice with targeted knockout of Phex in osteoblasts (OC-Cre- PhexDflox/y) and osteocytes (DMP1-Cre-PhexDflox/y), which challenge a singular role for FGF23 in the regulation of bone mineralization. Despite an identical biochemical phenotype of hypophosphatemia, renal phosphate wasting, and abnormal vitamin D metabolism, and an equally elevated serum Fgf23 level in each of these murine models, only the Hyp mice and those with a targeted knockout of Phex in osteoblasts had severe rickets and osteomalacia, which did not ameliorate in response to phosphate loading. In contrast, mice with targeted knockout of Phex in osteocytes had a very mild mineralization disorder, which was significantly different from that in the Hyp and OC-Cre- PhexDflox/y mice and, in contrast, completely resolved following phosphate loading. These observations challenge the assumption that increased circulating levels of FGF23 underlie the abnormal bone and cartilage mineralization in XLH. Rather, the data suggest that a factor(s) downstream of Phex, other than FGF23 and the serum phosphorus concentration, is variably controlled by the inactivating PHEX mutation and plays a pivotal role in the regulation of bone mineralization in XLH. Preliminary studies by Yuan et al. [101] indicate that elevated Sclerostin, which is present in Hyp and OC-Cre-PhexDflox/y mice, but not in DMP1-Cre-PhexDflox/y mice, may serve as the pivotal factor underlying the abnormal bone mineralization in XLH. This possibility is supported by recent investigations that document increased Sclerostin is likewise present in the Dmp1-null mouse, the murine model for ARHR. Moreover, the 57 kDa C-terminal rescue of the Dmp1-null mouse phenotype is associated with normalization of the elevated Sclerostin, consistent with the role that this protein may play in abnormal bone mineralization (Fig. 63.3). Further, although De Beur et al. [41] only identified FGF23 and sFRP-4 as the candidate genes for the proteins underlying TIO, because only 67 of the original 364 candidate genes identified by SAGE from the tumors evaluated were validated by array analysis or RT-PCR, it is plausible that the SOST gene, encoding Sclerostin, is overexpressed in TIO and possibly the cause of abnormal mineralization in this syndrome. Interestingly, the anticipated effects of phosphatonins (most notably FGF23) on vitamin D metabolism are also controversial. Although phosphate-mediated enhancement of calcitriol production and serum levels is
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DISORDERS OF PHOSPHATE HOMEOSTASIS
uniformly inhibited in ADHR, TIO, ARHR, and XLH, the cause of this abnormality in each disorder is less certain. Since regulation of calcitriol production occurs primarily at the transcription level, several groups have anticipated that a phosphatonin would inhibit renal 1a-hydroxylase transcription. In accord, current studies indicate that FGF23 fulfills this criterion both in vitro and in vivo (Table 63.2). However, investigations in the Hyp mouse indicate that renal 1a-hydroxylase mRNA is, in fact, elevated under basal conditions and following PTH stimulation [102]. Therefore, the inhibition of calcitriol production occurs at the translational level [103]. As shown in Table 63.2, available data have not linked any of the phosphatonins to this phenotypic characteristic of XLH. Confounding this issue further, Yuan et al. [104] recently reported that the effects of FGF23 on 1a-hydroxylase activity are not direct but are mediated by abnormal renal phosphate transport. Thus, Hyp mice, with transgenic overexpression of Npt2a mRNA and consequent normal serum phosphorus levels, do not exhibit aberrant regulation of 1,25(OH)2D production. These observations suggest that the effects of FGF23 on vitamin D metabolism are mediated by a hormone-induced decrease in renal phosphate transport, which results in abnormal translational regulation, likely due to inadequate P450 phosphorylation and consequent ineffective bimodal targeting of 1a-hydroxylase protein to mitochondria, the site of enzyme activity [105e110]. These remarkable advances in understanding the abnormal regulation of vitamin D metabolism in Hyp mice and likely patients with XLH do not explain the discrepant observations regarding FGF23 effects on 1,25(OH)2D production in vitro and in vivo, which are mediated by decreased 25(OH)D-1a-hydroxylase gene transcription. Thus, further studies are essential in order to determine which elements of the HYP phenotypic milieu alter the effects of FGF23 on vitamin D metabolism and ascertain if regulation of 1,25(OH)2D production in other hypophosphatemic disorders is similar to that in XLH. Over the past decade, the progress made in defining a common pathogenetic mechanism for these hypophosphatemic diseases has been truly remarkable and enhanced our understanding of the various disorders of phosphate homeostasis. Indeed, based on these advances, many investigations are under way to create new treatment strategies for these hypophosphatemic disorders. Moreover, further progress in the next decade will undoubtedly resolve the details of the common FGF23-dependent pathogenetic mechanism underlying ADHR, TIO, ARHR, and XLH. Consideration of these diseases in the remainder of this chapter will highlight many of these advances. Chapter 26 and Chapter 42 have further discussion of the phosphatonins.
1165
X-linked Hypophosphatemia (XLH) XLH is the prototypic renal phosphate wasting disorder, characterized by progressively severe skeletal abnormalities and growth retardation. The syndrome occurs as an X-linked dominant disorder with complete penetrance of a renal tubular abnormality resulting in phosphate wasting and consequent hypophosphatemia (Table 63.3). The clinical expression of XLH varies widely, ranging from a mild presentation with apparently isolated hypophosphatemia, to severe rickets and/or osteomalacia [111]. In children, the most common clinically evident manifestations include short stature and limb deformities. This height deficiency is more evident in the lower extremities, since they represent the fastest-growing segment before puberty. In contrast, upper segment growth is generally less affected. The majority of children with the disease exhibit enlargement of the wrists and/or knees secondary to rickets, as well as bowing of the lower extremities. Additional signs of the disease may include late dentition, tooth abscesses secondary to poor mineralization of the interglobular dentine, and premature cranial synostosis. Many of these features are not apparent until 6 to 12 months of age or older [112]. Despite marked variability in the clinical presentation, bone biopsies in affected children and adults invariably reveal low turnover osteomalacia without osteopenia. The severity of the bone disorder has no relationship to gender, the extent of the biochemical abnormalities, or the degree of the clinical disability. In untreated youths and adults, the serum 25(OH)D levels are normal and the concentration of 1,25(OH)2D is in the lowenormal range [113e115]. The paradoxical occurrence of hypophosphatemia and normal serum calcitriol levels is due to aberrant regulation of renal 25(OH)D1a-hydroxylase activity. Studies in Hyp and Gy mice, the murine homologs of the human disease, have established that defective regulation is confined to the enzyme localized in the proximal convoluted tubule, the site of the abnormal phosphate transport [116e119]. PATHOPHYSIOLOGY
Investigators generally agree that the primary inborn error in XLH results in an expressed abnormality of the renal proximal tubule that impairs Pi reabsorption. This defect has been indirectly identified in affected patients and directly demonstrated in the brush border membranes of the proximal nephron in Hyp mice. Whether this renal abnormality is primary or secondary to the elaboration of a humoral factor was controversial. In this regard, demonstration that renal tubule cells from Hyp mice maintained in primary culture exhibit a persistent defect in renal Pi transport [120,121], likely due to decreased expression of NPT2a mRNA and
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1166 TABLE 63.3
63. CLINICAL DISORDERS OF PHOSPHATE HOMEOSTASIS
Biochemical/genetic characteristics of the prototypic phosphopenic disorders in humans FS XLH
TIO
ADHR
ARHR
HHRH
I
II
TC
Serum P
Z
Z
Z
Z
Z
Z
Z
\
Renal TmP/GFR
Z
Z
Z
Z
Z
Z
Z
\
GI P absorption
Z
Z
Z
Z
\
Z
\
\
FGF-23
N or \
\
\
\
?
?
?
?
Serum Ca
N
N
N
N
N
N
N
N
Urine Ca
Z
Z
Z
Z
\
Z
\
\
Nephrolithiasis
e
e
e
þ
e
e
e
e
GI Ca absorption
Z
Z
Z
Z
\
Z
\
\
Serum PTH
N
N
N
N
N
N
N
N
P HOMEOSTASIS
Ca HOMEOSTASIS
VITAMIN D METABOLISM 25(OH)D
N
N
N
N
N
N
N
N
1,25(OH)2D
N/Z
Z
N/Z
Z
\
N/Z
\
\
Serum Alk Phos
N/\
N/\
N/\
N/\
N/\
N/\
N/\
N
Serum NPT
N
N
N
N
N
N
N
?
Familial
þ
e
þ
þ
þ
Variable
þ
þ
Transmission
X-linked dominant
e
Autosomal dominant
Autosomal recessive
Autosomal recessive
Variable
?
Variable
Abnormal gene
PHEX
e
FGF-23
DMP1
NPT2c
Variable
?
?
BONE METABOLISM
GENETICS
XLH, X-linked hypophosphatemia; TIO, tumor-induced osteomalacia; HHRH, hereditary hypophosphatemic rickets with hypercalciuria; FS I, Fanconi syndrome, type I; FS II, Fanconi syndrome, type II; ADHR, autosomal dominant hypophosphatemic rickets; ARHR, autosomal recessive hypophosphatemic rickets; TC, tumoral calcinosis. N, normal; Z, decreased; \, increased. Modified from [237].
immunoreactive protein [122e124], supported the presence of a primary renal abnormality. In contrast, transfer of the defect in renal Pi transport to normal and/or parathyroidectomized normal mice, parabiosed to Hyp mice, implicated a humoral factor in the pathogenesis of the disease [58,125]. Current studies, however, have provided compelling evidence that the defect in renal Pi transport in XLH is secondary to the effects of a circulating hormone or metabolic factor. In this regard, immortalized cell cultures from the renal tubules of Hyp and Gy mice exhibit normal Naþ-phosphate transport, suggesting that the paradoxical effects observed in primary cultures may represent the effects of impressed memory and not an intrinsic abnormality [126,127]. Moreover, the report that cross-transplantation of kidneys in normal and Hyp mice results in neither transfer of the mutant phenotype or its correction
unequivocally established the humoral basis for XLH [62]. Subsequent efforts, which resulted in localization of the gene encoding the Naþ-phosphate co-transporter to chromosome 5, further substantiated the conclusion that the renal defect in brush border membrane phosphate transport is not intrinsic to the kidney in XLH [128]. These data established the presence of a humoral abnormality in XLH. Subsequently, several investigators identified and characterized the biological activities of a variety of phosphaturic factors (inhibitors of Naþdependent phosphate transport) and mineralization inhibitory factors, which may play a role in the pathogenesis of XLH (see above). Moreover, several reports have documented production of phosphaturic and mineralization inhibitory factors by Hyp mouse osteoblasts maintained in culture [61,89,93,126,129]. Therefore, as noted above, these studies argue that
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DISORDERS OF PHOSPHATE HOMEOSTASIS
a circulating factor(s), phosphatonin(s), FGF23, plays an important role in the pathophysiological cascade responsible for X-linked hypophosphatemia. GENETIC DEFECT
Efforts to better understand XLH have led to identification of the genetic defect underlying this disease. In 1986 Read et al. [130] and Machler et al. [131] reported linkage of the DNA probes DXS41 and DXS43, which had been previously mapped to Xp22.31-p21.3, the HYP gene locus. In subsequent studies Thakker et al. [132,133] reported linkage to the HYP locus of additional polymorphic DNA, DXS197, and DXS207 and, using multipoint mapping techniques, determined the most likely order of the markers as Xpter-DXS85-(DXS43/ DXS197)-HYP-DXS41-Xcen and Xpter-DXS43-HYP(DXS207/DXS41)-Xcen, respectively. The relatively small number of informative pedigrees available for these studies prevented definitive determination of the genetic map along the Xp22-p21 region of the X-chromosome and only allowed identification of flanking markers for the HYP locus 20 centimorgans (cM) apart. However, the independent and collaborative efforts of the HYP consortium resulted in the study of 13 multigenerational pedigrees and consequent refined mapping of the Xp22.1-p21 region of the X chromosome, identification of tightly linked flanking markers for the HYP locus, construction of a YAC contig spanning the HYP gene region, and eventual cloning and identification of the disease gene as PHEX, a Phosphate regulating gene with Homologies to Endopeptidases located on the X-chromosome. In brief, these studies ascertained a locus order on Xp22.1 of: Xcen DXS451 ðDXS41=DXS92Þ DXS274 DXS1052 DXS1683 PHYP DXS7474 DXS365 ðDXS443=DXS3424Þ DXS257 ðGLR=DXS43Þ DXS3 15 Xtel Moreover, the physical distance between the flanking markers, DXS1683 and DXS7474, was determined as 350 kb and their location on a single YAC ascertained. Subsequently, a cosmid contig spanning the HYP gene region was constructed and efforts directed at discovery of deletions within the HYP region. Identification of several such deletions permitted characterization of cDNA clones that mapped to cosmid fragments in the vicinity of the deletions. Database searches with these cDNAs detected homologies at the peptide level to a family of endopeptidase. These efforts clearly established PHEX as the candidate gene responsible for XLH [134e139]. Identification of the gene associated with XLH as PHEX [134] has facilitated efforts to better understand
1167
this disease. The gene codes for a 749-amino-acid protein, consisting of three domains: (1) a small aminoterminal intracellular tail; (2) a single, short transmembrane peptide; and (3) a large carboxyterminal extracellular peptide, which, typical of zinc metalloproteases [140], has 10 conserved cysteine residues and a HEXXH pentapeptide motif. The homology of PHEX with metalloproteases resulted in inclusion of this protein in the M13 family of membrane-bound metalloproteases, also known as neutral endopeptidases [141e143]. M13 family members, including neutral endopeptidase 24.11 (NEP), endothelin-converting enzymes 1 and 2, the Kell blood group antigen, neprilysin-like peptide (NL1), and endothelin converting enzyme-like 1 [140,142,144e150], degrade or activate a variety of peptide hormones. Preservation in the PHEX structure of catalytic glutamate and histidine residues (equivalent to Glu648 and His711 of NL1) argues for similar protease activity, as does alignment of PHEX mutations with regions required for peptidase activity in NL1 [151]. Further, like other neutral endopeptidases, immunofluorescent studies reveal a cell-surface location for PHEX in an orientation consistent with a type II integral membrane glycoprotein [151]. In any case, cloning the PHEX gene led relatively rapidly to cloning the homologous murine Phex gene and identification of the mutations in the murine homologs of XLH, the Hyp and Gy mice [64,152,153]. Unlike 97% of known genes, neither the human nor murine gene has a Kozak sequence, a purine at the e3 position before the ATG initiation sequence [152e154]. Since such genes are often posttranscriptionally regulated, this anomaly may impact understanding the hormonal and metabolic regulation of PHEX/Phex. Many investigators [63e69,134,154e162] have used Phex/Phex localization and mutation detection to help formulate the pathogenetic scheme for XLH. Investigation of murine tissues and cell cultures revealed that Phex is predominantly expressed in bones and teeth [63,64,66e68,153], while mRNA, protein or both have also been found in lung, brain, muscle, gonads, skin, and parathyroid glands [151,163]. Experiments in neonatal and adult mice further documented that the cellular locations of Phex in bone and teeth are the osteoblast/osteocyte and the odontoblast/ameloblast, respectively, while subcellular locations are the cell-surface membrane, the endoplasmic reticulum, and the Golgi compartment. Notably Phex expression is absent in the visceral abdominal organs, including the kidney, liver hepatocytes, and intestine, and in cardiac and skeletal muscle. In any case, the ontogeny of Phex expression reveals that the protein is expressed in osteoblasts at both primary and secondary ossification centers, suggesting a possible role in mineralization in vivo.
VIII. DISORDERS
1168
63. CLINICAL DISORDERS OF PHOSPHATE HOMEOSTASIS
To date >200 different PHEX mutations, consisting of deletions, frameshifts, insertions, and duplications, as well as splice site, frameshift, nonsense, and missense mutations, have been documented in >250 patients with XLH [134,154e161,164] and are scattered throughout exons 2e22, which encode the 749-amino-acid extracellular protein domain. In addition, a single mutation within the 50 untranslated region has been identified [157]. Although these mutations invariably cause loss of function, the mechanism by which such loss of activity occurs is unclear. However, preliminary data indicate that missense mutations interfere with protein trafficking, resulting in protein sequestration in the endoplasmic reticulum [160]. Since PHEX coding region mutations had not been detected in ~35% of patients, Christie et al. [161] explored whether such subjects have intronic mutations that result in mRNA splicing abnormalities. They found in one patient a unique mutation in intron 7 that created a novel donor splice site, which interacts with three naturally occurring acceptor splice sites, leading to the incorporation of three pseudoexons in PHEX transcripts. Translation of these pseudoexons results in the inclusion of missense amino acids into the PHEX protein or a truncated protein, lacking five of ten conserved cysteine residues and the pentapeptide zinc-binding motif. These observations suggest that intron mutations may represent a proportion of the gene abnormalities undiscovered in one-third of patients with XLH. In order to confirm the possibility that diminished PHEX/Phex expression in osteoblasts initiates the cascade of events responsible for the pathogenesis of XLH, several investigators have used targeted overexpression of Phex in attempts to normalize osteoblast mineralization, in vitro, and rescue the Hyp phenotype in vivo. Surprisingly, however, these studies [90,165] revealed that restoration of Phex expression and enzymatic activity to immortalized Hyp mouse osteoblasts, by retroviral-mediated transduction, does not restore their capacity to mineralize extracellular matrix in vitro, under conditions supporting normal mineralization. Moreover, in complementary studies Liu et al. [90] and Bai et al. [89] found transgenic Hyp mice (OscPhex-Hyp; pOb2.3[ColIaI]-Phex-Hyp), despite expressing abundant Phex mRNA and enzyme activity in mature osteoblasts and osteocytes, exhibited hypophosphatemia and persistently abnormal vitamin D metabolism. In the setting of P depletion, although exhibiting a modest improvement in bone mineralization, the transgenic mice maintained histological evidence of osteomalacia, similar to that in non-transgenic Hyp mice. These observations are consistent with several possibilities, acknowledged by Liu et al. [90] and Bai et al. [89], which suggest experimental design flaws in the study.
First, despite theoretical evidence to the contrary (see above), extraosseous Phex expression may play an important role in the modulation of phosphatonin activity. In support of this option, Miyamura et al. [166] using syngeneic bone marrow transplantation, were able to partly reverse the biochemical abnormalities in Hyp mice with an engraftment that was not restricted to cells of the osteoblast lineage, but included donor cells to alternate tissues, in many of which PHEX transcripts were detected [64e67]. Second and more likely, the temporal and developmental expression of the Osc and pOb2.3 promoter-driven Phex expression may not mimic the endogenous regulation of Phex. In this regard, the transgenic animals may experience PHEX expression later than, or in osteoblast-related cell subpopulations different from those in normal animals. In fact, neither promoter is expressed in the preosteoblast and the osteocalcin promoter appears at least 4 days later than PHEX in normally developing osteoblasts [89,90]. Thus, lack of Phex activity early in osteoblast development (in pre-osteoblasts or preceding osteocalcin expression in osteoblasts) may result in failure to alter an otherwise immutable osteoblast dysfunction, the continued presence of which contributes to the impairment of mineralization. In accord, later expression of PHEX may not rescue the phenotype, as the immutable change is refractory to endopeptidase activity. However, Erben et al. [91] reported that ubiquitous overexpression of Phex under the control of the bactin promoter in two different mice did not completely resolve the bone mineralization defect and failed to alter the abnormal phosphate homeostasis, raising significant further questions regarding the interaction between the PHEX gene defect and phenotypic expression of the disease. Regardless, in no way do these observations exclude a role for inadequate Phex expression in the genesis of XLH or more particularly in the mineralization process in the mature osteoblast. To further explore the role of the osteoblast and osteocyte in the pathogenesis of XLH, Yuan et al. [100] created mice with a global PHEX knockout (Cre-PhexDflox/y mice) and mice with a conditional osteocalcin-promoted PHEX inactivation limited to osteoblasts (OC-Cre-PhexDflox/y mice). Phenotype and bone histology were compared among the two knockout mouse strains, as well as Hyp and normal mice. Elevated Fgf23 levels, reduced brush border membrane phosphate transport, low renal NPT2a protein concentration, and decreased serum phosphorus levels were present in Hyp, Cre-PhexDflox/y and OC-CrePhexDflox/y mice when compared to normal mice. Moreover, Hyp, Cre-PhexDflox/y and OC-Cre-PhexDflox/y mice all demonstrated comparable osteomalacia, providing evidence that abnormal Phex function in osteoblasts alone is sufficient to create the HYP phenotype.
VIII. DISORDERS
1169
DISORDERS OF PHOSPHATE HOMEOSTASIS
PATHOGENESIS
In spite of the remarkable advances that have been made in understanding the genetic abnormality and pathophysiology of XLH, the detailed pathogenetic mechanism underlying this disease remains incompletely defined (see above). While as related previously, the Phex substrate remains unknown, a role for FGF23 in the pathogenesis of the disease seems certain. Moreover, it is now clear that a defect in the PHEX gene has downstream effects that result in overproduction and enhanced circulating levels of FGF23 and consequent inhibition of renal Naþ-phosphate transport, the likely operative scenario in the pathogenesis of XLH. The coexistence of osteoblast defects in XLH further confounds understanding the pathophysiology of this disorder. Elegant experiments, which documented the abnormal mineralization of periostea and osteoblasts of Hyp mice following transplantation into the muscle of normal mice, provide clear evidence for intrinsic defects in the bone of mutants [167]. Indeed, proof of specific osteoblast abnormalities has been provided by studies that show decreased phosphorylation of osteopontin and increased osteocalcin levels in cells from Hyp mice [168]. Based on these observations, it is tempting to speculate that FGF23 is not the sole abnormality resulting in abnormal bone mineralization. Indeed, a wide array of studies support this conclusion (see above). Thus, the precise mechanism underlying the abnormal bone mineralization in XLH is not definitively proven. In any case, it is evident that further information is requisite to enhance our understanding of the pathogenesis of XLH and, in turn, regulation of phosphate homeostasis, osteoblast function, vitamin D metabolism, and osteoid mineralization. Such data are not only critical to understanding the pathogenesis of XLH and the regulation of mineral homeostasis, but may have significant impact upon determination of optimal treatment strategies for many of the vitamin-D-resistant diseases. TREATMENT
In past years, physicians employed pharmacologic doses of vitamin D as the cornerstone for treatment of XLH. However, long-term observations indicate that this therapy fails to cure the disease and poses the serious problem of recurrent vitamin D intoxication and renal damage. Therefore, choice of therapy for this disease has been remarkably influenced by the increased understanding of the pathophysiological factors, which affect phenotypic expression of the disorder. Thus, current treatment strategies for children directly address the combined calcitriol and phosphorus deficiency characteristic of the disease. Generally, the regimen includes a period of titration to achieve a maximum dose of calcitriol, 1e3 mg/d in two divided doses and phosphorus,
1e4 g/d in 4e5 divided doses [169,170]. Such combined therapy often improves growth velocity, normalizes lower extremity deformities, and induces healing of the attendant bone disease. Refractoriness to the growth-promoting effects of treatment, however, is often encountered particularly in youths presenting at <5th percentile in height [171]. For that reason, the use of recombinant growth hormone as an additional treatment component has been advocated. Definite positive effects have been observed in young patients with XLH [172,173]. Of course, treatment involves a significant risk of toxicity that is generally expressed as abnormalities of calcium homeostasis, most notably secondary hyperparathyroidism that may become autonomous and require surgery. Detrimental effects on renal function secondary to abnormalities such as nephrocalcinosis are also possible. Thus, frequent monitoring of the phosphate and calcitriol dosage during growth is mandatory. Therapy in adults is reserved for episodes of intractable bone pain and refractory nonunion bone fractures. More recently, addition of calcimimetics as adjuvant therapy in patients with XLH has been advocated [174]. Preliminary observations indicate that inclusion of cinacalcet in the traditional treatment regimen abrogates the anticipated phosphorus-dependent increase in the serum PTH concentration, leading to an increase in the treatment-dependent renal TmP/GFR and serum phosphorus. Such modulation of treatment effects may allow use of lower doses of phosphate and calcitriol. As a consequence the incidence of secondary and tertiary hyperparathyroidism and nephrocalcinosis, known complications of standard therapy, may decrease [175]. Hereditary Hypophosphatemic Hypercalciuria (HHRH)
Rickets
with
This rare genetic disease is marked by hypophosphatemic rickets with hypercalciuria [176]. The cardinal biochemical features of the disorder include hypophosphatemia due to increased renal phosphate clearance and normocalcemia. In contrast to other diseases in which renal phosphate transport is limited, patients with HHRH exhibit increased 1,25(OH)2D production [176e180] (Table 63.3). The resultant elevated serum calcitriol levels enhance the gastrointestinal calcium absorption, which in turn increases the filtered renal calcium load and inhibits parathyroid secretion. These events cause the characteristic hypercalciuria observed in affected patients. The clinical expression of the disease is heterogeneous. In general, children become symptomatic between the ages of 6 months and 7 years. Initial symptoms consist of bone pain or deformities of the lower limbs (or both), which progressively interfere with gait
VIII. DISORDERS
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and physical activity. The bone deformities vary from genu varum or genu valgum to anterior external bowing of the femur and coxa vara. Additional features at presentation include short stature with disproportionately short lower limbs, muscle weakness, and radiological signs of rickets or osteomalacia (or both). These various symptoms and signs may exist separately or in combination and may be present in a mild or severe form. A large number of apparently unaffected relatives of patients with HHRH exhibit an additional mode of disease expression [180]. These subjects, although without evidence of bone disease, manifest idiopathic hypercalciuria (IH), most evident in postprandial periods, as well as a pattern of biochemical abnormalities similar to those of children with rickets and osteomalacia. Quantitatively, however, the abnormalities are milder, and the relevant biochemical values intermediate between those observed in family members with HHRH and those in normal relatives. The absence of bone disease in these patients may be explained by relatively mild hypophosphatemia compared to the severe phosphate depletion evidenced in patients with the full spectrum of the disorder [180]. Relatively few unrelated kindreds with HHRH have been described, including an extended family of Bedouin origin that includes 13 patients with HHRH and 42 with hypercalciuria; a smaller kindred of oriental Jewish origin with five affected members; and a family of Yemenite Jewish origin that includes two patients with HHRH and two with hypercalciuria [178]. However, several small families with similar genetic abnormalities as those established in patients with classical HHRH have presented with phenotypic variants of the HHRH disease. The variations include: (1) presentation with hypercalciuria and nephrocalcinosis, as well as rickets/osteomalacia, but an associated normal serum calcium and phosphorus and elevated serum calcitriol concentration [181] and (2) a phenotypically similar disorder, including mild childhood idiopathic hypercalciuria with bone lesions (rickets) and stunted linear growth, which is associated with a heterozygotic mutation and variable penetrance [182]. Moreover, several patients with a sporadic occurrence of HHRH have been recognized. Studies are generally incomplete, however, and the presence of hypercalciuria in relatives has not been excluded. PATHOPHYSIOLOGY
Liberman, Tieder, and associates [176,180,183] have presented data that indicate the primary inborn error underlying this disorder is an expressed abnormality in the renal proximal tubule, which impairs phosphate reabsorption. They propose that this pivotal defect in turn stimulates renal 25(OH)D-1a-hydroxylase, thus
promoting the production of 1,25(OH)2D and increasing its serum and tissue levels. As a result intestinal calcium and phosphorus absorption is augmented and the renal filtered calcium load consequently increased. The enhanced intestinal calcium absorption also suppresses parathyroid function. In addition, prolonged hypophosphatemia diminishes osteoid mineralization, resulting in rickets and/or osteomalacia. The proposal that abnormal phosphate transport results in increased calcitriol production remains untested. Indeed, the elevation of 1,25(OH)2D in patients with HHRH is a unique phenotypic manifestation of the disease that distinguishes it from other disorders in which abnormal phosphate transport is likewise manifest. Such heterogeneity in the phenotype of these disorders suggests that disease at variable anatomical sites along the proximal convoluted tubule or involving compensatory enhancement of sodium-dependent phosphate cotransporters, such as that in the Npt2ae/e mouse [28,29], while uniformly impairing phosphate transport, may not necessarily inhibit 25(OH)D-1ahydroxylase activity. GENETIC DEFECT
Autosomal recessive transmission of HHRH seems consistent with the inheritance pattern in the described kindreds. However, if HHRH is an autosomal recessive disease and individuals with idiopathic hypercalciuria (IH) are heterozygous for the mutant allele, IH must be an incompletely penetrant trait because not all obligate heterozygotes manifest hypercalciuria. Alternatively, it has been suggested that HHRH and IH could be the result of mutations in two different genes [180]. The strongest support against this hypothesis is that when individuals with HHRH are treated with oral Pi, both the hypophosphatemia and the hypercalciuria are corrected. Moreover, recent studies have identified mutations in the SLC34A3 gene, coding the Npt2c protein, as causal of the HHRH, albeit the Npt2c protein was thought to have only a minor role in renal Pi transport. In fact, mice that are null for Npt2c (Npt2c/) reportedly show no evidence for renal phosphate wasting when maintained on a diet with a normal phosphate content. However, double knockout mice (Npt2a/ and Npt2c/) do exhibit a phenotype (hypophosphatemia, hypercalciuria, and rickets) substantially more severe than that of Npt2a and Npt2c knockout mice. Such observations lend credence to a substantial role for the Npt2c protein in the regulation of renal phosphate transport and maintenance of phosphate homeostasis [14]. TREATMENT
In accord with the hypothesis that a defect in renal phosphate transport alone underlies HHRH, patients
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have been treated successfully with high-dose phosphorus (1e2.5 g/d in five divided doses) alone. Within several weeks after initiation of therapy, bone pain disappears and muscular strength improves substantially. Moreover, the majority of treated subjects exhibit accelerated linear growth and radiological signs of rickets are completely absent within 4e9 months. Concordantly, serum phosphorus values increase towards normal, the 1,25(OH)2D concentration decreases, and alkaline phosphatase activity declines to normal. Despite this favorable response, limited investigation indicates that the osteomalacic component of the bone disease does not exhibit normalization. Further studies will be necessary, therefore, to determine if phosphorus alone will be sufficient treatment for this rare disorder. In any case, administration of phosphorus in patients with this disease does not result in the same spectrum of complications encountered upon its use in other disorders. Most notably, nephrocalcinosis, a common complication in treated patients with XLH, occurs infrequently in subjects with HHRH. In fact, the rare occurrence of this complication is associated with a history of vitamin D intoxication prior to initiation of treatment with phosphorus. Similarly, the development of secondary hyperparathyroidism in treated patients with HHRH has not been reported, although expectation of this complication is high since oral administration of phosphate does diminish the circulating calcium concentration and, in turn, stimulates parathyroid function. Fanconi Syndrome (FS) Rickets and osteomalacia are frequently associated with Fanconi syndrome, a disorder characterized by hyperphosphaturia and consequent hypophosphatemia, hyperaminoaciduria, renal glycosuria, albuminuria, and proximal renal tubular acidosis [184e187]. Damage to the renal proximal tubule, secondary to genetic disease or environmental toxins, represents the common underlying mechanism of this disease [188]. Resultant dysfunction causes renal wasting of those substances primarily reabsorbed at the proximal tubule. The inherited form may occur in isolation (in the absence of any other metabolic disease) or secondary to various primary Mendelian diseases. The associated bone disease in this disorder is likely secondary to hypophosphatemia and/or acidosis, abnormalities that occur in association with aberrantly (Fanconi syndrome, type I) or normally regulated (Fanconi syndrome, type II) vitamin D metabolism. FANCONI SYNDROME, TYPE I
Renal phosphate wasting and hypophosphatemia are the hallmark abnormalities of this disease, which
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resembles in many respects the more common genetic disease, XLH. In this regard, occurrence of abnormal bone mineralization appears dependent upon the prevailing renal phosphate wasting and resultant hypophosphatemia. Indeed, disease subtypes in which isolated wasting of amino acids, glucose, or potassium occur, are not associated with rickets and/or osteomalacia. Further, in the majority of patients studied, affected subjects exhibit abnormal vitamin D metabolism, characterized by serum 1,25(OH)2D levels that are overtly decreased or abnormally low relative to the prevailing serum phosphorus concentration [189e191]. Although the aberrantly regulated calcitriol biosynthesis may be due to the abnormal renal phosphate transport, as suspected in patients with XLH, proximal tubule damage and acidosis may play important roles. A notable difference between this syndrome and XLH is a prevailing acidosis, which may contribute to the bone disease. In this regard, several studies indicate that acidosis may exert multiple deleterious effects on bone. Such negative sequelae are related to the loss of bone calcium that occurs secondary to calcium release for use in buffering [184,192]. Alternatively, several investigators [193,194] have reported that acidosis may impair bone mineralization secondary to direct inhibition of renal 1a-hydroxylase activity. Others dispute these findings and claim that acidosis does not cause rickets or osteomalacia in the absence of hypophosphatemia. Indeed, Brenner et al. [195] reported that the rachitic/osteomalacic component of this disorder occurs only in patients with type 2 renal tubular acidosis and phosphate wasting. In contrast, those with types 1 and 4 renal tubular acidosis displayed no evidence of abnormal bone mineralization. Thus, the interplay of acidosis and phosphate depletion on bone mineralization in this disorder remains poorly understood. Most likely, however, hypophosphatemia and abnormally regulated vitamin D metabolism are the primary factors underlying rickets and osteomalacia in this form of Fanconi syndrome. Although the genetic defect resulting in this disease has been unknown, recent studies have identified a homozygous duplication in SLC34A1, encoding Npt2a, as the cause of autosomal recessive Fanconi syndrome associated with hypophosphatemic rickets and renal failure. This finding provides a human model for the loss of function of Npt2a, constitutes previously unavailable evidence for the inclusion of Npt2a in the list of proteins involved in human hypophosphatemic rickets and proximal tubulopathy, and validates the pivotal role of the human Npt2a cotransporter in renal phosphate handling and in the maintenance of wholebody phosphate homeostasis. The mechanism whereby intracellular accumulation of a membrane-targeted transporter such as Npt2a may induce a more
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generalized proximal-tubule dysfunction and renal failure is currently unknown but is likely to become the subject of further research [196]. FANCONI SYNDROME, TYPE II
Tieder et al. [197] have described two siblings (from a consanguineous mating) who presented with classic characteristics of Fanconi syndrome, including renal phosphate wasting, glycosuria, generalized aminoaciduria, and increased urinary uric acid excretion. However, these patients had appropriately elevated (relative to the decreased serum phosphorus concentration) serum 1,25(OH)2D levels and consequent hypercalciuria despite normal serum parathyroid hormone levels and cyclic AMP excretion. Moreover, treatment with phosphate reduced the serum calcitriol in these patients into the normal range and normalized the urinary calcium excretion. In many regards, this syndrome resembles HHRH and represents a variant of Fanconi syndrome, referred to as type II disease. The bone disease in affected subjects is likely due to the effects of hypophosphatemia. In any case, the existence of this variant form of disease is probably the result of renal damage to a unique segment of the proximal tubule or involvement of a different mechanism at the same site [197]. Further studies will be necessary to distinguish these possibilities. TREATMENT
Ideally, treatment of the bone disease in this disorder should offset the pathophysiological defect influencing proximal renal tubular function. In many cases, however, the primary abnormality remains unknown. Moreover, efforts to decrease tissue levels of causal toxic metabolites by dietary (such as in fructose intolerance) or pharmacological means (such as in cystinosis and Wilson syndrome) have met with variable success. Indeed, no evidence exists that indicates if the proximal tubule damage is reversible upon relief of an acute toxicity. Regardless, in instances when specific therapies are not available or do not lead to normalization of the primary defect, therapy must be directed at raising the serum phosphorus concentration, replacing calcitriol (in type I disease) and reversing the associated acidosis. However, use of phosphorus and calcitriol in this disease has been limited. In general, such replacement therapy leads to substantial improvement or resolution of the bone disease [198]. Unfortunately, growth and developmental abnormalities, more likely associated with the underlying genetic or acquired disease, remain substantially impaired [198]. More efficacious therapy, therefore, is dependent upon future research into the causes of the multiple disorders that underlie this syndrome.
X-linked Recessive Hypophosphatemia (XLRH) The initial description of X-linked recessive hypophosphatemic rickets involved a family in which males presented with rickets or osteomalacia, hypophosphatemia, and a reduced renal threshold for phosphate reabsorption. In contrast to patients with XLH, affected subjects exhibited hypercalciuria, elevated serum 1,25 (OH)2D levels (Table 63.1), and proteinuria of up to 3 g/day. Patients also developed nephrolithiasis and nephrocalcinosis with progressive renal failure in early adulthood. Female carriers in the family were not hypophosphatemic and lacked any biochemical abnormalities other than hypercalciuria. Three related syndromes have been reported independently: X-linked recessive nephrolithiasis with renal failure, Dent’s disease, and low-molecular-weight proteinuria with hypercalciuria and nephrocalcinosis. These syndromes differ in degree from each other, but common themes include proximal tubular reabsorptive failure, nephrolithiasis, nephrocalcinosis, progressive renal insufficiency, and, in some cases, rickets or osteomalacia. Identification of mutations in the voltage-gated chloride-channel gene CLCN5 in all four syndromes has established that they are phenotypic variants of a single disease and are not separate entities [199,200]. However, the varied manifestations that may be associated with mutations in this gene, particularly the presence of hypophosphatemia and rickets/osteomalacia, underscore that environmental differences, diet, and/or modifying genetic backgrounds may influence phenotypic expression of the disease.
Disorders of Renal Phosphate Transport: Hyperphosphatemic Diseases (Hyperphosphatemic) Tumoral Calcinosis (TC) Tumoral calcinosis is a rare genetic disease characterized by periarticular cystic and solid tumorous calcifications. Most patients in North America with this disorder are black and about one-third of the cases are familial. There is no gender preference. Biochemical markers of the disorder include hyperphosphatemia and an elevated serum 1,25(OH)2D concentration in many patients. Using these criteria, evidence has been presented for autosomal recessive inheritance of this syndrome. The hyperphosphatemia characteristic of the disease results from an increase in capacity of renal tubular phosphate reabsorption [201,202]. Hypocalcemia is not a consequence of this abnormality, however, and the serum parathyroid hormone concentration is normal. Moreover, the phosphaturic and urinary cAMP responses to parathyroid hormone are not disturbed.
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Interestingly, affected patients manifest increased circulating 1,25(OH)2D levels despite hyperphosphatemia, underlining the fact that it is TmP/GFR rather than the serum phosphorus concentration that controls 1a-hydroxylase activity (Fig. 63.1). Undoubtedly, the calcific tumors result from the elevated calciumephosphorus product. The observation that long-term phosphorus depletion alone [203] or in association with administration of aluminum hydroxide [204] or acetazolamide, a phosphaturic agent [205], leads to resolution of the tumor masses supports this possibility. Furthermore, reduction of phosphate levels in extracellular fluid helps prevent reformation of mineral deposits [203]. In addition, preliminary studies indicate that calcitonin therapy may also be efficacious by enhancing phosphaturia [206]. An acquired form of this disease is rarely seen in patients with end-stage renal failure. Affected patients manifest hyperphosphatemia in association with either: (1) an inappropriately elevated calcitriol level for the degree of renal failure, hyperparathyroidism or hyperphosphatemia; or (2) long-term treatment with calcium carbonate, calcitriol, or high-calcium-content dialysates. Again, calcific tumors likely result from an elevated calciumephosphorus product. Indeed, complete remission of the tumors occurs with treatment with vinpocetine, a mineral scavenger drug. A series of recent studies have documented that the autosomal recessive disorder is due to loss-of-function mutations in FGF23, as well as GALNT3 and Klotho, all causing familial tumoral calcinosis because of inadequate intact FGF23 concentrations or action. FGF23 MUTATIONS
It is not surprising that a genetic defect underlying familial tumoral calcinosis is an FGF23 loss-of-function mutation, since the phenotype of the disorder is the metabolic mirror image of that in patients with ADHR, which results from a gain-of-function mutation. Indeed, several reports have documented that affected individuals with familial tumoral calcinosis had biallelic mutations in the FGF23 gene [207e210]. The mutations resulted in various abnormalities including: (1) destabilization of full-length FGF23; and (2) retention of the fulllength FGF23 in the Golgi complex and secretion of only the biologically inactive C-terminal fragment. Hence, the syndrome resembles that evident in FGF23 knockout mice. GALNT3 MUTATIONS
Novel biallelic mutations affecting UDP-N-acetyl-aD-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc transferase 3) have been found in patients of Middle Eastern and European descent [211e213]. This protein is a Golgi-associated biosynthetic
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enzyme, which initiates mucin-type O-glycosylation of proteins. O-glycosylation of FGF23 by the GalNAc transferase 3 is essential for the secretion of intact FGF23 because O-glycosylation at a subtilisin-like proprotein convertase recognition sequence motif prevents cleavage of FGF23. The absence of secretion underlies the loss-offunction nature of the mutation. In these studies of patients with GALNT3 mutations have indicated phenotypic variability in affected individuals. While the mutations invariably result in hyperphosphatemia as a result of low circulating FGF23 concentrations, other clinical manifestations of the disorder were variable. Indeed, the spectrum of phenotypic abnormalities ranged from those typical of familial tumoral calcinosis to those associated with hyperostosisehyperphosphatemia (recurrent long bone lesions with hyperostosis). Therefore, tumoral calcinosis and hyperostosisehyperphosphatemia syndrome likely represent a continuous spectrum of the same disease caused by increased phosphate levels, rather than two distinct disorders. KLOTHO MUTATIONS
Recently Ichikawa et al. [214] reported a homozygous missense mutation in the KLOTHO gene in a patient who presented with severe tumoral calcinosis. The mutation attenuated production of the membrane bound Klotho, resulting in diminished ability of FGF23 to signal via its cognate FGFR1 receptors. As a consequence, FGF23 resistance resulted in a syndrome reflecting absent FGF23 action. See Chapter 42 for further discussion of the role of Klotho in FGF23 action. The genetic abnormalities underlying familial tumoral calcinosis add to the evolving story of the role that FGF23 plays in the evolution of genetic disorders of hypophosphatemic and hyperphosphatemic diseases. Indeed, the data available argue forcefully for the central role of this phosphatonin in the pathophysiology of these diseases.
DISORDERS RELATED TO AN ALTERED PHOSPHATE LOAD Decreased Phosphate Load Phosphate Deprivation Hypophosphatemia and phosphate depletion due to inadequate dietary intake are rare. However, studies indicate that hypophosphatemia is a relatively frequent occurrence in children under 5 years of age presenting with kwashiorkor or marasmic kwashiorkor [215]. With a decline in ingested phosphate, the renal TmP increases and urinary phosphate excretion decreases [216]. In addition, gastrointestinal phosphate secretion gradually lessens. However, severe dietary deprivation
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(less than 100 mg/d) leads to a prolonged period of negative phosphate balance and total body depletion. Affected females may display hypophosphatemia (1.4 to 2.5 mg/dl). Interestingly, in contrast, males generally do not manifest a decreased serum phosphate concentration in response to dietary deprivation. Nevertheless, attempts to maintain phosphate homeostasis in both sexes include suppression of the serum PTH concentration and increased 1,25(OH)2D production by an unknown mechanism. Thus, hypercalciuria may be associated with the syndrome. Whether the efficiency of calcitriol responsiveness or differential effects on end organs, such as the gastrointestinal tract or bone, underlies the noted gender difference remains unclear. Total starvation does not cause profound hypophosphatemia. The catabolic effects of total food deprivation result in the release of phosphate from intracellular stores, which compensates for the negative phosphorus balance. However, refeeding of the starved person can precipitate hypophosphatemia if phosphate deprivation is maintained. Indeed, this “refeeding syndrome” was observed by Kimutai et al. [215] when patients with phosphate depletion due to kwashiorkor and marasmic kwashiorkor received nutritional intervention. A decrease in serum phosphorus levels to levels as low as 0.63 mmol/liter (~2.0 mg/dl) occurred, resulting in deaths amongst 15% of the population. Gastrointestinal Malabsorption Gastrointestinal absorption of phosphorus may be decreased with the use of aluminum- or magnesiumcontaining antacids, as well as other drugs, including calcium acetate/magnesium carbonate and sevelamer hydrochloride; prolonged use of these drugs in large amounts has been associated with hypophosphatemia and a negative phosphorus balance [217]. Long-term reduction of the serum phosphorus concentration owing to chronic, excessive use of antacids leads to frank osteomalacia and myopathy. The osteomalacia results as a direct consequence of the phosphate depletion and in spite of normal vitamin D stores and an increased serum 1,25(OH)2D concentration, which occurs in response to the hypophosphatemia. In contrast, the pathophysiology of variably occurring osteomalacia secondary to gastrointestinal diseases that cause steatorrhea or rapid transit time (e.g., Crohn’s disease, postgastrectomy states, and intestinal fistulas) is significantly different [218]. In this case, mild to moderate hypophosphatemia occurs due to vitamin D malabsorption/deficiency and resultant secondary hyperparathyroidism and renal phosphate wasting. Further, the relation between the metabolic bone disease, vitamin D deficiency, and hypophosphatemia is
complex and likely involves the influence of phosphopenia and vitamin-D-dependent calciopenia on bone mineralization. However, the impact of vitamin D deficiency may be overriding since osteomalacia may occur in the absence of hypophosphatemia. Regardless, in the presence of hypophosphatemia (and/or phosphate depletion), an elevated serum calcitriol level is part of this syndrome, likely due to elevated serum parathyroid hormone and has no apparent effect on the evolution of the bone disease. Many other causes of malabsorption may likewise influence phosphate and vitamin D homeostasis. See Chapter 69 for a review of these GI diseases.
Transcellular Shift In a large proportion of clinically important cases of hypophosphatemia, a sudden shift of phosphorus from the extracellular to the intracellular compartment is responsible for the decline of the serum phosphate concentration. This ion movement occurs in response to naturally occurring disturbances and after the administration of certain compounds. Alkalosis Alkalosis secondary to intense hyperventilation may depress serum phosphate levels to less than 1 mg/dl [219]. A similar degree of alkalemia from excess bicarbonate also causes hypophosphatemia, but of a much lesser magnitude (2.5 to 3.5 mg/dl). The disparity between the effects of a respiratory and metabolic alkalosis is related to the more pronounced intracellular alkalosis that occurs during hyperventilation. The phosphate shift to the intracellular compartment results from the utilization attendant on glucose phosphorylation, a process stimulated by a pH-dependent activation of phosphofructokinase. Glucose Administration The administration of glucose and insulin often results in moderate hypophosphatemia [220]. Endogenous or exogenous insulin increases the cellular uptake not only of glucose, but also of phosphorus. The most responsive cells are those of the liver and skeletal muscle. The decline of the serum phosphate concentration generally does not exceed 0.5 mg/dl. A lesser decrease is manifest in patients with type 2 diabetes mellitus and insulin resistance or those with a disease causing a diminished skeletal mass. The administration of fructose and glycerol similarly reduces the serum phosphorus concentration. In contrast to glucose, fructose administration may be associated with more pronounced hypophosphatemia; the more striking effect is due to unregulated phosphorus uptake by the liver.
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Combined Mechanisms There are special clinical situations in which an altered phosphate and consequent hypophosphatemia result from both a transcellular shift of phosphorus and phosphate deprivation or renal phosphate wasting. These disorders represent some of the more common and profound causes of a decreased serum phosphorus concentration. Alcoholism Alcoholic patients frequently enter the hospital with hypophosphatemia. However, many do not exhibit a decreased serum phosphate concentration until several days have elapsed and refeeding has begun. The multiple factors that underlie the hypophosphatemia include poor dietary intake, use of phosphate binders to treat gastritis, excessive urinary losses of phosphorus, and shift of phosphorus from the extracellular to the intracellular compartment, owing to glucose administration and/or hyperventilation that occurs in patients with cirrhosis or during alcohol withdrawal [220]. Moreover, many alcoholic patients are hypomagnesemic, which potentiates renal phosphate wasting by an unclear mechanism. Burns Within several days after sustaining an extensive burn, patients often manifest severe hypophosphatemia. The initial insult induces a transient retention of salt and water. When the fluid is mobilized, significant urinary phosphorus loss ensues. Coupled with the shift of phosphorus to the intracellular compartment, which occurs secondary to hyperventilation, and the anabolic state, profound hypophosphatemia may result. Nutritional Recovery Syndrome Refeeding of starved individuals or maintaining nutritional support by parenteral nutrition or by tube feeding, without adequate phosphorus supplementation, may also cause hypophosphatemia [221]. A prerequisite for the decreased serum phosphate concentration in affected patients is that their cells must be capable of an anabolic response. As new proteins are synthesized and glucose is transported intracellularly, phosphate demand depletes reserves. Several days are generally required after the initiation of refeeding in order to establish an anabolic condition. In patients receiving total parenteral nutrition, serum phosphate levels may be further depressed if sepsis supervenes and a respiratory alkalosis develops. Diabetic Ketoacidosis Poor control of blood glucose and consequent glycosuria, polyuria, and ketoacidosis invariably cause
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renal phosphate wasting [220]. The concomitant volume contraction may yield a normal serum phosphate concentration. However, with insulin therapy, the administration of fluids, and correction of the acidosis, serum and urine phosphate fall precipitously. The resultant hypophosphatemia may contribute to insulin resistance and slow the resolution of the ketoacidosis. Hence, the administration of phosphate may improve the capacity to metabolize glucose and facilitate recovery.
Increased Phosphate Load Vitamin D Intoxication An increase of the phosphate load from exogenous sources generally does not cause hyperphosphatemia because the excessive phosphorus is excreted by the kidney. However, an increased serum phosphate concentration may occur in vitamin D intoxication when the gastrointestinal absorption of phosphate is markedly enhanced. Increased phosphate mobilization from bone and a reduction of GFR, secondary to hypercalcemia and/or nephrocalcinosis, may also contribute to the evolution of the hyperphosphatemia. The chronic ingestion of large doses of vitamin D, in excess of 100 000 IU/day, is generally required to cause intoxication. Suspected hypervitaminosis D may be investigated using specific assays, which can document excessive amounts of vitamin D and its metabolites in the circulation (see Chapters 45 and 72 for a discussion of vitamin D intoxication). Rhabdomyolysis Because muscle contains a large amount of phosphate, necrosis of muscle tissue may acutely increase the endogenous phosphate load and result in hyperphosphatemia. Such muscle necrosis (rhabdomyolysis) may complicate heat stroke, acute arterial occlusion, hyperosmolar non-ketotic coma, trauma, toxic agents such as ethanol and heroin, and idiopathic paroxysmal myoglobinuria [222,223]. Muscle biopsy often reveals myolytic denervation. As a consequence of muscle destruction, acute renal failure caused by myoglobin excretion frequently complicates the clinical presentation and contributes to the hyperphosphatemia. However, an elevated serum phosphate concentration may precede evidence of renal failure, or occur in its complete absence when rhabdomyolysis is present. The diagnosis is confirmed by elevated serum creatine phosphokinase, uric acid, and lactate dehydrogenase concentrations, and the demonstration of heme-positive urine in the absence of red blood cells. Therapy is directed at the underlying disorder with maintenance of the extracellular volume to avoid volume depletion
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and alkalinization of the urine to prevent uric acid accumulation and consequent acute tubular necrosis. Cytotoxic Therapy Cytotoxic therapy often causes cell destruction and liberation of phosphorus into the circulation [224]. The lysis of tumor cells begins within 1 to 2 days after initiating treatment, and is followed quickly by an elevation of the serum phosphate concentration. Hyperphosphatemia supervenes, however, only when the treated malignancies have a large tumor burden, rapid cell turnover, and substantial intracellular phosphorus content. Such malignancies include lymphoblastic leukemia, various types of lymphoma, and acute myeloproliferative syndromes. Hyperkalemia and hyperuricemia also occur. Indeed, uric acid nephropathy may cause renal insufficiency that predisposes to further phosphate retention and worsening of the hyperphosphatemia. Malignant Hyperthermia Malignant hyperthermia is a rare familial syndrome characterized by an abrupt rise in body temperature during the course of anesthesia [225]. The disease appears to be autosomal dominant in transmission, and an elevated serum creatine phosphokinase concentration is found in otherwise normal family members. Hyperphosphatemia results from shifts of phosphate from muscle cells to the extracellular pool. A high mortality rate accompanies the syndrome.
Clinical Signs and Symptoms of Abnormal Serum Phosphorus in Diseases Caused by an Altered Phosphate Load As related above, a wide variety of diseases and syndromes with varying clinical manifestations have the characteristic biochemical abnormalities of hyperphosphatemia or hypophosphatemia. A unique complex of disturbances often is directly related to the abnormal phosphate homeostasis. The recognition of these signs and symptoms may lead to appropriate biochemical testing, the diagnosis of an unsuspected disease, and initiation of lifesaving or curative treatment. Hypophosphatemia Hypophosphatemia secondary to impaired expression or function of phosphate transporters frequently causes renal stones in affected mice and humans [5]. Indeed, a decreased TmP/GFR (<0.70 mmol/liter), indicative of impaired renal phosphate transport, is present in 20% of subjects in whom renal stones form but in only 5% of control subjects [226]. Renal phosphate
wasting is also associated with bone demineralization as evident in innumerable diseases discussed above. More profound consequences of a low serum phosphorus level occur if the hypophosphatemia is associated with concomitant phosphate depletion. Such phosphate deficiency may cause widespread disturbances. This is not surprising, since severe hypophosphatemia/phosphate deficiency causes two critical abnormalities that impact on virtually all organ systems. First, a deficiency of 2,3-diphosphoglycerate (2,3-DPG) occurs in red cells, which is associated with an increased affinity of hemoglobin for oxygen and, therefore, tissue hypoxia. Second, there is a decline of tissue ATP content and a concomitant decrease in the availability of energyrich phosphate compounds that are essential for cell function [227,228]. The major clinical syndromes resulting from these abnormalities include nervous system dysfunction, anorexia, nausea, vomiting, ileus, muscle weakness, cardiomyopathy, respiratory insufficiency, hemolytic anemia, and impaired leukocyte and platelet function. Additionally, phosphate deficiency causes osteomalacia and bone pain, clinical sequelae that are probably independent of the aforementioned abnormalities. Central nervous system dysfunction has been well characterized in severe hypophosphatemia/phosphate depletion, especially in patients receiving total parenteral nutrition for diseases causing severe weight loss. A sequence of symptoms compatible with a metabolic encephalopathy usually begins one or more weeks after the initiation of therapy with solutions that contain glucose and amino acids, but lack adequate phosphorus supplementation to prevent hypophosphatemia. The onset of dysfunction is marked by irritability, muscle weakness, numbness, and paresthesias, with progression to dysarthria, confusion, obtundation, coma, and death [229]. These patients have a profoundly diminished red cell 2,3-DPG content. Both biochemical abnormalities and clinical symptoms improve as patients receive phosphorus supplementation. Peripheral neuropathies, Guillain-Barre-like paralysis, hyporeflexia, intention tremor, and ballismus have also been described with hypophosphatemia and phosphate depletion. The effects of hypophosphatemia on muscle depend on the severity and chronicity of the deficiency. Chronic phosphorus deficiency results in a proximal myopathy with striking atrophy and weakness. Osteomalacia frequently accompanies the myopathy, so patients complain of pain in weight-bearing bones. Normal values for serum creatine phosphokinase and aldolase activities are characteristically present. Rhabdomyolysis does not occur with chronic phosphate depletion. In contrast, acute hypophosphatemia can lead to rhabdomyolysis with muscle weakness and pain. Most
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cases occur in chronic alcoholics or patients receiving total parenteral nutrition. In both groups of patients, muscle pain, swelling, and stiffness occur 3 to 8 days after the initiation of therapies that do not contain adequate amounts of phosphorus. Muscle paralysis and diaphragmatic failure may occur. Studies of muscle tissue from chronically phosphate-depleted dogs made acutely hypophosphatemic show a decrement in cellular content of phosphorus, ATP, and adenosine diphosphate (ADP). Rhabdomyolysis occurred in these muscle fibers. The laboratory findings in patients with hypophosphatemic myopathy and rhabdomyolysis include elevated serum creatine phosphokinase levels; however, serum phosphate levels may become normal if enough necrosis has occurred with subsequent phosphorus release. Also, renal failure and hypocalcemia can be associated with the syndrome. Myocardial performance can be abnormal at serum phosphate levels of 0.7 to 1.4 mg/dL, which reflect profound phosphate depletion. This occurs when ATP depletion causes impairment of the actinemyosin interaction, the calcium pump of the sarcoplasma, and the sodiumepotassium pump of the cell membrane [230]. The net result is reduced stroke work and cardiac output, which may progress to congestive heart failure. These problems are reversible with phosphate replacement. Respiratory failure can occur owing to failure of diaphragmatic contraction in hypophosphatemic patients. When serum phosphate levels are raised, diaphragmatic contractility improves. The postulated mechanism for the respiratory failure is muscle weakness secondary to inadequate levels of ATP and decreased glycolysis as the result of phosphate depletion [231]. Two disturbances of red cell function may occur secondary to phosphorus deficiency. First, as intraerythrocyte ATP production is decreased, the erythrocyte cell membrane becomes rigid, which can cause hemolysis [232]. This is rare and is usually seen in septic, uremic, acidotic, or alcoholic patients, when serum phosphate levels are less than 0.5 mg/dl. Second, the limited production of 2,3-DPG causes a leftward shift of the oxyhemoglobin curve and impairs the release of oxygen to peripheral tissues. Such a consequence of chronic hypophosphatemia has been documented in children with XLH and proposed as one factor underlying retarded growth stature [233]. Leukocyte dysfunction, which complicates phosphate deficiency, includes decreased chemotaxis, phagocytosis, and bactericidal activity [234]. These abnormalities increase the host susceptibility to infection. The mechanism by which hypophosphatemia impairs the various activities of the leukocyte probably is related to impairment of ATP synthesis. Decreased availability of energy impairs microtubules that regulate
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the mechanical properties of leukocytes and limits the rate of synthesis of organic phosphate compounds that are necessary for endocytosis. Abnormal platelet survival, causing thrombocytopenia, profuse gastrointestinal bleeding, and cutaneous bleeding, has also been described in association with phosphate depletion in animal studies. Despite these abnormalities, there is little evidence that hypophosphatemia is a primary cause of hemorrhage in humans. Perhaps the most consistent abnormalities associated with phosphate depletion are those on bone. Acute phosphate depletion induces dissolution of apatite crystal from the osseous matrix. This effect may be due to 1,25(OH)2D3, which is increased in response to phosphate depletion in both animals and humans. More prolonged hypophosphatemia leads to rickets and osteomalacia. This complication is a common feature of phosphate depletion. However, the ultimate cause is variable. While simple phosphate depletion alone may underlie the genesis of the abnormal mineralization, in many disorders the defect is secondary to phosphate depletion and commensurate 1,25(OH)2D3 deficiency. Thus, treatment of this complication may often require combination therapy, phosphate supplements, and calcitriol. Hyperphosphatemia Hypocalcemia and consequent tetany are the most serious clinical sequelae of hyperphosphatemia [235]. The decreased serum calcium concentration results from the deposition of calcium phosphate salts in soft tissue, a process that may lead to symptomatic ectopic calcification. The dystrophic calcification is frequently seen in acute and chronic renal failure, hypoparathyroidism, pseudohypoparathyroidism, and tumoral calcinosis. Indeed, deposition of calciumephosphate complexes in the kidney may predispose to acute renal failure. When the calciumephosphate product exceeds 70, the probability that soft tissue calcification will occur increases sharply. In addition, local factors, such as tissue pH and injury (e.g., necrotic or hypoxic tissue), may predispose to precipitation of the calciumephosphate salts. In chronic renal failure, calcification occurs in arteries, muscle tissue, periarticular spaces, the myocardial conduction system, lungs, and the kidney. Affected patients may also have ocular calcification, causing the “red eye” syndrome of uremia and subcutaneous calcification, which also plays a role in uremic pruritus. On the other hand, a predisposition to calcification of periarticular surfaces of the hips, elbows, shoulders, and other large joints occurs in tumoral calcinosis. In some disease states, hyperphosphatemia may also play an important role in the development of secondary hyperparathyroidism [236]. A decrement in the serum calcium concentration secondary to hyperphosphatemia
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stimulates the release of PTH. Furthermore, hyperphosphatemia decreases the activity of 1a-hydroxylase. The consequent diminished production of 1,25(OH)2D3 impairs the gastrointestinal absorption of calcium and induces skeletal resistance to PTH, influences that augment the development of hyperparathyroidism. Thus, hyperphosphatemia triggers a cascade of events that impact on calcium homeostasis at multiple sites. Therefore, the prevention of secondary hyperparathyroidism, metabolic bone disease, and soft tissue and vascular calcification in affected patients depends on ultimately controlling the serum phosphate concentration.
Treatment of Abnormal Serum Phosphorus in Diseases Caused by an Abnormal Phosphate Load Treatment of the myriad of diseases that characteristically display hyperphosphatemia or hypophosphatemia depends on determining the mechanism underlying their pathogenesis. The cause can almost always be ascertained by assessment of the clinical setting, determination of renal function, measurement of urinary phosphate excretion, and analysis of arterial carbon dioxide tension and pH. Therapy is aimed at correcting both the serum phosphate concentration and associated complications. The treatment of phosphate depletion depends on replacing body phosphorus stores. Preventive measures, however, will preclude the onset of phosphate depletion. Thus, appropriate monitoring of patients taking large doses of aluminum-containing antacids and provision of phosphorus intravenously to patients with diabetic ketoacidosis will preserve phosphate stores. Alternatively, the treatment of established phosphate depletion may require 2.5 to 3.7 g of phosphate daily, preferably administered orally in at least four equally divided doses. Providing K-Phos Neutral tablets, which contain 250 mg of elemental phosphorus per tablet, will fulfill this goal. If oral therapy is not tolerated and the serum phosphate shows a downward trend approaching dangerous levels (<1.2 mg/dl), intravenous phosphate supplementation at a dose of 10 mg/kg body weight/day may be administered. Such therapy should be discontinued when the serum phosphate reaches values >2.0 mg/dl. However, therapy is not required for many of the conditions resulting in phosphate depletion. Only when the consequences of severe depletion are manifest should treatment be initiated. Theoretically, treatment of an elevated phosphate load and lowering the attendant serum phosphorus concentration depend upon decreasing the TmP, increasing the GFR, or diminishing the phosphate load
(exogenous or endogenous). There are no generally available pharmacologic means of acutely altering the GFR or reducing the TmP. However, chronic use of drugs, such as acetazolamide, which decreases TmP and induces phosphaturia, is effective as ancillary treatment of disorders such as tumoral calcinosis. More recently, attention has turned to increasing endogenous FGF23 or providing pharmacological preparations to lower the serum phosphorus over prolonged periods of time. Nevertheless, regulation of hyperphosphatemia is most often achieved by reducing the renal phosphate load. In tumoral calcinosis and chronic renal failure, such an effect is obtained by restricting the dietary phosphate intake or by administering phosphate binders. Alternative strategies for management of load-dependent hyperphosphatemia include the administration of intravenous calcium or intravenous glucose and insulin. The consequence of such intervention is sequestration of phosphate in bone or soft tissues. Dialysis can also be used for the acute management of load-dependent disorders, or the chronic maintenance of phosphate overload such as those that complicate chronic renal failure. The problem of phosphate overload in renal disease is discussed in Chapter 70.
CONCLUDING REMARKS Over the past several decades it has become abundantly clear that an alteration of phosphate homeostasis can impact the function of a wide variety of tissues. As the control mechanisms for phosphate homeostasis have been discovered, specific abnormalities in phosphate transport have been identified and linked to a variety of diseases. Particular evolution of concepts regarding the role of phosphate in human bone diseases has been quite remarkable. Indeed, the similarities between various bone diseases can now be traced to variable mechanisms that alter the regulatory factors governing renal phosphate reabsorption. Perhaps most importantly, the advances recorded in understanding this regulatory process have led to emerging concepts for potentially effective treatment strategies. The enhanced information regarding diseases of phosphate homeostasis has also charted a better understanding of disorders related to an altered phosphate load and the potential consequences of such disorders. As a result appreciation of the available treatment strategies has improved and the therapy offered for these disorders is currently more effective. The material offered in this chapter is intended not only to provide up-to-date information but to begin formulating central theses and hypotheses into which forthcoming information is integrated.
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[180] M. Tieder, D. Modai, U. Shaked, R. Samuel, R. Arie, A. Halabe, et al., Idiopathic" hypercalciuria and hereditary hypophosphatemic rickets. Two phenotypical expressions of a common genetic defect, N. Engl. J. Med. 316 (1987) 125e129. [181] A.L. Tencza, S. Ichikawa, A. Dang, D. Kenagy, E. McCarthy, M.J. Econs, et al., Hypophosphatemic rickets with hypercalciuria due to mutation in SLC34A3/type IIc sodium-phosphate cotransporter: presentation as hypercalciuria and nephrolithiasis, J. Clin. Endocrinol. Metab. 94 (2009) 4433e4438. [182] N. Mejia-Gaviria, H. Gil-Pena, E. Coto, T.M. Perez-Menendez, F. Santos, Genetic and clinical peculiarities in a new family with hereditary hypophosphatemic rickets with hypercalciuria: a case report, Orphanet. J. Rare Dis. 5 (2010) 1. [183] U. Liberman, Inborn errors in vitamin D metabolism e their contribution to the understanding of vitamin D metabolism, in: A.W. Norman, SK, H.-G. Grigoleit, D. Herrath (Eds.), Vitamin D Molecular, Cellular, and Clinical Endocrinology, Walter de Gruyter, Berlin, 1988, pp. 935e947. [184] J.C. Chan, U. Alon, Tubular disorders of acid-base and phosphate metabolism, Nephron 40 (1985) 257e279. [185] R. Chesney, Faconi syndrome and renal tubular acidosis, in: M. Favus (Ed.), Primer on Metabolic Bone Diseases and Disorders of Mineral Metabolism, first ed., American Society of Bone and Mineral Research, Kelseyville, CA, 1990. [186] De Toni G, Remarks on the relations between renal rickets (renal dwarfism) and renal diabetes, Acta. Paediatr. Scand. 16 (1933) 479e484. [187] D.J. McCune, H.H. Mason, H.T. Clarke, Intractable hypophosphatemic rickets with renal glycosuria and acidosis (the Fanconi syndrome), Am. J. Dis. Child. 65 (1943) 81e146. [188] M. Bergeron, A. Gougoux, P. Vinay, The renal Fanconi syndrome, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), The Metabolic and Molecular Bases of Inherited Disease, seventh ed., McGraw-Hill, New York, 1995, pp. 3691e3704. [189] R. Steinherz, R.W. Chesney, J.D. Schulman, H.F. DeLuca, M. Phelps, Circulating vitamin D metabolites in nephropathic cystinosis, J. Pediatr. 102 (1983) 592e594. [190] R.W. Chesney, B.S. Kaplan, M. Phelps, H.F. DeLuca, Renal tubular acidosis does not alter circulating values of calcitriol, J. Pediatr. 104 (1984) 51e55. [191] R.W. Chesney, J.F. Rosen, A.J. Hamstra, H.F. DeLuca, Serum 1,25-dihydroxyvitamin D levels in normal children and in vitamin D disorders, Am. J. Dis. Child. 134 (1980) 135e139. [192] R.L. Chevalier, Hypercalciuria in a child with primary Fanconi syndrome and hearing loss, Int. J. Pediatr. Nephrol. 4 (1983) 53e57. [193] S.W. Lee, J. Russell, L.V. Avioli, 25-hydroxycholecalciferol to 1,25-dihydroxycholecalciferol: conversion impaired by systemic metabolic acidosis, Science 195 (1977) 994e996. [194] E.D. Brewer, H.C. Tsai, K.S. Szeto, R.C. Morris Jr., Maleic acidinduced impaired conversion of 25(OH)D3 to 1,25(OH)2D3: implications for Fanconi’s syndrome, Kidney Int. 12 (1977) 244e252. [195] R.J. Brenner, D.B. Spring, A. Sebastian, E.M. McSherry, H.K. Genant, A.J. Palubinskas, et al., Incidence of radiographically evident bone disease, nephrocalcinosis, and nephrolithiasis in various types of renal tubular acidosis, N. Engl. J. Med. 307 (1982) 217e221. [196] D. Magen, L. Berger, M.J. Coady, A. Ilivitzki, D. Militianu, M. Tieder, et al., A loss-of-function mutation in NaPi-IIa and renal Fanconi’s syndrome, N. Engl. J. Med. 362 (2010) 1102e1109.
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C H A P T E R
64 Pseudo-vitamin D Deficiency Francis H. Glorieux, Thomas Edouard, Rene´ St-Arnaud Shriners Hospital for Children, Montreal, Quebec, Canada
INTRODUCTION Following the description by Albright et al. in 1937 of “rickets resistant to vitamin D therapy,” a number of observations were published [1,2] that indicated that there was a variant of resistant rickets which differed from the classic hypophosphatemic type (X-linked hypophosphatemic rickets, or XLH, see Chapter 63) by its clinical and biological symptoms and response to therapy. In 1961, Prader et al. showed that this form of rickets differed from XLH by its early onset (within the first year of life), the development of severe hypocalcemia with tetany, moderate hypophosphatemia, and hyperaminoaciduria both reflecting secondary hyperparathyroidism, enamel hypoplasia, and the complete correction of all clinical and biochemical evidence of rickets with daily administration of large amounts of vitamin D [3]. In view of the latter, the term “vitamin D dependency” was proposed to describe the new syndrome [4]. In 1973, through the availability of 1a,25-dihydroxyvitamin D3 (1,25(OH)2D, calcitriol) as a therapeutic agent, came the demonstration that this rare form of rickets was an inborn error of vitamin D metabolism involving the defective conversion of 25hydroxyvitamin D (25(OH)D, calcidiol) to 1,25(OH)2D (Fig. 64.1) [5]. For this reason, it seems to be more appropriate to return to the original terminology of Prader and use the term “Pseudo-vitamin D Deficiency Rickets (PDDR)” to describe this form of rickets. In 1997, remarkable progress was made in the understanding of the molecular etiology of PDDR through the identification of mutations in the gene encoding the 25-hydroxyvitamin D-1a hydroxylase (1a-OHase) in PDDR patients, leading to an inability to synthesize 1,25 (OH)2D [6]. In 1978, another inborn error of vitamin D metabolism was recognized in which a clinical picture of pseudo-vitamin D deficiency developed despite
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10064-2
high circulating levels of endogenously produced 1,25(OH)2D [7]. In some pedigrees, the phenotype was compounded with complete alopecia [8]. This second variety of pseudo-deficiency, also referred to as “vitamin D dependency type II,” is caused by a spectrum of mutations affecting the vitamin D receptor (VDR) causing true resistance to 1,25(OH)2D action [9]. This condition is discussed in detail in Chapter 65 where it is currently referred to as hereditary vitamin D resistant rickets or HVDRR.
CLINICAL MANIFESTATIONS Patients with PDDR are healthy at birth and the first symptoms usually appear within the first year of life. The distribution of presenting signs and symptoms seems to differ somewhat between PDDR and vitamin D deficiency rickets (Table 64.1) [10,11]. The majority of patients (85%) present with neurological signs: delayed gross motor development and hypotonia (70%), or hypocalcemic seizures (15%). Growth retardation is a common manifestation (70%) and height at the time of diagnosis is reduced for age (Fig. 64.2). Pathological fractures may occur. Infant death by hypocalcemia or pulmonary infections was not infrequent in the past when the diagnosis was either missed or made too late to intervene. A history of adequate mineral and vitamin D intake, without evidence of intestinal malabsorption, is a constant finding. Physical examination reveals a short, hypotonic child with features of rickets. There is a wide anterior fontanel with frontal bossing and frequent craniotabes (easy depression of the softened parieto-occipital area). Tooth eruption is delayed, and erupted teeth show evidence of enamel hypoplasia. A “rachitic rosary” (enlargement of the costochondral junctions along the anterolateral area of the chest) is either visible
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Liver: Vitamin D 25-hydroxylase
Cytoplasm 25(OH)D3
VDR
VDR
PDDR
DBD LBD
1,25(OH)2D3 DNA
RXR Transcription
Kidney: 25-hydroxyvitamin D 1a-hydroxylase
Translation
mRNA
Nucleus
FIGURE 64.1 Schematic representation of the main steps of the vitamin D biosynthetic pathway, where genetic aberrations may lead to rickets and osteomalacia. The renal defect in PDDR is indicated by the break in the 1,25(OH)2D arrow arising from the kidney. The mutations lead to insufficient synthesis of 1,25(OH)2D.
or palpable. The development of a Harrisson sulcus caused by the muscular pull of the diaphragmatic attachments to the lower ribs can be observed. In the appendicular skeleton, enlargement of the metaphyseal areas is more evident in the wrists and ankles, and there is a variable degree of deformity (bowing) of long bone diaphyses. The site and type of deformity of the extremities depend upon the age of the child and the weight-bearing patterns in the limbs. Thus, deformities of the forearms and anterior bowing of the distal tibia are found more commonly in infants, whereas an exaggeration of the normal physiological bowing of the legs (genu varum), rarely seen in patients with PDDR, is a characteristic finding in the toddler who has started to walk. The Chvostek sign (twitching of the upper lip on light finger tapping of the facial nerve) reflects
TABLE 64.1
nerve irritability, a consequence of a rapid drop in serum calcium. Radiological examination of the skeleton reveals diffuse osteopenia and the classic metaphyseal changes of vitamin D deficiency. There is a fraying, cupping, widening, and fuzziness of the zone of provisional calcification immediately under the growth plate. These changes are seen better and detected earlier in the
Presenting Signs and Symptoms at Diagnosis in Patients with PDDR Versus in Patients with Vitamin D Deficiency Rickets PDDR [11]
Vitamin D deficiency rickets [10]
Short stature
70%
3%
Motor delay
70%
3%
Seizures
15%
25%
Clinical signs of rickets
100%
70%
Height z-score according to age at time of diagnosis in young children with PDDR, from [11] with permission.
FIGURE 64.2
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Hypocalcemia is the cardinal feature in PDDR. Serum calcium concentration will drop below 2 mmol/l (8 mg/ dl). This, particularly if the decrease is rapid, may give rise to tetany and convulsions, which may occur prior to any radiological evidence of rickets. Persistent hypocalcemia triggers secondary hyperparathyroidism and hyperaminoaciduria [12]. Urinary calcium content is low, whereas fecal calcium is high, reflecting impaired intestinal calcium absorption. Serum phosphate concentration may be normal or low. Hypophosphatemia, when present, is usually of a lesser degree than in XLH (see Chapter 63). It is the result of both impairment of intestinal absorption and increased urinary loss induced by secondary hyperparathyroidism. Serum alkaline phosphatase activity is consistently elevated (over 800 IU/l in our experience). Its increase often precedes the appearance of clinical symptoms. The calcemic response to PTH is usually, but not necessarily, absent [13]. Studies of circulating vitamin D metabolites have provided a key insight into the pathogenesis of PDDR. Serum levels of 25(OH)D are normal in the untreated patient. These results indicate that intestinal absorption of vitamin D and its hydroxylation in the liver are not impaired in PDDR. On the contrary, circulating levels of 1,25(OH)2D are low [13e15]. This is evident rapidly after birth, months before any clinical or radiological signs of rickets develop. Even when patients are treated with large doses of vitamin D, causing major increases in the circulating levels of 25(OH)D, 1,25(OH)2D levels do not reach the normal range (Fig. 64.3). This clearly identifies defective activity of the 1a-OHase enzyme as the basic abnormality in PDDR and differentiates it from vitamin D dependency type II (HVDRR). Although 1,25(OH)2D serum levels are low, they are not undetectable. This finding, coupled with the observation that serum levels of 1,25(OH)2D are positively correlated to the serum concentration of 25(OH)D in PDDR patients (either untreated or treated with large amount of vitamin D), suggests that the renal 1a-OHase is not totally absent in PDDR [14].
200 80
150 60
100 40
Serum 1,25(OH)2D3 (pmol/l)
BIOCHEMICAL FINDINGS
100
Serum 1,25(OH)2D3 (pg/ml)
most active growth plates, namely, the distal ulna and femur and the proximal and distal tibia. Changes in the diaphyses may not be evident when metaphyseal changes are first detected. However, they will appear a few weeks later as rarefaction, coarse trabeculation, cortical thinning, and subperiosteal erosion. The latter reflects the increased resorption induced by secondary hyperparathyroidism. When performed, bone mineral density (BMD) of the lumbar spine is very low.
50
20
0
0 Control Untreated
+D2 +1,25(OH)2D3
PDDR
FIGURE 64.3 Serum 1,25(OH)2D concentrations in control children and in PDDR patients either untreated or treated with high doses of vitamin D or calcitriol.
Circulating levels of 24,25(OH)2D are normal in PDDR patients and are highly correlated with those of 25(OH)D, indicating a fully functional 25(OH)D-24hydroxylase (24-OHase) enzyme [16]. These findings, as well as the observation that modulation of the expression of the 24-OHase is regulated independently from that of the 1a-OHase [17], strongly suggest that the two renal hydroxylases are distinct gene products (see Chapters 3 and 4).
GENETIC AND MOLECULAR STUDIES PDDR is inherited as a simple autosomal recessive trait [4]. No phenotypic abnormalities have been observed in presumed obligate heterozygotes [12]. Although identified in several ethnic groups, PDDR occurs at an unusually high frequency in the French Canadian population originating from the CharlevoixSaguenay-Lac Saint Jean region in Quebec [18,19]. With the cooperation of several large families, we set out to map the PDDR locus by using DNA markers and linkage analysis to approach the primary defect in PDDR. It was found that the gene responsible for the disease was linked to polymorphic restriction fragment length polymorphism (RFLP) markers in region 14 of the long arm of chromosome 12 (12q14) [20]. Multipoint linkage analysis and studies of haplotypes and recombinants strongly suggest the localization of the PDDR locus between the collagen type II alpha 1 (COL2A1)
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64. PSEUDO-VITAMIN D DEFICIENCY
locus and a cluster of three anonymous probes (D12S14, D12S17, and D12S6) which segregate as a three-marker haplotype. Linkage disequilibrium between PDDR and this three-marker haplotype supports the notion of a founder effect that had taken place in the second half of the 17th century (about 12 generations ago), when a small number of colonists settled in this area of Quebec. This is consistent with the present-day prevalence of 1 in 2400 births and a carrier rate of 1 in 26 individuals in northeastern Quebec [21]. Remarkable progress was made in the understanding of the molecular etiology of PDDR through the cloning of the gene encoding the 1a-OHase (CYP27B1), from rat [22,23] and mouse [24] kidney, and human keratinocytes and kidney [6]. The human gene was also cloned, sequenced, and mapped to chromosome 12q13.1-13.3 by fluorescence in situ hybridization [24e26], consistent with the earlier mapping of the disease by linkage analysis. The definite proof that mutations in the 1a-OHase gene are responsible for the PDDR phenotype comes from the identification of such mutations in PDDR patients and obligate carriers [6]. To date, many mutations in the CYP27B1 gene that codes for the 1a-OHase enzyme have been identified, including missense mutations, deletions, duplications, and splice mutations [27,28]. These mutations are dispersed throughout the CYP27B1 gene, affecting all exons (Table 64.2). Mutations are numbered following the convention which starts with the translation initiator methionine as amino acid þ1, and the A of the ATG codon as nucleotide þ1. All patients have mutations on both alleles, but some cases are due to compound heterozygosity, a different mutation on each allele. The two most frequently observed mutations are a deletion of guanine at nucleotide 958 (g.958delG) in exon 2, commonly found in French Canadian patients, and a 7 bp duplication in exon 8, that arose independently in several populations [28]. The g.958delG mutation (which corresponds to the deletion of a guanine in position 958 of the genomic DNA) is predicted to result in a frameshift leading to a premature stop codon [29]. Most mutations associated with PDDR lead to a total loss of 1a-OHase activity when expressed in vitro [29], but some mutations seem to retain partial enzyme activity in vivo [28]. An important aspect of the identification of mutations in the CYP27B1 gene is to correlate genotype and phenotype, i.e. the severity of the disease and the circulating levels of 1,25(OH)2D. However, in our experience, the clinical presentation of patients with missense mutations is indistinguishable from that of the patients with frameshift mutations (personal observations). An animal model of PDDR was engineered independently by two laboratories using targeted inactivation of the gene of interest in mice [30,31]. A detailed description of the model is presented in Chapter 33.
TABLE 64.2 Known Mutations in the 1a-OHase Gene (CYP27B1) Detected in PDDR Patients Nucleotide change
Amino acid change
Exon
Missense mutation 246G>T 913G>C 1016G>A 1031C>T 1070G>A 1634C>T 1696G>A 1772A>G 1771G>A 2337C>G 2546C>A 2582G>C 2605C>T 2925C>T 2946C>T 2946C>G 2947G>A 3299C>T 331A>G 3430G>A 3359G>C 3430C>T 3680T>G 3917C>G 246G>T
Q65H G73R R107H P112L G125E P143L D164N E189G E189K T321R S323Y R335P L343F P382S R389C R389G R389H T409I Y413G R453H R429P R453C V478G R492P P479R
1 2 2 2 2 3 3 3 3 5 6 6 6 7 7 7 7 8 8 8 8 8 9 9 9
Nonsense mutations 2014G>A 2561G>A 3372G>A
W241X W328X W433X
4 6 8
Deletions 212delG 958delG 1609delC 1921delG 1984delC 3922delA
Frameshift after 55K Frameshift after 87Y Frameshift after 135K Frameshift after 209C Frameshift after 230V Frameshift after 498E
1 2 3 4 4 9
Insertions 3392-3398dup 3398-3406insCCCACCC 3398-3408insCCCACACCC
Frameshift after 443P Frameshift after 441H Frameshift after 441H
8 8 8
Frameshift after 66V
2
Frameshift Frameshift Frameshift Frameshift
Intron 2 Intron 3 Intron 6 Intron 7
Deletioneinsertion 897-901delGGGCG; 897-902insCTTCGG Splice-site mutations IVS2þ1 G to A (1083G>A) IVS3þ1 G to A (1796G>A) IVS6þ1 G to A (2715G>T) IVS7þ1 G to A (1083G>A)
after 129A after 196E after 379R after 405N
Sequence traces were aligned with the GenBank reference sequences of the CYP27B1 genomic DNA (AF027152). Mutations were numbered following the convention (http://www.hgvs.org/mutnomen/recs.html), which starts with the translation initiator methionine as amino acid þ1, and the A of the ATG codon as nucleotide þ1.
VIII. DISORDERS
1191
TREATMENT
TREATMENT Historically, patients with PDDR were treated with high doses of vitamin D (calciferol, 20 000 to 100 000 IU/day), in an attempt to overcome 1a-OHase deficiency, with a certain success [12,32]. Under such treatment, circulating levels of 25(OH)D increase sharply, with only minor changes in the levels of 1,25(OH)2D (Fig. 64.3). It’s likely that massive concentrations of 25(OH)D are able to bind to the VDR and induce the response of the target organs to normalize calcium homeostasis. However, because such therapy leads to progressive accumulation of vitamin D in fat and muscle tissues, adjustment in case of overdose is difficult and slow to come into effect. Furthermore, the therapeutic doses are close to the toxic doses and place the patient at risk for nephrocalcinosis and impaired renal function. There have been reports on the use of 25(OH)D3 as therapeutic agent in PDDR [33]. The doses used are smaller than those of vitamin D and induce a similar response. The action of 25(OH)D3 is likely to be similar to the one of vitamin D itself, by maintaining high serum concentrations of 25(OH)D3. The low availability and high cost of such a preparation have discouraged its widespread use as a long-term therapy for PDDR. The treatment of choice is replacement therapy with calcitriol. Before the compound became available from commercial sources, several investigators used the
monohydroxylated analog 1a-hydroxyvitamin D (1aOHD), which requires only liver hydroxylation at the 25 position (a step not affected by the PDDR mutation) to fully mimic 1,25(OH)2D [34]. The response is rapid with healing of rickets in 7e9 weeks, requiring a daily dosage of 2e5 mg. The maintenance dose is about half the initial dose. Withdrawal induces a reappearance of symptoms within 3 weeks. Thus, long-term compliance is a more important consideration than in the case of vitamin D treatment. On a weight basis, 1a(OH)D3 is about half as potent as 1,25(OH)2D, nullifying any possible economic advantage in favor of the monohydroxylated form. The reason for this difference in potency has not been investigated, but may be related to a difference in intestinal absorption or to a variable degree of 25-hydroxylation of 1a(OH)D3. Since 1,25(OH)2D3 (calcitriol) became commercially available in 1973, the treatment of choice for PDDR patients is replacement therapy with calcitriol. Treatment with calcitriol (either in liquid form or in capsules) is started at a dose of 1.0 mg per day, given in two doses of 0.5 mg. Subsequently, the calcitriol dose is modified according to the results of biochemical analyses. The aims of the treatment are to achieve normocalcemia, to maintain PTH levels within normal limits and to avoid hypercalciuria. In our experience, the median daily calcitriol dose is 0.50 mg per day (range: 0.2 mg to 1.0 mg) after 3 months of treatment, 0.25 mg after 1 year (range
A.D. 25
2.4
PTH (pmol/l)
Ca (mmol)
2.8
2.0 1.6
10
0 Aug
Sep
Oct
Nov
Dec
Jan
Feb
Mar
Aug
Sep
Oct
Nov
Dec
Jan
Feb
Mar
Aug
Sep
Oct
Nov
Dec
Jan
Feb
Mar
Nov
Dec
Jan
Feb
Mar
0 BMD (Z score)
3000 P’ase Alc (U/L)
15
5
1.2
2000
1000
-2 -4 -6 -8
Aug 1.0
Sep
Oct
Nov
Dec
Jan
Feb
Mar
Ca (300 mg/d)
0.5
Rocaltrol Rx
0.0 Aug
Sep
Oct
Nov
Dec
Jan
Feb
Mar
Rocaltrol ( g/d)
0 Rocaltrol ( g/d)
20
1.0
Ca (300 mg/d)
0.5
Rocaltrol Rx
0.0 Aug
Sep
Oct
FIGURE 64.4 Biochemical response to treatment in a patient with PDDR treated with calcitriol.
VIII. DISORDERS
1192
64. PSEUDO-VITAMIN D DEFICIENCY
supplemented to ensure a daily supply of around 1 g of elemental calcium.
EVOLUTION OF PDDR UNDER TREATMENT FROM CHILDHOOD TO ADULTHOOD Short-term Effects of Treatment with Calcitriol
Radiographs of the right wrist (upper panel) and knee (lower panel) of a patient with PDDR, before treatment (left panel) and after only 3 weeks of treatment (right panel); healing of rickets is well under way.
FIGURE 64.5
0.1 mg to 1.0 mg), and 0.5 mg after 2 years (range 0.25 mg to 1.0 mg) (personal data). An important component of treatment is to ensure adequate calcium intake during the bone-healing phase. Dietary sources are
Replacement therapy with calcitriol results in rapid and complete correction of the abnormal phenotype, eliminating hypocalcemia, secondary hyperparathyroidism, and radiographic evidence of rickets within 3 months (Figs 64.4 and 64.5) [14]. Lumbar spine areal BMD also normalizes within 3 months (Fig. 64.6) [11], as described in children and adults who are treated for vitamin D deficiency [10]. The rapidity of the increase in BMD suggests that calcitriol treatment of PDDR patients initially leads to the mineralization of pre-existing unmineralized osteoid rather than the production of new bone matrix. Histological evidence of healing has been documented in PDDR patients [14] as well as in the animal model of PDDR [35]. The height deficit persists somewhat longer than the low areal BMD, but after 2 years of calcitriol treatment, height is also normalized (catch-up growth) and remains so until adulthood (Fig. 64.6) [11]. Severe enamel hypoplasia is only partially corrected if treatment, as is usually the case, is started around 12e15 months of age when permanent tooth enamel has already started to develop (Fig. 64.7).
From Childhood to Adulthood From childhood to adulthood, calcitriol doses are increased as needed to maintain PTH levels within
Evolution of height (in 12 patients) and areal BMD of the lumbar spine (in five patients) during the first 2 years of treatment with calcitriol in young children with PDDR, from [11] with permission.
FIGURE 64.6
VIII. DISORDERS
EVOLUTION OF PDDR UNDER TREATMENT FROM CHILDHOOD TO ADULTHOOD
FIGURE 64.7 Permanent incisors of a 9-year-old patient with PDDR in whom calcitriol treatment was initiated at age 14 months. The part of the enamel that was formed before treatment remains hypoplastic. Subsequent to treatment, normal enamel was produced.
1193
treatment history. With regard to serum levels of vitamin D metabolites under calcitriol treatment, 25(OH)D levels and 1,25(OH)2D are normal in most patients. As previously discussed, dental structural and/or developmental abnormalities are documented in adult patients treated late with calcitriol. Hypercalciuria is not infrequent during treatment with calcitriol, and changes in urinary calcium excretion are used to adjust the daily calcitriol dose. High levels of calcium excretion may lead to calcium deposition in tissues especially in cornea and kidneys. In our treatment protocol, renal ultrasound and slit-lamp examinations of the cornea are performed every 2 years to assess for the presence of nephrocalcinosis and corneal calcium deposits. This screening for potential adverse events of hypercalcemia revealed that one patient (4% of the study population of 25) had mild corneal calcium deposits and four patients (16%) had mild nephrocalcinosis on renal ultrasound.
Pregnancies in Women with PDDR
FIGURE 64.8 Final height according to age of onset of calcitriol treatment in adult patients with PDDR, from [11] with permission.
normal limits. In our experience, in the period from 4 to 9 years of age, the median daily calcitriol dose increases from 0.25 mg to 0.50 mg. From 11 to 15 years of age, during the pubertal growth spurt, median calcitriol doses increase from 0.50 mg per day to 0.75 mg per day and remain so until adulthood [11].
Adult Patients with PDDR Adult height is significantly associated with the age at which calcitriol treatment is started (Fig. 64.8) [11]. The height of adult patients who receive calcitriol before the pubertal growth spurt is normal whereas patients who receive calcitriol only after the pubertal growth spurt are significantly shorter (Table 64.3). Lumbar spine areal BMD is normal in all adult patients whatever the
Female mice that are deficient in 1a-OHase (and that do not receive calcitriol) have uterine hypoplasia, absent corpora lutea, and thus are infertile [31]. This defect is at least partly due to hypocalcemia. Moreover, the 1aOHase gene is expressed in human endometrial stromal cells independent of the cycle phase but with a significant increase in early pregnant deciduas, suggesting a potential role of local production of 1,25(OH)2D in pregnancy establishment or maintenance [36]. The study of decidual tissues from two PDDR patients showed that it did not have the capacity to produce 1,25(OH)2D, indicating that decidua is a target for the PDDR mutation [37]. However, the physiologic importance of this defect is unclear. The importance of non-renal 1a-OHase and the role of local 1,25(OH)2D is discussed in Chapter 45. In our experience, pubertal development seemed to be normal in treated PDDR children. In our cohort, nine of 13 women with PDDR who were above 20 years of age have had 19 documented pregnancies [11]. It therefore appears that local production of 1,25(OH)2D in female reproductive organs is not critical for fertility, as systemic supplementation with calcitriol and normalization of serum calcium was sufficient to achieve fertility. During normal pregnancy, 1,25(OH)2D circulating levels steadily increase to about twice the control values. This adaptation to the specific needs of pregnancy can be mimicked in pregnant women with PDDR by increasing the daily calcitriol dose during the second half of pregnancy. During pregnancy, doses of calcitriol are adjusted according to the results of biochemical analyses (obtained every 4 weeks) to
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1194 TABLE 64.3
64. PSEUDO-VITAMIN D DEFICIENCY
Auxological, Biochemical, and Radiological Data of Adult Patients with PDDR Group 1
Group 2
Group 3
p
N
Value (range)
N
Value (range)
N
Value (range)
Number (female/male)
7
7 (1/6)
4
4 (4/0)
14
14 (9/5)
Age (years)
7
20.0 (17.0; 27.0)
4
20.5 (18.0; 26.0)
14
31.0 (24.0; 45.0)a,b
0.0009
Age of onset of calcitriol treatment (years)
7
1.0 (0.1; 1.6)
4
7.0 (4.0; 10.0)
14
23.0 (12.0; 31.0)
0.0001
Duration of calcitriol treatment (years)
7
19.1 (15.2e26.2)
4
14.8 (11.2e18.2)
14
11.0 (3.5e24.1)a
0.0025
Dose of calcitriol treatment (mg/day)
7
0.75 (0.75; 1.00)
3
1.25 (0.50; 1.50)
14
0.5 (0.50; 1.00)
Height (z-score)
7
0.3 (0.9; 1.0)
4
1.3 (1.6; 0.9)
14
2.2 (5.5; 0.9)
0.0008
Weight (z-score)
7
0.5 (1.4; 1.4)
4
1.0 (0.43; 2.18)
14
1.3 (3.0; 1.4)
0.011
Total calcium (mmol/l) (Norm: 2.25e2.63)
7
2.37 (2.29; 2.50)
4
2.33 (2.26; 2.55)
12
2.32 (2.10; 2.45)
0.39
Phosphate (mmol/l) (Norm: 1.23e1.62)
7
1.24 (0.87; 1.36)
4
1.07 (0.75; 1.22)
12
0.87 (0.74; 1.13)a
0.008
Alkaline phosphatase (UI/l) (<300)
7
86 (47; 130)
4
85 (39; 158)
12
61 (35; 103)
0.36
PTH (% normal maximal value)
7
82 (42; 166)
3
90 (50; 90)
11
60 (50; 150)
0.99
Urinary calcium/creatinine ratio
7
0.4 (0.1; 0.7)
4
0.3 (0.1; 0.6)
11
0.5 (0.1; 1.1)
0.50
LS volumetric BMD (z-score)
7
1.1 (1.9; 2.7)
3
0.9 (0.0; 3.0)
14
0.4 (0.9; 2.3)
0.42
0.24 a
a,b
a
Significantly different between groups 1 and 3. b Significantly different between groups 2 and 3. p values were calculated using the Kruskal-Wallis test. Group 1: Exclusively treated with calcitriol. Group 2: Initially treated with high doses of vitamin D, but started calcitriol before the pubertal growth spurt. Group 3: Initially treated with high doses of vitamin D, but started calcitriol after the pubertal growth spurt.
maintain serum calcium levels within normal limits. After delivery, calcitriol treatment is returned to prepregnancy doses. All pregnancies were without complications. In particular, there is no case of intra-uterine growth retardation and all newborns were normocalcemic at birth.
correction of all clinical, biochemical, and radiological abnormalities related to PDDR, without serious adverse events. Because of the easiness and efficacy of the replacement therapy, it is unlikely that any form of gene-based therapy will be considered anytime soon.
References CONCLUSION Pseudo-vitamin D deficiency rickets is a rare autosomal recessive disorder caused by mutations in the gene encoding 1a-OHase (CYP27B1), leading to an inability to synthesize 1,25(OH)2D. This disease was the first described inborn error of vitamin D metabolism and has contributed in a major way to our understanding of vitamin D biology. To date, close to 100 patients with PDDR have been reported in the literature. Although identified in several ethnic groups, PDDR occurs at an unusually high frequency in the French Canadian population originating from the CharlevoixSaguenay-Lac Saint Jean region in Quebec. The treatment of choice for PDDR patients is replacement therapy with small daily doses of calcitriol. Started in infancy, this treatment results in short- and long-term
[1] D. Fraser, R.B. Salter, The diagnosis and management of the various types of rickets, Pediatr. Clin. North Am. (1958) 417e441. [2] P. Royer, [Study on idiopathic hypophosphatemic vitaminresistant rickets.], Acta Clin. Belg. 15 (1960) 499e517. [3] A. Prader, R. Illig, E. Heierli, [An unusual form of primary vitamin D-resistant rickets with hypocalcemia and autosomaldominant hereditary transmission: hereditary pseudo-deficiency rickets.], Helv. Paediatr. Acta 16 (1961) 452e468. [4] C.R. Scriver, Vitamin D dependency, Pediatrics 45 (1970) 361e363. [5] D. Fraser, S.W. Kooh, H.P. Kind, M.F. Holick, Y. Tanaka, H.F. DeLuca, Pathogenesis of hereditary vitamin-D-dependent rickets. An inborn error of vitamin D metabolism involving defective conversion of 25-hydroxyvitamin D to 1 alpha,25dihydroxyvitamin D, N. Engl. J. Med. 289 (1973) 817e822. [6] G.K. Fu, D. Lin, M.Y. Zhang, D.D. Bikle, C.H. Shackleton, W.L. Miller, Portale AA 1997 Cloning of human 25-hydroxyvitamin D-1 alpha-hydroxylase and mutations causing vitamin D-dependent rickets type 1, Mol. Endocrinol. 11 (1997) 1961e1970.
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REFERENCES
[7] S.J. Marx, A.M. Spiegel, E.M. Brown, D.G. Gardner, R.W. Downs Jr., M. Attie, et al., A familial syndrome of decrease in sensitivity to 1,25-dihydroxyvitamin D, J. Clin. Endocrinol. Metab. 47 (1978) 1303e1310. [8] J.F. Rosen, A.R. Fleischman, L. Finberg, A. Hamstra, H.F. DeLuca, Rickets with alopecia: an inborn error of vitamin D metabolism, J. Pediatr. 94 (1979) 729e735. [9] M.R. Hughes, P.J. Malloy, D.G. Kieback, R.A. Kesterson, J.W. Pike, D. Feldman, et al., Point mutations in the human vitamin D receptor gene associated with hypocalcemic rickets, Science 242 (1988) 1702e1705. [10] S. Ladhani, L. Srinivasan, C. Buchanan, J. Allgrove, Presentation of vitamin D deficiency, Arch. Dis. Child 89 (2004) 781e784. [11] T. Edouard, N. Alos, G. Chabot, P. Roughley, F.H. Glorieux, F. Rauch, Short- and long-term outcome of patients with pseudo-vitamin D deficiency rickets treated with calcitriol, J. Clin. Endocrinol. Metab. 96 (2011) 82e89. [12] C. Arnaud, R. Maijer, T. Reade, C.R. Scriver, D.T. Whelan, Vitamin D dependency: an inherited postnatal syndrome with secondary hyperparathyroidism, Pediatrics 46 (1970) 871e880. [13] J.F. Rosen, L. Finberg, Vitamin D-dependent rickets: actions of parathyroid hormone and 25-hydroxycholecalciferol, Pediatr. Res. 6 (1972) 552e562. [14] E.E. Delvin, F.H. Glorieux, P.J. Marie, J.M. Pettifor, Vitamin D dependency: replacement therapy with calcitriol? J. Pediatr. 99 (1981) 26e34. [15] C.R. Scriver, T.M. Reade, H.F. DeLuca, A.J. Hamstra, Serum 1,25dihydroxyvitamin D levels in normal subjects and in patients with hereditary rickets or bone disease, N. Engl. J. Med. 299 (1978) 976e979. [16] S. Mandla, G. Jones, H.S. Tenenhouse, Normal 24-hydroxylation of vitamin D metabolites in patients with vitamin D-dependency rickets type I. Structural implications for the vitamin D hydroxylases, J. Clin. Endocrinol. Metab. 74 (1992) 814e820. [17] A. Arabian, J. Grover, M.G. Barre, E.E. Delvin, Rat kidney 25hydroxyvitamin D3 1 alpha- and 24-hydroxylases: evidence for two distinct gene products, J. Steroid Biochem. Mol. Biol. 45 (1993) 513e516. [18] G. Bouchard, C. Laberge, C.R. Scriver, [Hereditary tyrosinemia and vitamin-dependent rickets in Saguenay. A genetic and demographic approach], Union Med. Can. 114 (1985) 633e636. [19] M. De Braekeleer, J. Larochelle, Population genetics of vitamin D-dependent rickets in northeastern Quebec, Ann. Hum. Genet. 55 (1991) 283e290. [20] M. Labuda, D. Labuda, M. Korab-Laskowska, D.E. Cole, E. Zietkiewicz, J. Weissenbach, et al., Linkage disequilibrium analysis in young populations: pseudo-vitamin D-deficiency rickets and the founder effect in French Canadians, Am. J. Hum. Genet. 59 (1996) 633e643. [21] De Braekeleer M, Hereditary disorders in Saguenay-Lac-St-Jean (Quebec, Canada), Hum. Hered. 41 (1991) 141e146. [22] T. Shinki, H. Shimada, S. Wakino, H. Anazawa, M. Hayashi, T. Saruta, et al., Cloning and expression of rat 25-hydroxyvitamin D3-1alpha-hydroxylase cDNA, Proc. Natl. Acad. Sci. USA 94 (1997) 12920e12925. [23] R. St-Arnaud, S. Messerlian, J.M. Moir, J.L. Omdahl, F.H. Glorieux, The 25-hydroxyvitamin D 1-alpha-hydroxylase
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gene maps to the pseudovitamin D-deficiency rickets (PDDR) disease locus, J. Bone Miner. Res. 12 (1997) 1552e1559. K. Takeyama, S. Kitanaka, T. Sato, M. Kobori, J. Yanagisawa, Kato S 1997 25-Hydroxyvitamin D3 1alpha-hydroxylase and vitamin D, synthesis Science 277 (1997) 1827e1830. S. Kitanaka, K. Takeyama, A. Murayama, T. Sato, K. Okumura, M. Nogami, et al., Inactivating mutations in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene in patients with pseudovitamin D-deficiency rickets, N. Engl. J. Med. 338 (1998) 653e661. T. Yoshida, T. Monkawa, H.S. Tenenhouse, P. Goodyer, T. Shinki, T. Suda, et al., Two novel 1alpha-hydroxylase mutations in French-Canadians with vitamin D dependency rickets type II, Kidney Int. 54 (1998) 1437e1443. C.J. Kim, L.E. Kaplan, F. Perwad, N. Huang, A. Sharma, Y. Choi, et al., Vitamin D 1alpha-hydroxylase gene mutations in patients with 1alpha-hydroxylase deficiency, J. Clin. Endocrinol. Metab. 92 (2007) 3177e3182. X. Wang, M.Y. Zhang, W.L. Miller, A.A. Portale, Novel gene mutations in patients with 1alpha-hydroxylase deficiency that confer partial enzyme activity in vitro, J. Clin. Endocrinol. Metab. 87 (2002) 2424e2430. J.T. Wang, C.J. Lin, S.M. Burridge, G.K. Fu, M. Labuda, A.A. Portale, et al., Genetics of vitamin D 1alpha-hydroxylase deficiency in 17 families, Am. J. Hum. Gene 63 (1998) 1694e1702. O. Dardenne, J. Prud’homme, A. Arabian, F.H. Glorieux, R. StArnaud, Targeted inactivation of the 25-hydroxyvitamin D(3)-1 (alpha)-hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D-deficiency rickets, Endocrinology 142 (2001) 3135e3141. D.K. Panda, D. Miao, M.L. Tremblay, J. Sirois, R. Farookhi, G.N. Hendy, et al., Targeted ablation of the 25-hydroxyvitamin D 1alpha -hydroxylase enzyme: evidence for skeletal, reproductive, and immune dysfunction, Proc. Natl. Acad. Sci. USA 98 (2001) 7498e7503. R. Hamilton, J. Harrison, D. Fraser, I. Radde, R. Morecki, L. Paunier, The small intestine in vitamin D dependent rickets, Pediatrics 45 (1970) 364e373. S. Balsan, M. Garabedian, V. Courtecuisse, J. Gueris, J.P. Dommergues, L. Creignou, et al., Long-term therapy with 1alpha-hydroxyvitamin D3 in children with "pseudo-deficiency" rickets, Clin. Endocrinol. (Oxf.) 7 (Suppl.) (1977) 225se230s. T.M. Reade, C.R. Scriver, F.H. Glorieux, B. Nogrady, E. Delvin, R. Poirier, et al., Response to crystalline 1alpha-hydroxyvitamin D3 in vitamin D dependency, Pediatr. Res. 9 (1975) 593e599. O. Dardenne, J. Prud’homme, F.H. Glorieux, R. St-Arnaud, Rescue of the phenotype of CYP27B1 (1alpha-hydroxylase)deficient mice, J. Steroid Biochem. Mol. Biol. 89e90 (2004) 327e330. P. Vigano, D. Lattuada, S. Mangioni, L. Ermellino, M. Vignali, E. Caporizzo, et al., Cycling and early pregnant endometrium as a site of regulated expression of the vitamin D system, J. Mol. Endocrinol. 36 (2006) 415e424. E.E. Delvin, A. Arabian, F.H. Glorieux, O.A. Mamer, In vitro metabolism of 25-hydroxycholecalciferol by isolated cells from human decidua, J. Clin. Endocrinol. Metab. 60 (1985) 880e885.
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C H A P T E R
65 Hereditary 1,25-Dihydroxyvitamin-DResistant Rickets Peter J. Malloy 1, Dov Tiosano 2, David Feldman 1 1
2
Stanford University School of Medicine, Stanford, CA, USA Meyer Children’s Hospital, Rambam Medical Center, Haifa, Israel
INTRODUCTION Vitamin D, the primary regulator of calcium homeostasis in the body, is particularly important in skeletal development and in bone mineralization. The active form of vitamin D, 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3 or calcitriol), functions by binding with high affinity to the vitamin D receptor (VDR). The VDR is a member of the steroidethyroideretinoid receptor gene superfamily of nuclear transcription factors that regulate the expression of specific target genes in response to hormone binding. Hereditary 1,25-dihydroxyvitamin-D-resistant rickets (HVDRR) is a rare genetic disease that is due to a generalized resistance to 1,25(OH)2D3. HVDRR is caused by heterogeneous mutations in the VDR gene that cause loss of function of the receptor ultimately leading to complete or partial target organ resistance to 1,25(OH)2D3. In this chapter, we update our previous reviews on HVDRR [1e6] and describe the clinical manifestations of the disease and discuss the genetic defects in the VDR underlying the molecular basis for HVDRR. Over the years a number of different names have been used to describe the condition caused by 1,25(OH)2D resistance. In addition to HVDRR, the disease has been referred to as vitamin-D-dependent rickets type II (VDDR-II), pseudovitamin D deficiency rickets type II (PDDR-II), calcitriol-resistant rickets (CRR), and hypocalcemic vitamin-D-resistant rickets (HVDRR). In the Online Mendelian Inheritance in Man website (http://www.ncbi.nlm.nih.gov/omim/) this disease is referred to as vitamin-D-resistant rickets with end-organ unresponsiveness to 1,25-dihydroxycholecalciferol, ricketsealopecia syndrome, VDDR-II with alopecia,
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10065-4
and hypocalcemic vitamin-D-resistant rickets. We prefer to use the designation “Hereditary 1,25-dihydroxyvitamin-D-Resistant Rickets” (HVDRR) since this disease is now known to be caused by genetic defects in the VDR that lead to resistance to the action of 1,25(OH)2D3 [3e6]. The notion that rickets could be due to hormone resistance was put forward in 1937 by Albright et al. [7]. In their report they described a patient with rickets and normal serum calcium levels but low phosphate levels who responded to treatment with abnormally high doses of vitamin D. This led the authors to suggest that the cause of the condition was due to end-organ resistance to vitamin D and thus the concept of hormone resistance evolved. The patient they described appears to have had what is now known as X-linked hypophosphatemic rickets (XLH, described in Chapter 63). Twenty-four years later, Prader et al. [8] reported on two patients with rickets who were hypocalcemic and hypophosphatemic. These patients also responded to high doses of vitamin D and they referred to this condition as vitamin-D-dependent rickets type I (VDDR-I). The cause of rickets in these individuals was due to an inborn error in the conversion of vitamin D to the hormonally active form 1,25(OH)2D. It is now well established that VDDR-I arises from mutations in the CYP27A1 gene [9] that encodes the enzyme 25hydroxy-1a-hydroxylase (CYP27B1) that converts 25hydroxyvitamin D (25(OH)D) to 1,25(OH)2D [10,11]. This disease is described in Chapter 64. In 1978, the first cases of HVDRR were reported by Brooks et al. [12] and Marx et al. [13]. The patient in the Brooks et al. study was both hypocalcemic and hypophosphatemic and had secondary hyperparathyroidism. The clinical findings were similar to patients
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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65. HEREDITARY 1,25-DIHYDROXYVITAMIN-D-RESISTANT RICKETS
with VDDR-I except that the patient had markedly increased serum levels of 1,25(OH)2D. Brooks et al. postulated that the rickets was due to end-organ resistance to 1,25(OH)2D3 and they named the syndrome vitamin-D-dependent rickets, type II (VDDR-II). Marx et al. reported similar findings in two children and also suggested that the disease was due to end-organ resistance to 1,25(OH)2D. Since these initial studies there have been many reports of patients with apparent target organ resistance to 1,25(OH)2D [3,6]. In this chapter, we
A
THE CLINICAL FEATURES OF HVDRR Clinical and Biochemical Findings HVDRR is manifested by a constellation of signs and symptoms caused by a generalized resistance to
C
B
D
F
will review the clinical features and the genetic basis underlying the disease.
E
G
H
FIGURE 65.1 Children with HVDRR. A, Patient F70 with total alopecia; B, the child exhibited bowed legs; C, X-ray of wrist; D, X-ray of bowed legs; E, X-ray of legs after 4 years of calcium and calcitriol therapy. HVDRR children with partial alopecia. F, Patient F69; G, Patient F78; H, Patient F79. Panels (AeE) reproduced from [171] with permission of the American Society for Bone and Mineral Research. Panel (F) reproduced with permission from Molecular Genetics and Metabolism [177]. Panels (GeH) reproduced with permission from the Journal of Pediatric Endocrinology and Metabolism [149]. Please see color plate section.
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THE CLINICAL FEATURES OF HVDRR
1,25(OH)2D and by a loss of ligand-dependent and ligand-independent actions of the VDR. The main features of HVDRR are severe rickets with osteomalacia, hypocalcemia, secondary hyperparathyroidism, hypophosphatemia, and elevated alkaline phosphatase. Some patients have partial or total alopecia. At birth or soon after, partial or total alopecia may be the only obvious symptom (Fig. 65.1); calcium levels are often within normal limits at first, due to adequate transfer of calcium from the mother to the fetus. Biochemically, the earliest evidence of HVDRR is often elevated serum 1,25(OH)2D concentration. This is an inherent part of the disease due to loss of the downregulating effect that calcitriol has on 25(OH)D-1a-hydroxylase through the VDR as well as the development of secondary hyperparathyroidism due to falling serum calcium levels and PTH stimulation of 1a-hydroxylase [14]. During the first months of life, phosphaturia, due to secondary hyperparathyroidism, is followed by a decline in serum phosphorus. In the following months, severe rickets, with hypocalcemia and hypophosphatemia, develop rapidly. The potential role of FGF23 or other phosphatonins in the phosphate abnormality or in the elevated serum 1,25(OH)2D concentration has not been studied but these effects seem clearly to be related to the secondary hyperparathyroidism and resistance to 1,25 (OH)2D action. If not treated, growth of the child is severely attenuated and bowing of the long bones and fractures may develop. X-ray studies may show “transparent” bones, like black holes, which have been referred to as “hungry bones” [15,16]. In HVDRR signs of rickets generally appear early, usually within months of birth. The rickets is usually severe and affected children suffer from bone pain, muscle weakness, and hypotonia. In the worst cases convulsions due to the hypocalcemia have occurred. Children are usually growth retarded and they often develop severe dental caries or exhibit enamel hypoplasia of the teeth [17e23]. Some infants have died from pneumonia as a result of poor respiratory movement due to severe rickets of the chest wall [18,21,24]. In many cases, children with HVDRR have sparse body hair and some have total scalp and body alopecia including eyebrows and in some cases eyelashes (Fig. 65.1). Alopecia will be discussed in more detail below. An example of the serum biochemistry levels found in an HVDRR patient is shown in Table 65.1. The abnormalities include low serum concentrations of calcium and phosphate and elevated serum alkaline phosphatase activity. The hypocalcemia leads to secondary hyperparathyroidism. The elevated parathyroid hormone (PTH) level then contributes to the hypophosphatemia. These clinical and biochemical findings are also common to patients with 1a-hydroxylase deficiency (Table 65.2) as
TABLE 65.1
Biochemical Profile of a Patient with HVDRR Unresponsive to Calcitriol and Calcium
Biochemical marker
Normal values
Calcium (mmol/liter)
2.2e2.6
1.86
1.77
1.80
1.71
Phosphate (mmol/liter)
1.4e2.2
1.0
1.0
1.0
0.9
ALP (IU/liter)
145e320
3056
3991
3800
3609
25(OH)D (nmol/liter)
25e85
30
37.4
250
211
1,25(OH)2D (pmol/liter)
40e105
521
953
1830
1560
e
34.2
69.9
64.5
PTH (pmol/liter) <8
Referral values 40 daysa 80 daysb 100 days
Treatment: 250 mg elemental calcium 4 times per day and 0.5 mg calcitriol (Rocaltrol) twice per day and 20 000 IU vitamin D3 daily. Treatment: 250 mg elemental calcium 4 times per day and 5 mg calcitriol twice per day. ALP, alkaline phosphatase. Adapted from [145] with permission of the American Society for Bone and Mineral Research.
a
b
described in Chapter 64. In HVDRR patients the serum 25(OH)D values may be normal and the 1,25(OH)2D levels are elevated. This singular feature distinguishes HVDRR from 1a-hydroxylase deficiency (VDDR-I) where the serum 1,25(OH)2D values are depressed or absent. When analyzed, the 24,25(OH)2D levels have been normal or low [18,21,25e31]. Patients with 1ahydroxylase deficiency (VDDR I) can be successfully treated with physiologic doses of calcitriol that circumvent the 1a-hydroxylase deficiency and restore the circulating 1,25(OH)2D levels to normal. In contrast, patients TABLE 65.2 A Comparison of 1a-Hydroxylase Deficiency and HVDRR Feature
1a-Hydroxylase deficiencya
HVDRR
Gene mutated
CYP27B1
VDR
Autosomal recessive
Yes
Yes
Manifested at early age
Yes
Yes
Rickets
Yes
Yes
Hypocalcemia
Yes
Yes
Alopecia
No
Sometimes
PTH
Elevated
Elevated
25(OH)D levels
Normal
Normal
1,25(OH) 2D levels
Low
Elevated
Response to physiological doses of 1,25(OH)2D3
Yes
No
a
1a-Hydroxylase deficiency also known as VDDR I or PDDR is caused by mutations in the CYP27B1 gene.
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65. HEREDITARY 1,25-DIHYDROXYVITAMIN-D-RESISTANT RICKETS
with HVDRR do not respond to physiologic doses of calcitriol and most patients are resistant to even extreme supra-physiologic doses of all forms of vitamin D therapy (Table 65.2). HVDRR is inherited as an autosomal recessive disease. The recessive nature of the disease is evident from the patient’s parents and siblings who are heterozygous for the genetic trait but most often show no symptoms of the disease and have normal bone development. In most cases, consanguinity in the family lineage can be found and intermarriage is highly associated with the disease. Males and females are equally affected and often a family has several affected children [32].
Pathophysiology In HVDRR, the intestine, and other vitamin D target organs including bone, the parathyroid glands and kidneys, are resistant to 1,25(OH)2D action. Without vitamin D action, the intestine becomes less efficient in promoting calcium and phosphate absorption into the circulation. It is now well established that the biological actions of 1,25(OH)2D are mediated by the VDR, a nuclear transcription factor that regulates gene expression in 1,25(OH)2D-responsive cells (see several chapters in Section II of this volume). As will be discussed in detail below, the hallmark of the HVDRR syndrome is resistance to 1,25(OH)2D action. It is now clear that the usual cause of HVDRR is due to mutations in the VDR that render the receptor non-functional or less functional than the wild-type VDR. The primary biological process attributed to vitamin D is maintenance of calcium and bone homeostasis. 1,25(OH)2D is essential for promoting the transport of calcium and phosphate across the small intestine and into the circulation (see Chapter 19). Adequate delivery of calcium and phosphate to the bone is essential for the normal mineralization of bone (see Chapter 21). Approximately half of the total calcium absorption by the intestine is attributed to 1,25(OH)2D action while passive absorption accounts for the remaining half [33,34]. Since vitamin D regulates the translocation of calcium, conditions that adversely affect the 1,25(OH)2D action pathway cause a decrease in mineral transport leading to hypocalcemia. The hypocalcemia and the resistance of the parathyroid gland to suppression by 1,25(OH)2D because of defective VDR within the gland, in turn, cause secondary hyperparathyroidism. The increase in circulating 1,25(OH)2D levels is due to an increase in renal 1a-hydroxylase activity caused by both elevated PTH and hypophosphatemia to upregulate 1a-hydroxylase gene expression as well as failure of elevated 1,25(OH)2D to suppress 1ahydroxylase. The hypophosphatemia results from the elevated PTH downregulating the Na/P cotransporter
and/or by the loss of a functional VDR in the kidney as well as decreased intestinal absorption. The absence of an effect of 1,25(OH)2D to normally regulate FGF23 or other phosphatonins to normalize phosphaturia and 1a-hydroxylase activity may also play a role but it has not been studied. The calcium and phosphate deficiencies compromise normal bone mineralization leading to rickets in children and osteomalacia in adults.
Alopecia Alopecia (sometimes called atrichia) is a clinical feature that is found in many patients with HVDRR (Fig. 65.1). Some patients have sparse body hair and some exhibit total scalp and body alopecia [27,35,36]. Children with extreme alopecia often lack eyebrows and in some cases eyelashes. Hair loss may be evident at birth or occurs during the first few months of life as some hair the child is born with falls out and is not replaced. An analysis of HVDRR patients shows that there is some correlation between the severity of rickets and the presence of alopecia [36]. Patients with alopecia are generally more resistant to calcitriol therapy than those without alopecia. In families with a prior history of the disease, the absence of scalp hair in newborns provides initial diagnostic evidence for HVDRR. The mechanism causing alopecia is unknown but VDRs are present in the hair follicle [37,38]. A skin biopsy from one patient with HVDRR and alopecia showed apparently normal follicles with no hair [35] while other patients exhibited an absence of normal hair follicles and the presence of follicular remnants and cysts [39,40]. This latter observation led the authors to conclude that the alopecia in HVDRR patients was a phenocopy of atrichia with papular lesions (APL), a genetic disorder caused by mutations in the hairless (HR) gene. They further speculated that the VDR and HR converge to regulate similar pathways in the hair cycle [39,40]. A ligand-independent action of the VDR has been proposed to be critical during hair follicle development and therefore mutations in the VDR that disrupt these ligandindependent actions are suspected to cause alopecia [41,42]. On the other hand, patients with mutations in the 1a-hydroxylase gene (CYP27B1) or with other causes of vitamin D deficiency do not have alopecia, further supporting the role of a ligand-independent action of the VDR during the hair cycle. Alopecia is also discussed in detail in Chapter 30.
Other Aspects of HVDRR As mentioned above and discussed extensively in this volume, in addition to maintaining calcium homeostasis, 1,25(OH)2D has been shown to regulate a number of biological processes in many tissues [43e49].
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MECHANISM OF 1,25(OH)2D ACTION
Although there are multiple pleiotropic tissue responses regulated by 1,25(OH)2D, children with HVDRR appear relatively normal except for the constellation of features that relate to their calcium deficiency, rickets, and alopecia. VDRs have been found in endocrine glands such as pituitary, pancreas, parathyroid, gonads, and placenta, and 1,25(OH)2D3 regulates hormone synthesis and secretion from these glands [43e48]. Hochberg et al. [50] examined insulin, thyrotropin (TSH), prolactin (PRL), growth hormone (GH), and testosterone levels in sera from patients with HVDRR and found no abnormalities in hormone secretion. Furthermore, Even et al. [51] showed that urinary cAMP and excretion of potassium, phosphorus, and bicarbonate were normal in HVDRR patients following a PTH challenge. However, PTH failed to decrease urinary calcium and sodium excretion in these patients to the extent found in the control patients. This suggests that 1,25(OH)2D may selectively modulate the renal response to PTH and facilitate the PTH-induced reabsorption of calcium and sodium [51]. VDRs have also been found in hematolymphopoietic cells and 1,25(OH)2D has been shown to regulate cell differentiation and the production of interleukins and cytokines [52] (see Chapters 91 and 92). Neutrophils isolated from HVDRR patients exhibit only minor aberrations in their fungicidal activity [53], and HVDRR patients have no clinically apparent immunologic defects. In the light of the diverse actions of 1,25(OH)2D demonstrated in many tissues, the absence of related findings in children with HVDRR suggests that the pleiotropic responses regulated by 1,25(OH)2D in non-osteogenic tissues are redundant in vivo and that other factors or compensatory mechanisms subsume the role of vitamin D in such a way that abnormalities are not clinically manifested. This possibility does not necessarily minimize the contribution of 1,25(OH)2D to these systems under normal physiologic conditions. Similarly, the VDR knockout mouse displays the same phenotypic and physiologic patterns as patients with HVDRR [54,55]. The VDR knockout mouse model can be used to analyze the abnormalities caused by the loss of VDR action in detail not possible in the HVDRR patients (see Chapter 33). The clinical findings in a limited number of children and young adults who have been followed medically will be further discussed in “Therapy of HVDRR,” below.
MECHANISM OF 1,25(OH)2D ACTION The Vitamin D Receptor The human VDR gene is located on chromosome 12q13.11 and is composed of 14 exons spanning
1201
~64 kbp of DNA (Fig. 65.2). The human VDR protein contains either 427 or 424 amino acids depending upon the presence of a T to C polymorphism (ATG to ACG) in a translational start site known as the FokI site [56,57]. However, differential splicing of exon 1D appears capable of producing even longer isoforms of 450 and 477 amino acids [58]. The overall structure of the VDR protein is similar to the other members of the steroidethyroideretinoid receptor superfamily. At the N-terminus the VDR has a highly conserved DNA-binding domain (DBD) and in the C-terminal half of the protein a more variable ligand-binding domain (LBD) (Fig. 65.2). The DBD is encoded by exons 2 and 3 and is composed of two finger-like modules of 12e13 amino acids each. Four cysteine residues in each finger-like module bind one zinc atom to form the two zinc-finger configuration [59]. Specific regions of the DBD are critical for DNA binding, as well as providing a dimerization interface for interaction with the retinoid X receptor (RXR). A nuclear localization signal is also present in the DBD [60e65]. The hinge region (amino acids 88e122) connecting the DBD and LBD is important in providing the proper spacing between these domains as well as for transcriptional activation [66,67]. Details about the VDR and its mechanism of action are covered in Chapters 7 and 8. The VDR LBD is encoded by exons 4e9 and is composed of amino acids 123 to 427. X-ray crystallography of the VDR showed that the LBD contains 12 a-helices (H1eH12) and three b-sheets (S1eS3) [68] (see Chapter 9). Helix H12 forms a retractable lid that traps and holds the ligand in position. Ligand binding causes a conformational change in the VDR that promotes heterodimerization with RXR. VDR heterodimerization with RXR involves residues in H9, H10 and an E1 domain that overlaps H4 and H5 within the LBD. An activating function domain 2 (AF-2 domain) residues 416e424 of helix H12 and the region between amino acids 232e272 encompassing H3 and H4 are essential for transactivation [68]. The repositioning of helix H12 after ligand binding is critical for the formation of a hydrophobic groove that binds LxxLL motifs (where L is leucine and x is any amino acid) in the nuclear receptor interacting domains of coactivator proteins such as the g160 family of coactivators (SRC-1, SRC-2, SRC-3) and DRIP205 a member of the DRIP complex (see Chapter 10). Other regions of the VDR also act to recruit coactivator proteins or facilitate contact with proteins associated with the core transcriptional machinery such as TFIIB or the TAFs [69,70]. As discussed below, mutations in the VDR LBD that cause HVDRR have been shown to affect ligand binding, RXR heterodimerization or interactions with coactivators.
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65. HEREDITARY 1,25-DIHYDROXYVITAMIN-D-RESISTANT RICKETS
Arrangement of the chromosomal gene and domains of the VDR. The structural organization of the human VDR gene which spans approximately 64 kilobases of DNA is shown [219]. The locations of the start (ATG) and termination (TGA) codons are indicated. The exons encoding the various domains and structural motifs in the VDR protein are shown. The VDR DNA-binding domain is comprised of two zinc-finger modules each of which contains four invariant cysteine residues that function to coordinate a single zinc atom. Two a-helices (helix A and B) shaded in the diagram are located on the carboxy terminal side of each zinc module. Amino acid residues essential to functional interaction of these a-helices with either DNA or with RXR are boxed and designated the P-box and D-box, respectively. In the ligand-binding domain the position of the a-helices (H1eH12) and b-turns (S1eS3) are shown as shaded and hatched boxes respectively. The E1 and AF-2 regions are indicated.
FIGURE 65.2
Regulation of Gene Expression by 1,25-Dihydroxyvitamin D The mechanism of action of vitamin D is detailed in multiple chapters in Section II of this book. Thus, we will briefly review only those aspects of vitamin D action particularly relevant to understanding HVDRR and the mutations responsible for causing this syndrome. The biological actions of 1,25(OH)2D in tissues and cells are orchestrated through complex changes in gene expression [44,71e73]. These changes lead to cellspecific alterations in the level of proteins directly responsible for a myriad of differentiated cell functions, as well as in proteins that function as transcription factors or as signaling molecules to regulate secondary and tertiary levels of gene expression [74]. In the latter case, these molecules may function directly within the cell or indirectly via additional cellular signaling pathways in either autocrine or paracrine fashion. As indicated earlier, most, if not all, of the molecular actions of 1,25(OH)2D in the nucleus are mediated by the VDR. A simplified model of 1,25(OH)2D activated gene transcription is shown in Figure 65.3 that points out the areas of HVDRR mutations. A more comprehensive analysis of 1,25(OH)2D action is presented in several chapters in Section II of this volume.
In brief, after 1,25(OH)2D is synthesized in the kidney, it circulates in the blood mostly bound to DBP and perhaps other carriers with a small fraction of hormone in the free state (see Chapter 5). The free, fat-soluble hormone is believed to enter target cells through the lipid bilayer of the cell membrane although additional complex mechanisms may play a role (see Chapter 14). Once inside the cell, 1,25(OH)2D3 binds to the VDR, prompting its translocation to the nucleus whereupon a series of changes occurs that enables the VDR to activate gene transcription [75]. Ligand binding also promotes VDR-RXR heterodimerization. Once inside the nucleus the VDR-RXR heterodimer binds with high affinity through their respective DBDs to vitamin D response elements (VDREs) located in regulatory regions of target genes [72]. The typical VDRE contains two imperfect hexanucleotide segments arranged as direct repeats separated by a 3-nucleotide base spacer (DR3). In the LBD, 1,25 (OH)2D3 makes contact with specific amino acid residues lining the ligand-binding pocket [68]. 1,25 (OH)2D3 binding triggers helix H12 to fold back upon the LBD enclosing the ligand. A coactivator-binding cleft is formed by the repositioning of helix H12 together with helix H3 allowing the recruitment of coactivators and other transcription factors [76]. The VDR
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CELLULAR BASIS OF HVDRR
1203
FIGURE 65.3 Mechanism of 1,25-dihydroxyvitamin D3 action. 1,25-Dihydroxyvitamin D3 circulates in the blood mainly bound to the
vitamin D binding protein (DBP) with a small amount in equilibrium with the free or unbound state. The free lipophilic ligand diffuses through the lipid bilayer of the cell membrane and binds with high affinity to the VDR. The ligand-bound VDR heterodimerizes with RXR and in the nucleus binds to VDREs via the two zinc-finger modules of the DNA-binding domain of the receptors. Ligand binding also induces changes in the VDR that allows it to recruit coactivators. The VDReRXRecoactivator complex interacts with the general transcription apparatus (GTA) and initiates gene transcription. The newly synthesized proteins that then elicit intracellular or extracellular activities manifest the physiologic response to the hormone.
complex together with the general transcription apparatus then drives the transcription of 1,25(OH)2Dresponsive genes that ultimately determine the cellular response to the hormone. Proof that the cause of HVDRR was defective regulation of gene expression by VDR was initially developed through studies of HVDRR patients where natural mutations in the VDR gene prevented 1,25(OH)2D3 induction of target genes such as 24-hydroxylase [26,28,77]. Further studies demonstrated that 1,25(OH)2D3 could regulate promoter activity and that specific DNA sequences within the promoter were required for recognition by the VDR-RXR heterodimer. Direct regulation of gene expression by liganded VDR has been demonstrated for many genes such as
osteocalcin [78e83], osteopontin [84], calbindin [85,86], and CYP24A1 [87e89]. Mutant VDRs identified in HVDRR patients were incapable of activating VDREpromoter constructs supporting the critical role of functional VDR in transactivation as well as defining the defects causing HVDRR [90e92].
CELLULAR BASIS OF HVDRR Initial Studies using Cultured Skin Fibroblasts The syndrome of HVDRR was first recognized as an entity in 1978e79 [12,13,17,93]. Since that time more than 100 patients with vitamin D resistance have been
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65. HEREDITARY 1,25-DIHYDROXYVITAMIN-D-RESISTANT RICKETS
reported [3]. An updated summary of these cases is shown in Table 65.3. Throughout this chapter, the HVDRR cases are denoted by a family number, e.g. F1, F2, etc. Studies to elucidate the nature of the defect in HVDRR cases began soon after receptors for 1,25(OH)2D3 were found in skin [37,94e96]. Feldman et al. [95] demonstrated that the VDR was present in fresh and cultured human foreskin as well as in cultured keratinocytes and dermal fibroblasts grown from adult skin biopsies. The studies to unravel the basis of the disease began in earnest in 1981 when Eil et al. [97] showed in cultured skin fibroblasts that the disease was associated with defective nuclear uptake of 1,25(OH)2D3 although the cause of this defect was not characterized (families F1 and F3). The following year, Feldman et al. [26] analyzed the VDR in cultured skin fibroblasts from two siblings with HVDRR (F11). In this study, they demonstrated that cytosolic extracts of cultured fibroblasts from the HVDRR patients had undetectable levels of [3H]1,25(OH)2D3 binding. Furthermore, when cultured fibroblasts from normal subjects were treated with 1,25(OH)2D3 they were able to demonstrate an increase in 24-hydroxylase activity, a well-characterized marker of 1,25(OH)2D3 responsiveness. However, the patients’ fibroblasts failed to induce 24-hydroxylase activity following hormone treatment demonstrating that HVDRR fibroblasts were resistant to high concentrations of 1,25(OH)2D3 due to defective VDR binding. Subsequently, a number of other HVDRR cases were examined using cultured skin fibroblasts [21,98e100] or cells derived from bone [101]. Some patients’ fibroblasts lacked specific [3H]1,25(OH)2D3 binding [21,98e101] similar to the cases reported by Feldman et al. [26]. On the other hand, some fibroblasts exhibited normal [3H]1,25(OH)2D3 binding but were nevertheless unresponsive to 1,25(OH)2D3 treatment, suggesting defects beyond ligand binding [21,28,99,101e103]. The conclusion reached was that the HVDRR syndrome was caused by cellular resistance to 1,25(OH)2D3 action and was due to at least two types of defects in the VDR. One defect affects 1,25(OH)2D3 binding and the other causes resistance by a defect downstream of 1,25(OH)2D3 binding. Griffin and Zerwekh [99] and Liberman et al. [100,101] also measured 1,25(OH)2D3induction of 24-hydroxylase activity in patients’ cells to demonstrate 1,25(OH)2D3 resistance. Clemens et al. [98], on the other hand, showed that fibroblasts from HVDRR patients were resistant to 1,25(OH)2D3 by demonstrating a loss of the growth-inhibitory effects of 1,25(OH)2D3 treatment in contrast to fibroblasts from healthy individuals that were growth arrested. These early observations demonstrated that cells from HVDRR patients were resistant to 1,25(OH)2D3 and
that a spectrum of abnormalities in the VDR could cause resistance. As the number of reports on HVDRR increased, the heterogeneous nature of the defects in the VDR became more apparent. Hochberg et al. [27,35] reported the clinical findings in four patients from two unrelated families of Arab origin (F11, F18) who exhibited HVDRR and alopecia. A follow-up study by Chen et al. [77] showed that fibroblasts from three of these patients and a patient from an unrelated family from Germany (F17) had no detectable [3H]1,25(OH)2D3 binding and 1,25(OH)2D3 treatment failed to induce 24-hydroxylase activity. Pike et al. [104] used a radioligand immunoassay [105] and a monoclonal antibody to the chick VDR [106e108], to demonstrate the presence of an immunoreactive protein in cell extracts from fibroblasts of HVDRR patients that exhibited no 1,25(OH)2D3 binding. The authors speculated that the lack of [3H]1,25(OH)2D3 binding in these patients was not due to defective synthesis of the VDR protein but was due to defects in the VDR LBD that abolished 1,25(OH)2D3 binding [104]. Liberman et al. [102] described four cases (F1, F3, F5, F20) with normal ligand binding and 1,25(OH)2D3 resistance. In two families (F5, F20) the VDRs exhibited a low affinity for DNA. Gamblin et al. [109] examined 1,25(OH)2D3 induction of 24-hydroxylase activities and showed that the F5 and F20 fibroblasts were completely resistant to 1,25(OH)2D3 while the F1 and F3 fibroblasts were able to induce 24-hydroxylase activity when treated with high concentrations of 1,25(OH)2D3. Liberman et al. [102] further showed that the VDRs from the F1 and F3 fibroblasts had a reduced ability to localize to the nucleus despite showing a normal affinity for DNA. The F1 and F3 patients also exhibited a calcemic response to high doses of calciferols. Castells et al. [110] also reported on a patient (F22) who had sparse hair, rickets, and high circulating 1,25(OH)2D3 levels. Studies of the VDR from the patient’s fibroblasts showed that the VDR had a decreased affinity for [3H]1,25(OH)2D3. The patient showed a marked improvement after treatment with extremely high doses of 1,25(OH)2D3 apparently overcoming the low affinity binding abnormality in the VDR. Based on the hypothesis that VDR binding to DNA was essential for its activity, Hirst et al. [28] examined whether defective VDR binding to DNA could be the cause of resistance in cases where the VDR exhibited normal ligand binding. In a study of a family from Haiti (F19) with two sisters with HVDRR and an unaffected sister, they showed that the fibroblasts from the two affected sisters had normal [3H]1,25(OH)2D3 binding but were resistant to 1,25(OH)2D3 treatment. The authors further demonstrated that the VDR from the patients’ fibroblasts exhibited a significant decrease
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TABLE 65.3 Compilation of HVDRR Cases Family
Patient name/description
F1
Ethnic origin
Alopecia
1,25D binding
2
No
þ
VDR mutation
Male
IIB, patient 1, 1a
No
20 months
1
IIC, patient 2, 1b
No
5 months
1
F2
patient
No
15 years
1
1
No
F3
patient 1, patient 2a, 2a
Yes
1 year
1
2
Yes
patient 2, patient 2b, 2b
Yes
1 year
1
F4
patient
No
2 years
1
1
No
F5
patient, patient 3, 3, kindred 3, P3
Yes
10 months
1
1
Yes
þ
F6
K.N.
Yes
15 months
1
1
Yes
F7
patient
No
18 months
1
1
Yes
[19]
F8
patient
?
45 years
1
1
?
[188]
F9
M.A., kindred 6, patient 6, 6
No
1 year
1
1
Yes
F10
patient
No
12 years
1
1
No
F11
I.H., A1, patient 2, I.K., case 1
Yes
1 year
2
Yes
Tyr295stop
[26,27,77,140]
R.K., patient 1, A2, case 2
Yes
<1 year
1
Yes
Tyr295stop
[27,35,77,140]
F12
patient 4
Yes
?
1
Yes
[100,101]
F13
patient 5
Yes
5 months
1
Yes
[100,101]
F14
patient
Yes
2 years
1
1
No
F15
patient A, patient 5
Yes
19 months
{2}
1 {1}
4
Yes
[21]
F16
patient B, patient 4
Yes
8 months
{1}
1
2
Yes
[21]
F17
B patient
?
1
Yes
[27]
F18
S.H., patient 3, case 3, C1
2
Yes
Tyr295stop
[27,35,77,91]
Yes
Tyr295stop
[27,35,77,91]
Yes
þ
Arg73Gln
[28,90]
Yes
þ
Arg73Gln
[28,90]
2
Yes
þ
Arg 80Gln
[109,129,144]
Arg274Leu
[24,143,155]
Arab
Arab
Arab
R.H., patient 4, case 4, C2 F19
D1
Haitian
D2
1 1
No
6 months
1
No
1 month
1
Yes
?
1 {1}
Yes
?
1
Reference [13,97,109] [13,97]
3
[12] þ
[17,97,100,144]
þ
[17,97,100,109] Ile314Ser
[93,99,156]
Arg80Gln
[18,100,109,129] [110,187]
[100,220] [20]
[221]
kindred 7, patient 7, 7, P7
F21
I.S., patient
Kuwaiti
Yes
1 year
1
1
No
F22
patient
Hispanic
No
1 year
1
1
Yes
F23
patient
Saudi
Yes
1
1
Yes
þ
[110] Gly46Asp
[135,136] (Continued)
1205
F20
CELLULAR BASIS OF HVDRR
VIII. DISORDERS
Onset age
Japanese
Female
Total
Consanguinity
Compilation of HVDRR Casesdcont’d Alopecia
1,25D binding
1
Yes
1
2
Yes
1
1
Yes
1
1
Yes
9 months
1
1
Yes
13 months
1
2
Yes
Tyr295stop
[23,141,144]
Tyr295stop
[141]
Yes
þ
Gly33Asp
[90,103]
Yes
þ
Gly33Asp
[90,103]
Yes
Gln152stop
[143,144,189]
Yes
Gln152stop
[141,189]
Yes
þ
Arg50Gln
[57,130]
Yes
þ
Arg50Gln
[57,130]
1
Yes
Tyr295stop
[32,91]
Ethnic origin
Consanguinity
Onset age
Male
F24
patient 1, N.D.
Arab
Yes
4 months
1
F25
patient 1
Japanese
Yes
2 months
F26
patient 2
Japanese
No
2 months
F27
patient 3
Japanese
No
2 months
F28
patient 2, M.T.
PersianeJewish
Yes
F29
patient, line 10
Saudi
Yes
F30
line 15
Saudi
F31
G1
Arab
F32
line 11, patient 1
Turkish
line 11b, patient 2 F33
patient 1, patient 1a, patient 4a
Yes
1
Yes
1
Yes
5 weeks
Yes Japanese
patient 2, patient 1b,patient 4b
Female
2
1
2
1
Yes
16 months
Yes
16 months
1
2 1
1
Reference [111,114] [29]
þ
Arg50Gln
[29,57] [29] [111,114]
F34
E1
Arab
Yes
F35
F1
Arab
Yes
1
1
Yes
Tyr295stop
[32]
F36
H1
Arab
No
1
1
Yes
Tyr295stop
[32,91]
F37
J1
Arab
No
1
1
Yes
Tyr295stop
[32]
F38
K1
Arab
No
1
1
Yes
Tyr295stop
[32]
F39
L1
Arab
Yes
1
Yes
[32]
F40
Ro-VDR, brother
2
No
[115]
No
[115]
Yes
[115]
1 1
Al-VDR, sister
1
F41
Ab-VDR
1
1
F42
patient
1
F43
child
1
F44
patient II, case 2
Tunisian
Yes
1
1
Yes
F45
propositus
JapaneseeBrazilian
Yes
1
1
F46
line 14
Moroccan
Yes
F47
patient I
Mauritius
No
1
exon 7e9 deletion
[153]
Cys190Trp
[153]
þ
Lys45Glu
[132,134]
Yes
þ
His35Gln
[30]
2
Yes
Arg73stop
[141]
1
Yes
þ
Phe47Ile
[132,133] (Continued)
65. HEREDITARY 1,25-DIHYDROXYVITAMIN-D-RESISTANT RICKETS
Patient name/description
G2
VIII. DISORDERS
Total
Family
VDR mutation
1206
TABLE 65.3
TABLE 65.3
Compilation of HVDRR Casesdcont’d Total
Alopecia
1,25D binding
VDR mutation
Reference
1
1
Yes
þ
None
[178,179]
2
Yes
þ
Arg80Gln
[31]
Yes
þ
Arg80Gln
[31]
eliminate splice
[150]
Patient name/description
Ethnic origin
Consanguinity
Onset age
F48
J.K.
English
No
16 months
F49
N1
TunisianeJewish
Yes
7 months
Yes
6 month
1 1
N2 F50
patient
Greek
No
9 months
F51
patient
Turkish
Yes
3 months
sister
Male
Female
Family
1
1
Yes
1
Yes
2
No
þ
His305Gln
[157,158]
No
þ
His305Gln
[157,158]
1
Yes
þ
Arg391Cys
[156]
1
Yes
Arg30stop
[145]
2
Yes
nd
Arg30stop
[146]
1
patient 2
1
F53
patient
FrencheCanadian
F54
patient
Brazilian
Yes
F55
patient 1, B.G.
Greek
No
2 years
1
Yes
Arg73stop
[142]
F56
patient 2, A.H.
German
Yes
7 months
1
Yes
create splice
[142]
F57
patient 3, A.J.
Indian
Yes
15 months
1
Yes
þ
Gln259Pro
[142]
patient 4, U.A.
Indian
Yes
2.5 years
Yes
þ
Gln259Pro
[142]
F58
patient
Hmong
Yes
þ
Phe251Cys
[165]
F59
patient 1
Algerian
Yes
4 months
No
Trp286Arg
[160]
patient 2
Algerian
Yes
5 months
No
Trp286Arg
[160]
F60
patient
Caucasian
Yes
No
þ
Glu420Lys
[41]
F61
patient
Iranian
Yes
þ
Gln317stop
[148]
F62
patient
Caucasian
No
F63
patient
Saudi
Yes
F64
patient
Saudi
Yes
F65
F.C.
Arab
Yes
F66
patient
Chilean
F67
patient
Bedouin
Yes
3
F68
patient
French
No
1
1 1
1
2 1
1 1
2 1
1 1
20 months
1
1
Yes
nd
Glu329Lys/ c.366delC
[40]
2
3
No
þ
Ile268Thr
[161]
Yes
Arg30stop
Unpublished
Yes
Tyr295stop
Unpublished
1
No
5 bp deletion/8 bp insertion
[172]
4
Partial
Val246Met
[167]
1
Yes
Leu263Arg/ Arg391Ser
[170]
1 1
1
(Continued)
1207
1
CELLULAR BASIS OF HVDRR
VIII. DISORDERS
F52
1208
TABLE 65.3
Compilation of HVDRR Casesdcont’d
Patient name/description
Ethnic origin
Consanguinity
Onset age
Male
Female
F69
patient
Caucasian
Yes
F70
patient
Czech/Indonesian
F71
patient
Jamaican
?
F72
patient
Hispanic
Yes
F73
patient 1
Brazilian
F74
patient 2
Brazilian
F75
patient 3
Brazilian
F76
patient 4
Brazilian
Yes
1
1
F77
patient
Iranian
Yes
2
2
Yes
F78
patient #1
Hispanic
Yes
1
1
Partial
F79
patient #2
Caucasian
?
1
1
Partial
F80
J.P.
Sardinian
No
1
1
F81
patient
India
Yes
1
2
F82
patient
Thai
1
1
Total
Alopecia
1
Partial
1,25D binding
VDR mutation
Reference
Val26Met
[138]
Arg30stop/ 6Lys246
[171]
1
Partial
103bp insertion/ duplication/ Tyr401stop
[173]
1
Partial
Splice site/ del exon 8
[152]
1
1
Yes
Gln259Asp
[164]
1
1
Yes
Gly319Val
[164]
1
Yes
Gln259Asp
[164]
Arg73stop
[164]
Cys41Tyr
[137]
Arg50stop
[149]
Nd
Arg50stop
[149]
Partial
Cys84Arg
[139]
Yes
c.716delA
[154]
Partial
Intron retention
[151]
1
1
1
65. HEREDITARY 1,25-DIHYDROXYVITAMIN-D-RESISTANT RICKETS
VIII. DISORDERS
Family
MOLECULAR BASIS FOR HVDRR
in its affinity for DNA. Using calf thymus DNAcellulose chromatography, the VDR from the unaffected sister bound strongly to DNA eluting at a high concentration of KCl (170e173 mM KC1) whereas the patients’ VDR bound weakly to DNA and eluted at lower concentrations of KCl (105e109 mM). A subsequent study by Malloy et al. [103] demonstrated a similar DNA-binding defect in the VDR from HVDRR patients (F31) who had normal [3H]1,25(OH)2D3 binding. DNA-cellulose chromatography clearly revealed that the patient’s VDR had a low affinity for DNA. Furthermore, the cells from the parents expressed two forms of the VDR, one with a high affinity for DNA (eluting at 200 mM KC1) and the other with a low affinity for DNA (eluting at 100 mM KC1). This was the first evidence demonstrating the presence of the defective VDR in the cells from parents of HVDRR children. It was hypothesized that the VDR defects in these cases would likely be due to mutations in the DBD [28,103], which later proved to be correct [90].
Studies in Other Cell Types In addition to studying cultured skin fibroblasts, a number of other cell types have been used to study HVDRR cases. These include peripheral mononuclear cells [111], phytohemagglutinin (PHA)-stimulated lymphocytes [112,113], myeloid progenitor cells [114], Epstein-Barr virus (EBV) immortalized B lymphoblasts [32,90,91,103], and HTLV-1 virus immortalized T lymphoblasts [115]. It is interesting to note that although EBV immortalized B lymphoblasts from normal subjects express wild-type VDR, they failed to induce 24-hydroxylase activity and their growth was not inhibited by 1,25(OH)2D3 [32]. A possible mechanism for these early findings was recently elucidated. Yenamandra et al. showed that EBNA-3, a member of the EBNA-3-protein family that regulates transcription of cellular and viral genes, blocked the activation of VDR-dependent genes and protected the EBV immortalized lymphoblasts against vitaminD3-induced growth arrest [116]. In contrast, PHA-stimulated lymphocytes and HTLV-1 immortalized T lymphoblasts from normal subjects do respond to 1,25(OH)2D3 [115,117]. Takeda et al. [113] used PHAstimulated lymphocytes from HVDRR patients to demonstrate 1,25(OH)2D3 resistance by its failure to inhibit DNA synthesis or induce 24-hydroxylase activity [112,113]. Takeda et al. [113] also showed that PHA-stimulated lymphocytes from parents of children with HVDRR expressed intermediate levels of 24hydroxylase in response to 1,25(OH)2D3 compared to normal cells.
1209
MOLECULAR BASIS FOR HVDRR First Description of a Genetic Defect in the Nuclear Receptor Superfamily The biochemical and cellular data obtained from the earlier studies of HVDRR patients provided a framework to begin the search for the molecular cause of this disease. Investigations to determine the specific mutations in the VDR that cause HVDRR began shortly after the human VDR cDNA sequence was elucidated by Baker et al. [118]. During this same time period the polymerase chain reaction (PCR) technique [119] was developed that provided a method to rapidly amplify genes for sequence analysis. The amino acid sequence of the VDR cDNA suggested the presence of highly conserved zinc-finger structures that were thought to be involved in DNA binding. The initial focus was HVDRR patients that had normal [3H]1,25(OH)2D3 binding but abnormal binding to DNA since it was suspected that this defect would arise from mutations in the VDR DBD. In 1988, Hughes et al. [90] used PCR to amplify exons of the VDR gene from DNA isolated from the F19 and F31 families that were defective in DNA binding [28]. The patients in the F19 family were shown to have a unique G to A single base change in exon 3 that encodes the second zinc module of the DBD. This missense mutation replaced arginine with glutamine at amino acid residue 73 (Arg73Gln) (Fig. 65.4). In the F31 family a G to A transition was identified in exon 2 that encodes the first zinc module of the DBD. This missense mutation changed glycine to aspartic acid at amino acid residue 33 in the first zinc-finger module (Gly33Asp) (Fig. 65.4). Only the mutant sequences were found in the children with HVDRR. Their parents had a normal and a mutant sequence demonstrating the genetic transmission and recessive nature of the disease. The study by Hughes et al. [90] was the first description of a genetic defect in any member of the steroidethyroideretinoid receptor gene superfamily. Mutations have now been found in many of the classical receptors including thyroid receptor (TR) [120,121], androgen receptor (AR) [122,123], estrogen receptor (ER) [124], glucocorticoid receptor (GR) [125,126], and mineralocorticoid receptor (MR) [127,128]. To determine whether the missense mutations found in these families were the cause of 1,25(OH)2D3 resistance in the HVDRR patients, the Arg73Gln and the Gly33Asp mutations were generated in the wild-type VDR cDNA by site-directed mutagenesis [92]. The recreated mutant VDRs were expressed in COS-1 cells and exhibited normal [3H] 1,25(OH)2D3 binding and weak binding to DNA similar to the VDR in the patient’s fibroblasts. In addition, Sone et al. [92] demonstrated that the mutant VDRs were transcriptionally inactive in cotransfection
VIII. DISORDERS
1210
65. HEREDITARY 1,25-DIHYDROXYVITAMIN-D-RESISTANT RICKETS
FIGURE 65.4 Model of the DNA-binding domain of the VDR and location of mutations causing HVDRR. The two zinc-finger modules and the amino acid composition of the DBD are shown. Conserved amino acids are depicted as shaded circles. Numbers specify amino acid number.
experiments in CV-1 cells. Using an osteocalcin-CAT reporter plasmid, they showed that CAT activity could be induced by the wild-type VDR but not by the Arg73Gln or the Gly33Asp mutant VDRs [92]. These data proved that the missense mutations caused the VDR to be transcriptionally inactive and were the cause of 1,25(OH)2D3 resistance in the patients. Since the original report by Hughes et al. [90] a number of VDR mutations have been identified in patients with HVDRR. Over 100 cases of HVDRR have been recorded and a number of these have been analyzed at the biochemical and molecular level [3] (Table 65.3). Multiple genetic abnormalities have been found in the VDR gene, mainly missense and nonsense mutations, but also deletions and splice site mutations. A description of these mutations and the consequences of the abnormality in the VDR will be discussed below.
Mutations in the VDR DNA-Binding Domain (DBD) Analyses of DBD Mutations Since the initial report by Hughes et al. [90], a number of mutations have been identified in the VDR DBD. The location of these mutations within the DBD is illustrated schematically in Fig. 65.4. Sone et al. [129] examined the VDR from two unrelated patients (F5 and F20) previously shown to exhibit a ligand-binding positive and low-affinity DNA-binding phenotype by Liberman et al. [100,102]. In both patients, a G to A missense mutation was identified in exon 3. The mutation changed arginine to glutamine at amino acid 80 in the second
zinc-finger module (Arg80Gln). The recreated Arg80Gln mutant receptor had a high affinity for [3H]1,25(OH)2D3, but had a lower affinity for DNA and was unable to activate gene transcription from a reporter plasmid demonstrating that this molecular defect was the cause of HVDRR in these cases [129]. Malloy et al. [31] also identified the same Arg80Gln mutation in two siblings with HVDRR (F49). The F49 family and the families (F5, F20) described by Sone et al. [129] both had origins in North Africa; however, no genetic relationship between these families could be established. Saijo et al. [57] described a DBD mutation in three HVDRR patients from two unrelated families (F26 and F33) of Japanese origin. Earlier investigations showed that fibroblasts from the patients had normal [3H]1,25(OH)2D3 binding but the VDR exhibited abnormal nuclear binding [112,130,131]. A G to A mutation was found in exon 3 that converted arginine to glutamine at amino acid 50 in the second zinc-finger module (Arg50Gln). The children’s parents were identified as carriers of both the normal and mutant alleles using single-strand conformational polymorphism (SSCP) [57]. No data were provided to confirm the functional consequences of this defect on the VDR. A missense mutation in the first zinc module of the VDR DBD that changed a histidine to glutamine at amino acid 35 (His35Gln) was identified by Yagi et al. [30]. The mutation replaced a positively charged amino acid with a neutral one. The VDR from the patient’s cells (F45) exhibited normal 1,25(OH)2D3 binding but the receptors exhibited low-affinity binding to DNA. The patient’s fibroblasts were transiently transfected with a VDRE-CAT reporter plasmid to test for 1,25(OH)2D3
VIII. DISORDERS
MOLECULAR BASIS FOR HVDRR
responsiveness. The transformed cells were unable to induce gene transcription. However, when the patient’s fibroblasts were cotransformed with the wild-type VDR cDNA and the reporter plasmid the cells acquired the ability to respond to hormone. Two mutations in the VDR DBD were reported by Rut et al. [132]. One patient (F47) described previously by Lin and Uttley [133] had an A to G mutation in exon 2. The missense mutation resulted in lysine being replaced by glutamic acid at amino acid 45 (Lys45Glu). In the same study, Rut et al. [132] examined the VDR gene in a patient (F44) described previously by Simonin et al. [134]. They identified a unique T to C base change in exon 2 that resulted in phenylalanine being replaced by isoleucine at amino acid 47 (Phe47Ile). The recreated mutant VDRs exhibited normal 1,25(OH)2D3 binding but were transcriptionally inactive [132]. Lin et al. [135] examined the VDR gene for mutations in a patient (F23) with HVDRR previously described by Sakati et al. [136]. A unique G to A base change was found in exon 2. The mutation resulted in a glycine being changed to an aspartic acid at amino acid 46 (Gly46Asp). The recreated Gly46Asp mutant VDR exhibited the characteristics of a DBD mutation in that the mutant receptor bound [3H] 1,25(OH)2D3 normally but displayed a reduced affinity for DNA. The mutant receptor was also shown to be transcriptionally inactive in reporter gene assays. The authors used PCR and a restriction fragment length polymorphism (RFLP) generated by the mutation to demonstrate that the patient was homozygous for the mutation and the patient’s father was a carrier of the mutant allele. In contrast to the other DBD mutations described above, the mutation at Gly46 occurs in an amino acid that is not well conserved in the steroidethyroideretinoid receptor superfamily. However, Gly46 is conserved among receptors that form heterodimers with RXR proteins such as TR and RAR. A young Iranian boy (F77) with HVDRR and alopecia was found to have a G to A substitution in exon 2 that resulted in a Cys41Tyr change in the first zinc-finger module [137]. An older brother with similar signs and symptoms died at the age of 2 years 8 months. A young Caucasian boy (F69) with HVDRR and alopecia was shown to have a G to A mutation that changed the codon for valine to methionine (Val26Met) in the first zinc-finger module [138]. The Val26Met mutant VDR was able to heterodimerize with RXR and bind to the coactivator DRIP205 and to the corepressor HR. However, the Val26Met mutant VDR failed to form a stable complex with a VDRE and was transcriptionally inactive. A 7-year-old Sardinian boy (F80) with HVDRR and alopecia had a T to C substitution in exon 3 that changed cysteine to arginine at amino acid 84 (Cys84Arg) [139].
1211
Structural Analysis of DBD Mutations The Lys45Glu and Gly46Asp mutations are located in the P-box (Fig. 65.2). This region of the VDR DBD is likely important in contacting the DNA bases and determining the specificity of the receptor for specific VDREs. Rut et al. [132] proposed that the Lys45Glu mutation would disturb the hydrogen bonding between Lys45 and a guanine nucleoside in the VDRE half-site. The conversion of glycine to aspartic acid, a bulky, charged amino acid, probably leads to unfavorable electrostatic interactions with the negatively charged phosphate backbone of the DNA helix that may prevent the receptor from contacting specific nucleotide bases in the VDRE. Alternatively, the Gly46Asp mutation may eliminate the ability of the VDR to specifically recognize VDREs [135]. Similarly, the Gly33Asp mutation is expected to repel the negatively charged phosphate backbone due to the negatively charged aspartic acid [132]. On the other hand, the substitution of glycine for histidine in the His35Gly mutation most likely eliminates a hydrogen bond donated from the positively charged histidine to the phosphate of a guanine nucleoside in the VDRE [132]. In the Phe47Ile mutation, the loss of the phenylalanine ring structure may disrupt the integrity of the hydrophobic core of the DBD. The mutation may obstruct the formation of the a-helical structure h-1 proposed at the base of the first zinc finger such that the VDR could not bind normally to its VDRE (Fig. 65.4) [132]. The Cys41Tyr mutation would destroy the coordination of the zinc ion in the first zinc-finger module and the Arg50Gln mutation would be expected to inhibit the translocation of the VDR to the nucleus since the mutation occurs in the nuclear localization signal (NLS) [63]. The Val26Met mutation may disrupt the topography of the first zinc-finger module while the Cys84Arg mutation may disrupt the a-helical structure of h-2 proposed at the base of the second zinc-finger module (Fig. 65.4).
Mutations Causing Premature Termination of the VDR Nonsense Mutations Nonsense mutations in the VDR gene that introduce a premature stop signal cause early termination of the mature VDR protein. The first molecular analysis of three HVDRR cases (F18, F34, F36) that exhibited no [3H]1,25(OH)2D3 binding was reported by Ritchie et al. [91]. A single unique C to A base change was found in exon 8 that changed the codon for tyrosine (TAC) to an ochre termination codon (TAA) (Tyr295stop) (Fig. 65.5). The location of the premature stop at amino acid 295 truncates 132 amino acids of the carboxy terminus of the VDR that results in the deletion
VIII. DISORDERS
1212
65. HEREDITARY 1,25-DIHYDROXYVITAMIN-D-RESISTANT RICKETS
Mutations that cause premature termination of the VDR. The locations of the nonsense mutations causing HVDRR are illustrated in the VDR protein. The location of the splice site mutations and the single base deletion are depicted in the VDR gene.
FIGURE 65.5
of a major portion of the LBD thereby creating the ligand-binding negative phenotype. The recreated mutant VDR with a molecular size of 32 000 daltons was unable to bind [3H]1,25(OH)2D3 and failed to activate gene transcription demonstrating that this mutation was the cause of the hormone-resistant state in the three patients. The Tyr295stop mutation was the first nonsense mutation identified in the VDR. The location of this mutation and other nonsense mutations that cause premature termination of the VDR is shown in Figure 65.5. The three families studied by Ritchie et al. [91] and four additional families (F35, F37, F38, and F39) that comprised a large kindred in a region where consanguineous marriages were common was analyzed by Malloy et al. [32] (Table 65.3). A total of eight children from this kindred exhibited HVDRR with alopecia. All of the affected children were homozygous for the Tyr295stop ochre mutation and their parents were heterozygous as determined by analysis of a RsaI restriction fragment length polymorphism (RFLP) created by the mutation [32]. Interestingly, the 32 000 molecular weight truncated protein that is predicted to be produced by this mutation could not be detected in extracts of cultured dermal fibroblasts using Western blots. In addition, in all but one case, the VDR mRNA was undetectable on Northern blot using RNA isolated from the cultured fibroblasts or EBV-transformed lymphoblasts from the patients. The absence of VDR mRNA indicated that the Y295X mutation led to nonsense-mediated mRNA decay and, in this case, accounts for the absence of the mutant VDR protein.
Since the reports by Ritchie et al. [91] and Malloy et al. [32] a number of other premature stop mutations have been identified in the VDR from patients with HVDRR [3]. The locations of these mutations are shown in Figure 65.5. Family F11 described in earlier papers [26,27,35,77] had two affected children with HVDRR who exhibited the ligand-binding negative phenotype. This family of Christian Arabs lives in the same town as the extended kindred (F18, F34eF39) described above who are Muslim Arabs. Although there is no known genetic relationship between family F11 and the large kindred, the Tyr295stop mutation is the cause of HVDRR in this family as well [140]. The Tyr295stop mutation was also identified by Wiese et al. [141] in two related patients (F29, F30) from Saudi Arabia who were previously studied by Bliziotes et al. [23]. These patients are apparently unrelated to the other families with the same mutation. It is not clear whether Tyr295stop mutation represents a mutational “hot spot” in the VDR gene or whether these cases all descended from a common founder mutation. A patient with HVDRR from a Moroccan family (F46) was examined for mutations in the VDR gene by Wiese et al. [141]. At the cellular level, no [3H]1,25(OH)2D3 binding was detected in the patient’s cells. The authors discovered an opal mutation (CGA to TGA) in which a C to T substitution introduced a premature stop codon at amino acid 73 (Fig. 65.5). The Arg73stop mutation truncates the receptor in the middle of the second zinc-finger module. Interestingly, the Arg73stop mutation occurs in the same codon that gives rise to the Arg73Gln mutation but at a different nucleotide base
VIII. DISORDERS
MOLECULAR BASIS FOR HVDRR
[90]. In the Arg73stop mutation, CGA is mutated to TGA while in the Arg73Gln mutation the CGA is mutated to CAA. The Arg73stop mutation has also been identified in a young boy (F56) from Greece who had HVDRR with alopecia [142]. A Turkish patient (F32) with HVDRR studied by Kristjansson et al. [143] was found to harbor an amber mutation that changed the codon for Gln152 (CAG) to a stop mutation (TAG) in exon 4 (Fig. 65.5). Previous studies of fibroblasts from this patient showed an absence of ligand binding and 1,25(OH)2D3 responses [144]. As expected, the Gln152stop mutant VDR was unresponsive to 1,25(OH)2D3 in gene transactivation assays. Wiese et al. [141] also identified the Gln152stop mutation in a patient (F32) with HVDRR previously reported by Barsony et al. [144]. Zhu et al. [145] analyzed the VDR in fibroblasts from a young boy (F53) of French-Canadian origin with HVDRR and alopecia. The patient’s fibroblasts lacked specific [3H]1,25(OH)2D3 binding and failed to induce 24-hydroxylase mRNA after treatment with up to 100 nM 1,25(OH)2D3. Northern blotting showed that the cells expressed a normal-sized VDR mRNA, but Western blotting failed to detect any protein. A C to T base substitution was located in exon 2 which changed the codon for arginine (CAG) at amino acid 30 to an opal stop codon (TAG) (Arg30stop) (Fig. 65.5). The same Arg30stop mutation was also identified in two children with HVDRR from a family (F54) living in Brazil [146]. One child died at 4 years of age due to cardiorespiratory insufficiency. Interestingly, the parents, who were first cousins, were phenotypically normal but had slightly elevated levels of serum 1,25(OH)2D. The mean value for the father was 73 pg/ ml and for the mother 93 pg/ml (normal range 20e80 pg/ml). The elevated 1,25(OH)2D values raise the possibility of mild vitamin D resistance in the heterozygotic parents, a finding that had not been documented previously in other parents of HVDRR children. Bouillon and Vainsel [147] analyzed the VDR in fibroblasts from a young Iranian girl (F61) with HVDRR and alopecia. They showed that the patient’s fibroblasts had no demonstrable VDRs by sucrose density ultracentrifugation and by ligand binding. A follow-up study by Malloy et al. [148] showed that the patient’s fibroblasts expressed the VDR mRNA but had no VDR protein and were unresponsive to high doses of 1,25(OH)2D3. Sequence analysis of the VDR gene uncovered an opal mutation in exon 8 that changed the codon for glutamine to a termination codon (Gln317stop) (Fig. 65.5) [148]. A sister that also had rickets and alopecia had died earlier. Forghani et al. [149] studied two unrelated patients (F78, F79) with HVDRR and alopecia living in different parts of the USA. Both patients had a C to T base change
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in exon 3 that changed the codon for arginine at amino acid 50 to a stop codon (Arg50stop). Mutations that disrupt MRNA splicing Hawa et al. [150] examined the VDR in a young Greek girl (F50) with HVDRR and alopecia. Using RT-PCR and DNA sequencing, they showed that the patient’s RNA sequence diverged from the wild-type sequence at nucleotide 147. The sequence from exon 3 that encodes the second zinc-finger module was deleted and the sequence that followed was from exon 4. Sequence analysis of the VDR chromosomal gene found no mutations in the exons; however, a G to C base change was found in the 5’ end of intron E (Fig. 65.5). This single nucleotide change converted the wild-type sequence from GTGAGT to GTGACT and eliminated the 5’ donor splice site. The loss of the 5’ donor splice site caused exon 3 to be skipped in the processing of the VDR transcript and introduced a reading frameshift that resulted in a premature stop codon. The mutant protein contained 92 amino acids of the wild-type sequence plus six amino acids due to the frameshift (Glu92fs) (Fig. 65.5). The shortened VDR had no [3H]1,25(OH)2D3 binding and failed to induce 24-hydroxylase activity. A splice site mutation was also identified in a German patient (F56) with HVDRR and alopecia [142]. Studies of the patient’s fibroblasts showed absent [3H]1,25(OH)2D3 binding and failure to induce 24-hydroxylase activity with 1,25(OH)2D3 treatment. In this case, a cryptic 5’ donor splice site was generated in exon 6. The mutation in this case, a C to G transition, changed the sequence from GTCAGT to GTGAGT. This single base change did not alter the amino acid coding sequence in exon 6 but introduced a splice site that could be recognized by the spliceosome complex during RNA processing. As a result, the mutation caused a 56 bp deletion in exon 6 which led to a frameshift 15 bases into exon 7. The mutant protein contained 233 amino acids of the wild-type sequence and an additional four amino acids due to the frameshift (Leu233fs) (Fig. 65.5). The mutation caused the truncation of 194 amino acids of the VDR leading to a loss of 1,25(OH)2D3 binding and hormone responsiveness. A young Thai girl (F82) with HVDRR and partial alopecia had a single base substitution in the 5’-donor splice site at the exon 4eintron F junction [151]. The mutation caused 254 base pairs of intron F to be incorporated into the VDR mRNA. The encoded VDR protein contained 154 amino acids of the wild-type sequence and an additional 23 amino acids from the unspliced intron F. A splice site mutation was also identified in a young Hispanic girl (F72) with HVDRR and partial alopecia. A novel G to T substitution was identified in the 5’-splice site in the exon 8eintron J junction [152]. Sequence
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analysis of the VDR cDNA amplified by RT-PCR showed that exon 8 was skipped and that exons 7 and 9 were fused in frame. However, the mutation caused a 39-amino-acid deletion (6Ala303-Pro341) in the VDR LBD. The recreated mutant VDR6Ala303-Pro341 was unable to activate gene transcription (Malloy, unpublished study). Deletions In a preliminary report of an additional patient (F42), a major structural defect in the VDR gene was described as the cause of HVDRR [153]. The defect, found by PCR and Southern blotting, was a deletion in the VDR gene that eliminated exons 7, 8, and 9. A young girl from India with HVDRR and alopecia was found to have a 1 bp deletion in exon 6 (c.716delA) [154]. The mutation caused a shift in the reading frame that introduced a premature stop codon and resulted in the truncation of the VDR LBD. An older brother with similar symptoms of HVDRR died of pneumonia. All of the nonsense mutations and all splice site mutations that lead to frameshifts, as well as the gene deletion mutations result in truncated VDRs. Although some of the mutants VDRs may have intact DBDs, the loss of the LBD and therefore its ability to bind 1,25(OH)2D3, associate with RXR, and interact with coactivators makes the mutant receptors non-functional and causes complete hormone resistance.
Mutations in the VDR Ligand-binding Domain (LBD) Mutations that Affect 1,25(OH)2D3 Binding Rut et al. [155] and Kristjansson et al. [143] identified the first missense mutation in the VDR LBD. The patient from Kuwait (F21) had HVDRR but did not
have alopecia. Preliminary studies by Fraher et al. [24] on the patient’s fibroblasts showed absent [3H]1,25(OH)2D3 binding. However, a later study by Rut et al. [155] showed that the fibroblasts contained normal amounts of [3H]1,25(OH)2D3 binding but the affinity of the receptor for 1,25(OH)2D3 was significantly reduced (Kd 1 109 M) compared to normal controls (Kd 0.7 1010 M). They showed that the patient’s fibroblasts were resistant to vitamin D by their failure to induce 24-hydroxylase activity when treated with high doses of 1,25(OH)2D3 [24,155]. Molecular analysis of the VDR gene identified a unique G to T missense mutation in exon 7 [143,155]. This mutation resulted in replacement of a positively charged arginine residue by a neutral charged leucine at amino acid 274 (Arg274Leu) (Fig. 65.6). The Arg274Leu mutation is located in helix H5. As described by Rochel et al. [68] and Chapter 9, Arg274 is a contact point for the 1ahydroxyl group in 1,25(OH)2D3. In transactivation assays, the recreated Arg274Leu mutant VDR was relatively resistant to vitamin D requiring approximately 1000-fold more 1,25(OH)2D3 to activate gene transcription than the wild-type receptor [143]. Although the resistance caused by the defective VDR could be overcome by treating with high concentrations of 1,25(OH)2D3 in vitro, the patient failed to respond to massive does of the hormone and eventually died of pneumonia. Whitfield et al. [156] analyzed the VDR from a girl (F4) who had the classic symptoms of HVDRR but without alopecia. The patient’s fibroblasts were originally examined by Griffin and Zerwekh [99] who showed that the cells had normal 1,25(OH)2D3 binding but had defective induction of 24-hydroxylase activity. Sequencing of the VDR gene uncovered a T to G substitution in exon 8 that changed isoleucine to serine at amino acid 314 (Ile314Ser) (Fig. 65.6). The Ile314Ser
Schematic illustration of the ligand-binding domain of the VDR and location of amino acid substitutions causing HVDRR. The a-helices (H1eH12) of the VDR LBD are depicted as shaded rectangles and the b-turns are drawn as a hatched rectangle. The loops connecting the helices are drawn as solid lines. The E1 and AF-2 regions are shown below the a-helices.
FIGURE 65.6
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mutation occurs in H7. The mutation causes a subtle defect in heterodimerization with RXR and decreased response to 1,25(OH)2D3 in transactivation assays. This patient showed a nearly complete cure when treated with pharmacological doses of 25-hydroxyvitamin D3. A missense mutation in the VDR LDB was described by Malloy et al. [157]. The patient, a Turkish boy (F51), had HVDRR and two other rare disorders, congenital generalized lipoatrophic diabetes (Berardinelli-Seip syndrome), and persistent Mu¨llerian duct syndrome [158]. The patient had rickets and high 1,25(OH)2D3 levels but did not have alopecia. He was treated with extremely high doses of calcitriol (Rocaltrol 12.5 mg/day) that eventually normalized his serum calcium and ultimately improved his rickets. However, the child died of apparently unrelated problems. The patient’s fibroblasts expressed normal VDR levels but the affinity for 1,25(OH)2D3 was decreased by about two-fold. The patient’s fibroblasts required approximately a five-fold more 1,25(OH)2D3 to induce 24hydroxylase mRNA compared to control cells. Sequence analysis of the VDR gene uncovered a C to G missense mutation in exon 8. This mutation led to the replacement of histidine by glutamine at amino acid 305 (His305Gln) (Fig. 65.6). The His305Gln mutation occurs in the interhelical loop between H6eH7. Interestingly, [3H]1,25(OH)2D3-binding studies of the reconstructed mutant protein demonstrated an eight-fold lower affinity for 1,25(OH)2D3 compared to the wild-type VDR when the assays were performed at 24 C (versus two-fold at 0 C). In gene transactivation assays, the His305Gln mutant VDR required five-fold more 1,25 (OH)2D3 to achieve the same level of activity as the wild-type VDR. RFLP analysis with AlwNI showed that the patient and a sibling with HVDRR were homozygous for the mutation and that their parents were heterozygous. As shown by Rochel et al. [68], His305 is a contact point for the 25-hydroxyl group in 1,25 (OH)2D3. The boy’s sister, who also had HVDRR and the same mutation in the VDR, did not exhibit the other genetic defects. No explanation was forthcoming for the presence of three genetic defects in a single individual. It is unclear how the His305Gln mutation in the VDR is related, if at all, to the two other genetic abnormalities that were present in this child. The congenital total lipodystrophy was subsequently shown to be due to a mutation in the seipin gene (BSCL2) [159]. A genetic cause for the persistent Mu¨llerian duct syndrome was not determined. An Algerian boy and his younger sister (F59) both with HVDRR without alopecia were shown to have a T to C mutation in exon 7 that changed tryptophan to arginine at amino acid 286 (Trp286Arg) (Fig. 65.6) [160]. Trp286 is the only tryptophan in the VDR. The patient’s fibroblasts expressed a normal-size VDR
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protein and normal-length VDR mRNA but no specific [3H]1,25(OH)2D3 binding was observed and the cells were totally unresponsive to 1,25(OH)2D3 treatment. The Trp286Arg mutation is located in the b1 sheet of the three-stranded b sheet between helices H5 and H6 (Fig. 65.6). A young Saudi Arabian girl (F63) with HVDRR but without alopecia was homozygous for a unique T to C mutation in exon 7 of the VDR gene that changed the codon for isoleucine to threonine at amino acid 268 (Ile268Thr) [161]. Based on the crystallographic studies of the VDR LBD, Ile268 located in helix H5 directly interacts with 1,25(OH)2D3. The Ile268Thr mutant VDR exhibited an ~10-fold lower affinity for [3H]1,25 (OH)2D3 compared to the WT VDR consistent with its interaction with the ligand. However, in transactivation assays, the Ile268Thr mutant required ~100-fold higher concentrations of 1,25(OH)2D3 to stimulate gene transcription compared to the WT VDR. The Ile268Thr mutant also exhibited a marked decrease in RXR binding compared to the WT VDR. Ile268 is also involved in the hydrophobic stabilization of helix H12. Consistent with this activity, the Ile268Thr mutant required ~100-fold more 1,25(OH)2D3 to promote binding to the coactivators SRC-1 and DRIP205. These cumulative defects caused 1,25(OH)2D3 resistance and resulted in the syndrome of HVDRR in the patient [161]. A missense mutation was also described in the VDR LBD by Thompson et al. [153]. The HVDRR patient (F43) was shown to have a mutation in exon 5. In this case, a cysteine was changed to tryptophan at amino acid 190 (Cys190Trp). The Cys190Trp mutation occurs in the loop between H1 and H3. Further details about this case were not included in this preliminary report; however, deletion of this region of the VDR LBD has been shown to have no effect on ligand binding or transactivation [162]. Also, we have recreated the Cys190Trp mutation and have shown that it does not affect transactivation (Malloy, unpublished study). These data suggest that the Cys190Trp substitution was unlikely to be the sole cause of HVDRR in this case. STRUCTURAL ANALYSIS OF LBD MUTATIONS THAT AFFECT 1,25(OH)2D3 BINDING
Since the crystal structure of the holo-VDR has been reported [68], a more definitive explanation of the effects of the LBD mutations on the VDR is now possible. In one patient (F21) Arg274 was mutated to leucine (Arg274Leu) and resulted in a 1000-fold decrease in ligand-binding affinity [143]. This can now be explained by the fact that Arg274 is a contact point for the 1ahydroxyl group of 1,25(OH)2D3 [68]. Thus the mutation to leucine alters the contact point and lowers the binding affinity of the VDR for 1,25(OH)2D3. In a second patient (F51), a His305Gln mutation was shown to lower the
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binding affinity for 1,25(OH)2D3 by about 5e10-fold [157]. The crystal structure of the His305Gln mutant VDR bound to 1,25(OH)2D3 has recently been elucidated [163]. This analysis showed that the His305Gln mutant VDR adopts the active conformation of the wild-type liganded VDR; however, the mutation introduces a local conformational change that results in the loss of two hydrogen bonds between Ser306-Gln400 and Gln400His305. This local conformational change around Gln305 weakens the hydrogen bond between the 25hydroxyl group of 1,25(OH)2D3 with Gln305 and lowers the affinity of the mutant protein for 1,25(OH)2D3 [163]. In a third patient (F59) Trp286 was mutated to arginine (Trp286Arg) that totally abolished ligand binding [160]. Trp286 makes contact with the alpha face of the C ring in 1,25(OH)2D3 and is involved in forming the hydrophobic channel where the conjugated triene connecting the A and the C rings fits [68]. The Trp286Arg mutation alters the contact point with the ligand and causes resistance to 1,25(OH)2D3 [160]. It is evident from these data that the disruption of a ligand contact point can be the basis for HVDRR. Mutations that Affect VDR-RXR Heterodimerization As mentioned above, the VDR requires heterodimerization with RXR for activity. Disruption of this proteine protein interaction can thereby cause 1,25(OH)2D3 resistance. The first patient found to have a mutation in the VDR that disrupted VDR-RXR heterodimerization was described by Whitfield et al. [156]. The patient was a young girl (F52) who had HVDRR and alopecia. Sequencing showed that the patient had a C to T base change in exon 9 that converted an arginine to cysteine at amino acid 391 (Arg391Cys) (Fig. 65.6). The Arg391Cys mutation is located in helix H10. Ligand binding was unaffected but transactivation of a reporter gene by the Arg391Cys mutant VDR required higher than normal concentrations of 1,25(OH)2D3 for activity. However, when RXR was co-transfected in the assays the transactivation activity could be restored to normal levels. VDR-RXR interactions and VDRE binding were further examined using electrophoretic mobility shift assays (EMSA). The Arg391Cys mutant exhibited a lower capacity for forming a VDR-RXR-VDRE complex than the wild-type VDR. The Arg391Cys mutation thus reduces the interaction between the VDR and RXR. By increasing the RXR protein concentration the affinity defect could be overcome and the transactivation rescued. This study demonstrated the importance of both 1,25(OH)2D3 binding and RXR heterodimerization in VDR-mediated gene transactivation. This was the first report of a mutation in the VDR that interfered with RXR binding and caused HVDRR.
Two siblings, a brother and sister from India (F57) that had HVDRR with alopecia, were studied by Cockerill et al. [142]. The children’s parents were first cousins. Using cultured fibroblasts, [3H]1,25(OH)2D3 binding was found to be normal but 1,25(OH)2D3 induction of 24-hydroxylase activity was absent. DNA sequence analysis revealed a single base alteration in exon 7 that changed glutamine to proline at amino acid 259 (Gln259Pro) (Fig. 65.6). The Gln259Pro mutation occurs in helix H4. Although Gln259Pro had no apparent affect on ligand binding there was evidence of impaired VDRRXR-VDRE formation. Whereas the wild-type VDR formed two complexes (complex A and complex B) in the EMSA, the Gln259Pro mutant VDR showed a reduction in the formation of complex B and an enhancement in complex A. The authors speculated that the Gln259Pro mutation in the VDR affected proteineprotein interactions possibly by increasing the affinity of the receptor for an unidentified protein. In transactivation assays the recreated Gln259Pro mutant VDR was functionally inactive. In two Brazilian patients (F73, F75) with HVDRR a missense mutation in exon 7 was found that changed Gln259 to aspartic acid (Gln259Asp) [164]. Like the Gln259Pro mutation, the Gln259Asp mutation would also be predicted to affect heterodimerization with RXR. Malloy et al. [165] examined the VDR in a young Hmong boy (F58) with HVDRR and alopecia. Analyses of the VDR demonstrated a normal-size receptor on Western blots; however, the amount of VDR was decreased compared to normal. Northern blot analysis of 24-hydroxylase mRNA induction showed that the patient’s fibroblasts were approximately 1000-fold less responsive to 1,25(OH)2D3 than control fibroblasts confirming target organ resistance. A unique missense mutation was found in exon 6 that changed a phenylalanine to cysteine at amino acid 251 (Phe251Cys) (Fig. 65.6). In [3H]1,25(OH)2D3 binding experiments, the recreated Phe251Cys mutant VDR exhibited a lower affinity for the ligand than wild-type VDR when assayed at 24 C. Using GST pull-down assays and yeast twohybrid constructs, the Phe251Cys mutant VDR was shown to have reduced capacity to bind RXR. In transactivation assays, cotransfection of RXR partially restored the activity of the mutant receptor. The Phe251Cys mutation occurs in the E1 region (amino acids 244e263) of the VDR LBD. The E1 region overlaps the C-terminal portion of helix H3, loop 3e4 and the N-terminal portion of helix H4. This structural motif is highly conserved throughout the nuclear receptor superfamily. A cluster of hydrophobic amino acids within the E1 region is critical to the three-dimensional folding and formation of the ligand-binding pocket [166]. At the center of this region is an invariant aromatic phenylalanine residue that corresponds to Phe251 in the
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VDR. Since Phe251 is in such a critical site in the LBD, replacing the aromatic amino acid phenylalanine with a small hydrophilic cysteine residue likely disrupts the ligand-binding pocket of the VDR and interferes with the fundamental conformation required for optimal function. From the crystallographic studies, Phe251 does not form a direct contact point with the bound ligand. Indeed, at 4 C normal ligand binding was observed. However, at elevated temperatures, the binding affinity was severely decreased suggesting that the Phe251Cys mutation disrupted the folding of the ligand-binding pocket [165]. A Val346Met mutation was identified in a Bedouin boy with HVDRR and alopecia [167]. Val346 is located in the inter-helical loop between helix H8 and H9 and may be important in RXR heterodimerization. The patient also had papular lesions on the scalp and face. Two brothers and one sister were also affected. The affected children also had mild hearing loss and secondary speech abnormalities. Although hearing loss had not been noted before in other HVDRR patients, progressive hearing loss was found in VDR knockout mice [168]. STRUCTURAL ANALYSIS OF VDR-RXR HETERODIMERIZATION MUTATIONS
Although the VDR-RXR dimer crystallographic studies have not been performed as of this writing, studies of RXRa, RAR-RXRa, and PPARg-RXRa, show that the dimer interface is formed from helix H9 and helix H10 and the interhelical loops between H7eH8 and H8eH9 [68]. The Arg391Cys mutation occurs in helix H10 [156]. The mutation lowers the binding affinity for RXR causing 1,25(OH)2D3 resistance. However, in transactivation assays, the 1,25(OH)2D3 resistance could be overcome by overexpression of RXR [156]. The Phe251Cys mutation reduces the affinity of the VDR for 1,25(OH)2D3 and alters RXR binding [165]. The Phe251Cys mutation is located in the E1 region in the interhelical loop between H3 and H4. This region of the VDR is not part of the dimer interface; however, the loop does appear to be positioned beneath the interhelical loop between H8eH9 and helix H9. The mutation apparently disrupts the formation of the dimer interface of the receptor and results in a decreased ability to heterodimerize with RXRa. The Gln259Pro mutation is also located in the E1 region in helix H4. The Gln259Pro mutant did not affect 1,25(OH)2D3 binding but exhibited an abnormality in forming a complex on VDREs in EMSA [142]. A study by Whitfield et al. [169] has shown that a Gln259Gly mutant VDR binds ligand normally but is defective in its ability to form a complex with RXR. They also showed that addition of increasing amounts of RXR
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could restore the transactivation capacity of the Gln259Gly mutant to near wild-type levels. These single amino acid changes demonstrate the interdependency of ligand binding and heterodimerization for transactivation. Mutations that Affect Coactivator Binding As noted above, the VDR also must recruit coactivator proteins for transcriptional activity. It is now clear from crystallographic studies of the VDR and other members of the steroid receptor superfamily that repositioning of helix H12 is an essential event that occurs as a consequence of ligand binding and is essential for transactivation [68]. The repositioning of helix 12 leads to the formation of the hydrophobic cleft critical for coactivator binding. Therefore disruption of this surface interface may cause hormone resistance and HVDRR. A study by Malloy et al. [41] examined the VDR in a young boy (F60) with HVDRR. The patient did not have alopecia. The patient’s fibroblasts exhibited normal ligand binding but the cells were totally resistant to 1,25(OH)2D3. A novel missense mutation was found in exon 9 that changed a glutamic acid to lysine at amino acid 420 (Glu420Lys) (Fig. 65.6). The Glu420Lys mutation is located in helix H12. The recreated Glu420Lys mutant VDR showed no defects in VDR-RXR heterodimerization or binding to VDREs. However, the mutation prevented the coactivators SRC-1 and DRIP205 from binding to the VDR. In transactivation assays, cotransfection of SRC-1 failed to restore transactivation by the mutant VDR. This case represented the first description of a naturally occurring mutation in the VDR that disrupts coactivator interactions and causes HVDRR [41]. We analyzed the effects of this mutation in some detail. The polar interactions that stabilize the repositioning of helix H12 involve a conserved salt bridge between Lys264 in helix H4 and Glu420 in helix H12 and a hydrogen bond between Ser235 in helix H3 and Thr415 in helix H12 [68]. The substitution of the negatively charged glutamic acid (Glu420) with a positively charged lysine residue (Lys420) would prevent the polar interaction with the positively charged lysine (Lys264) salt bridge partner. The charge clamp formed by Lys264 and Glu420 enables the VDR to recruit and bind coactivators through their LxxLL motifs. The Glu420Lys mutation prevents the correct repositioning of helix H12 after binding the ligand that disrupts coactivator binding and causes the hormone resistance seen in the patient.
Compound Heterozygous Mutations in the VDR There are several reports of patients with HVDRR with compound heterozygous mutations, i.e. a different
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mutation in each VDR allele. In these cases there is no consanguinity and the asymptomatic parents, each carrying a different heterozygous mutation in one allele of their VDR gene, unfortunately both pass the defective allele to their offspring. One patient (F62) with HVDRR and alopecia was heterozygous for a missense mutation in exon 8 that caused a Glu329Lys substitution (Fig. 65.6) [40]. The patient was also heterozygous for a mutation in exon 4 that deleted a single cytosine at nucleotide 366 (c.366delC) in the VDR cDNA (Fig. 65.5). The deletion of the cytosine resulted in a frameshift that led to a premature termination signal in the VDR mRNA that truncated most of the LBD. Although the effects of the Glu329Lys mutation on VDR function were not reported, the mutation occurs in helix H8 that is important in heterodimerization with RXR and would likely disrupt this activity. Thus, both VDR alleles were non-functional and resulted in HVDRR in this child. Compound heterozygous mutations were also identified in the VDR gene in a child (F68) with HVDRR, total alopecia, and early childhood-onset type 1 diabetes [170]. A missense mutation was found in exon 7 that caused a Leu263Arg substitution in helix H4. A second missense mutation was found in exon 9 that caused an Arg391Ser substitution in helix H10. As described above, an Arg391Cys mutation was previously shown to affect RXR heterodimerization thus it was likely that the arginine to serine substitution also affected heterodimerization [156]. Interestingly, the Leu263Arg mutant and the Arg391Ser mutant VDRs exhibited differential effects on 24-hydroxylase and RelB promoters. The 24hydroxylase responses were abolished in the Leu263Arg mutant but only partially altered in the Arg391Ser mutant. On the other hand, RelB responses were normal for the Leu263Arg mutant but the Arg391Ser mutant was defective in this response [170]. Compound heterozygous mutations were also found in the VDR gene in a young girl (F70) with HVDRR and alopecia [171]. One mutation changed the codon for arginine to a nonsense mutation at amino acid 30 (Arg30stop). The second mutation was an in-frame 3 bp deletion in exon 6. The 3 bp deletion removed the codon for lysine at amino acid 246 (6K246) but did not alter the reading frame. The patient’s fibroblasts expressed the VDR6K246 mutant protein but were unresponsive to 1,25(OH)2D3. The 6K246 mutation abolished heterodimerization with RXR and binding to coactivators.
Other Mutations in the VDR A young boy (F66) from Chile with HVDRR without alopecia was found to have a unique 5 bp deletion/ 8 bp insertion in exon 4 of the VDR gene [172]. The
mutation in helix H1 of the LBD deleted His141 and Tyr142 and inserted three amino acids in their place (Leu141, Trp142, and Ala143). The patient’s fibroblasts had no demonstrable [3H]1,25(OH)2D3 binding and were resistant to 1,25(OH)2D3 treatment. When the effects of the three individual mutations were analyzed only the insertion of Ala143 into the WT VDR disrupted VDR transactivation to the same extent observed with the natural mutation. A young Jamaican boy (F71) with HVDRR and patchy alopecia was found to have a 102 bp insertion/duplication in the VDR gene that introduced a premature stop (Tyr401stop) [173]. The mutation deleted a part of helix H11 and all of helix H12. The patient’s fibroblasts expressed the truncated VDR, but were resistant to 1,25(OH)2D3. The truncated VDR weakly bound [3H]1,25(OH)2D3 and was able to heterodimerize with RXR, bind to DNA and interact with the corepressor HR. However, the truncated VDR failed to bind coactivators and was transactivation defective.
HVDRR in Cats and Dogs There are several reports of HVDRR in animals. In one report, a 4-month-old male domestic shorthair cat was examined because of lethargy, vomiting, diarrhea, muscle tremors, and mydriasis. Laboratory evaluation revealed hypocalcemia, hypophosphatemia, and high serum 1,25(OH)2D and PTH levels. The cat was treated with oral calcium and calcitriol supplements. After 18 months of treatment, the cat was clinically normal. After the treatment was discontinued, the cat was able to maintain normal serum calcium levels [174]. A second report described the biochemical abnormalities and radiographic changes in a 4-month-old kitten with symptoms of HVDRR [175]. The cat was treated with calcium and vitamin D. The treatment failed to increase the serum calcium levels or reverse the lateral antebrachial bowing, lumbar spinal lordosis and costochondral beading. The cat was euthanized at 9 years of age as a result of refractory hip pain [176]. HVDRR was also diagnosed in a Pomeranian dog [177]. On physical examination, the dog exhibited lateral bowing of the antebrachium of both forelimbs and generalized non-pruritic partial alopecia. Serum biochemistry revealed marked hypocalcemia, hyperphosphatemia, and increased alkaline phosphatase. Although her alopecia was resolving, the dog’s coat was of poorer quality than that of her half-sibling. At 8 months of age, while receiving 1,25(OH)2D3, the dog’s serum 1,25(OH)2D3 level was very high (1017 pg/ml; normal 25e50 pg/ml) despite persistent hypocalcemia (total calcium 5.4 mg/dl; normal 8.9e11.4 mg/dl) indicative of HVDRR. Analysis of the VDR gene revealed a single base deletion in exon 4.
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THERAPY OF HVDRR
Sequencing of the VDR cDNA showed that the single guanine nucleotide deletion led to a frameshift that caused the amino acid sequence to diverge after Arg175. An additional 41 amino acids were in frame before a downstream termination signal occurred. The mutation deleted a major part of the LBD and caused the VDR to be non-functional. The dog was eventually treated with intravenous (IV) calcium gluconate. After initial stabilization, a neurologic examination revealed signs attributable to diffuse forebrain disease and hindlimb paresis. Spinal radiographs and computed tomography confirmed a fracture of T11 and showed accompanying spinal cord compression. Flaring of the ribs at the costochondral junctions (so-called rachitic rosary in people) was also readily apparent on radiographs. Given the severity of the dog’s spinal injury the decision was made to euthanize the animal. This was the first molecular description of HVDRR in a mammal other than humans.
HVDRR without Mutations in the VDR Since the initial description of HVDRR as a genetic disorder, mutations in the VDR gene were suspected as the likely cause of 1,25(OH)2D3 resistance. Although the VDR is the principal determinant of the 1,25(OH)2D3 action pathway, it is possible that target organ resistance to 1,25(OH)2D3 may result from mutations in other proteins that are essential to the transactivation process. The following cases highlight this possibility and are considered in more detail in Chapter 14. Hewison et al. [178] have described a case of HVDRR in which a mutation could not be found in the VDR. The patient (F48), a young girl of English descent, exhibited all of the hallmarks of HVDRR including alopecia. The patient’s fibroblasts expressed a normal-sized VDR mRNA and exhibited normal [3H]1,25(OH)2D3 binding. However, no induction of 24-hydroxylase activity was observed when the fibroblasts were treated with up to 1 mM 1,25(OH)2D3. Although the fibroblasts were clearly resistant to 1,25(OH)2D3, no mutations were found within the coding region of the VDR gene. The patient’s VDR mRNA was reverse transcribed and amplified by PCR and then expressed in CV-1 cells. In transactivation assays, the patient’s VDR exhibited a normal transactivation response to 1,25(OH)2D3. These results indicated that the patient’s VDR DNA sequence was normal. The authors suggested that the tissue resistance was not due to a defect in the VDR and that the hormone resistance causing HVDRR was most likely the result of a mutation in an essential protein that participates in the 1,25 (OH)2D3 action pathway. In a follow-up study of this interesting case, Chen et al. [179] proposed that the cause of 1,25(OH)2D3 resistance was due to the abnormal expression of a VDRE-
interacting hormone response element-binding protein. This protein is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that binds to double-stranded DNA and modulates RNA processing. In New World primates the hormone response elementbinding proteins have been shown to compete with the VDR for binding to VDREs located near regulated genes and are believed to cause target organ resistance for adrenal, gonadal, and vitamin D sterol/steroid hormones [180e185]. The fact that the patient had HVDRR without a mutation in the VDR highlights the importance of the complex machinery involved in VDR transactivation and that mutations in the hormone-signaling pathway may result in 1,25(OH)2D3 resistance (see Chapter 14). In Cauca, Colombia, more than 200 patients have been diagnosed with a disease that somewhat resembles HVDRR without alopecia [186]. The patients exhibit lower limb deformities due to rickets but are otherwise in good physical condition. Rickets limited to the lower extremities, as in these cases, has not been reported in other HVDRR families. The affected individuals have serum calcium levels that are within the normal range but their serum 1,25(OH)2D levels are unusually high, suggesting target-organ resistance. Fibroblasts from two of the most severely affected patients showed a low induction of 24-hydroxylase activity by 1,25(OH)2D3. However, no mutations were found in the VDR gene. Since the cause of vitamin D resistance in this instance was not due to mutations in the VDR and a functional response to 1,25(OH)2D3 was demonstrated, it has not been clearly ascertained whether this entity is a variant of HVDRR. The high prevalence of the disease in the population and the localized distribution of the rickets raise the possibility of an environmental cause. These cases support the concept that target organ resistance to 1,25(OH)2D3 may be due to mechanisms other than mutations in the VDR or overexpression of components such as an hnRNP that can inhibit 1,25(OH)2D3 signaling.
THERAPY OF HVDRR Vitamin D As detailed in the preceding sections, HVDRR is almost always caused by heterogeneous mutations in the VDR that result in partial or total resistance to 1,25(OH)2D3. Partially or totally inactive VDRs reduce calcium and phosphate absorption from the intestine into the circulation and result in hypocalcemia. Low serum calcium levels lead to secondary hyperparathyroidism that exacerbates the hypophosphatemia and cumulatively these effects result in decreased bone
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mineralization and cause rickets. In order to cure the rickets, calcium and phosphate levels must be normalized. A number of therapies using calcium and active vitamin D metabolites aimed at increasing the serum calcium levels have been tried and the responses have varied widely. For the most part, it was thought that patients with HVDRR without alopecia (usually LBD mutations with VDR present but with decreased affinity for calcitriol) were better responders to treatment with vitamin D preparations than those patients with alopecia [36]. In several of the earlier cases that were reported, patients without alopecia showed improvement both clinically and radiologically to the administration of pharmacological doses of vitamin D ranging from 5000 to 40 000 IU/day [12,13,93]; 20 to 200 mg/ day of 25(OH)D3; and 17e20 mg/day of 1,25(OH)2D3 [13]. Of the patients with HVDRR without alopecia, patient (F51) with the His305Gln mutation in the VDR LBD, a contact point for the 25-hydroxyl group of 1,25(OH)2D3, responded to 12.5 mg/day calcitriol [157,158]. The treatment overcame the low-affinitybinding defect and achieved adequate VDR occupancy to mediate normal 1,25(OH)2D3 responses. Patient (F4) with the Ile314Ser mutation in the VDR LBD was treated with 1 mg/day of vitamin D2 (40 000 IU) from age 2 to age 18 [93]. At age 20 following an uneventful pregnancy the patient developed hypocalcemia and was treated successfully with 50 mg/day of 25(OH)D3. On the other hand, the patient (F21) with the Arg274Leu mutation in the VDR LBD, a contact point for the 1ahydroxyl group of 1,25(OH)2D3, failed to respond to treatment with 600 000 IU vitamin D; up to 24 mg/ day of 1,25(OH)2D3 (calcitriol); and 12 mg/day 1a(OH)D3. The patient later died of pneumonia [24]. Fibroblasts from this patient exhibited no detectable [3H]1,25(OH)2D3 binding and were unresponsive to hormone treatment. However, the recreated Arg274Leu mutant VDR did exhibit transactivation activity at high doses of 1,25(OH)2D3 [143]. In general, HVDRR patients with alopecia are more resistant to treatment with vitamin D metabolites. However, some of these patients have been treated successfully using vitamin D. Two patients showed signs of improvement when given vitamin D or 1a-(OH)D3 [20,187] and one patient responded to 25(OH)D as well as 1a-(OH)D3 [21]. 1a-(OH)D3 and 1,25(OH)2D3 also were effective treatments in other cases [28,29,110,130,188] including patients with the Arg50Gln and Arg73Gln mutations [57,90]. Two siblings (F32), with the Glu152stop mutation, showed no increase in serum calcium during high-dose vitamin D treatment despite raising their circulating 1,25(OH)2D levels to more than 100 times the mean normal range. However, notwithstanding their low serum calcium concentrations, healing of rickets and suppression of
PTH was evident [189]. In one case, where vitamin D and 1,25(OH)2D3 therapies failed, the patient responded to oral phosphorous treatment [17]. The molecular cause of HVDRR in this case has not been elucidated. In many cases when patients fail to respond to 1,25(OH)2D3, intensive calcium therapy is used as described below.
Calcium Balsan et al. [190] first reported the most significant development in the treatment of HVDRR. In their insightful study, they used long-term IV calcium infusions to successfully treat a child with HVDRR who had failed prior treatments with large doses of vitamin D derivatives and/or oral calcium [21]. This novel therapy by-passed the calcium absorption defect in the intestine caused by the mutant VDR. The patient was infused with high IV doses of calcium during the nocturnal hours over a 9-month period. Clinical improvement including relief of bone pain was observed within the first 2 weeks of therapy. Within 7 months, the child gained both weight and height. Eventually, the serum calcium normalized, the secondary hyperparathyroidism was reversed, and the rickets ultimately resolved as assessed by X-ray and bone biopsy. However, when the IV infusions were discontinued the hypocalcemia and rickets returned. Several other groups have reported using IV calcium infusions as a therapy for HVDRR [23,191,192]. Weisman et al. [191] treated two patients with IV calcium and showed a decrease in their serum alkaline phosphatase activity and an increase in their serum calcium and phosphate over a 1-year period [191]. X-ray analysis showed resolution of the rickets with the appearance of normal mineralization of bone. In some cases, after radiological healing of the rickets has been achieved with IV calcium infusions, high-dose oral calcium therapy has been shown to be effective in maintaining normal serum calcium concentrations [192]. For those HVDRR children that do not respond to high-dose calcitriol, this two-step protocol is initiated at an early age. Much of the following description is based on the experience with the Israeli cohort of HVDRR cases (and one of the co-authors of this chapter, D.T.). The Israeli cohort is composed of the largest group of patients with defined VDR mutations that has been followed for many years. The experience to be described has been gleaned from 28 patients, belonging to four families, each with a different mutation. The patients currently range from newborn to 36 years of age. Their phenotypes and genotypes have been previously described [14,51]. One family (F11) has a Tyr295stop nonsense mutation in the LBD that truncates the VDR
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rendering it devoid of biological function [26,32]. Two families have missense mutations in VDR DBD. In one family (F31), a Gly33Asp mutation was identified near the tip of the first zinc finger [90,103]. In the other family (F49), an Arg80Gln mutation was identified in the base of the second zinc finger [31]. In the fourth family, the patient’s cultured skin fibroblasts failed to bind [3H]1,25(OH)2D3, and 1,25(OH)2D3 treatment failed to stimulate 24-hydroxylase activity. Molecular analysis of the VDR defect was not performed in this patient [192,193]. Based on this experience, the Israeli group feels that in children beyond infancy presenting with severe rickets and bone deformities, the only reliably effective therapeutic measure is long-term IV calcium infusion to heal the “hungry bones,” bypassing intestinal calcium absorption. The infusions require establishing a central IV port to allow continuous infusions over prolonged periods. In the hands of the Israeli group the following plan has proved successful. The initial infusion solution of 100 mg of elemental calcium (calcium gluconogalactogluconate, Sandoz Laboratories, Basel, Switzerland) in 500 ml isotonic sodium chloride is increased gradually to doses of 500 to 1000 mg of elemental calcium. The infusion is administered continually, 24 hours daily during the first month, and gradually reduced to 12 hours daily given overnight. The dosage must be adjusted frequently to avoid severe hypercalciuria, i.e. keep the ratio of urine calcium to urine creatinine less than 3 (Ucal/Ucrea <0.3), and to achieve a serum calcium concentration of 2.1 to 2.5 mmol/l (8.4 to 10 mg/dl). Cardiac monitoring is important to prevent bradycardia. Clinical improvement is expected within a week of starting IV therapy, at which time bone pain recedes. Some young patients started to walk for the first time within a week of starting IV therapy. Once the “hungry bones” are corrected, transition to therapy with high-dose oral calcium can be successful and is recommended. The Israeli group advises a large dose of 5 to 6 grams per meter square body surface of elemental calcium. This dose has been shown to be required to maintain normal calcium and phosphorus levels, thus preventing rickets and promoting normal linear growth. The detailed use of IV calcium in the pediatric population has also been reported by Malloy et al. [138], Forghani et al. [149], and Ma et al. [152]. Delivery of calcium through a central vein, over an extended period of time, has been associated with significant morbidity. During the initial week of therapy, cardiac arrhythmias, mostly bradycardia, have been observed. Reduction in the infusion rate has been shown to eliminate the bradycardia. Episodes of septicemia occur frequently and require hospitalization, cessation of calcium therapy, removal of the central catheter, and IV antibiotics [138]. Whether this problem
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is due to decreased ability of HVDRR patients to fight infection or is a problem inherent with long-term IV infusions, especially in the home setting, is not clear [138]. The Israeli group has recently found that oral calcium in high doses may be successful as initial therapy and may allow avoidance of ever requiring IV calcium therapy. The dose required is 5 to 6 grams per meter square body surface of elemental calcium. If successful in restoring normocalcemia and reversing secondary hyperparathyroidism, IV infusions with all of their associated problems may be avoided. On the other hand, if the patient has required IV calcium to replenish calcium deficits, a transition to oral calcium should be attempted. After IV calcium is transitioned to oral calcium, substantial oral doses of calcium are required. Treatment is recommended with the same large dose of 5 to 6 grams per meter square body surface of elemental calcium to maintain normal calcium and phosphorus levels, thus preventing return of rickets and promoting normal linear growth. Others also have used administration of oral calcium salts to restore serum calcium as a therapy for HVDRR patients. In a study by Sakati et al. [136], a patient (F23) who failed to respond to calciferols received 3e4 grams of elemental calcium orally per day and showed clinical improvement during 4 months of therapy. The patient cells were later shown to be totally resistant to 1,25(OH)2D3 due to a Gly46Asp mutation in the VDR DBD [135]. During and after puberty and into adulthood many HVDRR patients are able to maintain normal serum calcium levels with more modest oral calcium supplements or, in some cases, even without calcium supplements. Many have near normal PTH levels with normal bone mineral density although some continue to show elevated serum 1,25(OH)2D levels suggesting residual target organ resistance. This phenomenon of spontaneous healing in the presence of non-functioning VDR is not understood. We have speculated previously [3,4] that once the skeleton is fully formed the demand for calcium decreases and this lower requirement can possibly be met by oral calcium absorption by vitaminD-independent intestinal pathways. Patients who start treatment during early childhood and continue therapy and medical follow-up can achieve normal adult height; whereas patients who start treatment during late childhood and early puberty, typically only reach very short final heights (3, 4.42, and 5.5 Ht SD score, respectively). Although many vitamin D actions have been described in bone cells (see Chapter 16) and the parathyroid glands (Chapter 27), the administration of calcium alone is sufficient to reverse the rickets and secondary hyperparathyroidism. The correction of bone mineralization suggests that 1,25(OH)2D action is not essential
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for normalization of rickets and osteomalacia as along as mineral supply is normalized. The correction of the hyperparathyroidism by IV calcium without additional phosphate and return of phosphate to normal suggests that the hypophosphatemia was due to the secondary hyperparathyroidism and that 1,25(OH)2D action is not required to suppress PTH levels to normal. In the VDR knockout mice, a high-calcium rescue diet also corrects the bone deformities although subtle defects may remain. Only the alopecia is not reversed in either the human disease or the mouse models. These findings suggest that the critical defect in HVDRR is defective intestinal absorption of calcium and that once normal serum concentrations of calcium are restored, PTH and phosphate return to normal and the rickets heals even without VDR action. Beyond effects on bone, the HVDRR cases raise interesting questions about vitamin D actions throughout the body. The medical literature (as described in this volume) is flooded with reports of the various effects of calcitriol on multiple tissues, including the heart, blood pressure, the immune system, endocrine glands, etc. Thus, it is very intriguing and confounding that successfully treated HVDRR patients whose calcium and phosphate are normalized do not suffer from problems in any of these tissues. With calcium therapy, the HVDRR children appear to grow and mature normally, their hearts function well, as does their immune systems and they do not appear to have hypertension. Immunologically it has been shown that other than slightly impaired phagocytosis and Candida killing, the immune system functions quite well in the absence of VDR action [53]. Endocrine glands, including pancreatic b-cells, respond normally to routinely used stimuli, provided that serum calcium is normal [50]. Two male patients in the Israeli cohort have married and fathered children. All females have normal and regular menses, none have married yet. So far there is no apparent increased risk of cancer. Thus although the number of HVDRR patients is very small, and most are still relatively young, their lack of increased risk for many of the diseases discussed in this book is of great interest and major consternation. The main unresolved medical problem of HVDRR patients is persistent alopecia despite normalization of serum biochemistry tests and rickets. The number of hair follicles seems to be normal. However, hair does not grow during childhood and the hair follicles are empty. In some cases papular lesions develop on the face and shoulders, but usually not on the scalp. Both the clinical presentation and the hair histology are similar to those found in the autosomal recessive disease total alopecia known as “atrichia with papular lesions” [194] due to mutations in the hairless (hr) gene [206,207]. The entire Israeli cohort of HVDRR patients maintains a normal lifestyle. All have normal liver and
kidney functions, none have diabetes, or require treatment with any medication other than calcium. The alopecia is troublesome and is sometimes made more acceptable using a wig.
Prenatal Diagnosis In affected families, HVDRR can be diagnosed during pregnancy by chorionic villus sampling (CVS) or amniocentesis. In some cases defects in the unborn child’s VDR have been detected using [3H]1,25(OH)2D3binding assays and 1,25(OH)2D3 induction of 24hydroxylase activity in cultured cells obtained from CVS or amniotic fluid [193]. In other cases, a prenatal diagnosis of HVDRR was determined by examining DNA from chorionic villus samples for RFLPs generated by the known mutation in the VDR gene present in other family members [195]. Early diagnosis prompts timely treatment, before bones become undermineralized and severe rickets develops.
Spontaneous Healing An interesting dilemma regarding HVDRR was that several patients have had a spontaneous improvement in their rickets but not their alopecia [27,28,77]. Spontaneous healing of rickets usually happens between 7 and 15 years of age and was not necessarily associated with the time of puberty. Sometimes the spontaneous recovery occurs after the patient has undergone a relatively ineffective long-term treatment with vitamin D metabolites and mineral replacement. The healing process arises spontaneously and does not appear to be related to the treatment. In some patients, spontaneous improvement occurred after treatment was discontinued [28]. The patients appear to remain eucalcemic without therapy and show no evidence of osteomalacia or rickets. Interestingly, cultured fibroblasts obtained from a skin biopsy of a patient taken after spontaneously healing of the rickets had occurred were still resistant to 1,25(OH)2D3 [28]. Spontaneous improvement has been observed in patients (F18) [27,77] with the Tyr295stop mutation [31,91] and in patients (F19) [28] with the Arg73Gln mutation [90]. Despite the patient’s improvement in their hypocalcemia and rickets, the alopecia remained [27,28,77]. It is not uncommon for children to “outgrow” genetic diseases and, perhaps after skeletal growth has been completed, the body can more easily compensate for the defective VDR gene by other mechanisms. Many patients can be maintained on oral calcium; however, occasionally some patients fall back to hypocalcemia due to decreased compliance or lack of tolerance to the high doses of calcium. Intravenous calcium
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therapy is then reinstituted. After IV therapy normalizes the rickets and metabolic abnormalities, the patients can be switched to oral calcium. The hypocalcemia and secondary hyperparathyroidism can often be controlled with high-dose oral calcium.
Future Therapy using Vitamin D Analogs Vitamin D analogs have been proposed as a potential therapy for patients with HVDRR especially those with mutations in the VDR LBD [196e199]. The use of vitamin D analogs is based on the rationale that they bind to the VDR at different amino acids than 1,25(OH)2D3. Using cultured fibroblasts from patients and in vitro transactivation assays, the vitamin D analogs 20-epi-1,25(OH)2D3 and 1b-hydroxymethyl-3epi-16-ene-26a,27a-bishomo-25(OH)D3 were shown to partially or completely restore the responsiveness to the Arg274Leu and His305Gln mutant VDRs but were less effective in activating the Ph2251Cys mutant [196]. These results suggest that amino acids that are involved in ligand binding rather than amino acids that are involved in heterodimerization or coactivator binding are more likely to respond to analogs. Future therapy using vitamin D analogs could be based on a rationale drug design for individual patients with specific types of VDR mutations.
ALOPECIA The mechanism of how the VDR regulates hair growth is still unclear. Insights gained from the molecular analysis of the VDR from HVDRR patients with and without alopecia along with studies in VDR knockout mice has revealed a number of interesting findings concerning the role of the VDR and vitamin D in regulating hair growth (see Chapter 30). First, alopecia is not found in patients with 1a-hydroxylase deficiency (VDDR I) and other forms of vitamin D deficiency. In the 1a-hydroxylase knockout mouse model, abnormalities develop in skeletal, reproductive, and immune function [200]. However, the 1a-hydroxylase knockout mice do not develop alopecia. These findings suggest that 1,25(OH)2D3 itself is not required for hair development. On the other hand, VDR knockout mice develop alopecia indicating that the VDR itself (unliganded) is essential for hair growth [54,55]. Furthermore, targeting of the WT VDR to keratinocytes of the VDR knockout mouse prevents alopecia [201]. These findings raise the question of how vitamin D and the VDR are involved in regulating hair growth. Patients with DBD mutations and premature stop mutations all have alopecia and are totally hormone-resistant. Alopecia remains unchanged even after patients
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undergo successful therapy or that shows spontaneous improvement in rickets. The patients that did not develop alopecia all had missense mutations in the VDR LBD. Some of these mutations affect ligand binding (Arg274Leu, His305Gln, Ile314Ser, and Trp286Arg). Three of these amino acids, Arg274, His305, Trp286, are contact points for 1,25(OH)2D3. Since the patients with the His305Gln and Ile314Ser mutations were somewhat responsive to vitamin D therapy, it is reasonable to speculate that the limited VDR function may have prevented the development of alopecia after birth. On the other hand, the patients with the Arg274Leu and Trp286Arg mutations that severely diminished or abolished [3H]1,25(OH)2D3 binding were totally resistant to 1,25(OH)2D3 yet did not have alopecia. In contrast, patients with LBD mutations that reduced heterodimerization with RXRa (Phe251Cys, Gln259Pro, and Arg391Cys), but had little or no effect on ligand binding, alopecia was present. Although these mutations caused 1,25(OH)2D3 resistance in the patient, the function of the mutant VDRs could be restored in vitro by supra-physiological doses of the hormone and addition of excess RXR. Perhaps most interestingly was the patient (F60) with the Glu420Lys mutation that prevents coactivator binding but not ligand binding or RXR heterodimerization [41]. The patient did not have alopecia yet was clearly resistant to high doses of hormone. Also, when ligand-binding defective or coactivator-binding defective mutant VDRs were specifically expressed in keratinocytes in VDR knockout mice that have alopecia, hair growth was fully or partially restored [42]. Based on these findings one can conclude that mutations that affect DNA binding, VDR-RXR heterodimerization or that truncate the LBD are linked to alopecia while mutations that affect ligand binding or coactivator binding are not. This suggests that VDRRXR heterodimerization and DNA binding are critical for VDR function in hair development. These findings also suggest that ligand binding and coactivator binding are not essential functions of the VDR for hair growth and/or to prevent alopecia. A role for RXR is clearly demonstrated since targeted inactivation of RXRa in keratinocytes also causes alopecia [202]. The alopecia associated with HVDRR is clinically and pathologically indistinguishable from the generalized atrichia with papules found in patients with mutations in the hr gene [39,40,203,204]. The hr gene is expressed in many tissues especially in the skin and brain [205]. The hr gene product, HR, acts as a corepressor and directly interacts with the VDR and suppresses 1,25(OH)2D3-mediated transactivation [206e208]. Like the VDR, HR is a zinc-finger protein suggesting that it interacts with DNA. It has been hypothesized that the role of the VDR in the hair cycle is to repress the expression of a gene(s) in a ligand-independent manner
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[5,41,42,172,206,208]. The ligand-independent activity requires that the VDR heterodimerize with RXR and bind to DNA even if it failed to activate gene transcription [5,41]. The corepressor actions of HR may also be required in order for the unliganded VDR to repress gene transcription during the hair cycle. Mutations in the VDR that disrupt the ability of the unliganded VDR to suppress gene transcription are hypothesized to lead to the derepression of a gene(s) whose product, when expressed inappropriately, disrupts the hair cycle that ultimately leads to alopecia [5,41,42,172,206,208]. Potential candidates include inhibitors of the Wnt signaling pathway [209,210] and PTHrP. Overexpression of PTHrP causes alopecia indicating its involvement in the regulation of the hair cycle [211,212]. Since the VDR is a negative regulator of the PTHrP gene expression [213], loss of VDR regulation of the PTHrP gene due to mutations in the VDR may lead to the development of alopecia in HVDRR patients. The data on the VDR mutations combined with the findings in the 1a-hydroxylase knockout mouse model suggest that the role of the VDR in the hair cycle is to repress the expression of some gene(s) in a ligand-independent manner. This activity requires RXR heterodimerization and DNA binding but not interaction with coactivators. HR may also be required as a co-repressor for this negative regulatory activity of the VDR. The VDR is a negative regulator of a number of genes and the loss of a negative regulatory activity by the unliganded VDR could potentially lead to the derepression of those genes that could ultimately lead to alopecia.
CONCLUDING REMARKS HVDRR is a rare recessive genetic disorder caused by heterogeneous mutations in the VDR that result in endorgan resistance to 1,25(OH)2D3 action. The major manifestation of the defective VDR on the vitamin D endocrine system is to decrease intestinal calcium and phosphate absorption that results in decreased bone mineralization and rickets. Secondary hyperparathyroidism results as a consequence of the low serum calcium. The classical role of 1,25(OH)2D is to regulate mineral homeostasis, achieved through its coordinated actions on intestine, kidney, bone, and parathyroid gland [214,215]. The VDR is also expressed in a wide variety of tissues, including kidney, skin, liver, pancreas, muscle, breast, prostate, adrenal, thyroid, and cells of mesenchymal or hematopoietic origin [37,38,45,46, 94,216,217]. From the ubiquitous nature of the VDR it appears that the role of the VDR in cellular function is not homeostatic but rather pleiotropic. The expanded scope of vitamin D actions includes stimulation of differentiation, inhibition of cell proliferation and modulation
of the immune response [45,46,49,217]. In addition, the regulation of cellular proliferation and differentiation by 1,25(OH)2D is a common feature in many tissues examined and it is likely that this regulatory feature is a fundamental component of all biological responses to 1,25(OH)2D. Notwithstanding the complexity and diversity of biological responses elicited by 1,25(OH)2D, the profound skeletal abnormalities demonstrated in patients with HVDRR emphasizes the fundamental and essential role of 1,25(OH)2D in calcium homeostasis. It is interesting to note that despite the many pleiotropic processes regulated by 1,25(OH)2D3, children with HVDRR exhibit only symptoms that relate to their calcium deficiency and/or alopecia. After normalization of calcium they appear normal in all respects except for the alopecia. Analysis of HVDRR patients provides many interesting insights into vitamin D physiology and the role of the VDR in mediating 1,25(OH)2D3 action. Since 1978, more than 80 families with HVDRR have been reported (Table 65.3). A number of cases have been examined for mutations in the VDR. However, since some of these earlier cases of HVDRR presented late in life, it is possible that they may have been due to other conditions unrelated to genetic defects in the VDR or to some form of acquired resistance to 1,25(OH)2D3. Presently, 40 unique mutations have been identified in the VDR gene. Eleven missense mutations have been identified in the VDR DBD. All of the DBD mutations prevent the VDR from binding to DNA causing total 1,25(OH)2D3 resistance even though 1,25(OH)2D3 ligand binding is normal. Fifteen missense mutations have been identified in the LBD. These mutations disrupt ligand binding, VDR-RXR heterodimerization, or modify coactivator-binding sites. They result in partial or total hormone resistance. In addition, five nonsense mutations, four splice site mutations and three deletions have been identified that truncate the VDR and cause total 1,25(OH)2D3 resistance. One unique deletion/ substitution and one unique insertion/duplication have also been identified as the molecular basis for HVDRR. The Israeli group has recently emphasized that starting therapy with very high-dose oral calcium may be sufficient to treat many cases of HVDRR precluding the requirement for IV infusions. If IV infusions are required, transition to oral calcium replacement should be considered at an early time-point. The successful use of calcium therapy, whether oral or IV, raises interesting questions about the role of vitamin D in bone homeostasis. Correction of hypocalcemia and associated secondary hyperparathyroidism leads to healing of the rickets as assessed by X-ray and bone biopsy. However, careful analysis of bone histomorphometry has not been accomplished in HVDRR cases. The bone biopsy data in the VDR knockout mice is described in Chapter 33.
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Thus, although there are many well-defined actions of vitamin D on osteoblasts, the response to normalization of serum calcium and phosphate suggests that 1,25 (OH)2D3 action on osteoblasts is not essential to form bone and heal rickets although the bone may not be completely normal. The implication is that 1,25 (OH)2D3 action on bone is mainly due to its effects on intestinal mineral absorption to provide calcium and phosphate for bone formation. The same conclusion was reached by Underwood and DeLuca [218] who showed that rickets could be prevented in severely vitamin-D-deficient rats by calcium and phosphate infusions. Also, normalizing serum calcium by oral or IV infusion is sufficient to suppress PTH overproduction in HVDRR children and correct the hypophosphatemia. This suggests that the hypophosphatemia in these patients is mainly the result of secondary hyperparathyroidism and not inadequate intestinal phosphate absorption. The mechanism for the spontaneous improvement exhibited by some HVDRR children as they get older is an interesting dilemma. One hypothesis that explains the normalization of the 1,25(OH)2D3 endocrine system in the face of an inactive VDR is that some other transcription factor substitutes for the defective vitamin D system. Possibly RAR, RXR, or TR can substitute for a non-functional VDR and activate the appropriate target genes to reverse the hypocalcemia and restore the bones to normal. In this context, Whitfield et al. [156] have shown in vitro that addition of RXR can rescue mutant VDR with defects in the heterodimerization domain and restore hormone responsiveness. Another possibility is that a diet sufficient in calcium may bypass the need for vitamin D action once the demand for calcium for bone growth is diminished at older age. Finally, the lack of any disease in the HVDRR young adults after calcium has been restored to normal raises interesting questions about the role of the VDR and vitamin D action in the multiple areas throughout the body being studied and considered as having important vitamin D actions. In conclusion, the biochemical and genetic analysis of the VDR in HVDRR patients has yielded important insights into the structure and function of the receptor in mediating 1,25(OH)2D3 action. Similarly, studies of the affected children with HVDRR continue to provide further insight into the biological role of 1,25(OH)2D3 in vivo. However, the findings that many young adults with HVDRR, essentially a human VDR knockout, are functioning normally without apparent abnormality or disease raises important and difficult questions about vitamin D actions throughout the body. A concerted investigative approach of HVDRR at the clinical, cellular, and molecular level has proven exceedingly valuable in understanding the mechanism of action of
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1,25(OH)2D3 and improving the diagnostic and clinical management of this rare genetic disease.
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[192] Z. Hochberg, D. Tiosano, L. Even, Calcium therapy for calcitriol-resistant rickets, J. Pediatr. 121 (1992) 803e808. [193] Y. Weisman, N. Jaccard, C. Legum, Z. Spirer, G. Yedwab, L. Even, et al., Prenatal diagnosis of vitamin D-dependent rickets, type II: response to 1,25-dihydroxyvitamin D in amniotic fluid cells and fetal tissues, J. Clin. Endocrinol. Metab. 71 (1990) 937e943. [194] W. Ahmad, A.D. Irvine, H. Lam, C. Buckley, E.A. Bingham, A.A. Panteleyev, et al., A missense mutation in the zinc-finger domain of the human hairless gene underlies congenital atrichia in a family of Irish travellers, Am. J. Hum. Genet. 63 (1998) 984e991. [195] Y. Weisman, P.J. Malloy, A.V. Krishnan, N. Jaccard, D. Feldman, Z. Hochberg Prenatal diagnosis of calcitriol resistant Rickets (CRR) by 1,25(OH)2D3 binding, 24-hydroxylase induction and RFLP analysis. Ninth workshop on Vitamin D, Orlando, 1994 p. 106. [196] S.A. Gardezi, C. Nguyen, P.J. Malloy, G.H. Posner, D. Feldman, S. Peleg, A rationale for treatment of hereditary vitamin Dresistant rickets with analogs of 1 alpha,25-dihydroxyvitamin D3, J. Biol. Chem. 276 (2001) 29148e29156. [197] S.L. Swann, J. Bergh, M.C. Farach-Carson, C.A. Ocasio, J.T. Koh, Structure-based design of selective agonists for a rickets-associated mutant of the vitamin d receptor, J. Am. Chem. Soc. 124 (2002) 13795e13805. [198] S.L. Swann, J.J. Bergh, M.C. Farach-Carson, J.T. Koh, Rational design of vitamin D3 analogues which selectively restore activity to a vitamin D receptor mutant associated with rickets, Org. Lett. 4 (2002) 3863e3866. [199] A. Kittaka, M. Kurihara, S. Peleg, Y. Suhara, H. Takayama, 2Alpha-(3-hydroxypropyl)- and 2alpha-(3-hydroxypropoxy)1alpha,25-dihydroxyvitamin D(3) accessible to vitamin D receptor mutant related to hereditary vitamin D-resistant rickets, Chem. Pharm. Bull. (Tokyo) 51 (2003) 357e358. [200] D.K. Panda, D. Miao, M.L. Tremblay, J. Sirois, R. Farookhi, G.N. Hendy, et al., Targeted ablation of the 25-hydroxyvitamin D 1alpha-hydroxylase enzyme: evidence for skeletal, reproductive, and immune dysfunction, Proc. Natl. Acad. Sci. USA 98 (2001) 7498e7503. [201] C.H. Chen, Y. Sakai, M.B. Demay, Targeting expression of the human vitamin D receptor to the keratinocytes of vitamin D receptor null mice prevents alopecia, Endocrinology 142 (2001) 5386e5389. [202] M. Li, H. Chiba, X. Warot, N. Messaddeq, C. Gerard, P. Chambon, et al., RXR-alpha ablation in skin keratinocytes results in alopecia and epidermal alterations, Development 128 (2001) 675e688. [203] W. Ahmad, M. Faiyaz ul Haque, V. Brancolini, H.C. Tsou, S. ul Haque, H. Lam, et al., Alopecia universalis associated with a mutation in the human hairless gene, Science 279 (1998) 720e724. [204] A. Zlotogorski, Z. Hochberg, P. Mirmirani, A. Metzker, D. BenAmitai, A. Martinez-Mir, et al., Clinical and pathologic correlations in genetically distinct forms of atrichia, Arch. Dermatol. 139 (2003) 1591e1596. [205] S. Cichon, M. Anker, I.R. Vogt, H. Rohleder, M. Putzstuck, A. Hillmer, et al., Cloning, genomic organization, alternative transcripts and mutational analysis of the gene responsible for autosomal recessive universal congenital alopecia, Hum. Mol. Genet. 7 (1998) 1671e1679. [206] J.C. Hsieh, J.M. Sisk, P.W. Jurutka, C.A. Haussler, S.A. Slater, M.R. Haussler, et al., Physical and functional interaction between the vitamin D receptor and hairless corepressor, two proteins required for hair cycling, J. Biol. Chem. 278 (2003) 38665e38674.
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65. HEREDITARY 1,25-DIHYDROXYVITAMIN-D-RESISTANT RICKETS
[207] P.J. Malloy, J. Wang, K. Jensen, D. Feldman, Modulation of vitamin d receptor activity by the corepressor hairless: differential effects of hairless isoforms, Endocrinology 150 (2009) 4950e4957. [208] J. Wang, P.J. Malloy, D. Feldman, Interactions of the vitamin D receptor with the corepressor hairless: analysis of hairless mutants in atrichia with papular lesions, J. Biol. Chem. 282 (2007) 25231e25239. [209] G.M. Beaudoin 3rd, J.M. Sisk, P.A. Coulombe, C.C. Thompson, Hairless triggers reactivation of hair growth by promoting Wnt signaling, Proc. Natl. Acad. Sci. USA 102 (2005) 14653e14658. [210] C.C. Thompson, J.M. Sisk, G.M. Beaudoin 3rd, Hairless and Wnt signaling: allies in epithelial stem cell differentiation, Cell Cycle 5 (2006) 1913e1917. [211] J.J. Wysolmerski, A.E. Broadus, J. Zhou, E. Fuchs, L.M. Milstone, W.M. Philbrick, Overexpression of parathyroid hormone-related protein in the skin of transgenic mice interferes with hair follicle development, Proc. Natl. Acad. Sci. USA 91 (1994) 1133e1137. [212] Y.M. Cho, G.L. Woodard, M. Dunbar, T. Gocken, J.A. Jimenez, J. Foley, Hair-cycle-dependent expression of parathyroid hormone-related protein and its type I receptor: evidence for regulation at the anagen to catagen transition, J. Invest. Dermatol. 120 (2003) 715e727. [213] K. Ikeda, C. Lu, E.C. Weir, M. Mangin, A.E. Broadus, Transcriptional regulation of the parathyroid hormone-related peptide gene by glucocorticoids and vitamin D in a human C-cell line, J. Biol. Chem. 264 (1989) 15743e15746.
[214] M.R. Haussler, T.A. McCain, Basic and clinical concepts related to vitamin D metabolism and action, N. Engl. J. Med. 297 (1977) 1041e1050. [215] H.F. DeLuca, The vitamin D system in the regulation of calcium and phosphorus metabolism, Nutr. Rev. 37 (1979) 161e193. [216] T.L. Clemens, K.P. Garrett, X.Y. Zhou, J.W. Pike, M.R. Haussler, D.W. Dempster, Immunocytochemical localization of the 1,25dihydroxyvitamin D3 receptor in target cells, Endocrinology 122 (1988) 1224e1230. [217] R. Skowronski, D. Peehl, D. Feldman, Vitamin D and prostate cancer: 1,25-dihydroxyvitamin D3 receptors and actions in prostate cancer cell lines, Endocrinology 132 (1993) 1952e1960. [218] J.L. Underwood, H.F. DeLuca, Vitamin D is not directly necessary for bone growth and mineralization, Am. J. Physiol. 246 (1984) E493e498. [219] K. Miyamoto, R.A. Kesterson, H. Yamamoto, Y. Taketani, E. Nishiwaki, S. Tatsumi, et al., Structural organization of the human vitamin D receptor chromosomal gene and its promoter, Mol. Endocrinol. 11 (1997) 1165e1179. [220] S. Beer, M. Tieder, D. Kohelet, O.A. Liberman, E. Vure, G. BarJoseph, et al., Vitamin D resistant rickets with alopecia: a form of end organ resistance to 1,25 dihydroxy vitamin D, Clin. Endocrinol. (Oxf.) 14 (1981) 395e402. [221] J.S. Adams, M.A. Gacad, F.R. Singer, Specific internalization and action of 1,25-dihydroxyvitamin D3 in cultured dermal fibroblasts from patients with X-linked hypophosphatemia, J. Clin. Endocrinol. Metab. 59 (1984) 556e560.
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C H A P T E R
66 Glucocorticoids and Vitamin D Philip Sambrook University of Sydney, Sydney, NSW, Australia
MECHANISMS OF GLUCOCORTICOID OSTEOPOROSIS Glucocorticoid-induced osteoporosis (GIO) is the most common cause of secondary osteoporosis, and bone loss associated with therapeutic use of exogenous glucocorticoids or endogenous production in Cushing’s syndrome can occur rapidly leading to fracture and disability. This chapter provides an overview of the current understanding of glucocorticoid effects on the skeleton especially with respect to vitamin D metabolism. The chapter also includes a review of steroid hormone receptor expression in bone cells with specific reference to glucocorticoid and vitamin D hormones and their interactions. Clinical aspects of GIO and the role of vitamin D metabolites compared to other therapeutic agents in GIO will also be discussed. Bone loss resulting in fractures from long-term glucocorticoid therapy is a common clinical problem. The deleterious effects of glucocorticoids result primarily from
direct effects on all three bone cells, osteoblasts and their progenitors, osteocytes and osteoclasts. Glucocorticoids have direct inhibitory effects on osteoblastogenesis, as well as stimulating osteoblast and osteocyte apoptosis (leading to reduced bone remodeling and diminished repair of microdamage in bone) [1]. The pro-apoptotic effect of glucocorticoids on osteocytes appears to be initiated by detachment of their cellular processes from the lacunar or canalicular walls as a result of disengagement of integrins from proteins of the extracellular matrix [2]. Glucocorticoid effects on bone resorption include increased production of macrophage colony-stimulating factor and receptor activator of nuclear factor KB ligand (RANKL) by osteoblasts and downregulation of osteoprotegerin, resulting in increased osteoclastogenesis and prolongation of osteoclast lifespan [3]. Glucocorticoids have other effects on calcium metabolism (Fig. 66.1) that indirectly increase the risk of GIO including (1) antagonizing gonadal function and inhibiting the osteoanabolic action of sex steroids; 66.1 Mechanisms induced osteoporosis.
FIGURE
Glucocorticoids
Osteoblast Proliferation Apoptosis
Adrenal & Testis Androgens
RANKL OPG
GIT
of
glucocorticoid-
Kidney
Ca Absorption
Ca Reabsorption
Parathyroid PTH
BONE FORMATION
BONE RESORPTION
BONE LOSS
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10066-6
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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66. GLUCOCORTICOIDS AND VITAMIN D
(2) increasing renal excretion and reducing intestinal absorption of calcium leading to negative calcium balance that can promote secondary hyperparathyroidism and (3) effects on vitamin D metabolism [2,4] discussed in more detail later. 11b-Hydroxysteroid dehydrogenases are known to be key regulators of glucocorticoid pre-receptor metabolism. 11b-HSD1 facilitates the regeneration of biologically active cortisol and corticosterone by its oxidoreductase (11b-reductase) action. Through 11b-dehydrogenation, the same enzyme can affect the reverse reaction, to inactive forms. 11b-HSD2 has dehydrogenase activity only, that is it unidirectionally catalyses the conversion of active glucocorticoids to their inactive metabolites, namely, cortisone and 11-dehydrocorticosterone. The balance between 11b-HSD type 1 and type 2 appears to modulate intracellular glucocorticoid concentrations and sensitivity to exogenous glucocorticoids and have been associated with phenotypic and functional cell changes [5]. It has been reported that 1a,25-dihydroxyvitamin D can stimulate 11b-HSD1 expression and thereby promote the conversion of the glucocorticoid precursor cortisone to cortisol [6]. 1a,25-Dihydroxyvitamin D ([1,25(OH)2D) stimulation of corticosteroid synthesis via the 11b-HSD has been reported to provide an indirect positive feedback on the nuclear vitamin D receptor (VDR) [7].
STEROID RECEPTORS AND ACTIONS IN BONE Receptors Steroid hormones including glucocorticoids and vitamin D all act via structurally homologous nuclear receptors that form part of the steroid/thyroid receptor superfamily. The action of all these hormones is mediated by hormone binding to nuclear receptors, which act as ligand-dependent transcription factors to either activate or repress target gene expression [8,9]. By this classic genomic mechanism, lipophilic glucocorticoids diffuse across the cell membrane, attach to the cytosolic GR and after dimerization, the GR binds to conserved sequence motifs (glucocorticoid response elements or GREs) in the promoter region of glucocorticoid-responsive genes to positively or negatively regulate specific gene transcription [9,10]. Activation of glucocorticoid-responsive genes can also occur via an interaction between the DNA-bound GR and transcriptional coactivator molecules (such as cyclic AMP response element binding protein), which have intrinsic histone acetyltransferase activity and cause acetylation of core histones. It is also recognized that some biological activities of glucocorticoids may be mediated via other
transcription factors, such as AP-1 and NFkB, independent of GR binding to DNA but dependent upon interaction with these factors [10]. Similarly the diverse biological activities of vitamin D, which include effects on bone, cancer and immune cells, are mediated by binding of the ligand, calcitriol or 1,25(OH)2D3, to the ubiquitously expressed VDR. The VDR functions as a heterodimer with retinoid X receptors (RXR) to bind vitamin D response elements (VDREs) within gene promoters and interacts with a number of cofactors that are critical for transcriptional activation (VDR action is discussed in detail in Section II of this volume). It appears that both GR and VDR share a large number of co-activators and co-repressors, raising the possibility that shared components for certain functions could exert a limiting effect. In humans, alternative splicing of GR mRNA produces two similar isoforms, GRa and GRb. However, GRb does not bind ligand and, although it may act as a dominant negative regulator of GRa activity, most studies consequently have focused on the GRa isoform [8,9]. In terms of glucocorticoid effects on bone cells, cytolosolic-binding studies have shown specific glucocorticoid-binding sites predominantly in cells of osteoblastic lineage. This glucocorticoid binding occurs in animal and human osteoblasts and human osteosarcoma cells [11e18]. The effects of glucocorticoid on bone cells appear species specific. For example in rats, glucocorticoids increase differentiation of osteoblasts and osteoblast progenitors [19e21]. The increase in osteoblast differentiation is associated with induction of osteoblast marker genes such as alkaline phosphatase, osteocalcin, osteopontin, and bone sialoprotein [22e24]. In mice, however, the principal effect of glucocorticoids is to stimulate bone resorption and osteoclast formation [25e26]. In human osteoblast cultures, glucocorticoids appear necessary for osteoblast differentiation and may induce some osteoblast genes such as alkaline phosphatase [26e29], while inhibiting others such as osteocalcin [26e29], although these effects may differ depending upon the age and stage of differentiation of the cultured cells [29,30]. From a mechanistic point of view, most of the effects of glucocorticoids have been thought to be mediated at the molecular level exclusively by these genomic actions. However, there is increasing evidence of glucocorticoid effects that are not compatible with this classical mode of action [9,31]. These rapid non-genomic effects appear to be mediated by glucocorticoid interactions with biological membranes, either through binding to membrane receptors or by physicochemical interactions [9,31].
Calcitriol Vitamin D has predominant effects on osteoblasts and modulates the expression of a number of osteoblast
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GLUCOCORTICOIDS, VITAMIN D, AND INFLAMMATION
genes including alkaline phosphatase, osteopontin, and osteocalcin [32e36] as well as inhibiting collagen synthesis [37e39]. However, the osteoblastic response to 1,25(OH)2D3 appears to vary in relation to the stage of osteoblast development [30]. Because osteocalcin is induced by 1,25(OH)2D3 but suppressed by glucocorticoids, this gene has provided many insights into the interactions between vitamin D and glucocorticoids at the genomic level. For example, transcription of the rat osteocalcin gene is controlled by basal and hormone response elements located in proximal and distal sites respectively [40]. 1,25(OH)2D3 acts on the osteocalcin gene via the distal VDRE within the promoter at nucleotides e465 to e437. This VDRE functions as an enhancer and cannot induce transcription but requires basal expression. It has been shown that glucocorticoids repress both 1,25(OH)2D3 induction and basal activity of the osteocalcin promoter through a region distinct from the VDRE [41]. Indeed, the rat and human osteocalcin promoters contain multiple GREs [42e45]. In the rat, GREs in the distal (nucleotides e697 to e683) and proximal promoter regions (e16 to e1) of the osteocalcin gene bind GR and suppress 1,25(OH)2D3-induced transcription of osteocalcin [45]. Shalhoub et al. [24] have shown the influence of glucocorticoids on osteocalcin transcription is dependent on the stage of osteoblast maturation. In proliferating early-stage osteoblasts, dexamethasone increased osteocalcin transcriptional rates, although not to the extent of vitamin D, and only in cells continuously treated with dexamethasone for prolonged periods. In mature cells, acute treatment with dexamethasone resulted in decreased transcription and mRNA levels. The results indicate that transcriptional control of basal and hormone-regulated osteocalcin expression predominates in immature osteoblasts prior to matrix mineralization. However, in mature osteoblasts, osteocalcin expression is controlled primarily by posttranscriptional mechanisms. Vitamin D, alone or in combination with dexamethasone, was a significant factor contributing to mRNA stabilization in mature osteoblasts with a mineralized extracellular matrix. Transcriptional modifications in response to dexamethasone were reflected by quantitative differences between proliferating and mature osteoblasts in the formation of GR binding complexes at the proximal osteocalcin GRE. Both VDR and GR basal mRNA levels were significantly higher in mature osteoblasts than in early-stage bone cells. These results suggest there are developmental, stage-specific effects of steroid hormone on transcriptional regulation of bone-expressed genes and inverse relationships between levels of transcription and cellular representation of mRNA with osteocalcin message stabilized in mature osteoblasts. Binding of the VDR by 1,25(OH)2D induces conformational changes in the receptor that enable it to interact
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with several types of cofactors that are necessary for transcriptional activation. It has been shown that osteocalcin gene activation by 1,25(OH)2D at the VDRE region is accompanied by changes in chromatin structure and that such chromatin remodeling is a prerequisite for transcription [46]. The effects of glucocorticoids on transcriptional events related to vitamin D metabolism are complex. For example, glucocorticoids can potentiate the effect of 1,25(OH)2D3 by increasing VDR at the transcriptional level [47]. This rapid increase in VDR transcript levels may indicate glucocorticoids can directly induce de novo VDR transcription. However, glucocorticoids can also reduce the transcriptional levels of duodenal calcium-processing genes [48], which may explain the negative effects of glucocorticoids on calcium absorption discussed later. Glucocorticoids can also induce transcription of 24-hydroxlase [49], the kidney enzyme that initiates the catabolism of 1,25(OH)2D and may explain why reduced 25(OH)D levels are not uncommonly reported in patients receiving glucocorticoids, as discussed later.
GLUCOCORTICOIDS, VITAMIN D, AND INFLAMMATION Recently there has been increasing interest in the role of vitamin D in non-inflammatory diseases including asthma and airways disease, with emerging evidence of an interaction between vitamin D metabolism and glucocorticoid effects. Patients with asthma exhibit a variable response to inhaled glucocorticoids and vitamin D may exert effects on phenotype and glucocorticoid response in asthma. Indeed a proportion of patients with severe asthma fail to demonstrate clinical improvement upon glucocorticoid therapy and their asthma is characterized as glucocorticoid-resistant. Chemokines are known to play a critical role in the pathogenesis of inflammation in asthma and facilitate the recruitment of inflammatory cells in the airways. Evidence now suggests that airway smooth muscle may serve as a source of chemokines in inflamed airways and vitamin D may interact with glucocorticoids to affect airway smooth muscle cells. Banerjee investigated whether 1,25(OH)2D3 modulated chemokine production in airway smooth muscle [50]. In TNFa-treated cells, 1,25(OH)2D3 inhibited RANTES (a chemokine that attracts monokines, eosinophils, and T cells during inflammation) and IP-10 secretion in a concentration-dependent manner. In TNFa/IFNgtreated cells, the glucocorticoid fluticasone or 1,25(OH)2D3 alone partially inhibited RANTES secretion (by 38 and 20%, respectively) whereas the combination of both drugs additively inhibited RANTES secretion
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66. GLUCOCORTICOIDS AND VITAMIN D
(by 60%). While fluticasone enhanced fractalkine (the transmembrane form of the CX3CL1 chemokine) secretion (by 50%), 1,25(OH)2D3 significantly decreased fractalkine levels (by 50%). 1,25(OH)2D blocked the stimulatory effect of fluticasone on fractalkine secretion, which was inhibited by 60% with the combination of calcitriol and fluticasone. A related study by Damera et al. [51] has shown that 1,25(OH)2D3 can inhibit thrombin and platelet-derived growth-factor-induced airway smooth muscle cell proliferation. Interestingly Sutherland et al. [52] reported that in asthmatics reduced vitamin D levels are associated with impaired lung function and reduced glucocorticoid response, suggesting that supplementation of vitamin D levels in patients with asthma may improve multiple parameters of asthma severity and treatment response. Consistent with this observation, Xystrakis et al. [53] have shown here that addition of vitamin D with dexamethasone to cultures of glucocorticoid-resistant CD4þ T cells enhanced IL-10 synthesis to levels observed in cells from glucocorticoid-sensitive patients cultured with dexamethasone alone. Vitamin D significantly overcame the inhibition of glucocorticoid-receptor expression by dexamethasone, while IL-10 upregulated glucocorticoid-receptor expression by CD4þ T cells, consistent with the suggestion that vitamin D analogs could potentially increase the therapeutic response to glucocorticoids in resistant patients. TABLE 66.1
EFFECT OF GLUCOCORTICOIDS ON VITAMIN D METABOLISM Glucocorticoid therapy appears to increase renal expression of 24-hydroxylase and decrease expression or renal 1a-hydroxylase deceasing levels of 1,25(OH)2D3 [49,54]. Kurahashi et al. [55] investigated the mechanisms of this in UMR-106 osteoblast-like cells and found dexamethasone dose-dependently increased 24hydroxylase mRNA expression and enzymatic activity in the presence of 1,25(OH)2D3. The mechanism of this effect appeared to involve activation of the AP-1 site by increased c-fos protein. However, the significance of this observation is uncertain since whether changes in circulating vitamin D metabolites occur with glucocorticoid therapy is not clearly established (Table 66.1). However, it must be acknowledged that interpretation of the published data is difficult because many of the studies have been cross-sectional in nature with small samples, although some prospective studies have been performed. Long-term excess glucocorticoids have been reported to produce varied effects on vitamin D metabolites such as 25(OH)D or 1,25(OH)2D3, including reductions [56e61], no change [62e64], or small increases [65e68]. Chesney et al. [56] reported reductions in serum 1,25(OH)2D in 22 children receiving long-term glucocorticoid treatment for various glomerular diseases,
Studies of Serum Vitamin D Metabolites in Glucocorticoid-induced Osteoporosis Sample size
Reference
Type
Disease population
Chesney [56]
Cross-sectional
Renal, pediatric
Chesney [57]
Cross-sectional
Klein [69] Seeman [62]
25(OH)D
1,25(OH)2D
Findings
22
N
L
Renal, pediatric
21
L if edema
Cross-sectional
Rheumatic
27
L
e
Cross-sectional
Mixed
14
L
N
Slovik [63]
Cross-sectional
Asthma
48
LeN
e
Cannigia [61]
Cross-sectional
Not stated
15
LeN
e
Hahn [64]
Prospective
Normal
12
N
small H
Hahn [65]
Prospective
Rheumatic
17
N
e
Findling [66]
Prospective
Cushing’s
7
N
N
Morris [58]
Cross-sectional
Adult, miscellaneous
60
e
LeN
Prummel [67]
Prospective
Graves disease
10
N
N
Cosman [68]
Prospective
Multiple sclerosis
56
N
H
Oetinger-Barak [59]
Prospective
Liver transplant
21
L
e
Searing [60]
Prospective
Asthma, pediatric
100
L
e
Toloza [61]
Prospective
SLE
124
L
e
L ¼ low; N ¼ normal; H ¼ high or elevated.
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EFFECT OF GLUCOCORTICOIDS ON VITAMIN D METABOLISM
including nephrotic syndrome (mean SD serum 1,25(OH)2D: 20 4 pg/ml versus 53 5 pg/ml in controls, p < 0.005). Moreover the reduction in concentration of serum 1,25(OH)2D correlated with the dose of corticosteroid administered and with reduction in forearm bone mineral content. In contrast, in ten children with chronic glomerulonephritis not treated with glucocorticoids who had similar serum creatinine to those children treated with glucocorticoids, serum 1,25(OH)2D concentrations were similar to those in 18 healthy controls, indicating that glomerular renal disease per se did not account for the observed reduction. In a subsequent study, Chesney et al. [57] measured vitamin D metabolites in eight children with chronic glomerulonephritis not treated with prednisone (group 1), in nine non-edematous children with nephrotic syndrome treated with prednisone for more than 18 months (group 2) and in five children with nephrotic edema also treated with prednisone (group 3). Reductions in serum calcium, albumin and 25(OH)D were found in group 3 only, whereas both group 2 and group 3 patients showed reduced values of 1,25(OH)2D (p < 0.001 versus group 1 or controls). It was concluded that chronic glucocorticoid administration in children with glomerulonephritis and minimally impaired renal function (group 2) was associated with a reduction in the circulating level of 1,25(OH)2D, since children with comparable type and degree of renal disease but nonglucocorticoid treatment (group 1) had normal 1,25(OH)2D values. Children with nephrotic edema (group 3) had greater reduction of 1,25(OH)2D values, as well as lower 25(OH)D values and serum calcium values, possibly related to a urinary loss of vitaminD-binding protein. No changes in PTH were evident in either glucocorticoid-treated or edematous patients. Morris et al. [58] examined serum 1,25(OH)2D levels in 60 postmenopausal women on glucocorticoid therapy (29 with and 31 without vertebral compression fractures) and compared these results with those from 31 normal age-matched postmenopausal women. Serum 1,25(OH)2D levels were slightly reduced in both glucocorticoid-treated groups (mean SE: 80 8.4 and 92 7.9 pmol/l, respectively) compared to normal subjects (107 7.3 pmol/l), but these differences were not significant. Low 25(OH)D levels have also been reported in a study of 21 subjects undergoing liver transplantation [59], in 100 children with childhood asthma [60], and in a large cohort comprising 124 subjects with lupus [61]. In the lupus patients, 66.7% had 25(OH)D levels below 80 nmol/l and 18% below 40 nmol/l. In a multivariate logistic regression 25(OH)D levels were related to cumulative glucocorticoid exposure. Seeman et al. [62] studied circulating levels of vitamin D metabolites in six patients with endogenous
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Cushing’s syndrome and eight patients treated with prednisone (mean dose 50 mg/day, range 30e60 mg/ day) for 1 month for various connective tissue diseases. Comparing “euglucocorticoid” levels (i.e., after surgical correction in the Cushing’s patients or before treatment in the prednisone therapy group) with the hyperglucocorticoid state (i.e. before surgery or after prednisone therapy, respectively), they observed a non-significant reduction in serum 1,25(OH)2D (mean SD: 32 8 versus 23 6 pg/ml), but significantly lower serum 25(OH)D (22 2 versus 18 2 ng/ml) in the hyperglucocorticoid state. Kinetic studies using tritiated 1,25(OH)2D in ten hyperglucocorticoid patients and 14 normal controls revealed no evidence for altered production or degradation. Slovik et al. [63] observed mean serum 25(OH)D and PTH levels in 48 adult asthmatic patients on chronic glucocorticoid therapy were not significantly different from a disease control group of 12 asthmatic patients not on glucocorticoids, but nine such patients had abnormally low 25(OH)D levels. As noted above, Sutherland et al. [52] have reported that in asthma patients, low serum 25(OH)D was associated with poorer lung function, increased airway hyper-responsiveness and reduced glucocorticoid responsiveness. Hahn et al. [64] studied 12 normal adults treated with prednisone 20 mg daily for 2 weeks. Serum 25(OH)D did not change significantly but serum 1,25(OH)2D rose significantly as did serum PTH, but the latter rise was not significant. In another prospective study of the effect of administration of 25(OH)D in GIO (discussed below), Hahn et al. [65] observed no difference in baseline serum 25(OH)D in 17 patients treated with chronic glucocorticoids (dose greater than 7.5 mg prednisone equivalent for at least 18 months) compared to 15 normal subjects. Thus cross-sectional studies have generally reported no change in vitamin D metabolites in response to glucocorticoid therapy. Findling et al. [66] studied vitamin D metabolites in seven patients with spontaneous ACTH-dependent Cushing’s syndrome. Remission of hypercortisolism resulted in a significant increase in tubular reabsorption of phosphate and serum phosphate. Serum PTH levels were normal during Cushing’s syndrome, but fell significantly after remission. Plasma 25(OH)D and 1,25(OH)2D did not differ from measurements in 97 normal subjects. After treatment, serum 25 (OH)D did not change, but serum 1,25(OH)2D fell (mean 44 to 22 pg/ml, p < 0.02) and was inversely correlated with serum phosphate (r ¼ 0.59; p < 0.01), but did not correlate with serum PTH. It was concluded that impairment of intestinal calcium absorption in Cushing’s syndrome could not be attributed to any decrease in the circulating levels of 1,25(OH)2D. In a small study, Prummel et al. [67] prospectively measured biochemical markers of bone turnover in ten
VIII. DISORDERS
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66. GLUCOCORTICOIDS AND VITAMIN D
euthyroid patients with Graves ophthalmopathy treated with tapering prednisone (initial dose 60 mg/day) over 19 weeks. There were no significant changes in serum 25(OH)D or 1,25(OH)2D, although serum intact PTH fell slightly. In another larger prospective study, Cosman et al. [68] studied various biochemical markers of bone turnover in a population of 56 patients with multiple sclerosis, also treated with glucocorticoids (initially intravenous methylprednisolone 1 g daily for 10 days then 500 mg/day for 2 days, 250 mg/day for 2 days followed by oral prednisone 80 mg/day reducing in dose over 28 days). During and after treatment there were no changes in serum 25(OH)D, but serum 1,25(OH)2D increased and serum phosphate decreased within 3 days of commencing corticosteroid. Serum PTH increased later to a peak at 2 weeks and serum 1,25(OH)2D subsequently declined. The early changes in serum 1,25(OH)2D were considered to represent a direct effect of glucocorticoids on the kidney, rather than being secondary to the decrease in serum phosphate, with the later decline secondary to the change in PTH. Taken overall, these studies suggest modest reductions in 25(OH)D levels are often associated with chronic glucocorticoid therapy, possibly due to increased catabolism.
VITAMIN D AS TREATMENT FOR GIO Even though the literature is controversial as to whether circulating metabolites of vitamin D are affected by glucocorticoid therapy, the original rationale for use of vitamin D in GIO was based upon the demonstration of impaired calcium absorption in glucocorticoid-treated subjects and that this could be reversed by vitamin D [69e72]. Therapeutic studies of vitamin D have examined both changes in calcium absorption and bone mass or density. Some of the earlier studies have been performed with calciferols; however, the active metabolites, calcitriol and alfacalcidol (1ahydroxyvitamin D), have also been studied.
Calcium Absorption Klein et al. [69] compared fractional calcium absorption in 27 patients receiving prednisone compared to 27 age- and sex-matched controls. In patients receiving high doses of prednisone (15e100 mg/day) calcium absorption and serum 25(OH)D were decreased. However, in patients receiving low doses (8e10 mg/ day) or high doses (30e100 mg) on an alternate day schedule, both of these parameters were normal. Calcium absorption correlated inversely with daily prednisone dose. Administration of 0.4 mg of calcitriol
daily for 7 days in five patients led to an increase in calcium absorption. A study by Hahn et al. [65] examined the effect of treatment with calcidiol (25 hydroxyvitamin D) 40 mg/day plus calcium in 17 patients with GIO compared to 15 controls. The glucocorticoid group had reduced calcium absorption and increased serum PTH, but similar serum 25(OH)D levels and reduced forearm bone mass compared to controls. Treatment increased calcium absorption by 46%. Caniggia et al. [70] reported that low to normal serum 25(OH)D in patients treated with chronic glucocorticoids and impaired calcium absorption could be reversed by treatment with either calcidiol or calcitriol. Braun et al. [72] performed a double-blind, placebocontrolled study of alfacalcidol 2 mg daily for 6 months in 14 patients receiving long-term glucocorticoid therapy. Treatment with alfacalcidol increased calcium absorption at 3 and 6 months and, on repeat iliac crest biopsy, showed a decrease in active resorption and a trend for increased trabecular bone volume, suggesting no suppression of bone formation was occurring. Morris et al. [58] examined the relation between calcium absorption and serum 1,25(OH)2D levels in a set of 60 postmenopausal women on glucocorticoid therapy (29 with and 31 without vertebral compression fractures) and compared these results with those from 31 normal postmenopausal age-matched women. Calcium absorption was reduced in glucocorticoidtreated patients and shown to be linearly related to serum 1,25(OH)2D in both glucocorticoid-treated groups and in the glucocorticoid set as a whole. However, only about one-third of the impairment in calcium absorption was accounted for by serum 1,25(OH)2D levels. This apparent resistance to the intestinal action of 1,25(OH)2D was quantified by a z-score analysis which expressed the difference between the measured calcium absorption and that predicted from the 1,25(OH)2D level. The calcium absorption was significantly reduced in the fracture group by e0.52 of a standard deviation. Calcium transport proteins regulate calcium influx in the gastrointestinal tract by mediating the transport of calcium to the bloodstream. Although the mechanism of the negative effects of glucocorticoids on calcium absorption remain unclear, Kim et al. [48] recently observed in an animal model that 5 days of treatment with dexamethasone reduced the transcriptional levels of duodenal TRPV6 (a highly selective calcium channel on the apical side of gut cells) and CaBP-9k (an intracellular calcium-ion-binding protein involved in shuttling calcium ions from the apical to the basolateral membrane) by 60%. Although the demonstration of impaired calcium absorption with glucocorticoids provided a rationale for use of vitamin D in GIO, human studies also suggest a role for vitamin D as a therapeutic agent.
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VITAMIN D AS TREATMENT FOR GIO
Bone Mass or Density These observations led inevitably to studies of the effects of vitamin D on bone mass. Vitamin D in various formulations, usually in combination with calcium, has been studied as a treatment for GIO in studies dating from the 1970s. These studies can be broadly divided into primary prevention (in patients commencing glucocorticoid therapy) or treatment (in patients on chronic glucocorticoids) (Table 66.2). In one of the first trials of vitamin D in GIO, Hahn et al. [73] examined the effect of treatment with calcium 500 mg/day and vitamin D 50 000 IU per week on bone mass in patients on chronic glucocorticoid therapy. A significant increase in forearm bone mass was observed with vitamin D treatment, but the study was not randomized. Another study by Hahn et al. [65] examined the effect of treatment with calcidiol (40 mg/day) plus calcium in 17 patients receiving chronic glucocorticoids compared to 15 controls. Treatment with vitamin D improved bone mass over 12 months by 13.2% at the metaphyseal site and 2.1% at the diaphyseal site. Bijlsma et al. [74] studied the effect of calcium 500 mg daily versus calcium plus a vitamin D preparation (dihydrotachysterol 4000 IU on alternate days) in 21 patients on long-term glucocorticoid therapy (mean daily TABLE 66.2
prednisone dose 14 mg/day) for 2 years. A small increase in lumbar spine BMD was noted in both groups, but there was no significant difference between groups. Adachi et al. [75] compared a combination of calcium plus vitamin D (1000 mg daily plus 50 000 IU weekly, respectively) against placebo over 3 years in 62 patients initiating corticosteroids (i.e., primary prevention). Bone loss at the lumbar spine was reduced by treatment with calcium/vitamin D, but the difference was not significantly different from placebo. However, a secondary prevention study by Buckley et al. [76] in 65 patients receiving chronic low-dose corticosteroids for rheumatoid arthritis observed an annual spinal loss of 2.0% in placebo-treated patients compared to 0.7% gain in calcium/vitamin-D3-treated patients (1000 mg plus 500 IU/day, respectively). Sambrook et al. [77] examined the effect of 12 months of calcium, calcitriol, or calcitonin in 103 patients starting corticosteroids. Patients treated with calcium lost bone rapidly at the lumbar spine (4.3% in the first year) whereas patients treated with either calcitriol alone (mean dose 0.6 mg/day) or calcitriol plus calcitonin lost at a much reduced rate (1.3% and 0.2% per year, respectively). Both groups were significantly different from the calcium group. Approximately 25% of patients developed mild hypercalcemia, probably
Trials of Vitamin D Metabolites on Bone Mineral Density BMD effect size (% change vs. control)*
Reference
Type
Prevention/Treatment
Agent
Sample size
Hahn [69]
Non-randomized, open
Treatment
Vit D 50 000 IU/wk
26
4.4
e
Hahn [56]
Non-randomized, open
Treatment
Calcidiol 40 mg/d
32
5.8
e
Dykman [80]
Randomized, DB
Treatment
Calcitriol 0.4 mg/d
23
NS**
e
Biljsma [74]
Non-randomized, open
Treatment
Vit D 2000 IU/d
21
e
5.4
Sambrook [77]
Randomized, DB
Prevention
Calcitriol 0.75 mg/d
103
e
3.0
Adachi [75]
Randomized,
Prevention
Vit D 50 000 IU/wk
62
e
0.69
Buckley [76]
Randomized,
Treatment
Vit D 500 IU/d
65
e
2.65
Reginster [78]
Randomized,DB
Prevention
Alfacalcidol 1 mg/d
145
e
6.06
Lambrindouki [81]
Randomized,DB
Treatment
Calcitriol 0.5 mg/d
81
e
1.6
Ringe [84]
Non-randomized, open
Treatment
Alfacalcidol 1 mg/d*
85
e
2.0
Sambrook [86]
Randomized, open
Mixed
Calcitriol 0.5 mg/d*
198
e
0.2
De Nijs [87]
Randomized, DB
Mixed
Alfacalcidol 1 mg/d
201
e
1.9
Ringe [85]
Randomized, open
Treatment
Alfacalcidol 1 mg/d*
204
e
3.2
Yamada [88]
Randomized, open
Treatment
Alfacalcidol 0.5 mg/d
12
e
2.5
Fujii [89]
Randomized, open
Treatment
Alfacalcidol 0.4 mg/d
114
e
1.0
* vs. D2 or D3. ** small increase both groups, % difference not stated. Transplant studies are not included as immunosuppressives other than glucocorticoids are administered complicating the picture.
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Forearm
Spine
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66. GLUCOCORTICOIDS AND VITAMIN D
related to co-administration of calcium, which settled with reduction in calcitriol dosage. A randomized, double-blind, controlled trial in 145 patients starting corticosteroids compared 1 mg/day of alfacalcidol with calcium [78]. After 12 months the change in spinal bone density with alfacalcidol was þ0.4% compared to e5.7% with calcium. Hypercalcemia occurred in 6.7% of alfacalcidol-treated patients. A randomized, doubleblind, prospective trial found that prophylactic treatment with calcitriol in 66 patients undergoing cardiac or single lung transplantation was able to prevent or markedly reduce bone loss over 24 months [79]. Not all studies with active metabolites have shown positive effects on bone density. Dykman et al. [80] studied 23 rheumatic disease patients with GIO in an 18-month, double-blind, randomized study to assess the effect of oral calcium and calcitriol (mean dose 0.4 mg/day) or calcium versus placebo. Intestinal calcium absorption was increased and serum PTH levels were suppressed by calcitriol; however, no significant gain in forearm bone mass occurred, and fractures were as frequent in both groups. In the calcitriol group, histomorphometric analysis of iliac crest biopsy specimens demonstrated a decrease in osteoclasts/mm2 of trabecular bone (p < 0.05) and parameters of osteoblastic activity (p < 0.05), indicating that calcitriol reduced both bone resorption and formation. Lambrinoudaki et al. [81] studied the effect of calcitriol in GIO in a double-blind, placebo-controlled study of 81 premenopausal women with systemic lupus erythematosus on chronic steroid therapy (mean cumulative prednisone dose of 28 g). They were randomly allocated to three groups: Group 1: 0.5 mg calcitriol and 1200 mg calcium daily; Group 2: 1200 mg calcium and placebo calcitriol; and Group 3: both placebo calcitriol and placebo calcium. At the end of 2 years, patients in the calcitriol group exhibited a significant increase of 2.1% in BMD at the lumbar spine compared to baseline value (p < 0.05). This change was not significantly different from the respective change in either calcium or placebo group (0.4% and 0.3%, respectively). No significant changes were observed in any treatment group in BMD at the hip or radius. It was concluded that premenopausal women with lupus taking prolonged glucocorticoid therapy had reduced BMD (probably due to disease effects) but showed no significant bone loss over the 2-year study period. The beneficial effect of calcitriol treatment in these premenopausal women was small when it was instituted late in the course of glucocorticoid therapy. The results of many of these studies have been summarized in meta-analyses which generally had consistent findings [82,83]. For example, one of these meta-analyses concluded that in GIO vitamin D had a modest beneficial effect on BMD (effect size 0.35;
95% CI 0.18e0.52) and was associated with a reduction in vertebral fracture risk (pooled risk reduction 0.56; 95% CI 0.34e0.92) [83].
Efficacy of Different Vitamin D Preparations Whether cholecalciferol (D3) or ergocalciferol (D2) is less efficacious than active metabolites in GIO remains unclear, but six studies have addressed this question. Ringe et al. [84] evaluated the efficacy of alfacalcidol compared with D3 in patients on chronic corticosteroids. Eighty-five patients on long-term glucocorticoid therapy were allocated to either 1 mg alfacalcidol plus calcium 500 mg daily or 1000 IU vitamin D3 plus 500 mg calcium in a non-randomized parallel group study. The two groups were similar in age, sex, underlying diseases, initial BMD (lumbar spine: mean T-score 3.28 and 3.25, respectively) and rates of vertebral and non-vertebral fractures. During the 3-year study, a small but significant increase was seen in lumbar spine BMD in the alfacalcidol group (þ2.0%, p < 0.0001) with no significant changes at the femoral neck. In the vitamin D group, there were no significant changes at either site. Subsequently, Ringe et al. [85] repeated this study with the same treatment regimen in a larger study sample and as a randomized open label trial. In 204 patients on chronic glucocorticoids over 3 years, significant increases in BMD were seen at lumbar spine (þ2.4%) and the femoral neck (þ1.2%) in the alfacalcidol group with no significant changes in the plain vitamin D group at either site (0.7% and þ0.7%, respectively). By the end of the study, 16 new vertebral fractures had occurred in ten patients of the alfacalcidol group and 25 in 25 patients of the vitamin D group. These studies suggest alfacalcidol is superior to cholecalciferol in the treatment of established glucocorticoid osteoporosis. In contrast, Sambrook [86] compared treatment with calcitriol with ergocalciferol (D2) or alendronate in 198 patients commencing or already being treated with chronic glucocorticoids. Patients were randomized to one of three groups: calcitriol 0.5 to 0.75 mg/day; ergocalciferol 30 000 IU weekly plus calcium carbonate (600 mg daily); or alendronate 10 mg/day plus calcium carbonate (600 mg daily). Over 2 years mean lumbar BMD change was þ5.9% with alendronate, 0.5% with ergocalciferol and e0.7% with calcitriol (p < 0.001). At the femoral neck, there was no significant difference in BMD change between the treatments over 2 years; alendronate (þ0.9%), ergocalciferol (2.2%) and calcitriol (3.2%). The calcitriol group was treated with higher cumulative glucocorticoid dose but after adjustment, no significant difference was seen between calcitriol or ergocalciferol in prevention of bone loss but both were inferior to alendronate.
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SUMMARY
Effects of 1,25(OH)2D3 compared to alendronate in patients on oral glucocorticoids over 18 months on serum carboxyterminal propeptide of type 1 procollagen (PICP). Reproduced from [87] with permission.
FIGURE 66.2
Effects of 1,25(OH)2D3 compared to alendronate in patients on oral glucocorticoids over 18 months on lumbar spine bone mineral density. Reproduced from [87] with permission.
FIGURE 66.3
De Nijs et al. [87] also compared the effect of alfacalcidol with alendronate in 201 patients starting glucocorticoids at a daily dose of 7.5 mg prednisone over 18 months. Although serum carboxy-terminal propeptide of type 1 procollagen (PICP) improved more with alfacalcidol than alendronate (Fig. 66.2), lumbar spine BMD increased by 2.1% in the alendronate group and decreased by 1.9% in the alfacalcidol group (Fig. 66.3). Three patients with alendronate had new vertebral fractures compared with eight patients in the alfacalcidol group. These results were interpreted as indicating alendronate was more effective than alfacalcidol in the prevention of glucocorticoid-induced bone loss. Consistent with these findings, Yamada et al. [88] performed a small study in Japanese patients (12 patients with rheumatoid arthritis) randomized to
either low-dose risedronate (2.5 mg daily) or alfacalcidol 0.5 mg daily for 48 weeks. Lumbar spine BMD increased by 5.2% with risedronate compared to 2.5% with alfacalcidol. Urinary N telopeptides and deoxypyridinoline were significantly suppressed after risedronate but not with alfacalcidol. Okada et al. [90] also compared the efficacy of bisphosphonates with active vitamin D in 47 premenopausal women commencing high-dose glucocorticoids. Patients were randomized to be treated with prednisolone and alfacalcidol 1 mg/day alone or prednisolone and alfacalcidol 1 mg/day with low-dose alendronate (5 mg/day) for 18 months. The percentage change in lumbar spine BMD after 6 months of the therapy was 10.5% in the alfacalcidol group, but only 2.1% in the combined group. The rate of bone loss in the lumbar spine was significantly lower in the combined group than in the alfacalcidol group at 6 months. At 12 months, the percentage change in lumbar spine BMD was increased by 1.7% in the combined group, but decreased by 9.9% in the alfacalcidol group. Fractures occurred at 12 months or later in four patients in the alfacalcidol groups, but not in the combined group. Although oral bisphosphonates show efficacy and tolerability in osteoporotic patients without renal impairment, their combination with active vitamin D analogs in chronic kidney disease (CKD) patients taking glucocorticoids remains unknown. Fujii et al. [89] conducted a prospective open label study of 114 CKD patients receiving glucocorticoid therapy for 6 months. Eighty-eight subjects who had received alfacalcidol (mean dose 0.4 mg daily) were randomly assigned to either a group treated with alfacalcidol only (group A), or to a group also receiving risedronate 2.5 mg/day (group B). The remaining patients (group C) received risedronate only. Lumbar BMD significantly increased over 12 months by 2.8 and 2.5% in groups B and C, respectively, but decreased by 1.0% in group A. Serum N-terminal telopeptides of type I collagen (NTX) and bone alkaline phosphatase (BALP) fell significantly in groups B and C at 3 and 6 months, respectively, while in group A, NTX remained unchanged and BALP significantly increased. There was no significant difference between groups B and C regarding BMD and bone markers. It was concluded that risedronate was effective in increasing BMD with or without active vitamin D metabolites in CKD patients receiving long-term glucocorticoid therapy.
SUMMARY To summarize, evidence from all these randomized trials taken together suggests that patients receiving glucocorticoids, who are at risk of rapid bone loss and
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66. GLUCOCORTICOIDS AND VITAMIN D
consequent fracture, should be actively considered for prophylactic measures that include a vitamin D metabolite. However, based upon the available evidence, firstline therapy would be a bisphosphonate, with vitamin D as adjunctive therapy or second-line therapy. Moreover, although vitamin D metabolites appear to confer an additional therapeutic effect in GIO, it remains unclear whether the active metabolites are superior to D2 or D3 in this context and further studies are required.
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[78] J.Y. Reginster, D. Kuntz, W. Verdickt, M. Wouters, L. Guillevin, C.J. Menkes, et al., Prophylactic use of alfacalcidol in corticosteroid-induced osteoporosis, Osteop. Inter. 9 (1999) 75e81. [79] P.N. Sambrook, N.K. Henderson, A. Keogh, P. MacDonald, A. Glanville, P. Spratt, et al., Effect of calcitriol on bone loss after cardiac or lung transplantation, J. Bone Miner. Res. 15 (2000) 1818e1824. [80] T.R. Dykman, K.M. Haralson, O.S. Gluck, W.A. Murphy, S.L. Teitelbaum, T.J. Hahn, et al., Effect of oral 1,25-dihydroxyvitamin D and calcium on glucocorticoid-induced osteopenia in patients with rheumatic diseases, Arthritis Rheum. 27 (1984) 1336e1343. [81] I. Lambrinoudaki, D.T. Chan, C.S. Lau, R.W. Wong, S.S. Yeung, A.W. Kung, Effect of calcitriol on bone mineral density in premenopausal Chinese women taking chronic steroid therapy. A randomized, double blind, placebo controlled study, J. Rheumatol. 27 (2000) 1759e1765. [82] F. Richy, O. Ethgen, O. Bruyere, J.Y. Reginster, Efficacy of alphacalcidol and calcitriol in primary and corticosteroidinduced osteoporosis: a meta-analysis of their effects on bone mineral density and fracture rate, Osteoporosis International 15 (2004) 301e310. [83] R.N. de Nijs, J.W. Jacobs, A. Algra, W.F. Lems, J.W. Bijlsma, Prevention and treatment of glucocorticoid-induced osteoporosis with active vitamin D3 analogues: a review with metaanalysis of randomized controlled trials including organ transplantation studies, Osteoporosis International 15 (2004) 589e602. [84] J.D. Ringe, A. Coster, T. Meng, E. Schacht, R. Umbach, Treatment of glucocorticoid-induced osteoporosis with alfacalcidol/ calcium versus vitamin D/calcium, Calcif. Tiss. Int. 65 (1999) 337e340. [85] J.D. Ringe, A. Dorst, H. Faber, E. Schacht., Treatment of established glucocorticoid-induced osteoporosis with alfacalcidol or plain vitamin D, Calcif. Tiss. Int. 72 (2003) 4. [86] P.N. Sambrook, M. Kotowicz, P. Nash, C.B. Styles, V. Naganathan, K.N. Henderson-Briffa, et al., Prevention and treatment of glucocorticoid induced osteoporosis: a comparison of calcitriol, vitamin D plus calcium and alendronate plus calcium, J. Bone Miner. Res. 18 (2003) 919e924. [87] R.N.J. de Nijs, J.W.G. Jacobs, W.F. Lems, et al., Alendronate or alfacalcidol in glucocorticoid-induced osteoporosis, New Engl. J. Med. 355 (2006) 675e684. [88] S. Yamada, H. Takagi, H. Tsuchiya, T. Nakajima, H. Ochiai, A. Ichimura, et al., Comparative studies on effect of risedronate and alfacalcidol against glucocorticoid-induced osteoporosis in rheumatoid arthritic patients, Yakugaku. Zasshi. e J. Pharma. Soc. Japan 127 (2007) 1491e1496. [89] N. Fujii, T. Hamani, S. Mikami, Y. Nagasawa, Y. Isaka, T. Moriyama, et al., Risedronate, an effective treatment for glucocorticoid-induced bone loss in CKD patients with or without concomitant active vitamin D (PRIUS-CKD), Nephrol. Dial Transplant. 22 (2007) 1601e1607. [90] Y. Okada, M. Nawata, S. Nakayamada, K. Saito, Y. Tanaka, Alendronate protects premenopausal women from bone loss and fracture associated with high-dose glucocorticoid therapy, Nat. Clin. Pract. Rheumatol. 5 (2009) 74e75.
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C H A P T E R
67 Drug and Hormone Effects on Vitamin D Metabolism Barrie M. Weinstein, Sol Epstein Mount Sinai School of Medicine, New York, NY, USA
INTRODUCTION This chapter will discuss the effects that drugs and hormones have on vitamin D metabolism. The level of active vitamin D metabolite and the activity of the renal hydroxylase enzymes that are responsible for regulating its production are governed by a variety of hormones and cations. Parathyroid hormone (PTH), phosphate, and calcium are the principal controlling factors of the renal hydroxylases (discussed in Chapters 3 and 4) yet several other hormones may also play a role in the metabolism of vitamin D. Among those discussed here are parathyroid hormone-related protein (PTHrP), fibroblast growth factor 23 (FGF23), leptin, calcitonin, prolactin, growth hormone and insulin-like growth factor (IGF), sex steroids (estrogen, testosterone, and progesterone), insulin, thyroid hormone, prostaglandins, interferon-g, and tumor necrosis factor-a. The findings are summarized in Table 67.1. Along with the endogenous regulators of the levels of the vitamin D metabolites, some exogenous drugs and hormones are also capable of exerting an effect on the vitamin D endocrine system. Drugs are being increasingly recognized in this regard. Three that have received the most attention are the anticonvulsants, ethanol, and corticosteroids, but many others have also been implicated. The mechanisms by which these drugs affect vitamin D metabolism are discussed in detail and the findings summarized in Table 67.2 (see “Hormone effects on vitamin D metabolism,” below). It should be realized that the introduction of new drugs and molecular technology will likely expand this area considerably.
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10067-8
HORMONE EFFECTS ON VITAMIN D METABOLISM Parathyroid Hormone and Parathyroidhormone-related Protein This section summarizes the relationship between exogenous PTH or stimulated PTH secretion and vitamin D metabolism. For a more extensive review, refer to Chapter 41. A large body of evidence has established PTH as one of the main regulating hormones of vitamin D metabolism. Early rodent and chicken work demonstrated that PTH was necessary for the stimulation of 1a-hydroxylase (CYP27B1) and synthesis of 1,25(OH)2D3 [1e7]. Similarly, most in vitro studies have shown increased production of 1,25(OH)2D3 [8e10], stimulation of 1a-hydroxylase, and inhibition of 24-hydroxylase in the presence of PTH [11e15]. The mechanism by which PTH enhances renal 1ahydroxylase and inhibits 24-hydroxylase is most probably via second messengers. PTH increases cyclic AMP (cAMP) production in proximal tubules [16e18] and in renal slices from adult rats treated with PTH [12,13,15]. Further evidence for a role of cAMP emerged following studies by Horiuchi and co-workers [19]. They found that an infusion of cAMP and dibutyryl cAMP into vitamin-D-deficient rats increases the conversion of tritiated 25-hydroxyvitamin D3 (25(OH)D) to 1,25(OH)2D3 [7,19]. Furthermore, cAMP [8,9,14] and forskolin [20,21], a direct activator of adenylate cyclase which increases intracellular cAMP levels [22], have been shown, in vitro, to enhance the production of 1,25(OH)2D3 and inhibit 24-hydroxylase activity [7]. The application of molecular technology has led to
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
1246 TABLE 67.1
67. DRUG AND HORMONE EFFECTS ON VITAMIN D METABOLISM
Hormone Effects on Vitamin D Metabolism
Hormone
Study
PTH
In vitro
\
\
Animal
\
\
Human
\
\
In vitro
\
Animal
\
Human
\
In vitro
Z
Animal
Z
Human
Z
In vitro
Z
Animal
Z
PTHrP
FGF23
Leptin
25(OH)D 1,25(OH)2D3 24,25(OH)2D3
In vitro Animal
5
\
Z
Human
5
\5
\\
Human IGF-1
Prolactin
Estrogen
Testosterone
Progesterone
Thyroid hormone
5
Human
Z
Z
\
In vitro
\
Z
Animal
\5
Human
\
Tumor necrosis factor-a
In vitro
\
Interferon-g
In vitro
\
Animal
5
\
\Z
In vitro
\
Animal
\
TABLE 67.2
In vitro
\
Drug
Study
25(OH)D 1,25(OH)2D3
24,25(OH)2D3
Bird
\
Anticonvulsants
Animal
5
Z
Mammal
\5
Human Insulin
\
25(OH)D 1,25(OH)2D3 24,25(OH)2D3
important mechanistic insights into PTH action. The promotor region of the gene for 1a-hydroxylase has been shown to contain cAMP-response elements which respond to PTH [23,24]. PTH has also been shown to decrease 24-hydroxylase activity through suppression of CYP24 expression, the 24-hydroxylase gene [25], thus strengthening the association of PTH, cAMP, and 1,25(OH)2D3. Another mechanism of downregulation of 24-hydroxylase appears to be by altering the stability of its mRNA [26].
5
Animal
Study
Symbols denote effects on serum levels of vitamin D metabolites: \, increased; Z, decreased; 5 unchanged. See text for details and references
Z\
Growth hormone In vitro
Hormone
Prostaglandins
Human Calcitonin
TABLE 67.1 Hormone Effects on Vitamin D Metabolismdcont’d
5
In vitro
\
Z
Animal
5
\
Z
Human
5
5\
5Z
In vitro
\Z5
Animal
5
\5
Human
5
\5
In vitro
\
Animal
5
5\
Human
5
\
In vitro
\
Z\5
Z
5
Human Z5
\5Z
Animal
Z5
5
Human Z5
Z5\
5
Ethanol
Animal
Z
Human Z5
Z5
Ketoconazole
In vitro
Z
Corticosteroids
5\
Drug Effect on Vitamin D Metabolism
5
\
5
Human
Z
Statins
Human 5
5
Cholestyramine
Animal
5
Z
Z
Human Z5
\
Animal
\
5
Human
5
5\
In vitro
\
Z
\
Animal
5
Z
\
Fibric acid
Human Z
Ezetimibe
Animal
Bisphosponates
In vitro
Z
Animal
\
Human 5Z
\Z
Z
Human \
Z
\
Thiazide diuretics (Continued)
DISORDERS
\
5
(Continued)
HORMONE EFFECTS ON VITAMIN D METABOLISM
TABLE 67.2
Drug Effect on Vitamin D Metabolismdcont’d
Drug
Study
Calcium channel blockers
Animal
Z
Human 5
5
Heparin
Animal
Z
Human 5
Z
5
Human 5Z
5
5
Aluminum
Animal
5Z
Oral parenteral
Human 5
Z
Human
\
Antituberculous agents
Human 5Z
5Z
Caffeine
In vitro
Z
Animal
5\
In vitro
Z
Cimetidine
Theophylline
Animal
Animal
25(OH)D 1,25(OH)2D3 24,25(OH)2D3
Z
5
Z
5
Immunosuppressants
Animal
5\Z
Human 5
5
Flouride
Animal
5
5
Human 5
5
Olestra
Animal
\
\
5
Human 5 Orlistat
Human 5Z
Lithium
Human 5
5Z
Symbols denote effects on serum levels of vitamin D metabolites: \, increased; Z, decreased; 5 unchanged. See text for details and references.
Along with cAMP, investigators have demonstrated that the phospholipase C/protein kinase C (PKC) second messenger system may also mediate PTH stimulation of 1,25(OH)2D3 secretion by mammalian renal proximal tubules in vitro [27,28]. This occurs at concentrations of PTH that are insufficient to raise cAMP content [27]. It is conceivable that PTH activation of 1,25(OH)2D3 may involve both signaling pathways, with the PKC pathway being responsive to lower concentrations of PTH [27,29]. Clinically, patients with primary hyperparathyroidism have mean plasma concentrations of 1,25(OH)2D that are significantly increased in approximately one-third of cases versus controls [30e33]. In patients with Paget’s bone disease, 1,25(OH)2D3 elevation in response to mithramycin-induced hypocalcemia also corresponds to an increase in PTH secretion [34].
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In addition, treatment with 1,25(OH)2D3 will correct the hypocalcemia found in hypoparathyroid patients [35e37]. Moreover, patients with pseudohypoparathyroidism, a disease characterized by metabolic unresponsiveness to PTH, have low circulating levels of 1,25(OH)2D3 [38]. 1,25(OH)2D3 is also widely used to suppress PTH levels in uremic patients with secondary hyperparathyroidism [39]. Another major influence on vitamin D metabolism is PTHrP (see also Chapter 41). PTHrP is a protein that is produced by a number of solid tumors, especially squamous and renal cell carcinomas, and is thought to be responsible for many cases of hypercalcemia of malignancy. However, it is also seen in the normal physiological situation [40]. PTHrP binds to the classic type I PTH/PTHrP receptor, which is expressed in many tissues, such as bone and lung, as well as to alternative type II receptors in several non-classic PTH target tissues, such as keratinocytes and squamous carcinoma cell lines [41]. PTHrP has a similar activity to PTH [42,43]; however, unlike in primary hyperparathyroidism where patients have normal or elevated 1,25 (OH)2D3 levels (see above), 1,25(OH)2D3 is not elevated and may be decreased in patients with hypercalcemia of malignancy and increased urinary cAMP excretion [44,45]. Thus, the hypercalcemia associated with humoral hypercalcemia of malignancy (HHM) is thought to be caused by increased renal calcium reabsorption [46,47] and/or increased bone resorption [33,48], rather than increased intestinal calcium absorption. Unlike the human scenario of HHM, rodent studies have shown an elevation in serum calcium and 1,25(OH)2D levels following tumor transplantation [49] and infusion of synthesized N-terminal fragments of PTHrP [50,51]. Walker et al. [51] also demonstrated direct stimulation of 1a-hydroxylase in rodent kidney slices in vitro by PTHrP (le36). Conversely, Michigami et al. recently described an animal model of HHM in which serum 1,25(OH)2D levels were markedly reduced in the setting of severe hypercalcemia [52]. In this model, PTHrP-producing infantile fibrosarcomas were inoculated into nude rats. Administration of a bisphosphonate to these rats normalized their serum calcium and increased their 1,25(OH)2D levels. Moreover, 1ahydroxylase activity was decreased in the hypercalcemic rats and rose after treatment with a bisphosphonate. In contrast, administration of a neutralizing antibody to PTHrP led to a reduction in serum calcium without an increase in 1,25(OH)2D levels [52]. These results suggest that PTHrP stimulates 1a-hydroxylase in this animal model. Because of the differences observed between HHM and infusion of synthesized PTHrP, additional factors, other than PTHrP, may be involved in the inhibition of renal 1,25(OH)2D3 production associated with HHM
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67. DRUG AND HORMONE EFFECTS ON VITAMIN D METABOLISM
[53]. It has been suggested that hypercalcemia itself may be the cause of low 1,25(OH)2D3 levels [54]. However, Nakayama et al. [33] reported that low 1,25(OH)2D3 levels were demonstrated at any serum calcium level in patients with HHM compared to patients with primary hyperparathyroidism. This suggests that the reduction in serum 1,25(OH)2D3 observed in these patients cannot be explained solely by an elevation in serum calcium [33]. As in animals, a study by Everhart-Caye et al. [55] revealed a dose-related increase in renal production of 1,25(OH)2D following a 6 h infusion of human PTHrP (1e36) into healthy subjects. This confirms a previous report by Fraher and co-workers [56], who observed a similar increase in 1,25(OH)2D3 production following human PTHrP (1e34) infusion in healthy subjects. Although the in vivo human studies of Fraher and Everhart-Caye do indeed demonstrate PTHrP stimulation of 1,25(OH)2D3 production, they were conducted in normal healthy subjects and do not mirror the clinical situation as seen in HHM, in which a host of factors may be at play (as discussed below). They were also short-term infusion studies, and it is quite possible that equivalent findings may not be produced with longer-term studies. More recently, Horowitz et al. [57] directly compared the effects of 48 h infusions of human PTHrP (1e36) versus human PTH (1e34) in healthy subjects. Although the calcemic, renal handling of calcium and phosphaturic effects were similar, PTH was significantly more effective at stimulating renal 1,25(OH)2D than PTHrP [57]. In a similar, more recent, study Horowitz et al. confirmed their findings of a discordant effect of PTH and PTHrP on 1,25(OH)2D stimulation [58]. These findings, however, do not explain the low 1,25(OH)2D3 levels seen in HHM. Possible explanations for the disparate effect of PTHrP in HHM are that other regions, or alternatively the full length of the PTHrP [1e141] molecule, may act differently from the foreshortened synthetic molecules administered in previous studies [56]. This may modify or impair the capacity of the N-terminal moiety of PTHrP to stimulate 1a-hydroxylase, as might other factors also produced by tumors, such as the cytokines, interleukin-1 (IL-1), and tumor necrosis factor, and/or growth factors [59]. The other clinical scenario in which PTHrP may play an important role is during lactation. PTHrP may regulate mammary blood flow, and is a potential mediator of the changes in calcium metabolism seen during lactation [60]. Mather et al. [61] reported a case of a woman with hypoparathyroidism whose need for calcium and calcitriol supplementation abated during lactation. Her 1,25(OH)2D3 levels remained in the normal range, despite a cessation of supplemental calcitriol and a lack of endogenous PTH. This was associated with a rise in PTHrP and suggests that PTHrP was able to
stimulate production of 1,25(OH)2D3 in this setting. This lends further support to the hypothesis that the decrease in 1,25(OH)2D3 seen in HHM is related to other tumor-related factors. In summary, available evidence, both in vitro and in vivo in animal models and humans, clearly defines the role of PTH as an activator of 1,25(OH)2D3 synthesis and an inhibitor of 24,25(OH)2D3 production. Aminoterminal fragments of PTHrP also stimulate 1,25(OH)2D3 production experimentally, both in vitro and in vivo in animals and humans. However, it is as yet unclear as to why patients with HHM and elevated PTHrP have reduced 1,25(OH)2D3.
Fibrobalst Growth Factor 23 (FGF23) A review of the recent literature reveals new understanding about an important factor in vitamin D metabolism, FGF23. FGF23 is a glycoprotein comprising 251 amino acids. It is secreted by osteocytes and osteoblasts in response to elevated serum phosphorus levels [62,63]. It was first identified from mouse embryos by homology-based PCR [64]. When loss-of-function mutations of FGF23 were shown to cause autosomal dominant hypophosphatemic rickets (ADHR), its importance as a regulator of phosphate and bone metabolism became apparent [65,66]. Studies by Shimada et al. then found that FGF23 was found in tumors causing tumor-induced osteomalacia (TIO) [67]. Recombinant FGF23 injected into mice resulted in renal phosphate wasting, hypophosphatemia, decreased 1,25(OH)2D and osteomalacia; the clinical picture seen in TIO [67]. Further studies by Bai et al. found, using an in vivo overexpression system, that FGF23 resulted in hypophosphatemia, low 1,25(OH)2D3, decreased expression of renal 1a-hydroxylase, and increased expression of 24hydroxylase [68]. Serum PTH was elevated, with evidence of parathyroid hyperplasia, but the authors attributed this to the low 1,25(OH)2D3 [68]. In another study by Shimada et al., when recombinant FGF23 was injected into normal and parathyroidectomized animals, serum phosphorus and 1,25(OH)2D decreased, no change in PTH levels, and FGF23 decreased renal mRNA for 1a-hydroxylase and increased it for 24hydroxylase [69]. Supporting the importance of FGF23 in regulation of phosphate and vitamin D, studies of FGF23 null mice and humans with homozygous missense mutations revealed hyperphosphatemia and excessive levels of 1,25(OH)2D [69e73]. In conclusion, FGF23 appears to be an important regulator of renal vitamin D metabolism, independent of PTH. Elevated FGF23 results in low 1,25(OH)2D levels, likely due to inhibition of renal 1a-hydroxylase and stimulation of 24-hydroxylase. Although much has been learned about FGF23 in the past few years, its
DISORDERS
HORMONE EFFECTS ON VITAMIN D METABOLISM
complete role in vitamin D metabolism still needs to be investigated. Further discussion of FGF23 can be found in Chapters 42 and 63.
Leptin Leptin, the white adipose tissue hormone that has been shown to have both peripheral and central effects [74], has been found in recent studies to play a role in renal vitamin D metabolism [75]. Leptin-deficient mice (ob/ob mice) have been shown to have low BMD, and elevated serum calcium, phosphorus, and 1,25(OH)2D3 levels compared to lean mice [76]. In addition, renal expression of 1a-hydroxylase mRNA and its activity, as well as 24-hydroxylase mRNA expression, were increased in leptin-deficient mice [76]. Injecting these mice with murine leptin corrected the electrolyte disturbance as well as decreased the expression of the renal hydroxylases [76]. Further work by Matsunuma et al. confirmed the above findings, and found that leptin did not inhibit 1a-hydroxylase expression as earlier work had suggested; other factors were proposed as being involved [77]. They hypothesized that this factor was FGF23 and that administration of leptin could suppress renal 1a-hydroxylase expression via bone-derived FGF23 [78]. In vivo studies using ob/ob mice, leptin injected at 200 ng/ml for 24 hours, stimulated FGF23 expression in primary cultured rat osteoblasts, and at higher doses significantly increased serum FGF23 levels while decreasing serum calcium, phosphorus, and 1,25OH2D3 levels [78]. Administration of FGF23 to leptin-deficient mice resulted in suppression of renal 1a-hydroxylase mRNA expression, with the main site of FGF23 expression in bone [78]. Leptin appears to have an indirect effect on vitamin D metabolism by stimulating osteoblast expression of FGF23. This is an example of how complex the regulation of vitamin D is, with many factors playing a role. It is unclear, however, if this is all species-specific. More clinical studies are needed to test the effect of leptin on vitamin D metabolism in humans. Chapter 44 goes into more detail about the connections between adipose tissue and bone.
Calcitonin The role of calcitonin in vitamin D metabolism has been thoroughly investigated in both in vivo and in vitro studies (see Chapter 41). Initial in vivo studies demonstrated a pronounced increase in 1,25(OH)2D3 levels and decrease in 24,25(OH)2D3 levels after the administration of synthetic salmon calcitonin to vitamin-D-deficient rats [79]. Subsequent studies attempted to clarify if this effect was independent of PTH. Lorenc and co-workers [80] found that following
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thyroparathyroidectomy (TPTX), the calcitonin effect was eliminated. In contrast, subsequent studies involving vitamin-D-deficient [81] and vitamin-D-replete [82] TPTX rats demonstrated increased production of 1,25(OH)2D3 following calcitonin administration. Furthermore, the actions of PTH and calcitonin were additive, suggesting independent actions [81]. This effect was seen in rats fed regular diets as well as calcium-free diets. There were no significant changes in 25(OH)D levels [82]. However, Shinki et al. [83] demonstrated that calcitonin administered to sham and TPTX normocalcemic rats caused an increase in the expression of renal 25hydroxylase and increased the conversion of 25(OH)D to l,25(OH)2D3. This effect was not seen in hypocalcemic rats whose 25-hydroxylase increased in response to PTH injection [83]. Calcitonin also appears to act as a negative regulator of intestinal 24-hydroxylase. The inhibition of intestinal 24-hydroxylase activity and expression may spare 1,25(OH)2D3 from deactivation and thereby result in enhanced l,25(OH)2D3 - mediated activity [84]. The majority of in vitro data have supported the positive effect of calcitonin on 1,25(OH)2D3 levels [85,86]. Similar to in vivo data, 1a-hydroxylase was stimulated by calcitonin in vitamin-D-deficient rat kidneys, post-TPTX [86]. This effect was independent of the adenylate cyclase system which, as mentioned earlier, is believed to be the mechanism of PTH stimulation of 1a-hydroxylase [19,81]. These findings suggest that calcitonin acts independently of PTH. In human studies, patients with medullary carcinoma of the thyroid and excess calcitonin had serum 1,25(OH)2D levels that were elevated [87]. However, therapeutic use of calcitonin has produced mixed results. Injectable [88] and nasal calcitonin [80,81] treatment daily for postmenopausal osteoporosis produce either no changes [88e90] or an increase in 1,25(OH)2D3 levels at 6 months, which returned to normal at 1 year, with PTH and 25(OH)D levels being unchanged during the study period [91]. No changes in 1,25(OH)2D3 levels were observed in patients with Paget’s disease of bone treated for 3 months with calcitonin, although in these patients 24,25(OH)2D3 was elevated, most probably due to decreased consumption by osteoclasts and osteoblasts [92]. Other studies involving the treatment of Paget’s disease of bone with nasal calcitonin did show a rise in 1,25(OH)2D3 after 18 months duration, yet longerterm therapy had no effect. Levels of other vitamin D metabolites such as 24,25(OH)2D3 and 25(OH)D were unchanged [93]. In summary, in vivo and most in vitro work suggests that calcitonin plays an important role in stimulating 1,25(OH)2D3 production. The less than convincing
DISORDERS
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67. DRUG AND HORMONE EFFECTS ON VITAMIN D METABOLISM
evidence seen in clinical studies may be related to the doses used or the route of calcitonin administration.
Growth Hormone and Insulin-like Growth Factor Growth hormone (GH) and its intermediary, insulinlike growth factor (IGF-1), have been shown to play a role in bone metabolism. One of the many functions of GH is to stimulate intestinal calcium absorption, which has been shown in both rats [94] and humans [95,96]. Although the exact mechanism of this effect is unknown, it may be a l,25(OH)2D3-dependent phenomenon. Hypophysectomy and subsequent GH deficiency in rats was shown to reduce the level of 1,25(OH)2D3 and to increase the conversion of 25(OH)D to 24,25(OH)2D3 [97e100]. GH replacement led to the restoration of normal levels of vitamin D metabolites [97e100]. No increase in the metabolic clearance or increase in tissue metabolism of 1,25(OH)2D3 occurred; thus, the most likely explanation is that hypophysectomy decreases the renal 1a-hydroxylase activity [99]. Furthermore, hypophysectomized rats fed a lowphosphate diet and replaced with rat GH had increased levels of 1,25(OH)2D3, as a result of stimulation of 1ahydroxylase activity [101]. The work by Zoidis et al., in hypophysectomized rats infused with both GH and IGF-1, showed that 1,25(OH)2D3 levels significantly increased, along with 1a-hydroxylase expression in the kidney, with downregulation of 24-hydroxylase mRNA [102]. A study in growing dogs given supraphysiologic doses of porcine GH found a significant increase in 1,25(OH)2D3 and in renal 1a-hydroxylase expression, with no difference in PTH levels between treated and control groups [103]. In other animal work, exogenous porcine GH also increased 1,25(OH)2D in intact pigs [104]. Clinical studies have yielded conflicting results. Acromegalic subjects, with endogenous GH excess, have increased levels of 1,25(OH)2D3 [32,105e108], which are reduced by treatment with bromocriptine, which decreases GH [105e108]. However, in a recent study of acromegalic patients treated with the somatostatin analog lanreolide, no effect was seen on 1,25(OH)2D levels [109]. Short-term recombinant GH therapy in healthy young [110] or elderly people [111] caused significant increases in 1,25(OH)2D, and increased vitamin D levels in response to phosphate depletion were found to be dependent on the presence of GH [112]. Two recent studies in which adult GH-deficient patients (Ahmad et al.) and postmenopausal women with osteoporosis (Joseph et al.) were treated with 12 months of GH found a significant elevation in 1,25 (OH)2D and decrease in PTH secretion [113,114]. However, other studies, have found that chronic GH
therapy does not seem to cause a rise in 1,25(OH)2D levels [115,116]. GH replacement given to children with GH deficiency did not alter 25(OH)D, 1,25(OH)2D, or PTH levels [117]. Interestingly, a study in children with end-stage renal disease found that higher doses of calcitriol were needed in those children treated with GH [118]. If GH does, in fact, increase 1,25(OH)2D levels in humans, the possible mechanism remains controversial. Although Marcus et al. [111] found that the increase is mediated by an increase in PTH, other studies have not arrived at the same conclusion [116,119]. More recent studies continue to find the effect to be independent of PTH. Joseph et al. found that despite the fall in PTH after GH administration in 14 postmenopausal women with osteoporosis, 1,25(OH)2D, urine phosphorus, and bone turnover markers all increased [114], likely reflecting GH/IGF-1 actions on peripheral tissues [120]. In the study by Ahmad et al., GH decreased PTH secretion and in some tissues (such as bone and kidney) enhanced PTH action [113]. Wei et al. [121] reported that children treated with GH experienced an increase in 1,25(OH)2D3 after 1 month of therapy, which subsequently decreased at 3 months. 24,25-Dihydroxyvitamin D decreased at both 1 and 3 months and returned to baseline at 6 months. No change in 25(OH)D was seen. IGF-1 levels increased in this study, while PTH levels declined, suggesting the effect of GH on vitamin D metabolism may be mediated by IGF-I [121]. A small, randomized crossover study of IGF-I versus GH found that IGF-I increased the free calcitriol index, while calcium, phosphate, and PTH levels were unchanged [122]. Similarly, Wright et al. [123] found that GH’s positive effect on 1,25(OH)2D levels was independent of PTH and likely mediated by IGF-1. In vitro evidence supports the above clinical findings and suggests that the effects of GH in vivo on 1,25(OH)2D3 production are indirect, as GH fails to stimulate 1,25(OH)2D3 production in chick renal preparations [124]. IGF-I receptors are present on the basolateral membrane of renal proximal tubules [125], and low concentrations of exogenous IGF-I enhance 1,25(OH)2D3 synthesis when phosphate concentrations are low [126]. In vivo animal studies also show that the GH-dependent increases in serum 1,25(OH)2D3 levels induced by dietary phosphate restriction may be mediated by IGF-I, as administration of IGF-I to hypophysectomized rats increases serum 1,25(OH)2D3 to approximately the same degree as GH [127,128]. In addition, Gray [129] showed that serum concentrations of 1,25(OH)2D3 are directly related to the serum levels of IGF-I in rats fed a low-phosphate diet. IGF-I was also shown to stimulate renal 1a-hydroxylase activity in a time- and dose-dependent manner in weanling mice that were phosphate-depleted [130], but this was
DISORDERS
HORMONE EFFECTS ON VITAMIN D METABOLISM
also demonstrated to be independent of changes in serum calcium or phosphate [130]. Currently, no data suggest that IGF-II affects vitamin D metabolism. The majority of in vivo data seem to suggest that GH, via IGF-I and especially during hypophosphatemia, is an important regulator of serum 1,25(OH)2D levels. Data from human clinical studies are more conflicting but increasingly support the theory that GH, via IGF-I, regulates serum 1,25(OH)2D. This may partially explain the positive effects of GH on bone mass in patients with GH deficiency.
Prolactin Initial work involving prolactin (PRL) as a possible stimulator of 1,25(OH)2D3 synthesis came from in vitro studies of chick cell cultures. Spanos et al. [131e134] and Bikle et al. [124] found that ovine PRL could stimulate 1a-hydroxylase. Unlike the case in birds, however, in mammals, fish, and amphibians PRL does not seem to play a significant role in vitamin D metabolism [97,101,134,135]. In certain physiological situations, such as lactation, however, PRL may play a role in vitamin D metabolism. In rats, PRL stimulates intestinal transport of calcium [136e139], especially during lactation, when increased serum levels of 1,25(OH)2D3 are found [140,141]. Suppression of PRL by bromocriptine decreases 1,25(OH)2D levels in lactating rats, but the drug has no effect in non-lactating controls [142]. A study by Robinson et al. showed that when bromocriptine was given to lactating rats 1,25(OH)2D3 decreased to non-lactating levels but when PRL was given with bromocriptine, levels of 1,25(OH)2D3 increased back to baseline lactation levels [143]. The authors therefore suggested that PRL could stimulate 1,25(OH)2D3, resulting in enhanced absorption of calcium [143]. Other studies showed that prolactin may stimulate intestinal calcium absorption independent of 1,25(OH)2D3 [144,145]. However, a recent study by Ajibade et al. has shown that PRL may modulate the effects of 1,25(OH)2D3 in rats via multiple mechanisms, including a direct effect on the transcription of the 1a-hydroxylase gene [146]. The results in rats are likely species-specific. Although a study of rural Mexican women found that women who were lactating had higher 1,25(OH)2D than age-matched non-lactating women [147], many studies show that the elevated levels of 1,25(OH)2D3 seen in pregnancy decrease back to normal levels [148,149]. However, in support of the role of prolactin in vitamin D metabolism during lactation, a patient with hypoparathyroidism was found to require less exogenous vitamin D supplementation during lactation than at other times [150]. Conversely, a study of postpartum women, followed for 18 months, failed to find a relationship between
1251
vitamin D levels and serum prolactin, estradiol, lactation status, or PTHrP [151]. Another study of pregnant women did not find a relationship between PRL levels and 1,25(OH)2D3 [152]. The rise in PRL associated with lactation may, however, lead to a release of PTHrP, which is secreted by the breast during lactation into the circulation and into breast milk (see Chapter 41). Other than in lactating females, PRL most probably has no effect on plasma 1,25(OH)2D3 levels or calcium metabolism, as patients with hyperprolactinemia due to functioning pituitary adenomas had 25(OH)D and 1,25(OH)2D levels in the normal range versus age-matched controls, as well as similar serum calcium, phosphate, and PTH levels [32,153e157]. Although PRL seems to play a role in vitamin D metabolism in birds, this effect is likely species-specific. The majority of animal and clinical data suggest its positive effect on 1,25(OH)2D3 production is limited to states of physiological elevation in PRL, such as lactation. The recent study by Ajibade suggests that further investigation into the mechanisms involving PRL effects on calcium homeostasis are needed. For a more detailed discussion of the effects of lactation on vitamin D metabolism the reader is referred to Chapter 38.
Insulin The majority of recent literature on this topic addresses the role of vitamin D deficiency in the development of diabetes and insulin resistance, in addition to the immunomodulatory role of vitamin D on insulin resistance. This section, however, will be limited to the effect of insulin on vitamin D metabolism (see Chapter 94 for a discussion of vitamin D effects on the immune system in the prevention of type 1 diabetes). Patients with insulin-dependent diabetes mellitus (type 1) suffer from a number of disturbances in bone mineral metabolism, including alterations in vitamin D metabolism [158]. Work in streptozotocin- and alloxan-induced diabetic rats by Schneider and co-workers [159,160] first suggested a role for insulin in the production of 1,25(OH)2D. This experimental diabetic model is associated with a reduction in duodenal calcium absorption, calcium-binding protein, and total and ionized calcium levels [159,160]. Treatment with 1,25(OH)2D3 but not 25(OH)D corrects subnormal calcium absorption in these rats [161], as does insulin replacement [162]. These data suggest that a lack of insulin in diabetes impairs 1ahydroxylation. Further support of this concept emerged after finding that 1,25(OH)2D levels in streptozotocininduced diabetic rats were depressed to one-eighth the level in control rats and were returned to control values with insulin treatment [163]. No changes in serum 25(OH)D were found, suggesting either decreased 1a-hydroxylation of 25(OH)D or increased catabolism
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67. DRUG AND HORMONE EFFECTS ON VITAMIN D METABOLISM
of 1,25(OH)2D. A recent study in diabetic rats also found decreased 1,25(OH)2D levels compared to control, with no change in serum 25(OH)D levels [164]. It is possible that a change in PTH is responsible for decreased conversion of 25(OH)D. It has been shown that PTH levels increase in diabetic rats, probably secondary to calcium malabsorption resulting from decreased 1,25(OH)2D [165e168]; however, some studies have reported low PTH levels in streptozotocin-induced diabetic rats [169]. To further localize the site at which insulin is proposed to act, Spencer et al. [170] studied the conversion of 3H-25(OH)D to 3H-1,25(OH)2D3, as well as the metabolic clearance of 3H-1,25(OH)2D3 in control, streptozotocin-induced diabetic and insulintreated streptozotocin-diabetic rats. The results showed that the metabolic clearance was not increased in diabetic rats and that the in vivo conversion of 3H-25(OH)D to 3H-1,25(OH)2D3 was reduced by 60% in diabetic rats, which normalized with insulin therapy. No intrinsic intestinal mucosal defect in the incorporation of 3H-1,25(OH)2D3 was evident [170]. These results further support the notion that a lack of insulin impairs 1a-hydroxylation. The osteopenia seen in streptozotocin-induced diabetic rats [168,169] is also significantly attenuated by treatment with 25(OH)D, suggesting that vitamin D deficiency contributes to this bone loss [171]. Other studies in diabetic animals have demonstrated a decrease in 1a-hydroxylase activity and an increase in 24-hydroxylase activity [172,173]. Another study in the diabetic animal model found that insulin deficiency directly inhibited 25-hydroxylase activity despite finding normal levels of serum 25(OH)D [174]. Insulin may also play an important role in the stimulation of renal 1,25(OH)2D synthesis in response to phosphate deprivation. In streptozotocindiabetic rats, 1,25(OH)2D rose only slightly following phosphate deprivation, as compared with a marked response in rats replaced with insulin [175]. Decreased serum vitamin-D-binding protein (DBP) has also been reported in experimentally induced diabetic rats, resulting in decreased total 1,25(OH)2D and normal free 1,25(OH)2D [176]. In vitro evidence suggests a permissive role of insulin in vitamin D metabolism. Primary cultures of chick kidney cells in serum-free medium respond to PTH with increased production of 1,25(OH)2D3 only when exposed to insulin [177]. Confirming in vitro work, Wongsurawat et al. [167], using a renal slice technique, showed reduced 1,25(OH)2D3 and increased 24,25(OH)2D3 levels in streptozotocin-induced diabetic rats, which reversed with insulin therapy. PTH levels were found to be higher in diabetic rats relative to controls [167]. Renal resistance to PTH has been suggested as the mechanism by which 1a-hydroxylase is
depressed in these rats [178]. However, Wongsurawat et al. showed a normal cAMP response to PTH in streptozotocin-induced diabetes [167], which makes this theory unlikely. There are also in vitro data to suggest that insulin increases the capacity of PTH to increase in the activity of 24-hydroxylase [179]. Clinical studies have shown decreased 1,25(OH)2D3 concentrations with increased 24,25(OH)2D3 and normal 25(OH)D levels in insulin-dependent diabetic children [180,181] and in poorly controlled African diabetics [182]. However, no abnormalities of calcium or vitamin D metabolism have been found in adult insulin-dependent diabetics [183]. In summary, diabetic animals show abnormalities in vitamin D metabolism with depressed levels of 1,25(OH)2D3, increased 24,25(OH)2D3, and normal levels of 25(OH)D. The decrease in 1,25(OH)2D3 may be secondary to decreased 1a-hydroxylase activity or depressed DBP; the effects on PTH levels vary [184]. In human studies findings have been inconsistent, which may be due to variations among the study populations. Alternatively, the animal model of diabetes may differ from diabetes in the clinical setting with regard to changes in vitamin D.
Sex Steroids Estradiol The possible role of estrogen in vitamin D metabolism was first demonstrated in avian studies involving egglaying Japanese quail [185]. Increased estradiol, either via ovulation [185] or exogenous administration [186], resulted in enhanced production of 1,25(OH)2D3. In females 24,25(OH)2D3 was stimulated, but in males the concentration of this metabolite was reduced. Further studies supported the notion that estradiol might be a regulator of vitamin D. Castillo et al. [187] administered 5 mg of estradiol to mature male quail and found that it markedly stimulated 1a-hydroxylase and suppressed 24-hydroxylase activity 24 hours after administration. In castrated male chickens, estradiol injections stimulated 1,25(OH)2D3 production in vitro, but only in the presence of testosterone or progesterone [188]. Similarly, the injection of stilbestrol, an estrogenic drug, into immature male chickens stimulated 1ahydroxylase and suppressed 24,25(OH)2D3 production in chick kidney homogenates [189]. Recent in vitro work supports the above findings of increased 1,25 (OH)2D synthesis and upregulation of 1a-hydroxylase in response to estradiol [190e192]. In addition, multiple studies have shown that treatment with estradiol results in increased expression of the vitamin D receptor (VDR); in the uterus [193,194], liver [195], and in human breast cancer and colon cancer cells [196].
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In mammalian studies, female rats treated with estradiol benzoate daily for 8 days were found to have increased 1,25(OH)2D3 concentrations in plasma, gut mucosa, and kidneys [197]. Others have not found a rise in 1,25(OH)2D levels in rats treated with estradiol, but have recorded increased in vivo intestinal absorption of calcium [198e200]. In keeping with these findings, another study in rats found that estradiol’s ability to increase intestinal calcium absorption was independent of 1,25(OH)2D3 [201]. A study by Criddle et al. [202] suggests the decrease in renal calcium excretion associated with estrogen replacement is independent of 1,25(OH)2D3 and may be dependent on increased expression of calbindin D28k protein, located in the distal renal tubule. Furthermore, Liel et al. [203] found that the administration of estrogen to ovariectomized rats was found to increase intestinal calcium absorption by increasing the activity of duodenal vitamin D receptors (VDRs), rather than increasing levels of 1,25(OH)2D3. However, in a study by Ash and Goldin [204], both young and old ovariectomized rats administered 3H-25(OH)D had reduced 3H-1,25(OH)2D3 production, which was increased with estradiol replacement. In the same study, parathyroidectomy eliminated estradiol’s therapeutic effect on 3H-1,25(OH)2D3 recovery, which implicates PTH. However, in a recent study by Carillo-Lopez et al. using a rat model with CKD with ovariectomy and exogenous administration of estradiol, no ERa or ERb mRNA protein was found in the parathyroids, suggesting an indirect effect of estrogens on PTH regulation [205]. Estradiol may either act directly on the parathyroid gland or act by decreasing bone resorption and lowering serum calcium, thus triggering PTH secretion [204]. Despite the data, these findings have not been consistently demonstrated in ovariectomized rats by others [198,206]. Of note, similar to in vitro work mentioned previously, in vivo data also demonstrate that estrogen increases the expression of VDR in bone [207]. Clinically, plasma 1,25(OH)2D3 levels are elevated in human pregnancy and remain high postpartum in lactating women [147,148] (see Chapter 38). Levels of 25(OH)D in pregnant women are similar to those of controls [208]. However, another study did find a 39% increase in 25(OH)D levels in premenopausal women receiving oral contraceptives compared with non-users [209]. Despite an expected increase in DBP, free 1,25(OH)2D3 levels are also elevated in the pregnant state [210]. Low [211,212] and normal levels [213] of 1,25(OH)2D have been reported in early postmenopausal women. The increased bone resorption associated with the postmenopausal period leads to a slight elevation of serum calcium, which decreases PTH secretion, and subsequently reduces renal 1a-hydroxylase activation and 1,25(OH)2D production. This ultimately
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results in decreased calcium absorption and negative calcium balance [214]. The fall in calcium absorption seen during this period, however, appears to be only partially explained by this fall in 1,25(OH)2D levels [215]. An alternative explanation for the decline in calcitriol is that estrogen deficiency may alter the responsiveness of PTH to changes in calcium. However, in a study of 16 women who were rendered estrogen deficient via administration of a GnRH analog, no change in the ability of PTH to respond to changes in calcium or stimulate 1,25(OH)2D was observed [216]. Serum levels of 25(OH)D have been reported as unchanged [211,213] or increased [217] in various studies. Studies in postmenopausal women have demonstrated increased 1,25(OH)2D levels after both shortterm and long-term estrogen therapy [212,218e220]. In women with postmenopausal osteoporosis, estrogen replacement results in decreased bone resorption that lowers serum calcium, which subsequently stimulates PTH and renal 1a-hydroxylase leading to increased 1,25(OH)2D production. The latter increases intestinal calcium absorption [221]. However, Stock et al. [222], using sensitive assays for PTH, failed to show a rise in PTH levels accompanying the elevated 1,25(OH)2D in postmenopausal women treated with estradiol. In fact 2 weeks of treatment with estradiol decreased PTH levels. This suggests that the estrogen effect on vitamin D metabolism may not only be secondary to a change in PTH [222]. Estradiol-induced elevation of serum 1,25(OH)2D may occur as a result of increased DBP levels. A higher concentration of DBP would increase total 1,25(OH)2D3, but leave free 1,25(OH)2D3 unchanged [223,224]. However, as in pregnancy, free levels of 1,25(OH)2D3 were shown to be elevated in response to estradiol treatment [225]. While the majority of recent in vitro work points to a direct effect of estrogen on vitamin D metabolism, the evidence in mammals is inconclusive. However, the majority of studies have found a positive effect of estrogen on 1,25(OH)2D levels. This effect may be indirect as estrogen alters intestinal calcium absorption, bone resorption, and PTH levels which increases, in turn, 1,25(OH)2D levels. Testosterone Evidence for a role for testosterone in vitamin D metabolism was first demonstrated in avian studies. An interdependence and synergism between testosterone and estradiol were observed during the in vitro stimulation of 1a-hydroxylase in castrated male chickens [188]. Castillo et al. [187] also demonstrated that testosterone alone administered to the male quail suppressed 24-hydroxylase but produced little change in 1a-hydroxylase. Again the data among species are not uniform. Hypoandrogenemia as a result of
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orchiectomy in rats has produced mixed findings, with either no change in total or free vitamin D metabolites [226] or decreased 1,25(OH)2D3 and DBP with normal free 1,25(OH)2D3 levels [227,228]. A recent study in orchidectomized mice, either treated with or without testosterone, found no difference in 1,25(OH)2D3 or PTH levels [229]. Castrated male guinea pigs demonstrated a 50% decline in 1a-hydroxylase activity, which was reversed with testosterone replacement. Ovariectomized female guinea pigs also responded to testosterone therapy with a 50% increase in 1a-hydroxylase activity. Both groups, however, had similar serum levels of 1,25(OH)2D3, DBP, and free 1,25(OH)2D levels versus controls [230]. Of note, testosterone administration to sexually immature, vitamin-D3-replete male chicks was shown to decrease circulating levels of 1,25(OH)2D3, while intestine and bone concentrations were significantly increased [231]. There are limited data on the effect of testosterone on 25(OH)D levels. One study demonstrated an elevation in serum 25(OH)D levels following androgen treatment in ultraviolet-irradiated rats compared to those that did not receive testosterone [232]. Studies in humans have been focused on hypogonadal men. A recent prospective study of 2310 men investigating the association of vitamin D and serum androgen levels in men found that in the 18% that were hypogonadal, there was a significantly lower mean 25(OH)D level compared to men with sufficient levels [233]. Hagenfeldt et al. [234] studied hypogonadal men before and after treatment with testosterone enanthate every 3e4 weeks, for varying lengths of time. They found that basal serum 1,25(OH)2D, DBP and free 1,25(OH)2D concentrations were similar to those of controls; however, testosterone treatment still increased total 1,25(OH)2D and free 1,25(OH)2D significantly. In contrast, Morley et al. [235] looked at the effects of testosterone replacement therapy in elderly hypogonadal males (mean age 77.6 2.3 years) and found no effect on PTH or serum vitamin D metabolites pre- or post-treatment. Studies in hypogonadal men with osteoporosis have found both normal [236,237] and low [238] serum 1,25(OH)2D levels. In the latter study by Francis et al. [238], testosterone replacement therapy increased both total and free 1,25(OH)2D levels. However, patients treated with orchidectomy for prostate cancer were found to have no changes in total 1,25(OH)2D or DBP concentrations [239]. In studies involving pubertal boys, results have been equivocal. Krabbe et al. [240] found no changes in the serum levels of vitamin D metabolites before and after peak pubertal testosterone surges, whereas Aksnes et al. [241] did find an increase in vitamin D. It is interesting to note that androgens have been shown to regulate vitamin D receptors in epithelial
and stromal cells of human prostate cells [242]. Moreover, genetic polymorphisms in the vitamin D receptor have been shown to be associated with prostate cancer [243,244]. Please refer to Chapters 53, 82 and 56 for detailed discussions of these topics. In summary, it is unlikely that testosterone is a major controlling factor in vitamin D metabolism, but it may play a minor role in overall vitamin D homeostasis. Hypogonadal men, especially those at risk for osteoporosis, should be monitored for vitamin D deficiency and receive supplementation if necessary. Progesterone Work by Tanaka et al. [188] demonstrated that in castrated male chickens progesterone, like testosterone, supported the stimulation of 1a-hydroxylase by estradiol. As previously mentioned, further marked stimulation of 1a-hydroxylase activity occurred with combined progesterone, testosterone, and estradiol treatment [188], demonstrating pronounced synergy among the sex steroids. In Japanese female quail treated with progesterone in vitro, renal production of 1,25(OH)2D3 was stimulated; however, this was significantly less than that of estradiol [186]. In the same study, immature male quail treated with progesterone had increased 24,25(OH)2D3 production [186]. Similar findings were recorded by Castillo et al. [187]. Unlike in birds, treatment of ovariectomized rats with progesterone led to an increase in 25(OH)D, while 1,25(OH)2D3 levels were similar to controls [199]. In postmenopausal women, progesterone in combination with estrogen was found to lower estradiolstimulated increases in total and free vitamin D levels [245], and norethisterone treatment caused a slight decrease in free and total vitamin D [224]. No effects on vitamin D metabolites following medroxyprogesterone therapy were seen in male patients treated for glucocorticoid-induced osteoporosis [246]. Although the role of progesterone in vitamin D metabolism has been less extensively investigated than the other sex steroids, the available literature suggests that it likely has a minor function, if any.
Thyroid Hormone The effect of thyroid hormone on vitamin D metabolism is of particular interest given the concern over the impact of this hormone on bone metabolism. In an extensive review on bone and mineral metabolism in thyroid disease, Auwerx and Bouillon [247] described the changes encountered in this disease. In hyperthyroidism, excess thyroid hormone stimulates bone resorption [247e250], which increases serum calcium and phosphate concentration with resultant suppression of PTH [249,251e253] and a decrease in
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1,25(OH)2D3 production. This leads to lower intestinal calcium absorption [254,255], which Peerenboom et al. [255] had earlier demonstrated to be reversible after treatment of the thyroid abnormality. However, recent studies by Zaidi et al. demonstrate that TSH itself directly regulates skeletal remodeling and, in addition to elevated thyroid hormone levels, a low TSH also plays a role in bone metabolism [256,257]. Another possible factor contributing to low plasma 1,25(OH)2D3 levels found in hyperthyroidism is that of enhanced metabolic clearance of 1,25(OH)2D3. Karsenty et al. [258] studied seven hyperthyroid patients and found increased 1,25(OH)2D clearance after administration of tritiated 1,25(OH)2D3. Hyperthyroidism has been reported to be associated with either unchanged [255,259e261] or decreased [262,263] levels of 25(OH)D. Yamashita et al. found that 68% of men and 29% of women who were undergoing subtotal thyroidectomy for Grave’s disease had vitamin D deficiency, defined as 25(OH)D <25 nmol/l [264]. In fact, despite normal bone mass and markers of bone turnover, 25(OH)D and 1,25(OH)2D levels have been shown to be persistently reduced in euthyroid patients who were 6 years post-treatment of hyperthyroidism [265]. Dietary vitamin D intake and exposure to sunlight may contribute to these differences in 25(OH)D. Circulating levels of 24,25(OH)2D3 are generally increased in hyperthyroid patients [260,261]. Hypothyroidism is associated with decreased bone turnover [266] and low serum calcium levels that activate PTH, which, in turn, enhances 1a-hydroxylase activity [253,259,267]. Finally, serum-binding protein, DBP, does not appear to be affected by thyroid status [259]. Similar to human hyperthyroidism, daily injections of L-thyroxine to rats decreased 1,25(OH)2D3 and increased 24,25(OH)2D3 and 25(OH)D levels [268]. Hypothyroidism in the rat induced by the drug propylthiouracil, however, produces confusing results in that it is associated with low calcium and phosphate levels and decreased 1,25(OH)2D3 concentration [269]. There is some in vitro evidence to suggest that thyroid hormone directly affects renal 25(OH)D metabolism. Kano and Jones [270] found that thyroxine, triiodothyronine, and thyrotropin (TSH) decreased 1,25(OH)2D3 synthesis in perfused rat kidneys from vitaminD-deplete rats, whereas 24,25(OH)2D3 synthesis was increased in kidneys from vitamin-D-replete rats. Miller and Ghazarian [271] also demonstrated a 50% reduction in 1a-hydroxylase activity in vitamin-D-deficient chicks and stimulation of 24-hydroxylase activity in vitaminD-replete chicks following thyroxine administration. The majority of evidence suggests that thyroid hormone indirectly affects vitamin D metabolism via alterations in serum calcium, phosphate, and PTH. A direct action of thyroid hormone on renal hydroxylase
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activity, however, has been suggested by in vitro work. Again, the clinical significance in vitamin-D-replete states is unknown.
Prostaglandins Much of the recent literature on prostaglandins and vitamin D focuses on the effect of calcitriol on prostaglandins and subsequent antitumor effect in prostate, and more recently, breast cancer cells. However, this section will focus solely on the effect of prostaglandins on vitamin D metabolism. This relationship has been investigated in vitro and in vivo. Work by Trechsel et al. [272] in primary chick kidney cell cultures found that the addition of prostaglandin E2 (PGE2) and prostaglandin F2a (PGF2a) stimulated 1a-hydroxylase activity in a dose-dependent manner. PGE2 also significantly decreased 24-hydroxylase activity [272]. A proposed mechanism of action is that prostaglandins act through an increase in cAMP [272,273], but as discussed below this has not been proven. Further in vitro work by Wark et al. [274] in isolated renal tubules, prepared from vitamin-D-deficient chicks, showed that the addition of PGE2 to the tubule incubation medium in the presence of 1,25(OH)2D3 increased 1,25(OH)2D3 production. Acetylsalicylic acid, which inhibits prostaglandin synthesis, decreased the prostaglandin content of the tubule medium and subsequently inhibited 1,25(OH)2D3 production. Furosemide raised prostaglandin content in a dose-dependent manner, which led to a significant increase in 1,25(OH)2D3 production and decreased 24,25(OH)2D3 synthesis [256]. Kurose et al. [275] found similar results in vitro. in vivo work involving vitamin-D-deficient TPTX rats by Yamada et al. [276] found that, after an intra-arterial infusion of PGE2, 1,25(OH)2D3 production was significantly stimulated. No changes in plasma calcium or phosphate levels or urinary cAMP excretion were observed, suggesting that the effects of prostaglandins are independent of the cAMP system [276]. In a further rat study, the effects of PGE2 on the actions of PTH and calcitonin and the conversion of 3 H-25(OH)D to 3H-1,25(OH)2D3 were investigated. PGE2 inhibited calcitonin stimulation of 1,25(OH)2D3 but had no effect on PTH-stimulated 1,25(OH)2D3 production. This suggests that PGE2 may modulate the actions of calcitonin, but not those of PTH on 1a-hydroxylase [277]. Reduced plasma levels of 25(OH)D and 1,25(OH)2D were also found following the administration of indomethacin, a potent inhibitor of prostaglandin synthesis, to pregnant rabbits [278]. In contrast to the above findings, a lack of effect of prostaglandin in vivo has been shown by Katz et al. [279], who administered PGE2 by subcutaneous injection daily for 3 weeks to rats. They found no change in
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1,25(OH)2D or PTH levels, although a significant increase in bone mass in PGE2-treated rats was demonstrated [279]. Furthermore, long-term subcutaneous administration of indomethacin failed to alter 1,25(OH)2D or affect histomorphometric indices of bone formation and resorption [280]. The majority of studies look at the effects of PGE2 on vitamin D metabolism; however, recent in vitro and in vivo work using rabbits, by Velasquez-Forteo et al., showed that prostaglandin E1 (PGE1) affects vitamin D biosynthesis [281]. Significant elevations in 1,25(OH)2D3 and calcium were found in rabbits treated with PGE1 at 50 mg/d for 20 days. No effect was seen on either PTH or 25(OH)D [281]. In vitro data using rabbit proximal renal tubules showed similar results with increased 1,25(OH)2D3 synthesis after administration of PGE1 [263]. Limited clinical data are available to assess the role of PGE2 in humans. In one study, elevated levels of PGE2 were believed to be the cause of hypercalcemia and enhanced 1a-hydroxylase activity in children with Bartter’s syndrome [282]. Another study of children with idiopathic hypercalcuria found a positive correlation between 1,25(OH)2D3 and prostaglandin E2 activity [283]. Overall, although it would seem that prostaglandins stimulate 1a-hydroxylase in vitro, variable results in vivo create doubt over the exact contribution that prostaglandins make in vitamin D metabolism. Again, the problem of which particular animal species is used may confound the results. For a discussion of the tissue-specific effects of vitamin D on COX-2 and prostaglandin synthesis and signaling, see Chapter 86.
Tumor Necrosis Factor-a and Interferon-g Recently many papers have been published about the effects of vitamin D on cytokines and the immune system. This section will focus, however, on the effects of cytokines on vitamin D metabolism. The majority of information regarding these effects has been derived from in vitro studies. Pryke et al. [284] examined the effects of tumor necrosis factor-a (TNFa) on 1a-hydroxylase activity in cultured alveolar macrophages. Incubation for 6 days with 50 IU TNFa resulted in an average four-fold increase in 1,25 (OH)2D3 production. An increase in 1a-hydroxylase activity was maximally reached at 72 hours. This finding may account for the “spontaneous” or local production of 1,25(OH)2D by non-renal cell 1a-hydroxylase activity, in this case the sarcoid macrophages encountered in sarcoidosis [284] (see Chapter 45). A further study by Bikle et al. [285] provided evidence that TNFa stimulates 1,25(OH)2D production in human keratinocytes. In preconfluent cells, TNFa
stimulates 1,25(OH)2D production; however, this ceased once the keratinocytes achieved confluence [285]. TNFa has also been shown to induce 1a-hydroxylase activity in human endothelial cells [286], and in human peripheral blood monocytes [287]. No effect of TNFa has been shown on bone cell lines, but TNFa was shown to inhibit VDR content in osteoblastic cells [288]. TNFa, via activation of NF-kB, has also been shown to decrease osteoblast transcriptional responsiveness to 1,25(OH)2D3 [289]. This may be one of the mechanisms by which TNFa contributes to bone loss in disease states such as postmenopausal osteoporosis and inflammatory arthritis. However, the predominant role of TNFa may be independent of 1,25(OH)2D3 and result from osteoclastic effects on bone resorption. Interferon-g (IFN-g) also appears to play a similar role in vitamin D metabolism. Cultured normal human pulmonary alveolar macrophages in the presence of IFN-g increased 1,25(OH)2D3 production in a dosedependent manner [290]. Bikle et al. [291] provided confirmatory findings showing that IFN-g stimulated preconfluent keratinocytes to produce 1,25(OH)2D. This increase in 1,25(OH)2D, as with TNFa, is likely due to upregulation of 1a-hydroxylase activity. Stoffels et al. found that when IFN-g was added to human peripheral blood monocytes, 1a-hydroxylase activity was induced; and this effect was even greater with the co-addition of lipopolysaccharide (LPS) [292]. Bone marrow-derived macrophages were also demonstrated to respond to IFN-g with enhanced 1,25(OH)2D3 production [293]. Because lung T lymphocytes from sarcoidosis patients produce IFN-g, this may play a role in extrarenal 1,25(OH)2D3 production in vivo in sarcoidosis [294]. Little is known about the in vivo effect of IFN-g on vitamin D metabolism. Mann et al. [295] studied the effect of IFN-g on bone mineral metabolism in rats and reported no changes in serum ionized calcium, PTH, or 1,25(OH)2D3 levels, whereas IFN-g had a significant osteopenic effect. Human studies on the effects of these cytokines are lacking, and the underlying disease for which these factors are therapeutically administered may influence the results (e.g., chronic active hepatitis and malignancy). In summary, these inflammatory cytokines may not change blood levels of 1,25(OH)2D but by increasing local production at sites of inflammation, they may have important local/paracrine effects, not just in sarcoid but, for example, rheumatoid arthritis, inflammatory bowel disease, and other inflammatory diseases. The reader should refer to Chapter 45 for further discussion of extra-renal 1a-hydroxylase activity in inflammatory diseases and sarcoidosis.
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DRUG EFFECTS ON VITAMIN D METABOLISM The effects of drugs on vitamin D metabolism are summarized in Table 67.2.
Anticonvulsants Anticonvulsants have long been recognized to cause a number of alterations in bone mineral metabolism. The following drugs will be discussed in this section: phenobarbital, phenytoin, carbamazepine, oxcarbazepine, primidone, valrpoic acid, ethosuxamide, dilantin, clonazepam, toparimate, gabapentin, aborigine, lamotrigine, levetiracetam, and zonisamide. In 1968 Kruse [296] first reported osteomalacia resulting from the use of anticonvulsants, and since then there have been numerous documentations of this finding [297e299]. However, the effect of these agents on vitamin D metabolism remains controversial and animal and clinical studies have yielded conflicting results. Animal studies have demonstrated enhanced metabolism [300] and biliary excretion [301] of vitamin D. Although Hahn et al. [302] showed an initial increase in 25(OH)D after phenobarbital treatment in the rat, this was followed by a subsequent decline in levels. However, Ohta et al. [303] reported no effect of lowdose phenytoin treatment on serum 25(OH)D or 1,25(OH)2D3 under conditions where the rats were fed a vitamin-D-supplemented diet. More recent animal data revealed that administration of phenytoin to growing rats for 5 weeks led to a significant decrease in osteocalcin levels, while there were no significant changes in serum calcium, pyridinoline, 25(OH)D, or PTH compared with vehicle-treated rats [304]. This same study showed decreased trabecular bone volume and trabecular thickness, without significant change in osteoid thickness in phenytoin-treated animals. Another study also showed affected bone quality in rats treated with phenytoin, valproic acid or levetiracetam: treatment with phenytoin and valproic acid resulted in decreased bone mineral density and content while low-dose levetiracetam treatment led to decreased biomechanical strength in the femoral neck [305]. However, in this study osteocalcin levels were not significantly affected in the phenytoin group, but they were increased in rats treated with valproic acid and increased with low-dose levetiracetam [305]. Although clinical studies have shown that these effects on bone density may be attenuated by vitamin D3 treatment [306], more studies are needed to assess the effects of anticonvulsants on bone strength [307]. A number of biochemical changes have been demonstrated in humans after the use of anticonvulsants such as phenobarbital, phenytoin, carbamazepine,
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primidone, valproic acid, ethosuxamide, dilantin or those mentioned in combination. Serum calcium is reduced [298,308e310], which leads to the development of secondary hyperparathyroidism [298,310,316,318]. However, serum phosphate levels have been reported to be unchanged [298,310,314,316,318]. Levels of 1,25(OH)2D have been reported to be high [319,335,326], normal [317,319,321], or low [318], while 24,25(OH)2D3 levels have been shown to be decreased [326,327] following long-term anticonvulsant use. An increase in 1,25(OH)2D concentration may be secondary to increased PTH secretion. Levels of DBP are reported as unchanged [318], and intestinal absorption of dietary vitamin D is not altered by anticonvulsant drugs [298]. The overwhelming majority of reports demonstrate low serum levels of 25(OH)D [298,310e320]; however, there are some studies in which 25(OH)D levels are unchanged [299,321e324]. These conflicting data have been thought to be due to differences in study design and, in particular, differences in the ambulatory status of subjects [328,307]. One might hypothesize that the variation in vitamin D levels may be more related to exposure to ultraviolet light than to the direct effect of these agents on vitamin D metabolism. To control for this potential important confounder, more recent studies have attempted to characterize the skeletal health of ambulatory patients. A study of 30 ambulatory adult patients treated with phenytoin, carbamazepine, or valproate found significantly lower 25(OH)D levels than age- and sex-matched controls [329]. The decrease in 25(OH)D was independent of anticonvulsant type. In contrast, a study of 18 ambulatory pediatric patients on valproate or carbamazapine found normal levels of vitamin D and PTH [330]. Another study of 60 pediatric patients receiving carbamazapine also found evidence of normal vitamin D metabolism [331]. Anticonvulsants are unlikely to have uniform effects on vitamin D metabolism. Phenobarbital, phenytoin, carbamazepine, and primidone are well-known inducers of hepatic cytochrome P450 enzymes [332e334]. The observed reduction in 25(OH)D levels with these agents is thought to arise from their enhancing the hepatic breakdown of vitamin D into inactive polar metabolites other than 25(OH)D [301,312]. Hahn et al. [312] demonstrated, both in vivo in humans and in vitro in rat liver, that phenobarbital stimulated the conversion of 25(OH)D to more polar metabolites. Serum 25(OH)D levels are clearly decreased in patients receiving phenobarbital [311e314,320,335,336] and phenytoin [311,313,314, 317,320,326,337]. Conflicting evidence, however, exists regarding the effect of carbamazepine on 25(OH)D levels. Some reports show no change [328,338,340], and some show decreased levels [311,339e341, 343e345] with carbamazepine. Oxcarbazepine, a newer
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medication which acts similarly to carbamazepine, seems to be associated with an overall decrease in 25(OH)D levels [346e348]. Other newer anticonvulsants such as valproate, aborigine, clonazepam, gabapentin, topamirate, zonisamide, and levetiracetam are non-inducers of cytochrome P450 enzymes. Although Nicolaidou et al. found a decrease in 25(OH)D levels in patients on valproic acid [341], the majority of studies report no reduction of 25(OH)D concentration with the use of sodium valproate [311,313]. Yet, osteoporosis has been reported in children on chronic valproic acid therapy [349,350], and long-term use of valproate and lamotrigine has been shown to be associated with reduced bone formation [351]. Animal studies using zonisamide have shown a significant decrease in BMD [352,353]; however, this has been attributed to carbonic anhydrase inhibition [346]. Limited data exist on the effects of levetiracetam on vitamin D metabolism. Of note, a study of 71 patients on chronic anticonvulsant therapy found no significant difference in 25(OH)D levels between patients on CYP P450 inducers versus non-inducers; despite this, patients on inducers did have lower BMD than patients on non-inducers [354]. Various factors that have been shown to be associated with more severe changes in vitamin D metabolism include: polytherapy [308,311,315,320,355], larger total daily dose [308,314,317,320], duration of therapy [314], and female sex [318]. Treatment of anticonvulsantinduced alterations of vitamin D with replacement therapy of 400e4000 IU/day of vitamin D3 has been shown to be effective in normalizing parameters of mineral metabolism and improving bone mass [306,357,358]. In addition, treatment of anticonvulsant osteomalacia with small doses of oral 25(OH)D has also proved effective [315]. In summary, the overall results demonstrate that most anticonvulsants increase the metabolic degradation of 25(OH)D, and therefore it is recommended that vitamin D metabolites be consistently monitored. The prophylactic use of vitamin D supplements should be considered for patients on chronic anticonvulsant therapy. Those especially at risk include long-term institutionalized patients, those with reduced ultraviolet light exposure, those with poor dietary intake of vitamin D, and those receiving long-term treatment with multiple anticonvulsant drugs [357].
Corticosteroids Corticosteroid use is among the most important causes of secondary osteoporosis (see Chapter 66). Pharmacological levels of corticosteroids impair the intestinal absorption of calcium [359e362] and induce hypercalciuria [363,364]. Both effects lead to a state of
secondary hyperparathyroidism [364e367]. Hyperparathyroidism, however, does not appear to explain the observed effects of corticosteroids on the skeleton. The histology of bone exposed to glucocorticoids reveals decreased bone remodeling, while that of bone exposed to hyperparathyroidism shows increased bone remodeling [368]. It is now apparent that abnormal PTH regulation does not play a significant role in the development of glucocorticoid-induced osteoporosis [369,370]. In this section, we focus on potential actions of corticosteroids on vitamin D metabolism. There are multiple other pathways postulated for the effect of these agents on bone, including: decreased differentiation, number and function of osteoblasts, i.e. increased apoptosis, changes in synthesis and binding of skeletal growth factors, enhanced activity of 11-beta hydroxysteroid dehydrogenase type 1 in osteoblasts, decreased function and increased apoptosis of osteocytes and decreased apoptosis of osteoclasts [371,372]. Levels of 25(OH)D were initially shown to be low in glucocorticoid-treated men. Avioli and co-workers [373] reported evidence of impaired hepatic conversion of vitamin D to 25(OH)D. Furthermore, Klein et al. [361] demonstrated low 25(OH)D levels following high-dose prednisone therapy, although in patients receiving low or alternate-day doses 25(OH)D levels were unchanged. Similar findings were reported by Seeman et al. [365] in patients receiving high-dose glucocorticoids for the treatment of connective tissue disorders. These findings are plausible in light of the fact that corticosteroids are known to induce hepatic microsomal oxidase enzymes [332], and hence chronic glucocorticoid use could produce effects similar to chronic anticonvulsant use (see “Anticonvulsants,” above). However, other studies have failed to show a decrease in 25(OH)D concentrations. No differences in serum 25(OH)D levels have been shown in patients with corticosteroid-induced osteopenia [366,374,375] or in healthy [361] or diseased subjects treated with corticosteroids [376e378]. Patients treated with inhaled steroids for asthma also fail to demonstrate any alterations in 25(OH)D, 1,25(OH)2D3, or PTH levels [379,380]. Furthermore, patients with Cushing’s disease were also found to have normal levels of 25(OH)D [381,382]. Similarly, in animal studies, no effect on the conversion of vitamin D to 25(OH)D or 25(OH)D to 1,25(OH)2D3 has been reported [260,383,384]. Carre et al. [385] suggested that use of prednisolone may enhance the inactivation of 1,25(OH)2D3 to more polar biologically inactive metabolites at the tissue level in rats. The same study showed no alteration in the conversion of 3H-25(OH)D3 to 3H-1,25(OH)2D3 or 3 H-24,25(OH)2D3. This is in accordance with the findings of Favus et al. [384], who demonstrated normal
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conversion of tritiated 25(OH)D3 to 1,25(OH)2D3 and normal subcellular localization of 1,25(OH)2D3 in the intestinal mucosa of glucocorticoid-treated rats. In children treated with glucocorticoids for glomerulonephritis, 1,25(OH)2D3 concentrations have been shown to be reduced [386]. However, these children had proteinuria, which could decrease the levels of DBP. Adolescents with systemic lupus erythematosus also had low 1,25(OH)2D3 levels following treatment with glucocorticoids [387]. However, the effect of corticosteroids on 1,25(OH)2D levels remains controversial. Seeman et al. [365] reported no changes in 1,25(OH)2D3 levels in 14 patients with either endogenous or exogenous glucocorticoid excess. They also showed no differences in the production or metabolic clearance rate of 1,25(OH)2D [365]. Others have confirmed these findings of unchanged 1,25(OH)2D levels [359,375e377,383]. However, certain studies have shown increased 1,25(OH)2D values following subacute prednisone use [360,388e390], and in patients with Cushing’s disease 1,25(OH)2D levels are in the normal range but decrease following remission. This suggests that higher, though normal, values of 1,25 (OH)2D3 are present in the untreated state [381]. Increased levels of 1,25(OH)2D3 may be due to the state of secondary hyperparathyroidism produced by glucocorticoids. Cortisol has been shown to stimulate PTH secretion by rat parathyroid glands in vitro [391]. In vivo this has been demonstrated both in rats [392] and in humans following acute and chronic administration of glucocorticoids [364e367]. Elevated PTH is not a consistent finding, however, as normal PTH levels have been reported in both endogenous [381] as well as exogenous [360,365,375e377] glucocorticoid excess. Studies have also demonstrated no differences in levels of DBP [359] or concentrations of 24,25(OH)2D3 [360,375e377]. The exact effect that corticosteroids have on vitamin D metabolism remains controversial. The conflicting evidence may reflect the differences in experimental conditions including variations in dosages, dietary intake, and/or sunlight exposure, and very importantly the effect of the underlying disease for which the glucocorticoids were given. Peripheral enzymes, 11b-hydroxysteroid dehydrogenase (11bHSD) that interconverts active and inactive glucocorticoid molecules, may also play a role [371]. The elderly have been found to have enhanced sensitivity to glucocorticoids on bone, and this may be in part due to an increase in 11b-HSD type I activity, which occurs with aging [393]. Another explanation might be variation in genetic susceptibility to the effects of glucocorticoids among individuals. Recent studies show an association between glucocorticoid receptor polymorphisms and differences in BMD and body composition [394e396].
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The current literature, however, does not support an association between vitamin D receptor gene polymorphisms and corticosteroid-induced osteoporosis [397]. Chapter 66 provides a detailed discussion of other aspects of glucocorticoids and vitamin D.
Ethanol It is well known that chronic alcoholics suffer from bone disease, and abnormalities in vitamin D metabolism have been well described. Despite this, the most common histomorphometric abnormality seen in these patients is osteoporosis, rather than osteomalacia [398]. Serum levels of 25(OH)D have been reported to be low [399e406] or normal [407e409] among alcoholic patients. Serum 1,25(OH)2D has also been shown to be reduced [405,410,411] or unchanged [407]. This seems to be due to a combination of factors. First, many alcoholics have an inadequate dietary supply of vitamin D. A cross-sectional study of 181 male alcoholics revealed a small but significant association between bone mass and 25(OH)D; however, a marked association between nutritional status and bone mass was observed [409]. Second, many have insufficient exposure to the ultraviolet light of the sun for adequate synthesis of vitamin D. Malabsorption [413] and increased biliary excretion of 25(OH)D [414] are other possible factors involved. However, intestinal absorption of vitamin D has been shown to be normal in patients with alcoholic liver disease [409,415]. Induction by alcohol of the cytochrome P450 system may also occur with subsequent increase in the degradation of vitamin D metabolites in the liver [416]. Ethanol may also inhibit hydroxylase activity in the kidney [417] or liver. However, hepatic hydroxylation was normal in cirrhotic alcoholics [409,416,418]. Decreased DBP may lead to low levels of vitamin D metabolites in patients with cirrhotic liver disease [419,420]. It is important to note that acute ethanol ingestion has not been shown to cause changes in vitamin D metabolism [421]. One might hypothesize that PTH mediates the effect of ethanol on vitamin D metabolism. However, human and animal data on the effects of ethanol on PTH are conflicting. Acute ethanol loading in rats has produced both elevated [422] and suppressed [423] serum levels of PTH, whereas alcohol has been shown to stimulate PTH release from bovine parathyroid cells in vitro [424]. In humans, acute alcohol ingestion produces unchanged [425] or increased PTH [426] levels. Serum levels of PTH in chronic alcoholics have been normal [406,408e411,418], decreased [412], or increased [340,404,407,412], the latter probably secondary to diminished intestinal absorption of calcium [431e434]. Increased PTH secretion leads to inhibition of tubular reabsorption of phosphate and lower serum phosphate
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levels [435]. However, 1,25(OH)2D3 levels are not increased despite increased PTH and low phosphate, which suggests a direct inhibition of 1a-hydroxylase by ethanol [436]. The decreased PTH levels reported in some human studies may be related to hypomagnesemia [437]. In contrast to human studies, ethanol in rats seems to increase the production of 25(OH)D by the liver and lowers 1,25(OH)2D levels [438e441]. In a recent study by Shankar et al., chronic ethanol administration in rats was found to decrease 1,25(OH)2D3 levels as a result of both decreased synthesis at the level of the kidney and increased inactivation [442]. The difference in vitamin D metabolite levels between humans and rats is due to the presence of both mitochondrial and microsomal hydroxylases in the rat, as opposed to only mitochondrial enzymes in humans [443,444]. Ethanol inhibits mitochondrial enzymes, but induces microsomal ones. This might lead to increased 25(OH)D in rats by microsomal induction and to decreased 25(OH)D in humans by mitochondrial suppression [445]. Thus, the rat does not appear to be an appropriate model for assessing changes in vitamin D metabolism induced by ethanol. In summary, in chronic alcoholics, the circulating concentrations of vitamin D metabolites likely depend on the overall nutritional and health status of the individual [428]. A well-functioning alcoholic with satisfactory dietary ingestion and adequate sunlight exposure might be expected to have normal levels of circulating 25(OH)D or 1,25(OH)2D3. In contrast, non-functioning alcoholics with poor nutrition and reduced sunlight exposure are likely to have low 25(OH)D and 1,25(OH)2D3 levels [446].
Ketoconazole The imidazole antifungal agent ketoconazole has been shown to inhibit cytochrome P450-dependent enzymes [447], and thus might be expected to alter vitamin D metabolism. In vitro data demonstrated a dose-dependent reduction in 24-hydroxylase activity [448]; which was also seen with the imidazole derivative liarozole [449]. Additional in vitro data revealed that both ketoconazole and a related azole antifungal, miconazole, behave as competitive inhibitors of 1ahydroxylation of 25(OH)D [450]. Brown et al. was the first to observe, in studies using bovine parathyroid cell cultures, that ketoconazole alone can inhibit PTH secretion [451]. This was again shown by work by Segersten et al. [452]. It is unclear what the mechanism of the suppression is. Clinically, a reduction in 1,25(OH)2D was demonstrated in healthy men treated for 1 week with ketoconazole [453]. No changes in 25(OH)D, PTH, or serum calcium or phosphate levels were shown, suggesting
a direct inhibitory effect of the drug on renal 1a-hydroxylase activity [453]. Subsequently, Glass and Eil [454] treated patients with primary hyperparathyroidism and hypercalcemia for 1 week with ketoconazole. The treatment produced a reduction in 1,25(OH)2D levels. Serum total calcium but not serum ionized calcium levels fell, with no changes in 25(OH)D, PTH, or serum phosphate [454]. A slightly longer study of 2 weeks’ duration, also in hyperparathyroid patients, confirmed low 1,25(OH)2D3 levels but also demonstrated a nonsignificant fall in 25(OH)D [455]. This raises the possibility that one of the cytochrome-P450-dependent enzymes involved in 25-hydroxylation may be inhibited. Perhaps studies of a longer duration will demonstrate significant falls in 25(OH)D as well. Finally, ketoconazole has been shown to be effective in decreasing serum 1,25(OH)2D and calcium concentrations, both in cultured pulmonary alveolar macrophages taken from patients with sarcoidosis [456] and in vivo in sarcoid patients [456,457]. This suggests that ketoconazole also inhibits the extrarenal production of 1,25(OH)2D3 known to occur in sarcoidosis (see Chapter 45). Current data on the effects of ketoconazole on vitamin D metabolism suggest that the drug inhibits PTH secretion and that it decreases 1,25(OH)2D3 levels by directly inhibiting 1a-hydroxylase activity. The effects of ketoconazole on 25-hydroxylase activity remain to be determined. During treatment with ketoconazole for any of its indications, susceptible individuals should be monitored and supplemented with vitamin D, if necessary. Of note, combination therapy with ketoconazole and vitamin D analogs enhances the inhibitory effects of vitamin D on prostate cancer cell growth by inhibiting 24-hydroxylase, thus slowing the degradation and thereby increasing the activity of calcitriol or analogs [458]. See Chapter 86 for a discussion of ketoconazole effects on vitamin D action in prostate cancer.
Hypolipidemics Drugs that inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) are the most commonly prescribed agents for the treatment of hypercholesterolemia. Because HMG-CoA reductase inhibitors (or statins, as they are more commonly known) are potent inhibitors of cholesterol synthesis, they may impact vitamin D production because cholesterol is the vitamin D3 precursor. However, the effect of these medications on vitamin D levels is conflicting. Wilczek et al., in two separate, small studies, found that lovastatin, one of the earlier HMG-CoA reductase inhibitors, increased 25(OH)D levels after 3 months of treatment, and that simvastatin, another HMG-CoA reductase
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inhibitor, increased 25(OH)D levels in a dose-dependent manner [459,460]. In a larger double-blind randomized control trial of 280 healthy black postmenopausal women, a subgroup of 51 women who were getting a statin Perez-Castrillon et al. found that, in 83 patients treated with atorvastatin for 12 months, vitamin D3 levels increased by 33% [461]. A recent study by Yavuz et al. involving the newest statin, rosuvastatin, found a significant increase in both 25(OH)D and 1,25(OH)2D levels [462]. This is in contrast to the Rejnmark et al. randomized control trial of 77 healthy postmenopausal women taking simvastatin 40 mg versus placebo, which found no effect on either vitamin D or PTH levels [463]. Ismail et al. [464] studied 40 hypercholesterolemic patients treated for 24 weeks with one of the HMGCoA reductase inhibitors, pravastatin (40e80 mg daily). Results showed that although levels of total and lowdensity lipoprotein (LDL) cholesterol were significantly reduced, no changes in 25(OH)D, 1,25(OH)2D3, or PTH levels were observed [464]. The capacity of the skin to synthesize vitamin D3 after ultraviolet light exposure also showed no changes after 3 months of pravastatin therapy [465]. Lack of effect of HMG-CoA reductase inhibitors on vitamin D metabolism has been confirmed by others [466], and indeed combination therapy using both pravastatin (40 mg/day) and the bile acid sequestrant cholestyramine (24 g/day) failed to affect levels of vitamin D metabolites or PTH [464]. The evidence for an effect of statins on vitamin D metabolism is unclear. Most likely, variations in the data depend on which statin is being studied since there are major differences in their handling and degradation. Different effects of the various statins on vitamin D levels may in part depend on which cytochrome P450 enzyme the specific statin is metabolized by and whether that enzyme system may also affect vitamin D metabolism. Also many genetic variations in different individuals (single nucleotide polymorphisms or SNPs) alter the ability of the complex transport and degradation system of the statins to function so that the pharmacokinetic interactions between these drugs and the vitamin D synthetic and degradation pathway will require more careful study to elucidate the drug interactions for specific individuals. Although the literature suggests that statins may have an anabolic effect on bone [467] and may be protective against fracture [468,469], studies show discrepant results. In a prospective double-blind randomized controlled trial of 626 postmenopausal women treated with atorvastatin 10e80 mg, Bone et al. found no effect on bone mineral density at the lumbar spine [470]. Rejnmark et al., in a double-blind randomized controlled trial of 82 patients treated with simvastatin 40 mg for 1 year, also found no effect on bone mineral density [471]. Another study by Rejnmark et al. also
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showed no effect of statins on bone mineral density [472]. However, in observational studies by Edwards et al. and Lupatelli et al., a significant increase in BMD was found at the lumbar spine and hip in statin users versus controls [473,474]. Whether or not statins are protective against hip fractures remains to be seen. Observational studies have shown statistically significant improvements in hip fracture risk [475e478] yet this trend has not been seen in other randomized controlled trials [477]. More randomized controlled trials are needed to assess the effect of statins on fracture risk. Absorption of vitamin D from the gut requires the presence of bile acid. Moreover, vitamin D is excreted in the bile [479], and some degree of enterohepatic circulation of vitamin D occurs [480,481]. Thus, it stands to reason that cholestyramine might adversely affect vitamin D absorption. There have been reports in rats of decreased vitamin D absorption following treatment with cholestyramine [482]. Moreover, there are isolated case reports of osteomalacia associated with long-term cholestyramine use, which were attributed to cholestyramine-induced vitamin D deficiency [483,484]. Compston and Thompson [485] also reported decreased levels of 25(OH)D and reduced intestinal absorption of vitamin D in patients with primary biliary cirrhosis treated with cholestyramine for greater than 2 years. However, a large double-blind randomized trial of patients treated with cholestyramine (24 g/day) for 4 months showed similar levels of vitamin D metabolites, PTH, and serum calcium and phosphate compared with placebo-treated patients [486]. Ismail et al. showed equivalent findings after 6 months of therapy [464], as did long-term studies using another bile acid sequestrant, colestipol, for the treatment of children with familial hypercholesterolemia [487,488]. Ezetimibe, a novel cholesterol absorption inhibitor, has been shown not to inhibit the absorption of vitamin D across the intestinal wall in rodents [489]. The effect of this agent on vitamin D absorption, calcium metabolism, or bone mass has not been reported in humans. A single report of the effect of fibrates on vitamin D metabolism showed a decline in 25(OH)D and a rise in 1,25(OH)2D3 levels [490]. It seems, therefore, that vitamin D may be affected by the statin drugs and is unaffected by short-term cholestyramine treatment. Long-term cholestyramine therapy may affect 25(OH)D, and levels should be routinely monitored in susceptible individuals and vitamin D supplementation provided, if necessary. Larger studies are needed to assess the effects of statins on bone and fracture risk. The effect of cholestyramine on bone mass and fracture has not been investigated. Research into the effects of ezetimibe on vitamin D metabolism in humans is warranted.
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Bisphosphonates Bisphosphonates are widely used for the treatment of osteoporosis, Paget’s bone disease, hypercalcemia of malignancy, and metastatic bone disease. By inhibiting osteoclastic bone resorption [491,492], these agents cause an increase in the calcium mineral content of bone and a decrease in serum calcium levels [493]. This fall in serum calcium results in the stimulation of PTH secretion. PTH reduces the renal tubular reabsorption of phosphate, which reduces serum phosphate levels. Both increased PTH and low phosphate levels can lead to increased 1,25(OH)2D3 production [494e496]. The first-generation bisphosphonate, ethane-1hydroxy-1,1-diphosphate (EHDP), also known as etidronate, was initially shown to cause a reduction of 1,25(OH)2D levels at high doses in vivo [497,498]. This effect was probably an indirect one as EHDP failed to stimulate or inhibit 1a- or 24-hydroxylase in primary chick kidney cell cultures [10]. Later, evidence emerged to suggest that bisphosphonates indirectly stimulate 1a-hydroxylase at least in part by some unknown humoral factor. An experimental bisphosphonate compound, YM175, was shown in TPTX and sham-operated rats to increase 1,25(OH)2D and 1ahydroxylase in a dose-dependent manner; however, no increase in 1a-hydroxylase activity was demonstrated in vitro [499]. In clinical studies, Paget’s disease patients treated with etidronate had higher 1,25(OH)2D3 levels without significant changes in serum PTH, ionized calcium, or phosphate [85,500]. Lawson-Matthew et al. [501] found increased 1,25(OH)2D3 concentrations following shortterm oral etidronate treatment but decreased 1,25(OH)2D3 levels after high-dose intravenous etidronate. The effects of etidronate are thus most probably dose-related. In rats low-dose etidronate inhibits bone resorption and calcium release from bone and stimulates dietary calcium absorption. The lower calcium concentration, in turn, probably either directly or indirectly, via PTH, mediates 1a-hydroxylase activation. However, high-dose etidronate inhibits bone formation as well as resorption, and decreased calcium absorption from the gut is found [493]. Paget’s disease patients treated with another bisphosphonate, aminohydroxy-propylidene bisphosphonate (APD), known as pamidronate, also had elevated 1,25(OH)2D levels following short-term intravenous [502,503] and oral [506] administration. PTH rose to twice pretreatment levels in a response to a fall in ionized calcium levels [502,504]. 24,25(OH)2D3 concentration declined, but 25(OH)D remained unchanged [502]. Concentrations of 1,25(OH)2D [505,506], as well as PTH [506e508], were also elevated after treatment with pamidronate in patients with tumor-associated hypercalcemia.
The third-generation bisphosphonate 4-aminohydroxybutylidene-1,1-bisphosphonate, or alendronate, is a 100- to 500-fold more potent inhibitor of bone resorption than is etidronate [509]. Postmenopausal women treated with alendronate showed initial rises in 1,25(OH)2D and PTH that normalized after chronic administration of the drug [495,510,512], probably because of inhibition of bone resorption and decreased serum calcium concentrations. 1,25(OH)2D and PTH levels also rose following intravenous alendronate infusion for the treatment of hypercalcemia of malignancy [506]. Zoledronic acid (ZA) is the newest and most potent bisphosphonate [512]. In a randomized placebocontrolled trial assessing the efficacy of ZA in liver transplant patients at a dose of 4 mg every 3 months for 1 year, levels of 1,25(OH)2D were increased at 1 month in the ZA group. PTH levels increased at 1 month and at 3 months; there was no significant difference in 25(OH)D levels [513]. Of note, in a recent study by Simmons et al. of 46 patients with breast cancer and bone metastases on long-term bisphosphonate therapy, PTH levels were higher compared to controls at similar calcium levels [514]. The authors speculate that this is likely due to secondary hyperparathyroidism in the setting of bisphosphonate use [514]. Bisphosphonates may have an effect on 25(OH)D levels. A randomized controlled trial of 20 breast cancer patients treated with clodronate versus calcitonin revealed decreased 25(OH)D levels in addition to decreased calcium levels and increased PTH and 1,25(OH)2D3 levels in the clodronate-treated subjects [515]. However, in a small trial of 17 patients evaluating the safety of IV pamidronate in patient with osteoporosis, there was no significant change in 25(OH)D levels or serum calcium after 1 year [516]. Additionally, there was no difference in 25(OH)D levels in the ZA trial mentioned above [513]. In general, bisphosphonates do appear to influence vitamin D metabolism. The most common finding is an elevation in 1,25(OH)2D levels, which is most likely due to secondary alterations in serum-ionized calcium, phosphate, and PTH levels. These alterations generally appear to be short term, but may theoretically also confer some initial benefit in regard to an anabolic action of the elevated PTH levels. It is advisable to rule out vitamin D deficiency prior to initiation of bisphosphonate therapy and to supplement all patients with calcium and vitamin D.
Thiazide Diuretics Clinically, the use of thiazide diuretics has been associated with favorable effects on bone mineral density [518e520] and hip fracture rate [521e523]. These beneficial effects may be related to its ability to decrease
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PTH-stimulated bone resorption and decrease bone remodeling [524]. Thiazide use leads to a number of alterations in bone mineral metabolism. Thiazides are well known to decrease urinary calcium excretion [517,523,525,526], with resultant increases in serum calcium concentration [525,527]. This leads to reduced PTH levels [519,524], which in turn decreases 1,25(OH)2D synthesis [519,524,528,529] and leads to a reduction in intestinal calcium absorption [519]. Clinical studies on the effect of hydrochlorothiazide on vitamin D metabolism have shown a consistent decrease in 1,25(OH)2D3 levels. The administration of 50 mg/day of hydrochlorothiazide to postmenopausal women significantly decreased 1,25(OH)2D3 and increased 25(OH)D and 24,25(OH)2D3 levels [519]. Similar findings of low 1,25(OH)2D levels were reported by Sowers and co-workers [528]. Riis and Christiansen [530] conducted a double-blind, long-term controlled trial in early postmenopausal women and demonstrated a trend toward lower 1,25(OH)2D with a significantly elevated 24,25(OH)2D3 concentration [530]. No evidence of a direct effect on the synthesis or degradation of 1,25(OH)2D3 by thiazides is currently available. It thus seems that changes in vitamin D metabolites found after the use of thiazide diuretics are most likely secondary to alterations in serum calcium concentrations and PTH levels subsequent to actions to reduce urinary calcium excretion.
Calcium Channel Blockers Calcium channel blockers, of which nifedipine, amlodipine, verapamil, and diltiazem are the most well recognized and frequently prescribed, are chemically dissimilar. As such they have different effects on PTH secretion. In vitro experiments show that verapamil both increases and decreases PTH secretion, depending on extracellular calcium concentrations [531e533]. Diltiazem inhibited PTH release in bovine parathyroid cells by 40% and in human parathyroid cells by 20% [534]. A similar inhibition of PTH secretion was seen with nitrendipine, an analog of nifedipine [535]. There are no studies evaluating the effect of amlodipine on PTH. It has been suggested that in certain circumstances calcium channel blockers can act as calcium agonists, thus leading to an increase rather than a decrease in intracellular calcium concentration with associated inhibition of PTH secretion [534]. Verapamil has been reported to inhibit PTH secretion from rat parathyroid glands in vitro [536] and from goat parathyroid glands perfused in vivo [533]. In conflicting in vivo animal studies, verapamil stimulated PTH secretion in rats [537,538], whereas 1,25(OH)2D3 levels were decreased [537].
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Clinical studies, however, have shown no change in PTH levels using either verapamil [539] or nifedipine [540]. Only a short-term (3-day) study with diltiazem reported decreased PTH levels with normal ionized calcium and phosphate concentrations [534]. Studies evaluating vitamin D metabolites showed that 16 weeks of diltiazem administration had no effect on either PTH or 1,25(OH)2D3 levels [541]. Long-term use of nifedipine also failed to alter PTH or 25(OH)D levels or to affect serum parameters of bone turnover or bone mineral density in a group of males treated with calcium channel blockers for coronary heart disease [532]. Finally, calcium channel blockers are known inhibitors of hepatic microsomal cytochrome P450-dependent enzymes, and thus have the potential to cause alterations in vitamin D metabolites [533]. Despite possible interactions with PTH release, all past and recent clinical data have failed to demonstrate any effect on vitamin D metabolites, and thus calcium channel blockers cannot be considered to have a major influence on vitamin D metabolism.
Heparin Long-term use of heparin has been associated with the development of osteopenia in humans [544e547] and in rats [548e550]. Chronic heparin use is most frequently encountered in pregnant women with venous thrombosis. Pronounced bone loss and low 1,25(OH)2D levels have been seen in such patients, while serum 25(OH)D and 24,25(OH)2D3 levels and calcium and phosphate concentrations have been unchanged [551,552]. Heparin decreases bone formation in cultured fetal rat calvaria [553,554] and stimulates bone resorption by increasing the number and activity of osteoclasts in vitro [555]. Mutoh et al. [548] treated 4-week-old vitamin-D-deficient rats with heparin (2000 IU/day). Significant bone loss developed after 2 weeks, which peaked at 4 weeks. No change in serum total or ionized calcium was observed, but a significant elevation of serum PTH was seen. Furthermore, 1,25(OH)2D levels were decreased by 54% versus controls [548]. Although in the Mutoh et al. study no change in serum ionized calcium was demonstrated, this is presumed to be the mechanism by which heparin increases serum PTH. Heparin has been reported to have a high affinity for calcium ions [556]. This may lead to lower calcium levels, which would stimulate PTH release. However, this is a doubtful mechanism for heparin-induced osteopenia, as calcium salts of heparin are as effective in inducing osteoporosis as the corresponding sodium salts [557]. The reasons for the low 1,25(OH)2D3 values with heparin are entirely
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speculative, but may involve direct inhibition of the 1a-hydroxylase system, with the low 1,25(OH)2D3 levels in turn influencing receptors on the parathyroid gland, which induces PTH stimulation. The use of low-molecular-weight heparins (LMWH) in the treatment of thrombotic disorders has steadily grown over the past decade. LMWH seem to have a similar effect on bone compared to unfractionated heparin, but the degree of bone loss is less [558,559]. The long-term effect, however, of these agents on bone has yet to be determined. In vitro data suggest that LMWHs inhibit osteoblast proliferation [560]. Delayed fracture repair has also been reported in rabbits treated with the LMWH, enoxaparin [561]. A prospective study of 16 pregnant women who received 19e32 weeks of enoxaparin revealed a significant decrease in bone mineral density of the proximal femur at 6 months postpartum; however, the authors speculate that this is more likely an effect of lactation rather than continued effect of enoxaparin [562]. A non-randomized, open-label, prospective study investigating the effects of anticoagulation on bone mineral density found that in the 42 patients (pregnant women were excluded) who were treated with enoxaparin for 3e24 months, the BMD decreased by 3.1% at 1 year follow-up and by 4.8% at 2 years follow-up [563]. However, in a randomized substudy looking at the effects of the low-molecular-weight heparin dalteparin, no significant loss of bone mineral density was found at 6 weeks postpartum [564]. The real risk of long-term treatment with LMWH is largely unknown. The majority of studies are small, primarily looking at women during pregnancy. Larger clinical trials are needed to assess the effects of these medications [558]. The effect of LMWH on vitamin D metabolism has not been studied.
Cimetidine and Anti-acids Cimetidine, a histamine H2 receptor antagonist, is frequently used for the treatment of peptic ulcer disease. In vitro studies have demonstrated histamine H2 receptors in both normal and adenomatous parathyroid gland tissue [565], and stimulation of these receptors by histamine increases PTH release [566]. Cimetidine has been shown to decrease serum PTH in patients with either parathyroid adenoma [567,568] or secondary hyperparathyroidism due to chronic renal insufficiency [569,570]. However, there have been equivocal findings regarding the effects on serum calcium accompanying the changes in PTH, with both low [567] and unchanged levels [568,570,571] being found. Cimetidine was also shown to significantly decrease net intestinal calcium transport either secondary to its effect on PTH or via changes in vitamin D [572]. Cimetidine is an inhibitor of microsomal drug metabolism
[573,574], and thus one might expect this agent to inhibit hepatic vitamin D 25-hydroxylase, a cytochrome P450dependent enzyme. Animal studies have shown a dose-dependent decrease in 25-hydroxylase activity in the presence of increasing concentrations of cimetidine [575]. Cimetidine was also shown to reduce 25(OH)D levels in hens [576]. In humans, short-term use of cimetidine (800 mg/day for 4 weeks) did not decrease the level of 25(OH)D but prevented the expected seasonal rise in 25(OH)D [425]. After cessation of cimetidine therapy, levels rose significantly. Levels of 1,25(OH)2D3 and 24,25(OH)2D3 were not affected, and serum calcium and phosphate concentrations remained normal [577]. There are conflicting reports about the effect of H2 receptor antagonists on fracture risk. Grisso et al. first reported a 2.5-fold increase in hip fracture risk in cimetidine users [578]. A larger study by Yang et al. reported a dose-dependent increase in fractures with use of H2 receptor antagonists [579]; a finding consistent with de Vries et al., which showed an increased risk of osteoporotic, hip, and vertebral fracture in current users of H2 receptor antagonists [580]. However, they found that the effect attenuates over time with prolonged use. This study also found an increase in vertebral fractures in patients on both H2 receptor antagonists and bisphosphonates versus bisphosphonates alone [580]. These findings are in contrast with the Danish study which found a dosedependent decrease in fracture risk [581] and the study by Yu et al., which did not find a significant effect upon fracture rate in men or women using H2 receptor antagonists [582]. A small study by Adachi et al., looking at the effects of cimetidine, ranitidine, and famotidine in 33 patients on these medications for at least 2 years, found that there was no significant effect on the bone density of L2eL4 by DXA [583]. The authors of these studies speculate that the difference in findings is likely due to study design. In summary, cimetidine has multiple effects on calciotropic hormones: PTH levels are decreased, and 25(OH)D is reduced by inhibition of hepatic 25-hydroxylase activity. Despite low PTH levels, however, no changes in serum 1,25(OH)2D or serum calcium or phosphate concentrations are evident. Monitoring of 25(OH)D levels is indicated only in susceptible individuals, such as those with hepatic insufficiency, poor nutrition, or the elderly. Other H2 receptor antagonists, such as ranitidine, have less effect on hepatic drug metabolism [574] and thus are less likely to interfere with vitamin D metabolism. No studies employing ranitidine or other H2 receptor antagonists and their effects on vitamin D metabolism are currently available.
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Proton Pump Inhibitors (PPIs) In May 2010 the US Food and Drug Administration (FDA) announced that prescription and over-thecounter labels for PPIs would be revised to include new safety information about the possible risk of hip, wrist, and spine fractures with the use of these medications [584]. This announcement came in the wake of several epidemiological studies published in the past several years showing an association of PPI use and increased fracture risk [579,581,582,585e587]. In a caseecontrol study using the General Practice Research Databse (GPRD), Yang et al. showed a 44% increase in hip fracture with PPI use for 1 year, and the risk increased with larger doses and greater duration of use [579]. A prospective analysis of 161 806 postmenopausal women without history of fracture enrolled in the Women’s Health Initiative focused on 3396 women who used PPIs from <1 year to >3 years. PPI use was not related to hip fracture, but was related to clinical spine, forearm, or wrist fracture, and 25% increased risk for total fracture was seen. In those with no history of fracture, fracture risk was 32% higher in PPI users compared to non-users [587]. In a retrospective caseecontrol study Targownik et al. found a 92% increase in the risk of any osteoporotic fracture with PPI use of at least 7 years, and a 62% increased risk for hip fracture with at least 5 years of use [585]. More recently, Targownik et al. did not find an association between PPI use and osteoporosis at the hip or spine in a cross-sectional study or an association between PPI use and BMD decline in a longitudinal study [588]. In addition, like Yang et al., Kaye et al. used the GPRD, but did not find an association with PPI use and hip fractures in those without other risk factors for fracture [589]. It is speculated that the discrepancy between these findings may be due to unmeasured cofounders, such as smoking, alcohol use, and family history of osteoporosis [584,588]. The mechanism most commonly postulated in the past for the association of PPIs and fracture is the negative effect of the hypochlorhydria associated with these medications on calcium absorption, but the data in this area are conflicting. Two studies by Graziani et al. (one using eight healthy males and the other with 30 dialysis patients) using omeprazole 20 mg every 8 hours for 3 days showed the lack of a postprandial increase in calcium, suggesting a decrease in calcium absorption [590,591]. O’Connell et al. studied 18 postmenopausal women taking omeprazole 20 mg for 7 days and found a 41% decrease in calcium absorption of radioactive labeled calcium carbonate in the fasting state [592]. However, results from a prospective study by Hansen et al. showed no decrease in fractional calcium absorption, using dual stable calcium isoptopes,
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in 21 postmenopausal women taking omeprazole 40 mg daily for 30 days [593]. PTH, serum, and urine calcium remain unchanged after omeprazole; also there was no significant difference in serum 25(OH)D levels during the study [593]. A randomized placebo-controlled double-blind cross-over study of 12 young healthy adults taking omeprazole 20 mg daily showed no effect on intestinal calcium absorption in the fed state using dual stable calcium isotopes and no significant difference in total serum calcium, PTH, or 1,25(OH)2D [594]. The above studies all differ in study design, including differing in duration of PPI treatment and in the method that calcium absorption was measured. It is therefore unclear whether the hypochlorhydria associated with PPI use does decrease calcium absorption. Although many epidemiologic studies have found an association between PPI use and fracture risk [579,581,582,585,587], there is much debate about whether PPIs are the cause of the observed fractures [584,595]. It may be that the underlying disease state places patients at increased risk [593], or that PPIs affect bone and calcium/vitamin D metabolism independent of its potential effect on calcium absorption. More prospective studies are needed in the future to help answer these questions.
Aluminum Aluminum toxicity is known to cause metabolic bone disease. Although there has been a decline in the rates of aluminum accumulation in populations at risk, such as those with renal insufficiency, those on chronic total parenteral nutrition (TPN), and chronic antacid users, these patient populations are still at risk for aluminum toxicity [597,598]. Aluminum contamination of parenteral nutrition (PN) solutions is still a concern, primarily in neonates [599]. The main sources of aluminum in PN solutions are calcium gluconate, phosphate salts, and acetate [600,601]. The FDA established a safe upper limit for intravenous aluminum intake at 5 mg/kg/d [602]; however, multiple studies have shown that contamination of TPN products results in intakes of more than 5 mg/kg/d [597,599,603]. Prior to the FDA regulations TPN was associated with low levels of 1,25(OH)2D3 [582], and patients developed a low-turnover osteomalacia with aluminum accumulation in bone [585]. There are no recent studies in adults on the effects of aluminum exposure from TPN on bone or vitamin D metabolism, but Fewtrell et al. found that neonatal exposure may be associated with decreased hip bone mineral content, highlighting the need for more longterm studies in this population [604]. Patients with renal insufficiency are at risk from aluminum toxicity from occasional dialysate contamination, intermittent use of aluminum-based phosphate
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binders, and environmental exposure [596,605]. Patients undergoing hemodialysis with water containing high levels of aluminum have a high incidence of aluminum bone disease [606,607]. Aluminum content in bone is elevated and correlates positively with the development of osteomalacia [608e612]. Patients with renal failure are also susceptible to aluminum accumulation in bone [611,613]. There is some evidence that aluminum may act indirectly on bone by suppressing PTH release. Hemodialysis patients with osteomalacia have demonstrated lower mean PTH values than hemodialysis patients with normal 25(OH)D levels [610,612]. In rats, aluminum was shown to accumulate in parathyroid tissue [614], and reports show that aluminum impairs PTH release in vitro [615]. Studies using a rat model with chronic kidney disease have described a dose-dependent effect of aluminum on PTH secretion and synthesis [616] and found an inhibitory effect on parathyroid cell proliferation [617,618]. This may lead to low 1,25(OH)2D3 production. High-dose aluminum injections have also caused osteomalacia [611,619] and lowered PTH levels in rats [619]. However, in another similar experiment in rats using equivalent doses of aluminum, no skeletal changes or alterations in serum vitamin D metabolites were observed [620]. In dogs, serum calcium increased and serum phosphate and PTH did not change significantly following 5 weeks of parenteral aluminum administration daily [621]. 25(OH)D was normal, but a marked decline in 1,25(OH)2D was demonstrated. Renal function also declined, and this may account for the changes in 1,25(OH)2D. However, the reduction in 1,25(OH)2D occurred prior to the appearance of renal impairment, suggesting a direct inhibitory effect on the synthesis of 1,25(OH)2D [622]. Opposite effects on vitamin D metabolites seem to occur following the oral ingestion of aluminum hydroxide as an antacid. Increased values of 1,25(OH)2D3 may occur with hypophosphatemia induced by oral aluminum salts, which in turn increases 1a-hydroxylase activity in the kidney [623]. Aluminum is absorbed from the gut and deposits in bone in patients both with [624,625] and without [626] renal impairment. Day-long intake by a group of postmenopausal women resulted in a fall in serum phosphate, which correlated significantly with a rise in 1,25(OH)2D3 levels. Total and ionized calcium and PTH levels were unchanged [627]. This study was very short in duration, however, and longer studies would be required to assess the effects of oral aluminum on vitamin D metabolism. Nevertheless, chronic use of high doses of aluminumcontaining antacids has been reported to induce osteomalacia and rickets [628e630]. In addition, using aluminum-based antacids on a regular basis has been associated, in women 65 years old, with an increased risk of first vertebral fracture [631].
Antituberculous Agents Anecdotal case reports of rifampicin (rifampin)induced osteomalacia [632] led to further investigation of the effect of antituberculous agents on vitamin D metabolism. Studies by Brodie and colleagues [633] initially suggested that the frequently used first-line antituberculous drugs rifampicin and isoniazid, alone or in combination, could affect vitamin D metabolism. In the first study, short-term use of rifampicin (600 mg/day for 2 weeks) in healthy subjects reduced plasma 25(OH)D levels by as much as 70%, whereas 1,25(OH)2D3 and PTH remained unchanged [633]. This same group later showed decreased 1,25(OH)2D levels with rifampicin [634]. Decreased levels of 25(OH)D and 1,25(OH)2D may likely be due to increased hepatic, and possibly renal, metabolism. Rifampicin is a known inducer of the hepatic cytochrome P450s (otherwise known as CYPs) [635]. Multiple enzymes have been shown to have 25-hydroxylation activity, for example CYP3A4, CYP27A1, CYP2J2, CYP2R1 [636,637]. (See Chapter 3 for a more detailed discussion of this complex enzyme.) In a study by Zhou et al. rifampicin was found to upregulate CYP3A4, thereby converting vitamin D to more polar inactive metabolites [638]. However, Pascussi et al. showed that rifampicin upregulates CYP24 (25-hydroxyvitamin D3-24-hydroxylase) gene expression, thereby converting 25(OH)D to inactive 24,25-dihydroxyvitamin [635]. More studies are needed with rifampicin to clarify the effect on vitamin D metabolism. Short-term use of isoniazid (300 mg/day) in healthy subjects also produced a decrease in 25(OH)D and 1,25(OH)2D3 levels, accompanied by a fall in serum calcium and phosphate and rise in PTH [639]. Inhibition by isoniazid of hepatic enzyme activity [639] could explain the decreased levels of 25(OH)D and 1,25(OH)2D3. For example, Gupta et al. found that the CYP3A4, an enzyme with many metabolic actions including 25-hydroxylating activity, was inhibited by isoniazid [640]. A further study using both rifampicin and isoniazid also led to decreased 25(OH)D and 1,25(OH)2D levels together with raised PTH [634]. Similar short-term effects of rifampicin and isoniazid have been reported by others [641,642]. Despite evidence of short-term derangements in vitamin D metabolism, it seems that long-term studies reveal no significant effects [643,644]. One study reported that treatment of tuberculous patients with both rifampicin and isoniazid for 9 months produced no significant alterations in 25(OH)D and 1,25(OH)2D levels [644]. Another study found that tuberculous patients had low baseline 25(OH)D and PTH levels and high urinary calcium and 1,25(OH)2D3 levels, presumably due to granulomatous synthesis of 1ahydroxylase. After treatment with isoniazid and
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rifampin for 9 months, these parameters normalized [645]. Thus, overall it seems that only in the short-term are patients treated with these agents susceptible to vitamin D deficiency. Patients in developing countries with insufficient vitamin D intake and others at risk for vitamin D deficiency may require vitamin D supplementation, but it is unlikely that antituberculous drugs contribute to the development of overt metabolic bone disease. In fact, a recent randomized double-blind placebo-controlled trial comparing the effects of supplentation with 100 000 IU of cholecalciferol in patients undergoing antituberculous therapy, found that at 2 months 25(OH)D levels increased in both the treatment group and the control group [646]. Although the placebo group may have been exposed to more sun or foods rich in vitamin D, the authors speculate that antituberculous therapy may lead to a correction of the abnormalities of vitamin D in these patients as it treats the underlying condition [646].
Caffeine The first mention of a potential effect of caffeine on bone metabolism is found in a study by Daniell [647], who noted a higher caffeine intake in osteoporotic patients compared to age-matched controls. Heaney and Recker [648], in studies of perimenopausal women, were the first to show a negative calcium balance in association with caffeine intake. Additional controlled trials of caffeine use indicate that in individuals ingesting inadequate dietary calcium, caffeine ingestion leads to a small negative calcium balance via a small but significant decrease in calcium absorption efficiency [649]. Barger-Lux and Heaney have estimated that this small effect on calcium balance can be offset by the addition of 1e2 tablespoons of milk per cup of coffee [650]. The above results are in conflict with other studies which examine the effects of caffeine on the skeleton. The Framingham Osteoporosis Study failed to show an effect of caffeine intake on bone mass in elderly men and women [651]. Rico et al. failed to show a relationship between caffeine intake on bone mass as measured by quantitiative phalangeal bone ultrasound in 93 healthy postmenopausal women [652]. Similarly, Lloyd et al. [653] did not find an association between dietary caffeine intake and total body or hip BMD. However, another study found that elderly women who consumed >300 mg/d of caffeine had higher rates of bone loss than women with an intake of <300 mg/d [654]. These conflicting reports may be due to differences in determination of caffeine intake and methods of assessing bone density [652]. In addition, in studies where an association between caffeine consumption (at least two cups per day) and decreased BMD were found, the effect
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was not seen in women with increased calcium intake (either one glass of milk per day or at least 800 mg of calcium per day) [655,656]. Caffeine has been reported to enhance hepatic microsomal drug metabolism in rats and mice [657,658]. An inhibitory effect on the conversion of 25(OH)D to 1,25(OH)2D3 was reported in isolated renal tubules from vitamin-D-deficient chicks [659]. Studies in rats following chronic caffeine administration showed normal serum calcium but increased urinary excretion, and intestinal endogenous excretion of calcium, as well, increased intestinal absorption of calcium [660]. Yeh and Aloia [661] studied serial changes of serum calcium, PTH, 1,25(OH)2D, and calcium balance in young and old adult rats following daily caffeine administration for 4 weeks. In young rats, urinary calcium excretion increased and serum calcium decreased initially, but then returned to control levels. Serum PTH and 1,25(OH)2D increased after 2 weeks, and intestinal absorption of calcium remained unchanged. In adult rats similar changes occurred except that 1,25(OH)2D levels were similar to those in controls [661]. One drawback to this study is that the caffeine content that these rats received is the equivalent of 16 cups of coffee each day, which would be rather excessive for human consumption. A further study demonstrated no effect on bone histomorphometry after administration of caffeine to rats [662]. In addition to the animal studies mentioned, a recent study by Rapuri et al. found that, in osteoblast cells treated with physiologic doses of 1,25(OH)2D3 and increasing doses of caffeine, there was a dose-dependent effect on 1,25(OH)2D3-induced VDR expression; higher doses of caffeine resulted in decreased 1,25(OH)2D3-induced VDR expression [663]. Thus, caffeine seems to have some effect on calcium homeostasis in humans and animals. Its impact is likely more substantial in patients who are already at nutritional risk, such as the elderly. A clear effect on vitamin D metabolism has not been demonstrated.
Theophylline Theophylline, a once common treatment for reactive airway disease, has been shown to have an impact on vitamin D metabolism. An in vitro study showed an inhibitory effect of theophylline on the conversion of 25(OH)D to 1,25(OH)2D3 [659]. This occurred despite an increase in renal tubule cAMP levels, which have been shown to enhance 1,25(OH)2D3 formation in vivo [19]. Furthermore, there are reports of enhanced 24hydroxylase activity by aminophylline in normal birds [664] and rats [665]. In rat studies, the effect of longterm constant subcutaneous theophylline infusion was assessed. Increased urinary calcium excretion was demonstrated together with a reduction in total body
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calcium. Serum 25(OH)D was decreased, but no changes in 1,25(OH)2D3 or PTH levels were observed [666]. One possible explanation for the effects on 25(OH)D is that theophylline, an agent that has been previously demonstrated to induce hepatic microsomal enzymes [658,667], enhances conversion of 25(OH)D to other metabolites. In humans, theophylline increased phosphate and calcium urinary excretion in healthy males [668]. McPherson et al. [669] found that patients hospitalized for theophylline toxicity had hypercalcemia that normalized after theophylline was discontinued. PTH levels were unchanged, which implies that theophylline may act by enhancing the action of available PTH. Although animal data suggest that theophylline may alter vitamin D metabolism and clinical studies demonstrate a change in calcium balance, a direct effect on vitamin D has not been demonstrated in humans.
Immunosuppressants Potent immunosuppressants, which are widely used in the management of transplant patients and patients suffering from autoimmune conditions, have altered the prognosis of these disorders. A recent prospective study of 120 post-renal transplant patients on various immunosuppressive therapies found no alteration in the serum levels of 1,25(OH)2D3 or any significant difference between the groups [670]. Additional studies have also shown that post-transplantation, the use of CsA [671e675], or azathioprine [673e678] does not appear to result in any changes in 1,25(OH)2D3 levels compared to control subjects. The reader is referred to Elizabeth Shane’s Chapter 68 on transplantation for a detailed review of the effects of immunosuppressants on bone and mineral metabolism.
Fluoride Fluoride is no longer used as a treatment for osteoporosis; however, it is commonly found in drinking water at varying concentrations. Although studies in rats have found reduced calcium and phosphate levels when fluoride was added to drinking water at the 2.0 mM level, no changes in either serum 25(OH)D or 1,25(OH)2D concentrations were reported [679]. A recent study using rats showed evidence that fluoride affects calcium metabolism and stimulates secretion of PTH; however, the study did not look at vitamin D levels [680]. In human studies, no changes in serum PTH, 1,25(OH)2D3, 25(OH)D, or calcium and phosphate levels were found after 1 to 2 years of fluoride treatment for osteoporosis in both men and women [681,682]. Thus, these data suggest that fluoride does not interfere with vitamin D metabolism.
Olestra Olestra, formerly known as sucrose polyester (SPE), is a non-absorbable mixture of hexa-, hepta-, and octacarbon fatty acid esters of sucrose. It is an edible material that can be incorporated into the diet as a fat substitute. Olestra has physical properties similar to those of conventional dietary fats [683,684]; however, it is not absorbed [685] or hydrolyzed by gastric lipases [686]. As dietary vitamin D is absorbed from the intestine in association with dietary fats [687] and has an enterohepatic circulation, these processes may be altered by the presence of non-digestible lipid. Although there have been reports of reduced absorption of the fat-soluble vitamins A and E [683,684,688e690], all human studies have thus far have shown no significant effects on serum 25(OH)D levels [688,689,691,692] or dietary vitamin D absorption [691]. The most recent results from the Olestra Post-Marketing Surveillance Study are consistent with its earlier report that showed no association between olestra consumption and concentrations of fat-soluble vitamins [690,693], though there was a slight reduction in serum carotenoids. A 20-month feeding study in dogs also showed no effects on vitamin D status following olestra ingestion [694]. Since photoinduced cutaneous synthesis of vitamin D is the major factor determining vitamin D status [695e697], olestra ingestion would not be expected to adversely affect vitamin D nutritional status.
Orlistat Orlistat is a reversible inhibitor of gastric and pancreatic lipases. It is widely used in the management of obesity. Given its inhibition of absorption of lipids from the gastrointestinal tract, it has the potential to inhibit the absorption of fat-soluble vitamins. A small prospective study found a significant decrease in vitamin D levels after 1 month of therapy with orlistat in adolescents, despite the use of a multivitamin [698]. However, a randomized, double-blinded study found no significant difference in vitamin D levels between orlistat users and controls [699]. Additional studies examining the effect of this agent on vitamin D metabolism are warranted. In light of the fact that there is an increased incidence of vitamin D deficiency in obese people, it may be prudent in the meantime to monitor vitamin D levels in patients using this drug [700].
Lithium Lithium is a monovalent cation and is widely used in the management of bipolar affective disorders. It is well known to have an array of endocrine and metabolicrelated side effects, including the alteration of systemic
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calcium metabolism. In humans, lithium has been shown to lower serum phosphate [700e703] and reduce urinary calcium excretion [703e708]. This occurs as a result of increased tubular reabsorption of calcium, and it results in hypercalcemia [704e706,709e713]. Increases in PTH levels and parathyroid volume have been attributed to lithium therapy [702,704,705, 709e713,715]. Drug withdrawal reverses these effects [649]. There are a couple of proposed mechanisms for the elevation in PTH seen in lithium therapy. In vitro and in vivo evidence has shown that lithium directly stimulates the release of PTH from human parathyroid tissue [657]. Parathyroid adenoma and multiglandular hyperplasia have been reported in several patients with lithium-induced hyperparathyroidism [716,717]. Studies have also demonstrated a lithium-induced shift in the PTH-calcium set-point to the right [708,710, 718,719] as well as non-suppressibility of PTH levels [718] and altered calcium sensing [719]. Another possible mechanism by which lithium may elevate PTH is secondary to lithium-induced nephropathy [720,721]. However, serum creatinine levels were found to be normal in a patient with increased PTH [649]. Renal tubular acidosis has also been associated with lithium use [714]. There is limited knowledge about the effects of lithium on vitamin D as most metabolic studies following lithium administration in humans and animals have not measured vitamin D metabolites. One study found elevated PTH levels, yet normal 1,25(OH)2D3 levels after either short-term (mean, 1.7 months) or long-term (mean, 103 months) lithium carbonate administration [651]. Another study of ten patients treated for 1 month with lithium carbonate noted elevated serum PTH and reduced 1,25(OH)2D3 levels, although 25(OH)D calcitonin and serum calcium levels remained unchanged [722]. This is surprising as the elevation of PTH levels would be expected to increase rather than decrease 1,25(OH)2D3 levels. The authors hypothesize that lithium may act by inhibiting renal 1a-hydroxylase. The sample size and mean serum lithium levels were similar in both of the above studies; thus, the conflicting observations may relate to differences in the patient populations. Although lithium use can affect serum calcium, phosphate, and PTH levels, a direct effect on vitamin D metabolism has not been demonstrated thus far.
CONCLUSION The effects of various endogenous and exogenous hormones and drugs on the metabolism of vitamin D are complex. PTH and PTHrP are clearly potent stimulators of 1,25(OH)2D3 production. Past and current
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literature suggest that growth hormone, via IGF-1, and estrogen increase production of 1,25(OH)2D3. Longterm use of most anticonvulsants leads to increased 25(OH)D catabolism which is likely the main cause of the low bone mass observed in these patients. A strong association exists between the use of corticosteroids and immunosuppressants and the development of significant bone loss; however, the effect of these agents on vitamin D metabolism in humans is less clear. Although statins do not appear to affect vitamin D metabolism, long-term use of cholestyramine may decrease 25(OH)D levels. Parenteral aluminum appears to decrease 1,25(OH)2D3 levels, while its oral use results in hypophosphatemia and increased 1,25(OH)2D3 levels. The evidence on the effect of ethanol and commonly used agents such as heparin, lithium, theophylline, and cimetidine on vitamin D metabolism is inconclusive. More careful screening of susceptible individuals, such as those with poor nutritional status or who lack exposure to ultraviolet light, is likely indicated.
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[698] J.R. McDuffie, K.A. Calis, S.L. Booth, G.I. Waifo, J.A. Yanovski, Effects of orlistat on fat soluble vitamins in obese adolescents, Pharmacotherapy (2002) 814e822. [699] A. Gotfredsen, H. Westergren Hendel, T. Anderson, Influence of orlistat on bone turnover and body composition, Int. J. Obes. Relat. Metab. Disord. 25 (2001) 1154e1160. [700] L.M. Kaplan, Pharmacologic therapies for obesity, Gastroenterol Clin. North Am. 39 (2010) 69e79. [701] E.T. Mellerup, B. Lauritsen, H. Dam, O.J. Rafaelson, Lithium effects on diurnal rhythms of calcium, magnesium, and phosphate metabolism in manic-melancholic disorder, Acta Psychiat. Scand. 53 (1976) 360e370. [702] B.M. Davis, A. Pfefferbaum, S. Krutzik, K.L. Davis, Lithium’s effect on parathyroid hormone, Am. J. Psychiatry. 138 (1981) 489e492. [703] P. Plenge, O.J. Rafaelsen, Lithium effects on calcium, magnesium, and phosphate in man: effects on balance, bone mineral content, faecal, and urinary excretion, Acta Psychiat. Scand. 66 (1982) 361e373. [704] L.E. Mallette, K. Khouri, H. Zengotita, B.W. Hollis, S. Malini, Lithium treatment increases intact and midregion parathyroid hormone and parathyroid volume, J. Clin. Endocrinol. Metab. 68 (1989) 654e660. [705] J.L. Nielsen, M.S. Christiansen, E.B. Pedersen, S. Darling, A. Amdisen, Parathyroid hormone in serum during lithium therapy, Scand. J. Clin. Lab Invest. 37 (1977) 369e372. [706] P.D. Miller, S.L. Dubovsky, K.M. McDonald, C. Arnaud, R.W. Schrier, Hypocalciuric effect of lithium on man, Miner. Electrolyte Metab. 1 (1978) 3e11. [707] N. Bjorum, I. Hornum, E.T. Mellerup, P.K. Plenge, O.J. Rafaelsen, Lithium, calcium, and phosphate, Lancet 1 (1975) 1243. [708] J. Birnbaum, H. Klandorf, A. Giuliano, A. Van Herle, Lithium stimulates the release of human parathyroid hormone in vitro, J. Clin. Endocrinol. Metab. 66 (1988) 1187e1191. [709] C. Christiansen, P.C. Baastrup, I. Transbol, Lithium, hypercalcemia, hypermagnesaemia, and hyperparathyroidism, Lancet 1 (1976) 969. [710] F.-H. Shen, D.J. Sherrard, Lithium-induced hyperparathyroidism: an alteration of the "set point, Ann. Intern. Med. 96 (1982) 63e65. [711] C. Christiansen, P.C. Baastrup, P. Lindgreen, I. Transol, Endocrine effects of lithium: II. Primary hyperparathyroidism, Acta. Endocrinol. 88 (1978) 528e534. [712] C. Christiansen, P. Baastrup, I. Transol, Development of primary hyperparathyroidism during lithium therapy, Neuropsychobiology 6 (1980) 280e283. [713] J. Nordenstrom, M. Elvius, M. Bagedahl-Strindlund, B. Zhao, O. Torring, Biochemical hyperparathyroidism and bone mineral status in patients treated long-term with lithium, Metabolism 43 (1994) 1562e1567. [714] G.O. Perez, J.R. Oster, C.A. Vaavioucle, Incomplete syndrome of renal tubular acidosis induced by lithium carbonate, J. Clin. Lab. Med. 86 (1975) 386e394. [715] J. Nordenstrom, K. Strigard, L. Perbeck, J. Willems, M. Bagedahl-Strindlund, J. Linder, Hyperparathyroidism associated with treatment of manic-depressive disorders by lithium, Eur. J. Surg. 158 (1992) 207e211. [716] L.E. Mallette, E. Eichhorn, Effects of lithium carbonate on human calcium metabolism, Arch. Intern. Med. 146 (1986) 770e776. [717] T. Dwight, S. Kytola, B.T. Teh, G. Theodosopoulos, A.L. Richardson, J. Philips, et al., Genetic analysis of lithium-associated parathyroid tumors, Eur. J. Endocrinol. 146 (2002) 619e627. [718] S.T. Haden, A.L. Stoll, S. McCormick, J. Scott, Gel-H Fuleihan, Alterations in parathyroid dynamics in lithium treated subjects, J. Clin. Endocrinol. Metab. 82 (1997) 2844e2848.
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68 Vitamin D and Organ Transplantation Emily M. Stein, Elizabeth Shane Columbia University Medical Center, New York, NY, USA
INTRODUCTION The many skeletal and extra-skeletal sequelae of vitamin D deficiency are of particular consequence in organ transplant patients. Vitamin D deficiency can result in secondary hyperparathyroidism, bone loss, and fracture, as well as muscle weakness and falls [1,2]. Insulin resistance, hypertension and malignancy are important extra-skeletal sequelae [3,4]. The role of vitamin D in regulating immune function is of potential importance among transplant patients [5]. In this chapter, we will review the role of vitamin D in immune function as it relates to the transplant population. The prevalence of vitamin D deficiency in organ transplant candidates and in long-term transplant recipients will be examined, with assessment of vitamin D status based upon 25(OH)D measurements, the most reliable and stable indicator of vitamin D stores. Finally, we will summarize interventional trials evaluating vitamin D, 1,25(OH)2D and its analogs for the prevention and treatment of bone loss following solid organ transplantation.
EFFECTS OF VITAMIN D ON IMMUNITY AND GRAFT REJECTION Role of Vitamin D in Allograft Rejection The role of vitamin D in the regulation of immune cell proliferation, differentiation and responsiveness has been demonstrated by recent studies [5]. In animal studies administration of 1,25(OH)2D3 or calcitriol prevented acute allograft rejection following kidney [6], heart [7], and liver [8,9] transplantation. Data from human studies are limited. In kidney transplant recipients, calcitriol supplementation was associated with fewer episodes of acute cellular rejection [10], reduced
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10068-X
glucocorticoid requirements [11], and decreased expression of co-stimulatory and HLA-DR molecules, suggesting a possible mechanism for allograft survival [12]. Briffa et al. found that patients treated with calcitriol following heart transplantation had a reduction in their requirement for cyclosporine [13]. However, in our clinical trial of heart transplant recipients treated with calcitriol, we did not observe a reduction in cyclosporine or prednisone dose [14]. In a retrospective study, patients with lower pre-operative 25(OH)D had more frequent moderate to severe rejection episodes in the first 2 months after cardiac transplantation [15]. In a clinical non-experimental setting, patients who were supplemented with cholecalciferol (vitamin D3) had fewer rejection episodes [15]. Further prospective human studies are needed to explore the role of 1,25(OH)2D and of parent vitamin D in prevention of graft rejection and infection after transplantation.
Immune Effects of Vitamin D Vitamin D potentiates the innate immune system, and protects against bacterial infections and tuberculosis [16]. Monocytes and macrophages produce 1,25(OH)2D, which has intracellular antimicrobial effects and can also interact with and govern the cytokine profiles of activated T and B lymphocytes in the local environment [17]. The ability of monocytes and macrophages to synthesize sufficient 1,25(OH)2D is dependent on availability of adequate serum concentrations of 25(OH)D and therefore increases in response to vitamin D supplementation [18]. When there is insufficient 25(OH)D immunity may be impaired because local production of 1,25(OH)2D will decline. Subsequent decreased binding of 1,25(OH)2D to the macrophage vitamin D receptor (VDR) will result in reduced antimicrobial activity against ingested microbes [17]. The
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anti-microbial actions of 1,25(OH)2D also occur in barrier epithelial cells of the skin [19,20], gut [21], and lungs [22], sites which may be of particular importance to transplant recipients. Infections with herpes simplex virus-1 and Candida albicans, two common opportunistic infections in transplant patients, were reduced in animals treated with calcitriol [23]. In a population study, upper respiratory tract infections were more common in individuals with lower 25(OH)D [24].
VITAMIN D DEFICIENCY PRIOR TO ORGAN TRANSPLANT Vitamin D insufficiency and deficiency have been described in patients with end-stage pulmonary disease [25], liver failure [26,27], congestive heart failure [28], and chronic kidney disease [29,30]. Several factors place these patients at particular risk for vitamin D deficiency, including limited sunlight exposure and low dietary intake of vitamin-D-containing foods. Further, hepatic dysfunction, which can result from intrinsic liver disease or from hepatic congestion in heart failure patients, may contribute (Table 68.1). In this chapter, we will define insufficiency as 25(OH)D <30 ng/ml, deficiency as 25(OH)D <20 ng/ml, and severe deficiency as 25(OH)D <10 ng/ml.
Vitamin D Deficiency in Patients with End-stage Pulmonary Disease Patients with end-stage pulmonary disease have widespread 25(OH)D deficiency. Severe vitamin D deficiency has been reported in 20e50% of subjects TABLE 68.1 Risk Factors for Vitamin D Deficiency in Organ Transplant Patients African-American race Limited sunlight exposure, northern latitude, and winter months Low dietary intake of vitamin D Low fat mass Low serum albumin Hepatic dysfunction Obstructive pulmonary disease Renal insufficiency Diabetes Malabsorption Poor general health Female sex* Glucocorticoid use* e increases catabolism of 25(OH)D Organ transplanted* e liver transplant recipients may be at increased risk Recent transplantation* Proteinuria* Use of ACE inhibitors or aldosterone receptor blockers* * Risk factor specifically demonstrated following transplantation.
[25,31,32]. In cystic fibrosis (CF), a common indication for lung transplantation, we have observed that vitamin D deficiency is extremely common, related in part to pancreatic insufficiency and impaired dietary vitamin D absorption. Despite supplementation, bone density is significantly lower in the D-deficient patients [33]. Further, vitamin D deficiency is an important factor associated with osteoporosis and fractures among patients with CF [33e35]. In patients with advanced pulmonary disease, low 25(OH)D was associated with lower fat mass, obstructive pulmonary disease, low dietary vitamin D intake and was a predictor of decreased walking distance [36].
Vitamin D Deficiency in Patients with Liver Failure Among liver transplant candidates, vitamin D deficiency is also prevalent. In 45 patients awaiting transplantation, mean 25(OH)D was in the severely deficient range, 9 ng/ml [26]. In cirrhotic patients referred for liver transplantation [37], serum 25(OH)D, 1,25(OH)2D, intact parathyroid hormone (iPTH), and osteocalcin were lower and urinary hydroxyproline excretion was higher than in controls. Another study found that a model for end-stage liver disease (MELD) score of >15, indicative of worse disease and poorer health, was associated with lower serum 25(OH)D [15]. It is conceivable that free 25(OH)D may not be as low as suggested by total serum 25(OH)D measurements in patients with severe liver disease who may have lower levels of vitamin-D-binding protein, as a result of reduced synthetic capacity.
Vitamin D Deficiency in Patients with Congestive Heart Failure We found that patients with congestive heart failure had vitamin D deficiency (mean 25(OH)D 18 ng/ml); 18% of patients had severe deficiency (<9 ng/ml). Patients with lower 25(OH)D had lower serum calcium, phosphorus and albumin and higher total alkaline phosphatase activity and bone resorption markers. No association between 25(OH)D and 1,25(OH)2D was found. Serum iPTH was significantly lower in those patients in the highest tertile of 25(OH)D [28]. In another study of patients with end-stage heart failure, lower circulating calcitriol was associated with the need for transplantation and death [38].
Vitamin D Deficiency in Patients with Chronic Kidney Disease (CKD) In patients with CKD, glomerular loss leads to declining renal function and subsequent calcitriol
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deficiency [39,40]. Patients with CKD also have 25(OH)D deficiency [29,30,40], and lower 25(OH)D is associated with poorer kidney function [41]. In a population-based study, 71% of patients with stage 3 and 83% with stage 4 CKD had 25(OH)D insufficiency; 14% of patients with stage 3 and 26% with stage 4 CKD had severe deficiency [30]. Similarly, in a recent study 39% of CKD patients had 25(OH)D between 16 and 30 ng/ ml, 33% less than 16 ng/ml and 6% less than 5 ng/ml [29]. Factors associated with low 25(OH)D in CKD patients include female sex, African-American race, latitude, season, diabetes, and low serum albumin [40]. Baseline 25(OH)D was an independent predictor of death over 6 years in patients with CKD [41]; however, this finding likely reflects the poorer general health of the subjects with vitamin D deficiency at baseline. Comparison of serum 25-hydroxyvitamin D levels at the time of organ transplantation in heart and liver transplant recipients. Adapted from [42].
FIGURE 68.1
VITAMIN D DEFICIENCY FOLLOWING ORGAN TRANSPLANT Vitamin D insufficiency has been reported in 51e97% of organ transplant recipients, and severe deficiency in 26e33% [42e46]. Variability in these estimates relates to the organ transplanted, patient population, and assay utilized for measurement of 25(OH)D [47e49]. Poor health following transplant can lead to decreased dietary intake of vitamin-D-containing foods and increases risk of vitamin D deficiency. Many organ transplant recipients dramatically limit their sun exposure because of increased skin cancer risk [50,51]. Further, animal studies suggest that glucocorticoids, commonly used for immunosuppression, may increase catabolism of 25(OH)D [52,53]. Other factors associated with vitamin D deficiency after transplant include African-American race [46,54], avoidance of sun, low dietary intake [45], and transplant during winter months [54]. Variations in 25(OH)D related to sun exposure have not been observed in all studies [42], perhaps because in the most severely ill patients sun exposure is so limited.
Vitamin D Deficiency at the Time of Organ Transplantation Few studies have examined vitamin D levels at the time of transplantation. Recently, we evaluated heart and liver transplant recipients at the time of transplantation and found that 91% of patients had vitamin D insufficiency, 55% had deficiency (25(OH)D 10e20 ng/ml) and 16% had severe deficiency. Vitamin D levels were significantly lower among liver compared to heart transplant recipients (Fig. 68.1). This finding was likely related to malabsorption, and impaired hepatic 25hydroxylation of vitamin D in liver transplant patients
[42]. In patients at the time of renal transplant, vitamin D insufficiency was found in 59% and severe deficiency in 29% [54].
Vitamin D Deficiency in Long-term Transplant Recipients Vitamin D deficiency is common and severe in patients several years after kidney transplantation [43,46,55]. In one study, transplant recipients, who were an average of 7 years post-transplant, had significantly lower levels of 25(OH)D than age-matched controls [43]; among transplant patients mean serum 25(OH)D was 10 ng/ml and one-third of patients had undetectable levels [43]. Factors associated with low 25(OH)D in renal transplant recipients include African-American race [46], female sex, measurement in autumn and winter months [46], recent transplantation [55], inadequate dietary vitamin D intake, reported in 87e91% of patients [56], proteinuria [57], and use of ACE inhibitors or aldosterone receptor blockers [55]. Persistent elevations in FGF-23 after kidney transplantation may result in lower calcitriol levels [58]. In long-term liver transplant recipients, 65e68% had 25(OH)D levels below 15 ng/ml [44,59]. Patients with lower 25(OH)D had lower age-matched scores for bone mineral density (BMD) at the femoral neck (FN) by DXA [59]. Many authors report increases in both 25(OH)D and iPTH following liver transplantation [26,27,60,61]. However, some have not found significant changes [62e64]. Reported increases appear to be sustained for at least 3 to 4 years following transplantation [26,60]; most studies have found that, despite these increases, 25(OH)D remains in the insufficient range.
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Kaplan-Meier survival estimates in cardiac transplant recipients according to categories of serum calcitriol concentrations 21 days after transplant (log-rank test p < 0.001). From [69].
FIGURE 68.2
Factors associated with vitamin D deficiency in transplant recipients are detailed in Table 68.1.
Vitamin D Insufficiency/Deficiency in Cardiac Transplant Recipients In a recent study, 97% of cardiac transplant recipients had insufficient 25(OH)D levels. Though not directly associated with vertebral fractures, low 25(OH)D was associated with higher iPTH, which was significantly associated with vertebral fractures [65]. After cardiac transplantation sustained increases in serum creatinine [66e68] and decreases in 1,25(OH)2D are observed [67,69]. Further, low concentrations of 1,25(OH)2D measured 21 days post-transplantation were associated with 1-year mortality (Fig. 68.2) [69]. Whether this observation reflects the role of low calcitriol as a marker of renal dysfunction or poorer health or a causal relationship between calcitriol and mortality requires further investigation.
TREATMENT OF POST-TRANSPLANT BONE LOSS WITH VITAMIN D AND ANALOGS Vitamin D and its analogs are often used to prevent or treat osteoporosis after transplantation [70]. These agents may influence post-transplantation bone loss through several mechanisms. They may overcome glucocorticoid-induced decreases in intestinal calcium absorption, reduce secondary hyperparathyroidism, promote differentiation of osteoblast precursors into
mature cells, or influence the immune system and potentiate the immunosuppressive action of calcineurin inhibitors or prednisone, thus reducing the required dose of immunosuppressive drugs [13,71,72]. The majority of observational studies of bone loss after organ transplantation have included at least 400 IU of parent vitamin D in the post-transplant regimen, and thus it is clear that the RDA for vitamin D is not sufficient to prevent transplantation osteoporosis. In two recent studies, parent vitamin D, at doses of 800 IU daily [73] or 25 000 IU monthly [74] did not prevent bone loss after kidney transplantation. Active forms of vitamin D may have greater efficacy. Calcidiol (25(OH)D) prevented bone loss and increased lumbar spine (LS) BMD after cardiac transplantation [75]. When administered immediately after kidney transplantation, alfacalcidiol (1-a-(OH)D) prevented or attenuated bone loss at the LS and FN [76e78]. De Sevaux and colleagues [77] found that alfacalcidiol treatment during the first 6 months following renal transplant attenuated bone loss at the LS and greater trochanter and prevented loss at the FN (Fig. 68.3). In kidney transplant patients, calcitriol production by the transplanted kidney may be inadequate to suppress excess PTH secretion by hyperplastic parathyroid tissue [79]. Calcitriol treatment may be of particular benefit to these patients [11]. Treatment with calcitriol may prevent hyperparathyroidism after cardiac transplant as well [14]. Studies have found beneficial effects on BMD at doses greater than 0.5 mg per day, although this finding is not uniform. Calcitriol given during the first year after kidney transplantation was associated with an increase in LS, FN, and forearm BMD [80]. In another study of renal transplant recipients, intermittent calcitriol and calcium prevented bone loss at the total hip (TH) but not LS [81]. In a stratified, placebocontrolled randomized study, in which heart and lung transplant recipients received calcitriol or placebo for 12 or 24 months after transplantation [82], LS bone loss was equivalent between groups. At the FN, bone loss at 24 months was significantly reduced only in the group that received calcitriol for the entire period. In contrast to these results, which suggest that the protective effects of calcitriol are not sustained after cessation of treatment, we found no bone loss when calcitriol was discontinued after the first post-transplant year [83]. In contrast to the above findings, other studies have failed to find any benefit of calcitriol [84e86]. Vitamin D analogs have been compared to bisphosphonates in several studies, with mixed results. In a randomized trial, we found that alendronate (10 mg daily) and calcitriol (0.25 mg twice daily) given immediately after cardiac transplant provided similar protection against bone loss at the LS, FN, and TH 1 year after transplant (Fig. 68.4); both were superior
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FIGURE 68.3 Change in bone mineral density in renal transplant recipients treated with calcitriol or placebo (% from baseline SE; significance shown for differences between groups at 6 months). From [77].
FIGURE 68.4 Comparison of mean (SE) percent change in bone mineral density from baseline in cardiac transplant subjects treated with alendronate or calcitriol, and untreated reference group. From [14].
compared with a reference group receiving only calcium and vitamin D [14]. During the second year after cardiac transplant, BMD remained stable after discontinuation of both drugs [83]. During the first 6 months after heart or lung transplantation, calcitriol prevented bone loss at the spine and hip and was as effective as cyclic etidronate [87]. Kidney transplant patients treated with alendronate, calcitriol, and calcium had increases in LS BMD compared to decreases in those who received only calcium and calcitriol [88]. In another trial of long-term kidney transplant patients started on alendronate, calcitriol, and calcium or only calcitriol and calcium approximately 5 years after transplantation,
those in the alendronate group had significant improvements in LS and FN BMD, BMD in the other group was stable [89]. In a randomized trial that compared alendronate and alfacalcidiol plus alendronate for 1 year of long-term kidney transplant recipients, BMD improved at the LS and FN in both groups. The increase was only significant in the combination alendronatee alfacalcidiol group, however, likely because of inadequate power in this small study [90]. The major side effects of therapy with active vitamin D and analogs are hypercalcemia and hypercalciuria, which may develop at any time during the course of treatment necessitating frequent urinary and serum
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monitoring. We believe these agents should be adjunctive rather than primary therapy for the prevention and treatment of transplantation osteoporosis, given the narrow therapeutic window with respect to hypercalcemia and hypercalciuria, and monitoring requirements as well as the demonstrated efficacy of bisphosphonates to prevent post-transplantation bone loss [14,91]. Treatment of bone loss following transplantation may be complicated by 25(OH)D deficiency. Bisphosphonates may not be optimally effective in the setting of severe vitamin D deficiency. Further, intravenous bisphosphonate treatment has been reported to precipitate symptomatic hypocalcemia in patients with severe, unrecognized vitamin D deficiency [92].
CONCLUSIONS Vitamin D deficiency is extremely prevalent in patients with organ failure prior to and following transplantation. Pharmacologic doses of vitamin D and its analogs have utility after renal transplant, but should be utilized as adjunctive rather than primary therapy for osteoporosis in patients after other types of solid organ transplantation. All patients should be assessed for vitamin D insufficiency and deficiency before transplantation and receive treatment. If present, long-term transplant recipients should be monitored and treated for vitamin D deficiency as part of broader management of bone disease. Treatment of vitamin D deficiency may reduce skeletal and extra-skeletal morbidity in transplant patients. Interventional studies are needed to elucidate optimal repletion regimens after transplantation and to determine whether restoring 25(OH)D at the time of transplant reduces the development of infectious complications and immunosuppressant requirements.
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69 Vitamin D and Bone Mineral Metabolism in Hepatogastrointestinal Diseases Daniel D. Bikle Veterans Affairs Medical Center and University of California, San Francisco, CA, USA
PHYSIOLOGIC CONSIDERATIONS The mineral constituents of bone come from the diet and must be absorbed from the ingested food in the intestine. Vitamin D, through its active metabolite 1,25dihydroxyvitamin D (1,25(OH)2D), regulates the intestinal absorption of the two major mineral constituents of bone, calcium and phosphate. Although synthesized in the skin under the influence of ultraviolet light, vitamin D is also an important dietary constituent especially in circumstances of reduced exposure to ultraviolet light. Therefore, the bone is dependent on the adequate supply of calcium, phosphate, and vitamin D from the diet, and abnormalities of the hepatogastrointestinal tract, which impair their absorption and further metabolism, cause bone disease.
Calcium Absorption Intestinal calcium absorption occurs throughout the intestine, although the highest rates of absorption are found in the duodenum [1]. Calcium absorption occurs through both transcellular and paracellular pathways. In the duodenum, the transcellular pathway dominates, but in the jejunum and ileum, the paracellular pathway accounts for the bulk of calcium absorption [2]. However, surgical procedures which exclude the duodenum from the stream of ingested food as in the Roux-en-Y gastric bypass procedure for obesity lead to a reduction in true fractional calcium absorption [3]. Net absorption is reduced by calcium secretion and endogenous calcium losses associated with the sloughing of cells into the lumen [4]. Vitamin D, through its active metabolite 1,25(OH)2D, controls the transcellular pathway. Thus any process that reduces vitamin D intake, absorption,
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10069-1
or conversion to 1,25(OH)2D will reduce this process. This pathway is primarily mediated by three 1,25(OH)2D-induced proteins: TRPV6, a calcium channel in the brush border, regulates calcium entry into the cell; calbindin, a calcium-binding protein thought to transport the calcium through the cell; and PMCa1b, an ATP-dependent calcium pump at the basolateral membrane that transports calcium from the cell into the blood stream [2]. The sodium/calcium exchanger at the basolateral membrane also participates in transporting calcium out of the cell, but its regulation by 1,25(OH)2D is less well established. Surprisingly, however, deletion of both TRPV6 and calbindin fail to eliminate active calcium transport from the duodenum or its stimulation by 1,25(OH)2D, suggesting other as yet unknown mechanisms are also participating in transcellular calcium transport [5]. Moreover, intestinal calcium transport increases during pregnancy even in animals lacking the vitamin D receptor gene, indicating that calcium transport is not totally dependent on 1,25(OH)2D [6]. At low calcium intakes, the transcellular pathway dominates and provides a highly efficient means of absorption. However, as calcium intake increases, non-saturable but less-efficient pathways come into play, and calcium absorption falls to approximately 10% of the amount ingested at calcium intakes above 500 mg/day [7,8]. With age the efficiency of calcium absorption and the ability of the intestine to adapt to decreased calcium intake fall. Most reports using radioisotope absorption techniques have documented a reduction of intestinal calcium absorption with age especially in osteoporotic patients [9e16], although this was not observed in a study by Eastell et al. [17] using a stable isotope procedure which traced all meals. Balance studies by Heaney
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et al. [18] indicate that with age and menopause the amount of dietary calcium required to maintain a positive balance increases, again consistent with the concept that calcium absorption efficiency decreases with age. Surgical induction of menopause (ovariectomy) results in a fall in intestinal calcium absorption which can be prevented by estrogen replacement [19]. 1,25(OH)2D corrects the decrease in calcium absorption with age [15,16,20]. Postmenopausal women who respond to the precursor of 1,25(OH)2D, namely 25-hydroxyvitamin D (25(OH)D), with a rise in intestinal calcium transport also show an increase in 1,25(OH)2D concentrations following 25(OH)D administration, whereas the nonresponders do not [21]. Estrogen administration to postmenopausal women raises circulating 1,25(OH)2D [15,19,22] and increases intestinal calcium transport [15,19]. Although estrogen also increases the concentration of the vitamin-D-binding protein, this does not account for the entire increase in 1,25(OH)2D since both free and total 1,25(OH)2D levels are raised by estrogen administration [23,24]. The stimulation of calcium absorption by estrogen may not be limited to increased 1,25(OH)2D production since estrogen receptors have been described in intestinal epithelial cells, which may respond directly to estrogen with increased calcium transport [25]. Thus, a fall in 1,25(OH)2D levels, perhaps secondary to a fall in 1,25(OH)2D production by the aging or estrogen-deprived kidney, could account for the decrease in intestinal calcium absorption with age. On the other hand, the aging intestine may become more resistant to 1,25(OH)2D action with respect to calcium absorption [26], as is suggested by the age-related fall in vitamin D receptor and TRPV6 expression in the intestinal epithelium [27] despite normal or even elevated 1,25(OH)2D levels [28]. Other dietary constituents alter calcium absorption. Lactose increases calcium absorption in a number of animals including humans [29,30], and lactase deficiency has been associated with an increased risk for osteoporosis [31e33]. To the degree that lactase deficiency would reduce calcium intake by creating an aversion to dairy product ingestion, the loss of this enzyme could predispose to osteoporosis. However, it is not clear that lactase deficiency per se reduces the efficiency of calcium absorption [13,34e36]. Phosphate in the diet increases fecal loss of calcium in part by increased endogenous intestinal calcium secretion [37]. However, phosphate reduces urinary loss of calcium [37e39], so the net effect of phosphate on calcium balance is not obviously harmful [40]. Nevertheless, at least in shortterm studies, increased dietary phosphate can result in increased FGF23 production suppressing 1,25(OH)2D production [41], hormonal changes which decrease intestinal calcium absorption. A diet rich in fiber and phytates could reduce calcium absorption by chelating
calcium and other cations [42,43] and so decrease calcium balance [44,45]. However, a high-fiber diet has not been correlated with the development of bone disease.
Vitamin D Absorption Although the skin has the capability to produce adequate amounts of vitamin D given enough sunlight of sufficient intensity, because of our indoor lifestyle, modesty with respect to amount of skin exposed, and the use of sunscreens because of concern for skin cancer and photoaging, this biologic pathway does not always suffice. Thus, dietary intake and intestinal absorption of vitamin D become important. Vitamin D is absorbed in the jejunum and ileum [46,47] by a mechanism capable of absorbing approximately 75% of the vitamin D administered [48]. Vitamin D appears in both the portal venous system and lymphatics, indicating that both the venous return (blood) and lymph pathways are utilized [46,47], although the lymphatic route may be preferred in humans [49]. In lymph, approximately 50% of vitamin D is found in the chylomicron fraction [47]. Thus, absorption of vitamin D resembles that of cholesterol and other lipids. Fatty acids reduce vitamin D absorption, but this can be reversed with the addition of bile acids [46,47]. 25(OH)D (calcifediol) is better absorbed than vitamin D [47,50], especially in patients with steatorrhea (fat malabsorption) [51,52], but this form of vitamin D is no longer available in the USA. Vitamin D metabolites also undergo enterohepatic circulation. Arnaud et al. [53] noted the appearance in the duodenal lumen of 33% of the label 24 h after an intravenous dose of radiolabeled 25(OH)D. Nearly all of the secreted vitamin D metabolites were reabsorbed. The appearance of label following the intravenous administration of the dihydroxylated metabolites 1,25(OH)2D [54] and 24,25 (OH)2D [55] in bile is even faster than that following 25(OH)D administration. In contrast, the appearance of radiolabel in the bile following the administration of radiolabeled vitamin D is slower and less extensive [56]. Primary biliary cirrhosis (PBC) further reduces the appearance of vitamin D metabolites in the bile [56,57]. However, Clements et al. [57a] demonstrated that most of the vitamin D administered to patients with t-tube biliary drainage after cholecystectomy was excreted as highly polar metabolites, suggesting that the enterohepatic circulation contributed little to vitamin D homeostasis or when disrupted would be a cause for vitamin D deficiency. Thus it is not clear whether PBC results in vitamin D malabsorption, but patients with pancreatic disease (e.g., cystic fibrosis) require much higher doses of vitamin D supplementation to maintain normal 25(OH)D levels than do normal individuals [58] supporting the concept that disruption
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of vitamin D absorption as in pancreatic insufficiency is an important cause of vitamin D deficiency.
Hepatic Vitamin D Metabolism Following its absorption from the intestine, vitamin D is hydroxylated to 25(OH)D primarily in the liver, although a number of other tissues express this enzymatic activity. 25(OH)D is the major circulating form of vitamin D and provides a clinically useful marker for vitamin D status. 25-hydroxylase activity has been found in both the liver mitochondria and endoplasmic reticulum, and the enzymatic activities differ indicating different proteins. At this point most attention has been paid to the mitochondrial CYP27A1 and the microsomal CYP2R1. However, in mouse knockout studies and in humans with mutations in these enzymes, only CYP2R1 loss is associated with substantial changes in vitamin D metabolite production. These are mixed function oxidases, but differ in their apparent affinity and specificity for substrate in addition to their differences in intracellular location. The mitochondrial 25-hydroxylase is now well accepted as CYP27A1, an enzyme first identified as catalyzing a critical step in the bile acid synthesis pathway. This is a high-capacity, low-affinity enzyme consistent with the observation that 25-hydroxylation is not generally rate limiting in vitamin D metabolism [59e61]. CYP27A1 is widely distributed throughout different tissues with highest levels in liver and muscle, but also in kidney, intestine, lung, skin, and bone [59e62]. Mutations in CYP27A1 lead to cerebrotendinous xanthomatosis [63,64] and are associated with abnormal vitamin D and/or calcium metabolism in some but not all of these patients [64e66]. However, it is not clear that mutations in or even absence of CYP27A1 necessarily lead to cessation of vitamin D 25-hydroxylase activity despite leading to abnormalities in bile acid synthesis [67]. CYP27A1 can hydroxylate vitamin D and related compounds at the 24, 25, and 27 positions. However, D2 appears to be preferentially 24-hydroxylated, whereas D3 is preferentially 25-hydroxylated [68]. The 1a(OH) derivatives of D are more rapidly hydroxylated than the parent compounds. These differences between D2 and D3 and their 1a(OH) derivatives may explain the differences in clearance rates [68a] between D2 and D3 or between 1a(OH)D2 and 1a(OH)D3. The major microsomal 25-hydroxylase is CYP2R1, although other enzymes have been shown to have 25hydroxylase activity in vitro. This enzyme like that of CYP27A1 is widely distributed, although it is most abundantly expressed in liver, skin, and testes [69]. Unlike CYP27A1, CYP2R1 25-hydroxylates D2 and D3 equally [69]. A patient with an inactivating mutation in CYP2R1 has been described with rickets and reduced
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25(OH)D levels, reduced serum calcium and phosphate, but normal 1,25(OH)2D levels [70]. The subject responded to D2 therapy [70]. No other phenotype was reported in this subject, in particular no abnormalities in bile acid synthesis. Thus, neither CYP27A1 nor CYP2R1 by themselves account for all 25-hydroxylase activity in the body, but each most likely contributes and together may account for most if not all of the 25hydroxylase activity in humans. Studies of the regulation of 25-hydroxylation have not been completely consistent, most likely because of the initial failure to appreciate that at least two enzymatic activities were involved and because of species differences. In general, 25-hydroxylation in the liver is affected little by vitamin D status. However, CYP27A1 expression in the intestine [71] and kidney [72] is reduced by 1,25(OH)2D. Not surprisingly bile acids decrease CYP27A1 expression [73] as does insulin [74] through an unknown mechanism. Dexamethasone, on the other hand, increases CYP27A1 expression [75]. The regulation of CYP2R1 has been less well studied. Whether any of these manipulations alters 25-hydroxylase activity in the liver remains unknown. Estrogen given to male rats increases 25-hydroxylase activity, whereas testosterone given to female rats has the opposite effect [76]. However, evidence for such sex steroid hormone regulation of 25-hydroxylase activity in humans is lacking. This chapter will emphasize the bone disease resulting from vitamin D and calcium malabsorption that complicates disorders in the hepatogastrointestinal tract. Many of these disorders result in both osteoporosis and osteomalacia. Figure 69.1 shows six points at which vitamin D and/or calcium absorption could be affected by such disorders. First, adequate intake of vitamin D and calcium is required especially in an individual who otherwise fails to synthesize sufficient quantities of vitamin D in the skin. Milk and other dairy products are a good source of both, if these products are supplemented with vitamin D. Second, vitamin D absorption requires an intact small intestine, pancreas, and liver to provide the milieu (lipase, bile acids) required for vitamin D absorption. Partial gastrectomy, chronic pancreatic insufficiency, intrinsic small bowel disease, disorders of the biliary tract, and surgical bypass procedures of the stomach and small intestine can all cause problems here. Third, vitamin D that enters the body must be further metabolized to active metabolites. Diseases of the liver, where the first step in bioactivation takes place, or drugs such as phenytoin, which alter this first metabolic step, may lead to deficiency of the active metabolites although the liver and other tissues have substantial reserves such that only with profound liver damage is this likely to lead to decreased 25(OH)D production. Fourth, the
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thus decreasing the epidermal production of vitamin D. If this patient were also intolerant of milk products or had a condition in which malabsorption of calcium and vitamin D was present, the stage would be set for vitamin D deficiency. Glucocorticoid therapy is used for a number of conditions discussed in this chapter. Glucocorticoid therapy by itself leads to osteoporosis, and when used to treat a disease which has already jeopardized skeletal integrity because of an abnormality in calcium and vitamin D absorption or metabolism, such therapy can be especially detrimental. In this chapter, specific disease entities will be discussed individually even though different diseases may impact vitamin D function by similar or identical mechanisms.
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FIGURE 69.1 Six steps in vitamin D and calcium absorption and
handling that may be altered by hepatogastrointestinal disorders and so lead to bone disease. (1) Decreased intake of vitamin D and calcium. (2) Decreased absorption of vitamin D secondary to disorders in biliary secretion, pancreatic enzymes, enterocyte function, or intestinal anatomy. (3) Reduced production of 25(OH)D by the liver secondary to hepatic parenchymal disease. (4) Disruption in the enterohepatic circulation of vitamin D metabolites and conjugates secondary to disorders in biliary secretion. (5) Reduced delivery of vitamin D metabolites to target tissues secondary to decreased DBP and albumin synthesis. (6) Decreased response of the diseased intestine to 1,25(OH)2D with respect to Ca and P absorption. This figure is reproduced from [306].
Gastrointestinal diseases lead to bone disease primarily through the malabsorption of vitamin D and calcium, although the presence of disease may itself lead to reduced intake of vitamin D and calcium or limited exposure to sunlight. Furthermore, chronic inflammation as in the inflammatory bowel diseases may contribute to increased bone resorption in a potentially vitamin-D-regulated fashion. Each disease discussed below has its own subtle variations of these prevailing themes (Table 69.1).
Postgastrectomy Bone Disease Incidence and Prevalence
vitamin D metabolites undergo an enterohepatic circulation, being secreted in bile in conjugated form with subsequent reabsorption in the small intestine. Disruption of this pathway may contribute to vitamin D deficiency in certain diseases of the liver and small intestine. Fifth, vitamin D and its metabolites are poorly soluble in water and must be transported in blood bound to proteins, vitamin-D-binding protein (DBP), and albumin, which are synthesized in the liver. Decreased synthesis of these proteins may impair the delivery of the vitamin D metabolites to the target tissues. Finally, the diseased or surgically altered intestine may fail to respond normally to the active vitamin D metabolites with respect to calcium and phosphate absorption. Clearly one disease may impact adversely on bone by several mechanisms involving aberrations in vitamin D and calcium absorption, metabolism, or function. Furthermore, the systemic effects of the disease or its treatment may aggravate the abnormalities in vitamin D and calcium absorption, metabolism, or function. For example, chronic illness may limit the ability of the patient to get outdoors into the sunlight,
In a large (9704 subjects) study of older women, gastrectomy correlated with an 8.2% decrease in bone density [77]. In another study comparing 342 postgastrectomy patients with 180 unoperated patients of similar age with peptic ulcer disease, Eddy [78] observed osteopenia of the spine in 24% of the postgastrectomy patients compared to 4% of the unoperated controls. Fractures were found in 2.4 and 5.2% of gastrectomized patients, respectively, whereas none were seen in the controls. Bone pain or tenderness was observed in 26% of the gastrectomized patients compared to 4% of controls. Bone biopsies of 84 gastrectomized patients showed widened osteoid seams in 32% compared to none of the nine controls biopsied. Mellstrom et al. [79] reported spinal fractures in 19% of males with partial gastrectomies compared to 4% of age-matched controls. Smokers were at particularly high risk. An even higher prevalence of spinal osteopenia (69% of females, 41% of males) was observed by Deller et al. [80,81] in a study of 100 unselected patients following partial gastrectomy; however, 41% of females and 13% of males in the age-matched unoperated
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TABLE 69.1
Bone Disease Associated with Gastrointestinal Disorders
Prevalence
Clinical features
Pathogenesis
Treatment
Older adults, females, males, bone pain common; Y Ca, P, 25(OH)D, [ alk. phosphatase, normal PTH, Y urine Ca; osteopenia by X-ray; [ osteoid, Y TBV
Y Ca, vit. D absorption, 2 to Y acid, duodenal bypass, increased motility, steatorrhea
Vitamin D and calcium
Children, younger adults, growth retardation, steatorrhea, response to gluten-free diet; Y 25(OH)D, [ alk. phosphatase, may be occult
Y Ca, vit. D absorption 2 abnormal enterocyte function from gliadin toxicity
Gluten-free diet
Younger adults, steatorrhea, frequent ileal resection, frequent glucocorticoid use, Y 25(OH)D; Ca, P, alk. phosphatase often normal; osteopenia by X-ray; [ osteoid, Y TBV
Y Ca, vit. D absorption 2 disruption of enterohepatic circulation and abnormal jejunal-ileal function; glucocorticoid use
Vitamin D and calcium, minimize steroids and bile acid binders
Y Ca, vit. D absorption 2 partial gastric bypass and bypass of proximal small intestine
Vitamin D and calcium, restore normal anatomy
1. POSTGASTRECTOMY Up to 70%, increase with age 2. CELIAC DISEASE Up to 80% if untreated 3. CROHN’S DISEASE ~30%
4. BARIATRIC SURGERY Up to 60%
Y Ca, Mg, albumin, Y 25(OH)D, [ alk. phosphatase, PTH; osteopenia uncommon; [ osteoid, Y bone formation
TBV, total bone volume; 2 , secondary. This table is modified from [306].
control group with peptic ulcer disease also had spinal osteopenia. Twelve of 20 patients were selected for bone biopsy because the severity of their bone disease had increased osteoid seam width (12 mm), and 17 of 20 had decreased trabecular bone volume. In contrast to the high prevalence of bone disease among gastrectomized patients in these studies, two British studies showed a lower prevalence. In a survey of 1241 patients following partial gastrectomy, Morgan et al. [82,83] identified only six who had symptoms, biochemical features, and bone biopsy evidence of bone disease. However, these investigators were looking for osteomalacia, not osteoporosis. Tovey et al. [84,85] found evidence for osteomalacia (increased osteoid, decreased calcification front) in only 10 of 240 postgastrectomy patients followed over a 25-year period (only 23 patients were selected for biopsy, however). Most patients identified as having osteomalacia were females. In contrast, 22% of the males and up to 86% of the females (percentage increased with age) had radiologic evidence of osteopenia. Thus, osteopenia appears to be quite common in patients following partial gastrectomy especially as they age, although frank osteomalacia is much less frequently seen. Females in particular are predisposed to developing bone disease following gastrectomy. With the use of potent antacids the number of gastrectomies performed has decreased substantially. However, with the increase in obesity, gastric bypass procedures are increasingly popular. Such procedures also result in decreased bone mass associated with increased bone turnover as recently shown by Coates et al. [86] and
Marceau [87]. This will be discussed further under bariatric surgery as well in Chapter 55. Clinical Features Peptic ulcer leading to gastrectomy is a problem primarily of middle-aged adults, and bone disease is not likely to develop until several years after the procedure. Therefore, the clinical presentation is often that of osteoporosis in the elderly. Distinguishing between the bone disease accompanying the aging process and that due to gastrectomy is not always obvious even with a bone biopsy unless the biopsy shows frank osteomalacia. In a recent study of 471 patients following operation for peptic ulcer disease, Melton et al. [88] noted increased fracture risk but concluded that associated conditions including age, use of glucocorticoids, thyroid hormone replacement, and chronic anticoagulation could account for these findings. They found no relationship between the type of operation and fracture risk. Bone pain or tenderness is generally found in patients in whom osteomalacia is eventually diagnosed but is not a reliable indicator. Symptomatic patients often provide a history of at least modest fat malabsorption [80,82,89,90] and milk intolerance [91]. Routine laboratory assessment of patients following partial gastrectomy reveals a reduction in serum calcium and phosphate concentrations (albeit generally within the normal range) and an increase in alkaline phosphatase activity and osteocalcin in 10e25% [79,81,82]. Urinary calcium excretion tends to be low and phosphate clearance increased [81]. PTH values
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may be elevated but are normal in most patients [79,92,93], although urinary cAMP levels may be increased [94], suggesting mild hyperparathyroidism. 25(OH)D levels tend to be reduced in most studies [79,92,93,95], 24,25(OH)2D concentrations are even further reduced [93,95], while 1,25(OH)2D levels are normal or slightly increased [92,93,95,96]. The significance of these changes is obscured by concomitant reductions in the transport proteins of the vitamin D metabolites, vitamin-D-binding protein and albumin [97e99], and no correlation between 25(OH)D level and bone disease has been established [84]. In subjects undergoing complete gastrectomy for gastric cancer, Baek et al. [100] noted rapid bone loss from 5.7% in the spine to 8.7% in the hip (intertrochanter) with increased levels of bone markers and PTH but no change in 25(OH)D. This profile suggests mild hyperparathyroidism secondary to calcium deficiency, but the contribution of vitamin D deficiency is less clear. As described above, radiologic assessment demonstrates the frequent association of osteopenia and spinal fractures with gastrectomy [78e80,85,88]. Pseudofractures (condition observed in X rays as a thickening of the periosteum and formation of new bone over a region of incomplete fracture) and fractures of the hip [101] are less common but occur more frequently in these patients than in aged-matched controls [78,86,102]. The type of bone disease reported on bone biopsy varies widely from study to study, reflecting the often subtle nature of the findings and the lack of tetracycline labeling in earlier reports to determine the dynamic parameters associated with bone formation. Osteoporosis, as indicated by reduced trabecular bone volume, and osteomalacia, as indicated by increased osteoid volume and a reduced calcification front, frequently coexist [78,81,90,103]. A more recent report [95] using double-label tetracycline found normal mineral apposition rates, normal mineralization lag time, and slightly increased bone formation rates along with increased osteoid volume in 16 asymptomatic patients with partial gastrectomies. This suggests that most “osteomalacia” diagnosed in other studies not using tetracycline labeling may represent early vitamin D deficiency and/or secondary hyperparathyroidism. Some patients have increased marrow fibrosis and osteoclast numbers, clearly indicating that secondary hyperparathyroidism occurs at least occasionally [89]. Pathogenesis Absorption of vitamin D [104e106] and calcium [91,92,107,108] is reduced in postgastrectomy patients, especially those who have evidence of bone disease. Such patients tend to have mild degrees of fat malabsorption [78,80,104] in the absence of small bowel disease [78]. Milk intolerance contributes to the reduced oral intake of vitamin D and calcium in at least some
patients [109]. Normal calcium absorption has been thought to require the acid environment of the stomach to solubilize calcium salts prior to their absorption in the small intestine. Thus, procedures which reduce acid output would reduce calcium absorption. Supporting this concept is the increased relative risk of hip fractures (RR ¼ 1.44) in subjects on long-term proton pump inhibitors [110]. However, this concept has been tested directly [111,112], and it appears that gastric acid is not required for normal calcium absorption when given with food. The duodenum plays an important role in the vitamin-D-regulated absorption of calcium [1,2], so that with duodenal bypass as in the Billroth II procedure, calcium absorption is likely to be reduced. Fat malabsorption would be expected to reduce calcium absorption both directly by the formation of calcium complexes as well as indirectly by the accompanying malabsorption of vitamin D. Inadequate mixing of bile and pancreatic enzymes with the luminal contents, which could occur in duodenal isolation procedures such as Billroth-II operations, would be expected to decrease absorption of fat-soluble vitamins such as vitamin D. However, evidence supporting a greater incidence of low 25(OH)D levels or bone disease in general in subjects with Billroth-II procedures than in those with other surgical procedures is not strong [85,88,92]. Treatment The osteomalacic component of the bone disease responds to vitamin D and calcium supplements [83], but the osteoporotic component may not [113,114]. Distinguishing between these two components without a bone biopsy is difficult, and unless a biopsy is obtained to exclude osteomalacia, a clinical trial with vitamin D and calcium is indicated especially if the serum 25(OH)D concentration is low. Since malabsorption of these substances variably accompanies gastrectomy, the amount of either agent required to correct the deficiency will vary and needs to be individually established. Serum 25(OH)D concentrations are good indicators of vitamin D absorption adequacy; therefore, they can be used to titrate the amount of supplementation required. Since renal function is normal in most patients, treatment with calcitriol (1,25(OH)2D) or its analogs is seldom indicated.
Celiac Disease Incidence and Prevalence Celiac disease is not rare. The estimated prevalence is 1%, with a peak in the 4the5th decades, but may be as high as 10% in premenopausal females presenting with osteoporosis [115e117]. Most patients lack diarrhea, so the disease is often occult. The prevalence of
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bone disease among patients with celiac disease depends on the age at which the diagnosis was made and treatment with a gluten-free diet started. Most untreated adults have reduced bone mineral density at the time of diagnosis [118e124]. Similarly, 60% of children have bone growth retardation at the time of diagnosis [125]. Those who fail to respond to a gluten-free diet with improved intestinal morphology continue to have decreased bone density in comparison to their successfully treated peers [126]. Treatment started after childhood, because of delayed recognition of the disease, may be less successful and lead to persistent osteopenia [120,127]. Lindh et al. [128] found that 11 of 92 individuals with osteoporosis had IgA antibodies to gliadin compared to 3% of age-matched controls, suggesting that occult, untreated celiac disease contributes to the development of osteoporosis in a substantial portion of the population. Stenson et al. [117] found 4.5% of subjects with osteoporosis (12/266) tested positive by serologic screening, compared to 1% of non-osteoporotic controls (6/574), and of those with positive antibodies, nine with osteoporosis but only one without osteoporosis had biopsy-proven celiac disease. However, Drummond et al. [129] found no correlation between IgA endomysial and tissue transglutaminase antibodies and bone mineral density in their patient population screened for osteoporosis. The bone disease associated with celiac disease can present as osteoporosis or osteomalacia or both [121,130e132]. A recent survey found osteomalacia in three and osteoporosis in 32 of 56 patients with celiac disease [133]. Clinical Features The finding of bone disease in patients with celiac disease is usually made in association with malabsorption, although steatorrhea may be occult [130] or absent [122,131,134]. The upper small intestine is usually more affected than the ileum. Untreated patients tend to have reduced serum and urine calcium levels and elevated values for serum alkaline phosphatase, PTH, and urine hydroxyproline [120e122,127,134]. Net calcium absorption can be reduced, in part due to increased endogenous fecal calcium losses [125]. Of nine untreated patients with celiac disease Dibble et al. [97] found two with low (5 ng/ml) 25(OH)D concentrations. A higher (3/7) prevalence of low 25(OH)D levels was found by Arnaud et al. [135] using a higher value of 25(OH)D as the lower limit of normal. Bone mineral density (BMD) measurements have been found to correlate directly with circulating 25(OH)D levels [117,122]. Non-spine fracture risk is increased 1.5-fold in women over 50 years of age [136], although not in younger patients [136,137]. Serum 1,25(OH)2D levels tend to be elevated [134]. With gluten-free diet these biochemical abnormalities improve even if BMD does not
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[97,121,127]. Radiologic evidence for osteopenia is common in untreated individuals, but if treatment is started in childhood and successfully reverses villous atrophy, peak bone mass can be achieved and maintained [126]. Spinal and rib fractures occur, but pseudofractures are uncommon even in individuals with osteomalacia on bone biopsy [121]. Studies incorporating bone biopsies have found evidence for both osteomalacia and osteoporosis [121,133]. Seven of nine untreated patients studied by Melvin et al. [121] had increased osteoid, three had a diminished calcification front, and none had decreased bone volume on bone biopsy. In contrast, other studies found that osteoporosis was more common than osteomalacia [133]. Pathogenesis Celiac disease is now recognized as an autoimmune disorder associated with other autoimmune diseases such as diabetes mellitus and thyroid disorders [138]. The inflammatory component may also contribute to bone loss in that IL-1 and IL-6 levels are increased [139], whereas growth factors such as IGF-I are reduced [140]. Vitamin D and calcium absorption are abnormal in untreated patients with celiac disease [48,121] as part of their general disorder in enterocyte function. Part of the increased fecal losses of calcium may result from increased endogenous secretion through the deranged epithelium [121]. Treatment The treatment of choice is a gluten-free diet. This will correct the disorder in calcium metabolism in most cases. Vitamin D and calcium supplementation should be reserved for the individual on a gluten-free diet who fails to normalize serum calcium, phosphorus, alkaline phosphatase, and 25(OH)D levels or urine calcium excretion [141].
Inflammatory Bowel Syndromes Incidence and Prevalence Of the two major forms of inflammatory bowel disease (IBD), Crohn’s disease and ulcerative colitis, severe bone disease is most frequently associated with Crohn’s disease especially when treated with ileal resection and glucocorticoids [142]. Twenty-three subjects of an unselected series of 75 patients with inflammatory bowel disease had BMD >2 SD below normal, demonstrated by single photon absorptiometry of the radius or quantitative computed tomography of the spine [142,143]. Six of these subjects had spinal fractures. Eighteen had ileal resections. In a separate study, Bernstein et al. [143] found that glucocorticosteroid use correlated better with the bone
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loss than did disease diagnosis (Crohn’s disease versus ulcerative colitis). In an earlier retrospective study of 700 patients with IBD, osteoporosis was noted in only 3% [144], but sensitive methods to make the diagnosis were not used in that study. Osteomalacia may also be present [145]. The clinical syndrome of bone pain, weakness, elevated alkaline phosphatase activity, and radiologic features were found in 5% of subjects with Crohn’s disease [145]. Six of nine patients studied by Driscoll et al. [146] and nine of 25 patients studied by Compston et al. [147] had increased osteoid on bone biopsy despite the fact that most of these patients had few clinical features of osteomalacia. On the other hand, Ward et al. [148] noted decreased osteoid and evidence of low turnover bone disease in 20 children with IBD. Adolescents have a high likelihood of developing osteopenia and retarded bone growth [149]. Clinical Features The clinical features of bone disease in patients with Crohn’s disease are usually subtle. Most patients are young adults with a variety of gastrointestinal and extragastrointestinal concerns that obscure symptoms of bone disease. However, in a large epidemiologic study of the Danish population a slight increase in fracture risk (RR 1.15) was found in subjects with Crohn’s disease but not in those with ulcerative colitis [137]. Ileal resection, malabsorption, and glucocorticoid treatment are common and relevant to the bone disease that ensues. Routine serum biochemical measurements are generally normal but calcium, phosphorus, and magnesium may be low and the alkaline phosphatase activity may be high [146,150]. The level of alkaline phosphatase activity may correlate negatively with the degree of osteopenia [151]. IGF-1 levels have also been found to be reduced in IBD [152]. However, serum 25(OH)D concentrations are reduced in up to 65% of patients [97,142,146,153], especially those who have undergone ileal resection [97,142], but the correlation with BMD can be weak [153]. In contrast to the low 25(OH)D levels, 1,25(OH)2D3 levels are often elevated despite normal PTH levels. Colonic biopsies of these patients demonstrated increased CYP27B1 (25(OH)D-1a-hydroxylase) expression in the involved mucosa [154]. Osteopenia is commonly observed both in cortical [142,150] and cancellous [142] bone, but less than 10% of patients will have fractures or pseudofractures [142,145,146]. Bone biopsy is the only means of diagnosing osteomalacia in most of these patients. Bone biopsies frequently show reduced trabecular bone volume and increased osteoid [142,149,150]. However, one study [155] using double tetracycline labeling failed to show a reduction in bone formation, mineral apposition, or mineralization lag time in 30 unselected patients despite decreased
trabecular bone volume, suggesting that osteomalacia is less common than osteoporosis. These patients also had normal vitamin D metabolites, inactive disease, and were not on glucocorticoid therapy, so these results may not be applicable to sicker patients. Pathogenesis Patients with Crohn’s disease have multiple reasons for developing bone disease. Vitamin D [51] and calcium [156] absorption are reduced. Vitamin D is absorbed primarily in the jejunum and ileum via a process expedited by bile salts [157]. Therefore, disease or resection of this portion of the intestine will result in reduced vitamin D absorption. Concurrent use of cholestyramine or the development of hepatobiliary complications will reduce the availability of bile salts for vitamin D absorption. Vitamin D metabolites undergoing enterohepatic circulation [53] cannot be reabsorbed by a diseased or resected ileum. Calcium malabsorption reflects both the state of vitamin D insufficiency and the steatorrhea. Low dietary intake of nutrients including milk products often compounds the problem of absorption. Glucocorticoid therapy is frequently used during active disease and can contribute to the calcium malabsorption and bone loss [142,143,146]. As in celiac disease, increased levels of cytokines have been shown to stimulate bone resorption and reduce IGF-I levels at least in animal studies [158]. Additional information regarding the interplay between vitamin D and inflammatory bowel disease can be found in Chapter 96. Treatment Vitamin D in doses of 4000 to 12 000 units per day is generally adequate therapy for patients with low serum 25(OH)D levels, although the appropriate dose must be determined for each patient [150]. Calcitriol (1,25(OH)2D3) therapy would not be appropriate especially if these levels are already elevated. Dietary counseling to ensure that adequate calcium is also being ingested should be performed. Serum 25(OH)D and urine calcium levels provide good markers of treatment. Vitamin D treatment will reduce the osteomalacic component of the bone disease [146], but available data are not sufficient to determine whether the osteoporotic component will improve.
Bariatric Surgery Incidence and Prevalence There are two general types of gastrointestinal operations used in the management of obesity [159] (Fig. 69.2). Restrictive procedures such as circumgastric banding or vertical banded gastroplasty seek to reduce the capacity of the stomach leading to an obligatory
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FIGURE 69.2 Restrictive procedures such as circumgastric banding or vertical banded gastroplasty seek to reduce the capacity of the stomach leading to an obligatory restriction in oral intake of solids. Bypass procedures are designed to create various levels of malabsorption and greater weight loss. The jejunoileal bypass connects the jejunum to the ileum e the extent of the bypass can vary. The Roux-en-Y gastric bypass (RYGB) procedure combines a restricted procedure and a bypass procedure by anastomosing a small pouch of stomach into the transected jejunum attached to the duodenojejunal limb. The size of the gastric pouch and length of the bypassed limb can vary. Biliopancreatic diversion (BPD) involves a partial gastrectomy or bypass connected to the proximal ileum/distal jejunum with reanastomosis of the duodenojejunal limb into the ileum distal to the connection with the stomach.
restriction in oral intake of solids; these procedures result in less weight loss than intestinal bypass procedures, and are less likely to have much impact on bone mineral homeostasis. Bypass procedures, on the other hand, are designed to create various levels of malabsorption and greater weight loss. There are three main bypass procedures. The jejunoileal bypass connects the jejunum to the ileum e the extent of the bypass can vary. The Roux-en-Y gastric bypass (RYGB) procedure combines a restricted procedure and a bypass procedure by anastomosing a small pouch of stomach into the transected jejunum attached to the duodenojejunal limb. The size of the gastric pouch and length of the bypassed limb can vary. Biliopancreatic diversion (BPD) involves a partial gastrectomy or bypass connected to the proximal ileum/distal jejunum with reanastomosis of the duodenojejunal limb into the ileum distal to the connection with the stomach. As for RYGB, the extent of bowel bypassed correlates both with the amount of weight loss and the degree of malabsorption. All of these bypass procedures produce both substantial weight loss and malabsorption of vitamin D and calcium. The popularity of jejunoileal bypass as treatment for massive obesity has waned because of the large number of undesirable side
effects, of which bone disease is one. Initially following this operation, nearly all patients undergo at least a transient change in calcium homeostatic mechanisms [160]. Recovery occurs such that little or no osteopenia or osteoporosis can be appreciated by routine radiologic procedures [160e163]. However, osteomalacia and/or reduced trabecular bone volume on bone biopsy have been found in up to 60% of unselected individuals evaluated several years after bypass surgery [164e168]. The newer bypass procedures, reviewed more extensively in Chapter 55, have received less study and few long-term studies. However, BPD appears to have a greater impact on vitamin D levels than RYGB; for example, in one study of 82 patients, 50% were found to have 25(OH)D levels less than 14 nM (5.2 ng/ml), a level commonly associated with osteomalacia on bone biopsy, and 63% had elevated PTH levels [169]. In contrast, in a smaller study of patients treated with the RYGB, increased markers of bone turnover and reduced BMD were observed, but no changes in 25(OH)D or PTH were seen either in a 9-month prospective trial or in comparison with obese controls who did not undergo the operation (25(OH)D levels were low in both groups before and after the operation [86]).
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Clinical Features A reduction in BMD is frequently found after gastric bypass procedures accompanied by an increase in bone markers indicating high turnover [170e172]. Weight loss per se has been associated with decrements in BMD in the absence of surgery, suggesting that at least part of the bone loss is due to the reduced load on the skeleton [173]. Reductions in serum calcium and magnesium concentrations with an increase in serum alkaline phosphatase activity are observed in most subjects within 3 months of the operation and persist in approximately half the patients for years [160e163,174,175]. Urine calcium excretion tends to decrease [171] consistent with the reduction in intestinal calcium absorption [3]. Serum phosphorous levels are generally normal. At least part of the fall in serum calcium can be attributed to the fall in albumin concentration. Serum 25(OH)D levels, which tend to be reduced in the obese patient prior to operation, were found to fall even further in earlier studies [160e163,175], but more recent studies in patients supplemented with vitamin D after their procedure have not shown this decrease, and may even show an increase [170,171,176]. This increase in 25(OH)D does not prevent the fall in BMD. 1,25(OH)2D levels are often normal but may also be reduced [161,165,175]. PTH levels tend to be increased [161,165,166,175]. In most studies osteomalacia is the dominant lesion found on bone biopsy [161e164], even when more restrictive criteria incorporating the results from double tetracycline labeling are used [165e167]. Clinical features do not predict the existence of bone disease on bone biopsy [164]. Pathogenesis In the jejunoileal bypass the duodenum and much of the jejunum are effectively cut off from the flow of nutrients. RYGB restricts the size of the stomach and bypasses much of the duodenum, whereas BPD in addition bypasses much of the jejunum. Thus, these procedures would be expected to decrease calcium and vitamin D absorption, and as noted above, this has been demonstrated. However, normalizing the 25(OH)D after RYGB or BPD has not prevented the bone loss, raising the question as to whether other factors are in play. Adiponectin increases and leptin falls following surgery, either or both of which could signal to the bone to increase resorption [172,177]. Fatty infiltration of liver is often found following jejunoileal bypass, but the degree to which liver disease contributes to the bone disease is not clear. Treatment Vitamin D and the analog of 1,25(OH)2D, 1aOHD, have been used successfully to treat the bone disease following jejunoileal bypass [178,179]. However, since 1,25(OH)2D levels are usually normal in these patients,
vitamin D should be tried first using doses which normalize serum 25(OH)D levels. As noted above normalizing the 25(OH)D after RYGB or BPD has not prevented bone loss. Nevertheless this should be done given the lack of long-term follow-up studies following these procedures and the likely benefits overall of adequate vitamin D nutrition. Calcium supplementation to normalize urine calcium excretion is also indicated. Treatment failures may respond to antibiotic treatment of bacterial overgrowth in the bypassed segment [179a]. If these measures fail, reanastomosis of the bypassed segment may be required [180].
PANCREATIC DISEASES Incidence and Prevalence Clinically significant bone disease in patients with isolated pancreatic insufficiency is unusual [181], although a modest reduction in BMD has been observed in chronic pancreatitis [182]. However, reduced bone density and a high rate of fractures have been found in studies of children and young adults with cystic fibrosis [183e188]. These patients have multiple risk factors including poor nutrition, pancreatic insufficiency, reduced absorption of calcium and vitamin D, reduced physical activity, pulmonary disease, delayed and reduced production of sex steroids, use of corticosteroids, and increased circulating concentrations of osteoclast-activating cytokines [189]. These studies demonstrate a progressive decrement in bone mass relative to age group as these individuals pass through puberty into young adulthood. The loss of bone predisposes these individuals to fractures [190]. Clinical Features The clinical features of pancreatic insufficiency include diabetes mellitus and steatorrhea. Although diabetes mellitus could contribute to the reduction in bone mass, steatorrhea is the feature that should most affect vitamin D and calcium absorption. However, the link between steatorrhea and bone disease is not established for this condition. 25(OH)D and serum and urine calcium values are generally in the low or low normal range [184,185,188], although normal values have been found in some series [191]. The 25(OH)D concentrations are more likely to be reduced if the pancreatic disease is associated with cholestasis, small bowel, and/or liver disease [97]. The presence of bone disease in a patient with malabsorption thought to be secondary to pancreatic insufficiency should lead to a search for complicating features such as alcohol abuse, cholestasis, cirrhosis, or intrinsic small bowel involvement. Case reports have been published demonstrating
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osteomalacia in patients with cystic fibrosis, each of whom had liver involvement [181,192]. Nevertheless, current data are insufficient to determine the extent to which the osteopenia observed radiologically in most cases of pancreatic insufficiency represents osteoporosis versus osteomalacia. Pathogenesis The infrequency of serious bone disease in patients with pancreatic insufficiency and steatorrhea who do not also have other risk factors indicates that the steatorrhea resulting from decreased pancreatic enzyme secretion is not sufficient to cause major impairment of vitamin D and calcium absorption. However, the lack of pancreatic enzymes appears to be synergistic with disruption of bile secretion and/or intrinsic small bowel disease, producing bone disease when these other risk factors are present. The increased bone disease in patients with cystic fibrosis is better correlated with chronic inflammation than malabsorption and vitamin D deficiency [193]. Treatment Patients with low 25(OH)D concentrations should be given sufficient amounts of vitamin D to restore their 25(OH)D levels to normal. The dose will vary from patient to patient, and a number of trials have shown that even relatively high doses of vitamin D (e.g., 2000 Iu/day or 50 000 Iu/week for 8 weeks) do not normalize 25(OH)D levels in patients with cystic fibrosis [194,195]. The diet should be supplemented with enough calcium to raise urinary calcium excretion to above 150 mg/day (adults). These patients are likely to require pancreatic enzyme replacement and supplementation with other fat-soluble vitamins. TABLE 69.2
HEPATIC DISEASES For this discussion on the relationship of liver disease to vitamin D insufficiency and bone disease, four categories of liver disease will be considered: chronic cholestatic disease of which the most common is primary biliary cirrhosis, chronic active hepatitis, viral hepatitis, and alcoholic cirrhosis (Table 69.2). The major themes linking liver disease to reduced vitamin D levels are: (1) the ability of the liver to convert vitamin D to 25(OH)D; (2) the role of the liver to produce vitamin-D-binding proteins, albumin and DBP, required to transport the vitamin D metabolites to their target tissues; (3) the degree to which the enterohepatic circulation of the vitamin D metabolites contributes to the maintenance of vitamin D metabolite levels; and (4) the role of bile in promoting vitamin D and calcium absorption. Each type of liver disease has its own nuances, which contribute to these major themes such as the use of bileacid-binding resins in cholestatic diseases, the use of glucocorticoids and other immunosuppressives to treat chronic active hepatitis, and the direct skeletal toxicity of alcohol in subjects with alcoholic cirrhosis. The recent increase in liver transplantation procedures, the immunosuppression following which can accelerate bone loss at least initially, makes it important to prevent or treat these disorders at an early stage before substantial bone loss occurs.
Chronic Cholestatic Diseases Incidence and Prevalence Of the various chronic cholestatic diseases, bone disease in primary biliary cirrhosis (PBC) has been best studied. PBC is a disease primarily of middle-aged
Bone Disease Associated with Liver Disorders
Prevalence
Clinical features
Pathogenesis
Treatment
1. Primary biliary cirrhosis Up to 80%
1 females, bone pain, jaundice; Y Ca, 25(OH)D, PTH; [[ alk. phosphatase (liver); osteopenia by X-ray; Fxs uncommon, Y TBV more common than [ osteoid, generally low turnover osteoporosis
Y Ca, P, vit. D absorption; 2 , [ urinary losses of vit. D conjugates, 25 hydroxylation of vit. D intact
Osteomalacia responds to vit. D, osteoporosis does not
2. Chronic active hepatitis ~50%
Patients often on glucocorticoids; bone disease usually asymptomatic; Y 25(OH)D, osteopenia by X-ray; Y TBV
Bone disease 2 to glucocorticoid use more than to liver disease
Ensure adequate nutrition, limit glucocorticoid dose
3. Alcoholic cirrhosis Most alcoholics with 10 years abuse, bone disease and fx increase with age
Back pain, fractures; Y Ca, Mg, P, albumin, Y 25(OH)D, [ PTH; osteopenia, fractures by X-ray; Y TBV, Y bone formation, ETOH
Poor diet, ETOH induced [ urinary losses of Ca, Mg, possible direct toxic effects of ETOH on bone
Stop ETOH, [ Ca, Mg, P in diet; vit. D if Y 25(OH)D and osteomalacia on biopsy
TBV, total bone volume; 2 , secondary; ETOH, ethanol. This table is modified from [306].
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women, an age at which postmenopausal osteoporosis is common and not readily distinguished from the osteoporosis of liver disease. Both osteomalacia and osteoporosis occur in PBC, but estimates of the prevalence of these forms of bone disease vary widely. Some studies indicate that patients with PBC have primarily osteomalacia [196,197], others find mostly osteoporosis [198e201], some find a high prevalence of both osteomalacia and osteoporosis [202,203], and still others find little of either [204e207]. Although studies differ somewhat with respect to the severity of the disease in the study population or in the criteria used to diagnose the bone disease, these differences do not fully account for the differences in results. Newly diagnosed patients who have received no treatment and have mild cholestasis appear to have less bone disease than those with more severe liver disease of greater duration [97,205,208]. Although a common thread in most studies is a low 25(OH)D level [209], the 25(OH)D levels do not correlate with the BMD, PTH or findings of osteomalacia on bone biopsy [209,210]. The likelihood of developing osteomalacia as well as or instead of osteoporosis may depend on the prevalence of osteomalacia in the population since the reports of osteomalacia in PBC tend to come from the UK and Scandinavian countries where osteomalacia is more likely to be found in the general population [211,212]. Since the more recent studies show the least amount of bone disease, it may be that with the heightened awareness of the potential for bone disease in patients with PBC more attention is being paid to nutritional factors, which can prevent or forestall this complication. In contrast to PBC, biliary atresia is a disease of infants and children. Children with this condition, even if surgically corrected, have a high likelihood of developing rickets [213], which is readily treated with vitamin D. Clinical Features Patients with PBC are often asymptomatic, although bone pain is common in patients subsequently shown to have osteoporosis or osteomalacia on bone biopsy [196,199]. Laboratory assessment tends to show normal or slightly reduced serum and urine calcium, low normal serum phosphorus, and normal serum magnesium levels. PTH concentrations may be low even in subjects with decreased circulating calcium and 25(OH)D [196,199,204,214,215], although reports of elevated PTH can also be found [198,201,206,216]. Alkaline phosphatase activity in serum is increased, but the source is the liver, not bone. Osteocalcin and urinary hydroxyproline levels are normal [196,204,216,217]. Serum 25(OH)D levels can be normal in asymptomatic patients but fall as the disease progresses [197,199,218]. The 25(OH)D level is not a good predictor of bone disease, however [196,199]. 1,25(OH)2D concentrations are generally
normal [214e217,219]. Subjects with PBC have an increased prevalence of fractures and decreased bone mineral density [199,204,208,220e222], although pseudo-fractures are rare. Children with biliary atresia often present with florid rickets [213,223]. Bone biopsy is required to make a definitive diagnosis of osteomalacia, a finding which was commonly described in the early reports from the UK and Scandinavia [196,211,212]. However, the most common lesion seen in more recent studies using double label tetracycline is reduced trabecular bone volume with normal or low amounts of osteoid, reduced bone formation rates, and increased mineralization lag time e characteristics of low-turnover osteoporosis [198e200,204,214]. High-turnover osteoporosis has also been found in a subset of patients [217], which may account in part for the surprisingly rapid loss of bone seen by Matloff et al. [199] and Herlong et al. [200] during a 1-year follow-up period. Pathogenesis Bone disease in PBC has several potential etiologies. Intestinal malabsorption of calcium [199,200,214,224], phosphate [225], and vitamin D [197,224,226] has been demonstrated to occur. Vitamin D absorption is further impaired in patients treated with cholestyramine [227]. Although some reports [226,227] indicate that the hepatic hydroxylation of vitamin D to 25(OH)D is impaired, this does not appear to be a problem in most patients. 25(OH)D concentrations are readily increased with vitamin D therapy [197,228,229], although some patients have required 1,25(OH)2D to treat the osteomalacic component of the bone disease [230]. Disruption of the enterohepatic circulation of vitamin D metabolites with increased losses in the urine has been postulated to lead to vitamin D deficiency in PBC [53,54], but it has also been proposed that the lack of biliary secretion of 1,25(OH)2D accounts for the normal level of 1,25(OH)2D and decreased concentrations of PTH seen in many patients [57]. Finally, an abnormality in the bone-forming cell itself, the osteoblast, has been postulated to account for the failure of adequate vitamin D and mineral levels to correct the reduction in bone formation seen in patients with PBC [198e200,204]. Treatment When present, osteomalacia responds readily to vitamin D, calcifediol (25(OH)D), calcitriol (1,25(OH)2D), or 1aOHD [203,230]. However, osteoporosis has not been successfully treated with vitamin D or its metabolites, and calcifediol may even be detrimental [199,200,230]. If the serum 25(OH)D concentration is reduced, vitamin D should be given to restore the level to normal, although it is not clear that this will improve BMD in most patients. As will be discussed below, part of the reason is that many patients are likely
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to have low 25(OH)D levels because of a reduction in the vitamin-D-binding proteins, not vitamin D deficiency or failure to convert vitamin D to 25(OH)D, and their free 25(OH)D levels may be normal. The use of vitamin D for patients with normal 25(OH)D values is not justified. The rationale for using calcitriol or 1aOHD in the absence of renal failure is weak. Supplementing this regimen with calcium may increase the effectiveness of vitamin D therapy [231].
Chronic Active Hepatitis Incidence and Prevalence Patients with chronic active hepatitis may not have the same increased prevalence of fractures and decreased bone density as those with other forms of chronic liver disease unless they are treated with glucocorticoids [208,232]. Osteopenia of the distal radius or reduced trabecular bone volume on bone biopsy was found in 47% of patients with chronic active hepatitis treated with glucocorticoids [233], but the prevalence of bone disease in the absence of such treatment is not established. Clinical Features The bone disease associated with chronic active hepatitis is usually asymptomatic. Many patients are treated with glucocorticoids, which may account for much of the bone disease which manifests primarily as osteopenia or osteoporosis, although osteomalacia has been described [234]. Patients with chronic active hepatitis tend to have 25(OH)D concentrations below the normal range and comparable to those seen in patients with alcoholic cirrhosis or PBC [97,218,235]. The reduction in 25(OH)D is accompanied by a reduction in DBP levels [97], suggesting that the free 25(OH)D concentration may be normal in a number of patients whose total 25(OH)D values are low. No osteomalacia was observed by Stellon et al. [233] in bone biopsies from 36 patients with chronic active hepatitis, although reduced trabecular bone volume was frequently seen. In contrast Dibble et al. [234] reported the presence of osteomalacia in the bone biopsies from two of seven patients with chronic active hepatitis. Pathogenesis The scarcity of data specific to the impact of this disease on bone mineral metabolism makes problematic the compilation of a pathogenetic mechanism for the bone disease in chronic active hepatitis. As for other liver diseases calcium and vitamin D deficiency secondary to malabsorption or impaired hepatic conversion of vitamin D to 25(OH)D may be implicated but
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appears not to play a major role. Glucocorticoid therapy is likely to be more important in the pathogenesis of the bone disease in these patients than abnormalities in vitamin D absorption and metabolism. Treatment Limiting the use of glucocorticoid therapy, ensuring adequate nutrition, and encouraging sunlight exposure are recommended first steps. The role of vitamin D and calcium supplementation in patients requiring glucocorticoid therapy is not clear and cannot be recommended unless malabsorption of these substances is strongly suspected.
Viral Hepatitis Incidence and Prevalence Decreased bone mineral density was reported in 40 consecutive patients with viral hepatitis compared to healthy volunteers by Gonzalez-Calvin et al. [236]. However, in a subsequent publication this group noted that when comparing 84 postmenopausal patients with viral hepatitis to 96 healthy postmenopausal women, no differences in the numbers with osteoporosis were found [237]. Thus it is unclear whether viral hepatitis increases the risk of bone disease at least in the older population. Clinical Features In one recent study [238] 32 males mean age 58 with hepatitis B or C were evaluated. Cirrhosis on liver biopsy was documented in 25. No differences in biochemistries were found between the two forms of hepatitis. BMD of the lumbar spine and femoral neck correlated with disease severity (Child-Pugh score) in this study but not in others [237]. 25(OH)D and PTH concentrations decreased with increasing severity of the liver disease while the marker of bone resorption, urine deoxypyridinoline cross-links (D-pyr), increased [238,239]. D-pyr correlated negatively with BMD. Insulin-like growth factor I (IGF-I) also decreased with increasing severity of the liver disease, and correlated with the fall in bone mineral density. These data suggest a high state of bone turnover leading to net bone loss, but the role of vitamin D is not clear. The reported cases of osteosclerosis associated with hepatitis C also demonstrated increased bone turnover demonstrated by increased level of bone markers and by bone histomorphometry [240e242]. However, in these cases bone mass is increased, not decreased. Pathogenesis The finding of increased bone turnover in this disease suggests that the chronic inflammation in the
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liver is accompanied by increased cytokines systemically that serve to stimulate bone cell activity. Supporting this hypothesis is a study in which circulating levels of TNF receptor subunit p55 were increased [236]. The cause of osteosclerosis in some patients remains obscure. The role of vitamin D in this disease seems limited. Treatment Treatment of the underlying hepatitis is likely to prove most efficacious. Bisphosphonate therapy may prove useful in reducing what appears to be increased bone resorption, but this has not yet been tested in clinical trials. In one trial calcitriol appeared to slow the progression of bone loss [243].
Alcoholic Cirrhosis Incidence and Prevalence Alcohol-induced bone disease is not restricted to those individuals who develop cirrhosis. However, the role of vitamin D in alcohol-induced bone disease is implicated in those with cirrhosis. Saville [244] was the first to call attention to the high prevalence of osteopenia in alcoholics in his study of bone biopsies from cadavers in the New York City morgue. Spinal osteopenia may be observed in 50% of ambulatory male alcoholics by routine X-ray procedures [245], and fractures of ribs or vertebrae occur in nearly 30% of this population [246,247]. Caucasians may be more susceptible to alcohol-induced bone disease than AfricanAmericans [248], perhaps owing to the presence of a relatively higher peak bone mass in African-Americans. This prevalence of fractures is much higher than in other types of liver disease [246]. The likelihood of developing a fracture increases rapidly beyond age 45 [249]. Partial gastrectomy increases the likelihood of developing osteopenia and fractures [250,251]. Bone densitometry and bone biopsy have demonstrated osteopenia in most patients with a prolonged history of heavy alcohol abuse [252e254]. Osteoporosis is the disease usually found histologically [252e256], although osteomalacia does occur [257,258] and may be more likely in patients who have had a partial gastrectomy [259]. Clinical Features Alcoholism can be a subtle disease and may be undetected unless and even if the patient is specifically questioned about alcohol intake. The presentation is often that of idiopathic osteoporosis, discovered by chance on radiologic assessment for low back pain or pulmonary complaints. Aseptic necrosis of the hip is associated with alcoholism, but the incidence of this disease
in alcoholics is low [258,260,261]. Serum concentrations of calcium, phosphorus, and magnesium tend to be low normal in ambulatory alcoholics [252,253,256]. However, following a binge or when other alcoholrelated medical problems are serious enough to require hospitalization, serum levels of these minerals can be sufficiently reduced to cause neuromuscular disturbances and rhabdomyolysis [262]. Part of the reduction in serum calcium is accounted for by a reduction in serum albumin concentration. Serum PTH and urinary cAMP levels may be elevated or high normal in part because of the lowered calcium and magnesium levels [252,256,263], although acute administration of alcohol can lower the PTH level [264]. 25(OH)D levels are usually low [196,216,239,252,257,263,265] and correlate with the low albumin and DBP concentrations [266,267]. 1,25(OH)2D concentrations have been variably reported as low [257,268], normal [252], or high [263]. Low levels of 1,25(OH)2D are found in the alcoholics with the severest liver disease, and like 25(OH)D levels, correlate with serum albumin and DBP [267]. The free or unbound concentrations of the vitamin D metabolites are generally normal [267,268] (Table 69.3). It is now appreciated that much of the reduction in the total concentrations of the vitamin D metabolites is a direct result of the reduction in the circulating levels of the carrier proteins [99,269]. The radiologic assessment of bone reveals osteopenia or osteoporosis. Cancellous bone is more affected than cortical bone. Fractures are common, often following minimal trauma, but pseudofractures are rare in this population. The bone biopsy usually reveals reduced trabecular bone volume with normal or decreased amounts of osteoid [249,252,256], although a few patients will have increased osteoid volume [257,258]. Marrow fibrosis is uncommon. Bone formation and active bone resorption are generally reduced
TABLE 69.3 Total and Free Vitamin D Metabolite Levels in Subjects with Alcoholic Liver Disease Liver disease
Normal
Total (ng/ml)
10.9 9.5*
19.2 6.6
Free (pg/ml)
6.6 4.6
5.9 2.3
Total (pg/ml)
22.6 12.5*
41.5 11.5
Free (fg/ml)
209 91
174 46
DBP (g/ml)
188 105*
404 124
Albumin (g/dl)
2.8 0.7*
4.5 0.2
25(OH)D
1,25(OH)2D
* Significantly lower than normal. Data taken from [268] and [267].
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[252e258], although in younger patients high rates of bone turnover may be observed [249]. Pathogenesis The original reports of low 25(OH)D levels in alcoholics led to suggestions that poor nutrition [270], decreased sunlight [271], vitamin D malabsorption [272], or defective hydroxylation of vitamin D to 25(OH)D [266,273] might be involved in alcoholic bone disease. Vitamin D deficiency, therefore, could account for the osteomalacia seen in some alcoholic patients. Hypophosphatemia due to poor intake, malabsorption, concomitant use of aluminum-containing antacids, or increased renal excretion [274] could enhance the mineralization defect. However, the infrequency of osteomalacia [252], the finding of normal free levels of the vitamin D metabolites [267], and the realization that the low total concentrations of the vitamin D metabolites reflect decreased hepatic production of DBP and albumin not decreased hepatic production of 25(OH)D [275e277] all indicate that for most individuals the bone disease is not one of vitamin D deficiency in that the free 25(OH)D levels are likely to be normal. Calcium deficiency from poor intake, malabsorption [278,279], or increased urinary excretion [279] could lead to osteoporosis especially if associated with secondary hyperparathyroidism [252,256,263]. Mild degrees of hypomagnesemia could aggravate this picture (very low magnesium levels cause hypoparathyroidism). However, evidence for hyperparathyroidism is seldom seen on bone biopsies; rather the picture is usually one of inactive bone at least in the older individual. Failure to explain the bone disease of alcoholics based on changes in the calciotropic hormones has led to the hypothesis that the prime offender is alcohol or one of its metabolites such as acetaldehyde causing direct inhibition of bone cell activity [252,256,264]. Treatment Cessation of alcohol consumption appears to arrest the progression of the bone disease, and may reverse it [280,281]. Vitamin D therapy should be considered if the 25(OH)D levels are lower than what would be expected for the reduction in albumin and DBP. Such therapy will reverse osteomalacia if present and may help to restore bone mass [256]. Vitamin D itself is effective in most subjects since malabsorption is usually not severe [276], and 25(OH)D production is usually intact [276,277]. Ensuring adequate nutrition including calcium, magnesium, and phosphate also is appropriate. However, the degree to which osteoporosis can be reversed with current therapeutic measures remains unclear.
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Liver Transplantation Incidence and Prevalence As the survival of patients undergoing liver transplantation improves (currently approximately 75% 5-year survival) [282], the metabolic complications of this procedure become more important. Osteoporosis is one such complication (see Chapter 68). In a series of 146 patients surviving for at least 1 year, Porayko et al. [232] found accelerated bone loss in nearly all patients, but the degree of morbidity in the skeletal system depended on the underlying liver disease prior to transplantation. Patients with PBC and primary sclerosing cholangitis (PSC) had the highest prevalence of osteopenia and osteoporosis prior to surgery (54% of 78 subjects had spinal bone densities below the fracture threshold of 0.98 g/cm2, five of whom had fractures) compared to patients with chronic active hepatitis (n ¼ 44) and a miscellaneous group (n ¼ 24) (15% of whom had spinal bone densities below the fracture threshold, one of whom had a fracture). Similar results were found by others [283]. Following transplantation the patients with PBC and PSC showed accelerated bone loss (up to 30 times normal) for the first 3 months, and 29 (37%) of these patients developed new fractures. In the 68 non-PBC/PSC patients, bone loss was more gradual, and only three of these patients developed new fractures. Of the 12 patients who developed aseptic necrosis, 11 were in the PBC and PSC group. Most of the fractures occur in the first 6 months following transplantation [232,284]. The accelerated bone loss appears to be due to increased bone turnover as demonstrated histomorphometrically by Vedi et al. [285]. However, if liver function is restored and adequate levels of 25(OH)D are maintained (pre and post transplantation) recovery of BMD over the subsequent 4e24 months is generally good [283]. Clinical Features Many patients being considered for liver transplantation already have bone disease [286]. Such patients are started on high doses of glucocorticoids and immunosuppressives such as cyclosporine and azathioprine. Following transplantation osteocalcin levels may increase [287,288] and vitamin D metabolite concentrations fall [288]. Bone mineral density falls rapidly [232,282,284]. Fractures and aseptic necrosis appear within months [232,284]. These fractures tend to occur primarily in the spine and ribs, although hip fractures are also observed, and a single patient may have several fractures in rapid succession [289]. Bone biopsy data show high-turnover osteoporosis [285]. Pathogenesis The drugs used to prevent and treat rejection are almost certainly the cause of the rapid loss of bone,
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leading to aseptic necrosis and fracture. Patients with preexisting bone disease and low 25(OH)D levels are particularly susceptible. Contributing factors include immobilization and wasting, which accompany any major surgical procedure. Treatment Prior to transplantation all factors predisposing to bone disease should be corrected if possible, including calcium and vitamin D deficiencies. Newer immunosuppressive agents may permit lower doses of glucocorticoid hormones to be used, but it remains to be seen whether such changes will alter the rapidity and extent of bone loss following transplantation. Calcium and 1a(OH)D did not prevent bone loss in one study [284]. However, antiresorptive agents such as bisphosphonates in combination with calcium and vitamin D have been shown to prevent bone loss following transplantation in more recent studies [290,291].
TOTAL PARENTERAL NUTRITION Incidence and Prevalence At the time the relationship between total parenteral nutrition (TPN) and bone disease was first described [292,293], 20e30% of patients on long-term TPN complained of bone pain often occurring within 1 year of beginning treatment. Infants similarly treated presented with radiologic and biochemical evidence of rickets [294]. Most of the patients who were biopsied showed evidence of osteomalacia [292,293] regardless of the presence of bone pain. In these early studies, casein hydrolysate was used as the source of amino acids. Casein hydrolysate was subsequently shown to contain high concentrations of aluminum [294], a contaminant strongly implicated in the osteomalacia associated with hemodialysis and which was found in high concentrations in the bone of patients on long-term TPN [295]. Substituting purified amino acids for casein hydrolysate has markedly reduced the incidence of bone pain and prevalence of osteomalacia on bone biopsy, although osteopenia still occurs and may affect approximately 50% of patients on long-term therapy [296e298]. Although TPN appears to result in progressive loss of bone, patients requiring TPN often have preexisting bone disease [299]. Clinical Features When initially described, TPN-induced bone disease resulted in severe bone pain primarily affecting the lower extremities, lower back, and ribs. Some patients could not walk as a result of the pain. These symptoms resolved when TPN was discontinued. This clinical
picture is seldom seen today with newer formulations of TPN solutions. In the initial studies [292,293, 300,301], serum calcium, alkaline phosphatase, and phosphorus levels were elevated. At least part of the increased alkaline phosphatase was hepatic in origin as other liver function tests were abnormal [302]. Hypercalciuria exceeding the infused amount of calcium was observed. PTH and 1,25(OH)2D levels were low despite normal levels of 25(OH)D and adequate amounts of vitamin D in the TPN solution. Substituting purified amino acids for casein hydrolysate [296,297] resulted in lower serum calcium levels and normal serum phosphorus, PTH, 25(OH)D, and 1,25(OH)2D concentrations. Alkaline phosphatase activity continued to be elevated in these patients, and osteopenia was still found radiologically [299]. In the original reports bone biopsies showed reduced trabecular bone volume, increased osteoid, and decreased mineralization characteristic of osteomalacia [292e295]. More recent reports of patients on TPN supplemented with purified amino acids rather than casein hydrolysate show normal levels of osteoid and normal bone formation rates, although reduced trabecular bone volume is still seen [297,298]. Pathogenesis In the original studies by Shike et al. [293,301], vitamin D itself was implicated in the genesis of the bone disease, although the mechanism for this was obscure. This explanation has given way to the hypothesis that aluminum is the likely culprit for many of the abnormalities. Aluminum contaminates not only casein hydrolysate, but also albumin, phosphate, and calcium solutions [303,304]. However, casein hydrolysate appears to be the major source of aluminum contamination, and replacing this with purified amino acids has resulted in a marked reduction in aluminum concentrations in the blood, urine, and bone of patients receiving TPN [294]. Changing from casein hydrolysate to purified amino acids has reduced the amount of clinically evident bone disease and altered the morphologic picture from osteomalacia to osteopenia. The reduction in aluminum has also corrected the low levels of PTH and 1,25(OH)2D and improved the hypercalciuria which characterized the original syndrome. The reasons for the persistence of the bone disease in patients receiving the newer formulations of TPN are not yet clear. At least some of the patients have bone disease and vitamin D deficiency before they begin TPN because of the underlying gastrointestinal disorder that leads them to require TPN. Furthermore, the amino acids in the TPN solution may induce hypercalciuria and subtle hyperparathyroidism if the infused amounts are high [305].
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Treatment Discontinuing TPN, when feasible, may correct the bone disease [292]. Adjusting the vitamin D, amino acid, and calcium concentration to achieve a positive calcium balance needs to be done in those patients who cannot discontinue TPN. Reducing the aluminum contamination of the solutions to the lowest possible level has proven to be of great importance. Other trace contaminants or deficiencies which impact on the skeleton may be found in the future.
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C H A P T E R
70 Vitamin D and Renal Disease Adriana S. Dusso 1, Eduardo Slatopolsky 2 1
Experimental Nephrology Laboratory, IRB Lleida, Lleida, Spain, 2 Washington University School of Medicine, St. Louis, MO, USA
INTRODUCTION The kidney is a central component of the powerful endocrine system that has evolved to maintain extracellular calcium and phosphate within narrow limits, a process vital for normal cellular physiology and skeletal integrity. Indeed, in the course of kidney disease, abnormalities in calcium, phosphate, parathyroid hormone (PTH), and vitamin D metabolism cause alterations in bone turnover, mineralization, volume, linear growth or strength, vascular and soft tissue calcifications, and high mortality rates [1]. Therefore, the Global Mineral and Bone Initiative to improve outcomes in renal disease has recommended in its KDIGO (Kidney Disease: Improving Global Outcomes) guidelines to replace the term “renal osteodystrophy” with “chronic kidney disease-mineral and bone disorder” (CKDMBD) to better describe the broader syndrome that affects kidney disease patients [1]. The integrity of the vitamin D endocrine system plays a central role in the tight control of complex calcium and phosphate fluxes, secretory control, and hormonal interactions between the kidney, the parathyroid glands, bone, and intestine. Normal renal synthesis and secretion of the potent calcitropic hormone 1,25-dihydroxyvitamin D (1,25(OH)2D or calcitriol) [2] is critical to integrate the calcium (Ca)/parathyroid hormone (PTH) axis as well as the phosphate (P)/fibroblast growth factor 23 (FGF23) loop, in order to ensure normal Ca and P homeostasis and skeletal integrity while preventing an excess of Ca and P ions that could lead to overmineralization of bone or ectopic calcification (reviewed in [3,4] and depicted in Fig. 70.1). Briefly, in normal individuals, a decrease in serum Ca is sensed by the parathyroid glands, which to restore Ca balance rapidly enhance the secretion and/or synthesis of PTH. Elevations in serum PTH induce Ca resorption
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10070-8
from the bone and stimulate the expression and activity of renal cyp27B1, also called 25-hydroxyvitamin D 1ahydroxylase (1a-hydroxylase), the enzyme responsible for the conversion of 25-hydroxyvitamin D (25(OH)D) to calcitriol. The increases in serum calcitriol induce intestinal Ca absorption, and upon Ca normalization, both Ca and calcitriol close the loop by suppressing PTH synthesis and renal 1a-hydroxylase expression and activity [3]. Calcitriol also elevates serum P levels by promoting intestinal absorption, renal reabsorption and bone resorption, and both high P and calcitriol induce FGF23 synthesis in bone [5]. Elevations in FGF23 close the P/ calcitriol/FGF23 loop by suppressing renal calcitriol production through a direct inhibition of 1a-hydroxylase expression [6] and through the induction of cyp24A1 (24hydroxylase) [7], the enzyme responsible for calcitriol degradation, and by lowering serum P through the inhibition of renal P reabsorption [6]. FGF23 also affects the Ca/calcitriol/PTH loop by a direct inhibition of PTH secretion by the parathyroid glands [8]. In CKD, the progressive loss of renal capacity to synthesize calcitriol impairs intestinal calcium absorption. The low serum calcitriol and calcium levels are key contributors to elevations in PTH and eventually to an induction of hyperplastic parathyroid growth in an effort to re-establish calcium balance. High circulating levels of PTH cause bone loss, osteitis fibrosa and various degrees of skeletal abnormalities [9], but fail to induce renal calcitriol synthesis [10]. Similarly, the increases in FGF23 that are induced by the high serum P levels due to renal P retention further aggravate calcitriol deficiency through direct inhibition of renal calcitriol synthesis and through the induction of calcitriol catabolism, but fail to suppress PTH secretion and to induce hypophosphatemia. The combination of high serum levels of PTH, low serum calcitriol, and impaired
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FIGURE 70.1 Central role of renal calcitriol production in mineral and skeletal homeostasis. Renal calcitriol (1,25D) production tightly controls the complex hormonal feedback loops between PTH and FGF23, as well as Ca (black arrows) and P (gray arrows) fluxes between the intestine, bone, and the kidney that ensure normal mineral homeostasis and skeletal integrity, while preventing the excess of both ions predisposing to ectopic calcifications. Kidney disease impairs both the renal uptake of 25(OH)D and calcitriol synthesis causing severe abnormalities in the calcitriol/FGF23/PTH loops (see text for details) predisposing to bone and mineral disorders, soft tissue calcifications, renal and cardiovascular damage, and high mortality rates.
Parathyroid glands
PTH Blood 1-hydroxylase
1,25D
25D
Ca P
FGF23
Kidney
P
Bone
(Urine)
Intestine
FGF23 actions causes an excess of Ca and P ions and skeletal abnormalities predisposing to renal and cardiovascular damage, ectopic calcifications, and high mortality rates [1]. Only recently the impact of increases in FGF23 in the P/calcitriol/PTH loop both systemically and in the parathyroid glands has been under extensive scrutiny. Therefore, for almost 30 years the main therapeutic approach to improve outcomes in CKD has been based upon the safe and effective correction of calcitriol deficiency to suppress PTH [9]. However, two critical findings of recent years have challenged the adequacy of the exclusive control of the Ca/calcitriol/ PTH axis with calcitriol replacement therapy to improve outcomes in CKD. First, interventions with either calcitriol or its less calcemic analogs improve outcomes in CKD patients through renal and cardioprotective actions which cannot be fully accounted for by its efficacy for PTH suppression [11]. This finding has rendered serum PTH an inappropriate marker of the efficacy of calcitriol replacement therapy to moderate renal and cardiovascular lesions. Second, a striking
80% incidence of “nutritional” vitamin D deficiency (serum levels of 25(OH)D below 20 ng/ml) occurs in CKD patients [12]. This is intriguing because it is in the liver, and not in the kidney, where the conversion of inactive vitamin D to 25(OH)D takes place [3]. More significantly, vitamin D deficiency is a risk factor higher than calcitriol deficiency for the progression of renal damage to end-stage renal disease and death [13]. Thus, the safe correction of vitamin D deficiency/insufficiency in CKD, and a better understanding of the mechanisms underlying the defects of a failing kidney to maintain a normal vitamin D status and its prosurvival benefits have become a high priority for nephrologists. This chapter presents the current understanding of the molecular mechanisms causing vitamin D and calcitriol deficiency as well as resistance to calcitriol actions in CKD. The pathophysiological implications of these defects are examined in classical vitamin D target organs (intestine, parathyroid glands, bone, and the kidney), and special focus is directed to tissues unrelated to
VIII. DISORDERS
RENAL MAINTENANCE OF THE VITAMIN D ENDOCRINE SYSTEM
calcium homeostasis, such as the renin/angiotensin system and the immune system, whose malfunction compromise the renal and cardiovascular protective actions of the vitamin D endocrine system, and therefore the well-being of CKD patients in a PTH-independent manner. The last section presents the current clinical and preclinical evidence and some mechanistic considerations on the vitamin D metabolite to use and the dosage and timing to safely and efficaciously correct vitamin D and calcitriol deficiency/insufficiency and halt/ moderate skeletal, renal, and cardiovascular protection in early and advanced CKD and in kidney transplant recipients.
RENAL MAINTENANCE OF THE VITAMIN D ENDOCRINE SYSTEM The integrity of the vitamin D endocrine system is essential for human health and disease prevention [14]. Vitamin D deficiency in otherwise healthy individuals has been associated not only with the expected reductions in intestinal calcium absorption causing elevations in PTH and bone loss, but also with increased risk of hypertension, glucose intolerance, certain infectious diseases, autoimmune disorders such as multiple sclerosis and rheumatoid arthritis, cancer [14] and, more significantly for this chapter, with albuminuria, a hallmark of an abnormal kidney function [15] and with cardiovascular disease, the main cause of the high mortality of CKD patients. The kidney is essential for the integrity of the vitamin D endocrine system as the frequency and severity of all of these serious health disorders is markedly enhanced in CKD. The regulation of the expression of the more than 200 genes that mediate the health benefits of a normal vitamin D status requires the activation of the vitamin D receptor (VDR), a member of the nuclear superfamily of transcriptional regulators, by calcitriol, the most active vitamin D metabolite in the blood (reviewed in [3,14]). The kidney proximal convoluted tubule is the principal site for the more critical of the two steps that convert inactive vitamin D to calcitriol: the 1a-hydroxylation of 25-hydroxyvitamin D [2]. Therefore, normal renal function to ensure normal serum calcitriol levels was considered “the” essential role of the kidney for the health benefits of the vitamin D endocrine system. If so, why is vitamin D deficiency a better predictor than low serum calcitriol of the risk for disease progression to end-stage renal disease and death in pre-dialysis patients [13], and for early mortality in hemodialysis patients [16]? Why do vitamin-D-deficient individuals with normal kidney function have a higher risk for allcause mortality in spite of normal serum calcitriol
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[17]? These reports suggest a critical role of a defective local production of calcitriol by non-renal cells, and the resulting impairment in autocrine/paracrine activation of the VDR in skeletal, renal, and cardiovascular damage, disease progression, and death in CKD. Since the liver is the main site for the conversion of vitamin D to 25(OH)D [3], and kidney cells are not unique in their capacity to convert 25(OH)D to calcitriol [18], why are all the health disorders caused by vitamin D deficiency exacerbated in CKD? Because normal kidney function is also essential in the maintenance of serum levels of 25(OH)D for its local activation to calcitriol by non-renal 1a-hydroxylases and, consequently, for the health benefits of autocrine/paracrine VDR activation. This section presents the current understanding of the abnormalities in renal and extrarenal calcitriol production that compromise calcitriol endocrine and autocrine/paracrine actions in CKD.
Alterations in Renal Vitamin D Bioactivation to Calcitriol in CKD In health, the expression and activity of renal 1a-hydroxylase, a mitochondrial cytochrome-P450 mixed-function oxidase, is tightly regulated to maintain extracellular calcium and phosphate levels within narrow limits. PTH, hypocalcemia, and hypophosphatemia are the major inducers of 1a-hydroxylase expression and activity whereas FGF23, hyperphosphatemia, hypercalcemia, and calcitriol are the main repressors [3]. CKD impairs both the expression and the tight regulation of renal 1a-hydroxylase. Decreased Renal Mass Martinez et al. [19] and other investigators have shown that in the course of CKD the levels of calcitriol in the blood remain in the normal range until the glomerular filtration rate (GFR) falls below 50% of normal (see Fig. 70.2) [20e23]. However, plasma levels of calcitriol below normal with creatinine clearances between 50 and 80 ml per minute were also reported [24]. Importantly, even normal levels of calcitriol should be considered abnormally low for the elevated circulating concentrations of PTH at these early stages of kidney disease. The progressive impairment in calcitriol synthesis by the remnant renal 1a-hydroxylase of a failing kidney is aggravated by: (1) an impaired delivery of 25(OH)D to mitochondrial-renal 1a-hydroxylase; (2) a blunted induction of enzymatic activity in response to PTH, calcium, or dietary phosphate restriction; and (3) a direct inhibition of the expression and/ or activity of remnant renal 1a-hydroxylase by Nterminally truncated PTH fragments or C-terminal PTH fragments [25], by hyperphosphatemia, by elevations
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FIGURE 70.2 Serum calcitriol correlates directly with renal function. The reductions in serum calcitriol levels in 165 patients with different degrees of CKD parallel the decreases in glomerular filtration rates (GFR). * indicates GFR levels at which the decreases in calcitriol reached statistical significance. Adapted from [3].
in FGF23, acidosis, and/or the accumulation of uremic toxins. Reduced Substrate Availability to Renal 1a-hydroxylase In hemodialysis patients with a GFR below 25 ml/ min and therefore very limiting renal-1a-hydroxylase activity, serum calcitriol levels are low only if serum 25(OH)D concentrations are low or normal [26]. However, serum levels of calcitriol can be normalized simply by increasing the serum concentrations of 25(OH)D to 100e200 ng/ml through oral supplementation [26]. Furthermore, a strong correlation exists between serum levels of 25(OH)D and calcitriol which does not occur in individuals with normal kidney function (see Fig. 70.3, left panel), and which is believed to result from an impaired 25(OH)D availability to renal 1a-hydroxylase. Studies in the megalin-null mice [27] have provided important mechanistic insights on the role of the kidney in 25(OH)D availability for calcitriol production. The concept that renal 25(OH)D uptake by proximal tubular cells occurs through simple diffusion of the sterol through the basolateral membrane was challenged. In fact, simple diffusion of 25(OH)D through the cell membrane upon its dissociation from its main carrier in circulation, the vitamin-D-binding protein (DBP), cannot explain the impaired substrate availability to mitochondrial 1a-hydroxylase of CKD. Instead (see Fig. 70.3, right panel), the 25(OH)D/DBP complex in the circulation is filtered through the glomerulus and is actively endocytosed into the proximal tubular cell by the apical-membrane receptor, megalin [27], a member of the LDL receptor superfamily, which is also involved in the renal reabsorption of albumin and several other low-molecular-weight
proteins. In CKD, the lower the GFR, the lower is the amount of 25(OH)D/DBP complex in the glomerular ultrafiltrate. Reduced filtered 25(OH)D limits its uptake by proximal tubular cells, and consequently its intracellular content not only for its conversion to calcitriol by renal 1a-hydroxylase, but also for its recycle to the circulation for its delivery to non-renal 1a-hydroxylases (see Chapter 45) for autocrine/paracrine VDR activation as will be described below. In CKD, regardless of the GFR, ergocalciferol, cholecalciferol, or 25(OH)D supplementation increases the proportion of DBP carrying 25(OH)D in the blood, and consequently the amount of filtered 25(OH)D/DBP complex available for megalin-mediated endocytosis. However, renalmegalin content is reduced in CKD, which worsens the already low uptake of 25(OH)D caused by the decreases in GFR. In rats, marked reductions in renal megalin-mRNA levels are evident by 2 weeks after the induction of renal failure [28]. The low circulating calcitriol of CKD may contribute to the reduction in megalin expression in cells of the proximal convoluted tubules, as calcitriol upregulates megalin expression in renal cells in culture [29]. Thus, calcitriol deficiency in CKD not only generates a vicious cycle for progressive reductions in renal megalin and in 25(OH)D uptake which further aggravates renal calcitriol production, but also enhances the urinary loss of 25(OH)D. The latter could partially account for the high incidence of vitamin D deficiency of CKD (reviewed in [12]), and for the difficulties in normalizing serum 25(OH)D levels in these patients [30]. However, we lack direct measurements of urinary DBP and 25(OH)D in CKD to validate the accuracy of these two parameters as early predictors of the loss of kidney function. Mechanistically, the combination of vitamin D supplementation and calcitriol replacement therapy should simultaneously correct renal megalin levels for 25(OH)D uptake and renal calcitriol production, and enhance the levels of serum 25(OH)D available for renal and extrarenal 1a-hydroxylases. Blunted Induction of Renal 1a-Hydroxylase by PTH, Calcium and Phosphate Restriction PTH is the most potent stimulator of renal calcitriol synthesis. Patients with hypoparathyroidism have low calcitriol levels despite persistent hypocalcemia [31]. Furthermore, parathyroidectomy severely blunts the induction of renal 1a-hydroxylase by hypocalcemia [32]. Mechanistically, PTH activates 1a-hydroxylasegene transcription through an incompletely characterized cAMP-mediated mechanism [33e35]. In CKD, abnormalities in PTH induction of renal calcitriol synthesis prevent serum calcitriol to raise above normal levels in response to the elevations in serum PTH that are present at early stages of renal disease
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FIGURE 70.3 Defective renal 25(OH)D uptake limits renal calcitriol synthesis in CKD. Left panel: The strong correlation between serum levels of 25(OH)D and calcitriol (1,25(OH)2D) in hemodialyis patients (GFR <25 ml/min), which is absent in normal individuals (dotted line), demonstrates impaired 25(OH)D availability for its bioactivation to calcitriol by the remnant renal 1-hydroxylase in CKD. Only supraphysiological concentrations of 25(OH)D normalize serum calcitriol levels. Adapted from [26]. Right panel: circulating 25(OH)D (25D) bound to its carrier vitamin-D-binding protein (DBP) is filtered by the kidney and internalized into proximal tubular cells via megalin-mediated endocytosis. Upon its release from DBP, 25(OH)D is either delivered to 1-hydroxylase by intracellular vitamin-D-binding protein 3 (IDBP3) for its bioactivation to calcitriol (1,25D), or it re-enters the circulation. Calcitriol induces renal megalin expression thereby generating a cycle that ensures normal systemic 25(OH)D and calcitriol levels as well as the reabsorption of low-molecular-weight proteins from the glomerular filtrate, including albumin. In CKD, decreases in GFR and low megalin content contribute to impair 25(OH)D uptake and protein reabsorption.
(GFR between 50 and 80 ml/min). In fact, Ritz and collaborators demonstrated an impaired increase in serum calcitriol in response to pharmacological doses of PTH in patients with a GFR of 70 ml/min and normal calcitriol levels when compared to individuals with normal kidney function [36]. As will be discussed below, increases in FGF23 in the course of kidney disease could contribute to impair the net calcitriol production in response to PTH. Acidosis, a common feature in kidney disease contributes to the impaired induction of renal calcitriol synthesis by high PTH levels. In dogs, acidosis blunts the action of PTH on the 1a-hydroyxlase of the proximal convoluted tubules. This effect can be overcome by cAMP and is unrelated to a loss of renal PTH receptors suggesting that acidosis induces an altered coupling of adenylate cyclase with the PTH receptor [10] that impedes PTH induction of renal calcitriol synthesis. Since acidosis also blunts the response to PTH on phosphate reabsorption [37], it could affect PTH induction of renal 1a-hydroxylase indirectly through concurrent effects on PTH/cAMP signaling and phosphate retention. The latter is a common feature in kidney disease and a potent inhibitor of renal 1ahydroxylase [38]. CKD also blunts the increases in serum calcitriol in response to calcium restriction [39]. In spite of the increasing use of calcimimetic drugs to control secondary hyperparathyroidism in CKD, the role of the calcium-sensing receptor (CaSR) in Ca regulation of renal 1a-hydroxylase remains unclear at present.
In individuals with normal kidney function, phosphate restriction increases renal calcitriol production and serum calcitriol levels in spite of decreases in serum PTH [40]. In intact mice and rats, dietary phosphate restriction not only enhances the activity but also the mRNA levels of renal 1a-hydroxylase [41,42]. Growth hormone plays a role in the induction of renal-1ahydroxylase mRNA levels by dietary phosphate restriction which is blunted by kidney disease. Whereas hypophysectomy blocks the stimulating action of dietary phosphate restriction on 1a-hydroxylase activity, injection of growth hormone or IGF-I to hypophysectomized rats restores the induction of renal calcitriol production in response to a low dietary phosphate intake [43]. Patients with end-stage renal disease [44] or severely uremic dogs [45] do not increase serum calcitriol in response to P restriction. It is possible, however, that the induction of the very low amounts of remnant 1a-hydroxylase by phosphorus restriction fail to result in measurable increases in serum calcitriol. Transepithelial inorganic phosphate transport by the renal tubule was suggested as an additional mechanism for low phosphate induction of renal-1a-hydroxylase. However, studies in a mouse lacking the phosphateregulated renal sodium (Na)/P co-transporter 2a (NaPT2a) demonstrated that an intact renal-Na/P co-transport is not required for the regulation of 1ahydroxylase mRNA levels and activity by dietary phosphate restriction [46]. The lower serum levels of FGF23 in the NaPiIIa null mouse compared to those in
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wild-type mice suggest a role for reduced FGF23 in the enhanced calcitriol synthesis [47]. Direct Inhibition of 1a-Hydroxylase Expression and Activity by PTH Fragments, Accumulation of Uremic Toxins, Acidosis, Hyperphosphatemia, FGF23, Klotho, and Calcitriol Replacement Therapy The accumulation of N-terminally truncated PTH fragments or C-terminal PTH fragments that directly inhibit renal 1a-hydroxylase [25] may account for the blunted response to PTH induction of calcitriol synthesis in CKD. The accumulation of uremic toxins contributes to the reduced renal calcitriol synthesis of CKD. In normal rats, the infusion of uremic plasma ultrafiltrate markedly decreases calcitriol production rates. Low-molecularweight compounds from uremic plasma ultrafiltrate act as potent direct inhibitors of 1a-hydroxylase activity in vitro [48]. More importantly, in partially nephrectomized rats, the increases in the production of uremic toxins, which can be induced by enhancing dietary protein intake, markedly suppress calcitriol synthesis. The net direct impact of acidosis on renal 1ahydroxylase is controversial, as it has been shown to decrease, increase or not to change serum calcitriol levels [49e53]. As described earlier, metabolic acidosis could indirectly inhibit calcitriol synthesis through a blunted response to PTH/cAMP, and also through acidosis-mediated increases in serum phosphate and in ionized calcium, all of which are known inhibitors of renal 1a-hydroxylase expression and activity. In contrast to the stimulatory effects of phosphate restriction, oral phosphate supplementation reduces serum calcitriol concentrations in patients with idiopathic hypercalciuria or in children with moderate renal insufficiency [54]. The induction of the phosphaturic factors or phosphatonins FGF-23, frizzled-related protein 4 (FRP-4), and matrix extracellular phosphoglycoprotein (MEPE) is the key determinant of the suppressive effects of high P (reviewed in [55]) on renal calcitriol production. Transgenic mice that constitutively express FGF-23 have reduced calcitriol levels in spite of low plasma phosphate [56]. FGF23 was shown to suppress 1a-hydroxylase mRNA levels dose dependently [47] and dietary and serum high P directly induces FGF23 expression. Proximal tubular cells express klotho [57], a protein essential for FGF receptor activation by FGF23 [58]. In fact, in spite of high serum FGF23 levels, the klotho-null mouse has the same phenotype as the FGF23-null mouse [59]. The role of soluble klotho in FGF23 suppression of renal 1a-hydroxylase has not been firmly established. Similar to high levels of FGF23, FRP-4 [60] and MEPE overexpression in vivo produce hypophosphatemia and
reduced calcitriol levels [61]. The klotho gene product, which encodes a b-glucuronidase and is implicated in aging and in facilitating FGF receptor 1 activation by FGF23, is a potent negative regulator of 1a-hydroxylase per se. Klotho-null mice have elevated calcitriol levels, high plasma calcium and phosphate, and die prematurely due to ectopic calcifications [62]. Basal 1ahydroxylase mRNA levels in these mice are increased in spite of hypercalcemia, hyperphosphatemia, and low PTH [58]. Calcitriol itself is a key determinant of its circulating levels. Calcitriol feedback inhibition of 1a-hydroxylase minimizes the potential for vitamin D intoxication. Calcitriol directly suppresses 1a-hydroxylase activity in kidney cell culture [63,64], and reduces 1a-hydroxylase mRNA [65e68]. However, there is no conclusive evidence of a direct suppression of the 1a-hydroxylase gene promoter by the calcitriol/VDR complex. Instead, calcitriol appears to inhibit the PTH/cAMP induction of 1a-hydroxylase gene expression [69]. Several indirect mechanisms are responsible for calcitriol inhibition of 1a-hydroxylase in vivo, including calcitriol-mediated increases in serum Ca and P levels, decreases in serum PTH, and the recently identified induction of bone synthesis of FGF23 [4,5] and renal klotho expression [58]. As described in “Alterations in calcitriol and 25 (OH)D catabolism in CKD,” below, calcitriol induction of 24-hydroxylase and, consequently, of its own degradation contributes to further reduce net 1-hydroxylase activity. The relative contribution of each of these suppressors of renal 1a-hydroxylase to the low serum calcitriol of kidney disease patients is unknown. However, a simple therapeutic maneuver, as is the enhancement of serum 25(OH)D concentrations to increase substrate availability for calcitriol production, appears sufficient to effectively counteract them all, including the strong inhibition of renal 1a-hydroxylase caused by the high serum FGF23 levels in advanced CKD, as conclusively demonstrated in hemodialysis patients [26]. Importantly, as will be discussed below, non-renal 1ahydroxylases also contribute to the correction of the low serum calcitriol to normal levels upon 25(OH)D supplementation. The relative contribution of renal and non-renal 1a-hydroxylases to the increases in calcitriol production rates with 25(OH)D supplementation cannot be easily measured. Alterations in Calcitriol and 25(OH)D Catabolism in CKD The induction of calcitriol catabolism provides a fine tuning not only of serum calcitriol levels but also of the response to calcitriol in target organs [2]. Cyp 24A1 or 24-hydroxylase is an enzyme constitutively expressed in the kidney and induced ubiquitously by calcitriol.
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24-Hydroxylase catalyzes most steps in the major pathway responsible for calcitriol metabolic inactivation. To tightly maintain normal serum calcitriol levels, 24-hydroxylase expression and activity are regulated by calcitriol, PTH, and FGF23 in an opposing fashion to their regulation of renal 1a-hydroxylase [70]. 24Hydroxylase is induced by calcitriol and FGF23, and inhibited by PTH. In fact, in normal individuals the metabolic clearance rate of calcitriol is accelerated when its production rate increases [71]. In CKD, reduced catabolic rates of calcitriol were expected from the low circulating levels of calcitriol and the high PTH. However, decreased, unaltered, and also increased rates of calcitriol catabolism were reported. Hsu and collaborators showed decreases in calcitriol metabolic clearance rates in patients with CKD [72] and in rats with renal insufficiency [73]. These reductions were considered a compensatory mechanism to maintain normal serum calcitriol levels at early stages of kidney disease. In contrast to these findings, in dogs, calcitriol metabolic clearance rates remained unchanged with the progression of kidney disease from mild to severe. Furthermore, the changes in serum levels of calcitriol reflected the progressive decreases in production rates [74]. Importantly, the decreases in calcitriol synthesis can be reversed to a two-fold increase in production rates with 25(OH)D supplementation, raising serum calcitriol to normal levels [75], as described earlier in hemodialysis patients [26]. In relation to the reports of enhanced calcitriol catabolism in CKD, enhanced rather than reduced intestinal 24-hydroxylase was found in renal failure patients [76]. This increase in intestinal calcitriol degradation could partially account for the blunted response to calcitriol treatment to increase intestinal calcium absorption [77]. It is unclear whether CKD increases 24-hydroxylase activity in tissues other than the intestine, and whether the increases in 24-hydroxylase result from a blunted suppression by high PTH, similar to that described earlier for PTH induction of renal 1ahydroxylase [10]; from a direct induction of 24-hydroxylase by metabolic acidosis [78], by increases in FGF23 [7]; or in response to therapy with calcitriol or its less calcemic analogs. Enhanced expression of the pregnane X receptor, an important xenobiotic and drug-detoxifying receptor, and a potent inducer of 24-hydroxylase, could also contribute to aggravate calcitriol deficiency in CKD in a calcitriol (analog)-independent manner [79]. An important consideration for therapy regarding 24-hydroxylase is that this enzyme also inactivates 25(OH)D. Therefore, in CKD, increases in 24-hydroxylase induced by treatment with calcitriol or its analogs, by the increases in FGF23, acidosis, and/or calcitriol
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(analog)-independent mechanisms may contribute to further reduce the low serum levels of 25(OH)D; to the difficulties in raising 25(OH)D above 35 ng/ml; to a defective non-renal calcitriol production, and to impair autocrine/paracrine VDR activation in a tissue-specific manner. Alterations in Calcitriol Production by Non-renal 1a-Hydroxylases in CKD 1a-hydroxylase is expressed in numerous non-renal cells that contribute to systemic calcitriol levels in CKD, as conclusively demonstrated in bilaterally nephrectomized patients undergoing hemodialysis [80]. Importantly, in these anephric patients in hemodialysis, CKD also impairs 25(OH)D availability to non-renal 1-hydroxylases, as suggested by the strong correlation between serum levels of 25(OH)D and calcitriol depicted in Figure 70.4, left panel. In fact, in the absence of renal mass, the very low serum calcitriol levels can be raised to normal levels with 25(OH)D supplementation [80]. The impact of CKD on calcitriol production by nonrenal 1a-hydroxylases was examined in monocyte/ macrophages, as the enzyme is identical to the renal 1a-hydroxylase though more readily accessible. Surprisingly, a higher rather than a reduced expression of 1ahydroxylase was found in monocytes derived from peripheral blood mononuclear cells from hemodialysis patients compared to that in monocytes from normal individuals [81]. This suggests a role for the 1a-hydroxylase of monocytes in compensating for the defective calcitriol production by the proximal tubule cells of a damaged kidney. The high monocyte 1-hydroxylase content appears to reflect a defective feedback inhibition of the expression of the enzyme by the low serum calcitriol levels, as monocytes lack PTH receptors and monocyte 1a-hydroxylase does not respond to changes in serum Ca and P levels as the renal enzyme does [81]. In fact, the Vmax of monocyte 1a-hydroxylase, a measure of cell content of the enzyme, correlates inversely with serum calcitriol, and is exquisitely sensitive to downregulation by physiological levels of calcitriol [82]. The tight control of monocyte-macrophage calcitriol production by physiological concentrations of serum calcitriol in CKD patients differs markedly with that reported in monocytes from patients with sarcoidosis and other granulomatoses. In these disorders, hypercalcemic episodes result from the inability of the high serum calcitriol concentrations to either suppress calcitriol synthesis by the disease-activated macrophages or to induce 24-hydroxylase [83]. It is unclear at present whether the high levels of FGF23 of CKD compromise or enhance monocyte/ macrophage calcitriol production because, contrary to the inhibition of renal 1-hydroxylase, FGF23 enhances
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FIGURE 70.4 Calcitriol corrects the impaired 25(OH)D availability to non-renal 1-hydroxylases in CKD. Left panel: the strong correlation between serum levels of 25(OH)D and calcitriol (1,25(OH)2D) in bilaterally nephrectomized patients (anephrics) demonstrates impaired 25(OH)D availability for its bioactivation to calcitriol by non-renal 1-hydroxylases. Only supraphysiological concentrations of 25(OH)D normalize serum calcitriol levels. Adapted from [260]. Right panel: 25(OH)D uptake (30 min) at day 0 (left bar) and at day 15 (right bar) by peripheral blood monocytes from normal individuals (Normal) and hemodialysis patients (Uremic) receiving either vehicle (Controls) or intravenous calcitriol (1,25(OH)2D) three times a week for 15 days. Modified from [82].
1a-hydroxylase mRNA levels in cells from normal parathyroid glands [8]. The decreases in parathyroid klotho content induced by CKD [84] appear to limit FGF23 induction of parathyroid calcitriol production. The distinct regulation of renal and parathyroid 1a-hydroxylase by FGF23, and that of monocyte/ macrophage 1a-hydroxylase by CKD or inflammatory disorders provide clear examples of the complexity behind the design of effective vitamin D interventions that simultaneously improve endocrine and autocrine/ paracrine VDR actions in the most relevant target cells. CKD also impairs 25(OH)D uptake by non-renal cells with the capacity to produce calcitriol [85]. Peripheral blood monocytes from hemodialysis patients elicit ex vivo a markedly impaired uptake of 25(OH)D compared to monocytes from normal individuals (Fig. 70.4, right panel) [82]. This defective uptake can be corrected by normalizing the low serum calcitriol levels of hemodialysis patients through intravenous calcitriol supplementation for 15 days (Fig. 70.4, right panel). Neither the mechanisms mediating the impaired 25(OH)D uptake nor those involved in its correction by calcitriol treatment are known. Calcitriol was shown to modulate LDL-receptor expression and function in the human monocytic cell line HL60 [86,87]. A similar mechanism could partially account for calcitriol-induced correction of the impaired uptake of 25(OH)D by peripheral blood monocytes from hemodialysis patients.
In the parathyroid glands, a defective uptake of 25(OH)D by parathyroid cells, which express 1ahydroxylase [88,89] and megalin [90], appears to limit the local activation of 25(OH)D to calcitriol [89] and therefore the autocrine/paracrine suppression of even PTH at early stages of CKD. Indeed, in CKD stages 3 and 4, ergocalciferol administration effectively reduces serum PTH exclusively in those patients achieving serum 25(OH)D levels above 30 ng/ml [30,91,92]. Unfortunately, the minute size of the rat parathyroid glands precludes a direct assessment of the relative contribution of the low serum 25(OH)D, or of a defective uptake of 25(OH)D to the impaired PTH suppression by the parathyroid-produced calcitriol. The failing kidney appears to contribute to the impaired autocrine/paracrine calcitriol actions, as the difficulties in normalizing serum 25(OH)D in these patients associate directly with the degree of proteinuria [12]. In summary, in the course of kidney disease, progressive reductions in serum 25(OH)D levels are key determinants of the severity of the defects in renal and extrarenal calcitriol synthesis causing low serum calcitriol and impaired calcitriol endocrine and autocrine/ paracrine actions. Vitamin D supplementation can improve the impaired substrate availability to renal and non-renal 1a-hydroxylases and normalize serum calcitriol levels. Calcitriol replacement therapy can correct the reduced renal megalin content and the
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defective 25(OH)D uptake by non-renal cells. Thus, the combination of nutritional vitamin D and calcitriol (analog) replacement therapy should simultaneously enhance 25(OH)D availability and improve renal and non-renal 25(OH)D uptake for endocrine and/or autocrine/paracrine actions. The safety and efficacy of this combination to improve outcomes need to be tested in prospective trials.
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Alterations in Calcitriol/VDR Endocrine and Autocrine/Paracrine Actions Most if not all of calcitriol-biological actions are mediated by a high-affinity receptor, the vitamin D receptor (VDR), which acts as a ligand-activated transcription factor (see Chapters 7, 8, and 10). There is compelling evidence from patients and experimental animal models that kidney disease not only impairs renal and extrarenal calcitriol synthesis, but also reduces VDR content in target tissues and induces abnormalities in calcitriol/VDR regulation of the expression of vitamin-D-responsive genes, all of which severely impair calcitriol endocrine and autocrine/ paracrine actions. This section presents the current understanding of the mechanisms underlying the defects in VDR expression and function, and the potential use of polymorphisms in the VDR gene for the early identifications of patients at a higher risk for disease progression and death due to defects in VDR content or function. Defective Homologous Upregulation of VDR The best-known regulator of VDR expression in target tissues is calcitriol itself. Calcitriol upregulation of VDR expression involves dual mechanisms: it increases VDR mRNA levels and/or VDR-protein stability [93,94]. The latter is the most common process in calcitriol upregulation of VDR content. Calcitriol binding to the VDR prevents the degradation of the receptor by the proteasome complex, thereby enhancing the half-life of the ligand-bound VDR compared to that of the unliganded VDR [94]. In renal failure, the low serum calcitriol levels contribute to the decreases in VDR content, as demonstrated in parathyroid glands from normal and uremic rats. Not only does parathyroid VDR content strongly correlate with serum calcitriol levels [95] (see Fig. 70.5), but, more significantly, the reductions in parathyroid VDR content could be prevented by prophylactic administration of calcitriol or its less calcemic analog 22-oxa-calcitriol (see Fig. 70.6) [95]. Furthermore, calcitriol-binding studies in the parathyroid glands of humans [96,97], rats [98], and dogs [99] support a reduction in VDR number in uremia without changes in the affinity of the receptor for calcitriol. In
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Parathyroid VDR content correlates directly with serum calcitriol. The reductions in parathyroid VDR in uremic rats compared to normal controls parallel the progressive decreases in serum calcitriol levels induced by CKD. Adapted from [54].
FIGURE 70.5
human secondary hyperparathyroidism, the lowest VDR content, both protein and mRNA levels, occurs in areas of nodular growth, the most aggressive form of parathyroid hyperplasia in advanced kidney disease [96]. The lower the parathyroid VDR the poorer is the response of these patients to calcitriol replacement to suppress PTH. Controversial reports exist on calcitriol binding to and regulation of intestinal VDR. Increased and reduced VDR binding was found in uremic rats compared to normal animals. In normal rats, calcitriol has no effect on VDR mRNA levels but markedly increase VDR protein by 12 to 24 h [100]. In contrast, in uremic rats, there is a blunted response to calcitriol. Despite an increase in VDR mRNA levels by 5 h, there is only a mild increase in VDR protein after 12 to 24 h of calcitriol exposure [100]. High PTH levels or hypocalcemia could mediate the impaired calcitriol upregulation of intestinal VDR, as the induction of hyperparathyroidism by calcium restriction was shown to prevent calcitriol upregulation of intestinal VDR [101]. As described earlier, PTH induction of 24-hydroxylase and calcitriol degradation could contribute to the defective upregulation of the VDR in the intestine. In contrast to these reports, Patel and co-workers demonstrated similar increases in VDR binding after 18 h exposure to calcitriol that were preceded by transient increases in VDR mRNA [102]. In the kidney, vitamin D or calcitriol supplementation to rats with a normal calcium intake increases VDR content up to five-fold [103e105]. While Brown and collaborators reported a mild induction of kidney VDR mRNA by calcitriol in rats fed a high-calcium diet [106], other investigators found no induction of renal VDR mRNA by calcitriol [94,107]. In peripheral blood monocytes, reduced and normal VDR levels were reported in monocytes from hemodialysis patients compared to monocytes from normal individuals [82,108].
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VDR binding (fmol/mg protein)
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70.6 Ligand binding prevents decreases in parathyroid VDR content. Prophylactic calcitriol (1,25(OH)2D3) and 22-oxacalcitriol (OCT) supplementation prevented the reductions in VDR content in the parathyroid glands of uremic rats receiving vehicle (0 ng). Parathyroid VDR levels in the treated uremic rats were similar to those in rats with normal renal function (Normal).Adapted from [54]. FIGURE
1000
*
*
*
*
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400
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Calcitriol is known to upregulate VDR mRNA levels in the parathyroid glands and the kidney through mechanisms that cannot be fully accounted for by a classical induction of VDR gene expression by the calcitriol/ VDR complex, as there are no vitamin-D-responsive elements in the VDR promoter. In addition to the existence of distant VDRE enhancers in the VDR gene, several lines of evidence support a role of the transcription factor C/EBPb (CAAT-enhancer-binding protein b) in calcitriol upregulation of VDR mRNA levels: (1) C/ EBPb induces the expression of the VDR gene upon binding to a C/EBP-binding site in the VDR promoter to enhance gene transcription [109]; (2) calcitriol induces C/EBPb expression in several cell types including renal and parathyroid cells [109e111]; (3) inhibition of C/ EBPb induction of VDR gene expression mediates the progressive reduction in parathyroid VDR levels with the severity of parathyroid hyperplasia in human and rat secondary hyperparathyroidism [111]. The molecular bases for the strong association between the severity of parathyroid hyperplasia and the reductions in parathyroid VDR have been delineated (see Fig. 70.7). In rat and human secondary hyperparathyroidism, elevations in the parathyroid expression of the potent growth promoter transforming growth factor-a (TGFa) in response to renal injury, low dietary calcium, high dietary P and/or calcitriol deficiency, and TGFa self-induction are sufficient to generate a feed-forward loop for TGFa activation of its receptor, the epidermal growth factor receptor (EGFR), which aggravates growth and reduces parathyroid VDR content [111,112]. In fact, interrupting this loop with
the use of highly specific EGFR-tyrosine kinase inhibitors, which impede TGFa activation of the EGFR, not only prevents further increases in parathyroid TGFa levels and growth rates, but also prevents VDR reduction, hence restoring the response to calcitriol replacement therapy for PTH suppression [111]. Enhanced parathyroid TGFa/EGFR signaling induces the synthesis of the truncated, dominant negative isoform of C/EBPb, also called LIP for liver inhibitory protein. LIP is a potent mitogen and its increases in the parathyroid gland aggravate the hyperplastic growth and also reduce parathyroid VDR mRNA and protein by antagonizing C/EBPb induction of VDR gene expression [111]. In fact, in human secondary hyperparathyroidism, the highest parathyroid TGFa and LIP levels of nodular hyperplasia coincide with an 80% reduction in VDR mRNA [111]. The translational implications of a direct cause/effect relationship between EGFR activation and VDR reductions extend beyond parathyroid hyperplasia to other benign and malignant hyperproliferative disorders driven by EGFR activation and are of relevance in the progression of renal and cardiovascular injury and in the onset of resistance to vitamin D renal and cardiovascular protection. Specifically, in the mouse kidney enhanced renal TGFa/EGFR activation is induced by nephron reduction, prolonged renal ischemia, or prolonged exposure to angiotensin II [113] and is a main determinant of the severity of the damage to the renal parenchyma also in human kidney disease [114]. Therefore, increases in renal TGFa/EGFR-driven induction of LIP may generate a vicious cycle similar to that of nodular hyperplasia; that is, progressive renal damage
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FIGURE 70.7 Pathogenesis of parathyroid hyperplasia and calcitriol resistance in CKD. Increases in parathyroid TACE, TACE-mediated release of TGFa, TGFa activation of the EGFR, and TGFa/EGFR induction of the synthesis of the potent mitogen LIP cause a feed-forward loop for exacerbated growth and propensity to nodular hyperplasia. Because LIP also exerts a dominant negative inhibition of VDR gene transcription, the reductions of parathyroid VDR content causing resistance to calcitriol therapy are directly proportional to the severity of the hyperplastic growth.
TGFα
EGFR Pro-TGFα P-EGFR
Translocation tion
tabiliza
rotein s
TACE p
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P-ERK1/2
LIP
TACE (active)
Growth exacerbation Inhibition of VDR gene transcription
Parathyroid cell
and reductions of calcitriol synthesis, as well as a LIPdriven antagonism on C/EBPb induction renal VDR gene expression thereby aggravating the reductions in renal VDR content resulting from the low serum calcitriol. Potential Contribution of VDR Polymorphisms to Reduced VDR Expression and Function The human VDR is encoded by a gene, which localizes in chromosome 12. The gene has 11 exons and the first three are variably present in VDR mRNAs. There are normal genetic variants or polymorphisms of the human VDR [115,116] and their association with human disease is the focus of Chapter 56. This section summarizes the existing evidence of the failure of the analysis of single nucleotide polymorphisms (SNP) in the VDR gene in CKD, as well as the potential for linkage disequilibrium analysis to identify sequences in normal VDR variants that effectively predict which patients are at higher risk of poor responses to calcitriol replacement therapy. The most frequently studied of these polymorphisms are located at the 30 untranslated region in the intron separating exon VIII and IX and defined by the restriction enzymes BmsI, ApaI, and TaqI. Controversial reports exists on the association between the frequency of these SNPs and: (1) the propensity for the onset [117,118] and progression of secondary hyperparathyroidism [119]; (2) the responsiveness of the parathyroid glands to changes in ionized calcium [120] or vitamin D therapy [121]; (3) the time these patients can remain in hemodialysis before they need parathyroidectomy [122]; (4) the susceptibility to bone loss [123]; and (5) the faster loss of bone mineral density after
transplantation resulting from a more severe secondary hyperparathyroidism [124]. The cause of these associations is still unclear because the encoded VDR protein remains unchanged and there are no changes in VDR mRNA stability to affect VDR expression or an accelerated targeting of the VDR for proteasomal degradation. These findings leave open the possibility that the observed correlations might be due to another nearby site or even to a different gene. In fact, recent linkage disequilibrium analysis has suggested that these 30 anonymous, non-functional SNPs may be marking the location of a functional VDR sequence that confers immune tolerance. In a highly homogeneous population of patients with diabetes type 1, a rare BaT haplotype was identified which seems to protect the kidney from the development of diabetic nephropathy [125]. The translational significance of this finding is emphasized by a prior demonstration that the same rare haplotype also confers immune protection in patients with Hashimoto thyroiditis [126]. In view of the increasing incidence of diabetes in the world population and its impact on renal damage, linkage disequilibrium analysis between SNPs could provide an effective and inexpensive approach to facilitate the identification of causal VDR gene variants predisposing to renal damage to effectively customize early vitamin D interventions. Additional Mechanisms for Calcitriol Resistance in CKD The calcitriol synthesized in the kidney by the mitochondrial 1a-hydroxylase is transported in the blood by carrier proteins. Vitamin-D-binding protein (DBP) is the main carrier. However, calcitriol also binds albumin
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and lipoproteins [2]. As for the cellular uptake of 25(OH)D, simple diffusion of the free form of calcitriol through the cell membrane, upon its dissociation from DBP may not be the only mechanism to initiate calcitriol biological actions. As indicated earlier, megalin is expressed in key targets of calcitriol actions in kidney disease, including parathyroid cells [88] and osteoblasts [127]. More significantly, megalin-mediated endocytosis has also been implicated in the uptake and biological responses to other steroid hormones such as androgens and estrogens in primary and secondary sex organs [128]. Therefore, uremia-induced reductions in megalin expression could constitute an additional mechanism for the development of tissue-specific resistance to calcitriol therapy, in a manner that is independent of abnormalities in VDR content or function. The LDL receptor has also been implicated in calcitriol uptake by human fibroblasts [129]. However, the impact of uremia on LDL receptor expression and function is unclear at present. In CKD, the low serum 25(OH)D could affect calcitriol dissociation from DBP for cellular uptake. 25(OH)D binds DBP with an affinity up to 600 times stronger than that of calcitriol [130]. In fact, in vitamin D intoxication, the displacement of calcitriol from DBP to increase “free” calcitriol levels to enter target cells was considered a mechanism for vitamin D toxicity as total serum calcitriol concentration remained unchanged [131]. Although only 5% of circulating DBP suffices to bind all vitamin D metabolites combined in normal individuals [3], the combination of lower DBP levels in patients with proteinuria and bolus doses of nutritional vitamin D could compromise safety in patients receiving active vitamin D therapy. The demonstration that, in normal women, the survival benefits associated with the correction of vitamin D deficiency appear to start reversing to show mild enhancements in mortality rates for 25(OH)D concentrations above 50 ng/ml [17] pose an important challenge to define the threshold for serum 25(OH)D levels that can safely overcome the impaired uptake and enhance local calcitriol production and autocrine/ paracrine pro-survival actions. Once inside the target cell, calcitriol binding to the VDR activates the receptor to translocate from the cytosol to the nucleus where it heterodimerizes with its partner the retinoid X receptor, RXR. The VDR/ RXR complex binds specific sequences in the promoter region of target genes, called vitamin D response elements (VDRE), and recruits basal transcription factors and co-regulator molecules to either increase or suppress the rate of gene transcription by RNApolymerase II [2]. More than 200 genes transcriptionally induced or suppressed by the calcitriol/VDR complex mediate the efficacy of calcitriol replacement therapy to improve outcomes in kidney disease.
The intracellular levels of both calcitriol and the VDR are the main determinants of the magnitude of calcitriol/VDR induction or suppression of gene transcription, and both are reduced in CKD. The marked decreases in the formation of the calcitriol/VDR complex are aggravated by uremia-induced reductions in RXR/VDR heterodimerization, and in the binding of the VDR/RXR heterodimer to DNA, with the consequent decrease in calcitriol/VDR regulation of gene expression (see Fig. 70.8). Studies in unilaterally nephrectomized rats demonstrated a reduction in the content of a 50 kDa RXR isoform in cell extracts from the remnant kidney. This decrease in RXR results in a reduction of the binding of the endogenous VDR/RXR heterodimer to the VDRE of the mouse osteopontin promoter [132]. A similar reduction of RXR content in the parathyroid glands of these rats could explain their enhanced serum PTH levels in the absence of hypocalcemia or hypophosphatemia [132], as RXR mediates both VDR and retinoic acid suppression of PTH mRNA levels and protein expression [133]. The accumulation of uremic toxins contributes to calcitriol resistance [134]. Ultrafiltrate from uremic plasma causes a dose-dependent inhibition of VDR/RXR binding to VDRE and calcitriol/VDR-transactivating function [135]. Increases in nuclear calreticulin also impair calcitriol/VDR action. Calreticulin is a cytosolic protein that binds integrins in the plasma membrane and the DNA-binding domain of nuclear receptors, including the VDR, thus interfering with receptor-mediated transactivation [136]. Hypocalcemia, commonly present in renal failure and caused by either low serum calcitriol levels or hyperphosphatemia, enhances nuclear levels of parathyroid calreticulin. In vitro studies demonstrated that increases in nuclear calreticulin inhibit VDR/RXR binding to VDRE in a dosedependent manner and totally abolish calcitriol suppression of PTH gene transcription [136]. However, there is no direct evidence that experimental CKD induces increases in the nuclear levels of calreticulin in the parathyroid glands ex vivo. Impaired binding of the VDR/RXR complex to DNA and a concomitant reduction in calcitriol/VDR-mediated transcription also occur as a result of activation of VDR-unrelated pathways. In human monocyte/macrophages, activation by the cytokine gamma interferon of its signaling molecule, Stat1, induces physical interactions between Stat1 and the DNA-binding domain of the VDR that impair VDR/RXR binding to VDRE and consequently calcitriol transactivation of the 24hydroxylase and osteocalcin genes [137]. Therefore, the higher levels of inflammatory cytokines after hemodialysis could contribute to vitamin D resistance.
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VDR
Kidney disease
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Reduced 1,25D/VDR-complex formation 1,25D
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Impaired VDR/RXR binding to DNA Uremic toxins Calreticulin Squelching (VDR-unrelated pathways)
RXR
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DNA binding RXR
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Transactivation/transrepression RNA Pol II CoReg
mRNA
B
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FIGURE 70.8 Abnormal VDR regulation of gene transcription in CKD. Calcitriol (1,25D)/VDR-transcriptional control of the expression of
vitamin-D-responsive genes involves ligand binding to VDR; VDR heterodimerization with RXR; DNA binding of the VDR/RXR complex, and recruitment of basal transcription factors (B), co-regulator molecules (Co-reg), and RNA polymerase II to activate or repress gene transcription. Kidney disease induces several mechanisms (listed on the left) responsible for impairing critical steps (indicated by blue arrows) in calcitriol/ VDR transcriptional activity.
Kidney disease may also impair the recruitment of coactivator molecules that act synergistically with the VDR to markedly amplify calcitriol/VDR-mediated transactivation or transrepression of vitamin-Dresponsive genes [138e140]. The reductions in megalin induced by CKD could affect VDR regulation of gene transcription, as megalin sequesters Skp [141], a component of the VDR-transcriptional complex. The low serum calcitriol of CKD could also affect the expression of essential coactivator or corepressor molecules [142], or the proper translational modifications required for their recruitment to the VDR transcription pre-initiation complex. Indeed, selective recruitment of co-repressor molecules by calcitriol analogs contributes to their diverse potency in suppressing PTH gene expression “ex vivo” [143]. Rapid, non-genomic actions of the calcitriol/ membrane-associated VDR are known to regulate the phosphorylation status and activity, as well as the subcellular location of nuclear co-regulators (see Chapter 10). Because 25(OH)D is well known for its potency to induce non-genomic vitamin D actions (see Chapter 15) [144]), the low serum 25(OH)D levels in CKD could contribute to the defective VDR control of gene expression in response to calcitriol or its lesscalcemic analogs. The section below presents the most current mechanistic understanding of the impact of abnormalities in the vitamin D endocrine system induced
by the failing kidney on the progression of CKDMBD to end-stage renal disease. Special attention is directed to the preclinical and clinical evidence of the distinct contribution of autocrine/paracrine versus endocrine calcitriol/VDR actions in attenuating mineral, skeletal, renal, and cardiovascular damage in CKD that can help expedite the design of vitamin D interventions that safely and effectively improve outcomes.
CONTRIBUTION OF THE LOW 25(OH)D AND 1,25(OH)2D LEVELS AND ABNORMAL VDR ACTIONS TO MINERAL AND SKELETAL ABNORMALITIES, RENAL, AND CARDIOVASCULAR DAMAGE In “Vitamin D maintenance of mineral and skeletal health” an update on the abnormalities in the classical vitamin D actions in the intestine, parathyroid glands, bone, and the kidney that maintain mineral homeostasis and skeletal integrity predisposing to the onset and progression of CKD-MBD is presented. In “Renal and cardiovascular protective actions of vitamin D unrelated to PTH suppression” the focus is on the abnormalities in the vitamin D endocrine system causing the loss of renal and cardioprotective properties unrelated to the suppression of secondary hyperparathyroidism (SH).
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Vitamin D Maintenance of Mineral and Skeletal Health Intestine A defect in intestinal calcium absorption caused by vitamin D and/or calcitriol deficiency in patients with renal disease [145,146] and in animal models of renal failure [147,148] initiates the vicious cycle of hypocalcemia, elevations in serum PTH, and disease progression. Indeed, the stimulation of the small intestine to absorb calcium and phosphate is the most critical calcitriol action in vivo, as conclusively demonstrated in the VDR-null mice [149]. Calcitriol induces intestinal calcium absorption through transcellular and paracellular transport mechanisms. For the transcellular transport, active-calcium uptake requires the epithelial calcium channel TRPV6 and, to a lesser extent, TRPV5. Thereafter, calbindin 9K transports calcium across the cell, and the final delivery to the bloodstream involves the plasma membrane calcium pump. The initial active calcium uptake is the rate-limiting step in intestinal calcium absorption and is highly dependent on vitamin D [150]. Whereas the VDR-null mice elicit a reduced expression of both channels [150,151], the mRNA levels for both channels are upregulated upon calcitriol supplementation [152]. Importantly, a differential modulation of the expression of these channels partially explains the less calcemic properties of the calcitriol analog, 19nor-1,25(OH)2D2, as only calcitriol upregulates channel expression [153]. Recent studies in transgenic mouse models have demonstrated, however, that calcitriol induction of intestinal Ca absorption does not require either calbindin 9K [154] or TRPV6 [155], but a novel paracellular process that involves calcitriol induction of the tight junction proteins claudin 2 and 12. Claudins form paracellular Ca channels in the intestinal epithelia [156]. It is unclear at present whether a distinct upregulation of claudin expression contributes to the lower potency of calcitriol analogs to induce hypercalcemic episodes. Although calcitriol treatment increases intestinal calcium transport in CKD patients [157], there is a blunted response [158] caused by enhanced calcitriol catabolism and impaired VDR function. As stated earlier, CKD could induce intestinal 24-hydroxylase [76] either through the high levels of FGF23 or simply as a direct result of calcitriol (analog) therapy for SH. The contribution of reduced intestinal VDR content to the blunted response to calcitriol to absorb calcium cannot be ruled out, as there are no comparative studies on intestinal VDR content in normal individuals and patients with CKD. Also, there are no epidemiological studies in the hemodialysis population suggesting a higher frequency of the 50 polymorphism affecting Cdx-2 binding to the VDR promoter, known to result
in an intestine-specific reduction in VDR expression [159]. The increases in calcium absorption following dialysis [77,160] support an impaired VDR function, as either the removal of uremic toxins, or other dialysisdriven changes such as a decrease in serum phosphate or volume depletion suffice to restore the response to serum calcitriol [160]. As with intestinal calcium absorption, phosphate absorption is also decreased in CKD and can be enhanced by administration of calcitriol or the prohormone 1a-hydroxyvitamin D3. An average ten-fold reduction in intestinal calcium and phosphate absorption by the calcitriol analog paricalcitol partially accounts for its less hypercalcemic and hyperphosphatemic properties in spite of a similar potency to suppress PTH in the rat model of kidney disease [161]. Parathyroid Glands Nearly all patients with end-stage renal disease develop secondary hyperparathyroidism, a condition characterized by parathyroid hyperplasia and increased synthesis and secretion of PTH. Hypocalcemia, hyperphosphatemia due to phosphate retention, vitamin D, and calcitriol deficiency are the main causes of secondary hyperparathyroidism [162]. Hyperphosphatemia and calcitriol deficiency also enhance parathyroid function indirectly by lowering serum calcium [162]. Because of the severe adverse impact of secondary hyperparathyroidism on outcomes in CKD patients, the parathyroid gland is one of the best-studied targets of the vitamin D endocrine system. The ability of calcitriol to inhibit PTH synthesis and to arrest parathyroid cell growth in vivo and in vitro has been known for many years [2]. The mechanisms mediating calcitriol transcriptional repression of the PTH gene are well characterized and described in Chapter 27. The elevations in serum PTH levels in vitamin-Ddeficient individuals with normal kidney function [30] as well as the reductions in serum PTH upon vitamin D supplementation at early stages of kidney disease [12] or in renal transplant recipients [163] support a role for parathyroid 1a-hydroxylase and autocrine/ paracrine VDR activation in PTH suppression. Calcitriol induction of FGF23 synthesis in bone also provides an additional indirect control of PTH synthesis and secretion [84] and parathyroid cell growth [164]. In normal parathyroid glands, FGF23 not only directly suppresses PTH mRNA levels and secretion even with coexisting hyperphosphatemia or hypocalcemia [164], but also induces parathyroid 1a-hydroxylase mRNA levels [8] for an autocrine/paracrine suppression of PTH by locally produced calcitriol. In addition to a direct suppression of PTH gene expression, calcitriol appears to be critical for the response of the parathyroid gland to calcium. In
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vitamin-D-deficient rats with normal renal function, parathyroid levels of the calcium-sensing receptor (CaSR) mRNA are 40% lower than normal [165], and are enhanced by calcitriol treatment in a time- and dosedependent manner. Functional vitamin-D-responsive elements were identified in both promoters of the human CaSR gene [166]. In advanced renal failure, a strong association exists between defective levels of parathyroid CaSR and low VDR in areas of high proliferative activity [167]. Upregulation of parathyroid CaSR by calcitriol treatment could explain the decrease in the set point for PTH suppression by calcium [168], and also the higher levels of the CaSR in surgically removed parathyroid glands from patients receiving calcitriol compared to those from untreated patients [169]. At present, calcimimetic drugs provide an effective therapeutic tool to compensate for the abnormalities in the response of the parathyroid glands to calcium that are induced by calcitriol deficiency. Recent advances in our understanding of the pathogenesis of parathyroid hyperplasia have identified novel antiproliferative properties of vitamin D as well as the most effective vitamin D interventions to maximize their potency. As mentioned earlier, in rat and human SH, elevations in the parathyroid expression of TGFa and TGFa self-induction are sufficient to generate a feedforward loop for the activation of TGFa/EGFR signaling, which aggravates growth and reduces parathyroid VDR content [111,112]. Low dietary calcium, high dietary P and/or calcitriol deficiency aggravate the increases in parathyroid TGFa and the hyperplastic growth triggered by kidney disease. In contrast, high dietary Ca, dietary phosphate restriction, or prophylactic administration of calcitriol or its less calcemic analog, paricalcitol, mimic highly specific EGFR-tyrosine kinase inhibitors in halting parathyroid cell growth. In rat SH, however, by day 7 after the induction of kidney injury, the rapid parathyroid cell growth causes a reduction in VDR content sufficient to make the prophylactic doses of calcitriol or paricalcitol no longer effective to suppress further parathyroid gland enlargement [111]. The need to overcome the resistance to calcitriol replacement therapy caused by a similar drastic reduction of parathyroid VDR by enhanced TGFa/EGFR in advanced human SH led to the successful development of less calcemic vitamin D analogs. The survival advantage of paricalcitol over calcitriol reported in large observational trials in hemodialysis patients [170,171] was initially attributed to paricalcitol efficacy to suppress PTH while causing fewer hypercalcemic or hyperphosphatemic episodes [172]. A recent report has identified tumor necrosis factora converting enzyme (TACE, also called ADAM17) as the key therapeutic target to overcome the tremendous
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adverse impact of parathyroid hyperplasia on the response to calcitriol (analog) replacement therapy. ADAM17 is a metalloproteinase essential for EGFR signaling because it releases the mature isoforms of TGFa and several other EGFR-activating ligands thereby enhancing autocrine/paracrine EGFR activation [173,174]. In rat SH, increases in parathyroid content of ADAM17 in response to high dietary phosphate are sufficient to trigger the release of parathyroid TGFa that starts the vicious cycle for increases in parathyroid growth rates and reductions in VDR content [175]. Also important was the demonstration that the efficacy of calcitriol and/or analog therapy in preventing the onset as well as further enlargement of the parathyroid gland in established secondary hyperparathyroidism also involves direct suppression of ADAM17 expression [175]. More significantly, the combination of nutritional vitamin D and small doses of the calcitriol analog paricalcitol, at dosages ineffective to suppress PTH or growth rates when given as monotherapy after 1 week of the onset of renal injury, mimics the efficacy of small molecule EGFR tyrosine kinase inhibitors in preventing increases in parathyroid ADAM17, TGFa/EGFR activation, and growth rates. This finding supports a role for autocrine/paracrine calcitriol actions in the control of parathyroid function, and it also raises the interesting possibility that the safe correction of vitamin D deficiency can help overcome resistance and improve the responses to lower doses of calcitriol or its analogs. If parathyroid ADAM17 can release soluble klotho from its membrane-bound precursor as demonstrated in renal distal tubule cells [176], a defective inhibition of parathyroid ADAM17 expression and/or activity caused by vitamin D and/or calcitriol deficiency/insufficiency could partially account for the reduced klotho levels in the hyperplastic parathyroid glands that impede the suppression of PTH and growth rates by the high FGF23 in CKD [84,164,177]. Deregulation of the ADAM17, TGFa/EGFR growth signals due to vitamin D and/or calcitriol deficiency could account for the switch from diffuse to nodular hyperplasia in human SH, as well as for the increased expression of parathyroid cyclin D1, a common feature in human hyperparathyroidism [178]. In fact, in normal and carcinogenic cell lines, overexpressing EGFR, the potent vitamin-D-antiproliferative actions, also involve inhibition of EGFR growth-promoting signals from the plasma membrane as well as EGFR-transactivation of the cyclin D1 gene [179]. Calcitriol antiproliferative actions in hyperplastic human parathyroid glands also involve the induction of anti-mitogenic signals such as the cyclin-dependent kinase inhibitors p21 and p27 [180,181]. A strong direct correlation exists between parathyroid VDR levels and p21 and p27 content in human secondary
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1,25D
Role of calcitriol deficiency in the pathogenesis of secondary hyperparathyroidism in chronic kidney disease.
FIGURE 70.9
VDR 1,25D/VDR
Suppression of PTH gene
Induction of CaSR gene
CaSR Ca inhibition of PTH gene expression
Suppression of mitogenic signals
Induction of Antimitogenic signals p21 p27 anti-EGFR LAP
TACE /EGFR LIP
Set Point
PTH
Hyperplastic growth Propensity to nodularity
hyperparathyroidism. The p21 gene is under direct transcriptional induction by the calcitriol/VDR complex [182]. In rats, prophylactic administration of calcitriol or its analog 19-nor-1,25-dihydroxyvitamin D2 (paricalcitol) also prevents the development of parathyroid hyperplasia through the induction of the expression of the cell-cycle inhibitor p21 [180]. Calcitriol reduction of parathyroid c-myc expression also contributes to calcitriol antiproliferative properties in human secondary hyperparathyroidism [183]. Figure 70.9 summarizes the multiple direct effects of low serum calcitriol on the parathyroid glands leading to parathyroid hyperplasia and secondary hyperparathyroidism. It is clear from these actions of calcitriol that early interventions with vitamin D supplementation, low doses of calcitriol or its less calcemic analogs, or the combination should prevent the decreases in VDR and CaSR responsible for the reduced sensitivity of hyperplastic parathyroid glands to control PTH synthesis and secretion in response to calcium, calcitriol or analog therapy, and possibly for the maintenance of the parathyroid responses to FGF23. The prevention/moderation of increases in parathyroid TACE/TGFa signaling at early stages in CKD should decrease the incidence of the most aggressive forms of secondary hyperparathyroidism that are resistant to therapy. Bone The combination of low vitamin D and/or calcitriol levels and high serum PTH contributes to the wide range of bone disorders of CKD. The essential role of vitamin D in the development and maintenance of a mineralized skeleton has been known for centuries. However, initial studies in the VDR knockout mice suggested that vitamin D is not required for the ossification process [149] other than for the adequate supply of calcium and phosphate. In fact, in spite of low serum
calcitriol, only a fraction of renal failure patients show evidence of defective mineralization [184]. As extensively discussed earlier, in CKD, calcitriol deficiency causes decreased intestinal calcium absorption and hypocalcemia, a potent stimulus for parathyroid gland hyperplasia and, consequently, increases in circulating PTH responsible for the increases in osteoclastogenesis and osteoclast activation, osteitis fibrosa and bone loss. Conversely, treatment with calcitriol or its less calcemic analogs is the therapy of choice to control the skeletal abnormalities caused by hyperparathyroidism in advanced kidney disease [161]. An important consideration when using calcitriol/analog therapy is to avoid oversuppression of PTH with the consequent decrease in bone turnover to abnormally low levels that cause adynamic bone disease and predispose to vascular calcifications [185]. More significantly, comprehensive studies in the VDR-null mice, 1a-hydroxylase-null mice, PTH-null mice, and multiple double knockout combinations (see Chapter 33) have conclusively shown that calcitriol actions in bone are not limited to providing an adequate supply of Ca and P for bone mineralization. Calcitriol also induces osteoblastogenesis, skeletal anabolism, and the proper coupling of osteoblastic and osteoclastic activities [3] in a PTH-independent manner [186]. Of great translational importance were the studies in mouse [187] and rat [188] models demonstrating that in spite of a ten-fold lower potency of the less calcemic calcitriol analog paricalcitol to induce osteoclastic bone resorption compared to that of calcitriol, their efficacy to maintain/promote bone anabolism was not compromised. Kidney In CKD, vitamin D and/or calcitriol deficiency compromises the uptake of 25(OH)D from the glomerular ultrafiltrate to induce calcitriol production and megalin content, as well as the renal handling of Ca
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and P. In health, calcitriol suppression of 1a-hydroxylase and the stimulation of 24-hydroxylase were considered the most important actions of calcitriol in the kidney due to their major impact on serum calcitriol levels, and consequently in calcium and phosphate homeostasis. In addition to decreasing 1a-hydroxylase activity by reducing serum PTH, calcitriol induction of the expression of the CaSR in the proximal tubules could also mediate the sensitivity of the renal 1a-hydroxylase to calcium. Calcitriol involvement in the renal handling of calcium and phosphate continues to be controversial due to the simultaneous effects of calcitriol on intestinal calcium and phosphate absorption, which affects the filter load of both ions, and on serum PTH. Calcitriol enhances renal calcium reabsorption, and accelerates PTH-dependent calcium transport in the distal tubule [189], the site with the highest VDR content and the determinant of the final excretion of calcium into the urine. Similar to calcitriol induction of intestinal transepithelial calcium transport, calcitriol enhancement of renal calcium reabsorption involves direct induction of calbindin and the epithelial calcium channel TRPV5 [190]. Several putative VDR-binding sites have been located in the human promoter of the renal epithelial calcium channel. In CKD, calcitriol deficiency results in a marked decline in the expression of the channel at the protein and mRNA levels [190]. Calcitriol also increases renal calcium channel activity indirectly through inhibition of the expression of SBPRY (B-box and SPRY domain-containing protein), a TRPV5 and TRPV6 interacting protein, which inhibits TRVP5 calcium channel activity at the cell surface [191]. Calcitriol induction of Ca channel activity confers two important properties to renal Ca influx, namely a high calcium selectivity and a negative feedback regulation of transepithelial Ca transport to prevent calcium overload [192]. This mechanism, however, may not be the only contributor to calcitriol induction of renal Ca reabsorption. Calcitriol induction of the tight junction protein claudin 2 could also contribute to paracellular renal Ca reabsorption, as described for the intestine because claudin 2 is expressed from the early proximal tubule to the early descending limb of Henle’s loop [193]. The relative potencies of calcitriol and its analogs to induce renal claudin expression and paracellular Ca transport are unknown at present. The net effects of calcitriol or its analogs on renal P reabsorption are also controversial. Calcitriol directly increases renal P reabsorption by inducing NPT2c [4], a renal sodiumephosphate co-transporter with a minor role in renal phosphate reabsorption in rodents. However, mutations in this gene cause the human P wasting disorder of hereditary hyperphosphatemic rickets with hypercalciuria [194,195]. On the other
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hand, calcitriol also induces serum FGF23 and klotho levels, and both are potent inducers of phosphaturia and suppressors of renal 1a-hydroxylase [196]. In CKD, defective calcitriol induction of renal megalin may contribute indirectly to the impaired phosphaturic responses to PTH and FGF23. Megalin is essential for the maintenance of adequate steady-state expression of the sodium (Na)/P co-transporter NaPiIIa, and for the capacity of proximal tubular cells to react to PTH-driven inactivation of NaPi-IIa by endocytosis and intracellular translocation [197]. Kidney-specific inactivation of the megalin gene results in markedly reduced phosphaturia due to enhanced steady-state levels of NaPiIIa in the brush border membrane, and a defective retrieval and impaired degradation of NaPiIIa in response to PTH [197]. The correction of renal megalin levels by interventions with paricalcitol at early stages of kidney disease could partially account for the efficacy of the analog in correcting low-molecular-weight proteinuria [198] and also for the low phosphatemic activity of the analog. The relative potencies of calcitriol and its less calcemic analogs to induce renal megalin for protein reabsorption or for PTH or FGF23-driven phosphaturia are unknown. A defective maintenance of renal megalin expression in proximal tubular cells, caused by an impaired autocrine/paracrine upregulation in vitamin-D-deficient individuals with normal kidney function and/or in vitamin-D-deficient renal graft recipients, predispose not only to the development of proteinuria but also to abnormalities in renal megalin/NaPi2a handling of P. The contribution of proteinuria to renal and cardiovascular lesions is well known. P retention also has severe direct repercussions not only in the development of secondary hyperparathyroidism [199], but also in abnormalities in the vasculature predisposing to soft tissue calcifications [200].
Renal and Cardiovascular Protective Actions of Vitamin D Unrelated to PTH Suppression A large retrospective study in hemodialysis patients in the USA has suggested that patients treated with intravenous administration of either calcitriol or its less calcemic analogs live longer compared to those not receiving any active vitamin D metabolites [11]. This survival advantage cannot be totally accounted for by PTH suppression. Similarly, in a large historical cohort of patients from South America, oral active vitamin D metabolites also conferred pro-survival benefits. Interestingly, the best outcomes with oral active vitamin D therapy were achieved for the lowest doses [171]. At present, there are no prospective studies that conclusively establish the beneficial outcomes reported
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for active vitamin D therapy in CKD. However, these observational studies prompted intensive research that identified the important renal and cardiovascular protective properties of calcitriol and its analogs that are presented in this section. Intriguingly, however, low serum 25(OH)D levels are better predictors than low serum calcitriol of the risk for disease progression to end-stage renal disease and death in pre-dialysis patients [13], and for early mortality in hemodialysis patients [16]. These reports suggest an important contribution of autocrine/paracrine VDR activation to the renal and cardiovascular protective actions of a normal vitamin D status. In fact, induction of 25(OH)D catabolism to impair local calcitriol production could contribute to the lower efficacy of higher oral paricalcitol dosage [171]. None of these epidemiological studies proves that vitamin D deficiency “causes” renal disease progression or death. Prospective studies should examine whether the prevention of the onset of vitamin D deficiency or its correction improves clinical outcomes in patients receiving high dosage of calcitriol or its analogs. Vitamin D Deficiency in the Onset and Progression of Renal Lesions Urinary protein is a well-recognized determinant of renal disease progression and higher risk for cardiovascular complications [201]. This section presents potential defects in autocrine/paracrine mechanisms that may explain why the degree of vitamin D deficiency strongly correlates with the severity of albuminuria in a large population of individuals with normal kidney function [15]. In vitamin-D-deficient individuals, in spite of a normal GFR, the low serum 25(OH)D levels will markedly reduce the filtered 25(OH)D available for uptake by renal proximal tubular cells thereby compromising its activation to calcitriol, and calcitriol-VDR induction of renal megalin for urinary protein reabsorption in an autocrine/paracrine manner [202]. Indeed, the lowmolecular-weight proteinuria of Dent’s disease [203] and Fanconi syndrome [204] results from reduced megalin expression in renal proximal tubular cells and/or abnormalities in the recycling of cytosolic megalin to the brush border membrane. In addition, vitamin D deficiency could also impair calcitriol autocrine/paracrine maintenance of a tolerogenic phenotype [205] in dendritic cells, thereby contributing to non-immune-mediated proteinuric nephropathy. Indeed, the cross-presentation of antigenic peptides from tubular-cell-processed albumin by proximal tubule-associated dendritic cells reinforces tolerance in a normal kidney, and an active immune response in the proteinuric kidneys [206]. Once the albumin-driven inflammatory response is initiated, vitamin D deficiency will limit 25(OH)D uptake by
monocyte-macrophages for its conversion to calcitriol. Reduced calcitriol autocrine/paracrine anti-inflammatory properties [207] could result in enhanced macrophage infiltration and lesions to the renal parenchyma. There is a precedent for these mechanisms, as vitamin D deficiency is sufficient to disable macrophage antimycobacterial activity in the African-American population thereby enhancing the risk of tuberculosis [208] (see Chapter 93), a defect that can be reversed with appropriate vitamin D supplementation. Similarly, in renal transplant recipients, an early correction of serum 25(OH)D levels, either before or at transplantation, could help maintain renal megalin levels, 25(OH)D uptake and protein reabsorption, as well as the tolerogenic state of renal dendritic cells, in addition to the reported benefits of the correction of vitamin D deficiency “after” transplantation in preventing elevations in serum PTH, Ca and P levels [163]. Vitamin D Renoprotective Actions Contrary to the initial fears on the potential adverse renal effects of the hypercalcemia or hyperphosphatemia induced by active vitamin D therapy, preclinical and clinical data support a renoprotective role for therapy with calcitriol and its analogs against renal injury. In animal models of kidney disease, the renoprotective actions of active vitamin D therapy include: (1) suppression of the renin/angiotensin system (RAS); (2) prevention/amelioration of chronic renal inflammation; and (3) attenuation of glomerular injury, tubule-interstitial fibrosis, and proteinuria through direct regulation of specific functions of mesangial cells, podocytes, tubular cells, fibroblasts, resident or infiltrating macrophages, and immune cells, all of which are essential in maintaining a normal kidney. Active vitamin D metabolites directly suppress the RAS through a VDR-mediated repression of the expression of the renin gene [209,210]. In the glomerulus, prolonged RAS activation has been implicated in the development of the glomerular hemodynamic adaptation leading to fibrogenic responses, glomerulosclerosis, tubular hyperplasia, fibrosis, mononuclear cell infiltration, interstitial inflammation, and severe proteinuria [113]. In the rat model of remnant kidney after subtotal nephrectomy (SNX) that recapitulates the renal insufficiency of advanced stages of kidney disease, treatment with calcitriol and its analogs not only effectively decreases albuminuria, glomerulosclerosis, and glomerular cell proliferation [211e213], but also serum creatinine, suggesting a role for these active vitamin D metabolites in the preservation of renal function [213]. Calcitriol administration effectively reduces podocyte loss and hypertrophy, improves podocyte ultrastructure, and suppresses the expression of desmin, a marker
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of podocyte injury [212,214]. In immune and nonimmune rat models of mesangial proliferative glomerulonephritis, calcitriol reduces mesangial cell proliferation [215]. Accordingly, a diabetic mouse model null for the VDR develops a more severe form of albuminuria and glomerulosclerosis, with reduced nephrin expression in podocytes, a protein essential for the proper functioning of the renal filtration barrier, and with enhanced fibronectin content in mesangial cells [216]. Calcitriol renoprotection also involves the prevention/amelioration of chronic inflammation, a disorder characterized by infiltration of inflammatory cells into the glomeruli and tubulointerstitium with a major role in the decline in renal function. Calcitriol elicits potent anti-inflammatory properties [207], and the VDR is present in most cells of the immune system including macrophages, dendritic cells, and both CD4þ and CD8þ T cells [217,218]. In fact, in the subtotal nephrectomy SNX rat model, prophylactic interventions with paricalcitol inhibit macrophage infiltration via suppression of macrophage chemoattractant protein 1 (MCP-1), an effect that is enhanced using a combination of paricalcitol and renineangiotensin blockage with the ACE inhibitor enalapril [219]. In the rat anti-thy-1 model of mesangial proliferative glomerulonephritis, in which a single intravenous injection of anti-rat thymocyte antiserum induces complement-dependent mesangiolysis followed by mesangial cell reappearance, proliferation and matrix deposition, administration of calcitriol or its less calcemic vitamin D analog, 22-oxa-calcitriol, prevents albuminuria, inflammatory cell infiltration, and the extracellular matrix accumulation that accompanies myofibroblast activation [220,221]. Also, calcitriol treatment almost completely abrogates the glomerular infiltration of neutrophils in the anti-thy-1 model [220]. Calcitriol and its analogs may also attenuate proteindependent interstitial inflammation by reducing proteinuria, and by inducing tolerance in the renal tubule-associated dendritic cells [205] thereby reducing active immune responses triggered by filtered albumin in the damaged kidney [206]. Calcitriol inhibition of myofibroblast activation contributes to attenuate/suppress interstitial fibrosis. Inhibition of the expression of TGF-b1, a well-known mediator of the onset and progression of various forms of renal fibrotic lesions, can partially account for calcitriol renoprotection [214]. In the obstructed kidney, paricalcitol elicits a dose-dependent reduction in interstitial volume and the deposition of interstitial matrix components, through inhibition of renal mRNA expression of fibronectin, type I and III collagen, and fibrogenic TGF-b1 [222]. In primary cultures of rat renal interstitial fibroblasts, calcitriol suppresses TGF-b1-induced
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a-smooth muscle actin (a-SMA) expression and the resulting increases in type I collagen and thrombospondin-1 in a dose-dependent manner through upregulation of mRNA levels and protein secretion of hepatocyte growth factor [223]. The calcitriol analog, paricalcitol, directly blocks the epithelial mesenchymal transition of tubular epithelial cells to become myofibroblasts, a process induced by TGF-b1 and common in the fibrotic kidney that follows sustained injury. Indeed, in in vivo models of obstructive nephropathy, paricalcitol preserves the integrity of the tubular epithelium by restoring E-cadherin and VDR expression, and also by attenuating interstitial fibrosis through the suppression of a-SMA and fibronectin expression [222]. Intriguingly, paricalcitol treatment increases instead of reducing perivascular fibrosis in small cardiac arteries in the rat model of renal insufficiency. This adverse effect of the analog was attributed to suppression of local calcitriol synthesis [224], as it is associated directly with a marked reduction in serum calcitriol and a mild decrease in 25(OH)D levels. These results raise important translational considerations. One is the potential for nutritional vitamin D supplementation to induce “autocrine/paracrine” antifibrotic and other renoprotective actions of calcitriol at early stages of kidney disease. Another is that it supports the importance of avoiding excessive dosage of calcitriol analogs that could compromise VDR activation by locally produced calcitriol in advanced kidney disease either through an inhibition of local 1a-hydroxylase, or due to the induction of 25(OH)D or calcitriol catabolism through the enhancement of 24-hydroxylase expression. Finally, it supports the potential for combination therapy with nutritional and active vitamin D metabolites to effectively improve VDR activation using lower dosages of calcitriol, or its analogs, as recently demonstrated for the inhibition of renal ADAM17 expression and ADAM17-driven proteinuria in the rat model of kidney disease [175]. Inhibition of renal and monocyte-macrophage ADAM17 activation by calcitriol or its analogs provides a mechanistic understanding for the renal and cardioprotective properties of vitamin D, and also for the synergy between vitamin D interventions and anti-angiotensin II therapy. The activation of ADAM17 in response to angiotensin-II and/or nephron reduction is crucial for the development of renal lesions in mice [113], and associates with structural and functional damage in various human renal inflammatory and fibrotic diseases [225]. In mice, increased renal expression of ADAM17, and not hypertension, causes the renal lesions induced by prolonged exposure to angiotensin-II (Ang II) [113]. As summarized in Figure 70.10, Ang II binding to and activation of Ang II-receptor 1 (AT-R1) enhance TACE expression and activity to release mature TGFa from its transmembrane precursor. Soluble TGFa binds to and activates the
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TGFα
g An
EGFR
II AT
TNFα
Pro-TGFα
- R1
P-EGFR
Translocation
TACE (active)
ICAM-1
Proteinuria Glomerulosclerosis Tubular hyperplasia Mononuclear cell infiltration Fibrosis
Renal cells Increases in renal TACE mediate Ang II-driven renal lesions and systemic inflammation in CKD. Ang II binding to its AT1 receptor induces increases in cytosolic TACE, and its translocation to the plasma membrane. TACE releases TGFa from its trans-membrane precursor, which then binds and activates the EGFR causing proteinuria, tubular hyperplasia, fibrosis, mononuclear cell infiltration. Kidneydisease-induced increases in TACE and TACE-mediated release of the soluble pro-inflammatory molecules TNFa, ICAM-1, and VCAM-1 contribute to aggravate systemic inflammation and renal and cardiovascular damage.
FIGURE 70.10
EGFR [113] causing renal parenchymal lesions, including glomerulosclerosis, tubular atrophy and/or dilation with microcyst formation, mild interstitial fibrosis, and multifocal mononuclear cell infiltration accompanied by severe proteinuria. EGFR activation is known to stabilize ADAM17 protein [226] thereby generating a vicious cycle that aggravates ADAM17 activation and renal damage. In humans, ADAM17 is constitutively expressed in renal distal tubules and podocytes, and CKD induces de novo ADAM17 expression in proximal tubules, peritubular capillaries and glomerular mesangium, and upregulates podocyte ADAM17 content [225]. The increases in glomerular and interstitial ADAM17 expression enhance TGFa expression and associates directly with structural damage, including mesangial matrix expansion, focal glomerulosclerosis, glomerular macrophage infiltration, as well as interstitial fibrosis, interstitial macrophage infiltration, and decreased renal function, as demonstrated by lower estimated GFR and higher serum creatinine [225]. Defective inhibition of ADAM17 by calcitriol deficiency could also compromise graft survival, as renal EGFR transactivation is a main determinant of the accelerated progression of renal lesions upon prolonged renal ischemia, in a mouse model that mimics the arterial stenosis during transplantation [227].
Calcitriol inhibition of EGFR activation in the kidney could also account for its renoprotective effects. In fact, in severe proteinuric rats, EGFR activation was shown to be an important mediator of the endothelial dysfunction that causes the decline in renal blood flow during hypertension-induced CKD [228]. Inhibition of EGFR activation with small molecule tyrosine kinase inhibitors can restore renal hemodynamics and moderate microvascular hypertrophy, but it fails to completely reverse the disease because it has little effect on inflammation [228]. Calcitriol inhibition of renal ADAM17 activation contributes to calcitriol anti-inflammatory properties. Renal ADAM17 activation also releases two other potent pro-inflammatory and pro-fibrotic molecules: the soluble forms of intercellular adhesion molecule 1 (ICAM-1) [229] and vascular adhesion molecule 1 (VCAM1) [230,231] (see Fig. 70.10). In kidney disease, whereas elevated plasma levels of soluble ICAM1 and VCAM1 correlate indirectly with GFR, soluble VCAM1 correlates directly with fibrinogen expression [232]. Furthermore, elevated serum levels of soluble ICAM1 and VCAM1 are more accurate than proteinuria as markers of endothelial dysfunction and in predicting mortality in renal transplant recipients [233]. Increases in renal ADAM17 activation in kidney disease also provide a novel mechanistic understanding for the higher risk of cardiovascular mortality in these
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patients. ADAM17 also releases the soluble form of the potent inflammatory cytokine TNFa (see Fig. 70.10), a critical contributor to systemic inflammation, a wellrecognized leading cause of renal and cardiovascular damage and arterial calcification [234]. In fact, a higher risk for cardiovascular mortality also occurs in normal individuals carrying an ADAM17 polymorphism that results in a higher TNFa release [235]. A similar inhibition of renal ADAM17 by nutritional and/or active vitamin D therapy in humans could render the reductions in serum levels of the markers of ADAM17 activation, the soluble forms of TGFa, TNFa, ICAM1, and VCAM1, appropriate markers to customize interventions with either vitamin D alone or in combination with anti-angiotensin II therapy to improve renal and cardiovascular outcomes in CKD in a PTH-independent manner. Vitamin D Cardioprotective Actions At least half of the deaths among dialysis patients are caused by cardiovascular disease. Therefore, it is not surprising that most of the reported survival benefits of therapy with calcitriol or its analogs are associated with downregulation of vascular calcification, the RAS, systemic inflammation, atherosclerosis and cardiac dysfunction, all of which are important contributors to the development and progression of cardiovascular damage. Vitamin D and calcitriol deficiency impair cardiovascular health in spite of normal renal function. In mice with intact kidneys, the absence of either the VDR or the 1a-hydroxylase results in upregulation of the renineangiotensin system, cardiac hypertrophy, and impaired cardiac function [209,236]. All of these defects can be corrected by calcitriol treatment in the 1a-hydroxylase-null mice. Calcitriol (analog) suppression of the renine angiotensin system (RAS) is an essential contributor to cardiovascular protection regardless of kidney function. In spontaneously hypertensive heart failure rats that carry two copies of a mutant form of the leptin gene, increased dietary salt intake induces left ventricular hypertrophy (LVH) and fibrosis with severe hypertension [237]. Treatment of these rats with calcitriol reduces heart weight, myocardial collagen levels, ventricular diameter, and improves cardiac output. In the Dahl-salt-sensitive rat model [238], in which a high-salt diet induces hypertension, paricalcitol therapy also prevents cardiac hypertrophy and cardiac dysfunction independently of blood pressure control through the reduction of serum levels of brain natriuretic peptide, and of the cardiac mRNA expression of brain natriuretic peptide, atrial natriuretic factor, and renin. In humans, serum vitamin D and calcitriol levels are inversely correlated with coronary calcification [239],
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symptoms of heart failure [240], and LVH [241]. More importantly, active vitamin D treatment downregulates plasma renin activity [209], LVH [242], and systemic inflammation [243] suggesting that early interventions with active vitamin D therapy may either prevent the development of clinically adverse cardiovascular signs and symptoms or reverse them. Preclinical studies in rats have reproduced the findings of human CKD. Calcitriol and its analogs attenuate vascular calcification through mechanisms unrelated to their efficacy in suppressing SH, or in preventing increases in serum P, Ca, or Ca X P product. In uremic rats, administration of the analog 22-oxa calcitriol, at doses 50-fold higher than those of calcitriol, conferred a protection from vascular calcification superior to that achieved with calcitriol, in spite of similar increases in serum Ca and P [213]. Also, calcitriol and doxercalciferol increased aortic calcification compared to paricalcitol through mechanisms unrelated to a distinct regulation of serum P, Ca, or Ca X P product [244]. Paricalcitol prevented the increases in Runx2 and osteocalcin mRNA levels in the vasculature, two genes required for vascular smooth muscle cells to acquire the osteoblastic phenotype that prompts calcification. Direct inhibition of oxidative stress and inflammation by calcitriol and its analogs may also provide cardiovascular protection regardless of the efficacy in the control of blood pressure. Activation of the RAS induces oxidative stress in the cardiovascular system, which in turn initiates membrane lipid peroxidation leading to inflammation responsible for cardiovascular dysfunction. In 5/ 6 nephrectomized rats, paricalcitol is as effective as the angiotensin II converting enzyme inhibitor enalapril affording protection against cardiac oxidative stress [245]. Also, the synergy demonstrated by combined therapy with angiotensin II converting enzyme inhibitors and paricalcitol in attenuating the progression of monocyte infiltration and interstitial inflammation of the renal parenchyma, in spite of a defective control of blood pressure [219], could certainly operate in the cardiovascular system, in which increased TNFa is a key contributor to atherosclerosis and vascular calcification [234]. In fact, while renal ablation in the LDL receptor null mice markedly aggravates the calcification of the atherosclerotic plaques [246], neointimal vascular calcium content was reduced by calcitriol and paricalcitol treatment. Intriguingly, however, high paricalcitol dosage resulted in enhanced rather than reduced calcification. Paricalcitol inhibition of autocrine/paracrine VDR actions could contribute to the reversal of the protective effects of active vitamin D metabolites as vascular smooth muscle and aortic endothelial cells express 1a-hydroxylase. Indeed, not only serum calcitriol but also serum 25-hydroxyvitamin D levels were correlated negatively with pulse wave velocity and
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positively correlated with brachial artery distensibility and flow-mediated dilation [247], supporting a role for autocrine/paracrine activation of the VDR by locally produced calcitriol to the cardiovascular protection conferred to these patients by a normal vitamin D status. In CKD studies by Wolf et al. in hemodialysis patients they report a stronger association of increased risk of allcause early mortality with severe vitamin D deficiency than with calcitriol deficiency [16]. Low 25(OH)D levels also affect mortality rates in CKD patients independently of vascular calcification and stiffness [248]. The reversal of the antifibrotic properties of calcitriol to an induction of perivascular fibrosis by paricalcitol treatment in uremic rats was associated with a reduced local calcitriol production [224]. It is unclear whether inhibition of autocrine/paracrine VDR activation mediated the enhanced aortic calcification observed with high paricalcitol dosage in mice [246]. Vascular inflammation enhances the production of matrix metalloproteinases that remodel the vascular wall and the myocardium, destabilizing atherosclerotic plaques and causing thrombosis. The prevention of thromboses is another mechanism by which active vitamin D therapy can reduce mortality, as demonstrated by the development of arterial thrombosis in the VDR-null mice in association with the downregulation of antithrombin and thrombomodulin [249]. Active vitamin D attenuation of proteinuria may also protect the cardiovascular system through a reduction in filtered protein-induced inflammation [206]. In humans, studies in two CKD patient populations demonstrated an inverse correlation between serum calcitriol levels and coronary artery calcification and moderate risk for coronary heart disease [239,250]. Also, in a small trial in 24 patients (22 CKD stage 3, and two CKD 2), oral paricalcitol administration, at doses of 1 and 2 mg, reduced albuminuria and systemic inflammation, as demonstrated by serum levels of C reactive protein, through mechanisms unrelated to the control of blood pressure or serum PTH [243]. A double-blind randomized study utilizing paricalcitol is testing the hypothesis that treatment with active vitamin D metabolites can moderate the progression of LVH (PRIMO study, www.clinicaltrials.gov, NCT00497146 and NCT00616902).
SAFETY AND EFFICACY OF VITAMIN D AND 1,25(OH)2D THERAPY TO IMPROVE OUTCOMES IN CHRONIC RENAL FAILURE This section summarizes the evidence collected from the clinical outcomes of almost three decades of vitamin
D, vitamin D metabolite, and vitamin D analog interventions in CKD patients these studies have provided a better understanding of the interactions between the kidney and vitamin D endocrine system responsible for the efficacy or the failure of interventions with nutritional and/or active vitamin D metabolites to improve clinical outcomes. As indicated earlier in this chapter, calcitriol deficiency was considered the main defect in the vitamin D endocrine system in CKD, and for many years, the main therapeutic approach has been the safe correction of the progressive reductions in renal calcitriol production in the course of kidney disease with calcitriol replacement therapy to moderate the elevations in serum PTH, and the consequent mineral and skeletal abnormalities predisposing to extra-osseous calcifications and increased mortality rates [199]. The development of less calcemic calcitriol analogs with a wider therapeutic window to suppress PTH with a better control of serum calcium and phosphate [251] provided a safer alternative to calcitriol replacement in advanced kidney disease, in which the high doses of calcitriol required to suppress PTH in glands with a markedly reduced VDR content caused hypercalcemia and hyperphosphatemia. The promising preclinical studies on the advantage of these less calcemic analogs over calcitriol to suppress PTH with less calcemic and hyperphosphatemic episodes were supported by large epidemiological studies in hemodialysis patients reporting a survival advantage in patients receiving the calcitriol analog paricalcitol compared to those receiving calcitriol [170,252]. The next important hallmark in support for interventions with calcitriol or its less calcemic analogs (now referred to as “active” vitamin D therapy, as these are the most active vitamin D metabolites) came from the demonstrations from a large observational study in hemodialysis patients in the USA that patients treated with intravenous administration of active vitamin D metabolites not only live longer than those not receiving any active vitamin D metabolites [11], but more significantly, that the survival advantage of active vitamin D therapy could not be totally accounted for by PTH suppression. Similar survival benefits of active vitamin D therapy were also reported in a large historical cohort of patients from South America receiving oral active vitamin D metabolites exclusively [171]. These highly relevant epidemiological data of the “pro-survival” actions of “active” vitamin D therapy and/or the preclinical and clinical evidence of the renal and cardioprotective properties of active vitamin D therapy [211,212,216,222] suggested the importance to either intervene earlier in the course of kidney disease, or to extend the use of active vitamin D metabolites regardless of serum PTH, calcium, and phosphate levels in
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advanced kidney disease. However, two recent reports by the Cochrane Renal Group, using current epidemiological standards to assess the efficacy of “active” vitamin D interventions in pre-dialysis and dialysis patients, have concluded that currently there are no well-designed, adequately powered studies in support of the beneficial effects of “active” vitamin D interventions on clinical outcomes [253,254] reported for active vitamin D therapy in large epidemiological studies in CKD patients, and in preclinical studies in experimental models of renal injury. Also intriguing was the report that the greatest benefits of oral “active” vitamin D therapy were seen for the lowest doses [171]. Recent epidemiological studies have demonstrated a striking incidence of vitamin D deficiency in CKD patients and that vitamin D deficiency associates more strongly than calcitriol deficiency with a higher risk for disease progression and death. These findings have provided a potential explanation for the failure of exclusive “active” vitamin D therapy to improve clinical outcomes in CKD. They can also explain the attenuation of the pro-survival actions of “active” vitamin D interventions with the largest doses of paricalcitol in preclinical studies on vascular calcification [246] and perivascular fibrosis [224] in mice and in the large epidemiological cohort of pre-dialysis patients receiving oral paricalcitol [171]: “Active” vitamin D therapy is insufficient to correct the vitamin D deficiency that causes a defective local calcitriol production by non-renal cells, a requirement for the autocrine/ paracrine activation of the VDR that mediates survival. Also, high doses of calcitriol/analog can directly inhibit local calcitriol production by non-renal 1-hydroxylases and/or aggravate vitamin D deficiency through the induction of 24-hydroxylase and 25(OH)D catabolism. Indeed, 25(OH)D supplementation is sufficient to enhance renal and non-renal calcitriol production and normalize serum calcitriol levels (a) in uremic dogs with moderate to severe renal injury, (b) in hemodialysis patients in which the high serum FGF23 directly inhibits renal calcitriol synthesis, and (c) in bilaterally nephrectomized patients in which only non-renal 1-hydroxylases contribute to circulating calcitriol. These findings emphasize the importance of the safe correction of vitamin D deficiency and consequently of the impaired substrate availability to renal and non-renal 1-hydroxylases required to improve calcitriol endocrine or autocrine/paracrine VDR activation. The safety and efficacy of the simultaneous correction of 25(OH)D and calcitriol deficiency/insufficiency to improve autocrine and endocrine VDR actions need to be examined in prospective, well-powered clinical trials. An additional consideration to improve current recommendations for vitamin D interventions relates to the adequate translation to the bed site of the existing
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evidence from studies in normal humans on the best approach to correct vitamin D deficiency in CKD.
ADEQUACY OF CURRENT RECOMMENDATIONS TO CORRECT VITAMIN D DEFICIENCY In view of the impact of vitamin D deficiency on mortality risks in individuals with normal kidney function, the issue of optimal dosage and timing for vitamin D supplementation for its correction in kidney disease patients is of high translational relevance. According to the KDOQI guidelines, in CKD stages 2 or 3, high serum PTH is an indicator to evaluate vitamin D status, and give weekly bolus oral doses of 50 000 IU of either ergocalciferol (vitamin D2) or cholecalciferol (D3). However, these recommendations for vitamin D supplementation often fail in raising serum 25(OH)D levels above 30 ng/ml, a requirement to effectively suppress PTH [12]. Simple modifications in timing and dosage should markedly improve the efficacy in reaching the threshold required to overcome the defective uptake of 25(OH)D for local calcitriol production. In regards to the efficacy of vitamin D supplementation with timing, cholecalciferol and ergocalciferol are not equivalent. Upon a bolus administration of 50 000e100 000 IU, the initial increases in serum vitamin D and 25(OH)D levels for either metabolite are identical only over the first 3 and 7 days, respectively [255]. Serum 25(OH)D levels continue to rise in cholecalciferoltreated subjects and peak by day 14. In ergocalciferoltreated individuals, serum 25(OH)D levels at day 14 are not different from baseline. The shorter duration of bolus ergocalciferol dosage relative to cholecalciferol in correcting 25(OH)D deficiency [255] can be resolved simply through daily oral administration of 2000e4000 IU of either metabolite, as it results in identical increases in serum 25(OH)D levels from baseline in normal individuals [256]. In regards to the efficacy of vitamin D supplementation with dosage, in normal individuals, the rates of conversion of vitamin D to 25(OH)D are rapid at lower doses of vitamin D, but much lower at higher vitamin D doses, and reach a plateau at dosages of 2000 to 4000 IU per day [257]. This suggests that high doses are stored, likely in the body fat, and are slowly released to be converted to 25(OH)D [257]. The impact of uremia on vitamin D storage and conversion rates to 25(OH)D is unknown. In Europe, direct supplementation with 25(OH)D is available and should allow the desired raise in serum 25(OH)D above 35 ng/ml in CKD. However, extreme caution is necessary with 25(OH)D dosage and timing to avoid hypercalcemia and hyperphosphatemia.
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25(OH)D can directly activate the VDR, and even though its affinity is 100e200 times lower than that of calcitriol, it circulates at concentrations in the order of ng/ml, 1000-fold higher than serum calcitriol levels. More importantly, the half-life of 25(OH)D is of 15 to 18 days, and therefore daily or weekly regimens could rapidly increase serum concentrations to toxic levels (above 200 ng/ml [258,259]).
SUMMARY The integrity of the vitamin D endocrine system is important for human health, as suggested by the epidemiological association between vitamin D deficiency and a high risk for all causes of mortality in the general population. Normal kidney function is critical to maintain the health benefits of a normal vitamin D status, as demonstrated by almost 30 years of therapy directed to correct calcitriol deficiency in kidney disease. Renal maintenance of serum 25-hydroxyvitamin D levels and calcitriol synthesis is essential not only for the tight and coordinated regulation of the complex PTH/FGF23/calcitriol loops that maintain calcium and phosphate homeostasis, serum levels of PTH and skeletal health thereby preventing soft tissue calcification, but also for an increasing number of anti-inflammatory, renal, and cardioprotective mechanisms including the control of proteinuria and the renineangiotensin system, all of which contribute to the pro-survival advantage reported for interventions with calcitriol and its analogs in large epidemiological studies in CKD patients. In chronic kidney disease, the progressive reduction in kidney function causes a defective renal handling of calcium and phosphorus and also a decrease in renal calcitriol synthesis proportional to the reduction in functional renal mass. The reductions in renal 1ahydroxylase expression are aggravated by an impaired availability of 25(OH)D to the remnant enzyme, as well as by the inhibition of its expression and activity by hyperphosphatemia-induced increases in FGF23, metabolic acidosis, the accumulation of uremic toxins and PTH fragments, and by a blunted response to PTH. These decreases in 1a-hydroxylase expression and activity were considered the main determinants of the reductions in serum calcitriol in the course of CKD. However, correction of 25(OH)D deficiency is sufficient to normalize serum calcitriol at early stages of kidney disease, in hemodialysis patients with very limiting remnant renal 1a-hydroxylase and high levels of FGF23, and more significantly in anephric patients. Thus, vitamin D deficiency is an important contributor to the calcitriol deficiency of CKD. Kidney disease causes decreases in renal megalin content and
proteinuria, both of which enhance the urinary loss of 25(OH)D and make it difficult to maintain normal serum 25(OH)D. Kidney disease also causes a defective uptake of 25(OH)D by renal and non-renal 1-hydroxylases, thereby compromising the autocrine/paracrine activation of the VDR by locally produced calcitriol. The striking incidence of vitamin D deficiency in CKD, which in turn correlates more strongly than calcitriol deficiency with disease progression and death, supports an important contribution of autocrine rather than endocrine VDR activation in the mineral, skeletal, renal, and cardiovascular protection associated with a normal vitamin D status in individuals with normal kidney function and normal serum calcitriol. Interestingly, whereas normal 25(OH)D levels are required for renal and extrarenal calcitriol synthesis, normal serum calcitriol levels are required to correct the impaired uptake of 25(OH)D by monocyte 1ahydroxylase [82]. Thus, the simultaneous correction of vitamin D and calcitriol deficiency could be more effective to improve clinical outcomes in CKD. In CKD, the impact of an impaired renal and extrarenal calcitriol production on biological actions is aggravated by reductions in VDR content in target tissues and by an impaired activity of the calcitriol/VDR complex as a transcriptional regulator of the expression of calcitriol responsive genes. The high dosage of calcitriol or its analogs required to overcome the resistance to therapy induced by reductions in VDR content and actions could aggravate vitamin D deficiency and reduce net local calcitriol production through the induction of 24-hydroxylase, the enzyme responsible for the degradation of calcitriol and its analogs and also of 25(OH)D and through a direct inhibition of 1ahydroxylase. In the parathyroid glands, the reduction in calcitriol/ VDR expression and transcriptional activity results in a defective inhibition of PTH synthesis as well as of the potent mitogenic signals emerging from increases in ADAM17/TGFa activation of EGFR signals; in a defective induction of the antiproliferative molecules LAP, p21 and p27, and of the expression of the CaSR. These defects cause parathyroid hyperplasia, high serum PTH, and reduced sensitivity of the parathyroid gland to suppress growth and PTH secretion in response to calcitriol or calcium. High serum PTH levels lead to osteitis fibrosa, bone loss soft tissue calcifications, all of which increase morbidity and mortality in patients with CKD. After almost three decades with calcitriol replacement therapy with calcitriol or its less calcemic analogs to treat secondary hyperparathyroidism, the adequacy of monitoring the efficacy of current vitamin D and “active” vitamin D interventions through PTH measurements has been challenged. Large
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epidemiological studies in hemodialysis patients have shown that calcitriol analogs confer a greater survival benefit than calcitriol, and that mortality is highest in patients not receiving any vitamin D therapy. More importantly, the survival benefits of calcitriol and its less calcemic analogs can be only partially accounted for by their efficacy in suppressing PTH. Furthermore, the highest benefits of oral therapy with paricalcitol were achieved for the lower doses, thereby supporting a potential adverse effect on high dosage of calcitriol or its analogs on autocrine/paracrine VDR activation by locally produced calcitriol. Taken together these findings and the high incidence of vitamin D deficiency in CKD, the safe correction of vitamin D deficiency/insufficiency has become a high priority among nephrologists, and has posed important challenges regarding: (1) the adequacy of dosage and timing of current recommendations for vitamin D supplementation; (2) the efficacy of exclusive calcitriol (analog) therapy to fully compensate for autocrine VDR actions; (3) the potential adverse effects of interventions with high doses of calcitriol and its analogs to aggravate vitamin D deficiency and the impaired autocrine VDR actions through the induction of 25(OH)D catabolism; and (4) the safety and efficacy of the combined correction of 25(OH)D and calcitriol deficiency/insufficiency to maximize autocrine and endocrine VDR actions. Prospective clinical trials need to address these complex but highly relevant translational issues. The identification of accurate markers of renal and cardiovascular lesions should help monitor the efficacy of vitamin D interventions to improve renal and cardiovascular protection in a PTH-independent manner.
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Acknowledgments This work was supported in part by grants to ASD from the Center for D-receptor Activation Research, Massachusetts General Hospital, Boston, MA, USA, and from Abbott Laboratories, as well as by a Washington University grant for Research in Renal Disease to Dr. Slatopolsky.
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C H A P T E R
71 Idiopathic Hypercalciuria and Nephrolithiasis Murray J. Favus, Fredric L. Coe University of Chicago Pritzker School of Medicine, Chicago, IL, USA
INTRODUCTION The inclusion of a review of idiopathic hypercalciuria (IH) in a book devoted to vitamin D is entirely appropriate given that all of the metabolic changes of IH can be reproduced in healthy volunteers by the addition of doses of calcitriol that are so small as to not cause hypercalcemia. Hypercalciuria occurs in 5 to 7% of children and adults and is the single most common cause of calcium oxalate kidney stones. The chapter focuses on IH as a major cause of hypercalciuria and nephrolithiasis and the potential role of vitamin D. Less frequent causes of hypercalcemia and hypercalciuria may also promote renal stone formation and are discussed in Chapters 45 and 72. Idiopathic hypercalciuria is characterized by normocalcemia in the absence of known systemic causes of hypercalciuria. Intestinal calcium (Ca) absorption is almost always increased, and serum 1,25(OH)2D levels are elevated in some but not all patients. Serum parathyroid hormone (PTH) levels are elevated in less than 5%. The pathogenesis of IH is unknown but several models have been offered from observations in patients including: a primary increase in intestinal Ca absorption; a primary overproduction of 1,25(OH)2D; and a primary renal tubular Ca transport defect or “renal leak” of Ca. Evidence for each model can be found in some patients with IH, suggesting the disorder may be heterogeneous. One-half to two-thirds of IH patients have elevated serum 1,25(OH)2D levels. The remainder with normal serum 1,25(OH)2D levels cannot be distinguished from those with elevated levels as intestinal Ca absorption is just as high, and negative Ca balance may develop during low Ca intake. Of particular importance is the observation that all of the changes in Ca metabolism characteristic of IH can be induced by the administration of small doses of 1,25(OH)2D3 to healthy volunteers.
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10071-X
An animal model of genetic hypercalciuria has been developed in Sprague-Dawley rats by breeding hypercalciuric males and females. The hypercalciuria is now present in all offspring and all form Ca-containing kidney stones. The hypercalciuria in genetic hypercalciuric stone-forming (GHS) rats is due to increased intestinal Ca absorption and bone resorption and decreased renal Ca reabsorption. Elevated vitamin D receptor (VDR) content in intestinal mucosa, renal tubules, and bone cells strongly supports the concept that the genetic hypercalciuria is a state of excess vitamin D receptor. A post-transcriptional dysregulation of VDR is suggested by increased VDR mRNA and VDR protein that has normal binding affinity for 1,25(OH)2D3. The nature of the genetic defect in GHS rats and in human IH remains unknown.
IDIOPATHIC HYPERCALCIURIA Overview Hypercalciuria is common among patients with Ca oxalate nephrolithiasis and is thought to contribute to stone formation by increasing the state of urine supersaturation with respect to Ca and oxalate. Flocks [1] first commented on the frequency of hypercalciuria among patients with Ca stones; however, it was not until the mid-1950s that Albright and Henneman [2,3] defined the condition of IH as hypercalciuria with normal serum Ca, no systemic illness, and no clinical skeletal disease. The definition of hypercalciuria is arbitrary and based on the distribution of urine Ca excretion values among unselected populations of healthy men and women in Western countries [4,5]. The distribution of urine Ca forms a continuum that is clustered about a mean with a long tail of higher values. IH patients are those whose urine Ca exceeds the arbitrary upper limit of normal,
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71. IDIOPATHIC HYPERCALCIURIA AND NEPHROLITHIASIS
which is most commonly defined as greater than 300 mg/24 h for men, greater than 250 mg/24 h for women, or greater than 4 mg/kg body weight or 140 mg Ca per gram urine creatinine for either sex [6]. Using these definitions, hypercalciuria is found in about 50% of calcium oxalate stone-formers [6,7] and is the most common cause of normocalcemic hypercalciuric stone formation [7]. The diagnosis of IH requires the exclusion of known causes of normocalcemic hypercalciuria (see Table 71.1). Surveys of stone-formers attending kidney stone clinics report a high proportion with kidney stone formation among first-degree relatives [8,9]. A genetic basis of IH was further suggested by subsequent surveys [10e12] that revealed a strong familial occurrence of IH with high rates of vertical and horizontal penetrance (see Fig. 71.1) consistent with an autosomal dominant mode of inheritance. IH also occurs in children with the same frequency of occurrence as in adults [13]. That hypercalciuria can have a genetic origin has been clearly demonstrated by breeding experiments in which the offspring of spontaneously hypercalciuric male and female SpragueDawley rats are intensely hypercalciuric [14e16]. Other human hypercalciuric genetic disorders have been described (Chapter 63), but they differ from IH in having a renal phosphate leak [17] that may lead to rickets including renal tubular acidosis [18] and X-linked recessive stone formation with early renal failure [19]. Idiopathic hypercalciuria is a common disorder that affects 5 to 7% of otherwise healthy men and women [4]. If 50% of all stone-formers have IH [6,7], and the frequency of stone disease among adults is 0.5%, then 80 to 90% of IH is asymptomatic and never associated with stone formation. The increased frequency of osteopenia in IH patients (see “Low bone mass,” below) suggests that hypercalciuria may be an important pathogenetic factor for development of low bone mass even among those who do not form stones.
TABLE 71.1
Causes of Normocalcemic Hypercalciuria
Paget’s disease Sarcoidosis Hyperthyroidism Renal tubular acidosis Cushing’s syndrome Immobilization Malignant tumor Furosemide administration
Pathogenesis of Human Idiopathic Hypercalciuria Renal Histopathology in Calcium Oxalate Nephrolithiasis Interstitial crystal deposition at or near the tips of papillae is found in 100% of kidneys of Ca oxalate stone-formers who have IH and no systemic cause of hypercalciuria or other cause of stone formation (Table 71.1). Less frequently (43%), non-stone-formers may have such papillary depositions [20]. On biopsy, these lesions first described by Randall [21] have recently been found to be composed of Ca phosphate (apatite) and contain no Ca oxalate [22]. The plaques originate in the basement membrane of the thin loops of Henle and spread through the interstitium to just beneath the urothelium. There is no inflammatory cell infiltrate to suggest tissue reaction or cell injury. On transmission electron microscopy, lesion particles are laminated microspheres composed of apatite crystals alternating with organic matrix that comes together to form a syncytium. The syncytium extends to the sub-urothelial space and can be seen from the urolithelial side as the white plaque Randall described. Osteopontin has been found in the plaque and may play a role in possible tissue injury response or mineral deposition [23]. The absence of Ca phosphate or Ca oxalate crystal deposition within the renal tubule lumen in IH stoneformers strongly suggests that the Ca phosphate plaque appears to serve as a site onto which Ca oxalate crystals attach, grow, and form clinical Ca oxalate stones [22,24]. The role of hypercalciuria in the development of the Ca phosphate interstitial lesions of Randall’s plaques remains unknown; however, the Ca phosphate plaques are rather specific for Ca oxalate stone-formers, as the interstitial lesions are absent in intestinal bypass patients who form Ca oxalate stones [22]. Sub-urothelial plaque is also found in stone-formers with primary hyperparathyroidism, ileostomy, and small bowel resection, and in brushite stone-formers. Brushite (CaHPO4 2H2O) is a unique form of Ca phosphate stone that has a tendency to recur if patients are not aggressively treated. In five patients with primary hyperparathyroidism and Ca phosphate stones, renal cortical and papillary histopathology showed both dilation and plugging of ducts and papillary deformity characteristic of Ca phosphate stone-formers. Interstitial plaque and stone anchoring characteristic of IH Ca oxalate stoneformers were also present. Thus, the two different pathogenetic processes of stone formation may occur in the same individual [25,26].
$
Increased Intestinal Calcium Absorption Normally, the quantity of Ca absorbed is determined by dietary Ca intake and the efficiency of intestinal Ca
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Family pedigrees of nine probands with idiopathic hypercalciuria. Solid circles and solid squares are females and males with hypercalciuria; S is stone formation; * indicates children (younger than age 20). Arrows indicate probands from each family. Dashed symbols are relatives who were not studied. Hypercalciuria occurred in 11 of 24 siblings, 7 of 16 offspring, and 1 of 3 parents of probands. Reprinted by permission of The New England Journal of Medicine [10]. Copyright 1979, Massachusetts Medical Society.
FIGURE 71.1
absorption [27]. Absorption of Ca across the intestine is the sum of two transepithelial transport processes: a non-saturable paracellular pathway and a saturable, cellular active transport system [28,29] (see also Chapters 19 and 34). Absorption via the paracellular path is diffusional and driven by the lumen-toblood Ca gradient [27]. The cellular pathway is vitamin D dependent and is regulated by the ambient concentration of 1,25(OH)2D. Thus, intraluminal Ca concentration and tissue 1,25(OH)2D levels are the driving forces for Ca translocation via the paracellular and cellular pathways, respectively. Increased intestinal Ca absorption has been found in most patients with IH [30e38]. Using either a single oral dose of Ca isotope to measure fecal isotope excretion or double Ca isotope administration in which the intravenous dose adjusts for isotope distribution, IH patients have an increase in the Ca absorptive flux (Fig. 71.2). External Ca balance studies conducted while IH patients and normal non-stone-formers ingested diets containing comparable amounts of Ca show net intestinal Ca absorption rates to be greater in IH
patients [39]. Biopsies of proximal intestinal mucosae following oral Ca isotopic administration reveal increased mucosal accumulation of isotope compared
FIGURE 71.2 Intestinal Ca absorption in healthy volunteers and patients with IH. Absorption rates are expressed as percentages of dietary Ca absorbed as calculated from the appearance of Ca isotopes in blood or fecal collections. Values are means (horizontal bar) 2 standard deviations. Names indicate references: Birge [31]; Wills [32]; Pak [35]; Kaplan [36]; and Shen [37].
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to normocalciuric non-stone-formers [40]. Thus, by all techniques used, IH is characterized by increased intestinal Ca absorption. Elevated 1,25(OH)2D Kaplan and colleagues [36] first reported elevated serum levels of 1,25(OH)2D in a group of patients with IH. Subsequently, others have confirmed that, on average, serum 1,25(OH)2D levels are higher in IH (Fig. 71.3). Increased in vivo conversion of tritiated 25hydroxyvitamin D3 (3H-25(OH)D3) to 3H-1,25(OH)2D3 with normal metabolic clearance [41] in a group of IH patients with elevated serum 1,25(OH)2D levels indicate that the increase in serum 1,25(OH)2D levels in some IH patients results from increased production by increased conversion of 25(OH)D to 1,25(OH)2D. Of note is the considerable overlap of serum 1,25(OH)2D levels between IH patients and non-stone-formers (Fig. 71.3). Thus, for about 50% of IH patients, increased intestinal Ca transport may be driven by increased circulating 1,25(OH)2D. For the remainder, other mechanisms of increased Ca absorption must be considered in the presence of normal circulating 1,25(OH)2D. The mechanism whereby 1,25(OH)2D production is increased in IH is unknown. The major regulators of renal proximal tubule mitochondrial 25-hydroxyvitamin D 1a-hydroxylase (1a-hydroxylase) activity include PTH, phosphate depletion, and insulin-like growth factor-I (IGF-I) (see Chapters 3 and 45). However, only 5% of IH patients have elevated circulating PTH levels [35,42], and urinary cAMP levels,
a surrogate measure of PTH, are normal in most patients [35,43,44]. Mild hypophosphatemia with reduced renal tubular phosphate reabsorption has been described in as many as one-third of IH patients [36,37,42,45]. A strong inverse association between serum 1,25(OH)2D levels and renal tubular phosphate reabsorption has been reported [37,44]. As elevated PTH or hypophosphatemia accompany elevated serum 1,25(OH)2D in only a minority of patients; the cause of increased serum 1,25(OH)2D in most patients with IH remains unknown. Detailed studies of IGF-I have not been performed. The description of mutations in the q23.3eq24 region of the first chromosome in three kindreds with absorptive IH [46] involves a region containing a gene that is analogous to the rat soluble adenylate cyclase gene. This first description of specific base pair substitutions suggests the possibility of a gene defect associated with IH that may involve altered receptor signaling. Whether this mutation alters functions related to the regulation of the 1a-hydroxylase remains to be determined. However, caution has been expressed in accepting this report as conclusively demonstrating that the substitutions or a mutation of this gene causes IH [47]. Serum 1,25(OH)2D values in normal subjects and IH patients overlap extensively in each series reported (Fig. 71.3). Kaplan et al. [36] found that in patients with absorptive IH (defined as normal fasting urine Ca and normal or elevated serum 1,25(OH)2D) intestinal Ca absorption measured by fecal excretion of orally administered 47Ca was increased out of proportion to the simultaneously measured serum 1,25(OH)2D concentration (Fig. 71.4B). In contrast, a strong positive correlation between intestinal Ca absorption and serum 1,25(OH)2D is found in normal volunteers, normocalciuric stone-formers, patients with primary hyperparathyroidism, and IH patients with fasting hypercalciuria (Fig. 71.4A). The high intestinal Ca absorption rates with either normal or elevated serum 1,25(OH)2D levels suggest that the pathogenesis of IH is heterogeneous, with at least one phenotype resulting from 1,25(OH)2D overproduction. Decreased Renal Calcium Reabsorption
Plot of means 2 SD of serum 1,25(OH)2D in IH patients and non-stone-formers. Horizontal bar is mean of group. Names indicate references: Kaplan [36]; Shen [37]; Insogna [41]; Coe [43]; Gray [45]; Van Den Berg [72]; and Breslau [73].
FIGURE 71.3
A defect in the tubular reabsorption of Ca, a so-called renal leak of Ca, has been postulated as a primary event in the development and maintenance of hypercalciuria in IH. Two reports [48,49] found a greater fraction of filtered Ca excreted in the urine of IH patients compared to non-stone-formers. The values were calculated from inulin clearance or creatinine clearance and used blood ionized Ca as an estimate of ultrafilterable Ca. Although urinary sodium (Na) excretion is a major determinant of Ca excretion in normal and IH patients, there is no evidence that patients over-ingest or over-excrete Na.
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FIGURE 71.4 Relationship of calcium absorption to 1,25(OH)2D levels. (A) Fractional intestinal absorption of oral
47
Ca versus serum 1,25(OH)2D level in normal controls (open circles), normocalciuric stone-formers (NN, filled circles), IH stone-formers who have fasting hypercalciuria and elevated PTH (RH, filled squares), and patients with primary hyperparathyroidism (PHPT, open squares). (B) Fractional calcium absorption of IH patients with absorptive hypercalciuria (normal fasting urine Ca) superimposed on the 95% confidence limits for the relationship in controls. Reproduced from [36] by copyright permission of the American Society for Clinical Investigation.
Hydrochlorothiazide and acetazolamide increase urine Ca, Na, and magnesium (Mg) excretion in IH compared to normals [50], suggesting a generalized defect in proximal tubule electrolyte and water transport in IH patients. Studies of the patterns and timing of urine Ca excretion in IH stone-formers and controls while fed three meals daily demonstrate that hyperabsorption of dietary Ca is rapidly followed by enhanced excretion of Ca into the urine with the bulk of 24-hour urine Ca excretion occurring postprandially during the waking hours. The marked increase in urine Ca excretion after meals is due to decreased fractional tubular Ca reabsorption in IH and control subjects with greater changes in IH subjects. The increased Ca excretion is independent of sodium excretion, and serum PTH levels do not differ between control and IH subjects and cannot explain the greater prandial fall in tubule Ca reabsorption in IH. Serum magnesium and phosphorus levels in IH are below controls throughout the day, and tubule phosphate reabsorption is lower in IH after meals. The studies indicate that the primary mechanism whereby postprandial urine Ca increases is reduced tubule Ca reabsorption, and IH differ from controls in the magnitude of the response [51]. In IH stone-formers, the reduction in postprandial proximal tubule reabsorption of sodium and Ca is matched by increased distal reabsorption so that urine sodium excretion is not different between normals and IH. Distal Ca reabsorption is not sufficiently increased to match Ca delivery, so hypercalciuria results. Urine Ca excretion and overall renal fractional Ca reabsorption are high in IH when adjusted for distal Ca delivery, which strongly suggests a reduction in both distal
as well as proximal Ca reabsorption. These new findings indicate that IH results from a multi-site, presumably genetic-mediated alteration in tubule transport. The increased Ca delivery into the distal nephron may be involved in apatite plaque and stone formation through deposition of apatite within the papillary interstitium [52]. Ca oxalate stone formation depends on urine ion concentrations and state of supersaturation, which are heavily influenced by an imbalance between water excretion and the excretion of the insoluble stoneforming salts and subsequent high concentrations that supersaturate the urine and inner medullary collecting duct (IMCD) fluid. Activation of the IMCD apical membrane calcium-sensing receptor (CaSR) has been postulated to modulate urine volume, and the VDR is a regulator of CaSR gene expression. However, CaSR activation does not appear to be a key regulator in protecting against stone formation [53]. The CaSR is described in detail in Chapter 24. Low Bone Mass Abnormal skeletal metabolism in IH has been demonstrated by low bone mineral density of the distal radius [54,55] and lumbar spine [56e58] and by lower skeletal Ca content by neutron activation analysis [59]. Reports differ as to possible pathogenesis, with low bone density found only in those with renal leak hypercalciuria in one study [55], and in those with absorptive hypercalciuria in another study [57]. Information on bone dynamics is limited to one early study in which 47Ca labeling showed increased bone turnover, with bone resorption and formation both enhanced [60]. Two studies of bone histology showed reduced bone apposition rate,
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delayed mineralization of osteoid seams, and prolonged mineralization lag time and formation period [61,62]. These observations suggest defective mineralization, which may be caused by hypophosphatemia in some patients. The observations are also consistent with a defect in osteoblastic function. Measurements of biochemical markers of bone turnover reveal increased urine hydroxyproline excretion in unselected IH patients [63] and increased serum osteocalcin in IH patients with renal but not absorptive hypercalciuria [64]. Whether the low bone density is a result of the lifelong hypercalciuria, habitual low Ca intake, or a genetic defect in osteoblast or osteoclast function independent of urine Ca excretion remains to be determined. In a study of 59 subjects from 11 families with at least one member a hypercalciuric Ca oxalate stone-former [65], lumbar spine and femoral neck bone density z-scores varied inversely with urine Ca and urine ammonium in the stone-formers but not in the non-stone-formers (Fig. 71.5). There were no correlations of z-score for bone turnover markers or serum 1,25(OH)2D levels. Ca consumption was lower in stone-formers, suggesting that the admonition to ingest a low Ca diet to avoid more stones in fact predisposes to bone loss. A 3-year follow-up of BMDs of the same subjects showed that the changes over time in BMD femoral neck z-score and to a lesser extent in spine z-score can be predicted by the baseline 24-hour urine calcium excretion in hypercalciuric stone-formers and their first-degree relatives (Fig. 71.6). The strong predictive value of urine Ca excretion strengthens the concept that BMD changes in IH are indeed linked closely with hypercalciuria itself through mechanisms that are yet to be determined. Markers of bone turnover, serum 1,25(OH)2D levels and urine ammonium and sulfate did not predict bone loss, nor did Ca intake. Early identification of those stone-formers at greatest risk of bone loss may provide greater incentive for the treating physician to monitor BMD over time and provide dietary and pharmacologic intervention as needed. In the current study, bone turnover markers including serum bone-specific alkaline phosphatase (BSAP), urine hydroxyproline (OHP), and urine collagen breakdown products, were normal and not useful in predicting bone loss. Other studies of IH bone disease have reported either normal or slightly elevated turnover markers; however, the inconsistency of the bone marker data across studies of IH patients differs from the highenormal or modestly elevated bone marker levels found in untreated postmenopausal women. In general, metabolic bone diseases with high bone turnover are associated with high rates of bone resorption and formation rates that lag behind, thereby permitting net bone loss. In IH bone disease, levels of bone turnover markers
are not elevated systematically and suggest that bone formation may be suppressed and bone resorption may not be elevated. Therefore, bone turnover rates may not play as critical a role in creating low bone mass in IH as it does in postmenopausal osteoporosis and other metabolic bone disorders. Cvijetic et al. [67] followed BMD over time in 34 male recurrent stone-formers, nine of whom were hypercalciuric, and compared them to 30 normal male subjects. One year later BMDs of the spine and femoral neck were not different between the groups. Significant correlations were noted between loss of BMD at the femoral neck and diet calcium intake, urinary uric acid excretion, and age of the subjects. The 1-year data are consistent with IH bone disease being a state of low bone turnover that may require some time to manifest low bone mass and increased fracture risk. The well-documented low bone mass in IH patients is associated with increased fracture risk [68]. Reduction of urine Ca during thiazide therapy has been studied in a small number of IH patients and found to be effective in improving bone mass (see “Thiazides,” below). Genetic Contributions to Stone Disease Genetic studies have been conducted on small numbers of subjects often with familial nephrolithiasis, and allelic variants of VDR and CaSR have covaried with stones in some studies but not others. However, the associations are weak, and reproducibility has been difficult. A recent genome-wide association study has yielded potential new important insights [69]. The study of 3773 cases and 42 510 controls from Iceland and the Netherlands reveals common, synonymous variants in the CLDN14 gene that are associated with kidney stones. The claudin genes encode a family of proteins important in tight junction formation and function in a number of tissues as diverse as the kidney and cochlear structures of the inner ear. Sixty-two percent of the general population are homozygous for rs219780[C] and this variant increases the stone risk 1.64-fold compared to noncarriers. The same variants were also found to associate with reduced BMD at the hip. These findings suggest that reduced claudin protein function in kidney and perhaps intestine may alter Ca transport and predispose to Ca kidney stone formation. The data also suggest that both Ca stone formation and low BMD are specifically mediated through the variants in CLDN14 rather than a more general relationship between the two phenotypes. However, the presence and location of CLDN14 expression in bone is unknown, and an alternative explanation is that altered renal Ca transport and urine Ca losses are proximate events in development of low bone mass independent of a direct involvement of the CLDN14 gene in bone. In the kidney CLDN14 is expressed in the proximal convoluted tubule and loop
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FIGURE 71.5 Femoral neck and lumbar spine bone density z-scores and urine calcium excretion in IH stone-formers and non-stone-formers. (A). Non-stone-formers femoral neck z-scores. (B). Non-stone-formers lumbar spine z-scores. (C). IH femoral neck z-scores. (D). IH lumbar spine z-scores. Men are solid circles and women are sold triangles. For IH, BMD at both sites varied inversely with urine calcium. Reproduced from [65] with permission from Nature Publications Group.
Relationship between change in bone density z-score and urine calcium excretion in idiopathic hypercalciuria. Left panel: Change in femoral neck z-score over 3 years inversely correlated (r ¼ 0.37, p ¼ 0.02) with initial urine calcium excretion (mg/day). Right panel: Change in lumbar spine z-score inversely correlated (r ¼ 0.28; p ¼ 0.08) with urine calcium excretion. Ellipses of containment are 1.0 standard deviation. Reproduced from [66] with permission from Nature Publications Group.
FIGURE 71.6
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of Henle and has been shown to selectively decrease cation paracellular permeability [70]. If defective CLDN14 increases proximal convoluted tubule cation paracellular permeability, then greater distal Ca delivery may be anticipated, where transcellular transport of monovalent potassium ion creates an electrical gradient that favors retention of Ca within the tubule lumen and increased urine Ca excretion. It would be predicted that altered CLDN14 protein would have little or no effect on distal Ca reabsorption. Further studies of CLDN14 in well-characterized phenotypes of kidney stone-formers will be informative. Proposed Pathogenetic Models of Idiopathic Hypercalciuria On the basis of the consistent increase in intestinal Ca absorption, normal or elevated serum 1,25(OH)2D levels, and normal or elevated fasting urinary Ca, Pak and colleagues [71] separated IH into three groups: absorptive, renal, and resorptive. In the first, primary intestinal Ca hyperabsorption (Fig. 71.7A) would transiently raise postprandial serum Ca above normal and increase ultrafilterable Ca. Postprandial hypercalcemia would transiently suppress PTH secretion, resulting in reduced tubular Ca reabsorption and hypercalciuria.
In the second model, a primary renal tubular leak of Ca (Fig. 71.7B) would cause hypercalciuria and a transient reduction in serum Ca. Secondary hyperparathyroidism would normalize serum Ca and increase proximal tubule 1,25(OH)2D synthesis, which would stimulate intestinal Ca absorption. PTH secretion would then decline to the extent that serum Ca is normalized. This scenario predicts that serum 1,25(OH)2D would be elevated in renal IH and normal or elevated in absorptive IH [72,73]. A third possibility is based on a primary overproduction of 1,25(OH)2D3 that increases intestinal Ca absorption and bone resorption (Fig. 71.7C) while PTH remains normal and fasting urine Ca excretion may be normal or elevated. Tests of the Models Knowledge of the pathophysiology of IH is fundamental to developing rational therapy for the prevention of recurrent kidney stones. If the model of primary intestinal overabsorption were correct, then dietary Ca restriction would reduce the amount of Ca absorbed and Ca excreted in the urine without altering bone mass. If a renal leak of Ca were the primary event, or if urinary Ca originates from bone rather than diet, then restricting dietary Ca will have little effect on
FIGURE 71.7 Three proposed models of IH. (A) Absorptive IH with primary intestinal overabsorption, postprandial hypercalcemia, sup-
pressed PTH, and normal fasting urine Ca. Serum 1,25(OH)2D is normal. (B) Primary renal tubular leak of Ca leads to a transient decrease in serum Ca and elevated PTH with secondary increases in serum 1,25(OH)2D and intestinal Ca absorption. Fasting urine Ca is elevated. (C) Primary overproduction of 1,25(OH)2D increases serum 1,25(OH)2D and stimulates intestinal Ca absorption and bone resorption. Serum PTH is normal or decreased, and fasting urine Ca is normal or elevated.
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urinary Ca excretion, while worsening Ca balance and promoting bone loss. Testing certain predictions has assessed the accuracy of the absorptive and renal models. FASTING SERUM PTH AND URINE CA
If repeated episodes of postprandial hypercalcemia suppress PTH secretion sufficiently to cause chronic hypoparathyroidism, fasting serum PTH and urine cAMP would be low and fasting urine Ca elevated. Transient suppression of PTH would permit normal serum PTH, urine cAMP, and fasting urine Ca. In contrast, renal IH requires increased PTH and urine cAMP and increased fasting urine Ca [74]. As existing PTH radioimmunoassays do not differentiate normal from low values, most IH patients have been found to have PTH levels in the normal range. The recent study of prandial urine Ca excretion in IH subjects found serum PTH to be within the normal range and slightly lower than controls, which argues against a regulatory role of PTH in the decreased renal tubule Ca reabsorption [51,52]. Although normal fasting urine Ca is not unusual among IH patients, only 5% have elevated PTH and, therefore, fail to meet the criteria for renal IH [35,71]. Thus, a primary renal leak of Ca with a secondary increase in PTH cannot account for fasting hypercalciuria in a majority of patients. About 24% of patients meet the criteria of absorptive hypercalciuria by reducing urine Ca during fasting to maintain neutral Ca balance [72]. EXTERNAL CA BALANCE
The relationship between net intestinal Ca absorption and 24-hour urine Ca excretion calculated from 6-day balance studies is different in IH patients compared to normal subjects (Fig. 71.8) [75e81]. In non-stoneformers, urinary Ca excretion is positively correlated with net absorption, and overall Ca balance is positive when net absorption is greater than 200 mg per 24 h (see 95% confidence limits calculated from balance studies on normal subjects in Fig. 71.8). Net Ca absorption tends to be greater in IH patients, and for every level of net absorption, 24-hour urine Ca excretion is higher in the patients compared to healthy subjects. In IH patients, a greater portion of absorbed Ca is excreted in the urine. In normal subjects, net Ca absorption exceeds urine Ca excretion, and balance is positive when net absorption exceeds 200 mg/24 h. In contrast, almost 50% of the IH patients have urine Ca excretion in excess of net absorption and are in negative Ca balance, even when allowance is made for some variability in the balance data (50 mg). Thus, at all levels of net Ca absorption, negative Ca balance (above the zero balance or above the line of identity) is common in IH patients but not in healthy subjects. Negative Ca
Urinary Ca excretion as a function of net intestinal Ca absorption. Data are derived from 6-day external mineral balance studies. Solid lines indicate the 95% confidence limits about the mean regression line derived from the data on 195 adult non-stone-formers. Individual balance studies performed on 51 patients with IH are shown as open circles. The dashed line represents equivalent rates of urinary Ca excretion and net intestinal Ca absorption (the line of identity). Normal values are from [50,63,77,78]. Values from patients are from [50,62,80e84] and J. Lemann (personal communication, 1992). Adapted from [110].
FIGURE 71.8
balance in the presence of adequate Ca intake is incompatible with a primary hyperabsorption of dietary Ca or absorptive hypercalciuria and cannot, by itself, account for the hypercalciuria. URINE CA AND CA BALANCE DURING LOW-CA DIET
The hypothesis of primary intestinal Ca overabsorption predicts that dietary Ca restriction would reduce the amount of Ca absorbed and would therefore reduce urinary Ca excretion. Like normal subjects, IH patients would be in positive or neutral Ca balance when net absorption is above 200 mg/24 h (Fig. 71.8). A low-Ca diet would reduce urine Ca excretion through an increase in PTH secretion, which would promote distal tubular Ca reabsorption. In contrast, patients with a primary renal Ca leak would be unable to conserve urine Ca at any level of Ca intake and would maintain an excessive or inappropriately high urine Ca excretion even during low-Ca diet. As a result, Ca balance during low-Ca diet would shift from positive or neutral to negative or become more negative. Serum PTH would be expected to increase to high levels during a low-Ca diet. To test whether the responses of IH patients fit these predictions, Coe et al. [43] fed a low-Ca diet (2 mg/kg/day) to nine normal volunteers and 26 unselected IH stone-formers. After 10 days on the
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detectable bone loss. The data also suggest that some patients with IH may have diet-dependent hypercalciuria, whereas others have diet-independent hypercalciuria. The two proposed mechanisms cannot be readily distinguished by any clear discontinuity in the distribution of urine Ca values, and serum PTH and 1,25(OH)2D levels do not predict the urine Ca responses during a low-Ca diet. Patients with the highest urine Ca and most extreme negative Ca balance had serum PTH and 1,25(OH)2D levels that were not different from patients who conserved Ca to the levels found in normal subjects. ROLE OF 1,25(OH)2D EXCESS
Calcium intake and urinary excretion in patients with IH and normal subjects. Urine Ca excretion (UCa) and Ca intake (Cal) are during a low-Ca diet. Mean Ca intakes for patients and controls (2.29 0.15 versus 2.31 0.05 mg/kg/day) were not different. Mean urine excretion rates during low Ca intake and values of Cal-CaE (an index of Ca balance) differed significantly between normals and IH patients. Subjects and patients are arranged in ascending order of urinary Ca excretion. Reprinted by permission of the publisher from [43]. Copyright 1982 by Excerpta Medica Inc.
FIGURE 71.9
diet, urine Ca excretion decreased to 2.0 mg/kg body weight or less in both patients and controls, but 17 of the 26 IH patients (Fig. 71.9) showed values greater than the highest value in normal controls. In patients, urine Ca excretion (CaE) ranged from normal to persistently high levels. It exceeded Ca intake (CaI) (Fig. 71.9, CaI e CaE) in 11 of the 26 patients and none of the non-stone-forming controls. Thus, almost 50% of the patients had more Ca in the urine than was provided by the diet and were clearly in negative Ca balance. The results indicate that a chronic low-Ca diet may be detrimental for some patients, as the inability to conserve urine Ca would eventually lead to clinically
The majority of patients are classified as having absorptive hypercalciuria [71,74], yet negative Ca balance during low-Ca diet [43] without elevated PTH, or 1,25(OH)2D, is not predicted by the absorptive model (Fig. 71.7A). Patients who meet the criteria of renal hypercalciuria tend to have higher serum 1,25(OH)2D levels, but only a small portion have elevated PTH levels. Further, serum 1,25(OH)2D levels do not predict whether patients will be classified as absorptive or renal, and at least one-third of patients have normal serum 1,25(OH)2D levels despite intestinal Ca hyperabsorption. For them, the mechanism of intestinal Ca hyperabsorption remains unexplained. One study of ten IH stone-formers and ten age-matched normal subjects found a two-fold elevation of peripheral blood monocyte (PBM) VDR [82]. As PBM may be stimulated to differentiate into mature osteoclasts, in part mediated by VDR, the results suggest that elevated tissue VDR concentrations in the presence of normal circulating 1,25(OH)2D levels may mediate increased vitamin D biologic actions. The model of primary vitamin D excess (Fig. 71.7C) is supported by elevated 1,25(OH)2D production rates and enhanced biological actions of 1,25(OH)2D, including increased intestinal Ca absorption and bone resorption. Creation of a mild form of 1,25(OH)2D excess was achieved by the administration of pharmacological doses of 1,25(OH)2D3 (3.0 mg/day) to healthy men for 10 days while Ca intake varied from low (160 mg) to normal (372 mg) or high (880 mg) [83e85]. Increased urine Ca excretion and net intestinal Ca absorption led to negative Ca balance as calculated from 6-day metabolic balance studies (Fig. 71.10). Dietary Ca strongly influenced the response to 1,25(OH)2D3, as Ca balance was more negative during low Ca intake, and the increase in urine Ca resulted primarily from accelerated bone resorption. At lowenormal or normal Ca intake, 1,25(OH)2D3 administration increased urine Ca and net intestinal Ca absorption, and Ca balance remained neutral. During normal-Ca diet, 1,25(OH)2D3 administration maintained neutral or positive Ca balance.
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FIGURE 71.10 Intestinal Ca absorption, urine Ca excretion, and Ca balance in normal men receiving 1,25(OH)2D3 (hatched bars) or controls (open bars) at varying levels of dietary Ca. Values (mg/24 h) are means SEM for six men per group. For Ca balance, values above the horizontal line indicate positive balance and those below the line, negative balance. Data from [84] and [85]. Reprinted with permission from [83].
Thus, 3 mg/day of 1,25(OH)2D3, which was insufficient to cause hypercalcemia, during the 10-day study has profound effects on intestinal Ca absorption, urine Ca excretion, and Ca balance. Further, 1,25(OH)2D3 administration caused negative Ca balance only during low Ca intake. l,25(OH)2D3-induced changes in Ca balance in normal subjects are similar to those observed in IH patients at comparable levels of Ca intake. In other experiments, ketoconazole administration to IH patients inhibited renal 1,25(OH)2D biosynthesis [73] and decreased serum 1,25(OH)2D levels, intestinal Ca absorption, and urine Ca excretion. Although ketoconazole has many actions in multiple tissues, it appears that by inhibiting 1a-hydroxylase activity, it can reverse the Ca metabolic defects in IH patients. The results of the effects of 1,25(OH)2D3 treatment and the response to ketoconazole provide further support for a primary 1,25(OH)2D excess in at least some patients with IH. The nature of the disordered regulation of renal 1,25(OH)2D production or action remains to be determined, as neither elevated PTH nor hypophosphatemia were present in responders or were absent in nonresponders to ketoconazole.
GENETIC HYPERCALCIURIC RATS Clinical studies that test the proposed models of human IH including primary intestinal Ca overabsorption, decreased renal Ca reabsorption, and excess vitamin D have been complicated by difficulty in
controlling for potential variables such as genetic and environmental factors that may influence dietary patterns. The availability of an animal model of IH would permit the testing of the three hypotheses under conditions that exclude or control for genetic and dietary influences. The strong familial occurrence of IH in humans and the high frequency of elevated urine Ca in adult men and women suggested that spontaneous hypercalciuria might also be found in animals.
Establishment of a Colony of Genetically Hypercalciuric Rats The distribution of urine Ca excretion in a population of male Sprague-Dawley (SD) rats fed a normal-Ca diet (0.8% Ca) followed a non-Gaussian distribution, which was similar to that found in a population of healthy humans in that values were clustered about the mean with a long tail of higher values [14]. Hypercalciuria (in mg Ca/24 hour) defined as urine Ca greater than two standard deviations above the mean value identified 5 to 10% of male and female rats as spontaneously hypercalciuric. Mating males and females with the most severe hypercalciuria resulted in offspring with hypercalciuria. The most hypercalciuric offspring were used for repeated matings, creating a colony with hypercalciuria that has increased in intensity and frequency with each successive generation [15]. By the twentieth generation, over 95% of males and females were hypercalciuric. By the fortieth generation, mean urine Ca
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excretion was 7.0 0.3 mg/24 h compared to the stable mean excretion of less than 0.75 mg/24 h by wild-type SD rats [87]. Hypercalciuria is detectable soon after weaning at about 6 weeks (50 g body weight) and persists lifelong. Weight and growth of the hypercalciuric rats are comparable to wild-type SD rats obtained from the same supplier that provided the original spontaneously hypercalciuric animals. No anatomical or structural abnormalities have been identified in GHS rats; however, by 18 weeks of age 100% of the animals have multiple bilateral Ca-containing kidney stones in the upper and lower urinary tracts [87]. No stones are found in the kidney or urinary tract of wild-type SD rats. In a first approach to mapping the genes contributing to hypercalciuria in GHS rats, quantitative trait locus (QTL) mapping of F2 rats from a cross between GHS and normocalciuric WKY rats was undertaken [88]. A Ca excretion QTL (hypercalciuria 1 (HC1)) was identified with a logarithm of odds (LOD) score of 2.91 on chromosome 1. The microarray data for the HC1 congenic rats clearly demonstrate the perturbation of Ca metabolism and transport pathways defined in the GHS rats. The HC1 QTL region contains at least one gene that contributes either directly or indirectly to hypercalciuria in the GHS rat model, which was previously estimated to contribute 7% of the variation in Ca excretion. To date a putative HC1 gene or other genes that may contribute to the hypercalciuria has yet to be identified.
Serum and Urine Chemistries Serum Ca and Mg are within the normal range in the colony now referred to as genetic hypercalciuric stoneforming (GHS) rats [15]. Serum phosphate is lower in female rats, and there is no difference between GHS and wild-type males and females. Serum PTH levels in GHS rats are not different from controls. Urine volumes are greater in the GHS rats.
Renal Ca Handling During an adequate Ca diet, urine Ca excretion was greater in the prandial (0e3 h) and postprandial (3e6 h) periods when the GHS rats were offered their daily food as a bolus compared to when given three equal portions. Total 24-hour urine Ca excretion was greater in the GHS rats fed as a bolus compared to Ca excretion by rats given a similar amount of food in divided doses [90]. Of particular note is that the bolusfed rats excreted more Ca in the urine over the entire 24 hours than excreted by rats fed the same amount of Ca in three divided portions. Urine Ca excretion increased after meals in GHS and NC rats due to a fall in the fractional reabsorption of Ca and fell further in the GHS rats just as the idiopathic stone-formers had
greater reductions in renal tubule Ca reabsorption than normal controls. If peak supersaturation drives Ca oxalate or Ca phosphate stone formation, then it would be predicted that less stone formation would appear in GHS rats fed in a divided manner. Calcimimetics, such as cinacalcet (Cin), are small organic molecules that act as allosteric activators of the CaSR, which increases sensitivity of the CaSR to serum Ca and substantially lowers PTH levels. While Cin reduced serum PTH and Ca in the GHS and control rats, it did not alter urine supersaturation with respect to CaOxalate or phosphate (CaHPO4) in either strain [91]. Similar responses have been observed in humans treated with Cin. In rats fed a diet low in Ca (0.02% Ca), serum PTH was lower in GHS rats than in NC rats, suggesting that even during reduced dietary Ca intake, hypercalciuria in GHS rats persisted and was driven more by enhanced intestinal Ca absorption and/or bone resorption than by reduced renal tubular Ca reabsorption.
Mineral Balance Six-day external balance studies performed while the animals were fed a normal-Ca diet showed the animals to be in positive balance for Ca, Mg, and phosphorus [15] with greater net Ca absorption in GHS rats. The GHS rats maintained positive Ca balance because the increased urine Ca excretion was matched by a greater net intestinal Ca absorption.
Intestinal Calcium Transport To investigate the mechanism of the increased net Ca absorption, segments of proximal duodenum were mounted in vitro in modified Ussing chambers, and transepithelial bidirectional fluxes of Ca were measured in the absence of electrochemical gradients [22]. Under these conditions [15], duodenal segments from GHS rats had a five-fold increase in the mucosal-to-serosal (absorptive) transepithelial flux of Ca (Jms), whereas the secretory flux of Ca from serosa to mucosa (Jsm) was only mildly increased compared to wild type (Table 71.2). As Ca Jms was 10 to 12 times higher than Ca Jsm, changes in Jsm had a non-significant effect on net Ca absorption (Ca Jnet).
Serum 1,25(OH)2D Circulating 1,25(OH)2D levels were lower in the fourth-generation GHS rats; however, the differences disappeared by the tenth generation (at 190 g, mean SD serum 1,25(OH)2D was 135 12 versus 174 19 pg/ml (P,NS), and no subsequent differences in serum 1,25(OH)2D levels have been observed [16]). In vitro
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TABLE 71.2
In vitro Bidirectional Duodenal Calcium Active Transport*
Flux
NM
GHM
NF
GHF
Jms
51 þ 12
264 þ 27
29 þ 9
258 þ 40
Jsm
11 þ 2
19 þ 2
14 þ 2
23 þ 2
Jnet
40 þ 11
245 þ 28
14 þ 8
235 þ 40
* Values are means SE for 5 to 11 rats per group. NM and NF are normocalciuric (wild-type) male and female rats, respectively. GHM and GHF are genetic hypercalciuric male and female rats, respectively. Jms and Jsm are mucosal-to-serosal and serosal-tomucosal fluxes of Ca, respectively. Jnet is net Ca absorption, where Jnet ¼ Jms e Jsm. Adapted from [16] and reproduced with permission from The Journal of Clinical Investigation, by copyright permission of the American Society for Clinical Investigation.
duodenal net flux (Jnet, equal to Jms Jsm) for Ca was positively correlated with serum 1,25(OH)2D in normocalciuric and GHS male and female rats (Fig. 71.11). However, the regression coefficients were different for the wildtype and GHS rats, with the latter having a steeper slope. Ca Jnet was greater in GHS rats with serum 1,25(OH)2D levels comparable to the wild-type rats, strongly suggesting that duodenal Ca-transporting cells in GH rats are more sensitive to 1,25(OH)2D.
Vitamin D Receptor The increased intestinal Ca transport and normal serum 1,25(OH)2D levels in GHS rats suggested either that Ca transport was being stimulated by an unidentified, vitamin D-independent process or that 1,25(OH)2D action was being amplified at the level of vitamin D target tissues. As 1,25(OH)2D stimulates Ca
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transport by binding to the vitamin D receptor (VDR) that in turn upregulates vitamin-D-dependent genes that encode for proteins involved in transepithelial Ca transport, and because the biological actions of 1,25(OH)2D are directly related to the tissue VDR content [92e94], VDR binding in intestinal epithelial cells was measured. Duodenal cytosolic fractions prepared in high-potassium buffer from male GHS rats bound more 3 H-1,25(OH)2D3 than comparable fractions from wildtype control rats [16] (Fig. 71.12). Cytosolic fractions from kidney cortex and from splenic monocytes also exhibited greater specific binding of 1,25(OH)2D3. Scatchard analysis of the specific binding curves revealed a single class of VDR-binding sites in tissues from both wild-type and GHS rats. The number of VDR-binding sites in GHS rat duodenal cells was double that found in cells from wild-type rats (536 73 versus 243 42 fmol/mg protein; n ¼ 8 and n ¼ 14; p < 0.001), with comparable affinity of the receptor for its ligand (0.33 0.01 versus 0.49 0.01 nM; non-significant). A two-fold increase in VDR-binding sites was also found in GHS rat renal cortical homogenates [16]. Using Western blotting, homogenates of duodenal mucosa from GHS rats contained a band at 50 kDa that comigrated with duodenal extracts from wild-type rats and with recombinant human VDR. The bands from the GHS rat tissues were more intense compared to controls, confirming that the increase in specific 3 H-1,25(OH)2D3 binding was due to an increase in VDR protein. Northern analysis of RNA extracts from GHS and wild-type rat tissues revealed a single species of VDR mRNA at 4.4 kb with no difference in migration between the two groups [16]. Duodenal extracts from
FIGURE 71.11 Duodenal Ca net flux (Jnet) as a function of serum
1,25(OH)2D for hypercalciuric and normocalciuric male (open and filled squares, respectively) and female (open and filled circles, respectively) rats. Jnet and serum 1,25(OH)2D were correlated for male and female normocalciuric rats (r ¼ 0.789, n ¼ 12, p < 0.001, solid line) and for male and female GHS rats (r ¼ 0.500, n ¼ 17, p < 0.03, dotted line). The regressions were different (F ratio ¼ 5.469, p < 0.015). Reproduced from [15] by copyright permission of the American Society for Clinical Investigation.
FIGURE 71.12 Specific binding of 3H-1,25(OH)2D3 to duodenal cytosolic fractions (VDR) prepared from GHS rats (filled circles) and wild-type controls (open circles) while fed a normal Ca diet. Values are means SEM for four observations per concentration point. *, p < 0.05; **, p < 0.01; ***, p < 0.005 versus controls. Reproduced from [16] by copyright permission of the American Society for Clinical Investigation.
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71. IDIOPATHIC HYPERCALCIURIA AND NEPHROLITHIASIS
GHS rats contained less VDR mRNA than controls. Estimates of duodenal cell transcription rates using standard nuclear run-on assays found no clear difference between GHS rats and controls [16]. The in vivo halflife of the VDR mRNA in GHS rat duodenum was comparable to that of controls (6 h). Administration of a small dose of 1,25(OH)2D3 (30 ng as a single dose) resulted in a significant elevation of VDR message and prolongation of message half-life in GHS rats but not controls [95,96]. Thus, in GHS rat intestine, the increased VDR level is not due to an increase in VDR gene transcription. The data are consistent with either an increase in VDR mRNA translation efficiency or changes that result in a prolongation of the VDR half-life. The increased accumulation of the vitamin-D-dependent calbindin-D9K found in GHS rat duodenum [16] is evidence that the increased level of VDR is functional and that the increased Ca transport is likely a vitaminD-mediated process. VDR protein levels in GHS rat duodenum and kidney (Fig. 71.13) in GHS rats from generation 80e90 are greater [97] than reported from earlier generations [16,95,96]. In the recent studies [97], VDR protein levels were elevated 9.9-fold in jejunum and 6.2-fold in ileum from GHS rats compared with NC rats (Fig. 71.13A). GHS rats had elevated VDR protein levels in duodenum, jejunum, ileum, and kidney cortex. In contrast to the initial measurements of VDR mRNA in the early generations of offspring [16,95,96], the current study of generations 80e90 [97] reveals significant increases in VDR gene expression. While the early generations of GHS rats had low levels of VDR mRNA by Northern blotting, the most recent generations of GHS rats have duodenal VDR mRNA levels that are increased 3.1-fold above controls [97]. The increases in VDR gene expressions from the early to the recent generations accompanies the increases in urine Ca excretion that ranged from 2.0 mg per 24 h in the early
generations to 8 to 12 mg/24 h in the current generations (control rats are 0.5 to 1.0 mg/24 h urine Ca). Whether the increases in urine Ca excretion are mediated by the greater expressions of VDR is plausible but remains to be determined. Compared to VDR gene exression rates, duodenal VDR protein levels are now increased 6.3-fold. In GHS rat kidney, VDR mRNA and protein levels were raised 2.5- and 5.5fold, respectively. The greater magnitude increase in VDR protein compared with mRNA in GHS rat duodenum and kidney supports previous observations [16,96] that increased VDR levels may be due to a combination of enhanced transcriptional regulation, greater efficiency of VDR translational events, and prolonged half-life and stability of the VDR protein. Indeed, prolonged half-life of the GHS rat duodenal and renal cortical VDR protein was described in a previous in vivo study [96]. To assess the genetic molecular basis for the elevated VDR, kidney genomic DNA from four GHS and two NC rats were subjected to DNA sequencing. The results revealed no mutation, polymorphism, or splicing variant in the 50 UTR exons, exons, intron/exon boundary regions, or 30 UTR exons of the VDR. There was no difference in the DNA sequence of the proximal 2 kb of the VDR proximal promoter region between the two groups. Therefore, the higher mRNA levels of VDR in GHS rats may not result from mutation or allelic variation in the cDNA or proximal promoter regions. Nevertheless, it is possible that differences exist further upstream than 2 kb. Snail nuclear protein has been recognized as a negative regulator of VDR in neoplastic cells and tissues [98]. In GHS rats, Snail mRNA levels were suppressed 7.5-fold in duodenum, 2.2-fold in jejunum, 2.0-fold in ileum, and 6.7-fold in kidney, with only the duodenal and kidney suppressions being statistically significant [97]. In colon cancer cells, upregulated Snail is inversely
FIGURE 71.13 VDR protein and mRNA levels in GHS rat intestine and kidney. (A) Representative immunoblot of VDR in total nuclear protein extracts from GHS and NC (control) rat intestine and kidney. Lanes 1e4 are NC and lanes 5e8 are from GHS rats. Lanes are: duodenum (1 and 5); jejunum (2 and 6); ileum (3 and 7); and kidney cortex (4 and 8). (B) VDR and GAPDH mRNA by real-time PCR for GHS and NC intestinal segments and kidney. Results are individual values with mean as horizontal bars (n ¼ 4 per group). *, p ¼ 0.027; **, p ¼ 0.034; and ***, p ¼ 0.036. Reproduced from [97] by copyright permission of the American Society for Bone and Mineral Research.
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correlated with cellular dedifferentiation and low VDR expression. The mechanism whereby suppression of Snail in duodenum and kidney of GHS rats may enhance VDR expression is centered on the interaction of Snail and E-box binding sites within the VDR proximal promoter [97]. In the GHS rats, we postulate that suppression of Snail removes tonic repression of VDR and permits its overexpression. The cause of reduced Snail expression in GHS rat tissues is not known; however, Snail binding to the VDR promoter in NC was weaker in GHS rats (Fig. 71.13). These results indicate that under the pathologic condition in GHS rats, lower levels of Snail reduced repression of VDR expression. Major questions remain as to the genetic basis of the reduced Snail levels and increased VDR activity. The cumulative evidence suggests that the primary genetic defect does not directly involve the VDR gene.
Low Bone Density In vitro studies of bone resorption using neonatal calvariae from normal and GHS rats show that Ca efflux (a measure of bone resorption) increases in a dose-dependent manner in the presence of 1,25(OH)2D3 or PTH [99]. The doseeresponse curve is much steeper for 1,25(OH)2D3 in calvariae from GHS rats, whereas the doseeresponse curves for PTH-stimulated Ca efflux are not different between control and GHS calvariae. Western blotting showed a four-fold increase in VDR protein from GHS neonatal rat calvariae [99]. Thus, the increase in target tissue VDR exerts biological actions that increase l,25(OH)2D3-dependent bone resorption, which likely contributes to the hypercalciuria. In studies of bone composition, GHS rats fed a diet high in Ca (2.0% Ca) had reduced cortical (humerus) and trabecular (L1eL5 vertebrae) BMDs, whereas lowcalcium diet (LCD) reduced BMDs to a similar extent in both GHS and NC rats [100]. In GHS rats fed HCD, trabecular volume and thickness decreased, whereas LCD increased both osteoid surface and volume 20fold. GHS rats fed HCD had no change in vertebral strength (failure stress), ductibility (failure strain), stiffness (modulus), or toughness, whereas in the humerus, there was reduced ductibility and toughness and an increase in modulus, indicating that the defect in mechanical properties is mainly manifested in cortical, rather than trabecular, bone. GHS rat cortical bone is more mineralized than trabecular bone and LCD decreased the mineralization profile. While fed an adequate Ca diet (0.6%), GHS rats have reduced BMD with reduced trabecular volume, mineralized volume, and thickness, and their bones are more brittle and fracture prone, indicating that GHS rats have an intrinsic disorder of bone that is not secondary to diet.
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Response to Low-calcium Diet To test whether the hypercalciuria in GHS rats is the result of a primary overabsorption of dietary Ca, GHS and wild-type control rats were fed diets either normal (0.6% Ca) or low (0.02% Ca) with respect to Ca. During the LCD, urine Ca excretion decreased in both groups (Fig. 71.14); however, urine Ca remained higher in GHS rats and resulted in negative Ca balance [101]. The inability of GHS rats to conserve Ca during low Ca intake excludes overabsorption of dietary Ca as the sole cause of hypercalciuria in GHS rats.
Summary of Pathogenesis in the Genetic Hypercalciuric Rat Figure 71.15 summarizes current knowledge of the pathogenesis of hypercalciuria in the GHS rats. Breeding by selection for hypercalciuria has emphasized a trait in the offspring that likely involves the expression of several genes for full phenotypic expression. To date, none of the genes has been identified. Studies demonstrate increased VDR concentration in intestine, kidney, and bone which may be part of the primary event(s) and cause the hypercalciuria; however, a secondary adaptive increase in VDR to compensate for urinary Ca losses has not been excluded. The elevated VDR gene expression is accompanied by suppression of SNAIL gene, which is known to suppress VDR gene expression. The role of the suppressed SNAIL is not known, but appears to be pivotal in permitting VDR to increase and hence the GHS rats phenotype. Further information is required regarding the renal handling of Ca in GHS rats and whether the GHS genotype results in a primary defect in renal Ca transport.
Daily urine Ca excretion in nineteenth-generation GHS rats (open symbols) or wild-type control rats (filled symbols) fed a normal Ca diet (NCD, 0.6% Ca, triangles) during days 1e10 followed by either continuation of the NCD (triangles) or feeding of a low Ca diet (LCD, 0.02% Ca, circles). Rats were pair-fed to 13 g of diet per day. Reprinted with permission from [101].
FIGURE 71.14
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71. IDIOPATHIC HYPERCALCIURIA AND NEPHROLITHIASIS
Breeding for hypercalciuria
Genomic events
Vitamin D receptor
Intestinal Ca absorption
Bone resorption
?
Renal tubular Ca transport
Hypercalciuria
FIGURE 71.15 Proposed series of events that result from breeding selection for hypercalciuric rats. The renal handling of Ca by GHS rats and the role of increased VDR content, if any, in the transport process remain unknown.
CURRENT VIEW OF HUMAN GENETIC HYPERCALCIURIA Striking similarities in Ca metabolism between GHS rats, IH patients, and human volunteers treated with 1,25(OH)2D3 (Table 71.3) strongly support a primary role of excess 1,25(OH)2D biological action in the pathogenesis of human IH. When deprived of dietary Ca, few IH patients conserve Ca to the extent that normals do (Fig. 71.9). The renal IH model predicts ongoing urinary losses of Ca independent of Ca intake, and negative Ca balance during a low-calcium diet. However, most patients have normal, not elevated PTH, as renal IH would require. Therefore, the absorptive and renal models of hypercalciuria cannot explain the response of most patients to a low-Ca diet. In nonstone-formers, 1,25(OH)2D3 administration changes
TABLE 71.3
Pathophysiology of Genetic Hypercalciuria
Parameter
Human
Human
GHS rats
Serum Ca
N
N
N
Serum phosphate
N
N-D
N
Serum 1,25(OH)2D
I
N-I
N
Urinary Ca on NCD
I
I
I
Urinary Ca on LCD
I
N-I
I
Intestinal Ca absorption
I
I
I
Ca balance on NCD
Pos-N
N-Neg
Pos
Ca balance on LCD
N-Neg
N-Neg
Neg
Values for human controls are responses to treatment with 3 mg 1,25(OH)2D3 daily for 7 days compared to pretreatment. GHS, genetic hypercalciuric stoneforming; NCD, normal Ca diet; LCD, low Ca diet; N, normal; I, increased; D, decreased; Pos, positive; Neg, negative.
urine Ca and Ca balance to those observed in a majority of IH patients who have either normal or elevated serum 1,25(OH)2D levels. For some patients, elevated serum 1,25(OH)2D3 increases in intestinal Ca hyperabsorption and urine Ca excretion, and causes negative Ca balance during low Ca intake. The source of 1,25(OH)2D excess is more elusive in patients with normal serum 1,25(OH)2D levels. They may be more similar to the GHS rats in that both have normal serum 1,25(OH)2D, increased intestinal absorption and bone resorption during a low-Ca diet, and low bone density. Whether these changes in human IH are due to increased intestinal, renal, and bone cell VDR content that can amplify the biological actions of normal circulating 1,25(OH)2D levels remains to be determined.
THERAPEUTICS OF IDIOPATHIC HYPERCALCIURIA AND EFFECTS ON CALCIUM METABOLISM Dietary Calcium Restriction Hypercalciuria promotes urine calcium oxalate supersaturation and increases spontaneous crystal formation [102]. The goals of preventive therapy are to reduce Ca oxalate supersaturation by increasing urine volume and decreasing urine Ca excretion. If the pathophysiologic role of 1,25(OH)2D excess or VDR excess is borne out, then ideal therapy may eventually include either a specific 1,25(OH)2D antagonist or an inhibitor of VDR function. In the absence of such agents, therapies will continue to concentrate on lowering urine Ca excretion using thiazide agents to enhance renal tubular reabsorption. Restriction of dietary Ca does not reduce urine Ca oxalate supersaturation but can promote bone loss. Increasing dietary Ca intake may reduce urine oxalate excretion but the rise in urine Ca excretion prevents a decrease in urine Ca oxalate supersaturation. Since the description of IH, dietary Ca restriction has been recommended to lower urine Ca. Dietary Ca restriction or the use of Ca-binding resin to prevent absorption [103] could be efficacious for patients with primary intestinal Ca hyperabsorption (absorptive hypercalciuria). However, it appears that many patients develop negative Ca balance during low Ca intake. For them, chronic dietary Ca restriction and negative Ca balance would eventually cause bone loss, osteoporosis, and increased fracture risk. Reports of lower bone density in IH patients suggest that Ca restriction may only worsen the existing reduction in bone mass. Therefore, treatment with Ca restriction requires knowledge that the patient will likely not conserve urine Ca and will develop negative Ca balance.
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SUMMARY
Thiazides Thiazide and the related chlorthalidone diuretics reduce urine Ca excretion by inducing an NaCl diuresis, which causes volume contraction and decreased Ca delivery to the distal tubule segments [104]. These agents also stimulate distal tubule Ca reabsorption through a direct interaction with the tubule cells [103e105]. Thiazides may decrease or have no effect [34,38,106] on intestinal Ca transport in IH patients, and serum 1,25(OH)2D and PTH levels are not changed by thiazides. In one study, IH patients treated with chlorthalidone for 6 months improved Ca balance to or toward positive by decreasing both urine Ca and intestinal Ca absorption [107], with urine Ca declining to a greater extent than intestinal absorption. The epidemiological studies suggesting that chronic thiazide therapy reduces fracture risk [108,109] may result from druginduced improvement in Ca balance [106] and reduced bone turnover and improved mineralization [64]. The effects of thiazide on urine Ca and bone metabolism are accompanied by a decrease in new Ca stone formation compared to placebo controls [110]. The beneficial effect of thiazide is evident during the second and third year of therapy, when stone recurrence is reduced by about 50 to 80%. The reduction in new stone formation is due to a decrease in urine Ca oxalate supersaturation, as urine Ca declines while oxalate is unchanged. As thiazides can reduce urine Ca excretion and stone formation rates in all forms of IH [111], knowledge of the pathogenesis of IH in each patient may not be required prior to selecting thiazide therapy. Additional management includes adequate hydration to reach 2.0 liters of urine per 24 h. Decreased urine oxalate can decrease urine Ca oxalate supersaturation; however, a low-oxalate diet is difficult for patients to follow, and reduction in intestinal oxalate absorption enhances Ca absorption and urine Ca excretion, resulting in no net change in urine Ca oxalate supersaturation. Urine Ca excretion may be reduced by sustained alkalinization of the urine through increasing citrate delivery. Potassium citrate in multiple divided doses can raise urine pH and reduce urine Ca by creating a soluble Ca citrate complex that lessens Ca available for complexation with oxalate.
RISK OF STONE FORMATION USING VITAMIN D ANALOGS A growing research interest in the cell differentiation and immune modulator effects of vitamin D and analogs may result in their use in a variety of disorders [112e114] (also see chapters in Section IX “Analogs”). However, the development of hypercalciuria and hypercalcemia
may limit the use of the naturally occurring vitamin D metabolites, as well as synthetic analogs [115,116]. While some vitamin D analogs are reported to have little or no hypercalcemic action, hypercalcemia and hypercalciuria may appear at higher doses through the classic vitamin D actions on intestine, kidney, and bone [114,115]. LowCa diets have had only modest beneficial effects to limit hypercalciuria and hypercalcemia and could promote bone loss. It remains to be determined whether the newer vitamin D analogs with less calcemic activity will, in practice, cause less calciuria and a lower risk of kidney stone formation. Until such actions of the vitamin D analogs are known, standard approaches to minimize stone formation should be followed. These include (1) assuring sufficient fluid intake to maintain at least 1.5 to 2.0 liters urine output per day; and (2) if necessary, increasing urine citrate excretion to normal in those with low citrate [102]. While discontinuation or reduction in vitamin D analog treatment would reverse the hypercalciuria, the beneficial effect of vitamin D analog therapy to control tumor growth may well be greater than the relative importance of reversing hypercalciuria and preventing kidney stones. The addition of a thiazide may avoid or minimize hypercalciuria, but hypercalcemia may occur because of thiazide-induced Ca retention.
SUMMARY Idiopathic hypercalciuria (IH) affects 5 to 7% of adults and children and is the single most common cause of Ca oxalate kidney stone formation and also causes low bone mass. All of the metabolic features of IH can be reproduced by the administration of calcitriol to normal adults. There is also evidence of 1,25(OH)2D3 overproduction in some IH subjects. Excess 1,25(OH)2D3 action appears responsible for the intestinal Ca hyperabsorption, increased bone resorption, and decreased renal tubule Ca reabsorption. Serum 1,25(OH)2D3 is elevated in about 60% of patients. Patients with normal serum 1,25(OH)2D3 levels have comparable elevations in intestinal Ca absorption, and the possibility of elevated tissue vitamin D receptor found in GHS rats is suggested by one study in IH subjects which found elevated peripheral blood monocyte levels [82]. Further, GHS rats and IH patients share changes in Ca transport in intestine, bone, and kidney. The GHS model of human IH permits studies of the cellular and molecular mechanisms of hypercalciuria found in humans. In this model, increased levels of VDR protein in duodenum, kidney, and bone may explain the hyperabsorption of Ca, increased bone resorption, and decreased renal tubule Ca reabsorption even in the presence of normal circulating concentrations of 1,25D.
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C H A P T E R
72 Hypercalcemia Due to Vitamin D Toxicity Natalie E. Cusano, Susan Thys-Jacobs, John P. Bilezikian Columbia University College of Physicians and Surgeons, New York, NY, USA
INTRODUCTION Vitamin D toxicity is not a common cause of hypercalcemia. In the differential diagnosis of hypercalcemia, it is often buried amidst a long list of other more and less common causes (Table 72.1). Among the more common causes, primary hyperparathyroidism and hypercalcemia of malignancy are the principal etiologies. Together, primary hyperparathyroidism and hypercalcemia of malignancy constitute the overwhelming majority of causes of hypercalcemia. They are in fact so common that the practical issue in the diagnosis of a hypercalcemic individual is to distinguish between these two etiologies first and not to consider other etiologies until these two have been ruled out. Patients with primary hyperparathyroidism (PHPT) tend to be asymptomatic, whereas patients with hypercalcemia of malignancy tend to be ill. The diagnosis of primary hyperparathyroidism is established by a frankly elevated concentration of parathyroid hormone (PTH), an association that is made in over 80% of patients with PHPT. The remaining subjects with PHPT have levels of circulating PTH that are in the upper range of normal, a decidedly abnormal concentration in the face of hypercalcemia. In contrast, patients with hypercalcemia of malignancy, including those whose hypercalcemia is due to the elaboration of parathyroid hormone-related protein (PTHrP), show levels of PTH that are typically suppressed. If the PTH level is suppressed, the diagnosis of PHPT is ruled out. The diagnosis of malignancy, however, is not necessarily ruled in. Certainly, if a malignancy is detected that is classically associated with hypercalcemia, such as squamous cell carcinoma of the lung, the etiology becomes clear. However, the longer list of other causes of hypercalcemia is also associated, with rare exceptions, with reduced levels of PTH. This situation, namely, elevated serum calcium concentration with reduced or
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10072-1
undetectable levels of PTH and PTHrP, is seen in the various forms of vitamin D toxicity. If PHPT is ruled out, malignancy is not obviously apparent, and PTHrP is not elevated, the likelihood of vitamin D toxicity looms as a consideration in the differential diagnosis of hypercalcemia. In that long list of other causes, vitamin D toxicity now becomes an important diagnostic consideration (Table 72.1). This chapter reviews the various forms of vitamin D toxicity, mechanisms of hypercalcemia due to vitamin D toxicity, clinical manifestations, diagnosis, and management.
FORMS OF EXOGENOUS VITAMIN D TOXICITY Vitamin D toxicity can be life-threatening and associated with substantial morbidity, if not identified quickly. Hypervitaminosis D with hypercalcemia may be secondary to excessive intake of parent vitamin D, its metabolites 25-hydroxyvitamin D (25(OH)D), 1,25dihydroxyvitamin D (1,25(OH)2D), or vitamin D analogs; to increased production of 25(OH)D or 1,25(OH)2D from exogenous substrate; and even to topical applications of potent vitamin D analogs.
Vitamin D and 25-Hydroxyvitamin D Toxicity The most common etiology of vitamin D toxicity is inadvertent or improper oral use of pharmaceutical preparations. Excessive ingestion of vitamin D (usually greater than 10 000 IU daily; see below) can cause vitamin D intoxication that is recognized by markedly elevated levels of 25(OH)D (usually >150 ng/ml) in association with levels of 1,25(OH)2D that are only slightly elevated. Hyperphosphatemia typically accompanies the hypercalcemia [1e3]. The hyperphosphatemia can be a clue to the etiology of the hypercalcemia
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TABLE 72.1
Differential Diagnosis of Hypercalcemia
Primary hyperparathyroidism Sporadic (adenoma, hyperplasia, or carcinoma) Familial Isolated Cystic Multiple endocrine neoplasia type I or II Malignancy Parathyroid hormone-related protein Excess production of 1,25-dihydroxyvitamin D Other factors (cytokines, growth factors) Disorders of vitamin D Exogenous vitamin D toxicity e parent D compound, 25(OH)D, 1,25(OH)D, vitamin D analogs Endogenous production of 25-hydroxyvitamin D (may be seen in Williams syndrome) Endogenous production of 1,25-dihydroxyvitamin D Granulomatous diseases a. Sarcoidosis b. Tuberculosis c. Histoplasmosis d. Coccidioidomycosis e. Leprosy f. Others Lymphoma Non-parathyroid endocrine disorders Thyrotoxicosis Pheochromocytoma Acute adrenal insufficiency Vasoactive intestinal polypeptide hormone-producing tumor (VIPoma) Medications Thiazide diuretics Lithium Estrogens/antiestrogens, testosterone in breast cancer Milk-alkali syndrome Vitamin A toxicity Familial hypocalciuric hypercalcemia Immobilization Parenteral nutrition Aluminum excess Acute and chronic renal disease
as due to vitamin D toxicity since PTH and PTHrPrelated disorders usually are associated with reduced serum phosphate or frank hypophosphatemia. The usual setting of vitamin D toxicity is in its use as a therapy for the hypocalcemic disorders: hypoparathyroidism, pseudohypoparathyroidism, osteomalacia, or renal failure. Ingestion of excessive quantities of 25(OH)D, 1-alpha-hydroxyvitamin D, 1,25(OH)2D, dihydrotachysterol, or exuberant use of the topical calcipotriene (Dovonex) for psoriasis can also cause vitamin D intoxication [4] and are further discussed in the
succeeding sections. Health-conscious adults have been reported to ingest large doses of megavitamins from over-the-counter supplements, in amounts that may exceed 2 million IU of vitamin D daily [5]. Cancer patients, in particular, have been observed to consume excess nutritional supplements such as calcium, vitamin D, and shark cartilage [6]. Moreover, individuals have mistakenly ingested ergocalciferol 50 000 IU daily instead of weekly [6a,6b]. An over-the-counter supplement called SoladekÒ has been implicated in vitamin D toxicity in a published case report and our own personal experience. This supplement, readily available in the Dominican Republic and in urban areas such as Manhattan, contains over 500 000 IU of vitamin D3 and 120 000 IU of vitamin A per 5 mg vial. The package label for SoladekÒ lists a number of indications for its use, including “hypo and avitaminosis, rickets, growth, dentition, lactation, fractures, infections, convalescence, protection and regeneration of certain epithelium (bronchial, glandular, ocular, cutaneous), corticotherapy, aging, pregnancy.” In the case report by Leu et al. [7], a 60-year-old female with a medical history significant only for osteoarthritis presented with symptoms of hypercalcemia and a serum calcium of 15.2 ng/dl in the setting of recent SoladekÒ use. Her 25(OH)D was >150 ng/ml and PTH and PTH-RP levels were undetectable. CT of the chest, abdomen, and pelvis, skeletal survey, and bone marrow biopsy were all negative for malignancy. However, colonoscopy revealed an anal squamous cell carcinoma which was resected. The patient’s hypercalcemia resolved after discontinuation of SoladekÒ in addition to treatment with intravenous fluids, intermittent furosemide, and a single dose of pamidronate. In our own case series of nine patients, the majority of patients with hypercalcemia in the setting of SoladekÒ use had a second condition that could have contributed to the development of hypercalcemia. No patient had previously experienced hypercalcemia before ingesting SoladekÒ, however, indicating that the secondary condition was likely necessary, but not perhaps sufficient to produce hypercalcemia outside of excessive vitamin D supplementation from SoladekÒ. Hypercalcemia resolved in all patients after stopping the preparation [8]. It is commonly perceived that excessive sunlight exposure can be associated with vitamin D toxicity. However, studies have documented that full summerlong unprotected sun exposure cannot raise serum concentrations of 25(OH)D much more than 70e80 ng/l (nl: 30e100) [9,10]. These recent observations help to document the widely held belief that sun alone cannot cause vitamin D toxicity. In situations where there is excessive conversion of 25(OH)D to 1,25(OH)2D such as in sarcoidosis or other granulomatous diseases,
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however, sun exposure can lead to hypercalcemia. Natural foods, in general, other than fatty fishes, eggs, milk, and liver do not contain much vitamin D. Hypervitaminosis D has been associated with drinking milk when inadvertently fortified with massive concentrations of vitamin D. One investigation of eight patients manifesting symptoms of nausea, vomiting, weight loss, hyperirritability, or failure-to-thrive revealed markedly elevated mean concentrations of 25(OH)D of 293 174 ng/ml [3]. Analysis of the milk production facility at the local dairy revealed excessive vitamin D fortification of milk with up to 245 840 IU per liter (232 565 IU of vitamin D3 per quart). Usual fortification of milk in the USA and Canada is 400 IU per quart. Milk is not fortified with vitamin D in most other parts of the world. Generally, milk is the only dairy product that is fortified with vitamin D in the USA. Some yogurts in the USA, however, are now fortified with vitamin D. In addition to milk, vitamin D fortification of natural foods includes certain breakfast cereals, pasta, baked goods, fats, and orange juice [11]. Since the amount of vitamin D fortification is very modest, these foods are not generally suspected as a cause, bearing in mind of course the rare but important example of inadvertent toxic amounts of vitamin D fortification in milk. Of note, in addition, industrial contamination of table sugar with vitamin D3 and consequent severe vitamin D toxicity (25(OH)D 623 ng/ml) has been reported [12]. Vitamin D2 and vitamin D3, although used interchangeably in the treatment of metabolic bone diseases, may differ in toxic potential at higher doses. Prior research has led to the established view that D3 is more potent and effective than D2. As reported by Armas et al. [13] the potency of vitamin D3 is three times that of D2. These investigators found that D2 has a shorter duration of action and that it accelerates the metabolism of D3 in human subjects. However, controversy exists in the literature and more recently Holick et al. [14] argue that both D2 and D3 are bioequivalent. The current, officially recommended dietary allowance (RDA) for vitamin D is 400 IU for infants 0e12 months of age and 600 IU subsequently throughout life. Many experts and authoritative bodies have been calling for increases in these recommended amounts [15e17]. These guidelines are controversial and it is uncertain as to how the recommendations will be adopted by the scientific community [17a,17b,17c]. Although this chapter concerns itself with vitamin D toxicity, the reason for the trend to increase recommendations for vitamin D intake is the large numbers of free-living adults who are being shown worldwide to have vitamin D deficiency (see Chapter 52) [18,19]. It thus remains to be seen whether a future increase in
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the RDA for vitamin D will enhance the potential for vitamin D toxicity. While primary hyperparathyroidism in the developed world has become a disorder of mild hypercalcemia with few, if any, symptoms, the clinical picture of PHPT in the developing world, in which vitamin D deficiency is more common, remains a disease with classic signs and symptoms, including osteitis fibrosa as well as other skeletal and renal complications. Vitamin D deficiency in the setting of PHPT is associated with a more severe phenotype [20], and many studies show larger adenoma size, higher serum parathyroid hormone and alkaline phosphatase levels, lower bone mineral density, and higher rates of fracture [21e25]. Moreover, patients with vitamin D deficiency are at greater risk to develop hypocalcemia due to “hungry bone” syndrome after parathyroidectomy. Patients with PHPT and comorbid vitamin D deficiency present a therapeutic dilemma due to the potential risk of worsening hypercalcemia with vitamin D supplementation. Grey et al. have provided recent data that address this challenge [26]. They evaluated 21 patients with mild PHPT (serum calcium 10.8 0.5) and coexistent vitamin D deficiency (25(OH)D <20 ng/ml) with ergocalciferol 50 000 IU weekly for 4 weeks then 50 000 IU monthly for the remainder of a year. With a rise in 25(OH)D levels, PTH levels fell by 25%. The reduction in PTH levels by approximately 25% suggests that this was the component of the elevation due not to the endogenously overactive parathyroid gland but rather to the secondary hyperparathyroidism of the vitamin D deficiency state. Of note, in one patient the serum calcium increased from 10.5 to 11.9 mg/dl and in another patient the 24-hour urinary calcium excretion increased to over 400 mg/d. In the remaining patients, there were no substantial changes in serum calcium or urinary calcium excretion. Tucci [27] subsequently showed no overall increase in serum calcium with doses of ergocalciferol 50 000 IU weekly for 8 weeks. Grubbs et al. [28] also showed no change in serum calcium with a dosing regimen of 50 000 IU weekly or twice weekly for a mean duration of 4 weeks. However, three of 56 patients, and six of 112 patients, respectively, did show increases in serum calcium during the follow-up period. Tucci found no increase in urinary calcium excretion. Isidro et al. [29] studied 27 patients with PHPT administered calcifediol (25(OH)D3) supplementation of up to 960 IU daily and found no increase in serum calcium levels. However, up to one-third of patients became hypercalciuric. These studies, in the aggregate, make two points. First, vitamin D supplementation in PHPT seems to be well tolerated and does not lead, in general, to worsening hypercalcemia or hypercalciuria. Second, the studies also highlight the importance of careful monitoring.
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The smallest dose of parent vitamin D (cholecalciferol or ergocalciferol) in healthy adults that can produce toxicity and hypercalcemia is not known, but is clearly much higher than the RDA [30]. The current tolerable upper intake level (UL) recommended by the Food and Nutrition Board is 1000 IU daily for infants 0e6 months of age, 1500 IU daily for infants 6e12 months of age, 2500 IU daily for children 1e3 years of age, 3000 IU daily for children 4e8 years of age, and 4000 IU daily subsequently throughout life. Again, controversy exists regarding these guidelines [17a,17b,17c]. Two small well-conducted clinical trials by Heaney et al. [31] and Barger-Lux et al. [32] showed that vitamin D3 10 000 IU daily for 8 and 20 weeks, respectively, did not cause an increase in serum calcium or any adverse effects in the combined cohort of 26 healthy men. In these subjects, mean 25(OH)D levels rose to 85 ng/ml (n ¼ 10) and 88 ng/ml (n ¼ 16) in the two studies, respectively. A review by Hathcock et al. [33] supports the selection of 10 000 IU daily as the UL based on these studies. Other studies of much shorter duration or concurrent treatment with prednisone and/or sodium fluoride have also shown that higher doses of vitamin D (up to 100 000 IU per day) were well tolerated. However, these vitamin D studies were not considered in their risk assessment due to short study lengths and potential confounders. Individuals manifest wide variations both in their response to hypercalcemic doses of vitamin D and in the duration of the effect. This variation in individual responsiveness might reflect differences in intestinal absorption and vitamin D metabolism, in the concentration of free vitamin D metabolites, in the rate of degradation of the metabolites and conversion to inactive metabolites, and in the capacity of storage sites for 25(OH)D [34]. Factors that enhance susceptibility to vitamin D toxicity and hypercalcemia include increased dietary calcium intake, reduced renal function, coadministration of vitamin A, and granulomatous disorders such as sarcoidosis that render subjects more sensitive to vitamin D (see Chapter 45) [2]. Hypercalciuria in hypervitaminosis D usually presents earlier than hypercalcemia, but it is easily missed for the obvious reason that urinary calcium is not routinely measured.
1,25-Dihydroxyvitamin D Toxicity The greater potency of 1,25(OH)2D3 and its direct actions on target tissues, in addition to its ability to inhibit PTH synthesis and secretion, has made 1,25 (OH)2D3 and its analogs useful agents in patients with renal osteodystrophy and secondary hyperparathyroidism [35] (see Chapters 70 and 81). 1,25(OH)2D3 has also been found to inhibit the growth of human cancer
cells in vitro [36]. As 1,25(OH)2D3 is increasingly recognized for its anti-proliferative, prodifferentiating, and immunomodulatory actions, its potential therapeutic use is expanding [37]. Mechanisms associated with the hypercalcemia due to 1,25(OH)2D3 are increased intestinal absorption of calcium and potentiation of osteoclastic activity in bone. Dosages of 1,25(OH)2D3 considerably above 0.75 mg/day have been associated with toxicity, whereas dosages at or below 0.5 mg/day rarely result in toxicity. One investigation showed that over 90% of patients on doses of 1,25(OH)2D3 between 1.0 and 2.0 mg/day became hypercalcemic, and all had hypercalciuria when calcium intake was set at 1000 mg per day [38]. Accelerated deterioration of renal function was recorded in a number of reports in patients with renal insufficiency receiving 1,25(OH)2D3 therapy [39]. Compared to oral therapy, intravenous administration of 1,25(OH)2D3 to renal dialysis patients induces hypercalcemia less frequently, with a smaller increment in the serum calcium concentration and a more effective reduction of PTH levels [40]. Other studies, however, suggest that intermittent oral pulse administration of 1,25(OH)2D3 may be effective, though not as effective as intravenous 1,25(OH)2D3, in suppressing PTH in uremic patients with secondary hyperparathyroidism [41e43] (see Chapters 70 and 81).
Toxicity Due to Synthetic Analogs In one investigation, oral pulse therapy with 1ahydroxyvitamin D3 (1a-OHD3) resulted in a rapid control of secondary hyperparathyroidism without causing hypercalcemia or hyperphosphatemia [44]. However, 1a-OHD3 may harbor potential calcemic effects similar to 1,25(OH)2D3 in the treatment of renal osteodystrophy. Crocker et al. [45] investigated the comparative toxicity of vitamin D, 1a-OHD3, and 1,25(OH)2D3 in weanling male mice at three different doses over a 4-week period. 1a-OHD3 appeared to be more toxic in the high-dose group only, with significantly higher serum calcium levels, higher urinary calcium excretion, and severe nephrocalcinosis. 1aOHD3 has been described as less potent than 1,25 (OH)2D3 at low doses but equipotent at doses greater than 2.0 mg/day. At the higher doses, there is a delayed onset of action and a prolonged half-life, suggesting a potential for cumulative toxicity in renal insufficiency [46,47]. The potential for hypercalcemia, hypercalciuria, and soft tissue calcifications limits the clinical usefulness of 1a-OHD3. Mortensen and colleagues compared the toxicity of both 1a-OHD3 and 1,25(OH)2D3 in rats fed standard or low-calcium diets. High doses of either
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compound resulted in severe hypercalcemia, with retarded growth, nephrosis, and structural bone changes in the rats fed the standard diet. On the low-calcium diet, however, slight hypercalcemia occurred, but without growth retardation or bone changes. There was minimal effect on the kidney. Calcium restriction again proved effective in protecting the animals against the toxic effects of the vitamin D analogs. Animals fed the low-calcium diet tolerated 1a-OHD3 at dose levels up to 10 times higher than rats on the standard diets [48]. In human subjects, 1a-OHD3 can be associated with hypercalcemia at doses above 1.0 mg/day, but amounts between 0.5 and 1.0 mg/day appear to be safe. Because of the relatively narrow therapeutic window of vitamin D3 1-hydroxylated compounds, a synthetic analog of vitamin D2, 1a-OHD2 (doxercalciferol) was developed with the concept that the window of therapeutic efficacy to toxicity would be wider. Doses of doxercalciferol ranging from 1.0 to 5.0 mg/day were administered to 15 female postmenopausal osteopenic subjects. There was no evidence of vitamin D toxicity manifesting as either hypercalciuria or hypercalcemia, whereas significant therapeutic effects on osteoblastic activity were demonstrated [49]. Similar to the 1aOHD3, doxercalciferol requires obligatory hepatic 25hydroxylation for activation. However, doxercalciferol is able to activate its catabolic pathway via hepatic 24hydroxylation with a lower potential for toxicity [50]. Because of our emerging greater understanding of the non-classic target tissue effects of vitamin D in the modulation of hormones and cytokines, and in the regulation of cellular differentiation and proliferation, newer clinical uses have been developed (see Section XII of this book). The clinical applications of these newer properties of vitamin D, however, have also been tempered by the potential for complications, such as hypercalcemia and hypercalciuria. These concerns have prompted the development of additional analogs to better distinguish calcemic from antiproliferative effects [51] discussed in Section IX of this book. Depending on the chemical modification of the basic structure of vitamin D, some analogs do demonstrate reduced calcemic activity, but others have been developed that exhibit increased calcemic activity due to enhanced intestinal calcium absorption and bone mineral mobilization. Fluorination of C-24, C-26, or C-27 apparently results in markedly increased calcemic activity resulting from reduced enzymatic degradation of the side chain. Calcemic potency of 1,25(OH)2D3 and its analogs can be also enhanced at least two- to five-fold by epimerization at the C-20 site [52]. The vitamin D analogs in use for secondary hyperparathyroidism in the USA include doxercalciferol and paricalcitol (19-nor-1,25-dihydroxyvitamin D2). Alfacalcidol, falecalcitriol, and 22-oxacalcitriol are in use outside the USA. Each analog retains suppressive action on PTH
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and parathyroid gland growth, but has less calcemic and phosphatemic activity than calcitriol. Paricalcitol and doxercalciferol appear to be equally effective in suppressing PTH; however, paricalcitol has been found to be less hypercalcemic [53]. Overall, the effect of vitamin D analogs to minimize the calciumephosphate product might reduce vascular calcification and mortality in the renal failure population; however, observational and animal data [54,55] have yet to be confirmed by randomized control trial [56]. Of additional potential importance may be the decreased likelihood of low bone turnover, or adynamic bone disease, with the use of these agents [57e59]. The mechanism for the differential actions of vitamin D analogs is not completely understood. Oxacalcitriol, for example, has a low affinity for vitaminD-binding protein, so more of the drug circulates in the free form, allowing it to be more rapidly metabolized than calcitriol [60]. This leads to a shorter half-life, which could explain the small and transient stimulation of intestinal calcium absorption. It does not, however, seem to account for the prolonged inhibition of PTH release. The use of analogs to treat chronic kidney failure is discussed in Chapter 81). Other vitamin D analogs, such as topical calcipotriol, have proved very effective in the treatment of psoriasis (see Chapter 97). Because of its low absorption rate and rapid degradation, calcipotriol is believed to have negligible effects on systemic calcium homeostasis when administered topically. However, isolated cases of hypercalcemia and hypercalciuria have been reported [61], even in patients using prescribed doses [62]. In one investigation, Bourke and colleagues noted suppression of serum PTH concentrations in all patients within 2 weeks of treatment with topical calcipotriol. Mean serum and urine calcium levels increased during treatment and fell following withdrawal [63]. The authors concluded that although this particular synthetic analog alters serum and urinary calcium with a dose-dependent effect on systemic calcium homeostasis, it is well tolerated and effective for mild to moderate chronic plaque psoriasis. However, it is potentially hazardous in extensive, unstable, exfoliative disease where increased absorption by inflamed and damaged skin can cause hypercalcemia [64].
FORMS OF ENDOGENOUS VITAMIN D TOXICITY Endogenous Production of 25-Hydroxyvitamin D Endogenous dysregulation of vitamin D metabolites may be seen in Williams-Beuren syndrome (WBS), an idiopathic infantile form of hypercalcemia. WBS is also associated with elfin facies, late psychomotor
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development, selective mental deficiency, and supravalvular aortic stenosis [65]. Hypercalcemia occurs in up to 15% of infants and children with WBS. The hypercalcemia has been reported to range widely from 12 to 19 mg/dl and usually subsides by 4 years of age; however, it may recur during puberty. Early reports suggested an exaggerated production of 25(OH)D with small doses of vitamin D as a possible cause of the hypervitaminosis D [66]. The underlying source of the hypercalcemia remains unknown, however. More recent cases have shown normal 25(OH)D levels which may indicate an etiology unrelated to abnormal vitamin D metabolism [67].
Production of 1,25-Dihydroxyvitamin D Granulomatous Diseases In contrast to the megadosages of vitamin D that are usually required to produce vitamin D toxicity, patients with granulomatous diseases can develop hypercalcemia rather easily without excessive intake of exogenous vitamin D. They are said to be hypersensitive to vitamin D. The etiology of the vitamin D toxicity in this syndrome is due to poorly regulated extrarenal synthesis of 1,25(OH)2D by the granulomatous tissue itself (as described in detail in Chapter 45). In contrast to the various presentations of vitamin D toxicity described earlier, the responsible metabolite in granulomatous disease is quite different. In the case of vitamin D toxicity due to overdosage of vitamin D or 25(OH)D, 25(OH)D is the active metabolite; renal production of 1,25(OH)2D in this setting is highly regulated and not excessively high due to suppression of PTH by the slightest rise in serum calcium and feedback inhibition of 1a-hydroxylase by the rising levels of 1,25(OH)2D. In granulomatous tissue, however, 1,25(OH)2D formation is not subject to control by any recognized regulators, such as PTH, phosphorus, or calcium. Thus, this syndrome is due to ectopic production of 1,25(OH)2D by the granulomatous tissue itself. The mechanisms by which hypercalcemia occurs, however, are similar to all other vitamin D toxic states, namely, increased intestinal calcium absorption and enhanced osteoclastic bone resorption [68,69]. Many studies have led to greater understanding of the pathophysiology and immunological features associated with this syndrome. SARCOIDOSIS
Abnormalities in calcium metabolism have long been noted in patients with sarcoidosis [70]. Sarcoidosis is also the most common granulomatous disease associated with hypercalcemia. Approximately 10% of patients with sarcoidosis may develop hypercalcemia [71]; however, severe hypercalcemia is uncommon [72].
Approximately 50% of patients will experience hypercalciuria at some time during the course of the disease, with hypercalciuria invariably present when patients develop hypercalcemia [58]. In the 1950s, studies had already revealed similarities between hypercalcemia of sarcoidosis and the hypercalcemia of vitamin D toxicity, namely, increased intestinal absorption of calcium, hypercalciuria, and therapeutic efficacy of glucocorticoids [73,74]. The major distinguishing feature was in the amount of vitamin D associated with the hypercalcemia and/or hypercalciuria. Seasonal variation of the serum calcium level in sarcoidosis was correlated with availability of sunlight as a source of vitamin D [75]. In the late 1970s, two independent groups showed that the vitamin-D-like principle that appeared to be responsible in sarcoidosis was, in fact, the active metabolite of vitamin D, 1,25(OH)2D3 [69,76]. Ectopic production of 1,25(OH)2D3 was confirmed by demonstrating high circulating concentrations of 1,25(OH)2D3 in anephric patients with sarcoidosis on hemodialysis during hypercalcemic episodes [77,78]. This observation showed unequivocally that the kidney, usually the predominant source of 1,25(OH)2D3 in nonpregnant individuals, could not be the source of 1,25 (OH)2D3 in these patients. The serum calcium and 1,25 (OH)2D3 levels were positively correlated with indices of disease activity [79e81], namely, the extent of granuloma formation and the angiotensin-converting enzyme level. It was subsequently shown that the granulomatous tissue was, in fact, the site of 1,25(OH)2D3 production. The la-hydroxylase enzyme responsible for formation of 1,25(OH)2D3 was present in lymph node homogenates [82]. Moreover, pulmonary alveolar macrophages [83] could be shown to catalyze the formation of a 3H-labeled 25(OH)D3 metabolite. This metabolite was definitively identified as 1,25(OH)2D3 by high-performance liquid chromatography (HPLC), by the chick intestinal receptor assay for 1,25(OH)2D3, by UV spectroscopy, and by mass spectrometry [84]. The production of mRNA for 1a-hydroxylase is markedly increased in alveolar macrophages isolated from hypercalcemic patients with sarcoid [85]. Importantly, control of the macrophage 1a-hydroxylase enzyme differs from that of the renal 1a-hydroxylase. The renal 1a-hydroxylase is regulated at the level of transcription by calciotropic hormones, especially PTH, and is exquisitely autoregulated by 1,25(OH)2D3 itself [86]. In contrast, the macrophage 1a-hydroxylase mRNA expression is potently stimulated by inflammatory agents, such as g-interferon [87], and shows no feedback control in response to 1,25(OH)2D3 [88]. Communication between signaling pathways of g-interferon and the vitamin D receptor has been reported [89]. These mechanisms account for the uncontrolled synthesis of 1,25(OH)2D3 and the characteristic finding of increased
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sensitivity to vitamin D in these patients [90,91]. Another property of the macrophage 1a-hydroxylase enzyme is that it is inhibited in a dose-dependent fashion by dexamethasone and chloroquine that do not influence the renal 1a-hydroxylase enzyme that catalyzes synthesis of 1,25(OH)2D3 [92]. These in vitro observations have direct clinical relevance and suggest possible treatments of hypercalcemia due to sarcoidosis. There are several mechanisms by which calcium metabolism is disturbed in sarcoidosis [93]. First, 1,25(OH)2D3 causes hypercalcemia, in part, by stimulating intestinal calcium absorption. A low-calcium diet [94,95], alone or in association with cellulose phosphate [96], was found to normalize the calcium level in some patients with sarcoidosis. Second, 1,25(OH)2D3 directly stimulates osteoclastic-mediated bone resorption; skeletal granulomas are not required for this effect [97e99]. The increased flux of calcium into the extracellular space by these gastrointestinal and skeletal mechanisms, aided by suppression of PTH [76e78], leads to hypercalciuria. Chronic hypercalciuria favors nephrocalcinosis and renal stone formation [100]. When the kidneys are unable to excrete the calcium presented to them, because of either declining renal function, enhanced bone resorption, a sudden influx of dietary calcium, dehydration, or any combination of these events, hypercalcemia ensues [101]. Granulomatous production of PTHrP may also play a role in abnormal calcium metabolism, where TNFa and interleukin-6, produced by macrophages, increase PTHrP gene expression. PTHrP was reported in one series to be present in 85% of biopsies of granulomatous tissue from patients with sarcoidosis [102]. TUBERCULOSIS
Longitudinal studies from the USA [103] and India [104] suggested that 16 to 28% of patients with tuberculosis develop hypercalcemia. However, in these early studies, vitamin D supplements were employed, increasing the risk and severity of hypercalcemia. A similar study from Greece [105] reported a figure as high as 48% when serum calcium was corrected to a normal albumin level. Other studies from the UK [106], Belgium [107], Hong Kong [108], and Malaysia [109] have shown a much lower prevalence of hypercalcemia, in the range of 0 to 2.3%. It is likely that hypercalcemia is not as common in tuberculosis as was previously thought [110]. This discrepancy might be attributable to regional differences in calcium and vitamin D intake, which can unmask hypercalcemia [111], along with increased sun exposure. Reports of high circulating levels of 1,25(OH)2D3 in three anephric patients with tuberculosis support an extrarenal source of the active vitamin D metabolite
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[112,113]. Positive correlation of the albumin-adjusted calcium level with the radiographic extent of the disease has been shown [108]. Hypercalcemia in tuberculosis may occur weeks to months after starting antituberculosis chemotherapy [103,104]. Thus, the hypercalcemia is not related to the presence of viable acid-fast bacilli, but rather to the granulomatous process and associated reactions. As with sarcoidosis, hypercalcemia in tuberculosis can be controlled by administration of glucocorticoids [114]. In patients with tuberculous pleuritis, the mean free 1,25(OH)2D3 concentration in pleural fluid was selectively concentrated by 5.3-fold over that in serum [115]. Positive correlation between the concentrations of substrate (25(OH)D3) and product (1,25(OH)2D3) in pleural fluid supported the idea that 1,25(OH)2D3 was produced locally by activated inflammatory cells in or adjacent to the pleural space. The pleural fluid was found to have high concentrations of g-interferon, a cytokine known to stimulate activated macrophages in vitro to synthesize 1,25(OH)2D3 [116]. Cells obtained from bronchoalveolar lavage in patients with tuberculosis were also found to synthesize 1,25(OH)2D3 in vitro. An important source of the active vitamin D metabolite appears to be the CD8þ T lymphocytes at the granulomatous sites [117]. If one wonders etiologically about the production of 1,25(OH)2D3 under these circumstances, the immunomodulatory functions of 1,25(OH)2D3 acting as a beneficial local paracrine factor could be pertinent (see Chapter 95). In fact, increased levels of 1,25(OH)2D3 have been found to result in the production of cathelicidin, a peptide with antimicrobial properties against Mycobacterium tuberculosis, by macrophages [118]. Viewed in this context, hypercalcemia occurs when 1,25(OH)2D3 is produced in such quantities that it gains entry into the circulation. Hypercalcemia in tuberculosis is usually mild and asymptomatic. Besides glucocorticoids, ketoconazole administration has been associated with a rapid decline in 1,25(OH)2D3 and normalization of serum calcium levels, through inhibition of 1a-hydroxylase [119]. Long-term antituberculosis therapy with isoniazid and rifampin can also be effective in treating the hypercalcemia by controlling the disease. OTHER GRANULOMATOUS DISEASES
Hypercalcemia in other granulomatous disorders is relatively rare and the subject of case reports, described in infectious etiologies including leprosy [120], coccidioidomycosis [121], histoplasmosis [122], candidiasis [123], cat-scratch disease [124], Pneumocystis jirovecii pneumonia [125], Mycobacterium avium complex [126], and paracoccidioidomycosis [127]. Hypercalcemia was also reported as a systemic reaction to Calmette-Gue´rin bacillus (BCG) therapy for bladder cancer complicated by disseminated granulomatosis [128]. Non-infectious
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associations, aside from sarcoidosis, have been reported with Wegener’s granulomatosis [129], Langerhans’ cell granulomatosis [130], Crohn’s disease [131], infantile fat necrosis [132], giant cell polymyositis [133], in addition to foreign body reactions such as berylliosis [134], silicone-induced granuloma [135], paraffin-induced granulomatosis [136], and talc granulomatosis [137]. The mechanism of increased production of the active vitamin D metabolite is believed to be shared by all of these granulomatous disorders. Spontaneous idiopathic excess production of calcitriol in the absence of granulomatous disease has also been reported in patients with elevated angiotensin converting enzyme levels [138], presumably with increased calcitriol production by macrophages. A possible role for 1,25(OH)2D3 in these granulomatous disorders as noted above includes immunomodulatory features, which is discussed in Chapter 45. Interesting, an experimental analog of cAMP (8-Cl-cAMP) given to patients with advanced solid malignancies was shown to cause hypercalcemia associated with elevated levels of 1,25-dihydroxyvitamin D not clearly associated with a granulomatous reaction [139]. Lymphoma Hypercalcemia has been reported to occur in up to 5% [140] and 15% [141] of patients with Hodgkin’s disease and non-Hodgkin’s lymphoma (NHL), respectively. Up to 80% of patients with human T-cell leukemia virus type 1 (HTLV-1)-associated adult T-cell lymphoma/ leukemia (ATLL) may develop hypercalcemia [142]. As is the case with other malignancies, hypercalcemia is a poor prognostic feature in lymphoma [143], adding substantially to morbidity and mortality. The humoral mediators of hypercalcemia in lymphoma are multiple and heterogeneous. However, evidence has shown 1,25(OH)2D3 to be an important factor in many cases. Hypercalcemia development in Hodgkin’s disease is most consistently associated with 1,25(OH)2D3. Since the first report of hypercalcemia complicating Hodgkin’s disease in 1956 [144], more than 60 cases have been described. In a retrospective review of the literature [145], 84% of patients had a peak serum calcium above 12 mg/dl, 74% of the patients had Ann Arbor stage III or IV disease, and 68% were symptomatic with night sweats, fever, and weight loss. Only three of 23 patients had radiological evidence of lytic bone lesions. In 17 hypercalcemic patients, all but one patient had an elevated 1,25(OH)2D3 level. There is no evidence to implicate parathyroid hormone-related peptide (PTHrP) as a mediator of hypercalcemia in Hodgkin’s disease. Two patients with Hodgkin’s disease [146,147] were reported to have intermittent hypercalcemia during two consecutive summers or on vitamin D challenge. There was a close association between
hypercalcemia and the abnormally raised 1,25(OH)2D3 level, but serum 25(OH)D3 was within the normal range. These observations support the idea that the mechanism of the hypercalcemia in Hodgkin’s disease is similar to that of the granulomatous diseases, namely, production by the lymphomatous tissue of 1,25(OH)2D3. A number of cases of 1,25(OH)2D3-induced hypercalcemia in non-Hodgkin’s lymphoma have been described [145]. Most patients had bulky or advanced-stage disease, but no clinically or radiographically evident bone lesions. In one case, the 1,25(OH)2D3-mediated hypercalcemia was associated with transformation from a chronic lymphocytic leukemia to an aggressive high-grade non-Hodgkin’s lymphoma [148]. Seymour et al. [149] prospectively studied patients with nonHodgkin’s lymphoma and found that 12 of 22 patients (55%) with hypercalcemia had elevated serum calcitriol levels above their reference range, defined by a control group of patients with hypercalcemia mediated by osteolytic metastases in multiple myeloma. They also found that among 25 normocalcemic patients, 71% were hypercalciuric and 18% had elevated serum 1,25(OH)2D3 levels. Data supporting extrarenal synthesis of 1,25(OH)2D3 include: the presence of severe renal failure in a number of instances [150,151]; the demonstration of in vitro conversion of 25(OH)D3 to 1,25(OH)2D3 by excised lymph node homogenates [152]; the prompt decline of 1,25(OH)2D3 levels to normal after excision of an isolated splenic lymphoma [153]; and a primary ovarian lymphoma [154]; and sensitivity to glucocorticoid suppression [151]. Five of ten patients with either HIV or non-HIV-associated nonHodgkin’s lymphoma and hypercalcemia had frankly elevated serum 1,25(OH)2D3 concentrations [155]. Other malignant lymphoproliferative diseases associated with 1,25(OH)2D3-mediated hypercalcemia include lymphomatoid granulomatosis [156], dysgerminoma [157], and an inflammatory myofibroblastic tumor [158]. The precise cell type responsible for the extrarenal synthesis of 1,25(OH)2D3 in lymphoma remains to be established. There are two possibilities. One is the tumor-infiltrating reactive macrophage, recognized by a “starry-sky” appearance [159] in intermediate and high-grade lymphomas, in which hypercalcemia is also most common. Alternatively, it may be that a particular clone of the malignant lymphoma cell synthesizes 1,25(OH)2D3 [160]. Immunohistochemical analysis of the enzyme 1a-hydroxylase in a B-cell lymphoma associated with hypercalcemia and raised circulating levels of 1,25(OH)2D3 suggests that the tumor itself is not a source of the steroid hormone. Rather, macrophages adjacent to the tumor are likely to be the major site of ectopic 1,25(OH)2D3 synthesis [161]. 1,25(OH)2D3 is only one cause of hypercalcemia in lymphoma. About half of the patients with non-
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Hodgkin’s lymphoma and hypercalcemia have suppressed 1,25(OH)2D3 levels. Additional circulating or local osteolytic factors are likely to be involved. Two of 22 patients in the study by Seymour et al. had elevated PTHrP levels. A few other cases of hypercalcemia in non-Hodgkin’s lymphoma associated with elevated levels of PTHrP have been reported [162e164]. Cytokines such as interleukin-1, tumor necrosis factora (TNFa), and transforming growth factor (TGFb) may also play a role in the pathogenesis of lymphomaassociated hypercalcemia. Although HTLV-1-transformed lymphocytes were shown in vitro to possess the capacity to convert 25(OH)D3 to 1,25(OH)2D3 [165], most studies have shown reduced 1,25(OH)2D3 levels in hypercalcemia associated with HTLV-1-related adult T-cell leukemia/ lymphoma [166,167]. PTHrP is most strongly implicated as the major mediator in this syndrome [168]. PTHrP messenger RNA has been demonstrated in HTLV-1infected T cells [169] and tumor cells from adult T-cell lymphoma/leukemia (ATLL) patients with hypercalcemia [170]. Nevertheless, there are two welldocumented instances of elevated 1,25(OH)2D3 levels in ATLL [142,150]. In the first case, a PTHrP level was not available. In the second case, concomitant elevation of 1,25(OH)2D3 and PTHrP was shown, suggesting the possibility of increased renal 1a-hydroxylase activity secondary to PTHrP. Alternatively, the tissue could be the site of both PTHrP and 1,25(OH)2D3 formation. Other malignancies associated with 1,25(OH)2D3-mediated hypercalcemia include seminoma [171], leiomyoblastoma [172], and squamous cell bronchogenic carcinoma [173]. Most patients with hypercalcemia due to classic squamous cell carcinoma have elevated PTHrP levels and either suppressed or normal 1,25(OH)2D3 levels [174].
MECHANISMS OF VITAMIN D TOXICITY General Mechanisms Vitamin D toxicity may occur in patients due to any one of the three forms of vitamin D, namely, the vitamin D parent compound, 25(OH)D, or 1,25(OH)2D. Multiple factors may influence susceptibility to vitamin D toxicity and include the concentration of the vitamin D metabolite itself, vitamin D receptor (VDR) number, activity of 1a-hydroxylase, the metabolic degradation pathway, and the capacity of the vitamin-D-binding protein (DBP). Vitamin D2 or D3 toxicity is more difficult to manage than toxicity due to its metabolites 25(OH)D or 1,25(OH)2D. In part, this is due to the extensive lipid solubility of the parent compound in liver, muscle, and fat tissues and corresponding large storage capacity.
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As a result, the half-life of vitamin D ranges from 20 days to months. In contrast, the biological half-life of the less lipophilic compound 25(OH)D is shorter, approximately 15 days [175]. The biological half-life of the least lipophilic compound 1,25(OH)2D, is much shorter, approximately 15 hours [176]. In general, duration of toxicity is related to the half-life of the vitamin D compound. Thus, the hypercalcemia of parent vitamin D overdose can theoretically last for as long as 18 months, long after dosing is discontinued, because of its slow release from fat deposits. Overdosage of 25(OH)D can persist for weeks also, but excessive 1,25(OH)2D toxicity is more rapidly reversed because 1,25(OH)2D is not stored in appreciable amounts in the body [80]. The toxicity of either parent vitamin D or 25(OH)D is due to 25(OH)D. In an investigation examining the concentrations of vitamin D3 and its metabolites in the rat as influenced by various intakes of vitamin D3 or 25(OH)D, Shepard and DeLuca found that intakes of vitamin D3, ranging from 1 to 10 000 IU daily (0.65 to 6500 nmol/day), resulted in excessive concentrations of vitamin D3 and 25(OH)D3 but not in 1,25(OH)2D3 [177]. Similarly, increased dosages of 25(OH)D3 ranging from 0.46 to 4600 nmol/day resulted in excessive amounts of 25(OH)D3, but not of vitamin D3 or 1,25(OH)2D3. Unlike 1,25(OH)2D whose production is tightly regulated in the kidney, the production of 25(OH)D is not tightly controlled by the liver. The high capacity for 25-hydroxylation of vitamin D in the liver as well as loose regulation at this site allows for massive amounts of 25(OH)D to be generated from large amounts of vitamin D. Thus, excessive concentrations of 25(OH)D are typically measured in vitamin D toxicity. Hypercalcemia appears to result only when 25(OH)D concentrations are consistently above 150 ng/ ml (375 nmol/l) in normal individuals [2,178]. As would be expected, PTH levels are suppressed in this form of hypercalcemia. In the setting of toxicity due to overadministration of 1,25(OH)2D3, the active metabolite itself is responsible for the hypercalcemia [179].
Role of Vitamin D Receptor (VDR) in Vitamin D Toxicity Various investigations have helped to shed light on the interrelationship among vitamin D metabolites, the VDR, and PTH in vitamin D toxicity. The biologically active form of vitamin D, 1,25(OH)2D, as is typical of other steroid hormones, binds to a specific intracellular receptor protein (VDR) within its target tissues. The hormoneeVDR complex then triggers subsequent transcriptional events by binding to DNA elements. Regulation of cellular VDR numbers is believed to be an important mechanism by which cellular responsiveness to 1,25(OH)2D is modulated, because the
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biological activity of 1,25(OH)2D is proportional both to tissue VDR number and concentration of 1,25(OH)2D. Increased VDR concentrations imply enhanced tissue responsiveness to 1,25-dihydroxyvitamin D, whereas decreased receptor numbers indicate reduced tissue responsiveness. Several investigations have suggested that exogenous 1,25(OH)2D3 can lead to homologous upregulation of VDR in vitro and in vivo, in contrast to endogenous production of 1,25(OH)2D3. In vitro and in vivo administration of 1,25(OH)2D3 to rats has been shown to increase VDR content. In vitro exposure of human skin fibroblasts and osteosarcoma cells to 1,25(OH)2D3 has been shown to result in a three- to five-fold increase in VDR number [180]. Similarly, in vivo studies have shown increased VDR with exogenous administration of 1,25(OH)2D3. Costa and Feldman administered 1500 pmol/kg of 1,25(OH)2D3 daily to rats and found a 30% increase in intestinal VDR and a three-fold increase in renal VDR concentration [181]. Reinhart et al. infused rats with 250 pmol/kg of 1,25(OH)2D3 daily for 6 days and noted a 22% increase in VDR levels in the intestine and a 37% increase in bone [182]. Goff and colleagues infused 36 ng of 1,25(OH)2D3 to rats over 7 days and found a 1.5-fold increase in duodenal VDR content and a three-fold increase in renal VDR content [183]. Goff et al. [183] also demonstrated that endogenously produced 1,25(OH)2D3 has a different effect than exogenous administration of 1,25(OH)2D3 on tissue VDR content. Rats fed a calcium-restricted diet resulting in “nutritional” hyperparathyroidism achieved a similar increase in endogenous 1,25(OH)2D3 concentration as rats administered exogenous 1,25(OH)2D3. However, calcium-restricted rats failed to upregulate VDR content in the duodenum or kidney, presumably a consequence of the negative control of VDR by PTH [184]. This point has at least conceptual relevance in the case of vitamin D toxicity. Rather than downregulation occurring during hypervitaminosis D, which is a more typical regulatory and protective event to limit tissue responsiveness, exposure of cells to exogenous 1,25(OH)2D results in enhanced responsiveness by virtue of upregulation. Such a mechanism would be of particular clinical relevance if the toxicity were due to overexposure of 1,25(OH)2D. Moreover, in this setting, the associated suppression of PTH would prevent the regulatory mechanism from being operative. Evidence suggests that in parent vitamin D toxicity, target tissues are responding to high concentrations of 25(OH)D, not 1,25(OH)2D. Concentrations of 1,25(OH)2D are typically only slightly increased, if at all. The hypercalcemia is due to the effects of pharmacologically high levels of 25(OH)D, even though in physiological settings, 25(OH)D is relatively weak. At high concentrations, 25(OH)D can compete for binding at
VDR sites, and thereby produce biological effects similar to those of 1,25(OH)2D on intestine and bone [185]. Beckman and colleagues [186] suggested, furthermore, that hypervitaminosis D, like excessive exogenous 1,25(OH)2D, is associated with homologous upregulation of intestinal VDR. Their investigation demonstrated that supraphysiological amounts of vitamin D2 or vitamin D3 administered to rats at doses of 25 000 IU daily for 6 days resulted in increasing plasma 25(OH)D concentrations with significant upregulation of intestinal VDR concentration and hypercalcemia. Plasma 1,25(OH)2D levels were not altered substantially. A comparison between hypervitaminosis D3 and D2 was also made [186]. No differences in 25(OH)D and plasma calcium concentrations were noted between either of the preparations. Concentrations of 25(OH)D in each case were markedly higher than the control group. The concentration of 1,25(OH)2D was observed to be only slightly greater in the vitamin-D3-treated group than the vitamin-D2-treated group. Because the 25(OH)D concentrations were elevated 20- to 25-fold, whereas 1,25(OH)2D showed only minimal increases, the biochemical and clinical changes associated with parent vitamin D toxicity were attributed to 25(OH)D. The data provided further support for the importance of 25(OH)D as the major toxic metabolite in vitamin D-associated hypercalcemia, as well as for the importance of increased intestinal VDR in the pathophysiological process that leads to enhanced effects of this metabolite.
Control of Renal 1a-Hydroxylase in Vitamin D Toxicity Some investigators have suggested that toxic effects of excessive concentrations of 25(OH)D may result from PTH suppression and downregulation of 1ahydroxylase with increased concentrations of 25(OH)D. PTH and 1,25(OH)2D have known reciprocal actions on 1a-hydroxylase and 24-hydroxylase activities. PTH stimulates 1a-hydroxylase activity and downregulates 24-hydroxylase activity; 1,25(OH)2D, on the other hand, downregulates 1a-hydroxylase activity and stimulates 24-hydroxylase activity. Beckman and colleagues [187] studied the effects of an excess of vitamin D3 and dietary calcium restriction on tissue 1a-hydroxylase and 24-hydroxylase activity in rats. Four groups of rats with different dietary calcium and vitamin D3 concentrations were studied (normal calcium, NC; low calcium, LC; and the excess vitamin D groups with normal or low calcium, NCT and LCT). The data showed that in the setting of a calcium-restricted diet, a nutritional hyperparathyroidism ensued. Under conditions of excess vitamin D3
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at doses of 75 000 IU per week and on a calciumrestricted diet, elevations in PTH facilitated the elimination of 25(OH)D3 through its metabolism to 1,25(OH)2D3 and/or degradation to 24,25(OH)2D3. The elevation in PTH was accompanied by increased activation of renal 1a-hydroxylase activity, lower concentrations of 25(OH)D3, increased activation of intestinal 24-hydroxylase activity, and lower renal VDR content compared to the normal calcium group. In contrast, the normal calcium diet in the vitamin D3 excess group contributed to the toxicity by virtue of suppressed PTH concentrations resulting in downregulation of renal 1a-hydroxylase and decreased 24-hydroxylase activity, and, thus, higher 25(OH)D3 concentrations. On the other hand, dietary calcium restriction in the setting of vitamin D3 excess seemed to be protective, providing less biological stimulation due to higher PTH concentrations with reduced VDR, increased activation of both 1a-hydroxylase and 24-hydroxylase activities, greater reductions in 25(OH)D3 concentrations, and lower concentrations of total calcium resulting in a less toxic state. So the lowcalcium diet protects, not only by contributing to less hypercalcemia, but also by facilitating metabolic pathways of vitamin D inactivation.
Inhibition of the Catabolic Pathway of 24-Hydroxylase Others have proposed that inhibition of the enzymes that degrade the vitamin D metabolites may have a role in the pathogenesis of hypervitaminosis D. 1,25(OH)2D is a known regulator of its own catabolism and an inhibitor of its synthesis. In the kidney, intestine, and other targets 1,25(OH)2D induces the enzyme 24-hydroxylase. This enzyme initiates a catabolic cascade that ultimately causes side chain oxidation, cleavage, and metabolic elimination of both 1,25(OH)2D and 25(OH)D, and it accounts for 35e40% of the catabolism of 1,25(OH)2D [188]. The remainder of the metabolic degradation is due to other side chain oxidations and biliary clearance. Reinhart and Horst [189] initially proposed that blunting of the catabolic pathway of 1,25(OH)2D3 with high concentrations of 24,25(OH)2D3 in rat cells would competitively inhibit further inactivation of 1,25(OH)2D3, resulting in an accumulation of 1,25 (OH)2D3 and toxicity. Clinical investigations of the downregulation of rat intestinal 24-hydroxylase and its inhibition by calcitonin may help to elucidate a role of this hormone in potentiating the toxicity of vitamin D. 24-Hydroxylation is important in the inactivation of both 1,25(OH)2D3 and 25(OH)D3, and in the kidney is largely regulated inversely by 1a-hydroxylation [190]. In a study examining the effects of dietary calcium and vitamin D status on the regulation of intestinal 24-hydroxylase enzyme
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and mRNA expression, rats were fed normal or lowcalcium diets with variable amounts of vitamin D [191]. Half of the rats on the normal and low-calcium diets were administered pharmacological doses of vitamin D3 (25 000 IU three times weekly). Excess vitamin D3 resulted in significant elevations in plasma 25(OH)D3 in both calcium groups (LCT and NCT), with a much larger increase noted in the normal calcium group (NCT). Hypercalcemia was most severe in the NCT group, whereas rats in the low-calcium and vitamin D3 excess group (LCT) had plasma calcium levels similar to the NC group. Because the NCT was accompanied by an increased calcitonin concentration compared to the LCT, the authors suggested that the increased calcitonin in the NCT group may have suppressed 24-hydroxylase activity, with resultant higher 25(OH)D3 and calcium concentrations [191]. This concept was further supported when rats, subjected to thyroparathyroidectomy (TPTX), which eliminated endogenous calcitonin, were found to have higher concentrations of 24-hydroxylase activity than the NCT group. Through inhibition of intestinal 24-hydroxylase activity, calcitonin could be associated with reduced turnover and catabolism of 25(OH)D3, thereby potentiating its toxicity. Thus, increased expression of 24hydroxylase activity in cases of pharmacological amounts of 25(OH)D3 may be an important mechanism to counteract vitamin D toxicity. A key role for 24hydroxylase in preventing the development of vitamin D toxicosis was found in a recent animal study [192]. Growing dogs given 135-fold vitamin D3 supplementation actually had a decrease in plasma 1,25(OH)2D3 levels by 40% as compared to controls, despite an increase in 1,25(OH)2D3 production. This was attributed to an upgraded catabolism of 1,25(OH)2D3 by 24hydroxylase, as evidenced by increased gene expression of renal and intestinal 24-hydroxylase, thus providing an efficient hormonal counteraction [192]. More recently, St-Arnaud et al. [192a] and Masuda et al. [192b] showed that a targeted inactivating mutation of the 24hydroxylase gene in mice resulted in impaired catabolism of 1,25(OH)2D3. Absence of the 24-hydroxylase gene during development also resulted in abnormal bone mineralization, with rescue of the phenotype by subsequent inactivation of the VDR. These experiments support a critical role of 24-hydroxylase in preserving vitamin D homeostasis.
Vitamin-D-binding Protein and the Level of Free Metabolite in Vitamin D Toxicity The vitamin-D-binding protein (DBP) is a specific transport protein that binds large quantities of the circulating vitamin D metabolites (see Chapter 5). Similar to the situation for other steroid hormones, fat-soluble
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compounds, and thyroid hormones, only a small fraction of the metabolites circulate free in plasma. The binding affinity of the protein for the vitamin D metabolites is moderate and the capacity is great (only 5% of binding sites on DBP are normally occupied). In addition, the various metabolites have different binding affinities for the protein, in the following sequence: 25(OH)D > 24,25(OH)2D > 1,25(OH)2D [193]. Of note is the fact that the potent metabolite 1,25(OH)2D has the least affinity for DBP, but the highest affinity for the intracellular VDR that triggers subsequent transcriptional events. Therefore, freeing bound 1,25(OH)2D metabolite from DBP could promote its entry into various tissues and promote biological activity [194]. In states of vitamin D toxicity, the presence of elevated free 1,25(OH)2D levels despite normal total 1,25 (OH)2D levels suggests that 1,25(OH)2D is displaced from DBP by 25(OH)D, resulting in a rise of serum free calcitriol [195]. Evidence indicates that the biologically active form of the vitamin D steroid hormone is the free hormone that is accessible to cells [196]. Because of technical difficulties in measuring the free hormone, the determination of vitamin D status involves a measurement combining free vitamin D and DBP concentrations. In normal individuals, 85% of the total 1,25(OH)2D is bound to DBP, 15% is bound to albumin, and 0.4% is free [197]. However, under conditions of altered or reduced albumin and DBP concentrations, as in liver or kidney disease, the free hormone may provide different information compared to the total measured concentration of vitamin D. Theoretically, total hormone concentration in such settings may erroneously suggest deficiency of vitamin D with needless institution of replacement therapy. Bikle and colleagues noted that subjects with liver disease have reduced DBP concentrations with low total 1,25(OH)2D and 25(OH)D levels, whereas free forms are normal [198,199]. Similarly, in certain forms of renal disease, the concentrations of DBP and vitamin D metabolites are reduced, thus measurements of total hormone may provide an inaccurate reflection of vitamin D status. Koenig et al. [200] investigated free and total 1,25(OH)2D concentrations in subjects with renal disease. Patients with nephrotic syndrome, with varying degrees of renal failure, and on chronic hemodialysis and peritoneal dialysis were studied. The serum concentrations of total and free 1,25(OH)2D correlated well with one another in the patients with renal failure and those undergoing hemodialysis. The concentrations of DBP and 25(OH)D, thus, were unaffected by renal function. The concentrations of total 1,25(OH)2D accurately reflected free 1,25(OH)2D in patients with varying degrees of renal failure when DBP levels remained normal. However, this did not hold true for the subjects
with nephrotic syndrome or those on chronic peritoneal dialysis, who lost DBP and bound vitamin D metabolites into the urine or peritoneal fluid, respectively, with a rise in the percentage free 1,25(OH)2D (also low-density-lipoprotein-related protein 2 or megalin may play a role as discussed in Chapter 14). Measurement of free metabolite in these particular patients may be important to avoid vitamin D toxicity when supplementation is instituted. Thus, in this context, the binding proteins of the vitamin D metabolites not only serve a transport function, but also may provide a buffering mechanism to protect against toxicity [201].
CLINICAL MANIFESTATIONS The clinical manifestations of vitamin D toxicity result from hypercalcemia and reflect the essential role of calcium in many tissues and targets, including bone, the cardiovascular system, nerves, and cellular enzymes. Initial signs and symptoms of hypervitaminosis D may be similar to other hypercalcemic states and include generalized weakness and fatigue. Central nervous system features may include confusion, difficulty in concentration, drowsiness, apathy, and coma [202]. Neuropsychiatric symptoms include depression and psychosis, which resolve following improvement of the hypercalcemia. Hypercalcemia can affect the gastrointestinal tract and cause anorexia, nausea, vomiting, and constipation. It can also induce hypergastrinemia. There is no evidence that peptic ulcers are more common in any other form of hypercalcemia. Rarely, pancreatitis may be a presentation of either acute or chronic hypercalcemia. In the heart, hypercalcemia may shorten the repolarization phase of conduction reducing the QeT interval on the electrocardiogram (EKG). EKG changes in vitamin D toxicity have been mistaken for myocardial ischemia [203]. A more accurate EKG indication of the level of hypercalcemia is the QeT interval corrected for rate. Bradyarrhythmias and first-degree heart block have been described, but are rare. Hypercalcemia may potentiate the action of digitalis on the heart [204]. Kidney function is affected because high concentrations of calcium alter the action of vasopressin on the renal tubules. The net result is reduced urinary concentrating ability and a form of nephrogenic diabetes insipidus. This usually presents as polyuria, but rarely is the volume as high as that associated with central diabetes insipidus. Symptoms may include polydipsia, which is an expected consequence of polyuria. Hypercalciuria is one of the earliest signs of vitamin D toxicity and precedes the occurrence of hypercalcemia. The initial
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hypercalciuria may be ameliorated as renal failure progresses because of reduced calcium clearance. The pathophysiology of hypercalcemia can be rapidly worsened when dehydration develops. When reduced renal blood flow occurs, less calcium is presented to the renal glomerulus, and hypercalcemia can rapidly progress. Renal impairment from the hypercalcemia is reversible if of short duration. Chronic, uncontrolled hypercalcemia can lead to deposition of calcium phosphate salts in the kidney and permanent damage with eventual nephrocalcinosis. In an investigation of vitamin-Dinduced nephrocalcinosis, Scarpelli and colleagues [205] noted that cell damage, specifically in mitochondria, preceded intracellular calcium deposition. The hypercalcemia induced in rats by excessive vitamin D administration caused mitochondrial swelling, cell injury, and subsequent calcification. Ectopic soft tissue calcification can be a particular problem in hypervitaminosis D. The tendency towards soft tissue calcification is compounded by the combination of hypercalcemia and hyperphosphatemia, often exceeding the solubility product of the two ions [206e208]. In rats exposed to excessive vitamin D, Hass and colleagues demonstrated that the pathological processes of vitamin D toxicity were related to dosage, length of time between doses, and duration of exposure [209]. For rats subjected to sublethal doses, generalized calcinosis was seen after only 8 days, when a total of 300 000 units of ergosterol was administered. Pathologically, bones appeared more brittle than normal, with increased cortical bone resorption, increased numbers of osteoclasts, and reduced numbers of osteoblasts. Abnormal calcium deposits were noted in the aorta and its major branches, heart, kidney, muscle, and respiratory tract. The earliest evidence of hypervitaminosis D was in the proximal aorta. The subject of vascular calcification is discussed in detail in Chapter 73. Muscle tissue was the least resistant to calcification, with the order of decreasing susceptibility being smooth muscle > cardiac muscle > skeletal muscle [210]. The liver, brain, and pituitary were not affected by high doses of vitamin D. Permanent dental changes have also been reported with hypervitaminosis D, including enamel hypoplasia and focal pulp calcification [211]. Bone mineral density can be decreased due to excessive bone resorption [207,212], changes which can be reversed when vitamin D levels return to normal [213].
DIAGNOSIS OF VITAMIN D TOXICITY With modern assays for calciotropic hormones, PTH, 25(OH)D, and 1,25(OH)2D (see Chapter 47), one can
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readily differentiate vitamin D metabolite-mediated hypercalcemia from other causes of hypercalcemia. The circulating intact PTH level, measured by the two-site immunoradiometric assay (IRMA) or immunochemiluminometric assay (ICMA), should be suppressed in virtually all hypercalcemic disorders with the exception of primary hyperparathyroidism, familial hypocalciuric hypercalcemia, administration of lithium or thiazides, and renal failure. Although patients with malignancy-associated hypercalcemia tend to have a higher serum calcium concentration than those with other causes of hypercalcemia, diminished glomerular filtration rate and subsequent reduction in renal calcium excretion can dramatically increase the serum calcium level in any hypercalcemic patient. In contrast to the low serum phosphorus level in patients with hypercalcemia due to PTH or PTHrP, the serum phosphorus level is at the upper limit of normal or frankly elevated in patients with vitamin D metabolite-mediated hypercalcemia. This is due to increased intestinal absorption and reduced renal clearance of phosphate. An elevated 25(OH)D concentration with normal 1,25 (OH)2D level is indicative of toxicity with exogenously administered vitamin D or 25(OH)D. The serum 1,25 (OH)2D level may be normally increased in patients with primary hyperparathyroidism due to the induction of renal 1a-hydroxylase by PTH. Abnormally high 1,25(OH)2D levels, in the setting of suppressed PTH and hypercalcemia, indicate dysregulated production of 1,25(OH)2D due to either granulomatous diseases, lymphoma, or toxicity with exogenous 1,25 (OH)2D or la-OHD. In cases of hypercalcemia due to PTHrP or local osteolytic factors, the serum 1,25 (OH)2D concentration is usually suppressed. In patients with hypercalcemia due to toxicity with other vitamin D analogs such as dihydrotachysterol (DHT) [214] and calcipotriol, the active metabolites may not be recognized by the antibodies used in the conventional assays for 1,25(OH)2D. The diagnosis of vitamin D toxicity can be made on clinical grounds. Detailed clinical and drug history are of paramount importance in order to make an early diagnosis. Most patients who are suffering from vitamin D toxicity are taking vitamin D for osteoporosis, hypoparathyroidism, pseudohypoparathyroidism, hypophosphatemia, osteomalacia, or renal osteodystrophy in excessive dosages or at too frequent dosing intervals. With the recent idea that vitamin D is protective of many diseases, vitamin D therapy is becoming very widespread in otherwise normal subjects. Therefore, one should have a high index of suspicion in patients who are being treated with pharmacological dosages of vitamin D or its metabolites. History should be obtained to rule out use of over-the-counter supplements such as SoladekÒ which may contain suprapharmacologic
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72. HYPERCALCEMIA DUE TO VITAMIN D TOXICITY
amounts of vitamin D at manufacturer-recommended dosages. Patients with granulomatous diseases or lymphoma usually have widespread active disease when hypercalcemia develops. In such cases, the diagnosis is obvious at the time of presentation. However, exceptions do exist. In patients with unexplained hypercalcemia, if the 1,25(OH)2D level is elevated and other more easily identifiable causes for this elevation such as primary hyperparathyroidism, pregnancy, and exogenous toxicity (by history) are excluded, measurement of angiotensin converting enzyme level and a systemic search for lymph node enlargement, pulmonary, renal, hepatosplenic, ocular, central nervous system, and bone marrow granulomas or lymphoma should be made.
TREATMENT OF VITAMIN D TOXICITY Dietary calcium and vitamin D restriction and avoidance of exposure to sunlight and other ultraviolet light sources should be advised to patients at high risk to develop vitamin D metabolite-mediated hypercalcemia. Those at risk include patients with granulomatous diseases and lymphoma whose disease
TABLE 72.2
is widespread and active and patients who are already hypercalciuric. Fluid intake should be encouraged. Daily dietary calcium intake should be minimized to approximately 400 mg or less in these patients. Any use of vitamin D supplements should be discontinued. The patient should be encouraged to use sunscreen (sun protection factor (SPF) >15) as much as possible when out doors. The calcium level should be monitored closely in patients who have a previous history of hypercalcemia or hypercalciuria, or who have recently taken diets enriched in vitamin D and calcium, or who have a recent history of excessive sunlight exposure. A reduction in oxalate intake may also be advisable, so as to prevent an increase in oxalate absorption and hyperoxaluria, which may increase the risk of kidney stone formation, despite a reduction in urinary calcium excretion [215]. Patients with isolated hypercalciuria without other indications for corticosteroid therapy can be considered for a therapeutic trial of a thiazide diuretic. Thiazide diuretics inhibit calcium excretion from the distal convoluted tubule and have been shown to decrease the risk of recurrent nephrolithiasis in idiopathic hypercalciuria [215a,215b]. When hypercalcemia develops, the aforementioned preventive measures will help to ameliorate the severity
Treatment of Vitamin D Toxicity
Intervention
Mechanism of action
Onset of action
Duration of action
Treatment of the underlying disease process
Variable
Variable
Variable
Isotonic intraveneous saline
Restoration of intravascular volume Increases urinary calcium excretion
Within hours
During infusion
Loop diuretics
Increase urinary calcium excretion via inhibition of calcium reabsorption in the loop of Henle
Within hours
During therapy
Glucocorticoids
Decrease intestinal calcium absorption Decrease 1,25-dihydroxyvitamin D production by activated mononuclear cells May alter hepatic vitamin D metabolism to favor the production of inactive vitamin D metabolites In some cases, direct anti-tumor effect (e.g., lymphomas, multiple myeloma)
2e5 days
Days to weeks
Bisphosphonates
Inhibit bone resorption via interference with osteoclast function
24e72 hours
2e4 weeks
Ketoconazole [119,214,226e228]
Decrease 1,25-dihydroxyvitamin D production by activated mononuclear cells through inhibition of cytochrome P450
2e4 days
During therapy
Aminoquinolones (chloroquine and hydroxychloroquine) [221e225]
Decrease 1,25-dihydroxyvitamin D production by activated mononuclear cells through unknown mechanism in granulomatous disease (shown to be ineffective in lymphoma)
Days to weeks
During therapy
FIRST-LINE TREATMENTS
SECOND-LINE TREATMENTS
Adapted from [229].
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REFERENCES
of hypercalcemia. Treatment measures are summarized in Table 72.2. Treatment of the underlying disease process is essential. General measures in those who are symptomatic include hydration with normal saline followed by the judicious use of a loop diuretic, like furosemide, in certain cases. Specific inhibitors of bone resorption, such as bisphosphonates [207,212] and calcitonin, can be helpful. Glucocorticoids have proved to be particularly effective in vitamin D intoxication, granulomatous diseases, and lymphoma (see also Chapter 45). The precise mechanism of action of glucocorticoids in calcium homeostasis is not known. Nonetheless, they are useful because they (1) directly inhibit gastrointestinal absorption of calcium by decreasing the synthesis of calcium-binding protein (calbindin-D) and may downregulate intestinal VDR [215c] and decrease active transcellular transport [216], (2) increase urinary excretion of calcium [217], and (3) may alter hepatic vitamin D metabolism to favor the production of inactive vitamin D metabolites, resulting in lower concentrations of 25(OH)D [218]. Evidence also suggests that they may increase the degradation of 1,25(OH)2D at the receptor sites [219]. Glucocorticoids may also limit osteoclastic bone resorption [220]. Institution of glucocorticoid therapy results in prompt decline of the circulating 1,25(OH)2D concentrations within 3 to 4 days [80]. Patients with non-hematological malignancies and those with primary hyperparathyroidism do not usually respond to glucocorticoids. Aminoquinolones (chloroquine and hydroxychloroquine) are also capable of reducing the 1,25(OH)2D and calcium concentrations in patients with sarcoidosis [221e224]. The theoretical advantage of aminoquinolones over glucocorticoids is that correction of the 1,25(OH)2D should result in rapid recovery of at least some of the bone density lost to the disease [213]. In lymphoma cells, however, aminoquinolones do not have the same regulatory effects on the excess 1,25(OH)2D as they do in granulomatous disease. In the presence of lymphoma, it is preferable to use steroid-containing antitumor regimens [225]. Owing to the limited experience with aminoquinolone drugs as antihypercalcemic agents and their potential side effects, they should be reserved for patients in whom steroid therapy is unsuccessful or specifically contraindicated. Ketoconazole, an antifungal agent, in high dosages can inhibit the mitochondrial cytochrome P450-linked 25(OH)D 1a-hydroxylase irrespective of whether it is renal [214] or extrarenal as in sarcoidosis [226] and tuberculosis [119]. Case reports of ketoconazole use in conjunction with glucocorticoids in sarcoidosis suggest that the addition of ketoconazole may reduce steroid requirements [227,228].
SUMMARY AND CONCLUSIONS Vitamin D toxicity is not a common cause of hypercalcemia, but it can be life-threatening if not identified promptly. The major causes of hypercalcemia are primary hyperparathyroidism and malignancy. If these two etiologies are excluded, vitamin D toxicity becomes an important diagnostic consideration. There are many forms of exogenous and endogenous vitamin D toxicity. Inadvertent excessive use of pharmaceutical preparations is the most common etiology of exogenous toxicity. Excessive amounts of the parent compound, vitamin D, can be most difficult to manage as compared to toxicity due to the metabolites 25(OH)D or 1,25(OH)2D. Extensive lipid solubility of vitamin D accounts for its long half-life and tendency for prolonged hypercalcemia. New clinical applications of 1,25(OH)2D and its synthetic analogs have been accompanied by the increased potential for toxicity. Endogenous etiologies may result from ectopic production of 1,25(OH)2D in granulomatous diseases, such as sarcoidosis and tuberculosis, or in lymphoma. Many different mechanisms have been proposed to account for vitamin D toxicity, including the vitamin D metabolite itself, VDR number, activity of 1a-hydroxylase, inhibition of vitamin D metabolism, and the capacity of DBP. Mounting evidence that higher levels of vitamin D may have beneficial effects on bone and cellular health may predispose to enhanced administration of vitamin D in the future and thereby increased frequency of vitamin D toxicity.
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dietary calcium restriction, on vitamin D receptors, Endocrinology 126 (2) (1990) 1031e1035. T.A. Reinhardt, R. Horst, PTH down-regulates 1,25(OH)2D receptors and VDR mRNA in vitro and blocks homologous up-regulation of VDR in vitro, Endocrinology 127 (1990) 942e948. M.R. Haussler, P.E. Cordy, Metabolites and analogs of vitamin D. Which for what? JAMA 247 (6) (1982) 841e844. M.J. Beckman, R.L. Horst, T.A. Reinhardt, D.C. Beitz, Upregulation of the intestinal 1,25-dihydroxyvitamin D receptor during hypervitaminosis D: a comparison between vitamin D2 and vitamin D3, Biochem. Biophys. Res. Commun. 169 (3) (1990) 910e915. M.J. Beckman, J.A. Johnson, J.P. Goff, T.A. Reinhardt, D.C. Beitz, R.L. Horst, The role of dietary calcium in the physiology of vitamin D toxicity: excess dietary vitamin D3 blunts parathyroid hormone induction of kidney 1-hydroxylase, Arch. Biochem. Biophys. 319 (2) (1995) 535e539. M.R. Haussler, Vitamin D receptors: nature and function, Annu. Rev. Nutr. 6 (1986) 527e562. T.A. Reinhardt, R.L. Horst, Self-induction of 1,25-dihydroxyvitamin D3 metabolism limits receptor occupancy and target tissue responsiveness, J. Biol. Chem. 264 (27) (1989) 15917e15921. T. Shigematsu, N. Horiuchi, Y. Ogura, T. Miyahara, T. Suda, Human parathyroid hormone inhibits renal 24-hydroxylase activity of 25-hydroxyvitamin D3 by a mechanism involving adenosine 3’,50 -monophosphate in rats, Endocrinology 118 (4) (1986) 1583e1589. M.J. Beckman, J.P. Goff, T.A. Reinhardt, D.C. Beitz, R.L. Horst, In vivo regulation of rat intestinal 24-hydroxylase: potential new role of calcitonin, Endocrinology 135 (5) (1994) 1951e1955. M.A. Tryfonidou, M.A. Oosterlaken-Dijksterhuis, J.A. Mol, T.S. van den Ingh, W.E. van den Brom, H.A. Hazewinkel, 24Hydroxylase: potential key regulator in hypervitaminosis D3 in growing dogs, Am. J. Physiol. Endocrinol. Metab. 284 (3) (2003) E505eE513. R. St-Arnaud, A. Arabian, R. Travers, F. Barletta, M. RavalPandya, K. Chapin, et al., Deficient mineralization of intramembranous bone in vitamin D-24-hydroxylase-ablated mice is due to elevated 1,25-dihydroxyvitamin D and not to the absence of 24,25-dihydroxyvitamin D, Endocrinology 141 (7) (2000) 2658e2666. S. Masuda, V. Byford, A. Arabian, Y. Sakai, M.B. Demay, R. StArnaud, et al., Altered pharmacokinetics of 1alpha,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 in the blood and tissues of the 25-hydroxyvitamin D-24-hydroxylase (Cyp24a1) null mouse, Endocrinology 146 (2) (2005) 825e834. J.P. Mallon, D. Matuszewski, H. Sheppard, Binding specificity of the rat serum vitamin D transport protein, J. Steroid Biochem. 13 (4) (1980) 409e413. J.S. Adams, Specific internalization of 1,25-dihydroxyvitamin D3 by cultured intestinal epithelial cells, J. Steroid Biochem. 20 (4A) (1984) 857e862. J.M. Pettifor, D.D. Bikle, M. Cavaleros, D. Zachen, M.C. Kamdar, F.P. Ross, Serum levels of free 1,25-dihydroxyvitamin D in vitamin D toxicity, Ann. Intern. Med. 122 (7) (1995) 511e513. R.F.A. Bouillon, H. Van Assche, Van Baelen, W. Heyns, P. De Moor, Influence of the vitamin D-binding protein on the serum concentration of 1,25-dihydroxyvitamin D3. Significance of the free 1,25-dihydroxyvitamin D3 concentration, J. Clin. Invest. 67 (3) (1981) 589e596.
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[197] D.D. Bikle, P.K. Siiteri, E. Ryzen, J.G. Haddad, Serum proteinbinding of 1,25-dihydroxyvitamin D: a reevaluation by direct measurement of free metabolite levels, J. Clin. Endocrinol. Metab. 61 (5) (1985) 969e975. [198] D.D. Bikle, E. Gee, B. Halloran, J.G. Haddad, Free 1,25dihydroxyvitamin D levels in serum from normal subjects, pregnant subjects, and subjects with liver disease, J. Clin. Invest. 74 (6) (1984) 1966e1971. [199] D.D. Bikle, B.P. Halloran, E. Gee, E. Ryzen, J.G. Haddad, Free 25-hydroxyvitamin D levels are normal in subjects with liver disease and reduced total 25-hydroxyvitamin D levels, J. Clin. Invest. 78 (3) (1986) 748e752. [200] K.G. Koenig, J.S. Lindberg, J.E. Zerwekh, P.K. Padalino, H.M. Cushner, J.B. Copley, Free and total 1,25-dihydroxyvitamin D levels in subjects with renal disease, Kidney Int. 41 (1) (1992) 161e165. [201] C.M. Mendel, The free hormone hypothesis: a physiologically-based mathematical model, Endocr. Rev. 10 (3) (1989) 232e274. [202] G.W. Edelson, M. Kleerekoper, Hypercalcemic crisis, Med. Clin. North Am. 79 (1) (1995) 79e92. [203] N. Ashizawa, S. Arakawa, Y. Koide, G. Toda, S. Seto, K. Yano, Hypercalcemia due to vitamin D intoxication with clinical features mimicking acute myocardial infarction, Intern. Med. 42 (4) (2003) 340e344. [204] S.R. Nussbaum, Pathophysiology and management of severe hypercalcemia, Endocrinol. Metab. Clin. North Am. 22 (2) (1993) 343e362. [205] D.G. Scarpelli, G. Tremblay, A.G. Pearse, A comparative cytochemical and cytologic study of vitamin D-induced nephrocalcinosis, Am. J. Pathol. 36 (1960) 331e353. [206] K.R. Shetty, K. Ajlouni, P.S. Rosenfled, T.C. Hagen, Protracted vitamin D intoxication, Arch. Intern. Med. 135 (7) (1975) 986e988. [207] R. Rizzoli, C. Stoermann, P. Ammann, J.P. Bonjour, Hypercalcemia and hyperosteolysis in vitamin D intoxication: effects of clodronate therapy, Bone 15 (2) (1994) 193e198. [208] E. Hefti, U. Trechsel, H. Fleisch, J.P. Bonjour, Nature of calcemic effect of 1,25-dihydroxyvitamin D3 in experimental hypoparathyroidism, Am. J. Physiol. 244 (4) (1983) E313eE316. [209] G.M. Hass, R.E. Trueheart, C.B. Taylor, M. Stumpe, An experimental histologic study of hypervitaminosis D, Am. J. Pathol. 34 (3) (1958) 395e431. [210] J. Swierczynski, G. Nagel, M.M. Zydowo, Calcium content in some organs of rats treated with a toxic calciol dosis, Pharmacology 34 (1) (1987) 57e60. [211] J.L. Giunta, Dental changes in hypervitaminosis D, Oral. Surg. Oral. Med. Oral. Pathol. Oral. Radiol. Endod. 85 (4) (1998) 410e413. [212] P.L. Selby, M. Davies, J.S. Marks, E.B. Mawer, Vitamin D intoxication causes hypercalcemia by increased bone resorption which responds to pamidronate, Clin. Endocrinol. (Oxf.) 43 (5) (1995) 531e536. [213] J.S. Adams, G. Lee, Gains in bone mineral density with resolution of vitamin D intoxication, Ann. Intern. Med. 127 (3) (1997) 203e206. [214] A.R. Glass, C. Eil, Ketoconazole-induced reduction in serum 1,25-dihydroxyvitamin D and total serum calcium in hypercalcemic patients, J. Clin. Endocrinol. Metab. 66 (5) (1988) 934e938. [215] B.A. Kogan, J.W. Konnak, K. Lau, Marked hyperoxaluria in sarcoidosis during orthophosphate therapy, J. Urol. 127 (2) (1982) 339e340.
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[215a] B. Ettinger, J.T. Citron, B. Livermore, L.I. Dolman, Chlorthalidone reduces calcium oxalate calculous recurrence but magnesium hydroxide does not, J. Urol. 139 (4) (1988) 679e684. [215b] E. Laerum, S. Larsen, Thiazide prophylaxis of urolithiasis. A double-blind study in general practice, Acta. Med. Scand. 215 (4) (1984) 383e389. [215c] M. Hirst, D. Feldman, Glucocorticoids down-regulate the number of 1,25-dihydroxyvitamin D3 receptors in mouse intestine, Biochem. Biophys. Res. Commun. 105 (4) (1982) 1590e1596. [216] J.J. Feher, R.H. Wasserman, Intestinal calcium-binding protein and calcium absorption in cortisol-treated chicks: effects of vitamin D3 and 1,25-dihydroxyvitamin D3, Endocrinology 104 (2) (1979) 547e551. [217] Y. Suzuki, Y. Ichikawa, E. Saito, M. Homma, Importance of increased urinary calcium excretion in the development of secondary hyperparathyroidism of patients under glucocorticoid therapy, Metabolism 32 (2) (1983) 151e156. [218] B.P. Lukert, L.G. Raisz, Glucocorticoid-induced osteoporosis: pathogenesis and management, Ann. Intern. Med. 112 (5) (1990) 352e364. [219] M. Carre, O. Ayigbede, L. Miravet, H. Rasmussen, The effect of prednisolone upon the metabolism and action of 25hydroxy- and 1,25-dihydroxyvitamin D3, Proc. Natl. Acad. Sci. USA 71 (8) (1974) 2996e3000. [220] J.P. Bilezikian, Etiologies and therapy of hypercalcemia, Endocrinol. Metab. Clin. North Am. 18 (2) (1989) 389e414. [221] T.J. O’Leary, G. Jones, A. Yip, D. Lohnes, M. Cohanim, E.R. Yendt, The effects of chloroquine on serum 1,25-dihydroxyvitamin D and calcium metabolism in sarcoidosis, N. Engl. J. Med. 315 (12) (1986) 727e730.
[222] P.E. Barre, M. Gascon-Barre, J.L. Meakins, D. Goltzman, Hydroxychloroquine treatment of hypercalcemia in a patient with sarcoidosis undergoing hemodialysis, Am. J. Med. 82 (6) (1987) 1259e1262. [223] J.S. Adams, M.M. Diz, O.P. Sharma, Effective reduction in the serum 1,25 dihydroxyvitamin D and calcium concentration in sarcoidosis-associated hypercalcemia with short course chloroquine therapy, Ann. Intern. Med. 111 (5) (1989) 437e438. [224] B.J. Hunt, E.R. Yendt, The response of hypercalcemia in sarcoidosis to chloroquine, Ann. Intern. Med. 59 (4) (1963) 554e564. [225] J.S. Adams, V. Kantorovich, Inability of short-term, low-dose hydroxychloroquine to resolve vitamin D-mediated hypercalcemia in patients with B-cell lymphoma, J. Clin. Endocrinol. Metab. 84 (2) (1999) 799e801. [226] J.S. Adams, O.P. Sharma, M.M. Diz, D.B. Endres, Ketoconazole decreases the serum 1,25-dihydroxyvitamin D and calcium concentration in sarcoidosis-associated hypercalcemia, J. Clin. Endocrinol. Metab. 70 (4) (1990) 1090e1095. [227] C. Young, R. Burrows, J. Katz, H. Beynon, Hypercalcaemia in sarcoidosis, Lancet 353 (9150) (1999) 374. [228] M. Conron, C. Young, H.L. Beynon, Calcium metabolism in sarcoidosis and its clinical implications, Rheumatology (Oxford) 39 (7) (2000) 707e713. [229] E. Shane, D. Irani, Hypercalcemia: pathogenesis, clinical manifestations, differential diagnosis, and management, in: M.J. Favus (Ed.), Sixth ed., Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, 179 American Society for Bone and Mineral Research, Washington, 2006.
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73 Vitamin D: Cardiovascular Effects and Vascular Calcification Dwight A. Towler Washington University in St. Louis, St. Louis, MO, USA
INTRODUCTION The role of vitamin D in skeletal physiology and calcium/phosphate homeostasis has been well recognized. Rachitic musculoskeletal frailty arising from vitamin D deficiency e and the threshold-dependent, beneficial responses of repletion e has appropriately held our attention for five decades. However, with our increasingly aged, obese, and dysmetabolic Westernized societies, the role of vitamin D insufficiency in the pathogenesis of cardiovascular disease has begun to be appreciated and considered [1]. Unlike the repletion regimen for rickets treatment, the regimen and pharmacokineticepharmacodynamic (PKePD) relationship for accrual of cardiovascular benefit with vitamin D repletion has yet to be established. This is an important shortcoming. Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in our aging population [2]. Sedentary lifestyle, obesity, diabetes, dyslipidemia, and declining renal function all increase the risk of atherosclerosis, arteriosclerosis, peripheral vascular disease, vascular dementia, stroke, myocardial infarction, and heart failure with or without valvular heart disease. Diabetes and obesity are the major contributors [3]. Our current understanding of how “diabesity” impairs cardiovascular health now encompasses the global contribution of lower-grade systemic inflammation with oxidative stress to CVD processes e processes modulated by vitamin D receptor agonists [4e6]. At the far end of the spectrum, chronic kidney disease (CKD) represents a “perfect storm” of risk factors for CVD [7]. Five-year mortality rates for CKD3 and CKD4 are approximately 25% and 45%, respectively, largely from CVD. In CKD5, the annual CVD mortality approaches 20% [8].
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10073-3
Of note, the accelerated CVD of CKD5 has been refractory to intervention with lipid-lowering agents and anti-hypertensive strategies successfully used to decrease disease burden in those without CKD [9e12]. The marked perturbations in mineral homeostasis with CKD prompted careful reassessment of the contributions of calcium, phosphate, and calciotropic hormones e including vitamin D e to cardiovascular health [13,14]. Much of the available clinical data demonstrating the benefit of calcitriol-based VDR agonism (VDRA) in cardiovascular disease arises in patients with chronic kidney disease (CKD) on renal replacement therapy (RRT) [15]. However, the major perturbations in mineral metabolism, osteotropic hormone signaling, and cardiovascular function in uremia preclude simple extrapolation of PKePD relationships for VDRA from this select population to our general populace. Moreover, prior to recent years the concern of cardiovascular toxicity dominated our thinking of vitamin D in vascular biology; indeed, several well-established rodent models utilize vitamin D toxicity to study vascular calcification and the impact of arteriosclerotic vessel “hardening” on myocardial hypertrophy [16e20]. Vitamin D toxicity can and does occur in humans, more commonly in children than adults, and highly concentrated formulations of vitamin D3 are marketed as rodenticides [21]. The bimodal theoretical response of cardiovascular health to vitamin D tone presented previously (Fig. 73.1) has been confirmed by clinical epidemiology at least for children with CKD [22]. Thus, the cardiovascular toxicities of vitamin D excess must also be considered in addition to cardiovascular benefits of vitamin D sufficiency and/ or repletion. At the first version of this chapter (second edition of this book), a woefully small number of patient-oriented
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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73. VITAMIN D: CARDIOVASCULAR EFFECTS AND VASCULAR CALCIFICATION
The bimodal response of cardiovascular health to vitamin D tone. Copyright Ó 2005, 1997, Elsevier Inc. All rights reserved.
FIGURE 73.1
and preclinical studies addressed the role for vitamin D in cardiovascular health. Since then, the number of studies addressing this unmet scientific need have increased. The detrimental cardiovascular consequences of vitamin D deficiency and extreme vitamin D excess have become clear. In Westernized societies, many of us are vitamin-D-insufficient [23], and increasing daily intakes to 2000e5000 IU per day is likely to be well tolerated with accrual of benefit and with little downside potential in healthy adults [23]. Yet, many important questions remain to be answered, including the actual cardiovascular benefit of aggressive vitamin D supplementation on public health and healthcare costs. The goal of this chapter is to provide an updated but abbreviated overview of vitamin D in cardiovascular physiology and toxicology. Emphasis is placed upon congestive heart failure (CHF), atherosclerosis, and arteriosclerotic calcification e i.e. the prevalent cardiovascular disease processes potentially remediable in part via consideration of vitamin D endocrinology and pharmacology. Other chapters relevant to this subject in this volume include: vitamin D toxicity (Chapter 72), vitamin D in heart (Chapter 31) and in kidney disease (Chapter 70), use of vitamin D and analogs in renal failure patients (Chapter 81) and vitamin D deficiency and cardiovascular risk (Chapter 102).
VITAMIN D SIGNALING IN CARDIOVASCULAR REMODELING AND MYOCARDIAL FUNCTION McGonigle and colleagues were among the first to describe a pharmacologically relevant relationship
between vitamin D signaling and congestive heart failure (CHF) [24]. Thirty years ago, in a study of a dozen patients on dialysis, they assigned beneficial actions of 1a-hydroxycholecalciferol (1a-(OH)D3) on ventricular inotropy to the concomitant reductions in secondary hyperparathyroidism [24]. Severe hypocalcemia and hypophosphatemia associated with vitamin D deficiency of rickets suppresses myocardial contractility, and several cases of calcium and VDR agonistresponsive dilated cardiomyopathy have been reported [25e32]. Burch and colleagues recently presented a series of 16 infants with severe, life-threatening cardiomyopathy due to hypocalcemia and hypovitaminosis D seen over a 7-year period in southeastern England [33]. Maternal vitamin D insufficiency was noted in this study and in preclinical rodent models’ maternal insufficiency during gestation induces cardiac hypertrophy. Early studies indicated that cell-autonomous actions of vitamin D signaling were responsible for improving cardiomyocyte calcium handling and contractility independent of effect upon ambient calcium and PTH tone. In the 1980s Simpson first provided evidence that the vitamin D receptor was expressed in rat cardiomyocytes, and that ventricular and vascular hypertrophic remodeling arising from nutritional vitamin D deficiency occurs independent of ambient serum calcium [34,35]. Yet, it was not until the era of the VDR “knockout” mouse e ushered in by Li and Demay and colleagues [36] e that the multiple, direct actions of vitamin D signaling on myocardial maturation and function were more widely appreciated and embraced (Table 73.1). This was due to the subsequent discovery that vitamin D regulates the renineangiotensinealdosterone (ReAeA) axis (see also Chapter 40) [37].
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TABLE 73.1
Selected Mechanisms Whereby Vitamin D Receptor Agonists Benefit Cardiovascular Physiology
Action
Citations
Decreased cardiac hypertrophy. Suppressed local renin production and paracrine angiotensin II signals that drive myocardial hypertrophic remodeling and vascular fibrosis. Preserved systolic and diastolic function
[40,42,44,45,49,104, 248,249]
Decreased systemic activation of the renineangiotensinealdosterone axis, reduced blood pressure, decreased arteriosclerotic remodeling, improved endothelial function
[49,52,53,243]
Preservation of MHC-alpha expression in response to myocardial stress and/or increased afterload. Maintain chamber-specific MHC expression
[45,47,250e252]
Enhanced cardiomyocyte contractility/inotropy via non-genotropic signaling (caveolae and T-tubule SERCA-2)
[35,54,55,253]
Enhanced myocardial relaxation kinetics via PKC activation and troponin I phosphorylation, improved diastolic function
[57]
Transcriptional suppression of myocardial atrial natriuretic peptide expression, reduced permeabilitydependent volume expansion
[45,49,254,255]
Inhibition of c-myc, cdk2 expression, and proliferative hyperplasia in cardiovascular myocytes. Inhibition of prohypertrophic endothelin signaling
[254,256,257]
Restoration of endothelial function, vasodilatory responses and NO production
[91,92,106,109]
Reduced circulating TNF and other inflammatory cytokines
[100,118]
Inhibition of TNF signaling in endothelial cells and monocyte adherence
[5,103,107,108,110,243, 255,258,259]
Downregulation of thrombomodulin and anti-thrombin, maintenance of anti-thrombotic homeostasis
[122]
Increased circulating anti-inflammatory cytokine IL10, reductions in IL12
[4]
Reduced cardiovascular oxidative stress
[6]
Decreased foam cell formation, inhibit oxidized LDL uptake by macrophages, reduced pro-calcific macrophage TNF production
[89,90,123]
Upregulation of osteopontin production by macrophages and inhibition of arteriosclerotic calcium accrual
[119]
Suppressed VSMC proliferation, decreased intimal-medial thickness
[256,260]
Prevention of hyperparathyroidism-induced endothelial dysfunction, diastolic dysfunction
[249,261e265]
The ReAeA axis plays a critical role in control not only of blood pressure, but also the pro-fibrotic hypertrophic remodeling responses that arise in the myocardium with increased afterload and/or ischemia [38,39]. In 2002, Li et al. reported on the negative regulation of the ReAeA axis by VDR agonists via the VDR [37]. First, they demonstrated the presence of hypertension and myocardial hypertrophy in VDR-null mice, with concomitant increases in activity of the ReAeA axis and plasma angiotensin II (AT-II) levels. In addition, administration of calcitriol to wild-type mice reduced renal renin expression. Moreover, inhibition of endogenous calcitriol synthesis with dietary strontium upregulated renin mRNA accumulation. Subsequent studies demonstrated that genetic lesioning of CYP27B1, the 25(OH)D 1a-hydroxylase, phenocopied the myocardial hypertrophic responses of VDRKO mice [40,41], confirming the relevance of vitamin D endocrinology to cardiovascular physiology. Importantly, myocardial hypertrophy persisted in the presence of “rescue diets”
that normalized serum calcium and phosphate levels, indicating that anti-hypertrophic actions of vitamin D signaling are in part independent of the systemic calcium phosphate milieu. This VDR-dependent control of cardiovascular physiology was independent of changes in ambient calcium levels, and ligand-activated VDR directly suppressed renin promoter activity by antagonizing CREB-dependent support of transcription in renal juxtaglomerular cells [42]. Furthermore, Ren1c-VDR transgenic mice, possessing augmented levels of VDR selectively within the juxtaglomerular cell, exhibit significantly reduced plasma renin activity, although blood pressure was not significantly altered [43]. Overlapping yet distinct results were reported by Simpson and colleagues (see Chapter 104). While myocardial hypertrophy with myocardial fibrosis was observed, significant changes in the ReAeA system and blood pressure were not noted in their studies of VDRKO mice [44]. Collagen gene expression and
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73. VITAMIN D: CARDIOVASCULAR EFFECTS AND VASCULAR CALCIFICATION
MMP9 activity e key targets of AT-II-dependent hypertrophic remodeling within the cardiovascular system e were upregulated in VDRKO mice. Thus, Simpson et al. emphasized that the ability of VDR to negatively regulate myocardial hypertrophic remodeling is cellautonomous e which is indeed the case (see below) e but independent of the systemic ReAeA endocrine axis. Support for the role of local tissue renin signaling in VDR regulation of hypertrophic remodeling has been independently forthcoming from studies of saltsensitive Dahl hypertensive rats [45]. In this model, high-salt diets increase blood pressure and myocardial expression of the renin gene. Paricalcitol, a vitamin D receptor agonist with reduced calcemic activity, suppresses hypertrophy and renin expression in this diet-induced hypertensive model. Cardiac brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) levels were also reduced, but consistent with Simpson et al., paricalcitol did not reduce blood pressure. Importantly, paracalcitol treatment also preserved alpha-myosin heavy chain (MHC) expression [45] e a physiologically desirable response that reverses maladaptive, hypertension-induced MHC isoform switching [46e48]. Of note, myocardial renin is dramatically upregulated in VDRKO mice [49] as observed in hypertensive Dahl rats [45]. Furthermore, distressed cardiomyocytes express not only renin but the prorenin receptor ATP6AP2 [50] and tissue angiotensin converting enzyme (ACE) [38] e and myocardial stretch upregulates expression of angiotensinogen [51]. Furthermore, ACE inhibitors improve myocardial hypertrophy in VDRKO and CYP27B1-null mice within the setting of elevated plasma renin, independent of calcium and phosphate homeostasis [40,52]. Thus, inhibition of local activation of the renineangiogensin system and subsequent downregulation of paracrine AT-II actions are likely critical to VDR-dependent reductions in hypertrophic remodeling and myocardial fibrosis [51]. Nevertheless, recent data from LURIC (Ludwigshafen Risk and Cardiovascular Health) demonstrate that circulating 25(OH)D levels are independently and inversely related to plasma renin activity in humans [53]. Circulating angiotensin II and aldosterone levels are clearly elevated in VDRKO mice [37]. Therefore, vitamin-D-dependent downregulation of systemic ReAeA activity may also contribute to reductions in hypertrophic cardiovascular remodeling. Studies examining vascular responses to myocardialspecific conditional VDR deletion in mice promise to be enlightening. In addition to impacting the ReAeA system and regulating MHC isoform expression, vitamin D exerts direct, rapid, non-genotropic actions on cardiomyocytes that may augment inotropy (cardiac pumping force). Simpson and colleagues identified that a distinct pool
of VDR was associated with plasma membrane and contiguous myocardial T-tubule caveolar complexes [54]. In ex vivo experiments of single cell inotropic imaging studies of cardiomyocytes, calcitriol treatment accentuates excitation-induced contractility as evidenced by sarcomere and cell shortening [55,56]. VDR interacts with SERCA2, the sarcoplasmic reticulum (SR) Caþþ ATPase, and directly modulates the kinetics of SR calcium uptake and egress that determine relaxation and contractility, respectively. Transient increases in cytoplasmic calcium induced by calcitriol activate protein kinase C (PKC) which helps to accelerate the subsequent relaxation phase of the myocardial contractionerelaxation cycle [57]. The precise composition, role, and regulation of the non-nuclear VDR complexes relevant to cardiomyocyte function in vivo has yet to be explored (see also Chapter 31).
VITAMIN D ACTIONS IN ATHEROSCLEROSIS AND ARTERIOSCLEROSIS The first studies of vitamin D in cardiovascular disease, atherosclerosis and arteriosclerosis emphasized toxicity [16,60,61] (see “The impact of calcium, phosphate, and vitamin D excesses on smooth muscle matrix vesicle physiology and vascular calcification,” “The FGF23/Klotho/vitamin D axis, nephrocalcinosis, and cardiovascular calcification,” and “Vitamin D, vascular PTH receptor signaling and arteriosclerotic calcium accrual: lessons learned from preclinical models and patients with end stage renal disease,” below). Vitamin D intoxication in nicotine-treated rats induces arteriosclerotic calcification [16]. Elastinolysis [62] and perturbed elastin biogenesis figure prominently [63e65], and proteolysis generates bioactive elastin degradation products that augment trans-differentiation of vascular smooth muscle cells (VSMCs) to the mineralizing osteochondrogenic lineage [66e68]. Proteolytic degradation also stiffens the vessel wall and provides foci for elastocalcinotic mineralization. Elastocalcinotic vessel stiffening [16,20] impairs conduit artery Windkessel function [69], thus increasing cardiac workload and inducing myocardial hypertrophy [70,71] even in the absence of overt hypertension [16]. (In this setting, Windkessel refers to the rubbery elasticity of conduit vessels that provides a functional reservoir that dampens fluctuations in blood pressure and thus organ perfusion throughout the cardiac cycle.) The precise mechanisms whereby vitamin D agonists promote arterial calcification are as yet unknown but multifactorial; contributions include increased bone resorption and the release of mineralizing serum calciprotein particles [19,72e74], frank elevations in serum phosphate and
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VITAMIN D ACTIONS IN ATHEROSCLEROSIS AND ARTERIOSCLEROSIS
calcium [75], and the downregulation of VSMC PTHrP [76]. Vascular PTH/PTHrP receptor activation reduces pro-fibrotic and pro-calcific signaling in the vessel wall and reduces vascular stiffness [77]. Extracellular PTHrP, released by VSMCs in response to mechanical stretch, functions as a paracrine stimulus for vascular PTH1R activation [76]. Jono and colleagues were the first to demonstrate that 1,25(OH)2D3 promotes VSMC alkaline phosphate expression and mineralization in part by downregulating VSMC PTHrP, and that PTHrP treatment suppress calcitriol-mediated activation of VSMC mineralization [76]. Furthermore, vitamin D toxicity increases circulating phosphate calcium levels, another potent stimulus for vascular calcification via SCL20A1/PiT1-mediated activation of BMP2, Runx2, and Msx2 osteogenic signaling [75,78e81]. Of note, inhibition of MMP9, a collagenase with elastase bioactivity, reduces arteriosclerotic calcification [82]. Similarly, in models of uremic atherosclerosis, cathepsin S elastase activity plays an important role [83]. Whether these proteases are important in the pathobiology of vitamin D intoxication has yet to be examined. Thus, the arteriosclerotic disease of vitamin D intoxication is well appreciated, and will be considered in detail in subsequent sections. However, it has become increasingly apparent that vitamin D insufficiency also conveys significant risk for atherosclerotic and arteriosclerotic disease (Table 73.1). A recent prospective epidemiological study of Framingham offspring demonstrated that serum 25hydroxyvitamin D levels under 15 ng/ml (37.5 nmol/l) are associated with a 60% increase in the risk of incident cardiovascular disease in those individuals without known prior CVD [1]. This significant risk relationship was identified in a multivariate adjusted model that accounted for the traditional Framingham risk factors. Moreover, risk of CVD was even greater for those with 25(OH)D levels under 10 ng/ml (p ¼ 0.01 for trend) [84]. Events existed primarily of fatal or non-fatal MI (>50%) or fatal or non-fatal CVA (~30%, all CVAs nonhemorrhagic), indicative of atherosclerotic and/or arteriosclerotic disease. Along with the NHANES prospective study [85], the Framingham offspring analysis provides important support for causation suggested by the cross-sectional associations identified in LURIC between vitamin D deficiency and CVD (angiographic CAD, CHF, sudden death) [53]. Lower serum 25(OH)D levels are also associated with higher prevalence of peripheral arterial disease (PAD) [86e88]. Why might this occur? Normal vitamin D tone plays a critical role in limiting foam cell formation, and vascular inflammation at least in preclinical and translational studies models of atherosclerosis [89]. Bernal-Mizrachi and colleagues recently demonstrated that VDR agonists such as calcitriol suppress uptake of cholesterol,
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diminish foam cell formation, and suppress macrophage activation [90]. Using primary human blood mononucleocytes from obese T2DM patients e a cohort at increased risk for macrovascular disease e they demonstrated that uptake of oxidized low-density-lipoprotein (LDL) cholesterol was markedly reduced by 10 nM calcitriol. Similar results were obtained using peritoneal macrophages obtained from LDL receptor (LDLR)/ mice fed high-fat diets either sufficient or insufficient for nutritional vitamin D supplementation [90]. The macrophage endoplasmic reticulum (ER) stress response, indexed by GADD34 and CHOP1 expression, was also diminished by vitamin D supplementation cells obtained from diabetic patients. ER stress elicits many of its negative effects on cellular physiology via impaired mitochondrial function and increased reactive oxygen species (ROS) generation [90]. Vitamin D insufficiency results in remediable endothelial dysfunction [91,92] which is reminiscent of that seen with vascular inflammation and oxidative stress [93e96]. Indeed, vitamin D insufficiency is associated with increased levels of circulating inflammatory cytokines such as TNF that induce ROS production [97,98], and vitamin D replacement reduces TNF levels [99,100]. Since ROS figure prominently in the pathobiology of endothelial dysfunction, neointima formation, vascular arteriosclerosis, and atherosclerotic calcification [101,102], the vascular “senescence” phenotype [71] of vitamin D insufficiency may relate in part to worsening mitochondrial dysfunction and ROS generation [38,101]. However, these notions have yet to be directly assessed in vivo. The beneficial actions of vitamin D signaling in the endothelium have only recently been examined in any detail. Paricalcitol, a clinically useful VDRA with reduced calcemic actions, restores endothelial function and cholinergic-dependent vasodilation in uremic rats [103]. This effect was independent of changes in serum calcium and phosphate, but fully dependent upon paracrine nitric oxide (NO) actions. Moreover, improvement of endothelial function was also independent of changes in PTH levels, since reduction of secondary hyperparathyroidism with a type II CaSR calcimimetic did not improve endothelial vasodilatory responses [103]. Vitamin D3 supplementation in a spontaneously hypertensive rat model e a model with impaired calcitriol biogenesis [104,105] also restores endothelial function [106], pointing to the generality of the response. Activation of the VDR inhibits NFkB signaling in endothelial cells (ECs) [107,108], and prevents TNF and advanced glycosylation product inhibition of eNOS [109]. Inflammatory cytokines induce EC CYP27B1-dependent production of calcitriol by ECs, and thus may participate in the autocrine/intracrine feedback inhibition of cytokine actions [110]. Thus, by maintaining robust eNOS
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activity, VDR tone preserves vascular endothelial function and decreases proinflammatory cytokine signaling in the vasculature. Importantly, vitamin D supplementation in asymptomatic individuals with vitamin D insufficiency restores flow-mediated dilation in humans, an assay of endothelium- and NO-dependent signaling [91]. Similar observations have been reported in vitamin-D-insufficient patients with type II diabetes [92]. Thus, preclinical and clinical studies converge to highlight the important role for endocrine and autocrine vitamin D signaling in the regulation of endothelial function. However, the study of EC VDR signaling and autocrine/intracrine actions of vascular calcitriol production is in its infancy. In addition to actions on cardiac remodeling emphasized above, AT II is a potent vascular pressor that drives oxidative stress of the tunica adventitia (outermost arterial wall layer) [111e113], promotes hyperplastic vascular remodeling [114], and induces mitochondrial dysfunction [38]. AT II upregulates Nox2-based arterial superoxide production in adventitial myofibroblasts [115]. Superoxide reacts with nitric oxide to form peroxynitrite; thus, AT II generates an adventitial-to-medial superoxide gradient that antagonizes vasodilatory intimal-to-medial nitric oxide signals via chemical nullification [115]. As such vitamin D downregulation of vascular renineangiotensin signaling also contributes to improvements in endothelial function. AT-II [116] and TNF [117] -regulated adventitial inflammation may also be enhanced with vitamin D insufficiency [118]. Vitamin D is an immune modulator (detailed in Section XI of this volume) that controls atherosclerotic Th1/Th2 T cell subsets, mediated in part by enhancing elaboration of anti-inflammatory IL-10 to IL-12 ratios by dendritic cells [4]. Therefore, vascular insult that arises from vitamin D insufficiency/deficiency may also reflect exaggerated adventitial oxidative stress and mural inflammation. Indeed, the VDRA paricalcitol reduces cardiovascular oxidative stress in uremic rats [6]. The molecular mechanisms of these effects, however, have yet to be detailed. Very recently, Giachelli and colleagues [119] identified a novel mechanism whereby VDR signaling in the macrophage directly inhibits arteriosclerotic calcification via enhanced osteopontin (OPN) production [119]. OPN is both an extracellular matrix adhesion molecule and inflammatory cytokine. The poly-phosphorylated form of OPN is a potent inhibitor of matrix mineralization, and enhances monocyte/macrophagemediated calcium egress from sites of ectopic mineral deposition [120]. However, when dephosphorylated by alkaline phosphatase [121], extracellular OPN loses the capacity to inhibit mineralization e and becomes susceptible to thrombin proteolysis [120] that generates pro-inflammatory peptide fragments [98]. As
mentioned above, the diverse mechanisms whereby vitamin D signaling interacts with proteolytic cascades that control extracellular matrix remodeling relevant to arteriosclerotic disease e including OPN processing and elastinolysis e has yet to be fully explored. Importantly, however, in addition to enhancing macrophage OPN production, vitamin D increases anti-thrombin thrombomodulin [122]. Thus, net actions of physiological vitamin D tone are predicted to inhibit thrombin generation, reduce thrombogenicity, augment fulllength OPN production by monocyte/macrophages, and diminish vascular calcium accrual as described in a number of preclinical disease models. At this point, the relationships between cells of the monocyte/macrophage lineage and ectopic mineral deposition should be discussed. As first noted by Demer, oxidized LDL stimulates mineralization of calcifying vascular cells (a type of pericytic myofibroblast), augmented by signals provided by the monocyte/ macrophage cells that include TNF and other unidentified paracrine signals that upregulate AKP2/ALP expression [123]. Once ectopic mineral is deposited in the vasculature, it provides a pro-inflammatory stimulus for monocyte/macrophage release of TNF [124]. Hence, independent of the initiating mechanism of deposition (diabetes, renal failure, vitamin D intoxication), calcium phosphate accrual in the vasculature may elicit a second phase of inflammation, and subsequent vascular mineralization that is dependent upon monocyte/macrophage-derived signals. Yet, cells of the monocyte/macrophage lineage also enhance calcium egress via carbonic anhydrase-based matrix acidification [125]. In a rat model of calcitriol-induced intoxication, arterial calcium deposition was identified as being reversible and associated with CD68þ cells as vascular calcium deposits; intact renal function was a critical consideration and required for reversibility of vascular mineralization [126]. Whether different monocyte/macrophage subsets e e.g. M1, M2, CD1, osteoclast-like cells, dendritic cells e differentially contribute to ectopic mineral deposition and mineral egress is as yet unknown.
VITAMIN D AND HUMAN CARDIOVASCULAR DISEASE: COMPELLING EPIDEMIOLOGY AND PHYSIOLOGY, EMERGING BUT LESS COMPELLING EVIDENCE OF INTERVENTIONAL BENEFIT IN 2010 NHANES, LURIC, and most convincingly Framingham Offspring Study have provided compelling evidence for the positive predictive value of vitamin D insufficiency for CVD even after adjusting for traditional
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cardiovascular risk factors [53,84,85]. Moreover, the implementation of VDR agonists has had a significant impact upon cardiovascular survival in the setting of end-stage renal disease [15]. Common genetic polymorphism in the CYP27B1 gene that impairs 1,25(OH)2D3 biosynthesis increases the risk of CHF in patients with hypertension [127]. Moreover, vitamin D supplementation improves endothelial dysfunction [91,92] and insulin sensitivity [128,129] in those with hypovitaminosis D. Yet, in the absence of end-stage renal disease (ESRD), the clinical cardiovascular benefits of vitamin D repletion or supplementation have been remarkably equivocal. In a recent randomized, double-blind, placebo-controlled trial of elderly patients with systolic heart failure and vitamin D deficiency (25(OH)D <20 ng/ml), oral supplementation with 100 000 IU ergocalciferol (D2) every 10 weeks (approximately 1400 IU/day on average) had no effect upon physical performance as an index of CHF severity [130]. Standardized 6-minute walk and timed up and go tests did not improve, even though serum natriuretic peptide (BNP) levels were significantly reduced by D2 [130]. This observation was all the more disappointing since low 25(OH)D and elevated C-reactive protein (CRP) are associated with reduced functionality in this same physiological assay of frailty [84,131]. However, the intervention and evaluation only spanned a 20-week timeframe, and no changes in circulating TNF were observed in this D2-based intervention. Previously, Schleithoff and colleagues demonstrated that supplementation with 50 micrograms (2000 IU) of daily cholecalciferol (D3) with 500 mg of calcium for 36 weeks significantly reduced circulating TNF levels versus calcium supplementation alone [100]. A reciprocal change was also observed in the anti-inflammatory cytokine IL10, and BNP was significantly suppressed. However, clinically relevant functional endpoints were not assessed. Moreover, as divulged by Heaney [132], Romagnoli [133] and colleagues, D3 is at least 2e3 times as potent as D2 with respect to achieving serum 25(OH)D levels and regulating serum PTH e and differences in biological potency not yet reflected in “units” of activity as prescribed. The Women’s Health Initiative (WHI) evaluated the impact of calcium plus 400 IU of daily D3 supplementation on cardiovascular health endpoints (events or blood pressure control), and found no evidence of benefit CVD [134]. Yet, once again, the PKePD relationships for D3 and D2 for cardiovascular endpoints have yet to be established e and the 400 IU (10 micrograms) of D3 given daily in the WHI is likely to be inadequate for robust nutritional responses in middle-aged to older individuals. Calcium was co-administered e a potentially confounding variable [135] e and compliance is also an issue; indeed, the primary endpoint response of musculoskeletal frailty (fracture reduction) was only observable in
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a subset of patients that took 80% of the calcium þ D3 [136]. Supplementation clearly improves endothelial function (see above), but as our public health guidelines for daily recommended intakes of vitamin D increase, as a practicality it will become increasingly difficult to robustly establish the benefit of nutritional vitamin D supplementation on cardiovascular disease endpoints in randomized prospective studies. Moreover, a recent meta-analysis suggests that calcium supplementation may negatively impact and confound interpretation of many of the current studies evaluating the impact of vitamin D intervention on CVD endpoints [135]. Prospective studies implementing new non-calcemic VDRAs in early-stage CHF with cardiovascular phenotyping in addition to clinical endpoints may also provide this much needed information. Additionally, a recently initiated study of 20 000 adults (The Vital Trial), randomized to be treated with either 2000 IU of daily vitamin D3 with or without 1 g daily omega-3 fatty acid versus control (800 IU of D3) will do much to improve our understanding of how higher-dose D3 supplementation impacts CVD as a primary endpoint [137]. In MESA, the Mutliethnic Study of Atherosclerosis, 25(OH)D levels under 15 ng/ml (37.5 nmol/l) were predictive of incident development of coronary artery calcification (CAC) in a 3-year follow-up period [138]. This inverse relationship appeared to be even stronger in those with reduced renal function (GFR <60 cc/min/1.73 m2). Vascular calcification is clinically predictive in both PAD [139] and CVD [140], and these observations are consistent with the protective role of adequate vitamin D tone on vascular health as outlined above. Of note, MESA had approximately 30% AfricanAmerican participants, 20% Hispanic participants, 10% Chinese participants, and 40% Caucasian participants [141]. However, a very recent paper from Freedman and colleagues reported a positive relationship between 25(OH)D levels and abdominal or carotid calcification in African-Americans [142]. Furthermore, no relationship to coronary artery calcification was observed in the African-American Diabetes Heart Study [142]. Taken together, these reports indicate that marked ethnic, gender, and histoanatomic differences will exist in the regulation of vascular calcium accrual e as previously noted [143]. These clinically important differences in arteriosclerotic disease have only begun to be appreciated, and are poorly understood.
VITAMIN D INTOXICATION AND CARDIOVASCULAR CALCIFICATION: PHARMACOLOGICAL CONSIDERATIONS A discussion of vitamin D toxicity must take into account the major differences in pharmacology of
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vitamin D, its metabolites, and pharmacokinetice pharmacodynamic (PKePD) relationships with respect to calcium homeostasis [144] (see also Section I of this volume). Those features most relevant to subsequent consideration of tissue calcinosis, including cardiovascular calcification, are emphasized here. Vitamin D toxicity is also discussed in Chapter 72. Two significant sources of vitamin D exist in our society: (1) photoisomerization of 7-dehydrocholecalciferol by UVB light in the skin to generate vitamin D3; and (2) diet and dietary supplementation with either vitamin D3 or D2. Both cholecalciferol and ergocalciferol are hydroxylated by a low affinity/high capacity 25hydroxylase of the hepatic endoplasmic reticulum, CYP2R1, to generate 25-hydroxyvitamin D (25(OH)D). Although mitochondrial P450s (e.g., CYP27A1) can and do generate 25(OH)D, proof that microsomal CYP2R1 is the physiologically relevant hepatic 25hydroxylase comes from studies of CYP2R1-deficient humans with rachitic phenotype [145]. CYP2R1 is constitutive and unregulated and has a Km (500 nM) for vitamin D that is high when compared to physiological ranges of substrates. This means that 25(OH)D formation is substrate (vitamin D) limited, and the 25(OH)D product is produced as an approximately linear function of substrate availability as follows from Michaelis-Menton kinetics (e.g., 25(OH)D formation rate f [S] [E]/Km, where [E] ¼ hepatic CYP2R1 enzyme concentration and Km >> [S] ¼ vitamin D concentration, even with fluctuations in the latter). One can appreciate from this relationship how severe chronic liver disease causes physiological vitamin D insufficiency e but this relationship only tangentially hints as to how vitamin D toxicity unfolds. The relatively long-lived pro-hormone 25(OH)D is highly bound by circulating vitamin-D-binding protein. DBP-bound 25(OH)D is taken up by target tissues (e.g., kidney, bone, intestine, endothelium, and hematopoietic cells) and converted by a highly specific 1a-hydroxylase, CYP27B1, to a much more potent VDR agonist, 1a,25(OH)2D. In the circulation, both 1a,25(OH)2D and 25(OH)D are bound to DBP [146]; however, 1a,25(OH)2D has lower affinity for DBP and thus a much shorter half-life (~1 day, Kd ~200 nM) than 25(OH)D (~1 month; Kd ~50 nM), and is catabolized by multiple side-chain hydroxylases to generate inactive metabolites [144]. Although 25(OH)D is two orders of magnitude less potent than 1a,25(OH)2D as a vitamin D receptor agonist, 25(OH)D does suppress PTH production by bovine parathyroid cells with an IC50 of 2 nM [147]; given its relatively higher serum concentration, this is a meaningful response with respect to vitamin D toxicology (see below). In general, in healthy individuals with intact renal parenchyma, circulating 1a,25(OH)2D levels primarily
reflect PTH tone and actions on the renal proximal convoluted tubule. Indeed, in children with rachitic bone e or in the CYP2R1-deficient genetic state e low 25(OH)D, elevated PTH (secondary hyperparathyroidism), low normal serum calcium, hypophosphatemia with relative phosphaturia and upper-limit-normal or elevated 1a,25(OH)2D levels are noted; the latter are due to secondary elevations in PTH [145]. However, in the setting of hypercalcemia, this very same level of 1a,25(OH)2D e with suppressed serum PTH, elevated or upper-limit-normal serum phosphorus and hypercalciuria e would be taken to indicate a vitamin-Dmediated toxicity! For example, in hypercalcemia arising from granulomatous sources of 1a-hydroxylase (e.g., sarcoid) or B cell lymphoma e associated macrophages [148], this biochemical pattern provides the robust clinical evidence that calcitriol excess is the endocrine etiology of hypercalcemia e and can be observed even in the setting of mild 25(OH)D insufficiency with seasonality [149]. Such endocrine physiology points to the problem of simply relating measurements of 1a,25(OH)2D levels and pharmacologically less potent 25(OH)D levels with respect to toxicity thresholds [144]; clinical interpretation of the vitamin D serum biochemistries currently measured is almost entirely context-specific. Consideration of toxicity threshold is also complicated by the physiology of vitamin-D-binding protein (DBP), a molecule that clearly contributes to the pharmacokineticepharmacodynamic (PKePD) relationships for vitamin D. Like sex steroid hormones, the vitamin D secosteroids (D2 and D3), calcifidiol, and calcitriol are all carried in the blood stream on a hepatocytederived, secosteroid-specific binding protein, DBP [146] described in detail in Chapter 5. Haddad and colleagues were among the first to discover DBP, and actually implemented it in clinically useful assays of serum 25(OH)D [150,151]. DBP circulates at remarkably high concentrations, ca. 5 mM, in great molar excess to all known ligands [152]. Cooke, Haddad and colleagues subsequently identified the surprising fact that while murine DBP deficiency increased the risk for secondary hyperparathyroidism on vitamin-Ddepleted diets, DBP deficiency concomitantly decreased sensitivity of mice to vitamin-D-intoxication and hypercalcemia [153]. Moreover, the kinetics of calcitriol actions on induction of key regulators of intestinal calcium transport were accelerated and exaggerated. Thus DBP emerges as a key regulator of vitamin D PKePD e e.g. metabolite bioavailability, metabolism and activation, and target organ sensitivity as well as toxicity [153]. The function of DBP as a “buffer” for storing 25(OH)D and providing regulated rapid delivery for 1a-hydroxylation in target tissues is intuitive with respect to
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modifying risk for secondary hyperparathyroidism. But how would absence of the DBP “buffer” decrease sensitivity to vitamin D intoxication while increasing intestinal sensitivity to calcitriol [144]? This relates in part to the significant difference that exists between calcifidiol (25(OH)D), calcitriol (1a,25(OH)2D), and vitamin D (D2, D3), for DBP, with Kd values of approximately 50 nM, 200 nM, and 1000 nM, respectively [144]. Intoxication with vitamin D displaces bioactive 25(OH)D and 1a,25(OH)2D from DBP e a “feedforward” process that occurs because (1) the hepatic 25-hydroxylase is constitutive as noted above and monotonically increases 25(OH)D production with increases in vitamin D availability; and (2) activated VDR induction of 24-hydroxylase generates increasing amounts of 24,25(OH)2D. Why is the latter relevant? Although inactive for classic pro-calcemic VDR activation, like 25(OH)D the inactive metabolite 24,25(OH)2D also exhibits high affinity for DBP e approximately 50 nM [144]. Thus, vitamin D intoxication involves the activity of both 1a,25(OH)2D and 25(OH)D displaced from DBP stores. In the absence of DBP, susceptibility to acute intoxication is reduced e but vitamin D stores are also concomitantly reduced and the consequences of nutritional deficiency are accelerated [153]. The PKePD relationship for vitamin D toxicity is clearly dependent upon DBP. Once again, however, as noted above the relationships between total, bound and free levels of 25(OH)D, 1a,25(OH)2D, and tissue calcinosis are not well established e and may not predictably correlate. Moreover, current clinical assays do not discriminate between bound and free forms of vitamin D and its bioactive metabolites. What are the lessons to be learned? First, the dynamic interactions between the parathyroids, kidneys, bone, and DBP determine the toxicity threshold for vitamin D e i.e. whether hypervitaminosis D is associated with actual toxicity. In the otherwise healthy individual, the dynamic interactions between the parathyroid gland, kidney, and bone set the context, since it is these interactions that control minute-to-minute calcium homeostasis and that are exquisitely responsive to vitamin D and calcium intake. The inability to adjust urinary excretion of calcium and bone formation rates to compensate for increases in bone resorption and intestinal calcium absorption driven by vitamin D excess ultimately determine toxicity threshold. Moreover, while protective in the short term, in the long term compensatory hypercalciuria becomes nephrotoxic and thus “resets” the threshold for calcium- and phosphate-mediated vitamin D toxicity to a lower level. Second, consideration of risk thresholds for vitamin D toxicity must encompass this integrated physiology e i.e. serum and urinary calcium flux, serum phosphate levels, bone turnover, extant renal function and vascular
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disease, and vitamin-D-mediated perturbations in PTH/PTH1R and FGF23/Klotho signaling (see below). These physiological parameters change with growth, maturation, and aging. Moreover, ectopic mineralization is not simply a uniform reflection of elevations in calcium phosphate product as once thought; now well debunked by O’Neill [154]. Ectopic mineral deposition is an actively regulated process [155], and mechanistic heterogeneity exists in tissue calcinosis. Finally, this physiology closely relates to, but is not precisely co-registered with, our current serum measurements of vitamin D and its metabolites. The clinical interpretation of serum 25(OH)D and calcitriol levels with respect to toxicity is completely dependent on physiological context. These three lessons, combined with the heterogeneous pathobiology of tissue calcinosis, highlight why a uniform tolerable upper intake limit for vitamin D (and calcium) has been difficult to assign with precision.
THE IMPACT OF CALCIUM, PHOSPHATE, AND VITAMIN D EXCESSES ON SMOOTH MUSCLE MATRIX VESICLE PHYSIOLOGY AND VASCULAR CALCIFICATION Vitamin-D-driven tissue calcinosis e whether associated with excess ingestion, or dysregulated 1ahydroxylase activity e is associated with elevation in serum calcium and phosphorus. Our understanding of how changes in serum calcium and phosphate contribute to tissue calcification has largely been oriented by the needs of our patients with end-stage renal disease (ESRD). In the setting of declining renal function, multiple major perturbations in calcium phosphate homeostasis increase the risk of cardiovascular disease and correlate with arterial calcium deposition [14]. Arterial calcium deposition contributes to vascular stiffening in valves and conduit vessels, impairs blood flow and increases myocardial workload [69,71,156]. Since 40% of patients that develop ESRD do so because of antecedent diabetes, many of these patients will have significant arterial- calcium loads even prior to declines in renal function. At every level of renal function, glycemic control contributes to the extent of vascular calcification [157]. However, beginning at CKD3, FGF23 and PTH levels rise and postprandial fractional excretion of calcium becomes impaired [158] e and the attendant impact that even transient elevations in serum calcium may have on VSMC mineralization [159] and vascular function [160] have become increasingly important to consider. Shanahan, Giachelli, and their colleagues have performed the most enlightening studies with respect to calcium- and phosphate-driven vascular calcification. Giachelli and Moe first identified that inorganic
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phosphate (Pi) could actively promote the osteochondrogenic trans-differentiation of VMSCs in culture [79]. Signaling via the sodium phosphate symporter SLC20A1 (PiT1, Glvr1) upregulates VSMC expression and activity of Runx2/Cbfa1, the master osteochondrogenic transcription fact of skeletal development that drives the osteogenic trans-differentiation program [79,80]. However, Shanahan, Jahnen-Dechent et al. recently identified an additional component of VSMC physiology highly relevant to the pathobiology of vitamin D intoxication [159,161]. When exposed to elevated levels of either extracellular calcium or phosphate, the arterial VSMC elaborates extracellular matrix vesicles (MVs) and apoptotic bodies that can nucleate hydroxyapatite mineral deposition [159]. Moreover, in a process that requires both MV-associated matrix gla protein (MGP) and serum fetuin, VSMCs can clear MVs from the extracellular milieu via a newly described pinocytotic process [159,161]. The regulation of elaboration and uptake of VSMC matrix vesicles is poorly understood, and has not been studied in great detail. Additionally, calcitriol treatment of VSMCs upregulates “bone” alkaline phosphatase (AKP2) [76], an ectoenzyme known to be associated with MVs bound to elastin in vascular lesions in vivo [162]. In toto, these data point to the importance of the VSMC in regulating vascular mineralization in the setting of calcium phosphate extremes and vitamin D excess. With dialysis-induced oxidative stress, fetuin reductions, transient elevations in serum calcium and phosphate, and VSMC apoptosis [163], the balance favors net vascular mineral accrual over time [7]. Should similar MV clearance functions be shared by pericytes (multipotent microvascular smooth muscle cells) as well as by VSMCs, the impairment of this important cell physiology may be highly relevant to soft tissue calcification in addition to macrovascular mineralization. In a small subset of patients with chronic kidney disease who are treated with warfarin anticoagulation (also inhibits MGP gamma-carboxylation, osteogenic BMP inhibition, and MV uptake) [161,164], lifethreatening widespread tissue calciphylaxis and calcific uremic arteriolopathy occur [165,166]. In addition to amorphous calcium phosphate deposition in pulmonary, intestinal, and dermal parenchyma, arteriolar (<0.6 mm diameter) medial calcification arises with fibroproliferative occlusion that infarcts dermal fat and leads to skin necrosis. Similarly, fetuin-deficient mice develop pulmonary and intestinal calcification with nephrocalcinosis e but only on certain genetic backgrounds and in the absence of significant aortic calcification [167]. Additionally, Price et al. have demonstrated that with vitamin D intoxication, fetuin:calciprotein complexes increase in circulation that can nucleate mineralization in elastin-rich matrices such as lung,
artery, and dermis [74]. Whether vitamin-D-intoxicationinduced bone resorption increases formation of fetuin:calciprotein complexes at the expense of fetuin:MV complexes that help VSMCs to clear extracellular MVs is unknown at this point. The functional relationships between fetuin, MGP, and MV biology and vitamin D intoxication remain to be studied. At this point, it should be emphasized that arterial calcification of aging, diabetes, dyslipidemia, and uremia in the absence of vitamin D toxicity is an overlapping yet distinct process [155,168]. Once considered only a passive process of dead and dying cells, data from laboratories worldwide have identified that vascular calcification is an actively regulated process, characterized by arterial elaboration of osteochondrogenic gene regulatory programs that participate in skeletal mineral deposition [155]. Based upon histoanatomic and molecular criteria, at least five types of vascular calcification can be identified: atherosclerotic intimal calcification, medial artery calcification, calcific aortic valve disease, calcification of chronic kidney disease (CKD), and calcific uremic arteriolopathy [155,168]. Detailed analyses of these disorders have recently been published [155,169]. In many substantive ways, the heterogeneity elaborated by vascular calcification reflects mechanistic heterogeneity in orthotopic mineralization programs e e.g. membranous ossification, endochondral ossification, dentinogenesis, amelogenesis, etc. e that contribute to skeletogenesis [170]. In general, a minimum of two processes contribute to the accumulation of actively osteogenic cells within the vessel wall: (1) osteochondrogenic lineage allocation of multipotent vascular mesenchymal progenitors; and (2) osteochondrogenic trans-differentiation of VSMC to an osteogenic phenotype [169]. The former mechanism contributes substantially to initiation and progression of diabetic medial artery calcification, while the latter is a key component of atherosclerotic vascular calcium accrual and disease progression. Both mechanisms appear to be active in calcific aortic stenosis, a vascular mineralization process that afflicts >2% of our population over age 60 and can result in vascular deposition of woven bone formation e complete with bone marrow and hematopoietic elements in ~15% of specimens [171]! A circulating bone-marrow-derived osteoprogenitor may also contribute to the osteogenic regulation of vascular calcification [172], but this has yet to be studied in detail. Common to all five of these vascular calcification processes is low-grade arterial/arteriolar inflammation and oxidative stress that activate ectopic osteogenic Msx2-Wnt/b-catenin and Runx2/Cbfa1 gene regulatory programs, with induction of bone alkaline phosphatase (AKP2, or ALP) [155]. AKP2 is of particular importance since this osteogenic ectoenzyme degrades extracellular inorganic pyrophosphate, a key paracrine inhibitor of
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vascular mineralization [173]. Important cytokine signals provided by TNF and RANKL mediate early [97] and late [174], respectively, inflammatory cues that promote vascular mineralization programs. Recent data have shown that TNF, hydrogen peroxide and oxidized lipid constituents of LDL cholesterol activate Msx2-Wnt/b-catenin and Runx2 osteogenic programs [97,123,175,176], while BMP4/ALK2 and hydrogen peroxide propagate RANKL- and Runx2- osteochondrogenic trans-differentiation [177,178]. In type II diabetes, a continuum of TNF-dependent initiation phases and RANKL-dependent progression phases are currently posited to drive vascular calcium accrual [155,168]. By increasing serum calcium phosphate, inducing VSMC apoptosis, suppressing of VSMC PTH/PTHrP receptor activation, increasing VSMC Runx2 signaling and AKP2 expression, and perturbing arterial elastin integrity, vitamin D intoxication promotes arteriosclerotic calcification by multiple mechanisms. This multifactorial process is somewhat similar to the calcification of CKD, and likely narrows the therapeutic window for VDR agonists in ESRD.
THE FGF23/KLOTHO/VITAMIN D AXIS, NEPHROCALCINOSIS, AND CARDIOVASCULAR CALCIFICATION The kidney receives about 20% of total cardiac output every minute, and of the ~10 000 mg or so of calcium filtered by the kidney each day, ~9800 mg is reabsorbed. About 85% is passively handled by the proximal tubule. The last 10e15% of filtered load is transported in the distal tubule via TRPV5, and is actively regulated by the calcium-sensing receptor (CaSR; inhibits transport) and PTH1R (stimulates transport) signaling at this site [179]. While elevation in serum calcium suppresses distal tubule uptake, PTH increases calcium uptake in distal tubule and is a critical determinant of hypercalciuria. (The importance of PTH on distal tubule renal calcium handling is evident in patients with congenital or surgical hypoparathyroidism.) Treatment of hypocalcemia in these individuals requires titrating albumincorrected calcium levels to slightly hypocalcemic levels but without tetany. This is because of the significant hypercalcuria and nephrocalcinosis risk associated with fully normalized serum calcium levels in the complete absence of endogenous PTH tone [180]. By contrast, the hypercalciuria of hyperparathyroidism occurs due to increased bone resorption and intestinal calcium absorption; with hypercalcemia the total amount of calcium filtered exceeds threshold capacity for distal tubule uptake even in the presence of enhanced PTH tone. Effects of CaSR activation exacerbate the hypercalciuria. Of note, excessive calcitriol
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dosing profoundly suppresses PTH [181], and increases both bone resorption and intestinal calcium absorption directly. Hence, the hypercalcemia arising with vitamin D toxicity suppresses PTH production, inhibits distal tubule calcium resorption, and promotes hypercalciuria via synergistic direct (increased serum calcium) and indirect (biochemical hypoparathyroidism) actions. While the hypercalciuria so induced protects against hypercalcemia, when long-standing this protective mechanism increases the risk for nephrocalcinosis as discussed next. The kidney, in doing its job to protect against cardiovascular and neuromuscular consequences of systemic calcium excess, places itself in a precarious position with prolonged hypercalciuria from any cause. Nephrocalcinosis comes in two anatomically defined variants: interstitial nephrocalcinosis and intratubular nephrocalcinosis [182,183]. Unlike the vascular calcification of atherosclerosis and diabetes [155,168] (see preceding section), active osteochondrogenic transcriptional regulatory programs have NOT been described for either type of nephrocalcinosis [182]. Rather, failure of the renal mechanisms that inhibit nucleation and propagation of the common calcium salts e calcium phosphate, calcium oxalate, and mixed salts due to epitaxial deposition e have emerged as a key component in pathobiology. Tamm-Horstfall protein (THP) and osteopontin (OPN) have been best studied as reviewed by Sakhaee, and OPN is in fact a direct genomic target of vitamin D signaling in most tissues [184]. However, major changes do occur in the tubular renal epithelial injury that promote calcium binding and nucleation. The histopathological hallmark of the earliest lesion in interstitial nephrocalcinosis is Randall’s plaque, a mixed apatitic calcium phosphate e calcium carbonate tissue mineralization that initiates near the thin segments of Henle’s loops and within the basement membrane of the associated vasa recta [185]. However, the source of these early lesions is unclear; lesions may in fact arise via the “trans-cytosis” of intratubular microcrystals with deposition and epitaxial propagation of mineralization within the interstitium [182]. In intratubular nephrocalcinosis, there is a failure in the defense mechanisms that inhibit nucleation and propagation of calcific microcrystals that form in concentrated urine. Moreover, tubule epithelial responses to metabolic, inflammatory, or ischemic insult upregulate hyaluronan and annexin V e calcium-binding proteins that can interact with microcrystals (normally not expressed on luminal surfaces). Current working models suggest that epithelial translocation (a.k.a. exotubulosis) to the interstitium permits disintegration and solubilization [182,183]. Autopsy studies demonstrate these lesions in over 50% of individuals using high-resolution calcium imaging techniques [185]; thus it is highly likely that
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such renal microcrystals are continuously made and dissolved throughout our lives [182,183]. The processes that determine and regulate rates of translocation and dissolution of renal calcinotic microcrystals are not known e but are presumably overwhelmed with time by long-standing hypercalciuria. Kidney stones are discussed in Chapter 71. Recently, a series of elegant clinical and preclinical genetic studies have identified that FGF23 signaling plays a critical role in the regulation of ectopic tissue calcification and nephrocalcinosis e and that calcitriol formation via CYP27B1/1a-hydroxylase activity figures prominently in this biology [186e188] (see also Chapter 42). FGF23 was originally identified as a tumor-derived phosphaturic factor that causes oncogenic osteomalacia; it does so not only by directly enhancing phosphate secretion, but also by inhibiting the 1a-hydroxylation of 25(OH)D and intestinal phosphate absorption. Subsequently, the role of FGF23 as an osteoblast-derived hormone [189] e important to both boneeparathyroid and boneerenal endocrine axes that control calcium phosphate homeostasis e became apparent [190]. PTH(1-34) stimulates bone formation and RANKL production reduces FGF23, while inhibition of RANKL-mediated bone turnover increases FGF23 production [191]. Osteoblast-derived FGF23 enhances phosphaturia, reduces active calcitriol by inhibiting renal 1a-hydroxylase and increasing vitamin D 24-hydroxylase-mediated vitamin D catabolism. These actions of FGF23 presumably avoid toxicity of hyperphosphatemia during states of high bone turnover. Physiology is still being studied, but data suggest that FGF23 also directly inhibits parathyroid PTH production in a negative feedback loop [192]. The role for FGF23 in soft tissue calcinosis became apparent from studies of humans and mice with inactive FGF23 or Klotho allele (Klotho is a co-receptor of FGF23, and signals via renal FGFR1(IIIc) to promote phosphaturia) [186]. In patients with tumoral calcinosis due to FGF23 deficiency, extensive tophus-like periarticular calcium phosphate concretions, dermal and internal organ soft tissue calcification, nephrocalcinosis and medial artery calcification accrue, the latter similar to pseudoxanthoma elasticum [193,194]. Elevated serum calcitriol levels and phosphorus levels are key biochemical features e with suppression of PTH production presumed to be due to the constitutively produced and longer-lived calcitriol. Indeed, elegant studies by Razzaque, Lanske, St-Arnaud and colleagues have demonstrated that 1a-hydroxylase induction and subsequent calcitriol production are key contributors to the pathophysiology of FGF23-deficient states [195e198]. FGF23/ and klotho/ mice phenocopy key features of the human tumoral calcinosis syndrome, including nephrocalcinosis, hyperphosphatemia, and
elevated calcitriol levels [196e198]. However, mice lacking both FGF23 and 1a-hydroxylase (murine CYP27B1) or klotho and 1a-hydroxylase are not only protected from FGF23 deficiency-induced cardiac calcification and nephrocalcinosis, but also exhibited reduced serum phosphate and calcium levels [195,196,198]. This strongly suggests that most of the soft tissue calcification and nephrocalcinosis observed with FGF23 deficiency and hyperphosphatemia is related to excessive calcitriol actions. Importantly, in preclinical models, Price et al. have clearly demonstrated that increased bone resorption e arising from vitamin D upregulation of RANKL and subsequent osteoclast activity e is required to drive soft tissue calcification and nephrocalcinosis of vitamin D intoxication accentuated by warfarin [19,72,73,199]. Increases in bone resorption are presumed to contribute to calcitriol-dependent soft tissue calcinosis in FGF23deficient mice as observed in vitamin-D-intoxicated rats, but this has yet to be confirmed. A direct role for FGF23-klotho signaling that helps protect against calcitriol-mediated toxicities cannot be excluded. For example, Bindels and Hoendroop have recently demonstrated that absence of klotho impairs TRPV5-mediated calcium transport in the distal tubule [200]. Calcitriol upregulates FGF23 production by osteoblast and osteocytes [189], and novel klotho-dependent actions such as just described e independent of phosphatonin function and calcitriol inhibition [200] e may also convey physiological mechanisms that mitigate nephrocalcinosis. Of note, pyrophosphate and fetuin have emerged as particularly important regulators of soft tissue calcification, including arteries and kidneys. The regulation of pyrophosphate and fetuin by FGF23 remains incompletely studied; however, in patients with intact renal function, FGF23 and fetuin levels do not correlate with coronary artery measures of calcium load [201].
VITAMIN D, VASCULAR PTH RECEPTOR SIGNALING AND ARTERIOSCLEROTIC CALCIUM ACCRUAL: LESSONS LEARNED FROM PRECLINICAL MODELS AND PATIENTS WITH END-STAGE RENAL DISEASE The PTH/PTHrP receptor (PTH1R) and its ligands PTH (parathyroid hormone) and PTHrP (parathyroid hormone-related protein) globally control calcium homeostasis in vertebrates. While sustained PTH or PTHrP administration suppresses bone formation, pulsatile administration promotes bone formation and bone mass accrual [202]. The first robust evidence that signaling via PTH1R would play an important role in the biology of arterial calcification arose from elegant
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patient-oriented studies performed by Gerard London at Manhes Hospital in Fleury-Me´rogis [156,203]. His early studies had first established that atherosclerotic intimal calcification and medial artery calcification both conveyed increased mortality risk in patients with ESRD [156,203]. Because of the prototypic contributions of PTH as a calciotropic hormone and bone anabolic stimulus, London related the extent of vascular calcification to PTH levels and bone turnover as assessed by dynamic histomorphometry [156,203]. In these dialysis patients, those individuals with the lowest serum PTH values and lowest level of bone turnover e reduce bone formation and bone resorption e had the most extensive arterial calcification [203]. Several possibilities exist that may explain the relationship, including: (1) the important role of PTH-maintained bone formation as a calcium phosphate “buffer” that mitigates pro-calcific actions of elevated calcium and phosphate on VSMC physiology [159]; (2) boneelaborated endocrine cues [189] such as serum OPN that limit vascular calcium accrual and are upregulated by PTH [204]; or (3) actions of PTH signaling that inhibit arterial calcification by limiting osteogenic lineage allocation and/or trans-differentiation [169]. When fed high-fat diets characteristic of Westernized societies (42% of calories from fat), the male LDLR/ mouse develops insulin-resistant diabetes and aortic calcification; this model recapitulates key features of aortic calcification identified in humans with type II diabetes and in clinical pathology specimens [204]. We identified that at the same time intermittent (pulsatile) PTH(1-34) stimulates bone formation; PTH(1-34) also suppresses vascular calcification and aortic osteogenic gene expression programs in diabetic LDLR/ mice [204]. Subsequently, Friedman and colleagues demonstrated that, in a uremic rat model of vascular calcification, PTH(1-34) administration again reduced arterial calcification while stimulating bone formation [205]. Serum calcitriol levels were not measured in either of these studies, but are presumed to have increased with PTH(1-34) administration. Recall that London and colleagues highlighted that in ESRD, those patients with lowest levels of endogenous PTH and low bone formation had the greatest extent of arterial calcification [203]. Thus, while high-turnover bone disease driven by hyperparathyroidism may sometimes drive soft tissue calciphylaxis (calcific arteriolopathy) in ESRD [206], low PTH levels have significant negative consequences as well with respect to macrovascular calcification [203]. In toto, excessive vitamin D and calcium intake e stimuli that can profoundly and tonically reduce PTH tone e likely exert pro-calcific actions in the vasculature in part via enhanced bone resorption [207e209], chronic suppression of PTH and reduction in VSMC PTH1R signaling [76].
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Jono and colleagues were the first to relate the expression and regulation of VSMC PTHrP by calcitriol to potential vascular toxicology of excessive vitamin D [76]. Like PTH, PTHrP is a potent ligand for the PTH1R. However, unlike PTH, it is highly expressed in VSMC and is upregulated in response to biomechanical force and oxidized LDL [210e212]. Jono demonstrated that treatment of VSMC with 1,25(OH)2D dose dependently increased VSMC AKP2/ALP and mineral deposition, and concomitantly reduced PTHrP expression [76]. Adding either exogenous synthetic PTH(1-34) or PTHrP(1-34) prevented calcitriol-induced mineralization and AKP2/ALP induction. As such, calcitriol toxicity directly compromises local VSMC defenses against mineralization e while indirectly increasing VSMC vesicle elaboration via elevations in serum calcium and phosphate (see “Vitamin D intoxication and cardiovascular calcification: pharmacological considerations,” above). Thus, PTH1R signaling in VSMC provides a cell-autonomous signal that inhibits the initiation of VSMC-mediated calcium deposition. Based upon our studies, this protective action of PTH1R occurs via downregulation of pro-osteogenic and pro-fibrotic b-catenin signaling in VSMCs [77,169]. However, the regulation of VSMC matrix vesicle release and uptake by PTH, PTHrP, and 1,25(OH)2D has yet to be investigated.
DEFINING CARDIOVASCULAR TOXICITY THRESHOLDS FOR VITAMIN D: CRITICAL CLUES FROM THE CLINICAL LITERATURE As discussed above, it is the dynamic and agedependent physiological interactions between parathyroids, kidneys, and bone that determine toxicity thresholds for vitamin D e i.e. whether hypervitaminosis D or excessive calcium intake is associated with toxicity [144,213]. Moreover, this physiology closely relates to, but is not precisely co-registered with, our current serum measurements of vitamin D and its metabolites. Furthermore, although one can relate that with vitamin D toxicity concentrations of serum 25(OH)D are usually >220 nM (88 ng/ml) [144,213], biochemical hypervitaminosis D modestly exceeding this threshold does not always give rise to toxicity, even in the elderly [214]. Thus, definition of a tolerable upper intake level (TUIL) that avoids toxicity by extrapolation from levels of 25(OH)D alone is difficult. Somewhat more useful insights can be obtained from other clinical data, where systematic vitamin D3 administration without toxicity e from either UVB or oral dosing e has been accompanied by longitudinal observation that includes biochemical phenotyping (serum
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and urinary calcium, and PTH levels in addition to 25(OH)D levels). Haddad and colleagues were among the first to demonstrate that summer sun-exposed lifeguards have serum levels of 25(OH)D of about 150 nM (60 ng/ml) without toxicity [213]. Of note, dependent upon latitude and pigmentation, a full body dose of exposure to 15 to 20 minutes of summer sunlight provides approximately 10 000 IU of vitamin D3, equivalent to 250 mg of oral vitamin D3 [215]. Longer exposure to UVB provides no additional boost to 25(OH)D levels due to photoinactivation. As a fatsoluble vitamin, oral vitamin D is absorbed and ferried on chylomicrons [216]. Thus, the PKePD relationships for daily oral administration of 10 000 IU of vitamin D3 are not certain to be the same for cutaneous vitamin D3 synthesis. This issue was addressed by Heaney and colleagues; they examined the doseeresponse for 5 months of daily vitamin D3 e up to 10 000 IU/ 250 mg of vitamin D3 per day e in healthy men during winter months in Omaha, a time when little if any UVB-generated D3 contributes to vitamin D nutrition [59]. With that dosing, serum levels of 25(OH)D achieved were 150 nM for 5000 IU D3 (125 mg) and 200 nM for 10 000 IU D3 (250 mg). No evidence of toxicity was evident in these individuals, and serum calcium levels were unchanged [59]. However, 10 000 IU/250 mg of D3 per day reduced PTH by 25% from the baseline of ~30 pg/ml, while the intermediate doses of 5000 IU/125 mg had no significant effect [59]. Recall that the 5000 IU/125 mg per day oral dosing of vitamin D3 safely delivered serum levels of 25(OH)D that are also achievable by non-toxic daily sun exposure. Thus, the TUIL probably exists at or slightly above 5000 IU per day for a healthy male adult with normal renal function [59]. Given the important roles of vascular PTH/PTHrP receptor signaling to the arteriosclerotic defense mechanisms discussed in the preceding section, the lowest dose that does not excessively reduce PTH levels [217] yet achieves healthy serum 25(OH)D levels [218] would seem most appropriate. However, as commented above, GFR estimate and urinary calcium clearance would have been helpful to better co-register potential nephrotoxicity risk. Reid and colleagues recently published a highly enlightening study that provided a strategy for safe oral replacement of vitamin D in the setting of mild primary hyperparathyroidism with co-existing vitamin D insufficiency [58]. However, this same study also provides data that can help to refine the TUIL for vitamin D e in a population at high risk for hypercalcemia and hypercalciuria [58]. This has added relevance, since up to 0.5% of our female patients over 60 years of age may have asymptomatic primary hyperparathyroidism [219]. In primary hyperparathyroidism,
PTH drives high bone turnover, stimulates renal 1ahydroxylase, and conveys risk for hypercalciuria with attendant hypercalcemia. Thus, there has been concern that replacement protocols for vitamin D insufficiency in setting of otherwise asymptomatic primary hyperparathyroidism might precipitate clinical worsening of hypercalcemia and hypercalcuria. Using a regimen of 50 000 IU of oral ergocalciferol per week for 4 weeks (i.e., ~6700 IU per day on average for one month) followed by 50 000 IU per month for another year (i.e., ~1650 IU per day for 1 year), Reid demonstrated that this at-risk population could be successfully repleted without worsening hypercalcemia [58]. This subject is also discussed in Chapter 72. Moreover, of the 21 hyperparathyroid patients so treated, only three developed transient hypercalciuria (>400 mg/24 hours) at 6 months, with only two remaining hypercalciuric at 12 months (~10%). No nephrolithiasis or renal insufficiency was observed. Averaged over the entire year, the mean daily dose of vitamin D2 was 2050 IU per day in this at-risk population [58]. Thus, once again, the TUIL for healthy adults appears to be above 2000 IU per day, and this daily dosing appeared to be acceptable even for individuals at risk due to asymptomatic primary hyperparathyroidism. In sum, daily recommended intakes of vitamin D between 2000 and 5000 IU per day are likely to be acceptable without toxicity for the vast majority of adults. However, the relationship of the tolerable upper limit of dosing to the anti-rachitic 400 IU per day dose for children [220] e particularly young children [221] or very petite adults e is much less clear. The subject of optimal dosing of vitamin D is considered in Chapters 57 and 58 where multiple arguments for high or low recommended dosing are presented.
VITAMIN D, THE CALCIUM-SENSING RECEPTOR (CASR), AND CARDIOVASCULAR DISEASE Vitamin D intoxication occurs in part via absorptive and resorptive hypercalcemia. Thus, the contributions of calcium physiology and calcium-sensing receptor signaling must also be considered. Daily recommended intakes of 1000 to 1500 mg of elemental calcium appear safe for most of our society, routinely achieved by supplementation in lieu of calcium-rich food intake. However, a recent meta-analysis of calcium supplementation in the absence of vitamin D supplementation suggests that the risk of myocardial infarction is increased by approximately 30% with use of calcium supplements to achieve this intake [135]. Why might this occur? With aging-related accrual of medication use and declines in renal function, some consideration
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of dose and formulation should be made. Beginning at a glomerular filtration rate (GFR) of 60 cc/min/1.73 m2 e i.e. CKD3 e the postprandial urinary excretion of calcium delivered in standardized meals is reduced by more than 50% [158]. Depending upon the bioavailability of the calcium (formulation dependent and modified by dietary phytates), postprandial serum calcium transients will vary. Impaired renal calcium clearance not only arises because of reduced GFR, but will likely be related to chronically elevated levels of PTH and FGF23 in those with CKD3 (or worse); both of these hormones enhance calcium resorption from glomerular filtrate that reaches the distal renal tubule [222]. As highlighted by Chertow and colleagues [223], vascular calcium “overload” can occur in patients with CKD5 e a condition where postprandial calcium clearance is completely dependent upon dialysis and tissue deposition. Precious little is known of how vascular mineralization is regulated and perturbed by moderate renal insufficiency in humans, but preclinical and clinical data strongly indicate that calcific vascular disease rapidly progresses with chronic declines in renal function [14]. Very recently, an autopsy study from the National Cardiovascular Center in Osaka, Japan, confirmed that prevalence and extent of atherosclerotic intimal calcification was approximately doubled in the left anterior descending artery of patients with CKD3 versus those without renal insufficiency [224]. Although medial artery calcification is highly prevalent in aortofemoral and distal conduit arteries of patients with diabetes and/or CKD, medial artery calcification of the coronary arteries was much less common. However, the use of calcium-based phosphate binders increased by 20-fold the relative risk of proximal coronary artery medial calcification (p < 0.05; RR 21.0, range 1.9e236.1) [224]. Most individuals receiving calciumbased phosphate binders at autopsy were CKD4 or worse (GFR <15 cc/min/1.73 m2). Nevertheless, this pathological study and Chertow’s observations [223] converge with the patient-oriented research of Wolf and colleagues [158] to highlight even moderate reductions in the impact of GFR on calcium and calciotropic hormone physiology. Thus, given that 7% of our population between age 60 and 69 and 20% of our population over age 70 have a GFR under 60 cc/min/ 1.73 m2 e i.e. CKD3 afflicts one-quarter of our populace >60 years old [225] e recommended daily intakes for calcium intake must consider impact of aging, renal function, calcium formulation, and concurrent medications. The differences between the data of Reid and colleagues [226] and the WHI outcomes [134] with respect to the potential cardiovascular risks of calcium supplementation have yet to be resolved, but may relate to the genotype environment age-
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dependent interactions that influence renal calcium homeostasis and cardiovascular risk. In the age of modern genomics, the goal to predict, prevent, and preempt on a personalized basis has been outlined as the strategy to reduce disease burden in our population [227]. The same should hold for nutritional genomics and recommendations for calcium and vitamin D intake with respect to beneficial impact versus toxicity. An explosion of information has occurred surrounding the genetics and physiology of calcium nutrient sensing relevant to this discussion [228]. This relates to the cloning of the calcium-sensing receptor (CaSR) and characterization of its role in calcium homeostasis and the PTHerenal axis e one of the most significant contributions to the field of mineral metabolism in the last half of the 20th century [229]. A primary role for the CaSR in calcium homeostasis exists in at least three tissues: (1) within the parathyroid chief cells, where CaSR signaling inhibits PTH release and promotes intraglandular degradation [229]; (2) in the kidney, where CaSR signaling inhibits calcium and water resorption by the distal tubule [228]; and (3) in the developing skeleton [230]. In the kidney, CaSR signaling provides a direct mechanism to mitigate hypercalcemia by promoting calciuresis, and provides tubular water content necessary for brisk urine flow to minimize risk of intratubular crystal deposition. Although adaptive in the short term, the latter “diuretic” actions of CaSR signaling can worsen pre-renal azotemia and contraction alkalosis that hampers further calciuretic responses as outlined above. Work from multiple laboratories worldwide has now converged to indicate that CaSR signaling plays a role in cardiovascular physiology. The CaSR is expressed in autonomic nerve endings [231], within arterial smooth muscle and endothelial cells [232e234] and by calcifying microvascular pericytes [222]. Synthetic calcimimetic compounds reduce blood pressure while calcium receptor antagonists e “calcilytics” e increase blood pressure [235]. However, the responses of vascular functions with respect to perturbations in serum calcium, alterations in CaSR genetics, and newly possible CaSR-specific pharmacologic manipulation are very complex [235]. For example, correction of hypercalcemia of primary hyperparathyroidism with adenoma resection improves flow-mediated dilatation (FMD), an index of endothelial function [236]. Likewise, FMD is reduced in dialysis patients with high-calcium as compared to low-calcium dialysates [160]. These responses do not co-register well with the pharmacology of CaSR ligands [235]. In vitro, calcifying microvascular pericytes e contributors to osteogenic vascular calcium load e express the CaSR and downregulate its expression with mineralization [222]. Moreover, siRNA “knockdown” of the CaSR accelerates mineralization in vitro, suggesting that the CaSR
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play a protective role in vascular mineralization [222]. Consistent with this, administration of a calcimimetic reduces arterial calcium accrual in rodent models of renal insufficiency [237]. Yet, the Nuf/þ mouse, containing one hyperactive CaSR (L723Q) allele, exhibits reduced PTH, hypocalcemia, phosphate retention, and increased cardiovascular calcification and mineral deposition in striated muscle (tongue), kidney tubules, and vibrissae dermal sheaths [238]. Cardiovascular calcification was more extensive in homozygous Nuf/Nuf mice [238]. Genetic studies of the CaSR in humans point to contributions in CVD risk. In LURIC, a German study of 3259 human subjects [239], genotypeephenotype relationships between the CaSR (986S) allele, myocardial infarction, and angiographically proven coronary artery disease have been reported [240]. The prevalence of the CaSR (986S) allele is high, with an allelic frequency of ca. 18e20% [240]. As compared to those individuals homozygous for the CaSR (A986) allele, humans either homozygous or heterozygous for the CaSR (986S) allele were at a 20e30% increase for both MI and angiographic CVD [240]. The population with the CaSR (986S) allele had slightly higher serum calcium levels as well as slightly higher PTH levels, consistent with a mild “loss-of-function” phenotype for this allelic variant [240]. The pathophysiologic mechanisms of this genotypeephenotype association are not known, however. Given the profound impact of calciotropic hormones and calcium homeostasis on cardiovascular physiology, a better understanding of CaSR-regulated physiology should provide insights useful for guiding recommendations concerning calcium supplementation. Because of the relatively high allelic frequency of CaSR(986S) [240], polymorphism at CaSR codon 986 may potentially impact risk of hypercalcemia and attendant cardiovascular risks associated with excessive calcium supplementation either with or without vitamin D. The CaSR is fully discussed in Chapter 24.
SUMMARY, CONCLUSIONS, AND FUTURE DIRECTIONS Vitamin D plays a biphasic role in human health, with both insufficiency and intoxication compromising cardiovascular physiology. Vitamin D agonists (VDRAs) are vasculotropic hormones, and vitamin D insufficiency is associated with significant, undesirable changes in cardiovascular physiology and structure. In end-stage renal disease, benefits of pharmacological enhancement of VDR tone with VDRAs has emerged [15], likely via improvements in endothelial function [91,92] and reductions in myocardial hypertrophic
responses [241]. However, data demonstrating unequivocal clinical cardiovascular benefit of vitamin D supplementation and/or VDRAs in individuals without ESRD are still meager in 2010 [134,137]. Furthermore, vitamin D intoxication also exerts significant undesirable actions on the cardiovascular system via elevations in serum phosphate and serum calcium/calciproteins, impairment of VSMC elastin biology, and inhibition of protective vascular PTH/PTHrP receptor signaling. Data forthcoming from studies of the renineangiotensin system and its regulation by vitamin D signaling has begun to reveal unexpected complexities in cardiovascular endocrinology [38] e and highlighted the need to implement a “systems biology” approach in order to better identify opportunities for intervention. Histoanatomic, molecular, and ethnic heterogeneities are emerging in the pathobiology of arteriosclerotic disease; these heterogeneities confound straightforward extrapolation of the downsides of insufficiency to benefits with repletion or supplementation. However, in the USA many of us are vitamin-D-insufficient [23], and increasing daily intakes to 2000e5000 IU per day is likely to be well tolerated with accrual of benefit and little downside potential in healthy adults [23], even in the presence of mild asymptomatic primary hyperparathyroidism [58]. Based upon current physiological and pharmacological data, tissue calcinosis, hypercalcemia, and hyperphosphatemia e the primary toxicities of vitamin D excess e are not seen and probably will not be seen until doses of vitamin D3 exceed 5000e10 000 IU daily in most healthy adults [59]. However, doses for children are much less clear, and indiscriminate vitamin D administration by parents and physicians can induce nephrocalcinosis [221,242]. Dynamic interactions between the parathyroids, kidneys, and bone determine the toxicity threshold for vitamin D and calcium e i.e. whether hypervitaminosis D or excessive calcium intake is associated with toxicity. Age-dependent declines in renal function afflict ~25% of our population over age 60 and impair postprandial calcium excretion e and awareness of chronic renal insufficiency is very poor. Because vitamin D toxicities relate in part to abnormal serum and vascular calcium phosphate homeostasis, recommended daily intakes of vitamin D and VDRA treatment should incorporate consideration of this important physiological decline commonly seen in aging. CaSR polymorphisms that also alter calcium homeostasis and cardiovascular risk are also very prevalent [240], and may impact outcomes of interventional studies. The net interactions of calcium supplementation and VDRA treatments with adequate vitamin D nutrition upon cardiovascular outcomes have not been assessed. Patient-oriented research is needed that helps develop age-, ethnicity-, genotype-, and GFR-specific guidelines for recommended calcium and vitamin D
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intake based upon physiological fluxes of calcium into skeletal, renal, and vascular mineral compartments. Basic and translational research is required to better define how parathyroidekidneyebone interactions impact cardiovascular responses to VDRAs e and to determine the role of vascular cell-autonomous VDR expression in VDRA physiology [103,119,243] and toxicology [244]. Moreover, Cre-Lox technology should be implemented to evaluate how cell-autonomous VDR signaling in VSMCs, cardiomyocytes, macrophages, endothelial cells, and T cells contributes to cardiovascular physiology and pathobiology in murine models. The relationships between the VDR, its non-nuclear role in mitochondrial function [38,245e247] and cardiovascular oxidative stress signaling have yet to be detailed. Much work remains to be done e but assiduous attention and implementation of the lessons learned hold promise for tremendous benefit to human health.
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Acknowledgment D.A.T. is supported by NIH grants HL69229, HL81138, HL88651, and the Barnes-Jewish Hospital Foundation.
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[238] T.A. Hough, D. Bogani, M.T. Cheeseman, J. Favor, M.A. Nesbit, R.V. Thakker, et al., Activating calcium-sensing receptor mutation in the mouse is associated with cataracts and ectopic calcification, Proc. Natl. Acad. Sci. USA 101 (2004) 13566e13571. [239] B.R. Winkelmann, W. Marz, B.O. Boehm, R. Zotz, J. Hager, P. Hellstern, et al., Rationale and design of the LURIC study e a resource for functional genomics, pharmacogenomics and long-term prognosis of cardiovascular disease, Pharmacogenomics 2 (2001) S1e73. [240] W. Marz, U. Seelhorst, B. Wellnitz, B. Tiran, B. ObermayerPietsch, W. Renner, et al., Alanine to serine polymorphism at position 986 of the calcium-sensing receptor associated with coronary heart disease, myocardial infarction, all-cause, and cardiovascular mortality, J. Clin. Endocrinol. Metab. 92 (2007) 2363e2369. [241] A. Covic, L. Voroneanu, D. Goldsmith, The effects of vitamin D therapy on left ventricular structure and function e are these the underlying explanations for improved CKD patient survival? Nephron. Clin. Pract. 116 (2010) c187ec195. [242] M. Riordan, G. Rylance, K. Berry, Poisoning in children 3: common medicines, Arch. Dis. Child 87 (2002) 400e402. [243] M. Freundlich, Y. Quiroz, Z. Zhang, Y. Zhang, Y. Bravo, J.R. Weisinger, et al., Suppression of renin-angiotensin gene expression in the kidney by paricalcitol, Kidney Int. 74 (2008) 1394e1402. [244] V. Shalhoub, E. Shatzen, S. Ward, J.I. Young, M. Boedigheimer, Twehues L., et al., Chondro/osteoblastic and cardiovascular gene modulation in human artery smooth muscle cells that calcify in the presence of phosphate and calcitriol or paricalcitol. J. Cell Biochem. 111 (2010) 911e921. [245] F. Silvagno, E. De Vivo, A. Attanasio, V. Gallo, G. Mazzucco, G. Pescarmona, Mitochondrial localization of vitamin D receptor in human platelets and differentiated megakaryocytes, PLoS. One 5: e8670. doi: 10.1371/journal.pone.0008670. [246] V. Gonzalez Pardo, R. Boland, A.R. de Boland, Vitamin D receptor levels and binding are reduced in aged rat intestinal subcellular fractions, Biogerontology 9 (2008) 109e118. [247] V.F. Sahach, P. Korkach Iu, A.V. Kotsiuruba, O.V. Rudyk, H.L. Vavilova, [Mitochondrial permeability transition pore opening inhibition by ecdysterone in heart mitochondria of aging rats], Fiziol. Zh. 54 (2008) 3e10. [248] A. Rahman, S. Hershey, S. Ahmed, K. Nibbelink, R.U. Simpson, Heart extracellular matrix gene expression profile in the vitamin D receptor knockout mice, J. Steroid. Biochem. Mol. Biol. 103 (2007) 416e419. [249] M.D. Walker, J.B. Fleischer, M.R. Di Tullio, S. Homma, T. Rundek, E.M. Stein, et al., Cardiac structure and diastolic function in mild primary hyperparathyroidism, J. Clin. Endocrinol. Metab. 95 (2010) 2172e2179. [250] R. Rice, P. Guinto, C. Dowell-Martino, H. He, K. Hoyer, M. Krenz, et al., Cardiac myosin heavy chain isoform exchange alters the phenotype of cTnT-related cardiomyopathies in mouse hearts, J. Mol. Cell Cardiol. 48 (2010) 979e988. [251] T.D. O’Connell, R.E. Weishaar, R.U. Simpson, Regulation of myosin isozyme expression by vitamin D3 deficiency and 1,25dihydroxyvitamin D3 in the rat heart, Endocrinology 134 (1994) 899e905.
[252] G.F. Wang, W. Nikovits Jr., Z.Z. Bao, F.E. Stockdale, Irx4 forms an inhibitory complex with the vitamin D and retinoic X receptors to regulate cardiac chamber-specific slow MyHC3 expression, J. Biol. Chem. 276 (2001) 28835e28841. [253] R.U. Simpson, R.E. Weishaar, Involvement of 1,25-dihydroxyvitamin D3 in regulating myocardial calcium metabolism: physiological and pathological actions, Cell. Calcium. 9 (1988) 285e292. [254] K.A. Nibbelink, D.X. Tishkoff, S.D. Hershey, A. Rahman, R.U. Simpson, 1,25(OH)2-vitamin D3 actions on cell proliferation, size, gene expression, and receptor localization, in the HL1 cardiac myocyte, J. Steroid Biochem. Mol. Biol. 103 (2007) 533e537. [255] J.R. Wu-Wong, M. Nakane, J. Ma, X. Ruan, P.E. Kroeger, Effects of vitamin D analogs on gene expression profiling in human coronary artery smooth muscle cells, Atherosclerosis 186 (2006) 20e28. [256] S. Chen, C.S. Law, D.G. Gardner, Vitamin D-dependent suppression of endothelin-induced vascular smooth muscle cell proliferation through inhibition of CDK2 activity. J. Steroid Biochem. Mol. Biol. 118 (2010) 135e141. [257] T.D. O’Connell, R.U. Simpson, 1,25-Dihydroxyvitamin D3 regulation of myocardial growth and c-myc levels in the rat heart, Biochem. Biophys. Res. Commun. 213 (1995) 59e65. [258] J.R. Wu-Wong, M. Nakane, J. Ma, Vitamin D analogs modulate the expression of plasminogen activator inhibitor-1, thrombospondin-1 and thrombomodulin in human aortic smooth muscle cells, J. Vasc. Res. 44 (2007) 11e18. [259] J.R. Wu-Wong, M. Nakane, J. Ma, Effects of vitamin D analogs on the expression of plasminogen activator inhibitor-1 in human vascular cells, Thromb. Res. 118 (2006) 709e714. [260] G. Targher, L. Bertolini, R. Padovani, L. Zenari, L. Scala, M. Cigolini, et al., Serum 25-hydroxyvitamin D3 concentrations and carotid artery intima-media thickness among type 2 diabetic patients, Clin. Endocrinol. (Oxf.) 65 (2006) 593e597. [261] T. Neunteufl, R. Katzenschlager, C. Abela, K. Kostner, B. Niederle, F. Weidinger, et al., Impairment of endotheliumindependent vasodilation in patients with hypercalcemia, Cardiovasc. Res. 40 (1998) 396e401. [262] M. Kosch, M. Hausberg, K. Vormbrock, K. Kisters, K.H. Rahn, M. Barenbrock, Studies on flow-mediated vasodilation and intima-media thickness of the brachial artery in patients with primary hyperparathyroidism, Am. J. Hypertens. 13 (2000) 759e764. [263] M. Kosch, M. Hausberg, K. Vormbrock, K. Kisters, G. Gabriels, K.H. Rahn, et al., Impaired flow-mediated vasodilation of the brachial artery in patients with primary hyperparathyroidism improves after parathyroidectomy, Cardiovasc. Res. 47 (2000) 813e818. [264] M. Baykan, C. Erem, T. Erdogan, A. Hacihasanoglu, O. Gedikli, A. Kiris, et al., Impairment of flow mediated vasodilatation of brachial artery in patients with primary hyperparathyroidism, Int. J. Cardiovasc. Imaging. 23 (2007) 323e328. [265] J. Hjelmesaeth, D. Hofso, E.T. Aasheim, T. Jenssen, J. Moan, H. Hager, et al., Parathyroid hormone, but not vitamin D, is associated with the metabolic syndrome in morbidly obese women and men: a cross-sectional study, Cardiovasc. Diabetol. 8 (2009) 7.
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C H A P T E R
74 Alterations in 1,25-Dihydroxyvitamin D3 Structure that Produce Profound Changes in in Vivo Activity Hector F. DeLuca, Lori A. Plum University of Wisconsin-Madison, Madison, WI, USA
INTRODUCTION The early attempts at the preparation of analogs of the hormonally active form of vitamin D were driven primarily by the convenience of the chemistry, known requirements of in vivo 25-hydroxylation, or the availability of simple modifications of the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) molecule [1e3]. Their preparation was not driven by in vivo biology or by in vitro binding measurements. However, some of the analogs prepared as a result of this effort have provided interesting biological properties. The basis for these properties is often not known but may be the result of different metabolic inactivation reactions or delivery to target organs [4,5]. This chapter will deal with selected modifications of the 1,25(OH)2D3 molecule and their impact on in vivo biological activity where the results are available. The geometry of the binding pocket of the vitamin D receptor (VDR) is adequately described elsewhere in this series (Chapters 7e9). Furthermore, many laboratories have been working on modeling systems that would describe how an analog might interact with the VDR. Although these are all extremely worthwhile endeavors and, in fact, our laboratory has often been involved with similar studies, this review will not rely on modeling but on in vivo biological activity and, if known, the reason for the biological profile.
THE NON-CALCEMIC HOLY GRAIL OF VITAMIN D ANALOGS The discovery of previously unknown functions of vitamin D has led to an interest in synthesizing analogs
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10074-5
of 1,25(OH)2D3 that would not cause hypercalcemia while providing for the desired activities, as, for example, anti-proliferation in cancer cells [7]. The calcemic actions of vitamin D can only be ascertained by in vivo measurements in experimental animals. Clearly, analogs differ in their potency in elevating serum calcium, either by intestinal absorption or by bone calcium mobilization. There are literally many analogs that would fit this criteria, i.e. on a weight basis, they are clearly less calcemic than 1,25(OH)2D3 [1e8]. However, the real question is whether that analog has the expected activities that are not related to calcemia when measured in vivo. Very often the activities of an analog are measured for calcemia in vivo and measured in vitro for binding to the receptor, for inhibition of proliferation of cancer cells, the differentiation of HL-60 and other cells, or some other similar in vitro measurement. This comparison can indeed be quite misleading. In the experience of these authors, all vitamin D compounds are potentially calcemic, i.e. they will upon increasing doses eventually cause hypercalcemia. A good example is 2-methylene-19nor-1a-hydroxy-bishomopregnacalciferol (Fig. 74.1). This compound when studied in vitro is able to bind to the receptor, albeit at a one-half log higher concentration. It stimulates transcription of a VDR-reporter gene system as well as in vitro differentiation and suppression of proliferation of HL-60 cells [9]. However, when tested in vivo in vitamin-D-deficient rats, this compound appears to have virtually no activity on intestine and bone calcium mobilization at doses where the vitamin D hormone is clearly active [9]. This compound can truly be considered
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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74. ALTERATIONS IN 1,25-DIHYDROXYVITAMIN D3 STRUCTURE THAT PRODUCE PROFOUND CHANGES IN IN VIVO ACTIVITY
ANALOGS DIRECTED TO SPECIFIC STRUCTURES The Cis-triene Structure (Fig. 74.2)
HO
OH
74.1 Structure of 2-methylene-19-nor-1a-hydroxybishomopregnacalciferol (2MbisP).
FIGURE
non-calcemic in the sense that it does not raise serum calcium at doses similar to the vitamin D hormone. However, if the dose is increased 1000-fold, serum calcium will begin to rise (Plum LA, DeLuca HF, unpublished results). In fact, hypercalcemia can be achieved with this compound. The real question is whether it can cause other activities at the lower doses in vivo. Another excellent example is the EB-1089 compound of Leo Pharmaceuticals [10]. This compound appeared at least 300 times more active than 1,25(OH)2D3 on anti-cancer cell proliferation and differentiation in vitro and had much less activity in raising serum calcium in vivo [10]. Reports of inhibition of in vivo animal models of cancer also appeared [11e13]. However, the anti-cancer activities failed to materialize in vivo in controlled clinical trials [10]. A similar experience can easily be established with a well-known compound that is used clinically, MC903, known as DovonexÒ for the topical treatment of psoriasis [14e16]. In vitro, this compound is almost as effective as 1,25(OH)2D3 in causing cellular differentiation of HL-60 cells and keratinocytes but in vivo it is much less calcemic but nevertheless hypercalcemia can be achieved by giving higher doses of this compound [17,18]. The primary reason for this behavior is that the MC-903 has a very short lifetime in plasma because it is rapidly metabolized and excreted. Is this compound truly a non-calcemic analog; is it a selective analog; or is its selectivity because it is applied topically where less than 5% enters the circulation which results in its rapid inactivation?
Is the triene system found in 1,25(OH)2D3 essential for its biological activity? For binding to the VDR, it is well known that the diene bridge must align with the sole tryptophan residue of the VDR [19]. It is reasonable to expect that the diene/triene system is essential for the function of vitamin D. However, elimination of the 10,19-portion does not eliminate vitamin D activity [20]. In fact, one of the most used analogs is the 19-nor-1a,25-dihydroxyvitamin D2 known as ZemplarÒ [21,22]. 19-Nor-1,25(OH)2D3 and 19-nor-1,25(OH)2D2 bind equally well as the native 1,25(OH)2D3 to the VDR, but are very much less effective on raising serum calcium and phosphate. 19-nor-1,25(OH)2D2 retains most of the activity of 1,25(OH)2D3 in suppressing PTH and parathyroid proliferation in chronic renal failure patients [22]. Thus, this compound has a wider therapeutic window than 1,25(OH)2D3 in suppressing secondary hyperparathyroidism of renal failure patients and has resulted in improved survival of these patients [23,24]. That the 5,7-diene structure is essential is also illustrated by replacing the 6- and 7-carbons with nitrogens. This analog is biologically inactive [25]. Catalytic reduction of the triene system of 1,25(OH)2D3 also destroys vitamin D activity (DeLuca HF, unpublished results). Thus, the alignment of Trp of the receptor with the diene must be considered of central importance to the function of vitamin D.
A-ring Hydroxyls (Fig. 74.3) That the 1-hydroxyl must be in the a-configuration is clear because 1b-hydroxylated 25(OH)D is essentially inactive [26,27]. Replacing the 1a-hydroxyl with a fluoro
OH
7 6 5
19
10
HO
FIGURE 74.2 triene system.
IX. ANALOGS
OH 1a,25-Dihydroxyvitamin D3 highlighting the cis-
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ANALOGS DIRECTED TO SPECIFIC STRUCTURES
calcium absorption with much less activity on bone [38]. Modification of the 2-carbon with a hydroxypropoxy group imparts bone selectivity [39]. OH
The C,D-ring (Fig. 74.4)
3
HO
FIGURE 74.3 highlighted.
1
OH 1a,25-Dihydroxyvitamin D3 with A-ring hydroxyls
group markedly reduces biological activity [28], essentially reducing activity to a level produced by 25(OH)D3 itself in 1a-hydroxylase knockout animals [29]. Difluoro-1,25(OH)2D3 is devoid of all activity, demonstrating clearly the need for 1a-hydroxylation and 25-hydroxylation [30]. On the other hand, the 3hydroxyl is not required if the 1a-hydroxyl is already in place, although the absence of the 3-hydroxyl does reduce biological activity [31]. Okamura first suggested that the 1a-hydroxyl must be in the equatorial position when interacting with the VDR [32]. This idea has been confirmed with the synthesis of analogs where the 1a-hydroxyl is apparently restricted to the axial configuration [33,34].
A-ring Modifications A large number of analogs modified at the 2-carbon have been prepared. Two a-methyl substitutions on 19nor-(20S)-1a,25-dihydroxyvitamin D3 increase biological activity especially on bone where it is at least 100 times more effective at osteoclast-mediated bone calcium mobilization [35,36]. The b-methyl is essentially inactive [35]. Takayama’s group has demonstrated similar results with the 2a-methyl 1,25(OH)2D3 [37]. This also supports the idea that the 1a-hydroxyl must be in the equatorial configuration since the 2a-methyl substitution results in 90% of the 1a-hydroxyl in the equatorial position [35]. Other 2-carbon derivatives in this series have proved interesting. 2-Methylene-19nor-(20S)-1a,25-dihydroxyvitamin D3 (2MD) is at least two orders of magnitude more active than 1,25 (OH)2D3 on osteoclastogenesis, about two orders of magnitude more active in bone calcium mobilization, while being equally active in intestinal calcium mobilization [36]. This analog is clearly selective for bone activity in vivo. The 2-ethylidine derivatives in the Econfiguration have selective activity for intestinal
At least three laboratories have attempted to evaluate the importance of the C,D-ring structure for vitamin D activity. Kutner et al. first made a 1,25(OH)2D3 analog with an absent C,D-ring structure [40]. It was virtually devoid of vitamin D activity. The De Clercq group has made a number of compounds without the C,D-ring resulting in essentially an absence of vitamin D activity [41]. More recently in our own research group, compounds without the C,D-ring but having the backbone that should provide for binding at the 25- and 1-hydroxyl-positions have been prepared [42,43]. Only the compounds with methyl groups in the position of the C-ring showed significant binding activity and activity on cellular differentiation [42,43]. Currently the idea is that the C,D-rings are required for activity of the vitamin D compounds. On the other hand, a group at Ligand Corp. prepared a non-steroidal group of compounds that bind the VDR but have weak vitamin D activity [44]. A follow-up by Eli Lilly scientists on the topical application revealed compounds without a C,D-ring that would increase epidermal thickness without raising serum calcium of mice [45]. Very likely many of these compounds do not survive once they appear in the circulation, similar to the MC-903 or DovonexÒ [14,15] or the 22-oxa compound of Chugai Pharmaceuticals [39]. Since compounds without the C,D-ring do not exert the classical actions of vitamin D in vivo, it is not certain whether the absence of the C,D-ring results in an in vivo biologically effective compound. The current evidence suggests that removal of the C,D-ring almost completely eliminates vitamin D
18
OH 14
HO
FIGURE 74.4 ring structure.
IX. ANALOGS
OH 1a,25-Dihydroxyvitamin D3 highlighting the C,D-
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activity and even a manipulation as simple as 14epimerization results in markedly reduced in vivo biological activity [46,47]. There is, therefore, little evidence to support the idea that the C,D-ring is not essential for vitamin D activity unless one is willing to accept unproven functional activities or is willing to accept simple binding to the receptor in vitro as proof of biological effectiveness. Removal of the C-18-methyl group from the 1,25(OH)2D3 molecule results in little change in biological activity [48]. Thus, this methyl group does not appear essential for the known functions of vitamin D.
Side Chain Modifications (Fig. 74.5) There are an enormous number of 1,25(OH)2D analogs with a variety of side chain modifications. The first question that can be addressed is in the 1,25(OH)2D3 molecule: is the side chain required for vitamin D activity at all? If the side chain is reduced to an ethyl group, binding to the receptor is reduced by at least two orders of magnitude [49] and in vivo activity is absent [50]. This profile is improved by 2-C substitution with a methylene group [51] but is still quite inactive. As carbons are added, as, for example, an ethyl group or an isopropyl group, there is increasing biological activity [51,52]. The longer the hydrocarbon side chain, the greater the calcemic activity. As a reference point, the 2-methylene-19nor-bishomopregnacalciferol compound when given at 300 mg/kg will begin to raise serum calcium in rats (Plum LA, DeLuca HF, unpublished results). However, this compound at very much lower doses will suppress PTH levels in intact and 5,6-nephrectomized rats [51,53]. Clearly for maximal vitamin D activity, a full side chain is required together with a 25-hydroxyl group. A 25-methyl or 26,27-dimethyl that sterically hinders 25-hydroxylation markedly
reduces in vivo activity [54,55]. Altering the C-20 configuration to the unnatural S-configuration will increase biological activity [56,57]. In the case of the 2-methylene-19-nor-1,25(OH)2D3 series, the 20-S configuration confers a marked increase in bone resorption activity as much as 100-fold while not increasing intestinal calcium transport [36]. This compound proved to be anabolic on bone [58,59] but because of its high bone resorption activity, it failed to increase bone mass in postmenopausal women [60]. Nevertheless, it has potential because of the stimulation of bone remodeling. Thus, changing the 20carbon configuration to the unnatural or S-configuration increases biological activity and in combination with the 19-nor and 2-methylene modification, preferential activity on bone synthesis and resorption results [35]. Other modifications of the side chain can provide a diversity of biological activity. Most notable is the first true antagonist of vitamin D activity in vitro. The Schering-Plough chemists have synthesized two strong antagonistics in both binding to the receptor and in anti-transcription [61]. The structures of these are shown in Fig. 74.6. A similar side chain structure in the 2-methylene-19-nor series of compounds has been accomplished in our laboratory [62]. These
OH
O
O
HO
OH
ZK159222
21 22
OH
20 25
O
26 OH
27
O
HO
HO
FIGURE 74.5
droxyvitamin D3.
OH
ZK168281
OH
Highlighted side chain structure of 1a,25-dihy-
FIGURE 74.6 Potent in vitro antagonists of 1a,25-dihydroxy-
vitamin D3.
IX. ANALOGS
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REFERENCES
compounds are all esters and thus are not in vivo antagonists because likely the ester group is hydrolyzed rendering it no longer an antagonist. To date, only one true in vivo antagonist of 1,25(OH)2D3 has been reported and the antagonistic effects were at best limited and only to intestine [63]. Yamada’s group has published a series showing that alteration of the 22-configuration markedly affects biological activity [64]. The orientation of the side chain then within the pocket has a significant influence on biological activity. A very dramatic demonstration of this has been the synthesis of the two compounds shown in Fig. 74.7 in which a double bond has been inserted between the 17- and 20-carbons of the 2methylene-19-nor series and produced in either the cis- or trans-configurations [65]. The cis-configuration produces a strong calcemic, highly biologically active compound, whereas the trans-configuration results in
OH
HO
OH
Trans
HO
a compound with weak calcemic activity but retains in vitro activities such as cell differentiation or in vivo suppression of PTH levels. The importance of the methyl groups at 21, 26, and 27 for in vivo activity has been examined in the 2-methylene-19-nor-(20S)-1,25-(OH)2D3 series. Elimination of the 21-methyl markedly reduces bone calcium mobilization with a minimal effect on calcium absorption [66]. On the other hand, removal of either the 26- or 27-methyls equally affect both intestinal calcium absorption and bone calcium mobilization [67].
SUMMARY Many extremely interesting analogs have been made by several excellent chemical laboratories, but unfortunately we are unable to provide a clear pattern of in vivo structureeactivity relationship. The molecular and even physiological basis of biological activity in the multiple actions of vitamin D have not been adequately described and thus in vivo measurements that would indicate activity in one aspect of vitamin D function versus another are not yet available. Certainly in vivo evaluation of calcemic activity is available but even in this circumstance, the measurements of calcemic activity differ widely among research groups, making comparisons difficult. Nevertheless, some facts have emerged that can provide a basis for design of compounds with expected outcomes. For example, the 5,7-diene, the 1a-hydroxyl, and the C,D-ring structure are required for in vivo activity, while the 20-C configuration, the 21-methyl, 2-C substitutions and a wide diversity of side chains are alterations that may produce selective in vivo activity. It seems possible that analogs that are selective for specific functions of vitamin D will be possible. If realized, this will result in important therapeutic vitamin D compounds for the treatment of human disease.
Acknowledgment
H
This work was supported by the Wisconsin Alumni Research Foundation.
References HO
OH
Cis FIGURE 74.7 E and Z isomers of 17,20-dihydro-2-methylene-19nor-1a,25-dihydroxyvitamin D3.
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74. ALTERATIONS IN 1,25-DIHYDROXYVITAMIN D3 STRUCTURE THAT PRODUCE PROFOUND CHANGES IN IN VIVO ACTIVITY
[4] G. Jones, Analog metabolism, in: D. Feldman, F.H. Glorieux, J.W. Pike (Eds.), Vitamin D. Academic Press, San Diego, 1997, pp. 973e994. [5] N. Rochel, J.M. Wurtz, A. Mitschler, B. Klaholz, D. Moras, The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand, Molec. Cell 5 (2000) 173e179. [6] J.L. Vanhooke, M.M. Benning, C.B. Bauer, J.W. Pike, H.F. DeLuca, Molecular structure of the rat vitamin D receptor ligand binding domain complexed with 2-carbon-substituted vitamin D3 hormone analogues and a LXXLL-containing coactivator peptide, Biochemistry 43 (2004) 4101e4110. [7] L. Binderup, E. Binderup, W.O. Godtfredsen, Development of new vitamin D analogs, in: D. Feldman, F.H. Glorieux, J.W. Pike (Eds.), Vitamin D, Academic Press, San Diego, 1997, pp. 1027e1043. [8] G. Jones, Analog metabolism, in: D. Feldman, F.H. Glorieux, J.W. Pike (Eds.), Vitamin D, Volume 2, Elsevier Academic Press, San Diego, 2005, pp. 1423e1448. [9] L.A. Plum, J.M. Prahl, X. Ma, R.R. Sicinski, S. Gowlugari, M. Clagett-Dame, et al., Biologically active non-calcemic analogs of 1a,25-dihydroxyvitamin D with an abbreviated side chain containing no hydroxyl, Proc. Natl. Acad. Sci. USA 101 (2004) 6900e6904. [10] L. Binderup, E. Binderup, W.O. Godtfresen, M. Kissmeyer, Development of new vitamin D analogs, in: D. Feldman, F.H. Glorieux, J.W. Pike (Eds.), Vitamin D, Volume 2, Elsevier Academic Press, San Diego, 2005, pp. 1423e1448. [11] K.W. Colston, S.K. Chander, A.G. Mackay, R.C. Coombes, Effects of synthetic vitamin D analogs on breast cancer cell proliferation in vivo and in vitro, Biochem. Pharmacol. 44 (1992) 693e702. [12] S.Y. James, E. Mercer, M. Brady, L. Binderup, K.W. Colston, EB 1089, a synthetic analog of vitamin D, induces apoptosis in breast cancer cells in vivo and in vitro, Br. J. Pharmacol. 125 (1998) 953e962. [13] B.L. Lokeshwar, G.G. Schwartz, M.G. Selzer, K.L. Burnstein, S.H. Zhuang, N.L. Block, et al., Inhibition of prostate cancer metastasis in vivo: a comparison of 1,25-dihydroxyvitamin D (calcitriol) and EB 1089, Cancer Epidemiol. Biomark. Prev. 8 (1999) 241e248. [14] M.J. Calverley, Synthesis of MC 903, a biologically active vitamin D metabolite, Tetrahedron. 43 (1987) 4609e4619. [15] L. Binderup, E. Bramm, Effects of a novel vitamin D analogue MC 903 on cell proliferation and differentiation in vitro and on calcium metabolism in vivo, Biochem. Pharmacol. 37 (1988) 889e895. [16] K. Kragballe, The use of vitamin D3 analogues in dermatology, Dermatology 2 (1995) 198e203. [17] G. Jones, M.J. Calverley, A dialogue on analogs; newer vitamin D drugs for use in bone disease, psoriasis, and cancer, Trends Endocrinol. Metab. 4 (1993) 297e303. [18] A.M. Kissmeyer, L. Binderup, Calcipotriol (MC 903): pharmacokinetics in rats and biological activities of metabolites. A comparative study with 1,25(OH)2D3, Biochem. Pharmacol. 41 (1991) 1601e1606. [19] W. Sicinska, W.M. Westler, H.F. DeLuca, NMR assignments of tryptophan residue in Apo and Holo LBD-rVDR, Proteins 61 (2005) 461e467. [20] K.L. Perlman, R.R. Sicinski, H.K. Schnoes, H.F. DeLuca, 1a,25Dihydroxy-29-nor-vitamin D3, a novel vitamin D-related compound with potential therapeutic activity, Tetrahedron. Lett. 31 (1990) 1823e1824. [21] E. Slatopolsky, J. Finch, C. Ritter, M. Dena, J. Morrissey, A. Brown, et al., A new analog of calcitriol, 19-nor-1,25-(OH)2D2, suppresses parathyroid hormone secretion in uremic rats in the absence of hypercalcemia, Am. J. Kidney Dis. 26 (1995) 852e860.
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C H A P T E R
75 Mechanisms for the Selective Actions of Vitamin D Analogs Alex J. Brown Washington University School of Medicine, St. Louis, MO, USA
INTRODUCTION Vitamin D was first identified as an essential factor for normal mineral metabolism and skeletal development. These actions are now known to be mediated by metabolites of vitamin D, primarily 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), that act as steroid hormones, and which bind to a well-characterized intracellular receptor that is localized in the nucleus and regulates gene transcription by binding to specific motifs within target genes (see Section II of this volume). There is also in vitro evidence that 1,25(OH)2D3 and other vitamin D metabolites can interact with cell surface receptors and rapidly stimulate signaling from the cell membrane [1] (see Chapter 15). At present, the mechanisms, potential roles for these non-genomic actions and their relevance in vivo are less well understood. Finally, 1,25(OH)2D3 and its analogs, at high doses or levels, have been reported to be active in VDR null mice [2,3], suggesting the existence of another receptor that can recognize higher, non-physiologic concentrations of active vitamin D compounds. The most essential function of 1,25(OH)2D3 is to increase calcium and phosphate levels by enhancing their intestinal absorption and renal reabsorption, and modulating bone mineralization. However, research from many laboratories has demonstrated that the actions of the vitamin D system extend beyond a role restricted to bone and mineral metabolism. Many of these activities suggested potential therapeutic applications for 1,25(OH)2D3. The native vitamin D hormone is currently in use for the treatment of secondary hyperparathyroidism in renal failure patients [4], psoriasis [5], and X-linked hypophosphatemic rickets [6]. In addition, the ability of 1,25(OH)2D3 to block proliferation of many cell types, including neoplastic cells,
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10075-7
in vitro has indicated the potential of this compound for treating various types of cancer [7] (see Section X of this volume). However, a major limitation to 1,25(OH)2D3 therapy is its potent calcemic and phosphatemic activities. Studies to date suggest that the doses of 1,25(OH)2D3 necessary to block cell proliferation in vivo may also produce hypercalcemia and hyperphosphatemia. 1,25(OH)2D3 can also modulate the immune system, but this activity is also limited by its potent actions on mineral metabolism. Clearly, there is a need for vitamin D analogs with wider therapeutic windows, but to develop suitable analogs, it is necessary to understand potential mechanisms that underlie cell or gene selectivity.
IDENTIFICATION OF SELECTIVE VITAMIN D ANALOGS The major goal of vitamin D therapeutics is to develop vitamin D analogs that retain clinically useful activities of 1,25(OH)2D3 for the newly discovered indications, but with minimal or tolerable calcemic and phosphatemic activities [8]. The promising analogs identified initially displayed high differentiating and antiproliferative activities in vitro but were found to have low calcemic activity in vivo [8,9]. However, subsequent investigation found that many of these analogs were generally less active than 1,25(OH)2D3 in vivo due to reduced bioavailability. Although this may be the case for some analogs, several “non-calcemic” analogs (actually, the analogs are all calcemic but to a lesser degree than 1,25(OH)2D3) have been found to retain some of the activities of 1,25(OH)2D3 in animal models. Selective actions of several analogs have now
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75. MECHANISMS FOR THE SELECTIVE ACTIONS OF VITAMIN D ANALOGS
been demonstrated in vivo, and are discussed in several chapters of this volume. Secondary hyperparathyroidism in chronic renal failure has been treated with 1,25(OH)2D3 or its synthetic precursor 1a(OH)D3 for many years, but the potent calcemic activities of these compounds often produces hypercalcemia. Several analogs have been developed that retain the PTH suppressive effect of 1,25(OH)2D3 but with lower calcemic activity. Four analogs are currently available to patients: l,25(OH)2-19-nor-D2 (paricalcitol) and 1a(OH)D2 (doxercalciferol) in the USA and 22-oxa1,25(OH)2D3 (maxacalcitol) and 1,25(OH)2-26,27-F6-D3 (falecalcitriol) in Japan [10] (see Chapters 70 and 81). Preclinical studies revealed that 22-oxa-1,25(OH)2D3 effectively suppressed parathyroid hormone (PTH) levels in rats and dogs with renal insufficiency at doses that had minimal effects on serum calcium and phosphate levels [11e13]. Data from the study of Hirata et al. [13] demonstrating the selectivity of 22-oxa1,25(OH)2D3 are illustrated in Fig. 75.1. Similarly, 1,25(OH)2-19-norD2 was found to be about three times less active than 1,25(OH)2D3 in suppressing PTH in patients, but ten times less calcemic and phosphatemic [14e18]. Selectivity of 1a(OH)D2 and 1,25(OH)2-26,27F6-D3 on the parathyroid glands in animal models has not been reported. Other analogs shown to display
parathyroid selectivity in the uremic rat model include 1,25-dihydroxy-dihydrotachysterol [19], the 20-epi analogs CB 1093, EB 1213, and GS 1725 [20], and 2-methylene-19-nor-20(S)-1a-bishomopregnacalciferol (2MbisP) [21e23]. Clearly, it is possible to develop analogs selectively for suppression of PTH. Psoriasis is also a target disease for vitamin D therapy, and several analogs are now available for treatment of this disorder. Calcipotriol, the first of the “non-calcemic” analogs to be approved for clinical use, is effective in treating psoriatic lesions by topical application, and has minimal calcemic activity, even when administered systemically [24,25]. 1,24(OH)2D3 and 22-oxa1,25(OH)2D3 are also available in Japan for psoriasis [26,27]. Although toxicity of topically applied vitamin D compounds is less compared to systemic administration, excessive application can cause hypercalcemia and therefore the lower calcemic activities of the new analogs provide a safer means to treat psoriasis. The greatest potential for vitamin D analog therapy is in the treatment of various types of cancer, as discussed in greater detail in Section X of this volume. 22-Oxa1,25(OH)2D3 and the analog EB1089 (Leo Pharmaceuticals) suppressed PTH-related peptide in cancer cells in vivo [28,29], suggesting their use in the treatment of hypercalcemia of malignancy. 22-Oxa-1,25(OH)2D3
FIGURE 75.1 Selectivity of OCT for suppression of PTH in uremic rats. Uremic rats were treated with OCT or 1,25(OH)2D3 at the specified IV
doses every other day for 2 weeks, and PTH and calcium were determined 24 hours after the final injection. The doses of each compound that produced a 40% suppression of PTH or an increase in calcium to 11.5 mg/dl are shown. OCT was 8.3 times less potent in suppressing PTH, but 47.6 times less calcemic, indicating that the therapeutic window is approximately 5.7 times wider for OCT than for 1,25(OH)2D3. Adapted from [13].
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IDENTIFICATION OF SELECTIVE VITAMIN D ANALOGS
[30,31], calcipotriol [32] and EB1089 [33], l,25-(OH)2-16ene-23-yne-26,27-F6-vitamin D3 [34], 1a(OH)D5 [35], and Gemini analogs [36] inhibited the proliferation of breast cancer cells in vivo. The analogs KH1060, EB1089, and Ro 26-9114 reduced prostate cancer cell (LNCaP) growth in nude mice more effectively than 1,25(OH)2D3, inducing tumor necrosis and calcification, but with no hypercalcemia [37]. EB1089 also blocked growth of hepatocarcinoma (SKHEP-1 cells) in mice with no hypercalcemia [38]. The analog l,25-(OH)2-16ene-23-yne-26,27-F6-vitamin D3 was effective in the control of androgen-induced carcinoma of the prostate and seminal vesicles [39,40]. 22-Oxa-1,25(OH)2D3 [41] and 1,25-(OH)2-16-ene-23-yne-26,27-F6-vitamin D3 [42], and 1,25(OH)2-16-ene-19-nor-24-oxo-D3 [43] inhibited growth of experimentally induced tumors of the small and large intestine. Another derivative, l,25-(OH)2-16ene-23-yne-vitamin D3, prolonged the survival of mice injected with leukemia cells [44]. EB1089 was more effective than 1,25(OH)2D3 in reducing tumor size in a mouse model of head and neck squamous cell carcinoma without producing hypercalcemia [45]. Many other analogs with low calcemic activity that have been shown to inhibit proliferation of cancer cells in vitro have not yet been demonstrated to exert selectivity in vivo. The analog BXL0124 (1a,25-dihydroxy-20R-21(3hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne26,27-hexafluoro-cholecalciferol) inhibited mammary tumor growth in the ErbB2/Her-2/neu overexpressing mouse model of mammary tumorigenesis without raising serum calcium [46]. PRI-2208, the (5E,7E) analog of calcitriol, reduced tumor growth in the LLC mouse model with no effect on serum calcium [47]. At present, no successful clinical trials have been reported for vitamin D analogs in the treatment of cancer. The immunomodulatory actions of vitamin D compounds have suggested therapeutic applications for autoimmune disorders and transplantation. Modulation of the immune system with little hypercalcemia has now been demonstrated for several analogs. Abe et al. found that OCT was 50 times more potent than 1,25(OH)2D3 in augmenting a primary immune response in mice, but was 100 times less calcemic [48]. Lemire et al. demonstrated that 1,25(OH)2-16-ene-24oxo-D3 was more potent than 1,25(OH)2D3 or 1,25(OH)2-16-ene-D3 in suppressing experimental autoimmune encephalomyelitis, but, unlike the other two compounds tested, did not increase serum calcium [49]. KH1060 was shown by Mathieu et al. to reduce the incidence of diabetes in NOD mice at doses that did not increase serum or urinary calcium [50]. Zugel et al. showed that the vitamin D analog ZK 191784 effectively inhibited contact hypersensitivity in mice [51]. Although it was approximately 100 times less active than 1,25(OH)2D3, its effect on urinary calcium was
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more than 3000 times lower. More recently, the analog ZK203278 was found to inhibit allergic contact dermatitis at 100 times below the calcemic dose [52], and BXL-628 was shown to inhibit prostate inflammation and growth at non-calcemic doses [53]. These studies illustrate the selectivity of the analogs on the immune system in animal models, but successful trials in humans have not been reported. Osteoporosis is another therapeutic target for vitamin D analogs. 1,25(OH)2D3 and its synthetic analog 1a(OH)D3 have been used with success to slow mineral loss, but analogs are being developed that increase bone mineral. One such analog with clinical potential is 1,25(OH)2-2-(3-hydroxypropoxy)-D3 (ED-71). This analog was much more effective than 1,25(OH)2D3 and 1a(OH)D3 in stimulating bone mineralization in ovariectomized rats [54,55] and in corticosteroid-treated rats [56], and was recently found to be superior to 1a(OH) D3 in clinical trials. Another promising new analog for osteoporosis is Ro-26-9228. This highly modified compound (1F,25(OH)-20-epi-23-ene-25,26-dimethyl-D3) is bone protective in vivo, and has been shown to be very active in bone but not in the duodenum [57]. Similar selectivity was noted for 2-methylene-19-nor-(20S)1,25(OH)2D3 (2MD), which showed greater relative activity in bone than in the intestine. In OVX rats, 2MD increased bone mass whereas 1,25(OH)2D3 only prevented loss of bone mineral [58]. Another 2-substituted analog, 19-nor-14-epi-2a(3-hydroxypropyl)-1,25(OH)2D3 was shown to increase bone mineral density in OVX rats at non-calcemic doses [59]. The non-secosteroidal VDR ligand VDRM2 was shown to increase bone mass in OVX rats at doses that did not raise serum calcium, and although it was less potent than other vitamin D analogs used for osteoporosis, VDRM2 displayed a wider safety margin in this model [60]. The mechanism(s) responsible for the cell/tissue specificity of these analogs is under investigation, but a closer examination of their specific actions and protein binding may reveal key properties that elicit a positive effect on bone mineral. To summarize, there is considerable evidence, mostly from animal models, that vitamin D analogs can display selective actions in vivo. These analogs have relatively high affinity for the vitamin D receptor (VDR), usually within one order of magnitude, and would be expected to mimic the actions of 1,25(OH)2D3 in vivo. Most commonly, they retain the desired therapeutic effect, but are less calcemic. Some analogs, however, have higher calcemic activity than 1,25(OH)2D3 , but exert greater selectivity on the target tissue. The selectivity is not always cell or tissue specific, but can be gene or process specific within the same tissue. For example, several analogs (l,25(OH)222-ene-24-dihomo-D3, l,25(OH)222-ene-24-trihomo-D3, and 1,25,28(OH)3D2) have been shown to induce the
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vitamin-D-dependent calcium-binding protein (calbindin-D9k) in the intestine without increasing intestinal calcium transport [61,62]. Similarly, 20-epi1,25(OH)2D3 was found to be 1000 times more potent than 1,25(OH)2D3 in inducing cell differentiation in a human leukemia cell line (HL-60), but it was equipotent in activating transcription of a vitamin-Dresponsive reporter construct in the same cells [63]. The remainder of this chapter discusses potential mechanisms for the selective actions of vitamin D analogs, presenting examples when available.
five, or possibly six, classes of proteins: the nuclear vitamin D receptor, transport proteins (primarily the serum DBP), metabolic enzymes (the vitamin D-24hydroxylase and the 3-epimerase), cell surface membrane receptors, intracellular vitamin-D-binding proteins, and in at least one case cyclooxygenase-2. The following sections discuss the structural modifications in vitamin D analogs that can affect these interactions and, ultimately, the biological profile of the compounds.
Vitamin D Receptor (VDR) THE IN VIVO SELECTIVITY OF VITAMIN D ANALOGS IS DETERMINED BY MULTIPLE PROTEIN INTERACTIONS The potential mechanisms through which selectivity can be achieved are summarized schematically in Figure 75.2. These include (1) interactions with DBP or other serum proteins including lipoproteins, (2) cellular uptake and interaction with intracellular binding proteins, (3) intracellular metabolism to inactive endproducts (4) or to active intermediary metabolites (5) that, like 1,25(OH)2D3, enter the nucleus and bind to the VDR, eliciting a conformational change in the VDR (6) that promotes formation of the VDReRXR complex, binding to the activated complex to DNA and formation of the preinitiation complex RNA polymerase II (RNApol), and (7) activation of the non-genomic pathway through a putative membrane vitamin D receptor (mVDR). The factors that contribute to selectivity of only a few vitamin D analogs have been identified, and in some cases multiple factors may be involved. As a general concept, the actions of vitamin D compounds are determined by their interactions with
Most of the biological activities of vitamin D compounds are mediated by a nuclear receptor that binds 1,25(OH)2D3 with high affinity. The observation that some vitamin D analogs exerted selective actions in vivo led to early speculation that there could be multiple forms of the VDR with differing specificities. Presently, there is no evidence that multiple forms of the VDR are responsible for the altered actions of selective analogs. However, the report by Crofts et al. [64] revealed that the VDR gene is driven by multiple promoters that incorporate different 5’ exons into the VDR mRNA, which could result in VDR proteins with different N-termini. The expression of the bulk of these transcripts was low, however, and varied with cell type and tissue. It is unlikely that cell-specific expression of these forms of the VDR account for the differential effects of vitamin D analogs observed in vivo. Vitamin D ligands induce a conformational change in the receptor upon binding. This is discussed in detail in several other chapters in this volume. The altered structures of vitamin D analogs have been shown to contact the VDR differently than 1,25(OH)2D3, which may lead to subtle changes in the active VDR conformation that Potential sites of differential actions of 1,25(OH)2D3 and its analogs. Possible steps in the vitamin D activation pathways at which differences in vitamin D analog action could lead to selective activities in vivo are shown. The steps diagrammed include: (1) interactions with DBP or other serum proteins including lipoproteins, (2) cellular uptake and interaction with intracellular binding proteins, (3) intracellular metabolism to active intermediary metabolites, or (4) to inactive end-products, or (5) nuclear uptake and VDR binding (6) formation of the VDReRXR complex, binding to the activated complex to DNA and formation of the preinitiation complex RNA polymerase II (RNApol), and (7) activation of the non-genomic pathway through a putative membrane vitamin D receptor (mVDR).
FIGURE 75.2
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THE IN VIVO SELECTIVITY OF VITAMIN D ANALOGS IS DETERMINED BY MULTIPLE PROTEIN INTERACTIONS
could potentially produce cell- or gene-specific selectivity [9]. Receptors for steroid hormone are complex molecules that interact with small molecules (ligands), many other proteins (kinases, motor proteins, other transcription factors, and components of the transcriptional initiation complex) and specific DNA sequences. Not surprisingly, VDR and the members of this receptor superfamily contain multiple functional domains. The locations and functions of these domains in the VDR molecule are discussed in detail elsewhere in this volume (see Section II). The following sections present potential mechanisms by which differential interactions of vitamin D analogs with the VDR could lead to selective actions at the cellular or gene level. Ligand Binding Vitamin D analogs must bind well to the VDR to exert therapeutic actions. The key structural portion of the vitamin D compound for VDR binding is the A-ring containing the 1a-hydroxyl group. Until recently, all evidence indicated that the 1a-hydroxyl group was essential for VDR binding and activation. Modification of other parts of the molecule, however, appears capable of restoring biological activity in the absence of the 1ahydroxyl group. Gardner and coworkers [65] reported that hexafluorination of carbons 26 and 27 can partially restore the HL-60 differentiating activity of several 1desoxy analogs. The basis for these findings is unclear, but 1-hydroxylation of the analogs by these cells cannot be excluded. This explanation cannot account for the observation by Peleg et al. [66] that epimerization of carbon 20 can restore the transcriptional activity of 1bhydroxymethyl-1,25(OH)2D3. These findings indicate that other modifications appear to be capable of compensating for the lack of a functional 1a-hydroxyl group. However, 25(OH)D3 itself can activate the receptor. Direct suppression of PTH by 25(OH)D3 was demonstrated even when 1-hydroxylation was blocked with clotrimazole [67], and 19-nor-25(OH)D3, which is not 1-hydroxylated, inhibited growth of prostate cancer cells [68]. Other portions of the vitamin D backbone, notably the side chain, can be greatly modified with minimal effect on VDR binding. In fact, a recently reported analog lacking most of the side chain ((20S)-1ahydroxy-2-methylene-19-nor-bishomopregnacalciferol or 2MbisP) retains 15% of the binding affinity of calcitriol and is able to effectively suppress PTH in vivo with virtually no calcemic or phosphatemic activity [21,22]. Thus, while the lack of a hydroxyl group in the side chain of 1a(OH)D3 reduces VDR affinity by 400-fold compared to 1,25(OH)2D3, removal of most of the side chain partially restores binding even with
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no hydroxylation. The selectivity of 2MbisP, however, is likely due to its very low DBP affinity and altered pharmacokinetics [22] (see below). Other analogs that lack the C ring have been shown to retain high VDR affinity and activity [69]. Inversion of the stereochemistry at carbon 20 has been shown to greatly enhance activity [70e72]. The greater potency of the 20-epi analogs is not due to increased VDR affinity [73,74], but has been attributed to a number of other factors, including induction of a different VDR conformation [73,75], altered metabolism [76e79], reduced rate of VDR degradation [80,81], differential coactivator recruitment [82,83] and a more stable complex with the VDR [84]. This last property and the ability of 20epi analogs to slow the degradation of the VDR may reflect a slow rate of dissociation from the receptor. Typical measurements of the interaction of analogs with the VDR yield an equilibrium dissociation constant (Kd), which is simply a ratio of the rate constants for dissociation and association (Kd ¼ kdissoc/ kassoc). Vitamin D compounds can have similar Kd but different kdissoc and kassoc. These rate constants are rarely measured, but would be useful for interpreting the activities of vitamin D analogs in vitro and in vivo. Whether these rates are influenced by the formation of secondary complexes with co-regulators at target genes remains to be determined. VDR affinity may also differ between cell types in vivo. Koike et al. [85] performed autoradiography on tissues from rats injected with varying doses of [3H] OCT. They observed saturation of the tritium localization to the parathyroid glands at lower concentrations than in other tissues. The mechanism for this lower apparent Kd for the VDR in the parathyroid gland is unclear. The VDR in the parathyroid glands is identical to that in other tissues, and therefore the higher apparent affinity is likely due to other factors, e.g. the intracellular binding proteins that appear to play a role in the delivery of ligands to the VDR (see below). The ligand-binding domain (LBD) is located in the carboxy-terminal half of the VDR. At the C-terminal end of the LBD is the activation function-2 (AF-2) domain. This terminal a-helix (helix 12) undergoes a dramatic conformational shift upon binding of the ligand, a crucial step in receptor activation. As discussed below and elsewhere in this volume (see Section II), several vitamin D analogs have been shown to induce a different VDR conformation than 1,25(OH)2D3 [86]. As a result, each of the following ligand-dependent processes may be differentially affected by vitamin D analogs. It is worth noting, however, that many of the differential conformations identified above have not been confirmed directly through VDR protein crystallization (see Chapter 9).
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Translocation of the VDR to the Nucleus Ligand binding leads to nuclear translocation of the VDR from the cytoplasm to the nucleus. Two potential nuclear translocation motifs have been identified in the VDR molecule. One is a bipartate motif consisting of basic residues at each end of the sequence spanning amino acids 79 to 105 in the human VDR. The other is a basic sequence of seven amino acids (49e55) that is unique to the VDR. Point mutations in these sequences impair VDR translocation and activity. The VDR is rapidly shuttled to the nucleus along microtubules in response to 1,25(OH)2D3 in cultured fibroblasts and monocytes [87,88]. This was confirmed by following, in real time, the ligand-dependent translocation of a fusion protein of VDR with green fluorescent protein (GFP) [89]. Structural analysis revealed the requirement of the AF-2 domain of the VDR. It is likely that the VDR interacts with a motor protein that directs it to the nucleus. However, it is unclear whether this interaction is stimulated by a ligand-induced conformational change in the VDR or by activation of the process by ligand-dependent signaling via a membrane receptor (e.g., stimulation of kinases that activate the motor proteins). By either mechanism vitamin D analogs could differentially affect the rate of nuclear uptake of the VDR and, therefore, its ability to control gene transcription.
transactivation by 1,25(OH)2D3, but mutation of the PKA site on the VDR did not affect the inhibitory effect of PKA, suggesting that phosphorylation of other proteins, not the VDR, is responsible [97]. Phosphorylation of Ser208 by casein kinase-II has been shown to increase the transcriptional activity of the VDR [98]. The overall role of serine phosphorylation of the VDR appears to be stimulatory. Okadaic acid, an inhibitor of the serine/threonine protein phosphatase-1, enhances VDR-mediated transactivation [99,100]. Tyrosine phosphorylation of the VDR has also been reported. In skeletal muscle cells, 1,25(OH)2D3 has been shown to rapidly activate Src kinase leading to Src interaction with and phosphorylation of the VDR [101]. The effect of tyrosine phosphorylation on VDR activity is not clear, but it may allow interaction of the receptor with proteins involved in rapid signaling. Thus, phosphorylation of the VDR may regulate its activity. At present there is no evidence that vitamin D analogs differentially affect VDR phosphorylation. However, given that 1,25(OH)2D3 may alter the activity of the kinases responsible for VDR phosphorylation by stimulation of nongenomic pathways mediated by membrane receptors with clearly different ligand specificities, it seems likely that analogs would vary in their abilities to alter the phosphorylation state, and therefore activity, of the VDR independently of their actual affinity for the VDR.
VDR Phosphorylation The VDR becomes hyperphosphorylated in response to ligand [90] (see Section II), but the role of phosphorylation on VDR activity in vivo is not fully understood. The major ligand-dependent phosphorylation sites have been identified as Ser51 and Ser208, although other potential phosphorylation sites may be present. As for stimulating nuclear translocation, it is unclear whether the ligand induces VDR phosphorylation by changing the conformation of the receptor to allow access to protein kinases or by stimulating specific kinase cascades through activation of the membrane receptor. The effect of phosphorylation on VDR activity appears to vary with the site modified. In vitro studies have shown that phosphorylation of Ser51 by protein kinase C (PKC) [91] leads to a decrease in VDR activity [92,93]. 1,25(OH)2D3 also has been shown to rapidly activate PKC through interaction with a membrane receptor, suggesting that activation of PKC may operate to attenuate the genomic activity of hormone. The VDR also contains a consensus site for protein kinase A [94], but the role of phosphorylation of this site is unclear. Initial studies reported enhancement [95,96] and inhibition [94] of transcriptional activity. In support of the latter observation, co-expression of the catalytic subunit of PKA with the VDR in HeLa and Saos-2 cells inhibited
Heterodimerization of the VDR with RXR Ligand-activated VDR binds to DNA motifs (vitamin D response elements or VDREs) as part of a multi-protein complex. Binding to most known VDREs is greatly enhanced by the ligand-dependent interaction of the VDR with another nuclear transcription factor, the retinoid X receptor (RXR), but some VDREs may recognize a VDR complex lacking RXR [102]. It has been postulated that VDR can also form homodimers, but this has been demonstrated only in vitro with much higher, non-physiological concentrations of VDR. The portion of the VDR that interacts with RXR involves the ligand-binding domain and the interaction could be greatly affected by the type of conformational change induced by 1,25(OH)2D3 or its analogs, or by steric hindrance by the ligand itself. Liu et al. found that 20-epi-1,25(OH)2D3 is more potent than 1,25(OH)2D3 in promoting VDReRXR dimerization, in keeping with the higher activity of this analog [103]. Enhanced promotion by 20-epi analogs of VDR homodimerization has also been reported [104]. While the role of VDR homodimers is uncertain, this provides a potential mechanism for analog-enhanced activation of genes with VDREs that respond to the homodimer.
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THE IN VIVO SELECTIVITY OF VITAMIN D ANALOGS IS DETERMINED BY MULTIPLE PROTEIN INTERACTIONS
DNA Binding Promoter analysis of genes regulated transcriptionally by 1,25(OH)2D3 has identified many VDREs with similar but distinct structure [105,106] (see Section II). The most common VDRE type, designated DR3, contains two direct repeats of six nucleotide bases separated by a 3nucleotide spacer. The sequence of the hexameric repeats, or half-sites, varies considerably, but a general consensus sequence of AGGTCA has been established. In general, the RXR binds to the upstream half-site and the VDR to the downstream half-site. Another type of VDRE, the IP9, consists of two inverted palindromic sequences separated by nine base pairs [107]. Vitamin D ligands have been shown to influence both the strength and specificity of the interaction of the VDR/RXR heterodimer with DNA. Analogs with the 20-epi configuration induce a stronger interaction between the VDR and RXR in vitro, leading to a potentially more stable association of the heterodimer with VDREs. Studies by Peleg and coworkers [73] demonstrated that several 20-epi vitamin D analogs have higher activity than predicted from their VDR affinities. Using electrophoretic mobility shift assays (EMSA), which measure binding of proteins to small DNA molecules, these authors found increased binding of VDR/ RXR to the osteocalcin VDRE in nuclear extracts from cells treated with 20-epi analogs than in extracts from cells treated with 1,25(OH)2D3. Furthermore, they provided evidence that the 20-epi analogs produced a conformation in the VDR that was distinct from that induced by 1,25(OH)2D3, using an in vitro protease clipping assay in which recombinant 35S-labeled VDR bound to various ligands is treated with a protease and the fragments are resolved by electrophoresis. The 20-epi analogs protected the VDR from proteolysis in a way different from that of 1,25(OH)2D3, suggesting an altered conformational state. As stated above, the C-terminus of the VDR (AF-2 domain) is essential for binding of other critical proteins and for transcriptional activity. Peleg et al. reported that elimination of AF-2 domain prevented VDR activation by 1,25(OH)2D3 but not by the 20-epi analogs [108]. The explanation for these findings remains unclear. Ryhanen et al. [72] reported that the 20-epi analogs MC1288 and KH1060 were more potent inducers of alkaline phosphatase and osteocalcin in osteoblastic cells than 1,25(OH)2D3 and that the effects were of longer duration. They demonstrated more stable DNA binding of VDR/RXR from nuclear extracts from analog-treated than from 1,25(OH)2D3treated osteoblasts. Similar findings have been reported for other 20-epi analogs [109]. At least part of this enhanced stability induced by the 20-epi analogs may be attributed to stabilization of the VDR as discussed below.
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While these properties of the 20-epi analogs could endow them with greater potency in stimulating VDRmediated responses, they do not necessarily confer selectivity since the VDR is responsible for both antiproliferative and calcemic activities of vitamin D compounds. For example, despite the extremely potent immunosuppressive potential of KH1060 observed in vitro, the analog did not prevent renal allograft rejection or the development of hypercalcemia [110]. The vitamin D ligand may not only affect the strength of VDR binding to the VDRE but its specificity as well. Analogs containing a 20-methyl group were shown to preferentially activate VDR binding to the IP9 type of VDRE [111]. The analog EB1089 was shown to promote preferential interaction of the VDR with the IP9 type of VDRE, while CB1093 promoted binding preferentially to the DR3 type [112]. This could account for the differential effects of the two analogs on cell growth. While the two compounds are equipotent inhibitors of cell proliferation, CB1093 is ten times more active in inducing apoptosis. Thus, an analog may determine the activity of the VDR on a gene-specific basis by altering the interaction with specific VDRE motifs. Coactivator/Corepressor Recruitment Steroid receptor coactivators and corepressors interact with the VDR in a ligand-dependent fashion. Binding of the ligand promotes a dramatic conformational change in the VDR in which helix 12 folds over the ligandbinding site exposing a surface that binds coregulators. Mutations in this region abrogate the activity of 1,25(OH)2D3 by influencing both heterodimerization with RXR and the coactivator binding [113,114]. Although these mutations do not affect VDReRXR heterodimerization induced by the 20-epi-1,25(OH)2D3, they hamper SRC-1 binding and reduce transcriptional activity. Subtle differences in this conformational shift by vitamin D analogs could affect coactivator binding and, therefore, activity. A number of vitamin D analogs have been shown to differ from 1,25(OH)2D3 in their recruitment of these coactivators. The higher potencies of 20-epi analogs in the induction of the cell cycle inhibitor p21 have been attributed, at least in part, to their enhanced abilities to recruit the DRIP coactivator complex to the initiation complex [82]. Again, this could be due to enhanced stabilization of the VDR. On the other hand, the 20-epi analogs were found to be equipotent to 1,25(OH)2D3 in the recruitment of the coactivators SRC-1 and GRIP-1 [113]. A more recent study found that 20-epi compounds were more active in recruiting several coactivators (eEF1A, hsp84, eIF5B, TIF2, DRIP205, and DRIP240) [115], and 2-methylene-19-nor-20-epi-1,25(OH)2D3 was more potent in stimulating VDR binding to SRC-1 and
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DRIP205 [116]. Differential effects on ligand-induced coactivator binding to VDR have been reported for 22oxa-1,25(OH)2D3 as well [117]. While 1,25(OH)2D3 could promote interaction of the VDR with SRC-1, TIF2, and AIB1, 22-oxa-1,25(OH)2D3 supported only interaction with TIF2. The superagonist activities of the 14-epi analogs TX527 and TX522 could also be due to the potent induction of VDR binding to TIF2, SRC-1, and DRIP205 [118]. Issa et al. investigated 12 analogs for their abilities to mediate recruitment of the coactivators GRIP1 and RAC3 in vitro [83]. There was considerable ligandspecific variability in the strength of the VDRe coactivator interaction that was not correlated with the affinity of the ligands for the VDR. Analogs lacking a typical C-ring exhibit higher activity than 1,25(OH)2D3 and induce greater interaction of the VDR with SRC-1 [119]. Co-crystallization of one of these analogs (CD578) with zebrafish VDR revealed several novel contacts with the receptor that enhance the stability of helix 12. The Gemini analogs with dual side chains were shown to possess agonist activity similar to that of 1,25(OH)2D3 in the presence of the normal complement of coactivators and corepressors in MCF-7 cells, but with overexpression of corepressor CoR, the analog acted as an inverse agonist, recruiting CoR to the VDR and inducing super-repression [120]. Thus, cell-specific differences in coactivatore corepressor levels or ratio may influence the mode of action of a vitamin D analog. Ligand-dependent VDR Regulation Binding of 1,25(OH)2D3 to the VDR reduces its rate of intracellular degradation, as discussed above and in greater detail in Chapter 7. Masuyama and MacDonald [121] demonstrated that the VDR is degraded primarily by the proteasome complex, a large macromolecular structure that degrades most cellular proteins. Inhibition of proteasome activity prevented VDR turnover and increased VDR content. However, control of VDR levels by vitamin D compounds appears to be complex, since ligands may not only stabilize the VDR, but also target the receptor for degradation by promoting binding to TRIP1(SUG1), a subunit of the proteasome complex [121]. Ligands for steroid hormone receptors that dissociate more slowly have been shown to better protect the receptor from degradation [122]. Vitamin D analogs have differing abilities to stabilize the VDR, and in a few cases, this has been attributed to altered dissociation rates. The best-studied examples are the 20-epi compounds. Van den Bemd et al. [81] reported that KH1060 was much more effective than 1,25(OH)2D3 in slowing the rate of VDR degradation in osteoblastic cells. This is consistent with the finding that nuclear extracts from cells treated with the 20-epi analog
CB1093 showed more VDRE binding than nuclear extracts from 1,25(OH)2D3-treated cells, but when these vitamin D compounds were added to nuclear extracts of untreated cells ex vivo, similar VDRE binding was observed. Thus, it appears that the higher potency of 20-epi analogs cannot be attributed solely to the distinct conformational change in the VDR, but appear also to involve changes observed only in whole cells, most likely through the increased ability to stabilize the VDR. Additional evidence that slowly dissociating vitamin D ligands are superior in stabilizing the VDR was reported by Peleg et al. [123]. They found that the analog 1b-hydroxymethyl-3-epi-1,25(OH)2D3 was more sensitive to changes in the AF-2 domain, tended to dissociate more readily from the VDR and was less potent than 1,25(OH)2D3. Modifying this analog to contain 1bhydroxymethyl and dimethyl groups at carbons 26 and 27 enhanced transcriptional activity, increased the stability of the VDR to proteolysis and reduced the dissociation rate. Addition of a 16-ene to the molecule enhanced these properties even further. Stabilization leads to an increase in VDR content in the cell, and this would largely explain the greater activity of certain analogs, notably those with the 20epi configuration. Clearly, the degree of VDR upregulation would be related to the activity of the degradative pathway in the cell. In cells with a very high rate of VDR turnover, ligand-dependent stabilization would produce a larger increase in VDR content. Thus, analogs with slow dissociation rates would be expected to be more active in these cells. This could provide a mechanism for cell-selective actions of vitamin D analogs. A further complexity in VDR regulation was revealed in the recent report by Masuyama and MacDonald [121]. The VDR binds in a ligand-dependent manner to TRIP1, a component of the proteasome complex. Overexpression of TRIP1 increased VDR degradation and reduced 1,25(OH)2D3-mediated activity. These results suggest that vitamin D ligands, by promoting interaction with TRIP1, may target the VDR for degradation. The ligand specificity for TRIP1 binding has not been rigorously examined, but since this interaction requires the AF-2 domain of the VDR [121], it is likely that analogs with altered interaction with this domain would differentially promote TRIP1 binding. Thus, there appear to be counteracting effects of vitamin D compounds on VDR regulation, and the relative abilities of vitamin D compounds to stabilize the VDR or target the receptor for degradation may determine the VDR content, and hence biological activity, in a specific cell. In summary, there is abundant evidence that vitamin D analogs can induce distinct conformational changes in the VDR that can affect heterodimerization, DNA binding and coactivator/corepressor recruitment. Nonetheless, several studies that compared gene expression profiles
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of cells treated with 1,25(OH)2D3 or various vitamin D analogs found only quantitative differences, with the same set of genes regulated by both 1,25(OH)2D3 and the analog [124e127]. These critical findings suggest that analog selectivity observed in vivo may not be attributable to differences in VDR conformation, but to additional factors presented below.
Interaction with the Serum Vitamin-D-Binding Protein (DBP) and Other Transporters Vitamin D compounds are relatively hydrophobic with little solubility in aqueous solution and must be transported in vivo attached to proteins. The major carrier of vitamin D compounds in the circulation is the serum vitamin-D-binding protein (DBP). This protein binds all of the natural vitamin D metabolites with high affinity and circulates at a concentration of 5 mM, compared to picomolar levels of 1,25(OH)2D3 (see Chapter 5). Other abundant proteins such as albumin and lipoproteins may bind lesser amounts of the natural vitamin D compounds, but with much lower affinity. However, they may play a role in transporting analogs that bind poorly to DBP. In the case of 1,25(OH)2D3, over 99% is protein bound, mostly to DBP. The structural elements of vitamin D compounds that affect DBP binding are different than for interaction with the VDR. The 1a-hydroxyl group is not required for DBP binding, and 1-desoxy compounds have a higher affinity than their 1-hydroxylated counterparts. On the other hand, DBP affinity is greatly affected by changes in the side chain, a common site of modification in the therapeutically important vitamin D analogs [128]. In addition, inversion of the stereochemistry of carbon 20 dramatically reduces DBP affinity [76,129]. Altered DBP binding has been shown to play a critical role in the selectivity of several vitamin D analogs. DBP performs two major functions with respect to vitamin D compounds: it enhances their circulating half-life and, importantly in the case of 1,25(OH)2D3 and its analogs, it decreases tissue accessibility. In this way DBP acts as a reservoir and plays a key role in guarding against vitamin D intoxication. Thus, the DBP affinity will affect both the clearance rate and tissue uptake of vitamin D analogs. Altered pharmacokinetics plays a central role in determining the unique biological profile of several analogs. The side chain modifications of most of the vitamin D compounds in development reduce DBP affinity, and cause the analog to be more rapidly cleared or poorly absorbed into the circulation. The best-studied example of an analog that exerts its selectivity through this pharmacokinetic mechanism is 22-oxa1,25(OH)2D3. The affinity of 22-oxa-1,25(OH)2D3 for DBP is about 500 times lower than that of 1,25(OH)2D3
Plasma levels and intestinal localization of [3H]1,25(OH)2D3 (1,25D3) and [3H]OCT following a single intraperitoneal injection. Upper panel: Plasma levels of HPLC-purified [3H]1,25(OH)2D3 and [3H]OCT at various times post-injection. Lower panel: Duodenal VDR content of [3H]1,25(OH)2D3 and [3H]OCT at various time post-injection. From [132].
FIGURE 75.3
[130]. As a result, this analog is cleared from the circulation more rapidly than 1,25(OH)2D3 and achieves lower peak levels following injection as shown in Fig. 75.3, upper panel [131,132]. Despite the lower peak levels of 22-oxa-1,25(OH)2D3 in the blood, the peak levels of the analog were greater than those of 1,25(OH)2D3 in most target tissues, including the intestine as shown in Fig. 75.3, lower panel. This increased tissue content was short-lived, falling rapidly as the analog was cleared from the circulation. This “pulse” of 22-oxa1,25(OH)2D3 in the intestine elicited only a transient increase in calcium transport which fell to basal levels soon after 22-oxa-1,25(OH)2D3 disappeared from the circulation (Fig. 75.4, upper panel) [132]. The effects of 22-oxa-1,25(OH)2D3 on bone were also short-lived compared to those of 1,25(OH)2D3 (Fig. 71.4, lower panel). Further evidence for pharmacokinetics being responsible for the low calcemic activity of OCT was the sustained increases in intestinal calcium transport and bone mobilization with constant infusion of the analog, and the rapid return (within 24 hours) to baseline with cessation of the infusion [132]. 22-Oxa-1,25(OH)2D3 has been approved in Japan for treatment of secondary hyperparathyroidism in renal
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75. MECHANISMS FOR THE SELECTIVE ACTIONS OF VITAMIN D ANALOGS
FIGURE 75.4 Effects of OCT and 1,25(OH)2D3 on intestinal
calcium transport and bone mobilization. Vitamin-D-deficient rats were fed a 0.02% Ca diet for 2 days and then injected IP with 250 ng of 1,25(OH)2D3 or OCT or vehicle. Intestinal calcium absorption was determined by the isolated duodenal loop method using 45Ca. Bone calcium mobilization was estimated by the increase in serum calcium. From [132].
failure patients and psoriasis. In the parathyroid glands, 22-oxa-1,25(OH)2D3 treatment produces a prolonged suppression of parathyroid hormone (PTH) gene transcription [133]. The initial findings of Kobayashi et al. [131] indicated that injected [3H]22-oxa-1,25(OH)2D3 rapidly disappears from the parathyroid glands once the analog is cleared from the circulation, similar to the time course observed in the intestine and other tissues. However, the recent autoradiography study of Koike et al. [134] found that [3H]22-oxa-1,25(OH)2D3 persists in parathyroid cell nuclei. Furthermore, the nuclear binding of [3H]22-oxa-1,25(OH)2D3 in the parathyroid glands was saturated at a lower dose than the other tissues examined [85]. These findings would support a pharmacokinetic mechanism for the selectivity of 22-oxa-1,25(OH)2D3 in the parathyroid glands. This rapidly cleared analog exploits the differences in the half-lives of the therapeutic response (prolonged PTH suppression) and the toxic responses (short-lived stimulation of intestinal calcium transport and bone mobilization). The molecular basis for the different durations of these
responses is unclear. Enhancement of intestinal calcium transport and bone mobilization appears to require continuous exposure to vitamin D compounds, possibly because the inducible proteins mediating these responses have relatively short half-lives. Alternatively, the transient responses could be due to a cessation of stimulation by 22-oxa-1,25(OH)2D3 of the non-genomic pathway of vitamin D action as discussed below. The relatively long-lasting effects of all vitamin D compounds, including 22-oxa1,25(OH)2D3, on PTH gene expression may be attributable to their slower metabolism in parathyroid cells as discussed in the next section. The same pharmacokinetic mechanism may explain the effectiveness of 22-oxa-l,25(OH)2D3 in the treatment of cancer. Abe et al. showed that 22-oxa-l,25(OH)2D3 could effectively inhibit the growth of breast cancer cells in mice without producing hypercalcemia [30]. The short half-life of 22-oxa-l,25(OH)2D3 did not prevent its beneficial anti-proliferative activity. It is likely that this pharmacokinetic mechanism for analog selectivity will apply, with varying degrees, to all analogs with low DBP affinity. This mechanism may also apply to VDR ligands with non-vitamin-D structures. Boehm et al. [135] screened a chemical library and found a nonsteroidal biphenyl compound that could activate the VDR. This compound was very potent in inhibiting cancer cell growth in vitro, but had very low calcemic activity in vivo. Further analysis showed that the new vitamin D ligand did not bind to DBP. Therefore, it is likely that the compound is rapidly cleared and unable to sustain calcemic effects in the intestine and bone. As mentioned earlier, the analog 2MbisP, which lacks most of the side chain, can still bind to the VDR and suppresses PTH in vivo, but with very low calcemic and phosphatemic activities [21e23]. The truncated side chain of 2MbisP prevents DBP binding, and the analog is cleared with a half-life of only 10 minutes [22]. Thus, its selectivity for the parathyroid glands appears to involve the same pharmacokinetic mechanism as 22-oxa-l,25(OH)2D3. On the other hand, analogs with higher DBP affinity than 1,25(OH)2D3 tend to have longer circulating halflives and less accessibility to target tissues. An example is ED-71 (2-(3-hydroxypropoxy)-1,25(OH)2D3), which is in development for osteoporosis [136] (see Chapter 77). This analog increases bone formation and reduces bone resorption by mechanisms that are still unclear. ED-71 is more potent than 1,25(OH)2D3 in stimulating calcium and phosphate absorption, and this may be due, in part, to its four-fold higher DBP affinity and longer half-life [137], but resistance to metabolism may also contribute. Finally, the recent development of VDR ligands with dramatically altered structures (e.g., non-steroidal analogs) would likely have very low
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THE IN VIVO SELECTIVITY OF VITAMIN D ANALOGS IS DETERMINED BY MULTIPLE PROTEIN INTERACTIONS
affinities for DBP, which could explain their low calcemic activities. Although these studies indicate a key role of DBP in the clearance and distribution of vitamin D compounds, the recent study by Zella et al. found similar pharmacokinetics for exogenous 1,25(OH)2D3 in wild-type and DBP-null mice [138] instead of the expected rapid clearance in the knockout mice. The explanation for the disparity between these findings and those for analogs with low DBP affinities requires further investigation. Another consideration with respect to DBP affinity is its influence on analog activity in cells cultured in serum-containing medium. Analogs with lower DBP affinity than 1,25(OH)2D3 will be taken up more readily in the presence of serum and will be more active under these conditions [138]. This may often be the explanation for why some analogs have higher potency than predicted by their VDR affinity. A number of other studies have documented this serum effect on the relative activities of 1,25(OH)2D3 and its analogs [76,139,140]. Nonetheless, the most relevant system to assess the activity and selectivity of vitamin D analogs is in vivo where the serum concentration is essentially 100%. Another consequence of low DBP binding is the freedom of the analogs to associate with other serum proteins. This could potentially alter the delivery of vitamin D analogs to various target tissues. Currently, there is considerable disagreement as to the degree of binding of vitamin D metabolites and analogs to the various potential carriers. Okano et al. [130] and Kobayashi et al. [141] reported differences in the association of 22-oxa-1,25(OH)2D3 and 1,25-(OH)2D3 with serum lipoproteins. When 22-oxa-1,25(OH)2D3 was mixed in vitro with human plasma, it bound almost exclusively (99%) with the lipoprotein fraction, mainly with chylomicrons and low-density lipoproteins (LDL), whereas less 1,25(OH)2D3 was bound to lipoprotein (60%). A similar study by Teramoto et al. [142] reported much less binding of these two compounds to lipoprotein fractions. The significance of the lipoprotein binding in the selectivity of 22-oxa1,25(OH)2D3 is unclear, as the role of carrier proteins in the delivery of vitamin D compounds has received only slight attention [143e146]. At present, a comparison of binding of 22-oxa-1,25(OH)2D3 and 1,25(OH)2D3 to lipoproteins has been done only in human plasma and only in vitro. Therefore, the relevance to ligand selectivity observed in experimental animals is not clear. Better evidence for a mechanism for selectivity involving carrier protein distribution would be the demonstration of differences in the binding of vitamin D analogs and 1,25(OH)2D3 to rat lipoproteins in vivo.
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Cellular Metabolism by the Vitamin D-24-hydroxylase and Other Enzymes Cellular metabolism has been shown to play an important role in several steroid hormone systems [147]. In mineralocorticoid-responsive tissues, the receptor that mediates the action of aldosterone binds glucocorticoids with equal affinity. Selectivity is achieved in these tissues by efficient degradation of glucocorticoids, rendering them unavailable to the receptor. Alternatively, cells can convert hormones to more active metabolites as in the conversion of T4 to T3 or estrone (E1) to estradiol (E2). Vitamin D compounds are metabolized primarily, but not exclusively, by the vitamin-D-24-hydroxylase which catalyzes a series of oxidations at carbons 24 and 23 in the side chain of the molecules (see Chapter 4). Oxidative cleavage between C23 and C24 yields calcitroic acid, probably via an aldehyde intermediate. The 24hydroxylase is highly induced by 1,25(OH)2D3 and its analogs, and it is generally believed that side chain metabolism has an attenuating effect on vitamin D action. Intermediary metabolites of 1,25(OH)2D3 produced by this pathway have lower VDR affinity and are usually less active than the parent, but exceptions have been noted and are discussed below. Since the most common site of modification of vitamin D analogs is in the side chain, it would be expected that their metabolism would differ from that of 1,25(OH)2D3. In fact, differences in the rates of catabolism and the end-products of metabolism have been shown to account, at least in part, for the unique properties of several vitamin D analogs. Analogs that are more quickly degraded within target cells would have lower overall biological activity. This effect was illustrated in a study by Zhao et al. in cell culture [148]. They examined the anti-proliferative effects of 1,25(OH)2D3 and several analogs on cultured MCF-7 breast cancer cells (Fig. 75.5). The ED50 values for each analog were reduced by co-treatment with ketoconazole, a cytochrome P450 inhibitor that blocks 24-hydroxylase activity. The higher ED50 in the absence of the inhibitor was due to the influence of target cell catabolism of the vitamin D compounds. The degree of the reduction of the ED50 by ketoconazole co-treatment varied for each analog, likely due to the differential rates of catabolism of the compounds. Other examples of analogs whose activity may be influenced by their catabolic rate include EB1089 and 20-epi1,25(OH)2D3. These compounds have higher biological activity than 1,25(OH)2D3 despite similar VDR affinities. The disparity has been attributed, at least in part, to their very slow rates of intracellular catabolism [76,149e151]. Lin et al. showed that EB1089 produced the same changes in gene expression as 1,25(OH)2D3 in squamous
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75. MECHANISMS FOR THE SELECTIVE ACTIONS OF VITAMIN D ANALOGS
FIGURE 75.5 Influence of catabolism on the biological activities of vitamin D analogs. Left panel: The anti-proliferative effects of 1,25(OH)2D3 and its analog KH1060 on MCF-7 breast cancer cells was measured in the absence or presence of the cytochrome P450 inhibitor ketoconazole. Blocking the catabolic enzyme (24hydroxylase) enhanced the anti-proliferative activities. Right panel: The ratio of the ED50 in the presence (þketo) versus absence (keto) of ketoconazole. The activity is enhanced by ketoconazole in all cases, but the variation between analogs likely reflects differences in their rates of catabolism. From [148].
carcinoma cells, but that the effects were more prolonged [152]. The difference was abolished when 1,25(OH)2D3 was co-incubated with ketoconazole. It is important to note that rapid disappearance of an analog in the target cell could produce the same differential effects as accelerated clearance as described for analogs with low DBP affinity. The selectivity of vitamin D analogs in vivo may also be attributed to cell-specific differences in the rates of catabolism of an analog. For example, 22-oxa1,25(OH)2D3 appears to be degraded at the same rate as 1,25(OH)2D3 in parathyroid cells [153], more rapidly in keratinocytes [154] and more slowly by monocytes [155]. The rates of catabolism are consistent with the similar activities of 22-oxa-1,25(OH)2D3 and 1,25(OH)2D3 in parathyroid cells [153] and lower activity of 22-oxa-1,25(OH)2D3 in keratinocytes [154]. The explanation for this cell-specific catabolism is unclear but certainly deserves further study since exploitation of the catabolic differences between vitamin D compounds could produce highly selective therapeutic agents. There are also differences in the amount of catabolic activity in various cell types. In the study by Zhao et al. described above [148], ketoconazole had no effect on the activities of the analogs in the osteoblastic cell line MG-63, indicating that the catabolic activity in these cells is very low. This variation in catabolic activity could lead to cell-specific differences in the actions of 1,25(OH)2D3 and its analogs. In cells with high catabolic activity, analogs that are better substrates for the 24hydroxylase would be rapidly degraded and less active, whereas ligands that are poor substrates would be more active. By this mechanism alone, each analog could have a unique biological profile. Structural modifications in some vitamin D analogs prevent the completion of the side chain cleavage pathway and yield stable active intermediates that
may accumulate in target cells. One example is 1,25(OH)2-16-ene-D3. This analog is catabolized by the 24-hydroxylase to the 24-oxo intermediate [156], but further oxidation occurs very slowly. The 1,25(OH)216-ene-24-oxo-D3 retains significant biological activity in vitro [157] and in vivo [49]. In a murine model of autoimmune encephalitis, 1,25(OH)2-16-ene-24-oxo-D3 was as active as 1,25(OH)2-16-ene-D3 and 1,25(OH)2D3 in suppressing the immune response, but it was substantially less calcemic. A similar interruption in side chain catabolism was noted for 20-epi-1,25(OH)2D3. The 24oxo metabolite of this analog accumulates in target cells and may be responsible in part for the high biological activity of the parent 20-epi compound [76]. KH1060, which has been shown to be much more active than 1,25(OH)2D3 in slowing cell proliferation, is rapidly metabolized to 24- and 26-hydroxylated metabolites that are also more potent than 1,25(OH)2D3 [158]. These two metabolites have potencies similar to the parent KH1060 in the induction of osteocalcin expression by osteoblast-like cells, in enhancing VDR stability by increasing resistance to proteolysis, and in binding of the VDR to the osteocalcin VDRE [159]. Thus, the 24and 26-hydroxy metabolites contribute significantly to the overall effects of the parent KH1060. Komuro et al. [160] found that 1,25(OH)2-26,27-F6-D3 is converted to 1,23,25(OH)3-26,27-F6-D3 which is resistant to further metabolism and retains significant activity. This metabolite was found to accumulate in parathyroid glands where it could repress PTH expression, but it also accumulates in the intestine and kidney where it could increase serum calcium [161]. Thus, it appears unlikely that resistance to metabolism would confer selectivity to this analog. Similarly, 1,25(OH)2-16ene-20-cycloproply-D3 is converted to a stable 24-oxo metabolite that retains anti-inflammatory activity, but is less calcemic than the parent compound [162].
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THE IN VIVO SELECTIVITY OF VITAMIN D ANALOGS IS DETERMINED BY MULTIPLE PROTEIN INTERACTIONS
Recent development of analogs with substitutions at C-2 revealed that modification of this site in the A-ring affects 24-hydroxylation. Flanagan et al. reported that 19-nor-2a-hydroxypropyl-1,25(OH)2D3 was much more potent than calcitriol in inhibiting prostate cell growth in vitro, which they attributed to the 50-fold slower metabolism of this analog by the 24-hydroxylase [163]. Saito et al. found that 2a-propoxy-1,25(OH)2D3 was metabolized more slowly than 1,25(OH)2D3 [164]. Several groups have demonstrated that 2a-substituted vitamin D analogs are more active than predicted by their VDR affinity, even when tested in vitro [163e168], probably because the stability of these compounds. We found a similar resistance to metabolism of 2b-(3-hydroxypropyloxy)-1,25(OH)2D3 (ED-71) [168a], indicating that substitutions in both orientations impede the 24-hydroxylase. A 24-phenylsulfone analog of 1,25(OH)2D3 was shown to be active and also to inhibit 24-hydroxylase activity [169], allowing activity in tumor cells with high catabolic activity. Other metabolic enzymes may play a key role in analog selectivity. Reddy and his colleagues recently discovered that 1,25(OH)2D3 can undergo epimerization at carbon 3 to change the 3b-hydroxyl to the 3a configuration [170]. The enzyme responsible for 3-epimerization is not ubiquitously expressed. This reaction has been documented in keratinocytes, parathyroid cells, osteoblastic cells, and colon cancer (Caco-2) cells, but not in the kidney or myeloid leukemia (HL-60) cells. In parathyroid cells, 3-epi-1,25(OH)2D3 has nearly the same potency as 1,25(OH)2D3 in suppressing PTH, but is catabolized more slowly by the 24-hydroxylase and may accumulate in these cells [171]. The slower inactivation, if it occurs in parathyroid glands in vivo, could be responsible, at least in part, for the prolonged effects of 1,25(OH)2D3 on PTH secretion. An example of cell-specific metabolism was reported by Kamao et al. [172]. 22-Oxa-1,25(OH)2D3 was metabolized mainly by the 24-hydroxylase in intestinal (Caco-2) cells and renal (LLC-PK1) cells, whereas in osteoblastic (UMR-106) cells, 22-oxa-1,25(OH)2D3 was metabolized by the 3-epimerization and 25-dehydration pathways. Differences between 1,25(OH)2D3 and its analogs in their relative rates of metabolism through these pathways could lead to preferential action in one cell over the other. The structural requirements for substrates of the enzyme responsible for 3-epimerization are under investigation. Reddy et al. found that the rate of 3-epimerization of 1,25(OH)2-16-ene-23-yne-20-epi-D3 was 10 times faster than for 1,25(OH)2-16-ene-23yne-D3 [173]. It is likely that the 20-epi modification facilitates 3-epimerization of other analogs as well. Structural alterations that facilitate the conversion to the 3-diastereomer may further slow the rate of metabolism
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in cells with high 3-epimerase activity and therefore enhance the analog action in a cell-specific manner. Differential metabolism may also play a role in the calcemic actions in vivo of 19-nor-1,25(OH)2D2 and 20-epi-1,25(OH)2D3. Both compounds produced the same effect as 1,25(OH)2D3 on intestinal calcium absorption and gene regulation at 24 hours after a single treatment. With repeated dosing, the effects of 19-nor1,25(OH)2D2 diminished compared to 1,25(OH)2D3 [15]. In contrast, the activity of 20-epi-1,25(OH)2D3 continued to increase at 48 hours post-dose while the effects of 1,25(OH)2D3 did not [79]. In both cases differential rates of catabolism compared to 1,25(OH)2D3 in the intestine were suggested as a potential mechanism. Finally, the heavily altered non-steroidal VDR ligands are probably not substrates for the 24-hydroxylase or the 3-epimerase and are likely metabolized by other enzymes. This could have a major influence on their activities in a cell-specific manner. The examples presented in this section illustrate the diverse modifications that influence metabolism of vitamin D compounds. Alterations of the side chain may be expected to interfere with the 24-hydroxylase, but substituents at other sites, notably the 2 position, block side chain metabolism as well. Resistance to metabolism is likely a common feature of many of the superagonists described in the literature, and may allow for selectivity in tissues with high rates of catabolism.
Rapid Actions Mediated by a Cell-surface Receptor Vitamin D compounds can activate a number of signaling pathways, apparently by activating a receptor at the plasma membrane [1,174] (see Chapter 15). These effects are observed within seconds to minutes following exposure to 1,25(OH)2D3, too quickly to involve changes in gene transcription, although at least some of the target alterations within the cell may ultimately be dependent upon changes in gene expression. One of the best-characterized rapid actions is the stimulation of calcium movement across the intestinal epithelium, a process termed transcaltachia. Nemere and coworkers perfused chick duodenum with 1,25(OH)2D3 and observed increased calcium movement from the lumen to the perfusate within minutes [175]. Others have noted rapid stimulation by 1,25(OH)2D3 of PKC activation and translocation, phosphate fluxes, alkaline phosphatase, cGMP, and phosphoinositide metabolism in cell culture. 1,25(OH)2D3 increases cytosolic calcium within minutes in a number of cell types, including osteoblasts, parathyroid cells, human myeloid leukemia cells, enterocytes, and myocytes. 1,25(OH)2D3 has been shown to rapidly activate mitogen-activated
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protein kinase (MAPK) [176,177] and stimulate opening of chloride channels [178] (see Chapter 15). The nature of the receptor(s) that mediates these rapid actions remains controversial. Early evidence suggested that the rapid effects of calcitriol may be mediated by the VDR. 1,25(OH)2D3 cannot rapidly increase cGMP in skin fibroblasts from patients with vitamin-D-resistance rickets that lack a functional VDR. In addition, calcitriol has also been shown to stimulate phosphoinositide metabolism in isolated enterocytes from adult rats, but not in those from 16-day-old rats that do not express intestinal vitamin D receptors [179]. However, the ligand specificity for the rapid actions is different to that for the genomic response [128,180,181]. Stimulation of the non-genomic actions does not require the 1a-hydroxyl group; 25(OH)-23-yne-D3 and its 16-ene counterpart can stimulate transcaltachia, but do not bind the VDR [180]. In contrast, 1,25(OH)-16-ene-23-yne-2D3 and calcipotriol, which have high VDR affinity, do not produce transcaltachia [180]. Further evidence for the distinct nature of the nuclear and membrane receptors is the finding that 22-oxa-1,25(OH)2D3 has genomic but not non-genomic activity in rat osteoblastic cells [182]. More recent studies have demonstrated that vitamin D analogs that are locked in the 6-cis configuration act only as agonists for the putative membrane receptor and do not bind to the VDR [181]. On the other hand, in silico modeling of the VDR has suggested the existence of a separate, but overlapping, binding site for non-genomic ligands [183], suggesting that the VDR itself may mediate the rapid actions of vitamin D (see Chapter 15). However, it is not clear why binding to this site has not been detectable with the non-genomic ligands. There is also considerable evidence that rapid actions are mediated by a distinct plasma membrane protein, originally isolated from chick duodenum, that binds vitamin D analogs with affinities that correlate with their activation of transcaltachia [184]. Antibodies raised against the protein recognized primarily a 66 kDa peptide by immunoblot analysis, blocked binding to the plasma membrane and inhibited the 1,25(OH)2D3 stimulation of PKC activity in chondrocyte membranes [185]. This receptor, 1,25D3-MARRS (membrane-associated, rapid response steroid binding), has now been identified as ERp57 [174]. The functions of the non-genomic actions of 1,25(OH)2D3 in most cell types and their relevance in vivo are still unclear. In the intestine, it is well established that exposure of the basolateral membrane to 1,25(OH)2D3 stimulates transcaltachia [180]. In chondrocytes, non-genomic actions of 1,25(OH)2D3 and 24,25(OH)2D3 alter membrane lipid turnover, prostaglandin production, and protease activity that leads to modification of bone matrix and calcification [186].
In other cells, the non-genomic events have been proposed to modulate the genomic actions of 1,25(OH)2D3 [187,188], but this remains controversial. Numerous studies have presented evidence that the non-genomic actions may not be critical for gene activation [98,182, 1,25(OH)2D3-mediated 189e192] or inhibition of cell proliferation [191,193]. However, non-genomic stimulation of protein kinases could potentially influence the VDR-mediated effects of 1,25(OH)2D3. As introduced earlier, genomic activity of the VDR is decreased by PKC which is stimulated by 1,25(OH)2D3 activation of the membrane receptor. Thus, differential effects of vitamin D analogs on the membrane receptor could influence its genomic actions as well as the VDR-independent effects of the signaling pathways. A recent example of this was the observation that silencing of the 1,25D3-MARRS in breast cancer (MCF-7) cells enhanced the growth-inhibitory effects of 1,25(OH)2D3, but not those of other inhibitors (retinoic acid, paclitaxel, or pomiferin), indicating that the membrane receptor interferes with the antiproliferative actions of the VDR [194]. This observation implies that analogs that bind the VDR but not the 1,25D3-MARRS would be more effective anti-cancer agents than 1,25(OH)2D3. The low calcemic activity of two analogs, 1,25(OH)216-ene-23-yne-D3 and calcipotriol, could be due to their inability to stimulate transcaltachia in the intestine [180]. Several other analogs, including 1,25,28trihydroxyvitamin D2, 1,25(OH)2-24-dihomo-22-ene-D3, 1,25(OH)2-24-trihomo-22-ene-D3, that stimulate genomic responses (calbindin D9k) in the intestine but not calcium transport [61,62] may lack non-genomic activity, but these analogs have not been tested. On the other hand, 22-oxa-1,25(OH)2D3 has been shown to stimulate transcaltachia in the intestine [182]. The inability of 22oxa-1,25(OH)2D3 to sustain a high rate of calcium transport after its disappearance from the circulation (see Fig. 75.3) could be due to the loss of stimulation of the non-genomic pathway(s). The physiologic role of the non-genomic pathway in bone is not fully understood. As described above, the non-genomic effects of 1,25(OH)2D3 and 24,25(OH)2D3 may play a role in bone formation [186]. 1,25(OH)2D3 is known to stimulate both the production and phosphorylation of an osteoblast-derived protein, osteopontin. Safran et al. [195] found that the analog 25(OH)16-ene-23-yne-D3, which is known to stimulate only the non-genomic pathway (no VDR affinity), cannot induce osteopontin but does promote its phosphorylation. Clearly, non-genomic activities could play a role in the vitamin D effects on bone formation. On the other hand, the role of non-genomic effects on bone resorption is not known. It is possible that analogs that are incapable of activating or sustaining a non-genomic
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CONCLUDING REMARKS
response are unable to efficiently induce or maintain bone mobilization. Despite remaining questions concerning the physiologic relevance of the non-genomic pathways, these membrane-binding sites could potentially offer new pharmacologic targets that could produce cellspecific or even process-specific effects of vitamin D analogs.
Intracellular Vitamin-D-Binding Proteins Proteins of the heat shock-70 family are now known to bind vitamin D compounds and appear to play a determining role in their metabolism and actions [196e203]. These proteins, discovered during the investigation of the vitamin D resistance observed in New World primates, were found to be overexpressed in a marmoset B-lymphoblast cell line B95-8. Unfractionated B95-8 cytosol containing a mixture of intracellular vitamin-D-binding proteins (IDBPs) was used in a competitive binding assay with [3H]25(OH)D3 to assess binding affinities of various vitamin D ligands. 25(OH)D3 and 25(OH)D2 bound most avidly. 1,25(OH)2D3 and 1,25(OH)2D2 had about one-third the affinity of 25(OH)D3, and 1a(OH)D3, which lacks a 25hydroxyl group, did not bind at all [196,200]. Overexpression of IDBP-1, later identified as hsc70, stimulated the transactivation by 1,25(OH)2D3 [201] and enhanced the conversion of 25(OH)D3 to 1,25(OH)2D3 [202]. Subsequently, it was reported that IDBP-1 predominantly directs delivery of ligands to the VDR, while IDBP-3 facilitates mitochondrial delivery of substrates via an N-terminal targeting signal for the inner mitochondrial membrane where it interacts directly with the vitamin D-1a-hydroxylase [203]. The mitochondrial delivery by IDBP-3 of active vitamin D compounds (i.e., VDR ligands) may accelerate their metabolism and inactivation. More recently, the hsc70 co-chaperone Bcl2associated athanogene (BAG)-1 was also found to enhance VDR transcriptional activity, but not metabolism [204]. A more thorough discussion of the IDBP and their roles in vitamin D metabolism and action can be found in Chapter 14. Only a few vitamin D analogs have been tested for their binding to the IDBPs. Calcipotriol, EB1089, and KH1060 were found to have no apparent binding affinity for the IDBPs in unfractionated supernatants from B95-8 cells [199] when competing with [3H]25(OH)D3 as ligand. These three analogs are modified in their side chains, indicating the importance of this portion of the vitamin D structure in binding to the IDBPs. In addition, the initial studies used unfractionated cytosol as a source of a mixture of IDBPs; the interaction of the analogs with the individual IDBPs has not been reported.
The finding that distinct IDBPs may be involved in enhancement of transactivation and the facilitation of metabolism suggests a complex means for potentially affecting analog selectivity. High-affinity binding to IDBP-1 would increase the potency whereas binding to IDBP-3 would facilitate metabolic inactivation. Relative differences in the interactions of analogs in binding to the IDBPs along with cell-specific differences in the relative expressions of the IDBPs could contribute to the unique biological profiles of the analogs. Future studies are required to assess the impact of the IDBPs on the actions of 1,25(OH)2D3 and its analogs.
Cyclooxygenase-2 The immunomodulatory actions of vitamin D compounds involve regulated expression of a number of genes, including cyclooxygenase-2 (COX-2). Repression of COX-2 contributes to the inhibitory action of vitamin D analogs on prostate cancer [205]. However, the recent study of Aparna et al. demonstrated that COX-2 activity is directly inhibited by 1,25(OH)2D3 and even more dramatically by 1,25(OH)2-16-ene-23-yne-D3 (IC50 ¼ 5.8 nM) [206]. COX-1 activity was not affected. This novel finding revealed another direct target that can be differentially regulated by vitamin D analogs.
CONCLUDING REMARKS Discovery of the non-classical actions of 1,25(OH)2D3 has spawned new interest in vitamin D therapy for the treatment of hyperproliferative diseases such as cancer and psoriasis, and endocrine disorders including hyperparathyroidism, as well as for immunosuppression to prevent autoimmunity and transplant rejection. Hypercalcemia has limited or precluded the use of 1,25(OH)2D3 for most of these applications. Several new vitamin D analogs that retain the desired therapeutic activity but with less toxic (calcemic) side effects have now reached the clinic. Calcipotriol is available for psoriasis, and four analogs, 19-nor-1,25(OH)2D2, 22-oxa1,25(OH)2D3, 1a(OH)D2, and 1,25(OH)2-26,27-F6-D3, have been approved for secondary hyperparathyroidism. Many other analogs are in development for other applications. Perhaps the most critical of these is cancer. The initial approach for developing therapeutic vitamin D analogs involved testing of hundreds of compounds in cell culture and animal models. As selective analogs were discovered, the mechanisms responsible were investigated, and a few general concepts have begun to emerge. For hyperparathyroidism, a wider therapeutic window was found for compounds that are rapidly cleared such as 22-oxa-1,25(OH)2D3 and
IX. ANALOGS
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75. MECHANISMS FOR THE SELECTIVE ACTIONS OF VITAMIN D ANALOGS
2MbisP, although this does not appear to hold for 19nor-1,25(OH)2D2. Several analogs for osteoporosis that increase bone density are modified at C2, but the mechanism for their actions in bone are not yet clear. Our current knowledge of vitamin D physiology and biochemistry indicates that the overall activities of vitamin D compounds are primarily determined by the combined interactions with several key proteins: the nuclear vitamin D receptor, the serum vitamin-Dbinding protein, the 24-hydroxylase and a membrane receptor, although other proteins may be involved. A major goal is to understand how the integrated interactions lead to the in vivo actions of the analogs. This requires an understanding of both the structureeactivity relationships for each of these interactions and the role that these proteins play in the desired and undesired activities. As our knowledge of these two areas increases, so does the reality of designing vitamin D analogs with precise target specificity.
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1457 M.L. Siu-Caldera, J.W. Clark, A. Santos-Moore, S. Peleg, Y.Y. Liu, M.R. Uskokovic, et al., 1Alpha,25-dihydroxy-24-oxo16-ene vitamin D3, a metabolite of a synthetic vitamin D3 analog, 1alpha,25-dihydroxy-16-ene vitamin D3, is equipotent to its parent in modulating growth and differentiation of human leukemic cells, J. Steroid Biochem. Mol. Biol. 59 (1996) 405e412. F.J. Dilworth, G.R. Williams, A.M. Kissmeyer, J.L. Nielsen, E. Binderup, M.J. Calverley, et al., The vitamin D analog, KH1060, is rapidly degraded both in vivo and in vitro via several pathways: principal metabolites generated retain significant biological activity, Endocrinology 138 (1997) 5485e5496. G.C. van den Bemd, F.J. Dilworth, H.L. Makin, J.M. Prahl, H.F. DeLuca, G. Jones, et al., Contribution of several metabolites of the vitamin D analog 20-epi-22-oxa24a,26a,27a-trihomo1,25(OH) 2-vitamin D3 (KH1060) to the overall biological activity of KH1060 by a shared mechanism of action, Biochem. Pharmacol. 59 (2000) 621e627. S. Komuro, H. Kanamaru, I. Nakatsuka, A. Yoshitake, Distribution and metabolism of F6-1,25(OH)2 vitamin D3 and 1,25(OH)2 vitamin D3 in the bones of rats dosed with tritiumlabeled compounds, Steroids 63 (1998) 505e510. S. Komuro, M. Sato, H. Kanamaru, Disposition and metabolism of F6-1alpha,25(OH)2 vitamin D3 and 1alpha,25(OH)2 vitamin D3 in the parathyroid glands of rats dosed with tritium-labeled compounds, Drug. Metab. Dispos. 31 (2003) 973e978. G. Laverny, G. Penna, M. Uskokovic, S. Marczak, H. Maehr, P. Jankowski, et al., Synthesis and anti-inflammatory properties of 1alpha,25-dihydroxy-16-ene-20-cyclopropyl-24-oxovitamin D3, a hypocalcemic, stable metabolite of 1alpha,25dihydroxy-16-ene-20-cyclopropyl-vitamin D3, J. Med. Chem. 52 (2009) 2204e2213. J.N. Flanagan, S. Zheng, K.C. Chiang, A. Kittaka, T. Sakaki, S. Nakabayashi, et al., Evaluation of 19-nor-2alpha-(3hydroxypropyl)-1alpha,25-dihydroxyvitamin D3 as a therapeutic agent for androgen-dependent prostate cancer, Anticancer. Res. 29 (2009) 3547e3553. N. Saito, Y. Suhara, D. Abe, T. Kusudo, M. Ohta, K. Yasuda, et al., Synthesis of 2alpha-propoxy-1alpha,25-dihydroxyvitamin D3 and comparison of its metabolism by human CYP24A1 and rat CYP24A1, Bioorg. Med. Chem. 17 (2009) 4296e4301. M. Shimizu, Y. Miyamoto, E. Kobayashi, M. Shimazaki, K. Yamamoto, W. Reischl, et al., Synthesis and biological activities of new 1alpha,25-dihydroxy-19-norvitamin D3 analogs with modifications in both the A-ring and the side chain, Bioorg. Med. Chem. 14 (2006) 4277e4294. A. Glebocka, R.R. Sicinski, L.A. Plum, M. Clagett-Dame, H.F. DeLuca, New 2-alkylidene 1alpha,25-dihydroxy-19-norvitamin D3 analogues of high intestinal activity: synthesis and biological evaluation of 2-(30 -alkoxypropylidene) and 2(30 -hydroxypropylidene) derivatives, J. Med. Chem. 49 (2006) 2909e2920. E. Kobayashi, M. Shimazaki, Y. Miyamoto, H. Masuno, K. Yamamoto, H.F. DeLuca, et al., Structureeactivity relationships of 19-norvitamin D analogs having a fluoroethylidene group at the C-2 position, Bioorg. Med. Chem. 15 (2007) 1475e1482. A. Glebocka, R.R. Sicinski, L.A. Plum, H.F. DeLuca, 2-(30 -Hydroxypropylidene)-1alpha-hydroxy-19-norvitamin D compounds with truncated side chains, J. Steroid. Biochem. Mol. Biol. 103 (2007) 310e315.
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C.S. Ritter, A.J. Brown, Suppression of PTH by the vitamin D analog eldecalcitol is modulated by its high affinity for the serum vitamin D binding protein and resistance to metabolism, J. Cell. Biochem. (2011) in press. D. Lechner, T. Manhardt, E. Bajna, G.H. Posner, H.S. Cross, A 24-phenylsulfone analog of vitamin D inhibits 1alpha,25dihydroxyvitamin D(3) degradation in vitamin D metabolism-competent cells, J. Pharmacol. Exp. Ther. 320 (2007) 1119e1126. H. Sekimoto, M.L. Siu-Caldera, A. Weiskopf, P. Vouros, K.R. Muralidharan, W.H. Okamura, et al., 1Alpha,25dihydroxy-3-epi-vitamin D3: in vivo metabolite of 1alpha,25-dihydroxyvitamin D3 in rats, FEBS. Lett. 448 (1999) 278e282. A.J. Brown, C. Ritter, E. Slatopolsky, K.R. Muralidharan, W.H. Okamura, G.S. Reddy, 1Alpha,25-dihydroxy-3-epivitamin D3, a natural metabolite of 1alpha,25-dihydroxyvitamin D3, is a potent suppressor of parathyroid hormone secretion, J. Cell Biochem. 73 (1999) 106e113. M. Kamao, S. Tatematsu, S. Hatakeyama, K. Ozono, N. Kubodera, G.S. Reddy, et al., Two novel metabolic pathways of 22-oxacalcitriol (OCT). C-25 dehydration and C-3 epimerization and biological activities of novel OCT metabolites, J. Biol. Chem. 278 (2003) 1463e1471. G.S. Reddy, D.S. Rao, M.L. Siu-Caldera, N. Astecker, A. Weiskopf, P. Vouros, et al., 1a25-Dihydroxy-16-ene-23-ynevitamin D3 and 1a,25-dihdyroxy-16-ene-23-yne-20-epivitamin D3: analogs of 1a25-dihdroxyvitamin D3 that resist metabolism through the C-24 oxidation pathway are metabolized through the C-3 epimerization pathway, Arch. Biochem. Biophys. 383 (2000) 197e205. R. Khanal, I. Nemere, Membrane receptors for vitamin D metabolites, Crit. Rev. Eukaryot. Gene. Expr. 17 (2007) 31e47. I. Nemere, Y. Yoshimoto, A.W. Norman, Calcium transport in perfused duodena from normal chicks: enhancement within fourteen minutes of exposure to 1,25-dihydroxyvitamin D3, Endocrinology 115 (1984) 1476e1483. D.W.A. Beno, L.M. Brady, M. Bissonnette, B.H. Davis, Protein kinase C and mitogen-activated protein kinase are required for 1,25-dihydroxyvitamin D3-stimulated Egr induction, J. Biol. Chem. 270 (1995) 3642e3647. X. Song, J.E. Bishop, W.H. Okamura, A.W. Norman, Stimulation of phosphorylation of mitogen-activated protein kinase by 1a,25-dihydroxyvitamin D3 in promyelocytic NB4 leukemic cells: a structureefunction study, Endocrinology 139 (1998) 457e465. L.P. Zanello, A.W. Norman, 1 Alpha,25(OH)2 vitamin D3mediated stimulation of outward anionic currents in osteoblast-like ROS 17/2.8 cells, Biochem. Biophys. Res. Commun. 225 (1996) 551e556. M. Lieberherr, B. Grosse, P. Duchambon, T. Drueke, A functional cell surface type receptor is required for the early action of 1,25-dihydroxyvitamin D3 on the phosphoinositide metabolism in rat enterocytes, J. Biol. Chem. 264 (1989) 20403e20406. L.X. Zhou, I. Nemere, A.W. Norman, 1,25-Dihydroxyvitamin D3 analog structureefunction assessment of the rapid stimulation of intestinal calcium absorption (transcaltachia), J. Bone Min. Res. 7 (1992) 457e463. A.W. Norman, X. Song, L. Zanello, C. Bula, W.H. Okamura, Rapid and genomic biological responses are mediated by different shapes of the agonist steroid hormone, 1alpha,25(OH)2vitamin D3, Steroids 64 (1999) 120e128. M.C. Farach-Carson, J. Abe, Y. Nishii, R. Khoury, G.C. Wright, A.W. Norman, 22-Oxacalcitriol: dissection of
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1459 intracellular vitamin D binding protein-1, Endocrinology 143 (2002) 4135e4138. J.S. Adams, H. Chen, R.F. Chun, L. Nguyen, S. Wu, S.Y. Ren, et al., Novel regulators of vitamin D action and metabolism: lessons learned at the Los Angeles zoo, J. Cell Biochem. 88 (2003) 308e314. R.F. Chun, M. Gacad, L. Nguyen, M. Hewison, J.S. Adams, Co-chaperone potentiation of vitamin D receptor-mediated transactivation: a role for Bcl2-associated athanogene-1 as an intracellular-binding protein for 1,25-dihydroxyvitamin D3, J. Mol. Endocrinol. 39 (2007) 81e89. A.V. Krishnan, S. Srinivas, D. Feldman, Inhibition of prostaglandin synthesis and actions contributes to the beneficial effects of calcitriol in prostate cancer, Dermatoendocrinol. 1 (2009) 7e11. R. Aparna, J. Subhashini, K.R. Roy, G.S. Reddy, M. Robinson, M.R. Uskokovic, et al., Selective inhibition of cyclooxygenase2 (COX-2) by 1alpha,25-dihydroxy-16-ene-23-yne-vitamin D3, a less calcemic vitamin D analog, J. Cell Biochem. 104 (2008) 1832e1842.
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C H A P T E R
76 Analogs of Calcitriol Lieve Verlinden 1, Guy Eelen 1, Roger Bouillon 1, Maurits Vandewalle 2, Pierre De Clercq 2, Annemieke Verstuyf 1 1
2
Katholieke Universiteit Leuven, Leuven, Belgium Universiteit Gent, Vakgroep voor Organische Chemie, Gent, Belgium
the structure of 1,25(OH)2D3: a central rigid CD-bicyclic ring portion to which are connected at C-17 the flexible side chain and at C-8 the flexible seco-B,A-ring system. Many scientists focused on the introduction of chemical modifications into these flexible parts of the secosteroidal structure of 1,25(OH)2D3 such as elongation, unsaturation, epimerization of the side chain or alterations of the A-ring such as the successful 19-nor modification which resulted in a 10-fold decreased calcemic activity of 1,25(OH)2D3 [2,3]. Next to the introduction of structural modifications in the side chain or the A-ring, an extensive study of the structureefunction relationship was performed focusing on the least-studied CD-ring region. In this context the CD-cis fused (14-epi analogs) analogs were synthesized. Furthermore, the central CD-region has been fundamentally changed by replacing the natural hydrindane CD-ring fragment into a decalin CD-ring fragment. Moreover, by stripping the molecule to its five-carbon backbone (C-8 to C-20, KS 018, Fig. 76.1) and resubstituting it again in various ways different classes of non-steroidal analogs were
INTRODUCTION The paracrine function of 1,25(OH)2D3 has stimulated an elaborate exploration of the structureefunction relationships of this secosteroid molecule in order to develop analogs with a clear dissociation between the anti-proliferative, prodifferentiating and immunomodulatory properties on the one hand and calcemic in vivo activity on the other hand [1]. By introducing chemical modifications into the flexible molecule 1,25(OH)2D3 (Fig. 76.1), an extensive generation of vitamin D analogs was created [1]. The characteristic seco-structure of vitamin D originates from the photolytic cleavage of the bond between C-9 and C-10 upon UV irradiation of the provitamin 7-dehydrocholesterol. The so-generated previtamin triene then undergoes a reversible thermal [1,7]-sigmatropic hydrogen shift leading to the vitamin form in its less stable 6-s-cis conformation. Rapid rotation around the C-6, C-7 single bond brings vitamin D3 in its more stable extended 6-s-trans conformation. Three different parts can be distinguished in
21
20
18 11 9
12
24 23
17 16 15
13
22
26 25
20
B
7
OH 13
27
H
HO
17
21
26 25
20 18
OH
27
12
10 A 1 2
B
7
4 3
OH
HO
OH
27
7
H
6 19
19
26 25
8
6 5
24 23
17
13
22
C 14 D
14
8
6
4 3
24 23
C 14 D 8
22
5
10 A 1 2
4 3
OH
HO
5
10 A 1 2
19
OH
FIGURE 76.1 Structure of 1,25(OH)2D3 and numbering of the vitamin D ring system used in this chapter.
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10076-9
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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76. ANALOGS OF CALCITRIOL
developed; the six-membered C-ring analogs, the fiveor six-membered D-ring analogs, CF-ring analogs and the E-ring analogs. Modifications in the central CDregion of 1,25(OH)2D3 led to the development of analogs with a clear dissociation between the anti-proliferative, prodifferentiating and immunomodulatory effects on the one hand and the calcemic effects on the other hand. Due to these interesting biological properties these analogs have the potential to be used for the treatment of bone disorders but also for non-classical applications such as treatment of autoimmune disorders, cancer, or psoriasis. Finally, non-secosteroidal compounds without any basic structural features of 1,25(OH)2D3 have been characterized as vitamin D mimics and further modified to enhance their anti-proliferative and immunomodulatory capacity. In this chapter we will give an overview of non-classical vitamin D receptor modulators.
14-EPI ANALOGS OF 1,25(OH)2D3 14-epi stereoisomers of 25(OH)D3 and 1,25(OH)2D3 have the tendency to isomerize via [1,7]-sigmatropic hydrogen shifts towards their corresponding previtamin forms [4]. The first synthesized vitamin D analogs with a reorientation of the hydrogen on C14 into the cis configuration demonstrated lower affinity to VDR and vitamin-D-binding protein (DBP) but also markedly decreased calcemic effects when compared to 1,25(OH)2D3 [4]. Within the cis-fused series, we synthesized 14-epi analogs lacking the A-ring methylene group at C-10 in order to avoid the ready thermal conversion into previtamin derivatives which is known to occur via [1,7]-sigmatropic shift (Fig. 76.2). Whereas in the natural trans-fused perhydrindane series the thermal equilibrium is in favor of the vitamin derivative, in the cis-fused series the opposite is true. The latter observation is in line with the more substituted nature of the triene system. On the other hand, the torsion constraint that is imposed on the six-membered C-ring by the trans-fused five-membered D-ring in the natural series is in part relieved in the vitamin form [5]. The 14-epi-19-nor analog KS 532 with the natural
R OH 14
H HO
R OH
14-epi
H
14-nat
HO
FIGURE 76.2 14-Epi analogs lack the A-ring methylene group at
C-10 in order to avoid the thermal conversion into previtamin derivatives which is known to occur via [1,7]-sigmatropic shift.
side chain of 1,25(OH)2D3 had reduced VDR and vitamin-D-binding protein (DBP) affinity and was more than 300 times less calcemic than 1,25(OH)2D3 [6] (Table 76.1). The antiproliferative activity of KS 532 on keratinocytes was two-fold higher than 1,25(OH)2D3 and its differentiating capacity on MG63 osteosarcoma cells was comparable to the potency of the parent compound. The 23-yne modification, known to be successful in the parent steroid [1,7], was introduced in the side chain of KS 532 leading to the analog 14-epi-19-nor-23-yne-1,25(OH)2D3 (TX 522, Inecalcitol) [6,8]. The anti-proliferative and prodifferentiating capacity was strongly enhanced (5 to 17 times more potent than 1,25(OH)2D3) while the calcemic effects remained very low (400 times less calcemic than 1,25(OH)2D3) (Table 76.1). The selectivity profile of TX 522 based on data obtained in vitro on breast cancer MCF-7 cells compared to its actual in vivo calcemic effect in mice (serum calcium levels after 7 days of treatment) exceeds several fold that of the best analogs of 1,25(OH)2D3 yet published when measured with the same methods in the same laboratory. The 20-epimer of TX 522, 19-nor-14,20-bis-epi-23-yne-1a,25(OH)2D (TX 527) is even more potent than TX 522 to inhibit cell proliferation or induce cell differentiation but its calcemic activity is also increased (Table 76.1). The superagonistic action of both compounds correlated with their ability to induce coactivatoreVDR interactions [9]. Mammalian two-hybrid assays with VP16-fused VDR and GAL4-DNA-binding-domainfused steroid receptor coactivator 1 (SRC-1), transcriptional intermediary factor 2 (TIF2), or vitamin D receptor interacting protein (DRIP) 205 demonstrated that TX522 and TX527 were more potent to induce VDR interaction with each of the three coactivators compared to the interaction induced by 1,25(OH)2D3. For SRC-1, the higher induction was most pronounced at 1010 M and 1011 M concentrations of TX 522 and TX 527 (Fig. 76.3), for TIF2 at concentrations of 109 M and 1010 M and for DRIP205 at 108 M, 109 M and 1010 M. The differences in VDRecoactivator interaction could only be partially explained by a difference in metabolic stability between both 14-epi analogs and 1,25(OH)2D3 [9]. Co-crystallization studies of the human VDR-LBD with TX 522 indicated that C-12 of TX 522 shows a closer interaction with Val300 (H6) because of a shift of the CD-ring caused by the 14-epimerization. Moreover, the 23-yne side chain is forced to take another orientation in the ligand-binding pocket and makes an additional contact with the CD1 atom of Ile 268 (H5) [9]. To support the clinical applicability of these analogs both compounds were further tested in cancer and autoimmune mouse models. TX 522 and TX 527 were able to retard tumor growth in a xenograft model of
IX. ANALOGS
1463
14-EPI ANALOGS OF 1,25(OH)2D3
14-Epi Analogs
TABLE 76.1
Affinity studies
In vitro studies
In vivo studies
VDR (%)
DBP (%)
HL-60 (%)
MCF-7 (%)
MG-63 (%)
KERAT (%)
Calcium serum (%)
1,25(OH)2D3
100
100
100
100
100
100
100
KS 532
20
7
50
30
100
260
0.33
TX 522
20
0.3
560
1100
190
1760
0.25
TX 527
25
0.1
2200
5300
3200
4400
1
OH
H
HO
OH
OH H
HO
OH
OH H
HO
OH
Summary of the in vitro effects of 14-epi analogs of 1,25(OH)2D3 on vitamin D receptor (VDR) and vitamin-D-binding protein (DBP) binding, promyelocytic HL-60 (nitroblue tetrazolium assay) or osteosarcoma MG-63 (osteocalcin measured by radioimmunoassay) differentiation together with the anti-proliferative effects (3Hthymidine incorporation assay) on MCF-7 breast cancer cells and primary keratinocytes (Kerat). The in vitro effects are expressed as percentage activity (at EC50) in comparison with 1,25(OH)2D3 (¼100% activity). The in vivo activity of 14-epi analogs is determined in mice by intraperitoneal injections during 7 consecutive days, using serum calcium concentration as parameter. A doseeresponse curve for the analogs is compared with the doseeresponse curve for 1,25(OH)2D3 (¼100% activity). The activity of all other analogs described in Tables 76.2e76.11 is determined and expressed in the same way as in Table 76.1. ND ¼ not determined.
breast cancer without major calcemic side effects for TX 522 (Fig. 76.4) [6]. The tumor volume of prostate cancer PC-3 xenografts was also reduced by daily oral treatment with TX 522 and demonstrated additive effects with docetaxel [10]. TX 522 (Inecalcitol, Hybrigenics) is currently in phase II in patients with hormone-refractory prostate cancer. Preliminary results demonstrated that 27 of the 31 patients treated with Inecalcitol (at different doses up to 600 mg/day) and Taxotere during 18 weeks showed a decrease in prostate-specific antigen (PSA) levels of more than 30% within 3 months of initiation of treatment without any changes in calcium parameters [11]. TX 527, the 20-epi counterpart of TX 522, has potent immunomodulatory properties and is extensively discussed in Chapter 94.
14-Epimerization was also introduced in the potent C2-modified 19-nor vitamin D3 analogs 2a-(3-hydroxypropyl)-1,25(OH)2D3 (MART10) and 2b-(3-hydroxypropyl)-1,25(OH)2D3 (MART11) [12]. Whereas the affinity of MART10 for VDR was comparable with that of 1,25(OH)2D3, the 14-epimer of MART10 (14-epi MART10) displayed a lower VDR affinity [12,13]. The biological activity of MART10 and MART11, as measured by the capacity to inhibit invasiveness and proliferation of PZ-HPV-7 prostate cells, was 10- to 1000-fold higher than that of 1,25(OH)2D3 [13]. Introduction of the 14-epi modification in MART10 did not enhance this biological activity but the compound was still much more active than the natural hormone 1,25(OH)2D3. Moreover, this analog enhanced bone mineral density in ovariectomized rats without
IX. ANALOGS
1464
76. ANALOGS OF CALCITRIOL
120 vehicle 1,25(OH)2 D3
100
TX522 TX527
RCA
80
60
40
20
0
Dose (M)
FIGURE 76.3 Effect of 1,25(OH)2D3 and 14-epi analogs TX 522 and TX 527 on the interaction VDR-SRC-1. COS-1 cells were transfected with pVPVDR (encoding VP16-fused VDR), pSG424SRC1NIR (encoding a fragment of SRC-1 (AA 570-782) containing the nuclear receptor interacting domain fused to GAL4 DNA-binding domain), and the pG5CAT reporter and treated with 1,25(OH)2D3, TX 522 or TX 527 at the indicated concentrations, or with vehicle [9]. CAT accumulation was normalized to total protein content and expressed as relative CAT amounts (RCA). Results shown are the mean SEM of at least three independent experiments performed in triplicate. *RCA significantly different from RCA for 1,25(OH)2D3-treated samples; p < 0.05 according to Fisher’s LSD multiple-comparison test.
(A)
(B)
350 300
significant calcemic effects. Introduction of the 14-epi modification in MART11 led to a reduced affinity for VDR as well as to a decreased potency to inhibit proliferation of PZ-HPV-7 prostate cells so that 14-epiMART11 was slightly less potent than 1,25(OH)2D3 [12]. As outlined above, vitamin D3 is present in thermal equilibrium with previtamin D3 via [1,7]-sigmatropic rearrangement. In this equilibrium, the vitamin D3 form with the 6-s-trans triene structure is more stable than the previtamin form. Therefore, the vitamin D3 form, rather than the previtamin D3 form, has been the focus for the development of novel analogs with possible therapeutic applications. However, epimerization at C14 reverses the equilibrium and as a result the previtamin D3 form is more abundant [4]. Recently, a number of modifications were introduced in the A-ring of 14-epi-1,25(OH)2-previtamin D3 in order to evaluate the therapeutic potential of analogs with a previtamin D3 skeleton [14e16]. As crystallographic studies revealed the presence of several water molecules near the A-ring which link the ligand C-2 position to the protein surface [17], the effect of C-2 substitutions in 14-epi-1,25(OH)2-previtamin D3 was evaluated. Several analogs with different substituents at C-2 were synthesized and their affinity to VDR as well as their potency to transactivate the osteocalcin promoter was evaluated [15,16]. 14-Epi-1,25(OH)2-previtamin D3 itself had a 200fold lower affinity for VDR and the EC50-concentration for transactivation of the osteocalcin promoter was 15fold higher than that of 1,25(OH)2D3. All the analogs
vehicle 1,25(OH)2D3 (5 µg/kg/d)
Calcium serum (mg/dl)
Tumor volume (mm3)
TX522 (80 µg/kg/d)
250 200
*
150 100
*
12
*
10 8 6 4 2
50
0
0 14
22 Time (days)
26
vehicle
1,25 D3
TX522
FIGURE 76.4 In vivo anti-proliferative effects of 1,25(OH)2D3 and TX 522. (A) Evolution of tumor volume during time. Nude mice were treated every other day with vehicle (control), 5 mg/kg 1,25(OH)2D3 or 80 mg/kg TX 522. An example of a representative experiment is shown (14 tumors per group were measured). Results shown are the mean SEM. * Tumor volume of TX522-treated mice is significantly smaller than that of control mice, p < 0.05 according to Fisher’s LSD multiple-comparison test. (B) In vivo calcemic activity of 1,25(OH)2D3 or TX 522. Serum calcium levels were measured at the end of the experiment (26e28 days after MCF-7 breast tumor transplantation). Results shown are the mean SEM of at least three independent experiments. * Serum calcium in 1,25(OH)2D3-treated mice is significantly higher than in control mice; p < 0.05 according to Fisher’s LSD multiple-comparison test.
IX. ANALOGS
1465
C- AND D-RING ANALOGS
with C-2 b-substitutions and most of the analogs with asubstitutions were characterized by a low affinity to VDR and a reduced transactivating activity when compared with 14-epi-1,25(OH)2-previtamin D3. However, introduction of a C-2 a-methyl moiety showed a considerable increase in VDR binding affinity and the transactivating capacity was significantly enhanced. In a second series of analogs substituents were introduced at the C-4-position of 14-epi-1,25(OH)2-previtamin D3 as it was hypothesized that 4-oxy-substitution could lead to a new hydrogen bond in the VDR ligand-binding domain [14]. However, the VDR-binding affinity and the transactivating potency of 14-epi-1,25(OH)2-previtamin D3 analogs with C-4 hydroxy and C-4 methoxy moieties was lower than that of 14-epi-1,25(OH)2-previtamin D3 itself.
DECALIN ANALOGS The introduction of a six-membered D-ring instead of the natural five-membered ring in the CD-region gave rise to trans-fused [18] or cis-fused decalin 1,25(OH)2D3 analogs (Table 76.2) [19]. The VDRbinding affinity of the trans-fused decalin analog CY 10012 with the natural side chain of 1,25(OH)2D3 is nearly the same as the binding affinity of 1,25(OH)2D3. CY 10012 is at least 10-fold more potent than 1,25(OH)2D3 to inhibit cell proliferation (MCF-7 breast cancer cells, keratinocytes) or to induce cell differentiation (HL-60 leukemia cells) but is also twice as calcemic as 1,25(OH)2D3 [20]. The introduction of a 19-nor A-ring (CY 10010) reduced, as expected, the calcemic activity of CY 10012 two times (Table 76.2). 20-Epimerization of CY 10012 and CY 10010 diminished drastically their in vitro biological and in vivo calcemic activity. This group of trans-decalin analogs represents one of the exceptions where 20-epimerization shows a reduced biological activity when compared to the derivative with the natural configuration. In the context of the study of structureefunction relationships the 20-epimerization modification has taken a unique position. Following the 1991 report by Binderup about the increased overall activity of 20epi-1,25(OH)2D3 [21] it became clear that the orientation in space of the side chain plays an important role. Okamura developed a dot-map approach in order to visualize the portions in space that were accessible for the side chain, with focus on the 25-hydroxyl group [22,23]. This approach was further used by Yamada in a comprehensive structureefunction relationship study [24,25]. In particular, it was found that analogs with preferred side-chain orientation in a particular region consistently possess a higher biological activity (except the affinity for DBP). A similar procedure for defining
a volume in space was developed in Ghent, the preferred occupation of which would correspond to high prodifferentiating (HL-60) and anti-proliferative activity (MCF-7) activity. Interestingly, it was found that the preferred orientation in space of the side chain of trans-decalin analogs with the natural configuration matches the one occupied by the side chain of the 20epimer of 1,25(OH)2D3 rather than of the natural hormone ([18] and references therein). Interestingly, the previtamin configuration of the biologically potent 19-nor-trans-decalin-1,25(OH)2D3 (19nor-trans-decalin-1,25(OH)2-previtamin D3, GAO182) was able to inhibit cell proliferation and induce cell differentiation to the same extent as 1,25(OH)2D3 [26]. This previtamin analog represents the first previtamin with potent vitamin-D-like activity which acts through the genomic signal transduction pathway as it could bind to VDR and was equipotent to 1,25(OH)2D3 in inducing interaction with VDREs and the coactivator TIF-2. Mutational analysis and in silico docking suggested that this 19-nor-trans-decalin-1,25(OH)2-previtamin D3 made similar contacts with the ligand-binding pocket as 1,25(OH)2D3 [26]. Another series of decalin analogs is characterized by the presence of a symmetrical central bicyclic core but two identical entities are now connected serving as A-ring and side-chain surrogates. This type of analog featuring structural symmetry could bind in two different and opposite directions within the LBD. Figure 76.5 presents pseudo-symmetrical analogs with a trans-fused decalin CD-core with local S2-symmetry possessing identical seco-B,A-ring structures (GM 7, GM 8, GM 15, and GM 14) [27]. The side chain section contains structural modifications similar to the so-called arocalciferols [28] and 16-ene analogs [29]. Figure 76.5 includes also C2-symmetrical analogs with a cis-fused decalin CD-core (GM 38A and GM 38B). None of these decalin analogs showed affinity for the VDR or demonstrated antiproliferative effects.
C- AND D-RING ANALOGS Over the years numerous analogs were developed featuring a more substantially modified CD-region such as the six-membered C-ring analogs (lacking the D-ring, Tables 76.3e76.4) [30,31], five-membered D-ring analogs (lacking the C-ring, Tables 76.5e76.8) [32,33] and six-membered D-ring analogs (lacking the C-ring and possessing an enlarged D-ring, Table 76.9) [34,35]. In comparison with the natural compound 1,25(OH)2D3, the non-steroidal analogs described in this section possess a greater flexibility. There are five freely rotatable CeC bonds in the side chain of 1,25(OH)2D3 excluding the A-ring and the C-6, C-7
IX. ANALOGS
1466
76. ANALOGS OF CALCITRIOL
TABLE 76.2
Decalin Analogs
H
OH
H
OH
H
OH
H
OH
H
OH
Affinity studies
In vitro studies
In vivo studies
VDR (%)
DBP (%)
HL-60 (%)
MCF-7 (%)
KERAT (%)
Calcium serum (%)
1,25(OH)2D3
100
100
100
100
100
100
CY 10012
90
3
1000
2250
1400
200
CY 10010
50
2
700
5000
1000
100
CY 943
80
1
70
85
70
<0.1
CY 941
8
0
10
30
30
<0.1
CY 1006
40
0
85
100
70
< 0.1
GAO 182
3
1
90
70
ND
2
H
HO
OH
H
HO
OH
H
HO
OH
H
HO
OH
H
HO
OH
OH OH H HO
IX. ANALOGS
1467
C- AND D-RING ANALOGS
HO
OH
OH
H
H OH OH H
H HO
HO
OH
GM 7
HO
HO
GM 8
HO
OH
OH
HO
OH
HO
H
H
H
H
H
H
H
H
OH
GM 15
HO
OH
HO
GM 14
OH
GM 38A
HO
OH
OH
GM 38B
FIGURE 76.5 Chemical structure of pseudo-symmetric and C2-symmetric analogs.
bond. In the C-ring analogs there is an extra rotatable CeC bond between C-13 and C-17, whereas in the Dring analogs this extra bond is between C-8 and C-14.
C-ring Analogs The C-ring analog with the natural side chain of 1,25(OH)2D3 (ZG 1368) possessed about 60% and 20% of the affinity for VDR and DBP, respectively, compared to 1,25(OH)2D3 (100% binding, Table 76.3) [32]. Introduction of unsaturation (23-yne) in the side chain (XM 612) or a double bond between C-16 and C17 (CY 625) whether or not in combination with an homologation of the side chain at C-26, C-27 (CY 628) decreased the VDR and DBP affinity but increased the anti-proliferative activity compared to 1,25(OH)2D3 (Table 76.3). In contrast to the situation in the natural 1,25(OH)2D3, epimerization of C-20 decreased the antiproliferative effect of the C-ring analog with the natural side chain of 1,25(OH)2D3 (XM 806). When the A-ring of the C-ring analog ZG 1368 was changed into a
19-nor-A-ring (ZG 1423), the VDR affinity and DBP binding decreased, similar to previous observations for 19-nor-1,25(OH)2D3. The anti-proliferative potency of ZG 1368 was not enhanced by this 19-nor modification but this compound (ZG 1423) was still 20 to 50 times better than the parent compound to inhibit cell proliferation or stimulate cell differentiation (Table 76.4). When the stereochemistry of C-20 in ZG 1423 was altered (XM 804) its potency diminished 50- to 100-fold. Also 20-epimerization of ZG 1423 in combination with a 23-yne side chain (XM 720) exhibited lower anti-proliferative and prodifferentiating effects. The biological activity of other chemical modifications of 19-nor C-ring analogs were investigated such as 23-yne (XM 615), 16-ene (CY 613), and 16-ene in combination with 26-27 homologation (CY 616). These analogs were equipotent or even superagonistic compared to 1,25(OH)2D3. The C-ring analog with the natural side chain of 1,25(OH)2D3 (ZG 1368) was two times less calcemic than 1,25(OH)2D3 (Table 76.3) [32]. Further reduction of calcemic activity could be obtained by 20-epimerization
IX. ANALOGS
1468
76. ANALOGS OF CALCITRIOL
TABLE 76.3
C-ring Analogs Affinity studies
In vitro studies
In vivo studies
VDR (%)
DBP (%)
HL-60 (%)
MCF-7 (%)
KERAT (%)
Calcium serum (%)
1,25(OH)2D3
100
100
100
100
100
100
ZG 1368
60
20
1000
6000
6000
50
XM 806
70
0
60
75
85
0.25
XM 612
80
0
450
1000
4250
10
XM 723
40
0
10
70
50
1
CY 625
80
5
150
200
200
1
CY 628
85
0
85
600
700
13
OH
HO
OH
OH
HO
OH
OH
HO
OH
OH
HO
OH
OH
HO
OH
OH
HO
OH
IX. ANALOGS
1469
C- AND D-RING ANALOGS
TABLE 76.4
19-Nor C-ring Analogs Affinity studies
In vitro studies
In vivo studies
VDR (%)
DBP (%)
HL-60 (%)
MCF-7 (%)
KERAT (%)
Calcium serum (%)
ZG 1423
45
3
1000
2000
5000
13
XM 804
10
0
10
40
80
0.25
XM 615
60
0
850
1750
1500
2
XM 720
4
0
10
60
80
2
CY 613
20
6
100
200
300
0.5
CY 616
40
0
400
1275
600
1
OH
HO
OH
OH
HO
OH
OH
HO
OH
OH
HO
OH
OH
HO
OH
OH
HO
OH
IX. ANALOGS
1470
76. ANALOGS OF CALCITRIOL
TABLE 76.5
Five-membered D-ring Analogs Affinity studies
In vitro studies
In vivo studies
VDR (%)
DBP (%)
HL-60 (%)
MCF-7 (%)
KERAT (%)
Calcium serum (%)
1,25(OH)2D3
100
100
100
100
100
100
SL 117
80
10
85
85
90
0.3
SL 137
70
9
100
85
100
3
WU 442
40
0.9
100
200
75
0.25
SL 142
40
0.9
100
150
200
0.5
WU 515
70
3
1000
5000
3000
6
WU 507
70
1
1000
5000
2000
10
OH
HO
OH OH
HO
OH
OH
HO
OH
OH
HO
OH
CF3 OH CF3
HO
OH CF3 OH
CF3
HO
OH
IX. ANALOGS
1471
C- AND D-RING ANALOGS
TABLE 76.6
17-Methyl Five-membered D-ring Analogs Affinity studies
In vitro studies
In vivo studies
VDR (%)
DBP (%)
HL-60 (%)
MCF-7 (%)
KERAT (%)
Calcium serum (%)
1,25(OH)2D3
100
100
100
100
100
100
WY 1036
6
10
50
60
100
0.25
WY 821
85
9
800
2000
1000
8
WY 10061
60
0
80
600
500
3
WY 9361
75
4
800
1250
3250
3
WY 1038
30
40
150
2500
2400
10
WY 1112
60
6
400
30000
20000
> 100
OH
HO
OH
OH
HO
OH
OH
HO
OH
OH
HO
OH
CF3 OH CF3
HO
OH
CF 3 OH CF 3
HO
OH
(Continued)
IX. ANALOGS
1472
76. ANALOGS OF CALCITRIOL
TABLE 76.6
17-Methyl Five-membered D-ring Analogsdcont’d
WY 10071
Affinity studies
In vitro studies
In vivo studies
VDR (%)
DBP (%)
HL-60 (%)
MCF-7 (%)
KERAT (%)
Calcium serum (%)
60
0
250
4500
3600
6
CF 3 OH CF 3
HO
OH
TABLE 76.7
17-Methyl 19-nor Five-membered D-ring Analogs Affinity studies
In vitro studies
In vivo studies
VDR (%)
DBP (%)
HL-60 (%)
MCF-7 (%)
KERAT (%)
Calcium serum (%)
1,25(OH)2D3
100
100
100
100
100
100
WY 1037
3
70
10
8
80
0.12
WY 1116
40
6
600
400
600
0.5
WY 1046
7
5
85
70
200
0.12
WY 1106
40
0.8
350
1300
2000
4
OH
HO
OH
OH
HO
OH
OH
HO
OH
OH
HO
OH
(Continued)
IX. ANALOGS
1473
C- AND D-RING ANALOGS
TABLE 76.7
17-Methyl 19-nor Five-membered D-ring Analogsdcont’d
CF 3
Affinity studies
In vitro studies
In vivo studies
VDR (%)
DBP (%)
HL-60 (%)
MCF-7 (%)
KERAT (%)
Calcium serum (%)
WY 1039
9
70
100
600
1500
50
WY 1113
40
4
400
20000
16000
100
WY 1048
50
7
300
1500
3000
3
OH CF 3
HO
OH
CF3 OH
CF 3
HO
OH
CF 3 OH CF 3
HO
OH
TABLE 76.8
17-Methyl 21-nor Five-membered D-ring Analogs and 17-methyl 19-nor 21-nor Five-membered D-ring Analogs Affinity studies
In vitro studies
In vivo studies
VDR (%)
DBP (%)
HL-60 (%)
MCF-7 (%)
KERAT (%)
Calcium serum (%)
1,25(OH)2D3
100
100
100
100
100
100
KS 699
30
12
40
80
40
<1
WY 722
9
0.2
100
150
400
0.1
OH
HO
OH
OH
HO
OH
(Continued)
IX. ANALOGS
1474
76. ANALOGS OF CALCITRIOL
TABLE 76.8
17-Methyl 21-nor Five-membered D-ring Analogs and 17-methyl 19-nor 21-nor Five-membered D-ring Analogsdcont’d Affinity studies
In vitro studies
In vivo studies
VDR (%)
DBP (%)
HL-60 (%)
MCF-7 (%)
KERAT (%)
Calcium serum (%)
WY 718
80
0.1
215
3500
3500
1
WY 619
80
10
150
1250
750
10
WY 838
40
9
9
30
10
0.25
WY 906
60
<1
60
70
85
0.25
CD 578
100
0
300
2000
4500
1
CF 3 OH CF 3
HO
OH
CF 3 OH
CF 3
HO
OH
OH
HO
OH
OH
HO
OH
CF 3 OH CF 3
HO
OH
(XM 806), unsaturation of the side chain (XM 612), and introduction of a double bond between C-16 and C-17 (CY 625). Since the calcemic activity of 1,25(OH)2D3 was decreased 10 times when the A-ring of 1,25(OH)2D3 was modified to a 19-nor A-ring, a similar modification was introduced in the C-ring analog with
the side chain of 1,25(OH)2D3. As a result, ZG 1423 displayed only 13% of the calcemic activity of 1,25 (OH)2D3 (Table 76.4). Some side chain modifications of the 19-nor C-ring analog led to even lower calcemic effects such as the 19-nor modification in combination with 23-yne (XM 615), 16-ene-26,27 bishomo (CY 616)
IX. ANALOGS
1475
C- AND D-RING ANALOGS
TABLE 76.9
Six-membered D-ring Analogs Affinity studies
In vitro studies
In vivo studies
VDR (%)
DBP (%)
HL-60 (%)
MCF-7 (%)
KERAT (%)
Calcium serum (%)
1,25(OH)2D3
100
100
100
100
100
100
BL 269
125
80
90
300
300
4
BL 314
0.9
25
<1
0
8
< 0.1
BL 562
60
5
85
100
150
0.5
SG 396
20
0.6
90
200
ND
4
SG 402
2
7
9
30
ND
< 0.25
SG 340
2
0
8
70
ND
< 0.25
OH
HO
OH
OH
HO
OH
OH
HO
HO
HO
HO
OH
H
OH
H
OH
H
OH
OH
OH
OH
(Continued)
IX. ANALOGS
1476
76. ANALOGS OF CALCITRIOL
TABLE 76.9
Six-membered D-ring Analogsdcont’d
OH
HO
In vitro studies
In vivo studies
VDR (%)
DBP (%)
HL-60 (%)
MCF-7 (%)
KERAT (%)
Calcium serum (%)
FV 708
35
3
ND
90
ND
0.5
FV689
0
4
ND
0
ND
ND
FV721
0
0
ND
0.3
ND
ND
FV 521
2
2
ND
90
ND
< 0.25
FV 604
0.3
0
ND
3
ND
ND
FV 639
0
0
ND
0
ND
ND
OH
OH
HO
Affinity studies
OH
OH
HO
OH
OH
HO
OH
OH
HO
OH
OH
HO
OH
IX. ANALOGS
1477
CF-RING ANALOGS
or 20-epi (XM 804) being 100 times less toxic than 1,25 (OH)2D3 (Table 76.4).
D-ring Analogs Most D-ring analogs displayed a good affinity for the VDR (40%) and demonstrated low binding to hDBP (<10%) [32,33]. The five-membered D-ring analogs (lacking the C-ring) displayed in vitro effects comparable with the anti-proliferative activity of 1,25(OH)2D3, except the 23-yne-26,27-F6 (WU 515) and 20-epi-23yne-26,27-F6 (WU 507) D-ring analogs that were 10 (HL-60) to 50 (MCF-7) times more potent than 1,25(OH)2D3 (Table 76.5) [32]. Epimerization of C-20 did not increase cell-differentiating activity of the Dring analogs (SL 137, SL 142, WU 507) compared to similar analogs with normal C-20 stereochemistry (SL 117, WU 442, WU 515). An extensive collection of fivemembered D-ring analogs was synthesized with a methyl group substituted on C-17 (Tables 76.6e76.8) [36,37]. The introduction of 17-methyl (WY 1036) in the five-membered D-ring analog with the natural side chain of 1,25(OH)2D3 (SL 117) decreased its binding to VDR and its anti-proliferative and prodifferentiating effects [37]. However, when a methyl group was introduced at C-17 of the 20-epi D-ring analog (SL 137), the 23-yne D-ring analog (WU 442) or of the D-ring analog with both modifications 20-epi-23-yne (SL 142), the in vitro biological activity increased respectively nineto 23-fold (WY 821), seven-fold (WY 10061) and nineto 36-fold (WY 9361) (Table 76.6). Fluorination of the side chain makes it more resistant to metabolic degradation by the 24-hydroxylase (CYP24) enzyme. As a result, the 17-methyl-26,27-F6 D-ring analog (WY 1038) inhibited breast cancer MCF-7 cell proliferation 25 times more efficiently than 1,25(OH)2D3. Moreover, the most successful modification was 26,27-hexafluorination in combination with 20-epimerization (WY 1112). The anti-proliferative activity of WY 1112 was 300 times enhanced compared to 1,25(OH)2D3 and is comparable with the in vitro activity of known “top” analogs such as KH 1060 (20-epi-22-oxa-24,26,27-trihomo1,25(OH)2D3) [21]. All described five-membered D-ring analogs, with or without a 17-methyl group, demonstrated decreased calcemic effects compared to 1,25(OH)2D3, except for WY 1112 which was even more calcemic than 1,25(OH)2D3 (Tables 76.5e76.8). When the A-ring of the 17-methyl D-ring analogs was changed into a 19-nor A-ring the calcemic activity decreased, except for WY 1106 (20-epi-23-yne) and WY 1039 (26,27-F6) (Table 76.7) [37]. When the 21-methyl side chain was replaced by a 21-nor side chain in the 17-methyl D-ring analogs or in the 17-methyl-19-nor D-ring analogs the in vitro activity decreased for most of the analogs (Table 76.8) [36].
The replacement of the five-membered D-ring of analog SL 117 by a six-membered ring (BL 269) resulted in a more potent analog (three-fold increased inhibition of MCF-7 or keratinocytes proliferation compared to 1,25(OH)2D3) (Table 76.9) [34,35]. The introduction of other modifications (20-epi; 16-ene-23-yne) did not enhance the potency of BL 269. As already mentioned before (see “Decalin analogs,” above) different studies have demonstrated that a correlation exists between biological potency and the preferred spatial orientation of the side chain [24,25]. 6D-ring analogs were synthesized with biased spatial orientations of the side chain (Table 76.9). In particular, the relative orientation of the methyl substituents at C-13, C-16, or C-20 determines the distinct conformation of the side chain at C-17. The most biologically active compounds of this series of D6-ring analogs were the ones with the side chain associated with the active region (based on calculated occupancies of the side chain) [35,38]. We demonstrated furthermore that the 26,27-hexafluorinated non-steroidal D-ring analogs CD 578, WU 515, and WY 1113 have a more potent prodifferentiating action on human SW480-ADH colon cancer cells than 1,25(OH)2D3 [39]. The increased prodifferentiating potential of these analogs correlated with an increased potency to induce expression of the invasion suppressor E-cadherin and a more pronounced repression of the c-Myc oncogene. The c-Myc oncogene is a downstream target of the Wnt/b-catenin signaling pathway and we demonstrated that the analogs were more potent to inhibit the transcriptional activity of b-catenin/TCF complexes compared to 1,25(OH)2D3. This superagonistic activity profile may be attributed to an increased VDR-based transactivating potency and a stronger induction of the interaction between VDR and coactivators SRC-1, TIF2, and DRIP205. Co-crystallization of analog CD 578 with the zebrafish (z)VDR and an LXXLL-motif containing SRC-1 peptide demonstrated that the side chain fluorine atoms on analog CD 578 make additional contacts with activation helix 12 of the VDR and with the loop between helix 11 and helix 12 [39]. These additional contacts stabilize the active conformation of VDR and consequently favor coactivator recruitment which can explain the increased potency of the analog [39].
CF-RING ANALOGS This particular series of CF analogs is characterized by a spiro-ring system which is the result of the deletion of C-15 and C-16, and of the connection between C-18 and C-21. We can distinguish within this group of analogs the spiro[5.5]undecane CF-ring analogs [40] and spiro[4.5]decane CF-ring analogs [41] involving,
IX. ANALOGS
1478
76. ANALOGS OF CALCITRIOL
TABLE 76.10
CF Analogs Affinity studies
O
In vitro studies
In vivo studies
VDR (%)
DBP (%)
HL-60 (%)
MCF-7 (%)
Calcium serum (%)
1,25(OH)2D3
100
100
100
100
100
SW 931
< 0.1
0
8
60
0.5
SW 123
10
3
200
450
< 0.5
FDB 187E
2
0
ND
80
< 0.13
FDB 198E
8
0
200
500
0.25
OH
HO
OH
OH
HO
OH
O OH
HO
OH
O
HO
CF3 OH CF3
OH
respectively, a six-membered or five-membered F-ring. Table 76.10 summarizes the biological activity of the most potent CF-ring analogs. The binding affinity of the spiro[5.5]undecane CF-ring analogs for the VDR was very low (<1%) together with the prodifferentiating and anti-proliferative effects (SW 931). The spiro[4.5] decane CF-ring analogs possessed higher VDR affinity and two (HL 60) to five (MCF-7) times increased in vitro biological effects compared to 1,25(OH)2D3 (Table 76.10). The spiro analogs showed low calcemic effects. The group of Mourin˜o synthesized non-steroidal analogs with locked spatial orientations of the side chain by the introduction of a new cycle between the C-18 and C-21 positions (Fig. 76.6) [42,43]. C-18 can be connected to C-21 by means of an ether bridge (Fig. 76.6A), which results in an analog with a restricted mobility in the side chain. Another possibility is the introduction of an
additional five-carbon-membered ring structure that links C-18 and C-21. An example of such a compound is depicted in Fig. 76.6B and this analog is characterized by fixed torsion angles between C-16eC-17eC-20eC-22 and as consequence possesses a side chain with a locked spatial orientation at C-20.
E-RING ANALOGS A very far-reaching modification consisted in the replacement of the natural CD-region by an unnatural five-membered ring resulting in non-steroidal E-ring analogs [44,45]. These molecules exhibit an artificial E-ring characterized by a direct linkage between C-12 and C-21 (Table 76.11). The E-ring analog with the natural side chain of 1,25(OH)2D3 (KS 176) displayed
IX. ANALOGS
1479
ACYCLIC ANALOGS
A
B
O OH
H
H
HO
fluorination (CD 503, CD 509), and unsaturation (CD 483, CD 504) of the side chain. The successful 26,27-hexafluorinated E-ring analog (CD 503) was as active as 1,25(OH)2D3 to stimulate HL-60 differentiation and approximately nine times more potent to inhibit cell proliferation (MCF-7, keratinocytes). The calcemic activity remained very low (<0.1%) when the side chain was homologated and increased by the introduction of fluor atoms into the side chain although the calcemic activity was only 2 to 3% of the calcemic effects induced by 1,25(OH)2D3. When the five-membered E-ring was changed into a six-membered E-ring (Fig. 76.7) the biological activity was completely lost. In addition, introduction of the Cring in the five-membered or six-membered E-ring analog resulted in CE-ring analogs (Fig. 76.8) [46] that had a very low or undetectable biological activity.
OH
H
OH
HO
OH
FIGURE 76.6 Chemical structures of analogs with locked spatial orientations of the side chain.
about 10% of the VDR and 19% of the DBP affinity and 10e30% of the prodifferentiating and anti-proliferative effects of 1,25(OH)2D3 (Table 76.11). Moreover, KS 176 was in vivo 1000 times less calcemic than 1,25(OH)2D3 [45]. The biological activity of this analog proved that the full central CD-region in the parent compound is not necessary to be biologically active. Structural modifications were introduced in KS 176 to increase its biological potency such as homologation (KS 291), 20-epimerization together with homologation (KS 512), TABLE 76.11
ACYCLIC ANALOGS The most profound modification of 1,25(OH)2D3 is the complete removal of the CD-ring region by stripping
E-ring Analogs Affinity studies
In vitro studies
In vivo studies
VDR (%)
DBP (%)
HL-60 (%)
MCF-7 (%)
KERAT (%)
Calcium serum (%)
1,25(OH)2D3
100
100
100
100
100
100
KS 176
10
19
20
30
10
< 0.1
KS 291
36
2
70
150
90
< 0.1
KS 512
15
0
12
100
30
< 0.1
OH
HO
OH
OH
HO
OH
OH
HO
OH
(Continued)
IX. ANALOGS
1480
76. ANALOGS OF CALCITRIOL
TABLE 76.11
E-ring Analogsdcont’d Affinity studies
In vitro studies
In vivo studies
VDR (%)
DBP (%)
HL-60 (%)
MCF-7 (%)
KERAT (%)
Calcium serum (%)
CD 483
10
3
25
215
400
0.2
CD 503
80
40
100
850
900
3
CD 504
65
9
80
550
500
2
CD 509
10
0
80
200
250
2
CF3 OH CF3
HO
OH
CF3 OH CF3
HO
OH
CF3 OH CF3
HO
OH
CF3 CF2 OH CF2 CF3
HO
OH
the molecule to its five-carbon backbone C-8eC-14eC13eC-17eC-20 [45,47] (KS 018, Fig. 76.9). The acyclic analog KS 018 had no binding at all to VDR or DBP and showed no differentiating effect on promyelocytic HL-60 cells or osteosarcoma MG63 cells. Other acyclic analogs were also synthesized such as the retiferols RAD1 and RAD2 which possessed a C-20 methyl group with or without 1a-OH (Fig. 76.9) [48,49]. Furthermore, des-CD 19-nor vitamin derivatives were described (Fig. 76.9) [50e52], some of which demonstrated VDR transactivation potency and antipsoriatic potential as demonstrated by epidermal thickening in hairless mice [52]. Des-CD analogs of the potent 2-methylene-19-nor-
R
HO
OH
Chemical structure of six-membered E-ring analogs; R ¼ natural side chain.
FIGURE 76.7
IX. ANALOGS
NON-SECOSTEROIDAL COMPOUNDS
R
R
H
HO
H
OH
FIGURE 76.8 Chemical R ¼ natural side chain.
HO structure
OH of
CE-bicyclic
analogs;
(20S)-1,25(OH)2D3 (2MD) analog [53] retained some VDR-binding affinity and transcriptional (CYP-24 promoter driving luciferase reporter gene system) or HL-60 prodifferentiating activity but two orders of magnitude lower in comparison with 1,25(OH)2D3 (Fig. 76.9) [54,55]. The conformation of the des-CD2MD analog was further adapted with a C-13 methyl group (13R or 13S) (Fig. 76.9) [54,56]. The 13R-des-CD2MD analog was the most potent analog but the VDR-binding affinity, prodifferentiating and CYP24 transcriptional activity was respectively 30-, twoand 15-fold decreased in comparison with 1,25(OH)2D3 [54]. In general, the acyclic des-CD analogs demonstrated decreased VDR affinity and in vitro biological activity but all these compounds were poorly calcemic compared to the natural hormone.
NON-SECOSTEROIDAL COMPOUNDS A novel class of non-secosteroidal analogs without any basic structural features of 1,25(OH)2D3 was developed by Ligand Pharmaceuticals and Galderma that independently characterized bis-phenyl derivatives as new VDR agonists. Ligand Pharmaceuticals identified the nonsecosteroidal activators of VDR upon screening of a compound library in cells, which were transfected with a reporter construct that contained a vitamin-Dresponsive element of the CYP24 promoter [57] (Table 76.12). A bis-phenyl derivative, LG190090, was characterized that was able to transactivate the reporter construct in a VDR-dependent manner (1000-fold less potent than 1,25(OH)2D3). Further analysis demonstrated that LG190090 bound weakly to VDR, inhibited proliferation of LNCaP prostate cancer cells and normal epidermal keratinocytes (17 to 1000 times less potent than 1,25(OH)2D3) and also induced differentiation of HL-60 leukemia cells (200 times less potent than 1,25(OH)2D3) in vitro. Chemical modifications of the central moiety in the lead compound and addition of polar groups led to increased VDR
1481
binding affinity and enhanced growth-inhibitory and pro-differentiation capacity and the most potent of these analogs (LG19078) was only 10 times less potent than the natural hormone [57]. Interestingly, these bis-phenyl compounds did not bind to DBP, which suggested that they might be less calcemic in vivo than 1,25(OH)2D3. Indeed, a greater separation between induction of kidney CYP24 mRNA and calcemic effects was demonstrated for these non-secosteroidal analogs than for 1,25(OH)2D3. The antineoplastic activity of LG190119, in which a diethyl group was added to the bridgehead of LG190090, was evaluated in an in vivo model of prostate cancer [58]. LG190119 was able to retard prostate xenograft tumor growth without increased serum calcium levels both in a prevention model, where treatment was initiated before tumor development, and in a model of established cancer, where treatment was started when tumors had an average size of 150 mm3. LG190178, the most potent bis-phenyl analog originally synthesized by Boehm et al., includes four stereo-isomers [59]. The biological activity of those isomers was evaluated and only the (2S, 20 R)-analog of LG190178 (YR301) showed potent anti-proliferative effects. The crystal structure of YR301 complexed with the rat VDR-ligand-binding domain revealed that YR301 and 1,25(OH)2D3 occupied a similar space in the ligandbinding pocket and that the diethylmethyl group took a comparable position to the C- and D-rings of 1,25(OH)2D3 [60]. The symmetric ligand placed one of the 3,3-dimethyl-2-butanone groups in the same space occupied by the C-22eC-25 side chain of 1,25(OH)2D3 whereas the other 3,3-dimethyl-2-butanone group occupied the same space usually occupied by C-3eC-5 of the A-ring of 1,25(OH)2D3. The two characteristic hydroxyl groups of YR301, 20 -OH and 2-OH, played exactly the same role as the 25-OH and 1-OH groups in 1,25(OH)2D3, respectively, and contributed to the potent activity of YR301. Indeed, the 20 -OH group formed hydrogen bonds to the NE2 atoms of both His301 and His393 just as the 25-OH group did. The other hydroxyl group, 2-OH, interacted with Ser233 OG and Arg270 NH1, while the 1-OH group in 1,25(OH)2D3 interacted with Ser233 OG and Arg274 NH1. The terminal hydroxyl group (3-OH) was directly hydrogen bonded to Arg270 and also interacted indirectly with Tyr232 OH and the backbone NH of Asp144 through water molecules. The finding that VDR liganded with LG190178 recruited a large number of coregulators, among which SRC-1, SRC-2, SRC-3, and DRIP 205, with the same affinities as VDR bound to 1,25(OH)2D3. This suggested that VDR after binding to LG190178 adopted an agonistic conformation and that the downstream signaling cascade was similar to that induced by 1,25(OH)2D3 [61]. The fact that these non-secosteroidal bis-phenyl vitamin D3 mimics did not interact with Arg274 was taken advantage of in the structural design of analogs
IX. ANALOGS
1482
76. ANALOGS OF CALCITRIOL
OH
OH
OH
H
HO
HO
OH
1,25(OH)2D3
X
HO
OH
HO
KS 018
R
F 3C
R OH
RAD 1 (R = H) RAD 2 (R = OH)
OH CF3 OH
HO
OH HO
des-CD-19-nor R = Me, X = CH2 R = Et, X = CH2 R = Me, X = O R= Et, X = O
R
OH
OH
des-CD-19-nor-20-dimethyl22-ene-27,27-F6
des-CD-2-MD
OH
HO
OH
HO
13R-des-CD-2-MD FIGURE 76.9
OH
OH
13S-des-CD-2-MD
Chemical structure of acyclic analogs.
that complement the Arg274/Leu mutation which, analogous to some other mutations in the VDR, results in hereditary vitamin-D-dependent rickets [62]. Indeed, the missense mutation Arg274/Leu causes a more than 1000-fold reduction in responsiveness to 1,25(OH)2D3 and is, therefore, no longer regulated by physiological concentrations of the hormone. Computer-aided
molecular design was used to generate a focused library of non-steroidal analogs of the VDR agonist LG190155 that were predicted to bind to mutant VDR (Arg274Leu) and to transactivate an osteopontin-driven reporter construct in a VDR (Arg274Leu)-dependent manner [63]. The non-secosteroidal scaffold of LG190155, originally described by Boehm et al., was modified such
IX. ANALOGS
1483
NON-SECOSTEROIDAL COMPOUNDS
Vitamin D Mimics
TABLE 76.12
VDR binding (%)
Cell proliferation (%)
1,25(OH)2D3
100
100
LG 190090
< 0.005a
0.07c
Ligand Pharmaceuticals
LG 190119
< 0.005a
0.1c
Ligand Pharmaceuticals
LG 190155
< 0.005a
0.1c
Ligand Pharmaceuticals
LG 190178
0.3a
10c
Ligand Pharmaceuticals
CH 5036249
37b
100d
Chugai Pharmaceutical
LY 2108491
na
850e
Eli Lilly and Company
LY 2109866
na
1700e
Eli Lilly and company
Compound
O
Company
O
O
Cl
O
Cl
O
O
O
O
O
O
O
O
OH O
O
OH
OH
O
OH
N
NaO
OH
S O
OH
O S O
S OH
O O a
Ki (equilibrium dissociation constant) value was expressed relative to Ki value of 1,25(OH)2D3 (100%; 0.5 nM) and was determined in an in vitro competition binding assay to yeastexpressed hVDR. b Binding affinity was expressed relative to 1,25(OH)2D3 and was determined in an in vitro competition binding assay to hVDR. c Relative percent activity versus 1,25(OH)2D3 in inhibiting proliferation of human LNCaP prostate cancer cells as determined by BrdU incorporation with 100% (2 nM) indicating the EC50 value of 1,25(OH)2D3. d Relative percent activity versus 1,25(OH)2D3 in inhibiting proliferation of human prostate stromal cells as determined by BrdU incorporation with 100% (100 nM) indicating the EC50 value of 1,25(OH)2D3. e Relative percent activity versus 1,25(OH)2D3 in inhibiting proliferation of human transformed skin keratinocytes as determined by BrdU incorporation with 100% (153 nM) indicating the EC50 value of 1,25(OH)2D3.
IX. ANALOGS
1484
76. ANALOGS OF CALCITRIOL
A
o o
O
OH
OH
B
o o
O HO HO
OH OH
FIGURE 76.10
Chemical structures of 1,25(OH)2D3 analogs with a carborane cluster.
that one of the 3,3-dimethyl-2-butanone groups was substituted with a limited set of commercially available alkylating agents. Thirteen analogs with the lowest calculated apparent association energies were selected for synthesis and more than half of the designed analogs were identified as promising leads as they showed greater activity than LG190155. Significantly, three of the analogs represented highly active agonists for the mutant VDR (Arg274Leu) and were able to induce activities in the mutant that were greater than that obtained with 1,25(OH)2D3 in wild-type VDR. Interestingly, none of these compounds induced significant increases in 45Ca2þ influx in MC3T3-E1 preosteoblastic cells. Although the above-described non-secosteroidal compounds with a bis-phenylmethane skeleton did not show cross-reactivity with retinoid X and retinoid acid receptors in cotransactivation assays, they were shown to possess androgen-antagonistic activities as determined by their potential to inhibit androgen-induced cell growth of Shionogi Carcinoma SC-3 cells [64]. Indeed, compounds with a bis-phenylmethane skeleton that were derived from the analogs LG190155 and LG190178 could be categorized in three groups, that is, vitamin D3 agonists, androgen antagonists, and dual ligands. The compound CH5036249, developed by Chugai Pharmaceutical, represents another non-secosteroidal structure with a bis-phenyl scaffold that showed significant VDR binding and was as potent as 1,25(OH)2D3 in inhibiting the growth of human prostate stromal cells [65] (Table 76.12). Moreover, this analog showed a high bioavailability and good metabolic stability whereas the calcemic effects were reduced in comparison with the natural ligand, 1,25(OH)2D3. An additional class of non-secosteroidal compounds with an original phenylthiophene skeleton was described and proposed to possess tissue-selective activity. Compounds LY2108491 and LY2109866 were synthesized by Eli Lilly and Company and showed weak VDR binding despite the observation that the crystal structure of the VDR-ligand-binding domain complexed to LY2108491 revealed that the ligand was
bound in the same space that is occupied by 1,25(OH)2D3 [66] (Table 76.12). Furthermore, these novel vitamin D receptor modulators were potent inhibitors of keratinocyte proliferation and were efficacious in shifting the balance of helper T cells from pathologic Th1 to anti-inflammatory Th2 cells, as evidenced by their ability to inhibit interleukin (IL)-2 and augment IL-4 and IL-10 expression in activated peripheral blood mononuclear cells. Strikingly, the transcriptional activity of these compounds was attenuated in intestinal cells as demonstrated by their low potency to induce the expression of endogenous calcium transport protein 1 (Cat1), Calbindin 9k, and CYP24 in intestinal cell cultures. These observations suggest that the analogs LY2108491 and LY2109866 are cell-context-specific vitamin D receptor modulators with limited calcemic effects and a high therapeutic potential. Indeed, these non-secosteroidal analogs showed significantly improved therapeutic indices relative to 1,25(OH)2D3 in a surrogate in vivo model for psoriasis. Finally, novel non-steroidal analogs were developed in which the hydrophobic core was replaced with a carborane cage, a chemical and thermal stable cluster of boron and carbon atoms [67]. In a first set of analogs the CD-ring region of 19-nor-1,25(OH)2D3 analog was replaced by a carborane group and several modifications were introduced in the side chain (Fig. 76.10A). This approach yielded interesting compounds with a similar activity to that of 19-nor-1,25(OH)2D3. Second, a series of non-secosteroidal analogs of 1,25(OH)2D3 was synthesized as dialkylcarborane derivatives, which have three hydroxyl groups corresponding to those of 1,25(OH)2D3 and lack the A-ring and the conjugated diene. The potency of some of these analogs was again comparable to that of 19-nor-1,25(OH)2D3 (Fig. 76.10B).
CONCLUSION The modes of action of these superanalogs with increased anti-proliferative, prodifferentiating effects
IX. ANALOGS
REFERENCES
and decreased calcemic side effects are not yet fully understood and are probably based on a combination of several phenomena. First, differences in extracellular pharmacokinetics may contribute to lower calcemic activity because most of these analogs display a low affinity for DBP and therefore their free concentration approaches their total plasma concentration which results in rapid extracellular clearance. Moreover, altered pharmacokinetics may also explain the enhanced anti-proliferative effects since the analog will reach fast high peak levels in target tissues in comparison to the slow rise and drop of 1,25(OH)2D3. Also, the intracellular metabolism might be different from that of 1,25(OH)2D3 due to chemical modifications (fluorination, 23-yne, 20-epimerization) that make the analogs more resistant to metabolic degradation so that the cells are exposed for a longer period to the analog. At the cellular level, the anti-proliferative activity seems to associate well with the ability of an analog to promote interaction between VDR and coactivator proteins [68]. Crystallographic studies provided a more in-depth view of analogeVDR binding. The non-steroidal 14-epi analog TX 522 was co-crystallized with VDR-LBP [9] and the 17-methyl D-ring analog CD 578 with VDR-LBP together with an SRC-1 coactivator peptide with second LXXLL-motif containing NR box [39]. Both studies indicated that the analogs did not induce major differences in the protein conformation upon binding with the LBD as compared to binding with 1,25(OH)2D3 but induce rather subtle differences such as closer or additional contacts to certain amino acid residues allowing more stable interactions with coactivators (see “14-Epi analogs of 1,25(OH)2D3” and “D-ring analogs,” above). The involvement of different phenomena (DBP affinity, VDR binding, recruitment of transcriptional machinery, metabolism) in the superagonistic activity profile of these non-steroidal analogs is not only true for these particular analogs but also for analogs with a more classical 1,25(OH)2D3 structure (see Chapter 75).
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76. ANALOGS OF CALCITRIOL
[19] Y.J. Chen, P. De Clercq, M. Vandewalle, Synthesis of new vitamin D3 analogues with a decalin-type CD-ring, Tetrahedron Letters 37 (1996) 9361e9364. [20] G. Eelen, L. Verlinden, J. Laureys, S. Marcelis, P. De Clercq, C. Mathieu, R. Bouillon, A. Verstuyf, Antiproliferative and calcemic actions of trans-decalin CD-ring analogs of 1,25-dihydroxyvitamin D-3, Anticancer Res. 29 (2009) 3579e3584. [21] L. Binderup, S. Latini, E. Binderup, C. Bretting, M. Calverley, K. Hansen, 20-Epi-vitamin-D3 analogs e a novel class of potent regulators of cell-growth and immune-responses, Biochem. Pharmacol. 42 (1991) 1569e1575. [22] M.M. Midland, J. Plumet, W.H. Okamura, Studies of vitamin-D (calciferol) and its analogs. 46. Effect of C20 stereochemistry on the conformational profile of the side-chains of vitamin-D analogs, Bioorg. Med. Chem. Lett. 3 (1993) 1799e1804. [23] W.H. Okamura, J.A. Palenzuela, J. Plumet, M.M. Midland, Vitamin-D e structureefunction analyses and the design of analogs, J. Cell Biochem. 49 (1992) 10e18. [24] S. Yamada, K. Yamamoto, H. Masuno, M. Ohta, Conformationefunction relationship of vitamin D: conformational analysis predicts potential side-chain structure, J. Med. Chem. 41 (1998) 1467e1475. [25] K. Yamamoto, W.Y. Sun, M. Ohta, K. Hamada, H.F. Deluca, S. Yamada, Conformationally restricted analogs of 1 alpha,25dihydroxyvitamin D-3 and its 20-epimer: compounds for study of the three-dimensional structure of vitamin D responsible for binding to the receptor, J. Med. Chem. 39 (1996) 2727e2737. [26] L. Verlinden, S. Verstuyf, C. Verboven, G. Eelen, C. De Ranter, L.J. Gao, et al., Previtamin D-3 with a trans-fused decalin CDring has pronounced genomic activity, J. Biol. Chem. 278 (2003) 35476e35482. [27] G. Minne, L. Verlinden, A. Verstuyf, P.J. De Clercq, Synthesis of 1 alpha,25-dihydroxyvitamin D analogues featuring a S-2symmetric CD-ring core, Molecules 14 (2009) 894e903. [28] B. Figadere, A.W. Norman, H.L. Henry, H.P. Koeffler, J.Y. Zhou, W.H. Okamura, Studies of vitamin-D (calciferol) and its analogs. 39. Arocalciferols e synthesis and biological evaluation of aromatic side-chain analogs of 1-alpha,25-dihydroxyvitaminD3, J. Med. Chem. 34 (1991) 2452e2463. [29] J.Y. Zhou, A.W. Norman, D.L. Chen, G.W. Sun, M. Uskokovic, H.P. Koeffler, 1,25-Dihydroxy-16-ene-23-yne-vitamin-D3 prolongs survival-time of leukemic mice, Proc. Natl. Acad. Sci. USA 87 (1990) 3929e3932. [30] G.D. Zhu, Y.J. Chen, X.M. Zhou, M. Vandewalle, P.J. De Clercq, Synthesis of CD-ring modified 1 alpha,25-dihydroxy vitamin D analogues: C-ring analogues, Bioorg. Med. Chem. Lett. 6 (1996) 1703e1708. [31] X.M. Zhou, G.D. Zhu, D. Van Haver, M. Vandewalle, P.J. De Clercq, A. Verstuyf, et al., Synthesis, biological activity, and conformational analysis of four seco-D-15,19-bisnor-1 alpha,25dihydroxyvitamin D analogues, diastereomeric at C17 and C20, J. Med. Chem. 42 (1999) 3539e3556. [32] A. Verstuyf, L. Verlinden, E. Van Etten, L. Shi, Y.S. Wu, C. D’Halleweyn, et al., Biological activity of CD-ring modified 1 alpha,25-dihydroxyvitamin D analogues: C-ring and fivemembered D-Ring analogues, J. Bone Min. Res. 15 (2000) 237e252. [33] W. Yong, S. Ling, C. D’Halleweyn, D. Van Haver, P. De Clercq, M. Vandewalle, et al., Synthesis of CD-ring modified 1,25dihydroxy vitamin D analogues: five-membered D-ring analogues, Bioorg. Med. Chem. Lett. 7 (1997) 923e928. [34] B. Linclau, P. DeClercq, M. Vandewalle, The synthesis of CDring modified 1 alpha,25-dihydroxy vitamin D analogues: sixmembered D-ring analogues. 1, Bioorg. Med. Chem. Lett. 7 (1997) 1461e1464.
[35] F. Vrielynck, D. Van Haver, M. Vandewalle, L. Verlinden, A. Verstuyf, R. Bouillon, et al., Development of analogues of 1 alpha,25-dihydroxyvitamin D-3 with biased side-chain orientation: C20 methylated des-C, D-homo analogues, Eur. J. Org. Chem. (2009) 1720e1737. [36] Y.S. Wu, K. Sabbe, P. De Clercq, M. Vandewalle, R. Bouillon, A. Verstuyf, Vitamin D-3: synthesis of seco C-9,11,21-trisnor-17methyl-1 alpha, 25-dihydroxyvitamin D-3 analogues, Bioorg. Med. Chem. Lett. 12 (2002) 1629e1632. [37] Y.S. Wu, P. De Clercq, M. Vandewalle, R. Bouillon, A. Verstuyf, Vitamin D-3: synthesis of seco-C-9,11-bisnor-17-methyl-1 alpha, 25-dihydroxyvitamin D-3 analogues, Bioorg. Med. Chem. Lett. 12 (2002) 1633e1636. [38] S. Gabriels, D. Van Haver, M. Vandewalle, P. De Clercq, A. Verstuyf, R. Bouillon, Development of analogues of 1 alpha,25-dihydroxyvitamin D-3 with biased side chain orientation: methylated des-C, D-homo analogues, ChemistrydA European Journal 7 (2001) 520e532. [39] G. Eelen, N. Valle, Y. Sato, N. Rochel, L. Verlinden, P. De Clercq, et al., Superagonistic fluorinated vitamin D-3 analogs stabilize helix 12 of the vitamin D receptor, Chem. Biol. 59 (2008) 1029e1034. [40] W. Schepens, D. Van Haver, M. Vandewalle, P.J. De Clercq, R. Bouillon, A. Verstuyf, Synthesis and biological activity of 22oxa CD-ring modified analogues of 1 alpha,25-dihydroxyvitamin D-3: spiro[5.5]undecane CF-ring analogues, Bioorg. Med. Chem. Lett. 14 (2004) 3889e3892. [41] F. De Buysser, L. Verlinden, A. Verstuyf, P.J. De Clercq, Synthesis of 22-oxaspiro[4.5]decane CD-ring modified analogs of 1 alpha,25-dihydroxyvitamin D-3, Tetrahedron Letters 50 (2009) 4174e4177. [42] I. Cornella, S.J. Perez, A. Mourino, L.A. Sarandeses, Synthesis of new 18-substituted analogues of calcitriol using a photochemical remote functionalization, J. Org. Chem. 67 (2002) 4707e4714. [43] C. Varela, K. Nilsson, M. Torneiro, A. Mourino, Synthesis of tetracyclic analogues of calcitriol (1alpha,25-dihydroxyvitamin D3) with side-chain-locked spatial orientations at C(20), Helv. Chim. Acta 85 (2002) 3251e3261. [44] K. Sabbe, C. DHallewyn, P. De Clercq, M. Vandewalle, Synthesis of CD-ring modified 1 alpha,25-dihydroxy vitamin D analogues: E-ring analogues, Bioorg. Med. Chem. Lett. 6 (1996) 1697e1702. [45] A. Verstuyf, L. Verlinden, H. Van Baelen, K. Sabbe, C. D’Hallewyn, P. De Clercq, et al., The biological activity of nonsteroidal vitamin D hormone analogs lacking both the Cand D-rings, J. Bone Min. Res. 13 (1998) 549e558. [46] S. Demin, D. Van Haver, M. Vandewalle, P.J. De Clercq, R. Bouillon, A. Verstuyf, Synthesis and biological activity of 22oxa CD-ring modified analogues of 1 alpha,25-dihydroxyvitamin D-3: cis-perhydrindane CE-ring analogues, Bioorg. Med. Chem. Lett. 14 (2004) 3885e3888. [47] P. De Clercq, R. Bouillon, M. Vandewalle, Novel structural analogs of vitamin D. WO 9501960 (1995). [48] A. Kutner, H. Zhao, H. Fitak, S.R. Wilson, Synthesis of retiferol Rad(1) and Rad(2), the lead representatives of a new class of des-Cd analogs of cholecalciferol, Bioorg. Chem. 23 (1995) 22e32. [49] A. Kutner, H. Zhao, H. Fitak, M. Chodynski, S. Halkes, S.R. Wilson, et al., New pharmacotherapeutically active compounds. WO 9519963 (1995). [50] Y. Hu, J.A. Porco, J.W. Labadie, O.W. Gooding, B.M. Trost, Novel polymer-supported trialkylsilanes and their use in solid-phase organic synthesis, J. Org. Chem. 63 (1998) 4518e4521.
IX. ANALOGS
REFERENCES
[51] H.F. Deluca, K. Plonska-Ocypa, R.R. Sicinski, P. Grzywacz, L.A. Plum, M. Clagett-Dame Des-C, D analogs of 1alpha,25-dihydroxy-19-norvitamin D3. WO 2007028000 (2007). [52] P. Barbier, F. Bauer, P. Mohr, M. Muller, W. Pirson Cyclohexanediole derivatives. WO 9943646 (1999). [53] H.Z. Ke, H. Qi, D.T. Crawford, H.A. Simmons, G. Xu, M. Li, et al., A new vitamin D analog, 2MD, restores trabecular and cortical bone mass and strength in ovariectomized rats with established osteopenia, J. Bone Min. Res. 20 (2005) 1742e1755. [54] K. Plonska-Ocypa, R.R. Sicinski, L.A. Plum, P. Grzywacz, J. Frelek, M. Clagett-Dame, et al., 13-Methyl-substituted des-C, D analogs of (20S)-1 alpha,25-dihydroxy-2-methylene-19-norvitamin D-3 (2MD): Synthesis and biological evaluation, Bioorg. Med. Chem. 17 (2009) 1747e1763. [55] K. Plonska-Ocypa, P. Grzywacz, R.R. Sicinski, L.A. Plum, H.F. Deluca, Synthesis and biological evaluation of a des-C, Danalog of 2-methylene-19-nor-1 alpha,25-(OH)(2)D-3, J. Steroid Biochem. Mol. Biol. 103 (2007) 298e304. [56] H.F. Deluca, M. Clagett-Dame, L.A. Plum, R.R. Sicinski, K. Plonska-Ocypa, N.J. 13,13-Dimethyl-des-C, D analogs of 1alpha,25-dihydroxy-19-nor-vitamin D3 compounds and topical composition dosage forms and methods of treating skin conditions. WO2009094426 (2009). [57] M.F. Boehm, P. Fitzgerald, A. Zou, M.G. Elgort, E.D. Bischoff, L. Mere, et al., Novel nonsecosteroidal vitamin D mimics exert VDR-modulating activities with less calcium mobilization than 1,25-dihydroxyvitamin D3, Chem. Biol. 6 (1999) 265e275. [58] T.C. Polek, S. Murthy, S.E. Blutt, M.F. Boehm, A. Zou, N.L. Weigel, et al., Novel nonsecosteroidal vitamin D receptor modulator inhibits the growth of LNCaP xenograft tumors in athymic mice without increased serum calcium, Prostate 49 (2001) 224e233. [59] W. Hakamata, Y. Sato, H. Okuda, S. Honzawa, N. Saito, S. Kishimoto, et al., (2S,20 R)-analogue of LG190178 is a major active isomer, Bioorg. Med. Chem. Lett. 18 (2008) 120e123. [60] S. Kakuda, K. Okada, H. Eguchi, K. Takenouchi, W. Hakamata, M. Kurihara, et al., Structure of the ligand-binding domain of rat
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1487 VDR in complex with the nonsecosteroidal vitamin D3 analogue YR301, Acta Crystallogr. Sect. F. Struct. Biol. Cryst. Commun. 64 (2008) 970e973. A. Teichert, L.A. Arnold, S. Otieno, Y. Oda, I. Augustinaite, T.R. Geistlinger, et al., Quantification of the vitamin D receptorecoregulator interaction, Biochemistry 48 (2009) 1454e1461. K. Kristjansson, A.R. Rut, M. Hewison, J.L. O’Riordan, M.R. Hughes, Two mutations in the hormone binding domain of the vitamin D receptor cause tissue resistance to 1,25 dihydroxyvitamin D3, J. Clin. Invest. 92 (1993) 12e16. S.L. Swann, J. Bergh, M.C. Farach-Carson, C.A. Ocasio, J.T. Koh, Structure-based design of selective agonists for a rickets-associated mutant of the vitamin D receptor, J. Am. Chem. Soc. 124 (2002) 13795e13805. S. Hosoda, A. Tanatani, K. Wakabayashi, Y. Nakano, H. Miyachi, K. Nagasawa, et al., Ligands with dual vitamin D3-agonistic and androgen-antagonistic activities, Bioorg. Med. Chem. Lett. 15 (2005) 4327e4331. K. Taniguchi, K. Katagiri, H. Kashiwagi, S. Harada, Y. Sugimoto, Y. Shimizu, et al., A novel nonsecosteroidal VDR agonist (CH5036249) exhibits efficacy in a spontaneous benign prostatic hyperplasia beagle model (2010). J. Steroid Biochem. Mol. Biol. 121 (2010) 204e207. Y. Ma, B. Khalifa, Y.K. Yee, J. Lu, A. Memezawa, R.S. Savkur, et al., Identification and characterization of noncalcemic, tissueselective, nonsecosteroidal vitamin D receptor modulators, J. Clin. Invest. 116 (2006) 892e904. S. Fujii, A. Kano, R. Sekine, E. Kawachi, H. Masuno, T. Hirano, et al., Development of novel non-secosteroidal vitamin D receptor ligands based on carborane as a hydrophobic core structure, p. 6 (2009). Abstract book “14th workshop on Vitamin D”, october 4e8, 2009, Brugge, Belgium. G. Eelen, L. Verlinden, M. Van Camp, F. Claessens, P. De Clercq, M. Vandewalle, et al., Altered vitamin D receptorecoactivator interactions reflect superagonism of vitamin D analogs, J. Steroid Biochem. Mol. Biol. 97 (2005) 65e68.
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C H A P T E R
77 Analogs for the Treatment of Osteoporosis* Noboru Kubodera, Fumiaki Takahashi Chugai Pharmaceutical Co., Ltd, Tokyo, Japan
INTRODUCTION Osteoporosis is a disease caused by bone resorption overtaking bone formation and the creation of an imbalance between the two processes that is normally due to aging. Women in particular develop the disease more frequently than men because their bone mineral density (BMD) is rapidly reduced after menopause. In Japan, active vitamin D3 (calcitriol) (1) and its synthetic prodrug (alfacalcidol) (2) have been widely used for the treatment of osteoporosis for more than 25 years [1]. Calcitriol and alfacalcidol have been recognized as very safe medicines showing mild increase in BMD in osteoporotic patients. In the USA and Europe, on the other hand, bisphosphonates such as sodium alendronate, sodium risedronate and sodium ibandronate or selective estrogen receptor modulators (SERM) such as raloxifene, have been mainly used for the treatment of osteoporosis because of their strong increment of BMD. This is contrary to Japanese clinical practice, which focuses on active vitamin D3 therapy [2]. Various reasons for this have been enunciated which include differences in the history of osteoporosis therapy development, the amount of calcium intake, the ratio of responders to non-responders due to gene polymorphism of vitamin D receptor (VDR), and culture. Of these reasons, the most significant is that in Western countries, the average amount of calcium intake is higher than in Japan, and vitamin D is added to milk and milk products. Hence, in Western countries, active vitamin D3 as a treatment might induce hypercalcemia rather than have therapeutic effects on bone [3]. Although currently SERMs and bisphosphonates are also gaining ground as an accepted form of therapy in Japan, there is still intense interest in obtaining active
vitamin D3 analogs more potent than calcitriol/alfacalcidol or comparable to bisphosphonates/SERM in increasing BMD and preventing bone fracture with relatively less hypercalcemic character than that observed for calcitriol/alfacalcidol [4]. Eldecalcitol, 1a,25-dihydroxy-2b-(3-hydroxypropoxy) vitamin D3 (developing code: ED-71) (3), is such an analog that possesses a hydroxypropoxy substituent at the 2b-position of the A-ring of calcitriol and shows potent effects on bone therapy [5e7]. Eldecalcitol has been shown to be more effective than alfacalcidol/calcitriol in increasing BMD and mechanical strength in ovariectomized (OVX) model rats for osteoporosis [8e9]. Moreover, recent completion of phase III clinical trials with eldecalcitol in comparison with alfacalcidol for bone fracture prevention of osteoporotic patients produced excellent results and eldecalcitol is now ready for marketing as a promising medicine for the treatment of osteoporosis in Japan. In this chapter we focus our attention on active vitamin D3 analogs for osteoporosis treatment, namely the initial recognition of alfacalcidol as a therapeutic agent for osteoporosis, research and development of eldecalcitol, and clinical comparison between alfacalcidol and eldecalcitol [10] (Fig. 77.1).
ALFACALCIDOL Development of Alfacalcidol It is well established that cholecalciferol (4) ingested into foods or synthesized in the skin is metabolized to calcifediol (5) in the liver, which is further hydroxylated at the 1a-position in the proximal renal tubules to active vitamin D3, calcitriol (1) (Fig. 77.2). Patients with renal
*
This chapter is dedicated to the memory of the late Dr. Etsuro Ogata, Professor Emeritus, Tokyo University/Director Emeritus, Cancer Institute Hospital, Japan, who passed away on November 1, 2009.
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10077-0
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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77. ANALOGS FOR THE TREATMENT OF OSTEOPOROSIS
OH
HO
OH
OH
OH
HO
OH
HO
O calcitriol (1)
FIGURE 77.1
Chemical structures of calcitriol, alfacalcidol, and eldecalcitol.
25
OH
liver
HO
HO cholecalciferol (4)
calcifediol (5) kidney
25
OH
liver/bone
1
OH
alfacalcidol (2)
eldecalcitol (3)
alfacalcidol (2)
damage who require artificial kidney dialysis are expected to develop vitamin D deficiency due to a disorder of vitamin D activation caused by insufficient hydroxylation in the kidney. An effective treatment for such individuals with renal impairment is through chemical treatment, specifically the administration of a vitamin D analog with a hydroxyl group at the 1a-position of the A-ring. As part of these efforts, alfacalcidol (2) was developed in Japan in 1981 as the first prodrug of calcitriol (1) for medical use. Since hydroxylation in the liver is comparatively unregulated, alfacalcidol (2) is efficiently activated to calcitriol (1) in the body. In addition, it has been revealed that this activation also takes place in the bone [11] (Fig. 77.2). A key question is why the prodrug alfacalcidol was developed as a pharmaceutical product, rather than calcitriol itself? There are two reasons for this; one is that it was difficult to develop calcitriol for medical use due to
HO
OH
HO
1
OH
calcitriol (1)
FIGURE 77.2 Activation of cholecalciferol and prodrug (alfacalcidol) to calcitriol.
issues associated with patent rights; the other is that alfacalcidol had some advantages with respect to the cost of industrial synthesis, compared to calcitriol. Specifically, inexpensive and easily available cholesterol can be used as the starting material because alfacalcidol does not have a hydroxyl moiety at the 25-position [12,13].
Practical Synthesis of Alfacalcidol Figure 77.3 shows an example of the improved practical synthesis of alfacalcidol. In brief, the salient features are: 1. Inexpensive and readily available cholesterol is used as the starting material. If 25-hydroxylated cholesterol is used, basically a similar reaction to give calcitriol would follow, but with much higher costs than alfacalcidol. 2. 1a-Hydroxylation, which is done in the kidney, is replaced by stereoselective formation of an a-epoxide and subsequent regioselective epoxide cleavage by hydride reduction. 3. Introducing a biomimetic method, i.e. using ultraviolet irradiation and thermal isomerization to obtain the same reaction in the skin caused by sunlight following the synthesis of the 5,7-diene segment. Thus, cholesterol (6) was oxidized with aluminum iso-propoxide (Al(Oi-Pr)3) in 80% yield to 4-en-3-one (7), which was further oxidized with 2,3-dichloro-5,6dicyano-1,4-benzoquinone (DDQ) to 1,4-dien-3-one (8) in 75% yield. Treatment of (8) with sodium ethoxide (NaOEt) gave 1,5-dien-3-one (9) in 53% yield, which was reduced with sodium borohydride (NaBH4) yielding 3b-hydroxy-1,5-diene (10) in 78% yield. After protection of hydroxyl moiety in (10) as acetate (11), the 5,7-diene system in (12), required for ultraviolet irradiation, was introduced by bromination with N-bromosuccinimide (NBS)/2,20 -azobisisobutyronitrile (AIBN)
IX. ANALOGS
ALFACALCIDOL
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FIGURE 77.3 An example of the improved practical synthesis of alfacalcidol from cholesterol.
in hexane and dehydrobromination with g-collidine in toluene after deacetylation. The 5,7-diene moiety in (12) was protected by adduct formation with 4-phenyl1,2,4-triazoline-3,5-dione (PTAD) to give PTAD adduct (13) in 80% yield from (11). The hydroxyl group in (13) was protected as its tert-butyldimethylsilyl (TBS) ether (14) in 95% yield, which was then regio- and stereoselectively epoxidized with m-chloroperbenzoic zcid (MCPBA) to give 1,2a-epoxide (15) in 78% yield. Retrocycloaddition of the PTAD adduct (15) to regenerate the 5,7-diene system in (16) in 75% yield was carried out by simply heating (140 C) (15) in 1,3-dimethyl-2-imidazolidinone (DMI). The 3b-hydroxy moiety in (17), obtained by deprotection of TBS group in (16) with tetrabutylammonium fluoride (TBAF), contributed to the regio- and stereoselective cleavage of epoxide with NaBH4 to produce diol (18), quantitatively. Finally, diol (18) was subjected to photolysis and thermal isomerization to afford alfacalcidol (2) (Fig. 77.3). The yields for converting steroidal frameworks to secosteroids by photolysis and thermal isomerization are usually moderate or low, since several structural byproducts such as lumisterol, tachisterol, etc. are formed [14].
Character of Alfacalcidol as an Anti-osteoporotic Agent Although the initial indications of alfacalcidol included vitamin D deficiency, hypocalcemia, and
others, osteoporosis was added to the list of indications in 1983. Clinically, alfacalcidol has been the first-line treatment for osteoporosis in Japan for more than a quarter of a century [15]. For the prevention of osteoporosis, it is very important to ingest calcium and get exposure to sunlight in order to form sufficient vitamin D from an early age. It seems unproven, however, that the administration of calcium and vitamin D is effective for the treatment of fractures in patients with osteoporosis. The administration of high-dose calcium is a burden on these advanced-age patients because the VDR level in their small intestine is low. Furthermore, if high-dose vitamin D is administered, sufficient activation would not be expected due to reduced renal hydroxylase activities. The author and colleagues recently found that the administration of high-dose cholecalciferol only increases bone strength to a certain level in OVX rats, while alfacalcidol treatment improves both strength and density in a dose-dependent manner [16]. Although an explanation for such a phenomenon has not yet been elucidated, it is suggested that only calcitriol is produced from prodrug alfacalcidol for blood circulation, while a number of minor metabolites are produced in addition to calcitriol from cholecalciferol. Almost no roles for these metabolites have been clarified. It should be acknowledged that the physiological need of ingesting cholecalciferol is significantly different from the administration of alfacalcidol/calcitriol for the treatment of osteoporosis.
IX. ANALOGS
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77. ANALOGS FOR THE TREATMENT OF OSTEOPOROSIS
In clinical use, the intestinal calcium absorption effect of alfacalcidol becomes active in a daily dosage of 0.25 to 0.5 mg and becomes saturated with higher dose. At dose levels between 0.75 and 1 mg, alfacalcidol suppresses parathyroid hormone (PTH) and inhibits bone resorption in adults who ingest a normal amount of calcium. These findings suggest a therapeutic threshold for alfacalcidol, wherein bone is intensified. On the other hand, bone resorption becomes encouraged at a dosage of 1.5 mg or more, which is the threshold to develop adverse effects. It has been pointed out that alfacalcidol has a narrow therapeutic window. Thus, it is important to note (see below) that eldecalcitol promotes potent bone formation in lower doses than alfacalcidol and has a wider window between therapeutic and bone resorption than alfacalcidol, and is therefore expected to be a more useful therapy for osteoporosis.
ELDECALCITOL Separation of Biological Activities of Calcitriol As described above, alfacalcidol was first introduced to the Japanese pharmaceutical market in 1981. Following the elucidation of the activation pathway of vitamin D and the development of alfacalcidol/calcitriol for supplementing vitamin D deficiency, research and development toward vitamin D was generally believed to have achieved its goal. Coincident with the market release of alfacalcidol, Suda and colleagues made the momentous discovery that calcitriol differentiates myelogenous leukemia cells to normal macrophages in mice and humans, opening a new page in the history of vitamin D research [17]. At the same time, it was also discovered that the vitamin D receptor exists in various organs and tissues in the body. Thus, in addition to our understanding that active vitamin D is involved in bone and calcium metabolism, studies revealed that this substance also contributes to numerous other physiological reactions, such as differentiation-inducing activity, cell growth inhibition, and immunomodulation. Furthermore, there was an increasing number of patients given alfacalcidol for the treatment of vitamin D deficiency and hypocalcemia, whose prognosis for rheumatoid arthritis (RA) and psoriasis vulgaris (PV), a therapyresistant dermal disease, was clinically promising, potentially as a result of alfacalcidiol treatment. Unfortunately, it was found that increasing the alfacalcidol dosage to enhance the therapeutic effects of this drug on these comorbidities did not achieve enhanced target effects but rather produced a side effect of increased blood calcium. Although it was not known why alfacalcidol was effective for RA and PV, there
was an increasing number of cases discovered in basic research showing the involvement of the differentiation-inducing actions of calcitriol. Worldwide, new analogs were actively synthesized in drug development research that focused on separating the biological actions between in vitro differentiation-inducing effects and in vivo calcemic actions [18,19]. The authors began early in this research area, and invented maxacalcitol (19), an analog whose side chain is modified to have potent differentiation-inducing effects but weak blood calcium raising effects. Maxacalcitol (19) was released into the market as an injectable for treating secondary hyperparathyroidism (2HPT) in 2000 and as an ointment for PV in 2001 and is now in clinical practice [20,21] (Fig. 77.4). Figure 77.4 shows active vitamin D analogs marketed currently, namely maxacalcitol (19) [22], calcipotriol (20) [23], tacalcitol (21) [24], paricalcitol (22) [25e26], doxercalciferol (23) [27], and falecalcitriol (24) [28]. Most are analogs that have a similar balance of biological action to maxacalcitol (19), and are used mainly to treat 2HPT or PV. Although scientists have reportedly attempted to identify and develop active vitamin D analogs for the treatment of cancer, Alzheimer’s disease, and prostate hyperplasia, none have yet been released into the market. Please refer to relevant reviews for the detailed and industrial syntheses and characteristics of these six analogs [29] (Fig. 77.4).
Exploratory Research for Eldecalcitol Interestingly, an analog with the opposite balance of biological actions to maxacalcitol was found among A-ring modified candidates. Thus, modification of the A-ring led to the discovery of an analog with weak in vitro differentiation-inducing properties but potent calcemic actions in vivo. It was reasoned that the steroidal A-ring bearing 1,2a-epoxide (17) in the abovementioned process of industrial synthesis of alfacalcidol might avail itself as an abundant starting material for synthetic studies (17 / 18 / 2). Initially, we explored model experiments for the feasibility of 1,2a-epoxide (17) used in the synthesis of alfacalcidol (2). The nucleophiric reaction of (17) with alkoxides, derived from glycols and potassium tert-butoxide (t-BuOK), resulted in regio- and stereoselective introduction of the hydroxyl-ether group at the 2b-position of (25), which was then irradiated and thermally isomerized to (26). The method was then extended to 1,2a-epoxide (29) with a hydroxyl group at C-25, prepared from lithocholic acid (27) via 25-hydroxycholesterol (28) by 24-step [30]. Eldecalcitol (3), which possesses a hydroxypropoxy group at the 2b-position, was finally selected as an analog with potent calcemic actions [5] (Fig. 77.5).
IX. ANALOGS
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CLINICAL COMPARISON BETWEEN ALFACALCIDOL AND ELDECALCITOL
OH
OH
O OH
HO
OH
OH
HO
maxacalcitol (19) (2HPT and PV)
calcipotriol (20) (PV)
HO
OH
tacalcitol (21) (PV)
CF3 OH CF3
OH
H H HO
OH
paricalcitol (22) (2HPT)
OH
HO
doxercalciferol (23) (2HPT)
HO
OH
falecalcitriol (24) (2HPT)
FIGURE 77.4 Chemical structures and indications of commercially available active vitamin D analogs, maxacalcitol, calcipotriol, tacalcitol, paricalcitol, doxercalciferol, and falecalcitriol.
Distinctive Features of Eldecalcitol Eldecalcitol has a strong affinity for vitamin D binding protein (DBP) and its affinity is about 2.7-fold greater than that of calcitriol. This resulted in longer sustained plasma levels of eldecalcitol than calcitriol. Assuming that potent calcemic activity is accompanied by intense effect on bone, such effects of eldecalcitol were examined using OVX rats; potent effects on bone were discovered. Please refer to the first and second editions of this series, Vitamin D, for details of the basic study on eldecalcitol, preclinical comparison between eldecalcitol and alfacalcidol using OVX rats, phase I study, early phase II and late phase II studies with eldecalcitol [20,21].
CLINICAL COMPARISON BETWEEN ALFACALCIDOL AND ELDECALCITOL Randomized Open-label Clinical Comparison of Eldecalcitol with Alfacalcidol Although eldecalcitol increases lumbar and hip BMD in a dose-dependent manner in osteoporotic patients with or without vitamin D supplementation, there has been no direct clinical comparison of the effects of
eldecalcitol with alfacalcidol on bone and calcium metabolism in patients. Therefore, a randomized openlabel clinical trial was conducted to compare the effect of eldecalcitol on bone turnover markers and calcium metabolism in 59 Japanese postmenopausal women. Patients were randomly assigned to receive 1.0 mg alfacalcidol, 0.5 or 1.0 mg eldecalcitol once a day for 12 weeks. There was almost no increase in serum calcium throughout the study period. Eldecalcitol treatment from 0.5 to 1.0 mg increased daily urinary calcium excretion in a dose-dependent manner, and 1.0 mg eldecalcitol increased urinary calcium to a similar extent to 1.0 mg alfacalcidol. Both 0.5 and 1.0 mg eldecalcitol suppressed urinary type 1 collagen N-terminal telopeptide (NTX) stronger than 1.0 mg alfacalcidol (6%, 30%, and 35% in 1.0 mg alfacalcidol, 0.5 and 1.0 mg eldecalcitoltreated groups, respectively, at 12 weeks). In contrast, changes in serum bone-specific alkaline phosphatase (BALP) were similar among the three groups (22%, 22%, and 29% in 1.0 mg alfacalcidol, 0.5 and 1.0 mg eldecalcitol-treated groups, respectively, at 12 weeks) (Fig. 77.6). These results demonstrate that 0.5 to 1.0 mg eldecalcitol can effectively inhibit bone resorption with greater efficacy than alfacalcidol with a similar effect on bone formation. A comparable effect on urinary calcium excretion suggests that eldecalcitol may have better osteoprotective effects than alfacalcidol [31].
IX. ANALOGS
1494
Exploratory research for eldecalcitol.
Randomized Double-blind Clinical Comparison of Eldecalcitol with Alfacalcidol The late phase II clinical trial with eldecalcitol, namely a randomized placebo-controlled double-blind clinical trial, revealed that treatment of osteoporotic patients with 0.75 mg/day eldecalcitol for 12 months increased lumber and hip BMD by 3.4% and 1.5%, respectively, without causing sustained hypercalcemia [32]. In order to compare the anti-fracture efficacy of
(A)
(B)
30
Change in urinary NTX (%)
20 10
eldecalcitol with that of alfacalcidol, we performed a randomized, active comparator, double-blind phase III clinical trial. Osteoporotic patients were randomly assigned to receive either 1.0 mg alfacalcidol or 0.75 mg eldecalcitol once a day for 36 months. The increase in the lumbar and hip BMD was significantly larger in the eldecalcitol-treated group than in the alfacalcidoltreated group. The reduction in bone resorption markers as well as in bone formation markers was significantly lower in the eldecalcitol-treated group than in the
Time (Weeks) 0
4
0 –10 –20 –30 –40
8
12
Change in serum BALP (%)
FIGURE 77.5
77. ANALOGS FOR THE TREATMENT OF OSTEOPOROSIS
30 20 10
0
4
8
12
0 –10 –20 –30 –40
–50
–50
–60
–60 1.0 µg alfacalcidol
Time (Weeks)
0.5 µg eldecalcitol
1.0 µg eldecalcitol
Changes in urinary NTX (A) and BALP (B) in postmenopausal women given eldecalcitol or alfacalcidol for 12 weeks. Data represent mean percent changes from baseline and mean þ SEM.
FIGURE 77.6
IX. ANALOGS
REFERENCES
alfacalcidol-treated group. After 36 months of treatment, the eldecalcitol-treated group exhibited a significantly lower incidence of new vertebral fractures compared with the alfacalcidol group, especially in patients with BMD T-score below 2.5 and those with multiple vertebral fractures. These results demonstrate that eldecalcitol can increase the lumbar and hip BMD better than alfacalcidol, and that eldecalcitol shows superior antifracture efficacy to alfacalcidol especially in those patients with established osteoporosis [33]. Detailed results of the phase III clinical trial with eldecalcitol will soon be published elsewhere.
CONCLUSION
calcitriol/alfacalcidol cholecalciferol
BMD increase
highlights the need for new improvements to achieve a more effective and safer active vitamin D analog for osteoporosis based on the assessment of its limitation. Nevertheless, it is expected that eldecalcitol, a promising new analog, will contribute to the treatment of patients with osteoporosis in the near future.
Acknowledgments We are grateful to Professor David Horne of Division of Molecular Medicine, City of Hope, for reading of the manuscript and helpful suggestions.
References
Figure 77.7 illustrates the basic relationship between calcemic activity (serum calcium level) and the effect on bone (increase in BMD) in cholecalciferol, alfacalcidol/calcitriol, and eldecalcitol. The potential effect on bone is highest with eldecalcitol followed by alfacalcidol/calcitriol and then cholecalciferol, at doses that induce approximately the same level of calcemic activity (Fig. 77.7). What is the different mode-of-action between alfacalcidol/calcitriol and eldecalcitol? The long duration of eldecalcitol in the blood stream arises from its strong affinity for DBP and might explain, in part, the enhanced activity of eldecalcitol over alfacalcidol/calcitriol in bone. Interestingly, it was recently suggested that calcitriol may be responsible for calcium metabolism whereas the strong binding of calcifediol to DBP may be responsible for its anabolic effect on bone [34]. There are still many challenges ahead in attempting to gain a full understanding of the mode-of-action of eldecalcitol with the objective of developing an even more effective and sophisticated pharmaceutical product. This
eldecalcitol
1495
normal range serum calcium
FIGURE 77.7 Basic relationship between calcemic activity (serum calcium level) and effect on bone (BM increase) in cholecalciferol, calcitriol/alfacalcidol, and eldecalcitol.
[1] R. Eastell, B.L. Riggs, Vitamin D and osteoporosis, in: D. Feldman, F.H. Glorieux, J.W. Pike (Eds.), "Vitamin D Second Edition", Elsevier Academic Press, Burlington, 2005, pp. 1101e1120. [2] S.E. Papapoulos, Pharmacology and use in the treatment of osteoporosis, in: R. Marcus, D. Feldman, J. Kelsey (Eds.), "Osteoporosis", Academic Press, San Diego, 1996, pp. 1209e1234. [3] Y. Nishii, T. Okano, History of the development of new vitamin D analogs: studies on 22-oxacalcitriol (OCT) and 2b-(3hydroxypropoxy)calcitriol (ED-71), Steroids 66 (2001) 137e146. [4] K. Eto, A. Fujiyama, M. Kaneko, K. Takahashi, J. Ishihara, S. Hatakeyama, et al., An improved synthesis of 1-epi-ED-71, a biologically interesting diastereomer of 1a,25-dihydroxy-2b-(3hydroxypropoxy)vitamin D3 (ED-71), Heterocycles 77 (2009) 323e331. [5] K. Miyamoto, E. Murayama, K. Ochi, H. Watanabe, N. Kubodera, Synthetic studies of vitamin D analogues. XIV. Synthesis and calcium regulating activity of vitamin D3 analogues bearing a hydroxyalkoxy group at the 2b-position, Chem. Pharm. Bull. 41 (1993) 1111e1113. [6] Y. Ono, H. Watanabe, A. Shiraishi, S. Takeda, Y. Higuchi, K. Sato, et al., Synthetic studies of active vitamin D analogs. XXIV. Synthesis of active vitamin D3 analogs substituted at the 2bposition and their preventive effects on bone mineral loss in ovariectomized rats, Chem. Pharm. Bull. 45 (1997) 1626e1630. [7] Y. Ono, H. Watanabe, A. Shiraishi, S. Takeda, Y. Higuchi, K. Sato, et al., Syntheses and preventive effects of analogues related to 1a,25-dihydroxy-2b-(3-hydroxypropoxy)vitamin D3 (ED-71) on bone mineral loss in ovariectomized rats, Bioorg. Med. Chem. 6 (1998) 2517e2523. [8] T. Kobayashi, T. Okano, N. Tsugawa, M. Murano, S. Masuda, A. Takeuchi, et al., 2b-(3-Hydroxypropoxy)-1a,25-dihydroxyvitamin D3 (ED-71), preventive and therapeutic effects on bone mineral loss in ovariectimized rats, Bioorg. Med. Chem. Lett. 3 (1993) 1815e1819. [9] Y. Uchiyama, Y. Higuchi, S. Takeda, T. Masaki, A. Shira-Ishi, K. Sato, et al., ED-71, a vitamin D analog, is a more potent inhibitor of bone resorption than alfacalcidol in an estrogendeficient rat model of osteoporosis, Bone 30 (2002) 582e588. [10] N. Kubodera, D-Hormone derivatives for the treatment of osteoporosis: from alfacalcidol to eldecalcitol, Mini-Reviews Med. Chem. 9 (2009) 1416e1422. [11] F. Ichikawa, K. Sato, M. Nanjo, Y. Nishii, T. Shinki, N. Takahashi, et al., Mouse primary osteoblasts express vitamin D3 25hydroxylase mRNA and convert 1a-hydroxyvitamin D3 into 1a,25-dihydroxyvitamin D3, Bone 16 (1995) 129e135.
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[12] N. Kubodera, Search for and development of active vitamin D3 analogues, J. Syn. Org. Chem. Jpn. 63 (2005) 137e146. [13] N. Kubodera, Search for and development of active vitamin D3 analogs, Curr. Bioact. Compd. 2 (2006) 301e315. [14] N. Kubodera, A new look at the most successful prodrugs for active vitamin D (D hormone): alfacalcidol and doxercalciferol, Molecules 14 (2009) 3869e3880. [15] Y. Nishii, Active vitamin D and its analogs as drugs for the treatment of osteoporosis: advantages and problems, J. Bone Miner. Metab. 20 (2002) 57e65. [16] A. Shiraishi, S. Higashi, H. Ohkawa, N. Kubodera, T. Hirasawa, I. Ezawa, et al., The advantage of alfacalcidol over vitamin D in the treatment of osteoporosis, Calcif. Tissue Int. 65 (1999) 311e316. [17] E. Abe, C. Miyaura, H. Sakagami, M. Takeda, K. Konno, T. Yamazaki, et al., Differentiation of mouse myeloid leukemia cells induced by 1a,25-dihydroxyvitamin D3, Proc. Natl. Acad. Sci. USA 78 (1981) 4990e4994. [18] R. Bouillon, W.H. Okamura, A.W. Norman, Structureefunction relationships in the vitamin D endocrine system, Endocr. Rev. 16 (1995) 200e257. [19] G.H. Posner, M. Kahraman, Overview: rational design of 1a,25dihydroxyvitamin D3 analogs (deltanoids), in: D. Feldman, F.H. Glorieux, J.W. Pike (Eds.), "Vitamin D Second Edition", Elsevier Academic Press, Burlington, 2005, pp. 1405e1422. [20] N. Kubodera, K. Sato, Y. Nishii, Characteristics of 22-oxacalcitol (OCT) and 2b-(3-hydroxypropoxy)calcitriol (ED-71), in: D. Feldman, F.H. Glorieux, J.W. Pike (Eds.), "Vitamin D", Academic Press, San Diego, 1997, pp. 1071e1086. [21] N. Kubodera, Development of OCT and ED-71, in: D. Feldman, F.H. Glorieux, J.W. Pike (Eds.), "Vitamin D Second Edition", Elsevier Academic Press, Burlington, 2005, pp. 1525e1541. [22] H. Shimizu, K. Shimizu, N. Kubodera, T. Mikami, K. Tsuzaki, H. Suwa, et al., Industrial synthesis of maxacalcitol, the antihyperparathyroidism and antipsoriatic vitamin D3 analogue exhibiting low calcemic activity, Org. Process. Res. Dev. 9 (2005) 278e287. [23] M.J. Calverley, Synthesis of MC 903, a biologically active vitamin D metabolite analog, Tetrahedron 43 (1987) 4609e4619. [24] M. Morisaki, N. Koizumi, N. Ikekawa, T. Takeshita, S. Ishimoto, Synthesis of active forms of vitamin D. Part IX. Synthesis of
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IX. ANALOGS
1a,24-dihydroxycjolecalciferol, J. Chem. Soc. Perkin Trans. 1 (1975) 1421e1424. E. Slatopolsky, J. Finch, M. Ritter, M. Denda, J. Morrissey, A. Brown, A new analog of calcitriol, 19-nor-1,25-(OH)2D2, suppresses parathyroid hormone secretion in uremic rats in the absence of hypercalcemia, Am. J. Kidney Dis. 26 (1995) 852e860. F. Takahashi, J.L. Finch, M. Denda, A.S. Dusso, A.J. Brown, E. Slatopolsky, A new analog of 1,25-(OH)2D2, suppresses serum PTH and parathyroid gland growth in uremic rats without elevation of intestinal vitamin D receptor content, Am. J. Kidney Dis. 30 (1997) 105e112. H.Y. Lam, H.K. Schnoes, H.F. DeLuca, Synthesis of 1a-hydroxyergocalciferol, Steroids 30 (1977) 671e677. Y. Kobayashi, T. Taguchi, S. Mitsuhashi, T. Eguchi, E. Ohshima, N. Ikekawa, Studies on organic fluorine compounds. XXXIX. Studies on steroids. LXXIX. Synthesis of 1a,25-dihydroxy26,26,26,27,27,27-hexafluorovitamin D3, Chem. Pharm. Bull. 30 (1982) 4297e4303. N. Kubodera, Pharmaceutical studies on vitamin D derivatives and practical syntheses of six commercially available vitamin D derivatives that contribute to current clinical practice, Heterocycles 80 (2010) 83e98. K. Miyamoto, N. Kubodera, E. Murayama, K. Ochi, T. Mori, I. Matsunaga, Synthetic studies on vitamin D analogues VI. A new synthesis of 25-hydroxycholesterol from lithocholic acid, Synth. Commun. 16 (1986) 513e521. T. Matsumoto, T. Takano, S. Yamakido, F. Takahashi, N. Tsuji, Comparison of the effects of eldecalcitol and alfacalcidol on bone and calcium metabolism, J. Steroid Biochem. Mol. Biol. 2 (2010) 261e264. T. Matsumoto, T. Miki, H. Hagino, T. Sugimoto, S. Okamoto, T. Hirota, et al., A new active vitamin D, ED-71, increases bone mass in osteoprotic patients under vitamin D supplementation: a randomized, double-blind, placebo-controlled clinical trial, J. Clin. Endocrinol. Metab. 90 (2005) 5031e5036. T. Matsumoto, N. Kubodera, The ED-71 Study Group 2009 Vitamin D analogs for fracture prevention in osteoporosis. In "Abstracts of Fourteenth Workshop on Vitamin D, Brugge, Belgium, October 2009" pp. 126. A.G. Need, B.E.C. Nordin, Misconceptions e vitamin D insufficiency causes malabsorption of calcium, Bone 42 (2008) 1021e1024.
C H A P T E R
78 Non-secosteroidal Ligands and Modulators Keith R. Stayrook 1, Matthew W. Carson 2, Yanfei L. Ma 2, Jeffrey A. Dodge 2 1
Indiana University School of Medicine, Indianapolis, IN, USA 2 Lilly Research Laboratories, Indianapolis, IN, USA
INTRODUCTION The core chemical structures of secosteroids are similar to a steroid except that the two B-ring carbon atoms at positions C9 and C10 of the traditional four steroid rings are not joined. Vitamin D is a secosteroid of utmost physiological importance as vitamin D deficiency causes osteomalacia and rickets and significantly increases risk of osteoporosis, autoimmune disease, infections, various cancers, and cardiovascular disease [1]. Vitamin D is produced from steroidal precursors in the skin via the action of UVB-containing sunlight. Vitamin D produced by the skin as either vitamin D2 or vitamin D3 is physiologically inactive and must undergo a series of successive hydroxylation in the liver, kidney, and/or various target tissues to become the biologically active 1a,25-dihydroxyvitamin D (1a,25(OH)2D3) [2e4] (see Chapter 3). The pleiotropic actions of hormonally active 1a,25(OH)2D3 (calcitriol) are mediated by the nuclear vitamin D receptor (VDR). VDR is a ligand-dependent transcription factor that belongs to the superfamily of steroid/thyroid hormone receptors and classically functions as a heterodimer with its cognate co-receptor retinoid X receptor (RXR) to control expression of genes involved in calcium and phosphorus homeostasis and bone mineral content. However, the scope of vitamin D and VDR biology has expanded to include a wide range of physiological cellular responses including proliferation, differentiation, and immunomodulation. In addition to its welldescribed transrepression and transcriptional regulatory activities, VDR also has the ability to rapidly initiate several key signal transduction pathways in a liganddependent non-genomic manner that does not require its co-receptor partner RXR [5,6]. While driving many cellular activating events through VDR, 1a,25(OH)2D3 levels are regulated by a finely tuned series of regulatory
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10078-2
mechanisms. These regulatory mechanisms include the catabolic inactivation of 1a,25(OH)2D3 via 24-hydroxylase/CYP24A1 enzymatic activity, regulation of 1ahydroxylation via 1a-hydroxylase/CYP27B1 enzymatic activity, and alterations in serum 1a,25(OH)2D3 half-life or target tissue uptake via vitamin-D-binding protein (DBP) binding (see Chapter 5). It is the cooperative complexity of these multifaceted ligand-dependent actions of VDR coupled to regulatory feedback mechanisms that ultimately produce the natural physiological activity of the secosteroid vitamin D and its metabolites. The therapeutic benefit and application of pharmacological doses of 1a,25(OH)2D3 has been used clinically for the treatment of renal osteodystrophy, secondary hyperparathyroidism, psoriasis, osteoporosis, cancer, and autoimmune disease, but its use is limited due to excessive absorption of mineral and pathological tissue hypermineralization [7]. Therefore, the synthesis and design of novel secosteroidal analogs exhibiting beneficial physiologic function with concomitant lower capacity for toxicity has been undertaken in the last two decades. More than 2000 secosteroidal analogs have been synthesized with modifications to the A and/or CD rings or the aliphatic side chains (see Chapters 76, 77, and 81). Many of these molecules display unique VDR modulating behavior in various biological assays and/or preclinical pharmacology models [8]. However, very few of these analogs have displayed the appropriate enhanced therapeutic margin of safety for the treatment of human disease for which they were designed. Furthermore, secosteroidal-based chemical synthesis has proven to be difficult and costly. Upon the identification, development, and proven clinical effectiveness of the synthetic non-steroidal tissue-selective modulators of estrogen receptor, raloxifene and tamoxifen, numerous novel synthetic chemical series for many of the receptors of the nuclear hormone
1497
Copyright Ó 2011 Elsevier Inc. All rights reserved.
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78. NON-SECOSTEROIDAL LIGANDS AND MODULATORS
receptor superfamily have now been pursued and described [9,10]. This chapter is dedicated to highlighting the identification, synthesis, biological and pharmacological activities of non-secosteroidal ligands and/or modulators that are capable of binding in the traditional ligand-binding pocket of VDR to manipulate its physiologic and homeostatic functions in the various target tissues where it is expressed. The two main reasons for pursuing the design and development of novel non-secosteroidal ligands include the need for new and effective pharmacological agents for human disease treatment as well as the identification of research tools to further elucidate the biological function of vitamin D and VDR. To this end, there are relatively few published reports or descriptions of non-secosteroidal VDR ligands by comparison to those described for the classical steroid hormone receptor family. However, it is believed that the unique structural features of nonsecosteroidal ligands may afford advantageous properties that are inherently different from the liabilities surrounding 1a,25(OH)2D3 or its secosteroidal analogs. These properties may include altered DBP binding, reduced 24-hydroxylase metabolism, distinct VDR modulating behavior, or enhanced pharmacokinetic and pharmacodynamic profiles. There are currently five classes of compounds with published data that can be chemically classified as non-secosteroids. These include molecules based on a diarylmethane chemical scaffold (LG190119, VDRM-2), C/D ring-modified scaffolds, bis- and tris-aromatic triols (CD4420), podocarpic acid derivative (VDRL-1), and steroidal compounds such as the secondary bile acid lithocholic acid and/or its derivatives (Fig. 78.2). A review of the structures and activities of the diarylmethanes, C/D ring modified chemistry, bis- and tris-aromatic triols, and podocarpic acid derivative classes are included in this chapter. A review of steroidal lithocholic acid and its derivatives can be found in Chapter 79 and is not included here.
DIARYLMETHANE LIGANDS Identification, Structure, and Biological Characterization The hypothesis that non-secosteroidal analogs could mimic the effects of 1a,25(OH)2D3 (Fig. 78.1) prompted the search for novel chemistry in the hopes of obtaining VDR ligands with improved tissue selectivity relative to calcitriol or secosteroidal analogs. The diarylmethane chemical scaffold was one of the first non-secosteroidal series to be identified and shown to exhibit both in vitro and in vivo vitamin D pharmacology [11]. The original screening hit LG190090 as shown in Figure 78.3 was able to induce activity in a VDR-dependent manner in
20 25 17
C
D
14
OH
H
19
A HO
3
1
OH
FIGURE 78.1 Structure of 1a,25(OH)2D3 (calcitriol). A-, B-, and Drings and carbons mentioned in the text are marked. Aliphatic alcohol side chain is C20eC25.
a cotransfection (CTF) assay using HepG2 cells transfected with a VDRE-based luciferase reporter. Structuree activity relationship (SAR) optimization led to identification of LG190178 (Fig. 78.3), a potent VDR agonist with an EC50 value of 40 nM in the CTF assays coupled with good binding affinity to the vitamin D receptor (Ki ¼ 150 nM) [11]. In biochemical assays measuring SRC peptide coregulator interaction, LG190178 was comparable to 1a,25(OH)2D3 [12]. The individual enantiomers of LG190178 have been prepared and evaluated for VDR binding and transcriptional activity using human osteosarcoma and colon carcinoma cell lines [13]. Overall, one stereoisomer, YR301 (2S,20 R) (Fig. 78.3), demonstrates high binding affinity to VDR (RBA ¼ 28.3%) and strong transcriptional activity in human osteosarcoma cells (MG-63 EC50 ¼ 0.78 nM) and in human colon cells (Caco2 EC50 ¼ 1.8 nM). Interestingly, the remaining stereoisomers (2S,20 R), (2R,20 R), and (2S,20 S), were significantly less active in binding and VDR functional activity indicating ligand stereo-sensitivity of VDR binding pocket with these compounds. Docking studies with all four stereoisomers in VDR indicated that the (2S,20 R)-isomer was the most stable having hydrogen bonds between the 2-OH and Arg274, Ser237 and between the 20 -OH and His305 and His397. Further SAR studies with LG190178 resulted in nitrogen-linked side chains on the diphenylmethane core [14]. One such analog, DPP-1023 (Fig. 78.3), binds with high affinity to VDR (Ki ¼ 52 nM) and displays potent agonism in HL60 cell differentiation with an EC50 of 48 nM. The optically pure isomers of DPP-1023 were obtained and characterized. The (R,R) configuration displays the most potent activity, relative to 1a,25(OH)2D3, having a Ki of 9.5 nM coupled with a >10-fold increase in cellular potency as measured by the induction of HL-60 cell differentiation (i.e., EC50 ¼ 4.1 nM for (R,R)-DPP-1023 compared to 59 nM for 1a,25(OH)2D3). This compound binds to the androgen receptor (AR) with a Ki of 910 nM thereby demonstrating dual VDR-AR activity. Other compounds in this series bind to AR in the 400 nM range while
IX. ANALOGS
1499
DIARYLMETHANE LIGANDS
H OH
O
OH
N OH
O O OH
H HO
VDRM-2
F3C
JN
OH
F F
CF3
F OH F F F
O
O
NH
H OH
OH OH
VDRL-1
CD4420
H OH OH
HO
OH
OH
HO
des CD analogs
CF-ring analogs
FIGURE 78.2 Structures of various non-secosteroid VDR ligands. The five classes of non-secosteroid molecules are represented by the selected structures and include the diarylmethanes (VDRM-2), steroidal (JN), podocarpic acid derivative (VDRL-1), bis- and tris-aromatic triols (CD4420), and deconstructive secosteroidal compounds such as des-CD and CF-ring analogs.
demonstrating anti-androgen activity by inhibiting testosterone-dependent proliferation in SC-3 cells. No in vivo data have been reported to elaborate on the dual intrinsic activity for these ligands. Further elaboration of the SAR within this chemical series finally led to the identification of LG190119 (Fig. 78.3) which demonstrated the first described separation between the induction of certain VDR target genes and serum calcium concentration in mice [17]. LG190119 and LG190178 constitute the first disclosed selective non-secosteroidal vitamin D receptor modulators (VDRMs).
Additional reported synthesis efforts within the diarylmethane chemical series resulted in the identification and description of an analog that demonstrates improved efficacy on bone-related endpoints relative to hypercalcemia in rodent models. Referred to as VDRM2 (Fig. 78.3), it contains a novel amide side chain, displays good affinity for VDR (Ki ¼ 57.8 nM), and is a potent inducer of RXRVDR heterodimerization (EC50 ¼ 7.1 nM). VDRM2 induces osteocalcin-promoter activity with an EC50 of 1.9 nM as compared to 1.3 nM for 1a,25(OH)2D3, while being less potent in calcium channel TRPV6 upregulation
IX. ANALOGS
1500
78. NON-SECOSTEROIDAL LIGANDS AND MODULATORS
O
O O
Cl
O
O
O
Cl
O
O
LG190090
LG190119
original screening hit
OH
2S
OH
OH
OH
O
O
O
O
2'R OH
OH
YR301
LG190178
O N
O
OH
O
OH
OH
VDRM2
HO
S
OH
O
N
OH
H
DPP-1023
HO
O S O O
S OH
O O
LY2109866
LY2108491
Representative structures of the non-secosteroidal diarylmethane ligand class. Select structures include original screening hit LG190090, diphenylmethanes (LG190119, LG190178, YR301, VDRM2, DPP-1023) and phenyl-thiophenes (LY2108491 and LY2109866).
FIGURE 78.3
than 1a,25(OH)2D3 with an EC50 of 37.2 nM compared to that of 0.6 nM for 1a,25(OH)2D3 [15]. Derivatives of the diphenylmethane structure have been identified. In one such series, one of the phenyl groups in LG190119 has been replaced by a thiophene [16]. These ligands, LY2108491 and LY2109866 (Fig. 78.3), contain a traditional hydroxyalkyl side chain on the novel thiophene ring while the adjacent phenyl ring has a sulfate ester in the case of LY2108491 and a carboxylic acid in the case of LY2109866. Both compounds are potent agonists of VDR-RXR heterodimerization in human SaOS-2 cells with an EC50 of 11 nM and 13 nM, respectively. These molecules induce VDRE-dependent expression of a rat osteocalcin
reporter in rat osteoblast cells (ROS17/2.8) with an EC50 of 25 nM and 3 nM. In contrast, LY2108491 and LY2109866 are poor inducers of intestinal vitamin-Dresponsive genes relative to 1a,25(OH)2D3 that include TRPV6, CYP24, and calbindin-D9k, but are potent agonists in keratinocytes and peripheral blood mononuclear cells. Additionally, phenyl-thiophenes with carboxamide side chains have also been disclosed with VDR-modulating activities (US patent 7601850B2). Other analogs related to the diarylmethane chemical scaffold have also been disclosed in the patent literature. These include compounds represented by phenyl-furan, phenyl-benzofuran, and phenyl-benzothiophene (US patents 7468449B2, 7579488B2, 7582775B2).
IX. ANALOGS
1501
DIARYLMETHANE LIGANDS
14
800
750
13
700 12 650
625 mg/cc
s 11.20 mg/dL 11
600 s o o
550
Serum Ca (mg/dl)
The primary goal of identifying and characterizing the biological activities of non-secosteroidal VDR ligands is ultimately to design therapeutics that may serve to treat various disease states sensitive to the benefits of vitamin D physiology. This is manifested by the clinically proven lack of hypercalcemia/hypercalciuria risk while displaying significant disease efficacy at a given dose or exposure of VDR ligand. Currently, no clinical validation of non-secosteroidal VDR ligands demonstrating separation of hypermineralization risk has been reported. Several reports of preclinical validation to this effect have been observed with compounds from the diarylmethane chemical class. Boehm et al. reported that the first non-secosteroidal VDR ligand diphenylmethane LG190119 displays pharmacologic separation as measured by VDR target gene induction versus serum calcium concentrations in vivo. Oral gavage dosing for 3e5 days of LG190119 at 10 or 30 mg/kg/d to Balb/c mice was able to induce 30-fold expression of VDR target gene 24(OH)ase RNA in kidney without significantly increasing serum calcium. Although 1a,25(OH)2D3 5 mg/kg/day was able to induce a similar fold elevation of kidney 24(OH)ase RNA in the same study, it significantly increased serum calcium at this dose [11]. Furthermore, LG190119 was reported to be effective in inhibiting tumor growth while displaying no significant hypercalcemia at the dose used over a 12-week treatment period in an LNCaP-derived prostate tumor xenograft athymic mouse model. This efficacy compared favorably to EB1089, a secosteroidal analog which has been previously shown to be less calcemic but more potent in inhibiting LNCaP cell growth than 1a,25(OH)2D3 in vitro. In the same study, 1a,25(OH)2D3 dosed at 0.5 mg/kg was too low to see significant tumor suppression in this model [17]. The authors claimed that the fewer in vivo calcium effects of their diphenylmethane vitamin D mimics may be attributable to less binding to serum D-binding protein. Sato et al. reported that amide side-chain-containing VDRM2 displays significant pharmacological separation of bone efficacy versus serum calcium in an ovariectomized (OVX), osteopenic rat model. Oral treatment of VDRM2 administered for 8 weeks to 7-month-old, 1-month post-ovariectomized (OVX) rats dose dependently increased bone mineral density of lumbar vertebrae (LVBMD), and increased trabecular bone volume of the proximal tibial metaphyses from OVX control back to Sham levels at a dose of 0.08 mg/kg/day. At doses above 0.03 mg/kg/day, VDRM2 restored bone strength parameters back to Sham levels or above in the vertebra, femoral neck, and femoral midshaft.
Hypercalcemia in those animals was not observed until 4.6 mg/kg/day, indicating a therapeutic safety margin of 57-fold between bone efficacy and hypercalcemia (Fig. 78.4). Histomorphometric analysis showed that VDRM2 increased periosteal bone formation rate resulting in increased cortical bone properties, while functioning as a weak antiresorptive on trabecular bone surfaces. In comparative studies, 1a,25(OH)2D3, ED71, and alfacalcidol displayed a therapeutic safety window of 7.3, 4.9, and 5, respectively, based on the ratio between the Sham level threshold BMD efficacy dose and the hypercalcemic dose from the same animals (Table 78.1). These data suggest that the non-
Vertebral BMD (mg/cc)
In Vivo Characterization of Diarylmethane Ligands
10
9
500 0.001
0.01
0.1
1
0.081 µg/kg
10
100
4.6 µg/kg
Therapeutic safety window between Sham level threshold bone mineral density efficacy dose and hypercalcemia dose for VDRM2 from the reproducible doseeresponse studies of 8 weeks’ treatment on 7-month-old, 1-month post-ovariectomized (Ovx) rats. Plotted are mean SEM. Modified from [15].
FIGURE 78.4
TABLE 78.1
Comparison of VDRM2 to Vitamin D3 Analogs on Bone Efficacy to Hypercalcemia Ratio (NOEL Based)
Compound (mg/kg/d) VDRM 2 1,25-OH2D3 Alfacalcidol ED-71 LVBMD (mg/cc)*
0.081
0.03
0.046
0.0055
Serum calcium**
4.6
0.22
0.23
0.027
Serum Ca/LVBMD
57
7.3
5
4.9
* Minimal efficacy dose which restores LVBMD from Ovx to Sham levels. ** Minimal hypercalcemia dose which reaches 97.5th percentile of the historical Ovx rat value (11.2 mg/dl). Modified from [15]. LVBMD ¼ lumbar vertebral bone mineral density. Seven-month-old rats were permitted to lose bone due to ovariectomy for 1 month before dosing with compounds for 8 weeks. Data presented were averaged from 2e5 assays for each compound.
IX. ANALOGS
1502
78. NON-SECOSTEROIDAL LIGANDS AND MODULATORS
secosteroidal compound VDRM2 has an increased safety margin versus its secosteroidal counterpart molecules in osteopenic ovariectomized rats [15]. It remains unknown whether this rodent pharmacological distinction is directly translatable to the human clinical setting. Pharmacology studies with phenyl-thiophene derivatives LY2108491 and LY2109866 (Fig. 78.3) have also been reported. These molecules were found to be significantly less hypercalcemic as compared to 1a,25(OH)2D3 in vivo when administered via oral or topical application routes. In a mouse 6-day oral dosing study, 1a,25(OH)2D3 resulted in a significant increase in blood ionized calcium at a 1 mg/kg/day dose. In contrast, oral dosing of LY2108491 resulted in no significant hypercalcemia up to 3000 mg/kg/day, while LY2109866 treatment similarly showed no evidence of hypercalcemia at a dose of 1000 mg/kg/day [16]. Using a hairless mouse model of epidermal proliferation via a topical application route, Ma et al. calculated the therapeutic indices (TI) of these ligands and compared them head-to-head with 1a,25(OH)2D3. They found that calcitriol had a 0.3 TI ratio between the threshold minimum effective dose (TMED) for hypercalcemia and the TMED of keratinocyte proliferation in the same animal. In contrast, the calculated TI ratio for LY2108491 was found to be greater than 81 and LY2109866 was more than 27. Therefore, LY2108491 and LY2109866 were greater than 270- and 90-fold, respectively, better than calcitriol in terms of the TI in this topically applied surrogate model of psoriasis. This model is regarded as a surrogate in vivo preclinical model of psoriasis since VDR ligands (calcitriol and calcipotriol) and retinoids that inhibit keratinocyte proliferation in psoriatic lesions also induce epidermal proliferation when applied topically to normal skin [16,18,19]. These data indicate that non-secosteroidal phenyl-thiophene derivatives may exhibit less hypercalcemia liability when administered orally but also may extend to topically applied pharmacological settings as well. However, no clinical development information has been reported for these compounds.
C/D-RING MODIFIED LIGANDS Identification, Structure, and Biological Characterization Since the strong calcemic effects of the secosteroid natural hormone calcitriol limit its therapeutic potential, a number of efforts have been carried out to find a safe VDR ligand. Early work in the field involved modifications of either the secosteroid A-ring or the aliphatic side chain [20]. A-ring modified 2MD and
aliphatic alcohol analog calcipotriol exhibit improved margins of safety for bone versus hypercalcemia, but their similarity in structure to calcitriol suggested that the therapeutic index may not have been optimized. To improve upon this, a deconstructive approach of the secosteroid scaffold was undertaken (Fig. 78.5) [21]. These studies commenced with the removal of the C/D rings of calcitriol yielding the retiferols [23]. Retiferols and their 19-nor versions were reported in the patent literature (WO199943646) which revealed that these compounds exhibit selectivity for cell differentiation versus hypercalcemia. Further SAR efforts involved replacement of the fused C/Drings of secosteroids with either a 5- or 6-membered ring [24]. These series of compounds are referred to as the C-ring, D-ring, and E-ring analogs. The C/Dring modification was later expanded to CF-spiro[4.5] decanes and [5.5]undecanes (WO 2006060884 and WO 2006060885). The synthesis of the latter has been reported [25,26]. Investigators have also combined structural elements of the 2-methylenes with the desCD ring scaffold to give a molecule with binding affinity two orders of magnitude greater than 2MD. More recently, it was reported that activity of this compound is improved by increased van der Waals interactions with the central hydrophobic channel of the ligand-binding domain of VDR. This was accomplished by incorporating methyl groups at the C13 position of des-CD-2-methylene-19 norvitamin D compounds [27]. The absolute configuration of C13 was determined by analyzing circular dichromism spectra of g-lactone precursor diastereomers. Biological characterization of C/D-ring modified non-secosteroids clearly demonstrate that full C/Drings are not required for the biological activity of 1a,25(OH)2D3 as C/D-ring replacements in many cases retain full or modulatory 1a,25(OH)2D3 activities in various VDR binding, cellular activity and pharmacological settings. However, for the various reported C/D-ring modified compounds there do appear to be important spatial and geometric requirements that new ring replacements must generate an appropriate spacing of the A-seco B-rings in relationship to the particular side chain used. Several reports including Verstuyf et al. have described in detail the structuree activity relationships of C-ring, D-ring, and E-ring ligand classes and their VDR modulating behavior [21,22,28,29] (see Chapter 76). This was performed by analyzing VDR and DBP binding, in vitro studies using several cell lines, and in vivo calcemia studies in mice. Table 78.2 is a summarized table of biological activities from select ring-modified compounds from each of the C/D- and E-ring SAR efforts [22]. Currently, while these data support the potential therapeutic capacity of C/D-ring modified
IX. ANALOGS
1503
BIS- AND TRIS-AROMATIC TRIOLS
OH
X
X=CH2 retiferol analog X=H,H 19-nor retiferol analog
6 HO
OH R R
FIGURE 78.5 A retiferol and 19-nor retiferol; 19-nor refers to the lack of methylene group at C6. C- and D-ring analogs represent disconnected D- and C-rings, respectively. Ering analogs are retiferols with a gem dimethylcyclopentane spacer element (R ¼ aliphatic alcohol chain). CF-spiro derivatives are C-ring analogs with spiro-fused five- or sixmembered ring spacers between the aliphatic alcohol chain and cyclohexane core. Des-CD-2methylene analogs of 2MD are similar to the retiferol series except that the methylene group on the A-ring is located at the 2-position rather than the C-6. The diastereomeric C-13 methyl substituted analogs are shown.
R
HO
HO
OH
C-ring analogs
HO
OH
D-ring analogs
OH
E-ring analogs
O
OH OH 13
2 HO
OH
HO
OH
(R)-13-methyl (S)-13-methyl
spiro [5.5]undecane
non-secosteroid ligands, no clinical development information has been reported for this compound class.
BIS- AND TRIS-AROMATIC TRIOLS Identification, Structure, and Biological Characterization Bis- and tris-aromatic triols were one of the first non-secosteroidal VDR ligand classes reported. These compounds were originally developed for the treatment of dermatological diseases. The common fragment among the triols is the dibenzyl alcohol located
in the hydrophilic region of the molecule. The synthesis of these tris-aromatics and the phenyl ether-linked bis-aromatic compounds (Fig. 78.6) are covered along with their biological evaluation in the patent literature (WO200138303 and WO2004020379). The biological evaluation of tris-aromatics was conducted in a cotransfected HeLa cell line. The preparation of the bis-aromatic analogs along with their biological characterization on the proliferation of human keratinocytes was reported in these patents as well. Another subseries of the bis-aromatic triols containing a dienyl alcohol chain was reported by Pera¨kyla¨ et al. [30]. The most potent dienyl bis-aromatic
IX. ANALOGS
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78. NON-SECOSTEROIDAL LIGANDS AND MODULATORS
TABLE 78.2
Biological Activities of Various C/D-modified Analogs
Compound
VDR1 binding (%)
HL-602 diff (%)
MCF-73 prolif (%)
Kerat4 prolif (%)
Serum Ca5(%)
Calcitriol
100
100
100
100
100
10
20
30
10
<0.1
60
1000
6000
6000
50
80
85
85
90
0.3
OH
HO
OH
KS 176 KS 176
OH
HO
OH
ZG 1368 ZG 1368
OH
HO
OH
SL 117 SL 117 1
Competitive VDR binding. Differentiation of HL-60 cells (NBT tetrazolium assay). Proliferation of breast cancer MCF-7 cells ([3H]thymidine incorporation assay). 4 Proliferation of human keratinocytes ([3H]thymidine incorporation assay). 5 Determined by intraperitoneal injections for 7 consecutive days in mice. Table modified from [22]. In vitro data expressed as percentage of calcitriol. In vivo data are comparison of doseeresponse curves versus calcitriol (calcitriol ¼ 100%). 2 3
IX. ANALOGS
MISCELLANEOUS NON-SECOSTEROIDS
MISCELLANEOUS NON-SECOSTEROIDS
OH OH O
O
OH
OH
Tris-aromatic triol
1505
OH
OH
Ether-linked bis-aromatic triol
FIGURE 78.6 The tris- and ether-linked bis-aromatic triols. The core is an aromatic ring containing a straight chain hinge-region.
compounds CD4409, CD4420, and CD4528 were evaluated in a variety of in vitro and in vivo assays. In addition, molecular dynamic (MD) simulations demonstrated that these dienyl bis-aromatic ligands occupy the VDR ligand-binding pocket in a similar fashion as calcitriol. During the MD simulation study, distances between the polar anchoring points (Y147, S237, R274, S278, H305, and H397) in the VDR LBD and the hydroxyl groups of the ligand were measured and determined to be comparable to the VDR-bound structure of 1a,25(OH)2D3. The docking of these ligands suggests that the dibenzyl alcohol sits in the same region of the VDR LBD as the cyclohexanediol of 1a,25(OH)2D3 and the branched alcohol interacts with H305 and H397. The combination of MD simulation and single point mutation of the above residues suggests that CD4528 most closely resembles the structure of calcitriol. In VDR biochemical and functional studies, this class of ligands displays the capacity to bind within the VDR ligand-binding pocket and can drive VDReRXR heterodimer complex formation on a DR3 element in gel-shift experiments. In addition, they display potent cellular activity as they induced Gal4-VDR LBD mediated-luciferase activity in Hela cells comparable to 1a,25(OH)2D3 (Fig. 78.7). Furthermore, CD4409, CD4420, and CD4528 demonstrate VDR pharmacological capacity as measured in male BALB/c mice. Interestingly, in 8week-old Balb/c mice, oral gavage with these ligands for 3 or 5 days at non-calcemic doses stimulated higher VDR target gene CYP24 mRNA expression in renal tissue than that of a non-calcemic dose of calcitriol (1 mg/kg) [31]. Representation of summarized functional and pharmacological data generated with CD4409, CD4420, and CD4528 can be found in Fig. 78.7 [31]. However, this non-secosteroidal class continues to be represented by a limited number of peer-reviewed disclosures.
Identification, Structure, and Biological Characterization A survey of the literature reveals that there are a few known singleton non-secosteroids reported. For example, Chen et al. reported on VDRL-1, a novel VDR ligand derived from podocarpic acid (Fig. 78.8) [31]. This compound’s activity was evaluated for VDR binding and transcriptional activity in several cell-based settings. It displays considerably weaker binding affinity as compared to calcitriol (Ki ¼ 1.4 mM versus 0.15 nM for 1a,25(OH)2D3) but is able to drive efficient VDR transactivation at notably higher concentrations. Using computer-assisted molecular docking studies, VDRL-1 conformation and interaction with the VDR LBD was analyzed in both agonist and antagonist modes. VDRL1 bound to the agonist conformation exhibited little strain energy for the top two poses, therefore suggesting that it does not displace helix 12. As is the case with nuclear receptors such as GR, AR, and ER, the position of helix 12 is important for agonist activity due its close proximity to AF-2. Pose 1 docks the ligand so that the hexafluoroisopropanol (HFiP) moiety interacts with H305/397 residues which directly or indirectly influence the position of H12. Pose 2 is flipped relative to pose 1 to allow for the phenolic OH to interact with H305/397 and the HFiP to interact with S237 and R274. The less favorable interaction energies of poses of VDRL-1 in the antagonist conformation of VDR LBD suggest that VDRL-1 likely occupies the same space and interacts with the same polar anchoring points as calcitriol. In addition to mineral and bone homeostasis, VDR biology plays important roles in immunomodulation, antimicrobial defense, xenobiotic detoxification, anticancer actions, control of insulin secretion, and cardiovascular benefits. In a landmark paper, Makishima et al. reported that activated VDR detoxifies bile acid lithocholic acid (LCA) via CYP3A4 activation. This pathway is initiated by the weak binding of LCA or its 3-substituted esters to the VDR [32,33]. LCA-related VDR biology is covered in detail in Chapter 79. However, recent evidence suggests that other non-secosteroidal nutritional ligands exist, albeit with low affinity for VDR, but in high local concentration could function as sensors for extraosseous VDR-mediated pathways. These putative VDR ligands, as illustrated in Figure 78.9, include turmeric-derived curcumin, vitamin E derivative c-tocotrienol, and polyunsaturated acids docosahexaenoic and arachidonic acid. Haussler et al. reported that curcumin, c-tocotrienol, docohexaenoic, and arachidonic acids compete with tritiated calcitriol for VDR with similar affinity as LCA. Curcumin was also shown to activate VDR in a responsive reporter gene assay at
IX. ANALOGS
1506
78. NON-SECOSTEROIDAL LIGANDS AND MODULATORS
GAL4 luciferase in HeLa EC50, nM (no CYP24 inhib)
GAL4 luciferase in HeLa EC50, nM (+ CYP24 inhib)
Ligand-dep gel shift EC50, nM
calcitriol
1
0.12
0.2
CD4409
8
10
6
CD4420
5
6
4.8
CD4528
1.7
2
9
In vitro profile Compounds
In vivo profile Compounds vehicle
Dose, μg/kg
Treatment, days 5
Kidney CYP24 mRNA induction/serum calcium ratio 1
calcitriol
1
5
6.6
CD4409
500
3
53.6
CD4409
500
5
62.6
CD4420
500
3
0.5
CD4420
500
5
1.2
CD4528
500
3
80.7 2.7
CD4528
50
5
CD4528
150
5
8.5
CD4528
500
5
59.3 F
F F
OH
F
F OH
F OH F
F S F
CD4409
O
O
OH
F
F F
CD4528
CD4420
OH
OH
OH
OH
OH
FIGURE 78.7 Structures and summarized data tables of the in vitro and in vivo activities of dienyl bis-aromatic ligands CD4409, CD4420, and CD4528. Data modified from [31].
F3 C
O
OH CF3
NH
H
OH
FIGURE 78.8 Merck’s VDRL-1, a podocarpic acid core containing hexafluoro-isopropanol and phenolic anchoring points.
concentrations four orders of magnitude higher than calcitriol [34]. Curcumin and bisdemethoxycurcumin also display significant activity in VDR-mediated nongenomic chloride channel opening in TM4 Sertoli cells [35]. The large volume of the VDR ligand-binding domain coupled with multiple polar residues deep in the pocket may explain how VDR accommodates these nutritional lipophilic acids and phenols. Like LCA, 1a,25(OH)2-lumisterol D3 (JN) is a steroid with demonstrable poor affinity for VDR in competitive binding experiments with 1a,25(OH)2D3. However, JN functions as a highly potent agonist of non-genomic and rapid signaling events mediated by VDR. It has been hypothesized that the impetus for the rapid nongenomic response of JN/VDR is the fact that it prefers to bind in an alternative binding pocket (AP) which contains more hydrophilic and p-bond residues than the genomic pocket (GP). Evidently, the dihydroxy and
IX. ANALOGS
1507
PERSPECTIVES O
O
HO O O
HO
OH
curcumin
O
gamma-tocotrienol O O
OH
OH
ϖ-3-docosahexaenoic acid
ϖ-6-docosahexaenoic acid
O H
OH H OH
H O R
H
R=H lithocholic acid R= -C(O)Me R= -C(O)Et
OH H
HO
JN
FIGURE 78.9 Nutritional and steroidal ligands.
double bonds, as well as the overall planarelinear shape of JN, drive the kinetically driven binding to AP. Alternatively, this is compared to the bowl-shaped calcitriol and many of the other non-secosteroidal ligands described in this chapter which likely prefer the hydrophobic GP. The vitamin D sterol-vitamin D receptor ensemble model described by Mizwicki et al. offers unique dynamic insights into both genomic and rapid-response signaling by these steroidal ligands [5,6,35] (see Chapter 15). Whether other physiological-relevant steroids are capable of this unique VDR activation remain to be determined. Nonetheless, they remain a potentially viable class of ligand for the vitamin D receptor.
PERSPECTIVES The numerous studies performed by both applied and basic researchers into the molecular physiology of vitamin D signaling have greatly enhanced our understanding of this hormone and its metabolites. In particular, the creative work of drug discovery scientists and medicinal chemists in the design and synthesis of vitamin D receptor ligands has paved the way for potential therapeutic exploitation of these compounds in the numerous disease states where classical vitamin D has a proven clinical benefit. The significant hurdle of excessive hypermineralization in vivo at efficacious pharmacologic doses of these compounds continues to hamper their development as useful therapeutic agents. To this end, several hundred secosteroidal analogs have been synthesized and well characterized over the past two decades, with comparably few reports for non-secosteroidal VDR ligands.
Most notably, the diarylmethanes, bis- and trisaromatic triols, and the podocarpic derivative VDRL-I are non-secosteroids not found and based on the core secosteroidal template. By comparison, C/D-ring modified and the newly described steroidal ligands are conceptually derived from classical vitamin D chemistry but claim technical classification as non-secosteroids. Nonetheless, the non-secosteroidal ligands described in this chapter display all of the binding, cellular, and pharmacological characteristics of their secosteroidal counterparts. However, they also display a number of mechanistic and therapeutic properties that may prove valuable including reduced DBP binding, enhanced ADME properties, unique VDR modulating behavior, and scalability of chemical synthesis. Furthermore, many of these ligands demonstrate potential for less calcemic risk while maintaining desired pharmacology in vivo as compared to calcitriol or select analogs. Despite much promise, it remains to be seen whether the non-calcemic behavior of these ligands in preclinical studies using non-human species will readily transfer to the human clinic. In summary, while the number of described and characterized non-secosteroidal chemical scaffolds continues to be small, they continue to hold promise in an everexpanding number of human disease states where vitamin D physiology has demonstrable importance. Additionally, the search and identification of new novel non-secosteroidal chemical scaffolds remains a valuable ongoing yet challenging goal. The growing molecular and structural understanding of the vitamin D receptor ligand-binding pocket in conjunction with availability of superior new research tools will prove useful in this endeavor.
IX. ANALOGS
1508
78. NON-SECOSTEROIDAL LIGANDS AND MODULATORS
References [1] M.F. Holick, Vitamin D deficiency, N. Engl. J. Med. 357 (2007) 266e281. [2] M.F. Holick, Calcitropic hormones and the skin: a millennium perspective, J. Cell Biochem. 88 (2003) 296e307. [3] R. Bouillon Vitamin D: from photosynthesis, metabolism and action to clinical applications. in: L.L. Degroot, J.L. Jameson (Eds.), Endocrinology, 2001 vol. 2, Fourth ed., pp.1009e1028. [4] H.F. Deluca, M.T. Cantorna, Vitamin D: its role and uses in immunology, FASEB 15 (2001) 2579e2585. [5] M.T. Mizwicki, D. Keidel, C.M. Bula, J.E. Bishop, L.P. Zanello, J. Wurtz, et al., Identification of an alternative ligand-binding pocket in the nuclear vitamin D receptor and its functional importance in 1a,25(OH)2-vitamin D3 signaling, PNAS 101 (2004) 12876e12881. [6] M.T. Mizwicki, A.W. Norman, The vitamin D sterol-vitamin D receptor ensemble model offers unique insights into both genomic and rapid-response signaling, Science Signaling 2 (2009) 1e15. [7] R. Vieth, Vitamin D toxicity, policy, and science, J. Bone Miner Res. 22 (2007) V64eV68. [8] C. Carlberg, Molecular basis of selective activity of vitamin D analogues, J. Cell Biochem. 88 (2003) 274e281. [9] H. Hansdottir, Raloxifene for older women: a review of the literature, Clin. Interv. Aging 3 (1) (2008) 45e50. [10] D.P. McDonnell, C.E. Connor, A. Wijayaratne, C.Y. Chang, J.D. Norris, Definition of the molecular and cellular mechanisms underlying the tissue-selective agonist/antagonist activities of selective estrogen receptor modulators, Recent Prog. Horm. Res. 57 (2002) 295e316. [11] M.F. Boehm, P. Fitzgerald, A. Zou, M.G. Elgort, E.D. Bischoff, L. Mere, et al., Novel nonsecosteroidal vitamin D mimics exert VDR-modulating activities with less calcium mobilization than 1,25-dihydroxyvitamin D3. Chem. Biol. 6 (1999) 265e275. [12] A. Teichert, L.A. Arnold, S. Otieno, Y. Oda, I. Augustinaite, T.R. Geistlinger, et al., Quantification of the vitamin D receptor e coregulator interaction, Biochemistry 48 (7) (2009) 1454e1461. [13] W. Hakamata, Y. Sato, H. Okuda, S. Honzawa, N. Saito, S. Kishimoto, et al., (2S,20 R)-Analogue of LG190178 is a major active isomer, Bio. Med. Chem. Lett. 18 (2008) 120e123. [14] S. Hosada, A. Tanatani, K. Wakabayashi, M. Makashima, K. Imai, H. Miyachi, et al., Ligands with a 3,3-diphenylpentane skeleton for nuclear vitamin D and androgen receptors: dual activities and metabolic activation, Bioorg. Med. Chem. 14 (2006) 5489e5502. [15] M. Sato, J. Lu, S. Iturria, K.R. Stayrook, L. Burris, Q.Q. Zeng, et al., A non-secosteroidal vitamin D receptor ligand with improved therapeutic window of bone efficacy over hypercalcemia, J. Bone Min. Res. (2010). [E-pub]-In press. [16] Y. Ma, B. Khalifa, Y.K. Yee, J. Lu, A. Memezawa, R.S. Savkur, et al., Identification and characterization of noncalcemic, tissueselective, nonsecosteroidal vitamin D receptor modulators, J. Clin. Invest. 116 (2006) 892e904. [17] T.C. Polek, S. Murthy, S.E. Blutt, M.F. Boehm, A. Zou, N.L. Weigel, et al., Novel nonsecosteroidal vitamin D receptor modulator inhibits the growth of LNCaP xenograft tumors in athymic mice without increased serum calcium, Prostate 49 (2001) 224e233. [18] R. Gniadecki, J. Serup, Stimulation of epidermal proliferation in mice with 1-alpha, 25-dihydroxyvitamin D3 and receptor-active 20-epi analogues of 1-alpha, 25-dihydroxyvitamin D3, Biochem. Pharmacol. 49 (1995) 621e624. [19] C. Lutzow-Holm, P. De Angelis, O.P. Clausen, Calcitriol and its analog KH 1060 induce similar changes in keratinocyte cell cycle
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
IX. ANALOGS
progression after topical application to mouse skin. A bromodeoxyuridine pulse-chase flow cytometric study, J. Investig. Dermatol. Symp. Proc. 1 (1996) 54e59. R. Bouillon, W.H. Okamura, A.W. Norman, Structure function relationships in the vitamin D endocrine system, Endocr. Rev. 16 (2) (1995) 200e257. P.J. De Clercq, F. De Buysser, G. Minne, W. Schepens, F. Vrielynck, D. Van Haver, et al., The development of CD-ring modified analogs of 1a,25-dihydroxyvitamin D, J. Steroid. Biochem. Mol. Biol. 103 (3e5) (2007) 206e212. R. Bouillon, L. Verlinden, G. Eelen, P. De Clercq, M. Vandewalle, C. Mathieu, et al., Mechanisms for the selective action of vitamin D analogs, J. Ster Biochem. Mol. Bio. 97 (1e2) (2005) 21e30. A. Kutner, H. Zhao, H. Fitak, H. Wilson, Synthesis of Retiferol RAD1 and RAD2, the lead representatives of a new class of desCD analogs of cholecalciferol, Bioorg. Chem. 23 (1995) 22e32. K. Sabbe, P. D’Hallewyn, P.J. De Clercq, M. Vandewalle, Synthesis of CD-ring modified 1a,25-dihydroxy vitamin D analogues: E-ring analogues, Bioorg. Med. Chem. Lett. 6 (1996) 1697e1702. W. Schepens, D. Van Haver, M. Vandewalle, R. Bouillion, A. Verstuyf, P.J. De Clercq, Synthesis of spiro[4.5]decane CF-ring analogues of 1a,25-dihydroxy vitamin D3, Organic Let. 8 (2006) 4247e4250. F. De Buysser, L. Verlinden, A. Verstuyf, P.J. De Clercq, Synthesis of 22-oxaspiro[4.5]decane CD-ring modified analogs of 1a,25dihydroxy vitamin D3, Tetrahedron. Lett. 50 (2009) 4174e4177. K. Plonska-Ocypa, R.R. Sicinski, L.A. Plumn, P. Grzywacz, J. Frelek, M. Clagett-Dame, et al., 13-Methyl-substituted des-C, D analogs of (20S)-1a,25-dihydroxy-2-methylene-19-norvitamin D3 (2MD): synthesis and biological evaluation, Bioorg. and Med. Chem. 17 (2009) 1747e1763. A. Verstuyf, L. Verlinden, E. Van Etten, L. Shi, Y. Wu, C. D’Halleweyn, et al., Biological activity of CD-ring modified 1a,25-dihydroxy vitamin D analogues: C-ring and fivemembered D-ring analogues, J. Bone Miner. Res. 15 (2) (2000) 237e252. A. Verstuyf, L. Verlinden, H. Van Baelen, K. Sabbe, C. D’Hallewyn, P. De Clercq, et al., The biological activity of nonsteroidal vitamin D analogs lacking both the C- and D-rings, J. Bone Miner. Res. 13 (1998) 549e558. M. Pera¨kyla¨, M. Malinen, K.H. Herzig, C. Carlberg, Gene regulatory potential of nonsteroidal vitamin D receptor ligands, Mol. Endocrinol 19 (2005) 2060e2073. F. Chen, Q. Su, M. Torrent, N. Wei, N. Peekhaus, D. McMasters, et al., Identification and characterization of a novel nonsecosteroidal vitamin D receptor ligand, Drug. Dev. Res. 68 (2007) 51e60. M. Makishima, T.L. Lu, W. Xie, G.K. Whitfield, H. Domoto, R.M. Evans, et al., Vitamin D receptor as an intestinal bile acid sensor, Science 296 (5571) (2002) 1313e1316. M. Ishizawa, M. Matsunawa, R. Adachi, S. Uno, K. Ikeda, H. Masuno, et al., Lithocholic acid derivatives act as selective vitamin D receptor modulators without inducing hypercalcemia, J. Lipid Res. 49 (4) (2008) 763e772. M.R. Haussler, C.A. Haussler, L. Bartik, G.K. Whitfield, J.C. Hsieh, S. Slater, et al., Vitamin D receptor: molecular signaling and actions of nutritional ligands in disease prevention, Nutr. Rev. 66 (2009) S98eS112. M.T. Mizwicki, D. Menegaz, S. Yaghmaei, H.L. Henry, A.W. Norman, A molecular description of ligand binding to the two overlapping binding pockets of the nuclear vitamin D receptor (VDR): structure-function implications, J. Steroid. Biochem. Mol. Biol. [E-pub] (2010). In press.
C H A P T E R
79 The Bile Acid Derivatives Lithocholic Acid Acetate and Lithocholic Acid Propionate are Functionally Selective Vitamin D Receptor Ligands Makoto Makishima, Sachiko Yamada Nihon University School of Medicine, Tokyo, Japan
THE VITAMIN D RECEPTOR IS A DUAL-FUNCTIONAL RECEPTOR FOR VITAMIN D AND BILE ACIDS Vitamin D plays a regulatory role in numerous physiological processes, including bone and calcium metabolism, cellular growth and differentiation, immunity, and cardiovascular function [1,2]. Vitamin D is a secosteroid, in which the B ring of the canonical steroid structure is ruptured. It is synthesized from 7-dehydrocholesterol, an intermediate metabolite in cholesterol synthesis, or derived directly from dietary sources [3]. Ultraviolet irradiation of sunlight-exposed skin induces a photochemical reaction of 7-dehydrocholesterol to produce the secosteroid vitamin D3 (cholecalciferol) (see Section I of this volume). Vitamin D3 is hydroxylated at the 25-position by the hepatic vitamin-Dhydroxylases, sterol 27-hydroxylase (cytochrome P450 27A1; CYP27A1) and, perhaps more likely, vitamin D 25-hydroxylase (CYP2R1), to yield 25-hydroxyvitamin D3 (25(OH)D3; 25-hydoxycholecalciferol), the major circulating form of vitamin D [4] (see Chapter 3). 25(OH)D3 is further hydroxylated at the 1a-position by 25-hydroxyvitamin D 1a-hydroxylase (CYP27B1). This reaction occurs mainly in the kidney to yield the active metabolite 1,25-dihydroxyvitmain D3 (1,25(OH)2D3; 1,25-dihydroxycholecalciferol or calcitriol) (Fig. 79.1). 1,25(OH)2D3 is also synthesized by CYP27B1 which is expressed in extrarenal tissues such as macrophages, and has been suggested to exert non-calcemic effects
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10079-4
[5] (see Chapter 45). Dietary vitamin D2 (ergocalciferol) and vitamin D3 are metabolized through the same activation process, involving 25-hydroxylation in the liver and subsequent 1a-hydroxylation in the kidney [6]. 1,25(OH)2D2 has a different catabolic pathway than 1,25(OH)2D3 [7], and vitamin D2 is less effective than vitamin D3 in maintaining serum 25(OH)D levels [8]. Vitamin D deficiency, caused primarily by inadequate sun exposure, results in rickets and osteomalacia, and is also associated with increased risk of cancer, autoimmune disease, infection, and cardiovascular disease [2,9] (see Sections X, XI, and XII of this volume). 1,25(OH)2D3 and its synthetic derivatives exert their physiological and pharmacological effects by binding to the vitamin D receptor (VDR; NR1I1), a member of the nuclear hormone receptor superfamily of transcription factors [1,10] (see Chapters 7e10). Forty-eight human nuclear receptors have been identified, including the endocrine receptors for steroid and thyroid hormones, metabolic sensors for fatty acids, bile acids, oxysterols and xenobiotics, and orphan receptors whose natural ligands are unknown [11,12]. Like other nuclear hormone receptors, VDR is activated in a liganddependent manner (see Chapters 7e9). Upon ligand binding, VDR undergoes a conformational change in the cofactor binding site and activation function 2 (AF2) domain, a structural rearrangement that results in the dynamic exchange of cofactor complexes [13]. In the absence of ligand, corepressors bind to the AF2 surface, composed of portions of helix 3, loop 3e4,
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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79. THE BILE ACID DERIVATIVES LITHOCHOLIC ACID ACETATE AND LITHOCHOLIC ACID PROPIONATE
25
O
OH
X12
X24 X23
3 RO
X7 H
X6
Bile acid
FIGURE 79.1
3
Bile acid
HO
2
1,25(OH)2D3
1 OH
X7
X12
R
X6
X23
X24
Cholic acid (CA)
α-OH
OH
H
H
H
OH
Deoxycholic acid (DCA)
H
OH
H
H
H
OH
Chenodeoxycholic acid (CDCA)
α-OH
H
H
H
H
OH
Ursodeoxycholic acid
β-OH
H
H
H
H
OH
Lithocholic acid (LCA)
H
H
H
H
H
OH
Glyco-LCA
H
H
H
H
H
NHCH2COOH
3-Keto-LCA
H
H
C(3)=O
H
H
OH
LCA acetate
H
H
CH3CO
H
H
OH
LCA propionate
H
H
C2H5CO H
H
OH
LCA formate
H
H
HCO
H
H
OH
7ξ-Methyl-LCA
CH3
H
H
H
H
OH
6α-Ethyl-CDCA
α-OH
H
H
C2H5
H
OH
6α-Ethyl-23S-methyl-CA
α-OH
OH
H
C2H5
CH3
OH
Structures of bile acids, bile acid derivatives, and 1,25(OH)2D3.
helices 4/5 and helix 11. Ligand binding alters the AF2 surface by repositioning helix 12, reducing the affinity for corepressors and increasing the affinity for coactivator recruitment, resulting in nuclear-receptor-mediated induction of target gene transcription. Cofactor complexes have been classified into at least three functional categories [14] (see Chapters 10 and 12). Members of the first cofactor complex class regulate transcription directly via interactions with general transcription factors and RNA polymerase II. Members of the second cofactor complex class modify histone tails by acetylation or deacetylation. The third class of complexes facilitates ATP-dependent dynamic chromatin remodeling. Ligand-bound VDR is not only involved in transactivation, but in some contexts can also mediate transrepression [15] (see Chapter 12). A variety of posttranslational modifications of nuclear proteins, such as methylation, phosphorylation, ubiquitination, sumoylation, ADP ribosylation, and glycosylation, also regulate nuclear receptor signaling [16e18]. Dynamic modification is required for the coordinated interaction of VDRecofactor complexes and for efficient regulation of transcription [19]. VDR binds preferentially to a vitamin D response element (VDRE), consisting of a two hexanucleotide
(AGGTCA or a related sequence) direct repeat motif separated by three nucleotides (direct repeat 3; DR3), as a heterodimer with isoforms of the retinoid X receptor (RXR), RXRa (NR2B1), RXRb (NR2B2), and RXRg (NR2B3), the receptors for 9-cis retinoic acid [10,20] (see Section II of this volume). DR3-type VDREs have been identified in the regulatory regions of many target genes, including 25-hydroxyvitamin D 24-hydroxylase (CYP24A1), calbindin D9k (CaBP-9k), transient receptor potential vanilloid type 6 (TRPV6), receptor activator of nuclear receptor kB ligand and cathelicidin antimicrobial peptide (CAMP) [2,21,22]. An everted repeat of the hexanucleotide motif separated by six nucleotides (everted repeat 6; ER6) is an alternately configured VDRE that regulates the human CYP3A4 gene [23]. Several VDREs, including DR4, ER7, ER8, and ER9 motifs, have been identified in p21waf1/cip1, cyclin C, and insulin-like growth-factor-binding protein genes [19,24e26]. Genes involved in inflammation and cell growth, such as interleukin-2, tumor necrosis factor a, and c-Myc, are negatively regulated by VDR activation [2]. Although the potential therapeutic effects of VDR ligands in the treatment of autoimmune diseases and cancer are thought to be based on negative regulation of these genes, underlying molecular mechanisms
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BILE ACIDS AND NUCLEAR RECEPTORS
remain unknown. VDR activation by 1,25(OH)2D3 induces negative feedback regulation of vitamin D signaling by inducing the catabolizing enzyme CYP24A1 and inhibiting the expression of CYP27B1 and parathyroid hormone (PTH) [9,10]. CYP27B1 is a key enzyme in 1,25(OH)2D3 biosynthesis and PTH promotes 1,25(OH)2D3 production by inducing CYP27B1 expression. 1,25(OH)2D3-induced transrepression of CYP27B1 and PTH expression is due to VDRmediated recruitment of a repressor complex to these promoters [14,27]. 1,25(OH)2D3 induces DNA methylation of the CYP27B1 gene promoter, a repressive modification reversed by PTH-induced active demethylation through a mechanism involving protein kinase C activation and a base-excision repair process mediated by a DNA glycosidase [28] (see Chapter 12). Over 2000 vitamin D derivatives have been synthesized and evaluated for potential therapeutic application [29]. Although they have been used successfully in the treatment of bone, mineral and skin disorders, adverse effects, particularly hypercalcemia, limit their clinical application in the management of other diseases, such as cancer, autoimmunity, and infection [30]. Therefore, the development of VDR ligands that do not increase serum calcium levels is required to create novel therapies for VDR-related diseases. Since its initial characterization, VDR has been thought of primarily as a receptor for 1,25(OH)2D3. In a 2002 discovery, VDR was found to act as a receptor for secondary bile acids, including lithocholic acid (LCA) and 3-ketocholanic acid (3-keto-LCA) (Fig. 79.1) [31] (see Chapter 43). VDR induces the expression of CYP3A enzymes that catabolize toxic secondary bile acids [32,33]. Thus, VDR has dual functions as an endocrine receptor for 1,25(OH)2D3 and as a metabolic sensor for bile acids. These findings provide a rationale for the development of novel VDR ligands derived from bile acids.
BILE ACIDS AND NUCLEAR RECEPTORS Bile Acid Metabolism Bile acids are the end products of hepatic cholesterol catabolism, and are essential detergents required for the ingestion and intestinal absorption of hydrophobic nutrients, such as cholesterol, fatty acids, and lipidsoluble vitamins, including vitamin D [34] (see Chapter 43). Primary bile acids, such as cholic acid (CA) and chenodeoxycholic acid (CDCA) (Fig. 79.1), are generated from cholesterol by the sequential actions of liver enzymes and are secreted in bile as glycine or taurine conjugates [35]. After assisting in lipid digestion and absorption, most bile acids are reabsorbed in the
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intestine and recirculate to the liver through the portal vein in a circuit known as the enterohepatic circulation. Bile acids that escape reabsorption are converted to secondary bile acids, such as deoxycholic acid (DCA) and LCA (Fig. 79.1), by the intestinal microflora [36]. A portion of secondary bile acids enter the enterohepatic circulation, where under pathological conditions, they are thought to participate in the pathogenesis of liver disease and colon cancer [37]. The synthesis of bile acids from cholesterol is mediated by at least 17 different enzymes [35]. The first step is initiated by the 7a-hydroxylation of sterol precursors, and is mediated by two metabolic pathways. In the classic pathway, cholesterol is converted to 7a-hydroxycholesterol by cholesterol 7a-hydroxylase (CYP7A1), a microsomal cytochrome P450 enzyme. Under normal conditions, this pathway is responsible for 50% or more of bile acid production in humans. In the second pathway, called the alternative or acidic pathway, cholesterol is converted into one of several oxysterols prior to being 7a-hydroxylated by oxysterol 7a-hydroxylase (CYP7B1). The oxysterol 24-hydroxycholesterol, 25-hydoxycholesterol and 27-hydroxycholesterol can serve as substrates for bile acid synthesis [38]. 27-Hydroxycholesterol, which is the most abundant oxysterol in plasma, is synthesized from cholesterol by a mitochondrial cytochrome P450, CYP27A1. This enzyme can also hydroxylate cholesterol to 24hydroxycholesterol and 25-hydroxycholesterol [35], and vitamin D3 to 25(OH)D3 [4]. A microsomal cytochrome P450, CYP46A1, and a non-P450 microsomal enzyme, cholesterol 25-hydroxylase, participate in the conversion of cholesterol to 24-hydroxycholesterol and 25-hydroxycholesterol, respectively [35]. The preferred substrates for 7a-hydroxylation by CYP7B1 are 25-hydroxycholesterol and 27-hydroxycholesterol. An additional microsomal cytochrome P450, CYP39A1, has oxysterol 7a-hydroxylase activity on the 24hydroxycholesterol substrate. The CYP7B1 oxysterol 7a-hydroxylase pathway likely synthesizes 25 to 30% of total bile acids in mice, and 5 to 10% of the bile acid pool in humans. The initial step of 7a-hydroxylation of sterol precursors is followed by the modification of ring structures. 7a-Hydroxylated intermediates derived from cholesterol and oxysterols are next converted into their 3-oxo,D4 forms by a microsomal 3b-hydroxy-D5-C27steroid oxidoreductase. The resulting products take one of two routes in the subsequent steps of bile acid synthesis. The reaction catalyzed by sterol 12a-hydroxylase (CYP8B1) initiates one cascade that results in the formation of CA. In the absence of CYP8B1, the intermediates are ultimately converted into CDCA (in rat, human, and hamster), muricholic acid (in mouse), ursodeoxycholic acid (in bear) (Fig. 79.1), or
IX. ANALOGS
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hydrodeoxycholic acid (in pig). The products of ring modification next undergo oxidation and side chain shortening. The first few steps in this pathway are performed by CYP27A1, the same enzyme involved in the alternative pathway of bile acid synthesis and in 25hydroxylation of vitamin D. The side chain oxidation steps catalyzed by CYP27A1 are involved in the synthesis of all bile acids, regardless of their source. As a consequence, disruption of the Cyp27a1 gene in mice results in severe derangements in bile acid synthesis [39]. In contrast, vitamin D metabolism is unimpaired in these mice, a finding which led to the discovery of the microsomal vitamin D 25-hydroxylase CYP2R1 [40]. After oxidation by CYP27A1, bile acid intermediates are subjected to side chain shortening and finally to conjugation with an amino acid, usually glycine or taurine [35]. Conjugation increases the amphipathicity of bile acids and enhances their solubility, alterations that make them impermeable to cell membranes. The enterohepatic circulation of bile acids begins in the hepatocyte canaliculus. Canalicular membrane-associated transport systems are required to carry conjugated and some free bile acids across the cell membrane [41]. The bile salt export pump (BSEP; ABCB11), an ATP-binding cassette (ABC) transporter, is localized in the canalicular membrane of hepatocytes and transports monovalent bile salts, such as taurocholate, glycocholate, and taurochenodeoxycholate [42]. The canalicular multidrug resistance-associated protein 2 (MRP2; ABCC2) accounts for the transport of divalent bile salts, such as sulfated tauro- or glycolithocolate, and other amphipathic conjugates including bilirubin diglucuronide. As the major components in bile, bile acids solubilize dietary lipids and promote their absorption in the small intestine. Most conjugated bile acids are reabsorbed in the terminal ileum via an Naþ-dependent mechanism mediated by the apical sodium-dependent bile salt transporter (ASBT; SLC10A2). Bile acids are then shuttled to the basolateral membrane and effluxed into the portal circulation by a heterodimeric transporter, organic solute transporter (OST) a/b [43]. The final step in the enterohepatic circulation, uptake of bile acids by hepatocytes from the portal blood, is mediated by the sodium taurocholate-transporting polypeptide (NTCP; SLC10A1) and organic anion transporting polypeptides (OATPs). After uptake and, if needed, reconjugation, bile acids are transported to the canalicular pole of the hepatocytes to be secreted again into the bile. Several hundred milligrams of bile acids escape the enterohepatic circulation daily and become substrates for commensal bacterial reactions in the colon [36]. Bile acids are metabolized in a variety of ways by intestinal bacteria, including deconjugation, epimerization of hydroxyl groups at C-3, C-7, and C-12, and the
7a-hydroxylation of CA and CDCA, yielding the secondary bile acids DCA and LCA, respectively. Secondary bile acids and metabolites are passively absorbed from the colon and are returned to the liver via the portal vein. DCA, and to a much lesser extent LCA, accumulate in the bile acid pool due to inability of hepatocytes to 7a-hydroxylate them to primary bile acids. DCA increases the hydrophobicity of the bile acid pool, which is associated with greater toxicity and increased cholesterol secretion from the liver. LCA is sulfated in the liver at the 3-hydroxy position, conjugated at C-24, and excreted back into bile. 3-Sulfo-LCA is poorly reabsorbed from the gut. Although 3-sulfoLCA glycine and taurine conjugates are deconjugated and to some extent desulfated by intestinal bacteria, 3sulfo-LCA and LCA are lost in feces and do not normally accumulate in the enterohepatic circulation. Since LCA is very toxic, it must be rapidly eliminated through the feces to reduce its pathogenesis [44]. Bile acids that escape first-pass clearance by the liver or are actively excreted by hepatocytes into sinusoidal blood are filtered at the glomerulus from plasma into urine [42]. Bile acids are reabsorbed by ASBT, localized to the apical membrane of proximal renal tubular cells, and are excreted into the systemic circulation most likely by basolateral OSTa/b. Under cholestatic conditions, hepatocellular efflux from the basolateral membrane and renal excretion are the major alternative elimination route for accumulating bile acids [42]. The basolateral transporters MRP3 (ABCC3), MRP4 (ABCC4), and OSTa/b play roles in the excretion of bile acids from hepatocytes into the systemic circulation. MRP2 and MRP4 are localized to the apical tubular membrane and may stimulate urinary excretion of bile acids.
Regulation of Bile Acid Metabolism by Nuclear Receptors Bile acid metabolism is regulated at several levels, including gene transcription, RNA translation, and protein stability. Bile acids have been identified as regulatory signals for the transcription of genes involved in their synthesis (e.g., CYP7A1, CYP8B1) and transport (e.g., BSEP, NTCP). Nuclear receptors play an important role in the regulatory network by acting as sensors for the bile acid metabolic environment. Liver X receptor a (LXRa; NR1H3), originally identified as an orphan receptor, is chiefly expressed in liver, adipose tissue, intestine, kidney, and macrophages [45,46]. By examining lipid extracts from a variety of tissues, oxysterols were found to be the natural ligands for LXRa, a key discovery in lipid metabolism research [47]. LXRa is activated by oxysterol intermediates in bile acid synthesis, such as 7a-hydroxycholesterol,
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BILE ACIDS AND NUCLEAR RECEPTORS
24-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol, while the most potent oxysterol for LXRa is 24(S),25-epoxycholesterol, which is derived from squalene in a shunt pathway of cholesterol biosynthesis [38,48]. LXRa stimulates the transcription of mouse Cyp7a1, the rate-liming enzyme in the classical bile acid synthetic pathway. LXRa plays a role in the feed-forward induction of bile acid synthesis in rodents. The ABC transporters ABCG5 and ABCG8 mediate biliary excretion of cholesterol and these proteins are also induced by LXRa activation [49]. LXRa heterodimerizes with RXR, and LXRa-RXR functions as a permissive heterodimer, in that the receptor complex can be activated by ligands for either LXRa or RXR [12]. Contrary to the prediction that treatment of mice with an RXR agonist could induce Cyp7a1, its expression was dramatically decreased in both wild-type and LXRa-null mice [50]. This unexpected finding led to the hypothesis that a distinct RXR heterodimeric partner might respond to bile acids, since bile acids have been known to induce feedback regulation in their synthesis by inhibiting Cyp7a1 transcription [35]. Indeed, farnesoid X receptor (FXR; NR1H4) was found to be a bile acid receptor [51]. FXR was originally identified as an orphan receptor that is weakly activated by farnesol, an intermediate in cholesterol synthesis, and juvenile hormone III, an insect hormone [52,53]. FXR is expressed in liver, intestine, kidney, and the adrenal gland, heterodimerizes with RXR, and responds to both primary and secondary bile acids in their free and conjugated forms [51,54,55]. Among the major bile acids, CDCA is the most potent FXR agonist. FXR regulates the synthesis and enterohepatic circulation of bile acids by both direct and indirect mechanisms [56,57]. The nuclear receptors hepatocyte nuclear factor 4a (HNF4a; NR2A1) and liver receptor homolog-1 (LRH-1; NR5A2) are involved in transcription of the bile acid synthetic enzymes CYP7A1 and CYP8B1 [58e60]. FXR represses the expression of CYP7A1 and CYP8B1 by inducing transcription of small heterodimer partner (SHP; NR0B2) in hepatocytes. SHP is an unusual nuclear receptor in that it lacks a DNAbinding domain, and suppresses transactivation of HNF4a and LRH-1 through direct interaction [57]. FXR also represses hepatic CYP7A1 expression via an SHPindependent mechanism. FXR induces the expression of fibroblast growth factor 19 (FGF19) in human hepatocytes and of FGF15 (the mouse ortholog of FGF19) in murine intestine. FGF15/19 represses the expression of CYP7A1 by binding to FGF receptor 4 (FGFR4) in hepatocytes [61,62]. The FGF15/19-FGFR4 signaling pathway also increases stability of SHP protein by inhibiting degradation [63]. Thus, FXR activation suppresses bile acid synthesis through multiple mechanisms. The hepatic bile acid transport system is also regulated by FXR. The basolateral bile salt uptake transporter NTCP
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is negatively regulated by FXR through a SHP-mediated mechanism [64,65]. FXR-null mice exhibit reduced fecal bile acid excretion due to decreased expression of the target gene Bsep, which is involved in canalicular bile salt export in hepatocytes [64]. FXR activation by a synthetic ligand protects hepatocytes from cholestatic liver damage by repressing bile acid synthesis and hepatocellular uptake and stimulating bile acid export from cells [66]. Interestingly, FXR-null mice are protected from obstructive cholestasis [67]. This may be due to decreased BSEP expression and biliary pressure and enhanced elimination of bile acids through a xenobiotic metabolic pathway in FXR-null mice. FXR activation induces the bile acid transporters Osta and Ostb mRNA in intestine as well as in the kidney [68,69]. The ileal bile-binding protein (I-BABP; also called fatty acid binding protein 6) is the first FXR target gene to be identified [51]. I-BABP, an abundant cytosolic protein in the ileal mucosa, is functionally associated with FXR in the nucleus and with ASBT on the membrane, where it stimulates FXR transactivation and ASBT-mediated ileal conjugated bile acid uptake [70]. FXR-null mice demonstrate efficient intestinal bile salt absorption without I-babp expression, suggesting that the physiological role of I-BABP in the enterohepatic circulation is limited. Bile acids have bacteriostatic effects [71], and also protect the intestine from bacterial invasion in an FXR-dependent mechanism [72]. The pregnane X receptor (PXR; NR1I2) is activated by toxic secondary bile acids, including DCA and LCA, and induces their elimination through xenobiotic metabolism pathways [73,74]. PXR, a nuclear receptor that is expressed in the liver and intestine and binds to DNA sequences as a RXR heterodimer, senses numerous structurally diverse drugs and environmental contaminants, induces expression of transporters and a battery of genes encoding phase I and II metabolic enzymes, and plays an important role in the detoxification and clearance of xenobiotics [75]. PXR agonists enhance bile acid detoxification by inducing the import transporter Oatp1a4, the detoxifying enzyme Cyp3a11, and the basolateral export transporter Mrp3, resulting in decreased serum bile acids and increased urinary bile acid excretion [76]. CYP3A4, a human ortholog of Cyp3a11, is involved in the metabolism of 50e60% of pharmaceuticals as well as natural compounds such as steroids and herbal supplements [75]. CYP3A4 metabolizes LCA into 3-keto-LCA (3-dehydro-LCA) by 3-oxidation, hydrodeoxycholic acid by 6ahydroxylation, 1b,3a-dihydroxy-5b-cholanoic acid by 1b-hydroxylation, while it catalyzes the 3-oxidation and 1b-hydroxylation of DCA [33,73]. CYP3A4 can also catalyze 3-oxidation of CDCA, CA, and ursodeoxycholic acid. 3-Keto-LCA is a more potent PXR ligand than LCA, indicating that LCA enhances detoxification
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79. THE BILE ACID DERIVATIVES LITHOCHOLIC ACID ACETATE AND LITHOCHOLIC ACID PROPIONATE
via a PXR-mediated feed-forward mechanism. Sulfation of LCA is mediated primarily by dehydroepiandrosterone sulfotransferase 2 (SULT2), and SULTs catalyze the transfer of a sulfonyl group from the donor molecule (PAPS) to 30 -phosphoadenosine-50 -phosphosulfate hydroxyl or amino groups of lipophilic molecules, forming sulfate or sulfamate conjugates, respectively. The expression of Sult2 and PAPS synthase 2 (Papss2) is induced by PXR [77]. PXR also induces the basolateral bile salt efflux transporter Mrp3 [78]. The constitutive androstane receptor (CAR; NR1I3) is another nuclear receptor that regulates the transcription of genes involved in xenobiotic metabolism and is abundantly expressed in the liver and intestine [79]. CAR and PXR bind to response elements with overlapping specificity, and the two receptors coordinately regulate xenobiotic metabolism [75]. Although there is no evidence to date that endogenous bile acids are ligands for CAR, comparison of Car/, Pxr/ and Pxr/Car-double knockout mice shows that CAR plays a role in the elimination of toxic bile acids through a xenobiotic metabolism pathway [80]. VDR, a receptor for 1,25(OH)2D3, also acts as a bile acid receptor with specificity for LCA and its derivatives. Sequence and structure analyses reveal that VDR is the nuclear receptor most closely related to PXR [81e83] (see Chapters 7 and 8). When activated by LCA, and more effectively by 3-keto-LCA [31], VDR induces the expression of Cyp3a11 in the mouse intestine and CYP3A4 in human intestinal cells [84]. SULT2A and MRP3 are VDR targets in mouse and human cells [85,86]. Epidemiological studies have established that dietary uptake of vitamin D reduces the risk of colon carcinogenesis [87]. In addition to direct VDR effects on oncogenic mechanisms, VDR-mediated detoxification of LCA may contribute to colon cancer prevention by vitamin D. Both VDR and PXR induce expression of multidrug resistance 1 (MDR1; ABCB1) [88e91]. Pglycoprotein, which is encoded by the MDR1 gene, functions as a biological barrier to the systemic exposure of chemical substances by effluxing a broad range of hydrophobic compounds from the intracellular to the extracellular compartment [92]. Thus, the NR1I family of nuclear receptors (PXR, VDR, and CAR) has overlapping functions in xenobiotic metabolism. Although the roles of VDR in calcium and bone homeostasis have been investigated for decades, an understanding of VDR regulation of bile acid metabolism is newly emerging. Whereas administration of high concentrations of LCA restores serum calcium levels to the normal range in vitamin-D-deficient rats by increasing VDR target gene expression and bone calcium mobilization, it does not affect rats with normal vitamin D levels, indicating that LCA can substitute for vitamin D in calcium homeostasis only in vitamin-D-deficient rats [93].
Pharmacological activation of VDR enhances the metabolism of bile acids, particularly urinary excretion, by increasing the expression of bile acid transporter genes in mice [94]. 1,25(OH)2D3 does not induce hepatocyte target gene expression due to low VDR expression [95]. Similarly, VDR activation does not alter bile acid accumulation in bile-duct-ligated mice [96]. Thus, VDR regulates xenobiotic metabolism in the intestine and kidney but does not play a major role in the liver. The VDRs of non-mammalian species, such as lamprey, zebrafish and Xenopus laevis, are insensitive to bile acids and bile alcohols. The ability of VDR to bind bile acids appears, therefore, to be a more recent evolutionary development and may be an adaptation in response to changes in intestinal anatomy and microbial colonization [97]. In addition to nuclear receptors, the G-proteincoupled receptor TGR5 (also known as M-BAR, BG37, or GPBAR1) is activated by bile acids [98,99]. TGR5 is expressed in brown adipose tissue and muscle as well as in enteroendocrine L cells [100]. Stimulation of the TGR5 signaling pathway modulates energy metabolism by controlling the activity of type 2 deiodinase and the subsequent activation of thyroid hormone in brown adipose tissue and muscle [101]. TGR5 signaling also induces intestinal glucagon-like peptide-1 release from enteroendocrine L cells, leading to enhanced glucose tolerance [102]. Bile acids have been known to activate other cell-signaling pathways, including protein kinase C, c-Jun N-terminal kinase and epidermal growth factor receptor, by poorly defined mechanisms [44]. Thus, bile acids function not only as lipid detergents in intestinal digestion but also as signaling molecules. The development of synthetic bile acid derivatives targeting the bile acid receptors should provide effective treatment for a variety of disorders related to bile acids or their receptors.
DEVELOPMENT OF BILE ACID DERIVATIVES Bile Acid Derivatives Acting on FXR and TGR5 FXR regulates lipid and glucose handling as well as bile acid metabolism [57]. Additionally, FXR modulates immunity in the liver and intestine [103,104]. Since the clinical utilization of natural bile acids is limited due to their toxic effects, bile acid derivatives that more potently activate FXR with reduced off-target effects have been developed. In a screen of synthetic bile acid derivatives, 6a-methyl-CDCA was found to be a more potent FXR agonist than CDCA, a finding that led to the discovery of 6a-ethyl-CDCA (Fig. 79.1), the most
IX. ANALOGS
DEVELOPMENT OF BILE ACID DERIVATIVES
potent steroidal FXR agonist reported to date [105]. In the crystal structure, 6a-ethyl-CDCA is bound to the FXR ligand-binding domain (LBD) with ring A directed toward helices 11/12, while the side chain carboxylic acid approaches the rear of the entry pocket [106]. This side chain orientation allows conjugated bile acids to activate FXR. While linear alkyl or alkyl-substituted groups at the 6a-position of CDCA, such as 6a-ethyl, 6a-allyl, or 6a-propargyl group, induce high potency and efficacy, more polar substituents such as the hydroxyl or methoxy group reduce efficacy [107]. Although all natural bile acids contain a 3a-hydroxyl group in their A ring, the 3a-hydroxyl group can be eliminated without reducing FXR activation [106,107]. The introduction of bulky substituents in the 3b position decreases activity [108]. While the 5b-(A/B cis) bile alcohols 5b-cyprinol and bufol are potent FXR agonists, their 5a-(A/B trans) counterparts act as FXR antagonists [109]. Derivatization of the C-24 group by carbamate moieties generates both FXR agonists and antagonists [110]. Docking studies show that the side chain of the derivatives projects toward loop 1e2 without stabilizing a conformational change in helices 3 and 12, suggesting a functional role of loop 1e2 in selective FXR modulation. The membrane bile acid receptor TGR5 is emerging as an attractive drug target in the treatment of obesity, diabetes, and the metabolic syndrome [111]. The synthetic FXR agonist 6a-ethyl-CDCA also activates TGR5 with a TGR5/FXR EC50 ratio ¼ 2.1 [112]. 23(S)-Methyl-CDCA has no FXR activity up to 100 mM at concentrations, but activates TGR5 with an EC50 ¼ 3.58 mM (TGR5/FXR EC50 ratio <0.036). The introduction of the 6a-methyl group and 6a-ethyl group to 23(S)-methyl-CDCA increases the potency of TGR5 activation to EC50 ¼ 0.012 mM and 0.0081 mM, respectively. LCA is the most active natural bile acid ligand of TGR5 and the LCA derivative 7x-methyl-LCA (Fig. 79.1) has been found to be one of the most active and selective TGR5 agonists [113]. Insertion of a fluorine atom in the 7a position of LCA results in slightly augmented TGR5 activity but the ligand selectivity for TGR5 over FXR is significantly enhanced. The introduction of a C23(S)-methyl group in the side chain of CDCA and 6a-ethyl-CDCA increases selectivity for TGR5, and 6a-ethyl-23(S)-methyl-CA (Fig. 79.1) is a potent TGR5 agonist with no FXR activity [114]. Treatment of obese mice with 6a-ethyl-23(S)-methyl-CA has been shown to improve insulin sensitivity [102].
Bile Acid Derivatives that Act on VDR Secondary bile acids, such as LCA and 3-keto-LCA, have been identified as endogenous non-secosteroidal
1515
VDR ligands [31] (see Chapters 8 and 43). Bile acids are the major catabolic detergents that are required for the ingestion and intestinal absorption of hydrophobic nutrients such as cholesterol, triacylglycerol, and lipidsoluble vitamins, including vitamin D. Although the physiological role of VDR in bile acid metabolism is still under investigation, mounting evidence suggests that VDR acts as a bile acid sensor as well as an endocrine receptor for vitamin D signaling [93,94]. Apart from their direct effects on vitamin D absorption, bile acids have not been demonstrated to regulate calcium metabolism. Accordingly, bile-acid-derived VDR ligands may exhibit selective VDR activity without inducing hypercalcemia [30]. In a crystal structure of the VDRe1,25(OH)2D3 complex, Y143 and S278 interact with the 3b-hydroxyl group of 1,25(OH)2D3, S237 and R274 hydrogen bond with the 1a-hydroxyl group, H305 and H397 coordinate the 25-hydroxyl group, and S275, L233, V234, and W286 mediate hydrophobic interactions with 1,25(OH)2D3 (Table 79.1) [82]. Ala scanning mutational analysis of residues of the VDR ligand-binding pocket has demonstrated importance of these residues in VDR transactivation by 1,25(OH)2D3 [115,116]. We carried out ab initio calculations of interaction energies between 1,25(OH)2D3 and residues in the VDR ligand-binding pocket by fragment molecular orbital analysis at the Møller-Plesset second-order perturbation level and provided physicochemical insight into the role of these residues [117,118]. We generated VDR mutants predicted to modulate ligand specificity based on sequence homology to PXR, another bile-acid-responsive nuclear receptor, and found that VDReS278V is activated by 1,25(OH)2D3, but not by LCA, whereas VDReS237M can respond to LCA but not to 1,25(OH)2D3 [81]. LCA binds to VDR with binding modes that differ from the 1,25(OH)2D3eVDR structure [81,115]. We performed Ala scanning mutational analysis to study VDR transactivation by LCA and 3-keto-LCA and modeled these ligands in the VDReLBD using FlexX software [81,115]. The docking models of LCA and 3-keto-LCA reveal that these compounds are more weakly coordinated in the VDR ligand-binding pocket than 1,25(OH)2D3, suggesting that modification of these bile acids could increase the VDR transactivation activity. Esterification of the LCA side chain carboxyl group with methyl, ethyl, and benzyl moieties drastically decreases transactivation, and modification of the 3ahydroxyl group increases VDR activity [84]. LCA formate and LCA acetate (Fig. 79.1) activate VDR with three times and 30 times the potency of LCA, respectively. The VDR Y143A and W286A mutations inhibit activation by LCA, LCA acetate, and 1,25(OH)2D3. The effect of S237A is modest on LCA, LCA acetate, and 1,25(OH)2D3 activity. Whereas the S275A and S278A mutations almost completely abolish the activity of
IX. ANALOGS
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79. THE BILE ACID DERIVATIVES LITHOCHOLIC ACID ACETATE AND LITHOCHOLIC ACID PROPIONATE
TABLE 79.1
1,25(OH)2D3-interacting residues in the VDR ligand-binding pocket and their predicted functions
Residues
Position
Functions
Y143
Helix 1
Ligand-mediated folding of the b-turn side and/or hydrogen bond with 3b-OH
L233
L229
Helix 3
Hydrophobic interaction with ligand
S237
S233
Helix 3
Assistant hydrogen bond with 1a-OH
V234
V230
Helix 3
Hydrophobic interaction with ligand
R274
R270
Helices 4/5
Hydrogen bond with 1a-OH
S275
S271
Helices 4/5
Hydrophobic interaction with ligand
S278
S274
Helices 4/5
Hydrogen bond with 3b-OH
W286
W282
b sheet
Hydrophobic interaction with ligand
H305
H301
Loop 6-7
Assistant hydrogen bond with 25-OH
H397
H393
Helix 11
Hydrogen bond with 25-OH
V418
V414
Helix 12
Ligand-mediated folding of the AF2 surface
F422
F418
Helix 12
Packing of helix 12 against helix 3, helices 3/4, and helix 11 and folding of the AF2 surface
Human
Rat
Y143
LCA, these mutants are still activated by LCA acetate and 1,25(OH)2D3. Although H305A has significant effects on LCA and 1,25(OH)2D3 activity, this mutation had little effect on activation by LCA acetate or 3-ketoLCA [115]. The S237M mutation weakly diminishes LCA acetate and LCA activity, and S278V drastically decreases LCA acetate activity [84]. Structureefunction analysis and docking models show that LCA acetate interacts with the VDR ligand-binding pocket in a mode distinct from 1,25(OH)2D3, particularly in its contacts with helix 3 and helices 4/5 residues, a structural surface that functions in dynamic cofactor protein recruitment [116]. LCA derivatives may induce a VDR conformation distinct from 1,25(OH)2D3 and its analogs and mediate selective physiological function. LCA propionate (Fig. 79.1) is as potent a VDR agonist as LCA acetate [119]. LCA propionate is a weaker FXR agonist than LCA acetate, and LCA acetate and LCA propionate do not activate PXR. While LCA effectively induces TGR5 activation, LCA acetate and LCA propionate have no TGR5 specificity [113,119]. Since LCA propionate activates VDR at concentrations that are not effective on other bile acid receptors (FXR, PXR, and TGR5), this compound is a VDR-selective bile acid derivative. While LCA acetate and LCA propionate induce the expression of TRPV6 as effectively as CYP24A1, 1,25(OH)2D3 more effectively upregulates TRPV6 than CYP24A1 in intestinal cells [119]. Administration of LCA acetate or LCA propionate induces tissue VDR
activation in mice, resulting in kidney Cyp24a1 induction, without causing hypercalcemia. Treatment with 1a-hydroxyvitamin D3 (1a(OH)D3), which is rapidly converted to 1,25(OH)2D3 in vivo [120], increases plasma calcium levels, decreases body weight and induces renal expression of Cyp24a1, CaBP-9k, Trpv6, and Trpv5 genes [119]. Treatment of mice with LCA acetate or LCA propionate induces the expression of Cyp24a1 as effectively as 1a(OH)D3, but these LCA derivatives do not change the plasma calcium level, body weight, or expression of CaBP-9k, Trpv5, and Trpv6. Therefore, LCA acetate and LCA propionate are functionally selective VDR ligands and the underlying molecular mechanisms of this specificity are under active investigation. 1,25(OH)2D3 is known as an inducer of myeloid leukemia differentiation [10] (see Chapter 88). LCA acetate inhibits proliferation of human monoblastic leukemia THP-1 cells more effectively than the natural bile acids LCA and 3-ketoLCA [84]. LCA acetate induces myeloid differentiation makers in THP-1 cells, while LCA and 3-keto-LCA are not able to induce this activity even at concentrations that completely inhibit cell proliferation. LCA acetate also induces the differentiation of other leukemia cell lines, such as HL60, U937, and NB4 cells, along with myeloid leukemia cells freshly isolated from patients [121]. LCA acetate activates mitogen-activated protein kinase before inducing differentiation and the underlying molecular mechanism remains unknown. LCA propionate, like LCA acetate, induces the differentiation of THP-1, U937, and HL60 leukemia cells [119].
IX. ANALOGS
X-RAY CRYSTAL STRUCTURES OF VDR IN COMPLEX WITH LCA DERIVATIVES
Induction of the cathelicidin antimicrobial peptide by VDR activation plays a role in the innate immune response to infections, such as tuberculosis [122] (see Chapters 91 and 93). LCA acetate and LCA propionate increase CAMP transcription in myeloid THP-1, U937, and HL60 cells and immortalized keratinocyte HaCaT cells [119]. Thus, LCA derivatives are VDR ligands that induce the differentiation of myeloid leukemia cells and enhance innate immunity. Like 1,25(OH)2D3, LCA and its derivatives, such as LCA acetate, promote osteoblast maturation, suggesting that LCA and its derivatives act as a 1,25(OH)2D3 surrogate in osteoblast differentiation [123].
X-RAY CRYSTAL STRUCTURES OF VDR IN COMPLEX WITH LCA DERIVATIVES X-ray crystal structures of ternary complexes of rat VDReLBD with LCA and its derivatives (3-keto-LCA, LCA acetate and LCA propionate), and the coactivator DRIP205 peptide have been solved [124]. The results provide insight into the structureefunction relationships of LCA derivatives and VDR.
VDReLBD Complexed with LCA and DRIP205 The rat VDReLBD complexed with LCA adopts a canonical active conformation (Fig. 79.2A). In the crystal structure, LCA is accommodated in the VDR ligand-binding pocket with an orientation opposite to that of 1,25(OH)2D3 in both the horizontal and vertical planes (Figs 79.2A and B). This orientation is the same as that of 6a-ethyl-CDCA docked in the FXR ligandbinding pocket [106]. The side chain carboxyl group is directed toward the b-turn side, the A ring faces helix 12 and the b-face of the steroid is directed toward helix 7 in the bottom of the ligand-binding pocket. As shown in Figure 79.2C, the non-planar cis A/B steroid skeleton fits closely with the curved 9,10-secosteroid framework of 1,25(OH)2D3. While 1,25(OH)2D3 is strongly anchored in the VDR ligand-binding pocket with three pairs of hydrogen bonds at its three hydroxyl groups [82,125], LCA forms only one pair of hydrogen bonds at the carboxyl oxygen (Fig. 79.2B). This carboxyl oxygen occupies the same position as the 3b-oxygen of 1,25(OH)2D3 and forms hydrogen bonds with Y143 ˚ ) and S274 (corresponding to S278 of human (2.46 A ˚ ) (Table 79.1). These two residues are essenVDR) (2.76 A tial for VDR activation by LCA, because Ala mutation of either human VDR Y143 or S278 (corresponding to S274 of rat VDR) abolishes the activity [115,116]. Human VDR
1517
S278 (corresponding to S274 of rat VDR) is particularly important for LCA and its derivatives, while the human VDR S278A mutation does not affect the potency of 1,25(OH)2D3 and its derivatives. Five water molecules are incorporated into the ligand-binding pocket at the b-turn side, a feature known as the water channel (Fig. 79.2D), and one of them is involved in a hydrogen bond network around the 24-carboxyl group (Fig. 79.2B). R270 in helix 5 and S233 in helix 3 form hydrogen bonds with an ordered water molecule, which in turn interacts with the other oxygen of carboxyl group. The hydrogen bond of the 1a-hydroxyl group with the human VDR R274 and S237 residues (corresponding to R270 and S233 of rat VDR, respectively) are essential for VDR activation by 1,25(OH)2D3 and its derivatives (Table 79.1), and elimination of the 1a-hydroxyl group decreases the binding affinity by two orders. By contrast, the interaction of LCA with the R270 and S233 residues occurs via a water molecule. Due to the low occupancy of this water channel, glycine-conjugated LCA (Fig. 79.1) can be readily accommodated. This structure explains why glycoLCA has similar activity to LCA on VDR [31]. The water channel is occupied by substituents at the C-2 position of vitamin D derivatives, which greatly increase VDR transactivation [126,127]. Three water molecules are inserted into the ligand-binding pocket at the helix 12 side (Fig. 79.2D), and the 3a-hydroxyl group of LCA participates in a hydrogen bond network including the water molecules and two His residues, H301 in helix 6 and H393 in helix 11 (Fig. 79.2B). 1,25(OH)2D3 and its potent derivatives make critical interactions with these His residues that place helix 11 in the active AF2 surface conformation [116]. The imidazole ring of H301 makes close van der Waals contacts with the A ring of LCA. Interactions mediated by water molecules seem to be less important in stabilizing the VDReLCA complex. The binding affinity of LCA for VDR is much lower than that of 1,25(OH)2D3 [31]. Since the surface area of ˚ 2) is smaller than that of 1,25(OH)2D3 LCA (371 A 2 ˚ (442 A ), the total interaction energy between the ligand-binding pocket residues and LCA would be expected to be less than that of 1,25(OH)2D3. The VDR ligand-binding pocket accommodating LCA is packed loosely, specifically at the helix 12 side, as shown by the Connolly channel surface of this complex (Fig. 79.2D). LCA is a weak VDR agonist and does not act as an antagonist. LCA induces VDR transactivation with the same efficacy as 1,25(OH)2D3 although its potency is 100e500 times weaker than that of 1,25(OH)2D3 [81]. LCA makes a weak interaction with F418, a residue on helix 12, at ˚ , while the terminal methyl group of a distance of 4.80 A ˚) 1,25(OH)2D3 makes intimate interactions (within 4.3 A with two human VDR residues, F422 and V418 at helix 12 (corresponding to F418 and V414 of rat VDR,
IX. ANALOGS
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79. THE BILE ACID DERIVATIVES LITHOCHOLIC ACID ACETATE AND LITHOCHOLIC ACID PROPIONATE
(A)
Helix 9 Helix 8
Helix 1
Helix 7 Helix 3 Helices 4/5 DRIP205 H393 F418 Helices 10/11
Helix 12
(B)
R270
S233 3.01 H393 2.89 2.59
2.90
2.69
Y143 2.48
3.75 3.86
2.71
3.72 3.53
S274
H301
Crystal structure of the rat VDR ligand-binding domain complexed with LCA and DRIP205. (A) Overall structure of VDReLBD (ribbon) complexed with LCA (atom-type line) and DRIP205 peptide (ribbon). The residues (H393 at helix 11 and F418 at helix 12) forming the Hep bond are shown with stick model (atom-type color) and highlighted with a blue circle. The insert is a close-up view of the Hep ˚ from each carbon of the benzene interaction. An imidazole proton of H393 is placed on the center of the p electron cloud of F418 within 2.9e3.2 A ring. A ribbon model is shown with secondary structure colors (red helix and cyan b-sheet) except for magenta helix 12 (H12) and green DRIP205 peptide. (B) Hydrogen bond network of LCA. LCA and its interacting amino acid residues (stick model, atom-type color) and 1,25(OH)2D3 (blue line with only oxygen shown in blue ball) are overlaid with the superimposed protein. Water molecules incorporated into the ligand-binding pocket are shown as red balls. Hydrogen bonds among LCA, the ligand-binding pocket residues and the water molecules are shown with red dotted lines and hydrophobic interactions with blue dotted lines. (C) Side view of overlaid LCA (stick, atom-type color) and 1,25(OH)2D3 (stick, blue carbon and red oxygen). (D) LCA (CPK model with white carbon and red oxygen) and water (CPK, magenta) molecules in the Connolly channel surface (transparent yellow) of rat VDR-LBD. Please see color plate section.
FIGURE 79.2
IX. ANALOGS
X-RAY CRYSTAL STRUCTURES OF VDR IN COMPLEX WITH LCA DERIVATIVES
1519
imidazole ring of H301 intimately interacts with the A ring of 3-keto-LCA (Fig. 79.3A). Ala mutation of human VDR H305 (corresponding to H301 of rat VDR) increases the transactivation potency of 3-keto-LCA [115,116], likely due to reduced steric congestion between the His and the A ring of the ligand.
VDReLBD Complexed with LCA Acetate/LCA Propionate and DRIP205 LCA acetate and LCA propionate are accommodated in the ligand-binding pocket of VDR with the same orientation as that of LCA (Fig. 79.3B). Interaction modes at the carboxyl group of these LCA derivatives are the same as those of LCA and 3-keto-LCA. LCA acetate and LCA propionate interact with ligandbinding pocket residues at the helix 12 side differently from LCA and 3-keto-LCA. No water molecule is inserted at this position and the acyl oxygen interacts directly with H301 and H393. The alkyl part of the acyl group interacts with residues V414 and F418 at helix 12, stabilizing the AF2 conformation. This structure may contribute to the increased potency of the ligands on VDR transactivation [84,119].
FIGURE 79.2 (continued).
respectively) (Table 79.1). However, the protonep (Hep) interaction between F418 and H393 in helix 11 is structured normally (Fig. 79.2A). This type of Hep (or cationep) interaction works as an AF2 switch in FXR and LXRb [106,128]. In order to form the Hep switch and support AF2 function, H393 in helix 11 and F418 in helix 12 must be appropriately positioned. The Hep interaction is accurately conserved in all the known crystal structures of the complexes of VDReLBD with ligands, including 1,25(OH)2D3 [82,125], superagonists [125,129], and partial agonists [130].
VDReLBD Complexed with 3-keto-LCA and DRIP205 3-Keto-LCA is docked in the ligand-binding pocket of VDR in a manner similar to LCA (Fig. 79.3A). The 24-carboxyl group forms hydrogen bonds with Y143 and S274. These residues are essential for the action of 3-keto-LCA as shown by the Ala scanning mutation study [115,116]. R270 and S233 interact with the carboxyl group via an ordered water molecule as seen in the LCA complex. One water molecule is involved in the hydrogen bond network formed among the 3-ketone and two His residues, H301 and H393. 3-Keto-LCA is a more potent VDR agonist than LCA, most likely because the hydrogen bond network around the 3-oxygen is tighter than in the LCA complex. The
Docking Models of the VDR-LBD Bound to LCA and LCA Derivatives We previously reported docking models of LCA, 3-keto-LCA and LCA acetate in the VDR ligand-binding pocket using the software Flex X [81,84,115]. Flex X successfully suggested the correct docking modes for the complexes of VDR with 3-keto-LCA and LCA acetate [84,115]. For the VDReLCA complex, Flex X proposed two models, one was nearly the same as that found in the X-ray crystal structure and the other has the ligand in a reversed orientation [81,115]. The distinct response of LCA and 3-keto-LCA in Ala scanning mutational analysis support the latter model [115,116]. Ala mutation of human VDR H305 (corresponding to H301 of rat VDR) abolishes the activity of LCA and increases the activity of 3-keto-LCA. We assumed that this difference occurred because these bile acids are accommodated in the ligand-binding pocket with different docking modes, suggesting that 3-keto-LCA is sterically hindered by H305 (human VDR) at the A ring. The former docking model shows that LCA has similar steric congestion with H305 (human VDR) as 3-keto-LCA, although the latter model does not. Although the crystal structures indicate that LCA and 3-keto-LCA bind to VDR in the former mode, LCA in physiological conditions may interact with VDR in the second orientation. Steroid ligands are accommodated in the ligandbinding pocket with their D ring or the side chain
IX. ANALOGS
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79. THE BILE ACID DERIVATIVES LITHOCHOLIC ACID ACETATE AND LITHOCHOLIC ACID PROPIONATE
(A)
R270 S233
H393
2.84
2.97 2.78
2.73 2.59
Y143
2.84 3.41
2.43
3.61 2.69 3.55 S274 H301
(B) Helix 12
R270 V414 F418 3.87 3.51 H393
Y143
S233 2.92
3.96
3.37 2.84
4.78 2.42
3.70 2.92
3.53 3.60 H301
S274
FIGURE 79.3 3-Keto-LCA and LCA acetate docked in the rat VDR ligand-binding pocket and their interacting residues. (A) 3-Keto-LCA and
interacting residues. (B) LCA acetate and interacting residues. The ligands and ligand-binding pocket residues are shown with stick and line models, respectively, both in atom-type color. Hydrogen bonds are shown with red dotted lines and hydrophobic interactions with blue dotted lines. Please see color plate section.
directed toward helix 12 in most nuclear receptors (except for FXR), such as oxysterols in LXRb [128], steroid hormones in estrogen receptor a [131], progesterone receptor [132], glucocorticoid receptor [133], androgen receptor [134], and vitamin D derivatives in VDR [82,125,127,129,130].
PERSPECTIVES Bile is placed at the center of the traditional medical models of many cultures and bile acids are its major
components [135]. An appreciation is emerging that bile acids function as signaling molecules via bile acid receptors, such as VDR, and play a regulatory role in a diverse variety of metabolic processes ranging from lipid homeostasis to glucose metabolism and energy expenditure [136]. Vitamin D is a well-known lipidsoluble vitamin and its active form, 1,25(OH)2D3, is a hormone that regulates bone and calcium homeostasis. An improved understanding of the various physiological and pharmacological properties of 1,25(OH)2D3 indicates that its receptor VDR is a promising drug target in the treatment of cancers, autoimmune diseases,
IX. ANALOGS
REFERENCES
infection and cardiovascular disease, as well as bone and mineral disorders. Thus, VDR has dual functions as an endocrine receptor for 1,25(OH)2D3 and as a metabolic sensor for bile acids, and plays a role in linking bile acid metabolism and vitamin D physiology. LCA derivatives are useful tools in the elucidation of the calcemic and non-calcemic actions of VDR and in the further development of non-calcemic VDR ligands.
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[126]
[127]
[128]
[129]
[130]
[131]
[132] [133]
[134]
[135] [136]
IX. ANALOGS
acetate, a bile acid derivative, and cooperative effects with another differentiation inducer, cotylenin A, Leuk. Res. 32 (2008) 1112e1123. P.T. Liu, S. Stenger, H. Li, L. Wenzel, B.H. Tan, S.R. Krutzik, et al., Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response, Science 311 (2006) 1770e1773. J.P. Mansell, D. Shorez, D. Farrar, M. Nowghani, Lithocholate e a promising non-calcaemic calcitriol surrogate for promoting human osteoblast maturation upon biomaterials, Steroids 74 (2009) 963e970. H. Masuno, D. Morizono, T. Ikura, N. Ito, S. Yamada, H.F. DeLuca, et al., Crystal structure of vitamin D receptor in complex with lithocholic acid derivative. Program of the 13th Workshop on Vitamin D, Victoria, Canada, 2006, p. 144 (Abstract). J.L. Vanhooke, M.M. Benning, C.B. Bauer, J.W. Pike, H.F. DeLuca, Molecular structure of the rat vitamin D receptor ligand binding domain complexed with 2-carbon-substituted vitamin D3 hormone analogues and a LXXLL-containing coactivator peptide, Biochemistry 43 (2004) 4101e4110. N. Saito, S. Honzawa, A. Kittaka, Recent results on A-ring modification of 1a,25-dihydroxyvitamin D3: design and synthesis of VDR-agonists and antagonists with high biological activity, Curr. Top. Med. Chem. 6 (2006) 1273e1288. S. Hourai, T. Fujishima, A. Kittaka, Y. Suhara, H. Takayama, N. Rochel, et al., Probing a water channel near the A-ring of receptor-bound 1a,25-dihydroxyvitamin D3 with selected 2a-substituted analogues, J. Med. Chem. 49 (2006) 5199e5205. S. Williams, R.K. Bledsoe, J.L. Collins, S. Boggs, M.H. Lambert, A.B. Miller, et al., X-ray crystal structure of the liver X receptor b ligand binding domain: regulation by histidine-tryptophan switch, J. Biol. Chem. 278 (2003) 27138e27143. G. Tocchini-Valentini, N. Rochel, J.M. Wurtz, D. Moras, Crystal structures of the vitamin D nuclear receptor liganded with the vitamin D side chain analogues calcipotriol and seocalcitol, receptor agonists of clinical importance. Insights into a structural basis for the switching of calcipotriol to a receptor antagonist by further side chain modification, J. Med. Chem. 47 (2004) 1956e1961. M. Nakabayashi, S. Yamada, N. Yoshimoto, T. Tanaka, M. Igarashi, T. Ikura, et al., Crystal structures of rat vitamin D receptor bound to adamantyl vitamin D analogs: structural basis for vitamin D receptor antagonism and partial agonism, J. Med. Chem. 51 (2008) 5320e5329. A.M. Brzozowski, A.C.W. Pike, Z. Dauter, R.E. Hubbard, T. Bonn, O. Engstrom, et al., Molecular basis of agonism and antagonism in the oestrogen receptor, Nature 389 (1997) 753e758. S.P. Williams, P.B. Sigler, Atomic structure of progesterone complexed with its receptor, Nature 393 (1998) 392e396. R.K. Bledsoe, V.G. Montana, T.B. Stanley, C.J. Delves, C.J. Apolito, D.D. McKee, et al., Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition, Cell 110 (2002) 93e105. X.E. Zhou, K.M. Suino-Powell, J. Li, Y. He, J.P. MacKeigan, K. Melcher, et al., Identification of SRC3/AIB1 as a preferred coactivator for hormone-activated androgen receptor, J. Biol. Chem. 285, 9161e9171. D.D. Moore, Does loss of bile acid homeostasis make mice melancholy? J. Clin. Invest. 110 (2002) 1067e1069. W.C. Duane, Bile acids: developments new and very old, J. Lipid. Res. 50 (2009) 1507e1508.
C H A P T E R
80 CYP24A1 Regulation in Health and Disease Martin Petkovich 1, 2, Christian Helvig 1, Tina Epps 1 1 2
Cytochroma Inc., Markham, Ontario, Canada Queen’s University, Kingston, Ontario, Canada
INTRODUCTION Vitamin D is best known for its essential role in regulating bone and mineral homeostasis; however, a growing body of evidence indicates that a number of important physiological processes require adequate vitamin D status. Such requirements are best revealed through the spectrum of disorders associated with vitamin D deficiency, including autoimmune disorders, infectious diseases, cancer, cardiovascular disease, as well as some neurological disorders. Vitamin D deficiency can arise primarily through reduced sun exposure along with lack of adequate nutritional supplementation. More recently, it has become apparent that vitamin D deficiency may also arise from certain diseases, including diabetes, cancer, chronic kidney disease (CKD), and genetically linked hypophosphatemia [1]. Although there may be several root causes for the deficiency associated with these diseases, abnormally elevated levels of 24-hydroxylase (CYP24A1), a cytochrome P450 enzyme uniquely responsible for the catabolism of vitamin D, has been observed in various types of cancer, including breast, prostate, esophageal, colon, and lung [2], genetically linked hypophosphatemia [3,4], and more recently, in diabetic nephropathy [5,6] and CKD [7,8]. For such diseases, blocking CYP24A1 activity may be a viable therapeutic strategy to minimize target tissue resistance and limit vitamin D depletion. Understanding the mechanisms giving rise to elevated CYP24A1 and related pathophysiology will also enable the development of agents which can selectively modify vitamin D metabolism.
CYP24A1: PROPERTIES, FUNCTION AND EXPRESSION Whether vitamin D is produced in the skin following UV exposure or obtained from diet, the production and
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10080-0
regulation of vitamin D hormone are tightly controlled by a number of cytochrome P450 enzymes (Fig. 80.1). The first step in this process occurs in the liver where vitamin D3 is hydroxylated at the carbon-25 position. This step is effected primarily by the vitamin D-25hydroxylase (CYP27A1) and possibly by other nonspecific cytochrome P450s including CYP2R1 [9,10] to form the prohormone, 25-hydroxyvitamin D3 (25(OH)D3), the major systemic form of the vitamin. Circulating 25(OH)D3, delivered to tissues by vitaminD-binding proteins (DBP), is converted to the biologically active hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) by 1a-hydroxylase (CYP27B1). This enzyme is highly expressed in the kidney [11,12], but is also found in many other tissues permitting local generation of hormone [13e15]. Both local and systemic levels of 1,25(OH)2D3, as well as 25(OH)D3, are tightly controlled by the CYP24A1 enzyme encoded by the CYP24A1 gene. CYP24A1 is the primary catabolic enzyme responsible for the conversion of 25(OH)D3 and 1,25(OH)2D3 to the less active metabolites 24,25dihydroxyvitamin D3 (24,25(OH)2D3) [16e18] and 1,24,25-trihydroxyvitamin D3 (1,24,25(OH)3D3) [19]. The regulation of CYP24A1 expression and enzyme activity in various tissues plays an important role in preventing excess vitamin D hormone exposure; however, in certain disease states, dysregulated CYP24A1 expression can have a significant impact on vitamin D status and responsiveness. A comprehensive understanding of the structure and function of CYP24A1 and its regulation in health and disease will enable the rational development of therapeutic strategies to block CYP24A1 activity.
CYP24A1 Structure Knutson and DeLuca first established that the activity responsible for carbon-24 hydroxylation of vitamin D metabolites was a mitochondrial enzyme requiring
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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80. CYP24A1 REGULATION IN HEALTH AND DISEASE
UV
Provitamin D3
Skin
Previtamin D3
Isomerization
Dietary Sources Vitamin D3 Milk Eggs Fish Supplement
Vitamin D3
CYP27A1 Liver
25(OH)D3
Extra-Renal Tissue: Prostate Breast Colon Bone
Local 1,25(OH)2D3
CYP27B1
CYP24 Kidney 24,25(OH)2D3
Regulation of Cellular Processes
CYP24 1,24,25(OH)2D3
1,25(OH)2D3
Excretion
CYP24
Systemic Increase Intestinal Absorption of Ca2+ and Pi
FIGURE 80.1 Vitamin D signaling pathway. Schematic representation of the systemic and localized biosynthesis and catabolism of 25(OH)D3 and 1,25(OH)2D3.
NADPH and molecular oxygen [18]. This finding indicated that, like all mitochondrial P450 enzymes, the function of 24-hydroxylase requires additional electron transport proteins to enable the catalysis of monooxidation reactions from NADPH via an electron transfer system consisting of the soluble matrix proteins adrenodoxin and NADPH-adrenodoxin reductase that binds NADPH [20]. This differs from microsomal P450s which require only a single membrane-bound protein, NADPH-P450 reductase, to receive electrons from NADPH [20]. Primary DNA sequence data initially derived from rat [21,22], and later from human [23,24], mouse [25], chicken [26] and pig [27], provided structural evidence supporting Knutson’s findings. Sequence analysis indicated functionally conserved domains characteristic of mitochondrial P450 enzymes, including the heme-binding site, the mitochondrial-targeting sequence at the N-terminus, as well as amino acids necessary for interaction with the electron-transferring machinery [21,24,28,29]. While the protein size of the 24-hydroxylase (~53 kDa) is consistent with mitochondrial P450s, the derived sequence exhibited less than 40% similarity to other known cytochrome P450s, leading to the identification of a new mitochondrial P450 family in which the newly abbreviated CYP24A1 was assigned [21]. CYP24A1 is responsible for the multiple step side chain oxidation of 25(OH)D3 and 1,25(OH)2D3 to inactive metabolites via C24- or C23-hydroxylation pathways. Affinity of the CYP24A1 enzyme for the
prohormone 25(OH)D3 is generally observed to be lower (Km ~0.5e3.0 mM) than that for the active hormone (Km ~0.02e0.25 mM), indicating an enzyme preference for 1,25(OH)2D3 [30e33]. However, evidence suggests that both 25(OH)D3 and 1,25(OH)2D3 are metabolized at a similar rate by the catabolic enzyme CYP24A1 in vitro [34]. The C24 pathway begins with 24-hydroxylation of 25(OH)D3 and 1,25(OH)2D3 to the initial metabolites 24,25(OH)2D3 and 1,24,25(OH)3D3, respectively. This is followed by oxidation to C24-oxo and 23-hydroxylation to C23-OH/C24 oxo, side chain cleavage to a 23-alcohol product and oxidation to C-23 carboxylic acid, leading to the end product calcitroic acid which is ultimately excreted by the kidney [35]. Conversely, the C23 pathway involves initial hydroxylation at C23, followed by 26-hydroxylation, leading to the formation of a 26,23lactol and the final 1,25(OH)2D3-26-23-lactone product [33,36e38]. C23- or C24-hydroxylase activity can be preferentially effected by the CYP24A1 enzyme depending on the species-specific isoform involved. For example, in humans [33,38] and the guinea pig [37], the C23pathway is predominantly expressed over the C24pathway, whereas rat [39] and mouse [40] express primarily C24-hydroxylase activity with almost no detectable C23-oxidation. Similar to endogenous substrates 25(OH)D3 and 1,25(OH)2D3, differences between rats and humans in the CYP24A1-dependent metabolism of various synthetic vitamin D compounds have also been reported [41e44]. Using the recombinant
IX. ANALOGS
CYP24A1: PROPERTIES, FUNCTION AND EXPRESSION
E. coli system expressing either human or rat CYP24A1, metabolism of various vitamin D analogs yielded species differences in the number and type of metabolites generated [43,44], the degree of analog inactivation [41], as well as differences in the ratio between C23- and C24-oxidation pathways [42]. Species differences in the amino acid residues at positions 416 and 500 in the substrate binding and catalytic site of CYP24A1 may influence substrate alignment and promote C23- or C24-hydroxylation [45]. Metabolites derived from CYP24A1-mediated catabolism of 25(OH)D3 and 1,25(OH)2D3 are generally thought to be inactive intermediates of vitamin D degradation and thus serve no physiological function. Although biological relevance has not yet been convincingly demonstrated for any of these intermediates, there is some in vivo and in vitro evidence indicating unique activity for the most abundant metabolite of 25(OH)D3, 24,25(OH)2D3, in bone physiology, including growth plate chondrocyte differentiation [46e48], bone fracture repair [49,50], and osteogenesis [51e53]. Regulation of growth plate chrondrocytes by 24,25(OH)2D3 may involve protein kinase C (PKC)-activated mitogen-activated protein kinases (MAPK) (i.e., MAP kinase kinase and extracellular signal-regulated kinases (ERK) 1/2) pathways through a non-1,25(OH)2D3-nuclear vitamin D receptor (VDR) [48,54e57]. A similar pathway has also been reported to contribute to 24,25(OH)2D3induced differentiation of osteoblasts in vitro [58]. Although a role for 24,25(OH)2D3 in cartilage maturation has been suggested, mutant mice having a deletion of the CYP24A1 gene exhibit normal growth plate development indicating that the CYP24A1-induced metabolite 24,25(OH)2D3 may not be essential for chondrocyte maturation [59]. However, CYP24A1-null mice also display a delay in the mineralization of cartilaginous tissue of the soft callus and aberrant expression of chondrocyte marker genes [60]. Treatment with 24,25(OH)2D3 effectively rescued gene expression and delay in mineralization, suggesting a possible role for 24,25(OH)2D3 in fracture healing [60]. While the role of 24,25(OH)2D3 in bone physiology remains controversial, establishing whether CYP24A1 has an anabolic role, in addition to its catabolic one, is an important consideration for understanding the physiological consequences of CYP24A1 over expression in disease, as well as the expected outcome of therapeutic CYP24A1 inhibition. CYP24A1 has been classified as a mitochondrial protein belonging to Class I of the P450 superfamily associated with the biosynthesis of steroid hormones and vitamin D3 [23,61,62]. This family also includes genes encoding the CYP27 subfamily A1 and B1 enzymes important in metabolic conversion of vitamin D3 to 25(OH)D3 and 1,25(OH)2D3, respectively. Overall sequence identity of CYP24A1 to that of other
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vitamin-D-related enzymes is approximately 30% [21,63e66], while the heme- and substrate-binding domains for the CYP24A1 and CYP27B1 enzymes have been shown to have a high sequence homology [66]. The key to the development of specific CYP24A1 inhibitors is to identify compounds which do not also inhibit those cytochrome P450 enzymes that bear close structural and/or functional similarity. Unlike xenobiotic metabolizing cytochrome P450s, whose substratebinding pockets can accommodate a wide range of unrelated substrates, the vitamin-D-metabolizing enzymes are highly specific for their respective vitamin D metabolites, thus facilitating the development of specific inhibitors to CYP24A1. Identification of putative residues involved in the binding and catalytic activity of CYP24A1 is of fundamental importance for understanding the structuree function of CYP24A1 necessary for the rational design of biologically efficacious vitamin D analogs and selective CYP24A1 inhibitors for the treatment of diseases characterized by vitamin D insufficiency. Cytochrome P450s are hemoproteins with well-conserved a helices (termed A-L) and b-sheet structures. The protein core surrounding the heme region that includes the hemebinding loop, helix K Glu-X-X-Arg motif and helix I proton transfer groove consensus sequence has the highest structural conservation among all P450 proteins [67,68]. Substrate recognition sites are among the most variable regions associated with helices A (N-terminus), B and B0 , F (C-terminus) and G, as well as their adjacent loops [67]. Characterization of the substrate binding and active sites (heme-center) of CYP24A1 has largely depended on homology modeling derived from crystallized microsomal CYP structures (i.e., CYP3A4) [69,70] and mutational assays. Using a homology model, Gomma et al. found that the heme center of human CYP24A1 contains several conserved key residues (Leu-148, Glu-322, Ala-326, Thr-330, Val-391) similar to the three template models CYP3A4, CYP2C8, and CYP2C9 [71]. Of these models, however, only CYP24A1 demonstrated an ability to accommodate its endogenous substrate 1,25(OH)2D3 at the heme-center ˚ [71]. Mutational assays found at a distance of 6.1 A that active-site mutations in the F-helix (C-terminus) residue Phe-249, the b3a strand residue Val-391, and the b5 hairpin residue Ile-500 significantly lowered substrate binding affinity and recognition, as well as impaired the hydroxylation of 1,25(OH)2D3 to inactive metabolites [72,73]. Additional mutations in conserved residues in the B0 helix/B0 -C loop (Ile-131, Trp-134, and Leu-148), F helix (Met-246), and b5 hairpin (Gly-499) comprising the substrate recognition site have also been found to markedly impair catalytic activity of the CYP24A1 enzyme towards its endogenous substrate 1,25(OH)2D3 [74].
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However, information derived from homology modeling of CYP24A1 based on mammalian microsomal structures cannot accurately account for membrane insertion, substrate binding, and catalytic activity of the CYP24A1 enzyme, which can only be fully achieved with the crystallization of CYP24A1 or other vitamin-D-related mitochondrial CYP enzymes. This issue was resolved earlier this year by Annalora et al. with the first successful crystallization of the CYP24A1 enzyme in the open conformation [75]. The canonical fold of the rat CYP24A1 structure is similar to that of other P450s, consisting of 12 a-helices, four b-sheets, along with the conserved heme-binding domain. The substrate access channel, referred to as pathway 2a (pw2a), is comprised of a cluster of aromatic residues highly conserved across all mitochondrial P450s. The aromatic cluster may contribute to the stability of this region. The substrate-binding cavity of CYP24A1 is most similar to CYP3A4 [70], particularly the wider substrate-binding cavity in close proximity to the catalytic center and a larger surface exposure of the heme domain. Association of CYP24A1 with the lipid bilayer is achieved through hydrophobic membrane insertion sequences (MIS-1 and MIS-2), along with conserved residues surrounding helices A0 and G0 . The crystal structural determination of CYP24A1 will permit elucidation of mechanism of action of existing inhibitors and enable the rational design of novel CYP24A1 inhibitors.
Function of CYP24A1 Establishing the function(s) of CYP24A1 is essential for understanding the potential benefit and consequences of therapeutic inhibition of CYP24A1. The principal and most well-established physiological function of CYP24A1 is to limit tissue exposure to 1,25(OH)2D3. Accordingly, CYP24A1 expression rapidly changes in response to rising or falling vitamin D hormone levels. The CYP24A1 gene promoter contains vitamin D response elements (VDRE) which respond strongly and rapidly once 1,25(OH)2D3 levels reach a certain threshold (see “Regulation of CYP24A1 mRNA expression and stability,” below) [76,77]. This forms an effective negative autoregulatory feedback loop which can rapidly neutralize excess vitamin D hormone. Conversely, when vitamin D status is low, as in a state of deficiency, CYP24A1 levels drop dramatically to preserve any residual hormone produced. Since CYP24A1 also acts on 25(OH)D3, this feedback loop also limits availability of substrate for CYP27B1, thereby reducing 1,25(OH)2D3 production. The physiological importance of CYP24A1 in regulating vitamin D homeostasis is clearly demonstrated in CYP24A1-null mice in which high circulating levels of both 1,25(OH)2D3 and
25(OH)D3 are observed, accompanied by overt signs of vitamin D hypersensitivity and toxicity [60,78]. On the other hand, transgenic rats that constitutively express the CYP24A1 gene display lower levels of circulating 25(OH)D3 and the metabolite 24,25(OH)2D3, thus confirming the importance of CYP24A1 in vitamin D homeostasis [79,80]. Recently, CYP24A1 splice variants (CYP24A1-SV) have been identified in human kidney, placenta, keratinocytes, macrophages, male reproductive tract, and colon tissue [81e83]. CYP24A1-SVs have also been characterized in cancer cell lines of human myelomonocytic leukemia [81], prostate [84], and colon adenocarcinoma [82], as well as in colorectal tumors obtained from patients [82]. Two (SV-1 and SV-2) of the identified CYP24A1-SV proteins have lost their mitochondrial-targeting sequence and likely localized exomitochondrial [81,82], whereas the third CYP24A1-SV (SV-3) lacks the heme-binding domain [82]. At present, it is not clear what physiological consequences follow from high-level expression of these variants. A fourth CYP24A1 splice variant in which introns 9 and 10 are not spliced has been reported in prostate cancer cells lines [84], and more recently, ovary, epididymis, and the seminal vesicle [83]; however, the functional significance of this variant is also unknown.
CYP24A1 Expression Profile The CYP24A1 enzyme is expressed in essentially all vitamin D target tissues. Constitutive expression of CYP24A1 is generally low in vitamin D target tissues, in particular in a state of vitamin D insufficiency, but undergoes a large and rapid induction in response to 1,25(OH)2D3. While the most abundant expression of induced CYP24A1 is localized to the proximal and distal tubules of the kidney [85,86], the primary endocrine organ responsible for the production of systemic 1,25(OH)2D3, CYP24A1 has also been detected in a variety of extra-renal tissues that include the small intestine, skin, heart, lung, brain, placenta, testis, and bone, among others [87e89]. Induced expression of CYP24A1 is primarily a 1,25(OH)2D3-VDR-dependent process. Expression of CYP24A1 is linked to cellular and systemic levels of 1,25(OH)2D3, as animals deficient in vitamin D do not express this enzyme, whereas treatment with 1,25(OH)2D3 potentiates enzyme levels. Lower levels of CYP24A1 metabolites were previously reported in mice lacking the VDR, suggesting that CYP24A1 expression is dependent, at least to some extent, on VDR-mediated regulation [90]. Expression levels of CYP24A1 in rat kidney are developmentally regulated such that levels are low at birth and continue to decline for about 2 months [91,92]. Following this period, renal CYP24A1 levels rise
IX. ANALOGS
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REGULATION OF CYP24A1 mRNA EXPRESSION AND STABILITY
substantially, peaking at approximately 1 to 2 years of age [92e94]. A similar expression pattern has been noted for the VDR in the maturing kidney, suggesting that developmental CYP24A1 expression may be 1,25(OH)2D3-VDR-mediated [92,94,95]. In contrast to both CYP24A1 and the VDR, CYP27B1 levels peak within the first month and decline thereafter [92]. Developmental changes in the expression patterns of CYP24A1 and CYP27B1 are likely associated with an age-dependent decline in serum levels of 1,25(OH)2D3 in rats [92,94,96] and humans [93,97], particularly in those over the age of 65 [98,99]. Regulation of CYP24A1 and association with 1,25(OH)2D3 in the maturing bone is distinct from that observed in kidney. While the age-dependent expression of CYP27B1 mRNA resembles that of the kidney, CYP24A1 and VDR mRNA in bone peaks in young animals (between 3 and 15 weeks) and declines thereafter [92]. In contrast to the inverse relationship found in the kidney, bone CYP24A1 exhibits a strong positive correlation with CYP27B1 [92]. This finding indicates that vitamin D metabolism is differentially regulated in kidney compared to bone. Moreover, the expression of CYP24A1 in bone tissue is independent of circulating levels of 1,25(OH)2D3, indicating that the local production of 1,25(OH)2D3 by CYP27B1 in bone cells [100,101] may determine CYP24A1 expression levels rather than systemic 1,25(OH)2D3 derived from the kidney.
have been identified on the CYP24A1 promoter (Fig. 80.2). CYP24A1 is dysregulated in a number of different diseases. However, the mechanisms underlying disease-related expression of CYP24A1 are varied and may involve transcriptional, post-transcriptional, and in the case of certain cancers, may involve gene amplification.
Transcription Factor Binding Sites on the CYP24A1 Promoter VDREs: VDR and Vitamin D Hormones Vitamin-D-related compounds are used in the treatment of a number of diseases. Unfortunately, the use of these active forms of vitamin D can result in the induction of CYP24A1, resulting in the depletion of vitamin D stores. CYP24A1 expression is most highly induced by 1,25(OH)2D3 and related analogs, which can significantly increase gene expression on average from 10- to 100-fold above basal levels in most cell types [24,76,102], and approximately 20 000-fold in human skin-derived fibroblasts [103]. Although the prohormone 25(OH)D3 has a lower affinity for the VDR compared to 1,25(OH)2D3, physiological concentrations of 25(OH)D3 may significantly enhance CYP24A1 transcription and induce biological effects in vitro [104e107]. This effect of 25(OH)D3 is not dependent on CYP27B1 and may involve direct activation of the VDR [107]. Strong transcriptional upregulation of CYP24A1 is dependent on binding of the 1,25(OH)2D3/ VDR/retinoid X receptor (RXR) complex to two functional VDREs, the proximal VDRE-1 at 136/150 and the distal VDRE-2 at 244/258, in the rat CYP24A1 gene promoter [102,108,109]. Similar VDREs located within 293 bp from the transcription start site were also reported for the human CYP24A1 promoter [76].
REGULATION OF CYP24A1 mRNA EXPRESSION AND STABILITY Various functional transcription-binding sites and corresponding signaling pathways that underlie both the constitutive and induced expression of CYP24A1
1,25D3
1,25D3
1,25D3+PMA 1,25D3
MAPK or c-jun
PI3+PKC
Ets-1
Sp-1
EBS
GC
CCAAT
Sp-1
NF-Y
JNK C/EBβ
C/EBP
VDRE-2 VDR RXR
PKA
GR
VSE
VDRE-1
CYP24A1
VDR RXR SXR
Retinoic Acid PTH
c-fos Glucocorticoids
PKA & PKC Anti-Epileptics Antimicrobial
Calcitonin
FIGURE 80.2 Proximal CYP24A1 promoter elements. Schematic of the relative locations of VDRE-1, VDRE-2, VSE, EBS, GC, CCAAT, C/EBP, and coding region on the proximal promoter of CYP24A1. Molecular pathways and corresponding binding sites involved in the regulation of CYP24A1 are illustrated.
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80. CYP24A1 REGULATION IN HEALTH AND DISEASE
Despite a greater affinity of VDRE-2 for the VDR/RXR heterodimer, VDRE-1 is a stronger contributor to 1,25(OH)2D3-inductive response in various cell types [77,110]. Transcriptional synergism has been demonstrated between the VDREs at high concentrations of 1,25(OH)2D3 [77] which may be important to ensure maximal induction of CYP24A1 and rapid inactivation of 1,25(OH)2D3 to prevent vitamin D toxicity. The proximal VDRE-1 is preferentially employed at lower concentrations of 1,25(OH)2D3 [77]. Furthermore, Tashiro et al. reported that a distal upstream sequence of the human CYP24A1 promoter between nucleotides 548 and 294, which contains three potential Sp-1 sites, acts synergistically with the nearby VDRE-1 and VDRE-2 and may be necessary for maximal induction of CYP24A1 by 1,25(OH)2D3 [111]. In addition to the VDRE-mediated induction of CYP24A1 activity, other signaling pathways appear to have influence on vitamin D metabolism, some of which are most affected in disease states, such as cancer and diabetes. Several DNA motifs which are implicated in integrating signals from these other pathways are present in the promoter of the CYP24A1 gene and have been evaluated for their influence on CYP24A1 gene expression. Ets-1 Binding Site (EBS) The transcription factor Ets-1 has been identified to potentiate 1,25(OH)2D3-mediated induction of CYP24A1 via binding to the Ets-binding site (EBS; 128/119) located downstream of the proximal VDRE-1 [112]. Transcriptional synergism between EBS and VDRE-1, as well as a direct proteineprotein interaction in vivo between Ets-1 and the VDR suggests that EBS is localized close to the proximal VDRE-1 and therefore may likely interact with the 1,25(OH)2D3/VDR/RXR complex bound to VDRE-1 to upregulate CYP24A1 [112,113]. The EBS site is not involved in regulating the basal expression of the CYP24A1 promoter [112]. MAPK KINASE PATHWAYS
In COS-1 monkey kidney fibroblast cells, rapid activation of the Ras-MAPK-ERK5 pathway by 1,25(OH)2D3 enhances CYP24A1 promoter activation via phosphorylation/activation of Ets-1/EBS [112,113]. In addition to ERK5, activation of the MAPK ERK1/2 pathway by 1,25(OH)2D3, in conjunction with ERK2 phosphorylation of RXRa, is also critical for the induction of the CYP24A1 promoter by the active hormone in COS-1 cells [113]. In HEK293 cells, however, c-Jun N-terminal kinase (JNK) is necessary for activation of CYP24A1 by 1,25(OH)2D3 and not ERK1/2 activity, indicating that the CYP24A1 promoter may show cell-type specificity [114]. Involvement of specific MAPK-ERK signaling pathways in 1,25(OH)2D3-mediated activation of the CYP24A1 promoter may also be specific to the stage of
cell differentiation. Cui et al. reported that ERK1/2 signaling, but not ERK5, primarily regulates 1,25(OH)2D3-induced CYP24A1 mRNA expression in differentiated Caco-2 cells [115]. This contrasts with proliferating Caco-2 cells in which ERK5-mediated phosphorylation of Ets-1/EBS mainly potentiates 1,25(OH)2D3-mediated CYP24A1 gene transcription [115]. It will be interesting to establish the signaling pathways that utilize the interaction between the VDR and Ets-1 binding to modify vitamin D metabolism in disease states. One possibility is that fibroblast growth factor 23 (FGF23) may facilitate MAPK phosphorylation of Ets-1/EBS signaling to enhance VDR-mediated regulation of CYP24A1 in disease states characterized by aberrant FGF23 expression, such as X-linked hypophosphatemia (XLH) [116,117]. Vitamin D Stimulatory Element (VSE) PMA-PKC PATHWAY
The phorbol ester PMA via a PKC-dependent mechanism acts synergistically with 1,25(OH)2D3 to upregulate CYP24A1 gene transcription in rat renal and intestinal cells [114,118e122]. Nutchey et al. reported that the synergistic induction of CYP24A1 by combined 1,25(OH)2D3 and PMA was shown to be dependent on both the EBS and a newly identified site containing the sequence 5’-TGTCGGTCA-3’ located approximately 30 bp upstream of VDRE-1 at nucleotides 171/163 in the rat CYP24A1 proximal promoter [114]. The critical role of this site in 1,25(OH)2D3-mediated induction of CYP24A1, as well as the synergistic induction with PMA, lead to its identification as the vitamin D stimulating element (VSE). Similar to EBS, VSE does not contribute to the basal expression of the CYP24A1 promoter [114]. In HEK-293T cells, PMA enhancement of 1,25(OH)2D3-induced upregulation of the rat CYP24A1 promoter involves the activation of the MAPK ERK 1/2, as well as JNK. Synergistic contribution of JNK is partially mediated by the VSE [114]. A conserved VSE sequence of six nucleotides (5’-CGGTCA) between VDRE-1 and VDRE-2 was recently identified in the human CYP24A1 promoter [123]. Despite the presence of relevant transcription factors in human (HEK293T) nuclear extracts which were capable of binding to the rat VSE, the VSE motif in the human CYP24A1 promoter is non-functional and not involved in CYP24A1 activation by 1,25(OH)2D3 [123]. The authors suggest that the first three nucleotides (5’-TGT), which are highly conserved among murine species but not in human (5’-CCC), are necessary for 1,25(OH)2D3-induction of CYP24A1 [123]. GC and CCAAT Box While the VSE and the EBS are mainly involved in 1,25(OH)2D3 induction of CYP24A1, basal expression
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of the CYP24A1 promoter is regulated by an inverted CCAAT box at 62/51 and a GC box at 114/101 located downstream of the EBS in vitro [124,125]. The ubiquitous transcription factors Sp-1 and NF-Y acting through the GC and CCAAT box, respectively, contribute to the activation of basal transcriptional activity of the CYP24A1 promoter [124,125]. Molecular components involved in Sp-1/GC and NF-Y/CCAAT regulation of CYP24A1 basal expression are presently not known; however, recent evidence indicates that the PI-3-kinase-PKCx module is not required [125]. Recently, Dwivedi et al. have demonstrated that Sp-1 acting through the GC box is critical not only for regulating the basal expression of CYP24A1, but is also involved in 1,25(OH)2D3-mediated induction of the CYP24A1 promoter in HEK293 cells [125]. In this cell line, 1,25(OH)2D3 can activate the PKC isozyme PKCx via stimulation of the upstream regulator phosphatidylinositol 3-kinase (PI3-kinase) to regulate the CYP24A1 promoter. Phosphorylation of Sp-1 in the presence of 1,25(OH)2D3 is dependent on PI3-kinase and PKCx activation, suggesting a potential regulatory role of the PKCx isozyme in Sp-1 phosphorylation by 1,25(OH)2D3, acting via the GC box in HEK293 cells [125]. CALCITONIN
A similar pathway is reported to contribute to the substantial induction of CYP24A1 by the peptide hormone calcitonin in HEK293 cells [124]. Calcitonin is primarily secreted by the thyroid parafollicular C cells in response to increasing calcium concentrations and is involved in calcium homeostasis by inhibiting bone resorption and modulating calcium excretion from the kidney [126]. Comparable with other regulators of basal CYP24A1 expression, calcitonin-mediated induction of the CYP24A1 promoter is dependent on the transcription factors Sp-1 and NY-F acting through the GC and CCAAT box sites, respectively [124]. PKC and PKA signaling pathways are involved in calcitonin-mediated induction of CYP24A1, possibly acting through Sp-1 and NY-F [124]. Despite activation of ERK1/2 by calcitonin previously demonstrated in HEK293 cells [127,128], enhancement of the CYP24A1 promoter by calcitonin may not necessarily involve ERK1/2 signaling [124]. Calcitonin-stimulated expression of renal CYP24A1 may be important for decreasing circulating levels of 1,25(OH)2D3, which in turn contributes to lowering intestinal calcium absorption primarily regulated by the active hormone. In contrast to the kidney, however, calcitonin suppresses CYP24A1 mRNA and activity in rat intestine [129]. The negative action of calcitonin on CYP24A1 would be expected to prolong intestinal 1,25(OH)2D3 availability and enhance the biological effects of 1,25(OH)2D3 in this tissue, possibly increasing the uptake of calcium and phosphorus. Consequently,
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regulation of vitamin D metabolism, and thus bone and mineral metabolism, may be the net effect of multiple signaling pathways that influence CYP24A1 regulation, either at the transcriptional or post-transcriptional level. C/EBP Binding Sites The transcriptional activator CCAAT enhancerbinding protein b (C/EBPb) is involved in regulating cell proliferation and differentiation, as well as inflammation [130]. In mouse kidney and primary murine osteoblasts, C/EBPb is induced by 1,25(OH)2D3 and is a strong enhancer of CYP24A1 transcription mediated through 1,25(OH)2D3/VDR activation [131,132]. Transactivation of CYP24A1 by C/EBPb occurs via a C/EBP binding site at positions 395 to 388 (TTGGCAA) in the rat CYP24A1 promoter [132]. Functional cooperation between C/EBPb, the co-activator CBP (CREB-binding protein), as well as the VDR, are necessary for the transcriptional regulation of CYP24A1 in vitro [132,133]. Moreover, SWI/SNF complexes containing the homologous ATPase Brahma (Brm) also participate in the C/ EBPb-mediated enhancement of 1,25(OH)2D3-induced CYP24A1 transcription. Effects of Brm may occur via C/EBPb interaction and association with the C/EBP site on the CYP24A1 promoter [133]. GLUCOCORTICOIDS
Glucocorticoid therapy is often used to treat inflammation; however, prolonged use increases the risk of bone loss believed to be due to decreasing bone formation and increasing bone resorption [134,135]. Glucocorticoids may also induce bone loss by reducing intestinal calcium absorption and increasing calcium excretion from the kidney, leading to the development of secondary hyperparathyroidism [134]. Changes in calcium metabolism suggest a possible regulatory effect of glucocorticoids on vitamin D metabolism. Serum levels of vitamin D metabolites in animal models and patients exposed to glucocorticoids have yielded conflicting results, showing either an increase [136], decrease [137e139], or no change [140,141]. Despite these variations, disruption in 25(OH)D3 and 1,25(OH)2D3 homeostasis indicates a potential interaction between glucocorticoids and enzymes responsible for the synthesis and/or catabolism of 1,25(OH)2D3. The potent glucocorticoid, dexamethasone, increases the renal expression of CYP24A1 mRNA and enzymatic activity in vivo [139,141e143], and can enhance 1,25(OH)2D3-mediated induction of CYP24A1 mRNA in various cells lines, including osteoblast-like cells, as well as in the kidney [143e145]. Stimulation of c-fos protein expression by dexamethasone may be required for the promotion of CYP24A1 gene expression in vitro [144]. New evidence suggests that glucocorticoids
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directly enhance VDR-mediated CYP24A1 transcription through the functional cooperation between the glucocorticoid receptor and C/EBPb-binding motif on the CYP24A1 promoter [143]. Glucocorticoid-induced increase in VDR expression and VDR-mediated transcription of the CYP24A1 promoter may also indirectly enhance enzyme levels in vitro [145]. These findings suggest that exposure to glucocorticoids may result in increased rates of 1,25(OH)2D3 catabolism both in the kidney and osteoblasts, thus contributing to lower circulating levels of 1,25(OH)2D3 and consequent bone loss. Downstream Enhancer Sequences Emerging evidence suggests that the expression of numerous genes can be regulated by multiple enhancers located at sites not only proximal to the transcriptional start site, but far distal as well [146]. The induction of CYP24A1 by 1,25(OH)2D3 is highly dependent on VDR/RXR interaction with VDRE-1 and VDRE-2 located within an enhancer site near the transcriptional start site of the proximal promoter and recruitment of coregulatory complexes. While upregulation of CYP24A1 by 1,25(OH)2D3 is understood to be mediated through VDREs located near the proximal promoter, a recent study by Meyer et al. reports that a cluster of approximately four enhancer regions located considerably downstream of the CYP24A1 gene may also be involved [146]. Several enhancer regions located þ50 to þ69 kb and þ35 to þ45 kb downstream of the human CYP24A1 gene and mouse CYP24A1 gene, respectively, demonstrate VDR/RXR binding activity and are independently active in mediating the 1,25(OH)2D3 response. Two downstream human enhancer regions contain at least two functional VDREs, both of which contribute to 1,25(OH)2D3 induction of CYP24A1 responses. Recruitment of coregulators (i.e., MED1, SRC-1, and SMRT) to these regions, as well as increasing histone acetylation levels and RNA polymerase II activity by 1,25(OH)2D3 provides additional support for a role of downstream enhancers in the 1,25(OH)2D3-mediated upregulation of CYP24A1. Therefore, these findings indicate that, in addition to VDREs located in the proximal CYP24A1 promoter close to the transcriptional start site, enhancer regions downstream of the CYP24A1 gene may also play a significant role in regulating 1,25(OH)2D3-induced increase in CYP24A1 transcription.
Retinoic X Receptors and Retinoic Acid Receptors Retinoic acid is a biologically active derivative of vitamin A involved in a variety of cellular processes, including cell proliferation and differentiation. Retinoid X receptors (RXRs) and retinoic acid receptors (RARs)
are nuclear transcription factors that are activated by retinoid ligands, 9-cis-retonoic acid (9-cis-RA) and alltrans-retinoic acid (all-trans-RA). While both natural ligands bind to RARs, RXRs are activated by 9-cis-RA only [147,148]. Along with the intracellular VDR, RXR heterodimerizes with other nuclear receptors, such as RARs and thyroid hormone receptors, to activate a number of target genes responsive to 1,25(OH)2D3, tRA, or thyroid hormone [149]. Natural and synthetic RXR and RAR compounds increase CYP24A1 expression in mouse kidney and in renal cell lines independent of 1,25(OH)2D3, possibly through the binding of RXR/VDR or RXR/RAR heterodimers to previously identified VDRE sequences on the CYP24A1 promoter [150,151] and recruitment of coactivators by the VDR/RXR bound to DNA [152]. By contrast, in rat kidney, and in several extra-renal tissues, such as epidermal keratinocytes from human skin, CYP24A1 transcription is not influenced by 9-cis-RA or all-trans-RA alone [153e155]. The ability of retinoids to regulate CYP24A1 gene expression in the absence of 1,25(OH)2D3 in some tissues, while not in others, indicates that CYP24A1 gene regulation by retinoids is tissue specific and that other factors, such as tissue-restricted coactivators or corepressors, in addition to the receptors and their cognate ligands may be involved. The combined presence of liganded VDR and RXR can synergistically or additively enhance the expression of CYP24A1 mRNA in vivo, as well as in various cell lines, including HEK293 and MCF-7 cells [151e155]. The enhanced induction of CYP24A1 transcription by 1,25(OH)2D3 and RXR ligands is mediated by RXR/ VDR and RXR/RAR heterodimers [150e152]; however, the contribution of the liganded RXR/RAR complex to synergistic responses observed in epidermal human keratinocytes and HEK293 cells is minimal or entirely absent compared to RXR/VDR [153,154]. Mechanism(s) responsible for the synergistic effect of retinoids on CYP24A1 gene expression are currently unknown; however, a conformational change in the RXR/VDR VDRE-1 and VDRE-2 complex in the presence of both 9-cis-RA and 1,25(OH)2D3 has been reported which may influence coactivator binding and CYP24A1 transcription [155]. As demonstrated above, both 9-cis-RA, as well as alltrans-RA, are commonly reported to increase transcription of CYP24A1 in the absence of or accompanied by 1,25(OH)2D3 in vivo and in a range of cell lines; however, retinoic acid induction of CYP24A1 is not observed in all cell types. In human prostate stromal cell lines P29SN and P32S, for example, administration of all-trans-RA markedly suppresses the expression of CYP24A1 mRNA induced by 25(OH)D3 and 1,25(OH)2D3 via RARa-dependent signaling pathway [156]. By contrast, all-trans-RA did not hinder the induction of CYP24A1
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by either 25(OH)D3 or 1,25(OH)2D3 in human prostate cancer cells LNCaP and PC3, in addition to primary culture of human prostatic epithelial cells PrEC [156]. Cell growth is strongly inhibited by the combined treatment of 1,25(OH)2D3 with the RARa agonist Am80 in P32S cells [156], which supports a vital role for CYP24A1 as a powerful determinant of biological action of vitamin D metabolites in the prostate.
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CYP3A4 by SXR agonists in the liver and small intestine suggests that the catabolism of 1,25(OH)2D3, and druginduced osteomalacia is likely dependent on CYP3A4 rather than CYP24A1 [142,165,166]. Thus, CYP24A1specific inhibitors may not be useful in preventing osteomalacia associated with drug-induced metabolism of vitamin D hormone.
Post-transcriptional Regulation: MicroRNAs Steroid and Xenobiotic Receptors (SXR) Anti-epileptics and Antimicrobial Agents Prolonged use of some antiepileptics (AEDs), including phenobarbital, phenytoin, and carbamazepine, and the antimicrobial agent rifampicin (RIF) can lead to the development of osteomalacia, a metabolic bone disease characterized by bone loss and increased risk of fractures [157e161]. Osteomalacia is associated with biochemical abnormalities, such as hypocalcemia, hypophosphatemia, hyperparathyroidism, and lower levels of circulating vitamin D metabolites, in some patients utilizing AEDs and RIF [161,162]. Similar side effects have also been observed with vitamin D deficiency, indicating a potential interaction between AEDs and the vitamin D signaling pathway. Several pharmacological agents that induce osteomalacia activate the human steroid and xenobiotic receptor (SXR), also known as the pregnane X receptor (PXR). SXRs form heterodimers with RXR and regulate the transcription of genes primarily involved in xenobiotic and drug metabolism (i.e., CYP2 and CYP3), and thus, xenobiotic clearance, in the small intestine and liver [163]. The expression of CYP24A1 in kidney and liver cells is enhanced by the AEDs phenobarbital and carbamazepine, as well as the antimicrobial agent RIF possibly through direct binding of SXR/RXRa to the proximal VDREs on the CYP24A1 promoter in the absence of 1,25(OH)2D3 [142,164]. Since the SXR is expressed at very low levels in the kidney relative to CYP24A1, the renal expression of CYP24A1 is postulated to play an insignificant role in development of druginduced osteomalacia [165,166]. Induction of CYP24A1 by SXR agonists in hepatic cells is inconsistent with findings reported by Zhou et al. which failed to demonstrate SXR-mediated transactivation of the CYP24A1 promoter or induction of CYP24A1 mRNA by RIF and the antifungal agent clotrimazole in human primary hepatic and intestinal cells and in vivo [166]. Conversely, SXR agonists were shown to inhibit VDR-mediated CYP24A1 promoter activity in kidney, liver, and intestinal cells and in vivo, perhaps by preventing the 1,25(OH)2D3-dependent dissociation of the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) [164,166]. Induction of
MicroRNAs (miRNAs) are a conserved class of regulatory RNA molecules primarily involved in suppressing protein expression or mRNA degradation [167]. miRNAs have a key role in a wide range of biological processes, including development, cellular differentiation, apoptosis, and proliferation [168e170]. Aberrant expression of miRNA, and to a lesser extent mutations in miRNAs, has been implicated in the pathogenesis of various human diseases, such as cancer, kidney pathology, and cardiovascular disease [171e175]. In addition to the transcriptional activation of the CYP24A1 gene by the liganded VDR/ RXR heterodimer, CYP24A1 may be post-transcriptionally regulated by miRNAs. Overexpression of the miRNA 125b (miR-125b) represses CYP24A1 protein expression, possibly through binding to a miR-125b recognition element (MRE125b) in the 3’ untranslated region of human CYP24A1 mRNA, whereas inhibition of miR125b leads to elevated protein levels in vitro [176]. These findings indicate a role for miR-125b in the regulation of human CYP24A1.
Methylation State Methylation of mammalian DNA is an epigenetic event involving the methylation of cytosine residues of CpG dinucleotides and is often equated with transcriptional repression of the promoter regions of genes, leading to gene silencing [177]. Hypermethylation of CpG islands identified in the 5’ end of the mouse and human CYP24A1 promoter correlates with a lack of CYP24A1 expression despite intact VDR signaling in tumor-derived endothelial cells (TDECs), osteoblastic ROS17/2.8 cells, prostate cancer cell lines, and malignant prostate tissue from human, as well as colon cells derived from differentiated tumors [178e182]. Although epigenetic silencing of the CYP24A1 gene has been primarily reported in cancerous cell lines and tissue from humans, CYP24A1 gene promoter methylation has also been reported in placental tissues from healthy patients, as well as in normal prostate cells, indicating that hydroxylases regulating the vitamin D signaling pathway in healthy tissue may be under epigenetic control [183,184]. CpG regions are located in close proximity to VDREs on the CYP24A1 promoter [182]. Methylation of these regions is suggested to prevent VDR binding to the
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VDREs and thus may account for a lack of CYP24A1 transactivation by 1,25(OH)2D3 in some cell types, such as TDECs and prostate cancer cell lines [179,181,182]. Cells exhibiting methylation silencing of CYP24A1 display greater growth inhibition by 1,25(OH)2D3 compared to those in which CYP24A1 remains highly inducible [179,181]. Conversely, the absence of and/or low methylation status is associated with higher basal levels and inducible transactivation of CYP24A1 by 1,25(OH)2D3, which may account for a loss of sensitivity to the anti-proliferative effects of 1,25(OH)2D3 in these cells [179,181,182]. Methylation status of the CYP24A1 promoter may be one contributory factor regulating the differential expression of this gene, as well as cellular sensitivity to 1,25(OH)2D3-mediated anti-proliferative effects in diseased and healthy tissue.
Hormones: Parathyroid Hormone and FGF23 Parathyroid Hormone Parathyroid hormone (PTH) is primarily secreted in response to low circulating calcium [185]. To correct for low calcium levels, PTH upregulates renal CYP27B1, and thus stimulates the production of 1,25(OH)2D3, the active hormone responsible for increasing calcium absorption from the intestine [186,187]. CYP24A1 promoter activity is enhanced by PTH through a cAMP-dependent process in kidney cell lines [27,188]. Despite an increase in renal CYP24A1 gene transcription, 1,25(OH)2D3-mediated induction of CYP24A1 mRNA and activity is significantly attenuated by PTH in vitro and in vivo [27,189,190] due to destabilization and increased degradation of CYP24A1 mRNA by PTH [191]. In vitro studies demonstrate that PTH-mediated suppression of CYP24A1 mRNA occurs independently of the VDR in kidney cells and may act through the cAMP/PKA signaling pathway [27]. Sequences in the 3’ untranslated region commonly involved in regulating mRNA stability, along with the 5’ untranslated region, do not affect the stability of CYP24A1 mRNA by PTH, indicating that the site of PTH action is to be found elsewhere in the coding region [191]. In contrast to CYP24A1 suppression in the kidney, PTH enhances 1,25(OH)2D3-mediated induction of CYP24A1 transcription, mRNA and protein through the cAMP signaling pathway in osteoblastic cells [192e194]. PKA-mediated induction of VDR transcription through C/EBPb contributes to PTH enhancement of CYP24A1 transcription by 1,25(OH)2D3 [132,193]. PTH induction of CYP24A1 in bone cells may prevent aberrant elevations in 1,25(OH)2D3 and resultant bone formation abnormalities [59], whereas in kidney, suppression of CYP24A1 by PTH would be expected to increase systemic 1,25(OH)2D3 and subsequent levels of calcium in circulation.
FGF23 FGF23 plays a central role in the regulation of mineral homeostasis affecting both the expression of genes regulating serum phosphorus, as well as those controlling vitamin D metabolism [195e197]. Stimulated in osteocytes and osteoblasts by rising serum phosphate levels, FGF23 reduces renal phosphate reabsorption by inhibiting Na/Pi co-transporter activity [198,199] and indirectly suppresses intestinal phosphate absorption through the lowering of systemic 1,25(OH)2D3 levels by reducing the renal expression of CYP27B1 [199e201]. FGF23 also controls 1,25(OH)2D3 levels by inducing expression of CYP24A1 mRNA in the kidney in vivo [199e204]. This effect of FGF23 in kidney may be partially dependent on the VDR; however, this remains controversial [201,204]. FGF23 induction of CYP24A1 has also been demonstrated in renal proximal tubule cells, albeit of lower magnitude than stimulated levels observed in vivo [200]. While these findings confirm that FGF23 plays an essential role in the regulation of CYP24A1, the mechanisms underlying this elevation remain relatively unknown yet important to understand the relationship between FGF23 and vitamin D status in diseases, such as hypophosphatemia, CKD, and cancer. It is clear from the above discussion that the level of CYP24A1 expression is determined by the net influence of many signaling pathways; therefore, normal or disease-related changes in the activity of these pathways can have an effect on CYP24A1 levels and hence vitamin D status. Since vitamin D status may influence patient outcomes, understanding how certain diseases can affect CYP24A1 will be important to optimize treatment strategies.
DYSREGULATION OF CYP24A1 AND ROLE IN THE PATHOGENESIS OF HUMAN DISEASE Importance of Vitamin D in Health and Disease The major physiological function of 1,25(OH)2D3 is to maintain bone and mineral metabolism in the body through binding to its cognate VDR in classic target tissues, including the kidney, intestine, bone, and parathyroid. Vitamin D deficiency leads to impaired skeletal mineralization and markedly increases PTH secretion from the parathyroid gland, causing secondary hyperparathyroidism. Chronic elevation of PTH exacerbates 1,25(OH)2D3 deficiency, facilitates calcium mobilization from bone, and increases phosphate wasting in the kidney, resulting in the precipitation and exacerbation of rickets in children and osteomalacia in adults [205,206].
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Moreover, there are other complications associated with vitamin D deficiency which appear unrelated to bone and mineral metabolism. The ubiquitous expression of the VDR in a wide range of tissue in addition to the kidney such as skin, pancreas, reproductive organs, cardiovascular and immune system, highlight an autocrine role for vitamin D [207]. Many of these tissues also express the CYP27B1 enzyme, indicating a capacity to locally synthesize 1,25(OH)2D3 from the prohormone 25(OH)D3 [13,14,208]. The non-calcitropic actions of 1,25(OH)2D3 in peripheral tissue may therefore be extensive; playing a role not only in cell proliferation and differentiation, but also hormone secretion, immunomodulation, blood pressure regulation, brain development, and central nervous system function [15]. Moreover, evidence is mounting suggesting a potential link between vitamin D deficiency and some pathophysiological conditions, including autoimmune diseases (i.e., multiple sclerosis, inflammatory bowel disease, type-1 diabetes), endocrine disorders (i.e., hyperparathyroidism), cardiovascular disease and central nervous system disorders (i.e., Alzheimer’s and Parkinson’s disease) [208e211]. A large volume of evidence suggests that vitamin D deficiency may be an important factor in the genesis and progression of various types of cancer, such as prostate, colon, and breast [212,213]. Clinical trials are currently under way to determine the therapeutic potential for vitamin D in the treatment of cancer [214]. The relationship between vitamin D deficiency and disease is complicated since in some instances, vitamin D deficiency may play a causal role in the disease and, conversely, the disease state may exacerbate vitamin D deficiency. External factors, such as lack of sunlight and inadequate vitamin D intake, are recognized as important factors contributing to vitamin D deficiency; however, disturbances in the regulation of key P450 enzymes involved in the synthesis (CYP27B1) and catabolism (CYP24A1) of vitamin D metabolites may also be implicated. Aberrant expression of CYP24A1 has been demonstrated in certain disease states in experimental models and patients, such as genetically linked hypophosphatemia, CKD, and diabetes, and is proposed to be a major mechanism underlying accelerated degradation of 1,25(OH)2D3 in these pathologies [3,4,8,215]. Growing evidence indicates that basal CYP24A1 expression can also be abnormally high in various forms of cancer and may be a primary determinant of vitamin D deficiency and decreased responsiveness of tumor cells to 1,25(OH)2D3 treatment [2]. The use of CYP24A1 inhibitors alone or in combination with other vitamin D analogs may provide a means of overcoming such resistance and maintaining optimal levels of vitamin D metabolites in these patients.
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X-linked Hypophosphatemia The link between vitamin D enzymes and disease was first established in the hypophosphatemic mouse model (Hyp), a murine homolog of X-linked hypophosphatemic rickets (XLH) in humans, initially characterized by Eicher et al. in 1976 [216]. Deletion in the 3’ region of the Phex gene [217,218] is associated with hypophosphatemia and impaired bone metabolism arising, in part, from defective renal reabsorption of inorganic phosphate at the brush border membrane [216,219,220]. Similar to human patients with XLH, hypophosphatemic mice exhibit normal levels of 1,25(OH)2D3 in spite of significant hypophosphatemia, a condition normally associated with elevated concentrations of 1,25(OH)2D3 in animals and humans [221,222]. Moreover, serum levels of 1,25(OH)2D3 decrease or remain constant with further phosphate deprivation in Hyp mice and patients with XLH [222,223], respectively, whereas phosphate supplementation markedly increases 1,25(OH)2D3 levels [223,224]. In normal mice, serum levels of 1,25(OH)2D3 increase with phosphate deprivation, while remaining unchanged with supplementation [221,223]. Abnormal regulation of vitamin D metabolism indicated by the paradoxical response of 1,25(OH)2D3 to phosphate observed in mutant mice and XLH patients is partly attributed to low CYP27B1 activity and possible changes in mRNA expression in the proximal convoluted tubule [225e228], as well as a blunted response to regulators of CYP27B1 activity, including hypophosphatemia [221,225] and PTH [229,230]. However, levels of 1,25(OH)2D3 in serum are also correlated with the rate of renal catabolism of 1,25(OH)2D3 in Hyp mice as evidence by an increased production of CYP24A124-oxodependent metabolites 1,24,25(OH)3D3, 1,25(OH)2D3, and 24-oxo-1,23,25(OH)3D3 [231e233]. Accelerated catabolism of 1,25(OH)2D3 in Hyp mice has been ascribed to an increase in CYP24A1 enzyme activity in renal mitochondria [233], as well as elevated levels of CYP24A1 mRNA and protein expression in the kidney proximal tubules [4,227,234]. Mechanisms giving rise to the aberrant expression of CYP24A1 in the Hyp mouse model are not well understood. Dysregulation of renal CYP24A1 in the Hyp mouse may involve PKC phosphorylation [235] and growth hormone [236], as well as FGF23. The phosphatauric hormone, FGF23, plays a central role in the regulation of phosphorus homeostasis. While levels of FGF23 are normally low, serum concentrations of this hormone are generally high in Hyp mice [237] and hypophosphatemic patients, including some subjects with XLH [238e240]. In addition to lowering 1,25(OH)2D3 production by suppressing CYP27B1, FGF23 increases the renal expression of CYP24A1 in cells [200] and in vivo [199,200,241], and therefore, may be partially responsible
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for the elevated basal expression of CYP24A1 observed in this mouse model of XLH.
Cancer Interest in using vitamin D and vitamin D analogs in the treatment and prevention of cancer has grown steadily over the past 25 years, sparked initially by epidemiological studies demonstrating a link between low UV exposure and increased incidence of cancer [242,243] (see Chapter 53). The cancer-protective effects of vitamin D were first demonstrated in 1980, when Garland and Garland, while studying geographical trends in cancer incidence, observed an inverse association between latitude and risk of developing colon cancer in the USA [244]. They found that colon cancer mortality rates were highest in places where people were exposed to lower amounts of sunlight. A similar relationship between the development of rickets, a childhood disease primarily caused by vitamin D deficiency, and geographic location was known previously, and led Garland and Garland to propose that low levels of vitamin D led to an increased risk of developing colon cancer. Since then, studies have provided some support for a link between circulating levels of the vitamin D metabolite 25(OH)D3 and risk of developing colon cancer [245e248], and have further identified an association for additional cancer types that include prostate [249e251], breast [252e255], and ovarian [256,257] (see Chapter 82). However, the available epidemiological evidence for these cancers other than colon remain inconclusive, showing negligible support for an association between vitamin D and risk of breast [258,259], prostate [260,261], and ovarian [262] cancers. Epidemiological and experimental data linking vitamin D and cancer risk has provided compelling evidence for the potential use of vitamin D therapeutics in cancer prevention and treatment. In addition to the classical role of 1,25(OH)2D3 in the intestinal regulation of calcium and phosphate transport and bone mineralization, several non-classical actions of 1,25(OH)2D3 unrelated to calcium homeostasis have been identified that are fundamental in carcinogenesis (see many other chapters in this volume). It has been over 25 years since the seminal discovery elucidating the ability of 1,25(OH)2D3 to repress human melanoma cell proliferation in vitro [263] and promote cell differentiation of immature cultured myeloid leukemia cells derived from human [264] and mouse [265]. Since then, an abundance of experimental in vivo and in vitro evidence has further demonstrated anti-proliferative effects of 1,25(OH)2D3 in a wide variety of tumor models, including breast, colon, prostate, bladder, pancreas, lung, as well as in healthy tissue [212]. Growth-inhibitory effects of 1,25(OH)2D3 have been attributed to its ability
to induce cell-cycle arrest, cellular differentiation and apoptosis [212]. Anti-angiogenic activity and inhibition of extracellular matrix degradation underlying tumor invasiveness and metastasis induced by 1,25(OH)2D3 may also contribute to its anti-tumor effects [212]. Cancer cells exhibit a wide range of sensitivities to the growth-inhibitory action of 1,25(OH)2D3 and its analogs (see Chapters 82e90). Dysregulation of the vitamin D signaling pathway contributes to limited sensitivity and responsiveness of tumor cells to the anti-cancer effects of 1,25(OH)2D3 and vitamin D analogs. Tumor resistance to 1,25(OH)2D3 may involve multiple factors, including a functional VDR and changes in the expression of extra-renal CYP27B1 enzyme, important for synthesis of local 1,25(OH)2D3 [14,266]. The functional importance of VDR expression is supported by an increased susceptibility of tissue to cancer development and tumor angiogenesis in mice lacking the VDR [267e269]. Downregulation of CYP27B1 and/or VDR expression has been observed in some types of cancer cells [270e273], suggesting that perturbations in these mechanisms may contribute to cancer pathogenesis and progression. However, a number of cancer cell lines exhibit a lack of growth inhibition by 1,25(OH)2D3 irrespective of VDR expression [274e278], indicating that additional intrinsic mechanisms may be involved in regulating growth-inhibition responsiveness to 1,25(OH)2D3. Apart from disturbances in 1,25(OH)2D3 action and synthesis, accelerated catabolism may also play a significant role in lowering vitamin D levels and limiting responsiveness of tumor cells to 1,25(OH)2D3. Overexpression of CYP24A1 mRNA is reported in a wide variety of human cancers, including breast [14,279], lung [273,280], colon [273,281,282], cervical [279], ovarian [273,279], and esophageal [283], and in some cases is linked to a poor prognosis and overall reduced survival [283]. Overexpression of CYP24A1 increases the growth potential of tumor cells and lowers the responsiveness of tumors to the anti-cancer effects of endogenous 1,25(OH)2D3 [273,280]. Inhibition of CYP24A1 prolongs the biological half-life of 1,25(OH)2D3 and slows the loss of 1,25(OH)2D3 in human cancer cells, presumably increasing exposure of target tissues to 1,25(OH)2D3 [280,284e287]. Increase in the systemic exposure to 1,25(OH)2D3, as well as a delay in the clearance of serum 1,25(OH)2D3, is observed in normal mice treated with CYP24A1 inhibitors [288]. Moreover, administration of the non-specific CYP24A1 inhibitor ketoconazole, together with dexamethasone, enhances 1,25(OH)2D3-mediated growth inhibition in a human prostate cancer PC3 xenograft model [288]. These findings strongly suggest that inhibition of CYP24A1 activity may provide a novel therapeutic approach to maximize the anti-proliferative activity
IX. ANALOGS
DYSREGULATION OF CYP24A1 AND ROLE IN THE PATHOGENESIS OF HUMAN DISEASE
and effectiveness of vitamin D in the prevention and treatment of cancer. Amplification at the 20q.13 chromosomal locus that encodes CYP24A1 [289] has been identified in a number of tumor types, including breast [289], lung [290], colon [291], and esophageal [292], and may be one factor contributing to the aberrant expression of CYP24A1 in some cancers. An increase in CYP24A1 mRNA stability, number of functional VDREs within the CYP24A1 gene [293], epigenetic mechanisms [178e182], as well as microRNAs [176] may serve as additional genomic mechanisms regulating CYP24A1 expression and 1,25(OH)2D3 inactivation in malignant cells. Therefore, perturbations in multiple pathways implicated in cancer cell growth can potentially influence CYP24A1 expression in tumor cells.
Diabetes and Nephropathy Diabetes affects over 7% of the population in the USA and is rapidly becoming a global epidemic [294]. Diabetes is accompanied by characteristic long-term complications, including retinopathy, neuropathy, cardiovascular disease, and cerebrovascular disease, as well as nephropathy which is the most common renal complication of diabetes mellitus and a leading cause of renal failure in these patients [295e297] (see Chapters 98 and 102). Vitamin D deficiency is prevalent in patients with type 1 (T1) [298e301] and type 2 (T2) diabetes [299,302e304] and in some animal models of T1, including the Streptozotocin (STZ) mouse model [305e307], BioBreeding (BB) rats [308], and in the Goto-Kakizaki (GK) rats, a genetic model of T2 diabetes [309]. Vitamin D status is reported to be inversely associated with risk of developing T1 [310,311] and T2 [312e314] and may contribute to allcause mortality in T2 patients [315]. A reduction in the incidence of insulitis and diabetes following chronic administration of 1,25(OH)2D3 or vitamin D analogs has also been reported in the non-obese diabetic (NOD) mouse model of T1 diabetes [316e318]; however, in the BB rat model, diabetic incidence was not affected [319]. Secondary complications arising from long-term diabetes, including cardiovascular disease and mortality [315,320,321], retinopathy [322], and diabetic nephropathy [323], may also be linked to vitamin D deficiency in patients with T2 diabetes. While these studies suggest that vitamin D may be an important player in the pathogenesis in both forms of diabetes and long-term complications, emerging evidence derived from animal models and intervention studies with diabetic patients also indicates a potential role for vitamin D in the treatment of this disease. Administration of 1,25(OH)2D3 has been shown to improve diabetes and assist in the recovery of bone mineral density in the STZ mouse model
1537
[324], whereas in patients with T1 diabetes, total insulin dose was reduced for a period of time with 1,25(OH)2D3 therapy [325]. Vitamin D3 supplementation given to T2 diabetic Wistar and spontaneously hypertensive rats treated with STZ resulted in reduced blood glucose levels [326]. Moreover, treatment of T2 diabetic patients with vitamin D3 or 1,25(OH)2D3 improved insulin sensitivity and secretion [303,327]. Although the mechanisms of these associations are not yet entirely clear, the ability of vitamin D and its metabolites to stimulate insulin synthesis and secretion, influence insulin action and effect components involved in inflammation may, in part, contribute to its reno-protective effects in the prevention and treatment of diabetes [319,328]. Underlying mechanisms contributing to vitamin D deficiency in diabetes are not well understood, but may be multi-factorial, resulting from diet and/or reduced exposure to sunlight. In diabetic patients and animal models of diabetes, impaired renal function is associated with a greater reduction in 25(OH)D3 levels [305,323,329,330], as well as 1,25(OH)2D3 [330,331], indicating that disturbances in the regulation of metabolic enzymes responsible for the renal synthesis (CYP27B1) and catabolism (CYP24A1) of vitamin D metabolites may also contribute to declining systemic levels of vitamin D in this population. In the STZ rodent model of T1 diabetic nephropathy, chronic insulin deficiency decreases CYP27B1 activity in the kidney, while that of CYP24A1 is significantly elevated [5,6]. Elevated CYP24A1 activity has also been reported in the NOD T1 diabetes model in which mild kidney abnormalities are evident [332]. Evidence also indicates that renal expression of CYP24A1 mRNA is also elevated in the db/db genetic model of T2 diabetes [333], suggesting that accelerated catabolism of vitamin D metabolites by CYP24A1 may occur in both forms of diabetes in these animal models of diabetic nephropathy. CYP24A1 mRNA extracted from blood mononuclear cells is unchanged in T1 diabetic patients [334]; however, our group recently reported a significant upregulation in the constitutive expression of CYP24A1 protein in renal tissue biopsied from T2 diabetic patients with nephropathy [8] (Fig. 80.3). Overexpression of CYP24A1 may play an important role in the etiology of diabetic nephropathy. Early intervention with CYP24A1 inhibitors may limit the damage caused by extended vitamin D deficiency in CYP24A1 and overexpressing tissues may halt or slow the progression from diabetic nephropathy to kidney failure.
Chronic Kidney Disease Vitamin D insufficiency and deficiency is commonly observed in patients with CKD and is causally related to secondary hyperparathyroidism, a disorder
IX. ANALOGS
1538
80. CYP24A1 REGULATION IN HEALTH AND DISEASE
Control
(A)
(B)
(C)
CKD
a
b
a
b
a
b
FIGURE 80.3 Immunohistochemisty demonstrating elevated CYP24A1 protein expression in renal tissue biopsied from patients with type II diabetes and diabetic nephropathy. Immunperoxidase staining of CYP24A1 protein in the (A) renal artery, (B) medulla, and (C) cortical tubules from DN and age-matched controls is presented. Arrows indicate localized CYP24A1 staining to the apical membrane of the proximal tubules in control tissue (arrows; Aa). DN tissue showed marked and diffuse cytoplasmic staining in the proximal tubular (Arrows; Ab), as well as cortical (Bb) and medullary (Cb) distal tubules. Original magnification was 400 for artery and 200 for medulla and cortical tubules. From [8]. Please see color plate section.
characterized by elevated serum intact PTH levels, parathyroid gland hyperplasia and imbalances in bone and mineral metabolism [335e337] (see Chapters 70 and 81). Declining renal mass and concomitant loss of renal CYP27B1 capacity in CKD is commonly associated with reductions in circulating levels of both 1,25(OH)2D3 and 25(OH)D3 [338,339]. However,
observations of low serum 1,25(OH)2D3 have not been consistently linked with decreases in renal CYP27B1 expression since levels of CYP27B1 mRNA may in some cases remain unchanged in CKD patients deficient in 1,25(OH)2D3 [7]. Moreover, diminishing CYP27B1 expression levels cannot directly account for the progressive loss of 25(OH)D3. These findings suggest
IX. ANALOGS
1539
CYP24A1 INHIBITORS
that additional intrinsic mechanisms may underlie declining vitamin D metabolites, 25(OH)D3 and 1,25(OH)2D3, in renal disease. Using the adenine rat model of CKD, we investigated the regulation of CYP27B1 and CYP24A1 in uremia [8]. In vitamin D target tissues, including the kidney, it is well established that CYP24A1 is inversely correlated with vitamin D status [59,285,340]. Along with a decline in serum 25(OH)D3 and 1,25(OH)2D3 levels in uremic animals, we also observed a marked elevation in CYP24A1 mRNA and protein expression in the uremic kidney in the absence of a concomitant decline in CYP27B1. While CYP24A1 mRNA levels were low or non-detectable in the kidney of vitamin-D-deficient animals, expression of CYP24A1 mRNA remained markedly elevated in uremic kidneys despite animals being rendered vitamin-D-deficient (Fig. 80.4). These findings suggest that factors other than vitamin D determine the expression levels of CYP24A1 in the uremic state. A similar pattern of renal enzyme expression was recently reported in promycin aminonucleoside (PAN) nephrosis rats which exhibit proteinuria and podocytes injury [341]. In agreement with animal models of CKD, aberrant expression of CYP24A1 was also recently reported in CKD patients with acute renal inflammation [7], as well as in renal tissue biopsied from patients with diabetic nephropathy [8] (Fig. 80.3). Non-uremic
Relative mRNA expression
4
Uremic
*
CYP24
2
1
* 0 Vitamin D deficient
The relationship between CYP24A1 expression and disease suggests that this enzyme might be a useful therapeutic target. A number of inhibitors have been synthesized to explore their utility in the treatment of diseases associated with elevated vitamin D catabolism (Fig. 80.5). Most progress in this regard has been directed toward blocking CYP24A1 activity in tumors overexpressing the enzyme.
Azoles
3
Normal
CYP24A1 INHIBITORS
*
**
CYP27B1
Although CYP24A1 elevation appears to be strongly correlated with kidney injury, whether induced by hypophosphatemia, diabetes, or exposure to kidneydamaging agents, the consequences of elevated CYP24A1 are not known. If the elevated fraction of CYP24A1 is functional, it is likely that the overexpressing cells would be less responsive to vitamin D hormones. This is supported by observations that high-level expression of CYP24A1 in tumor cells can effectively block the anti-proliferative effects of vitamin D agonists susceptible to CYP24A1-based catabolism (see “Cancer,” above). Since CYP24A1 expression acts as a barrier which can significantly restrict vitamin D hormone activity [59,280,342], overexpression of CYP24A1 may deprive kidney tissue of vitamin D, creating a localized state of deficiency. This local depletion of vitamin D signaling may have a direct effect on kidney disease progression by promoting inflammation and fibrosis [343].
Normal
Vitamin D deficient
FIGURE 80.4 Upregulation of basal CYP24A1 mRNA expression is independent of vitamin D status in uremic rats. Rats were fed a normal or vitamin-D-deficient diet for 6 weeks, with both groups dosed orally with adenine (uremic) or vehicle (non-uremic) from week 4 to 6. Vitamin D status was measured 1 week and kidneys were harvested 2 weeks after adenine treatment. Summary plot of CYP24A1 and CYP27B1 mRNA expression in non-uremic and uremic rats fed a normal or vitamin-D-deficient diet. Relative mRNA values are normalized to vehicle (relative expression ¼ 1). Non-uremic vitaminD-deficient, uremic-normal and uremic-vitamin-D-deficient rats were compared to non-uremic normal rats. Statistical difference (*) p < 0.05 (**) p < 0.01 relative to non-uremic normal. Values represent mean SEM. From [8].
The possible involvement of CYP24A1 in the etiology and progression of a number of diseases supports the development of inhibitors designed to effectively block CYP24A1 catalytic activity, thereby facilitating the activity of either endogenous vitamin D hormone or administered analog thereof. A number of CYP24A1 inhibitors have been identified, some of which act specifically and many of which do not. Being a member of the cytochrome P450 family of enzymes, CYP24A1 is equipped with a heme moiety at the catalytic core of the protein. Azole-based compounds have long been known to functionally inhibit a wide range of cytochrome P450 enzymes. The binding of such inhibitors occurs through the azole nitrogen of such compounds which coordinates heme iron [344]. Azoles bind heme with significantly stronger binding energies than the hydrogen-bonded water molecule which it displaces to bind to heme; consequently, these inhibitors block the catalytic cycle of the enzyme and prevent oxygen binding required for substrate oxidation. A number of such compounds including ketoconazole, econazole,
IX. ANALOGS
1540
80. CYP24A1 REGULATION IN HEALTH AND DISEASE
(B) (R)-VID-400
(A) Ketoconazole
N
O H3C
N
N
H N
O
N
N N
O
O
Cl
O Cl
Cl
(D) Sulfone CTA018
(C) Sulfoximine CTA091
24 O
HN O
22 H
S
23
23
S
O
Ph 16 H
H O NH 23 H3C
O
Ph
1 HO
S
24 H3C
S
O
1 OH
HO
OH
(E) 2-ethyl benzyl tetralone O
R
H3CO
R=CH2CH3
FIGURE 80.5 CYP24A1 inhibitors. (A) Ketoconazole; (B) (R)-VID-400; (C) sulfoximine CTA091; (D) sulfone CTA018; (E) 2-ethyl benzyl
tetralone.
fluconazole, metyrapone, itraconazole, and liarazole have been well studied. Some of these compounds, such as ketoconazole and fluconazole, have been widely used in the clinic mainly as antifungal agents because of their ability to inhibit the function of cytochrome P450 enzymes important for maintaining the structural components of fungal cell walls. Because of their mechanism of inhibition, many of these compounds also inhibit other cytochrome P450s, including CYP24A1. Liarazole has been shown to increase the half-life
of 1,25(OH)2D3 in prostate cancer cells (DU145) which exhibit CYP24A1 activity [285]. Similarly, studies have also demonstrated that ketoconazole can also significantly increase the half-life of co-administered 1,25(OH)2D3 [288]. Although inhibition of CYP24A1 by these compounds may prolong the biological activity of 1,25(OH)2D3 in vitro, both of these compounds are also potent inhibitors of CYP27B1, and therefore would not be useful candidates to facilitate the activity of endogenously produced 1,25(OH)2D3.
IX. ANALOGS
CYP24A1 INHIBITORS
Genistein Genistein (4,5,7-trihydroxyisoflavone), a plantderived isoflavonoid, has been widely suspected as one of the key dietary components linking consumption of soy products with lower cancer incidence [345]. Genistein and other isoflavonoids have been shown to inhibit vitamin-D-metabolizing enzymes in human prostate cancer cells DU145 [346], suggesting that at least some of its anti-cancer properties may stem from modulation of vitamin D signaling. Attempts to understand the activity of genistein have revealed that it may limit the expression, at the transcriptional level, and/or inhibit the catalytic activity of vitamin-D-metabolizing enzymes [347]. Genistein has been shown to inhibit transcription of both the CYP24A1 and CYP27B1 gene [347]. In the case of CYP27B1, the decrease in gene transcription is associated with histone deacetylase [347]. Moreover, when genistein was combined with a histone deacetylase inhibitor trichostatin, a synergistic effect in reducing CYP24A1 transcription was observed [346]. More recently, Swami et al. demonstrated that the use of a combination of 1,25(OH)2D3 and genistein could enhance the anti-cancer properties of 1,25(OH)2D3 [348]. The active vitamin D hormone 1,25(OH)2D3 can inhibit the growth of malignant pancreatic cells by affecting the prostaglandin (PG) signaling pathway. 1,25(OH)2D3 decreases cyclooxygenase-2 (COX-2) expression, stimulating 15-hydroxyprostaglandin dehydrogenase expression, and decreasing EP (PGE2) and FP (PGF2a) receptors, leading to cell growth inhibition [348]. Genistein itself can also interfere with the PG pathway by inhibiting both COX-2 expression and activity, as well as by inhibiting EP and FP prostaglandin receptors, leading to reduced biological activity of PGE2. Synergy between 1,25(OH)2D3 and genistein has also been demonstrated on adipogenesis and apoptosis in adipocytes [349]. A combination therapy using genistein and 1,25(OH)2D3 could be beneficial for the treatment of cancer where high CYP24A1 levels are present.
VID400 A number of lipophilic imidazole derivatives have been synthesized to achieve selectivity in the inhibition of CYP24A1. One such compound is VID400 which was first developed to enhance vitamin D signaling in keratinocytes by inhibiting CYP24A1 for the treatment of skin disorders, such as psoriasis [350]. VID400 has an estimated 40-fold selectivity for CYP24A1 over CYP27B1 and could be shown to enhance the effect of 1,25(OH)2D3 on keratinocytes by approximately two orders of magnitude [351]. The striking potentiation of vitamin D hormone activity by the administration of CYP24A1 inhibitors, such as VID400, clearly indicates
1541
that CYP24A1 can form a significant and effective barrier to block vitamin D signaling in target cells. Such compounds may have utility in the treatment of other diseases where CYP24A1 overexpression is implicated in the etiology or progression of disease.
CYP24A1 Specific Inhibitors The development of CYP24A1 inhibitors has typically exploited azole chemistry to specifically target heme. Consequently, such inhibitors inherently have the potential to inhibit other heme-containing cytochrome P450s and suffer from non-selectivity. While attempts have been made to increase the specificity of such compounds by making modifications to the scaffold attached to the azole moiety, exquisite selectivity has not yet clearly been demonstrated for such compounds. While many of the xenobiotic metabolizing P450s possess large substrate-binding pockets which can accommodate a wide range of substrates, CYP24A1 is much more selective for vitamin-D-related analogs reflecting a binding pocket more cognately related to its natural substrates, 25(OH)D3 and 1,25(OH)2D3. The selective nature of this binding pocket has been exploited to generate inhibitors which do not rely on heme coordination to block CYP24A1 activity. CTA091 (MK-24(S)-S(O)(NH)-Ph-1), a non-azole type CYP24A1 inhibitor, is both potent and highly selective [352]. This 1,25(OH)2D3-based inhibitor contains a 24(S)-NH phenyl sulfoximine D-ring side chain modification. Although the mechanism of CYP24A1 inhibition by this compound is not known, it does not detectably bind to the VDR (Fig. 80.6A) nor does it activate VDRmediated transcription (Fig. 80.6B), and therefore could be classified as a “pure” CYP24A1 inhibitor. It inhibits CYP24A1 with an IC50 in the low nanomolar range (~7.5 nM) and does not appreciably inhibit CYP27B1 or CYP27A1 [352] (Fig. 80.6C). Studies evaluating the impact of elevated CYP24A1 in lung cancer cells demonstrated that blockade of CYP24A1 with CTA091 resulted in decreased catabolism of 1,25(OH)2D3 and enhanced the sensitivity of cells to the anti-proliferative effects of 1,25(OH)2D3 by approximately 50-fold [280]. Synergistic inhibition of human breast tumor-derived MCF-7 cell proliferation has also been demonstrated by co-administration of CTA091 with 1,25(OH)2D3 (Fig. 80.6D). More recently, it has been shown that CTA091 can lower PTH levels in an animal model either by increasing the half-life of circulating 1,25(OH)2D3 and/or by reducing the impact of expressed CYP24A1 in target parathyroid gland cells [353]. Since it has also recently been demonstrated that CYP24A1 is elevated in kidney from uremic rats or CKD patients, such compounds may be useful alternatives to active vitamin D compounds for the treatment of secondary hyperparathyroidism [8].
IX. ANALOGS
1542
80. CYP24A1 REGULATION IN HEALTH AND DISEASE
(B)
100
50
0 10-11
110 100 90 80 70 60 50 40 30 20 10 0
Relative CYP24 Induction (%)
B/Bmax (%)
(A)150
10-10
10-9 10-8 Concentration (M)
10-7
1,25(OH)2D3 CTA091
0
10-6
1 10 Log Concentration (nM)
100
1,25(OH)2D3 B50 ~ 59 nM CTA091 B50 >2000 nM
(D) Incorporation (cpm)
(C)
100 75 50
-
3H-thymidine
CYP24 Activity (%)
125
25 0 0-
10-10
10-9
10-8
10-7
10-6
0.1 nM
14000
1 nM
12000
10 nM
10000
50 nM
8000 6000 4000 2000
10-5
0 0
Concentration (M)
10-11 10-10 10-9 10-8 10-7 1,25(OH)2D3 (10xM)
10-6
Ketoconazole IC50 ~ 265 nM CTA091 IC50 ~ 7 nM
FIGURE 80.6 CTA091 is a highly selective and potent CYP24A1 inhibitor. (A) Percent B/Bmax for 1,25(OH)2D3 and CTA091 are plotted as
a function of increasing concentration (106e1010 M). B50 values for 1,25(OH)2D3 and each analog are shown below each figure. (B) Relative induction of CYP24A1 mRNA (%) caused by 1,25(OH)2D3 and CTA091 at 1, 10, and 100 mM. Data are presented as mean SD. (C) Relative values of CYP24A1 activity (%) are plotted as a function of inhibitor concentration: ketoconazole (105e109 M), CTA091 (106e1010 M). Data are presented as mean SD. IC50 values for ketoconazole and each analog are shown below each figure. (D) MCF-7 cells were treated with 1,25(OH)2D3 (106e1011) in the presence or absence of CTA091 (0.1, 1.0, 10, and 50 nM). 3H-thymidine incorporation was used to evaluate relative rates of cell proliferation. Data are presented as mean SD. Panels (AeC) are from [353].
Compounds which can both inhibit CYP24A1 and activate VDR-mediated signaling are another approach to avoid the problems associated with disease-related expression of CYP24A1. The sulfone GHP-GH-16,23diene-25S02-1 (CTA018) is a potent activator of VDRmediated transcription not readily affected by the intracellular expression of CYP24A1. CTA018 inhibits CYP24A1 with an IC50 27 6 nM, about 10 times more potent than the non-selective CYP24A1 inhibitor ketoconazole (253 20 nM). Unlike CTA091, CTA018 readily binds the VDR and induces VDR-mediated gene expression. Although CTA018 binds to the VDR with an affinity that is 15-fold lower than that for 1,25(OH)2D3, this analog is approximately 10-fold more potent than
1,25(OH)2D3 in activating VDR-mediated transcription. When administered to uremic rats, CTA018 has been shown to effectively suppress elevated plasma intact PTH at doses which have no effect on serum calcium or phosphorus. Therefore, this analog has potential utility in treating secondary hyperparathyroidism in CKD [8]. A phase II clinical trial with CTA018 is currently in progress in patients with end-stage renal disease and secondary hyperparathyroidism. Vitamin D analogs such as CTA018 which can avoid CYP24A1 metabolism could be useful in the treatment of cancers overexpressing CYP24A1 since it has recently been demonstrated that blocking CYP24A1 can enhance tumor responsiveness to 1,25(OH)2D3 [288].
IX. ANALOGS
REFERENCES
Tetralone-based Inhibitors Recently, a series of non-vitamin-D-related, nonazole-based inhibitors have been synthesized which have been derived from 2-substituted tetralone derivatives [354]. In a second-generation series of these compounds, investigators were able to identify a 2ethyl-substituted benzyl tetralone (IC50 ~2.0 mM) comparable in potency to ketoconazole (IC50 ~0.5 mM) [355]. However, at this early stage of development such compounds did not exhibit selectivity for CYP24A1 over CYP27A1. The recent crystallization of CYP24A1 [75] and the generation of models of CYP24A1 selectivity [351] may be useful in guiding the synthesis of more selective derivatives of these molecules. These studies hold promise that further generations of non-vitaminD-related compounds which are not azole based may yield potent and selective inhibitors of CYP24A1.
CONCLUSION AND FUTURE DIRECTIONS A number of diseases are associated with vitamin D deficiency. Although external factors, such as nutrition and sun exposure, have a major impact on vitamin D status, emerging evidence suggests that accelerated catabolism may also play a key role in vitamin D depletion, as well as influence the pathogenesis and progression of these disease states. Aberrant expression of CYP24A1 may be a critical factor contributing to the depletion of systemic levels of vitamin D in diseases characterized by vitamin D deficiency, but also may deprive CYP24A1-expressing tissues of the important anti-proliferative and anti-inflammatory activities of vitamin D. From recent studies it seems plausible that CYP24A1 expression associated with kidney disease caused by kidney injury or diabetes may contribute to the progressive deterioration of kidney function, ultimately leading to renal failure. Similarly, abnormally elevated levels of CYP24A1 observed in a number of different cancers may become resistant to the growth-suppressive effects of 1,25(OH)2D3. These resistant tumors may arise through a process of selection whereby subpopulations of cells within tumors that can rapidly catabolize 25(OH)D3 and 1,25(OH)2D3 will emerge as a result of their growth advantage. If the tumor burden is sufficient, such CYP24A1-expressing tumor cells may contribute to systemic depletion of vitamin D stores. It is clear that elevation of CYP24A1 can arise via multiple mechanisms, whether through gene amplification or dysregulation of the numerous signaling pathways described above (Fig. 80.2). Resolving the roles of these additional pathways in CYP24A1 regulation may lead to novel therapeutic strategies to block the deleterious
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effects of accelerated vitamin D catabolism; however, direct blockade of CYP24A1 using specific inhibitors alone or in combination with 1,25(OH)2D3 may be the optimal therapeutic approach. Such inhibitors would have a wide applicability in various diseases where CYP24A1 expression contributes to the disease state. The recent development of novel classes of CYP24A1 inhibitors will be valuable tools in establishing CYP24A1 as a viable target in the treatment of a number of important diseases.
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Human Services, Centers for Disease Control and Prevention, 2008, Atlanta, GA, 2007. D.M. Nathan, Long-term complications of diabetes mellitus, N. Engl. J. Med. 328 (1993) 1676e1685. E. Ritz, S.R. Orth, Nephropathy in patients with type 2 diabetes mellitus, N. Engl. J. Med. 341 (1999) 1127e1133. J.R. Sowers, M. Epstein, E.D. Frohlich, Diabetes, hypertension, and cardiovascular disease: an update, Hypertension 37 (2001) 1053e1059. P. Pozzilli, S. Manfrini, A. Crino, A. Picardi, C. Leomanni, V. Cherubini, et al., Low levels of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 in patients with newly diagnosed type 1 diabetes, Horm. Metab. Res. 37 (2005) 680e683. D.J. Di Cesar, R. Ploutz-Snyder, R.S. Weinstock, A.M. Moses, Vitamin D deficiency is more common in type 2 than in type 1 diabetes, Diabetes Care 29 (2006) 174. B. Littorin, P. Blom, A. Scholin, H.J. Arnqvist, G. Blohme, J. Bolinder, et al., Lower levels of plasma 25-hydroxyvitamin D among young adults at diagnosis of autoimmune type 1 diabetes compared with control subjects: results from the nationwide Diabetes Incidence Study in Sweden (DISS), Diabetologia 49 (2006) 2847e2852. A. Bener, A. Alsaied, M. Al-Ali, A. Al-Kubaisi, B. Basha, A. Abraham, et al., High prevalence of vitamin D deficiency in type 1 diabetes mellitus and healthy children, Acta Diabetol. 46 (2009) 183e189. G. Isaia, R. Giorgino, S. Adami, High prevalence of hypovitaminosis D in female type 2 diabetic population, Diabetes Care 24 (2001) 1496. A.M. Borissova, T. Tankova, G. Kirilov, L. Dakovska, R. Kovacheva, The effect of vitamin D3 on insulin secretion and peripheral insulin sensitivity in type 2 diabetic patients, Int. J. Clin. Pract. 57 (2003) 258e261. A.G. Pittas, J. Lau, F.B. Hu, B. Dawson-Hughes, The role of vitamin D and calcium in type 2 diabetes. A systematic review and meta-analysis, J. Clin. Endocrinol. Metab. 92 (2007) 2017e2029. H. Ishida, Y. Seino, K. Tsuda, J. Takemura, S. Nishi, S. Ishizuka, et al., Effects of streptozotocin-induced diabetes on circulating levels of vitamin D metabolites, Acta. Endocrinol. (Copenh.) 104 (1983) 96e102. H. Ishida, Y. Seino, S. Nishi, N. Kitano, M. Seno, T. Taminato, et al., Effects of insulin on altered mineral and vitamin D metabolism in streptozotocin-induced diabetes, Acta. Endocrinol. (Copenh.) 108 (1985) 231e236. T. Matsumoto, Y. Kawanobe, I. Ezawa, N. Shibuya, K. Hata, E. Ogata, Role of insulin in the increase in serum 1,25-dihydroxyvitamin D concentrations in response to phosphorus deprivation in streptozotocin-induced diabetic rats, Endocrinology 118 (1986) 1440e1444. J. Verhaeghe, E. van Herck, W.J. Visser, A.M. Suiker, M. Thomasset, T.A. Einhorn, et al., Bone and mineral metabolism in BB rats with long-term diabetes. Decreased bone turnover and osteoporosis, Diabetes 39 (1990) 477e482. E. Ishimura, Y. Nishizawa, H. Koyama, S. Shoji, M. Inaba, H. Morii, Impaired vitamin D metabolism and response in spontaneously diabetic GK rats, Miner. Electrolyte Metab. 21 (1995) 205e210. The EURODIAB Substudy 2 Study Group, Vitamin D supplement in early childhood and risk for type I (insulin-dependent) diabetes mellitus, Diabetologia 42 (1999) 51e54. E. Hypponen, E. Laara, A. Reunanen, M.R. Jarvelin, S.M. Virtanen, Intake of vitamin D and risk of type 1 diabetes: a birth-cohort study, Lancet 358 (2001) 1500e1503.
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[347] H. Farhan, K. Wahala, H.S. Cross, Genistein inhibits vitamin D hydroxylases CYP24 and CYP27B1 expression in prostate cells, J. Steroid Biochem. Mol. Biol. 84 (2003) 423e429. [348] S. Swami, A.V. Krishnan, J. Moreno, R.B. Bhattacharyya, D.M. Peehl, D. Feldman, Calcitriol and genistein actions to inhibit the prostaglandin pathway: potential combination therapy to treat prostate cancer, J. Nutr. 137 (2007) 205Se210S. [349] S. Rayalam, M.A. Della-Fera, J.Y. Yang, H.J. Park, S. Ambati, C.A. Baile, Resveratrol potentiates genistein’s antiadipogenic and proapoptotic effects in 3T3-L1 adipocytes, J. Nutr. 137 (2007) 2668e2673. [350] I. Schuster, H. Egger, N. Astecker, G. Herzig, M. Schussler, G. Vorisek, Selective inhibitors of CYP24: mechanistic tools to explore vitamin D metabolism in human keratinocytes, Steroids 66 (2001) 451e462. [351] I. Schuster, H. Egger, P. Nussbaumer, R.T. Kroemer, Inhibitors of vitamin D hydroxylases: structureeactivity relationships, J. Cell Biochem. 88 (2003) 372e380.
[352] M. Kahraman, S. Sinishtaj, P.M. Dolan, T.W. Kensler, S. Peleg, U. Saha, et al., Potent, selective and low-calcemic inhibitors of CYP24 hydroxylase: 24-sulfoximine analogues of the hormone 1a,25-dihydroxyvitamin D3, J. Med. Chem. 47 (2004) 6854e6863. [353] G.H. Posner, C. Helvig, D. Cuerrier, D. Collop, A. Kharebov, K. Ryder, et al., Vitamin D analogues targeting CYP24 in chronic kidney disease, J. Steroid Biochem. Mol. Biol. 121 (2010) 13e19. [354] S.W. Yee, C. Simons, Synthesis and CYP24 inhibitory activity of 2-substituted-3,4-dihydro-2 H-naphthalen-1-one (tetralone) derivatives, Bioorg. Med. Chem. Lett. 14 (2004) 5651e5654. [355] A.S. Aboraia, B. Makowski, A. Bahja, D. Prosser, A. Brancale, G. Jones, et al., Synthesis and CYP24A1 inhibitory activity of (E)-2-(2-substituted benzylidene)and 2-(2-substituted benzyl)-6-methoxy-tetralones, Eur. J. Med. Chem. 45 (2010) 4427e4434.
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C H A P T E R
81 Calcitriol and Analogs in the Treatment of Chronic Kidney Disease Ishir Bhan, Ravi Thadhani Harvard Medical School, Massachusetts General Hospital, Boston, MA, USA
INTRODUCTION While vitamin D deficiency is common in the general population, chronic kidney disease (CKD) represents a particular challenge in management of this condition. While readily available therapies such as ergocalciferol and cholecalciferol can be employed in individuals with normal renal function, the decline in renal 1ahydroxylase activity that accompanies CKD renders these therapies less effective. Without the ability to convert nutritional forms of vitamin D to the hormonally active form, patients with CKD develop impaired ability to mobilize calcium this leads to hypocalcemia and, consequently, secondary hyperparathyroidism (sHPT). Since the underlying metabolic deficit is the inability to convert 25(OH)D to 1,25(OH)2D, a rational therapy is to supplement with vitamin D that is pre-hydroxylated at the first position, so as to bypass the requirement for the renal 1a-hydroxylase. The most common form of this therapy is calcitriol, or 1,25(OH)2D3, the active metabolite of cholecalciferol. Calcitriol suppresses parathyroid hormone (PTH) production and consequently decreases the risk of bone disease such as osteitis fibrosa [1]. However, use of this therapy is not without limitations. Calcitriol can promote intestinal phosphate absorption, which can lead to hyperphosphatemia. Hyperphosphatemia itself can further stimulate the parathyroid gland, counterproductively worsening the sHPT that calcitriol aimed to address [2]. While calcitriol can effectively treat hypocalcemia, the high doses sometimes needed to suppress profound elevations in parathyroid hormone can be complicated by hypercalcemia. These metabolic consequences of calcitriol use are particularly concerning in light of the fact that advanced CKD is marked by excessive soft tissue and vascular calcification; the latter of
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10081-2
these likely contributes to the profound excess cardiovascular mortality observed in this population. Management of both sHPT and complications of the treatments designed to address it represent a significant clinical challenge in the treatment of kidney disease. The subject of vascular calcification is discussed in Chapter 73 and the general subject of vitamin D and the kidney is covered in Chapter 70. Several studies have linked both sHPT and abnormalities in calcium and phosphorus to poor clinical outcomes, particularly in individuals with end-stage renal disease (ESRD) on hemodialysis. In one large cohort of over 58 000 maintenance hemodialysis patients, hypercalcemia, hyperphosphatemia, and hyperparathyroidism were all significant predictors of mortality [3]. Another study of over 40 000 hemodialysis patients also found that higher calcium and phosphate levels were independently linked to death, along with severe uncontrolled sHPT (parathyroid hormone 600 pg/ml) [4]. A recent study of non-dialysis-dependent patients with CKD similarly found that higher serum calcium was associated with long-term mortality [5]. Nephrologists must therefore take great care to balance active vitamin D therapy with other measures to prevent excessive calcium and phosphate loading. Oral phosphate binders, particularly those lacking calcium, have been viewed as a helpful adjunct in this regard, but management remains difficult. Since the initial adoption of calcitriol to treat sHPT, several analogs have come into clinical use, many of which are suspected to have safety profiles and metabolic properties distinct from calcitriol. The ideal analog, or vitamin D receptor (VDR) activator, would likely retain the beneficial effects of calcitriol on sHPT (and potentially on pathways outside the traditional axis of bone
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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metabolism) while minimizing excess calcium and phosphate absorption. In this chapter, we will review the data surrounding the use of calcitriol and its analogs in the management of CKD, discuss how these analogs compare with calcitriol, and evaluate their potential role in ameliorating CKD-associated cardiovascular disease.
another synthetic derivative of calcitriol, with an oxygen replacing the carbon at position-22. Falecalcitriol is a third D3 derivative, a 25-hexafluorinated calcitriol. Alfacalcidol, maxacalcitol, and falecalcitriol are not available in the USA, but have been extensively studied in other parts of the world. Section IX of this book discusses various analogs under development and their use in multiple clinical settings.
ANALOGS OF VITAMIN D USED IN CKD The nutritional forms of vitamin D, ergocalciferol (D2) and cholecalciferol (D3), are not considered analogs of active vitamin D since they still require conversion by 1a-hydroxylase. Deficiency of 25(OH)D is present in the majority of patients with ESRD, but the clinical significance of this remains unclear. Some studies have suggested that, while nutritional forms of vitamin D may help ameliorate sHPT in stage 3 CKD, their efficacy appears limited in more advanced CKD [6]. The presence of extra-renal 1a-hydroxylase raises exciting questions surrounding the potential action of these therapies outside of the traditional axis of PTH (including paracrine actions where conversion to 1,25(OH)2D takes place in target tissues). Co-treatment with active and nutritional forms of vitamin D has gained popularity in recent years for this reason. While randomized trials to assess the clinical efficacy and safety of this therapy in advanced kidney disease are underway [7], there are little currently available data to guide therapy of patients with ESRD or late CKD. For this reason we will focus on data surrounding the use of active vitamin D analogs. Three forms of active vitamin D are available in the USA. Calcitriol, which is equivalent to the endogenous 1,25(OH)2D3, has the longest history. Two synthetic analogs are now in use in the USA. The best studied of these is paricalcitol (ZemplarÔ), or 19-nor-1,25(OH)2D2, a derivative of activated ergocalciferol (vitamin D2). Although less thoroughly examined in CKD than paricalcitol, several studies have now also evaluated another derivative of ergocalciferol, doxercalciferol (HectorolÔ), or 1a(OH)D2. Since doxercalciferol is pre-hydroxylated at the first carbon position, it requires only hydroxylation in position-25 by the liver to obtain biological activity. As doxercalciferol and paricalcitol are the best studied of the synthetic analogs, this chapter will focus on these agents in comparison to calcitriol. Three additional analogs of vitamin D have been studied in CKD, though less widely than paricalcitol and doxercalciferol. Alfacalcidol is a synthetic prohormone, 1a(OH)D3, or cholecalciferol pre-hydroxylated in the first position. As the D3 analog of doxercalciferol, it too requires hepatic 25-hydroxylation to obtain full biologic activity. Maxacalcitol (22-oxacalcitriol) is
ANALOGS IN ANIMAL MODELS OF UREMIA Many of the findings surrounding differences in activity between calcitriol and the synthetic vitamin D analogs are derived from animal models of uremia. Studies of these agents in humans are often complicated by the effects of other medications (such as phosphorus binders and calcimimetics), heterogeneity in the population, diet, and variation in clinical practice, confounding factors that can be controlled and therefore do not usually affect animal studies. However, there are important limitations in extending the results of these studies to the management of human CKD. Most of these studies use a rat model of CKD in which 5/6th of renal mass is surgically removed (5/6th nephrectomy model), which may differ from human disease. These agents are typically studied over a substantially shorter time period than in humans, and treatments such as hemodialysis are not available in animals. Furthermore, there may be important fundamental differences in vitamin D biology between rodents and humans. For example, the antimicrobial peptide cathelicidin is under regulation by vitamin D in humans, but not in mice [8]; other clinically significant differences may also be present. Despite this, these studies present a useful biological complement to the more limited human data. The majority of research in uremic animals on paricalcitol, the best studied of these analogs, has been done by the Slatopolsky laboratory. One early study used a 5/6th nephrectomy rat model of uremia to compare the actions of paricalcitol (25e100 ng, three times/week) to those of calcitriol (2 or 6 ng, three times/2 weeks) [9]. In this study, paricalcitol and calcitriol both suppressed PTH, but paricalcitol did not increase serum calcium or phosphorous while calcitriol did. Of note, calcitriol appeared to further increase intestinal concentrations of the VDR relative to paricalcitol, suggesting a potential mechanism for this difference. Intestinal RNA for proteins suspected to be involved (CaT1, Calbindin D9K, and PCMA1) was also lower in paricalcitol-treated animals compared with their calcitriol-treated counterparts [10]. This research group also found that, in vitamin-Ddeficient rats, the differences in calcium absorption between calcitriol and paricalcitol appeared to increase
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with time [10]. In addition to these models, a study involving rats that had undergone parathyroidectomy found that, in this situation as well, paricalcitol had markedly reduced effects on serum calcium and phosphorus when compared with calcitriol [11]. Recently, other groups have also performed extensive animal studies addressing the differential effects of calcitriol and other analogs. Lopez and colleagues studied a rat model of uremia and sHPT and examined the effects of both calcitriol and paricalcitol over 28 days. Rats underwent a 5/6th nephrectomy and were placed on a low-calcium, high-phosphate diet. Animals were then given intraperitoneal (IP) monotherapy with either calcitriol (80 ng/kg q 48 h), paricalcitol (240 ng/kg q 48 h), an experimental calcimimetic drug (AMG 641), or given AMG 641 in combination with one of the vitamin D analogs [12]. The calcimimetic had no significant effects on 28-day survival but, in contrast to effects suggested by other studies in humans (see below), both calcitriol and paricalcitol were associated with a significant decrease in survival. The effect was more dramatic in calcitriol (28-day survival: 18%) versus paricalcitol (50%), although both groups performed significantly worse than vehicle-treated rats (100% survival). While co-treatment with AMG 641 helped to ameliorate this increase in mortality in both groups, calcitriol-treated rats continued to fare worse than their paricalcitoltreated counterparts. Compared with paricalcitoltreated rats, calcitriol-treated animals had significantly higher plasma calcium and significantly lower PTH. Serum phosphorous was also lower with paricalcitol, although this did not reach statistical significance. Differences between the two VDR analogs were more striking when vascular calcification was more directly measured. Aortic calcium and phosphorus content was significantly lower at both 14 and 28 day time points in rats treated with paricalcitol versus calcitriol. While it is tempting to take these data as evidence of superiority of the paricalcitol over calcitriol, there are several caveats to this interpretation. Both agents were associated with a dramatic increase in mortality when compared with animals that received no treatment, an observation that is not generally seen in humans. Phosphorous levels in all nephrectomized animals were poorly controlled, with vehicle-treated rats reaching mean levels of over 12 mg/dl (compared with shamoperated rats, which had phosphorous levels less than half this level); calcitriol-treated animals reached levels of nearly 20 mg/dl. These are markedly abnormal levels of phosphorus that, in typical human treatment protocols, warrant avoidance of VDR agonists until phosphate control has been achieved. In addition, mean ionized calcium levels were universally low in this study, below 1 mM in all nephrectomized animals except those receiving calcitriol (sham-operated rats
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had levels of 1.2 mM). Even ignoring likely differences in the biology of vitamin D between rodents and humans, generalizability of the findings is likely limited by the markedly ineffective calcium and phosphorous control. Differences between the calcitriol and paricalcitol in this state of metabolic disarray may thus not persist in a metabolically controlled environment. Lastly, any comparison of vitamin D analogs may be affected by relative dosing of the agents. These authors employed a 3:1 ratio of paricalcitol:calcitriol whereas many others have used a 4:1 dosing regimen, based largely on equivalency studies of comparing the two analogs with respect to PTH suppression [13]; in a metabolic environment favoring complications of vitamin D therapy, this lower ratio may favor calcitriol. Further information on the differential actions of analogs can be found in Chapter 75. Cardus et al. attempted to study the differential effects of calcitriol and paricalcitol both in a 5/6th nephrectomy model and also by assessing vascular smooth muscle cell (VMSC) calcification in vitro [14]. At drug concentrations of 100 and 300 nM, cultured VSMC incorporated calcium when treated with calcitriol, but not paricalcitol, suggesting a direct effect on calcium deposition in the vasculature. At 100 nM, calcitriol also was more than twice as potent at increasing transcription of RANKL in these cells, though this may have been due to the relative potencies of these agents. In a rat model of CKD with 5/6th nephrectomy (but without a low-calcium, high-phosphate diet), effects of these analogs were assessed. Both calcitriol (1 mg/kg, three times/week) and paricalcitol (3 mg/kg, three times/week) increased aortic calcification over an 8-week period, but this effect was more dramatic with calcitriol (Fig. 81.1). Despite the observed difference in vascular calcification, the drugs appeared similar with respect to effects on mineral metabolism. While both treatments increased calcium levels and, to a nonsignificant degree, phosphate, there was no significant difference between calcitriol and paricalcitol. PTH levels were also similar between the two groups. In addition, while both calcitriol and paricalcitol increased systolic blood pressure to a similar degree, paricalcitol treatment was accompanied by a greater increase in diastolic pressure than calcitriol. As a result, pulse pressure, a measure that is believed to correspond to vascular stiffness and poor cardiovascular outcomes in humans, was greater in calcitriol-treated animals. While survival was not analyzed by these authors, there are some marked differences from the study by Lopez et al. While rats in the prior study were generally hypocalcemic, those in this study were mildly hypercalcemic. While hyperphosphatemia was also present here, it was considerably milder than in the study by Lopez, with mean phosphorous levels between 6 and 7 mg/dl. PTH levels
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(A)
81. CALCITRIOL AND ANALOGS IN THE TREATMENT OF CHRONIC KIDNEY DISEASE
(B)
(C)
FIGURE 81.1 In an animal model of uremia, Cardus et al. found that, when compared with control animals (A), both paricalcitol (B; 3 mg/kg) and calcitriol (A; 1 mg/kg) increased aortic calcification over an 8-week period; this effect was more marked with calcitriol. Reproduced with permission from [14]. Please see color plate section.
were also suppressed to a greater degree by both calcitriol and paricalcitol, with mean levels below 100 pg/ml with both treatments. These differences are likely explained by the different diets and drug dosages used in each protocol and highlight the limitations in extending the interpretation of these studies to real-world clinical practice in CKD patients. As a phenomenon potentially associated with vitamin D analog treatment, vascular calcification has been of particular interest because of the possible causative link to left ventricular hypertrophy (LVH), diastolic dysfunction, and increased cardiovascular mortality that is commonly observed in dialysis patients. While the prior two studies in rats found that VDR agonist use contributed to the development of vascular calcification in their CKD models, another study found the opposite by using a different model of CKD in mice [15]. Mice lacking the low-density lipoprotein receptor (LDLR/) fed a high-fat diet develop CKD, hyperphosphatemia, and vascular calcification. Administration of low-dose calcitriol (20 ng/kg, three times/ week) and paricalcitol (100 ng/kg, three times/week) was associated with significantly less aortic calcification compared to untreated mice; there was no difference between the two VDR agonists at low doses, though high-dose paricalcitol (400 ng/kg) led to increased vascular calcification. At all doses, paricalcitol increased bone volume while calcitriol did not. Paradoxically, both agents decreased serum phosphate levels compared with untreated animals. Mizobuchi et al. explored this relationship further in 5/6th nephrectomized rats comparing three times/ week regimens of calcitriol (40 ng/kg), doxercalciferol (160 ng/kg), and paricalcitol (160 ng/ml) [16]. While both calcitriol- and doxercalciferol-treated rats developed significantly increased aortic calcification, paricalcitol-treated animals did not. The authors also observed that paricalcitol’s effects on both calcium and phosphorus levels were similarly blunted when compared with either doxercalciferol or calcitriol, although its
suppression of PTH was also slightly (though nonsignificantly) less potent. However, even after the doxercalciferol dose was decreased (to 100 ng/kg) and that of paricalcitol increased (to 240 ng/kg), doxercalciferol continued to cause greater elevation of both ionized and total calcium levels, as well as significantly more aortic calcification. Comparative studies such as this, which contrast two or more non-calcitriol vitamin D analogs, are uncommon. Noonan et al. also compared paricalcitol and doxercalciferol (at doses of 83, 167, and 333 ng/kg, three times/week) and found that doxercalciferol induced significantly more calcium and phosphorus deposition in the aorta, with a relative increase in pulse wave velocity [17]. A recent study examined 5/6th nephrectomized rats comparing doxercalciferol and paricalcitol [18]. Paricalcitol was less potent at increasing serum calcium and more effective at inducing the calcium-sensing receptor compared with doxercalciferol. Neither had a significant effect on serum phosphate. An earlier study had suggested that paricalcitol was less potent than alfacalcidol at promoting calcium and phosphorous in normal and uremic rats [19] (Fig. 81.2). Given observations that vitamin D analogs may suppress the renineangiotensinealdosterone axis, Kong et al. compared the effect of paricalcitol or doxercalciferol (both 400 ng/kg, three times/week over 2 months), alone and in combination with losartan, in spontaneously hypertensive rats (a model of hypertension and left ventricular hypertrophy) [20]. Both vitamin D analogs reduced wall thickness, an effect that was potentiated by co-treatment with losartan. Despite differences in their metabolism, the performance of paricalcitol and doxercalciferol was similar, with neither agent demonstrating a distinct advantage. No comparison with calcitriol, however, was performed. In summary, a range of animal studies have compared the efficacy and safety of vitamin D analogs in models of CKD, often yielding conflicting results
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FIGURE 81.2 One study compared the effects of calcitriol (1,25D3), paricalcitol (19-nor D2), and alfacalcidol (1a(OH)D2) over a 14-day period in rats. Calcitriol and alfacalcidol demonstrated significantly increased calcium and phosphorus absorption while paricalcitol’s effects on mineral absorption were not significantly different from vehicle alone. * p < 0.01 versus control rats; y p < 0.01 versus 19-nor treated rats. Reproduced with permission from [19].
depending on dosing and dietary factors. In several studies, paricalcitol appeared to be less potent than calcitriol or doxercalciferol at promoting vascular calcification. These findings, however, may not be directly generalizable to human disease, where calcium and phosphorous are typically monitored and controlled during treatment with vitamin D analogs.
CKD STAGES 3e4 While studies of calcitriol and its analogs are most well established in the ESRD literature, these agents are also used in patients with CKD who have not yet been initiated on dialysis. sHPT, the primary clinical indication for use of these agents, begins long before renal replacement is needed. While correction of 25(OH)D is an important component of sHPT in stage 3 CKD, its contribution to the management of this disorder is generally thought to diminish as CKD progresses, so active analogs are often instituted [6]. Stages 3 and 4 CKD (Table 81.1) affect a much larger number of individuals than are afflicted with ESRD, and hence the impact of calcitriol and its analogs would be broader if instituted in this population. The impact of differences TABLE 81.1
Stages of Chronic Kidney Disease (CKD)
Stage
Description
GFR
1
Kidney damage with normal or increased GFR
90*
2
Kidney damage with mild decrease in GFR
60e89*
3
Moderate decrease in GFR
30e59
4
Severe decrease in GFR
15e29
5
Kidney failure
<15 (or dialysis)
* Stages 1 and 2 require other evidence of kidney damage (e.g., proteinuria) whereas stages 3e5 can be defined by GFR alone. GFR ¼ glomerular filtration rate (ml/min/1.73 m2). Adapted from [50].
in safety or effectiveness between agents would be similarly magnified. Oral calcitriol is used extensively in predialysis CKD, and there is a long history of experience with this agent [21]. Alfacalcidol, paricalcitol, and doxercalciferol are also available in oral formulations, enabling use in early stages of CKD. A double-blind placebo-controlled randomized trial of patients with stages 3e4 CKD compared the effects of alfacalcidol versus placebo at 17 centers in Europe [22]. Of the 176 patients who enrolled in the study, the majority had histologically demonstrated bone disease (predominantly osteitis fibrosa) prior to initiating therapy. A titration regimen was used to adjust dose based on the serum calcium level. Over the 2-year follow-up period, patients treated with placebo had increases in both alkaline phosphatase and PTH, while these levels remained unchanged with alfacalcidol. Subjects with abnormal bone histology prior to treatment were also more likely to have this resolve with alfacalcidol. As might be expected, calcium levels were significantly higher with alfacalcidol. The study protocol adjusted drug dose to maintain calcium at the upper limit of normal. Overt hypercalcemia occurred more frequently in the alfacalcidol group, though was infrequent in both groups; the difference was not statistically significant. This study suggested that alfacalcidol was an effective means of treating bone disease in pre-dialysis CKD, though there was no direct comparison with calcitriol. A smaller 3-month study compared doxercalciferol to placebo in 55 subjects with stages 3e4 CKD and sHPT [23]. In this double-blind-controlled trial, subjects were randomized to 1 mg per day of doxercalciferol or placebo, with a dose adjustment protocol if PTH failed to decline by at least 30% over 1 month in the absence of hypercalcemia or hyperphosphatemia. Subjects treated with doxercalciferol had significantly lower PTH levels after 24 weeks than those treated with placebo, with the majority (74%) achieving an over 30% reduction in PTH as opposed to only 7% of placebo-treated individuals. Total and bone-specific
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alkaline phosphatase levels also decreased significantly with doxercalciferol, but not placebo. There was no difference in 24-hour urinary calcium excretion, change in creatinine clearance, or phosphate binder usage. While phosphate levels were higher in those treated with doxercalciferol at 24 weeks, the fraction of patients with overt hypercalcemia or hyperphosphatemia was not significantly different. Thus, doxercalciferol appeared to be effective at influencing biochemical markers of bone disease without a marked adverse impact on calcium and phosphorus metabolism. Paricalcitol was also studied in CKD using an oral formulation. Coyne et al. studied 220 patients with stages 3e4 CKD as part of three randomized placebocontrolled trials [24]. Like previous studies, a dosetitration protocol was used. As with doxercalciferol treatment in the prior study, paricalcitol was significantly more likely than placebo to produce sustained reductions in PTH. There was no difference in the incidence of hypercalcemia, hyperphosphatemia, or urinary excretion of either calcium or phosphorous, though the paricalcitol group was noted to have a significantly lower serum calcium level at baseline. Changes in renal function were similar in both groups. Thus, paricalcitol, like the other analogs, appeared to effectively suppress PTH without inducing significantly more adverse effects on mineral metabolism than placebo. Effects of vitamin D analogs on outcomes outside of mineral metabolism are likely to be an increasingly common focus of future studies. PRIMO is a randomized-controlled trial (currently ongoing) comparing the effects of paricalcitol to placebo on cardiac morphology and functions in patients with stages 3 to 4 chronic kidney disease and LVH [25]. VITAL is a recently published randomized controlled trial which found that paricalcitol reduced albuminuria in patients with type 2 diabetes and diabetic nephropathy [26]. Direct comparisons between different agents are uncommon in the CKD literature. While some reviews suggest that paricalcitol and doxercalciferol appear to have similar efficacy [27], there is no evidence comparing these agents as part of a single study. A recent, albeit small (n ¼ 47) randomized trial compared doxercalciferol with cholecalciferol in stages 3e4 CKD. Subjects had PTH levels above the KDOQI target ranges and were largely vitamin-D-insufficient (mean 25(OH)D 14.6 ng/dl). While only doxercalciferol significantly suppressed PTH, the difference between agents was not statistically significant, likely owing to the small size of the study. This study also contrasted an active analog (doxercalciferol) that requires only hepatic hydroxylation with a nutritional form of vitamin D that requires both renal and hepatic metabolism; the results for cholecalciferol could not be generalized to calcitriol.
Enthusiasm for the use of newer analogs in CKD must be tempered by the limited clinical data, particularly comparisons with calcitriol. Of note, recent data suggest that oral calcitriol is associated with improved survival in CKD [28,29]. As effects on survival may act through mechanisms other than changes in mineral metabolism, effects on mortality and other clinically relevant outcomes in CKD will be important in informing the selection of agents for treatment.
ESRD Calcitriol has become a widely adopted therapy in the management of sHPT in ESRD. As calcitriol’s use increased, there was a significant drop in the rates of parathyroidectomy in this population [30]. Newer analogs, which some animal studies suggested might have equivalent or superior suppression of PTH with less promotion of calcium or phosphorus absorption, are becoming increasingly popular. In contrast to studies of stage 3 and 4 CKD, there is considerably more human research comparing different analogs in ESRD.
Effects on Mineral Metabolism As with the animal literature, most studies have focused on mineral metabolism as a measure of safety and efficacy. One small double-blind randomized crossover trial (n ¼ 22) compared paricalcitol with calcitriol [31]. In this brief study, ESRD patients on hemodialysis underwent washout of any active vitamin D for 1 week and then were randomized to either paricalcitol (6 mg, given intravenously three times per week at each dialysis) or calcitriol (2 mg) for the next six dialysis sessions. This was followed by another washout period and six more treatments with the crossover drug. Fractional intestinal calcium absorption, measured by a tracer-laden oral calcium load, was 14% lower in paricalcitol, though the authors noted that the overall absorption was low with calcitriol as well. There were no differences in PTH or phosphate levels, and the short study duration made long-term projections impossible. Another small randomized trial that used weight-based dosing in a 4:1 ratio of paricalcitol to calcitriol found that paricalcitol, but not calcitriol, significantly reduced PTH while serum calcium was only increased by calcitriol, lending support to the suggestion that paricalcitol may be a less calcemic analog [32]. Sprague et al. performed a larger, multi-center, double-blind randomized controlled trial comparing paricalcitol and calcitriol over 32 weeks in prevalent hemodialysis patients, also using a dosing ratio of 4:1 [33]. The therapies were titrated to a 50% reduction
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ESRD
in PTH levels. A 2-week washout period was used for subjects receiving calcitriol, dihydrotachysterol, alfacalcidol, or calcitonin. All subjects had a PTH 300 pg/ml. During treatment (up to 32 weeks), doses of drugs were escalated every 4 weeks up to five times. Paricalcitol achieved the PTH target faster than calcitriol; paricalcitol-treated subjects achieved persistent suppression of PTH by 87 days (median) versus 108 days for the calcitriol group. However, there was no difference in the primary endpoint, the proportion of subjects achieving 50% reduction in PTH by the end of the study. There was no difference in the fraction of patients who developed hypercalcemia, hyperphosphatemia or an elevated calcium x phosphorus product, but the authors noted that persistent hypercalcemia was nearly half as common in the paricalcitol group (18% versus 33%). This decreased predilection for promoting calcium absorption by paricalcitol was noted in a prospective crossover study of 10 hemodialysis patients on a lowcalcium and low-phosphorus diet compared high doses of calcitriol (20 mg) and very high doses of paricalcitol (120 and 160 mg) [34]. Calcium and phosphate levels were measured every 6 hours for the subsequent 36 hours. Despite the higher dosing of paricalcitol, calcitriol provoked a greater rise in the calcium-phosphate product compared with either dose of paricalcitol, an effect that appeared to be driven largely by an increase in phosphate. PTH suppression appeared to be greater with paricalcitol, particularly at the higher dose. Of note, the doses of both agents used in this study are considerably higher than those typically used in clinical practice. One multi-center randomized controlled trial compared calcitriol and maxacalcitol in 91 hemodialysis patients with secondary hyperparathyroidism [35]. All patients in this study used a relatively high calcium dialysis bath of 3 meq/l. There were no significant differences between the agents in calcium, phosphorus, and several measures of PTH. While this small study may not have had adequate power to detect subtle differences between the agents, no clear advantage of one agent over the other was observed. Another small crossover study yielded similar results [36]. A 24-week study of 46 hemodialysis patients randomized to intravenous maxacalcitol or “pulse” oral calcitriol (given twice weekly). After 4 weeks, PTH levels were significantly lower and calcium levels significantly higher in the calcitriol arm, though both these effects waned with time. Few clinical studies have looked at falecalcitriol, an analog currently unavailable in the USA. A small crossover trial from Japan compared oral falecalcitriol (0.3 mg/d) with intravenous calcitriol (1 mg, three times/week) in 21 relatively stable hemodialysis patients with controlled calcium and phosphorous
1561
levels at the start of treatment [37]. All subjects were being treated with a VDR agonist at the start of the study, though there was a 4-week run-in period and 4-week washout period between the two 12-week treatment periods. Both treatments reduced PTH, and there was no significant difference between the treatments with respect to calcium, phosphorus, or PTH. Calcitriol, but not falecalcitriol, significantly increased osteocalcin and significantly decreased cross-linked N-telopeptide of type 1 collagen, both bone turnover markers, though the clinical significance of this is unclear. Might newer VDR agonists have a particular role in a subgroup of CKD patients? One open-label trial studied 37 hemodialysis patients who had persistent hyperparathyroidism (iPTH 600) despite treatment with calcitriol [38]. Considered to have “calcitriol-resistant” sHPT, these individuals were then switched to paricalcitol (at a 1:4 dosing regimen for initial subjects, and 1:3 for later subjects). While there was no significant change in either calcium or phosphorus levels over the subsequent 16 months, there was a substantial reduction in iPTH (from a mean of 901 pg/ml to 165 pg/ml over 16 months) and similar reduction in alkaline phosphatase. Although comparisons between non-calcitriol VDR activators are uncommon, one small study (n ¼ 13) compared the effects of large, single doses of paricalcitol and doxercalciferol [39]. On different occasions, each subject received extremely high doses of either paricalcitol (160 mg) or doxercalciferol (120 mg). Over the ensuing 36 hours, the authors found that doxercalciferol treatment led to a more rapid rise and higher peak in serum phosphorous, while PTH suppression was similar between the two agents. While this would seem to support data that doxercalciferol may be more tightly linked with increased gastrointestinal mineral absorption, the significance of these findings with such nonstandard dosing (as with the similar study that compared paricalcitol and calcitriol) [34] is unclear. There are little data to allow for a direct comparison between doxercalciferol and calcitriol with respect to mineral metabolism in ESRD.
Effects on Survival While metrics such as PTH, calcium, and phosphorus are rational measures for assessing differences between analogs, recent research has focused on survival. Mortality rates on dialysis remain high, and any several treatments that are beneficial in the general population, such as statins, have failed to have a measurable impact on survival in ESRD. In 2003, Teng et al. published a retrospective cohort study and examined over 67 000 dialysis patients at Fresenius Medical Care North America treated with either calcitriol or paricalcitol [40].
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81. CALCITRIOL AND ANALOGS IN THE TREATMENT OF CHRONIC KIDNEY DISEASE
The authors found a 16% lower mortality rate among those treated with paricalcitol. Since the study was not randomized, this analysis included adjustment for multiple potential confounders including baseline laboratory values, duration of dialysis prior to treatment initiation, dialysis access, and dialysis center standardized mortality rate. This relationship persisted across a range of calcium and phosphorus levels, as well as demographic and clinical subgroups. Strengthening these observations was the crossover population that switched analogs during their time on dialysis. Those that switched from calcitriol to paricalcitol achieved improved survival while those who switched from paricalcitol to calcitriol had lower survival (Fig. 81.3). At 1-year time points, increases in calcium and phosphorus levels were also muted in the paricalcitol arm. This paper was followed by another study which showed a highly significant 26% mortality reduction in individuals who received any active vitamin D analog versus those who received none [41]. Another group studied over 55 000 maintenance hemodialysis patients,
Survival (%)
Survival (%)
over two-thirds of whom received paricalcitol. At any dose, paricalcitol was associated with a survival advantage (adjusted HR 0.6e0.9, depending on dose) versus no vitamin D treatment. Doses 15 mg/week were associated with the least survival advantage, but this may have been influenced by the increased mortality associated with higher PTH levels, the primary indication for paricalcitol treatment and the reason for increased dosage [3]. One study compared mortality rates between the three most popular analogs in the USA: calcitriol, paricalcitol, and doxercalciferol [42]. This observational study followed 7731 incident dialysis patients for a median of 37 weeks. Either paricalcitol or doxercalciferol was associated with an ~22% reduction in mortality versus calcitriol in unadjusted analyses. There was no difference between doxercalciferol and paricalcitol, and the survival benefit associated with these agents compared with calcitriol was no longer significant after adjustment for potential confounders (including laboratory values and standardized mortality rates). Of note, the groups had significantly different clinical characteristics with respect to markers of mineral metabolism. Calcium, phosphate, and PTH levels at the time of treat(A)100 ment initiation were considerably lower in subjects who 90 received calcitriol. While those treated with calcitriol were witnessed to have a more rapid rise in calcium Paricalcitol 80 than those who received other treatments, which might 70 support others’ findings of lower calcium absorption 60 Calcitriol with non-calcitriol analogs, these data are difficult to 50 interpret given the baseline metabolic differences. 40 Shinaberger and colleagues studied 34 307 mainte30 nance hemodialysis patients from across the USA, P<0.001 20 examining the relationship between paricalcitol admin10 istration and mortality [43]. To address the previously 0 noted paradox that observed a decline in benefit with 15 25 0 5 10 20 30 35 40 increasing doses of paricalcitol, they attempted to Months address the potential problem of confounding by indica(B) 100 tion. Instead of measuring absolute dosage of paricalci90 Switch to paricalcitol tol, they created an index that represented the ratio of 80 average quarterly paricalcitol to average quarterly 70 PTH over the same period and assigned individuals to Switch to calcitriol 60 one of three categories based on this value. After adjust50 ing for standard demographic and clinical covariates, 40 they found that the death rate ratio steadily improved 30 (from 0.99 to 0.92) as the paricalcitol:PTH ratio 20 increased, suggesting that doses adjusted for PTH level 10 P=0.04 were important. Patients who had been administered 0 15 25 0 5 10 20 30 35 40 calcitriol represented only 4e7% of the total population and hence were excluded from this study. Thus, the Months comparisons herein were between paricalcitol and no FIGURE 81.3 A retrospective cohort study by Teng et al. showed analog at all and between different paricalcitol doses that dialysis patients treated with paricalcitol had better survival than their counterparts who received calcitriol (A). Those who switched relative to time-averaged PTH. These analyses bring us closer to linking paricalcitol agents took on the survival characteristic of the new drug (B). Reproduced with permission from [37]. dosage to survival, though do not contrast paricalcitol’s
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1563
CONCLUSIONS
actions with those of calcitriol. They do, however, provide another piece of evidence that paricalcitol itself, rather than confounding factors, may be the reason for the improved survival. However, studies such as this one may be difficult to interpret. The authors used an index to essentially control for potential independent effects of PTH level on mortality to better isolate direct effects of paricalcitol. If paricalcitol dosage was truly an independent factor, and variations in dosage at given levels of PTH were truly random, then this study would indeed suggest that paricalcitol might be mediating an improvement in survival. The increased dosage required for a given level of PTH may in fact be a measure of resistance to vitamin D’s actions, or at least those actions that affect the calciumephosphorusePTH axis. Indeed, if individuals vary in their response to a given vitamin D analog, this may be as important, or even more important, than differences between the analogs themselves. As always, it is impossible in a non-randomized study to know whether this relative dosing of paricalcitol is in fact a proxy for some other measure, such as physicians’ therapeutic aggressiveness, which may affect outcome.
CARDIOVASCULAR DISEASE IN CKD AND DIFFERENT FORMS OF VITAMIN D Cardiovascular disease (CVD) is the leading cause of death in both pre-dialysis CKD and ESRD. The observed correlations between use of VDR agonists and survival have raised questions about a specific role for these agents in the amelioration of CVD. The question of nutritional forms of vitamin D and CVD outcomes has also been of considerable interest. Given the disproportionate contribution of CVD to morbidity and mortality in advanced CKD and ERSD, it seems likely that any benefit of one VDR agonist over another in overall survival would be mediated through either direct or indirect influences on CVD. 1a-Hydroxylase knockout mice develop hypertension and LVH with impaired systolic function that can be rescued by exogenous 1,25(OH)2D3 [44]. This animal model may be analogous to CKD, where renal 1a-hydroxylase activity declines significantly. Although knockout mice were given a rescue diet to normalize calcium and phosphorus levels, and 25(OH)D3 levels were not low, these factors failed to abrogate the deleterious cardiovascular phenotype. Only administration of 1,25(OH)2D3 was able to normalize blood pressure as well as cardiac structure and function. Plasma levels of renin, angiotensin II, and aldosterone were also suppressed by 1,25(OH)2D3 treatment and a similar phenotypic effect was seen with administration of either the angiotensin converting enzyme inhibitor
(ACE-inhibitor) captopril or the angiotensin receptor blocker (ARB) losartan, suggesting that vitamin D may exert beneficial effects by directly influencing the renineangiotensinealdosterone (RAA) axis. The central role for vitamin D analogs in cardiac hypertrophy and the RAA axis has been also demonstrated in animal studies using vitamin D receptor (VDR) knockout mice [45]. These studies demonstrated that VDR knockout animals developed left ventricular cardiomyocyte hypertrophy, increased levels of atrial natriuretic peptide, and increased expression of cardiac renin. Further information on cardiovascular effects of calcitriol can be found in Chapters 31, 40, and 102. Other studies have linked vitamin D metabolism to cardiac structure and function in dialysis patients. In one study of subjects with ESRD, both 25(OH)D3 and 1,25(OH)2D3 levels were negatively correlated with aortic pulse wave velocity, a measure of arterial stiffness that has been linked to diastolic dysfunction and LVH [46]. This effect appeared to be independent of vascular calcification, a feared complication of vitamin D treatment. Indeed, the desire to identify less calcemic analogs has been driven in part by the desire to minimize the risk of vascular calcification; this study suggested that an excessive focus on calcium and phosphorus absorption as a metric of potential cardiovascular risk may be misplaced. On the other hand, patients with ESRD are known to be at high risk of calcific atherosclerotic lesions [47], and the causative association between the development of vascular calcification and mortality is the topic of ongoing investigation [48] (see also Chapter 73). Finally, studies such as these also leave open the question of whether 25(OH)D3 replenishment will have an effect on cardiac phenotype. Few studies have focused on the role of specific analogs in the amelioration of left ventricular hypertrophy. Bodyak et al. examined the role of paricalcitol in an animal model of vitamin D deficiency and left ventricular hypertrophy: the Dahl salt-sensitive rat [49]. In rats fed a high-salt diet, concomitant treatment with paricalcitol reduced cardiac and pulmonary mass, left ventricular mass, and diastolic pressures while improving diastolic function. Atrial and brain natriuretic peptide levels as well as renin were also reduced. A randomized trial studying the effects of paricalcitol in humans with CKD stages 3e4 and left ventricular hypertrophy is currently under way [25].
CONCLUSIONS For many years, calcitriol has been central to the treatment of sHPT in ESRD and earlier stages of CKD. While calcitriol has been demonstrated to suppress production of PTH, concern has emerged around calcitriol’s
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predilection for stimulating absorption of calcium and phosphate. Analogs of calcitriol, most prominently paricalcitol and doxercalciferol, have gained increasing popularity as animal and human studies have suggested that these agents may suppress PTH at doses that drive calcium and phosphorus to a lesser degree than calcitriol. Retrospective studies suggest that these agents may offer a survival advantage over calcitriol, and this has fueled their popularity, particularly in the management of dialysis patients. However, there are limited head-to-head data comparing analogs in CKD or ESRD, as well as comparisons with calcitriol itself. The clinician currently must ask about the potential for harm, and assess the totality of data. Based on currently available data, there is no conclusive evidence that any vitamin D analog is superior to calcitriol in patients, particularly with respect to hard outcomes such as survival. Randomized controlled trials in patients are needed to provide clearer guidance for clinicians. As evidenced by considerable variability in animal studies, factors beyond the analogs themselves may influence their effect, including diet and other methods of controlling calcium and phosphorus. Increased adoption and availability of calcimimetics and phosphate binders may change the relative benefits and risks of these agents. Finally, the questions about the role for nutritional forms of vitamin D are now being addressed, both in the CKD/ESRD populations and in the general population at large.
References [1] D.L. Andress, K.C. Norris, J.W. Coburn, E.A. Slatopolsky, D.J. Sherrard, Intravenous calcitriol in the treatment of refractory osteitis fibrosa of chronic renal failure, N. Engl. J. Med. 321 (1989) 274e279. [2] E. Slatopolsky, J. Finch, M. Denda, C. Ritter, M. Zhong, A. Dusso, et al., Phosphorus restriction prevents parathyroid gland growth. High phosphorus directly stimulates PTH secretion in vitro, J. Clin. Invest. 97 (1996) 2534e2540. [3] K. Kalantar-Zadeh, N. Kuwae, D.L. Regidor, C.P. Kovesdy, R.D. Kilpatrick, C.S. Shinaberger, et al., Survival predictability of time-varying indicators of bone disease in maintenance hemodialysis patients, Kidney Int. 70 (2006) 771e780. [4] G.A. Block, P.S. Klassen, J.M. Lazarus, N. Ofsthun, E.G. Lowrie, G.M. Chertow, Mineral metabolism, mortality, and morbidity in maintenance hemodialysis, J. Am. Soc. Nephrol. 15 (2004) 2208e2218. [5] C.P. Kovesdy, O. Kuchmak, J.L. Lu, K. Kalantar-Zadeh, Outcomes associated with serum calcium level in men with non-dialysis-dependent chronic kidney disease, Clin. J. Am. Soc. Nephrol. CJASN 5 (2010) 468e476. [6] A.L. Zisman, M. Hristova, L.T. Ho, S.M. Sprague, Impact of ergocalciferol treatment of vitamin D deficiency on serum parathyroid hormone concentrations in chronic kidney disease, Am. J. Nephrol. 27 (2007) 36e43. [7] DIVINE: Dialysis Infection and Vitamin D In New England, http://clinicaltrials.gov/ct2/show/NCT00892099.
[8] M. Zasloff, Fighting infections with vitamin D, Nat. Med. 12 (2006) 388e390. [9] F. Takahashi, J.L. Finch, M. Denda, A.S. Dusso, A.J. Brown, E. Slatopolsky, A new analog of 1,25-(OH)2D3, 19-NOR-1,25(OH)2D2, suppresses serum PTH and parathyroid gland growth in uremic rats without elevation of intestinal vitamin D receptor content, Am. J. Kidney Dis. 30 (1997) 105e112. [10] A.J. Brown, J. Finch, E. Slatopolsky, Differential effects of 19-nor1,25-dihydroxyvitamin D(2) and 1,25-dihydroxyvitamin D(3) on intestinal calcium and phosphate transport, J. Lab. Clin. Med. 139 (2002) 279e284. [11] E. Slatopolsky, M. Cozzolino, Y. Lu, J. Finch, A. Dusso, M. Staniforth, et al., Efficacy of 19-nor-1,25-(OH)2D2 in the prevention and treatment of hyperparathyroid bone disease in experimental uremia, Kidney Int. 63 (2003) 2020e2027. [12] I. Lopez, F.J. Mendoza, E. Aguilera-Tejero, J. Perez, F. Guerrero, D. Martin, et al., The effect of calcitriol, paricalcitol, and a calcimimetic on extraosseous calcifications in uremic rats, Kidney Int. 73 (2008) 300e307. [13] K.J. Martin, E.A. Gonzalez, M.E. Gellens, L.L. Hamm, H. Abboud, J. Lindberg, Therapy of secondary hyperparathyroidism with 19-nor-1alpha,25-dihydroxyvitamin D2, Am. J. Kidney Dis. 32 (1998) S61e66. [14] A. Cardu´s, S. Panizo, E. Parisi, E. Fernandez, J.M. Valdivielso, Differential effects of vitamin D analogs on vascular calcification, J. Bone Miner. Res. 22 (2007) 860e866. [15] S. Mathew, R.J. Lund, L.R. Chaudhary, T. Geurs, K.A. Hruska, Vitamin D receptor activators can protect against vascular calcification, J. Am. Soc. Nephrol. 19 (2008) 1509e1519. [16] M. Mizobuchi, J.L. Finch, D.R. Martin, E. Slatopolsky, Differential effects of vitamin D receptor activators on vascular calcification in uremic rats, Kidney Int. 72 (2007) 709e715. [17] W. Noonan, K. Koch, M. Nakane, J. Ma, D. Dixon, A. Bolin, et al., Differential effects of vitamin D receptor activators on aortic calcification and pulse wave velocity in uraemic rats, Nephrol. Dial. Transplant. 23 (2008) 3824e3830. [18] J.R. Wu-Wong, M. Nakane, G.D. Gagne, K.A. Brooks, W.T. Noonan, Comparison of the pharmacological effects of paricalcitol and doxercalciferol on the factors involved in mineral homeostasis, Int. J. Endocrinol. 2010 (2010) 621687. [19] E. Slatopolsky, M. Cozzolino, J.L. Finch, Differential effects of 19-nor-1,25-(OH)(2)D(2) and 1alpha-hydroxyvitamin D(2) on calcium and phosphorus in normal and uremic rats, Kidney Int. 62 (2002) 1277e1284. [20] J. Kong, G.H. Kim, M. Wei, T. Sun, G. Li, S.Q. Liu, et al., Therapeutic effects of vitamin D analogs on cardiac hypertrophy in spontaneously hypertensive rats, Am. J. Pathol. 177 (2010) 622e631. [21] L.R. Baker, L. Abrams, C.J. Roe, M.C. Faugere, P. Fanti, Y. Subayti, et al., 1,25(OH)2D3 administration in moderate renal failure: a prospective double-blind trial, Kidney Int. 35 (1989) 661e669. [22] N.A. Hamdy, J.A. Kanis, M.N. Beneton, C.B. Brown, J.R. Juttmann, J.G. Jordans, et al., Effect of alfacalcidol on natural course of renal bone disease in mild to moderate renal failure, BMJ 310 (1995) 358e363. [23] J.W. Coburn, H.M. Maung, L. Elangovan, M.J. Germain, J.S. Lindberg, S.M. Sprague, et al., Doxercalciferol safely suppresses PTH levels in patients with secondary hyperparathyroidism associated with chronic kidney disease stages 3 and 4, Am. J. Kidney Dis. 43 (2004) 877e890. [24] D. Coyne, M. Acharya, P. Qiu, H. Abboud, D. Batlle, S. Rosansky, et al., Paricalcitol capsule for the treatment of secondary hyperparathyroidism in stages 3 and 4 CKD, Am. J. Kidney Dis. 47 (2006) 263e276.
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REFERENCES
[25] The PRIMO Study-Paricalcitol Capsules Benefits in Renal Failure Induced Cardiac Morbidity in Chronic Kidney Disease Stage 3/4, http://clinicaltrials.gov/ct2/show/NCT00497146. [26] D. de Zeeuw, R. Agarwal, M. Amdahl, P. Audhya, D. Coyne, T. Garimella, et al., Selective vitamin D receptor activation with paricalcitol for reduction of albuminuria in patients with type 2 diabetes (VITAL study): a randomised controlled trial, Lancet 376 (2010) 1543e1551. [27] A.J. Brown, Vitamin D analogs for secondary hyperparathyroidism: what does the future hold? J. Steroid Biochem. Mol. Biol. 103 (2007) 578e583. [28] A.B. Shoben, K.D. Rudser, I.H. de Boer, B. Young, B. Kestenbaum, Association of oral calcitriol with improved survival in nondialyzed CKD, J. Am. Soc. Nephrol. 19 (2008) 1613e1619. [29] C.P. Kovesdy, S. Ahmadzadeh, J.E. Anderson, K. KalantarZadeh, Association of activated vitamin D treatment and mortality in chronic kidney disease, Arch. Intern. Med. 168 (2008) 397e403. [30] B. Kestenbaum, S.L. Seliger, D.L. Gillen, H. Wasse, B. Young, D.J. Sherrard, et al., Parathyroidectomy rates among United States dialysis patients: 1990-1999, Kidney. Int. 65 (2004) 282e288. [31] R.J. Lund, D.L. Andress, M. Amdahl, L.A. Williams, R.P. Heaney, Differential effects of paricalcitol and calcitriol on intestinal calcium absorption in hemodialysis patients, Am. J. Nephrol. 31 (2010) 165e170. [32] A.H. Abdul Gafor, R. Saidin, C.Y. Loo, R. Mohd, S. Zainudin, S.A. Shah, et al., Intravenous calcitriol versus paricalcitol in haemodialysis patients with severe secondary hyperparathyroidism, Nephrology (Carlton., Vic.) 14 (2009) 488e492. [33] S.M. Sprague, F. Llach, M. Amdahl, C. Taccetta, D. Batlle, Paricalcitol versus calcitriol in the treatment of secondary hyperparathyroidism, Kidney Int. 63 (2003) 1483e1490. [34] D.W. Coyne, M. Grieff, S.N. Ahya, K. Giles, K. Norwood, E. Slatopolsky, Differential effects of acute administration of 19nor-1,25-dihydroxy-vitamin D2 and 1,25-dihydroxy-vitamin D3 on serum calcium and phosphorus in hemodialysis patients, Am. J. Kidney Dis. 40 (2002) 1283e1288. [35] M. Hayashi, Y. Tsuchiya, Y. Itaya, T. Takenaka, K. Kobayashi, M. Yoshizawa, et al., Comparison of the effects of calcitriol and maxacalcitol on secondary hyperparathyroidism in patients on chronic haemodialysis: a randomized prospective multicentre trial, Nephrol. Dial. Transplant. 19 (2004) 2067e2073. [36] T. Mochizuki, S. Naganuma, Y. Tanaka, Y. Iwamoto, C. Ishiguro, Y. Kawashima, et al., Prospective comparison of the effects of maxacalcitol and calcitriol in chronic hemodialysis patients with secondary hyperparathyroidism: a multicenter, randomized crossover study, Clin. Nephrol. 67 (2007) 12e19. [37] H. Ito, H. Ogata, M. Yamamoto, K. Takahashi, K. Shishido, J. Takahashi, et al., Comparison of oral falecalcitriol and intravenous calcitriol in hemodialysis patients with secondary
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1565 hyperparathyroidism: a randomized, crossover trial, Clin. Nephrol. 71 (2009) 660e668. F. Llach, M. Yudd, Paricalcitol in dialysis patients with calcitriolresistant secondary hyperparathyroidism, Am. J. Kidney Dis. 38 (2001) S45e50. H.E. Joist, S.N. Ahya, K. Giles, K. Norwood, E. Slatopolsky, D.W. Coyne, Differential effects of very high doses of doxercalciferol and paricalcitol on serum phosphorus in hemodialysis patients, Clin. Nephrol. 65 (2006) 335e341. M. Teng, M. Wolf, E. Lowrie, N. Ofsthun, J.M. Lazarus, R. Thadhani, Survival of patients undergoing hemodialysis with paricalcitol or calcitriol therapy, N. Engl. J. Med. 349 (2003) 446e456. M. Teng, M. Wolf, M.N. Ofsthun, J.M. Lazarus, M.A. Herna´n, C.A. Camargo, et al., Activated injectable vitamin D and hemodialysis survival: a historical cohort study, J. Am. Soc. Nephrol. 16 (2005) 1115e1125. F. Tentori, W.C. Hunt, C.A. Stidley, M.R. Rohrscheib, E.J. Bedrick, K.B. Meyer, et al., Mortality risk among hemodialysis patients receiving different vitamin D analogs, Kidney Int. 70 (2006) 1858e1865. C.S. Shinaberger, J.D. Kopple, C.P. Kovesdy, C.J. McAllister, D. van Wyck, S. Greenland, et al., Ratio of paricalcitol dosage to serum parathyroid hormone level and survival in maintenance hemodialysis patients, Clin. J. Am. Soc. Nephrol. CJASN 3 (2008) 1769e1776. C. Zhou, F. Lu, K. Cao, D. Xu, D. Goltzman, D. Miao, et al., dependent regulation of the renin-angiotensin system in 1alphahydroxylase knockout mice, Kidney Int. 74 (2008) 141e143. W. Xiang, J. Kong, S. Chen, L.-P. Cao, G. Qiao, W. Zheng, et al., Cardiac hypertrophy in vitamin D receptor knockout mice: role of the systemic and cardiac renin-angiotensin systems, Am. J. Physiol. Endocrinol. Metab. 288 (2005) E125e132. G.M. London, A.P. Gue´rin, F.H. Verbeke, B. Pannier, P. Boutouyrie, S.J. Marchais, et al., Mineral metabolism and arterial functions in end-stage renal disease: potential role of 25hydroxyvitamin D deficiency, J. Am. Soc. Nephrol. 18 (2007) 613e620. U. Schwarz, M. Buzello, E. Ritz, G. Stein, G. Raabe, G. Wiest, et al., Morphology of coronary atherosclerotic lesions in patients with end-stage renal failure, Nephrol. Dial. Transplant. 15 (2000) 218e223. I. Bhan, R. Thadhani, Vascular calcification and ESRD: a hard target. Clin. J. Am. Soc. Nephrol. CJASN. 4 (Suppl. 1) (2009) S102e105. N. Bodyak, J.C. Ayus, S. Achinger, V. Shivalingappa, Q. Ke, Y.-S. Chen, et al., Activated vitamin D attenuates left ventricular abnormalities induced by dietary sodium in Dahl salt-sensitive animals, Proc. Natl. Acad. Sci. USA 104 (2007) 16810e16815. National Kidney Foundation, K/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification, Am. J. Kidney Dis. 39 (2002) S1e266.
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C H A P T E R
82 The Epidemiology of Vitamin D and Cancer Risk Edward Giovannucci Harvard School of Public Health, Boston, MA, USA and Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA
INTRODUCTION The hypothesis that vitamin D has anti-cancer effects was spawned from clinical and epidemiologic observations decades ago. In 1937, Peller and Stephenson hypothesized that sunlight exposure, by inducing skin cancer, could induce immunity against internal cancers [1]. In 1941, Apperly demonstrated an association between latitude and cancer mortality, leading him to hypothesize a direct benefit of sun exposure on cancer mortality that was not dependent on inducing skin cancer [2]. These observations and hypotheses were largely ignored by the medical community for the next four decades. In the early 1980s, Garland and Garland hypothesized that inadequate vitamin D status resulting from lower solar UV-B radiation exposure accounted for the association between higher latitudes and increased mortality rates of various cancers, including colon cancer [3], breast cancer [4], and ovarian cancer [5]. The hypothesis that vitamin D deficiency was related to carcinogenesis was then extended to prostate cancer [6,7] and to various other cancers [8] by others. These initial observations largely formed the basis of the hypothesis that vitamin D status could influence cancer incidence or mortality. The sunlight hypothesis is further detailed in Chapter 53. In the past several decades, laboratory studies have discovered and confirmed various anti-carcinogenic properties of vitamin D. In the 1970s and 1980s, many discoveries indicated that the role of vitamin D was pervasive, and extended beyond the traditional effects on calcium and phosphorus homeostasis [9]. The current
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10082-4
understanding is that cells in various tissues have the biochemical machinery to convert 25-hydroxyvitamin D (25(OH)D) to 1,25(OH)2D, the active form that binds the vitamin D receptor (VDR). The activated VDR then influences a variety of transcriptional changes. Levels of 25(OH)D that are inadequate to maintain physiologic concentrations of 1,25(OH)2D in cells cause aberrations in pathways including differentiation, proliferation, invasiveness, angiogenesis, and metastatic potential. Thus, individuals or populations with inadequate or deficient 25(OH)D levels may in the course of time, be pre-disposed to a higher risk of cancer and possibly more advanced or aggressive forms of cancers. Over the past several decades, a variety of epidemiologic study designs have been implemented to test the hypothesis that vitamin D is associated with risk of cancer incidence or mortality. Meanwhile, only limited data from randomized interventional trials have emerged on this topic. This chapter will review the epidemiologic evidence of the association between vitamin D status and cancer risk, including studies directly measuring circulating levels of 25(OH)D, and surrogates or determinants of 25(OH)D level, such as dietary intake or sun exposure, or composite scores that take multiple factors that influence vitamin D status into account. Both caseecontrol and cohort studies, examining the hypothesis at the individual risk level, and ecologic studies, assessing risk at the population level, will be reviewed. Before the specific studies are reviewed, the major strengths and limitations of the various approaches to assess vitamin D status in relation to cancer risk that have been used will be discussed.
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82. THE EPIDEMIOLOGY OF VITAMIN D AND CANCER RISK
OVERVIEW OF EPIDEMIOLOGIC STUDY DESIGNS FOR THE STUDY OF VITAMIN D AND CANCER Randomized Trials A double-blinded, placebo-controlled, randomized intervention trial (RCT) is the “gold standard” in establishing a causal association. The main strength from this study design is that, through effective randomization, confounding by other causal factors can be largely eliminated as an explanation of a positive result in a particular study. Because of the expense of performing randomized interventional studies of vitamin D and cancer incidence or mortality, randomized studies are rare. Although considered the gold standard in addressing causality, in practice, randomized studies have a number of practical limitations. The selection of the effective dose may be problematic. Unlike a drug, everyone has an underlying baseline level of 25(OH)D, and thus the potential for benefit likely varies among the subjects; those with adequate vitamin D may not benefit from the study intervention and may contribute to null results. In addition, there may be poor compliance, and contamination by the placebo group adopting the change (for example, taking vitamin D supplements outside of the study protocol). In some cases, the necessary duration for the study may not be adequate to show an effect for a cancer outcome. Finally, the period of time during the long natural history of cancer where vitamin D action is relevant is unknown. The National Institutes of Health has decided to fund a new RCT known as the VITAL Trial that will prospectively follow thousands of subjects treated with 2000 IU of vitamin D or placebo for 5 years. The trial is powered to examine total cancer incidence, and some of the major cancer types including colorectal, breast, and prostate cancer. The details of the trial are described in Chapter 81. When a well-designed and effectively executed RCT shows a positive finding, this result may provide strong or even compelling evidence of support for the hypothesis. However, because of the limitations enumerated above, when these studies show a null association, caution must be given not to overinterpret the results. Besides the absence of a true association, one or more of the limitations mentioned above could produce a null association. Historically, such limitations have likely frequently contributed to many null results in the study of nutritional factors in relation to cancer risk [10].
Prospective Studies of Circulating 25(OH) Vitamin D and Cancer Risk Some studies have examined plasma or serum 25(OH) level in relation to cancer risk. When inadequate
randomized data are available, studies based on circulating 25(OH)D and cancer risk are generally believed to provide the most reliable human evidence regarding the vitamin Decancer hypothesis. The 25(OH)D level is presumably the relevant factor for influencing cancer risk. A measurement of 25(OH)D incorporates the many aspects that influence 25(OH)D status, both cholecalciferol production from skin exposure to UV-B radiation and intake. However, how effectively a single measurement of 25(OH)D predicts cancer risk depends on how well the single measure is correlated with long-term exposure. 25(OH)D has a relatively long half-life (t1/2) in the circulation of about 2e3 weeks, but average cumulated exposure over many years or even decades may be required to appreciably influence a chronic disease such as cancer. Limited data address the critical issue of how well a single measure is a good indicator of long-term vitamin D status. In one study of middle-aged to elderly US male health professionals, the correlation of two 25(OH)D measurements approximately 3 years apart was 0.7 [11]. In another study of US men and women, the Spearman rank correlation coefficients comparing values at baseline to those taken within 1 year, 1 year apart, and 5 years apart were 0.65, 0.61, and 0.53, respectively [12]. In a Norwegian study, the correlation coefficient between serum 25 (OH)D measurements taken at specific intervals from 1994 and 2008 ranged from 0.42 to 0.52 [13]. These findings suggest that 25(OH)D levels correlate reasonably well over time (say 5e15 years) to provide meaningful results for epidemiologic studies, though some degree of measurement error, which attenuates associations, is inevitable. An additional important issue regards complexities in 25(OH)D measures using different assays, and even within batches using the same assay. Some caution should be used when considering the absolute levels of 25(OH)D from a study. Nonetheless, if cases and controls are matched and assayed in the same batch, then the relative ranking is valid if the assay has high inter-batch reliability. A specific complexity in studies of 25(OH)D is that levels fluctuate seasonally throughout the year due to variances in sun exposure, so cases and controls are often matched for season of blood draw. While matching or controlling for season reduces some extraneous variation in 25(OH)D level, it cannot overcome some potential limitations; for example, if the nadir of 25(OH)D level in winter months is etiologically most relevant for an outcome, the samples that were collected in non-winter months may be less informative and contribute noise. In epidemiologic studies, circulating 25(OH)D has typically been based on a measurement in archived blood samples using a nested caseecontrol study design. Because the sample was taken before the
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OVERVIEW OF EPIDEMIOLOGIC STUDY DESIGNS FOR THE STUDY OF VITAMIN D AND CANCER
diagnosis of cancer, it is unlikely that any association observed results spuriously from the cancer influencing the blood level (frequently called “reverse causation”). Reverse causation could occur, for example, if the cancer itself or treatments cause metabolic changes that affect the 25(OH)D level, if cancer leads to changes in behaviors (e.g., sun exposure due to treatment or disability) that influence 25(OH)D levels, or if cancer tissues directly increase or decrease levels of 25(OH)D or 1,25D. Thus, studies that have been based on the measurement of 25(OH)D in individuals already diagnosed with cancer need to be interpreted very cautiously because of the potential for the phenomenon of reverse causation. Another potential limitation of epidemiologic studies is that the investigator is limited to studying the range of 25(OH)D in the specific study population, which may have a limited range of 25(OH)D that may not encompass the etiologically relevant range. For example, in a population with relatively low sunshine exposure and low intakes of vitamin D, few individuals may achieve adequately high levels of 25(OH)D to elicit an association if a high threshold exists. On the other hand, if the inverse association between 25(OH)D and cancer risk is most apparent at the lower range with a leveling of benefits at higher ends, in some populations with relatively adequate vitamin D status it is possible that few individuals achieve low enough levels to demonstrate an association.
Studies of Vitamin D Intake Because of the scarcity of vitamin D in natural foods, in most populations vitamin D intakes are relatively low compared to levels that are hypothesized to minimize cancer risk. Moreover, fortification of vitamin D is limited. For example, a glass of fortified milk (in the USA) contains only 100 IU vitamin D, a level that is almost trivial compared to amounts of vitamin D that can be formed from exposure to UV-B radiation in some circumstances [14,15]. In most populations, probably considerably more vitamin D is made from sun exposure than is ingested. Nonetheless, vitamin D intake is an important contributor to 25(OH)D levels, especially in winter months in regions at high latitudes. During these time periods, intake may be the sole source, contributing to the depleting sources from summer UV-B exposure. One important consideration of studies of vitamin D intake is that, depending on the specific population, intake of vitamin D may be predominantly from one or a few sources, such as fatty fish, fortified milk, or supplements. Thus, dietary vitamin D intake will tend to be highly correlated with other dietary factors (e.g., omega-3 fatty acids in fish, calcium in milk, and other
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vitamins and minerals in supplements). If the correlation is very high (e.g., r > 0.7), separating the influence of vitamin D from the correlated factor may be difficult. Finally, ergocalciferol (D2) is often used in supplements, and ergocalciferol has been estimated to be less potent than cholecalciferol (D3) in raising 25(OH)D level [16]. Most databases do not take into account whether the source of vitamin D in supplements is from D2 or D3.
Studies of Predicted 25(OH)D Level A study can use known predictors of 25(OH)D level based on data on the individual level to formulate a predicted 25(OH)D score. For example, based on individuals’ reported vitamin D intake, region of residence (surrogate for UV-B exposure), sun exposure behaviors, outdoor activity level, skin color, and body mass index, a quantitative estimate of the expected vitamin D level can be made. This approach can be considered as an extension of that using dietary intake, or region, as a surrogate of 25(OH)D, and by using multiple determinants should in theory provide a better estimate of 25(OH)D status than using a single determinant. The predicted 25(OH)D approach may have some advantages and disadvantages compared to the use of a single measurement of circulating 25(OH)D in epidemiologic studies. The measurement of 25(OH)D is more direct, intuitive, and incorporates some of the sources of variability of 25(OH)D not taken into account by the score. However, the predicted 25(OH)D measure may provide a reasonable measure of vitamin D status over a long period of time because some factors accounted by the predicted 25(OH)D score are immutable (for example, skin color) or relatively stable (region of residence, body mass index), and these can be updated periodically. In contrast, circulating 25(OH)D level has a halflife of 2 to 3 weeks, and thus a substantial proportion of variability picked up by a single blood measure would likely be due to relatively recent exposures, which may not be representative of long-term exposure. Some investigators have argued that predictors of 25(OH)D should not be given as much weight in epidemiologic studies as measures of 25(OH)D because the variance of a 25(OH)D measurement explained by the predicted score is relatively low (e.g., 21% in one study) [17]. While this conclusion may be true, the issue is more complex because the relevant exposure is long-term 25(OH)D status, not that from a single measure. The argument assumes a single measure of 25(OH)D is a perfect assessment of long-term 25(OH)D status, say over a 10- or 20-year period, but as summarized above, correlations between measures of 25(OH)D over a period of 3e10 years can be as low as 0.4 to 0.5. For a true “gold
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standard,” this correlation should be 1. Frequently, all of the measurement error is assumed to be in the predicted score, but the inherent measurement error in a single 25(OH)D assessment to estimate long-term 25(OH)D status will dampen the correlation between the 25(OH)D measurement and the predicted score; thus, the R-square between the 25(OH)D measurement and the predicted score cannot be simply used to assess the ability of the predicted 25(OH)D measurement to estimate long-term 25(OH)D status.
Studies of Sun Exposure as a Surrogate of Vitamin D Status Since the major source of vitamin D is from sun exposure, some studies have used sun exposure as a surrogate of vitamin D status. Ecologic studies have examined geographical location as a surrogate of 25(OH)D status, and examined cancer incidence or mortality rates by region on a population level, typically within a country. In ecologic studies, exposure is inferred e for example, presumably living in regions with greater UV-B exposure may allow for greater opportunity for sun exposure, but actual exposure depends on the individuals’ behaviors. One potentially important advantage of these studies is that most serum-based and dietary cohorts are composed of middle-aged individuals, and the assessment of past sun exposures allows for estimating vitamin D status at points earlier in life, at least based on region of residence. For some cancers, these earlier time periods could be most relevant. These studies are also feasibly done, and can be utilized in many settings, providing a broad set data. On the downside, this approach does not directly assess vitamin D status directly. Region is not a perfect surrogate of vitamin D status. The main limitation for some ecologic studies may be the difficulty in controlling for various potential confounding factors. In some caseecontrol studies, region- and some questionnaire-based measures of sun exposure have been used to assess potential vitamin D status on an individual basis. Some surrogates that have been used (such as sunburns) may represent acute short-term exposures to sun rather than chronic exposures, which may be more relevant for vitamin D synthesis. Measurement error and possibly recall bias in caseecontrol studies in assessing past exposures are issues that may potentially influence validity. This study design is strengthened if there is some evidence supporting the finding that the surrogate used is an actual measure of 25(OH)D status in the study setting. Some objective methods to assess sun exposure, such as the use of reflectometry, have been utilized.
STUDIES OF 25(OH)D, PREDICTED 25(OH)D, VITAMIN D INTAKE AND CANCER RISK BY CANCER SITE Colorectal Cancer or Adenoma Colorectal cancer was the first cancer hypothesized to be associated with vitamin D status, and has been relatively well studied. Studies that have examined circulating 25(OH)D levels prospectively in relation to risk of colorectal cancer have generally supported an inverse association [18e26]. In an initial meta-analysis of the colorectal cancer studies, based on 535 cases, individuals with 82 nmol/l (33 ng/ml) serum 25(OH)D level had 50% lower incidence of colorectal cancer (p < 0.01) compared to those with levels of less than 30 nmol/l [27]. The doseeresponse appeared to be linear up to a 25(OH)D level of at least approximately 90 nmol/l, with no obvious threshold or non-linear relationship. Controlling for multiple covariates, including physical activity, body mass index, and various dietary factors, had little influence on the findings. These results were confirmed in a more recent updated meta-analysis based on 2630 colorectal cancer cases [28]. In that study, based on the literature up to December 2009, the summary relative risk (RR) was 0.85 (95% confidence interval (CI) 0.790.91) for a 25 nmol/l increment in 25(OH)D. The results are somewhat inconsistent in distinguishing whether the association is stronger for colon cancer or for rectal cancer, possibly due to small numbers, but in general the association has been observed for both anatomic sites. Many of the existing large cohort studies with plasma or serum biomarkers have published on this association, and most found an inverse association as reflected in the meta-analysis [28]. In the Nurses’ Health Study [20], based on 193 incident cases of colorectal cancer, after adjustment for age, body mass index, physical activity, smoking, family history, use of hormone replacement therapy, aspirin use, and dietary intakes of various factors, the RR for quintile 5 versus 1 was 0.53 (95% CI 0.271.04). The doseeresponse appeared linear across levels of 25(OH)D. The Health Professionals Follow-up Study [29], a large cohort of men, showed a non-statistically significant inverse association between higher plasma 25(OH)D concentration and risk of colorectal cancer and a statistically significant inverse association for colon cancer (highest versus lowest quintile: RR 0.46; 95% CI 0.240.89; p trend ¼ 0.005). After the results from the Health Professionals Follow-up Study and the Nurses’ Health Study were pooled, higher plasma 25(OH)D levels were associated with decreased risks of both colorectal cancer (RR 0.66; 95% CI 0.421.05; p trend ¼ 0.01) and colon cancer (RR 0.54; 95% CI 0.340.86;
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p trend ¼ 0.002). The results for rectal cancer were inconsistent, though the number of cases was small. In the Women’s Health Initiative, based on a total of 322 cases of colorectal cancer [26], an inverse association was observed between baseline 25(OH)D level and colorectal cancer risk; however, detailed analyses on potential confounders were not included in the report. The Japan Public Health Center-based Prospective Study [30] provided results from a nested caseecontrol study of 375 incident cases of colorectal cancer during 11.5 years of follow-up after blood collection. After adjustment for smoking, alcohol consumption, body mass index, physical exercise, vitamin supplement use, and family history of colorectal cancer, plasma 25(OH)D was not significantly associated overall with colorectal cancer risk. However, the lowest category of plasma 25(OH)D was associated with an elevated risk of rectal cancer in both men (RR 4.6; 95% CI 1.020) and women (RR 2.7; 95% CI 0.947.6), compared with the other quartiles combined. The association between 25(OH)D and colorectal cancer mortality was examined in 16 818 participants followed from 1988e1994 through 2000 in the Third National Health and Nutrition Examination Survey (NHANES) [31]. Serum 25(OH)D level measured at baseline was inversely associated with colorectal cancer mortality (n ¼ 66 cases). Specially, those with levels of 80 nmol/l or higher had a 72% risk reduction (RR 0.28; 95% CI 32e89) compared with those whose levels were <50 nmol/l (p trend ¼ 0.02). The largest study of 25(OH)D and colorectal cancer was based on the European Prospective Investigation into Cancer and Nutrition study (EPIC), a cohort of more than 520 000 participants from ten Western European countries [32]. This study was based on 1248 participants who developed colorectal cancer after enrollment, who were then matched to 1248 controls. Baseline 25(OH)D concentration demonstrated a strong inverse association with risk of colorectal cancer (p trend <0.001). Compared with a concentration of 25(OH)D (50.075.0 nmol/l), lower levels were associated with higher colorectal cancer risk (<25.0 nmol/l: incidence rate ratio 1.32 (95% CI 0.872.01); 25.0e49.9 nmol/l: 1.28 (95% CI 1.051.56), and higher concentrations associated with lower risk (75.0e99.9 nmol/l: 0.88 (95% CI 0.681.13); 100.0 nmol/l: 0.77 (95% CI 0.561.06). Subgroup analyses showed a strong association for colon but not for rectal cancer (p heterogeneity ¼ 0.048). The Multiethnic Cohort including men and women of Japanese, Latino, African-American, White, and Native Hawaiian ancestry also reported on plasma 25(OH)D level in relation to colorectal cancer in a nested casee control study of 229 cases and 434 controls [33]. In this study, an inverse trend was observed (the RR (per doubling of 25(OH)D) ¼ 0.68; 95% CI 0.510.92; p ¼ 0.01). However, the increased risk was observed
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primarily in those with deficient levels of 25(OH)D. When examined in quintiles, relative to the first quintile (<16.8 ng/ml), the RRs in all other quintiles were similarly reduced between 37 and 46%. The association was not significantly heterogeneous among the four largest ethnic groups (p heterogeneity ¼ 0.46). Several studies have examined circulating 25(OH)D levels on the risk of colorectal adenoma. Adenomas are well-established precursors to colorectal cancer, and most colorectal cancers are thought to arise from adenomas. Because adenomas are largely asymptomatic, most of the studies compared 25(OH)D levels in adenoma cases to controls who were adenoma-free on colonoscopy or sigmoidoscopy. The studies were based either on initially diagnosed adenomas in the participants, or on adenomas among individuals who had had an adenoma and were then followed for subsequent or recurrent adenomas. In general, these studies suggest an inverse association with 25(OH)D and possibly 1,25(OH)2D [21e24,34], particularly for advanced adenomas [24]. In a recent meta-analysis that summarized the results for circulating 25(OH)D and adenoma risk [35], higher circulating 25(OH)D was associated with lower risk of colorectal adenomas; the RR ¼ 0.70 (95% CI 0.560.87) for high versus low circulating 25 (OH)D. The inverse associations were stronger for advanced adenoma (RR 0.64; 95% CI 0.450.90), though only a fraction of the studies reported on advanced adenomas. In the studies, multivariate analysis generally did not change the relative risk appreciably. Although most of the studies can be considered as cross-sectional rather than truly prospective, most adenomas are asymptomatic, are unlikely to cause dietary and lifestyle changes before diagnosis, and probably do not influence vitamin D metabolism given that they are relatively small and localized. Thus, reverse causation is unlikely to be of major concern in these studies. Arguably, physical activity is the most relevant potential confounding factor to consider for studies of colorectal cancer and adenoma. Physical activity is consistently associated with lower risk of colon cancer (though not rectal cancer) [36], and it is associated with 25(OH)D status, probably indirectly as an indicator of outdoor activities which lead to sun exposure. Physical activity has been measured in most epidemiologic studies of 25(OH)D and colorectal cancer risk, and has not been found to be an important confounding factor. Typically, adjustment did not appreciably alter the main results for 25(OH)D and cancer or adenoma risk. However, if measurement error of physical activity is very high (which is plausible in at least some studies, depending on the method of assessment), residual confounding could occur. Arguing against residual confounding from physical activity entirely accounting for
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the association between 25(OH)D and colorectal cancer is the fact that the association is observed also with rectal cancer, which appears unrelated to physical activity level. In addition, the associations observed with vitamin D intake and solar UV-B exposure based on residence (discussed below) are unlikely to be appreciably confounded by physical activity. One study used an estimate of circulating 25(OH)D based on how various factors predicted 25(OH)D levels. The predicted 25(OH)D score was then examined in association with risk of colorectal cancer in men of the Health Professionals Follow-up Study [37]. First, in a sample of 1095 men, actual plasma 25(OH)D levels were the dependent variable in a multiple linear regression. The independent (predictor) variables were geographical region, skin pigmentation, dietary intake, supplement intake, body mass index, and leisure-time physical activity (a surrogate of potential exposure to sunlight UV-B). Based on the regression coefficients, a score was calculated for each of approximately 47 000 cohort members who had information on these variables. This variable was then examined in relation to subsequent risk of incident colorectal cancer cases (n ¼ 691). In the multivariate analysis, a 25 nmol/l increment in 25(OH)D was associated with a 37% reduced risk of colorectal cancer (RR 0.63; 95% CI 0.480.83). This association persisted after controlling for body mass index and physical activity. The association between colorectal cancer risk and dietary or supplementary vitamin D has been investigated in cohort studies of men [38,39] and women [40e42] or both sexes [43,44], and in caseecontrol studies [45e52]. As summarized above, the main limitation of these studies is that vitamin D intake in most populations accounts for a relatively small proportion of the variation in level of 25(OH)D. Despite this limitation, the majority of these studies found inverse associations for either colon or rectal cancer, or both subsites [38e41,44,46,48,50,51,53]. This finding was especially evident in studies that took into account supplementary vitamin D and in populations where milk is fortified with vitamin D. In the studies conducted in the USA, where milk is fortified with vitamin D and vitamin supplement use is relatively high, the cutpoint for the top intake category was from approximately 500 to 600 IU/day, with an average intake in this category of approximately 700e800 IU/day. Intakes of this magnitude are expected to increase circulating 25(OH)D level by about 15e20 nmol/l, a reasonably high increment that is expected to show a moderate but detectable influence on risk based on the doseeresponse seen in the studies based on 25(OH)D and cancer risk. The risk reduction of the colorectal cancer in the top versus bottom category was generally marked (in the various studies, risk reduction: 46% [40], 34% [39], 58% [41],
24% [42], 30% [51], 29% male, 0% female [44], 50% males, 40% females [52], and 28% male, 11% female [53]). Risk reductions, though somewhat weaker, were also observed for colorectal adenoma [35]. A recent metaanalysis of 12 studies based on vitamin D intake [35] found that compared with the lowest quantile of vitamin D intake, the highest quantile was associated with an 11% marginally decreased risk of colorectal adenomas (odds ratio (OR) ¼ 0.89; 95% CI 0.781.02) and recurrent adenomas (OR 0.88; 95% CI 0.721.07). The inverse association was stronger for advanced adenoma (OR 0.77; 95% CI 0.630.95). An important consideration of studies in the USA is that high vitamin D intakes are generally associated with high calcium intakes. Thus, some of the apparent benefit may be related to calcium intake, although some evidence shows that vitamin D intake remains associated with lower risk even after statistical adjustment for calcium intake [54]. Some studies indicate that vitamin D and calcium may interact and both may be required to minimize risk of colorectal cancer. In a randomized trial of calcium intake and risk of recurrent adenoma, 25(OH)D concentration was much more strongly associated with a reduced risk of adenoma only among subjects randomized to receive calcium [24]. Some of the proposed anti-cancer mechanisms are based on interactive functions of vitamin D and calcium [55]. The subject of vitamin D and colon cancer is discussed in Chapter 87.
Prostate Cancer Most of the studies of circulating 25(OH)D level and prostate cancer risk have not found clear risk reductions associated with higher 25(OH)D levels, although some of these studies suggested weak inverse associations [25,56e60]. Two studies [61,62] that tend to support an inverse association between 25(OH)D and prostate cancer risk were conducted in Nordic countries. In these geographic regions at higher latitudes, 25(OH)D levels may be particularly low due to low solar UV-B exposure. However, even the findings from these studies were equivocal. In fact, one of these studies also found an increased risk in men with the lowest and also with the highest 25(OH)D values, suggesting a U-shaped relationship between vitamin D and prostate cancer risk, with lowest risk observed in those in the intermediate range within that study [62]. Several studies found supportive [56] or suggestive [57] inverse associations for circulating 1,25(OH)2D levels and prostate cancer risk, especially for aggressive prostate cancer. In the Physicians’ Health Study, the men who had both low 25(OH)D and 1,25(OH)2D were at about a two-fold higher risk of being diagnosed with aggressive prostate cancer [63]. Because they are tightly regulated, levels of
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1,25(OH)2D are difficult to interpret in this context. In the Health Professionals Follow-up Study, both lower 25(OH)D and 1,25(OH)2D levels were surprisingly associated with reduced prostate cancer risk [59], although the cancers in this study were mostly organ-confined prostate cancers that had been detected through prostate-specific antigen (PSA) testing. In the same study population, a suggestive inverse association between 25(OH)D levels and risk of advanced-stage prostate cancer was observed, although numbers of advanced cases were limited (n ¼ 60) [59]. Recent large studies also found no association between higher 25(OH)D levels and lower risk for prostate cancer. In the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial, an analysis based on 749 cases and 781 controls found no association, and even a suggestively increased risk of aggressive prostate cancer (Gleason sum 7 or clinical stage III or IV) among men with higher circulating 25(OH)D levels [64]. However, this population by nature of the study design is extensively screened with PSA, so the vast majority of the prostate cancers were not of advanced stage (e.g., metastatic), and aggressive prostate cancers were mostly high-grade but organ-confined cancers. In the European Prospective Investigation into Cancer and Nutrition study (EPIC), serum 25(OH)D levels were measured in 652 prostate cancer cases matched to 752 controls from seven European countries [65]. The median follow-up time for the study was 4.1 years. No significant association was found between 25(OH)D concentration and risk of prostate cancer (RR for highest versus lowest quintile ¼ 1.28; 95% CI 0.881.88; p trend ¼ 0.188). Subgroup analyses showed no significant heterogeneity by cancer stage or grade. In Europe, there is relatively low screening for prostate cancer by PSA testing, so these cases were likely of a relatively more advanced stage than in the studies based in the USA. The lack of an association between circulating 25(OH)D and risk of prostate cancer was confirmed in a recent meta-analysis [28]. Based on 3956 cases in 11 identified studies, the summary RR for a 25 nmol/L increment in circulating 25(OH)D was 0.99 (95% CI 0.951.03). Clearly, studies of circulating 25(OH)D have tended not to support an association for prostate cancer, or at best have yielded equivocal results, with suggestive results possibly at the very low end of 25 (OH)D (e.g., <20 nmol/l). The predicted 25(OH)D level was examined in relation to advanced-stage prostate cancer in the Health Professionals Follow-up Study. The method for this analysis was summarized above [37]. Over follow-up from 1986 to 2002, 461 cases of advanced-stage prostate cancer were identified. In the multivariate model, a 25 nmol/l increment in predicted 25(OH)D level was associated with a non-significant 20% reduction
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in risk of advanced-stage prostate cancer. This finding suggests at best a modest association. Studies that have examined vitamin D intake in relation to prostate cancer incidence have also not tended to support any benefit of vitamin D [66e69]. Two of these studies [66,69] assessed supplemental vitamin D in addition to diet. Data on intake and advanced-stage or fatal prostate cancer are relatively sparse. The subject of vitamin D and prostate cancer is discussed in Chapter 86.
Breast Cancer Several large prospective studies have examined circulating 25(OH)D levels in relation to breast cancer risk. The first of these was the Nurses’ Health Study, which was a prospective nested caseecontrol study based on 701 breast cancer cases and 724 controls [70]. The results were suggestive of a moderate-sized association; women in the highest quintile of 25(OH)D had an RR of 0.73; 95% CI 0.491.07 (p trend ¼ 0.06) when compared with women in the lowest quintile of 25(OH)D. In a subgroup analysis, this inverse association was primarily observed in women of ages 60 years and older, but not in younger women. This finding may suggest that vitamin D may be a more important factor for postmenopausal than for premenopausal breast cancer. Another large prospective study of 25(OH)D level and breast cancer risk was based on the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial study. In this study, 1005 incident cases of breast cancer were identified in follow-up from 1993 to 2005; the mean time between blood draw and diagnosis was relatively low at 3.9 years [71]. In this cohort, women with 25(OH)D levels in the highest quintile were not at lower risk for breast cancer when compared to women with values in the lowest quintile (RR 1.04; 95% CI 0.751.45). In addition, no evidence of any trend was observed (p trend ¼ 0.81). In contrast to the Nurses’ Health Study results, the risk of breast cancer was not reduced even in the stratum of older women. The range of 25(OH)D was comparable to that in the Nurses’ Health Study. Further prospective data on 25(OH)D and postmenopausal breast cancer risk come from the Cancer Prevention Study-II (CPS-II) Nutrition Cohort [72]. The study was a prospective nested caseecontrol study based on 516 incident breast cancer cases and 516 matched controls. The investigators observed no association between 25(OH)D and breast cancer (OR 1.09; 95% CI 0.701.68, p ¼ 0.60) for the top versus bottom quintile, or using a priori cutpoints (OR 0.86 (95% CI 0.591.26), for 75 versus <50 nmol/l). A small nested casee control study, based on only 28 cases, reported a nonsignificant inverse association for risk of breast cancer mortality [31].
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A recent meta-analysis found different results for circulating 25(OH)D and breast cancer risk based on study design [28]. For prospective studies, no significant association was found (for a 25 nmol/l increment in 25(OH)D, the summary RR was 0.97 (95% CI 0.921.03) based on 3145 cases). However, for caseecontrol (retrospective studies), an inverse association was found (RR 0.83; 95% CI 0.790.87) based on 3030 cases. For the caseecontrol studies, a number of issues were noted, including assessing blood status long after the diagnosis (allowing more potential for changes in vitamin D status that do not reflect pre-diagnostic values), and low participation rates among controls (allowing for potential selection bias). As discussed above, retrospective caseecontrol studies of 25(OH)D and cancer are prone to biases, and the divergent results from prospective and retrospective studies for breast cancer may well reflect these biases. A number of studies have examined vitamin D intake in relation to breast cancer risk. A meta-analysis for studies identified six such studies that were conducted up to June 2007 [73]. In the meta-analysis, vitamin D intake was not associated with risk of breast cancer (summary RR 0.98; 95% CI 0.931.03). However, significant heterogeneity (p < 0.01) was apparent, which was due mostly to the level of vitamin D intake in the different studies. When the studies were stratified into those either with vitamin D intakes higher than 400 IU or with intakes lower than this amount, a modest inverse association was observed only in the three studies where intakes were 400 IU (summary RR 0.92; 95% CI 0.870.97; p heterogeneity ¼ 0.14). One of these studies, the Nurses’ Health Study, is of special interest because vitamin D intake was updated by questionnaires administered every 2 to 4 years, which allowed for an improved estimate of long-term intake [74]. That study, which was based on 3482 cases of breast cancer, found that total (dietary plus supplementary) vitamin D intake was inversely associated with risk of incident breast cancer (multivariate RR 0.72; 95% CI 0.550.94 for >500 versus 150 IU/day of vitamin D). Of note, in that study, inverse associations were also observed with other components of dairy foods, including lactose and calcium. Nonetheless, total vitamin D intake had a stronger inverse association than did either dietary or supplemental vitamin D intake individually. Thus, although it is difficult to tease out the independent effects of correlated nutrients, this study suggested that vitamin D may have been the relevant causal factor. The subject of vitamin D and breast cancer is discussed in Chapter 85.
Other Cancers For cancers other than prostate, breast, and colorectal, until very recently, only sparse data on circulating
25(OH)D in relation to risk existed. Other than the most common cancers, most epidemiologic studies have not had adequate numbers to examine 25(OH)D level in relation to cancer risk. To address 25(OH)D in relation to risk of rarer cancers, the Cohort Consortium Vitamin D Pooling Project of Rarer Cancers (VDPP) was formed [75]. This consortium brought together ten cohorts to conduct a prospective study of the association between concentrations of 25(OH)D and risk of seven rarer cancer sites. These cohorts had stored plasma or serum samples and long-term follow-up for cancer. The malignancies studied in the VDPP included endometrial, esophageal, gastric, kidney, non-Hodgkin’s lymphoma, ovarian, and pancreatic cancers. For this consortium, seven of the cohorts were based in the USA, one in Finland and two in China. The participants were from a wide range of latitudes in various countries, potentially broadening the range of exposure to solar UV-B and thereby vitamin D. The results were published in multiple reports in a single issue of the American Journal of Epidemiology. The total numbers of cases for each of the cancer sites were as follows: endometrium, 830, upper gastrointestinal (esophagus/stomach), 1065, kidney, 775, non-Hodgkin’s lymphoma, 1353, ovary, 516, and pancreas, 952. The consortium used a standard laboratory to examine 25(OH)D in a nested caseecontrol sample in the various studies, and then pooled the results. The overall design, description of the cohorts, and statistical methodology are outlined in a methods paper [75]. For all studies, the reported categories of serum 25(OH)D concentration were: <25, 25e<37.5, 37.5e<50, 50e<75, 75e<100, and 100 nmol/l. In general, no overall association was observed in these studies, with no trends approaching statistical significance [75e82]. Some notable subgroup findings are discussed below. For all the cancers, compared to the reference category of 50e75 nmol/l, no comparison group was statistically different, except for an increased risk of pancreatic cancer associated with concentrations of 25(OH)D greater than 100 nmol/l (adjusted odds ratio 2.12; 95% CI 1.233.64). There were 39 total cases in the upper category (100 nmol/l). This finding was driven largely by the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Cohort (male Finnish smokers) [83] and the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial study. In the Health Professionals Follow-up Study and Nurses’ Health Study, predicted 25(OH)D was examined in relation to pancreatic risk during 20 years of follow-up for 575 incident pancreatic cancer cases [84]. Higher 25(OH)D score was associated with a significant reduction in pancreatic cancer risk; compared with the lowest quintile, participants in the highest quintile of 25(OH)D score had an adjusted RR of 0.65 (95% CI 0.500.86; p trend ¼ 0.001). The results
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were not appreciably different when they were further adjusted for BMI and physical activity. In addition, in these cohorts, a significant reduction in risk of pancreatic cancer was observed when comparing vitamin D intakes of 600 IU/day to total vitamin D intake <150 IU/day (multivariate RR 0.59; 95% CI 0.400.88; p trend ¼ 0.01) [85]. Why these results differ markedly from those based on circulating 25(OH)D in the VDPP is unclear. The predicted score did not directly assess UV-B exposure, and limitations in the predicted score as described above, should be considered to possibly contribute to differences in the results. However, although the predicted score or intake does not assess the entire variance in 25(OH)D status, the score does account for approximately a 25 nmol/l difference in 25(OH)D level. If higher 25(OH)D truly increased risk of pancreatic cancer, one would expect that higher predicted 25(OH)D would be associated with an increased risk, rather than a decreased risk. For ovarian cancer, as for the other cancers, no overall association was observed, although there was statistically significant interaction with body mass index (BMI) (p < 0.01), with an inverse association suggested only for women with a BMI 25 kg/m2 (p trend ¼ 0.01). A similar interaction with BMI was reported previously in a report using data from three prospective cohorts: the Nurses’ Health Study, the Nurses’ Health Study II, and the Women’s Health Study [86]. The Nurses’ Health Study was included in the VDPP, but excluding it did not change the results appreciably. Thus, overall, the data suggest that higher 25(OH)D may be associated with a lower risk of ovarian cancer among overweight and obese women only, although a biologic explanation for this finding is not yet apparent. Two prospective nested caseecontrol studies conducted in the Finnish Maternity Cohort provide evidence of an inverse association between 25(OH)D and ovarian cancer for a relatively young age-at-onset group of ovarian cancer. In the first report, the RR among women with insufficient (<75 nmol/l) serum 25(OH)D concentration was 2.7 (95% CI 1.07.9) compared to that among those with sufficient (>75 nmol/l) serum 25(OH)D concentration [87]. In the second report, having sufficient compared to insufficient serum 25(OH)D levels was associated with a significantly decreased risk of ovarian cancer (RR 0.32; 95% CI 0.120.91) [88]. In the VDPP, overall, no association was observed between 25(OH)D level and risk of upper gastrointestinal cancer. However, some difference appeared by race. Among Caucasians, no overall trend was observed; but, above levels of 25 nmol/l of 25(OH)D, a suggestive inverse trend was observed with increasing levels, though those with levels <25 nmol/l were not at higher risk. In contrast, among Asians, a significant positive trend was noted with higher circulating
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25(OH)D (p trend ¼ 0.003). However, in one of the two main Asian cohorts (Shanghai Men’s Health Study), the median follow-up time was only 1.7 years, suggesting the possibility of reverse causation. Further, treated as a continuous variable, a 1 log unit increment of 25(OH)D was suggestively associated with increased risk in follow-up less than 2 years (RR 1.19; 95% CI 0.831.71) but not in follow-up time 2 years (RR 1.10; 95% CI 0.841.28), also suggestive of reverse causation. This positive trend in the VDPP for upper gastrointestinal cancers is noteworthy given that in a previous study nested in randomized trial of micronutrients [89] conducted in Linxian, China, for squamous cell carcinomas of the esophagus, a positive association was found (RR 1.77; 95% CI 1.162.70; p trend ¼ 0.0033) when comparing men in the fourth quartile of serum 25(OH)D concentrations to those in the first. The study included 545 squamous cell carcinomas of the esophagus, 353 adenocarcinomas of the gastric cardia, and 81 gastric non-cardia adenocarcinomas diagnosed over 5.25 years of follow-up. In contrast, no association was found in women (RR 1.06 (95% CI 0.711.59), p trend ¼ 0.70), or for gastric cardia or non-cardia adenocarcinoma. Notably, the cutpoint for the top quartile was only 48.7 nmol/l, suggesting that the overall vitamin D status may be relatively poor in this population. In a cross-sectional analysis of 720 subjects from Linxian, China, who underwent endoscopy and biopsy, and were categorized by the presence or absence of histologic esophageal squamous dysplasia, those in the highest compared with the lowest quartile of 25(OH)D were at a significantly increased risk of squamous dysplasia (RR 1.86; 95% CI 1.352.62 overall, RR 1.74; 95% CI 1.082.93 in men, and RR 1.96; 95% CI 1.283.18 in women) [90]. The mean level of 25(OH)D in this population was only 35 nmol/l. In this area at high risk for squamous esophageal cancer, 230 of 720 subjects were diagnosed with squamous dysplasia.
Total Cancer Total cancer risk was examined in relation to circulating 25(OH)D in three studies. One of these studies was conducted using data from the Third National Health and Nutrition Examination Survey [31]. In this analysis, 16 818 participants were followed from 1988e1994 through 2000, over which 536 cancer deaths were identified. Baseline vitamin D status was not significantly associated with total cancer mortality in men and women combined. A non-significant inverse trend (p ¼ 0.12) was observed in the women only. In general, the numbers of cases were too few to examine specific cancer sites. As discussed above, colorectal
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cancer mortality was strongly associated inversely to serum 25(OH)D level, and a non-significant inverse association was observed for breast cancer risk. Two small studies of 25(OH)D and total cancer risk were conducted in specialized populations. In the Ludwigshafen Risk and Cardiovascular Health study, 25(OH)D was measured in 3299 patients who provided a blood sample in the morning before a scheduled coronary angiography [91]. These cardiovascular patients were followed for a median period of about 8 years, over which 95 cancer deaths were identified. The multivariate RR 0.45; 95% CI 0.220.93, for the fourth quartile versus the first quartile of 25(OH)D, adjusting for age, sex, body mass index, smoking, retinol, exercise, alcohol, and diabetes history. The risk decrease had a monotonic doseeresponse (RR per increase of 25 nmol/l ¼ 0.66; 95% CI 0.490.89). Another study was based on renal transplant patients in France. Pre-transplant 25(OH)D levels in 363 renal transplant recipients at Saint-Jacques University Hospital at Besancon, France, were measured [92]. Thirty-two cancers were diagnosed over 5 years of follow-up following the renal transplant. The risk of total cancer increased by 12% for each 2.5 nmol/l decrement in 25(OH)D (RR 1.12; 95% CI 1.041.23; p ¼ 0.021). This association appears stronger than in most other studies, possibly due to chance from small numbers, the particularly low 25(OH)D levels as renal transplant patients are advised to avoid sun exposure, and the special nature of this population (i.e., renal transplant patients). It is also possible that the subjects were treated with vitamin D, glucocorticoids, and other anti-rejection medications that would interfere with vitamin D metabolism. In the Health Professionals Follow-up Study cohort, predicted 25(OH)D levels were examined in relation to risk of total cancer in men. The methods for this analysis were discussed above. From 1986 through January 31, 2000, 4286 incident cancers (excluding organ-confined prostate cancer and non-melanoma skin cancer) and 2025 cancer deaths from cancer were identified. An increment of 25 nmol/l in predicted 25(OH)D level was associated with a 17% reduction in total cancer incidence (multivariate RR 0.83; 95% CI 0.740.92) and a 29% reduction in total cancer mortality (multivariate RR 0.71; 95% CI 0.60e0.83). The reduction in incidence was largely confined to cancers of the digestive tract system, including esophagus, stomach, pancreas, colon, and rectum. For these digestive system cancers, an increment of 25 nmol/l in the predicted 25(OH)D level was associated with a 43% reduction in incidence (RR 0.57; 95% CI 0.460.71) and a 45% reduction in mortality (RR 0.55; 95% CI 0.410.74). Controlling for BMI and physical activity did not alter these results appreciably.
STUDIES OF SUN EXPOSURE Ecologic Studies of Regional UV-B Exposure The relationship between solar UV-B and risk of colon cancer initially proposed by Garland and Garland [3] has been confirmed in subsequent analyses. Grant demonstrated that regional solar UV-B radiation correlated inversely with mortality rates of numerous cancers, particularly digestive organ cancers [8]; the strongest association (in terms of number of cancers potentially preventable) was for colorectal cancer. In another ecologic study of solar UV-B and colorectal cancer, an inverse association was stronger for colorectal cancer mortality than for incidence [93]. Importantly, the association has been observed in different populations outside of the USA. For example, Mizoue examined averaged annual solar radiation levels for the period from 1961 through 1990 in relation to cancer mortality in the year 2000 in 47 prefectures in Japan [94]. Adjusting for regional per capita income and dietary factors, an inverse correlation was found between averaged annual solar radiation levels and mortality from gastrointestinal cancers, but not from breast cancer. Cancer mortality data were obtained from the Second National Death Survey conducted in a sample of 263 counties in China from 1990 to 1992. National cancer registration data from 1998e2002 in China were used for estimation of cancer incidence [95]. Satellite measurements of cloud-adjusted ambient UV-B intensity at 305 nm were obtained from a NASA database used to estimate the average daily irradiance for the 263 counties in 1990. Mortality rates for all cancers and cancers of the esophagus, stomach, colon and rectum, liver, lung, breast, and bladder were inversely correlated with ambient UV-B. This correlation was present in men and women and rural residents for all these cancers but not urban residents for cancers of the esophagus, colon and rectum, and liver. Lung cancer mortality showed the strongest inverse correlation per unit increase in UV-B irradiance. Only incidence rates for cancers of the esophagus, stomach, colon and rectum, and cervix were inversely correlated with ambient UV-B.
CaseeControl Studies of Sun Exposure The largest caseecontrol study of sun exposure and cancer risk was a US death-certificate-based casee control study [96], which examined mortality from some cancers (colon, breast, prostate, ovarian, nonHodgkin’s lymphoma (NHL)) in relation to residential and occupational exposure to sunlight. Non-melanoma skin cancer served as a positive “control” as excessive sun exposure is expected to be associated positively with risk for skin cancer. The cases consisted of cancer
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deaths between 1984 and 1995 in 24 states. The controls were ageefrequency matched (deaths from cancer and certain neurological diseases were excluded because of possible relationships with sun exposure). The multivariate analyses controlled for age, sex, race, and mutual adjustment for residence, occupation (outdoor versus indoor), occupational physical activity levels, and socioeconomic status. Based on 153 511 colon cancer deaths, those with high compared to low exposure to sun based on residence were at decreased risk (RR 0.73; 95% CI 0.710.74). In addition, individuals who had had outdoor occupations (RR 0.90; 95% CI 0.860.94) and who had been in occupations that required more physical activity (RR 0.89; 95% CI 0.860.92) were at lower risk. The inverse association with outdoor occupation was strongest among those living in the highest sunlight region. Based on 97 873 prostate cancer deaths, greater residential exposure to sunlight had an inverse association with prostate cancer mortality, though the magnitude of this association was modest (RR 0.90; 95% CI 0.860.91). Further, occupation exposure to sunlight was found not to be associated with fatal prostate cancer risk (RR 1.00; 95% CI 0.961.05). For female breast cancer deaths (n ¼ 130 261 cases), greater residential exposure to sunlight (RR 0.74; 95% CI 0.720.76) and occupational exposure to sunlight (RR 0.82; 95% CI 0.700.97) were associated with reduced risk [96]. In addition, the association between outdoor employment and reduced breast cancer mortality was strongest in regions of greatest residential sunlight (OR 0.75; 95% CI 0.551.03), suggesting that sunlight exposure was the primary reason underlying the reduced risk with outdoor employment. Based on over 33 000 fatal cases of NHL, a 17% reduction in risk was found for those residing in states with the highest sunlight exposure (multivariate RR 0.83; 95% CI 0.810.86) [97]. The lower risk was particularly notable for individuals under 45 years of age (RR 0.44; 95% CI 0.280.67). The risk of NHL mortality was also reduced with higher occupational sunlight exposure (RR 0.88; 95% CI 0.810.96). For ovarian cancer mortality (n ¼ 39 002 cases), residential (RR 0.84; 95% CI 0.810.88), but not occupational exposure to sunlight was inversely associated with risk. In several caseecontrol and cohort studies, surrogates of sun exposure were examined in relation to prostate cancer risk. One caseecontrol study of advanced prostate cancer was based on use of a reflectometer to measure overall sun exposure [98]. This method assesses the difference between facultative skin pigmentation on the forehead (a sun-exposed site) and constitutive pigmentation on the upper underarm (a sun-protected site). The difference between facultative and constitutive pigmentation is used to estimate sun exposure. In this
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study, sun exposure estimated by reflectometry was inversely associated with risk of advanced prostate cancer (RR 0.51; 95% CI 0.330.80). Further, this study found that high occupational outdoor activity level was associated with a suggestively reduced risk of advanced prostate cancer relative to low exposure (RR 0.73; 95% CI 0.481.11). A cohort study was based on 5811 non-Hispanic white men using National Health and Nutrition Examination Survey I data; of these men, 151 (102 non-fatal, 59 fatal) were diagnosed with prostate cancer over followup from 1971 to 1992. Several measures of presumed sun exposure were associated with significantly lower risk of prostate cancer; these were longest residence in regions with high solar radiation (RR 0.66; 95% CI 0.470.93), and high solar radiation in the state of birth (RR 0.49; 95% CI 0.270.90) [99]. The associations were stronger for fatal prostate cancer. Frequent recreational sun exposure in adulthood was associated with a lower risk of fatal prostate cancer only (RR 0.47; 95% CI 0.230.99). Based on these findings, the authors hypothesized that both early-life and adult exposure to sun are critical for prostate carcinogenesis, although the study did not have adequate power to simultaneously adjust for adult and early-life residences. Studies in the UK are noteworthy given the low sun exposure in that region. Several caseecontrol studies in the UK reported on factors such as childhood sunburns, holidays in a hot climate, and skin type in relation to prostate cancer risk. Rather striking findings were found in subgroups characterized by childhood sunburns, holidays in a hot climate, and skin type; specifically, a significant 13-fold higher risk of prostate cancer was observed in men with combinations of high sun exposure/light skin compared to low sun exposure/darker skin type [100,101]. Solar vitamin D effective UV-radiation (VD-dose), dietary vitamin D, sun-seeking holidays, use of solarium, and frequency of sunburn were studied in relation to breast cancer risk in 41 811 women from the prospective Norwegian Women and Cancer Study [102]. The women were aged 40e70 years at baseline, and were followed for 10 years, over which 948 new cases of breast cancer were diagnosed. This study found no significant associations with breast cancer risk for VD-dose (RR for highest versus lowest category ¼ 1.17 (0.95e1.44)), vitamin D intake (RR 0.95; 95% CI 0.751.21), sun-seeking holidays (RR 1.07; 95% CI 0.871.32), use of solarium (RR 0.93; 95% CI 0.761.14) and frequency of sunburn (RR 1.10; 95% CI 0.891.36). A population-based caseecontrol study of 972 cases and 1135 controls conducted in Canada examined selfreported sun-exposure behaviors at different age periods in relation to risk of breast cancer [103]. The study found a significantly reduced risk of breast cancer
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associated with increasing estimated sun exposure from ages 10 to 19 (RR 0.65; 95% CI 0.500.85 for the highest quartile of outdoor activities versus the lowest; p trend ¼ 0.0006). Notably, the associations from ages 20 to 29 years were weaker, and no evidence was observed for exposures for ages 45 to 54 years. These results suggest that the relevant time for vitamin D exposure and reduced breast cancer risk occurs primarily or solely during adolescence. A population-based caseecontrol study was conducted based on 1788 incident cases of advanced breast cancer and 2129 controls over the years 1995e2003 among Hispanic, African-American, and non-Hispanic White women from California [104]. In this study, among women with light constitutive skin pigmentation, those with high sun exposure index based on reflectometry had a reduced risk of advanced breast cancer (RR 0.53; 95% CI 0.310.91). However, among women with medium or dark pigmentation, high sun exposure index was not associated with risk. To explain these discordant findings, the investigators speculated that these measures based on reflectometry may reflect vitamin D status better in more lightly pigmented women than in darker-skinned women. Finally, in a relatively small cohort of 5009 women, among whom 190 women developed incident breast cancer, several measures of sunlight exposure and dietary vitamin D intake showed a moderate inverse association with risk of breast cancer [105]. The International Lymphoma Epidemiology Consortium (InterLymph) examined the association between sun exposure and NHL risk in a pooled analysis of 10 caseecontrol studies [106], comprising 8243 cases and 9697 controls of European origin. Four measures of self-reported personal sun exposure were assessed at interview; these included time (1) outdoors and not in the shade in warmer months or summer, (2) in the sun in leisure activities, (3) in sunlight, and (4) sun bathing in summer. The risk of NHL declined with the composite measure of increasing recreational sun exposure; the multivariate pooled RR (adjusting for smoking and alcohol) ¼ 0.76 (95% CI 0.630.91) for the highest exposure category (p trend ¼ 0.005). For increasing total sun exposure, a non-significant inverse trend was observed with NHL risk (RR 0.87; 95% CI 0.711.05; p ¼ 0.08). In addition, the inverse association between recreational sun exposure and NHL risk was statistically significant at 18e40 years of age and in the 10 years before diagnosis, and statistically significant for B cell lymphomas, but not for the rarer T cell lymphomas. Of note, besides its effects on vitamin D levels, chronic UV exposure has effects on the immune system [107]. Consideration of the sunlight hypothesis and cancer risk is further discussed in Chapter 53.
RANDOMIZED CONTROLLED TRIALS Data from randomized controlled trials may more definitively establish a causal association. However, the current randomized data on vitamin D and cancer risk are largely from three studies and each has important limitations. The largest relevant study is the Women’s Health Initiative. This investigation was a randomized placebo-controlled trial of 400 IU vitamin D3 plus 1000 mg/day of elemental calcium in 36 282 postmenopausal women [26]. The incidence of colorectal cancer over a 7-year period did not differ between women assigned to calcium plus vitamin D supplementation and those assigned to placebo (168 and 154 cases; RR 1.08; 95% CI 0.861.34; p ¼ 0.51). For breast cancer there were 528 cases in the supplement group and 546 in the placebo group (RR 0.96; 95% CI 0.851.09) [108]. A suggestive interaction was observed based on baseline vitamin D intake; among women in the highest quartile of reported total vitamin D intake (diet plus supplement) at baseline, more breast cancers were seen in the supplement group than in the placebo group (HR 1.34, 95% CI 1.011.78); among women in the lowest baseline vitamin D intake quartile, fewer cancers were seen in the supplement group (HR 0.79, 95% CI 0.650.97) (p interaction ¼ 0.003). However, no interaction was observed with baseline 25(OH)D level (p ¼ 0.99), so the interaction with vitamin D intake is difficult to interpret as circulating 25(OH)D status is likely to be a more reliable indicator of vitamin D status. The Women’s Health Initiative study had some important limitations. First, the vitamin D dose of 400 IU/day was probably inadequate to yield a substantial contrast between the treated and the control groups, as the expected increase of serum 25(OH)D level would be approximately 7.5 nmol/l. Given that the adherence for this trial was suboptimal and a high percentage of women took non-study supplements, the actual contrast of 25(OH)D tested was likely further reduced. In comparison, in the epidemiologic studies of 25(OH)D and colorectal cancer, the contrast between the high and low quintiles of 25(OH)D was generally at least 50 nmol/l. Second, whether the 7-year duration for the trial was sufficiently long to show an effect is unclear, as one epidemiologic study suggested that at least 10 years may be required for an effect of calcium and vitamin D intake for colorectal cancer to emerge [41]. Finally, the study was based on a factorial design along with hormonal replacement use, and a post-hoc analysis for colorectal cancer suggested that women on hormones did not benefit from the vitamin D and calcium, but women not taking hormones may have benefited [109]. Although this apparent interaction could have been a chance finding, some subsequent observational studies support this interaction. For
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example, in the Nurses’ Health Study of colorectal adenomas, no material association with vitamin D intake was observed for premenopausal women or for current users of postmenopausal hormones (HRT), but adenoma risk was significantly reduced among past users of HRT (RR 0.56; 95% CI 0.360.89; p trend ¼ 0.03) and suggestively so among never users (RR 0.82; 95% CI 0.541.27; p trend ¼ 0.37) [54]. In addition, one study found no association between outdoor time or ambient UV measure and colorectal cancer risk in current HRT users, but in never/past HRT users, an inverse association with higher ambient UV exposure was found (RR for highest versus lowest tertile ¼ 0.40; 95% CI 0.170.93; p trend ¼ 0.04) [110]. Of interest, a recent study of global gene expression in rectal mucosal biopsies suggested that the preventive action of HRT on colon neoplasia results, at least in part, from changes in vitamin D activity [111]. Another randomized controlled trial with relevant data on this topic was a UK study of 2686 subjects 65e85 years old who received 100 000 IU of vitamin D3 every 4 months for 5 years [112]. The amount of vitamin D averages to 820 IU of vitamin D daily. After treatment, the 25(OH)D level was 74.3 nmol/l in the vitamin D group and 53.4 nmol/l in the placebo group (a 21 nmol/l difference in 25(OH)D). There were 188 incident cancer cases in the vitamin D group and 173 in the placebo group, and no overall reduction was observed for cancer risk (RR 1.09; 95% CI 861.36). Based on 53 cases of colorectal cancer, there was no association with treatment relative to placebo (RR 1.02; 95% CI 0.601.74). The main limitations of this study were that it was relatively small and the follow-up was only for 4 years. The final relevant study was a 4-year, communitybased, double-blind, placebo-controlled randomized trial of vitamin D and calcium of 1179 US women aged >55 years living in Nebraska. The primary outcome of the study was fracture incidence and the principal secondary outcome was cancer incidence [113]. The subjects were randomly assigned to receive daily 1400e1500 mg supplemental calcium alone (Ca-only), supplemental calcium plus 1100 IU vitamin D (Ca þ D), or placebo. The achieved 25(OH)D level after treatment was 96 nmol/l in the vitamin-D-treated group and 71 nmol/l in the non-vitamin-D groups (a 25 nmol/l difference). Relative to the placebo group, the RR of incident cancer was 0.40 (p ¼ 0.01) in the Ca þ D group and 0.53 (p ¼ 0.06) in the Ca-only group. In a subanalysis confined to cancers diagnosed after the first year, the RR for the Ca þ D group was 0.23 (95% CI 0.090.60; p < 0.005); no significant risk reduction was observed for the Ca-only group. In multivariate models, both vitamin D treatment and higher 25(OH)D levels each were significantly associated with reduced cancer
incidence. This study was limited by the small number of total cancers (n ¼ 50 in total, n ¼ 37 excluding the first year cases). Although these results are provocative, the increment in 25(OH)D and the duration of follow-up in this study was comparable to the UK study described above, which was larger and of similar duration and showed no benefit on incidence. The 25(OH)D levels achieved in the Nebraska study, however, were higher (96 versus 74.3 nmol/l) so it is plausible that optimal risk reduction is observed only at these high levels.
MORTALITY/SURVIVAL While vitamin D status may influence the risk of developing cancer, it is plausible that some or most of the effect of vitamin D is on tumor biologic aspects of aggressive behavior, such as advanced stage or high grade, metastasis, and survival. Late-stage anti-cancer effects of vitamin D, such as reduction in metastases, are observed in numerous animal models. Although cancer incidence is the major determinant of cancer mortality, other factors may influence survival. Prostate cancer is an example for which determinants of cancer mortality may differ extensively from those for cancer incidence [37]. Various lines of evidence from human studies suggest that vitamin D may be related to cancer progression. This evidence includes ecologic and casee control studies of UV-B exposure and cancer mortality rates, studies examining season of diagnosis in relation to cancer survival, studies that have examined circulating 25(OH)D pre-diagnostic or around the time of diagnosis in relation to survival, and some limited data from randomized interventional studies of vitamin D supplementation. This evidence is summarized in this section. The geographical association between UV-B exposure and cancer was stronger for or limited to mortality than for incidence for many cancers in the USA and China [93,95]. In the study from China, regional cancer mortality rate was associated inversely with UV-B but cancer incidence rate was not [95]. Many ecologic studies of UV-B exposure have relied primarily on cancer mortality than incidence so it is possible that some of the associations were driven by survival rather than solely by incidence. In the large caseecontrol study of sun exposure and cancer mortality risk in the USA (based on death certificates) [96], cancers of the colon, breast, prostate, ovary, and NHL were related to residential and occupational exposure to sunlight. Since this study examined only cancer mortality, the association with incidence is unknown so it is plausible that some or most of the association was driven by survival. In one UK study of prostate cancer, self-reported solar UV-B exposure and skin type in 553 patients were
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studied in association with stage, Gleason score, and survival after starting hormonal therapy [114]. UV-B exposures 10, 20, and 30 years before diagnosis were inversely associated with stage; the RR for UV-B exposure 10 years before diagnosis was lowest ¼ 0.69 (95% CI 0.560.86), suggesting that vitamin D status within the decade before diagnosis predicted stage of prostate cancer at diagnosis. The relative risks were also lower in men with (lighter) skin types I/II than III/IV, and men with skin types I/II experienced longer survival after beginning hormone therapy (RR 0.62; 95% CI 0.400.95). These findings also support that vitamin D may influence prostate cancer mortality, since lighter skin types are more amenable to vitamin D production. One line of evidence suggests that vitamin D status assessed pre-diagnostically or at the time of diagnosis/ treatment is associated with better survival. In one study of 447 patients with early-stage (stages I and II) nonsmall-cell lung cancer, after adjusting for age, sex, stage, smoking, and treatment, a 26% borderline statistically significant better overall survival was observed for those in the highest versus lowest quartile of circulating 25(OH)D levels that was measured at the time of diagnosis. When stratified by stage, a 55% statistically significant reduction was observed among stage IBeIIB patients [115,116]. No association was observed for advanced-stage non-small-cell lung cancer in the same population [117]. In one study conducted in Norway, cod liver oil, an important source of vitamin D, was associated with improved survival for total solid tumors, especially for lung cancer, among those who developed cancer [118]. However, other supplements also were associated with improved survival, and cod liver oil has components other than vitamin D. For breast cancer, in one cohort of 512 women in Canada with early-stage (T1 to T3, N0 to N1, M0) breast cancer diagnosed in 1989 to 1996, 25(OH)D levels were measured in stored blood, and the women were followed with a mean follow-up duration of 11.6 years [119]. The plasma sample was taken before the onset of systematic therapy. Over this time period, 116 of the women had distant recurrences, and 106 of the women died. Women with deficient vitamin D levels (defined as <50 nmol/L) had a statistically significant 94% increased risk of distant recurrence and a 73% increased risk of death, compared with those with sufficient levels. The association remained after individual adjustment for key tumor and treatment-related factors but was slightly attenuated in multivariate analyses for distant recurrence and for death. A breast cancer survivor cohort (the Health, Eating, Activity, and Lifestyle Study) examined 25(OH)D levels in 790 breast cancer survivors from the western USA, and cancer treatment data were obtained from Surveillance, Epidemiology, and End Results registries and
medical records [120]. Women with localized (n ¼ 424) or regional (n ¼ 182) breast cancer had lower serum 25(OH)D than did women with in situ disease (n ¼ 184) (p ¼ 0.05 and p ¼ 0.03, respectively). Multivariate regression models controlled for age, body mass index (in kg/ m2), race-ethnicity, geography, season, physical activity, diet, and cancer treatments showed that stage of disease independently predicted serum 25(OH)D (p ¼ 0.02). A limitation of this study was that blood samples were drawn more than 2 years after diagnosis, so reverse causation is a possibility. The Women’s Health Initiative Trial of vitamin D and calcium and cancer described above reported no reduction in breast cancer incidence, and no reduction in breast cancer mortality, though based on only 23 deaths each in the treatment and placebo groups [108]. However, there were some suggestive indications of less-aggressive disease in the women receiving vitamin D and calcium. For example, the mean tumor size was smaller in the treated group (mean, 1.54 versus 1.71 cm, p ¼ 0.05), and hazard ratios were nonsignificantly inverse for 4 lymph nodes and distant disease (SEER stage). One study suggested that vitamin D may protect against recurrence of melanoma. The Leeds Melanoma Cohort examined melanoma relapse in 872 melanoma patients [121]. Over a median follow-up of 4.7 years, there were 173 relapses and 141 deaths. The study had both a retrospective and a prospective component. In the retrospective component, self-reports of taking vitamin D supplements were suggestively associated with a reduced risk of melanoma relapse (RR 0.6; 95% CI 0.41.1; p ¼ 0.09). In the prospective study, higher 25(OH)D levels were associated with lower Breslow thickness at diagnosis (p ¼ 0.002), and were independently protective of relapse and death in multivariate analysis. The hazard ratio for relapse-free survival was 0.79 (95% CI 0.640.96; p ¼ 0.01) for a 20 nmol/l increase in serum 25(OH)D level, and the multivariate HR was 0.57 (95% CI 0.330.97) for the upper versus low tertile (>61.4 versus 41.3 nmol/l). For overall survival, the RR was 0.83 (95% CI 0.681.02) for a 20 nmol/l increase. Chapter 89 discusses vitamin D and skin cancer including melanoma. One study of head and neck patients did not support a prognostic role of vitamin D [122]. The study assessed pretreatment 25(OH)D in 522 patients who had enrolled in a randomized trial of vitamin E and beta-carotene. During active follow-up (median 4.4 years), 119 patients had a recurrence, 113 were diagnosed with a second primary cancer (53 were lung cancers); during a longer follow-up for mortality (median 8.0 years), 223 patients died, 62 from their initial cancer, and 81 for secondary primary (52 lung cancers). No association was found between dietary vitamin D or 25(OH)D status with
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head and neck cancer recurrence, second primary cancer incidence, and overall mortality. Prediagnostic 25(OH)D levels were examined in relation to mortality among 304 participants with colorectal cancer in the Nurses’ Health Study and the Health Professionals Follow-up Study cohorts [115]. The patients were diagnosed with colorectal cancer from 1991 to 2002 and were then followed until 2005. Patients diagnosed within 2 years of blood collection were excluded from the analysis to reduce the possibility of reverse causation. In multivariate analyses, compared with those in the lowest quartile, participants in the highest quartile had a multivariate adjusted RR 0.52 (95% CI 0.290.94) for overall mortality and RR 0.61 (95% CI 0.311.19) for colorectal cancer-specific mortality. The results persisted after excluding patients diagnosed within 5 years of blood collection. In a small Japanese study of 257 colorectal cancer patients undergoing surgery with 39 deaths (30 of which were colorectal cancer-specific deaths), higher serum 25(OH)D levels were associated with better survival (p ¼ 0.027) in multivariate analysis [123]. For prostate cancer, 160 patients (123 patients with a pretreatment serum sample and 37 patients who had received hormone therapy prior to the blood collection) in the JANUS serum bank were followed for a median period of 44 months [124]. During follow-up, 61 of the men died, 52 from prostate cancer. Serum 25(OH)D >50 nmol/l was associated with reduced mortality (RR 0.33; 95% CI 0.140.77; RR 0.16; 95% CI 0.050.43) compared to lower levels. However, the analysis restricted to patients receiving hormone therapy showed a stronger association, possibly suggesting that treatment may have influenced 25(OH)D levels. Thus, reverse causation cannot be excluded. Although the results from studies in which 25(OH)D is measured in cancer patients are provocative, potential reverse causation in the studies that assessed 25(OH)D after diagnosis need to be considered. First, sicker patients with a worse prognosis may have lower vitamin D levels (for example, due to less sun exposure or effects of the disease), even after adjustment for other prognostic indicators. That the results were stronger when limiting the analyses to early-stage cases or in studies that only examined early-stage cases tends to argue against this possibility, though it cannot be excluded entirely. In addition, some studies assessed 25(OH)D in the pre-diagnostic period, so reverse causation was unlikely. In these studies, we cannot definitively determine the timing of the association as pre-diagnostic and post-diagnostic 25(OH)D levels are likely to be correlated. It is possible that vitamin D status before the diagnosis, when the tumor is developing, may lead to less aggressive cancers, or that 25(OH)D after the diagnosis improves prognosis. Of course, effects of
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vitamin D on both pre-diagnostic and post-diagnostic stages are possible. Predicted 25(OH)D level was examined in relation to mortality among 1017 participants in the Nurses’ Health Study and the Health Professional Follow-up Study cohorts who were diagnosed with colorectal cancer from 1986 to 2004 [125]. Higher predicted 25(OH)D levels were associated with a significant reduction in colorectal cancer-specific and overall mortality; compared with those with levels in the lowest quintile, participants with predicted 25(OH)D levels in the highest quintile had an adjusted RR ¼ 0.50 (95% CI 0.260.95) for cancer-specific mortality and 0.62 (95% CI 0.420.93) for overall mortality. These associations persisted even after adjusting for pre-diagnostic predicted 25(OH)D level. Although predicted 25(OH)D level has limitations as described above, this study suggests that post-diagnostic 25(OH)D levels may influence prognosis. In some studies, those diagnosed with cancer during the summer and autumn months, when vitamin D status is higher, have a better prognosis than those diagnosed and/or treated during the winter months [126]. The effect of season of diagnosis on survival from colon, breast, and prostate cancers was studied in Norway, where solar-generated vitamin D is minimal during the winter months [127]. This study included 45 667 men and women with colon, breast, or prostate cancer, and the period of observation was from 1964 to 1992. There was no significant seasonal variation in the incidence rates of these cancers, with 25% of the cancers diagnosed in each season. Death rates at 18 months, 36 months, and 45 months were significantly lower in those diagnosed in autumn months compared with those diagnosed in the winter months. The magnitude was about 20 to 30% lower, with maximal benefit at 18 months. Similar findings from this group were later reported additionally for lung cancer and Hodgkin’s lymphoma [128]. Similar findings for breast and lung cancer were noted in the UK [129], though not in Sweden, where the structure of the health care system and vacationing patterns tend to cause later-stage disease in the summer months [130]. Overall, these finding suggest that high vitamin D level at the time of diagnosis, and presumably treatment, may improve survival from various cancers. Late anti-cancer effects of vitamin D, such as reduction in metastases, are observed in numerous animal models. Some evidence from animal models suggests that vitamin D analogs may improve tumor control by radiation treatment, in part by promoting apoptosis [131]. Although these data are provocative, other micronutrients related to fruits and vegetables may be consumed in the summer months only, so an effect cannot necessarily be attributed solely to vitamin D.
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The suggested relationship between vitamin D and cancer mortality and survival prompted an examination of the results from the Women’s Health Initiative and the UK randomized trials for total cancer mortality and colorectal cancer morality. These studies (described above) tended to show no appreciable association with cancer incidence, though there were limitations regarding dose, duration, and power. In the Women’s Health Initiative, women randomized to vitamin D and calcium had a reduced risk of cancer mortality (344 cancer deaths in the treatment group versus 382 in the placebo group (RR 0.89; 95% CI 0.771.03). In the UK study, overall there were 63 cancer deaths in the treatment group and 72 in the placebo group (RR 0.86; 95% CI 0.611.20; for men RR 0.93; 95% CI 0.641.34; for women RR 0.53; 95% CI 0.211.33). In a post-hoc pooled analysis, the pooled RR ¼ 0.885 (95% CI 0.781.01) for total mortality for these two studies. In addition, although based on small numbers, a suggestive reduction in colorectal cancer mortality was observed in those randomized to vitamin D (pooled RR 0.78; 95% CI 0.521.17). These results should be treated with caution, given that they are from a posthoc analysis of only two studies, each with limitations. Nonetheless, they are consistent with an important effect of vitamin D in reducing cancer mortality independently of any effect on incidence. Because the doses and duration were unlikely to be optimal for cancer prevention, if this association is causal, then the possible effect of vitamin D on cancer mortality could be considerably stronger.
SUMMARY AND SYNTHESIS OF THE EPIDEMIOLOGY OF VITAMIN D AND CANCER Since the vitamin D and cancer hypothesis was formulated in the 1980s, a number of studies have addressed this hypothesis using various approaches. Although this chapter focused specifically on the epidemiologic evidence, from a broader perspective other types of studies inform on the vitamin D cancer relationship, including animal models and in vitro studies. The mechanistic evidence supporting a role for vitamin D in carcinogenesis is extensive and compelling (for example, see Chapters 88, 89, 90). Most human epidemiologic data are based on vitamin D exposure over a relatively short timeframe, generally relatively late in the carcinogenesis process, and within the range of vitamin D inherent in the population. RCTs, while better at avoiding confounding, may be even more restricted in timeframe and range of vitamin D exposure, and stage of disease examined. Despite the limitations, the human data are necessary in ultimately informing about issues
concerning dose and duration required to observe effects, and magnitudes of effects. Interpretation of the results from studies to date is complicated because the strengths and limitations of the various types of study designs tend to differ, and each particular study may have its own nuances to consider. The evidence may be considered strongest when different types of studies in various settings tend to yield consistent results. Ecologic studies that compared cancer mortality rates in different regions within the USA formed the basis of the hypothesis that high vitamin D levels may lower risk of various cancers. Colorectal cancer was the first malignancy linked to vitamin D status [3], perhaps because the association with vitamin D is strongest for this cancer [8]. For example, based on an estimate of preventable premature deaths from cancer due to insufficient UV-B radiation in US whites, for men, Grant estimates 60.2% of these would be from colorectal cancer and 39.8% from all the rest combined; for women, 35.5% would be from colorectal cancer, 42.1% from breast cancer and 23.4% from the rest combined. Since colorectal cancer accounts for approximately 10% of the total cancer in the USA (excluding non-melanoma skin cancers), the ecologic data would support a much stronger effect of vitamin D on colorectal cancer than for other cancers. Ecologic studies of UV-B and mortality due to various cancers (particularly for colorectal and other gastrointestinal cancers) have been seen in diverse countries. Confounding cannot be excluded, but the presence of an association in diverse populations is striking as it would appear somewhat unlikely that some confounding factor would be consistently observed in these diverse settings. Further epidemiologic study based on other study designs has strongly supported that vitamin D status influences colorectal cancer. Specifically, with remarkable consistency, a higher level of circulating 25(OH)D has been consistently associated with a decreased risk of colorectal cancer and adenoma, a finding confirmed in meta-analyses. Individuals in the high quartile or quintile of 25(OH)D had a 40 to 50% reduction of risk of colon and/or rectal cancer relative to those in the lowest group. Inverse associations for colorectal cancer and adenoma have also been observed for predicted vitamin D, UV-B exposure (at the individual level) and dietary and supplementary intake. The inverse association observed for dietary and supplementary intake may be even stronger than anticipated, possibly because higher calcium intake is associated with a reduced risk of colorectal cancer and fortified milk is a major source of both dietary vitamin D and calcium. The consistency of the association with direct or indirect measures of vitamin D in diverse circumstances indicates that a factor associated with vitamin D and causally with cancer risk (confounder) is unlikely to account for these
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SUMMARY AND SYNTHESIS OF THE EPIDEMIOLOGY OF VITAMIN D AND CANCER
associations. The evidence from RCTs is limited; the largest study (WHI) had a very limited dose and did not show an overall benefit of vitamin D on colorectal cancer for postmenopausal women, but a post-hoc analysis indicated a potential benefit in women not on hormonal replacement therapy. Thus, the current epidemiologic evidence overall strongly supports an important role for vitamin D on colorectal cancer. A role for vitamin D and colorectal cancer is also strongly supported in animal studies (see Chapter 87). Ecologic data of UV-B exposure support a link with breast cancer in some populations (especially the USA), but not in Japan or China. Possibly, the predominance of different etiologic factors among these populations (e.g., obesity, diet) could account for the variable effects. The evidence from epidemiologic studies of vitamin D status and breast cancer is somewhat conflicting. One caseecontrol study provided intriguing findings: breast cancer cases reported less sun exposure primarily during ages 10e19, but not at other ages, than controls. Because recall bias is a possible explanation for this finding in a retrospective study, replicating these results in prospective settings is important. Interestingly, adolescent exposures have often been found to be critical in determining subsequent breast cancer risk, probably because the breast tissue is rapidly developing over this time period. Most prospective data based on 25(OH)D measurement do not support an association when study results are aggregated; however, inverse associations have been observed in some studies, and effects in certain susceptible groups are plausible. Caseecontrol studies of 25(OH)D and breast cancer risk are prone to reverse causation, as disease status could influence 25(OH)D levels. The theoretical concern of reverse causation has apparently been borne out given the divergent results between the cohort and the caseecontrol studies, the latter tending to show an inverse association. The studies of vitamin D intake are modestly supportive, but interpretation is limited by the generally low intakes of vitamin D in most populations. Ecologic studies of regional UV-B and cancer mortality find an inverse association with prostate cancer mortality, but this association appears much weaker than that observed for colorectal or breast cancer [8], and perhaps limited to counties north of 40 N latitude in the USA [132]. The studies of circulating 25(OH)D have found no or relatively weak non-significant associations and vitamin D intake studies are not supportive of any association between 25(OH)D and total prostate cancer risk. Some very limited evidence suggests that perhaps a higher risk occurs only with very low levels of 25(OH)D (for example, <20 nmol/l). It is possible that the vitamin D pathway is relevant for prostate carcinogenesis, but prostate cancer cells
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apparently lose 1a-hydroxylase activity early in the carcinogenesis process, possibly making some cancers relatively insensitive to circulating 25(OH)D levels [133,134]. Prostate cancer is known to be a very heterogeneous malignancy in terms of biologic and clinical behavior, and risk factors for advanced-stage prostate cancer incidence may differ from those for incident prostate cancer [37]. Some data suggest that vitamin D may be more relevant for advanced or aggressive prostate cancer than for total prostate cancer. For other cancer sites, the data have generally been too sparse to support strong conclusions. A recent set of studies on the pooled analyses of 25(OH)D in relation to incidence of various malignancies, including endometrial, esophageal, gastric, kidney, non-Hodgkin’s lymphoma, ovarian, and pancreatic cancers, has been published (Cohort Consortium Vitamin D Pooling Project of Rarer Cancers (VDPP)) [75]. The results from the VDPP have been disappointing, with no clear risk reductions with higher 25(OH)D noted, except perhaps in some subgroups (e.g., ovarian cancer in overweight/obese women). More worrisome, a higher risk was suggested at the highest level of 25(OH)D for pancreatic cancer. Although this unexpected result does not seem to be supported by other data, it bears further study. While reasonably powered, as the number of endpoints for each cancer ranged from about 500e1000 cases, modest associations could have been missed, and the use of a single blood assessment of 25(OH)D could incur a considerable amount of measurement error. The etiologically relevant time period could have been missed. Nonetheless, these results suggest that 25(OH)D levels in middle-aged individuals may not be a very potent determinant for incident cancer risk for these malignancies. From an epidemiologic perspective, more promising is the evidence that poor vitamin D status may be related to more aggressive forms of cancer and prognosis independent of any effects on occurrence of the cancer. This evidence is from diverse lines of investigation, and while not definitive, suggests that effects of vitamin D on cancer progression may be a potentially fruitful avenue to pursue. From available evidence, it is unclear if the potential benefit of better vitamin D status on cancer mortality is in pre-diagnostic stages by influencing tumor aggressive behavior, during treatment through positive interactions with therapies, or in post-diagnostic stages by enhancing survival. Of course, these potential benefits are not mutually exclusive and more than one may be operative. Intervention studies, with reasonably sized samples and relatively short follow-up times, could provide a relatively feasible test of the hypothesis that administering vitamin D after diagnosis improves survival. It is also important for future studies to examine markers in the vitamin D
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pathway in the cancer pathway in relation to survival. For example, a recent study in the Health Professionals Follow-up Study found that higher expression of the vitamin D receptor in prostate cancer strongly predicted better survival [135]. In conclusion, a broad body of evidence supports an important role of vitamin D on prevention of cancer incidence and mortality. To date, the data are most compelling for a strong role of vitamin D for colorectal cancer, a malignancy that accounts for approximately one million cases annually worldwide. Given current knowledge, targeting a 25(OH)D level of at least 80 nmol/l is prudent for prevention of cancer incidence and mortality, given that this is a typical level of 25(OH)D in “natural” settings and has generally not been associated with any adverse effects. It is possible that higher levels may be more desirable, but given limited ranges of 25(OH)D in most epidemiologic studies to date, further study would be required. Ongoing and future epidemiologic studies and RCTs are likely to provide further data as to optimal 25(OH)D levels. Future epidemiologic should also incorporate cancer tissue markers, such as expression of the vitamin D receptor or enzymes that influence vitamin D metabolism.
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of the Cohort Consortium Vitamin D Pooling Project of Rarer Cancers, Am. J. Epidemiol. 172 (2010) 10e20. K.J. Helzlsouer, Overview of the cohort consortium vitamin D pooling project of rarer cancers. VDPP Steering Committee, Am. J. Epidemiol. 172 (2010) 4e9. C.C. Abnet, Y. Chen, W.H. Chow, Y.T. Gao, K.J. Helzlsouer, L. Le Marchand, et al., Circulating 25-hydroxyvitamin D and risk of esophageal and gastric cancer: Cohort Consortium Vitamin D Pooling Project of Rarer Cancers, Am. J. Epidemiol. 172 (2010) 94e106. A. Zeleniuch-Jacquotte, L. Gallicchio, V. Hartmuller, K.J. Helzlsouer, M.L. McCullough, V.W. Setiawan, et al., Circulating 25-hydroxyvitamin D and risk of endometrial cancer: Cohort Consortium Vitamin D Pooling Project of Rarer Cancers, Am. J. Epidemiol. 172 (2010) 36e46. L. Gallicchio, L.E. Moore, V.L. Stevens, J. Ahn, D. Albanes, V. Hartmuller, et al., Circulating 25-hydroxyvitamin D and risk of kidney cancer: Cohort Consortium Vitamin D Pooling Project of Rarer Cancers, Am. J. Epidemiol. 172 (2010) 47e57. W. Zheng, K.N. Danforth, S.S. Tworoger, M.T. Goodman, A.A. Arslan, A.V. Patel, et al., Circulating 25-hydroxyvitamin D and risk of epithelial ovarian cancer: Cohort Consortium Vitamin D Pooling Project of Rarer Cancers, Am. J. Epidemiol. 172 (2010) 70e80. R.Z. Stolzenberg-Solomon, E.J. Jacobs, A.A. Arslan, D. Qi, A.V. Patel, K.J. Helzlsouer, et al., Circulating 25-hydroxyvitamin D and risk of pancreatic cancer: Cohort Consortium Vitamin D Pooling Project of Rarer Cancers, Am. J. Epidemiol. 172 (2010) 81e93. M.P. Purdue, D.M. Freedman, S.M. Gapstur, K.J. Helzlsouer, F. Laden, U. Lim, et al., Circulating 25-hydroxyvitamin D and risk of non-Hodgkin lymphoma: Cohort Consortium Vitamin D Pooling Project of Rarer Cancers, Am. J. Epidemiol. 172 (2010) 58e69. R.Z. Stolzenberg-Solomon, R. Vieth, A. Azad, P. Pietinen, P.R. Taylor, J. Virtamo, et al., A prospective nested caseecontrol study of vitamin D status and pancreatic cancer risk in male smokers, Cancer Res. 66 (2006) 10213e10219. Y. Bao, K. Ng, B.M. Wolpin, D.S. Michaud, E. Giovannucci, C.S. Fuchs, Predicted vitamin D status and pancreatic cancer risk in two prospective cohort studies, Br. J. Cancer 102 (2010) 1422e1427. H.G. Skinner, D.S. Michaud, E. Giovannucci, W.C. Willett, G.A. Colditz, C.S. Fuchs, Vitamin D intake and the risk for pancreatic cancer in two cohort studies, Cancer Epidemiol. Biomarkers Prev. 15 (2006) 1688e1695. S.S. Tworoger, I.M. Lee, J.E. Buring, B. Rosner, B.W. Hollis, S.E. Hankinson, Plasma 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D and risk of incident ovarian cancer, Cancer Epidemiol. Biomarkers Prev. 16 (2007) 783e788. A.T. Toriola, H.M. Surcel, C. Agborsangaya, K. Grankvist, P. Tuohimaa, P. Toniolo, et al., Serum 25-hydroxyvitamin D and the risk of ovarian cancer, Eur. J. Cancer 46 (2010) 364e369. A.T. Toriola, H.M. Surcel, A. Calypse, K. Grankvist, T. Luostarinen, A. Lukanova, et al., (Epub ahead of print) Independent and joint effects of serum 25-hydroxyvitamin D and calcium on ovarian cancer risk: a prospective nested caseecontrol study, Eur. J. Cancer (2010 Jun 18). W. Chen, S.M. Dawsey, Y.L. Qiao, S.D. Mark, Z.W. Dong, P.R. Taylor, et al., Prospective study of serum 25(OH)-vitamin D concentration and risk of oesophageal and gastric cancers, Br. J. Cancer 97 (2007) 123e128. C.C. Abnet, W. Chen, S.M. Dawsey, W.Q. Wei, M.J. Roth, B. Liu, et al., Serum 25(OH)-vitamin D concentration and risk of
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[123] H. Mezawa, T. Sugiura, M. Watanabe, C. Norizoe, D. Takahashi, A. Shimojima, et al., Serum vitamin D levels and survival of patients with colorectal cancer: post-hoc analysis of a prospective cohort study, BMC Cancer 10 (2010) 347. [124] S. Tretli, E. Hernes, J.P. Berg, U.E. Hestvik, T.E. Robsahm, Association between serum 25(OH)D and death from prostate cancer, Br. J. Cancer 100 (2009) 450e454. [125] K. Ng, B.M. Wolpin, J.A. Meyerhardt, K. Wu, A.T. Chan, B.W. Hollis, et al., Prospective study of predictors of vitamin D status and survival in patients with colorectal cancer, Br. J. Cancer 101 (2009) 916e923. [126] A. Porojnicu, T.E. Robsahm, J.P. Berg, J. Moan, Season of diagnosis is a predictor of cancer survival. Sun-induced vitamin D may be involved: a possible role of sun-induced Vitamin D, J. Steroid. Biochem. Mol. Biol. 103 (2007) 675e678. [127] J. Moan, A.C. Porojnicu, T.E. Robsahm, A. Dahlback, A. Juzeniene, S. Tretli, et al., Solar radiation, vitamin D and survival rate of colon cancer in Norway, J. Photochem. Photobiol. B. 78 (2005) 189e193. [128] J. Moan, A. Dahlback, Z. Lagunova, E. Cicarma, A.C. Porojnicu, Solar radiation, vitamin D and cancer incidence and mortality in Norway, Anticancer Res. 29 (2009) 3501e3509. [129] R. Roychoudhuri, D. Robinson, V. Coupland, L. Holmberg, H. Moller, Season of cancer diagnosis exerts distinct effects upon short- and long-term survival, Int. J. Cancer 124 (2009) 2436e2441.
[130] L. Holmberg, J. Adolfsson, L. Mucci, H. Garmo, H.O. Adami, H. Moller, et al., Season of diagnosis and prognosis in breast and prostate cancer, Cancer Causes Control 20 (2009) 663e670. [131] S. Sundaram, A. Sea, S. Feldman, R. Strawbridge, P.J. Hoopes, E. Demidenko, et al., The combination of a potent vitamin D3 analog, EB 1089, with ionizing radiation reduces tumor growth and induces apoptosis of MCF-7 breast tumor xenografts in nude mice, Clin. Cancer Res. 9 (2003) 2350e2356. [132] G.G. Schwartz, C.L. Hanchette, UV, latitude, and spatial trends in prostate cancer mortality: all sunlight is not the same (United States), Cancer Causes Control 17 (2006) 1091e1101. [133] L.W. Whitlatch, M.V. Young, G.G. Schwartz, J.N. Flanagan, K.L. Burnstein, B.L. Lokeshwar, et al., 25-Hydroxyvitamin D1alpha-hydroxylase activity is diminished in human prostate cancer cells and is enhanced by gene transfer, J. Steroid Biochem. Mol. Biol. 81 (2002) 135e140. [134] T.C. Chen, L. Wang, L.W. Whitlatch, J.N. Flanagan, M.F. Holick, Prostatic 25-hydroxyvitamin D-1alpha-hydroxylase and its implication in prostate cancer, J. Cell Biochem. 88 (2003) 315e322. [135] W.K. Hendrickson, R. Flavin, J.L. Kasperzyk, M. Fiorentino, F. Fang, R. Lis, et al., Vitamin D receptor protein expression in tumor tissue and prostate cancer progression, J. Clin. Oncol. in press (2011).
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C H A P T E R
83 Vitamin D: Cancer and Differentiation Johannes P.T.M. van Leeuwen 1, Marjolein van Driel 1, David Feldman 2, Alberto Mun˜oz 3 1
2
Erasmus Medical Center, Rotterdam, The Netherlands Stanford University School of Medicine, Stanford, CA, USA 3 Instituto de Investigaciones Biomedicas, Madrid, Spain
INTRODUCTION The secosteroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is the most potent metabolite of vitamin D3 and is an important regulator of calcium homeostasis and bone metabolism via actions in intestine, bone, kidney, and parathyroid glands. 1,25(OH)2D3 exerts its effects via an intracellular receptor that is a member of the steroid hormone receptor family (see chapters in Section II on Mechanism of Action). Throughout the last few decades it has become evident that the vitamin D receptor (VDR) is not limited to cells and tissues involved in regulation of calcium and bone metabolism but is also present in a wide variety of other cells and tissues including cancer cells of various origins. This has led to a vast series of studies on the role of vitamin D in tumor cell growth regulation, treatment of cancer and development of potent synthetic vitamin D analogs. Various specialized chapters will discuss in detail the effect of vitamin D on specific cancers (see chapters in Section X) and the development of analogs (see chapters in Section IX). In this chapter our goal is to set the stage by providing an overview of the history and current state of knowledge of the field. We will address several areas: recent developments in studies of vitamin D and cancer, regulation of tumor cells, possible mechanisms, and clinical applications.
VITAMIN D AND CANCER Vitamin D Receptor As exemplified in Table 83.1, the VDR has been demonstrated in a broad range of tumors and malignant
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10083-6
cell types. For colon and breast cancer cells an inverse relationship between VDR level and degree of differentiation has been described by some investigators [1,2]. VDR level is increased in ovarian carcinoma compared to normal ovarian tissue [3]. For colorectal cancer it was shown that VDR expression is associated with the degree of tumor differentiation [4] and with a more favorable prognosis [5]. A VDR immunoreactivity score showed an increase in VDR in breast carcinoma specimens compared to normal breast tissue but no clear relation with proliferative status could be assessed [6]. A later study by the same group showed that VDR expression is not a prognostic factor for breast cancer but the strong VDR immunoreactivity in the breast cancer specimens supports the evidence that it may be a target for intervention [7]. Also in other studies no associations between VDR and clinical and biochemical parameters of breast cancer were found [8e13]. Albeit that the association studies on VDR expression and predictive and/or prognostic characteristics for cancer are so far not conclusive, the widespread distribution of the VDR in malignant cells indicates that regulation of cancer cell function might be a new target in the action of 1,25(OH)2D3 and provides a biological basis for the epidemiological observations discussed below. An interesting recent observation has put the VDR in relation to cancer in a whole new perspective. It was shown that VDR can function as a receptor for the secondary bile acid lithocholic acid (see Chapter 43). This compound is hepatotoxic and a potential enteric carcinogenic. Interestingly, binding of both lithocholic acid and vitamin D to the VDR results in induction of CYP3A, the enzyme that detoxifies lithocholic acid in the liver and intestine [14,15] (see also Chapter
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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VDR in Tumors and Malignant Cell Types
Basal cell carcinoma Breast carcinoma Bladder cancer Cervical carcinoma Colonic adenocarcinoma Colorectal carcinoma Gall bladder carcinoma Glioma cells Karposi sarcoma Lung carcinoma Lymphocytic leukemia Malignant B-cell progenitors Malignant melanoma Medullary thyroid carcinoma
Myeloid leukemia Myeloma Osteogenic sarcoma Ovarian carcinoma Neuroblastoma Non-Hodgkin’s lymphoma Pancreatic carcinoma Parathyroid adenoma Pituitary adenoma Prostate carcinoma Renal cell carcinoma Squamous cell carcinoma Transitional cell bladder carcinoma Uterine carcinosarcoma
79). It is postulated that vitamin D and lithocholic acid, by binding to the VDR, activate a feed-forward catabolic pathway that increases CYP3A expression leading to detoxification of carcinogenic bile acids. A relationship between the presence of VDR and carcinogenesis was recently also shown for the skin. Absence of VDR increased the sensitivity for chemically induced tumorigenesis [16]. Moreover, in mice the vitamin D analog EB1089 prevents b-catenin-induced trichofolliculomas, while low levels of VDR associate with the induction by b-catenin of infiltrative basal cell carcinomas [17]. Although cellular effects of 1,25(OH)2D3 traditionally have been attributed to activation of the nuclear VDR, over the years research has been performed to identify a membrane 1,25(OH)2D3 receptor (see also Chapter 15). As discussed in Chapter 15, the best evidence suggests that this rapid-acting membrane receptor is related to the VDR. Another presumed membrane receptor called 1,25D3-MARRS (membrane-associated, rapid response steroid-binding) has also been studied and recently identified. The 1,25D3-MARRS protein was found to be expressed in MCF-7, MDA MB 231, and MCF-10A breast cancer cells by qRT-PCR and Western blotting [18]. Interestingly knockdown of 1,25D3-MARRS receptor increased the sensitivity of MCF-7 cells to the vitamin D analogs KH1060 and MC903, but not to unrelated agents such as all-trans retinoic acid and paclitaxel. These results implicate that 1,25D3-MARRS receptor expression interferes with the growth-inhibitory activity of 1,25(OH)2D3 in breast cancer cells via the nuclear VDR [18]. A future challenge will be to confirm and eventually assess the clinical implications of this bi-directional regulation of tumor cell growth by 1,25(OH)2D3.
Epidemiology The first to document an association of cancer mortality with sun exposure and latitude was Hoffman
in 1915 [19]. More recent studies in 1980 by Garland et al. provide additional data showing that death rates from colon cancer tended to increase with increasing latitude and decreasing sunlight [20]. Schwartz discusses the sunlight hypothesis in Chapter 53. Later more direct evidence about a correlation between vitamin D and colon cancer came from the inverse relationship between levels of serum 25-hydroxyvitamin D3 (25(OH)D3) and the incidence of colon cancer [21,22]. In a meta-analysis Gorham estimated that an increase of 84 nmol/l (33 ng/ ml) in serum 25(OH)D3 level would lead to a 50% reduction in the incidence of colon cancer [23]. A study of National Health and Nutrition Examination Survey III (NHANES III) data also found an association between 25(OH)D3 and colorectal cancer mortality. Individuals with a 25(OH)D3 level over 80 nmol/l (32 ng/ml) had a 75% lower risk of death from colorectal cancer than those with lower levels of 25(OH)D3. A level over 95 nmol/l correlated with a 55% reduction in colon cancer risk compared to those with a level below 40 nmol/l [24]. In addition, a similar relationship between sunlight exposure, vitamin D, and the risk for fatal breast and prostate cancer also has been suggested [25e29]. From the NHANES III study it was reported that women with a serum level of 25(OH)D3 more than 62 nmol/l had a 75% decrease in mortality due to breast cancer [24]. From two other studies the authors concluded that there was a 58% lower risk of breast cancer in women with 25(OH)D3 levels more than 95 nmol/l compared to women with levels lower than 37.5 nmol/l [30,31]. In a meta-analyses 1750 women were stratified into five groups of 25(OH)D3 levels ranging from high to low and this showed a clear doseeresponse association [32]. The highest breast cancer rates were found in the group with the lowest 25(OH)D3 levels (<32 nmol/l) while the cancer rates were lower at higher levels (>130 nmol/l). For prostate cancer the incidence of prostate cancer was twice as high in men with a 25(OH)D3 level below 70 nmol/l and 1,25(OH)2D3 levels below 77 pmol/l. A full discussion of the epidemiologic data linking vitamin D and cancer can be found in Chapter 82. Recent analyses showed that the association between UVB irradiance and prostate cancer incidence depends on the season of irradiance [33]. However, a large prospective study by Ahn et al. did not support the hypothesis that vitamin D is associated with decreased risk of prostate cancer; in contrast higher circulating 25(OH)D3 concentrations may be associated with increased risk of aggressive disease [34]. Also for ovarian cancer a similar discrepancy was observed. For example, Grant et al. reported a strong association between vitamin D levels, geographical latitude and ovarian cancer mortality [35,36] while more recently Toriola et al. in a caseecontrol study with the Finnish
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Maternity Cohort did not find significant association between ovarian cancer and serum 25(OH)D3 levels [37]. The relationship between sunlight exposure and cancer, especially with respect to vitamin D, has been carefully reviewed by Studzinski and Moore [38]. The dual relationship between sunlight and cancer is of interest and remains the subject of many studies [35,39,40]. A relation between skin type and prostate cancer has been described [41e43] and an article discussing the skin, sunlight, vitamin D and cancer from an evolutionary perspective has been published [44]. Grant et al. estimated that between 50 000 and 63 000 Americans and between 19 000 and 25 000 adults from the UK die every year from cancer due to vitamin D deficiency [45]. An analysis of the economic burden due to vitamin D insufficiency from inadequate exposure to solar UVB, diet and supplements was $40e56 billion in 2004 versus an economic burden for excess UV irradiation of $6e7 billion [46]. Also, the relationship between cancer, diet, and calcium intake and vitamin D has been addressed in several studies [47e52]. A Canadian study noted similar vitamin D intakes in breast cancer patients and control subjects [53]. Moreover, in a mouse model no relationship was found between dietary intake of a wide range of doses of calcium or vitamin D on carcinogen-induced skin tumors [54]. A large Finnish epidemiological study showed an association of low serum 25(OH)D3 with prostate cancer [55,56]. A study on intake of micronutrients suggested that vitamin D and calcium might interact with antioxidants like vitamin C and E in reducing colorectal cancer risk [57]. It is clear that sunlight exposure, vitamin D intake, and other dietary components such as calcium and fat should be considered as possibly interacting with one another when the relationship between vitamin D and cancer risk is assessed. The data on VDR as bile acid sensor and its postulated role in detoxification provide a direct biological basis for the relation between increased colon cancer and high-fat diets [58] and that colon cancer occurs in areas with higher prevalence of rickets [51]. In addition, mice lacking VDR have been reported to have a higher proliferation rate in the colon [59,60]. A survey of mutations in the VDR in osteosarcomas, several other sarcomas, non-small-cell lung cancers, and a large number of cell lines representing many tumor types did not show that mutations or rearrangements in the VDR gene play a role in these cancers [61]. Aspects of sunlight and the epidemiology of vitamin D and calcium will be discussed in greater detail in Chapters 53 and 56. A concluding comment is that a high number of, but not all, observational, epidemiological and preclinical studies suggest a protective anticancer action of vitamin D. A recent report published by the International
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Agency for Research on Cancer of the World Health Organization considers the risk of colorectal cancer to be associated with vitamin D deficiency. However, pending larger epidemiological studies and prospective clinical trials, the relationship is less clear for other types of cancer [62]. Vitamin D Receptor Gene Polymorphisms Several polymorphisms have been identified in the VDR gene and studied in relation to various endpoints including osteoporosis and other diseases (discussed in Chapter 56). Over the last 15e20 years an increasing number of studies have examined the association of polymorphisms in the VDR and cancer. The first study showed an association between polymorphisms at the 3’ end of the VDR gene and prostate cancer [63]. This was shortly followed by a study showing an association of prostate cancer with variations in the 3’ poly-A stretch in the VDR gene [64]. Subsequently several other studies also showed associations of polymorphisms in the 3’ region of the VDR gene and prostate cancer risk [65e71] albeit other studies did not confirm this association [72e76]. For the Cdx-2 VDR promoter polymorphism an increased risk for prostate cancer was reported to be dependent on UV radiation exposure [77]. For breast cancer both the presence [78e83] and absence [84] of an association with polymorphisms in the VDR gene have been reported. Also for colon cancer both presence [85,86] and absence [87] of an association with VDR polymorphisms have been reported. In a recent study that compared cases to unaffected sibling controls, no association between any of the VDR single nucleotide polymorphisms and risk for colorectal cancer was observed [88]. No association of VDR polymorphisms with basal cell carcinoma was reported [89]. An association with the aggressive renal cell carcinoma was found for the TaqI VDR polymorphism [90], while the FokI but not the TaqI polymorphism was associated with altered risk for malignant melanoma [91]. Another study on rectal cancer reported a correlation between VDR gene polymorphisms and erbB-2/HER-2 expression [92]. It can be concluded that so far the studies searching for a link between VDR gene polymorphisms and cancer risk are far from conclusive with some studies finding a relationship to cancer risk and others failing to find one. A major reason might be the limited size of most of the studies so that they do not have the power to identify with statistical significance a small increase in risk. In the absence of a large definitive study, more association studies of VDR gene polymorphisms and specific cancers are needed, which should be followed by a meta-analysis to more definitively assess whether there is an association and, if so, what the size of the effect is.
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In studies of VDR gene polymorphisms it also is important to take into account the potential impact of environmental factors interacting with the genetic variance. Diet, vitamin D intake, and sun exposure may modify the association with cancer risk. Interaction between vitamin D and calcium intake and cancer was found in some of the VDR gene polymorphism studies [85,93e95]. They reported decreased risk of prostate cancer [93] and colorectal adenomas [94] in those with lower vitamin D levels and a particular VDR gene polymorphism. However, results of these studies are unusual in light of the fact that higher calcium and vitamin D intake are generally associated with a modestly reduced risk of colorectal neoplasia. In the recent study by Poynter et al. calcium and vitamin D intake derived from the food frequency questionnaire did not change their observation about the absence of an association between VDR gene variations and colorectal cancer [88]. Finally, and most importantly, it should be realized that except for the FokI translational start site polymorphism, all other polymorphisms analyzed so far are anonymous with no change in the coded protein. Thus functionality of the polymorphism, or linkage with other polymorphisms that may be functional, still needs to be proven. The 3’ polymorphisms have been shown to be in linkage with 3’-UTR polymorphisms but no relation with VDR mRNA stability could be demonstrated [96]. In the VDR promoter region 1a two functional polymorphisms have been identified. The Cdx-2 promoter polymorphism has been reported to lead to different VDR gene expression [97,98] and the G-1521-C polymorphism to binding of different complexes in gel shift analyses [99,100]. Further detailed discussion of possible functional consequences of VDR gene polymorphisms and impact of vitamin D levels is beyond the scope of this chapter but will be addressed in Chapter 56.
of osteoclasts, and 1,25(OH)2D3 induces differentiation of immature myeloid cells toward monocytese macrophages and also stimulates the activation and fusion of some macrophages (discussed in Chapters 91, 92, and 93). From these results it has been postulated that 1,25(OH)2D3 stimulates differentiation and fusion of osteoclast progenitors into osteoclasts (104e106). Also, in the intestine, 1,25(OH)2D3 has important effects on cellular proliferation and differentiation [107]. Thus the differentiation-inducing capacity of bone and interstitial cells, 1,25(OH)2D3 may play an important role in the regulation of calcium and bone metabolism. These in vitro findings were followed by the in vivo observation that 1,25(OH)2D3 prolongs the survival time of mice inoculated with myeloid leukemia cells [108]. As shown in Table 83.2, over the years 1,25(OH)2D3 has been shown to have beneficial effects in several other in vivo animal models of various types of cancers [109e131]. An important aspect and limitation of the treatment of cancer with 1,25(OH)2D3 was revealed by this limited set of clinical trials (see “Cinical studies,” below); to achieve growth inhibition, high doses of 1,25(OH)2D3 are needed (confirming the in vitro data), which can cause the side effect of hypercalcemia. This has prompted the development of analogs of 1,25(OH)2D3 in order to dissociate the anti-proliferative effect from the calcemic and bone metabolism effects (see Section IX in this book). Although the precise mechanism for this dissociation of activities is not completely understood, at the moment several 1,25(OH)2D3 analogs are available that seem to fulfill these criteria. In Table 83.3 the in vivo animal studies using 1,25(OH)2D3 analogs on various cancer types are summarized [119,125,126,128e150] and more fully discussed in Section IX of this volume.
Clinical Studies Growth and Development In addition to the epidemiological studies and demonstration of VDR in cancer cells, since the early 1980s there has also been an increasing amount of cell biological data supporting a role for vitamin D as an inhibitor of cancer growth. Multiple studies have shown that at high concentrations (10 9e10 7 M), 1,25(OH)2D3 inhibits the growth of tumor cells in vitro. It was demonstrated as early as 1981 that 1,25(OH)2D3 inhibits the growth of malignant melanoma cells and stimulates the differentiation of immature mouse myeloid leukemia cells in culture [101e103]. 1,25(OH)2D3 also induces differentiation of normal bone marrow cells. Immature bone marrow cells of the monocytee macrophage lineage are believed to be the precursors
Only a limited number of clinical trials of vitamin D in cancer have been performed up to now which may be attributed to the calcemic activity of 1,25(OH)2D3. Alfacalcidol (1a-hydroxyvitamin D3; 1a-(OH)D3), which is converted to 1,25(OH)2D3 in vivo, caused a beneficial response in low-grade non-Hodgkin’s lymphoma patients (151,152). Also, in a study treating patients with myelodysplasia with alfacalcidol, transient improvement in peripheral blood counts was seen; however, half of the patients developed hypercalcemia [153]. Another study reported a sustained hematological response in six myelodysplasia patients treated with high doses of alfacalcidol [154]. These patients were restricted in their dietary calcium intake; nevertheless, four patients developed hypercalcemia due to increased bone resorption. With respect to treatment of cutaneous
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TABLE 83.2
In vivo Effects of 1,25(OH)2D3 and 1a-(OH)D3 in Animal Models of Cancera
Tumor
Model
Effect
Refs
Adenocarcinoma
CAC-8 cells injected in nude mice
Reduction in tumor volume
[129]
Breast
NMU- and DMBA-induced breast cancer in rats
Tumor suppression
[115,118]
Colon
Human colon cell line implanted into nude mice; DMH-induced colon cancer in rats; APCmin mice
Tumor suppression; reduction of the incidence of colon adenocarcinomas; decrease in polyp number and tumor load
[112,114,117,471]
Karposi sarcoma
KS Y-1 cells implanted in nude mice
Tumor growth retardation
[127]
Leydig tumor
Leydig cell tumor implanted into rats
Tumor suppression
[119]
Lung
Implantation of Lewis lung carcinoma into mice
Reduction of the number of metastases (without suppression of primary tumor); tumor suppression; increased anti-tumor immunity
[109,123,382,472]
Melanoma
Human melanoma cells implanted into nude mice
Tumor suppression
[112]
Osteosarcoma
Human osteosarcoma cells implanted into nude mice
Tumor suppression
[120]
Prostate
Dunning MAT LyLu rat prostate model; LNCaP xenografts in nude mice; PAIII tumors in Lobund-Wistar rats
Reduction in lung metastasis; tumor suppression
[125,126,128,130,131]
Retinoblastoma
Retinoblastoma cell line implanted into nude mice; transgenic mice with retinoblastoma
Tumor suppression
[113,116]
Walker carcinoma
Walker carcinoma cells injected in rats
Tumor suppression
[122]
Skin
DMBA/TPA-induced skin tumors in mice
Inhibition of tumor formation
[110,111]
a
The dosage, duration of treatment, diet, and effects on serum/urinary calcium vary among the studies. NMU, nitrosomethylurea; DMBA, 7,12-dimethylbenz[a]anthracene; DMH, 1,2-dimethylhydrazine dihydrochloride; TPA, 12-O-tetradecanoylphorbol-13-acetate
T-cell lymphoma with a combination of 1,25(OH)2D3 and retinoids, contrasting results have been obtained. It has been suggested that the variability was due to differences in phenotype of the various lymphomas [155e159]. A study on early recurrent prostate cancer showed that daily treatment with 1,25(OH)2D3 slowed the rise in prostate-specific antigen (PSA), but treatment coincided with hypercalcemic affects [160]. Using a regimen of once weekly treatment with very highdose calcitriol was found to be safe but did not result in a significant reduction in PSA in prostate cancer cells [161]. Two studies were specifically designed to examine the route and schedule of administration and calcemic response in patients with advanced malignancies [162,163]. The complicated set of trials using very high-dose 1,25(OH)2D3 plus taxotere in advanced prostate cancer is discussed in Chapter 86 on prostate cancer and has recently been reviewed [164]. Further discussion on clinical trials can be found in the chapters on the specific malignancies that follow. Clinical trials using vitamin D analogs have been initiated over recent years. However, these were mostly limited clinical trials focusing on small groups of patients for whom regular treatment had failed. Only a relatively few studies have been published. The analog
calcipotriol (Daivonex/Dovonex/MC903) has been used for topical treatment of advanced breast cancer; however, several of the patients still developed hypercalcemia [165]. More recent studies have been carried out in advanced breast cancer [166] and pancreatic cancer [167], but the clinical results were limited. In a single case of Karposi sarcoma and topical application of calcipotriol good success in tumor regression was reported [127]. Also the impact of inhibition of CYP24 to enhance the anti-cancer activity of vitamin D has been studied and a potentiation of the vitamin D effect was found as had been shown in cell work previously [168]. Data on clinical studies with vitamin D and vitamin D analogs are reviewed by Vijayakumar [169,170] and by Krishnan et al. [164]. In Chapter 90 the current clinical status of 1,25(OH)2D3 and its analogs as therapeutic agents for cancer will be discussed in greater detail.
Angiogenesis and Metastasis For the tumor-suppressive activity of vitamin D3 compounds in vivo, besides growth inhibition and differentiation, two additional aspects contribute to
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83. VITAMIN D: CANCER AND DIFFERENTIATION
In vivo Effects of 1,25(OH)2D3 Analogs in Animal Models for Cancer
Analog
Model
Antitumor effect
Refs
1,25(OH)D2
Retinoblastoma
Tumor suppression
[148]
1,25(OH)D5
Breast
Tumor suppression
[149]
CB966
Breast
Tumor suppression
[134]
CB1093
Prostate
Tumor suppression No effect on angiogenesis
[130]
DD-003
Colon
Tumor suppression
[140]
EB1089
Adenocarcinoma
Tumor suppression
[129]
EB1089
Breast
Tumor suppression
[134,137,145,400]
EB1089
Colon
Tumor suppression
[144]
EB1089
Hepatocellular carcinoma
Inhibition of tumor incidence
[473]
EB1089
Leydig cell tumor
Tumor suppression
[119]
EB1089
Prostate
Tumor suppression Reduction lung metastases No effect on angiogenesis
[126,128,130,131,146,147]
KH1060
Prostate
Tumor suppression
[131]
LG190119
Prostate
Tumor suppression
[128]
OCT
Breast
Tumor suppression
[133,138]
OCT
Breast
Tumor suppression
[135]
OCT
Breast
Tumor suppression
[138]
OCT
Colon
Decreased tumor incidence
[141]
MC903
Breast
Tumor suppression
[136]
Ro 23-7553
Prostate
Tumor suppression
[142]
Ro 23-7553
Leukemia
Increased survival
[132]
Ro 24-5531
Breast
Decreased tumor incidence
[139]
Ro 24-5531
Colon
Decreased tumor incidence
[143]
Ro-25-6760
Prostate
Tumor suppression
[125]
Ro-26-9114
Colon
Decrease in polyp number and tumor load
[471]
Ro-26-9114
Prostate
Tumor suppression
[131]
MC903, 1,24-dihydroxy-22-ene-24-cyclopropyl-vitamin D3; CB966, 24a,26a,27a-tri-homo-1a,25-dihydroxyvitamin D3; CB1093, 20-epi-22(S)-ethoxy-23yne-24a, 26a,27atrihomo-1a,25-dihydroxyvitamin D3; DD-003,22(S)-24-homo-26,26,26,27,27,27-hexafluoro-1a,22,25-trihydroxyvitamin D3; EB1089, 22,24-diene-24a,26a,27a-trihomo1a,25-dihydroxyvitamin D3; OCT, 22-Oxacalcitriol; Ro 23-7553, 1,25dihydroxy-16-ene-23-yne-vitamin D3; Ro 24-5531, 1,25dihydroxy-16-ene-23-yne-26,27-hexafluorovitamin D3. Ro 26-9114, 1a,25-(OH)2-16-ene-19-nor-24-oxo-D3.
potential benefits including: (1) effects to inhibit angiogenesis, and (2) actions that inhibit invasion and metastasis. First, we will discuss vitamin D and angiogenesis. Angiogenesis is an essential requirement for the growth of solid tumors. Compounds that inhibit angiogenesis might therefore contribute to anti-tumor therapy. Antiangiogenic drugs may lead to inhibition of tumor progression, stabilization of tumor growth, tumor regression, and prevention of metastasis. Antiangiogenic effects may play a role in the tumor-suppressive
activity of vitamin D3 compounds. Two studies reported an antiangiogenic effect of 1,25(OH)2D3 and the analog 22-oxacalcitriol using different experimental model systems [135,171]. In addition, it was shown that 1,25(OH)2D3 inhibits angiogenesis induced by the human papilloma virus type 16 (HPV16)- or HPV18-containing HeLa cell lines, Skv-e2, and Skv-el2, when intradermally injected into immunosuppressed mice. Also, with the non-virus-transformed human cell lines T47-D (breast carcinoma) and A431 (vulva carcinoma), similar results
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were obtained [172]. In these studies the mice were treated for 5 days with 1,25(OH)2D3 prior to the injection of tumor cells. The effect of 1,25(OH)2D3 on angiogenesis may be due to inhibition of tumor cell proliferation, resulting in fewer angiogenic cells. However, inhibition of angiogenesis could also be observed when the tumor cells were treated in vitro with 1,25(OH)2D3 and, after cell washing, were injected into mice [172]. Under these conditions both control and 1,25(OH)2D3-treated mice were injected with similar numbers of cells. Therefore, these data indicate that 1,25(OH)2D3 inhibits the release of angiogenic factors (vascular endothelium growth factor, transforming growth factor-a, basic fibroblast growth factor, epidermal growth factor, etc.) or stimulates antiangiogenic factors. 1,25(OH)2D3 treatment caused a reduction in the angiogenic signaling molecule, angiopoietin-2 in squamous cell carcinoma and radiation-induced fibrosarcoma-1 cells [173]. In retinoblastomas in mice, 1,25(OH)2D3 has also been shown to reduce angiogenesis [174]. A study by Oades et al., however, showed that the 1,25(OH)2D3 analogs EB1089 and CB1093 inhibited tumor growth in two prostate animal models but did not inhibit angiogenesis in a rat aorta assay [130]. Whether this implicates that vitamin D affects angiogenesis in a tumor situation and not in a non-malignant condition is not clear. This may resemble the effects of endostatin which inhibits pathological but not normal vascularization [175,176]. In support of this possibility is the finding that 1,25(OH)2D3 and its analogs EB1089, Ro-25-6760, and ILX23-7553 potently inhibit growth of endothelial cells derived from tumors but less potent against normal aortic or yolk sac endothelial cells [173]. In SW480ADH colon cancer cells 1,25(OH)2D3 has a complex regulatory effect on the angiogenic phenotype: it increases the expression of VEGF and TSP-1, but not that of PDGF-B, through the activation of their respective promoters [177]. Finally, an interesting observation is deglycosylated vitamin-D-binding protein (DBPmaf) has also been reported to inhibit angiogenesis [178,179] and to inhibit growth of pancreatic tumor in nude mice [179]. Whether 1,25(OH)2D3 may interfere with DBP-maf in tumor growth inhibition and antiangiogenesis remains to be established. Interaction with another factor, interleukin-12, in the inhibition of angiogenesis has been reported [180]. The second mechanism of antitumor activity to be discussed, and one that is related to angiogenesis, is invasion and metastasis. Metastasis is the primary cause of the fatal outcome of cancer diseases. A study by Mork Hansen et al. indicated that 1,25(OH)2D3 may be effective in reducing the invasiveness of breast cancer cells [181]. They showed that 1,25(OH)2D3 inhibited the invasion and migration of a metastatic human breast cancer cell line (MDA-MB-231) using the Boyden chamber
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invasion assay. In support of this, it was shown that 1,25(OH)2D3, and the analogs KH1060, EB1089, and CB1093, all inhibited secretion of tissue-type and urokinase plasminogen activator and increased plasminogen activator inhibitor 1 (PAI-1) in the MDA-MB-231 metastatic breast cancer cell line [182]. Moreover, the vitamin D analog EB1089 also prevented skeletal metastasis in vivo and prolonged survival time in nude mice transplanted with human breast cancer cells [183]. Vitamin D also inhibited the invasive ability of human prostate cancer cell lines, LNCaP, PC-3, and DU 145. 1,25(OH)2D3 decreased MMP-9 and cathepsins, but not plaminogen activities, while it increased the activity of tissue inhibitors of metalloproteinase-1 (TIMP-1) and cathepsin inhibitors [184]. In an in vivo study it was shown that 1,25(OH)2D3 reduces the metastasis to the lung of subcutaneously implanted Lewis lung carcinoma cells [123]. In two animal models of prostate cancer 1,25(OH)2D3 and the analogs EB1089 and RO256760 inhibited lung metastases [125,126]. In these models the tumors were implanted subcutaneously and therefore, in contrast to the model of direct tumor cell injection in the left ventricle [185], no bone metastases occurred. Interestingly, it was shown that vitamin D deficiency promotes the growth of human breast cancer cells in the bones of nude mice [186]. A fact to be considered in relation to metastasis is that bone is the most frequent site of metastasis of advanced breast and prostate cancer. There are some indications from clinical studies that bone metastases develop preferentially in areas with high bone turnover [187,188]. In contrast, agents that inhibit bone resorption have been reported to reduce the incidence of skeletal metastasis [189]. Akech et al. showed findings that indicate that Runx2 is a key regulator of events associated with prostate and breast cancer metastatic bone disease [190]. Runx2 is intimately involved in vitamin D actions in osteoblast development [191]. As 1,25(OH)2D3 may stimulate bone turnover, treatment of cancer with 1,25(OH)2D3 might theoretically increase the risk of skeletal metastases. This aspect of 1,25(OH)2D3 therapy certainly needs further study. Considering the use of vitamin D3 analogs with reduced calcemic activity or treatment with vitamin D3 in combination with other compounds to reduce bone turnover (see “Combination therapy,” below) may be helpful. The data obtained so far on angiogenesis and metastasis show that these two processes contribute to the multiple mechanisms by which vitamin D3 exerts anticancer activity.
Parathyroid Hormone-related Peptide 1,25(OH)2D3 and parathyroid hormone (PTH) mutually regulate synthesis and secretion of one another
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(see Chapter 27). Production and secretion of PTH are inhibited by 1,25(OH)2D3 via a transcriptional effect, and a vitamin D responsive element (VDRE) in the promoter of the PTH gene has been identified [192,193]. Parathyroid hormone-related peptide (PTHrP) was initially isolated from several carcinomas and is responsible for the syndrome of humoral hypercalcemia of malignancy [194)] (see Chapter 41). Although originally identified in carcinomas, PTHrP has also been identified in normal cells. As will be discussed now, vitamin D effects to inhibit PTH and PTHrP may have a role in its anticancer actions and in reducing metastases to bone [195,196]. In normal human mammary epithelial cells, 1,25(OH)2D3 did not affect basal but inhibited growthfactor-stimulated PTHrP expression via an effect on transcription [197]. In normal keratinocytes 1,25(OH)2D3 had no effect on PTHrP secretion in basal culture conditions [198] but did inhibit growth-factor-stimulated PTHrP production as well [199]. Likewise, 1,25(OH)2D3 as well as the analogs 22-oxacalcitriol and MC903 inhibited PTHrP secretion in immortalized human keratinocytes (HPK1A), but this inhibition was less in the more malignant ras-transfected clone HPK1A-ras [200,201]. 1,25(OH)2D3 and the analogs EB1089 and 22-oxacalcitriol inhibit the PTHrP gene transcription in and release from the squamous cancer cell line NCI H520 [202]. In addition, in the human T-cell lymphotrophic virus type I (HTLV-I)-transfected T-cell line MT-2, 1,25(OH)2D3 and 22-oxacalcitriol inhibited PTHrP gene expression and PTHrP secretion [203] and in rat H-500 Leydig tumor cells [204], and 1,25(OH)2D3 inhibited PTHrP secretion by PC-3 prostate cancer cells. However, another study demonstrated a prostate-cancer-specific or cell-specific effect. Vitamin D and the analog EB1089 inhibit the PTHrP expression via a negative VDRE in LNCaP but not PC3 prostate cancer cells [205,206]. It was suggested that this might play a role in the growth inhibition by vitamin D as PTHrP stimulates prostate cancer growth, tumor invasion and metastasis [207e209]. In vivo observations comparable to these in vitro observations have also been made. When H-500 Leydig tumor cells were implanted in Fisher rats, treatment with 1,25(OH)2D3 and the analog EB1089 resulted in reduced levels of tumor PTHrP mRNA and PTHrP serum levels [119]. EB1089 also reduced serum levels of PTHrP in nude mice implanted with squamous cancer cells [210]. In Fisher rats implanted with the Walker carcinoma, 1,25(OH)2D3 caused a decrease in serum PTHrP but the ratio of PTHrP levels and tumor weight was similar in rats receiving vehicle or 1,25(OH)2D3. The data point to an indirect effect on PTHrP via growth inhibition. However, the PTHrP mRNA levels appeared to be decreased by 1,25(OH)2D3 [122]. In nude mice bearing
the FA-6 cell line of a pancreas carcinoma lymph node metastasis, 22-oxacalcitriol inhibits PTHrP gene expression, which is related to inhibition of tumor-induced hypercalcemia [211]. Together, the overall picture that emerges from these studies is that an important additional anticancer effect of vitamin D3 and analogs could be the inhibition of the humoral hypercalcemia of malignancy. In contrast to these inhibitory effects in human tumor cells and tumor models, a stimulatory effect of 1,25(OH)2D3 and EB1089 on PTHrP gene transcription and PTHrP production by a canine oral squamous carcinoma cell line (Sec 2/88) has been observed [212,213]. Also in vivo with the canine adenocarcinoma CAC-8 in nude mice stimulation of PTHrP by 1,25(OH)2D3 and EB1089 was observed [213]. These data indicate that the effect of vitamin D and analogs on canine tumors differs from that on human tumors.
VITAMIN D EFFECTS ON TUMOR CELLS Cell Cycle It has now been well established that vitamin D inhibits growth of cells by interfering with the cell cycle (see Chapter 84). Proliferating cells progress through the cell cycle, which comprises the G0/G1 phase (most differentiated, non-dividing cells are in the G1 phase), the S phase in which new DNA is synthesized, and the G2 phase, which is followed by mitosis (M phase) whereon the cells reenter the G0/G1 phase. In most of the cells studied so far treatment with 1,25(OH)2D3 and its analogs results in a blockade at a specific check-point, i.e. the restriction point (R), in the G1 phase limiting the transition of G1 to S and reducing the number of cells in S phase. Some studies also have examined the effect on the G2 phase, but these results are somewhat more diverse. In general it can be concluded that blocking the transition from the G0/G1 phase to the S phase plays an important role in the growth-inhibitory effect of 1,25(OH)2D3. Numerous genes and proteins have been described that participate in the regulation of the cell cycle. It is beyond the scope of this chapter to discuss in detail the regulation of all of the genes/proteins by vitamin D. In Figure 83.1, an overview is given of the interacting genes/proteins that are involved in intracellular signaling and regulating the cell cycle. These genes and proteins are part of the cascade of events on which vitamin D exerts its effects. The components shown to be regulated by vitamin D are indicated. Figure 83.1 is a compilation of data presented thus far and it is important to realize that probably not all of the genes/proteins are affected by vitamin D in all tumor cells. However, in
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this way, one can get an overview and appreciate the broad range of effects mediated by vitamin D on intracellular signaling pathways involved in regulation of (tumor) cell growth. Regulation will be discussed in more detail in various other chapters especially Chapter 84. Besides effects on cell cycle regulation vitamin D has recently been implicated to be involved in control of genomic stability [214]. 1,25(OH)2D3 has been reported to inhibit hepatic chromosomal aberrations and DNA strand breaks [215]. This is supported by the finding that 1,25(OH)2D3 and EB1089 stimulated the expression of GADD45 which stimulates DNA repair [216] and might be coupled to release of p53 from Mdm2 (see Fig. 83.1). Notably, a recent study has shown that supplemental vitamin D3 and calcium, separately but not together, decreased the level of the DNA damage marker 8-hydroxy-2’-deoxyguanosine in normal colorectal mucosa in a randomized clinical trial [217]. (Proto)-oncogenes and Tumor Suppressor Genes Oncogenes and tumor suppressor genes generally are involved in control of the cell cycle and apoptosis. One of the most widely studied oncogenes in relation to vitamin D is c-myc. c-Myc suppresses expression of cell cycle/growth arrest genes gas1, p15, p21, p27, and gadd34, -45, and -153 [218] and has been postulated to play an early role in the following cascade of events in G1: cyclins activate cyclin-dependent kinases (CDKs) which in turn can phosphorylate the retinoblastoma tumor suppressor gene product (p110RB), resulting in transition from G1 to S phase (see Fig. 83.1). In HL-60, breast cancer, and several other cell types 1,25(OH)2D3 has been reported to decrease c-myc oncogene expression [219e224]. Analysis of HL-60 sublines showed a relation between reduction of c-myc expression and inhibition of proliferation [225]. Similar observations were made for neuroblastoma cells treated with 1,25(OH)2D3, EB1089, and KH1060 [226]. The mechanism of c-myc inhibition appears to be both direct, by inducing the binding of proteins to an intron element and the involvement of HOXB4 [227,228], and at least in colon cancer cells also indirect via the inhibition of the transcriptional activity of b-catenin and T cell factor (TCF) complexes [229]. In earlier studies, we did not observe a 1,25(OH)2D3-induced change in c-myc expression in MCF-7 and ZR-75.1 breast cancer cells while they were both growth inhibited [230], and a similar observation has been made for the colon-adenocarcinoma CaCo-2 cell line [231]. Non-transformed embryonic fibroblasts are growth inhibited by 1,25(OH)2D3, whereas c-myc is not changed or is even increased [232,233]. In the MG63 osteosarcoma cell line, 1,25(OH)2D3 has been shown to enhance c-myc expression [234], whereas we observed growth inhibition by 1,25(OH)2D3 [235]. Likewise,
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1,25(OH)2D3 inhibits proliferation and increases c-myc expression in fibroblasts from psoriatic patients [236]. In a recent study inhibition of c-myc was implicated as playing a major role in the ability of 1,25(OH)2D3 to inhibit prostate cancer proliferation [237]. Collectively, these data show that regulation of c-myc expression may be part of growth inhibition by vitamin D but that this is not generally applicable to all cells. 1,25(OH)2D3 has also been reported to regulate expression of other oncogenes, like c-myb, c-fos, c-fms, c-fra1, c-jun, junD , c-Ki-ras, N-ras, c-src [224,238e243]; however, these data are rather limited. Nevertheless, it is clear that 1,25(OH)2D3 has effects on the expression of various proto-oncogenes. The data so far are not conclusive with respect to which genes are crucial in the growth-inhibitory action of 1,25(OH)2D3. This can be attributed to the fact that these (proto)oncogenes encode for transcription factors, growth factor receptors or components or intracellular signaling cascades. The effects of these genes may differ between cells dependent on the presence or absence of additional celltype-specific conditions. Therefore, their postulated role is often complex. For example, increased c-myc expression can be related to induction of apoptosis but also to stimulation of cell cycle progression. In contrast to the oncogenes, the effect of 1,25(OH)2D3 on tumor suppressor genes like the retinoblastoma gene is much clearer. This may be related to the fact that, in contrast to oncogenes, retinoblastoma and p53 take well-defined positions in the control of cell cycle and DNA repair (see Fig. 83.1). The p110RB retinoblastoma gene product can either be phosphorylated or dephosphorylated. In the phosphorylated form it can activate several transcription factors and cause transition to S phase and DNA synthesis [244]. In human chronic myelogenous leukemia cells [245], breast cancer cells [246], and HL-60 cells [247,248], 1,25(OH)2D3 caused a dephosphorylation of p110RB, which is related to growth inhibition and cell cycle arrest in G0/G1 and in one study also in G2 [248]. In the leukemic cells 1,25(OH)2D3 also caused a reduction in the cellular level of p110RB [245,247]. In non-transformed keratinocytes 1,25(OH)2D3 induced dephosphorylation of p110RB as well [249]. The other major tumor suppressor gene is p53 (TP53 in humans). For leukemic U937 cells it was reported that presence of p53 is important for 1,25(OH)2D3-induced differentiation [250]. In rat glioma cells 1,25(OH)2D3 induces expression of p53 [251]. However, 1,25(OH)2D3 can inhibit cell growth and induce differentiation in cancer cells with defective p53 [252] and also p53-independent induction of apoptosis by EB1089 has been demonstrated [253]. These latter observations might be explained by the fact that vitamin D also interferes at levels in the cascade of cell cycle control downstream of p53 (see Fig. 83.1).
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FIGURE 83.1 Schematic representation summarizing the intracellular pathways and signaling pathways involved in regulation of the cell cycle shown to be regulated by 1,25(OH)2D3 and 1,25(OH)2D3 analogs in regulating cell proliferation. Targets shown to be affected by 1,25(OH)2D3 and/or its analogs are indicated in the bold boxes and ovals. Bold arrows and fine dotted lines indicate stimulation and inhibition, respectively. Coarse dotted lines indicate processing to the proteasome. p indicates phosphorylation. The effects on these cellular targets are not demonstrated in all types of cancer cells but this diagram is aimed to give an overview of demonstrated targets and potential targets.
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Recently, novel interesting data were added to the story of p53 and 1,25(OH)2D3 [254]. It was shown that a mutant p53, often present in tumors, physically and functionally interacts with VDR. Mutant p53 is recruited to vitamin D target genes and can stimulate gene expression and relieve suppression of other genes. Mutant p53 increases nuclear accumulation of VDR and transforms vitamin D into an antiapoptotic agent [254]. An interesting unique relationship between tumor suppressor genes and vitamin D has recently been shown for the Wilms’ tumor suppressor gene WT1. This zinc-fingercontaining transcription factor induces transcription of the VDR gene [255]. Several interesting additional genes, interactions, and vitamin D targets in cancer treatment should be mentioned. It has been demonstrated that 1,25(OH)2D3 can trigger NF-kB activity through PI3K/Akt pathways [256,257] and also treatment of NB4 leukemic cells with vitamin D causes a rapid phosphorylation of IkBa [258]. Contrary to these observations, vitamin D has been shown to inhibit NF-kB activity by increasing IkBa expression in different cell lines [259e261]. Sun et al. [262], using mouse embryonic fibroblasts derived from VDR / mice, demonstrated that VDR plays an inhibitory role in NF-kB activation by regulating IkBa levels and VDRep65 interaction. This role for VDR was supported by a recent study that also demonstrated that 1,25(OH)2D3 inhibits transcriptional activity of NF-kB in breast cancer cells via histone deacetylase (HDAC3 and SMRT) mediated p65 transrepression [263]. Kovalenko et al. showed direct transcriptional regulation by 1,25(OH)2D3 of NF-kB in RWPE1 immortalized but non-tumerigenic prostate cells cells [264]. 1,25(OH)2D3 indirectly inhibits NF-kB by directly stimulating expression of IGFBP-3, an inhibitor of NF-kB [265]. Interestingly, in relation to NF-kB regulation, as early as 1994, Chen and DeLuca isolated and characterized a vitamin-D-induced gene in HL-60 cells [266]. The encoded protein, named vitamin-D-upregulated protein-1 (VDUP1), is a thioredoxin-binding protein-2 [267]. Thioredoxin has several roles in processes such as proliferation or apoptosis. It also promotes DNA binding of transcription factors such as NF-kB, AP-1, p53, and PEBP2. In addition, overexpression of thioredoxin suppresses the degradation of IkB and the
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transactivation of NF-kB, whereas overexpression of nuclear-targeted thioredoxin exhibits enhancement of NF-kB-dependent transactivation [268]. However, it is in only more recent studies that a coupling between VDUP1 and cancer has been made. The expression of VDUP1 was found to correlate with malignant status of colorectal and gastric cancers [269]. 5-Fluorouracil, which is widely used for treatment of colon cancer, induces VDUP1 expression in the SW620 colon cancer cell line [270]. In smooth muscle cells and cardiomyocytes VDUP1 inhibits proliferation and is involved in induction of apoptosis [271,272]. A relation with vitamin D effects on cancer is made by two recent studies showing induction of VDUP1 by 1,25(OH)2D3 in tumor cells and that VDUP1 induces cell cycle arrest [273,274]. Moreover, interaction with histone deacetylase (HDAC; see Fig. 83.1), and promyelocytic leukemia zinc finger (PLZF) was demonstrated. Interestingly and further complicating the story, PLZF inhibits 1,25(OH)2D3-induced differentiation of U937 leukemic cells by binding to the VDR and inhibiting gene transcription [275,276]. Also, a new related gene, DRH1, was cloned and its expression was found to be strongly reduced in hepatocellular carcinoma tissue compared to normal liver [277]. DRH1 is 41% homologous with VDUP1. Whether this points to a new family of cancer genes remains to be established but it certainly opens new avenues for intervening in cancer cell growth. Several alternate therapeutic targets for vitamin D anticancer activity can be mentioned here which are discussed in more detail in the following various chapters on specific cancers. One is vitamin D regulation of enzymes involved in estrogen and androgen synthesis and metabolism since these pathways drive the growth of breast and prostate cancer, respectively [278e282]. Next, telomerase activity provides a mechanism for unlimited cell division. In HL-60 cells 1,25(OH)2D3 inhibits telomerase activity [283]. Additionally, whether the homeobox genes will prove to be a major target for vitamin D action in cancer remains to be elucidated but in a differential expression screen in the human U937 leukemic cells the HoxA10 gene was shown to be regulated by 1,25(OH)2D3 [284]. A final area is the anti-inflammatory activity of vitamin D, especially its ability to inhibit COX-2 and the prostaglandin pathway
=
NOTE: Dependent on the site of phosphorylation proteins can either be destabilized or degraded or be stabilized and activated. For example: phosphorylation of p21 at T145 by AKT leads to degradation while phosphorylation of S146 by AKT leads to increased stability. Abbreviations used in Fig. 83.1: AKT (PKB), Protein kinase B; Bad, BCL2-antagonist of cell death; Bcl2, B-cell leukemia/lymphoma 2; Cdk, Cyclin-dependent kinase; CKS-1, Cyclin kinase subunit 1; DG, Diacylglycerol; E2F, Transcription factor; ERK, Extracellular-signal regulated kinase; FHKR (AFX/FOX), Forkhead family of transcription factors; GSK-3, Glycogen synthase kinase-3; HDAC, Histone deacetylase; IKK, I-kB kinase; IP3, Inositol 1,4,5-trisphosphate; Mdm2, Mouse double minute 2; MEK, Raf-1-MAPK/ERK kinase; PDK1, Phosphatidylinositoldependent kinase 1; PI3-K, Phosphatidylinositol 3 kinase; PIP2, Phosphatidylinositol (4,5)-phosphate; PIP3, Phosphatidylinositol (3,4,5)-phosphate; PKC, Protein kinase C; PLC, Phospholipase C; PLD, Phospholipase D; PP!, Protein phosphatase 1-like protein; pRB, Retinoblastoma protein; PTEN, Phosphatase and tensin homolog; SHIP 1 and 2, Src homology 2 (SH2) containing phosphatases 1 and 2; SKP2, Ubiquitin ligase; VDR, Vitamin D receptor.
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[285]. Inflammation and carcinogenesis are intimately related and vitamin D inhibits many pro-inflammatory pathways perhaps contributing to its chemoprevention as well as its therapeutic activity [265]. It is to be expected that as a result of the increasing application of large-scale microarray gene expression analyses a vast number of new cell cycle and vitaminD-regulated genes will be identified and these additional findings will add to the unraveling and further understanding of the mechanism of vitamin D control of cancer cell proliferation [286e290].
Apoptosis The blockade in the cell cycle that prevents transition into S phase may cause cells to either go into apoptosis (programmed cell death) or enter a specific differentiation pathway. What exactly determines the decision between apoptosis or differentiation remains to be elucidated. It is suggested that early G1 phase may be the point at which switching between cell cycle progression and induction of apoptosis occurs [291,292]. Induction of apoptosis by 1,25(OH)2D3 is an orderly and characteristic sequence of biochemical, molecular, and structural changes resulting in the death of the cell [293]. Apoptosis is a mechanism by which 1,25(OH)2D3 inhibits tumor cell growth and may be the explanation for the tumor suppression and reduction in tumor volume found in various in vivo animal studies (see “Growth and development,” above). 1,25(OH)2D3 has been shown to regulate expression of apoptosis genes and to induce apoptosis of cancer cells of various origins. For example, 1,25(OH)2D3 and the analog Ro 25-6760 induce a cell cycle blockade in HT-29 human colon cancer cells causing growth inhibition and induction of apoptosis [294]. The bcl-2 oncogene decreases the rate of programmed cell death [295,296]. However, protection of HL-60 cells against apoptosis occurred despite downregulation of bcl-2 gene expression [297]. In several breast cancer cell lines (MCF-7, BT-474, MDA-MB-231) 1,25(OH)2D3 and the analogs KH1060 and EB1089 decreased bcl-2 expression [252,298] and also CB1093 reduced bcl-2 expression in MCF-7 cells related to induction of apoptosis [299]. However, only in MCF-7 cells was this change in bcl-2 expression accompanied by apoptosis. The apoptosis induced by 1,25(OH)2D3 and the analogs EB1089 and CB1093 in MCF-7 and T47D breast cancer cells does not involve caspases or p53 activation [300]. 1a,25(OH)2D3 induced apoptosis in MCF-7 cells via disruption of mitochondrial function, which is associated with Bax translocation to mitochondria, cytochrome c release, and production of reactive oxygen species [301]. It was shown that for MCF-7 cells calpain,
a calcium-dependent cysteine protease, may take over the role of the major execution protease in apoptosislike death induced by vitamin D and EB1089 [302]. In B-cell chronic lymphocytic leukemia cells in vitro (B-CLL), the vitamin D3 analog EB1089 also induces apoptosis via a p53-independent mechanism involving p38 MAP kinase activation and suppression of ERK activity [253]. In prostate cancer, the effects of vitamin D on apoptosis of tumor cells is caspase-dependent and the human VDR is a target of caspase-3, suggesting that activation of caspase-3 may limit VDR activity [303]. Effects on other apoptosis genes/proteins such as BAX and BAK have been reported [304] and microarray gene expression analyses and differential screening will also definitively reveal additional vitamin D targets involved in regulating apoptosis [290,305]. Remarkably, treatment of patients with vitamin D3 and calcium increased BAK immunostaining in the interior of colonic polyps [306] without affecting BCL2 expression in the same polyps [306] or in normal colon mucosa [307]. A central role for apoptosis in the action of 1,25(OH)2D3 is unclear because growth inhibition of several other breast cancer cells besides MCF-7 cells appeared to be independent of apoptosis [252]. Also, MCF-7 cells that showed growth inhibition by 1,25(OH)2D3 could, after removal of the hormone, again be stimulated to grow, implying transient growth inhibition and not cell death [230]. Stable transfection of leukemic U937 cells with the wild-type p53 tumor suppressor gene resulted in a reduced growth rate and produced cells that can undergo either apoptosis or maturation. In these cells 1,25(OH)2D3 protects against p53-induced apoptosis and enhances p53-induced maturation [250]. In two independent studies with HL-60 cells, 1,25(OH)2D3 was found either to protect against or to have no effects on apoptosis [297,308]. Vitamin D protection against apoptosis was also detected in human U937 leukemic cells treated with tumor necrosis factor a [309]. Absence of a vitamin D effect on apoptosis might be explained by the expression of the anti-apoptotic protein BAG-1 p50 isoform. This protein has been shown to bind to the VDR and block vitamin-D-induced transcription [310]. Presence of additional interacting factors might also be important for the eventual effect on apoptosis as in the study with HL-60 cells that, in the presence but not the absence of 9-cis-retinoic acid, 1,25(OH)2D3 did induce apoptosis [308]. Role of vitamin D interaction with other factors will be discussed in more detail in “Combination therapy,” below. In summary, the data obtained so far show that 1,25(OH)2D3-induced growth inhibition can be related to apoptosis in some cases but that growth inhibition
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also can be observed independent of apoptosis. Possibly in these latter cases induction of differentiation is more prominent. The factor(s) that decide whether cells undergo apoptosis or differentiation is (are) unclear but is probably dependent on cell cycle stage, presence of other factors, and levels of expression of various oncogenes and tumor suppressor genes. These variables contribute to what appears to be cell-specific actions of vitamin D to induce apoptosis. An interesting phenomenon to be studied concerning vitamin D and apoptosis is calbindin 28K. Calbindin 28K is a well-known vitaminD-induced protein which has recently been shown to inhibit apoptosis [311]. It is tempting to speculate that calbindin 28K plays a role in the decision whether vitamin D induces cells to differentiate or to go into apoptosis or that it is involved when 1,25(OH)2D3 protects against apoptosis. Additionally, EB1089 induces lysosomal changes and autophagic cell death in human MCF-7 breast cancer cells [312,313].
Differentiation In addition to proliferation and apoptosis, the third major cellular process in the array of vitamin D anticancer actions is differentiation. As described above for the classic actions of 1,25(OH)2D3 related to calcium homeostasis, effects on cell differentiation and proliferation are involved. There is a considerable body of evidence that the principal human cancer cells can be suitable candidates for chemoprevention or differentiation therapy with vitamin D. However, different mechanisms of 1,25(OH)2D3-induced differentiation are cell-type and cell-context specific [314,315]. The coupling between proliferation and differentiation has been most widely studied for cells of the hematopoietic system and keratinocytes. In general, 1,25(OH)2D3 inhibits proliferation and induces differentiation along the monocyteemacrophage lineage. Rapidly proliferating and poorly differentiated keratinocytes can be induced to differentiate by 1,25(OH)2D3. A further relationship between the vitamin D3 system and differentiation is demonstrated by the fact that in poorly differentiated keratinocytes 1,25(OH)2D3 production and vitamin D receptor levels are high, whereas after induction of differentiation these levels decrease [316]. In melanoma cells, in addition to growth inhibition [101], 1,25(OH)2D3 stimulates melanin production [317]. Effects on differentiation have also been reported for other cell types. Inhibition of prostate cancer cell proliferation is paralleled by an increased production of PSA per cell, a sign of differentiation [318e321]. In the BT-20 breast cancer cells 1,25(OH)2D3 induced morphological changes indicative for differentiation [322]. In several breast cancer cell lines the stimulation of differentiation has been established by determining
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lipid production by the cells [252]. In this study, Elstner et al. demonstrated an uncoupling between effects on proliferation and differentiation. In two breast cancer cell lines 1,25(OH)2D3 and various analogs induced differentiation even though the cells were resistant to cell cycle and anti-proliferative effects. This together with data obtained with human myelogenous leukemia cells [245] suggest a dissociation between the cellular vitamin D3 pathways involved in regulation of differentiation and proliferation (see also “Resistance and vitamin D metabolism,” below). For an HL-60 subclone a similar observation was made [225], and in another HL-60 subclone the induction of differentiation was found to precede the G0/G1 cell cycle blockade. In contrast to the above-mentioned observations on stimulation of differentiation, 1,25(OH)2D3 inhibits erythroid differentiation of the erythroleukemia cell line K562 [221] and 1,25(OH)2D3 inhibits Activin A-induced differentiation of murine erythroleukemic F5-5 cells [323]. Paracalcitol, a vitamin D2 analog, converted committed myeloid hematopoietic stem cells from wild-type but not from VDR knockout mice to differentiate into macrophages [324]. In an early paper Shabahang et al. found that the level of vitamin D receptor correlated with the degree of differentiation in human colon cancer cell lines and suggested it might serve as a useful biological marker in predicting clinical outcome in patients [1]. Differentiation of rapidly dividing HT-29 colon cancer cells to differentiated slowly proliferating cells was associated with decreased VDR abundance, loss of VDR homologous upregulation, and the development of hormone unresponsiveness to 1,25(OH)2D3 [297]. 1,25(OH)2D3 induces an adhesive phenotype typical of the differentiated epithelial cells that is mostly based on the upregulation of E-cadherin and other plasma membrane adhesion proteins of adherens junctions (acatenin) and tight junctions (occludin, claudins, ZO-1) [229,325]. Also, 1,25(OH)2D3 regulates the phenotype of human breast cancer cells. Thus, it increases the expression of E-cadherin, claudin-7 and occludin and of proteins such as paxillin, focal adhesion kinase and av and b5 integrins that are involved in adhesion to the substratum [326]. Moreover, 1,25(OH)2D3 represses several markers of the basal/myoepithelial phenotype (P-cadherin, smooth muscle a-actin and a6 and b4-integrins), the pro-invasive and pro-angiogenic protein tenascin-C protein, and the mesenchymal marker N-cadherin that are associated with aggressiveness and poor prognosis in breast cancer [327,328]. Another pro-differentiation action of vitamin D, which may be beneficial in breast cancer, is the differentiation of preadipocytes that express high levels of aromatase, to differentiated adipocytes that express much lower levels of aromatase [282]. Although precise relationships among growth inhibition, cell cycle effects, and apoptosis are not
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entirely clear, it can be concluded that an important effect of vitamin D3 on both normal and malignant cells is induction of differentiation.
Growth Factors and Growth Factor Receptors Besides regulation of cell-cycle-related oncogenes and tumor suppressor genes, interaction with tumor- or stroma-derived growth factors is important for growth inhibition. Stimulation of breast cancer cell proliferation by co-culture with fibroblasts is inhibited by 1,25(OH)2D3 [329]. A good candidate to interact with the 1,25(OH)2D3 action is transforming growth factor-b (TGFb). TGFb is involved in cell cycle control and apoptosis [330,331]. TGFb can interfere with the cascade of events in the GI phase described above and inhibit the ability of cells to enter S phase when it is present during the GI phase. TGFb has been shown to suppress c-myc, cyclin A, cyclin E, and cdk2 and cdk4 expression [331]. In line with this, TGFb has been reported to inhibit phosphorylation of p110RB [332]. Vitamin D3 compounds induce dephosphorylation of the retinoblastoma gene product, and vitamin D3 growth inhibition of MCF-7 breast cancer cells is inhibited by a TGFb neutralizing antibody [333]. 1,25(OH)2D3 and several analogs stimulated the expression of TGFb mRNA and secretion of active and latent TGFb1 by the breast cancer cell line BT-20 [179]. 1,25(OH)2D3 enhanced TGb1 gene expression in human keratinocytes [334] and the secretion of TGFb in murine keratinocytes [335]. In both studies antibodies against TGFb inhibited the growth inhibitory effect of vitamin D3. Further evidence for a vitamin D3eTGFb interaction is that bone matrix of vitamin-D-deficient rats contains substantially less TGFb than controls [336]. It has been shown for the interaction between TGFb signaling pathways and vitamin D that the cross-talk may be mediated by Smad3. Smad3, one of the SMAD proteins downstream in the TGFb signaling pathway, was found in mammalian cells to act as a coactivator specific for ligand-induced transactivation of VDR by forming a complex with a member of the steroid receptor coactivator-1 protein family in the nucleus [337]. However, Smad3 is not of itself sufficient to coactivate VDR in TGFb/vitamin D3-resistant MCF7L cells and other factors are required. It was found that the PI 3-kinase pathway inhibitor LY29004 inhibited the synergy of TGFb and EB1089 on VDR-dependent transactivation activity. This indicates that the crosstalk between TGFb and vitamin D signaling is also PI 3kinase pathway dependent [338]. Therefore, on the basis of these consistent findings, TGFb is a likely candidate to play a role in the 1,25(OH)2D3-induced growth inhibition [338]. Interactions with the insulin-like growth factor [254] system have also been described. IGFs are potent growth
stimulators of various cells, and their effect is regulated via a series of IGF-binding proteins (IGFBPs). The IGFBPs, especially IGFBP-3, have potent anti-proliferative and pro-apoptotic actions [339]. These effects include both IGF-dependent actions, by sequestering the potent growth factor, and IGF-independent actions, having direct actions via its own receptor [340,341]. Among the many ways vitamin D inhibits prostate cancer growth, stimulation of IGFBP-3 may be a major contributor [342]. 1,25(OH)2D3 and the analog EB1089 inhibit the IGF-Istimulated growth of MCF-7 breast cancer cells [343]. In prostate cancer cell lines, 1,25(OH)2D3 induced expression of IGFBP-6 but not IGFBP-4 [344]. In human osteosarcoma cell lines, 1,25(OH)2D3 and the analog 1a-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol potently stimulated the expression and secretion of IGFBP-3 [345e347]. In one study an association has been made between increased IGFBP-3 levels and 1,25(OH)2D3 growth inhibition [345]. Recent observations that antisense oligonucleotides to IGFBP-3 prevented growth inhibition of prostate cancer cells by 1,25(OH)2D3 [289] provided further evidence for an interplay between 1,25(OH)2D3 and IGFBP-3. Interestingly, in the human osteosarcoma cell line MG-63, 1,25(OH)2D3 and TGFb synergistically increased IGFBP-3 secretion [347]. IGF-II is also a growth and survival factor for colorectal cancer cells and 1,25(OH)2D3 and several analogs interfere with IGF-II signaling. They upregulate IGFBP-6, which inhibits IGF-II signaling, and type II IGF receptor (IGF-R-II) that also blocks this pathway and accelerates IGF-II degradation [348,349]. An example of growth factor receptor regulation by 1,25(OH)2D3 concerns the epidermal growth factor receptor (EGFR). This receptor is downregulated in T47-D breast cancer cells and upregulated in BT-20 breast cancer cells. Nevertheless, 1,25(OH)2D3 inhibits the growth of both cell lines [350,351]. These data provide evidence that interactions with growth factors are part of the 1,25(OH)2D3 action on tumor cells. In primary colon adenocarcinoma cells as well as in the colon cancer Caco-2 cell line 1,25(OH)2D3 inhibits EGF mitogenic signaling and a mutual modulation of receptor expression between 1,25(OH)2D3 and EGF has been proposed [352,353]. In A431 epidermoid cells 1,25(OH)2D3 alters EGFR membrane trafficking and inhibits EGFR signaling [354]. Recently it was found that TCF-4, a transcriptional regulator and beta-catenin binding partner is an indirect target of the VDR pathway. TCF-4 functions as a transcriptional repressor that restricts breast and colorectal cancer cell growth. 1,25(OH)2D3 increases TCF-4 at the RNA and protein levels in several human colorectal cancer cell lines, the effect of which is completely dependent on the VDR. This 1,25(OH)2D3/VDR-mediated
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increase in TCF-4 may have a protective role in colon cancer as well as other diseases [355]. As described above, it is clear that 1,25(OH)2D3 has effects on the expression of various oncogenes and tumor suppressor genes and that multiple interactions with various growth factors exist. However, the data on these aspects separately as well as in combination are still too limited to define the total mechanism of action for the 1,25(OH)2D3 anticancer effects. However, with respect to growth inhibition, at this time two models of action can be postulated. In the first one 1,25(OH)2D3 directly interferes with a crucial gene(s) involved in the control of the cell cycle. In this case, in view of the general pattern of the genes involved in cell cycle control, this mechanism of action will be similar in all types of cancer cells. However, the effect on cell cycle genes will be dependent on the presence or absence of additional growth factors. This will eventually determine, depending on which growth factors are present, the differences in 1,25(OH)2D3 action between cancer types of different origin but also within cancer types of similar origin. In the second model 1,25(OH)2D3 may regulate cell cycle indirectly via changing the production of growth factors, growth factor signaling, growth-factor-binding protein levels, or receptor regulation. It is conceivable that a combination of both models forms the basis of 1,25(OH)2D3 regulation of tumor cell growth.
COMBINATION THERAPY The data obtained with 1,25(OH)2D3 and its analogs on growth inhibition and stimulation of differentiation offer promise for their use as an endocrine anticancer treatment. Single-agent treatment with low calcemic 1,25(OH)2D3 analogs could be useful; however, combination therapy with other tumor-effective drugs may provide an even more beneficial effect. Up to now several in vitro and in vivo studies have focused on possible future combination therapies with 1,25(OH)2D3 and 1,25(OH)2D3 analogs. For breast cancer cells, the combination of one of the most widely used endocrine therapies, the antiestrogen tamoxifen, with 1,25(OH)2D3 or 1,25(OH)2D3 analogs resulted in a greater growth inhibition of MCF-7 and ZR-75-1 cells than treatment with either compound alone [138,356]. In combination with tamoxifen, the cells were more sensitive to the anti-proliferative action of 1,25(OH)2D3 and the analogs; that is, the EC50 values of the vitamin D3 compounds in the presence of tamoxifen were lower than those in the absence of tamoxifen. Studies with MCF-7 cells suggested a synergistic effect of 1,25(OH)2D3 and tamoxifen on apoptosis [357]. In addition, in in vivo breast cancer models, a synergistic
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effect of the tamoxifenel,25(OH)2D3 analogs combination was observed [138,139]. Another interesting interaction relevant to breast cancer is that vitamin D inhibits aromatase thus reducing the estrogenic stimulus for proliferation [282]. Combination of 1,25(OH)2D3 and aromatase inhibitors also showed synergistic activity in breast cancer cells. 1,25D also downregulates the estrogen receptor, again reducing the ability of estrogens to stimulate breast cancer growth [358]. Additional data on the interaction between the estrogen/antiestrogen system and vitamin D comes from studies showing the presence of an estrogenresponsive element in the VDR promoter and regulation of VDR by estradiol in breast cancer cells [359]. It is intriguing that the stimulator of breast cancer cell growth induces the expression of the receptor for a growth inhibitor. VDR upregulation in breast cancer cells and increased transcriptional activity was mimicked by the phytoestrogens resveratrol and genistein and blocked by tamoxifen [360]. Estradiol induces metastasis-associated protein (MTA)-3, a component of the Mi-2/NuRD transcriptional co-repressor complex that inhibits Snail1, which is in turn a repressor of VDR gene expression [361,362]. In this way, estradiol may increase VDR levels in breast cells. In colon cancer also VDR upregulation by estradiol has been reported; however, in colon it was hypothesized to contribute to the protective effect of estradiol on chemically induced colon carcinogenesis [363]. These important and complex interactions between the vitamin D and estrogen endocrine systems in the regulation of cancer [281] are promising and warrant further detailed analyses, e.g. regarding tissue (cancer)-specific effects. In addition, the estrogen endocrine system may regulate the metabolism of 1,25(OH)2D3 in cancer cells and thereby affect its action (see “Resistance and vitamin D metabolism,” below). Interaction with another sex steroid, testosterone, has been described for ovarian cancer. Vitamin D inhibits dihydrotestosterone (not convertible to estradiol) growth stimulation of ovarian cancer cells [364]. Intriguingly, also here the growth stimulator and growth inhibitor mutually upregulate their receptors. Also in prostate cancer cells it has been shown that 1,25(OH)2D3 while inhibiting androgenstimulated growth upregulates the androgen receptor [365]. Interaction with another steroid in regulating cancer cells has already been reported in 1983. The synthetic glucocorticoid, dexamethasone and 1,25(OH)2D3 synergistically induced differentiation of murine myeloid leukemia cells [366]. This was supported by in vitro and in vivo data that dexamethasone enhanced the effect of vitamin D on growth inhibition, cell cycle arrest and apoptosis of squamous carcinoma cells [367,368]. A
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possible mechanism is the upregulation of VDR by dexamethasone [367]. An interesting aspect of this combination is not only the direct interaction at cancer cell level, but also in the control of the calcemic action of 1,25(OH)2D3. Glucocorticoids inhibit intestinal calcium absorption and increase renal calcium excretion and in this way it may limit the hypercalcemic action of 1,25(OH)2D3 [369]. Combination of vitamin D3 and retinoids has been examined in various systems. A combination of retinoic acid and 1,25(OH)2D3 resulted in a more profound growth inhibition of both T47-D breast cancer cells [370] and LA-N-5 human neuroblastoma cells [371]. 9-cis-Retinoic acid augmented l,25(OH)2D3-induced growth inhibition and differentiation of HL-60 cells [372]. Besides growth inhibition and differentiation effects, the combination of 1,25(OH)2D3 and various isomers of retinoic acid were more potent in reducing angiogenesis than either compound alone [171,172,373]. The background of the interaction between retinoids and 1,25(OH)2D3 may be attributed to heterodimer formation of the respective receptors [374]. For several cytokines, interactions with 1,25(OH)2D3 have been described. Interferon-g and 1,25(OH)2D3 synergistically inhibited the proliferation and stimulated the differentiation of HL-60, WEHI-3, and U937 myeloid leukemia cells [375e378]. Treatment of LLC-LN7 tumor cells with 1,25(OH)2D3 with IFN-g synergistically reduced tumor granulocyteemacrophage colonystimulating factor (GM-CSF) secretion and a blockage in the capacity of the tumor cells to induce granulocyte-macrophage-suppressor cells [121]. In the mouse myeloid leukemia cell line Ml interleukin-4 enhanced 1,25(OH)2 D3-induced differentiation [224,379,380]. Also with interleukin-1b, interleukin-3, interleukin-6, and interleukin-12 interactions with 1,25(OH)2D3 have been reported [381e383]. 1,25(OH)2D3 and tumor necrosis factor synergistically induced growth inhibition and differentiation of HL-60 [384]. For MCF-7 cells an interaction between 1,25(OH)2D3 and tumor necrosis factor has also been reported [383,385]. In the presence of GM-CSF lower concentrations of 1,25(OH)2D3 could be used to achieve a similar anti-proliferative effect in MCF-7 cells [386] and to induce differentiation of U937 myeloid leukemic cells [387]. Other factors shown to interact with 1,25(OH)2D3 are butyrate [388e390], melatonin [391], and factors described in “Differentiation,” above. Furthermore, combinations of vitamin D3 compounds with cytotoxic drugs, antioxidants, and radiation have been studied. In vivo adriamycin and in vitro carboplatin and cisplatin, doxorubicin interacted synergistically with 1,25(OH)2D3 to inhibit breast cancer cell growth [133,392e395]. In a carcinogen-induced rat mammary tumor model, treatment with 1a-(OH)D3
and 5-fluorouracil, however, did not result in enhanced anti-tumor effects [118]. Recently interactions with a plant-derived polyphenolic antioxidant, carnosic acid, were demonstrated in the differentiation of HL-60 cells, which was related to a decrease in the intracellular levels of reactive oxygen species [396,397]. Also interaction with radiation therapy in breast cancer has been described [398e400]. The data on combinations of 1,25(OH)2D3 and 1,25(OH)2D3 analogs with various other anticancer compounds are promising and merit further analyses. The development of effective combination therapies may result in better response rates and lower required dosages, thereby reducing the risk of negative side effects. An additional benefit is that some direct actions of 1,25(OH)2D3 may reduce side effects of toxic chemotherapy drugs when given in combination [401].
RESISTANCE AND VITAMIN D METABOLISM Classic vitamin D resistance concerns the disease hereditary vitamin-D-resistant rickets, which is characterized by the presence of a non-functional VDR and consequently aberrations in calcium and bone metabolism (see Chapter 65). For cancer cells the presence of a functional VDR is also a prerequisite for a growth regulatory response, and a relationship between VDR level and growth inhibition has been suggested for osteosarcoma, colon carcinoma, breast cancer, prostate cancer cells, and rat glioma [1,2,130,149,246,251,402e406]. Cell lines established from DMBA-induced breast tumors in VDR knockout mice are insensitive to growth arrest and apoptosis by 1,25(OH)2D3, EB1089, and CB1093 [407]. Albeit that VDR is a prerequisite for tumor cell growth regulation, the presence of the VDR is not always coupled to a growth-inhibitory response of 1,25(OH)2D3. Results from studies with transformed fibroblasts [232], myelogenous leukemia cells [225,245,408], transformed keratinocytes [222], and various breast cancer cell lines [252,409] demonstrated a lack of growth inhibition by 1,25(OH)2D3 even in the presence of VDR. In this situation the designation “resistant” is based on the lack of growth inhibition, even though, as discussed earlier in “Differentiation,” above, some of these cells are still capable of being induced to differentiate [245,252]. This points to a specific defect in the growth-inhibitory pathway. In the resistant MCF-7 cells this defect is not located at a very common site in the growth-inhibitory pathway of the cell, because the growth could still be inhibited with the antiestrogen tamoxifen (409). For myelogenous leukemia cells similar observations have been made [410]. Human VDR gene is transcriptionally repressed by SNAIL1 and SNAIL2/SLUG in human colon cancer
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cells leading to decreased levels of VDR RNA and protein and unresponsiveness to 1,25(OH)2D3 effects [411e413]. SNAIL1 causes also a decrease in VDR RNA stability [411]. Also, Snail1 represses VDR in mouse osteoblasts and SNAIL2/SLUG in human breast cancer cells [414]. In addition, Snail1 is probably mediating the decrease in VDR mRNA stability induced by oncogenic Ha-ras in mouse NIH-3T3 cells [415,416]. For VDR-independent resistance to growth inhibition and in general to 1,25(OH)2D3 effects several underlying mechanism(s) have been proposed: increased levels of VDR co-repressors, reduced bioavailability of 1,25(OH)2D3 due to either or both 24-hydroxylase (CYP24) upregulation and 25-hydroxyvitamin D3 1ahydroxylase (CYP27B1) downregulation, and disruption or phosphorylation of VDReRXR dimers. Resistance to 1,25(OH)2D3 in breast and prostate cancer cells has also been found to be a consequence of increased levels of the VDR co-repressors NCoR or SMRT [417,418]. This is in line with the reported synergistic effect on the proliferation of prostate cancer cells of combined treatment with 1,25(OH)2D3 and the histone deacetylase inhibitor trichostatin [388]. For the resistant MCF-7 clone this is not related to upregulation of the P-glycoprotein [409]. Interestingly, these vitamin-D-resistant MCF-7 clones can be sensitized to vitamin D by activation of protein kinase C, resulting in induction of apoptosis and transcriptional activation, suggesting that alterations in phosphorylation may affect vitamin D sensitivity [419]. Hansen et al. described an interesting growth-inhibition-resistant MCF-7 cell clone. This clone was not growth inhibited while VDR was still present and CYP24 could still be induced [420]. Another example of vitamin D resistance is HL60 cells that have been cultured for 4 years in the presence of 1,25(OH)2D3 resulted in clones that are resistant to differentiation induction and growth inhibition. They became not only resistant to vitamin D but also to 5-beta-D-arabinocytosine, suggesting a common metabolic pathway being responsible [421]. Whether this relates to the upregulation of the multi-drug resistance proteins is not clear. In the resistant leukemia JMRD3 cell line, altered regulation and DNA-binding activity of junD as part of the AP-1 complex has been reported [240]. Resistance to growth inhibition in the presence of VDR has also been linked to disruption of the VDReRXR complex [422] and increased RXR degradation [423]. In addition, other factors, like the acute myeloid leukemia translocation products (e.g., PLZF) may contribute to resistance to vitamin D by sequestering the VDR [275,276]. More recently it was shown that alterated corepressor and co-activator interaction with VDR and that epigenetic preferential suppression of antiproliferative gene promoters can explain the resistance to growth
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inhibition [424]. Resistance has also been linked to epigenetic changes in the VDR promoter leading to suppressed or absent expression of VDR [425]. A unique mechanism for vitamin D resistance in immortalized cells has very recently been uncovered. Epstein-Barr virus (EBV) has been used to transform and immortalize lymphoblasts that can grow as cell lines in vitro. EBNA-3 is an EBV-encoded protein that can regulate transcription of cellular and viral genes. EBNA3 binds the VDR and blocks the activation of VDR-dependent genes and protects transformed cell lines against vitamin-D3-induced growth arrest and/or apoptosis [426]. The 1,25(OH)2D3-sensitive and -resistant cell clones provide interesting models to examine the molecular mechanisms of 1,25(OH)2D3-induced growth inhibition. For example, lack of p21 results in no cell cycle block [427] and no apoptosis was detected with a mutated p53 [252]. Finally, the recent identification of cellular proteins that are involved in the vitamin D resistance in New World primates might add to the understanding of tumor cell resistance to vitamin D [428,429]. At the moment the major mechanism for vitamin D resistance or reduced sensitivity in VDR containing tumor and cancer cells is 1,25(OH)2D3 catabolism via the C24-hydroxylation pathway. An inverse relationship between cellular metabolism of 1,25(OH)2D3 via 24hydroxylation and growth inhibition of prostate cancer cells has been suggested [403]. The latter observation is intriguing, the more so as an inverse relationship between VDR level and induction of CYP24 activity was reported. In general, there may exist a direct relationship between VDR level and induction of CYP24 activity [404,430]. An important role in the control of 1,25(OH)2D3 action on cancer cells was provided by studies with the 1,25(OH)2D3-resistant prostate cancer cell line DU145. It was shown that 1,25(OH)2D3 did inhibit the growth of these cells when it was combined with the 24-hydroxylase inhibitor Liazorole [431]. 1,25(OH)2D3 activity was likewise enhanced by combination with ketoconazole, a drug commonly used to treat prostate cancer that inhibits CYP24 activity [432,433]. Inhibition of CYP24 activity in HL-60 cells also altered the effect of 1,25(OH)2D3 and 20-epi analogs [434]. Recently, epigenetic silencing of the CYP24 gene modulates the growth response of tumor-derived endothelial cells [435]. The action of the analog EB1089 was also limited by hydroxylation at the C24 position [436]. However, it was suggested that the increased potency of EB1089 is at least partly due to resistance to CYP24 [288]. Alternatively, 24-hydroxylation of the analog KH1060 has been implicated as one of the mechanisms to explain the potency of this analog. The 24-hydroxylated metabolites of this analog are very stable and remain biologically active [437,438]. It has been shown
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that the naturally occurring 24-hydroxylated metabolite of vitamin D3 (24R,25(OH)2D3) also has a preventive effect on chemically induced colon cancer [439]. Interaction between the estrogen system and CYP24 is also of importance. Recent data have shown that the phytoestrogen genistein inhibits CYP24 activity in prostate cancer cells and thereby increases the responsiveness to 1,25(OH)2D3 [440,441]. A role for CYP24 as oncogene is suggested by data showing amplification of the CYP24 locus on chromosome 20q13.2 [442]. In contrast to degradation of 1,25(OH)2D3 by CYP24 in cancer cells recently it has become clear that tumor cells contain CYP27B1 activity and thereby are able to locally generate 1,25(OH)2D3. Expression of 1a-hydroxylase has been demonstrated in colorectal cancer [443e445]. It was postulated that in early stages tumor cells respond by upregulating 1a-hydroxylase activity to counteract neoplastic growth while at later stages of tumor development this is lost [443]. Also in prostate cancer [446] and inflammatory myofibroblastic tumor [447], CYP27B1 has been detected, albeit that in the latter case the tumor contains large numbers of macrophages. It can be anticipated that in the coming years investigation of the expression of both CYP24 and CYP27B1 in tumors will add to understanding the role of vitamin D in inhibiting the initiation and progression of cancer.
STIMULATION OF PROLIFERATION Over the years a limited number of studies have demonstrated that, in contrast to growth inhibition, 1,25(OH)2D3 can also stimulate tumor cell growth and tumor development. In several cells 1,25(OH)2D3 has been reported to have a biphasic effect, that is, at lower concentrations (<10 9 M) it stimulates proliferation and at higher concentrations (10 9 to 10 7 M) it inhibits proliferation. However, clear growth stimulation can sometimes be observed not only at low concentrations but also at the concentrations generally found to inhibit tumor cell proliferation and tumor development. 1,25(OH)2D3 has been shown to stimulate the growth of a human medullary thyroid carcinoma cell line [448]. Not only cancer cells but also several normal cells, for example human monocytes [449], smooth muscle cells [450], and alveolar type II cells [451], are stimulated to grow by 1,25(OH)2D3. Skin is another organ in which different effects of 1,25(OH)2D3 have been observed. In vivo studies demonstrated that 1,25(OH)2D3 and analogs stimulate keratinocyte proliferation in normal mice [452e455] and enhance anchorage-independent growth of preneoplastic epidermal cells [456]. In contrast, other studies showed 1,25(OH)2D3 inhibition of proliferation of mouse
and human keratinocytes [457,458], and 1,25(OH)2D3 is also effective in the treatment of the hyperproliferative disorder psoriasis [459]. Moreover, in vivo studies demonstrated that, depending on the carcinogen, 1,25(OH)2D3 can either reduce [110] or enhance the induction and development of skin tumors in mice [460,461]. In addition, 1,25(OH)2D3 enhances the chemically induced transformation of BALB 3T3 cells and hamster embryo cells [462,463]. 1,25(OH)2D3 also enhanced 12-O-tetradecanoylphorbol-13-acetate-induced tumorigenic transformation of mouse epidermal JB6 Cl41.5a cells [464,465]. Another example comes from research on osteosarcoma cells. In 1986 it was shown that 1,25(OH)2D3 stimulated the growth of tumors in athymic mice inoculated with the ROS 17/2.8 osteosarcoma cell line [466]. Earlier the same group reported growth stimulation in vitro of these osteosarcoma cells at low concentrations but growth inhibition by 10 8 M [402]. They speculated that this discrepancy resulted from limited in vivo availability of 1,25(OH)2D3 for the tumor cells, resulting in concentrations shown to be growth stimulatory in vitro. However, in other experiments with nude mice the availability of 1,25(OH)2D3 did not seem to be a factor, as growth inhibition was observed (see Table 83.2). In particular, in nude mice implanted with human osteosarcoma cells (MG-63), growth inhibition and tumor suppression by 1,25(OH)2D3 were observed [120]. In two different in vitro studies, growth inhibition of MG-63 and growth stimulation of ROS 17/2.8 cells was reported [467,468]. For smooth muscle cells it has been demonstrated, for example, that growth inhibition or stimulation can depend on the presence of additional growth factors in the culture medium [450]. We followed up on this concept by comparing the effects of 1,25(OH)2D3 and analogs on the growth and osteoblastic characteristics of the two osteosarcoma cell lines under identical culture conditions. At concentrations 10 10 to 10 7 M 1,25(OH)2D3 caused an increase in cell proliferation by 100% in ROS 17/2.8 cells, whereas the proliferation of MG-63 cells was inhibited (Fig. 83.2) [235]. In contrast, in both cell lines 1,25(OH)2D3 stimulated osteoblastic differentiation characteristics such as production of osteocalcin and alkaline phosphatase activity [235,467]. Analyses with another steroid hormone demonstrated that glucocorticoids inhibited the growth of both osteosarcoma cell lines [469,470]. These data indicate specific differences between these cell lines, especially with respect to the 1,25(OH)2D3 growth regulatory mechanisms. In addition to these biological data in cells, an epidemiological study also showed an increased risk of aggressive prostate cancer with higher levels of 25hydroxyvitamin D3 [34]. Taken together, the data on growth stimulation and tumor development, although
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is needed so that therapeutic modalities may be employed more effectively.
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Effect of 1,25(OH)2D3 on proliferation of the osteosarcoma cell lines ROS 17/2.8 and MG-63. Effects on proliferation were examined as described by van den Bemd et al. [235].
FIGURE 83.2
detected in only a small minority of cancer cells, demonstrate that treatment with 1,25(OH)2D3 or analogs may not always cause growth inhibition and tumor size reduction. It is therefore of utmost importance to identify the mechanism(s) by which 1,25(OH)2D3 exerts its inhibitory and stimulatory effects on cell growth. This may provide tools to assess whether treatment of a particular tumor will be beneficial. Moreover, purely from a mechanistic point of view, further study of growth-stimulated and growth-inhibited cells, like the 1,25(OH)2D3-sensitive and -resistant cells, may provide tools to examine the 1,25(OH)2D3 mechanism of growth regulation.
CONCLUSIONS The data obtained so far, on (1) the distribution of the VDR in a broad range of tumors and (2) the inhibition of cancer cell growth, angiogenesis, metastasis, inflammation and PTHrP synthesis as well as the stimulation of differentiation and apoptosis by 1,25(OH)2D3, all hold promise for the development of treatment strategies based on vitamin D3 use in a wide range of cancers. Moreover, combination of vitamin D3 with other antitumor drugs, hormones, or growth factors is an important additional therapeutic option. Throughout the previous decade data have accumulated on the cellular targets and mechanism of action of 1,25(OH)2D3induced cancer growth inhibition. The clinical application is enhanced by the development of 1,25(OH)2D3 analogs with potent growth-inhibitory actions and reduced hypercalcemic activity. Nevertheless it is crucial for the coming years to deliver strong clinical trials to support the potential of vitamin D in cancer treatment uncovered by investigation of cultured cells, animal models, and epidemiological studies. In the meantime continuing research to understand the mechanisms by which vitamin D3 exerts its effects on tumor cell growth
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1623 human circulating monocytes in vitro, FEBS Lett. 185 (1985) 9e13. T. Mitsuhashi, R.C. Morris Jr., H.E. Ives, 1,25-Dihydroxyvitamin D3 modulates growth of vascular smooth muscle cells, J. Clin. Invest. 87 (1991) 1889e1895. J.D. Edelson, S. Chan, D. Jassal, M. Post, A.K. Tanswell, Vitamin D stimulates DNA synthesis in alveolar type-II cells, Biochim. Biophys. Acta. 1221 (1994) 159e166. C. Lutzow-Holm, P. De Angelis, H. Grosvik, O.P. Clausen, 1,25Dihydroxyvitamin D3 and the vitamin D analogue KH1060 induce hyperproliferation in normal mouse epidermis. A BrdUrd/DNA flow cytometric study, Exp. Dermatol. 2 (1993) 113e120. R. Gniadecki, A vitamin D analogue KH 1060 activates the protein kinase C-c-fos signalling pathway to stimulate epidermal proliferation in murine skin, J. Endocrinol. 143 (1994) 521e525. R. Gniadecki, J. Serup, Stimulation of epidermal proliferation in mice with 1 alpha, 25-dihydroxyvitamin D3 and receptoractive 20-EPI analogues of 1 alpha, 25-dihydroxyvitamin D3, Biochem. Pharmacol. 49 (1995) 621e624. R. Gniadecki, M. Gniadecka, J. Serup, The effects of KH 1060, a potent 20-epi analogue of the vitamin D3 hormone, on hairless mouse skin in vivo, Br. J. Dermatol. 132 (1995) 841e852. J. Hosoi, E. Abe, T. Suda, N.H. Colburn, T. Kuroki, Induction of anchorage-independent growth of JB6 mouse epidermal cells by 1 alpha,25-dihydroxyvitamin D3, Cancer Res. 46 (1986) 5582e5586. Y. Kitano, N. Ikeda, M. Okano, Suppression of proliferation of human epidermal keratinocytes by 1,25-dihydroxyvitamin D3. Analysis of its effect on psoriatic lesion and of its mechanism using human keratinocytes in culture, Eur. J. Clin. Invest. 21 (1991) 53e58. J. Hosomi, J. Hosoi, E. Abe, T. Suda, T. Kuroki, Regulation of terminal differentiation of cultured mouse epidermal cells by 1 alpha,25-dihydroxyvitamin D3, Endocrinology 113 (1983) 1950e1957. K. Kragballe, Vitamin D3 and skin diseases, Arch. Dermatol. Res. 284 (Suppl. 1) (1992) S30eS36. T. Kuroki, K. Sasaki, K. Chida, E. Abe, T. Suda, 1 Alpha,25dihydroxyvitamin D3 markedly enhances chemically-induced transformation in BALB 3T3 cells, Gann. 74 (1983) 611e614. K. Sasaki, K. Chida, H. Hashiba, N. Kamata, E. Abe, T. Suda, et al., Enhancement by 1 alpha,25-dihydroxyvitamin D3 of chemically induced transformation of BALB 3T3 cells without induction of ornithine decarboxylase or activation of protein kinase C1, Cancer Res. 46 (1986) 604e610. C.A. Jones, M.F. Callaham, E. Huberman, Enhancement of chemical-carcinogen-induced cell transformation in hamster embryo cells by 1 alpha,25-dihydroxycholecalciferol, the biologically active metabolite of vitamin D3, Carcinogenesis 5 (1984) 1155e1159. A.W. Wood, R.L. Chang, M.T. Huang, E. Baggiolini, J.J. Partridge, M. Uskokovic, et al., Stimulatory effect of 1 alpha, 25-dihydroxyvitamin D3 on the formation of skin tumors in mice treated chronically with 7,12-dimethylbenz[a]anthracene, Biochem. Biophys. Res. Commun. 130 (1985) 924e931. P.L. Chang, C.W. Prince, 1 Alpha,25-dihydroxyvitamin D3 enhances 12-O-tetradecanoylphorbol-13-acetate-induced tumorigenic transformation and osteopontin expression in mouse JB6 epidermal cells, Cancer Res. 53 (1993) 2217e2220. P.L. Chang, T.F. Lee, K. Garretson, C.W. Prince, Calcitriol enhancement of TPA-induced tumorigenic transformation is mediated through vitamin D receptor-dependent and -independent pathways, Clin Exp. Metastasis 15 (1997) 580e592.
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[466] K. Yamaoka, S.L. Marion, A. Gallegos, M.R. Haussler, 1,25Dihydroxyvitamin D3 enhances the growth of tumors in athymic mice inoculated with receptor rich osteosarcoma cells, Biochem. Biophys. Res. Commun. 139 (1986) 1292e1298. [467] R.T. Franceschi, W.M. James, G. Zerlauth, 1 Alpha, 25-dihydroxyvitamin D3 specific regulation of growth, morphology, and fibronectin in a human osteosarcoma cell line, J. Cell Physiol. 123 (1985) 401e409. [468] G. Gronowicz, J.J. Egan, G.A. Rodan, The effect of 1,25-dihydroxyvitamin D3 on the cytoskeleton of rat calvaria and rat osteosarcoma (ROS 17/2.8) osteoblastic cells, J. Bone Miner. Res. 1 (1986) 441e455. [469] B.O. Hodge, B.E. Kream, Variable effects of dexamethasone on protein synthesis in clonal rat osteosarcoma cells, Endocrinology 122 (1988) 2127e2133. [470] Z. Abbadia, J. Amiral, M.C. Trzeciak, P.D. Delmas, P. Clezardin, The growth-supportive effect of thrombospondin (TSP1) and
the expression of TSP1 by human MG-63 osteoblastic cells are both inhibited by dexamethasone, FEBS Lett. 335 (1993) 161e166. [471] S. Huerta, R.W. Irwin, D. Heber, V.L. Go, F. Moatamed, C. Ou, et al., Intestinal polyp formation in the Apcmin mouse: effects of levels of dietary calcium and altered vitamin D homeostasis, Dig. Dis. Sci. 48 (2003) 870e876. [472] M.R. Young, J. Halpin, J. Wang, M.A. Wright, J. Matthews, A.S. Pak, 1 Alpha,25-dihydroxyvitamin D3 plus gamma-interferon blocks lung tumor production of granulocyte-macrophage colony-stimulating factor and induction of immunosuppressor cells, Cancer Res. 53 (1993) 6006e6010. [473] D. Sahpazidou, P. Stravoravdi, T. Toliou, G. Geromichalos, G. Zafiriou, K. Natsis, et al., Significant experimental decrease of the hepatocellular carcinoma incidence in C3H/Sy mice after long-term administration of EB1089, a vitamin D analogue, Oncol. Res. 13 (2003) 261e268.
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C H A P T E R
84 Vitamin D Effects on Differentiation and Cell Cycle George P. Studzinski 1, Elzbieta Gocek 1, 2, Michael Danilenko 3 1
UMD-New Jersey Medical School, Newark, New Jersey, USA 2 University of Wroclaw, Wroclaw, Poland 3 Ben-Gurion University of the Negev, Beer-Sheva, Israel
INTRODUCTION In general, cell cycle control is extremely well conserved throughout the eukaryotic species. The basic machinery consists of several cyclin-dependent kinases (Cdks) and cyclins that pair with each other, sometimes changing partners, to drive the cell towards and through mitosis [1e4]. This basic arrangement seems almost monotonously similar in all cells, yet in multicellular organisms control of cell proliferation must be, and is, cell-type-specific. The required control is provided by proteins that regulate kinase activity of Cdk/cyclin pairs, most often in a negative fashion, and regulation usually occurs in response to cues from the environment. Importantly, cell cycle changes are also triggered in cells undergoing differentiation. Differentiation can be considered to be, in essence, a persistent pattern of expression of previously dormant genes, which results in new functional capabilities of the differentiated cell. The new functions require cellular resources that compete with and finally titrate out the resources required for proliferation, and allow an accumulation of negative regulators of the cell cycle (e.g., p27/Kip1 [5]), which then predominate over the positive regulators. Thus, there is a reciprocal relationship between cellular differentiation and cell cycle progression/proliferation [6e8], though there is also evidence that differentiation and cycle arrest need not be strictly coupled [9e12]. Cell cycle changes in differentiating cells need not take place immediately e in some cells there is at first a boost of proliferation e as in normal hematopoiesis, or in HL60 [11e13] and U937 [14] cells differentiating
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10084-8
in response to derivatives of vitamin D3. However, even in these cases, there is an eventual slowdown of the cell cycle traverse and cessation of proliferation of differentiated cells. Consequently, numerous attempts are being made to exploit the differentiating actions of vitamin D and its analogs to induce proliferative quiescence of neoplastic cells, and thus increase the range of options for optimal therapy of human cancer. This is also addressed in the other chapters in Section X on Cancer. The term “differentiation” is also often used in a rather specialized way to imply the acquisition of new functional properties by a cell that already appears to be mature and capable of function. Examples are monocytes differentiating into macrophages, or naı¨ve T lymphocytes becoming helper or cytotoxic cells. Little is known, however, of cell cycle alterations in this form of differentiation.
INDUCTION OF DIFFERENTIATION BY 1,25(OH)2D3 AND ITS ANALOGS Principal Models In 1981 Suda Laboratory reported that an in vitro exposure of M1 mouse myeloid leukemia cells to 1,25(OH)2D3 induces these immature cells to differentiate into functional macrophages [15]. This seminal finding was followed by numerous reports not only confirming that leukemic cells, murine or human [16e19], differentiate in response to treatment with 1,25(OH)2D3 or its analogs, but several other types of
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84. VITAMIN D EFFECTS ON DIFFERENTIATION AND CELL CYCLE
neoplastic cells show similar responses. These include colon cancer, breast cancer, prostate cancer, neuroblastoma, osteosarcoma, squamous cell carcinoma (SCC), and malignant melanoma [20]. In addition, normally developing immature cells can be induced to differentiate by 1,25(OH)2D3, for example keratinocytes, myoblasts and perhaps hematopoietic cell precursors. Specific markers by which these phenotypes can be recognized are summarized, and sample references are provided in Table 84.1. It is apparent that 1,25(OH)2D3 is a powerful differentiating agent that targets diverse cell types, but its physiological role in development remains to be fully determined. Although data from vitamin D receptor (VDR) knockout mice suggest that this is the case, most in vitro studies with normal immature cells used high concentrations of 1,25(OH)2D3, which were in great excess over the physiological levels. While it can be argued that concentrations of 1,25(OH)2D3 in specific tissue niches in which precursor cells differentiate can be higher than the concentrations found in the plasma, a clearer assessment of the physiological role of 1,25(OH)2D3 in normal differentiation may be achieved by studies that utilize low nanomolar or subnanomolar concentrations of 1,25(OH)2D3 (e.g., [21,22]).
Initial Signals for Differentiation; Effects of 1,25(OH)2D3 Cells that differentiate when exposed to 1,25(OH)2D3 usually express VDR [23e27]. In several studies, evidence that VDR is strictly required for the 1,25(OH)2D3-induced differentiation was obtained. For instance, transfection of the VDR conferred differentiation responsiveness to 1,25(OH)2D3 in WEHI-3B Dþ murine myelomonocytic cells, which lack inducible VDR expression [28]. In VDR knockout mice, 12-O-tetradecanoylphorbol-13-acetate (TPA), but not 1,25(OH)2D3 [29] or 19-nor-1,25-dihydroxyvitamin D2 [30], induced differentiation of bone-marrow-committed myeloid stem cells to monocytes/macrophages, which indicates the requirement of VDR for 1,25(OH)2D3-induced monocyte/macrophage differentiation. Similarly, VDR is required for the morphogenesis and negative growth regulation in the mammary gland [31,32]. However, a cell-type- or species-specific involvement of VDR in vitamin D analog-induced growth inhibition of breast cancer cells has recently been revealed in studies by Welsh and co-workers. For instance, in contrast to VDR expressing WT145 murine mammary tumor cells, KO240 cells isolated from tumors that developed in VDR knockout mice were not growth inhibited nor rendered apoptotic by any of the tested vitamin D compounds [33]. On the other hand, knockdown of VDR by siRNA did not affect the anti-proliferative
effects of 1,25(OH)2D3 in the most widely studied human breast cancer cell line, the MCF-7 cells [34]. Furthermore, genome-wide arrays demonstrated that neither VDR, CYP24A1, nor other known vitamin D signaling pathway genes were altered in MCF-7 cells made resistant to 1,25(OH)2D3 by long-term exposure to this agent [34]. VDR is a nuclear protein that may also shuttle to and from the cytoplasm [35,36]. Thus, liganded VDR, often acting as a heterodimer with one of the three members of the retinoid X receptor (RXR) family is the principal mediator of the effects of vitamin D, as described in detail in Chapter 8. There is, however, intriguing evidence that 1,25(OH)2D3 and especially some analogs have direct cell membrane effects that modify, or enhance, the VDR-transmitted signals. This is extensively discussed in Chapter 15. Although the membrane effects originally described in the enterocytes are able to account for the very rapid calcium transport in the intestine (“transcaltachia” [37,38]), several reports suggest that the increased therapeutic ratios (i.e., the ratios of differentiation to calcemic potencies) of 1,25(OH)2D3, and some low-calcemic analogs may be due to the auxiliary effects of nonVDR-mediated actions of these compounds [39e41]. Nonetheless, it is most likely that the gene expressionmodulating actions of VDReRXR heterodimers transmit the differentiation signals to the basal transcription machinery by interacting with vitamin-D response element (VDRE) sequences of the DNA molecule [42,43]. A large assortment of nuclear receptor co-activators, such as DRIP/Mediator and SRC/p160 [44e47] and co-repressors (e.g., SMRT and N-CoR) [46,48e50] has been identified, which provides positive or negative regulation of the VDR transcriptional activity. In addition to the “classic” VDRE sequences that transactivate vitamin-D-responsive gene transcription, the “negative” VDREs have also been characterized that inhibit transcription of certain genes, e.g. parathyroid hormone gene [51e53]. Interestingly, in myeloid leukemia cells, promyelocytic leukemia zinc finger (PLZF) and the chromosomal translocation products, promyelocytic leukemia-retinoic acid receptor alpha (PML/RARa) and PLZF/RARa also repress the differentiating action of VDR by binding and sequestering the VDR [54e56]. The regulatory effects of VDR’s co-modulators are discussed in detail in Chapter 10 and the nature of VDREs is discussed in Chapter 11. How the initial vitamin-D-induced gene expression leads to the acquisition of a new functional phenotype, i.e. differentiation, is one of current mysteries, as among the known direct target genes of VDR only a few have a possible relevance to the differentiating actions of 1,25(OH)2D3 in tissues other than bone. These include p21/Cip1 [14,57e59] and KSR1 and KSR2 [60,61].
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INDUCTION OF DIFFERENTIATION BY 1,25(OH)2D3 AND ITS ANALOGS
TABLE 84.1
Examples of Cellular Models of Differentiation Induced by 1,25(OH)2D3 and Analogs
Differentiation marker
Known function
Comments
References
HEMATOPOIETIC CELLS (HL60, U937, THP-1, UF-1, WEHI-3) CD14
Cell-surface LPS binding protein
Early monocytic differentiation
[40,146,362e364]
CD11b
Cell-surface protein (integrin aM)
General myeloid differentiation
[287,365,366]
Non-specific esterase
Cytoplasmic hydrolytic enzyme
Monocytic differentiation
[16,40,331,367,368]
Component of oxidative burst
Phagocytic activity
[369e372]
Superoxide anion
[16,131,368]
Morphologic changes
a
COLON CANCER CELLS (Caco-2, SW480-ADH, PRIMARY ADENOMA AND CARCINOMA LINES) Alkaline phosphatase
Brush border-associated hydrolase
Intestinal and placental isozymes
[24,115,373e375]
Carcinoembryonic antigen (CEA)
Adhesion molecule
Early development protein
[376,377]
E-cadherin
Calcium-dependent cell adhesion molecule
Invasion suppressor
[312,329]
OSTEOBLAST-LIKE CELLS (MG-63, ROS 17/2.8, MC3T3-E1) Osteopontin
Bone sialoprotein I (BSP-1), adhesion molecule
Early osteoblastic differentiation
[378,379]
Osteocalcin
Osteoblast-specific non-collagenous protein
Late osteoblastic differentiation
[380e382]
Alkaline phosphatase
Hydrolytic enzyme
Bone mineralization
[380,383,384]
PROSTATE CANCER CELLS (LNCaP, PC-3) Prostate-specific antigen (PSA)
Serine protease
Secreted by prostate epithelial cells
[385e389]
Prostate-specific acid phosphatase
Protein tyrosine phosphatase
Prostate growth-regulating enzyme
[387]
E-cadherin
Calcium-dependent cell adhesion molecule
Major epithelial cadherin
[388,390]
BREAST CANCER CELLS (MCF-7, T47D, MDA-MB-231, MDA-MB-436, BT-20, SK-BR-3, UISO-BCA-4) Intracellular lipid droplets
Storage/precursor material
[391e394]
Casein
Major milk protein
[392,393]
Reverts the myoepithelial features associated with more aggressive breast cancer
[395]
Morphologic changes
a
NEUROBLASTOMA (LA-N-5) Acetylcholine esterase
Serine hydrolase
May regulate neurite outgrowth
Neurite outgrowth
[396e398] [397e399]
MELANOMA CELLS (B16) Tyrosinase
Copper-containing oxidase
Key enzyme in melanin synthesis
[400,401]
SQUAMOUS CELL CARCINOMA (SCC13, SCC25, SCC 2/88) Keratin 1
Fibrous scleroprotein
Structural skin component
[402]
Transglutaminase
Calcium-dependent crosslinking enzyme
Keratinocyte-specific form
[403]
Involucrin
Glutamine-rich transglutaminase substrate
Cornified cell envelope constituent
[402,404e406]
Transglutaminase
Calcium-dependent crosslinking enzyme
Keratinocyte-specific form
[403,407e410]
Keratin 1
Fibrous scleroprotein
Early differentiation marker
[409,410]
Keratin 10
Fibrous scleroprotein co-expressed with keratin 1
Early differentiation marker
[409,410]
KERATINOCYTES
(Continued)
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1628 TABLE 84.1
84. VITAMIN D EFFECTS ON DIFFERENTIATION AND CELL CYCLE
Examples of Cellular Models of Differentiation Induced by 1,25(OH)2D3 and Analogsdcont’d
Differentiation marker
Known function
Comments
References
Involucrin
Glutamine-rich transglutaminase substrate
Cornified cell envelope constituent
[150,404,408e411]
Cystatin A
Cysteine proteinase inhibitor
Cornified cell envelope constituent
[412,413]
Loricrin
Late differentiation marker
[409,410]
Filaggrin
Late differentiation marker
[409,410]
Cornified envelope formation
[407,414]
MUSCLE CELLS (C2C12) Myosin
Contractile protein
Creatine kinase
ATP metabolizing enzyme
Late differentiation
[63,415] [63,416]
VASCULAR SMOOTH MUSCLE CELLS (PRIMARY CULTURES) Osteopontin
Bone sialoprotein I (BSP-1), adhesion molecule
Osteoblastic differentiation
[417]
Alkaline phosphatase
Hydrolytic enzyme
Osteoblastic differentiation
[417,418]
a
Morphologic changes can be recognized in many forms of differentiation.
However, with regard to differentiation of most cell types, the links from VDR-initiated events to the downstream targets of 1,25(OH)2D3 still need to be found.
Pathways that Participate in 1,25(OH)2D3 Signal Propagation Pharmacologic inhibitors of several signaling pathways reduce, to varying extents, 1,25(OH)2D3-induced differentiation in diverse model systems [13,62e70] encouraging the belief that these pathways participate in generating signals to cells to differentiate. Protein Kinase C A number of early studies linked several isoforms of PKC to differentiation [71e75]. Following the observation by Martell, Simpson and Taylor [76] that treatment of HL60 cells by 1,25(OH)2D3 increases cellular TPA receptors, which implies increased PKC abundance, the Hannun Laboratory showed that 1,25(OH)2D3 increases the mRNA for isoforms a and b of PKC in these cells [77]. These results were duplicated in many other laboratories, and PKC inhibitors or antisense oligonucleotides to this enzyme were demonstrated to interfere with 1,25(OH)2D3-induced differentiation [63,65,78], further implying a role for at least some isoforms of PKC in differentiation induced by 1,25(OH)2D3. If its role could be established, PKC would provide a central position in a logically pleasing sequence of events that lead from an exposure of a cell to 1,25(OH)2D3 towards differentiation. First, the lipidsoluble 1,25(OH)2D3 may interact with cell membrane lipids or activate membrane-associated phospholipases
(e.g., [79]) directly or through the still elusive membrane receptor, to generate a phospholipid second messenger, such as inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG). IP3 then releases calcium from the endoplasmic reticulum and thus promotes an increase in intracellular calcium ([Ca2þ]i). As the result of raised [Ca2þ]i and DAG concentrations several PKC isoforms can be activated (e.g., [80e82]), and the signal can be propagated further by activating regulators of other signaling pathways, such as the mitogen-activated protein kinase (MAPK) pathway. Known examples of such links are the phosphorylation of Raf1 by PKCa in colon cancer CaCo-2 cells [83], and the translocation of MAPK ERK1/2 to the nucleus [84]. Another potential link is the regulation of VDR by PKC activation [85e88]. However, several major difficulties have so far precluded a full assessment of the role of PKC activity in differentiation. These include its presence in multiple isoforms with overlapping properties, and the fact that full inhibition of cellular PKC activity is usually incompatible with cell survival. Phosphatidylinositol 3-kinase/Akt Pathway Cell membrane-originating effects of 1,25(OH)2D3 are also linked to several other signaling pathways in still ill-defined ways. The phosphatidylinositol 3-kinase (PI3-K)/Akt signaling has been implicated in 1,25(OH)2D3-induced differentiation of THP-1 human leukemia cells [62,89], HL60 cells [70,89e92] and keratinocytes, in which the membrane receptor for 1,25(OH)2D3 is reported to be annexin II [67]. While in several studies [70,92] 1,25(OH)2D3 was found to
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The MEK/ERK MAPK module is activated by a sequence of kinase reactions that are initiated at the cell membrane by extracellular signals which activate receptors that include growth factor and cytokine receptors (reviewed in [98e101]). This cascade of phosphorylations leads to DNA replication, differentiation and enhanced cell survival, the outcomes depending on the duration of the signal as well as the activity of other signaling pathways. The current paradigm is that these membrane events activate the small G protein Ras which sequentially activates Raf1, MEK1, then ERK1/2, which translocates to the nucleus and phosphorylates transcription factors, thus increasing their activity [98e102]. Recently, however, another component, kinase suppressor of Ras-1 (KSR1), has been shown to be an intermediary between Ras, Raf, and MEK [64,102e105]. Although its function has been argued to be a kinase that phosphorylates and thus activates Raf1 [106e108], or a scaffold that brings Ras, Raf, and MEK together [109,110], it is clear that KSR1 facilitates the actions of the ERK MAPK pathway irrespective of its mode of action. Interestingly, KSR1 is upregulated by 1,25(OH)2D3 in HL60 cells, and appears to amplify the differentiation signal provided by nanomolar concentrations of 1,25(OH)2D3 [60,64,111]. 1,25(OH)2D3 can amplify the Raf1eMEKeERK pathway by direct transcriptional upregulation of KSR1 [60]. KSR2, a close homolog of KSR1 which is also directly upregulated by 1,25(OH)2D3 [61], may have a similar role in the activation of the Raf1eMEKeERK pathway [112].
upregulate Akt in HL60 cells following 8e48 h incubations, Wang at al. [93] have presented results suggesting that 1,25(OH)2D3 can downregulate Akt in these cells at a later stage. Furthermore, overexpression of Akt blunted differentiation in response to 1,25(OH)2D3 whereas knockdown of Akt by RNA interference had an opposite effect [93]. Likewise, vitamin D analogs downregulated Akt in SCC cells [94] and ErbB2 overexpressing mammary tumors [95], which was associated, respectively, with the anti-proliferative effect and inhibition of tumor growth. Thus, the involvement of Akt in the differentiation and anti-proliferative effects of 1,25(OH)2D3 appears to be complex and is likely to be dependent on both the tissue type and treatment conditions. MAP Kinase Pathways Although it is possible that MAPK activation also originates at the cell membrane, particularly in intestinal cells, osteoblasts, and chondrocytes, where the activation of MAPKs is very rapid [96], in other cell systems upregulation of MAPKs can take place in a matter of hours and days, not only seconds or minutes, and genomic actions of 1,25(OH)2D3 appear to be essential. In these model systems, which include HL60 leukemia cells, three MAPK pathways have been extensively studied. These pathways utilize ERKs, JNKs, and p38 MAPKs as the principal signal transmitters [97], as illustrated in Figure 84.1.
1,25(OH)2D3
An upstream regulator PKC ?
p38αβ
MKK4
MEK1/2
JNK
ERK1/2
p38γδ
KSR1/2
Raf1 MKK3/6
P c-Jun
ATF-2 Fos family
p38 targets ?
ERK targets Differentiation
AP-1
p27 Cell cycle arrest
1629
VDR
TRE
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FIGURE 84.1 The MAP kinase pathways,
which are upregulated by 1,25(OH)2D3 in leukemia cells. The ERK and JNK pathways have positive effects on differentiation [13,68], while the p38 MAPK pathway may have a dual effect on differentiation. Several lines of evidence demonstrate that p38a and p38b have an inhibitory effect on monocytic, but not granulocytic, differentiation of HL60 cells [119,120], while p38g and d may positively modulate monocytic differentiation of these cells [452]. 1,25(OH)2D3 can amplify the Raf1eMEKeERK pathway by direct transcriptional upregulation of kinase suppressor of Ras-1 (KSR1) [60], which acts as a scaffold that coordinates signaling along the Ras/ERK signaling module [110] or as an active kinase that phosphorylates Raf1 [108]. KSR2, a close homolog of KSR1 which is also directly upregulated by 1,25(OH)2D3 [61], may have a similar role in the activation of the Raf1-MEKERK pathway [112]. Also shown is the potential role of the AP-1 transcription factor, which acts as an intermediary positive effector of 1,25(OH)2D3 signals by upregulating the expression of VDR [97].
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Activation of ERK2 by 1,25(OH)2D3 has been found in many differentiation systems. For instance, “rapid and transient” activation has been reported in NB4 acute promyelocytic leukemia cells [113], normal human keratinocytes [67,114], CaCo-2 colon cancer cells [115], and HL60 human myeloid leukemia cells [13,89,116]. While all these examples were found to be transient, the scale of “rapidity” varied from 30 s [113], to several hours [13]. In the case of HL60 cells, Wang and Studzinski [13] found that the period of ERK2 activation following exposure to 1,25(OH)2D3 corresponded to the time that these cells continued to proliferate before 1,25(OH)2D3induced cell cycle block became apparent. Similarly, Gniadecki [114] reported that in normal human keratinocytes 1,25(OH)2D3 stimulated the activity of MAPKs and DNA synthesis. Together with the finding by Song et al. [113] of MAPK activation by 1,25(OH)2D3 in NB4 leukemia cells in which 1,25(OH)2D3, when administered alone, does not induce differentiation, these observations raise the question regarding the nature of the association between ERK activation and 1,25(OH)2D3induced differentiation. It is possible that ERK facilitates the early, proliferative phase of differentiation [9,11,12,117], thus increasing the numbers of differentiated cells, rather than being related to the expression of the differentiated phenotype. This is consistent with the observation that the MEK/ERK inhibitor PD98059 substantially reduces, but does not totally prevent, 1,25(OH)2D3-induced differentiation (e.g., [13]), and the report that transformation of immortalized keratinocytes with Ha-Ras oncogene, an upstream regulator of the MEK/ERK pathway, results in resistance to the anti-proliferative action of 1,25(OH)2D3 [118]. The stress and proinflammatory cytokine-activated p38 MAPK has a complex relationship to 1,25(OH)2D3induced differentiation [97,119e122], as well as to apoptosis (e.g., [123]). It has been reported that p38mediated signals are necessary for induction of osteoclast differentiation, but not for osteoclast function [122], and that in prostate cancer cells [124], and in keratinocytes 1,25(OH)2D3 inhibits its activation [121]. In contrast, in HL60 cells 1,25(OH)2D3 and analogs activate p38, though inhibition of its activity by specific inhibitors (SB202190 or SB203580) actually increases differentiation [69,97,119,120,125]. It was postulated that this paradoxical effect is due to the presence of a negative feedback mechanism that regulates several MAPK pathways (Fig. 84.1), and this explanation is consistent with the finding of increased JNK pathway activity, and ERK activation, as well as enhanced differentiation, in HL60 cells treated with the p38 MAPK inhibitors SB202190 or SB 203580 and 1,25(OH)2D3 [119,120,126]. The JNK MAPK pathway activation by 1,25(OH)2D3 in general has a positive effect on differentiation. In
addition to HL60 cells [68,119,120,127], this pathway has been shown to be involved in stimulation of CaCo2 cell differentiation [115], and together with p38, in sensitization of human breast cancer cells MCF-7 to 1,25(OH)2D3-induced growth inhibition [128]. In contrast to ERK MAPK pathway activation which is transient, the JNK pathway activity increases more slowly after an exposure to 1,25(OH)2D3, and in HL60 cells parallels the growth-inhibitory effects of 1,25(OH)2D3 [68]. Thus, in HL60 cells, enhanced JNK activity is a feature of late stages of monocytic differentiation, and perhaps is responsible for its maintenance. The Cdk5/p35 Pathway The pathways described above have been implicated in 1,25(OH)2D3-induced differentiation by showing that their inhibition by pharmacological agents and dominant-negative or antisense constructs diminishes differentiation, suggesting that they are necessary for, or contributory to, signaling of differentiation. Whether the enhanced activity of any of these pathways is sufficient to induce the differentiated phenotype has not been unequivocally shown. One exception is the demonstration that transfection of a cyclin-dependent kinase 5 (Cdk5)-expressing plasmid can lead to the expression of markers of early monocytic differentiation (CD14 and NSE) if a cyclin-like protein p35Nck5a (p35), the activator of Cdk5, is present [129]. Since the expression of both Cdk5 and p35 is upregulated by 1,25(OH)2D3 [129e132], this provides a mechanism by which 1,25(OH)2D3 can signal the early stages of monocytic differentiation. A confirmation of this role of the Cdk5/p35 module was obtained by finding that monocytes in p35 knockout mice were deficient in NSE, a monocyte-specific esterase used as a cell-specific marker [133]. The role of the ERK pathways as the upstream regulators of Cdk5/p35 was first studied by Harada et al. [134] in differentiating neurons. The authors reported that the nerve-growth-factor-induced differentiation in these cells was associated with a strong, sustained expression of p35 through activation of the ERK pathway, and that constitutive activation of ERK was necessary and sufficient for p35 induction. Furthermore, the mechanism by which activation of ERK induces expression of p35 involves the transcription factor Egr1, which was in these experiments induced by nerve growth factor through the ERK pathway and appeared to mediate the induction of p35 by ERK [134]. In leukemia cells induced to differentiate by 1,25(OH)2D3, Egr1 is upregulated and promotes p35 gene transcription [135]. The Cdk5/p35 complex can phosphorylate MEK1 on Thr286, which is an inhibitory phosphorylation. This mechanism can contribute to inhibition of cell proliferation by 1,25(OH)2D3, and most likely involves p27/Kip1, which in this situation
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accumulates in the cell nucleus and arrests the cells in the G1/S phase [135].
[67,119,141,151]. Thus, AP-1 transcription factor appears to be an important integrator of converging differentiation pathways.
Role of General Transcription Factors in 1,25(OH)2D3-induced Differentiation
Sp1 Transcription Factor The Spl, a 95e105 kDa protein, is ubiquitously expressed in growing cells, and, usually in combination with other factors, acts as a transcriptional activator of many housekeeping genes [152e156]. Its role in 1,25(OH)2D3-induced differentiation has been suggested in human myeloid leukemia cells. Specifically, the DNA binding of Spl was found to be increased following an exposure of HL60 cells to 1,25(OH)2D3 [10,146,157], while in U937 cells Spl may participate in the 1,25(OH)2D3-induced expression of CD14 monocyte marker, which has several Spl sites in its promoter [158,159], as well as the CD11b promoter [160]. It was also shown that upregulation of p27/Kipl in 1,25(OH)2D3-treated U937 [161] and LNCaP cells [137] can be mediated by Spl, as discussed below.
In addition to directly activating VDR to heterodimerize with a member of RXR family, which results in transcription of VDRE-containing genes, several ubiquitous transcription factors appear to be involved, perhaps in a contributory way, in 1,25(OH)2D3-induced differentiation. These may act by interacting with the adjacent VDREs [136], by upregulating VDR expression through its promoter region (e.g., [128]), by complexing with other transcription factors (e.g., [137]) and in other ways, many of which remain to be elucidated. Activator Protein-1 (AP-1) ERK and JNK pathways activate members of the Fos and Jun families, as well as some related proteins, which dimerize in various combinations to form the AP-1 transcription factor [138,139]. Thus, AP-1 can integrate and transmit signals transduced by the MAPK pathways previously discussed. Several studies have shown, for instance, that the expression of c-Jun is increased during the 1,25(OH)2D3-induced differentiation of human myeloid cells [68,127,140,141], and coordinate occupancy of AP-1 sites and VDRE elements by their cognate transcription factors provides a possible model for the reciprocal relationships between different cellular phenotypes and functional activities such as those that occur during differentiation [142,143]. For example, a composite AP-1 steroid hormone element that responds to 1,25(OH)2D3 mediates differentiationspecific gene expression of human keratin-1 [144]. There is also evidence for functional cooperation between VDR and Ras-activated Ets transcription factors in 1,25(OH)2D3-mediated induction of CYP24 gene expression [145]. Liu and Freedman [136] conducted an extensive study of such transcriptional synergism between VDR and non-receptor transcription factors, and concluded that the functional basis for such synergism appears to be at the level of cooperative DNA binding. AP-1 activation by 1,25(OH)2D3 has been described in diverse differentiation systems. These include HL60 cells [141,146], colon cancer CaCo-2 cells [115], osteoblastic cells [147,148], keratinocytes [149,150], and breast cancer cells [128]. Interestingly, the pathways that signal AP-1 activation are apparently cell-type-specific. For instance, the p38 and JNK MAPK pathways cooperate to activate VDR by c-Jun/AP-1 in breast cancer cells [128], while in keratinocytes and HL60 leukemia cells AP-1 activation is attributed to both the ERK and JNK pathways in the presence of plant antioxidants
Other Transcription Factors Undoubtedly, many other transcription factors, including the CAAT Enhancer Binding Protein b (C/ EBPb), contribute to 1,25(OH)2D3-induced differentiation [102,162,163]. It has been reported that C/EBPb isoforms are activated by phosphorylation by ERK [164] and by RSK [165], and interacts directly with the promoter of one of the principal markers of monocytic differentiation e CD14 [166]. In myeloid leukemia cells, hematopoietic progenitors are unable to undergo granulocytic differentiation, as a result of various mutations, which alter the proper balance of transcription factor activity. Elevated expression of C/EBPb induced by 1,25(OH)2D3, allows the cells to bypass this block by switching the lineage of differentiation to monocytelike cells instead of granulocytes [102]. Other transcription factors may also contribute to the eventual cell cycle arrest and some of these, notably cMyc, will be discussed relative to cell cycle control. The important distinction between the transcription factors which regulate the expression of new genetic programs, and those which control functions of the differentiated cells, is not easy to make at present.
CELL CYCLE CONSEQUENCES OF VITAMIN-D-INDUCED DIFFERENTIATION General Features of Cell Cycle Machinery Cell Cycle Compartments and Checkpoints The consecutive progression through four distinct phases of the cell cycle called G1, S, G2, and M results
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Terminal differentiation
Terminal differentiation
Polyploidy
Licensing factors
p130Rb2- E2F complex G0
Cdk1-Cyc B
M p21Cip1 p27Kip1
G2
pRb-P < pRb/E2F
G1
Cdk4/6-Cyc D Cdk2-Cyc A/E S
c-myc
Cdk2-Cyc E R
Post-RC ORC
pRb-P > pRb + E2F
Pre-RC ORC Cdc 6/18 MCM Cdt1
FIGURE 84.2 The general concept of the cell cycle, and examples of factors that control DNA replication and the cell cycle traverse. The principal locus for terminal differentiation is in G0 phase, but can occur in G2, with polyploidy. In early G1 the level of phosphorylated retinoblastoma protein (P-pRb) is less (<) than the level of hypophosphorylated pRb protein complexed with transcription factor E2F (pRb/E2F), while in late G1 the level of P-pRb is high, and it is not complexed with E2F. This and the increasing Cdk2/cyclin E activity allow passage through the Restriction point (R). Additional details of the controls of cell cycle progression are provided in the text and illustrated in Figs 84.3e84.7. RC, replicative complex; ORC, origin recognition complex; MCM, mini-chromosome maintenance complex; licensing factors, ORC, MCM, and other components of the pre-RC complex; Cyc, cyclin.
in proliferation of eukaryotic cells (Fig. 84.2). DNA replication occurs during the S phase; chromosome separation (karyokinesis) takes place during the M phase, and is followed by cell division (cytokinesis); G1 and G2 are gap or growth phases. The G1 phase can be further subdivided into early G1, or post-mitotic G1, mid-G1, in which principal cell growth takes place, and late G1, in which final preparations for DNA replication occur. The G2 phase is thought to be necessary for monitoring of chromosome replication and preparations for mitotic spindle assembly [167e169]. Cells that are not actively dividing may either be permanently removed from these cycling phases by terminal differentiation, senescence or apoptosis, or be temporarily arrested in a non-cycling quiescent state known as G0 if the cells have the G1 DNA content, though quiescence can also occasionally take place in the G2 phase (G2 arrest). As mentioned above, specific nuances have been described, but the remarkable feature of the cell cycle is the conservation, from yeast to mammalian cells, of the basic regulatory mechanisms and components. A series of regulatory steps, referred to as checkpoints, control the traverse of the various compartments of the cell cycle [170,171]. A simple definition of a checkpoint is that it is a mechanism that prevents progression to the next part of the cell cycle unless and until the
preceding part has been satisfactorily completed. Such checkpoints operate in each phase of the cell cycle, and although the precise mechanisms are in most cases unclear, DNA damage is known to activate two protein kinases, Chk1 and Chk2, which then mediate cell cycle arrest [172e174]. In addition, several regulators downstream from Chk1 or Chk2, such as p53, have been shown to have importance in checkpoint control. Interestingly, these regulators of cell cycle progression may also influence cellular decisions to differentiate [174,175]. The forerunner of checkpoints was described as the restriction (R) point in mid to late G1 phase. Based on work in his own and in other laboratories, Pardee defined a transition in G1 phase which commits a cell to initiate DNA replication [167]. Additional work indicated that phosphorylation of the retinoblastoma susceptibility protein (pRb) may provide the principal mechanism for the transition through the R point [176e178]. Subsequent passage through the S phase can also be controlled by the S phase checkpoints (e.g., [179]). The final result of the cell cycle traverse is a faithful replication and accurate partitioning of genetic information. The fidelity of this partitioning is maintained by the G2 and the M phase checkpoints. The G2 phase checkpoints monitor the integrity of DNA and the accuracy of DNA replication, and the M phase checkpoints ensure correct chromosomal segregation and alignment. Each of these checkpoints arrests cell cycle progress to allow editing and repair of genetic information. Overall, the checkpoints assure that each daughter cell receives a full complement of genetic information intact and identical to the parental cell. Mechanisms that Drive Cell Cycle Progression Passage through the restriction points is propelled by the activity of a group of enzymes known as cyclindependent kinases (Cdks). Cdks are usually present throughout the cell cycle, and work in concert with cyclins, which are nuclear proteins whose levels oscillate in a cell-cycle-dependent manner [180,181]. In general, there are at least nine levels at which the activity of Cdks can be controlled, as detailed in Figure 84.3. The primary regulator of Cdks activity is cyclin binding, because Cdks and cyclins need to form a complex prior to activation. Since the protein levels of the cyclins dramatically change during the cell cycle, the binding of Cdks and cyclins is related to cyclin availability. The abundance of cyclins, like all proteins, is dependent on the balance between their synthesis and their degradation, the latter occurring by ubiquitindependent proteolysis [182]. The activity of the cyclineCdk complexes also depends on both activating and inhibitory
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The central paradigm for the control of the cycle traverse by cyclin-dependent kinases (Cdks). Cdks activity is primarily activated by cyclin binding, phosphorylation of Thr160 by cyclin activating kinase (CAK), and dephosphorylation of Thr14 and Tyr15 by Cdc25 phosphatases. Phosphorylation of Thr14 and Tyr15 by Wee1 and Mik1 kinases as well as binding of Cdk inhibitors (CDKIs) results in Cdk inhibition. In this figure, the centrally placed canonical Cdk/cyclin complex is the driving force for cell cycle progression.
FIGURE 84.3
Degradation
2
CAK
3
CDK7 MAT1
Cyclin H
Cyclin
CDK
CDKI
4
P
9
T 160
CDKI
CDK
1
Y 15
P 7 5
6
Wee1 Mik1
Cyclin
T 14
Cyclin
Chk1/2
CDK
CDC25
8
T 160
CDK
T 14 Y 15
P P
Cyclin
Degradation
phosphorylations. A known kinase, which can phosphorylate Cdks is cyclin activating kinase (CAK). CAK is comprised of cyclin H (regulatory subunit), Cdk7 (catalytic subunit), and MAT1 (assembly factor) [183,184]. This complex phosphorylates the threonines (Thr) 160/161 on Cdk1 (formerly Cdc2) and Cdk2, respectively [185]. Phosphorylation of this site is necessary for the cyclineCdk complex to be activated. Cdk inhibitory phosphorylation sites include Thr14 and Tyr15. Regulation of these sites is achieved by a group of proteins that include Cdc25A, B, C (phosphatases), as well as wee1 and mik1 (kinases). The phosphatases activate the cell cycle by cleaving the phosphate groups on Thr14 and Tyr15 residues of the Cdk, while the kinases are inhibitory by phosphorylating the same sites. Cdc25A is involved in the G1/S checkpoint [186], while Cdc25B and C, and human wee1-like kinase, regulate the traverse through the G2/M phases [187e189]. If DNA damage occurs prior to or in the S phase, Chk2 phosphorylates Cdc25A and targets it for degradation, while G2/M transition is regulated by Chk1 and Chk2 by phosphorylation of Cdc25C at Ser216 [190]. Another level of Cdk regulation is provided by the Cdk inhibitory proteins (CDKIs), which prevent Cdk activation, generally by binding to the kinases, and thus preventing their activation by cyclins, as described below.
(Fig. 84.4). Specifically, cyclin D-, E-, and A-dependent kinases are negatively regulated by a family of CDKIs that consists of p21/Cip1, p27/Kip1, and p57/Kip2. Although all three of these inhibitors block progression through the G1 phase, each is usually activated by different stimuli. The expression of p21/Cip1 can be
The G1 to S Phase Transition While the cyclin DeCdk 4/6 and cyclin EeCdk2 complexes control the entry into the S phase, these complexes are in turn controlled by families of G1/S regulatory polypeptides, the CDKIs [180,191]
Other kinases p27Kip1
p21Cip1
pRb-E2F INK4
CDK4,6
CDK2
Cyclin D
Cyclin E P
pRb + E2F
c-Myc
G1
ODC; Cdc25A; Cyclins D, E, A
S
Control of G1 to S phase transition by the pRbeE2F pathway. Phosphorylation of pRb by active Cdks releases E2F transcription factors, which activate, directly or indirectly, genes whose products are required for DNA replication [211,213]. The activity of Cdks can be controlled by factors shown in Fig. 84.3, and these include CDKIs belonging to the INK4 and Cip/Kip families. ODC, ornithine decarboxylase. Only a few examples of over 1600 known c-Myc target genes are shown here; this growing list can be accessed at www. myccancergene.org.
FIGURE 84.4
Regulation of Cell Cycle Progression
Other signals
p53
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1,25(OH)2D3
Mitogenic stimuli
Cdk2, ERK (?), Other kinases
Akt
FOXO TFs
P
miR-181a
+ G1/S arrest
p27Kip1 Skp2
p27Kip1 3
14.3.
Transcriptional and post-transcriptional regulation of p27/Kip1 expression during G1/S progression. Proteasome-dependent protein degradation is the main form of regulation of the p27/Kip1 expression in most mammalian cells. In proliferating cells, p27/Kip1 is phosphorylated by several kinases (originally proposed to be Cdk2 and ERKs [198]), then ubiquitinated by Skp2 [296], which leads to its degradation. It can be also destabilized by phosphorylation on non-Cdk sites [300e302] or by cytoplasmic localization through binding to proteins such as 14-3-3 [451]. Additionally, in myeloid cells translation of p27/Kip1 mRNA can be inhibited by miR181a, resulting in G1/S block [21,303]. Conversely, upregulation of p27/Kip1 expression can be transcriptionally activated by the forkhead transcription factors (TFs), such as AFX (FOXO4) [204,205]. In myeloid cells, 1,25(OH)2D3 reduces the level of miR181a, and leads to an increase in p27/Kip1 level resulting in G1/S arrest [21].
FIGURE 84.5
under the transcriptional control of the p53 tumor suppressor gene, activated by DNA damage [58,192], but may also be independent of p53 [193,194]. The increase in p21/Cip1 protein levels may lead to the inhibition of cyclin D-Cdk4/6 activity, which contribute to the G1 arrest. A second mechanism of action of p21/ Cip1 may be related to its ability to bind to the proliferating cell nuclear antigen (PCNA), a molecule involved in DNA replication and repair [195,196]. Like p21/Cip1, p27/Kip1 inhibits the activity of the G1/S cyclineCdk complexes. For instance, p27/Kip1 participates in G1 arrest produced by the exposure of fibroblasts derived from mink lung to the transforming growth factor b (TGFb), and by cellecell contact [197]. There are multiple levels of control of p27/Kip1 abundance in mammalian cells (Figs 84.4 and 84.5). In actively dividing cells, p27/Kip1 is phosphorylated by cyclin EeCdk2 complex in the nucleus [198], and its abundance can then be reduced by ubiquitin-mediated degradation in the proteosomal system, regulated by p45/Skp2 [199,200]. The stability of p27/Kip1 can also be regulated by Cdk2 independent processes, such as the destabilization by phosphorylation on non-Cdk sites, or by cytoplasmic localization by binding to proteins such as 14-3-3 [201e203]. The forkhead family
of transcription factors upregulate transcriptional expression of p27/Kip1 through FOXO binding sites in the p27/Kip1 promoter [204,205], but the serineethreonine kinase Akt can phosphorylate and thus turn off the FOXO factors [206,207]. Vitamin-D-derived compounds can influence several of these control points, as discussed below. Another family of regulatory peptides are the INK4 proteins, which include p16(INK4A), p15(INK4B), p18 (INK4C), and p19(INK4D). These proteins specifically block cyclin D-Cdk 4/6 activity leading to a G1 phase arrest (e.g., [208]). The INK4 proteins inhibit Cdk4 and 6 by preventing the binding of cyclin D, but also inhibit the activation of the formed Cdk 4/6ecyclin D complexes. Treatment of human keratinocytes with TGF-b results in an increase in p15(INK4B) expression and its association with Cdk4 and 6 [209]. While there is a large body of evidence showing the regulation of Cip/Kip CDKIs by 1,25(OH)2D3 and its analogs (see below), the control of INK proteins by vitamin D derivatives has not been established. An important event for the G1eS transition appears to be the phosphorylation of the tumor suppressor, pRb, as shown in Figure 84.4 [210,211]. pRb, and other pRb-like “pocket” proteins (p130/Rb2, p107), are
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believed to control the entry into the S phase by interacting with a member of the E2F transcription factors family. This family is composed of at least five proteins (E2F 1e5) which are active when they form heterodimeric complexes with one of the E2F-related transcription factors, DP-1, 2, or 3. In its simplest form, the current hypothesis is that hypophosphorylated pRb binds to E2F, preventing cell entry into the S phase. Upon increased level of phosphorylation, pRb frees E2F, which in its heterodimeric complex with DP is capable of activating genes necessary for S phase initiation in part mediated by c-Myc/max transcription factor [212e216]. However, the actual situation appears to be more complicated, since gene repression by pRb also involves modulation of chromatin architecture [217,218]. The proposed mechanism rests on the finding that histone deacetylase HDAC1 physically interacts with pRb through the “pocket” domain, and recruits HDAC1 to E2F. The complex of these three proteins binds to E2F target promoters. HDAC1 may then facilitate the removal of highly charged acetyl groups from core histones, leading to a tight association between the nucleosomes, which prevent the access of transcription factors to their cognate elements in the gene promoters (e.g., [208]). Interestingly, recent studies have shown an extensive evolutionary conservation of pRb repressor complexes that contain specific sets of E2F targets as well as Myb-interacting proteins [219]. In this context, in Drosophila pRb represses differentiation-specific genes by a mechanism of repression that differs from that of cell-cycle-regulated genes [220]. Mitogenic signals (e.g., growth factors, serum compounds) that stimulate cell progress through G1 coincide with an increased expression of cyclin D. This extracellular regulation of cyclin D isoforms is not observed with other cyclin proteins. The current classical scenario is that cyclin D then forms a complex with Cdk4 and/or Cdk6, and its activity is regulated by the mechanisms described above. The activated cyclin DeCdk 4/6 complexes then phosphorylate pRb and release E2F, or relieve the chromatin configuration constraints described above, leading to G1 progression. The cyclin D complex alone does not dictate control of progression through G1. Cyclin E is another G1 cyclin, which is synthesized later in the cell cycle than cyclin D, peaking at the late G1/S phase boundary. The expression of cyclin E is mitogen independent, and cyclin E forms an active complex with Cdk2. One level of regulation for the cyclin EeCdk2 complex is through protein phosphatase Cdc25A which cleaves the phosphate groups on the Thr14 and Tyr15 residues of Cdk2, and activates the Cdk2ecyclin E complex [221]. Cdc25A activity is in turn regulated by phosphorylation by Chk1 [222]. c-Myc has been shown to regulate the
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expression and phosphorylation, and therefore the activity, of Cdc25A [223]. Cyclin DeCdk4 and cyclin DeCdk6 complexes are believed to trigger pRb phosphorylation, but cyclin EeCdk2 complex also contributes to the phosphorylation of pRb in late G1, leading to cell entry into the S phase, where pRb phosphorylation is maintained by cyclin EeCdk2 or cyclin AeCdk2. This is illustrated in outline in Figure 84.4. Control of DNA Replication Licensing The S phase is the period where DNA replication takes place, and the basic machinery for this process is well conserved from yeast to mammals [224]. It is permitted by the rising level of Cdk activity, and is initiated on many sites on chromosomes, designated as “replication origins,” which can exist in two states (e.g., [225,226]). In G1 phase a multiprotein complex, the pre-replicative complex (pre-RC) is assembled, but once DNA replication is initiated, the complex has fewer components (post-RC), which persists to the end of mitosis, and does not permit re-replication of DNA. At that time point proteolytic activity destroys the cyclins and other nuclear proteins, and Cdk activity becomes low. These two states of replication origins, separated by Cdk activity, generally ensure that the S phase and mitosis alternate. When chromatin becomes competent for DNA replication by the presence of pre-RC on the replication origin, it is considered to be “licensed.” Components of the pre-RC, the “initiator” proteins, include the Origin Recognition Complex (ORC), Cdc6/18, Cdt1, and MiniChromosome Maintenance 2-7 (MCM 2-7) proteins. Although their DNA consensus sequences have not been found in organisms other than yeast, ORC proteins have been found in several eukaryotes, and all have ATP binding sites, consistent with ATP requirement for the initiation of DNA replication [227,228]. Cdc6/18 mammalian homologs may be related to M checkpoint control [229], while Cdt1 (Cdc10-dependent transcript 1), which also has peak protein levels at G1/S boundary [230], is associated with DNA replication checkpoint control. The six MCM proteins form a hexameric complex, approximately 600 kDa in size, which may function as a replicative helicase [231]. Importantly, Cdt1 binds tightly to a DNA replication initiation inhibitor (Geminin) and this inhibits MCM loading [232]. Licensing in G1 phase is permitted after the end of mitosis, when Geminin is destroyed by the Anaphase Promoting Complex (APC)eubiquitin system [233]. In yeast, the ORC remains associated with the replication origins throughout the cell cycle, while in mammalian cells chromatineORC interactions appear more dynamic with ORC subunits cycling on and off replication initiation sites [234,235]. When the cells exit mitosis Cdc6/18 and Cdt1 are loaded on chromatin, and in turn
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84. VITAMIN D EFFECTS ON DIFFERENTIATION AND CELL CYCLE
aid loading of MCM on the pre-RC complex, thus completing licensing (e.g., [230]). The licensed complex can now be activated for DNA replication by a protein kinase, such as cyclin E/Cdk2 or Dbf4-dependent kinase (DDK) [236], and the DNA replicating machinery (e.g., Cdc45, replication protein A (RPA), DNA polymerase a and e) is recruited to the initiation sites [237e240]. To further facilitate replication, a SCFeubiquitination complex, which destroys Cdk inhibitors, can be recruited to the pre-RC by a cyclin-binding site on Cdc6/18 [241]. The G2 and M Phase Transition Once the cell has faithfully replicated its genome, the next cellular function is to segregate this DNA into equivalent, or nearly equivalent, daughter cells. The central regulation for the transition from G2 to mitosis is by the cyclin BeCdk1 complex, initially called maturation (mitosis) promoting factor, or MPF [242]. In general, the activity of this complex is governed by factors similar to those responsible for the G1eS transition, including cyclineCdk association and phosphorylation by Cdk-activating kinase (CAK) (Fig. 84.6). The CDKIs were not known to play a major role in the control of the G2/M traverse, but studies in Reed’s laboratory indicate that the situation is more complex than previously believed [243,244]. Regulation of the cyclin
Wee1
Mik1
Cdc25C Activating phosphatase
Inactivating kinases
CAK Chk2 Plk1
Activating kinase
G2
Y15
T161
T14
Cdk1 Cyclin B
M
Ubiquitination Control of G2 to M phase transition and the completion of mitosis. Cyclin BeCdk1 complex is a central regulator of the transition from G2 to M. The activity of this complex is governed by factors similar to those responsible for the G1eS transition (see Fig. 84.4), including Cdkecyclin association, activating phosphorylation of Thr161 as well as phosphorylation/dephosphorylation of Thr14 and Tyr15. See text for additional details. CAK, cyclin activating kinase.
FIGURE 84.6
BeCdk1 complex includes the G2/M specific phosphatase/kinase cell cycle regulatory proteins. The three Cdc25 isoforms A, B, and C are protein phosphatases, which cleave the inhibitory phosphate groups at both Tyr15 and Thr14 on Cdk1, and are required for timely assembly of the cyclin BeCdk1 complex [245e247]. Cdc25C itself requires phosphorylation to be activated, and recent data support that Cdc25C is phosphorylated and activated by the cyclin BeCdk1 complex, thus forming a positive feedback loop [248]. On the other hand, wee1-like tyrosine kinase phosphorylates these same sites, and thus acts as an inhibitor of the progression into mitosis [249]. Conversely, mitotic exit can be fine tuned by such inhibitory phosphorylations of Cdk1, perhaps by wee1 and myt1, thus maintaining the cell in the G1 phase [250]. In addition to the accurate duplication of the genetic material, cell cycle controls ensure its correct segregation into the daughter cells. This occurs at two levels: regulation of proteins that bind together the two chromatids, and control of centrosome duplication and spindle assembly. The sister chromatids adhere to one another by the adhesive properties of a multi-subunit complex composed of several proteins, so far best characterized in yeast, collectively called “cohesin” [251,252]. Cohesin is dissolved by proteolytic cleavage of one of its subunits, Scc1/Med1, by a calcium-activated cysteine protease, related to caspases, known as “separase” [253,254]. In animal cells the dissolution of cohesion between the chromatids occurs in two steps, one at prophase, the other at anaphase, and only the latter requires separase [255]. Interestingly, separase is subjected to multiple levels of regulation. These include its phosphorylation by cyclin BeCdk1 [256], and the inhibition of separase catalytic activity by securin [257]. Since both cyclin B and securin are ubiquitinated by the anaphase promoting complex (APC) and destroyed at the end of mitosis, this ensures orderly and precisely timed separation of the chromosomes at telophase. The precise timing of entry into the M-phase may depend on the co-competing levels of the APC substrates, cyclin B and securin [258]. Polo-like kinase (Plk1) also regulates chromosome adhesion and other aspects of mitosis, including centrosome maturation and orientation [259]. Plk1 has been reported to phosphorylate cyclin B1 and target it to the nucleus during prophase [260], and the finding of co-localization of Plk1 and Chk2 suggests that there is a lateral communication between the mitotic checkpoint and the DNA integrity checkpoint [261]. The changes in cell cycle traverse and DNA replication that occur in numerous forms of differentiation have been previously reviewed [8]. Changes that
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specifically follow exposure to vitamin D derivatives have been less extensively studied, and with this background these will now be described.
Modulation of Cell Cycle Events by Vitamin D and Its Analogs The inhibition of cell cycle traverse by 1,25(OH)2D3 and analogs has been investigated in normal and malignant keratinocytes [262,263], and in many other types of tumor cells, with myeloid leukemia providing an excellent in vitro model system for this purpose [30,264]. The G1/S block Vitamin D and its analogs inhibit proliferation of diverse types of mammalian cells by arresting them in the G1/G0 phase of the cell cycle. While the exact sequence of events from VDR activation to G1/G0 arrest remains to be elucidated, and may not be exactly the same in all cell types, several pathways and cell cycle arrest effectors are already known to be involved. These are the upregulation of protein levels of the Cdk inhibitors p21/Cip1 and/or p27/Kip1, upregulation of the Rb gene expression and phosphorylation of pRb protein, and the inhibition of c-Myc expression (e.g., [97,117,265e268]). UPREGULATION OF p21/Cip1 and p27/Kip1
Elevated protein levels of the Cip/Kip family of CDKIs result from the exposure to 1,25(OH)2D3 and other analogs in many cell types (Table 84.2), and may be a near-universal feature of the anti-proliferative effects of these compounds. In view of the importance of Cdk activity as the driving force for cell cycle progression, it is not difficult to understand that increased levels of CDKIs can target Cdk2 complexed to cyclin D, E, or A, and grind the cell cycle traverse to a halt. However, the mechanisms of the CDKI upregulation are not entirely clear, and there are subtle differences between the antiproliferative effects of p21/Cip1 and p27/Kip1. In contrast, p57/Kip2 was seldom found to have a role in the anti-proliferative effects of vitamin D and its analogs, apart from the possible involvement in the transit of osteoblasts from proliferation to differentiation [269]. Considerable excitement was generated when p21/ Cip1 was found to be upregulated in a number of differentiation systems, including HL60 cells and SCC cells treated with 1,25(OH)2D3 [59,193,270] (see also Table 84.2). It was suggested that p21/Cip1, and/or p27/Kip1, not only promote the G1 arrest but also contribute to differentiation (e.g., [271]). It seems, however, that while these Cdk inhibitors may not be
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solely responsible for the G1 block, the data regarding their role in differentiation are conflicting. For instance, mice lacking p21/Cip1 undergo normal development [272], even though p21/Cip1/ embryonic fibroblasts show impaired arrest in G1 in response to DNA damage. An imbalance between growth and differentiation can be demonstrated in these cells, and in other in vitro cell differentiation systems with p21/Cip1 knockouts. Keratinocytes which are p21/Cip1/, and to a lesser extent those with p27/Kip1 knockouts, have an increased proliferative potential [273]. With regard to differentiation, however, Harvat et al. [274] showed that growth arrest resulting from overexpression of p21/Cip1 in mouse primary keratinocytes is not sufficient to induce the expression of markers of differentiation. Further, in malignant counterparts of these cells, the SCC cells, not even growth arrest is clearly linked to p21/Cip1, as 1,25(OH)2D3 inhibited growth but reduced p21/Cip1 levels in vitro and in SCC tumors [275]. In another system, the myelomonocytic cell line U937, Freedman’s group noted transcriptional activation of the p21/Cip1 gene by 1,25(OH)2D3, and suggested that this is linked to differentiation of these leukemia cells [57]. Importantly, they identified a functional vitamin D response element (VDRE) in the promoter of the p21/ Cip1 gene, and noted that the p21/Cip1 transcript can be detected as early as 2 h after 1,25(OH)2D3 addition, consistent with p21/Cip1 being a direct mediator of 1,25(OH)2D3 action. However, in this system the upregulation of p21/Cip1 after exposure to 1,25(OH)2D3 is transient and accompanied by a proliferative burst [14], which does not correlate with the onset of the G1 block that is observed 24e48 h later, and is accompanied by markedly increased levels of p27/Kip1 [57,117,267]. Thus, although p21/Cip1 may initiate a cascade of unknown events that lead to the expression of the differentiated monocytic phenotype, it is unlikely to be directly responsible for the G1 arrest in leukemia, or SCC cells. Such function, however, has been attributed to p21/Cip1 in other cells, including prostate cancer [276e278], breast cancer [279e281], and parathyroid cells [282]. The finding that p21/Cip1 binds to PCNA [195], the processive factor for DNA replication, suggests that this complex might play an important role in maintaining the integrity of the genome [283]. The first demonstration that upregulation of p27/ Kip1 is associated with G1 arrest which takes place following 1,25(OH)2D3-induced differentiation was reported by Wang et al. in 1996 [117]. They showed a sustained increase in p27/Kip1 protein abundance that coincided with the appearance of the 1,25(OH)2D3induced G1 block in HL60 cells, and correlated with reduced kinase activity of Cdk6 and Cdk2 [117,267]. Further, reductions of the levels of p27/Kip1 by several
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TABLE 84.2
Examples of Upregulation of CDKI Levels by 1,25(OH)2D3 and Analogs
CDKI
Cell type
Functional effect/Comment
References
HEMATOPOIETIC CELLS p21/Cip1
HL60
Immediate early gene induced during monocytic differentiation/G1 arrest
[193,270,419,420]
p21/Cip1
U937
Transcriptional activation of p21/Induces monocytic differentiation
[57]
p21/Cip1
U937
Cytoplasmic localization/Anti-apoptotic effect
[421]
p21/Cip1
U937
Antisense to p21 decreases differentiation
[422]
p27/Kip1
HL60
Proliferation block/G1 arrest
[117,267,372,423]
p27/Kip1
HL60
Antisense to p27 reverses G1 arrest
[264]
p27/Kip1
HL60
miR-181a abrogates p27 expression and inhibits G1 arrest and differentiation
[21]
p27/Kip1
U937
Proliferation block
[372]
p27/Kip1
U937
Vitamin D receptor-independent upregulation of p27 gene
[161]
p27/Kip1
UF-1
Stabilization of p27 protein
[297]
p27/Kip1
B cells, primary
Inhibition of proliferation and differentiation of activated B cells/Apoptosis
[424]
p21/p27
HL60
Little change in CDK4 activity/G1 arrest
[425]
p21/p27
U937
Early proliferative burst followed by growth arrest and differentiation
[14]
p21/p27
UF-1
Granulocytic differentiation/Proliferation block/G1 arrest
[287]
p21/p27
HL60, AML3 Expression of CDKIs is not regulated via p53
[426]
& MOLM-13 PROSTATE CANCER CELLS p21/Cip1
LNCaP
Proliferation block/G1 arrest
[276e278]
p21/Cip1
ALVA-31
Proliferation block/G1 arrest
[278]
p21/Cip1
PC-3
Proliferation block/Differentiation
[388]
p21/Cip1
DU-145
Proliferation block
[388]
p27/Kip1
LNCaP
Increased association of p27 with Cdk2/Stabilization of p27 protein/G1 arrest
[427,428]
p27/Kip1
LNCaP
Induction of p27 expression requires VDR/Sp1 interaction
[137]
p21/p27
LNCaP
Proliferation block/Differentiation
[388]
Transcriptional activation of p21/Proliferation block/G1 arrest/Apoptosis
[429e431]
BREAST CANCER CELLS p21/Cip1
MCF-7
p21/Cip1
MCF-7E
Proliferation block/G1 arrest
[279]
p21/Cip1
MCF7/LCC2
Proliferation block/G1 arrest/Transient upregulation of com1
[432]
p21/Cip1
MDA-MB453
Transcriptional activation and cyclical accumulation of p21
[433]
p21/Cip1
Tumor tissue
Suppression of ER-positive and ER-negative breast tumor growth in micea
[434]
p27/Kip1
MCF-7
Proliferation block/G1 arrest
[435]
p27/Kip1
SK-BR-3
Proliferation block
[435]
p21/p27
MCF-7
Proliferation block/G1 arrest/Downregulation of c-Myc
[280,281,394,436,437]
p21/p27
BT20
Proliferation block/G1 arrest
[279]
p21/p27
BT-474
Proliferation block/G1 arrest
[437]
p21/p27
ZR75
Proliferation block/G1 arrest
[279]
p21/p27
SUM-159PT
Proliferation block/Apoptosis
[438] (Continued)
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CELL CYCLE CONSEQUENCES OF VITAMIN-D-INDUCED DIFFERENTIATION
TABLE 84.2
Examples of Upregulation of CDKI Levels by 1,25(OH)2D3 and Analogsdcont’d
CDKI
Cell type
Functional effect/Comment
References
PANCREATIC CANCER CELLS p21/p27
BxPC-3
Early transient CDKI upregulation/Proliferation block/G1 arrest
[284,439]
p21/p27
Hs 700T
Early transient CDKI upregulation/Proliferation block/G1 arrest
[284,439]
p21/p27
SUP-1
Early transient CDKI upregulation/Proliferation block/G1 arrest
[284]
p21/p27
AsPC-1
Upregulation of CDKIs and proliferation block in vitro and in vivo
[439]
G1 arrest/Apoptosis
[440]
Stabilization of p27 protein/G1 arrest
[299]
INSULINOMA CELLS p21/Cip1
Beta TC(3)
HEPATOMA CELLS p27/Kip1
HepG2
COLON CANCER CELLS p21/Cip1
HT-29
Proliferation block/G1 arrest/Apoptosis
[441]
p21/p27
Caco-2
Co-association between Cdk2, p27Kip1 and cyclin E/Proliferation block
[442e444]
p21/p27
CBS
Differentiation
[445]
OSTEOSARCOMA CELLS p21/Cip1
SaOS-2
Proliferation block
[148]
p27/Kip1
MG-63
Increased Sp1þNF-Y binding to the p27 promoter/Proliferation block/G1 arrest
[446]
Stabilization of p27 protein
[297]
SQUAMOUS CELL CARCINOMA p27/Kip1
AT-84
p27/Kip1
SCC-VII/SF
Proliferation block/G1 arrest/Apoptosis
[94]
p27/Kip1
TDEC, primary
Proliferation block/G1 arrest/Apoptosis
[447]
b
THYROID FOLLICULAR CARCINOMA CELLS p27/Kip1
WRO, tumor tissue
Suppression of tumor growth and metastasis/Differentiation
[448]
OVARIAN CANCER CELLS p27/Kip1
2008, CAOV3
Proliferation block
[298]
p27/Kip1
OVCAR3
Stabilization of p27 protein/Proliferation block/G1 arrest
[298]
NEUROBLASTOMA CELLS p21/Cip1
SH-SY5Y
Downregulation of Myc and Id2/Induction of RARb/Proliferation block
[449,450]
p21/Cip1
NB69
Downregulation of Myc and Id2/Induction of RARb/Proliferation block
[449,450]
p21/Cip1
SK-N-AS
Downregulation of Myc/Induction of RARb/Proliferation block
[449,450]
p21/Cip1
IMR5
Downregulation of Myc and Id2/Induction of RARb/Proliferation block
[449,450]
p21/Cip1
CHP134
Downregulation of Id2/Induction of RARb/Proliferation block
[449,450]
p21/Cip1
NGP
Downregulation of Myc and Id2/Induction of RARb/Proliferation block
[449,450]
a
ER, estrogen receptor. b TDEC, tumor-derived endothelial cells.
independent approaches reversed the G1 block, but not the differentiated phenotype [264]. Accordingly, these data clearly show that, at least in HL60 cells, p27/Kip1 controls the 1,25(OH)2D3-induced G1 block, but not the differentiated phenotype.
Similar findings have been obtained in several other systems (Table 84.2), although the data cannot always be so clearly interpreted. For instance, the upregulation of p27/Kip1 is often accompanied by an upregulation of p21/Cip1 (e.g., [279,284e286]). However, even in
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situations where p21/Cip1 and p27/Kip1 are both upregulated by 1,25(OH)2D3 or its analogs, increased levels of p27/Kip1 correlate better with the onset of G1 block than the upregulation of p21/Cip1 (e.g., [57,117,275,287,288]). This, however, is subject to cell context, a striking example being a recent report that p27/Kip1 is essential for the anti-proliferative action of 1,25(OH)2D3 on primary, but not on immortalized, mouse embryonic fibroblasts [289]. The role of CDKIs in 1,25(OH)2D3-induced cell cycle arrest is also difficult to assess because, when present at relatively low levels, p21/Cip1 and p27/Kip1 serve to facilitate complex formation of cyclins D with Cdks, and their transport to the nucleus [290,291], and only high levels of CDKIs are inhibitory [290,292,293]. Thus, one possible explanation for the upregulation of p21/Cip1 which may not correlate with G1 arrest is that p21/Cip1 simply serves to facilitate cyclin DeCdk complex formation. More likely, however, is that elevated levels of p21/Cip1 inhibit cyclin EeCdk2 activity and therefore block cyclin EeCdk2 phosphorylation of p27/Kip1, which then leads to degradation of p27/Kip1 in proliferating cells [198,294,295]. Unlike p21/Cip1, which can be directly upregulated by 1,25(OH)2D3 through a VDRE in p21/Cip1 promoter [57,59], p27/Kip1 has no VDR-binding element in its promoter, and may be regulated in a cell-type and condition-specific manner by several mechanisms, which include both transcriptional and post-transcriptional levels as discussed above and illustrated in Figure 84.5. The known transcriptional regulation of p27/Kip1 expression includes at least two mechanisms that can be influenced by 1,25(OH)2D3, though in opposing ways. One proposal is that transcription factors Sp1 and NF-Y can synergistically mediate the 1,25(OH)2D3induced expression of p27/Kip1 by acting directly on the p27/Kip1 promoter, as shown in transiently transfected U937 leukemia cells [161]. In these experiments, deletion and mutational analysis revealed that p27/ Kip1 promoter activation required both GGGCGG (Spl binding) and CCAAT (NF-Y binding) sequences. Although p27/Kip1 promoter does not contain VDRE, Huang et al. [137] have shown that transfection of VDR in SW620 colon cancer cells, which express low level of endogenous VDR, enhances 1,25(OH)2D3induced p27/Kip1 promoter activation, and that VDR and Sp1 cooperate to activate p27/Kip expression in LNCaP prostate cancer cells. Particularly, it was demonstrated that VDR physically interacts with Sp1 to activate p27/Kip1 promoter via a GC-rich Sp1 site [137]. As presented above (see “Sp1 transcription factor”), Sp1 transcription factor is activated in 1,25(OH)2D3treated leukemia cells, thus, these data could potentially be a plausible mechanism for the induction of G1 arrest by 1,25(OH)2D3-induced p27/Kip1, at least in myeloid
leukemia cells. The second transcriptional mechanism that can be regulated by 1,25(OH)2D3 is based on the demonstration that in a number of cell types p27/Kip1 is transcriptionally activated by the forkhead transcription factors, such as AFX (FOXO4) [204,205], and Akt can phosphorylate and thus turn off the FOXO factors [206], allowing cell cycle progress. Interestingly, Akt can be upregulated by 1,25(OH)2D3 [70], and thus actually provide an opportunity for the cells to mitigate the signals that tend to upregulate p27/Kip1 expression. However, the dominant form of regulation of p27/ Kip1 expression by 1,25(OH)2D3 and analogs appears to be by proteasome-dependent protein degradation. For instance, in mouse SCC AT-84 cells, an analog, EB1089 did not change p27/Kip1 mRNA levels, but reduced the mRNAs for p45/Skp2, which ubiquitinates p27/Kip1, and for Cks1, which targets p45/Skp2 to p27/Kip1 [296,297]. A similar decrease in p45/Skp2 expression and stabilization of p27/Kip1 protein was demonstrated in acute promyelocytic leukemia cells [297], ovarian cancer cells [298], and human hepatoma cells [299]. Since these changes become evident at about 48 h of the exposure to the analog, there is good correlation with the onset of the G1 block. The latent period of 24e48 h for p27/Kip1 upregulation may also be needed to inactivate the cyclin EeCdk2 complex, which phosphorylates Thr187 of p27/Kip1 that under some conditions is required for ubiquitination of p27/Kip2 by p45/Skp2 [198]. Inhibition of cyclin EeCdk2 activity following exposure of HL60 cells to 1,25(OH)2D3 has been demonstrated [267], and this may contribute to 1,25(OH)2D3-induced increase in p27/Kip1 levels. However, p27/Kip1 can be phosphorylated on nonCdk sites [300e302], so this form of control may have redundancy. Recently, another form of regulation of p27/Kip1 was discovered, so far limited only to myeloid cells. Wang et al. found that the members of the microRNA 181 family can inhibit the expression of p27/Kip1, but exposure of the cells to 1,25(OH)2D3 reduces the levels of these microRNAs, and thus lead to an increase in the levels of p27/Kip1 and cell cycle arrest [21] (Fig. 84.5). Independently, this role of microRNA 181a was confirmed by Cuesta et al. in myeloid cells induced to macrophage differentiation by TPA [303]. It seems that such multiple controls by 1,25(OH)2D3 of p27/Kip1 expression signify a specially critical role of 1,25(OH)2D3 in cell cycle regulation. RETINOBLASTOMA PROTEIN CONTROL OF 1,25(OH)2D3INDUCED G1 BLOCK
The suggested placement of the inactivation of the cyclin EeCdk2 complex upstream of p27/Kip1 upregulation raises the question of how this complex is inactivated in 1,25(OH)2D3-treated cells. One possible
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1,25(OH)2D3 miR-106b
Differentiation
?
p53, other
Skp2
p21Cip1
miR-181a p27Kip1 protein
CDK4,6
CDK2
Cyclin D
Cyclin E pRb
E2F
× G1
c-Myc
normal human adult hematopoiesis [307]. Thus, pRb appears to have a role in the early stage of 1,25(OH)2D3-induced differentiation, and to contribute to changes in cellular transcriptional and kinase activities that lead to G1 arrest at a later stage. DOWNREGULATION OF c-MYC EXPRESSION IN 1,25(OH)2D3-INDUCED DIFFERENTIATION AND G1 ARREST
pRb-E2F INK4
1641
× S
FIGURE 84.7 Inhibitory effects of 1,25(OH)2D3 on the G1 traverse, which result in G1/S block. The key events in leukemia cells include: (1) an early upregulation of RB gene expression, which sequesters E2F necessary for the expression of cyclin E; (2) inhibition of p45/Skp2 and miR-181a expression, which, together with reduced cyclin E levels and consequent reduced phosphorylation of p27/Kip1, lead to increased levels p27/Kip1; (3) progressively increasing sequestration of E2F resulting in further increase in p27/Kip1 and decreases in c-Myc and other proteins required for DNA replication. The direct 1,25(OH)2D3induced activation of p21/Cip1 gene, which can also be upregulated by p53 or downregulated by miR106b, may have an effect on other pathways that lead to differentiation, or contribute to the effects of 1,25(OH)2D3 on the pRb/E2F pathway.
answer is provided by the finding that the Rb gene is upregulated early in 1,25(OH)2D3-induced differentiation of HL60 cells [97]. The increased levels of pRb can then bind and inactivate E2F transcription factors necessary for the expression of cyclin E, and thus the activity of the cyclin EeCdk2 complex (Fig. 84.7). Accordingly, the phosphorylation of Thr187 on p27/ Kip1 is reduced, allowing the accumulation of this Cdk inhibitor, and a further increase in the hypophosphorylated forms of pRb, also a substrate for the cyclin EeCdk complex. Hypophosphorylated pRb now further binds E2F, and thus reduces cyclin E expression to the point that p27/Kip1 is no longer phosphorylated and degraded, as the result of this positive feedback loop, leading to G1 arrest. It is known that in HL60 cells the expression of pRb normally occurs primarily during G1 phase [304], and can be detected at both mRNA and protein levels within 10 h of exposure to 1,25(OH)2D3 [97] although the mechanism of its upregulation remains to be determined. These in vitro studies are supported by the finding of gross defects in the development of the hematopoietic system in Rb knockout mice [305,306], and by the transcriptional studies which show that the Rb gene plays a role in
The pRb/E2F pathway also controls the expression of c-Myc, as E2F transcription factors upregulate the c-Myc gene, and inhibition of c-Myc expression by 1,25(OH)2D3 may be responsible, at least in part, for the G1 block in differentiating cells [308]. Indeed, the association between c-Myc downregulation and 1,25(OH)2D3induced differentiation of human leukemia cells was one of the earliest findings that initiated the studies of the molecular basis of the cellular changes that follow exposure to this hormone [16,265,309,310]. Subsequently, several studies have shown a marked reduction in c-Myc expression in response to 1,25(OH)2D3 and analog treatment of colon cancer cells [311,312] and prostate cancer cells [268,313], and this was associated with the induction of differentiation [312] and G1 arrest [313]. The intense interest in c-Myc as a potential negative regulator of differentiation was fueled largely by its deregulated expression in several types of human neoplasia [314,315]. c-Myc is known to promote cell cycle progression mostly through coordinated transcriptional regulation of target genes (e.g., [316]). These include the DNA replication and cell cycle traverse-promoting genes such as ornithine decarboxylase, Cdc25A, and cyclins E and A [314]. Conversely, c-Myc inhibits the transcription of cell cycle inhibitor p21/Cip1 [317], and it has been suggested that this is due, at least in part, by sequestering the Sp1 transcription factor, which is required for p21/Cip1 transcription [318,319]. While the above considerations present an almost complete sequence of events that can explain the G1 arrest induced by 1,25(OH)2D3 (summarized in Fig. 84.7) an additional level of control of c-Myc expression by 1,25(OH)2D3 is provided by studies of Simpson et al. [320]. They found that in differentiating HL60 cells 1,25(OH)2D3 increased the expression and DNAbinding activity of HOXB4, a product of a homeobox gene, and that HOXB4 binds to the sites in the c-Myc gene which are involved in blocking by 1,25(OH)2D3 of the elongation of c-Myc transcripts [321]. Further, these authors demonstrated that a HOXB4 antisense oligonucleotide partially inhibited the 1,25(OH)2D3induced decrease in c-Myc protein levels [322]. While they observed reduction of 1,25(OH)2D3-induced differentiation in these experiments, the effect of HOXB4 antisense on the G1 block was not reported. Nonetheless, these studies are significant, as members of the HOX
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gene family are known to be involved in hematopoiesis and leukemogenesis [323e325]. Also, other HOX genes participate in 1,25(OH)2D3-induced differentiation; HOXB7 was reported to increase in HL60 cells [326], while in U937 and MCF-7 cells 1,25(OH)2D3 increased expression of HOXA10 [327], and the iroquois homeobox gene 5 (Irx5) is regulated by 1,25(OH)2D3 in LNCaP human prostate cancer cells, and controls the cell cycle as well as apoptosis [328]. Current data suggest that vitamin D may inhibit colon cancer cell proliferation by inhibiting the APC/b-catenin pathway. For instance, in SW480 cells 1,25(OH)2D3 promotes VDR/b-catenin interaction and prevents b-catenin nuclear translocation, leading to inhibition of TCF-4 responsive genes such as c-Myc [329], a protooncogene required for formation of tumors in many settings [330]. Thus, transcriptional blockage of c-Myc expression in these cells after exposure to 1,25(OH)2D3 [311] may be associated with modulation of the APC/ b-catenin signaling pathway. Another mechanism by which 1,25(OH)2D3 can downregulate c-Myc expression was suggested by Rohan et al. [268], who demonstrated that treatment of C4-2 prostate cancer cells with 1,25(OH)2D3 resulted in a 50% decrease in c-Myc mRNA but a much more extensive reduction in c-Myc protein. Further experiments showed that decreased c-Myc stability was due to an increase in the proportion of c-Myc phosphorylated on Thr58, a glycogen synthase kinase-3b site that serves as a signal for ubiquitinmediated proteolysis. Thus it appears that 1,25(OH)2D3 regulates c-Myc levels in cancer cells by several different pathways, which involve both a reduction in c-Myc gene expression and a decrease in c-Myc protein stability. The G2/M Retardation and Polyploidization The occurrence of abnormalities in G2/M transition in 1,25(OH)2D3-treated cells has been observed infrequently, with a general consensus that the G1 phase is the principal target of the anti-proliferative actions of vitamin D and its analogs. However, in early studies of 1,25(OH)2D3 action Abe et al. [331] detected an increase in the G2þM compartment in WEHI murine myelomonocytic cells, also described in HL60 cells by Godyn et al. [332]. The basis for this increase may be a reduction in the levels of Cdk1 in these cells [333], although the roles of cohesin, separase, or Plks remain to be investigated in the light of the recently accumulating knowledge of mitotic controls (see “The G2 and M phase transition,” above). One consequence is the higher ploidy of HL60 cells exposed for prolonged periods of time to 1,25(OH)2D3, observed as an increased number of bi-nucleated cells [332], or as nearly doubled DNA content of these cells [146]. Interestingly, polyploidization of 1,25(OH)2D3-treated cells is an alternative to differentiation, as these cells over-ride the
anti-proliferative actions of 1,25(OH)2D3 and do not express differentiated phenotype [146]. Thus, HL60 cells can have a 1,25(OH)2D3-induced defect in completion of mitosis that allows one round of DNA endoreduplication. Whether osteoclast, or perhaps megakaryocyte, polyploidization is also influenced by 1,25(OH)2D3 remains a possibility.
CELL-TYPE SPECIFICITY OF INHIBITION OF CELL PROLIFERATION BY VITAMIN D AND ANALOGS WITHOUT EVIDENCE OF DIFFERENTIATION While some effects of 1,25(OH)2D3 and its analogs can be recognized in a variety of cell types, there is remarkable cell-type specificity in most of such effects, and it is important to realize that only a few generalizations can be made regarding the anti-proliferative actions of these compounds. However, it appears to be true that 1,25(OH)2D3-induced differentiation is not simply a consequence of inhibited proliferation, as differentiation often precedes the G1 block [9,12], and 1,25(OH)2D3 can inhibit cell proliferation with only minimal, or absent, evidence of differentiation. Indeed, the anti-proliferative effect of 1,25(OH)2D3 on cultured melanoma cells was recognized by Colston et al. [334] in 1981, at the same time as the differentiation-inducing action of 1,25(OH)2D3 was described in myeloid leukemia cells by Abe et al. [15]. A recent example of inhibition of growth without differentiation is the report that in human airway smooth muscle cells 1,25(OH)2D3 decreased PDGF-induced cell growth by inhibiting pRb and Chk1 phosphorylation [335]. Cell specificity of responses to 1,25(OH)2D3 is also illustrated by the finding that 1,25(OH)2D3 can cause a G1 block in cultured thyroid carcinoma and pituitary corticotroph, but not lactotroph, cells [336,337]. Interestingly, while in thyroid carcinoma cells the mechanisms of the antiproliferative effects include dephosphorylation of p27/ Kip1 in a PTEN-dependent manner, leading to a diminished association between p45/Skp2 and p27/Kip1 with its consequent accumulation [336], in pituitary corticotroph cells the mechanism appears to be a diminished association of p27/Kip1 with p45/Skp2 and Cdk2, without an involvement of PTEN [337]. This illustrates not only the exquisite cell-type specificity of the mechanisms involved in the anti-proliferative actions of vitamin D and its analogs, but also that the upregulation of p27/Kip1 is unrelated to differentiation, as demonstrated previously in leukemia cells [264]. Another mechanism for the anti-proliferative actions of 1,25(OH)2D3 on cell types that show only minimal evidence of differentiation is provided by the apoptosis-inducing actions of vitamin D and its analogs,
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as described in other chapters in this volume (e.g., Chapter 89). Again, cell type determines this response, as in contrast to various carcinomas, e.g. breast cancer cells [338], 1,25(OH)2D3 protects HL60 leukemia cells from apoptosis, as first demonstrated by Xu et al. [339] and further confirmed by subsequent studies in both HL60 cells [70,340e342] and other cancer [343e345] and non-cancer [346e350] cell types. Thus, the activity of survival pathways may determine whether differentiation can take place in the presence of 1,25(OH)2D3, or whether a potential default pathway will lead to apoptosis, perhaps as the result of prolonged residence in a cell cycle compartment other than G0. In any case, 1,25(OH)2D3 can be an effective anti-proliferative agent in many cell types that express VDR.
CONCLUSIONS 1,25(OH)2D3 and its analogs present new therapeutic options for treatment of human malignancies due to their demonstrated anti-proliferative actions in a wide variety of cell types. While the mechanisms vary, G1 block produced by upregulation of p27/Kip1 is an almost constant feature of the cell cycle effects of 1,25(OH)2D3. Further studies are needed on the multiple mechanisms that upregulate p27/Kip1, although the control of its degradation by p45/Skp2 that is influenced by p27/Kip1-Thr187 phosphorylation by still not fully elucidated pathways, and inhibition of p27/Kip1 mRNA translation by microRNAs, present exciting possibilities. Also, in view of the ability of cells treated with 1,25(OH)2D3 to develop resistance to its anti-proliferative actions [351,352], synergistic effects of vitamin D analogs combined with other agents should be further explored [126,353e361] as should the pathways that transmit other extracellular signals to the cell nucleus in concert with the vitamin D receptor-initiated signals.
Acknowledgments We thank Dr. F. Coffman for comments on the manuscript. We also acknowledge the support for experimental work performed in our laboratories by the National Cancer Institute (Grants RO1-CA44722, to G.P.S., RO1-CA-117942-3, to G.P.S. and M.D.), the Israel Science Foundation (Grant 778/07 to M.D.), the Polish Ministry of Science and Higher Education (Grant 2132/B/P01/2008/34 to E.G.), and the Foundation for Polish Science (E.G.).
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[362] M. Hewison, S. Barker, A. Brennan, D.R. Katz, J.L. O’Riordan, Modulation of myelomonocytic U937 cells by vitamin D metabolites, Bone Miner. 5 (1989) 323e333. [363] S.M. Kelsey, H.L. Makin, A.C. Newland, Functional significance of induction of differentiation in human myeloid leukaemic blasts by 1,25-dihydroxyvitamin D3 and GM-CSF, Leuk. Res. 16 (1992) 427e434. [364] I. Molnar, T. Kute, M.C. Willingham, B.L. Powell, W.H. Dodge, G.G. Schwartz, 19-Nor-1alpha,25-dihydroxyvitamin D2 (paricalcitol): effects on clonal proliferation, differentiation, and apoptosis in human leukemic cell lines, J. Cancer Res. Clin. Oncol. 129 (2003) 35e42. [365] C. Rius, P. Aller, Modulation of ornithine decarboxylase gene transcript levels by differentiation inducers in human promyelocytic leukemia HL60 cells, Cell Differ. Dev. 28 (1989) 39e46. [366] D. Brackman, F. Lund-Johansen, D. Aarskog, Expression of leukocyte differentiation antigens during the differentiation of HL-60 cells induced by 1,25-dihydroxyvitamin D3: comparison with the maturation of normal monocytic and granulocytic bone marrow cells, J. Leukoc. Biol. 58 (1995) 547e555. [367] L.T. Yam, C.Y. Li, W.H. Crosby, Cytochemical identification of monocytes and granulocytes, Am. J. Clin. Pathol. 55 (1971) 283e290. [368] T. Saito, R. Okamoto, T. Haritunians, J. O’Kelly, M. Uskokovic, H. Maehr, et al., Novel Gemini vitamin D3 analogs have potent antitumor activity, J. Steroid Biochem. Mol. Biol. 112 (2008) 151e156. [369] I. Olsson, U. Gullberg, I. Ivhed, K. Nilsson, Induction of differentiation of the human histiocytic lymphoma cell line U937 by 1 alpha,25-dihydroxycholecalciferol, Cancer Res. 43 (1983) 5862e5867. [370] O.C. Blair, R. Carbone, A.C. Sartorelli, Differentiation of HL-60 promyelocytic leukemia cells monitored by flow cytometric measurement of nitro blue tetrazolium (NBT) reduction, Cytometry 6 (1985) 54e61. [371] L.M. Sly, M. Lopez, W.M. Nauseef, N.E. Reiner, 1Alpha,25dihydroxyvitamin D3-induced monocyte antimycobacterial activity is regulated by phosphatidylinositol 3-kinase and mediated by the NADPH-dependent phagocyte oxidase, J. Biol. Chem. 276 (2001) 35482e35493. [372] M. Shiohara, M. Uskokovic, J. Hisatake, Y. Hisatake, K. Koike, A. Komiyama, et al., 24-Oxo metabolites of vitamin D3 analogues: disassociation of their prominent antileukemic effects from their lack of calcium modulation, Cancer Res. 61 (2001) 3361e3368. [373] J.R. Gum, W.K. Kam, J.C. Byrd, J.W. Hicks, M.H. Sleisenger, Y.S. Kim, Effects of sodium butyrate on human colonic adenocarcinoma cells. Induction of placental-like alkaline phosphatase, J. Biol. Chem. 262 (1987) 1092e1097. [374] A.R. Giuliano, R.T. Franceschi, R.J. Wood, Characterization of the vitamin D receptor from the Caco-2 human colon carcinoma cell line: effect of cellular differentiation, Arch. Biochem. Biophys. 285 (1991) 261e269. [375] T. Gaschott, J. Stein, Short-chain fatty acids and colon cancer cells: the vitamin D receptor-butyrate connection, Recent Results Cancer Res. 164 (2003) 247e257. [376] K. Saini, G. Steele, P. Thomas, Induction of carcinoembryonicantigen-gene expression in human colorectal carcinoma by sodium butyrate, Biochem. J. 272 (1990) 541e544. [377] R.R. Buras, M. Shabahang, F. Davoodi, L.M. Schumaker, K.J. Cullen, S. Byers, et al., The effect of extracellular calcium on colonocytes: evidence for differential responsiveness based upon degree of cell differentiation, Cell Prolif. 28 (1995) 245e262.
[378] A. Staal, A.J. van Wijnen, J.C. Birkenhager, H.A. Pols, J. Prahl, H. DeLuca, et al., Distinct conformations of vitamin D receptor/retinoid X receptor-alpha heterodimers are specified by dinucleotide differences in the vitamin D-responsive elements of the osteocalcin and osteopontin genes, Mol. Endocrinol. 10 (1996) 1444e1456. [379] R. Kommagani, A. Whitlatch, M.K. Leonard, M.P. Kadakia, p73 is essential for vitamin D-mediated osteoblastic differentiation, Cell Death Differ. 17 (2010) 398e407. [380] R.T. Franceschi, W.M. James, G. Zerlauth, 1 Alpha, 25-dihydroxyvitamin D3 specific regulation of growth, morphology, and fibronectin in a human osteosarcoma cell line, J. Cell Physiol. 123 (1985) 401e409. [381] G.J. van den Bemd, H.A. Pols, J.C. Birkenhager, W.M. Kleinekoort, J.P. van Leeuwen, Differential effects of 1,25dihydroxyvitamin D3-analogs on osteoblast-like cells and on in vitro bone resorption, J. Steroid Biochem. Mol. Biol. 55 (1995) 337e346. [382] N. Fratzl-Zelman, H. Glantschnig, M. Rumpler, A. Nader, A. Ellinger, F. Varga, The expression of matrix metalloproteinase-13 and osteocalcin in mouse osteoblasts is related to osteoblastic differentiation and is modulated by 1,25-dihydroxyvitamin D3 and thyroid hormones, Cell Biol. Int. 27 (2003) 459e468. [383] N. Pernalete, T. Mori, Y. Nishii, E. Slatopolsky, A.J. Brown, The activity of 22-oxacalcitriol in osteoblast-like (ROS 17/2.8) cells, Endocrinology 129 (1991) 778e784. [384] Y. Li, C.M. Backesjo, L.A. Haldosen, U. Lindgren, Species difference exists in the effects of 1alpha,25(OH)2D3 and its analogue 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 (2MD) on osteoblastic cells, J. Steroid Biochem. Mol. Biol. 112 (2008) 110e116. [385] R.J. Skowronski, D.M. Peehl, D. Feldman, Vitamin D and prostate cancer: 1,25 dihydroxyvitamin D3 receptors and actions in human prostate cancer cell lines, Endocrinology 132 (1993) 1952e1960. [386] M. Esquenet, J.V. Swinnen, W. Heyns, G. Verhoeven, Control of LNCaP proliferation and differentiation: actions and interactions of androgens, 1alpha,25-dihydroxycholecalciferol, alltrans retinoic acid, 9-cis retinoic acid, and phenylacetate, Prostate 28 (1996) 182e194. [387] T.E. Hedlund, K.A. Moffatt, M.R. Uskokovic, G.J. Miller, Three synthetic vitamin D analogues induce prostate-specific acid phosphatase and prostate-specific antigen while inhibiting the growth of human prostate cancer cells in a vitamin D receptordependent fashion, Clin. Cancer Res. 3 (1997) 1331e1338. [388] M.J. Campbell, E. Elstner, S. Holden, M. Uskokovic, H.P. Koeffler, Inhibition of proliferation of prostate cancer cells by a 19-nor-hexafluoride vitamin D3 analogue involves the induction of p21waf1, p27kip1 and E-cadherin, J. Mol. Endocrinol. 19 (1997) 15e27. [389] T.M. Beer, M. Garzotto, B. Park, M. Mori, A. Myrthue, N. Janeba, et al., Effect of calcitriol on prostate-specific antigen in vitro and in humans, Clin. Cancer Res. 12 (2006) 2812e2816. [390] T. Otto, K. Rembrink, M. Goepel, M. Meyer-Schwickerath, H. Rubben, E-cadherin: a marker for differentiation and invasiveness in prostatic carcinoma, Urol. Res. 21 (1993) 359e362. [391] E. Elstner, M. Linker-Israeli, J. Said, T. Umiel, S. de Vos, I.P. Shintaku, et al., 20-Epi-vitamin D3 analogues: a novel class of potent inhibitors of proliferation and inducers of differentiation of human breast cancer cell lines, Cancer Res. 55 (1995) 2822e2830. [392] G. Lazzaro, A. Agadir, W. Qing, M. Poria, R.R. Mehta, R.M. Moriarty, et al., Induction of differentiation by 1alphahydroxyvitamin D5 in T47D human breast cancer cells and its
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85 Vitamin D Actions in Mammary Gland and Breast Cancer JoEllen Welsh University at Albany, Rensselaer, NY, USA
INTRODUCTION The mammary gland is composed of a branching network of ducts embedded in an adipose-rich stroma. Adenocarcinoma of the breast arises when epithelial cells or their precursors become transformed through a series of genetic and epi-genetic events. Multiple initiating factors, including genetics, environment and diet, are thought to impact on breast cancer risk. Genomic profiling has identified many subtypes of breast cancers (luminal, ductal, basal, triple negative, HER2 positive, inflammatory, etc.), which likely result from different initiating agents acting on diverse target cells in the gland [1]. It is also clear from laboratory studies that there are critical time windows over the lifespan of an individual during which exposure to harmful or preventive factors is more likely to impact on breast cancer development [2]. For these reasons, identification of specific factors that may trigger or prevent human breast cancer is extremely challenging, and multiple lines of evidence (laboratory, clinical, population) are employed in riskebenefit analyses. Despite the aforementioned experimental limitations, evidence strongly suggests that hormones such as estrogen and progesterone, which drive mammary epithelial cell proliferation and may give rise to genotoxic metabolites, are important etiologic factors for human breast cancer. Therapies that block estrogen synthesis (aromatase inhibitors) or target estrogen receptors (anti-estrogens, including tamoxifen) are highly effective for both treatment and prevention of estrogen-dependent breast cancer. Tamoxifen represents the best-characterized selective estrogen receptor modifier (SERM), a class of synthetic compounds that interact with the nuclear estrogen receptors alpha (ERa) and beta
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10085-X
(ERb) in a cell-type-specific manner. SERMS designed for breast cancer therapy antagonize ERa signaling in breast tissue but not in bone, thus limiting proliferation of estrogen-dependent breast cancer cells without adversely affecting bone mass. Although SERMs are often effective in the initial treatment of estrogenresponsive disease, tumor progression may be associated with resistance to anti-estrogens. Thus, there is a need both for improved treatments of estrogendependent breast cancer as well as for alternative therapies that target estrogen-independent cells and that minimize the progression of estrogen-responsive disease to hormone independence. Other nuclear receptors present in breast cancer cells, such as the progesterone receptor, retinoid receptors, and the vitamin D receptor (VDR), have emerged as promising therapeutic targets for breast cancer. Based on the importance of nuclear receptors in mediating expression of genes involved in proliferation, differentiation and apoptosis, synthetic structural analogs of nuclear receptor ligands which exhibit biological properties distinct from the natural ligands represent a feasible approach to manipulation of nuclear receptor activity. Structurally, most nuclear receptor ligands have the additional advantage of being orally active. In the case of the VDR, many synthetic analogs with desirable therapeutic profiles have been developed, and some are in clinical trials for various indications, including cancer (discussed in Section IX of this volume). However, further development of synthetic ligands for either treatment or prevention of breast cancer requires more accurate understanding of the role(s) of their cognate nuclear receptors in both normal and transformed mammary cells. In this chapter, we will review the extensive literature documenting the effects of 1,25(OH)2D3 (the natural
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ligand for VDR) and numerous bio-active vitamin D analogs on breast cancer cells and tumors. Furthermore, we will highlight emerging data on the role of the vitamin D endocrine system in the normal mammary gland and the possibility that vitamin D signaling may influence breast cancer development.
VITAMIN D ACTIONS ON BREAST CANCER CELLS IN VITRO Histological analysis has demonstrated that the majority of human breast cancers (up to 80%) express immunoreactive VDR protein. However, the expression and function of VDR in established breast cancer cell lines is quite variable, with high expression of transcriptionally active VDR in some lines (i.e., MCF-7) but low levels in others (i.e., MDA-MB-231). Although, as further discussed below, VDR is required for the antitumor effects of vitamin D steroids, the presence of VDR is not sufficient for a cellular response, as some lines with detectable VDR expression fail to respond to 1,25(OH)2D3. Since the identification of VDR in breast cancers [3], extensive research has been directed towards elucidation of the effects of 1,25(OH)2D3 on breast cancer cells, identification of VDR targets that mediate these effects, and mechanisms of sensitivity and resistance. Several reviews on this topic are available [4,5] and this section provides a concise summary of the effects of 1,25(OH)2D3 and other VDR agonists on proliferation, differentiation, apoptosis, angiogenesis, and invasion of breast cancer cells that represent different stages of disease progression (Table 85.1).
Regulation of Breast Cancer Cell Proliferation and Survival by 1,25(OH)2D3 Cell Cycle Characterization of the effects of vitamin D signaling on cell cycle kinetics has primarily been conducted under in vitro conditions using established human breast cancer cell lines actively undergoing proliferation. Under these conditions, most breast cancer cells treated with nanomolar concentrations of 1,25(OH)2D3 undergo cell cycle arrest in G0/G1 within 48 h [6,7]. G1 arrest is associated with dephosphorylation of the retinoblastoma protein [7] and increases in genes that code for the cyclin-dependent kinase inhibitors CDKN1A (p21) and CDKN1B (p27) [8e10]. Direct effects of ligandbound VDR on the promoter activity of CDKN1A via vitamin D response elements [11,12] and CDKN1B via interaction with Sp1 [6,13] have been described. Although cell cycle arrest is a common consequence of 1,25(OH)2D3 treatment, the stage, kinetics and target
TABLE 85.1
Stage-specific Effects of Vitamin D Signaling on Breast Cancer. Summary of Effects of Natural and Synthetic Vitamin D Compounds that Act as VDR Agonists on Breast Cancer in Relation to Tumor Progression. Details and References are Noted in Text
Model
Effects of VDR agonists
Early stage breast cancers ERa positive High VDR expression
Reduce growth of primary tumor via: Y mitogenesis (ERa; CYP19A1; IGFBP3,5; EGF) [ apoptosis/autophagy (TGFb; bcl-2; ROS; Caþþ) Y inflammation (Cox-2; NFkB; CD14/ TLR) Y angiogenesis (VEGF) target endothelial and cancer cells
Advanced breast cancers ERa negative VDR usually reduced
Inhibit invasion in vitro: [ E-cadherin, Ycellecell contacts [ PAI-1, YuPA Reduce metastatic spread in nude mouse models: Y number of secondary lesions Y skeletal metastases [ survival time Dietary vitamin D deficiency enhances bone metastases
proteins appear to be cell-type specific. Upregulation of CDKN1A and CDKN1B by VDR agonists is generally associated with G1 arrest due to inhibition of cyclindependent kinase (CDK) activity, including CDK2associated histone H1 kinase, cyclin D1/CDK4, and cyclin A/CDK2 [14]. VDR also interacts with protein phosphatases PP1c and PP2Ac to inactivate the p70 S6 kinase which is essential for G1/S phase transition [15]. Thus, the net result of vitamin D signaling is to prevent entry into S phase, leading to accumulation in G1. In some breast cancer cells, vitamin-D-mediated G1 arrest is associated with induction of differentiation markers such as lipid and casein [16e18]. Additional changes linked to cell cycle arrest include reorganization of the actinecytoskeletal network, promotion of cellecell contacts and enhanced adhesion in association with altered activity of integrins and focal adhesion kinase signaling [19]. Cell Death: Apoptosis and Autophagy In addition to their anti-proliferative effects, 1,25(OH)2D3 and other VDR agonists induce morphological and biochemical features of apoptosis (cell shrinkage, chromatin condensation, and DNA fragmentation) in breast cancer cells [20e22]. Other markers of apoptosis induced by 1,25(OH)2D3 include reorientation of phosphatidylserine (PS) to the exterior of the cell, PARP cleavage and upregulation of apoptotic-related proteins, such as clusterin, cathepsin B, and TGFb [22].
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Although caspases are activated by vitamin D steroids in some cells [23], it is clear that MCF-7 cells undergo cell death in the presence of caspase inhibitors, indicating that the commitment to 1,25(OH)2D3-mediated cell death is caspase-independent [25,26]. Furthermore, 1,25(OH)2D3 exerts additive or synergistic effects in combination with other triggers of apoptosis, such as anti-estrogens, TNFa, radiation, and chemotherapeutic agents [17,20,24e27]. It is not quite clear whether these synergistic effects result from effects of 1,25(OH)2D3 on agonist-specific signaling pathways or on components of a common apoptotic pathway. Several independent studies have reported that sensitivity to 1,25(OH)2D3-mediated apoptosis reflects the relative expression and/or subcellular localization of the Bcl-2 family of pro- and anti-apoptotic proteins. Treatment of MCF-7 cells with 1,25(OH)2D3 or the analog EB1089 induces redistribution of the proapoptotic Bcl-2 family member, Bax, from the cytosol to the mitochondria and downregulates the antiapoptotic protein Bcl-2 [7,20,28,29]. Furthermore, overexpression of Bcl-2 renders MCF-7 cells resistant to 1,25(OH)2D3-mediated apoptosis [30]. Since Bcl-2 and Bax act antagonistically to one another in the regulation of apoptosis, these data suggest that translocation of Bax in conjunction with downregulation of Bcl-2 may be apoptosis. necessary for 1,25(OH)2D3-mediated Vitamin-D-mediated Bax translocation triggers reactive oxygen species (ROS) generation, dissipation of the mitochondrial membrane potential, and release of cytochrome c into the cytosol [28,29], features of the intrinsic (mitochondrial) pathway of apoptosis. 1,25(OH)2D3 also enhances mitochondrial ROS generation and cytochrome c release in MCF-7 cells treated with TNFa [31]. Of particular interest, neither Bax translocation, ROS generation, mitochondrial membrane potential dissipation, nor cytochrome c release are induced by 1,25(OH)2D3 in MCF-7DRES cells, a variant of MCF-7 cells selected for vitamin D resistance [28,29]. Another pathway implicated in vitamin-D-mediated apoptosis of MCF-7 cells involves calcium release from the endoplasmic reticulum and activation of m-calpain; this process can be prevented by either calpain inhibitors or calcium-buffering agents such as calbindin D28K [32]. In addition to apoptosis, the release of calcium from the endoplasmic reticulum and the resulting increase in cytosolic calcium in response to VDR agonists activates autophagy, a catabolic process involving the degradation of cellular components via lysosomes. Using electron microscopy, Hoyer-Hansen et al. demonstrated that the vitamin D analog EB1089 induces the accumulation of autophagosomes in MCF7 cells [33]. EB1089-mediated autophagy is associated with increased intracellular calcium, activation of Ca2þ/calmodulin-dependent kinase-b and inhibition of
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the mammalian target of rapamycin (mTOR). This process is dependent on both beclin-1 and Atg7 and can be blocked by ectopic bcl-2 targeted to the endoplasmic reticulum [34]. Induction of autophagy in p53positive breast cancer cells exposed to ionizing radiation is also enhanced by D analog EB1089 [35]. Collectively, these data suggest that cell death induced by VDR agonists likely involves interactions between both apoptotic and autophagic signals emanating from the mitochondria and the endoplasmic reticulum.
Target Genes and Pathways Regulated by 1,25(OH)2D3 in Breast Cancer Cells Estrogen Metabolism and Signaling 1,25(OH)2D3 and EB1089 downregulate ERa and suppress estrogen action in MCF-7 cells [7,36]. Since estrogen is mitogenic for most breast cancer cells, downregulation of estrogen-regulated pathways may contribute to cell cycle arrest in response to 1,25(OH)2D3. Sensitivity to 1,25(OH)2D3 is generally higher in breast cancer cells which express ERa, such as MCF-7 and T47D, than in those that do not [37,38]. Downregulation of ERa by vitamin D compounds attenuates both the mitogenic effects of estrogen and the induction of target genes such as the progesterone receptor and pS2 [36]. Co-treatment of ERa-positive breast cancer cells with 1,25(OH)2D3 or EB1089 and the anti-estrogens tamoxifen or ICI 182,780 inhibits proliferation more than either compound alone [21,25,36,39,40]. Sequence analysis of the ERa gene promoter has identified a functional consensus VDRE, suggesting a direct regulatory effect of 1,25(OH)2D3 on ERa gene transcription [41,42]. Although it is clear that vitamin D compounds exert multiple effects on estrogen-induced gene expression and block estrogen-driven proliferation, the effects of VDR agonists and ERa antagonists can be mechanistically dissociated [25]. In addition, 1,25(OH)2D3 and its analogs also inhibit growth of estrogen-independent breast cancer cells, ERa signaling is not required for the anti-proliferative effects of vitamin D compounds [10]. Furthermore, in some cases, breast cancer cells selected for anti-estrogen resistance show increased sensitivity to vitamin-D-mediated growth inhibition [43]. Another mechanism by which 1,25(OH)2D3 interacts with estrogen signaling in breast cancer cells is at the level of steroid metabolism [44]. In MCF-7 cells, 1,25(OH)2D3 downregulates CYP19A1, which codes for the aromatase enzyme that catalyzes synthesis of estrogen from testosterone. Mechanistically, 1,25(OH)2D3 directly represses transcription via VDREs in the promoter II region (which is highly active in breast cancers) and also reduces the activity of prostaglandins (which stimulate aromatase
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transcription). Since aromatase inhibitors are effective clinically to reduce breast cancer recurrence in postmenopausal women, these dual actions of vitamin D to suppress aromatase could substantially contribute to its anti-proliferative effects in estrogen-dependent cancers. Growth Factor Signaling Insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) are potent mitogens for breast cancer cells and their bioactivity can be enhanced or inhibited by specific IGF-binding proteins (IGFBPs). In particular, IGFBP-3 and IGFBP-5 are secreted proteins that bind IGF-1, reducing its availability to the membrane IGF-1 receptor. The accumulation of IGFBP-5 in conditioned media from MCF-7 cells treated with 1,25(OH)2D3 or analogs EB1089 and KO1060 was initially reported by Pollak’s group [45]. Subsequent studies confirmed that growth inhibition mediated by vitamin D steroids involves disruption of the mitogenic signals from extracellular IGFs via modulation of both IGFBP-5 and IGFBP-3 and inhibition of IGF-1 receptor signaling [46,47]. Direct binding of VDR/RXR heterodimers to a consensus VDRE in the IGFBP-3 promoter has been confirmed by ChIP assays [48]. Since IGF-1 promotes survival of most breast cancer cells, disruption of IGF-1 signals likely also contributes to the induction of apoptosis in response to 1,25(OH)2D3 [49]. The epidermal growth factor (EGF) family includes ligands (EGF, amphiregulin, epiregulin, neuregulins, etc.) and receptors (EGFR, HER2, etc.) which promote proliferation and survival of epithelial cells. In MCF7, T47D, and BT549 breast cancer cells, 1,25(OH)2D3 reduces EGFR mRNA and protein via direct repression of the EGFR proximal promoter [50]. Tumor cells derived from MMTV-neu mice, which display constitutive receptor activity and model Her2-positive human breast cancer, express VDR and retain growth inhibitory responses to 1,25(OH)2D3 in vitro, although the mechanisms by which vitamin D signaling inhibits neu signaling has yet to be explored [51]. Several genome-wide profiling studies have identified genes in the TGFb family as vitamin D inducible in breast cancer cells [52e54]. VDREs have been identified in the TGFb2 gene promoter, suggesting at least some of these genomic changes could be directly mediated at the level of transcription [55]. Although TGFb family members can exert diverse effects during the carcinogenic process, most act as negative growth regulators in early-stage breast cancer cells. In MCF-7 cells, 1,25(OH)2D3 enhances the expression of TGFb1 as well as its latent form binding protein [56]. The anti-proliferative effect of VDR agonists is partially abrogated by neutralizing antibodies to TGFb [57,58], indicating a functional link between growth inhibition by 1,25(OH)2D3 and TGFb in vitro.
Inflammatory Pathways Multiple interactions between vitamin D signaling and inflammation have been described in various tissues, but few studies have specifically focused on breast cancer. In vitro, 1,25(OH)2D3 inhibits the synthesis and biological actions of pro-inflammatory prostaglandins by suppression of cyclooxygenase-2 expression, upregulation of 15-hydroxyprostaglandin dehydrogenase and downregulation of prostaglandin receptors. In prostate cancer cells, the combination of 1,25(OH)2D3 with non-steroidal anti-inflammatory drugs synergistically inhibits cell growth, supporting the concept that simultaneously targeting VDR and inflammatory pathways could be of benefit therapeutically [59,60]. In addition, 1,25(OH)2D3 exerts antiinflammatory activity through the inhibition of NF-kB, which is often constitutively expressed in breast cancer cells and promotes both survival and the invasive phenotype. Treatment of Hs578T and Her-2/neu-driven NF639 cells with 1,25(OH)2D3 decreased expression of the NF-kB subunit RelB and downstream pro-survival targets survivin, MnSOD, and Bcl-2 [61]. Given the emerging importance of inflammation in cancer development and progression, further clarification of these interactions is clearly warranted. More recently, several genes involved in the innate immune system have been identified as potential vitamin D targets in mammary cells. Of particular interest is Cluster of Differentiation 14 (CD14), which is highly induced by 1,25(OH)2D3 in normal and transformed breast cells [51,53]. CD14 codes for a pattern recognition receptor which acts as a co-receptor (along with the Toll-like receptor TLR 4) for the detection of lipopolysaccharide and thus aids in protection against bacterial infections. Regulation of innate immune responses in mammary epithelial cells by 1,25(OH)2D3 likely represents another example of vitamin D’s role in maintenance of barrier integrity, as reported for the gastrointestinal tract and skin [62]. In addition to its function in innate immunity, CD14 is highly induced during post-lactational mammary gland remodeling [63] and functions in clearance of apoptotic cells [64]. Thus, regulation of CD14 and other immune regulatory cytokines/receptors by 1,25(OH)2D3 in breast cancer cells and tumor-infiltrating macrophages may contribute to its overall anti-cancer effects.
Stress Pathways Cellular responses to stresses such as DNA damage include activation of cell cycle arrest, apoptosis or senescence, which provide protection against cancer development. The p53 tumor suppressor gene is a crucial sensor of cellular stress that transcriptionally activates genes
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involved in repair and response to multiple types of damage. It is clear that the effects of 1,25(OH)2D3 are not strictly p53-dependent, since VDR agonists inhibit growth of some breast cancer cell lines that express mutant p53 such as T47D [28,61,62]. However, it is likely that 1,25(OH)2D3 signaling contributes to cellular stress responses, as p53 and VDR cooperate at multiple sites in the CDKN1A gene promoter to induce transcription of p21, which has been linked to cell cycle arrest [11,12]. Furthermore, VDR has been identified as a direct transcriptional target of the p53 family of tumor suppressors [65]. In addition, p53 mutations that are commonly found in cancer alter VDR expression, subcellular localization and target gene regulation, resulting in altered biological responses to 1,25(OH)2D3 [66]. These data suggest that complex interactions between VDR, p53 and DNA damage signaling could be important determinants of breast cancer development and/or progression, but further studies are needed to substantiate these interactions and their implications. BRCA1, another tumor suppressor gene that functions in DNA repair, is induced by 1,25(OH)2D3 in MCF-7 cells [37]. BRCA1 promoter analysis indicated that the effects of 1,25(OH)2D3 are indirectly mediated by the VDR. Sensitivity to 1,25(OH)2D3-mediated growth inhibition correlates with induction of BRCA1 and is highest in well-differentiated breast cancer cells. These data suggest that hereditary breast cancers which develop in patients with germ-line mutations in BRCA1 might be less responsive to vitamin-D-mediated growth inhibition.
Other Candidate Genes and Pathways Linked to Vitamin-D-mediated Growth Control In addition to the genes and pathways discussed above, various high-throughput approaches, including genomic and proteomic screening, have been used to identify 1,25(OH)2D3/VDR targets in breast cancer cells [51,53]. In the first screen for vitamin-D-regulated genes in breast cancer cells, Swami et al. [67] profiled ERapositive MCF-7 cells (with high VDR expression) and ERa-negative MDA-MB-231 cells (with low VDR expression) after treatment with 50 nM 1,25(OH)2D3 for 6 and 24 hours utilizing a CMT Cancer Array format that contained 2000 cancer-related genes. As expected based on the differential VDR expression, the number of genes altered two-fold or more by 1,25(OH)2D3 treatment was greater in MCF-7 cells (51 increased/19 decreased) than in MDA-MB-231 cells (16 increased/14 decreased). Also, as expected based on the distinct properties of the two cell lines, only eight genes were similarly regulated by 1,25(OH)2D3 in MCF-7 and MDA-MB-231 cells (VDR, CYP24, thioredoxin reductase I, retinoblastoma-like 2,
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retinoblastoma binding protein-6, vitronectin receptor, TGFb2 and RAB5A). Using a whole-genome array, Lee et al. [53] profiled the effects of a synthetic vitamin D analog (Ro3587, 1 nM, 4 and 12 hours) in premalignant (MCF10AT1) and fully malignant (MCF10CA1) human breast cells, with similar results: the VDR agonist triggered more changes in gene expression in the premalignant MCF10AT1 cells than in the metastatic MCF10CA1 cells (391 versus 156 altered two-fold or more). Interestingly, the regulation of many genes, including CYP24, remained intact in the metastatic cells, thus the transformation process appears to selectively affect VDR gene regulation. Comparative analysis of gene profiles in sensitive and resistant MCF-7 variants has yielded a number of candidates that confer sensitivity to vitamin-D-mediated growth inhibition, including BAX, GADD45a, IGFBP-3, EGFR, MAPK4, and TGFb [52]. Using a proteomics approach, Byrne and Welsh [68] profiled 270 apoptosis-related proteins in sensitive and resistant MCF-7 cells. Ten proteins were differentially expressed in sensitive MCF-7 cells treated with 100 nM 1,25(OH)2D3, including GRIM-19, cyclin A, cyclin D3, SHC, GSK3b, Rho-DGI (downregulated) and cathepsin B and PTEN (upregulated). For the most part, the changes in expression of these proteins are consistent with anti-proliferative and pro-apoptotic actions of 1,25(OH)2D3 in these cells. Comparison of the proteome in sensitive versus resistant MCF-7 cells identified a distinct set of 14 proteins that may underlie 1,25(OH)2D3 resistance, including those involved in cell motility and cytoskeletal dynamics (ACAP2, CapZ2), apoptosis (caspase 7, 14, Bid), and mitosis (phospho-p38, MEK2, cdk2). In another approach, murine mammary cells that differentially express VDR were used to profile VDRdependent 1,25(OH)2D3-mediated changes in gene expression [51]. Transformed mammary cells isolated from wild-type mice and VDR-null mice were treated for 24 h with 100 nM 1,25(OH)2D3 and processed for microarray profiling. No significant changes in gene expression were detected in VDR-null cells, whereas >80 genes were significantly altered in the VDRpositive WT145 cells derived from wild-type mice. In VDR-null cells engineered to stably express human VDR (KOhVDR cells), more than 200 genes were significantly altered in response to 1,25(OH)2D3. In both WT145 cells and KOhVDR cells, the most highly induced gene in response to 1,25(OH)2D3 was Cyp24A1 (which was not induced by 1,25(OH)2D3 in VDR-null cells); other highly regulated genes in WT145 cells are presented in Table 85.2. Again, there is limited overlap between this VDR-driven genomic profile obtained with murine mammary cancer cells and that reported for human-derived cells [53,67].
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TABLE 85.2
List of Genes Altered >Three-fold in VDR-positive Murine Mammary Tumor Cells Treated with 1,25(OH)2D3
Gene symbol
Gene name
Fold change
Cyp24a1
Cytochrome P450, family 24a1
[ 50.3
Enpp1
Ectonucleotide pyrophosphatase/ phosphodiesterase 1
[ 6.7
Prelp
Proline arginine-rich end leucine-rich repeat
[ 5.2
Cib2
Calcium and integrin binding family member 2
[ 4.6
Elovl7
ELOVL family member 7
[ 4.3
Islr
Immunoglobulin superfamily containing leucine-rich repeat
[ 4.3
Postn
Periostin, osteoblast-specific factor
Y 4.3
Crabp2
Cellular retinoic acid binding protein II
[ 4.0
Apcdd1
Adenomatosis polyposis coli downregulated 1
[ 3.8
Sepp1
Selenoprotein P
[ 3.4
Rassf2
Ras association (RalGDS/AF-6) domain family member 2
[ 3.4
Grk5
G protein-coupled receptor kinase 5
[ 3.0
RNA isolated from VDR-positive WT145 tumor cells treated in triplicate with vehicle or 100 nM 1,25(OH)2D3 for 24 h was analyzed on Affymetrix expression arrays and analyzed with Genespring X. Shown are the 12 genes which were significantly different (t-test) in 1,25(OH)2D3-treated cells that exhibited threefold change.
While only a small subset of the targets discussed above have been mechanistically studied in relation to growth arrest/apoptosis, it is clear that, despite similar anti-proliferative effects of 1,25(OH)2D3, the specific target genes regulated by vitamin D signaling vary considerably in different model systems. However, pathways that drive mitosis, apoptosis, and differentiation, alter metabolic flux, remodel the extracellular matrix and trigger innate immunity appear to be commonly regulated by VDR agonists in mammary tissue. Further analysis of these genomic and proteomic datasets, including validation of 1,25(OH)2D3 regulation, pathway analysis, and mechanistic studies are necessary to evaluate the significance of these potential VDR targets in relation to the anti-tumor effects of vitamin D.
Angiogenesis, Invasion and Metastasis Metastasis, the process by which tumor cells invade secondary sites, requires degradation of the extracellular matrix and is facilitated by angiogenesis, the growth of new blood vessels into developing tumors. Effects of
vitamin D signaling on late-stage breast cancer have been studied in ER-negative breast cancer cell lines, such as MDA-MB-231 and SUM159PT cells, which are invasive in vitro and metastatic in vivo. In these cell lines, 1,25(OH)2D3 and EB1089 inhibit invasion as measured by the in vitro Boyden chamber assay [10]. Inhibition of invasion by vitamin D compounds can be dissociated from effects on proliferation, and may be linked to regulation of extracellular proteases such as MMP-9, urokinase-type plasminogen activator (uPA), and tissue type plasminogen activator (tPA). In MDAMB-231 cells, these effects may result from vitamin-Dmediated upregulation of protease inhibitors PA inhibitor 1 and MMP inhibitor 1 [69]. The anti-tumor effects of 1,25(OH)2D3 may also involve regulation of angiogenesis, since 1,25(OH)2D3 inhibits angiogenesis in the chick embryo chorioallantoic membrane assay [70] and in tumor-cell-induced angiogenesis assays in mice [71]. Moreover, vitamin D analogs reduce angiogenesis of MCF-7 breast tumors overexpressing vascular endothelial growth factor (VEGF) and inhibit VEGF expression in MDA-MB-231 xenografts [72,73]. VDR is expressed in endothelial cells, and 1,25(OH)2D3 blocks both basal and VEGF-induced endothelial cell sprouting, elongation and proliferation [72,74,75]. Anti-proliferative effects of 1,25(OH)2D3 are maintained in tumor-derived endothelial cells and are VDR dependent [76]. Collectively, these studies indicate that vitamin D signaling likely inhibits tumor angiogenesis via VDRs expressed on both the transformed mammary epithelial cells and the tumor-associated endothelial cells.
DETERMINANTS OF BREAST CANCER SENSITIVITY TO VITAMIN D Ligand Availability and Metabolism Circulating vitamin D metabolites are delivered to cells via the serum vitamin D binding protein (DBP), but little is known about uptake, metabolism or half life of 25(OH)D3 or 1,25(OH)2D3 in breast cells. Catabolism of 1,25(OH)2D3 is initiated via hydroxylation at the 24 position in the side chain, a reaction catalyzed by the 25(OH)D3 24-hydroxylase (CYP24). Comparative genome hybridization studies have reported CYP24 amplification in human breast cancer [77], suggesting that enhanced catabolism of 1,25(OH)2D3 by the 24-hydroxylase, leading to reduced ligand availability to the VDR, could contribute to breast cancer. Some, but not all, studies in human tumor tissue and cell lines have supported this concept [78e80]. Furthermore, variants of CYP24 which may have altered function seem to be expressed in some cancer cells [78].
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In addition to uptake of 1,25(OH)2D3 from the circulation, some breast cancer cells retain expression of the 25(OH)D3 1a-hydroxylase (CYP27B1) and can produce 1,25(OH)2D3 from 25(OH)D3. The 1a-hydroxylase mRNA has been detected in MCF-7 and T47D cells and in human breast tumors [79,81]. However, MCF-7 cells are not growth inhibited by 25(OH)D3, suggesting that although 1a-hydroxylase is detectable in these cells, its presence is not sufficient to support growth inhibition by 25(OH)D3 [82]. One explanation might be that the relative activities of CYP24 versus CYP27B1 result in net 24-hydroxylation of 25(OH)D3, producing 24,25(OH)2D3 which is unable to trigger VDR-mediated growth inhibition. In fact, CYP27B1 splice variants that code for proteins that lack 1a-hydroxylase activity have been described in MCF-7 cells [81]. Another explanation is that delivery of the substrate 25(OH)D3 to the mitochondrial 1a-hydroxylase enzyme is limiting in MCF-7 cells. In support of this concept, in vitro uptake studies suggest that internalization of the DBPe 25(OH)D3 complex by mammary cells is facilitated by the megalinecubilin complex, which is defective in MCF-7 cells [82]. In contrast to MCF-7 cells, T47D breast cancer cells express megalin and cubilin, internalize DBP and are growth inhibited by nanomolar concentrations of 25(OH)D3 [83]. These data suggest that functional machinery to internalize serum-derived DBP enhances mammary cell response to 25(OH)D3; however, further studies to assess changes in uptake and metabolism of both 25(OH)D3 and 1,25(OH)2D3 in mammary cells and tissue as a function of transformation are necessary.
Expression, Activity, and Regulation of VDR in Breast Cancer Cells A recent study has clarified the importance of the nuclear VDR in mediating the effects of 1,25(OH)2D3 and its analogs in breast cancer cells. Zinser et al. [85] utilized cell lines derived from carcinogen-induced mammary tumors generated in VDR-null mice and wild-type control littermates to demonstrate that cells lacking nuclear VDR fail to respond to natural or synthetic vitamin D compounds. Thus, if rapid/nongenomic actions contribute to the anti-proliferative effects of 1,25(OH)2D3, the nuclear VDR is necessary for the function of those pathways. In addition to establishing that the nuclear VDR is required for the antitumor effects of 1,25(OH)2D3, these data emphasize the importance of identifying factors that govern VDR expression, function, and regulation in mammary cells. VDR abundance is affected by many physiological factors and is achieved through a variety of mechanisms, including alterations in transcription and/or
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mRNA stability, post-translational effects and ligandinduced stabilization. Expression of the VDR in cultured cells and in vivo is regulated by many physiological agents, including 1,25(OH)2D3 itself, estrogens, retinoids, and growth factors [86e90]. Thus, breast cancer cell sensitivity to 1,25(OH)2D3-mediated growth regulation may in part reflect the activity of other hormone signaling pathways through their impact on VDR expression. Comparison of a panel of breast cancer cells indicates that ERa-positive cells tend to express higher levels of VDR than ERa-negative cells [38]. Furthermore, in vitro studies demonstrate that estrogen upregulates the VDR, and anti-estrogens such as tamoxifen downregulate the VDR, in ERa-positive breast cancer cells [89,91]. In MCF-7 and T47D cells, estrogen transcriptionally upregulates the VDR promoter upstream of exon 1c [89,90]. Collectively, these data support the concept that estrogen and anti-estrogens are important regulators of VDR expression in breast cancer cells, a concept with clinical implications arising from the potential use of SERMs for prevention and/or treatment of breast cancer and osteoporosis. The efficacy and toxicity of vitamin D compounds in vivo is determined, in part, by the level of VDR in target tissues, and thus it will be important to determine the degree to which estrogen status influences VDR abundance in different 1,25(OH)2D3 target cells in vivo (i.e., breast, bone, uterus). In this respect, it will also be important to assess whether novel SERMs or phytoestrogens currently utilized by postmenopausal women act as estrogen agonists or antagonists in regulation of VDR expression. The phytoestrogen resveratrol has recently been shown to upregulate VDR and sensitize breast cancer cells to vitamin-D-mediated growth inhibition, offering proof of principle that dietary factors can impact cellular sensitivity to 1,25(OH)2D3 through regulation of VDR [89]. Additional factors that directly regulate VDR at the transcriptional level include members of the p53 family [65,92,93], the pituitary transcription factor pit-1 [94] and the transcriptional repressor slug [95]. As discussed above, tumor suppressor p53 (which is frequently mutated to loss-of-function in breast cancer) directly induces VDR transcription, and thus breast cancers that retain wild-type p53 would be predicted to express high levels of VDR and remain sensitive to vitamin-Dbased therapeutics. In contrast, slug (which is upregulated during epithelialemesenchymal transition) binds E-box sequences in the VDR promoter to directly repress transcription, thus, advanced breast cancers that have acquired the invasive phenotype would be predicted to express low levels of VDR and be less sensitive to vitamin-D-based therapeutics. Quantitative analysis of VDR, p53, and slug expression in relation to markers of tumor stage, as well as clinical trials, will be necessary to test these predictions.
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Vitamin D Resistance As noted above, the VDR is required for breast cancer cell responsiveness to vitamin D compounds, and numerous factors have been identified that alter VDR expression. Introduction of SV40 transforming proteins or oncogenic versions of ras into non-transformed mammary cell lines leads to loss of VDR function through as yet undefined mechanisms, and induces resistance to the growth-inhibitory effects of 1,25(OH)2D3 [87,96]. These data provide proof of principle that carcinogenesis may be facilitated by corruption of VDRmediated negative growth regulation and may explain why supra-physiologic concentrations of 1,25(OH)2D3 are required to induce growth arrest and differentiation in cells derived from established cancers. In fact, comparison of clinical specimens and breast cancer cell lines indicates that reduced expression of VDR coupled with increased expression of the co-repressor NCoR1 correlates with suppressed regulation of VDR target genes and is a common feature of advanced malignancy [97]. In cases where these cancer-associated changes are epigenetic, restoration of VDR expression and 1,25(OH)2D3 sensitivity may be possible with agents that target chromatin structure or DNA methylation [98]. In an effort to create a model for studying the cellular basis for insensitivity to vitamin D in the presence of VDR, Narvaez et al. [91] selected and characterized 1,25(OH)2D3-resistant subclones of MCF-7 cells. The resulting MCF-7DRES cells express VDR, but do not undergo growth arrest or apoptosis in response to 1,25(OH)2D3. MCF-7DRES cells are highly resistant to 1,25(OH)2D3 and its structural analogs, yet retain responsiveness to other anti-proliferative agents [25,91,99]. Similar results have been obtained in an independently derived 1,25(OH)2D3 resistant subclone of MCF-7 cells, labeled MCF-7/VDR [100]. The mechanisms underlying vitamin D resistance in these MCF-7 clones are incompletely understood. Theoretically, selective insensitivity to 1,25(OH)2D3 could be secondary to defective VDR, reduced availability of ligand, or uncoupling of functional VDR from growth arrest/apoptosis. While resistance could be associated with elevated expression of CYP24, which inactivates 1,25(OH)2D3, this does not appear to be the case for either of the vitamin-D-resistant MCF-7 variants. Both MCF-7DRES and MCF-7/VDR cells contain transcriptionally active VDRs when measured with consensus VDREs; however, VDR activity is lower in both resistant cell lines than in parental MCF-7 cells. In MCF-7DRES cells, 1,25(OH)2D3 comparably upregulates the steady-state level of the VDR protein in both sensitive and resistant cell lines [101]. MCF-7DRES cells can be sensitized to the growthinhibitory effects of 1,25(OH)2D3 by co-treatment with low concentrations of the phorbol ester TPA, suggesting
that phosphorylation pathways may be altered in this cell line [28,101]. Genomic and proteomic profiling supports the notion that pathways unrelated to VDR itself are disturbed during selection for 1,25(OH)2D3 resistance [52,68]. Further studies with these interesting cell lines will be necessary to resolve the mechanism(s) of vitamin D resistance, in particular the relative contribution of genetic versus epi-genetic changes. Significantly, MCF-7DRES cells display resistance to the vitamin D analog EB1089 when grown as xenografts in nude mice [102], providing an in vivo model system for studying vitamin D resistance.
Prognostic Significance of Breast Cancer VDR Expression A high proportion (>80%) of breast cancer biopsy specimens contain immunoreactive VDR [103e105] but there are no consistent correlations between VDR expression and ER expression, lymph node status or tumor grade [106,107]. VDR status does not appear to be related to overall survival or to survival after relapse; however, in a study of 136 patients with primary breast cancer, it was found that women with VDR-negative cancer relapsed significantly earlier than women with VDR-positive cancer [108].
VITAMIN D METABOLITES AND ANALOGS: PRECLINICAL AND CLINICAL TRIALS While the beneficial effects of 1,25(OH)2D3 on cancer cells support its use as a therapeutic agent, toxic side effects related to calcium handling can occur with chronic administration at the doses required for anticancer effects. Precursor metabolites (i.e., 1a(OH)D3) which are converted to 1,25(OH)2D3 in vivo have shown efficacy to inhibit tumor growth but again the therapeutic window is extremely narrow [9]. In efforts to alleviate potential calcemic toxicity, synthetic analogs of 1,25(OH)2D3 have been generated that display enhanced growth-regulatory effects with less calcium-mobilizing activity. Several vitamin D analogs have been tested in preclinical and clinical models of breast cancer including those developed by drug companies (Leo Pharmaceuticals, Chugai Pharmaceutical Co. Ltd, Hoffmann-LaRoche, BioXell) and those developed in academia [109,110]. For the most part, these vitamin D analogs display reduced calcemic activity in rodent models and inhibit growth of human breast xenografts in nude mice, chemically induced mammary tumors in rats and oncogene-driven tumors in transgenic mice [111e113].
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As an example, the well-studied Leo analog Seocalcitol (EB1089) contains a conjugated double bond system and is approximately 50 times more potent than 1,25(OH)2D3 in vitro, and when given orally it dose dependently inhibits growth of nitrosomethyl urea (NMU)-induced rat mammary tumors and MCF-7 xenografts without increasing serum calcium [102,114]. In both model systems, EB1089 induces tumor regression through inhibition of proliferation and induction of DNA fragmentation indicative of apoptotic cell death [102,115]. This analog also inhibits development of bone metastases and increases survival of mice following intra-cardiac inoculation of ER-negative MDA-MB-231 cells [116]. The beneficial effects of EB1089 on primary tumor growth in the MCF-7 xenograft model are enhanced when co-administered with paclitaxel [117], retinoic acid [118], or radiation [119]. Clinical evaluation of EB1089 was initiated with a dose-finding study in healthy volunteers, which indicated tolerance to doses in the range of 5e20 mg/day. However, a phase I trial of oral EB1089 in patients with advanced breast and colorectal cancer failed to demonstrate anti-cancer effects, although several patients (two colorectal, four breast cancer) showed disease stabilization. These and other studies provided evidence that vitamin D compounds with less calcemic activity than 1,25(OH)2D3 could be tolerated in cancer patients, but the therapeutic window for most analogs remained narrow, especially with chronic administration. The majority of ongoing clinical trials examining vitamin-D-based approaches for breast cancer therapy are employing natural vitamin D compounds rather than synthetic analogs (www.clinicaltrials.gov). Newer strategies include supplementation with vitamin D2 or D3, intermittent administration of active metabolites (including 1,25(OH)2D3), and combining vitamin-Dbased approaches with established therapies. The renewed focus on supplementation with vitamin D2 or D3 reflects the demonstration that breast cancer cells express CYP27B1, thus, increasing serum 25(OH)D3 to optimize generation of 1,25(OH)2D3 within the neoplastic tissue might inhibit disease progression. This concept has gained credence with the recognition that vitamin D insufficiency (when defined as serum 25(OH)D3 <32 ng/ml) is as high as 75% in women living with breast cancer and is more prevalent in women with advanced disease, African-Americans, and Hispanics [142,143]. In one study stratified by disease subtype, women with triple negative breast cancer (a very aggressive form of the disease) had the lowest average 25(OH) D3 levels, and the highest percentage of patients with frank deficiency were in this group [144]. In a prospective study that followed women with breast cancer for 11þ years after diagnosis, women with insufficient/deficient serum 25(OH)D3 at diagnosis had a significantly
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increased risk of distant recurrence and death compared to those with sufficient vitamin D status [143]. The subject is more fully discussed in Chapter 82. Vitamin D supplementation may also be beneficial for alleviating side effects of estrogen ablation therapies, which adversely affect bone and muscle metabolism. Khan et al. [145] examined whether 50 000 IU vitamin D weekly given to breast cancer patients with suboptimal serum 25(OH)D3 levels impacted musculoskeletal symptoms associated with the aromatase inhibitor letrozole. After 16 weeks, vitamin D intervention significantly increased serum 25(OH)D3 and alleviated joint pain with no side effects. Although data are limited at this point, high-dose vitamin D supplementation appears to be safe in breast cancer survivors, and may provide relief from side effects of anti-estrogen therapies. No studies have yet evaluated whether high-dose vitamin D supplementation alters disease progression or improves the efficacy of adjuvant therapies for breast cancer. Studies to test this concept in animal models of breast cancer are also lacking. Ooi et al. [120,121] demonstrated that severe vitamin D deficiency promotes the growth of breast cancer cell lines (MCF-7 and MDAMB-231) within the bone microenvironment, most likely via enhancement of bone resorption [120]. This degree of vitamin D deficiency, however, is more severe than that observed in women with breast cancer, and significantly disrupts the calciumePTH axis. Thus, further studies are needed to clarify the role of vitamin D status per se on the bone metastatic process.
VITAMIN D AND PREVENTION OF BREAST CANCER Expression and Role of VDR in Normal Mammary Gland A potential role of vitamin D in breast cancer prevention has been suggested based on animal, epidemiological and genetic studies (Table 85.3). In contrast to breast cancer cells, where VDR function is often disrupted, the receptor is present at high level in normal mammary gland, and its expression is developmentally regulated [3,122]. VDR expression is high throughout puberty, pregnancy, and lactation, periods of maximal tissue growth and remodeling [123,124]. The VDR has been identified in all major cell types of the gland (basal and luminal epithelial cells, cap cells, stromal cells) and is highest in differentiated cells of the ductal epithelium. The dynamic regulation of VDR in mammary gland during the reproductive cycle suggests that hormones and/or growth factors which impact on glandular development may modulate VDR expression under physiologic conditions. Indeed, lactogenic hormones
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TABLE 85.3 Summary of Evidence Linking Vitamin D to Prevention of Breast Cancer. Summary of Data Generated in Animal, Epidemiological and Genetic Studies. See Text for Details and References Approaches
Observations
Animal models
VDR and CYP27B1 are expressed in mammary epithelial cells in vitro and in vivo 1,25(OH)2D3 and 25(OH)D3 induce quiescence of human mammary epithelial cells 1,25(OH)2D3 inhibits hormonestimulated ductal growth and branching in glands from wild-type but not VDRnull mice 1,25(OH)2D3 and 25(OH)D3 inhibit carcinogen-induced pre-neoplastic lesions in mouse mammary organ culture VDR-null mice exhibit accelerated mammary gland development and increased hyperplasia in response to carcinogens/oncogenes
Human studies Clinical Epidemiological Genetic
Meta-analysis indicates significant associations between dietary vitamin D/ calcium, serum 25(OH)D3 and breast cancer risk FokI and BsmI polymorphisms in VDR linked to breast cancer risk Vitamin D insufficiency (serum 25(OH)D3 <32 ng/ml) is highly prevalent in breast cancer survivors Low serum 25(OH)D3 is associated with increased risk of recurrence and reduced survival in women living with breast cancer Women with VDR negative tumors relapse earlier than women with VDR positive tumors Vitamin D supplementation improves musculoskeletal symptoms associated with anti-estrogen therapy
upregulate VDR in normal mammary gland and nontransformed mammary cells in vitro [87,125]; however, the specific factors responsible for VDR regulation in the normal mammary gland have yet to be defined. Developmental regulation of VDR in mouse mammary cells implies that vitamin D signaling may be involved in the regulation of glandular function or protection from carcinogenesis. Non-transformed human mammary epithelial cells, which retain characteristic morphology of primary mammary epithelial cell cultures, express high levels of VDR and are dosedependently growth inhibited by 1,25(OH)2D3 [87,126]. However, in contrast to breast cancer cells, nontransformed mammary cells exhibit markers of
differentiation rather than apoptosis [16,87]. This has led to the suggestion that 1,25(OH)2D3 and the VDR induce a program of genes that inhibit proliferation and maintain differentiation in the normal gland [127]. This suggestion is supported by organ culture studies which demonstrate effects of 1,25(OH)2D3 on calcium transport, casein expression and branching morphogenesis [122,128,129]. Furthermore, mammary glands from VDR-null mice exhibit accelerated growth during puberty and pregnancy and delayed post-lactational involution compared to glands from wild-type mice [124], suggesting that VDR transmits negative growthregulatory signals under physiologic conditions. In organ culture, incubation with 1,25(OH)2D3 inhibits branching of mammary glands from wild-type mice but has no effect on glands from VDR-null mice. In addition to VDR, CYP27B1 is expressed in normal human mammary epithelial cells and in mouse mammary gland [124,126,130]. In human mammary cells, 25(OH)D3 induces CYP24 gene expression and inhibits growth at concentrations as low as 10 nM, suggesting conversion of 25(OH)D3 to 1,25(OH)2D3 by functional CYP27B1 in these cells. This concept is consistent with the lack of effect of up to 100 nM 25(OH)D3 on VDR-mediated Cyp24 gene expression in epithelial cells isolated from mammary gland of Cyp27b1-null mice [126]. However, at higher concentrations (250 nM), 25(OH)D3 does induce Cyp24 even in mammary glands from Cyp27b1-null mice, indicating that very high serum levels of 25(OH)D3 may directly trigger VDR signaling [130]. Genomic profiling conducted on non-transformed human mammary epithelial cells identified over 200 genes whose expression was altered two-fold or more after 24 h treatment with 100 nM 1,25(OH)2D3. As expected, Cyp24A1 was the most highly induced gene (>200-fold induction) and ten additional highly upand downregulated genes from this analysis are shown in Table 85.4. Several of these genes were followed up by real-time PCR analysis in an in vitro model of HME cell transformation [80] to assess the effect of transformation on cellular responsiveness to 1,25(OH)2D3. VDR expression is downregulated in HME cells expressing SV-40 large T antigen (HMESV40) and HME cells expressing both SV-40 large T antigen and activated ras (HMESV40þRas). Consistent with the reduced VDR content, the regulation of CD14 and Il1Rl1 by 1,25(OH)2D3 was significantly blunted in HMESV40 and HMESV40þRas cells relative to HME cells. Surprisingly, 1,25(OH)2D3 induction of Bmp6 was comparable in HME, HMESV40 and HMESV40þRas cells despite the reduced VDR content. These data suggest that transformation selectively alters the induction of a subset of VDR target genes in mammary cells, consistent with the genomic profiling data discussed earlier [53].
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TABLE 85.4 Top Five Up- and Downregulated Genes in Nontransformed Human Mammary Epithelial Cells Treated with 1,25(OH)2D3 Gene symbol
Gene name
Fold change
Cyp24a1
Cytochrome P450, family 24a1
206
CD14
CD14
63
ITGB3
Integrin beta 3
19
DHRS9
Dehydrogenase/reductase (SDR) family member 9
18
BMP6
Bone morphogenic protein 6
15
Upregulated genes
Downregulated genes KDR
Kinase insert domain receptor
8
RGS2
Regulator of G-protein signaling 2
5
BIRC3
Baculoviral IAP repeat containing 3
5
GLUL
Gutamine synthetase
4
SCNN1G
Sodium channel, nonvoltage-gated 1, gamma
4
RNA isolated from VDR-positive human mammary epithelial cells treated in triplicate with vehicle or 100 nM 1,25(OH)2D3 for 24 h was analyzed on Affymetrix expression arrays and analyzed with Genespring X. Shown are the top five up- and downregulated genes (out of >200 genes altered two-fold).
Collectively, these data from human and mouse systems provide evidence that 25(OH)D3, 1,25(OH)2D3, and the nuclear VDR impact on ductal elongation, branching morphogenesis and hormonal sensitivity during mammary gland development, and support the concept that 1,25(OH)2D3 and the VDR participate in pathways that inhibit proliferation and induce differentiation in the mammary gland. Furthermore, several oncogenes and tumor suppressor genes abrogate VDR function, resulting in desensitization of mammary cells to 1,25(OH)2D3 during the cancer development process.
Prevention of Breast Cancer by Vitamin D: Preclinical Studies Identification of 1,25(OH)2D3 and the VDR as components of a signaling network that impacts proliferation and differentiation in the normal mammary gland raises the possibility that optimal vitamin D status may protect against mammary transformation. In support of this suggestion, rats fed diets high in calcium and vitamin D develop fewer mammary tumors in response to the carcinogen dimethylbenzanthracene (DMBA) than
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mice fed diets low in calcium and vitamin D [131]; however, whether this difference specifically reflects vitamin D signaling is unclear since vitamin D deficiency is associated with multiple metabolic disturbances. Data from VDR-null mice (maintained on rescue diet to normalize serum calcium), however, does suggest that vitamin D signaling is tumor suppressive in mammary gland. VDR-null mice exhibit a higher incidence of mammary hyperplasia and altered tumor spectrum compared to wild-type mice when exposed to DMBA [132]. Furthermore, mice with haploinsufficiency of VDR exhibit increased tumor burden when bred onto the MMTV-neu background, a model of HER2-positive human breast cancer [133]. Vitamin D analogs, including Ro245531 (1a,25-dihydroxy-16-ene-23-yne-26-27-hexafluorocholecalciferol) and 1a-hydroxyvitamin D5, are effective in prevention of NMU-induced mammary tumors, providing further support that vitamin-D-regulated pathways may protect against breast cancer [134,135]. Direct effects of 25(OH)D3, 1,25(OH)2D3, and 1a-hydroxyvitamin D5 on the sensitivity of the mammary gland to transformation is suggested by studies indicating that all three vitamin D compounds prevent DMBA-induced pre-neoplastic lesions in organ culture [130,136].
Epidemiological Studies on Vitamin D Status and Breast Cancer The majority of women who develop breast cancer are of postmenopausal age, and estrogen deficiency and aging are often associated with vitamin D deficiency. Several recent meta-analyses have evaluated dietary calcium/vitamin D and serum vitamin D metabolites in relation to breast cancer risk. Chen et al. [137] evaluated 11 studies on dietary vitamin D and 15 studies on dietary calcium and found significant inverse relationships between both nutrients and breast cancer risk. Three meta-analyses focused on studies in which serum 25(OH)D3 was measured in relation to breast cancer risk, and all three reported an overall significant inverse relationship [137e139]. However, the data appear stronger in caseecontrol studies than in prospective studies. Clearly, data from large randomized vitamin D supplementation trials will be highly informative in elucidating the relationship between vitamin D and breast cancer risk. In the absence of such trials, two intervention studies are worth mentioning e a small pilot study by Lappe et al. in which healthy postmenopausal women were supplemented with vitamin D (1100 IU/day) and/or calcium (1500 mg/day) for 4 years [140] and an analysis of the Women’s Health Initiative to ascertain whether supplementation with vitamin D (400 IU/day) and calcium (1000 mg/day) for 7 years
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affected the development of invasive breast cancer [141]. The Lappe study reported a significant decrease in the development of all types of cancer, including breast cancer, with calcium/vitamin D intervention, although the number of cancers that developed in the study population was quite low. The Women’s Health Initiative failed to show any reduction in invasive breast cancer incidence with vitamin D/calcium supplementation. The negative results from the Women’s Health Initiative trial may relate to the relatively low dose of vitamin D supplementation used, which had limited effect on serum 25(OH)D3 in the study population.
VDR Polymorphisms and Breast Cancer Risk A number of common polymorphisms in the human VDR gene have been examined in relation to risk of breast cancer. The best-studied VDR polymorphisms include a start codon polymorphism (FokI) in exon 2, BsmI and Apa I polymorphisms in an intronic region between exons VIII and IX, a TaqI variant in exon IX and a singlet (A) repeat in exon IX. A recent meta-analysis of 67 independent studies on VDR polymorphisms and cancer indicated significant associations for the FokI and BsmI variants and breast cancer [146]. An independent meta-analysis focusing solely on breast cancer caseecontrol studies confirmed the link with FokI (eight studies) but not Bsm1 (14 studies) [147]. Although these findings are certainly intriguing, the underlying basis for an association between VDR polymorphisms and breast cancer susceptibility is currently unclear. The BsmI variant does not alter the amount, structure or function of the VDR protein produced. However, the FokI polymorphism determines the translation initiation site and therefore dictates the size of the VDR protein (424 versus 427 amino acids). In transient transfection assays with a vitamin-D-responsive reporter gene, the shorter VDR variant interacted more strongly with the transcription factor TFIIB and displayed higher potency in suppression of beta-catenin signaling than the longer VDR variant [148,149]. These data support the concept that functionally relevant polymorphisms in the VDR exist, and further studies will be required to determine how VDR genotype interacts with other indices of vitamin D status and risk factors for breast cancer. VDR polymorphisms are more fully discussed in Chapter 56.
SUMMARY AND OUTSTANDING RESEARCH QUESTIONS VDR and 1,25(OH)2D3, its natural ligand, act through multiple signaling pathways to induce growth arrest,
differentiation and apoptosis in mammary epithelial cells. Synthetic analogs of 1,25(OH)2D3 which have potent anti-cancer effects with reduced calcemic activity in animal models of breast cancer provide proof of principle that vitamin D signaling can inhibit the growth of established tumors. Ongoing clinical trials are using new strategies to target the vitamin D pathway in women living with breast cancer, a population with a high incidence of vitamin D insufficiency. In addition, laboratory and animal data support a role for vitamin D in breast cancer prevention. Studies with VDR-null mice indicate that vitamin D signaling impacts mammary gland function in a physiological context, and genomic profiling has identified hundreds of vitaminD-regulated genes in normal human mammary cells. Clinical studies and epidemiological approaches suggest that optimal vitamin D signaling may reduce risk for breast cancer in human populations. Challenges for the future include better understanding of the transport, uptake and metabolism of vitamin D steroids in breast cancer cells, the molecular mechanism of VDR action in normal mammary gland, windows of development when optimal vitamin D signaling exerts critical influences on breast cancer risk, and the importance of genetic differences in the VDR on an individual’s response to vitamin D compounds. Such understanding should provide improved insight into the design of vitamin-D-based strategies to reduce breast cancer development or improve therapeutic options for women living with the disease.
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[139] L. Yin, N. Grandi, E. Raum, U. Haug, V. Arndt, H. Brenner, Meta-analysis: Serum vitamin D and breast cancer risk, Eur. J. Cancer 1 (2010) 188e191. [140] J.M. Lappe, D. Travers-Gustafson, K.M. Davies, R.R. Recker, R.P. Heaney, Vitamin D and calcium supplementation reduces cancer risk: results of a randomized trial, Am. J. Clin. Nutr. 85 (2007) 1586e1591. [141] R.T. Chlebowski, K.C. Johnson, C. Kooperberg, M. Pettinger, J. Wactawski-Wende, T. Rohan, et al., Calcium plus vitamin D supplementation and the risk of breast cancer, J. Natl. Cancer Inst. 100 (2008) 1581e1591. [142] M.L. Neuhouser, B. Sorensen, B.W. Hollis, A. Ambs, C.M. Ulrich, A. McTiernan, et al., Vitamin D insufficiency in a multiethnic cohort of breast cancer survivors, Am. J. Clin. Nutr. 88 (2008) 133e139. [143] P.J. Goodwin, M. Ennis, K.I. Pritchard, J. Koo, N. Hood, Prognostic effects of 25-hydroxyvitamin D levels in early breast cancer, J. Clin. Oncol. 27 (2009) 3757e3763. [144] C. Rainville, Y. Khan, G. Tisman, Triple negative breast cancer patients presenting with low serum vitamin D levels: a case series, Cases J. 2 (2009) 8390.
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[145] Q.J. Khan, P.S. Reddy, B.F. Kimler, P. Sharma, S.E. Baxa, A.P. O’Dea, et al., Effect of vitamin D supplementation on serum 25-hydroxy vitamin D levels, joint pain, and fatigue in women starting adjuvant letrozole treatment for breast cancer, Breast Cancer Res. Treat. 119 (2010) 111e118. [146] S. Raimondi, H. Johansson, P. Maisonneuve, S. Gandini, Review and meta-analysis on vitamin D receptor polymorphisms and cancer risk, Carcinogenesis 30 (2009) 1170e1180. [147] C. Tang, N. Chen, M. Wu, H. Yuan, Y. Du, Fok1 polymorphism of vitamin D receptor gene contributes to breast cancer susceptibility: a meta-analysis, Breast Cancer Res. Treat. 117 (2009) 391e399. [148] G.K. Whitfield, L.S. Remus, P.W. Jurutka, H. Zitzer, A.K. Oza, H.T. Dang, et al., Functionally relevant polymorphisms in the human nuclear vitamin D receptor gene, Mol. Cell Endocrinol. 177 (2001) 145e159. [149] J.B. Egan, P.A. Thompson, M.V. Vitanov, L. Bartik, E.T. Jacobs, M.R. Haussler, et al., Vitamin D receptor ligands, adenomatous polyposis coli, and the vitamin D receptor FokI polymorphism collectively modulate beta-catenin activity in colon cancer cells, Mol. Carcinog. 49 (2010) 337e352.
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C H A P T E R
86 Vitamin D and Prostate Cancer Aruna V. Krishnan, David Feldman Stanford University School of Medicine, Stanford, CA, USA
INTRODUCTION Scope of the Problem Adenocarcinoma of the prostate gland is the most commonly diagnosed malignancy in American men, excluding skin cancer [1]. The numbers of cases may actually be substantially underestimated since clinically silent prostate cancer (PCa) is very common. In men over the age of 50, subclinical PCa is found in as many as 40% of individuals [2]. Although PCa is generally a slow-growing malignancy, mortality from this disease is nonetheless considerable. In contrast to many other malignancies, the incidence of PCa has continued to rise each year and currently PCa is the second leading cause of cancer death among US men [1]. Over the last two decades the age-adjusted mortality rate from PCa increased 7% among US Caucasian men. Since PCa rates increase with advancing age, one can expect that PCa will become an even greater problem as life expectancy continues to increase. As a result PCa has rapidly become a major public health concern not only in the USA but also worldwide.
Etiology, Treatment, and the Role of Hormonal Factors The etiologic factors associated with PCa are varied and include age and race as well as genetic, dietary, and hormonal influences [3]. Androgens are integrally involved in the regulation of prostate growth and cell proliferation [4]. Several studies have demonstrated a correlation between serum testosterone levels and increased risk of PCa [5]. Circulating androgens may serve as factors that promote tumor growth and perhaps neoplastic transformation [4]. Indeed, the withdrawal of androgens causes prostate involution and apoptosis of
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10086-1
normal as well as malignant prostatic epithelial cells [5,6]. This provides the basis for the use of androgen ablation in the treatment of clinically advanced PCa and androgen deprivation therapy (ADT) remains the mainstay of PCa treatment after failure of primary therapy [7]. At the outset of this chapter, a brief overview of therapeutic considerations for PCa would be useful. Early diagnosis of PCa is often made by screening for serum prostate-specific antigen (PSA) level, one of the most useful cancer biomarkers. A digital rectal exam (DRE) may also indicate prostate enlargement and/or abnormal texture. When indicated, prostate biopsy is performed to confirm the presence of cancer. If the cancer is diagnosed as being confined within the prostate gland, choices for primary therapy are prostatectomy or radiation using external beam or radioactive seed implantation. If the biopsy reveals minimal disease with low invasive potential, or if the cancer is diagnosed in elderly men or in those with serious co-morbidities, the therapeutic choice may be the careful monitoring of progress (watchful waiting/active surveillance) with assessment of serum PSA level and re-biopsy over time. If the cancer is within the prostate capsule, surgery or radiation may lead to a cure. If the cancer had already escaped the capsule, primary therapy will likely fail and eventually serum PSA will again be found to rise indicating that the cancer has spread. ADT almost always leads to cancer regression, whether treating cancer recurrence following primary therapy or in those men with metastatic cancer. Deprivation of androgens can be accomplished by orchiectomy or more commonly today by pharmacological means using gonadotropinreleasing hormone analogs such as lupron, zoladex, or other drugs that inhibit leutinizing hormone release. Additional therapy may include the use of antiandrogen drugs (androgen antagonists) such as
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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flutamide (Eulexin) or bicalutamide (Casodex) that block binding of androgens to the androgen receptor (AR). A recently completed trial suggested that the 5a-reductase inhibitor finasteride (Proscar), which blocks the synthesis of the potent androgen dihydrotestosterone (DHT) from testosterone, might reduce PCa incidence although the cancers that did occur might be more aggressive [8]. Recent follow-up did not indicate increased aggressiveness, although the debate about the role of 5a-reductase inhibitors in PCa prevention and treatment continues [9]. Initially almost all patients respond to ADT. However, many if not all patients eventually fail as the cancer develops the ability to grow in the absence of androgens [10,11]. This is called androgen-independent prostate cancer (AIPC) or hormone refractory prostate cancer (HRPC) or, more recently, castrate-resistant prostate cancer (CRPC). We will use the term CRPC while referring to studies in PCa patients and animal models and the term AIPC while referring to observations in PCa cell cultures. CRPC is the progressive and metastatic form of the disease and unfortunately it is not amenable to current therapies. This transition of PCa to CRPC remains both a therapeutic as well as an experimental challenge [12]. Growth of the normal or malignant prostate is dependent on androgens. Circulating testosterone enters the prostate through the megalin-dependent endocytic pathway [13] and is converted to the more potent DHT within the prostate by the enzyme 5a-reductase. Testosterone or DHT acts via the classical AR to stimulate growth, cell survival and the expression of PSA and other androgen-regulated genes. Most cases of CRPC retain AR and AR remains the “driver” of PCa growth. There are several molecular mechanisms underlying CRPC development including: (1) amplification of the AR gene which increases AR sensitivity to even low levels of androgens; (2) mutations in the AR gene that broaden ligand specificity so that non-androgens or even androgen antagonists can stimulate prostate growth; (3) androgen-independent activation of the AR by other growth factor signaling pathways or activation of AR coactivators; (4) synthesis of androgens locally within the prostate driving AR and growth in a paracrine manner; and (5) pathways that are independent of AR [11]. Evidence is now accumulating for the etiologic role of AR gene mutations in the pathogenesis of many cases of CRPC [10e12,14]. The presence of missense mutations in the AR gene renders the AR “promiscuous” and allows the inappropriate activation of the AR by nonandrogen steroids as well as AR antagonists leading to CRPC [10e12,14e19]. Non-androgen steroid hormones such as estrogens, progestins as well as glucocorticoids may activate AR containing mutations in its ligandbinding domain and thereby stimulate the growth of
PCa cells that harbor such promiscuous mutant ARs [11,20e23]. Some of these AR mutations also enable AR antagonists to acquire agonistic activity leading to a failure of androgen ablation therapy [24,25]. New pathways for the development of CRPC continue to be defined [26]. Other steroid hormones such as progestins and estrogens may play a role in PCa. Receptors for both progesterone and estrogen have been observed in prostatic tumors [27]. The presence of estrogen receptor (ER)-a and -b has been demonstrated in normal prostate, dysplasia and cancer [28]. Estrogens and selective estrogen receptor modulators (SERMs) have been shown to modulate growth and apoptosis in PCa cell lines [29]. Estrogens stimulate the growth of the LNCaP, human PCa cell line [30] by acting as pseudo-androgens through the mutated AR present in these cells. A selective ER antagonist ICI 182,780 fulvestrant (Faslodex) inhibits the growth of human PCa cells [31,32] and significantly downregulates AR expression in these cells [31]. Phytoestrogens such as genistein, daidzein, and equol also inhibit the growth of human PCa cells [33,34]. However, in addition to acting through their own receptors, estrogens, SERMs, progestins, glucocorticoids and other steroid hormones can activate mutated ARs in PCa cells or tumors harboring promiscuous AR mutations, and thereby modulate cancer cell growth [11]. One of the goals of current research on PCa and CRPC is the identification of new agents that would prevent and/or slow down the progression of this disease. In recent years vitamin D has emerged as a promising therapeutic agent [35e45].
Epidemiology Sunlight Exposure Based upon epidemiological studies, several risk factors for PCa have been identified including age, race, and genetics [6,46]. It has long been appreciated that solar radiation can decrease the mortality rates of non-cutaneous malignancies [47,48]. Age is the strongest risk factor for PCa, and the elderly are frequently vitamin-D-deficient due to several factors including less exposure to UV radiation (see Chapter 53). Of particular interest is the hypothesis put forward by Garland and Garland [49] and Schwartz and colleagues [50,51] suggesting a role for sunlight (a surrogate for vitamin D) in decreasing the risk of developing different cancers. These hypotheses are based upon the observation that colon cancer and PCa mortality rates are inversely proportional to the geographically determined incident UV radiation exposure from the sun, and that UV light is essential for vitamin D synthesis [51,52]. It may also offer a potential explanation of why African-American men have a higher incidence of PCa than Caucasian
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PROSTATE AS A TARGET FOR VITAMIN D
men [53]. African-American individuals have lower serum 25(OH)D levels as a result of their darker skin pigmentation, because melanin in darkly pigmented skin absorbs UV radiation, inhibiting the formation of vitamin D3 in the basal layer of the epidermis [54]. The findings of a study of patients with PCa and benign prostatic hypertrophy (BPH) [55] suggest that susceptibility to PCa is in part determined by the extent of exposure to UV radiation and that the ability to form pigment in the skin modulates this effect. Diet and Serum Levels of Vitamin D Metabolites Dietary forms of vitamin D include vitaminD-supplemented milk and other foods, ergocalciferol (vitamin D2) in plants, and vitamin D3 in animal products. Diet is considered to be a risk factor for PCa, and it has been proposed that the low risk of PCa for indigenous Japanese is related to their traditional diet [56]. This diet, among other attributes, is rich in oily fish, which is an important dietary source of vitamin D3. Some epidemiological studies show that high levels of dietary calcium are a significant risk factor for PCa [57,58]. This may be relevant to the relationship of vitamin D and PCa because high levels of serum calcium suppress parathyroid hormone and reduce the renal production of 1a,25-dihydroxyvitamin D3 (1,25(OH)2D, calcitriol), the active hormonal form of vitamin D. These observations provide indirect support for the possible protective role of high vitamin D levels on PCa. However, some studies do not find an association between vitamin D intake and PCa risk [59]. Corder et al. [60] undertook a prospective caseecontrol study and determined the levels of vitamin D metabolites in stored sera collected in the San Francisco Bay area between 1964 and 1971 and matched for age, race, and day of serum storage. Mean levels of calcitriol, the active metabolite of vitamin D, were slightly but significantly lower in men who went on to develop PCa when compared to controls who did not develop cancer. But other pre-diagnosis studies [61,62] did not find a correlation between levels of circulating vitamin D metabolites and subsequent development of PCa. However, in a further analysis of their data, Corder et al. [63] subsequently found that calcitriol levels showed a seasonal variation in PCa cases but not in controls, with a nadir in summer months. This may explain the lack of an effect in the Braun study due to the fact that serum collections occurred mainly in the fall [61], and as a result might have missed a nadir in levels among individuals that went on to develop PCa. A nested caseecontrol study in Japanese-American men conducted in Hawaii [64] also did not find a strong association between serum calcitriol levels and the incidence of PCa. Many later studies suggest that vitamin D deficiency, characterized by low
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circulating levels of the pro-hormone 25-hydroxyvitamin D (25(OH)D), is associated with increased PCa risk and progression [65e68], although all studies do not support such a correlation [69,70]. Several more recent studies also do not support a correlation between serum 25(OH)D levels and the risk of developing PCa [71e73]. Other studies also find no evidence of increased risk for multiple other cancers in vitamin-D-deficient populations compared to sufficient subjects [74,75]. However, it should be noted that these types of studies all compare the lowest levels found in their population with the highest levels found and do not necessarily have a comparator to a cohort with high enough 25(OH)D levels to inhibit cancer development. The vitamin-D-binding protein (DBP) may modulate vitamin D action by controlling the levels of free 25(OH) D or calcitriol available to activate the VDR. As such, it may play a role in the etiology of PCa. However, studies in this area have also produced divergent results. Corder et al. [63] failed to find an association with DBP and PCa in their cohort of subjects. In a group of 68 men with PCa, Schwartz et al. [76] found that DBP levels were significantly higher in individuals with PCa compared to controls. Polymorphisms in the DBP molecule may alter its ability to bind 25(OH)D and play a role in altering cancer risk (see Chapter 5). As in other target tissues, the mechanism of action of calcitriol in the prostate involves hormone action through the classical ligand-dependent activation of genes via the vitamin D receptor (VDR), although some actions are proposed to be mediated via a membrane receptor [77]. Several polymorphisms have been identified in the VDR gene that may contribute to the risk of osteoporosis and some of these polymorphisms may contribute to PCa risk as well [78] (see also Chapter 56).
PROSTATE AS A TARGET FOR VITAMIN D Vitamin D is an Anti-proliferative and Pro-differentiation Agent Although the role of calcitriol in maintaining calcium homeostasis has been known for a long time, it is only recently that investigators have begun to understand the broader scope of calcitriol actions [79]. Calcitriol exhibits anti-proliferative and pro-differentiation actions in a number of tumors and malignant cells including PCa cells (Chapter 83), raising the possibility of its use as a therapeutic agent in PCa [35e45]. The finding by Miller et al. [80] of the presence of VDR in LNCaP human PCa cells and the demonstration of VDR and the anti-proliferative actions of calcitriol in
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three different PCa cell lines including LNCaP, PC-3, and DU 145 cells by Skowronski et al. [81] were important early findings that suggested that calcitriol might play a direct role in prostate biology and might be beneficial in PCa.
VDR in the Normal Prostate Although the initial description of VDR in the prostate and most of the subsequent investigation of calcitriol actions have centered around PCa cell lines, calcitriol also appears to play an important role in normal prostate tissue. Peehl et al. [82] reported the presence of VDR in freshly obtained surgical prostate specimens as well as primary cultures of epithelial and stromal cells of the prostate. Characterization of these cultures revealed that the primary epithelial cells exhibited features of both basal cells and differentiated secretory cells of the benign prostatic epithelium [83]. Although PCa is characterized by luminal cell expansion and the absence of basal cells, a recent study shows that basal cells from primary benign prostate tissue can initiate PCa in immunodeficient mice, demonstrating that histological characterization of PCa does not necessarily correlate with the cellular origins of PCa [84]. Primary cultures from surgical specimens of benign prostatic hyperplasia (BPH) also expressed VDR [82]. Although VDR was present in both epithelial and stromal cells cultured separately, lower levels of VDR were seen in the stromal fibroblasts compared to cells of the glandular epithelium. The region of origin within the prostate tissue did not influence the abundance of VDR, as both the peripheral zone and central zone cultures had similar amounts of VDR [82]. Krill et al. [85] studied VDR expression in normal prostate glands from donors of various age groups and found that VDR expression changed with age with peak levels in the fifth decade and a decline thereafter. Calcitriol exerts anti-proliferative effects on rat neonatal prostatic epithelial cells [86] and human prostatic epithelial cells [87]. Konety et al. [86,88] showed that exposure of rat pups in utero to calcitriol influenced prostatic growth and differentiation throughout the lives of the animals. In general, all these studies support a role for vitamin D in normal prostate physiology and growth.
calcitriol at concentrations from 1011 to 109 M was slightly stimulatory to cell growth, when the cells were cultured in media supplemented with charcoal-stripped serum depleted of endogenous androgens, conditions under which LNCaP cells grew very poorly. A subsequent study by Skowronski et al. [81] demonstrated the presence of VDR in LNCaP cells as well as two other human prostate cancer cell lines DU 145 and PC-3 and found calcitriol exerted growth-inhibitory effects upon these cell lines (Fig. 86.1). The growth inhibition due to calcitriol in LNCaP cells was dose-dependent and quite striking (~60%) in regular growth medium containing 5% fetal bovine serum (FBS) that supported robust cell growth. This study provided the basis for considering calcitriol an inhibitor of PCa growth. The discrepancy between calcitriol effects on the growth of LNCaP cells reported in these studies compared to the Miller study may be due to differences in the culture conditions. Indeed as shown in a later study by Zhao et al. [89], androgens present in serum in the growth medium influenced the effect of calcitriol on LNCaP cell growth (see “Calcitriol and androgen interactions,” below). Esquenet et al. [90] also reported significant inhibition of LNCaP cell growth by calcitriol in the presence of androgens. The study by Skowronski et al. [81] demonstrated growth inhibition by calcitriol in other PCa cells such as PC-3 and DU 145 (Fig. 86.1). The magnitude of growth inhibition was less in PC-3 cells (~40e50%) when compared to LNCaP cells, and the growth inhibition seen in DU 145 cells was minimal. Interestingly, the 120 DNA content (% control)
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VITAMIN D AND INHIBITION OF PROSTATE CANCER GROWTH In Vitro Studies in Prostate Cells Cancer Cell Lines Miller and coworkers [80] demonstrated the presence of VDR in LNCaP human PCa cells. In their study,
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0.1 1 10 1,25(OH)2D3 (nM)
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FIGURE 86.1 Calcitriol inhibits the growth of three human prostate cancer cell lines. LNCaP (circles), PC-3 (triangles) and DU-145 (squares) cells growing in their respective growth media containing FBS were treated for 6 days with either vehicle alone or indicated concentrations of calcitriol. Proliferation rate was assessed by determination of DNA content. Values shown are means SD. From [71] with permission.
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vitamin D catabolic enzyme 25-hydroxyvitamin D 24-hydroxylase (24-hydroxylase or CYP24) was induced by calcitriol maximally in DU 145 and substantially in PC-3 cells while the induction of 24-hydroxylase mRNA was not detected in LNCaP cells [81]. Thus as discussed in “Cellular responsiveness to vitamin D compounds e role of enzymes involved in vitamin D metabolism” below, there appears to be an inverse correlation between calcitriol induction of 24-hydroxylase and calcitriol-mediated growth inhibition in PCa cells [81,91]. Since then, calcitriol has been shown to inhibit the growth of a number of other androgen-dependent and androgen-independent PCa cell lines such as ALVA 31 and PPC-1 [91], MDA PCa 2a and 2b [92], LAPC-4 [93,94] and C4-2 [93,95]. The recently identified TMPRSS:ERG gene fusion occurs in a high percent of prostate cancers and plays a role in PCa growth and invasiveness [96]. Weigel and colleagues examined the effect of calcitriol on the growth of VCaP cell line that contains a fusion of TMPRSS:ERG as well as a normal copy of each gene [97] and found that calcitriol and its synthetic analog EB 1089 inhibited the growth of VCaP cells in spite of increasing TMPRSS:ERG mRNA levels in these cells [98]. Primary Prostate Cells The use of primary cultures of prostate cells from prostatectomy samples provides an important tool to study cancer biology as they may be more closely related to the clinical setting than cancer cell lines established from metastases. Peehl et al. [82] generated epithelial cultures from surgical specimens obtained during prostatectomy. Variable levels of VDR abundance were seen among the different cultures, which did not correlate with histological grade of the tumors. As shown in Figure 86.2, in clonogenic growth assays, calcitriol exhibited a substantial inhibition (ED50 ¼ 0.25 1 nM) of the growth of these primary epithelial cells derived from the peripheral and central zones of normal prostate as well as from BPH and cancer. Interestingly the growth inhibition was irreversible after withdrawal of calcitriol from the medium [82]. In contrast, the growth inhibition seen in LNCaP cells was reversible [90]. The abundance of VDR was less in the stromal fibroblasts and the growth inhibition by calcitriol was also less when compared to the epithelial cultures [82]. The significance of the presence of VDR in the stromal fibroblastic component of PCa is uncertain. However, there is evidence that the stromal component may influence prostate epithelial cell growth through the production of growth factors that act in a paracrine manner [99,100]. Calcitriol may potentially regulate stromal production of some of these growth factors and thereby control the growth of prostatic epithelium [101,102].
120 P e r 100 c e n t 80
E-PZ-2 E-PZ-6 E-CZ-3 E-CZ-4 E-BPH-2 E-BPH-3 E-CA-11
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0.25 2.5 25 0.0025 1,25-(OH)2 VITAMIN D3 (nM)
Calcitriol inhibits the growth of normal and malignant primary prostatic epithelial cells. Primary prostate epithelial cell strains were inoculated into collagen-coated dishes containing serumfree medium with the indicated amounts of calcitriol. After 10 days of incubation, the cells were fixed and stained, and growth was measured with an Artek image analyzer. Growth of each cell strain in medium without vitamin D was standardized as 100%. Points represent average of duplicate dishes. E-PZ and E-CZ are normal prostatic epithelial cell strains. E-BPH represents epithelial cell cultures derived from BPH specimens. E-CA refers to epithelial cell cultures generated from prostatic carcinoma. From [72] with permission.
FIGURE 86.2
Virally Transformed Prostate Cells The large T antigen of simian virus 40 (SV40) and the E6 and E7 proteins of the human papilloma virus (HPV) cause disruptions in the p53 and retinoblastoma (Rb) genes [103,104] and have been used to transform prostate cells into immortalized cell lines [105,106]. Investigators have evaluated VDR status and calcitriol actions in both SV40- and HPV-transformed cell lines and have shown that high-affinity VDRs were present in the SV40 and HPV cell lines and that 24-hydroxylase mRNA is induced by calcitriol treatment of these cell lines [105,107,108]. However, the effect of calcitriol on growth is different between these transformed cell lines. Whereas the growth of HPV-transformed cell lines is inhibited by calcitriol, the SV40-transformed cell lines are resistant to calcitriol action in terms of an anti-proliferative response. As discussed in the following section, the expression of the large T antigen appears to cause resistance to calcitriol action on proliferation in prostate epithelial cells due to an inhibition of the transcriptional activity of VDR [109]. Resistance of Some Prostate Cells to Growth Inhibition by Calcitriol While many of the established PCa cell lines or primary cultures of adenocarcinoma-derived cells from PCa patients respond to calcitriol, there is evidence
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that some PCa cells may become resistant to the anti-proliferative activity of calcitriol. This resistance can develop due to several possible mechanisms: (1) the loss or decreased expression of VDR [110], (2) VDR polymorphisms that diminish its function, (3) elevated expression of VDR co-repressors, (4) increased expression of enzymes that metabolize calcitriol [81,91], or (4) by other unknown means. An example of a loss of response to calcitriol due to the loss of VDR is seen in the cancer cell line JCA-1, which does not respond to calcitriol with growth inhibition [110]. VDR is not detectable in these cells and stable transfection of the cells with VDR cDNA makes these cells sensitive to growth inhibition by calcitriol [110]. As discussed above, transformation of prostatic epithelial cells by SV40 but not HPV results in a loss of growth-inhibitory effect of calcitriol in the transformed cells. Human breast epithelial cells transformed by the SV40 large T antigen also became resistant to calcitriol [109], demonstrating the generality of this finding. In breast epithelial cells the expression of the large T antigen strongly inhibited calcitriol-induced VDR transcriptional activity in a dose-dependent manner and increasing the VDR concentration could reverse the inhibitory effect of the large T antigen and restore the sensitivity of the cells to growth inhibition by calcitriol [109]. Recently Epstein-Barr virus (EBV) encoded EBNA-3 protein was found to bind to the VDR and block its activation [111] explaining earlier findings that showed that EBV-immortalized cells also failed to respond to calcitriol [112].
Cellular Responsiveness to Vitamin D Compounds e Role of Enzymes Involved in Vitamin D Metabolism The key enzymes involved in vitamin D metabolism are 24-hydroxylase (CYP 24), which catalyzes the initial step in the conversion of calcitriol to less active metabolites (see Chapter 4) and 1a-hydroxylase (CYP 27B1), which catalyzes the synthesis of calcitriol from 25(OH)D3 (see Chapter 3). As discussed in the following sections, the level of expression of these enzymes in target cells such as PCa cells influences the magnitude of the growth-inhibitory responses to vitamin D metabolites. 25-Hydroxyvitamin D3 24-hydroxylase (CYP24) Calcitriol induces the expression of 24-hydroxylase in target cells, which catalyzes the initial step in the conversion of the active molecule calcitriol into less active metabolites. The enzyme also converts 25(OH)D substrate to 24,25(OH)2D preventing its activation to 1,25(OH)2D. In prostate cells, the degree of growth
inhibition by vitamin D appears to be inversely proportional to the 24-hydroxylase activity in the cells. Among the human PCa cell lines DU 145, PC-3, and LNCaP, DU 145 cells exhibit the highest level of 24-hydroxylase and are the least responsive to calcitriol in terms of growth inhibition [81,91]. On the other hand, the basal and induced expression of 24-hydroxylase is very low in LNCaP cells and growth inhibition by calcitriol is substantial. In DU 145 cells, addition of liarozole (an imidazole derivative that inhibits P450 hydroxylases) causes significant inhibition of 24-hydroxylase activity. This action leads to an increase in calcitriol half-life in DU 145 cells and thereby allows a substantial calcitriol anti-proliferative effect [113]. In many target cells, including PCa cells [114], calcitriol causes homologous upregulation of VDR levels [115,116]. Prolongation of calcitriol half-life (due to the inhibition of 24-hydroxylase by liarozole) also leads to an enhanced upregulation of VDR in DU 145 cells making them more responsive to calcitriol [113]. Thus the combination of calcitriol and liarazole resulted in a substantial inhibition of DU 145 cell growth as a result of both an extension of the calcitriol half-life and enhanced VDR upregulation. Miller et al. [91] also demonstrated that the differences in calcitriol-mediated growth inhibition between various PCa cell lines correlated inversely to 24-hydroxylase expression in these cells. In primary human PCa cells, the use of the P450 inhibitor ketoconazole [117] potentiates the growth-inhibitory effects of calcitriol or its structural analog EB 1089 by inhibiting the 24-hydroxylase activity in these cells [118]. The soy component genistein and other isoflavanoids modulate the availability of calcitriol in DU 145 cells by reducing the expression of 24-hydroxylase [119]. The suppression of 24-hydroxylase expression appears to be due to regulation at the transcriptional level [119]. Interestingly, genistein also causes a direct non-competitive inhibition of the enzymatic activity of 24-hydroxylase and a prolongation of calcitriol half-life in DU 145 cells, thereby rendering these cells sensitive to growth inhibition by calcitriol [114]. Thus, combinations of calcitriol with inhibitors of 24-hydroxylase may enhance its anti-tumor effects in PCa therapy (see “Calcitriol in combination with other agents”). Muindi et al. recently showed that a combination of calcitriol, ketoconazole, and dexamethasone suppressed the clonogenic survival and enhanced the growth inhibition observed with calcitriol alone in the PC-3 human PCa xenograft mouse model [120]. In this model, dexamethasone also served to minimize calcitriol-induced hypercalcemia. Dexamethasone further served as glucocorticoid replacement for the Addisonian state induced by ketoconazole inhibition of steroidogenic biosynthesis due to its inhibition of cytochrome P450 enzymes in the adrenal glands [117,120]. A new class of 24-hydroxylase inhibitors is being
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developed to enhance the actions of vitamin D analogs to inhibit cancer growth and to treat chronic renal disease [121,122] (see Chapter 80). An interesting study that used array comparative genomic hybridization to facilitate oncogene identification resolved two regions of amplification within an approximately 2 Mb region of recurrent aberration at chromosome 20q13.2 in breast cancer. A known putative oncogene ZNF217 mapped to one peak, and CYP24A1 (encoding vitamin D 24hydroxylase), whose overexpression is likely to lead to abrogation of growth control mediated by vitamin D, mapped to the other [123]. These findings led the authors to suggest that the gene encoding 24-hydroxylase should be regarded as an oncogene. 25-Hydroxyvitamin D3 1a-hydroxylase (CYP27B1) The active hormone calcitriol is formed in the kidney by the hydroxylation of 25(OH)D3 at the C-1 position by the enzyme 1a-hydroxylase (see Chapter 3). The kidneys are the major source of circulating calcitriol in the body. In recent years, however, the presence of extra-renal 1ahydroxylase has been demonstrated which contributes to the local production of calcitriol within various tissues (see Chapter 45). Schwartz et al. [124] showed that normal human prostatic epithelial cells express 1a-hydroxylase. They raised the possibility that treatment with 25(OH)D3 could potentially inhibit the growth of PCa, due to local production of calcitriol within the prostate, thus avoiding the systemic side effect of hypercalcemia due to calcitriol administration. The ability of 25(OH)D3 to cause hypercalcemia is much reduced compared to calcitriol because of its much lower affinity for the VDR. Results of a study by Barreto et al. [125] support this hypothesis by demonstrating the growth-inhibitory effect of 25(OH)D3 in primary epithelial cell strains derived from normal human prostatic peripheral zone. Hsu et al. [126] showed that epithelial cells from normal prostate had more 1a-hydroxylase activity than those derived from BPH or cancer. The anti-proliferative effect of 25(OH)D3 correlated with the endogenous 1a-hydroxylase activity in these cells. The growth of primary epithelial cells from normal tissue or BPH was inhibited by 25(OH)D3 to an extent similar to calcitriol as it could be converted to calcitriol by endogenous 1a-hydroxylase activity. In contrast, in primary epithelial cells from cancer or in the LNCaP human PCa cell line, with very low endogenous 1a-hydroxylase activity, the anti-proliferative action of 25(OH)D3 was much less pronounced compared to calcitriol. Whitlatch et al. [127] similarly found reduced 1a-hydroxylase activity in PCa cells compared to normal prostatic cells. Although these studies reported that 1a-hydroxylase activity was reduced in malignant prostate cells, it was still measurable (except in LNCaP cells) and the biological effect of
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25(OH)D in a reporter assay at concentrations similar to calcitriol was demonstrated in PC-3 cells transfected with the VDR [128]. Later studies found equivalent levels of 1a-hydroxylase mRNA and protein but not enzyme activity in normal versus malignant primary prostate cells [129]. Chen et al. suggested that the reduced enzyme activity might result from decreased 1a-hydroxylase promoter activity in the cancer cells [130]. In contrast, 1a-hydroxylase activity is higher in some other malignancies such as parathyroid carcinoma [131] and colon cancer [132]. The finding of reduced 1a-hydroxylase activity in cancer-derived prostatic epithelial cells suggests that this difference may endow the malignant cells with an intrinsic growth advantage because of the resultant decrease in the local production of the growth-inhibitory molecule calcitriol allowing cellular de-differentiation and invasion that are the hallmarks of malignancy. The presence of 1a-hydroxylase in prostate cells raises the possibility that treatment with vitamin D could potentially inhibit PCa growth due to its conversion within the prostate to calcitriol, which would then act in an autocrine/paracrine manner to exert antiproliferative effects. The presence of 1a-hydroxylase in the prostate therefore is the critical factor in resolving the conundrum of why circulating 25(OH)D level and not calcitriol level is the major factor correlated with reducing PCa risk. Unlike the renal enzyme, the prostate 1a-hydroxylase is not regulated by PTH and calcium [133]; therefore circulating 25(OH)D levels determine the extent of its conversion to calcitriol within the prostate and thereby the magnitude of local paracrine actions. These paracrine actions would likely occur without increasing circulating calcitriol levels. Thus, this hypothesis provides the basis for the protective effect of sunlight and dietary vitamin D in PCa, where serum concentrations of 25(OH)D and not calcitriol are relevant to reduced risk and benefit. This hypothesis also raises the possibility that dietary vitamin D will be useful in the chemoprevention and/or treatment of PCa. Along these lines, Chen discusses the possibility that 1a-hydroxylase is actually a tumor suppressor gene [134].
Calcitriol and Androgen Interactions Androgens acting through the AR regulate prostate growth and play an important role in the development and progression of PCa [4]. In vitro studies have shown that there is cross talk between calcitriol and androgen signaling in the androgen-responsive PCa cell line LNCaP [89,135]. Calcitriol upregulates AR gene expression at both mRNA and protein levels and also increases PSA expression in LNCaP cells [135,136]. The secretion of PSA by LNCaP cells is synergistically enhanced
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when the cells are exposed to a combination of androgens and calcitriol, probably due, in part, to the upregulation of AR by calcitriol [135]. The anti-proliferative action of calcitriol in LNCaP cells appears to be androgen dependent as it could be blocked by the AR antagonist bicalutamide [89]. Bicalutamide reduces the induction of APRIN (AS3) by calcitriol, suggesting that it may be responsible for bicalutamide reversal of calcitriol-mediated growth inhibition in LNCaP cells [93]. cDNA microarray analyses of treated LNCaP cells reveal that several of the calcitriol-regulated target genes in these cells that modulate cell growth are also androgen-responsive genes [137]. These observations might suggest the possibility that prior or concomitant androgen ablation might make calcitriol or its analogs less effective in the treatment of CRPC. The androgen-dependent mechanism of calcitriol action may be specific to LNCaP cells. Calcitriol also inhibits the growth of several other PCa cells that do not express the AR [81,82]. Calcitriol inhibits the growth of other AR-positive PCa cells such as MDA PCa 2a and 2b [92] and LAPC-4 and 22Rv1 [93]. The AR blocker bicalutamide does not reverse the calcitriol-mediated growth inhibition in these cells, indicating that calcitriol action through the VDR is independent of AR activity in these cells. Further, calcitriol inhibits the proliferation of LNCaP-derived androgen-independent sublines (capable of tumor formation in castrated nude mice) such as LNCaP-104R1 [138], C4-2 and LN3 [93] growing in medium containing charcoal-stripped serum depleted of endogenous androgens. Thus the mechanism of calcitriol-mediated growth inhibition appears to be cell-specific and depends on a combination of AR-dependent and AR-independent pathways, suggesting that androgen ablation will not eliminate the utility of calcitriol in the treatment of most PCa.
Studies in Animal Models Although several rodent models of PCa have been developed [139e142], there is still a lack of a perfect model for human PCa. Several researchers have developed human PCa xenograft models by transplanting clinical specimens of human PCa or cultured human PCa cells into immunodeficient mice [143]. Using these animal models investigators have attempted to validate the in vitro observations on the growth-inhibitory actions of vitamin D compounds in PCa cells [35e45]. As discussed in detail in several sections of this book, the concentrations of calcitriol required for a significant anti-proliferative effect in vivo can cause hypercalcemia as a side effect. Therefore, investigators have used structural analogs of calcitriol, which exhibit reduced hypercalcemic effects, in several in vivo animal studies as well as in clinical trials. Schwartz et al. [144] showed that
administration of the vitamin D analog 1,25-dihydroxy16-ene-23-yne-vitamin D3 to mice bearing PC-3 xenografts resulted in a 15% decrease in tumor volume without significant increases in serum calcium levels. Blutt et al. [145] demonstrated that intraperitoneal injections of the analog EB 1089 reduced the growth of LNCaP tumors in a nude mouse xenograft model. EB1089 also retards the growth of tumors in the androgen-independent LNCaP C4-2 tumor xenograft model [146]. Vegesna et al. reported inhibition of LNCaP xenograft growth in nude mice by three different vitamin D analogs without increases in serum calcium levels [147]. Other models of PCa have been developed in rats, mice, and dogs (reviewed in [142]). One of the first established and widely used models is the Dunning rat model [148]. The original Dunning R-3327 was a spontaneous rat prostatic adenocarcinoma and a variety of sublines have been developed from it including the highly metastatic MAT LyLu subline which when injected into rats formed highly metastatic tumors [149]. Getzenberg et al. [139] demonstrated that calcitriol and the analog RO 23-6760 inhibited the growth of the MAT LyLu tumors in rats. Other analogs of calcitriol have also been shown to retard tumor growth and metastasis in MAT-LyLu tumor model [150,151]. Oades et al. [152] tested the effects of calcitriol and its analogs EB1089 and CB1093 to retard PCa growth in vivo. Calcitriol and both analogs inhibited the growth of MAT LyLu tumors in Copenhagen rats (Fig. 86.3A). The effect of EB1089 on the growth of LNCaP xenografts in athymic nude mice was also evaluated, and the results showed an ~50% reduction in tumor volume in mice treated with EB 1089 for 3 weeks (Fig. 86.3B). The calcemic effect of the analogs was significantly less than calcitriol [152]. Investigators have attempted to explore the chemopreventive activity, if any, of vitamin D compounds in animal models. Transgenic models of PCa have been developed in mice to explore molecular processes underlying PCa initiation, progression and metastasis and to evaluate the effects of potential therapeutic and chemopreventive agents (reviewed in [142,153]). In the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model, the prostate-specific rat probasin promoter is used to drive the expression of the SV 40 large-T antigen coding region and mice expressing the transgene display progressive forms of prostatic disease that histologically resemble human PCa [140]. Another model that has been used to test the chemopreventive effect of vitamin D metabolites and analogs is the G gamma/T-15 transgenic mouse line containing the human fetal globin promoter linked to SV40 T antigen (Tag), which drives the development of CRPC. Although the administration of the analog EB1089 did not alter the onset of tumors in these mice, it slowed the rate of tumor
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growth [154]. Xue et al. [155] fed rats a high-fat, lowcalcium and low-vitamin-D diet which resulted in the hyperproliferation of the dorsal prostate epithelium which could promote tumorigenesis. Increasing the
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FIGURE 86.3A Calcitriol and analogs inhibit the growth of Mat LyLu tumors in Copenhagen rats. The tumor volume in Copenhagen rats inoculated with 1 105 Mat LyLu cells that were treated from 1 day afterward with 1 mg/kg calcitriol (closed circles) in sesame oil, or 2.5 mg/kg CB1093 (closed square) or EB1089 (open square) in propylene glycol:Na2HPO4 (80:20) intraperitoneally for 5 days/week. Control animals (open circles) received vehicle alone. There were ten animals in each group and the error bars represent SEM, with ) p < 0.001 by ANOVA. From [145] with permission.
levels of calcium and vitamin D in the diet inhibited hyperproliferation, providing evidence for the antitumor activity of calcium and/or vitamin D in the diet. Studies using Nkx3.1; Pten mutant mice revealed that calcitriol administration significantly decreased the formation of the pre-cancerous lesions known as prostate intraepithelial neoplasia (PIN) in the mutant mice [156]. Furthermore calcitriol was maximally effective when delivered before rather than subsequent to the occurrence of PIN. Prevention of the development of PIN lesions was interpreted as calcitriol blocking tumor progression and demonstrating chemopreventive activity in this mouse model of PCa [156]. The analog elocalcitol (BXL-628) has been shown to reduce ventral prostate growth in rat and dog models of BPH [157] (see also Chapter 99). Some studies have used calcitriol or its analogs in combination with other therapeutic agents and demonstrated their anti-tumor activity in vivo (for a detailed discussion of the combination approach see “The role of calcitriol in prostate cancer chemoprevention,” below). Thus in vivo models provide a valuable tool to demonstrate the anti-tumor activity of calcitriol and its analogs while monitoring their tendency to elevate serum calcium levels and validate their use in clinical trials.
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FIGURE 86.3B The calcitriol analog EB1089 inhibits the growth of LNCaP xenografts in athymic nude mice. Growth inhibition of LNCaP tumors in male athymic nu/nu mice, showing the tumor volume of the mice inoculated with 2 106 LNCaP cells in Matrigel basement membrane matrix. The treatment group received 2.0 mg/kg EB1089 (closed circles) in propylene glycol:Na2HPO4 (80:20) intraperitoneally for 5 days/week. Control animals (open circles) received injections of vehicle alone. There were eight animals in each group; the error bars represent SEM, with ) p < 0.05, Mann-Whitney U-test. From [145] with permission.
The concentrations of calcitriol used to elicit significant anti-proliferative effects in cultured PCa cells are often much higher than circulating levels of calcitriol; whether those levels can be safely achieved in vivo is being investigated (see Chapter 90). Use of high doses of calcitriol as a treatment for PCa and other cancers predictably results in hypercalcemia and hypercalciuria. Potential hypercalcemia, renal stone formation, and soft tissue calcification limit the concentration of calcitriol that can be safely administered to patients. Consequently structural analogs of calcitriol that effectively activate the VDR, but which are less hypercalcemic, are being developed and evaluated for their potency as anti-proliferative agents in vitro and in vivo. Section IX of this book extensively reviews the currently available vitamin D analogs and the structureefunction relationships that determine the relative hypercalcemic and differentiating potencies of these analogs. Several mechanisms could contribute to the differential activity of analogs on calcium metabolism and proliferation control, which are discussed in detail in Chapter 75. However, a complete understanding of the mechanistic basis for the separation of hypercalcemic and antiproliferative activities has not yet been realized.
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Many investigations have shown that a number of these analogs can inhibit the growth of PCa cells in culture at concentrations lower than for calcitriol [145,158e164]. Several of these analogs have also been shown to slow the growth of PCa xenografts in animals (as discussed above in “Studies in animal models”). For example, Schwartz et al. [158] showed that the 16-diene analogs were more effective in inhibiting the growth of PCa cells than calcitriol. Unlike calcitriol, derivatives that render the analog resistant to 24-hydroxylase degradation inhibited the growth of DU 145 cells as well. The analogs EB 1089, MC903, 22-oxacalcitriol, and RO24-2637 significantly inhibit the growth of PCa cells with ED50 values lower than that of calcitriol [159]. EB1089 decreases migration and invasion potential of the LNCaP-derived AIPC cell line C4-2 through downregulation of the expression of PTHrP in these cells [146]. As shown in Figures 86.3A and 86.3B, EB1089 also demonstrates tumor-inhibitory effects in animal models of PCa [145,152]. 24-Oxo metabolites of vitamin D [160] and fluorine derivatives of the calcitriol side-chain [160] also exhibited enhanced antiproliferative potencies. Hisatake et al. [165] reported that the vitamin D analog (1,25(OH)2-16-ene-5,6-trans-D3 (Ro 25-4020)), which has a novel 5,6-trans motif, exhibits a 10- to 100-fold increase in anti-proliferative activity in breast cancer and PCa cells while exhibiting at least 40fold less hypercalcemic activity in mice. Substitution at the C-2 position of the 19-nor-1a-25(OH)2D3 molecule with a hydroxypropyl group greatly increases the antiproliferative and anti-invasive potencies [166]. 19-Nor2a-(3-hydroxypropyl)-1a-25 (OH)2D3 (MART-10) and the 14-epimer of MART-10 exhibit greater anti-proliferative, anti-invasive and pro-differentiation activities in prostate cells compared to calcitriol [167,168]. Derivatives of vitamin D that have been shown to be less hypercalcemic than calcitriol, exhibit anti-proliferative effects in PCa cells [128,169]. A new class of vitamin D3 analogs that has two side-chains attached to C20 (Gemini analogs), first designed by Milan Uskokovic [170], exhibit enhanced anti-proliferative activity in PCa cells [171]. Calcitriol analogs that function as ligands capable of activating mutant VDRs are also being synthesized and evaluated [172]. Interestingly Polek et al. [173] showed that LG190119, one of a series of novel non-steroidal VDR modulators, also inhibits LNCaP xenograft growth in mice without causing hypercalcemia. Non-steroidal analogs are discussed in Chapter 76. Calcitriol analogs have also been used to treat BPH (Chapter 99).
MECHANISMS OF THE ANTI-CANCER ACTIONS OF CALCITRIOL Many studies have investigated the mechanisms by which calcitriol exerts growth-inhibitory effects
on cancer cells including PCa cells. As described below, a number of molecular pathways are involved in the anti-cancer actions of calcitriol in PCa cells [45,174]. Investigators have also attempted to identify novel target genes that mediate the various anticancer actions of calcitriol [137,175,176]. These calcitriol actions are often cell-specific and include regulatory effects on cell cycle progression, apoptosis, inflammatory pathways, angiogenesis, invasion, and metastasis.
Cell Cycle Arrest Treatment of many cancer cells with calcitriol or its analogs results in the accumulation of cells in the G0/ G1 phase of the cell cycle [177]. The effects of vitamin D on the cell cycle are detailed in Chapter 84. This mechanism is an important component of the antiproliferative actions of calcitriol in PCa. Treatment of LNCaP cells with calcitriol or the analog EB1089 causes an increase in the percentage of cells accumulating in the G1 phase of the cell cycle [178]. The combination of calcitriol and 9-cis retinoic acid results in synergistic growth inhibition and causes more cells to accumulate in G1 when compared to calcitriol alone [177,178]. The molecular mediators of calcitriol-induced cell cycle arrest have been well characterized. Rb is a key regulator of G1 to S phase transition. Hyperphosphorylation of the Rb protein by G1 cyclins and their cyclin-dependent protein kinase (CDK) partners inactivates the Rb protein and releases the repression on E2F transcriptional activity allowing cells to progress from G1 to S phase. Zhuang and Burnstein [179] have shown that in LNCaP cells calcitriol exerts its effects on some of these key steps. Calcitriol treatment of LNCaP cells causes an increase in the expression of the CDK inhibitor p21, a decrease in CDK2 activity leading to a decrease in the phosphorylation of Rb and repression of E2F transcriptional activity resulting in G1 arrest of the cells. Liu et al. [180] have shown that calcitriol directly upregulates p21 gene expression in U937 leukemia cells, acting through a putative vitamin D response element (VDRE) in the promoter of the p21 gene. However, in LNCaP cells, the regulation of p21 gene expression appears to be indirect [177,179]. Boyle et al. [181] have shown that the induction of insulin-like growth factor binding protein-3 (IGFBP-3) gene expression in these cells by calcitriol results in increased p21 levels. The induction of p21 expression appears to be necessary to mediate the anti-proliferative effect of calcitriol in ALVA-31 cells [182]. However, calcitriol does not increase p21 expression in PC-3 cells, which is consistent with the lack of G1 accumulation of these cells following calcitriol treatment. Thus, the regulation of cell cycle distribution by calcitriol appears to be cell-specific and
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may involve multiple molecular mediators. p53 is not required to induce growth inhibition or cause accumulation of cells in the G1 phase by calcitriol in LNCaP cells [183]. However, elimination of p53 function in these cells reduces G0 arrest as measured by the loss of Ki67 expression, allowing the cells to recover from calcitriolmediated growth arrest [183]. Substantial decreases in the activities of several cyclineCDK complexes are seen in MCF-7 breast cancer cells treated with calcitriol, preventing the entry of cells into S-phase [184]. Further, calcitriol appears to regulate the nuclearecytoplasmic trafficking of CDK2 and causes cytoplasmic mislocalization of CDK2 in PCa cells leading to growth arrest and inhibition of cell proliferation [185]. In several PCa cell lines including the androgen-independent, LNCaPderived C4-2 cells, calcitriol reduces the levels of c-MYC, a transcription factor known to promote G0/ G1 to S phase transition, by downregulating c-MYC mRNA levels and decreasing the stability of the c-MYC protein [186]. As the loss of the expression of cell cycle regulators has been associated with a more aggressive cancer phenotype as well as decreased prognosis and poorer survival, these observations suggest that calcitriol may be a suitable therapy to inhibit cancer progression. Loss of Rb occurs frequently in PCa raising the possibility that prostate tumors lacking functional Rb may not be responsive to calcitriol. Lack of a functional Rb gene in DU 145 cells does not appear to be the critical reason for their reduced sensitivity to growth inhibition by calcitriol. As detailed earlier in “Cellular responsiveness to vitamin D compounds e role of enzymes involved in vitamin D metabolism,” a combination of calcitriol and the 24-hydroxylase inhibitor liarozole [113] or genistein [114] causes appreciable inhibition of growth in these cells. Also, transfection of a functional Rb into DU 145 cells did not render the cells more sensitive to growth inhibition by calcitriol in vitro [108], even though the Rb-transfected DU 145 cells exhibited reduced tumorigenicity in vivo as xenografts in nude mice [187]. In the C4-2 AIPC cells, calcitriol downregulates c-MYC leading to reduced E2F levels, which is sufficient to cause a G1 arrest even in the absence of a functional Rb [95], suggesting that prostate tumors with a loss of heterozygosity of Rb or Rb inactivation should still be responsive to calcitriol or its analogs. Targeting cyclin-dependent kinase 2 (CDK2) and its nuclear localization appears to be another critical mechanism for vitamin-D-mediated growth inhibition [185].
Apoptosis Induction of apoptosis or programmed cell death by calcitriol is not uniformly seen in all cancer cells.
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In the case of PCa, investigators have mostly focused on LNCaP cells and the findings have been variable. Zhuang and Burnstein [179] did not detect apoptosis using terminal transferase labeling in an adherent population of LNCaP cells treated with 10 nM calcitriol. Hsieh and Wu [188] examined the non-adherent portion of an LNCaP cell population and found a small (35%) increase in hypodiploid cells following calcitriol treatment that was characteristic of apoptosis. Fife et al. [189] demonstrated DNA fragmentation in LNCaP cells treated with 10 nM calcitriol for 4 days. Blutt et al. [145] showed evidence of apoptosis in LNCaP cells treated with calcitriol and associated downregulation of the pro-apoptotic proteins Bcl-2 and Bcl-XL. They went on to demonstrate the involvement of Bcl-2 in calcitriol-mediated apoptosis by stably transfecting the Bcl-2 gene into LNCaP cells and showing the loss of an apoptotic response to calcitriol in LNCaP cells that overexpressed Bcl-2. Further, the tumor suppressor p53 is not absolutely required for the induction of apoptosis in LNCaP cells by calcitriol and the induction appears to be caspasedependent [183]. In LNCaP cells, therefore, calcitriol stimulates not only growth arrest but also apoptosis, although to a much lesser extent. There are reports of inhibition of anti-apoptotic proteins by calcitriol in other PCa cells. Guzey et al. [190] showed that in LNCaP and ALVA-31 cells, calcitriol decreased the expression of several anti-apoptotic proteins such as Bcl-2, Bcl-XL, and Mcl-1 leading to the activation of the mitochondrial pathway of apoptosis. The vitamin D analog V decreases Bcl-2 expression in DU 145 cells [191]. Some calcitriol analogs have been shown to induce caspase-dependent apoptosis in several PCa cells [94,192]. Induction of apoptosis by calcitriol, however, appears to be cell-specific as it is not uniformly evident in all the cells that respond to calcitriol with growth inhibition. Even in LNCaP cells, where apoptosis has been demonstrated by some studies, the major action of calcitriol to inhibit cell growth appears to be cell cycle arrest [145].
Differentiation Calcitriol has been shown to induce the differentiation of a number of normal and malignant cells [193]. Peehl et al. [82] did not find any changes in cell morphology or in the expression of various keratins as markers of epithelial cell differentiation when primary human prostatic epithelial cells were exposed to calcitriol. Konety et al. [194] harvested prostate tissue from castrated rats treated with vehicle, testosterone (T), calcitriol, or a combination of T and calcitriol; histological examination of the prostate tissue revealed a greater degree of epithelial cellular differentiation in rats treated
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with T and calcitriol compared to rats treated with T alone. In the PCa cells LNCaP and MDA PCa 2a and 2b, calcitriol increases the expression of PSA [92,195], which is regarded as a differentiation marker for prostatic epithelial cells. However, the effect appears to be much smaller compared to the induction of PSA expression by androgens and the synergistic increase in PSA in cells treated with both calcitriol and androgens is in part due to the upregulation of AR levels by calcitriol [92]. In a microarray analysis of calcitriol regulation of gene expression in LNCaP cells, Krishnan et al. [137] reported that calcitriol upregulated the expression of “prostate differentiation factor,” a member of the bone morphogenetic protein family, which is known to be involved in the differentiation of both embryonic and adult tissues [196]. Based on the available data, there is no strong evidence as yet supporting a role for vitamin D as a differentiation-promoting agent in PCa. However, vitamin-D-induced differentiation in cancer in general is an important component of the anti-cancer actions [193] and is discussed in detail in Chapter 84.
Modulation of Growth Factor Actions Growth factors play an important role in the regulation of prostate epithelial cell growth by autocrine and paracrine mechanisms. Prostatic stromal cells are capable of modifying the epithelial cell environment through paracrine production of peptide growth factors that can act on the basal epithelium [10,197]. The stromal cells are androgen-responsive [197] and the expression of VDR has also been demonstrated in the stromal fibroblasts although at levels lower than the epithelial cells [82]. Expression of autocrine growth factors by the epithelium may contribute to the progression of PCa through the development of independence from epithelialestromal interactions that modulate the growth and development of the normal prostate gland. Some important growth factors that regulate prostate epithelial growth include epidermal growth factor (EGF), keratinocyte growth factor (KGF), basic fibroblast growth factor (bFGF), transforming growth factors (TGFs), and insulin-like growth factors (IGFs). Some of these growth factors also appear to play an important role in the establishment and growth of metastatic PCa cells in bone [198,199]. Targeting of the EGF receptor (EGFR) pathway might be a promising strategy in the treatment of CRPC. A combination of the EGFR blocking drug cetuximab and calcitriol efficiently suppressed the growth of the hormone refractory DU 145 PCa cells [200]. In PC-3 and ALVA 31 cells, calcitriol decreases the availability of IGF by increasing the expression of its
binding proteins IGFBP-3 and IGFBP-5 [201,202]. Boyle et al. [181] provided evidence that the upregulation of IGFBP-3 expression by calcitriol is a necessary component of calcitriol-mediated inhibition of LNCaP cell growth. In these cells, IGFBP-3 induction appears to be necessary for the upregulation of p21 expression by calcitriol [181]. A microarray analysis of gene expression in LNCaP cells revealed a major upregulation of IGFBP-3 following calcitriol treatment [137]. Calcitriol increases IGFBP-3 expression by a direct transcriptional stimulation of the IGFBP-3 gene through a VDRE identified and characterized in the IGFBP-3 promoter [203]. In LNCaP cells, siRNA knockdown of IGFBP-3 expression partially reverses the growth inhibition due to calcitriol or high-dose androgens (which anomalously are growth inhibitory), suggesting a significant role for IGFBP-3 in the anti-proliferative effects of these agents [204]. However, calcitriol upregulation of IGFBP-3 is cellspecific. For example, in primary prostate epithelial cells calcitriol-mediated growth inhibition does not involve IGFBP-3 as these cells lack IGFBP-3 expression [176]. Nickerson and Huynh [205] demonstrated prostate regression in rats following the administration of the vitamin D analog EB1089, which was associated with increases in the expression of several IGFBPs including IGFBP-3. TGFb and IGFBP-3 are pleiotropic factors that play an important role in the regulation of growth and differentiation in many cells [206e208]. They inhibit proliferation and induce apoptosis in prostate epithelial cells [206,208]. In PC-3 human PCa cells, TGFb has been shown to increase the expression of IGFBP-3 leading to growth arrest and apoptosis [207,208]. A significant proportion of calcitriol-mediated growth inhibition of PC-3 cells appears to be mediated by the TGFb pathway [209]. In NRP-152 cells, a non-tumorigenic epithelial line derived from rat dorsolateral prostate, calcitriol induces the expression of TGFb-2 and TGFb-3 [210]. In these cells TGFb appears to mediate growth inhibition and certain biological responses due to both retinoic acid and calcitriol [210]. The presence of VDRE sequences has been demonstrated in the promoter of the human TGFb-2 gene [211].
Anti-inflammatory Effects Chronic inflammation has been recognized as a risk factor for the development of many cancers [212,213]. Several studies support a link between inflammation and the development of PCa [3,214,215]. De Marzo et al. [216] postulate that exposure to infectious agents, hormonal alterations and dietary carcinogens could cause injury to the prostate epithelium leading to inflammation and the formation of lesions referred to
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as proliferative inflammatory atrophy (PIA), which are the precursors of prostatic intraepithelial neoplasia (PIN). PIN lesions are characterized by abnormalities that are intermediate between normal prostatic epithelium and cancer, while progression to high-grade PIN is the most likely precursor of prostate carcinoma [217]. Cancer-related inflammation is characterized by the presence of inflammatory cells at the tumor sites and the overexpression of inflammatory mediators such as cytokines, chemokines, prostaglandins (PGs), reactive oxygen species (ROS), and nitrogen oxide species in the tumor tissue [213,218e220]. Many of these pro-inflammatory mediators activate angiogenic switches usually under the control of vascular endothelial growth factor (VEGF) and thereby promote tumor angiogenesis, metastasis and invasion [221,222]. Recent research suggests that calcitriol also exerts anti-inflammatory actions that may contribute to its beneficial effects in PCa in addition to its known anti-proliferative actions described above [40,223,224]. Studies employing cDNA microarrays have identified many novel calcitriol target genes, some of which could be important molecular mediators of its potent anti-inflammatory activity [137,175,176]. In the following sections we will discuss some of the molecular mechanisms underlying the anti-inflammatory actions of calcitrol. Regulation of Prostaglandin (PG) Metabolism and Signaling PGs have been shown to play a role in the development and progression of many cancers by multiple mechanisms such as stimulating cellular proliferation, inhibiting apoptosis, promoting angiogenesis and activating carcinogens [225,226]. Cyclooxygenase-2 (COX2), the enzyme that catalyzes PG synthesis, is an important molecular target in cancer therapy [227e229]. Overexpression of COX-2 has been demonstrated in PIA lesions [230] and in prostate adenocarcinoma in some studies [231,232]. COX-2 expression in prostate biopsy cores and PCa surgical specimens is inversely correlated with disease-free survival [233], serving as an independent predictor of PCa recurrence [234]. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH), the physiological antagonist of COX-2, is the enzyme that catalyzes the conversion of PGs to their corresponding 15-keto derivatives which exhibit greatly reduced biological activity. 15-PGDH plays a tumor-suppressive role in colon cancer [235,236] and breast cancer [237]. PGs stimulate the proliferation of PCa cells by binding to G-protein-coupled membrane receptors [238,239]. Local production of PGs at the tumor sites by infiltrating inflammatory cells also increases the risk of carcinogenesis and/or cancer progression [215,216,229,240]. PG receptors are
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also expressed in most endothelial cells, macrophages, and stromal cells found in the tumor microenvironment. PG interaction with its receptors can send a positive feedback signal to increase COX-2 mRNA levels [238,241,242]. Therefore, irrespective of the initial trigger of COX-2 expression, PGs could mediate a wave of COX-2 expression at the tumor sites thereby promoting tumor progression. Calcitriol regulates the expression of several genes in the PG pathway in PCa cell lines as well as in cultured primary prostate epithelial cells established from surgically removed prostate tissue from PCa patients [239]. Calcitriol treatment of these cells decreases the levels of COX-2 mRNA and protein and increases 15-PGDH mRNA and protein expression [239]. By inhibiting COX-2 and stimulating 15-PGDH expression in PCa cells, calcitriol decreases the levels of biologically active PGs, thereby reducing the growth stimulation due to PGs. Further, calcitriol also decreases the expression of the PG E receptor (EP) and the PG F receptor (FP) mRNA in PCa cells [239]. Calcitriol suppresses the induction of the immediate-early gene c-fos and the growth stimulation seen following the addition of exogenous PGs or the PG precursor arachidonic acid to PCa cell cultures [239]. The downregulation of PG receptors by calcitriol would inhibit the positive feedback exerted by PGs on COX-2, thereby limiting the wave of COX-2 expression at the tumor sites and slowing the rate of tumor progression. Thus, calcitriol inhibits the PG pathway in PCa cells by three separate mechanisms: decreasing COX-2 expression, increasing 15-PGDH expression and reducing PG receptor levels and thereby suppress the proliferative and angiogenic stimuli provided by PGs in PCa. Further, combinations of calcitriol with non-steroidal anti-inflammatory drugs cause synergistic inhibition of PCa cell growth [239] suggesting that the combination might be therapeutically useful in PCa (for a detailed discussion see “Calcitriol in combination with other agents” and “Clinical trials,” below). The calcitriol analog elocalcitol also exhibits antiinflammatory effects on human BPH cells and causes significant decreases in COX-2 mRNA levels and PGE2 synthesis resulting in the suppression of cytokine-stimulated production of the pro-inflammatory chemokine interleukin-8 (IL-8), which is involved in BPH pathogenesis [243]. The calcitriol analog 1a,25-dihydroxy-16-ene-23-yne-vitamin D3 exhibits anti-inflammatory effects causing a reduction in the expression of inflammatory markers such as COX-2, inducible nitric oxide synthase (iNOS), and IL-2 in experimental models of inflammation [244]. Interestingly this study demonstrated that calcitriol and also 1a,25-dihydroxy-16-ene-23-yne-vitamin D3 directly inhibit COX-2 enzymatic activity [244].
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Induction of MKP5 (Mitogen-activated Protein Kinase Phosphatase 5) and Inhibition of Pro-inflammatory Cytokine Production In normal human prostate epithelial cells calcitriol upregulates the expression of MKP5, also known as DUSP10 [176]. MKP5 is a member of the dual-specificity MKP family of enzymes that dephosphorylate, and thereby inactivate, mitogen-activated protein kinases (MAPKs). MKP5 specifically dephosphorylates p38 MAPK and the stress-activated protein kinase JunN-terminal Kinase (JNK), leading to their inactivation. In primary cultures of normal prostatic epithelial cells from the peripheral zone, calcitriol increases MKP5 transcription through a VDRE identified in the MKP5 promoter [245]. MKP5 upregulation by calcitriol inhibits the subsequent phosphorylation and activation of p38 stress-induced kinase [245]. A consequence of p38 stress-induced kinase activation is an increase in the production of pro-inflammatory cytokines that sustain and amplify the inflammatory response [246]. Interleukin-6 (IL-6) is one such p38-regulated pro-inflammatory cytokine, which is implicated in PCa progression [247e249]. Calcitriol also reduces the production of IL-6 by interfering with the signaling of the pleiotropic inflammatory cytokine tumor necrosis factor a (TNFa) [245]. Calcitriol-mediated upregulation of MKP5 leading to downstream anti-inflammatory effects in prostate cells supports a role for calcitriol in the prevention and/or early treatment of PCa [40,223]. Interestingly, calcitriol upregulation of MKP5 expression is only seen in primary cells derived from normal prostatic epithelium and primary, localized adenocarcinoma but not in the established PCa cell lines derived from PCa metastasis [245]. This observation raises the speculation that a loss of MKP5 expression might occur during PCa progression as a result of a selective pressure to eliminate the tumor-suppressor activity of MKP5 and/or calcitriol. Inhibition of NFkB Activation and Signaling NFkB comprises a family of inducible transcription factors, ubiquitously present in all cells, that are important regulators of innate immune responses and inflammation and play a role in cancer development and progression [250,251]. In contrast to normal cells, many cancer cells have elevated levels of active NFkB [252,253] and constitutive activation of NFkB has been observed in AIPC/CRPC [254e256]. The NFkB protein RelB is uniquely expressed at high levels in PCa with high Gleason scores [257]. NFkB plays a major role in the control of immune responses and inflammation and promotes malignant behavior by increasing the transcription of the anti-apoptotic gene BcL-2 [258], cell cycle progression factors such as c-MYC and Cyclin
D1, proteolytic enzymes such as matrix metalloproteinase 9 (MMP-9) and urokinase-type plasminogen activator (uPA) and angiogenic factors such as VEGF and interleukin-8 (IL-8) [256,259]. Calcitriol and its analogs are known to directly modulate basal and cytokineinduced NFkB activity in many cells including human lymphocytes [260], fibroblasts [261], peripheral blood monocytes [262] and in different types of immune cells [263e265]. There is considerable evidence for the inhibition of NFkB signaling by calcitriol in PCa cells [243,266]; on the contrary, a reduction in the levels of the NFkB inhibitory protein IkBa has been reported in fibroblasts derived from VDR-knockout mice [267]. Calcitriol decreases the levels of the angiogenic and proinflammatory cytokine IL-8 in immortalized normal human prostate epithelial cell lines (HPr-1 and RWPE-1) and established PCa cell lines (LNCaP, PC-3, and DU145) through the inhibition NFkB subunit p65 nuclear translocation leading to the suppression of NFkB-directed signaling [266]. The calcitriol analog elocalcitol inhibits the production of IL-8 by human BPH cells by arresting NFkB p65 nuclear translocation [243]. NFkB also provides an adaptive response to PCa cells against cytotoxicity induced by redox active therapeutic agents and is implicated in radiation resistance of cancers [268,269]. Calcitriol significantly enhances the sensitivity of PCa cells to ionizing radiation by selectively suppressing radiation-mediated RelB activation [270]. Thus, calcitriol may serve as an effective agent for sensitizing PCa cells to radiation therapy via suppression of the NFkB pathway. In addition to direct inhibition, calcitriol also indirectly inhibits NFkB signaling by upregulating the expression of other proteins that interfere with NFkB activation such as IGFBP-3 [271].
Inhibition of Angiogenesis Angiogenesis is the process of formation of new blood vessels from existing vasculature and is a crucial step in continued tumor growth, progression and metastasis [272]. VEGF is the most potent stimulator of angiogenesis. PGs, as discussed below, are also important pro-angiogenic factors. The initiation of angiogenesis is controlled by local hypoxia, which induces the synthesis of pro-angiogenic factors that activate signaling pathways leading to the structural reorganization of endothelial cells favoring new capillary formation [273]. Stimulation of angiogenesis in response to hypoxia is mediated by hypoxia-inducible factor 1 (HIF-1), which directly increases the expression of several pro-angiogenic factors including VEGF [274,275]. Early studies indicate that calcitriol is a potent inhibitor of tumorcell-induced angiogenesis in experimental models [276]. Calcitriol inhibits VEGF-induced endothelial
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cell tube formation in vitro and decreases tumor vascularization in vivo in mice bearing xenografts of breast cancer cells overexpressing VEGF [277]. Calcitriol and its analogs also directly inhibit the proliferation of endothelial cells [278e281] leading to the inhibition of angiogenesis. At the molecular level, calcitriol exerts its anti-angiogenic effects by regulating the expression of key factors that control angiogenesis. Calcitriol reduces the expression of VEGF in several malignant cells including PCa cells through transcriptional repression of HIF-1 [278]. Further, as discussed above, calcitriol inhibits PCa cell-induced angiogenesis by suppressing the expression of the pro-angiogenic factor IL-8 in an NFkB-dependent manner [266]. Chung et al. established TRAMP-2 tumors in wild-type mice and VDRknockout mice and found enlarged vessels and increased vessel volume in TRAMP tumors in the VDR-knockout mice, suggesting an inhibitory role for VDR and calcitriol in tumor angiogenesis [280]. The study further showed increased expression of proangiogenic factors such as HIF-1a, VEGF, angiopoietin-1, and platelet-derived growth factor (PDGF) in the tumors in the VDR-knockout mice [280]. Interestingly, another important mechanism by which COX-2 promotes tumor progression is through the stimulation of angiogenesis [282]. The pro-angiogenic effect of COX-2-generated PGE2 might be due to its action to increase HIF-1a protein synthesis [283] as well as the translocation of HIF-1a protein to the nucleus [284]. The selective COX-2 inhibitor NS398 suppresses the growth of PC-3 xenografts in vivo by a combination of the direct induction of tumor cell apoptosis and the inhibition of angiogenesis as demonstrated by decreased microvessel density of the tumors [285]. An immunohistochemical analysis of human PCa specimens reveals that increased COX-2 immunostaining is associated with an increased infiltration of T-lymphocytes and macrophages and increased CD31-marked microvessel density, indicating a positive correlation between COX2 expression and inflammation and angiogenesis [229]. The pro-inflammatory cytokines released by tumor adjacent inflammatory cells such as T-lymphocytes and macrophages appear to induce COX-2 in the epithelial cells in prostate atrophic lesions promoting tumor progression [286]. Suppression of COX-2 expression by calcitriol therefore provides an important indirect mechanism by which calcitriol inhibits angiogenesis, in addition to its direct suppressive effects on pro-angiogenic factors such as HIF-1 and VEGF. It has been suggested that VEGF induction of p38 and JNK pathways is necessary for COX-2 expression in endothelial cells [287]. As discussed above calcitriol inactivates the p38 pathway by inducing MKP5 expression. Thus, MKP5 induction and VEGF suppression by calcitriol could further
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contribute to its anti-angiogenic effects through p38 inactivation. Matrix metalloproteinases (MMPs) promote angiogenesis by mediating the degradation of the basement membrane of the vascular epithelium and the extracellular matrix, thereby creating a passageway in these barriers for the formation of new capillaries [273]. In human PCa cells, calcitriol decreases the expression of MMP-9, while increasing that of its counterpart tissue inhibitor of metalloproteinase-1 (TIMP-1) [266]. Figure 86.4 summarizes the key molecular mediators of the anti-inflammatory and anti-angiogenic actions of calcitriol.
Inhibition of Invasion and Metastasis Calcitriol reduces the invasive and metastatic potential of many malignant cells including PCa cells. The mechanisms underlying this effect include the inhibition of angiogenesis (discussed above) and regulation of the expression of key molecules involved in invasion and metastasis [288]. Calcitriol decreases tumor size (Fig. 86.3) and lung metastasis of the highly metastatic Mat-lylu and R 3327-AT-2 Dunning PCa cells in vivo [139]. Calcitriol inhibits the invasiveness and migration potential of metastatic PCa cells such as DU145 and PC-3, due in part to decreasing the expression of a6 and b4 integrins [289]. In PCa cells, calcitriol and its analogs also increase the expression of E-cadherin, a tumor suppressor gene whose expression is inversely correlated to metastatic potential [177]. Calcitriol and the analog 1,25-dihydroxy-16-ene-23-yne-cholecalciferol markedly inhibit the invasiveness of DU 145 human PCa cells causing selective decreases in the secreted levels of MMP-2 and MMP-9 [290]. Calcitriol-mediated suppression of MMP-9 expression and upregulation of TIMP-1 levels also decrease the invasive potential of several PCa cells [291].
THE ROLE OF CALCITRIOL IN PROSTATE CANCER CHEMOPREVENTION The development of PCa is a multistep process that transforms normal glandular epithelium to preneoplastic lesions that progress on to invasive carcinoma. This transformation generally progresses very slowly, likely over decades, before symptoms become clinically detectable and a diagnosis is made [292e295]. The observed latency in PCa development provides a long window of opportunity for intervention by chemopreventive agents that would reverse, suppress or prevent the carcinogenic process [296]. Several drugs and nutritional agents have been or are being tested in PCa chemoprevention trials such as the PCPT (Prostate Cancer
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Calcitriol
MKP5
PG synthesis
IGFBP-3
HIF-1
NFκB activation
PG actions
MMP-9 E-cadherin
p38 activation
PGE2
Pro-inflammatory cytokines
Pro-inflammatory cytokines
IL-6
IL-8
VEGF
Inflammation
Proliferation
Apoptosis
Invasion
Metastasis
Angiogenesis
Summary of molecular pathways mediating the anti-inflammatory and anti-angiogenic actions of calcitriol. Several new calcitriol target genes have been identified revealing multiple molecular pathways of anti-inflammatory and anti-angiogenic actions of calcitriol in prostate cells. These include (1) the inhibition of PG synthesis and biological actions, (2) the induction of MKP5 expression and the subsequent inhibition of p38 stress kinase activation and the production of pro-inflammatory cytokines such as IL-6, (3) the inhibition of NFkB signaling resulting in the attenuation of the synthesis of pro-inflammatory cytokines such as IL-8, (4) upregulation of the expression of IGFBP-3, which suppresses proliferation and inhibits NFkB activation, (5) inhibition of tumor angiogenesis due to suppressive effects on the expression of proangiogenic factors such as HIF-1, VEGF, and IL-8, and (6) the regulation of the expression of molecular mediators such as MMP-9 and E-cadherin to inhibit invasion and metastasis. Solid lines indicate direct actions of calcitriol and dotted lines indicate downstream effects of calcitriol. From [215] with permission.
FIGURE 86.4
Prevention Trial), the SELECT (Selenium and Vitamin E Cancer Prevention Trial) and the REDUCE (Reduction by Dutasteride of Prostate Cancer Events) trials [297]. Inflammation in the prostate is proposed to be an etiological factor in the development of PCa [215,216]. De Marzo et al. [216] postulate that injury to the prostate epithelium caused by exposure to infectious agents, hormonal alterations and dietary carcinogens leads to inflammation and the formation of PIA lesions, which are the precursors to PIN. Progression to high-grade PIN is the most likely precursor of prostate carcinoma [217]. The epithelial cells in PIA lesions have been shown to exhibit many molecular signs of stress including elevated expression of COX-2 [214,230,298]. Thus mediators of inflammation appear to be involved in triggering the process of prostate carcinogenesis. Treatment with COX-2-selective NSAIDs such as celecoxib has been shown to suppress prostate carcinogenesis in the TRAMP model [299]. As discussed in the preceding section, calcitriol exhibits significant anti-inflammatory effects in experimental models of PCa. Overproduction of ROS causes the induction and accumulation of DNA damage contributing to carcinogenesis. Calcitriol demonstrates anti-oxidant activity by protecting non-malignant human prostate
epithelial cells from oxidative stress-induced cell death [300]. The mechanisms appear to be the induction of the expression of the key anti-oxidant enzyme glucose6-phosphate dehydrogenase and increases in glutathione levels resulting in the scavenging of cellular ROS [300]. Inhibition of HIF-1 and subsequent reduction in the expression of HIF-1 target genes such as VEGF, ET-1, and Glut-1 by calcitriol also contributes to the suppression of tumorigenesis and angiogenesis [278]. Therefore, calcitriol has the potential to be useful as a chemopreventive agent in PCa [36,156,223,224]. The efficacy of calcitriol as a chemopreventive agent has been examined in Nkx3.1; PTEN mutant mice, which recapitulate stages of prostate carcinogenesis from PIN lesions to adenocarcinoma [156]. As mentioned above, the data reveal that calcitriol significantly reduces the progression of PIN from low-grade to high-grade lesions. Calcitriol is more effective when administered before, rather than subsequent to, the initial occurrence of PIN. These animal studies as well as in vitro observations suggest that clinical trials in PCa patients with PIN or early disease to evaluate calcitriol and its analogs as agents that prevent PCa development and/or delay PCa progression are warranted.
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CALCITRIOL IN COMBINATION WITH OTHER AGENTS The efficacy of calcitriol in PCa therapy is no doubt directly related to the dose of calcitriol administered. Hypercalcemia becomes more frequent at higher concentrations of calcitriol, which limits the maximum dose that can be given safely. Although transient hypercalcemia is not necessarily a limiting toxicity in individuals with lethal cancer, severe hypercalcemia, renal stone formation and vascular calcification are potential side effects of calcitriol therapy that limit the dosages used. Several calcitriol analogs (discussed in Section IX of this book) exhibit increased potency as anti-proliferative agents and have fewer hypercalcemic effects and therefore form a class of agents with a higher therapeutic index than the parent compound, calcitriol. Another avenue to increase efficacy and decrease toxicity is to use a combination of agents that act by different mechanisms, at doses in the combination being less than the maximal doses of the individual agents. This drug combination strategy has the advantage of limiting the toxicity associated with the individual drugs while obtaining additive and potentially synergistic therapeutic effects. Preclinical [301] and clinical studies [302] in colon and other cancers have successfully employed the strategy of combining low doses of two active drugs to achieve a more effective chemoprevention and therapeutic outcome compared to the individual agents [303]. Investigators have been studying the anti-proliferative effect of calcitriol in PCa in combination with other agents [45,304,305]. This section discusses some of the agents that can be used in combination with calcitriol to improve its therapeutic utility.
24-Hydroxylase Inhibitors As detailed in “Cellular responsiveness to vitamin D compounds e role of enzymes involved in vitamin D metabolism,” above, azole antagonists of the primary vitamin D catabolic enzyme 24-hydroxylase enhance the anti-tumor effects of calcitriol. Combination of calcitriol with azole inhibitors of 24-hydroxylase such as liarozole [113] or ketoconazole [118] increases the halflife of calcitriol and enhances VDR upregulation, thereby sensitizing the cells to the biological actions of calcitriol. The combination of calcitriol with 24-hydroxylase inhibitors, therefore, would allow the use of lower doses of calcitriol to achieve significant anti-proliferative effects. However, increased biological activity of calcitriol may not be selective for anti-proliferative effects but may also include hypercalcemia. Ketoconazole, the most readily available imidazole derivative that inhibits mammalian P450 enzymes [117], exhibits growth inhibitory activity by itself in PCa cells [306]
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as well as in primary prostatic epithelial cells [118]. Ketoconazole enhances the inhibitory effects of calcitriol on the growth of PC-3 cells in culture as well as their growth as xenografts in nude mice [120]. Ketoconazole is known to block testicular synthesis of androgens and therefore has been frequently used as second-line therapy to ablate androgen biosynthesis in PCa patients [117]. However, ketoconazole, in addition to inhibiting 24-hydroxylase [307] also inhibits the activity of 1a-hydroxylase (Chapter 3) leading to low levels of calcitriol, a major risk factor for osteoporosis and osteomalacia [79]. Indeed administration of ketoconazole to normal men has been shown to result in a dose-dependent decrease in serum levels of calcitriol [308]. Patients receiving ketoconazole-based androgen ablation therapy would therefore be at a greater risk for metabolic bone disease and other side effects of vitamin D deficiency. Adding calcitriol or analogs to therapeutic regimens that include ketoconazole would be beneficial since it would restore circulating levels of active vitamin D [118]. However, the clinical use of ketoconazole and calcitriol combination needs to be approached with caution. Ketoconazole inhibits multiple P450 enzymes including those in the steroidogenic pathways leading to the synthesis of testosterone, cortisol and aldosterone [117]. Adrenal insufficiency would exacerbate hypercalcemia. Therefore, patients undergoing this combination therapy require careful monitoring for hypercalcemia and adrenal insufficiency in addition to the assessment of PCa progression. Trump and colleagues have added dexamethasone to a combination of ketoconazole and calcitriol in experimental models of PCa [120]. The rationale for this approach is that dexamethasone serves to minimize calcitriol-induced hypercalcemia (due possibly to a downregulation of VDR in the intestine [116,309]) as well as serve as a glucocorticoid replacement for ketoconazole inhibition of cytochrome P450 enzymes involved in adrenal steroid biosynthesis. Currently more specific inhibitors of 24-hydroxylase, both azoles and cholecalciferol analogs, are being developed (see Chapter 80). For example, 2-(4-hydroxybenzyl)-6-methoxy-3,4-dihydro-2H-naphthalen-1-one (tetralone) is a potent 24hydroxylase inhibitor and its co-addition sensitizes DU145 cells to calcitriol [310]. 24-Sulfoximine [311] and 24-phenylsulfone [312] analogs of calcitriol are potent inhibitors of 24-hydroxylase and inhibit the degradation of calcitriol in many tumor cells including PCa cells, suggesting their combinations with calcitriol might exhibit enhanced anti-tumor activity. But the approach requires some caution since amplification of calcitriol activity by 24-hydroxylase inhibition would also increase its hypercalcemic effects since thus far there are no methods to specifically enhance the antiproliferative actions of calcitriol.
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Retinoids, Glucocorticoids and Peroxisome Proliferator-activated Receptor (PPAR) Ligands The retinoid compounds all-trans retinoic acid (ATRA), 9-cis retinoic acid (9-cis RA), and 13-cis retinoic acid (13-cis RA) are ligands for the retinoid receptors (RARs and RXRs). RXRs are transcription factors that heterodimerize with retinoic acid receptor (RAR), VDR and other nuclear transcription factors (see Chapters 7 and 8). Because of their role in controlling growth and differentiation, retinoids have been examined for potential anti-cancer activity in several malignancies including PCa [313e315]. ATRA, 9-cis RA and various other retinoids when used in combination with calcitriol exhibit enhanced growth-inhibitory effects in PCa cells [135,178,316e320]. Several mechanisms might account for the cooperative effects of calcitriol and retinoids in inhibiting PCa cell growth including: the upregulation of VDR levels by retinoids [116], cooperative activation of common target genes involved in growth control such as TGFb [210], and the regulation of VDR signaling and cellular sensitivity to calcitriol by increased VDR levels [321], as well as modifications of its heterodimeric partner RXR [322,323]. Further, transcriptional activation through the VDR correlates with the potency of VDReRXR heterodimerization [324]. A combination of calcitriol and 9-cis RA inhibits human telomerase reverse transcriptase transcription and telomerase activity in PCa cells, a mechanism whereby this combination can potentially induce cellular senescence [325]. These studies provide a rationale for testing the use of combinations of vitamin D compounds with retinoic acid derivatives in PCa therapy. Glucocorticoids have been widely used in the treatment of advanced PCa and have been part of PCa therapy in several randomized trials (for a review see [326]). Glucocorticoids exhibit growth-inhibitory effects in and of themselves [327,328]. However, in PCa cells carrying promiscuous AR mutations (see “Etiology, treatment and the role of hormonal factors,” above), glucocorticoids can inappropriately act through the mutant AR and stimulate cell growth [20,21,23]. Glucocorticoids modulate VDR expression in a tissue- and species-specific manner [116,309]. Because of their ability to mitigate hypercalcemia induced by vitamin D compounds, combinations of glucocorticoids with vitamin D compounds may be beneficial in PCa treatment. Johnson and co-workers have observed cooperative anti-tumor effects of calcitriol and glucocorticoids. In a murine squamous cell carcinoma model system, dexamethasone has been shown to reduce calcitriol-mediated hypercalcemia and enhance the antitumor activity of calcitriol in vitro and in vivo, possibly
due to the upregulation of VDR levels [329,330]. The combination of calcitriol with mitoxantrone and dexamethasone causes significantly greater tumor regression in PC-3-xenograft-bearing mice [331]. These observations have prompted Trump and colleagues to test the combination of calcitriol and dexamethasone in PCa clinical trials (see “Clinical trials,” below). Ligands for the peroxisome proliferator-activated receptor g (PPARg) exhibit growth-inhibitory effects in cell culture and animal models of PCa [332e334]. PPARg ligands inhibit the activation of the PSA gene by androgens in LNCaP cells [335]. A phase II clinical study in patients with advanced PCa showed an unexpectedly high incidence of prolonged stabilization of PSA in patients treated orally with troglitazone [336], a drug that has since been withdrawn from the market because of toxicity. Combinations of vitamin D compounds and PPARg ligands need to be further tested in experimental models of PCa before evaluating their therapeutic utility in patients.
Calcitriol and Soy Isoflavones Soy is a rich source of isoflavones such as genistein and daidzein. An inverse correlation between consumption of soy in the diet and PCa incidence has been shown in epidemiological studies [337]. Soy-derived phytoestrogens inhibit the PCa growth in cell culture models [33,338] and in animal models [339,340]. Genistein decreases the expression of AR mRNA and protein and inhibits the transcriptional activation of the PSA gene in LNCaP cells [341]. Genistein also decreases AR expression in the dorsolateral prostate of rats [342]. Several studies have shown interactions between phytoestrogens and the vitamin D axis in prostate cells. Calcitriol and genistein synergistically inhibit the growth of human primary prostatic epithelial cells and LNCaP cells, causing the cells to arrest in the G0/G1 phase as well as the G2M phase of the cell cycle [343]. Farhan et al. [119] have reported that genistein and other isoflavanoids modulate the availability of calcitriol in DU 145 cells by reducing the expression of 24-hydroxylase and 1a-hydroxylase. The suppression of 24-hydroxylase expression appears to be due to regulation at the transcriptional level while that of 1a-hydroxylase involves deacetylation. Swami et al. have shown that genistein causes a direct non-competitive inhibition of the enzymatic activity of 24-hydroxylase and a prolongation of calcitriol half-life in DU 145 cells, thereby rendering these cells sensitive to growth inhibition by calcitriol [114]. In addition to inhibiting CYP24 enzyme activity, genistein has its own independent actions to inhibit the PG pathway in PCa cells. In a recent study Swami et al. showed that, similar to calcitriol, genistein decreased COX-2 expression, leading to decreased
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synthesis of PGE2. Genistein also induced downregulation of the expression of EP and FP receptors thereby reducing the biological functions of PGE2 [34]. This report also included a pilot randomized double-blind clinical study examining the effects of soy isoflavone supplements given to PCa patients in the neo-adjuvant setting before prostatectomy. Soy caused significant decreases in COX-2 mRNA and increases in p21 mRNA in the prostatectomy surgical specimens in patients treated with soy [34]. Furthermore, the combination of calcitriol and genistein acted additively to inhibit the PG pathway in PCa cells [34]. Thus, a combination of vitamin D compounds and soy or phytoestrogens such as genistein may be beneficial in PCa treatment as genistein can potentiate the growthinhibitory activity of vitamin D by the various mechanisms mentioned above [34,344]. However, as in the case of the azole compounds, combination with genistein by extending calcitriol half-life might also enhance the calcemic effects and toxicity of calcitriol.
genotoxic agent [350]. Another molecular mechanism underlying the enhanced effect of the combination therapy involves an increase in the expression of the p53 homolog p73 by calcitriol in many cell types, which in turn makes these cells more susceptible to the cytotoxic effects of platinum compounds [351]. Docetaxel-induced apoptosis in PC-3 cells is enhanced by the co-addition of calcitriol [352]. Thus, the ability of calcitriol to reduce cell survival signals and activate pro-apoptotic signals may explain why it can enhance the anti-tumor activity of mechanically diverse cytotoxic agents such as platinum compounds and taxol derivatives. These data clearly support the combined use of vitamin D compounds and cytotoxic drugs in the treatment of solid tumors such as PCa. The section “Clinical trials,” below, includes a detailed discussion of the AIPC Study of Calcitriol Enhancing Taxotere (ASCENT) clinical trials testing the combination of high-dose calcitriol and docetaxel in patients with CRPC unresponsive to other therapy.
Calcitriol and Chemotherapeutic Drugs
Calcitriol and Non-steroidal Anti-inflammatory Drugs
Several studies in cell cultures and animal models as well as clinical trials (see “Clinical trials” below) have demonstrated the potential utility of calcitriol and its analogs as agents that can enhance the anti-proliferative and cytotoxic effects of conventional chemotherapeutic drugs. Combined administration of calcitriol or its analogs with platinum compounds such as carboplatin or cisplatin has been shown to result in a marked enhancement of growth inhibition in PCa cell lines [345] over that seen with the platinum compound alone. Hershberger et al. [346] showed that treatment of murine squamous carcinoma cells (SCC) and PC-3 PCa cells in vitro with calcitriol prior to paclitaxel caused significantly greater growth inhibition than either agent alone. In mice bearing PC-3 xenograft tumors, calcitriol treatment in advance of engraftment, similarly enhanced the tumor-inhibitory effect of paclitaxel [346]. The molecular basis for the enhanced anti-tumor activity of this combination appears to be the increase in the expression of the cell cycle inhibitor p21 by calcitriol, rendering the cells more sensitive to chemotherapeutic agents such as paclitaxel [346]. Paclitaxel cytotoxicity has been similarly shown to be increased in breast and colon cancer cells when p21 expression is specifically perturbed [347,348]. Calcitriol upregulates the expression of the pro-apoptotic signaling molecule mitogen-activated protein kinase kinase kinase 1 (MEKK1) in SCC cells [349], and this upregulation is potentiated by co-treatment with cisplatin, suggesting that calcitriol pretreatment commits the cells to undergo apoptosis through specific molecular pathways, which is enhanced by the treatment with an additional
Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to reduce inflammation and prevent PCa development in a rat model of prostate carcinogenesis [353]. The use of NSAIDs such as aspirin lowers serum PSA levels in men with latent PCa [354,355]. In PCa patients a strong association between the levels of serum C-reactive protein (CRP), a non-specific marker of inflammation, and serum PSA has been reported [356]. Recent studies also show that an elevated serum CRP level is a strong predictor of poor prognosis in patients with metastatic CRPC [357,358]. NSAIDs are a class of drugs that decrease PG synthesis by inhibiting COX-1 and COX-2 enzymatic activities. Several NSAIDs non-selectively inhibit both the constitutively expressed COX-1 and the inducible COX-2, while others have been shown to be more selective in preferentially inhibiting COX-2 enzymatic activity. Moreno et al. showed that the combinations of calcitriol with the COX-2-selective NSAIDs NS398 and SC-58125 as well as the nonselective NSAIDs naproxen and ibuprofen caused a synergistic enhancement of the inhibition of PCa cell proliferation compared to the individual agents [239,359]. These results raise the possibility that the combination of calcitriol and NSAIDs may have clinical utility in PCa therapy [359]. The combination strategy allows the use of lower concentrations of NSAIDs thereby minimizing their undesirable side effects. It has recently become clear that the long-term use of COX-2-selective inhibitors such as rofecoxib (Vioxx) causes an increase in cardiovascular complications in patients [360e363]. Even the use of non-selective
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NSAIDs has been shown to increase cardiovascular risk in patients with heart disease [364]. However, in comparison to some COX-2-selective inhibitors, nonselective NSAIDs such as naproxen may be associated with fewer cardiovascular adverse effects [364,365]. Preclinical data [239] suggest that the combination of calcitriol with a non-selective NSAID would be a useful therapeutic approach in PCa that would allow both drugs to be used at reduced dosages leading to increased efficacy as well as improved cardiovascular safety [359,366]. As discussed in “Clinical trials,” below, a phase II trial evaluating the combination of the nonselective NSAID naproxen and high-dose calcitriol in patients with early recurrent PCa demonstrated some benefit in terms of reduction in PSA doubling time [367].
Other Agents Co-activator and co-repressor proteins that modulate the transcriptional activity of VDR exhibit histone-acetyl transferase (HAT) and HDAC activities, respectively. The decreased sensitivity of some PCa cells to growth inhibition by calcitriol or its analogs was restored by co-treatment with HDAC inhibitors such as sodium butyrate and trichostatin A [368], suggesting that this combination might be therapeutically useful since dysregulation of co-activators and co-repressors frequently occurs in cancer. The use of proteasome inhibitors for cancer therapy has been gaining interest. Proteasome inhibitors may have a role in combination therapy with a vitamin D compound and/or retinoids by inhibiting the degradation of VDR or its heterodimeric partner RXR. In support of this hypothesis, a study in osteosarcoma cells showed that proteasome inhibitors increased the expression of VDR and RXR and sensitized the cells to the growth-inhibitory effects of calcitriol and 9-cis retinoic acid [321].
CLINICAL TRIALS Calcitriol is an FDA-approved drug (for other indications) and its therapeutic utility has been evaluated in clinical studies in PCa patients. The anti-proliferative effects of calcitriol on cultured cells have been observed at high concentrations. Achievement of adequate concentrations of calcitriol in patients runs the risk of causing hypercalcemia and hypercalciuria leading to renal stone formation [369,370]. In patients with chronic renal disease, it has been suggested that calcitriol treatment may be associated with soft tissue calcification [371,372]. However, in some studies calcitriol analogs have been shown to inhibit vascular calcification (see Chapters 73 and 81). Animal studies also raise the
possibility that vitamin-D-induced hypercalcemia might lead to cardiovascular and renal calcifications [373e375]. In contrast, it should be recognized that chemotherapy drugs are usually associated with drastic and life-threatening toxicity. Hypercalcemia, although potentially serious, might be tolerable compared to the alternatives. The benefits of calcitriol should not necessarily be avoided due to transient hypercalcemia in cancer patients who face limited survival (for a discussion see Chapter 90). As an alternate approach several academic investigators and pharmaceutical companies have focused their efforts on designing calcitriol analogs that exhibit decreased calcemic effects while maintaining equal or increased anti-proliferative activity. For a discussion of analogs see Section IX of this book. The following is a discussion of the clinical trials in patients with PCa including: (1) the effects of vitamin D3 (the dietary supplement which is the precursor to 25(OH)D and calcitriol), (2) the active hormone calcitriol, (3) calcitriol analogs, and (4) combinations of calcitriol with other agents or therapy regimens.
Vitamin D3 (Cholecalciferol) in PCa Trials The presence of 1a-hydroxylase in prostate cells and the paracrine anti-proliferative actions of locally synthesized calcitriol [376,377] raise the possibility that increasing the circulating concentrations of 25(OH)D by dietary administration of precursor vitamin D2 (ergocalciferol) or vitamin D3 (cholecalciferol) will exert an anti-cancer effect due to its local conversion to calcitriol within the cancer cells (see Chapter 45). In addition, 1ahydroxylase present in multiple cell types in the tumor microenvironment such as tumor infiltrating macrophages, endothelial cells, etc., will contribute to calcitriol production at the tumor site [377]. Eliciting an anticancer effect by treating with vitamin D3 and allowing the local production of calcitriol in the tumor site to generate paracrine actions of calcitriol might be safer than raising circulating calcitriol levels with calcitriol administration, which can be associated with hypercalcemia, hypercalciuria, development of renal stones and possibly ectopic calcifications. An alternate and complementary hypothesis is that very high doses of vitamin D3 will generate increased concentrations of circulating 25(OH)D that achieve high enough levels to directly bind to and activate the VDR [378]. However, a recent assessment of the use of vitamin D supplementation in clinical trials indicates that although vitamin D supplementation does not cause hypercalcemia, it causes modest increases in serum calcium levels resulting in high normocalcemia (serum calcium levels that are high but fall within the normal reference range) [379]. High normocalcemia may represent a more
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sensitive index of potential vitamin-D-related toxicity, which theoretically may be associated with increased risk of mortality due to cardiovascular disease [379]. In a small pilot clinical study cholecalciferol was given to PCa patients at 2000 IU per day and monitored prospectively every 2e3 months over a period of 21 months. The results showed a beneficial effect of prolongation of serum PSA doubling time in 14 out of 15 patients [380]. The serum PSA level is a useful biomarker of prostate cell abundance and its rate of rise is often used as a surrogate for tumor growth. Although also synthesized by normal or BPH cells, after primary therapy has removed or irradiated the prostate, PSA is more specific as a biomarker for PCa growth.
Calcitriol in Clinical Trials The therapeutic utility of calcitriol has been evaluated in clinical studies in PCa patients. A decrease in the rate of rise of serum PSA levels was observed in PCa patients following modest but supra-physiological daily doses (2e2.5 mg/day) of calcitriol suggesting a beneficial effect in slowing the progression of the disease [370,381]. However, in addition to decreasing or stabilizing the rate of rise of PSA, the objective benefits were small and the risk of renal stones was significant [370]. To minimize these toxicities some investigations have followed the approach of administering very high doses of calcitriol intermittently in an attempt to reach higher peak concentrations of calcitriol to achieve greater efficacy while causing only transient hypercalcemia. Schedules have been developed using high-dose calcitriol three times a week [382] or very high doses once weekly [369,381], where it apparently can still elicit its antiproliferative effects but results in only transient hypercalcemia and infrequently causes renal stones.
Calcitriol Analogs An alternate approach is the development and use of calcitriol analogs that exhibit equal or even increased anti-proliferative activity while exhibiting reduced tendency to cause hypercalcemia. Preclinical data have shown that the calcitriol analog elocalcitol (BXL-628) reduces BPH partly through the inhibition of intraprostatic growth factors and a clinical study testing the effects of this analog in BPH patients has demonstrated the arrest of prostate growth in these patients [383] (see Chapter 99). Schwartz et al. evaluated the effect of intravenous administration of the calcitriol analog paricalcitol (19-nor-1a-25-dihydroxyvitamin D2) three times a week on an escalating schedule of 5 to 25 mg to patients with CRPC [384]. The primary endpoint of a sustained 50% decrease in serum PSA was not met. However,
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paricalcitol significantly reduced the elevated serum PTH levels seen in these patients, suggesting that the analog may be beneficial in reducing skeletal morbidity in patients with advanced PCa [384].
Calcitriol as a Part of Combination Therapy Calcitriol is also being used in combination therapy with other agents to achieve enhanced anti-cancer effects [385,386]. Administration of escalating intermittent doses of calcitriol along with the bisphosphonate zoledronic acid in men with progressive PCa did not demonstrate an anti-tumor effect [387]. A phase II trial in patients with CRPC using high-dose oral calcitriol (12 mg/day three times per week) with dexamethasone (4 mg/day four times per week) showed a 50% reduction in PSA in 28% of the patients and no symptomatic hypercalcemia [382]. Another phase II trial tested the combination of calcitriol, dexamethasone, and carboplatin in patients with CRPC and found a PSA response in 13 out of 34 patients [388]. Calcitriol administered intravenously at a high dose of 74 mg weekly in combination with dexamethasone (4 mg twice weekly) was well tolerated but failed to produce a clinical or PSA response in patients with CRPC [389]. The results of the ASCENT I clinical trial in advanced CRPC patients, who failed other therapies, demonstrated that extremely high doses (45 mg) of a formulation of calcitriol (DN-101, Novacea) administered orally once a week along with the usual regimen of the chemotherapy drug taxotere caused a statistically significant improvement in overall survival and time to progression of the disease [385]. These findings suggested that calcitriol might enhance the efficacy of active drugs in cancer patients and provide a survival advantage. Interestingly patients in the calcitriol plus docetaxel arm showed a statistically significant reduction in the incidence of venous and arterial thrombosis compared to docetaxel alone [390]. The ASCENT I trial did not meet its primary endpoint, i.e. a lowering of serum PSA. However, based on the promising survival results, a larger phase III trial (ASCENT II) with survival as an endpoint was initiated. A new, improved docetaxel regimen (every 3 week dosing) was used in the control arm of the ASCENT II trial, which was compared to DN-101 plus the older docetaxel dose regimen (once a week), resulting in an asymmetric study design. Unfortunately, the improved survival due to the combination demonstrated in the ASCENT I trial could not be confirmed in the ASCENT II trial [391]. In fact, the trial was prematurely stopped by the data safety monitoring committee. The FDA issued a temporary hold on DN101 when an excess number of deaths were noted in the study arm (DN-101 plus old docetaxel regimen) versus the control arm (placebo plus new docetaxel
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regimen). After the trial was stopped, further analysis [392] suggested that the increased deaths in the treatment arm compared to the control arm were not due to calcitriol toxicity but due to better survival in the control arm that received the new and improved docetaxel regimen with known better survival than the older docetaxel regimen. Another recent study tested the combination of highdose calcitriol (DN-101) with mitoxantrone and prednisone in patients with metastatic CRPC without previous chemotherapy and concluded that calcitriol did not significantly add to the activity of mitoxantone and prednisone as assessed by the decline in serum PSA levels [393]. In a randomized, double-blind, phase II study in a similar patient population, the addition of daily doses of doxercalciferol (1a-hydroxyvitamin D2) to weekly docetaxel did not enhance the PSA response rate or survival [394]. Interestingly, another study testing the effect of the combination of weekly high-dose calcitriol and docetaxel in PCa patients, whose disease progressed after resistance developed to first-line chemotherapy using docetaxel alone, showed that high-dose calcitriol restored the sensitivity to chemotherapy with docetaxel [395]. Based on preclinical observations in PCa cells [239], a single-arm, open-label phase II study evaluating the combination of the non-selective NSAID naproxen (375 mg naproxen twice a day) and high-dose calcitriol (45 mg of calcitriol (DN101) orally once a week) was carried out in patients with early recurrent PCa [367]. The trial was based on the rationale that NSAID inhibition of COX-2 activity and PG synthesis could be synergistically enhanced by co-administration of calcitriol [239]. The trial was prematurely stopped after 21 patients had been enrolled when the FDA put a temporary hold on DN-101 based on the data from the ASCENT II trial described above. The therapy was well tolerated by most patients. A prolongation of the PSA doubling time was achieved in 75% of the patients, suggesting a beneficial effect of the combination therapy [366,367].
Summary of Clinical Trial Data The results of clinical trials using vitamin D, calcitriol or various vitamin D analogs in cancer patients have thus far been somewhat disappointing. Based on some of the epidemiological findings and preclinical data from cell culture and animal models showing substantial benefits, the modest efficacy thus far achieved in patients is not as impressive as had been hoped. The trials in patients with early recurrent PCa show stabilization of the disease based on slowing or stopping the rise in serum PSA levels. However, many of the clinical trials including the ASCENT trials have been carried out
in patients with far-advanced cancer who have failed multiple other therapies. Preclinical observations demonstrating significant inhibitory effects of calcitriol in initial stages of cancer development suggest that calcitriol may be more effective when used in early disease and/or in chemoprevention. However, it would be premature to conclude from the limited number of clinical trials completed thus far that calcitriol has little efficacy in advanced-cancer patients. The following important criteria to achieve optimal efficacy remain to be elucidated: (1) what is the optimum dose or schedule of calcitriol to use; (2) which form of the drug is most effective and least toxic (dietary vitamin D3, calcitriol, or an analog); (3) when in the course of cancer would it be most effective to administer calcitriol; and (4) what drug combinations would generate the most benefit. Although calcitriol does induce apoptosis in some cancer cells, its major actions are anti-proliferative, anti-inflammatory and pro-differentiating effects. Considering that its actions are more cytostatic than cytolytic, we believe that calcitriol would be most effectively used in chemoprevention or inhibiting or delaying the progression of cancer in patients with early disease. Administered at the highest dose that can be tolerated without serious side effects and probably in combination with other drugs, we believe vitamin D, calcitriol, or its analogs will augment other therapies and be a useful addition to cancer treatment [223,304].
SUMMARY AND CONCLUSIONS Many epidemiologic studies indicate that vitamin D deficiency increases the risk of a variety of cancers and that higher levels of vitamin D are associated with better prognosis and improved outcomes. However, many studies do not find a benefit of vitamin D sufficiency [71e75] and the reasons for these discrepancies are not clear. Extensive cellular and in vivo research provides a strong rationale and evidence for benefit based on the anti-proliferative, anti-inflammatory and pro-differentiation effects of calcitriol. The anti-cancer effects in cell culture and animal models of cancer strongly support the utility of calcitriol in cancer prevention and treatment. Although we have emphasized its anti-inflammatory activity in this chapter because of relatively new data on this activity, multiple molecular pathways of calcitriol action in cancer cells have been identified providing a mechanistic basis for its potential efficacy in cancer. The data suggest that calcitriol has therapeutic and cancer-preventive effects in several malignancies including PCa. Although the preclinical data are persuasive and the epidemiologic data intriguing, but inconsistent, no well-designed clinical trial of optimal administration
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of vitamin D as a cancer therapy has ever been conducted [304]. The preclinical data provide considerable rationale for continued development of calcitriol or its analogs for cancer therapy. Future clinical trials should be designed using good clinical trial design principles. Such studies may finally provide compelling data to prove whether or not there is a role for calcitriol or its analogs in cancer prevention or therapy [223, 304]. The recently approved VITAL trial will be an NIH-sponsored prospective randomized trial following thousands of participants receiving therapeutic or maintenance doses of vitamin D (see Chapter 105). The trial will enroll 20 000 participants including women over 65 and men over 60 with no prior history of cancer, heart disease or stroke who will be followed for 5 years. The participants will be treated with 2000 IU of vitamin D, 800 IU of vitamin D, and/or omega-3 fish oil. Hopefully this trial will resolve some of the outstanding questions about vitamin D efficacy in cancer prevention as well as reduction of the risk of cardiovascular and other diseases. Although skepticism remains in some quarters, from an analysis of the available data, we have reached several conclusions about vitamin D efficacy in cancer. (1) Vitamin D deficiency is a risk factor for a number of cancers and vitamin D supplementation to increase serum levels of 25(OH)D appears to exert chemopreventive actions on cancer development. (2) The avoidance of vitamin D deficiency should be an important goal for reducing cancer incidence as well as reducing the risk of other diseases including osteoporosis. (3) It is unclear what the optimum target level of vitamin D supplementation should be to reduce cancer risk; however, we believe that it will turn out to be higher than the current normal range cut-off point of 25(OH)D at 30 ng/ml. This may explain why some population studies fail to see increased risk of cancer in the vitamin-D-deficient group since the comparator cohort does not have elevated levels of 25(OH)D to reduce cancer risk. (4) Anti-inflammatory activity is an additional important pathway for calcitriol to exert chemopreventive and/ or therapeutic anti-cancer activity. (5) Vitamin D, calcitriol, or its analogs may have best utility as cancer therapeutic agents when used in cancer patients with early disease and perhaps in combination with other drugs. (6) We believe that calcitriol and its analogs should therefore be further evaluated in clinical trials in patients with early or precancerous disease. (7) Higher concentrations of calcitriol in the circulation may have to be achieved to obtain convincing proof of efficacy, which may require very high-dose intermittent therapy or the use of less-calcemic analogs. (8) In the case of established cancer, it is reasonable to consider that combination therapy will be required and vitamin D, calcitriol, or an analog added to other effective therapies will likely
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increase the benefit of the standard therapy and perhaps reduce some side effects. New clinical trials in PCa patients with minimal or early disease, treating with high doses of calcitriol, dietary vitamin D, or new potent analogs as well as combination therapy may finally demonstrate the promise of the benefit of vitamin D in PCa prevention and treatment.
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C H A P T E R
87 The Vitamin D System and Colorectal Cancer Prevention Heide S. Cross Vienna Medical University, Vienna, Austria
COLORECTAL CANCER EPIDEMIOLOGY AND PATHOGENESIS Epidemiology Colorectal cancer (CRC) is the third most common malignancy in developed countries with 147 500 new cases and 57 100 deaths in the USA in 2003 [1]. In statistical models to estimate incidence and mortality data for 25 cancers in 40 European countries for 2008, similar data were obtained: the most common cancer was CRC, and the second most common cause of cancer mortality was again CRC [2]. Among men 40 to 79 years old, CRC is the second most common fatal cancer [3], indicating the age-specific incidence of this disease: 90% of cases occur in persons 50 years or older. However, also intriguing variations with respect to gender, race, and geographic distribution are observed. It is quite evident that age-conditional morbidity as well as mortality for CRC is considerably less in women than in men. Also, survival time in years after the diagnosis of CRC is longer in women than in men [4]. While there are high CRC morbidity and mortality rates in socio-economically developed areas like the USA, Europe, and Australia, underdeveloped areas such as Africa and large parts of Asia have much lower incidence. However, when Asians migrate to mainland USA, they show rapid adaptation to incidence levels there. While CRC is a rarity in black Africans, CRC and colon polyps occur more frequently in AfricanAmericans than in any other ethnic group in the USA [5]. These geographic differences may indicate prevalence of some etiological factors that are preventive, such as lifestyle and diet, but not the relevance of a certain genetic background.
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10087-3
While it is recognized that CRC carcinogenesis results from the accumulation of multiple sequential mutations and alterations in nuclear and cytoplasmic molecules leading to invasive neoplasia [6], it also should be recognized that only up to 10% of CRC cases are estimated to be a direct consequence of inherited genetic alterations. Such familial CRC occurs at a relatively young age and cannot be treated by causal therapy and/or primary prevention. Already in 1975, Muto et al. were the first to establish the term “polypecancer sequence” meaning a multi-step carcinogenesis from normal colon mucosa through the stages of hyperproliferative epithelium, polyps (or adenomas, the classical precursor lesion of CRC), carcinoma in situ, to invasive cancer and metastasis [7]. However, there is increasing insight that many factors have a major influence on the risk to develop non-familial (sporadic) CRC during advancing age, and up to 70% of these are estimated to be preventable by changes in diet and lifestyle [8]. Since it can take more than two decades to complete the carcinogenetic process of sporadic CRC, there is a wide time window not only for the colorectal mucosa to be directly exposed to fecal irritating and mutagenic substances, but also for preventive substances to become effective. This may explain the well-known fact that at least 20% of the population between 50 and 60 years is positive for colonic microadenomas [9], while certainly not that many present with outright CRC later.
Risk Factors that can Enhance Colorectal Cancer Incidence While the role of actual mutagens as an etiological factor in human CRC is as yet undefined, it is well
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known that high amounts of carcinogens such as fecapentaenes or heterocyclic amines are present in the feces of persons living on a typical rich Western diet [10]. Also, physical inactivity, excess body weight and a central deposition of adiposity are considered risk factors. Dietary fats (especially saturated and transfats) may induce secretion of bile acids such as deoxycholic and cholic acid, which are converted to secondary and tertiary bile acids by colonic bacteria. Such bile acids may promote tumors by increasing colonic cell proliferation or by mutagenesis [11]. A consequence of some aspects of the Western diet (high in meat, saturated fats, and refined carbohydrates), i.e. excess energy intake, would be insulin resistance and subsequent hyperinsulinemia. A mechanism involving high levels of insulin-like growth factor-1, and alterations in signaling by nuclear transcription factor kappa B as well as by peroxisome proliferator-activated receptor gamma could lead to altered colonocyte growth kinetics [12]. Also, smoking, especially early in life, and high alcohol consumption leading to low serum levels of micronutrients such as folate and methionine could increase risk of CRC [13] (see “Folate consumption and epigenetic regulation of the vitamin D system,” below). There is strong evidence that insufficiency of vitamin D and of nutritional calcium may contribute to CRC pathogenesis. This evidence will be discussed in more detail in “Regulation of vitamin D metabolism in the gut mucosa by calcium,” below.
Some Potentially Protective Factors in CRC Pathogenesis Fiber presumably dilutes fecal carcinogens and reduces colonic transit time resulting in reduced mucosal exposure. However, recent epidemiological studies have cast some doubt on the long-standing concept that fruits and vegetables protect against CRC (see, e.g., [14]). Strong evidence is accumulating for a CRC protective effect of female sex hormones: women of all ages are less likely than men to develop colon cancer. Postmenopausal hormone replacement therapy is associated with decreased incidence and death rate of colon cancer in epidemiologic studies and intervention trials (see, e.g., [15]). Recently, an apparent interaction of estrogen therapy with calcium and vitamin D supplementation was found in a reanalysis of WHI data [16]. Although the precise mechanism for this is not clear yet, colon tissue is known to be positive primarily for estrogen receptor (ER)-b regardless of gender [17]. The ER-a/ER-b ratio has been identified as a possible determinant of the susceptibility of a tissue to estrogen-induced carcinogenesis. Thus, binding of estrogen to ER-a could induce cancer-promoting effects, whereas binding to ER-b exerts a protective action. Loss
of expression of ER-b is associated with advanced Duke’s staging in colon cancer [18]. It is significant that several studies have reported a lowered CRC risk associated with enhanced phytoestrogen intake. Phytoestrogens are plant-derived compounds (heterocyclic non-steroid phenols, isoflavones) that structurally and functionally act as estrogen agonists, but preferentially via ER-b: their affinity for ER-b is higher than that of estradiol itself, and is lower for ER-a [19]. Isoflavones are primarily found in soy bean products and other legumes. In Asian countries, populations with high consumption of soy foods have a clearly reduced risk of colorectal cancer incidence irrespective of gender (see, e.g., [20]). The relevance of sex hormones and phytoestrogens for optimization of the colonic vitamin D system is discussed in “Regulation of vitamin D metabolism and receptor expression by sex hormones,” below. In Western affluent societies there is widespread use of non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, sulindac, etc., and such substances were consistently shown to reduce incidence of CRC (see, e.g., [21]). While there is considerable debate about the exact mechanism of action, it is evident that cyclooxygenase-2 (COX-2) expression is increased in up to 90% of sporadic CRC, but not in normal mucosa from non-cancer patients. NSAIDs are COX-2 inhibitors and celecoxib, a COX inhibitor, was shown to reduce number and size of adenomas [22]. COX-2-dependent enhanced prostaglandin E2 (PGE2) production could eventually support increased cellular proliferation. A role for colonic vitamin D in detoxification for cancer prevention is discussed in “Regulation of vitamin D metabolism in the gut mucosa by calcium,” below and in Fig. 87.6 (see also Chapters 43 and 96 for further discussion on the role of vitamin D in detoxification and anti-inflammation). Calcium has a direct growth-restraining and differentiation- and apoptosis-inducing action on diverse normal and malignant cells, including colonocytes [23]. The antimitotic activity of dietary calcium on intestinal mucosal cells may be by binding and eliminating irritating bile acids and fatty acids in the feces [24]. However, since intestinal mucosa is exposed to dietary calcium in the feces, it also could have an antiproliferative effect directly on cells [25]. Findings from large prospective cohort studies have been notably consistent in finding inverse associations between dietary calcium and risk of colorectal carcinoma (for review see, e.g., [26]). Particularly the study of Garland et al. implied that CRC incidence could be significantly reduced by increasing daily calcium intake from 800 to 1400 mg [27]. However, in many of these studies vitamin D and calcium were supplemented together and, when calcium alone was provided, prevention of colorectal
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adenomas was only marginally reduced [28]. In this respect it is interesting that there seems to be a vitamin-D-dependent intracellular calcium gradient along the colon crypt axis with the highest calcium concentration to be found at the crypt surface [29]. The apparent dependence of calcium action on vitamin D for CRC prevention will be discussed in “Regulation of vitamin D metabolism in the gut mucosa by calcium,” below (see also Fig. 87.5). Folate, a water-soluble vitamin of the B family, is essential for synthesis, repair and methylation of DNA. Humans are unable to synthesize folate, therefore it must be provided in the diet. Important sources include citrus fruits, dark-green vegetables, and dried beans. DNA methylation of cytosine residues of CpG islands in the promoter region of genes is associated with transcriptional silencing of gene expression in mammalian cells, while decreased methylation of CpG islands enhances gene activity. Intake levels of folate and of other methyl-related nutrients as well as polymorphisms of methylenetetrahydrofolate reductase (MTHFR) are well known to contribute to cancer risk. MTHFR is required for the synthesis of methionine and S-adenosylmethionine, the DNA methyl donor. MTHFR polymorphisms, in association with low folate status, are frequently associated with modified colon cancer risk (see, e.g., [30]). There is a positive association between alcohol consumption and CRC risk, which may be related to the role of alcohol as a folate antagonist. Folate status in individuals who chronically consume moderate (15 g/day) alcohol may be impaired: alcohol consumption may contribute to folate malabsorption, enhanced excretion and abnormal metabolism [31]. The risk of colorectal adenoma incidence may be significantly modified by folate status. Colorectal adenoma risk was 30e40% lower in individuals with a median folate intake of 800 mg/day as compared with intake of around 200 mg/day [32]. Such high folate intake can be reached only by taking supplements. It has been suggested that daily ingestion of 400 mg as present in supplements, produces a sustained level of plasma folic acid [33]. Giovannucci et al. [34] and others demonstrated that prolonged intake of folate above currently recommended levels significantly reduced the risk not only of adenomas but of colorectal cancer as well (see “Folate consumption and epigenetic regulation of the vitamin D system,” below). Vitamin D is considered to be a micronutrient with high anticarcinogenic potential, especially in the colon. Recently, the concept of vitamin D insufficiency and its causal relationship with CRC pathogenesis has gained increasing support (for review see [35]). Some aspects of its CRC preventive mechanism will be discussed in “Colonic synthesis of 1,25-(OH)2D3,” below, and
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emphasis will be on its interrelationship with some other protective factors cited above (see “Regulation of CYP27B1 and of CYP24A1 expression by nutrition,” below).
VITAMIN D AND COLORECTAL CANCER PATHOGENESIS In the last decades there has been growing appreciation for the multitude of physiological roles that vitamin D has in many body tissues. As early as 1979 Stumpf et al. demonstrated that cells from heart, stomach, pancreas, colon, brain, skin, gonads, and many more, have the nuclear receptor for 1,25(OH)2D3, the so-called vitamin D receptor (VDR), and such tissues are potential targets for 1,25(OH)2D3 activity [36]. Many of these VDR-positive tissues are also positive for CYP27B1, i.e. the enzyme that can convert 25(OH)D3 to the active metabolite [37], and many of these tissues are known to be targets for development of malignancies. Regulation of CYP27B1 in these non-renal tissues differs from that observed in the kidney and, importantly in contrast to the renal enzyme, its activity may depend on high substrate concentration. This led to the novel concept that maintenance of adequate serum 25(OH)D3 levels would be essential for providing the substrate for the synthesis of the active metabolite at extrarenal sites which in turn would have physiological functions apart from those involved in bone mineral metabolism. There is increasing evidence that function and regulation of vitamin D synthesizing and catabolic hydroxylases, i.e. CYP27B1 and CYP24A1, respectively, in colorectal cells could affect pathogenesis during advancing age.
25(OH)D Insufficiency and Colorectal Cancer Pathogenesis Serum levels of 25(OH)D (i.e., the sum of 25(OH)D3 and 25(OH)D2, derived from synthesis in the skin and from dietary sources, respectively) is considered a reliable indicator of the vitamin D status of a given individual. Interestingly, a recent study undertaken to investigate long-term variation (over 5 years) of the serum 25(OH)D concentration among participants in the colorectal cancer screening trial indicated such low coefficients of variation, that a single time point of measurement may already be a useful biomarker to base disease prediction [38]. There is agreement that outright vitamin D deficiency or depletion, causing rickets or osteomalacia, is defined by 25(OH)D concentrations below 10 nM. Conservative calculations of the cut-off point between vitamin D insufficiency, i.e. suboptimal vitamin D supply, and
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vitamin D sufficiency arrived at a value of 30 nM [39]. However, there is good reason to believe that serum 25(OH)D should be maintained at much higher levels, i.e. between 60 and 100 nM, in order to prevent a variety of diseases, such as CRC and other malignancies [40]. Vitamin D insufficiency is frequently observed in individuals with limited sun exposure, such as in chronically ill, immobilized or housebound elderly people. Moreover, a compromised vitamin D status is a common phenomenon in the free-living normal population at any age. Even in Mediterranean countries with high UV-B irradiation, there is a risk for hypovitaminosis D because of specific lifestyle factors and socio-economic status. Many other studies indicate that hypovitaminosis D is a widespread phenomenon in the adult population of Central, Western and Northern Europe as well as in North America (see Chapter 52 on worldwide vitamin D deficiency and Chapter 56 on vitamin D and cancer risk). In 1980, Garland et al. were the first to propose that vitamin D is protective against CRC [41]. Highest death rates from CRC occur in regions distinguished by lack of winter UV-B radiation (generally due to a combination of high latitude, air pollution, ozone thickness and cloud cover). While global differences in CRC incidence may be primarily determined by nutrition and lifestyle, it is evident also that differences in UV-B radiation are a strong contributing factor. However, even in areas with ample sunshine like in the Mediterranean or in Australia, vitamin D insufficiency is frequently observed due to work/lifestyle and fear of skin damage by UV rays. It should be stressed again that, with low UV-B exposure, levels of the vitamin D precursor deemed sufficient for prevention of rickets may, however, not be high enough to support colonic synthesis of 1,25(OH)2D3 adequately to reach concentrations for malignancy prevention. This hypothesis is strongly supported by the linear concentration dependence for CRC incidence and serum 25(OH)D concentration in meta-analyses (see, e.g., [35,42]).
Some Mechanisms of Action by Vitamin D for CRC Prevention 1,25(OH)2D3 controls growth of normal and neoplastic cells by modulating the transcriptional activity of key genes involved in control of cellular proliferation, differentiation and apoptosis (for review see [43]). We have provided ample evidence for in vitro (see, e.g., [44,45]) as well as in vivo [46] inhibition of colonocyte proliferation and induction of differentiation by a functional vitamin D system. In vitro, 1,25(OH)2D3 concentrations of at least 10 nM or more are needed to inhibit growth significantly. However, it has to be stressed that, even in vitro, elevated calcium
concentrations in the medium reduced the amount of 1,25(OH)2D3 necessary for antimitotic action [47]: while 10 nM 1,25(OH)2D3 given to colonic cells cultured in a low calcium medium (0.25 mM) reduced growth only by 20%, cells grown in a normal calcium medium (1.80 mM) and treated with the same concentration of vitamin D showed reduced growth by 60% (see also “Regulation of vitamin D metabolism in the gut mucosa by calcium,” below, and Fig. 87.5). Recently it has been claimed that also the precursor 25(OH)D3 may be an agonistic vitamin D receptor ligand: apparently 400 nM 25(OH)D3 induced CYP24A1 expression significantly in 1a-hydroxylase-negative cells [48]. Since physiological 25(OH)D3 concentrations in human serum are considerably lower, ranging from 10 to a maximum of 80 nM, which is rarely reached [49], this probably does not play a role in human health and disease prevention. Studies from our laboratory identified c-Myc protooncogene and cyclin D1 expression as key targets of growth-inhibitory signaling from 1,25-(OH)2D3/VDR [50]. Since a number of intracellular proliferative pathways, namely the Raf-1/MEK1/ERK and STAT-3, converge at c-myc and engage cyclin D1, a key element in cell cycle control, as a common downstream effector, 1,25(OH)2D3 must be considered a potent general inhibitor of mitogenesis. Vitamin D induces the expression of the cyclin-dependent kinase inhibitors p21/Waf1 and p27Kip1 [51], which both are involved in G1 phase arrest. Another anti-proliferative mechanism of 1,25(OH)2D3 in human CRC cells involves direct interaction with growth factor receptor-activated pathways: 1,25(OH)2D3 reduces the number of ligand-occupied epidermal growth factor receptors (EGF-R) [52,53]. Palmer et al. [54,55] analyzed the complicated network of intracellular pathways by which signaling from 1,25(OH)2D3/VDR is transduced into cellular differentiation: induction of E-cadherin and inactivation of the b-catenin/T cell transcription factor-4 (TCF-4) complex are the key actions of 1,25(OH)2D3 in promoting differentiation of human colon carcinoma cells. In colorectal cells it also has been demonstrated that apoptosis was induced by vitamin D [56]. Another important role of vitamin D for cancer prevention may be the involvement of 1,25(OH)2D3 as a VDR agonist in detoxification of xenobiotics and carcinogens. It is especially the intestinal mucosa that is exposed directly to irritating and mutagenic substances. Kutuzova et al. demonstrated a significant increase in expression of phase I and phase II biotransformation enzymes by 1,25(OH)2D3 [57]. Also, the realization that the VDR can bind lithocholic acid, a carcinogenic bile acid, and that 1,25(OH)2D3 could act as a detoxifying agent, supports the concept that the vitamin D system protects the colonic
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mucosa against incipient malignancy [58,59] (see also Chapter 43). Ulcerative colitis (UC), an inflammatory bowel disease (IBD), is an important precursor lesion for colorectal cancer: risk is increased at least two-fold compared with the normal population [60]. It has been demonstrated that the expression of the VDR was significantly decreased in mucosal cells of UC patients [61]. Laverny et al. recently showed the efficacy of a new vitamin D analog with low calcemic activity to have higher anti-inflammatory potency than 1,25(OH)2D3 in cells derived from IBD patients. Also, the therapeutic efficacy of this compound was demonstrated in mice with experimental colitis [62].
COLONIC SYNTHESIS OF 1,25(OH)2D3 We have demonstrated previously the differential regulation of vitamin D hydroxylases in kidney and colon by using VDR knockout (KO) and wild-type (WT) mice: as demonstrated in Figure 87.1, both CYP27B1 and CYP24A1 mRNA are low in colon mucosa regardless of whether they are derived from WT or KO animals. In the kidney, however, the major site of 1,25(OH)2D3 synthesis, the expression of CYP27B1 is hugely increased in KO mice and CYP24A1 is downregulated in comparison with WT indicating the overproduction of the hormone due to the malfunction of the vitamin D system. This was used as a physiological indicator to clone the gene [63]. Such data strongly support the concept of differential regulation of 1,25 (OH)2D3 synthesis at extrarenal sites. Such differential regulation of 1,25(OH)2D3 production at multiple levels is a crucial determinant of nonclassical aspects of 1,25(OH)2D3 function. When we showed that normal and neoplastic human colon epithelial cells are endowed with a functional 25-hydroxyvitamin D-1a-hydroxylase and can thus convert 25(OH)D3
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to 1,25(OH)2D3 [64e66], we hypothesized that adequate accumulation of the active metabolite could slow down or inhibit progression of malignant disease by promoting differentiation and apoptosis and by suppressing antimitotic activity locally. While renal CYP27B1 activity is tightly regulated by serum Caþþ and parathyroid hormone (PTH) as well as by feedback inhibition from 1,25(OH)2D3, CYP27B1 expression in colonocytes is relatively insensitive to modulation via the PTH/[Caþþ]o axis [67]. Intracellular synthesis of 1,25(OH)2D3 at extra-renal sites depends largely on ambient 25(OH)D3 levels and is not influenced by plasma levels of 1,25(OH)2D3 (see, e.g., [68]). This may explain why the incidence of CRC is correlated with low serum 25(OH)D3 rather than with serum concentrations of 1,25(OH)D3 [69]. Moreover it is amply demonstrated that for growth suppression of colon cancer cells nanomolar concentrations of the active hormone are needed, a thousand-fold more than the physiological serum concentration. Strong support for the importance of 1,25(OH)D3 produced in colonocytes over that circulating in serum for regulation of cell functions comes from a study by Lechner et al. [70]: the characteristic anti-mitogenic effect of 1,25(OH)2D3 was induced in differentiated human colon carcinoma cells also when treated with 10 nM 25(OH)D3, but only when they were CYP27B1-positive. However, at low serum levels of 25(OH)D3, CYP27B1 activity in colonic cells may be not high enough since, in normal colonic mucosa without hyperproliferative signaling, positivity for CYP27B1 is extremely low [66] (see Fig. 87.2). Therefore, those steady-state tissue concentrations of 1,25(OH)2D3 necessary to maintain normal cellular growth cannot be achieved to counteract mitotic signaling, unless activity of the 1a-hydroxylating enzyme increases. It also has to be considered that 1,25(OH)2D3 itself is an important regulator of CYP27B1 gene expression since it downregulates the CYP27B1 gene via a negative vitamin D response element [71].
Evaluation of CYP24A1 and CYP27B1 mRNA by RT-PCR in colon and kidney of VDRþ/þ and VDR/ mice. Note the differential regulation of CYP24A1 and CYP27B1 mRNA in the two organs. Insert: Serum concentration of calcium and of 1a,25(OH)2D3. Note the exorbitant production of serum 1a,25(OH)2D3 in KO mice due to the non-functioning of the vitamin D system. KO ¼ VDR/ genotype. WT ¼ VDRþ/þ genotype.
FIGURE 87.1
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FIGURE 87.2 Immunofluorescent staining of CYP27B1 protein in normal colonic tissue from a non-cancer patient (left) and in a human colon adenoma (right). Magnification 20 (courtesy G. Bises, unpublished).
Expression of CYP27B1 and of the Vitamin D Receptor during Hyperproliferation and Tumor Progression We demonstrated the relevance of an intact vitamin D/vitamin D receptor (VDR) axis for maintenance of normal epithelial cell turnover in the large intestine in mice, which were genetically altered to block 1,25(OH)2D3/VDR signaling: the colon mucosa of VDR-null (VDR/) mice shows a pattern of increased DNA damage and cell division, the former probably due to formation of reactive oxygen species [72]. Interestingly, the large intestine reacts to inflammatory and hyperproliferative conditions with upregulation of the VDR and of its ligand-synthesizing enzyme, CYP27B1. In a mouse model of ulcerative colitis, a precursor lesion to CRC, it was reported that expression of CYP27B1 was increased four-fold [73]. We showed in human colon cancer tissue that expression of CYP27B1 rises about four-fold in the course of progression from normal mucosa to adenomas (see Fig. 87.2) and to well and moderately differentiated (G1 and G2) tumors, and then substantially declines during further progression [74]. Expression of the VDR showed the same dependence on tumor cell differentiation [75]. However, cells from poorly differentiated (G3) colonic lesions frequently are devoid of immunoreactivity for VDR and CYP27B1 while, at the same time, epidermal growth factor (EGF) receptor mRNA can be detected by in situ hybridization in almost any cancer cell [76]. This suggested that the 1,25(OH)2D3/VDR system can be activated in colon epithelial cells in response to mitogenic stimulation. However, further tumor growth could be retarded as long as cancer cells retain a certain degree of differentiation and, consequently, high levels of CYP27B1 activity and of VDR
expression. During progression to high-grade malignancy, when CYP27B1 activity and VDR expression are repressed by whatever mechanism, signaling from the vitamin D system would be too weak to effectively counteract proliferative effects from, for example, enhanced EGF-R activation. These hypotheses were confirmed in vitro by demonstrating that, in differentiated colon cells, EGF stimulates expression of VDR and CYP27B1, whereas after mitogenic stimulation of a primary culture derived from an advanced tumor, expression of VDR and of CYP27B1 was actually reduced by EGF treatment [77]. Induction of the adhesion protein E-cadherin expression by vitamin D enhances differentiation of colon cancer cells and opposes hyperproliferation. This indicates the importance of vitamin D activity for normal maintenance of the Wnt pathway. It is significant that repression of E-cadherin and of the VDR, with parallel enhanced expression of the transcription factor SNAIL, was found in patients with aggressive tumor characteristics [78] (see Chapter 13).
Colonic Expression of CYP24A1 during Hyperproliferation and Tumor Progression As already mentioned, besides substrate availability also additional regulatory factors may determine effective tissue concentration of 1,25(OH)2D3: (1) in colonocytes, 1,25(OH)2D3 downregulates CYP27B1 as well as VDR mRNA (see, e.g., [70]); (2) accumulating 1,25(OH)2D3 leads to activation of CYP24A1-encoded 25(OH)D3-24-hydroxylase expression, the enzyme that initiates stepwise degradation of the hormone [70]; and (3) at least in colon tumors, expression of CYP24A1 increases dramatically during progression to a poorly differentiated state (G3eG4) though CYP27B1
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that a large part of normal colonic mucosa was expressing CYP24A1 not at all or at low levels, whereas in the majority of adenocarcinomas the enzyme was present at high concentrations and expression of the proliferation marker Ki-67 was increased in parallel [81] (see also Fig. 87.4). These data suggest that, in patients with such basally high expression of CYP24A1 during advanced malignancy, effective treatment with vitamin D or certain vitamin D analogs will not be possible since these will be efficiently degraded via the C-24 pathway. However, this also clearly shows that inhibition of CYP24A1 activity in tumor cells could be of primary importance for cancer therapy. One example of CYP24A1 hydroxylase inhibiting compounds would be the 24-sulfone analogs of 1,25(OH)2D3 [82]. Interestingly, an analog from this group rapidly and potently inhibited 24-hydroxylase activity in human colon cancer cells by direct interaction with the enzyme, and this resulted in anti-proliferative activity of 1,25(OH)2D3 in colon cancer cell lines inherently positive for CYP24A1 and negative for CYP27B1 [66]. However, Lechner et al. [83] also demonstrated that the analog binds to the VDR and potently induces CYP24A1 mRNA, but for reasons unknown this is not translated into enzymatic activity. As mentioned above, and as shown by HPLC, cells isolated from well-advanced colon tumors express extremely high levels of 24-hydroxylase activity, whereas they are negative for that of the 1a-hydroxylase [70] (see also Fig. 87.3). While the exact mechanism of this upregulation is still not quite clear, this aspect will be discussed further in the section on epigenetic regulation of CYP24A1 (see below).
expression is diminished [79]. Therefore, not only in renal cells but also in colonic cells, sufficiency of tissue/serum 1,25(OH)2D3 concentration leads to induction of the vitamin-D-inactivating enzyme 1,25(OH)2D324-hydroxylase (CYP24A1), though during colon cancer progression when there is diminished expression of colonic CYP27B1, CYP24A1 is substantially induced [79]. The latter observation strongly suggests that in advanced tumors regulation of CYP24A1 expression is uncoupled from the vitamin D system. Therefore, a major mechanism for vitamin D resistance or reduced sensitivity in VDR-positive cancer cells is 1,25(OH)2D3 catabolism via the C-24 hydroxylation pathway. Colon cells isolated from well-advanced (G3) tumors express extremely high levels of CYP24A1, and cannot be growth-inhibited by 1,25(OH)2D3. As demonstrated in Figure 87.3, differentiated colon cells Caco-2 (A) initially synthesize 1,25(OH)2D3 efficiently (white columns) when they are exposed to 16.6 nmol 25(OH)D3 and catabolize it slowly (about 50% catabolized after 24 h, black column). The CYP27B1-negative cells COGA-13 (B), however, will efficiently use up the precursor within 12 h for 24,25(OH)2D3 production (black columns) and further degradation [70] (see Fig. 87.3). This demonstrates, at least in colon cells, an uncoupling of 1,25(OH)2D3 action from expression of CYP24A1 during advancing malignancy. Anderson et al. found an upregulation of CYP24A1 and a modest downregulation of the VDR in colon tumors when compared with normal tissue, though grading of tumors was not taken into consideration [80]. Very recently, an important paper by Horvath et al. demonstrated by real time (RT)-PCR as well as by immunohistochemistry
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FIGURE 87.3 Vitamin D metabolism in a differentiated (Caco-2) and an undifferentiated (COGA-13) colon tumor-derived cell line. (A) The active 1a,25(OH)2D3 is synthesized from its serum precursor 25(OH)D3 if cells are positive for CYP27B1. Sufficient 1a,25(OH)2D3 will induce colonic CYP24A1 expression and 1a,25(OH)2D3 produced is converted to less active metabolites by CYP24A1 (black columns). (B) In COGA-13 cells there is obviously no conversion of 25(OH)D3 to the active metabolite, but very rapidly the precursor is metabolized to 24,25(OH)2D3 and further (black columns). Modified from [70].
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Immunofluorescent staining of CYP24A1 (left) and Ki-67 (right) in a colon adenoma, magnification 20 (courtesy HC Horvath and E Kallay, unpublished).
FIGURE 87.4
Regulation of VDR, CYP27B1, and CYP24A1 Expression by Polymorphisms or Splicing Alternative gene splicing affects up to 70% of human genes and enhances genetic diversity by generating proteins with distinct new functions. In line with many cytochrome P450s, CYP27B1 is known to exhibit alternative splicing and, in kidney cells, this led to modified 1,25(OH)2D3 synthesis [84]. There have been several reports on differential expression of splice variants for CYP27B1 also in cancerous cells, for instance mammary cells where at least six splice variants of CYP27B1 were detected resulting in at least six protein variants [85]. Such splice variants present during tumor progression could lack 1a-hydroxylation activity. Though this has not been investigated yet in colon tumor cells, it does suggest a role for gene splicing in tissue-specific regulation of 1,25(OH)2D3 production. Unbalanced high levels of the candidate oncogene CYP24A1 were found in a variety of human malignancies, e.g. colorectal, ovary, breast, and lung tumors (see, e.g., [79,80]). It was observed that in colon tumors a CYP24A1 splice variant at 754 bp was much more prominent in differentiated (G1) than in undifferentiated tumors [65]. However, the activity of CYP24A1 is highly variable and does not correlate always with the expression level of the mRNA or protein [86]. Recently, new data on post-transcriptional modifications of CYP24A1 mRNA in human myelomonocytic [87] and prostate adenocarcinoma cell lines [75] suggested that alternative splicing might be the cause of the observed differences in CYP24A1 activity, and thus of inconsistent vitamin D effects in growth inhibition. Splice variants of CYP24A1 could lead to abnormal vitamin D catabolism and reduced or enhanced 1,25(OH)2D3 accumulation, respectively (see, e.g., [87]). The extremely high constitutive activity of the 24-hydroxylase in undifferentiated colon cancer cells such as COGA-13 (see Fig. 87.3) and
the only moderately enhanced mRNA expression when compared with differentiated cells, does suggest this alternative regulation [70]. Lately it was definitively shown that there are discrepancies between mRNA and protein expression levels of CYP24A1 in normal human colon mucosa compared with benign lesions and adenocarcinomas: more positivity for the degrading enzyme was demonstrated than for the mRNA species [81]. Further investigation provided strong evidence for alternative splicing of the CYP24A1 gene in human colon cancer cell lines and tissue samples [88] though the impact of such splice variants for colorectal tumorigenesis is not clear yet. However, catalytically dysfunctional isoenzymes could cause alterations in 1,25(OH)2D3 levels in the colonic cell microenvironment. In colon cancer patients, genetic variants of several markers, among them the VDR, were investigated to explore associations with microsatellite instability (MSI) or the CpG Island methylator phenotype (CIMP). Fok1 VDR polymorphism was associated with CIMP-positive tumors [89]. This polymorphism alters the start codon. Absence of this site results in a shorter VDR protein with higher biological activity [90]. However, presence of this site is linked to decreased calcium absorption, and low dietary calcium increases colon cancer risk in individuals with increasing copies of this particular VDR allele [91]. Recently, a caseecontrol study was conducted nested within the European Prospective Investigation into Cancer and Nutrition. The VDR Bsmi polymorphism was associated with a reduced risk of colon but not rectal cancer, and there was no association with Fok1 or the level of dietary calcium in this study [92]. Studies of genetic polymorphisms with respect to vitamin D hydroxylases are rare. As yet there is no consistent epidemiological evidence for substantial influence of SNP variants of vitamin D metabolism genes on risk of colorectal cancer. However, recently
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several SNPs were investigated for CYP24A1 and for CYP27B1. There was a statistically significant association between a particular CYP24A1 polymorphism and the risk of proximal colon cancer. A possible interaction between a CYP27B1 polymorphism, sun exposure, and proximal colon cancer risk was observed as well [93].
REGULATION OF CYP27B1 AND OF CYP24A1 EXPRESSION BY NUTRITION The colorectum, as part of the digestive system, clearly is particularly affected by nutritional components. Among these are substances supporting pathogenesis, as well as those contributing to prevention of malignancies, as outlined in “Risk factors that can enhance colorectal cancer incidence” and “Some potentially protective factors in CRC pathogenesis,” above. Particularly interesting, also for future preventive measures, is regulation of vitamin D hydroxylases by nutrients (see, e.g., [94]). It is becoming evident that, for prevention of sporadic CRC, average 25(OH)D3 levels at or above at least 50 nM need to be achieved in the general population, though there is still some discussion whether these levels should not be higher. As outlined previously, precursor levels in serum need to be high to adequately support extrarenal synthesis of 1,25(OH)2D3. However, there is apparently also potential to enhance or reduce activity of colonic vitamin D hydroxylases by physiological measures. Experimental proof is accumulating that nutrient factors like calcium, phytoestrogens, and folate could regulate expression of vitamin D hydroxylases.
Regulation of Vitamin D Metabolism in the Gut Mucosa by Calcium While in the past several groups found no evidence that increasing dietary calcium by enhanced intake of dairy foods had an inverse association with sporadic CRC incidence (a disease of advancing age) [95], it was accepted that there is age-related regulation of intestinal calcium absorption by vitamin D [96]. However, it now has finally been established that there is indeed a preventive effect of dietary calcium on CRC incidence. Some suggested mechanisms of calcium for CRC prevention include protection of colonocytes against bile acids and fatty acids [97], direct effects on cell proliferation [25] and modulation of the APC colon carcinogenesis pathway [98]. In addition, novel evidence from a recent pilot trial provided information that enhancing calcium and vitamin D intake over 6 months resulted in increased DNA mismatch repair system activity in the normal colorectal mucosa of sporadic adenoma patients [99].
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It was recognized that not only vitamin D but also calcium regulates Wnt signaling, and therefore adhesion and differentiation of cells: 1,25(OH)2D3 and calcium can control expression of E-cadherin and can regulate both the canonical (b-catenin-dependent) and non-canonical Wnt pathway [100]. b-Catenin is a multifunctional protein that is normally required both for cellecell adhesion and for regulation of gene expression in response to Wnt signaling [101]. Activation of the Wnt/b-catenin pathway by mutation of intracellular components or by epigenetic alteration of several Wnt inhibitors is one of the initial steps in colorectal tumorigenesis. The interference of 1,25(OH)2D3 with the canonical Wnt pathway may rely on the rapid induction of VDR/ b-catenin complexes that titrate out b-catenin, thus preventing formation of b-catenin/TCF complexes that regulate transcription of genes involved in tumorigenesis, e.g. c-myc and cyclin D1 [102,103,100] (see Fig. 87.6). Thus, our observation that 1,25(OH)2D3 downregulates c-myc and Cyclin D1 expression [47,50,53,104] can be explained through the modulation of Wnt signaling by vitamin D. Recently it has been show that 1,25(OH)2D3 activated different kinases involved in inhibition of the Wnt pathway [105]. The first step in this process was induction of Ca2þ influx by 1,25(OH)2D3, through L-type voltage-gated calcium channels [106]. While in normal colorectal mucosa human pilot trials indicated separate beneficial effects of both vitamin D and calcium supplementation, respectively, on proliferation, differentiation and a marker of oxidative DNA damage, there was no additional effect when both substances were given together [107,108]. Interestingly, however, during incipient malignancy, vitamin D in combination with high intake of calcium is much more effective in reducing risk than when vitamin D is provided alone [109,110]. Grau et al. [110] demonstrated that 25(OH)D3 levels were associated with a reduced risk of adenoma recurrence only among subjects with high calcium intake. An interventional trial conducted by Holt et al. [109], in which adenomatous polyp patients received high doses of supplemental calcium in combination with vitamin D for 6 months, resulted in a significant reduction in the rate of polyp formation and an increase in expression of apoptotic markers. Cho et al. [111] concluded from an analysis of pooled primary data from 10 cohort studies, in which more than half a million individuals were followed up for 6e16 years, that optimal risk reduction for colorectal cancer necessitates high intake levels of both vitamin D and calcium. Peterlik et al. [35] demonstrated in their analysis of primary data that efficiency of vitamin D in reducing the risk of colorectal cancer depends very much on the calcium status of an individual and vice versa (see Fig. 87.5).
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1.1
Adjusted hazard ratio
1.0 Vitamin D Intake Tertiles (IU/day)
0.9
1 (92–315)
0.8
2 (315–538) 3 (538–760)
0.7 0.6 0.5
200
400 600 800 1000 Calcium intake (mg/day)
FIGURE 87.6 Cooperative signaling from 1,25(OH)2D3/VDR and
Relative risk of colorectal cancer for total calcium intake by levels of total vitamin D intake. With permission from [35]. Data are from Table 4 in [173].
FIGURE 87.5
The observation that the efficiency of the antimitogenic action of 1,25(OH)2D3 in the colon is enhanced by adequate calcium nutrition can be explained on a molecular basis, at least in part: human neoplastic colonocytes express the parathyroid-type extracellular calcium-sensing receptor (CaR) at the mRNA and protein level as long as they retain a certain degree of differentiation [112e114]. The CaR is a G-protein-coupled membrane receptor which transduces minute changes in extracellular fluid Caþþ concentration into various intracellular signaling pathways. Activation of the CaR in human colon adenocarcinoma-derived cells causes inhibition of phospholipase A2 activity, which, in turn, would reduce the amount of arachidonic acid available for the synthesis of proliferation-stimulating prostaglandins. Further downstream, CaR-activated anti-proliferative signaling targets the canonical Wnt/ b-catenin pathway and thereby induces downregulation of T cell transcription factor (TCF)-4 and induction of E-cadherin expression [115] (see Fig. 87.6). In addition, it stimulates secretion of Wnt5a [115]. The secreted Wnt5a inhibits b-catenin signaling by increasing expression of a ubiquitin ligase that is involved in degradation of b-catenin [115]. Wnt5a protein expression in primary Dukes B colon cancer [116] is associated with a good prognosis. This suggests that activation of the CaR will enhance anti-proliferative and pro-differentiating signaling from 1,25(OH)2D3/VDR via the Wnt/ b-catenin pathway. Experimental studies in mice provided some in vivo evidence for the beneficial role of combining calcium and vitamin D for preventing colon cancer [117]. Normal C57Bl/6 mice, when fed for 2 years a Western-style diet consisting of high fat, decreased calcium, vitamin D and methyl donor nutrients, developed colonic tumors.
Caþþ/CaR inhibits proliferation and promotes differentiation of human colon cancer cells via the Wnt pathway (with permission from [35]).
However, formation of tumors could be prevented if the diet was supplemented with high levels of both calcium and vitamin D not only in normal [117,118] but also in APC-mutated mice [119]. To investigate this further on a molecular level, male and female mice were fed an AIN76 minimal diet containing 0.04% calcium compared with 0.9%. Animals on a low-calcium diet presented with enhanced positivity for PCNA (proliferating cell nuclear antigen) and for cyclin D1 in their colonic mucosa, while that for p21, a cyclin-dependent kinase inhibitor, was diminished. Mice on a calcium-deficient diet also expressed CYP24A1 mRNA at a six- to eight-fold higher level than their counterparts on a 0.9% calcium diet [67]. It is recognized that a lack of calcium increases concentrations of free bile acids in the gut lumen. Of these lithocholic acid, by binding to the VDR, can induce expression of CYP24A1 [59]. Interestingly, CYP27B1 mRNA was significantly upregulated in the proximal colon of mice fed 0.04% compared to 0.9% calcium, though in females only [120]. Importantly, measurement of 1,25(OH)D3 concentrations in mucosal homogenates by a newly developed assay [120] indicated that upregulation of CYP27B1 by low calcium is translated into increased CYP27B1 protein activity causing enhanced accumulation of 1,25(OH)D3 in colonic mucosal cells derived from female mice. In parallel, in these cells apoptotic pathways, i.e. expression of the downstream effector proteases, caspase-3 and caspase-7, are stimulated. This suggests that enhanced synthesis of 1,25(OH)D3 in females overrides the gender-independent stimulatory effect of low calcium on CYP24A1-mediated vitamin D catabolism, thereby providing protection against incipient hyperproliferation induced by inadequate calcium nutrition. This enhanced synthesizing activity occurred
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in the proximal colon only and suggests that there is site-specific action of 17b-estradiol. It is significant in this respect that the estrogen receptor is more methylated (inactivated) in the human distal than in the proximal colon [121] and that the incidence of advanced (G3) cancer of the proximal colon occurs 10e15 years later in female compared with male patients [122] (see also the following section). This also provides experimental support for the observation provided by the WHI, that HRT significantly reduced the incidence of CRC in postmenopausal women [123]. Our experiments with mice further confirmed the hypothesis on differential regulation of CYP27B1 and CYP24A1 in renal compared to colonic cells (see also Fig. 87.1 for our observations in VDR/KO mice): while low dietary calcium reduced CYP24A1 expression in renal, but increased it in colonic, cells, it elevated CYP27B1 in renal cells and did not affect colonic expression [67], except for that in female mice [120]. Interestingly, another extrarenal CYP27B1 gene, that in osteoblasts, was differentially and specifically regulated by dietary calcium similar to that in colonocytes: there was strong induction of mRNA expression by high dietary calcium. Unfortunately only female mice were used for these experiments [124]. Our results suggest that also in humans, calcium supplementation could lower the risk of colorectal cancer, because high dietary calcium suppresses vitamin D catabolism and thus would favor accumulation of 1,25(OH)D3 in the colon mucosa. Furthermore, 1,25(OH)D3 could increase expression of the CaR by binding to a vitamin D response element in its promoter region [125]. However, it is clear that there could be additional factors: evidence is accumulating that the VDR genotype and its influence on CRC incidence may be strongly modified by the level of dietary calcium [126]. In another large study of the European population polymorphisms of the VDR as well as of the CaR were evaluated in relation to incidence of CRC. Interestingly, only the VDR BsmI polymorphism, but neither Fok1 nor CaR genotypes, was associated with CRC risk [92].
Regulation of Vitamin D Metabolism and Receptor Expression by Sex Hormones Although men and women suffer from similar rates of colorectal cancer deaths in their lifetime, the ageadjusted risk for colorectal cancer is less for women than for men [127]. Recently, using population-based cancer registry data from the USA as well as national mortality statistics from different countries, it was demonstrated that CRC incidence and death occurred 4 to 8 years later in women than in men. This difference in age was increasing with advancing seniority [128].
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However, evidence from meta-analyses is increasing that men are actually at a greater risk for advanced colorectal neoplasia across all age groups [129]. We have recently performed a cross-sectional study on a cohort of CRC patients recruited in Vienna, Austria, to reveal the influence of age, gender, and subsite on grades of malignancy. Women diagnosed with high-grade cancer in the proximal and distal colon were at a median age of 75 years and were thus 10e15 years older than their male counterparts. This suggests that, premenopausally, female sex hormones could postpone progression of the disease. In contrast, high-grade rectal cancer developed in both genders around the early age of 60 years [122]. This, and other evidence, indicates a protective role of female sex hormones, particularly of estrogens, against colorectal cancer (see, e.g., [3,130]). In observational studies postmenopausal hormone therapy is associated with a lower risk for colorectal cancer and a lower death rate in women [131]. A metaanalysis of studies showed a 34% reduction in the incidence of this tumor in postmenopausal women receiving hormone replacement therapy (HRT) [123]. Further investigation confirmed that in a majority of clinical studies either HRT or estrogen replacement therapy (ERT) significantly reduced the risk of colon cancer in postmenopausal women (see, e.g., [132]). While a precise mechanism of action for estrogens to decrease colon cancer risk is not known yet, estrogen receptors are present in both normal intestinal epithelium and in colorectal cancer which suggests that the hormone is probably protective through these receptors and resultant post-receptor cellular activities. While the colon cannot be considered an estrogendependent tissue, it must be defined as an estrogenresponsive organ. Whereas human colon mucosa expresses primarily the ER-b type regardless of gender [133], ER-a is mainly expressed in the breast and the urogenital tract [134]. Both receptors bind estrogen, but they activate promoters in different modes. Studies of breast and prostate carcinogenesis suggested opposite roles for ER-a and ER-b in proliferation and differentiation [135]. A protective role of ER-b was suggested since decreasing levels of the receptor were reported during colonic tumorigenesis compared with expression in the adjacent normal mucosa from the same patient [17]. Using ER-b knockout and wild-type mice, it was recently confirmed that estradiol protected the colonic mucosa from development of preneoplastic lesions via an ER-b-dependent mechanism [136]. Furthermore, an ER-b-selective agonist has been reported to be an effective treatment in animal models of IBD [137], and IBD patients are known to have an at least two-fold increased risk of CRC incidence. Interestingly, inflammationassociated CRC occurs in males at least twice as frequently compared with females [138].
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A potential mechanism of the protective action of estrogens may be via regulation of the vitamin D system. As early as 1986 a study on the effect of endogenous estrogen fluctuation with respect to 25(OH)D3 metabolism was published [139]. This study in healthy premenopausal women suggested that 25(OH)D3 was metabolized predominantly to 24,25(OH)2D3 at low estrogen, but to 1,25(OH)2D3 at higher serum estrogen concentrations. While this, in 1986, primarily concerned renal synthesis of vitamin D metabolites, it was the first suggestion that estrogen elevates CYP27B1 expression. Liel et al. [140] reported that estrogen increased VDR activity in epithelial cells of the gastrointestinal tract. In the colon adenocarcinoma-derived cell line Caco-2, which is ER-b positive but negative for ER-a, Lechner et al. [141] demonstrated an increase of CYP27B1 mRNA expression and also of enzymatic activity after treatment with 17b-estradiol. Based on these findings a clinical pilot trial was designed, in which postmenopausal women with a past history of rectal adenomas were given 17b-estradiol daily for 1 month to reach premenopausal serum levels. Rectal biopsies were obtained at the beginning and end of the trial. A predominant result was the elevation of VDR mRNA [142]. We also observed significant induction of CYP27B1 mRNA in parallel with a decrease in COX-2 mRNA expression in those patients who had particularly high levels of the inflammatory marker at the beginning of the trial (Fig. 87.7). To study modification of vitamin D hydroxylase activity by 17b-estradiol further, a mouse model was used to measure actual 1,25(OH)2D3 synthesis and accumulation in colonic mucosa. In female compared with male mice, CYP27B1 mRNA was doubled and 1,25(OH)2D3 concentration in the mucosa was increased by more than 50%. This occurred in the proximal colon only and suggested that there may be site-specific action
Quantitative real-time PCR analysis of CYP27B1 and COX-2 mRNA levels in rectal mucosa. Ten postmenopausal women at risk for colorectal neoplasia were treated for 4 weeks with 0.5 mg 17-b estradiol. Biopsies were taken at the beginning and end of the pilot trial (courtesy E Kallay and HS Cross, unpublished).
FIGURE 87.7
of 17b-estradiol [120]. In this respect it is significant that the estrogen receptor has been shown to be more methylated (inactivated) in the human distal than in the proximal colon [121]. It is of interest for CRC prevention by nutrition that in East Asian populations the risk of cancers not only of the breast and prostate gland but also of the colorectum is clearly lower than elsewhere. It has been suggested that this is due to the typical diet in this part of the world, which is rich in soy products and therefore contains high amounts of phytoestrogens. Genistein could also have anti-inflammatory properties in the colon: When mice were fed low dietary calcium (0.04%), COX-2 mRNA and protein were increased two-fold in colon mucosa. Supplementation of genistein to the diet lowered COX-2 expression to control levels (0.5% dietary calcium) in both genders [143]. This suggests that genistein could have a beneficial effect on colonic inflammation similar to that seen with 17b-estradiol in the human pilot study described above. Since genistein preferentially activates ER-b [19,144], which is equally expressed in the colon of women and men, low rates of colorectal cancer incidence in both genders in soy-consuming populations could be due to appropriate modulation of the anti-inflammatory and anti-cancer potential of vitamin D by phytoestrogens. In an in vitro study comparing breast cancer with colon cancer cells we investigated whether extrarenal vitamin D metabolism could be involved in the protective action of phytoestrogens. Whereas genistein induces CYP27B1, and reduces CYP24 expression as well as activity in human colon adenocarcinomaderived cell lines [145], daidzein, another phytoestrogen prominent in soy and, importantly, its metabolite equol which is strongly active in other biological systems, did not affect colonic or mammary cell vitamin D hydroxylases [141]. It has been claimed that the biological effects of soy foods are mainly due to equol produced by fecal bacteria [146]. Equol is bacterially derived from daidzein as two distinct diastereoisomers. The S-equol enantiomer is apparently the active metabolite since it has a high affinity for ER-b, whereas R-equol is rather inactive [147]. Since only about 30e50% of the human population are equol producers this could potentially account for discrepancies in human nutritional studies with soy [148]; however, obviously not with respect to the vitamin D system. In a recent meta-analysis, soy consumption was not associated with colon cancer risk, nor with that for rectal cancer. However, when data were analyzed separately for gender a significant reduction of risk became apparent in women only [149]. In view of the fact that both men and women have similar distributions of estrogen receptors in the colon, additional modifying factors certainly must exist.
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Folate Consumption and Epigenetic Regulation of the Vitamin D System Intake levels of folate and of other methyl-related nutrients as well as polymorphisms of methylenetetrahydrofolate reductase (MTHFR) are well known to contribute to cancer risk. MTHFR is required for the synthesis of methionine and S-adenosylmethionine, the DNA methyl donor. MTHFR polymorphisms, in association with low folate status, are frequently associated with modified colon cancer risk (see, e.g., [30]). In addition, methionine synthase is a key enzyme in the pathway leading to DNA methylation, and its polymorphisms are related to protection against colon adenoma risk in the presence of a good folate status [150]. There is also a positive association between alcohol consumption and cancer risk, which may be related to the role of alcohol as a folate antagonist. Folate status in individuals who chronically consume moderate amounts (15 g/day) of alcohol may be impaired: alcohol consumption may contribute to folate malabsorption, enhanced excretion and abnormal metabolism [31,151]. The risk of colorectal adenoma may be significantly modified by folate status [152]. Colorectal adenoma risk was 30e40% lower in individuals with a median folate intake of around 800 mg/day compared with intake around 200 mg/day [32]. Such high folate intake can be reached only by taking supplements. It has been suggested that daily ingestion of 400 mg as present in supplements produces a sustained level of plasma folic acid [33]. Giovannucci et al. [34] and others demonstrated that prolonged intake of folate above currently recommended levels significantly reduced the risk not only of adenomas but of colorectal cancer as well. Higher folate intake also significantly reduced the excess risk of colon cancer among women with a family history, especially if there was use of multivitamins for longer than 5 years [153]. However, it has to be recognized that optimal folate supplementation may depend not only on dose but also on timing: at least in rodent models very high folate supplementation may promote instead of suppress colorectal carcinogenesis [154]. Therefore a “dual-modulator” role was suggested for folate in colorectal cancer, with a protective influence when ingesting moderate amounts before development of aberrant crypt foci [155]. Progression from the normal to the malignant cell phenotype is associated with various genetic and epigenetic alterations [156]. The latter regulate gene expression by DNA methylation and histone acetylation/ deacetylation, without changing the dinucleotide sequence. Repeat CpG residues (CpG islands) are localized predominantly in the promoter regions of genes: methylation of cytosine residues of CpG dinucleotides
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is catalyzed by several methyltransferases and this is thought to control gene transcription [157]. Such epigenetic modulations are implicated in aging and in tumor development. Aberrant DNA methylation has been linked to diverse human pathologies [158] such as colorectal cancer [159,160]. Bariol et al. [161] have shown that proliferation in small adenomas and hyperplastic polyps correlated with extent of DNA demethylation. In contrast, hypermethylation has been reported to silence a variety of genes implicated in tumor growth and cell proliferation [162]. The CpG island methylator phenotype (CIMP) is a distinct phenotype in sporadic colorectal cancer. For instance, a CIMP-high status is significantly associated with tumors of the proximal colon. Also relative survival can be associated with methylation status [163]. Also histone acetylation/deacetylation regulates transcription epigenetically: histone acetyltransferases (HAT) are associated with increased transcriptional activity, whereas deacetylation by histone deacetylases (HDAC) causes the condensation of chromatin, making it inaccessible to transcription factors; genes are therefore silenced [164]. Genes modified by epigenetic events could be those coding for the vitamin D system. In a mouse model of chemically induced colon cancer, protection against tumor incidence by estrogen was associated with decreased CpG island methylation of the VDR promoter and enhanced VDR expression [165]. Kim et al. [166] demonstrated that the negative response element in the CYP27B1 promoter is regulated by the ligandactivated vitamin D receptor through recruitment of histone deacetylase, a critical step for chromatin structure remodeling in suppression of the CYP27B1 gene. In addition, this transrepression by VDR requires DNA methylation in the CYP27B1 gene promoter. This, however, was demonstrated in kidney cells and not in tumor-derived cells. Another study highlighted the relevance of different microenvironments (tumor versus normal) for the regulation of CYP24A: CYP24A1 promoter hypermethylation was present in endothelial cells derived from tumors, but not from normal tissue [167]. To investigate the potential relevance of folate intake for regulation of the vitamin D system, C57/BL6 mice were fed a semisynthetic AIN76A diet, which contained, among others, 5% fat, 0.025 mg/g vitamin D3, 5 mg/g calcium and 2 mg/g folic acid [97,168]. When this basal diet was modified to contain high fat, low calcium, low vitamin D3 and low folic acid, mice exhibited signs of hyperplasia and hyperproliferation in the colon mucosa [168], which were accompanied by a more than 2.5-fold elevated CYP24A1 mRNA expression [169]. When calcium and vitamin D3 in the diet were optimized while fat was still high and folic acid low,
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CYP24A1 mRNA expression fell by 50%, but was still higher than in the colon mucosa of mice fed the basal (control) diet. Finally, when the diet contained high fat, low calcium and low vitamin D, but folic acid content was optimized, only then was any increment in colonic CYP24A1 due to dietary manipulations completely abolished [169]. This suggests that, at least in mice, a so-called “Western diet,” resembling the high-fat, lowvitamin-D and calcium diet prevailing in affluent Western countries, leads to degradation of colonic 1,25(OH)2D3, and it is only by folate optimization that this degradation can be stopped. It also suggests that folate optimization overrides the negative effects of low vitamin D and calcium intake. Differences in expression of vitamin D hydroxylases in the course of tumor progression as observed in colon cancer patients [74,170] could be caused by epigenetic regulation of gene activity via methylation/demethylation processes as well as histone acetylation/deacetylation. In early malignant lesions, CYP27B1 expression is exceedingly high compared to normal mucosa in noncancer patients [66]. If the ambient concentration of 25(OH)D3 is sufficient, this could potentially result in enhanced synthesis and accumulation of 1,25(OH)2D3 in the colon mucosa, and this in turn would be responsible for autocrine/paracrine inhibition of tumor cell growth. We suggest that enhanced expression of CYP27B1 during early malignancy could be due, at least in part, to epigenetic regulation, i.e. demethylation, while raised CYP24A1 expression in such tumors probably results from the normal regulatory loop following accumulation of 1,25(OH)2D3 in colonic mucosa [70]. However, in highly malignant tumors, an efficient antimitogenic effect by 1,25(OH)2D3 is unlikely, because expression of the catabolic vitamin D hydroxylase by far exceeds that of CYP27B1 [81,171]. We therefore postulated that also the potential oncogene CYP24A1 could be under epigenetic control [94]. This hypothesis was initially confirmed in prostate cancer cell lines that express high vitamin D catabolic activity during progression as well [172]. Studies in colon tumor-derived primary cultures and cell lines subsequently indicated that this might also be the case in colonocytes: Constitutive CYP24A1 expression is extremely high in COGA-13 cells derived from an advanced colon tumor, but not apparent in differentiated Caco-2 cells (see also Fig. 87.3). Addition of the methyltransferase inhibitor 5-aza-20 -deoxycytidine induced CYP24A1 mRNA expression significantly in Caco-2 cells while, in COGA-13 cells, the methyltransferase inhibitor did not further raise the already high basal CYP24A1 expression. Interestingly, even the totally repressed CYP27B1 expression in COGA-13 cells appeared to be under epigenetic control, since there was distinct elevation of CYP27B1 mRNA after
treatment with 5-aza-20 -deoxycytidine (Khorchide et al., manuscript submitted). This suggests that, during cancer progression, CYP27B1 expression could be inactivated by epigenetic mechanisms, whereas that of CYP24A1 would be activated. We therefore studied expression of vitamin D hydroxylases in 105 colon tumor patients entering a Viennese hospital for tumor resection. Uncoupling of CYP24A1 expression from regulation by colonic 1,25(OH)2D3 would lead to vitamin D hydroxylase expression in opposite directions during progression to a highly malignant state. This is actually the case: transition from low- to high-grade cancers is associated with a further highly significant rise in CYP24A1 and a simultaneous decline of CYP27B1 mRNA expression [171]. These changes in expression levels are not only colon site specific but also gender specific (Brozek et al., manuscript submitted).
Acknowledgments I would like to thank my graduate students and postdoctoral collaborators for their contributions to these studies that were performed primarily between 1995 and 2007. I also would like to thank my long-time technician Teresa Manhardt for honesty and perseverance. The financial support I have obtained over the years from the Austrian National Bank, the American Institute for Cancer Research, the World Cancer Research Fund, and the European Community Research Funds is gratefully acknowledged.
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colorectal adenomas: results of a randomized trial, J. Natl. Cancer Inst. 95 (2003) 1765e1771. E. Cho, S.A. Smith-Warner, D. Spiegelman, W.L. Beeson, P.A. van den Brandt, G.A. Colditz, et al., Dairy foods, calcium, and colorectal cancer: a pooled analysis of 10 cohort studies, J. Natl. Cancer Inst. 96 (2004) 1015e1022. E. Ka´llay, E. Bajna, F. Wrba, S. Kriwanek, M. Peterlik, H.S. Cross, Dietary calcium and growth modulation of human colon cancer cells: role of the extracellular calcium-sensing receptor, Cancer Detect Prev. 24 (2000) 127e136. E. Kallay, E. Bonner, F. Wrba, R.V. Thakker, M. Peterlik, H.S. Cross, Molecular and functional characterization of the extracellular calcium-sensing receptor in human colon cancer cells, Oncol. Res. 13 (2003) 551e559. Y. Sheinin, E. Kallay, F. Wrba, S. Kriwanek, M. Peterlik, H.S. Cross, Immunocytochemical localization of the extracellular calcium-sensing receptor in normal and malignant human large intestinal mucosa, J. Histochem. Cytochem. 48 (2000) 595e602. R.J. MacLeod, M. Hayes, I. Pacheco, Wnt5a secretion stimulated by the extracellular calcium-sensing receptor inhibits defective Wnt signaling in colon cancer cells, Am. J. Physiol. Gastrointest. Liver Physiol. 293 (2007) G403eG411. J. Dejmek, A. Dejmek, A. Safholm, A. Sjolander, T. Andersson, Wnt-5a protein expression in primary dukes B colon cancers identifies a subgroup of patients with good prognosis, Cancer Res. 65 (2005) 9142e9146. H. Newmark, K. Yang, N. Kurihara, K. Fan, L. Augenlicht, M. Lipkin, Western-style diet-induced colonic tumors and their modulation by calcium and vitamin D in C57Bl/6 mice: a preclinical model for human sporadic colon cancer, Carcinogenesis 30 (2009) 88e92. K. Yang, N. Kurihara, K. Fan, H. Newmark, B. Rigas, L. Bancroft, et al., Dietary induction of colonic tumors in a mouse model of sporadic colon cancer, Cancer Res. 68 (2008) 7803e7810. K. Yang, S.A. Lamprecht, H. Shinozaki, K. Fan, W. Yang, H.L. Newmark, et al., Dietary calcium and cholecalciferol modulate cyclin D1 expression, apoptosis, and tumorigenesis in intestine of adenomatous polyposis coli1638N/þ mice, J. Nutr. 138 (2008) 1658e1663. T. Nittke, E. Kallay, T. Manhardt, H.S. Cross, Parallel elevation of colonic 1,25-dihydroxyvitamin D3 levels and apoptosis in female mice on a calcium-deficient diet, Anticancer Res. 29 (2009) 3727e3732. J. Horii, S. Hiraoka, J. Kato, K. Harada, K. Kuwaki, H. Fujita, et al., Age-related methylation in normal colon mucosa differs between the proximal and distal colon in patients who underwent colonoscopy, Clin. Biochem. (2008) 1440e1448. W. Brozek, S. Kriwanek, E. Bonner, M. Peterlik, H.S. Cross, Mutual associations between malignancy, age, gender, and subsite incidence of colorectal cancer, Anticancer Res. 29 (2009) 3721e3726. R.T. Chlebowski, J. Wactawski-Wende, C. Ritenbaugh, F.A. Hubbell, J. Ascensao, R.J. Rodabough, et al., Estrogen plus progestin and colorectal cancer in postmenopausal women, N. Engl. J. Med. 350 (2004) 991e1004. P.H. Anderson, S. Iida, J.H. Tyson, A.G. Turner, H.A. Morris, Bone CYP27B1 gene expression is increased with high dietary calcium and in mineralising osteoblasts, J. Steroid Biochem. Mol. Biol. 121 (2010) 71e75. L. Canaff, G.N. Hendy, Human calcium-sensing receptor gene. Vitamin D response elements in promoters P1 and P2 confer transcriptional responsiveness to 1,25-dihydroxyvitamin D, J. Biol. Chem. 277 (2002) 30337e30350.
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C H A P T E R
88 Hematological Malignancy Ryoko Okamoto 1, H. Phillip Koeffler 1, 2 1
Division of Hematology and Oncology, Cedars-Sinai Medical Center, Los Angeles, CA, USA, 2 National University of Singapore, Singapore
INTRODUCTION Vitamin D3 is produced in skin and is sequentially metabolized by the liver and kidney to the biologically active form 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). This secosteroid hormone regulates calcium homeostasis within the body. In addition, 1,25(OH)2D3 has a role in normal hematopoiesis, inducing myeloid progenitor cells to differentiate into monocytesemacrophages. This chapter focuses on the interaction of 1,25(OH)2D3 with the normal and the malignant hematopoietic system, especially examining the ability of this secosteroid to induce differentiation and inhibit proliferation of normal and leukemic myeloid cells. The development and testing in vitro and in vivo of vitamin D analogs is discussed. Although these compounds, used either alone or combined with other agents, have preclinical activity, their efficacy in clinical trials has thus far appeared to be limited.
OVERVIEW OF HEMATOPOIESIS Hematopoiesis is the process that leads to the regulated formation of the highly specialized circulating blood cells from pluripotent hematopoietic stem cells (HSCs) in the bone marrow. The HSCs are the most primitive blood cells, and they have the ability for both selfrenewal and pluripotency (Fig. 88.1). They differentiate to more mature “committed” cells including the common lymphoid progenitor (CLP) and the common myeloid progenitor (CMP). The CLP population produces dendritic cells and mature T or B lymphocytes. CMP cells differentiate into megakaryocyteeerythroid progenitors (MEP), granulocyteemacrophage progenitors (GMP), mature mast cells, eosinophils and basophils. The MEP
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10088-5
cells eventually differentiate into functional red blood cells and platelets. The GMPs give rise to either neutrophils or monocytes. Differentiation toward monocytes is induced synergistically by interleukin (IL)-3, granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF) in vivo. This process can be fostered by either all-trans-retinoic acid (ATRA) or 1,25(OH)2D3 in vitro (see “Effects of vitamin D compounds on normal hematopoiesis,” below). Monocytes differentiate into either dendritic cells or macrophages. The differentiation and proliferation of hematopoietic stem cells as well as their more mature precursor cells are highly controlled by cytokines from the extracellular environment. Each of these stem cells has cell surface receptors for specific cytokines. Binding of cytokines to these receptors stimulates secondary intracellular signals that deliver a message to the nucleus to enhance proliferation, differentiation, and/or activation. The growth factors acting primarily on the granulocyteemacrophage pathway are GM-CSF, G-CSF, and macrophage colony-stimulating factor (M-CSF). GMCSF also stimulates eosinophils, enhances megakaryocytic colony formation, and increases erythroid colony formation in the presence of erythropoietin (Epo). In vivo, GM-CSF causes an increase in granulocytes, monocytes, and eosinophils. GM-CSF can activate monocytes and granulocytes to efficiently kill invading microbes. GM-CSF stimulates the formation of granulocyte colonies in vitro, synergistically with IL-3 and M-CSF. This GM-CSF is active in vivo, stimulating an increase of peripheral blood granulocytes. M-CSF stimulates the formation of macrophages, maintains the survival of differentiated macrophages, increases their antitumor activities and their secretion of oxygen reduction products, as well as plasminogen activators. M-CSF binds
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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88. HEMATOLOGICAL MALIGNANCY
IL-3
Mast cell
IL-3, IL-4, GM-CSF
EoP
IL-3, IL-4
BoP EPO
miR-451 SCF-IL-3 MeP
miR-155
SCF IL-3
GM-CSF IL-3, G-CSF miR-223
NeP
GMP SCF MPP HSC
SCF
IL-3 GFI C/EBPα GM-CSF MDP
RUNX1 SCL
CLP
Basophil
RCP
MEP
CMP
Eosinophil
SCF, GM-CSF, IL-3, TPO Megakaryocyte / Platelet miR-150, -155, -451 GM-CSF, IL-3, G-CSF GFI, C/EBPε
PU.1, IRF8 GM-CSF IL-3, G-CSF miR-17, -18 -20, -106, -155
Mo
GM-CSF IL-4 GATA-1
SCF, IL-7
PreB
Dendritic cell
T lymphocyte
miR-142, -181
ProB
Neutrophil
M-CSF Macrophage MafB C/EBPβ miR-424
IL-1, IL-2, IL-3, IL-6, IL-7
PreT
Red blood cell
Myelopoiesis
MCP
IL-6, IL-4
B lymphocyte
miR-150
FIGURE 88.1 Scheme of hematopoiesis. Myelopoiesis is the regulated formation of myeloid cells, including eosinophilic granulocytes, basophilic granulocytes, neutrophilic granulocytes, and monocytes. This occurs in the bone marrow. Monocytopoiesis is the process which leads to the production of monocytes, subsequently, macrophages. It is one component of myelopoiesis. Abbreviation: HSC, hematopoietic stem cell; MPP, multipotential progenitor; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; MCP, mast cell precursor; EoP, eosinophil precursor; BoP, basophil precursor; MEP, megakaryocyteeerythroid progenitor; GMP, granulocyteemacrophage progenitor; RCP, red blood cell precursor; MeP, megakaryocyte precursor; NeP, neutrophil precursor; MDP, macrophage and dendritic-cell progenitor; Mo, monocyte; PreT, precursor of T lymphocyte; PreB, precursor of B lymphocyte; M-CSF, macrophage colony-stimulating factor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyteemacrophage colony-stimulating factor; IL, interleukin. Black box shows upregulated transcriptional factors. Gray box shows involving miRNAs. Of note, miR-17, -18, -20, -106 are downregulated during monocytic differentiation.
to a receptor that is the product of the proto-oncogene cfms. IL-3 has multilineage stimulating activity and acts directly on the granulocyteemacrophage pathway, but also enhances the development of erythroid, megakaryocytic, and mast cells, and possibly T lymphocytes. In synergy with Epo, IL-3 stimulates the formation of early erythroid stem cells, promoting the formation of colonies of red cells known as BFU-E in soft gel culture. In addition, IL-3 supports the formation of early multilineage cells in vitro. IL-3 also induces early progenitor cells to enter the cell cycle, and in combination with other growth factors, stimulates the production of all the myeloid cells in vivo. Stem cell factor (SCF) promotes survival, proliferation, and differentiation of hematopoietic progenitor cells. It synergizes with other growth factors such as IL-3, GM-CSF, G-CSF, and Epo to support the clonogenic growth of HSCs in vitro. SCF is a ligand for the c-kit receptor, a tyrosine kinase receptor that is expressed in hematopoietic progenitor cells. The growth factor Epo stimulates the formation of erythroid colonies
(CFU-E) in vitro and is the primary hormone of erythropoiesis in animals and humans. It binds to a specific receptor (Epo-R). Production of erythroblasts is regulated by Epo which is modulated by the amount of tissue oxygenation of Epo-producing cells in the kidney. Oxygen-carrying hemoglobin in the red blood cells is the physiologic rheostat determining the amounts of circulating Epo. Anemia causes tissue hypoxia, resulting in an increase in serum Epo levels. During differentiation, specific transcriptional factors are expressed at crucial developmental stages. Transcription factors bind to specific nucleotide sequences called response elements on either the enhancer or the promoter regions of DNA adjacent to the genes, regulating transcription levels of these targeted genes. HSCs express runt-related transcription factor 1 (RUNX1; also known as AML1) and stem-cell leukemia factor (SCL; also known as TAL1). During myelopoiesis, these transcription factors are downregulated; on the other hand, PU.1, CCAAT/enhancer binding protein
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(C/EBP) a followed by C/EBPb and C/EBPe, growthfactor independent 1 (GFI1) and interferon-regulatory factor 8 (IRF8; also known as ICSBP) are upregulated [1]. Macrophage differentiation depends on PU.1 and IRF8. GFI1 and C/EBPe are crucial for neutrophil differentiation. These transcription factors regulate the expression of many myeloid genes, such as those encoding receptors for M-CSF, G-CSF, and GM-CSF [2]; GATA1 is induced by IL-4 during dendritic cell differentiation from monocytes [3]. MicroRNAs (miRs, 19e24 nucleotides) are small noncoding RNAs that act at the post-transcriptional level and bind to complementary sequences of target messenger RNA, resulting in regulating protein expression. miRs have an important role in normal hematopoiesis. For example, miR-155 targets PU.1 [4] and negatively regulates normal myelopoiesis and erthryopoiesis [5]. Forced expression of miR-155 into human CD34þ HSCs blocks clonogenic growth of myeloid and erythroid precursor cells. Levels of miR-451 increase during human erythroid differentiation [6]. miR-223 and NFI-A are reported as novel players in controlling human granulopoiesis [7,8]. PU.1, nuclear factor I-A (NFI-A) and C/EBPa regulate miR-223 during retinoic acid (RA)-induced myeloid differentiation. miR-223 itself targets NFI-A which competes with C/EBPs in binding the CCAAT element. NFI-A transcription factor blocks granulocytic differentiation. c-MYB RNA is a direct and functional target of miR-150. miR-150 is highly expressed in naı¨ve and memory B cells [9] and megakaryocytes [10]. It is weakly expressed in erythroid cells and moderately expressed in MEPs. Forced expression of miR-150 in human CD34þ HSCs leads to an 8fold enrichment of megakaryocytes compared with control HSCs. miRs 17-5p, 18a, 20a, and 106a are downregulated during monocytic differentiation of CD34þ HSCs [11]. These miRs function to suppress RUNX1 expression, leading to downregulation of M-CSFR. RUNX1 is critical for myeloid differentiation. Expression of miR-424 is induced by the transcription factor PU.1 during monocyteemacrophage differentiation of human CD34þ cells, and miR-424 subsequently represses translation of NFI-A [12]. miR-181a and miR142-5 are detectable in T lymphocytes, suggesting that their function might be involved in developing T lymphocytes [7].
Vitamin D Receptors in Blood Cells The genomic actions of 1,25(OH)2D3 are mediated by the intracellular vitamin D receptor (VDR), which belongs to a large family of nuclear receptors [13]. VDR forms a heterodimer with the retinoid X receptor (RXR); this complex regulates expression of target genes by binding to vitamin-D-responsive elements (VDREs)
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in the promoter regions of their target genes [14]. The interaction of 1,25(OH)2D3 with the VDR is discussed in Chapters 7 and 8. Expression of VDR has been detected in bone-marrow-derived stromal cells, as well as various normal and leukemic hematopoietic cells [15,16]. Patients with hereditary vitamin-D-resistant rickets type II (HVDRR) (discussed in Chapter 65) have various mutations of the VDR resulting in prominent skeletal abnormalities and subtle hematopoietic abnormalities [17,18].
Vitamin D Receptors in Myeloid Cells VDR is expressed in monocytes, neutrophils, platelets, and antigen-presenting cells such as macrophages and dendritic cells (DCs) [15,19e22]. Circulating monocytes have higher levels of VDR than tissue macrophages [23]. Mature DCs showed lower levels of VDR than immature DCs or monocytes [21]. VDR protein levels of peripheral blood monocytes have been reported to be two-fold higher in patients with idiopathic hypercalciuria with normal serum 1,25(OH)2D3 levels compared to monocytes from normal individuals [24]. On the other hand, fewer receptors have been detected in the peripheral blood mononuclear cells of patients with X-linked hypophosphatemic rickets [25]. These individuals have a significant positive correlation between VDR concentration in their mononuclear cells and their serum phosphate levels. Examination of a large number of myeloid leukemia cell lines blocked at various stages of maturation showed that they all expressed VDR, albeit at different levels [15]. Treatment of HL-60 myeloblastic leukemia cells with 1,25(OH)2D3 (107 M) decreased their VDR protein levels by 50% at 24 hours and levels return to normal after 72 hours. No change of VDR mRNA expression occurred in the cells [15,26], suggesting that one of the major sites of regulation of expression of VDR occurs at the post-transcriptional level. Exposure to 1,25(OH)2D3 induces the VDR to move from the cytoplasm to the nucleus, and this translocation is prevented by treatment with inhibitors of the PI3-K (LY294002) and the MAPK (PD98059) pathways [27]. The monocyte-like differentiation of HL-60 cells treated with 1,25(OH)2D3 may require functional activator protein-1 (AP-1) complexes which bind to the TRE of the promoter region in human VDR [28]. MEG-01 cells, a human megakaryoblastic leukemia cell line, matured to megakaryocytes by 12-O-tetradecanoylphorbol-13acetate (TPA) (1.6 107 M, 8 days) with increasing VDR expression [22]. In platelets, VDR is localized in the mitochondrial compartment suggesting that activation of calcium-dependent platelets might be regulated by rapid non-genomic effects of VDR in mitochondria.
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Vitamin D Receptors in Lymphoid Cells Subsets of thymocytes, including both resting T lymphocytes (expressing either CD8 or CD4) and activated T lymphocytes express VDR [15,29,30]. VDR mRNA expression increases when these cells are stimulated to proliferate, for example after their exposure to phytohemagglutinin-A (PHA) for 24 hours in vitro. Another major site of regulation of VDR expression in these cells is at the transcriptional level [15,29]. No VDR mRNA or protein is detected in resting B lymphocytes, for example in normal human B cells from tonsils, until their cellular activation [29,31]. 1,25(OH)2D3 inhibits the synthesis of immunoglobulins (Ig) by B lymphocytes in vitro [32]. Their inhibition may be mediated through activation of VDR/RXR in these cells, and/ or through the inhibition of T-helper activity [33]. Production of lymphokines, including IL-2, is markedly decreased by 1,25(OH)2D3 in activated T lymphocytes, and this could cause the suppression of Ig synthesis [34e37]. Recently, VDR was detected by RT-PCR in murine intraepithelial lymphocytes and regulatory T cells expressing CD4 and CD25 [38]. The effects of vitamin D on the immune system are discussed in Chapters 91 and 92. Levels of VDR mRNA in leukocytes from healthy individuals after the oral administration of 1,25(OH)2D3 increased an average of 1.2- to 11.1-fold [39]. The maximum increase of VDR mRNA levels occurs over 1 and 5 hours, with a mean of 3.6 hours. Expression of VDR is induced in the lymphocytes of patients with rheumatoid arthritis and in pulmonary lymphocytes of patients with tuberculosis and sarcoidosis [40e42]. Non-Hodgkin’s lymphoma and Burkitt’s B-cell lines have low levels of VDR; but nevertheless, 1,25(OH)2D3 (107 M) can decrease the proliferation of these cells in vitro [43]. Surprisingly, few studies have examined the anti-proliferative effect of 1,25(OH)2D3 on fresh lymphoma cells obtained from patients.
Hematopoiesis in VDR Knockout Mice Studies by our group using VDR knockout (KO) mice indicated that expression of VDR is dispensable for normal hematopoiesis [44]. No difference in the numbers and percentages of red and white cells were found between VDR KO and wild-type (WT) mice. Similarly, children with HVDRR and defective VDR have normal hematopoeisis (Chapter 65). Committed myeloid stem cells from the bone marrow of VDR KO mice cultured in methylcellulose formed similar numbers of colonies as cells from intact WT mice when grown in the presence of various cytokines including GM-CSF, G-CSF, M-CSF either alone or in combination with IL-3. Furthermore, bone marrow
progenitor cells from VDR KO and WT mice formed a similar number and percentage of granulocyte, macrophage, and granulocyteemacrophage mixed colonies when cultured in methylcellulose with GM-CSF and IL-3. Under these conditions, treatment with 1,25(OH)2D3 dramatically increased the percentage of macrophage colonies derived from WT but not VDR KO bone marrow cultures. This observation demonstrates the requirement of VDR expression for 1,25(OH)2D3 induction of bone marrow progenitors into monocytes/ macrophages (Fig. 88.1). The proportions of T and B cells were normal in the VDR KO mice. However, the antigen-stimulated spleen cells from VDR KO mice produced less interferon (IFN)-g and more IL-4 than those from WT mice, indicating impaired Th1 differentiation. Additionally, IL-12 stimulation induced a weaker proliferative response in VDR KO splenocytes as compared to those in WT mice, and expression of STAT4 was reduced. These results suggest that VDR plays an important role in the Th1-type immune response but not T cell development. In addition, regulatory T cells (CD4þ/CD25þ/FoxP3þ) are phenotypically, functionally and quantitatively normal in VDR KO mice [38]. However, the number of intraepithelial lymphocytes is reduced in VDR KO mice, resulting in failure of T-cell homing into the gastrointestinal tract possibly increasing a risk of developing inflammatory bowel disease [38]. Another report using VDR KO mice showed that VDR is required for normal development and function of Va14 invariant natural killer T cells which are involved in immune regulation, host defense against pathogens and tumor surveillance[45].
EFFECTS OF VITAMIN D COMPOUNDS ON NORMAL HEMATOPOIESIS 1,25(OH)2D3 modulates the differentiation of normal hematopoietic progenitors. Normal human bone marrow committed stem cells (HSCs) cultured in either soft agar or liquid culture with 1,25(OH)2D3 differentiate into macrophages. Likewise, monocytes cultured in serum-free medium with 1,25(OH)2D3 become macrophages within 7 days [46e51]. These macrophages are functionally competent [49]. Concentrations of 1,25(OH)2D3 causing this differentiation range between 1010 M (slightly higher than physiological serum level) to 107 M. For example, daily treatment with 1010 M of 1,25(OH)2D3 for 14 days induced a differentiation of CD34þ HSCs to monocyte/macrophages [51]. During differentiation of CD34þ HSCs by 1,25(OH)2D3, upregulation of CD14, PPARd, Hox-A10, and MafB was observed [52,53]. MafB transcription factor is directly regulated by Hox-A10 transcription factor and
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stimulates monocytic differentiation [54]. On the other hand, 1,25(OH)2D3 (109 to 107 M) can inhibit the differentiation into CD1aþ dendritic cells [55]. As mentioned earlier, 1,25(OH)2D3 is able to inhibit both the synthesis of IL-2 and the proliferation of peripheral blood lymphocytes [33e36]. Indeed, 1,25(OH)2D3 can regulate the expression of many lymphokines, such as GM-CSF, IFN-g, and IL-12 [33,56,57]. For example, Tobler et al. showed that expression of the lymphokine GM-CSF is regulated by 1,25(OH)2D3 through VDR by a process independent of IL-2 production. In particular, 1,25(OH)2D3 was able to inhibit both mRNA and protein expression of GM-CSF in PHA-activated normal human peripheral blood lymphocytes (PBLs). The former occurred at least in part by destabilizing and shortening the half-life of the GM-CSF mRNA [56]. The downregulation of GM-CSF was achieved at concentrations similar to those reached in vivo, with a 50% reduction of GM-CSF activity occurring at 1010 M 1,25(OH)2D3. In addition, IL-2 did not affect the modulation of GM-CSF production by PBLs which were co-cultured with 1,25(OH)2D3 (1010 to 107 M). Recent studies have shown that 1,25(OH)2D3 in combination with Epo can synergistically stimulate proliferation of CD34þ HSCs to differentiate to erythroid cells [58].
EFFECTS OF VITAMIN D COMPOUNDS IN LEUKEMIC CELLS A characteristic abnormality of leukemic cells is that they are blocked at an early stage of their development
TABLE 88.1
and fail to differentiate into functional mature cells. During the 1970s and 1980s, several scientific achievements popularized the strategy of inducing differentiation and apoptosis as an alternative to killing cancer cells by cytotoxic therapies. The potential for differentiating therapy to improve cure rates in leukemia is exemplified by the development of ATRA for the targeted treatment of acute promyelocytic leukemia (APL, also known as AML3). ATRA was specifically effective in APL cells carrying a typical chromosomal translocation between chromosomes 15 and 17 (t[15;17][q22; q21]), which fusion product is PMLeRARa, but not in other leukemias. 1,25(OH)2D3 was first noted to induce leukemia cell differentiation in the M1 murine myeloid cell line [59]. Moreover, 1,25(OH)2D3 extended the survival of mice inoculated with the M1 leukemia cells [60]. In spite of the promising data obtained from in vitro and animal studies, results of clinical trials of 1,25(OH)2D3 in leukemia are limited in scope and the clinical improvements such as blood counts or survival were limited [61,62]. Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder with a risk of transformation to acute myelogenous leukemia (AML); and affected individuals often exhibit anemia, thrombocytopenia, and/or leukopenia as well as an increased number of myeloid progenitor cells in their bone marrow. Administration of vitamin D3 analogs (19-nor1,25-dihydroxyvitamin D2 (paricalcitol) or 1(OH)D2 (doxercalciferol)) produced only minor clinical improvement of these patients at best (Table 88.1) (discussed in “Vitamin D analogs activity against leukemic cells,” below) [63,64].
Trials of Vitamin D Compounds in MDS Treatment duration (months)
No. of patientsa Efficiency
Reference
Compound
Another name
Hyper-calcemia
Dose /day (microgram)
Vitamin D3
Cholecalciferol
e
50e100
5
26
No therapeutic effect
[182]
1,25(OH)2D3
Calcitriol
þ (50%)
2
3
18
Occasional minor response
[61]
1,25(OH)2D3
Calcitriol
e
0.25e0.75
9e27
14
10/14 (71%) respondedb
[62]
1(OH)D2
Doxercalciferol
e
12.5
3
15
No therapeutic effect
[64]
1(OH)D3
Alfacalcidol
þ (13%)
4e6
17c
15
Markedly decreased [180] progression to AML
19-Nor1,25(OH)2D2
Paricalcitol
e
8e56
9
12
Occasional minor response
Only trials with 12 patients are listed. Criteria for response was not stringent. c Median. a
b
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[63]
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Cellular Effects of Vitamin D Compounds in Leukemic Cells
Molecular Mechanisms of Action of Vitamin D Compounds in Leukemic Cells
AML arises from neoplastic transformation of myeloid stem cells. Most acute myelogenous leukemia cells are unable to undergo terminal differentiation. A number of hematopoietic leukemia cell lines of myeloid origin can be inhibited in their proliferation and/or induced to undergo differentiation by 1,25(OH)2D3 [65,66] such as HL-60 (human promyelocytes), U937 (human myelomonoblasts), THP-1 (human monoblasts), HEL (human monoblast and erythroblast characteristics) and to a lesser extent NB4 (human promyelocyte) cells. In contrast, many immature myeloid leukemia cell lines such as KG-1 (human myeloblasts), KG-la (human early myeloblasts), and K562 (early myeloiderythroid blasts) are not responsive to vitamin D compounds. Constitutive expression of VDR mRNA is detectable in all these leukemic cell lines, although expression is relatively low in THP-1 and K562. Vitamin D analogs inhibit leukemic cell growth by inducing cell cycle arrest. The cells accumulate in the G0/G1 and G2/M phase of the cell cycle, with a concomitant decrease in the proportion of cells in S-phase [67e69]. These effects on the cell cycle are discussed in Chapter 84.
Vitamin D compounds can exert their biological effects by genomic (nuclear) (discussed in “Molecular mechanisms of genomic action of 1,25(OH)2D3 in leukemic cells,” below) and/or non-genomic (extra-nuclear) (discussed in “Modulation of kinase activities by 1,25(OH)2D3 in leukemic cells,” below) pathways. Both pathways require ligand binding to the VDR. The former pathway relies on a 1,25(OH)2D3-activated VDR/RXR complex binding to VDREs in order to modulate the transcription of various target genes. The latter increases rapid intracellular Ca2þ influxes resulting in activation of kinases and phosphatases within seconds to minutes [73,74]. It is still unknown whether the non-genomic actions are mediated through the classical nuclear VDR, a membrane-associated VDR or other proteins. Exposure of hematopoietic cells to 1,25(OH)2D3 controls a myriad of genes, including those responsible for the regulation of cellular proliferation, differentiation, apoptosis, and angiogenesis. Unsupervised microarray analysis using HL-60 cells shows that at least 612 genes are induced by 1,25(OH)2D3 (2.5 107 M, 48 hours) [75]. Modulation of these genes by 1,25(OH)2D3 may not always be a direct effect on transcription of target genes, but can reflect the entire process of differentiation associated with a series of interacting transcription factors. Nonetheless, 1,25(OH)2D3-activated intracellular signaling pathways require the presence of VDR to stimulate monocyte/macrophage differentiation, as demonstrated by studies on bone marrow cells from VDR KO mice [44] and cells from patients with vitamin-D-dependent rickets type II (HVDRR) [18,76]. The rapid, nongenomic activities of vitamin D are described in detail in Chapter 15. The molecular targets of vitamin D compounds in a variety of AML cell lines including HL-60, U937, NB4, and THP-1 cells are summarized in Table 88.2 (discussed below).
Differentiation Markers HL-60 cells cultured with 1,25(OH)2D3 (1010e107 M for 7 days) morphologically and functionally differentiate towards macrophages, expressing CD11 and CD14 (cell surface markers of early macrophage differentiation), becoming adherent to charged surfaces, developing pseudopodia, staining positively for non-specific esterase (NSE), reducing nitroblue tetrazolium (NBT), and acquiring the ability to phagocytose yeast [50,70,71]. In addition, these cells develop the ability to degrade bone marrow matrix in vitro, raising the possibility that the cells may have acquired some osteoclast-like characteristics. Leukemic cells from AML patients respond to vitamin D compounds when cultured in vitro; however, they are often less sensitive to this secosteroid than are the HL-60 cell lines. They frequently undergo partial monocytic differentiation as assessed by NBT reduction, morphology, and phagocytic ability. Furthermore, their clonal growth is often inhibited [50,61]. A recent study suggested that samples of leukemic blasts having deletion of chromosome 7 partially differentiated towards monocytes when cultured with 1,25(OH)2D3; in contrast, those leukemic samples having mutations of FLT-3 (internal tandem duplication or missense mutation) did not differentiate when cultured with 1,25(OH)2D3 [72].
Molecular Mechanisms of Genomic Action of 1,25(OH)2D3 in Leukemic Cells CELL CYCLE
Cell division is governed by the concerted action of cyclins and cyclin-dependent kinases (CDKs). Myeloid leukemic cell lines cultured with 1,25(OH)2D3 undergo an initial proliferative burst, which is followed by growth inhibition, terminal differentiation, and subsequent apoptosis [77,78]. Levels of cyclin A1, D1, and E increase in the U937 myelomonoblastic leukemia cells within 24 hours of 1,25(OH)2D3 treatment and then expression decreases after 48 hours [77]. The CDK inhibitors CDKN1A (p21Waf1) and CDKN1B (p27Kip1) are important regulators of the cell cycle that are elevated during periods of both proliferation and growth
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TABLE 88.2
Molecular Targets of Vitamin D Compounds in Leukemic Cellsa
TABLE 88.2 Molecular Targets of Vitamin D Compounds in Leukemic Cellsadcont’d
Cell cycle/Apoptosis
junD binding activity [
Cyclin A1 Y
DRIP [
Cyclin D1 Y
ETV7 Y
Cyclin E Y
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Feedback control Waf1
)[
Cyp24 [
Kip1
)[
Immunity
CDKN1A (p21 CDKN1B (p27
CDKN2A (p16-INK4A) [
CAMP [
CDKN2B (p15-INK4B) [
b-defensin [
CDKN2C (p18-INK4C) [
a
Regulation of expression or activity may occur either directly or as a consequence of differentiation. See text for details. b Putative components of AP-1 complex are c-jun, ATF-2, jun-B and fos-B.
Bcl-2 Y Differentiation markers
inhibition. Expression of these proteins, as well as CDKN2A (p16-INK4A), CDKN2B (p15-INK4B), and CDKN2C (p18-INK4C) CDK inhibitors are increased in a time-dependent manner after exposure of U937 cells to 1,25(OH)2D3 [79]. A strong correlation exists between early induction of p21Waf1 and the beginning of the differentiation program. The upregulation of p21Waf1 mRNA occurred within 4 hours of the exposure to 1,25(OH)2D3 independent of de novo protein synthesis, suggesting a direct transcriptional activation by VDR [79]. Indeed, the p21Waf1 promoter contains a vitamin D response element, and induction requires the presence of VDR. Nevertheless, some data have suggested that the marked increase of p21Waf1 protein expression in response to 1,25(OH)2D3, may also be due to enhanced post-transcriptional stabilization of p21Waf1 mRNA [80]. The transcription factor p53 is a strong inducer of p21Waf1; but 1,25(OH)2D3 can elevate p21Waf1 levels independently of p53 activity. A strong upregulation of p27Kip1 protein expression was evident after 72 hours’ exposure of HL-60 cells to the compound, and levels of the protein were dependent on the concentration of 1,25(OH)2D3 [81]. This upregulation was also associated with increased levels of cyclin D1 and E, coinciding with a G1 arrest. These results suggested a prominent role of p27Kip1 in mediating the anti-proliferative activity of 1,25(OH)2D3 in this cell line.
CD11b [ CD14 [ NSE [ NBT Y Oncogenes c-myc Y DEK Y FLI1 Y c-fms (M-CSFR) [ Tumor suppressors PTEN [ BTG [ Kinases PKC levels [ PI3-K activity [ Akt activity [ MAPK activity [ ERK 1/2 activity [ KSR-1,-2 activity [ Transcription factors C/EBP b [ PU.1 [
APOPTOSIS
IRF8 b [
AP-1b [
1,25(OH)2D3 can induce apoptosis in HL-60 cells. This is associated with downregulation of the anti-apoptotic gene Bcl-2 after HL-60 cells were treated with 1,25 (OH)2D3 or its analogs [82,83].
PPARd[
PROTO-ONCOGENES
HoxA10 [ HoxB4 [
(Continued)
Activation of the proto-oncogene c-myc is a typical feature of human leukemias. The HL-60 leukemia cell
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line is characterized by high levels of expression of cmyc due to gene amplification [84,85]. Treatment of this cell line with 1,25(OH)2D3 results in a downregulation of expression of c-myc associated with cell differentiation [86]. This suppression of c-myc occurs at the transcriptional level in HL-60 cells [87,88]. Exposure of HL-60 cells to 1,25(OH)2D3 induces the expression of the proto-oncogene c-fms, which encodes the receptor for M-CSF. It occurs in parallel with the induction of CD14 expression and a block of the cell cycle in the G0/G1 phase [89]. cDNA microarray analysis showed that at early times, the putative oncogenes DEK and FLI1 were downregulated and the anti-proliferative gene BTG1 was upregulated. TRANSCRIPTION FACTORS
1,25(OH)2D3 upregulates the protein encoded by the homeobox gene HOXB4 that binds to the first exon/ intron border of MYC to prevent transcriptional elongation, a process dependent on activation of PKC-b [90,91]. Another homeobox gene, HOXA10, was found by differential display to be a gene that is transcriptionally induced by 1,25(OH)2D3 through binding to the VDRE in the promoter during differentiation of U937 cells [92,93]. Besides MYC and HOX genes, 1,25(OH)2D3 can induce other transcription factors and coactivators to regulate gene expression. For example, exposure of U937 cells to 1,25(OH)2D3 induced the expression of PU.1, IRF8, and C/EBPb [94]. In contrast, exposure of U937 cells to 1,25(OH)2D3 (108 M) downregulated the expression of ETV7 (also known as TEL2), which is a member of the ETS family [95]. Interestingly, forced overexpression of ETV7 inhibited 1,25(OH)2D3-induced differentiation by 1,25(OH)2D3. Also, high expression levels of PPARd are detected in the AML cells with an M5 (monoblastic) phenotype. Furthermore, the forced overexpression of PPARd surpressed the 1,25(OH)2D3mediated monocyteemacrophage differentiation [52]. The ligand-activated VDR can bind to the AP-1 complex. Exposure of the chronic myelogenous leukemia (CML) cell line RWLeu-4 to 1,25(OH)2D3 inhibited their proliferation and enhanced the binding activity of the proto-oncogene jun D to the VDRE [96]. Exposure of HL-60 cells to 1,25(OH)2D3 upregulated expression of genes that code for the AP-1 complex including c-jun, ATF-2, jun-B, and fos-B [28,97]. Moreover, 1,25(OH)2D3 (107 M) induced within 6 hours the expression of the subunits of the transcriptional coactivator, vitamin D receptor-interacting proteins (DRIP, also called thyroid hormone receptor-associated polypeptide (TRAP)), in the HL-60 cells [98]. The DRIP complex plays a role in direct communication between the nuclear receptors and the general transcriptional machinery through direct interaction with RNA
polymerase II [99]. DRIP knockdown HL-60 cells as well as murine Trap220/ yolk sac hematopoietic progenitor cells are resistant to induction of differentiation by 1,25(OH)2D3. Fusion proteins involving the retinoic acid receptor alpha (RARa) with either the PML or PLZF nuclear proteins are the genetic markers of acute promyelocytic leukemias (APLs). APL cells expressing PMLeRARa fusion are sensitive to retinoid-induced differentiation to granulocytes in the presence of RA. In contrast, forced expression of either PMLeRARa or PLZFeRARa in either U937 or HL-60 cells blocked their terminal differentiation after exposure to 1,25(OH)2D3 [100]. Both PMLeRARa and PLZFeRARa can bind to VDR in U937 cells and sequester VDR away from activation of its normal DNA targets [101]. Overexpression of VDR overcomes the block in 1,25(OH)2D3-stimulated differentiation caused by the fusion proteins. Of note, PLZF itself can interact directly with VDR, and overexpression of PLZF can inhibit the 1,25(OH)2D3-induced differentiation of U937 cells [102]. IMMUNITY
About 160 years ago, sunlight or cod liver oil (both abundant sources of vitamin D) were used as treatment of tuberculosis [103,104]. Recent in vitro studies suggest that 1,25(OH)2D3 can have a role in activating human macrophages in host defenses against mycobacterium tuberculosis [105]. Moreover, screening of the human genome for VDREs showed that the human cathelicidin antimicrobial peptide (CAMP) gene has a VDRE in its promoter; and exposure of myeloid cells to 1,25(OH)2D3 and its analogs induced expression of CAMP [106e108]. Induction of CAMP by 1,25(OH)2D3 has been described in hematopoietic cell lines including myeloid leukemias (U937, HL60, NB4, K562, KG-1, and THP-1) and primary hematopoietic cells including leukocytes (monocytes, neutrophils, and macrophages) and bone marrow cells of both normal and leukemic individuals [109]. Interestingly, the VDRE for CAMP only appears in a transposable short-interspersed nuclear element, and these sequences occur only in primates [107]. 1,25(OH)2D3 treatment induces both phagocytic [71] and autophagocytic [110,111] activity. CAMP induced by 1,25(OH)2D3 is localized at autophagosomes in human monocyte and leukemia cells. The formation of autophagosomes occurs in a CAMP-dependent fashion. Moreover, 1,25(OH)2D3 directly stimulates expression level of nucleotide-binding oligomerization domain protein 2 (NOD2) [112]. This protein recognizes bacterial products, works as an intracellular receptor and activates NFkB signaling. Muramyl dipeptide, NOD2 ligand, in combination with 1,25(OH)2D3 enhances the expression of CAMP as well as the antimicrobial peptide defensin b2. However, no induction of
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antimicrobial peptides by the combination was observed in macrophages from individuals with Crown disease who have a homozygous R702W mutation of NOD2. Induction of these anti-microbial agents by combination of 1,25(OH)2D3 with infectious pathogen may provide significant cellular protection against various microbial infections.
Modulation of Kinase Activities by 1,25(OH)2D3 in Leukemic Cells Data suggest that both the anti-proliferative and differentiation-inducing effects of vitamin D compounds may, in addition to the actions already described, also require the modulation of the intracellular kinase pathways, including PKC, PI3-K, Akt, p38 MAPK, and ERK. This modulation probably occurs too quickly to be attributed to the genomic actions of vitamin D. Activation of PKC by the phorbol diesters such as TPA promotes monocyte differentiation of leukemic cell lines [113,114]. Differentiation of HL-60 cells in response to 1,25(OH)2D3 is accompanied by increased levels of PKC-b; and this differentiation can be inhibited by a specific PKC inhibitor, chelerythrine chloride [115]. Other vitamin D analogs have been shown to stimulate expression and translocation of PKC-a and -d during NB4 monocytic differentiation [116]. 1,25(OH)2D3 probably activates the PI3-K/Akt pathway in both non-genomic and genomic fashions. Activation of PI3-K may be required for the 1,25(OH)2D3stimulated myeloid differentiation, as monitored by induction of CD14 expression [117]. For example, PI3-K was activated by 1,25(OH)2D3 in THP-1 cells within 20 minutes. Pretreatment with the PI3-K inhibitors, LY 294004 or wortmanin, inhibited monocytic differentiation in response to 1,25(OH)2D3 in HL-60 and THP-1 cells, as well as peripheral blood monocytes [117,118]. Antisense oligonucleotides against PI3-K blocked induction of CD14 expression in THP-1 and HL-60 cells. PI3-K activates (phosphorylates) Akt, as well as its downstream targets, within 6 to 48 hours of exposure to 1,25(OH)2D3 in HL-60 cells [119]. PI3-K inhibitors synergized with 1,25(OH)2D3 to induce cell cycle arrest of HL-60 cells, associated with a synergistic upregulation of p27 Kip1. On the other hand, treatment with 1,25(OH)2D3 for 4 days induced the expression of PTEN, which could block the PI3-K/ Akt pathway, resulting in differentiation, cell death or inhibition of growth of HL-60 cells [69]. Also, exposure to 1,25(OH)2D3 (5 109 M, 72 hours) decreased both phosphorylated and total Akt in HL-60 cells [120]. In addition, Akt is dissociated from Raf1 in the presence of 1,25 (OH)2D3, and the Raf1 makes a complex with MEK1/2 and ERK1/2. Therefore, 1,25(OH)2D3 treatment enhances the Raf1/MEK/ERK pathway.
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The MAPK pathway can also be activated by 1,25(OH)2D3, and this probably involves genomic and non-genomic mechanisms. Exposure of either HL-60 or NB4 cells to differentiation-inducing concentrations of vitamin D compounds causes activation and nuclear translocation of MAPK [121e123]. Rapid changes of MAPK phosphorylation occurred within 30 seconds of exposure to 1,25(OH)2D3 in NB4 cells [122]. In addition, the vitamin D3 analog EB1089 was demonstrated to induce apoptosis of B-cell chronic lymphocytic leukemia cells from patients, an event preceded by stimulation of p38 MAPK and suppression of ERK activity [124]. 1,25(OH)2D3 stimulated the transient (24e48-hour) phosphorylation of ERK1/2 in HL-60 cells. After 24 hours, the level of phosphorylated ERK decreased to basal levels, while differentiation continued over an additional 48 hours [125]. In another report, phosphorylated -Raf, -MEK1/2, and -ERK1/2 are detected in HL-60 at least 72 hours after treatment with 5 109 M of 1,25(OH)2D3 [126]. Furthermore, PD98059, an ERK1/2 inhibitor, blocked the 1,25(OH)2D3-stimulated differentiation of HL-60 cells [126], kinase suppressor of Ras-1 and -2 (KSR-1, -2) which phosphorylate Raf-1 and act as scaffolds, increases the efficiency of signaling by Raf-1 [127,128]. These two genes have an upstream promoter containing a functional VDRE motif. Knocked-down of KSR-2 blocked 1,25(OH)2D3-induced myeloid differentiation. Signaling by Raf-1 is required for the later stage of 1,25(OH)2D3induced differentiation and requires p90 RSK which is either directly or indirectly phosphorylated by Raf-1 [129].
Vitamin D Compounds in Combination with Other Agents Because of the potential toxicity of 1,25(OH)2D3 and its analogs at the concentrations required in vivo to inhibit proliferation and/or induce differentiation of leukemia cells, various attempts have been made to use them with other compounds that might act synergistically yet have an acceptable toxicity. The mechanism of action and toxicity (hypercalcemia) of vitamin D compounds differ from chemotherapeutic agents. A variety of agents, including ATRA, iron chelating agents, arsenic trioxide (As2O3), non-steroidal anti-inflammatory drugs (NSAIDs), carnosic acid, bufalin, MAPK and mTOR inhibitors, cisplatin, taxol, paclitacel, doxorubicin, MDM2 antagonist, a HIV-protease inhibitor, demethylating agents, and histone deacetylase inhibitors have been combined in vitro with vitamin D compounds in a variety of cancers including leukemia. ATRA We and others have shown that the combination of a vitamin D compound and either ATRA or 9-cis-RA
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can potentiate the terminal monocytic differentiation of HL-60, NB4, and U937 cells [130e133]. These combinations included ATRA (109 M) and either 1,25(OH)2-16ene-23-yne D3, 1,25(OH)2-23-yne D3 (109 to 1010 M), or 9-cis-RA and KH1060 (a 20-epi-vitamin D3 analog) [66,134e137]. These cells often differentiate atypically, having a neutrophilic morphology, but acquiring other properties typical of monocytes (e.g., CD14 expression, ability to bind to bacterial LPS, and express lineage specific enzymes like non-specific acid esterase [132,133]. The combination of ATRA with 1,25(OH)216-ene-23-yne D3 enhanced the decreased expression of c-myc. Interestingly, U937 cells exposed to a moderate thermal stress responded with increased differentiation after the addition of 1,25(OH)2D3 and ATRA, suggesting that induction of heat-shock protein may be sequestering a protein that may favor proliferation or differentiation [138]. Iron Chelating Agents Iron chelating agents have anti-proliferation activities against cancer cells including AML cells that overexpress the transferrin receptor-1 by inhibiting uptake of iron in these highly propagating cells [139,140]. Moreover, the AML cells exposed to iron chelating agents can differentiate towards monocytes/macrophages associated with alteration of expression of 105 genes [75]. Combination of iron depletion and 1,25(OH)2D3 enhanced the differentiation of both HL-60 cells and AML primary blasts in vitro and suppressed the growth of HL-60 xenografts in mice.
Natural Products Vitamin D compounds have also been combined successfully with natural products. One of these is bufalin, a major component of toad venom. A low dose (10 nM) of this compound induced growth arrest and differentiation of leukemia cell lines towards monocytesemacrophages. A combination of 1,25(OH)2D3 with bufalin enhanced differentiation and expression levels of cyclin-dependent kinase inhibitor 1A. Another interesting compound, carnosic acid, is a plant-derived polyphenol antioxidant which can potentiate the prodifferentiative effects of 1,25(OH)2D3 [145e147]. Differentiation was correlated with anti-oxidant activity, and was associated with activation of the Raf-ERK pathway and increased binding of the AP-1 transcription factor to the promoter of VDR. MAPK and mTOR Inhibitors A p38 MAPK inhibitor (SB202190) enhanced the ability of 1,25(OH)2D3 to induce differentiation of HL60 cells [148]. In addition, the combination of the three agents (SB202190, carnosic acid, and 1,25(OH)2D3) further potentiated the anti-leukemic activity against HL-60 cells and primary AML blasts [149]. This augmented potency was associated with increased activation of the JNKeMAP kinase pathway. An mTOR inhibitor, RAD001 (everolimus), also enhanced the ability of 1,25(OH)2D3 to increase p21Waf1 expression in U937 cells [150]. Chemotherapy Agents (Cisplatin, Taxol, Paclitacel, Doxorubicin)
Arsenic Trioxide (As2O3) 19-Nor-1,25-dihydroxyvitamin D2 (paricalcitol) and As2O3 are both approved Food and Drug Administration drugs. Their combination resulted in a strong antiproliferative effect on HL-60, NB4, and PMLeRARa overexpressing U937 cells [141]. As2O3 decreased the levels of both the repressive PMLeRARa fusion protein and the vitamin-D-metabolizing protein 25-hydroxyvitamin D3-24-hydroxylase, which had been increased by paricalcitol. This combination may be effective for ATRA-resistant APL patients, as well as those with other types of AML.
Combining vitamin D compounds with traditional chemotherapy agents such as cisplatin, etoposide, and doxorubicin reduces the concentration of chemotherapy required for their anti-leukemic effects. Also, studies have suggested that the sequential order that the compounds are given may be important [151,152]. For example, pretreatment with etoposide enhanced the subsequent action of 1,25(OH)2D3, but pretreatment with 1,25(OH)2D3 had little effect on the activity of etoposide. The explanation for this observation is unclear now.
NSAIDs
MDM2 Antagonist
NSAIDs enhance the differentiation of HL-60 cells in response to 1,25(OH)2D3 and its analogs [142,143]. This effect may occur because of a block of NF-kB activation. Bhatia et al. showed that the combination of 1,25(OH)2D3, TPA, and M-CSF resulted in a synergistic response in NB4 cells, causing a complete differentiation to fully functional adherent macrophages with a rapid arrest of cell growth in the first 24 hours [144].
Nutlin-3a, an inhibitor of p53eMDM2 interaction, binds to the p53 pocket of MDM2 and stabilizes wildtype p53, resulting in cell cycle arrest and increase of apoptosis. A combination of nutlin-3a with 1,25(OH)2D3 enhanced apoptosis in wild-type p53 expressing MOLM-13 and OCI-AML3 cells in vitro by suppressing anti-apoptitic protein BCL-2 and p90RSK and by inducing the pro-apoptotic protein PIG-6 [153].
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HIV-protease Inhibitor One of the human immunodeficiency virus type I protease inhibitors, ritonavir, can enhance the antileukemic potency of 1,25(OH)2D3 [154]. Ritonavir inhibits Cyp24 expression. This enzyme normally metabolizes 1,25(OH)2D3 resulting in decreased levels of the active secosteroid. By blocking this enzyme, ritonavir increases the amount of active, intracellular 1,25(OH)2D3. Demethylating Agent and Histone Deacetylase The combination of a demethylating agent with a vitamin D3 compound can have enhanced activity [94]. For example, when the demethylating agent decitabine was combined with 1,25(OH)2D3, they synergistically induced monocytic differentiation of U937 cells and primary patient AML blast cells in vitro. Valproic acid (VPA) is an inhibitor of histone deacetylase which can also change the epigenetic landscape by acetylating histones and other proteins. This compound can induce myeloid differentiation [155]. In one clinical study of 19 MDS patients treated with the combination of VPA, 9-cis-RA, and 1,25(OH)2D3, three patients (16%) responded to treatment. A cautionary note, eight patients (42%) had toxicity from the combination [156]. The investigators did not find any correlation between histone acetylation and clinical response. Clearly, further studies are required using less-toxic histone deacetylating agents. In summary, treatment of leukemia or MDS with a vitamin D compound is unlikely, by itself, to be successful; but when given either in the maintenance phase of therapy after the leukemic patient is placed into remission or combined with other agents, vitamin D compounds may be useful therapeutically. Furthermore, 1,25(OH)2D3 can induce the expression of the antimicrobial peptide CAMP (see discussion above), which may afford the cancer patient some protection from lifethreatening infections while receiving aggressive chemotherapy.
VITAMIN D ANALOGS ACTIVITY AGAINST LEUKEMIC CELLS Synthesis of Vitamin D Analogs and their Functional Analysis A major drawback of using 1,25(OH)2D3 is its calcemic effect, which prevents pharmacological doses of the compound from being given. Vitamin D analogs have been synthesized that have enhanced potency to inhibit proliferation and promote differentiation of cancer cells, with less calcemic activity as compared with 1,25(OH)2D3 (see Section IX in this volume for
1741
a discussion of analogs). Many of these analogs in vitro are between 10- and 1000-fold more active than the parental 1,25(OH)2D3 in their growth-suppressive activity. These novel analogs can provide a larger therapeutic window for the treatment of hematologic malignancies and should be considered for the selected trials in hematologic malignancies either alone or in combination with other therapies. A comparison of the relative anti-leukemic potencies of some of these vitamin D compounds is provided in Table 88.3. 1a-Hydroxyvitamin D3 The first attempts using analogs focused on 1ahydroxyvitamin D3 (1a(OH)D3), a vitamin D3 compound that is efficiently converted to 1,25(OH)2D3 in vivo by D3-25-hydroxylase. This compound was administered to mice previously inoculated with the M1 leukemia cell line, and it showed greater activity than 1,25(OH)2D3 [60]. Its conversion to the active form resulted in a more prolonged elevation of plasma levels of 1,25(OH)2D3, and the dose (25 pmol, every other day) produced only a slight increase of the serum calcium. In addition, survival of the leukemic mice was increased by 50e60%; nevertheless, the more effective doses did cause hypercalcemia. Also, the administration of 1a(OH)D3 produced tumor regression in follicular NHLs in rats, but hypercalcemia was the dose-limiting factor [43]. Calcipotriol Calcipotriol (MC903) has a cyclopropyl group at the end of the side chain formed by the fusion of C-26 and C-27, a hydroxyl group at C-24, and a double bond at C-22. This compound is equipotent to 1,25(OH)2D3 in inhibiting the proliferation and inducing the differentiation of the monoblastic cell line U937 in culture [157,158]. In bone marrow cultures, the analog promotes the formation of multi-nucleated osteoclast-like cells, a vitamin D function. The effects of this compound on the immune system are very similar to those produced by 1,25(OH)2D3. By interfering with T-helper cell activity, calcipotriol reduces immunoglobulin production and blocks the proliferation of thymocytes induced by IL-1 [159,160]. Exposure of the follicular NHL B-cell lines SU-DUL4 and SU-DUL5, carrying the t(14;18) translocation characteristic of the disease, to calcipotriol inhibited their proliferation, but only at high concentrations of the compound (107 M) [43]. At the same time, calcipotriol was 100-fold less active than 1,25(OH)2D3 in inducing hypercalcemia and mobilizing bone calcium in rats [161]. However, the analog is rapidly inactivated in the intact animal, and therefore has been developed as a topical agent for skin diseases like psoriasis.
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88. HEMATOLOGICAL MALIGNANCY
Effect of Vitamin D Compounds on Both Clonal Proliferation of HL-60 Cells in Soft Agar and Calcium Levels in Mice
Compounds
ED50a (310L9 mol/l)
MTDb (mg)
Reference
1,25(OH)2D3
0.074e900
0.0625
[68,69, 162e164, 166e169, 175,176]
1,25(OH)2-16-ene-D3
0.015
0.125
[162]
1,25(OH)2-16-ene-23-yne-D3
3
2
[162,164]
1,25(OH)2-16-ene-5,6-trans-D3
0.03
4
[163]
1,25(OH)2-16-ene-24-oxo-D3
0.2
NDc
[167]
1,25(OH)2-16-ene-19-nor-D3
0.8
0.5
[167]
1,25(OH)2-16-ene-24-oxo-19-nor-D3
0.1
6
[166]
1,25(OH)2-20-epi-D3
0.006
0.00125
[164,168,169]
1,25(OH)2-20-epi-22-oxa-24,26,27-trishomo-D3d
0.001
0.0125
[168]
1,25(OH)2-diene-24,26,27-trihomo-D3
0.23
0.25
[164]
2.4
0.1
[68]
0.28
30
1,25(OH)2-21-(3-methyl-3-hydroxy-butyl)-19-nor D3h
0.17
ND
1,25(OH)2-20S-21(3-methyl-3-hydroxy-butyl)-23yne-26,27-hexafluoro-D3i
4
0.0625j
[175]
1,25-Dihydroxy-20R-(4-hydroxy-4-deuteromethyl5,5,5-deuteropentyl)-23-yne-26,27-hexafluoro-D3k
0.00014
NDc
[176]
19-Nor-1,25(OH)2D2
e
f
19-Nor-14-epi-23-yne-1,25(OH)2D3
g
Under review c
[69]
a
ED50 represents the effective dose achieving 50% growth inhibition of HL-60 cells. MTD, Maximally tolerated dose; highest dose reported that did not produce hypercalcemia or other noticable toxicities in mice when injected intraperitoneally, three times per week. c ND, not done. d Leo Pharmaceutical code name is KH 1060. e Leo Pharmaceutical code name is EB 1089. f Abbott Laboratories code name is Paricalcitol. g Hybrigenics Pharmaceutical code name is Inecalcitol. h Gemini-19-nor D3. i Gemini-23-yne-26,27-hexafluoro-D3. j At least mice that received the 0.0625 mg/mouse of Gemini-23-yne-26,27-hexafluoro-D3 had normal serum calcium levels. k Hoffman-LaRoche Inc. code name is BXL-01-0120. b
C-16-ene Vitamin D3 Analogs Introduction of a double bond at carbon 16 of 1,25 (OH)2D3 (C-16-ene) has proved to be an effective modification, particularly when combined with other motifs to generate a series of analogs with potent anti-proliferative and pro-differentiation promoting activities, with decreased calcemic effects. Prior studies by our group have shown that vitamin D3 analogs having the C-16ene motif were almost 100-fold more potent than 1,25 (OH)2D3 at inhibiting growth of HL-60 leukemia cells, but the calcemic activity was the same or markedly less than 1,25(OH)2D3 [162,163]. Combination of the C-16edouble bond and the C-23-triple bond (C-23-yne) (1,25(OH)2-16-ene-23-yne-D3) produces a compound that is a more potent inducer of growth inhibition and differentiation of HL-60 cells than 1,25(OH)2D3, and is 15-fold less hypercalcemic in mice. This analog has potent anti-proliferative and pro-differentiating effects
on leukemic cells in vitro [164]. In blocking HL-60 clonal growth, l,25(OH)2-16-ene-23-yne D3 has a potency of about four times higher than 1,25(OH)2D3. This compound administered to vitamin-D-deficient chicks is about 30 times less effective than 1,25(OH)2D3 in stimulating intestinal calcium absorption and about 50 times less effective in inducing bone calcium mobilization [158]. Further experiments have demonstrated the therapeutic potential of l,25(OH)2-16-ene-23-yne D3 by its ability to prolong markedly the survival of mice that had been inoculated with the myeloid leukemic cell line WEHI 3BDþ and treated with a high dose (1.6 mg, every other day) of the compound [165]. The 1,25(OH)216-ene-19-nor-24-oxo-D3 was synthesized as a result of previous studies that isolated 24-oxo metabolites of potent vitamin D3 analogs, which were formed in a rat kidney perfusion system [166]. We found that these 24oxo metabolites had markedly reduced calcemic activity,
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but possessed at least an equal ability as the unmetabolized analogs to inhibit the clonal growth of breast and prostate cancer cells and myeloid leukemia cells in vitro. Taken together, these findings prompted the chemical synthesis of a series of vitamin D3 analogs with 1,25(OH)2-16-ene-19-nor-24-oxo-D3 being one of the more exciting compounds, having the ability to inhibit acute myeloid leukemia cells in the range of 1010 M [167]. Remarkably, this compound had very little calcemic activity even when 6 mg was administered intraperitoneally to the mice, three times a week. 20-Epi Vitamin D3 The compound 1,25(OH)2-20-epi D3 is characterized by an inverted stoichiometry at C-20 of the side chain. The monoblastic cell line U937 cultured with this compound showed a strong induction of differentiation [168]. It was also a potent modulator of cytokine-mediated T-lymphocyte activation and exerted calcemic effects comparable to 1,25(OH)2D3 in rats. A study by our group suggested that l,25(OH)2-20-epi D3 is a potent vitamin D3 compound at inhibiting the clonal growth of HL-60 cells and at inducing cell differentiation. In fact, it is about 2600-fold more potent than 1,25(OH)2D3 in inhibiting the clonal growth of HL-60 cells and about 5000-fold more effective in preventing clonal proliferation of fresh human leukemic myeloid cells [169]. 1,25(OH)2-20-epi D3 exerts its effects by binding directly to VDR as shown by a T-lymphocytic cell line established from a patient with HVDRR. These cells with a dysfunctional VDR were no longer able to have a biologic effect. KH1060 is a potent vitamin D3 20-epi analog with an oxygen in place of C-22 and three additional carbons in the side chain. It is about 14 000-fold more potent than 1,25(OH)2D3 in inhibiting the growth of the monoblastic cell line U937 [168] and S-LB1 cells (human T-cell lymphotropic virus type 1 immortalized human T-lymphocyte cell line) and leukemic cells from patients with AML [133,169]. Although many hold more potent than 1,25(OH)2D3, in its anti-leukemic properties, both have the same receptor binding affinity and the same hypercalcemic activity as 1,25(OH)2D3. Paricalcitol 19-nor 1,25(OH)2D2 Paricalcitol has been approved by the Food and Drug Administration for the clinical treatment of secondary hyperparathyroidism. Clinical trials have demonstrated that it possesses reduced calcemic activity [170,171]. Studies by our group and another group have demonstrated that paricalcitol has anti-proliferative, pro-differentiation activities against myeloid leukemia and myeloma cell lines at a clinically achievable concentration [68,172,173]. Paricalcitol activity was dependent on the presence of VDR, as it was unable to induce differentiation of mononuclear bone marrow cells from
1743
VDR knockout mice, whereas cells from WT mice were differentiated towards monocytes/macrophages [68]. Furthermore, paricalcitol was able to inhibit tumor growth without causing hypercalcemia in immunodeficient mice. These observations prompted us to begin a clinical trial to treat patients with MDS with paricalcitol. A clinical trial of oral paricalcitol was conducted on 12 MDS patients. Although paricalcitol was well tolerated in all patients at the doses (8e56 mg/day) used, it had only minimal activity against MDS [63]. Another 19-nor compound, 19-nor-14-epi-23-yne-1,25 (OH)2D3 (inecalcitol), has been recently highly purified and analyzed in detail (Okamoto et al., manuscript under review). Inecalcitol is 11-fold more potent than 1,25(OH)2D3 in inhibiting proliferation of HL-60 cells. The maximal tolerated dose (MTD) before development of hypercalcemia is 30 mg/mouse, i.p. injection, every other day. Therefore, inecalcitol is 480 times less hypercalcemic than 1,25(OH)2D3. Within this context, a clinical trial has been initiated in prostate cancer. Gemini Vitamin D3 Analogs 1a,25-Dihydroxy-21-(3-hydroxy-3-methylbutyl) vitamin D3 (Gemini) compounds, having two side-chains attached to carbon-20, increases the anti-tumor activities against HL-60 and NB4 compared to 1,25(OH)2D3 [69,174]. Gemini-19-nor stimulated expression of the potential tumor suppressor PTEN [69]. Gemini-23-yne26, 27-hexafluoro-D3 is approximately 225-fold more potent than 1,25(OH)2D3 in inhibiting the clonal growth of HL-60 cells [175]. This compound produces hypercalcemia at the same concentrations as 1,25(OH)2D3 in mice with an MTD of 0.0625 mg per mouse (intraperitoneal injections, three times per week) which is the same MTD as 1,25(OH)2D3. A newer generation of Gemini compounds has a deuterium substitution on one side-chain, for example 1,25-dihydroxy-20R-(4-hydroxy4-deuteromethyl-5,5,5-deuteropentyl)-23-yne-26,27-hexafluoro-D3 (BXL-01-0120). This compound has one side-arm with a deuterium and the other has a double bond and a fluorine. BXL-01-0120 is 529-fold more potent than 1,25(OH)2D3 in its ability to inhibit the growth of HL60 cells [176]. The structural changes with the fluorine and the deuterium on the side-chains probably slow metabolism of the active compound [177,178]. Therefore, the Gemini compounds possess greater anti-proliferative activity than 1,25(OH)2D3, and yet produce hypercalcemia at similar concentration as occurs with 1,25(OH)2D3, thus resulting in a larger therapeutic window.
Trials of Vitamin D Compounds in MDS As shown earlier in Table 88.1, vitamin D compounds have been studied for MDS patients. In one study, six patients with MDS were treated with 1a((OH)D3 at
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88. HEMATOLOGICAL MALIGNANCY
1 mg/day for a minimum of 3 months, but neither a good clinical response nor toxicity was observed in these individuals [179]. In another clinical study, 30 MDS patients were divided into two different groups: one group received 1a((OH)D3 at 4e6 mg/day and the other group received placebo; the patients were treated for a median of 17 weeks [180]. An improvement of hematologic parameters was detected in only one patient, and the investigators believed that the treated group had a greater proportion of patients who did not progress to leukemia as compared to the control group. Doxercalciferol is in clinical use for the treatment of secondary hyperparathyroidism for reduction of elevated parathyroid hormone levels with acceptable mild hypercalcemia and hyperphosphatemia [181]. Recently, a phase II trial of doxercalciferol was conducted in 15 patients with MDS [75]. Each received 12.5 mg/day of 1a((OH)D2 for 12 weeks; the individuals did not develop hypercalcemia, but they also did not obtain a clinical response. Potential mechanisms by which vitamin D analogs have increased biological activity compared to 1,25(OH)2D3 include: reduced affinity to the serum vitamin-D-binding protein; decreased catabolism by 24-hydroxylase; retention of biological activities by metabolic products of vitamin D analogs; increased stability of the ligandeVDR complex; increased dimerization with RXR associated with increased affinity for its VDRE in the region of target genes; and enhanced recruitment of the DRIP coactivator complex. These topics are covered in detail in Chapter 75.
combination, working through different pathways, could lead to synergistic activity. Proof of principle that 1,25(OH)2D3 and its analogs are beneficial in leukemia has been shown in experiments conducted in vitro and in laboratory animals, but to date their therapeutic value in patients is unproven. Of interest, 1,25 (OH)2D3 and its analogs can stimulate synthesis of CAMP and b-defensins in myeloid cells in humans and may represent an evolutionary important pathway to protect humans against microbes.
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CONCLUSIONS The secosteroid hormone 1,25(OH)2D3 has a role in normal hematopoiesis, enhancing the activity of monocyteemacrophage differentiation. It also has anti-proliferative and pro-differentiation effects against various myeloid leukemia cell lines by both genomic and nongenomic pathways. Nevertheless, hematopoiesis in vitamin D3 receptor deletional mice is fairly normal, suggesting the vitamin D3 pathway has an adjunctive role in blood formation in mammals. The anti-proliferative activities of 1,25(OH)2D3 in vivo require supraphysiological levels of the secosteroid. Limited clinical trials with 1,25(OH)2D3 have been performed for the treatment of preleukemia/myelodysplastic syndrome; but the in vitro effective dose caused hypercalcemia in vivo. Since the mid-1980s, many vitamin D analogs have been synthesized that possess reduced hypercalcemic activity and increased ability to induce cell differentiation and to inhibit proliferation of leukemic cells. Further studies have been performed in vitro and in vivo using these analogs with other differentiating and/or anti-proliferative agents in the hopes that their
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R. Masciulli, U. Testa, T. Barberi, P. Samoggia, E. Tritarelli, R. Pustorino, G. Mastroberardino, A. Camagna, C. Peschle, Combined vitamin D3/retinoic acid induction of human promyelocytic cell lines: enhanced phagocytic cell maturation and hybrid granulomonocytic phenotype, Cell Growth Differ. 6 (1995) 493e503. E. Elstner, M. Linker-Israeli, T. Umiel, J. Le, I. Grillier, J. Said, I. Shintaku, S. Krajewski, J. Reed, L. Binderup, H. Koeffler, Combination of a potent 20-epi-vitamin D3 analogue (KH 1060) with 9-cis-retinoic acid irreversibly inhibits clonal growth, decreases bcl-2 expression, and induces apoptosis in HL-60 leukemic cells, Cancer Res. 56 (1996) 3570e3576. M. Taimi, M. Chateau, S. Cabane, J. Marti, Synergistic effect of retinoic acid and 1,25-dihydroxyvitamin D3 on the differentiation of the human monocytic cell line U937, Leuk. Res. 15 (1991) 1145e1152. B. Dore´, M. Uskokovı`c, R. Momparler, Interaction of retinoic acid and vitamin D3 analogs on HL-60 myeloid leukemic cells, Leuk. Res. 17 (1993) 749e757. A. Muto, M. Kizaki, K. Yamato, Y. Kawai, M. Kamata-Matsushita, H. Ueno, M. Ohguchi, T. Nishihara, H. Koeffler, Y. Ikeda, 1,25-Dihydroxyvitamin D3 induces differentiation of a retinoic acid-resistant acute promyelocytic leukemia cell line (UF-1) associated with expression of p21(WAF1/CIP1) and p27(KIP1), Blood 93 (1999) 2225e2233. J. Bastie, N. Balitrand, I. Guillemot, C. Chomienne, L. Delva, Cooperative action of 1alpha,25-dihydroxyvitamin D3 and retinoic acid in NB4 acute promyelocytic leukemia cell differentiation is transcriptionally controlled, Exp. Cell. Res. 310 (2005) 319e330. M. Cellier, M. Taimi, M. Chateau, A. Cannat, J. Marti, Thermal stress as an inducer of differentiation of U937 cells, Leuk. Res. 17 (1993) 649e656. K. Bridges, A. Cudkowicz, Effect of iron chelators on the transferrin receptor in K562 cells, J. Biol. Chem. 259 (1984) 12970e12977. D. Richardson, P. Ponka, E. Baker, The effect of the iron(III) chelator, desferrioxamine, on iron and transferrin uptake by the human malignant melanoma cell, Cancer Res. 54 (1994) 685e689. T. Kumagai, L. Shih, S. Hughes, J. Desmond, J. O’Kelly, M. Hewison, H. Koeffler, 19-Nor-1,25(OH)2D2 (a novel, noncalcemic vitamin D analogue), combined with arsenic trioxide, has potent antitumor activity against myeloid leukemia, Cancer Res. 65 (2005) 2488e2497. C. Bunce, P. French, J. Durham, R. Stockley, R. Michell, G. Brown, Indomethacin potentiates the induction of HL60 differentiation to neutrophils, by retinoic acid and granulocyte colony-stimulating factor, and to monocytes, by vitamin D3, Leukemia 8 (1994) 595e604. J. Sokoloski, A. Sartorelli, Induction of the differentiation of HL-60 promyelocytic leukemia cells by nonsteroidal antiinflammatory agents in combination with low levels of vitamin D3, Leuk. Res. 22 (1998) 153e161. M. Bhatia, J. Kirkland, K. Meckling-Gill, M-CSF and 1,25 dihydroxy vitamin D3 synergize with 12-O-tetradecanoylphorbol-13-acetate to induce macrophage differentiation in acute promyelocytic leukemia NB4 cells, Leukemia 8 (1994) 1744e1749. M. Danilenko, X. Wang, G. Studzinski, Carnosic acid and promotion of monocytic differentiation of HL60-G cells initiated by other agents, J. Natl. Cancer Inst. 93 (2001) 1224e1233. M. Danilenko, Q. Wang, X. Wang, J. Levy, Y. Sharoni, G. Studzinski, Carnosic acid potentiates the antioxidant and
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1749 prodifferentiation effects of 1alpha,25-dihydroxyvitamin D3 in leukemia cells but does not promote elevation of basal levels of intracellular calcium, Cancer Res. 63 (2003) 1325e1332. H. Sharabani, E. Izumchenko, Q. Wang, R. Kreinin, M. Steiner, Z. Barvish, M. Kafka, Y. Sharoni, J. Levy, M. Uskokovic, G. Studzinski, M. Danilenko, Cooperative antitumor effects of vitamin D3 derivatives and rosemary preparations in a mouse model of myeloid leukemia, Int. J. Cancer 118 (2006) 3012e3021. X. Wang, J. Rao, G. Studzinski, Inhibition of p38 MAP kinase activity up-regulates multiple MAP kinase pathways and potentiates 1,25-dihydroxyvitamin D(3)-induced differentiation of human leukemia HL60 cells, Exp. Cell Res. 258 (2000) 425e437. Q. Wang, J. Harrison, M. Uskokovic, A. Kutner, G Studzinski, Translational study of vitamin D differentiation therapy of myeloid leukemia: effects of the combination with a p38 MAPK inhibitor and an antioxidant, Leukemia 19 (2005) 1812e1817. J. Yang, T. Ikezoe, C. Nishioka, L. Ni, H. Koeffler, A. Yokoyama, Inhibition of mTORC1 by RAD001 (everolimus) potentiates the effects of 1,25-dihydroxyvitamin D(3) to induce growth arrest and differentiation of AML cells in vitro and in vivo, Exp. Hematol. 38 (2010) 666e676. R. Torres, C. Calle, P. Aller, F. Mata, Etoposide stimulates 1,25-dihydroxyvitamin D3 differentiation activity, hormone binding and hormone receptor expression in HL-60 human promyelocytic cells, Mol. Cell Biochem. 208 (2000) 157e162. A. Siwi nska, A. Opolski, A. Chrobak, J. Wietrzyk, E. Wojdat, A. Kutner, W. Szelejewski, C. Radzikowski, Potentiation of the antiproliferative effect in vitro of doxorubicin, cisplatin and genistein by new analogues of vitamin D, Anticancer Res. 21 (2001) 1925e1929. T. Thompson, M. Andreeff, G. Studzinski, L. Vassilev, 1,25dihydroxyvitamin D3 enhances the apoptotic activity of MDM2 antagonist nutlin-3a in acute myeloid leukemia cells expressing wild-type p53, Mol. Cancer. Ther. 9 (2010) 1158e1168. T. Ikezoe, K. Bandobashi, Y. Yang, S. Takeuchi, N. Sekiguchi, S. Sakai, H. Koeffler, H. Taguchi, HIV-1 protease inhibitor ritonavir potentiates the effect of 1,25-dihydroxyvitamin D3 to induce growth arrest and differentiation of human myeloid leukemia cells via down-regulation of CYP24, Leuk. Res. 30 (2006) 1005e1011. M. Go¨ttlicher, S. Minucci, P. Zhu, O. Kra¨mer, A. Schimpf, S. Giavara, J. Sleeman, F. Lo Coco, C. Nervi, P. Pelicci, T. Heinzel, Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells, EMBO J. 20 (2001) 6969e6978. T. Siitonen, T. Timonen, E. Juvonen, V. Tera¨va¨, A. Kutila, T. Honkanen, M. Mikkola, H. Hallman, M. Kauppila, P. Nyla¨nden, E. Poikonen, A. Rauhala, M. Sinisalo, M. Suominen, E. Savolainen, P. Koistinen, Valproic acid combined with 13-cis retinoic acid and 1,25-dihydroxyvitamin D3 in the treatment of patients with myelodysplastic syndromes, Haematologica 92 (2007) 1119e1122. L. Binderup, E. Bramm, Effects of a novel vitamin D analogue MC903 on cell proliferation and differentiation in vitro and on calcium metabolism in vivo, Biochem. Pharmacol. 37 (1988) 889e895. A. Brown, A. Dusso, E. Slatopolsky, Selective vitamin D analogs and their therapeutic applications, Semin. Nephrol. 14 (1994) 156e174.
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C H A P T E R
89 Vitamin D and Skin Cancer Jean Y. Tang 1, Ervin H. Epstein, Jr. 2 1
2
Stanford University Medical Center, Redwood City, CA, USA Children’s Hospital Oakland Research Institute, Oakland, CA, USA
TWO SOURCES OF VITAMIN D: DIET OR SUNLIGHT Vitamin D can be obtained from the diet or sunlight. The two most important dietary sources are D2 (ergocalciferol), which is plant derived, and D3 (cholecalciferol), which can be obtained through ingestion of select animal products such as oily fish (salmon, cod). Vitamin D3 is also obtained via endogenous synthesis by keratinocytes. 7-Dehydrocholesterol is converted to vitamin D3 in keratinocytes in a photo-chemical reaction triggered by ultraviolet B light at wavelengths 290e315 nm. Whether obtained from sun exposure, food, or supplements, vitamins D2 and D3 undergo two hydroxylations in the body to form the physiologically active 1,25-dihydroxyvitamin D (1,25(OH)2D), also known as calcitriol. 1,25(OH)2D exerts its effects on target organs by binding to its intracellular nuclear vitamin D receptor (VDR) to regulate gene transcription. In addition to its production in the kidney, 1,25(OH)2D can also be produced in various tissues, including keratinocytes [4e6]. However, keratinocyte-produced 1,25(OH)2D does not contribute to circulating levels suggesting the existence of a local autocrine or paracrine regulatory pathway [7]. More recent studies show that keratinocyte-synthesized 1,25(OH)2D may modulate epidermal cellular proliferation, differentiation, and apoptosis [4e6] (see also Chapter 30). UVB is responsible for cutaneous vitamin D synthesis [8] as detailed in Chapter 2. However, the same portion of the UVB spectrum necessary for vitamin D synthesis coincides with wavelengths implicated in photocarcinogenesis [9]. Thus what exactly constitutes a “safe” dose of UVB exposure which also maximizes cutaneous vitamin D synthesis remains controversial. While some studies [4,5] have attempted to better quantify UV dosage in terms of energy thresholds, others have sought to quantify dosage in terms of
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10089-7
exposure duration. Studies have concluded that exposure of just 15% of total body surface to a single minimal erythemal dose (MED) of sunlight is equivalent to oral ingestion of 1500e3750 IU of vitamin D3 [10]. Holick has been perhaps the most vocal proponent of UV exposure as the primary source of vitamin D, and he has recently suggested that 5e30 minutes biweekly during midday hours would be sufficient [1]. Webb et al. have expanded on Holick’s recommendations by analyzing, among other factors, effects of solar zenith angles, air ozone composition, and time of day. Studies [7,8,11,12] suggest that midday sunlight exposure corresponding with low solar zenith angles results in optimal vitamin D synthesis. At greater solar zenith angles, which occur during the winter season as well as at more northern latitudes, a greater amount of exposure is needed to achieve the standard vitamin D effective dose (SDD) which is the UV equivalent of an oral dose of 1000 IU vitamin D [9]. Thus, current recommendations to avoid midday sun exposure may in fact hinder adequate vitamin D synthesis if subjects obtain exposure at “safer” hours such as before 10 am or after 4 pm, when high solar zenith angles result in suboptimal vitamin D synthesis. However, the relative cost:benefit (i.e., skin carcinogenesis:vitamin D production) of midday versus early or late sun exposure is unknown. Interestingly, the effect of UVB broadband exposure and subsequent cutaneous D3 synthesis seems to follow an exponential function [13]. With longer exposure to UVB rays, the vitamin begins to degrade as fast as it is generated; thus equilibrium is achieved in the skin. Production appears to level off after approximately 10e20% of epithelial 7-dehydroxycholesterol stores are used; this maximum level of synthesis is reached after only a sub-MED dose of sunlight; and further exposure transforms newly synthesized vitamin D into inactive metabolites [14e17]. Meanwhile, prolonged UVB
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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exposure leads to further DNA damage and potential for carcinogenesis. Taken together, the data suggest that, at least theoretically, the relative costly:beneficial effects of sunlight exposure follow a J-curve in which an initial amount of beneficial sunlight (producing vitamin D) gives way to harmful effects (causing mutagenesis) after a prolonged period. Further studies must look to define the distinct boundaries of the curve, but the current literature does not offer compelling data to reject the sun exposure guidelines proposed by Holick and others. However, the matter remains controversial. Many in the field advocate avoidance of increased sun exposure as a safe source of vitamin D because of the skin cancer risk, photoaging, and difficulty in giving sun exposure advice across various geographies and cultures, etc. and instead advocate supplements as a safe and effective means of supplying vitamin D. The length of sun exposure needed to generate 1 SDD will vary depending on latitude, season, skin pigment, skin thickness, age, and sunscreen use [18,19]. Maximum endogenous production with full body exposure to sunlight is 10 000 IU (250 mg) per day [20]. Studies suggest that clinically significant increases in serum 25(OH)D levels can be seen with UVB doses small enough to produce minimal tanning noticeable only to a colorimeter [19]. Armas et al. found that subjects’ serum levels of 25(OH)D increased an average of 12 ng/ml after 4 weeks of regular, measured exposure to UVB light. Despite the studies suggesting that casual sun exposure can lead to clinically significant increases in serum 25(OH)D, more recent work by Rhodes et al. found that white subjects who received a simulated summer’s sunlight exposure as recommended by UK guidelines were able to attain sufficient (>20 ng/ml) serum levels of 25(OH)D but were unable to reach optimal (>30 ng/ ml) levels [21]. The authors concluded that short, midday exposure to sunlight in the summer, while wearing summer clothing, is not enough to produce and maintain optimal serum 25(OH)D levels. Thus it can be inferred that for most individuals, sunlight exposure year-round may not be sufficient to attain optimal levels of serum 25(OH)D, although cutaneous vitamin D synthesis may indeed be a good adjunct source of vitamin D. However, it is difficult to recommend a standard and safe dose of sunlight to boost 25(OH)D levels given the great number of factors that affect vitamin D synthesis from the sun (subject age, pigment, BMI, season, latitude). Constitutive factors such as skin pigmentation and age also affect vitamin D synthesis. Those with darker skin types have greater difficulty synthesizing vitamin D from sunlight, as increased melanin in the epidermis absorbs much of the UVB radiation needed for vitamin D production [22,23]. In contrast, Caucasians and those with lighter skin types can obtain maximum vitamin D synthesis per cm2 of skin exposed with as little as 5
minutes of exposure to the hands [23]. There are conflicting data on how serum 25(OH)D levels differ by skin pigmentation e Armas et al. suggest that individuals with lighter skin tones have higher 25(OH)D levels than darker skin types, but another recent study by Bataille et al. found that lighter skin types actually have lower 25(OH)D levels [24,33]. However, the authors of the latter study attribute this counterintuitive result to increased sun-avoidance behavior in lighterskinned individuals. Therefore, our interpretation is that individuals with darker skin types are still at increased risk of vitamin D deficiency. Average skin thickness (both dermal and epidermal) decreases with age [24e26], and may contribute to the decreased cutaneous vitamin D synthesis due to smaller stores of the precursor 7-dehydroxycholesterol [26]. The elderly may be at further risk of deficiency due to decreased mobility and consequently decreased sun exposure [27]. Given the evidence, it would seem that cautious increased cutaneous UVB exposure can be beneficial. However, several studies have noted that living in a sunny climate does not guarantee optimum levels of 25(OH)D. Studies performed in Hawaii, southern Arizona, southern Florida, and Chile have all observed that even in sunny environments, deficiency is still noticeable among the population; darker-skinned individuals and individuals with lower amounts of sun exposure seem at particular risk of deficiency [28e31]. This, again, concurs with the findings from Rhodes et al. [21]. Factors such as BMI, skin thickness, and sunscreen application and sun avoidance behavior are likely also important for understanding these findings.
VITAMIN D AND INDOOR TANNING Deliberate tanning is a common practice and 17e29% engage in deliberate tanning in the USA [32]. In 1988, as few as 1% of American adults reported using indoor tanning facilities; by 2007 that number had increased to 27% [33]. While indoor tanning is becoming more popular, many tanning devices are employing more powerful UV lamps: a recent study from England found that 83% of tanning beds evaluated exceeded European standards for UV-B radiation levels [34]. Tanning is most common among lighter-skinned individuals, and this is the group that is most at risk for skin cancer [35]. Indoor tanning represents a growing, multibillion-dollar industry, though its growth comes at a time when there is scientific consensus that exposure to UV increases the risk of developing skin cancer [36]. The WHO recently classified tanning bed exposure as “carcinogenic to humans.” A caseecontrol study that included 603 basal cell carcinoma (BCC) case patients and 293 squamous cell carcinoma (SCC) case patients
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found that use of tanning devices was significantly associated with increased risk; a 50% increase in BCC risk and a two-fold increase in SCC risk [37]. Age at first use of a tanning device was also a significant risk factor, with age less than 20 years associated with the highest risk for both BCC and SCC. The tanning industry has attempted to downplay the significance of the elevated BCC and SCC risk, arguing that these forms of skin cancer are generally less aggressive than melanoma. Such an argument ignores the fact that non-melanoma skin cancer, especially SCC, does have metastatic potential and it also ignores the morbidity and cost associated with surgical excision of non-lethal BCC and SCC. A recent meta-analysis of 19 published reports since 2007 confirmed the association between indoor tanning and melanoma [38]. Ever-use of a tanning bed was associated with an increased risk of melanoma, and use prior to age 35 increased the risk of melanoma by 75%. This study provides the most extensive evidence to date of the risk of melanoma associated with indoor tanning, especially as it pertains to youth tanning. There was no evidence of any protective effect of the use of sunbeds against damage to the skin from subsequent sun exposure. Several studies have also shown that a subset of frequent tanners may show signs of addiction to tanning especially among adolescents which may or may not be related to endogenous opioids [39]. Some in the tanning industry have suggested that indoor tanning can raise vitamin D levels. However, most tanning beds primarily emit UVA rays (97%) [40] which do not contribute to vitamin D synthesis in the skin. However, some tanning beds do emit a low amount of UVB (about 3%) which can raise 25(OH)D levels after many weeks of treatment. In a caseecontrol study of 50 subjects who chronically used indoor tanning versus non-tanners, UV tanners had twice the 25(OH)D levels as non-tanners [41]. However, it is unclear if the higher 25(OH)D level in tanners is due to their use of indoor tanning beds versus their overall higher use of outdoor sun exposure. In a pilot study, a portable tanning device emitting UVB was shown to improve 25(OH)D levels in patients with cystic fibrosis and short-bowel syndrome who could not absorb vitamin D [42]. Eight weeks of sunlamp exposure increased 25(OH)D levels by 25%. However, most individuals do not have malabsorption and their vitamin D status can be repleted with oral vitamin D. Although indoor tanning can lead to the endogenous synthesis of previtamin D3, several studies in human skin have shown that total previtamin D3 production in the skin plateaus with exposure time [28]. Further increases in UV exposure will not increase the total amount of previtamin D3. One MED of UVB exposure to 6% of the body surface area is equivalent to the ingestion of 600e1000 IU of vitamin D in most light-skinned
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individuals. This translates to roughly 10e20 minutes of sun exposure 2e3 times per week. Moderate sun exposure is as efficient as prolonged sun exposure for previtamin D production. However, sunlight exposure as the only source of vitamin D may be impractical in cold weather and for those with darker skin types [30]. Therefore, moderate sunlight exposure should be considered in combination with a diet fortified with vitamin D for optimal vitamin D status.
UV EXPOSURE CONTRIBUTES TO NMSC AND MELANOMA DEVELOPMENT The incidence of non-melanoma skin cancer (NMSC) is now estimated at over one million new cases a year and approximately equals all other cases of human malignancies combined (American Cancer Society Facts and Figures [43]). This may be due to increased UV exposure, a larger elderly population, and lifestyle changes with increased outdoor activities and a desire to tan. The majority of NMSC cases are basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) in a ratio of approximately 4:1. Incidence rates vary significantly depending on the ethnicity and geographic location of subjects, but despite this variability, most studies show that NMSC rates are increasing worldwide, and it is estimated that nearly 30% of Caucasians living in areas of high ambient sun exposure will develop a BCC [1]. Furthermore, the incidence of BCC is rising in younger populations, especially among women [44]. The highest incidence rates are in Australia with 1e2% of the population developing an NMSC each year. Although the case fatality rate of NMSC is low, the high incidence and frequent occurrence of multiple primary tumors in affected individuals leads to significant morbidity. The large number of NMSC cases makes it the fifth most costly cancer to treat in the Medicare population. NMSCs characteristically arise in sunexposed body areas, most commonly on the head and neck, but also occur on the trunk and extremities. UV exposure in the UVB wavelength is the most accepted environmental insult for NMSC development; however, inter-individual differences in the susceptibility to NMSC development have been recognized for many years. Epidemiologic studies have identified phenotypic features such as fair skin and freckling tendency that are associated with an increased susceptibility to NMSC [45]. Additional studies have shown that sun-exposed human keratinocytes and melanocytes harbor signature UVB mutations in known oncogenes and tumor suppressor genes, strongly supporting the role of UV radiation in causing NMSC. Furthermore, UV radiation drives NMSC and melanoma formation in mouse models [46,47].
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While the risk of SCC is strongly related to cumulative sun exposure, sunlight’s exact role in BCC development remains less clear [48]. Thus, epidemiologic studies have shown that BCC risk correlates better with intermittent sun exposure (i.e., childhood sunburns, weekend sun exposure) than with cumulative lifetime sun exposure [49]. Hence, the timing, dose, and duration of UV exposure are critical to carcinogenesis, but UV seems to have a different role in SCC versus BCC carcinogenesis development. Consistent with this, a randomized clinical trial of daily sunscreen use found that sunscreen reduced the incidence of SCC but not BCC e the daily application of an SPF 15 sunscreen to the head, neck, arms, and hands over a 4- to 5-year period had no effect on the risk of basal cell carcinoma development at these sites but did reduce the risk of SCC by 40% [50]. Malignant melanoma is one of the fastest-growing cancers worldwide; studies from Europe [51], Singapore, Canada, and the USA [52] suggest consistent and dramatic increases in incidence since the 1950s. Yet the underlying causes of these observed trends are widely debated, with some authors attributing the rapid rises to environmental risk factors and sun exposure behavior and others maintaining they result from expanded screening and biopsy. To date, it is generally accepted that the risk of melanoma is determined through the interplay between genetic factors and UV exposure as intermittent sun exposure and sunburn history have been identified as risk factors. Melanocytic nevi (moles) have been identified as the most important risk factor for melanoma, and sun exposure, sunburns, and light pigmentation have been found to be associated with nevi development in childhood. The pathogenic effects of UV exposure are likely due to its DNA-damaging effects leading to genetic mutations in melanocytes or local immunosuppression in the epidermis. In fact, a comprehensive catalog of somatic mutations from a human cancer genome obtained from a metastatic melanoma from a 43-year-old male clearly revealed UV signature mutations in DNA from melanoma tumor samples implicating the important role of UV in melanoma development [53].
VITAMIN D AND VISCERAL CANCER RISK AND ITS POTENTIAL ROLE IN REDUCING SKIN CANCER RISK Multiple epidemiological studies show an inverse association between sun exposure, vitamin D sufficiency, intake of vitamin D, and risk of developing and/or surviving cancer, with the strongest such evidence showing an inverse relationship between vitamin D levels and risk of colorectal cancer [54e56]. The Nurses’ Health Study, with 32 826 subjects, showed a roughly
50% reduction in risk of colon cancer with a two-fold rise in serum 25(OH)D (OR 0.53, p < 0.01) [57]. Similarly, Women’s Health Initiative participants who had 25(OH) D levels below 12 ng/ml at baseline had a 253% increase in developing colorectal cancer during an 8-year followup period [58]. The subject of sun exposure and cancer risk is further discussed in Chapters 53 and 82. Epidemiologic studies also show an association between latitude and incidence and mortality; living at lower latitudes has been associated with decreased risk of developing and/or dying from several types of cancer, including prostate and ovarian [59e65]. Studies have also found cancer patients who have surgery or treatment in the summer, when they are presumed to be making more vitamin D, have a better chance of surviving than those who undergo treatment in the winter. These studies posit that this seasonal variation in cancer survival may be due to the seasonal 25(OH)D gradient [66e70]. The suggested protective effects of vitamin D may result from its role as a nuclear transcription factor that regulates cell growth and apoptosis, promotes cellular differentiation, and inhibits proliferation, angiogenesis, invasiveness e all cellular mechanisms central to the development and persistence of cancer [71] and discussed in detail in other chapters in this section of this volume. However, the translation of these promising findings in cells and animal models may not translate to cancer prevention in people, and no clear conclusion is available. The association between vitamin D levels and cancer risk is not without significant controversy. A large, recently published randomized controlled trial of 1000 mg of calcium and 400 IU of vitamin D found that supplementation did not reduce the incidence of colorectal or breast cancer in postmenopausal women [72]. Another smaller, randomized trial gave subjects 1000 IU of vitamin D and 1500 mg of calcium daily and found that supplementation decreased the risk of developing any cancer during the trial by 60% [58]. However, the study was designed to examine fracture risk, and the results regarding malignancies were seen in post-hoc analysis. More data are needed before a clearly defined relationship between development of cancer and vitamin D status can be confirmed. A randomized control trial has recently been initiated including 20 000 subjects to test the effects of taking omega-3 fatty acids (fish oil) or 2000 IU of vitamin D daily or a combination, on the risk of developing heart disease, cancer, and diabetes (VitalStudy. org). The control group will receive 800 IU of vitamin D.
Vitamin D in Skin Cancer Despite the growing body of epidemiologic evidence suggesting an inverse relationship between vitamin D and cancer risk in various visceral organs, few studies
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ROLE OF VITAMIN D IN SQUAMOUS CELL CARCINOMAS (SCC)
have assessed a possible relationship with skin cancer risk, perhaps because UV is a primary risk factor for skin cancer development. Also, epidemiologic studies on NMSC are difficult in the USA because NMSC cases are not collected in most national registries, such as the Surveillance, Epidemiology, and End Results (SEER) program. However, there is growing evidence to support a relationship between vitamin D levels and risk of both non-melanoma and melanoma skin cancers.
ROLE OF VITAMIN D IN KERATINOCYTES At high levels, 1,25(OH)2D3 inhibits keratinocyte proliferation in vitro and interacts with calcium to regulate keratinocyte differentiation [73,74] (see also Chapter 30). Several cofactor proteins that modulate the interaction between VDR and the transcription machinery are differentially associated with the VDR in proliferating versus differentiating keratinocytes and also have a different profile in early and late differentiation [75]. The two primary cofactor complexes that interact with the VDR in keratinocytes are the D receptor-interacting protein (DRIP) complex and the steroid receptor cofactor (SRC) complex. The differential use of the coactivator complexes allows keratinocytes to fine-tune their response to 1,25(OH)2D3. It is thought that vitamin D, in its role in keratinocyte homeostasis, plays a key defense role against skin carcinogenesis. Evidence for the role of vitamin D in keratinocyte homeostasis comes from studies in cultured keratinocytes and in knockout mice devoid of functional VDR. Keratinocytes lacking VDR are hyperproliferative and exhibit decreased apoptosis in response to UV [76]. VDR knockout mice have reduced expression of epidermal differentiation markers and abnormal hair follicles indicative of a role of VDR in keratinocyte differentiation [77]. VDR knockout mice have alopecia, dermal cysts and utriculi, and epidermal proliferation at twice the normal rate [78]. In contrast, mice lacking the CYP27B1 gene (thus lacking the ability to synthesize 1,25(OH)2D ligand) have normal hair-follicle cycling and normal keratinocyte proliferation. Similarly patients with defective VDR exhibit the syndrome of hereditary vitamin-D-resistant rickets (HVDRR) and many have alopecia (Chapter 65) while children with CYP27B1 mutations, who cannot synthesize 1,25(OH)2D, do not have alopecia (Chapter 64). Clinically, vitamin D’s anti-proliferative effect in humans has been demonstrated in psoriasis, a hyperproliferative keratinocyte disorder, which can be successfully treated by the topical application of 1,25(OH)2D3 (calcitriol, Vectical) or its synthetic analog (calcipotriene, Dovonex) [79] (see Chapter 97).
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UVB radiation damages keratinocyte DNA, in particular causing mutagenic cyclobutane pyrimidine dimers (CPDs). There is evidence that keratinocyte-produced vitamin D may protect against UVR-induced dimer formation. Thus topically applied 1,25(OH)2D3 blocks the formation of CPDs [80]. Similarly, 1,25(OH)2D3 treatment of keratinocytes in vitro enhances expression of two DNA repair genes, XPC and DDB2, suggesting 1,25(OH)2D3 may enhance DNA repair. More recent studies have shown a protective effect of topical vitamin D compounds against DNA photodamage in mice in vivo [81] that is dose-dependent [82,83]. At lower doses of UVB radiation (30e40 mJ/cm2), pretreatment with vitamin D decreased photodamage, but this photoprotective effect was not seen at higher doses of UVB (50 mJ/cm2). Similar findings have been reported in rat keratinocytes [84]. These findings suggest that 1,25(OH)2D exerts its photoprotective effect against a moderate range of UVB irradiation, but that at higher doses, this effect is lost, possibly explaining why chronic, high-dose UVB exposure is associated with increased skin cancer risk. Vitamin D and sunlight photoprotection is more fully discussed in Chapter 100.
ROLE OF VITAMIN D IN SQUAMOUS CELL CARCINOMAS (SCC) UVR leads directly to characteristic DNA mutations that can be identified within the SCC tumors. While these mutations occur throughout the genome, SCC tumor cells frequently harbor UV-signature mutations in specific pathways. The most frequent UV-signature mutations occur in the p53 tumor suppressor gene (53%), the H-ras proto-oncogene (12%), and at the INK4a-ARF locus (54%) whose two overlapping genes encode critical proteins which indirectly regulate p53 and Rb. Mouse model systems for SCC rely on chemical carcinogens (DMBA and TPA) or UVR. VDR knockout mice develop an increased number of skin tumors (primarily papillomas and BCCs) when exposed to oral DMBA [85] or SCC tumors when exposed to UVR [86]. These studies demonstrate a key role for the VDR in keratinocytic tumor formation. Exogenous 1,25(OH)2D3 and its analogs reduce SCC formation. In mice, topically applied 1,25(OH)2D3 inhibits chemically induced tumor formation in a dose-dependent manner [87], and 1,25(OH)2D3 inhibits the growth of SCC tumors in vivo and in vitro by inducing cell cycle arrest [88]. Analogs of 1,25(OH)2D3 also inhibit cell growth by inhibiting DNA synthesis and inducing apoptosis in SCC cell lines [89]; it also reduces chemically induced SCC formation [90]. In humans, VDR polymorphisms are associated with increased development of solar keratoses [91].
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Like many steroid hormones, 1,25(OH)2D3 appears to exert the full spectrum of its functional effects through two main pathways. One is the classical or genomic pathway, in which the hormone binds to the vitamin D receptor (VDR) which then dimerizes with the retinoid X receptor, and the complex then binds to the vitamin D response element on the promoter region of target genes, resulting in an increase or a decrease in transcription of those genes. The second major pathway, the rapid or non-genomic pathway, involves the hormone binding to a membrane receptor, the identity of which is still not completely resolved, stimulating a variety of intracellular signaling pathways such as intracellular calcium, protein kinase C and MAP kinase (see Chapter 15). Dixon et al. showed that vitamin D analogs that only stimulate the rapid, non-genomic pathway are photoprotective and reduce UVR-induced CPDs and apoptosis in human keratinocytes and in mouse skin [92] (see Chapter 100). The underlying molecular mechanisms of the non-genomic pathway have not been fully elucidated, but may involve tumor suppressor genes such as p53 or p63 and their interaction with the VDR, or nuclear excision repair genes. 1,25(OH)2D can induce DNA repair enzymes such as XPC and DDB2 following exposure to UVR via p53. This response may differ between normal keratinocytes and SCCs. Molecular studies have shown that the VDR is induced by one of the isoforms of p63, potentially linking the VDR to the requirement for p63 in the increase of XPC and DDB2 following UVR exposure. Indeed, p63, rather than p53, is critical in keratinocytes for initiating nucleotide excision repair following UVR exposure; its deletion results in failure to increase XPC and DDB2 levels and reduces cell survival after UVR exposure [93].
ROLE OF VITAMIN D IN BASAL CELL CARCINOMAS (BCC) The pivotal molecular abnormality in BCC carcinogenesis is inappropriate activation of the Hedgehog (HH) signaling pathway [94]. Essentially all BCC tumors have an overactive HH pathway, commonly due to mutational inactivation of the tumor suppressor gene, Patched1 (Ptch1). Ptch1 is a transmembrane receptor for the HH ligand, and is the key inhibitor of HH signaling (Fig. 89.1). Ptch1 inhibits HH target genes by repressing Smoothened (Smo), rendering downstream Gli transcription factors inactive. The HH ligand binds to the Ptch receptor, thus relieving inhibition of Smo and activating Gli transcription factors [95]. Gli transcription factors are then continuously active and alter the transcriptional profile thereby enhancing growth and inhibiting differentiation, increasing the expression
Model of Hedgehog signaling pathway in vertebrates. (A) Ptch inhibits Smo in the absence of Hh. Smo is unable to activate Gli transcription factors as Sufu binds Gli and prevents the transcription of Hh target genes. Hh ligand is bound by Hip on the membrane surface. (B) Binding of the Hh ligand inhibits Ptch, and relieves the inhibition of Smo by Ptch. Smo localizes to the membrane and is now able to activate Gli transcription factors. Hh target genes are transcribed.
FIGURE 89.1
of genes responsible for tumor growth and proliferation or decreasing inhibitors of proliferation. 1,25(OH)2D3 has been shown to inhibit the HH pathway directly by binding to Smo [96] thus suggesting that vitamin D could be used to inhibit or prevent BCC tumors in vivo [97,98]. Indeed, topical application of vitamin D3 (cholecalciferol) in mice reduces BCC cell proliferation and reduces Gli1 mRNA in vitro and in vivo [97]. Vitamin D could also inhibit BCC tumors by activating the canonical vitamin D receptor pathway that regulates keratinocyte differentiation. Like the keratinocytes from which they are derived, BCCs also express VDR, as evidenced from studies using immunostaining and mRNA expression quantification [99]. In one study, human BCC tumors showed pronounced expression of the VDR at the periphery of tumor cells. In an animal model, VDR knockout mice developed more skin tumors (primarily BCCs) when exposed to a carcinogen (oral DMBA) than did their wild-type littermates [85]. The development of BCCs in these mice lacking
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ROLE OF VITAMIN D IN MELANOMAS
functional VDR suggests the importance of the vitamin D pathway in regulating genes downstream of the HH pathway [100]. Further evidence of the role of vitamin D in BCCs comes from clinical studies of BCC patients (Table 89.1). In a nested caseecontrol study of elderly men with NMSC (n ¼ 178) or without skin cancer (N ¼ 930) enrolled in an osteoporosis study, higher levels of 25(OH)D were associated with a decreased risk of skin cancer (ptrend ¼ 0.04). Men in the highest quintile of 25(OH)D (>30 ng/ml) had 47% lower odds of NMSC (95%, CI 0.30e0.93, p ¼ 0.026) compared to those in the lowest quintile. These results suggest that a diagnosis of NMSC is not a marker for adequate 25(OH)D levels and that high 25(OH)D levels may be associated with a reduced risk of NMSC [101]. In contrast, another caseecontrol study from the Kaiser population showed that higher prediagnostic serum 25(OH)D levels were associated with a small increased risk of BCC [102] likely due to increased sun exposure causing both higher 25(OH)D levels and BCC risk. Finally, one prior prospective cohort study on vitamin D intake from dietary questionnaires found no association between vitamin D and BCC risk [24]. These studies are difficult to compare as the study subjects differed in age and geography, measurements of vitamin D (25(OH)D3 versus total 25(OH)D which includes 25(OH)D3 and 25(OH)D2 versus dietary intake), and ascertainment of BCC outcome (self-report versus pathology records). Thus, in vitro and mouse studies provide a mechanism by which vitamin D may reduce or prevent NMSC, but prospective studies in humans need to be done to
TABLE 89.1
Association of Increasing Serum 25(OH)D Levels with Non-melanoma Skin Cancer Odds ratio (OR) and 95%CI
25(OH)D (ng/ml)
Base model*
Fully adjusted modely
Q1 (<16)
1.00 (referant)
1.00 (referant)
Q2 (16e20.8)
0.94 (0.56e1.55)
0.94 (0.56e1.55)
Q3 (20.9e25.1)
0.91 (0.55e1.55)
0.93 (0.56e1.54)
Q4 (25.2e29.8)
0.84 (0.50e1.42)
0.86 (0.51e1.45)
Q5 (29.9e58.3)
0.53 (0.30e0.93)
0.54 (0.31e0.96)
P trend
0.032
0.044
>29.9 ng/ml (Q5 vs. Q1eQ4)
0.57 (0.35e0.93)
0.60 (0.37e0.98)
>32.0 ng/ml
0.57 (0.33e0.98)
0.59 (0.34e1.04)
* Adjusted for age (continuous variable), BMI (continuous variable), season of blood draw, and clinic site. y Adjusted for age, BMI, season of blood draw, clinic site, outdoor walking activity (continuous variable), and cigarette smoking (yes/no).
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determine the true relationship between vitamin D and NMSCs.
ROLE OF VITAMIN D IN MELANOMAS Vitamin D may affect melanoma development and/or progression through various mechanisms, principally via the VDR, which is present to a varying degree in melanoma cells [103]. Stimulation of the VDR has been shown to inhibit cell growth and invasion and to inhibit the G1/S cell cycle checkpoint in melanoma cells [104]. Like keratinocytes, melanocytes also have the capacity for autonomous local production of 1,25(OH)2D [105,106]. The anti-proliferative and pro-differentiative effects of vitamin D and its metabolites have been demonstrated in multiple melanoma cell lines in vitro [105e107]. Eisman et al. showed 1,25(OH)2D3 suppresses growth in human melanoma-derived xenografts in immunosuppressed mice [108]. Inhibition of melanoma migration, invasion and metastasis is another potential mechanism by which vitamin D may inhibit progression of melanoma. One study showed that pretreating melanoma cells with 1,25(OH)2D3 inhibited in vitro invasiveness and in vivo murine pulmonary metastasis [109]. Lastly, vitamin D may promote differentiation by maintaining calcium gradients in the epidermis [4]. In humans, there is accumulating evidence that the vitamin D pathway may reduce melanoma progression. One population-based study (N ¼ 500 cases) of survival from melanoma suggested that some factors associated with high levels of sun exposure, such as solar elastosis (i.e., dermal changes associated with sun seen in histology sections of skin) and sunburns/intermittent sun exposure, are inversely associated with death from melanoma. The association between survival and solar elastosis was not explained by confounding with early detection or screening behaviors or education level [110]. A recent observational study reported an inverse association between levels of 25(OH)D with melanoma stage. Thus patients with advanced-stage IV melanoma had lower 25(OH)D levels compared to patients with stage I disease [111]. A large cohort study from the UK also showed that melanoma patients with lower 25(OH)D were more likely to have thicker tumors (Breslow depth) and were more likely to relapse from melanoma [112]. Observational studies of dietary or supplemental intake of vitamin D in melanoma are less consistent. One caseecontrol study found that high vitamin D intake from food was associated with a reduction in melanoma risk [113] while another caseecontrol study [114] and a large prospective study did not find any evidence of protection [115]. Again, these conflicting findings may be due to differences in prior UV exposure since UV is a primary risk factor for skin cancer development.
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89. VITAMIN D AND SKIN CANCER
PHOTOPROTECTION AND VITAMIN D LEVELS Sun protection is widely recommended to prevent both melanoma and non-melanoma skin cancer. The American Medical Association [116], the World Health Organization [117], and the American Academy of Dermatology [118] advise individuals to limit midday sun exposure, seek shade, wear protective clothing, including hat and sunglasses, and use broad-spectrum sun protection factor (SPF) 30þ sunscreen liberally. Several studies show that regular use of sunscreen correlates with lower circulating levels of 25(OH)D. The use of SPF 15 sunscreens inhibits more than 90% of vitamin D production in the skin [11]. In one of the earliest studies on the subject, Matsuoka et al. showed that vitamin D levels fall with uniform, whole-body application of an SPF 15-equivalent sunscreen compound [119]. Later studies have not found a significant difference in 25(OH)D between sunscreen and non-sunscreen users, even in a randomized controlled trial of sunscreen on 25(OH)D levels [120e123]. These results suggest that typical sunscreen use is not nearly as efficient as Matsuoka’s controlled experiments, with proper application technique and whole-body application being unlikely. Use of other photoprotective measures (shade and long-sleeve use) as recommended by the successful Australian sun safety health campaign may contribute to an increased number of Australians becoming vitamin-D-deficient [124]. Similarly, a study by Tang et al. (Fig. 89.2) on 41 patients with basal cell nevus syndrome (BCNS) showed that these patients, who exhibit higher levels of sun-protective behavior, are three times more likely to be vitamin-D-deficient (57% versus 18%, p < 0.001) compared to the general population. BCNS patients inherit a mutated copy of the PTCH1 tumor suppressor gene [125] and develop tens to hundreds of BCCs in their lifetime. Similar to other UVsensitive patients such as those with xeroderma pigmentosum (XP), patients with BCNS generally try to photoprotect themselves by avoiding the sun during peak hours in an attempt to prevent BCCs, which can be locally destructive and can cause significant morbidity. Furthermore, those BCNS patients who were more overweight had lower levels of 25(OH)D, showing that obesity can also contribute to vitamin D deficiency in these patients as in the general population [126]. Two other studies even question the notion that individuals with fairer skin are more likely to have adequate vitamin D levels since they can synthesize the molecule more efficiently than those with darker skin; one study found that fairskinned subjects were more likely to be deficient and attributed the differences to differing sunscreen use and sun-avoiding behavior [24,127].
FIGURE 89.2 Percentage of BCNS (N ¼ 41) or NHANES (N ¼ 360) subjects with vitamin D deficiency (25(OH)D levels 20 ng/ml) or severe deficiency (10 ng/ml). NHANES controls were matched by age, gender, race, skin type, and season/geography.
Are we doing harm to patients by advocating strict sunscreen use and sun avoidance? Given the great number of factors that affect vitamin D synthesis from the sun (subject age, pigment, season, latitude, etc.) it is difficult to recommend a standard dose of sunlight to boost 25(OH)D levels, especially for a population as diverse as that in the USA. Yet the sun is not to be altogether avoided, as current recommendations of restricted sun exposure may essentially rob the individual of easy and free vitamin D synthesis. Moreover, the role of sunscreen may need to be re-evaluated, as the data show its proper use may block vitamin D synthesis while still allowing certain portions of the erythema action spectrum to transmit through to the skin [11]. In light of these findings, a combination of vitamin-D-rich dietary sources, supplements, and measured and limited incidental sunlight exposure is more likely to be the best current recommendation for adequate intake than any of these sources alone.
Serum Levels of Vitamin D: How Much is Enough? The serum concentration of 25(OH)D is currently the best indicator of vitamin D status given its long half-life of over several weeks. It does not reflect total tissue stores, but shows the amount of available precursor to the hormonally active form of vitamin D. Circulating levels of active 1,25(OH)2D are generally not good indicators of vitamin D status because of its short half-life and tight regulation; its levels do not typically decrease until vitamin D deficiency is severe. The optimal amount of serum vitamin D levels for health maintenance and disease prevention is still being defined. A concentration of <20 ng/ml (<50 nmol/l) is generally considered inadequate for maintaining bone health. Taking into account the multiple health outcomes such as autoimmune conditions, cancer prevention, and bone health,
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REFERENCES
adequate serum concentrations of 25(OH)D may range between 28 and 40 ng/ml [128]. An intake of an additional 100 IU of vitamin D per day increases serum 25(OH)D by an average of 1 ng/ml [129,130]. Thus, if the lower limit of 30 ng/ml is regarded as the minimum desirable target, then the current adequate intake levels of 200 IU/day for young adults, 400 IU/day for those aged 51e70 years, and 600 IU/day for those aged greater than 70 years is inadequate. The Canadian Cancer Society has recommended that adults consider taking 1000 IU of vitamin D per day during the fall and winter. They base this recommendation on the growing evidence for a link between vitamin D and a reduced risk for colorectal, breast, and prostate cancers. Similarly, the American Cancer Society in 2008 stated that the current nutritional guidelines for vitamin D are likely too low and the optimal intake of vitamin D for cancer prevention is still uncertain but may be somewhere between 1000 and 2000 IU. The American Academy of Pediatrics recently doubled its recommended intake from 200 to 400 IU daily for children and adolescents [131] while the American Academy of Dermatology and the National Council on Skin Cancer Prevention both recently recommended a daily intake of 1000 IU, especially for people who practice photoprotection. However, the 2010 Institute of Medicine recommends 600 IU for adults.
CONCLUSIONS As for the role of vitamin D in skin cancer treatment or prevention, vitamin D has been shown to upregulate DNA repair, inhibit tumor cell proliferation, and inhibit the Hedgehog signaling pathway. In vitro and murine data offer great biologic plausibility for a role for vitamin D in preventing melanoma and non-melanoma skin cancers; however, more data are needed from human studies. We await results for the effect of vitamin D on cancer prevention from the first randomized, controlled trial of 2000 IU, which is currently enrolling subjects (the VITAL trial is discussed in Chapter 105). The relative contributions of diet, supplementation and cutaneous vitamin D synthesis require further study. It may be that given UV exposure’s postulated riskebenefit J-curve, small quantities of exposure may actually protect against skin cancer. However, UV rays are known to be carcinogenic, and it is still unclear how to recommend a uniform and healthy dose of UV at the population level given the multiple factors (age, pigment, season, geographic latitude) that impact an individual’s ability to synthesize vitamin D. Indoor tanning beds contain lamps that primarily emit UVA radiation that is the wrong wavelength for synthesis of vitamin D and are carcinogenic.
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The American Academy of Dermatology (AAD) recommends that an adequate amount of vitamin D should be obtained from a healthy diet that includes foods naturally rich in vitamin D, foods and beverages fortified with vitamin D, and/or vitamin D supplements; it should not be obtained from unprotected exposure to UV radiation (AAD position statement, June 2009). Groups known to be at particular risk for deficiency such as the elderly, the obese, individuals with dark skin, and individuals with limited sun exposure due to photoprotection would especially benefit from supplementation.
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C H A P T E R
90 The Anti-tumor Effects of Vitamin D in Other Cancers Donald L. Trump, Candace S. Johnson Roswell Park Cancer Institute, Buffalo, NY, USA
INTRODUCTION Vitamin D compounds have been evaluated for their potential role in cancer causation and treatment. A substantial portion of this work has been directed toward studies in prostate, breast, colorectal cancer, and hematologic malignancies, especially acute leukemias. Studies in these tumors are reviewed in Chapters 85, 87, 86, and 88. This chapter will review data in other tumor types, concentrating on those tumors for which there are the most detailed studies. We advance the unifying hypothesis for the study of vitamin D and cancer that, given the ubiquity of expression of the vitamin D receptor (VDR) in most human tissues and cancers that arise from these tissues, there is as yet no reason to believe that the development and/or growth of any particular cancer is more, or less, likely to be modulated by vitamin D. The mechanisms determining resistance to vitamin D effects are varied and all tumor types studied express sensitivity to vitamin D effects.
THYROID CANCER Epidemiologic studies suggest a link between several variables which are associated with variations in light exposure and hence vitamin D synthesis in the skin and thyroid cancer. Examples of such variables are latitude of residence and measured or estimated geographic variations in ultraviolet B light exposure. Thyroid cancer is reported to have increased incidence mortality among women who are exposed to lower levels of UV light [1e3].
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10090-3
In preclinical studies, the VDR and 1a-hydroxylase (CYP27B1) are increased in expression in papillary thyroid cancer compared to “normal,” adjacent thyroid [4]. In this study, it was also noted that VDR and 1a-OHase were increased “especially in areas of lymphocyte infiltration,” suggesting this cell population may contribute to these findings. RXR isoform expression has been noted to be low and localized in the cytoplasm, rather than the nucleus among thyroid cancers [5]. These data suggest that changes in vitamin D signaling may be important in thyroid cancer. In models of human thyroid cancer, Asa and colleagues at the University of Toronto have shown, in vitro and in vivo, that 1,25-dihydroxyvitamin D3 (1,25 (OH)2D3) increases p27 expression, reduces proliferation, reduces in vivo tumor growth, increases differentiation and restores thyroid cancer cell adhesiveness, the latter effect being associated with PTEN/Pi3kinase signaling and fibronectin expression [6e8]. Similar in vitro and in vivo anti-proliferative effects have been reported by others in papillary, follicular, anaplastic and medullary histologies [9e13]. There are few clinical studies of the vitamin D axis in thyroid cancer patients. Stepien et al. reported lower circulating levels of 1,25(OH)2D among patients with papillary, follicular and anaplastic thyroid cancer [14]. We are aware of only a single clinical report of vitamin D administration in the treatment of thyroid cancer in patients. Morishita et al. described a patient with locally extensive thyroid cancer (increased thyroglobulin level, no biopsy) in whom administration of AlpharolÒ (alphacalcidol, 1a-(OH)D3) at a dose of 0.5 mg per day was associated with “stability” of the advanced cancer for 2 years [15].
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BONE AND SOFT TISSUE SARCOMAS
GASTRO-ESOPHAGEAL CANCERS
There are extensive in vitro data evaluating the effect of vitamin D analogs on the biochemistry, molecular biology, metabolism and growth of cells derived from human and murine osteogenic sarcomas as well as immortalized osteoblasts [16e20]. There are insufficient data to draw any conclusions regarding the role of vitamin D in the etiology and progression of bone and soft tissue sarcomas and no data, of which we are aware, on treatment of patients or in vivo anticancer models.
For both gastric and esophageal cancers, there are epidemiologic data e similar to those reported for many other cancers e which suggest that the risk of cancer in each of these sites is inversely related to estimates of UV light exposure and other potential surrogate measures of vitamin D levels [30e32]. Data are less consistent for these two tumor sites, at least in part because of the substantially reduced frequency of the tumors. For esophageal tumors, there are additional intriguing experimental data. Mimori and colleagues reported an inverse relationship between the expression of CYP24, the primary calcitriol degrading enzyme, and patient survival among those with esophageal cancer [33]. This observation is consistent with the observation that at least progression of esophageal cancer is facilitated by degradation of vitamin D and hence reduction in vitamin D signaling. Interestingly, similar data have been reported in lung cancer [34e36]. Preclinical studies indicate that treatment of esophageal cancer (squamous and adenocarcinoma histology) and gastric cancer cells in vitro and in vivo with vitamin D analogs will reduce proliferation and reduce tumor growth [37,38]. It is of interest and raises the possibility of another anticancer mechanism that vitamin D analogs may block the effect of carcinogens in inducing gastric cancer in the rat [39]. There are no clinical data of which we are aware evaluating clinical cancer treatment with vitamin D analogs.
MELANOMA Considerable epidemiologic data link the risk of cutaneous melanoma and exposure to sunlight [21,22]. Vitamin D and melanoma is also discussed in Chapter 89. Interestingly, the incidence of vulvar melanoma appears unrelated to the rising incidence of cutaneous melanoma. The data which suggest that the incidence of vulvar melanoma is less in more equatorial latitudes consistent with the hypothesis that light exposure may reduce the risk of melanoma in areas of the body not exposed to sunlight [23]. Several groups have reported that based on population studies, higher 25(OH)D levels are associated with melanomas with a better prognosis (lesser Breslow depth) and that certain VDR polymorphisms are associated with a lesser risk of melanoma, better prognosis and lower mortality (e.g., Fok I T e increased risk and Bsm I A reduced risk). While there are studies which do not support these hypotheses, a substantial body of epidemiologic data does support the hypotheses that reduced vitamin D signaling is associated with melanoma with a worse prognosis [24e28]. As in other tumor systems, there are extensive data evaluating the anti-proliferative effects of vitamin D analogs in melanoma models in vitro and in vivo. Anti-proliferative and molecular effects are readily demonstrable [25e28]. Essa and colleagues have made the intriguing observation that in vivo responsiveness among seven melanoma cell lines was associated with higher VDR expression and that such expression was inversely linked to expression of miRNA 125b (MeWo and SK-Mel128 lines) [29]. These investigators also showed that the histone deacetylase inhibitor trichostatin A and the DNA methyltransferase inhibitor 5-azacytidine potentiated the anti-proliferative effects of 1,25(OH)2D3. These data suggest that there may be biomarkers of vitamin D responsiveness. There are no clinical trials, of which we are aware, testing the clinical effectiveness of vitamin D analogs in patients with melanoma.
LUNG CANCERS Epidemiologic data indicate, with moderate uniformity, that low vitamin D serum concentrations and factors which are associated with low D serum levels are associated with higher risks of lung cancer [40e45]. In addition, lung carcinogenesis can be blocked in experimental models by vitamin-D-based therapies. Mernitz et al. reported that a combination of 1,25(OH)2D3 þ 9-cis retinoic acid in the A/J mouse reduces the frequency of lung tumor development. Similarly, experimental carcinogenesis in the lung can be substantially enhanced by VDR ablation [46,47]. In preclinical lung cancer models in vitro and in vivo invasiveness and metastases can be suppressed by vitamin D analog therapy or by VDR knockout [48e51]. As with many other tumor types in vitro and in vivo vitamin D analog treatment of lung cancer cell lines reduces proliferation, reduces tumor growth and induces molecular changes consistent with those seen in other tumor types. There are no data to suggest specific lung histologic types are more or less modulated by vitamin D [52e59].
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In lung cancer there are more data than in other tumors that suggest that vitamin D catabolism may play a role in enhanced tumor growth mediated through disrupted vitamin D signaling [60e63]. As in all other human tumor systems, there is very limited information regarding the role of vitamin-D-based therapies in patients with lung cancer. It is worth emphasizing that in lung cancer models as well as all other tumor models combinations of vitamin D analogs and cytotoxic agents are often substantially additive or synergistic. There are many clinical trials which establish that chemotherapy agents can be safely administered in combination with high doses of vitamin D analogs [64e69].
HEPATOCELLULAR CARCINOMA (HCC) While HCC is a relatively uncommon tumor in Western countries, it is a very common cause of death in Asia. A number of studies have evaluated the role of vitamin D not only in the development and progression of HCC, but also in those factors predisposing to HCC. Considerable circumstantial and epidemiologic data suggest that vitamin D may play a role in development of viral infections [70,71]. Huang and colleagues have reported that some VDR polymorphisms are associated with distinct clinical phenotypes in the Taiwanese population who are carriers of hepatitis B [72]. Falleti et al. have noted that certain VDR polymorphisms (bb/Bsml and TT/Taql) were significantly associated with HCC among individuals with alcoholic cirrhosis; the role of coexisting viral infection was not reported [73]. Chen et al. note an increased incidence and mortality rate for liver cancer related to reduced ambient UV irradiance in China [74]. Vitamin D analogs have been reported to reduce the development of liver tumors in a number of animal carcinogenesis models. Bankar and colleagues reported a protective effect of 1,25(OH)2D3 against “nodulogenesis” as well as reduction in DNA damage (as assessed by comet assay) in rats treated with diethylnitrosamine (DEN) þ phenobarbital [75]. In C3H/5y mice, a strain which develops at high frequency spontaneous HCC, the vitamin D analog EB1089 (seocalcitol) reduces the occurrence of HCC 10-fold [76]. Saha et al. note that 1,25(OH)2D3 reduces HCC occurrence in DEN as well as streptozotocin-induced rat liver cancer, DNA damage and chromosomal aberrations [77,78]. Many preclinical studies demonstrate the antiproliferative effects of 1,25(OH)2D3 and EB1089 in vitro and in vivo [79e83]. By contrast, Huynh et al. found no anti-tumor effects of EB1089 among seven xenografts established directly from human HCC [84]. These contrasting results suggest that xenografts may more closely
represent clinically relevant preclinical models than live cell-based studies in vitro and in vivo. As demonstrated in other tumor systems, Miyaguchi and Watanabe showed that the anti-proliferative effects of 1,25(OH)2D3 were closely related to VDR expression in two human HCC cell lines [85]. p27 induction, cell cycle G1 arrest and ERK {1/2} and c-met suppression are associated with vitamin D anti-proliferative effects in Hep-G2 and MHCC 97 lines, respectively [86,87]. No careful studies have been done in vivo which explore optimal dose or schedule of 1,25(OH)2D3 or other vitamin D analogs. In contrast to most other tumors discussed in this chapter, clinical trials of vitamin D analogs have been conducted in HCC. Morris and colleagues studied 1,25(OH)2D3 in vitro (along and with the vehicles, medium-chain triglycerides or lipiodol) and in vivo. The primary focus of these studies was on regional treatment by hepatic artery infusion [88e90]. Anti-proliferative effects were demonstrated and substantial doses of 1,25(OH)2D3 administered (10 mg) without any toxicity. Subsequent trials in pigs demonstrated “first pass” extraction of 1,25(OH)2D3 by the liver [91]. Clinical trials have been completed which demonstrate that 100 dose escalation can be achieved without toxicity if 1,25(OH)2D3 is administered by hepatic artery infusion rather than by IV infusion [92e94]. Clinical trials to evaluate the efficacy of this approach have not been reported. Dalhoff et al. conducted a phase II trial of daily, oral EB1089, 10 mg, in 56 patients with HCC [95]. Among 33 patients evaluable for response by imaging studies, two had complete resolution of tumor lasting 29 and >36 months. No significant toxicity was noted. The overall benefit of this treatment is difficult to judge and no further studies of EB1089 in HCC have been conducted. Since HCC is almost uniformly fatal, the two long-term survivors treated with EB1089 are of great interest. Based on extensive preclinical studies and intriguing, albeit limited, clinical data, determination of maximally tolerated or optional biologic doses of a vitamin D analog in patients with HCC and subsequent clinical efficacy evaluation appears warranted.
BLADDER CANCER Bladder cancer, the fourth most common cancer, usually presents as superficial transitional cell carcinoma with a local recurrence rate of 88% at 15 years with 10e30% progressing to invasive disease [96,97]. Risk factors include cigarette smoking, exposure to aromatic amines and chronic bladder infections. Epidemiologic data suggest that low vitamin A consumption and low serum levels of carotene and retinol are
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associated with the incidence of bladder cancer [96]. Studies are inconclusive with regard to the association of vitamin D in the diet and bladder cancer [96,98]. A recent study demonstrated a higher intake of vitamin D that is inversely correlated with bladder cancer risk in older individuals suggesting further studies are warranted [99]. Transitional cell carcinoma cell lines from canine, rat, and human express significant levels of VDR [100,101]. When patient tumor samples were examined, over 85% of bladder tumors expressed VDR with no correlation between VDR expression and pathologic stage and grade. Progression in pathologic stage, however, is associated with higher VDR [102]. Hermann and Andersen demonstrated a higher level of VDR expression in bladder tumors from patients with advanced disease as compared to lower-stage disease with normal urothelium expressing VDR with low intensity [101]. VDR polymorphisms and bladder cancer risk have demonstrated a significant difference in the frequency of the VDR Fok1 polymorphism, when compared to bladder cancer patients and normal controls with no effect on smoking status with these patients [103]. Therefore, level of VDR expression and VDR polymorphisms may be useful as a prognostic indicator in bladder cancer. Preclinical studies have demonstrated anti-proliferative activity of 1,25(OH)2D3 and its analog seocalcitol (EB1089) in canine and human bladder cancer cell lines [100]. In vivo studies using an N-methylnitrosoureainduced model of bladder cancer demonstrated that intravesical 1,25(OH)2D3 after carcinogen resulted in fewer tumors with little or no toxicity [104]. Studies by Ma et al. [105] demonstrated that 1,25(OH)2D3 significantly enhanced the anti-tumor activity in vitro and in vivo of gemcitabine and cisplatin, the current standard chemotherapy regimen for invasive bladder cancer [106,107]. Interestingly, 1,25(OH)2D3 upregulates the expression of p73, which sensitizes the tumor cells to the anti-tumor effects of these cytotoxic agents [108,109]. Loss of p73 is associated with advanced tumor stage in bladder cancer [110], therefore 1,25(OH)2D3mediated upregulation could sensitize tumor cells to the effects of chemotherapy. Despite potentially significant preclinical studies, 1,25(OH)2D3 or its analogs have not been given to bladder cancer patients either as a single agent or in combination with cytotoxic drugs.
GLIOMAS Gliomas are the most common malignant tumors in the brain [111]. Vitamin D is suggested to be involved in brain function because VDR expression was found not only in gliomas and glial tumors [112], but also in
normal neurons and glial cells [113,114]. Neuroprotective and immunomodulatory effects are also associated with 1,25(OH)2D3 in a number of experimental models [114] (see also Chapter 32). Using human glioblastoma cell lines, both 25(OH)D3 and 1,25(OH)2D3 resulted in a significant reduction in cell proliferation and induction of apoptosis [115e117]. 1,25(OH)2D3 inhibits tenascin-C expression in rat glioma cells where tenascin-C is an extracellular matrix protein with growth-potentiating and angiogenic properties [118]. 1,25(OH)2D3-mediated cell death in glioma cells is dependent on protein synthesis and upregulation of c-myc expression, IL-6, vaso-endothelial growth factor and gadd45 gene expression [117]. In glioblastoma multiforme cell lines that were resistant to 1,25(OH)2D3, resistance cannot be overcome by epigenetic silencing and does not support the modulation of the notch-signaling pathway, which plays a role in Ras-induced transformation in glial cells [119]. Preclinical in vitro studies with glioma cell lines demonstrate an anti-proliferative effect with 1,25(OH)2D3 and its analogs; however, in vivo studies in animal models or clinical studies are yet to be performed.
HEAD AND NECK Cancer of the mouth and oropharynx is the 10th most common cancer worldwide which increases in incidence with age [120]. Studies to correlate low serum vitamin D levels in head and neck cancer and cancers of the oral cavity with poor prognosis have been mixed [121,122]. Studies by Lipworth et al. [122] demonstrate an inverse relationship between dietary intake of vitamin D and risk of squamous cell carcinoma (SCC) of the head and neck. Conversely, vitamin D status did not affect disease outcome in patients with head and neck cancer despite cancer stage or season of initial treatment [121]. Polymorphisms of the VDR gene are associated with a decreasing risk of head and neck cancer and suggest that the VDR f and t alleles and their genotypes may protect against this disease [123]. Studies in head and neck cancer are complicated due to patients who are heavy smokers and consumers of alcohol which may affect serum vitamin D status [124]. Numerous in vitro studies exist in a variety of rodent and human SCC tumor cell lines which demonstrate an anti-proliferative effect with 1,25(OH)2D3 and its analogs [125e130]. Anti-proliferative activity occurs through modulation of cell cycle inhibitors p18, p27, and p21 [127,129]. Studies have also shown that the analog EB1089 inhibits the proliferation of human laryngeal SCC through the cyclin-dependent kinase inhibitor p57 [130]. In vivo, enhanced anti-tumor activity is observed in syngeneic and xenograft rodent SCC models with 1,25(OH)2D3 and its analogs can also
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inhibit carcinogenesis in the hamster buccal punch model suggesting a chemo-preventive effect [134]. Synergy with cytotoxic drugs such as cisplatin, docetaxel, and paclitaxel and 1,25(OH)2D3 and its analogs have also been observed both in vitro and in vivo in a number of head and neck model systems [131e133]. Treatment of CD34þ progenitor cells with 1,25(OH)2D3 induces differentiation into immune stimulatory dendritic cells [135]. In a clinical study, patients with head and neck cancer were treated for 3 weeks with or without vitamin D3 before surgery. In the patients treated with vitamin D3, intratumor levels of CD34þ cells and immature dendritic cells declined and levels of mature dendritic cells increased significantly suggesting that vitamin D can enhance immune competence and could play a role in the augmentation of immunebased therapeutic approaches [136].
RENAL CELL CARCINOMA The kidney is the major site for vitamin D metabolism and calcium-related homeostasis [137]; however, the role of vitamin D in renal cell carcinoma (RCC) has not been adequately addressed. In a recent study in men, there was an inverse correlation between occupational UV light exposure and the risk of RCC [138]. Studies have suggested that a decreased serum 25(OH)D level increases the risk of RCC [139]; however, other studies examining circulating levels found no correlation between 25(OH)D levels and the risk of RCC [140]. Despite this controversy, it is clear that normal kidney strongly expresses the VDR and the vitamin D metabolizing enzymes, CYP27B1 and CYP24A1 [141]. In RCC, VDR expression may be decreased as compared to normal kidney [141,142] and this has been suggested to limit responsiveness of RCC to therapy with 1,25(OH)2D3. Correlation has been observed between expression of RXRg and the tumor stage or 5-year survival rate in RCC [143]. VDR polymorphisms of the AA genotype at the Apal site may be associated with an increased risk and poor prognosis for RCC in Japan [144]. In in vitro model systems, RCC tumor cell lines are growth-inhibited by 1,25(OH)2D3 and its analogs [145,146]; in vivo in the murine renal cell cancer Renca model, 1,25(OH)2D3 and vitamin D3 resulted in the inhibition of tumor growth and prolonged the lifespan in a dose-dependent manner [146]. In this model, vitamin D also inhibited angiogenesis and resulted in fewer pulmonary and hepatic metastatic nodules. Treatment of a single patient with RCC with 1 mg of 1a-(OH)D3 (alphacalcidol) plus 3 106 units of interferon-a (INFa), resulted in the resolution of metastatic bone lesions [147]. In a phase II clinical trial of metastatic
RCC, patients received 1 mg of alphacalcidol daily with 3 106 units of INFa three times a week. The therapy was well tolerated with only a modest effect on response. With only 16 patients that were treated in the phase II setting, further studies are needed to determine the potential efficacy of this approach [148].
PANCREATIC CANCER Pancreatic cancer is ranked fourth among cancer deaths in the USA and has a 5-year survival rate of less than 5% [149]. Numerous epidemiologic studies suggest a relationship between vitamin D status and incidence as well as mortality of pancreatic cancer [149,150,156]. In the USA, relatively high pancreatic cancer rates are observed in states where UV exposure is low [151] and in Japan an inverse correlation is observed between solar radiation and pancreatic cancer risk [152]. African-Americans have an increased risk of pancreatic cancer and a higher mortality rate as compared to Caucasians [153]. African-Americans have serum 25(OH)D3 levels around 10 ng/ml lower than white Americans due to dark pigmentation of the skin [154]. Studies in both men and women observed a higher intake of vitamin D was associated with a decreased risk for pancreatic cancer [150]. In contrast, in a Finnish study, pancreatic cancer occurred in men with the highest serum 25(OH)D3 levels as compared to those with low levels [155]. Differences in these studies may relate to differences in these two populations; however, further studies may be required to determine the extent of how vitamin D status impacts the risk of pancreatic cancer. The VDR is expressed in freshly isolated normal pancreatic cancer cells and in pancreatic tumor cell lines [157,158]. In addition, normal and malignant pancreatic cells both express 1a-hydroxylase that converts 25(OH)D to 1,25(OH)2D and these cells are growth-inhibited by 25(OH)D [149,159]. In vitro studies demonstrate that freshly isolated pancreatic tumor cells have a higher level of VDR expression as compared to normal cells and the pancreatic cancer cells responded to growth inhibition to vitamin D3 in vitro [157]. 1,25(OH)2D3 and its analogs demonstrate significant anti-proliferative activities in a number of in vitro pancreatic cancer cell lines [160e165]. The 19-nor-1a-25(OH)2D2 (paricalcitol) has been extensively characterized preclinically in pancreatic model systems [166,167]. Paricalcitol and other vitamin D analogs upregulate p21 and p27 in human pancreatic cell lines [168]. Besides the effects on cell cycle, vitamin D and its analogs also can induce apoptosis and differentiation in pancreatic cell lines [160,167e169].
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Synergy was observed in combination with retinoids [169] which may have therapeutic implications. Enhanced anti-tumor effects are also observed when 1,25(OH)2D3 is combined with gemcitabine in pancreatic models in vitro and in vivo [160]. Gemcitabine has activity in pancreatic cancer and is a part of the initial “upfront” therapy in patients with pancreatic cancer [170] suggesting that combination therapy with 1,25(OH)2D3 may offer benefit in this disease. A phase II trial of EB1089 (seocalcitol) in patients with inoperable pancreatic cancer resulted in no objective responses with minimal toxicity. The optimal maximum tolerated dose (MTD), however, was not determined in this study [171]. Vitamin D3 has also been given as a single injection at 200 000 IU in patients with operable pancreatic cancer [172]. Twenty-nine patients were treated with no observed toxicity and further studies are needed to determine the therapeutic efficacy in this patient population. Additionally, a phase II study of 1,25(OH)2D3 and docetaxel was performed in patients with untreated metastatic or locally advanced pancreatic cancer [173]. Twenty-five patients were treated with oral 1,25(OH)2D3 at 0.5 mg/kg on day 1 with docetaxel at 36 mg/m2 on day 2 repeated weekly for 3 weeks without significant 1,25(OH)2D3-related toxicity. The primary endpoint of this phase II trial was time-to-progression where 12% of the patients had a partial response with a median overall survival of 24 weeks, results not significantly different than standard therapy alone. These studies demonstrate the safety of the approach and suggest 1,25(OH)2D3 may have activity in the enhancement of docetaxel-mediated anti-tumor activity in pancreatic cancer.
CONCLUSIONS Extensive epidemiologic data exist to demonstrate a role for vitamin D status on incidence, progression, and mortality in cancers of the breast, colon, and prostate as well as in hematologic malignancies. We have examined here, the potential role of vitamin D and its analogs across the less commonly studied tumors for a vitamin D effect (thyroid cancer, bone and soft tissue sarcoma, melanoma, gastro-esophageal cancers, lung cancers, heptocellular carcinoma, bladder cancer, glioma, head and neck cancer, renal cell carcinoma, and pancreatic cancer) where the data are not as extensive or as well characterized. VDR expression is found across all these tumor types and differences between the level of expression and cancer grade/pathologic type have been identified with the need for further studies warranted. Anti-proliferative activities of vitamin D and its analogs are observed across all of these tumors with many in vivo studies in relevant tumor
models showing a vitamin D benefit. However, a number of early-phase clinical studies have been performed without significant beneficial responses observed. These clinical studies are limited, as in the other more commonly studied tumor types, in that the optimal dose is yet to be determined and the availability of vitamin D preparations for clinical use is also limited. The breadth and depth of the studies presented, however, suggests that vitamin D status and the antitumor activity of vitamin D/vitamin D analogs in these less-studied tumor types offers promise for development and further analysis.
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[124] K.A. Johnson, M.A. Bernard, K. Funderburg, Vitamin nutrition in older adults, Clin. Geriatr. Med. 18 (4) (2002) 773e799. [125] M.C. McElwain, R.A. Modzelewski, W.D. Yu, D.M. Russell, C.S. Johnson, Vitamin D: an antiproliferative agent with potential for therapy of squamous cell carcinoma, Am. J. Otolaryngol. 18 (5) (1997) 293e298. [126] P.A. Hershberger, R.A. Modzelewski, Z.R. Shurin, R.M. Rueger, D.L. Trump, C.S. Johnson, 1,25-Dihydroxycholecalciferol (1,25D3) inhibits the growth of squamous cell carcinoma and downmodulates p21 (Waf1/Cip1) in vitro and in vivo, Can. Res. 59 (11) (1999) 2644e2649. [127] T.F. McGuire, D.L. Trump, C.S. Johnson, Vitamin D3-induced apoptosis of murine squamous cell carcinoma cells: selective induction of caspase-dependent MEK cleavage and upregulation of MEKK-1, J. Biol. Chem. 276 (28) (2001) 26365. [128] J. Wietrzyk, M. Milczarek, A. Kutner, The effect of combined treatment on head and neck human cancer cell lines with novel analogs of calcitriol and cytostatics, Oncol. Res. 16 (11) (2007) 517e525. [129] C. Gedicla, G. Hager, M. Weissenbock, W. Gedlicka, B. Knerer, J. Kornfehl, et al., 1,25(OH)2Vitamin D3 induces elevated expression of the cell cycle inhibitor p18 in a squamous cell carcinoma cell line of the head and neck, J. Oral Pathol. Med. 35 (8) (2006) 472e478. [130] L. Lu, J. Qiu, S. Liu, W. Luo, Vitamin D3 analogue EB1089 inhibits the proliferation of human laryngeal squamous carcinoma cell via p57, Mol. Can. Ther. 7 (5) (2008) 1268e1274. [131] P.A. Hershberger, T.F. McGuire, W.-D. Yu, E.G. Zuhowski, M.J. Egorin, D.L. Trump, et al., Cisplatin potentiates 1,2-dihydroxyvitamin D3-induced apoptosis, Mol. Can. Ther. 1 (2002) 821e829. [132] B.W. Light, W.-D. Yu, M.C. McElwain, D.M. Russell, D.L. Trump, C.S. Johnson, Potentiation of cisplatin antitumor activity using a vitamin D analogue in a murine squamous cell carcinoma model system, Can. Res. 57 (17) (1997) 3759e3764. [133] M.R. Young, D.M. Lathers, Combination docetaxel plus vitamin D(3) as an immune therapy in animals bearing squamous cell carcinomas, Otolaryngol. Head Neck Surg. 133 (4) (2005) 611e618. [134] J.D. Meier, D.J. Enepekides, B. Poirier, C.A. Bradley, J.S. Albala, D.G. Farwell, Treatment with 1-alpha,25-dihydroxyvitamin D3 (vitamin D3) to inhibit carcinogenesis in the hamster buccal pouch model, Arch. Otolaryngol. Neck Surg. 133 (11) (2007) 1149e1152. [135] T. Garrity, R. Pandit, M.A. Wright, J. Benefield, S. Keni, M.R. Young, Increased presence of CD34þ cells in the peripheral blood of head and neck cancer patients and their differentiation into dendritic cells, Int. J. Cancer 73 (5) (1997) 663e669. [136] J.S. Kulbersh, T.A. Day, B. Gillespie, M.R. Young, 1a,25-Dihydroxyvitamin D3 to skew intratumoral levels of immune inhibitory CD34þ progenitor cells into dendritic cells, Otolaryngol. Head Neck Surg. 140 (2009) 235e240. [137] S. Christakos, D.V. Ajibade, P. Dhawan, A.J. Fechner, L.J. Mady, Vitamin D: metabolism, Endocrinol. Metab. Clin. North Am. 39 (2) (2010) 243e253. [138] S. Karami, P. Boffetta, P. Stewart, N. Rothman, K.L. Hunting, M. Dosmeci, et al., Occupational sunlight exposure and risk of renal cell carcinoma, Cancer 116 (8) (2010) 2001e2010. [139] T. Fujioka, Y. Suzuki, T. Okamoto, N. Mastushita, M. Hasegawa, S. Omori, Prevention of renal cell carcinoma by active vitamin D3, World J. Surg. 24 (10) (2000) 1205e1210. [140] L. Gallicchio, L.E. Moore, V.L. Stevens, J. Ahn, D. Albanes, V. Hartmuller, et al., Circulating 25-hydroxyvitamin D and risk
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S E C T I O N
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C H A P T E R
91 Vitamin D and Innate Immunity John H. White McGill University, Montreal, Canada
INTRODUCTION AND HISTORICAL BACKGROUND Vitamin D was originally discovered for its critical role in calcium homeostasis. However, we now know that vitamin D has pleiotropic actions, and one of the more active areas of recent vitamin D research has focused on understanding its role as a modulator of immune system function. The immune system in vertebrates can be divided into innate and adaptive arms. The role of vitamin D in regulating adaptive immunity is covered by Dr. Luciano Adorini in the following chapter. Unlike the adaptive immune system, which is present only in vertebrates, innate immune responses are found in a wide variety of plant and animal life and provide front-line defenses to pathogenic challenge [1e3]. The innate immune system defends the host from infection in a non-specific manner but, unlike adaptive immunity, has no memory and does not confer long-lasting immunity against specific pathogens. Stimulation of the innate immune system leads to the production of cytokines and chemokines, which act to communicate with other components of the immune system, including cells essential for eventual adaptive responses. As developed further below, it also leads to the production of antimicrobial peptides (AMPs), which represent the first wave of defense against invading pathogens [4]. We now know that vitamin D can be obtained from limited dietary sources as well as photochemical and thermal conversion of cutaneous 7-dehydrocholesterol in the presence of sufficient solar ultraviolet B radiation (see relevant chapters in Section VII). Several chapters in this book also describe the discovery of vitamin D as the cure for nutritional rickets, as well as the early and more recent work on vitamin D function as a regulator of calcium homeostasis and bone metabolism. However, there are threads of evidence linking vitamin D to
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10091-5
immune system function that extend back over millennia, and closely parallel the emergence of its role in calcium homeostasis. Vitamin D therapy can be traced back to Hippocrates, the father of medicine, who used heliotherapy, or exposure to sunlight, to treat phthisis (tuberculosis; TB) [5]. Nutritional vitamin D therapy arose from the early medicinal use of cod liver oil, which was first described as an agent for treatment of chronic rheumatism in 1789. Throughout the next century, the medical literature documented its effectiveness for treating a number of prevalent conditions such as gout and scrofula, a form of tuberculosis which infects the lymph nodes [6]. The use of cod liver oil as an anti-rachitic agent dates from the 1820s [6,7]. By 1849, the list of conditions treatable with cod liver oil would grow to include TB infection [8,9]. Links between sun exposure, UV light and treatment of rickets date from the 1820s [7]. Sun exposure became a popular therapy for treatment of TB in the mid-19th century with the emergence of sanitoria, and Niels Finsen won the 1903 Nobel prize for showing that UV light could be used to treat cutaneous TB (lupus vulgaris) e Hippocrates would surely have approved.
Vitamin D and Infectious Diseases Vitamin D2 or D3 from dietary sources or vitamin D3 formed by photochemical conversion in skin (see Section VII in this volume) must undergo two modifications to become biologically active. Vitamin D compounds are constitutively hydroxylated largely in the liver to form 25-hydroxyvitamin D2 or D3 (25(OH)D2 or 25(OH)D3), the major circulating forms and markers of vitamin D status. These precursors are then 1a-hydroxylated locally by the enzyme CYP27B1 to produce the hormonal 1,25(OH)2D2 or 1,25(OH)2D3. While there is no strict definition, prior to the November 2010 report from the
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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91. VITAMIN D AND INNATE IMMUNITY
Institute of Medicine (IOM) vitamin D deficiency was widely defined as circulating 25(OH)D levels of less than 20 ng/ml (50 nM) [10e12], whereas one was generally considered to be vitamin D sufficient with circulating 25(OH)D concentrations of greater than 30e32 ng/ml (75e80 nM) [13,14]. The IOM report defines less than 20 ng/ml as deficient, 20e50 ng/ml (125 nM) as normal, and greater than 50 ng/ml as excessive. However, it can be argued that defining 30e32 ng/ml as sufficient is more reflective of human physiology and may indeed be more beneficial for long-term health than maintaining circulating 25(OH)D levels 10 ng/ml lower, the bottom end of the “normal” range. Vitamin D intoxication is not observed until 25(OH)D levels reach 150 ng/ml (375 nM) or more [12], and is associated with hypercalcemia, which if chronic can result in urinary calculi (renal or bladder stones) and renal failure. Cases of vitamin D toxicity do occur, but are far less frequent than vitamin D insufficiency/deficiency, which is quite common. With increasing latitude, surface solar UVB irradiation is insufficient to induce cutaneous vitamin D3 synthesis for periods around the winter solstice of 6 months or even more at the latitudes of northern Europe or Scandinavia [15], a period that is known as vitamin D winter. UVB-induced vitamin D synthesis is also strongly influenced by skin color [16]. Lack of cutaneous vitamin D3 synthesis because of lack of sun or sun avoidance, coupled with vitamin-D-poor diets, has contributed to widespread vitamin D insufficiency or deficiency [11,12,15,17]. There is a wealth of epidemiological data linking vitamin D deficiency to increased rates of a range of life-threatening diseases including digestive tract cancers and leukemias, as well as autoimmune and infectious diseases [18]. US rates of bladder, breast, colon, ovary, and rectal cancer increase two-fold from south to north [19], and vitamin D deficiency is associated with autoimmune conditions such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and type 1 diabetes [20e23].
Tuberculosis As documented above, there is a long history of links between vitamin D deficiency and elevated rates of TB through the therapeutic use of sun exposure or cod liver oil [5e7]. Associations between clinical vitamin D deficiency and TB susceptibility were made over 20 years ago [24,25], and evidence continues to accumulate (e.g., [26]). More importantly, we have known for over 20 years that hormonal 1,25(OH)2D3 inhibits the growth of M. tuberculosis in cultured human macrophages [27,28], thus providing evidence that 1,25(OH)2D3 directly enhances host innate immune responses to infection. As detailed below, we are now starting to establish the molecular genetic links between vitamin
D signaling and the induction of antimycobacterial innate immune responses. Interest in the connection between vitamin D supplementation and treatment of TB has been rekindled lately by several studies (e.g., [28e31]), not least observations that a single dose of 100 000 IU of vitamin D3 (2.5 mg) enhanced anti-mycobacterial immunity in healthy tuberculin skin test-positive donors in a double-blind randomized controlled trial [31]. A small trial in which subjects received 10 000 IU/d (0.25 mg) or placebo provided evidence for radiological improvement in the vitamin D group [32]. On the other hand, three bolus doses of 100 000 IU of vitamin D3 at onset, and after 5 and 8 months of a 12-month trial (~820 IU/d) had no effect on the clinical severity of patients with TB, suggesting that the equivalent of moderate daily doses may not be therapeutically effective [33].
Other Infectious Diseases Data are accumulating from several sources that maintaining vitamin D sufficiency may also be beneficial in combating a range of other infectious agents of bacterial or viral origin. One small but intriguing study worthy of follow-up found that elderly women undergoing longterm treatment with vitamin D as an anti-osteoporosis agent had a significantly lower rate of Helicobacter pylori infections than an untreated control group [34]. A recently completed clinical trial showed that supplementation with 2000 versus 800 IU reduced the risk of hospital readmission significantly by 39% in elderly patients recovering from fractures. Remarkably, there was a 90% reduction in readmissions due to infections [35]. There are also a number of studies examining the potential role of vitamin D in protection against upper and lower respiratory tract infections, which can be caused by a variety of etiological agents, many of them viral in origin [35e38]. Subclinical vitamin D deficiency was associated with severe lower respiratory tract infection in an Indian study [39], and clinical vitamin D deficiency was associated with a 13-fold increased risk of pneumonia in Ethiopian children [40]. A Finnish study found an association between serum 25(OH)D concentrations of less than 40 nM (16 ng/ml) and a range of acute respiratory infections (sinusitis, tonsillitis, otitis, bronchitis, pneumonia, pharyngitis, and laryngitis) in young army recruits [41]. There are also epidemiological data that cutaneous vitamin D production provides the “seasonal stimulus” associated with solar radiation that underlies the seasonality of epidemic influenza [42,43]. Indeed, a recent randomized double-blind placebocontrolled trial [44] conducted between December 2008 and March 2009 found that vitamin D supplementation of school children (1200 IU/d) significantly reduced rates
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VDR GENE POLYMORPHISMS AND INFECTIOUS DISEASES
of influenza A (relative risk (RR) 0.58; 95% CI 0.34, 0.99; p ¼ 0.04). The effect of supplementation was even more pronounced in children not previously receiving vitamin D supplements (RR 0.36; 95% CI 0.17, 0.79; p ¼ 0.006). However, 12 weeks of treatment with 2000 IU/d of vitamin D3 had no effect on frequency of severity of upper respiratory tract infections in adults [45]. Clinical and genetic evidence is also accumulating that vitamin D may play a role in modulating human immunodeficiency virus (HIV) infection. Vitamin D insufficiency or deficiency is widespread in urban European HIV patients (e.g., [46e48]). One study established positive correlation between vitamin D supplementation and CD4-positive T cell counts in seropositive individuals [49], whereas an analysis of 884 HIV-infected pregnant women in Tanzania did not reveal any association between vitamin D status and T cell counts [50]. However, the authors did conclude that “vitamin D status had a protective association with HIV disease progression, all-cause mortality, and development of anemia during follow-up in HIV-infected women,” and encouraged confirmation of the results in randomized trials [50]. Moreover, analysis of the same cohort showed that 347 of the women had a low vitamin D status and provided evidence that low maternal vitamin D levels were associated with enhanced risk of motherto-child transmission of HIV [51].
VDR GENE POLYMORPHISMS AND INFECTIOUS DISEASES The physiological actions of 1,25(OH)2D3 can be largely explained by its binding to and regulation of the vitamin D receptor (VDR; see Chapters 7e9), which is a member of the nuclear receptor family of ligand-regulated transcription factors. The hormonal bound VDR recognizes cognate DNA motifs called vitamin D response elements (VDREs) with high affinity. As detailed below, genes implicated in immune system function whose expression is regulated by 1,25(OH)2D3 were identified through the presence of VDREs in their regulatory regions. Given the connection between VDR function and disease, the VDR gene itself has been the subject of considerable analysis. Numerous genetic studies over the years have provided links between common polymorphisms in the VDR gene and susceptibilities to a number of diseases associated with (lack of) vitamin D signaling, including infectious diseases [52e54]. There are several VDR polymorphisms, including a common Fok1 restriction fragment length polymorphism (RFLP) that shifts translational initiation to an ATG 3 codons downstream, Taq1 and Bsm1 RFLPs in the 30 untranslated region, and an intronic Apa1 RFLP. VDR polymorphisms have been studied most
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intensively in connection with TB incidence and outcome. A recent meta-analysis of genetic studies linking VDR polymorphisms with TB surveyed 23 studies [54]. The authors concluded that the assembled studies reached heterogeneous results, which could be attributed at least in part to the heterogeneity of the populations studied. The strongest associations were observed between variants at the Fok1 and Bsm1 loci and TB susceptibility in Asian populations, whereas none of the common polymorphisms were associated with disease susceptibility in African or South American studies [54]. VDR polymorphisms have also been linked to leprosy, which is caused by a distinct mycobacterial agent Mycobacterium leprae [55]. Tuberculoid leprosy is characterized by few bacilli in macrophages and a strong cell-mediated response, whereas bacilli are far more numerous in the more severe lepromatous leprosy and the cellular response is much weaker. The Taq1-associated tt VDR polymorphism was associated with tuberculoid leprosy whereas the TT genotype was associated with lepromatous leprosy in Bengali patients [56]. The tt genotype was also associated with susceptibility to leprosy in a caseecontrol study of patients in the Karonga district of Malawi [57]. However, the expected frequency of tt homozygotes was low (5%) and apparent differences between patient and control populations could have been due to chance. Consistent with studies described above linking vitamin D deficiency to respiratory tract in infections, the ff genotype was associated with an adjusted relative odds of acute lower respiratory tract infection (predominantly viral bronchiolitis) in young children that was seven times that of the FF genotype [58]. VDR polymorphisms have been linked to HIV infection, although clear conclusions regarding the role of vitamin D signaling in controlling HIV infection have been difficult to draw. No associations were found between Bsm1 polymorphisms and HIV infection, whereas an association was established between the BB genotype and disease progression based on several criteria [59]. However, it is difficult to ascribe variations in the Bsm1 genotype to changes in VDR function. Another study found no association between a specific polymorphism and protection against HIV infection in a population of injection drug users, but did find a correlation between specific VDR haplotypes (blocks of polymorphisms) [60]. The authors concluded that protective VDR polymorphisms were associated with reduced VDR function, consistent with vitamin D signaling promoting HIV infection, and noted in an in vitro study that the 1,25D-bound VDR could activate the HIV1 long terminal repeat [61]. Given the recent studies linking vitamin D sufficiency to reduced HIV progression and mother-to-child transmission [50,51] further work regarding links between VDR polymorphisms and HIV appears warranted.
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MOLECULAR EVENTS UNDERLYING INNATE IMMUNE REGULATION BY VITAMIN D Vitamin D Signaling in the Immune System The VDR is widely expressed in the immune system, including in T lymphocytes, neutrophils, and antigen presenting cells such as macrophages and dendritic cells [62e65]. In addition, CYP27B1, the enzyme that converts circulating the major circulating precursor 25(OH)D3 to hormonal 1,25(OH)2D3, is expressed in cells such as macrophages and dendritic cells [66e69]. Moreover, unlike the renal enzyme, CYP27B1 expression in the immune system is not regulated by Caþþ homeostatic signals, but primarily by immune inputs, rendering the immune system responsive to circulating levels of 25(OH)D3 upon appropriate stimulation. In the last few years, researchers in the vitamin D field, and particularly those interested in its immunomodulatory functions, have come to appreciate the important contributions of extra-renal 1a-hydroxylase (CYP27B1) to vitamin D physiology (see Chapter 45). Microarray-based mRNA expression profiling of cultured human macrophages revealed that signaling through human macrophage TLR1/2 toll-like receptor heterodimers stimulated with bacterial lipopeptides induced expression of both CYP27B1 and the VDR [29] (Fig. 91.1). TLRs are so-called pattern recognition receptors, which are essential for innate immune responses as they are stimulated by a variety of molecular motifs characteristic of invading pathogens. In TLR2/1stimulated human macrophages cultured in the presence of human serum, downstream VDR-driven transcriptional responses were strongly dependent on
serum 25(OH)D3 concentrations. VDR-driven responses were strongly attenuated in serum from vitamin-Ddeficient individuals; a defect that could be overcome by 25(OH)D3 supplementation. Consistent with previous findings [16,17], 25(OH)D levels serum from AfricanAmericans used in the study were markedly lower than those of Caucasian Americans [29]. These results provided a clear demonstration of the dependence of the magnitude of immune responses on circulating 25(OH)D levels, thus providing a molecular basis for the links between vitamin D deficiency and elevated rates of infectious diseases. Similarly, stimulation of the TLR4/ CD14 receptor complexes by bacterial lipopolysaccharide (LPS) induced CYP27B1 expression [69,70], consistent with correlations others have seen between TLR4 and CYP27B1 expression [71,72]. We have known since the early 1990s that expression of the coreceptor of TLR4, CD14, is strongly induced by 1,25(OH)2D3 in human cells [73], providing early evidence that 1,25(OH)2D3 signaling regulated innate immune function (Fig. 91.1). Regulation of CD14 expression is conserved in the mouse; for example, induction of CD14 by 25(OH)D3 was abrogated in mice lacking CYP27B1 [74]. The study also showed that vitamin D signaling enhanced the expression of TLR2 approximately two-fold in human keratinocytes. Given that signaling through either TLR2 or TLR4 enhances vitamin D signaling by upregulating expression of the VDR and CYP27B1, the effects of 1,25D on TLR2 and CD14 expression in keratinocytes constitutes a positive feedback loop. However, recent findings suggest that such a loop is cell-specific. Treatment of human monocytes with 1,25D suppressed expression of both TLR2 and TLR4 mRNA and protein in a time- and dosedependent manner [75]. Signaling through TLR2 was Schematic representation of the signaling pathways discussed in the text. The stimulation of CYP27B1 and VDR gene expression via TLR signaling, resulting in enhanced intracellular production of 1,25(OH)2D3 from circulating 25(OH)D3 is shown. This leads to activation of the VDR and transactivation of target gene transcription by VDR/RXR heterodimers bound to VDREs. Regulation of VDR target genes presented is discussed in the text. These include CAMP, whose product colocalizes with mycobacteria in phagolysosomes. Stimulation of NOD2/CARD15 expression leads to stimulation of activity of the transcription factor NF-kB upon binding of NOD2 agonist muramyl dipeptide (MDP). MDP is a product of lysosomal breakdown of bacterial peptidoglycan of pathogens such as M. tuberculosis (Mtb). DNA-bound NF-kB and the VDR cooperate to induce transcription of the gene encoding antimicrobial peptide DEFB2/ HBD2. See text for details and appropriate references.
FIGURE 91.1
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MOLECULAR EVENTS UNDERLYING INNATE IMMUNE REGULATION BY VITAMIN D
suppressed in 1,25(OH)2D3-treated monocytes, as was signaling through TLR4 in the presence of LPS, even though CD14 expression was induced by 1,25(OH)2D3. It was speculated that downregulation of pattern recognition receptors by 1,25(OH)2D3 in antigen-presenting cells may contribute to its capacity to attenuate excessive T helper Th1-driven inflammatory responses and potential downstream autoimmunity [75].
Direct and Indirect Induction by the 1,25(OH)2D3-bound VDR of Antimicrobial Innate Immunity Given that 1,25(OH)2D3 binds to and regulates the function of a transcription factor, its signaling is ideally suited to analysis using genomic approaches. 1,25(OH)2D3regulated gene expression has been studied using a combination of microarrays and in silico screens for VDREs, leading to the identification of several hundred target genes. Among other findings, these techniques have provided numerous insights into regulation of immune system function by 1,25(OH)2D3 [76e78]. For example, CD14 expression was induced 27-fold by 1,25(OH)2D3 in well-differentiated squamous epithelial cell line [77], and in silico analysis identified an upstream VDRE in the human CD14 gene [78]. In silico screening for VDREs revealed consensus response elements in two genes encoding antimicrobial peptides CAMP (cathelicidin antimicrobial peptide, hCAP18, LL37) and human b-defensin 2 (DEFB2, DEFB4, HBD2) contained promoter-proximal consensus DR3-type response elements [79] (Fig. 91.1). AMPs are vanguards of innate immune responses against bacterial, fungal, and viral attack, and many act directly by disrupting the integrity of pathogen membranes [4,80e82]. Further analysis of the CAMP and DEFB2 VDREs showed that both elements bound the VDR in a 1,25(OH)2D3-dependent manner in vitro and in cells in culture, and functioned in reporter gene assays [96]. DEFB2 expression was modestly induced by 1,25(OH)2D3 in cells of epithelial origin. In contrast, CAMP expression was strongly stimulated in all cell types examined (epithelial cells, macrophages/monocytes, and neutrophils). The strong induction of CAMP by 1,25D was subsequently observed by others in a range of cell types [29,83], including in 1,25(OH)2D3-treated or UVB-irradiated human skin biopsies [84], clearly indicating that 1,25(OH)2D3 is a primary inducer of the gene. Recently, microarray studies have also revealed that 1,25(OH)2D3 induces expression of the gene encoding the pattern recognition receptor NOD2/CARD15 (nucleotide oligomerization domain protein 2/caspase recruitment domain-containing protein 15) [70]. NOD2 is an intracellular protein that is a member of a family of pattern recognition receptors distinct from the TLRs.
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It recognizes muramyl dipeptide (MDP), a lysosomal breakdown product of bacterial peptidoglycan. Regulation of NOD2 expression by 1,25(OH)2D3 was conserved in human cells of both epithelial and myeloid origin and occurred through binding of the VDR to distal VDREs in the gene. Regulation of NOD2 by 1,25(OH)2D3 is noteworthy for several reasons. Signaling through NOD2 induces the function of the NF-kB transcription factor and induction of DEFB2/HBD2 expression [85] (summarized in Fig. 91.1), and pretreatment with 1,25(OH)2D3 to induce NOD2, followed by incubation with MDP led to synergistic induction of DEFB2/HBD2 [70]. The observed synergism is also consistent with other studies showing combined effects of 1,25(OH)2D3 and interleukin 1b on DEFB2/HBD2 expression, interleukin 1b also induces NF-kB activity [79,86]. The induction of NOD2 expression by 1,25(OH)2D3 is of considerable clinical significance as attenuated expression of NOD2 or DEFB2/HBD2 is associated with the pathogenesis of Crohn’s disease (CD), a chronic incurable inflammatory condition [87,88]. Northesouth gradients in rates of CD have been described in Europe and North America [89e91], although data concerning seasonal variations in CD relapse rates are conflicting [92e94]. In addition, VDR gene polymorphisms correlate with susceptibility to CD [95]. Genetic studies have identified several CD-susceptibility loci and have provided abundant evidence that CD arises from defects in intestinal innate immunity [87]. The regulation of NOD2 expression by 1,25(OH)2D3 thus provides further evidence that vitamin D sufficiency is essential for optimal innate immune responses in humans. Note that CD is widely thought of as an autoimmune condition. However, evidence for autoimmunity is lacking and recent publications have argued that defects in innate immune responses are sufficient to explain the pathogenesis of the disease [96,97]. The induction of NOD2 and AMP expression by 1,25(OH)2D3 also consolidates the molecular basis for the associations described above between vitamin D sufficiency and reduced incidences of infectious diseases. For example, NOD2 is particularly sensitive to the N-glycolyl form of MDP produced by mycobacteria, providing a further clue to associations between vitamin D signaling and reduced rates of TB [98]. 1,25(OH)2D3-induced CAMP colocalized with mycobacteria in infected macrophages [29], and subsequently knockdown of CAMP expression in TB-infected human THP-1 macrophage-like cells, confirmed that its induction is essential for 1,25(OH)2D3stimulated anti-mycobacterial activity [28]. 1,25(OH)2D3-induced CAMP expression has also been linked to enhancement of autophagy in M. tuberculosisinfected macrophages [99], although it is not clear whether the stimulation was a direct effect of CAMP on autophagy itself or an indirect effect of reduced mycobacterial viability due to AMP activity. Autophagy is
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essential for capture and destruction of immature phagosomes that harbor M. tuberculosis in infected cells. In this regard, it is intriguing that many of the loci, including NOD2, associated with susceptibility to CD encode proteins implicated in autophagy [87,100], indicating that robust autophagic responses to phagocytosed pathogens are essential components of innate immunity. In addition, DEFB2/HBD2 expression was induced in response to H. pylori infection in the gastric mucosa [101], and rhinovirus infection in airway epithelia [102]. Finally, while the relationship between vitamin D signaling in controlling HIV infection remains to be clarified, it is noteworthy that human cathelicidin inhibited the replication of a number of HIV isolates [103], and that the human and porcine homologs reduced the infectivity of lentiviral vectors [104], suggesting that vitamin D signaling may indeed induce antiretroviral activity.
Regulation of AMP Expression by 1,25(OH)2D3 is Species Specific There is considerable interspecies variation in both gene sequence and number, and in tissue distribution and regulation of expression of AMPs. Cathelicidins are present in a variety of species, and their name reflects the dual properties of members of the family. Mice and humans have single cathelicidin genes, whereas there are multiple cathelicidin peptides in bovine species [105]. Cathelicidins are derived in part from the conserved N-terminal region known as the cathelin domain, which has the capacity to inhibit cathepsin-L cysteine protease activity. The suffix reflects the AMP activity of family members. Cathelicidin precursor proteins are proteolytically processed to release C-terminal peptides, whose sequences are poorly conserved among species [4]. The human gene encodes
a precursor called hCAP18, which is cleaved to release LL37, a cationic 37 a.a. AMP bearing tandem N-terminal leucine residues (Fig. 91.2). a, b and q defensins contain six disulfide bond-forming cysteines [106], with subclasses distinguished by different spacings of Cys residues. While b-defensins are widespread in vertebrates, a-defensins are mammalian, and q-defensins are primate specific. The five human a-defensins are expressed in myeloid or enteric tissues, whereas the 19 murine genes (cryptdins) are enteric only. Apart from variations in gene number and tissue distribution of expression, there are also differences in gene regulation between species. Notably, neither of the VDREs in the CAMP or DEFB2 genes are conserved in mice, and Gombart et al. [83] noted that the CAMP VDRE is imbedded in an Alu repeat, which is a humanor primate-specific transposable element. The VDREcontaining Alu repeat in the CAMP gene originated in the primate lineage leading to humans and apes, as well as Old World and New World monkeys [107], dating the insertion event back at least 55e60 million years. This conservation strongly suggests that the insertion led to a selective advantage and was therefore of considerable functional significance in innate immune responses in these species. In other words, the conserved element pointed to an enhanced role for vitamin D signaling in regulating innate immunity in humans/ primates. The insertion also represented a rare event. Alu repeats represent 10% of the human genome and are a rich source of consensus DR2-type (PyG(G/T) TCAnnPyG(G/T)TCA) hormone response elements recognized by related retinoic acid receptors [108]. However, Alu repeats containing DR3 motifs recognized by the VDR, which would require a precise single nucleotide insertion sequence in the DR2 element, are far less common [108]. FIGURE 91.2 Structure and functions of antimicrobial peptide CAMP/LL37. A schematic representation of the hCAP18 precursor protein is shown on top, with the N-terminal signal peptide, the conserved cathelin domain and the poorly conserved CAMP/LL37 cleavage product possessing AMP activity. The hCAP18 precursor is cleaved by proteinase-3 to release CAMP/LL37 [127]. The sequence of the cleavage product is presented below. The N-terminal tandem leucines are underlined and the basic amino acids are in bold. The functions of CAMP/ LL37 discussed in the text are presented at the bottom of the figure. See text for appropriate references.
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PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL RAMIFICATIONS OF 1,25(OH)2D3-REGULATED CAMP EXPRESSION
PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL RAMIFICATIONS OF 1,25(OH)2D3REGULATED CAMP EXPRESSION Initial work [79,83] showed that 1,25(OH)2D3 induced CAMP expression strongly and in a wide variety of cell human types in vitro, suggesting that the regulation is widespread in vivo. This has since been borne out in studies performed in several tissues. CAMP expression in skin is of broad (patho)physiological significance. It has been speculated that regulation of CAMP transcription by 1,25(OH)2D3 was retained in primates and humans because of the importance of cutaneous UVBinduced vitamin D synthesis in these organisms [109]. CAMP is strongly induced in human keratinocytes by UV light [84], and under conditions of epithelial wound healing [110e112], providing a molecular basis for the stimulatory role of vitamin D in the process. LL37 produced during wound healing induces cleavage and release of membrane-bound heparin-binding-EGF (HBEGF), which in turn stimulates the EGFR to enhance keratinocyte migration underlying re-epithelialization of the wound [113]. Notably, CYP27B1 expression is also induced in keratinocytes during wound healing, leading to induction of CAMP by locally produced 1,25(OH)2D3 [74]. While the above would be beneficial, the expression of cathelicidin in skin has emerged as a double-edged sword. AMP expression is decreased in atopic dermatitis (AD), leading to increased rates of infection of affected skin [114]. However, elevated 1,25(OH)2D3 signaling in AD would not be beneficial because it would skew the T helper response towards a Th2 phenotype, due in part to the induction of thymic stromal lymphopoietin, which is a 1,25(OH)2D3 target gene in human and mouse [78,115]. In contrast, the persistent inflammatory skin disorder rosacea is characterized by elevated expression of CAMP, which is abnormally processed and contributes to inflammation [116]. 1,25(OH)2D3-induced CAMP expression may contribute to aggravation of rosacea caused by exposure to UV light. It has been noted that azole antimycotics (ketoconazole, itraconazole, metronidazole), used in dermatology as antifungal agents in treatment of inflammatory conditions such as rosacea, also block cytochrome P450-driven vitamin D metabolism [116]. Intriguingly, expression of cathelicidin is also elevated in psoriasis, another inflammatory skin condition [114], where vitamin D analogs are therapeutically effective. 1,25(OH)2D3-dependent CAMP expression is enhanced by proinflammatory cytokine IL-17A produced by psoriatic lesional Tcells [66]. Moreover, CAMP triggers an autoimmune response in psoriasis by activating TLR signaling in plasmatoid dendritic cells (pDCs) of the skin, which are
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specialized for sensing viral and certain microbial infections. CAMP binds directly to DNA in pDCs of psoriatic skin forming condensed aggregates that are delivered to TLR9 receptors, triggering an immune response [118]. The DNA-binding capacity of CAMP arises from its cationic, amphipathic structure (Fig. 91.2). It is likely that anti-psoriatic activity of vitamin D analogs results from their anti-proliferative activities and their capacity to suppress Th1-driven immune responses. Both the VDR and CAMP were highly expressed in biliary epithelial cells in human liver [119]. Unlike the lumen of the intestines, the biliary tract is normally free of microbes, consistent with robust antimicrobial defenses. Remarkably, CAMP expression in biliary epithelial cells is regulated by physiological and therapeutic bile salts via signaling through the VDR and the related farnesoid X receptor (FXR) [119]. FXR is a physiological bile acid receptor. However, there is also strong evidence that the VDR can function as a bile acid sensor [120], and the secondary bile acid lithocholic acid was found to be an efficacious inducer of CAMP mRNA and protein in normal human keratinocytes [121]. Notably, CAMP expression induced by bile acids could be further enhanced by 1,25(OH)2D3, and therapeutic bile acid treatment of inflammatory biliary disease enhanced expression of both the VDR and CAMP [119]. Similarly, antimicrobial defenses are essential for maintaining the integrity of the epithelial lining of the lungs. We found that 1,25(OH)2D3 strongly induced CAMP expression in human Calu-3 lung carcinoma cells, a line that retains characteristics of upper airway epithelial cells [79]. Calu-3 cells treated with 1,25(OH)2D3 released antimicrobial activity against Pseudomonas aeruginosa, a pathogen responsible for chronic infections in patients with cystic fibrosis (CF). More recent work showed that 1,25(OH)2D3 induced CAMP transcription 10-fold in normal human bronchial epithelial (NHBE) cells [122]. This regulation was conserved in bronchial epithelial cells derived from CF patients homozygous for an inactivating mutation of the CFTR gene, encoding the cystic fibrosis transmembrane conductance regulator, an epithelial chloride channel. Moreover, NHBE cells could be induced to release antimicrobial activity against P. aeruginosa that could be partially blocked by preincubation with an anti-LL37 antibody [122]. The endometrium and placenta during pregnancy are also active sites of vitamin D metabolism and signaling. Placental CYP27B1 is produced early in gestation, and Hewison and colleagues showed that 25(OH)D is converted to 1,25(OH)2D3 in cultured maternal decidual cells from first-trimester human pregnancies, and drives induction of CAMP expression [123]. CAMP is also robustly induced from 1,25(OH)2D3 produced in situ in placental trophoblast cells and promotes antibacterial responses [124]. CYP27B1 expression in trophoblasts in
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this study appeared to be independent of TLR signaling pathways. Collectively, these results show that CAMP expression can be induced in both maternal and fetal cells during gestation by intracrine vitamin D signaling. There is some evidence that trophoblasts can be phagocytic, and Liu et al. [125] hypothesized that intracrine production of CAMP may lead to its delivery to phagocytic vesicles, similar to what is observed in macrophages [29].
CONCLUSIONS AND FUTURE DIRECTIONS The past few years have provided a wealth of data to support a fundamental role for vitamin D signaling in regulating innate immunity in humans and a firm molecular basis for the numerous studies linking vitamin D deficiency with increased rates of infectious diseases. We now know that stimulation of naı¨ve macrophages through TLRs strongly enhances vitamin D signaling through induction of CYP27B1 and VDR expression [29], which leads to production of hormonal 1,25(OH)2D3, and that the magnitude of this production is strongly dependent on ambient 25(OH)D3 concentrations. 1,25(OH)2D3 thus produced signals via the VDR to induce expression of key components of innate immune responses such as AMPs and the pattern recognition receptor NOD2 [70,79,83]. Follow-up studies have revealed that regulation of AMP expression by 1,25(OH)2D3 is widespread in vivo. However, as 1,25(OH)2D3 signaling may regulate as much as 1% of the genome there is much work to be done to fully understand the scope of its actions in innate immune regulation. For example, autophagy has emerged as a key component of mechanisms of pathogen containment and killing [87,100,126], and recent work has provided initial evidence that 1,25(OH)2D3 plays a key role in enhancing the process in part through induction of CAMP and NOD2 expression [70,99,100]. There is likely more to uncover in this area. In addition, the recognition of the importance of vitamin D in controlling innate immune function should spur renewed interest in developing vitamin D analogs that may find roles, if not as front-line therapies for infectious disease, as components of combined therapies to treat emerging antibiotic-resistant disease. Similarly, analogs that mimic many of the actions of 1,25(OH)2D3 but are poor or weak inducers of CAMP expression may find a role in treatment of psoriasis.
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C H A P T E R
92 Control of Adaptive Immunity by Vitamin D Receptor Agonists Luciano Adorini Intercept Pharmaceuticals, Corciano (Perugia), Italy
INTRODUCTION The raison d’ eˆtre of the immune system is to control the biological integrity of the individual. This is accomplished by two layers of immune responses, innate and adaptive, which are tightly interconnected [1]. Innate immune responses can be induced in virtually any cell, but they are primarily mediated by specialized cell types, such as dendritic cells (DCs), macrophages, neutrophils, and natural killer cells. Innate immunity is characterized by rapid, local responses, largely based on the production of pro-inflammatory mediators, in particular cytokines, chemokines, and reactive oxygen species. Production of these mediators is triggered by recognition of stereotyped patterns conserved in infectious microorganisms via toll-like receptors (TLRs), surface molecules able to recognize distinct structural components of pathogens [2]. Thus, the innate immune system is genetically programmed to detect invariant features of invading microbes. Activation of signal transduction pathways by TLRs leads to upregulation of different genes that operate in host defense, including co-stimulatory molecules, cytokines and chemokines [3]. Adaptive immune responses are primarily induced by cells specialized in antigen processing and presentation, in particular DCs, and are mediated by cells specialized in antigen recognition, namely T and B lymphocytes. Lymphocytes employ antigen receptors that are not encoded in the germ line but are generated de novo in each organism, rendering adaptive immune responses highly specific. Adaptive immune responses are primarily orchestrated by CD4þ T lymphocytes. Triggered by microbial pathogens, naı¨ve CD4þ T cells orchestrate immune responses by differentiating into T helper (Th) cell populations that secrete distinct sets of cytokines [4]. Tailoring their responses to the character
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10092-7
of the threat encountered, Th cells differentiate to provide help to B lymphocytes and CD8þ cytotoxic T cells, and to activate cells of the innate immune system. CD4þ T cells can be distinguished, based on their pattern of cytokine production, into three major effector cell types, Th1, Th2, and Th17 cells. Th1 cells are characterized by secretion of interferon-g (IFN-g), IL-2, and TNF-b, and they promote cell-mediated immunity able to eliminate intracellular pathogens. Th2 cells selectively produce IL-4, IL-5, and IL-13, and are involved in the development of humoral immunity protecting against parasites. Th17 cells, characterized by production of IL-17, IL-21, and IL-22, are involved in host defense against extracellular pathogens. All these effector T cell types can also mediate pathogenic immune responses, such as autoimmunity (Th1 and Th17) and allergy (Th2). Peripheral naı¨ve CD4þ T precursor cells can differentiate not only into the three main subsets of effector T cells but also into several subsets of regulatory T cells (Treg), including induced Treg cells (iTreg), Tr1 cells, and Th3 cells. Naturally occurring Treg cells (nTreg) are generated from CD4þ thymic T cell precursors. The differentiation of these subsets is governed by selective cytokines and transcription factors, but their differentiation into lineages with distinct effector functions is not necessarily an irreversible event and a great degree of flexibility in their differentiation options exists, with an exquisite plasticity characterizing iTreg and Th17 cells [5,6].
VITAMIN D RECEPTOR AGONISTS AS IMMUNOREGULATORY AGENTS 1,25(OH)2D3, the activated form of vitamin D, is a secosteroid hormone that regulates, in addition to
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calcium and bone metabolism, growth and differentiation of many cell types, and displays pronounced immunoregulatory properties [7e11]. The biological effects of 1,25(OH)2D3 are mediated by the vitamin D receptor (VDR), a member of the superfamily of nuclear hormone receptors [12]. Ligand binding induces conformational changes in the VDR, which promote heterodimerization with the retinoid X receptor (RXR) and recruitment of a number of corepressor and coactivator proteins, including steroid receptor coactivator family members and a multimember coactivator complex, D receptor interacting proteins (DRIP). These coactivators induce chromatin remodeling through intrinsic histone-modifying activities and direct recruitment of key transcription initiation components at regulated promoters. Thus, the VDR functions as a ligandactivated transcription factor that binds to specific DNA sequence elements (vitamin D responsive elements, VDRE) in vitamin D responsive genes and ultimately influences the rate of RNA polymerase IImediated transcription [13]. The discovery of VDR expression in most cell types of the immune system [14], in particular in APCs such as macrophages [14] and DCs [15], as well as in both CD4þ and CD8þ T lymphocytes (reviewed in [16]), prompted the investigation of VDR ligands as agents able to modulate T cell responses [17]. Data accumulated in the last few years clearly demonstrate that the vitamin D endocrine system is involved in a variety of biological processes able to modulate immune responses, and plays an important role in the control of autoimmune diseases [10,18e20]. In addition to exerting direct modulatory effects on T and B cell function, VDR agonists shape phenotype and function of dendritic cells (DCs), promoting tolerogenic properties that favor the induction of regulatory rather than effector T cells [21]. These intriguing actions of VDR agonists have been demonstrated in several experimental models and could be exploited, in principle, to treat a variety of autoimmune diseases and other immuno-mediated pathologies characterized by chronic inflammatory responses. Equally important, accumulating data document the capacity of 1,25(OH)2D3, which is produced by macrophages [22,23], DCs [24,25], T [26], and B [27] cells, to physiologically contribute, via the VDR expressed in all these cell types, to regulate via autocrine and paracrine effects both innate and adaptive immune responses. The tight control of bioactive hormone production by cells of the immune system itself further supports the relevance of the vitamin D endocrine system in the modulation of immune responses in health and disease. This appealing concept, although still speculative, is mostly based on epidemiological data and is indirectly supported by the observation that VDR-deficient, compared to
wild-type mice, show hypertrophy of subcutaneous lymph nodes with an increase in mature DCs [28].
DENDRITIC CELLS AS TARGETS FOR IMMUNOREGULATION BY VDR AGONISTS DCs, a highly specialized antigen-presenting cell system critical for the initiation of CD4þ Tcell responses, are present, in different stages of maturation, in the circulation as well as in lymphoid and non-lymphoid organs. After antigen uptake, DCs migrate through the afferent lymph to T-dependent areas of secondary lymphoid organs where they can prime naı¨ve Tcells. During migration to lymphoid organs, DCs mature into potent APCs by increasing their immunostimulatory properties while decreasing antigen-capturing capacity [29]. DCs are heterogeneous in terms not only of maturation state, but also of origin, morphology, phenotype, and function [30]. Two distinct DC subpopulations were originally defined in human blood based on the expression of CD11c, and they have been subsequently characterized as belonging to the myeloid or lymphoid lineage, and defined as myeloid (M-DCs) and plasmacytoid (P-DCs) DCs [31,32]. M-DCs are characterized by a monocytic morphology; express myeloid markers like CD13 and CD33, the b2 integrin CD11c, the activatory receptor ILT1 and low levels of the IL-3 receptor a chain CD123. Conversely, P-DCs have a morphology resembling plasma cells, are devoid of myeloid markers, and express high levels of CD4, CD62L, and CD123. M-DCs produce high levels of IL-12, while PDCs secrete high levels of IFN-a [33], cytokines with clearly distinct effects on T cell activation and differentiation. DCs can induce or tolerize T cells, and tolerogenic DCs can promote the development of Treg cells [34]. Treg cells fail to proliferate and to secrete cytokines in response to polyclonal or antigen-specific stimulation, and are not only anergic but also inhibit the activation of responsive T cells. Although CD25, CD152, and glucocorticoid-induced TNF-related protein (GITR) are markers of CD4þCD25þ Treg cells, they are also expressed by activated T cells [35]. A more accurate marker distinguishing CD4þCD25þ Treg cells from recently activated CD4þ T cells is Foxp3, a member of the forkhead family of transcription factors required for CD25þ Treg development and sufficient for their suppressive function [36]. Foxp3þ CD4þCD25þ Treg cells play an important role in controlling immune responses in diverse experimental and clinical settings [37]. The clinical relevance of CD4þCD25þ Treg cells has also been shown in patients affected by type 1 diabetes, rheumatoid arthritis, and multiple sclerosis,
XI. IMMUNITY, INFLAMMATION, AND DISEASE
DENDRITIC CELLS AS TARGETS FOR IMMUNOREGULATION BY VDR AGONISTS
offering interesting potential opportunities for immunointervention [38]. DCs modulate T cell development and they can be not only immunogenic but also tolerogenic, both intrathymically and in the periphery [34]. In particular, immature DCs have been found to have tolerogenic properties, and to induce Treg cells. However, the simplistic concept that immature DCs are intrinsically and uniquely able to induce Treg cells has been dispelled by their very efficient induction by mature DCs [39], a property already noted for semi-mature DCs [40]. Tolerogenic DCs are often characterized by reduced expression of costimulatory molecules, in particular CD40, CD80, CD86, although this is not an absolute requirement [41]. The local microenvironment plays an important role in the induction of tolerogenic DCs [42], together with several other mechanisms such as indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme of tryptophan catabolism [43], and immunoglobulin-like transcripts (ILTs) [44]. Cytokines are key factors shaping DC tolerogenicity, as shown by the phenotypic and functional properties expressed by DCs differentiated in the presence of IL-10, TGF-b, TNF-a, or G-CSF [34]. More recently, additional cytokines have been shown to promote DC tolerogenicity, including vasoactive intestinal peptide [45], hepatocyte growth factor [46], IL-21 [47], and thymic stromal lymphopoietin (TSLP) [48]. Treg cells themselves induce and maintain DCs into a tolerogenic state [49]. Treg cells can inhibit myeloid DC maturation, reduce their antigen-presenting function, and decrease IL-12 secretion [50,51]. In addition, Treg cells can induce IL-10 production by DCs, which is maintained following LPS stimulation, suggesting the induction of stable tolerogenic properties [51]. Treg cells not only trigger high levels of IL-10 production by DCs, but induce expression of B7-H molecules with suppressive effects on T cell activation [49]. Treg cells also induce and sustain DC tolerogenicity in vivo, as shown in rheumatoid arthritis patients, highlighting the functional relevance of the cross-talk between Treg cells and DCs [52]. Manipulation of DC function to favor the induction of DCs with tolerogenic properties leading to the development of Treg cells could be exploited to modulate immune responses [29]. Among agents able to promote induction of tolerogenic DCs, VDR agonists have attracted considerable attention, also due to their potential in clinical translation. DCs are key targets for the immunomodulatory effects of VDR agonists, which shape DC phenotype and function enhancing their tolerogenicity in adaptive immune responses. As detailed below, tolerogenic DCs induced by a short treatment with VDR agonists promote CD4þCD25þFoxp3þ Treg cells that are able to mediate transplantation tolerance and to arrest the development of autoimmune diseases.
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VDR agonists not only favor induction of CD4þCD25þ Treg cells, but can also enhance their recruitment at inflammatory sites. The tolerogenic properties induced by VDR agonists in DCs, leading to enhanced Treg cell development, likely contribute to the beneficial activity of these hormone-like molecules in autoimmune disease and graft rejection models, highlighting their applicability to the treatment of chronic inflammatory conditions sustained by autoreactive or alloreactive immune responses.
Common Features of Pharmacological Agents Promoting Induction of Tolerogenic DCs The mechanism of action of major immunosuppressive drugs, like the calcineurin inhibitors cyclosporine A and tacrolimus, has been understood only after almost 20 years of clinical use [53]. Thus, it is perhaps not surprising that a novel mechanism of action shared by many immunosuppressive and anti-inflammatory drugs, based on the induction of DCs with tolerogenic properties, has only recently emerged [54e58]. Immunosuppressive and anti-inflammatory drugs can directly target both DCs and T cells, leading to the inhibition of pathogenic effector T cells and enhancing the frequency of T cells with suppressive properties, effects that appear to be largely mediated via induction of tolerogenic DCs. Notable examples of anti-inflammatory and immunosuppressive agents shown to induce DCs with tolerogenic phenotype and function are glucocorticoids [59], mycophenolate mofetil (MMF) [60], and sirolimus [58]. These agents impair DC maturation and inhibit upregulation of costimulatory molecules, secretion of pro-inflammatory cytokines, in particular IL-12, and allostimulatory capacity. Sirolimus appears to be quite tolerogenic, because it induces tolerogenic DCs, and sirolimus-treated alloantigen-pulsed DCs infused 1 week before transplantation inhibit antigen-specific T cell responsiveness and prolong graft survival [58]. Conversely, controversial effects of calcineurin inhibitors, like cyclosporine A and tacrolimus, have been reported on DC maturation, although these drugs have a clear inhibitory effect on DC, decreasing their cytokine production and allostimulatory capacity [61]. Other immunosuppressive agents, like desoxyspergualin, also inhibit the allostimulatory capacity of DCs, impairing their maturation and IL-12 production as well [62]. Similar effects are exerted on DCs by anti-inflammatory agents, such as acetylsalicylic acid [63], butyric acid [64], niflumic acid [65], and N-acetyl-L-cysteine [66]. Interestingly, aspirin-modified DCs, expressing a tolerogenic phenotype and high levels of ILT3, are potent inducers of allo-specific Treg cells [63]. The pro-tolerogenic effects of several pharmacological agents on DCs have been explored for their capacity
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to induce Treg cells promoting transplantation tolerance. MMF monotherapy can induce limited levels of transplantation tolerance even if no induction of tolerogenic DCs was observed in vivo [67]. Conversely, calcineurin inhibitors have been reported to prevent transplantation tolerance induced by costimulation blockers, although the issue is still unresolved [68], but successful establishment of alloantigen-specific hyporesponsiveness by NF-kB inhibitor-treated DCs was not inhibited by concomitant calcineurin inhibition [69]. In addition, the sirolimus derivative everolimus did not hamper in vitro the suppressive activity of CD4þCD25þ Treg cells, suggesting that these cells may still exert suppressive activity in transplant recipients treated with drugs interfering with IL-2 signaling [70]. Finally, as discussed in detail below, VDR agonists promote DC tolerogenicity. These tolerogenic DCs show decreased capacity to stimulate alloreactive T cells, and enhance the differentiation of CD4þCD25þ Treg cells. Common features shared by biological and pharmacological agents favoring the induction of tolerogenic DCs are their capacity to inhibit differentiation, maturation, costimulatory molecule expression, and IL-12 production, leading to decreased allostimulatory capacity [57,58]. Costimulatory molecule expression is almost invariably reduced in tolerogenic DCs induced by anti-inflammatory and immunosuppressive drugs. In addition, all the tolerogenic agents tested inhibit DC maturation and reduce their capacity to stimulate alloreactive T cells in a mixed leukocyte reaction assay [57,58]. Another common feature of DC-targeting drugs is the inhibition of IL-12, a cytokine critically involved in the development of Th1-dependent diseases [71]. In contrast, only vitamin D receptor (VDR) agonists, among the agents tested, are able to enhance the secretion by DCs of IL-10, a cytokine favoring the induction of regulatory T cells [57]. Several of these effects could be mediated by NF-kB, a signal transduction pathway crucially involved in the inflammatory response [72]. The NF-kB family member RelB is required for myeloid DC differentiation, and antigen-pulsed DCs in which RelB function is inhibited can induce regulatory CD4þ T cells able to transfer tolerance to primed recipients in an IL-10dependent fashion [73]. Interestingly, both dexamethasone [74] and VDR agonists [75] upregulate the transcription of Nfkbia, which results in increased rate of IkBa synthesis and in reduced NF-kB translocation to the nucleus. The promoter of the Nfkbia gene encoding IkBa contains, as the Relb gene [76], several vitamin D responsive elements, suggesting a direct transcriptional regulation of IkBa by VDR agonists. The direct targeting of NF-kB components by VDR agonists exemplified by arrest of NF-kBp65 nuclear translocation in M-DCs [77] contributes to explain
their capacity to induce tolerogenic DCs, as well as their inhibitory effects on pro-inflammatory cytokine and chemokine production by DCs. Thus, VDR agonists share with several immunomodulatory agents the capacity to target DCs, rendering them tolerogenic and fostering the induction of regulatory rather than effector T cells. Multiple mechanisms contribute to induction of DC tolerogenicity by VDR agonists, from downregulation of costimulatory molecules, both membrane-bound as CD40, CD80, CD86, and secreted as IL-12, to upregulation of inhibitory molecules like ILT3 and IL-10, to modulation of chemokine secretion, enhancing the production of chemokines able to recruit Treg cells, and inhibiting production of chemokines recruiting pathogenic cells by the target organ in inflammatory conditions.
INDUCTION OF TOLEROGENIC DENDRITIC CELLS BY VDR AGONISTS APCs, and notably DCs, express the VDR and are key targets of VDR agonists, both in vitro and in vivo. A number of studies have clearly demonstrated that 1,25(OH)2D3 and its analogs markedly modulate DC phenotype and function [21]. These studies have consistently shown that in vitro treatment of DCs with VDR agonists leads to downregulated expression of the costimulatory molecules CD40, CD80, CD86, and to decreased IL-12 and enhanced IL-10 production, resulting in decreased T-cell proliferation (Fig. 92.1). The arrest of maturation, coupled with abrogation of IL-12 and strongly enhanced production of IL-10, highlight the important functional effects of VDR agonists on DCs and are, at least in part, responsible for the induction of DCs with tolerogenic properties. However, recent data indicate that exogenous or endogenously generated 1,25(OH)2D3 regulates in DCs a large set of target genes involved in tolerogenicity autonomously rather than via inhibition of differentiation and maturation, suggesting that the tolerogenic program initiated in DCs by VDR agonists is largely independent of their inhibitory effects on DC differentiation and maturation [78]. DCs are able to synthesize 1,25(OH)2D3 in vitro as a consequence of increased 1a-hydroxylase expression [24,25], and this could contribute to promote regulatory T cell induction. It is also possible that 1,25(OH)2D3 may contribute to the physiological control of immune responses, and possibly be also involved in maintaining tolerance to self antigens, as suggested by the enlarged lymph nodes containing a higher frequency of mature DCs in VDR-deficient mice [28]. This intriguing concept has been recently highlighted by the observation that vitamin D3 induced by sunlight in the skin is hydroxylated by local DCs into the active hormone, which in turn
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INDUCTION OF TOLEROGENIC DENDRITIC CELLS BY VDR AGONISTS
CCL17
Inhibition
CCL22
Upregulation
IL-10 CD152
Activation
CD28
MHC II
TCR
CD54
CD4+ T Cell
Recognition
CD11a/CD18 Adhesion
CD40
CD154 ILT3
Activation
?
EFFECT
MATURATION MARKER EXPRESSION
Inhibition
CD80/86
M-DC
TABLE 92.1 Phenotypic and Functional Modifications Induced by VDR Agonists in Human Myeloid Dendritic Cells PHENOTYPE
IL-12/IL-23p40
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Inhibition
VDR agonists target dendritic cells modulating T cell responses. VDR agonists inhibit in myeloid dendritic cells (MDC), but not in plasmacytoid DCs, expression of surface costimulatory molecules, e.g. CD40, CD80, CD86, as well as MHC class II and CD54 molecules. Production of cytokines affecting T cell differentiation into Th1 and Th17, IL-12 and IL-23, respectively, are also inhibited. Conversely, expression of surface inhibitory molecules like ILT3, and of secreted inhibitory cytokines like IL-10 are markedly upregulated. Chemokines potentially able to recruit CCR4þ regulatory T cells like CCL22 are also upregulated, whereas the CCR4 ligand CCL17 is downregulated. Upon interaction with M-DCs, CD4þ T cells upregulate expression of the inhibitory molecule CD152 (CTLA-4). DCs expressing low levels of costimulatory molecules, secreting IL-10, and expressing high levels of inhibitory molecules (e.g., ILT3) favor the induction and/or the enhancement of Treg cells.
FIGURE 92.1
upregulates on activated T cells expression of the epidermiotropic chemokine receptor CCR10, a primary VDRresponsive gene, enabling them to migrate in response to the epidermal chemokine CCL27 [25]. Thus, the autocrine production of 1,25(OH)2D3 by DCs can program the homing of skin-associated T cells, which could include regulatory T cells able to counteract the proinflammatory effects induced in the skin by sun exposure. Interestingly, B cells can also synthesize 1,25(OH)2D3 [27] and can preferentially expand Foxp3þ Treg cells [79], suggesting that the tolerogenic potential of B cells could perhaps be associated with their capacity to produce 1,25(OH)2D3.
CD83
decreased
DC-LAMP
decreased
ANTIGEN UPTAKE Mannose receptor expression
increased
COSTIMULATORY MOLECULE EXPRESSION CD40
decreased
CD80
decreased
CD86
decreased
INHIBITORY MOLECULE EXPRESSION ILT3
increased
ILT4
unmodified
B7-H1
unmodified
CHEMOKINE RECEPTOR EXPRESSION CCR7
decreased
CYTOKINE PRODUCTION IL10
increased
IL12
decreased
IL23
decreased
CHEMOKINE PRODUCTION CCL2
increased
CCL17
decreased
CCL18
increased
CCL20
decreased
CCL22
increased
APOPTOSIS Maturation induced
increased
T-CELL ACTIVATION
Tolerogenic Dendritic Cells Induced by VDR Agonists Lead to Enhancement of Regulatory T Cells The prevention of DC differentiation and maturation as well as the modulation of their activation and survival leading to DCs with tolerogenic phenotype and function play an important role in the immunoregulatory activity of VDR agonists, and appear to be critical for the capacity of this hormone to induce CD4þCD25þ Treg cells that are able to control autoimmune responses and allograft rejection (Table 92.1).
Response to alloantigens
decreased
Compiled from [130,210] and from the author’s unpublished data.
VDR agonists enhance CD4þCD25þ Treg cells and promote tolerance induction in transplantation and autoimmune disease models. A short treatment with 1,25(OH)2D3 and MMF, a selective inhibitor of T and B cell proliferation that also modulates APCs, induces tolerance to islet allografts associated with an increased frequency of CD4þCD25þ Treg cells able to adoptively
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transfer transplantation tolerance [67]. The induction of tolerogenic DCs could indeed represent a therapeutic strategy promoting tolerance to allografts [80] and the observation that immature myeloid DCs can induce T cell tolerance to specific antigens in human volunteers represents an important proof of concept for this approach [81]. CD4þCD25þ Treg cells able to inhibit the T cell response to a pancreatic autoantigen and to significantly delay disease transfer by pathogenic CD4þCD25 T cells are also induced by treatment of adult non-obese diabetic (NOD) mice with the VDR agonist BXL-219 [82]. This treatment arrests insulitis, blocks the progression of Th1 cell infiltration into the pancreatic islets, and inhibits type 1 diabetes development at non-hypercalcemic doses [82]. Although the type 1 diabetes and islet transplantation models are quite different, in both cases administration of VDR agonists doubles the number of CD4þCD25þ Treg cells, in the spleen and pancreatic lymph nodes, respectively [67,82]. Topically applied 1,25(OH)2D3 enhances the suppressive capacity of draining lymph node CD4þCD25þ Treg cells, but the involvement of tolerogenic DCs has not been determined [83]. Conversely, 1,25(OH)2D3 has also been shown to prevent and treat TNBS-induced colitis by reducing Th1 and Th17 cells while upregulating Foxp3þ Treg cells, associated with significant reduction of IL12p75, IL-23p19, and IL-6 production by DCs [84]. However, tolerogenic DCs may not always be necessarily involved in the generation of Treg cells by VDR agonists. A combination of 1,25(OH)2D3 and dexamethasone has been shown to induce human and mouse naı¨ve CD4þ T cells to differentiate in vitro into Treg cells, even in the absence of APCs [85]. These Treg cells produced IL-10, but no IL-5 nor IFN-g, thus distinguishing them from the previously described Tr1 cells [86]. Upon transfer, the IL-10-producing Treg cells could prevent central nervous system inflammation, indicating their capacity to exert a suppressive function in vivo [85]. Thus, although DCs appear to be primary targets for the immunomodulatory activities of VDR agonists, they can also act directly on T cells, as expected by VDR expression in both cell types and by the presence of common targets in their signal transduction pathways, such as the nuclear factor (NF)-kB that is downregulated in both APCs and T cells. Interestingly, agonists of the peroxisome proliferatoractivated receptor g (PPARg), a member of the steroid hormone receptor superfamily, also inhibit DC maturation and Ag-presenting capacity in an NF-kB-dependent fashion [87]. Unlike its downregulatory effect on other cells of the immune system, the PPARg agonist ciglitazone has been found to exert an enhancing effect on both inducible and natural Treg cells [88], similarly to VDR agonists. In addition, using PPARg-deficient
CD4þ T cells obtained from tissue-specific PPARg null mice it has been suggested that endogenous PPARg activation represents a Treg intrinsic mechanism for downregulation of effector CD4þ T cell function and prevention of colitis [89]. Thus, PPARg as well as VDR agonists can promote Treg activity, likely involving in both cases induction of tolerogenic DCs.
Upregulation of Inhibitory Receptor Expression in Dendritic Cells by VDR Agonists Most cell types involved in innate or acquired immune responses, including myeloid, lymphoid, and dendritic cells, express at least one member of the immunoglobulin-like transcripts (ILTs) family, receptors structurally and functionally related to killer cell inhibitory receptors [90] that have been shown to be involved in immunoregulation [91]. ILT family members can be subdivided into two main types. The first one, comprising ILT1, ILT7, ILT8, and leukocyte Ig-like receptor 6, is characterized by a short cytoplasmic tail delivering an activating signal through the immunoreceptor tyrosine-based activatory motif (ITAM) of the associated common g chain of the Fc receptor. Members of the second type, including ILT2, ILT3, ILT4, and ILT5, contain a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) transducing a negative signal [44]. When inhibitory ILTs are activated, their ITIM domains become phosphorylated and recruit p56lck and SH2-containing proteinetyrosineephosphatase 1 (SHP-1), leading to downstream events and gene modulation, as exemplified by the inhibitory receptor ILT3 which negatively regulates activation of antigen-presenting cells [92]. A connection between ILTs and tolerance induction has been established by the observation that CD8þCD28 suppressor T cells upregulate ILT3 and ILT4 expression on DCs, rendering them tolerogenic [93]. Such tolerogenic DCs have been reported to anergize alloreactive CD4þCD45ROþCD25þ T cells converting them into regulatory T cells which, in turn, continue the cascade of suppression by tolerizing other DCs [94]. Alloantigen-specific CD8þCD28Foxp3þ T suppressor cells have also been shown to induce ILT3þ ILT4þ tolerogenic endothelial cells, inhibiting alloreactivity [95]. Consistent with these results, rat CD8þFoxp3þ Treg cells have been shown to induce PIR-B, a hortolog of inhibitory ILTs [96], in DCs and in heart endothelial cells, and to mediate tolerance to allogeneic heart transplants [97]. ILT3 appears to be responsible for induction of CD8þ suppressor T cells in cancer patients, suggesting that depletion and/or blockade of soluble ILT3 may be crucial to the success of anti-tumor immunotherapy [98]. ILT3 may actually act as a master switch in the regulation of antigen-specific responses mediated
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INDUCTION OF TOLEROGENIC DENDRITIC CELLS BY VDR AGONISTS
by CD8þ and CD4þ T cells not only in cancer but also in transplantation, autoimmunity, and allergy [99]. To characterize mechanisms accounting for the induction of DCs with tolerogenic properties by VDR agonists, we have examined ILT expression by 1,25(OH)2D3-treated DCs, and found that incubation of monocyte-derived human DCs, either immature or during maturation, with 1,25(OH)2D3 leads to a selective upregulation of ILT3 [57]. Analysis of DC subsets revealed a higher ILT3 expression on P-DCs compared to M-DCs [100,101]. CD40 ligation reduced ILT3 expression on M-DCs but had little effect on P-DCs [102]. Thus, analysis of DC subsets revealed a differential regulation of ILT3 expression by 1,25(OH)2D3, with a marked upregulation in M-DCs but no effect on its high expression by P-DCs. Maintaining high ILT3 expression on P-DCs matured via CD40 ligation is of interest, because this cell population has been shown to induce CD8þ regulatory T cells able to suppress the proliferation of naı¨ve CD8þ cells through an IL-10-dependent pathway [103]. While incubation with 1,25(OH)2D3 did not affect the already high ILT3 expression by P-DCs, it increased its expression on M-DCs considerably [102]. The downregulation of ILT3 on M-DCs by T-cell-dependent signals, and the upregulation of this inhibitory receptor by 1,25(OH)2D3 in DCs suggests a novel mechanism for the immunomodulatory properties of this hormone that could play a role in the control of T cell responses. As tolerogenic DCs induced by different pharmacological agents share several properties, we have analyzed upregulation of ILT3 expression in immature and mature DCs by selected immunomodulatory agents. 1,25(OH)2D3 markedly upregulates ILT3 expression on both immature and mature DCs, whereas IL-10 has a much less pronounced effect, and dexamethasone no observable activity. In the same experiment, all the three agents inhibited DC maturation, as shown by decreased CD83 expression [57]. An in vivo correlate could be established by the marked upregulation of ILT3 expression in DCs of psoriatic lesions treated with the VDR agonist calcipotriol, whereas no ILT3 expression was induced by topical treatment of psoriatic plaques with the glucocorticoid mometazone [102]. These results indicate that drug-induced ILT3 upregulation is not a general feature of tolerogenic DCs, and are consistent with the view that VDR agonists and glucocorticoids modulate DCs using distinctive pathways [104]. A regulatory role for ILT3 expressed on DCs was shown by the increased IFN-g secretion promoted by anti-ILT3 addition to cultures of DCs and T cells, which was blunted in 1,25(OH)2D3-treated DCs, suggesting ILT3-independent mechanisms able to regulate T cell activation. Although ILT3 expression by DCs is required for induction of regulatory T cells, DC pretreatment with
1795
1,25(OH)2D3 leads to induction of CD4þ Foxp3þ T cells with suppressive activity irrespective of the presence of neutralizing anti-ILT3 mAb, indicating that ILT3 expression is dispensable for the capacity of 1,25(OH)2D3treated DCs to induce regulatory T cells [102]. A lack of correlation between ILT3 and inhibition of T cell responses was also observed with sirolimus, which downregulates the inhibitory receptors ILT2, ILT3, and ILT4 on human DCs [105] and yet sirolimus-treated DCs retain the ability to stimulate and enhance Treg cells [106].
Modulation of Chemokine Production by VDR Agonists can Affect Recruitment of Effector T Cells and CD4DCD25D Treg Cells to Inflammatory Sites In both islet transplantation and type 1 diabetes models, treatment with VDR agonists has a profound effect on the migration of effector T cells, preventing their entry into the pancreatic islets [67,82]. The VDR agonist BXL-219 significantly downregulates in vitro and in vivo proinflammatory chemokine production by islet cells, inhibiting T cell recruitment into the pancreatic islets and T1D development [75]. The inhibition of CXCL10 is particularly relevant, consistent with the decreased recruitment of Th1 cells into sites of inflammation by treatment with an anti-CXCR3 antibody [107], and with the substantial delay of T1D development observed in CXCR3-deficient mice [108]. The inhibition of islet chemokine production by BXL-219 treatment in vivo is associated with upregulation of IkBa transcription, an inhibitor of nuclear factor kB (NF-kB), and with arrest of NF-kBp65 nuclear translocation [75], highlighting a novel mechanism of action exerted by VDR agonists potentially relevant for the treatment of T1D and other autoimmune diseases. Both human [109] and mouse [110] CD4þCD25þ Treg cells express CCR4, and selectively migrate in response to CCR4 agonists like CCL22. An interesting confirmation to this finding is provided by the observation that human ovarian tumors produce CCL22, the cognate ligand of the CCR4 receptor, promoting the recruitment of CCR4þCD4þCD25þ Treg cells that act as a tumorprotective mechanism [111]. We have found that, in contrast to the high production by circulating human M-DCs, the CCR4 agonists CCL17 and CCL22 are poorly produced by P-DCs [112]. It is noteworthy that blood-borne M-DCs, in contrast to P-DCs, constitutively produce CCL17 and CCL22 ex vivo [112]. This selective constitutive production of CCR4 agonists by immature M-DCs could lead to the preferential attraction of CD4þCD25þ Treg cells, a mechanism expected to favor tolerance induction.
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This has been observed in ovarian carcinoma patients, in which Foxp3þCCR4þCD25þ Treg cells are selectively recruited by tumor-produced CCL22, and suppress anti-tumor responses leading to reduced patient survival [111]. Intriguingly, the production of CCL22 is markedly enhanced by 1,25(OH)2D3 in blood M-DCs but not P-DCs [77]. Besides maintaining peripheral immunological tolerance in homeostatic conditions, CD4þCD25þ Treg cells could turn off and limit ongoing inflammatory responses. Inflammatory signals strongly induce maturation and influx of both M-DCs and PDCs to secondary lymphoid tissues [31], and maturation of M-DCs and P-DCs enhances their production of several pro-inflammatory chemokines that can potentially attract different T-cell subsets. Interestingly, maturing P-DCs, similarly to activated B cells, produce large quantities of the CCR5 agonist CCL4 [112]. Thus, in analogy with the proposed role for CCL4 in CD4þCD25þ Treg cells attraction by activated B cells, mature P-DCs could recruit these cells to limit ongoing inflammatory responses.
VDR Agonists Selectively Modulate Tolerogenic Properties in Myeloid but not Plasmacytoid Dendritic Cells Although the immunomodulatory effects of VDR agonists on DCs are well established, the capacity of this hormone to modulate DC subsets has only recently been addressed. M-DC and P-DC subsets play complementary roles in the induction and regulation of innate and adaptive immune responses [113]. M-DCs are the most efficient APCs directly able to prime naı¨ve T cells and can become, under different conditions, immunogenic or tolerogenic, whereas P-DCs, under steady-state conditions, appear to play a key role in maintaining peripheral immune tolerance, and can be considered naturally occurring tolerogenic DCs [30]. Analysis of immunomodulatory effects exerted by 1,25(OH)2D3 on human blood M-DCs and P-DCs demonstrates a differential capacity of this hormone to modulate cytokine and chemokine production in DC subsets, showing marked effects in M-DCs and negligible ones in P-DCs [77]. In addition to CCL22 and CCL17, neither IFN-a, the signature cytokine produced by P-DCs, nor expression of MHC class II molecules or CCR7, a key regulator of DC migration to secondary lymphoid organs, are affected in P-DCs by 1,25(OH)2D3 treatment [77]. Conversely, production of IL-12, the M-DC signature cytokine, as well as MHC class II and CCR7 expression are markedly inhibited by 1,25(OH)2D3 in M-DCs [77]. All these molecules are controlled by NF-kB, a signal transduction pathway crucially involved in the inflammatory response [114]. Our data showing inhibition of RelA nuclear
translocation by 1,25(OH)2D3 in M-DCs but not P-DCs demonstrate a mechanism of action selectively targeting NF-kB in DC subpopulations [77]. The selective targeting of NF-kB components by VDR agonists in M-DCs could thus contribute to explain the lack of activity of these agents on cytokine and chemokine production by P-DCs. These cells, however, respond to 1,25(OH)2D3, as shown by CYP24 upregulation, although less markedly than M-DCs, as indicated by the three-fold reduction in the number of modulated genes by microarray analysis. Also inhibition of Th1 development and enhancement of CD4þ suppressor T cell activity are selectively induced by 1,25(OH)2D3 in M-DCs but not P-DCs. This differential capacity of DC subsets to respond to 1,25(OH)2D3 is not due to a diverse VDR expression or VDR-dependent signal transduction, as shown by the marked upregulation of CYP24 [77], a primary VDR response gene rapidly induced following exposure to 1,25(OH)2D3. Thus, both M-DCs and P-DCs express similar VDR levels and respond equally well to VDR ligation. To further assess responsiveness of DC subsets to 1,25(OH)2D3, we determined its effects at the genome level. Microarray analysis of ex vivo purified DC subpopulations incubated for 24 h with or without 100 nM 1,25(OH)2D3 revealed a three-fold lower number of modulated genes in P-DCs compared to M-DCs. The cut-off for gene modulation was a two-fold difference, marked by the outer diagonal lines in the scatter plots [21]. P-DCs can therefore respond to VDR agonists, which, however, do not appear to modify their tolerogenic potential. Thus, 1,25(OH)2D3 appears to upregulate tolerogenic properties selectively in M-DCs, downregulating IL-12 and Th1 cell development, while promoting CD4þ suppressor T cell activity and enhancing the production of CCL22, a chemokine able to recruit regulatory T cells. In contrast, no immunomodulatory effects appear to be induced by 1,25(OH)2D3 in P-DCs, a DC subset prone to favor tolerance [33]. P-DCs, characterized by an intrinsic ability to prime naı¨ve CD4þ T cells to differentiate into IL-10-producing T cells and CD4þCD25þ regulatory T cells, and to suppress immune responses, may represent naturally occurring regulatory DCs [33,115], and the lack of P-DC modulation by 1,25(OH)2D3 would thus leave this tolerogenic potential unmodified.
MODULATION OF T AND B LYMPHOCYTES BY VDR AGONISTS VDR agonists modulate DC function, thus shaping T cell activation and development, but they can also have direct effects on T and B lymphocytes. T and B cells, like macrophages and DCs, express the VDR and can
XI. IMMUNITY, INFLAMMATION, AND DISEASE
MODULATION OF T AND B LYMPHOCYTES BY VDR AGONISTS
synthesize 1,25(OH)2D3 able to exert autocrine and paracrine regulatory actions. VDR agonists primarily inhibit proinflammatory, pathogenic effector T cells like Th1 and Th17 cells and, under appropriate conditions, may favor a deviation to the Th2 pathway. These effects are partly due to direct T cell targeting [116], in addition to modulation of DC function [21]. Thus, VDR agonists can target T cells both directly and indirectly, selectively inhibiting T cell subsets able to mediate chronic inflammation and tissue damage (Table 92.2). 1,25(OH)2D3 has also potent direct effects on B cell responses, inducing apoptosis and inhibiting proliferation, generation of memory B cells, plasma cell differentiation, and Ig production [27]. A novel connection between the vitamin D system and T cell receptor (TCR) signaling pathways has recently been revealed, showing its capacity to control human T cell activation [117]. Coupling of the TCR to its signaling pathway, where phospholipase C (PLC)g1 has a central role, is more efficient in antigen-primed T cells than in naı¨ve T cells but, using an alternative TCR signaling pathway, Zap70 can directly phosphorylate and activate MAPK p38. This alternative TCR signaling pathway is independent of Lat and thus of PLC-g1, and the classical Ras-MAPK cascades. Naı¨ve human T cells express low levels of PLC-g1, with consequent impairment of TCR signaling via the classical PLC-g1-dependent pathway. However, TCR signaling via the alternative p38 pathway is intact in naı¨ve T cells, and it induces VDR expression. Following interaction with 1,25(OH)2D3, VDR activates the gene encoding PLCg1, resulting in upregulation of PLC-g1 protein
TABLE 92.2
Effects of VDR Agonists on T Cells
Effect
References
Inhibition of T cell proliferation
[17]
Induction of hyporesponsiveness to allo and self antigens
[130,137,211e214]
Inhibition of IL-2 production
[123,124]
Inhibition of IFN-g production
[125,134]
Inhibition of IL-17 production
[84,122,135,138]
Inhibition of Th1 cell development
[120,121]
Inhibition of Th17 cell development
[84,138]
Variable effects on IL-4 production and deviation to Th2
[121,126,131e134]
Increased production of IL-10
[85]
Increased expression of CD152
[67,122,130]
Downregulation of CD95 expression
[127]
Enhanced frequency of regulatory T cells
[67,82,85,137]
1797
expression by ~75-fold and allowing TCR signaling via the classical PLC-g1-dependent pathway [117]. Thus, initial TCR signaling via p38 leads to successive induction of VDR and PLC-g1, which are required for subsequent classical TCR signaling and T cell activation, explaining the higher sensitivity of primed T cells to antigen stimulation and highlighting an intriguing new facet of the complex regulatory mechanisms exerted by the vitamin D system in T lymphocytes.
Modulation of T Cells by VDR Agonists Soon after the discovery of VDR expression in T cells [14,118], 1,25(OH)2D3 was shown to inhibit antigeninduced T cell proliferation [17] and cytokine production [119]. Later studies demonstrated selective inhibition of Th1 cell development [120,121], although it was not clarified how much of this effect could be accounted for by modulation of DC functions, in particular inhibition of IL-12 production. 1,25(OH)2D3 can act directly on CD4þ T cells to influence their phenotype, downregulating production of the effector cytokines IFN-g, IL-17, and IL-21 [122]. Following 1,25(OH)2D3 treatment, T cells adopt the phenotypic and functional properties of adaptive Treg cells, expressing high levels of CTLA4 and FoxP3. T cells cultured in the presence of both 1,25(OH)2D3 and IL-2 expressed the highest levels of CTLA-4 and FoxP3 and possessed the ability to suppress proliferation of resting CD4þ T cells [122]. Indeed, several key cytokines in T lymphocytes are direct targets of VDR agonists, in particular Th1-type cytokines such as IL-2 and IFN-g. 1,25(OH)2D3 inhibits IL-2 secretion by impairing the transcription factor NFAT complex formation, because the ligand-bound VDR complex binds to the distal NF-AT binding site of the human IL-2 promoter [123,124]. Another key T cell cytokine, the Th1 signature IFN-g, is directly inhibited by 1,25(OH)2D3 through interaction of the ligand-bound VDR complex with a VDRE in the promoter region of the cytokine [125]. Progressive deletion analysis of the IFN-g promoter revealed that negative regulation by 1,25(OH)2D3 is also exerted at an upstream region containing an enhancer element [125]. However, some in vivo studies have failed to support a direct effect of 1,25(OH)2D3 on IFN-g production by T cells [126]. 1,25(OH)2D3 inhibits in T cells activation-induced cell death by downregulating the expression of CD95L, a cell surface molecule that activates apoptosis in CD95 (Fas)expressing target cells, via repression of CD95L promoter activity through an AF-2-dependent mechanism [127]. Downregulation of CD95L expression may have functional consequences, because CD95L costimulates the in vivo proliferation of CD8þ T cells [128] and the activated CD95 (Fas) induces DC maturation and a preferential T cell polarization towards the Th1
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92. CONTROL OF ADAPTIVE IMMUNITY BY VITAMIN D RECEPTOR AGONISTS
pathway [129]. This could be one of the mechanisms VDR ligands utilize to arrest indirectly DC maturation, although they directly promote [130], rather than inhibit, apoptosis in DCs. 1,25(OH)2D3 has been also shown to enhance the development of Th2 cells via a direct effect on naı¨ve CD4þ cells [131], and this could account for the beneficial effect of VDR agonists in the treatment of autoimmune diseases. The capacity of 1,25(OH)2D3 to skew T cells towards the Th2 pathway had been previously suggested [126,132], but could not be confirmed by other studies [121,133]. 1,25(OH)2D3 has also been shown to inhibit both IFN-g and IL-4 production in T cells [134]. According to this study, the inhibition of IL-4 production in naı¨ve T cells does not result from a cell cycle block or from inhibition of Th2 transcription factor expression, but rather from a VDR-induced direct downregulation of IL-4 transcription. It is puzzling to note that 1,25(OH)2D3 can apparently upregulate, downregulate or have no effect on IL-4 production, and consequently on Th2 cell development. These disparate results may reflect the different conditions tested, but also illustrate the complex immunoregulatory pathways set in motion by 1,25(OH)2D3. Treatment with VDR agonists also inhibits T cell production of IL-17 [135], the signature cytokine of Th17 cells, pathogenic effector T cells in various models of organ-specific autoimmunity [136]. Interestingly, IL17 production is sustained by IL-23, an IL-12 family member inhibited by 1,25(OH)2D3 [137]. 1,25(OH)2D3 is able to prevent and to partially reverse experimental autoimmune uveitis (EAU). This is accompanied by reduced production of IL-17 via two independent mechanisms, direct inhibition of IL-17 production by CD4þ T cells and indirect inhibition of IL-17 lineage commitment by downregulation of the ability of DCs to support priming of T cells towards the Th17 effector pathway [138]. Lineage commitment, as judged by induction of the Th17 lineage-specific transcription factor RORgt, is not impaired by the direct inhibitory effect on T cells, but is downregulated by the indirect effect mediated by DCs, thus permitting to distinguish between these two effects. In addition, development of Th17 cells requires interferon-regulatory factor 4 (IRF4) [139], a transcription factor that is strongly inhibited by 1,25(OH)2D3 in DCs [140]. Thus, 1,25(OH)2D3 may act at several levels to suppress the pro-inflammatory uveitogenic Th17 response by affecting functions of both DCs and CD4þ T cells. In conclusion, VDR agonists appear primarily to inhibit proinflammatory, pathogenic T cells like Th1 and Th17 cells and, under appropriate conditions, may favor a deviation to the Th2 pathway. These effects are partly due to direct T cell targeting, but modulation of DC function by VDR agonists plays an important role
in directing T cell responses. Thus, VDR agonists can target T cells both directly and indirectly, selectively inhibiting T cell subsets able to mediate chronic inflammation and tissue damage.
Modulation of B Cells by VDR Agonists Mature human lymphocytes are unique targets of 1,25(OH)2D3 because VDR is not constitutively expressed in these cell types, and specific cellular activation signals are required for both the upregulation of VDR and establishment of reactivity to the ligand. Treatment of B lymphocytes with IL-4, in the absence of prior activation, induces a weak upregulation of VDR expression but fails to generate VDRE-reactive nuclear protein complexes [141]. Stimulation of B lymphocytes by either ligation of CD40 Ag or cross-linking the Ig receptor is also insufficient to render B lymphocytes responsive to 1,25(OH)2D3. However, this apparent lack of response to 1,25(OH)2D3 can be overcome by stimulation of B lymphocytes with a combination of these cellular activation signals, which are sufficient to lead to G1 cell cycle progression [141]. In the presence of 1,25(OH)2D3, cellular activation associated with stimulation of such a progression appears to be sufficient for the upregulation of VDR message and protein and necessary for the establishment of VDRE binding complexes. Furthermore, biologic functions are modulated, as 1,25(OH)2D3 inhibits proliferation in a subset of activated B cells. These observations suggest that reactivity to 1,25(OH)2D3 is tightly regulated in B lymphocytes, requiring specific signals for its initiation, but once activated B cells, like macrophages, DCs and T cells, express the VDR and can synthesize 1,25(OH)2D3 able to exert autocrine and paracrine regulatory actions [141]. These findings were confirmed by the observation that resting B cells fail to express VDR, but this can be induced by stimulation with anti-CD40 mAb and IL-4 [142]. While VDR agonists did not inhibit B cell proliferation in this study, synthesis of IgE, but not of IgA and IgG, was markedly inhibited by VDR agonists [142]. In addition, 1,25(OH)2D3 enhanced IL-10 expression in activated B cells more than three-fold, both by recruiting the VDR to the IL-10 promoter, and to a lesser extent by modulation of calcium-dependent signaling [143]. The molecular link in activated B cells between vitamin D signaling, expression of IgE and IL-10, and their ability to produce 1,25(OH)2D3 from its precursor, suggest that VDR agonists could be used to modulate allergic responses [143]. In the B cell lineage, CCR10 is considered to play an essential role in the common mucosal immune system by guiding IgA-secreting cells to various mucosal tissues via its ligand CCL28, which is constitutively expressed by epithelial cells in various mucosal tissues
XI. IMMUNITY, INFLAMMATION, AND DISEASE
MECHANISMS FOR THE IMMUNOMODULATORY EFFECTS OF VDR AGONISTS IN AUTOIMMUNE DISEASES
[144]. 1,25(OH)2D3 markedly increases the proportion of CD19þIgDCD38þ cells expressing high levels of CCR10 [145]. Furthermore, the human CCR10 promoter is cooperatively activated by Ets-1 and VDR in the presence of 1,25(OH)2D3 [145]. Thus, 1,25(OH)2D3 may promote mucosal immunity partly by inducing CCR10 expression in terminally differentiating B cells, although this may not be sufficient to promote mucosal IgA responses in vivo. Potent direct effects of 1,25(OH)2D3 on B cell responses, inhibiting proliferation, generation of classswitched memory B cells, plasma cell differentiation, and Ig production, were also observed by Chen and coworkers [27]. In addition, the reduced levels of 1,25(OH)2D3 in systemic lupus patients, particularly in those with high disease activity scores and anti-nuclear antibodies, suggest that vitamin-D-dependent B cell regulation may play an important role in maintaining normal B cell homeostasis and that decreased levels of 1,25(OH)2D3 may contribute to B cell hyperactivity in systemic lupus patients [27].
VITAMIN D DEFICIENCY IN AUTOIMMUNE DISEASES Research on the relation between vitamin D exposure and disease in population-based studies is increasing exponentially [146]. Although there is no firm consensus on optimal serum levels of 25(OH)D3, the accepted biomarker of vitamin D status, vitamin D deficiency is usually defined by 25(OH)D3 levels below 20 ng/ml [147,148]. Mounting evidence indicates a high prevalence of vitamin D deficiency in the general population, and this has been linked to increased frequency of autoimmune diseases, in addition to bone diseases and cancer [149,150]. Epidemiologic analysis shows strong ecologic and caseecontrol evidence that vitamin D reduces the risk of several autoimmune diseases, including multiple sclerosis (MS), type 1 diabetes (T1D), inflammatory bowel disease (IBD), rheumatoid arthritis (RA), osteoarthritis, and SLE [151]. A large prospective study has confirmed that high circulating levels of 25(OH)D3 are associated with a lower risk of MS [152], suggesting that dietary vitamin D supplementation may help prevent the development of MS and may represent a useful addition to therapy in this indication [153,154]. Consistent with this possibility, vitamin D supplementation during infancy significantly reduced T1D incidence evaluated 30 years later [155], and was confirmed by a meta-analysis of data from caseecontrol studies [156]. In addition, vitamin D intake has been found to be inversely correlated with the risk of developing RA [157], and caseecontrol studies have shown significantly lower 25(OH)D3 levels
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in SLE patients [158]. However, in large prospective cohorts of women, increasing levels of vitamin D intake were not associated with the relative risk of developing either SLE or RA [159]. Vitamin D deficiency has been reported to contribute to IBD development [160], and lower amounts of 1,25(OH)2D3 are synthesized from sunlight exposure in areas in which IBD occurs most often, such as North America and Northern Europe, a situation common to other autoimmune diseases [19]. Collectively, these data indicate vitamin D status as a key environmental factor affecting autoimmune disease prevalence. VDR polymorphisms have also been repeatedly correlated with increased frequency of autoimmune diseases, but so far no association has been described with functional phenotypes [161].
MECHANISMS FOR THE IMMUNOMODULATORY EFFECTS OF VDR AGONISTS IN AUTOIMMUNE DISEASES The immunoregulatory properties of VDR ligands have been studied in different models of autoimmune diseases (Table 92.3). The capacity of VDR agonists to inhibit autoimmune diseases has been studied in different experimental models, including collageninduced arthritis, Lyme arthritis, SLE in MRLlpr/lpr mice, T1D in non-obese diabetic (NOD) mice, experimental allergic encephalomyelitis (EAE), and colitis, reviewed in [10,18,19]. VDR agonists are able not only to prevent but also to treat ongoing autoimmune diseases, as demonstrated by their ability to inhibit T1D development in adult NOD mice, and to ameliorate established chronic-relapsing EAE [10]. In addition, additive and even synergistic effects have been observed between VDR agonists and immunosuppressive agents, such as cyclosporin A and sirolimus, in autoimmune diabetes and EAE models [18]. Distinct regulatory mechanisms induced by VDR agonists may predominate in different autoimmune disease models but a common pattern, characterized by induction of tolerogenic DCs, inibition of Th1 and Th17 cell development, and enhancement of CD4þCD25þ Treg cells has been frequently observed (Fig. 92.2). In the following sections, only selected autoimmune diseases will be reviewed. More analytical reviews on treatment of autoimmune diseases by VDR agonists can be found in references [10,18e20,162].
Rheumatoid Arthritis Rheumatoid arthritis (RA) is an immune-mediated disease characterized by articular inflammation and
XI. IMMUNITY, INFLAMMATION, AND DISEASE
1800 TABLE 92.3
92. CONTROL OF ADAPTIVE IMMUNITY BY VITAMIN D RECEPTOR AGONISTS
Efficacy of VDR Agonists in the Treatment of Experimental Autoimmune Diseases
Experimental models
Main effects
References
Arthritis
Decreased incidence and severity of collageninduced or Lyme arthritis, also when given at disease onset
[163,164]
Type 1 diabetes
Inhibition of insulitis and reduction of type 1 diabetes, even when given after islet infiltration
[18,82,215e218]
Experimental allergic encephalomyelitis
Prevention and treatment of disease, inhibition of relapses
[121,133,185,186, 219,220]
Inflammatory bowel disease
Significant amelioration of symptoms, block of disease progression
[84,190,195,221]
Systemic lupus erythematosus
Inhibition of proteinuria, prevention of skin lesions
[171,222,223]
Immunomediated prostatic diseases
Inhibition of experimental autoimmune prostatitis, arrest of benign prostatic hyperplasia
[135,200,202,206]
subsequent tissue damage leading to severe disability and increased mortality. VDR agonists have been tested in two RA models, namely Lyme arthritis and collageninduced arthritis [163]. Infection of mice with Borrelia burgdorferi, the causative agent of human Lyme arthritis, produces acute arthritic lesions with footpad and ankle swelling. Supplementation with 1,25(OH)2D3 to mice infected with B. burgdorferi minimized or prevented these symptoms, and the same treatment could also inhibit collagen-induced arthritis, preventing the progression to severe arthritis when given to mice with early symptoms [163]. In a separate study, VDR agonists displayed a similar capacity to prevent and to suppress already established collagen-induced arthritis without inducing hypercalcemia [164]. 1,25(OH)2D3 contributes to the regulation of MMPs and PGE2 production by human articular chondrocytes in osteoarthritic cartilage [165], suggesting immunomodulatory effects also in human RA. In addition, 1,25(OH)2D3 directly modulates human Th17 polarization, accompanied by suppression of IL-17A, IL-17F, TNF-a, and IL-22 production by memory T cells sorted by FACS from patients with early RA [166]. VDR agonists show potential in RA treatment [167,168], as indicated by the beneficial effects of alphacalcidiol in a 3-month open-label trial on 19 RA patients [169].
Peripheral lymphoid organ
Target tissue Treg
Th17
CD40
M-DC
IL-12
CD80
L22
CTL
IL-21
Th1
2 IL-
Th1
Th1
TN Fα γ
IFN -
ILT3
Target cell
Inflammatory chemokines
Th1
CD86
CC
IL-17
NO
2
IL-6
E
IL-23
PG
B
MΦ Φ COX-2
IL-10
/= Th2
iNOS
Treg
Mechanisms involved in the regulation of adaptive immune responses by VDR agonists. VDR agonists modulate adaptive immune responses via several mechanisms in secondary lymphoid organs and in target tissues. M-DC modulation by VDR agonists inhibits development of Th1 and Th17 cells while inducing CD4þCD25þFoxp3þ Treg cells and, under certain conditions, Th2 cells. VDR agonists can also inhibit the migration of Th1 cells, and they upregulate CCL22 production by M-DC, enhancing the recruitment of CD4þCD25þ Treg cells and of Th2 cells. In addition, VDR agonists exert direct effects on T cells by inhibiting IL-2 and IFN-g production, and on B cells inducing apoptosis and inhibiting proliferation, generation of memory B cells, plasma cell differentiation, and Ig production. In target tissues, pathogenic Th1 cells, which can damage target cells via induction of cytotoxic T cells (CTL) and activated macrophages (MF), are reduced in number and their activity is further inhibited by CD4þCD25þ Treg cells and by Th2 cells induced by VDR agonists. IL-17 production by Th17 cells and Th17 cell development are also inhibited. In MF, important inflammatory molecules like cyclo-oxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) are inhibited by VDR agonists, leading to decreased production of nitric oxide (NO) and prostaglandin E2 (PGE2). MFs, as well as DCs, T and B cells, can synthesize 1,25(OH)2D3 which contributes to the regulation of local immune responses. Blunted arrows indicate inhibition, and broken arrows cytotoxicity.
FIGURE 92.2
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MECHANISMS FOR THE IMMUNOMODULATORY EFFECTS OF VDR AGONISTS IN AUTOIMMUNE DISEASES
Systemic Lupus Erythematosus Systemic lupus erythematosus (SLE) is a T-celldependent antibody-mediated autoimmune disease, and the mouse strain MRLlpr/lpr spontaneously develops an SLE-like syndrome sharing many immunological features with human SLE [170]. Administration of VDR agonists significantly prolonged the average lifespan of MRLlpr/lpr mice and induced a significant reduction in proteinuria, renal arteritis, granuloma formation, and knee joint arthritis [171,172]. In addition, dermatological lesions, like alopecia, necrosis of the ear, and scab formation, were also completely inhibited by 1,25(OH)2D3 therapy [172]. Preclinical models and epidemiological data suggest a beneficial role of VDR agonists in the treatment of human SLE [173]. VDR agonists can significantly reduce cell proliferation and IgG production, both polyclonal and anti-dsDNA, while enhancing B cell apoptosis in lymphocytes from SLE patients [174]. Vitamin D deficiency is a risk factor for SLE [158,175], and reduced levels of 1,25(OH)2D3 in SLE patients may contribute to B cell hyperactivity in this disease [27].
Type 1 Diabetes The non-obese diabetic (NOD) mouse, which spontaneously develops type 1 diabetes with a pathogenesis similar to the human disease, represents a useful model for the study of autoimmune diabetes [110]. VDR agonists have been extensively tested for their capacity to inhibit T1D in the NOD mouse, and 1,25(OH)2D3 itself has been found to reduce the incidence of insulitis and to prevent T1D development but only when administered to NOD mice starting from 3 weeks of age, before the onset of insulitis, whereas a combined treatment of 8-week-old NOD mice with the VDR agonist MC1288 and cyclosporine A reduced T1D incidence [18]. In contrast, the VDR agonist BXL-219 is able, as a monotherapy at non-hypercalcemic doses, to treat T1D in the adult NOD mouse, arresting its immunological progression and preventing clinical onset [82]. A short treatment with non-hypercalcemic doses of BXL219 inhibits IL-12 production and pancreatic infiltration of Th1 cells while increasing the frequency of CD4þCD25þ Treg cells in pancreatic lymph nodes [82]. Efficacy is likely due, at least in part, to the increased metabolic stability of this analog against the inactivating C-24 and C-26 hydroxylations, and the C-3 epimerization [176], resulting in a 100-fold more potent immunosuppressive activity compared to 1,25(OH)2D3 [177]. BXL-219 inhibits in vitro and in vivo pro-inflammatory chemokine production by islet cells, associated with upregulation of IkBa transcription and arrest of NF-kB p65 nuclear translocation, inhibiting T cell recruitment
1801
into the pancreatic islets and T1D development [75]. These findings highlight the capacity of VDR agonists to inhibit secretion of pro-inflammatory chemokines by target organs of autoimmune attack, a potentially relevant mechanism for the treatment of T1D and other chronic inflammatory conditions. However, VDR disruption does not alter T1D presentation in NOD mice, in contrast to the more aggressive disease observed in vitamin-D-deficient NOD mice [178]. Thus, although 1,25(OH)2D3 is a pharmacological and possibly a physiological immunomodulator in T1D development, its function appears to be redundant to control this disease in the NOD mouse. The observation that ongoing T1D in the adult NOD mouse can be arrested by a relatively short course of treatment with a 1,25(OH)2D3 analog suggests that a similar treatment may also inhibit disease progression in prediabetic patients. Although polymorphisms of the VDR gene could not be associated with T1D [179], common inherited variation in the vitamin D metabolism has been shown to affect susceptibility to T1D [180], and epidemiological studies have shown an association between ultraviolet B irradiance, vitamin D status and incidence rates of T1D [181], suggesting a possible involvement of the vitamin D system in T1D pathogenesis. This is further supported by a large population-based caseecontrol study [182] and by a birthe cohort study [155] showing that dietary vitamin D supplementation contributes to a significantly decreased risk of T1D development.
Multiple Sclerosis Experimental allergic encephalomyelitis (EAE), an autoimmune disease resembling multiple sclerosis (MS) characterized by Th1 and Th17-type cells specific for myelin antigens [136], can be ameliorated by treatment with VDR agonists [19,183]. 1,25(OH)2D3 and the non-hypercalcemic analog Ro 63-2023 have been shown to be selective and potent inhibitors of Th1 development in vitro and in vivo [121]. Administration of 1,25(OH)2D3 or its analog could prevent chronic-relapsing experimental allergic encephalomyelitis (CR-EAE) induced by the MOG peptide 35-55 in Biozzi AB/H mice, and this was associated with a profound reduction of MOG35-55-specific proliferation and Th1 cell development. Importantly, the non-hypercalcemic analog Ro 63-2023 also provided long-term protection from EAE relapses induced by immunization with spinal cord homogenate when administered for a short time either at symptom onset or even after the first peak of disease [121]. Neuropathological analysis showed a significant reduction of inflammatory infiltrates, demyelinated areas and axonal loss in brains and spinal cords of treated mice. Thus, inhibition of IL-12-dependent Th1
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92. CONTROL OF ADAPTIVE IMMUNITY BY VITAMIN D RECEPTOR AGONISTS
cell development is associated with effective treatment of CR-EAE. The mechanisms involved have been subsequently shown to include modulation of JAK-STAT signaling pathways in the IL-12/IFN-g axis, leading to reduced Th1 differentiation [184]. IL-10 signaling is also essential for 1,25(OH)2D3-mediated inhibition of EAE, as shown by the hormone capacity to significantly inhibit EAE in wild-type mice, but not in mice with disrupted IL-10 or IL-10R genes [185]. Thus, a functional IL-10eIL-10R pathway appears to be essential for 1,25(OH)2D3-mediated EAE inhibition. Activated inflammatory cells in EAE produce 1,25(OH)2D3, which enhances the apoptotic death of inflammatory CD4þ T cells, thus dampening the driving force for continued inflammation [186]. Strikingly, 1,25(OH)2D3 treatment significantly reduced clinical EAE severity within 3 days, preceded by sharp declines in chemokines, inducible iNOS, and CD11bþ monocyte recruitment into the central nervous system [186]. Levels of 25(OH)D3 and 1,25(OH)2D3 are significantly lower in relapsingeremitting MS patients than in controls [187]. In addition, levels in patients suffering relapse are lower than during remissions. In contrast, primary progressive MS patients showed similar values to controls [187]. Proliferation of both freshly isolated CD4þ T cells and MBP-specific T cells was significantly inhibited by 1,25(OH)2D3. Moreover, 1,25(OH)2D3 enhanced the development of IL-10 producing cells, and reduced the number of IL-6 and IL-17 secreting cells. 1,25(OH)2D3 also increased the expression and biological activity of indoleamine 2,3-dioxygenase (IDO), mediating significant increase in the number of CD4þCD25þ Treg cells [187]. Collectively, these data suggest that 1,25(OH)2D3 plays an important role in T cell homeostasis during the course of MS, highlighting its potential in treatment of the disease [188].
Inflammatory Bowel Diseases Inflammatory bowel diseases (IBDs) are immunemediated diseases affecting the gastrointestinal tract. Two distinct forms of IBDs have been defined, ulcerative colitis and Crohn’s disease. These are chronic recurring illnesses most commonly involving inflammation of the terminal ileum and colon, although they can also affect many sites throughout the alimentary tract [189]. In addition to genetic factors, including also VDR gene polymorphisms, the environment contributes to IBD development, and vitamin D may be an important environmental component in this respect. Epidemiological evidence supports a pathogenetic link between vitamin D deficiency and the risk of IBD [160]. Vitamin D deficiency may compromise the mucosal barrier, leading to increased susceptibility to mucosal damage and
increased risk of IBD, as suggested by studies in VDRdeficient mice indicating a critical role for the VDR in mucosal barrier homeostasis, by preserving the integrity of junction complexes and the healing capacity of the colonic epithelium [190,191]. In IBD models, the immune-mediated attack against the gastrointestinal tract has been shown to be mediated by Th1 and Th17 cells, and the production of Th1-type cytokines has also been found associated with human IBDs [192]. Animal models have been developed in which IBD symptoms occur spontaneously, and a wellstudied one is the IL-10 knockout (KO) mouse [193]. In conventional animal facilities, IL-10 KO mice develop enterocolitis within 5 to 8 weeks of life, and approximately 30% of these mice die of severe anemia and weight loss [193]. IL-10 KO mice were made vitamin D deficient, vitamin D sufficient or supplemented with 1,25(OH)2D3 [19]. Vitamin D-deficient, in contrast to vitamin D-sufficient, IL-10 KO mice rapidly developed diarrhea and a severe wasting disease. Administration of 1,25(OH)2D3 significantly ameliorated IBD symptoms in IL-10 KO mice, and treatment for as little as 2 weeks blocked disease progression and ameliorated symptoms in mice with already established IBD [19]. VDR expression is required to control inflammation in the IL-10 KO, because colitis is exacerbated in IL-10/VDR double-deficient mice associated with high local expression of IL-2, IFN-g, IL-1b, TNF-a and IL-12, and VDRdeficient mice are extremely sensitive to dextran sodium sulfate (DSS)-induced colitis [190]. These results point to an important role for the vitamin D system in the control of innate immunity and gastrointestinal homeostasis. Dietary calcium and 1,25(OH)2D3 treatment directly and indirectly inhibit the TNF-a pathway reducing colonic inflammation in IL-10 deficient mice [194], and VDR agonists delivered rectally can decrease the severity and extent of DSS-induced colitis in wild-type mice [190,195]. TNF-a represents a validated target in IBD, since this cytokine plays an important role in the initiation and perpetuation of intestinal inflammation in IBD, and anti-TNF-a antibodies are approved therapies also for this indication. The VDR agonist TX527 (19-nor-14,20bisepi-23-yne-1,25(OH)2D3) inhibits proliferation and TNF-a production by peripheral blood mononuclear cells from CD patients [196]. Activation of NF-kB stimulated by TNF-a and its nuclear translocation together with the degradation of IKB-a were blocked by TX527, further indicating anti-inflammatory properties of VDR agonists. In addition, treatment of TNBS-induced colitis with the VDR agonist 22-ene-25-oxa-vitamin D (ZK156979) inhibited disease at normocalcemic doses, accompanied by downregulation of myeloperoxidase activity, TNF-a, IFN-g, and T-bet expression, whereas local tissue IL-10
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and IL-4 protein levels increased [197]. In the same model of TNBS-induced colitis, 1,25(OH)2D3 promoted Treg cells, as indicated by a marked increase of IL-10, TGF-b, FoxP3, and CTLA4. Furthermore, analysis of DC mediators responsible for a pro-inflammatory differentiation of T cells revealed a significant reduction of IL12p70 and IL23p19 as well as IL-6 and IL-17 [84].
Immuno-mediated Prostatic Diseases The prostate has been recognized as a target organ of VDR agonists and represents an extra-renal synthesis site of 1,25-dihydroxyvitamin D3 [198]. Neurological, immunological, and endocrine dysfunctions have been proposed to be involved in the pathogenesis of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), with emerging evidence for an important autoimmune component [199]. Based on the marked inhibitory activity of the VDR agonist elocalcitol on basal and growth factor-induced proliferation of human prostate cells [200], we have tested its anti-inflammatory properties in the treatment of experimental autoimmune prostatitis (EAP) induced by injection of prostate homogenate-CFA in NOD male mice [135]. Administration of elocalcitol, at normocalcemic doses, for 2 weeks in already established EAP inhibits significantly the intra-prostatic cell infiltrate, with reduced cell proliferation and increased apoptosis of resident and infiltrating cells [135]. Th1 cell responses are decreased as well as production of IL-17 [135], a cytokine involved in prostate inflammation [201], emphasizing the potential of VDR agonists in the treatment of immuno-mediated diseases of the prostate [202]. This is also supported by the observation that VDR-KO NOD mice develop a more aggressive form of EAP, characterized by a greater lymphoproliferative response against prostate antigen in vitro and higher levels of specific INF-g secretion, accompanied by more severe lesions and augmented mononuclear cell infiltration in the prostate gland [203]. We have also analyzed the capacity of VDR agonists to treat benign prostatic hyperplasia (BPH), a complex syndrome characterized by a static component related to prostate overgrowth, a dynamic component responsible for urinary irritative symptoms, and an inflammatory component [204]. VDR agonists, and notably elocalcitol, reduce the static component of BPH by inhibiting the activity of intra-prostatic growth factors downstream the androgen receptor [200], and the dynamic component by targeting bladder cells [205]. Elocalcitol inhibits spontaneous BPH development in beagle dogs [202], a finding observed also with the non-secosteroidal VDR agonist CH5036249 [206]. In addition, elocalcitol inhibits production of pro-inflammatory cytokines and chemokines by human BPH cells [207]. These inhibitory
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activities are accompanied by decreased COX-2 expression and PGE2 production and by arrest of NF-kB p65 nuclear translocation, associated with inhibition of the RhoA/ROCK pathway, providing a mechanistic explanation for the anti-proliferative and anti-inflammatory properties of elocalcitol in BPH cells [207]. Also the prostatic urethra is a target for VDR agonists, as shown by the capacity of elocalcitol to inhibit ROCK activity and to limit inflammatory responses in human primary urethra cells [208]. A proof-of-concept phase II study has successfully shown arrest of prostate growth in BPH patients treated with elocalcitol [209], highlighting its potential for clinical translation in this indication.
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type 1-mediated colitis in mice, J. Pharmacol. Exp. Ther. 319 (2006) 622e631. J.N. Flanagan, M.V. Young, K.S. Persons, L. Wang, J.S. Mathieu, L.W. Whitlatch, et al., Vitamin D metabolism in human prostate cells: implications for prostate cancer chemoprevention by vitamin D, Anticancer Res. 26 (2006) 2567e2572. R.D. Motrich, M. Maccioni, C.M. Riera, V.E. Rivero, Autoimmune prostatitis: state of the art, Scand. J. Immunol. 66 (2007) 217e227. C. Crescioli, P. Ferruzzi, A. Caporali, M. Scaltriti, S. Bettuzzi, R. Mancina, et al., Inhibition of prostate cell growth by BXL628, a calcitriol analogue selected for a phase II clinical trial in patients with benign prostate hyperplasia, Eur. J. Endocrinol. 150 (2004) 591e603. G.E. Steiner, B. Djavan, G. Kramer, A. Handisurya, M. Newman, C. Lee, et al., The picture of the prostatic lymphokine network is becoming increasingly complex, Rev. Urol. 4 (2002) 171e177. L. Adorini, G. Penna, S. Amuchastegui, C. Cossetti, F. Aquilano, R. Mariani, et al., Inhibition of prostate growth and inflammation by the vitamin D receptor agonist BXL-628 (elocalcitol), J. Steroid Biochem. Mol. Biol. 103 (2007) 689e693. R.D. Motrich, E. van Etten, J. Depovere, C.M. Riera, V.E. Rivero, C. Mathieu, Impact of vitamin D receptor activity on experimental autoimmune prostatitis, J. Autoimmun. 32 (2009) 140e148. L. Adorini, G. Penna, B. Fibbi, M. Maggi, Vitamin D receptor agonists target static, dynamic, and inflammatory components of benign prostatic hyperplasia, Ann. NY Acad. Sci. 1193 (2010) 146e152. C. Crescioli, A. Morelli, L. Adorini, P. Ferruzzi, M. Luconi, G.B. Vannelli, et al., Human bladder as a novel target for vitamin D receptor ligands, J. Clin. Endocrinol. Metab. 90 (2005) 962e972. K. Taniguchi, K. Katagiri, H. Kashiwagi, S. Harada, Y. Sugimoto, Y. Shimizu, et al., A novel nonsecosteroidal VDR agonist (CH5036249) exhibits efficacy in a spontaneous benign prostatic hyperplasia beagle model, J. Steroid Biochem. Mol. Biol. 121 (2010) 204e207. G. Penna, B. Fibbi, S. Amuchastegui, E. Corsiero, G. Laverny, E. Silvestrini, et al., The vitamin D receptor agonist elocalcitol inhibits IL-8-dependent benign prostatic hyperplasia stromal cell proliferation and inflammatory response by targeting the RhoA/Rho kinase and NF-kappaB pathways, Prostate 69 (2009) 480e493. P. Comeglio, A.K. Chavalmane, B. Fibbi, S. Filippi, M. Marchetta, M. Marini, et al., Human prostatic urethra expresses Vitamin D receptor and responds to Vitamin D receptor ligation, J. Endocrinol. Invest. 33 (2010) 730e738. E. Colli, P. Rigatti, F. Montorsi, W. Artibani, S. Petta, N. Mondaini, et al., BXL628, a novel vitamin D3 analog arrests prostate growth in patients with benign prostatic hyperplasia: a randomized clinical trial, Eur. Urol. 49 (2006) 82e86. M. Vulcano, S. Struyf, P. Scapini, M. Cassatella, S. Bernasconi, R. Bonecchi, et al., Unique regulation of CCL18 production by maturing dendritic cells, J. Immunol. 170 (2003) 3843e3849.
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[211] L. Piemonti, P. Monti, M. Sironi, P. Fraticelli, B.E. Leone, E. Dal Cin, et al., Vitamin D3 affects differentiation, maturation, and function of human monocyte-derived dendritic cells, J. Immunol. 164 (2000) 4443e4451. [212] M.D. Griffin, W.H. Lutz, V.A. Phan, L.A. Bachman, D.J. McKean, R. Kumar, Potent inhibition of dendritic cell differentiation and maturation by vitamin D analogs, Biochem. Biophys. Res. Commun. 270 (2000) 701e708. [213] A. Berer, J. Stockl, O. Majdic, T. Wagner, M. Kollars, K. Lechner, et al., 1,25-Dihydroxyvitamin D(3) inhibits dendritic cell differentiation and maturation in vitro, Exp. Hematol. 28 (2000) 575e583. [214] M.O. Canning, K. Grotenhuis, H. de Wit, C. Ruwhof, H.A. Drexhage, 1-Alpha,25-dihydroxyvitamin D3 (1,25(OH)(2) D(3)) hampers the maturation of fully active immature dendritic cells from monocytes, Eur. J. Endocrinol. 145 (2001) 351e357. [215] M. Inaba, Y. Nishizawa, K. Song, H. Tanishita, S. Okuno, T. Miki, et al., Partial protection of 1 alpha-hydroxyvitamin D3 against the development of diabetes induced by multiple lowdose streptozotocin injection in CD-1 mice, Metabolism 41 (1992) 631e635. [216] C. Mathieu, M. Waer, J. Laureys, O. Rutgeerts, R. Bouillon, Prevention of autoimmune diabetes in NOD mice by 1,25 dihydroxyvitamin D3, Diabetologia 37 (1994) 552e558. [217] C. Mathieu, M. Waer, K. Casteels, J. Laureys, R. Bouillon, Prevention of type I diabetes in NOD mice by nonhypercalcemic doses of a new structural analog of 1,25-dihydroxyvitamin D3, KH1060, Endocrinology 136 (1995) 866e872. [218] K.M. Casteels, C. Mathieu, M. Waer, D. Valckx, L. Overbergh, J.M. Laureys, et al., Prevention of type I diabetes in nonobese diabetic mice by late intervention with nonhypercalcemic analogs of 1,25-dihydroxyvitamin D3 in combination with a short induction course of cyclosporin A, Endocrinology 139 (1998) 95e102. [219] J.M. Lemire, D.C. Archer, 1,25-Dihydroxyvitamin D3 prevents the in vivo induction of murine experimental autoimmune encephalomyelitis, J. Clin. Invest. 87 (1991) 1103e1107. [220] D. Branisteanu, M. Waer, H. Sobis, S. Marcelis, M. Vandeputte, R. Boullion, Prevention of murine experimental allergic encephalomyelitis: cooperative effects of cyclosporine and 1 a,25-(OH)2D3, J. Neuroimmunol. 61 (1995) 151e160. [221] M.T. Cantorna, C. Munsick, C. Bemiss, B.D. Mahon, 1,25Dihydroxycholecalciferol prevents and ameliorates symptoms of experimental murine inflammatory bowel disease, J. Nutr. 130 (2000) 2648e2652. [222] T. Koizumi, Y. Nakao, T. Matsui, T. Nakagawa, S. Matsuda, K. Komoriya, et al., Effects of corticosteroid and 1,24R-dihydroxy-vitamin D3 administration on lymphoproliferation and autoimmune disease in MRL/MP-lpr/lpr mice, Int. Arch. Allergy Appl. Immunol. 77 (1985) 396e404. [223] J.M. Lemire, Immunomodulatory role of 1,25-dihydroxyvitamin D3, J. Cell Biochem. 49 (1992) 26e31.
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C H A P T E R
93 The Role of Vitamin D in Innate Immunity: Antimicrobial Activity, Oxidative Stress and Barrier Function Philip T. Liu 1 University of California at Los Angeles, CA, USA
INNATE IMMUNITY Tuberculosis Tuberculosis has plagued humans throughout history with prehistoric fossil evidence as well as written recordings of the disease in ancient Egyptian and Chinese manuscripts [1]. In 1882, Robert Koch first described Mycobacterium tuberculosis, the etiological agent of tuberculosis, which primarily infects lung macrophages leading to pathogenesis of the disease. More than a century later, tuberculosis remains a leading cause of morbidity and mortality worldwide, with one third of the world’s population infected and eight million new cases of tuberculosis appearing each year [2]. Even developed countries are not spared by this pandemic; an estimated 10e15 million people residing in the USA are infected with M. tuberculosis [3,4]. The World Health Organization recently reported the incidence of multidrug-resistant (MDR) M. tuberculosis is at the highest in recorded history, and extensively drug-resistant (XDR) strains, which have no effective treatment, are also rapidly emerging [5]. In addition to its importance with respect to global health, tuberculosis provides an important model for investigation of the human immune response to an intracellular pathogen. Studies of tuberculosis helped delineate several basic immunological paradigms such as the role of Toll-like receptor 2 (TLR2) in recognition of microbial lipoproteins [6], leading to (1) instruction of the adaptive immune response [6e8], (2) macrophage differentiation [7], (3) a nitricoxide-dependent antimicrobial pathway in mice [9]
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10093-9
and (4) a vitamin-D-dependent antimicrobial pathway in humans [10]. TLR2 has been shown to be important for resistance to M. tuberculosis in mouse models [11e13], and polymorphisms in both the vitamin D receptor (VDR) and TLR2 are associated with human susceptibility to tuberculosis [14e21]. This association of tuberculosis with both the innate immune system (via infection of macrophages) and vitamin D led to a delineation of the molecular mechanisms by which an individual’s vitamin D status could alter their ability to combat pathogens.
Pathogen Detection Rapid detection and antimicrobial activity against microbes are considered to be key direct functions of innate immune cells in relation to infection control. However, the mechanism of innate immune cells used to detect invading pathogens remained a mystery for many years; Charles Janeway proposed the existence of evolutionarily primitive receptors which bind conserved microbial constituents, termed pattern recognition receptors [22]. In 1996, Lemaitre et al. reported Toll-deficient adult Drosophila were more susceptible to fungal infection [23]. Activation of Toll results in production of antimicrobial peptides [24], thus implicating Toll as a player in a primitive immune system. One year later, Medzhitov et al. demonstrated that a human Toll homolog, or a Toll-like receptor (TLR), modulates the adaptive immune response by inducing cytokines secretion and co-stimulatory molecule expression [25]. Together, these reports first established the importance of Toll and TLRs in host defense.
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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M. tuberculosis is known to activate at least two different families of pattern recognition receptors: TLRs and the nucleotide oligomerization domain (NOD)-like receptors (NLR). The TLR2 and TLR1 heterodimer recognizes a triacylated lipoprotein derived from M. tuberculosis, leading to activation of NF-kB and to the production of inflammatory cytokines and direct antimicrobial activity [6,9,10]. NOD2 recognizes muramyl dipeptide (MDP), which is a peptidoglycan present on M. tuberculosis [26,27]. Triggering NOD2 similarly leads to a NF-kB-mediated inflammatory response; however, in contrast to TLRs, NOD2 also results in activation of the inflammasome [28]. The inflammasome is a protein complex whose function is to cleave and activate the pro-IL-1b protein into the active IL-1b cytokine through the enzymatic actions of caspase-1.
Toll-like Receptors TLRs have been shown to recognize microbial ligands and mediate immune functions of the innate immune system. To date, 11 mammalian TLRs have been identified in both the human and murine genomes. Although TLR1-9 are conserved between humans and mice, the murine Tlr10 gene is non-functional, and the human TLR11 gene harbors a premature stop codon preventing its expression [29]. All the mammalian TLRs share a highly similar cytosolic Toll/IL-1 receptor (TIR) domain, which triggers several signaling pathways including the transcription factor: NF- [30]. The extracellular TLR domains include multiple leucine-rich repeat motifs and is responsible for recognition of conserved pathogen associated molecular patterns (PAMPs). TLR2 is known to heterodimerize with either TLR1 or TLR6, and the dimers mediate recognition of triacylated and diacylated bacterial lipoproteins respectively [31]. The remainder of the known TLR ligands are as follows: viral dsRNA (TLR3), lipopolysaccharide (LPS) (TLR4), bacterial flagellin (TLR5), single-stranded RNA (ssRNA) (TLR7 and TLR8), bacterial unmethylated CpG DNA (TLR9), and protozoan profilin-like molecule (TLR11) [29]. The ligand for TLR10 is still unclear. Thus, TLRs provide a rapid first line of defense against a variety of microbial invaders through the recognition of a milieu of pathogen associated molecules. Activation of TLRs induces a variety of effects, including enhancement of macrophage phagocytosis [32], endosomal/lysosomal fusion [33], production of antimicrobial peptides [34,35], as well as induction of direct antibacterial [9,34] and antiviral activity [36e38]. M. tuberculosis-infected macrophages can induce a direct antimicrobial activity upon TLR2/1 activation. In a murine macrophage cell line, this activity is dependent
on the generation of nitric oxide (NO) through inducible nitric oxide synthease (iNOS) activity. Addition of the iNOS inhibitors L-NIL and L-NAME ablated the murine TLR2/1-mediated antimicrobial activity; however, neither has an effect on human monocytes, suggesting human TLR2/1-induced antimicrobial activity is fundamentally different from murine cells [9]. This is correlated with the finding that, upon TLR2/1 activation, human monocytes do not generate detectable levels of NO [39]. Accordingly, the mechanism by which human macrophages kill intracellular M. tuberculosis intrigued immunologists for many years; the surprising role of the vitamin D synthetic/metabolic pathway in this mechanism is detailed below.
Nucleotide Oligomerization Domain There is building evidence for the participation of the NLR family of pattern recognition receptors in the host defense response against M. tuberculosis infection. Studies have demonstrated the resultant gene program elaborated by TLR and NOD2 are distinct, despite both receptor families activating the transcription factor NF-kB [40,41]. Of the NLRs, NOD2 has been the most extensively studied, and recognizes muramyl dipeptide (MDP), which is a peptidoglycan present on M. tuberculosis [26,27]. In 2005, Ferwerda et al. published a study providing evidence for the role of NOD2 in the recognition of M. tuberculosis infection [41]. However, conflicting results have emerged regarding the role of NOD2 in control of M. tuberculosis infection. One study by Gandortra et al. demonstrated NOD2 knockout mice are able to control M. tuberculosis infection [42], whereas Divangahi et al. showed that NOD2 deficient mice have impaired resistance [43]. In humans, an epidemiological study identified NOD2 polymorphisms which are associated with both increased resistance and susceptibility to tuberculosis in an African-American cohort [44], but a separate study conducted in Gambia was unable to identify any NOD2 polymorphisms associated with tuberculosis [45]. One potential reason for these conflicting results in humans is neither study accounted for the vitamin D levels of the patients, an important factor when considering disease linkage in relation to the vitamin D pathway [21]. Furthermore, the vitamin D levels of the patients may regulate NOD2 expression as shown by Wang et al. in 2010. Their study demonstrated, through binding of the VDR to distal VDR response elements (VDREs), that 1,25(OH)2D3 strongly induces the expression of NOD2, which then mediates the expression of the antimicrobial peptide hBD2 (DEFB4) [46]. Taken together, these studies provide sufficient in vitro evidence for the role of NOD2 in the host response against M. tuberculosis infection; however, the in vivo
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data would indicate there exist additional layers of complexity yet to be clarified.
IMMUNE ACTIVITY OF 1,25DIHYDROXYVITAMIN D There have been many studies on the role of 1,25(OH)2D on innate and adaptive immune responses [47e49]. Insight into vitamin-D-induced antimicrobial activity by human monocytes and macrophages against M. tuberculosis was first suggested by experiments in the labs of Rook in 1986 [50] and Crowle in 1987 [51]. These experiments were performed by adding 1,25(OH)2D3 to the extracellular medium of M. tuberculosis-infected human monocytes and macrophages in vitro, which resulted in reduction of the intracellular bacterial load. However, the authors noted that “concentrations of 1,25(OH)2D near 4 mg/ml were needed for good protection, these levels seemed unphysiologically high compared with 26 to 70 pg/ml being in the normal circulating range”. Nevertheless, these studies opened new questions regarding the role of vitamin D in the physiological response to M. tuberculosis and the identity of the vitamin D-dependent antimicrobial effectors. Nearly a decade later, the vitamin D-induced antimicrobial activity of the macrophage began to be elucidated. One study by Sly et al. reported 1,25(OH)2D3-induced antimicrobial activity is regulated by phosphatidylinositol 3-kinase and mediated through the generation of oxygen intermediates via NADPH-dependent phagocyte oxidase [52]. Interestingly, these authors observed 1,25(OH)2D3-induced oxidative burst occurred earlier than the resultant antimicrobial activity, thus leading the authors to postulate there was another key factor [52]. Anand et al. proposed an alternative mechanism in their study demonstrating 1,25(OH)2D3-induced antimicrobial activity was associated with downregulated transcription of the host protein, tryptophan-aspartate containing coat protein (TACO) [53]. They also demonstrated that TACO plays an important role in M. tuberculosis entry and survival in human macrophages [54]. In 2005, using a genome-wide scan for vitamin D response elements (VDREs), Wang et al. reported the genes encoding antimicrobial peptides, cathelicidin and hBD2, were regulated by the VDR [55]. Prior to this study, human macrophages were not thought to utilize antimicrobial peptides as a defense mechanism; however, in the same year human monocytes were demonstrated to express cathelicidin at both the mRNA and protein levels when stimulated with 1,25(OH)2D3 [10,55,56]. Although monocytes could express cathelicidin, the role of cathelicidin expression in host defense against intracellular M. tuberculosis infection was not clear. Two years later, a critical role for cathelicidin in
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the 1,25(OH)2D3-induced antimicrobial activity against intracellular M. tuberculosis was shown in human monocytic cells using siRNA knockdowns [57]. In contrast to its effects on macrophages, many studies have reported 1,25(OH)2D3 induces immunosuppressive effects, including but not limited to (1) inhibition of IL-12 secretion, (2) inhibition of lymphocyte proliferation and immunoglobulin synthesis and (3) impairment of dendritic cell maturation, leading to the generation of tolergenic dendritic cells and T-cell anergy [58e61]. In particular, it was suggested that 1,25(OH)2D3 produced by the macrophage in granuloma-forming diseases, like tuberculosis and sarcoidosis, exerted a paracrine immune inhibitory effect on neighboring, activated lymphocytes expressing the VDR, which slows an otherwise “overzealous” adaptive immune response [62]. The physiological significance of this has been highlighted by the development of 1,25(OH)2D3-deficient mouse models where CYP27B1 has been knocked out [63,64]. A notable feature of these animals is that they present with enhanced adaptive immunity signified by multiple enlarged lymph nodes. However, whether this enhancement was due to loss of 1,25(OH)2D3-mediated suppression on the adaptive immune response remains to be tested.
Antimicrobial Peptides Antimicrobial peptides consist of a highly diverse family of small peptides which can function as chemoattractants [65,66], dendritic cell activators [67], and, importantly, direct antimicrobial effectors [68,69]. They exert antimicrobicidal activity by disrupting the pathogen membrane through electrostatic interactions with the polar head groups of membrane lipids [70], or the creation of membrane pores [68]. Given this mechanism of activity, antimicrobial peptides exhibit a wide range of microbial targets including bacteria [71], fungi [72,73], parasites [74,75], and enveloped virii [76]. Epithelial cells, at the interface between the outside and inside environment of the host, can express antimicrobial peptides [77]. However, it is the population of innate immune cells buttressing this barrier, such as neutrophils [78], mast cells [79] and monocytes/macrophages [56,80], that are recognized to be the major producers of antimicrobial peptides. Several antimicrobial peptides produced by macrophages have been demonstrated to have direct antimicrobial activity against M. tuberculosis, including but most likely not limited to LL-37 (cathelicidin) [10,81], hBD2 (DEFB4) [82], and hepcidin [83]. In humans, cathelicidin and DEFB4 were found to contain activating VDREs in their promoter regions; whether or not hepcidin is vitamin Dregulated at the level of transcription is unknown [55]. Activation of the VDR in monocytes/macrophages
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results in the expression of cathelicidin at both the mRNA and protein levels [10,55,81]. siRNA knockdown of cathelicidin in human monocytes results in complete loss of 1,25(OH)2D3-induced antimicrobial activity [57], suggesting the generation of antimicrobial peptides by the active vitamin D metabolite represents a major human macrophage host defense mechanism.
Oxidative Stress: Reactive Oxygen Species Generation of reactive oxygen species (ROS) is dependent upon the enzymatic activity of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and occurs predominately in neutrophils and to a lesser degree in macrophages [84]. NADPH oxidase is a complex of both membrane and cytosolic components assembled on the cell membrane. The membrane bound components of the complex include gp91phox and p22phox, the a/b subunits of flavocytochrome b558, respectively, and the cytosolic proteins include p47phox, p67phox, and Rac [85,86]. In addition, p40phox associates to the complex with a negative regulatory role [16]. The NADPH oxidase complex functions through the transfers of electrons, converting molecular oxygen (O2) into superoxide anion (O 2 ), which can then be converted to hydrogen peroxide (H2O2), the hydroxyl radical (OH) or other ROS [87]. ROS have been shown to cause microbial DNA damage, oxidative damage to proteins and disruption of membrane lipids as possible direct and indirect antimicrobial mechanisms [87,88]. All the products of this reaction exhibit varying degrees of antimicrobial ability. The earliest products O 2 and H2O2 exhibit the weakest antimicrobial activity, whereas downstream products, hypochlorites (OCl) and chloramines (RNH2Cl), are the most potent [88]. The assembly of the NADPH oxidase is regulated by the phosphatidylinositol 3-kinase (PI3K), which can be induced by certain pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-a) and granulocytemacrophage colony stimulating factor (GM-CSF) [89]. Induction of PI3K in neutrophils as part of a priming response will result in a stronger oxidative burst upon phagocytosis or encounter with microbial stimuli [89]. The induction of NADPH oxidase activity by 1,25(OH)2D3 on human monocytes is through the increased expression of the p47 subunit [90] and mediated through PI3K pathway [52]. Other immune stimuli, such as interferon gamma (IFN-g) and microbial products, can directly induce transcription of the NADPH oxidase complex components [91].
Oxidative Stress: Reactive Nitrogen Species Generation of reactive nitrogen species (RNS) is achieved through a family of nitric oxide syntheases
(NOS): inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS) [92]. The iNOS isoform is predominantly responsible for generation of nitric oxide radicals (NO) by cells of the innate immune system, which functions as a potent antimicrobial effector [93]. NOSs are multi-domain proteins with an aminoterminal oxidase domain containing a heme center, a calmodulin-binding domain, and a carboxy-terminal reductase domain. The enzymes contain binding sites for L-arginine and tetrahydrobiopterin, while the reductase domain contains binding sites for NADPH, FAD, and flavin mononucleotide (FMN). The transfer of electrons from NADPH to FAD, then FMN and finally to the heme iron in NOS, results in generation of NO and citrulline from L-arginine [94]. In the presence of oxygen, nitric oxide can be further catalyzed into nitrogen dioxide (NO2), nitrogen trioxide (N2O3), nitrate (NO 3 ) and other RNS [87]. NO can also interact with super oxide to form peroxynitrite (ONOO) and peroxynitrous acid (ONOOH) [84]. The RNS-mediated antimicrobial mechanisms are more complex than those of ROS. Nitric oxide can inhibit both microbial DNA replication and cellular respiration [95e97]. Through interactions with ribonuclease reductase, RNS can limit the precursors required for DNA replication and repair [98]. Nitrogen dioxide (NO2), nitrogen trioxide (N2O3), and nitrate (NO 3 ) can potentiate oxidative damage similar to damage by ROS [99e101]. Together with H2O2 and myeloperoxidase, ONOO can nitrate tyrosine residues on proteins [102]. Regulation of iNOS activity is predominantly at the transcriptional level. Inflammatory cytokines such as interferons (IFNs), IL-1b, and TNF-a can induce transcription of iNOS through the p38-MAPK, NF-kB, and Janus-activated kinase (JAK)-signal transducer and activator of transcription (STAT)-IRF3 signaling pathways [103e105]. Interestingly, despite numerous reports of human macrophages inducing the iNOS transcript and promoter activity [106e109], there have been a sparingly small number of publications that have detected robust levels of NO, comparable to other animal models [110]. The ability of 1,25(OH)2D3 to regulate iNOS activity in innate immune cells is complex. Using avian monocytic cells, 1,25(OH)2D3 synthesis through CYP27b-1hydroxylation of 25(OH)D3 was demonstrated to be dependent upon and regulated by the level of NO production by the same cells [111,112]. The authors hypothesized the NO produced in the cells acted as a source of electrons for the hydroxylation reaction. In rat and mouse monocytic cells, LPS-induced iNOS expression and function was inhibited by 1,25(OH)2D3 [113,114]; however, the role of NO in 1,25(OH)2D generation was not explored. In contrast, bovine monocytes stimulated with 1,25(OH)2D3 by itself induced expression of iNOS [115]. For human
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promyeloid cells, 1,25(OH)2D3 stimulation alone did not induce either iNOS mRNA expression or NO, whereas co-stimulated with both 1,25(OH)2D3 and phorbol 12-myristate 13-acetate (PMA) resulted in a robust induction of both iNOS mRNA and NO. These results suggest a more complex mechanism of iNOS regulation in humans involving multiple pathways which may include the vitamin D pathway. Primary human monocytes stimulated through TLR2/1 do not produce appreciable levels of NO [9]; whether or not sufficient bioavailability of 25(OH)D3 would alter this result remains to be tested.
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demonstrated lysosomal hydrolyzed ubiquitin peptides have direct antimicrobial activity against M. tuberculosis (Fig. 93.1A), and are delivered in an autophagy-dependent manner to phagosomes harboring mycobacterium [117]. Autophagy can be induced via a variety of methods in macrophages, most immunologically relevant includes IFN-g, activation of TLRs and 1,25(OH)2D3 [116,118e120]. In human macrophages, induction of autophagy is required for 1,25(OH)2D3mediated antimicrobial activity against M. tuberculosis [118].
Barrier Function
Autophagy Recent studies have demonstrated autophagy; the cellular process by which a cell degrades its own intracellular compartments, as a previously unappreciated innate immune defense mechanism [116]. Stimulation of mouse macrophages with the key immune cytokine, IFN-g, induces autophagy-dependent antimicrobial activity against M. tuberculosis [116]. A separate study
The first line of immune defense is the responsibility of the barrier cells, including cells of the skin, the corneal epithelium, the oral mucosa, the urinary tract, as well as the respiratory and digestive systems. In addition to the specialized functions each of these cell types performs, they also serve as the barrier to exclude invading pathogens, which is the most effective way of preventing infection. Vitamin D has been demonstrated to play an
The role of 25(OH)D in the innate immune response. (A) 1,25(OH)2D3 induction of autophagy leads to antimicrobial activity against intracellular M. tuberculosis (M.tb.) infection. (B) TLR activation results in (1) induction of expression of the CYP27B1-hydroxylase and vitamin D receptor (VDR) genes and (2) intracrine generation of 1,25-dihydroxyvitamin D (1,25(OH)2D), from substrate 25-hydroxyvitamin D (25(OH)D) when present in sufficient quantities. (C) 1,25(OH)2D triggering of the VDR leads to (1) transactivation of the cathelicidin gene via interaction of the 1,25(OH)2DeVDR complex with a VDR enhancer element in its promoter; (2) expression of the cathelicidin gene product; and (3) killing of ingested mycobacteria. (D) Activation of TLR2/1 induces both IL-15 and key components of its receptor, leading to downstream expression of CYP27B1 and VDR (E) TLR-induction of IL-1b activity converges with the VDR pathway, resulting in expression of DEFB4.
FIGURE 93.1
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integral role in the maintenance of this barrier [121]; however, there are emerging data implicating barrier cells as an active participant in the innate immune response in relation to the detection and elimination of invaders. Particularly with respect to the vitamin Dmediated innate immune response to tuberculosis, cells of respiratory lining play important roles. Several studies have demonstrated lung epithelial cells express functional Toll-like receptors capable of being triggered by their cognate ligands [122e124]. Notably, TLR2 expression in primary small airway epithelial cells was strongly induced following stimulation with cytokines IFN-g and TNF-a, both recognized to be important for resistance to tuberculosis [123]. Activation of TLR2 on lung epithelial cells results in both direct and indirect immune modulatory response, including the expression of the antimicrobial peptide hBD-2 and induction of chemokines [122]. In another study, 1,25(OH)2D3 stimulation of lung epithelial cells resulted in the expression of antimicrobial peptide cathelicidin [125]. Taken together, these studies indicate lung epithelial cells provide several key innate immune mechanisms for defense against infection through (1) barrier function, (2) recognition of pathogens through TLRs and (3) induction of direct antimicrobial effectors, i.e. antimicrobial peptides hBD-2 and cathelicidin.
VITAMIN D PATHWAY AND TUBERCULOSIS Genetics of Tuberculosis Many studies have identified genes which may confer some degree of susceptibility to tuberculosis, including: HLA-DR alleles [126e128], NRAMP1 [129], interferon-g signaling [130], SP110 [131], complement receptor-1 [132], and, notably, the VDR [17e21]. However, these studies did not identify a clear-cut host defense mechanism which explains the linkage. Several studies have linked serum levels of 25(OH)D to both tuberculosis disease progression and susceptibility [21,133]. In 1985, a study reported of 40 Indonesian patients with active tuberculosis were treated with anti-tuberculosis chemotherapy, the 10 patients with the highest 25(OH)D levels at the outset of therapy had “less active pulmonary disease” [133]. Another aspect of the vitamin D pathway which has been extensively studied is the VDR itself. There are two major VDR polymorphisms that have been studied in terms of tuberculosis susceptibility with conflicting results: TaqI [18e20] and FokI [20,134], located in exons nine and two of the VDR gene, respectively [135]. Bellamy et al. conclude the tt allele of the TaqI polymorphism protects against tuberculosis; however, studies by
two other groups report no such association [19,134]. Liu et al. report the FokI ff allele is associated with active tuberculosis among the Chinese Han population [20], but there are no other reports concluding an association for FokI ff and tuberculosis. These conflicting associations became clarified in a study examining the relationship between vitamin D deficiency and VDR polymorphisms with tuberculosis in the Gujarati Asians living in West London in the year 2000 [21]. The study reported both the TaqI (Tt/TT) and FokI (ff) alleles were associated with tuberculosis only when the individual exhibited serum 25(OH)D deficiency [21]. One problem with comparing the in vitro studies and these in vivo observations with regard to human tuberculosis is that previous in vitro studies used the active, 1,25(OH)2D metabolite to affect antimicrobial activity, while the association with tuberculosis was with serum 25(OH)D levels.
Role of 25-hydroxyvitamin D on the Innate Immune Response Relatively little is known about the direct effects of 25(OH)D on innate immunity. Hewison et al. found 25(OH)D at physiologic levels (100 nM) suppressed CD40L-induced IL-12 production in day-7 GM-CSF/ IL-4 derived DCs in vitro [136]. Other studies in vitro have shown intracrine metabolism of 25(OH)D to 1,25(OH)2D via endogenous expression of CYP27B1hydroxylase is a more efficient mechanism for modulating the phenotype of either DCs or monocytes compared to the exogenous addition of active 1,25(OH)2D itself [137]. In relation to the adaptive immune response, Yang et al. showed significant blunting of the cell-mediated immune response to cutaneous dinitrofluorobenzene (DNFB) challenge in mice with profound reduction of serum 25(OH)D levels [138]. Administration of 25(OH)D to humans with head and neck squamous cell carcinoma increases plasma IL-12 and IFN-g levels, and improves T-cell blastogenesis [139].
TLRs and Vitamin D In 2006, a potential mechanism by which the 25(OH) D status of an individual may alter their ability to mount an innate immune response against M. tuberculosis was reported. Activation of TLR2/1 on human monocytes and macrophages results in the induction of key genes in the vitamin D pathway (Fig. 93.1B), including the vitamin D receptor (VDR) and CYP27B1. Under conditions where the extracellular concentration of 25(OH)D is present at sufficient levels, TLR2/1 activation of monocytes results in a CYP27B1- and VDR-dependent
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VITAMIN D PATHWAY AND TUBERCULOSIS
expression of the antimicrobial peptide, cathelicidin, and direct microbicidal activity against intracellular M. tuberculosis (Fig. 93.1C). The induction of CYP27B1 and VDR in monocytes was subsequently demonstrated to be mediated through the actions of TLR2/1-induced IL-15 expression (Fig. 93.1D) [140]. Interestingly, the human but not the murine cathelicidin promoter contains an activating VDRE [56], perhaps suggesting a point of divergent evolution between mice and humans in the antimicrobial effectors used by the TLRmediated innate immune response. Given the fact that murine species are nocturnal and therefore will have limited opportunity for vitamin D synthesis, the importance of high vitamin D levels to their immune response is unclear. In addition to cathelicidin, TLR activation of human monocytes results in the vitamin D-dependent expression of DEFB4, an antimicrobial peptide gene also characterized with a VDRE in its promoter [55,141]. Convergence of IL-1b and vitamin D transcriptional activation was required for the TLR-induced expression of DEFB4 (Fig. 93.1E). Triggering of TLR2/1 was found to modulate IL-1b activity by increasing the cell’s responsiveness to IL-1b through the simultaneous secretion of IL-1b, upregulation of cell surface IL-1R1 and downregulation of baseline IL-1 receptor antagonist (IL-1RA) [141]. These findings provide a potential molecular mechanism for the previously known associations of IL-1b and IL1RA polymorphisms with tuberculosis [142], as well as the requirement for the IL-1R1 in host defense against M. tuberculosis [143]. Inhibition of the VDR, as well as knockdown of cathelicidin or DEFB4, resulted in ablation of the TLR2/1-induced antimicrobial activity, implicating VDR activation as a critical step in the innate immune response against M. tuberculosis [10,141]. This potentially explains the association of low 25(OH)D serum levels with susceptibility to tuberculosis; where low 25(OH)D levels in circulation cannot provide sufficient substrate 25(OH)D for CYP27B1-mediated production of 1,25(OH)2D and subsequent activation of the VDR-dependent antimicrobial. Interestingly, TLR activation of bovine macrophages in the presence of 25(OH)D3 resulted in CYP27B1 activity and expression of iNOS [115]. This demonstrates an evolutionary conservation of the TLR-induced vitamin D-dependent antimicrobial pathway as a mechanism of host defense with the exception of the end result, where humans induce antimicrobial peptides and ruminants utilize iNOS [10,115]. It remains to be seen if other diurnal species also maintain this conservation. Through rheostatic regulation of CYP27B1 activity and conversion of substrate 25(OH)D to produce 1,25(OH)2D, the macrophage directly controls its intracellular level of 1,25(OH)2D [144]. These data suggest it is serum 25(OH)D, and not the serum 1,25(OH)2D
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concentration, which controls the intracellular 1,25(OH)2D level that is essential for the TLR-induced antimicrobial activity. This explains why in previous experiments in vitro, a super-physiologic concentration of 1,25(OH)2D in the conditioning extracellular media was required to generate sufficient intracellular levels of the metabolite to affect the VDR and to achieve an antimicrobial effect in human macrophages [50,51].
Clinical Relevance This requirement of adequate 25(OH)D in the extracellular environment of the human macrophage for the induction of host defense mechanisms via TLR2/1 provided a link between two well-documented clinical observations: compared to lightly pigmented human populations, darkly pigmented black individuals are (1) more susceptible to virulent infections of tuberculosis and (2) have lower circulating serum 25(OH)D levels owing to their relatively diminished capacity to synthesize vitamin D in their skin during sunlight exposure. The biosynthetic pathway of 25(OH)D in humans involves the absorption of ultraviolet B (UVB) photons from sunlight by 7-dehydrocholesterol (7HDC) in the basal layer of the epidermis and its non-enzymatic conversion to a pre-vitamin D3 precursor in the skin; in fact, the melanin in pigmented skin will competitively absorb these UVB rays preventing this photoreaction [145]. In human monocytes cultured in sera from pigmented AfricaneAmerican subjects and stimulated with a TLR2/1 ligand, there was no upregulation of cathelicidin mRNA, whereas the same human monocytes conditioned in sera from lightly pigmented subjects did. Moreover, supplementation of the African-American sera with exogenous 25(OH)D3 restored the induction of cathelicidin mRNA [10]. In recent studies, the ability of monocytes from human subjects to mount a cathelicidin response following TLR challenge has been shown to be directly proportional to circulating levels of 25(OH)D but not 1,25(OH)2D [146]. Importantly, this study also demonstrated TLR-induction of cathelicidin was enhanced in subjects supplemented with vitamin D (500 000 IU vitamin D2 over 5 weeks), indicating the immunomodulatory effects of 25(OH)D also occur in vivo. Another study demonstrated a single oral dose of vitamin D was able to increase the in vitro response to mycobacterium infection in a whole blood assay [147]. Furthermore, the TLR2/1L-induced, vitamin-D-dependent antimicrobial pathway has been shown to involve autophagy mediated through the VDR [148], which highlights another key host defense pathway reliant upon the host’s vitamin D status. The effects of the host 25(OH)D levels could extend beyond the macrophage, as lung epithelial cells have
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been demonstrated to express CYP27B1, and convert 25 (OH)D into 1,25(OH)2D leading to expression of cathelicidin [125]. These studies demonstrate a host deficient in 25(OH)D could suffer impaired innate immune responses from the barrier and innate immune effector cells resulting in increased susceptibility to infection. Therefore, returning circulating levels of 25(OH)D to normal could potentially restore their host defense mechanisms.
HISTORY OF VITAMIN D, SUNSHINE, AND TUBERCULOSIS Establishment of vitamin D’s role in host defense against tuberculosis provides new insights into the historical understanding of tuberculosis treatment prior to the advent of antibiotics. In the late 19th century, two young physicians, who themselves had contracted tuberculosis, were instructed by their physicians to travel to mountainous regions of Europe during the summertime as part of their attempt to recover. Their trek into this high UVB environment led to the “remission” of their disease. As a consequence of this success, Hermann Brehmer built the world’s first high-altitude tuberculosis sanatorium in Germany, designed to allow patients to be exposed to “fresh air and sunlight.” At about the same time in the USA, Edward Livingston Trudeau of New York published his original scientific finding that rabbits infected with tuberculosis had a more severe course of disease if caged indoors in the dark as opposed to being kept outdoors on a remote island. These experimental observations led him to build the first sanatorium at Saranac Lake, NY. In fact, it was the success of treatment facilities like these which paved the way to the 1903 Nobel Prize in Medicine awarded to the Danish physician Niels Ryberg Finsen, who demonstrated that UV light was beneficial to patients with lupus vulgaris, a form of cutaneous M. tuberculosis infection. Despite widespread skepticism about the value of sanatoria at the time and since then, it is likely that the prolonged exposure to sunlight increased cutaneous vitamin D production, increased substrate 25(OH)D levels and enhanced innate immunity to combat tuberculosis. There is a long history of using vitamin D to treat mycobacterial infections with apparent success. In 1946, Dowling et al. reported the treatment of patients with lupus vulgaris with oral vitamin D [149]. Eighteen of 32 patients appeared to be cured, nine improved. Morcos et al. treated 24 newly diagnosed cases of tuberculosis in children with standard chemotherapy with and without vitamin D [150]; they noted more profound clinical and radiological improvement in the group treated with vitamin D [150]. Nursyam et al.
administered vitamin D or placebo to 67 tuberculosis patients following the 6th week of standard treatment [151]. Out of a total of 60 patients, the group with vitamin D had higher sputum conversion rate and radiological improvement (100%) than the placebo group (76.7%). This difference was statistically significant (p ¼ 0.002). Despite the clear benefits of vitamin D treatment for tuberculosis, the mechanism of action had not been elucidated. The fact that TLR-activated macrophages can convert vitamin D to produce antimicrobial peptides could be a possible mechanism by which supplementation of patients with inactive vitamin D leads to a positive therapeutic outcome. Progress in curtailing the human death rate from tuberculosis has been hampered by access to, cost and effectiveness of current antibiotic regimens [152]. Some of these problems could potentially be overcome by adding vitamin D to the treatment regimen of tuberculosis. Although the currently published studies on the effects of vitamin D supplementation are generally inadequate to evaluate the efficacy of such treatment [147], a single oral dose of 50 000 IU of vitamin D has been shown to enhance killing of mycobacteria by whole blood of healthy volunteers [153]. As such, knowledge of the role of human vitamin D metabolism and action in the basic innate immune defense mechanisms against mycobacterial infection provides hope in the development of safe, simple and cost-effective strategies in the near future to prevent and treat tuberculosis.
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C H A P T E R
94 Vitamin D and Diabetes Conny Gysemans, Hannelie Korf, Chantal Mathieu Katholieke Universiteit Leuven, Leuven, Belgium
INTRODUCTION Diabetes mellitus is a common disease in the Western world, with an estimated prevalence of 4 to 5%. The majority (95%) of diabetic patients suffer from type 2 diabetes or non-insulin-dependent diabetes, a metabolic syndrome characterized by insulin resistance and relatively inadequate insulin production by the beta cell in the pancreatic islets of Langerhans [1]. In this metabolic syndrome it is still unclear whether the primary dysfunction is situated in the peripheral insulin target organs (being mainly liver, fat, and skeletal muscle) [2] or in the beta cell itself [3,4]. Insulin resistance is induced by obesity and sedentary lifestyle and is also involved in the pathogenesis of cardiovascular disease. This insulin resistance is probably the major determinant of the disease, but beta cell dysfunction is always present and will determine the severity of the clinical presentation. Type 1 diabetes, also known as juvenile or insulin-dependent diabetes mellitus, is a totally different disease in etiology. It has become clear in recent years that this disease is an autoimmune disorder, characterized by a destruction of the insulin-producing beta cells in the pancreas by the body’s own immune system [5]. Whereas type 2 diabetes is a typical disease of the obese and aging patient, type 1 diabetes mainly occurs in children and adolescents. Since expression of the vitamin D receptor (VDR) has been described in the majority of immune and metabolic cell types involved in the pathogenesis of both types of diabetes [6,7], scientists and clinicians have been intrigued by a possible role for these molecules in the disease process, but even more so, their therapeutic potential in the prevention of disease progression [7e9]. In this chapter, the main effects of vitamin D, and its activated form, on the beta cell, with direct implications for the pathogenesis and prevention of mainly
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10094-0
type 2, but also type 1 diabetes, will be described and discussed. Furthermore, the role of vitamin D and 1,25(OH)2D3 in the prevention of type 1 diabetes and its effects on the immune system of the animal models for the disease will be addressed. Special attention will also be given to emerging evidence of correlations between polymorphisms in the vitamin D metabolism and risk for type 1 and type 2 diabetes.
VITAMIN D AND THE BETA CELL The actions of vitamin D are not limited to skeletal health benefits and may extend to preservation of insulin secretion and insulin sensitivity. Since the early observations in 1980 by Norman et al. [10], that pancreatic insulin secretion is selectively inhibited by hypovitaminosis D, several reports have demonstrated an active role for vitamin D and especially its bioactive form, 1,25(OH)2D3, in the regulation of endocrine pancreas function, especially the beta cell. An important role for vitamin D is suggested by the presence of the VDR in pancreatic beta cells [6], the expression of CYP27B1hydroxylase in pancreatic beta cells [11], and the presence of a vitamin D response element in the human insulin receptor gene promoter [12]. 1,25(OH)2D3 directly activates transcription of the human insulin receptor gene, stimulates the expression of the insulin receptor, and enhances insulin-mediated glucose transport in vitro [13]. Counter-regulatory mechanisms also exist, as illustrated by the fact that a rat insulinoma cell line RINm5F cell expresses the calcium-binding protein, calbindin-D28k (CaBP-D28k), in addition to the VDR [14]. CaBP-D28k may control the rate of insulin release via regulation of intracellular calcium. Presence of CaBP-D28k results in reduced calcium influx through voltage-dependent L-type calcium channels and
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enhanced sensitivity of the channels to calcium-dependent inactivation [15,16]. 1,25(OH)2D3 is known to stimulate a wide array of non-genomic responses including rapid insulin secretion by the pancreatic beta cells [17,18]. A controversy regarding the identity of receptors that mediate these non-genomic transcription-independent responses to steroids is presently attracting extensive scientific interest. In the case of the VDR, its ligand binding domain alone seems to be sufficient to exert some of these responses [19].
Effect of Vitamin D Metabolites on Beta Cell Function Effect of Vitamin D Metabolites in Vitro The beneficial effects of vitamin D on beta cell function were convincingly demonstrated by experiments showing that insulin secretion by islets from normal animals could be enhanced by glucose challenge in the presence of high doses of 1,25(OH)2D3 [17,20,21]. However, interpretation of these findings was sometimes obscured by the numerous different methodologies used (such as the duration of incubation with 1,25(OH)2D3, the animal source of the islets, and the type of glucose challenge); nevertheless, overall improved beta cell function was observed in the presence of exogenous 1,25(OH)2D3. Interestingly, some authors also studied the effects of 1,25(OH)2D3 on insulin synthesis. Beta cell function was also normalized or increased by 1,25(OH)2D3 [22,23]. These studies contributed to our current understanding of the mechanism by which 1,25(OH)2D3 acts on insulin secretion and additionally implicated that stimulation of islets by 1,25(OH)2D3 significantly increases the levels of cytosolic calcium, suggesting a possible link between these two parameters [24,25]. Controversy still exists on whether only an influx of extracellular calcium is responsible for this rise or whether also mobilization of intracellular calcium reserves is involved [20,24,26]. Calcium is known to be important for the secretion of insulin by the beta cell. The active vitamin D 1,25(OH)2D3 may also regulate insulin secretion and synthesis by facilitating the conversion of pro-insulin into insulin [27]; this reaction is known to be dependent on the cleavage by calcium-dependent endopeptidases in the pancreatic beta cells. It has been suggested that 1,25(OH)2D3 could directly modulate beta cell growth and differentiation [6,28] (Fig. 94.1). The effects of 1,25(OH)2D3 have been examined in the context of binding to the nuclear VDR as well as to a membrane VDR, through which genomic and non-genomic responses are proposed to be induced, respectively. Among these studies, Sergeev and Rhoten have reported that the administration of 1,25(OH)2D3
can evoke oscillations of intracellular calcium in a pancreatic beta cell line within a few minutes [20]. Also, the 6-s-cis analog, 1,25(OH)2lumisterol3, has a rapid insulinotropic effect, possibly by triggering non-genomic signal transduction pathways as a result of binding to a membrane-bound VDR, an event that depends on the augmentation of calcium influx through voltage-dependent calcium channels in the plasma membrane [29]. As type 2 diabetes has recently been linked to systemic inflammation, and consequently insulin resistance, 1,25(OH)2D3 may also improve insulin sensitivity and beta cell function by directly modulating the generation and effects of inflammatory cytokines [30], as will be discussed in “Effects of vitamin D metabolites on beta cell health,” below. Effect of Vitamin D Metabolites in Vivo e (Pre)clinical Application Based on the observations of vitamin D metabolites on beta cell function in vitro, both animal as well as human trials with vitamin D3 or 1,25(OH)2D3 on glucose metabolism have been performed. In the leptin-deficient ob/ob mouse model, treatment with 1a(OH)D3, a synthetic precursor of 1,25(OH)2D3 (also known as alfacalcidol), improved hyperglycemia, hyperinsulinemia, and responsiveness of fat to hormones [31]. Although vitamin D3 supplementation in the obese, spontaneously hypertensive rat (SHR) did not adjust glycemic status in every rat, 40% of them had a 60% reduction in their blood glucose levels [32]. In obese Wistar rats, a significant decline in glucose levels in all animals supplemented with vitamin D3 was found [32]. In addition, feeding of vitamin-Dcontaining cod liver oil to streptozotocin-induced diabetic rats partially improved their blood glucose levels as well as their cardiovascular and metabolic abnormalities [33]. From these preclinical data it is reasonable to consider that vitamin D and its metabolites have the potential to positively influence the glycemic status and as such prevent or ameliorate diabetes, at least in cases of (mild) vitamin D deficiency. In spite of this, trials in humans yielded conflicting results. Interesting are the studies on the effects of 1,25(OH)2D3 repletion in the highly 1,25(OH)2D3-deficient state of uremia [34]. For example, uremic patients with low 1,25(OH)2D3 serum levels were daily treated with 0.5 mg of 1,25(OH)2D3 combined with 500 mg of calcium during a period of 3 weeks. Remarkably, the treatment caused a rise in glucose-induced insulin release only in the first few minutes of the stimulation (in uremia especially this rapid phase of insulin release is disturbed). However, repletion of 1,25(OH)2D3 could not completely reverse glucose intolerance. On the other hand, Orwoll et al. investigated the possible clinical applications of
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inflammation ↓ ↓ insulin resistance ↓ glucose intolerance
chemokines ↓ MHC-I and -II ↓ Fas ↓ NO ↓ pancreas
adipose tissue
islet viability? insulin secretion ↑ insulin synthesis ↑ pro-insulin to insulin ↑
obesity? liver
skeletal muscle
1,25(OH)2D3 in a situation of impaired insulin secretion without vitamin D deficiency [35]. Type 2 diabetic patients received 1,25(OH)2D3 (1 mg/d during 4 days) in a double-blind, placebo-controlled cross-over survey. Clear effects of 1,25(OH)2D3 treatment were noted on parameters of calcium metabolism, but it could not be concluded if vitamin D deficiency increased the risk of developing type 2 diabetes. Although the latter result could be attributed to the short duration of 1,25(OH)2D3 treatment, it still provided valuable information. Recently, a New Zealand study found that South Asian women with insulin resistance improved markedly after taking vitamin D3 supplements (4000 IU daily) for period of 6 months [36]. Optimal vitamin D concentrations for reducing insulin resistance were shown to be between 32 and 48 ng/mL [36,37]. On the other hand, a randomized, double-blinded, placebocontrolled pilot study on 28 Asian Indian subjects with type 2 diabetes did not find beneficial effects of intramuscular vitamin D3 administration (300 000 IU) on glucose tolerance, insulin secretion or insulin sensitivity [38]. In this trial, basal serum 25(OH)D3 concentrations were ~15 ng/mL and increased to 42 ng/mL in the vitamin D3 supplemented group, while the placebo group remained vitamin D insufficient (30 ng/mL) until trial termination. Currently, serum levels of 30e50 ng/mL 25(OH)D meet the requirements for vitamin D sufficiency; however, the optimal serum 25(OH)D levels may be much higher. For instance, Jorde and Figenschau also failed to demonstrate increased insulin secretion in patients with type 2 diabetes after supplementation with 40 000 IU vitamin D3 per week [39] Moreover, the Women’s Health Initiative (WHI) reported that daily supplementation with 400 IU of vitamin D3 in combination with 1000 mg of calcium did not show protection against type 2 diabetes [40], although having a small effect on the prevention of
FIGURE 94.1 Vitamin D3 and its active metabolite 1,25(OH)2D3 play important roles in glucose homeostasis via different mechanisms. Vitamin D3 (solid line) is not only essential for normal beta cell functioning, but also improves insulin sensitivity of the target cells (liver, skeletal muscle, and fat tissue). In addition, 1,25(OH)2D3 (dashed line) protects the beta cell from detrimental immune attacks, by its direct actions on the beta cell. 1,25(OH)2D3 improves markers of systemic inflammation in type 2 diabetes but it is unclear whether vitamin D (deficiency) can be linked to obesity.
weight gain [41]. In this study, participants featured starting 25(OH)D3 levels of ~18 ng/mL, which increased by 28% in women within the active calcium plus vitamin D group. It was suggested that higher doses might be required to affect diabetes risk. However, a daily dose of 800 IU of vitamin D3 alone or combined with 1000 mg calcium also failed to prevent type 2 diabetes as reported by the RECORD survey group [42]. In three cases of British Asians with vitamin D deficiency and type 2 diabetes, vitamin D2 supplementation even led to increased insulin resistance and deteriorated glycemic control [43]. As for vitamin D2, it has several unknown metabolites with unknown effects. Nevertheless, the contradictory results of vitamin D supplementation in type 2 diabetes suggest that especially the dose, methodology, and duration of supplementation, as well as ethnicity and baseline vitamin D status, appear to be important for the protective efficacy of vitamin D supplementation against development of type 2 diabetes [44,45].
Effect of Vitamin D Deficiency on Beta Cell Function Vitamin D deficiency can be defined according to population-based reference limits for serum 25(OH)D and linked biological indices like PTH levels [46]. A link between vitamin D deficiency and the appropriate function of the pancreatic beta cells in animal models and humans has been described [22,47e50]. The earliest observations mention important effects of vitamin D deficiency on insulin synthesis, secretion and action, but not on the other pancreatic hormones, with restoration of these defects by exogenous administration of vitamin D [22]. In particular, hypovitaminosis D affects the glycolytic pathway after the D-glyceraldehyde step and mainly alters oxidative events within the
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tricarboxylic acid cycle (TCA), which could lead to beta cell dysfunction and death [51]. The effects of vitamin D deficiency and repletion on glucose homeostasis in vivo are diverse and involve not only the beta cell. Vitamin-D-deficient animals do not have a normal food intake, lose weight and cannot maintain normal mineral metabolism or bone formation and mineralization [52]. The most dramatic metabolic effect of vitamin D deficiency is impaired absorption of calcium from the intestinal lumen of the gut, possibly leading to secondary hyperparathyroidism; it is implicated that serum PTH is relatively high for the associated serum calcium concentration. As calcium is essential for intracellular processes associated with insulin secretion [53], it has been suggested that hypocalcemia by itself is able to reduce insulin secretion and, possibly, endogenous glucose production through a reduced drive for calcium entry following glucosestimulated closure of the ATP-sensitive potassium channels on the beta cells [25,54]. Yet, vitamin D deficiency (serum 25(OH)D levels of 15 ng/mL) has been associated with higher risks for metabolic syndrome and insulin resistance [55]. A large cross-sectional National Health and Nutrition Examination Survey (NHANES) demonstrated that serum 25(OH)D is inversely correlated with type 2 diabetes incidence and insulin resistance [56,57]. These data are in line with results from the Medical Research Council Ely Study 1990e2000 which also found an inverse relationship between 25(OH)D levels and blood glucose concentrations [58]. An extended investigation from New Zealand found decreased 25(OH)D levels in individuals with recently diagnosed glucose intolerance and type 2 diabetes after adjustment for obesity, gender, age, ethnicity, and season [59]. At present it is unclear whether vitamin D deficiency itself contributes to obesity [60]. Nonetheless, the absolute fat mass is inversely related to serum 25(OH)D levels [61] and correlates positively with serum PTH levels [62]. This may be due to the great capacity of adipose tissue to store vitamin D and thus make it biologically unavailable. Increased PTH levels and decreased 25(OH)D3 levels can increase intracellular calcium in adipocytes, consequently modulating lipolysis and predisposing to weight gain. Interestingly, leptin, the serum concentrations of which positively correlate with obesity, normalizes the 1a(OH)ase and 24(OH)ase activities and corrects elevated calcium, phosphate, and 1,25(OH)2D3 levels in ob/ob mice, implicating a role for leptin in downregulating 1,25(OH)2D3 synthesis [63]. Vitamin D deficiency in obese patients has been linked to secondary hyperparathyroidism [64] that might contribute to type 2 diabetes development, as elevated PTH levels are linked to glucose intolerance.
A recent Italian study suggests, however, that there is no causeeeffect correlation between vitamin D and insulin sensitivity and that in obese subjects, both low 25(OH)D3 levels and insulin resistance appear to be dependent on the increased body size [65]. On the other hand, current data suggest that type 2 diabetic patients with vitamin D deficiency have elevated C-reactive protein (CRP), fibrogen, and hemoglobin A1c (HbA1c) levels compared to healthy controls [66], indicating that inflammation elicited by immune cell subsets (e.g., possibly macrophages) are implicated in insulin resistance and type 2 diabetes. We reported that exogenous administration of vitamin D ameliorates markers of systemic inflammation, which are typically found in type 2 patients, thereby possibly improving beta cell function and survival [30]. Vitamin D deficiency is reported to be more common in type 2 diabetes than in type 1 diabetes, unrelated to age, sex, or insulin treatment [67]. Although studies examining vitamin D deficiency in type 1 diabetic patients have been limited, 25(OH)D levels were found to be lower in patients with type 1 diabetes at the time of diagnosis compared with control subjects [68]. A recent cross-sectional study demonstrated that of the 128 participants with type 1 diabetes, most had inadequate levels of 25(OH)D: 61% insufficiency and 15% deficiency compared to 24% sufficiency [69]. The criteria used to define vitamin D sufficiency, insufficiency, and deficiency were 25(OH)D levels of 30 ng/mL, 21e29 ng/mL, and 20 ng/mL, respectively. Moreover, inadequate 25(OH)D levels were most prevalent in the age group from 12 to 18 years, with 75% meeting criteria for vitamin D insufficiency or deficiency [69]. Most of these studies do not address the question whether the high frequency of vitamin D inadequacy has adverse effects on metabolic (beta cell) function of the study participants. In the type 1 diabetes-prone NOD mouse, a deficiency in vitamin D leads to impaired glucose tolerance, with decreased insulin secretion of the islets in vitro and earlier onset of diabetes in vivo, leading to a final increased diabetes incidence [70]. In this model, however, the beta cell as well as immune factors may contribute to the increase in diabetes incidence. Data from VDR knockout mice are conflicting, with some groups reporting impaired glucose tolerance [71], while in other reports no impairment of the glucose metabolism is described in mice with mutations in the VDR [72]. Here, the genetic background of the mouse in which the knockout for VDR was introduced is possibly of critical importance. Similar to the latter study, Hochberg et al. found glucose curves within the normal range when they performed oral glucose tolerance tests in a small number of patients with vitamin-D-dependent
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rickets type II (a rare genetic disorder caused by mutations in the VDR) [73].
Effects of Vitamin D Metabolites on Beta Cell Health In the pathogenesis of type 1 diabetes, considerable evidence indicates that the cytotoxic action of proinflammatory cytokines on pancreatic islets in vitro is mediated through generation of free radicals and induction of ER stress [74,75]. Yet, it is unclear how these cytokines act to promote beta cell destruction. We and others propose that these inflammatory cytokines serve to predispose pancreatic beta cells for lysis by auto-reactive T cells. Sandler et al. demonstrated that 1,25(OH)2D3 and some of its synthetic analogs counteract the suppressive effects of IL-1b on beta cell function, such as insulin synthesis as well as release [76]. On the other hand, Mauricio et al. could not reproduce the positive effects of 1,25(OH)2D3 and some of its analogs (KH1060 and MC1288) on IL-1b-induced beta cell dysfunction [77]. The main difference between the papers is the incubation time of the cytokines with the beta cells; in the former work incubation periods range between 48 and 72 hours, while in the latter experiments islets were only incubated with 1,25(OH)2D3 for 24 hours. Of note, in our hands 1,25(OH)2D3 does not provide protection against cytokine-induced beta cell death in vitro even when using variable incubation times and multiple cellular islet systems. We found that treatment with 1,25(OH)2D3 did not protect the rodent insulinoma INS-1E cell line and rat FACS-purified single beta cells against cytokine-induced cell death in vitro [78]. Not only were the effects of cytokines on beta cell function altered by 1,25(OH)2D3 (at concentrations ranging from 108 to 106 M), but also the induction of surface markers by these cytokines appears to be blocked. When neonatal rat islets were incubated with T helper (Th)-1 IFNg, several surface markers, including those linked to antigen presentation-like MHC-II molecules, were, as expected, upregulated, while co-incubating the islets with 1,25(OH)2D3 or some of its analogs decreased markedly the upregulation of MHC-II molecules after IFNg stimulation [79]. Moreover, treatment of beta cells with 1,25(OH)2D3 has been reported to directly protect against beta cell death by reducing density of MHC class I molecules [79], inducing expression of anti-apoptotic A20 protein [80] and decreasing expression of Fas [81] (Fig. 94.1). Complementing these findings, we observed that exposure to 1,25(OH)2D3 in three cell systems (e.g., INS-1E beta cell line, fluorescence-activated cell sorting purified rat beta cells, and NOD-severe combined
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immune-deficient islets) suppressed IP-10 and IL-15 expression in the beta cell itself but did not prevent cytokine-induced beta cell death [78]. These data showing beta cell protection against inflammatory agents involved in the pathogenesis of type 1 diabetes may have direct implications for the observed in vivo effects of 1,25(OH)2D3 and its analogs in prevention of type 1 diabetes in animal models. Indeed, we propose that this 1,25(OH)2D3-induced inhibition of chemokine expression in beta cells is associated with a decreased diabetes incidence by targeting early insulitis.
VITAMIN D AND THE IMMUNE SYSTEM IN TYPE 1 DIABETES The VDR is present in almost every cell of the immune system, including macrophages, dendritic cells (DC), B and T cells, allowing them to respond to the active ligand [7]. Therefore, a physiological role for 1,25(OH)2D3 as a messenger or cytokine-like molecule in the immune system is reasonable, and so far many studies yielded beneficial results using active vitamin D to prevent and/or intervene in different autoimmune disease models including type 1 diabetes [82,83]. Results in NOD mice are promising [84], but many obstacles to human application still exist. Clinical application of 1,25(OH)2D3 is hindered by toxicity, since the supra-physiological doses needed to shape the immune system elicit hypercalcemic side effects [85]. All studies involving long-term immune suppression are inconceivable as strategy for the prevention of a chronic disease striking mainly children; moreover, the preliminary results on the beneficial effects of these drugs in recent-onset diabetic subjects are disappointing. Interestingly, most of the aforementioned immune cells are able to convert 25(OH)D3 into 1,25(OH)2D3 as they express the CYP27BI gene (1a(OH)ase), while dendritic cells (DC) also express a 25-hydroxylase (25(OH)ase) [86e88]. Local processing of vitamin D precursors into the bioactive metabolite may represent an important mechanism by which immune cells can reach these supra-physiological levels of 1,25(OH)2D3 inside the cell, which are locally needed to modulate immune responses autonomously, without affecting systemic levels of this hormone [89]. These intracrine, autocrine, and paracrine signaling events are critically dependent on sufficient 25(OH)D levels available to the cell as substrate for the 1a(OH)ase. Therefore, supplementation with vitamin D represents an attractive strategy to both overcome vitamin D insufficiency and deficiency and ensure adequate immune cell function.
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Primary Prevention of Type 1 Diabetes in Animal Models by 1,25(OH)2D3 and its Analogs Early Intervention e Effects of 1,25(OH)2D3 on Type 1 Diabetes Prevention The non-obese diabetic (NOD) mouse and biobreeding (BB) rat are the two most commonly used animals that spontaneously develop diseases with similarities to human type 1 diabetes [90]. The BB rat is characterized by severe immune abnormalities (i.e., T cell lymphopenia in the circulating blood); therefore, most studies on the pathogenesis and development of therapeutic strategies have been performed in the NOD mouse. The NOD mouse was derived from a cataractdeveloping substrain of the outbred JcI-ICR mouse by selective inbreeding from 1974 to 1980. Insulitis, the prototypical (histo)pathological lesion of type 1 diabetes in the pancreas, is present when mice are 4e5 weeks of age. This is followed by subclinical beta cell destruction and decreasing insulin concentrations in the blood. Overt diabetes typically presents between the 12th and 30th weeks of age. Unlike human type 1 diabetes, ketoacidosis is less severe and diabetic animals can survive for weeks without exogenous insulin administration. Also, there is a clear gender difference with 70% of females, and only 40% of males developing diabetes in some colonies. Chronic administration of pharmacological doses of 1,25(OH)2D3 can diminish the incidence of both insulitis and diabetes in NOD mice [85,91,92]. When 1,25(OH)2D3 was administered at a dose of 5 mg/kg body weight or vehicle (arachis oil) i.p. every 48 hours from 3 weeks of age until adulthood (30 weeks of age), insulitis was significantly reduced from 80% in control to 58% in 1,25(OH)2D3-treated NOD mice. At the same time diabetes itself was reduced from 56% to 8%. Although the treatment was generally well tolerated, hypercalcemia and bone decalcification were observed; these data have been confirmed by others [93]. The basis for the diabetes protection was the restoration of suppressor cell function, which could be demonstrated both in vitro and in vivo [92]. Later, Adorini et al. demonstrated that the nature of the suppressor cell induced by 1,25(OH)2D3 (or its analogs) is most likely a CD4þCD25þ regulatory T cell (Treg) [94]. Interestingly, oral administration of 50 ng of 1,25(OH)2D3 (by incorporating the hormone into the diet;) was able to fully protect NOD mice from developing diabetes through 30 weeks of age [93]. Unfortunately, the serum calcium levels significantly increased in all mice fed with a daily dose of 50 ng 1,25(OH)2D3. Unfortunately, the serum calcium levels significantly increased in all mice fed with a daily dose of 50 ng 1,25(OH)2D3. This dose is the highest tolerable dose in NOD mice and at
the edge of toxicity. In this study, the spontaneous disease incidence of the control mice was unusually low, possibly because a low-calcium purified diet was fed which is often inherently diabetes-protective. When mice were given a normal-calcium full-grain diet, 1,25(OH)2D3 treatment did not reduce diabetes incidence to previously reported levels (32% diabetic for females at 20 weeks of age) [95]. The question remains, however, whether the restoration of suppressor cells is the main mechanism involved in protection against diabetes by 1,25(OH)2D3. Protection against the clinical disease and against insulitis was seen, pointing towards interference with the induction of (auto)immune responses themselves. Diabetes occurring in NOD mice after cyclophosphamide (CTX) administration is demonstrated to be due to a reduction in naturally occurring Tregs [96,97]. Tregs, characterized by a combination of surface markers including CD25, CD103, CTLA-4, CD62L, glucocorticoid-induced TNFR family related protein (GITR), glycoprotein-A repetitions predominant (GARP) as well as by expression of the forkhead/winged helix transcription factor (Foxp3), have been implicated as key players in immune tolerance. Shortly after CTX treatment, CD4þCD25þ Tregs are functionally impaired in their suppressive activity in vitro and display higher levels of apoptosis [96]. Whereas cell numbers recover in lymphoid tissues immediately before onset of type 1 diabetes, frequencies of Tregs in the pancreas remain low. Several potential preventive therapies for diabetes have already been tested in this CTX model of diabetes [98,99]. Protection against diabetes by therapeutic interventions that are believed to induce suppressor T cells, but that have no effect on auto-reactive T cells, can be broken by CTX. Casteels et al. demonstrated that 1,25(OH)2D3 can prevent diabetes induced by CTX in the NOD mouse [100]. Insulitis was significantly reduced from 100% in control to 42% in 1,25(OH)2D3-treated NOD mice, while diabetes itself was reduced from 78% to 17%. As such, this protection was achieved despite a total elimination of immunosuppressive cells in the 1,25(OH)2D3-treated group by CTX (as shown by co-transfer experiments). 1,25(OH)2D3 treatment also did not interfere with the quantitative and qualitative recovery of the major lymphoid-hematopoietic cells after CTX administration. Striking was the absence of insulitis in most animals treated with 1,25(OH)2D3. Combined, these data, resistance against CTX and reduction of insulitis together with the absence of protection in co-transfer experiments, suggested that CTX had eliminated Tregs. It appears that these suppressive cells are not the sole protective mechanism in the 1,25(OH)2D3-treated NOD mice. Recently, it was reported that also CD11bþ Ly6G Ly-6Cþ monocytes might contribute to the adjuvant effect of CTX, but no data are available on the
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FIGURE 94.2 At the level of the DC, 1,25(OH)2D3
inhibits the surface expression of MHC-II and of costimulatory molecules, in addition to production of the cytokine IL-12 (and IL-23), thereby indirectly shifting the polarization of T cells from a Th1 towards a Th2 phenotype. In addition, 1,25(OH)2D3 acts as immune modulator directly at the level of the T cell, by inhibiting the production of the Th1 cytokines IL-2 and IFNg, Th17 cytokine IL-17, and stimulating the production of Th2 cytokines like IL-4. Moreover, 1,25 (OH)2D3 favors the induction of Tregs. 1,25(OH)2D3 also improves chemotactic and phagocytic capacity of macrophage (adapted from [7]).
DC
CD
4
MHC-II↓
CD83 ↓ CD86 ↓ CD40 ↓
IL-12 ↓ IL-23 ↓ IL-10 ↑
IL-2 ↓ IFN-g ↓
M
IL-17 ↓
chemotaxis ↑ phagocytosis ↑ IL-4 ↑ CD25hi FoxP3+ IL-10 ↑
effects of 1,25(OH)2D3 on this myeloid cell population and its immunosuppressive capacities [101]. At present, we propose that the basis of diabetes protection by 1,25(OH)2D3 seems to be a reshaping of the immune repertoire (Fig. 94.2), with also direct protective effects of 1,25(OH)2D3 on the beta cell. At the level of the immune system, its regulatory actions involve a shift in T cell cytokine production from predominantly pro-inflammatory Th1 (e.g., IL2, IFNg, and TNFa) and Th17 (e.g., IL17) in control mice to anti-inflammatory Th2 (e.g., IL4, IL10) in 1,25(OH)2D3treated mice [94,102], with 1,25(OH)2D3 acting either directly on T cells [103] or indirectly via effects on antigen-presenting DC [104,105]. Indeed, 1,25(OH)2D3 is able to induce a reshaping of DC towards tolerogenic cells, less capable of stimulating auto-reactive T cells and even inducing anergy in T cells [106e110]. We demonstrated that DC generated in the presence of 1,25(OH)2D3 can redirect already committed T cell clones derived from a type 1 diabetic patient towards non-proliferation [108]. In the NOD mouse, the reshaping of the immune system already happens centrally, in the thymus, where treatment with 1,25(OH)2D3 restores the sensitivity of T lymphocytes towards apoptosis-inducing signals, allowing only “activationinduced cell death”-sensitive T cells to reach the periphery [100,111,112]. Moreover, it has been suggested
that 1,25(OH)2D3 triggers the induction of DC differentiation in the thymus of NOD mice, consistent with the modulation to a more pronounced lymphoid phenotype and upregulation of CD86 [112]. At the level of the beta cell, we observed that treatment of diabetes-prone NOD mice with 1,25(OH)2D3, in vivo, initiated before insulitis onset, not only diminished the severity of islet infiltration and preserved beta cell functionality during the course of insulitis but also decreased the levels of IL-1b, IL-15, and CXCL10 levels in the islet cells from NOD mice at 8 and 10 weeks of age [78]. We hypothesize that the reduced expression of inflammatory cytokines as well as inhibition of chemotaxis of auto-reactive T cells and macrophages to the islets by 1,25(OH)2D3 might limit the development of insulitis in vivo by affecting the migration and recruitment of inflammatory cells. Taken together, protection against insulitis and diabetes in NOD mice by 1,25(OH)2D3 may be due to a combination of effects on both effector (e.g., immune cells) and target (e.g., beta cells) cells of the autoimmune process culminating in type 1 diabetes. Studies on diabetes prevention in the NOD mouse are probably the most relevant for direct application of the findings in the human situation, but the fluctuating diabetes incidence of the stock colonies and the duration of most intervention studies make this model
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time-consuming for the screening of large groups of potentially new therapeutic agents. A faster-developing, more dependable model is the multiple lowdose streptozotocin model [113]. Streptozotocin is an antibiotic produced by Streptomyces achromogenes that has a specific beta cell toxic effect. A single high dose of streptozotocin (70e250 mg/kg body weight) causes a rapid and complete destruction of beta cells in most species, probably as a result of direct toxic effects. On the other hand, the administration of multiple subdiabetic doses of streptozotocin (e.g., 50 mg/kg on 5 consecutive days) in susceptible animals causes more subtle beta cell damage followed by insulin deficiency. A major criticism of this model is the fact that the complete scenario of beta cell destruction in this model is unclear and that diabetes is probably the result of non-specific inflammatory damage of the beta cell together with other islet cells [114]. Accordingly, this diabetes model is not considered a true autoimmune model. This is further supported by the fact that disease progression is not dependent on the presence of an autoimmune response, since administration of this agent results in diabetes induction even in the absence of functional T and B cells [115]. It remains questionable, however, whether type 1 diabetes is a true autoimmune disease and a consequence of non-specific beta cell destruction by one or other inflammation (e.g., viral infection) in some humans. In the model of low doses of streptozotocin (60 mg/ kg body weight) treatment with 1,25(OH)2D3 (250 ng, three times a week) reduced diabetes incidence from 85% in control to 45% in rats [116]. At the end of the experiment bone loss was observed in diabetic animals, an event that did not occur in control mice. Interestingly, when diabetic animals were treated with 1,25(OH)2D3, an increase in bone mineral density was observed in cured animals, reaching similar values to those of the control group. Inaba et al. used both the high- and the low-dose model of streptozotocin to test the effect of 1a(OH)D3 on diabetes prevention [117]. The rationale for utilizing 1a(OH)D3 is that a 25(OH)ase will convert it to the bioactive 1,25(OH)2D3 compound in vivo. When diabetes was chemically induced by a single injection (200 mg/kg body weight) of streptozotocin, no protection against diabetes was seen with 1a(OH)D3. When multiple low doses of streptozotocin were administered, however, 1a(OH)D3 reduced the diabetes incidence dramatically in a dose-dependent manner; control mice developed diabetes in 100% of cases, while administration of 0.4 mg/kg of 1a(OH)D3 reduced the diabetes incidence to 46%. Data on toxicity in this study were unfortunately limited to evolution of body weight (unchanged) and no indication of calcemic or skeletal outcomes was mentioned. Histological examination of the pancreas of
these experimental mice demonstrated that 1a(OH)D3 also reduced insulitis. The BB rat is the second spontaneous animal model for type 1 diabetes. The existence of different pathogenic mechanisms for autoimmune diabetes in animal models suggests the existence of a pathogenic scenario that is different in the human situation. In the BB rat, no difference in diabetes incidence between control (20%) and 1,25(OH)2D3-treated rats (29 and 39%) was observed when 1,25(OH)2D3 (0.2 mg/kg/d or 1 mg/kg/2d, respectively) was administered from 3 to 50 days of age [118]. These findings in the BB rat again confirm the basic differences in disease pathogenesis that can be found between the two available animal models for type 1 diabetes and indicate that caution is warranted when transferring findings from either of these models to the human situation. Late Intervention e Effects of 1,25(OH)2D3 on Type 1 Diabetes Progression A relevant question is whether long-term treatment with 1,25(OH)2D3 is necessary for disease protection or whether a short-term intervention would suffice, and, if so, when in the course of the disease this short-term treatment should be administered. Therefore, we designed an experiment in which NOD mice received 1,25(OH)2D3 (5 mg/kg/every 2 days or vehicle (arachis oil) i.p.) in different time windows: during whole lifespan (from 3 until 30 weeks of age); across childhood and puberty (from 3 until 14 weeks of age); and during adulthood (from 14 until 30 weeks of age) [78]. As expected clinical diabetes was reduced from 58% in controls to 20% in the 1,25(OH)2D3-treated group. Mice treated only during youth also had reduced diabetes (35%). When therapy was initiated after the majority of control mice already had insulitis, 1,25(OH)2D3 was unable to protect mice from diabetes development (40%). None of the different treatment regimens had an effect on body weight at the end of the experiment, and total serum calcium levels remained in the normal range. Bone turnover (reflected in serum osteocalcin levels) in the animals treated with 1,25(OH)2D3 from 21 until 200 days of age was, as previously demonstrated, increased. On the other hand, when treatment was either ended or initiated at 14 weeks of age, no bone loss under 1,25(OH)2D3 treatment was seen. Bone turnover is higher during childhood than adulthood and interference with bone remodeling during infancy will leave traces for the rest of life. Indeed, early adulthood is a critical time for peak bone mass accrual. Clinical data on diabetes intervention with 1,25(OH)2D3, starting when decline of beta cell function is already ongoing, are disappointing. A small intervention trial, in which new-onset diabetic children were given a small dose of 1,25(OH)2D3 (0.25 mg/2d) or
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nicotinamide (25 mg/kg/d), showed that they had no improvements of C-peptide levels, although insulin requirements decreased in the 1,25(OH)2D3-treated group [119]. Latent autoimmune diabetes in adults (LADA) is considered to be a subtype of type 1 diabetes, in which the clinical manifestation begins and progresses slowly in adulthood. The patients who received 1a(OH)D3, the synthetic precursor of 1,25(OH)2D3, exhibited a partial preservation of beta cell function in comparison to patients treated with insulin alone [120]. An open study on recent-onset diabetic patients with 1,25(OH)2D3 (0.25 mg daily for 9 months; probably the maximum tolerable dose) revealed no significant safety issues as a result of the therapy but the treatment failed to induce preservation of beta cell function [121]. No differences in AUC C-peptide, peak C-peptide, and fasting C-peptide levels between the treatment and placebo groups were observed at 9 and 18 months after study entry. Moreover, HbA1c and daily insulin requirement were comparable between control and 1,25(OH)2D3treated patients throughout the study follow-up period. The latter investigation is in line with findings in the NOD mouse, concluding that there was no benefit when 1,25(OH)2D3 was given late in the disease course (i.e., at diabetes onset). Analogs of 1,25(OH)2D3 A major obstacle to the human application of 1,25(OH)2D3 is its adverse effects on calcium and bone metabolism. New structural analogs of 1,25(OH)2D3 have been reported displaying a clear dissociation of calcemic and anti-proliferative effects [122,123]. Some chemical modifications of the side chain and A-ring result in “super-analogs” with 10- to 100-fold more activity on cell differentiation and the immune system than 1,25(OH)2D3. Lately, non-steroidal analogs of 1,25(OH)2D3, lacking either the full five-membered Dring (C-ring analogs) or the full six-membered C-ring (D-ring analogs) are designed and documented to be very effective inhibitors of cell proliferation or inducers of cell differentiation than is 1,25(OH)2D3 [123,124]. Interestingly, several of the vitamin D analogs possess more potent immunomodulatory properties, resulting in protection against autoimmunity and prolongation of allograft survival [104,125e127]. Some of the most promising analogs coming from different chemical laboratories have been tested in the NOD mouse [125] as well as in the BB rat [128]. The mechanism of protection against insulitis and diabetes appears to be similar to that of the mother compound, 1,25(OH)2D3. Effects of the analogs have been described on the morphology, cell surface marker expression, endocytic, and migratory capacity of DC, the induction of Treg cells (e.g., CD4þCD25þFoxP3þ), the redirection of T cells to sites of inflammation, and beta cell protection have been
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described [7]. Exposure to vitamin D analogs enhances both chemotactic and phagocytotic capacity of monocytes/macrophages, while their antigen-presenting cell function decreases [129]. Moreover, 1,25(OH)2D3 analogs alter significantly the antigen-presenting cell function of DC [130]. Treatment with vitamin D analogs during both in vitro and in vivo maturation inhibits the surface expression of MHC class II and of co-stimulatory molecules, such as CD86, CD80, and CD40 and inhibits immunity after stimulation with antigen. In these cells, production of IL-10 is stimulated and production of IL-12 inhibited, which leads to suppression of T cell activation [7,130]. In the search for the optimal analog, a combination of beta cell protection, immune modulation and low calcemic effects is sought after. Until now, several analogs are promising, but before embarking on long-term interventions in high-risk individuals for type 1 diabetes, longterm safety data will have to be gathered. Combination with Other Immune Modulators In animal models of type 1 diabetes, disease prevention can be achieved by chronic use of a myriad of different drugs like 1,25(OH)2D3, azathioprine, cyclosporine A, tacrolimus, nicotinamide, and anti-CD3 while disease intervention is only (partially) possible by some of these agents like anti-CD3 [131]. The development of drugs that are highly selective and yet produce minimal toxicity to the host remains one of the most complicated challenges in (autoimmune) disease protection. Moreover, since the majority of diseases are treated with drugs in combination rather than single agents, one practical approach to circumvent toxicity is to develop new therapeutic medication that will potentiate the effectiveness of current (clinical) protocols. This strategy would accelerate the acceptance of new drugs as adjunct strategies since these molecules could be used at concentrations well below their maximal tolerable doses. Azathioprine can cause pancreatitis and bone marrow suppression, which may increase the risk of infection or serious bleeding [132]. Common adverse effects of calcineurin inhibitors like cyclosporine A and tacrolimus are bowel disturbance, nephrotoxicity, neurotoxicity as well as serious side effects on pancreatic beta cell function, for example reduced insulin synthesis and beta cell toxicity [133,134]. Most immunosuppressive agents target T cells, or some, like mycophenolate mofetil, both T and B cells. Conversely, no immunomodulatory agent in clinical use specifically targets antigen-presenting cells and in particular DC, which are known to be involved in T cell activation and tolerance induction. In NOD mice, diabetes can be prevented by 1,25(OH)2D3 and its “super-analogs” when treatment is started before insulitis is present [125]. A critical
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question for the applicability of these analogs in the human situation is, however, whether the vitamin D analogs can arrest progression to overt diabetes, which is the situation in pre-diabetic subjects in whom immune intervention is considered. Casteels et al. demonstrated that some of these analogs when combined with a short induction course of cyclosporine A can arrest the progression of the disease when administered after autoimmunity has already started [135]. The mechanism of protection in our model was clearly not a generalized immune suppression nor the induction of different immune cell subsets, and co-transfer experiments with splenocytes from animals treated with the combination therapy did not reveal the presence of suppressor cells. Signs of local immunoregulation were, however, prominent locally in the islets. The approach of utilizing vitamin D analogs, at concentrations which produce limited hypercalcemia, as adjuncts to conventional immunotherapy, is very promising and might open new perspectives in the protection against autoimmune diabetes also in humans. Several combinations of vitamin D analogs with other immune modulators have been tested both in vitro and in vivo. We have tested five candidates for a combination therapy with 1,25(OH)2D3 (cyclosporine A, tacrolimus, rapamycin, leflunomide, and mycophenolate mofetil) and showed maximum synergism with cyclosporine A [136e138]. More novel immunomodulators such as type I interferons that exhibit a broader range of immunomodulatory properties (mainly restriction of T cell proliferation and IFNg production in part by inhibiting DC-mediated IL12 secretion while promoting IL-10 production) also gave interesting results in combination with less hypercalcemic vitamin D analogs in experimental models of multiple sclerosis and type 1 diabetes [139,140]. Effects of Vitamin D Supplementation on Type 1 Diabetes Development As already mentioned, production of vitamin D3 to reach supra-physiological levels of 1,25(OH)2D3 locally in the immune system may circumvent the need for systemic administration of high doses of 1,25(OH)2D3. As most cells of the immune system have the appropriate machinery to process vitamin D3 into bioactive compound, vitamin D3 may ultimately be more desirable for clinical use [7]. We have investigated whether supplementation with vitamin D during early life could prevent type 1 diabetes in vitamin-D-sufficient NOD mice and BB rats. Peritoneal injection of 1000 IU/d of solubilized vitamin D3 (cholecalciferolFNA from 3 till 70 days of age) was unable to provide protection against diabetes development in diabetes-prone NOD mice by age 30 weeks [141]. Interestingly, however, a major difference in pancreatic
insulin content was noted with a higher content in vitamin-D-treated mice. Also in the BB rat model, diabetes could not be prevented by early life treatment (from 3 to 50 days of age) with a similar dose of vitamin D3 [118]. Also others reported that vitamin D (16 IU by gavage) administered in utero and in the early stages of life at the dosage used did not change the incidence of diabetes or modify the disease process that leads to beta cell destruction in the NOD mouse [142]. In humans only epidemiological and retrospective data are available. In childhood diabetes, several epidemiological studies describe a correlation between a northesouth gradient and the incidence of disease as well as an inverse correlation between monthly hours of sunshine and the incidence of diabetes [143]. Also a seasonal pattern of disease onset is well described in type 1 diabetes [144]. Dietary vitamin D supplementation is often recommended in pregnant women and children to prevent vitamin D deficiency. Besides the study of Stene et al. who demonstrated that cod liver oil taken during pregnancy (and during the first year of life) could reduce the risk for type 1 diabetes in their offspring [145,146], the EURODIAB group suggested an association between vitamin D supplementation in infancy and a decreased risk for type 1 diabetes in a multi-center caseecontrol study [147]. The group of Virtanen did not find a positive correlation between maternal intake of vitamin D from food or supplements during pregnancy and increased risk for development of diabetes-associated auto-antibodies or clinical type 1 diabetes [148]. On the other hand, Hypponen et al. reported that intake of 2000 IU of vitamin D during the first year of life diminished the risk of developing type 1 diabetes [149]. This study also confirmed that suspected rickets was associated with higher incidence for childhood diabetes (odds ratio ¼ 2.6). A meta-analysis of data from four caseecontrol studies and one cohort study revealed lately that the risk of diabetes was significantly reduced (29% reduction) in infants who were supplemented with vitamin D compared to unsupplemented controls [150]. There was also some evidence of a doseeresponse effect, with those using higher amounts of vitamin D being at lower risk of developing type 1 diabetes. Taking into account that diabetes susceptibility is linked to specific HLA genotypes, Wicklow and Taback intend to pursue a trial using 2000 IU/d of cholecalciferol in newborn babies with increased HLA-associated risk [151]. To date, they have demonstrated in a few infants that this dose of supplementation seems safe and did not cause any changes in serum and urine calcium levels. Effects of Vitamin D Deficiency on Type 1 Diabetes Development Important advances on the role of vitamin D in the immune system came from studies of vitamin D
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deficiency in vitro and in vivo. In addition to its effects on innate immune responses (e.g., impaired macrophage function, including defective chemotaxis, phagocytosis, respiratory burst capacity, and proinflammatory cytokine production) [70,152], a growing amount of data strongly supports the proposed role of vitamin D as a regulator of adaptive immune responses. Geisler et al. reported that T cells isolated from patients with low 25(OH)D3 and 1,25(OH)2D3 levels exhibited a lower proliferation index after stimulation than T cells isolated from healthy controls with normal concentrations of these metabolites [153]. Importantly, the impaired T cell proliferation observed could be reversed by addition of 1,25(OH)2D3. These observations support the idea that (extra-renal) synthesis of 1,25(OH)2D3 is of great importance for the homeostasis in the immune system, and that this function of vitamin D is likely accountable for the numerous epidemiologic observations that persons living at higher latitudes, who are more prone to vitamin D deficiency, are at higher risk of developing autoimmune diseases, including type 1 diabetes [154,155]. Moreover, vitamin D deficiency during pregnancy may represent for the fetus a predisposing factor for the development of various disorders, including autoimmune diseases [156]. In NOD mice vitamin D deficiency in utero and early life increases the risk for type 1 diabetes development [70]. At the cellular level, vitamin-D-deficient NOD mice display decreased numbers of CD8þ T cells in the thymus, but increased numbers of immature CD4þCD8þ T cells, possibly pointing towards a T cell maturation defect. In addition, compared to their vitamin-D-sufficient counterparts, reduced numbers of Treg cells are present, both in the thymus and in the periphery of these mice, suggesting a defect in the maintenance of tolerance [70].
Prevention of Autoimmune Disease Recurrence after Islet Transplantation by Analogs of 1,25(OH)2D3 Type 1 diabetes is an autoimmune disease in which insulin-producing beta cells in pancreatic islets are destroyed by auto-reactive T cells. The long-lasting persistence of auto-reactive T cells in human blood, and the memory phenotype displayed by at least some of these immune cells, suggests the existence of autoimmune memory [157]. The latter phenomenon is responsible for the destruction of MHC-matched or syngeneic beta cells, transplanted under the form of isolated beta cells, islets or whole pancreas [158]. Successful transplantation requires the prevention of allograft rejection and the break of the autoimmune memory [159,160]. Some analogs of 1,25(OH)2D3 have been tested for their capacity to prevent disease recurrence after islet transplantation in spontaneously diabetic NOD mice.
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The most spectacular results were obtained with a combination of KH1060 (20-epi-22-oxa-24,26,27-trishomo-1,25(OH)2D3) and subtherapeutic doses of cyclosporine A [84,127]. In the group receiving KH1060 (0.5 mg/kg/2d) together with cyclosporine A (7.5 mg/kg/d) a synergistic effect between both drugs was observed: 4 out of 7 mice maintained a functioning graft for 60 days and did not show recurrence for at least 30 days after stopping the treatment. Treatment was administered from the day before transplantation until diabetes recurrence or in case of persistent normoglycemia until 60 days after transplantation. The subtherapeutic doses of KH1060 together with CsA were non-toxic and had minor effects on serum calcium levels although osteocalcin levels were clearly elevated and bone calcium content was decreased. A novel approach can be found in combinations of vitamin D analogs and other natural immune modulators. We demonstrated that subtherapeutic doses of recombinant (r)IFNb alone (1 105 IU/d) had minor effects on autoimmune diabetes recurrence after islet transplantation (20.8 14.2 days versus 10.8 2.9 days in controls). However, interestingly, a combination of rIFNb with TX527 (14-epi-19-nor-20-epi-23-yne1,25(OH)2D3) maintained islet graft function in 100% of mice during treatment and resulted in a marked delay of autoimmune diabetes recurrence (61.6 19.6 days) [140]. We also demonstrated that rIFNb in combination with TX527 results in an inhibition of the Th1 pathway (IL12, IL2, and IFNg), which is known to be associated with the pathogenesis of organ-specific autoimmune diseases. In addition, enhanced expression of the regulatory cytokine, IL-10, by rIFNb in combination therapy with the TX527 analog was observed. Recently, we reported that combining low doses of anti-CD3, TX527, and cyclosporine A can protect NOD mice from diabetes recurrence after syngeneic islet transplantation. Remarkably, mice receiving all three drugs survived longer (69 10 days) than mice receiving one or two agents, and remained normoglycemic during the whole treatment period. Combining these drugs enhances their individual potency, but also offers an interesting strategy to circumvent dose-related sideeffects of immunosuppressants currently used in clinical transplantation.
VITAMIN D AND GENETIC PREDISPOSITION TO DIABETES Novel insights on a possible role of vitamin D/ 1,25(OH)2D3 in the pathogenesis of type 1 and type 2 diabetes originates from epidemiological data on associations between polymorphisms of either the VDR, CYP27B1, CYP24A1, or DBP gene and the risk of either metabolic disease in certain ethnic communities.
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1,25(OH)2D3 exerts its genomic effects mainly via the nuclear VDR which heterodimerizes with retinoid X receptors (RXR) and translocates into the nucleus [161]. VDRs are present in different tissues, including the human pancreatic beta cells and basically every cell of the immune system, and have important physiological effects such as calcium transport as well as cell growth and differentiation [7]. The gene encoding the VDR is located in humans on chromosome 12q13.11 and has at least five promoter regions, eight proteincoding exons, and six untranslated exons. It shows extensive single nucleotide polymorphisms (SNPs) including a FokI polymorphism in exon 2, BsmI and ApaI polymorphisms in the intron between exons 8 and 9, a TaqI polymorphism in exon 9 (rs10735810, rs1544410, rs7975232, and rs731236, respectively) and a polyadenylic acid (poly(A)) mononucleotide repeat in the 30 untranslated region [162]. These polymorphisms seem to be in strong linkage disequilibrium, although there are ethnic variations [163,164]. Over the years, numerous reports in different human populations show correlations between some of these VDR polymorphisms and type 1 or type 2 diabetes [165e168], while there are a considerable number of reports in which no association of VDR polymorphisms with type 1 diabetes is observed [169]. McDermott et al. demonstrated that excessive transmission of the BsmI alleles affects South Indian subjects with type 1 diabetes, linking BsmI polymorphism of the VDR to an increased risk for type 1 diabetes [170]. A study by Ban et al. revealed an association between FokI polymorphism and GAD65 positivity in the Japanese population [171]. However, the Type I Diabetes Genetics Consortium did not find any association of VDR SNPs with type 1 diabetes in the overall sample set, or in any of the subgroups analysed of the parent-oforigin, sex of offspring and HLA risk [172]. Nevertheless, the FokI polymorphism of the VDR could have functional implications, altering ligand-mediated gene expression in beta cells or the immune system [173]. An explanation for associations with the polymorphisms located in introns of the VDR is more complex. CYP27B1 (1a(OH)ase) catalyzes the metabolism of 25(OH)D3 to 1,25(OH)2D3, the most active natural vitamin D metabolite, while CYP24A1 (24(OH)ase) inactivates 1,25(OH)2D3 through a series of events starting with 24-hydroxylation. Associations of type 1 diabetes with polymorphisms in the CYP27B1 gene located on chromosome 12q13.1-q13.3 are documented [174] and assumed to reduce the (local) expression of the 1a(OH)ase enzyme and consequently the conversion of 25(OH)D3 to 1,25(OH)2D3. At present, no associations between CYP24A1 gene polymorphisms and type 1 diabetes have been found. The meaning of these correlations between genetic markers of vitamin D metabolism and type 1 diabetes remains unclear
although an association of VDR genotypes with VDR mRNA as well as protein has been demonstrated in peripheral blood mononuclear cells providing functional relevance to the VDR polymorphisms [167]. Moreover, the protective effects of vitamin D on the beta cell should not be neglected and might be an explanation why correlations are observed not only between genetic polymorphisms of vitamin D metabolism and type 1 diabetes but also type 2 diabetes. Several observational studies have reported associations between VDR polymorphisms and type 2 diabetes, fasting glucose, glucose intolerance, insulin sensitivity, insulin secretion, and calcitriol levels [175e178]. Indeed, a study on Bangladeshi Asians demonstrated that the ApaI polymorphism influences insulin secretion in response to glucose [166]. Also, Oh and Barrett-Conner investigated VDR polymorphisms and susceptibility for type 2 diabetes in a community-based study of unrelated adults without confirmed diabetes [176]. Their research suggests that the ApaI polymorphism may be associated with higher fasting plasma glucose and prevalence of glucose intolerance. Moreover, genotyping for TaqI, ApaI, BsmI, and FokI polymorphisms revealed that the VDR polymorphism BsmI in young males with low physical activity (3 h per week) was associated with high levels of fasting glucose [179]. A recent study found that the BsmI polymorphism seemed to influence BMI, while the FokI appeared to affect insulin sensitivity and serum cHDL level [180]. Moreover, there are several indications that not only polymorphisms of the VDR but also of the DBP gene (Gc1F, Gc1S, and Gc2) are related to glucose intolerance and obesity [181e183]. Even though these correlations vary according to age, lifestyle, and ethnicity of the subjects, there seems to be a good amount of evidence to support this theory. Type 2 diabetic patients are reported to have a higher frequency of the Gc1S/Gc2 genotype and lower frequencies of the Gc1F allele in comparison to control subjects in the Japanese population but this result could not be reproduced in Caucasian patients of American or European origin. It was hypothesized that these polymorphisms in the DBP gene influence 25(OH)D3 levels through changes in the ratio of free versus bound metabolites, by a differential affinity, or through effects on concentrations of the DBP/ 25(OH)D3 complex that can be internalized by receptor-mediated endocytosis and activate the VDR pathway.
CLINICAL PERSPECTIVES Clear effects of 1,25(OH)2D3 and its non-calcemic analogs have been described on the different players in the pathogenesis of both type 1 and type 2 diabetes
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REFERENCES
[83,184]. In vitro as well as in vivo a modest stimulation of insulin synthesis and insulin secretion by 1,25(OH)2D3 is observed [18,20,22,54]. This positive effect is not only observed upon repletion of 1,25(OH)2D3 in the vitamin-D-deficient state, but can also be observed in the vitamin-D-sufficient state [20,21]. Moreover, a direct beta cell protection by 1,25(OH)2D3 and its analogs against metabolic and inflammatory stress has been demonstrated [78e81]. On the other hand, major effects on immune cells involved in the pathogenesis of type 1 diabetes have been described in vitro as well as in vivo [7]. Consequently, prevention of type 1 diabetes and its recurrence after islet transplantation by 1,25(OH)2D3 and its analogs can be achieved (alone or in combination with other immunomodulators) [124e127,135,140,185,186]. A major problem with using 1,25(OH)2D3 or the currently available analogs in prevention or cure of diabetes is their hypercalcemic and bone remodeling effects when administered in the doses needed for immune or beta cell protective effects [122,187]. Future applications of this therapy in human diabetes are conceivable, since through chemical alterations of the 1,25(OH)2D3 molecule, even better analogs with an optimal dissociation between calcemic and immunomodulatory effects can be synthesized [123,124]. A place for these analogs in the treatment (prevention or cure) of diabetes can be conceived first of all as beta cell protective and stimulating agents added to the current treatment modalities of type 2 diabetes. Furthermore, these substances could play a contributing role in prevention strategies for type 1 diabetes in humans, because of their ideal profile as beta cell protective and especially immunomodulatory drugs. However, prior to applying these drugs in humans additional information is needed not only on their mechanism of action but especially on the safety of these “natural” products in long-term use. At present the only solid conclusion from the data on vitamin D and diabetes is that vitamin D deficiency should be avoided at all cost. Long-term intervention studies are, however, needed to demonstrate whether vitamin D plays a crucial role in the epidemic of diabetes and whether supplementing individuals will decrease their risk for diabetes.
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onset type 1 diabetes (IMDIAB XI), Diabet. Med. 23 (2006) 920e923. X. Li, L. Liao, X. Yan, G. Huang, J. Lin, M. Lei, et al., Protective effects of 1-alpha-hydroxyvitamin D3 on residual beta-cell function in patients with adult-onset latent autoimmune diabetes (LADA), Diabetes Metab. Res. Rev. 25 (2009) 411e416. M. Walter, T. Kaupper, K. Adler, J. Foersch, E. Bonifacio, A.G. Ziegler, No effect of the 1{alpha},25-dihydroxyvitamin D3 on beta cell residual function and insulin requirement in adults with new-onset type 1 diabetes, Diabetes Care 33 (2010) 1443e1448. A. Verstuyf, S. Segaert, L. Verlinden, K. Casteels, R. Bouillon, C. Mathieu, Recent developments in the use of vitamin D analogues, Curr. Opin. Nephrol. Hypertens. 7 (1998) 397e403. A. Verstuyf, L. Verlinden, E. van Etten, L. Shi, Y. Wu, C. D’Halleweyn, et al., Biological activity of CD-ring modified 1alpha,25-dihydroxyvitamin D analogues: C-ring and fivemembered D-ring analogues, J. Bone Miner. Res. 15 (2000) 237e252. G. Eelen, L. Verlinden, J. Laureys, S. Marcelis, P. De Clercq, C. Mathieu, et al., Antiproliferative and calcemic actions of trans-decalin CD-ring analogs of 1,25-dihydroxyvitamin D3, Anticancer Res. 29 (2009) 3579e3584. C. Mathieu, M. Waer, K. Casteels, J. Laureys, R. Bouillon, Prevention of type I diabetes in NOD mice by nonhypercalcemic doses of a new structural analog of 1,25-dihydroxyvitamin D3, KH1060, Endocrinology 136 (1995) 866e872. S. Amuchastegui, K.C. Daniel, L. Adorini, Inhibition of acute and chronic allograft rejection in mouse models by BXL-628, a nonhypercalcemic vitamin D receptor agonist, Transplantation 80 (2005) 81e87. K. Casteels, M. Waer, J. Laureys, D. Valckx, J. Depovere, R. Bouillon, et al., Prevention of autoimmune destruction of syngeneic islet grafts in spontaneously diabetic nonobese diabetic mice by a combination of a vitamin D3 analog and cyclosporine, Transplantation 65 (1998) 1225e1232. M. Pedulla, V. Desiderio, A. Graziano, R. d’Aquino, A. Puca, G. Papaccio, Effects of a vitamin D3 analog on diabetes in the bio breeding (BB) rat, J. Cell Biochem. 100 (2007) 808e814. K.J. Zhu, W.F. Zhou, M. Zheng, [1 Alpha,25-dihydroxyvitamin D3 and its analogues modulate the phagocytosis of human monocyte-derived dendritic cells], Yao. Xue. Xue. Bao. 37 (2002) 94e97. G.B. Ferreira, E. van Etten, K. Lage, D.A. Hansen, Y. Moreau, C.T. Workman, et al., Proteome analysis demonstrates profound alterations in human dendritic cell nature by TX527, an analogue of vitamin D, Proteomics 9 (2009) 3752e3764. X. Luo, K.C. Herold, S.D. Miller, Immunotherapy of type 1 diabetes: where are we and where should we be going? Immunity 32 (2010) 488e499. C.J. Nitsche, N. Jamieson, M.M. Lerch, J.V. Mayerle, Drug induced pancreatitis, Best Pract. Res. Clin. Gastroenterol. 24 (2010) 143e155. G.B. Ippoliti, M. Vigano, [Calcineurin inhibitors and mechanisms that are responsible for the appearance of post-transplant diabetes mellitus], G. Ital. Nefrol. 20 (Suppl. 25) (2003) S11e14. M.C. Rauch, A. San Martin, D. Ojeda, C. Quezada, M. Salas, J.G. Carcamo, et al., Tacrolimus causes a blockage of protein secretion which reinforces its immunosuppressive activity and also explains some of its toxic side-effects, Transpl. Immunol. 22 (2009) 72e81. K. Casteels, M. Waer, R. Bouillon, K. Allewaert, J. Laureys, C. Mathieu, Prevention of type I diabetes by late intervention with nonhypercalcemic analogues of vitamin D3 in combination with cyclosporin A, Transplant. Proc. 28 (1996) 3095.
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[136] C. Mathieu, R. Bouillon, O. Rutgeerts, M. Vandeputte, M. Waer, Potential role of 1,25(OH)2 vitamin D3 as a dose-reducing agent for cyclosporine and FK 506, Transplant. Proc. 26 (1994) 3130. [137] D.D. Branisteanu, C. Mathieu, R. Bouillon, Synergism between sirolimus and 1,25-dihydroxyvitamin D3 in vitro and in vivo, J. Neuroimmunol. 79 (1997) 138e147. [138] E. van Etten, D.D. Branisteanu, A. Verstuyf, M. Waer, R. Bouillon, C. Mathieu, Analogs of 1,25-dihydroxyvitamin D3 as dose-reducing agents for classical immunosuppressants, Transplantation 69 (2000) 1932e1942. [139] E. van Etten, C. Gysemans, D.D. Branisteanu, A. Verstuyf, R. Bouillon, L. Overbergh, et al., Novel insights in the immune function of the vitamin D system: synergism with interferonbeta, J. Steroid Biochem. Mol. Biol. 103 (2007) 546e551. [140] C. Gysemans, E. Van Etten, L. Overbergh, A. Verstuyf, M. Waer, R. Bouillon, et al., Treatment of autoimmune diabetes recurrence in non-obese diabetic mice by mouse interferon-beta in combination with an analogue of 1alpha,25-dihydroxyvitaminD3, Clin. Exp. Immunol. 128 (2002) 213e220. [141] C. Mathieu, E. van Etten, C. Gysemans, B. Decallonne, R. Bouillon, Seasonality of birth in patients with type 1 diabetes, Lancet 359 (2002) 1248. [142] M.I. Hawa, M.G. Valorani, L.R. Buckley, P.E. Beales, A. Afeltra, F. Cacciapaglia, et al., Lack of effect of vitamin D administration during pregnancy and early life on diabetes incidence in the non-obese diabetic mouse, Horm. Metab. Res. 36 (2004) 620e624. [143] G. Dahlquist, L. Mustonen, Childhood onset diabetes e time trends and climatological factors, Int. J. Epidemiol. 23 (1994) 1234e1241. [144] E.V. Moltchanova, N. Schreier, N. Lammi, M. Karvonen, Seasonal variation of diagnosis of Type 1 diabetes mellitus in children worldwide, Diabet. Med. 26 (2009) 673e678. [145] L.C. Stene, J. Ulriksen, P. Magnus, G. Joner, Use of cod liver oil during pregnancy associated with lower risk of type I diabetes in the offspring, Diabetologia 43 (2000) 1093e1098. [146] L.C. Stene, G. Joner, Use of cod liver oil during the first year of life is associated with lower risk of childhood-onset type 1 diabetes: a large, population-based, caseecontrol study, Am. J. Clin. Nutr. 78 (2003) 1128e1134. [147] Vitamin D supplement in early childhood and risk for type I (insulin-dependent) diabetes mellitus. The EURODIAB Substudy 2 Study Group, Diabetologia 42 (1999) 51e54. [148] L. Marjamaki, S. Niinisto, M.G. Kenward, L. Uusitalo, U. Uusitalo, M.L. Ovaskainen, et al., Maternal intake of vitamin D during pregnancy and risk of advanced beta cell autoimmunity and type 1 diabetes in offspring, Diabetologia 53 (2010) 1599e1607. [149] E. Hypponen, E. Laara, A. Reunanen, M.R. Jarvelin, S.M. Virtanen, Intake of vitamin D and risk of type 1 diabetes: a birthecohort study, Lancet 358 (2001) 1500e1503. [150] C.S. Zipitis, A.K. Akobeng, Vitamin D supplementation in early childhood and risk of type 1 diabetes: a systematic review and meta-analysis, Arch. Dis. Child 93 (2008) 512e517. [151] B.A. Wicklow, S.P. Taback, Feasibility of a type 1 diabetes primary prevention trial using 2000 IU vitamin D3 in infants from the general population with increased HLA-associated risk, Ann. NY. Acad. Sci. 1079 (2006) 310e312. [152] J. Miller, R.L. Gallo, Vitamin D and innate immunity, Dermatol. Ther. 23 (2010) 13e22. [153] M.R. von Essen, M. Kongsbak, P. Schjerling, K. Olgaard, N. Odum, C. Geisler, Vitamin D controls T cell antigen receptor signaling and activation of human T cells, Nat. Immunol. 11 (2010) 344e349.
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[154] M.F. Holick, Sunlight and vitamin D for bone health and prevention of autoimmune diseases, cancers, and cardiovascular disease, Am. J. Clin. Nutr. 80 (2004) 1678Se1688S. [155] T.R. Merriman, Type 1 diabetes, the A1 milk hypothesis and vitamin D deficiency, Diabetes Res. Clin. Pract. 83 (2009) 149e156. [156] M.L. Mulligan, S.K. Felton, A.E. Riek, C. Bernal-Mizrachi, Implications of vitamin D deficiency in pregnancy and lactation, Am. J. Obstet. Gynecol. 202 (429) (2010) e421e429. [157] S. Tsai, A. Shameli, J. Yamanouchi, X. Clemente-Casares, J. Wang, P. Serra, et al., Reversal of autoimmunity by boosting memorylike autoregulatory T cells, Immunity 32 (2010) 568e580. [158] T. Okitsu, S.T. Bartlett, G.A. Hadley, C.B. Drachenberg, A.C. Farney, Recurrent autoimmunity accelerates destruction of minor and major histoincompatible islet grafts in nonobese diabetic (NOD) mice, Am. J. Transplant. 1 (2001) 138e145. [159] D. Pipeleers, B. Keymeulen, L. Chatenoud, C. Hendrieckx, Z. Ling, C. Mathieu, et al., A view on beta cell transplantation in diabetes, Ann. NY. Acad. Sci. 958 (2002) 69e76. [160] B.O. Roep, I. Stobbe, G. Duinkerken, J.J. van Rood, A. Lernmark, B. Keymeulen, et al., Auto- and alloimmune reactivity to human islet allografts transplanted into type 1 diabetic patients, Diabetes 48 (1999) 484e490. [161] G. Eelen, C. Gysemans, L. Verlinden, E. Vanoirbeek, P. De Clercq, D. Van Haver, et al., Mechanism and potential of the growth-inhibitory actions of vitamin D and analogs, Curr. Med. Chem. 14 (2007) 1893e1910. [162] A.G. Uitterlinden, Y. Fang, J.B. Van Meurs, H.A. Pols, J.P. Van Leeuwen, Genetics and biology of vitamin D receptor polymorphisms, Gene. 338 (2004) 143e156. [163] A.G. Uitterlinden, Y. Fang, J.B. van Meurs, H. van Leeuwen, H.A. Pols, Vitamin D receptor gene polymorphisms in relation to vitamin D related disease states, J. Steroid Biochem. Mol. Biol. 89e90 (2004) 187e193. [164] J.M. Valdivielso, E. Fernandez, Vitamin D receptor polymorphisms and diseases, Clin. Chim. Acta 371 (2006) 1e12. [165] M.A. Pani, H. Donner, J. Herwig, K.H. Usadel, K. Badenhoop, Vitamin D binding protein alleles and susceptibility for type 1 diabetes in Germans, Autoimmunity 31 (1999) 67e72. [166] G.A. Hitman, N. Mannan, M.F. McDermott, E. Aganna, B.W. Ogunkolade, C.N. Hales, et al., Vitamin D receptor gene polymorphisms influence insulin secretion in Bangladeshi Asians, Diabetes 47 (1998) 688e690. [167] B.W. Ogunkolade, B.J. Boucher, J.M. Prahl, S.A. Bustin, J.M. Burrin, K. Noonan, et al., Vitamin D receptor (VDR) mRNA and VDR protein levels in relation to vitamin D status, insulin secretory capacity, and VDR genotype in Bangladeshi Asians, Diabetes 51 (2002) 2294e2300. [168] D.B. Mory, E.R. Rocco, W.L. Miranda, T. Kasamatsu, F. Crispim, S.A. Dib, Prevalence of vitamin D receptor gene polymorphisms FokI and BsmI in Brazilian individuals with type 1 diabetes and their relation to beta-cell autoimmunity and to remaining betacell function, Hum. Immunol. 70 (2009) 447e451. [169] S.W. Guo, V.L. Magnuson, J.J. Schiller, X. Wang, Y. Wu, S. Ghosh, Meta-analysis of vitamin D receptor polymorphisms and type 1 diabetes: a HuGE review of genetic association studies, Am. J. Epidemiol. 164 (2006) 711e724. [170] M.F. McDermott, A. Ramachandran, B.W. Ogunkolade, E. Aganna, D. Curtis, B.J. Boucher, et al., Allelic variation in the vitamin D receptor influences susceptibility to IDDM in Indian Asians, Diabetologia 40 (1997) 971e975. [171] Y. Ban, M. Taniyama, T. Yanagawa, S. Yamada, T. Maruyama, A. Kasuga, Vitamin D receptor initiation codon polymorphism influences genetic susceptibility to type 1 diabetes mellitus in the Japanese population, BMC Med. Genet. 2 (2001) 7.
[172] H. Kahles, G. Morahan, J.A. Todd, K. Badenhoop, Association analyses of the vitamin D receptor gene in 1654 families with type I diabetes, Genes Immun. 10 (Suppl. 1) (2009) S60e63. [173] E. van Etten, L. Verlinden, A. Giulietti, E. Ramos-Lopez, D.D. Branisteanu, G.B. Ferreira, et al., The vitamin D receptor gene FokI polymorphism: functional impact on the immune system, Eur. J. Immunol. 37 (2007) 395e405. [174] R. Bailey, J.D. Cooper, L. Zeitels, D.J. Smyth, J.H. Yang, N.M. Walker, et al., Association of the vitamin D metabolism gene CYP27B1 with type 1 diabetes, Diabetes 56 (2007) 2616e2621. [175] F. Dilmec, E. Uzer, F. Akkafa, E. Kose, A.B. van Kuilenburg, Detection of VDR gene ApaI and TaqI polymorphisms in patients with type 2 diabetes mellitus using PCR-RFLP method in a Turkish population, J. Diabetes Complications 24 (2010) 186e191. [176] J.Y. Oh, E. Barrett-Connor, Association between vitamin D receptor polymorphism and type 2 diabetes or metabolic syndrome in community-dwelling older adults: the Rancho Bernardo Study, Metabolism 51 (2002) 356e359. [177] W.Z. Ye, A.F. Reis, D. Dubois-Laforgue, C. Bellanne-Chantelot, J. Timsit, G. Velho, Vitamin D receptor gene polymorphisms are associated with obesity in type 2 diabetic subjects with early age of onset, Eur. J. Endocrinol. 145 (2001) 181e186. [178] M.T. Malecki, J. Frey, D. Moczulski, T. Klupa, E. Kozek, J. Sieradzki, Vitamin D receptor gene polymorphisms and association with type 2 diabetes mellitus in a Polish population, Exp. Clin. Endocrinol. Diabetes 111 (2003) 505e509. [179] J.R. Ortlepp, J. Metrikat, M. Albrecht, A. von Korff, P. Hanrath, R. Hoffmann, The vitamin D receptor gene variant and physical activity predicts fasting glucose levels in healthy young men, Diabet. Med. 20 (2003) 451e454. [180] A. Filus, A. Trzmiel, J. Kuliczkowska-Plaksej, U. Tworowska, D. Jedrzejuk, A. Milewicz, et al., Relationship between vitamin D receptor BsmI and FokI polymorphisms and anthropometric and biochemical parameters describing metabolic syndrome, Aging Male 11 (2008) 134e139. [181] M.T. Malecki, T. Klupa, K. Wanic, K. Cyganek, J. Frey, J. Sieradzki, Vitamin D binding protein gene and genetic susceptibility to type 2 diabetes mellitus in a Polish population, Diabetes Res. Clin. Pract. 57 (2002) 99e104. [182] T. Klupa, M. Malecki, L. Hanna, J. Sieradzka, J. Frey, J.H. Warram, et al., Amino acid variants of the vitamin Dbinding protein and risk of diabetes in white Americans of European origin, Eur. J. Endocrinol. 141 (1999) 490e493. [183] W.Z. Ye, D. Dubois-Laforgue, C. Bellanne-Chantelot, J. Timsit, G. Velho, Variations in the vitamin D-binding protein (Gc locus) and risk of type 2 diabetes mellitus in French Caucasians, Metabolism 50 (2001) 366e369. [184] C. Mathieu, K. Badenhoop, Vitamin D and type 1 diabetes mellitus: state of the art, Trends Endocrinol. Metab. 16 (2005) 261e266. [185] C. Mathieu, K. Casteels, M. Waer, J. Laureys, D. Valckx, R. Bouillon, Prevention of diabetes recurrence after syngeneic islet transplantation in NOD mice by analogues of 1,25(OH)2D3 in combination with cyclosporin A: mechanism of action involves an immune shift from Th1 to Th2, Transplant. Proc. 30 (1998) 541. [186] C. Gysemans, M. Waer, J. Laureys, R. Bouillon, C. Mathieu, A combination of KH1060, a vitamin D(3) analogue, and cyclosporin prevents early graft failure and prolongs graft survival of xenogeneic islets in nonobese diabetic mice, Transplant. Proc. 33 (2001) 2365. [187] R. Bouillon, L. Verlinden, G. Eelen, P. De Clercq, M. Vandewalle, C. Mathieu, et al., Mechanisms for the selective action of vitamin D analogs, J. Steroid Biochem. Mol. Biol. 97 (2005) 21e30.
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C H A P T E R
95 Vitamin D and Multiple Sclerosis Colleen E. Hayes 1, Faye E. Nashold 1, Christopher G. Mayne 2, Justin A. Spanier 1, Corwin D. Nelson 1 1
University of Wisconsin-Madison, Madison, WI, USA Medical College of Wisconsin, Milwaukee, WI, USA
2
THE VITAMIN DeMULTIPLE SCLEROSIS HYPOTHESIS A link between vitamin D and multiple sclerosis (MS) was suggested in 1974 [1] and again in 1992 [2]. However, the first proposed mechanism, weak myelin formation in vitamin D-deficient youth, lacked experimental support and was not entirely consistent with the natural history of MS, so the hypothesis languished. In 1997, my colleagues and I noted the inverse correlation between ultraviolet light (UVL) exposure and MS disease prevalence and suggested that selective vitamin D3 endocrine system regulation of T cell-mediated autoimmune responses might explain this relationship [3]. We wrote: “It is our hypothesis that one crucial environmental factor [in MS etiology] is the degree of sunlight exposure catalyzing the production of vitamin D3 in skin, and, further, that the hormonal form of vitamin D3 is a selective immune system regulator inhibiting this autoimmune disease.” In support of the hypothesis, we offered evidence from experimental autoimmune encephalomyelitis (EAE), the animal model of MS [4]. With the introduction of a plausible biological mechanism and supporting evidence, the vitamin DeMS hypothesis took root and gained momentum rapidly. In the years that have followed, a wealth of supporting evidence has been gathered. This evidence is now sufficient to address the first seven of the Hill criteria for suggesting that causation is the most parsimonious explanation for the inverse association between vitamin D3 and MS (Table 95.1) [5]. With additional research, particularly directed to the eighth criterion, it may be possible to conclude that the vitamin D3 endocrine system selectively regulates T cell-mediated autoimmune responses to decrease MS risk and clinical disease activity.
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10095-2
ORIGINAL EVIDENCE UNDERLYING THE VITAMIN DeMULTIPLE SCLEROSIS HYPOTHESIS The original vitamin DeMS hypothesis was based on circumstantial but compelling epidemiological evidence [3]. First, data from geographical and climatological studies showed that MS prevalence increased from a low of 1e2 cases/105 population near the equator to a high of >200 cases/105 population at latitudes >50 , and this prevalence gradient appeared to relate to UVL exposure [6]. The association between UVL and MS was strongest where UVL fluctuations were largest. Swiss data correlating MS rates inversely with altitude reinforced the theory that UVL exposure might influence MS risk [7]. Moreover, epidemiological data indicated that migration from a low UVL to a high UVL region decreased the MS risk, and, conversely, migration from a high to a low UVL region increased the MS risk at the population level [8]. The migration data reduced genetics as a confounding variable in the geographical studies of MS prevalence, and provided a temporal sequence, since childhood UVL exposure appeared to influence the risk of MS decades later. Finally, epidemiological data showed reduced MS rates on the Norwegian coast, correlating with high fish consumption [9,10]. The hypothesis was built on knowledge that vitamin D3 is a common link between UVL and fish oil. The vitamin D endocrine system is exquisitely responsive to UVL, and fish oil is a rich vitamin D3 source. The high energy UVB photons cause the photolysis of cutaneous 7-dehydrocholesterol, forming vitamin D3 (also known as cholecalciferol) in the skin. The vitamin D3 is transported on the vitamin-D-binding protein (DBP) to
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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95. VITAMIN D AND MULTIPLE SCLEROSIS
TABLE 95.1
The Hill Criteria for Asserting Causation in the Study of Environmental Factors and Disease [5]
Number
Criterion
Description
1
Strength
The association between the factor and the disease is strong
2
Consistency
The association is demonstrated reproducibly in different people, places, and circumstances
3
Specificity
The association is limited to particular people or diseases
4
Temporality
The association shows a plausible chronology with respect to cause and effect
5
Biological gradient
The association shows a doseeresponse curve
6
Plausibility
There exists a biological mechanism for the association that is plausible within the context of current biological knowledge
7
Coherence
The causeeeffect explanation coheres to generally known facts of the natural history and biology of the disease
8
Experiment
The disease is prevented or changed by an environmental intervention
9
Analogy
The association holds for related diseases
the liver, where it is hydroxylated to 25-hydroxyvitamin D3 (25(OH)D3), an inactive metabolite that is widely used as an indicator of vitamin D3 levels. A tightly regulated enzyme encoded by the human CYP27B1 gene (Cyp27b1 in mouse), 25-hydroxyvitamin D3 1a-hydroxylase (1a-hydroxylase), converts 25(OH)D3 into a biologically active hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3, also known as calcitriol). The kidney is the major producer of plasma 1,25(OH)2D3. There is a feedback inhibition loop that holds the plasma 1,25(OH)2D3 concentration within very narrow limits [11]. In the kidney, 1,25(OH)2D3 strongly induces the human CYP24A1 gene (Cyp24a1 in mouse) encoding a 1,25-dihydroxyvitamin D3 24-hydroxylase (24-hydroxylase) that converts 1,25(OH)2D3 into the inactive metabolite, 1,24,25(OH)3D3. Thus, although 1,25(OH)2D3 can be produced from ingested vitamin D3, an estimated w90% of an individual’s vitamin D3 derives from UVL exposure so the vitamin D3 hormone system is unique among hormone systems in its responsiveness to UVL [12]. A third and critical underpinning for the vitamin DeMS hypothesis came from reports that activated human T lymphocytes expressed the vitamin D receptor (VDR) [13,14]. The VDR is a nuclear protein that dimerizes with the retinoid X receptor (RXR) to regulate gene expression through vitamin D-responsive elements (VDRE) in the promoters of 1,25(OH)2D3-responsive
genes. The binding of the biologically active hormone 1,25(OH)2D3 to the VDR activates the transcriptional regulatory functions of the RXReVDR complex [12]. The VDRE is composed of two hexameric half-sites, arranged as direct repeats separated by three random base pairs, for example GGTTCACGAGGTTCA [15]. The VDR is expressed in the cells that maintain mineral ion homeostasis and skeletal health [12], and in immune system and CNS cells [16]. The concept that VDR expression might allow selective regulation of activated T lymphocytes provided a plausible biological mechanism for the vitamin DeMS link. We tested this concept through experiments with 1,25(OH)2D3 in the EAE model and found the hormone prevented T cell-mediated autoimmunity [4]. In this way, we combined geographical, climatological, biochemical, nutritional, and immunological data to formulate a fundamentally new biological explanation involving selective regulation of autoimmune T lymphocytes to explain how UVL might determine MS risk.
MULTIPLE SCLEROSIS ETIOLOGY AND THE ASSOCIATION WITH VITAMIN D MS is a genetically and immunologically complex neurodegenerative disease that often strikes in young adulthood and causes significant disability in the 2.5 million individuals worldwide who are afflicted [17e19]. The MS disease process is believed to begin many years before disease signs become clinically evident. The first clinical manifestation of the disease is often a monophasic neurologic symptom of the type seen in MS, but with no history of neurologic dysfunction. Individuals with these signs are said to have clinically isolated syndrome (CIS). About half of CIS patients are later diagnosed with clinically definite MS. For w85% of MS patients, MS shows acute episodes of neurological dysfunction (e.g., weakness or diminished dexterity in limbs, visual and sensory impairment, gait instability, ataxia, cognitive dysfunction) followed by periods of partial or complete remission. This disease course, termed relapsing-remitting MS (RRMS), is observed two to three times more frequently in females than males. In the majority of RRMS patients, remissions eventually cease and the disease progresses relentlessly (termed secondary-progressive MS; SPMS). About 15% of MS patients (equal numbers of males and females) experience unrelenting neurological dysfunction from the outset (termed primary-progressive MS; PPMS). Disability is typically quantified using the Kurtzke Expanded Disability Status Scale (EDSS). As the disease inevitably progresses and the neurological dysfunctions become more incapacitating, most patients become wheelchair bound within 15e20 years of diagnosis
XI. IMMUNITY, INFLAMMATION, AND DISEASE
MULTIPLE SCLEROSIS ETIOLOGY AND THE ASSOCIATION WITH VITAMIN D
[20]. Suicide is 7.5-fold more common among MS patients than in the general population, testifying to the heavy burden of this disease [17]. Based on the high degree of variability in disease phenotype and course, it is reasonable to expect that distinct pathological mechanisms may apply in relapse, remission, and progressive disease, and these mechanisms may show varying degrees of dominance in individual patients. The characteristic central nervous system (CNS) lesions of MS are focal in nature and show T lymphocyte and mononuclear cell infiltration, oligodendrocyte loss, reactive astrocyte formation, demyelination, and axonal injury and loss [17,18]. Though there is as yet no clear explanation for the focal nature of MS lesions, the foci may represent sites of bloodebrain barrier breaching of infectious or non-infectious origin. The presence of activated T cells and inflammatory macrophages in the focal lesions has been interpreted as evidence of a T cell-mediated immune response believed to be directed to components of the myelin sheath surrounding the nerve fibers [17,18]. The current model suggests that myelin-specific T lymphocytes become activated outside the CNS (although the precise activating antigen is unclear) and migrate into the CNS. Once in the CNS the autoimmune T cells sustain their activation through interactions with CNS-resident antigen-presenting cells (APC) displaying myelin sheath peptides, although the stimulus for upregulation of APC function in the CNS is also unclear. The MS lesions are believed to form as the inflammatory cells release harmful inflammatory mediators, oxidizing free radicals, pro-apoptotic factors, and matrix-degrading proteases into the surrounding tissue. Demyelination and partial remyelination appear to occur in RRMS and to represent fluctuations in the inflammatory process [17,18]. Progressive axonal injury and loss appear to occur in SPMS and PPMS and to represent a neurodegenerative process that may not be directly attributable to inflammation [17,18]. Current efforts to prevent MS relapses and disease progression focus on eliminating the T cell-mediated autoimmune attack and preventing axonal damage and neurodegeneration.
Etiology The origins of the pathogenic autoimmune attack in MS are not known. Intensive efforts to understand MS etiology have yielded the paradigm that this immunologically complex neurodegenerative disease occurs when there is a confluence of incompletely defined genetic and environmental risk factors [17e19]. The high frequency (about 1e2 in 1000) of MS in some ethnic populations (e.g., those of northern European origin) and low frequency in others (e.g., those of African, Asian, southern or eastern European origin),
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familial aggregation, the 15e20-fold increased relative risk of MS among siblings, and the 25% disease concordance between monozygotic twins have been interpreted as evidence of a polygenic heritable component [21,22]. However, MS is clearly not inherited as a simple Mendelian trait, and there is no known combination of genes that causes MS with complete penetrance. Moreover, analyses of the genomes, epigenomes, and transcriptomes of a monozygotic twin pair who were discordant for MS did not reveal differences that could account for their discordant status [23]. Accordingly, multifaceted geneeenvironment interactions are now believed to determine MS disease risk. The view that environmental factors strongly influence MS risk at a population level is supported by data showing high disease discordance between monozygotic twins, geographical gradients in MS prevalence, and alteration of MS risk by migration [24]. In fact, when genetic factors are held constant, as in monozygotic twins, modifiable environmental factors appear to set the disease threshold and may hold the key to preventing w75% of MS cases. This finding demands close investigation of environmental influences on pathophysiological processes suspected in MS disease.
Epstein-Barr Virus Viral and bacterial infections are among the most biologically plausible environmental risk factors that have been investigated in conjunction with MS risk [17,24,25]. Several chronic viral infections are under investigation because no single microbial agent has been identified as causative. Epstein-Barr virus (EBV) is discussed briefly here, because there exists a possibility that EBV infection could undermine the beneficial effects of vitamin D3 in MS. Strong epidemiological data have associated infectious mononucleosis (symptomatic EBV infection) with MS [25]. Other current evidence in support of EBV as a contributor to MS risk includes longitudinal studies documenting an increase in serum IgG antibodies to the EBV nuclear antigen-1 (EBNA-1) preceding the onset of MS in cohorts of US nurses, US service men and women, Canadian pediatric MS patients, and Swedish adult MS patients [25]. Additionally, new data show that high immune responses to EBNA-1 correlated with conversion from CIS to MS, and with brain lesion load and EDSS score 5 years after the CIS event [26]. Other new data show an extremely low MS risk in EBV non-infected individuals that rises sharply after they become EBV infected [27]. However, EBV infection is not universal in pediatric MS cases [28]. As discussed later, HLA-DRB1*1501 is the strongest known MS susceptibility allele [29,30]. Among the carriers of HLA-DRB1*1501 (whose MS risk is three-fold
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higher than non-carriers), those with a history of mononucleosis have a 10-fold increased risk of MS compared to those with no such history [31,32]. Accounting for the EBVeMS association mechanistically has been challenging. The detection of active EBV infection in postmortem MS brain samples suggested that persistence and reactivation of EBV in the CNS might have a direct pathogenic role [33]. The failures to replicate this finding [34] indicate that this mechanism is not universal. There exists a structural similarity between a myelin basic protein (MBP) peptide and an EBV peptide that might allow EBV-specific T lymphocytes to ignite an autoimmune response to the MBP peptide in the CNS (termed molecular mimicry) [35,36]. In this case, the immune response to the virus rather than the virus itself would have a direct role in MS pathogenesis. Attempts to identify such T lymphocytes among the circulating T cells in MS patients have not been universally successful, casting doubt on the generality of this suggested mechanism [37]. A third possibility, yet to be investigated, points to the near identity between human interleukin (IL)-10 and an EBV protein termed viral IL-10, which might allow viral IL-10 to undermine the protective effects of human IL-10 [38], particularly those that are induced by sunlight and vitamin D3 [39]. This possibility is discussed later.
Ultraviolet Light Exposure A very strong inverse correlation between MS disease prevalence (people living with MS per 105 population) and UVL exposure throughout the world was first noted in 1960 [6]. Acheson and his colleagues wrote: sunshine “could conceivably act directly e a certain skin dose of sunshine per unit time protecting the individual in some way” [6]. These data were confirmed about four decades later [40,41]. Recent data extend these findings and focus attention on childhood and young adulthood as the periods of life showing the strongest correlation between UVL exposure and reduced MS risk. A Tasmanian caseecontrol study determined that 2e3 hours/ week of youthful sun exposure reduced the MS risk by 60% [42]. A study of MS disease-discordant monozygotic twins determined that differences in childhood sun exposure, not childhood infections, correlated with disease discordance in female twin pairs [43]. A Norwegian study found that increased summer outdoor activities particularly at ages 16e20 correlated with a decreased MS risk [44]. In those who reported low summer outdoor activities, fish or fish oil consumption correlated with a decreased MS risk. Among French farmers [24], residents of Newfoundland and Labrador [45], and residents of the continental USA [46], available UVL was the best correlate for regional variations in MS
prevalence, suggesting that UVL exposure is protective with respect to MS risk at the population level. Several longitudinal studies have found an inverse correlation between seasonal fluctuations in UVL and MS disease activity. The most detailed study was performed in southern Germany [47]. Investigators examined MS patients (n ¼ 53) with serial magnetic resonance imaging (MRI) scans (n ¼ 202) over a 3-year period. Plotting the average number of enhancing MRI lesions by month yielded a smooth biphasic curve with a peak (4.1 lesions) in April and a nadir (0.77 lesions) in October. This pattern closely resembled the pattern of onsets and exacerbations observed in Switzerland [48] and Arizona [49], and could not be explained by coincident infections. Later work correlated the MRI lesion number inversely (r ¼ 0.90; <0.001) with the monthly mean duration of sunlight hours in Munich, the change in UVL preceding the change in disease activity by several months [50]. A longitudinal prospective study of relapse rates in Australian MS patients recorded the lowest relapse rate, 0.5 per 1000 days, in midelate summer, 1.5 months after UVL levels peaked, consistent with a role for UVL-generated vitamin D3 as an inhibitor of relapses [51]. Another recent study investigated the relationship between past UVL exposure, melanocortin 1 receptor gene variants denoted as red hair color phenotype, and MS in Australia [52]. Nonred-haired individuals showed an inverse correlation between childhood summer sun exposure and reduced MS risk that was particularly evident in women. In contrast, red-haired individuals tended to avoid sun exposure and so did not show an association between summer sun exposure and MS risk. A Norwegian study reported a significant inverse correlation between UVL and MS risk in women but not in men, but the small sample size introduces uncertainty [44]. Together, these data reinforce the inverse correlation between UVL exposure and MS risk and suggest that there may be a female sex bias in UVL effects.
Vitamin D Analysis There is a wealth of evidence correlating low vitamin D3 levels with high MS risk and disease activity, and conversely high vitamin D3 levels with low MS risk and disease activity. This evidence has been reviewed recently [24,53e55]. Before summarizing the evidence, it is important to mention the complex considerations that apply to vitamin D3 studies and their interpretation. The vitamin D3 metabolite that best indicates near-term UVL exposure and vitamin D3 consumption is 25(OH)D3 [56]. Most methods for quantifying circulating 25(OH)D3 do not distinguish 25(OH)D3 from 25(OH)D2, so the analytical results are reported simply as 25(OH)D. The analytical methods have gradually evolved, and
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variation in results has been noted between assay methods and between laboratories performing similar assays [57,58]. Consequently, comparing 25(OH)D levels between studies can be problematic. The season of sampling is an important consideration, because an estimated 90% of vitamin D supplies derive from UVL exposure, and UVL availability varies seasonally at high but not low latitudes [59]. “Indeed for some 6 months of the year, lying naked outdoors in Scotland might get you arrested (if hypothermia did not supervene) but would not get you useful vitamin D synthesis in exposed skin” [60]. The population studied is also an important consideration, because skin pigmentation and genes independent of pigmentation influence circulating 25 (OH)D levels [61e67]. Finally, MS patients tend to limit their outside activities because of disability and heat intolerance, decreasing their circulating 25(OH)D levels and introducing the possibility of reverse causation [53]. Careful attention to these complex considerations has allowed robust conclusions to be drawn regarding the association between vitamin D and MS.
Vitamin D Deficiency in Multiple Sclerosis Patients The first evidence of vitamin D deficiency in MS patients came from studies of kidney function and skeletal maintenance [68e70]. These American MS patients had on average 10 nM of circulating 25(OH)D, significantly less than the 50 nM that was then considered sufficient [68]. In fact, most MS patients were classified as vitamin D insufficient or deficient. The MS patients also had significantly lower bone mineral densities than a healthy reference population [69], a finding that was recently confirmed [71,72]. Moreover, MS patients lost bone and suffered fractures at much higher rates than the reference population (bone loss 3% and 6% in pre- and postmenopausal women with MS versus 0.5% and 0.8% in controls; fractures 22% of MS patients versus 2% of controls) [70]. The MS patients had dietary vitamin D intakes below the recommended level, many reported no weekly sun exposure, most had mobility problems, and all had had corticosteroid treatments to manage attacks. Thus, sun avoidance, poor nutrition, immobility, and corticosteroid use accounted for vitamin D insufficiency and poor bone health in the MS patients. Data on Australian MS patients confirmed these conclusions [73]. More than half of the patients had vitamin D insufficiency; the most disabled patients obtained the least sun exposure and had the lowest 25(OH)D levels. In the Australian study, circulating 25(OH)D levels did not correlate with dietary vitamin D, emphasizing the importance of sun exposure for vitamin D synthesis, and the need to monitor circulating 25(OH)D rather than vitamin D intake when correcting
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vitamin D insufficiency in MS patients. Many other reports have also confirmed vitamin D insufficiency in MS patients [74,75]. An Irish study did not find differences between MS patients and controls, but recruitment occurred in the winter when both groups had low vitamin D levels [76]. In summary, studies performed worldwide have established that the majority of MS patients have vitamin D insufficiency, sometimes of long duration, and would benefit from comprehensive efforts to detect and address this problem.
Vitamin D and Multiple Sclerosis Risk The first concrete epidemiological evidence for an inverse correlation between vitamin D and MS risk derived from the prospective Nurses’ Health Studies I (1980e2000; 92 253 women) and II (1991e2001; 95 310 women) [77]. Nutritional data were collected before MS disease developed. Comparing women in the highest and lowest quintiles of vitamin D intake, or comparing women with >400 IU/d (40 IU ¼ 1 mg) of supplementary vitamin D to those who did not supplement, documented a 33% and 41% decreased relative risk of MS, respectively. A follow-up prospective study involved MS cases paired with controls matched for age, sex, race/ethnicity, and blood collection date from >7 million US military personnel, the majority of whom were men [78]. Serum samples collected and frozen on the enlistment date were later analyzed for 25(OH)D. The data showed an inverse correlation between 25(OH)D levels at enlistment and risk of MS years later that was particularly strong for Caucasian individuals 20 years of age at enlistment. Among the young Caucasian soldiers whose 25(OH)D levels were 100 nM (1 nM ¼ 2.5 ng/mL), the odds ratio of MS was just 0.09. Among the young soldiers of AfricaneAmerican ancestry, none had 25(OH)D levels 100 nM, so the quintile analysis did not include these subjects. Reverse causation does not apply in these two large studies, because data collection preceded the MS diagnosis. Accordingly, the data provide strong support for a possible causal relationship between high baseline circulating 25(OH)D levels, particularly in adolescence, and a subsequently reduced risk of MS. Circulating 25(OH)D also correlated inversely with the risk of first demyelinating events, implicating vitamin D in prevention of these events [78a]. Recent data from the Netherlands examined the association between vitamin D supplies and MS risk in the context of gender [79]. Serial blood samples were obtained from MS patients and healthy controls, and analyzed for 25(OH)D and 1,25(OH)2D3. Using logistic regression methods, the investigators found a 19% decrease in the odds of MS for each 10 nM increase in serum 25(OH)D that was restricted to women. They
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also observed a negative correlation between serum 25(OH)D and disability level that was restricted to women. Since data collection followed the MS diagnosis, there is a reverse causation concern regarding this study. The fact that the correlation was restricted to women somewhat mitigates this concern, because if reverse causation were the explanation it would also have applied to men. That the inverse correlations were restricted to women in the Dutch study but not in the study of US military personnel is intriguing and remains unexplained. The possible gender bias in vitamin D3 actions is discussed below and will no doubt be an active area of future inquiry.
Longitudinal Studies of Vitamin D and MS Disease Activity Serum 25(OH)D levels vary seasonally with insolation, reaching a nadir 2 months after the winter solstice, and a zenith 2 months after the summer solstice [80]. There was close correspondence between the periodicity of the near-sinusoidal fluctuations in the average number of enhancing MRI lesions [47] and circulating 25(OH)D [81]. The change in MRI lesions followed the change in 25(OH)D with a 2-month lag. A cautionary note applies to these data, because the 25(OH)D levels were measured in community controls not in the MS patients. Two later studies confirmed these relationships using data from MS patients [50,50a]. A longitudinal study showed a close correspondence between falling serum 25(OH)D levels, hypocalcemia, a blunted parathyroid hormone (PTH) response, and increasingly frequent MS relapses, again with w2-month lag [82]. The 2-month lag suggests a causeeeffect relationship. Other studies have not found an inverse correlation between seasonal variations in UVL and changes in MRI lesions [83,84]. Two prospective longitudinal studies involving widely different cohorts on two separate continents found nearly identical, linear, dose-dependent inverse relationships between serum 25(OH)D and MS attack rates [85,86]. A study performed in the USA evaluated pediatric MS patients (n ¼ 134; age 15 3 years; EDSS 1.5 to 2.0; disease duration w1 year; 66% female) [85]. Baseline serum samples were collected and frozen, then clinical disease activity was monitored for w1.7 years. Subsequently, circulating 25(OH)D was quantified and compared to attack rates. A very similar study performed in Australia evaluated adult MS patients (n ¼ 145; age 44.8 10.8 years; EDSS 2.8 1.6; disease duration 11.1 9.1 years; 75% female) [86]. Serum samples were collected biannually, and clinical disease activity was monitored for w2.3 years, with real-time reporting and validation of relapses. In both studies, the individual 25 (OH)D values were adjusted for season of sampling.
Remarkably consistent linear, dose-dependent inverse relationships between serum 25(OH)D 3 levels and MS attack rates were found [85,86]. Each 10 nM increase in adjusted serum 25(OH)D correlated with a 13.6% decrease in attacks for the pediatric MS patients [85], and a 12% decrease in attacks for the adult MS patients [86]. Key strengths of the American study were the elimination of investigator bias through the blinded study design, and elimination of reverse causality by enrollment of newly diagnosed patients with very mild disability. Key strengths of the Australian study were the prospective study design with multiple serum 25(OH)D analyses and real-time relapse reporting, and elimination of reverse causality by comparing results between patients with mild to moderate disability and patients with more severe disability. Thus, these two comprehensive longitudinal studies addressed the dual problems of investigator bias and reverse causality, providing strong evidence consistent with a causal role for elevated 25(OH)D in the reduction of MS clinical disease activity. Furthermore, assuming causality, these studies suggest that vitamin D3 benefits may accrue in children and adults up to the EDSS 3 to 4 stage of MS disability.
Vitamin D in RelapsingeRemitting Multiple Sclerosis Finnish investigators were the first to correlate low 25 (OH)D levels with MS relapses [87]. They evaluated MS patients (n ¼ 40) at the time of diagnosis and compared them to matched controls. The circulating 25(OH)D levels were not different between the two groups when winter samples were compared, but MS patients had significantly lower 25(OH)D levels than controls when samples from June to September were compared. This study was the first to observe lower 25(OH)D levels during MS relapses than during remission, although they were still within the reference range for normal bone metabolism. These data suggest that optimal 25 (OH)D levels for regulation of MS disease activity might be significantly higher than for bone metabolism. Investigators in Argentina confirmed these data [88]. They found lower 25(OH)D and 1,25(OH)2D3 levels in RRMS patients than controls, with the lowest levels observed during MS relapses compared to remissions. These patterns were not observed in PPMS patients. The investigators interpreted their data as evidence that vitamin D might be involved in the regulation of MS disease activity, a very important insight. A retrospective study of vitamin D metabolites in MS patients (n ¼ 267) found lower 25(OH)D and 1,25(OH)2D3 levels in progressive MS patients than in RRMS patients [89]. Moreover, high 25(OH)D levels
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were associated with a higher probability of a relapsefree disease course. However, since the most disabled patients have the least sun exposure and the lowest 25(OH)D levels [73], reverse causality is a concern when the MS diagnosis precedes sampling. A Norwegian study failed to detect an inverse correlation between serum 25(OH)D levels and MS disease activity [90]. This study was the first to quantify and compare the 25(OH)D and 1,25(OH)2D3 levels in the serum and cerebralspinal fluid (CSF) of MS patients, an important accomplishment. There was only a weak correlation (r ¼ 0.5, p ¼ 0.002) between the 25(OH)D levels in the serum (mean 63 nM) and CSF (mean 0.23 nM) in MS patients, because the CSF:serum ratio varied considerably between individuals. Given the individual variation in serum 25(OH)D levels, the seasonal variation in serum 25(OH)D (which was not adjusted), the variability in the natural history of MS, and the small number of MS patients (27 relapsing, nine remitting), it is perhaps not surprising that serum 25(OH)D and MS disease activity were not correlated.
Vitamin D Supplementation in RelapsingeRemitting MS The most significant evidence for a causeeeffect relationship between an environmental factor and disease comes from experimentation, from changing the environmental factor and observing a change in the disease outcome [5]. Several vitamin D3 supplementation studies have been performed in MS patients, and the most recent of them strongly suggested a causal link between vitamin D and MS disease activity. The first study evaluated cytokine changes pursuant to 6 months of supplementation [91]. Only vitamin Ddeficient MS patients with <50 nM of 25(OH)D were accepted into the study; 48% of MS patients were in this category. Control MS patients (n ¼ 22) were given calcium plus placebo, and treated MS patients (n ¼ 17) were given calcium plus 1000 IU/d of vitamin D3. The supplementation increased the 25(OH)D levels of the treated patients from 42 to 70 nM as expected. This increase was accompanied by a 28% increase in serum transforming growth factor-b1 (TGF-b1), but no changes in interferon (IFN)-g, IL-2, IL-13, or tumor necrosis factor-a (TNF-a). These cytokine changes are potentially important clues to biological mechanisms of vitamin D action in MS. A recent vitamin D3 supplementation study provided the first glimpse of clinical efficacy in MS. This study sought to define the safe upper limit of vitamin D3 supplementation in MS patients [92]. Patients with active MS disease (n ¼ 12) were given progressively increasing vitamin D3 doses from 7000 to 40 000 IU/d
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over a 28-week time period. The mean serum 25(OH)D was initially 78 35 nM and rose to 386 157 nM, twice the top of the physiologic range, without adverse clinical or biochemical events. The mean number of gadoliniumenhancing MRI lesions per patient decreased from 1.75 to 0.83 (p ¼ 0.03). Noteworthy in the study was the large variation in peak serum 25(OH)D, supporting the concept that this parameter may be genetically determined [65,67]. The study showed that a vitamin D intake much higher than the current upper limit (4000 IU/d) was safe by a wide margin in MS patients, at least in the short term. The safety of vitamin D3 supplementation in MS patients was further investigated in a follow-up study [93]. RRMS patients (n ¼ 49; 41 8 years old; EDSS 1.34 1.6; disease duration 7.8 6 years; 82% female) were randomized into two groups (the study was not blinded). Most patients (57%) were receiving immunomodulatory therapy. The control patients (n ¼ 24) were permitted to take up to 4000 IU/d of vitamin D3 and calcium at their discretion. The treated patients (n ¼ 25) were given calcium and an escalating dose of vitamin D3 that began at 4000 IU/d, escalated progressively to 40 000 IU/d, and decreased to 10 000 IU/ d and then to 0 IU/d over the course of a year. The mean serum 25(OH)D was initially 78 nM and rose to 413 nM. There were no adverse clinical or biochemical events in either group. This study was not blinded, so the results must be considered exploratory. In the control group, 37% of patients had at least one relapse and 38% completed the study with increased disability. Among the treated patients, only 16% (p ¼ 0.09) had at least one relapse and only 8% (p ¼ 0.019) completed the study with increased disability. The challenge now is to perform a larger and longer blinded investigation evaluating the efficacy of vitamin D3 supplementation in MS. The vitamin D3 supplementation study also included immunological assessments [93]. The T lymphocyte responses to a panel of test antigens, including myelin and glial antigens, were determined at the beginning and the end of the study. The results were expressed as a total T cell response score. This score decreased significantly in the vitamin D3 supplemented group (p ¼ 0.002), but not in the controls. Among individuals with decreased T cell response scores, a significantly larger proportion completed the trial with 25(OH)D 100 nM than with 25(OH)D <100 nM (p ¼ 0.032). These data suggest 25(OH)D 100 nM as a tentative target level in follow-up studies. No consistent changes were observed as regards matrix metalloproteases and their inhibitors, or cytokines (IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL12p40, IL-13, IFN-g, TNF-a). The simplest mechanistic interpretation of the immunological data is that the vitamin D3 supplementation led ultimately to elimination
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of the activated neural-antigen-specific Tcells, rather than to a change in their cytokine production patterns.
GENETIC EVIDENCE LINKING VITAMIN D AND MULTIPLE SCLEROSIS MS risk is modestly influenced by a polygenic heritable component as evidenced by familial clustering, the 15-20-fold increased relative risk of MS among siblings, and the 20e30% disease concordance between identical twins [22,94]. Genes in the chromosome 6p21 major histocompatibility complex (MHC) region are most consistently and strongly associated with MS susceptibility, clinical phenotypes, and disease progression [29,30]. In particular, the class II HLA-DR region of the human MHC confers a genetic risk (odds ratio 5.4, three-fold increased risk) greater than all other known loci combined. One of these alleles, HLADRB1*1501, was recently reported to be under VDR control [95]. Here we review evidence from genetic studies on class II HLA-DR region genes, and the VDR, CYP27B1, and GC genes, which is relevant to the vitamin DeMS hypothesis.
Class II HLA-DR Region Genes Research on class II HLA-DR region genes was recently reviewed [96]. Strong linkage disequilibrium exists between HLA-DRB1*1501 (encoding HLADR2b), HLA-DRB5*0101 (encoding HLA-DR2a), and HLA-DQB1*0702 (encoding HLA-DQ6) making the assignment of genetic risk to an individual locus and allele very difficult. Nevertheless, powerful genetic studies have suggested that HLA-DRB1*1501 is the strongest genetic determinant of MS risk, particularly in individuals of Northern European descent [29,30]. This allele was associated with earlier disease onset and a more severe disease course, particularly in women [97]. In one kindred with consistently high penetrance over four generations, linkage studies identified only HLA-DRB1*1501 as a candidate susceptibility allele [98]. Other HLA-DRB1 alleles account for a modest proportion of MS risk in other populations (odds ratio w1.7 to 2.2). Complex epistatic interactions between HLA-DRB1 alleles modify MS risk [99]. For example, inheriting HLA-DRB1*01 with HLA-DRB1*1501 reduces the relative MS risk to 1 (dominant negative epistasis), whereas inheriting HLA-DRB1*08 with HLA-DRB1*1501 increases the relative MS risk to 6 (synergistic epistasis). The strong association between HLA-DRB1*1501 and MS risk demands close study of HLA-DRB1*1501 gene expression control and HLA-DR2b protein function in the context of pathophysiological processes suspected in MS disease.
New genetic and biological evidence suggests that 1,25(OH)2D3 may positively influence HLA-DRB1*1501 gene expression, heralding a new paradigm for genee environment interactions in the determination of MS risk [95]. A scan of DNA sequences within and upstream of the DRB1, DQA1, and DQB1 genes revealed a potential VDRE immediately 50 to the DRB1*1501 transcriptional start site. This sequence, 50 -GGGTGGAGGGGTTCA-30 , was present without variation in 322 DRB1*1501 homozygous individuals, and absent in 98% of individuals carrying the non-MS-associated alleles DRB1*04, DRB1*07, and DRB1*09. Biochemical methods detected specific VDR protein binding to an oligonucleotide with this sequence. Moreover, 1,25(OH)2D3 modestly (1.6fold) increased expression of a VDREereporter gene construct and the HLA-DR2b protein in DRB1*1501 homozygous B lymphoblastoid cells. Thus, this sequence performed as a weak VDRE in vitro. For comparison, the two VDRE in the Cyp24a1 gene promoter increased reporter gene expression five-fold in response to 1,25(OH)2D3 [100]. Confirmation of the potential VDRE will require evidence that vitamin D3 status, 1,25(OH)2D3 and the VDR positively influence HLADR2b expression in vivo in tissues that express MHC class II molecules and have relevance to MS. A follow-up study identified potential VDRE in additional genes of autoimmune disease significance [101]. Genome-wide VDR occupancy, defined by chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq), was examined in B lymphoblastoid cell lines before and after 1,25(OH)2D3 treatment. Subsequently, microarrays were used to measure transcript abundance and regions with high VDR occupancy and transcriptional responses to 1,25(OH)2D3 treatment were selected for further study. Finally, these regions of interest were cross-referenced to genomic loci of importance for autoimmune disease, as defined by genome-wide association studies. This analysis highlighted a number of autoimmune gene loci that may be transcriptionally regulated by 1,25(OH)2D3 and the VDR, for example IRF8 of relevance to MS and PTPN2 associated with Crohn’s disease and type 1 diabetes. The mechanism(s) involved in the DRB1*1501-associated MS risk, and how vitamin D might modify the risk, are not known [102]. The HLA-DRB1 gene encodes the HLA-DR2b polypeptide chain of the hetero-dimeric MHC class II molecule. The MHC class II molecules present peptides derived from host (self) proteins to T lymphocytes during tolerance induction, and present peptides derived from foreign (non-self) proteins to T lymphocytes during an immune response [103]. Mechanisms of tolerance induction occur at the maternalefetal interface, in the embryonic and postnatal thymus, and in adult peripheral lymphoid tissues. Mechanisms involved in host defense occur in all secondary
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lymphoid tissues. Since vitamin D3 is a protective factor, and 1,25(OH)2D3 increased HLA-DRB1*1501 expression, the question is how reduced vitamin D3 and HLA-DRB1*1501 gene expression could contribute to a pathological process relevant to MS. One possibility relates to thymic tolerance. During thymopoiesis, a complex and critical step occurs in the mammalian thymus to eliminate potentially pathogenic self protein-specific T helper cells and develop potentially self protein-specific natural T regulatory (Treg) cells [104,105]. Insufficient vitamin D3 and reduced thymic expression of HLA-DRB1*1501 might result in a failure to purge from the developing T cell repertoire those CD4þ T cells that recognize neural peptides presented by HLA-DR2 molecules. Alternatively, it might disallow the development of natural Treg with similar specificity. The birth month effect in MS is consistent with this model. An excess of MS patients are born in the spring (the nadir of vitamin D3 supplies) and fewer are born in the fall (the apex of vitamin D3 supplies), an asymmetry that is most pronounced at high latitudes [106] and among individuals with the HLA-DRB1*1501 allele [107]. Twin data are also consistent with this model. Among monozygotic twins, greatest disease concordance was observed among those with Scandanavian/Celtic ancestry (high frequency of DRB1*1501), a high latitude of residence, and an early disease onset [108]. Disease discordance in the female monozygotic twins correlated with differences in childhood sun exposure [43]. Three studies linking youthful sun exposure with reduced MS risk are also consistent with this model [42,44,109]. Some TCR structural data also support this model. Analysis of the Ob.1A12 and Ob.2F3 TCR that were cloned from an MS patient’s T cells showed characteristics that would be expected for TCR that escaped thymic deletion [36]. These TCR had a markedly atypical binding topology to a myelin peptide presented by HLA-DRB1*1501-encoded HLA-DR2 molecules. They were asymmetrically oriented, contacted only the myelin peptide N-terminus, and bound with low affinity. Collectively, these studies point to a risk factor linked to latitude and season that interacts with HLADRB1*1501 during a biological process that is most pronounced in the young. Thymic tolerance induction is one possibility, but other explanations are also consistent with the data [96].
The VDR Gene Genetic epidemiology studies have associated some alleles at the VDR locus on chromosome 12q13.1 with MS in particular populations, reinforcing the concept that the vitamin D endocrine system plays a role in MS susceptibility. These studies have been reviewed recently [110]. An early genetic epidemiology study correlated the
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BsmIbApaIA allele of the VDR gene with MS risk in a Japanese population [111,112]. These restriction site polymorphisms occur in the intron between exons 8 and 9 and do not themselves alter VDR protein structure [110]. The BsmIbApaIA polymorphisms are in linkage disequilibrium with the TaqIt polymorphism, a silent T>C substitution within exon 9 [110]. The results were confirmed and extended in an Australian population, where the ApaIATaqIt VDR gene variant was associated with MS [113]. Those who carried the TaqIt VDR gene variant were two-fold more likely to have MS than those who did not. Interestingly, the association was strongest for the progressive forms of MS disease. However, genetic studies of Canadian [114], British [115], and American [116] populations, as well as a second study of Australians [109], found no associations between the ApaIATaqIt VDR gene variants and MS risk. The impact of the BsmIb ApaIATaqIt polymorphisms on VDR function is a subject of some controversy. Although these polymorphisms do not alter VDR protein structure, they may be in linkage disequilibrium with polymorphisms that alter VDR expression or function [110]. In regard to linkage disequilibrium, it may be important that a potential MS susceptibility region with an autosomal dominant inheritance pattern was identified on chromosome 12q12 [117], near the VDR (12q13.1) and CYP27B1 (12q13.1-q13.3) genes. The chromosome 12q12 susceptibility region showed a maximum multipoint LOD score of 2.71 in a Pennsylvania Dutch family (LOD scores are log10 of the ratio of the probability that the linkage data arose by true linkage to the probability that the data arose by chance alone). The association between this region and MS susceptibility was conditional on the presence of HLA-DRB1*15; all MS affected family members (eight of 18) carried the chromosome 12 and HLA-DRB1*15 haplotypes, whereas all unaffected individuals lacked one or both of these haplotypes (p ¼ 0.00011). Further refinement of the chromosome 12q12 MS susceptibility region has not been reported. The autosomal dominant inheritance pattern of the 12q12 MS susceptibility region, and its relatively high LOD score, provide strong justification for further research to identify the affected gene. The linkage pattern found in the Pennsylvania Dutch family is reminiscent of the association found between the VDR and HLA-DRB1*15 genes in Japanese MS patients [112], and suggests a possible epistatic interaction between genes on chromosomes 6 and 12. It might be interesting to reinvestigate the HLA-DRB1*15 subset of Canadian [114] and British [115] MS patients in search of a conditional association between this HLA-DRB1 allele and ApaIATaqIt VDR gene variants and MS risk. The VDR gene FokIF variant has also been investigated in MS patients and an intriguing geneeenvironment interaction has emerged. The FokI polymorphism
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introduces a second start codon in the VDR gene [118]. The FokIF variant encodes a long VDR of 427 amino acids, whereas the FokIf variant encodes a short VDR of 424 amino acids. The short VDR encoded by the FokIf variant reportedly has higher transcriptional activation function [119,120]. In British populations, the Fok1Fencoded long VDR was associated with higher MS risk in males, but not females [121], whereas the FokIf-encoded short VDR was associated with reduced disability a decade after disease onset [122]. In what appeared to be a paradoxical finding, the FokIf-encoded short VDR was associated with lower 25(OH)D3 levels in a Canadian MS twin study [65], and lower 25(OH)D3 and 1,25(OH)2D3 levels in Dutch MS patients and healthy controls [123]. The explanation for this apparent paradox may derive from the higher transcriptional activation function of the short VDR variant [119,120]. The feedback loop that regulates serum 25(OH)D3 and 1,25(OH)2D3 levels involves VDRmediated transcriptional activation of the CYP24A1 gene in kidney cells, and 24-hydroxylase-mediated catabolism of both 25(OH)D3 and 1,25(OH)2D3 [12]. Thus, the finding of lower 25(OH)D3 and 1,25(OH)2D3 levels in individuals who are homozygous for the VDR FokIf variant suggests that the negative feedback loop is activated at a lower 1,25(OH)2D3 level, potentially confirming the higher transcriptional activation function of the VDR FokIf variant in vivo. Vitamin D intake (400 IU/d) was protective in women who were homozygous for the VDR FokIf variant, since their MS risk was 80% reduced compared to women who did not use vitamin D supplements, but this protective effect was not observed in women who were homozygous for the VDR FokIF variant [116]. An additional VDR geneeenvironment interaction involved the VDR gene Cdx-2G variant. The Cdx-2 polymorphism (A>G substitution) is located within the core binding element of the Cdx-2-encoded transcription factor in the 50 UTR of the VDR gene [124]. The Cdx-2G variant reduced the transcriptional activity at the VDR gene promoter by 70%. This allele conferred an increased risk of MS in individuals who had 2 h/day of winter sun exposure during childhood (age 6e10), but no increase in individuals who had 2 h/day [109]. These results suggest that a minimum threshold of sun exposure is needed to overcome the effect of reduced VDR gene expression on an event of relevance to MS that occurs in childhood.
The CYP27B1 Gene Genetic epidemiology studies have also associated some CYP27B1 alleles on chromosome 12q13.1-q13.3 with MS. The CYP27B1 gene encodes the 1a-hydroxylase that catalyzes the rate-limiting step of 1,25(OH)2D3
biosynthesis. The improbable discovery that all three women diagnosed with vitamin D-dependent rickets type I (VDDR I) in a Norwegian population also developed MS raised the possibility that deleterious mutations in the CYP27B1 gene might be causal for both diseases [125]. VDDR I (also termed pseudovitamin D deficiency rickets) is a very rare disease caused by loss-of-function CYP27B1 mutations [126,127]. Two of the women had homozygous c.1166 G>A CYP27B1 mutations and the third was heterozygous for the c.1166 G>A and c.1320_1321ins CCCACCC CYP27B1 mutations. All of the women carried the HLA-DRB1*15 gene [128]. If the association between VDDR I and MS were to be confirmed, then deleterious CYP27B1 mutations would be the first examples of dominant genetic risk factors for MS. A large genome-wide association study involving Australian and New Zealand MS patients of European ancestry also found evidence that may link CYP27B1 with MS risk [129]. In this study of w4000 patients and w8000 controls, three single nucleotide polymorphisms on chromosome 12q13-14 were significantly associated at the genome-wide level with MS. This association was confirmed in a Swedish cohort [129a]. These polymorphisms were also associated with type 1 diabetes, suggesting the existence of a common causal autoimmune disease allele. The authors identified CYP27B1 as the strongest candidate for the common causal autoimmune disease allele. Previously, investigators searching human chromosomal regions syntenic to rodent autoimmune disease genes found a weak MS susceptibility region on human chromosome 12q13.3 in Swedish families [130]. This region has been implicated in rodent EAE, arthritis, diabetes, and lupus. Further refinement of this putative susceptibility region has not been reported, but could be informative, since CYP27B1 appears to be in the region of interest. In contrast, no associations of CYP27B1 polymorphisms with MS were observed in Canadian [114] or American MS patients [116].
The GC Gene and the Vitamin-D-binding Protein The GC gene (group-specific component) on human chromosome 4q12-q13 encodes the DBP. The DBP is the plasma carrier protein for vitamin D metabolites [131]. It circulates in w100-fold molar excess of 25(OH) D3, its primary bound metabolite, and it has w10-fold higher affinity for 25(OH)D3 than for 1,25(OH)2D3. Interestingly, the hepatic synthesis of DBP is under 17b-estradiol (E2) control in women [132]. The GC gene is one of the most polymorphic genes in the human genome [131]. No associations between GC variants and MS risk were reported in early studies of Scandinavians [133,134], Canadians [114], or Japanese [135], or a recent
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study of Americans [116]. A weak association was reported in Italians [136]. Since the DBP is the plasma carrier protein for vitamin D metabolites, the GC variants might well be expected to regulate plasma 25(OH)D3 levels. A comparative study of monozygotic and dizygotic twins indicated that genotype significantly influenced plasma 25(OH)D3 levels [65]. Confirming and extending those results, a large genome-wide association study involving 4501 persons of European ancestry identified GC polymorphisms as important determinants of the plasma 25(OH)D3 level [67]. These findings are consistent with the suggestion that variation in GC alleles according to skin pigmentation evolved to facilitate prolongation of vitamin D supplies in light-skinned populations living in geographic regions with limited winter UVL availability [131]. Recently, associations between DBP levels and MS disease have been sought. Expression profiling of plasma proteins from pediatric MS patients identified DBP as a significantly upregulated protein in pediatric MS subjects compared to healthy controls [137]. However, all subjects in this study were female, and the average age of the pediatric MS subjects was 15.6 years compared to 12.2 years for the controls. Thus, the increased DBP in the female MS subjects could reflect E2-mediated enhancement of DBP synthesis in the more sexually mature MS subjects [132]. Expression profiling of CSF proteins identified DBP in MS patient samples [138]. Two studies found that DBP was w50% decreased in the CSF of MS patients in relapse [139] and CIS patients [140] compared to controls. A third study reported the opposite, that DBP was 35% elevated in the CSF of SPMS patients compared to controls [141]. Other similar studies reported no significant differences in DBP levels in the CSF of CIS or MS patients [142e144]. The inconsistencies in these studies remain unexplained. They may be attributable to methodological differences in these complex analyses, or differences in patient populations. It would be interesting to know the amount of 25(OH)D3 bound to DBP in the CSF samples during MS relapses, in addition to quantifying the DBP itself [145].
EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS, A MULTIPLE SCLEROSIS MODEL EAE is an induced paralytic autoimmune disease that has been intensively studied as an animal model of MS [146,147]. The origins of EAE can be traced to Louis Pasteur’s 1885 rabies vaccination studies, when sporadic cases of ascending paralysis were observed in patients who had been vaccinated with spinal cord preparations
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from rabies virus-infected rabbits. Histological analysis of CNS tissue from these patients showed lymphoid infiltrates and focal demyelination. Decades later, a similar disease was induced in animals by repeated immunizations with rabbit brain tissue. Now, EAE is induced most commonly in rodents by immunizing them with CNS tissue, proteins, or peptides emulsified in complete Freund’s adjuvant (containing heatkilled Mycobacterium tuberculosis), or by adoptive transfer of encephalitogenic T lymphocytes isolated from an EAE-diseased animal into a non-diseased normal animal. The EAE disease course and severity depends on the species and strain used. Clinical EAE severity is determined by daily examination of affected animals, and is scored on a disability scale of 1 to 6 (0, no disease; 1.0, limp tail; 2.0, weakness in hind legs; 3.0, ataxia; 4.0, paralysis of both hind legs; 5.0, hind and foreleg paralysis; 6.0, moribund/dead). Histopathological EAE severity is determined by examination of spinal cord sections for focal lesions that show inflammatory cell infiltration, demyelination, and axonal damage. Immunological EAE severity is determined by analysis of immune responses (e.g., lymphocyte proliferation, cytokine production) to neural antigens. Because EAE is so thoroughly characterized neurologically, immunologically, and genetically, and because it has proven to be extremely useful as a model in which to address complex questions concerning immunological function and mechanisms of central and peripheral immunological self tolerance, it has become the most intensively studied animal model of autoimmune disease. Our understanding of how autoimmune T cells orchestrate damage to CNS tissue has broadened and deepened through analysis of T cell-mediated mechanisms in EAE [148]. Myelin-specific CD4þ T cells activated outside the CNS must gain access to CNS tissue, which is normally protected from cellular infiltration by several barriers. Tight junctions between the endothelial cells of the bloodebrain barrier and the epithelial cells of the bloodeCSF barrier limit blood cell access to CNS tissue. The T cells that have encountered an antigen outside the CNS express adhesion molecules, chemokine receptors, and integrins that allow them to gain access to the CNS for immune surveillance. If a peripherally activated T cell migrates into the CNS and is reactivated through an encounter with an APC presenting peptides derived from the myelin sheath components, the T cell will induce and sustain a CNS inflammation. The details of the two-step process of invasion and reactivation are still poorly understood. Nevertheless, once in the CNS parenchymal space, the autoimmune T cells produce chemokines that attract other inflammatory cells, and pro-inflammatory cytokines that activate the parenchymal microglia and drive reactive astrocyte
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formation. The activated macrophages and microglia produce cytokines that sustain T cell activation (e.g., IL-12, IL-23), and neurotoxic chemicals (e.g., NO and oxygen radicals) that cause demyelination and axonal damage. The similarities between EAE and the human demyelinating disease MS were first noted in 1947 [149] and have since been widely documented [146,147]. The common clinical features of EAE and MS include either a relapsingeremitting or an unremitting disease course, depending on the individual patient or the experimental animal strain, the progressive accumulation of neuromuscular dysfunction, beginning with paraparesis and leading to ataxia and finally paralysis, visual impairments (optic neuritis), and other physical manifestations of neurologic dysfunction (dizziness and loss of balance, loss of bladder and bowel function). The common histological features of EAE and MS include meningitis, the formation of focal, perivascular lesions in the white matter (and occasionally the gray matter, brain stem, and optic nerves) that are disseminated in time and space, gliosis, destruction of the myelin sheaths surrounding axons with relative sparing of the neurons, and partial remyelination in older lesions. The common immunological features of EAE and MS include the appearance in the newly formed lesions of activated, neural antigen-specific T lymphocytes, activated macrophages and microglia, inflammatory cytokines, chemokines, reactive oxygen species, and the presence of oligoclonal immunoglobulins in the CNS and CSF. These histological and immunopathological changes represent a stereotypic response of CNS tissue to inflammation and immune cell-mediated injury. Although there are strong clinical, histopathological, and immunological similarities between EAE and MS which support the use of EAE as an experimental MS model, there are also differences between EAE and MS that require caution in extrapolating data from the animal model to the human disease. The most significant difference is that MS is a spontaneous disease of uncertain etiology, whereas EAE is an induced disease with a defined autoimmune origin. Genetic and epidemiological studies have implicated some combination of MS susceptibility and resistance genes, immune system dysfunction(s), microbial infection(s), insufficient UVL exposure, diet, and many other factors in MS etiology. Thus, MS may have a nearly unique etiology in each affected individual, due to her or his genotype, immune system development, past microbial exposures, lifestyle choices in relation to sunlight and dietary preferences, etc. In sharp contrast, EAE reflects an experimental model with genetic homogeneity, defined immune system function, specific pathogenfree living conditions, purified laboratory diets, and uniform exposure to light and dark cycles. The EAE
model has been useful in studying MS etiology from the perspective of immune system dysfunction(s), susceptibility and resistance genes, UVL exposure and diet, but not from the perspective of possible microbial contributions to MS etiology. The strong parallels between EAE and MS have allowed EAE researchers to develop three approved MS therapeutics. Glatiramer acetate (Copaxone) [150], mitoxantrone [151], and humanized monoclonal antibody specific for a4b1-integrin (natalizumab or Tysabri) [152] were all developed in the EAE model. Moreover, EAE has allowed researchers to examine the mechanisms of action of the anti-viral cytokine IFN-b [153], an approved MS therapeutic [154,155]. However, some agents capable of inhibiting EAE under well-defined and restricted circumstances have failed to show reproducibly beneficial effects in individuals with MS. Examples are induction of immunological self tolerance with orally administered neural antigens [156] or with altered peptide ligands [157]. Remarking on the successes and failures of the EAE model, one author wrote “the greatest value to come from our experience with EAE will result from attempting clinical translation while keeping an eye on the sum of knowledge accrued from experimentation e a sort of holistic reductionism e which is, after all, the basis for good clinical practice” [147].
VITAMIN D AND PREVENTION OF EAE Groundbreaking research in the EAE model of MS provided firm support for the vitamin DeMS hypothesis and suggested credible mechanisms of vitamin D hormone action. The evidence from the EAE model is reviewed here.
1,25(OH)2D3 and EAE Prevention Research on vitamin D and EAE began in 1991, with disease prevention studies in SJL mice [158]. The SJL strain has been extensively studied as a model of relapsingeremitting MS disease [159]. When alternate day 1,25(OH)2D3 injections were initiated 3 days before the female SJL/J mice were immunized with spinal cord homogenate to induce EAE (and continued for 15 days), the treatments decreased the morbidity and mortality of the disease [158]. A subsequent study confirmed and extended these findings to show synergy between 1,25(OH)2D3 and cyclosporin A [160]. Cyclosporin A in complex with cyclophilin is believed to inhibit the Ca-dependent phosphatase termed calcineurin that is essential for T lymphocyte activation. Both of these studies were performed using mice fed a low Ca diet (0.2% w/w), and neither study showed
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complete 1,25(OH)2D3-mediated inhibition of disease morbidity and mortality. The first study to demonstrate complete 1,25(OH)2D3mediated inhibition of disease morbidity and mortality used male and female B10.PL mice [4]. The B10.PL strain is a widely used model of relapsingeremitting MS. Feeding these mice a high Ca diet (1% w/w) with a small amount of 1,25(OH)2D3 (20 ng/d), beginning 1 day before the mice were immunized with MBP to induce EAE, completely prevented the disease. Administering an injection of 1,25(OH)2D3 at the first signs of EAE, and providing 1,25(OH)2D3 in the diet, completely inhibited EAE disease progression. However, the disease progressed when the hormone was removed. Finally, feeding animals a vitamin-D-deficient diet prior to EAE induction accelerated the disease onset. The effect of 1,25(OH)2D3 has also been examined in the adoptive-transfer model of EAE that has been widely used to examine the functions of pathogenic IFN-g-producing T helper type 1 (Th1) cells. The CD4þ T cells were collected from B10.PL mice with induced EAE disease, reactivated in vitro, and transferred into naı¨ve B10.PL animals to elicit EAE disease. Unlike the induced EAE model, where 1,25(OH)2D3 completely prevented disease morbidity and mortality, the 1,25(OH)2D3 treatment had no significant effect on EAE disease in the adoptive-transfer model [161]. Moreover, in the induced EAE model, the 1,25(OH)2D3treated mice had CD4þ T cells with a non-activated phenotype in the CNS, whereas in the adoptive-transfer model, the 1,25(OH)2D3-treated mice had activated pathogenic CD4þ Th1 cells in the CNS. One explanation for this critical difference may be the development of regulatory T or B cells capable of enforcing a non-activated phenotype on the neural antigen-specific CD4þ Th1 cells that migrate into the CNS in the induced EAE model but not in the adoptive-transfer model (see below).
Vitamin D3 and EAE Prevention The vitamin DeMS hypothesis predicts that a vitamin D3-deficient diet would accelerate or worsen disease, and conversely that a high vitamin D3 diet would inhibit the induction of disease [3]. These predictions were tested in the B10.PL model of EAE [4,162]. In the first study [4], mice were fed a vitamin D3-deficient diet or the same diet with 20 ng/d of vitamin D3 (mouse chow provides 0.33 mg/d of vitamin D3), immunized with MBP, and examined for EAE signs. The mice lacking vitamin D3 in the diet had significantly lower serum Ca and 1,25(OH)2D3, confirming their vitamin D3 deficient status. They also had a more rapid disease onset (13 days) than the mice ingesting vitamin D3 (23 days); other disease
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parameters were unchanged. In another study [162], mice were fed a diet without vitamin D3, or with 1 mg/d of vitamin D3 (three times as much as mouse chow), immunized with MBP, and examined for EAE signs. This high-vitamin-D3 diet increased the serum 25(OH)D3 to w75 nM compared to w55 nM in chow-fed mice and w15 nM in mice fed the vitaminD3-deficient diet. Unexpectedly, the high vitamin D3 diet moderately reduced all EAE disease signs (incidence, mortality, peak disease severity, cumulative index, spinal cord pathology) in female but not male mice. This result contrasts with the strong inhibition of EAE by 1,25(OH)2D3 in both males and females [4,161]. The mechanistic explanation for the sex-based difference is discussed below. At 5 mg/d of vitamin D3, the males had signs of vitamin D toxicity but no lessening of EAE disease. These data have been confirmed in C57BL/6 mice [163]. The C57BL/6 mice are used as a model of chronic MS because they develop EAE without remissions [164]. A separate study evaluated the effect of 25(OH)D3 pretreatment on EAE in C57BL/6 female mice [165]. The lowest dose tested, 0.2 mg/d, yielded a serum 25(OH)D3 level of w187 nM and had no effect on EAE disease. The next dose tested, 10 mg/d, twice the vitamin D3 dose reported to be toxic in mice [162], yielded a serum 25(OH)D3 level of w1050 nM and caused signs of vitamin D toxicity. These 25(OH)D3 pretreatment experiments are difficult to interpret, because doses between 0.2 and 10 mg/d were not tested. Nevertheless, there was a discrepancy between the vitamin D3 and 25(OH)D3 pretreatment results in C57BL/6 mice that remains unexplained.
UVL and EAE Prevention The vitamin DeMS hypothesis also predicts that UVL exposure would inhibit the induction of disease [3]. The ability of UVL to prevent EAE was examined in female SJL mice [166]. The shaved mice were exposed to UVL irradiation (2.5 kJ/m2) daily for 7 days, and immunized with mouse spinal cord homogenate 1 day later. This UVL treatment significantly reduced the incidence, severity, and neuropathology of EAE. UVL treatment at the time of immunization or after immunization had no effect on disease development. Thus, this study documented a protective effect of repeated UVL exposure in EAE. A separate study of female SJL mice reported a different outcome [167]. The shaved mice were exposed to UVL irradiation (5.9 kJ/m2) once, and immunized with MOG92e106 peptide just 1 hour later. The UVL-exposed mice had a higher disease incidence and many had a progressive EAE disease course, compared to the unexposed controls. The authors attributed their
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results to a UVL-induced shift towards a T helper type 2 (Th2)-mediated response supporting B cell antibody production to MOG92e106 peptide and antibody-mediated CNS pathology. It is not clear why a single high dose of UVL followed 1 hour later by MOG92e106 peptide immunization exacerbated EAE in female SJL mice, whereas a week of low-dose UVL followed 1 day later with an immunization of spinal cord homogenate protected female SJL animals from EAE. One possibility is that the high-dose UVL may have damaged the skin and elicited a damage response that influenced the outcome of the immunization just 1 hour later, whereas a week of low-dose UVL may not have induced such damage. A third study examined UVL exposure in C57BL/6 mice [165]. The shaved mice were exposed to UVL irradiation (2.5 kJ/m2) daily for 7 days before immunization with MOG35e55 peptide, and on each second or third day thereafter. This protocol increased the serum 25(OH)D level at the time of immunization and decreased EAE disease as in the prior study [166]. Because the controls and UVL-treated mice did not differ in serum 25(OH)D on days 16 and 32 postimmunization, the authors concluded that UVL suppression was independent of vitamin D3 synthesis. An alternative interpretation is that the UVL treatments on each second or third day catalyzed sufficient vitamin D3 synthesis in the skin to sustain systemic immunosuppression via the skin draining lymph node [168], although not enough to elevate serum 25(OH)D. The investigators did not analyze vitamin D3 or any of its metabolites in the skin, the draining lymph node, or the CNS, so localized production of 1,25(OH)2D3 cannot be ruled out. Others have shown that UVLinduced systemic immunosuppression depended on 1,25(OH)2D3-mediated induction of CD4þCD25þ Treg cells in the skin [168], and this induction depended on VDR expression [169], supporting a role for highly localized production of 1,25(OH)2D3 in UVL-induced suppression. These caveats preclude drawing reliable conclusions as to whether UVL functions independently of the vitamin D endocrine system in the EAE model [170].
MECHANISMS OF EAE PREVENTION Several mechanisms are likely to contribute to 1,25(OH)2D3-mediated EAE prevention. Although a variety of experimental approaches have provided clear hints of these mechanisms, there are some ambiguities and inconsistencies in the data, and many important details of the suggested mechanisms are missing. Below is a summary of the current understanding of 1,25(OH)2D3-mediated EAE prevention.
Calcium and EAE Prevention The suppressive effects of 1,25(OH)2D3 in the EAE model correlate with moderate elevations in serum Ca [4,39,171]. In fact, studies in B10.PL mice documented a strong inverse relationship between dietary Ca (1.0%, 0.47%, 0.02% w/w) and the amount of 1,25(OH)2D3 needed to completely prevent EAE in males (100 ng/d, 400 ng/d, incomplete inhibition) and females (6 ng/d, 50 ng/d, incomplete inhibition) [171]. That more 1,25(OH)2D3 was needed to completely prevent EAE in males than females was noteworthy in this study. The explanation for this sex difference is not entirely clear, but may relate to sex differences in Cyp24a1 gene expression, which may be crucial to our understanding of sex differences in MS incidence and disability. Additional research has examined the effects of elevated Ca independently from 1,25(OH)2D3 using C57BL/6 animals with a disrupted Cyp27b1 gene [172]. The Cyp27b1-null mice cannot synthesize 1,25(OH)2D3. These mice were fed a diet with 0.87% Ca and 1 ng/ d of 1,25(OH)2D3 to maintain normal serum mineral homeostasis, immunized to induce EAE, and infused with PTH to induce hypercalcemia. This protocol completely prevented EAE in female mice, but not in male mice. When the study was repeated with a 2% Ca plus 20% lactose diet, no 1,25(OH)2D3, and PTH infusion after EAE induction, the PTH had no effect on Ca levels or EAE disease. The researchers interpreted their data as showing that hypercalcemia was sufficient to inhibit EAE in females. Another interpretation is that hypercalcemia reduced the amount of 1,25(OH)2D3 needed to inhibit EAE to a threshold that was effective in females but not in males [171]. Subsequent research showed that hypercalcemia occurred in 1,25(OH)2D3treated female C57BL/6-IL-10-null mice, but EAE was not prevented [39]. This result clearly established that hypercalcemia was not sufficient for EAE prevention in females. The peptide hormone calcitonin (CT) is produced in response to hypercalcemia, and acts to suppress Ca release from bone to re-establish Ca homeostasis. Studies of CT in the EAE model showed that it reduced the amount of exogenously supplied 1,25(OH)2D3 needed to prevent EAE in female C57BL/6 mice, particularly when combined with high dietary Ca [173]. Moreover, the CT-treated mice did not develop hypercalcemia. Neither CT nor CT-related peptide played an essential role in the 1,25(OH)2D3-mediated protective mechanism, since the 1,25(OH)2D3 inhibited EAE induction in mice with targeted disruptions of the CT gene and its splice variant, CT-related peptide [174]. To summarize the research on Ca and 1,25(OH)2D3 in the EAE model, the data show that a strong inverse relationship exists between dietary Ca and the amount of 1,25(OH)2D3
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needed to completely prevent EAE [171], but hypercalcemia alone was insufficient to inhibit the disease [39]. Direct participation of PTH [172] and CT [174] in the mechanism of protection has been ruled out. Further research will be needed to uncover the mechanistic explanation as to the why the inverse relationship between Ca and 1,25(OH)2D3 exists. In the context of the strong inverse relationship between Ca and 1,25(OH)2D3, it is noteworthy that Finnish investigators found evidence of dysregulated Ca homeostasis in MS patients [82]. As serum 25(OH)D levels fell to w45 nM through autumn to winter, the controls increased their circulating intact PTH (peak 4.5 pM) and maintained serum Ca homeostasis (2.36 mM). In stark contrast, the MS patients reached the same serum winter 25(OH)D nadir, but the intact PTH response was blunted and relative hypocalcemia developed. The MS attacks showed a distinct pattern; 66% occurred at the intact PTH peak, and 76% occurred at the lowest or decreasing 25(OH)D level. All attacks occurred at 25(OH)D <85 nM, intact PTH >20 ng/l, and Ca 2.24 mM. The correlation between low 25(OH)D, high intact PTH, low Ca and attacks was striking. Whether this correlation reflects causation or reverse causation is a question that will require further study.
1,25(OH)2D3 Synthesis during CNS Inflammation Extra-renal expression of CYP27B1 encoding the 1ahydroxylase that synthesizes 1,25(OH)2D3 from 25(OH)D3 has been recognized for many years. Nevertheless, the physiological relevance of extra-renal 1,25 (OH)2D3 synthesis continues to be debated. The discovery that immune system cells biosynthesize 1,25 (OH)2D3 arose ultimately from clinical observations made in sarcoidosis patients [175]. These patients had hypercalcemia that was aggravated by sunlight exposure or fish oil consumption. Analysis revealed that they had about two-fold more 1,25(OH)2D3 than healthy controls [176,177]. The 1,25(OH)2D3 production in sarcoidosis was traced to IFN-g-stimulated pulmonary alveolar macrophages [178,179]. Immunohistochemical techniques have also detected the 1a-hydroxylase in skin, placenta, colon, pancreas, vasculature, and brain [33,180,181], suggesting that widespread extra-renal 1,25(OH)2D3 synthesis may play a role in many biological processes. New evidence suggests that immune cell 1,25(OH)2D3 synthesis has important intracrine, autocrine, and paracrine functions in the regulation of immune responses. Induction of CYP27B1 gene expression in innate immune cells has been reviewed recently [181e183]. The known inducers of Cyp27b1 in innate
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immune cells are IFN-g, Toll-like receptor 4 ligands, and Toll-like receptor 2/1 ligands via IL-15. Also, human B cells activated through the immunoglobulin receptor and CD40 [184], and CD4þ T cells activated through the TCR and CD28 [88], both produced and responded to 1,25(OH)2D3. Langerhans cells produced 1,25(OH)2D3 and programmed T cells to migrate to the epidermis [185], and DC cells to migrate to lymph nodes [186]. Direct evidence for 1,25(OH)2D3 synthesis in the inflamed CNS was obtained in an EAE study [162]. Feeding a high vitamin D3 diet beginning prior to disease induction delayed the onset and reduced the severity of EAE in female but not male mice. In this study, the serum 1,25(OH)2D3 level did not vary by diet, sex, or EAE disease status. In sharp contrast, the spinal cord 1,25(OH)2D3 level varied significantly by diet, sex, and disease status. It was w30e50 fmol/g CNS tissue in non-immune males and females, vitamin D3-deficient males and females, and vitamin D3-fed males with EAE. However, it increased to w125 fmol/ g CNS tissue in vitamin D3-fed females primed with neural antigen. The 1a-hydroxylase:24-hydroxylase ratio controls the net rate of 1,25(OH)2D3 synthesis and hence the transcriptional activity of the VDR [11]. Consistent with an increase in the spinal cord 1,25(OH)2D3 level only in females, the females had a Cyp27b1:Cyp24a1 transcript ratio of 17:1, whereas this ratio was only 6:1 in males. Only the males had high levels of Cyp24a1 transcripts in the CNS. The fact that the spinal cord 1,25(OH)2D3 level increased over three-fold while the serum 1,25(OH)2D3 level remained constant suggests that 1,25(OH)2D3 was synthesized in situ in the CNS, and this synthesis correlated with disease inhibition. The cells that produced 1,25(OH)2D3 were not identified but might be glial cells [187] or neurons [188], or infiltrating macrophages.
Vitamin D Receptor in Immune System Cells The VDR was essential for the mechanism by which 1,25(OH)2D3 inhibited EAE disease induction, ruling out non-genomic mechanisms [189]. Several types of immune system cells express the VDR and could be directly involved in the protective mechanisms. With regard to human cells, a radioactive ligand binding assay detected few VDR molecules in non-activated B and T cells (<1000 VDR/cell), but w10-fold more VDR molecules in activated T cells [190]. Human B lymphocytes activated through the immunoglobulin receptor, CD21, and CD40 in the presence of 1,25(OH)2D3 also increased their VDR expression w10-fold [191]. The human macrophages had no detectable VDR, but dendritic cells (DC) had w6000 VDR/cell [190]. With
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regard to rodent cells, neither VDR mRNA nor protein levels have been reported in murine DC, although these cells are known to be responsive to 1,25(OH)2D3 (see below). The VDR protein was abundant in activated murine CD8þ T cells (w160 fmol/mg protein), less abundant in activated CD4þ T cells (w100 fmol/mg protein), present at trace levels in macrophages (40 fmol/mg), and undetectable in lipopolysaccharide-activated B cells [192]. Among subsets of fully differentiated murine CD4þ T cells, the IL-4-producing Th2 cells had abundant VDR mRNA, whereas the IFN-g-producing Th1 cells had intermediate VDR mRNA levels [161]. The VDR expression was high in IL-17-producing Th17 cells, but low in natural Treg cells [161a].
T Lymphocytes Because a T cell-mediated immune response directed to components of the myelin sheath surrounding the nerve fibers is believed to be pathogenic in MS, T lymphocytes have been studied intensively in EAE. The CD8þ T cells played no role in the suppressive mechanisms of 1,25(OH)2D3, as evidenced by the hormone’s efficacy in CD8-null mice [193]. However, a role for CD4þ T cells in the hormone’s mechanisms to prevent EAE disease induction is very likely. In one study, a few CD4þ T cell receptor transgenic (TCR-tg) T cells specific for MBP were used as a tracer for T cell responses in B10.PL mice [161]. The mice were given 1,25(OH)2D3 continuously beginning before EAE disease induction. The hormone dramatically inhibited clinical EAE disease signs, but it did not inhibit the differentiation or proliferation of pathogenic IFN-gproducing Th1 cells in the periphery. Moreover, 1,25(OH)2D3 did not prevent CD4þ TCR-tg T cell migration into the CNS. Instead, the 1,25(OH)2D3 prevented these cells from displaying an activated state in the CNS (placebo-treated, w33%-activated phenotype; 1,25(OH)2D3-treated, w5% activated), consistent with the lack of IFN-g transcripts in the CNS, and with the disease-free status of the animals. The mechanisms that account for these results are not known, but are believed to involve the participation of neural antigen-specific regulatory T or B lymphocytes [161]. Recent experiments have investigated the need for VDR expression in T lymphocytes in 1,25(OH)2D3-mediated prevention of EAE through analysis of bone marrow chimeric animals and conditional VDR targeting studies [161a]. Evaluation of bone marrow chimeric mice established that VDR expression was required in radio-sensitive hematopoietic cells but not in non-hematopoietic cells for 1,25(OH)2D3 pretreatment to reduce EAE disease induction. Further studies of mice with a conditionally targeted VDR in T lymphocytes
established that VDR expression was required in these cells for 1,25(OH)2D3 to inhibit EAE, although it was not required for T lymphocyte development, activation, or proliferation. These data support a model wherein 1,25(OH)2D3 acts directly on CD4þ T cells to inhibit EAE pathogenesis.
Interferon-g-producing T Helper Type 1 Cells The IFN-g-producing T helper (Th)1 cells are known to be pathogenic in MS [17,18] and in EAE [148]. Accordingly, investigators have evaluated whether 1,25(OH)2D3 might inhibit IFN-g-producing Th1 cell development or function as a mechanism of EAE prevention. Consistent with this possibility, administering 1,25(OH)2D3 to Biozzi AB/H mice beginning prior to EAE induction with MOG35e55 peptide, decreased the frequency of IFN-g-producing Th1 cells in peripheral lymphoid tissues correlating with disease inhibition [194]. Biozzi AB/H mice develop acute EAE, recover, then develop chronic EAE. Other in vitro studies showed 1,25(OH)2D3 inhibition of T cell proliferation, and of IFN-g and IL-12 production and signaling [195], extending the abundant literature on 1,25(OH)2D3 inhibition of T cell function in vitro [16,196]. In sharp contrast to the results obtained with Biozzi AB/H mice, administering 1,25(OH)2D3 to B10.PL mice beginning prior to EAE induction with MBP had no effect on the IFN-g-producing Th1 cell response in vivo, whether IFN-g protein or IFN-g mRNA was quantified [4,161,197]. It also had no effect on the peripheral T cell proliferative responses to MBP. Furthermore, the 1,25(OH)2D3 treatments did not affect the amount of IFN-g (or TNF-a) produced in response to MBP on a per cell basis at any stage of EAE disease. Finally, the 1,25(OH)2D3 treatments did not affect the amount of IFN-g produced by fully differentiated Th1 cells [161]. These data are consistent with data showing no effect of vitamin D3 or 1,25(OH)2D3 on Th1 cell IFN-g responses in humans [91,93,196]. Thus, with the exception of one report [194], abundant in vivo evidence in mouse and human has indicated that 1,25(OH)2D3 does not inhibit the development or function of IFN-gproducing Th1 cells in peripheral tissues.
Interleukin-4-producing T Helper Type 2 (Th2) Cells The IL-4-producing Th2 cells counteract the pathogenic Th1 cells [148], so researchers have investigated possible IL-4-producing Th2 cell enhancement by 1,25(OH)2D3 as a mechanism of action. In support of this mechanism, B10.PL mice treated continuously with 1,25(OH)2D3 and immunized to induce EAE had a significantly higher level of IL-4 transcripts in the lymph
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nodes and in the CNS than the placebo/EAE controls [197]. Moreover, targeted disruption of the IL-4 gene moderately reduced the 1,25(OH)2D3 efficacy against EAE [198]. Specifically, the hormone reduced the peak of disease from 2.5 to 1.4 in IL-4þ/-heterozygous mice, and from 2.3 to 0.9 in IL-4/-homozygous mice. However, contrary to the idea that 1,25(OH)2D3 enhances the Th2 cell response, a subsequent report found no significant differences between 1,25(OH)2D3-pretreated and placebo-pretreated B10.PL mice with regard to IL-4 mRNA in the lymph nodes or the CNS after immunization with MBP [161]. There was also no effect of 1,25(OH)2D3 on the amount of IL-4 protein produced by fully differentiated Th2 cells. Similarly, 1,25(OH)2D3 pretreatment of Biozzi AB/H mice immunized with MOG35e55 peptide had no effect on the IL-4-producing Th2 cell frequency as determined by flow cytometric analysis [194]. Thus, there are some inconsistencies regarding IL-4 that remain to be resolved. The moderately decreased 1,25(OH)2D3 efficacy against EAE in IL4-null mice argues for some IL-4 contribution [198]. However, since no increase in IL-4-producing Th2 cells was observed [161,194], the IL-4 source may not be these cells.
Interleukin-17-producing T Helper Type 17 (Th17) Cells The recently identified IL-17-producing CD4þ Th17 cells are particularly pathogenic in autoimmune disease [199]. Accordingly, possible 1,25(OH)2D3-mediated inhibition of Th17 cell differentiation and function has been studied in animal models of autoimmune disease. In experimental autoimmune uveitis (EAU), a disease induced by immunization with a retinal interphotoreceptor protein, 1,25(OH)2D3-mediated prevention of the disease was accompanied by decreased development of antigen-specific Th17 cells in vivo, an effect that was also observed in vitro [200]. Similar findings were reported in an animal model of colitis [201]. An inhibitory effect on IL-17 has also been observed in humans. Adding 1,25(OH)2D3 to purified and activated CD4þ T cells from both controls and relapsingeremitting MS patients significantly reduced the frequency of CD4þ Th17 cells [88]. Thus, there is accumulating evidence that 1,25(OH)2D3 acts directly on nascent and fully differentiated Th17 cells to inhibit IL-17 production as a mechanism of autoimmune disease inhibition.
Interleukin-10-producing T Regulatory Cells In the last decade, the importance of Treg cells in limiting the expansion of autoimmune T cells,
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preventing autoimmune-mediated pathology, and promoting recovery from autoimmune-mediated attacks has been widely appreciated [202]. A decade ago, researchers showed that T cell clones from (chronic progressive MS (CPMS)) patients were unresponsive to regulatory signals [203]. Similar findings were reported for RRMS patients [204]. More recently, a defect in Treg cell function was documented in MS patients [205], notably in the IL-10-producing Treg cell subset [206,207]. Additional studies have confirmed Treg cell defects in MS patients [208,209]. This subject has been reviewed [210]. New data show positive correlations between serum 25(OH)D3 and Treg cells in MS patients [211e213], consistent with the possibility that vitamin D enhancement of Treg function is an important mechanism in autoimmune disease inhibition. In the autoimmune EAE model, it was striking that 1,25(OH)2D3 treatments begun prior to neural antigen immunization in either SJL mice [158] or B10.PL mice [161] conferred residual protection from EAE disease after the treatments were discontinued. Complete eradication of potentially pathogenic T cells did not explain this residual protection from EAE, because tracer MBPspecific CD4þ TCR-tg T cells were present in the CNS of the 1,25(OH)2D3 pretreated animals [161]. Rather, residual protection appeared to reflect the induction of regulatory T or B lymphocytes capable of inhibiting the activation of these potentially pathogenic T cells. In favor of this hypothesis, the 1,25(OH)2D3 pretreatment inhibited EAE in TCR-tg mice with a functional Rag-1 gene, but not in TCR-tg mice lacking a functional Rag-1 gene [161]. Since the Rag-1 gene is essential for lymphocyte development, the latter mice have no lymphocytes other than the MBP-specific CD4þ TCRtg T cells. In particular, these mice lack regulatory lymphocytes. These studies were the first to implicate regulatory lymphocytes (B or T cells) as essential for 1,25(OH)2D3-induced residual protection from EAE disease [161]. Suppressor cells capable of protecting animals against EAE immunopathology were first described in 1975 [214]. The suppressor cells were subsequently shown to be CD4þ T cells [215e218]. They had the surface marker phenotype CD25þCD44highCTLA-4highGITRhigh and expressed Foxp3 as a lineage-specific transcription factor [219]. These CD4þ Treg cells were specific for neuro-antigens [220] and suppressed EAE by an IL-10-dependent mechanism [221e225]. The hypothesis that 1,25(OH)2D3 might enhance the frequency or function of these IL-10-producing Treg cells received some support with the demonstration that culturing T cells in vitro with dexamethasone and 1,25(OH)2D3 generated Treg cells capable of inhibiting EAE development [226].
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Additional support for the IL-10-producing regulatory cell hypothesis came from experiments in C57BL/ 6 mice with a targeted disruption of the IL-10 gene or the gene encoding the IL-10-receptor [39]. The female IL-10þ/þ and IL-10/ mice were fed a diet without vitamin D3, or with 1 mg/d of vitamin D3, immunized with MBP, and examined for EAE signs. The vitamin D3 diet decreased the incidence, peak severity, and cumulative disease in IL-10þ/þ mice, but had no effect on any EAE disease parameter in the IL-10/ mice. Furthermore, 1,25(OH)2D3 pretreatment also failed to inhibit EAE in the IL-10/ mice, and in mice with a targeted disruption of the IL-10R gene. The MBP-immunized IL-10/ mice had activated, IFN-g-producing Th1 cells in the CNS despite 1,25(OH)2D3 pretreatment consistent with a lack of regulatory cell protection, whereas these cells were absent in 1,25(OH)2D3-pretreated IL-10þ/þ mice consistent with regulatory cell protection. Reciprocal bone marrow chimera studies established that limiting IL-10 synthesis to either the radio-sensitive hematopoietic or the radio-resistant non-hematopoietic cells abrogated 1,25(OH)2D3-mediated protection. These data show that a bi-directional IL-10 signaling loop between hematopoietic and nonhematopoietic cells is essential for 1,25(OH)2D3-mediated protection in EAE. The above EAE studies did not identify the IL-10producing hematopoietic or non-hematopoietic cells needed for 1,25(OH)2D3-mediated protection. A variety of cells produce IL-10, most notably innate immune system cells (macrophages, DC, glial cells), CD4þ Treg cells, and B lymphocytes [227]. It is noteworthy that human CD4þ T cells obtained from healthy controls or relapsingeremitting MS patients also produced 1,25(OH)2D3 and responded to 1,25(OH)2D3 with an enhanced frequency of IL-10-producing CD4þ T cells when activated through the TCR and CD28 [88]. Similarly, human B cells obtained from healthy controls produced 1,25(OH)2D3 and responded to 1,25(OH)2D3 with threefold enhanced IL-10 synthesis when activated through the immunoglobulin receptor and CD40 [184]. Thus, it seems probable that direct or indirect 1,25(OH)2D3-mediated enhancement of IL-10 synthesis by hematopoietic and non-hematopoietic cells contributes to the hormone’s protective effects in autoimmune disease. It is useful to consider how the bi-directional IL-10 signaling loop might be disrupted, since disrupting this loop could undermine the protective functions of 1,25(OH)2D3 and precipitate autoimmunity [38]. One disruption might come from the ubiquitous human herpes virus EBV, which has been linked to MS risk [25]. This virus produces viral IL-10, a protein with 83% amino acid sequence identity to human IL-10 [228]. The viral and human IL-10 structures differ in two regions that contact the IL-10R; they compete for
receptor binding, but they signal differently [229]. Whereas human IL-10 inhibited Th1-type inflammatory responses in vivo in mice, viral IL-10 actually enhanced these inflammatory responses [230]. Thus, in EBVinfected humans, the infected B cells might produce viral IL-10 instead of human IL-10, blocking human IL10 from performing essential regulatory functions, and driving inflammatory responses that facilitate autoimmune disease [38]. In the absence of human IL-10 function, the IL-10-dependent protective effects of 1,25 (OH)2D3 could fail. Additional research will be needed to test this proposed mechanism linking two of the major environmental factors believed to be involved in MS etiology.
Transforming Growth Factor-b1 The regulatory cytokine, transforming growth factorb1 (TGF-b1), has a prominent function in maintaining T cell self tolerance via its direct support for the differentiation of the CD4þFoxp3þ Treg cells that control excessive inflammatory responses and limit immunopathology [231]. It is noteworthy that administering 1,25(OH)2D3 to B10.PL mice prior to EAE induction with MBP significantly enhanced the TGF-b1 response in vivo [197]. The TGF-b1 mRNA increased six-fold in CNS samples and in peripheral lymph node samples from 1,25(OH)2D3-treated mice compared to placebo-treated animals. Consistent with the rodent data, vitamin D3 supplementation of MS patients significantly increased serum TGF-b1 levels from 230 21 pg/ml at baseline to 295 40 pg/ml 6 months later [91]. These data suggest that 1,25(OH)2D3-mediated upregulation of TGF-b1 may contribute to Treg cell differentiation as a mechanism of autoimmune disease prevention.
Dendritic Cells Another suggested mechanism for autoimmune disease prevention is 1,25(OH)2D3 action directly on myeloid DC to establish a tolerogenic phenotype, and subsequent tolerogenic DC induction of CD4þFoxp3þ Treg cells [232,233]. Human [234e236] and murine [237,238] DC respond to 1,25(OH)2D3 in vitro with an increase in phagocytic function, a decrease in MHC class II molecules and costimulatory molecules, and downregulation of the capacity to produce IL-12 and present antigens to T lymphocytes [239]. These changes were not observed in plasmacytoid DC [240]. Moreover, the changes were not observed in myeloid DC from VDR-null mice, implicating the VDR in the response [238]. Studies of mouse diabetes [241] and colitis [201] have extended these in vitro findings by correlating 1,25(OH)2D3-mediated disease prevention with the
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MECHANISMS OF EAE AMELIORATION
appearance of tolerogenic DC (decreased MHC class II, CD40, CD80, CD86, and IL-12p75) and Treg cells in vivo. Quite recently, in vitro experiments established that culturing human DC with 1,25(OH)2D3 increased their expression of indoleamine 2,3-dioxygenase, the enzyme that degrades tryptophan to kynurenine [88]. Microenvironments rich in kynurenine and poor in tryptophan are believed to favor conversion of CD4þCD25Foxp3 T cells into CD4þCD25þFoxp3þ Treg cells capable of restoring immunological self tolerance [242,243]. Thus, 1,25(OH)2D3-mediated induction of indoleamine 2,3dioxygenase in DC could lead to development of Treg cells capable of arresting the autoimmune responses of encephalitogenic T cells producing inflammatory cytokines like IL-17 [88]. It appears that 1,25(OH)2D3 action directly on T cells is essential in addition to actions on DC for inhibition of autoimmunity. In the EAE model, 1,25(OH)2D3 action directly on myeloid DC was not sufficient to prevent disease in TCR-Tg mice lacking a functional Rag-1 gene [161], or in mice whose CD4þ T lymphocytes lacked VDR expression [161a] . In EAU, 1,25(OH)2D3mediated actions on DC and prevention of the disease were not accompanied by increased development of CD4þFoxp3þ Treg cells in vivo [200]. Collectively, the data suggest that 1,25(OH)2D3 acts on myeloid DC to induce a tolerogenic phenotype, and in some autoimmune model systems (diabetes, colitis) this action is sufficient to prevent disease. However, in other model systems (EAE, EAU), induction of tolerogenic DC must be accompanied by direct actions of the hormone on T lymphocytes to prevent autoimmunity.
Summary To summarize, the disease prevention data suggest a model wherein UVL and dietary vitamin D3 support 1,25(OH)2D3 synthesis in the CNS, and this in situ hormone synthesis initiates a disease prevention pathway that centers on containment of the autoimmune T cell-driven inflammatory cascade in the CNS. Precisely how in situ hormone synthesis leads to containment of CNS inflammation is not yet fully understood. The mechanisms do not involve 1,25(OH)2D3 inhibition of IFN-gproducing Th1 cell development, function, or migration into the CNS, or promotion of IL-4-producing Th2 cells. The mechanisms do involve the VDR (particularly in T lymphocytes) and Ca. They also require Rag-1-dependent regulatory lymphocytes, possibly increases in TGF-b1, IL4, and IL-10, and possibly decreases in IL-17. Together, the mechanisms lead to the inhibition of activated neural antigen-specific pathogenic T cell accumulation in the CNS. Three specific molecular mechanisms under consideration to explain EAE disease prevention are (1)
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1,25(OH)2D3 action directly on DC (specifically downregulation of MHC class II molecules, costimulatory molecules, and cytokines) rendering them tolerogenic and capable of inducing Treg cells [232,233], (2) an action directly on induced regulatory cells to promote their differentiation and/or function [39,161,226], and/or (3) an action on encephalitogenic T cells (e.g., Th1 and Th17 cells) enhancing their responsiveness to apoptotic signals [244,245]. These mechanisms are not mutually exclusive and all may apply.
VITAMIN D AMELIORATION OF ESTABLISHED EAE Several research groups have reported that administering 1,25(OH)2D3 to rodents continuously beginning at the first clinical signs of EAE inhibited the EAE disease course [4,246,247]. When B10.PL mice with a loss of tail tone were treated with 1,25(OH)2D3, disease progression was prevented, but the loss of tail tone was not reversed [4]. The EAE disease progression resumed when the 1,25(OH)2D3 treatments were stopped. Administering 1,25(OH)2D3 at the peak of acute EAE disability reversed the EAE disease signs [197,248]. For example, when mice with complete hind limb paralysis were treated with 1,25(OH)2D3, they began walking 3 days later, while the placebo-treated mice remained paralyzed [248]. A dramatic reduction in histopathology (inflammatory infiltrate, demyelination) accompanied the reversal of EAE. In this study, the 1,25(OH)2D3treated mice had only a residual loss of tail tone. Treatment of established EAE disease with 1,25(OH)2D3 has also been reported in rats [246,247]. The treatments began on day 11 after disease induction. Subsequently, the placebo and 1,25(OH)2D3-treated groups both went into remission, then appeared to diverge as the placebo mice relapsed, but the divergence did not reach statistical significance due to the short time frame. Treatment of established EAE disease with a 1,25(OH)2D3 analog (Ro 63-2023) was evaluated in Biozzi AB/H mice [194]. This oral treatment began at the first remission on day 25 and was discontinued on day 48. Thereafter, the treated group had lower disability and relapse rates than the placebo group for an entire year.
MECHANISMS OF EAE AMELIORATION T Lymphocytes The mechanisms involved in amelioration of established EAE center on eradication of the T-cell-mediated inflammatory cascade. In rats with EAE, an
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immunohistochemical analysis performed after 2 weeks of repeated 1,25(OH)2D3 treatments showed fewer CD4þ T cells in the CNS [246]. Subsequently, kinetic analysis of 1,25(OH)2D3-mediated EAE disease resolution showed that clinical recovery occurred rapidly, with a decrease from 3 to 2 on the disability scale after just 3 days of 1,25(OH)2D3 treatment [248]. Correlating with clinical recovery there was a 50% decrease in the degree of white matter and meningeal inflammation (smaller lesions, fewer inflammatory cells, and less tissue disruption). A gene expression analysis performed 6 hours post 1,25(OH)2D3 treatment found altered expression of only a few (0.7%) of the CNS genes analyzed [244]. Noteworthy were 1,25(OH)2D3-driven transcriptional changes favoring apoptosis (see below), and a substantial 32-fold decrease in the transcript for the CD3d component of the TCR. These data suggested the hypothesis that the 1,25(OH)2D3 may have sensitized CNS-resident pathogenic T cells to AICD signals. Additional evidence for this mechanism came from flow cytometric analyses showing a decrease in living CD4þ T cells accompanied by an increase in apoptotic CD4þCD44high T cells, immunohistochemical analyses showing dying cells with fragmented DNA in the meningeal lesions, and PCR analyses showing less IFN-g production, all of which occurred in the spinal cord within 6e12 hours of the 1,25(OH)2D3 treatment [244,245]. The fact that these changes occurred in the CNS but not in the lymph nodes of intact animals or in cell cultures suggests that the apoptosis signals derived from the intact CNS [245].
Sensitivity of T Cells to Activation-induced Cell Death The details of the AICD mechanism that may be triggered by 1,25(OH)2D3 are not known, but several possibilities merit consideration [249]. One possibility is an L-arg starvation and peroxynitrite-mediated death mechanism, consistent with the observed increase in arginase transcripts [244,245]. Myeloid lineage cells exert metabolic control over T cells by depleting L-arg and producing peroxynitrite, which is toxic to T cells [250]. Another possibility is a calciumand calpain-2-dependent apoptosis program [251], consistent with the observed increase in calpain-2 transcripts [244]. A third possibility is an extrinsic Fas death pathway mechanism, consistent with the observed increase in caspase-8-associated protein 2 and the decrease in the inhibitor of apoptosis protein-2, which promote and oppose the death pathway, respectively [244]. Significantly, research on the NOD mouse model of diabetes also found that 1,25(OH)2D3 increased the sensitivity of T lymphocytes to AICD in vivo. The suggested mechanism in NOD mice was enhancement of
the extrinsic FAS death pathway leading to the disruption of mitochondrial membrane integrity [252]. Also significant is the finding that glucocorticoids act directly on T lymphocytes to increase apoptosis as a mechanism to reverse established EAE [253]. In summary, data from two autoimmune models highlight sensitization of pathogenic T cells to AICD as an important primary mechanism by which 1,25(OH)2D3 inhibits autoimmune disease.
Inhibition of Chemokine Synthesis Activated T cells drive pathogenic autoimmune responses in part through the production of chemokines that recruit additional immune system cells to an inflamed site [254]. A kinetic analysis of 1,25(OH)2D3mediated EAE disease resolution showed that 12 hours after the activated CD4þ T cells died, there was a coordinated 80e90% inhibition of chemokine transcripts (CCL2, CCL3, CCL4, CXCL2, and CXCL10) in the CNS [245]. The 1,25(OH)2D3 treatment did not decrease chemokine transcripts or immune cell recruitment in peripheral lymphoid tissues, consistent with the continued presence of activated CD4þ T cells in these tissues [245]. These data rule out a direct effect of 1,25(OH)2D3 on chemokine transcription. The decline in chemokines coincided with an w80e90% reduction in immune cell recruitment into the CNS, and was followed by a 70% decrease in CD11bþ macrophages in the CNS, and then EAE symptom abatement [248]. Significantly, research on diabetes in the NOD mouse also found an inhibitory effect of 1,25(OH)2D3 on chemokines in vivo [255]. Thus, data from these autoimmune models suggest inhibition of chemokine synthesis and immune cell trafficking are important secondary mechanisms by which 1,25(OH)2D3 inhibits autoimmune disease.
Inhibition of Inducible Nitric Oxide Synthase Transcription Increased bloodebrain barrier permeability due to nitric oxide (NO) production is thought to facilitate inflammatory cell recruitment into MS and EAE lesions [256]. Therefore, it was not surprising that reduced iNOS and NO production coincided with 1,25(OH)2D3 inhibition of inflammatory cell recruitment into the CNS. Immunohistochemical data from the rat model of EAE showed that after 2 weeks of 1,25(OH)2D3 treatment there was decreased expression of iNOS protein in the CNS [247]. Additional experiments in the mouse model of EAE showed that the 1,25(OH)2D3 did not decrease inducible nitric oxide synthase iNOS mRNA 6 hours post-treatment relative to placebo [244], but within
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24 hours, iNOS mRNA declined by 87% and iNOS protein became undetectable by immunohistochemical methods in the hormone-treated CNS samples, probably due to the disappearance of iNOS expressing cells [245]. This decrease in iNOS would lower NO production and contribute to the restoration of the bloodebrain barrier.
Summary To summarize, the treatment data suggest a model wherein 1,25(OH)2D3 initiates a disease resolution pathway that centers on eradication of the autoimmune T cell-driven inflammatory cascade. The hormone rapidly (6 hours) induced transcriptional changes favoring the sensitization of CNS-resident pathogenic T cells to in situ AICD signals that may have included Larg starvation and peroxynitrite production, a calciumand calpain-2-dependent apoptosis program, and/or an extrinsic FAS death pathway mechanism. Important secondary mechanisms of EAE amelioration were inhibition of chemokine synthesis, a decrease in iNOS and NO production, and cessation of immune cell recruitment (24 hours). Finally, infiltrating macrophages were lost from the CNS (48 hours) and EAE symptoms abated (72 hours). It seems possible that a short exposure to 1,25(OH)2D3 might induce similar mechanisms in MS patients experiencing an acute relapse.
MULTIPLE SCLEROSIS, PREGNANCY, AND IMMUNE TOLERANCE MS mainly affects women in the child-bearing years. A curious and long-standing observation is that the rate of MS attacks declines by two-thirds during pregnancy and rapidly increases to the pre-pregnancy rate postpartum [257]. This observation suggests that the endocrine changes associated with pregnancy somehow exert a protective effect as regards MS. Maternal circulating 1,25(OH)2D3 triples and maternal estrogen increases more than 200-fold during pregnancy, due to synthesis of these hormones by placental and decidual tissue [258,259]. It is possible that the pregnancyinduced increases in 1,25(OH)2D3 and estrogen contribute to the protective effects of pregnancy in women with MS [163,260]. Early studies on VDR protein expression in uterus, oviduct, ovary, mammary gland, placenta, and fetal membranes suggested a role for 1,25(OH)2D3 in female reproduction [261]. While some essential 1,25(OH)2D3 functions in female fertility relate to reproductive tissue development [262,263], other functions probably relate to immune tolerance induction and are highly relevant to the protective effects of pregnancy in women with MS. The semi-allogeneic, fetal trophoblast cells must
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invade the uterine decidua to establish the fetal blood supply, but the uterine decidua harbors T lymphocytes capable of recognizing and attacking the fetal tissue bearing paternal antigens [264]. Incompletely understood mechanisms exist at the border between mother and fetus to promote maternal immune tolerance of the fetus without unduly compromising immune responses to infectious agents. Expression of the CY27B1 and VDR genes in placental tissue suggests that 1,25(OH)2D3 and VDR signaling may have a role in maternal immune tolerance. The 1a-hydroxylase enzyme and the VDR were expressed at high levels in the human placenta and decidua throughout gestation, particularly in the first and second trimesters [180]. The 1a-hydroxylase co-localized with decidual macrophages, but not lymphocytes [180]. In contrast to the CY27B1 and VDR genes, which are hypomethylated and expressed at high levels, the CYP24A1 gene is hypermethylated and silenced [265]. Methylation of the core CYP24A1 promoter silenced basal promoter activity and abolished 1,25(OH)2D3-mediated feedback activation. The induction of the 1a-hydroxylase in decidual macrophages [180], together with the epigenetic decoupling of vitamin D feedback catabolism [265], maximizes the bioavailability of 1,25(OH)2D3 at the border between mother and fetus. The 1,25(OH)2D3 (and estrogen) may modulate the activity of T lymphocytes specific for paternal antigens to maintain the immune tolerance needed for successful gestation [180,266]. The dramatic increase in circulating 1,25(OH)2D3 (and estrogen) during pregnancy may also modulate the activity of T lymphocytes specific for neural antigens, thereby restoring immune self tolerance and reducing MS attacks during pregnancy [163]. If this hypothesis is correct, then it may be possible to restore immune self tolerance and reduce MS attacks in non-pregnant women with MS by administering 1,25(OH)2D3 or vitamin D3 in combination with estrogen.
VITAMIN D, ESTROGEN, EAE, AND MULTIPLE SCLEROSIS MS shows a female sex bias that is increasing rapidly and is not understood [267,268]. Women and men were afflicted with MS in equal numbers until w1950, when prevalence surveys first revealed a 1.4:1 female bias [269]. Recently, the sex ratio has been estimated between w3:1 [270] and w5:1 [271], depending on the decade of birth; the younger the cohort, the greater the female sex bias. This distressing trend has been observed in the USA [272,273], Canada [270,271], Australia [274], the UK [275,276], Norway [277,278], and Sardinia [279]. The trend is due to a rising incidence among young
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women, rather than a decreasing incidence among young men [280]. The two explanations advanced to explain the female sex bias in MS invoke X-chromosome linked genes, and/or female sex hormones [267]. Extensive X-chromosome linkage studies have not revealed MS susceptibility loci, suggesting that X-linked risk factors probably do not explain the female sex bias [281]. Moreover, the three-fold increase in MS incidence among young women in the last half-century is not consistent with a genetic origin [270]. The fact that the female sex bias becomes apparent only after sexual maturity strongly suggests the involvement of sex hormones [282]. One suggested explanation for the female sex bias in autoimmune disease is that E2 in females promotes stronger immune responses than males [267], but this explanation is at odds with the documented inhibitory effects of E2 and estriol in female MS patients and rodents with EAE [283e287]. Thus, neither X-linked risk factors nor sex hormone-determined differences in immune responsiveness adequately explain the rapidly increasing female sex bias in MS disease. A newly discovered synergism between the vitamin D and estrogen endocrine systems as regards inhibition of autoimmunity in females may provide an explanation for the rising female sex bias in MS. High level vitamin D3 supplementation (three-fold the amount of vitamin D3 in standard laboratory mouse chow) inhibited EAE in intact adult female mice, but not in adult male mice [162]. Gonadectomy eliminated the beneficial effects of vitamin D3 in females, but did not enable these benefits in males. Thus, vitamin D3 provided a female-specific and sex hormone-dependent protective effect in EAE. A follow-up study confirmed these findings, and extended them to show that diestrus-level E2 therapy alone did not alter EAE disease, but in ovariectomized female mice it restored the ability of vitamin D3 to delay EAE onset and reduce EAE incidence, peak disease severity, and cumulative disease score [163]. These data were the first to show functional synergy between the vitamin D and estrogen endocrine systems in the inhibition of an autoimmune disease. Several mechanisms contributed to the vitamin D and estrogen synergy [163]. First, there was a stimulatory effect of dietary vitamin D3 on E2 biosynthesis, consistent with reports that the hormone 1,25(OH)2D3 enhanced transcription of the Cyp19 gene encoding estrogen synthase (aromatase), the rate-limiting enzyme in the production of estrogens from the C19 androgens. The 1,25(OH)2D3 stimulation of Cyp19 gene expression occurred in reproductive tissue [288], placenta [289], and glial cells of the brain [290]. A second mechanism of synergy appeared to be a female sex hormone contribution to repression of the mouse Cyp24a1 gene in the CNS, thereby minimizing 1,25(OH)2D3 inactivation
and maximizing 1,25(OH)2D3 bioavailability [162]. Effects of E2 on 1,25(OH)2D3 have also been noted in humans [291]. In placental tissue, repression of the human CYP24A1 gene was accomplished by hypermethylation [265], decoupling vitamin D feedback catabolism through an epigenetic mechanism to maximize 1,25(OH)2D3 bioavailability at the maternalefetal interface. Whether E2 plays a role in this epigenetic decoupling of vitamin D feedback catabolism either in the CNS, at the maternal-fetal interface, or in lymphoid cells remains to be determined. A third mechanism of synergy was E2-dependent transcriptional activation of the VDR gene in the spinal cord during an inflammation [163]. E2-mediated upregulation of the VDR gene has also been reported in duodenal mucosa [292,293], liver cells [294], and breast cells [295]. An E2-responsive sequence has been reported upstream of exon 1c in the VDR gene [296]. Collectively, these data suggest an amplification loop, with 1,25(OH)2D3 enhancing E2 biosynthesis through upregulation of Cyp19 and estrogen synthase, E2 enhancing 1,25(OH)2D3 bioavailability through repression of Cyp24a1 and 24-hydroxylase, and E2 enhancing 1,25(OH)2D3 function through upregulation of VDR and VDR protein. These mechanisms and possibly others allow the vitamin D and estrogen endocrine systems to function synergistically in females to control autoimmune responses. The functional synergy between the vitamin D and estrogen endocrine systems has important implications for MS. If vitamin D3 were to provide a female-biased and sex hormone-dependent protective effect in MS as in EAE, then this protective effect would become evident at sexual maturity, and after puberty, vitamin D3 insufficiency would increase MS risk in a femalespecific manner. The lifestyle changes that have decreased overall UVL exposure and serum 25(OH)D3 levels in women over the last half century [59] could be contributing to the rapidly increasing MS incidence in young women. Moreover, declining E2 biosynthesis due to ovarian failure or menopause could undermine the beneficial effects of vitamin D3 that depend on E2, contributing to the rapid increase in MS disability after menopause [297e299], particularly for women with vitamin D3 insufficiency. Emerging research suggests that UVL and vitamin D3 may indeed provide female-biased benefits in humans. A protective effect of childhood sun exposure was noted in female but not male monozygotic twin pairs [43]. There was an inverse correlation between UVL and MS risk especially in females [44,52]. The female to male sex ratio among MS cases was greatest in areas with low UVL exposure and high MS prevalence [300]. Moreover, high serum 25(OH)D3 levels correlated with a reduced MS risk in women but not men [79]. Lastly, there was a female-biased association of the
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HLA-DRB1*1501 allele with early onset MS and a severe disease course [97]. These new data are consistent with a possible female-biased and E2-dependent protective effect of sunlight and vitamin D3 in MS. Thus, the functional synergy that exists between the vitamin D and estrogen endocrine systems suggests that the trend towards increasing incidence of MS among young women might be reversible with vitamin D3 supplementation, and the trend towards increasing disability in menopausal women might be reversible with a combination of E2 hormone replacement and vitamin D3 supplementation.
VITAMIN-D-BASED THERAPY OF MULTIPLE SCLEROSIS 1,25(OH)2D3 and Alfacalcidol Therapy of RelapsingeRemitting MS Research exploring the relationship between vitamin D3 and MS led to two pilot studies of oral vitamin D3 metabolites in relapsing remitting MS patients. The first study tested 1a-hydroxyvitamin D3, which is metabolized to 1,25(OH)2D3 [301]. Five MS patients (40% female) with low relapse rates were recruited. They received 1a-hydroxyvitamin D3 (1.5 mg/d of Alphacalcidol) for 6 months; no adverse clinical or biochemical events were recorded. At the end of the study, one patient’s neurological status had improved, three were stable, and one had an acute relapse. The authors concluded that 1a-hydroxyvitamin D3 was safe and well tolerated. It is possible that the 1a-hydroxyvitamin D3 treatment had a role in stabilizing or reversing MS disease in four of the five patients, but the small sample size, the recruitment of patients with a very mild phenotype, and the unblinded nature of the study lend uncertainty to inferences from the study. The second pilot study was a 48-week test of oral 1,25(OH)2D3 in ambulatory MS patients (n ¼ 15; 18e65 years of age; 80% female; no immunomodulatory therapy) [302]. Patients with at least one clinical relapse in the previous year were recruited. Dietary Ca was restricted to 800 mg/day. Patients received 1,25 (OH)2D3 (Rocaltrol) beginning at 0.5 mg/d and escalating by 0.5 mg/d increments each 2 weeks to a target dose of 2.5 mg/d. Bi-monthly clinical and laboratory tests were performed to detect adverse events. Dietcompliant patients had no adverse events, but two diet-non-compliant patients withdrew with symptomatic hypercalcemia. The composite on-study relapse rate was 0.27, compared to a baseline relapse rate of 1.0, and a post-study relapse rate of 0.8. The on-study median disability score was 2.2, unchanged from baseline, and less than the post-study median disability score
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of 3.1. Thus, 1,25(OH)2D3 appeared to inhibit MS disease aggravation, but the small sample size and the unblinded nature of the study introduced uncertainty. The authors commented that daily 1,25(OH)2D3 therapy might not be sustainable due to hypercalcemia risks, but 1a-hydroxyvitamin D3 therapy might be a reasonable approach.
Glucocorticoids Compared to 1,25(OH)2D3 It is useful to compare the secosteroid 1,25(OH)2D3 to the glucocorticoids like methylprednisolone that are currently used to treat acute MS relapses [303e305]. The 1,25(OH)2D3 [248] and methylprednisolone [306] have similar efficacy as treatments for acute neural antigen-induced EAE. Both the secosteroid and the corticosteroid reversed the disease signs (clinical score, CNS inflammatory infiltrate, demyelination, axonal degeneration), but in both cases disease progression continued after treatment withdrawal [197,248,306]. A 500-fold greater amount of methylprednisolone than 1,25(OH)2D3 was needed to induce EAE remission. The 1,25(OH)2D3 and methylprednisolone can also be compared in MS patients. In relapsing MS patients, a few days of high dose glucocorticoid treatment slightly reduced MS disability (EDSS change 0.22; Ambulation Index change þ0.40) over the next 1 month [304]. For comparison, continuous 1,25(OH)2D3 treatment reduced the MS relapse rate >70% and slowed disability accumulation [302]. For methylprednisolone and 1,25(OH)2D3, disease activity returned to pretreatment levels within 2 years [302,303]. It would appear from this analysis that 1,25(OH)2D3 and glucocorticoids have similar efficacy as treatment for autoimmunemediated attacks, but short-term 1,25(OH)2D3 may have markedly fewer risks. Glucocorticoids act on a variety of immune system cells, broadly reducing immune responses and increasing susceptibility to infection. Glucocorticoids are produced in response to stress, and the stress response involves many tissues in addition to lymphoid tissues [305]. Consequently, glucocorticoids can cause substantial damage to non-immunologically relevant tissues. Glucocorticoids inhibit protein synthesis and stimulate protein breakdown, leading to muscle and skin atrophy and gastrointestinal problems. They favor bone breakdown over formation leading to osteoporosis. Their metabolic actions in the liver can lead to insulin resistance and diabetes. Their actions in the brain can cause sleep and mood disturbance and psychosis. The only known risk of long-term 1,25(OH)2D3 treatment is hypercalcemia. Short-term 1,25(OH)2D3 treatment does not carry a risk of hypercalcemia [307]. Moreover, vitamin D3 supplementation reportedly supports muscle strength, bone formation, diabetes prevention, and sense of well-being [59].
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It is not widely appreciated that glucocorticoids inhibit 1,25(OH)2D3 synthesis [177,308]. Glucocorticoid treatments reduced circulating 1,25(OH)2D3 levels 30e47% in sarcoidosis patients [179]. Since immune cell 1,25(OH)2D3 synthesis initiates a feedback antiinflammatory response in inflamed tissues, inhibiting this natural feedback anti-inflammatory response with glucocorticoids may be counterproductive. All things considered, it may be fruitful to investigate 1,25(OH)2D3 as a possible replacement for methylprednisolone for treatment of acute MS relapses.
Vitamin D Compared to Immunomodulatory Therapy of MS Vitamin D3 supplementation may prove to be an effective therapy for RRMS. In the longitudinal studies and the intervention study, 80% of the pediatric MS patients, 82% of the adult MS patients, and 57% of the vitamin D3 supplemented adult MS patients were receiving immunomodulatory therapy [85,86,93]. Nevertheless, a substantial decrease in MS clinical disease activity correlated with the increase in 25(OH)D, irrespective of immunomodulatory therapy. One author wrote, “Our data imply that increasing serum 25-hydroxyvitamin-D3 levels by 50 nmol/L could more than halve the risk of relapse, a reduction at least on par with most immunomodulatory therapies” [86]. For comparison, the FDA-approved, self-injectable IFN-b1 drugs reduced the relapse rate w35% in some MS patients, but had no significant effects on the accumulation of disability [154,155,309]. Moreover, these drugs carry warnings about allergic reactions, depression and suicide, seizures, and problems related to the heart, liver, and blood cells. Natalizumab (Tysabri), an infusable integrin-specific monoclonal antibody, slowed disability progression w42% over 2 years, but increased the risk of cardiotoxicity and fatal progressive multifocal leukoencephalopathy [310]. The infusable immunosuppressant mitoxantrone reduced relapses and delayed disability progression, but increased the risk of cardiotoxicity, myelosuppression, and secondary acute myelogenous leukemia [311]. Compared to the currently FDA-approved MS therapies, vitamin D3 supplementation may have a significantly more favorable benefit to risk ratio.
Costs of MS Therapy MS is a chronic and disabling disease, but most patients have a normal lifespan, so the disease takes a considerable toll on individuals, families, health care systems, and society. Worldwide, w2.5 million individuals have MS. In the USA, MS prevalence is w0.4
million patients with w200 patients newly diagnosed daily, and self-injectable MS drugs, at w$28Ke$36K/ patient annually [312], accounted for a remarkable 2.5% of all pharmacy expenditures [313]. Drugs represented 65% of direct MS patient care costs [314]. The 2004 annual care costs were w$47.2K/patient on average, or w$20 billion for the total US MS patient population [315]. Annual care costs for European MS patients varied from a low of V18K for the mildly disabled to V62K for the highly disabled [316]. The costs of adverse events attributable to the MS drugs (including deaths) are inestimable. Vitamin D3 supplementation would cost very little. If vitamin D3 supplementation were to slow the accumulation of disability as it appeared to do in a recently completed safety study [93], the potential benefits to individuals, families, health care systems, and society could be very significant.
CONCLUSIONS AND FUTURE RESEARCH DIRECTIONS Conclusions Although there is no doubt that genetic risk factors and other environmental factors contribute to MS etiology, the evidence supporting an inverse association between vitamin D3 and MS risk and severity is sufficiently strong and diverse to nearly satisfy the Hill criteria [5] for asserting that a causal relationship exists between insufficient vitamin D3 and MS disease risk and severity [3]. The vitamin D3 and MS association has been observed consistently on five of the seven continents, and is strongest where seasonal vitamin D3 fluctuations are greatest. Current estimates suggest w75% of MS disease risk might be attributable to this factor. The association is specific; the vitamin D3 and MS link is particularly strong for Caucasian women of Northern European ancestry, possibly owing to VDR control of the MS susceptibility locus, HLA-DRB1*1501. Moreover, genetic data show that VDR and CYP27B1 gene variants appear to be MS risk factors. Temporal studies support the causality for the vitamin D3 and MS link; fluctuations in UVL and vitamin D3 preceded MS disease activity changes by several months, and preceded alterations in MS risk by a decade or more. Regarding a biological gradient, the data show a dosedependent inverse relationship between vitamin D3 and MS risk and attack rates in widely disparate populations. Two vitamin D3 supplementation studies have provided preliminary evidence for a causeeeffect relationship, since decreases in MS disease activity and disability progression were observed in the supplemented subjects, but the unblinded nature of the study
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CONCLUSIONS AND FUTURE RESEARCH DIRECTIONS
introduces enough uncertainty that additional research is needed to satisfy the Hill criteria [92,93]. Collectively, this diverse body of evidence from laboratories around the world suggests that the sunshine vitamin may well be the long-sought protective environmental factor as regards MS. The vitamin D3 and MS hypothesis provides a coherent explanation for several puzzling facts of MS natural history and biology in women. Specifically, this hypothesis provides some explanation as to why the disease course commonly shows a relapsingeremitting pattern that later becomes progressive, why there is a rapidly increasing female sex bias, and why the disease remits during pregnancy. Seasonal fluctuations in UVL and vitamin D supplies in high latitude regions may contribute to the relapsingeremitting pattern. Moreover, the emerging concept of synergy between the vitamin D3 and E2 endocrine systems suggests that increasing prevalence of vitamin D insufficiency may adversely affect women more than men, contributing to the growing female sex bias. The increases in estriol and 1,25(OH)2D3 during pregnancy may reduce MS disease activity, whereas the decreases in E2 production at menopause may undermine the vitamin D3 protective effects and accelerate MS disease. If so, these insights suggest new approaches to alter the MS disease course in women. Plausible mechanisms to explain how vitamin D3 may function as a natural inhibitor of MS are emerging from EAE research. Vitamin D3 is needed to support 1,25(OH)2D3 synthesis in the CNS and initiation of a disease prevention pathway centering on containment of the autoimmune T cell-driven inflammatory cascade. The containment mechanisms are not yet fully understood. Research has firmly supported roles for Ca, the VDR (particularly in T lymphocytes), DC, Rag-1-dependent regulatory lymphocytes, increases in TGF-b1, IL-4, and IL-10, and decreases in IL-17, but not direct inhibition of IFN-g-producing Th1 cells or promotion of IL-4-producing Th2 cells. Three specific molecular mechanisms under current investigation are direct 1,25(OH)2D3 actions (1) on DC rendering them tolerogenic, (2) on induced regulatory cells supporting their suppressive function, and/or (3) on encephalitogenic T cells enhancing their apoptosis, mechanisms that may all apply.
Future Clinical Research There is strong justification for additional vitamin D3 supplementation studies in MS patients. Arguably the most urgent priority is to replicate the preliminary data showing reduced MS relapses and disease progression in vitamin D3-supplemented RRMS patients [92,93], using a larger double-blind study design and evaluating
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additional parameters related to suspected beneficial effects of vitamin D3 (e.g., muscle strength and bone health, infectious disease burden, cognition and memory, sense of well-being). The need to evaluate the potential of vitamin D3 supplementation to prevent disability progression in CIS, PPMS, and SPMS patients is also urgent. A longer-range but no less urgent priority is evaluating the potential for vitamin D3 supplementation to prevent MS in high risk populations, particularly girls and women who have minimal UVL exposure and/or carry the HLA-DRB1*1501 genetic risk factor. Additionally, it may be fruitful to investigate shortterm 1,25(OH)2D3 or 1a-hydroxyvitamin D3 as a substitute for methylprednisolone treatment for acute MS relapses in the expectation of a more favorable benefit to risk ratio. Clinical research is also needed to investigate the possible confluence of environmental and hormonal factors in the determination of MS risk and disease activity. In-depth research is merited to probe possible protective synergy between the vitamin D3 and E2 endocrine systems as regards MS in women. For example, the possible female sex bias in the immunological functions of vitamin D3 deserves in-depth analysis. Also, the temporal relationships between natural E2 production, vitamin D3 status, and MS disease activity and disability progression require careful evaluation. Moreover, detailed biochemical analyses are needed to determine if 1,25(OH)2D3 enhances E2 biosynthesis and E2 enhances 1,25(OH)2D3 bioavailability and function in women as in female mice. These studies could be a prelude to possible interventional studies using a combination of E2 and vitamin D3 to address RRMS progression to CPMS in menopausal women.
Future Basic Research The potential relationships between genetics, vitamin D3, sex hormones, symptomatic EBV infection, and MS risk require evaluation in the quest to understand MS etiology. For example, a temporal sequence of inheriting the VDR-controlled HLA-DRB1*1501 gene, childhood vitamin D3 insufficiency, a rise in E2 at puberty, and infectious mononucleosis (symptomatic EBV infection) appears to confer the highest known risk of MS in women [25,31,32]. These findings require replication, and subsequently in-depth analysis of possible biological mechanisms that could unfold in sequence to explain this high risk. Possibilities are adverse effects of the HLA-DRB1*1501 genotype plus vitamin D3 insufficiency on immune tolerance [161] or control of EBV infection [317], or EBV hindrance of vitamin D3 protective effects [38], and/or HLA-DRB2 presentation of related MBP and EBV peptides to potentially autoimmune T cells in the absence of a vitamin-D3-dependent
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anti-inflammatory feedback loop. With respect to EAE research, it will be important to develop a deeper and more mechanistic understanding of the protective synergy between the vitamin D3 and E2 endocrine systems. Also needed is a better definition of the three specific molecular mechanisms that are under current investigation, 1,25(OH)2D3 actions on DC, induced regulatory cells, and/or encephalitogenic T cells. Continued basic research in the EAE model and in MS patients will bring us closer to understanding MS etiology and devising more effective intervention strategies.
A Call to Action Having suggested that vitamin D3 insufficiency likely plays a causal role as regards MS risk and disease activity, we close this chapter with a quote from Sir Austin Bradford Hill [5]. Finally, in passing from association to causation I believe in “real life” we shall have to consider what flows from that decision. On scientific grounds we should do no such thing. The evidence is there to be judged on its merits and the judgment (in that sense) should be utterly independent of what hangs upon it e or who hangs because of it. But in another and more practical sense we may surely ask what is involved in our decision. In occupational medicine our object is usually to take action. If this be operative cause and that be deleterious effect, then we shall wish to intervene to abolish or reduce death or disease.
Acknowledgments We are grateful to Dr. J. Wesley Pike (University of Wisconsin), Dr. Halina Offner (Oregon Health and Sciences University), and Dr. Alberto Ascherio (Harvard University) for their thoughtful review and comments on the manuscript.
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C H A P T E R
96 Vitamin D and Inflammatory Bowel Disease Danny Bruce, Margherita T. Cantorna Pennsylvania State University, University Park, PA, USA
INTRODUCTION It is estimated that 1 million Americans have inflammatory bowel disease (IBD). IBD occurs more often in northern regions, and in North America and Europe approximately 1 in 1000 people is affected by IBD [1,2]. In addition the prevalence of IBD is increasing in regions of the world that have traditionally been considered “lowincidence areas” [3]. A study in 2006 designed to investigate the prevalence of IBD in Canada showed that 0.5% of Canadians have IBD, the highest percentage reported worldwide [4]. Many autoimmune diseases have been linked to vitamin D deficiency. These include multiple sclerosis (MS), type 1 diabetes, and IBD [5]. In this chapter the immunoregulatory role of vitamin D and its effect on the pathology of IBD will be reviewed. The epidemiological evidence connecting vitamin D deficiency to IBD severity and the data from animal models of experimental IBD will be discussed. Finally current treatment options for IBD patients will be reviewed and how vitamin D might be used as an alternative or a supplemental treatment for patients with IBD will be discussed.
WHAT IS IBD? IBD occurs due to complicated interactions between multiple genetic and environmental factors. Crohn’s disease (CD) and ulcerative colitis (UC) are the major forms of IBD. CD and UC are chronic inflammatory disorders of the gastrointestinal tract that are characterized by remitting and relapsing inflammation of the intestinal mucosa [6]. These diseases often result in abdominal pain, diarrhea, and fever. As the disease progresses, rectal bleeding, weight loss and severe fatigue can occur limiting the quality of life for patients with IBD. Men and women are equally affected by IBD. CD affects teens
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10096-4
and young adults with the majority of patients being diagnosed between the ages of 15 and 35 years and UC affects slightly older individuals; UC patients are usually diagnosed in their mid to late 30s [7]. Because the symptoms of CD and UC are similar, approximately 10% of patients cannot be definitively diagnosed and these cases are termed indeterminate colitis. Even though CD and UC share similarities, there are distinct differences in their pathology. The inflammation found in CD most commonly involves the terminal ileum of the small intestine (SI) but can be diffuse affecting areas from the esophagus all the way to the rectum [8]. CD is characterized by aggregates or clusters of immune cells, specifically macrophage and T cells that form granulomas; therefore, CD is sometimes described as a granulomatous or granuloma-forming disease [8]. Inflammation in CD can be patchy or segmental. These are referred to as “skip” lesions [8]. The lesions can become transmural, affecting the entire thickness of the intestinal wall. Unlike CD, inflammation in UC typically involves the rectum and extends proximally in a continuous lesion. Histopathology of UC discloses an increase in white blood cells in the lamina propria of the colon and the crypts, which often leads to the development of micro-abscesses [8]. Normally, the body’s immune system protects from invading pathogens such as bacteria, viruses and fungi but tolerates food antigens and microbes living in the lumen of the intestine. However, in diseases such as IBD the immune system is inappropriately activated by the microbes found in the gut. In patients with IBD the inflammation does not resolve but instead persists. A complicated interplay between genetics and the environment predisposes individuals to the development of IBD. Current research focuses on understanding how the balance between bacteria, the host, and the immune system is maintained, so that new strategies to prevent or treat IBD can be discovered.
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WHO GETS IBD? Genetic Factors There is clear evidence of a strong genetic component to IBD. IBD is significantly more prevalent within families. In fact, 20 to 25% of patients have a close relative with CD or UC [9]. People with a biological relative with IBD are 10 times more likely to develop the disease than the general population and that number increases to 30 times more likely if the relative is a brother or sister [10]. Advances in genetics research have led to the discovery of several IBD susceptibility genes. Single nucleotide polymorphisms (SNPs) found in genes of the immune system are prevalent in IBD. The major histocompatibility complex (MHC) is expressed on all nucleated cells and controls the generation of antigen-specific immunity. MHC genes regulate the ability of the immune response to distinguish between self antigens and foreign antigens. In autoimmune diseases including MS, rheumatoid arthritis, and IBD the immune response inappropriately targets host tissues. SNPs in MHC genes have been described in patients with autoimmunity. SNPs in MHC affect the nature of the immune response and are associated with IBD as well as other autoimmune diseases [3]. Additional IBD-associated genes have been identified that regulate immune cell development, function, or activation. Some genetic polymorphisms found in patients with IBD affect the function of the immune system by altering the immunomodulatory cytokines produced by it. Interleukin (IL)-10 is an important suppressive cytokine and SNPs in and around the IL-10 gene have been associated with UC [11]. SNPs in inflammatory cytokine genes or in genes for receptors of inflammatory cytokines, including tumor necrosis factor (TNF)-a, IL-8, IL-12, IL-18R, and IL-26, have been described in patients with CD and UC [3,11e 13]. Several SNPs in genes associated with IBD play a role in the development of inflammatory T cells. Subsets of these genes encode cytokines or cytokine receptors that affect Th17 differentiation (IL-23 receptor) [3]. SNPs have been described that can affect or disrupt the IL-23R signaling cascade including STAT3 and STAT4 (associated with CD and UC, respectively) and JAK2 (associated with both) [14]. Another IBD susceptibility gene is CTLA4 which can suppress Tcell activation [15]. An important IBD susceptibility gene is nod-like (NOD)2 (also known as CARD15). SNPs of NOD2 have been shown to limit the immune system’s ability to recognize bacteria [3]. NOD2 encodes a cytosolic microbial molecular pattern-recognition receptor (PRR) that belongs to a large group of innate immune receptors [3]. Like NOD2, NOD1 is also a PRR, but NOD1 SNPs
are more strongly associated with UC than CD [16]. Toll-like receptor (TLR) 4 recognizes bacterial lipopolysaccharides and helps activate the immune response to invading microbes. Polymorphisms in the gene encoding TLR4 have recently been shown to be associated with both CD and UC [17]. In each of these cases, failure to recognize bacteria properly may result in an abnormal immune response to commensal microflora. Other IBD susceptibility genes have roles in maintaining intestinal integrity. Extracellular matrix protein 1 is implicated in the interaction between the intestinal epithelium and the basal membrane, and SNPs in the gene encoding this protein have a strong association specific for UC [11]. Nod-like receptor protein 3 SNPs are associated with CD, and in mouse models loss of this protein has been shown to result in the loss of epithelial integrity [18]. Loss of intestinal integrity may result in systemic infiltration of commensal flora and increased inflammation in the underlying tissue. To date over 500 SNPs have been discovered in genes of the vitamin D pathway with 470 of the SNPs being found within the VDR gene [19]. The VDR gene maps to a region on chromosome 12 that has been functionally linked to IBD by genome-wide association [20]. Through the use of genome-wide association studies (GWAS) polymorphisms in the VDR gene were shown to increase susceptibility to CD and UC [20,21]. Additional studies are needed to determine the extent of VDR polymorphisms in patients with IBD and whether the polymorphisms result in functional outcomes in vitamin D signaling. the 1,25-Dihydroxyvitamin D (1,25(OH)2D), hormonal form of vitamin D, is known to be a transcriptional regulator that targets genes via interaction in trans with vitamin D response elements (VDREs). Several IBD-associated genes have VDREs. Cytokine genes such as IL-2, IFN-g, IL-12, and others are transcriptional targets of 1,25(OH)2D [22]. Sequence analysis of human MHC class II genes revealed VDREs in the promoter [23,24]. The IBD-associated gene NOD2 is regulated by vitamin D and has an enhancer VDRE [25]. Other pathways that control vitamin D function, beyond the VDR polymorphisms, have not been examined in relation to IBD. There is evidence that SNPs within the gene encoding the vitamin D1ahydroxylase (CYP27B1) that is required for production of 1,25(OH)2D is associated with MS [26]. SNPs in the genes of other components of the vitamin D pathway have been described including genes for vitamin D catabolism and the vitamin D binding protein [19]. The effects of vitamin D and it metabolites could be regulated by alterations in genes that affect vitamin D metabolism, catabolism, or function. To date these genes have not been studied in IBD patients.
XI. IMMUNITY, INFLAMMATION, AND DISEASE
THE “VITAMIN D HYPOTHESIS”
Environment Genetic polymorphisms account for only 10e20% of the overall risk in CD and even less for UC [27]. The concordance rate for IBD development in genetically identical twins is only 20% in UC and 50% in CD [5]. These findings indicate that important environmental factors affect the development of IBD. IBD is largely a disease of the developed world, with the majority of the cases being reported in North America and Europe. These diseases occur more often in urban than in rural areas, and within the northern hemisphere IBD is more prevalent in northern versus southern climates [3,28]. The prevalence of IBD worldwide and specifically in countries that were previously considered “low-incidence areas,” such as Japan and India, is also increasing [3]. The increased prevalence of IBD cannot be explained by genetics. First and second generation immigrants from “low-incidence areas” that have moved to countries with higher incidences adopt risk levels similar or higher than the residents of that country [3]. The environmental factors that may play a role in IBD are poorly defined and may be numerous. The incidence of IBD is higher in industrialized nations and even more so for urban areas within those nations. Environmental factors that may be different in IBD low versus high areas include: diet, lifestyle, pollution, exposure to potential harmful chemicals, and exposure to smoke. Smoking tobacco is one of the most highly associated risk factors for CD but interestingly has been shown to be helpful in patients with UC [29]. One critical environmental factor associated with IBD is bacterial exposure. Bacterial exposure can be shown to either induce or prevent experimental IBD. In one animal model of IBD germ-free mice are protected and in another experimental model germ-free mice develop more severe disease [11,30]. Several infections have been shown to increase IBD, and antibiotic treatment is of some benefit for treating a subset of patients with IBD [28]. Infection with Mycobacterium avium paratuberculosis and several species of Helicobacter is associated with IBD [31,32]. Others have proposed that decreased exposure to microbes in industrialized countries has led to the increased incidence of IBD in the developed world. The mechanism by which some bacteria may help and others may exacerbate IBD is an area of current research.
THE “HYGIENE HYPOTHESIS” The higher incidence of IBD in industrialized countries has led to the proposal of the “hygiene hypothesis.” The hygiene hypothesis states that due to the use of
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vaccines, improved sanitation, and reduced rates of infection the immune system does not receive critical signals and as a result autoimmunity increases [33]. The immune system overreacts and subsequently fails to shut down inflammation. Improvements in hygiene in developed and developing countries includes vaccination, access to clean water, improved sewer systems, the use of food preservatives, and personal hygiene products such as: antibacterial soap, toothpaste, and more recently hand sanitizers [28]. Rural areas have fewer cases of IBD. Several studies have shown that children that live on farms have a lower prevalence of immune-mediated diseases including: asthma, allergies, and IBD [33,34]. Data indicate that elements of the farm lifestyle may expose children to factors that activate the immune response but do not cause inflammation. Possibilities include exposure to endotoxins, contact with animals or soil, microbial exposure, and diets rich in dairy products [33]. Interestingly, in 2007 a casecontrolled study showed that exposure to farm animals, especially cattle, during the first year of life had a protective effect against developing IBD [34]. Improved hygiene in developed countries limits exposure to previously ubiquitous infectious agents such as several different types of worm (helminth) infection. Helminths are parasites that infect humans throughout the world and are thought to play an important immunoregulatory role in the intestine. The response of the immune system to helminths such as Shistosoma mansoni and Trichinella spiralis has been shown to be protective against experimental models of IBD and in patients with IBD [3,35]. In addition, infection with these organisms results in reduced inflammation and increased production of mucins and water secretion into the lumen of the gut that also reduces inflammation [28]. Epidemiological data suggest that helminth infection is inversely correlated with the economic status of the region as well as incidence of IBD and other immune-mediated diseases [3].
THE “VITAMIN D HYPOTHESIS” The vitamin D hypothesis has been proposed and suggests that vitamin D status may be an environmental factor involved in the development of IBD [35]. A major source of vitamin D comes from a photolysis reaction in the skin after exposure of skin to sunlight. Skin pigment, aging, time of day, season and latitude dramatically affect vitamin D synthesis [36]. The incidence of IBD is higher in more northern regions of the USA and Canada. Vitamin D status is especially low during winter months in areas with the greatest seasonal fluctuation [37]. Many of the environmental factors that are present in high risk areas for IBD would also result in decreased availability
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96. VITAMIN D AND INFLAMMATORY BOWEL DISEASE
of vitamin D. Factors such as air pollutants and decreased outdoor activity are known to reduce vitamin D synthesis [35]. Intentional avoidance of sunlight exposure of our skin also reduces vitamin D production in the skin. In developed countries people limit skin exposure to sunlight in order to avoid skin cancer. The use of high SPF sunscreens decreases the risk of skin cancer but also reduces the amount of vitamin D made in the skin [36]. The other source of vitamin D is the diet. Most diets are limited in natural sources of vitamin D. Vitamin D-fortified dairy and grain products [38], egg yolk, and ultraviolet B (UVB)-exposed mushrooms contain some vitamin D. Oily fish like salmon or cod are high natural sources of vitamin D [39]. However, more recently vitamin D deficiency has reemerged in urban areas [38]. Obesity also results in lower serum levels of 25-hydroxyvitamin D (25(OH)D) [40] and obesity in IBD patients is associated with increased severity of the disease [41]. Vitamin D insufficiency (25(OH)D 20e30 ng/ml) and deficiency (<20 ng/ml) are common in northern regions of the northern hemisphere, and several studies report an even higher prevalence of insufficiency and deficiency in adult and pediatric patients with IBD [42]. Vitamin D deficiency is common even when the patient is in remission [42,43]. This is probably due to residual fat, malabsorption, low dietary intake, and reduced outdoor activity of the patient. The prevalence of osteoporosis, osteopenia and low bone mineral density
(A)
Vitamin D Sufficiency – Healthy Intestine
(BMD) is also high in patients with IBD [44]. Similarly, IBD patients have a higher rate of bone fracture [45]. These trends may be a consequence of the disease and/or glucocorticosteroid treatment that has been shown to decrease BMD. Many newly diagnosed IBD patients have reduced BMD and this prevalence is slightly higher in patients with CD than UC [46]. In sum, these observations indicate that low serum 25(OH)D levels are a common finding in patients with IBD and may both predispose and exacerbate IBD development.
THE IMMUNE RESPONSE AND IBD The human body is composed of roughly 100 trillion cells and the lumen of the intestine harbors approximately ten times that many bacteria [47]. Many of these bacteria are commensal, living in the intestine but not causing harm. Other bacteria in the intestine are pathogenic and require an appropriate immune response to combat their invasion. The immune system must not react to commensal flora but still clear pathogenic organisms. In addition, once the pathogen is cleared the immune response must be turned off. In healthy intestine the immune response is balanced and tightly regulated to clear pathogens and then shut off once the infection is cleared (Fig. 96.1A). The innate immune system is the first line of defense against an invading pathogen. Innate immune cells such as
(B)
Vitamin D Deficiency/VDR KO – Inflammation
NK T cells
DC
NK T cells DC
CD4
Treg
Treg DC
TGF
DC
TGF
Th0
TGF IL-6 IL-23
CD8 IEL
CD4
IFNIL-12
IL-4
Th0 TGF IL-6 IL-23
CD8 IEL
IFNIL-12
IL-4
Th17 Th17
Th1
Th2
Th1
Th2
Vitamin D sufficiency results in a healthy intestine, while vitamin D or VDR deficiency results in inflammation. (A) Healthy gastrointestinal tract: DC in the IEL sample the lumen of the intestine looking for harmful bacteria. These cells become activated through recognition of the pattern recognition receptors expressed on the bacteria and phagocytose the bacteria for killing and presentation of bacteriaspecific antigens to naı¨ve T cells (Th0). In the healthy intestine T cells differentiate into effector T cells under the influence of Th1 (IFN-g and IL-12), Th2 (IL-4 and IL-10), or Th17 (TGF-b, IL-6 and IL-23) cytokines. In addition, regulatory T cells (iNKT cells) are induced to help moderate the resulting immune response. Other regulatory T cells (FoxP3þ Tregs, and CD8aaþ T cells) serve to shut off effector T cells and halt inflammation in the gut. (B) Vitamin D deficiency leads to increased DC activation, antigen presentation, and cytokine production. Th17 and Th1 cells overproduce IL-17 and IFN- g with few regulatory cells to shut off the response. Too few iNKT cells and CD8aa T cells result in the inability to turn off the Th17 and Th1 cell response to commensal bacteria in the gastrointestinal tract and as a result IBD develops.
FIGURE 96.1
XI. IMMUNITY, INFLAMMATION, AND DISEASE
VITAMIN D REGULATES T CELL RESPONSIVENESS
macrophage and dendritic cells (DC) recognize pathogens via PPRs (Toll-like or NOD-like receptors) and become activated. These activated cells then become antigen-presenting cells (APC); they process peptide components of the pathogen and present these antigen peptides to T cells in the local environment. The type of CD4þ T cell (Th cells) response that is generated will dictate the outcome of the infection (Fig. 96.1A). Different T cells are required to induce protection from different pathogens. Effector T cell responses including Th1 and Th17 cells are required to fight many different gastrointestinal infections (Fig. 96.1A). In IBD uncontrolled Th1 and Th17 immune responses occur and strategies to suppress the Th1 and Th17 cells effectively suppress experimental IBD (Fig. 96.1B). Patients with IBD often have increased Th1 cells in the intestine that produce high levels of the Th1 inflammatory cytokine IFN-g [48]. Th17 cells produce two inflammatory cytokines, IL-17 and IL-22, both of which have been shown to play a pathogenic role in several autoimmune diseases including MS, type 1 diabetes and IBD [49,50]. All IBD is not solely Th1- and Th17-mediated. Some patients with UC have an increased Th2 cell response that is characterized by the production of IL-4, IL-5, and IL-13 instead of the Th1 and Th17 cell cytokines. Such patients harbor increased serum levels of IL-5 and produce autoantibodies that can, in some cases, disrupt the integrity of the epithelium or impair the nature of a healthy immune response [51e53]. There are a number of T cells that play a role in turning off immune responses in the gut (Fig. 96.1A). The invariant natural killer T (iNKT) cells are cells that produce cytokines early during an immune response acting on the APC and influencing the development of the T cell responses. iNKT cells in the intestine become activated by the epithelial cells to produce inhibitory cytokines like IL-10 [54]. iNKT cells are early producers of many cytokines including IFN-g, IL-4, IL-13, and IL-10. The type of cytokines produced by the iNKT cells dictates the nature of the resulting T cell response (Fig. 96.1A, [54]). Activation of iNKT cells protects mice from experimental IBD and reduced numbers of iNKT cells have been shown in experimental models of autoimmunity including MS and type 1 diabetes [55]. IBD patients have decreased numbers of iNKT cells (Fig. 96.1B, [56]). FoxP3þ CD4 T cells (Treg) act to inhibit the proliferation of T cell responses both in vitro and in vivo (Fig. 96.1A [57]). Animals that lack Treg cells spontaneously develop experimental IBD [11]. The spontaneous development of IBD in mice that lack Tregs is due to the inability to turn off T cell responses to the commensal flora since germ-free mice do not develop IBD symptoms [11]. Tregs have been shown to induce programmed cell death or apoptosis in effector T cells
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(Th1 and Th17 cells), suppress proliferation and produce the suppressive cytokines IL-10 and TGF-b1 [57]. Transfer of Tregs from a mouse to a syngeneic host have been shown to suppress experimental IBD in vivo in the recipient animal [57]. iNKT cells and Tregs function to suppress Th1 and Th17 responses in the gastrointestinal tract. The normal gut-associated lymphoid tissue harbors gut-specific T cells that also have a regulatory function (Fig. 96.1A). These unique T cells express a homodimeric form of CD8 e CD8aa; the CD8aa on the T cells acts to dampen signals coming into the cell through the T cell receptor [58]; it is thought that CD8aa is expressed on T cells in the gut in order to keep the cells from responding to the bacteria and food antigens found there [59]. In addition, CD8aa Tcells in the intraepithelial lymphocyte (IEL) pool can also suppress inflammation by producing suppressive cytokines like IL-10 and TGF-b1 [60,61]. Similar to the case for Tregs, adoptive transfer of CD8aa T cells have been shown to suppress experimental IBD [60].
VITAMIN D REGULATES T CELL RESPONSIVENESS As discussed elsewhere in this volume, the VDR is expressed in all immune cells that have been examined. The level of VDR expression in certain types of immune cells including T cells increases after activation [62]. Vitamin D deficiency or VDR knockout (KO) mice have been shown to have increased susceptibility to several different experimental models of IBD (Fig. 96.1B, [63]). In addition, the favored ligand for the VDR, 1,25(OH)2D, suppresses experimental models of autoimmunity including IBD [64]. Treatment with 1,25(OH)2D3 in vitro has been shown to inhibit differentiation and activation of DC [65]. 1,25 (OH)2D3-treated DC stay in a more immature state characterized by reduced antigen presentation, decreased IFN-g production, and increased production of IL-10 [66,67]. Macrophage proliferation and differentiation is also inhibited by 1,25(OH)2D in vitro, and treatment of the macrophage after activation inhibits the production of IL-12 and TNF-a [68,69]. These effects on macrophage and DC result in reduced Th1 cell activation. T cells are also direct targets of 1,25(OH)2D. In the absence of other cells, 1,25(OH)2D3 inhibits proliferation and IL-2 and IFN-g production [70,71]. Conversely, CD4þ Th cells from VDR KO mice overproduce IFN-g and proliferate twice as quickly in mixed lymphocyte reactions [63]. Treatment of peripheral blood mononuclear cells (PBMC) from IBD patients with physiological levels of 1,25(OH)2D3 decreased the production of IFN-g [72].
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Th17 cells are also targets of 1,25(OH)2D. 1,25(OH)2D3 treatment of DC reduces Th17-inducing cytokines IL-6 and IL-23 [73]. Treatment of CD4þ Th cells with 1,25(OH)2D3 under Th17 polarizing conditions (e.g., under the influence of IL-6 and TGF-b1) reduces the number of Th17 cells that develop [74]. Human PBMC from IBD patients overproduce IL-17 and treating the PBMC with a vitamin D analog reduces the production of Th17 cells [75]. The effects of 1,25(OH)2D3 on Th2 responses are less clear. Several groups have shown that 1,25(OH)2D3 treatment enhances the production of IL-4 and IL-5 and augments the expression of the Th2specific transcription factor GATA-3 [76,77]. However, others have reported that 1,25(OH)2D3 treatment has no affect on the expression of Th2 genes and inhibits the production of IL-4 [78]. Vitamin D has been shown to control regulatory T cells including iNKT cells, Treg cells, and CD8aa T cells (Fig. 96.1A). Expression of the VDR is required for normal development and function of iNKT cells [79]. 1,25(OH)2D3 increases iNKT cell cytokine production but has no effect on the numbers of iNKT cells [79]. VDR KO mice essentially have no functional iNKT cells [79]. The absence of iNKT cells is partially responsible for the susceptibility of VDR KO mice to dextran sodium sulfate-induced colitis (unpublished data, [80]). The ability of 1,25(OH)2D3 to suppress several experimental autoimmune diseases occurs via the induction of Treg cells [81]. 1,25(OH)2D3 has been shown in vitro to increase Treg numbers (Fig. 96.1A) [82,83]. However, VDR KO mice have normal numbers of Treg cells and the VDR KO Tregs are functionally normal both in vitro and in vivo [82]. While vitamin D is not required for Treg development, it does increase the numbers of Tregs that develop during an immune response. In the intestinal intraepithelial lymphocytes (IEL) the CD8aa T cells also require vitamin D for normal development. Vitamin D deficient and VDR KO mice have half as many of the CD8aa T cells as their WT counterparts (Fig. 96.1B) [82]. In addition, the CD8aa T cells from VDR KO mice are functionally impaired; they produce less IL-10 than those from the WT mice [82]. 1,25(OH)2D3 enhances the development and function of iNKT cells, Tregs and CD8aa T cells. In the absence of 1,25(OH)2D or the VDR iNKT cells and CD8aa T cells are defective, resulting in the increased susceptibility of these mice to inflammation in the gut. The vitamin D system (25(OH)D status, 1,25(OH)2D3 and VDR receptor expression) influences both the innate and acquired immune system in the gut (Fig. 96.1). 1,25(OH)2D3 directly and indirectly inhibits pathogenic Th1 and Th17 cell responses in the gut (Fig. 96.1A). In addition, vitamin D regulates iNKT cells, Treg cells and CD8aa T cells to suppress the Th1 and Th17 cell response and suppress inflammation in the
gastrointestinal tract (Fig. 96.1A). When vitamin D is low there is a limiting amount of 1,25(OH)2D3 and therefore the VDR does not function. Low vitamin D leads to an absence of regulatory T cells and overactive Th1 and Th17 responses resulting in excessive inflammation in the gastrointestinal tract and IBD (Fig. 96.1B).
EXPERIMENTAL MODELS OF IBD Much of our knowledge about the mechanisms involved in IBD comes from experimental animal models of intestinal inflammation. While these models provide insight into the pathogenesis of IBD there is not a mouse model that completely mimics either CD or UC. The aim of these models is to provide tools to researchers that allow for investigation into specific aspects of the diseases. There are several different animal models of IBD. Some result spontaneously following KO of regulatory cytokines or cells, while others are induced following chemical injury or immunization. In each of the models there is interplay between the inability to control inflammation in the gut, the bacteria in the gut and loss of barrier function. Examination of several different experimental models of IBD can give a more comprehensive view of the effects of vitamin D on several important characteristics of the human disease. Deletion of the suppressive cytokine IL-10 results in spontaneous intestinal inflammation. The intestinal inflammation that develops in IL-10 KO mice is due to an uncontrolled immune response to the commensal microflora in the intestine [11]. IL-10 KO mice that are either vitamin D deficient or VDR/IL-10 “double KO” develop severe fulminating colitis that is characterized by epithelial hyperplasia, significant weight loss and premature mortality [63]. The severity of the inflammation is associated with an increase in the Th1 response to the bacteria in the intestine including increased IFN-g, IL-12, TNF-a, and IL-1b [84]. In contrast, feeding supplemental 1,25(OH)2D3 prevents inflammation in IL-10 KO mice and when given after the onset of intestinal inflammation can block the progression of the disease by reducing TNF-a through inhibition of several genes of the TNF-a pathway [64,85]. A T-cell-driven model of intestinal inflammation develops when naı¨ve CD4 T cells are transferred to mice that lack T and B cells (recombination activating gene (Rag) KO). Rag KO mice that receive the transferred T cells develop a wasting disease as a result of increased inflammation in the small intestine and colon that is driven by uncontrolled Th1 and Th17 responses [86,87]. Transferring naı¨ve VDR KO CD4 T cells to Rag KO mice increases the severity and induces a more rapid onset of the disease than WT CD4 T cells [63].
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CURRENT TREATMENTS FOR IBD
Co-transfer of several different regulatory cells suppresses IBD in this model [60,88]. VDR KO Treg cells are as good as WT Treg at suppressing IBD in the Rag KO transfer model [82]. Chemical treatment with either trinitrobenzene sulfonic acid (TNBS) or dextran sodium sulfate (DSS) is a way to induce inflammation in the gastrointestinal tract. 1,25(OH)2D3 administration to mice was effective at reducing TNBS-induced colitis [73]. However, the mice in these experiments did become hypercalcemic [73]. VDR KO mice were extremely susceptible to colitis induced with DSS [80]. VDR KO mice had increased mortality following low levels of DSS as a result of severe intestinal inflammation, loss of barrier function and endotoxemia [80,89]. 1,25(OH)2D3 reduced the severity and improved recovery from DSS and TNBSinduced colitis [73,80,89]. In addition to the immunoregulatory functions of vitamin D, 1,25(OH)2D3 treatments protected mice from chemical injury to the gut and in part this was a result of improved barrier function in the presence of 1,25(OH)2D3. There is one model of IBD that failed to show beneficial effects of 1,25(OH)2D3. IL-2 KO mice develop IBD because of an absence of Treg cells [90]. 1,25(OH)2D3 treatment had no effect on IBD symptoms in the IL-2 KO mice suggesting that the ability of 1,25(OH)2D3 to induce Treg cells or IL-2 production may be critical for suppression of IBD [90]. The success of 1,25(OH)2D3 treatment in vivo in two chemical injury models, a T cell transfer model and IL-10 KO mice, and lack of success in the IL-2 KO model give important insights into the mechanisms underlying the effects of vitamin D as a regulator of intestinal inflammation.
CURRENT TREATMENTS FOR IBD There is no cure for IBD. Immunosupressive drugs are used to induce remission but are not used as maintenance drugs because of their toxicity and side effects. For maintenance there are a number of different options ranging from immunomodulators to biological therapies. The use of these newer treatments has resulted in the improved quality of life for patients living with IBD. 5-Aminosalicylic acid (5-ASA) and corticosteroids are the standard first line therapies for inducing remission in patients with mild to moderate IBD. 5-ASA has been shown to inhibit the synthesis of prostaglandin, leukotrienes, and IL-1b and to suppress the activation of NFkB by TNF-a and IL-1 [91]. 5-ASA is a broad anti-inflammatory agent that has been shown to be effective for treating mild to moderate UC but is less effective for CD [92]. Glucocorticoids, such as prednisone, nonspecifically suppress the immune system and are used to treat moderate to severe CD and UC. Treatment
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with glucocorticoids is an effective way of inducing remission; however, prolonged use results in steroid dependence or resistance [93]. Although glucocorticoids and 5-ASA are effective treatments to induce remission, not all patients respond. Failure to respond to drug therapy is associated with a worse prognosis, including increased risk of surgery, risk of disability, and an increased risk of infection [94]. The development of other immunosuppressive drugs has improved treatment for maintaining remission of IBD and has been shown to be useful for limiting longterm use of glucocorticoids [7,95]. Azothioprine, methotrexate, and cyclosporin suppress inflammation by limiting T cell activity. It has been demonstrated that azothioprine treatment is effective for inducing remission in patients with mild IBD and maintaining remission [96]. Methotrexate inhibits the enzymes involved in the nucleoside synthesis and consequently suppresses T cell activation, proliferation and inhibits the expression of adhesion molecules [97]. Treatment with methotrexate is effective in CD but less so for UC [98,99]. Cyclosporin is a widely used immunosuppressant that inhibits T cell activation by suppressing calcium activation pathway via the inhibition of calcineurin [100]. Cyclosporin has been shown to be effective for treating patients with severe, steroid-refractory UC but has not proven to be effective in patients with severe CD [101]. Within the last 10 years a number of new biological therapies have been developed and found to be useful to treat IBD. Biologicals target specific aspects of the immune response that contribute to intestinal inflammation. Infliximab (Remicade) and adalimumab (Humira) are some of the first biologics that have been shown to be effective therapy options for patients with IBD [102]. Infliximab and adalimumab are both humanized antibodies that block the activity of TNF-a. These TNFa blocking drugs were first shown to be effective in patients with arthritis. In 1998, the FDA approved the use of infliximab to treat patients with moderately and severely active IBD that do not respond to conventional treatment. One of the main complications that exist with the use of this family of drugs is that patients can develop specific immunity against the therapy and stop responding [103]. Since the introduction of the TNF blockers there have been many other biologicals developed that target other inflammatory cytokines or the cytokine receptors including those for IL-12, IFN-g, and IL-6 [104]. There are now biologicals that antagonize T cells and some that target leukocyte migration [104,105]. These treatments are very expensive and it is estimated that the use of biologicals to treat IBD results in health care costs of more than $200 billion worldwide [106]. There are many drawbacks to the biologics including expense, required medical personnel to administer and increased
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susceptibility to opportunistic infection and cancer. Presently patients with IBD will require life-long treatment. Because of the expense and risks of all of the current therapies, IBD researchers and patients are interested in exploring alternative strategies to limit intestinal inflammation. One strategy involves modifying the bacteria found in the intestine. Studies that looked at the use of antibiotics produced controversial results. Oral antibiotic treatment can be beneficial in models of IBD by eliminating bacteria that is inducing an inappropriate immune response [107]. The use of antibiotics in one study was associated with an increased risk of inflammation, presumably because good microbial flora was eliminated in the gastrointestinal tract [28]. Antibiotic resistance would be an additional barrier to using antibiotics for long-term treatment of IBD. Prebiotics are non-digestible fibers that have been reported to have beneficial effects in IBD. Using prebiotics changes the composition of the intestinal microflora toward more protective bacteria and alters systemic and mucosal immune responses of the host [108]. Another way to shift the balance of good versus bad bacteria is the use of probiotics. Probiotics contain viable, defined microorganisms that, when administered in adequate amounts, alter the microflora of the host [108]. If found to be effective prebiotics and probiotics hold the promise of treatments that have few side-effects and could be used to augment or even replace some of the maintenance therapies for IBD patients.
VITAMIN D AS A TREATMENT OPTION FOR IBD Vitamin D, in vitamin-D-insufficient patients, and its active metabolites and analogs could be a safe and effective adjunct to the therapies available to treat or prevent IBD. Unfortunately, clinical interventions have not been done to look at the effects of vitamin D on IBD disease in humans; based on the extensive data in animal models several studies have been proposed and registered at Clinicaltrials.gov using vitamin D or vitamin D analogs as treatments. Issues that need to be addressed include: (1) what is the appropriate dose of the vitamin D compound to be used? (2) does it matter what other therapies a patient is on? and (3) can patients with either UC or CD or both benefit? Even in the absence of clinical data it seems reasonable to treat IBD patients with vitamin D to normalize the 25(OH)D to serum levels >30 ng/ml as a possible preventive or maintenance treatment. 1,25(OH)2D3 or analogs of 1,25(OH)2D3 have been shown to be effective treatments of experimental IBD. It is possible that 1,25(OH)2D3 rather than vitamin D itself could suppress mild to moderate IBD and perhaps be a glucocorticoid-sparing drug. The
benefits of vitamin D in IBD could be multiple, including a reduction in inflammation and the decreased severity of the disease as well as resolution of secondary hyperparathyroidism and improvement in BMD [109]. However, evaluation of 25(OH)D status has not been established as standard-of-care in patients with IBD. Even when vitamin D deficiency is detected, there are no established guidelines for treatment in children or adult IBD patients [42].
References [1] D.K. Podolsky, Inflammatory bowel disease (1), N. Engl. J. Med. 325 (1991) 928e937. [2] D.K. Podolsky, Inflammatory bowel disease (2), N. Engl. J. Med. 325 (1991) 1008e1016. [3] N.A. Braus, D.E. Elliott, Advances in the pathogenesis and treatment of IBD, Clin. Immunol. 132 (2009) 1e9. [4] C.N. Bernstein, A. Wajda, L.W. Svenson, A. MacKenzie, M. Koehoorn, M. Jackson, et al., The epidemiology of inflammatory bowel disease in Canada: a population-based study, Am. J. Gastroenterol. 101 (2006) 1559e1568. [5] M.T. Cantorna, Vitamin D and its role in immunology: multiple sclerosis, and inflammatory bowel disease, Prog. Biophys. Mol. Biol. 92 (2006) 60e64. [6] D.K. Podolsky, Inflammatory bowel disease, N. Engl. J. Med. 347 (2002) 417e429. [7] D.C. Baumgart, W.J. Sandborn, Inflammatory bowel disease: clinical aspects and established and evolving therapies, Lancet 369 (2007) 1641e1657. [8] R.J. Xavier, D.K. Podolsky, Unravelling the pathogenesis of inflammatory bowel disease, Nature 448 (2007) 427e434. [9] R. Cooney, D. Jewell, The genetic basis of inflammatory bowel disease, Dig. Dis. 27 (2009) 428e442. [10] J. Satsangi, D.P. Jewell, J.I. Bell, The genetics of inflammatory bowel disease, Gut. 40 (1997) 572e574. [11] E. Louis, C. Libioulle, C. Reenaers, J. Belaiche, M. Georges, Genetics of ulcerative colitis: the come-back of interleukin 10, Gut. 58 (2009) 1173e1176. [12] L. Jalocha, S. Wojtun, P. Dyrla, J. Gil, J. Korsak, A. Rzeszotarska, et al., [TNF alfa polymorphism and course of ulcerative colitis], Pol. Merkur. Lekarski. 26 (2009) 444e445. [13] K. Li, S. Yao, S. Liu, B. Wang, D. Mao, Genetic polymorphisms of interleukin 8 and risk of ulcerative colitis in the Chinese population, Clin. Chim. Acta. 405 (2009) 30e34. [14] J.H. Cho, The genetics and immunopathogenesis of inflammatory bowel disease, Nat. Rev. Immunol. 8 (2008) 458e466. [15] Y. Luo, B. Xia, C. Li, Z. Chen, L. Ge, T. Jiang, et al., Cytotoxic T lymphocyte antigen-4 promoter -658CT gene polymorphism is associated with ulcerative colitis in Chinese patients, Int. J. Colorectal. Dis. 24 (2009) 489e493. [16] R. Verma, V. Ahuja, J. Paul, Frequency of single nucleotide polymorphisms in NOD1 gene of ulcerative colitis patients: a caseecontrol study in the Indian population, BMC Med. Genet. 10 (2009) 82. [17] X. Shen, R. Shi, H. Zhang, K. Li, Y. Zhao, R. Zhang, The Tolllike receptor 4 D299G and T399I polymorphisms are associated with Crohn’s disease and ulcerative colitis: a meta-analysis. Digestion 81 (2010) 69e77. [18] M.H. Zaki, K.L. Boyd, P. Vogel, M.B. Kastan, M. Lamkanfi, T.D. Kanneganti, The NLRP3 inflammasome protects against loss of epithelial integrity and mortality during experimental colitis, Immunity 32 (2010) 379e391.
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[19] M.L. McCullough, R.M. Bostick, T.L. Mayo, Vitamin D gene pathway polymorphisms and risk of colorectal, breast, and prostate cancer, Annu. Rev. Nutr. 29 (2009) 111e132. [20] J.D. Simmons, C. Mullighan, K.I. Welsh, D.P. Jewell, Vitamin D receptor gene polymorphism: association with Crohn’s disease susceptibility, Gut 47 (2000) 211e214. [21] R. Dresner-Pollak, Z. Ackerman, R. Eliakim, A. Karban, Y. Chowers, H.H. Fidder, The BsmI vitamin D receptor gene polymorphism is associated with ulcerative colitis in Jewish Ashkenazi patients, Genet. Test 8 (2004) 417e420. [22] E. van Etten, K. Stoffels, C. Gysemans, C. Mathieu, L. Overbergh, Regulation of vitamin D homeostasis: implications for the immune system, Nutr. Rev. 66 (2008) S125e134. [23] S.V. Ramagopalan, N.J. Maugeri, L. Handunnetthi, M.R. Lincoln, S.M. Orton, D.A. Dyment, et al., Expression of the multiple sclerosis-associated MHC class II allele HLADRB1*1501 is regulated by vitamin D, PLoS Genet. 5 (2009) e1000369. [24] N. Israni, R. Goswami, A. Kumar, R. Rani, Interaction of vitamin D receptor with HLA DRB1 0301 in type 1 diabetes patients from North India, PLoS One 4 (2009) e8023. [25] T.T. Wang, B. Dabbas, D. Laperriere, A.J. Bitton, H. Soualhine, L.E. Tavera-Mendoza, et al., Direct and indirect induction by 1,25-dihydroxyvitamin D3 of the NOD2/CARD15-defensin beta2 innate immune pathway defective in Crohn disease, J. Biol. Chem. 285 (2010) 2227e2231. [26] S.M. Orton, A.P. Morris, B.M. Herrera, S.V. Ramagopalan, M.R. Lincoln, M.J. Chao, et al., Evidence for genetic regulation of vitamin D status in twins with multiple sclerosis, Am. J. Clin. Nutr. 88 (2008) 441e447. [27] C.A. Anderson, D.C. Massey, J.C. Barrett, N.J. Prescott, M. Tremelling, S.A. Fisher, et al., Investigation of Crohn’s disease risk loci in ulcerative colitis further defines their molecular relationship, Gastroenterology 136 (2009) 523e529. e523. [28] N.A. Koloski, L. Bret, G. Radford-Smith, Hygiene hypothesis in inflammatory bowel disease: a critical review of the literature, World J. Gastroenterol. 14 (2008) 165e173. [29] F. van der Heide, A. Dijkstra, R.K. Weersma, F.A. Albersnagel, E.M. van der Logt, K.N. Faber, et al., Effects of active and passive smoking on disease course of Crohn’s disease and ulcerative colitis, Inflamm. Bowel. Dis. 15 (2009) 1199e1207. [30] S. Kitajima, M. Morimoto, E. Sagara, C. Shimizu, Y. Ikeda, Dextran sodium sulfate-induced colitis in germ-free IQI/Jic mice, Exp. Anim. 50 (2001) 387e395. [31] R. Hansen, J.M. Thomson, E.M. El-Omar, G.L. Hold, The role of infection in the aetiology of inflammatory bowel disease, J. Gastroenterol. 45 (2010) 266e276. [32] E.S. Pierce, Where are all the Mycobacterium avium subspecies paratuberculosis in patients with Crohn’s disease? PLoS Pathog. 5 (2009) e1000234. [33] J.D. Bufford, J.E. Gern, The hygiene hypothesis revisited, Immunol. Allergy. Clin. North Am. 25 (2005) 247e262, vevi. [34] N.A. Koloski, L. Brett, Animal farm: do our four-legged friends hold the answer to inflammatory bowel disease? Inflamm. Bowel. Dis. 14 (2008) 1163e1164. [35] M.T. Cantorna, S. Yu, D. Bruce, The paradoxical effects of vitamin D on type 1 mediated immunity, Mol. Aspects. Med. 29 (2008) 369e375. [36] M.F. Holick, Sunlight, UV-radiation, vitamin D and skin cancer: how much sunlight do we need? Adv. Exp. Med. Biol. 624 (2008) 1e15. [37] H.F. DeLuca, Vitamin D, Nutrition. Today 28 (1993) 6e11.
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[57] D.A. Vignali, L.W. Collison, C.J. Workman, How regulatory T cells work, Nat. Rev. Immunol. 8 (2008) 523e532. [58] A.J. Leishman, O.V. Naidenko, A. Attinger, F. Koning, C.J. Lena, Y. Xiong, et al., T cell responses modulated through interaction between CD8alphaalpha and the nonclassical MHC class I molecule, TL. Science 294 (2001) 1936e1939. [59] H. Cheroutre, Starting at the beginning: new perspectives on the biology of mucosal T cells, Annu. Rev. Immunol. 22 (2004) 217e246. [60] P. Poussier, T. Ning, D. Banerjee, M. Julius, A unique subset of self-specific intraintestinal T cells maintains gut integrity, J. Exp. Med. 195 (2002) 1491e1497. [61] X. Tang, I. Maricic, V. Kumar, Anti-TCR antibody treatment activates a novel population of nonintestinal CD8 alpha alphaþ TCR alpha betaþ regulatory T cells and prevents experimental autoimmune encephalomyelitis, J. Immunol. 178 (2007) 6043e6050. [62] C.M. Veldman, M.T. Cantorna, H.F. DeLuca, Expression of 1,25-dihydroxyvitamin D(3) receptor in the immune system, Arch. Biochem. Biophys. 374 (2000) 334e338. [63] M. Froicu, V. Weaver, T.A. Wynn, M.A. McDowell, J.E. Welsh, M.T. Cantorna, A crucial role for the vitamin D receptor in experimental inflammatory bowel diseases, Mol. Endocrinol. 17 (2003) 2386e2392. [64] Y. Zhu, B.D. Mahon, M. Froicu, M.T. Cantorna, Calcium and 1alpha,25-dihydroxyvitamin D3 target the TNF-alpha pathway to suppress experimental inflammatory bowel disease, Eur. J. Immunol. 35 (2005) 217e224. [65] M.D. Griffin, R. Kumar, Effects of 1alpha,25(OH)2D3 and its analogs on dendritic cell function, J. Cell Biochem. 88 (2003) 323e326. [66] G. Penna, L. Adorini, 1 Alpha,25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation, J. Immunol. 164 (2000) 2405e2411. [67] A.G. van Halteren, E. van Etten, E.C. de Jong, R. Bouillon, B.O. Roep, C. Mathieu, Redirection of human autoreactive Tcells upon interaction with dendritic cells modulated by TX527, an analog of 1,25 dihydroxyvitamin D(3), Diabetes 51 (2002) 2119e2125. [68] H.P. Koeffler, T. Amatruda, N. Ikekawa, Y. Kobayashi, H.F. DeLuca, Induction of macrophage differentiation of human normal and leukemic myeloid stem cells by 1,25dihydroxyvitamin D3 and its fluorinated analogues, Cancer Res. 44 (1984) 5624e5628. [69] D.R. Clohisy, Z. Bar-Shavit, J.C. Chappel, S.L. Teitelbaum, 1,25Dihydroxyvitamin D3 modulates bone marrow macrophage precursor proliferation and differentiation. Up-regulation of the mannose receptor, J. Biol. Chem. 262 (1987) 15922e15929. [70] M. Cippitelli, A. Santoni, Vitamin D3: a transcriptional modulator of the interferon-gamma gene, Eur. J. Immunol. 28 (1998) 3017e3030. [71] H. Reichel, H.P. Koeffler, A. Tobler, A.W. Norman, 1 Alpha,25dihydroxyvitamin D3 inhibits gamma-interferon synthesis by normal human peripheral blood lymphocytes, Proc. Natl. Acad. Sci. USA 84 (1987) 3385e3389. [72] S. Ardizzone, A. Cassinotti, D. Trabattoni, G. Manzionna, V. Rainone, M. Bevilacqua, et al., Immunomodulatory effects of 1,25-dihydroxyvitamin D3 on TH1/TH2 cytokines in inflammatory bowel disease: an in vitro study, Int. J. Immunopathol. Pharmacol. 22 (2009) 63e71. [73] C. Daniel, N.A. Sartory, N. Zahn, H.H. Radeke, J.M. Stein, Immune modulatory treatment of trinitrobenzene sulfonic acid colitis with calcitriol is associated with a change of a T helper
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[90] C.J. Bemiss, B.D. Mahon, A. Henry, V. Weaver, M.T. Cantorna, Interleukin-2 is one of the targets of 1,25-dihydroxyvitamin D3 in the immune system, Arch. Biochem. Biophys. 402 (2002) 249e254. [91] G.R. Lichtenstein, M.A. Kamm, Review article: 5-aminosalicylate formulations for the treatment of ulcerative colitis e methods of comparing release rates and delivery of 5-aminosalicylate to the colonic mucosa, Aliment. Pharmacol. Ther. 28 (2008) 663e673. [92] A.A. van Bodegraven, C.J. Mulder, Indications for 5-aminosalicylate in inflammatory bowel disease: is the body of evidence complete? World J. Gastroenterol. 12 (2006) 6115e6123. [93] W.A. Faubion Jr., E.V. Loftus Jr., W.S. Harmsen, A.R. Zinsmeister, W.J. Sandborn, The natural history of corticosteroid therapy for inflammatory bowel disease: a population-based study, Gastroenterology 121 (2001) 255e260. [94] P. Munkholm, E. Langholz, M. Davidsen, V. Binder, Frequency of glucocorticoid resistance and dependency in Crohn’s disease, Gut 35 (1994) 360e362. [95] J. Cosnes, I. Nion-Larmurier, L. Beaugerie, P. Afchain, E. Tiret, J.P. Gendre, Impact of the increasing use of immunosuppressants in Crohn’s disease on the need for intestinal surgery, Gut 54 (2005) 237e241. [96] D.C. Pearson, G.R. May, G.H. Fick, L.R. Sutherland, Azathioprine and 6-mercaptopurine in Crohn disease. A meta-analysis, Ann. Intern. Med. 123 (1995) 132e142. [97] A. Johnston, J.E. Gudjonsson, H. Sigmundsdottir, B.R. Ludviksson, H. Valdimarsson, The anti-inflammatory action of methotrexate is not mediated by lymphocyte apoptosis, but by the suppression of activation and adhesion molecules, Clin. Immunol. 114 (2005) 154e163. [98] B.G. Feagan, J. Rochon, R.N. Fedorak, E.J. Irvine, G. Wild, L. Sutherland, et al., Methotrexate for the treatment of Crohn’s disease. The North American Crohn’s Study Group Investigators, N. Engl. J. Med. 332 (1995) 292e297.
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C H A P T E R
97 Psoriasis and Other Skin Diseases Jo¨rg Reichrath 1, Michael F. Holick 2 1 2
Universita¨tsklinikum des Saarlandes, Homberg, Germany Boston University School of Medicine, Boston, MA, USA
INTRODUCTION/HISTORICAL OVERVIEW
PATHOGENESIS OF PSORIASIS
In the last century, vitamin D3 was used in dermatology in huge pharmacological doses for the treatment of scleroderma, psoriasis, lupus vulgaris, and atopic dermatitis. A rationale for the use of vitamin D in psoriasis was the clinical observation that this disease in general markedly improves in the summer. In 1936 J. Krafka wrote in The Journal of Laboratory and Clinical Medicine under the headline “A simple treatment for psoriasis”: “A commonly observed fact in the South concerning this disease (psoriasis) is that it generally clears up in the summer sun. This led the author to the hypothesis that it might be cured with viosterol.A patient with a case of ten years’ standing, continuous duration was put on viosterol.Within sixty days from the beginning of the test, the skin of the patient was entirely clear” [1]. In 1950 H.W. Spier reported results of a study with 94 psoriasis patients performed in 1948e49 [2]. First, patients received orally 3 10 mg vitamin D2/week for 2e4 weeks, thereafter 2 10 mg vitamin D2/week until a total dose of approx. 300 mg vitamin D2 was reached. In this study, patients were treated for 3e4 months; 20% of patients showed a good response, 25% a satisfactory response, 25% a moderate response, and 30% a non-satisfactory response [2]. But these first attempts of vitamin D treatment in dermatology were abandoned because of severe vitamin D intoxications that caused hypercalcemia, hypercalciuria, and kidney stones when huge pharmacological doses of vitamin D (up to 1000-fold of the regular daily requirement of vitamin D) were used and because other new treatments, including corticosteroids and retinoids, were introduced for the therapy of these diseases.
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10097-6
Psoriasis: Pathogenesis, Immunology, and Histology of Skin Lesions Psoriasis is a chronic dermatosis of unknown etiology characterized by skin inflammation, hyperproliferation and altered differentiation of epidermal keratinocytes [3]. The most common form of the disease is plaque psoriasis, in which skin develops scaly, red lesions. The severity of chronic plaque psoriasis ranges from mild, when the disease has only a moderate impact on quality of life, to severe, when patients’ lives are significantly affected [4]. In severe cases, most of the body surface, including the scalp and nails, may be involved. The peak age of onset for this psychologically debilitating and disfiguring disease is the second decade, but psoriasis may first appear at any age from infancy into old age [5]. It is considered a multifactorial disease and has a prevalence of about 1e2% in the USA. Population, family and twin studies clearly demonstrate that there is a strong but very complex genetic component leading to the development of psoriatic skin lesions [6]. Most likely, multiple genes are involved in the pathogenesis of psoriasis. During the last years, molecular biology techniques have been developed that allow studies to analyze psoriasis susceptibility genes, but up to today, no specific genetic marker of the disease has been identified. Psoriasis has long been known to be associated with certain HLA antigens, particularly HLA-Cw6, although there is no evidence that a psoriasis susceptibility gene exists at this locus [7]. Until today, it is still unknown what cell types in human skin are primarily affected by the disease. Many studies support the hypothesis that epidermal hyperproliferation in psoriasis may be mediated by cells of the immune system, most likely T lymphocytes
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[3]. The vast majority of T cells in psoriatic lesions are situated in the perivascular area in the dermis; a high number are also found in the epidermis. Activated CD4þ and/or CD8þ T cells in psoriatic lesions express HLA-DR, the interleukin-2 receptor (CD 25), bear the CLAþ memory-effector CD45ROþ phenotype, and secrete specific immune mediators and cytokines, such as IL-2 and interferon-g [3,8e10]. Thus, psoriasis represents mainly a so-called Th1 profile disease (characterized by T lymphocyte secretion of IL-2, IL-12, and interferon-g) [11]. In contrast, atopic dermatitis represents a so-called Th2 profile disease, which is characterized by T cell secretion of IL-4, IL-5, and IL-10 [12]. The activation signal for the development of psoriatic lesions is still unknown, although there is increasing evidence that superantigens such as the N-terminal component of bacterial M-proteins may be of importance for the initiation of T cell proliferation in psoriasis [3,9e13]. It has also been hypothesized that psoriasis patients develop an effector-immune response to skin (auto)antigens, which have yet to be specifically identified. According to this model, the immunologic process underlying psoriasis begins with a sensitization-type phase during which the skin dendritic cells migrate to regional lymph nodes where they present these skin antigens to naı¨ve T cells. This sensitization phase occurs prior to the development of skin lesions [3]. When sensitization is obtained, the psoriasis skin lesion may develop as a result of the emigration of T cells in the skin where they are activated by antigen-presenting cells including Langerhans cells presenting self-skin antigens [3]. The precise appearances of the histology of the skin will depend upon the age of the psoriatic lesion and the site of the biopsy. In general, epidermal hyperplasia, in which the granular layer may be lost and the stratum corneum shows parakeratosis, can be found (Fig. 97.1). Typical lesions histologically will show elongation of the dermal papillae, with a relatively thin epidermis at the top of the papillae. The epidermis may show, in suprapapillar compartments, intercellular edema and infiltration with T lymphocytes and neutrophiles, which can extend into spongiform pustules of Kogoj or Munro microabscesses [14].
THE VITAMIN D SYSTEM IN NORMAL AND PSORIATIC SKIN Vitamin D is photochemically synthesized by UV-B action in the skin [15,16] (also see Chapter 2). It is known that the skin itself is a target tissue for the secosteroid hormone 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3, calcitriol), the biologically active vitamin D metabolite [17,18]. 1,25(OH)2D3 exerts genomic and non-genomic
effects. Non-genomic effects of calcitriol and analogs are in part related to effects on intracellular calcium [19,20]. In keratinocytes and other cell types, calcitriol rapidly increases free cytosolic calcium levels [19,20]. Genomic effects of 1,25(OH)2D3 are mediated via binding to a nuclear receptor protein that is present in target tissues and binds calcitriol with high affinity (KD 109e1010 M) and low capacity [21,22], the vitamin D receptor (VDR) [23e25]. In the skin, both VDR (Fig. 97.2) and RXR-a are expressed in keratinocytes, fibroblasts, Langerhans cells, sebaceous gland cells, endothelial cells and most cell types related to the skin immune system [26,27]. In vitro studies have revealed that 1,25(OH)2D3 is extremely effective in inducing the terminal differentiation and in inhibiting the proliferation of cultured human keratinocytes in a dose-dependent manner [28e30]. Additionally, 1,25(OH)2D3 acts on many cell types involved in immunologic reactions, including lymphocytes, macrophages, and Langerhans cells [31,32]. Data about the effects of 1,25(OH)2D3 on the melanin pigmentation system are still conflicting, but most studies do not support the possibility that calcitriol might regulate melanogenesis in human skin [33]. Sebocytes are sebum-producing cells that form the sebaceous glands. When the vitamin D endocrine system in human sebocytes was analyzed recently, it was shown that sebocytes represent target cells for biologically active vitamin D metabolites [34]. It was demonstrated that human SZ95 sebocytes express VDR and the enzymatic machinery to synthesize and metabolize biologically active vitamin D analogs [34]. The expression of the vitamin D receptor (VDR) and the metabolic enzymes, vitamin D-25-hydroxylase (CYP27A1, 25-OHase), 25-hydroxyvitamin D-1ahydroxylase (CYP27B1, 1a-OHase), and 1,25-dihydroxyvitamin D-24-hydroxylase (CYP24, 24-OHase) were all detected in human SZ95 sebocytes in vitro using realtime quantitative polymerase chain reaction [34]. Although several other splice variants of 1a-OHase were detected by nested touchdown polymerase chain reaction, it was demonstrated that the full-length product represents the major 1a-OHase gene product in SZ95 cells [34]. It was shown that incubation of SZ95 sebocytes with 1,25(OH)2D3 resulted in a cell culture condition-, time-, and dose-dependent modulation of cell proliferation, cell cycle regulation, lipid content, and interleukin-6/interleukin-8 secretion in vitro, while RNA expression of VDR and 24-OHase was upregulated along with vitamin D analog treatment [34]. The authors concluded that the vitamin D endocrine system is of high importance for sebocyte function and physiology, and that sebaceous glands represent potential targets for therapy with vitamin D analogs or
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THE VITAMIN D SYSTEM IN NORMAL AND PSORIATIC SKIN
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FIGURE 97.1 Histological demonstration of morphological changes in lesional psoriatic skin after 6 weeks of topical treatment with calcitriol (15 mg/g, b) and calcipotriol (50 mg/g, c). a ¼ lesional psoriatic skin before treatment. d ¼ non-lesional psoriatic skin. Notice strong reduction of epidermal thickness after topical treatment with vitamin D analogs. Hematoxylin-eosin staining. Original magnification 200. Please see color plate section.
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FIGURE 97.2 Immunohistochemical demonstration of 1,25-dihydroxyvitamin D3 receptors (VDR) in human skin. Notice strong nuclear VDR immunoreactivity in cells of all layers of the viable epidermis (arrows). Labeled avidin-biotin technique using mAb 9A7g directed against VDR. Original magnification 400. Please see color plate section.
for pharmacological modulation synthesis and metabolism [34].
of
1,25(OH)2D3
PHYSIOLOGICAL AND PHARMACOLOGICAL ACTIONS OF VITAMIN D ANALOGS IN NORMAL AND PSORIATIC SKIN Biological Effects of Vitamin D and Analogs in Psoriasis Up to today, the mechanisms underlying the therapeutic effectiveness of vitamin D analogs in psoriasis are still not completely understood. Results from immunohistochemical and molecular biology studies indicate that the anti-proliferative effects of topical calcitriol on epidermal keratinocytes are more pronounced as compared to effects on dermal inflammation. Modulation of various markers of epidermal proliferation (proliferating cell nuclear antigen (PCNA) and Ki-67 antigen), and differentiation (involucrin, transglutaminase K, filaggrin, cytokeratins 10,16) in lesional psoriatic skin after topical application of vitamin D analogs was shown in situ [35] (Fig. 97.3). Interestingly, effects of
topical treatment with vitamin D analogs on dermal inflammation are less pronounced (CD-antigens, cytokines, HLA-DR, etc.) as compared to effects on epidermal proliferation or differentiation. One reason for this observation may be that the bioavailability of this potent hormone in the dermal compartment may be markedly reduced as compared to the epidermal compartment [35]. Molecular biology studies have demonstrated that clinical improvement in psoriatic lesions treated topically with calcitriol correlates with an elevation of VDR mRNA [36]. It is well known that some patients suffering from psoriasis are resistant to topical calcitriol treatment. It was demonstrated that responders can be distinguished from the non-responders at the molecular level since non-responders show no elevation of VDR mRNA in skin lesions along with the treatment and express relatively low levels of VDR. These data suggest that the ability of calcitriol to regulate keratinocyte growth is closely linked to the expression of VDR. The target genes of topical calcitriol that are responsible for its therapeutic efficacy in psoriasis are still unknown. Major candidates for calcitriol target genes that are responsible for the calcitriol-induced terminal differentiation in keratinocytes are distinct cell-cycle-associated proteins (i.e., INK4 family), including p21/WAF-1 [37]. Data analyzing VDR expression and genotype in psoriasis are somewhat conflicting, some studies report a correlation between VDR expression or individual VDR genotypes and the skin eruptions of psoriasis, as well as with responsiveness to treatment with vitamin D analogs [36,38,39]. While no differences in VDR genotype between controls and psoriasis patients were reported at the BsmI site, some studies reported significant difference in terms of ApaI SNP [40] and FokI SNP [41]. Additionally, it has been shown that vitamin D receptor genotypes are not associated with clinical response to calcipotriol (a topical analog used in psoriasis) at least in Korean psoriasis patients [42]. Kontula et al. [43] and Mee et al. [44] studied the BsmI polymorphism and the response to calcipotriol treatment in psoriatic patients and found no association between them. According to Colin et al. [45], the FokI polymorphism was associated with the response to calcipotriol, and under conditions of vitamin D insufficiency this finding might have clinical implications. Data concerning serum levels of 1,25(OH)2D or 25(OH)D in psoriatic patients are conflicting. Some studies report reduced levels of 1,25(OH)2D in patients with manifest disease [46]. Additionally, the coincidence of pustular psoriasis with hypocalcemia [47] and the exacerbation of psoriasis under chloroquin therapy (thereby reducing 1,25(OH)2D levels via inhibition of 1a-(OH)ase) are well known [48].
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CLINICAL USE OF CALCITRIOL IN PSORIASIS
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Immunohistological detection of transglutaminase K in lesional psoriatic skin before treatment (a), lesional psoriatic skin after 6 weeks of topical treatment with calcipotriol (50 mg/g, b), and in non-lesional psoriatic skin (c). Notice strong staining for transglutaminase K in all epidermal cell layers of lesional psoriatic skin before treatment (a, arrows). In contrast, after 6 weeks of topical treatment with calcipotriol staining in lesional psoriatic skin (b, arrows) is restricted to the upper layers of the viable epidermis, a staining pattern that is characteristic for non-lesional psoriatic skin (c, arrows). Original magnification 160. Please see color plate section.
FIGURE 97.3
CLINICAL USE OF CALCITRIOL IN PSORIASIS Clinical Studies of Vitamin D and Analogs in Psoriasis and Other Skin Diseases The use of calcitriol and its analogs for the treatment of psoriasis resulted from two independent lines of investigation. Since psoriasis is a hyperproliferative skin disorder, it seemed reasonable that the anti-proliferative effects of 1,25(OH)2D3 could be used for the treatment of this disease. However, before launching clinical trials in 1985, MacLaughlin and associates reported the observation that psoriatic fibroblasts are partially resistant to the anti-proliferative effects of 1,25(OH)2D3 [49]. This observation prompted MacLaughlin and associates to speculate that calcitriol may be effective in the treatment of the hyperproliferative skin disease psoriasis and treatment might overcome the intrinsic defect. The other line of investigation resulted from a clinical observation. In 1985, Morimoto and Kumahara reported that a patient who was treated orally with 1a-(OH)D3 for osteoporosis had a dramatic remission of psoriatic skin lesions [50]. Morimoto et al. reported a follow-up study, demonstrating that almost 80% of 17 patients with psoriasis who were treated orally with 1a-(OH)D3 at a dose of 1.0 mg/day for up to 6 months showed clinically significant improvement [51].
Currently, numerous studies have reported that various vitamin D analogs, including calcitriol, calcipotriol, tacalcitol, hexafluoro-1,25-dihydroxyvitamin D3 [52], and maxacalcitol are effective and safe in the topical treatment of psoriasis [53e59]. It has been shown that topical calcitriol and its analogs are very effective and safe for the long-term treatment of psoriasis [58e60]. Applied twice daily topically in amounts of up to 100 grams of ointment (50 mg calcipotriol/g ointment) per week, calcipotriol, a synthetic analog of calcitriol, was shown to be slightly more effective in the topical treatment of psoriasis than betamethasone 17-valerate ointment [60]. Efficacy of topical treatment with maxacalcitol was compared with topical calcipotriol treatment [56]. In this study, investigators’ overall assessment suggests that maxacalcitol 25 mg/g may be more effective than once-daily calcipotriol (50 mg/g). It has been reported that a mild dermatitis can be seen in about 10% of patients treated with calcipotriol (50 mg/ g), particularly on the face [61]. This side-effect (mild dermatitis on the face) is not reported after topical treatment with calcitriol. Allergic contact dermatitis to vitamin D analogs is very rare; however, cases with allergic contact dermatitis to other ingredients of the ointment including propylene glycol have been reported [62e64]. The most common adverse event observed in psoriasis patients treated with maxacalcitol (6e50 mg/g maxacalcitol ointment) was a burning sensation of the
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target plaque [56]. In three out of four patients developing this side effect in one study, symptoms were severe enough to require discontinuation of the treatment [56]. A double-blind, right/left comparison, placebocontrolled evaluation demonstrated efficacy and safety of topical treatment with hexafluoro-1,25(OH)2D3 (5 mg/g) in psoriasis patients [57]. Adverse events included mild irritation. This irritation did not necessitate discontinuation of the study medication. During the large area topical administration study period a cobblestone appearance was initially noted in a few patients. This resolved with continued therapy after 3e4 weeks. Hexafluoro-1,25-dihydroxyvitamin D3treated plaques also developed very mild perilesional scales as observed with other vitamin D analogs [57]. Efficacy and safety of topical treatment with tacalcitol (4 mg/g and 20 mg/g) has been shown as well [58,59,65]. In one study [59], tacalcitol treatment was generally well tolerated and there were no serious or unexpected adverse events reported. However, discontinuation of treatment as a result of skin irritation was seen in 5.9% of these patients [59]. The greatest frequency of cutaneous side effects occurred during initial treatment and the incidence decreased markedly as the treatment was well tolerated with continued use [59]. The results of four separate studies designed to evaluate specific local-safety parameters of various vitamin D analogs including cumulative irritancy, cutaneous contact sensitization, photoallergic contact sensitization and phototoxicity were analyzed [66]. Calcitriol 3 mg/g ointment was classified as non-irritant when compared to calcipotriol, tacalcitol, and white petrolatum (control). Petrolatum and tacalcitol were slightly irritant and calcipotriol moderately irritant. No sensitization was observed with calcitriol 3 mg/g ointment. With regard to phototoxic potential, sites treated with calcitriol 3 mg/g ointment or vehicle ointment were less irritated than those treated with white petrolatum or those that were untreated. Using standard photoallergenicity testing methodology, there were no skin reactions of a photoallergic nature to the study material [66]. A long-term follow-up study demonstrated the efficacy and safety of oral calcitriol as a potential treatment of psoriasis [67]. Of the 85 patients included in that study who received oral calcitriol for 36 months, 88.0% had some improvement in their disease, while 26.5%, 26.3%, and 25.3% had complete, moderate, and slight improvement in their disease, respectively. Serum calcium concentrations and 24 h urinary calcium excretion increased by 3.9% and 148.2%, respectively, but were not outside the normal range. Bone mineral density of these patients remained unchanged. A very important consideration for the use of orally administered calcitriol is the dosing technique. To avoid its
effects on enhancing dietary calcium absorption, it is very important to provide calcitriol at night time. Perez et al. [67] showed that as a result of this dosing technique along with maintaining a calcium intake of no more than 1000 mg/daily, doses of 2 to 4 mg/night are well tolerated by psoriatic patients. Patients with psoriasis may need intermittent treatment for their entire lives. Vitamin D analogs have been shown not to exhibit tachyphylaxis during treatment of psoriatic lesions and topical treatment can be continued indefinitely. They are effective and safe for the topical treatment of skin areas that are usually difficult to treat in psoriatic patients and that respond slowly. Additionally, vitamin D analogs are effective in the treatment of psoriatic skin lesions in children and in HIV patients. Treatment of Scalp Psoriasis A double-blind, randomized multicenter study demonstrated that calcipotriol solution is effective in the topical treatment of scalp psoriasis [68e70]. Fortynine patients were treated twice daily over a 4-week period [68]. Sixty percent of patients on calcipotriol showed clearance or marked improvement versus 17% in the placebo group. No side effects were reported. Treatment of Nail Psoriasis The occurrence of nail psoriasis has been reported in up to 50% of patients. Nails in general are very difficult to treat and respond slowly. Up to now, there has been no consistently effective treatment for psoriatic nails. It has been reported that calcipotriol ointment is effective in the treatment of nail psoriasis [71]. Treatment of Face and Flexures Although the use of calcipotriol ointment is not recommended on face and flexures due to irritancy, most patients tolerate vitamin D analogs on these sites. It has been shown that calcitriol ointment (3 mg of calcitriol per gram of petrolatum) was found to be better tolerated and would appear to be more effective than calcipotriol ointment (50 mg of calcipotriol per gram of petrolatum) in the treatment of psoriasis in sensitive areas [72]. Treatment of Skin Lesions in Children During the past few years it has been shown that topical application of vitamin D analogs including calcitriol ointment (3 mg of calcitriol per gram of petrolatum) is an effective, safe and reliable therapy to cure psoriatic skin lesions in children [73e75]. Treatment of Psoriatic Lesions in HIV Patients We have treated an HIV-positive patient suffering from psoriatic skin lesions with topical and oral calcitriol. The patient responded well and there was no
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VITAMIN D ANALOG THERAPY IN OTHER SKIN DISEASES
evidence of enhancement in HIV disease activity or alterations in the number of T lymphocytes or CD4þ and CD8þ cells. Combination of Vitamin D Analogs with Other Therapies It has been reported that efficacy of topical treatment with vitamin D analogs in psoriasis can be increased by combination with other therapies, including methotrexate (MTX), very low-dose oral cyclosporine (2 mg/ kg/day), oral acitretin, topical dithranol, topical steroids, PUVA (psoralen plus UV-A) and UV-B or narrow-band UV-B phototherapy [76e84]. It has been shown that the combination of calcipotriol and MTX is safe and well tolerated [83]. The combination resulted in lower cumulative dosages of MTX compared with MTX and vehicle. Therefore the risk of MTX-induced side effects is substantially decreased [83]. Addition of calcipotriol ointment to oral application of acitretin (a vitamin A analog) was shown to produce a significantly better treatment response achieved with a lower cumulative dose of acitretin in patients with severe extensive psoriasis vulgaris, as compared with the group of patients treated with oral acitretin alone. The number of patients reporting adverse events was similar between the two treatment groups [78]. Complete clearing or 90% improvement in PASI (Psoriasis Area and Severity Index) was observed in 50% of patients treated with calcipotriol/cyclosporine versus 11.8% in the placebo/cyclosporine group. No difference was found in that study between the groups in short-term side effects. Kragballe and coworkers reported that efficacy of topical calcipotriol treatment in psoriasis can be improved by simultaneous ultraviolet-B phototherapy. Combination therapy of psoriasis with topical calcipotriol and narrow-band UV-B has been shown to be very effective for the treatment of psoriatic plaques [80]. One can speculate whether the therapeutic efficacy of UV-B in psoriasis may be at least in part due to increased cutaneous vitamin D synthesis. It has been shown that the combination of topical treatment with vitamin D analogs and UV radiation does not alter the tolerability or safety of therapy [85]. Vitamin D analogs may be topically applied at any time up to 2 hours before or immediately after UV radiation [85]. Results of a controlled, right/left study have demonstrated that pretreatment of psoriasis with the vitamin D3 derivative tacalcitol increases the responsiveness to 311 nm UV-B [86]. Additionally, it was shown that tacalcitol ointment (4 mg/g) and 0.1% tazarotene gel are both comparably effective in improving the therapeutic result of PUVA therapy in patients with chronic plaque-type psoriasis [87]. The treatment requirements to induce complete or near complete clearing were significantly lower for
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both combination treatments than for PUVA monotherapy (p < 0.01). The median cumulative UV-A dose and number of exposures were 30.6 J/cm2 (95% confidence interval, CI 22.5e71.2) and 14 (95% CI 11e16) for tacalcitol plus PUVA, 32.3 J/cm2 (95% CI 22.5e73.8) and 14 (95% CI 11e19) for tazarotene plus PUVA, and 37.0 J/ cm2 (95% CI 29.5e83.9) and 16 (95% CI 14e22) for PUVA monotherapy. No difference between the three regimens was observed with regard to duration of remission. Adverse reactions occurred more often with 0.1% tazarotene than with tacalcitol but were in general mild and completely reversible upon using a lower concentration of 0.05% tazarotene. It has been concluded that besides accelerating the treatment response, both agents, by virtue of their UV-A dose-sparing effect, might also help to reduce possible long-term hazards of PUVA treatment. Previously, a case report described two patients treated with a combination treatment of calcipotriol and bath psoralens and UV-A who developed hyperpigmentation at the lesional sites where calcipotriol ointment was applied [88]. Combined topical treatment with calcipotriol ointment (50 mg/g) and betamethasone ointment was shown to be slightly more effective and caused less skin irritation than calcipotriol used twice daily [79]. A new vehicle has been created with the objective of obtaining optimal stability of both calcipotriene and betamethasone dipropionate in the combination product. Early onset of action and efficacy of a fixed combination of calcipotriene and betamethasone dipropionate in this new vehicle in the treatment of psoriasis has been reported, making it a new standard therapy for the topical treatment of psoriasis [89].
VITAMIN D ANALOG THERAPY IN OTHER SKIN DISEASES Treatment of Other Skin Disorders with Vitamin D Analogs Vitamin D and Ichthyosis A double-blind, bilaterally paired, comparative study has demonstrated the effectiveness of topical treatment with calcipotriol ointment on congenital ichthyoses [90]. Reduction in scaling and roughness on the calcipotriol-treated side was seen in all patients with lamellar ichthyosis and bullous ichthyotic erythroderma of Brocq. The only patient treated with Comel-Netherton syndrome showed mild improvement, while the only patient suffering from ichthyosis bullosa of Siemens that was treated with calcipotriol did not show any change in severity on the calcipotriol-treated as compared to the vehicle-treated side. It has been reported that topical tacalcitol therapy was ineffective
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against ichthyoses that are characterized by retentive hyperkeratosis and a lack of epidermal hyperproliferation, including X-linked ichthyosis (XLI), ichthyosis vulgaris (IV), and acquired ichthyosis [91]. Vitamin D and Scleroderma Preliminary findings point to the efficacy of vitamin D analogs for the treatment of scleroderma. Humbert et al. [92] reported that oral administration of 1.0e2.5 mg/d calcitriol improves skin involvement, probably via inhibition of fibroblast proliferation and dermal collagen deposition. Vitamin D and Actinic Keratoses, Skin Cancer It has been reported in the literature that distinct polymorphisms of the VDR gene are associated with the presence of actinic keratoses and non-melanoma and melanoma skin cancer [93,94], indicating that the vitamin D endocrine system may be important for development of cutaneous malignancies. Moreover, some investigations indicate that topically applied vitamin D analogs may be effective in the treatment of actinic keratoses (cutaneous squamous cell carcinoma in situ). While it has been reported that actinic keratoses in renal transplant recipients do not improve with calcipotriol cream and all-trans retinoic acid cream as monotherapies or in combination during a 6-week treatment period [95], more recent reports, including an investigator-blinded, half-side comparison trial, indicate that topically applied vitamin D analogs may be effective in the treatment of actinic keratoses [96]. In this study [96], patients applied calcipotriol cream to one side and Ultrabase cream as placebo to the other side of the scalp and/or face for 12 weeks. The total number of actinic keratoses, diameters and total scores of the target lesions were determined at each visit. Nine patients were included, eight of whom completed the treatment. There was a statistically significant difference between the total number of actinic keratoses at baseline and at week 12 on the calcipotriol applied side whereas no difference was detected on the placebo-applied side (p ¼ 0.028 versus p ¼ 1.00). The mean total score of the target lesions reduced significantly at week 12 on the calcipotriol side; in contrast, no significant reduction was found on the placebo side (p ¼ 0.017 versus p ¼ 0.056). In conclusion, although the study was suggestive, the clinical efficacy of vitamin D analogs in the topical treatment of actinic keratoses remains to be elucidated in future investigations. In vitro studies have demonstrated strong antiproliferative and pro-differentiating effects of vitamin D analogs in many VDR-expressing tumor cell lines, including malignant melanoma, squamous cell carcinoma, breast and lung cancer, and leukemic cells [97e100]. In vivo studies supported these results and
showed that active vitamin D analogs block proliferation and tumor progression of epithelial tumors in rats [98]. Inhibition of tumor growth of human malignant melanoma and other cancer xenografts was also demonstrated in immune-suppressed mice, but only with high doses of calcitriol [101]. Little is known regarding the effects of calcitriol on the formation of metastases in patients with malignant melanoma or squamous cell carcinoma of the skin. This subject is discussed in Chapter 89. Vitamin D and Acne The therapeutic efficacy of vitamin D in acne was investigated in the middle of the last century [102e105] with limited success. However, more recent laboratory investigations and animal studies indicate that vitamin D compounds may be effective in acne treatment [102e106]. In the rhino mouse model, a comedolytic effect of topically applied active vitamin D3 analog maxacalcitol on pseudocomedones was demonstrated [106]. The rhino (hrrh/hrrh) phenotype is due to an autosomal recessive mutation in the hairless (hr) gene [106]. In the rhino mouse, utriculi are derived from the infundibular zone of the initial follicular units, and are histologically similar to comedones [106]. In that study [106], rhino mice were treated topically with tretinoin and maxacalcitol once daily for 2 and 4 weeks, respectively. The dermal side of the epidermal sheet was observed to determine the size of the utricle. Hematoxylin-and-eosin-stained vertical sections were used to measure utricle diameter and density and to evaluate histological changes. Maxacalcitol (25 mg/g) and tretinoin (0.1%) significantly decreased the size and the diameter of the utricle after 1 week of treatment. Histopathologically, maxacalcitol and tretinoin markedly induced epidermal hyperplasia accompanied by a minor accumulation of inflammatory cells in the dermis, with and without hypercornification, respectively. These results [106] indicate that maxacalcitol has an effect on comedolysis and that its mechanism of action may be different from that of retinoids. The clinical relevance of these observations remains to be elucidated in future investigations. Vitamin D and Cutaneous Wound Healing Laboratory investigations and animal studies indicate that calcitriol and its analogs may be effective agents to promote cutaneous wound healing [107]. The effect of vitamin D analogs on wound healing is associated with an upregulation of the antimicrobial peptide hCAP18/LL-37 [107] (cathelicidin). However, the clinical relevance of these promising results has to be further investigated.
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VITAMIN D ANALOG THERAPY IN OTHER SKIN DISEASES
Vitamin D and Other Skin Diseases A number of case reports demonstrate positive effects of topical treatment with vitamin D analogs in a variety of skin diseases such as transient acantholytic dermatosis (Grovers disease), inflammatory linear verrucous epidermal nevus (ILVEN), disseminated superficial actinic porokeratosis, pityriasis rubra pilaris, epidermolytic palmoplantar keratoderma of Vorner, confluent and reticulated papillomatosis (Gougerot-Carteaud syndrome) and Sjo¨gren-Larsson syndrome [108,109]. These promising observations will have to be further evaluated in clinical trials.
Perspectives for the Evaluation of New Vitamin D Analogs with Less Calcemic Activity, which can be used for the Treatment of Hyperproliferative Skin Disorders The use of vitamin D analogs in dermatology and other medical fields was shown to be limited, since serious side effects, mainly on calcium metabolism, may occur at supraphysiological doses needed to reach clinical improvement. The evaluation of new vitamin D compounds with strong immunosuppressive, antiproliferative, and differentiating effects but only marginal effects on calcium metabolism introduces new important therapies for the treatment of various skin diseases. The goal to create new vitamin D analogs with selective biological activity and no undesirable side effects has still not been reached, but recent findings introduce new and promising concepts. Section IX of this book discusses the development of analogs in detail. Calcipotriol (MC 903), a vitamin D analog with similar VDR binding properties compared to calcitriol, but low affinity for the vitamin D binding protein (DBP), is well known to be effective and safe in the topical treatment of psoriasis [60]. In vivo studies in rats showed that effects of calcipotriol on calcium metabolism are 100e200 times lower as compared to calcitriol, while in vitro effects on proliferation and differentiation on human keratinocytes are comparable [110]. These differential effects are probably caused by the different pharmacokinetic profiles of calcipotriol and calcitriol (different affinity for DBP). Serum half-life in rats of these vitamin D analogs was shown to be 4 min after treatment with calcipotriol in contrast to 15 min after treatment with calcitriol [110]. However, most of the calcium metabolic studies comparing calcitriol and calcipotriol were done in vivo while most studies analyzing proliferation or differentiation were done in vitro. The rapid degradation of calcipotriol after systemic administration has limited its oral use but made it an ideal drug for topical use.
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A different approach to create new vitamin D analogs that are effective in the topical treatment of hyperproliferative or inflammatory skin diseases is the goal to create new synthetic compounds that are metabolized in the skin and therefore exert only little systemic side effects. Vitamin D analogs, obtained by a combination of the 20-methyl modification with biologically interesting artificial side chain subunits [111] or 2bsubstituted calcitriols [112] are promising candidates for this approach. Another interesting approach to enhance the local concentration of calcitriol in the skin without obtaining systemic side effects is attempts to specifically inhibit the activity of vitamin D metabolizing enzymes, i.e. various hydroxylases (catabolic D3-OHases, i.e. 24hydroxylase for calcitriol) that are present in the skin and that are responsible for the catabolism of calcitriol [113]. It is known that various pharmacologically active compounds, including other steroidal hormones but also cytochrome P450 inhibitors such as ketoconazole, inhibit the activity of D3-OHases in the skin [114]. It may be possible to locally enhance the concentration of endogeneous calcitriol in the skin by the topical application of these compounds without obtaining systemic side effects. It can be speculated that the therapeutic effects of various antimycotic compounds including ketoconazole in the treatment of seborrheic dermatitis may at least in part be due to this mechanism. It is now known that VDR requires nuclear accessory proteins for efficient binding to vitamin D response elements in promoter regions of target genes thereby inducing VDR-mediated transactivation [115]. As a consequence, different vitamin D analogs may have (depending on their chemical structure) different affinities for the various homo- or heterodimers of VDR and nuclear cofactors including RXR-a [116]. The synthesis of new vitamin D analogs that activate different vitamin D signaling pathways may lead to the introduction of new therapeutics for the topical or oral treatment of various skin diseases. These new drugs may induce strong effects on target cell proliferation and differentiation in the skin or the immune system, but only marginal effects on calcium metabolism. Another approach to enhance the therapeutic effects of orally or topically administered calcitriol may be the combination with synergistic acting drugs. The recent discovery of different vitamin D signaling pathways that are determined and regulated by cofactors of VDR including RXR-a and their corresponding ligands suggests that 9-cis retinoic acid (RA) or all-trans RA may act synergistically with vitamin D analogs to induce VDR-mediated transactivation and regulate the transcriptional activity of distinct gene networks. Only a little is known about the effects of the combined application of vitamin D and vitamin A analogs under
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physiological or pathophysiological conditions in vivo [117]. This combination may selectively enhance or block different biological effects of vitamin D analogs that are mediated by different vitamin D signaling pathways.
CONCLUSION It can be speculated that innovative vitamin D analogs will introduce novel alternatives for the treatment of various skin disorders. If the final goal to create strong anti-proliferative and anti-inflammatory vitamin D analogs with only minor calcemic activity is reached, these new agents would herald a new era in dermatologic therapy, which possibly can be compared with the introduction of synthetic corticosteroids or retinoids. These new drugs that may activate selective vitamin D signaling pathways but may exert only negligible calcemic activity may also be effective in the systemic treatment of various malignant lymphomas of the skin, including lymphomas, squamous cell carcinoma, or basal cell carcinoma.
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G. Neef, G. Kirsch, K. Schwarz, H. Wiesinger, A. Menrad, M. Fa¨hnrich, et al., 20-Methyl vitamin D analogues, in: A.W. Norman, R. Bouillon, M. Thomasset (Eds.), Vitamin D. A Pluripotent Steroid Hormone: Structural Studies, Molecular Endocrinology and Clinical Applications, Walter de Gruyter, Berlin, 1994, pp. 97e98. B. Scho¨necker, M. Reichenba¨cher, S. Gliesing, R. Prousa, S. Wittmann, S. Breiter, et al., 2b-Substituted calcitriols and other A-ring substituted analogues e synthesis and biological results, in: Vitamin D. A Pluripotent Steroid Hormone: Structural Studies, Molecular Endocrinology and Clinical Applications, Walter de Gruyter, Berlin, 1994, pp. 99e100. I. Schuster, G. Herzig, G. Vorisek, A.W. Norman, R. Bouillon, M. Thomasset, Steroidal hormones as modulators of vitamin D metabolism in human keratinocytes, in: Vitamin D. A Pluripotent Steroid Hormone: Structural Studies, Molecular Endocrinology and Clinical Applications, Walter de Gruyter, Berlin, 1994, pp. 184e185. J. Zhao, S. Marcelis, B.K. Tan, A. Verstuaf, R. Boillon, Potentialisation of vitamin D (analogues) by cytochrome P-450 enzyme inhibitors is analog- and cell-type specific, in: A.W. Norman, R. Bouillon, M. Thomasset (Eds.), Vitamin D. A Pluripotent Steroid Hormone: Structural Studies, Molecular Endocrinology and Clinical Applications, Walter de Gruyter, Berlin, 1994, pp. 97e98. C. Carlberg, I. Bendik, A. Wyss, E. Meier, L.J. Sturzenbecker, J.F. Grippo, et al., Two nuclear signalling pathways for vitamin D, Nature 361 (1993) 657e660. M. Schra¨der, K.M. Mu¨ller, M. Becker-Andre, C. Carlberg, Response element selectivity for heterodimerization of vitmain D receptors with retinoic acid and retinoid X receptors, J. Mol. Endocrinol. 12 (1994) 327e333. D.M. Peehl, D. Feldman, The role of vitamin D and retinoids in controlling prostate cancer progression, Endocr. Relat. Cancer 10 (2) (2003) 131e140.
XI. IMMUNITY, INFLAMMATION, AND DISEASE
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C H A P T E R
98 The Role of Vitamin D in Type 2 Diabetes and Hypertension Anastassios G. Pittas, Bess Dawson-Hughes Tufts Medical Center, Boston, MA, USA
EPIDEMIOLOGY AND BURDEN OF TYPE 2 DIABETES AND HYPERTENSION Type 2 diabetes has become a significant global health care problem. In the USA alone, total prevalence is expected to more than double in the next few decades from 6% of the population in 2005 (16 million) to 12% in 2050 (48 million) [1]. From a worldwide perspective, the total number of people with diabetes is expected to rise from 171 million in 2000 to 366 million by 2030 [2]. Type 2 diabetes is associated with serious morbidity and increased mortality. It is the leading cause of blindness, kidney disease, heart disease and stroke. In the USA alone, each year up to 25 000 individuals lose their sight and as many as 28 000 initiate treatment for chronic kidney failure because of diabetes. People with diabetes are four times more likely to develop cardiovascular disease (coronary artery disease, peripheral vascular disease or stroke) compared to those without diabetes. Heart disease and stroke are the leading causes of death among people with diabetes. Beyond its devastating human toll, type 2 diabetes is also associated with increasing costs. In the USA alone, the total (direct and indirect) costs of diabetes were estimated at $132 billion in 2002 and are expected to increase to $192 billion by 2020 [3]. Hypertension is the most common and wellestablished risk factor for cardiovascular morbidity and mortality [4]. The prevalence of hypertension, defined as higher than 140/90 mm Hg, among US adults is estimated at approximately 30% [5,6]. Worldwide, more than a quarter of the adult population already has hypertension and the number is projected to increase to more than 30%, or 1.6 billion people, by 2025 [5,6]. Hypertension is even more prevalent among
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10098-8
patients with type 2 diabetes, affecting 60e90% of them depending on age and blood pressure cut-offs for defining hypertension [7]. Although therapies for type 2 diabetes and its complications have improved over the last few decades, the increasing burden of type 2 diabetes highlights the need for innovative approaches for the management and prevention of the disease. Epidemiologic data suggest that 9 out of 10 cases of type 2 diabetes could be attributed to modifiable habits and lifestyle [8]. In clinical trials, lifestyle changes aiming at weight loss are successful at reducing risk of type 2 diabetes [9e12]. However, long-term weight maintenance in the clinical setting has proved elusive. Moreover, even after successful weight loss, there is still significant residual risk (~40e50%). Similarly, despite the availability of a wide spectrum of effective well-tolerated pharmacologic agents for treatment of hypertension, less than one-third of patients with diagnosed hypertension are adequately treated, and the number of people with uncontrolled blood pressure is increasing [13,14]. Primary prevention of hypertension is hindered by the lack of easily identifiable causes and lack of effective comprehensive national and international prevention strategies. Therefore, identification of weight-independent and easily modifiable risk factors is urgently needed to prevent type 2 diabetes and hypertension and decrease patient and societal burden. Based on accumulating evidence from animal and human studies [15,16], as reviewed in this chapter, suboptimal vitamin D status has emerged as a probable risk factor for both type 2 diabetes and hypertension and vitamin D supplementation has been suggested as a potential intervention to prevent type 2 diabetes and hypertension.
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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98. THE ROLE OF VITAMIN D IN TYPE 2 DIABETES AND HYPERTENSION
BIOLOGIC PLAUSIBILITY OF AN ASSOCIATION BETWEEN VITAMIN D AND TYPE 2 DIABETES AND HYPERTENSION For glucose intolerance and type 2 diabetes to develop, impaired pancreatic beta cell function, insulin resistance and systemic inflammation are often present [17,18]. The pathogenesis of essential hypertension is multi-factorial, including increased adrenergic drive, alteration of the renineangiotensinealdosterone system (RAAS) and increased peripheral vascular resistance all of which may be heightened by an unfavorable lifestyle pattern (e.g., physical inactivity, high sodium intake) [5,19]. There is evidence that vitamin D influences many of these mechanisms, as described next.
Vitamin D and Pancreatic Beta Cell Function/Insulin Secretion There are several lines of evidence supporting a beneficial role for vitamin D in pancreatic beta cell function (Fig. 98.1). In in vitro and in vivo studies, vitamin D
deficiency impairs glucose-mediated insulin secretion from rat pancreatic beta cells [20e24], while vitamin D supplementation restores glucose-stimulated insulin secretion [20,23e27]. Vitamin D may have a direct effect on beta cell function mediated by binding of the circulating active form, 1,25(OH)2D, to the vitamin D receptor (VDR), which is expressed in pancreatic beta cells [28]. Furthermore, mice lacking a functional VDR show impaired insulin secretory response following a glucose load, attributed to a decrease in insulin synthesis resulting in a reduction in the amount of insulin stored in the beta cell [29]. Alternatively, activation of vitamin D also occurs within the pancreatic beta cell by the 25hydroxyvitamin D-1a-hydroxylase enzyme (CYP27B1), which is expressed in pancreatic beta cells [30]. Such mechanism allows for an important paracrine effect of circulating 25-hydroxyvitamin D (25(OH)D). An indirect effect of vitamin D metabolites on the pancreatic beta cell may be mediated via its regulation of extracellular calcium concentration and calcium flux through the beta cell [31]. Insulin secretion is a calciumdependent process [32], therefore, alterations in calcium flux can have an effect on insulin secretion [33e35].
FIGURE 98.1 Vitamin D and pancreatic beta cell function. Vitamin D can promote pancreatic beta cell function in many ways. The active form of vitamin D, 1,25(OH)2D, enters the beta cell from the circulation and interacts with the vitamin D receptor-retinoic acid x-receptor complex (VDR-RXR) to enhance the transcriptional activation of the insulin gene and increase the synthesis of insulin. Vitamin D may promote beta cell survival by modulating the generation (through inactivation of nuclear factor kappa B (NF-kB)) and effects of cytokines. The antiapoptotic effect of vitamin D may also be mediated by downregulating the Fas-related pathways (Fas/Fas-L). Activation of vitamin D also occurs intracellularly by 25-hydroxyvitamin D-1a-hydroxylase enzyme (CYP27B1), which is expressed in pancreatic beta cells. The effects of vitamin D may be mediated indirectly via extracellular calcium (Ca2þ), calcium flux through the beta cell and intracellular calcium (Ca2þ)i. Alterations in calcium flux can directly influence insulin secretion, which is a calcium-dependent process. Vitamin D also regulates calbindin, a cytosolic calcium-binding protein found in beta cells, which acts as a modulator of depolarization-stimulated insulin release via regulation of intracellular calcium. Calbindin may also protect against apoptotic cell death via its ability to buffer intracellular calcium.
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Vitamin D also regulates calbindin, a cytosolic calciumbinding protein found in many tissues including beta cells [28,36]. Calbidin is a modulator of depolarizationstimulated insulin release via regulation of intracellular calcium [37]. Finally, vitamin D may promote beta cell survival by modulating the generation (e.g., through inactivation of nuclear factor-kB (NF-kB)) and effects of cytokines [38,39]. In cross-sectional human studies, an association between the blood 25(OH)D concentration and insulin secretion has been reported in some [40,41] but not all studies [42].
Vitamin D and Insulin Sensitivity In peripheral insulin-target cells, active vitamin D metabolites may enhance insulin sensitivity in several ways (Fig. 98.2). 1,25(OH)2D appears to directly augment insulin sensitivity by stimulating the expression of insulin receptors [43e46]; 1,25(OH)2D enters insulin-responsive cells from the circulation and interacts with the VDR, activates the VDR-retinoic acid X-receptor (RXR) complex which, in turn, binds to a vitamin D response element found in the human
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insulin receptor gene promoter. The result is enhanced transcriptional activation of the insulin receptor gene, which increases the total insulin receptor number without altering receptor affinity. 1,25(OH)2D may also enhance insulin sensitivity by transcriptionally activating peroxisome proliferator-activated receptor delta (PPAR-d), a transcription factor implicated in the regulation of fatty acid metabolism in skeletal muscle and adipose tissue [47]. An indirect effect of 1,25(OH)2D on insulin sensitivity might also be exerted via its important and well-recognized role in regulating extracellular calcium concentration and flux through cell membranes. Calcium is known to be essential for insulin-mediated intracellular processes in insulin-responsive tissues such as skeletal muscle and adipose tissue [48,49], with a very narrow range of intracellular calcium needed for optimal insulin-mediated functions [50]. Changes in intracellular calcium in insulin target tissues may contribute to peripheral insulin resistance [50e57] via impaired insulin signal transduction [57,58] leading to decreased glucose transporter activity [57e59]. Hypovitaminosis D also leads to increased parathyroid hormone (PTH) concentration, which has been
FIGURE 98.2 Vitamin D and insulin action. In peripheral insulin-target cells, vitamin D may directly enhance insulin sensitivity in several ways. The active form of vitamin D, 1,25(OH)2D, enters the insulin-responsive cells from the circulation and interacts with the vitamin D receptoreretinoic acid x-receptor complex (RXReVDR). The complex binds to a vitamin D response element (VDRE), which is found in the human insulin receptor gene promoter, to enhance the transcriptional activation of the insulin receptor gene and increase the synthesis of insulin receptors (INS-R) which act to promote glucose uptake via the glucose transporter 4 (GLUT-4) receptor and/or by activating peroxisome proliferator-activated receptor delta (PPAR-d), a transcription factor implicated in the regulation of fatty acid metabolism in skeletal muscle and adipose tissue. The effects of vitamin D may be mediated indirectly via regulating extracellular calcium (Ca2þ), calcium flux through the cell and intracellular calcium (Ca2þ)i. Vitamin D may promote beta cell survival by modulating the generation (through downregulation) of nuclear factor-kB and effects of cytokines. Vitamin D may also affect insulin resistance indirectly through the renineangiotensin system (RAS) via the angiotensin 1 receptor (AT1R).
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98. THE ROLE OF VITAMIN D IN TYPE 2 DIABETES AND HYPERTENSION
associated with increased insulin resistance [60,61]. Vitamin D may also affect insulin resistance indirectly through the renineangiotensinealdosterone system (RAAS), as described below. Finally, vitamin D insufficiency is associated with increased fat infiltration in skeletal muscle, independent of body mass, which is thought to contribute to decreased insulin action [62]. In observational human studies, low vitamin D status (assessed by self-reported vitamin D intake or blood 25(OH)D concentration) has been associated with simple indices of insulin resistance, including measurements of fasting insulin and homeostasis model assessment (HOMA-IR) [40,41,63-70], but the association is not consistent [42,67,71].
Vitamin D and Systemic Inflammation Systemic inflammation, via an increase in proinflammatory cytokines, plays an important role in the pathogenesis of type 2 diabetes and metabolic syndrome, mostly by promoting insulin resistance; however, pancreatic beta cell function may also be affected via cytokine-induced apoptosis [17,72e74]. 1,25(OH)2D can lessen the effects of systemic inflammation on type 2 diabetes risk in several ways. 1,25(OH)2D may improve insulin sensitivity and protect against beta cell cytokine-induced apoptosis by directly modulating the expression and activity of cytokines [39, 75e77]. One such pathway may be through downregulation of NF-kB, which is a major transcription factor for TNF-a and other inflammatory mediators [78]. Another pathway that may, at least in part, mediate the anti-apoptotic effect of 1,25(OH)2D on beta cell is through counteracting cytokine-induced Fas expression [79]. Several other immune-modulating effects of 1,25(OH)2D (e.g., blockade of dendritic cell differentiation, inhibition of lymphocyte proliferation, inhibition of foam cell formation and cholesterol uptake in macrophages, enhanced regulatory T-lymphocyte development) [76,80] may provide additional pathways of protection against inflammation-induced type 2 diabetes risk. In observational human studies, low vitamin D status (assessed by self-reported vitamin D intake or blood 25(OH)D concentration) has been associated with elevated concentration of markers of systemic inflammation in some [70,81,82] but not all studies [63,83e85].
Vitamin D and the RenineAngiotensineAldosterone System (RAAS) Vitamin D may affect insulin resistance and hypertension indirectly through the RAAS. The role of RAAS in regulation of blood pressure and development of
hypertension is well recognized. Available antihypertensive agents targeting all phases of the system (direct renin inhibitors, angiotensin-converting enzyme inhibitors, angiotensin II-receptor blockers, aldosterone inhibitors) are well established in the clinical management of hypertension. These agents are also thought to have additional protective benefits on kidney and vascular disease beyond what might be predicted based on their blood pressure lowering effect. Angiotensin II is also thought to contribute to the development of insulin resistance by inhibiting the action of insulin in vascular and skeletal muscle tissue, leading to impaired glucose uptake, via a number of mechanisms including activation of NF-kB [86,87] . Based on recent studies in the mouse, RAAS appears to be modulated by vitamin D. Renin expression and angiotensin II production were increased several fold in vitamin D receptor-null mice, which was accompanied by elevated blood pressure, cardiac hypertrophy and other renal abnormalities while administration of 1,25(OH)2D suppressed renin biosynthesis [88e90]. Vitamin D, therefore, by acting as a negative endocrine regulation of RAAS, provides another potential mechanism linking vitamin D to a decreased risk of both type 2 diabetes and hypertension.
Other Potential Mechanisms for Vitamin D Action in Hypertension In addition to regulation of the renineangiotensine aldosterone system, 1,25(OH)2D may also have a direct effect on blood pressure through its inhibitory effects on vascular smooth muscle cell proliferation [91] mediated by binding to the VDR, which is expressed in vascular smooth muscle cells [92]. Alternatively, activation of 1,25(OH)2D also occurs in the vascular smooth muscle cell via expression of the CYP27B1 gene product, which is expressed in these cells [93]. 1,25(OH)2D also improves endothelial cell-dependent vasodilation [94,95]. Finally, 1,25(OH)2D may also have an indirect effect on blood pressure regulation through its role in calcium homeostasis [96].
EVIDENCE FROM HUMAN STUDIES FOR A LINK BETWEEN VITAMIN D AND TYPE 2 DIABETES Seasonal and Geographic Studies of Vitamin D and Type 2 Diabetes A potential role of vitamin D in type 2 diabetes is suggested by a reported seasonal variation in glycemic control in patients with type 2 diabetes, being worse in the winter [97,98] when hypovitaminosis D is highly
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prevalent due to decreased exposure to solar UVB light. A strong geographic variability has been described in relation to type 1 diabetes, with incidence rates approaching zero in regions worldwide with high UVB irradiance [99e101]; however, a similar geographic association between decreasing UVB exposure and prevalent type 2 diabetes has not been reported.
CaseeControl and Cross-sectional Studies of Vitamin D and Type 2 Diabetes Additional supportive evidence for a role of vitamin D in type 2 diabetes comes from a large number of casee control studies, the first of which was published in 1979 [102]. These caseecontrol studies have included small numbers of participants and most [67,103-113], but not all [67,102,109,114], have reported that patients with type 2 diabetes or glucose intolerance have lower blood 25(OH)D concentration compared to controls without diabetes. Several large cross-sectional studies have examined the association between vitamin D status (assessed by the blood 25(OH)D concentration or self-reported vitamin D intake) and prevalence of glucose intolerance, type 2 diabetes or metabolic syndrome. The latter describes the clustering of several cardiometabolic risk factors (abdominal obesity, hypertension, dyslipidemia defined as high triglycerides and low HDL-cholesterol, and hyperglycemia) and it is closely linked to type 2 diabetes [115]. Most of these studies have reported inverse associations between vitamin D status and glucose intolerance and type 2 diabetes [40,61,64,68,107,116e121], metabolic syndrome [40,117,121e123] or markers of insulin resistance [41,68] while others failed to show any associations [40,61,64,84,116,124e129] . In a large cross-sectional study with data from the USbased National Health Nutrition Examination Survey (NHANES), serum 25(OH)D concentration was inversely associated with prevalence of diabetes in a dose-dependent pattern in non-Hispanic whites and Mexican-Americans, after multivariate adjustment, including BMI [64]. In this study, there was no association in non-Hispanic blacks despite lower 25(OH)D concentration found in this racial group, which may be explained by the observation that non-whites exhibit a different vitamin D, calcium and PTH homeostasis compared to whites [130]. However, a subsequent analysis from NHANES did not find an interaction between blood 25(OH)D concentration and race/ethnicity on glycemic outcomes [68]. More recent studies using NHANES data have repeatedly confirmed the inverse association between 25(OH)D and glycemia [68,117,118,120,121], which has also been reported in other large cohorts from the USA [61], Europe [123], and China [69].
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Vitamin D status has also been inversely associated with the prevalence of the metabolic syndrome in some [40,66,67,69,117,122e124,127] but not all studies [61,67,125,131]. In the NHANES database, serum 25(OH)D concentration (after multivariate adjustment) was inversely associated with prevalent metabolic syndrome among both genders and all three major racial or ethnic groups [117,127]. A similar inverse association has also been reported in two cohorts from the UK [66,123] and in a cohort from China [69]. The clinical components of the metabolic syndrome that have consistently been independently associated with low 25(OH)D are abdominal obesity and hyperglycemia; therefore, the association between 25(OH)D status and metabolic syndrome reported in these cross-sectional studies may simply reflect the well-established inverse association between 25(OH)D status and body weight or fatness [64,132e134].
Longitudinal Observational Cohort Studies of Vitamin D and Type 2 Diabetes To overcome the inability from cross-sectional studies to establish the direction of the association between vitamin D (25(OH)D) status and type 2 diabetes-related parameters, longitudinal observational studies have been conducted where vitamin D status is assessed at baseline prior to the development of the outcome of interest, incident type 2 diabetes. Five studies with data from five different cohorts have reported on the prospective association between 25(OH)D status and risk of incident type 2 diabetes [124,135e138] (Table 98.1). The studies included 99 435 participants (~98% white) who were followed from 7 to 20 years for incident type 2 diabetes. Two studies assessed vitamin D status by self-reported total vitamin D intake [124,135]; one study calculated predicted 25(OH)D score [137] and two studies measured blood 25(OH)D concentration [136,138]. Ascertainment of type 2 diabetes was by validated self-report [124,135,138], by combination of selfreport and laboratory measurement [137] or by national registry-based data [136]. Four studies reported multivariable adjusted results [135e138]; one adjusted for age only [124]. In the Women’s Health Study, an intake of 511 IU/day or more of vitamin D was associated with 27% lower risk of developing type 2 diabetes compared with an intake of 159 IU/day or less (3.4 versus 4.6% of the cohort developed type 2 diabetes, respectively) [124]. However, this analysis is limited because it did not adjust for any risk factors of type 2 diabetes other than age. In the Nurses’ Health Study, after multivariate adjustment for age, BMI, and non-dietary covariates, women who reported consumption of more than 800 IU/day of vitamin D had a 23% lower risk for developing incident
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TABLE 98.1
Observational Longitudinal Cohort Studies of Vitamin D Status (Plasma or Serum 25(OH)D Concentration, Predicted 25(OH)D Concentration or Self-reported Vitamin D Intake) and Incident Type 2 Diabetes or Hypertension
Male, %
Mean baseline age (range), y
White, %
n*/N (incidence)
Vitamin D measure; comparisony
Mean follow-up, y (starte end)
Outcome (ascertainment method)
Adjustments
Study qualityz
TYPE 2 DIABETES Liu et al., 2005 [124] Women’s Health Study (US)
0
52 (45e75)
95
805/10 066 (8.0%)
Vitamin D intake (total); 511 vs. 159 IU/d
9 (ND)
73 (0.54, 0.99) x p ¼ 0.02
Type 2 diabetes (validated selfreport)
Age
Fair
Pittas et al., 2006 [135] Nurses’ Health Study (US)
0
46 (30e55)
98
4843/83 779 (5.8%)
Vitamin D intake (total); >800 vs. 200 IU/d
20 (1980e2000)
0.87 (0.69, 1.09) p ¼ 0.67
Type 2 diabetes (validated selfreport)
Age, BMI, exercise, residence, othersk
Fair
Knekt et al., 2008 [136] Finnish Mobile Clinic Health Examination Survey (Finland)
100
ND (40e74)
100
105/1628 (6.4%); nested caseecontrol study with 206 control participants
25(OH)D concentration; 30 vs. 10 ng/ ml (means)
22 (1973e1994)
0.49 (0.15, 1.64) p ¼ 0.06
Type 2 diabetes (medicationtreated, registry-based)
Age, BMI, exercise, season, others{
Good
0
ND (40e74)
100
125/1699 (7.4%); nested caseecontrol study with 246 control participants
25(OH)D concentration; 25 vs. 9 ng/ml (means)
0.91 (0.37, 2.23) p ¼ 0.66
Age, BMI, exercise, season, others{
98. THE ROLE OF VITAMIN D IN TYPE 2 DIABETES AND HYPERTENSION
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
Study, year (reference) cohort (country)
Results, adjusted RR, OR, or HR (95% CI) p for trend
Knekt et al., 2008 [136] Mini-Finland Health Survey (Finland)
Good
53 (40e69)
100
83/1948 (4.3%); nested caseecontrol study with 245 control participants
25(OH)D concentration; 31 vs. 9 ng/ml (means)
0
ND (40e69)
100
99/2228 (4.4%); nested caseecontrol study with 289 control participants
25(OH)D concentration; 25 vs. 8 ng/ml (means)
Liu et al., 2010 [137] Framingham Offspring Study (US)
54
60
~100
133/2956 (4.4%)
Predicted 25 (OH)D score; 22 vs. 17 ng/ ml (median)
7 (1991e2001)
0.60 (0.37, 0.97) p ¼ 0.03
Type 2 diabetes (medicationtreated, laboratorybased)
Age, sex, waist circumference**
Fair
Pittas et al., 2006 [138] Nurses Health Study (US)
0
46 (30e55)
98
608/32 826 (1.8%); nested caseecontrol study with 569 control participants
25(OH)D concentration; 33 vs. 14 ng/ ml (median)
14 (1990e2004)
0.52 (0.33, 0.83) p ¼ 0.008
Type 2 diabetes (validated selfreport)
Age, BMI, exercise, season, race, othersyy
Good
Forman et al., 2007 [173] Health Professionals Follow-up Study (US)
100
65 (40e75)
95
133/613 (22%)
25(OH)D concentration; <15 vs. 30 ng/ml
8 (1993e2001)
3.53 (1.02, 12.3) ND
Hypertension (validated selfreport without BP measurement)
Age, BMI, exercise, race, season
Fair
Forman et al., 2007 [173] Nurses Health Study 1 (US)
0
57 (30e55)
95
274/1198 (23%)
25(OH)D concentration; <15 vs. 30 ng/ml
8 (1989e1997)
1.70 (0.92, 3.16) ND
Hypertension (validated selfreport without BP measurement)
Age, BMI, exercise, season, race, postmenopausal status
Fair
17 (1978e1994)
0.17 (0.05, 0.52) p < 0.001
Type 2 diabetes (medicationtreated, registry-based)
1.45 (0.58, 3.62) p ¼ 0.83
Age, BMI, exercise, season, others{
Age, BMI, exercise, season, others{
HYPERTENSION
EVIDENCE FROM HUMAN STUDIES FOR A LINK BETWEEN VITAMIN D AND TYPE 2 DIABETES
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
100
(Continued)
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1914 TABLE 98.1 Observational Longitudinal Cohort Studies of Vitamin D Status (Plasma or Serum 25(OH)D Concentration, Predicted 25(OH)D Concentration or Self-reported Vitamin D Intake) and Incident Type 2 Diabetes or Hypertensiondcont’d
Male, %
Mean baseline age (range), y
White, %
n*/N (incidence)
Vitamin D measure; comparisony
Mean follow-up, y (starte end)
Outcome (ascertainment method)
Adjustments
Study qualityz
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
Forman et al., 2008 [172] Nurses Health Study 2 (US)
0
43 (32e52)
~100
742/ (ND); nested caseecontrol study with 742 control participants
25(OH)D concentration; <21 vs. 32 ng/ml
7 (1997e2005)
1.66 (1.11, 2.48) p ¼ 0.01
Hypertension (validated selfreport without BP measurement)
Age, BMI, exercise, season, race, otherszz
Fair
Wang et al., 2008 [174] Women’s Healthy Study (US)
0
54 (45)
95
8526/28 886 (30%)
Vitamin D intake (dietary) 110 vs. 381 IU/d (median)
10 (ND)
0.95 (0.8, 1.02) p ¼ 0.02
Hypertension (validated selfreport without BP measurement)
Age, BMI, exercise, race, othersxx
Fair
Vitamin D intake (from supplements) 0 vs. 800 IU/d (median)
10 (ND)
1.09 (0.93, 1.27) p ¼ 0.27
Hypertension (validated selfreport without BP measurement)
Age, BMI, exercise, race, othersxx
* Number of cases if nested caseecontrol study. y Highest/lowest risk category versus reference category. z Study quality is determined based on a three-category grading system. Good-quality studies adhere most closely to the commonly held concepts of high quality including clear descriptions of the population and setting; unbiased assessments of vitamin D status and outcomes; appropriate statistical analysis including multivariable analysis adjusting for age, race, weight, and sun exposure and intention-to-treat analysis; no obvious reporting omissions or errors; and <20% dropouts. Fair quality studies have some deficiencies in the above criteria, but these are unlikely to cause major bias. Studies that used vitamin D intake or predicted 25(OH)D score as the predictor are rated fair. Poor-quality studies have major deficiencies in design, analyses or reporting, such that major bias could not be excluded. x Estimated from reported data. k Family history of diabetes, hypertension. calcium intake, smoking, alcohol, coffee, other diet. { Smoking, education, medications. ** Family history of diabetes, hypertension, low HDL-cholesterol, high triglycerides, impaired fasting glucose, diet. yy Fasting status, latitude, hypercholesterolemia, hypertension, family history of diabetes, smoking, physical activity, alcohol, multivitamin use, diet. zz Family history of hypertension, menstrual cycle, oral contraceptive use, creatinine, parathyroid hormone, calcium, phosphate, uric acid. xx Postmenopause, diabetes, smoking, hypercholesterolemia, energy intake, alcohol, diet (other), multivitamin use, randomized treatment 25(OH)D, plasma or serum 25-hydroxyvitamin D; BMI, body mass index; BP, blood pressure; HR, hazard ratio; IU, international units; ND, no data; OR, odds ratio; RR, relative risk. To convert 25(OH)D concentration from ng/ml to nmol/l multiply by 2.459.
98. THE ROLE OF VITAMIN D IN TYPE 2 DIABETES AND HYPERTENSION
Study, year (reference) cohort (country)
Results, adjusted RR, OR, or HR (95% CI) p for trend
EVIDENCE FROM HUMAN STUDIES FOR A LINK BETWEEN VITAMIN D AND TYPE 2 DIABETES
type 2 diabetes compared to women who reported consumption of less than 200 IU/day [135]. The association, however, was attenuated and became non-statistically significant after adjusting for dietary factors. The dietary variables solely responsible for attenuation of the results were magnesium and calcium, which share dietary sources with vitamin D. In the same study, women who reported the highest calcium (>1200 mg/day) and vitamin D (>800 IU/day) intake (1.3% of the cohort) had a statistically significant 33% lower risk of incident type 2 diabetes compared to women with the lowest calcium (<600 mg/day) and vitamin D (<400 IU/day) intakes, which highlights a potentially important role for calcium intake in type 2 diabetes risk. In the Framingham Offspring Study (men and women), investigators used a subsample of the cohort to develop a regression model to predict blood 25(OH)D concentrations from age, sex, body mass index, month of blood sampling, total vitamin D intake, smoking status, and total energy intake [137]. Using this model, a predicted 25(OH)D score was calculated for each non-diabetic participant and the association between the predicted 25(OH)D score and incidence of type 2 diabetes was assessed during a mean follow-up period of 6 years. Compared to participants in the lowest tertile of the predicted 25(OH)D score, those in the highest tertile had a 40% lower incidence of type 2 diabetes after multivariate adjustment. There are two longitudinal observational studies that have reported the association between measured blood 25(OH)D concentration and incident type 2 diabetes [136,138], both caseecontrol studies nested within larger cohorts (Table 98.1). In these studies, after a specified period of follow-up, blood samples from cases (i.e., participants who develop type 2 diabetes) and matched controls (i.e., participants who did not develop diabetes during the follow-up period) are retrieved from stored samples and blood 25(OH)D is measured at a time when all participants were free of type 2 diabetes. Analyses then compare the 25(OH)D concentration between cases and controls. The study by Knekt et al., which pooled data from two cohorts in Finland (total of 7503 available participants) included 412 cases who developed type 2 diabetes during the follow-up period and 986 matched controls [136]. After multivariate adjustment including BMI, participants who were in the highest quartile of 25(OH)D at baseline (mean 25(OH)D 27.6 ng/ml) compared to those in the lowest quartile (mean 25(OH)D 8.9 ng/ml) had a 40% lower risk of developing incident type 2 diabetes, which was entirely attributed to a lower risk among men only. Men in the highest quartile (mean 25(OH)D >30 ng/ml) had a 72% lower risk of developing type 2 diabetes compared to men in the lowest quartile (mean 25(OH)D <10 ng/ml) after multivariate adjustment,
1915
while there was no significant association among women. The second nested caseecontrol study by Pittas et al. was conducted in the Nurses’ Health Study among 608 women with newly diagnosed type 2 diabetes and 559 controls [138]. After adjusting for matching factors and diabetes risk factors, including BMI, higher levels of plasma 25(OH)D were associated with a lower risk for type 2 diabetes. The odds ratio for incident type 2 diabetes in the top (median 25(OH)D 33.4 ng/ml) versus the bottom (median 25(OH)D 14.4 ng/ml) quartile was 0.52 (95% confidence interval, 0.33, 0.83). The associations were consistent across subgroups of baseline BMI, age, and calcium intake although the association appeared to be stronger among overweight/obese versus normal weight women (odds ratio 0.46 versus 0.63, respectively). In the same study, spline regression models showed no apparent threshold and no deviation from linearity for the relation between 25(OH)D and risk of incident type 2 diabetes, although the shape of the figure suggested a stronger decrease in risk within the higher range of plasma 25(OH)D concentration, above 30e35 ng/ml (Fig. 98.3). The inconsistent results among women in the Finish and US cohorts may be explained by the higher baseline 25(OH)D concentration in women in the Nurses’ Health Study compared to the Finnish cohort (23 versus 15 ng/ml, respectively), which suggests that a potential threshold for 25(OH)D concentration in relation to type 2 diabetes risk exists, above
FIGURE 98.3 Spline regression models examining the possible
non-linear relation between plasma 25(OH)D concentration and incident type 2 diabetes, after multivariate adjustment (see Table 98.1). Women with extremely low or high plasma 25(OH)D concentrations (>1st or 99th percentile) were excluded from the analyses. Solid lines represent odds ratios, and dotted lines represent 95% CI. To convert 25(OH)D concentration from ng/ml to nmol/l multiply by 2.459.
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
1916
98. THE ROLE OF VITAMIN D IN TYPE 2 DIABETES AND HYPERTENSION
which risk of type 2 diabetes declines. Another longitudinal observational study has also reported inverse associations between baseline 25(OH)D concentration and future glycemia and insulin resistance, but it did not report results on incident diabetes [66]. In meta-analysis (unpublished results), after combining data from all published longitudinal observational studies, there was a statistically significant favorable association between 25(OH)D concentration and incident type 2 diabetes (risk ratio 0.60; 95% CI 0.39e0.92 for the highest versus lowest 25(OH)D concentration; (Fig. 98.4).
Effect of Vitamin D Supplementation on Type 2 Diabetes Over the last 2e3 years, several trials have reported the effect of vitamin D (ergocalciferol (D2) or cholecalciferol (D3)) supplementation (with or without calcium) on glycemia [16,139e148] or incident diabetes by selfreport [142,144] (Table 98.2). Seven trials have included participants with normal glucose tolerance [139e142,144e147,149] and three trials had participants with stable type 2 diabetes [143,148,150]. Study duration varied from 2 months to 9 years and doses ranged from 400 to 8571 IU/day. In three studies, vitamin D3 was combined with calcium [140,142,147]. Only the Women’s Health Initiative and the RECORD trials were rated good quality [142]. Among seven trials of participants with normal glucose tolerance at baseline, vitamin D supplementation had no effect on glycemic measures [139e142,144e147,149] or incident diabetes [142,144]. However, five of the studies were designed for non-glycemic outcomes and the analyses on vitamin D and type 2 diabetes were post-hoc [139,140,142,144,147] and all trials, with the exception of the Women’s Health Initiative trial [142] and the RECORD trial [144], were underpowered for glycemic outcomes. In several trials,
including the two largest ones [142,144], adherence with supplementation was suboptimal, which may have limit drawing any conclusions. For example, in a post-hoc analysis of the RECORD study, a community-based effectiveness trial designed for bone outcomes [144], supplementation with 800 IU/day of vitamin D3 (given in a 2 2 factorial design with calcium carbonate) did not change risk of self-reported type 2 diabetes; however, among study participants who were highly compliant with supplementation, there was a notable trend towards reduction in type 2 diabetes risk with vitamin D3 (odds ratio 0.68; 95% CI 0.40, 1.16), which highlights the importance of efficacy versus effectiveness community-based trials. In efficacy trials, internal validity (e.g., did participants follow the intervention?, is there any proof that they did so?, is outcome measured in an objective way?) takes center stage and these studies are better designs at evaluating causality. In effectiveness trials, external validity (e.g., how does the intervention do in “real-life” situations) is emphasized and these studies are better at informing policy changes. Finally, several trials provided supplementation as large infrequent doses [143,147e149,151], a commonly used clinical approach whose benefit has been questioned lately in the setting of studies showing increased risk of adverse outcomes with large infrequent doses of vitamin D [152,153]. There are three trials where participants with established type 2 diabetes were randomized to high infrequent doses of vitamin D2 (100 000 IU given once [143]) or vitamin D3 (40 000 IU/week for 26 weeks [150]; 100 000 or 200 000 IU/week for 16 weeks [148]) (Table 98.2). In these trials, there was no change in fasting plasma glucose [148,150], hemaglobin A1c [143,148,150] or insulin resistance [150] with vitamin D supplementation after a follow-up period of 8e26 weeks. However, these studies were underpowered (only 16e20 participants per arm) to test the hypothesis that vitamin D may benefit patients with type 2 diabetes; FIGURE 98.4 Meta-analysis of longitudinal observational studies reporting on the association between 25(OH)D and incident type 2 diabetes.
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
TABLE 98.2
Study, first author, year (country)
Randomized Controlled Trials of the Effect of Vitamin D Supplementation (With or Without Calcium) on Glucose Tolerance or Hypertension
Men, %
Mean baseline age, (range), y
Participants
Mean baseline 25(OH)D concentration, ng/ml
Interventions (number of participants)
Study duration
Effect of vitamin D vs. placebo (p value)
Study quality
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
0
ND (45e54)
Postmenopausal, healthy
ND
D3, 2000 IU/d (n ¼ 25) vs. placebo (n ¼ 103). All received calcium, 500 mg/d
2y
FPG change, 2.16 vs. 2.34 mg/dl (p ¼ 0.97)
Fair
Pittas et al., 2007 [140] (USA)
38
71 (65)
Normal fasting glucose
30
D3, 700 IU/d plus calcium citrate, 500 mg/d (n ¼ 108) vs. placebo (n ¼ 114)
3y
FPG change, 2.70 vs. 2.16 mg/dl (p ¼ 0.55)
Fair
52
71 (ND)
Impaired fasting glucose
30
D3, 700 IU/d plus calcium citrate 500 mg/d (n ¼ 45) vs. placebo (n ¼ 47)
3y
FPG change, 0.36 vs. 6.13 mg/dl (p ¼ 0.042)
0
62 (50e79)
Postmenopausal without diabetes
<32
D3, 400 IU/d plus calcium carbonate 1000 mg/d (n ¼ 16 999) vs. placebo (n ¼ 16 952)
7y
Incidence of diabetes (selfreported), HR 1.01 (0.94 to 1.10) (p ¼ 0.95)
0
ND (50e79)
Normal fasting glucose
D3, 400 IU/d þ calcium carbonate 1000 mg/d (n ¼ 866) vs. placebo (n ¼ 771)
6y
FPG change, 3.80 vs. 4.61 mg/dl (p ¼ 0.32)
0
ND (50e79)
Impaired fasting glucose N ¼ 1457
D3, 400 IU/d þ calcium carbonate 1000 mg/d (n ¼ 718) vs. placebo (n ¼ 739)
6y
FPG change, 3.93 vs. 4.69 mg/dl (p ¼ 0.79)
Sugden et al., 2008 [143] (UK)
53
64 (ND)
Stable type 2 diabetes and 25 (OH)D < 20 ng/ ml
15
D2, 100 000 IU once (equivalent to 1785 IU/d) (n ¼ 17) vs. placebo (n ¼ 17)
8 wk
Hemoglobin A1c change, 0.01% vs. 0.05% (p ¼ 0.74)
Fair
Avenell et al., 2009 [144] (UK)
15
77 (70)
History of fracture
ND
D3, 800 IU/d (n ¼ 2649) vs. placebo (n ¼ 2643) (2 2 factorial design with calcium carbonate 1000 mg/d)
2e5 y
Incidence of diabetes (selfreported), HR 1.11 (0.77 to 1.62) (p ¼ 0.57)
Good
Zittermann et al., 2009 [145] (Germany)
33
48 (18e70)
Healthy, BMI > 27 kg/m2
12
D3, 3332 IU/d (n ¼ 100) vs. placebo (n ¼ 100). All received weight reduction advice for 24 wk
1y
Hemoglobin A1c change, 0.25% vs. 0.25% (p ¼ 0.96) FPG change, 3.8 vs. 4.9 mg/dl (p ¼ 0.39)
Good
De Boer et al., 2008 [142] (USA)
Good
(Continued)
1917
Nilas and Christiansen, 1984 [139] (Denmark)
EVIDENCE FROM HUMAN STUDIES FOR A LINK BETWEEN VITAMIN D AND TYPE 2 DIABETES
TYPE 2 DIABETES
1918
TABLE 98.2
Randomized Controlled Trials of the Effect of Vitamin D Supplementation (With or Without Calcium) on Glucose Tolerance or Hypertensiondcont’d
Men, %
Participants
Mean baseline 25(OH)D concentration, ng/ml
Interventions (number of participants)
Study duration
Effect of vitamin D vs. placebo (p value)
Study quality
Jorde and Figenschau 2009 [150] (Norway)
50
56 (21e75)
Stable type 2 diabetes
24
D3, 40 000 IU/wk (equivalent to 5714 IU/d) (n ¼ 16) vs. placebo (n ¼ 16)
26 wk
Hemoglobin A1c change, 0.2% vs. 0.2% (p ¼ 0.90) FPG change, 3.6 vs. 7.2 mg/dl (p ¼ 0.43)
Fair
Von Hurst et al., 2010 [146] (New Zealand)
0
42 (23e68)
Insulin resistance without diabetes and 25(OH)D < 20 ng/ml
8
D3, 4000 IU/d (n ¼ 42) vs. placebo (n ¼ 39)
26 wk
FPG change, 1.8 vs. 1.8 mg/dl (p ¼ 0.82)
Fair
Jorde et al., 2010 [147] (Norway)
36
ND (21e70)
Overweight/ obese without diabetes
23
D3, 40 000 IU/wk (equivalent to 5714 IU/d) (n ¼ 150) vs. D3, 20 000 IU/wk (equivalent to 2857 IU/d) (n ¼ 139) vs. placebo (n ¼ 149). All received calcium 500 mg/d
1y
Hemoglobin A1c change, 0.09% vs. 0.11% vs. 0.09% (p ¼ NS) FPG change, 0.4 vs. 1.4 vs. 1.4 mg/dl (p ¼ NS) 2hPG change, 2.7 vs. 6.5 vs. 0.4 mg/dl (p ¼ NS)
Fair
Witham et al., 2010 [148] (UK)
ND
65 (ND)
Type 2 diabetes and 25(OH)D < 40 ng/ml
18
D3, 100 000 IU orally once (equivalent to 892 IU/d) (n ¼ 19) vs. D3, 200 000 IU orally once (equivalent to 1785 IU/d) (n ¼ 20) vs. placebo (n ¼ 22)
16 wk
Hemoglobin A1c change, “no change” (data ND) FPG change, “no change” (data ND)
Fair
Scragg et al., 1995 [175] (UK)
46
70 (63e76)
Healthy
13
D3, 100 000 IU once (equivalent to 2857 IU/d) (n ¼ 95) vs. placebo (n ¼ 94)
5 wk
SBP change, 5 vs. 5 mm Hg DBP change, 1 vs. 1 mm Hg
Good
Krause et el., 1998 [162] (ND)
66
48 (26e66)
Hypertension
23
Whole body ultraviolet B irradiation, 3/week (n ¼ 9) vs. whole body ultraviolet A irradiation (n ¼ 9)
6 wk
SBP change, 6 vs. 0 mm Hg (p < 0.001) DBP change, 6 vs. 2 mm Hg (p < 0.001)
Poor
HYPERTENSION
98. THE ROLE OF VITAMIN D IN TYPE 2 DIABETES AND HYPERTENSION
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
Study, first author, year (country)
Mean baseline age, (range), y
0
74 (70)
Healthy and 25 (OH)D < 20 ng/ ml
10
D3, 800 IU/d (n ¼ 74) vs. placebo (n ¼ 74); all received calcium carbonate 1200 mg/d
8 wk
SBP change, 13.1 [ND] vs. 5.7 [ND] mm Hg (p ¼ 0.02) DBP change, 7.2 vs. 6.9 mm Hg (p ¼ 0.1)
Fair
Schleithoff et al., 2006 [177] (Germany)
83
~55 (NR)
Congestive heart failure
15
D3, 2000 IU/d orally (n ¼ 17) vs. placebo (n ¼ 17)
36 wk
SBP change, 3 vs. 4 mm Hg; (p ¼ NS) SBP change, 3 vs. 2 mm Hg; (p ¼ 0.08)
Fair
Major et al., 2007 [178] (Canada)
0
43 (NR)
Overweight
ND
D3, 400 IU/d plus calcium carbonate 1200 mg/d (n ¼ 30) vs. placebo (n ¼ 33); all participants followed energy-restricted diet
15 wk
SBP change, 4.1 vs. 1.6 mm Hg (p ¼ 0.18) SBP change, 3 vs. 3 mm Hg (p ¼ 1.0)
Poor
Sugden et al., 2008 [143] (UK)
53
64 (ND)
Stable type 2 diabetes and 25 (OH)D < 20 ng/ ml
15
D2, 100 000 IU orally once (equivalent to 1785 IU/d) (n ¼ 17) vs. placebo (n ¼ 17)
8 wk
SBP change, 7.3 vs. 6.6 mm Hg (p ¼ 0.001) SBP change, 2.2 vs. 2.3 mm Hg (p ¼ 0.08)
Fair
Margolis et al., 2008 [179] (USA)
0
ND (50e79)
Postmenopausal women without hypertension
ND
D3, 400 IU/d plus calcium carbonate 1000 mg/d (n ¼ 8597) vs. placebo (n ¼ 8525)
7y
Incidence of hypertension (selfreported), HR 1.01 (0.96e1.06)
Good
0
ND (50e79)
<32
D3 , 400 IU/d plus calcium carbonate 1000 mg/d (n ¼ 18 176) vs. placebo (n ¼ 18 106)
7y
SBP change, about 1 mm Hg in both arms DBP change, about 4 mmHg in both arms
Nagpal et al., 2009 [151] (India)
100
>35
Central obesity
15
D3, 120 000 IU orally three times (equivalent to 8571 IU/d) (n ¼ 35) vs. placebo (n ¼ 36)
6 wk
SBP change, 0.6 vs. 3.35 mm Hg (p ¼ 0.058) DBP change, 0.43 vs. 1.26 mm Hg (p ¼ 0.305)
Fair
Zittermann et al., 2009 [145] (Germany)
33
48 (18e70)
Healthy, BMI > 27 kg/m2
12
D3, 3332 IU daily (n ¼ 100) vs. placebo (n ¼ 100); all received weight reduction advice for 24 wk
1y
SBP change, 4 vs. 3 mm Hg (p ¼ 0.66) DBP change, 3 vs. 3 mm Hg (p ¼ 0.95)
Good
Jorde and Figenschau 2009 [150] (Norway)
50
56 (21e75)
Stable type 2 diabetes
24
D3 40 000 IU/wk (equivalent to 5714 IU/d) (n ¼ 16) vs. placebo (n ¼ 16)
26 wk
SBP change, 1.3 vs. 3.6 mm Hg (p ¼ 0.15) DBP change, 1.6 vs. 3.2 mm Hg (p ¼ 0.25)
Fair
EVIDENCE FROM HUMAN STUDIES FOR A LINK BETWEEN VITAMIN D AND TYPE 2 DIABETES
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
Pfeifer et al., 2001 [176] (Germany)
(Continued)
1919
1920
TABLE 98.2
Men, %
Mean baseline age, (range), y
Participants
Mean baseline 25(OH)D concentration, ng/ml
Interventions (number of participants)
Study duration
Effect of vitamin D vs. placebo (p value)
Study quality
Jorde et al., 2010 [147] (Norway)
50
56 (21e70)
Overweight/ obese without diabetes
23
D3, 40 000 IU/wk (equivalent to 5714 IU/d) (n ¼ 150) vs. D3, 20 000 IU/wk (equivalent to 2857 IU/d) (n ¼ 139) vs. placebo (n ¼ 149). All received calcium 500 mg/d
1y
SBP change, 1.2 vs. 3.5 vs. 1.1 mm Hg (p ¼ NS) DBP change, 1.0 vs.1.0 vs. 0.2 mm Hg (p ¼ NS)
Fair
Witham et al., 2010 [148] (UK)
ND
65 (ND)
Type 2 diabetes and 25(OH)D < 40 ng/ml
18
D3, 100 000 IU orally once (equivalent to 892 IU/d) (n ¼ 19) vs. D3, 200 000 IU orally once (equivalent to 1785 IU/d) (n ¼ 20) vs. placebo (n ¼ 22)
16 wk
SBP change, “no change” (data ND) (p ¼ NS) DBP change, “no change” (data ND) (p ¼ NS)
Fair
25(OH)D, plasma or serum 25-hydroxyvitamin D; 2hPG, plasma glucose 2 hours after 75 gram glucose load; D3, cholecalciferol; D2, ergocalciferol; FPG, fasting plasma glucose; HR, hazard ratio; ND, no data; NS, not significant; DBP, diastolic blood pressure; SBP, systolic blood pressure. To convert 25(OH)D concentration from ng/ml to nmol/l multiply by 2.459; to convert FPG from mg/dl to mmol/l, multiply by 0.0555.
98. THE ROLE OF VITAMIN D IN TYPE 2 DIABETES AND HYPERTENSION
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
Study, first author, year (country)
Randomized Controlled Trials of the Effect of Vitamin D Supplementation (With or Without Calcium) on Glucose Tolerance or Hypertensiondcont’d
EVIDENCE FROM HUMAN STUDIES FOR A LINK BETWEEN VITAMIN D AND HYPERTENSION
therefore firm conclusions cannot be drawn. Given the multiple factors than can influence glycemia in patients with diabetes, adequately powered study that controls and adjusts for potential confounders between active and placebo arms (e.g., concurrent hypoglycemic medications, lifestyle habits) is needed to test the effectiveness of vitamin D supplementation among those with established type 2 diabetes. All three trials also used large infrequent doses of vitamin D, whose benefit has been questioned [152,153]. Among five trials in participants with normal glucose tolerance that reported insulin resistance as outcome, four studies showed no difference with vitamin D supplementation [140,142,147,151]. In contrast, the study by Von Hurst et al. randomized non-diabetic insulin-resistant vitamin-D-deficient (25(OH)D <20 mg/dl) South Asian women 23e68 years old, to 4000 IU/day of vitamin D3 (n ¼ 42) or placebo (n ¼ 39) for 6 months. Significant improvement in insulin resistance, assessed by HOMA-IR, was seen with vitamin D supplementation compared with placebo, which was more evident when endpoint serum 25(OH)D reached 32 mg/dl. Similarly, in a post-hoc analysis of a trial designed to assess bone-related outcomes, Pittas et al. combined daily supplementation with 700 IU of vitamin D3 and 500 mg of calcium as carbonate improved insulin resistance, assessed by HOMA-IR, only among those with glucose intolerance at baseline [140] but had no effect among those with normal glucose tolerance [140]. Two trials have reported the effect of combined vitamin D3 and calcium supplementation versus placebo on type 2 diabetes risk, both post-hoc. In the Women’s Health Initiative trial, vitamin D3 (400 IU/ day) and calcium supplementation (1000 mg/day) failed to reduce the risk of developing self-reported diabetes over a 7-year period and there was also no significant effect of treatment on simple indices of insulin resistance [142]. The Women’s Health Initiative trial used a small dose of vitamin D and allowed all participants to take vitamin D supplements on their own during the trial. Although the effect of supplementation on 25(OH)D concentration was not reported, based on dose and compliance, it has been estimated to be only 2 ng/ml [154], which is an increment very unlikely to be associated with any change in risk of cardiometabolic disease, based on data from the observational studies. In the trial by Pittas et al., combined supplementation with vitamin D3 and calcium carbonate did not have an effect on change in fasting plasma glucose [140]. However, there was an interaction between baseline glycemia and supplementation on the outcome of interest, change in glycemia. In subgroup analysis, among participants with impaired fasting glucose at baseline, combined vitamin D3 and calcium carbonate supplementation attenuated the increase in fasting glycemia that occurs
1921
over time in this population, but it had no effect on glycemia among subjects with normal glucose tolerance at baseline [140], suggesting that vitamin D may benefit only individuals at high risk for type 2 diabetes. In this study, the effect size of combined vitamin D and calcium supplementation on fasting glycemia in the high-risk subgroup was similar to the effect size seen in the Diabetes Prevention Program trial with intensive lifestyle or metformin therapy [9]. However, in similar subgroup analyses in the Women’s Health Initiative trial and in a smaller 1-year trial with weekly high-dose vitamin D supplementation, there was no benefit of supplementation with vitamin D3 among those with glucose intolerance [142,147].
EVIDENCE FROM HUMAN STUDIES FOR A LINK BETWEEN VITAMIN D AND HYPERTENSION Seasonal and Geographic Studies of Vitamin D and Hypertension Seasonal variations in blood pressure have been described, with blood pressure being higher in the winter than in the summer [155e159]. A strong geographic variation for hypertension also exists, with higher prevalence seen at increasing distances from the equator [160,161]. In support of these ecological data, irradiation with solar UVB has been reported to lower blood pressure in patients with mild hypertension [162].
CaseeControl and Cross-sectional Studies of Vitamin D and Hypertension In caseecontrol and cross-sectional observational studies, vitamin D status, measured by self-reported vitamin D intake or blood 25(OH)D concentration, is inversely associated with systolic and diastolic blood pressure [118,124,163e166]. In the Women’s Health Study, an intake of 511 IU/day or more of vitamin D was associated with lower risk of prevalent hypertension compared with an intake of 159 IU/day or less [124]. However, this analysis did not adjust for any risk factors of hypertension other than age. In cross-sectional studies of data from NHANES, serum 25(OH)D concentration was inversely associated with mean systolic [118,127,166,167] and diastolic blood pressure [118,166], after adjustment for age, sex, ethnicity and leisure-time physical activity. The association was stronger among those older than 50 years and among non-whites. However, after further adjustment for BMI, the association was weakened for both systolic and diastolic blood pressure and became non-statistically significant for diastolic blood pressure. Also, there was no association
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
1922
98. THE ROLE OF VITAMIN D IN TYPE 2 DIABETES AND HYPERTENSION
between 25(OH)D and prevalent hypertension, indicating that those with hypertension had overlapping 25(OH)D distribution compared to those without hypertension. In other cross-sectional studies from a variety of databases, 25(OH)D was associated with prevalent hypertension in some studies from the UK [123], Norway [168] and Lebanon [70] but not others from the USA [61] or the Netherlands [169]. In a study from the Insulin Resistance Atherosclerosis Family Study database (USA), which consists of Hispanice and Africane American families, there was an inverse association between 25(OH)D and blood pressure, but after adjustment for BMI, the association was no longer statistically significant [170].
Longitudinal Observational Cohort Studies of Vitamin D and Hypertension Four studies reported data from four cohorts on the association between vitamin D status and risk of incident hypertension [171e174] (Table 98.1). Two studies assessed vitamin D status by self-reported vitamin D intake [171,174]; the other two measured 25(OH)D concentration [172,173]. In all studies, ascertainment of hypertension was by validated self-report, without measurement of blood pressure; therefore all are graded fair quality. All studies reported multivariable adjusted results, including adjustments for BMI. Forman et al. conducted a study to test the association between vitamin D intake and incident hypertension among participants of three large and independent prospective cohorts, the Nurses’ Health Study I, and Nurses’ Health Study II (women only) and Health Professionals’ Follow-up Study (men only). A total of 32 181 participants was included (98% white) with follow-up between 7 and 10 years. There was no association between vitamin D intake and risk of developing hypertension. Subsequently, Forman et al. examined the association between baseline blood 25(OH)D concentration and incident hypertension in the same three cohorts. In two cohorts (one of men [173], one of women [172]), there was a statistically significant association between lower 25(OH)D concentration and risk of incident hypertension after 7 or 8 years, while the third [173] reported an association in the same direction in women, which was not statistically significant at 8 years. In one study, the association was reported to be stronger for men and women after 4 versus 8 years of follow-up (RR 6.13; 95% CI 1.00, 37.8 versus 3.53; 95% CI 1.02, 12.3 in men and RR 2.67; 95% CI 1.05, 6.79 versus 1.70; 95% CI 0.92, 3.16 in women after 8 versus 4 years, respectively) [173]. Meta-analyses of these three cohorts [172,173] found a statistically significant association comparing the lowest (<15e21 ng/ml) versus the highest (>30e32eng/ml) category of 25(OH)D and incident
hypertension after 7 to 8 years (RR 1.76; 95% CI 1.27, 2.44) without heterogeneity among studies [16]. Another longitudinal observational study showed no associations between baseline 25(OH)D concentration and change in systolic and diastolic blood pressure during a 14-year follow-up period, but it did not report results on incident hypertension [168]. Wang et al. evaluated the association between selfreported vitamin D intake and incident hypertension in the Women’s Health Study (a randomized trial) [174]. A statistically significant trend across quintiles of dietary vitamin D intake was reported (p ¼ 0.02); however, there was no consistency in the direction of the adjusted relative risks across quintiles and all were close to 1.0 (0.95 to 1.04). No association was found with supplemental vitamin D intake.
Effect of Vitamin D Supplementation on Hypertension Twelve trials have reported on the effect of vitamin D supplementation on blood pressure [143,145,147,148,150,151,175e179] or incident hypertension [179] (Table 98.2). Vitamin D was given either alone [143,145,148,150,151,175,177] or in combination with calcium [147,176,178,179] at doses equivalent to 400 to 8571 IU/day. All studies used vitamin D3 except one which used D2 [143]. Another trial compared UVB (which increases cutaneous synthesis of vitamin D) with UVA exposure (which does not) [162]. Follow-up varied from 5 to 36 weeks in most studies with the exception of two studies, which followed participants for up to 1 year [147] or 7 years [179]. The total number of participants was 37 659, with the Women’s Health Initiative trial contributing 36 282 participants. The study populations were heterogeneous, including healthy postmenopausal women [179], participants with established hypertension [162], heart failure [177], type 2 diabetes [143,148,150] or overweight/obese [147,151]. Three trials are rated good [145,175,179], seven trials are rated fair [143,147,148,150,151,176,177] and two trials ware rated poor quality [162,178]. The majority of trials found no statistically significant effects on either systolic or diastolic blood pressure. Two trials reported relatively large net effects of vitamin D supplementation on systolic blood pressure, 7 mm Hg [176] and 14 mm Hg [143]. The trial that compared UVB with UVA exposure also reported a large net effect on systolic blood pressure favoring UVB (6 mm Hg), but it was not clear whether the net effect was statistically significant [162]. The latter study was the only one that found a large net difference in diastolic blood pressure with UVB exposure [162]. In the largest and longest duration trial, the Women’s Health Initiative, combined low-dose vitamin D3 (400 IU/day) and
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SUMMARY OF EVIDENCE FROM HUMAN STUDIES ON TYPE 2 DIABETES AND HYPERTENSION
calcium carbonate supplementation (1000 mg/day) had no effect on incident self-reported hypertension after 7 years of follow-up [179]. In subgroup analyses from this trial, supplementation increased the risk of incident hypertension among blacks (relative risk 1.2; 95% CI 1.0, 1.4) [179]. In a published meta-analysis of ten of these trials (not including the studies by Jorde et al. [147] and Witham et al. [148]), there was a trend for vitamin D supplementation to improve systolic blood pressure (weighted mean difference 1.9 mm Hg; 95% CI 4.2, 0.4), but with significant heterogeneity [16]). There was no effect on diastolic blood pressure (weighted mean difference 0.2 mm Hg; 95% CI 0.9, 0.6). Excluding the large, long-term Women’s Health Initiative trial, the weighted mean difference for systolic blood pressure was 2.8 mm Hg (95% CI 6.7, 1.1) and for diastolic blood pressure was 0.5 mm Hg (95% CI 1.9, 0.8) for vitamin D versus placebo over 5 to 36 weeks. In the same metaanalysis, there was no difference in systolic or diastolic blood pressure change with vitamin D in trials that provided higher (1000 IU/day) versus lower (<1000 IU/day) dose of vitamin D or in trials that provided vitamin D alone or in combination with calcium.
SUMMARY OF EVIDENCE FROM HUMAN STUDIES ON TYPE 2 DIABETES AND HYPERTENSION AND LIMITATIONS IN THE STUDY OF VITAMIN D Overall, the evidence from the mechanistic studies and human observational and ecologic studies suggests an association between low vitamin D status and risk of type 2 diabetes and hypertension. However, definite conclusions cannot be drawn based on these studies for a variety of reasons: (1) Ecologic studies provide interesting data and assist in generating hypotheses; however, they are likely to be confounded by a large number of variables. For example, worsening glycemic control in the winter may be due to decreased physical activity and weight gain, which has been documented to occur in the winter months. (2) In cross-sectional or caseecontrol studies, vitamin D was measured in patients with glucose tolerance or established diabetes; therefore, these measures may not reflect vitamin D status prior to diagnosis and, as a result the causative nature of the reported associations cannot be established. (3) There is considerable variability among the studied cohorts (normal glucose tolerance versus diabetes, newly diagnosed versus established, age, ethnicity, latitude, mean vitamin D status, etc.), which makes it difficult to compare, contrast and combine results. (4) In most studies, there is lack of adjustment
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for important confounders (e.g., adiposity, physical activity, calcium status), although most of the studies published recently have accounted for these variables.
Confounding The most important limitation of the observational studies is residual confounding, which may explain, at least in part, the observed inverse association between vitamin D status and type 2 diabetes and hypertension. Vitamin D status is an excellent marker of good health, as high 25(OH)D concentration is associated with young age, normal body weight and a healthy lifestyle, including good dietary and exercise behaviors. A lower vitamin D status may reflect chronic non-specific illness, which may prevent individuals from having outdoor activities and sun exposure. Vitamin D is also associated with smoking, parental history of cardiovascular disease, and less alcohol intake [180] which are all risk factors for type 2 diabetes and hypertension. Additional issues related to food synergy may further complicate the study of the association between vitamin D and type 2 diabetes/hypertension [181]. For example, a higher vitamin D intake is often associated with higher intake of certain food groups (e.g., dairy) or an overall “prudent” dietary pattern; therefore, additional components in foods that are consumed with vitamin D may have direct effects on type 2 diabetes and/or hypertension or, alternatively, foods rich in vitamin D may replace other foods that increase risk of these conditions.
Measurement of 25(OH)D Observational studies typically measure blood 25 (OH)D concentration as the exposure (independent) variable and trials have used 25(OH)D level to evaluate the success of the intervention with vitamin D supplementation. Although 25(OH)D is widely accepted as the best biochemical surrogate of vitamin D status in clinical practice and human research, polymorphisms in several enzymes involved in the vitamin D metabolism pathway (e.g., vitamin D binding protein, VDR, CYP24A1-hydroxylase) may result in different tissue exposure and action to vitamin D [182e184]. Therefore, additional biochemical and genetic markers of vitamin D status and how they relate to disease progression and development need to be identified, which will help individualize vitamin D-based interventions. Furthermore, observational studies also used single measurements of blood 25(OH)D as a proxy of vitamin D status, which may not reflect vitamin D status over long periods as risk factors for vitamin D deficiency increase with time (aging, declining physical activity, changes in dietary habits, appearance of comorbidities, etc.). Analyses within longitudinal observational studies
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where the exposure is quantified by repeated measurements of 25(OH)D over time to accurately estimate vitamin D status over long periods prior to outcome development may improve the validity of observational studies. Finally, until recently the measurement of 25(OH)D was not standardized across laboratories, which precluded comparisons between studies.
population to achieve and maintain a mean serum 25(OH)D concentration of 30 ng/ml, an intake of approximately 1000 IU/day of vitamin D is generally recommended [188,189]. Individuals at high risk for hypovitaminosis D (e.g., blacks, limited sun exposure) will often require doses higher than 1000 IU/day, probably closer to 2000 IU/day [190].
Blacks versus Whites The study populations in nearly all available studies are predominantly white. Although non-whites have higher prevalence of both vitamin D deficiency [185] and cardiometabolic disease [118], the relationship between these conditions may not be in the same direction as in whites. Indeed, in several observational studies that reported results separately by race, there was no association between vitamin D status and cardiometabolic outcomes among blacks, while in certain studies, high 25(OH)D concentration among blacks was associated with increased risk of prevalent type 2 diabetes and hypertension [64,118,167]. In the Women’s Health Initiative, combined vitamin D and calcium supplementation increased risk of incident hypertension among blacks [179]. The lack of association in blacks is often attributed to the low number of blacks sufficient to conduct powered analyses. Alternatively, this apparent paradox may be explained by blacks having a different vitamin D/parathyroid hormone/ calcium homeostasis, which may not require them to maintain a high vitamin D concentration to achieve optimal skeletal and non-skeletal vitamin D-related function [186]. Therefore, the results seen in whites cannot be generalized to non-whites and future studies should evaluate the potentially divergent effects of vitamin D in whites versus non-whites.
CONCLUSIONS The results seen in the observational studies are supported by biological plausibility, and raise the possibility that optimizing vitamin D status may have a role in reducing risk of type 2 diabetes and hypertension. The results from small underpowered trials and post-hoc analyses of large trials do not currently support a role of vitamin D supplementation for treatment of established type 2 diabetes or prevention of the disease among those with normal glucose tolerance; however, vitamin D sufficiency may have a role in preventing progression from glucose intolerance to clinical diabetes in high-risk populations or in early type 2 diabetes. The evidence from the available trials favor a small reduction in systolic blood pressure with vitamin D supplementation. Type 2 diabetes and hypertension are multifactorial diseases and it is unlikely that vitamin D deficiency would prove to be a central cause or a major therapeutic target; nevertheless, it is imperative that randomized trials are conducted in well-defined populations (e.g., pre-diabetes, early diabetes, pre-hypertension, whites versus non-whites) to test the hypothesis that vitamin D is a direct contributor to the pathogenesis of type 2 diabetes and hypertension and has a role in prevention or therapy.
References OPTIMAL INTAKE OF VITAMIN D IN RELATION TO TYPE 2 DIABETES AND HYPERTENSION Currently recommended intakes for vitamin D are 600 IU/day for those aged 51e70 years and 800 IU/ day for those aged >70 years [187]. However, there is growing consensus that vitamin D intakes above the current recommendations may be associated with better health outcomes. For a variety of skeletal and non-skeletal outcomes, the optimal 25(OH)D level appears to be 20e30 ng/ml [188]. In relation to type 2 diabetes and hypertension, it is difficult to draw definitive conclusion but the data suggest that serum 25(OH)D concentrations above 20 ng/ml are desirable, while a level above 40 ng/ ml may be optimal, although such a higher threshold needs to be demonstrated in trials. For the general
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C H A P T E R
99 Vitamin D Receptor Agonists in the Treatment of Benign Prostatic Hyperplasia Annamaria Morelli 1, Mario Maggi 1, Luciano Adorini 2 1
2
University of Florence, Florence, Italy Intercept Pharmaceuticals, Corciano (Perugia), Italy
BENIGN PROSTATIC HYPERPLASIA: AN INTRODUCTION The human prostate is a gland of the male urogenital tract surrounding the urethra below the neck of the bladder and producing the prostatic fluid, a secretion that contributes 30% to the total ejaculate. The prostatic fluid is rich in fibrinolytic enzymes, such as prostatespecific antigen (PSA), acid phosphatase, citric acid, and zinc. The morphological structure of the prostate gland includes 40 to 50 ducts distributed essentially in three anatomically distinct zones: peripheral, central, and transitional or periurethral. Prostate weight is only a few grams at birth and increases during puberty, reaching approximately 20 g in the young adult. In contrast to the pubertal growth phase, which involves the entire gland, in about 75% of men, during the fifth decade of life, there is a second growth phase selectively involving the periurethral zone [1]. Conversely, the peripheral and central zones, which constitute up to 95% of the entire prostate volume, are usually unaffected. While cell transformation in the peripheral zone gives rise to prostate cancer, cell growth in the periurethral zone leads to the most common age-related disease of the male: benign prostatic hyperplasia (BPH) [1]. The prevalence of BPH increases with age so that 50% of men aged 50 to 60 years and 90% of men over 80 years show histological evidence of BPH at autopsy [1,2]. In a subset of elderly men (27e35%), BPH can cause lower urinary tract symptoms (LUTS) that may require medical or surgical treatment [3]. LUTS in BPH patients are due to the compression by the enlarged prostate of the prostatic urethra which decreases bladder outflow. In the earliest stages, this obstruction is compensated by hypertrophy
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10099-X
and increased activity of the bladder smooth muscle system. Later on, the hypertrophic bladder becomes dysfunctional, characterized by non-voiding contractions during the filling phase and deterioration of the ability to generate adequate voiding pressure. Urinary obstruction and even renal insufficiency might follow, with further complications which may lead to emergency surgery for acute urinary retention [4e6].
PATHOGENESIS OF BPH The pathogenesis of BPH is still poorly understood. Like most chronic diseases, BPH is a progressive condition requiring a long period to evolve from earlier tissue alterations to clinical onset with LUTS [7e9]. Histologically, BPH can be defined as an enlargement of the transitional (or periurethral) prostatic zone associated with a nodular, androgen-dependent, tissue remodeling that involves both epithelium and fibromuscular compartment [10e12]. Compared to normal prostate tissue, hyperplastic nodules are characterized by reduced epithelium-to-stroma ratio, determined by an imbalance between growth and death programs of stromal cells [13e15], leading to increased final stromal volume. These changes in stromal architecture and homeostasis, and in the microenvironment of prostatic stromaleepithelial cell interactions, induce subsequent epithelial rearrangements and BPH progression [16e18]. The overall disease process and the development of BPH-related symptoms include at least three distinct components (Fig. 99.1): (1) a static component, related to the nodular enlargement of the prostate gland, which physically interferes with the free flow of urine through
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99. VITAMIN D RECEPTOR AGONISTS IN THE TREATMENT OF BENIGN PROSTATIC HYPERPLASIA
BPH COMPONENTS
PROSTATE HYPERPLASIA STATIC Bladder outlet obstruction INFLAMMATORY Compensatory response of bladder musculature
DYNAMIC
Urethra dysfunction
LOW URINARY TRACT SYMPTOMS
FIGURE 99.1 Pathogenetic components responsible for BPH-related symptoms. Three distinct pathogenetic components have been recognized in BPH. The recently recognized inflammatory component contributes with static (mechanical urethral obstruction by prostatic adenoma) and dynamic (detrusor hypertrophy and bladder neck smooth muscle failure; urethra dysfunction) components to BPH-related LUTS development.
the urethra and causes various degrees of bladder obstruction, known as bladder outlet obstruction (BOO), responsible for weak stream, intermittent urinary flow, and/or straining to void; (2) a dynamic component, associated with bladder smooth muscle hyperactivity, clinically responsible for storage (irritative) symptoms, such as urinary frequency, urgency and nocturia, referred to as overactive bladder (OAB); (3) a recently recognized inflammatory component related to prostatic inflammatory infiltrates, which were observed in a large subset of BPH patients [19]. During fetal and adult life, prostate development and trophism are regulated both directly and indirectly by androgens [12]. Testosterone and its metabolite dihydrotestosterone (DHT), locally generated by the enzymatic activity of 5a-reductase type II, promote prostate cell growth and differentiation by two different mechanisms: signaling via the androgen receptor (AR), expressed by epithelial and stromal cells, and induction of growth factor synthesis by stromal cells, acting on epithelial and stromal compartments in a paracrine and autocrine manner, respectively [11,12]. Among these intrinsic factors, a crucial role in embryologic differentiation and prostatic branching is exerted by fibroblast growth factors (FGFs), transforming growth factor b (TGFb) and insulin-like growth factors (IGFs), which are all markedly increased in hyperplastic glands. In particular, they have been proven to reawaken prostatic
developmental programs normally repressed in adult life [11,12,20e25]. The most represented FGFs in prostate are FGF-2 and FGF-7 (or keratinocyte growth factor, KGF). FGF-2 is actively synthesized by stromal and epithelial prostate cells, which express also its specific receptor FGFR1 [26e29]. FGF-2 acts as an autocrine inducer of stromal proliferation, able to maintain mesenchymal homeostasis in normal prostate and to promote the structural remodeling typical of the earliest stages in BPH development [20,30]. All these events are clearly androgen-dependent. Accordingly, BPH does not develop in hypogonadal men, and either surgical or pharmacological castration results in a decreased gland size [31e33]. However, BPH develops mainly in older men, when circulating testosterone, and in particular free testosterone, are progressively decreasing. Therefore, it is possible that sensitivity to androgens, rather than circulating androgen levels, is involved in BPH pathogenesis. A higher transcriptional activity of the androgen receptor (AR), due to a decreased number of CAG repeats in exon 1, has been reported in the majority of [34e36] but not in all studies [37,38]. Interestingly, in hypogonadal patients the effect of androgen substitution on prostate growth was inversely related to the extent of CAG residues [39]. In addition, a decreased expression of the AR co-repressor DAX-1 has been associated with BPH [40]. These studies support the view that AR activity is upregulated in the prostate of BPH patients.
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It has recently emerged that local production of mitogens responsible for BPH development is regulated by additional mechanisms, besides sexual hormones. Chronic prostatic inflammation appears to be responsible for several biological changes leading to prostate overgrowth [11,41,42], joining the static (mechanical urethral obstruction) and dynamic (detrusor hypertrophy and hypercontractility of urethra and bladder neck smooth muscle) components in LUTS development [43].
MEDICAL TREATMENT FOR BPH Current medical treatment options for symptomatic BPH include surgery and pharmacotherapy, but there are limitations in terms of efficacy and side effects [44,45]. Surgical treatment may generate post-procedure complications, such as irritation, urinary retention, erectile dysfunction, and alterations of ejaculatory process which overall raise interest in the development of pharmacological approaches for BPH treatment [46]. At present, the mainstay therapies for the treatment of BPH and associated LUTS include a-adrenoceptor blockers, which act on the dynamic component of the disease to regulate the increased adrenergic tone of the lower urinary tract smooth muscles, and 5a-reductase inhibitors, which control the overgrowth of the prostate and hence the static component through the regulation of androgen levels. However, both these strategies display several limitations in terms of side effects which compromise their efficacy and tolerability. In particular, although blocking DHT formation with a type 2 selective (finasteride) or with a dual (dutasteride) inhibitor of 5a-reductase isoforms is, indeed, an effective treatment for BPH [47,48], prostate size reduction obtained with this strategy is relatively limited (about 25%) and sexual side effects related to partial androgen deficiency (decreased libido and impotence) are often present [4,49]. Moreover, it is possible that the limited clinical response to 5a-reductase inhibitors is due to a compensatory increase in intra-prostatic growth factor (GF) receptors, following androgen deprivation [50,51]. Therefore, an alternative strategy to reduce age-related prostate overgrowth is to decrease the activity of androgeninduced prostatic GFs, which are considered to mediate, at least partially, the proliferative activity of sex steroids in the gland [52e54]. In the past few years, and as discussed in many chapters of this volume, there has been growing evidence for the pleiotropic roles of vitamin D and its metabolites in a large number of tissues, leading to the realization that this hormone has many diverse biological functions beyond the classical actions in bone and mineral homeostasis. Actually, it is well known that vitamin D regulates
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growth and differentiation of many cell types, and has pronounced immunoregulatory and anti-inflammatory properties [55e57]. In this context, the regulation of GFs and their signaling in the BPH development process through vitamin D receptor (VDR) agonists may represent a promising approach for the pharmacological management of the disease and associated LUTS. Indeed, proof of concept studies in animal models and BPH patients overall indicate that vitamin D and its analogs may positively impact all three of the components of BPH pathogenesis, the static, the dynamic, and the inflammatory component [58,59]. The following sections of this chapter summarize the main findings demonstrating the beneficial effects of VDR agonists on the pathogenetic mechanisms responsible for BPH, including prostate overgrowth, BPH-related LUTS, and intraprostatic inflammation.
VITAMIN D RECEPTOR AGONISTS CONTROL PROSTATE CELL GROWTH A relationship between the vitamin D system and the prostate has been well established by epidemiological correlations of increased prostate cancer incidence and mortality rates in patients with vitamin D insufficiency [60,61] (see Chapter 89). Accordingly, VDR expression in the urogenital tract is well documented. VDR has been detected in cultured smooth muscle cells derived from prostate [62], bladder [63], and urethra [64] of BPH patients. Moreover, cultured human epithelial cells from prostate gland express VDR at higher levels than in corresponding stromal cells [65]. Interestingly, epithelial prostate cells also express the enzyme 1a-hydroxylase, which is required for 1,25(OH)2D3 synthesis, and produce the active hormone [66,67]. The extra-renal synthesis of 1,25(OH)2D3 in the prostate could have a growth-regulating role, as suggested by the marked decrease of 1a-hydroxylase activity in prostate cancer cell lines [68]. Malignant prostate cells express the VDR, and treatment with calcitriol or less hypercalcemic analogs can inhibit prostate cancer proliferation and invasiveness [see 65, 69, for reviews]. Preclinical studies have shown reduced growth and survival of primary BPH stromal cells by two nonhypercalcemic VDR agonists, BXL-353 and BXL-628 (elocalcitol) [70]. The structures of these compounds compared to calcitriol are shown in Figure 99.2. In particular, elocalcitol, which is more potent than BXL353, decreased testosterone-induced BPH cell proliferation to a similar extent as finasteride and cyproterone acetate, prompting prostate cell apoptosis even in the presence of intraprostatic growth factors. Elocalcitol, at subpicomolar concentrations, completely antagonized the effect of androgenic stimulation. However, this
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OH
OH
OH
HO
OH Calcitriol
HO
OH BXL-353
HO
FIGURE 99.2 Structures of calcitriol (1,25-dihydroxycholecalciferol), BXL-353 (1,25-dihydroxy-16ene23yne-cholecalciferol), and BXL-628 (elocalcitol, 1a-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20epi-cholecalciferol).
F BXL-628 (elocacitol)
anti-androgenic action is not exerted through a direct interference with the AR signaling, as elocalcitol does not modify 5a-reductase type I and II activity, and does not bind the AR or affect its transcriptional activity [71]. Therefore, molecular mechanisms involved in the anti-proliferative and pro-apoptotic effects of VDR agonists in BPH cells appear to operate downstream of the AR. Evidence supporting this hypothesis includes decreased autophosphorylation of KGFR and IGF1R, arrest of cell cycle progression at G1, with a parallel increase in clusterin expression, and decreased expression of the anti-apoptotic factor bcl-2, which is positively modulated by androgens and KGF [71]. Elocalcitol significantly decreases prostate growth both in unmanipulated and in castrated T-replaced rats, with an effect comparable to finasteride but without affecting testis androgenic secretion or pituitary function [71]. Interestingly, both prostatic epithelial and stromal cells from elocalcitol-treated rats showed increased apoptotic rate and clusterin expression, confirming in vitro observations [71]. The capacity of elocalcitol to inhibit prostate growth was further tested in vivo in male beagle dogs, an animal model which spontaneously develops BPH. Chronic oral treatment for 9 months with 5 mg/kg/day elocalcitol in this model induced a reduction of prostate weight, more evident after a 2-month recovery, suggesting a prolonged pharmacological activity of this compound in the absence of side effects including hypercalcemia [59]. Similar results have been obtained in the same spontaneous BPH beagle model following chronic treatment for 11 months with a novel non-secosteroidal VDR agonist, CH5036249 [72]. At very low doses (0.03 mg/kg), the compound inhibited prostate growth in two out of three dogs compared with vehicle-treated dogs, without altering serum calcium levels and showed high bioavailability and stability upon oral administration [72]. Moreover, CH5036249 inhibited growth of human stromal prostate cells with an efficacy comparable to that of calcitriol [72]. Overall, these observations strengthen the importance to target VDR with non-hypercalcemic agonists as a novel therapeutic approach in the clinical management of BPH.
VITAMIN D RECEPTOR AGONISTS AND BPH-RELATED LUTS Hypertrophy and increased contractility of bladder musculature characterize the early stages of BPH-related bladder dysfunction, mainly representing a compensatory response to the urethral obstruction caused by the hyperplastic prostate. Several lines of evidence obtained in human and rat bladder have demonstrated that the VDR agonist elocalcitol exerts positive effects on bladder musculature and thereby it may be useful in BPH patients not only for the effect on the static component (urethral compression), but also on the dynamic component of LUTS pathogenesis [63,64,73]. Experiments performed with stromal cells derived from human bladder neck obtained at surgery from BPH patients demonstrated that elocalcitol may counteract KGF- and androgen-induced cell proliferation and stimulate apoptosis with a parallel reduced expression of the survival oncoprotein Bcl-2. Moreover, chronic exposure to elocalcitol inhibited the starvation-induced expression of activated myofibroblast markers, such as desmin and smoothelin, thus preventing those cell phenotype modifications which may underlie bladder overactivity [63]. Since anticholinergic drugs, first-line treatment for overactive bladder, induce unpleasant side effects (dry mouth, constipation, tachycardia, central nervous system symptoms) due to their incomplete selectivity [74], alternative therapeutic strategies based on these positive effects of VDR agonists could open interesting perspectives. Consistent with the role of RhoA/ROCK signaling in regulating human and rat bladder contraction and tone, in particular in generating involuntary contractions [75], we have also demonstrated that elocalcitol affects bladder contractility via inhibition of the calcium sensitizing RhoA/ROCK pathway by interfering with RhoA activation [73]. The reduction of RhoA/ROCK-mediated inappropriate bladder contraction induced by elocalcitol does not interfere with the overall detrusor motility [73], preventing urinary retention due to voiding impairment. In addition, the capacity of elocalcitol to
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VITAMIN D RECEPTOR AGONISTS INHIBIT INTRAPROSTATIC INFLAMMATION
upregulate Ca2þ entry through L-type Ca2þ channels in human bladder cells can balance its inhibitory effect on RhoA/Rho kinase signaling [76], further suggesting beneficial effects on the modulation of bladder contractile mechanisms. On the basis of these preclinical results it is plausible to hypothesize that possible beneficial effects of elocalcitol on bladder overactivity are exerted by two mechanisms: (1) counteracting the enhanced expression and signaling of growth factors involved in bladder smooth muscle hypertrophy and hyperplasia [63], and (2) by increasing the contractile efficiency of bladder muscle cells through the modulation of smooth muscle gene expression and the downregulation of smooth muscle myofilament sensitization to calcium [73], which can be compensated by a modulation of L-type channel-mediated calcium entry [76]. Preclinical studies performed in a rat model of bladder outlet obstruction (BOO) have indeed demonstrated the efficacy of elocalcitol in reducing the negative functional changes of the bladder smooth muscle associated with BOO, thus preserving the emptying ability of the bladder [77].
VITAMIN D RECEPTOR AGONISTS INHIBIT INTRAPROSTATIC INFLAMMATION The pleiotropic anti-inflammatory effects induced by VDR agonists could turn out to be beneficial in different pathologies mediated by chronic inflammatory responses [55]. Although BPH is primarily characterized by prostatic cell proliferation, an inflammatory component has been extensively documented in this condition [19,41e43]. Interestingly, analysis of prostate biopsies in a subgroup of over 1000 randomly selected patients from the Medical Therapy Of Prostatic Symptoms (MTOPS) study indicates that presence of inflammatory infiltrates in the prostate of BPH patients is associated with increased rate of disease progression and higher risk of acute urinary retention [78]. In addition, analysis of baseline data from the REduction by DUtasteride of prostate Cancer Events (REDUCE) trial indicates an association between inflammation and BPH symptoms [79]. Inflammatory infiltrates in BPH have been found to consist primarily of T cells, mostly CD4þCD45þROþ cells, B cells, and macrophages [41]. Upregulation of several proinflammatory cytokines has been described in BPH, in particular IL-2 and IFN-g [80], IL-15 [81], and IL-17 [82], leading to the hypothesis that BPH may represent an “immune inflammatory” disease [41]. Notably, upregulation of IL-17 in BPH tissues was associated with pathological conditions [82], well before the
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establishment of a pathogenetic role of Th17 cells in autoimmune diseases. Significantly increased levels of the proinflammatory cytokines IL-1a, IL-1b, IL-6, and IL-12p70, and the chemokines CCL1, CCL4, CCL22, and IL-8, are present in the seminal plasma from BPH patients [83]. Overall, the association of BPH with chronic inflammation offers a sound framework to understand the pathogenesis of the disease. The concomitant increase of several inflammatory cytokines and chemokines in BPH patients is consistent with an important chronic inflammatory component in disease pathogenesis, and expression profiling data demonstrate a strong correlation between inflammation and symptomatic BPH [84]. IL-8 is expressed in situ by epithelial and stromal prostate cells, and is functional, as shown by the recruitment of CXCR1 and CXCR2positive leukocytes, as well as CD15þ neutrophils [83]. The potential value of IL-8 as a surrogate marker of disease is further supported by the positive correlation of IL-8 levels with symptom scores and serum PSA values in BPH patients [83]. Therefore, IL-8 may not only serve as a reliable biomarker applicable to diagnosis, prognosis, and assessment of treatment efficacy in BPH as well as in CP/CPPS patients, but does actually represent an important driver of prostate inflammation and an interesting therapeutic target in itself for the treatment of this condition. IL-8-mediated BPH cell growth can be induced by a combination of IFN-g and IL-17, thus establishing a possible relationship between the T cell response induced by BPH cells and prostate cell growth (Fig. 99.3). Consistent with an IL-8-dependent link bridging inflammatory response and cell growth in BPH cells, the VDR agonist elocalcitol has been shown to inhibit IL-8-mediated prostate growth and inflammation through multiple mechanisms of action [73,85]. We have recently demonstrated that elocalcitol inhibits IL-8 production induced by proinflammatory cytokines secreted by prostate-infiltrating CD4þ T cells (IFN-g, IL-17, and TNF-a) in human prostatic stromal cells, accompanied by reduced COX-2 expression and PGE2 production, and by arrest of the nuclear translocation of NF-kB, a transcriptional factor which regulates also IL-8 production [86]. IL-8 promotes BPH cell growth, and elocalcitol dose-dependently counteracts IL-8-dependent BPH cell proliferation via a number of mechanisms, including those mentioned above and inhibition of the RhoA/ROCK pathway [86]. These effects are consistent with the role of this calciumsensitizing signaling pathway in the regulation of inflammatory processes [87], through the activation of NF-kB [88] and the induction of IL-8 secretion [89]. Therefore, these data provide a mechanistic explanation for the role of IL-8 in BPH pathogenesis, showing that a combination of Th1 and Th17 cell-derived
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Epithelium Th1 T 1
Th17 T
C R2 CXCR2 CXCR XCR R
CXCR1 R1
ILIL- 8 C XC CR1 C CXCR1 CXC
Th17 T
CXCR1 CXCR1 Th17
Th1
Th17
Th1
CXCR2
Stroma
Stromal and Epithelial Cell Proliferation
FIGURE 99.3 IL-8 is actively involved in BPH-associated chronic inflammation and mediates epithelial and stromal cell proliferation. In BPH, epithelial and stromal prostate cells actively secrete the proinflammatory chemokine IL-8 in response to different stimuli, including the proinflammatory cytokines IFN-g and IL-17 produced by prostateinfiltrating Th1 and Th17 cells, respectively. IL-8 recruits to the prostate lymphomononuclear cells expressing the cognate receptors CXCR1 and CXCR2, and promotes both directly (through an autocrine/paracrine action) and indirectly (through the induction of FGF2) prostate cell proliferation. Therefore, IL-8 appears to represent a key link between chronic inflammation and overgrowth of BPH tissues.
inflammatory cytokines can markedly stimulate its secretion by BPH stromal cells. Moreover, our results suggest an IL-8-dependent link bridging inflammatory response and cell proliferation in BPH pathogenesis, which can be targeted by elocalcitol via multiple mechanisms of action. Finally, the increased VDR expression promoted by T-cell-derived inflammatory cytokines in BPH cells renders them more susceptible to the inhibitory action of elocalcitol [86]. The anti-inflammatory activities of elocalcitol have also been demonstrated in models of prostate inflammation in vivo. Non-obese diabetic (NOD) mice develop experimental autoimmune prostatitis (EAP) after immunization with mouse prostate homogenate emulsified in complete Freund’s adjuvant (CFA) [90]. Administration of elocalcitol at normocalcemic doses in already established EAP for 2 weeks (orally 5 dose/week at 100 mg/ kg from day 14 to 28 postimmunization) reduces by 67% the number of the intraprostatic infiltrates compared to vehicle-treated mice. The marked decrease in intraprostatic inflammatory cell (CD4þ and CD8þ T cells, B cells, macrophages, and DCs) infiltration is characterized by a reduced proliferation and an enhanced activationinduced cell death, effects superior to those induced by an optimal dose of dexamethasone [85].
The molecular effect of elocalcitol is exerted through inhibition of CD4þ T cell responses in prostate-infiltrating cells and in prostate-draining lymph node cells, as demonstrated by reduced ex vivo production of IFNg and IL-17, and markedly decreased expression of iNOS, a key enzyme required for the synthesis of the inflammatory agent nitric oxide (NO), and of NO itself, in peritoneal macrophages [85]. The inhibitory action of elocalcitol on T cell responses is also confirmed in NOD mice immunized with prostatic steroid-binding protein (PSBP) or its immunodominant epitope PSBP21e40. In addition, a reduced response to PSBP is observed in NOD.SCID recipients transferred with CD4þ T cells from elocalcitol-treated NOD mice [85], consistent with the ability of VDR agonists to inhibit pathogenic mechanisms in several Th1-cell-dependent autoimmune diseases [55]. Elocalcitol is able to inhibit the proliferation and to induce the apoptosis not only of pathogenic T cells, but also of epithelial and stromal prostatic cells, suggesting a dual effect in promoting maintenance of normal glandular architecture. Finally, the dosee response analysis of elocalcitol effects demonstrates that about 60% reduction in the number of intraprostatic infiltrates, compared to vehicle-treated animals, is already obtained with 3 mg/kg, a dose 30-fold lower than the maximum tolerated dose of this compound in terms of hypercalcemia. Thus, the therapeutic window of elocalcitol in the treatment of EAP appears to be rather wide [85].
VITAMIN D RECEPTOR AGONISTS MODULATE PROSTATIC URETHRA DYSFUNCTION In BPH patients, LUTS are predominantly determined by prostate growth and inflammation, and by alteration of prostate and bladder neck smooth muscle tone. The contribution of prostatic urethra dysfunction to LUTS development and the importance in reducing its tone in order to improve the management of these patients are also indicated by urodynamic studies, which demonstrate a positive correlation between urethral function and clinical BPH [91]. The physiological pattern of voiding shows an initial decrease in urethral pressure followed by an increase in intravesical pressure [92,93], therefore an increased urethral smooth muscle tone takes an active part in the pathogenesis of BPH-associated LUTS [94]. Given the crucial involvement of chronic inflammation in the BPH pathogenesis, a BPH-associated inflammatory response could also trigger urethral dysfunction, thus contributing to the clinical progression of BPH symptoms. Human prostatic urethra displays higher VDR expression compared to prostate and bladder neck
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CONCLUDING REMARKS
FIGURE 99.4 Schematic representation of the different sites targeted by the VDR agonist elocalcitol and the proposed mechanisms of action.
BLADDER VDR
VDR
Modulation of smooth muscle gene expression, proliferation, apoptosis, and Ltype calcium channels
Inhibition of RhoA/ROCK signalling VDR
VDR
VDR
Inhibition of prostate cell proliferation
Inhibition of inflammatory response
PROSTATE
PROSTATIC URETHRA
tissues obtained from the same individual [64]. Interestingly, in isolated human prostatic urethra (hPU) smooth muscle cells, the VDR agonist elocalcitol partially reverts COX-2 and IL-8 mRNA upregulation induced by a mixture of proinflammatory cytokines (IL-17, IFN-g, TNF-a) which are secreted by prostate-infiltrating CD4þ cells in BPH patients [80]. The molecular mechanism which underlies these effects of elocalcitol seems to involve inhibition of IL8-mediated induction of RhoA/ ROCK signaling [64]. These results are consistent with an IL-8-dependent link bridging inflammatory response with cell growth and contractile activity in hPU cells, which similarly to prostatic stromal cells can be targeted by elocalcitol through multiple mechanisms of action [86]. Through its anti-inflammatory and anti-contractile properties, VDR activation may counteract these dysfunctions in different portions of the male urogenital tract, notably prostate, bladder, and urethra, which are overall responsible for BPH-related LUTS (Fig. 99.4).
CONCLUDING REMARKS A large proportion of aging males develop BPH and, until recently, the only options for treatment were surgical intervention or watchful waiting. Progress in medical therapy of BPH has resulted in effective treatment patterns leading to a significant improvement in the quality of life of affected patients. At present, two different classes of agents are available for BPH treatment: a-blockers and 5a-reductase inhibitors. Although sexual-related side effects are more often reported with 5a-reductase inhibitors than with a-blockers [95], the reverse is true for reduction in risk of BPH-related
surgeries [96]. A population-based study including more than 5000 patients receiving one of the two forms of treatment, a-blocker compared to 5a-reductase inhibitor, showed that the former patients had a higher risk of BPH-related surgery, most probably because 5a-reductase inhibitors, by inhibiting DHT formation, consistently reduced prostate volume [96]. Hence, the ideal treatment of BPH should include a medication that reduces prostate volume without interfering with androgen activity. Well-tolerated calcitriol analogs, such as elocalcitol, might represent such a new class of drugs, because they decrease ARmediated prostate growth by acting on GF-mediated proliferation pathways, downstream of the AR. In addition, VDR agonists can modulate the dynamic component of LUTS pathogenesis, and exert antiinflammatory activities by targeting not only the bladder but also the urethra. Thus, this class of agents could represent an interesting therapeutic option for the pharmacological treatment of clinical BPH, mainly caused by a combination of prostate overgrowth and smooth muscle overactivity of bladder and urethra musculature. Chronic inflammation can be considered the third component of BPH pathogenesis, taking part with the AR signaling in the induction of the tissue remodeling typical of the advanced stages of the disease. Prostatic stromal cells appear to play a critical role in the induction of inflammatory responses by activating CD4þ lymphocytes and favoring their differentiation into effector Th1 and Th17 cells [97]. Thus, BPH can be seen as a form of chronic prostatitis, whose pathogenesis may be triggered by infection. Among the proinflammatory cytokines and chemokines produced by the prostatic microenvironment, stromal-derived IL-8 may be
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considered a key link between chronic inflammation and stromal cell proliferation [97]. The ability of VDR activation to inhibit inflammatory response both in prostatic and urethra smooth muscle cells seems to be related to the downregulation of the RhoA/ROCK signaling [64,73,86]. The effect on this pro-contractile, calcium sensitizing pathway may represent a common denominator for the therapeutic efficacy of VDR agonists on all three components of BPH: static, dynamic, and inflammatory. Based on the preclinical premises reviewed above, a 12-week phase IIa, multicenter, double-blind, randomized, placebo-controlled clinical trial was performed with the aim of evaluating the efficacy and safety of elocalcitol administration (150 mg/day) in BPH patients [98]. Elocalcitol exhibited a 7.2% reduction in prostate volume, compared to placebo. Importantly, 92% of elocalcitol-treated patients did not experience a clinically significant growth in prostate volume compared with 48% in the placebo group. Thus, the reduction of prostate volume in the elocalcitol-treated group against its marked increase in the placebo group indicates the ability of this VDR agonist to block the ongoing BPH process. During the trial, no difference was observed in symptom score or urodynamic parameters, probably because of the short duration of this proof-of-concept study and because patients were not screened for symptoms but only for prostatic volume [98]. To elucidate this point, a 6-month phase IIb study was performed to measure maximum urinary flow rate and symptom severity as secondary endpoints in patients with at least moderate symptomatology. Elocalcitol was effective in improving maximum urinary flow rate (Qmax) and ameliorating LUTS, as well as arresting prostate growth and preventing the risk of urinary retention and need for surgery [99], all key parameters of BPH progression. The efficacy of elocalcitol for OAB was investigated in a phase IIa randomized, placebo-controlled trial, which enrolled 120 patients in 13 centers in Italy. The study has shown clear efficacy signals on the primary endpoint, mean volume voided per micturition (MVV), which is an objective and commonly used parameter for OAB trials. Elocalcitol-treated patients exhibited a 21.5% increase in MVV compared with a 10.9% increase in the placebo group. Moreover, elocalcitol also demonstrated significant efficacy on symptoms, including frequency, nocturia, and incontinence episodes. Elocalcitol was already effective after 4 weeks of treatment and showed a profile of adverse events comparable to placebo [100]. These promising clinical results were disappointingly negated by the urodynamic, multi-center, phase IIb trial. This urodynamic trial was conducted in 257 patients treated for 4 weeks in a multi-center, double-blind,
placebo-controlled study. A statistically significant effect on the primary endpoint, the “change in volume at first involuntary contraction,” was not achieved. A statistically significant improvement in “bladder volume at first desire to void” was observed in the intention-totreat population (ITT). Numerical improvements in most other urodynamic parameters compared to placebo were observed but did not reach statistical significance. Numerical improvements were also seen for the key symptoms of OAB in all treatment groups. A statistically significant improvement in incontinence episodes was observed in the modified-intention-totreat (MITT) population. There was a strong doserelated trend in the improvement in Patient’s Perception Bladder Condition (PPBC) in the ITT population which reached statistical significance in the per protocol (PP) analysis [101]. Based on these data, BioXell decided to terminate its clinical development activities for elocalcitol in OAB and BPH. Beforehand, BioXell had already communicated its decision to suspend the phase II trial of elocalcitol in male infertility. All activities related to the vitamin D programs being developed by BioXell were therefore terminated, and the rights for elocalcitol returned to Roche. Nevertheless, given the novel mechanism of actions and the proven tolerability of VDR agonists over existing drugs, the potential use of these compounds in the expanding therapeutic market of BPH and related LUTS symptoms should be further investigated.
References [1] S.J. Berry, D.S. Coffey, P.C. Walsh, L.L. Ewing, The development of human benign prostatic hyperplasia with age, J. Urol. 132 (1984) 474e479. [2] K.T. McVary, BPH: epidemiology and comorbidities, Am. J. Manag. Care 12 (2006) S122eS128. [3] S.J. Jacobsen, C.J. Girman, H.A. Guess, L.A. Panser, C.G. Chute, J.E. Oesterling, et al., Do prostate size and urinary flow rates predict health care-seeking behavior for urinary symptoms in men? Urology 45 (1995) 64e69. [4] A. Thorpe, D. Neal, Benign prostatic hyperplasia, Lancet 361 (2003) 1359e1367. [5] C.G. Roehrborn, Alfuzosin 10 mg once daily prevents overall clinical progression of benign prostatic hyperplasia but not acute urinary retention: results of a 2-year placebo-controlled study, BJU Int. 97 (2006) 734e741. [6] C.G. Roehrborn, P. Siami, J. Barkin, R. Damia˜o, K. MajorWalker, B. Morrill, et al., The effects of dutasteride, tamsulosin and combination therapy on lower urinary tract symptoms in men with benign prostatic hyperplasia and prostatic enlargement: 2-year results from the CombAT study, J. Urol. 179 (2008) 616e621. [7] S.J. Jacobsen, C.J. Girman, H.A. Guess, T. Rhodes, J.E. Oesterling, M.M. Lieber, Natural history of prostatism: longitudinal changes in voiding symptoms in community dwelling men, J. Urol. 155 (1996) 595e600. [8] R.O. Roberts, S.J. Jacobsen, D.J. Jacobsen, T. Rhodes, C.J. Girman, M.M. Lieber, Longitudinal changes in peak
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99. VITAMIN D RECEPTOR AGONISTS IN THE TREATMENT OF BENIGN PROSTATIC HYPERPLASIA
[46] A. Tiwari, Advances in the development of hormonal modulators for the treatment of benign prostatic hyperplasia, Expert Opin. Investig. Drugs 16 (2007) 1425e1439. [47] J.D. McConnell, R. Bruskewitz, P. Walsh, G. Andriole, M. Lieber, H.L. Holtgrewe, et al., The effect of finasteride on the risk of acute urinary retention and the need for surgical treatment among men with benign prostatic hyperplasia Finasteride Long-Term Efficacy and Safety Study Group, N. Engl. J. Med. 338 (1998) 557e563. [48] C.G. Roehrborn, P. Boyle, J.C. Nickel, K. Hoefner, G. Andriole, ARIA3001 ARIA3002 and ARIA3003 Study Investigators, Efficacy and safety of a dual inhibitor of 5-alpha-reductase types 1 and 2 (dutasteride) in men with benign prostatic hyperplasia, Urology 60 (2002) 434e441. [49] W. Steers, 5alpha-reductase activity in the prostate, Urology 58 (2001) 17e24. [50] G. Fiorelli, A. De Bellis, A. Longo, A. Natali, A. Costantini, M. Serio, Epidermal growth factor receptors in human hyperplastic prostate tissue and their modulation by chronic treatment with a gonadotropin-releasing hormone analog, J. Clin. Endocrinol. Metab. 68 (1989) 740e743. [51] G. Fiorelli, A. De Bellis, A. Longo, S. Giannini, A. Natali, A. Costantini, et al., Insulin-like growth factor-I receptors in human hyperplastic prostate tissue: characterization, tissue localization, and their modulation by chronic treatment with a gonadotropin-releasing hormone analog, J. Clin. Endocrinol. Metab. 72 (1991) 740e746. [52] M. Serio, G. Fiorelli, Dual control by androgens and peptide growth factors of prostatic growth in human benign prostatic hyperplasia, Mol. Cell Endocrinol. 78 (1991) C77eC81. [53] G.R. Cunha, Growth factors as mediators of androgen action during male urogenital development, Prostate 6 (1996) 22e25. [54] V.J. Gnanapragasam, P.J. McCahy, D.E. Neal, C.N. Robson, Insulin-like growth factor II and androgen receptor expression in the prostate, BJU Int. 86 (2000) 731e735. [55] L. Adorini, G. Penna, Control of autoimmune diseases by the vitamin D endocrine system, Nat. Clin. Pract. Rheumatol. 4 (2008) 404e412. [56] S. Sam, M.D. Sitrin, Vitamin’s D role in cell proliferation and differentiation, Nutrition Rev. 66 (2008) S116eS124. [57] D. Bickle, Nonclassic action of vitamin D, J. Clin. Endocrinol. Metab. 94 (2009) 26e34. [58] A. Tiwari, Elocalcitol, a vitamin D3 analog for the potential treatment of benign prostatic hyperplasia, overactive bladder and male infertility, IDrugs 12 (2009) 381e393. [59] L. Adorini, G. Penna, S. Amuchastegui, C. Cossetti, F. Aquilano, R. Mariani, et al., Inhibition of prostate growth and inflammation by the vitamin D receptor agonist BXL-628 (elocalcitol), J. Steroid. Biochem. Mol. Biol. 103 (2007) 689e693. [60] G.G. Schwartz, Vitamin D and the epidemiology of prostate cancer, Semin. Dial 18 (2005) 276e289. [61] B. Mikhak, D.J. Hunter, D. Spiegelman, E.A. Platz, B.W. Hollis, E. Giovannucci, Vitamin D receptor (VDR) gene polymorphisms and haplotypes, interactions with plasma 25hydroxyvitamin D and 1,25-dihydroxyvitamin D, and prostate cancer risk, Prostate 67 (2007) 911e923. [62] C. Crescioli, M. Maggi, M. Luconi, G.B. Vannelli, R. Salerno, A.A. Sinisi, et al., Vitamin D3 analogue inhibits keratinocyte growth factor signaling and induces apoptosis in human prostate cancer cells, Prostate 50 (2002) 15e26. [63] C. Crescioli, A. Morelli, L. Adorini, P. Ferruzzi, M. Luconi, G.B. Vannelli, et al., Human bladder as a novel target for vitamin D receptor agonists, J. Clin. Endocrinol. Metab. 90 (2005) 962e972.
[64] P. Comeglio, A.K. Chavalmane, B. Fibbi, S. Filippi, M. Marchetta, M. Marini, et al., Human prostatic urethra expresses vitamin D receptor and responds to vitamin D receptor ligation, J. Endocrinol. Invest. (2010). In press. [65] D.M. Peehl, R.J. Skowronski, G.K. Leung, S.T. Wong, T.A. Stamey, D. Feldman, Antiproliferative effects of 1,25dihydroxyvitamin D3 on primary cultures of human prostatic cells, Cancer Res. 54 (1994) 805e810. [66] G.G. Schwartz, L.W. Whitlatch, T.C. Chen, B.L. Lokeshwar, M.F. Holick, Human prostate cells synthesize 1,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3, Cancer Epidemiol. Biomarkers Prev. 7 (1998) 391e395. [67] T.C. Chen, L. Wang, L.W. Whitlatch, J.N. Flanagan, M.F. Holick, Prostatic 25-hydroxyvitamin D-1alpha-hydroxylase and its implication in prostate cancer, J. Cell Biochem. 88 (2003) 315e322. [68] A.V. Krishnan, D.M. Peehl, D. Feldman, The role of vitamin D in prostate cancer, Recent Results Cancer Res. 164 (2003) 205e221. [69] A.V. Krishnan, D.M. Peehl, D. Feldman, Inhibition of prostate cancer growth by vitamin D: regulation of target gene expression, J. Cell Biochem. 88 (2003) 363e371. [70] M. Maggi, C. Crescioli, A. Morelli, E. Colli, L. Adorini, Preclinical evidence and clinical translation of benign prostatic hyperplasia treatment by the vitamin D receptor agonist BXL628 (elocalcitol), J. Endocrinol. Invest. 29 (2006) 665e674. [71] C. Crescioli, P. Ferruzzi, A. Caporali, M. Scaltriti, S. Bettuzzi, R. Mancina, et al., Inhibition of prostate cell growth by BXL628, a calcitriol analogue selected for a phase II clinical trial in patients with benign prostate hyperplasia, Eur. J. Endocrinol. 150 (2004) 591e603. [72] K. Taniguchi, K. Katagiri, H. Kashiwagi, S. Harada, Y. Sugimoto, Y. Shimizu, et al., A novel nonsecosteroidal VDR agonist (CH5036249) exhibits efficacy in a spontaneous benign prostatic hyperplasia beagle model, J. Steroid Biochem. Mol. Biol. (2010). [Epub ahead of print]. [73] A. Morelli, L. Vignozzi, S. Filippi, G.B. Vannelli, S. Ambrosini, R. Mancina, et al., BXL-628, a vitamin D receptor agonist effective in benign prostatic hyperplasia treatment, prevents RhoA activation and inhibits RhoA/Rho kinase signaling in rat and human bladder, Prostate 67 (2007) 234e247. [74] K.E. Andersson, A. Arner, Urinary bladder contraction and relaxation: physiology and pathophysiology, Physiol. Rev. 84 (2004) 935e986. [75] S.L. Peters, M. Schmidt, M.C. Michel, Rho kinase: a target for treating urinary bladder dysfunction? Trends Pharmacol. Sci. 27 (2006) 492e497. [76] A. Morelli, R. Squecco, P. Failli, S. Filippi, L. Vignozzi, A.K. Chavalmane, et al., The vitamin D receptor agonist elocalcitol upregulates L-type calcium channel activity in human and rat bladder, Am. J. Physiol. Cell Physiol. 294 (2008) C1206eC1214. [77] A. Schroder, E. Colli, M. Maggi, K.E. Andersson, Effects of a vitamin D(3) analogue in a rat model of bladder outlet obstruction, BJU Int. 98 (2006) 637e642. [78] C.G. Roehrborn, Definition of at-risk patients: baseline variables, BJU Int. 97 (2006) 7e22. [79] J.C. Nickel, C.G. Roehrborn, M.P. O’Leary, D.G. Bostwick, M.C. Somerville, R.S. Rittmaster, The relationship between prostate inflammation and lower urinary tract symptoms: examination of baseline data from the REDUCE trial, J. Urol. 17 (2007) 34e35. [80] G.E. Steiner, U. Stix, A. Handisurya, M. Willheim, A. Haitel, F. Reithmayr, et al., Cytokine expression pattern in benign prostatic hyperplasia infiltrating T cells and impact of
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C H A P T E R
100 Sunlight Protection by Vitamin D Compounds Rebecca S. Mason, Katie M. Dixon, Vanessa B. Sequeira, Clare Gordon-Thomson University of Sydney, NSW, Australia
INTRODUCTION The same ultraviolet (UV) B radiation that converts 7-dehydrocholesterol to pre-vitamin D (see Chapter 2), together with UVA, causes damage to skin cells. This includes several types of DNA damage, immune suppression, inflammation (sunburn), and photoaging [1]. The DNA damage, if inadequately repaired, leads to mutations, which, together with immune suppression, result in photocarcinogenesis [2e4]. Several adaptive mechanisms are known, which increase the ability of skin to better withstand the next exposure to UV. These include increased production and distribution of UV-absorbing melanin pigment, at least in some individuals, as well as increased thickness of the cornified layer of skin, which also absorbs UV [1,3,4]. There is some evidence for enhanced DNA repair processes after UV exposure [2]. There is now increasing evidence that the production of vitamin D in skin and its local conversion to other metabolites, including 1,25(OH)2D3 [5,6], also contributes to photoprotection.
UV-INDUCED DNA DAMAGE AND REPAIR Two primary DNA lesions formed in human skin by UVR are cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts. CPDs occur when UVB cleaves DNA between carbon-5 and carbon-6 of adjacent pyrimidines. Subsequent dimerization of these pyrimidines produces a stable ring structure. The majority of CPDs occur at thymineethymine pairs resulting in thymine dimers. Thymineecytosine and cytosineecytosine dimers are also formed but at a lesser rate [7]. The stable ring configuration of CPDs makes this structure hard to
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10100-3
repair, while 6-4 photoproducts occur at a much lower frequency and are repaired more rapidly after UVR exposure [8]. For these reasons CPDs are the most frequent promutagenic DNA photoproduct in human skin. Inefficient repair of CPDs and 6-4 photoproducts can result in mutations in the DNA sequence known as UV “signature” mutations [1]. UVexposure (both UVB and UVA) also generates reactive oxygen species, such as superoxide and hydrogen peroxide, which, if inadequately processed by skin cells, results in oxidative DNA damage e the base guanosine is converted to 8-hydroxy-20 deoxyguanosine, which is mutagenic [9]. High levels of nitric oxide (NO) and its products are also potentially very important players in UV-induced DNA damage. UV activation of nitric oxide synthase increases NO levels in skin [10e12]. Enzymeindependent release of NO from preformed stores of NO can also increase cellular NO levels [13e15], for example by UVA photo-decomposition of nitrosothiols and nitrite [14,15]. Powerful oxidating and nitrating intermediates such as peroxynitrite and nitrous anhydride, which form when NO in excess combines with superoxide or oxygen, respectively, are known to attack DNA [16,17]. Peroxynitrite induces DNA base modifications by oxidation or nitration of the primary amine of guanine generating 8-hydroxy-20 deoxyguanosine or 8-nitroguanine respectively [16e19]. Residues of 8-nitroguanine undergo rapid depurination to form abasic sites which are promutagenic [20]. Deamination of purine and pyrimidine bases by nitrosation of primary amines by nitrous anhydride leads to DNA strand breaks [21]. DNA replication across non-coding sites of unrepaired base modifications can result in deletions, base mispairing, or substitutions where adenine is the default base added [22]. Nitrosative deaminations generate C to T transition mutations in isolated DNA
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[21]. G to T transversions are typically associated with the 8-hydroxy-2’deoxyguanosine lesions in the tumor suppressor protein p53 and correlate with malignant transformation in skin photocarcinogenesis [23]. Predominantly C:G to T:A transversions, which cause inactivating mutations in p53, have also been reported in isolated DNA exposed to peroxynitrite [24]. In human colorectal carcinoma cells G:C to A:T transitions are the most frequent mutation found in p53. These are positively associated with increased expression and activity of inducible nitric oxide synthase (iNOS) [25]. DNA repair pathways include photoreactivation, base excision repair, mismatch repair, double-stranded break repair and nucleotide excision repair. The pathway that is activated depends on the primary DNA lesion and the species [1]. UVR-induced pyrimidine dimers are predominantly repaired by nucleotide excision repair, a complex mechanism involving around 30 proteins [1,26]. The significance of this repair pathway in skin cancer is highlighted by the disorder xeroderma pigmentosum, an inherited disease in which there is some defect in an aspect of nucleotide excision repair. These patients have 1000-fold increased incidence of skin cancer [27]. The p53 tumor suppressor gene is one of the most commonly mutated genes in UVR-induced skin cancers [28e30]. The p53 gene codes for a 53 kDa protein involved in regulation of the cell cycle and facilitation of DNA repair [31]. UVR, among other DNA-damaging agents, leads to upregulation of transcription of p53 and the subsequent accumulation of p53 protein, reaching maximal levels around 12 hours after UVR [32]. p53 accumulation and activation are believed to be mediated through protein phosphorylations and acetylations. At least 20 sites in the human p53 protein are modified in response to the activation of different stress signaling pathways [32]. Under normal physiological conditions p53 is present in cells at very low levels due to its very short half-life of less than 30 mins [31,33]. In normal cells p53 is bound to its negative regulator, the MDM2 protein. MDM2 maintains p53 at low levels by increasing its susceptibility to proteolysis by the 26S proteosome. The MDM2 gene is a protooncogene which functions mainly to modulate the activity of p53 [34]. Following UVR, the accumulation of activated p53 protein and its subsequent translocation to the nucleus leads to transactivation of downstream genes to induce cell cycle arrest in the G1 phase, presumably to allow for DNA repair prior to DNA synthesis and mitosis. Within this pathway, p53 induces p21 which inhibits cyclin-dependent kinases required for cell cycle progression [22,35]. Furthermore, p21 can directly interfere with DNA synthesis by binding to proliferating cell nuclear antigen and blocking its interaction with DNA
polymerase [36]. If the DNA damage is too severe to be repaired, apoptotic pathways are activated to eliminate the damaged cell before it replicates. p53 can upregulate the expression of pro-apoptotic genes such as Bax and Fas/Apo-1, or can downregulate expression of antiapoptotic genes such as Bcl-2 [37]. In addition to disrupting the cell cycle to allow time for DNA repair, p53 directly affects nucleotide excision repair by inducing the transcriptional activation of downstream genes [38,39]. p53 also regulates the GADD45 protein, which has been observed in vitro to bind to UVR-damaged DNA [40]. As well as being pro-mutagenic, reactive nitrogen species also have been reported to inhibit DNA repair [41,42] and conversely nitric oxide synthase inhibitors enhance DNA repair mechanisms [43].
UV-INDUCED IMMUNE SUPPRESSION UV irradiation from sunlight suppresses the immune system, which facilitates skin carcinogenesis by reducing cell-mediated immune responses that normally destroy developing skin tumors [44e46]. Exposure to UV results in a substantial reduction in numbers of Langerhans cells, as well as reducing the antigen-presenting ability of those remaining [47e49]. UVR also increases the release of immunosuppressive cytokines such as interleukin-10 from keratinocytes, and possibly from mast cells [50], increases pro-inflammatory cytokines such as IL-6 [51] and suppresses the release of immunestimulating cytokines such as IL-12 [52]. A major contributor to UV-induced immunosuppression is DNA damage (reviewed in [22]). UV exposure suppresses both contact hypersensitivity reactions and delayedtype hypersensitivity reactions to viral, bacterial, and fungal antigens and causes both local and systemic immune suppression (reviewed in [53]).
PHOTOCARCINOGENESIS UVR acts as a complete carcinogen, due to its ability to cause skin cancers without additional initiators or promoters [54]. UVR-induced carcinogenesis commences with DNA damage, which then leads to a cascade of events resulting in tumor development. In cells that have DNA damage, the increase in p53 phosphorylation and translocation to the nucleus facilitates DNA repair or causes apoptosis of cells with severely damaged DNA. Excessive UVR exposure can disrupt DNA repair mechanisms, leading to p53 mutations, allowing damaged cells to resist apoptosis. Such cells can undergo clonal expansion, leading to formation of premalignant actinic keratoses and squamous cell
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carcinomas (SCCs) [54]. Mutational analyses from human actinic keratosis lesions and murine carcinogenesis studies have shown that p53 gene mutations appear to be a key event, possibly the initiating event, in skin carcinogenesis [28,55,56]. Loss or mutation of p53 has been shown in approximately 50% of all human cancers, although the mutational frequency varies depending on the type of cancer [22]. Many p53 mutations increase the half-life of the protein, allowing for cytosolic accumulation of mutant p53 protein [57]. A number of groups have detected p53 mutations in basal cell carcinomas (BCCs), squamous cell carcinomas (SCCs), and premalignant actinic keratosis (AK) lesions, considered precursors for SCCs [56,58]. Most forms of DNA damage, if inadequately repaired, can result in mutations, including pyrimidine dimers, oxidative and nitrosative damage [20e24,59,60]. Furthermore, excessive levels of nitric oxide and its products inhibit DNA repair [41,61]. As noted earlier, UV-induced immune suppression also contributes to photocarcinogenesis. This was demonstrated by experiments where skin tumors were induced in mice by chronic UVR exposure. The tumors were then excised and transplanted into normal nonirradiated, immune-competent mice. The tumors were consistently rejected due to their high antigenicity. On the other hand, if the recipient mice were UV-irradiated before transplantation, the tumors were not rejected and grew progressively [44]. The importance of immune factors in UVR-induced skin carcinogenesis is demonstrated by renal transplant patients, who were reported to have a seven-fold risk of skin cancer [62] and an 82-fold risk of invasive squamous cell carcinoma in comparison to non-transplanted controls [63]. Immunosuppressive chemotherapy also increases the risk of skin cancer [64].
LOCAL PRODUCTION OF VITAMIN D METABOLITES IN SKIN As discussed in Chapter 45, there is evidence that some of the vitamin D produced in skin is converted locally into the active hormone 1,25(OH)2D3. Bikle et al. [5] first showed 1a-hydroxylase activity in keratinocytes and a series of experiments by Lehmann and colleagues [6,65,66] demonstrated evidence of complete conversion of 7-dehydrocholesterol to 1,25(OH)2D3 in epidermal cells, skin equivalents and living skin, though the process takes several hours. Dermal fibroblasts may also exhibit 25-hydroxylase activity, but not 1a-hydroxylase, after UV [67]. Other photoproducts are also produced from 7dehydrocholesterol in skin, including the so-called
1945
“overirradiation products” as discussed in Chapter 2. One of these, produced by continued irradiation of pre-vitamin D3, is lumisterol3 [68]. Recently, new metabolic pathways have been described [69,70] that could result in the local conversion of lumisterol3 to 1,25dihydroxylumisterol3, also known as JN, a welldescribed agonist of the non-genomic signaling pathway of 1,25(OH)2D3 [71].
VITAMIN D COMPOUNDS AND PHOTOPROTECTION Sunburn Cells The first description that vitamin D compounds might protect against UV damage was published in 1995 and showed that systemic or topically applied 1,25(OH)2D3 reduced sunburn cell formation in mice after UVB and reduced UV-induced cytotoxicity in rat keratinocytes [72]. Epidermal apoptotic keratinocytes, commonly known as “sunburn cells” (SBCs), are a recognized characteristic of UV-exposed skin. The presence of SBCs suggests that cellular DNA has been irreparably damaged by UV, and it is surmised that their formation results in eradication of cells with damaged DNA before replication [73]. SBCs display a distinct morphology following haematoxylin and eosin staining: a pyknotic nucleus and a shrunken glassy, eosinophilic cytoplasm [74]. In 1997, Youn and colleagues showed that the low calcemic analog of 1,25(OH)2D3, calcipotriol, protected against a UV-induced reduction in DNA synthesis in cultured keratinocytes and increased the dose of UV that was required to just produce faint redness (the minimal erythemal dose) in half of the human subjects tested [75]. In the following year, the same group of researchers showed that survival of UVB-irradiated human keratinocytes in culture was enhanced in the presence of 1,25(OH)2D3 and this was accompanied by an induction of the oxygen radical scavenger metallothionein [76]. Reductions in UVinduced apoptosis of human keratinocytes by 1,25(OH)2D3 were then reported by several groups [77e80]. Protection from UV-induced apoptosis was mimicked by the low calcemic but transcriptionally active analog 1a-hydroxymethyl-16-ene-24,24-difluoro25-hydroxy-26,27-bis-homovitamin D3 (QW-1624F2-2; QW) [81]. Further studies in Skh:hr1 (hairless) mice showed reduction in UV-induced sunburn cells even when the 1,25(OH)2D3 was applied immediately after UV [81], similar to findings in cultured human keratinocytes [82]. A reduction in sunburn cells after UV in the presence of 1,25(OH)2D3 has now also been reported in human subjects [83]. Interestingly, the protection by 1,25(OH)2D3 of keratinocytes and other skin cells from UV-induced apoptosis [84] and apoptosis caused by
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other stressors [85,86] is in contrast to the tendency of 1,25(OH)2D3 to enhance apoptosis of some neoplastic cells exposed to cytotoxic agents [86,87].
Protection from DNA Damage Although it was possible that the reduced induction of UV-induced apoptosis might allow cells with substantial DNA damage to survive, in fact it became clear that the improved cell survival in 1,25(OH)2D3-treated skin cells was associated with reduced DNA damage after UV e as shown by reduced nuclear staining for cyclobutane pyrimidine dimers using a well-characterized monoclonal antibody [81,82,84,88,89], in mouse skin and in human skin treated topically with 1,25(OH)2D3 [83,89]. Indeed, it is likely that the reduced DNA damage contributed to reduced skin cell apoptosis. The reduction in DNA damage in human keratinocytes and mouse skin was reported to occur even when application of 1,25(OH)2D3 was immediately after UV irradiation [82,89], though this was not a universal finding [88]. The reduced DNA damage after UV was also seen in the presence of low-calcemic analogs [84,88,89].
Protection from Other UV Damage Both De Haes et al. [77] and Mitani et al. [90] reported a reduction in the inflammatory cytokine, interleukin-6, in skin cells after UV irradiation in the presence of D compounds, 1,25(OH)2D3 or ergocalciferol, respectively. Reduction in UV-induced interleukin-6 in mouse skin after topical 1,25(OH)2D3 treatment has also been reported [4]. Mitani et al. [90] also showed reduced photodamage, and lowered glycosaminoglycans in the skin of hairless mice exposed to chronic UV over 15 weeks and treated topically with ergocalciferol.
D Compounds and UV-induced Immune Suppression Vitamin D compounds have many immunomodulatory effects in skin. They inhibit antigen presenting cell maturation and function, induce activation of regulatory T cells, suppress T-helper type 1 (Th1) T cell responses and modify cytokine expression patterns [83]. More recently, 1,25(OH)2D3 was reported to increase IL-10 production, which is an immune suppressor, in mast cells [91]. Furthermore, the VDR has been identified in a number of different immune cell types, including monocytes, macrophages and activated T and B cells [92]. Thus, it is not surprising that the local production of active 1,25(OH)2D3 in skin cells has been proposed to play a role in UVB-mediated immune suppression. The effects of vitamin D to modulate the immune system are discussed in Chapters 91 and 92.
Even without UV exposure, murine and human studies of vitamin D and immunosuppression have led to an array of conflicting data (reviewed in [53]). In human subjects, the vitamin D analog calcipotriene suppressed contact hypersensitivity responses to dinitrochlorobenzene by 64%, a level similar to that caused by solar-simulated UV [93]. Moreover, a recent human study conducted by Damian et al. [83] showed immunosuppressive effects of 1,25(OH)2D3 in a recall delayedtype hypersensitivity Mantoux model when total 1,25 (OH)2D3 doses of 1 mg or higher were applied topically. Furthermore, Gorman et al. [94] showed increased suppressive capacity of CD4þCD25þ regulatory T cells in the skin-draining lymph nodes of BALB/c mice following a single topical application of 1,25(OH)2D3 [94]. Conversely, other studies in mice have shown that 1,25(OH)2D3 and analogs caused no direct change in immune responses [81,89]. As shown in Fig. 100.1a,c, 1,25(OH)2D3 caused no reduction in immune response (ear swelling) to the contact sensitizer oxazalone in hairless mice subjected to sham irradiation, but substantially reduced the immune suppression observed after UV irradiation (Fig. 100.1b,d). After UV irradiation, ear swelling in response to a challenge with oxazalone was reduced in the presence of vehicle, but in the presence of the vitamin D compound it was close to the degree of swelling seen in sham-irradiated mice (Fig. 100.1a,c). This was the case whether the mice had been sensitized to oxazalone 1 week after exposure to solar-simulated UV, followed by one topical treatment with vehicle or 1,25(OH)2D3 and rechallenged a week after that (Fig. 100.1a,b), or sensitized to oxazalone 1 week before UV exposure, followed by one topical treatment with vehicle or 1,25(OH)2D3), then rechallenged a week later (Fig. 100.1c,d). The non-genomic agonist 1,25-dihydroxylumisterol3 (JN) also conferred protection from UVinduced immune suppression in hairless mice [81]. Similar protection from UV-induced immunosuppression has also been observed in C3H mice (not shown).
Protection from Photocarcinogenesis Since DNA damage and immune suppression both contribute to photocarcinogenesis, and both are reduced by 1,25(OH)2D3, at least in some models, it is not surprising that there is a preliminary report indicating protection from chronic UV-induced tumor formation in mice treated topically immediately after each UV exposure with 1,25(OH)2D3 [4]. Conversely, in the presence of a mutated vitamin D receptor, photocarcinogenesis was enhanced [95]. Indeed, there is long-standing evidence that 1,25(OH)2D3 inhibits chemically induced skin carcinogenesis [96] and this has been confirmed with low calcemic vitamin D analogs [97].
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MECHANISMS OF PHOTOPROTECTION
(a)
(c) Sham vehicle
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Sham 22.8 ρmol/cm2 1,25(OH)2D3 2
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Contact hypersensitivity response after UV and 1,25(OH)2D3. (a) Contact hypersensitivity e normal protocol. Mice were irradiated with UV on day 1 and treated immediately afterwards with 1,25(OH)2D3 or vehicle. They were immunized with oxazalone on the abdomen on day 8 and challenged on the ear at day 15. Ear swelling was measured the next day. No reduction in immune response (ear swelling was similar to vehicle) was observed in SHAM-irradiated mice following treatment with 22.8 pmol/cm2 1,25(OH)2D3 compared to vehicle. There was a significant reduction in ear swelling in mice exposed to UV and treated with vehicle compared to SHAM vehicle-treated mice (p < 0.001). The ear swelling in mice exposed to UV and treated with 22.8 pmol/cm2 1,25(OH)2D3 was similar to that of the SHAM-irradiated mice. (b) Percent immunosuppression was calculated as 100% minus (difference between pre- and post-challenge ear thickness measurements of irradiated mice as a proportion of the difference between pre- and post-challenge ear thickness measurements for non-irradiated mice). There was a 26% reduction in percent immunosuppression in mice treated with 22.8 pmol/cm2 1,25(OH)2D3 compared to vehicle (p < 0.001). (c) Contact hypersensitivity e recall response. Mice were immunized with oxazalone on the abdomen on day 1, irradiated with UV and treated immediately afterwards with 1,25(OH)2D3 or vehicle on day 8 and challenged on the ear on day 15. There was no reduction in ear swelling in SHAM-irradiated mice following treatment with 22.8 pmol/cm2 1,25(OH)2D3 or 4.6 pmol/cm2 1,25(OH)2D3 compared to SHAM vehicle. After exposure to UV, ear swelling in mice treated with vehicle was significantly reduced compared to SHAM vehicle (p < 0.001). Ear swelling in mice treated with 22.8 pmol/cm2 1,25(OH)2D3 or 4.6 pmol/cm2 1,25(OH)2D3 and exposed to UV was not significantly different to SHAM-irradiated mice. (d) Percent immunosuppression was significantly reduced by 43% in mice treated with 22.8 pmol/cm2 1,25(OH)2D3 (p < 0.01) and by 33% in mice treated with 4.6 pmol/cm2 1,25(OH)2D3 (p < 0.01) compared to vehicle-treated mice.
FIGURE 100.1
MECHANISMS OF PHOTOPROTECTION Genomic and Non-genomic Pathways As discussed in Chapter 7, it is well established that 1,25(OH)2D3 can produce genomic biological responses via signal transduction pathways that utilize a nuclear vitamin D receptor (VDR) for 1,25(OH)2D3 to regulate gene transcription. However, as outlined in the chapter on “non-genomic actions” (Chapter 15), there is now considerable evidence to suggest that 1,25(OH)2D3 can
utilize other signal transduction mechanisms in order to generate non-genomic responses. Reasonable evidence has accumulated to support the proposal that the photoprotective effects of 1,25(OH)2D3 are mediated, at least in part, via a non-genomic mechanism. Two 6-scis locked rapid-acting agonists, JN (1a,25(OH)2lumisterol3) and JM (1a,25(OH)2-7-dehydrocholesterol), which have no gene transactivating capacity [71], entirely mimicked the photoprotective effects e reduction in UV-induced DNA damage and in UVinduced apoptosis e of 1,25(OH)2D3 in skin cells with
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similar potency to 1,25(OH)2D3 [84]. Furthermore, the non-genomic antagonist 1b,25-dihydroxyvitamin D3 (HL) completely abolished these photoprotective effects of 1,25(OH)2D3 in skin cells [84]. Moreover, an antagonist of the genomic pathway, (23S)-25-dehydro-1ahydroxyvitamin D3-26,23-lactone (TEI-9647), had no effect on the ability of 1,25(OH)2D3 to reduce UVinduced DNA damage [89]. The potential non-genomic 1,25(OH)2D3 pathways for UVR protection will be discussed next.
JNK, Akt, ERK and Chloride Channels It has been reported that 1,25(OH)2D3 inhibits UV-induced activation of the stress-activated c-Jun N-terminal kinase (JNK) in human keratinocytes, leading to decreased apoptosis [77]. Studies by another group using the transformed human keratinocyte line HaCaT also demonstrated inhibition of JNK by 1,25(OH)2D3 following cellular stressors, including, but not limited to, UVR [86]. It is likely that the inhibition of UV-induced JNK by 1,25(OH)2D3 is important in the reduction in UVinduced apoptosis, since preliminary studies showed a dose-dependent reduction in UV-induced cell loss using SP600125, which inhibits JNK activity by inhibiting phosphorylation of its downstream targets, such as c-Jun [98]. Preventing the 1,25(OH)2D3-induced suppression of JNK phosphorylation after UVR abolished the protective effect of 1,25(OH)2D3 on cell survival [98], but had no effect on the reduction in UVR-induced DNA damage by 1,25(OH)2D3 [99]. Treatment of primary human keratinocytes with 1,25(OH)2D3 enhanced basal levels of phosphorylation of Akt to levels comparable to well-known activators of the phosphatidylinositol 3 kinase (PI-3K)/Akt pathway, such as insulin-like growth factor 1 and epidermal growth factor [100]. Furthermore, treatment of keratinocytes with 1,25(OH)2D3 also enhanced phosphorylation of extracellular related-signaling kinase (ERK), a downstream target of the MEK pathway. In addition, 1,25(OH)2D3 treatment increased expression of the anti-apoptotic protein Bcl-2 and decreased levels of pro-apoptotic proteins Bax and Bad [100]. An inhibitor of the PI-3K/Akt pathway, LY294002, or an inhibitor of the MEK/ERK pathway, PD98059, combined with 1,25(OH)2D3 given prior to UVR, partially blocked the previously documented inhibition of UVR-induced apoptosis by 1,25(OH)2D3 [100]. One difficulty in interpreting these results is that UVR on its own also activates both Akt and ERK [101,102]. Furthermore, in preliminary studies, inhibitors of PI3K and ERK, in the presence of vehicle, reduced post-UVR-induced thymine dimers detected by the monoclonal antibody, so that it was not possible to interpret results in the
presence of 1,25(OH)2D3 [103]. In osteoblasts, the nongenomic effects of 1,25(OH)2D3 were reported to be inhibited by DIDS (4,40 -diisothiocyanatostilbene-2,20 disulfonic acid disodium salt hydrate), a chloride channel blocker [104]. There is a preliminary report that 1,25(OH)2D3-induced reductions in DNA damage were also decreased by DIDS [103].
Metallothionein and Sphingosine Metallothionein is a cysteine-rich protein involved in metal detoxification, which can be induced by many heavy metals including cadmium, and which is also photoprotective [105]. Metallothionein is also induced through a process of increased gene transcription by 1,25(OH)2D3 [106]. Increased metallothionein expression has been found in mice treated with 1,25(OH)2D3 along with a reduction in UV-induced sunburn cells [76]. This may indicate that some photoprotection is mediated through a genomic pathway. The relative contributions of genomic and non-genomic pathways to photoprotection are unclear, due to limited data at this stage. Preliminary unpublished data from photocarcinogenesis studies in mice indicate protection mediated by topically applied cis-locked analog, 1a,25(OH)2lumisterol3, but not to the same extent as that produced by 1,25(OH)2D3. A protective effect of 1,25(OH)2D3 in keratinocytes against not only UV but also treatment with TNFa or ceramides has been reported. This appeared to be mediated by production of sphingosine-1 phosphate, which prevented apoptosis [78]. However, relatively high doses of 1,25(OH)2D3 (approximately 107 M) were required to provide protection in this study [78].
Nitric Oxide and p53 As noted earlier, enhanced expression of nuclear p53 protein facilitates DNA repair [2] while increased nitric oxide and related reactive nitrogen species induce DNA damage which is mutagenic [20] and also inhibits DNA repair [41,42]. UV exposure increases nuclear expression of p53 in irradiated skin cells. Treatment of these cells with 1,25(OH)2D3 substantially enhances this increased nuclear p53 expression after UV exposure [82,89]. Conversely, 1,25(OH)2D3 reduces nitric oxide products after UV [82]. Although in that report, the nitric oxide products were measured by the relatively insensitive Griess reaction which picks up the stable end product, nitrite, the reduction in nitrite was statistically significant and was indistinguishable from the reduction caused by a well-known nitric oxide synthase inhibitor, aminoguanidine [82]. Nitric oxide synthase inhibitors such as aminoguanidine and L-N-monomethylarginine (L-NMMA) were also reported to reduce
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Mechanism of photoprotection by 1,25(OH)2D3
p53 expression
1,25(OH)2D3 (JN)
p53 expression
non-genomic pathway
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FIGURE 100.2 Proposed mechanism of photoprotection by vitamin D compounds. Exposure of skin cells to UV causes an increase in nuclear p53 expression, which NO products facilitates DNA repair and an increase in nitric oxide products which both reduce DNA repair and increase DNA damage. Application of 1,25(OH)2D3 immediately after UV exposure further enhances p53 expression and suppresses nitric oxide products. Both these actions would result in less DNA damage, with reduced CPD, reduced immunosuppression and reduced photocarcinogenesis and there is evidence for these effects in a number of systems. The non-genomic analog JN mimics the actions of 1,25(OH)2D3. Reprinted from [4] with permission from Elsevier. NO products
Less DNA damage
CPD human keratinocytes mice human subjects
immunosuppression mice
photocarcinogenesis mice
DNA damage, assessed by the monoclonal antibody to thymine dimers, to a similar extent to 1,25(OH)2D3 [82]. It seems likely that both the reduction in nitric oxide products and the further increase in p53 expression produced by 1,25(OH)2D3 contribute to reduced DNA damage (Fig. 100.2).
Hedgehog-patched Pathways Recently, an entirely different anti-cancer action of the parent compound vitamin D3 has been described. The hedgehog signal, which is important in patterning and development, is relayed through the interaction of two receptors, patched and smoothened. Binding of hedgehog to patched releases smoothened and its downstream transcription factor, glioma associated oncogene (Gli), from inhibition, allowing cell proliferation, amongst other effects [106a,106b]. Over-expression of hedgehog or mutations of patched, rendering it unabkle to bind smoothened, are associated with basal cell carcinomas of skin, as well as other tumor types [106a,106b]. There is now evidence that production or release of vitamin D3 under the influence of patched plays a role in the inhibition of smoothened and
subsequent inhibition of Gli [106a]. The suppression of Gli by vitamin D3 appears to be vitamin-D-receptorindependent, and depends on direct binding to smoothened [106a,106b]. While anti-proliferative activity of vitamin D3 has been demonstrated in some relevant cells in vitro, studies so far indicate little effect on tumor growth in vivo [106b].
PERSPECTIVES ON FUTURE USE OF VITAMIN D COMPOUNDS FOR SUN PROTECTION Virtually all of the studies showing photoprotection with vitamin D compounds have used topical applications of the active agents. There is one study, published in abstract form to date, indicating that UV-induced DNA damage (pyrimidine dimers detected by immunohistochemistry) was increased in vitamin-D-deficient neonatal mice, compared to vitamin-D-replete littermate controls [107]. The question of whether supplemental oral vitamin D might reduce UV-induced DNA damage in human skin is still open. There is no evidence that sun-induced skin cancer is increased in vitaminD-deficient people. The major pathogenetic factor in
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skin cancer is still UV exposure [108]. On the basis of experimental data, it is reasonable to propose that because vitamin D and its metabolites are produced in skin as a result of UVR exposure, the DNA damage from UVR is less than it would be otherwise. Supporting this proposal are reports that polymorphisms of the VDR gene affect the risk of solar keratoses [109], squamous cell carcinoma [110] as well as melanoma occurrence [111e113], thickness [114e116] and outcomes [111,117]. See Chapter 89 for further discussion of VDR, skin cancer and protection afforded by vitamin D. Because 1,25(OH)2D3 is produced in skin by the action of UV [6] and 1,25-dihydroxylumisterol3 (JN) may also be produced as the overirradiation product lumisterol3, which is converted into the 1,25-dihydroxylated derivative by local metabolic pathways [69,70], it is likely that accumulation of these compounds in skin contributes to protection against the next exposure to UV irradiation. These compounds, 1,25(OH)2D3 and JN, take several hours to be produced under normal conditions, which makes it possible to demonstrate active photoprotection when these compounds are provided locally at the time of irradiation or immediately afterwards. This means that it might be possible to reduce the DNA damage caused by UV that penetrates sunscreen preparations, which are often inadequately applied, by incorporating D compounds into sunscreens or into after-sun lotions. At present, this is impractical, because 1,25(OH)2D3 itself and JN are expensive to synthesize and susceptible to light and oxidative degradation. Less expensive, more stable vitamin D-like compounds need to be identified, which may confer photoprotection and may reduce the risk of skin cancers.
References [1] Y. Matsumura, H.N. Ananthaswamy, Short-term and long-term cellular and molecular events following UV irradiation of skin: implications for molecular medicine, Expert Rev. Mol. Med. 4 (2002) 1e22. [2] M.S. Eller, T. Maeda, C. Magnoni, D. Atwal, B.A. Gilchrest, Enhancement of DNA repair in human skin cells by thymidine dinucleotides: evidence for a p53-mediated mammalian SOS response, Proc. Natl. Acad. Sci. USA 94 (1997) 12627e12632. [3] I.E. Kochevar, M.A. Pathak, J.A. Parrish, Photophysics, photochemistry, and photobiology, in: Fitzpatrick’s Dermatology in General Medicine, fifth ed., McGraw Hill, Health Provisions Division, New York, 1999. [4] R.S. Mason, V.B. Sequeira, K.M. Dixon, C. Gordon-Thomson, K. Pobre, A. Dilley, et al., Photoprotection by 1alpha,25dihydroxyvitamin D and analogs: further studies on mechanisms and implications for UV-damage, J. Steroid Biochem. Mol. Biol. 121 (2010) 164e168. [5] D.D. Bikle, M.K. Nemanic, E. Gee, P. Elias, 1,25Dihydroxyvitamin D3 production by human keratinocytes. Kinetics and regulation, J. Clin. Invest. 78 (1986) 557e566.
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1 alpha,25-dihydroxyvitamin D3 in cultured cells and in mice, Proc. Natl. Acad. Sci. USA 84 (1987) 8810e8813. M.F. Bijlsma, C.A Spek, D. Zivkovic, S. van de Water, F. Rezaee, M.P. Peppelenbosch, Repression of smoothened by patcheddependent (pro-) vitamin D3 secretion, PLos Biol. 4 (2006) e232. L.W. Bruggemann, K.C.S Queiroz, K. Zamani, A. van Straaten, C.A. Spek, M.F. Bijlsma, Assessing the efficacy of the hedgehog pathway inhibitor vitamin D3 in a murine xenograft model for pancreatic cancer, Cancer Biol. Ther. 10 (2010) 79e88. T. Dharmadasa, R. Malley, G.M. Woods, Dietary vitamin D3 deficiency alters skin UVB-induced DNA damage in neonatal mice, Australian Health and Medical Research Congress, Melbourne, Australia, 2006, p 466. B.K. Armstrong, A. Kricker, The epidemiology of UV induced skin cancer, J. Photochem. Photobiol. B. 63 (2001) 8e18. M.A. Carless, T. Kraska, N. Lintell, R.E. Neale, A.C. Green, L.R. Griffiths, Polymorphisms of the VDR gene are associated with presence of solar keratoses on the skin, Br. J. Dermatol. 159 (2008) 804e810. J. Han, G.A. Colditz, D.J. Hunter, Polymorphisms in the MTHFR and VDR genes and skin cancer risk, Carcinogenesis 28 (2007) 390e397. J.A. Halsall, J.E. Osborne, L. Potter, J.H. Pringle, P.E. Hutchinson, A novel polymorphism in the 1A promoter region of the vitamin D receptor is associated with altered susceptibility and prognosis in malignant melanoma, Br. J. Cancer 91 (2004) 765e770. C. Li, Z. Liu, Z. Zhang, S.S. Strom, J.E. Gershenwald, V.G. Prieto, et al., Genetic variants of the vitamin D receptor gene alter risk of cutaneous melanoma, J. Invest. Dermatol. 127 (2007) 276e280. J.A. Randerson-Moor, J.C. Taylor, F. Elliott, Y.M. Chang, S. Beswick, K. Kukalizch, et al., Vitamin D receptor gene polymorphisms, serum 25-hydroxyvitamin D levels, and melanoma: UK caseecontrol comparisons and a meta-analysis of published VDR data, Eur. J. Cancer 45 (2009) 3271e3281. P.E. Hutchinson, J.E. Osborne, J.T. Lear, A.G. Smith, P.W. Bowers, P.N. Morris, et al., Vitamin D receptor polymorphisms are associated with altered prognosis in patients with malignant melanoma, Clin. Cancer Res. 6 (2000) 498e504. J.A. Newton-Bishop, S. Beswick, J. Randerson-Moor, Y.M. Chang, P. Affleck, F. Elliott, et al., Serum 25hydroxyvitamin D3 levels are associated with Breslow thickness at presentation and survival from melanoma, J. Clin. Oncol. 27 (2009) 5439e5444. C. Santonocito, R. Capizzi, P. Concolino, M.M. Lavieri, A. Paradisi, S. Gentileschi, et al., Association between cutaneous melanoma, Breslow thickness and vitamin D receptor BsmI polymorphism, Br. J. Dermatol. 156 (2007) 277e282. J.A. Halsall, J.E. Osborne, M.P. Epstein, J.H. Pringle, P.E. Hutchinson, The unfavorable effect of the A allele of the vitamin D receptor promoter polymorphism A-1012G has different mechanisms related to susceptibility and outcome of malignant melanoma, Dermatoendocrinol. 1 (2009) 54e57.
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C H A P T E R
101 The Role of Vitamin D in Osteoarthritis and Rheumatic Disease M. Kyla Shea 1, Timothy E. McAlindon 2 1
Wake Forest University School of Medicine, Winston-Salem, NC, USA 2 Tufts Medical Center, Boston, MA, USA
OSTEOARTHRITIS Osteoarthritis (OA) is the most common form of arthritis, affecting nearly 27 million Americans. The prevalence of OA increases with age. According to estimates from population-based analyses, over 37% of US adults over age 60 have radiographic OA and over 12% have symptomatic OA in at least one knee [1e4]. OA commonly affects joints of the hands and feet, the knees, hips, and spine. People with OA often have multiple-joint involvement. However, knee and hip OA are the leading cause of knee and hip replacements, and a leading cause of disability in older age. The estimated annual cost-burden associated with OA in the USA is now over $80 billion [5,6]. OA is characterized pathologically by focal damage to articular cartilage, ranging in severity from minor surface roughening to complete cartilage erosion, and accompanied by some form of reaction in the adjacent peri-articular bone [7]. Thus, the prevailing paradigm of OA during the last few decades has been primarily as a cartilage disorder with a secondary response in bone. However, recent insights into the biomechanical properties of the peri-articular bone and the material changes that occur in OA have highlighted its importance as a predicate or participant in the processes of OA progression. These observations, together with epidemiologic data indicating a complex relationship with systemic bone health, form the basis for the rationale that vitamin D might influence OA. Finally, the application of new imaging technologies has demonstrated that low-grade synovitis is present in a substantial proportion of OA joints and appears to be a factor in accelerated progression of structural joint damage. Thus, there are three broad pathological processes in
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10101-5
OA joints that could form the basis for therapeutic targets: (1) cartilage damage, (2) processes in subchondral/peri-articular bone, and (3) synovitis. Osteoarthritis is typically diagnosed by physical examination and radiographs of the affected joint, which are read for joint space narrowing (JSN) and presence of osteophytes. These radiologic features may be summarized using the Kellgren-Lawrence (K-L) scale, which ranges from 0 to 4 and increases as OA becomes more severe. A joint with a K-L score 2 is considered osteoarthritic [8]. The main risk factors for OA are age, gender, obesity, joint injury, and family history of OA. The risk of structural progression is strongly influenced by biomechanical factors, such as varus thrust at the knee, and possibly by some nutritional factors [9]. There is also a strong but complicated association of OA with systemic bone density. There are currently no accepted disease-modifying medical interventions for OA. The symptoms can be alleviated pharmaceutically (such as with non-steroidal anti-inflammatory drugs), and also with weight loss and regular exercise [10].
HYALINE CARTILAGE IN OA Biochemically, cartilage consists of a network of collagen fibrils (predominantly type II) which constrain an interlocking mesh of proteoglycans that resist compressive forces through their affinity for water. The tissue is relatively avascular and acellular. Turnover in healthy cartilage is slow and represents a balance between collagen and proteoglycan synthesis and degradation by enzymes such as metalloproteinases (MMPs).
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In early OA, the chondrocytes proliferate and become metabolically active. These hypertrophic chondrocytes express vitamin D receptors and produce cytokines (e.g., IL-1, TNF-a), degradative enzymes (e.g., MMPs), and growth factors. Proteoglycan production is increased in early OA, but falls sharply at a later stage when the chondrocyte “fails.” In an attempt to repair damage, chondrocytes proliferate and increase cartilage matrix synthesis. However, the newly synthesized cartilage is unorganized. As cartilage degrades, its surface becomes rough, fissures develop, and eventually the process progresses to regions of full-thickness erosion. With the disappearance of the collagen “scaffold,” the opportunity for matrix repair is lost. Ultimately, the opposing areas of cartilage loss cause “bone-on-bone” contact in the joint and acceleration of joint damage.
Potential of Vitamin D to Influence Cartilage Homeostasis It is well established that metabolites of 25(OH)D are involved in the transition from cartilage to bone in the developing growth plate. Adult chondrocytes express the 25-hydroxyvitamin D-1 a-hydroxylase enzyme (Cyp27b1), so are capable of converting the circulating vitamin D precursor to the active form (1,25(OH)2D), which can stimulate proteoglycan synthesis [11]. Absence of the Cyp27b1 enzyme leads to disrupted chondrocyte differentiation and growth plate hypertrophy, increased bone volume, and reduced osteoclastogenesis. Endochondral chondrocytes can also convert 25(OH)D to 24,25-dihydroxyvitamin D (24,25(OH)2D), which also may have a role in regulation of chondrocyte development and endochondral ossification [12] (see Chapter 28). The 24,25(OH)2D metabolite appears to be able to stimulate proteoglycan synthesis [12,13] and MMP production [14,15] in mature growth plate chondrocytes. These observations indicate that metabolites of 25(OH)D are involved in the transition from cartilage to bone in the developing growth plate. However, it is less clear if 1,25 (OH)2D and/or 24,25(OH)2D similarly influence articular cartilage and bone homeostasis in the development and/or progression of OA. It was thought that chondrocytes lost their vitamin D receptor (VDR) during bone development; however, more recent evidence indicates otherwise [16,17]. Chondrocytes from osteoarthritic knee cartilage specimens revealed an upregulation of the VDR, compared to chondrocytes from non-arthritic knee cartilage [17]. The presence of the VDR would indicate a role for 1,25(OH)2D directly, on chondrocyte function. Tetlow and Woolley suggest 1,25(OH)2D influences MMP and/or cytokine production [17,18]. MMPs, which are capable of degrading cartilage, are produced by chondrocytes [19]. In arthritic cartilage, MMPs 1, 3, and 9 were found to
colocalize to areas where articular chondrocytes expressed the VDR; however, this colocalization was not seen in healthy cartilage. Furthermore, 1,25(OH)2D3 enhanced MMP3 expression and reduced MMP9 and prostaglandin E2 expression [17]. Recent evidence demonstrates 1,25(OH)2D3 regulates MMP2 and MMP3 release from chondrocytes as well [15]. Tissue and circulating concentrations of MMPs are elevated in patients with OA [20,21]. Observational human studies showed vitamin D status to be inversely associated with circulating MMP9 in healthy adults, while vitamin D3 supplementation (3 monthly injections of either 500 IU or 50 000 IU) reduced MMP9 concentrations in adults with vitamin D insufficiency [22]. However, another study found no effect of vitamin D supplementation on MMP9 concentrations among young (20e40 years) and older (>64 years) men and women during winter [23]. However, the serum 25(OH)D levels in that study did not increase following supplementation (given orally, up to 600 IU/d for 22 weeks), so the opportunity for an effect may have been constricted.
BONE HEALTH AND OA Bone Changes in OA Animal models of OA show that subchondral bone thickening occurs early in, or even prior to, the development of cartilage loss [24,25]. The thickening is closely linked to cartilage destruction, although the temporality of this association is unclear [26e29]. The stiffness of the subchondral plate is related to its thickness, so thickening of the subchondral bone can impair its ability to dissipate compressive forces [30]. Consequently, increased subchondral bone stiffness has historically been suspected of contributing to the development and progression of OA [24,31e33]. On the other hand, the high bone turnover rate in OA also has the effect of reducing overall mineralization, so the bone may be less dense [34]. This would be expected to reduce its stiffness, elastic modulus, and strength. While the reduction in stiffness attributable to lower mineral content might offset the effects of subchondral plate thickening, the net effect of reduced material BMD is unclear. However, the observation that lower systemic BMD is strongly associated with knee OA progression suggests that lower mineral content might also be detrimental [35]. Many microstructural abnormalities are present in OA peri-articular trabecular bone. Trabeculae in subchondral bone from OA proximal tibiae are thicker, plate-like, and have lower mechanical competence [36]. Furthermore, the overall trabecular orientation in OA is altered and manifests regional differences across
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THE SYNOVIUM
the bone [37]. Studies of peri-articular trabecular bone tend to confirm reductions in biomechanical competence [38]. Day et al. found that the matrix modulus was reduced by 60% in the medial tibial condyle of knees with cartilage damage yet was associated with a higher trabecular bone volume fraction [26]. The apparent modulus was not significantly different from the control sites, but the ultimate stress was reduced. The increase in bone volume may be an attempt to compensate for the decrease in matrix modulus e models indicate that the stiffness of trabecular bone may increase in the setting of increased bone volume even if the matrix modulus decreases [38]. In population-based studies increased BMD at the hip has been associated with a higher prevalence of OA [39e41]. Two longitudinal studies of the influence of systemic BMD on knee OA suggest that, although higher BMD is associated with greater risk for incident OA, among individuals with established knee OA, lower BMD predisposes to progression [42,43]. Zhang et al. examined the relationship of femoral neck BMD with progression of radiographic knee OA among 473 women over 8 years [42]. Among knees with OA, the risk of progression was strongly and inversely related to femoral neck BMD. In the Chingford cohort, women with progressive knee OA (based on radiographic evidence of osteophytes or joint space narrowing) also had modestly lower baseline hip BMD (approx. 2.5%) [43]. These observations raise the possibility that poorer subchondral bone structure (reflected in a lower systemic BMD) results in impairment of its adaptive response to the mechanical stresses associated with knee OA. Of interest, in population-based analyses, the rates of non-vertebral and vertebral fracture are not lower among patients diagnosed with OA in spite of an increased BMD [44e47].
Vitamin D, Musculoskeletal Health, and Knee OA Progression Vitamin D’s function in maintaining skeletal health is well known (see Chapter 61). Since OA affects the whole joint, including the bone, it is plausible that vitamin D may influence OA indirectly through its influence on bone metabolism. Recently, knee subchondral BMD was found to be positively correlated with BMD of the femoral neck and lumbar spine, as well as with vitamin D status, cartilage defects, osteophytes, and joint space narrowing in older men and women [48]. Among older men and women with knee OA, the prevalence of insufficient vitamin D status (defined as <80 nmol/l or 32 ng/ml) was reported to be 66%, and femoral neck BMD was significantly higher among those who were vitamin-D-sufficient [49]. Overall the relationship between OA and BMD is complex, and
the precise mechanisms underlying the association between bone mineral and cartilage metabolism remain to be clarified. However, since vitamin D status is associated with both cartilage and skeletal outcomes, it is plausible that any influence of vitamin D on OA may be explained, at least in part, by its role in maintaining skeletal health. Vitamin D insufficiency has also been associated with muscle pain and weakness [50,51], so vitamin D may influence these disease elements of OA. VDRs have been identified in human skeletal muscle tissue [52,53], and are important for normal muscle function [54]. Bischoff et al. compared VDR expression in gluteal muscle tissue of patients undergoing hip replacement surgery, due to OA or to an osteoporotic hip fracture, to VDR expression of transversospinalis muscle in patients undergoing back surgery. The skeletal muscle tissue of hip surgery patients had significantly fewer VDR positive nuclei compared to back surgery patients, and VDR expression was inversely associated with age. However, there was no association between VDR expression and serum 25(OH)D and 1,25(OH)2D in these patients [52].
THE SYNOVIUM The prevailing view of knee OA has been one of a non-inflammatory disorder. However, chronic inflammatory changes in the synovium are seen in all stages of knee OA [55e57], appear to be of clinical significance [58e60], are associated with regions of cartilage damage [61], and predict structural progression [60]. Synovial biopsy studies of osteoarthritic knees have found a high prevalence of inflammation. One study of 63 patients with moderate to severe knee OA found thickening of the lining layer, increased vascularity, and inflammatory cell infiltration in synovial membranes in all knees, with the most prominent features in those with advanced disease [56]. However, synovitis appears common even in mild knee OA [55]. Another biopsy study of nine selected knees with mild OA and short symptom duration also showed a mild chronic synovitis in all cases [59]. Synovial changes in the knee also appear to be associated with pain and structural damage. An MRI study of over 400 individuals from both Veterans Affairs and community sources found more effusions and synovial thickening among those with knee pain [61]. Among the subset with symptomatic radiographic knee OA, synovial thickening was associated with the severity of knee pain. Loeuille et al., in scrutinizing the relationships between synovitis and cartilage damage among 39 patients with knee OA using arthroscopy, biopsy and MRI, found a correlation of synovitis with medial
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tibiofemoral cartilage damage [62]. There is also evidence that synovial inflammation participates in the structural progression of knee OA [60,63]. Ayral studied a cohort of 422 individuals with medial compartment tibiofemoral OA who participated in a (null) clinical trial that included a systematic assessment of synovitis and chondropathy using arthroscopy [60]. Twenty-one percent of participants had inflammatory changes in the synovium at baseline and these individuals were three times more likely to exhibit progression of cartilage damage. In a 2-month observational study of 29 knee OA patients with MRI-confirmed synovitis, Pelletier et al. found that the baseline synovitis score correlated significantly with the percentage loss in cartilage volume over this relatively short period [63]. IL-1 and TNF-a are the dominant cytokines in inflamed OA joints, of which IL-1 appears to have the greatest potential to mediate cartilage damage [64,65]. IL-1 is produced in considerable quantities in OA synovium [66,67] and appears to be the driving force in the production of destructive enzymes (such as MMPs) and interleukin-6 (IL-6) [68]. However, there are numerous other cytokines that may have specific and important roles in OA. IL-6, for example, may upregulate matrix metalloproteinase expression in cartilage, stimulate hepatic production of acute-phase proteins and mediate bone destruction [69]. In vitro and in vivo data have shown 1,25(OH)2D3 can suppress production of IL-1 and TNF-a from macrophages, monocytes, and other cell lines [70e73], but it is not known if 1,25(OH)2D3 affects chondrocytes the same way. While these data suggest an alternate mechanism by which vitamin D may influence cartilage degradation, additional studies are needed to elucidate whether or not 1,25(OH)2D affects inflammatory cytokine production by articular chondrocytes specifically.
VITAMIN D RECEPTOR (VDR) GENOTYPE AND OA PROGRESSION Genetic studies indicate that OA is transmitted as a complex, multi-factorial trait, and suggest heterogeneity in the nature of the encoded susceptibility [74]. Several candidate genes have been implicated in susceptibility, among which the VDR gene has been linked to OA development through mechanisms involving vitamin D and bone metabolism. VDR mediates the effects of calcitriol (1,25(OH)2D3) on the intestinal absorption of calcium and phosphate and on bone mineralization [75]. VDR genotype has been shown to be associated with circulating levels of 25(OH)D and 1,25(OH)2D in healthy populations [76], as both 1,25(OH)2D production and catabolism are VDR dependent [77]. VDR genotype is associated with BMD [78,79]
and is a risk factor for osteoporotic fracture [80] (see Chapter 56). In a population-based study of 846 people aged >55 years old, one VDR haplotype was associated with 2.3-fold increased risk of radiographic knee OA (95% CI 1.5e3.5), an effect largely driven by osteophytes [81]. In a study of 351 postmenopausal women, the VDR TaqI polymorphism was associated with a 2.8-fold (95% CI 1.2e6.9) increased risk of knee OA [82]. In another study, a VDR haplotype was associated with a significantly higher risk of knee OA in men (OR 2.0; 95% CI 1.5e2.8). A similar trend was seen in women, albeit non-significant (OR 1.3; 95% CI 1.0e1.9) [83]. However, other studies have been unable to reproduce such VDR genotype associations [84e87]. It is possible that the association between VDR genotypes, circulating vitamin D levels and OA is modulated by vitamin D and/or calcium intake. In fact, an interaction between VDR genotypes and vitamin D intake has been reported in some studies in other disorders/traits, including bone mass in children [88] and postmenopausal women [89], osteoporotic facture [90], prostate cancer [91], and colorectal cancer [92,93]. The genetic effects tend to be stronger among people with poor calcium/vitamin D status [80]. Considering the potential roles of BMD and vitamin D metabolism in OA progression and current evidence suggesting the links with VDR genotypes, it is plausible that VDR genotype could influence OA progression and that its effect may interact with vitamin D levels.
VITAMIN D STATUS AND OA PROGRESSION The prevalence of vitamin D insufficiency is reported to be between 15 and 50% in different studies of patients with OA [49,94e96]. The level of 25(OH)D considered to be insufficient is not clearly defined and the threshold used in these studies varied between <37.5 nmol/l [94] and <80 nmol/l [49]. The relationship between vitamin D status and OA in humans has been investigated in several observational studies. The design and results of these studies are summarized in Table 101.1. While some publications reported that vitamin D status was associated with prevalence and/or progression of OA [95,97e100], others found no association [101]. Among the studies that reported associations between vitamin D status and OA outcomes, the significant associations were not consistent across all disease measures [95,97e100]. When Framingham Heart Study participants (n ¼ 556) were categorized into tertiles according to serum 25(OH)D and vitamin D intake, neither serum 25(OH)D nor vitamin D intake were associated with
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TABLE 101.1
Observational Studies Assessing Associations between Vitamin D Status and OA OA measure
Vitamin D status measure
Results
Reference
1104 elderly men in the Osteoporotic Fractures in Men Study (mean SD age ¼ 77.2 5.3 years)
Crosssectional
Hip radiographs taken at baseline; hip OA defined according to summary score e for JSN or osteophytes
Serum 25(OH)D, measured at baseline
Inverse association with prevalent hip OA
[98]
880 men and women in the Tasmanian Older Adult Cohort, 353 at follow-up (mean SD age ¼ 61 7 years; 50% female)
Crosssectional and longitudinal
Knee radiographs taken at baseline; knee OA defined according to presence of JSN or osteophytes. Knee MRIs taken at baseline and 2.9 years later to measure cartilage volumes
Serum 25(OH)D measured at baseline and follow-up
Positive association between 25(OH)D and cartilage volume at baseline and between change in 25(OH)D and change in cartilage volume. No association between 25(OH)D and JSN or osteophytes
[95]
556 men and women (mean SD age ¼ 70.3 4.5 yrs; 63% female); Framingham Heart Study
Longitudinal
Knee radiographs taken between 1983 and 1985 and again between 1992 and 1993; knee OA defined according to change in KL grade, JSN, and osteophyte growth
Vitamin D intake and serum 25(OH)D, measured between 1988 and 1989
No association between vitamin D status and incident knee OA. No association between serum 25(OH)D and cartilage loss. Inverse association between serum 25(OH)D and change in KL grade, JSN, osteophyte growth, among those with pre-existing knee OA
[100]
237 postmenopausal women; 65 years old; Study of Osteoporotic Fractures
Longitudinal
Hip radiographs taken at baseline and 8 years later; hip OA defined according to JSN, osteophytes, and summary score
Serum 25(OH)D and 1,25(OH) 2D, measured at baseline
No association between serum 25(OH)D and development of osteophytes or radiographic summary score. No association between serum 1,25(OH)2D and any measure of hip OA. Inverse association between serum 25(OH)D and hip JSN
[99]
715 men and women in Framingham Offspring Cohort; (mean SD age ¼ 53.1 8.7 years; 53% female)
Longitudinal
Knee radiographs taken between 1993 and 1994 then again between 2002 and 2005; knee OA defined according to JSN
Serum 25(OH)D, measured between 1996 and 2000
No association between serum 25(OH)D and knee OA incidence or progression
[101]
253 men and women in Boston Osteoarthritis Knee Study, with pre-existing knee OA (mean SD age ¼ 66.4 9.4 years; 40% female)
Longitudinal
Knee radiographs and MRI taken at baseline, and at 15 and 30 months later; knee OA defined according to JSN or cartilage loss
Serum 25(OH)D, measured at baseline
No association between serum 25(OH)D and knee OA incidence or progression
[101]
1248 men and women, in the Rotterdam Study (mean SD age ¼ 66.2 6.7 years; 58% female)
Longitudinal
Knee radiographs taken at baseline and 6.5 years later; knee OA defined according to KL score
Vitamin D intake and serum 25(OH)D, measured at baseline
Inverse association between vitamin D intake and knee OA progression. Significant interaction between vitamin D intake and BMD with respect to incident knee OA. No association between serum 25(OH)D and incidence or progression of knee OA
[97]
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Design
VITAMIN D STATUS AND OA PROGRESSION
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Participants
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incident knee OA. However, among participants with knee OA at the first assessment (n ¼ 162), serum 25(OH)D and vitamin D intake were inversely associated with risk for OA progression, when defined as an increase of at least 1 on the K-L scale (OR (95% CIs) lowest versus highest tertile: serum 25(OH)D ¼ 2.89 (1.01e8.25); vitamin D intake ¼ 4.05 (1.40e11.6)). Also participants with prevalent OA in the lower tertiles of serum 25(OH)D were more likely to develop JSN and osteophytes (OR (95% CI) lowest versus highest tertile ¼ 2.3 (0.9e5.5) for JSN, 3.0 (1.3e7.5) for osteophyte growth). However, serum 25(OH)D was not significantly associated with cartilage loss [100]. In a cohort of older Australian adults (age range 51e79 years) examined cross-sectionally, those with serum 25(OH)D <50 nmol/l at baseline had significantly lower medial and lateral tibial cartilage volume (assessed by magnetic resonance imaging (MRI)) compared to those with 50 nmol/l serum 25(OH)D. Serum 25(OH)D levels were also positively associated with cartilage volume when analyzed as continuous measures. There were no differences in JSN or presence of osteophytes between those with <50 nmol/l and those with 50 nmol/l serum 25(OH)D. However, in a subgroup analysis of participants with KL grade 2 at baseline, those with <50 nmol/l 25(OH)D were 1.68 times more likely to have JSN, compared to those with 50 nmol/l. In the 353 participants who had a followup knee MRI 2.9 years later, baseline serum 25(OH)D and the change in serum 25(OH)D were positively associated with annual change in cartilage volume, and 25(OH)D <50 nmol/l was predictive of cartilage loss [95]. In post-menopausal women participating in the Study of Osteoporotic Fracture (n ¼ 237), women in the lower two tertiles of serum 25(OH)D were more likely to develop hip OA defined according to JSN. However, serum 25(OH)D was not associated with incident hip OA, defined according to an individual radiographic feature score 2 or presence of osteophytes, over 8 years of follow-up. In this same study, serum 1,25(OH)2D was not associated with any measure of hip OA [99]. In a cross-sectional analysis of elderly men, lower serum 25(OH)D was associated with higher prevalence of hip OA, defined as a summary score 2 (based on presence of JSN and/or osteophytes) (OR 1.39; 95% CI 1.11e1.74, per standard deviation decrease in serum 25(OH)D). Those men with serum 25(OH)D between 37.5 and 75 nmol/l were more likely to have hip OA than men with 75 nmol/l (OR 2.19; 95% CI 1.21e3.97), and men with <37.5 nmol/l tended towards a higher odds of hip OA (OR 1.99; 95% CI 0.83e4.74) [98]. In 715 participants of the Framingham Offspring Study, plasma 25(OH)D was not associated with joint space loss in the knee over 9.5 years of follow-up. These
findings were supported by a longitudinal analysis of knee OA progression, in which serum 25(OH)D was not associated with cartilage loss, assessed using MRI, among 211 men and women with pre-existing knee OA, over 30 months of follow-up [101]. Bergink et al. also found no association between serum 25(OH)D and incident knee OA among 1248 men and women in the Rotterdam Study over 6.5 years of follow-up. However, they reported that BMD at the lumbar spine influenced the association between vitamin D intake and incident knee OA, such that low vitamin D intake was associated with higher incidence of knee OA among those with lower lumbar spine BMD [97]. Conversely, Bischoff-Ferrari et al. reported a positive association between 25(OH) D and BMD in 228 patients with knee OA [49]. Considering vitamin D’s role in both cartilage and bone metabolism, the inter-relationship between OA, BMD, and vitamin D status is not unexpected, and merits clarification in future studies. Furthermore, the inconsistent results from observational studies may be clarified by outcomes of ongoing randomized controlled trials of vitamin D supplementation in patients with OA.
RHEUMATOID ARTHRITIS Rheumatoid arthritis (RA) is an auto-immune disease characterized by joint inflammation leading to chronic pain, functional impairment and disability. The prevalence of RA is between 0.5 and 1.0% of the adult population in developed countries. It is estimated that approximately 1.3 million adults in the USA suffer from RA [2], at a total annual cost burden of over $19 billion [102]. The etiology of RA is unknown. The pathophysiology of RA is complex and has been described in detail elsewhere [103e105]. Briefly, localization of lymphocytes to the synovial tissue triggers cytokine production and attracts other immune cells to the area. Activated T-cells, B-cells, macrophages, and synovial fibroblasts are present in the synovium of joints affected by RA. Unlike OA, inflammation is a prominent feature of RA. There is a substantial elevation in TNF-a, and other pro-inflammatory cytokines, such as IL-1b and IL-6, in the synovium. These cytokines promote MMP activation, leading to cartilage degradation. At the same time, osteoclast cells, which are responsible for bone resorption, are recruited in response to T-cells and cytokine signaling, and contribute to the bone destruction that occurs in RA.
Underlying Mechanisms: Vitamin D and RA Similar to osteoarthritic cartilage, chondrocytes, synoviocytes, and macrophages from rheumatoid synovial tissue express the VDR [18,106]. In vitro data demonstrate
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RHEUMATOID ARTHRITIS
that 1,25(OH)2D3 treatment of rheumatoid synovial fibroblasts suppresses an IL-1b-induced increase in MMP-1, MMP-3, and PGE2 production. However, there was no change in MMP-1, MMP-3, and PGE2 produced when fibroblasts were treated with 1,25(OH)2D3 in the absence of IL-1b, which suggests the vitamin D hormone may mitigate cytokine-induced upregulation of MMP and PGE2 by synovial fibroblasts in RA. Many immune system cells express the 25hydroxyvitamin D-1 a-hydroxylase enzyme, and can therefore produce the 1,25(OH)2D hormone (see Chapter 45). At the same time, the VDR is present in several cells of the immune system, including lymphocytes, monocytes, and macrophages. It is plausible, therefore, that vitamin D influences RA via its effect on immune function. This role of vitamin D is thoroughly described in Chapters 91 and 92. Briefly, 1,25(OH)2D inhibits the maturation of dendritic cells, suppresses T-cell activation and reduces production of pro-inflammatory cytokines (including TNF-a) by helper T-cells [107e110] in vitro. Monocytes and macrophages are also capable of producing 1,25(OH)2D [111], and 1,25(OH)2D suppresses inflammatory cytokine production by these cells as well [108]. In a recent ex vivo experiment, 1,25(OH)2D3 treatment of peripheral blood mononuclear cells from patients with early RA inhibited production of interferon-g, IL-17, IL-22, and TNFa by memory T-cells. Since elevated cytokines can have a negative influence on bone, the authors proposed that this mechanism may contribute to the favorable influence of active vitamin D and vitamin D analogs on bone loss in RA patients taking corticosteroids [112]. Patients with RA are at greater risk for low BMD and osteoporotic fracture [113,114]. This may be due, in part, to disease-related variables, as well as to treatment. Many pro-inflammatory cytokines (including TNFa, IL-1, IL-6, prostaglandin E2) can directly or indirectly promote the differentiation and activation of osteoclasts, the cells responsible for bone resorption [115,116], so the pro-inflammatory state that is characteristic of RA can promote bone loss [117e119]. Glucocorticoid medications, which are commonly used to reduce inflammation and pain in RA, reduce the proliferation and bone formation activity of osteoblasts, disrupting the balance between bone formation and bone resorption, in favor of bone loss [120,121]. In addition, glucocorticoid medications can inhibit intestinal calcium absorption [122]. Prolonged use of these steroid medications, as is common in RA, is associated with bone loss and fracture in patients with RA [123e126]. Vitamin D may, therefore, indirectly influence RA through an effect on bone loss. (Vitamin D’s role in glucocorticoid-induced bone loss is discussed in Chapter 66.) The effect of vitamin D supplementation (as cholecalciferol or ergocalciferol) to treat [127e130] and prevent [129,131e133] glucocorticoid-induced
1961
bone loss has been studied in patients with RA specifically [128] and other inflammatory diseases [127,129,129e132]. Some have compared the effect of vitamin D plus calcium to placebo [128,131,133], while others sought to determine if calcium supplementation with vitamin D was better than calcium supplementation without vitamin D [129,130,132]. The results of these studies, which utilized different interventions and patient populations, are inconsistent making it difficult to discern the efficacy of vitamin D in treating or preventing glucocorticoid-induced osteoporosis. However, outcomes of more recent trials and one meta-analysis suggest that active vitamin D analogs (such as alfacalcidol) are more effective than the hormonally inactive forms of vitamin D (such as cholecalciferol or ergocalciferol) in reducing osteoporosis in RA patients [134e137]. Patients with RA are also at greater risk for falling [138e141]. This increase in falls has been attributed to osteoporosis [139], swollen joints [138,140], postural instability [140], visual impairment [138], poorer physical performance, and greater disability [138,140,141]. Although results from randomized trials of vitamin D supplementation and fall outcomes are inconsistent [142e148], recent evidence from a meta-analysis suggests high-dose vitamin D (700 IU/day) given with calcium can reduce fall risk in older adults [149]. (The role of vitamin D in fall-risk is described in greater detail in Chapter 62.) Vitamin D’s role in improving fall risk may be related to its function in maintaining skeletal health and/or to its effect on skeletal muscle [54]. Therefore, an indirect role of vitamin D on RA may be through its function in lowering fall risk in RA patients, since several correlates of fall risk in this patient population are positively influenced by vitamin D. However, this has not yet been tested in intervention studies of RA patients. The effects of VDR polymorphisms have been studied in relation to RA susceptibility and to bone loss in patients with RA. Smaller caseecontrol studies report inconsistent associations between VDR allelic variation and RA [150e153], and larger genome-wide association studies have not identified the VDR region (which is located on chromosome 12) as being associated with RA [154e156]. Together these findings indicate that genetic variation in the VDR does not contribute to RA onset. However, the VDR polymorphisms that have been associated with osteoporosis (BsmI, ApaI, TaqI, FokI) have been studied in relation to bone loss specifically in RA patients, with inconsistent outcomes. Some have reported BsmI and TaqI variation is associated with increased bone turnover and accelerated bone loss in patients diagnosed with RA [151,157], while others report no association between VDR polymorphisms and bone loss in RA [153,158,159]. Considering
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101. THE ROLE OF VITAMIN D IN OSTEOARTHRITIS AND RHEUMATIC DISEASE
the small sample size used in these studies, the contribution of genetic variation in the VDR to bone loss in RA patients needs to be clarified.
Animal Studies of Vitamin D and RA In rats injected with type II collagen to induce RA, treatment with 1,25(OH)2D3 given orally each day for 60 days reduced the incidence of arthritis (measured by paw swelling), as well as the levels of anti-type II collagen IgG antibodies, synovial lining cell proliferation, and bone and cartilage destruction [160]. Similarly, dietary supplementation with 1,25(OH)2D3 (20 ng/day per mouse) for 21 days lowered the incidence of RA (based on ankle and paw inflammation) by 50% in mice also immunized with type II collagen to induce inflammatory arthritis. Mice supplemented with 1,25(OH)2D3 after they developed type II collagen-induced arthritis had a lower arthritis severity score compared to mice that did not receive 1,25(OH)2D3, suggesting 1,25(OH)2D3 reduced the progression of RA. The same 1,25(OH)2D3 dietary supplementation (20 ng/day for 21 days) reduced the number of arthritic lesions in Lyme arthritis-induced mice, another animal model of RA [161].
Human Studies of Vitamin D and RA The relationship between vitamin D status and RA in humans has been studied observationally, with inconsistent outcomes. The results of these studies are summarized in Table 101.2. In 1987 Als et al. compared serum 25(OH)D, 1,25(OH)2D and vitamin D metabolites (24,25(OH)2D and 25,26(OH)2D) between patients with RA and controls. RA patients were categorized according to current treatment (gold salts, penicillamine, or glucocorticoids). Vitamin D status was measured between November and January. Serum 25(OH)D was significantly lower in all RA patients, compared to controls, while 1,25(OH)2D concentrations were significantly lower among patients being treated with penacillamine or glucocorticoids. Serum 24,24(OH)2D and 25,26(OH)2D did not differ between patients and controls. These investigators concluded that the lower vitamin D status among patients was not related to disease pathology, but rather was the result of the disease, since patients with RA were less likely to be active outdoors [162]. However, more recent caseecontrol studies did not find differences in vitamin D status between patients with RA and controls [163e165]. There is disagreement among cross-sectional studies that have examined the association between vitamin D status and disease activity in patients with RA. Oelzner et al. found serum 1,25(OH)2D, but not 25(OH)D, to be inversely correlated with RA disease activity, assessed according to circulating concentrations of inflammatory
markers and erythrocyte sedimentation rate [96,166]. Low serum 25(OH)D was, however, associated with elevated disease activity and inflammation among 65 patients with RA from Turkey [165]. In a cross-sectional analysis of 206 patients with inflammatory polyarthritis from the UK, serum 25(OH)D was inversely associated with swollen joint count, DAS28, HAQ [167]. Yet, among 266 African-American RA patients from four clinical centers in the USA, serum 25(OH)D was not associated with disease activity, joint pain, or swollen joint count, after adjustment for age, gender, and season [94]. It is important to consider that 50% of African-Americans in this study had 25(OH)D <37.5 nmol/l and 99% had 25(OH)D <80 nmol/l, while the mean (SD) 25(OH)D concentration in the patients from Turkey was 104.9 (60.1) nmol/l [165]. Longitudinal associations between vitamin D intake and incident RA are similarly inconsistent. Women with a vitamin D intake >467 IU/ day (the highest tertile) had a lower risk of developing RA, compared to women who consumed <221 IU/day (RR 0.67; 95% CI 0.44e1.00) [168]. However, these results were not supported by a similar analysis of women in the Nurses Health Study [169]. The effect of vitamin D treatment on RA outcomes has not been studied extensively in clinical trials. The only known trial of vitamin D treatment in patients with RA is a small (n ¼ 19) 3-month open label trial, in which treatment with 1,25(OH)2D3 improved symptoms of disease activity in patients with RA [170]. Based on the available data, the association between vitamin D status and RA incidence or severity is not clear. According to the majority of caseecontrol comparisons [163e165], it does not appear that vitamin D status differs between RA patients and controls. Crosssectional analyses of patients with RA suggest lower vitamin D status may be associated with more severe RA. However, overall these studies have utilized small sample sizes, and therefore merit replication in larger cohorts. Likewise, while the association between vitamin D intake and incident RA has been assessed in large-scale epidemiological studies [168,169], the association between 25(OH)D and incident RA merits exploration in larger studies as well. Therefore, the role of vitamin D in RA progression needs to be investigated using cohort studies with larger sample sizes and in blinded, randomized, controlled trials.
SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) SLE is a chronic inflammatory autoimmune disease with a complex etiology, with both environmental and genetic contributions. According to NHANES III, SLE afflicts 54/100 000 adults and 100/100 000 adult women
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
TABLE 101.2
Observational Studies Assessing Associations between Vitamin D Status and RA Design
102 patients with RA
Caseecontrol
54 female patients with RA and 30 age-matched controls from Estonia; 64 female patients with RA and 35 age-matched controls from Italy
Caseecontrol
76 cases and 154 controls, considered to be at risk for RA
RA measure
Vitamin D status measure
Results
Reference
Serum 25(OH)D, 1,25(OH)2D, 24,25(OH)2D3, 25,26(OH)2D
RA patients had significantly lower 25(OH)D3; serum 25(OH)D3, 24,25(OH)2D3, 25,26(OH) 2D were inversely associated with functional class in RA patients
[162]
Disease Activity Score (DAS28)
Serum 25(OH)D, measured in winter and summer
No difference in 25(OH)D between RA patients and agematched controls; RA patient and controls from Estonia had lower 25(OH)D compared to patients and controls from Italy; 25(OH)D was inversely correlated with DAS28 in patients from Estonia during winter and in patients from Italy during summer
[163]
Caseecontrol
Cases identified as those positive for at least two rheumatic factors (RF) or anticyclic citrillinated antibodies; controls were negative for antibody tests
Serum 25(OH)D
No difference in serum 25(OH)D between cases and controls
[164]
65 patients with RA (mean age ¼ 46.3 years) and 40 healthy controls (mean age ¼ 44.8 years)
Caseecontrol and crosssectional
Disease activity measured according to DAS28, HAQ, CRP; RA patients categorized as low, moderate, high disease activity according to DAS28
Serum 25(OH)D
No difference in serum 25(OH)D between cases and controls; among RA patients, 25(OH)D was inversely correlated with DAS28, CRO, and HAQ; patients with high disease activity had significantly lower 25(OH)D compared to low and moderate disease activity groups
[165]
96 patients with RA (83 women, mean age ¼ 54.7 years)
Crosssectional
Disease activity measured according to serum CRP
Serum 25(OH)D, 1,25(OH)2D, PTH
1,25(OH)2D inversely correlated with CRP and PTH; 25(OH)D was not associated with CRP
[96]
121 patients with early inflammatory joint disease (85 with RA)
Crosssectional
Disease activity measured according to joint assessment, CRP, DAS28
Serum 25(OH)D
42% of patients were vitamin D deficient. Serum 25(OH)D was not associated with any measure of inflammatory joint disease
[204]
SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)
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Participants
(Continued)
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Observational Studies Assessing Associations between Vitamin D Status and RAdcont’d
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Participants
Design
RA measure
Vitamin D status measure
Results
Reference
206 patients with early inflammatory polyarthritis (132 women, median age ¼ 59 years, 73 with RA)
Crosssectional and longitudinal
Disease activity measured according to joint assessment, CRP, DAS28, Health Assessment Questionnaire (HAQ) at baseline and 1 year of follow-up
Serum 25(OH)D, 1,25(OH)2D
Cross-sectionally, serum 25(OH)D inversely associated with tender joint count, DAS28, HAQ; 1,25(OH)2D inversely associated with HAQ. Baseline 25(OH)D was inversely associated with HAQ and tender joint count at year 1
[167]
266 African-American men and women with RA (216 women, mean age ¼ 51.3 years)
Crosssectional and longitudinal
Disease activity measured according to joint assessment, CRP, DAS28, Health Assessment Questionnaire (HAQ) at baseline and 3 years of follow-up
Serum 25(OH)D
50% of patients had serum, 25(OH)D <37.5 nmol/L. Serum 25(OH)D was not associated with any measure of RA, after adjustment for age, sex, season, cross-sectionally or longitudinally
[169]
186 389 women in Nurses Health Study
Longitudinal
Incident RA, confirmed through medical review, between 1980 and 2002
Vitamin D intake
No association between vitamin D intake and incident RA
[169]
29 386 women from Iowa Women’s Health Study without RA (age range 55e69 years)
Longitudinal
Incident RA, confirmed through medical review, over 11 years of follow-up
Vitamin D intake
Higher vitamin D intake was associated with lower risk of developing RA
[168]
101. THE ROLE OF VITAMIN D IN OSTEOARTHRITIS AND RHEUMATIC DISEASE
TABLE 101.2
SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)
18 years and older [171]. The incidence of SLE is three times greater in African-Americans compared to Caucasians [172]. The dysfunctional immune response in SLE generates autoantibodies that form immune complexes and promote inflammation and damage to multiple organ systems, including skin, heart, lungs, kidneys, and/or nervous system. Circulating concentrations of pro-inflammatory cytokines (including TNF-a, IL-1, and IL-6) are elevated in patients with SLE and positively correlate with disease severity [173e176]. Although clinical features can vary from patient to patient, skin rashes, photosensitivity and joint swelling and pain are common features.
Underlying Mechanisms: Vitamin D and SLE A role for vitamin D in SLE is plausible, as it functions in both the adaptive and innate immune responses (as described in Chapters 91 and 92). In SLE, multiple aspects of the immune system become dysregulated. T-cells, B-cell, and dendritic cells play key roles in SLE. Treatment of B-cells from patients with SLE with 1,25(OH)2D3 inhibited B-cell proliferation and suppressed antibody and autoantibody production [177,178]. Improving vitamin D status in patients with SLE has been suggested to reduce B-cell overactivity that occurs in SLE and other autoimmune disorders [177]. SLE is also characterized by self-targeted T-cells that promote an inflammatory response. Vitamin D acts to inhibit proliferation of and cytokine production by Th1 cells and to overall suppress Th1 immune response [179e181]. Adequate levels of 1,25(OH)2D3 inhibit the maturation of dendritic cells [182]. Immature dendritic cells are less capable of stimulating T-cell activation and expanding the T-cell and B-cell reactivity. It has been shown that the response of monocyte-derived dendritic cells from patients with SLE to 1,25(OH)2D3 treatment does not differ from the response of the same cells taken from healthy individuals. Since the immune response to 1,25(OH)2D3 was shown to be adequate, improving vitamin D status may represent a means by which to improve immune function (via suppression of dendritic cell maturation) in SLE patients [183].
Animal Studies of Vitamin D and SLE Several rodent models of SLE exist [184] and they have been used to determine the effect of vitamin D treatment on SLE symptoms in a limited number of experiments. Injection of 1,25(OH)2D3 in MRL/lpr mice (which spontaneously develop SLE) daily for 4 weeks then every other day for 18 weeks, inhibited dermatologic lesions and serum antibody titers, compared to mice not injected with 1,25(OH)2D3 [185]. Similar improvements in kidney function and T-cell
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immune response were observed in MRL/lpr mice administered 1,24(OH)2D3 compared to MRL/lpr mice administered a corticosteroid [186]. MRL/lpr mice that received a diet containing 1,25(OH)2D3 with adequate calcium had improved SLE severity scores compared to mice not receiving the 1,25(OH)2D3. Interestingly, dietary 1,25(OH)2D3 supplementation in the absence of adequate calcium exacerbated SLE symptoms in MRL/ lpr mice [187]. However, weekly injection with cholecalciferol (vitamin D3) did not improve SLE symptoms in NZBxW mice (an alternate mouse model of SLE). These investigators observed a worsening of SLE severity in the mice injected with 3 mg/week compared to mice injected with 10 mg/week vitamin D3 and mice not injected with vitamin D3 [188].
Human Studies of Vitamin D and SLE Vitamin D insufficiency and deficiency are prevalent among patients with SLE [189e191], even among populations living in sunnier climates [192e194]. This may be attributed to a combination of factors, including sun avoidance due to photosensitivity, medication use, and/or kidney function [189,195]. While reports of vitamin D insufficiency among patients with SLE are fairly numerous, reports of associations between vitamin D status and SLE disease risk and severity are less numerous. Costenbader et al. did not find any association between dietary vitamin D intake and risk of developing SLE in over 180 000 women participating in the Nurses Health Study. Since only 190 incident cases of SLE were documented over the 22 years of follow-up, isolating the effect of an intake of a single nutrient assessed by questionnaire on disease risk would have been difficult, given the variability in nutrient intakes quantified using the food frequency questionnaire [169]. In a study of SLE patients from northern and southern Europe, serum 25(OH)D was found to be inversely correlated with disease severity (measured according to the European Consensus Lupus Activity Measurement and the SLE Disease Activity Index (SLEDAI)) [195]. In a pooled-cohort of 378 SLE patients from Israel and Europe, serum 25(OH)D was negatively correlated with disease severity, measured according to the SLEDAI or the European Consensus Lupus Activity Measurement [196]. However, among SLE patients from Spain (n ¼ 92), serum 25(OH)D was not associated with SLE disease duration or severity (also measured according to the SLEDAI). In this cohort, patients with severe vitamin D deficiency (25(OH)D <10 ng/ml) had higher scores on the fatigue scale [193]. In a 2-year follow-up of this cohort, 60 of the 80 patients who were available for follow-up reported taking vitamin D3 supplements (doses ranged from 400 to 1200 IU/day). Improved serum 25(OH)D was associated with
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101. THE ROLE OF VITAMIN D IN OSTEOARTHRITIS AND RHEUMATIC DISEASE
improved scores on the fatigue scale, among those with serum 25(OH)D <30 ng/ml at baseline. However, the non-significant association between 25(OH)D and disease severity persisted 2 years later [197]. Based on the available data, the association between vitamin D status and SLE remains unclear and merits further investigation in larger cohorts. Furthermore, randomized trials are needed to clarify whether or not treatment with vitamin D improves disease severity and/or progression in patients with SLE.
PSORIATIC ARTHRITIS (PsA) PsA is a unique form of inflammatory arthritis that may accompany psoriasis, which is an autoimmune disease affecting the skin (see also Chapter 97). It is estimated that 25e30% of people with psoriasis will develop PsA. Usually, but not always, psoriasis develops before the onset of PsA [198]. T-lymphocytes drive the pathology of psoriasis and PsA. In psoriasis, T-cells are activated and migrate to the skin, where the production of inflammatory cytokines (including TNF-a, IL-2, interferon-g) is increased, leading to a hyperproliferation of keratinocytes, causing skin lesions, scaling, and plaque. This dysregulated pattern of T-cell activation and cytokine production also plays a role central to joint inflammation in PsA. Synovial tissue in PsA is infiltrated with T-cells and B-cells, and expresses pro-inflammatory cytokines, including TNF-a, IL-1b, IL-6, and interferon-g [199,200]. Hence, the skin and joint pathology in PsA share common inflammatory pathways. Topical vitamin D, in the form of 1,25(OH)2D3, is an FDA-approved treatment for psoriasis in adults 18 years and older [201]. In ointment form, 1,25(OH)2D3 calcitriol suppresses the immune response and reduces inflammation in the skin, by locally regulating dendritic cells and modifying T-lymphocyte activation [202]. Therefore, vitamin D may influence both the skin and joint inflammation in PsA. The only intervention study of vitamin D treatment in PsA was an open-label trial in which ten patients with PsA were given 2 mg/day 1,25(OH)2D3 orally. Following 6 months of treatment, seven patients reported at least moderate improvement in tender joint count [203]. Although this evidence is suggestive, the association between vitamin D status and PsA severity and the effect of vitamin D treatment among patients with PsA merits further investigation in larger studies.
CONCLUSIONS Although there is a growing body of evidence that vitamin D is important in joint health and mechanisms
have been identified that support a protective role for vitamin D in OA and rheumatic diseases, much remains to be understood about vitamin D’s function in these diseases. While several observational studies suggest vitamin D insufficiency is associated with increased risk for OA progression [97,99,100], with more severe RA [96,165,166] and with SLE severity [195,196], there is still an overall lack of consistency among the available studies [94,99,101,169,197]. The results of ongoing and future randomized controlled intervention trials we hope will clarify the influence of vitamin D on disease progression in patients with OA and rheumatic disease.
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[177] S. Chen, G.P. Sims, X.X. Chen, Y.Y. Gu, S. Chen, P.E. Lipsky, Modulatory effects of 1,25-dihydroxyvitamin D3 on human B cell differentiation, J. Immunol. 179 (2007) 1634e1647. [178] M. Linker-Israeli, E. Elstner, J.R. Klinenberg, D.J. Wallace, H.P. Koeffler, Vitamin D(3) and its synthetic analogs inhibit the spontaneous in vitro immunoglobulin production by SLEderived PBMC, Clin. Immunol. 99 (2001) 82e93. [179] J.M. Lemire, D.C. Archer, L. Beck, H.L. Spiegelberg, Immunosuppressive actions of 1,25-dihydroxyvitamin D3: preferential inhibition of Th1 functions, J. Nutr. 125 (1995) 1704Se1708S. [180] A.K. Bhalla, E.P. Amento, B. Serog, L.H. Glimcher, 1,25-Dihydroxyvitamin D3 inhibits antigen-induced T cell activation, J. Immunol. 133 (1984) 1748e1754. [181] W.F. Rigby, S. Denome, M.W. Fanger, Regulation of lymphokine production and human T lymphocyte activation by 1,25dihydroxyvitamin D3. Specific inhibition at the level of messenger RNA, J. Clin. Invest. 79 (1987) 1659e1664. [182] M.D. Griffin, W. Lutz, V.A. Phan, L.A. Bachman, D.J. McKean, R. Kumar, Dendritic cell modulation by 1alpha,25 dihydroxyvitamin D3 and its analogs: a vitamin D receptor-dependent pathway that promotes a persistent state of immaturity in vitro and in vivo, Proc. Natl. Acad. Sci. USA 98 (2001) 6800e6805. [183] I. Ben-Zvi, C. Aranow, M. Mackay, A. Stanevsky, D.L. Kamen, L.M. Marinescu, et al., The impact of vitamin D on dendritic cell function in patients with systemic lupus erythematosus, PLoS One 5 (2010) e9193. [184] L. Morel, Mouse models of human autoimmune diseases: essential tools that require the proper controls, PLoS Biol. 2 (2004) E241. [185] J.M. Lemire, A. Ince, M. Takashima, 1,25-Dihydroxyvitamin D3 attenuates the expression of experimental murine lupus of MRL/l mice, Autoimmunity 12 (1992) 143e148. [186] T. Koizumi, Y. Nakao, T. Matsui, T. Nakagawa, S. Matsuda, K. Komoriya, et al., Effects of corticosteroid and 1,24R-dihydroxy-vitamin D3 administration on lymphoproliferation and autoimmune disease in MRL/MP-lpr/lpr mice, Int. Arch. Allergy Appl. Immunol. 77 (1985) 396e404. [187] H.F. Deluca, M.T. Cantorna, Vitamin D: its role and uses in immunology, FASEB J. 15 (2001) 2579e2585. [188] M.W. Vaisberg, R. Kaneno, M.F. Franco, N.F. Mendes, Influence of cholecalciferol (vitamin D3) on the course of experimental systemic lupus erythematosus in F1 (NZBxW) mice, J. Clin. Lab. Anal. 14 (2000) 91e96. [189] S.M. Toloza, D.E. Cole, D.D. Gladman, D. Ibanez, M.B. Urowitz, Vitamin D insufficiency in a large female SLE cohort, Lupus 19 (2010) 13e19. [190] P.W. Wu, E.Y. Rhew, A.R. Dyer, D.D. Dunlop, C.B. Langman, H. Price, et al., 25-hydroxyvitamin D and cardiovascular risk factors in women with systemic lupus erythematosus, Arthritis Rheum. 61 (2009) 1387e1395.
[191] A.M. Huisman, K.P. White, A. Algra, M. Harth, R. Vieth, J.W. Jacobs, et al., Vitamin D levels in women with systemic lupus erythematosus and fibromyalgia, J. Rheumatol. 28 (2001) 2535e2539. [192] V.Z. Borba, J.G. Vieira, T. Kasamatsu, S.C. Radominski, E.I. Sato, M. Lazaretti-Castro, Vitamin D deficiency in patients with active systemic lupus erythematosus, Osteoporos. Int. 20 (2009) 427e433. [193] G. Ruiz-Irastorza, M.V. Egurbide, N. Olivares, A. MartinezBerriotxoa, C. Aguirre, Vitamin D deficiency in systemic lupus erythematosus: prevalence, predictors and clinical consequences, Rheumatology (Oxford) 47 (2008) 920e923. [194] D.L. Kamen, G.S. Cooper, H. Bouali, S.R. Shaftman, B.W. Hollis, G.S. Gilkeson, Vitamin D deficiency in systemic lupus erythematosus, Autoimmun. Rev. 5 (2006) 114e117. [195] M. Cutolo, K. Otsa, Review: vitamin D, immunity and lupus, Lupus 17 (2008) 6e10. [196] H. Amital, Z. Szekanecz, G. Szu¨cs, K. Danko´, E. Nagy, T. Cse´pa ny, et al., Serum concentrations of 25-OH vitamin D in patients with systemic lupus erythematosus (SLE) are inversely related to disease activity: is it time to routinely supplement patients with SLE with vitamin D? Ann. Rheum. Dis. 69 (2010) 1155e1157. [197] G. Ruiz-Irastorza, S. Gordo, N. Olivares, M.V. Egurbide, C. Aguirre, Changes in vitamin D levels in patients with systemic lupus erythematosus: Effects on fatigue, disease activity and damage, Arthritis Care Res. (Hoboken.) 62 (2010) 1160e1165. [198] D.D. Gladman, C. Antoni, P. Mease, D.O. Clegg, P. Nash, Psoriatic arthritis: epidemiology, clinical features, course, and outcome, Ann. Rheum. Dis. 64 (Suppl. 2) (2005) ii14eii17. [199] D.J. Veale, C. Ritchlin, O. Fitzgerald, Immunopathology of psoriasis and psoriatic arthritis, Ann. Rheum. Dis. 64 (Suppl. 2) (2005) ii26eii29. [200] W.A. Myers, A.B. Gottlieb, P. Mease, Psoriasis and psoriatic arthritis: clinical features and disease mechanisms, Clin. Dermatol. 24 (2006) 438e447. [201] J.R. Sigmon, B.A. Yentzer, S.R. Feldman, Calcitriol ointment: a review of a topical vitamin D analog for psoriasis, J. Dermatolog. Treat 20 (2009) 208e212. [202] S. Gorman, M.A. Judge, P.H. Hart, Immune-modifying properties of topical vitamin D: focus on dendritic cells and T cells, J. Steroid Biochem. Mol. Biol. 121 (2010) 247e249. [203] D. Huckins, D.T. Felson, M. Holick, Treatment of psoriatic arthritis with oral 1,25-dihydroxyvitamin D3: a pilot study, Arthritis Rheum. 33 (1990) 1723e1727. [204] Y. Braun-Moscovici, K. Toledano, D. Markovits, A. Rozin, A.M. Nahir, A. Balbir-Gurman, Vitamin D level: is it related to disease activity in inflammatory joint disease? Rheumatol. Int. (2009).
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C H A P T E R
102 Vitamin D and Cardiovascular Disease Harald Sourij, Harald Dobnig Medical University of Graz, Graz, Austria
INTRODUCTION Thirty years ago a hypothesis was brought forward by Scragg and colleagues proposing that cardiovascular disease seasonality may not only be a consequence of variations in temperature or prevalence of respiratory diseases but may be due to seasonal variation in ultraviolet radiation. It was already speculated back then that decreased body levels of vitamin D in winter-time could be responsible for changes in platelet function and serum calcium regulation leading to an increased risk of thrombus formation [1]. Twenty years ago the same author reported that patients with myocardial infarction, after adjusting for age, sex and time of blood collection, had lower 25(OH)D levels when compared to controls in all seasons [2]. Today we have available a vast number of epidemiological and observational studies and a rather small number of interventional studies that were published over the past few years and we still cannot clearly confirm or reject the hypothesis formulated three decades ago. What makes it worse is that appropriate studies are either under way or in the planning stage and important answers still cannot be expected for the next 5 to 10 years to come. And even given the results of these studies, it is not certain from the present day’s perspective how valid the data will be because vitamin D may well prove to be a slow-acting, background modulator of various musculoskeletal and cardiovascular health aspects. The situation as it exists today is that various knockout animal models and other preclinical data suggest direct effects of vitamin D on the vasculature, cardiac myocytes, or the renineangiotensinealdosterone system (RAAS) (to name a few of them) in rather extreme and nonphysiological biological settings and it remains unknown to what extent these findings translate into clinically relevant outcomes for humans. Because observations such as those from nationwide cross-sectional investigations in
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10102-7
the US linking low 25(OH)D levels with important cardiovascular risk factors [3,4] have a plausible biological basis and the degree of excitement about such data is enormous especially in the light of the prospect that vitamin D deficiency or insufficiency could be treated at almost no cost. Because 25(OH)D status depends so much on lifestyle aspects, primarily on diet, physical activity and UV-light exposure, it can be expected that most if not all impairments to general health are potential confounders in the association between vitamin D and cardiovascular disease. Moreover, it can be expected that there are further not-negligible socioeconomic confounders that are difficult to impossible to control for even in prospective observational studies. Although subgroup analyses of various studies do suggest persisting differences, i.e. in the percentage of overall survival [5] or parameters reflecting cardiac output [6] also within groups of individuals that are less active, one still needs to be cautious of general conclusions because of the complex interactions of the aforementioned links between health and vitamin D status. As low vitamin D status represents a problem of global dimensions [7,8] even a modest association with the risk of cardiovascular disease might translate into a large burden of disease in the population. Since various aspects laid out in this chapter can be found in more detail throughout this book, the specific aim here is to present a compact overview of direct and indirect actions of vitamin D on components and risk factors of the cardiovascular system (Fig. 102.1) and to try to summarize respective clinical data on cardiovascular outcomes. Important other relevant chapters include Chapter 31 on the heart, Chapter 40 on the renineangiotensen system, Chapter 73 on vascular effects, and Chapter 98 on diabetes and the metabolic syndrome.
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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102. VITAMIN D AND CARDIOVASCULAR DISEASE
FIGURE 102.1
Overview on potential direct and indirect cardiovascular effects of vitamin D deficiency.
COMPONENTS OF DIRECT CARDIOVASCULAR EFFECTS General Aspects There is a broad spectrum of possibilities on how vitamin D may exert effects at the tissue level. First, there is the ligand itself. The final mediator for the effects of vitamin D is thought to be 1,25(OH)2D which is either produced in the kidney (and enters the circulation thereafter) or by extra-renal tissues such as the vasculature that express 1a-hydroxylase which converts 25(OH)D to 1,25(OH)2D at the cellular level [9]. In the latter setting locally produced 1,25(OH)2D may act in an autocrine or paracrine fashion to activate the vitamin D receptor (VDR) which can be found in most cells and tissues throughout the body [10]. Because serum levels of 25(OH)D are roughly 1000-fold higher and binding affinities for the VDR 100-fold lower compared to 1,25(OH)2D, there is also the possibility that direct effects of 25(OH)D on gene transcription are operative [11]. Although it is known that a decrease in 25(OH)D substrate is counter-regulated by an increase in parathyroid hormone (PTH) to keep circulating 1,25(OH)2D levels stable, we know very little about extra-renal 1ahydroxylase or 24-hydroxylase regulation in the event of compromised vitamin D status. Therefore, low circulating levels of 25(OH)D could in theory damage target tissues in two important ways: (1) by low substrate availability for renal and extra-renal 1a-hydroxylase and decreased local conversion to 1,25(OH)2D and
(2) by a diminished direct effect on VDR activation. Because the vitamin D system intimately affects calcium and phosphorous handling, it was important to investigate whether vitamin D effects are triggered by a change in calcium or phosphate metabolism. In a very elegant study Zhou et al. [12] could demonstrate that effects of 1,25(OH)2D are operative independent of changes in calcium and phosphorous regulation at least in mice. Mice with an ablation of the 1a-hydroxylase and receiving a rescue diet to prevent hypocalcemia developed hypertension, cardiac hypertrophy and impaired cardiac function after 4 weeks. Administration of 1,25(OH)2D not only normalized serum calcium and phosphorous levels but also normalized blood pressure, cardiac structure and activity of the RAAS. Not surprisingly, the VDR is another essential component in the translation of potential cardiovascular effects of vitamin D. This was convincingly demonstrated in animal as well as in human studies. VDR knockout mice also display hypertension, cardiac hypertrophy with myocyte enlargement and elevations of atrial natriuretic peptide expression [13]. In a more physiologic setting vitamin D deficiency in Sprague-Dawley rats was accompanied by similar although transient effects on blood pressure as well as cardiac muscle mass [14]. The VDR as another important regulatory component within the vitamin D regulative system was shown to be upregulated in the rat heart by induction of myocyte hypertrophy under in vitro as well as in vivo conditions proposing a role of an antihypertrophic system [15]. However, it should be mentioned that children and
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COMPONENTS OF DIRECT CARDIOVASCULAR EFFECTS
young adults with hereditary vitamin-D-resistant rickets (HVDRR) and defective or absent VDR do not appear to show hypertension or abnormalities of the RAAS (Chapter 65).
Specific Aspects Vascular Smooth Muscle Cells and the Vasculature Throughout the cardiovascular system various cell types express the VDR and respond to 1,25(OH)2D. Among these cells are the cardiomyocytes, vascular endothelial and smooth muscle cells [16,17]. Of special interest are also juxtaglomerular cells that secrete renin and are highly responsive to 1,25(OH)2D. 1,25(OH)2D and vitamin D metabolites exert vital actions on vascular smooth muscle cell (VSMC) function and pathology, including contractility, growth, migration and the evolution of vascular calcifications [9]. A number of studies have shown that vitamin D metabolites enhance expression of Ca-ATPase [18], raise cytosolic free calcium [19], induce the expression of contractile proteins [20] and accelerate formation of prostacyclin [21] thus affecting arterial tone. Calcitriol treatment of rings of spontaneously hypertensive rat aortas reduced acetylcholine-induced and ATP-induced endothelium-dependent contractions and also reduced the increase in cytosolic free calcium concentrations caused by acetylcholine [22]. These data demonstrate that 1,25(OH)2D modulates vascular tone by reducing calcium influx into the endothelium and hence decreases the production of endothelium-derived contracting factors. Somjen et al. provided first evidence for the expression of an enzymatically active 1a-hydroxylase system that could be increased in its expression by PTH and estrogenic compounds enabling human VSMCs to regulate local concentrations of 1,25(OH)2D in an autocrine manner and provide ligands for the intracellular VDR [9]. Importantly, 1,25(OH)2D inhibits VSMC proliferation and diminishes the mitogenic response to growth enhancers such as thrombin or platelet-derived growth factor [23,24]. Recently, the cyclin-dependent kinase 2 (Cdk2), a key cell-cycle kinase, was identified as a target of 1,25(OH)2D in VSMC [25] at least partly explaining the antiproliferative effects of 1,25(OH)2D in this tissue. The role of 1,25(OH)2D in terms of vascular calcification is less clear and will be reviewed elsewhere in this book (see Chapter 73 for a discussion of the effects of vitamin D on vascular calcification). Vascular calcification is now understood to be both a passive as well as an active process. Ectopic calcifications may partially represent a passive phenomenon due to oversaturation of Ca x P concentrations or an active process due to true cellular transformation of mesenchymal cells into osteoblast-like cells under conditions of excess 1,25
1975
(OH)2D or excess serum phosphate [26]. These processes are further supported by decreases in the local expression of vascular calcification inhibitors such as matrix Gla protein (MGP), osteopontin, and type IV collagen in VSMC [27]. Another important pathophysiologic aspect of atherogenesis is inflammation within the vasculature wall. An increase in pro-inflammatory cytokines such as IL-1, IL6, and TNF-a contributes to immune cell recruitment and modified LDL cholesterol deposition by increasing scavenger receptor expression and cholesteryl ester synthesis and by decreasing cholesterol efflux [28]. Activation of VDR signaling was recently shown to prevent foam cell formation by reducing modified LDL cholesterol uptake in macrophages from diabetic patients [29]. Through suppression of endoplasmic reticulum (ER) stress and JNK activation, calcitriol downregulates two critical scavenger receptors that play a decisive role in transforming macrophages into foam cells [29]. Endothelial Dysfunction and Atherosclerosis During atherogenesis, endothelial dysfunction is considered an early step that identifies patients at high cardiovascular risk [30,31]. In addition, endothelial dysfunction is at least in part reversible and has therefore been identified as an interesting target for therapy. Since vitamin D status has been associated with worse cardiovascular outcome, a possible relationship between hypovitaminosis D, endothelial dysfunction, and atherogenesis has been suggested. In 1993 Helen Wiseman showed that 25(OH)D as well as 1,25(OH)2D exhibit membrane antioxidative properties by inhibiting lipid peroxidation [32]. In addition Wakasugi et al. [21] demonstrated in cultured rabbit vascular smooth muscle cells an increase in prostacyclin synthesis in the presence of 1,25(OH)2D as well as 1a(OH)D, suggesting vitamin D as a vasoactive agent that may play a role in endothelial function and atherogenesis. Moreover, pre-treatment of human endothelial cells with 1,25(OH)2D inhibited the lipopolysaccharide activation of transcription factor NF-kB [33] and the induction of adhesion molecule expression by TNFa [34] which seems to be involved in early stages of atherogenesis. Kuzmenko and colleagues confirmed in an animal model the anti-free-radical properties of 25(OH)D on lipid peroxidation as previously observed in vitro [35]. Furthermore 1,25(OH)2D administration in alloxan-induced diabetic rats enhances superoxide dismutase, catalase and glutathione peroxidase suggesting a preventive effect against oxidative stress in these diabetic animals by reducing hyperglycemia-induced reactive oxygen species production [36]. Clinical data on endothelial function and vitamin D are quite sparse. There is only one study in 23 asymptomatic
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102. VITAMIN D AND CARDIOVASCULAR DISEASE
vitamin-D-deficient subjects (25(OH)D <10 ng/ml) who received 300 000 IU vitamin D3 intramuscularly monthly for 3 months [37]. Endothelial function was assessed by flow-mediated dilation (FMD) measurement. In comparison to a control group with a mean 25(OH)D level of 30 ng/ml FMD measurements were significantly lower in vitamin-D-deficient subjects (7.0 3.2% versus 11.2 5.2%; p ¼ 0.001). Furthermore supplementation increased FMD to 10.4 3.3%, p ¼ 0.002 indicating on the one hand a detrimental effect of vitamin D deficiency on endothelial function and on the other a beneficial effect of vitamin D supplementation to restore endothelial function as a surrogate parameter of cardiovascular outcome. Additionally the authors measured lipid peroxidation that was increased in patients with vitamin D deficiency and could be significantly lowered by supplementation. Unfortunately, this study was neither randomized nor placebo-controlled. Another small study that was a double-blind, placebo-controlled trial, randomized 34 type 2 diabetic patients with vitamin D insufficiency (mean 25(OH)D level 15.3 ng/ml) to a single dose of 100 000 IU vitamin D2 or matched placebo [38]. After 8 weeks and an increase in 25(OH)D level of 6 ng/ml in the treated group relative to placebo, FMD significantly improved by 2.3%, representing a clinically relevant extent. While endothelial dysfunction can be regarded as an early step in atherogenesis, carotid intima-media thickness represents a more advanced stage and another important cardiovascular surrogate parameter. Data on the impact of vitamin D levels on carotid intima-media thickness (IMT) are conflicting though. In 2006, Tharger and colleagues showed that vitamin D levels are independently associated with carotid intima-media thickness in type 2 diabetic patients (130 with hypovitaminosis D versus 260 vitamin-D-sufficient counterparts) [39]. Interestingly, patients with vitamin D insufficiency had also significantly higher haemoglobin A1c, fibrinogen and C-reactive protein concentrations when compared to vitamin-D-sufficient counterparts. Data from the Rancho Bernado Study [40], a crosssectional study including 654 community-dwelling older adults aged 55e96 years without a history of coronary heart disease or stroke, showed an inverse association of 25(OH)D concentration with mean internal carotid IMT (p for trend ¼ 0.02) while there was no association with common carotid IMT. In contrast, Pilz [41] and colleagues could not find a significant association in the Hoorn study population. Michos et al. investigated 650 Amish people and were also unable to show an association of 25(OH)D levels with carotid IMT [42]. Thus, this issue remains unclear and needs to be further elucidated. With regard to more advanced atherosclerosis Fahrleitner et al. described dramatically low levels of 25(OH)D in patients with peripheral arterial occlusive
disease stage IV [43]. However, although clinically important this association seems to be rather the result of immobilization and subsequent reduced sunlight exposure than an evidence for hypovitaminosis D as a causal factor for peripheral artery disease. With regard to coronary artery disease, recently the Multi-Ethnic Study of Atherosclerosis investigated whether circulating 25(OH)D concentrations were associated with coronary artery calcification, as a measure of coronary atherosclerosis. They included 1370 participants and followed them up for 3 years. In this study low 25(OH)D concentrations were not associated with prevalent coronary artery calcification but did correlate with increased risk for developing incident coronary artery calcification. Overall, the data on vitamin D and its putative impact on atherogenesis and atherosclerosis are sparse and controversial. Cardiomyocytes Cardiac myocytes express the vitamin D receptor (VDR) and 1a-hydroxylase and 24-hydroxylase, the enzymes required for the conversion of 1,25(OH)2D from 25(OH)D and its subsequent breakdown. Treatment with 1,25(OH)2D leads to increased expression and nuclear localization of the VDR, increased expression of myotrophin, and decreased expression of c-myc [44] and human B-type natriuretic peptide [15]. Calcitriol affects growth, proliferation and morphology also in murine cardiomyocytes and increases expression and nuclear localization of the VDR in such cells [44]. Isolated murine cardiomyocytes from VDR knockout mice show accelerated rates of contraction and relaxation as compared to wild-type mice and when exposed to calcitriol only wild-type but not knockout mice display changes in contractility [45]. Interestingly, matrix metalloproteinases (MMPs), connective tissue enzymes that are involved in remodeling of vascular walls and the myocardium are underexpressed in VDR knockout mice compared to wild type [46]. MMPs not only are able to destabilize atheromatous plaques which may rupture and lay the ground for thrombus development but also contribute to extracellular matrix remodeling which may lead to progressive left ventricular remodeling, dilation and congestive heart failure [47]. Taking the above effects together they may at least in part explain why calcitriol treatment prevents cardiac hypertrophy in various hypertensive rat models [48]. Using microarray analysis, studies revealed 45 specific genes that were altered in paricalcitoltreated rats fed a high-salt diet that showed significant reduced left ventricular mass, end-diastolic pressures and increased fractional shortening [49]. It has long been known that rachitic children may have symptoms attributable to congestive heart failure suggesting a link between vitamin D status and cardiac
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COMPONENTS OF DIRECT CARDIOVASCULAR EFFECTS
function [50]. In a large cohort of patients referred for coronary angiography, 25(OH)D and 1,25(OH)2D levels were negatively correlated with NT-pro-BNP (N-terminal pro-brain natriuretic peptide) and were inversely associated with impaired left ventricular (LV) function and higher New York Heart Association (NYHA) classes [51]. Moreover, multivariate-adjusted analyses showed a hazard ratio (HR) for death due to heart failure of 2.84 (1.20e6.74) in a group of patients with 25(OH)D levels <10 ng/ml when compared to a group with 25(OH)D levels of 30 ng/ml. Interestingly, this study also reported an HR of 5.05 (2.13e11.9) for patients with low 25(OH)D status to die from sudden cardiac death (SCD) and shed some additional light on another potentially important aspect of vitamin D effect on the cardiovascular system. Similar results were obtained in vitamin-D-deficient animal models where myocardial hypertrophy and fibrosis led to aberrant cardiac contractility and relaxation [45,49]. In a recent randomized study of elderly patients with heart failure and baseline vitamin D levels <20 ng/ml, mean 25(OH)D levels increased by 9 ng/ml over the time-course of 5 months by administering an oral dose of 100 000 IU vitamin D at baseline and at week 10. Although B-type natriuretic peptide decreased significantly in the treatment group of that study there was no improvement in the 6-minute walk test and a small significant worsening in quality of life [52]. An older, small, uncontrolled trial performed in hemodialysis patients treated with calcitriol at doses between 1 and 2 mg after dialysis sessions for 3e4.5 months did not find any changes in cardiac function. However, the authors described an improvement in a subset of patients with severe hyperparathyroidism [53]. Definitely more studies with longer treatment durations in patients at various stages of congestive heart failure are needed to be performed before the question can be resolved whether vitamin D could constitute a valuable add-on to existing treatment strategies. It also remains to be seen whether the multiethnic differences in the incidence of congestive heart failure and here, especially those between African-American and the white population [54] have something to do with the well-known differences in vitamin D status of these populations [55]. RenineAngiotensineAldosterone System (RAAS) The first and rate-limiting enzyme of this cascade is renin, a protease synthesized and secreted predominantly by the juxtaglomerular cell apparatus in the nephron. It regulates blood pressure, electrolyte and volume homeostasis. Renin is a protease that cleaves angiotensinogen to angiotensin I, which is subsequently converted to angiotensin II. Angiotensin II, through binding to its receptors, exerts important and physiologically relevant actions in this system.
1977
Despite the fact that ultraviolet light exposure (required for cutaneous vitamin D generation) was shown to have blood-pressure-lowering effects and is related to the prevalence of hypertension in the general population [56,57] the mechanisms underlying this relationship were unknown until a few years ago. Li and colleagues demonstrated that in VDR-null mice renin expression and plasma angiotensin II production were several-fold increased and led to hypertension, cardiac hypertrophy, and increased water intake [58]. In wild-type mice, calcitriol injections led to renin suppression. In supplemental studies using As4.1 juxtaglomerular cells calcitriol was shown to markedly suppress renin transcription by a VDR-mediated and calcium-independent mechanism. Later it was shown by the same group that 1,25(OH)2D suppresses renin gene transcription by blocking the activity of the cyclic AMP-response element in the renin gene promoter [59]. Similar to the VDR knockout mice 1a-hydroxylase knockout mice developed hypertension and cardiac hypertrophy and despite normalization of serum calcium and phosphorous levels on rescue diets these abnormalities remained. The use of angiotensin-converting enzyme blockers normalized blood pressure and cardiac structure function despite elevated renin expression [12]. See Chapter 40 for further discussion of the effects of vitamin D on the RAAS. Human data are scarce regarding an interaction between vitamin D and the RAAS but very recently Forman et al. examined the relationship between plasma 25(OH)D levels and elements of the RAAS in 184 normotensive individuals in high sodium balance. They found that lower 25(OH)D status was associated with higher circulating angiotensin II levels (p for trend ¼ 0.03) and more importantly that individuals with vitamin D deficiency (<15 ng/ml) had significantly blunted renal plasma flow responses to infused angiotensin II (p for trend ¼ 0.009) suggesting that in humans low plasma 25(OH)D levels may result in upregulation of the RAAS in otherwise healthy individuals [60]. Taken together, it appears that more studies in homogeneous study populations are needed before a firm conclusion can be reached as to whether vitamin D deficiency causes or aggravates hypertension and whether vitamin D supplementation is safe and exerts cardioprotective effects. The potential problems with bias and confounding factors present in previous epidemiological studies can only be overcome or minimized by well-designed randomized controlled intervention trials. Inflammation Inflammation arising from atherosclerosis and other chronic diseases likely contributes to many of the health problems that increase morbidity and mortality. Today
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many regard atherosclerosis as a state of chronic inflammation and even chronic heart failure is thought to be modulated by increased oxidative stress and inflammatory processes. Serum levels of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-a), interleukin-6 (IL-6), interleukin-1 (IL-1), and adhesion molecules were found to be elevated in states of chronic heart failure and were related to long-term outcome. For years it has been proposed that the vitamin D system can potentially interfere with inflammatory response and fibrogenesis; however, clinical studies looking at associations between vitamin D and markers or correlates of inflammation have been scarce until recently. Some of these studies were quite large and found inverse associations between 25(OH)D levels and markers of endothelial dysfunction, oxidative stress, or inflammatory activation potentially reflecting incremental risk of clinical vascular endpoints with low vitamin D status. Out of the three randomized controlled trials two were negative and did not find a difference in inflammatory markers. In one study no change was found in C-reactive protein (CRP) or IL-6 levels using daily doses of 700 IU vitamin D3 [61]. In the second study no change in various interleukins/hsCRP/ICAM-1 levels was found using 3000e6000 IU vitamin D3 per day over a treatment period of 3 years and 1 year, respectively [62]. Of note, these studies were performed in healthy individuals who either had baseline 25(OH)D levels between 28 and 32 ng/ml with a mere 10% decrease in PTH (parathyroid hormone) following vitamin D3 supplementation [61], or who were all overweight and had normal 25(OH)D levels [62]. In the third randomized study 123 patients with congestive heart failure were treated with a dose of 4000 IU vitamin D3 daily over 9 months. 25(OH)D levels rose from an average of 15 ng/ml to 41 ng/ml and TNF-a decreased by 10% and IL-10, an important antiinflammatory cytokine, showed an average 50% increase in the vitamin D3 supplemented group. Neither CRP nor NT-proBNP levels nor left ventricular ejection fraction changed in this study. Similar positive correlations between 25(OH)D and IL-10 levels have also been seen in other studies [63,64] and, interestingly, deficiency of this cytokine has led to severe atherosclerosis in animal studies [65]. A recent review stressed that an optimal balance between TNF-a and IL-10 may be of crucial importance in mitigating both inflammation and oxidative stress processes leading to heart failure [66]. Apart from these cross-sectional and interventional studies there are other clinical or in vitro studies that report on the potential anti-inflammatory actions of vitamin D. Giulietti et al. reported that freshly isolated and immune-stimulated monocytes from patients with type II diabetes mellitus exhibit a pro-inflammatory profile with elevated mRNA expression levels for TNF-a, IL-1, IL-6, and ICAM compared to controls or type I diabetics
[67]. Incubation with 1,25(OH)2D downregulated TNF-a, IL-1, and IL-6 suggesting that calcitriol was able to modulate inflammation in monocytes of such patients. Another in vitro study performed with human coronary arterial endothelial cells (HCAEC) of patients with Kawasaki disease, an acute febrile vasculitis in childhood where vascular inflammation results in damage to the coronary arteries, found that 1a,25(OH)2D treatment significantly inhibited NF-kB activation which is essential for the expression of pro-inflammatory cytokines [68]. Furthermore, E-selectin, another important key component of the inflammatory response that contributes to the recruitment of leucocytes to the site of injury, was also significantly inhibited during TNF-a stimulation. It is unclear how far the following study by Talmor-Barkan [69] is relevant to human physiology , but it is worth remembering that one consequence of advanced vitamin D deficiency is lowenormal or obviously low serum calcium levels. Changing the extracellular calcium concentration in human umbilical vein cord endothelial cells led to a diminished protein expression and activity of eNOS and increased ICAM as well as mRNA levels of RAGE and IL-6, suggesting that ambient calcium concentration may also modify endothelial cell function [69]. Platelet Function Platelets play a pivotal role in atherothrombotic events since platelet activation is the initial step in artery thrombus formation. There are very little data available on possible effects of vitamin D on platelet function. Normocalcemic VDR knockout mice showed a significantly enhanced platelet aggregation in comparison to wild-type mice indicating a suppressive effect mediated via VDR on platelet aggregation [70]. Inoue et al. investigated the effect of a synthetic vitamin D3 analog on the production of prostacyclin, an inhibitor of platelet aggregation, using rat aortic rings [71]. They showed a marked increase in prostacyclin synthesis by these aortic rings treated with 22-oxa-1,25-dihydroxyvitamin D3 analog in comparison to non-treated controls. However, much more data are needed in particular interventional trials to clarify the role of vitamin D in platelet aggregation and thrombus development. Coagulation System Venous thromboembolism including deep vein thrombosis and pulmonary embolism is associated with a markedly increased morbidity and mortality [72]. Interestingly, it has been shown that the risk of venous thromboembolism is increased in winter in comparison to the summer months [73,74] and a similar seasonal pattern could be observed for arterial thrombotic complications [75,76]. Since vitamin D levels have shown the same but inverse seasonal pattern with lowest 25(OH)D levels in
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COMPONENTS OF INDIRECT CARDIOVASCULAR EFFECTS
winter it has been hypothesized that vitamin D might play an important role as a causal factor. Interestingly, Lindqvist and colleagues have added additional information by reporting that women with more active sun exposure have a significantly lower risk for venous thromboembolic events [74]. Unfortunately, vitamin D levels were not measured in this study. In vitro studies have shown that 1,25(OH)2D upregulates thrombomodulin, a co-factor in the protein C activation and downregulates tissue factor in human leukemic cells [77]. Therefore, low 1,25(OH)2D levels are considered to correlate with hypercoagulation. In addition, in smooth muscle cells, paricalcitol and calcitriol downregulated the expression of plasminogen activator inhibitor-1 (PAI-1) and upregulated thrombomodulin, further underlining possible anticoagulant effects of vitamin D [78]. Puri et al. investigated the effects of 1,25(OH)2D on tissue plasminogen activator secretion from cultured rat heart microvascular cells and showed a significant induction of secretion [79]. In vivo these observations were supported by hemostatic studies in VDR knockout mice. These mice show a downregulated antithrombin as well as thrombomodulin gene expression and an upregulated tissue factor expression regardless of plasma calcium levels in contrast to wild-type mice. Taken together, these observations make it not surprising that VDR knockout mice suffered from multi-organ thrombus formation after lipopolysaccharide (LPS) injection [70]. As stated above, sun exposure habits were shown to be associated with increased risk for thromboembolic events [74] and Jorde at al. [80] demonstrated an increase in overall thrombotic activity measured by calibrated automated thrombogram with a decrease in 25(OH)D levels. In addition 25(OH)D levels are inversely associated with PAI-1 and tissue-type plasminogen activator antigen (t-PA Ag) levels [81]. This could be a further indication that vitamin D levels might be related to fibrinolytic activity and to endothelial function. One study investigated the effect of additional administration of 45 mg calcitriol versus placebo to docetaxel on thrombotic events in 250 patients with prostate cancer. Interestingly only two cases of thrombotic events occurred in the calcitriol group in contrast to 11 cases in the placebo group (p ¼ 0.01) [82]. Very recently an investigation in 158 overweight or obese subjects was performed during which participants received either 40 000 IU vitamin D3 per week for 1 year or matched placebo [80]. Outcome parameter was the calibrated automated thrombogram, a parameter of overall thrombotic activity. Despite the inverse relationship of 25(OH)D levels and thrombotic activity at baseline, the authors could not show an anticoagulatory effect of vitamin D3 supplementation.
1979
Sympathetic Nervous System Although information is very limited in terms of potential sympathetic nervous system regulation by the vitamin D system there are two interesting clinical trials reporting a reduction of pulse rate by vitamin D [83,84]. Recently such a relationship was also seen in the analysis of the National Health and Nutrition Examination Surveys (NHANES) data comprising 27 153 individuals aged 20þ [6]. Rateepressure product calculated as heart rate times systolic blood pressure is a measure of cardiac work and cardiac oxygen demand and is correlated with myocardial blood flow in healthy volunteers. Adjusted mean rate pressure product was 408 110 beats/min mm Hg higher (p < 0.001) for participants with 25(OH)D levels <10 ng/ml and 245 80 beats/ min mm Hg higher (p < 0.01) for participants with 25(OH)D levels of 10.0 to 14.9 ng/ml, compared to those with 25(OH)D concentrations 35 ng/ml suggesting that low vitamin D status may increase cardiac work. The data of these three studies suggest that vitamin D may have a direct effect on the heart, possibly through a negative correlation between vitamin D signaling in cardiomyocytes and the sympathetic nervous system. Such an hypothesis is supported by the evidence that 1,25(OH)2D regulates the production of tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine biosynthetic pathway, and raises the possibility that the vitamin D hormonal system may modulate functions of the peripheral and central catecholaminergic system [85].
COMPONENTS OF INDIRECT CARDIOVASCULAR EFFECTS Body Weight and Obesity Obesity as a cardiovascular risk factor has been associated on its own with 25(OH)D deficiency in several studies in adults [86e88] as well as in children and adolescents [89]. There were several possible explanations for this association proposed: since 25(OH)D is fat soluble, an increased storage capacity in adipose tissue is a plausible explanation for a deficiency in obese subjects [90]. Furthermore one can argue that obese patients have less outdoor activity than lean individuals and therefore less sunlight exposure. Alternatively it has been hypothesized that low vitamin D status leads to an increased parathyroid hormone level that causes calcium influx into adipocytes and thereby enhances lipogenesis [91]. There is also evidence that 1,25(OH)2D modulates adipogenesis by influencing PPARg ligand formation, an important player in preadipocyte differentiation [92]. Low 25(OH)D levels were associated with various clinical measurements of obesity such as body mass
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102. VITAMIN D AND CARDIOVASCULAR DISEASE
index [3,86,94,95], waist circumference [88,94,96], or waist to hip ratio [88,96]. Moreover the Insulin Resistance Atherosclerosis (IRAS) Family study investigated the association of 25(OH)D levels with abdominal subcutaneous and visceral adipose tissue determined by computed tomography in Hispanic and African-Americans [95]. Both measurements were inversely associated with 25(OH)D levels. These data were confirmed by an analysis of the Third Generation participants of the Framingham Heart Study [97]. These authors also demonstrated that the association between 25(OH)D and adiposity was stronger for visceral than for subcutaneous abdominal adiposity and in turn 25(OH)D deficiency was more frequent among subjects with high as compared to low visceral adipose tissue values (14.4% versus 5.7%, p < 0.0001). Freedman et al. extended these findings to a population of patients with type 2 diabetes [98]. There are some interventional studies focusing on the impact of vitamin D supplementation on weight reduction. Table 102.1 gives an overview on the available study outcomes. In almost all of these studies additional calcium was administered in the treated group. Daly et al. [99] investigated the effect of vitamin-D- and calcium-fortified milk administration (1000 mg calcium and 800 IU vitamin D3) in comparison to no additional milk, on bone mineral density in 167 men over a period of 2 years. Beside the primary outcome, data on weight changes were presented. There was no significant ingroup or between-group difference in body weight recorded in this study. Another, placebo-controlled study investigated the effect of 600 mg calcium and 200 IU vitamin D3 on lipids and body weight in a small population of 63 obese subjects [100]. No effect on body weight by vitamin D supplementation was seen in this short study of 15 weeks. Higher doses of vitamin D were administered in two other studies [101,102] that both could not demonstrate an additional weight-lowering
TABLE 102.1
effect in comparison to placebo. In contrast to all the studies cited above, an analysis of the Women’s Health Initiative showed a slightly favorable difference in weight change (mean difference 0.13 kg (0.21 to 0.05), p ¼ 0.001) after 3 years in the group receiving 1000 mg calcium plus 400 IU vitamin D3 daily in comparison to the placebo group [103]. In total 36 282 postmenopausal women were included in this study and therefore it is the largest database currently available. In addition, in this study women randomized to the treatment group had a slightly lower risk of gaining weight in comparison to those randomized to placebo. However, the small difference in weight change between both groups is in fact statistically significant but does not seem to be clinically relevant.
Arterial Hypertension Arterial hypertension has been established as one of the most important cardiovascular risk factors. In industrialized countries the risk for development of arterial hypertension (>140/90 mm Hg) during lifetime exceeds more than 90% according to some estimates and blood pressure criteria [104] and whatever the actual incidence, the number of hypertensive people worldwide is expected to increase further in the future. Arterial hypertension has been established as one of the most important risk factors for stroke, myocardial infarction, peripheral vascular disease, heart failure, kidney failure, or mortality [104]. Interestingly, the same conditions that are associated with reduced vitamin D production due to diminished UVB radiation (such as industrialization, high latitude or dark skin) have also been associated with increased blood pressure [57]. Therefore, it has been hypothesised that levels of 25(OH)D might be inversely associated with blood pressure. Krause and colleagues [56] have done a small proof of concept trial by investigating the effect of UVB radiation in 18 patients with untreated
Interventional Trials with Data on Body Weight
Author (reference)
Population
Active substance
Comparator
Duration (weeks)
Participants
Weight effect of vitamin D
Daly et al. [204]
Overweight
1000 mg calcium þ 800 IU vitamin D3 e fortified milk/d
No milk
104
167
none
Major et al. [100]
Obese
600 mg calcium þ 200 IE vitamin D/d
Placebo
15
63
none
Zittermann et al. [102]
Overweight
3332 IU vitamin D/d
Placebo
52
200
none
Sneve et al. [101]
Overweight
20 000 IE vitamin D twice a week þ 500 mg calcium/d
Placebo
52
445
none
Caan et al. [103]
Obese
1000 mg calcium þ 400 IE vitamin D
Placebo
364
36.282
mean difference, 0.13 kg [0.21 to 0.05]; p ¼ 0.001
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COMPONENTS OF INDIRECT CARDIOVASCULAR EFFECTS
essential hypertension. A drop of 6 mm Hg in systolic as well as diastolic blood pressure was observed, accompanied by a 162% rise in 25(OH)D plasma concentration. Recently Pilz and colleagues (105) reviewed this topic and summarized several potentially involved mechanisms by which vitamin D may influence arterial hypertension: putative suppression of the renine angiotensinealdosterone system, prevention of secondary hyperparathyroidism, anti-inflammatory effects, impact on insulin resistance and direct effects on vasodilatation. This topic and other potential mechanisms are reviewed more extensively in Chapter 40. There are several cross-sectional studies investigating the association of 25(OH)D levels with arterial blood pressure but again results are inconsistent. There are a number of investigations showing an inverse association of 25(OH)D levels with blood pressure [106e111] but not all [112e117]. The largest data set derives from the third National Health and Nutrition Examination Survey (NYHANES III) [106]. Among 12 644 participants a significant inverse association between vitamin D levels and systolic blood pressure was found. There are several controlled intervention trials reporting blood pressure values but most of them were not primarily designed to investigate blood pressure effects of vitamin D supplementation [100,102,118,119]. A small study in 189 elderly subjects from 1995 investigated in a randomized, placebo-controlled design the effect of a single 2.5 mg cholecalciferol dose (100 000 IU) on blood pressure during winter months [83]. No superior effect of vitamin D supplementation on blood pressure in comparison to placebo could be observed in this study. A German study enrolled 148 women with known vitamin D deficiency (25(OH)D <25 ng/ml) and randomized them to either 1200 mg calcium or 1200 mg calcium plus 800 IU vitamin D3 daily [84]. After 8 weeks of treatment serum 25(OH)D levels increased by 72% in the treated group (p < 0.01). Systolic blood pressure dropped by 9.3% (p < 0.01) in the calcium plus vitamin D group and by 4% (p < 0.01) in the calciumonly group. Compared to the calcium-only group, systolic blood pressure was significantly lower in the combination group (p ¼ 0.02). No difference in diastolic blood pressure was observed between the two groups. The largest trial again e the Women’s Health Initiative e assigned 36 282 postmenopausal women to receive either 400 IU vitamin D3 plus 1000 mg calcium or placebo daily [120]. No effect of vitamin D supplementation on systolic or diastolic blood pressure or incidence of new cases with hypertension was noted during a mean follow-up period of 7 years. A recent meta-analysis by Pittas et al. did not show an effect of vitamin D supplementation on systolic or diastolic blood pressure compared with placebo [121].
1981
Glucose Metabolism A more detailed overview on type 2 diabetes and the metabolic syndrome, and their association with vitamin D is given in Chapter 98. The incidence of disturbances of glucose metabolism is rapidly growing in the Western world. Patients with overt diabetes mellitus or even prediabetes face a markedly increased risk for cardiovascular events and mortality [122]. Vitamin D deficiency has been suggested as a risk factor for both type 1 as well as type 2 diabetes mellitus [123e125] by influencing autoimmune processes [126], beta-cell function [127], and insulin sensitivity [128]. Beta-cell dysfunction and insulin resistance are the main players in the pathogenesis of type 2 diabetes that are both present from the very beginning of the disease [129]. Numerous epidemiologic data showed an increased prevalence of disturbances of glucose metabolism in vitamin-D-deficient individuals [124,130,131] proposing a causal role for this association. There are in vitro data proposing an effect of vitamin D on both of the above-mentioned pathogenetic processes of type 2 diabetes. Vitamin D deficiency was shown to be paralleled by impaired glucose-mediated insulin secretion from pancreatic b-cells in vitro [132e134]. In addition, since insulin secretion is a calcium-dependent process, alterations in the calcium flux might therefore have an impact on insulin secretion [135]. This may be of physiological relevance because severely compromised vitamin D status is well known to be often accompanied by hypocalcemic and hypophospatemic states. On the other hand 1,25(OH)2D stimulates insulin receptor expression and insulin responsiveness for glucose transport in vitro [128] and calcium is not only essential for insulin secretion but also for insulin-mediated intracellular processes [136]. Nymba and colleagues performed intravenous glucose tolerance tests on rabbits before and after nutritional vitamin D deficiency [137]. A marked decrease in insulin sensitivity was observed in the vitamin-D-deficient state, while supplementation of either 1,25(OH)2D or vitamin D3 resulted in a significant improvement in glucosestimulated insulin release and glucose tolerance. Interestingly, an intravenous calcium infusion restored the serum calcium concentration of the vitamin-D-deficient rabbits but did not improve glucose-mediated insulin secretion. Cade and colleagues confirmed these observations in vitamin-D-deficient and vitamin-D-repleted rats [138]. There are several cross-sectional and observational studies reporting an association between vitamin D status and the prevalence of type 2 diabetes [139,140,141]. Chiu et al. [124] investigated the association of 25(OH)D concentrations with insulin sensitivity and b-cell function by using the hyperglycemic clamp
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1982
102. VITAMIN D AND CARDIOVASCULAR DISEASE
technique in 126 healthy normal glucose-tolerant subjects. They showed a positive correlation of 25(OH)D concentrations with insulin sensitivity and a negative effect of vitamin D deficiency on b-cell function. The largest currently available data set from the National Health and Nutrition Examination Survey (NHANES) showed an inverse association of 25(OH)D concentrations after multivariate adjustment with diabetes prevalence in non-Hispanic whites and MexicanAmericans, whereas no such effect could be observed in non-Hispanic blacks [140]. The same study also showed a significant inverse correlation between 25 (OH)D levels and insulin resistance, quantified by homeostatic model assessment (HOMA-R). This holds true again only for non-Hispanic whites and MexicanAmericans. In the Nurses’ Health study 83 779 women with no history of diabetes, cardiovascular disease, or cancer were followed [142]. Vitamin D and calcium intake were recorded every 2e4 years. An impressive 33% relative risk reduction in incident diabetes mellitus was shown in women with the highest calcium (>1200 mg/d) and vitamin D3 (>800 IU/d) intake compared to those with an intake of <600 mg calcium and 400 IU vitamin D3 daily. Some small intervention trials [130,143,144] but not all [145] showed an increase predominantly in insulin secretion measured by an oral glucose or an intravenous glucose tolerance test. Major limitations of all these studies are that they were not randomized or placebo controlled and included only 4 to 22 patients, respectively. There are a few randomized controlled studies focusing on the effect of vitamin D supplementation on insulin sensitivity and/or insulin secretion. Table 102.2 summarizes the available evidence. Overall the available intervention studies were rather small and at least in part performed in patients with sufficient 25(OH)D levels. Inomata et al. showed in a small study of 14 Japanese type 2 diabetic patients an improvement of insulin secretion and a decrease in serum free fatty acids by supplementation of 2 mg/d alphacalcidiol for 3 weeks [146]. Another study in 100 centrally obese, non-diabetic, vitamin-D-deficient Asian Indian people showed a borderline increase in postprandial insulin sensitivity, whereas no effect on fasting indices of insulin resistance or insulin secretion was observed [119]. Pittas and colleagues investigated the effect of 700 IU vitamin D3 plus 500 mg calcium versus placebo on fasting plasma glucose (FPG) or homeostasis model assessment of insulin resistance (HOMA-IR) in 314 non-diabetic subjects over a period of 3 years [61]. Ninety-two individuals of this cohort had evidence of impaired fasting glucose and demonstrated a lower rise in FPG (0.4 mg/dl versus 6.1 mg/dl; p ¼ 0.042) from baseline to study end when allocated to the vitamin D plus
calcium group. Moreover, the vitamin-D-treated group exhibited a significantly lower increase in HOMA-IR. In contrast the group of 222 subjects with normal fasting glycemia showed no such effects. Several other intervention studies investigating the effect of vitamin D supplementation on glucose metabolism failed to show a beneficial effect [147e151]. There is a strong need for further randomized, controlled, intervention trials to define the true role of vitamin D and/or calcium supplementation in the therapy or prevention of type 2 diabetes.
Lipid Changes Total cholesterol as well as LDL-cholesterol and triglycerides have been shown to be directly associated with cardiovascular events and mortality (for review see [152]). By discussing a potential beneficial effect of vitamin D on cardiovascular outcome, all established cardiovascular risk factors have to be considered and therefore a putative beneficial effect of vitamin D on plasma lipids needs to be addressed. There are only a few studies, however, focusing on this topic. Several animal studies have suggested that calcium supplementation might induce beneficial effects on plasma lipid levels [153,154]. Furthermore some [155,156] but not all [157,158] trials in humans showed a beneficial effect of calcium supplementation on lipid levels. One explanation for a lipid-lowering effect could be an enhanced formation of indigestible calciumefatty acid complexes in the gastrointestinal tract when calcium intake is increased. One could also speculate that calcium effects may also be operative via vitamin D which was shown to be involved in the regulation of adipocyte activity [159]. A randomized controlled trial in 464 postmenopausal women was performed with four intervention groups being compared [160]: (1) hormone replacement therapy (estradiol valerate (2 mg) on cycle days 1e21, cyproterone acetate (1 mg) on cycle days 12e21 and a treatment-free interval on cycle days 21e28), (2) 300 IU vitamin D3/day plus 500 mg calcium lactate daily, (3) hormone replacement therapy plus vitamin D3 300 IU and 500 mg calcium lactate per day, and finally (4) 500 mg calcium lactate. Vitamin D3 supplementation was associated with an unfavorable effect on lipid levels after 3 years. LDL cholesterol levels increased by 4.1% (p ¼ 0.023), whereas hormone replacement therapy only reduced LDL cholesterol by 10.1% (p < 0.001). A smaller 5.9% (p ¼ 0.005) reduction was observed in the group with the combination of hormone replacement and vitamin D3. HDL cholesterol concentrations decreased in the vitamin D3 supplementation group (5.2%; p ¼ 0.001). Neither of two additional studies supplementing vitamin D found a significant change in lipid levels
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
TABLE 102.2
Effects of Vitamin D Supplementation on Various Measurements of Insulin Sensitivity or Insulin Secretion. Randomized Controlled Trials 25-(OH)D level at baseline
Measurment of glucose metabolism
Intervention
Duration of intervention
Inomata et al. 1986 [146]
14 Japanese T2DM
n.r.
oGTT
2 mg/d alphacalcidiol vs. Placebo
3 weeks
Insulin secretion[, free fatty acids levelY
Nilas et al. 1984 [147]
151 non-diabetic postmenopausal women
n.r.
Fasting blood glucose
2000 IU 25(OH)D3 vs. 0.25 mg/d alphacalcidiol vs. placebo (þ500 mg/ d calcium in all groups)
2 years
No change on blood glucose
Orwoll et al. 1994 [205]
20 T2DM
14 ng/ml
Fasting plasma glucose and insulin. Meal challenge
1 mg/d 1,25(OH)2D vs. placebo (crossover design)
4 days
No effect on fasting or stimulated glucose or insulin concentrations. Improved insulin response in patients with short diabetes duration
Ljunghall et al. 1987 [148]
65 subjects with IGT
38 ng/ml
ivGTT
0.75 mg/d alphacalcidiol vs. placebo
3 months
No changes in glucose tolerance or insulin secretion
Fliser et al. 1997 [149]
18 healthy males
n.r.
Euglycemic clamp
1.5 mg 1,25(OH)2D/d vs. placebo
1 week
No effect on insulin sensitivity
Pittas et al. 2007 [61]
92 subjects with IFG
30 ng/ml
Fasting plasma glucose, fasting insulin
700 IU 25(OH)D þ 500 mg calcium vs. placebo
3 years
Lower rise in FPG (0.4 mg/dl vs. 6.1 mg/dl, p ¼ 0.042). Lower increase in HOMA-IR
222 subjects with NFG
30 ng/ml
Fasting plasma glucose, fasting insulin
700 IU 25(OH)D þ 500 mg calcium vs. placebo
3 years
No difference in changes of FPG or HOMA-IR
Jorde and Fischgenau 2009 [150]
36 T2DM with metformin and bed-time insulin
24 ng/ml
Fasting plasma glucose and insulin
40 000 IU 25(OH)D/week vs. placebo
6 months
No change on insulin secretion or insulin sensitivity parameter
Nagpal et al. 2009 [119]
100 centrally obese, nondiabetic men
13 ng/ml
Fasting plasma glucose and insulin
3 doses of 25(OH)D (120 000 IU each fortnightly vs. placebo)
6 weeks
Increase in OGIS but no changes in fasting insulin secretion or insulin sensitivity
De Boer et al. 2008 [206]
718e738 postmenopausal women with IFG
n.r.
Fasting plasma glucose and insulin
400 IU 25(OH)D þ 1000 mg calcium vs. placebo
6 years
No change in fasting glucose or HOMA-IR
Outcome
COMPONENTS OF INDIRECT CARDIOVASCULAR EFFECTS
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
Author (reference)
Subjects investigated
n.r. not reported, T2DM type 2 diabetes mellitus, IGT impaired glucose tolerance, oGTT oral glucose tolerance test, ivGTT intravenous glucose tolerance test, IFG impaired fasting glycemia, NFG normal fasting glycemia, HOMA-IR homeostasis model assessment of insulin resistance, OGIS oral glucose insulin sensitivity.
1983
1984
102. VITAMIN D AND CARDIOVASCULAR DISEASE
[161,162], nor a third intervention study in 47 healthy postmenopausal women with combined supplementation of 1000 mg calcium and 800 IU vitamin D3 could demonstrate any changes in lipid parameters [163]. Finally a very recent analysis from the Women’s Health Initiative evaluated the effect of 5-year calcium and vitamin D supplementation (1000 mg calcium and 400 IU vitamin D3/day) on lipid levels [164]. They again were unable to detect significant differences in the mean change of important lipid parameters (total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides) between the supplementation and the control group.
CARDIO- AND CEREBROVASCULAR MORTALITY AS ENDPOINT IN OBSERVATIONAL AND INTERVENTIONAL STUDIES Because of the above-mentioned possible pathophysiologic and biologically plausible links between vitamin D, TABLE 102.3
Prospective Cohort Studies Assessing the Association of Serum 25-hydroxyvitamin D Status with Cardiovascular or Cerebrovascular Mortality
Reference (first author) Cohort Dobnig 2008 [5]
Pilz 2008 [172]
the vasculature, and the heart it is tempting to take a closer look at studies that reported hard cardiovascular endpoints such as cardiovascular and cerebrovascular mortality rates (Table 102.3). Most of these prospective observational studies are quite large, population-based and typically individuals were followed up for 6 to 8 years. Two studies reported on secondary cardiovascular disease mortality [5,165]. They were all published between 2008 and 2010. Most studies were performed in Europe (Germany [5], the Netherlands [166], Finland [167], and Italy [168]) and one in the USA [169] for which extensive subgroup analyses are available [170,171]. Usually studies reported on cardiovascular death rates without separating between coronary heart disease and cerebrovascular deaths (or fatal strokes). A formal metaanalysis has not yet been performed likely due to heterogeneity between studies that seems significant. Most studies reported low vitamin D status to be significantly associated with an elevated risk for cardio- and cerebrovascular mortality either in the primary or subgroup analysis. In four studies mean 25(OH)D levels of the reference
LURIC study (Germany)
Study setting
Median Mean No. of followage events up Participants (yrs) (n) (years)
sympt. 3217 patients scheduled for coronary angiography
62
LURIC study
463
7.7
25(OH)D of reference Outcome group (ng/ml) parameter
25(OH)D of group with significant outcome (ng/ml)
28.4
Q2: 13.3
1.82 (1.29e2.58)
Q1: 7.6
2.22 (1.57e3.13)
42
cardiovascular death
fatal stroke
RR or OR(95% CI) for fully adjusted model
OR 0.67 (0.47e0.94) per Z-value
Melamed NHANES 2008 [169] III
populationbased
13 331
45
777
8.7
>32.1
cardiovascular death
not sign.
Ginde NHANES 2009 [170] III
subgroup 65þ
3408
73
767
7.3
>40
cardiovascular death
10e<20
1.76 (1.16e2.68)
<10
2.97 (1.53e5.78)
cardiovascular death
Q1: 13.9 vs Q2e4
1.40 (1.16e1.69)
cardiovascular death
Lowest quintile: 9
1.31 (1.05e1.63)
cerebrovascular Lowest death quintile: 9
2.08 (1.33e3.22)
cardiovascular death
2.64 (1.14e4.79)
Fiscella NHANES 2010 [171] III
subgroup blacks
ca. 1300
43
not given
Kilkkinen Mini2009 [167] Finland Health Survey
populationbased
6219
49
640
Semba InCHIANTI population2010 [168] based
1006
74
107
27.1
6.5
28.0
>26.5
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
Q1: <10.5
CARDIO- AND CEREBROVASCULAR MORTALITY AS ENDPOINT IN OBSERVATIONAL AND INTERVENTIONAL STUDIES
groups were between 24 and 28 ng/ml and in three studies 30 ng/ml. Typically, significant results were seen for cohorts with 25(OH)D levels 10 to 15 ng/ml. In two analyses from the LURIC cohort that in contrast to population-based studies looked at patients complaining of cardiovascular symptoms [5,172] the relationship of both 25(OH)D as well as 1,25(OH)2D levels with cardiovascular, all-cause and fatal stroke mortality rate was examined. It seems interesting that despite an overall weak correlation (r ¼ 0.36, p < 0.001) between the two D vitamin metabolites, either a low 25(OH)D or a low 1,25(OH)2D level were associated with increased hazard ratios for mortality (Fig. 102.2). This led the authors to conclude that both forms of the D vitamins may yield similar but independent biologic effects. This was also in line with their finding that, when both variables were considered concomitantly, a synergistic effect on mortality risk seems to be evident. It seems of interest that Fiscella et al. who performed a subgroup analysis of the black population participating in the NHANES III survey found an incident rate ratio (IRR) for cardiovascular death for the lowest compared with the higher three 25(OH)D quartiles of 1.40 (95% CI 1.16e1.69; p ¼ 0.001) that further increased when baseline cardiovascular-related morbidity parameters such as diabetes, cardiovascular disease, and impaired renal function were excluded [171]. Similarly in the study by Dobnig et al. where coronary angiograms at baseline allowed patients to be categorized into those with and without present coronary artery disease (CAD), associations for 25(OH)D and 1,25(OH)2D were consistently higher for patients with no evidence of significant CAD
1985
[5] (Fig. 102.3). It was concluded that both D vitamins seem to be important mediators of mortality even when there is little or no indication of overt vascular disease. Fiscella et al. interpreted their results by commenting that the full analysis seems overadjusted [171]. They suggested that if 25(OH)D were causally related to the onset of cardiovascular-related morbidity then those with cardiovascular-related morbidity at baseline presumably developed those conditions, in part, because of low 25(OH)D levels. Thus, including such participants in the analysis and adjusting for those conditions results in partially adjusting for cardiovascular effects of 25(OH)D that had already occurred at baseline. They further stated that the analysis excluding those with baseline cardiovascularrelated morbidity likely reflects a less overadjusted estimate of the effects of 25(OH)D on cardiovascular mortality [171]. Zittermann and Grant came to a similar conclusion when saying that it did not make sense to adjust for parameters that influence 25(OH)D levels if one wanted to elucidate the relationship between 25 (OH)D levels and morbidity or mortality risk [173]. Nevertheless, there are also caveats in case one is tempted to extrapolate findings of prospective cohort studies. There are several important sources of potential residual confounding in analysis of prospective observational studies because low 25(OH)D levels may represent a marker of poor health or reflect chronic non-specific illness which in turn may lead to reduced sunlight exposure and consequently lower 25(OH)D levels [174]. Some studies have not taken into account the seasonal variation of 25(OH)D levels or have attempted to adjust for it by grouping individuals by season of sampling, an FIGURE 102.2 25(OH)D and 1,25(OH)2D associations with mortality. These are unpublished data from a post-hoc analysis from an observational cohort study published by Dobnig et al. [5]. This graph illustrates multivariate-adjusted all-cause mortality rates over an observation period in median of 7.7 years of patients who were complaining of cardiovascular symptoms at baseline. Individuals were grouped in 1,25(OH)2D categories (quartiles) and further subdivided by 25(OH)D quartiles. It seems interesting that low 25(OH)D levels were independently associated with all-cause mortality even in patients with relatively high 1,25(OH)2D levels. Only patients of the highest 1,25(OH)2D quartile seemed relatively protected against preterm mortality in case they concomitantly had very low 25(OH)D levels.
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
1986
102. VITAMIN D AND CARDIOVASCULAR DISEASE
FIGURE 102.3 CAD categories and vitamin D. The 25-hydroxyvitamin D (A) and 1,25-dihydroxyvitamin D (B) patient quartiles and corresponding effects on multivariate-adjusted hazard ratios for all-cause mortality according to patients’ coronary artery disease (CAD) status as determined by coronary angiography at baseline. CAD 50% indicates patients with significant CAD; patients without significant CAD are presented as two categories (<50% or by the most stringent criteria of <20% narrowing of 1 of 15 coronary artery segments evaluated). * p < .05. ** p < .01. *** p < .005. **** p < .001 compared with the reference group. There was a more pronounced rise in all-cause mortality across 25(OH)D categories in patients with less severe CAD when compared to those with significant CAD. From [5].
approach that ignores the substantial non-linear changes of 25(OH)D concentrations that occur across seasons [175,176]. If the time of the year of measurement of 25(OH)D is not taken into account appropriately, it is possible that the vitamin D status of individuals is misclassified, especially when recruitment of participants is not uniformly distributed throughout the year [177]. In addition, there may be other less controllable risk factors that could confound the findings, or there could be misclassification of causes of death.
Intervention Studies with All-cause Mortality as Outcome There have been no formal interventional studies with vitamin-D-treated cohorts where mortality was a primary outcome. However, in the past a number of studies were undertaken that investigated the effects
of vitamin D2 or D3 (with or without accompanying calcium supplements) on various health outcomes, such as fractures, changes in bone mineral density, falls, survival of patients with congestive heart failure or colorectal cancer incidence. Autier and Gandini identified 18 such independent randomized controlled trials (up to November 2006) including 57 311 participants and examined the risk of dying from any cause in a metaanalysis [178]. Most trials included in this analysis were conducted in frail elderly people who were at high risk of falls or low-trauma fractures (Fig. 102.4). A total of 4777 deaths from any cause occurred during a trial size-adjusted mean of 5.7 years where daily doses of vitamin D supplements varied from 300 to 2000 IU (mean daily dose was 528 IU). For trials in which 25(OH)D measurements were available, the ratio for in-study 25(OH)D levels between intervention and control groups was between 1.4 and 5.2. Absolute
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
CLINICAL CARDIOVASCULAR OUTCOMES IN OBSERVATIONAL AND RANDOMIZED INTERVENTIONAL STUDIES
1987
FIGURE 102.4 Meta-analysis total mortality [178]. Meta-analysis of 18 randomized controlled trials reporting data on all-cause mortality in vitamin D-treated populations. The trial size-adjusted mean daily vitamin D dose was 528 IU. In nine trials, with available 25(OH)D measurements there was a 1.4- to 5.2-fold difference in serum 25(OH)D levels between the intervention and control groups. The summary relative risk for mortality from any cause was 0.93 (95% confidence interval, 0.87e0.99). There was neither indication for heterogeneity nor indication for publication biases. The summary relative risk (SSR) did not change according to the addition of calcium supplements in the intervention. Most of the trials had an osteoporosis-related primary study endpoint. Ref: M.C. Chapuy et al., N. Engl. J. Med. 327 (1992) 1637e1642; P. Lips et al., Ann. Intern. Med. 124 (1996) 400e406; M.C. Chapuy et al., Osteoporos. Int. 13 (2002) 257e264; H.E. Meyer et al., J. Bone Miner. Res. 17 (2002) 709e715; D.P. Trivedi et al., BMJ. 326 (2003) 469e472; J. Porthouse et al., BMJ. 330 (2005) 1003; A.M. Grant et al., Lancet 365 (2005) 1621e1628; L.Flicker et al., [abstract] J. Bone Miner. Res. 19 (Suppl. 1) (2004) S99; J. Wactawski-Wende et al., N. Engl. J. Med. 354 (2006) 684e696; L. Baeksgaard et al., Osteoporos. Int. 8 (1998) 255e260; M. Komulainen et al. J. Clin. Endocrinol. Metab. 84 (1999) 546e552; M.A. Krieg et al., Osteoporos. Int. 9 (1999) 483e488; N.K. Latham et al., J. Am. Geriatr. Soc. 51 (2003) 291e299; A. Avanell et al., Clin. Trials. 1 (2004) 490e498; R.H. Harwood et al., Age Ageing 33 (2004) 45e51; C. Meier et al., J. Bone Miner. Res. 19 (2004) 1221e1230; M. Brazier et al., Clin. Ther. 27 (2005) 1885e1893; S.S. Schleithoff et al., Am. J. Clin. Nutr. 83 (2006) 754e759.
25(OH)D levels in the intervention groups at the time of supplementation were between 24 and 42 ng/ml. The summary relative risk for mortality was 0.93 (95% CI 0.87e0.99) and this risk was independent from accompanying calcium treatment in the intervention cohorts. There was no indication for heterogeneity (p ¼ 0.37) or of publication bias (p ¼ 0.37) and a subgroup analysis did not show appreciable changes in standardized rate ratios according to trial duration and dose of vitamin D supplements used. Of note is that the reported compliance in the included trials was between 48 and 95%. Because of these large variations in compliance (changes in 25(OH)D levels were also unrelated to daily doses taken) drawing of any conclusion regarding the optimal daily vitamin D dose associated with mortality reduction was not possible. In contrast to a recent meta-analysis reporting that calcium supplements may have an adverse effect on the incidence of myocardial infarction and cardiovascular events [179] the results
of the meta-analysis of Autier and Gandini provide reassurance that ordinary doses of long-term vitamin D supplementation are not associated with an overall adverse effect. Together these findings highlight the urgent need for randomized controlled trials to assess the impact of vitamin D supplementation on the development of cardiovascular-related conditions and mortality.
CLINICAL CARDIOVASCULAR OUTCOMES IN OBSERVATIONAL AND RANDOMIZED INTERVENTIONAL STUDIES An overview of cardiovascular disease outcomes in observational cohort and controlled trials of vitamin D supplementation such as myocardial infarction, stroke, or related composite endpoints is given in Table 102.4.
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
1988
102. VITAMIN D AND CARDIOVASCULAR DISEASE
TABLE 102.4
Reference
Studies Assessing the Association of 25(OH)D Concentration with Incidence of Cardiovascular Events (Studies with Outcome Parameter Cardiovascular Mortality or Stroke were Excluded from this Table) and Randomized, Controlled Trials of Vitamin D Supplementation on Cardiovascular Outcome
Cohort
No. of events Analysis/ Participants (n, %) Intervention
Mean follow-up Outcome (years) parameter
Adjusted HR, RR (95% CI); p for trend
Adjustments
LONGITUDINAL OBSERVATIONAL STUDIES Marniemi et al. 2005 [180]
Finnish
755
130 (17)
1,25(OH)2D3 low vs. high
10 y
MI
0.77 (0.47e1.27)
Age, sex, smoking, functional capacity
Giovanucci et al. 2008 [181]
Health 18 225 professionals follow-up
454 (2.5)
25(OH)D < 15 ng/ml vs. >30 ng/ml
10 y
MI fatal or nonfatal
2.09 (1.24e3.54); p ¼ 0.02
Age, BMI, LDL, HDL, triglycerides, others*
Wang et al. 2008 [182]
Framingham Offspring study
120 (6.9)
25(OH)D <10 ng/ml vs. >15 ng/ml
5y
Composite CVendpoint#
1.81 (1.03e3.18); p ¼ 0.01
Age, sex, BMI and others**
36 282
974 (2.7)
D3, 400 IU/d, plus calcium carbonate, 1000 mg/d vs. placebo
7y
Non-fatal MI, fatal CHD nsult oder TIA
HR 1.04 (0.92 e 1.18]; p ¼ ns HR 1.02 (0.91e1.15]; p ¼ ns
302
22 (7.3)
D2, 1000 IU/d, 1 y vs. placebo, plus calcium citrate 1000 mg/d
Ischemic heart disease Insult
RR 0.67 (0.11e 3.93); p ¼ ns RR 1.00 (0.21e4.88); p ¼ ns
2686
218 (8.1)
D3, 100 000 IU every 4 months vs. placebo
5y
Cardiovascular death
RR 0.84 (0.65 e 1.10); p ¼ ns
192
8 (4)
D3, 800 IU/d, plus calcium carbonate 1000 mg/d vs. placebo
1y
MI, stroke, RR 1.21 (0.38 e pulmonary edema, 3.84); p ¼ ns atrial fibrillation, death from CVD
1739
RANDOMIZED, CONTROLLED TRIALS Hsia et al. Women’s 2007 [183] and Health LaCroix et al. Initiative 2009 [184] Prince et al. 2008 [207]
Fall and 25(OH)D <60 nmol/l
Trivedi et al. 2003 [188]
Healthy subjects
Brazier et al. 2005 [189]
Women, 25(OH)D level <30 nmol/l
* Family history of myocardial infarction, alcohol consumption, physical activity, history of diabetes mellitus and hypertension, ethnicity, region, marine u-3 intake. ** Systolic blood pressure, use of antihypertensive therapy, diabetes mellitus, cigarette smoking, total-to-high-density lipoprotein cholesterol ratio, body mass index, serum creatinine, C-reactive protein levels, use of vitamin-D-containing supplements, physical activity, educational attainment. # Myocardial infarction, coronary insufficiency, angina, stroke, transient ischemic attack, peripheral claudication, heart failure. MI myocardial infarction, CHD coronary heart disease. n.d. no data, ns not significant.
For studies dealing with other cardiovascular or cardiometabolic outcomes such as body weight, lipid changes, incidence rates of type 2 diabetes, or arterial hypertension the reader is referred to respective subsections of this chapter. One early study was performed in 755 elderly subjects from Finland where information on individual food consumption was elicited by means of dietary history interviews in addition to biochemical determinations of serum vitamins and minerals. In this population-based health survey with a follow-up of 10 years low intake of vitamin D (p ¼ 0.011) as well as low serum levels of 1,25(OH)2D (but not 25(OH)D) were significantly predictive of stroke after adjustment for important confounders [180]. Two other studies had superior
study quality and merit special attention. A nested caseecontrol study was conducted in 18 225 men in the Health Professionals Follow-up Study where men aged 40 to 75 years were free of diagnosed cardiovascular disease at baseline [181]. During 10 years of follow-up, 454 men developed non-fatal myocardial infarction or fatal coronary heart disease. After adjustment for matched variables and those being relevant for cardiovascular disease etiology, men deficient in 25(OH)D (15 ng/ml) were at increased risk for myocardial infarction compared to those being sufficient (>30 ng/ ml) (relative risk 2.09; 95% CI 1.24e3.54; p ¼ 0.02 for trend). Even men with intermediate 25(OH)D levels (22.6e29.9 ng/ml) were at increased risk for myocardial infarction. Finally, the third study reported on multiple
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
SITUATION IN PATIENTS WITH CHRONIC KIDNEY DISEASE OR ON HEMODIALYSIS
cardiovascular outcomes from the Framingham Offspring cohort that included myocardial infarction, coronary insufficiency, angina, stroke, transient ischemic attack, peripheral claudication, or heart failure [182]. During a mean follow-up of 5.4 years 120 individuals who initially were free of cardiovascular disease developed a first cardiovascular event. In this large ambulatory cohort, individuals with 25(OH)D baseline levels of <10 ng/mL and those men with levels of 10 to <15 ng/ml, showed an elevated incidence of cardiovascular events (HR 1.80 (1.05e3.08) and HR 1.53 (1.00e2.36), respectively) after adjustment for clinical covariates (that did, however, not include a measure of physical activity). In a subgroup analysis this effect was found to be limited to patients with arterial hypertension at baseline. The authors hypothesized that since vitamin D deficiency may also influence cardiac and vascular remodeling, hypertension could magnify the adverse effects of vitamin D deficiency on the cardiovascular system. Out of the three interventional randomized, controlled trials that looked at cardiovascular outcomes the most significant one is the analysis by Hsia et al. who reported on coronary and cerebrovascular events in the Women’s Health Initiative where 36 282 postmenopausal women 50 to 79 years of age were treated with either 1000 mg of calcium plus 400 IU of vitamin D3 per day or placebo in divided doses [183]. The results showed that neither coronary nor cerebrovascular risk was affected by the study medication. A later subgroup analysis of the Women’s Health Initiative study published by LaCroix et al. looking at cause-specific mortality rates by age and adherence [184] reported a close to significant reduction in total mortality for postmenopausal women <70 of age at study entry (HR 0.89 (0.79e1.01)) but not for older ones. Stroke and coronary heart disease mortality were not affected in either of the reported analyses. This apparently discrepant finding to observational studies could be attributable to several factors [185]. First, the Women’s Health Initiative was a fracture-prevention trial and not designed to evaluate cardiovascular risk [186]. Most importantly, from today’s perspective the dose of 400 IU of vitamin D3 in the treatment arm was below the amount necessary to correct vitamin D deficiency. It has been calculated on the basis of dose and adherence that the effect of supplementation on 25(OH)D levels must have been in the order of 2.5 ng/ml which would be too small an increase to expect a detectable change in clinical outcome [187]. Moreover, patients in the placebo arm were allowed to take vitamin D supplements, which resulted in a mean consumption of vitamin D in the placebo group of close to 400 IU per day. The vitamin D3 administration intervals of 4 months in another interventional study by Trivedi et al. would from
1989
today’s perspective be considered as too long in order to guarantee a stable vitamin D supply and this may have contributed to the negative result [188]. The third interventional study was also negative; however, it lasted for only 1 year and was definitely underpowered [189]. Summarizing the findings from both observational and interventional studies one has to acknowledge that the current level of evidence is too low to proclaim large-scale supplementation based on these results.
SITUATION IN PATIENTS WITH CHRONIC KIDNEY DISEASE OR ON HEMODIALYSIS In the presence of kidney failure and treatment with calcitriol or vitamin D analogs, serum 25(OH)D levels seemed of less significance for the vast majority of physicians. Vitamin D3 or ergocalciferol to treat vitamin D deficiency has received little or no attention by nephrologists caring for patients with CKD because of the widely held view that the kidneys are the only sites where 1a-hydroxylation takes place and that by supplementation with active vitamin D or analogs the “vitamin D” problem would be appropriately addressed anyway. As has been elucidated throughout this book there is now ample evidence for extra-renal 1a-hydroxylase activity, independent of renal conversion. Based on these data it seems reasonable to assume that it may be important not only to care for “endocrine” or systemic 1,25(OH)2D levels (PTH-lowering and increase of calcium) but also for “autocrine” or paracrine (antiinflammation, cell cycle regulation) aspects of the vitamin D system. As is the case in other populations there is also a paucity of studies looking at the effects of vitamin D3 in predialysis and dialysis cohorts. There are no data available where cardiovascular effects of cholecalciferol-treated patients with CKD (chronic kidney disease) stages 2e5 were the primary endpoint. Patients at all stages of CKD, particularly those on dialysis, have greatly increased mortality and morbidity compared to the general population. No randomized controlled trials evaluated mortality, cardiovascular events, hospitalizations, quality of life, or fractures as primary or secondary ends point [190]. See Chapter 81 for discussion of the available data. Because patients with impaired renal function stages I to III commonly have secondary hyperparathyroidism it is reasonable to assume that correction of widespread 25(OH)D deficiency in these patients ameliorates the degree of hyperparathyroidism since 25(OH)D either indirectly via local conversion into 1,25(OH)2D within the parathyroid gland or directly suppresses PTH synthesis [191]. In patients with even higher degrees of
XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
1990
102. VITAMIN D AND CARDIOVASCULAR DISEASE
renal function impairment other effects, i.e. PTH resistance and/or downregulation of VDR, may be important. A decrease in PTH secretion may also be relevant in terms of cardiovascular protection since PTH promotes myocyte hypertrophy [192], vascular remodeling [193] and is thought to exert pro-inflammatory effects, stimulating the release of cytokines and collagen production by vascular smooth muscle cells (VSMC) [194]. In a post-hoc analysis of the Vitamin D, Calcium, Lyon Study II (Decalyos II) Kooienga et al. assessed the impact of treatment with daily 800 IU vitamin D3 plus 1200 mg calcium versus placebo on biochemical parameters in 610 elderly ambulatory institutionalized women, of whom 322 had estimated glomerular filtration rate (eGFR) values <60 ml/min per 1.73 m2, using the MDRD (Modification of Diet in Renal Disease) formula [195]. In all kidney function groups similar improvements in the proportion of individuals achieving 25(OH)D levels >30 ng/ml were seen at 6 months. Of note, the proportion of individuals showing a 30% or more decrease in PTH levels was 50% in all GFR groups of the treatment arm as opposed to 6e9% in the placebo group (p < 0.001 for all). An important limitation of this study is the inability to distinguish between the effects of calcium and vitamin D, because of the combination treatment. According to a recent analysis of the Cochrane Collaboration Group [196] where the effects of “vitamin D compounds” were investigated in randomized controlled trials of CKD patients not requiring dialysis, the active treatment arm was found to decrease PTH on average by 49 pg/ml (95% CI 85 to 12). Patients were clearly more likely to reduce PTH >30% from baseline values versus controls (RR 7.8; 95% CI 4.8e2.7). In this meta-analysis no formulation, route, or schedule of vitamin D compounds was found to alter mortality risk or the need for dialysis. An important limitation of this analysis, however, is the number of available randomized, controlled trials and the small percentage of patients who have reached clinical outcomes in these studies. Most importantly, the bottom-line question remains whether a lowering of PTH levels per se confers any clinical cardiovascular benefits. The uncertainty regarding vitamin D treatment is also reflected by the newer KDIGO (Kidney Disease: Improving Global Outcomes) guidelines that do not give a recommendation regarding treatment with ergoor cholecalciferol supplements of either patients with CKD stages 2e5 or patients on hemo- or peritoneal dialysis [190]. There are at least five prospective, observational studies [197e201] looking at the effects of active vitamin D treatment on cardiovascular disease mortality. These studies were not controlled and two of them did not adjust the analyses for confounders [197,202]. In the
largest study 51 037 hemodialysis patients with a homogeneous age distribution (61e63 years) receiving activated intravenous vitamin D had a lower cardiovascular disease-related mortality (7.6 per 100 personyears) than those who did not receive the treatment (14.6 per 100 person-years, p < 0.001) [202]. The same authors that published the above-cited Cochrane Collaboration Database study performed a second meta-analysis in which 2773 hemodialysis patients participating in 66 randomized clinical trials were included and came to the conclusion that treatment with vitamin D compounds suppresses PTH, leads to elevations in serum calcium and phosphorous levels but does not alter the risk of death [203]. Clearly, the observational data showing vitamin D compounds to be associated with improved survival in CKD need to be confirmed or refuted in specifically designed randomized controlled trials.
SUMMARY AND CONCLUSIONS The data presented in this chapter suggest an important causal link between the vitamin D system and cardiovascular health that is scientifically based on epidemiological data, numerous prospective observational studies, also on some interventional studies (where cardiovascular endpoints, however, were not a dedicated study outcome) and certainly on observations from VDR- and 1a-hydroxylase knockout mouse models. Since the vasculature forms the “nourishing backbone” of all body tissue-specific effects on vasculature tone, inflammation processes within the vascular wall, effects on remodeling of VSMS and cardiac myocytes, thrombosis and finally the RAAS could be expected to have wide-ranging and profound consequences for our overall health. It may well be that all effects in concert or part of these have been responsible for some of the encouraging findings in analyses where all-cause and cardiovascular mortality data have been reported. We might already be much closer to a better understanding of the role of vitamin D within the context of cardiovascular health if we had begun at an earlier time to recognize its potential to have other effects than those few early recognized actions to protect bone. The “drug” vitamin D is cheap, has been around for many years, and yet there are hundreds of unresolved questions about its usefulness. Still, barriers to future trials of vitamin D exist. Bearing in mind that vitamin-D-deficient patients, with better coronary artery disease status, show more favorable mortality rates compared to those at more advanced stages [5], tells us that future intervention trials have to be carefully designed to sort out any potential primary from
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secondary preventive cardiovascular effects. There may be other important obstacles to clinical vitamin D research such as unknown 25(OH)D thresholds, which may well be different for different tissues and also for different patient populations (young versus intermediate versus elderly individuals; patients with different ethnic background and patients with or without CKD). Today, we already know that there will definitely be many answers awaiting us tomorrow. Peering at the site www.clinicaltrials.gov gives us an impression of the wealth of data that will be generated over the next several years. More than 1090 studies dealing with different vitamin D compounds have been registered at this NIH (National Institutes of Health) site and only months ago this number was 930. We definitely foresee an exciting period of time over the next 10 years during which a united effort accomplished by endocrinologists, biologists, epidemiologists, nutritionists, and clinicians will help to lift the fog that currently shrouds the role of vitamin D. By then we will know how relevant today’s “call for action” by leading vitamin D scientists throughout the world is, who demand raising serum 25(OH)D levels to 40 to 60 ng/ml in order to help improve public health status significantly.
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C H A P T E R
103 Vitamin D, Childhood Wheezing, Asthma, and Chronic Obstructive Pulmonary Disease Carlos A. Camargo Jr. 1, Adit A. Ginde 2, Jonathan M. Mansbach 3 1
Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA 2 University of Colorado Denver School of Medicine, Aurora, CO, USA 3 Children’s Hospital Boston, Harvard Medical School, Boston, MA, USA
INTRODUCTION For most of the 20th century, research on sunlight and vitamin D focused on calciumephosphate homeostasis and bone health, particularly the prevention and treatment of rickets. Nevertheless, one of the first Nobel Prizes in Physiology and Medicine was awarded in 1903 to Dr Niels Ryberg Finsen for his discovery of the beneficial effects of sunlight on cutaneous tuberculosis (TB) [1]. This novel infectious disease finding led to important changes in the clinical management of pulmonary TB worldwide, including the development of mountain clinics to provide afflicted patients with better access to sunlight and fresh air [2]. Although the links between ultraviolet B (UVB) exposure and vitamin D synthesis, and between vitamin D deficiency and TB, were developed over subsequent decades, the role of sunlight and vitamin D on acute respiratory infection was largely neglected during the latter half of the 20th century. Over the past decade, researchers around the world have again taken interest in the antimicrobial potential of sunlight and vitamin D [3e8]. For example, while initial studies demonstrated an association between nutritional rickets and respiratory infection among children in developing nations [9e11], more recent studies have found an association between sunlight/vitamin D and risk of respiratory infection in otherwise well-nourished individuals, of all ages, in developed nations [12e16]. This concept is discussed in detail in Chapter 93. With growing evidence that the vitamin D hormone has complex immunologic effects, it was not surprising that some investigators would raise new concerns. Most
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10103-9
notably, Wjst and Dold proposed in 1999 that vitamin D supplementation might be a cause of global increases in asthma and other allergic disorders [17]. Genetic association studies were undertaken to address this putative harm by examining if specific polymorphisms of the vitamin D receptor (VDR) gene were associated with increased risk of asthma. Although these asthma genetics studies showed some statistically significant associations, the results conflicted across individual datasets and studies [18e22]; the relationship between VDR polymorphisms and asthma remained unclear. In this context, Camargo and colleagues proposed the opposite hypothesis of Wjst and colleagues at the 2006 Annual Meeting of the American Academy of Allergy, Asthma, and Immunology [23] e i.e. that widespread vitamin D deficiency (not excess) might explain recent increases in childhood wheezing and asthma. The investigators presented original evidence for this hypothesis from a Boston birth cohort study involving almost 1200 motherechild pairs. In brief, the investigators found that maternal intake of vitamin D during pregnancy had a striking inverse association with risk of recurrent wheezing in offspring. Because most wheezing illness of childhood represents uncomplicated respiratory infection, rather than incident asthma [24], the authors cautioned that further research would need to distinguish between simple respiratory infections (with consequent wheezing) versus a true diagnosis of incident childhood asthma. Over the past 5 years, the relation of low vitamin D status with acute respiratory infections and asthma has become a focus of research teams worldwide. Indeed,
1999
Copyright Ó 2011 Elsevier Inc. All rights reserved.
2000
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most major professional society meetings today, in both the pulmonary and allergy/immunology communities, include lectures or entire symposia on vitamin D. In this chapter, we will examine the relation of vitamin D to childhood wheezing and asthma, along with emerging evidence on the role of vitamin D among patients with chronic obstructive pulmonary disease (COPD). To minimize overlap with other chapters in Section XI of this volume on Immunity and Inflammation, we will touch only briefly on the immunologic mechanisms that likely mediate these recent clinical and epidemiological observations. By contrast, we will describe in more detail the emerging clinical and epidemiologic data on the relation of vitamin D with atopy and allergic diseases (such as eczema and food allergy), given their well-known mechanistic links with childhood asthma [25,26].
COMMON RESPIRATORY DISORDERS To better understand the effect of vitamin D on common respiratory disorders, it is important to discuss, at least briefly, some of the methodological challenges of research in this area, especially the complex interrelations between these disorders. In the following pages, we will provide key background on respiratory infections, childhood wheezing, asthma, and COPD. We then will return our attention to the role of vitamin D on each of these disorders.
Respiratory Infections Respiratory infections are a diverse and complex group of infections that have always been a major cause of morbidity and mortality [27,28]. Although these infections are frequently categorized anatomically into upper and lower respiratory tract infections, respiratory infections may (and often do) involve multiple anatomic locations. Another method of categorization is according to pathogen, with infections commonly divided into viral versus bacterial. For specific infections with respiratory involvement, however, researchers and the general public commonly identify them by the specific pathogen alone, such as influenza and TB. There also are basic issues of disease chronicity (i.e., acute versus chronic infection). As a result of these and other considerations, clinical and epidemiologic research on the relationship between vitamin D and acute respiratory infections has proven challenging. Bronchiolitis provides an excellent example of the complexity of research on acute respiratory infections [29]. Although bronchiolitis is the leading cause of hospitalization in infants and has high annual costs [30], there is no global consensus regarding its definition. Because
bronchiolitis remains a somewhat vague clinical diagnosis [31e33], it should not surprise readers that clinicians differ on who exactly has the condition [34]. In 2006, an American Academy of Pediatrics (AAP) position paper described the typical child with bronchiolitis as being age <2 years and having “rhinitis, tachypnea, wheezing, cough, crackles, use of accessory muscles, and/or nasal flaring” [35]. In other countries, however, we are familiar with strong opinions that bronchiolitis only should be diagnosed among infants (defined as age <1 year), or that the presence of specific exam findings (e.g., crackles) should be mandatory. Although the microbiology of bronchiolitis should help with classification, there is a spectrum of clinical disease that is not understood. For example, while almost all children are infected by age 2 years with respiratory syncytial virus (RSV) [36,37], the leading cause of severe bronchiolitis [38,39], most children infected with RSV do not present to an acute care setting with clinical bronchiolitis [36,40]. Indeed, we estimate that <10% of US children infected with RSV will present to the emergency department with bronchiolitis, and that only 2e3% are hospitalized [41,42]. To further complicate matters, not only is there a growing list of other viruses linked to bronchiolitis (Table 103.1), including human rhinovirus [43,44], but it is clear that multiple pathogen infections are common [29]. Furthermore, an unknown percentage of cases may be caused by bacteria such as Mycoplasma pneumoniae [45]. Even without considering the different short-term and long-term clinical implications of these different forms of bronchiolitis, one can quickly understand the complexity of classifying this common respiratory infection of childhood. The complexity and diversity of “respiratory infections” makes it challenging to link an individual factor TABLE 103.1
Frequency of Common Viruses Linked to Bronchiolitis, According to Healthcare Setting (Adapted from [29]) Outpatienta
Emergencyb
Inpatientc
Respiratory syncytial virus
11e27%
64%
69%
Parainfluenza
5e13%
n/a
3%
Influenza
1e5%
4%
2%
Human rhinovirus
46e49%
16%
21%
Metapneumovirus
2%
7%
5%
Combination infections
10e17%
14%
16%
a Based on upper and lower respiratory infections during infants’ first winter [203] and first year [204] b Based on children presenting to 14 US emergency departments [205] c Based on children admitted to 15 US hospitals (Camargo et al., unpublished data) Abbreviation: n/a denotes not available.
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like vitamin D to the risk of developing this composite outcome. To be more specific, vitamin D might have an important association with some but not all specific types of respiratory infections, as defined by either specific anatomical areas or by specific pathogenic organisms. Vitamin D also might influence disease severity. For example, while all children may at some point be infected with RSV, only those with low 25(OH)D levels e or other major predisposing factors e develop severe enough RSV infections to require intensive care. If these different hypotheses are true, the common practice of collapsing diverse infections into one composite “respiratory infection” outcome would likely obscure real associations e i.e. one would conclude that there was no association when, in fact, there was one. Studies on the relation of sunlight and vitamin D status to respiratory infections (and associated disorders) need to be interpreted with this important caveat in mind.
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Hypothetical prevalence of three different wheezing phenotypes in childhood. For each age interval, the overall wheezing prevalence is the sum of the areas under each curve. The dashed lines emphasize that the possibility of different curve shapes due to many factors, including overlap between the three groups. (Adapted from [202]).
FIGURE 103.1
Childhood Wheezing The heterogeneity of respiratory infections is exceeded by that of childhood wheezing. After all, many different conditions can cause “wheezes,” which are just musical sounds caused by the passage of air through narrowed respiratory tract airways [46]. Across the lifespan, this airway narrowing probably is most often caused by respiratory infections. For individuals with asthma or COPD, the airway narrowing and associated wheezing results from a complex mixture of either baseline or exacerbated inflammation/edema and bronchoconstriction, often but not always with concurrent infection. In all ages, wheezing also can be caused by other, less common conditions. A classic cause of childhood wheezing unrelated to asthma is an airway foreign body [47]. Among adults, decompensated heart failure e with so-called “cardiac wheezing” e is a common asthma/COPD mimic in acute care settings [48]. Large cohort studies have provided important information about the natural history of childhood wheezing. For example, the landmark studies of Martinez and colleagues clearly demonstrate that many children who wheeze in early childhood have only transient episodes (during acute respiratory infections) and do not go on to develop asthma [24,49]. Accordingly, asthma researchers try, with varying levels of success, to divide wheezing children into different clinical groups. In one system, children are grouped into: (1) transient early wheezing, (2) late-onset wheezing, or (3) persistent wheezing (i.e., wheezing was present during both early and late periods of early childhood). In another system, children are grouped into: (1) transient early wheezers, (2) non-atopic wheezers, or (3) IgE-associated wheeze/asthma (Fig. 103.1). Although
these groupings help to divide children according to their likelihood of developing asthma by age 6 years, many children e in all groups e do not develop asthma. Studies have repeatedly affirmed the clinical adage that “All that wheezes is not asthma.” To further emphasize the difference between childhood wheezing and asthma, many young children with asthma present with recurrent nocturnal cough and lack any evidence of wheezing [50]. For all of these reasons, one should be cautious about generalizing from findings about childhood wheezing to actual asthma. This cautionary note applies even to recurrent wheezing, which may represent little more than recurrent respiratory infections in nonasthmatic children. Since young children are estimated to experience six to ten acute respiratory infections per year [51], clinicians (and researchers) might anticipate that attentive parents are likely to report recurrent “wheezing” in most children, and that this could be misdiagnosed as asthma.
Asthma Asthma is a common medical condition that also is associated with high morbidity and health care utilization [52,53]. Although defining asthma challenged clinicians for centuries, today there is general acceptance of the 1987 definition from the American Thoracic Society (ATS) [54] e i.e. asthma is a chronic lung disease characterized by: (1) airway narrowing that is reversible (though not always completely), either spontaneously or with treatment; (2) airway inflammation; and (3) bronchial hyper-responsiveness (BHR) to a variety of stimuli.
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Although the ATS asthma definition is clear, it has proven very difficult to apply in epidemiologic studies, where subjects may be dispersed over large geographic areas and where spirometry and hyper-responsiveness testing usually are not feasible. As a result, asthma epidemiologists have relied on much simpler definitions of asthma, such as an affirmative response to the question: “Do you have doctor-diagnosed asthma?” Although the potential limitations of this epidemiologic approach are self-evident, this approach actually performs with adequate accuracy [55] to allow epidemiologists to perform asthma surveillance and to examine potential risk factors for the disease. Similar questions have, in fact, contributed to the widespread concern about the dramatic rise in “asthma” over the past few decades [52]. While viral respiratory infections are common triggers of asthma exacerbations [56,57], and occur across the lifespan, the vast majority of asthma begins in early childhood. Estimates vary but approximately 80e90% of asthma begins before age 6 years, with 70% of asthmatic children having asthma symptoms before age 3 years [58,59]. The etiology of asthma has proven elusive, but we can infer from these clinical observations that the major risk factors must be present in early life e either in utero or at least in the first months/years of childhood [60]. Childhood atopy is one of the strongest risk factors for asthma [61], yet many asthmatic individuals are nonatopic [62]. Indeed, a growing number of asthma researchers acknowledge that the term “asthma” is likely a syndrome composed of heterogeneous diseases [63].
Chronic Obstructive Pulmonary Disease COPD is another common medical condition that is associated with high morbidity and mortality [64]. The most widely accepted definition for COPD comes from the Global Initiative for Chronic Obstructive Lung Disease (GOLD): COPD is “a preventable and treatable disease with some significant extrapulmonary effects that may contribute to the severity in individual patients. Its pulmonary component is characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lung to noxious particles or gases” [64]. Even more so than asthma, there have been significant disagreements about the definition of COPD over the past few decades. Classical descriptions of COPD have emphasized the two major types: “chronic bronchitis” and “emphysema.” Although these COPD types continue to be taught to healthcare professionals, there now is universal recognition of their considerable overlap. Uncertainty regarding COPD diagnosis has been compounded by the fact that chronic bronchitis was,
for many years, a clinical diagnosis (e.g., requiring that the patient have a daily cough productive of sputum for at least 3 months over at least 2 consecutive years) while emphysema was an anatomic diagnosis requiring actual parenchymal destruction. By contrast, the 2009 GOLD definition acknowledges that the chronic airflow limitation characteristic of COPD is caused by a mixture of small airway disease (obstructive bronchiolitis) and parenchymal destruction (emphysema), with the relative contributions of these pathological changes varying from person to person [64]. The evolving definition of COPD requires careful attention to case definition when integrating evidence across recent decades.
VITAMIN D AND RESPIRATORY INFECTION Vitamin D is thought to affect risk of infection through its myriad effects on the immune system. The connection between vitamin D and infection is covered in more detail in Chapter 93. Nevertheless, because the effect of vitamin D on infections is so central to understanding the relation of vitamin D with respiratory/ allergic disorders, we address the topic here, focusing on the clinical and epidemiologic evidence to date.
Tuberculosis The first clear link between vitamin D and infectious disease goes back more than a century to the TB research of Dr. Finsen, who discovered that concentrated light radiation was an effective treatment for lupus vulgaris (cutaneous TB) [1]. Although he was not aware of the exact mechanism for this therapeutic benefit, most scientists today would attribute his treatment successes, at least in part, to UVB-induced increases in vitamin D. In recent years, many research groups around the world have focused directly on the link between vitamin D and TB [65,66]. For example, a hospital-based caseecontrol study in London found that vitamin D deficiency was associated with an odds ratio (OR) of 2.9 (95% confidence interval (CI) 1.3e6.5) for having active TB [67]. Susceptibility to TB also has been linked to VDR polymorphisms, with the presence of FokI F allele protecting against TB infection, and the TaqI t allele protecting against active disease but not infection [68]. More recently, Martineau and colleagues reported that carriage of the Gc2/2 genotype, compared with Gc1/1 genotype, in the vitamin D binding protein (DBP), was strongly associated with susceptibility to active TB [69]. They also noted that the association was preserved if the serum 25(OH)D level was <8 ng/ml but not if serum levels
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were higher. DBP polymorphisms are discussed further in Chapter 5. With regard to clinical trials, Martineau and colleagues [70] demonstrated that a single dose of 2.5 mg (100 000 IU) of vitamin D2 (ergocalciferol) enhanced immunity to M. tuberculosis in London patients. By contrast, a recent randomized controlled trial (RCT) of 365 adults with TB in Guinea-Bissau found that vitamin D treatment did not improve clinical outcomes [71]. The vitamin D intervention in this TB trial was 100 000 IU at enrollment and again 5 and 8 months later, and the authors suggest that this dose may have been insufficient for this population. In brief, the relationship between vitamin D and TB remains an active area of research. Given the important connection between respiratory infections and other common respiratory disorders, this TB research should continue to provide important insights into how vitamin D may affect childhood wheezing, asthma, and COPD.
Observational Studies on Respiratory Infections In recent years, researchers have repeatedly observed that vitamin D appears to have antimicrobial activity, not only against TB but against a broad range of respiratory pathogens. Several studies have found that nutritional rickets is associated with respiratory infections [9e11], and worse treatment outcomes [72]. Although 25-hydroxyvitamin D (25(OH)D) levels of at least 10 ng/ml (25 nmol/l) are known to prevent most cases of nutritional rickets, the relationship between higher 25(OH)D levels and respiratory infection is of growing public health interest. Many countries now have a low population prevalence of rickets, but they continue to have high rates of vitamin D deficiency [73]. Indeed, recent clinical studies from developing nations around the world have demonstrated a fairly consistent association between lower, but non-rachitic 25(OH)D levels, and increased risk of respiratory infections in children [74e76]. In 2004, Wayse and colleagues reported findings from a caseecontrol study that Indian children age 2e60 months with serum 25(OH)D levels <9 ng/ml had more than ten-fold higher odds of acquiring a severe acute lower respiratory infection than those with higher 25(OH)D levels [74]. Likewise, a recent caseecontrol study by Karatekin and colleagues in Turkey found that mean serum 25(OH)D levels were lower in neonatal cases of acute lower respiratory infection (9.1 ng/ml) than in age-matched controls (16.3 ng/ ml) [75]. More recently, Roth and colleagues reported a caseecontrol study from rural Bangladesh which showed lower mean 25(OH)D levels among children aged 1e18 months hospitalized with acute lower respiratory infection (11.7 ng/ml), as compared to matched controls (15.7 ng/ml) [76].
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Roth and colleagues reported quite different findings in their earlier caseecontrol study of Canadian children ages 1 to 25 months [77]. The Canadian study showed no difference in mean serum 25(OH)D levels between patients with acute lower respiratory tract infection (30.8 ng/ml) and hospital controls (30.9 ng/ml). Of note, the average circulating 25(OH)D level of these children was >30 ng/ml because “virtually all of the infants.consumed vitamin D” through fortified infant formula or supplements. We believe that inadequate variation in the primary exposure (vitamin D) did not allow for proper testing of the study hypothesis. These data also demonstrate that severe bronchiolitis occurs even at serum 25(OH)D levels of 30 ng/ml; the impact of significantly higher levels (e.g., 40e50 ng/ml) on bronchiolitis risk is not known. In another Canadian caseecontrol study, McNally and colleagues compared the 25(OH)D levels of 105 children hospitalized with bronchiolitis or pneumonia with 92 control subjects without respiratory symptoms [78]. Although the mean level of 25(OH)D was not different between cases and controls (32 versus 33 ng/ ml, respectively), the mean level for cases admitted to the pediatric intensive care unit (20 ng/ml) was significantly lower than that observed for children admitted to the general pediatrics ward (35 ng/ml) or that observed among controls (33 ng/ml). Although these novel findings require replication, they suggest that the immunomodulatory properties of vitamin D might influence the severity of respiratory infections. The relation between vitamin D status and incident childhood wheezing was examined by Camargo and colleagues in two separate birth cohorts at higher latitudes: one in Massachusetts [79] and one in New Zealand [80]. In the Massachusetts cohort, Project Viva, the investigators found that lower maternal intake of vitamin D during pregnancy was associated with significantly increased risk of recurrent wheezing [79]. In these 1194 motherechild pairs, the mean (SD) total vitamin D intake during pregnancy was 548 (167) IU/ d. By age 3 years, 186 children (16%) had recurrent wheeze. Compared with mothers in the lowest quartile of daily intake (median: 356 IU), those in the highest quartile (724 IU) had a lower risk of having a child with recurrent wheeze (OR 0.39; 95% CI 0.25e0.62; p for trend < 0.001). A 100 IU increase in vitamin D intake was associated with lower risk (OR 0.81; 95% CI 0.74e0.89), regardless of whether vitamin D was from the diet (OR 0.81) or supplements (OR 0.82). Adjustment for 12 potential confounders, including maternal intake of other dietary factors, did not change the results. Although the “respiratory infection” outcome was crude, it also showed an inverse association and favored an infectious disease explanation for the study results [79]. Ongoing follow-up of these children to age 7 years
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103. VITAMIN D, CHILDHOOD WHEEZING, ASTHMA, AND CHRONIC OBSTRUCTIVE PULMONARY DISEASE
Associations between cord blood 25-hydroxyvitamin D (25(OH)D) levels with probabilities of cumulative wheeze or incident asthma by age 5 years (n ¼ 922 children in New Zealand). The vertical lines denote serum 25(OH)D benchmarks (in ng/ml). (Adapted from [82]).
FIGURE 103.2
suggests no association between maternal intake of vitamin D and risk of incident doctor-diagnosed asthma (Camargo et al., unpublished data). The Boston findings on maternal intake of vitamin D and childhood wheezing were replicated in a Scottish birth cohort by Devereux and colleagues [81]. In the 1212 motherechild pairs, the median (interquartile range) total vitamin D intake during pregnancy was 128 (103e165) IU/d, which was significantly lower than the intake of Boston mothers [79]. Nevertheless, compared to mothers in the lowest quintile of daily intake (median: 77 IU), those in the highest quintile (median: 275 IU) had lower risk for ever wheeze (OR 0.48; 95% CI 0.25e0.91), wheeze in the previous year (OR 0.35; 95% CI 0.15e0.83), and persistent wheeze (OR 0.33; 95% CI 0.11e0.98) in their 5-year-old children. In contrast to these impressive vitamin Dewheeze findings, there was no association between maternal vitamin D intake during pregnancy with doctor-diagnosed asthma at age 5 years (p ¼ 0.98). In the New Zealand Asthma and Allergy Cohort Study, Camargo and colleagues examined the association between low cord blood levels of 25(OH)D and subsequent risk of respiratory infections and childhood wheezing [80,82]. The availability of circulating 25(OH)D levels provided a distinct advantage to prior studies since it better assesses vitamin D status than self-reports of dietary intake alone. In this seemingly healthy birth cohort of 922 children with cord blood specimens available for assay, 20% of children had
a cord blood 25(OH)D level <10 ng/ml, and 73% (cumulative) had a 25(OH)D level <30 ng/ml. Cord blood 25(OH)D level was inversely associated with risk of respiratory infection by age 3 months: children with 25 (OH)D levels <10 ng/ml were at a two-fold higher risk than those with levels 30 ng/ml. These prospective findings were independent of season and other potential confounders and provide strong evidence for the “protective” effect of vitamin D on risk of respiratory infection. Indeed, cord blood 25(OH)D level also had an inverse association with risk of wheezing illness at ages 15 months, 3 years, and 5 years (all p < 0.05; see Fig. 103.2). By contrast, cord blood 25(OH)D level had no apparent association with doctor-diagnosed asthma at age 5 years (p ¼ 0.37). To further explore the role of vitamin D in respiratory infections, we analyzed nationally representative data from the Third National Health and Nutrition Examination Survey (NHANES-III) to examine the association between vitamin D status and upper respiratory infection [14]. In brief, we found that lower serum 25(OH)D levels were associated with an increased adjusted OR of recent upper respiratory infection (compared with 30 ng/ml: OR 1.36 for <10 ng/ml and 1.24 for 10e29 ng/ml groups) [14]. As shown in Figure 103.3, the inverse association was present in all seasons. We also found that the association between serum 25(OH)D <10 ng/ml and upper respiratory infection was stronger among individuals with asthma (OR 5.67) versus those without asthma (OR 1.24; p for interaction ¼ 0.007).
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VITAMIN D AND RESPIRATORY INFECTION
35
25(OH)D <10 ng/ml 25(OH)D 10–29 ng/ml 25(OH)D ≥30 ng/ml
30
Recent URI (%)
25
20
15
10
5
0
Winter
Spring
Summer
Fall
Season Association between serum 25-hydroxyvitamin D level and recent upper respiratory infection (URI), by season (n ¼ 18 883 participants in Third National Health and Nutrition Examination Survey). Error bars represent standard errors of the estimates. (Adapted from [14]).
FIGURE 103.3
The first large cohort study on vitamin D and acute respiratory infections in adults was published in 2007 by Laaksi and colleagues in Finland [12]. In a cohort of 800 young male soldiers, the investigators found that soldiers with serum 25(OH)D levels <16 ng/ml at baseline had 63% higher risk of absence from duty due to respiratory infection over the following 6 months, as compared to soldiers with levels of 16 ng/ml (p ¼ 0.004). Recently, Sabetta and colleagues reported similar results in a cohort of almost 200 healthy adults in Connecticut [16]. They found serum 25(OH)D levels of 38 ng/ml were associated with a two-fold reduction in risk of acute respiratory infection (p < 0.0001) and with a marked reduction in the percentage of days ill. To date, few groups have examined the effect of vitamin D pathway-related polymorphisms on the association between vitamin D status and acute respiratory infection. In 2007, Janssen and colleagues reported a genetic association study involving 470 children hospitalized for RSV bronchiolitis, their parents, and 1008 random population controls [83]. They analyzed 384 single-nucleotide polymorphisms (SNPs) in 220 candidate genes and found that SNPs in innate immune genes e including VDR (rs10735810) e are important in determining susceptibility to RSV bronchiolitis. In a brief report, Roth and colleagues compared two vitaminD-related genes among 56 young children hospitalized with acute lower respiratory infection to 64 controls [84]. Compared to the FokI FF, the FokI ff genotype was much more common among cases than controls (adjusted OR 7.38). The investigators also found a weaker association for TaqI polymorphisms. In another recent study, Chun and colleagues demonstrated that DBP plays a pivotal role in regulating the bioavailability of
25(OH)D to monocytes [85]; the authors suggest that vitamin-D-dependent antimicrobial responses are therefore likely to be strongly influenced by DBP polymorphisms. Although these provocative genetic findings require confirmation, they suggest that an improved understanding of the interplay between vitamin D metabolites and VDR on host response to infection may affect the results of studies on vitamin D and respiratory infections. Likewise, failure to control, or otherwise account for, these genetic factors may tend to obscure real differences between groups. VDR polymorphism data are discussed further in Chapter 56 and DBP polymorphisms in Chapter 5.
Interventional Trials on Respiratory Infections To date, there have been few RCTs on the effect of vitamin D on respiratory infection. Nevertheless, the preliminary data are promising. For example, two interventional cohort studies with 600 to 700 IU of vitamin D daily from cod liver oil/multivitamin supplementation [86] and 60 000 IU weekly from a vitamin D/calcium supplement [87] noted a decrease in respiratory infections in children receiving supplementation. One randomized, controlled trial of bone loss in postmenopausal black women found that 7.7% of women randomized to vitamin D, 800 to 2000 IU daily, reported respiratory symptoms over the 3-year follow-up, compared with 25.0% in the control group [88]. Another sub-study of a randomized, controlled trial for fracture prevention [89] found a possible reduction in wintertime infection in participants randomized to vitamin D, 800 IU/day (adjusted OR 0.90; 95% CI 0.76e1.07), though the result was not statistically significant.
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103. VITAMIN D, CHILDHOOD WHEEZING, ASTHMA, AND CHRONIC OBSTRUCTIVE PULMONARY DISEASE
The first interventional studies that were specifically designed to test the effect of vitamin D supplementation on risk of acute respiratory infection have yielded mixed results [90e92]. Possible explanations were discussed at a recent National Institutes of Health-sponsored workshop on the “Impact of Micronutrients and Respiratory Infection” (Taylor CE and Camargo CA Jr; May 2010; unpublished report). In addition to traditional concerns about sample size and adherence to protocol, participants focused on issues of vitamin D dosage and timing. Although randomized trials typically assign a uniform intervention dose to subjects (as compared to placebo), vitamin D trials probably would be more effective with an individualized approach that strived for a target level of serum 25(OH)D (e.g., 40 ng/ml). Logistical challenges, however, preclude this approach because real-time measurement of 25(OH)D levels at higher latitudes is likely to reveal vitamin D deficiency and, for ethical reasons, this would require treatment with vitamin D; such a co-intervention would seriously impair the ability of the trial to demonstrate vitamin D effects. Likewise, RCTs need to be of sufficient duration for serum levels of 25(OH)D to stabilize and, consequently, for vitamin D to exert its effect. Shorter trials (e.g., those <6 months) may indeed show clinical differences but they would do so despite a 2e3 month delay in the treatment group reaching the 25(OH)D level necessary for the hypothesized benefits. With this background in mind, we now will review the three published RCTs on vitamin D and risk of acute respiratory infection (as of mid-2010): 1. Urashima and colleagues compared the effect of vitamin D3 supplement (1200 IU/day) versus placebo on seasonal influenza A among 430 Japanese schoolchildren [90]. Children were in the trial for a total of 4 months (from December to March). In the 334 subjects with complete follow-up, influenza A occurred in 10.8% of children in the vitamin D group compared with 18.6% in the placebo group (RR 0.58, 95% CI 0.34e0.99). In subgroup analyses, the investigators found that the reduction in influenza A was more prominent in children who were not taking other vitamin D supplements (RR 0.36; 95% CI 0.17e0.78). Among children with a history of asthma, asthma exacerbations were less common among children on vitamin D supplements (RR 0.17; 95% CI 0.04e0.73). There was no significant difference in other secondary outcomes, including risk of influenza B, pneumonia, or other infection outcomes. 2. Li-Ng and colleagues randomized 162 adults in New York to vitamin D3 supplement (2000 IU/day) versus placebo for a total of 3 months between December to March [91]. The mean 25(OH)D level at baseline was
similar between the two groups (vitamin D 25.6 ng/ ml versus control 25.2 ng/ml) and increased to 35.5 ng/ml in the vitamin D group while staying “virtually constant” among controls. Despite this increase, the investigators found no benefit on incidence or severity of viral upper respiratory infections. For example, the vitamin D group had 48 upper respiratory infections versus 50 outcomes in the placebo group (p ¼ 0.57). The investigators called for further studies to determine the role of vitamin D on infection. 3. The third RCT on vitamin D supplementation and acute respiratory infections was performed in Finland by Laaksi and colleagues [92]. The investigators randomized 164 young Finnish men to vitamin D (400 IU/day) versus placebo for a total of 6 months (October to March). The mean 25(OH)D level at baseline was similar in a subset of subjects from the two groups (vitamin D 31.5 ng/ml versus control 29.8 ng/ml). After 6 months, the mean 25(OH)D levels were 28.7 ng/ml and 20.6 ng/ml, respectively. Although the difference was of borderline statistical significance, subjects on vitamin D supplement had a lower mean number of days absent from duty due to respiratory infection, as compared to controls (2.2 versus 3.0 days, p ¼ 0.06). Likewise, the proportion of men remaining healthy throughout the 6-month period was higher in the intervention group (51.3% versus 35.7%, p ¼ 0.045). Taken together, we believe that interventional studies to date, while not uniform in their results, support an inverse association between vitamin D supplementation and risk of acute respiratory infection. Two of the three available RCTs were seriously compromised by either the use of a low dose of vitamin D (400 IU/day [92]) or a relatively short intervention period (3 months [91]). The impact of raising the serum 25(OH)D level of most subjects to “optimal” levels (e.g., 40 ng/ml) still requires investigation. Vitamin D trials would be particularly attractive in high-risk populations, such as children in day-care centers, residents of nursing homes, hospital personnel, or individuals with asthma or COPD. Such trials already are under way in several countries worldwide e e.g. New Zealand (ACTRN12609000486224), the UK (clinicaltrials.gov NCT01069874), and the USA (clinicaltrials.gov NCT01102374).
VITAMIN D AND ASTHMA Asthma Pathogenesis Given the early age of asthma onset [93,94], and growing evidence on the developmental origins of health and disease [95], the role of maternal diet during
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pregnancy on the asthma risk of offspring is a particularly intriguing topic. Although investigators have debated the role of micronutrient intake on asthma risk for more than a decade, multiple reviews of diet and asthma e from 1997 to 2005 e said nothing about vitamin D as a potential risk factor for asthma [96e99]. As discussed by Camargo and colleagues [79], epidemiologic data suggest a possible association between vitamin D deficiency and the asthma epidemic. For example, the prevalence of both conditions is higher in racial/ethnic minorities, obese individuals, and Westernized populations [52,100]. Furthermore, large crosssectional studies of adolescents and adults have found that vitamin D insufficiency is correlated with lower pulmonary function, including forced expiratory volume in 1 second (FEV1) and forced vital capacity [101,102]. Of course, the causal nature of these crosssectional associations is uncertain. Although low vitamin D status may cause asthma (or lower pulmonary function), it is also possible that individuals with asthma are less likely to exercise outdoors and that simple lifestyle differences could cause vitamin D insufficiency. Along those same lines, lingering concerns about the adverse effect of milk on asthma may have led some asthmatic children to avoid milk intake (an important dietary source of vitamin D in US children [79]), and this food avoidance also could create a spurious inverse association between vitamin D intake and asthma. Prospective studies (especially RCTs) would help to address these important methodological concerns. As noted earlier, the hypothesis that vitamin D deficiency may cause asthma e and that vitamin D supplements may prevent asthma e is disputed by Wjst, who has repeatedly described vitamin D supplementation as an important cause of asthma [17,103,104]. The strongest support for this putative harm comes from a large birth cohort in northern Finland [105]. In this 2004 publication, Hypponen, Wjst and colleagues reported that regular vitamin D supplementation (2000 IU/d) in the first year of life increased the risks of developing atopy (OR 1.46), allergic rhinitis (OR 1.66), and asthma (OR 1.35), by age 31 years. For reasons that we will discuss later in this chapter, we suspect that this finding may be related to the very high dose of vitamin D supplementation used in these infants (i.e., that this may represent a dose-specific effect). Regardless, this retrospective cohort study also was limited by the absence of data on maternal intake of vitamin D and the inability to control for major confounders. Furthermore, recall bias may have affected the ascertainment of early-life asthma and allergies. Two other recent European publications also suggest that vitamin D supplements may increase asthma/ allergy risk [106,107]. Gale and colleagues studied child
2007
health outcomes in a UK birth cohort and found that higher levels of maternal 25(OH)D (measured at a median of 33 weeks gestation) were associated with increased risk of atopic outcomes [106]. More specifically, children whose mothers had 25(OH)D levels in pregnancy >30 ng/ml had increased risk of eczema at 9 months (OR 3.26) and asthma at 9 years (OR 5.40), compared to children whose mothers had levels <12 ng/ml. Unfortunately, this study also has several important methodological limitations, including small numbers of outcomes and poor follow-up (e.g., only 30% at age 9 years); the authors appropriately called for confirmation of these associations in other studies. The findings are similar, however, to those of Back and colleagues in Sweden [107]. In a small cohort of 123 children, the investigators found higher age 6 year prevalence of atopic conditions among children who had taken vitamin D supplements during infancy. In sum, although we have methodological concerns about all three of the aforementioned studies [105e107], we acknowledge that these European data raise important questions about the safety of high-dose vitamin D supplementation in late pregnancy or infancy. Our concerns are mitigated by other epidemiologic evidence that suggests that vitamin D has no apparent association with incident asthma or may even have a modest “protective” association. As described in the section on “Vitamin D and respiratory infection,” above, birth cohorts in both Boston [79] and Scotland [81] found an inverse association between maternal intake of vitamin D during pregnancy and risk of recurrent childhood wheezing, which is e as previously noted e not synonymous with true asthma. The Scottish investigators reported that lower vitamin D intake during pregnancy was associated with a borderline significant decrease in bronchodilator response (p ¼ 0.04), but there was no association with doctor-diagnosed asthma or other asthma-related outcomes, such as spirometry or exhaled nitric oxide concentration [81]. Both findings were limited, however, by the measurement of vitamin D status from diet and supplements alone. Thus, the unique contribution of the New Zealand birth cohort [82] is the concurrent demonstration that low cord blood 25(OH)D levels were associated with increased risk of respiratory infections and childhood wheezing, but had no association with risk of current asthma at age 5 years (Fig. 103.2). The lack of association between vitamin D and incident asthma was present for both atopic asthma and non-atopic asthma, with atopy defined by skin-prick testing for common allergens at age 15 months. Additional birth cohort findings were reported recently from Finland [108] and Japan [109]. Although both publications lacked data on circulating 25(OH)D levels, they do provide valuable insights. In Finland,
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Erkkola and colleagues reanalyzed data from a birth cohort of 1669 children with HLA-DQB1-conferred susceptibility for type 1 diabetes [108]. Although they found that maternal intake of vitamin D from foods was negatively associated with risk of childhood asthma (hazard ratio, 0.80) and allergic rhinitis (hazard ratio, 0.85), they also found that vitamin D supplements alone had no association with either outcome. In Japan, Miyake and colleagues examined the association between maternal diet and wheeze symptoms during childhood in a birth cohort with 763 motherechild pairs [109]. They found that higher maternal intake of various dairy foods was associated with less wheeze by age 16e24 months. When they classified maternal vitamin D consumption during pregnancy into two groups using a cut-off point at the 25th percentile, children whose mothers consumed 172 IU/day had significantly reduced risk of early childhood wheeze (adjusted OR, 0.64; 95% 0.43e0.97). The investigators appropriately caution that their wheeze findings may be explained by a vitamin-D-related prevention of respiratory infections, rather than an effect on asthma or other atopic disorders. Other recent epidemiologic studies have used a caseecontrol design to examine the association between circulating 25(OH)D levels and asthma [110,111]. In Washington DC, Freishtat and colleagues found a higher prevalence of vitamin D deficiency among inner-city African-American youth with asthma than among non-asthmatic controls [110]. By contrast, Devereux and colleagues found no association between serum 25(OH)D level and asthma in UK adults [111]. Although the utility of the caseecontrol study design for causal inferences is quite limited, neither study supports increased asthma risk at higher 25(OH)D levels e at least among older children and adults. The link between vitamin D and asthma also has been examined using genetic data. Although preliminary evidence from two family-based studies suggested that specific VDR gene polymorphisms were associated with asthma among North American children and adults [18,19], the results were inconsistent across cohorts/studies and were not confirmed [20,22]. A recent study by Bosse and colleagues reported that several genes involved in the vitamin D pathway (e.g., IL10, CYP24A1, VDR) had “modest levels of association” with asthma and atopy but the specific SNPs, or the orientation of the risk alleles, were different between populations [112]. Studies of VDR polymorphisms in Chinese individuals with asthma also have found contradictory results, with one study showing significant results for one of five SNPs within the VDR gene [113] and the other reporting no significant associations [114]. In sum, the role of genetic factors on the relation between vitamin D and asthma remains unclear.
Taking all of this evidence under consideration, we think it is unlikely that vitamin D insufficiency during pregnancy (or during early infancy) is a major contributor to the current global asthma epidemic. Other investigators, in both the USA [115] and the UK [116], have expressed similar skepticism. If a modest association exists between vitamin D deficiency and incident asthma, we hypothesize that it would be mediated through increased risk of specific respiratory infections in early life. In a recent study from the Childhood Origins of Asthma (COAST) birth cohort in Wisconsin, Jackson and colleagues [117] found that wheezing rhinovirus illness during infancy predicted development of asthma at age 6 years. Linking these rhinovirus data and the aforementioned birth cohort data, it is possible that vitamin D deficiency may contribute to the seasonal nature of the infectious bronchiolitis in infants [118]. Early viral respiratory infections in a genetically susceptible host may induce subsequent asthma development, and if vitamin D mediates some part of this pathway, this might provide a new strategy for preventing some cases of asthma. Likewise, chronic infection with atypical bacteria may participate in asthma pathogenesis for a subset of patients, in addition to playing a role in lung inflammation and corticosteroid resistance in asthma [119]. For these susceptible individuals, correction of vitamin D deficiency would not only provide overall health benefits [100] but it might contribute to asthma prevention. These speculative constructs must be balanced, however, against the reviewed evidence, including studies suggesting that excessive vitamin D supplementation may increase risk of asthma and allergies in some children [105e107]. The resolution of this important debate requires RCTs of maternal/infant supplementation with vitamin D versus placebo. Such trials already are under way in several countries worldwide e e.g. Denmark (clinicaltrials.gov NCT00856947), New Zealand (ACTRN12610000483055), and the USA (clinicaltrials.gov NCT00920621).
Asthma Control, including Exacerbations Although improved vitamin D status may not affect risk of incident asthma (disease prevention), that definitely does not mean that it has little role in asthma management (disease modification). On the contrary, we believe that vitamin D may become an important adjunct therapy for many people with asthma. The potential benefits are based on two inter-related concepts: (1) responsiveness to corticosteroids, and (2) risk of exacerbations. With regard to corticosteroids, most individuals with asthma respond well to inhaled corticosteroids, with demonstrated reductions in symptoms and serious exacerbations and improved pulmonary function and
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asthma-related quality of life [120]. For these reasons, the 2007 NIH asthma guidelines consider inhaled corticosteroids the “preferred” long-term control medication for asthma [120]. Nevertheless, asthma control on even “optimal” controller therapy often leaves room for improvement [121]. This is particularly true in a relatively small subset of asthmatic patients with glucocorticoid resistance [122]. In 2006, Xystrakis and colleagues in London reported the effect of calcitriol treatment in this asthma subgroup [123]. The investigators administered oral 1,25(OH)2D3 (0.5 mg/day for 7 days) to a small group of healthy individuals and glucocorticoid-resistant asthmatic patients and found that the intervention enhanced subsequent responsiveness to dexamethasone due to induction of interleukin (IL)-10. The investigators concluded that vitamin D could potentially increase the therapeutic response to corticosteroids in otherwise resistant asthma patients. Two recent studies from Colorado [124,125] have built on this important work. Sutherland and colleagues studied 54 adults with asthma to determine the relation of serum 25(OH)D levels to asthma phenotype and glucocorticoid response [124]. In this cross-sectional study, patients with lower serum 25(OH)D levels were more likely to have impaired lung function and increased airway hyper-responsiveness. Among the subset of patients not on inhaled corticosteroids, lower 25(OH)D levels were correlated with lower glucocorticoid response (as measured by dexamethasone-induced expression of mitogen-activated protein kinase phosphatase (MPK)-1 by peripheral blood cells); this finding was not seen among patients on inhaled corticosteroids. Searing and colleagues studied similar issues in 100 asthmatic children [125]. In the cross-sectional part of their study, lower serum 25(OH)D levels were associated with greater corticosteroid use and worse airflow limitation. Moreover, the investigators found the amount of MPK-1 and IL10 mRNA induced by dexamethasone plus 1,25(OH)2D3 was significantly greater than that induced by dexamethasone alone (p < 0.01). In an experimental model of glucocorticoid resistance in which dexamethasone alone did not inhibit T-cell proliferation, addition of 1,25(OH)2D caused significant dose-dependent suppression of cell proliferation. Although these preliminary findings require replication, especially with RCT designs, there is growing laboratory evidence to support a clinically significant hormonal interaction between corticosteroids and vitamin D. For further discussion of the potential interactions between glucocorticoids and vitamin D pathways, see Chapters 66 and 67. With regard to asthma exacerbations, it is important to know that viral respiratory infections, particularly human rhinovirus, are associated with 50e85% of exacerbations [56,57]. While most adults experience two to
2009
four upper respiratory infections per year, young children experience six to ten per year [51]. Furthermore, individuals with asthma may be more susceptible to respiratory infection and have increased frequency of lower respiratory tract symptoms of higher severity and duration [126]. The emerging role of vitamin D in innate immune responses may explain predisposition to infection and asthma exacerbation in vitaminD-insufficient populations. As presented earlier in this chapter, there is growing evidence of a “protective” association between vitamin D and respiratory infection in the general population. To date, however, there are only sparse data on this association in asthmatic individuals. In our 2009 publication [14], based on nationally representative data from NHANES-III, we found that the association between serum 25(OH)D levels (<10 ng/ml versus 30 ng/ml) and upper respiratory infection was much stronger among individuals with asthma (OR 5.67) compared to those without asthma (OR 1.24; p for interaction ¼ 0.007). These findings are consistent with a cross-sectional study by Brehm and colleagues in 616 Costa Rican children [127]. In multivariable models, lower serum 25(OH)D levels were associated with higher total IgE and eosinophil count, increased airway responsiveness, higher likelihood to have been hospitalized for asthma in the past year, and higher likelihood of having used an anti-inflammatory medication in the past year. Although these findings sound compelling, the cross-sectional design leaves room for reverse causality. While upper respiratory infections are unlikely to cause an acute reduction in concurrent serum 25(OH)D level, one might reasonably ask if children with severe asthma are more likely to stay indoors e and, as a result, have lower serum 25(OH)D levels? To that end, Brehm and colleagues [128] examined the prospective association between serum 25(OH)D levels and risk of severe asthma exacerbations (defined as an asthma-related emergency department visit or hospitalization). In 1022 asthmatic children in the Childhood Asthma Management Program (CAMP), the investigators found that children with low baseline 25(OH)D levels (<30 ng/ml) were more likely to have a severe asthma exacerbation over a 4-month followup period (OR 1.5; 95% CI 1.1e1.9). The original CAMP trial involved randomization to different asthma treatments and the investigators found a “protective” association between 25(OH)D level and severe exacerbation in children randomized to budesonide (OR 1.8; 95% 1.0e3.2), as well as those randomized to either placebo or nedocromil (OR 1.3; 95% CI 1.0e1.9). The CAMP data provide support for the importance of vitamin D as an adjunct treatment for asthmatic individuals with low baseline levels of 25(OH)D. Additional support may come from RCTs on the effect of
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vitamin D supplementation on acute respiratory infections in the general population. Already, the influenza RCT by Urashima and colleagues found a significantly lower risk of asthma exacerbations among those receiving vitamin D [90]. Nevertheless, to optimally address the issue and thereby provide the strongest evidence to support possible changes in current asthma guidelines and health policy, dedicated RCTs are required. Such trials already are under way in several countries worldwide e e.g. Switzerland (clinicaltrials. gov NCT00712205) and the UK (clinicaltrials.gov NCT00978315).
VITAMIN D AND COPD COPD Pathogenesis Although vitamin D deficiency also may play a role in COPD pathogenesis, there are little (if any) data to directly address this hypothesis. Because of vitamin D’s established role in preventing and treating osteoporosis (see Chapters 62 and 61), we have compiled osteoporosis studies to explore this idea. Indeed, numerous studies report a positive cross-sectional association between osteoporosis and COPD [129e134], usually independent of corticosteroid use. A recent study by Franco and colleagues in Brazil also found that COPD patients without chronic use of corticosteroids were more likely to have osteoporosis [135]. They also reported that 94% of their patients had 25(OH)D levels <30 ng/ml and that 25(OH)D levels were correlated with oxygen saturation. The authors acknowledged that patients with more severe COPD may be less active and, therefore, less exposed to sunlight. In 2005, Black and Scragg [101] reported a strong doseeresponse correlation in NHANES-3 between serum 25(OH)D levels and pulmonary function, including FEV1, which is a key marker for increased susceptibility and diagnosis of COPD. They found that the adjusted FEV1 was 126 ml lower in the lowest 25(OH)D quintile compared to the highest quartile. Moreover, this difference appeared higher in participants with doctor diagnosis of chronic bronchitis (248 ml) or emphysema (344 ml), although the test for interaction was underpowered and not statistically significant. Potential mechanisms were explored in the accompanying editorial [136] and include the role of 1,25(OH)2D in modulating the formation of matrix metalloproteinases, fibroblast proliferation, and collagen synthesis [137,138]. Additionally, vitamin-D-mediated immunomodulation could affect airway inflammation, a central process in the pathogenesis of COPD [139]. Although intriguing, these cross-sectional results
provide very limited evidence for a causal link between vitamin D and COPD. To better address this possibility, Kunisaki and colleagues examined serum 25(OH)D levels and longitudinal lung function decline in the Lung Health Study 3 cohort [140]. The investigators performed a nested (prospective) caseecontrol study involving 196 COPD patients with either rapid (case) or slow (control) decline in FEV1 over approximately 6 years of follow-up. Despite rapid and slow decliners experiencing strikingly different rates of FEV1 decline (151 versus 0 ml/year, p < 0.001), there was no significant difference in baseline 25(OH)D levels between the two groups (25.0 versus 25.9 ng/ml, p ¼ 0.54). While these findings require replication, they suggest that vitamin D has little (if any) impact on lung function decline among patients with mild-to-moderate COPD. In the genetic realm, several studies have suggested a possible association between vitamin D and COPD pathogenesis [21,141e143]. Specifically, DBP (also known as the group-specific component of serum globulin (Gc-globulin)) is the major carrier protein of 25(OH)D in blood (see Chapter 5 for more details). In a Japanese population, Ito and colleagues found a strong association between Gc-globulin polymorphism and susceptibility to COPD [143]. While these data appeared promising, the association between DBP and COPD progression, as measured by rate of decline in pulmonary function, has yielded mixed results [143,144]. Most recently, Janssens and colleagues in Belgium reported a cross-sectional study of 414 adults where they confirmed that most patients with COPD have vitamin D deficiency, and that 25(OH)D levels correlated with COPD severity [145]. More importantly, they observed lower levels of 25(OH)D in homozygous carriers of the rs7041 at-risk T allele in DBP, and that homozygous carriers were more likely to have COPD (adjusted OR 2.11). While many COPD patients warrant vitamin D supplementation for general health reasons, the role of vitamin D in COPD pathogenesis awaits data from large prospective studies and, if warranted, future RCTs.
Acute Exacerbations of COPD Similar to asthma, many acute exacerbations of COPD (AECOPD) are caused by common respiratory viral pathogens, such as human rhinovirus, influenza, parainfluenza, and RSV. While no data directly link vitamin D with AECOPD, it seems reasonable to hypothesize that vitamin-D-mediated immune mechanisms may play a role in prevention of respiratory infection and, therefore, AECOPD. In our analysis of the NHANES-III data [14], we found that the association between serum 25(OH)D levels (<10 ng/ml versus
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30 ng/ml) and upper respiratory infection might be stronger among individuals with COPD (OR 2.26) compared with those without COPD (OR 1.27); statistical power was limited, however, and the difference was not statistically significant (p for interaction ¼ 0.30). Further studies of the role of vitamin D in AECOPD are warranted. As with asthma, dedicated RCTs would be most helpful and such trials already are under way in several countries worldwide e e.g. Belgium (clinicaltrials.gov NCT00666367) and UK (clinicaltrials. gov NCT00977873).
POTENTIAL MECHANISMS Vitamin D and Lung Development Human lung development begins around 4 weeks’ gestation and continues until age 2e3 years [146]. Although there are limited data about the role of vitamin D in human lung development, rat models may provide insights into some of the critical steps in lung development across species. For example, pulmonary surfactant is a mostly phospholipid complex present in the alveolus to reduce surface tension and regulate innate immune function and is critical to lung development [147]. In a 1990 publication, Marin and colleagues demonstrated, in explanted fetal rat lung tissue, that 1,25(OH)2D stimulates the synthesis of pulmonary surfactant phospholipids and the release of surfactant from alveolar type II (AT II) cells [148]. A 1996 publication by the same group showed that fetal rat lung fibroblasts in late pregnancy produce 1,25(OH)2D, which binds to numerous vitamin D receptors on AT II cells and thereby stimulates the production of pulmonary surfactant [149]. These findings led the authors to propose the existence of a vitamin D paracrine system that regulates maturation of the rat lung. The vitamin D paracrine system is discussed in Chapter 45. While these rat lung data are intriguing, their relevance to humans is uncertain. For example, a recent study of human fetal lung tissue demonstrated that 1,25(OH)2D does not induce expression of surfactant proteins in a coordinated manner [150]. Moreover, lung development requires many other complex steps, including thinning of the alveolar septal wall to allow effective gas exchange [151]. This thinning is thought to occur via apoptosis of lung fibroblasts [152]. Sukurai and colleagues recently explored this issue in fetal rat lung and found that 1,25(OH)2D, acting via local effects, promotes alveolar epithelialemesenchymal interactions and inhibits lipofibroblast atoptosis [153]. Moreover, the investigators found that rat pups administered 1,25(OH)2D postnatally showed increased expressions of key lipofibroblast and AT II cell differentiation
2011
markers, decreased spontaneous alveolar lipofibroblast and ATII cell apoptosis, increased alveolar count, and increased septal thickness. Another recent study also suggests complex effects of 1,25(OH)2D on rat lung development during the postnatal period [154]. Nadeau and colleagues demonstrated a complex integration of the effects of glucocorticoids, retinoic acid, and 1,25(OH)2D on gene expression in the postnatal lung, specifically Lgl 1. The authors conclude that all three hormones probably contribute to the timely advance of alveolarization without attendant inflammation. In sum, studies over the past 20 years support the presence of a vitamin D paracrine system in both the fetal and postnatal rat lung. While much remains to be learned about the connection of vitamin D to human lung development, it seems likely that vitamin D is playing a role in this complex process. Very low or very high levels of vitamin D e either locally or systemically e might have lasting adverse effects on lung development and function. Although speculative, such effects would provide a mechanism for how gestational and even postnatal vitamin D status could influence respiratory health.
Vitamin D and Immunity A more likely mechanism for the associations reported in this chapter is vitamin D’s myriad effects on the immune system. Because this enormous topic is covered in more detail in other dedicated chapters in Section XI of this volume, we will only briefly touch on the topic here. Most importantly, there now is considerable evidence that vitamin D has an important role in the innate immune system, which helps to prevent infection without the need for immunologic memory from previous exposure to the pathogen [5]. Innate immunity includes the production of antimicrobial peptides that are important in host defenses against respiratory pathogens, such as viruses, bacteria, and fungi [155e159]. These peptides include b-defensins and cathelicidins (e.g., hCAP-18 or LL-37), which are produced on epithelial surfaces and within circulating leukocytes [5]. The only human cathelicidin, hCAP-18, provides a particularly attractive explanation for studies showing an inverse association between serum 25(OH)D levels and risk of respiratory infection. hCAP-18 enhances microbial killing in phagocytic vacuoles, acts as a chemoattractant for neutrophils and monocytes, and has a defined vitamin-D-dependent mechanism [159]. In a landmark 2006 publication, Liu and colleagues [156] reported that in Mycobacterium tuberculosis-infected macrophages, there was a 30-fold increased cathelicidin expression in 1,25(OH)2D3-treated cells compared with controls, which corresponded to a 50% reduction in
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M. tuberculosis viability at 3 days. The individuals with serum 25(OH)D levels less than approximately 10 ng/ml had the least efficient cathelicidin expression, and those with serum 25(OH)D levels above approximately 30 ng/ml had the highest induction of cathelicidin mRNA. Furthermore, African-Americans, known to have increased susceptibility to TB infection, had low serum 25(OH)D levels and inefficient cathelicidin mRNA induction. Among African-American subjects, vitamin D supplementation of 25(OH)D to normal range enhanced cathelicidin induction five-fold, to similar levels as the white patients. Liu and colleagues extended these findings to provide further evidence that cathelicidin is the mechanism that enhances vitamin-D-mediated antimicrobial activity against M. tuberculosis [157]. In these experiments, a short, interfering RNA was used specifically to block cathelicidin mRNA and protein expression, which eliminated vitamin-D-mediated enhanced intracellular killing of M. tuberculosis that was observed in controls. These findings are discussed in more detail in Chapter 93. Thus, vitamin-D-mediated increases in cathelicidin provide an excellent explanation for the large body of evidence linking sun exposure, vitamin D, and TB [65,66] e and presumably for more recent evidence linking vitamin D and other respiratory infections (as reviewed earlier in this chapter). In brief, pathogenic antigens interact with Toll-like receptors on macrophages to upregulate the expression of genes that code for the vitamin D receptor and for the 1a-hydroxylase enzyme that converts 25(OH)D into the biologically active 1,25(OH)2D [156]. In turn, 1,25(OH)2D interacts with the promoter on the cathelicidin gene and enhances hCAP-18 production in myeloid cells [155], bronchial epithelial cells [158], and keratinocytes [160]. Furthermore, Weber and colleagues found that 25(OH)D could induce intracellular hCAP-18 through the autocrine induction of the 1a-hydroxylase enzyme [160]. These diverse pathways may be of particular importance for the fetus and neonate [161], who face a complex set of immunological demands, including protection against infection and balancing the transition from a sterile intra-uterine environment to a world that is rich in foreign antigens. Another proposed mechanism for vitamin-D-mediated effects on respiratory disorders involves adaptive (rather than innate) immunity, including modulation of antigen-presenting cells such as macrophages [162,163]. Moreover, vitamin D also affects the generation of regulatory T cells (Treg) [164,165] that express potentially inhibitory cytokines (IL10 and TGFb), and the ability to potently inhibit antigen-specific T cell activation [166]. In 2003, Matheu and colleagues described several of these effects using a murine model
of pulmonary eosinophilic inflammation [167]. In brief, the investigators demonstrated that vitamin D supplementation of adult mice led to changes in cytokines, IgE levels, and airway eosinophilia during allergen sensitization. Indeed, many laboratory studies suggest that vitamin D induces a shift in the balance between Th1- and Th2type cytokines toward Th2 dominance [5,168], a profile that is thought to favor the development of asthma and other allergic disorders. However, Pichler and colleagues [169] found that in CD4þ and CD8þ human cord blood cells, vitamin D inhibits not only IL-12-generated interferon-gamma production (Th1 type) but also suppresses IL-4 and IL-4-induced expression of IL-13 (Th2 type). In theory, this balanced Th1eTh2 regulation may modulate asthma and other allergic diseases. The role of regulatory T cells and IL-10 in the balance of the T-helper type 1 (Th1)-type and Th2-type cytokines and asthma phenotype was recently reviewed [170]. Thus, the differences between the studies on the Th1eTh2 dominance may lie in the timing of exposure of the cells to vitamin D (i.e., prenatal versus postnatal); the response of naı¨ve T cells to vitamin D exposure may differ from that of mature cells when exposed to vitamin D [171]. Another likely possibility is that the association depends on the vitamin D status of the individual. In other words, lower vitamin D intakes (e.g., to correct a deficiency state) may have different consequences than relatively high-dose supplementation, where an excess of vitamin D may indeed have adverse effects. These mechanistic hypotheses merit further investigation.
Vitamin D, Atopy, and Allergies Although this chapter focuses on the respiratory system, the close link between allergy and asthma supports a brief discussion of how vitamin D may affect risk of so-called “atopic disorders.” To be clear, we define atopy as IgE sensitization to a variety of allergens, such as different foods (e.g., peanut) and insect venoms. Atopy is different from an allergic reaction, which requires actual clinical manifestations (e.g., atopic dermatitis, allergic rhinitis, and allergic asthma). Finally, anaphylaxis is the most severe form of an acute allergic reaction and requires involvement of multiple organ systems [172]. The biological mechanisms for how vitamin D might influence atopy and allergies are presented briefly above (see “Acute exacerbations of COPD”) and discussed in more detail elsewhere [5,162,168]. Although data linking vitamin D to these conditions remain limited, we will briefly summarize clinical and epidemiologic studies of direct relevance to the present discussion. Atopic dermatitis is an allergic condition of particular relevance to asthma because it is an early step in the
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classic “atopic march” [25,26]. The nosology of this and related conditions (e.g., eczema) is controversial but we consider eczema the more inclusive skin condition, of which atopic eczema (atopic dermatitis) is one example. Based on vitamin-D-mediated effects on innate immunity, and the anticipated reductions in bacterial colonization of the skin, we hypothesized a few years ago that vitamin D might have beneficial effects on both the incidence and severity of atopic dermatitis. Recent evidence that sunlight and vitamin D supplementation increases skin production of cathelicidin in patients with atopic dermatitis [173e175] supports this hypothesis. Indeed, ecologic studies suggest higher prevalence of eczema at higher latitudes [176,177]. Moreover, a recent caseecontrol study demonstrated that atopic dermatitis was more common among obese individuals with low 25(OH)D levels (<10 ng/ml), as compared to obese individuals with levels of 30 ng/ml [178]. Another epidemiologic study, however, suggested a potential increase in eczema risk [106], and two birth cohorts with crude outcome data found no association at all [79,81]. In the recently published birth cohort study of Miyake and colleagues in Japan [109], children whose mothers consumed 172 IU/day had significantly reduced risk of eczema (adjusted OR 0.63; 95% 0.41e0.98). This dietary intake finding matches an inverse association between cord blood 25(OH)D levels and incident eczema in the New Zealand birth cohort (Camargo et al., unpublished data). Additional prospective cohort studies would better define the relation of vitamin D with eczema, and e if statistically significant e might support the initiation of primary prevention trials. Either way, the ongoing maternal/infant RCTs on vitamin D supplementation versus placebo should help to resolve this issue. In terms of disease modification, our group performed the first RCT on the role of vitamin D supplementation on winter-related atopic dermatitis [179]. Despite a very small sample (11 children in the Boston area), predominantly mild atopic dermatitis, low vitamin D dose (1000 IU/day) and short study duration (1 month), we found evidence for a statistically significant skin improvement among those assigned to vitamin D. Using the same RCT protocol, we recently found similar results in 107 Mongolian children with winterrelated atopic dermatitis [180]. Of note, an earlier RCT by Byremo and colleagues did not discuss vitamin D but is of likely relevance [181]. The investigators demonstrated that exposure benefits patients with severe atopic dermatitis, and that the intervention caused decreased bacterial skin colonization with S. aureus. Whether sunlight has distinct effects above and beyond vitamin D synthesis is a topic for future research. There are sparse data on the relation of vitamin D with allergic rhinitis. The historical cohort study by
2013
Hypponen and colleagues suggested that supplementation of infants with high daily doses (2000 IU/d) increased risk of incident disease [105]. A cross-sectional study by Wjst and Hypponen also reported a positive association between serum 25(OH)D levels and prevalence of allergic rhinitis [182], a result consistent with their hypothesis that vitamin D supplementation causes allergic disease. Given the strong association between allergic rhinitis and risk of developing childhood asthma [61], it is important to establish the causal nature of these associations. Prospective cohort studies would better define the relation of vitamin D with allergic rhinitis, and e if statistically significant e might support the initiation of primary prevention trials. Either way, the ongoing maternal/infant RCTs on vitamin D supplementation versus placebo should help to resolve this issue. More evidence for a link between vitamin D and allergic conditions comes from our recent ecologic studies of EpiPen prescribing [183e185], a surrogate marker for anaphylaxis [186]. In 2007, Camargo and colleagues [183] reported a strong northesouth gradient for the prescription of EpiPens in the USA, with the highest rates found in New England. Although population-adjusted rates were positively associated with several factors (e.g., number of health care providers), a multivariate analysis suggested that these factors did not mediate the strong northesouth gradient. Camargo and colleagues recently confirmed this northesouth gradient using a similar study design but restricted to urban areas in order to better control for this important determinant of health care utilization [184]. In a third ecologic analysis, Mullin and colleagues recently demonstrated a southenorth gradient for prescription of self-injectable epinephrine in Australia [185], consistent with the distribution of UVB in the Southern Hemisphere. The Australian study extended the earlier US-based observations by also showing a southenorth gradient in anaphylaxis admissions [185]. Emerging evidence suggests that the above findings are driven largely by regional differences in food allergy. For example, Sheehan and colleagues used a large national billing database of US pediatric hospitals to study regional differences in anaphylaxis hospitalizations [187]. Although overall anaphylaxis admissions were more common in northern versus southern hospitals (RR 1.41; 95% CI 1.34e1.48), the finding was stronger for food-induced anaphylaxis (RR 1.81; 95% CI 1.66e1.98). Likewise, Rudders and colleagues reported a significant northesouth difference in US emergency department visits for acute allergic reactions, a finding that was stronger for food-induced reactions than all reactions combined [188]. Lastly, Mullins and colleagues recently reported a southenorth gradient in Australia for prescription of infant hypoallergenic
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103. VITAMIN D, CHILDHOOD WHEEZING, ASTHMA, AND CHRONIC OBSTRUCTIVE PULMONARY DISEASE
formula [189], another finding that provides indirect evidence for the vitamin Defood allergy hypothesis. Although the atopic dermatitis findings can be explained by vitamin-D-mediated effects on innate immunity [190], the above food allergy/anaphylaxis findings present a conundrum: How is it that vitamin D lowers risk of childhood wheezing (respiratory infections) and possibly food allergy but does not lower risk of actual asthma? The answer undoubtedly involves a number of host and environmental factors, as well as the heterogeneity of these respiratory/allergy outcomes. In a recent paper by Vassallo and Camargo [191], the authors propose a “multiple hit” model in which vitamin D deficiency in a developmentally critical period increases susceptibility to colonization with abnormal intestinal microbial flora and gastrointestinal infections, contributing to abnormal intestinal barrier permeability and excess and inappropriate exposure of the immune system to dietary allergens. A compounding effect (and additional “hit”) of vitamin D deficiency is the promotion of a pro-sensitization immune imbalance that might compromise immunologic tolerance and contribute to food allergy. Many of these pathophysiologic processes (e.g., vitamin D’s impact on organ development, innate immunity, and immunologic tolerance) are of likely relevance to our understanding of the relation of vitamin D to respiratory infections, asthma, and COPD.
FUTURE RESEARCH ON VITAMIN D AND RESPIRATORY/ALLERGIC DISORDERS Although it seems likely that vitamin D has important effects on risk of respiratory/allergy disorders, many scientific gaps remain. The totality of the evidence supports that low levels of 25(OH)D increase risk of infections, including the viral respiratory infections of childhood wheezing, asthma exacerbations, and possibly AECOPD. We believe that this immunologic effect helps to explain emerging evidence that optimization of vitamin D status may improve other related allergic conditions (e.g., winter-related atopic dermatitis, risk of food allergy). If vitamin D truly decreases risk of respiratory infections, this could e at least theoretically e provide a mechanism to reduce incident asthma (e.g., in those individuals who now develop asthma as a result of exposure to specific viruses, such as human rhinovirus, in early childhood). We suspect that the public health impact of this hypothetical pathway will be small. By contrast, vitamin D-mediated decreases in respiratory infections among asthmatic individuals might dramatically improve asthma control. Moreover, optimization of 25(OH)D levels in asthmatic patients may result in improved effectiveness
of inhaled corticosteroids, the primary treatment of asthma today. This hormonal interaction, if confirmed, would have major therapeutic implications for millions of patients with asthma. Moreover, confirmation of the favorable effect of vitamin D on respiratory infections and corticosteroid responsiveness also would have favorable implications for millions of patients with COPD. Although there are many possible “next steps,” an initial one would be to examine the association between vitamin D status at birth (e.g., using cord blood 25(OH)D levels) with respiratory infections in early childhood. It is important for other groups to confirm the New Zealand birth cohort findings [82] in diverse populations of children. Likewise, the prospective association between 25(OH)D and acute respiratory infections should be extended to other high-risk populations, such as hospital workers and the elderly. Another important step would be to further describe the association between vitamin D and respiratory infection as it relates to the development of asthma, chronic asthma control, and risk for asthma exacerbations. For each of these issues, comparable vitamin D work is required for COPD, a field that remains largely unexplored. In all of these epidemiologic studies, the issue of reverse causation should be carefully addressed, particularly in cross-sectional studies. Because prevalent asthma, worse asthma control, and increased frequency of asthma exacerbations probably contribute to decreased time outdoors (and, therefore, decreased sunlight exposure), the presence of vitamin D deficiency may actually follow, rather than cause, the study outcomes. The use of longitudinal cohort designs will help to address this very important methodological issue. Future epidemiologic studies also will need to carefully account for the likelihood of confounding from the many health-related factors associated with frequent sun exposure, such as outdoor physical activity [192]. Additionally, measurement of immune markers, including hCAP-18, regulatory T cells, IL-10, and other markers of the Th1eTh2 balance, may help to elucidate mechanisms for the observed associations and provide additional face validity. Animal models in which these relevant pathways can be manipulated or “knocked out” will provide additional support and rationale for the associations between vitamin D, respiratory infections, asthma, and COPD. However, animal models should be selected with care. For example, it appears that only primates have the vitamin D response element on the promoter of the cathelicidin gene. Accordingly, mouse, rat, and dog cell lines do not appear to require vitamin D for cathelicidin expression and thus have been unsuccessful in evaluating this vitamin-D-mediated pathway [155]. Thus, some popular animal models may
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2015
SUMMARY
be of limited utility in testing the hypothesized association between vitamin D status and respiratory infections. Emerging areas of likely relevance to our future understanding of vitamin D and respiratory disease are the cellular effects of different vitamin D metabolites on the airway. For example, airway remodeling in asthma is manifested, in part, as increased airway smooth muscle mass due to myocyte proliferation. Bosse and colleagues have shown that 1,25(OH)2D stimulation of bronchial smooth muscle cells induces autocrine, contractility and remodeling processes [193]. Likewise, Banerjee and colleagues have found that 1,25(OH)2D uniquely modulates human airway smooth muscle expression of chemokines [194], effects that may help explain the hypothesized benefits of vitamin D supplementation in patients with glucocorticoid resistance [123e125]. Recently, Damera and colleagues found that vitamin D inhibits growth of human airway smooth muscle cells through specific pathways [195]. Although the relevance of this laboratory work to the prevention and management of asthma/COPD remains uncertain, these areas merit further exploration. Ultimately, large randomized, controlled trials of vitamin D supplementation will be needed to confirm the ability to reverse the suboptimal outcomes associated with vitamin D insufficiency. In these trials, higher doses of supplementation (e.g., at least 1000 IU/d in children and at least 2000 IU/day in adults) probably are required to maximize the hypothesized benefit. The 1997 Institute of Medicine recommendations for vitamin D supplementation e i.e. 200e600 IU/d, depending on age [196] e are unlikely to achieve the serum 25(OH)D levels (e.g., 40 ng/ml) that are more closely linked with optimal health [100,197,198], including prevention of infections. We agree with many experts who already argue that current recommended doses of vitamin D supplementation are woefully inadequate to meet the need for higher serum 25(OH)D levels [100,197,198]. For example, to raise serum 25(OH)D from 20 to 32 ng/ml requires an additional 1700 IU of vitamin D per day [199]. Use of an adequate vitamin D dose in future RCTs, along with adequate duration of intervention and measurement of pre-/post-supplementation serum 25(OH)D levels, will help to optimize trial conditions. Also, as the relative importance of vitamin D in different age and racial/ethnic groups is unknown, diverse study participants or multiple studies of different demographic subgroups will help us to understand the potential interactions between supplement dosage, achieved level, and health outcomes. Progress in these diverse topics will undoubtedly suggest new research avenues. For example, there is a growing research effort on the importance of vitamin D supplementation in cystic fibrosis patients [200,201].
Confirmation of a vitamin D benefit for respiratory infections and asthma suggest that vitamin D may be of particular importance for diseases such as bronchiectasis, which could be described simplistically as a combination of recurrent infections and obstructive airway disease. Likewise, if vitamin D truly prevents acute respiratory infections it might also be helpful in the prevention and management of chronic rhinosinusitis and related conditions. Although we have not directly addressed these topics in this chapter, due to the almost complete lack of scientific evidence on these topics, we hope to comment on these areas in future reviews. The demonstration of vitamin-D-related benefits would add to the relatively few effective treatment options available for these conditions today.
SUMMARY Over the past decade, a growing number of studies have linked vitamin D with common respiratory disorders, such as childhood wheezing, asthma, and chronic obstructive pulmonary disease (COPD). Vitamin D deficiency appears to increase risk of acute respiratory infections, which cause incident childhood wheezing and asthma exacerbations. To date, there is little evidence that vitamin D deficiency affects risk of incident asthma. The role of vitamin D in the etiology of COPD and its exacerbations remain uncertain. Vitamin D also may contribute to human lung development. Vitamin D’s myriad effects on innate and adaptive immunity provide biological plausibility for how vitamin D deficiency: (1) increases risk of infection; (2) lowers therapeutic response to corticosteroids; and (3) increases risk of other atopic conditions, such as eczema and food allergy. Further studies, especially RCTs, are needed to better establish the effects of vitamin D on acute respiratory infections and other respiratory disorders, particularly in fall/winter at higher latitudes. Future trials will need to use higher-dose vitamin D supplementation (e.g., at least 1000 IU/day in children and at least 2000 IU/day in adults) and to have sufficient statistical power to definitively answer if vitamin D can prevent or reduce the severity of respiratory infections. If our current understanding is confirmed, improvements in vitamin D status will advance ongoing efforts to prevent asthma/COPD exacerbations and optimize disease control.
Acknowledgments Dr. Camargo was supported by the Massachusetts General Hospital Center for D-receptor Activation Research (Boston, MA). All authors were supported by the National Institutes of Health (Bethesda, MD): Dr. Camargo by grants R01 HL-64925; Dr. Ginde by grant KL2 RR-25779; and Dr. Mansbach by grant K23 AI-77801.
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smooth muscle cells through growth factor-induced phosphorylation of retinoblastoma protein and checkpoint kinase 1, Br. J. Pharmacol. 158 (2009) 1429e1441. Institute of Medicine Food and Nutrition Board, DRI: Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride, National Academies Press, Washington, DC, 1997. H.A. Bischoff-Ferrari, E. Giovannucci, W.C. Willett, T. Dietrich, B. Dawson-Hughes, Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health outcomes, Am. J. Clin. Nutr. 84 (2006) 18e28. H.A. Bischoff-Ferrari, A. Shao, B. Dawson-Hughes, J. Hathcock, E. Giovannucci, W.C. Willett, Benefit-risk assessment of vitamin D supplementation, Osteoporos. Int. 21 (2010) 1121e1132. M.J. Barger-Lux, R.P. Heaney, S. Dowell, T.C. Chen, M.F. Holick, Vitamin D and its major metabolites: serum levels after graded oral dosing in healthy men, Osteoporos. Int. 8 (1998) 222e230. D. Green, K. Carson, A. Leonard, J.E. Davis, B. Rosenstein, P. Zeitlin, et al., Current treatment recommendations for correcting vitamin D deficiency in pediatric patients with cystic fibrosis are inadequate, J. Pediatr. 153 (2008) 554e559. W.B. Hall, A.A. Sparks, R.M. Aris, Vitamin D deficiency in cystic fibrosis, Int. J. Endocrinol. 2010 (2010) 218691. R.T. Stein, C.J. Holberg, W.J. Morgan, A.L. Wright, E. Lombardi, L. Taussig, et al., Peak flow variability, methacholine responsiveness and atopy as markers for detecting different wheezing phenotypes in childhood, Thorax 52 (1997) 946e952. J.P. Legg, J.A. Warner, S.L. Johnston, J.O. Warner, Frequency of detection of picornaviruses and seven other respiratory pathogens in infants, Pediatr. Infect. Dis. J. 24 (2005) 611e616. M.M. Kusel, N.H. de Klerk, T. Kebadze, V. Vohma, P.G. Holt, S.L. Johnston, et al., Early-life respiratory viral infections, atopic sensitization, and risk of subsequent development of persistent asthma, J. Allergy Clin. Immunol. 119 (2007) 1105e1110. J.M. Mansbach, A.J. McAdam, S. Clark, P.D. Hain, R.G. Flood, U. Acholonu, et al., Prospective multicenter study of the viral etiology of bronchiolitis in the emergency department, Acad. Emerg. Med. 15 (2008) 111e118.
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C H A P T E R
104 Vitamin D and Skeletal Muscle Function Lisa Ceglia 1, Robert U. Simpson 2 1
2
Tufts Medical Center, Boston, MA, USA University of Michigan Medical School, Ann Arbor, MI, USA
INTRODUCTION Although vitamin D was first recognized as an essential nutrient to treat children with rickets and is classified as a fat-soluble vitamin, a more accurate description of vitamin D is that of a prohormone which is metabolized to a secosteroidal hormone [1]. Solar ultraviolet B radiation is the major provider of vitamin D for humans because, with adequate skin exposure, it photolyzes 7-dehydrocholesterol present in plasma membranes of skin cells [2] (Fig. 104.1). 7-Dehydrocholesterol is then converted to pre-vitamin D3, which undergoes a thermally induced transformation to vitamin D3 [3] (Fig. 104.1). Newly produced vitamin D3 formed in the skin, and vitamin D2 or D3 ingested through food or supplements, is removed by binding to the plasma transport protein, vitamin-D-binding protein, present in the capillary bed of the dermis and the intestinal epithelium. Vitamin-D-binding protein then enters the circulation and delivers vitamin D2 or D3 first to the liver where it undergoes 25-hydroxylation, and then to the kidney where it undergoes 1-hydroxylation [4] (Fig. 104.1). The most abundant metabolite of vitamin D in the human body is 25-hydroxyvitamin D3 (25(OH)D3), and it is the best clinical indicator of overall vitamin D status [4]. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is the active metabolite of the vitamin, and its receptor (vitamin D receptor, VDR) has been identified in human tissues [5]. Optimal serum 25(OH)D concentration has classically been considered to be the level at which parathyroid hormone (PTH) achieves maximal suppression. This threshold level has varied depending on the analytical approach used [6]. Some studies have estimated it to be around 50 nmol/l, while others around 75 nmol/l [7]. Vitamin D insufficiency has, therefore, been defined as a level below either 50 or 75 nmol/l [7]. However, most
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10104-0
experts agree that a 25(OH)D level less than 25 nmol/l is considered in the deficient range. Vitamin D plays a critical role in the regulation of calcium and phosphate homeostasis and in bone development and maintenance [1]. Classically, 1,25(OH)2D3 is known to exert its actions on target organs, such as the intestine, the kidney, and bone, and stimulates calcium transport from these organs to the blood. Yet, as a secosteroid hormone, 1,25(OH)2D3 can also regulate transcription of numerous genes and influence many biological processes in a wide range of tissues. There has been growing evidence that vitamin D plays an important role in skeletal muscle. Early clinical descriptions of a reversible myopathy associated with profound vitamin D deficiency and/or chronic renal failure recognized a potential association between vitamin D and muscle [8]. More recent studies have reported reduced muscle mass, strength and performance and an increased risk of falls in older individuals with vitamin D insufficiency. A well-recognized action of vitamin D is its effects on muscle growth and development [9]. Initially, it was speculated that the actions of vitamin D on muscle growth were secondary to actions on calcium homeostasis [10]. Early attempts to demonstrate 1,25(OH)2D3 receptors in muscle cells were not successful [11], thus suggesting that 1,25(OH)2D3 acts indirectly on muscle tissue. Other evidence, however, suggested that 1,25(OH)2D3 has direct effects on muscle metabolism and contractile activities [12e14]. Simpson et al. reported that two established myoblast cell lines (H9c2 and G-8) and excised muscle cells rinsed of serum contamination, possess the VDR [15]. VDR concentrations in the myoblast cells decreased after terminal differentiation of these cells to the fused myotube form. Additionally, Simpson et al. demonstrated that DNA synthesis and cell proliferation of the myoblast
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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104. VITAMIN D AND SKELETAL MUSCLE FUNCTION
Synthesis of vitamin D3 occurs in the skin where 7-dehydrocholesterol is converted to previtamin D3 in response to sunlight (ultraviolet B radiation) exposure. Vitamin D3 is produced from the isomerization of previtamin D3 in the skin or intestinal absorption of natural and fortified foods and supplements. Vitamin D3 (bound to vitamin-D-binding protein) circulates in the bloodstream, and is transported to the liver where it is hydroxylated by liver 25-hydroxylases. The resultant 25-hydroxyvitamin D3 is hydroxylated to the active secosteroid 1a,25(OH)2D3 in the kidney by 1a-hydroxylase. 1a,25(OH)2D3 acts on various target tissues via its receptor (VDR). 1a,25(OH)2D3 appears to affect other non-classical target tissues such as skeletal muscle possibly via the VDR.
FIGURE 104.1
cell line was inhibited by 1,25(OH)2D3. These data revealed that 1,25(OH)2D3 directly affects muscle cell biochemistry and physiology and could be involved in myocyte maturation. Therefore, the identification of the VDR in muscle cells [15,16] has provided further support for a role of vitamin D in skeletal muscle. Recent investigations in cell culture and animals have advanced our understanding of some of the molecular mechanisms through which vitamin D targets skeletal muscle; however, much remains to be characterized. This chapter summarizes the clinical evidence of an association between vitamin D status and physical performance and falls, describes how vitamin D affects muscle tissue morphology, considers the molecular mechanisms of vitamin D activity in skeletal muscle, outlines the lessons learned from the VDR knockout mouse model, discusses potential VDR polymorphisms and their relationship to muscle performance and falls, and touches on the effects of PTH on muscle.
MYOPATHY It has long been recognized that vitamin D affects muscle function [12,17e20]. Muscle weakness or myopathy is a prominent feature described in states of
vitamin D inadequacy. Moreover, several reports described a link between osteomalacia and vitaminD-reversible myopathy [21e24]. Some of these clinical observations reported that the myopathy could be independent of metabolic abnormalities such as hypocalcemia, hypophosphatemia, and hyperparathyroidism, and that vitamin D supplementation resulted in improvement of muscle strength. Others, however, reported that a correlation between hypovitaminosisD-induced metabolic abnormalities and muscle myopathy existed [25]. Muscle weakness in chronic renal failure, anticonvulsant therapy, and hemodialysis has also been noted and, in some instances, found to be responsive to treatment with 1,25(OH)2D3 [19,24,26]. Some of the early descriptions of muscle weakness from profound vitamin D deficiency were in infants, who presented with muscle weakness and hypotonia [23]. In adults, the presentation has been described as more variable, ranging from a proximal muscle weakness to diffuse skeletal or muscle pain [19,27]. Individuals may have difficulty in walking up stairs, in rising from a sitting or squatting position, and in lifting objects. Other typical clinical features include a waddling gait and uniform generalized muscle wasting with preservation of sensation or deep tendon reflexes. Aside from a low serum 25(OH)D level, clinical laboratory data
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were normal or associated with hypocalcemia, hypophosphatemia, and hyperparathyroidism. Electromyographic abnormalities, such as polyphasic motor unit potentials with shortened duration and decreased amplitude consistent with a myopathy, have also been reported [28]. In an uncontrolled study by Skaria et al., 13 of 14 patients with osteomalacia improved their electromyogram but not nerve conduction velocity during vitamin D treatment for several months, suggesting that vitamin D plays an etiological role [28]. However, these findings were non-specific and are, in fact, seen in other muscular diseases, such as polymyositis [8]. Research using experimental animals has been important in uncovering the potential mechanisms underlying the effects of vitamin D depletion on muscle contraction and function. In a pertinent animal study, Rodman and Baker [14] used an in situ rat soleus neuromuscular preparation to study changes in muscle contraction kinetics in response to vitamin D depletion. Muscle from vitamin-D-deficient animals revealed prolongation of the relaxation phase of contraction. This research suggested that vitamin D affects calcium handling in muscle sarcoplasmic reticulum. Other lines of evidence also suggested that muscle is a target tissue for a vitamin-D-derived hormone. A study of the muscles of rabbits fed a vitamin-D-deficient diet showed that the isolated sarcoplasmic reticulum had increased ability to bind calcium compared to that from control animals [12]. Matthews et al. [13] provided evidence that 1,25(OH)2D3 potentiates the ability of uremic rabbit sarcoplasmic reticulum to bind and store calcium. A period of time was required, in vivo, for the 1,25(OH)2D3 to be effective. These studies suggest that an effect of vitamin D on muscle is to increase calcium accumulation in the sarcoplasmic reticulum either by increasing the number of calcium-binding sites or by altering the efficiency of these sites for calcium uptake. In the intestine, for example, 1,25(OH)2D3 induces the synthesis, de novo, of several calcium-binding proteins. However, in skeletal muscle, the exact response to 1,25(OH)2D3 and the nature of regulation of calciumbinding proteins associated with the sarcoplasmic reticulum is at present not clearly understood. Other studies have investigated vitamin D’s impact on muscle protein metabolism. 25(OH)D3 has been shown to have a stimulatory effect on muscle protein metabolism. Birge and Haddad [20] found that vitamin D3 and 25(OH)D3 administration to vitamin-D-deficient rats resulted in increased ATP content and leucine incorporation in the isolated diaphragm muscle. This study also showed that 25(OH)D3 stimulates the incorporation of leucine into protein and raises the intracellular ATP content of rat epitrochlear muscle. The finding of 25(OH)D3-specific muscle activities is not fully congruous with the data of Matthews et al. which
demonstrated that 1,25(OH)2D3 is the agent ultimately responsible for increased calcium binding to muscle sarcoplasmic reticulum [13]. The presence of a muscle VDR supports this conclusion, but the effect of vitamin D analogs on skeletal muscle has not been well characterized. The data support four possible mechanisms whereby 1,25(OH)2D3 may alter muscle function: (1) it may act by controlling serum calcium concentrations, thereby suggesting that calcium would have a direct and primary action on muscle; (2) it may have direct actions on dividing myoblasts and induce differentiation to multinucleated non-dividing myotubes; (3) it may have direct actions to activate the vitamin D receptor in muscle to induce expression of specific genes (i.e., a vitamin-D-dependent calcium-binding protein or a specific Caþþ-ATPase enzyme), thereby altering calcium handling of muscle cells; and (4) 1,25(OH)2D3 may activate the VDR and initiate non-genomic regulation of gene products involved in muscle excitation contraction coupling. Prior to the identification of the VDR in muscle, only the first of these mechanisms was possible.
PHYSICAL PERFORMANCE Observational Studies Observational studies in community-dwelling older adults have shown an association between vitamin D status and parameters of physical performance, especially when 25(OH)D levels are less than 75 nmol/l. In an analysis of men and women age 60 and over who participated in the cross-sectional NHANES III survey, individuals with higher serum 25(OH)D levels up to 94 nmol/l were able to walk faster (8-foot walk test) and to get out of a chair faster (sit-to-stand test) than subjects with lower levels [29], particularly in the subset with 25(OH)D levels under 60 nmol/l. This association was not influenced by physical activity level. An analysis of the Longitudinal Study of Aging Amsterdam (LASA) indicated that lower serum 25(OH)D levels predicted decreased grip strength and appendicular muscle mass in older men and women over the subsequent 3 years [30]. This longitudinal study also measured physical performance by a short battery of tests consisting of a walk test, chair stand, and tandem stand [31]. In older adults (age 65 and over) with serum 25(OH)D below 50 nmol/l, there was an increased risk of a decline in physical performance over 3 years compared to individuals with levels of 75 nmol/l or greater [32]. Somewhat similar to the NHANES III findings, a cross-sectional analysis of the LASA cohort suggested that a 25(OH)D concentration of 60 nmol/l was the threshold level for improvement in physical performance [33].
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Some observational data have reported sex-specific findings. In an analysis of older men and women (mean age 75) who participated in the longitudinal observational Rancho Bernardo Study, performance on a timed up and go (TUG) test and chair stand was poorer and declined at a faster rate over a 2.5-year period in women with 25(OH)D levels 80 nmol/l compared to women with 25(OH)D levels 115 nmol/l [34]. This finding was not seen in older male participants in this study. Another study including only older women found that low 25(OH)D levels (defined as <50 nmol/ l) were associated with reductions in gait speed, balance, and knee extension and flexion strength [35]. The epidemiological InCHIANTI study, on the contrary, found an association between vitamin D status and five measures of frailty in older men, but not older women [36]. The association between vitamin D status and physical performance has also been reported in nonCaucasian older populations. A longitudinal study of community-dwelling older Japanese women with impairments in physical function at baseline, reported that higher baseline 25(OH)D levels (defined as >67.5 nmol/l) were associated with improvements in physical fitness after 3 months on an exercise program [37]. A cross-sectional study of a North Taiwanese elderly community showed a similar result with vitamin D insufficiency and measures of frailty [38]. The effect of vitamin D on muscle strength and performance may not be unique to older individuals. Ward et al. reported a direct relationship between 25(OH)D levels and muscle power, force, velocity, and jump height in 99 postmenarchal 12e14-year-old girls in the UK [39]. Of note, most of the girls had low 25(OH)D levels with a mean of 21.3 nmol/l and the analyses were not adjusted for physical activity [39]. Another study in 301 Chinese adolescent girls with a mean age of 15 years and serum 25(OH)D levels of 34 nmol/l, found a positive association between 25(OH)D levels and handgrip strength after adjusting for physical activity [40]. Studies in a mixed-age population of younger and older women [41] and in younger postmenopausal women (early 60s) did not find a correlation between vitamin D status and tests of muscle performance [42].
Randomized Controlled Studies Several randomized clinical trials have examined the effect of vitamin D supplementation on tests of physical performance (Table 104.1). The majority of intervention trials have found that vitamin D supplementation in doses that achieved mean serum 25(OH)D levels of 66 to 85 nmol/l in the treated group, improved measures of muscle strength and physical performance [43e47]. Specifically, vitamin D3 800 IU daily with calcium,
compared to calcium alone, improved body sway by 9% in ambulatory older women with serum 25(OH)D levels <50 nmol/l over 8 weeks [45], muscle performance measures (knee flexor and extensor strength, grip strength, and TUG test) by 4e11% in institutionalized elders with 25(OH)D levels <78 nmol/l over 12 weeks [46], and lower extremity muscle performance (quadriceps strength, body sway, and TUG test) by 8e28% in ambulatory older men and women with 25(OH)D levels <78 nmol/l over a 12e20-month period [44]. Similarly, in another study among elders with low serum 25(OH)D levels, compared to placebo, a single 600 000 IU intramuscular injection of vitamin D2 significantly improved choice reaction time, aggregate functional performance time (50 ft walk, rising from a chair and walking 50 ft, ascent and descent of 13 steps) and postural sway over 6 months [47]. However, not all studies have shown positive results [48e51] (Table 104.1). In older adults with 25(OH)D levels below 50 nmol/l, vitamin D3 8400 IU weekly, compared to placebo, did not show improvement in body sway or the short physical performance battery (SPPB) over 4 months [51]. One explanation for this null effect was that the study subjects were too physically fit at baseline and thus had little room for improvement. In a large multicenter study in New Zealand, a single oral dose of vitamin D3 300 000 IU, compared with placebo, did not alter physical performance measures in adults age 65 and older over 6 months [49]. Yet, this study included older individuals in unstable health following a recent hospitalization or hip fracture. In addition, it did not report 25(OH)D levels achieved at 6 months, which would raise the question of whether levels were trending back down by the 6-month mark in light of the single dose given. A few trials have examined whether an active form of vitamin D may impact muscle strength and other measures of muscle performance. A randomized trial found that 1,25(OH)2D3 reduced the decline in physical performance tests in older women as compared with placebo over 3 years [52]. A trial of 6 months of alfacalcidol versus placebo revealed improvements in lower-extremity muscle strength and walking distance in a small group of older women [53]. Some investigators have argued that supplementation with active forms of vitamin D are superior to parent compounds in certain pathologic conditions such as rheumatoid arthritis [54]. Attempting to pool these studies together is challenging given the different ways muscle strength and physical performance were measured in each of the trials, the varying interventions given in each study (i.e., vitamin D alone, vitamin D plus calcium, vitamin D3 versus D2, daily versus less frequent dosing, etc.) and the differing baseline 25(OH)D levels of the
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TABLE 104.1
Effects of Parent Vitamin D Supplementation on Measures of Muscle Performance and Falls in Randomized Controlled Trials
Source (1st author, year) Sex (\_) , Mean age (y) Baseline 25(OH)D (nmol/l)
Intervention
Duration
25(OH)D Achieved (Rx group)
Muscle performance (vs. placebo)
Falls reduction (vs. placebo)
Pfeifer 2000 \, mean age 74 25(OH)D <50 nmol/l
Ca 1.2 g/d þ Vit D3 800 IU/d vs. Ca 1.2 g/d
2m
66 nmol/l
Body sway Y 9%
Yes
Bischoff 2003 \, mean age 85.3 25(OH)D <78 nmol/l
Ca 1.2 g/d þ Vit D3 800 IU/d vs. Ca 1.2 g/d
3m
66 nmol/l
Knee flexor strength [ 7.6% Knee extensor strength [ 7.2% Grip strength [ 5.5% TUG test Y 7.9%
Yes
Dhesi 2004 \ _, mean age 77 25(OH)D <25 nmol/l
Vit D2 600,000 IU/ once i.m. vs. Placebo/once i.m.
6m
44 nmol/l
AFPT Y 12% Choice reaction time Y 18% Body sway Y 16% Quadriceps strength NS
No
Flicker 2005 \ _, mean age 83 25(OH)D <90 nmol/l
Ca 0.6 g/d þ Vit D2 10,000 IU/wk vs. Ca 0.6 g/d
2y
NA
NA
Yes
Sato 2005 \, mean age 74 25(OH)D <25 nmol/l
Ca 0.6 g/d þ Vit D2 10,000 IU/wk vs. Ca 0.6 g/d
2y
84 nmol/l
Lower extremity muscle strength [ 56%
Yes
Bischoff-Ferrari 2006 \ _, mean age 71 25(OH)D <125 nmol/l
Ca 0.5 g/d þ Vit D3 700 IU/d vs. Placebo/d
3y
107 nmol/l
NA
Yes
Broe 2007 \ _, mean age 89 25(OH)D <60 nmol/l
Vit D3 800 IU/d vs. Placebo/d
5m
75 nmol/l
NA
Yes
Prince 2008 \, mean age 77 25(OH)D <60 nmol/l
Ca 0.5 g/d þ Vit D2 1000 IU/d vs. Ca 0.5 g/d
12 m
45e60 nmol/l
NA
Yes
Pfeifer 2009 \ _, mean age 77 25(OH)D <78 nmol/l
Ca 1.0 g/d þ Vit D3 800 IU/d vs. Ca 1.0 g/d
12 m
85 nmol/l
Quadriceps strength [ 8% Body sway Y 28% TUG test Y 11%
Yes
Latham 2003 \ _, mean age 79 25(OH)D <55 nmol/l
Vit D3 300 000 IU/ once p.o. vs. Placebo/once p.o.
6m
78 nmol/l at 3 mos NA at 6 mos
Knee extensor strength NS Balance test NS TUG NS 4 meter walk NS
No
Kenny 2003 _, mean age 76 25(OH)D <60 nmol/l
Vit D3 1000 IU/d vs. Placebo/d
6m
91 nmol/l
Single leg stance NS Chair stand NS Timed up and go NS 8-foot walk NS Supine to stand NS
NA
POSITIVE STUDIES
NEGATIVE STUDIES
(Continued)
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2028 TABLE 104.1
104. VITAMIN D AND SKELETAL MUSCLE FUNCTION
Effects of Parent Vitamin D Supplementation on Measures of Muscle Performance and Falls in Randomized Controlled Trialsdcont’d
Source (1st author, year) Sex (\_) , Mean age (y) Baseline 25(OH)D (nmol/l)
Intervention
Duration
25(OH)D Achieved (Rx group)
Muscle performance (vs. placebo)
Falls reduction (vs. placebo)
Law 2006 \ _, mean age 85 25(OH)D <50 nmol/l
Vit D2 50 000 IU/3 mos vs. Placebo/d
10 m
74 nmol/l
NA
No
Brunner 2008 \, age 50e79 25(OH)D NA
Ca 1.0 g/d þ Vit D3 400 IU/d vs. placebo/d
2 y and 5 y
NA
Grip strength NS Chair stand NS Timed walk NS
NA
Lips 2010 \ _, mean age 78 25(OH)D 50 nmol/l
Vit D3 8400 IU/wk vs. Placebo/wk
4m
66 nmol/l
Body sway NS SPPB NS
NA
NS: not statistically significant; NA: not assessed; TUG: timed up and go test; AFPT: aggregate functional performance time; SPPB: short physical performance battery.
participants. In summary, however, among older women and men, studies using parent vitamin D compounds at daily doses of 800e1000 IU have suggested beneficial effects on muscle strength and performance.
FALLS Observational Studies Muscle strength and physical performance are linked with risk of falls in older individuals. In view of substantial data demonstrating a positive association between serum 25(OH)D concentrations and physical performance, a similar relationship between vitamin D status and fall risk would be expected. In the LASA study, low 25(OH)D levels (less than 25 nmol/l) were associated with an increased risk of repeated falling over the subsequent year, particularly in persons under 75 years of age [55]. A similar finding was shown in a large cohort study of older community-dwelling women where higher 25(OH)D levels were associated with a lower rate of falls over a 4-year period [56]. Other observational data in older adults have demonstrated similar results [57e60].
Randomized Controlled Studies In a randomized, controlled trial, Bischoff et al. showed that treatment with vitamin D3 and calcium (800 and 1200 mg per day) reduced the number of falls by 49% in comparison to calcium alone in older institutionalized women over a 3-month period [46]
(Table 104.1). A similar intervention study in community-dwelling older women showed a similar reduction in number of falls over a 2-month period [45] (Table 104.1). Data suggest greater effects in compliant participants, indicating a dose effect. A study by Broe et al. supports the hypothesis of a dose effect. This double-blind randomized trial among 124 nursing home residents examined the effect of 200, 400, 600, or 800 IU of vitamin D3 daily, compared to placebo daily, on falls risk over a 5-month period [61] (Table 104.1). Participants in the 800 IU group had a 72% reduction in rate of falls than those in the placebo or lower-dose groups. Furthermore, several longer-term studies have shown positive results (Table 104.1). Pfeifer et al. published a larger and longer trial over a 12e20-month period in 242 healthy older adults with 25(OH)D levels below 78 nmol/l [44]. This study determined that treatment with vitamin D3 and calcium (800 IU and 1200 mg per day) versus calcium alone reduced the number of first falls by 39% over a 20-month period. In an Australian study, treatment with vitamin D2 (initially 10 000 IU per week then 1000 IU per day) and calcium (600 mg per day) for 2 years reduced the risk of falls in the compliant group by 30% compared to calcium alone [58]. Over a 3-year period, 700 IU of vitamin D3 plus 500 mg of calcium reduced the odds of falling by 46% among community-dwelling older women [62]. Even a few trials using an active form of vitamin D have been published. In a large randomized double-blind study in 378 Swiss community-dwelling older adults, treatment with alfacalcidol, when compared to the group taking the placebo, significantly reduced the number of fallers and the number of falls over 9 months [63].
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However, as discussed in the section of physical performance, the positive effect of vitamin D supplementation on falls is not observed consistently. Two large trials found no effect of supplementation with vitamin D on the incidence of falls [64,65] (Table 104.1). One of these, the Randomized Evaluation of Calcium and Vitamin D (RECORD) Group Trial, was a secondary fracture prevention trial that also collected information on falls [65]. Participants were mobile before their initial fracture but the authors did not specify their residential status. In this 5-year trial, participants received their supplements (833 IU of vitamin D3 daily with or without 1000 mg of calcium) and medical history questionnaires asking about falls in the last week, through the mail every 4 months. The study reported no effect on fall rates. Notably, although the return rate of the questionnaires was 94%, the questionnaires captured information on falls over a narrow time period (1 week every 4 months for 5 years). Furthermore, compliance with the supplements in this trial was <50% at the 2-year point. Other experimental studies using vitamin D in various doses did not observe significant effects on falls [49,64]. Potential reasons given for the negative clinical results include inadequate vitamin D dose, study design differences, falls were not the primary outcome, and assessment of fall frequency in them was not always optimal. Several systematic reviews and meta-analyses on this topic have been published over the last decade [66e71]. The results have not been consistent across meta-analyses and, thus, there has not been a final consensus. The most recent meta-analysis considers TABLE 104.2
issues of dose effect, 25(OH)D level achieved, and adherence to supplementation [70]. Eight randomized controlled trials, including over 2000 ambulatory and institutionalized subjects, found that vitamin D supplementation lowered the risk of falling by 19%. A similar risk reduction was seen with use of active forms of vitamin D. Gillespie et al., on the other hand, found a reduction in falls in vitamin-D-insufficient older individuals only [71].
MUSCLE MORPHOLOGY Human skeletal muscle consists of different types of muscle fibers. There are two principal ways to categorize muscle fibers: the type of myosin (fast or slow) present, and the degree of oxidative phosphorylation that the fiber undergoes. Skeletal muscle can thus be broken down into two broad categories: Type I and Type II. Type I fibers appear red due to the presence of the oxygen-binding protein myoglobin. These fibers are suited for endurance and are slow to fatigue because they use oxidative metabolism to generate ATP. Type II fibers are white due to the absence of myoglobin and a reliance on glycolytic enzymes. These fibers are efficient for short bursts of speed and power and use both oxidative metabolism and anaerobic metabolism depending on the particular subtype. These fibers are quicker to fatigue. Type II muscle fibers are also the first to be recruited to prevent a fall [72]. The classification and characterization of the distinct muscle fibers is shown in Table 104.2.
Muscle Morphology: Classification and Characterization of Distinct Muscle Fibers
Fiber type
Type I fibers
Type IIa fibers
Type IIx fibers
Type IIb fibers
Contraction time
Slow
Moderately fast
Fast
Very fast
Size of motor neuron
Small
Medium
Large
Very large
Resistance to fatigue
High
Fairly high
Intermediate
Low
Activity used for
Aerobic
Long-term anaerobic
Short-term anaerobic
Short-term anaerobic
Maximum duration of use
Hours
<30 minutes
<5 minutes
<1 minute
Power produced
Low
Medium
High
Very high
Mitochondrial density
High
High
Medium
Low
Capillary density
High
Intermediate
Low
Low
Oxidative capacity
High
High
Intermediate
Low
Glycolytic capacity
Low
High
High
High
Major storage fuel
Triglycerides
Creatine phosphate, glycogen
Creatine phosphate, glycogen
Creatine phosphate, glycogen
Myosin heavy chain, human genes
MYH7
MYH2
MYH1
MYH4
Table adapted from [161].
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Histological analysis of muscle biopsy specimens in adults with profound vitamin D deficiency reveals a predominance of type II muscle fiber atrophy. Thus, the fact that primarily type II fibers are affected by vitamin D deficiency may help explain the clinical reports of higher falling tendency in vitamin-D-deficient older individuals [55]. Histological sections of vitaminD-deficient individuals also reveal enlarged interfibrillar spaces and infiltration of fat, fibrosis and glycogen granules [73]. In a recent cross-sectional study in 90 young women, Gilsanz et al. noted a greater degree of fat infiltration in muscle by CT scan in those with lower serum 25(OH)D levels [74]. Similar morphological features are noted in the vitamin-D-deficient myopathy associated with chronic renal failure as well [75,76]. It has been hypothesized that individuals with low vitamin D status who are placed on supplementation with vitamin D improve muscle strength and performance in part by an increase in muscle mass. To date, there have been two reports examining whether supplementation with a form of vitamin D impacts on muscle fiber size and composition. These studies appear to support a selective effect on type II muscle fibers. In 1979, a small uncontrolled study by Sorenson et al. obtained muscle biopsies from 11 older women after treatment with 1e2 mg of 1a-hydroxyvitamin D and 1 g of calcium daily for 3e6 months [77]. Results showed an increase in relative number and in the cross-sectional area of type IIa muscle fibers (a subtype of type II fibers). About 25 years later, a different randomized controlled study in 96 older Japanese female stroke survivors found that treatment with 1000 IU of vitamin D2 daily significantly increased mean type II muscle fiber diameter and percentage of type II fibers compared to placebo over a 2-year period [43]. There was also a correlation between serum 25(OH)D level and type II muscle fiber diameter both at baseline and after 2 years of followup. It remains unclear, however, if the increase in type II muscle fiber number is caused by new formation of type II fibers or a transition of already-existing fibers from type I to type II.
MOLECULAR MECHANISMS OF ACTION Muscle and Actions of 1,25(OH)2D3 via Receptor The biologically active form 1,25(OH)2D3 exerts its principal actions by binding to VDR. VDRs are expressed in muscle tissue at particular stages of differentiation from myoblasts (mononucleated myogenic cells) to myotubes (multinucleated cells). In 1985, Simpson et al. identified a binding protein consistent with the 1,25(OH)2D receptor in rodent skeletal muscle
cell lines [15]. At the same time, other reports demonstrated evidence of the VDR in chick monolayers of myoblasts [78], and in cloned human skeletal muscle cells [79]. Two different 1,25(OH)2D receptors have been described, one acting as a nuclear receptor and the other located at the cell membrane. The nuclear VDR is a ligand-dependent nuclear transcription factor, which belongs to the steroidethyroid hormone receptor gene superfamily [80e83]. Using immunohistochemical methods to analyze tissue from adult females, Bischoff et al. reported the first in situ detection of the VDR in human skeletal muscle tissue [16]. The data demonstrated intranuclear staining for the VDR providing evidence for the presence of the nuclear 1,25(OH)2D receptor. Once transported to the nucleus by an intracellular binding protein, 1,25(OH)2D3 binds to its nuclear receptor which results in changes in the gene transcription of mRNA and subsequent de novo protein synthesis [84]. At the nuclear level, the activation of VDR induces the heterodimerization between the active VDR and an orphan steroid receptor known as retinoic receptor (RXR). The formation of this heterodimer facilitates the interaction between the receptor’s zinc finger region with DNA activating the protein transcription process [85]. Figure 104.2 depicts the mature skeletal muscle cell. It is important to recognize that skeletal muscle cells have evolutionally been guided into a contractile machine in which any mechanism that affects its function must be related to its structure. Thus the very large muscle cell has multiple nuclei and extensive invaginations of the plasma membrane. The genomic pathway has been found to influence muscle calcium uptake, phosphate transport across the cell membrane, phospholipid metabolism, and muscle cell proliferation and differentiation. In vitro and in vivo experiments in chick skeletal muscle have shown that 1,25(OH)2D regulates muscle calcium uptake by modulating the activity of calcium pumps in sarcoplasmic reticulum and sarcolemma. It also regulates the calcium influx via voltage-sensitive calcium channels thereby altering intracellular calcium [8]. Modifications in intracellular calcium levels control contraction and relaxation of muscle, thus impacting muscle performance [86,87]. Other experiments in cultured myoblasts and myocytes further supported these findings by demonstrating increased 45Calcium uptake in cells exposed to physiological levels of 1,25(OH)2D [88,89]. These longer-term effects of 1,25(OH)2D are dependent on de novo RNA and protein synthesis via the activation of the nuclear VDR. In vitro studies have suggested that 1,25(OH)2D modulates intracellular calcium levels by stimulating the expression of a calcium-binding protein called calbindin-D9K. Western blots using an antibody to rat intestine calbindin D9K revealed presence of an
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FIGURE 104.2 The cellular structure of the skeletal muscle cell is shown. As evident from this diagram skeletal muscle is composed of specialized cells that possess a dominant organelle, the myofibril. The important cell structures that regulate muscle contraction are Myofibrils also shown, as is their association with the myofibril structures. These structures include the mitochondria, the sarcolemma, the transverse tubule (t-tubule), and the terminal cisternae. These evolved structures are all A band essential components for the required ATP synthesis and regulation of intracellular calcium essential for muscle excitationecontraction coupling. Interestingly, as shown, I band there are multiple nuclei present in the fused mature muscle cell. The nuclei are located outside the myofibril bundles and are shown as flattened structures adjacent to the sarcolemma. Please see color plate section. Z line
Sarcolomma
Terminal cisternae Transverse tubule
Sarcoplasmic reticulum
Mitochondria Nucleus
immunoreactive protein of 9 kDa in chick embryo myoblasts treated with 1,25(OH)2D [90,91]. Northern blot analysis using a specific rat calbindin D9K probe showed an increase in mRNA levels corresponding to calbindin D9K in chick muscle cells treated with 1,25(OH)2D. Additional evidence for the expression of calbindin D9K mRNA in chicken muscle cells was obtained by combined reverse transcription and polymerase chain reaction [92]. Prolonged treatment with 1,25(OH)2D has been shown to affect the synthesis of certain muscle cytoskeletal proteins which are important in controlling muscle cell surface properties [86]. One of these 1,25(OH)2Ddependent proteins has been described as a calmodulin-binding component of the myoblast cytoskeleton [93]. Calmodulin is a calcium-binding protein that regulates several cellular processes including muscle contraction. Experiments in mitotic myoblasts treated with 1,25(OH)2D revealed increased synthesis of calmodulin [94]. 1,25(OH)2D appears to play a role in the regulation of phosphate metabolism in myoblasts [86]. In skeletal muscle cells as in other cell types, phosphate in the form of ATP or inorganic phosphate is necessary for structural and metabolic needs of the cell. The clinical manifestations of hypophosphatemia can include a proximal myopathy. Exposure to 1,25(OH)2D stimulates accelerated phosphate uptake and accumulation in cells. Studies by Birge and Haddad [20] were the first to show that 25(OH)D3 increased the rate of accumulation of inorganic phosphate by diaphragmatic muscle tissue of vitamin D-deficient rats. In cultured chick embryonic muscle cells, preincubation with physiological levels of 1,25(OH)2D3 resulted in a significant accumulation of phosphate by the cells [95]. This effect
is thought to be mediated through the nuclear VDR resulting in de novo protein synthesis [86]. Finally, via its genomic pathway, 1,25(OH)2D appears to have a role in the regulation of muscle cell proliferation and differentiation. Studies in cultured chick embryo myoblasts demonstrated that up to 40 hours of treatment with 1,25(OH)2D at physiological levels increased both cell density and fusion [17]. 1,25(OH)2D was found to exert a biphasic effect on DNA synthesis [96]. Specifically, the hormone had a mitogenic effect in proliferating myoblasts followed by an inhibitory effect during the subsequent differentiation phase. Additionally, expression of cell cycle genes, such as c-myc and c-fos, and other skeletal muscle cell proteins were altered during 1,25(OH)2D’s stimulatory and inhibitory effects on proliferation. Overall, the regulation of gene products via the genomic pathway occurs over hours to days.
Non-genomic Effects of 1,25(OH)2D Like other steroid hormones, 1,25(OH)2D also elicits rapid responses occurring within seconds to minutes involving stimulation of trans-membrane signal transduction pathways through putative membrane-associated receptors [97e100]. These rapid actions involve initiation of signaling cascades within the cell that amplify and propagate the signal. Endpoints of signaling through a cell-surface receptor may also include change in gene expression through the activation or subcellular translocation of transcription factors. These rapid responses appear to influence muscle intracellular calcium regulation, muscle contractility and myogenesis [101,102]. Moreover, these rapid actions involve signaling components that
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also impact the nucleus through the regulation of transcription factors. The rapid, non-genomic actions of 1,25(OH)2D have been reported in several target tissues, including skeletal muscle, and it is thought that 1,25(OH)2D exerts its effects through a membrane-associated VDR. The characterization and mechanism of action of this putative non-nuclear receptor has been controversial. Some have proposed that the initiation of the fast 1,25(OH)2D signal may involve binding to a novel membrane receptor [102e103], while others have suggested a membrane associated calcium-binding protein that functions as a calcium-specific ion channel, annexin II [104,105]. It has also been hypothesized that protein kinase C is the cell surface receptor for 1,25(OH)2D [106]. However, recent studies have suggested that the cell-surface receptor is the intranuclear VDR itself, which translocates from the nucleus to the plasma membrane [99]. Chick myoblasts treated with 1,25(OH)2D for up to 10 minutes show translocation of the VDR from the nuclear to the microsomal fraction in the cell. Tyrosine kinase inhibitors and microtubular transport inhibitors block this process. The hypothesis of binding to the membrane receptor, 1,25(OH)2D, initiates a cascade leading to the formation of a second-messenger or phosphorylation of intracellular proteins resulting in cellular effects within seconds to minutes. Although the characterization remains incomplete, it has been shown that VDR is associated with caveolae microdomains in chick skeletal muscle cells, suggesting a possible mechanism for membrane localization [99]. The VDR has been found in isolated membrane fractions of both chick intestinal cells, and chick embryonic skeletal muscle cells [107]. A mechanism for the association of the VDR with the membrane has as yet not been clearly delineated. The Simpson laboratory reported that in adult rat and mouse cardiomyocytes, the VDR is located in the t-tubular membrane structures [108]. The t-tubules of mammalian skeletal and cardiac myocytes are invaginations of the plasma (sarcolemma) membrane. The development of t-tubules appears to depend on proteins and lipids and shows properties that are similar to the development of caveolae, which require cholesterol and caveolins [109]. Caveolins are the principal structural proteins that are both necessary and sufficient for the formation of caveolae membrane domains. These proteins function both in protein trafficking and in signal transduction, as well as in cholesterol homeostasis [110,111]. A mechanism for the association of the VDR with the membrane has not been delineated. The Simpson laboratory has reported that the t-tubule fraction of isolated rat cardiomyocytes is enriched in the VDR and Caveolin-3, the predominant caveolin subtype in cardiac myocytes [108]. This report demonstrated that the VDR is
physically associated with Caveolin-3 in the t-tubules and sarcolemma of the cardiomyocyte by confocal immunofluorescence microscopy studies and coimmunoprecipitation studies. Recently, it has also been shown that 1,25(OH)2D induces a rapid direct effect on contraction of isolated individual cardiomyocytes [112]. Moreover, the hormone was found to decrease the stimulated peak of contraction or sarcomere shortening of the isolated heart muscle cells [113]. It is known that the rate of calcium influx through calcium channels is responsible for the rate of myocyte contraction and these calcium channels are located primarily at the t-tubules. T-tubules are composed of interconnected caveolae-like elements [114]. As shown in Fig. 104.3 many of the proteins involved in excitation contraction coupling are concentrated at the t-tubules, including L-type calcium channels (LTCC), which play a critical role in regulating calciumdependent signaling in myocytes. Receptors, associated with caveolins in myocytes, are important in the regulation of a number of key gene products, in particular, the LTCC, the sarcoplasmic reticulum Ca2þ-ATPase, and the contractile proteins (via troponin I) [115]. Recently it has been shown that subcellular localization of LTCC to caveolar macromolecular signaling complexes is essential for regulation of the channels by the 2-adrenergic receptor [116]. Previous studies in the Simpson laboratory have shown an increased nuclear localization of VDR after 1,25(OH)2D treatment in cardiac muscle [108]. It is reasonable to hypothesize that vitamin D acting through 1,25(OH)2D could cause changes (increase or decrease the association) in the interaction between VDR and caveolins, thus causing a cascade effect on caveolin-associated signal transducing gene products that then affect the contraction of myocytes function. Caveolin-associated proteins include the Src-family tyrosine kinase, MAP kinase cascades, adenylyl cyclase, PKA, PKC, TGF receptor pathway, and endothelial nitric oxide synthase (eNOS) [117]. Caveolin holds these signal transducers in the sensitive or inactive conformation until activation by an appropriate stimulus. Thus, part of the functions of a membrane-associated VDR could well be through its interaction with caveolins by affecting caveolin-associated signal transduction pathways. In support of this scheme, a recent study showed 1,25(OH)2D-dependent modulation of Src, MAPK cascades and VDR localization in skeletal muscle cells [118]. Thus, it may be that the processes that initiate the translocation of the VDR to the nucleus to affect gene transduction and the genomic actions of 1,25(OH)2D are directly responsible for the non-genomic effects of the hormone. However, at present it is not known if the processes involved in the genomic and non-genomic actions of 1,25(OH)2D are coordinated or time ordered.
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Junctional α1-α2/δ-β-γ DHPR
Non-junctional DHPR
VDR
TT
FKBP12
SL
RyR JFP
NUC
VDR
JN
CaM TRI
CSQ-like proteins
CSQ
SR CAL
PMCA Na+/K+ ATPase
HCP
SAR SERCA
Na+/ca2+ exchanger
FIGURE 104.3 Diagrammatic presentation of skeletal muscle proteins involved in the excitationecontractionerelaxation cycle taken from a review by Froemming and Ohlendieck [114]. The muscle cell is composed of transverse tubules (TT) and two surrounding terminal cisternae of the sarcoplasmic reticulum (SR). Located in the TT is the multimeric dihydropyridine receptor (DHPR) with its five subunits, the voltage-sensing alpha-1 subunit, the transmembrane alpha-2/delta and gamma subunits, as well as the cytosolic beta subunit. The ryanodine receptor (RyR) Ca2þ-release channel is enriched in the junctional SR membrane. Tightly associated with the receptor are two auxiliary proteins, the immunophilin protein of 12 kDa (FKBP12) and the Ca2þ-binding component calmodulin (CaM), which both modulate the opening time of the RyR. The Ca2þ-binding proteins calsequestrin (CSQ), calreticulin (CAL), sarcalumenin (SAR) and histidine-rich Ca2þ-binding protein (HCP) are thought to be involved in Ca2þ storage and the fine regulation of the RyR. Junctin (JN) represents a CSQ-binding protein. Triadin (TRI) clusters and the 90 kDa junctional face protein (JFP) are probably involved in maintaining receptor interactions and the overall architectural arrangement of triad junctions. The SR Ca2þ-ATPase (SERCA) represents the major energy-dependent Ca2þ-reuptake mechanism in skeletal muscle fibers and this enzyme is responsible for initiating the muscle relaxation step. In addition, a surface Ca2þ pump (PMCA) and an Naþ/Ca2þ exchanger, which is indirectly driven by the sarcolemmal Naþ/Kþ-ATPase, exist in skeletal muscle. Complex interactions between these various Ca2þ channels, Ca2þ-binding proteins and Ca2þ pumps provide the molecular basis that regulates muscle Ca2þ homeostasis. Also shown are putative sites at which the vitamin D receptor (VDR) may exist. The action of the VDR to regulate the expression of the gene products, including ones shown here, could account for the actions of 1,25(OH)2D on skeletal muscle function. Moreover the activation of the VDR associated with membrane structures such as the TT could well account for the reported rapid or non-genomic effects of this hormone on muscle function.
In vitro research suggests that a non-genomic mechanism also mediates the actions of 1,25(OH)2D on calcium influx and muscle contractility. These non-genomic responses consist of G-protein-mediated activation of phospholipase C [119] which generates diacylglycerol (DAG) and inositol 1,4,5-trisphosphatase (IP3) and adenylyl cyclase with the simultaneous acute increase in cyclic AMP levels [120], leading to the activation of
protein kinase A and C [121e123], release of calcium from intracellular stores [124], and activation of voltage-gated and store-operated calcium channels [125,126]. Work in embryonic chick muscle cells described other mechanisms including activation of phospholipase A2 [127] and phospholipase D [98] and modulation of specific protein kinase C isoforms [128]. In vitro studies in vitamin-D-deficient chicks showed
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that 1,25(OH)2D added to skeletal muscle cells had a rapid (1e15 min) effect on calcium uptake [129,130]. Inhibitors of RNA and protein synthesis did not inhibit these rapid effects suggesting no involvement of the nuclear VDR. However, calcium channel blockers did suppress these effects indicating that 1,25(OH)2D was acting at the membrane level affecting calcium entry into the cell. Recent data indicate that downstream responses to 1,25(OH)2D also depend on fast activation of mitogenactivated protein kinase (MAPK) signaling pathways [97]. These pathways transmit extracellular signals to their intracellular targets that ultimately result in initiation of myogenesis, cell proliferation, differentiation, or apoptosis [131]. In mammalian cells, the MAPK family has four different subgroups: extracellular signal-regulated kinases (ERKs 1/2), c-Jun N-terminal kinases (JNK), ERK5, and p38 MAPK [132]. When activated, these MAPKs regulate cell processes through phosphorylation of other kinases, proteins, and transcription factors. The ERKs are key components of the signal transduction pathways in growth and differentiation responses [133,134]. In proliferating cultured myoblasts, 1,25(OH)2D rapidly (within 1 min) activates ERK-1/2, phospholipase C and the c-myc [97]. The ERK pathway is activated by 1,25(OH)2D through phosphorylation by several kinases, such as c-Src, Raf-1, Ras, and MAPKK [135]. Through these mechanisms, 1,25(OH)2D causes the translocation of ERK-1/2 from the cytoplasm to the nucleus in an active phosphorylated form and induces the synthesis of the growth-related protein, c-myc, and stimulation of muscle cell proliferation. The hormone also stimulates tyrosine phosphorylation and membrane translocation of phospholipase C in myoblasts [136]. Although considerable progress has been made in characterizing the metabolic pathways involved in 1,25(OH)2D’s action on skeletal muscle cells, more research is needed to clarify how 1,25(OH)2D is affecting these pathways and what the mechanisms are.
VDR KNOCKOUT MOUSE MODEL The VDR knockout mouse model, in which animals are placed on normal or rescue diets, provides evidence that vitamin D has a direct effect on skeletal muscle mass, morphology and performance via its receptor [137]. The VDR null mutant mice are characterized by alopecia, reductions in both body size and weight and impaired motor coordination [138]. Studies in VDRnull mutant mice show that these animals can grow normally until weaning and thereafter develop various metabolic abnormalities including hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and bone deformities similar to those features typical of
rickets [139]. Independent of the systemic metabolic changes, VDR-null mutant mice have muscle fiber diameters that are approximately 20% smaller and more variable in size than those of the wild-type mice at 3 weeks of age (prior to weaning) [137]. By 8 weeks of age, these muscle fiber changes are more prominent in the VDR null mutant mice on rescue diet, as compared to the wild type, suggesting either that these abnormalities progress over time or that as these mice age, the metabolic alterations that occur contribute to the morphological changes [137]. The muscle fiber abnormalities are noted diffusely without any preference for type I or II fibers, differing from the human hypovitaminosis D myopathy that is characterized by type II muscle fiber atrophy on histological sections. Interestingly, there is no evidence of degeneration or necrosis in the VDRnull mice [137]. Such morphological changes indicate that the VDR plays an important role in skeletal muscle fiber development and its maturation. Studies in VDR-null mutant mice at 3 weeks of age also demonstrate abnormally high expression of myogenic differentiation factors compared to wild-type mice [137]. Thus, Myf5, E2A, and myogenin, factors minimally expressed in wild-type mice, were found to increase in the VDR-null mutant mice. Embryonic and neonatal myosin heavy chain (MHC) isoforms were also noted to have increased expression, whereas the type II (adult fast twitch) MHC expression was similar to the wild-type mice [137]. The abnormal levels in these differentiation factors may explain, in part, some of the morphological abnormalities seen in the VDR-null mutant mice. As the differentiation pathways are altered, so are muscle fiber development and maturation. An additional feature of the VDR knockout behavioral phenotype is poor swimming ability (as assessed by the forced swimming test) [140]. This is a well-known method to assess motor and balance functions in rodents. This finding has been attributed to muscular or motor impairment in the mouse; however, a recent study [141] considers whether impaired vestibular function in these VDR-null mutant mice may be an important contributor to this finding. Via immunohistochemical analysis, Minyasan et al. localized VDR-positive nuclei in epithelium of different structures in the vestibular system in wild-type mice and a significantly reduced expression of VDR in these same structures in the VDR-null mutant animals [141]. To further support the presence of a vestibular impairment in the VDR knockout mouse, measurement of postural control on balance tests, such as the accelerating rotarod and tilting platform, revealed significantly greater abnormal results in the VDR knockout than wild-type mouse. These findings strongly suggest an additional mechanism, loss of vestibular function, in the pathway to poor physical performance (particularly postural sway
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and balance) and falls seen in older humans with low 25(OH)D levels.
VDR POLYMORPHISMS VDR polymorphisms, which are defined as subtle variations in DNA sequence of the VDR gene, have been associated with a wide range of biological characteristics including muscle strength and falls. One welldescribed single nucleotide polymorphism, FokI, is a polymorphism involving the translation start site resulting in a VDR protein shortened by three amino acids and not linked to any of the other VDR polymorphisms [142,143]. Individuals with the C allele (“F”) have a shorter 424-amino acid VDR than do those with the T (“f”) allele, the former having been associated with enhanced VDR transactivation capacity as a transcription factor [144]. In light of the clinical data reporting a positive association between vitamin D status and muscle strength, this would suggest that greater VDR activity could result in improved muscle strength. On the contrary, the C allele has been associated with reduced fat-free mass and quadriceps strength in healthy elderly men [145], healthy middle-aged women [146], and older individuals with COPD [147]. There are several studies examining potential associations between restriction fragment length polymorphisms in VDR gene and muscle mass and strength. BsmI is a restriction fragment length polymorphism in intron 8 at the 30 end of the VDR gene, where B allele indicates absence and the b allele the presence of the restriction site. In non-obese older women aged 70 and older, those with the bb genotype were found to have a 7% higher grip strength and a 23% higher quadriceps strength than those with BB genotype [148]. TaqI is another restriction fragment length polymorphism at the 30 end of the VDR gene in a silent site in exon 9. In men, Bt/Bt homozygotes for the BsmI-TaqI haplotype outperformed bT carriers in both isometric and concentric quadriceps torques at different contraction velocities [146]. There are two recent studies examining a relationship between VDR polymorphisms and the risk of falls in older individuals. A study in a small group (n ¼ 259) of very elderly (over 80 years) women [149] found an association between Bsm1 and falls within 90 days of assessment, with a reduction in falls for those carrying the bb genotype, while no association was found for Fok1. Another recent study in two separate population cohorts (the Aberdeen Prospective Osteoporosis Screening Study and the Osteoporosis and Ultrasound study) also found an association between falls and polymorphisms in Bsm1. In this analysis, postmenopausal women carrying the B allele were at higher risk of falls
in both cohorts [150] compared to those not carrying the B allele. The study also showed that the association was independent of serum 25(OH)D levels. However, across these and several other [146,147,151,152] observational studies on specific VDR polymorphisms and skeletal muscle outcomes, the findings are not consistent. Explanations for at least some of this variability may be found in a better understanding of how these polymorphisms are linked to and interact with other genetic variations and environmental factors.
PTH Clinically, patients with PTH excess share similar symptoms of muscle weakness and fatigue. This finding has been reported in primary hyperparathyroidism [153e155] and in secondary hyperparathyroidism induced by chronic renal failure [75,156]. Muscle biopsies demonstrate atrophy of type II muscle fibers as in vitamin D deficiency [157]. Furthermore, PTH has been shown to predict falls [59] and muscle strength independent of 25(OH)D level, age, and body mass index [60]. The question of whether vitamin D deficiency itself or secondary hyperparathyroidism is the primary cause of muscle tissue and functional abnormalities still has not been fully answered. In primary hyperparathyroidism, hypercalcemia is unlikely to mediate these effects since serum calcium can be normal or low in persons with renal failure. Low vitamin D levels stimulate PTH production and PTH may have direct effects on skeletal muscle by increasing free intracellular calcium concentrations [158]. In a state of chronically elevated intracellular calcium concentrations in a muscle cell, muscle contraction may be impaired or disrupted. Studies in rats have also demonstrated that PTH induces muscle catabolism [159], reductions in calcium transport (calciumeATPase activity) and impairment of energy availability (reduction in intracellular phosphate and mitochondrial oxygen consumption) and metabolism (reduction in creatinine phosphokinases and oxidation of long-chain fatty acids) in skeletal muscle [160].
CONCLUSION Vitamin D supplementation may be a promising nutritional therapeutic intervention for reducing loss of muscle mass and improving muscle strength and physical performance in aging adults. An association between vitamin D status and skeletal muscle health is described in case reports of a reversible proximal myopathy, in large population-based studies, and more recently in well-designed randomized clinical trials. Much of the published data on this association is in
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older healthy individuals, who are at risk for age-related loss of muscle mass, a reduced physical function, and an increased risk of falls and disability. Not all trials have shown significantly positive improvements in muscle performance or reductions in the risk of falls; however, in older adults with lower serum levels of 25(OH)D at baseline, supplementation with various forms of vitamin D has generally shown beneficial effects on muscle strength, tests of physical performance, and rate of falls. Thus, repleting vitamin D stores in an aging population may be important for the preservation of physical function. As a next step in determining whether this supplementation is beneficial, recent research has delved into understanding the underlying actions of vitamin D in human skeletal muscle. Some investigators hypothesize that improvements in muscle strength and performance are mediated through an increase in muscle fibers (size and number). These data, however, are very limited and need additional study. The identification of the VDR, which is present in human myonuclei and possibly on the cell surface of the myocyte, provides further insight into the role of vitamin D in skeletal muscle. Experimental studies in both cell culture and animals in the last two decades begin to identify the genomic effects of 1,25(OH)2D leading to the synthesis of new proteins that affect muscle cell contractility, proliferation, and differentiation. In addition, scientists are gradually constructing non-genomic pathways of 1,25(OH)2D activity in muscle cells that also impact on muscle contraction and possibly muscle cell development. However, much remains to be clarified. “Gradually” is the operative word here, as little mechanistic progress has been made in 30 years. The VDR knockout mouse model has revealed important functions of the VDR in the development and maturation of skeletal muscle and will continue to help improve our understanding of the underlying mechanisms. Other incoming research, such as the studies in VDR polymorphisms and PTH effects on muscle, are somewhat inconsistent with the clinical hypothesis that vitamin D, VDR, and optimal muscle health are directly linked; however, they are raising important questions that need to be investigated. Additional research is needed to determine whether further increasing mean serum 25(OH)D to concentrations above 84 nmol/l would lead to additional benefits for muscle strength, physical performance and falls in older adults. In addition, given some inconsistencies in data from clinical trial studies, further investigation should include a clearer understanding of the underlying actions of vitamin D in human skeletal muscle. It remains unclear if vitamin D’s purported beneficial muscle effects occur via a change in muscle mass or muscle activation or both. Furthermore, whether
vitamin D acts on muscle via the vitamin D receptor (VDR) and/or other pathways is not entirely established. Progress in these areas of investigation will provide important direction for future clinical research to improve functional outcomes in older adults.
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[124] G. Vazquez, A.R. de Boland, R. Boland, Stimulation of Ca2þ release-activated Ca2þ channels as a potential mechanism involved in non-genomic 1,25(OH)2-vitamin D3-induced Ca2þ entry in skeletal muscle cells, Biochem. Biophys. Res. Commun. 239 (1997) 562e565. [125] G. Vazquez, A.R. de Boland, R.L. Boland, 1Alpha,25-dihydroxy-vitamin-D3-induced store-operated Ca2þ influx in skeletal muscle cells. Modulation by phospholipase c, protein kinase c, and tyrosine kinases, J. Biol. Chem. 273 (1998) 33954e33960. [126] G. Vazquez, A.R. de Boland, Stimulation of dihydropyridinesensitive Ca2þ influx in cultured myoblasts by 1,25(OH)2vitamin D3, Biochem. Mol. Biol. Int. 31 (1993) 677e684. [127] A.R. de Boland, R.L. Boland, 1,25-Dihydroxyvitamin D-3 induces arachidonate mobilization in embryonic chick myoblasts, Biochim. Biophys. Acta 1179 (1993) 98e104. [128] D.A. Capiati, G. Vazquez, R.L. Boland, Protein kinase C alpha modulates the Ca2þ influx phase of the Ca2þ response to 1alpha,25-dihydroxy-vitamin-D3 in skeletal muscle cells, Horm. Metab. Res. 33 (2001) 201e206. [129] J. Selles, R. Boland, Rapid stimulation of calcium uptake and protein phosphorylation in isolated cardiac muscle by 1,25dihydroxyvitamin D3, Mol. Cell Endocrinol. 77 (1991) 67e73. [130] A.R. de Boland, R.L. Boland, Rapid changes in skeletal muscle calcium uptake induced in vitro by 1,25-dihydroxyvitamin D3 are suppressed by calcium channel blockers, Endocrinology 120 (1987) 1858e1864. [131] S.N. Wu, H.F. Li, H.T. Chiang, Characterization of ATP-sensitive potassium channels functionally expressed in pituitary GH3 cells, J. Membr. Biol. 178 (2000) 205e214. [132] C. Widmann, S. Gibson, M.B. Jarpe, G.L. Johnson, Mitogenactivated protein kinase: conservation of a three-kinase module from yeast to human, Physiol. Rev. 79 (1999) 143e180. [133] P.H. Sugden, A. Clerk, Regulation of the ERK subgroup of MAP kinase cascades through G protein-coupled receptors, Cell Signal 9 (1997) 337e351. [134] M.H. Cobb, D.J. Robbins, T.G. Boulton, ERKs, extracellular signal-regulated MAP-2 kinases, Curr. Opin. Cell Biol. 3 (1991) 1025e1032. [135] C.G. Buitrago, V.G. Pardo, A.R. de Boland, R. Boland, Activation of RAF-1 through Ras and protein kinase Calpha mediates 1alpha,25(OH)2-vitamin D3 regulation of the mitogen-activated protein kinase pathway in muscle cells, J. Biol. Chem. 278 (2003) 2199e2205. [136] C. Buitrago, V. Gonzalez Pardo, A.R. de Boland, Nongenomic action of 1 alpha,25(OH)(2)-vitamin D3. Activation of muscle cell PLC gamma through the tyrosine kinase c-Src and PtdIns 3-kinase, Eur. J. Biochem. 269 (2002) 2506e2515. [137] I. Endo, D. Inoue, T. Mitsui, Y. Umaki, M. Akaike, T. Yoshizawa, et al., Deletion of vitamin D receptor gene in mice results in abnormal skeletal muscle development with deregulated expression of myoregulatory transcription factors, Endocrinology 144 (2003) 5138e5144. [138] T.H. Burne, J.J. McGrath, D.W. Eyles, A. Mackay-Sim, Behavioural characterization of vitamin D receptor knockout mice, Behav. Brain Res. 157 (2005) 299e308. [139] Y. Song, S. Kato, J.C. Fleet, Vitamin D receptor (VDR) knockout mice reveal VDR-independent regulation of intestinal calcium absorption and ECaC2 and calbindin D9k mRNA, J. Nutr. 133 (2003) 374e380. [140] A.V. Kalueff, Y.R. Lou, I. Laaksi, P. Tuohimaa, Impaired motor performance in mice lacking neurosteroid vitamin D receptors, Brain Res. Bull. 64 (2004) 25e29.
[141] A. Minasyan, T. Keisala, J. Zou, Y. Zhang, E. Toppila, H. Syvala, et al., Vestibular dysfunction in vitamin D receptor mutant mice, J. Steroid Biochem. Mol. Biol. 114 (2009) 161e166. [142] S. Nejentsev, L. Godfrey, H. Snook, H. Rance, S. Nutland, N.M. Walker, et al., Comparative high-resolution analysis of linkage disequilibrium and tag single nucleotide polymorphisms between populations in the vitamin D receptor gene, Hum. Mol. Genet. 13 (2004) 1633e1639. [143] H. Arai, K. Miyamoto, Y. Taketani, H. Yamamoto, Y. Iemori, K. Morita, et al., A vitamin D receptor gene polymorphism in the translation initiation codon: effect on protein activity and relation to bone mineral density in Japanese women, J. Bone Miner. Res. 12 (1997) 915e921. [144] G.K. Whitfield, L.S. Remus, P.W. Jurutka, H. Zitzer, A.K. Oza, H.T. Dang, et al., Functionally relevant polymorphisms in the human nuclear vitamin D receptor gene, Mol. Cell Endocrinol. 177 (2001) 145e159. [145] S.M. Roth, J.M. Zmuda, J.A. Cauley, P.R. Shea, R.E. Ferrell, Vitamin D receptor genotype is associated with fat-free mass and sarcopenia in elderly men, J. Gerontol. A. Biol. Sci. Med. Sci. 59 (2004) 10e15. [146] A. Windelinckx, G. De Mars, G. Beunen, J. Aerssens, C. Delecluse, J. Lefevre, et al., Polymorphisms in the vitamin D receptor gene are associated with muscle strength in men and women, Osteoporos Int. 18 (2007) 1235e1242. [147] N.S. Hopkinson, K.W. Li, A. Kehoe, S.E. Humphries, M. Roughton, J. Moxham, et al., Vitamin D receptor genotypes influence quadriceps strength in chronic obstructive pulmonary disease, Am. J. Clin. Nutr. 87 (2008) 385e390. [148] P. Geusens, C. Vandevyver, J. Vanhoof, J.J. Cassiman, S. Boonen, J. Raus, Quadriceps and grip strength are related to vitamin D receptor genotype in elderly nonobese women, J. Bone Miner. Res. 12 (1997) 2082e2088. [149] G. Onder, E. Capoluongo, P. Danese, S. Settanni, A. Russo, P. Concolino, et al., Vitamin D receptor polymorphisms and falls among older adults living in the community: results from the ilSIRENTE study, J. Bone Miner. Res. 23 (2008) 1031e1036. [150] R. Barr, H. Macdonald, A. Stewart, F. McGuigan, A. Rogers, R. Eastell, et al., Association between vitamin D receptor gene polymorphisms, falls, balance and muscle power: results from two independent studies (APOSS and OPUS), Osteoporos Int. 21 (2010) 457e466. [151] E. Grundberg, H. Brandstrom, E.L. Ribom, O. Ljunggren, H. Mallmin, A. Kindmark, Genetic variation in the human vitamin D receptor is associated with muscle strength, fat mass and body weight in Swedish women, Eur. J. Endocrinol. 150 (2004) 323e328. [152] G. Bahat, B. Saka, N. Erten, U. Ozbek, E. Coskunpinar, S. Yildiz, et al., BsmI polymorphism in the vitamin D receptor gene is associated with leg extensor muscle strength in elderly men, Aging Clin. Exp. Res. 22 (2010) 198e205. [153] S.R. Deutch, M.B. Jensen, P.M. Christiansen, I. Hessov, Muscular performance and fatigue in primary hyperparathyroidism, World J. Surg. 24 (2000) 102e107. [154] E.B. Colliander, K. Strigard, P. Westblad, C. Rolf, J. Nordenstrom, Muscle strength and endurance after surgery for primary hyperparathyroidism, Eur. J. Surg. 164 (1998) 489e494. [155] A. Kristoffersson, A. Bostrom, T. Soderberg, Muscle strength is improved after parathyroidectomy in patients with primary hyperparathyroidism, Br. J. Surg. 79 (1992) 165e168. [156] T.S. Kim, Primary hyperparathyroidism, Orthop. Nurs. 13 (1994) 17e27, quiz 28.
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[157] B.M. Patten, J.P. Bilezikian, L.E. Mallette, A. Prince, W.K. Engel, G.D. Aurbach, Neuromuscular disease in primary hyperparathyroidism, Ann. Intern. Med. 80 (1974) 182e193. [158] N. Begum, K.E. Sussman, B. Draznin, Calcium-induced inhibition of phosphoserine phosphatase in insulin target cells is mediated by the phosphorylation and activation of inhibitor 1, J. Biol. Chem. 267 (1992) 5959e5963.
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[159] A.J. Garber, Effects of parathyroid hormone on skeletal muscle protein and amino acid metabolism in the rat, J. Clin. Invest. 71 (1983) 1806e1821. [160] M. Smogorzewski, G. Piskorska, P.R. Borum, S.G. Massry, Chronic renal failure, parathyroid hormone and fatty acids oxidation in skeletal muscle, Kidney Int. 33 (1988) 555e560. [161] K.S. Saladin, Anatomy and Physiology, fifth ed., Watnick, New York, 2010.
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C H A P T E R
105 The VITamin D and OmegA-3 TriaL (VITAL): Rationale and Design of a Large-Scale Randomized Controlled Trial Olivia I. Okereke, JoAnn E. Manson Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA
Disclosures: Dr. Manson is the Principal Investigator (PI) of the VITamin D and OmegA-3 TriaL (VITAL; U01-CA138962), a National Institutes of Health (NIH, including NCI, NHLBI, NINDS, ODS, NCCAM) sponsored trial of vitamin D supplementation in the prevention of cancer and cardiovascular disease. Dr. Okereke is the PI of Depression Endpoint Prevention in the VITamin D and OmegA-3 TriaL (VITAL-DEP; R01MH091448), an NIH sponsored trial of vitamin D supplementation for primary and secondary prevention of late-life depression, and is also funded by K08AG029813 from the National Institute on Aging of the NIH. VITAL study pills are donated by Pharmavite LLC of Northridge, CA (vitamin D) and Pronova BioPharma, Norway (omega-3s).
INTRODUCTION The goal of the VITAL trial is to evaluate the role of vitamin D and long-chain marine omega-3 polyunsaturated fatty acid supplements in the primary prevention of cancer and cardiovascular disease (CVD). Results of this 2 2 factorial trial are expected in 5 years. The following chapter will review the background and potential of supplemental vitamin D3 as an agent for cancer and CVD prevention and provide details on how VITAL will test this hypothesis.
Vitamin D, Third Edition DOI: 10.1016/B978-0-12-381978-9.10105-2
BIOLOGY OF VITAMIN D AND PROPOSED MECHANISMS FOR LOWERING CANCER AND CVD RISK Source and Synthesis Both medical professionals and the lay public have expressed heightened interest in recent years about the possible health benefits of vitamin D. While vitamin D is called a “vitamin,” it actually functions as a hormone. However, there are few natural dietary sources of vitamin D: other than fatty fish, egg yolk, and the unusual sunlight-exposed mushroom (vitamin D2), most other dietary sources of vitamin D are fortified foods, such as milk and other dairy products. In addition to these exogenous sources, vitamin D is synthesized endogenously in the body by the skin’s exposure to ultraviolet-B (UVB) radiation from sunlight. Consequently, vitamin D is also known as the “sunshine vitamin.” The sun’s UVB radiation changes 7-dehydrocholesterol, present in skin, into pre-vitamin D which then becomes vitamin D and circulates in the bloodstream. Vitamin D then undergoes 25-hydroxylation in the liver; this circulating 25-hydroxyvitamin D, 25(OH)D, is measured to evaluate a person’s vitamin D status. The 1a-hydroxylase enzyme (present in the kidney and other organs) converts 25(OH)D to the active hormone 1,25-dihydroxyvitamin D (1,25(OH)2D). In turn, 1,25(OH)2D acts at the cellular level, binding to the vitamin D receptors (VDRs) found in diverse cell
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Copyright Ó 2011 Elsevier Inc. All rights reserved.
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105. THE VITAMIN D AND OMEGA-3 TRIAL (VITAL): RATIONALE AND DESIGN
types throughout the body and controlling gene expression in that target cell [1,2]. These subjects are discussed in detail in other chapters throughout this book.
renin and, thus, lower activity of the renine angiotensinealdosterone system; and improving insulin sensitivity and glucose metabolism (Fig. 105.1) [18e22].
Proposed Mechanistic Pathways involving Vitamin D Vitamin D may have an important role in regulating cell growth and differentiation relevant to reducing cancer risk. As depicted in Fig. 105.1, abundant evidence from laboratory, cell culture, and animal studies suggests that vitamin D may lower cancer risk by inhibiting cell proliferation, angiogenesis, and metastasis; reducing inflammation; and inducing apoptosis and cell differentiation [3e15]. With regard to CVD risk, a key potential mechanism again involves reducing inflammation through strong modulatory effects on both the innate and acquired immune response e a shared mechanism also implicated in cancer risk reduction [16,17]. There are several other proposed pathways related to CVD, such as inhibiting vascular muscle proliferation and vascular calcification; decreasing blood pressure through lower gene expression of
7-Dehydrocholesterol in the skin
CURRENT EVIDENCE ON VITAMIN D AND RISK OF CANCER AND CVD Cancer Initial data suggesting that vitamin D reduces cancer risk arose from early ecologic studies showing higher cancer mortality in regions with less average exposure to UVB radiation from the sun, the stimulus of vitamin D synthesis in the skin. Studies [23e25] in the USA, Europe, and Japan found inverse associations between UVB radiation exposure and total cancer mortality, including deaths from cancers of the breast, colon, prostate, ovary, lung, pancreas, and other sites. In recent years prospective data from more rigorous investigations, such as large-scale observational studies, suggest decreased risk of cancer with higher vitamin D e but associations are inconsistent for total
Vitamin D
Diet and supplements
25 (OH)D (Liver) Various tissues (kidney, colon, breast, prostate, vascular smooth muscle, other) 1 -hydroxylase
1,25 (OH)2D (circulating or binding to vitamin D receptor)
P21 P27 EGFR TGF IGF1
Inhibit cell proliferation
BCL2 BAK TERT
Induce apoptosis/ differentiation
↓ VEGF ↓ IL-8
Inhibit angiogenesis/ metastasis
Cancer Prevention
↓ PG ↓ COX-2 ↓ CRP ↓ IL-6 ↑ IL-10 ↓ TNF ↓ MMP-9
Inhibit inflammation
Renin RAAS
Ca2+ cellular influx Matrix Gla protein
Inhibit vascular smooth muscle proliferation and vascular calcification
Regulate blood pressure/ volume homeostasis
↑ Insulin sensitivity
Regulate glucose metabolism
CVD Prevention
( = increase, ↓ = decrease expression or levels)
FIGURE 105.1 Mechanisms by which vitamin D may lower cancer and CVD risk. Source: Adapted from [91]. Key: 25(OH)D ¼ 25-hydroxy-
vitamin D; 1,25(OH)2D ¼ 1.25-dihydroxyvitamin D receptor ; P21, P27 ¼ cyclin-dependent kinase inhibitors; EGFR ¼ epidermal growth factor receptor; TGFb ¼ transforming growth factor-beta; IGF-1 ¼ insulin-like growth factor-1; BCL2 ¼ B-cell lymphoma 2, a pro-survival protein; BAK ¼ BCL2-Antagonist/Killer, a pro-apoptotic protein; TERT ¼ telomerase reverse transcriptase; VEGF ¼ vascular endothelial growth factor; IL-6, IL-8, IL-10 ¼ interleukins -6, -8 and -10; PG ¼ prostaglandin; COX-2 ¼ cyclooxygenase-2; CRP ¼ c-reactive protein; TNFa ¼ tumor necrosis factor a; MMP-9 ¼ matrix metallopeptidase-9; Ca2þ ¼ calcium cation; Gla ¼ g-carboxyglutamate; RAAS ¼ renineangiotensinealdosterone system.
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CURRENT EVIDENCE ON VITAMIN D AND RISK OF CANCER AND CVD
cancer and across different cancer types [26e30]. For example, prospective data from large-scale studies on both dietary intake of vitamin D and circulating blood levels of vitamin D strongly suggest a protective association between higher vitamin D and colorectal cancer [31,32] (see Chapter 82 by Giovannucci for complete discussion). Interestingly, recent work also suggests that loss-of-function VDR genetic polymorphisms that interfere with vitamin D utilization may increase colorectal cancer risk, particularly when blood 25(OH)D levels are low [33e35]. However, findings have been suggestive but inconsistent with regard to breast or prostate cancer risk, and some studies have even suggested an increased risk of other cancers, including pancreatic and esophageal, with higher levels of vitamin D [36]. Overall, the relationship between vitamin D and total cancer incidence and mortality remains inconclusive. Of note, an important issue in the literature is the non-uniformity of vitamin D exposure assessment e namely, whether the vitamin D exposure was dietary intake or blood the total 25(OH)D level that reflects both cutaneous synthesis and dietary intake. New data on the role of vitamin D in cancer have emerged during the past decade from randomized controlled trials involving vitamin D supplements, but no completed trials have assessed cancer as a primary pre-specified outcome [37]. Most past trials were designed to examine the effect of vitamin D on fracture prevention and only two recently completed trials had a sufficient number of cancer endpoints to allow for meaningful review of the issue: the Women’s Health Initiative (WHI) calciumevitamin D trial, in which 36 282 postmenopausal women were randomly assigned to a combination of calcium (1000 mg/d) and low-dose vitamin D3 (400 IU/d) or to placebo and followed for a mean of 7 years [38,39] and a British trial, in which 2686 men and women aged 65e85 years were randomized to 100 000 IU of vitamin D3 or placebo (one capsule every 4 months; an equivalent of about 800 IU/d) and followed for up to 5 years [40]. In the British trial, although treatment was not associated with a reduction in total or colorectal cancer incidence, there were statistically non-significant inverse associations for mortality from total cancer and colorectal cancer; the WHI, albeit with half the dose, reported a similar pattern of findings. Additionally, in a 4-year trial among 1179 postmenopausal women in Nebraska, women receiving calcium plus vitamin D (1100 IU/d) did not have a significantly lower risk of cancer than those receiving calcium alone (13 vs. 17 cases; RR 0.76; 95% CI 0.38e1.55), although risk was lower than for those receiving placebo [41]. Moreover, although there is one recent trial [41] and two ongoing trials [42,43] that specifically evaluate cancer risk using moderate-to-high-dose vitamin D
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supplements, their enrollment is on the order of 1000e2000 participants. Thus, VITAL, with 20 000 participants, is uniquely poised to examine the impact of long-term supplementation with a higher dose of vitamin D on total cancer risk and secondarily on (i) cancer death, (ii) individual cancer endpoints, and (iii) cancer risk by key factors, such as skin pigmentation, nutrient intake, and geographic latitude.
Cardiovascular Disease As with cancer, several lines of early evidence from ecologic studies suggested cardioprotective and cerebroprotective roles for vitamin D. For example, higher CVD mortality has been observed during the winter and in regions with less average exposure to UVB radiation from the sun [44]. However, compared to the evidence base on cancer, prospective epidemiologic data on the association between vitamin D and CVD events are relatively limited. Recent analyses from two larger studies e the Framingham Offspring Study [45] and the Health Professionals’ Follow-up Study [46] e suggest a significant relation between low serum 25(OH)D and nearly two-fold relative risk of incident CVD; a statistically significant, but more modest, positive association was also observed in the NHANES III (the Third National Health and Nutrition Examination Survey) cohort [47]. There have been no prior CVD primary prevention trials using vitamin D. However, some trial data are available from two completed RCTs of vitamin D supplementation; although CVD prevention was not a main trial aim. In the British trial [40] referenced above (see “Cancer”), there was a suggestion of mild relative risk reductions (10e15%) for CVD events and CVD death. Although these results were not statistically significant, they could be considered promising, given the smaller size of this trial and low power to detect effects on CVD. In the WHI, the 400 IU/d vitamin D3 intervention yielded no significant differences compared to placebo on coronary disease or stroke incidence [48,49].
Debate Regarding Adequate Levels of Vitamin D It is worth noting that controversy exists about the actual prevalence of deficient vitamin D levels in the USA: because of differences in 25(OH)D assay methods, population characteristics, and the limited research on vitamin D and extraskeletal health outcomes, there is no consensus on the cutoff values defining vitamin D insufficiency or deficiency [50]. Indeed, this is a knowledge gap highlighted in the recent report of the Institute of Medicine (IOM) on Vitamin D [50]. Nevertheless, vitamin D deficiency appears to be a common public health problem that has adverse consequences for bone health and possibly for extraskeletal outcomes
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such as cancer and heart disease, as discussed above [51,52]. Vitamin D deficiency is likely determined by many factors, such as increased age [53], female gender [54], darker skin pigmentation and race/ethnicity [54e61], winter season [62,63], geographic latitude (e.g., northern latitudes above the 37th parallel) [60], low intake of food and supplemental sources of vitamin D, especially during winter months [64], cultural factors (e.g., regular skin concealment) [52,65,66], and genetic predisposition [52]. Regardless of the thresholds at which vitamin D deficiency states are determined, the likelihood of deficient vitamin D levels in segments of the population necessarily raises important questions of how best to increase those levels. Vitamin D supplementation is clearly an attractive option, as it is inexpensive and much safer than the most obvious alternative e increasing unprotected sun exposure and, thus, risks of skin cancers. However, as is likely true for everything in biological systems, there appears to be an optimal level of vitamin D intake to meet the body’s needs. Indeed, recent data from the Framingham Offspring and NHANES cohorts suggest possible U- or reverse-J-shaped associations between vitamin D levels and CVD and mortality, such that risk is decreased at intermediateehigh levels, compared to low levels, but is increased again with the highest levels [45,47]. Such findings underscore the importance of a well-designed clinical trial in characterizing not only the health benefits of vitamin D but also the overall balance of benefits and risks.
RATIONALE FOR VITAL The limitations of existing observational and trial data clearly indicate that a large trial of vitamin D, dosed in amounts adequate to produce meaningful changes in serum 25(OH)D levels, is needed to assess its role in cancer and CVD prevention. First, the challenges in observational studies related to the vitamin D exposure assessment and residual confounding are all obviated by the randomized controlled trial design. Another benefit of VITAL is that the trial will test the independent effects of vitamin D, thus, disentangling vitamin D’s effects on disease risk from those of calcium or dairy intake: elevated calcium and high-fat dairy intakes may actually increase risk of some cancers, but both are highly correlated with vitamin D intake. Furthermore, the recent IOM report emphasized several major scientific gaps in our current understanding of vitamin D: the committee concluded that for extraskeletal outcomes, including cancer and CVD, current observational and trial evidence was inconclusive and insufficient to inform nutritional requirements [50]. VITAL is poised to address these gaps; hence, the timing of this study
is critical. As described earlier in this chapter and also noted by the IOM committee in its report, higher values of vitamin D are not consistently associated with greater benefit: for some outcomes, there appear to be increased risks at both low and high levels. The growing popularity and use of over-the-counter high-dose vitamin D supplements highlights the need for a definitive trial that rigorously tests both benefits and risks, and such a study must be conducted before uptake in the general public is so high that a randomized trial is no longer feasible. Finally, another key objective of VITAL as a large-scale trial is the recruitment of a diverse study population, with representation of ethnic and racial minority participants (including African-American participants, who are at especially increased risk of vitamin D deficiency yet often under-represented in clinical research) in sufficient numbers such that meaningful health effects of vitamin D can be detected in these special populations.
RATIONALE FOR THE SELECTED VITAMIN D DOSE IN VITAL The active vitamin D supplement in VITAL is 2000 IU (50 mg) per day of vitamin D3 (cholecalciferol). Careful investigation of dosing indicates that this amount of vitamin D3 will yield a large enough difference in total 25(OH)D status between the VITAL treatment and placebo groups to detect differences in primary outcomes. VITAL was designed when the Recommended Dietary Allowance (RDA) amounts were 400 IU/d for adults aged 50e70 years and 600 IU/d for adults aged >70 years [67]. Since then, the IOM committee charged with determining the North American population’s needs for vitamin D concluded in its 2011 report that RDAs of 600 IU/d for persons aged 1e70 years and 800 IU/d for adults aged 71 years and older e corresponding to a serum 25(OH)D level of at least 20 ng/ml (50 nmol/l) e would meet the requirements of at least 97.5% of the population [50]. However, in a review of studies of serum 25(OH)D in relation to a variety of health outcomes, including colorectal cancer, Bischoff-Ferrari et al. [68] found that advantageous 25(OH)D levels began at 75 nmol/l, and optimal levels were between 90 and 100 nmol/l. Furthermore, the average older adult requires oral vitamin D3 intake of at least 1000 IU/d to achieve a serum 25(OH)D of 75 nmol/l [69]. Finally, a recent study by Aloia et al. indicates that the doseeresponse for serum 25(OH)D with vitamin D intake is not linear, but that the rate of increase in serum levels decreases at higher levels of intake [70]. Extrapolation of these data in the VITAL trial population suggests that, with 2000 IU/d
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CORE ASPECTS OF TRIAL DESIGN
(50 mg/day), the 25(OH)D level would achieve a final mean of 80e90 nmol/l, which would be above the 75 nmol/l threshold level at which extraskeletal health benefits are believed to occur [68]. The delta in achieved 25(OH)D levels between the active treatment and placebo groups is expected to be 40e50 nmol/l. In addition to addressing the adequate dose for observing health benefits, there was careful consideration of any health risks, and few safety issues are associated with the vitamin D3 dose used in VITAL. Although potential side effects of vitamin D are rare, they may include gastrointestinal (GI) upset (presence or absence of symptoms of peptic ulcer, nausea, constipation, diarrhea) and physician diagnosis of hypercalcemia or kidney stones. Thus, to minimize the risks of hypercalcemia and kidney stones, VITAL will require that participants limit their total intake of calcium from all sources, including multivitamins, single supplements of calcium, and other drugs that contain calcium to 1200 mg or less a day. In addition, participants will be asked to consume no greater than 800 IU/d of vitamin D from all supplemental sources combined (individual vitamin D supplements, calcium þ vitamin D supplements, medications with vitamin D, and multivitamins) to avoid dilution of trial results and to ensure total supplemental vitamin D intake does not exceed the IOM’s current Tolerable Upper Intake Level of 4000 IU/d, which corresponds to the current safety limit (no-observed-adverse-effect
level (NOAEL)) set by the European Commission Scientific Committee on Food [71]. Of note, in addition to the goal of testing effects of vitamin D independent from those of calcium, supplemental calcium was deliberately not included as a component of the VITAL intervention for several reasons. First, to test the effects of vitamin D alone, calcium alone, and calcium-plus-vitamin D would require a factorial design with a much larger sample size and a much higher cost than the proposed trial. Second, combined calciumevitamin D supplementation was associated with a significantly increased risk of kidney stones in the WHI [38]. Third, a recent 5-year trial among 1471 initially healthy women (mean age 74) found an increased risk of MI (RR 2.12 (1.01e4.47)) and major CVD events (RR 1.47 (0.97e2.23)) with calcium supplementation (calcium citrate, 1 g/d) [72]. Fourth, the high prevalence of calcium supplement use in women [73] would reduce the pool of willing and eligible female participants since trial participants are required to limit total supplemental calcium intake to 1200 mg/d.
CORE ASPECTS OF TRIAL DESIGN The overall design scheme of VITAL is depicted in Fig. 105.2. VITAL will be conducted among 20 000 apparently healthy, community-dwelling participants.
The VITamin D and OmegA-3 TriaL (VITAL): Design 20000 Inially Healthy Men and Women (Men >60 years; Women >65 years)
Vitamin D3 (2000 IU/d) N=10000 EPA+DHA (1 g/d) N=5000
Placebo N=5000
Placebo N=10000 EPA+DHA (1 g/d) N=5000
Placebo N=5000
Mean Treatment Period = 5.0 years Blood collecon in ~12 000; follow -up bloods in 2000 Primary Outcomes: Cancer (total) and CVD (MI, stroke, CVD death) FIGURE 105.2 The VITamin D and OmegA-3 TriaL(VITAL): design.
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105. THE VITAMIN D AND OMEGA-3 TRIAL (VITAL): RATIONALE AND DESIGN
Participants will be men aged 60 and women aged 65 e ages at which rates of chronic disease increase substantially. The study features a 2 2 factorial design, and each of the active agents e vitamin D3 and fish oil (1 g/d omega-3 fatty acids as eicosapentaenoic acid (EPA) þ docosahexaenoic acid (DHA), in a 1:1 ratio) e has a matching inert placebo. Thus, 5000 participants will be randomized to each of the following conditions: active vitamin D and fish oil placebo; vitamin D placebo and active fish oil; both active vitamin D and fish oil; both vitamin D and fish oil placebos. Consequently, VITAL will be able to test the individual effects of vitamin D as well as joint effects (with fish oil) on the primary outcomes over a 5-year treatment and followup period. The omega-3 fatty acids were chosen as a second agent to test in VITAL due to their promising but inconclusive role in the primary prevention of CVD and cancer; the 2 2 factorial design allows for testing their independent and interactive effects with vitamin D at minimal incremental cost. Thus, VITAL will make a major contribution to knowledge on the role of both vitamin D and omega-3 fatty acid supplementation in cancer and CVD prevention, by addressing the following aims in an exceptionally cost-effective design:
Primary Aims 1. To test whether vitamin D3 reduces risk of (a) total cancer, (b) major CVD events (composite of MI, stroke, CVD death). 2. To test whether EPA þ DHA reduces risk of (a) total cancer, (b) major CVD events.
Secondary Aims 1. To test whether these agents lower risk of (a) colorectal cancer, (b) breast cancer, (c) prostate cancer, (d) total cancer mortality. 2. To test whether these agents lower risk of (a) secondary composite endpoint of major CVD event or revascularization, (b) individual components of CVD outcome.
Tertiary Aims 1. To explore whether vitamin D3 and EPA þ DHA have synergistic or additive effects on risk of (a) total cancer, (b) major CVD events, (c) secondary endpoints. 2. To explore whether effects of vitamin D3 or EPA þ DHA on cancer and CVD vary by (a) baseline blood levels of these nutrients, (b) race/ skin pigmentation (for vitamin D3), (c) BMI (for vitamin D3).
TABLE 105.1 Eligibility Criteria for VITAL (1) Aged 60 (men) or 65 (women) years (2) No history of cancer (except non-melanoma skin cancer), myocardial infarction (MI), stroke, transient ischemic attack (TIA), angina pectoris, coronary artery bypass grafting (CABG), or coronary angioplasty or intervention procedure (3) None of the following safety exclusions: use of anticoagulants at baseline, renal failure or dialysis, hypercalcemia, hypo- or hyperparathyroidism, severe liver disease, or sarcoidosis, tuberculosis, or other granulomatous diseases (4) No allergy to fish (5) No other serious illness that would preclude participation (6) Currently consuming no more than 800 IU/d of vitamin D from all supplemental sources combined, or, if taking, willing to decrease or forego such use during the trial (7) Currently consuming no more than 1200 mg/d of calcium from all supplemental sources combined, or, if taking, willing to decrease or forego such use during the trial (8) Not taking fish oil supplements, or, if taking, willing to forego their use during the trial
TRIAL ELIGIBILITY Detailed eligibility criteria for VITAL, including safety requirements, are provided in Table 105.1. Of note, some of the safety exclusions pertain to potential risks posed by the fish oil supplement (e.g., bleeding risk, fish allergy), not vitamin D.
RECRUITMENT, ENROLLMENT, RUN-IN AND RANDOMIZATION Not surprisingly, given the size of the undertaking, participant recruitment for VITAL will be accomplished in stages. The first step will involve initial recruitment using a master mailing tape containing over 2.5 million names and addresses that will be assembled from commercially available US mailing lists identifying likely participants (i.e., licensed health professionals, other professionals, AARP and seniors’ organization members), as well as subscription lists of magazines that appeal to professionals and college-educated persons. In addition, in order to enhance recruitment of African-American participants e who are at higher risk for vitamin D deficiency, as well as heart disease, hypertension, diabetes, stroke, and certain cancers e subscription lists will include magazines that are known to have high readership within the AfricanAmerican/black community and other targeted recruitment efforts will be pursued (goal is 25% cohort participation among African-Americans).
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DETAILED ASSESSMENT OF DIETARY AND SUPPLEMENT INTAKE IN VITAL
To advance to the enrollment phase, individuals from the mailing tape will be assigned a study identification number and mailed the following materials: • A letter that explains the rationale for VITAL, outlines what participation would entail, and provides sources for further information on relevant scientific issues. • An informed consent form. • A brief questionnaire with items on demographics (age, gender, race/ethnicity, education, occupation, income); medical history (cancer, CVD, kidney stones, hypercalcemia, kidney failure, sarcoidosis, other major illnesses); allergy to fish; current use of supplements containing vitamin D or fish oil; current use of other supplements or medications; dietary intake of vitamin D and consumption of fish; cancer and vascular risk factors (e.g., smoking, height, weight, blood pressure, cholesterol, diabetes, alcohol use, physical activity, and family history of cancer and CVD); and potential effect modifiers such as skin pigmentation and sunlight exposure. Changes in medications, supplement use, diet, and lifestyle factors over the course of the trial will be monitored. • A self-addressed, pre-paid envelope for returning study forms. Eligibility (as detailed in Table 105.1) will be determined among individuals who return responses to this enrollment inquiry. The next phase of cohort assembly e run-in period e is a crucial aspect of VITAL design, and the trial aims to enroll 40 000 persons in this phase. Decades of previous work in large-scale trials involving tens of thousands of participants [74e76] has demonstrated the success of a run-in period in identifying a group of excellent compliers for long-term follow-up [77]. Because the health experience of participants will be analyzed according to randomized treatment assignment, the use of a placebo run-in phase to exclude prior to randomization those who are likely to have poor study agent adherence will increase the study power [77]. Thus, a 3-month run-in phase will take place, during which all participants will take both placebo vitamin D and placebo fish oil. Advantages of using placebo during run-in are two-fold: as the effects of both agents on cancer prevention are likely to be chronic, it would not be scientifically appropriate to use active agent in the run-in and then randomize to placebo; the use of placebos will allow the clearest analysis of the side effects of the study agents during the randomized treatment period. To maximize ease for participants, each calendar pack will include a 31-day pill supply, with participants taking two pills per day. Once run-in is complete, the final phase of randomization will proceed. Participants will be randomized only if they: (1) demonstrate good
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compliance in pill taking, defined as taking at least two-thirds of the study pills during the run-in; (2) express willingness to continue in the trial; (3) report no new history of cancer (except non-melanoma skin cancer), cardiovascular disease, hypercalcemia, sarcoidosis, or other serious illness during the run-in; and (4) demonstrate continued willingness to limit supplemental vitamin D intake to 800 IU/d and to forego the use of fish oil supplements. Based on pilot work conducted among 5000 people prior to the establishment of VITAL, it is estimated that of the 40 000 initially willing and eligible individuals enrolled in the run-in, 50% (n ¼ 20 000) will be compliant and remain willing and eligible for randomization. Block randomization of willing and eligible participants will be conducted within 5-year age groups using a computer-generated table of random numbers. Within each age group, treatment assignments will be generated in blocks of eight individuals, with two individuals in each of the four treatment combinations. Of note, such use of age stratification during randomization will increase statistical efficiency and power.
CHARACTERISTICS OF PARTICIPANTS IN VITAL VITAL participants will be 20 000 apparently healthy individuals: 10 000 men aged 60 years and 10 000 women aged 65, ages at which rates of chronic disease increase substantially. In accordance with the eligibility criteria and safety exclusions (see Table 105.1), they will have no history of cancer (except non-melanoma skin cancer) or CVD (including MI, stroke, TIA, angina pectoris, or coronary revascularization procedures). Furthermore, pilot work demonstrated that high levels of ethnic and/or racial minority participation can be achieved. Based on the pilot results, with regard to ethnic distribution, VITAL will include 1400 (7%) Hispanic and 18 600 (93%) non-Hispanic participants. With regard to race, the cohort will include an anticipated 5000 (25%) African-American or black, 500 (2.5%) Asian, 400 (2%) American Indian, 80 (0.4%) Pacific Islander, and 14 020 (70.1%) white individuals (NB: ethnic and racial categories can overlap).
DETAILED ASSESSMENT OF DIETARY AND SUPPLEMENT INTAKE IN VITAL As trial participants will be using vitamin D3 in the context of background consumption of vitamin D from other sources, careful ascertainment of dietary intakes is essential. Classifying participants by baseline
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105. THE VITAMIN D AND OMEGA-3 TRIAL (VITAL): RATIONALE AND DESIGN
intake of various nutrients will allow trial investigators to evaluate whether effects of vitamin D vary by such intake. To achieve this, a validated, self-administered semi-quantitative food frequency questionnaire (FFQ) will be mailed to participants during the run-in (i.e., prior to randomization). This questionnaire, adapted from that developed by Dr. Walter Willett and colleagues, is an efficient, reliable, and accurate instrument for categorizing individuals according to their intake of 32 nutrients, including vitamin D [78e82]. Participants estimate their average intake over the past year of various foods, beverages, and supplements that contain vitamin D and many other nutrients. Dietary vitamin D will be determined from participants’ reported intakes of certain foods, including dairy products, fortified breakfast cereals, fortified juices, darkmeat fish, and cod liver oil; intake of vitamin D from food and supplements separately, and from both sources combined, will be calculated. A sample of participants will also complete the FFQ at 2 years and at the trial’s end, enabling an examination of dietary changes over time.
BLOOD COLLECTIONS IN VITAL A major goal of VITAL is to collect baseline blood samples from as many trial participants as are willing to provide them. The main reason for collecting prerandomization blood specimens is to allow an opportunity to assess whether treatment effects are modified by baseline blood levels of nutrient indicators e 25(OH)D for vitamin D and EPA þ DHA for fish oil. Thus, VITAL will be able to test whether reduction in cancer or CVD risk by long-term vitamin D supplementation is modified by insufficient or deficient levels of 25(OH)D at baseline. Follow-up fasting blood samples will be collected in trial year 2 and year 4 from a randomly selected subset of 2000 participants who provided a baseline fasting blood sample. Thus, it will be possible to assess repeated 25(OH)D levels in these participants according to active and placebo assignment. The follow-up samples will be collected using the same methods as for the baseline samples, and will facilitate assessment of: (1) pill-taking compliance, (2) changes in biomarkers with treatment, and (3) in the placebo group, the effect of changing trends in background fortification with vitamin D. The samples will be particularly important for an exploratory analysis of treatment-induced changes in blood 25(OH)D levels among black participants, as there are few data on this topic [83]. In addition, changes in blood calcium and parathyroid hormone (PTH) levels will be examined as markers of possible hypercalcemia, a potential side effect of high vitamin D intake.
From among those who return bloods, a sample for assays of baseline blood levels will be selected using a caseecohort design to maximize efficiency, while allowing the unbiased estimation of risk ratios as well as absolute risk for individuals [84]. A common subcohort sample can be used as a reference risk set for more than one outcome e in the case of VITAL, the primary outcomes of total cancer and total (composite) CVD. Over 5 years of follow-up, cancer and CVD cases will accrue, and two non-cases will be assigned to each case for each outcome. Control selection will be stratified by gender and baseline age (within 5-year age groups) to frequency-match the distribution in the total case group. The biochemical assays for blood levels of 25(OH)D, calcium and PTH will be performed by major laboratories with extensive experience in clinical chemistry and the conduct of these assays. The bloods collected in VITAL (from which plasma, serum, RBCs, and buffy coat will be extracted and stored) will be archived for future use in Human Subjects Research Committee-approved studies. Thus, in addition to the nested caseecohort studies as described above, the resource of a blood collection will continue to prove valuable for future ancillary studies exploring various genetic and biochemical hypotheses (e.g., VDR polymorphisms) in a well-characterized cohort of trial participants, and at very low incremental cost.
FOLLOW-UP AND ENPOINT DETERMINATION PROCEDURES The primary method of follow-up will be mailed questionnaires and review of medical records for confirmation of relevant clinical outcomes. At 6 months participants will be mailed a brief follow-up questionnaire, and at each anniversary date a more detailed questionnaire and a resupply of calendar packs. The questionnaire will include items on: compliance with randomized treatments; use of non-trial supplements of vitamin D and fish oil; use of calcium supplements; development of major illnesses, including the primary endpoints of cancer and CVD; dietary intakes of vitamin D and fish; updated lifestyle and medical risk factors related to cancer and CVD; and potential side effects of the study agents. For vitamin D, these side effects include GI symptoms (stomach upset or pain, nausea, constipation, diarrhea) and physician diagnosis of hypercalcemia or kidney stones. Non-responders will be mailed second and/or third requests, and will then be telephoned to collect study data; at the very least, vital status will be ascertained. Participants will be instructed to discontinue study pills if, during follow-up, they receive a diagnosis of
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TRIAL MONITORING AND SAFETY
hypercalcemia, sarcoidosis, or other safety-exclusion conditions. At 6-month intervals (i.e., between the annual followups), participants will be mailed an interim form with questions on the development of primary endpoints (cancer, MI, and stroke), difficulties with pill compliance, and address changes. The primary cancer endpoint of VITAL is total cancer incidence (excluding non-melanoma skin cancer); cancer mortality and the individual sites of colorectal, breast, and prostate cancer are secondary endpoints. For CVD, the primary endpoint is a composite endpoint of MI, stroke, and CVD mortality; secondary endpoints include a second composite endpoint adding revascularization procedures of CABG and PCI, as well as the individual endpoints of MI, stroke, revascularization, and CVD mortality. Participants who self-report one of the above study endpoints will be asked to sign a medical release form for hospital/physician/clinic records. After all records are obtained, an Endpoints Committee of physicians, blinded to the randomized treatment assignment, will review the file and, using a defined protocol, will confirm or disconfirm the case. A cancer diagnosis will be confirmed with histologic or cytologic evidence. In the absence of these diagnostic tests, strong clinical evidence accompanied by radiologic evidence or relevant laboratory markers will be used to confirm cancer occurrence. The histologic type, grade, and stage of cancer will also be recorded [85]. MI will be confirmed using Joint European Society of Cardiology/American College of Cardiology Foundation/American Heart Association/World Heart Federation (ESC/ACCF/ AHA/WHF) Task Force for the Redefinition of Myocardial Infarction criteria [86]. Stroke will be confirmed and categorized according to Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria [87]. Death due to a cardiovascular cause will be confirmed by convincing evidence of a CVD event from all available sources, including death certificates, hospital records, autopsy, and observer accounts (for deaths outside the hospital). For reported deaths, the validation process will be similar. Permission will be requested from families to obtain medical records and a copy of the death certificate. In lieu of a death certificate, a copy will be requested from the state vital records bureau where the participant died. The Endpoints Committee will review all records relevant to the death and assign an International Classification of Diseases (ICD) code. At the end of the trial the National Death Index Plus (NDI-Plus) database will be searched for known deaths for cases where records could not be obtained for participants who became lost to follow-up. Of note, the NDIPlus provides an ICD-coded cause of death based on death certificate information.
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MONITORING OF COMPLIANCE The primary measure of compliance will be the selfreported information provided on the annual followup questionnaires. Participants will be asked to provide information about adherence to the pill-taking regimen. Extensive data from prior, similar, large-scale clinical trials [74,75] indicates that this approach is both simple and highly effective: although most participants try diligently to adhere to the regimen, those who do not comply have no embarrassment about describing what they are actually doing. Thus, in similar past trials, blood levels have shown extremely strong correlations with self-reported questionnaire data on adherence [88]. In addition, at regular intervals, a random sample of local participants will be asked to provide on-thespot blood samples to be analyzed for 25(OH)D levels to directly assess compliance. In addition, at least 2000 participants will have follow-up blood collections 2e4 years post-randomization to assess changes in 25(OH)D levels in the treatment group, as well as changes in the placebo group that may be due to changes in background food fortification, diet, or supplementation practices.
TRIAL MONITORING AND SAFETY An independent Data and Safety Monitoring Board (DSMB), consisting of experts in clinical trials, epidemiology, biostatistics, relevant clinical areas of cancer and CVD, and National Institutes of Health (NIH) representatives, will monitor VITAL. The DSMB will annually examine the progress of the trial and the unblinded data on study endpoints and possible adverse effects to recommend continuation, alteration of study design, or early termination, as appropriate. Interim trial results will be assessed and guided by Haybittle-Peto rules that are appropriately conservative and require very strong evidence for stopping the trial [89,90]. Further, while monitoring rules will be applied for the primary endpoints, the goal of VITAL is to assess the overall balance of benefits and risks of the two agents in the primary prevention of cancer and CVD. Thus, consideration will also be given to the secondary endpoints that are needed in the interpretation of overall results. In addition, the monitoring rules will serve solely as guidelines in decisions regarding continuation or stopping of treatment arms. All decisions must be made after examining the totality of evidence, including other available trial data, on these agents. The study pills in VITAL have undergone extensive quality control testing for stability of nutrient content and other parameters at a range of temperatures and humidity levels.
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VITAL VALUE: ANCILLARY STUDIES AND ESTABLISHMENT OF A CLINICAL AND TRANSLATIONAL SCIENCE CENTER SUB-COHORT
failure; age-related cognitive decline; late-life depression; osteoporosis and fractures; asthma and respiratory diseases and infections; rheumatoid arthritis, systemic lupus erythematosus and other autoimmune diseases. Other ancillary studies in imaging are planned among participants who are available for inperson assessment: dual energy X-ray absorptiometry (DXA) scans to assess bone density and body composition; mammographic density, which is relevant in breast cancer risk; and non-invasive vascular imaging, which has potential applications in clarifying CVD risks and mechanisms. A final key component of VITAL is the establishment of a sub-cohort of 1000 participants who live in proximity to Clinical and Translational Science Centers (CTSCs). The CTSC sub-cohort of will be identified toward the end of the placebo run-in phase of VITAL. In addition to eligibility criteria for the main VITAL trial (Table 105.1), eligible CTSC participants must be in generally good health, mobile, and able to give informed consent. CTSC participants will be assessed both at baseline (pre-randomization) and 2 years later. At both visits, detailed assessments will be performed e providing basic health information from standard clinical assessments (e.g., medical histories and exams, height, weight, other anthropometric assessments, and blood pressure) as well as data on key measures related to the ancillary studies (e.g., glucose tolerance testing, physical performance batteries, lung function exams, cognitive assessments, and structured interviews for mood and other mental health disorders). The two CTSC visits will utilize the same protocol, and the scheduling of the second visit will be matched by month to the initial visit, so that the vitamin D assays will be performed in the same season. Importantly, this CTSC sub-cohort is expected to have the same diverse racial/ethnic composition as the overall VITAL cohort and will be randomized equally into the four treatment groups (i.e., 250 participants in each of the four factorial groups). Lastly, the 1000 participants will also have blood drawn at baseline and 2 years later (matched by season, by month). Pertaining to the ancillary studies, the specific aims of the CTSC component of VITAL are to test whether vitamin D3 and/or marine omega-3 fatty acid supplementation can:
VITAL will add value to its design and make major contributions to our understanding of the role of vitamin D in several other major health outcomes through the integration of ancillary studies. By coordinating with the design and infrastructure of VITAL, these studies will evaluate and test specific hypotheses regarding the role of vitamin D in preventing: diabetes and glucose intolerance; hypertension and heart
• Improve insulin sensitivity and b-cell function as assessed by 2-hour oral glucose tolerance testing (OGTT) in individuals without diabetes; lower hemoglobin A1c (HbA1c), fasting glucose and insulin; improve other derived indices such as the homeostasis model assessment of insulin resistance (HOMA-IR) and the homeostasis model assessment of b-cell function (HOMA-B);
STATISTICAL POWER Ensuring adequate statistical power is essential to the conduct of any trial, but particularly for one of this scale. Thus, extensive calculations were undertaken to determine whether the study, as designed, will have adequate power to test primary hypotheses. To calculate power, the following set of assumptions were made: (1) a 2 2 factorial trial with 10 000 men aged >60 years and 10 000 women aged >65 years at baseline; (2) independent and equal allocation of participants to each treatment arm (i.e., from the randomization scheme); (3) an age distribution based on that observed at baseline in past large-scale trials involving men aged 60 and women aged 65 years; (4) age-specific cancer and CVD event rates, also based on observed rates in the first 5 years of trial follow-up among participants in similar trials; (5) follow-up length of 5 years, with the minimal loss to follow-up as achieved in past trials; and (6) compliance levels (>80%) similar to that observed in previous similar trials and upon which the estimated rate ratio (RR) reductions in VITAL are based. Thus, in the power calculation the observed RR reductions are the effects as would actually be observed in the trial, assuming an average compliance of 80%. Power was calculated for a two-sided test with a significance (a) level of 0.05. Assuming that only vitamin D is effective (i.e., assuming that omega-3s have no effect), with 5 years of treatment and follow-up there will be 91% power to detect an observed RR of 0.85 for the primary endpoint of total cancer incidence and 92% power to detect an observed RR of 0.80 for the primary composite endpoint of MI, stroke, and cardiovascular mortality (99% power for the expanded CVD outcome that includes coronary revascularizations). For site-specific cancers and individual CVD outcomes, the trial will be powered to detect 25e30% reductions in risk. If the vitamin D and omega-3 interventions interact, power will be affected to the extent of the interaction.
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REFERENCES
• Reduce airflow obstruction and decline of lung function, chronic obstructive pulmonary disease (COPD) and asthma exacerbations, and risk for pneumonia; • Reduce risk of late-life depression and level of depressive symptoms among individuals at high risk for depression; reduce risk for major depressive disorder and level of depressive symptoms in individuals with subsyndromal depressive symptoms; • Reduce risk of age-related cognitive decline; • Reduce bone loss in the spine, hip and total body as assessed by dual X-ray absorptiometry (DXA); • Improve physical performance as assessed by grip strength, timed chair stands, and timed walk; • Reduce blood pressure (systolic and diastolic) and indices of adiposity (body mass index (BMI), waist circumference, waist-to-hip ratio); • Reduce the frequency of infection; • Reduce risk of autoimmune disorders. A built-in advantage of the CTSC component is the ability to conduct in-person validations of remote assessment methods that are utilized in the larger trial. For example, the in-person assessments of cognitive function will be used to validate the telephone-based testing in the cognitive function ancillary study. Similarly, the inperson structured diagnostic interviews will be used to validate the clinical depression cases in the late-life depression ancillary. Finally, because the main VITAL trial is being conducted via mailings and record requests, the CTSC visits will represent a valuable opportunity for direct contact with a subset of the study participants.
KEY POINTS • VITAL (VITamin D and OmegA-3 TriaL) is a largescale randomized controlled trial of vitamin D for primary prevention of cancer and cardiovascular disease. • This trial provides for sufficient dosing (2000 IU/d) and trial duration (5 years) for convincing hypothesis testing of health effects of vitamin D. • VITAL has numerous added value features, including blood collections to assess biomarkers, key ancillary outcome studies, and an in-person Clinical and Translational Science Center assessment component.
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[20] Y.C. Li, J. Kong, M. Wei, Z.F. Chen, S.Q. Liu, L.P. Cao, 1,25Dihydroxyvitamin D(3) is a negative endocrine regulator of the renineangiotensin system, J. Clin. Invest. 110 (2002) 229e238. [21] P.M. Timms, N. Mannan, G.A. Hitman, K. Noonan, P.G. Mills, D. Syndercombe-Court, et al., Circulating MMP9, vitamin D and variation in the TIMP-1 response with VDR genotype: mechanisms for inflammatory damage in chronic disorders? Q. J. Med. 95 (2002) 787e796. [22] K.T. Weber, R.U. Simpson, L.D. Carbone, Vitamin D and calcium dyshomoeostasis-associated heart failure, Heart 94 (2008) 540e541. [23] W.B. Grant, An estimate of premature cancer mortality in the U.S. due to inadequate doses of solar ultraviolet-B radiation, Cancer 94 (2002) 1867e1875. [24] W.B. Grant, Ecologic studies of solar UV-B radiation and cancer mortality rates, Recent Results Cancer Res. 164 (2003) 371e377. [25] T. Mizoue, Ecological study of solar radiation and cancer mortality in Japan, Health Phys. 87 (2004) 532e538. [26] E. Giovannucci, Y. Liu, E.B. Rimm, B.W. Hollis, C.S. Fuchs, M.J. Stampfer, et al., Prospective study of predictors of vitamin D status and cancer incidence and mortality in men, J. Natl. Cancer Inst. 98 (2006) 451e459. [27] D.M. Freedman, A.C. Looker, S.C. Chang, B.I. Graubard, Prospective study of serum vitamin D and cancer mortality in the United States, J. Natl. Cancer Inst. 99 (2007) 1594e1602. [28] D.H. Pilz S, B. Winklhofer-Roob, G. Riedmuller, J.E. Fischer, U. Seelhorst, B. Wellnitz, et al., Low serum levels of 25hydroxyvitamin D predict fatal cancer in patients referred to coronary angiography, Cancer Epidemiol. Biomarkers Prev. 17 (2008) 1228e1233. [29] Kj Helzlsouer, VDPP Steering Committee, Overview of the cohort consortium vitamin D pooling project of rarer cancers, Am. J. Epidemiol. 172 (2010) 4e9. [30] R.Z. Stolzenberg-Solomon, E.J. Jacobs, A.A. Arslan, D. Qi, A.V. Patel, K.J. Helzlsouer, et al., Circulating 25-hydroxyvitamin D and risk of pancreatic cancer: cohort consortium vitamin D pooling project of rarer cancers, Am. J. Epidemiol. 172 (2010) 81e93. [31] L. Yin, N. Grandi, E. Raum, U. Haug, V. Arndt, H. Brenner, Metaanalysis: longitudinal studies of serum vitamin D and colorectal cancer risk, Aliment. Pharmacol. Ther. 30 (2009) 113e125. [32] M. Jenab, H.B. Bueno-de-Mesquita, P. Ferrari, F.J. van Duijnhoven, T. Norat, T. Pischon, et al., Association between prediagnostic circulating vitamin D concentration and risk of colorectal cancer in European populations: a nested casee control study, BMJ 340 (2010) b5500. [33] M.L. Slattery, S.L. Neuhausen, M. Hoffman, B. Caan, K. Curtin, K.N. Ma, et al., Dietary calcium, vitamin D, VDR genotypes and colorectal cancer, Int. J. Cancer 111 (2004) 750e756. [34] M.L. Slattery, C. Sweeney, M. Murtaugh, K.N. Ma, B.J. Caan, J.D. Potter, et al., Associations between vitamin D, vitamin D receptor gene and the androgen receptor gene with colon and rectal cancer, Int. J. Cancer 118 (2006) 3140e3146. [35] M.L. Slattery, Vitamin D receptor gene (VDR) associations with cancer, Nutr. Rev. 65 (2007) S102eS104. [36] C.D. Toner, C.D. Davis, J.A. Milner, The vitamin D and cancer conundrum: aiming at a moving target, J. Am. Diet Assoc. 110 (2010) 1492e1500. [37] P. Autier, S. Gandini, Vitamin D supplementation and total mortality: a meta-analysis of randomized controlled trials, Arch. Intern. Med. 167 (2007) 1730e1737. [38] J. Wactawski-Wende, J.M. Kotchen, G.L. Anderson, A.R. Assaf, R.L. Brunner, M.J. O’Sullivan, et al., Calcium plus vitamin D supplementation and the risk of colorectal cancer, N. Engl. J. Med. 354 (2006) 684e696.
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XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
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XII. THERAPEUTIC APPLICATIONS AND NEW ADVANCES
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Index
Acidemia, radiographic findings, 880 Acne, vitamin D therapy, 1898 Actin, vitamin D binding protein complex, 65e66 Actinic keratosis, vitamin D therapy, 1898 Activator protein-1 (AP-1), cell differentiation induction via vitamin D, 1631 AD, see Alzheimer’s disease ADAM17, vitamin D renoprotective actions, 1342e1345 Adipocyte vitamin D effects, 770e773 vitamin D sequestration, 1019 Adolescent asthma and vitamin D status, 671 calcium absorption enhancers, 653e654 retention markers, 667 diabetes and vitamin D status, 671 influenza and vitamin D status, 671 pubertal bone acquisition, 657e659 vitamin D deficiency prevalence, 659e660 intake recommendations, 671e672 intervention trials bone response, 667e669 muscle response, 669 status influences age and pubertal maturation, 662 latitude and season, 661e662 obesity, 662 race and socioeconomic status, 662 sex differences, 662, 669e671 status markers bone turnover markers, 666 1a,25-dihydroxyvitamin D, 663e664 fractional calcium absorption, 664e665 25-hydroxyvitamin D, 659 intact parathyroid hormone, 664 AF-2 mutation, 239 vitamin D receptor activation, 105, 108, 110, 148, 151 AFM, see Atomic force microscopy Aging, see also Elderly; Falls; Fracture klotho studies, 754 metabolic bone disease, 807 vitamin D receptor as longevity nuclear receptor, 160e161 vitamin D synthesis effects, 16
Akt cell differentiation induction via vitamin D, 1628e1629 leukemia signaling and vitamin D effects, 1739 photoprotection mechanisms of vitamin D compounds, 1948 Alcohol alcoholism cirrhosis and bone loss clinical features, 1312 epidemiology, 1311e1312 pathogenesis, 1312e1313 treatment, 1313 hypophosphatemia, 1175 vitamin D metabolism effects, 1259e1260 Aldosterone, see Renineangiotensin system Alfacalcidol chemical synthesis, 1490e1491 history of development, 1489e1490 multiple sclerosis treatment, 1865 osteoporosis management, 1491e1495 Alien, vitamin D receptor repression, 202e203 Alkaline phosphatase, vitamin D effects in bone, 416 Alkalosis, hypophosphatemia, 1174 Allergic rhinitis, vitamin D studies, 2013 All-trans retinoic acid (ATRA), leukemia treatment with vitamin D combination therapy, 1739e1740 Alopecia hereditary vitamin-D resistant rickets association, 1200, 1223e1224 rickets association, 814 Aluminum toxicity, radiographic findings, 874e875 Alzheimer’s disease (AD), vitamin D studies, 575 5-Aminosalicylic acid (5-ASA), inflammatory bowel disease management, 1885 Analogs, see Vitamin D analogs Anaphylaxis, vitamin D studies, 2012e2013 Angiogenesis inhibition by vitamin D in cancer, 1595e1597 prostate cancer and vitamin D inhibition, 1688e1689 vitamin D receptor functions, 543e544 Angiotensin, see Renineangiotensin system Animal feed biofortification, 999 vitamin D intake recommendations, 87e88
2057
Anticonvulsants, vitamin D metabolism effects, 1257e1258 Antimicrobial peptides, vitamin D effects on expression, 1781e1783, 1813e1814 AP-1, see Activator protein-1 Apoptosis breast cancer cells, 1658e1659 calbindin D function, 369e370 cancer and vitamin D, 1602e1603 leukemia, 1737 osteoclast, 339 prostate cancer, 1685 Arsenic trioxide, leukemia treatment with vitamin D combination therapy, 1740 Arteriosclerosis calcium-sensing receptor defects, 1417e1418 vitamin D calcium, phosphate, and vitamin D excess effects, 1141 fibroblast growth factor-23/klotho/ vitamin D axis, 1413e1414 intervention studies animal models, 549e550 humans, 1408e1409 overview of actions, 1406e1408 parathyroid hormone receptor signaling, 1414e1415 toxicity and ectopic calcification, 590 toxicity studies, 1409e1411, 1415e1416 vitamin D analog risks, 1418e1419 Arthritis, see Osteoarthritis; Psoriatic arthritis; Rheumatoid arthritis Arthroplasty, see Orthopedics 5-ASA, see 5-Aminosalicylic acid Association studies candidate gene association studies, 1027e1028 genome-wide association studies, 1028e1029 overview, 1027 Asthma adolescent vitamin D status, 671 clinical features, 2001e2002 control, 2008e2010 intervention trials of vitamin D in acute respiratory infection, 2005e2006 observational studies of vitamin D, 2004 pathogenesis, 2006e2008 prospects for study, 2014 vitamin D deficiency during pregnancy and late effects on child, 687
2058 Atherosclerosis endothelial dysfunction, 1975e1976 optimal vitamin D status, 1079e1080 vitamin D intervention studies, 555e556, 1408e1409 observational studies, 555 overview of actions, 1406e1408 Vitamin D and Omega-3 Trial background, 2045 Atomic force microscopy (AFM), mineralization analysis, 387 Atopic dermatitis, vitamin D studies, 2012e2014 ATRA, see All-trans retinoic acid AUF-1, 430, 500 Autoimmune disease, see specific diseases Autoimmune polyglandular syndrome type 1, hypocalcemia, 1095 Autophagy breast cancer, 1658e1659 innate immunity, 1815 BAC, see Bacterial artificial chromosome Bacterial artificial chromosome (BAC), vitamin D receptor clones bone cell activity, 123 transgenes in mice, 123e124 Bariatric surgery, see Obesity Basal cell carcinoma, see Skin cancer B cell CYP27B1/vitamin D receptor knockout studies, 594e596 vitamin D receptor agonist effects, 1798e1799 Benign prostatic hyperplasia (BPH) epidemiology, 1931 medical treatment, 1933 pathogenesis, 1931e1933 vitamin D receptor agonist studies inflammation inhibition, 1935e1936 lower urinary tract symptoms, 1931e1933 overview, 1803 prospects for study, 1937e1938 prostate cell growth control, 1933e1934 urethra dysfunction, 1936e1937 Beta cell, see Diabetes Bile acids, see also Lithocholic acid derivatives metabolism and regulation by nuclear receptors, 1511e1514 pathophysiology, 777 structures, 1510 synthesis and metabolism, 760, 1511e1512 types, 775 vitamin A effects on synthesis, 764e765 vitamin D receptor bile acid synthesis regulation, 765e766 binding, 766 detoxification role, 765e766 interaction overview, 1509e1511 Biliopancreatic diversion, see Obesity Bisphosphonates
INDEX
glucocorticoid-induced osteoporosis therapy, 1240e1241 vitamin D metabolism effects, 1262 BL269, 1475 BL314, 1475 BL562, 1475 Bladder cancer, vitamin D studies, 1765e1766 BMD, see Bone mineral density BMPs, see Bone morphogenetic proteins Body size, vitamin D synthesis effects, 986e987 Bolus dosing, vitamin D, 1001e1002 Bone, see also Collagen; Fracture; Mineralization; Oral skeleton; Osteoblast; Osteoclast; Osteocyte; Rickets; Runx2 bariatric surgery effects, 1016e1018 biopsy, see Bone histomorphometry calbindin D function, 366e367 calcium-sensing receptor function, 437e438 b-cateninevitamin D receptor interactions, 156, 243e244 chromatin remodeling, 311e313 CYP27B1 function, 412e413, 587 epigenetic mechanism for lineage commitment, 309e310 fetal development, 626 fibroblast growth factor-23 coordination of renal phosphate handling with bone mineralization and turnover, 757e758 formation endochondral bone formation in development, 301e303 nuclear organization of regulatory machinery, 306e308 signaling in formation bone morphogenetic proteins, 304 fibroblast growth factors, 304 Wnt, 304e305 gene expression within context of nuclear architecture, 309e311 gene transactivation by vitamin D receptor, 113, 117e118 maternal vitamin D deficiency effects on child later in life, 687 mechanical load response, 325e326 metastasis and chemokines in osteoclast regulation, 340e341 osteoarthritis changes, 1956e1957 parathyroid hormone function, 730e732 parathyroid hormone-related protein function, 734e735 pregnancy and lactation effects, 682e684 pubertal bone acquisition, 657e658 remodeling, 323e325, 813 scaffolding of regulatory components for combinatorial control of gene expression, 313e315 turnover, 335 turnover markers as vitamin D status markers in adolescence, 666 vitamin D
metabolism, 411e413 sources, 412e413 vitamin D receptor bacterial artificial chromosome clones studies, 123 function, 437e438 Wnt in homeostasis, 242e243 Bone histomorphometry biopsy adverse effects, 846 indications, 846 technique, 845e846 bone turnover analysis activation frequency, 852 formation rates, 852 mineralizing perimeter, 852 remodeling periods, 853 limitations, 848 methodology, 846e847 mineralization assessment adjusted apposition rate, 850 mineral apposition rate, 849e850 mineralization lag time, 850 osteoid maturation time, 850 osteoid measurement, 849 tetracycline labeling, 848e849 osteomalacia focal osteomalacia, 852 generalized osteomalacia, 851e852 prospects, 857 referents, 847 remodeling balance assessment formation, 853e854 resorption, 854e855 indices, 846 structural assessment determinants of bone strength, 855 three-dimensional approaches, 856 two-dimensional approaches, 856 terminology, 847e848 theory, 846 Bone mineral density (BMD) genome-wide association studies, 1034e1035 glucocorticoid-induced osteoporosis and vitamin D response, 1238e1241 idiopathic hypercalciuria and reduction, 1363e1365 measurement, see Dual-energy X-ray absorptiometry; Quantitative computed tomography optimal vitamin D status, 1073 osteoporosis effects of calcium and vitamin D supplementation, 1133e1134 pubertal bone acquisition, 657e658, 660e661 Bone morphogenetic proteins (BMPs), bone formation signaling, 304 Bone scinitigraphy metabolic bone disease diagnosis, 817 overview, 883 Bone sialoprotein (BSP), mineralization role and vitamin D effects, 392e393, 416e417
INDEX
Bone volume, CYP27B1 knockout studies, 587 BPH, see Benign prostatic hyperplasia Brain calbindin D function, 369e370 developmental vitamin D deficiency behavioral effects in later life, 568e569 dietary restriction, 567e568 gene expression effects in later life, 568 structural alterations, 576 inflammation, 571 knockout mouse studies of vitamin D system, 569e570 neurological and psychiatric disorder studies of vitamin D, 570e575 vitamin D metabolites, 566 vitamin D receptor developmental expression, 567 distribution, 566 non-neuronal cells, 566e567 BRCA1, vitamin D induction, 1661 Breast, see Breast cancer; Lactation; Mammary gland Breast cancer cell culture effects of vitamin D apoptosis and autophagy, 1658e1659 overview, 1658 target genes and pathways angiogenesis, invasion and metastasis, 1662 estrogen metabolism and signaling, 1659e1660 growth factor signaling, 1660 inflammation pathways, 1660 miscellaneous genes and pathways, 1661e1662 stress pathways, 1660e1661 CYP27B1/vitamin D receptor knockout studies, 590e591 epidemiological studies of vitamin D status, 1667e1668 estrogen in pathogenesis, 1657 intervention trials with vitamin D and analogs, 1665 prevention with vitamin D, 1667 prospects for study, 1668 vitamin D intake and risk analysis, 1572e1578 vitamin D sensitivity determinants ligand availability and metabolism, 1662e1663 vitamin D receptor expression and regulation, 1663 polymorphisms and risk, 1668 prognostic significance, 1664 resistance, 1664 Brown tumor, radiographic findings, 870e871 BSP, see Bone sialoprotein Burn injury, hypophosphatemia, 1175 BXL-628, 1439 BXL0124, 1439 Caffeine, 1267 Calbindin D
apoptosis role, 369e370 calcium absorption role, 353 discovery, 363 forms, 363 functions bone, 366e367 intestine, 364e366 kidney, 366, 478e480 nervous tissue, 368e369 pancreas, 367e368 reproductive tissues, 368 genes calbindin-D9k genomic organization, 371 regulation by miscellaneous steroids, 371e372 vitamin D regulation, 371 calbindin-D28k genomic organization, 370 regulation by miscellaneous steroids, 370e371 vitamin D regulation, 370 knockout mice, 353, 365e366 prospects for study, 373e374 renal function, 366, 478e480 structure, 364e365 tissue distribution, 373 Calcipotriol cancer studies, 1438e1439 leukemia studies, 1741 modification for receptor antagonism, 184e186 psoriasis management, 1438 Calcitonin calcium-sensing receptor effects, 433 clinical applications, 739e740 CYP24A1 expression regulation, 1531 functional overview, 739 gene, 738 history of study, 737e738 pathophysiology, 739 receptor, 738 secretion regulation, 738e739 vitamin D metabolism effects, 1249e1250 Calcium, see also Hypercalcemia; Hypercalciuria; Hypocalcemia absorption, see Calcium absorption body compartments, 607e609 channel blockers and vitamin D metabolism effects, 1263 channels, see L-type calcium channels; Voltage-sensitive calcium channels colorectal cancer effects of intake, 1719e1721 experimental autoimmune encephalitis prevention, 1856e1857 extracellular fluid concentration regulation bone mineralization, 609 deficiency, 611 diet, 611e612 obligatory loss, 609e610 parathyroid hormone independent mechanisms, 612 mediated response, 610e611 fetal metabolism, 625e627
2059 hypocalcemia and dietary deficiency, 1099 influx and adipocyte metabolism, 769 intake developing countries, 652 food fortification, 653 recommendations in infants and children, 649e650 recommendations infants, 649e650 infants and children, 649e650 toddlers, 650e651 neuroprotective actions of vitamin D, 572 osteoporosis studies of supplementation bone mineral density effects, 1133e1134 fracture prevention calcium and vitamin D, 1137 meta-analysis, 1138e1139 safety of supplements, 1139 single annual dosing, 1136 secondary prevention, 1139e1140 parathyroid hormone expression regulation, 498e499 pregnancy and lactation effects on metabolism, 679e682 prostate cancer intake studies, 972e973 retention markers in adolescence, 66 rickets and deficiency, 1118e1120 Calcium absorption bariatric surgery effects, 1019e1021 CYP27B1 in intestine, 585 dietary calcium intake as regulator of efficiency, 350, 614e617 enhancers for children and adolescents, 653e654 fetal, 629e630 fractional calcium absorption as vitamin D status marker in adolescence, 664e665 glucocorticoid-induced osteoporosis, 1238 25-hydroxyvitamin D role, 620 idiopathic hypercalciuria intestinal increase, 1360e1362 renal reabsorption decrease, 1362e1363 infants, 650 kinetics, 349 location and timing in gut, 614 models active versus passive mechanisms, 617e618 facilitated diffusion, 352e353 paracellular movement through tight junctions, 355 transcaltachia, 354e355 vesicular transport, 353e354 optimal vitamin D status, 1071e1073 overview, 349e350, 1299e1300 premature infants, 648 prospects for study, 355 race effects, 651e652 renal tubular reabsorption calcium-sensing receptor regulation, 434e435 CYP27B1 role, 585e586 overview, 471e472 vitamin D effects, 473e475
2060 Calcium absorption (Continued ) sex and race differences, 669e670 vitamin D receptor control, 351 vitamin D status effects, 350e352 Calcium-sensing receptor (CaSR) calcimimetic activators for hyperparathyroidism treatment, 440e443 calcium binding, 426e427 calcium homeostasis maintenance hypercalcemia defense, 429 mechanisms, 429e430 overview, 425, 427e428 cardiovascular disease defects, 1417e1418 colon cancer, 444e446 expression regulation, 427 hypocalcemia gene mutations, 1095 keratinocyte function, 444 knockout mouse, 429, 433, 526 parathyroid hormone secretion regulation, 727e728 polymorphisms, 446e447 prospects for study, 447e449 prostate cancer, 446 renin secretion regulation, 443e444 structure, 426 tissue distribution and function bone, 437e439 breast, 440 cartilage, 435e437 intestine, 439 kidney, 433e435, 475e478 parathyroid gland, 430e433 placenta, 439e440 Calreticulin, parathyroid hormone expression regulation, 496 CAMP, see Cathelicidin antimicrobial peptide Cancer, see also Metastasis; specific cancers angiogenesis inhibition by vitamin D, 1595e1597 cell cycle effects of vitamin D breast cancer, 1658 cell-type specificity of inhibition, 1642e1643 G1/S block c-Myc downregulation, 1641e1642 p21 upregulation, 1637e1640 p27 upregulation, 1637e1640 retinoblastoma protein regulation, 1640e1641 G2/M retardation and polyploidization, 1642 overview, 1598e1602 prostate cancer, 1683 cellular effects of vitamin D apoptosis, 1602e1603 cell cycle, 1598e1602 differentiation, 1603e1604 growth factors and receptors, 1604e1605 proliferation inhibition and stimulation D, 1594, 1608e1609 clinical trials of vitamin D, 1594e1595 combination therapy with vitamin D, 1605e1606
INDEX
CYP24A1 dysregulation, 1535e1536 epidemiology of vitamin D and risk breast cancer, 1575e1576 colon cancer, 1572e1574 esophageal cancer, 1577 intake studies, 1571 miscellaneous cancers, 1576e1577 mortality/survival impact, 1581e1584 ovarian cancer, 1576e1577 overview, 1569, 1592e1593 predicted 25-hydroxyvitamin D levels, 1571e1572 prospects for study, 1584e1586 prostate cancer, 1574e1575 study design prospective studies, 1570e1571 randomized trials, 1570, 1580e1581 sun exposure caseecontrol studies, 1578e1580 ecological studies of regional exposure, 1578 surrogate of vitamin D status, 1572 total cancer risk, 1577 extrarenal vitamin D synthesis, 779 growth inhibition by vitamin D, 1594, 1608 knockout mice of vitamin D system studies, 590e593 metastasis inhibition by vitamin D, 1595e1597 oncogenes, 1599 optimal vitamin D status, 1077e1078 parathyroid hormone-related protein levels, 1597e1598 tumor suppressor genes, 1599, 1601e1602 vitamin D analog studies, 1437 Vitamin D and Omega-3 Trial background, 2044e2045 vitamin D receptor functional overview, 1591e1592 polymorphisms, 1593e1594 resistance and vitamin D metabolism, 1606e1608 Wnt signaling, 235e237, 238e241 Candidate gene association studies, 1027e1028 vitamin D binding protein, 1032e1034 vitamin D receptor, 1031e1032 CAR, see Constitutive androstane receptor Cardiac fibrosis, see Heart Cardiac hypertrophy, see Heart Cardiomyocyte, vitamin D function, 1976 Cartilage, see also Chondrocyte calcium-sensing receptor function, 435e437 CYP27B1 function, 587e588 mineralization, 384e385 osteoarthritis homeostasis and vitamin D, 1955e1956 properties, 507 vitamin D regulation 1,25-dihydroxyvitamin D3 function, 509e511 24,25-dihydroxyvitamin D3 function, 509e511 overview, 507e509 prospects for study, 515e516
rapid actions definition, 511 membrane signaling, 512e513 models, 511e512 physiological relevance of nongenomic regulation of matrix vesicles, 513e515 CaSR, see Calcium-sensing receptor Catechol-o-methyltransferase (COMT), developmental vitamin D deficiency effects in adult brain expression, 568 b-Catenin keratinocyte stem cell function, 536e537 oncogenic activity, 156 osteoblast signaling, 243e244 signaling via Wnt, 235e237 skin signaling, 244e245 vitamin D inhibition, 238e241 vitamin D receptor interactions bone, 156, 243e244 colon, 114, 156, 239 keratinocytes, 156e158 Cathelicidin antimicrobial peptide (CAMP) respiratory disease, 2011e2012 vitamin D effects on expression, 1781e1783, 2012 Cav, see Voltage-sensitive calcium channels Caveolin vitamin D effects, 2032 vitamin D plasma membrane receptor interactions, 283e284 CBP, see CREB-binding protein CC, see Chief complaint CCNC, vitamin D regulation of expression, 219 CD14 breast cancer expression, 1660 vitamin D effects on expression, 1780e1781 CD483, 1480 CD503, 1480 CD504, 1480 CD509, 1480 CD4409, 1506 CD4420, 1499 CD4420, 1506 CD4528, 1506 Cdks, see Cyclin-dependent kinases CDNK1A, see p21 CDNK1B, see p27 CDP, vitamin D receptor interactions, 201 C/EBP cell differentiation induction via vitamin D, 1631 CYP24A1 expression regulation, 1531 Celiac disease, bone loss clinical features, 1305 epidemiology, 1304e1305 pathogenesis, 1305 treatment, 1305 Cell cycle checkpoints, 1631e1632 driving mechanisms, 1632e1633 leukemia, 1736e1737 regulation
INDEX
DNA replication licensing control, 1635e1636 G1/S phase, 1633e1635 G2/M phase, 1636e1637 vitamin D effects in cancer breast cancer, 1658 cell-type specificity of inhibition, 1642e1643 G1/S block c-Myc downregulation, 1641e1642 p21 upregulation, 1637e1640 p27 upregulation, 1637e1640 retinoblastoma protein regulation, 1640e1641 G2/M retardation and polyploidization, 1642 overview, 1598e1602 prostate cancer, 1684 vitamin D receptor targets, 219 Cell differentiation keratinocyte, 533e534, 537e538, 594 leukemia, 1736 osteoblast, 305e306 osteoclast, 338e339 osteocyte, 327e328 vitamin D effects initial signals, 1626, 1628 c-Myc downregulation, 1641e1642 osteoblasts cell culture studies, 326e327 direct actions, 327e328, 414e417 transcriptional control, 305e306 voltage-sensitive calcium channels, 462e463 overview in cancer, 1603e1604 principal models in cancer, 1625e1626 prostate cancer, 1685e1686 signaling Akt, 1628e1629 mitogen-activated protein kinase, 1629e1630 p35, 1630e1631 protein kinase C, 1628 transcription factors activator protein-1, 1631 C/EBPb, 1631 Sp1, 1631 CF, see Cystic fibrosis CH5036249, 1483 Chemiluminescence assay, 25-hydroxyvitamin D, 829 Chemotherapy, hyperphosphatemia induction, 1176 Chief complaint (CC), metabolic bone disease, 809 Children calcium absorption enhancers, 653e654 calcium intake recommendations, 649e651 food fortification with calcium and vitamin D, 653 pseudo-vitamin D deficiency management, 1192e1193 soda intake effects on bone health, 652 vitamin D deficiency prevalence, 659e660
intake recommendations, 649e650 status influences age and pubertal maturation, 662 latitude and season, 661e662 obesity, 662 race and socioeconomic status, 661e662 sex differences, 662 ChIP, see Chromatin immunoprecipitation Cholecalciferol, see 1a,25-Dihydroxyvitamin D3 Chondrocyte CYP24A1 in maturation, 48 CYP27B1 function, 589e590 origins, 321 vitamin D receptor function, 436e437 Chromatin osteocalcin gene organization, 312e313 remodeling in bone, 311e313 Chromatin immunoprecipitation (ChIP) vitamin D receptor studies, 111e115, 199, 214, 220 vitamin D response element studies, 218 Chromatin-looping model, vitamin D receptor transactivation, 152e153 Chromatin unit, structure, 214 Chronic kidney disease (CKD) bone effects, 1340 calcium-sensing receptor calcimimetic activators for treatment of uremic hyperparathyroidism, 441e442 secondary hyperparathyroidism, 442e443 cardiovascular disease and vitamin D status, 1989e1990 CYP24A1 activity, 49e50 CYP24A1 dysregulation, 1537e1539 CYP27B1 direct inhibition, 1330 extrarenal enzyme alterations, 1331e1333 induction suppression, 1328e1330 overview of studies, 32e33 substrate availability, 1328 epidemiology, 716 intestine effects, 1338 parathyroid gland effects, 502, 1338e1340 peritonitis, 781 renal mass reduction, 1327e1328 renineangiotensin system in pathophysiology, 717e718 vitamin D analog therapy analog types, 1556 animal model studies, 1556e1559 cardiovascular disease outcomes, 1563 end-stage renal disease, 1560e1563 prospects for study, 1563e1564 stage 3-4 disease, 1559e1560 survival impact, 1561e1563 vitamin D catabolism alterations, 1330e1331 intervention studies, 557 observational studies, 556e557 synthesis defects, 1325e1333 therapy, 718, 1346e1348
2061 vitamin D deficiency overview, 717, 1342 kidney transplantation post-transplantation, 1294 pre-transplantation, 1292e1293 vitamin D receptor alterations homologous upregulation defects, 1333e1335 miscellaneous resistance mechanisms, 1335e1337 polymorphisms, 1335 Chronic obstructive pulmonary disease (COPD) acute exacerbations, 2010e2011 clinical features, 2002 pathogenesis, 2010 Chugaev reaction, 87 Chvostek’s sign, 813 Cimetidine, vitamin D metabolism effects, 1264 Cip1, see p21 Cirrhosis, see Alcohol; Primary biliary cirrhosis CKD, see Chronic kidney disease CLDN14, idiopathic hypercalciuria defects, 1364, 1366 Clinical and Translational Science Center (CTSC), Vitamin D and Omega-3 Trial, 2052e2053 Clothing, vitamin D synthesis effects, 16e17, 989 Cognition, vitamin D studies, 575 Collagen bone type I collagen expression and vitamin D regulation, 402e405, 415 mineralization role and vitamin D effects, 392 Colorectal cancer (CRC) calcium-sensing receptor, 444e446 CYP24A1 expression regulation calcium intake, 1719e1721 folate intake and epigenetic regulation, 1723e1724 hyperproliferation and tumor progression, 1716e1717 polymorphisms and RNA splicing, 1718e1719 sex hormones, 1721e1722 CYP27B1 expression regulation calcium intake, 1719e1721 folate intake and epigenetic regulation, 1723e1724 hyperproliferation and tumor progression, 1716 polymorphisms and RNA splicing, 1718e1719 sex hormones, 1721e1722 dietary factors, 1711e1712 epidemiology, 1711e1712 protective factors, 1712e1714 vitamin D chemoprevention mechanisms, 1714e1715 deficiency and pathogenesis, 1713e1714 intake and risk analysis, 1572e1574
2062 Colorectal cancer (CRC) (Continued ) vitamin D receptor expression, 444e446, 1718 target gene transactivation, 114e115 Wnt signaling, 237e238, 241e242 Colorimetric assay, vitamin D, 85 Commercial aspects, vitamin D assays 25-hydroxyvitamin D3, 87 provitamins, 86 vitamin D, 84e86 historical perspective, 73e74 market, 88e90 metabolite manufacture 1-hydroxyvitamin D3, 83 25-hydroxyvitamin D3, 83e84 provitamins photoconversion to vitamin D, 79e83 synthesis 7-dehydrocholesterol, 74e78 ergosterol, 78e79 storage and shipping, 90 Computed tomography (CT), see also Quantitative computed tomography microcomputed tomography applications, 899e900 biomechanical analysis, 898e899 image acquisition conventional, 894, 896 synchrotron radiation, 896 morphometric analysis, 898 quantitative analysis, 897e898 in vivo imaging, 896e897 mineralization analysis, 385 multidetector computed tomography applications, 892 biomechanical analysis, 894 densitometric analysis, 893e894 image acquisition, 892e893 morphometric analysis, 894 principles, 891e892 COMT, see Catechol-o-methyltransferase Congestive heart failure, see Heart failure Constitutive androstane receptor (CAR), vitamin D receptor comparison, 144e146 COPD, see Chronic obstructive pulmonary disease Coronary artery disease, see Atherosclerosis Cortical bone, magnetic resonance imaging applications, 915e916 contrast, 913 image analysis, 915 porosity, 913 pulse sequences, 914e915 water quantification, 913e914 Corticosteroids, see Glucocorticoids COX, see Cyclooxygenase CRC, see Colorectal cancer CREB-binding protein (CBP) Runx2 interactions, 314 vitamin D receptor activation, 108, 112, 115 Crohn’s disease, see Inflammatory bowel disease
INDEX
Crystal structure CYP27A1, 34e36 CYP27B1 and homology models, 34e36 peroxisome proliferator-activating receptor-g, 104 vitamin D receptor antagonism structural basis adamantyl analog complexes, 186 calcipotriol modification for receptor antagonism, 184e186 22-butyl-1,24-dihydroxyvitamin D3 derivative complexes, 182e185 C-2a-substituted vitamin D analog complexes, 179e181 hVDRD bound to vitamin D, 172e175, 177 ligand-binding domain bound to superagonists 14-epi analogs, 178e179 20-epi analogs, 176, 178 2-substituted, 19-nor analogs, 178 lithocholic acid derivative binding 3-ketolithocholic acid, 1519 lithocholic acid acetate/propionate, 1519 unmodified lithocholic acid, 1517e1519 nonsteroidal ligand complexes YR301erVDR ligand-binding domain complex, 181 zVDR ligand-binding domain bound to CD578 analog, 181e182 overview, 139, 148 zVDR ligand-binding domain vitamin D complex, 175 gemini complex, 182 CsA, see Cyclosporine A CT, see Computed tomography CTA018, CYP24A1 inhibition, 1542 CTA091, CYP24A1 inhibition, 1540e1542 CTSC, see Clinical and Translational Science Center Cubilin knockout mouse, 64 osteoblast production, 342 CY613, 1469 CY616, 1469 CY625, 1468 CY628, 1468 CY941, 1466 CY943, 1466 CY1006, 1466 CY10010, 1466 CY10012, 1466 Cyclin-dependent kinases (Cdks) cell cycle regulation, 1632e1636 vitamin D upregulation, 1637e1640 Cyclooxygenase (COX) prostate cancer and inflammation, 1687 vitamin D analog inhibition of COX-2, 1451 Cyclosporine A (CsA), vitamin D metabolism effects, 1268 CYP24A1 breast cancer expression, 1661 catalyzed reactions
C23 hydroxylation, 45 C24 oxidation, 43e46 multifunctional activity, 45e46, 1528 overview, 43e44 chondrocyte maturation role, 48 chronic kidney disease activity, 49e50 colorectal cancer expression regulation calcium intake, 1719e1721 folate intake and epigenetic regulation, 1723e1724 hyperproliferation and tumor progression, 1716e1717 polymorphisms and RNA splicing, 1718e1719 sex hormones, 1721e1722 crystal structure, 34e36 dental expression, 524 discovery, 28e29 dysregulation and disease cancer, 1536e1537 chronic kidney disease, 1537e1539 diabetes, 1537 overview, 1534e1535 X-linked hypophosphatemia, 1535e1536 expression regulation, 155, 219, 586, 747e748, 1529e1534 fracture repair role, 50e51 gene methylation, 1533e1534 glucocorticoid effects, 1236 inhibitors azoles, 1539e1540 CTA018, 1542 CTA091, 1540e1542 genistein, 1541 overview, 36, 51e52 tetralone-based inhibitors, 1543 VID400, 1541 knockout mouse, 155, 748 oncogenic activity, 48e49 overview of features, 23e26 overview of features, 23e26, 1525 physiological function, 30 post-transcriptional regulation, 1533 promoter and transcription factor binding sites, 1529e1532 prospects for study, 1543 prostate cancer expression, 1680e1681 inhibitor therapy, 1691 renal activity, 473, 482 structure, 36, 46e48, 1527e1528 structureefunction relationships, 46e48, 1526e1527 substrate specificity, 29e30 tissue distribution, 1528e1529 vitamin D analog metabolism, 1447e1449 vitamin D toxicity and inhibition, 1391 CYP27B1 bone, 412e413, 587, 784 brain, 568 calcium transport role, 584e586 chronic kidney disease direct inhibition, 1330 extrarenal enzyme alterations, 1331e1333
INDEX
induction suppression, 1328e1330 overview of studies, 32e33 substrate availability, 1328 colorectal cancer expression regulation calcium intake, 1719e1721 folate intake and epigenetic regulation, 1723e1724 hyperproliferation and tumor progression, 1716 polymorphisms and RNA splicing, 1718e1719 sex hormones, 1721e1722 deficiency and disease, 584, 750 dendritic cell, 783, 792e793 dental expression, 524 discovery, 31 DNA methylation, 230e232 expression regulation, 33, 229e232, 583, 586, 619, 747e748 extrarenal vitamin D synthesis, see also Vitamin D toxicity diseases cancer, 782 noninfectious granuloma-forming diseases, 780e781 overview, 779 sarcoidosis, 778 systemic lupus erythematosus, 781 tuberculosis, 780 peritonitis, 781 immune system, 782e783 intestine, 896 mammary gland, 786 ovary, 785 pancreas, 784e785 parathyroid gland, 784 placenta, 783e784 prostate, 785 regulation, 787e794 skin, 784 testes, 785 functional overview, 583 glucocorticoid effects, 1236 heart, 550 hypocalcemia and deficiency, 1098 immune system, 1780 inhibitors, 36 intestinal expression, 351e352 knockout mouse, 31, 327, 395e396, 549, 569, 584e597 macrophage enzyme cytokine regulation, 791 lack of CYP24A1 competition, 790e791 nitric oxide, 792 pathogen-associated molecular patterns, 792 regulation insensitivity to parathyroid hormone, calcium, and phosphate, 787e790 subcellular localization and kinetics, 787 multiple sclerosis polymorphisms, 1852 osteoclasts, 418 overview of features, 23e26 parathyroid hormone induction, 155
prostate cancer, 1681 pseudo-vitamin D deficiency mutations, 1189e1190 structure, 31 tissue distribution, 31e32 vasculature, 542e543 vitamin D toxicity control, 1390e1391 CYP2R1 discovery, 26, 28 mutation and disease, 28 overview of features, 23e26 physiological relevance, 26, 28 structure, 28 CYP3A4 overview of features, 23e26 physiological function, 30e31 substrate specificity, 30 vitamin D receptor induction, 216 Cystic fibrosis (CF), cathelicidin antimicrobial peptide expression, 1783 Cytochromes P450, see CYPs Dahl salt-sensitive rat, 547e548 DBP, see Vitamin D binding protein DC, see Dendritic cell Deficiency, see Vitamin D deficiency 7-Dehydrocholesterol assays, 86e87 chemical synthesis, 74e78 photochemical conversion to vitamin D, 79e83 structure, 272 Dementia, vitamin D studies, 575 Dendritic cell (DC) CYP27B1 expression, 783, 792e793 experimental autoimmune encephalitis function, 1860e1861 vitamin D receptor agonist effects chemokine production, 1795e1796 inhibitory receptor upregulation, 1794e1795 overview, 1790e1791 regulatory T cell enhancement, 1793e1794 tolerance induction, 1791e1793, 1796 Dental health, see Oral skeleton Dentin, mineralization, 381, 384e385 Dentin matrix protein 1 (DMP1), vitamin D regulation of expression in osteocytes, 418 Dentin sialophosphoprotein (DSPP), mutation, 524 Dent’s disease, 64 Deoxycorticosterone acetate-salt rat, 547 Depression, vitamin D studies, 574e575 Developing countries, calcium intake, 652 DEXA, see Dual-energy X-ray absorptiometry Diabetes adolescent vitamin D status, 671 beta cell, vitamin D effects deficiency effects, 1827e1828 metabolite studies animal models, 1826e1827
2063 cell culture, 1826, 1828 overview, 1825e1826 cardiovascular risks and vitamin D interactions, 1981e1983 classification, 1825 CYP24A1 dysregulation, 1537 epidemiology, 1825 gestational diabetes mellitus and vitamin D status, 702 25-hydroxyvitamin D measurement, 1923e1924 ketoacidosis and hypophosphatemia, 1175 optimal vitamin D intake and status, 1080e1081, 1924 type 1 disease clinical perspectives of vitamin D, 1836e1837 genetic predisposition studies, 1835e1836 immune function overview, 1829 islet transplantation and recurrence prevention with vitamin D, 1835 primary prevention in animal models by vitamin D analogs, 1833 combination studies, 1833e1834 early intervention, 1830e1832 late intervention, 1832e1833 supplementation studies in prevention, 1834 vitamin D deficiency, 1834e1835 vitamin D receptor agonist studies, 180 type 2 disease epidemiological studies of vitamin D case-control studies, 1911 confounding, 1923 cross-sectional studies, 1911 longitudinal observational cohort studies, 1911e1916 race differences, 1924 seasonal and geographic studies, 1910e1911 epidemiology, 1907 supplementation studies of vitamin D, 1916e1921 vitamin D interactions beta cell function, 1908e1909 inflammation, 1910 insulin sensitivity, 1909e1910 vitamin D deficiency during pregnancy and late effects on child, 687e688 intervention studies, 557 Diarylmethane vitamin D receptor ligands characterization in vivo, 1501e1502 identification, structure, and biological characterization, 1498e1500 DICKKOPF-1 (DKK-1), Wnt inhibition and vitamin D regulation, 241, 305 DICKKOPF-4 (DKK-4), Wnt inhibition and vitamin D regulation, 241e242 Differentiation, see Cell differentiation DiGeorge sequence, hypocalcemia, 1094e1095
2064 7,20-Dihydro-2-methylene-19-nor-1a, 25-dihydroxyvitamin D, isomers, 1433 1,25-Dihydroxy-26,27-F6-vitamin D3, see Falecalcitrol 1,25-Dihydroxy-19-nor-vitamin D2, see Paricalcitol 1,25-Dihydroxyvitamin D, see also Vitamin D; Vitamin D3 sterol activation pathway, 8, 23e24 cartilage function, 509e511 membrane signaling, 512e513 circulating concentrations in diseases, 839 detection overview, 833e834 radioimmunoassay, 836 radioreceptor assay, 834e836 dietary calcium response, 7 discovery as active form of vitamin D hydroxylation enzymes, see CYP2R1; CYP3A4; CYP24A1; CYP27B1; Hydroxylases stimulators, 7 inactivation, 23e24 optimal vitamin D status, 1070 pharmacokinetics, 1042 renal synthesis, 472e473 stability in serum or plasma, 841 toxicity, see Vitamin D toxicity vitamin D status marker in adolescence, 663e664 21,25-Dihydroxyvitamin D3, discovery, 8 24,25-Dihydroxyvitamin D3 cartilage function, 509e511 membrane signaling, 513 circulating concentrations in diseases, 839 detection extraction, 832e833 high-performance liquid chromatography, 833 overview, 832 radioimmunoassay, 833 discovery, 7e8 Diuretics, see Thiazide diuretics DKK-1, see DICKKOPF-1 DKK-4, see DICKKOPF-4 DMP1, see Dentin matrix protein 1 DNA methyltransferase CYP24A1 expression regulation, 1534 CYP27B1 expression regulation, 230e231 DNA microarray, vitamin D receptor target gene expression analysis, 219e220 DNA replication, licensing control, 1635e1636 Dot map, vitamin D3 sterol side-chain conformational dynamics, 274e276 Doxercalciferol activity, 1438 cancer studies, 1438e1439 DPP-1023, 1500 DRIP, see MED-1 complex DSPP, see Dentin sialophosphoprotein
INDEX
Dual-energy X-ray absorptiometry (DEXA) bone mineral density determination, 884 metabolic bone disease diagnosis, 817 Dyslipidemia, cardiovascular risks and vitamin D interactions, 1983 EB1089, breast cancer trials, 1665 EBV, see EpsteineBarr virus ED-71, 1439, 1446 E/F domain, homology between nuclear receptors, 102e103 EGF, see Epidermal growth factor Eldecalcitol development, 1492 osteoporosis management, 1492e1495 vitamin D binding protein affinity, 1493 Elderly, see also Aging; Falls; Osteoporosis optimal vitamin D status, 1069e1076 Endometriosis, vitamin D studies, 697, 700 Endothelial cell, see Vasculature Enhancer linking to regulated genes, 118 vitamin D receptor, 121e123 Epidermal growth factor (EGF), breast cancer signaling, 1660 Epilepsy, vitamin D studies, 571 EpsteineBarr virus (EBV), multiple sclerosis role, 1845e1846 ERE-BP, see Estrogen response element binding protein Ergocalciferol, see Vitamin D2 Ergosterol chemical synthesis, 78e79 photochemical conversion to vitamin D, 79e83 ERK, see Mitogen-activated protein kinases Esophageal cancer vitamin D intake and risk analysis, 1577 vitamin D studies, 1764 Estrogen breast cancer pathogenesis, 1657 vitamin D effects on metabolism and signaling, 1659e1660 estradiol and vitamin D metabolism effects, 1252e1253 multiple sclerosis sex differences, 1863e1865 rescue of blunted breast development and function with estradiol and tamoxifen, 265 Estrogen response element binding protein (ERE-BP), gene cloning and expression, 264 Ethanol, see Alcohol Ethnicity, see Race Ets-1, vitamin D receptor interactions, 200 Experimental autoimmune encephalitis, see Multiple sclerosis Ezetimibe, vitamin D metabolism effects, 1261 FAK, see Focal adhesion kinase Falecalcitrol activity, 1438
cancer studies, 1438e1439 Falls rheumatoid arthritis patient risks, 1961 vitamin D studies impact on risks, 936e937, 1145e1148 observational studies, 2028 randomized controlled studies, 2028e2029 Fanconi syndrome type, 1, 1171e1172 type, 2, 1172 treatment, 1172 Farnesoid X receptor (FXR) bile acids derivatives for modulation, 1514e1515 metabolism regulation, 1513 vitamin D receptor comparison, 144, 146 FDB187E, 1478 FDB198E, 1478 Fetus calcium metabolism, 625e627 heart development, 550e551 parathyroid gland development, 494e495 vitamin D studies animal studies, 627e628 human studies association studies, 630e632 intervention studies, 630 overview, 628e630 FGF, see Fibroblast growth factor FGF-23, see Fibroblast growth factor-23 Fibroblast growth factor (FGF), see also Fibroblast growth factor-23 bile acids and FGF15 bone formation signaling, 304 Fibroblast growth factor-23 (FGF23) chronic kidney disease response, 1325e1326, 1330 clinical significance, 754e755 CYP24A1 expression regulation, 1534 CYP27B1/vitamin D receptor interactions, 588 expression regulation, 155e156, 248 FGF23eklothoeboneekidney axis, 749e752 hypophosphatemia defects, 1160e1165 knockout mouse, 155e156, 626, 633 neonatal levels, 632e633 nephrocalcinosis and cardiovascular calcification role, 1413e1414 osteocyte production coordination of renal phosphate handling with bone mineralization and turnover, 751e752 parathyroid hormone expression regulation, 501e502, 752 phosphate extracellular fluid concentration regulation, 613 phosphate homeostasis regulation, 325, 358, 752e753 prospects for study, 754e755 receptor, see Klotho rickets mutations, 750 signaling, 156 synthesis, 749
INDEX
tumor calcinosis mutations, 1173 vitamin D counter-regulatory hormone activity, 752e753 metabolism effects, 1248e1249 regulation of expression in osteocytes, 417e418 Fish farming, biofortification, 999 Fluoride, vitamin D metabolism effects, 1268 c-Fms, osteoclast signaling differentiation, 338e339 overview, 337e338 precursor proliferation, 338 Focal adhesion kinase (FAK), bone mechanical load response, 325e326 Folate, intake and epigenetic regulation of vitamin D system in colon cancer, 1723e1724 Food allergy, vitamin D studies, 2013 Food fortification, calcium and vitamin D, 653, 992, 994, 997e998, 1000 FOXO1, osteoblast transcriptional control of differentiation, 306 FOXO3, vitamin D regulation of expression, 161 Fractal analysis, bone histomorphometry, 856 Fracture optimal vitamin D status and risk, 1073e1074, 1148 osteoporotic fracture prevention primary prevention calcium and vitamin D, 1137 meta-analysis, 1137e1139 safety of supplements, 1139 vitamin D, 1134e1137 prospects for study, 1140 secondary prevention, 1139e1140 vitamin D analog studies, 1140 repair, see also Orthopedics CYP24A1 role, 50e51 24-hydroxylated metabolite supplementation, 51 vitamin D supplementation, 936e937 trauma and impact of vitamin D fixation, 929 fragility fracture, 927e928 healing, 928e929 nonunion, 929e930 stress and insufficiency fractures, 931 vitamin D in prevention dosing, 1148, 1150 fall prevention, 1146e1148 mechanisms, 1145e1146 optimal vitamin D status, 1148 Functional genetic studies, 1030 FV521, 1476 FV604, 1476 FV639, 1476 FV689, 1476 FV708, 1476 FV721, 1476 FXR, see Farnesoid X receptor
GADD45A, vitamin D regulation of expression, 219 GALNT3, tumor calcinosis mutations, 1173 GAO182, 1466 Gastrectomy, see Postgastrectomy bone disease Gastric bypass, see Obesity Gastric cancer, vitamin D studies, 1764 GC, multiple sclerosis polymorphisms, 1852e1853 Gcm-2, parathyroid development, 494 GDM, see Gestational diabetes mellitus GDNF, see Glial-derived neurotrophic factor Gemini leukemia studies, 1745 transcriptional cycling studies, 222 zVDR ligand-binding domain gemini crystal structure, 182 Genetic hypercalciuric rat, see Idiopathic hypercalciuria Genistein CYP24A1 inhibition, 51, 541 prostate cancer management with vitamin D, 1692e1693 Genome-wide association study (GWAS) bone mineral density, 1034e1035 data analysis, 1035e1036 overview, 1028e1029 sequencing, 1029e1030 vitamin D levels in serum, 1036e1037 Gestational diabetes mellitus (GDM), vitamin D status, 702 GH, see Growth hormone Ghrelin, bariatric surgery effects, 1017e1018 Glial-derived neurotrophic factor (GDNF), vitamin D effects, 570, 573 Glioma, vitamin D studies, 1766 Glucocorticoids CYP24A1 expression regulation, 1531e1532 inflammation mediation, 1235e1236 inflammatory bowel disease management, 1885 multiple sclerosis treatment, 1865e1866 neuroprotective actions of vitamin D, 573 osteoporosis bisphosphonate therapy, 1240e1241 calcium absorption, 1238 induction mechanisms, 1234 serum vitamin D metabolites, 1236 vitamin D therapy bone mineral density response, 1238e1240 preparations, 1240 prostate cancer management with vitamin D, 1692 receptors, 1234 vitamin D metabolism effects, 1236e1238, 1258e1259 vitamin D receptor regulation, 1235 GM-CSF, see Granulocyteemacrophage colony-stimulating factor GM7, 1467 GM8, 1467 GM14, 1467
2065 GM15, 1467 GM38A, 1467 GM38B, 1467 G1 phase, see Cell cycle G2 phase, see Cell cycle Graft rejection, see Organ transplantation Granulocyteemacrophage colonystimulating factor (GM-CSF), functional overview, 1734 Growth, vitamin D deficiency during pregnancy and late effects on child, 687 Growth hormone (GH), vitamin D metabolism effects, 1250 GWAS, see Genome-wide association study Hair cycle, vitamin D receptor role, 157e158, 534e536, 593e594 Hairless (Hr) keratinocyte stem cell function, 536e537 vitamin D receptor repression, 202 HAT, see Histone acetytransferase HCC, see Hepatocellular carcinoma HDAC, see Histone deacetylase Head and neck cancer, vitamin D studies, 1766e1767 Heart, see also Atherosclerosis cardiomyocyte and vitamin D function, 1976 cardioprotective actions of vitamin D, 1345e1346 cardiovascular mortality as endpoint in vitamin D observational and interventional studies, 1983e1987 clinical cardiovascular outcomes in vitamin D observational and interventional studies, 1987e1989 CYP27B1 expression, 550 transplantation and vitamin D deficiency, 1292 vitamin D receptor developmental role, 550e551 expression, 550 fibrosis animal studies, 552e553 vitamin D intervention studies, 553e554 hypertrophy animal studies, 551e552 vitamin D intervention studies, 553 vitamin D signaling in cardiovascular remodeling and myocardial function, 1404e1406 Heart failure vitamin D intervention studies, 556 vitamin D status observational studies, 556 Hedgehog, keratinocyte stem cell function, 537 Helicobacter pylori, vitamin D and infection, 1778 Hematopoiesis growth factors, 1733e1734 overview, 1733e1734 vitamin D effects
2066 Hematopoiesis (Continued ) leukemia, see Leukemia normal hematopoiesis, 1734e1735 vitamin D receptor blood cells, 1733 knockout mouse studies, 1734 lymphoid cells, 1734 myeloid cells, 1733 Heparin, vitamin D metabolism effects, 1263e1264 Hepatitis, bone loss chronic active disease clinical features, 1311 epidemiology, 1310e1311 pathogenesis, 1311 treatment, 1311 viral disease, 1311 Hepatocellular carcinoma (HCC), vitamin D studies, 1765 Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) clinical presentation, 1169e1170 gene defects, 1170 pathophysiology, 1170 treatment, 1170e1171 Hereditary vitamin-D resistant rickets (HVDRR) alopecia association, 1200, 1223e1224 biochemical findings, 1199e1200 case examples, 1205e1208 cats, 1218 cell studies, 1203e1204, 1209 clinical presentation, 1198e1201 dogs, 1218e1219 history of study, 1197e1198 pathophysiology, 1200 prenatal diagnosis, 1222 prospects for study, 1224e1225 spontaneous healing, 1222e1223 synonyms, 1197 treatment calcium, 1220e1222 vitamin D, 1219e1220 vitamin D analogs, 1223 vitamin D receptor defects coactivator binding mutations, 1217 compound heterozygous mutations, 1217e1218 deletions, 1214 disease without mutations, 1219 DNA-binding domain mutations, 1210e1211 heterodimerization-affecting mutations, 1216e1217 ligand-binding domain mutations, 1214e1216 miscellaneous mutations, 1218 nonsense mutations, 1211e1213 overview, 1209e1210 RNA splicing-disrupting mutations, 1213e1214 Heterogeneous nuclear ribonucleoproteins (hnRNPs) C family, 258e262 domains, 260e261
INDEX
multi-level, multi-site cis-binding regulation of gene expression, 262e264 overview, 258e262 HHM, see Humoral hypercalcemia of malignancy HHRH, see Hereditary hypophosphatemic rickets with hypercalciuria High-performance liquid chromatography (HPLC) 24,25-dihydroxyvitamin D, 831 25-hydroxyvitamin D, 828e829 vitamin D assay, 85e86, 824e825 High-resolution peripheral quantitative computed tomography (HRpQCT), see Quantitative computed tomography Hip replacement, see Orthopedics Histomorphometry, see Bone histomorphometry Histone acetytransferase (HAT), see also specific enzymes transcriptional regulation in chromatin context, 211 vitamin D receptor regulation, 106, 194, 229 Histone deacetylase (HDAC), see also specific enzymes transcriptional regulation in chromatin context, 211 vitamin D receptor regulation, 194, 201, 230 Historical perspective, vitamin D studies commercial production, 73e74 discovery, 4 hormonal form, 6e7, 97e99 isolation of nutritional forms, 5 metabolites, 7e8 photobiology, 4e5, 13e14 physiological functions calcium mobilization from bone, 5e6 intestinal absorption of calcium and phosphorous, 5 renal reabsorption of calcium and phosphorous, 6 receptor discovery, 99 tuberculosis, 1818 HIV, see Human immunodeficiency virus hnRNPs, see Heterogeneous nuclear ribonucleoproteins HOXA10, vitamin D regulation of expression, 700e702 HPLC, see High-performance liquid chromatography Hr, see Hairless HR-pQCT, see High-resolution peripheral quantitative computed tomography 11b-HSD, see 11-b-Hydroxysteroid dehydrogenase Human immunodeficiency virus (HIV) protease inhibitors for leukemia treatment with vitamin D combination therapy, 1740e1741 psoriasis management in patients, 1896e1897 vitamin D and infection, 1779
Humoral hypercalcemia of malignancy (HHM), parathyroid hormonerelated protein function role, 737 HVDRR, see Hereditary vitamin-D resistant rickets Hydroxyapatite, see Bone; Mineralization 1a-Hydroxylase, see CYP27B1 24-Hydroxylase, see CYP24A1 Hydroxylases, see also CYP2R1; CYP3A4; CYP24A1; CYP27A1; CYP27B1 calcium-sensing receptor effects in proximal tubule, 433 crystal structures and homology models, 34e36 drug effects, 1257e1269 hormone effects, 1245e1256 inhibitors, 36 overview of features, 23e26 prospects for study, 36, 38 sequence alignment, 24, 27 stimulators, 7 11-b-Hydroxysteroid dehydrogenase (11b-HSD), vitamin D stimulation and glucocorticoid-induced osteoporosis role, 1233e1234 1a-Hydroxyvitamin D2, see Doxercalciferol 1a-Hydroxyvitamin D3 chemical synthesis, 83 leukemia studies, 1741 25-Hydroxyvitamin D adverse effects of large fluctuations, 1044, 1047 calcium-sensing receptor effects in kidney, 433e434 chemical synthesis, 83e84 circulating concentrations in diseases, 839 detection chemiluminescence assay, 831 commercial synthesis assays, 87 high-performance liquid chromatography, 828e829 liquid chromatographyemass spectrometry, 831e832 overview, 827e828 radioimmunoassay, 829e830 reporting, 840 discovery, 7 endogenous production, 1385e1386 intestinal calcium absorption role, 620 kidney, 482 measurement in hypertension and diabetes type, 2, 1923e1924 normal levels, 839e840 optimal level, 1048e1050, 1069, 1131e1133, 1148 pharmacokinetics, 1042 predicted levels in cancer epidemiology, 1571e1572 serum levels, 619e620 stability in serum or plasma, 841 toxicity, 1381e1384 vitamin D status marker in adolescence, 663 vitamin D supplementation effects on conversion, 1055e1056
INDEX
25-Hydroxyvitamin D-1a-hydroxylase, see CYP27B1 25-Hydroxyvitamin D-24-hydroxylase, see CYP24A1 Hygiene hypothesis, 1881 Hypercalcemia, see also Vitamin D toxicity clinical manifestations, 1392e1393 diagnosis of vitamin D toxicity, 1393e1394 differential diagnosis, 1382 endogenous vitamin D toxicity 25-hydroxyvitamin D production, 1385e1386 lymphoma, 1388e1389 prevention, 795 sarcoidosis, 1386e1387 treatment, 795e796 tuberculosis, 1387 exogenous vitamin D toxicity, 1381e1385 treatment, 1394e1395 Hypercalciuria, see also Hereditary hypophosphatemic rickets with hypercalciuria endogenous vitamin D toxicity treatment, 795e796 treatment in endogenous vitamin D intoxication, 795e796 Hypercalciuria, see Idiopathic hypercalciuria Hyperlipidemia, see Dyslipidemia Hyperparathyroidism calcium-sensing receptor calcimimetic activators for treatment secondary hyperparathyroidism in chronic kidney disease, 442e443 uremic hyperparathyroidism, 441e442 hypercalcemia, 1381e1382 primary disease and surgical guidelines, 732 radiographic findings renal osteodystrophy, 869 secondary hyperparathyroidism, 867e869 Hyperphosphatemia, see also Tumor calcinosis clinical signs and symptoms, 1176e1177 hypocalcemia induction, 1099 phosphate load disorders, 1175e1176 treatment from abnormal phosphate load, 1178 Hypertension, see also Renineangiotensin system cardiovascular risks and vitamin D interactions, 1980e1981 epidemiological studies of vitamin D case-control studies, 1921e1922 confounding, 1923 cross-sectional studies, 1921e1922 longitudinal observational cohort studies, 1922 race differences, 1924 seasonal and geographic studies, 1921 epidemiology, 1907 25-hydroxyvitamin D measurement, 1923e1924 optimal vitamin D
intake, 1924 status, 1079e1080 sunlight and blood pressure, 715 supplementation studies of vitamin D, 1922e1923 vitamin D deficiency, 715 interactions inflammation, 1910 renineangiotensinealdosterone system, 1910 intervention studies animal models, 548e549 clinical studies, 554e555, 715e716 status observational studies, 554 Hypocalcemia biochemical changes, 1093e1094 calcium deficiency, 1099 calcium-sensing receptor gene mutations, 1095 classification, 1094e1100 clinical manifestations, 1091e1092 hyperphosphatemia in induction, 1099 illness induction, 1100 management acute, 1100e1101 long-term, 1101e1103 medication induction, 1100 mineralization acceleration as cause, 1099e1100 neonatal, 1097, 1100e1101 parathyroid hormone defects in disease autoimmune polyglandular syndrome type, 1, 1095 DiGeorge sequence, 1094e1095 gene mutations, 1095 infiltrative disease, 1095 pseudohypoparathyroidism, 1096 radiation, 1095 surgical patients, 1095 prevention, 1092 signs and symptoms, 813 vitamin D deficiency in etiology CYP27B1 deficiency, 1098 malabsorption, 1098 malnutrition, 1097e1098 vitamin D receptor defects and resistance, 1098e1099 vitamin D in long-term maintenance of eucalcemia, 1092e1093 Hypomagnesemia, parathyroid hormone dysfunction, 1096e1097 Hypoparathyroidism, overview, 732 Hypophosphatemia, see also Fanconi syndrome; Hereditary hypophosphatemic rickets with hypercalciuria; X-linked hypophosphatemia clinical presentation, 1176e1177 common pathogenesis pathway, 1160e1165 glucose induction, 1174 treatment from abnormal phosphate load, 1178
2067 IBD, see Inflammatory bowel disease Ichthyosis, vitamin D therapy, 1897e1898 IDBPs, see Intracellular vitamin D binding proteins Idiopathic hypercalciuria (IH) genetic hypercalciuric rat bone density, 1373 establishment, 1369e1370 intestinal calcium transport, 1370 low calcium diet response, 1373 mineral balance, 1370 renal calcium handling, 1370 serum and urine chemistries, 1370 vitamin D levels, 1370e1371 vitamin D receptor expression, 1371e1373 overview, 1359e1360 pathogenesis bone mineral density reduction, 1363e1365 calcium absorption intestinal increase, 1360e1362 renal reabsorption decrease, 1362e1363 calcium balance, 1367e1368 genetics, 1364, 1366 models, 1366e1367 parathyroid hormone levels, 1367 renal histopathology in calcium oxalate nephrolithiasis, 1360 vitamin D elevation, 1362, 1368e1369 pathophysiology, 1374 treatment dietary calcium restriction, 1374 thiazide diuretics, 1375 vitamin D analogs and risks of stone formation, 1375 IEBP, see Intracellular estrogen-binding protein IFN-g, see Interferon-g IGF-I, see Insulin-like growth factor-I IGFBP genes cancer, 1605 vitamin D regulation, 216, 219 IH, see Idiopathic hypercalciuria Infant calcium intake recommendations, 649e650 metabolism, 632e633 developmental vitamin D deficiency behavioral effects in later life, 568e569 dietary restriction, 567e568 gene expression effects in later life, 568 structural alterations, 576 premature infants, 648e649, 702 vitamin D studies in development animal studies, 633e635 human studies overview, 635e637 intervention studies, 637e638 association studies, 638e640 vitamin D intake recommendations, 649e650 supplementation, 650e651
2068 Inflammation benign prostatic hyperplasia and vitamin D analog inhibition, 1935e1936 brain, 571 breast cancer pathways, 1660 central nervous system inflammation and vitamin D synthesis, 1857 glucocorticoid mediation, 1235e1236 pregnancy and immune tolerance, 1863 prostate cancer and vitamin D effects, 1686e1688 renoprotective actions of vitamin D, 1342e1345 vascular vitamin D receptor functions, 544 vasculature and vitamin D, 1977e1978 vitamin D binding protein role, 66e67 Inflammatory bowel disease (IBD) animal models, 1884e1885 classification, 1879 colorectal cancer risks, 1715 environmental factors, 1881 epidemiology, 1879 genetics, 1880 hygiene hypothesis, 1881 immune response overview, 1882e1883 T cell regulation by vitamin D, 1883e1884 treatment immunosuppression, 1885e1886 vitamin D, 1886 vitamin D hypothesis, 1881e1882 vitamin D receptor agonist studies, 1802e1803 Influenza, adolescent vitamin D status, 671 Infrared spectroscopy mineralization analysis, 396 vitamin D, 86 Innate immunity, see specific components Insulin, vitamin D metabolism effects, 1251e1252 Insulin-like growth factor-I (IGF-I) breast cancer signaling, 1660 vitamin D inhibition in cancer overview, 1605 prostate cancer, 1686 vitamin D metabolism effects, 1250e1251 Insulin resistance, see also Metabolic syndrome cardiovascular risks and vitamin D interactions, 1981e1983 vitamin D intervention studies, 557 vitamin D status observational studies, 557 Intact parathyroid hormone, see Parathyroid hormone Intake recommendations, see Vitamin D status Interferon-g (IFN-g), vitamin D metabolism effects, 1256 Intestine, see also Colorectal cancer absorption, see Calcium absorption; Phosphate absorption calbindin D function, 364e366 calcium-sensing receptor function, 439 CYP27B1, 894 vitamin D receptor function, 439
INDEX
vitamin D synthesis in colon, 1715 vitamin D absorption, 1300 Intracellular estrogen-binding protein (IEBP), 266e267 Intracellular vitamin D binding proteins (IDBPs) effect on vitamin D synthesis, 266 functions, 265e266 types, 265 vitamin D analog binding, 1451 Ion channels, vitamin D potentiation through nongenomic action, 279, 290 Islet transplantation, diabetes recurrence prevention with vitamin D, 1835 Jaw osteonecrosis risk factors, 527e528 vitamin D status and supplementation, 527e528 Jejunoileal bypass, see Obesity JN, 1499 JNK, see Jun N-terminal kinase Jun N-terminal kinase (JNK), photoprotection mechanisms of vitamin D compounds, 1948 Keratinocyte calcium-sensing receptor function, 444 b-cateninevitamin D receptor interactions, 156e158 CYP27B1 in differentiation, 594 differentiation, 533e534, 537e538, 594 stem cells quantification, 536 signaling pathways, 536e537 sunburn cells, see Sun exposure vitamin D in homeostasis, 1755 Ketoconazole CYP24A1 inhibition, 1540 vitamin D metabolism effects, 1260 K-homology splicing regulator protein (KSRP), 430, 501 Kidney, see also Chronic kidney disease calbindin D function, 366, 478e480 calcium tubular reabsorption calcium-sensing receptor regulation, 434e435 CYP27B1 role, 585e586 overview, 471e472 parathyroid hormone role, 729e730 vitamin D effects, 473e475 calcium-sensing receptor function, 433e435 cancer, see Renal cell carcinoma fibroblast growth factor-23 coordination of renal phosphate handling with bone mineralization and turnover, 753e754 functional overview in mineral homeostasis, 471 parathyroid hormone function, 729e730 phosphate tubular reabsorption defects, see Hyperphosphatemia; Hypophosphatemia overview, 472
parathyroid hormone role, 729e730 vitamin D effects, 473e475 plasma membrane calcium pump, 480 stones, see Idiopathic hypercalciuria transplantation and vitamin D deficiency, 1292e1293 TRPV5 and TRPV6 function, 480e482 vitamin D functions, 1340e1341 metabolism 24-hydroxylase activity, 473 vitamin D3 synthesis, 473 renoprotection, 1342e1345 vitamin D receptor function, 433e435, 475e478 Kip1, see p27 Klotho FGF23eklothoeboneekidney axis, 749e753 hormonal actions, 754e755 knockout mouse, 325 nephrocalcinosis and cardiovascular calcification role, 1413e1414 rickets mutations, 753 tumor calcinosis mutations, 1173 vitamin D regulation of expression, 160 Knockout mouse calbindin D, 353, 365e366 calcium-sensing receptor, 429, 436, 526 cancer and vitamin D system studies, 590e593 cubilin, 64 CYP24A1, 155, 748 CYP27B1, 31, 327, 395e396, 569, 584e597 fibroblast growth factor-23, 155e156, 626, 633 klotho, 325 low-density lipoprotein receptor, 550 MARRS, 460 MBD4, 233 MED-1 complex, 198e199 megalin, 63e65 parathyroid hormone, 429, 436, 586 parathyroid hormone-related protein, 302 TRPV5, 585e586 TRPV6, 585 vitamin D binding protein, 57, 62e63, 395 vitamin D receptor, 141, 143e144, 160, 481, 497e409, 523, 525e526, 536, 549, 570, 584e597, 627, 749, 772e774, 1076e1077, 1130, 1201, 1734, 1978, 2034e2035 Wnt, 242 KS018, 1482 KS176, 1479, 1504 KS291, 1479 KS512, 1479 KS532, 1462e1463 KS699, 1473 KSRP, see K-homology splicing regulator protein Lactation bone mineral content and density overview, 683
INDEX
weaning effects, 683e684 vitamin D and calcium metabolism overview, 680 weaning effects, 682 vitamin D deficiency effects on infant, 688e689 Latitude vitamin D status influences, 661e662, 987e988 vitamin D synthesis effects, 15, 17 LC/MS, see Liquid chromatographyemass spectrometry LCA derivatives, see Lithocholic acid derivatives LDLR, see Low-density lipoprotein receptor Left shift, resting membrane potential, 461 Left ventricular hypertrophy, see Heart Leptin bariatric surgery effects, 1017 bone effects, 1017 vitamin D metabolism effects, 1249 Leukemia combination therapy with vitamin D all-trans retinoic acid, 1739e1740 arsenic trioxide, 1740 chemotherapy, 1740 human immunodeficiency virus protease inhibitor, 1741 iron chelating agents, 1740 kinase inhibitors, 1740 MDM2 antagonists, 1740 nonsteroidal anti-inflammatory drugs, 1740 valproic acid, 1741 intervention trials in myelodysplastic syndrome, 1735, 1743 vitamin D effects analog studies C-16-enes, 1742e1743 calcipotriol, 1741 20-epi vitamin D3, 1743 gemini analogs, 1743 1a-hydroxyvitamin D3, 1741 paracalcitol, 1743 apoptosis, 1737 cell cycle, 1736e1737 cell differentiation, 1736 immune response, 1738e1739 kinases, 1739 transcription factors, 1738 LG190090, 1483, 1500 LG190119, 1500 LG190155, 1483 LG190178, 1483, 1500 LiebermaneBurchard reaction, 87 Linkage analysis animal studies, 1027 overview, 1026e1027 Liquid chromatographyemass spectrometry (LC/MS), 25-hydroxyvitamin D, 829e830 Lithium, vitamin D metabolism effects, 1268e1269 Lithocholic acid (LCA) derivatives, vitamin D receptor interactions
crystal structures 3-ketolithocholic acid, 1519 lithocholic acid acetate/propionate, 1519 unmodified lithocholic acid, 1517e1519 docking models, 1519e1520 overview, 1515e1517 Liver, see also Hepatitis; Primary biliary cirrhosis cancer, see Hepatocellular carcinoma transplantation bone loss, 1313 vitamin D deficiency, 1292 vitamin D metabolism, 1300e1302 Liver X receptor (LXR) bile acid metabolism regulation, 1512e1513 vitamin D receptor comparison, 144, 146 Loading dose concept, 1058e1059 toxicity, 1059e1060 vitamin D2, 1059 Low-density lipoprotein, see Dyslipidemia Low-density lipoprotein receptor (LDLR), knockout mouse, 550 Low-density lipoprotein receptor-related proteins (LRPs) bone formation signaling, 305 LRP5 bone health role, 243 vitamin D regulation of expression, 154 Wnt coreceptors, 235e237, 243, 251 Lower urinary tract symptoms, see Benign prostatic hyperplasia LTCC, see L-type calcium channels L-type calcium channels (LTCC), vitamin D effects, 2032 Lung developmental role of vitamin D, 2011 transplantation and vitamin D deficiency, 1292 Lung cancer, vitamin D studies, 1764e1765 LXR, see Liver X receptor LY2108491, 1483, 1500 LY2109866, 1483, 1500 Lymphoma endogenous vitamin D toxicity, 1388e1389 extrarenal vitamin D production, 779 M phase, see Cell cycle M-CSF, see Macrophage colony-stimulating factor Macrophage, CYP27B1 cytokine regulation, 791 lack of CYP24A1 competition, 790e791 nitric oxide regulation, 792 pathogen-associated molecular patterns, 792 regulation insensitivity to parathyroid hormone, calcium, and phosphate, 787e790 subcellular localization and kinetics, 787 Macrophage colony-stimulating factor (MCSF), functional overview, 1731e1733 Magnesium, see also Hypomagnesemia hypocalcemia management, 1101
2069 Magnetic resonance imaging (MRI) cortical bone imaging applications, 915e916 contrast, 913 image analysis, 915 porosity, 913 pulse sequences, 914e915 water quantification, 913e914 principles, 905e907 trabecular bone imaging image processing coil sensitivity correction, 909 segmentation, 909e910 image registration, 912e913 morphometric analysis, 910e912 pulse sequences, 908e909 signal-to-noise considerations, 906e907 spatial resolution, 907e908 Malignant hyperthermia, hyperphosphatemia, 1176 Mammary gland, see also Breast cancer; Lactation calcium-sensing receptor function, 440 CYP27B1 expression, 784e785 parathyroid hormone-related protein function, 733e737 vitamin D receptor expression and function, 440, 1665e1667 MAPK, see Mitogen-activated protein kinase Market, vitamin D, 88e90 MARRS, see Membrane-associated rapid response, steroid-binding protein Matrix extracellular phosphoglycoprotein (MEPE, hypophosphatemia role, 1162e1163 Matrix Gla protein (MGP), vitamin D effects in bone, 416 Maxacalcitol activity, 1438, 1445e1446 cancer studies, 1438e1439, 1446 protein binding, 1446e1447 MBD4 CYP27B1 expression regulation, 232 knockout mouse, 233 parathyroid hormone signaling-induced transcriptional derepression, 232e233 2MbisP, see 2-Methylene-19-nor-1ahydroxybishomopregnacalciferol 2MD, 1140, 1439 MDCT, see Multidetector computed tomography MDM2, antagonists for leukemia treatment with vitamin D combination therapy, 1740 MDS, see Myelodysplastic syndrome MED-1 complex knockout mice, 198e199 vitamin D receptor activation, 106, 108, 112, 115, 198e199, 534 Medical history, metabolic bone disease diagnosis chief complaint, 809 family history, 812 past history, 811
2070 Medical history, metabolic bone disease diagnosis (Continued ) present illness, 810e811 review of systems, 812 social history, 811e812 Megalin knockout mouse, 63e65 osteoblast production, 342 structure, 63 tissue distribution, 63e64 vitamin D transport, 64 Melanin, vitamin D synthesis effects, 14e15, 19, 984e986 Melanoma, see Skin cancer Membrane-associated rapid response, steroid-binding protein (MARRS) knockout mouse, 460 transcaltachia role, 354 vitamin D analog binding, 1450 vitamin D plasma membrane receptor, 283 Meningioma-1 (MN1), vitamin D receptor interactions, 201 MEPE, see Matrix extracellular phosphoglycoprotein MESA, see Multiethnic Study of Atherosclerosis Meta-analysis, genetic studies, 1030 Metabolic syndrome optimal vitamin D status, 1080e1081 vitamin D intervention studies, 557 vitamin D status observational studies, 557 Metallothionein, photoprotection mechanisms of vitamin D compounds, 1948 Metastasis breast cancer, 1662 inhibition by vitamin D, 1595e1597 parathyroid hormone-related protein in bone metastasis, 737 prostate cancer and vitamin D inhibition, 1689 radiographic findings in calcification, 869e872 2-Methylene-19-nor-1ahydroxybishomopregnacalciferol (2MbisP), activity, 1429e1430 MGP, see Matrix Gla protein Microcomputed tomography, see Computed tomography Mineralization analysis atomic force microscopy, 387 localization, 385 nuclear magnetic resonance, 389 quantification, 385, 387 small-angle X-ray scattering, 387 transmission electron microscopy, 387 in vitro studies, 389 X-ray diffraction, 387e388 bone histomorphometry assessment adjusted apposition rate, 848 mineral apposition rate, 847e848 mineralization lag time, 848 osteoid maturation time, 849 osteoid measurement, 847
INDEX
tetracycline labeling, 846e847 calcium extracellular fluid concentration regulation, 609 CYP27B1 function, 587 definition, 381 dentin, 381 enamel, 381 hydroxyapatite properties, 384 hypocalcemia from acceleration, 1099e1100 mechanisms in bone, cartilage, and dentin, 384e385 osteoporosis/ostemalacia defects, 396 pathology, 382 phosphate extracellular fluid concentration regulation, 612 physical chemistry, 382e384 vitamin D deficiency, animal model studies, 390e396 vitamin D regulation physicochemical effects, 389e390 protein mediators bone sialoprotein, 392e393 collagen, 392 extracellular matrix proteins, 383 knockout and transgenic mice, 386 osteocalcin, 393 osteopontin, 393 Mitogen-activated protein kinase (MAPK) bone mechanical load response, 326 cell differentiation induction via vitamin D, 1629e1630 cross-talk of vitamin D signaling, 283 CYP24A1 expression regulation, 1530 leukemia signaling and vitamin D effects, 1739 muscle signaling and vitamin D effects, 2034 osteoblast signaling and vitamin D effects, 328e329 photoprotection mechanisms of vitamin D compounds, 1948 vitamin D regulation through nongenomic action, 281 Mitogen-activated protein kinase phosphatase-5 (MKP5), vitamin D effects in prostate cancer, 1688 MKP5, see Mitogen-activated protein kinase phosphatase-5 MN1, see Meningioma-1 Monocyte, CYP27B1, 781 MRI, see Magnetic resonance imaging MS, see Multiple sclerosis Multidetector computed tomography (MDCT), see Computed tomography Multiethnic Study of Atherosclerosis (MESA), 1409 Multiple sclerosis (MS) central nervous system inflammation and vitamin D synthesis, 1857 course, 1844e1845 EpsteineBarr virus role, 1845e1846 etiology, 1845 experimental autoimmune encephalitis dendritic cell function, 1860e1861
overview of model, 1853e1854 prevention studies calcium, 1856e1857 cholecalciferol, 1854e1855 ultraviolet light, 1855e1856 vitamin D3, 1855 transforming growth factor-b expression, 1860 vitamin D amelioration and T cell mechanisms activation-induced cell death sensitivity, 1862 chemokine synthesis inhibition, 1862 nitric oxide synthase inhibition of induction, 1862e1863 overview, 1861e1862 vitamin D receptor expression on T cells overview, 1858 regulatory T cells, 1859e1860 Th1 cells, 1858 Th2 cells, 1858e1859 Th17 cells, 1859 genetics CYP27B1 polymorphisms, 1852 GC polymorphisms, 1852e1853 HLA-DR, 1850e1851 vitamin D receptor polymorphisms, 1851e1852 pregnancy and immune tolerance, 1863 prospects for study, 1866e1868 sex differences and estrogen, 1863e1865 treatment with vitamin D costs, 1866 glucocorticoid comparison, 1865e1866 immunomodulatory therapy comparison, 1866 relapsingeremitting multiple sclerosis, 1865 vitamin D binding protein levels, 1853 vitamin Demultiple sclerosis hypothesis, 1843e1844 vitamin D receptor agonist studies, 1801e1802 vitamin D status analysis, 1846e1847 deficiency in patients, 1847 longitudinal disease, 1848 relapse and remission, 1848e1850 risk analysis, 1847e1848 sun exposure inverse correlation, 1846 vitamin D supplementation and relapseeremitting disease, 1849e1850 Muscle optimal vitamin D status, 1081 parathyroid hormone actions, 2035 strength, orthopedic rehabilitation and vitamin D, 933e934 vitamin D falls studies observational studies, 2028 randomized controlled studies, 2028e2029 fracture prevention, 1145 intervention trials in adolescence, 669
INDEX
mechanisms of action genomic actions, 2030e2031 non-genomic actions, 2031e2034 morphology studies, 2029e2030 myopathy and deficiency, 2024e2025 overview of function, 2023e2024 physical performance studies observational studies, 2025e2026 randomized controlled studies, 2026e2028 prospects for study, 2035e2036 vitamin D receptor knockout mouse studies, 2034e2035 polymorphisms, 2035 Mushroom, irradiation for biofortification, 999 c-Myc, vitamin D downregulation and effects on cell cycle and differentiation, 1641e1642 Myelodysplastic syndrome (MDS), vitamin D effects, 1735, 1743e1744 NaPi IIb, see SLC34A2 NCoA62/SKIP, vitamin D receptor activation, 199e200 NCoR, vitamin D receptor repression, 201e202 Neonatal severe hyperparathyroidism (NSHPT), mouse model, 430 Neonate calcium metabolism, 632e633 hypocalcemia, 1097, 1100e1101 premature infants, 648e649, 702 vitamin D studies animal studies, 633e635 human studies association studies, 638e640 intervention studies, 637e638 overview, 635e637 Nephrolithiasis, see Idiopathic hypercalciuria Nerve growth factor (NGF), neuroprotective actions of vitamin D, 572e573 Neuroprotection, vitamin D mechanisms, 572e573 New World primates (NWPs) bone disease, 253e254 evolution, 253 intracellular estrogen-binding protein, 266e267 intracellular vitamin D binding proteins, 265e266 rickets, 251, 254e255 NF-kB, see Nuclear factor-kB NGF, see Nerve growth factor Nitric oxide (NO), CYP27B1 regulation in macrophages, 792 Nitric oxide synthase (NOS) photoprotection mechanisms of vitamin D compounds, 1948 vitamin D inhibition of induction, 1862e1863 NMR, see Nuclear magnetic resonance NO, see Nitric oxide
NOD2 innate immunity, 1812e1813 vitamin D effects on expression, 1781 Nonsteroidal anti-inflammatory drugs (NSAIDs) leukemia treatment with vitamin D combination therapy, 1740 prostate cancer management with vitamin D, 1693e1694 NOS, see Nitric oxide synthase NSAIDs, see Nonsteroidal antiinflammatory drugs NSHPT, see Neonatal severe hyperparathyroidism Nuclear factor-kB (NF-kB), vitamin D effects in prostate cancer, 1688 Nuclear magnetic resonance (NMR), mineralization analysis, 389 Nutrition, see also Vitamin D status biofortification, 998e999 early views, 3 food composition data, 995e996 food fortification with calcium and vitamin D, 653, 992, 994, 997e998, 1000 geographic differences, 957 lifestyle strategies for vitamin D status improvement, 100 natural food sources, 996e997 NWPs, see New World primates OA, see Osteoarthritis Obesity bariatric surgery bone loss clinical features, 1308 epidemiology, 1306e1307 overview, 1016e1018 pathogenesis, 1308 treatment, 1308 calcium absorption effects, 1019e1021 complications medical, 1015e1016 surgical, 1013e1015 epidemiology, 1009e1010 health benefits, 1015 indications, 1010e1011 types adjustable silicone gastric banding, 1013 biliopancreatic diversion, 1011e1011 biliopancreatic diversion with duodenal switch, 1012e1013 jejunoileal bypass, 1011 roux-en-Y gastric bypass, 1013 sleeve gastrectomy, 1013 vertical banded gastroplasty, 1013 vitamin D status, 1018e1019 vitamin D supplementation, 1020e1021 cardiovascular risks and vitamin D interactions, 1979e1980 epidemiology, 1009 etiology, 1009 pathophysiology, 1009 vitamin D deficiency and adiposity, 770e773
2071 vitamin D status, 662, 773e774 OCN, see Osteocalcin Olestra, vitamin D metabolism effects, 1268 OPG, see Osteoprotegerin OPN, see Osteopontin Optimal vitamin D status, see Vitamin D status Oral skeleton dental rickets, 522e524 dento-maxillofacial skeleton properties, 521e522 parathyroid hormone-related protein function in teeth, 737 rare diseases, 522 vitamin D bioactivation, 524e526 development role, 526 pathology, 526e529 prospects for study, 529e530 therapeutic applications, 527e528 Organ transplantation bone loss and treatment with vitamin D analogs, 1294e1296 graft rejection and vitamin D role, 1291 immune function of vitamin D, 1291e1292 vitamin D deficiency post-transplantation heart, 1294 long-term, 1293e1294 perioperative, 1293 pre-transplantation heart, 1292 kidney, 1292e1293 liver, 1292, 1313 lung, 1292 Orlistat, vitamin D metabolism effects, 1268 Orthopedics, see also Fracture falls and vitamin D impact, 936e937, 1145e1148 muscle strength and rehabilitation, 935e937 pediatric orthopedics rickets, 931e933 slipped capital femoral epiphysis, 933 total joint arthroplasty and vitamin D impact osteoarthritis, 934e935 periprosthetic fracture, 935 thigh pain, 935 trauma and vitamin D impact fixation, 929 fragility fracture, 927e930 healing, 928e929 nonunion, 929e930 stress and insufficiency fractures, 931 vitamin D supplementation, 937e938 Osteoarthritis (OA) bone changes, 1956e1957 cartilage homeostasis and vitamin D, 1955e1956 clinical features, 1955 progression and vitamin D status, 1957e1960 radiographic findings in X-linked hypophosphatemia, 875
2072 Osteoarthritis (OA) (Continued ) synovial changes, 1957e1958 total joint arthroplasty and vitamin D impact, 934e935 vitamin D receptor polymorphisms, 1958 Osteoblast bone remodeling, 323e325 calcium-sensing receptor function, 437 collagen type I expression and vitamin D regulation, 402e405 differentiation and vitamin D effects cell culture studies, 326e327 direct actions, 327e328, 414e417 voltage-sensitive calcium channels, 462e463 mechanical load response, 325e326 origins, 321, 330 parathyroid hormone function, 731 phosphate homeostasis regulation, 325 properties, 321e322 signaling pathway regulation by vitamin D mitogen-activated protein kinase, 328e329 Wnt, 329e330 survival and vitamin D influences, 463e464 transcriptional control of differentiation, 305e306 vitamin D receptor function, 437e438 Wnt signaling, 243e244 Osteocalcin (OCN) chromatin organization of gene, 312e313 mineralization role and vitamin D effects, 393 vitamin D regulation of expression, 106e107, 113, 154, 417, 1234e1235 Osteoclast adhesion and migration, 340 apoptosis, 339 bone metastasis and chemokines in osteoclast regulation, 340e341 calcitonin role, 739e740 calcium-sensing receptor function, 438 cannabinoid receptors, 341 functional overview, 336e337 fusion, 340 inhibition strategies, 341e342 origins, 335 osteoblast activator secretion, 340 parathyroid hormone function, 730e731 properties, 335e336 RANK signaling differentiation, 338e339 overview, 337e338, 731 precursor proliferation, 338 vitamin D effects endogenous vitamin D, 418e419 exogenous vitamin D, 418 vitamin D receptor function, 438e439 Osteocyte differentiation and vitamin D effects, 327e328 fibroblast growth factor-23 production coordination of renal phosphate
INDEX
handling with bone mineralization and turnover, 750 functions, 325e326 gene expression regulation by vitamin D dentin matrix protein, 1, 418 fibroblast growth factor-23, 417e418 parathyroid hormone function, 731 properties, 322e323 Osteoid maturation time, 850e851 measurement, 847 Osteomalacia, see also Vitamin D deficiency bone histomorphometry assessment focal osteomalacia, 852 generalized osteomalacia, 851e853 etiology, 817 mineralization defects, 396 radiographic findings overview, 815e816, 863e865 X-linked hypophosphatemia, 873 treatment, 816e820 Osteopenia, premature infants, 648 Osteopontin (OPN) arteriosclerosis studies, 1408 bariatric surgery effects, 1017 functions, 393 vitamin D effects, 416 Osteoporosis bone effects of vitamin D, 1129e1130 bone mineral density effects of calcium and vitamin D supplementation, 1133e1134 fracture prevention primary prevention calcium and vitamin D, 1137 meta-analysis, 1137e1139 safety of supplements, 1139 vitamin D, 1134e1137 secondary prevention, 1139e1140 vitamin D analog studies, 1140 prospects for study, 1140 genetic factors, 1130e1134 glucocorticoid-induced osteoporosis bisphosphonate therapy, 1240e1241 calcium absorption, 1238 mechanisms, 1234 serum vitamin D metabolites, 1236 vitamin D therapy bone mineral density response, 1238e1240 preparations, 1240 25-hydroxyvitamin D optimal levels, 1131e1133 mineralization defects, 396 parathyroid hormone analogs for management, 733 radiographic findings, 869 vitamin D analog management alfacalcidol, 1491e1495 eldecalcitol, 1492e1495 Osteoprotegerin (OPG) pregnancy, 680 RANKL binding, 730e731 Osteosclerosis, radiographic findings, 868
Ovarian cancer, vitamin D intake and risk analysis, 1576e1577 Ovary, CYP27B1 expression, 783 22-Oxa-1,25-dihydroxyvitamin D3, see Maxacalcitol Oxidative stress reactive nitrogen species, 1814e1815 reactive oxygen species, 1814 vitamin D effects, 1814e1815 p16, vitamin D regulation of expression, 219 p21 parathyroid tumor downregulation, 432 Runx2 interactions, 314 vitamin D regulation of expression, 160, 219 vitamin D upregulation in G1/S block, 1637e1640 p27 parathyroid tumor downregulation, 432 vitamin D regulation of expression, 219 vitamin D upregulation in G1/S block, 1637e1640 p35, cell differentiation induction via vitamin D, 1630e1631 p38, see Mitogen-activated protein kinases p53 photoprotection mechanisms of vitamin D compounds, 1949 vitamin D regulation of expression, 160 p160, vitamin D receptor activation, 106 p300 Runx2 interactions, 314 vitamin D receptor activation, 108, 112 PAD, see Peripheral arterial disease Palmitoylation, vitamin D plasma membrane receptor, 284 Pancreas, see also Diabetes bone loss in disease clinical features, 1308 epidemiology, 1308 pathogenesis, 1308 treatment, 1309 calbindin D function, 367e368 CYP27B1, 782e783 Pancreatic cancer, vitamin D studies, 1767e1768 Paracalcitol, leukemia studies, 1743 Parathyroid gland calcium-sensing receptor calcimimetic activators for hyperfunction treatment, 440e443 function, 430e433 CYP27B1, 782 development, 494e495 vitamin D receptor function, 430e433 knockout effects, 586 Parathyroid hormone (PTH), see also Hyperparathyroidism; Hypoparathyroidism analogs for osteoporosis management, 733 arteriosclerosis and receptor signaling, 1414e1415 biosynthesis, 493 bone function, 730e732, 748e749
INDEX
calcium extracellular fluid concentration regulation, 610e611 calcium-sensing receptor effects, 430e433, 727e728 chronic kidney disease, 1325e1326 collagen synthesis inhibition in bone, 304 CYP24A1 expression regulation, 1534 CYP27B1 induction, 155 development role, 625 discovery, 725 expression regulation calcium, 498e499 calreticulin, 496 feedback regulation, 137, 155 fibroblast growth factor-23, 501e502, 727 phosphate, 499, 727 prospects for study, 502e503 vitamin D, 495e498, 727 fibroblast growth factor-23 relationship, 752 gene promoter sequences, 494e495 structure, 493e494, 725e726 transcription factors, 725 glucocorticoid effects, 1237e1238 hypocalcemia defects in disease autoimmune polyglandular syndrome type, 1, 1095 DiGeorge sequence, 1094e1095 gene mutations, 1095 infiltrative disease, 1095 pseudohypoparathyroidism, 1096 radiation, 1095 surgery, 1095 prevention, 1092 idiopathic hypercalciuria levels, 1367 intact parathyroid hormone as vitamin D status marker in adolescence, 664 knockout mouse, 429, 436, 586 lactation, 681 MBD4 in signaling-induced transcriptional derepression, 232e233 messenger RNA binding proteins, 499e501 processing, 494 muscle actions, 2035 optimal vitamin D status, 1070e1071 phosphate homeostasis regulation, 325 pregnancy, 679 processing, 727 receptors, 728e729 renal function, 729e730 secretion regulation, 727e728 transcaltachia induction, 354e355 vitamin D metabolism effects, 1245e1247 vitamin D responsive element, 405, 495 Parathyroid hormone-related protein (PTHrP) lactation, 681 endochondral bone formation role, 302e303 knockout mouse, 302 development role, 626, 633 pregnancy, 679 history of study, 733
gene, 733 processing, 733e734 receptors, 734 nuclear hormone, 734 functions bone, 734e735 mammary gland, 735e736 placenta, 736 teeth, 737 vasculature, 736e737 pathophysiology, 737 cancer levels, 1597e1598 vitamin D metabolism effects, 1247e1248 Paricalcitol activity, 1438 cancer studies, 1438e1439 chronic kidney disease management animal model studies, 1556e1559 cardiovascular disease outcomes, 1563 end-stage renal disease, 1560e1563 stage 3-4 disease, 1559e1560 survival impact, 1561e1563 Parkinson’s disease (PD), vitamin D studies, 570 PBC, see Primary biliary cirrhosis PCOS, see Polycystic ovary syndrome PD, see Parkinson’s disease PDDR, see Pseudo-vitamin D deficiency Periodontal disease, vitamin D studies, 527 Periosteal reaction, radiographic findings, 871 Peripheral arterial disease (PAD), vitamin D status observational studies, 555 Peritonitis, extrarenal vitamin D production, 779 Peroxisome proliferator-activating receptord (PPAR-d), vitamin D regulation of expression, 219 Peroxisome proliferator-activating receptorg (PPAR-g) crystal structure, 104 prostate cancer management with ligands, 1692 PGC-1a, vitamin D receptor interactions, 201 PGE2, see Prostaglandin E2 Pharmacology, vitamin D body storage, 1055 clinical trials, 1045e1046 dosing adults, 1050e1052 infants, 1050 25-hydroxyvitamin D optimal level, 1048e1050, 1069, 1131e1133, 1148 ideal drug features, 1041 loading dose concept, 1058e1059 toxicity, 1059e1060 vitamin D2, 1059 metabolism overview, 821e823, 981e982, 1042e1044 pharmacokinetics, 1042, 1054e1056 prospects for study, 1060e1061 sun exposure as vitamin D dose, 1053e1054 toxicity and safety issues, 1056e1058
2073 vitamin D binding protein role, 1047 vitamin D2 supplementation, 1052e1053 PHEX dental rickets defects, 523e524 FGF23 regulation, 155 mineralization role, 394 X-linked hypophosphatemia defects, 1167e1168 Phosphate, see also Hyperphosphatemia; Hypophosphatemia body compartments, 612 disorder classification, 1159e1160 extracellular fluid concentration regulation, 612 fibroblast growth factor-23 role in homeostasis, 751e753 homeostasis regulation, 1155e1157 load disorders alcoholism, 1175 alkalosis, 1174 burns, 1175 clinical presentation, 1176e1178 diabetic ketoacidosis, 1175 glucose administration, 1174 malabsorption, 1174 nutritional recovery syndrome, 1175 phosphate deprivation, 1173e1174 treatment, 1178 parathyroid hormone expression regulation, 499 vitamin D metabolism modulation, 1157e1159 Phosphate absorption dietary phosphate intake as regulator, 356e357 malabsorption, 1174 overview, 618e619 phosphatonin regulation, 358 prospects for study, 358 renal reabsorption, see Kidney SLC34A2 role, 356 vitamin D receptor control, 357e358 vitamin D status effects, 357 Phosphoinositol 3-kinase signaling, see Akt Phosphoinositols, vitamin D receptor interactions, 284 Phospholipase A2 (PLA2), vitamin D regulation through nongenomic action, 281 Phospholipase C (PLC), vitamin D regulation through nongenomic action, 281e282 Photobiology, see also Sun exposure cancer risk assessment, 19 history of study, 4e5, 13e14 influences on vitamin D synthesis clothing, 16e17, 989 glass and Plexiglas, 16 latitude, 15, 17, 987e988 race, 19 season, 15, 17 sunscreen, 15e17 time of day, 15, 17 photolysis of provitamin D3 into previtamin D3, 14e15
2074 Photobiology (Continued ) skin synthesis of vitamin D, 981 sunlight and dietary vitamin D in health, 16, 18e19 tanning salons and vitamin D synthesis, 1752e1753 vitamin D synthesis assessment from sun exposure objective measures, 990e991 questionnaires, 991 Physical examination, metabolic bone disease diagnosis, 810e813 Physical performance, see Muscle PIC, see Preinitiation complex Pim-1, 430 Pin1, parathyroid hormone messenger RNA binding, 501 PKC, see Protein kinase C PLA2, see Phospholipase A2 Placenta calcium-sensing receptor function, 439e440 CYP27B1, 783e784 mineral transport, 626 parathyroid hormone-related protein function, 736 vitamin D receptor function, 439e440 Plasma membrane calcium pump renal function, 480 tissue distribution, 480, 482 Platelet function, vitamin D receptor knockout mouse studies, 1978 PLC, see Phospholipase C PMS, see Premenstrual syndrome Polycystic ovary syndrome (PCOS), vitamin D studies, 696e699 Postgastrectomy bone disease clinical features, 1303e1304 epidemiology, 1302e1303 pathogenesis, 1304 treatment, 1304 PPAR-d, see Peroxisome proliferatoractivating receptor-d PPAR-g, see Peroxisome proliferatoractivating receptor-g PPIs, see Proton pump inhibitors Preeclampsia, vitamin D status, 701e702 Pregnancy bone effects bone mineral content and density, 682e683 long-term effects, 684 immune tolerance, 1863 optimal vitamin D status, 1082 pseudo-vitamin D deficiency, 1193e1194 vitamin D and calcium metabolism, 679e680 vitamin D deficiency during pregnancy late effects on child asthma, 687 bone mass, 687 diabetes, 687e688 growth, 687 maternal effects, 685, 701e702 neonate effects, 685e687, 702e703
INDEX
vitamin D toxicity, 689 Pregnane X receptor (PXR) bile acid metabolism regulation, 1512e1514 vitamin D receptor comparison, 144e150 Preinitiation complex (PIC), vitamin D receptor interactions, 194, 198, 203 Premenstrual syndrome (PMS), vitamin D studies, 697 Primary biliary cirrhosis (PBC), bone loss clinical features, 1310 epidemiology, 1309e1310 pathogenesis, 1310 treatment, 1310e1311 Progesterone, vitamin D metabolism effects, 1254 Prolactin, vitamin D metabolism effects, 1251 Prostaglandin E2 (PGE2), vitamin D metabolism effects, 1255e1256 Prostaglandin synthesis, see Cyclooxygenase Prostate, see also Benign prostatic hyperplasia CYP27B1, 783 vitamin D receptor, 1678 vitamin D synthesis, 965e966 Prostate cancer androgen interactions with calcitriol, 1681e1682 animal models, 1682e1683 calcium-sensing receptor, 446 CYP24A1, 1680e1681 CYP27B1, 1681 epidemiology calcium intake studies, 974e975 prospects for study, 975 study types, 965e966 subclinical disease, 966 sunlight, 969, 972e973, 1676e1677 vitamin D receptor polymorphisms, 969e970 etiology, 1675 hormonal factors, 1676 treatment, 1675e1676 vitamin D analog studies, 1683e1684 angiogenesis inhibition, 1688e1689 anti-inflammatory effects, 1686e1688 anti-proliferative and pro-differentiation activity, 1677e1678 apoptosis, 1685 cell culture studies of growth effects cancer cell lines, 1678e1679 primary prostate cells, 1679 resistance, 1679e1680 virally transformed cells, 1679 cell cycle arrest, 1684e1685 cell differentiation, 1685e1686 chemoprevention studies, 1689e1690 clinical trials analogs, 1695 calcitriol, 1695 cholecalciferol, 1694e1695 summary, 1696e1697 combination therapy
calcitriol, 1695e1696 chemotherapy, 1693 CYP24A1 inhibitors, 1691 genistein, 1692e1693 glucocorticoids, 1692 nonsteroidal anti-inflammatory drugs, 1693e1694 peroxisome proliferator-activated receptor-g ligands, 1692 retinoids, 1692 growth factor modulation, 1686 intake and risk analysis, 1574e1575, 1677 metastasis inhibition, 1689 vitamin D deficiency hypothesis overview, 964e965 serological studies, 971e972 vitamin D receptor, 446 Protein kinase C (PKC) cell differentiation induction via vitamin D, 1630 CYP24A1 expression regulation, 1530 leukemia signaling and vitamin D effects, 1739 Protein phosphatases PP1c and vitamin D plasma membrane receptor interactions, 284 vitamin D regulation through nongenomic action, 279 Proton pump inhibitors (PPIs), vitamin D metabolism effects, 1265 PsA, see Psoriatic arthritis Pseudohypoparathyroidism, hypocalcemia, 1096 Pseudo-vitamin D deficiency (PDDR) biochemical findings, 1189 calcitriol therapy adults, 1193 children, 1192e1193 overview, 1191e1192 short-term effects, 1192 clinical manifestations, 1187e1189 CYP27B1 mutations, 1189e1190 history of study, 1187 pregnancy, 1193e1194 Psoriasis calcipotriol therapy, 1438 history of study, 1891 pathogenesis, 1891e1892 vitamin D system in skin, 1892e1894 vitamin D therapy biological effects, 1894e1895 children, 1896 clinical studies, 1895e1897 combination therapies, 1896 human immunodeficiency virus, 1896e1897 nails, 1896 scalp, 1896 Psoriatic arthritis (PsA) clinical features, 1966 vitamin D therapy, 1966 PTH, see Parathyroid hormone PTHrP, see Parathyroid hormone-related protein Puberty, see Adolescent
2075
INDEX
Pulse sequence, see Magnetic resonance imaging PXR, see Pregnane X receptor qBEI, see Quantitative backscattering electron imaging qCT, see Quantitative computed tomography Quantitative backscattering electron imaging (qBEI), mineralization analysis, 387 Quantitative computed tomography (qCT) metabolic bone disease diagnosis, 815 mineralization analysis, 385 Quantitative computed tomography bone mineral density determination, 884 high-resolution peripheral quantitative computed tomography applications cross-sectional studies, 901e902 longitudinal studies, 902e903 hardware, 900 image analysis biomechanical analysis, 903 densimetric analysis, 902 longitudinal analysis, 903 morphometric analysis, 902e903 overview, 901e902 positioning, 900e901 tomography, 901 RA, see Rheumatoid arthritis Race calcium absorption effects, 651e652 calcium metabolism effects, 669e670 diabetes type 2 differences, 1924 hypertension differences, 1924 Multiethnic Study of Atherosclerosis, 1409 skeletal accretion effects, 670e671 skin pigmentation and vitamin D synthesis effects, 14e15, 19, 984e986 vitamin D status influences in children and adolescents, 662, 955, 957 vitamin D synthesis effects, 14e15, 19 Radioimmunoassay (RIA) 1a,25-dihydroxyvitamin D, 836e837 24,25-dihydroxyvitamin D, 836 25-hydroxyvitamin D, 834e835 Radioreceptor assay (RRA)a,25dihydroxyvitamin D, 1, 835e837 RANK, osteoclast signaling differentiation, 338e339 overview, 337e338 precursor proliferation, 338 RANKL bone remodeling, 323e325 dental expression, 525 vitamin D regulation of expression, 98, 113, 152e154, 415 Rb, see Retinoblastoma protein RCC, see Renal cell carcinoma Reactive oxygen species (ROS), neuroprotective actions of vitamin D, 572
Regulatory T cells, see T cell Renal cell carcinoma (RCC), vitamin D studies, 1767 Renal osteodystrophy, see Chronic kidney disease Renineangiotensin system, see also Hypertension cascade, 707e708 chronic kidney disease, 716e718 CYP27B1/vitamin D receptor knockout studies, 589e590 functional overview, 707e708 inhibitors, 710e711 renin and regulation of production and secretion calcium-sensing receptor, 443e444 overview, 708e710 transcriptional regulation, 710 vitamin D receptor, 443e444, 589e590, 712 vitamin D regulation analogs as inhibitors, 713e714 inhibition mechanisms, 711e713 pathophysiological implications, 714e715 vitamin D status effects, 716, 1976e1977 Reproductive function, see also Lactation; Pregnancy animal models, 695e696 male reproduction, 700 vitamin D studies endometriosis, 697, 700 fertility, 700e701 polycystic ovary syndrome, 696e699 premenstrual syndrome, 697 Respiratory syncytial virus (RSV) clinical features of infection, 2000 intervention trials of vitamin D in acute respiratory infection, 2005e2006 observational studies of vitamin D, 2005 Response element, see Estrogen response element binding protein; Vitamin D response element Retinoblastoma protein (Rb), vitamin D regulation in G1/S block, 1640e1641 Retinoid X receptor (RXR) chronic kidney disease effects, 1336 CYP24A1 expression regulation, 1532e1533 keratinocyte stem cell function, 536 vitamin D analogs and heterodimerization response, 1442 vitamin D receptor transactivation role, 107e108, 110, 113e116, 140, 151e152, 1234 Rhabdomyolysis, hyperphosphatemia, 1175e1176 Rheumatoid arthritis (RA) bone loss, 1961 clinical features, 1960 fall risks, 1961 immune function, 1961 vitamin D receptor agonist studies, 1799e1800
polymorphisms, 1961e1962 vitamin D status animal studies, 1962 human studies, 1962e1964 RhoA vitamin D analog effects in benign prostatic hyperplasia, 1934 vitamin D transcriptional activity role, 239 RIA, see Radioimmunoassay Rickets, see also Hereditary vitamin-D resistant rickets; Hypophosphatemia; Vitamin D deficiency biochemical findings, 1111e1113 calcium deficiency, 1118e1120 clinical presentation, 1110e1111 dental rickets, 522e524 developmental studies, 629, 635, 652 epidemiology, 1108e1110 etiology, 817 fibroblast growth factor-23 mutations, 750 growth plate, 1114e1115 history of study, 4, 13e14, 1107e1108 klotho mutations, 753 New World primate, 251, 254e255 optimal vitamin D status in supplementation, 1068e1069 pathogenetic spectrum, 1120e1121 pediatric orthopedics, 929e931 prevention, 1116e1118 radiographic findings overview, 813e814, 860e863 X-linked hypophosphatemia, 875 radiological findings, 861e863, 1113e1114 treatment, 818e822, 1115e1116 vitamin D response element binding proteins humans, 255e258 New World primates, 255 Rifampicin, vitamin D metabolism effects, 1266e1267 RNA polymerase II heterogeneous nuclear ribonucleoprotein regulation, 262 Mediator-D complex recruitment, 198 RNA processing coupling, 200 ROCK, vitamin D analog effects in benign prostatic hyperplasia, 1934 ROS, see Reactive oxygen species Rosenheim reaction, 87 Roux-en-Y gastric bypass, see Obesity RRA, see Radioreceptor assay RSV, see Respiratory syncytial virus Runx2 bone gene expression within context of nuclear architecture, 311 coregulatory factor interactions, 314 endochondral bone formation role, 303, 313 epigenetic regulation, 312 osteoblast transcriptional control of differentiation, 305e306, 309 RXR, see Retinoid X receptor Salkowski reaction, 86 Sarcoidosis, vitamin D toxicity
2076 Sarcoidosis, vitamin D toxicity (Continued ) CYP27B1 role, 777e778 evidence for endogenous intoxication, 778e779, 1386e1387 extrarenal production, 778e779 SAXS, see Small-angle X-ray scattering SCF, see Stem cell factor SCFE, see Slipped capital femoral epiphysis Schizophrenia, vitamin D studies, 573e574 Scleroderma, vitamin D therapy, 1898 Sclerostin (SOST), bone health role, 243 Season vitamin D status influences in children and adolescents, 661e662 vitamin D synthesis effects, 15, 17 Sex differences calcium metabolism, 669e670 skeletal accretion, 670e671 vitamin D status in children and adolescents, 662 SFRP1 bone formation signaling, 305 bone health role, 243 SG340, 1475 SG396, 1475 SG402, 1475 Shipping, vitamin D, 90 SHR, see Spontaneously hypertensive rat Silica cartridge chromatography, vitamin D, 824, 826, 834 Skin diseasessee specific diseases vitamin D analogs for hyperproliferative skin disorder management, 1899e1900 Skin, see also Hair cycle; Keratinocyte barrier function and vitamin D role, 534 barrier function of vitamin D, 1815e1816 CYP27B1, 782 optimal vitamin D status in disease prevention, 1076e1077 pigmentation and vitamin D synthesis effects, 14e15, 19, 984e986 vitamin D synthesis, see Photobiology Skin cancer CYP27B1/vitamin D receptor knockout studies, 590 photocarcinogenesis, 1944e1946 sun exposure risk assessment versus vitamin D deficiency, 19, 1756e1757 ultraviolet exposure risks, 1751e1752 vitamin D effects basal cell carcinoma, 1754e1755 chemoprevention, 1752 keratinocyte function, 1753 melanoma, 1755e1756, 1764 prospects for study, 1757 risk potentiation studies, 1753 squamous cell carcinoma, 1755e1756 vitamin D therapy, 1898 SKIP, see NCoA62/SKIP SL117, 1470 SL137, 1470 SL142, 1470 SLC34A2
INDEX
phosphate absorption role, 356 vitamin D regulation of expression, 218 SLC34A3, hereditary hypophosphatemic rickets with hypercalciuria mutations, 1170 SLE, see Systemic lupus erythematosus Sleeve gastrectomy, see Obesity Slipped capital femoral epiphysis (SCFE), vitamin D and pediatric orthopedics, 933 Smad3, vitamin D receptor interactions, 200 Small-angle X-ray scattering (SAXS), mineralization analysis, 387 SMC, see Smooth muscle cell Smooth muscle cell (SMC), see also Vasculature calcium, phosphate, and vitamin D excess effects, 1141e1143 vitamin D function, 1974e1975 SMRT, vitamin D receptor repression, 201e202 Socioeconomic status, vitamin D status influences in children and adolescents, 662 Sodium-dependent phosphate transporters hereditary hypophosphatemic rickets with hypercalciuria defects, 1170 phosphate homeostasis, 1156e1157 Soft drinks, intake effects on bone health, 652 SOST, see Sclerostin Sp1, cell differentiation induction via vitamin D, 1631 S phase, see Cell cycle Sphingosine, photoprotection mechanisms of vitamin D compounds, 1948 Spontaneously hypertensive rat (SHR), 547e549 Squamous cell carcinoma, see Skin cancer Src vitamin D plasma membrane receptor interactions, 28 vitamin D regulation through nongenomic action, 281 SRC-1, vitamin D receptor activation, 106, 108, 111, 195e198 SRC-2, vitamin D receptor activation, 106, 108, 111, 195e198 SRC-3, vitamin D receptor activation, 106, 108, 111, 195e198 Star volume, bone histomorphometry, 856 Statins, vitamin D metabolism effects, 1260e1261 Status, see Vitamin D status Stem cell factor (SCF), 1732 Steroid and xenobiotic receptor (SXR), CYP24A1 expression regulation, 1533 Steroid hormone receptor coregulators, 252 mechanism of action, 254e255 Stomach, see Gastric cancer; Postgastrectomy bone disease Storage, vitamin D, 90
Stroke, cerebrovascular mortality as endpoint in vitamin D observational and interventional studies, 1983e1987 Strut analysis, bone histomorphometry, 856 Sun exposure, see also Photobiology; Skin cancer blood pressure studies, 715 cancer risk studies relative to vitamin D caseecontrol studies, 1578e1580 ecological studies of regional exposure, 1578 surrogate of vitamin D status, 1572 cultural practices and preferences, 989e990 DNA damage and repair, 1943e1944 immune suppression, 1944 influences on vitamin D synthesis clothing, 16e17, 989 glass and Plexiglas, 16 latitude, 15, 17, 987e988 race, 19 season, 15, 17 sunscreen, 15e17 time of day, 15, 17 multiple sclerosis inverse correlation, 1846 photocarcinogenesis, 1944e1945 photoprotection studies of vitamin D compounds DNA damage, 1946 immune suppression, 1946 mechanisms Akt, 1948 genomic and non-genomic pathways, 1947e1948 Jun N-terminal kinase, 1948 metallothionein, 1948 mitogen-activated protein kinase, 1948 nitric oxide synthase, 1948 p53, 1949 sphingosine, 1948 photocarcinogenesis, 1946 prospects, 1949e1950 sunburn cells, 1945e1946 prostate cancer, 967, 970e971 skin cancer risk assessment, 19 surrogate vitamin D dose, 1053e1054, 1572 tuberculosis historical perspective, 1818 vitamin D synthesis assessment from sun exposure objective measures, 990e991 questionnaires, 991 Suprasterols, photobiology, 14 SW123, 1478 SW931, 1478 SXR, see Steroid and xenobiotic receptor Systemic lupus erythematosus (SLE) clinical features, 1962, 1965 extrarenal vitamin D production, 779 immune function, 1965 vitamin D receptor agonist studies, 1801 vitamin D status animal studies, 1965 human studies, 1965e1966
INDEX
Tanning salons, vitamin D synthesis, 1750e1751 TC, see Tumor calcinosis T cell classification, 1789 CYP27B1/vitamin D receptor knockout studies, 594e596 experimental autoimmune encephalitis vitamin D amelioration mechanisms activation-induced cell death sensitivity, 1862 chemokine synthesis inhibition, 1862 nitric oxide synthase inhibition of induction, 1862e1863 overview, 1861e1862 vitamin D receptor overview, 1858 regulatory T cells, 1859e1860 Th1 cells, 1858 Th2 cells, 1858e1859 Th17 cells, 1859 optimal vitamin D status, 1078e1079 regulatory T cell enhancement by tolerogenic dendritic cells, 1793e1794 vitamin D receptor agonist effects, 1797e1798 T-cell factors (TCFs) bone formation signaling, 305 TCF-4 and cancer, 1605 TCF-4 and vitamin D regulation, 114, 117, 241e242 vitamin D inhibition of b-catenin complexes, 238e241 Wnt signaling, 236 TCFs, see T-cell factors TEM, see Transmission electron microscopy Testes, CYP27B1 expression, 783 Testosterone, vitamin D metabolism effects, 1253e1254 Tetracycline, labeling in bone histomorphometry, 846e847 TGF-b, see Transforming growth factor-b TGR5, bile acid studies derivatives for modulation, 1514e1515 metabolism regulation, 1514 Theophylline, vitamin D metabolism effects, 1267e1268 Thiazide diuretics idiopathic hypercalciuria management, 1375 vitamin D metabolism effects, 1262e1263 Thin-layer chromatography (TLC), vitamin D, 86 Thyroid cancer, vitamin D studies, 1763 Thyroid hormone, vitamin D metabolism effects, 1254e1255 Time of day, vitamin D synthesis effects, 15, 17 TIO, see Tumor-induced osteomalcia TLC, see Thin-layer chromatography TLRs, see Toll-like receptors TNF-a, see Tumor necrosis factor-a TNFSF11, vitamin D regulation of expression, 218e219
Toll-like receptors (TLRs) activation, 1812 classification, 1812 pathogen detection, 1811e1812 tuberculosis and vitamin D effects, 1816e1817 vitamin D effects on expression, 1780 TortellieJaffe reaction, 87 Total parenteral nutrition (TPN), bone loss clinical features, 1314 epidemiology, 1313e1314 pathogenesis, 1314 treatment, 1314 Toxicity, see Vitamin D toxicity TPN, see Total parenteral nutrition Trabecular bone, magnetic resonance imaging image processing coil sensitivity correction, 907 segmentation, 909e910 image registration, 912e913 morphometric analysis, 910e912 pulse sequences, 906e907 signal-to-noise considerations, 906e908 spatial resolution, 905e906 Trabecular bone pattern factor, bone histomorphometry, 854 Transcaltachia, 354e355 Transcriptional cycling impact, 221 models, 220e221 TRANSFAC, vitamin D response element discovery, 218 Transforming growth factor-b (TGF-b) breast cancer signaling, 1660 cancer growth inhibition via vitamin D, 1604 chronic kidney disease, 1334 experimental autoimmune encephalitis expression, 1860 prostate cancer, 1686 Transmission electron microscopy (TEM), mineralization analysis, 387 Transplantation, see Organ transplantation Trousseau’s sign, 811 TRPV4, osteoclast effects, 339 TRPV5 calcium renal tubular reabsorption, 435 functions, 372 gene genomic organization, 372 vitamin D regulation, 372 kidney expression, 366 knockout mouse, 585e586 osteoclast effects, 339 renal function, 480e482, 585e586 vitamin D regulation of expression, 114 TRPV6 calcium absorption role, 352, 585 developmental regulation, 627e628, 634 functions, 373 gene genomic organization, 372e373 vitamin D regulation, 373 knockout mouse, 585
2077 renal function, 480e482 tissue distribution, 372 vitamin D regulation of expression, 113e114, 153e154, 160, 218 Tuberculosis clinical relevance of vitamin D status, 1817e1818 drug effects on vitamin D metabolism, 1266e1267 endogenous vitamin D toxicity, 1387 extrarenal vitamin D production, 778e779 genetics, 1816 history of vitamin D and sunshine studies, 1818 innate immunity and vitamin D role, 1811, 1816e1817 vitamin D deficiency and therapy, 1778, 2002e2003 Tumor calcinosis (TC) clinical presentation, 1172e1173 gene mutations FGF23, 1173 GALNT3, 1173 klotho, 1173 Tumor-induced osteomalcia (TIO), 1159e1165 Tumor necrosis factor-a (TNF-a) inflammatory bowel disease therapeutic targeting, 1885e1886 vitamin D metabolism effects, 1256 TX522, 1462e1463 TX527, 1462e1463 Ulcerative colitis bone loss, see also Inflammatory bowel disease clinical features, 1306 epidemiology, 1305 pathogenesis, 1306 treatment, 1306 Ultraviolet radiation, see Photobiology; Sun exposure UNR, parathyroid hormone messenger RNA binding, 500e501 Valproic acid, leukemia treatment with vitamin D combination therapy, 1741 Vascular endothelial growth factor (VEGF), see also Angiogenesis endochondral bone formation role, 303 Vasculature, see also Angiogenesis; Arteriosclerosis; Atherosclerosis; Heart bimodal response of vitamin D dose, 1403e1404 clinical cardiovascular outcomes in vitamin D observational and interventional studies, 1987e1989 coagulation system and vitamin D effects, 1978e1979 CYP27B1 expression, 542e543 inflammation, 1977e1978 mortality as endpoint in vitamin D observational and interventional studies, 1983e1987
2078 Vasculature (Continued ) parathyroid hormone-related protein function, 736e737 platelet function, 1978 smooth muscle cells calcium, phosphate, and vitamin D excess effects, 1141e1413 vitamin D function, 1974e1975 sympathetic nervous system and vitamin D effects, 1979 vitamin D intervention studies in animal models arteriosclerosis, 549e550 hypertension, 548e549 vitamin D receptor functions animal studies Dahl salt-sensitive rat, 547e548 deoxycorticosterone acetate-salt rat, 547 salt-induced hypertension, 547e548 spontaneously hypertensive rat, 547 blood coagulation and fibrinolysis, 546e547 cell studies angiogenesis, 543e544 endothelium, 543 inflammation, 544 vasoactivity, 543 overview, 541e542, 1974 prospects for study, 557 smooth muscle studies calcium deposition, 546 cell proliferation, 545e546 vasoactivity, 544e545 VDR, see Vitamin D receptor VDRE, see Vitamin D response element VDRL-1, 1499, 1506 VDRM2, 1499e1501 VDS, see Vitamin D3 sterol VDUP1, see Vitamin D-upregulated protein1 VEGF, see Vascular endothelial growth factor Vertical banded gastroplasty, see Obesity VID400, 36, 1540e1541 VITAL, see Vitamin D and Omega-3 Trial Vitamin A bile acid synthesis suppression, 764e765 discovery, 3e4 excess considerations in vitamin D status, 1002 Vitamin B complex, discovery, 3e4 Vitamin D, see also specific forms circulating concentrations in diseases, 837 definition, 979 detection, see also specific forms chemical synthesis and assays, 84e87 extraction, 825e826 high-performance liquid chromatography, 85e86, 826e827 serum, 825 silica cartridge chromatography, 826
INDEX
food fortification, 653, 992, 994, 997e998, 1000 glucocorticoid effects on metabolism, 1236e1238 history of study commercial production, 73e74 discovery, 4 hormonal form, 6e7, 97e99 isolation of nutritional forms, 5 metabolites, 7e8 photobiology, 4e5, 13e14 physiological functions intestinal absorption of calcium and phosphorous, 5 calcium mobilization from bone, 5e6 renal reabsorption of calcium and phosphorous, 6 intake recommendations, see also Vitamin D status animals, 88 humans, 18, 20, 87 infants and children, 650e651 intake, see Vitamin D status intestinal absorption, 1300 intracellular vitamin D binding proteins, 265e266 metabolism aluminum toxicity effects, 1265e1266 calcitonin effects, 1249e1250 drug effects anticonvulsants, 1257e1258 bisphosphonates, 1262 caffeine, 1267 calcium channel blockers, 1263 cimetidine, 1264 cyclosporine A, 1268 ethanol, 1259e1260 fluoride, 1268 glucocorticoids, 1258e1259 heparin, 1263e1264 ketoconazole, 1260 lithium, 1268e1269 olestra, 1268 orlistat, 1268 proton pump inhibitors, 1265 rifampicin, 1266e1267 statins, 1260e1261 theophylline, 1267e1268 thiazide diuretics, 1262e1263 estradiol effects, 1252e1253 fibroblast growth factor-23 effects, 1248e1249 growth hormone effects, 1250 insulin effects, 1251e1252 insulin-like growth factor-I effects, 1250e1251 interferon-g effects, 1256 leptin effects, 1249 overview, 823e825, 981e982, 1042e1044, 1300e1302 parathyroid hormone effects, 1245e1247 parathyroid hormone-related protein effects, 1247e1248 progesterone effects, 1254 prolactin effects, 1251
prostaglandin E2 effects, 1255e1256 rifampicin, 1266e1267 testosterone effects, 1253e1254 thyroid hormone effects, 1254e1255 tumor necrosis factor-a effects, 1256 osteoporosis supplementation studies bone mineral density effects, 1133e1134 fracture prevention primary prevention, 1134e1139 secondary prevention, 1139e1140 pharmacology, see Pharmacology, vitamin D phosphate modulation of metabolism, 1157e1159 ring numbering, 1461 storage and shipping, 90 Vitamin D2 commercial applications, 88e89 dietary considerations, 1001 loading dose, 1059 pharmacokinetics, 1042 structure, 821 supplementation, 1052e1053 Vitamin D analogs, see also specific analogs acyclic analogs, 1479e1482 A-ring derivatives, 1431 hydroxyls, 1430 benign prostatic hyperplasia studies inflammation inhibition, 1935e1936 lower urinary tract symptoms, 1931e1932 overview, 1803 prospects for study, 1937e1938 prostate cell growth control, 1933e1934 urethra dysfunction, 1936e1937 bile acid derivatives, see also Lithocholic acid derivatives bone loss management in organ transplantation, 1294e1296 calcemic action minimization, 1429e1430, 1438 cardiovascular risks, 1418e1419 CD-ring analogs C-ring, 1467e1469, 1474, 1477 D-ring, 1470e1477 identification, structure, and biological characterization, 1502e1504 overview, 1431e1432, 1461, 1465, 1467 CF-ring analogs, 1477e1478 chronic kidney disease management analog types, 1556 animal model studies, 1556e1559 cardiovascular disease outcomes, 1563 end-stage renal disease, 1560e1563 prospects for study, 1563e1564 stage 3-4 disease, 1559e1560 survival impact, 1561e1563 cis-triene structure, 1430 clinical availability, 1438, 1492 cyclooxygenase inhibition, 1451 CYP24A1 metabolism, 1447e1449 decalin analogs, 1465e1466 diabetes type 1 prevention, 1833 14-epi analogs, 1462e1465
INDEX
E-ring analogs, 1478e1480 hyperproliferative skin disorder management, 1899e1900 hypocalcemia management, 1100e1103 intracellular vitamin D binding protein binding, 1451 kidney stone formation risks, 1375 leukemia studies C-16-enes, 1742e1743 calcipotriol, 1741 20-epi vitamin D3, 1743 gemini analogs, 1743 1a-hydroxyvitamin D3, 1741 paracalcitol, 1743 miscellaneous compounds, 1505e1507 non-genomic rapid actions, 1449e1451 non-secosteroidal compounds bis- and tris-aromatic triols, 1503, 1505 diarylmethane ligands characterization in vivo, 1501e1502 identification, structure, and biological characterization, 1498e1500 overview, 1481, 1483e1484, 1497e1498 osteoporosis management alfacalcidol, 1491e1495 eldecalcitol, 1492e1495 prostate cancer studies, 1683e1684, 1695 psoriasis management, 1894e1897 ring numbering, 1461 side chain modifications, 1432e1433 toxicity, 1384e1385 vitamin D binding protein interactions, 1445e1447 vitamin D receptor binding, see also Vitamin D receptor 22-butyl-1,24-dihydroxyvitamin D3 derivative complexes, 182e185 C-2a-substituted vitamin D analog complexes, 179e181 coactivator/corepressor recruitment response, 1443e1444 DNA binding response, 1443 ligand binding, 1441 ligand-binding domain bound to superagonists 14-epi analogs, 178e179 20-epi analogs, 176, 178 2-substituted, 19-nor analogs, 178 ligand-dependent regulation, 1444e1445 nuclear translocation effects, 1442 phosphorylation response, 1442 retinoid X receptor heterodimerization response, 1442 zVDR ligand-binding domain bound to vitamin D, 175 Vitamin D binding protein (DBP) actin complex, 65e66 arteriosclerosis studies, 1410e1411 candidate gene studies, 1032e1033 functions overview, 57 vitamin D transport, 60e65 gene, 57e58 inflammation role, 66e67 knockout mouse, 57, 62e63, 395
multiple sclerosis levels, 1853 polymorphisms, 65 prospects for study, 67e68 structure, 58e60 synthesis and turnover, 60 vitamin D analog interactions, 1445e1447 vitamin D pharmacology role, 1047 vitamin D toxicity and levels, 1391e1392 Vitamin D deficiency, see also Pseudovitamin D deficiency; Rickets; Vitamin D status; specific diseases adiposity effects, 770e773 age-dependent signs and symptoms, 807e808 atherosclerosis observational studies, 555 autoimmune disease, 1799 beta cell function, 1827e1828 brain effects of developmental vitamin D deficiency behavioral effects in later life, 568e569 dietary restriction, 567e568 gene expression effects in later life, 568 structural alterations, 576 calcium extracellular fluid concentration regulation, 611 cancer studies with knockout mice, 590e593 chronic kidney disease observational studies, 556e557, 717 colorectal cancer pathogenesis, 1713e1714 heart failure observational studies, 556 history of study, 861e862 hypertension observational studies, 554, 715 hypocalcemia etiology CYP27B1 deficiency, 1098 malabsorption, 1098 malnutrition, 1097e1098 vitamin D receptor defects and resistance, 1098e1099 infants, 636e637 insulin resistance and diabetes observational studies, 557 lactating mothers and effects on infant, 688e689 metabolic bone disease diagnosis bone scintigraphy, 817 dual-energy X-ray absorptiometry, 815 laboratory testing, 813, 815 medical history chief complaint, 809 family history, 812 past history, 811 present illness, 810e811 review of systems, 812 social history, 811e812 physical examination, 812e815 quantitative computed tomography, 817 X-ray, 815e816 mineralization animal model studies, 390e396 multiple sclerosis, 1847 myopathy, 2024e2025 optimal vitamin D status, see Vitamin D status
2079 organ transplantation studies pre-transplantation heart, 1292 kidney, 1292e1293 liver, 1292 lung, 1292 post-transplantation heart, 1294 long-term, 1293e1294 perioperative, 1293 peripheral arterial disease observational studies, 555 pregnancy late effects on child asthma, 687 bone mass, 687 diabetes, 687e688 growth, 687 maternal effects, 685, 701e702 neonate effects, 685e687, 702e703 prevalence in children and adolescents, 659e660 prostate cancer overview, 965e966 serological studies, 970e972 risk groups, 959 skin cancer risk assessment versus vitamin D deficiency, 18 sun exposure in prevention, 16e19 Vitamin D External Quality Assessment Scheme, 839e840 Vitamin D-25-hydroxylase, see CYP2R1; CYP3A4; CYP27A1 Vitamin D and Omega-3 Trial (VITAL) ancillary studies, 2052e2053 blood collection, 2050 compliance monitoring, 2051 design, 2047e2048 follow-up and endpoints, 2050e2051 goals, 2043 monitoring and safety, 2051 participants characteristics, 2049 eligibility criteria, 2048 recruitment and enrollment, 2048e2049 randomization, 2049 rationale, 2046 statistical power, 2052 vitamin D dose, 2046e2047 intake assessment, 2049e2050 Vitamin D receptor (VDR) activation coactivators, 106, 139, 195e201 differential activation, 106 integrated model of coregulator activity, 203e204 analog effects, see Vitamin D analogs bacterial artificial chromosome clones bone cell activity, 123 transgenes in mice, 123e124 bile acids binding, 765 detoxification role, 764e765 synthesis regulation, 764
2080 Vitamin D receptor (VDR) (Continued ) bile acids interactions, 1515e1517 metabolism regulation, 1514 biochemical properties, 100e101 brain function, 566e567 breast cancer expression and regulation, 1663 polymorphisms and risk, 1668 prognostic significance, 1664 resistance, 1664 breast function, 440 calcium absorption control in intestine, 351 candidate gene studies, 1031e1032 chondrocyte function, 436e437 chronic kidney disease alterations homologous upregulation defects, 1333e1335 miscellaneous resistance mechanisms, 1335e1337 polymorphisms, 1335 colon cancer, 444e446 conformational ensemble model alternative ligand-binding pocket evidence, 286e289 overlapping two-pocket model, 285e287 overview, 284e285 crystal structure antagonism structural basis adamantyl analog complexes, 186 calcipotriol modification for receptor antagonism, 184e186 22-butyl-1,24-dihydroxyvitamin D3 derivative complexes, 182e185 C-2a-substituted vitamin D analog complexes, 179e181 hVDRD bound to vitamin D, 172e175, 177 ligand-binding domain bound to superagonists 14-epi analogs, 178e179 20-epi analogs, 176, 178 2-substituted, 19-nor analogs, 178 lithocholic acid derivative binding unmodified lithocholic acid, 1517e1519 3-ketolithocholic acid, 1519 lithocholic acid acetate/propionate, 1519 nonsteroidal ligand complexes YR301erVDR ligand-binding domain complex, 181 zVDR ligand-binding domain bound to CD578 analog, 181e182 overview, 139, 148 zVDR ligand-binding domain gemini binding, 182 vitamin D binding, 175 discovery, 8e9, 99 domains DNA-binding domain, 103e104, 147e148, 213, 1201 E/F domain homology between nuclear receptors, 102e103 ligand-binding domain, 104e105, 148e150, 213, 1201 overview, 102e103, 139e140, 171e172
INDEX
experimental autoimmune encephalitis and expression on T cells overview, 1858 regulatory T cells, 1859e1860 Th1 cells, 1858 Th17 cells, 1859 Th2 cells, 1858e1859 functions bone and mineral regulatory gene expression, 154 feedback control of vitamin D actions, 154e156 longevity, 160e161 neoclassical functions of liganded and unliganded receptor, 158e160 overview, 97e99, 144e145 Wnt/b-catenin signaling crosstalk, 156e157 gene cloning, 101e102 enhancers, 121e123 intracellular receptor gene family, 102 organization, 120e121 genetic hypercalciuric rat activity, 1371e1373 genomic versus nongenomic signaling cross-talk, 282e283 discrimination, 271 nongenomic actions examples, 280, 289e291 ion channels, 279 kinases, 281 phosphatases, 279 phospholipases, 281e282 structural determinants, see Vitamin D3 sterol W286R mutation and calcium signaling, 290e291 genomics bone cell gene transactivation, 113, 117e118 chromatin immunoprecipitation, 111e115 colon cancer gene transactivation, 114e115 DNA binding and dynamics at target genes, 111e112 enhancer linking to genes, 118 intestinal gene transactivation, 113e114, 116e117 overarching principles of gene regulation, 116e118 glucocorticoid regulation, 1235 heart developmental role, 550e551 expression, 550 fibrosis animal studies, 552e553 vitamin D intervention studies, 554e555 hypertrophy animal studies, 551e552 vitamin D intervention studies, 553 hereditary vitamin-D resistant rickets defects
coactivator binding mutations, 1217 compound heterozygous mutations, 1217e1218 deletions, 1214 DNA-binding domain mutations, 1210e1211 heterodimerization-affecting mutations, 1216e1217 ligand-binding domain mutations, 1214e1216 miscellaneous mutations, 1218 nonsense mutations, 1211e1213 overview, 1209e1210 RNA splicing-disrupting mutations, 1213e1214 immunoregulation by agonists, 1789e1799 intestinal function, 439 keratinocyte function, 444 kidney function, 434e435 knockout mouse, 141, 143e144, 160, 481, 497e409, 523, 525e526, 536, 549, 570, 584e597, 627, 747, 772e774, 1076e1077, 1130, 1201, 1734, 1978, 2034e2035 ligand structures and specificity, 278, 291 membrane binding, 284 nuclear receptor superfamily, 144e147 osteoblast function, 437e438 osteoclast function, 438e439 parathyroid function, 430e433 parathyroid hormone expression regulation, 495e498 phosphate absorption control in intestine, 357 placenta function, 439e440 plasma membrane receptors palmitoylation, 284 structural/scaffolding proteins, 283e284 types, 283 polymorphisms, 148, 446, 527, 669, 966e970, 1593e1594, 1779, 1851e1852, 1958, 1961e1962, 2005, 2035 post-translational modification, 124 renin secretion regulation, 443e444, 589e590 repressors corepressors, 201e203 integrated model of coregulator activity, 203e204 response element, see Vitamin D response element stability, 124 superagonist structure-based design, 187e188 tissue distribution, 99e100, 137, 430e440 transcriptional regulation of expression heterologous transcriptional regulation, 119e120 homologous transcriptional regulation, 120 overview, 118e119 tissue-specific expression, 120
INDEX
transcriptional transactivation, see also specific genes; Vitamin D response element chromatin-looping model, 152e153 conformational change, 105, 151 coregulators, 108e110, 115, 193e204, 212e213 osteocalcin as model, 106e107 overview, 150e154, 1202e1203 polarity of response element binding, 108 response elements in genes, 140e143 retinoid X receptor role, 107e108, 110, 113e116, 140 target genes analysis, 220 cell cycle regulation, 219 classical targets, 218e219 relative expression, 219 transcriptional cycling, 220e222 vascular functions animal studies Dahl salt-sensitive rat, 547e548 deoxycorticosterone acetate-salt rat, 547 salt-induced hypertension, 547e548 spontaneously hypertensive rat, 547 blood coagulation and fibrinolysis, 546e547 cell studies angiogenesis, 543e544 endothelium, 543 inflammation, 544 vasoactivity, 543 overview, 541e542 smooth muscle studies calcium deposition, 546 cell proliferation, 545e546 vasoactivity, 544e545 vitamin D toxicity role, 1389e1390 Vitamin D response element (VDRE) binding proteins in vitamin D resistance humans, 255e258 New World primates, 255 bone type I collagen gene, 405 clusters, 215e216 CYP24A1, 1529e1530 DR3-type, 214e215 DR4-type, 215 DR6-type, 215 E-box negative elements coregulator switching in transrepression, 229 mediation of transrepression, 228e229 ER9-type, 215 gene type distribution, 140e143 genome-wide analysis, 116 genome-wide screening, 218 multiple elements per gene, 216e217 negative elements, 217, 228e229 parathyroid hormone, 405, 495 screening in silico, 217e218 vitamin D receptor binding overview, 214 polarity, 108, 152
Vitamin D status, see also Vitamin D deficiency adolescents, see Adolescent bariatric surgery effects, 1018e1019 breast cancer trials, 1667e1668 calcium absorption effects, 350e352 chronic kidney disease observational studies, 556e557 determinants, 980e982 diabetes, 55, 671, 687e688, 702 food fortification, 653, 992, 994, 997e998, 1000 genome-wide association studies of serum levels, 1036e1037 geographic studies Africa, 955, 957 Asia, 955e956 ethnic differences, 662, 957, 959 Europe, 950, 952e954 global studies using central laboratory facility, 957 Middle East, 954e955 North America, 947e950 nutrition, 959 Oceania, 957e958 overview, 947 South America, 950e951 intake assessment, 991e992 recommendations animals, 87e88 estimation, 982e984 humans, 18, 20, 87 overview, 992e993 United States, 993, 995 latitude influences, 661e662, 987e988 lifestyle strategies for improvement, 1002e1004 multiple sclerosis analysis, 1846e1847 deficiency in patients, 1847 longitudinal disease, 1848 relapse and remission, 1848e1850 risk analysis, 1847e1848 sun exposure inverse correlation, 1846 obesity, 662, 773e774 optimal vitamin D status adults and elderly, 1069e1076 cancer, 1077e1078 cardiovascular and metabolic disease, 1079e1080 diabetes, 1080e1081, 1924 25-hydroxyvitamin D optimal level, 1048e1050, 1069, 1131e1133, 1148 hypertension, 1924 immune function, 1078e1079 metabolic syndrome, 1080e1081 muscle function, 1081 musculoskeletal health, 1148 overview, 620e622, 1067e1068, 2045e2046 pregnancy, 1081 prospects for study, 1082e1083 rickets prevention with vitamin D supplementation, 1068e1069
2081 skin disease, 1076e1077 orthopedic impact, see Orthopedics osteoarthritis, 1955e1958 phosphate absorption effects, 357 physiological factors, 983e988 rheumatoid arthritis, 1962e1964 sex differences in children and adolescents, 662 social and cultural factors, 988e990 sun exposure as surrogate of vitamin D status, 1572 systemic lupus erythematosus, 1962e1965 vitamin D supplementation, 999e1002, 1020e1021 vitamin D synthesis assessment from sun exposure objective measures, 990e991 questionnaires, 991 Vitamin D toxicity, see also Hypercalcemia arteriosclerosis studies, 590, 1409e1411, 1415e1416 clinical manifestations, 1392e1393 diagnosis, 1393e1394 endogenous intoxication diagnosis, 794 diseases cancer, 781 lymphoma, 1388e1389 noninfectious granuloma-forming diseases, 780e781 overview, 779 peritonitis, 779 sarcoidosis, 778e780, 1386e1387 systemic lupus erythematosus, 779 tuberculosis, 780e781, 1387 25-hydroxyvitamin D production, 1385e1386 hypercalcemia prevention, 795 treatment, 795e796 hypercalciuria treatment, 793e794 prevention, 795 risk assessment, 794e795 screening, 795 treatment, 795e796 exogenous toxicity 1,25-dihydroxyvitamin D, 1384 25-hydroxyvitamin D, 1381e1384 vitamin D analogs, 1384e1385 hyperphosphatemia, 1172 loading dose, 1059e1060 mechanisms CYP24A1 inhibition, 1391 CYP27B1 control, 1390e1391 general mechanisms, 1389 vitamin D binding protein levels, 1391e1392 vitamin D receptor role, 1389e1390 pregnancy, 689 radiographic findings in intoxication, 880, 883 supplement pharmacology and safety issues, 1056e1058 treatment, 1394e1395
2082 Vitamin D3 sterol (VDS) chemistry, 271e272 conformational dynamics of seco-B-rings and A-rings, 272e274 conformational ensemble, 275e276 metabolism, 275 nongenomic activity structural features and molecular dynamics agonists, 276e278 antagonists, 277e278 pharmacokinetics, 1042 side-chain conformational dynamics, 274e276 structure, 823 Vitamin D-upregulated protein-1 (VDUP1), cancer role, 1602 Voltage-sensitive calcium channels (VSCCs) calcium homeostasis overview, 457 families, 458 osteoblast mediation via vitamin D differentiation, 462e463 survival, 463e464 subunits, 458e459 vitamin D actions, 460e461 VSCCs, see Voltage-sensitive calcium channels Weaning, see Lactation Wheezing clinical features in children, 2001 intervention trials of vitamin D in acute respiratory infection, 2005e2006 observational studies of vitamin D, 2003e2004 WINAC, vitamin D receptor interactions, 200e201, 227e230 Windaus synthesis, 7-dehydrocholesterol, 76 Wnt bone formation signaling, 304e305 bone homeostasis, 242e243 cancer role, 237e238, 241e242 coreceptors, 156, 235 DICKKOPF inhibition and vitamin D regulation, 241e242
INDEX
hair cycle, 157e158, 593 inhibitors, 237 keratinocyte stem cell function, 536 knockout mice, 242 osteoblast signaling, 243e244 osteoblast signaling and vitamin D effects, 329e330 signaling noncanonical pathways, 237 overview, 235e237 skin signaling, 244e245 vitamin D inhibition, 238e241 WU442, 1470 WU507, 1470 WU515, 1470 WY10061, 1471 WY10071, 1472 WY1036, 1471 WY1037, 1472 WY1038, 1471 WY1039, 1473 WY1046, 1472 WY1048, 1473 WY1106, 1472 WY1112, 1471 WY1113, 1473 WY1116, 1472 WY578, 1474 WY619, 1474 WY718, 1474 WY722, 1473 WY821, 1471 WY838, 1474 WY906, 1474 WY9361, 1471 XLH, see X-linked hypophosphatemia X-linked hypophosphatemia (XLH) clinical presentation, 1165 CYP24A1 dysregulation, 1535e1536 pathogenesis, 1169 pathophysiology, 1165e1167 PHEX defects, 1167e1168 radiographic findings bone modeling abnormalities, 876
extraskeletal ossification, 876e877 osteoarthritis, 877 osteomalacia, 875e876 rickets, 875 recessive disease, 1172 treatment, 1169 XM612, 1468 XM615, 1469 XM720, 1469 XM804, 1469 XM806, 1468 X-ray acidemia, 880 aluminum toxicity, 874e875 brown tumor, 870e871 differential diagnoses, 880, 882e883 hyperparathyroidism renal osteodystrophy, 869e870 secondary hyperparathyroidism, 867e869 metabolic bone disease diagnosis, 815e816 metastatic calcification, 871e874 oncogenic rickets/osteomalacia, 879e881 osteomalacia, 865e867 osteoporosis, 871 osteosclerosis, 870 periosteal reaction, 871 rickets, 862e865, 1113e1114 vitamin D intoxication, 880, 883 X-linked hypophosphatemia bone modeling abnormalities, 876 extraskeletal ossification, 876e877 osteoarthritis, 877 osteomalacia, 875e876 rickets, 875 X-ray diffraction (XRD) crystallography, see Crystal structure mineralization analysis, 387e388 XRD, see X-ray diffraction Yeast, irradiation for biofortification, 999 YR301, 1500 ZG1368, 1468, 1504 ZG1423, 1469
Color Plates
A
D
F
C
B
E
G
H
Children with HVDRR. A, Patient F70 with total alopecia; B, the child exhibited bowed legs; C, X-ray of wrist; D, X-ray of bowed legs; E, X-ray of legs after 4 years of calcium and calcitriol therapy. HVDRR children with partial alopecia. F, Patient F69; G, Patient F78; H, Patient F79. Panels (AeE) reproduced from [171] with permission of the American Society for Bone and Mineral Research. Panel (F) reproduced with permission from Molecular Genetics and Metabolism [177]. Panels (GeH) reproduced with permission from the Journal of Pediatric Endocrinology and Metabolism [149].
FIGURE 65.1
(A)
Helix 9 Helix 8
Helix 1
Helix 7 Helix 3 Helices 4/5 DRIP205 H393 F418 Helices 10/11
Helix 12
(B)
R270
S233 3.01 H393 2.89 2.59
2.90
2.69
Y143 2.48
3.75 3.86
3.72 3.53
H301
2.71 S274
Crystal structure of the rat VDR ligand-binding domain complexed with LCA and DRIP205. (A) Overall structure of VDReLBD (ribbon) complexed with LCA (atom-type line) and DRIP205 peptide (ribbon). The residues (H393 at helix 11 and F418 at helix 12) forming the Hep bond are shown with stick model (atom-type color) and highlighted with a blue circle. The insert is a close-up view of the Hep ˚ from each carbon of the benzene interaction. An imidazole proton of H393 is placed on the center of the p electron cloud of F418 within 2.9e3.2 A ring. A ribbon model is shown with secondary structure colors (red helix and cyan b-sheet) except for magenta helix 12 (H12) and green DRIP205 peptide. (B) Hydrogen bond network of LCA. LCA and its interacting amino acid residues (stick model, atom-type color) and 1,25(OH)2D3 (blue line with only oxygen shown in blue ball) are overlaid with the superimposed protein. Water molecules incorporated into the ligand-binding pocket are shown as red balls. Hydrogen bonds among LCA, the ligand-binding pocket residues and the water molecules are shown with red dotted lines and hydrophobic interactions with blue dotted lines. (C) Side view of overlaid LCA (stick, atom-type color) and 1,25(OH)2D3 (stick, blue carbon and red oxygen). (D) LCA (CPK model with white carbon and red oxygen) and water (CPK, magenta) molecules in the Connolly channel surface (transparent yellow) of rat VDR-LBD.
FIGURE 79.2
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FIGURE 79.2 (continued).
(A)
R270 S233
H393
2.84
2.97 2.78
2.73 2.59
Y143
2.84 3.41
2.43
3.61 2.69 3.55 S274 H301
(B) Helix 12
R270 V414 F418 3.87 3.51 H393
Y143
S233 3.96
2.92
3.37 2.84
4.78 2.42
3.70 3.53
2.92
3.60 H301 S274
FIGURE 79.3 3-Keto-LCA and LCA acetate docked in the rat VDR ligand-binding pocket and their interacting residues. (A) 3-Keto-LCA and interacting residues. (B) LCA acetate and interacting residues. The ligands and ligand-binding pocket residues are shown with stick and line models, respectively, both in atom-type color. Hydrogen bonds are shown with red dotted lines and hydrophobic interactions with blue dotted lines.
Control
(A)
(B)
(C)
CKD
a
b
a
b
a
b
FIGURE 80.3 Immunohistochemisty demonstrating elevated CYP24A1 protein expression in renal tissue biopsied from patients with type II diabetes and diabetic nephropathy. Immunperoxidase staining of CYP24A1 protein in the (A) renal artery, (B) medulla, and (C) cortical tubules from DN and age-matched controls is presented. Arrows indicate localized CYP24A1 staining to the apical membrane of the proximal tubules in control tissue (Arrows; Aa). DN tissue showed marked and diffuse cytoplasmic staining in the proximal tubular (Arrows; Ab), as well as cortical (Bb) and medullary (Cb) distal tubules. Original magnification was 400 for artery and 200 for medulla and cortical tubules. From [8].
(A)
(B)
(C)
FIGURE 81.1 In an animal model of uremia, Cardus et al. found that, when compared with control animals (A), both paricalcitol (B; 3 mg/kg) and calcitriol (A; 1 mg/kg) increased aortic calcification over an 8-week period; this effect was more marked with calcitriol. Reproduced with permission from [14].
Histological demonstration of morphological changes in lesional psoriatic skin after 6 weeks of topical treatment with calcitriol (15 mg/g, b) and calcipotriol (50 mg/g, c). a ¼ lesional psoriatic skin before treatment. d ¼ non-lesional psoriatic skin. Notice strong reduction of epidermal thickness after topical treatment with vitamin D analogs. Hematoxylin-eosin staining. Original magnification 200.
FIGURE 97.1
FIGURE 97.2 Immunohistochemical demonstration of 1,25-dihydroxyvitamin D3 receptors (VDR) in human skin. Notice strong nuclear VDR immunoreactivity in cells of all layers of the viable epidermis (arrows). Labeled avidin-biotin technique using mAb 9A7g directed against VDR. Original magnification 400.
FIGURE 97.3 Immunohistological detection of transglutaminase K in lesional psoriatic skin before treatment (a), lesional psoriatic skin after 6 weeks of topical treatment with calcipotriol (50 mg/g, b), and in non-lesional psoriatic skin (c). Notice strong staining for transglutaminase K in all epidermal cell layers of lesional psoriatic skin before treatment (a, arrows). In contrast, after 6 weeks of topical treatment with calcipotriol staining in lesional psoriatic skin (b, arrows) is restricted to the upper layers of the viable epidermis, a staining pattern that is characteristic for non-lesiona psoriatic skin (c, arrows). Original magnification 160.
The cellular structure of the skeletal muscle cell is shown. As evident from this diagram skeletal muscle is composed of specialized cells that possess a dominant organelle, the myofibril. The important cell structures that regulate muscle contracMyofibrils tion are also shown, as is their association with the myofibril structures. These structures include the mitochondria, the sarcolemma, the transverse tubule (ttubule) and the terminal cisternae. These evolved A band structures are all essential components for the required ATP synthesis and regulation of intracellular calcium essential for muscle excitationecontraction coupling. I band Interesting, as shown, there are multiple nuclei present in the fused mature muscle cell. The nuclei are located outside the myofibril bundles and are shown as flattened structures adjacent to the sarcolemma. Z line
FIGURE 104.2
Sarcolomma
Terminal cisternae Transverse tubule
Sarcoplasmic reticulum
Mitochondria Nucleus
