Ceramide Signaling

MOLECULAR BIOLOGY INTELLIGENCE UNIT 21

Anthony H. Futerman

Ceramide Signaling

MOLECULAR BIOLOGY INTELLIGENCE UNIT 21

Ceramide Signaling Anthony H. Futerman, PhD Department of Biological Chemistry Weizmann Institute of Science Rehovot, Israel

LANDES BIOSCIENCE / EUREKAH.COM GEORGETOWN, TEXAS U.S.A.

KLUWER ACADEMIC / PLENUM PUBLISHERS NEW YORK, NEW YORK U.S.A.

CERAMIDE SIGNALING Molecular Biology Intelligence Unit 21 Landes Bioscience / Eurekah.com and Kluwer Academic / Plenum Publishers Copyright ©2002 Eurekah.com and Kluwer Academic/Plenum Publishers All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system; for exclusive use by the Purchaser of the work. Printed in the U.S.A. Kluwer Academic / Plenum Publishers, 233 Spring Street, New York, New York, U.S.A. 10013 http://www.wkap.nl/ Please address all inquiries to the Eurekah.com / Landes Bioscience: Eurekah.com / Landes Bioscience, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081; www.Eurekah.com; www.landesbioscience.com. Landes tracking number: 1-58706-137-6 Ceramide Signaling edited by Anthony H. Futerman/CRC, 182 pp. 6 x 9/ Landes/Kluwer dual imprint/ Landes series: Molecular Biology Intelligence Unit 21, ISBN 0-306-47442-5 While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data Cip applied for, but not received at time of publication.

CONTENTS Foreword .............................................................................................. xii Preface ................................................................................................ xvi 1. Insights into the Modulation of Ceramide Metabolism by Naturally Occurring and Synthetic Sphingolipid Analogs as Monitored by Electrospray Tandem Mass Spectrometry .......................................... 1 Alfred H. Merrill, Jr., M. Cameron Sullards, Jeremy C. Allegood, Elaine Wang, Stephen C. Linn, Lindsay Andras, Dennis C. Liotta, Michaela Hartl and Hans-Ulrich Humpf Inhibition of Ceramide Synthase ........................................................... 1 Analysis of Sphingolipids in Fumonisin-Treated Cells ........................... 2 Analysis of Sphingolipids by Electrospray Tandem Mass Spectrometry .............................................................. 2 Effects of Fumonisin B1 on Sphingolipid Metabolism .......................... 3 Additional Findings with Aminopentols and Other Sphingoid Base- Analogs ................................................................... 5 Conclusions ........................................................................................... 5 2. Ceramide in Apoptosis: Possible Biophysical Foundations of Action ........................................... 9 Paavo K. J. Kinnunen and Juha M. Holopainen Summary ............................................................................................... 9 Introduction .......................................................................................... 9 Topology of Ceramide Formation ....................................................... 10 Ceramide—From Simple Structure to Complex Behavior ................... 11 Impact of Ceramide on Biomembranes—Further Hypotheses ............. 14 3. Ceramide-Mediated Receptor Clustering ............................................. 21 Erich Gulbins and Heike Grassmé Abstract ............................................................................................... 21 Introduction ........................................................................................ 21 Ceramide Functions in Sphingolipid Enriched Membrane Rafts ......... 22 Ceramide-Mediated Signaling Platforms as a General Signaling Motif? .............................................................................. 24 4. Ceramidases: Regulators of Turnover of Ceramide and Ceramide-Mediated Responses ...................................................... 29 Cungui Mao and Lina Obeid Abstract ............................................................................................... 29 Introduction ........................................................................................ 29 Acid Ceramidases ................................................................................ 31 Neutral/Alkaline Ceramidases ............................................................. 32 Alkaline Ceramidases ........................................................................... 35 Final Remarks ..................................................................................... 38

5. Molecular Evolution of Neutral Ceramidase: From Bacteria to Mammals .................................................................. 41 Makoto Ito, Nozomu Okino, Motohiro Tani, Susumu Mitsutake and Katsuhiro Kita Summary ............................................................................................. 41 Introduction ........................................................................................ 41 Classification of Ceramidases ............................................................... 41 Bacterial Ceramidase ........................................................................... 42 Neutral Ceramidase in Mammals ........................................................ 45 6. The Role of Serine/Threonine Protein Phosphatases in Ceramide Signaling .......................................................................... 49 Charles E. Chalfant and Yusuf A. Hannun Abstract ............................................................................................... 49 Identification of a Ceramide-Activated Protein Phosphatase (CAPP) ........................................................................ 49 In Vitro Regulation of CAPP by Ceramide ......................................... 50 Selective CAPP Substrates in Cells ...................................................... 51 Role of Endogenous Ceramide in Regulating CAPP Activity ............... 54 General Mechanisms Regulated by Ceramide-Activated Protein Phosphatases ....................................................................... 55 Regulation of mRNA Processing as a Specific Pathway Regulated by CAPP ......................................................................... 57 7. Kinase Suppressor of Ras as a Ceramide-Activated Protein Kinase ....... 63 D. Brent Polk, Jose Lozano and Richard N. Kolesnick Abstract ............................................................................................... 63 Introduction ........................................................................................ 63 KSR Structure and Activity ................................................................. 64 Mechanisms of KSR Activation ........................................................... 65 Mechanisms of Raf-1 Activation via KSR ............................................ 66 Subcellular Localization ....................................................................... 67 Role of KSR Phosphorylation Sites ...................................................... 68 Biological Effects of KSR ..................................................................... 68 Conclusions and Remaining Questions ............................................... 69 8. Ceramide in Apoptosis: The FAN Thesis, Not a Fantasy ..................... 73 Bruno Ségui, Olivier Cuvillier, Sophie Malagarie-Cazenave, Sophie Lévêque, Valérie Gouazé, Nathalie Andrieu-Abadie, and Thierry Levade Abstract ............................................................................................... 73 Apoptosis, Ceramide and Sphingomyelinases ...................................... 73 FAN: A Link Between TNF-R1 and Ceramide Production ................. 74 FAN and TNF-R1- and CD40-Mediated Apoptosis ........................... 74 FAN and Other Effects ....................................................................... 76 Conclusion: Is Ceramide in Apoptosis Still a FANtasy? ....................... 78

9. The Cross-Talk Between Nitric Oxide and Ceramide and Its Role in Apoptosis Regulation ................................................... 81 Rico Barsacchi, Clara Sciorati and Emilio Clementi Abstract ............................................................................................... 81 Introduction ........................................................................................ 81 Nitric Oxide Regulates Ceramide Generation ..................................... 82 Ceramide Regulates Nitric Oxide Generation ..................................... 82 The Cross-Talk Between Nitric Oxide and Ceramide in the Context of Apoptosis ............................................................. 84 10. Crosstalk of Ceramide with Cell Survival Signaling ............................. 91 Toshiro Okazaki, Tadakazu Kondo, Mitsumasa Watanabe, Yoshimitsu Taguchi and Takeshi Yabu Summary ............................................................................................. 91 Introduction ........................................................................................ 91 Regulation of PI-3 Kinase-Dependent Signaling by Ceramide ............. 94 Regulation of Ceramide-Related Signaling by PI-3 Kinase .................. 95 Concluding Remarks ........................................................................... 96 11. Ceramide in the Regulation of Neuronal Development: Two Faces of a Lipid .......................................................................... 101 Christian Riebeling and Anthony H. Futerman Abstract ............................................................................................. 101 Ceramide and Neuronal Development and Death ............................. 101 Neurotrophins as Modulators of Neuronal Survival and Death ......... 102 Sphingomyelinases and their Regulation ............................................ 103 Downstream Players in the Ceramide Response ................................ 104 A Role for de novo Ceramide Synthesis ............................................. 106 Signaling Through Ceramide Metabolites ......................................... 106 Conclusions ....................................................................................... 107 12. Neurons, Neurotrophins and Ceramide Signaling: Do Domains and Pores Contribute to the Dichotomy? ...................... 113 Rick T. Dobrowsky Introduction ...................................................................................... 113 Influence of Ceramide on Lipid Raft Domains .................................. 114 Effect of Ceramides on Sphingomyelin-Cholesterol Poor Membranes ........................................................................... 115 Are There any Relationships between Neurotrophins, Ceramide, Lipid Rafts and Neuronal Biology? ............................... 117 Conclusions ....................................................................................... 119 13. Ceramide Signaling in Cannabinoid Action ....................................... 125 Ismael Galve-Roperh, Cristina Sánchez, Teresa Gómez del Pulgar, Guillermo Velasco, Daniel Rueda, Cristina Blázquez and Manuel Guzmán Summary ........................................................................................... 125

Introduction ...................................................................................... 125 Ceramide Generation ........................................................................ 127 Ceramide Targets .............................................................................. 129 Ceramide Function ........................................................................... 129 Therapeutic Implications ................................................................... 130 Concluding Remarks ......................................................................... 130 14. Ceramide Glycosylation and Chemotherapy Resistance ..................... 133 Myles C. Cabot 15. Ceramide in Serum Lipoproteins: Function and Regulation of Metabolism ............................................ 141 Mariana N. Nikolova-Karakashian Abstract ............................................................................................. 141 Introduction ...................................................................................... 141 Ceramide is a Component of Serum Lipoproteins ............................. 142 Secretion of Ceramide in the Form of VLDL by the Liver ................. 142 Generation of Ceramide in LDL Particles ......................................... 143 Biological Consequences of Elevation of Ceramide Concentrations in LDL ................................................................. 145 Conclusions and Future Directions ................................................... 147 16. Therapeutic Implications of Ceramide-Regulated Signaling Cascades ............................................................................. 149 Mark Kester, Jong K. Yun, Tom Stover and Lakshman Sandirasegarane Abstract ............................................................................................. 149 The Bench—Ceramides and Signaling Cascades ............................... 149 The Bedside—Ceramides and Cardiovascular Disease ....................... 150 The Bedside—Ceramides and Cancer ............................................... 153 The Bedside—Other Potential Applications for Ceramide-Based Therapeutics .................................................. 155 Conclusions—Back to the Bench ...................................................... 155 Index .................................................................................................. 161

EDITOR Anthony H. Futerman, PhD Department of Biological Chemistry Weizmann Institute of Science Rehovot, Israel email: [email protected] Chapter 11

CONTRIBUTORS Jeremy C. Allegood School of Biology Petit Institute for Bioengineering and Biosciences Georgia Institute of Technology Atlanta, Georgia, U.S.A.

Myles C. Cabot John Wayne Cancer Institute Santa Monica, California, U.S.A. email: [email protected]

Chapter 1

Charles E. Chalfant Department of Biochemistry and Molecular Biology Medical University of South Carolina Charleston, South Carolina, U.S.A. email: [email protected]

Lindsay Andras School of Biology Petit Institute for Bioengineering and Biosciences Georgia Institute of Technology Atlanta, Georgia, U.S.A. Chapter 1

Nathalie Andrieu-Abadie Inserm U466 Laboratoire de Biochimie CHU Rangueil Toulouse, France Chapter 8

Rico Barsacchi DIBIT-H San Raffaele Institute Milano, Italy email: [email protected]

Chapter 14

Chapter 6

Emilio Clementi University of Calabria and DIBIT-H San Raffaele Institute Milano, Italy email: [email protected] Chapter 9

Olivier Cuvillier Inserm U466 Laboratoire de Biochimie CHU Rangueil Toulouse, France Chapter 8

Chapter 9

Cristina Blázquez Department of Biochemistry and Molecular Biology I School of Biology Complutense University Madrid, Spain Chapter 13

Rick T. Dobrowsky Department of Pharmacology and Toxicology University of Kansas Lawrence, Kansas, U.S.A. email: [email protected] Chapter 12

Ismael Galve-Roperh Department of Biochemistry and Molecular Biology I School of Biology Complutense University Madrid, Spain

Yusuf A. Hannun Department of Biochemistry and Molecular Biology Medical University of South Carolina Charleston, South Carolina, U.S.A. email: [email protected]

Chapter 13

Chapter 6

Teresa Gómez del Pulgar Department of Biochemistry and Molecular Biology I School of Biology Complutense University Madrid, Spain

Michaela Hartl Lehrstuhl für Lebensmittelchemie Universität Würzburg Am Hubland, Würzburg, Germany

Chapter 13

Juha M. Holopainen Helsinki Biophysics & Biomembrane Group Institute of Biomedicine University of Helsinki, Finland

Valérie Gouazé Inserm U466 Laboratoire de Biochimie CHU Rangueil Toulouse, France Chapter 8

Heike Grassmé Department of Immunology St. Jude Children's Research Hospital Memphis, Tennessee, U.S.A. Chapter 3

Erich Gulbins Department Molecular Biology University of Essen Hufelandstrasse, Essen, Germany email: [email protected]

Chapter 1

Chapter 2

Hans-Ulrich Humpf Lehrstuhl für Lebensmittelchemie Universität Würzburg Am Hubland, Würzburg, Germany Chapter 1

Makoto Ito Departments of Bioscience and Biotechnology Graduate School of Kyushu University Kyushu, Japan email: [email protected] Chapter 5

Chapter 3

Manuel Guzmán Department of Biochemistry and Molecular Biology I School of Biology Complutense University Madrid, Spain email: [email protected] and [email protected] Chapter 13

Mark Kester Department of Pharmacology Penn State College of Medicine Hershey, Pennsylvania, U.S.A. email: [email protected] Chapter 16

Paavo K. J. Kinnunen Helsinki Biophysics & Biomembrane Group Institute of Biomedicine University of Helsinki, Finland email: [email protected]

Stephen C. Linn School of Biology Petit Institute for Bioengineering and Biosciences Georgia Institute of Technology Atlanta, Georgia, U.S.A.

Chapter 2

Chapter 1

Katsuhiro Kita Departments of Bioscience and Biotechnology Graduate School of Kyushu University Kyushu, Japan

Dennis C. Liotta Department of Chemistry Emory University Atlanta, Georgia, U.S.A. Chapter 1

Chapter 5

Richard N. Kolesnick Laboratory of Signal Transduction Memorial Sloan-Kettering Cancer Center New York, New York, U.S.A. [email protected] Chapter 7

Tadakazu Kondo Department of Hematology/Oncology Graduate School of Medicine Kyoto University Sakyo-ku, Kyoto, Japan Chapter 10

Thierry Levade Inserm U466 Laboratoire de Biochimie CHU Rangueil Toulouse, France email: [email protected]

Jose Lozano Memorial Sloan-Kettering Cancer Center New York, New York, U.S.A. Chapter 7

Sophie Malagarie-Cazenave Inserm U466 Laboratoire de Biochimie CHU Rangueil Toulouse, France Chapter 8

Chungui Mao Departments of Medicine and Biochemistry and Molecular Biology Ralph H. Johnson Veterans Administration Hospital Medical University of South Carolina Charleston, South Carolina, U.S.A. Chapter 4

Sophie Lévêque Inserm U466 Laboratoire de Biochimie CHU Rangueil Toulouse, France

Alfred H. Merrill, Jr. School of Biology Petit Institute for Bioengineering and Biosciences Georgia Institute of Technology Atlanta, Georgia, U.S.A. email: [email protected]

Chapter 8

Chapter 1

Chapter 8

Susumu Mitsutake Departments of Bioscience and Biotechnology Graduate School of Kyushu University Kyushu, Japan

Christian Riebeling Department of Biological Chemistry Weizmann Institute of Science Rehovot, Israel Chapter 11

Chapter 5

Mariana N. Nikolova-Karakashian Department of Physiology University of Kentucky Medical Center Lexington, Kentucky, U.S.A. email: [email protected]

Daniel Rueda Department of Biochemistry and Molecular Biology I School of Biology Complutense University Madrid, Spain

Chapter 15

Chapter 13

Lina M. Obeid Departments of Medicine and Biochemistry and Molecular Biology Ralph H. Johnson Veterans Administration Hospital Medical University of South Carolina Charleston, South Carolina, U.S.A. email: [email protected]

Cristina Sánchez Department of Biochemistry and Molecular Biology I School of Biology Complutense University Madrid, Spain

Chapter 4

Lakshman Sandirasegarane Department of Pharmacology Penn State College of Medicine Hershey, Pennsylvania, U.S.A.

Toshiro Okazaki Department of Hematology/Oncology Graduate School of Medicine Kyoto University Sakyo-ku, Kyoto, Japan email: [email protected] Chapter 10

Nozomu Okino Departments of Bioscience and Biotechnology Graduate School of Kyushu University Kyushu, Japan Chapter 5

D. Brent Polk Vanderbilt Digestive Disease Research Center Vanderbilt University Nashville, Tennessee, U.S.A. email: [email protected] Chapter 7

Chapter 13

Chapter 16

Clara Sciorati DIBIT-H San Raffaele Institute Milano, Italy email: [email protected] Chapter 9

Bruno Ségui Inserm U466 Laboratoire de Biochimie CHU Rangueil Toulouse, France Chapter 8

Tom Stover Department of Pharmacology Penn State College of Medicine Hershey, Pennsylvania, U.S.A. Chapter 16

M. Cameron Sullards Department of Chemistry and Biochemistry Georgia Institute of Technology Atlanta, Georgia, U.S.A. Chapter 1

Elaine Wang School of Biology Petit Institute for Bioengineering and Biosciences Georgia Institute of Technology Atlanta, Georgia, U.S.A. Chapter 1

Yoshimitsu Taguchi Department of Hematology/Oncology Graduate School of Medicine Kyoto University Sakyo-ku, Kyoto, Japan Chapter 10

Mitsumasa Watanabe Department of Hematology/Oncology Graduate School of Medicine Kyoto University Sakyo-ku, Kyoto, Japan Chapter 10

Motohiro Tani Departments of Bioscience and Biotechnology Graduate School of Kyushu University Kyushu, Japan Chapter 5

Takeshi Yabu Department of Hematology/Oncology Graduate School of Medicine Kyoto University Sakyo-ku, Kyoto, Japan Chapter 10

Guido Tettamanti University of Milan Department of Medical Chemistry and Biochemistry Segrate, Milano, Italy email: [email protected] Foreword

Guillermo Velasco Department of Biochemistry and Molecular Biology I School of Biology Complutense University Madrid, Spain Chapter 13

Jong K. Yun Department of Pharmacology Penn State College of Medicine Hershey, Pennsylvania, U.S.A. Chapter 16

FOREWORD Thirty years have passed since Singer and Nicolson proposed the fluid mosaic model for cellular membrane structure, introducing the concept that both the protein and lipid components of membranes are in a dynamic and flexible state. This concept had a revolutionary effect on the study of membrane lipids. In fact, in addition to being the building blocks that contribute to basic membrane organization by supplying a multitude of hydrophobic interactions, lipids were recognized early to act as modulators of specific membrane proteins, such as receptors, carriers, pumps, and enzymes. Moreover, the ability of lipids to undergo lateral phase separation and to influence membrane curvature, asymmetry and strain within the two lipid layers, emerged as an important factor in the formation of the concept of membrane microdomains of different composition and function. The notion of “lipids rafts”, “caveolae”, etc., membrane microdomains which have a unique lipid composition, is surely one of the most stimulating new perspectives in membranology. Another fundamental achievement concerning the role of membrane lipids was the discovery that they (or some of them) can undergo selective hydrolysis following appropriate stimulation, with the liberation of fragments behaving as second messengers (“lipid second messengers”) or cellular bioregulators. The involvement of membrane lipids in transmembrane signalling pathways initially focused on phosphoinositides and phosphatidylcholine, and the metabolic fragments carrying biological activity were inositol-1,4,5 triphosphate, diacylglycerol, phosphatidic acid and arachidonic acid. Of course this kind of finding highlighted the critical issue of the connection between membrane lipid metabolism and enzymology, and signalling processes. Finally (at least so far), phosphoinositides were recognized as the linkers of some membrane surface proteins through the glycosylphosphoinositide anchors (GPI), from which proteins can be released by the action of specific phospholipases C, leaving the diacylglycerol moiety within the lipid bilayer. Are membrane sphingolipids, containing ceramide, involved in this complex and fascinating scenario? The response is yes. Sphingolipids have a compositional heterogeneity that is similar to that of glycerophospholipids with regard to the lipophilic portion (fatty acids and long chain bases) but show enormously greater complexity in the hydrophilic portion, especially in glycosphingolipids. The chemical complexity, particularly of glycosphingolipids, required time for proper assessment and contributed to the delay in understanding the role that sphingolipids play in the membrane. However, in the late 1970s and in the 1980s the availability of pure (glyco)sphingolipids of known structure permitted delineation of their metabolic routes and analysis of their functional implications.1,2,3 It became gradually clear that they are involved in a number of relevant biological events including immune-modulation, receptor activity, cell-cell interactions, cell

adhesion, neural differentiation and development, functional recovery of the damaged nervous system, and tumour growth. The strict relationship between sphingolipids and protein phosphorylation processes, control of intra-cytosolic calcium concentrations, and transduction phenomena (also implying gene expression) were also recognized. The need for a better knowledge of the enzymes involved in sphingolipid metabolism and of its regulation was clearly understood.4 Curiously, the peculiar sensitivity of brain membrane-bound sphingomyelinase to stress conditions, like halothane anesthesia5 and simple brain handling,6 was accurately recorded, but without proper consideration of the possible significance of increased ceramide production. In the late eighties, the sphingolipid scientific community was eager to acknowledge the molecular link between sphingolipid-related events occurring at the cell surface and intracellular responses. In 1986 the expected innovatory trend began. Yusuf Hannun and colleagues discovered that sphingosine, as well as other lysosphingolipids, are potent and reversible inhibitors of protein kinase C, in vitro and in vivo.7 Shortly later, Kolesnick8 and Okasaki et al9 observed that some cells responded to specific stimulation (diacylglycerol, vitamin D3, tumor necrosis factor-α, γ-interferon, and some cytokines) with a transient activation of sphingomyelinase followed by elevation of ceramide content, within the framework of profound phenotypic changes. The remarkable body of evidence that accumulated thereafter led to the proposal of a “sphingomyelin cycle or pathway” generating ceramide, conceptually similar to that of the well established lipid second messengers. In 1991 Zhang et al10 showed that sphingosine-1-phosphate is also involved as a mediator in the processes promoting cell proliferation. Glucosylceramide was also recognised to have a regulatory role in neuronal growth and differentiation.11 For sphingosine and, particularly, ceramide, the major requirements for classifying them as genuine “second messengers” seemed to be fulfilled,12,13 e.g., intermittent rise of concentration elicited by an extracellular ligand binding to cells, the identification of specific enzymes involved in their production and removal, the assessment of down-stream specific targets, implication in well established cellular events, and induction of these events by administration to cells of the same sphingoid molecules or suitable analogues. The cellular events where sphingoid messenger molecules appeared to be involved are remarkably important such as cell growth or arrest of proliferation, differentiation, and apoptosis, with the latter particularly important with respect to involvement of ceramide.14 Acceptance of the sphingoid family of second messengers by lipidologists and membranologists ranges, or ranged, from mild scepticism,15 with special regard to the ceramide role in apoptosis16 to cautious neutrality17,18 and optimistic interest.19 The interest of investigators in sphingolipids is growing at a fast rate. In the last two years (2000-2001) the total number of papers published in

this field was about 1400, out of which 370 were on ceramide and 330 on sphingosine and sphingosine-1-phosphate. At the cellular level, further and more specialised processes were showed to involve sphingoid molecules, such as endocytosis, cell migration, protein synthesis, ubiquitin-dependent proteolysis, protein anchoring to membranes by glycan bridges with a ceramide hydrophobic tail, and cross-talk between the sphingoid-mediated and other signalling pathways. At the level of integrated functions the importance of sphingolipids grew from the general areas of neuroscience, oncology and immunology to more specific sectors of pharmacology and therapeutics, clinical sciences, laboratory medicine, food science and nutrition, and environmental health and biotechnology. It seems clear that sphingolipids offer a range of “molecular tools” for cellular regulation that include second messenger functions but also go beyond the classical notion of second messengers. Let us consider, for example, ceramide, sphingosine, sphingosine-1-phosphate and glucosylceramide, all bioregulatory molecules that sometimes elicit opposing effects. They are metabolically connected at both the same and different subcellular sites. Therefore, the change of activity of a single enzyme affecting their metabolism can profoundly modify the local concentration ratio between the sphingoid bioregulators, leading one of them to prevail over the other and impose its peculiar effect. Hence the concept of “lipostats”. Likewise, the presence in membrane microdomains of a sphingoid molecule like ceramide can be responsible for triggering fusion of small microdomains into larger platforms, or the presence of glycosphingolipids can enable a specific interaction with an external ligand with consequent changes in microdomain size and mobility. As a result, in both cases initiation of specific signalling pathways might be promoted. The exciting story of the involvement of sphingolipids in trans-membrane signalling and regulation of cell function is, in my opinion, only at the beginning. The contributions contained in this book, all based on solid experimental evidence, bear witness to the amplitude of the field and emphasize the huge potential of novel and surprising discoveries of research in this area. Guido Tettamanti University of Milan Department of Medical Chemistry and Biochemistry Via Fratelli Cervi 93 20090 Segrate, Milano, Italy e-mail: [email protected] References 1. Hakomori SI. Glycosphingolipids in cellular interaction, differentiation and oncogenesis. Ann Rev Biochem 1981; 50:733-764. 2. Tettamanti G. Gangliosides and neuronal plasticity. Berlin: Liviana Press, Padua/ Springer Verlag.

3. Ledeen RW. New trends in ganglioside research. Neurochemical and neurogenerative aspects. Berlin: Liviana Press, Padua / Springer Verlag. 4. Radin NS. Biosynthesis of the sphingoid bases: a provocation. J Lipid Res 1984; 25:1536-1540. 5. Pellkofer R, Sandhoff K. Halothane increases membrane fluidity and stimulates sphingomyelin degradation by membrane-bound neutral sphingomyelinase of synaptosomal plasma membranes from calf brain already at clinical concentrations. J Neurochem 1980; 34:988-992. 6. Deshmukh GD, Radin NS. Formation of free fatty acid and ceramide during brain handling: lability of sphingomyelin. J Neurochem 1985; 44:1152-1155. 7. Hannun YA, Loomis CR, Merrill AHJ et al. Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets. J Biol Chem 1986; 261:12604-12609. 8. Kolesnick RN. 1,2-Diacylglycerols but not phorbol esters stimulate sphingomyelin hydrolysis in GH3 pituitary cells. J Biol Chem 1987; 262:16759-16762. 9. Okazaki T, Bell RM, Hannun YA. Sphingomyelin turnover induced by vitamin D3 in HL-60 cells . Role in cell differentiation. J Biol Chem 1989; 264:19076-19080. 10. Zhang H, Desai NN, Olivera A et al. Sphingosine-1-phosphate, a novel lipid, involved in cellular proliferation. J Cell Biol 1991; 114:155-167. 11. Schwarz A, Rapaport E, Hirschberg K et al. A regulatory role for sphingolipids in neuronal growth—Inhibition of sphingolipid synthesis and degradation have opposite effects on axonal branching. J Biol Chem 1995; 270:10990-10998. 12. Hannun YA. The sphingomyelin cycle and the second messenger function of ceramide. J Biol Chem 1994; 269:3125-3128. 13. Kolesnick R. Ceramide: a novel second messenger. Trends in Cell Biol 1992; 2:232-236. 14. Hannun YA, Obeid LM. Ceramide: An intracellular signal for apoptosis. Trends Biochem Sci 1995; 20:73-77. 15. Hofmann K, Dixit VM. Ceramide in apoptosis—does it really matter? Trends Biochem Sci 1998; 23:374-377. 16. Commentary. (2001) in The Biochemist. 11-13 July Dublin: 2001:31-32. 17. Michell RH, Wakelam MJO. Sphingolipid signalling. Current Biology 1994; 4:370-373. 18. Exton JH. Messenger molecules derived from membrane lipids. Curr Opin Cell Biol 1994; 6:226-229. 19. Liscovitch M, Cantley LC. Lipid second messengers. Cell 1994; 77:329-334.

PREFACE Editing a book on ceramide signaling has been both a challenging and rewarding experience. Challenging inasmuch as this field has developed so rapidly over the past few years that keeping abreast of developments is taxing even for those of us who spend our days and nights glued to our computer screens performing electronic searches of the recent literature. Rewarding inasmuch as editing the book has been an excellent way to actually keep abreast of the field without becoming a computer junkie. It is hard to believe that only 15 years have passed since the concept of ceramide signaling (or rather the concept of the sphingomyelin cycle) was first introduced by Yusuf Hannun and Rich Kolesnick. Their contribution to this field has been immense, and it is to their credit that their chapters in this book are not ‘overviews’ of all they have ever done, but rather concise summaries of the current state of art in their specific fields. Since the original suggestion of a sphingomyelin cycle, events have unfolded at a rapid pace, and over the past few years have drawn in researchers totally new to the sphingolipid field. Those who worked in this area during the dark ages of sphingolipid research would have found the idea that of the 21,000 citations to sphingolipids in Medline in mid 2002, more than 8,000 are to ceramide, heresy of the first order. After all, wasn’t ceramide simply the inert building block from which the really important complex sphingolipids and glycosphingolipids were made? It is of great credit to the sphingolipid community that the central place which ceramide has taken has not been to the detriment of other areas of sphingolipid biology, but has rather catalyzed new and exciting directions of research in the field in general. It is also gratifying to find ceramide and complex sphingolipids attracting great interest not only from lipid biochemists, but also from cell biologists, biophysicists, pharmacologists and even organic chemists. This book, by definition, focuses on ceramide. Due to this narrow focus, readers may be surprised to see the names of some major players in the sphingolipid world absent, and some subjects not covered in great depth. Prominent among these subjects are sphingosine-1-phosphate and other sphingolipid second messengers, sphingolipid storage diseases, ceramide metabolism to higher order glycosphingolipids and sphingomyelin, and aspects related to the intracellular transport of ceramide. The absence of these subjects is not meant to imply that they are second class citizens, but is rather due to pragmatic considerations in keeping the book focused, and in producing what is, as far as I know, the first volume ever to concentrate specifically on ceramide signaling. After three chapters addressing aspects of ceramide analysis and biophysics, the enzymes of ceramide metabolism and down-stream ceramide targets are discussed in detail. This order is of course not a coincidence— having precise tools to measure ceramide levels and ceramide classes in

signaling, such as the methods and tools described by Merrill and colleagues, and understanding how ceramide formation might induce or cause changes in membrane properties, are prerequisites for understanding the complex functions of ceramide at biochemical and cellular levels. The description of enzymes of ceramide metabolism, with two chapters focusing on ceramidases, is clearly essential for understanding the ways in which ceramide levels are regulated in cells, both in metabolic and in signaling pathways. Conspicuous by its absence is a chapter on neutral sphingomyelinase, and we all await the purification, isolation and characterization of a genuine neutral sphingomyelinase. The inclusion of a chapter on FAN (factor associated with neutral sphingomyelinase) should at least be of some comfort to those of us who believe that it is only a question of time before neutral sphingomyelinase comes of age. Finally in this section, the inclusion of only two chapters discussing down-stream targets of ceramide is probably testimony to the likelihood that many more down-stream targets are out there waiting to be discovered. One of the reasons that the ceramide field has become so popular recently is the realization that ceramide impacts upon multiple signaling pathways, and the cross-talk of ceramide with two of these pathways, those involving nitric oxide and the more general area of cell survival signaling, precedes two chapters focusing on the role of ceramide in neuronal development. The latter area historically had been one of the major areas of sphingolipid research, since the discovery many years ago that gangliosides are enriched in neuronal membranes. A new appreciation of the role of the simplest sphingolipid of them all, ceramide, has forced a major rethink in this area, and the challenge today is to distinguish between events regulated by ceramide in neuronal development and those regulated by complex sphingolipids such as gangliosides. It is entirely proper that the book should close with four excellent chapters discussing some of the therapeutic applications of ceramide action, with issues discussed ranging from the role of ceramide in cannabinoid action, to how chemotherapy resistance is achieved by suppressing ceramide levels, to the role of ceramide during the acute phase response to inflammation, and last but not least, in cardiovascular disease and cancer. Indeed, it is this marriage between basic science and medical research that make it such an exciting time to be involved in the ceramide field, and if some of this excitement rubs off on the readers of this book, then not only the editor, but all the contributors will feel that their time and effort has been well spent. Tony Futerman Department of Biological Chemistry Weizmann Institute of Science Rehovot, Israel

ABBREVIATIONS AP1 A-SMase

aminopentol acid sphingomyelinase

BEACH bFGF BDNF

beige and Chediak-Higashi basic fibroblast growth factor brain-derived growth factor

CAPK CAPP CCT Cer-LDL CDase CRD Cyt

ceramide-activated protein kinase ceramide activated protein phosphatase CTP:phosphatidylcholine cytidyltransferase ceramide-enriched LDL ceramidase cysteine rich domain cytochrome

DAG DAP DD DEPE DIGs DMPC D-PDMP DPPC

diacylglcerol death-associated protein death domain dielaidoylphosphatidylethanolamine detergent-insoluble glycosphingolipid-enriched membranes dimyristoylphosphocholine D-erythro-1-phenyl-2-decanoylamino-3-morpholino1-propanol dipalmitoylphosphocholine

eNOS ERK ESI EST

endothelial nitric oxide synthase extracellular signal-regulated kinase electrospray ionization expression tagged sequences

FADD FAN FB1

Fas-associated death domain protein factor associated with neural sphingomyelinase activation Fumonisin B1

GC GCS GEM GFP GUV

glucosylceramide glucosylceramide synthase glycosphingolipid-enriched membrane green fluorescent protein giant unilamellar vesicles

hAC haPHC

heterodimeric enzyme acid ceramidase human alkaline phytoceramidase

HDL HMEC 4-HPR hsp

high density lipoproteins human microvascular endothelial cells N-(4-hydroxyphenyl) retinamide heat shock protein

IGF-1 ISGS ISGT

insulin-like grwoth factor-1 insulin-stimulated glucose synthesis insulin-stimulated glucose transport

KSR

kinase suppressor of Ras

LC-MS/MS liquid chromatography electrospray tandem mass spectrometry LDL low density lipoproteins LPS lipopolysaccharide maCER MAP MDR

mouse alkaline ceramidase mitogen-activated protein multidrug resistance

NGF NO NOE NOS NSD N-SMase NT-3 NT-4

nerve growth factor nitric oxide N-oleoylethanolamine nitric oxide synthase neutral sphingomyelinase domain neutral sphingomyelinase neurotrophin-3 neurotrophin-4

PC-PLC PI PI-3K PKB PKC PLC PLD PP1 PP2A PPMP PPPP PS

phosphatidylcholine-specific phospholipase C phosphatidylinositol phosphatidylinositol-3 kinase protein kinase B protein kinase C phospholipase C phospholipase D protein phosphatase-1 protein phosphatase-2A 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol phosphatidylserine

RACE Rb

rapid amplification of cDNA ends retinoblastoma

SAPK SDS-PAGE SHR SM SMC SMase SOD SPT

stress-activating protein kinases polyacrylamine gel electrophoresis spontaneously hypertensive rats sphingomyelin smooth muscle cells sphingomyelinase superoxide dismutase serine palmitoyltransferase

TGF TNF TRADD TRAF

tranforming growth factor tumor necrosis factor TNF-R associated death domain protein TNF receptor-associated factor

VLDL

very low density lipoproteins

YDC1p YPC1p

dihydroceramidase phytoceramidase

CHAPTER 1

Insights into the Modulation of Ceramide Metabolism by Naturally Occurring and Synthetic Sphingolipid Analogs as Monitored by Electrospray Tandem Mass Spectrometry Alfred H. Merrill, M. Cameron Sullards, Jeremy C. Allegood, Elaine Wang, Stephen C. Linn, Lindsay Andras, Dennis C. Liotta, Michaela Hartl and Hans-Ulrich Humpf

F

umonisins have considerable structural similarity to sphinganine, as illustrated in Figure 1 for one of the most prevalent species (fumonisin B1, FB1) and its backbone aminopentol (AP1). Fumonisins inhibit ceramide synthase, and perturbation of sphingolipid metabolism accounts for many of the pathological effects of consumption of these mycotoxins.1,2 In part because it is commercially available, many labs have utilized FB1 to explore the functions of complex sphingolipids (see ref. 3, for examples). Nonetheless, the cellular effects of fumonisins can be complex—constituting not only suppression of complex sphingolipid formation but also the accumulation of sphinganine and other bioactive metabolites.1,4,5 To understand how fumonisins affect sphingolipid metabolism as well as gain better insight into the behavior of other commonly encountered species, we have employed a variety of methods, including liquid chromatography electrospray tandem mass spectrometric (LC-MS/MS), to analyze essentially all of the pertinent bioactive sphingolipids (e.g., sphingoid bases, sphingoid base 1-phosphates, ceramides, sphingomyelins, glucosylceramides, inter alia)5 and to characterize fumonisin metabolites.6 Some of the findings will be summarized in this review.

Inhibition of Ceramide Synthase

In the current model for how FB1 inhibits ceramide synthase, it has been proposed1 that the aminopentol backbone competes for binding of the sphingoid base substrate whereas the tricarballylic acid side-chain interferes with utilization of the co-substrate fatty acyl-CoA, as can be envisioned by overlaying the structures in Figures 1A and B. Some of the evidence that supports this model is: 1) the potency of inhibition by FB1 depends on the concentrations of both sphingoid bases and fatty acyl-CoA6,7; 2) removal of the tricarballylic acids diminishes the potency of ceramide synthase inhibition6,8 and, 3) upon removing the tricarballylic acids, AP1 becomes a substrate for acylation (whereas FB1 is not acylated).6 As will be discussed later, this acylation may explain why aminopentols remain toxic to animals despite the lower potency of these compounds as ceramide synthase inhibitors. Ceramide Signaling, edited by Anthony H. Futerman. ©2002 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Ceramide Signaling

Figure 1. Scheme for the acylation of sphinganine by (dihydro)ceramide synthase emphasizing the potential for the localization of the charged groups of the sphingoid base and fatty acyl-CoA (Panel A) resembling those found in fumonisin B1 (panel B). Panel C shows the structure of the fumonisin (aminopentol) backbone alone (AP1).

When added to mammalian cells in culture, FB1 is usually effective in blocking the formation of ceramide, SM and glucosylceramide, and more complex sphingolipids, measured by incorporation of radiolabelled serine7 or of mass5,9 (see below). Blockage of complex sphingolipid formation must be confirmed for each case because some organisms, such as yeast,10 take up fumonisins poorly unless treated specially and there are cases where other enzymatic reactions, such as reversal of ceramidase,11,12 can provide an alternative pathway for ceramide synthesis. It is also advisable to check the effectiveness of fumonisin stock solutions occasionally because potency is sometimes lost during storage, presumably due to hydrolysis of the ester-linked tricarballylic acid(s).

Analysis of Sphingolipids in Fumonisin-Treated Cells Conventional methods for analysis of sphingolipids are sufficient to determine if FB1 is blocking de novo synthesis. Among these, the most convenient are measurement of the incorporation of radiolabelled serine or palmitic acid into sphingolipids13,14 or elevation of sphinganine (by HPLC with a fluorescence detector).15,16 In addition to the relative convenience of the latter, it serves to remind the investigator that the elevation in sphinganine can be dramatic— as high as several nmol/106 cells—amounts that are high enough to affect signaling pathways, such as the inhibition of protein kinase C.17 These conventional assays are labor intensive, provide little information about structural variants within each class (for example, the different molecular subspecies of ceramides), and are not very efficient at extracting and detecting polar compounds such as the sphingoid base 1-phosphates.

Analysis of Sphingolipids by Electrospray Tandem Mass Spectrometry Heretofore, mass spectrometry has been used primarily for structural elucidation of sphingolipids (reviewed in ref. 18), however, the development of electrospray ionization (ESI) provides the ability to generate intact molecular ions of polar biomolecules directly from solution (i.e., the eluate from HPLC columns) and to perform quantitative analysis. Equally important has been the availability of a method to quantify different species (multiple reaction

Insights into the Modulation of Ceramide Metabolism

3

Figure 2. Pathway for sphingoid base biosynthesis with illustration of the side reactions (phosphorylation and acetylation) that can increase when sphinganine and sphingosine are elevated by (dihydro)ceramide synthase inhibition by fumonisins.

monitoring, illustrated in Fig. 3) and the commercial availability of internal standards for sphingoid bases (C20-sphingosine and –sphinganine), sphingoid base 1-phosphates (the C17 homologs), ceramides, sphingomyelins, glucosylceramide and lactosylceramide (all prepared as the C12-fatty acid homologs) (Avanti Polar Lipids, Alabaster, AL). With these capabilities, a step-by-step procedure has been developed for the extraction, analysis, and quantitation of all of these categories of sphingolipids (as well as lysosphingolipids and N-methylsphingoid bases) using HPLC electrospray tandem mass spectrometry.5 Its sensitivity allows analysis of all of these compounds in samples as small as a petri dish of cells in culture.

Effects of Fumonisin B1 on Sphingolipid Metabolism Analyses of sphingolipid metabolism using diverse methods, most of which have now been confirmed by mass spectrometry, reveal interesting changes in the amounts of sphingolipids in cells. Pathways of sphingolipid metabolism that participate in the response to fumonisins are shown in Figure 2.

Elevation of Sphinganine Elevations in cellular sphinganine remain the hallmark of fumonisin’s effects on cells in culture and in vivo (see ref. 1 for review). An example of the magnitude of the elevations in sphinganine and sphingosine is shown in Figure 3. The amount of sphinganine in these cells (ca 2 nmol/106 cells) is at or above the levels that affect diverse cell signaling pathways when added to cells exogenously.17,19 Sphingosine is also sometimes elevated (presumably due to inhibition of the recycling of sphingosine produced during complex sphingolipid turnover), but the amounts are typically smaller than sphinganine (c.f. Fig. 2).

4

Ceramide Signaling

Figure 3. Illustration of the advantage of using multiple reaction monitoring (MRM) in the quantitative analysis of sphingolipids. Data from ref. 5).

Depletion of More Complex Sphingolipids Fumonisins can completely block synthesis of new sphingolipids and severely deplete the total mass of cellular sphingolipids.1,7,20,21 As an example of the findings from mass spectrometric analysis of the effects of FB1 on cellular amounts of SM, glucosylceramide and ceramide in NIH3T3 cells is shown in Figure 4. These changes can disrupt cell functions dependent on complex sphingolipids; for example, FB1 treatment of intestinal cells in culture blocks folate uptake because the folate transporter is a glycosylphosphatidylinositol-anchored protein, which typically require sphingolipids and cholesterol to function normally.22

Production of Sphingoid Base 1-Phosphates and Down-Stream Metabolites (e.g., Ethanolamine Phosphate) Sphingoid bases are catabolized by phosphorylation and lytic cleavage to a fatty aldehyde and ethanolamine phosphate (see Fig. 2). FB1 has been shown to increase the amounts of cellular sphingoid base 1-phosphates4,5 (see also Fig. 5) as well as to increase the amount of sphingolipidderived ethanolamine phosphate that is incorporated into phosphatidylethanolamine.4

Formation of N-Acetyl-Derivatives of Sphingoid Bases The accumulation of free sphingoid bases allows them to “spill over” into other pathways to become substrates for N-acetyltransferases that participate in xenobiotic metabolism23 or that transfer the acetyl-group from platelet activating factor to sphingosine.24 In a recent study of this side pathway, the amounts of “C2-ceramides” were measured in livers from rats fed a fumonisin-free diet and rats fed 150 µg FB1/g diet. The amounts in the control rats were ca 0.6 nmol of N-acetylsphingosine/g and 0.3 nmol of N-acetyl-sphinganine; FB1 feeding had no effect on the amount of N-acetylsphingosine, but increased N-acetyl-sphinganine by 4-fold.1

Alteration of other Lipid Metabolic Pathways Changes in these sphingolipids can impact other important lipid metabolic/signaling pathways. For example, phosphatidic acid phosphatase is highly sensitive to cellular amounts of free sphingoid bases,10,25,26 and FB1 has been shown to alter this (and other) pathway(s) in yeast.27 Sphingoid bases28,29 and their 1-phosphates30 can also activate phospholipase D, and sphingoid bases inhibit monoacylglycerol acyltransferase.31

Insights into the Modulation of Ceramide Metabolism

5

Amounts of sphingomyelin, glucosylceramide and ceramide in NIH3T3 cells incubated with (+) or without (-) FB1 for 24 h

Figure 4. Amounts of ceramide (Cer), sphingomyelin (SM) and glucosylceramide (GlcCer) in NIH 3T3 cells incubated for 24 h with (or without) 50 µM FB1 and analyzed by electrospray tandem mass spectrometry.5

Additional Findings with Aminopentols and other Sphingoid Base- Analogs Animals (including humans) are naturally exposed to the fumonisin aminopentols because they are formed during the treatment of corn with lye, a common practice in the preparation of flour for tortillas; furthermore, the intestinal microflora of primates can hydrolyze FB1 to AP1. Although AP1 is a weak inhibitor of ceramide synthase, it is acylated to form derivatives such as the one shown in Figure 6 (N-palmitoyl-AP1 or PAP1).6 These acylated aminopentols are more potent inhibitors of ceramide synthase and are as, or more, cytotoxic than FB1.6 Structural differences between the aminopentols and sphinganine include the absence of a hydroxyl group at the 1-position, the presence of an additional hydroxyl at position 5, and a threo- versus erythro-stereochemistry at positions 2 and 3. When the toxicities of naturally occurring fumonisin aminopentols (e.g., AP1) are compared to sphinganine with cells in culture, AP1 is less toxic. This may be due to the lower hydrophobicity of the aminopentol because when 1-deoxy-sphinganine analogs have been synthesized and compared (K. Desai et al, manuscript in preparation), they are approximately 10-fold more toxic than sphinganine. These types of compounds offer promise as probes for the mechanisms of action of both fumonisins and sphingoid bases.

Conclusions It should be clear from this overview that perturbation of one aspect of sphingolipid metabolism has ramifications for other pathways. Of particular interest is the finding that fumonisins can increase not only sphinganine—a growth inhibitory and pro-apoptotic compound—but also sphinganine 1-phosphate, a mitogenic and anti-apoptotic sphingolipid. This may account for the puzzling research literature for fumonisins in which they have been studied by toxicologists for their toxicity, but have been recently used by other researchers to protect cells against apoptosis (indeed, some companies market FB1 as an inhibitor of apoptosis). This may also have an in vivo correlate since fumonisins are toxic in vivo but also carcinogenic for liver, kidney and some other organs. The possible mechanisms of action of fumonisins are probably quite complex, involving reductions in complex sphingolipids, elevations in sphingoid bases (sometimes counterbalanced with sphingoid base 1-phosphates), and formation of other interesting compounds such

Ceramide Signaling

6

Amounts of sphinganine, sphingosine and sphinganine 1-phosphate in NIH3T3 cells incubated with (+) or without (-) FB1 for 24 h

Figure 5. Amounts of sphinganine (Sa), sphingosine (So) and sphinganine 1-phosphate (SaP) in NIH 3T3 cells incubated for 24 h with (or without) 50 µM FB1 and analyzed by electrospray tandem mass spectrometry.5

as the C2-(dihydro)ceramides and acylated aminopentols. One way to resolve some of these discrepancies is to use other inhibitors of sphingolipid biosynthesis, such as ISP1 or myriosin.32 Nonetheless, this complexity probably explains why these mycotoxins show such a wide spectrum of pathologies—including neurotoxicity, pulmonary toxicity, liver and renal toxicity (and carcinogenicity) inter alia.1,2

Acknowledgements Work from the lab of the corresponding author was supported by grants from the NIH (GM46368 and ES09204). We are also grateful to the many valuable collaborators—and particularly Ron Riley and colleagues at the USDA laboratories in Athens, GA—who have contributed to findings discussed in this review.

References 1. Merrill AH Jr, Sullards MC, Wang E et al. Sphingolipid metabolism: Roles in signal transduction and disruption by fumonisins. Environ Health Perspect 2001; 109 Suppl 2:283-289. 2. Marasas WFO. Discovery and occurrence of fumonisins: A historical perspective. Environ Health Perspect 2001; 109(Suppl. 2):239–243. 3. Linn SC, Kim HS, Keane EM et al. Regulation of de novo sphingolipid biosynthesis and the toxic consequences of its disruption. Biochem Soc Trans 2001; 29:831-835. 4. Smith ER, Merrill AH Jr. Regulation of the “burst” of free sphingosine and sphinganine, and their 1-phosphate and N-acyl-derivatives, that occurs upon changing the medium of cells in culture: Differential roles of de novo sphingolipid biosynthesis and turnover in J774A.1 macrophages. J Biol Chem 1995; 270:18749-18758. 5. Sullards MC, Merrill AH Jr. Analysis of sphingosine 1-phosphate, ceramides and other bioactive sphingolipids by liquid chromatography-tandem mass spectrometry, Science Signal Transduction Environment (STKE) 2000. 6. Humpf HU, Schmelz EM, Meredith FI et al. Acylation of naturally occurring and synthetic 1-deoxysphinganines by ceramide synthase: Formation of N-palmitoyl-aminopentol (PAP1) produces a toxic metabolite of hydrolyzed fumonisin (AP1), and a new category of ceramide synthase inhibitor. J Biol Chem 1998; 273: 19060-19064.

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Figure 6. Acylation of AP1 and some of the properties of the original aminopentol and this metabolite. 7. Merrill AH Jr, van Echten G, Wang E et al. Fumonisin B1 inhibits sphingosine (sphinganine) Nactyltransferase and de novo sphingolipid biosynthesis in cultured neurons in situ. J Biol Chem 1993; 268:27299-27306. 8. Merrill AH Jr, Wang E, Gilchrist DG et al. Fumonisins and other inhibitors of de novo sphingolipid biosynthesis. Adv Lipid Res 1993; 26:215-234. 9. Yoo HS, Norred WP, Showker J et al. Elevated sphingoid bases and complex sphingolipid depletion as contributing factors in fumonisin-induced cytotoxicity. Toxicol Appl Pharmacol 1996; 138:211-218. 10. Wu W-I, Lin Y-P, Wang E et al. Regulation of phosphatidate phospatase activity fom the yeast Saccharomyces cerevisiae by sphingoid bases. J Biol Chem 1993; 268:13830-13837. 11. El Bawab S, Birbes H, Roddy P et al. Biochemical characterization of the reverse activity of rat brain ceramidase. A CoA-independent and fumonisin B1-insensitive ceramide synthase. J Biol Chem 2001; 276:16758-16766. 12. Mao C, Xu R, Bielawska A et al. Cloning of an alkaline ceramidase from Saccharomyces cerevisiae. An enzyme with reverse (CoA-independent) ceramide synthase activity. J Biol Chem 2000; 275:6876-6884. 13. Van Echten-Deckert G. Sphingolipid extraction and analysis by thin-layer chromatography. Meth Enzymol 2000; 312:64-79. 14. Wang E, Norred WP, Bacon CW et al. Inhibition of sphingolipid biosynthesis by fumonisins: Implications for diseases associated with Fusarium moniliforme. J Biol Chem 1991; 266:14486-14490. 15. Wang E, Ross PF, Wilson TM et al. Alteration of serum sphingolipids upon exposure of ponies to feed containing fumonisins, mycotoxins produced by Fusarium moniliforme. J Nutr 1992; 122:1706-1716. 16. Wang E, Riley RE, Meredith FI et al. Fumonisin B1 consumption by rats causes reversible, dosedependent increases in urinary sphinganine and sphingosine. J Nutr 1999; 129:214-220. 17. Smith ER, Jones PL, Boss JM et al. Changing J774A.1 cells to new medium perturbs multiple signaling pathways, including the modulation of protein kinase C by endogenous sphingoid bases. J Biol Chem 1997; 272:5640-5646. 18. Adams J, Ann Q. Structure determination of sphingolipids by mass spectrometry. Mass Spectrom Rev 1993; 12:51-85. 19. Stevens VL, Nimkar S, Jamison WC et al. Characteristics of the growth inhibition and cytotoxicity of long-chain (sphingoid) bases for Chinese hamster ovary (CHO) cells: Evidence for an involvement of protein kinase C. Biochim Biophys Acta 1990; 1051:37-45. 20. Wang E, Norred WP, Bacon CW et al. Inhibition of sphingolipid biosynthesis by fumonisins: Implications for diseases associated with Fusarium moniliforme. J Biol Chem 1991; 266:14486-14490.

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Ceramide Signaling 21. Merrill AH Jr, Schmelz E-M, Dillehay DL et al. Sphingolipids. The enigmatic lipid class: Biochemistry, physiology and pathophysiology. Toxicol & Appl Pharm 1997; 142:208-225. 22. Stevens VL, Tang J. Fumonisin B1-induced sphingolipid depletion inhibits vitamin uptake via the glycosylphosphatidylinositol-anchored folate receptor. J Biol Chem 1997; 272:18020-18025. 23. King CM, Land SJ, Jones RF et al. Role of acetyltransferases in the metabolism and carcinogenicity of aromatic amines, Mutat Res 1997; 376:123-128. 24. Lee TC, Ou MC, Shinozaki K et al. Biosynthesis of N-acetylsphingosine by platelet-activating factor: Sphingosine CoA-independent transacetylase in HL-60 cells. J Biol Chem 1995; 271:209-217. 25. Aridor-Piterman O, Lavie Y, Liscovitch M. Bimodal distribution of phosphatidic acid phosphohydrolase in NG108-15 cells. Modulation by the amphiphilic lipids oleic acid and sphingosine. Eur J Biochem 1992; 204:561-568. 26. Perry DK, Hand WL, Edmondson DE et al. Role of phospholipase D-derived diradylglycerol in the activation of the human neutrophil respiratory burst oxidase. Inhibition by phosphatidic acid phosphohydrolase inhibitors. J Immunol 1992; 149:2749-2758. 27. Wu W-I, McDonough M, Nickels JT et al. Regulation of lipid biosynthesis in Saccharomyces cerevisiae by fumonisin B1. J Biol Chem 1995; 270:13171-13178. 28. Lavie Y, Liscovitch M. Activation of phospholipase D by sphingoid bases in NG108-15 neuralderived cells. J Biol Chem 1990; 265:3868-3872. 29. Kiss Z, Crilly KS, Rossi MA et al. Selective inhibition by 4-hydroxynonenal of sphingosine-stimulated phospholipase D in NIH 3T3 cells. Biochim Biophys Acta 1992; 1124:300-302. 30. Desai NN, Zhang H, Olivera A et al. Sphingosine-1-phosphate, a metabolite of sphingosine, increases phosphatidic acid levels by phospholipase D activation. J Biol Chem 1992; 267:2312223128. 31. Coleman RA, Wang P, Bhat BG. Fatty acids and anionic phospholipids alter the palmitoyl coenzyme A kinetics of hepatic monoacylglycerol acyltransferase in Triton X-100 mixed micelles. Biochemistry 1996; 35:9576-9583. 32. Schmelz EM, Dombrink-Kurtzman MA, Roberts PC et al. Induction of apoptosis by Fumonisin B1 in HT-29 cells is mediated by the accumulation of endogenous free sphingoid bases. Toxicol Appl Pharmacol 1998; 148:252-260.

CHAPTER 2

Ceramide in Apoptosis: Possible Biophysical Foundations of Action Paavo K. J. Kinnunen and Juha M. Holopainen

Summary

O

ne of the conserved lipid signaling systems in multicellular organisms is the SM cycle. 1,2 The key molecule in this cascade is ceramide, which has been identified to serve as a second messenger for a variety of cellular processes, ranging from differentiation and proliferation to senescence and programmed cell death, apoptosis. The site and source, as well as the amount of ceramide formed, the phase of the cell cycle, and the activation state of downstream signaling molecules all play a role in the final outcome. This review summarizes the impact of ceramide on the biophysical properties of model biomembranes and discusses how these profound and somewhat unusual effects could perhaps be manifested in the different cellular processes triggered by this lipid second messenger.

Introduction The last two decades have witnessed major progress in the development of our understanding of the complex signaling mechanisms controlling cell behavior. Several cascades have been discovered and involve the rapid formation of signaling complexes harboring docking proteins and enzymes such as kinases and phospholipases, jointly launching a branched, yet interconnected network of signals, which configure the cell for a specific response or an altered behavior. Importantly, in addition to providing the basic bilayer structure of biomembranes, specific lipids are now recognized as reservoirs or precursors for the generation of a variety of second messengers, such as phosphatidylinositols, phosphatidic acid, diacylglycerol, and ceramide, generated in different cellular signaling mechanisms after the activation of specific lipid-modifying enzymes. Lipids represent the chemically most diverse class of biomolecules;3 however, the functional significance of this diversity remains poorly understood. Accordingly, the roles of lipids in cellular signaling mechanisms are of particular interest. Membranes are best described as highly dynamic supramolecular assemblies, the functions and contained activities of the embedded proteins being controlled by their 2-D and 3-D organization and the physical (phase) state of the membrane, further determined by factors such as lipid composition and temperature.3-5 We have postulated that the physiological state of the cell is, in part, controlled by the physical state of the membranes. The latter should thus be comprehended as an element in cellular signaling mechanisms. A good example is the control of the activity of phospholipase A2 by osmotic stretching of the substrate membrane.6,7 Another and a somewhat analogous case is provided by the control of phospholipase A2 by membrane potential.8 Ceramide has been confirmed to function as a second messenger in several cellular processes, including apoptosis, growth suppression, differentiation, and cell senescence.2,9-13 Interestingly, Thomas et al4 observed that induction of apoptosis coincides with augmented Ceramide Signaling, edited by Anthony H. Futerman. ©2002 Eurekah.com and Kluwer Academic / Plenum Publishers.

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cellular levels of C16:0-ceramide while only minor changes were evident in other ceramide species. The impact of ceramide seems to depend on the cell line used as well as on the experimental setup.11 Moreover, the cell cycle could influence the signaling processes. A signaling cascade involving MAP kinase and leading to the activation of NF-κB has been proposed,15,16 as well as selective induction of the SAPK/JNK pathway.17 Treatment of cells by heat or radiation, for instance, to induce apoptosis has been reported to produce high concentrations of ceramide, up to 10 mol% of total cellular lipids.11 In the light of this very high content, pronounced physicochemical effects can also be anticipated to be manifested in the membranes in question, in addition to the activation of specific proteins by this lipid. This review compiles relevant currently available data on the biophysical properties of ceramide and the impact this lipid imparts on model membranes. Together with recent insights derived from cell biological approaches, potential mechanisms and events can be delineated at the molecular level.

Topology of Ceramide Formation In eukaryotic cells ceramide is formed by the hydrolysis of the ubiquitous SM by sphingomyelinase (SMase)2,18 or, alternatively, by de novo synthesis of ceramide. Both pathways are used in signaling, the major difference being the time course of their action. In brief, the activation of SMase yields ceramide within a few minutes whereas de novo synthesis on the surface of the cytosolic leaflet of the endoplasmic reticulum by serine palmitoyltransferase19 produces ceramide in the time range of hours.11 Ceramide formed in the latter pathway is transported to the Golgi apparatus where SM and complex glycosphingolipids are made, with their subsequent vesicular transfer to the plasma membrane. The cellular levels of ceramide are further influenced by the rate of its removal by ceramide glycosyl transferase (forming glycosylceramide), and ceramidase (yielding sphingosine and fatty acid20). Importantly, ceramide is a highly hydrophobic amphiphile and thus remains in membranes, unlike soluble second messengers formed in signalling cascades.21-23 The action of ceramide can be anticipated to be topical, and restricted to the site(s) of its formation within the cell. In keeping with this view, ceramide generated by the acidic SMase in lysosomes does not exchange with other membranes, thus challenging the involvement of lysosomal ceramide in apoptotic signaling. It is currently believed that the death signal derives from the plasma membrane. Alternatively, ceramide produced by ceramide synthase has been suggested to activate the preapoptotic signaling cascade in mitochondria.25 A third possibility is indicated by observations on apoptotic hepatocytes revealing the involvement of nuclear ceramide produced by SMase.26 SMases are ubiquitous enzymes found in several tissues. However, the human isoenzymes are likely to differ in their catalytic properties, subcellular localization, and in their mode of regulation.20 At least six different human SMases have been identified so far. An acidic SMase is found in lysosomes and its genetic defect causes hereditary Niemann-Pick disease.27 The acidic SMase has been suggested to mediate biological responses such as apoptosis and cell differentiation,28 although controversial results have been published.29 A secretory acidic SMase is encoded by the same gene as the lysosomal enzyme.30,31 Interestingly, this SMase resides in specialized plasma membrane domains, caveolae.32,33 The secretory acidic SMase has been linked to the progression of atherosclerosis.31,34 SMase is also associated with the human plasma low density lipoprotein, LDL, and sequence comparison to bacterial enzymes suggests that this activity could be intrinsic to the apolipoprotein moiety of LDL, apoB-100.35 A role for this enzyme in LDL metabolism and progression of atherosclerosis has been suggested35,36 Finally, two forms of neutral SMase, viz. Mg2+-dependent and independent, have been identified. The former has been putatively identified as an integral membrane protein37,38 residing on the cytoplasmic face of the plasma membrane and is thus an obvious candidate for the production of the lipid second messenger ceramide.20 Soluble Mg2+-independent neutral SMase39 resides in the cytoplasm, where its function remains unknown. Secreted alkaline SMase is found in the gastrointestinal tract and has been suggested to be linked to hepatobiliary diseases.40

Cermide in Apoptosis: Possible Biophysical Foundations of Action

11

Ceramide—From Simple Structure to Complex Behavior Biophysical approaches on the effects of ceramide on cell behavior have relied on the use of various well defined model biomembranes, composed of binary or ternary mixtures of both natural and synthetic ceramides with phosphatidylcholines and SM. The advantage of synthetic lipids is that difficulties in the interpretation of results due to the variation in the ceramide N-acyl chain can be avoided. The effects of ceramide on model membranes can be divided into three distinct yet interconnected effects, as follows.

Increase in Acyl Chain Order

2 H-NMR studies on bovine brain ceramide/dipalmitoylphospholcholine (DPPC) membranes in the fluid state at 60oC revealed enhanced acyl chain order (reduced acyl chain trans→gauche isomerization) to be induced by ceramide.41 These authors further showed that C6:0, C8:0, and C16:0-ceramides, but not C2-ceramide, increased acyl chain order in DPPC bilayers at 45oC.41 Similar results were obtained by measuring fluorescence anisotropy for diphenylhexatriene (DPH) for binary membranes composed of dimyristoylphosphocholine (DMPC) and bovine brain ceramide.43 The above studies used the saturated phospholipids, DMPC and DPPC, which cannot be considered as representative for lipids in biological membranes. Importantly, increased acyl chain order was observed by DPH anisotropy measurements also for liposomes of unsaturated 1-palmitoyl-2-oleoyl-phosphocholine (POPC) and synthetic C16:0-ceramide.21 Augmented acyl chain order in membranes containing ceramide is in accordance with the strongly elevated transition temperatures recorded by differential scanning calorimetry (DSC).42,44 These data, the DSC observations in particular, readily imply that upon the formation of ceramide in biomembranes there is a pronounced increase in their ‘rigidity’. In other words, in keeping with the high melting temperatures of pure ceramides, observed at approx. 90-93oC,45 the membranes in apoptotic cells containing high amounts of ceramide become semi-crystalline.

Lateral Microdomain Formation

The fluid mosaic model of biomembranes proposed by Singer and Nicolson46 implicitly assumed the lateral lipid distribution of lipids to be random. However, a wealth of data has demonstrated that this is not the case and that biomembranes are organized.3 While ordering on several time- and length-scales is inherent to fluctuating many-body systems, several molecular level mechanisms have also been established.47 With the recent surge of interest in lipid trafficking and the organization of cellular signaling complexes in the plasma membrane, the mechanisms accounting for lateral lipid organization have become a subject of intense study. Phase diagrams best describe the cross properties of lipids. The partial phase diagram for C16:0-ceramide/DMPC membranes constructed on the basis of DSC and x-ray scattering data21,22,43,44 reveals that already low contents of ceramide become enriched and segregate into gel-state microdomains both in gel and fluid membranes and at physiological temperature. Formation of ceramide-enriched microdomains was first observed by Huang et al41 using 2 H-NMR spectroscopy. These authors showed that increasing the content of bovine brain ceramide in a DPPC matrix at 45oC induced gel-fluid coexistence, with ceramide preferentially partitioning into the ordered gel domains. Segregation of ceramide was observed in both fluid as well as gel state membranes. Microdomain formation was shown in DMPC/natural ceramide bilayers measuring the excimer/monomer fluorescence emission intensity ratio Ie/Im for the pyrene labeled lipid probe as well as by DSC.43 These results were confirmed by Veiga et al and Carrer and Maggio.48,49 Importantly, segregation of ceramide into microdomains could be demonstrated also in binary liposomes composed of ceramide and the unsaturated POPC, which can be regarded as a representative for phospholipids in fluid biomembranes.21 Formation of gel state microdomains enriched in ceramide could be directly observed in Langmuir films.49a Interestingly, variation in the N-acyl chain of ceramide affects the 2-D ordering of the DMPC/ceramide film. In brief, immiscibility of C16:0-ceramide is evident

12

Ceramide Signaling

already at low content of this lipid. However, C24:1-ceramide in low content is miscible in DMPC, while either an increase in lateral pressure or the content of this ceramide induces immiscibility. This difference can be rationalized in terms of the long hydrocarbon chain of C24:1-ceramide protruding above the DMPC monolayer and causing an additional repulsive potential between the ceramides. Our results thus reveal a significant role for the N-acyl chain of ceramides in influencing the lateral segregation and organization of sphingolipids in biomembranes. The above experiments involved model membranes, which can be assumed to be either at or close to thermodynamic equilibrium. In cellular membranes, ceramide is formed from SM, which is miscible and fluid at physiological temperatures in phosphatidylcholine membranes. In addition, regardless of the distribution of SM in the plasma membrane with a high content of cholesterol, the formation of ceramide from SM should have a major impact on its overall physical state and organization. The consequences of forming ceramide were initially studied by subjecting large unilamellar binary POPC/C16:0-SM vesicles (LUVs, diameter approx. 100 nm) to SMase.21 Monitoring DPH anisotropy revealed an immediate and pronounced increase in acyl chain order, concomitant with the formation of ceramide. However, microdomain formation observed using a pyrene-labeled lipid analog was significantly slower, requiring nearly two hours.21 This difference in the impact of ceramide formation on membrane dynamics and organization is likely to reflect the high curvature of the liposomes used. Accordingly, in a subsequent study employing so-called giant unilamellar vesicles (GUVs), liposomes with diameters in the range of 10 to 100 microns, revealed that following topical application of SMase onto the surface of fluid POPC/C16:0-SM (3:1, molar ratio) GUVs, very rapid (within ~10 seconds) formation of ceramide enriched microdomains was evident.49

Membrane Vesiculation The above results demonstrate the profound segregation of ceramide-enriched microdomains in binary and ternary model biomembranes, as well as upon enzymatic formation of ceramide. Another and perhaps rather unprecedented observation made in giant liposomes was the vectorial formation of ceramide-enriched vesicles following the exposure of fluid POPC/C16:0-SM bilayers to SMase (Fig. 1).22 Accordingly, immediately following the formation of bright fluorescent areas, enriched in the marker molecule, BODIPY-ceramide, in the outer leaflet of the vesicle, these domains formed numerous small vesicles inside the GUVs, resembling endocytosis. Likewise, when the enzyme was microinjected into the aqueous cavity of GUVs, small vesicles budded on the outer surface of the GUV. The above observations readily comply with geometrical considerations on the effective molecular shapes of ceramide50 as well as with the packing properties of ceramide inferred from its chemical structure and observed by X-ray diffraction of its crystals. More specifically, the polar headgroup of ceramide is small and self-associates laterally by intermolecular hydrogen bonding.51,52 The latter further reduces the interactions of the ceramide headgroup with water, resulting in weak hydration.45 Upon the action of SMase on membranes consisting of SM and phosphatidylcholine, the ceramide formed first segregates laterally into microdomains.22 Because of their negative spontaneous curvature these ceramide-enriched microdomains cusp, eventually forming smaller vesicles. These results imply two possible functions for ceramide formation in cells. Firstly, it seems feasible to suggest that the enzymatic formation of ceramide may well cause the observed membrane blebbing in apoptosis, in a manner not requiring metabolic energy. Therefore, ceramide would not only function as a lipid second messenger but its effects could also involve changes in the 3-D topology of the plasma membrane. Secondly, these results suggest that the physicochemical properties of ceramide could also provide the actual driving force for the transport of this lipid from the endoplasmic reticulum to the Golgi membranes as well as in endocytosis from the plasma membrane. Similar formation of ‘endocytotic’ vesicles could be demonstrated with human plasma LDL and GUVs.35 Based on these findings, we have suggested that the SMase activity of LDL could

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Figure 1. Invagination and membrane vesiculation driven by the asymmetric formation of ceramide in the bilayer, resulting in bending strain.

be involved in the nonreceptor mediated entry of this lipoprotein into cells.35,36 Yet, this mechanism is likely to be more generic and allow for the entry of SMase containing particles, such as microbes into mammalian cells, inducing the pinching of ‘autocytotic’ vesicles off the plasma membrane into the cytoplasm, with the contained pathogen. Evidence for the involvement of SMase and phosphatidylcholine-specific phospholipase C (PC-PLC) in the entry of Neisseria gonorrhea, Staphylococcus aureus, and species of mycobacteria into human cells was recently presented (see ref. 22 for a brief review). The role of PC-PLC could be associated with breakdown of the surrounding vesicle inside the cell, allowing for the subsequent passage of the microbe into the cytoplasm. The above mechanism may not be limited to bacteria, but could be responsible for the entry of viruses as well. To this end, Sindbis virus triggers apoptosis in cells, with the formation of ceramide.53 Detailed understanding of the role(s) of SMase as well as other phospholipid-degrading enzymes in the above processes may thus open novel possibilities for therapeutic intervention.

Promotion of Inverted Nonlamellar Phases

Most biological membranes are in the lamellar phase states.54 Interestingly, Gõni and Alonso and their coworkers have provided evidence that ceramide promotes the formation of inverted nonlamellar phases.55 In brief, a thermotropic lamellar to nonlamellar lipid phases transition in SM/phosphatidylethanolamine/cholesterol (2:1:1, molar ratio) and phosphatidylcholine/phosphatidylethanolamine/cholesterol (2:1:1, molar ratio) liposomes was observed when ceramide (mole fraction X=0.10) was present.55 Using DSC and NMR, Veiga et al48 further demonstrated that ceramide from egg yolk as well as from bovine brain promotes the formation of the inverted hexagonal (H II ) phase when added to liposomes composed of dielaidoylphosphatidylethanolamine (DEPE). Upon including ceramide (up to mole fraction X=0.15), the La→HII transition temperature of DEPE decreased with no major changes in the transition enthalpy. The two ceramides did not differ in this respect although their acyl chain lengths varied considerably. Small effective size of a lipid’s headgroup compared to the volume occupied by its acyl chains increases the packing pressure between the latter and provides a generic driving force for the formation of inverted nonlamellar phases.4 When the formation

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Ceramide Signaling

of a nonlamellar phase such as HII is not allowed, either due to high bending rigidity of the other leaflet of the bilayer or due to packing constraints (for instance, in small diameter vesicles with high positive curvature) there is a pronounced increase in the pressure within the hydrocarbon region of the bilayer. This state has been defined as “frustrated”.5 High packing pressure of the acyl chains can be reduced by lipids adapting the so-called extended conformation (Fig. 2).56,57 Yet, to avoid the exposure of hydrocarbon to water this requires the presence of an adjacent lipid bilayer, which can accommodate the protruding acyl chains without contacts of the latter with water molecules. Lipids in the extended conformation thus constitute an effective molecular ‘zipper’ linking two adjacent bilayers together, thus driving membrane hemifusion and fusion.58,59 To this end, ceramide has been reported to induce fusion of liposomes composed of stratum corneum lipids.60

Impact of Ceramide on Biomembranes—Further Hypotheses The headgroup of ceramide interacts only weakly with water. As a consequence, the concentration of this lipid in the cytosol is extremely low (with the possible exception of short-chain ceramides) and this lipid is concentrated in the cellular membranes where it is formed. Ceramides could influence the activities of membrane proteins by two principally different mechanisms: (i) by binding and (in) activating proteins due to specific ceramide-protein interactions, and (ii) by changing the physical properties of cellular membranes and thus the activity of membrane proteins as well as the organization of membranes. These mechanisms are not mutually exclusive and are likely to operate in unison, leading to changes in the physiological state of the cell.1,54 The formation of ceramide in membranes could facilitate the bilayer association of specific peripheral membrane proteins. Although several downstream effectors for ceramide have been proposed, only cathepsin D, protein kinase Cζ, phospholipase A2, and ceramide activated protein phosphatase (CAPP) have been demonstrated to be activated by this lipid in vitro.1,12 For atypical protein kinases the interaction has been suggested to be mediated by their cysteine-rich domains.61 However, peripheral proteins lacking selective interactions with ceramide could also be influenced by this lipid. As outlined above, the formation of ceramide in the membrane changes its bulk physical properties by increasing surface hydrophobicity as well as the propensity of the membrane to form inverted nonlamellar phases, changing the bilayer lateral pressure profile. This could result in the activation of proteins such as phospholipase C and protein kinase C.4,5,17,55 Augmented acyl chain packing can be expected to be particularly pronounced in the ceramide enriched microdomains. Accordingly, the observed stereospecificity of the effects induced by ceramide12 may also involve stereospecific hydrogen bond mediated ceramide-ceramide interactions. Yet this possibility remains unexplored. Formation of ceramide enriched microdomains also produces phase boundaries, which facilitate membrane absorption and activation of proteins such as phospholipase A2.41,42 The most conserved part of integral membrane proteins is their membrane spanning domain.3 Accordingly, specific lipid-protein interactions as well as mechanisms conveying changes in the physical state of membrane lipids to the conformation and activity of integral membrane proteins are likely to exist. Unfortunately, our understanding of these issues remains rather limited. An interesting concept is provided by the ‘mattress’-model introduced by Mouritsen and Bloom,62 which is based on the hydrophobic matching condition of the length of the membrane spanning part of integral membrane proteins and the thickness of the surrounding lipids (Fig. 3). To this end, the hydrophobic matching condition is applicable not only to lipid-protein63-64 interactions but also to lipid-lipid interactions.65 This mechanism could be relevant to the action of ceramide. Increased acyl chain order due to ceramide readily causes the membrane thickness to increase. Our x-ray scattering data revealed a difference of 6 Å between the thickness of fluid DMPC and gel-state ceramide-enriched bilayers.44 The thickness of ceramide-enriched microdomains thus exceeds that of the remaining membrane. Accordingly,

Cermide in Apoptosis: Possible Biophysical Foundations of Action

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Figure 2. Extended lipid anchorage for a peripheral membrane protein containing a hydrophobic, lipid acyl chain accommodating cavity.4,5,68,69 This allows for a local relief of the packing of the hydrocarbon chains and thus reduction on the total system free energy. This mechanism would be operative on surfaces with enforced positive curvature, such as for the adjacent monolayers of the other side of cusping ceramide-enriched microdomains with high negative curvature.

we can expect integral membrane proteins with long hydrophobic transmembrane segments to cosegregate into the ceramide-enriched membrane domains. This co-enrichment can be anticipated to be more pronounced if the said transmembrane domains do not contain amino acids with bulky side chains, thus allowing them to be accommodated into the lattice formed by the ordered ceramide acyl chains. Ceramide formation could thus result in its ‘capping’ together with integral membrane proteins66 with matching hydrophobic thicknesses. Enrichment of these proteins into the ceramide containing microdomains also results in their depletion from other regions of the membrane. One of the hallmarks of apoptosis is membrane blebbing.67 This process could be driven by the formation of ceramide in the cytoplasmic leaflet of the plasma membrane.22 In the light of the above it is likely that these ceramide-enriched domains also contain specific proteins. Accordingly, if these proteins function in cellular signaling,12 their shedding from the cells would make apoptotic cells nonresponsive to external stimuli, thus aiding their deletion. Finally, in spite of the fact that more thorough studies are warranted in order to verify the above putative mechanism, they fully comply with the biophysical mechanisms established in model systems. Integration of these and similar basic mechanisms with cell biological studies thus appears to represent a new and attractive research area, with a great potential in unraveling novel mechanisms controlling cell behavior.

Acknowledgements Helsinki Biophysics and Biomembrane Group is supported by Tekes and Finnish Academy (P.K.J.K.). J.M.H. acknowledges grants from Farmos Research Foundation, Paulo Research Foundation, and Emil Aaltonen Foundation.

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Ceramide Signaling

Figure 3. Requirement for matching hydrophobic thickness for lipids and transmembrane proteins driving the cosegregation of proteins into ceramide-enriched microdomains. See text for details.

References 1. Kolesnick RN, Golde DW. The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling. Cell 1994; 77:325-328. 2. Hannun YA. The sphingomyelin cycle and the second messenger function of ceramide. J Biol Chem 1994; 269:3125-3128. 3. Kinnunen PKJ. On the principles of functional ordering in biological membranes. Chem Phys Lipids 1991; 57:375-399. 4. Kinnunen PKJ. In: Lasic DD, Barenholz Y, eds. Nonmedical Applications of Liposomes. Boca Raton: CRC Press, 1996a:153-171. 5. Kinnunen PKJ. On the molecular-level mechanism of peripheral protein-membrane interactions induced by lipids forming inverted nonlamellar phases. Chem Phys Lipids 1996b; 81:151-166. 6. Lehtonen JYA, Kinnunen PKJ. Phospholipase A2 as a mechanosensor. Biophys J 1995; 68:1888-1894. 7. Kinnunen PKJ, Holopainen JM. Mechanisms of initiation of membrane fusion: Role of lipids. Biosci Rep 2000; 20:465-482. 8. Thuren T, Tulkki AP, Virtanen JA et al. Triggering of the activity of phospholipase A2 by an electric field. Biochemistry 1987; 26:4907-4910. 9. Chao MV. Ceramide: a potential second messenger in the nervous system. Mol Cellular Neurosci 1995; 6:91-96. 10. Gómez-Muñoz A. Modulation of cell signalling by ceramides. Biochim Biophys Acta 1998; 1391:92-109. 11. Hannun YA. Functions of ceramide in coordinating cellular responses to stress. Science 1996; 274:1855-1859. 12. Kolesnick RN, Gõni FM, Alonso A. Compartmentalization of ceramide signaling: physical foundations and biological effects. J Cell Physiol 2000; 184:285-300. 13. Spiegel S, Foster D, Kolesnick R. Signal transduction through lipid second messengers. Curr Opin Cell Biol 1996; 8:159-167.

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14. Thomas RL Jr, Matsko CM, Lotze MT et al. Mass spectrometric identification of increased C16 ceramide levels during apoptosis. J Biol Chem 1999; 274:30580-30588. 15. Davis RJ. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem 1993; 268:14553-14556. 16. Hannun YA, Obeid LM. Ceramide: an intracellular signal for apoptosis. Trends Biochem Sci 1995; 20:73-77. 17. Verheij M, Bose R, Lin XH et al. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature 1996; 380:75-79. 18. Jarvis WD, Grant S, Kolesnick RN. Ceramide and induction of apoptosis. Clin Cancer Res 1996; 2:1-6. 19. Weiss B, Stoffel W. Human and murine serine-palmitoyl-CoA transferase. Cloning, expression and characterization of the key enzyme in sphingolipid synthesis. Eur J Biochem 1997; 249:237-247. 20. Huwiler A, Kolter T, Pfeilschifter J et al. Physiology and pathophysiology of sphingolipid metabolism and signaling. Biochim Biophys Acta 2000; 1485:63-99. 21. Holopainen JM, Subramaniam M, Kinnunen PKJ. Sphingomyelinase induced lipid microdomain formation in a fluid phosphatidylcholine/sphingomyelin membrane. Biochemistry 1998; 37:17562-17570. 22. Holopainen JM, Angelova MI, Kinnunen PKJ. Vectorial budding of vesicles by asymmetric enzymatic formation of ceramide in giant liposomes. Biophys J 2000b; 78:830-838 . 23. Venkatamaran K, Futerman AH. Ceramide as a second messenger: sticky solutions to sticky problems. Trends Cell Biol 2000; 10:408-412. 24. Chatelut M, Leruth M, Harzer K et al. Natural ceramide is unable to escape the lysosome, in contrast to a fluorescent analogue. FEBS Lett 1998; 426:102-106. 25. Kroesen BJ, Pettus B, Luberto C et al. Induction of apoptosis through B-cell receptor cross-linking occurs via de novo generated C16-ceramide and involves mitochondria. J Biol Chem 2001; 276:13606-13614. 26. Tsugane K, Tamiya-Koizumi K, Nagino M et al. A possible role of nuclear ceramide and sphingosine in hepatocyte apoptosis in rat liver. J Hepatol 1999; 31:8-17. 27. Elleder M. Niemann-Pick disease. Pathol Res Pract 1989; 185:293-328. 28. Wiegmann K, Schutze S, Machleidt T et al. Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 1994; 78:1005-1015. 29. Bezombes C, Segui B, Cuvillier O et al. Lysosomal sphingomyelinase is not solicited for apoptosis signaling. FASEB J 2001; 15:297-299. 30. Schissel SL, Keesler GA, Schuchman EH et al. The cellular trafficking and zinc dependence of secretory and lysosomal sphingomyelinase, two products of the acid sphingomyelinase gene. J Biol Chem 1998a; 273:18250-18259. 31. Schissel SL, Jiang X-C, Tweedie-Hardman J et al. Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral pH. Implications for atherosclerotic lesion development. J Biol Chem 1998b; 273:2738-2746. 32. Liu P, Anderson RG. Compartmentalized production of ceramide at the cell surface. J Biol Chem 1995; 270:27179-27185. 33. Smart EJ, Graf GA, McNiven MA et al. Caveolins, liquid-ordered domains, and signal transduction. Mol Cell Biol 1999; 19:7289-7304. 34. Schissel SL, Tweedie-Hardman J, Rapp JH et al. Rabbit aorta and human atherosclerotic lesions hydrolyze the sphingomyelin of retained low-density lipoprotein. Proposed role for arterial-wall sphingomyelinase in subendothelial retention and aggregation of atherogenic lipoproteins. J Clin Invest 1996; 98:1455-1464. 35. Holopainen JM, Penate Medina O, Metso AJ et al. Sphingomyelinase activity associated with human plasma low density lipoprotein: Possible functional implications. J Biol Chem 2000a; 275:16484-16489. 36. Kinnunen PKJ, Holopainen JM. Sphingomyelinase activity of LDL: A link between apoptosis, atherosclerosis, and ceramide? Trends Cardiovasc Med 2002; 12:37-42. 37. Tomiuk S, Hofmann K, Nix M et al. Cloned mammalian neutral sphingomyelinase: functions in sphingolipid signaling? Proc Natl Acad Sci USA 1998; 95:3638-3643. 38. Tomiuk S, Zumbansen M, Stoffel W. Characterization and subcellular localization of murine and human magnesium-dependent neutral sphingomyelinase. J Biol Chem 2000; 275:5710-5717. 39. Okazaki T, Bielawska A, Domae N et al. Characteristics and partial purification of a novel cytosolic, magnesium-independent, neutral sphingomyelinase activated in the early signal transduction of 1-α,25-dihydroxyvitamin D3-induced HL-60 cell differentiation. J Biol Chem 1994; 269:4070-4077. 40. Duan RD, Nilsson A. Purification of a newly identified alkaline sphingomyelinase in human bile and effects of bile salts and phosphatidylcholine on enzyme activity. Hepatology 1997; 26:823-830.

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41. Huang HW, Goldberg EM, Zidovetzki R. Ceramide induces structural defects into phosphatidylcholine bilayers and activates phospholipase A2. Biochem Biophys Res Commun 1996; 220:834-838. 42. Huang HW, Goldberg EM, Zidovetzki R. Ceramides perturb the structure of phosphatidylcholine bilayers and modulate the activity of phospholipase A2. Eur Biophys J 1998; 27:361-366. 43. Holopainen JM, Lehtonen JYA, Kinnunen PKJ. Lipid microdomains in dimyristoylphosphatidylcholine-ceramide liposomes. Chem Phys Lipids 1997; 88:1-13. 44. Holopainen JM, Lemmich J, Richter F et al. Dimyristoylphosphatidylcholine/C16:0-ceramide binary liposomes studied by differential scanning calorimetry and wide- and small- angle x-ray scattering. Biophys J 2000c; 78:2459-2469. 45. Chen H, Mendelsohn R, Rerek ME et al. Fourier transform infrared spectroscopy and differential scanning calorimetry studies of fatty acid homogenous ceramide 2. Biochim Biophys Acta 2000; 1468:293-303. 46. Singer SJ, Nicolson GL. The fluid mosaic model of the structure of cell membranes. Science 1972; 175:720-731. 47. Mouritsen OG, Kinnunen PKJ. Role of lipid organization and dynamics for membrane functionality. In: Merz K Jr, Roux B, eds. Biological Membranes. Boston: Birkhäuser, 1996: 463-502. 48. Veiga MP, Arrondo JLR, Goñi FM et al. Ceramides in phospholipid membranes: effects on bilayer stability and transition to nonlamellar phases. Biophys J 1998; 76:342-350. 49. Carrer DC, Maggio B. Phase behavior and molecular interactions in mixtures of ceramide with dipalmitoylphosphatidylcholine. J Lipid Res 1999; 40:1978-1989. 49a. Holopainen JM, Brockman HL, Brown RE et al. Interfacial interactions of ceramide with dimyristoylphosphatidylcholine. Biophys J 2001; 80:765-775. 50. Israelachvili JN, Marcelja S, Horn RG. Physical principles of membrane organization. Q Rev Biophys 1980; 13:121-200. 51. Pascher I. Molecular arrangements in sphingolipids. Conformation and hydrogen bonding of ceramide and their implication on membrane stability and permeability. Biochim Biophys Acta 1976; 455:433-451. 52. Moore DJ, Rerek ME, Mendelsohn R. FTIR spectroscopy studies of the conformational order and phase behaviour of ceramides. J Phys Chem B 1997; 101:8933-8940. 53. Jan J-T, Chatterjee S, Griffin DE. Sindbis virus entry into cells triggers apoptosis by activating sphingomyelinase, leading to the release of ceramide. J Virol 2000; 74:6425-6432. 54. Kinnunen PKJ, Laggner P. Phospholipid phase transitions. Topical issue of Chem Phys Lipids 1991; 57:109-408. 55. Ruiz-Argüello MB, Basáñez G, Goñi FM et al. Different effects of enzyme-generated ceramides and diacylglycerols in phospholipid membrane fusion and leakage. J Biol Chem 1996; 271:26616-26621. 56. Kinnunen PKJ. Fusion of lipid bilayers: a model involving mechanistic connection to HII phase forming lipids. Chem Phys Lipids 1992; 63:251-258. 57. Holopainen JM, Lehtonen JYA, Kinnunen PKJ. Evidence for the extended phospholipid conformation in membrane fusion and hemifusion. Biophys J 1999; 76:2111-2120. 58. Kinnunen PKJ, Holopainen JM, Angelova MI. Giant liposomes as model biomembranes for roles of lipids in cellular signalling. In: Luisi PL, Walde P, eds. Giant Vesicles. New York: John Wiley & Sons, 2000:273-284. 59. Kinnunen PKJ. Lipid bilayers as osmotic response elements. Cell Physiol Biochem 2000; 10:243-250. 60. Abraham W, Wertz PW, Downing DT. Fusion patterns of liposomes formed from stratum corneum lipids. J Invest Dermatol 1988; 90:259-262. 61. van Blitterswijk WJ. Hypothesis: ceramide conditionally activates atypical protein kinases C, Raf-1 and KSR through binding to their cysteine-rich domains. Biochem J 1998; 331:679-680. 62. Mouritsen OG, Bloom M. Mattress model of lipid-protein interactions in membranes. Biophys J 1984; 46:141-153. 63. Lehtonen JYA, Kinnunen PKJ. Evidence for phospholipid microdomain formation in liquid crystalline liposomes reconstituted with Escherichia coli lactose permease. Biophys J 1997; 72:1247-1257. 64. Dumas F, Sperotto MM, Lebrun MC et al. Molecular sorting of lipids by bacteriorhodopsin in dilauroylphosphatidylcholine/distearoylphosphatidylcholine lipid bilayers. Biophys J 1997; 73:1940-1953. 65. Lehtonen JYA, Holopainen JM, Kinnunen PKJ. Evidence for the formation of microdomains in liquid crystalline large unilamellar vesicles caused by hydrophobic mismatch of the constituent phospholipids. Biophys J 1996; 70:1753-1760. 66. Cremesti A, Paris F, Grassme H et al. Ceramide enables Fas to cap and kill. J Biol Chem 2001; 276:23954-23961.

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67. Majno G, Joris I. Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol 1995; 146:3-19 68. Rytömaa M, Kinnunen PKJ. Reversibility of the binding of cytochrome c to liposomes. Implications for lipid-protein interactions. J Biol Chem 1995; 270:3197-3202. 69. Kinnunen PKJ, Koiv A, Lehtonen JYA et al. Lipid dynamics and peripheral interactions of proteins with membrane surfaces. Chem Phys Lipids 1994; 73:181-207.

CHAPTER 3

Ceramide-Mediated Receptor Clustering Erich Gulbins and Heike Grassmé

Abstract

M

any stress or pro-apoptotic stimuli such as irradiation, heat shock, UV light, bacterial or viral infections, ligation of CD95 or the tumor necrosis factor receptor have been shown to activate the acid and N-SMase to release ceramide from SM or to stimulate the de novo synthesis of ceramide via the ceramide synthase pathway. Ceramide seems to function as a central messenger in the cellular response to these stimuli and regulates a whole variety of intracellular proteins. In addition, ceramide has been recently shown to modify distinct membrane domains that are enriched with sphingolipids and cholesterol. Ceramide accumulation in those membrane domains triggers the fusion of small domain entities to larger signaling platforms and, second, the trapping of activated receptors in those platforms. This finally results in the clustering of activated receptor molecules, which seems to be a pre-requisite for the initiation of specific signaling.

Introduction SM was originally considered to be inert and to act as a structural element of the outer leaflet of the plasma membrane.1 In 1987, Kolesnick and co-workers discovered that 1,2diacylglycerols trigger an A-SMase mediated hydrolysis of SM suggesting that SM and ceramide function in cell signaling.2 Further evidence for a signaling role of ceramide was provided by studies showing that exogenous addition of SMase or sphingoid bases to GH3 cells prevented down-regulation of protein kinase C (PKC) after phorbolester treatment by inducing a redistribution of PKC from the cell membrane to the cytosol.3 Ceramide prevented TPA-induced growth inhibition of HL60-cells. Likewise, the morphological and biochemical conversion of HL-60 cells into macrophages was prevented by ceramide.4 These data indicated a function of ceramide in cell signaling. This notion was confirmed by studies from Hannun et al in 1989 showing that vitamin D3 induced SM hydrolysis to ceramide is involved in cell differentiation.5,6 Those initial studies strongly suggested that the hydrolysis of SM and the release of ceramide are involved in the cellular response to receptor activation or stress stimuli. They also implied that SM and ceramide do not only function as inert, structural parts of the cell membrane, but also as transmitters in cellular signaling pathways. These findings were confirmed by many publications in the last ten years, which showed that ceramide is able to regulate a whole variety of cellular signaling molecules. Ceramide binds to PLA2 and cathepsin D, which seems to trigger autocatalytic cleavage of cathepsin to its active form as well as activation of PLA2.7,8 Further, ceramide has been shown to regulate kinase suppressor of Ras (KSR; identical to ceramide-activated protein kinase),9 a ceramide-activated protein phosphatase,10 protein kinase C isoforms,11 c-Raf-1,12 the small G-proteins Ras and Rac,13,14 Src-like tyrosine kinases,15 Jun-N-terminal kinases16 and the ion channels Kv1.3 and CRAC.17,18 Although the exact

Ceramide Signaling, edited by Anthony H. Futerman. ©2002 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Ceramide Signaling

mechanism of ceramide-mediated regulation of these proteins is still unknown, this list nicely demonstrates the significance of ceramide as a cellular signaling molecule.

Ceramide Functions in Sphingolipid Enriched Membrane Rafts

We have recently suggested a novel concept for the signaling function of ceramide,19,20 which might integrate many of the biological and biophysical features of this molecule and shall be discussed. Eukaryotic cell membranes are primarily composed of sphingolipids, cholesterol and glycerophospholipids. SM is the most prevalent cellular sphingolipid and consists of a ceramide moiety and a phosphorylcholine headgroup. Ceramide itself is the amide ester of the sphingoid base D-erythro-sphingosine and a fatty acid usually of C16 through C26 chain length. Ceramide can be released from SM by the activity of three SMases that are characterized by their pH optima for enzymatic activity: acid, neutral, and alkaline SMase. Sphingolipids and cholesterol associate and separate from phospholipids within the bilayer membrane to form distinct membrane domains. This association is caused by hydrogen bonds and hydrophobic van der Waal forces between cholesterol and the sphingolipids. In addition, sphingolipids interact with each other by hydrophilic interactions between the headgroups, while cholesterol seems to function as a hydrophobic spacer between the bulky sphingolipids. The highly ordered biophysical phase of these SM/cholesterol-rich domains transforms these membrane domains into a distinct liquid ordered phase21-23 and induces a relative resistance to some detergents. Therefore, the glycosphingolipid-enriched membrane (GEM) domains have also been named detergent-insoluble glycosphingolipid-enriched membranes (DIGs).21,22 Since sphingolipid/cholesterol-rich membrane domains seem to float within the phospholipid portion of the cell membrane, they have been compared with rafts, an expression, which will be used. Recent studies suggested that the transformation of sphingolipid-enriched membrane rafts to signaling structures is mediated by ceramide. These studies demonstrated that stimulation of CD95 or CD40 with cognate ligand or stimulatory antibodies triggers a translocation of the acid sphingomyelinase (A-SMase) onto the extracellular leaflet of the cell membrane (Fig. 1).19,24,25 Electron microscopy studies localized the A-SMase to intracellular vesicles in unstimulated cells.20 Thus, it is likely that the activation of cells via CD95 or CD40 results in a mobilization of A-SMase containing vesicles, fusion of these vesicles with the cell membrane and exposure of the A-SMase to the extracellular space. This process occurs within a very short time and first traces of extracellularly oriented surface A-SMase were detected within seconds after receptor ligation.19 A-SMase functions best at an acidic pH. However, neutral pH values only increase the Km value and do not alter the activity [Vmax] of the enzyme.26 Since the activity of the A-SMase can be modified by environmental factors such as LDL or other extracellular membrane co-factors, those factors might compensate the reduction of substrate affinity and restore A-SMase activity at the cell surface even at neutral pH values.27 The translocation of the A-SMase onto the cell surface seems to be directed, since surface A-SMase localizes in small sphingolipid rich rafts (Fig. 1).16,20 This was shown by the co-localization of surface ASMase with fluorescence labeled cholera toxin,16,20 which is used as a marker for sphingolipidenriched membrane rafts. Since the outer leaflet of the plasma membrane contains the vast majority of cellular SM in mammalian cells28, the translocation of the A-SMase brings the enzyme into close proximity to its substrate. The final result of A-SMase-surface translocation is the release of extracellularly-oriented ceramide in very small membrane rafts. A function of A-SMase in lipid rafts has also been previously suggested by studies on signaling of the IL-1 and p75 nerve growth factor receptor,30,31 which demonstrated an A-SMase-mediated consumption of SM and release of ceramide in rafts upon cellular stimulation via those receptors. A-SMase-mediated release and accumulation of ceramide in very small membrane rafts may constitute the key step to transform these very small rafts into signaling structures in the cell membrane (Fig. 1).

Ceramide-Mediated Receptor Clustering

23

Figure 1. CD95 triggers translocation of the A-SMase onto the cell surface and clusters in membrane rafts. Stimulation via CD95 results in translocation of the A-SMase onto the cell surface and clustering of CD95. Surface A-SMase and clustered CD95 co-localize with FITC-cholera toxin indicating a clustering of CD95 in membrane rafts modified by the ASM. Cells were stimulated for 2 min via CD95, stained with FITCCholeratoxin, Cy3-labeled anti-CD95 and Cy5-coupled anti-A-SMase antibodies and analyzed by confocal microscopy. The right panel shows the overlay. Printed with permission from the Journal of Biological Chemistry.

Biophysical studies indicate that ceramide spontaneously self-aggregates into microdomains.31-34 Because ceramide-rich microdomains spontaneously fuse,34 the generation of ceramide in microdomains may trigger the formation of larger platforms. Both properties of ceramide might be central for the accumulation and clustering of receptor molecules in those signaling platforms. Receptor molecules that are constitutively present in membrane rafts are clustered simply by ceramide mediated fusion of very small rafts to larger platforms. Receptor molecules that might be primarily outside of rafts, but preferentially interact with ceramide or ceramide rich membrane domains, will be trapped in the ceramide rich signaling platforms and thereby also cluster (Fig. 1). Such a mechanism of ceramide mediated clustering of receptor molecules has been recently demonstrated for CD95 and CD40.19,20,24 The significance of ceramide mediated clustering of CD95 and CD40 for their function was analyzed in experiments using A-SMase deficient cells, or with reagents that destroy sphingolipid enriched rafts.19,24 These studies revealed that the inhibition of raft formation prevents CD95 or CD40 clustering as well as intracellular signaling initiated by these receptors.19,24 The function of clustering of receptor molecules within a defined area of the cell membrane is very likely to induce a high receptor density and a close proximity of receptor molecules. Since many intracellular signaling events seem to be facilitated by trans-activation of signaling molecules associating or interacting with a receptor, a close proximity of those molecules in rafts might be required and sufficient to initiate receptor specific signaling. In addition, ceramide-enriched platforms might recruit further intracellular signaling molecules, e.g., phospholipases or Src-like tyrosine kinases,35,36 to the clustered receptor; they may serve to exclude inhibitory pathways, directly alter the affinity/avidity of the receptor for its ligand and stabilize the receptor/ligand interaction by physical trapping of activated receptors, respectively. Finally, ceramide generated in rafts might also directly modify intracellular signaling molecules and, thus, interplay with receptor initiated signaling in rafts.37

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Ceramide Signaling

Figure 2. Ceramide alters membrane rafts and mediates clustering of receptor molecules. Primary stimulation via a receptor results in mobilization of intracellular vesicles containing the A-SMase, fusion of those vesicles with the cell membrane and exposure of the A-SMase on the cell surface. Surface A-SMase releases ceramide, which modifies preexisting small sphingolipid-enriched membrane rafts to fuse to larger signaling platforms and to trap activated receptor molecules in those platforms. The latter results in clustering of the activated receptor, which may function to amplify the primary signal to a level sufficient to mediate the biological function of the receptor.

In summary, the release of ceramide in preformed very small, sphingolipid-enriched membrane rafts seems to constitute the central step in the transformation of those primary very small rafts from an “inactive” to an “active” form involved in receptor mediated signaling (Fig. 2).

Ceramide-Mediated Signaling Platforms as a General Signaling Motif? The concept of ceramide-mediated transformation of very small “inactive” membrane rafts into large, “active” signaling platforms may also provide an explanation for the finding that A-SMase and ceramide are involved in multiple signaling pathways. Thus, A-SMase mediated ceramide release is triggered by the tumor necrosis factor receptor38, Interleukin-1 receptor,39 CD95,13,40,41 γ-irradiation,42 UV light,43,44 ischemia,45 infection of mammalian cells with pathogenic bacteria, e.g., Neisseriae gonorrhoeae46 or Staphylococcus aureus,47 or pathogenic viruses, e.g., Sindbis virus.49 Examples of receptors that activate the A-SMase and are involved in cell differentiation/proliferation are CD4024 and CD28.49 Finally, A-SMase is also involved in developmental processes and genetic deficiency of A-SMase prevents developmental apoptosis of oocytes and results in oocyte hyperplasia at birth of those mice.50 These studies suggest the concept that ceramide-enriched signaling platforms may not only be central in receptor-initiated signaling, but also in stress-mediated signaling or developmental processes. Finally, membrane rafts have emerged as central structure in the infection of mammalian cells with pathogenic bacteria, viruses, parasites and even prions. Pathogenic E. coli, Mycobacterium bovis, Campylobacter jejuni, Neisseria gonorrhoeae, simian virus 40, Measles-, Influenza, Sindbis-virus, Plasmodium falciparum and prions have been shown either to employ rafts for infection or to reside in rafts.46,51-59 At least Neisseria gonorrhoeae46 and Sindbis virus57 have already been demonstrated to activate the A-SMase, to release ceramide and, even more important, require expression of the A-SMase for internalization into the target cells. Therefore, it might be possible that at least some of those infective organisms trigger the formation of ceramideenriched membrane rafts to infect mammalian target cells.

Ceramide-Mediated Receptor Clustering

25

In summary, ceramide has been demonstrated to have a central role in the cellular response to a variety of stimuli. Ceramide functions as a messenger to directly regulate the activity of several cellular proteins. Second, it modifies sphingolipid enriched membrane rafts to form signaling platforms. The latter might be a comprehensive model for the biological effects and provide a biophysical explanation for the diverse functions of ceramide.

References 1. Barenholz Y, Thompson TE. Sphingomyelin in bilayers and biological membranes. Biochim Biophys Acta 1980; 604:129-158. 2. Kolesnick RN, Paley AE. 1,2-Diacylglycerols, but not phorbol esters stimulate sphingomyelin hydrolysis in GH3 pituitary cells. J Biol Chem 1987; 262: 9204-9210. 3. Kolesnick RN, Clegg S. 1,2-Diacylglycerols, but not phorbol esters activate a potential inhibitory pathway for protein kinase C in GH3 pituitary cells: evidence for involvement of a sphingomyelinase. J Biol Chem 1988; 263:6534-6537. 4. Kolesnick RN. Sphingomyelinase action inhibits phorbol ester-induced differentiation of human promyelocytic leukemic (HL-60) cells. J Biol Chem 1989; 264:7617-7623. 5. Okazaki T, Bell RM, Hannun YA. Sphingomyelin turnover induced by vitamin D3 in HL-60 cells. J Biol Chem 1989; 264:19076-19080. 6. Okazaki T, Bielawska A, Bell RM, Hannun YA. Role of ceramide as a lipid mediator of 1α, 25Dihydroxyvitamin D3-induced HL-60 cell differentiation. J Biol Chem 1990; 265:15823-15831. 7. Huwiler A, Johansen B, Skarstad A, Pfeilschifter J. Ceramide binds to the CaLB domain of cytosolic phospholipase A2 and facilitates its membrane docking and arachidonic acid release. FASEB J 2001; 15:7-9. 8. Heinrich M, Wickel M, Schneider-Brachert W et al. Cathepsin D targeted by acid sphingomyelinasederived ceramide. EMBO J 1999; 18:5252-5263. 9. Basu S, Bayoumy S, Zhang Y et al. BAD enables ceramide to signal apoptosis via Ras and Raf-1. J Biol Chem 1998; 273:30419-30426. 10. Dobrowsky RT, Hannun YA. Ceramide-activated protein phosphatase: partial purification and relationship to protein phosphatase 2A. Adv Lipid Res 1993; 25:91-104. 11. Muller G, Ayoub M, Storz P et al. PKC zeta is a molecular switch in signal transduction of TNFalpha, bifunctionally regulated by ceramide and arachidonic acid. EMBO J 1995; 14:1961-1969. 12. Yao B, Zhang Y, Delikat S et al. Phosphorylation of Raf by ceramide-activated protein kinase. Nature 1995; 378:307-310. 13. Gulbins E, Bissonnette R, Mahboubi A et al. FAS-induced apoptosis is mediated via a ceramideinitiated RAS signaling pathway. Immunity 1995; 2:341-351. 14. Brenner B, Koppenhoefer U, Weinstock C. et al. Fas- or ceramide-induced apoptosis is mediated by a Rac1-regulated activation of Jun N-terminal kinase/p38 kinases and GADD153. J Biol Chem 1997; 272:22173-22181. 15. Gulbins E, Szabo I, Baltzer K, Lang F. Ceramide-induced inhibition of T lymphocyte voltagegated potassium channel is mediated by tyrosine kinases. Proc Natl Acad Sci USA 1997; 94:7661-7666. 16. Westwick JK, Bielawska AE, Dbaibo G et al. Ceramide activates the stress-activated protein kinases. J Biol Chem 1995; 270:22689-22692. 17. Szabo I, Gulbins E, Apfel H et al. Tyrosine phosphorylation-dependent suppression of a voltagegate K+ channel in T lymphocytes upon Fas stimulation. J Biol Chem 1996; 271:20465-20469. 18. Lepple-Wienhues A, Belka C, Laun T et al. Stimulation of CD95 (Fas) blocks T lymphocyte calcium channels through sphingomyelinase and sphingolipids. Proc Natl Acad Sci U S A 1999; 96:13795-13800. 19. Grassmé H, Jekle A, Riehle A et al. CD95 signaling via ceramide-rich membrane rafts. J Biol Chem 2001; 276:20589-20596. 20. Grassmé H, Schwarz H, Gulbins E. Surface ceramide mediates CD95 clustering. Biochem Biophys Res Commun 2001; 284:1016-1030. 21. Harder T, Simons K. Caveolae, DIGs, and the dynamics of sphingolipid-cholesterol microdomains. Curr Opin Cell Biol 1997; 9:534-542. 22. Brown DA, London E. Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol 1998; 14:111-167. 23. Andersen RG. The caveolae membrane system. Annu Rev Biochem 1998; 67:199-225. 24. Grassmé H, Jendrossek V, Riehle A, Gulbins E. Ceramide-rich membrane rafts mediate CD40 clustering. J Immunol 2002; 168:298-307.

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25. Cremesti A, Paris F, Grassmé H et al. Ceramide enables Fas to cap and kill. J Biol Chem 2001; 276:23954-23961. 26. Callahan JW, Jones CS, Davidson DJ, Shankaran P. The active site of lysosomal sphingomyelinase: evidence for the involvement of hydrophobic and ionic groups. J Neurosci Res 1983; 10:151-63. 27. Schissel SL, Jiang X, Tweedie-Hardman J et al. Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral pH. Implications for atherosclerotic lesion development. J Biol Chem 1998; 273:2738-2746. 28. Emmelot P, Van Hoeven RP. Phospholipid unsaturation and plasma membrane organization. Chem Phys Lipids 1975; 14:236-246. 29. Liu P, Anderson RG. Compartmentalized production of ceramide at the cell surface. J Biol Chem 1995; 270:27179-27185. 30. Bilderback TR, Grigsby RJ, Dobrowsky RT. Association of p75 (NTR) with caveolin and localization of neutrophin-induced sphingomyelin hydrolysis to caveolae. J Biol Chem 1997; 272:1092210927. 31. Huang HW, Goldberg EM, Zidovetzki R. Ceramides modulate protein kinase C activity and perturb the structure of phosphatidylcholine/ phosphatidylserine bilayers. Biophys J 1999; 77:14891497. 32. Veiga MP, Arrondon JL, Goni FM, Alonso A. Ceramides in phospholipid membranes: effects on bilayer stability and transition to nonlamellar phases. Biophys J 1999; 76:342-350. 33. ten Grotenhuis E, Demel RA, Ponec M et al. Phase behavior of stratum corneum lipids in mixed Langmuir-Blodgett monolayers. Biophys J 1996; 71:1389-1399. 34. Holopainen JM, Subramanian M, Kinnunen PK. Sphingomyelinase induces lipid microdomain formation in a fluid phosphatidycholine/sphingomyelin membrane. Biochemistry 1998; 37:17562-17570. 35. Hamaguchi M, Hanafusa H. Localization of major potential substrates of p60v-src kinase in the plasma membrane matrix function. Oncogene Res 1989; 4:29-37. 36. Stauffer TP, Meyer T. Compartmentalized IgE receptor-mediated signal transduction in living cells. J Cell Biol 1997;139:1447-1454. 37. Zundel W, Swiersz LM, Giaccia A. Caveolin 1-mediated regulation of receptor tyrosine kinaseassociated phosphatidylinositol 3- kinase activity by ceramide. Mol Cell Biol. 2000; 20:1507-1514. 38. Schutze S, Potthoff K, Machleidt T et al. TNF activates NF-kappa B by phosphatidylcholinespecific phospholipase C-induced “acidic” sphingo-myelin breakdown. Cell 1992; 71:765-776 39. Mathias S, Younes A, Kan CC et al. Activation of the sphingomyelin signaling pathway in intact EL4 cells and in a cell-free system by IL-1 beta. Science 1993; 259:519-522. 40. Cifone MG, De Maria R, Roncaioli P et al. Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase. J Exp Med 1994; 180:1547-1552. 41. Brenner B, Ferlinz K, Grassme H et al. Fas/CD95/Apo-I activates the acidic sphingomyelinase via caspases. Cell Death Differ 1998; 5:29-37. 42. Santana P, Pena LA, Haimovitz-Friedman A et al. Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis. Cell 1996; 86:189-199. 43. Zhang Y, Mattjus P, Schmid PC et al. Involvement of the acid sphingomyelinase pathway in UVA-induced apoptosis. J Biol Chem 2001; 276:11775-11782. 44. Chatterjee M, Wu S. Involvement of Fas receptor and not tumor necrosis factor-alpha receptor in ultraviolet-induced activation of acid sphingomyelinase. Mol. Carcinog. 2001; 30:47-55. 45. Yu ZF, Nikolova-Karakashian M, Zhou D et al. Pivotal role for acidic sphingomyelinase in cerebral ischemia-induced ceramide and cytokine production, and neuronal apoptosis. J Mol Neurosci 2000; 15:85-97. 46. Grassmé H, Gulbins E, Brenner B et al. Acidic sphingomyelinase mediates entry of N. gonorrhoeae into nonphagocytic cells. Cell 1997; 91:605-615. 47. Esen M, Schreiner B, Jendrossek V et al. Mechanisms of Staphylococcus aureus induced apoptosis of human endothelial cells. Apoptosis 2001; 6:441-445. 48. Jan JT, Chatterjee S, Griffin DE. Sindbis virus entry into cells triggers apoptosis by activating sphingomyelinase, leading to the release of ceramide. J. Virology 2000; 74:6425-6432. 49. Boucher LM, Wiegmann K, Futterer, A et al. CD28 signals through acidic sphingomyelinase. J Exp Med 1995; 181:2059-2068. 50. Morita Y, Perez GI, Paris F et al. Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy. Nat. Med. 2000; 6:1109-1114. 51. Shin JS, Gao Z, Abraham SN. Involvement of cellular caveolae in bacterial entry into mast cells. Science 2000; 289:785-788. 52. Gatfield J, Pieters J. Essential role for cholesterol in entry of mycobacteria into macrophages. Science 2000; 288:1647-1650.

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53. Ferrari G, Langen H, Naito M, Pieters J. Host signal transduction and endocytosis of Campylobacter jejuni. Microb Pathog 1996; 21:299-305. 54. Norkin LC. Simian virus 40 infection via MHC class I molecules and caveolae. Immunol Rev 1999; 168:13-22. 55. Manie SN, Debreyne S, Vincent S, Gerlier D. Measles virus structural components are enriched into lipid raft microdomains: A potential cellular location for virus assembly. J Virol 2000; 74:305-311. 56. Scheiffele P, Rietveld A, Wilk T, Simons K. Influenza viruses select ordered lipid domains during budding from the plasma membrane. J Biol Chem 1999; 274:2038-2044. 57. Jan JT, Chatterjee S, Griffin DE. Sindbis virus entry into cells triggers apoptosis by activating sphingomyelinase leading to the release of ceramide. J. Virol. 200; 74:6425-6432. 58. Olliaro P, Castelli F. Plasmodium falciparum: an electronmicroscopy study of caveolae and trafficking between the parasite and the extracellular medium. Int J Parasitol 1997; 27:1007-1012. 59. Kaneko K, Vey M, Scott M et al. COOH-terminal sequence of the cellular prion protein directs subcellular trafficking and controls conversion into the scrapie isoform. Proc Natl Acad Sci USA 1997; 94:2333-2338.

CHAPTER 4

Ceramidases: Regulators of Turnover of Ceramide and Ceramide-Mediated Responses Cungui Mao and Lina Obeid

Abstract

C

eramide has been shown to mediate various stress-induced responses such as apoptosis, growth arrest, differentiation, inflammation, and heat stress response. Regulation of these responses may rely on the net cellular levels of ceramide, which are determined by a balance between the rate of its formation and that of its degradation. The formation of ceramide in response to agonists or chemotherapeutic agents appears to involve two distinct pathways, SM breakdown and de novo synthesis. The degradation of ceramide is mainly through ceramidases. Three types of ceramidase activities have been described. They are classified as acid, neutral, and alkaline types according to their pH optima for activity. Neutral and alkaline ceramidase activities appear to be regulated by growth factors, cytokines, lipoproteins, and nitric oxide (NO)- generating reagents. The mechanisms of regulation are largely unknown because the enzymes encoding the ceramidase activities have just recently been cloned. These cloned ceramidases can be divided into three groups based on sequence similarity. The first group of ceramidases including the human, mouse, and Drosophila acid ceramidases, which share significant sequence similarity, are mainly localized to lysosomes, and have pH optima of 4 to 5 for their activity. The second group of ceramidases, including the Pseudomonas and Mycobacterium alkaline ceramidases, mouse and rat brain neutral ceramidases, and human mitochondrial ceramidase, also share sequence similarity, but have broad pH optima of 7-10 and various cellular localizations. This group of ceramidases are reclassified as neutral/alkaline ceramidases. The third group of ceramidases, including the yeast alkaline phytoceramidase (YPC1p) and dihydroceramidase (YDC1p), human alkaline phytoceramidase (haPHC), and mouse alkaline ceramidase (maCER), share several conserved domains. Members of this group of enzymes have several transmembrane domains and an alkaline pH optimum, are localized to the Golgi apparatus, endoplasmic reticulum, or both, and have a more restricted substrate specificity than the first two groups of ceramidases. This group of ceramidases defines a strictly alkaline type of ceramidases. Ceramidases in one subgroup do not share any sequence similarity to those in the other subgroups although all ceramidases catalyze the same reaction, breaking down the amide linkage of ceramides. This review will focus on the cloning and characterization of these ceramidases, and their possible physiological roles.

Introduction Three major types of ceramides have been found in eukaryotic cells: unsaturated ceramide (traditionally called ceramide), dihydroceramide, and phytoceramide (see Fig. 1 for the chemical structures of ceramides and their breakdown products). They differ structurally in their Ceramide Signaling, edited by Anthony H. Futerman. ©2002 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Figure 1. Hydrolysis of ceramides by alkaline ceramidases. aCER, alkaline ceramidase; aDHC, alkaline dihydroceramidase; aPHC, alkaline phytoceramidase; SPHK, sphingosine kinase.

sphingoid base moieties: ceramide contains sphingosine which has a double bond at the C4-5 position in the sphingoid base backbone; dihydroceramide contains dihydropshingosine which does not have a double bond; and phytoceramide contains phytosphingosine which has an additional hydroxyl group, but no double bond. These ceramides are deacylated to generate sphingoid bases and fatty acids through the action of ceramidases. Sphingosine, dihydrosphingosine, and phytosphingosine in turn are phosphorylated by sphingosine kinases to generate sphingoid base phosphates. Ceramide, sphingoid bases, and their phosphates have been shown to play important roles in various cellular responses including apoptosis, growth, proliferation, differentiation, cell adhesion, cell motility, and angiogenesis. Ceramidases may regulate these responses by regulating the cellular levels of ceramides, sphingoid bases, and their phosphates. Ceramidases attenuate the ceramide-mediated signaling on one hand, and on the other hand generate sphingoid bases and their phosphates which mediate their own signals. To date, there are three different types of ceramidases which are classified as acid, neutral, and alkaline according to their pH optima for activity.1 Human acid ceramidase (hAC, Nacylsphingosine deacylase EC 3.5.123), a lysosomal enzyme, was the first ceramidase to be cloned.11 Its inherited deficiency leads to a sphingolipidoses disease, termed Farber’s disease.12 Very recently, several novel non-lysosomal ceramidases13-18 have been cloned and some of them have been carefully characterized in vitro. These include bacterial,13 mouse,14 rat19 and human neutral/alkaline ceramidases,15 and the yeast16,17 and human alkaline ceramidases.18 Based on sequence similarity, these non-lysosomal enzymes can be classified into two sub-groups. Members of the first sub-group of enzymes exhibit sequence similarity and share several conserved blocks, have 0-2 transmembrane domains and a broad neutral to alkaline pH optimum for their activity,13-15 hence are reclassified as neutral/alkaline ceramidases. The second sub-group of enzymes share several conserved domains mainly clustered at N-termini, have 4-5 transmembrane domains and a relatively narrow alkaline pH optima,16-18 hence are classified as alkaline ceramidases. Although these three types of ceramidases catalyze the same reaction, breaking down the amide linkage of ceramides, there is no homology across the three types of ceramidases. They also differ in cellular localization. These observations suggest that ceramides are hydrolyzed by different ceramidases in different cellular compartments. The physiologic significance behind this is largely unknown. Cloning and identification of these ceramidases opened the door to dissecting the regulation of ceramide turnover and to defining the involvement of ceramides and their metabolites in cellular responses in a more definitive way. These enzymes are important not only because they regulate the cellular levels of ceramide and responses to ceramide, but also because they regulate the cellular levels of sphingoid bases and their phosphates that have been demonstrated to be important bioactive molecules as well. In this Chapter, the cloning, characterization, and physiological function of the three types of ceramidases will be discussed.

Ceramidases: Regulators of Turnover of Ceramide and Ceramide-Mediated Responses

Acid Ceramidases

31

Acid ceramidase activity was first reported in rat brain by Gatt et al.20 The optimum pH for the ceramidase activity is around 5. It was suggested that the single enzyme had both forward ceramidase activity and reverse ceramide synthase activity (reverse ceramidase activity). The reverse activity, which catalyzes the formation of ceramide from sphingosine and a free fatty acid, but not acyl-CoA, was demonstrated in a semi-purified protein. This raised the concern that the reverse activity could be encoded by a co-purified protein. The enzyme encoding acid ceramidase activity was purified from human urine21 and its cDNA was cloned by screening a cDNA library using the oligonucleotide probes degenerated from the peptide sequences.21 The cDNA encodes a polypeptide of 395 amino acids (~53-55 kDa), which is processed into a mature heterodimeric enzyme (hAC) containing α (13 kDa) and β subunits (40 kDa). Complete deglycosylation reduces the apparent molecular weight of the β subunit to 28 kDa, but does not alter that of the α subunit, suggesting that the β subunit of hAC is highly glycosylated.21 hAC purified from placenta or urine has a pH optimum of 4 for the ceramidase activity and uses D-erythro-C12-ceramide as its best substrate. No cations are required for the in vitro activity of hAC. In vitro hAC activity is enhanced by phosphatydlinositol (PI) and bis(monoacylglycerol)phosphate (BMP). In cells, hydrolysis of ceramide by hAC in the lysosomes requires the presence of the sphingolipid activator proteins SAP-A, C, and D, which are believed to bring the membrane-bound ceramide to the active center of hAC. It is unclear whether the cloned hAC has the reverse ceramidase activity as proposed by Gatt et al.20 The human acid ceramidase gene (~ 30 kb), located on chromosome VIII, contains 14 exons. The 470 bp region upstream of the translational start codon ATG displays a promoter activity. This region has over 60% GC content, containing several putative binding sites of transcription factors.22 The full length cDNA of the murine acid ceramidase, which has 90% overall identity to hAC at the protein level, was cloned based on sequence similarity.23 The cDNA encodes a polypeptide of 394 amino acids. The mRNA for the enzyme is highly expressed in kidney and brain, but not expressed in testis and skeletal muscle. Its gene structure has been elucidated; it spans approximately 38 kb, consisting of 14 exons separated by 13 introns.23 The 497-bp region upstream of the first in-frame ATG possesses a promoter activity. This region contains several features of a housekeeping promoter, as well as several tissue-specific and/or hormoneinducible regulatory motifs. A hAC-like polypeptide of 359 amino acids was identified by computer-assisted database analysis and its cDNA was cloned.24 This polypeptide exhibits 33% identity to the hAC polypeptide, but its ceramidase activity has not been demonstrated. This human hAC-like polypeptide displays 80% identity to a mouse hypothetical protein (AK008776, unpublished data), suggesting the existence of its mouse counterpart. Two hypothetical proteins homologous to hAC were identified in C. elegans, one is more homologous to hAC, the other to the hAC-like protein, suggesting that acid ceramidase and acid ceramidase-like protein are evolutionarily conserved. Sequence alignment demonstrated several conserved regions that may be important for their catalytic functions (Fig. 2). Based on its lysosomal localization and genetic consequence of its deficiency, acid ceramidase was thought to be mainly responsible for catabolism of ceramide, but not for ceramide-mediated signaling processes. This hypothesis was supported by a study25 showing that 1) normal fibroblasts and those deficient for hAC had the same extent of apoptosis induced by TNFα and CD40 ligand; and 2) normal lymphoid cells and those deficient for hAC had the same magnitude of apoptosis induced by anti-CD95 (Fas) or anti-CD40 antibodies, TNFα, daunorubicin, and ionizing radiation. However, the study by Strelow et al26 points to a role for acid ceramidase in apoptosis. It was demonstrated that overexpression of hAC protects L929 cells from the TNFα-induced apoptosis by shifting the balance between intracellular levels of ceramide and sphingosine-1-P. Kanto et al27 also showed that the increase in ceramide by inhibiting hAC with its inhibitor N-oleoylethanolamine (NOE) enhances dendritic cell apoptosis induced by

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Figure 2. Alignment of protein sequences of acid ceramidases. Protein sequences were aligned using the Jellyfish program. Identical amino acids across all proteins are depicted in yellow. Identical amino acids among three to four proteins are depicted in blue. Similar amino acids are depicted in green. Underlined are conserved domains.

the treatment of the supernatants of murine tumor cell lines B16 (melanoma), MCA207, and MCA102 (fibrosarcoma). The contradictory role of hAC in apoptosis could be due to the difference in the cell types. Also, the specificity of NOE is not established. Acid ceramidase may have a role in cellular responses other than apoptosis. The study by Mandal et al28 showed that expression of hAC in NIH 3T3 cells appears to be induced by pancreatic phospholipase A2 (sPLA2) via its receptor, and interleukin 1β, suggesting a role of hAC in inflammation.

Neutral/Alkaline Ceramidases

A neutral ceramidase activity was described in numerous tissues and organs.29 The first neutral ceramidase was purified to homogeneity from the medium of Pseudomonas aeruginosa strain AN17.13 The purified enzyme has a pH optimum of 8 to 9. It hydrolyzes both ceramide and dihydroceramide efficiently, but phytoceramide poorly. Ceramide with C16-fatty acyl chain is a better substrate for the ceramidase than ceramides with C12- and C18-fatty acyl chain.

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The sequences of several peptides generated from the purified protein were determined and used to design the primers for the PCR amplification of a DNA probe (359 base pairs) from the genomic DNA of Pseudomonas. A DNA fragment covering the entire coding sequence (2010 bp) for the enzyme was cloned13 by hybridization screening of the genomic DNA library of Pseudomonas aeruginosa strain AN17 using the DNA probe. The enzyme consists of 670 amino acids, with a predicted molecular weight of 70.7 kDa and a pI of 5.7. A secretion signal peptide sequence is found at the N-termini of the protein, which is in the agreement with the Pseudomonas enzyme being secreted into medium. The enzyme had ceramidase activity at a broad pH range (from 7 to 10) when expressed in E.coli, with a pH optimum at 8 to 9.5. It is activated by calcium, and completely inhibited by EDTA, suggesting that calcium may be required for its activity. The same Pseudomonas enzyme showed a reverse activity of catalyzing the formation of ceramide from sphingosine and a free fatty acid.30 The reverse activity is distinct from that of the CoA-dependent ceramide synthase since it does not use acyl-CoA as a substrate. The reverse activity is highest at a neutral pH and in the presence of low concentration of detergent (0.05% Triton X-100). Although the Pseudomonas ceramidase does not share sequence similarity to the mammalian acid ceramidases and other known proteins, its homologous sequences were found among different species including Mycobacterium tuberculosis, Dictyostelium discoideum, Arabidopsis thaliana, mouse, rat, and human (Fig. 3). The mycobacterium homologue, which shares 40% identity to the Pseudomonas ceramidase, was also cloned by the same group,13 and shown to have ceramidase activity at neutral to alkaline pH. A mouse liver ceramidase with a molecular mass of 94 kDa was purified.31 Similar to the Pseudomonas ceramidase, the mouse enzyme hydrolyzes both ceramide and dihydroceramide, but not phytoceramide, and the C16-ceramide is a better substrate than C12- and C18-ceramide. However, in contrast to the Pseudomonas ceramide, the mouse enzyme does not require calcium for the activity. This enzyme has a pH optimum of around 7.5, hence belongs to the neutral/alkaline type of ceramidase. Similar to the Pseudomonas ceramidase, this purified mouse ceramidase also exhibited a considerable reverse activity. Deglycosylation by glycopeptidase F reduces the molecular weight of the enzyme significantly, suggesting that it is highly glycosylated. The cDNA of the mouse liver ceramidase was subsequently cloned from a liver cDNA library using a strategy similar to that for the cloning of the Pseudomonas ceramidase.14 The cDNA contains a 2,268 bp coding sequence which encodes a protein of 756 amino acids, with a predicted molecular weight of 83.5 kD and pI of 6.4. A hydrophobic motif predicted by hydropathy plot is located at the N-terminus of the ceramidase. This motif is predicted to be a putative ER transitional signal sequence, but the cellular localization of the mouse liver enzyme to the ER has not been demonstrated experimentally. Northern blot analysis showed that the mRNA of mouse neutral ceramidase is ubiquitously expressed, at the highest level in liver and kidney, a moderate level in brain, heart, lung, and the lowest level in spleen, skeletal muscle, and testis. The mouse neutral ceramidase displays 33, 28.3, 38.3% identity to the Pseudomonas, Mycobacterium, and Dictyostelium discoideum ceramidase respectively (Fig. 3). A rat brain nonlysosomal ceramidase with a pH optimum of 8-10 was purified.32 The purified ceramidase has an apparent molecular weight of 90 and 95 kDa as estimated by SDSpolyacrylamine gel electrophoresis (SDS-PAGE) and gel filtration chromatography respectively. The rat brain ceramidase prefers unsaturated ceramides with medium to long chain (C12-C24) acyl chains to the ceramides with short acyl chain (< C6) as substrates. It also hydrolyzes saturated ceramides (dihydroceramides), but with a lower efficacy. Acidic phospholipids including phosphatidylserine (PS) and phosphatidic acid (PA) stimulate the enzyme in a dose dependent manner. The purified rat brain ceramidase exhibited a considerable reverse activity of forming ceramide from sphingosine and a fatty acid.33 Acyl-CoA is not the substrate for this reverse activity, nor does fumonisin B1 (FB1) inhibit it, suggesting that the rat brain ceramidase is distinct from the CoA-dependent and FB1-inhibitable ceramide synthase. Phosphatidic acid and cardiolipin are inhibitory to the reverse activity. This reverse activity is highest in a neutral pH range, different from the ceramidase (forward) activity.

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Ceramide Signaling

Figure 3. Alignment of protein sequence of neutral/alkaline ceramidases. Protein sequences were aligned using the Jellyfish program. Identical amino acids across all proteins are depicted in yellow. Identical amino acids among three to four proteins are depicted in blue. Similar amino acids are depicted in green. Underlined are the conserved domains.

From the rat kidney, a membrane bound ceramidase with a molecular weight of 112 kDa was purified.19 The purified ceramidase exhibited a pH optimum of 6-7, lower than that of the rat brain neutral/alkaline ceramidase. The rat kidney neutral ceramidase prefers the unsaturated ceramide with a medium chain-length fatty acid (D-erythro-C12-ceramide) to long chain ceramides (C16- or C18-ceramide). The activity is inhibited by Zn2+, Cu2+, and Hg2+, but not affected by Ca2+, Mg 2+, and Mn2+. The cDNA of the rat neutral ceramidase was cloned according to the sequences of peptides derived from the purified protein . The cDNA encodes a 761 amino acid protein with a predicted molecular weight of 83.4 kDa and pI of 6.55. The rat recombinant ceramidase expressed in HEK293 cells has a pH optimum of 6-7, identical to that of the native enzyme purified from rat kidney. Western blot analysis revealed two isoforms (with molecular mass of 113 and 133 kDa) of the rat neutral ceramidase expressed in HEK293 cells. Nine glycosylation sites were predicted in this protein, suggesting that the rat enzyme is highly glycosylated. Deglycosylation by glycopeptidase F converted the 133 kDa isoform into 87 kDa. Block of glycosylation by tunicamycin in cells converted both of the isoforms into the 87 kDa polypeptide. These results suggest that the glycosylation contributes to the increased apparent molecular weight of the rat neutral ceramidase isoforms. Deglycosylation caused a decrease in the activity of the enzyme, suggesting that the glycosylation is important for the full activity of the enzyme. Combination of cellular fractionation and immuno-histochemical studies suggests that the rat neutral ceramidase is localized to the apical membrane of proximal and distal tubules, and collecting ducts in rat kidney, but in hepatocytes, it seems to be localized to endosome-like organelles. Surprisingly, northern blot analysis showed that the tissue distribution for the rat enzyme expression is different from that of the mouse neutral ceramidase. The

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35

rat neutral ceramidase is expressed in brain, heart, and kidney at a high level, but expression in other organs including liver, lung, spleen, skeletal muscle, and testis is low. The rat neutral ceramidase exhibits considerable homology to the human, mouse, and bacterial neutral/alkaline ceramidases. The sequences of several peptides derived from the purified rat brain protein were determined.15 These peptide sequences were blasted against the GenBank databases, and two human expression tag sequences (EST) homologous to the peptides were found. Based on these EST sequences, a human cDNA was amplified from the human kidney cDNA library using a rapid amplification of cDNA ends (RACE) strategy. The cDNA was predicted to contain a 2,289 bp coding sequence that encodes a 763 amino acid protein with a predicted molecular weight of 84 kDa and pI of 6.69. The protein fused with green fluorescent protein (GFP) exhibited ceramidase activity at a broad pH range (from 7 to 10) when expressed in HEK293 or MCF7 cells. The fusion protein was localized to the mitochondria as revealed by GFP tagging. The pseudomonas, mouse liver, rat brain, and human neutral/alkaline ceramidases share sequence similarity (Fig. 3) and display similar biochemical properties as summarized in Table 1. Their roles in regulating metabolism of ceramide and other metabolites of sphingolipids and cell responses are largely unknown. The pseudomonas ceramidase is secreted from Pseudomonas which causes dermatitis, suggesting a role in pathogenesis. Neutral/alkaline ceramidases seem to be regulated by growth factors, cytokines, and oxidized lipoproteins.34 The study by Coroneos et al35 demonstrated that a neutral/alkaline activity (at pH 7 and 9 respectively) is stimulated by PDGF in rat mesangial cells, leading to an increase in cellular levels of sphingosine and sphingosine-1-P and proliferation of the cells. The study by Nikolova-Karakashian et al showed that in rat hepatocytes, interleukin 1β, at a low concentration, activates ceramidase activity at acid, neutral, and alkaline pH as determined using NBD-C6-ceramide as a substrate in vitro. The activation of ceramidase activity is also seen in vivo as indicated by the release of NBD-C6-fatty acid from hydrolysis of NBD-C6ceramide in cells. The increased ceramidase activity leads to the elevation of cellular levels of sphingosine (or sphingosine-1-P), which may inhibit the expression of cytochrome P450 2C11. In smooth muscle cells (SMC), oxLDL induces co-activated SMase activity, alkaline ceramidase activity, and sphingosine kinase.34 The co-activation leads to both proliferation of smooth muscle cells and the increased cellular levels of sphingosine and sphingosine-1-P. Both the proliferation and the increase in S1P are inhibited by D-MAPP, an inhibitor of alkaline ceramidase, or dimethylsphingosine, an inhibitor of sphingosine kinase. In another study by Huwiler et al,37 TNFα was shown to sustain the prolonged activation of neutral ceramidase in mesangial cells. These observations suggest that the activation of neutral/alkaline ceramidase is able to modulate the biological response. These also suggest that neutral/alkaline ceramidase activity may be involved in cellular responses in response to growth factors and cytokines. However, it is unknown whether the ceramidase activity stimulated by the growth factor and cytokines was encoded by the cloned neutral/alkaline ceramidase or the alkaline ceramidase that will be discussed below. Since all cloned mammalian neutral/alkaline ceramidases possess both ceramidase and reverse activity in vitro, which activity the enzymes have in cells may determine the net outcome of their regulation of the turnover of ceramide and cellular levels of ceramides, sphingoid bases, and their phosphates. Which activity the enzymes prefer in cells may be one of mechanism of the regulation for the cellular levels of ceramide. To date, such data are lacking.

Alkaline Ceramidases The eukaryotic alkaline ceramidase (YPC1p) was cloned as a fumonisin B1 (FB1) resistant gene from the yeast Saccharomyces cerevisiae.17 FB1 is a specific inhibitor of (FB1 inhibitable) ceramide synthetase that catalyzes the synthesis of the yeast ceramides (phytoceramide and dihydroceramide) from sphingoid bases and fatty acyl-CoA.38 FB1 inhibits yeast growth by blocking the synthesis of yeast ceramides and ceramide-containing sphingolipids that are

Ceramide Signaling

36

Table 1. Comparison of acid, neutral/alkaline, and alkaline types of ceramidases Acid Ceramidases

Neutral/Alkaline Ceramidase

Alkaline Ceramidase

Subunit Molecular mass (kDa) pH optimum Best substrate Reverse activity

2 50 4-5 D-e-ceramide unknown

1 94-112 6-9 D-e-ceramide high

1 ~30 8-9.5 PHC, DHC, or CER high (YPC1p), non (others)

Activator Inhibitor TM Glycosylation Localization

SAP A, C, and D N/A 0 High Lysosomes

Ca2+ (bacterial enzymes) Zn2+ 1-2 High Mitochondria, endosomes,lysosomes, PM, ER, Golgi, caveolae, and extracellular

Ca2+ Zn2+ and Cu2+ 4-6 Unknown ER, Golgi, or Golgi/ER

TM, transmembrane domain; SAP, Saposin; PM, plasma membrane; ER, endoplasmic reticulum; PHC, D-ribo-phytoceramide; DHC, D-e-dihydroceramide; CER, D-e-ceramide.

essential for yeast survival.39 Overexpression of YPC1p rescued the yeast cells from the FB1 inhibition by generating yeast ceramides, as revealed by metabolic labeling of sphingolipids, through a pathway independent of the FB1 inhibitable ceramide synthase. In vitro activity assay shows that YPC1p has both ceramidase activity and reverse activity. In the absence of FB1, overexpression of YPC1p caused a decrease in complex sphingolipids and a concomitant increase in phytosphingosine and its phosphate in cells. In contrast, overexpression of YPC1p caused an increase in complex sphingolipids in the presence of FB1. These results suggest that YPC1p has both ceramidase activity and reverse activity in cells. The cellular levels of phytoceramide and phytosphingosine are altered by FB1 treatment. Therefore, the interchange between the two activities of YPC1p may depend on the availability of substrates in cells, which underscores the importance of YPC1p in regulating the cellular levels of ceramides and their metabolites. YPC1p exhibits its highest activity at around pH 9.5, hence is classified as an alkaline ceramidase. YPC1p prefers phytoceramide to dihydroceramide for substrate, but it does not hydrolyze unsaturated mammalian type ceramide. The ceramidase activity of YPC1p was enhanced by Ca2+, inhibited by Zn2+ and Cu2+, but not affected by Mg2+ (unpublished data). It was stimulated by phosphatidylserine slightly (unpublished data). YPC1p prefers phytosphingosine and dihydrosphingosine to sphingosine for its reverse activity. Free fatty acid but not fatty acyl-CoA is another substrate for the reverse activity. Its preference for the chain length of free fatty acids is unknown. Another yeast alkaline ceramidase YDC1p was identified in the Saccharomyces genome database by sequence similarity to YPC1p.16 YDC1p has a 50% overall identity with YPC1p over the entire protein sequence (Fig. 4). YDC1p exhibits a major ceramidase activity with a pH optimum of 9 and a minor reverse activity both in vitro and in vivo, but it has different substrate specificity from YPC1p. YDC1p prefers dihydroceramide to phytoceramide as its substrate. A BLAST search did not identify any proteins with annotated functions, which are homologous to the yeast alkaline ceramidase. However, the yeast enzymes are highly homologous

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37

to putative proteins of Arabidoposis and C. elegans, peptides deduced from EST sequences of human, mouse, pig, zebra fish, and human genomic sequences. The full-length cDNA of a human homologue was cloned from a human kidney cDNA library by RACE based on the EST sequence.18 When expressed both in the yeast and mammalian cells, the human homologue exhibits an alkaline ceramidase activity in alkaline pH range, with the phytoceramide as its best substrate, hence termed the human alkaline phytoceramidase (haPHC). HaPHC hydrolyzes ceramide and dihydroceramide less efficiently. Similar to YPC1p, haPHC is activated by Ca2+, but inhibited by Zn2+ and Cu2+. In contrast to YPC1p, haPHC does not have reverse activity. Northern blot analysis shows that aPHC is ubiquitously expressed, with the highest expression in placenta and least expression in skeletal muscle. HaPHC has a dual localization in both the Golgi apparatus and ER. Phytoceramide, the substrate of haPHC, was thought to exist in lower eukaryotes mainly, such as the yeast Saccharomyces, fungi, and plants. Its existence in mammalian cells has not been reported until recently. This is further confirmed by the identification of haPHC. Its cloning also provides an essential tool to dissect the physiological roles of phytoceramide and its metabolites in humans and animals. Very recently, we identified and cloned a mouse alkaline ceramidase (maCER) based on sequence similarity to haPHC and the yeast alkaline ceramidases (unpublished data). Substrate specificity showed that maCER hydrolyzed ceramide exclusively. These alkaline ceramidases constitute a big family. In addition to maCER, from the mouse EST database, we have identified another murine homologue that exhibits 88% identity to the human alkaline phytoceramidase haPHC, suggesting that it may be the murine counterpart of haPHC. These studies suggest that in yeast, human, and mice there are at least two members of the alkaline ceramidase family. Each member from this family in the same organism has its own preferred substrate, phytoceramide, dihydroceramide, or ceramide (Fig. 1), suggesting that the metabolism of each ceramide species is independently regulated, so is that of its product. The independent regulation of its metabolism indicates that each ceramide species may have distinct physiological functions. Their highly restricted substrate specificities underscore the importance of the alkaline ceramidases in regulating the cellular levels of different ceramide species, their breakdown products, sphingoid bases and sphingoid base phosphates, and cellular responses mediated by these lipids. The alkaline ceramidases appear to have an important role in regulating metabolism of sphingolipids including ceramides, sphingoid bases, and their phosphates. Overexpression of the yeast16,40 and human alkaline ceramidases18 causes the increased breakdown of ceramides in cells, leading to a decrease in the cellular levels of ceramides and complex sphingolipids and a concomitant increase in sphingoid bases and their phosphates, whereas deletion of the yeast alkaline ceramidases appears to have opposing effects. Importantly, synthesis of the yeast ceramides through the reverse action of the yeast alkaline ceramidases is a salvage pathway for de novo synthesis of yeast ceramides. Overexpression of the yeast alkaline ceramidase YPC1p rescues the defect in synthesis of ceramides resulting from either FB1 inhibition17 or deletion41,42 of the yeast protein LAG1 and LAC1 which are essential for CoA dependent ceramide synthase activity. The substitution for ceramide synthesis by YPC1p may be an important protective mechanism in yeast. The human and mouse alkaline ceramidases seem to lose this function because they have little or no reverse activity. Regulation of metabolism of ceramides by the alkaline ceramidases appears to yield physiological responses. Deletion of the yeast YDC1p reduced heat tolerance.16 The reduced heat tolerance could be due to the decrease in cellular levels of sphingoid base phosphates which are shown to enhance heat tolerance in yeast. 40 Overexpression of the human alkaline phytoceramidase in yeast suppresses yeast growth (unpublished data). The growth inhibition could be attributable to a decrease in the cellular levels of complex sphingolipids or an increase in the production of phytosphingosine or its phosphate, which is shown to be inhibitory to yeast growth.

Ceramide Signaling

38

Figure 4. Alignment of protein sequence of alkaline ceramidases. Protein sequences were aligned using the Jellyfish program. Identical amino acids across all proteins are depicted in yellow. Identical amino acids among three proteins are depicted in blue. Similar amino acids are depicted in green. Underlined are the conserved domains.

As discussed before, neutral/alkaline ceramidase activities increase in response to stimulation of growth factors and cytokines. It is unclear which of these ceramdase activities were encoded by which ceramidases. The cloning of the mammalian alkaline ceramidases should provide a very useful tool to address this issue.

Final Remarks In this review, we have focused on the cloning, characterization, and possible physiological roles of three types of ceramidases. Little is known about regulation of their expression and activity, and their roles in regulation of biological responses are under investigation. We expect that with the cloning and characterization, more discoveries on the physiological roles of ceramidases will emerge.

Acknowledgments This work was supported by a Veterans Affairs Merit Review Award to LMO.

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References 1. Hassler DF, Bell RM. Ceramidases: enzymology and metabolic roles. Adv Lipid Res 1993; 26:49-57. 2. Nagiec MM, Skrzypek M, Nagiec EE et al. The LCB4 (YOR171c) and LCB5 (YLR260w) genes of Saccharomyces encode sphingoid long chain base kinases. J Biol Chem 1998; 273:19437-19442. 3. Liu H, Sugiura M, Nava VE et al. Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform. J Biol Chem 2000; 275:19513-19520. 4. Kohama T, Olivera A, Edsall Let al. Molecular cloning and functional characterization of murine sphingosine kinase. J Biol Chem 1998; 273:23722-23728. 5. Merrill AH, Schmelz EM, Dillehay DL et al. Sphingolipids—the enigmatic lipid class: biochemistry, physiology, and pathophysiology. Toxicol Appl Pharmacol 1997; 142:208-225. 6. Spiegel S. Sphingosine 1-phosphate: a prototype of a new class of second messengers. J Leukoc Biol 1999; 65:341-344. 7. Hla T, Lee MJ, Ancellin N et al. Sphingosine-1-phosphate signaling via the EDG-1 family of G-protein-coupled receptors. Ann N Y Acad Sci 2000; 905:16-24. 8. Auge N, Negre-Salvayre A, Salvayre R et al. Sphingomyelin metabolites in vascular cell signaling and atherogenesis. Prog Lipid Res 2000; 39:207-229. 9. English D, Garcia JG, Brindley DN. Platelet-released phospholipids link haemostasis and angiogenesis. Cardiovasc Res 2001; 49:588-599. 10. Hannun YA, Luberto C, Argraves KM. Enzymes of sphingolipid metabolism: from modular to integrative signaling. Biochemistry 2001; 40:4893-4903. 11. Koch J, Gartner S, Li CM et al. Molecular cloning and characterization of a full-length complementary DNA encoding human acid ceramidase. Identification of the first molecular lesion causing Farber disease. J Biol Chem 1996; 271:33110-33115. 12. Jameson RA, Holt PJ, Keen JH. Farber’s disease (lysosomal acid ceramidase deficiency). Ann Rheum Dis 1987; 46:559-561. 13. Okino N, Ichinose S, Omori A et al. Molecular cloning, sequencing, and expression of the gene encoding alkaline ceramidase from Pseudomonas aeruginosa. Cloning of a ceramidase homologue from Mycobacterium tuberculosis. J Biol Chem 1999; 274:36616-36622. 14. Tani M, Okino N, Mori K et al. Molecular cloning of the full-length cDNA encoding mouse neutral ceramidase. A novel but highly conserved gene family of neutral/alkaline ceramidases. J Biol Chem 2000; 275:11229-11234. 15. El Bawab S, Roddy P, Qian T et al. Molecular cloning and characterization of a human mitochondrial ceramidase. J Biol Chem 2000; 275:21508-21513. 16. Mao C, Xu R, Bielawska A et al. Cloning and characterization of a Saccharomyces cerevisiae alkaline ceramidase with specificity for dihydroceramide. J Biol Chem 2000; 275:31369-31378. 17. Mao C, Xu R, Bielawska A et al. Cloning of an alkaline ceramidase from Saccharomyces cerevisiae. An enzyme with reverse (CoA-independent) ceramide synthase activity. J Biol Chem 2000; 275:6876-6884. 18. Mao C, Xu R, Szulc ZM et al. Cloning and characterization of a novel human alkaline ceramidase: a mammalian enzyme that hydrolyzes phytoceramide. J Biol Chem 2001; 276:26577-26588. 19. Mitsutake S, Tani M, Okino N et al. Purification, characterization, molecular cloning, and subcellular distribution of neutral ceramidase of rat kidney. J Biol Chem 2001; 276:26249-26259. 20. Gatt S. Enzymatic hydrolysis of sphingolipids. I. Hydrolysis and synthesis of ceramides by an enzyme from rat brain. J Biol Chem 1966; 241:3724-3730. 21. Bernardo K, Hurwitz R, Zenk T et al. Purification, characterization, and biosynthesis of human acid ceramidase. J Biol Chem 1995; 270:11098-11102. 22. Li CM, Park JH, He X et al. The human acid ceramidase gene (ASAH): structure, chromosomal location, mutation analysis, and expression. Genomics 1999; 62:223-231. 23. Li CM, Hong SB, Kopal G et al. Cloning and characterization of the full-length cDNA and genomic sequences encoding murine acid ceramidase. Genomics 1998; 50:267-274. 24. Hong SB, Li CM, Rhee HJ et al. Molecular cloning and characterization of a human cDNA and gene encoding a novel acid ceramidase-like protein. Genomics 1999; 62:232-241. 25. Segui B, Bezombes C, Uro-Coste E et al. Stress-induced apoptosis is not mediated by endolysosomal ceramide. Faseb J 2000; 14:36-47. 26. Strelow A, Bernardo K, Adam-Klages S et al. Overexpression of acid ceramidase protects from tumor necrosis factor-induced cell death. J Exp Med 2000; 192:601-612. 27. Kanto T, Kalinski P, Hunter OC et al. Ceramide mediates tumor-induced dendritic cell apoptosis. J Immunol 2001; 167:3773-3784. 28. Mandal AK, Zhang Z, Chou JY et al. Pancreatic phospholipase A2 via its receptor regulates expression of key enzymes of phospholipid and sphingolipid metabolism. Faseb J 2001; 15:1834-1836.

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29. Spence MW, Beed S, Cook HW. Acid and alkaline ceramidases of rat tissues. Biochem Cell Biol 1986; 64:400-404. 30. Kita K, Okino N, Ito M. Reverse hydrolysis reaction of a recombinant alkaline ceramidase of Pseudomonas aeruginosa. Biochim Biophys Acta 2000; 1485:111-120. 31. Tani M, Okino N, Mitsutake S et al. Purification and characterization of a neutral ceramidase from mouse liver. A single protein catalyzes the reversible reaction in which ceramide is both hydrolyzed and synthesized. J Biol Chem 2000; 275:3462-3468. 32. El Bawab S, Bielawska A, Hannun YA. Purification and characterization of a membrane-bound nonlysosomal ceramidase from rat brain. J Biol Chem 1999; 274:27948-27955. 33. El Bawab S, Birbes H, Roddy P et al. Biochemical characterization of the reverse activity of rat brain ceramidase: A CoA-independent and fumonisin B1 insensitive ceramide synthase. J Biol Chem 2001; 276:16758-16766. 34. Auge N, Nikolova-Karakashian M, Carpentier S et al. Role of sphingosine 1-phosphate in the mitogenesis induced by oxidized low density lipoprotein in smooth muscle cells via activation of sphingomyelinase, ceramidase, and sphingosine kinase. J Biol Chem 1999; 274:21533-21538. 35. Coroneos E, Martinez M, McKenna S et al. Differential regulation of sphingomyelinase and ceramidase activities by growth factors and cytokines. Implications for cellular proliferation and differentiation. J Biol Chem 1995; 270:23305-23309. 36. Nikolova-Karakashian M, Morgan ET, Alexander C et al. Bimodal regulation of ceramidase by interleukin-1beta. Implications for the regulation of cytochrome p450 2C11. J Biol Chem 1997; 272:18718-18724. 37. Huwiler A, Pfeilschifter J, van den Bosch H. Nitric oxide donors induce stress signaling via ceramide formation in rat renal mesangial cells. J Biol Chem 1999; 274:7190-7195. 38. Merrill AH Jr, van Echten G, Wang E et al. Fumonisin B1 inhibits sphingosine (sphinganine) N-acyltransferase and de novo sphingolipid biosynthesis in cultured neurons in situ. J Biol Chem 1993; 268:27299-27306. 39. Wu WI, McDonough VM, Nickels JT Jr et al. Regulation of lipid biosynthesis in Saccharomyces cerevisiae by fumonisin B1. J Biol Chem 1995; 270:13171-13178. 40. Mao C, Saba JD, Obeid LM. The dihydrosphingosine-1-phosphate phosphatases of Saccharomyces cerevisiae are important regulators of cell proliferation and heat stress responses. Biochem J 1999; 342:667-675. 41. Schorling S, Vallee B, Barz WP et al. Lag1p and Lac1p are essential for the Acyl-CoA-dependent ceramide synthase reaction in Saccharomyces cerevisae. Mol Biol Cell 2001; 12:3417-3427. 42. Guillas I, Kirchman PA, Chuard R et al. C26-CoA-dependent ceramide synthesis of Saccharomyces cerevisiae is operated by Lag1p and Lac1p. Embo J 2001; 20:2655-2665.

CHAPTER 5

Molecular Evolution of Neutral Ceramidase: From Bacteria to Mammals Makoto Ito, Nozomu Okino, Motohiro Tani, Susumu Mitsutake and Katsuhiro Kita

Summary

C

eramidase is a hydrolase capable of cleaving the N-acyl linkage between a sphingosine base and a fatty acid of ceramide. Recent extensive studies have revealed that ceramidases can be classified into three different families; acid, neutral and alkaline enzymes, based on not only their catalytic pH optima but also their primary structure. The genetic information of neutral ceramidases is highly conserved from bacteria to humans. This Chapter summarizes recent progress in the study of neutral ceramidases.

Introduction Sphingolipids and their metabolites have emerged as a new class of lipid biomodulators of various cell functions.1,2 Ceramide (N-acylsphingosine), a common lipid backbone of sphingolipids, functions as a second messenger in a variety of cellular events including apoptosis and cell differentiation.3,4 Sphingosine, the N-deacylated product of ceramide, exerts mitogenic and apoptosis-inducing activities, depending on the cell type and cell cycle,5,6 and can be converted by sphingosine kinase to sphingosine-1-phosphate (S1P), which functions as an intra- and intercellular second messenger to regulate cell growth, motility, and morphology.7,8 Sphingosine is not produced by de novo synthesis 9 but rather is thought to be produced from ceramide by the action of ceramidase (CDase; EC 3.5.1.23), which hydrolyzes the N-acyl linkage between a sphingosine base and a fatty acid. Thus, the activity of CDase is crucial in regulating the balance of the cellular content of ceramide/sphingosine/S1P. Since the first description of the activity of CDase by Shimon Gatt in 1963,10 two isoenzymes showing acidic and neutral to alkaline pH optima, have been recognized.11 The former (acid CDase) resides in lysosomes and is thought to function primarily to degrade ceramide in the process of sphingolipid catabolism. A genetic deficiency of acid CDase causes Farber disease, in which ceramide accumulates in lysosomes.12 The latter (this type of enzyme is designated a neutral CDase in this Chapter to distinguish it from the alkaline CDase described below) has been proposed to function in signal transduction pathways to produce sphingosine for regulatory purposes,13,14 although the subcellular location has not been determined. This Chapter describes recent progress in the elucidation of the structure, localization and function of neutral CDases.

Classification of CDases In the past few years, molecular cloning of CDases has been performed. Sandhoff and his associates succeeded in purifying an acid CDase from human urine15 and cloned the gene from human16 and mouse.17 Meanwhile, neutral CDases have been cloned from bacteria,18 slime mold, Drosophila, mouse,19 and rat20 by our laboratory, and from human21 by Hannun’s group, Ceramide Signaling, edited by Anthony H. Futerman. ©2002 Eurekah.com and Kluwer Academic / Plenum Publishers.

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respectively. Very recently, Obeid and her associates found a new alkaline CDase in yeast22 and cloned its human homologue,23 which prefers phyto-ceramide over normal ceramide containing sphingenine and shows a pH optimum that is extremely alkaline (9.0~9.5). The cloning clearly indicated three different families of CDases; acid, neutral and alkaline, considered to be generated from different ancestral genes (Fig. 1). The genetic information of the neutral CDases is conserved from bacteria to humans. In conclusion, CDases are classified into three families based not only on their pH optima but also on their primary structure. Table 1 summarizes the properties of the three different types of CDases. Acid CDases are composed of α (40 kDa) and β (13 kDa) subunits, while the neutral and alkaline enzymes are composed of a single polypeptide of 70-110 kDa and 30 kDa, respectively. The alkaline CDases from yeast and human efficiently hydrolyze phytoceramide, which is resistant to hydrolysis by acid and neutral enzymes. The intracellular distribution is also quite different; the acid enzymes are exclusively present in lysosomes, neutral enzymes in endosome-like organelles, mitochondria and plasma membranes (to be described later in this Chapter), and alkaline enzymes in the endoplasmic reticulum/Golgi. Both acidic and neutral CDases are also actively released from cells.24

Bacterial CDase Gene Cloning and Characterization

Since the first report of the presence of a CDase in rat brain,10 both acid and neutral types have been found in various mammalian tissues and cultured cells including cells of human origin. However, a prokaryotic CDase was reported only recently. In 1998, we found a neutral CDase in Pseudomonas aeruginosa which was isolated from a patient with atopic dermatitis25, and cloned its gene.18 The enzyme, composed of 670 amino acids including a signal sequence of 24 residues, shows a pH optimum of 7.5-9.0. This enzyme hydrolyzes various ceramides containing C12:0-C24:0 fatty acids; not only ceramide containing sphingenine (d18:1) or sphinganine (d18:0) but also phytosphingosine (t18:0) as the long-chain base, although the latter is quite resistant to hydrolysis compared to the former two. The bacterial CDase does not hydrolyze glycosphingolipids or SM, and thus was clearly distinguishable from a Pseudomonas ceramide N-deacylase (SCDase) which hydrolyzes the N-acyl linkage of ceramide in various glycosphingolipids and in SM.26 It is worth noting that free ceramide is quite resistant to hydrolysis by SCDase.

Frequency of CDase-Producing Bacteria in the Skin of Patients with Atopic Dermatitis Although the etiologic factors for atopic dermatitis have yet to be fully elucidated, dry and barrier-disrupted skin is a distinctive feature and could be evoked by a decrease of ceramide in the stratum corneum.27 Bacterial CDase was suspected to contribute either directly or indirectly to the abnormality, since P. aeruginosa was isolated from desquamated materials of a patient with atopic dermatitis.25 Thus, we examined whether or not atopic skin is infected with bacteria capable of producing CDase. It was found that significantly more CDase was secreted from bacterial flora obtained from both lesional and non-lesional skin of patients with atopic dermatitis than from the skin of healthy subjects (25 positives from 46 patients; 2 positives from 23 controls, p< 0.001), while SMase was secreted from the bacterial flora obtained from all types of skin at similar levels for the patients with atopic dermatitis and the controls (13 positives from 45 patients; 5 positives from 23 healthy controls, p>0.5).28 The frequency of the bacterial CDase on atopic or normal skin was examined by PCR using a set of primers specific to bacterial CDase. As a result, 20 positives were obtained from 27 patients with atopic dermatitis and 12 positives from 66 controls. These findings clearly indicate that the bacterial flora from atopic skin tends to contain CDase-producing bacteria and suggest that these bacteria are related to the decrease of ceramide in the horny layer of the epidermis, which increases the hyper sensitivity of skin in atopic dermatitis patients by impairing the permeability barrier.

Molecular Evolution of Neutral Ceramidase: From Bacteria to Mammals

43

Figure 1. Dendrogram of CDases.

Activation of Bacterial CDase by Anionic Glycerophospholipids Interestingly, CDase-producing bacteria were found in atopic skin with significant frequency.28 However, it has remained unclear how the ceramide in atopic skin is hydrolyzed by bacterial CDase, because in the absence of detergents, almost no hydrolysis occurs and detergents are unlikely to be found on the skin. We found that certain anionic glycerophospholipids, such as cardiolipin and phosphatidylglycerol, functioned to stimulate the activity of the bacterial CDase in place of detergents.29 Cardiolipin and phosphatidylglycerol are major glycerolipids of Staphylococcus aureus, which is the dominant species of microflora in atopic skin.30 Surprisingly, P. aeruginosa was found to produce not only CDase but also staphyrolytic proteases, which decomposed S. aureus cells to generate these anionic glycerolipids. Actually, we observed a release of cardiolipin and phosphatidylglycerol from S. aureus cells when they were treated with the culture supernatant of several type-strains of P. aeruginosa, all of which exhibited the release of CDase. It was also shown that normal ceramide, as well as human skin-specific esterified-ω-hydroxy-ceramide, was degraded by the action of Pseudomonas CDase in the presence of Staphylococcus-derived glycerophospholipids instead of detergents. These observations would in part explain how bacterial CDase hydrolyzes ceramide in atopic skin; glycerophospholipids, which would be generated from S. aureus by Pseudomonas proteases (staphylolytic proteases such as Las A, D 31), activate the bacterial CDase in the absence of detergents. It is of note that atopic skin tends to have a neutral pH whereas healthy skin is normally acidic.32 The pH shift for atopic skin optimizes the action of both bacterial CDase and staphylolytic proteases, since both enzymes exhibit neutral to alkaline pH optima.

CDase Homologue in Mycobacterium Tuberculosis A sequence homologous to the neutral CDase was found in hypothetical proteins encoded in Mycobacterium tuberculosis, which is known to cause tuberuculosis. We cloned the homologue of M. tuberculosis, expressed it in E. coli, and confirmed that cell lysates of the transfectants showed the CDase activity with an optimum at pH 8.5.18 The pathogenic significance of Mycobacterium CDase remains to be clarified.

44

Table 1. Classification of cloned ceramidases (CDases) Classification

Optimum pH

Sources

Intracellular localization

Structure and Molecular mass

Substrate specificity

Acid CDase

4.5

human,16 mouse17

Lysosomes,16 secreted24

Neutral CDase

6.5~8.5

P. aeruginosa,18 M. tuberculosis,18 mouse,19 rat,20,35

plasma membrane,20 endosome-like organelle,20 mitochondria,21 secreted24

α,β subunits15 50kDa (13 kDa + 40 kDa) Single polypeptide20,25,34,35 70 kDa~110kDa

ceramide> dihydroceramide ceramide> dihydroceramide> phytoceramide

Alkaline CDase

9.0~9.5

human21 S. cerevisiae,22 human23

ER/Golgi23

Single polypeptide22,23 30 kDa

phytoceramide> ceramide

Cer, ceramide; dihydroCer, dihydroceramide; phytoCer, phytoceramide

Ceramide Signaling

Molecular Evolution of Neutral Ceramidase: From Bacteria to Mammals

45

Future Prospects Recently, it has come to light that some bacterial pathogens mimic the function of host proteins to manipulate host physiology and cellular functions. For example, several strains of Salmonella produced protein tyrosine phosphatase to disrupt focal adhesions by dephosphorylating the focal adhesion kinase, leading to a paralysis of the macrophage attack on the bacterium.33 Since tyrosine phosphorylation does not commonly occur in bacteria, it is likely that these molecules have specifically evolved to modulate host cellular functions. P. aeruginosa and M. tuberculosis do not possess ceramide. It should be elucidated whether or not bacterial CDase interferes with the signal transduction system mediated by sphingolipids as a mimicker of the functional host CDases.

Neutral CDase in Mammals Molecular Cloning and Characterization

Neutral CDases have been purified from mouse liver,34 rat brain35 and kidney,20 and then their cDNAs were cloned using partial amino acid sequences of the corresponding purified enzymes.19,20 The mouse and rat CDases were composed of 756 amino acids (predicted molecular weight, 83,504) and 761 amino acids (predicted molecular weight, 83,483), respectively, in which nine putative N-glycosylation sites were present. Both mouse and rat neutral CDases are actually N-glycosylated, because treatment of the purified enzymes by glycopeptidase F greatly reduced their molecular masses and they were stained strongly with HRP-labeled concanavalin A and R. communis agglutinin I. Both mouse and rat enzymes have putative phosphorylation sites for casein kinase II, tyrosine-specific kinase, and protein kinase C. The CDase activity increased more than 1,000 times when CHOP cells were transfected with either mouse or rat CDase cDNA in comparison with mock transfectants or untransfected CHOP cells. The optimum pH of the recombinant murine CDases was found to be pH 6.5-7.5, which is consistent with the results obtained using the purified enzymes. CDase activity was greatly decreased when cells overexpressing the CDase were treated with tunicamycin. The recombinant CDase hydrolyzed various ceramides but not galactosylceramide, sulfatide, GM1 or SM. Ceramides containing sphingenine (d18:1) were hydrolyzed much faster than those containing sphinganine (d18:0). Ceramides containing phytosphingosine (t18:0) were strongly resistant to hydrolysis by the enzyme. Interestingly, the fluorescent substrate C12-NBD-ceramide (NBDdodecanoylsphingosine), in which NBD (4-nitrobenzo-2-oxa-1,3-diazole) is covalently coupled to the amino group of ω-aminododecanoic acid,36 was hydrolyzed much faster than native ceramide, whereas C6-NBD-ceramide (NBD-hexanoylsphingosine) was somewhat resistant to hydrolysis by the enzyme.37 Interestingly, both recombinant and purified enzymes catalyzed not only the hydrolysis but also the synthesis of ceramide.34,38 However, the enzyme does not catalyze the palmitic acid-transfer reaction from palmitoyl-CoA to sphingosine. The nucleotide sequences of neutral CDases have been submitted to the GenBankTM/EBI Data Bank with accession number, AB028646 (Pseudomonas aeruginosa), Z95972 (Mycobacterium tuberculosis), U82513 (mouse), Ab057433 (rat), and AF250487 (human).

Distribution and Intracellular Localization In mouse and rat, neutral CDase activities were shown to be distributed in various tissues such as liver, kidney, spleen and brain. The enzymatic activity was detected in both membrane and soluble fractions, although the former was predominant in all tissues. Northern blotting using cDNA encoding a neutral CDase revealed that in mice strong signals were observed in liver and kidney whereas in rats, they were detected in kidney and brain but not in liver.19,20 In both mice and rats, the liver neutral CDase was solubilized from the membrane fractions by freeze-thawing without use of detergents, whereas kidney enzyme was not.34 The kidney enzyme was solubilized using detergents such as Triton X-100.20 To address the difference in the association of CDase to membrane fractions, the subcellular localization of neutral CDase

46

Ceramide Signaling

was examined using a specific polyclonal antibody against neutral CDase. In rat, the neutral CDase was mainly localized at the apical membrane of proximal tubules, distal tubles, and collecting ducts in kidney whereas in liver the enzyme was distributed to endosome-like organelles in hepatocytes.20 Interestingly, the kidney CDase was found to be enriched in raft/ microdomains with cholesterol and GM1 ganglioside.20 These results may indicate why the liver CDase, but not the kidney enzyme, can be extracted by freeze-thawing in the absence of detergents: that is, some enzymes residing in endosome-like organelles such as late endosomes and lysosomes could be released from membrane fractions by freeze-thawing.39 Recently, it was revealed that caveolin-enriched light membranes of murine endothelial cells bear neutral CDase as well as acid and neutral SMases.40 Interestingly, treatment of endothelial cells with cyclodextrin, which depleted cell cholesterol, significantly enhanced neutral CDase activity, but not SMase activity, suggesting that cholesterol negatively regulates the neutral CDase. 40 Since SM as well as free ceramide are abundant in the microdomains,41 neutral CDase, together with SMase, could regulate the ceramide content of the microdomains.

Future Prospects Neutral CDases are actively detached from murine endothelial cells, macrophages and human fibroblasts.24 It is noteworthy that a neutral CDase was also released by CHOP and HEK293 cells overexpressing the enzyme, indicating a hydrophobic N-terminus of the enzyme is a typical signal sequence for secretion. A-SMase is also known to be released from cells.42 Since both CDase and A-SMase seem to be associated with microdomains in part by unknown mechanisms, it is speculated that they generate sphingosine from SM in microdomains. The biological significance of SM metabolism in microdomains should be addressed.

Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (B) (12140204) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

References 1. Hakomori S, Igarashi Y. Functional role of glycosphingolipids in cell recognition and signaling. J Biochem (Tokyo)1995; 118:1091-1103. 2. Spiegel S, Merrill AH Jr. Sphingolipid metabolism and cell growth regulation. FASEB J 1996; 10:1388-1397. 3. Okazaki T, Bielawska A, Bell RM et al. Role of ceramide as a lipid mediator of 1α,25dihydroxyvitamin D3-induced HL-60 cell differentiation. J Biol Chem 1990; 265:15823-15831. 4. Hannun YA. Functions of ceramide in coordinating cellular responses to stress. Science 1996; 274:1855-1859. 5. Zhang H, Buckley NE, Gibson K et al. Sphingosine stimulates cellular proliferation via a protein kinase C-independent pathway. J Biol Chem 1990; 265:76-81. 6. Ohta H, Sweeney EA, Gibson K et al. Induction of apoptosis by sphingosine in human leukemic HL-60 cells: A possible endogenous modulator of apoptotic DNA fragmentation occurring during phorbol ester-induced differentiation. Cancer Res 1995; 55:691-697. 7. Igarashi Y. Functional roles of sphingosine, sphingosine 1-phosphate, and methylsphingosines: In regard to membrane sphingolipid signaling pathways. J Biochem (Tokyo) 1997; 122:1080-1087. 8. Kupperman E, An S, Osborne N et al. A sphingosine-1-phosphate receptor regulates cell migration during vertebrate heart development. Nature 2000; 406:192-194. 9. Michel C, van Echten-Deckert G, Rother J et al. Characterization of ceramide synthesis: A dihydroceramide desaturase introduces the 4,5-trans-double bond of sphingosine at the level of dihydroceramide. J Biol Chem 1997; 272:22432-22437. 10. Gatt S. Enzymatic hydrolysis of sphingolipids. 1. Hydrolysis and synthesis of ceramides by an enzyme from rat brain. J Biol Chem 1963; 238:3131-3133. 11. Hassler DF, Bell RM. Ceramidase: Enzymology and metabolic roles. Adv Lipid Res 1993; 26:49-57. 12. Sugita M, Dulaney JT, Moser HW. Ceramidase deficiency in Farber’s disease (lipogranulomatosis). Science 1972; 178:1100-1102.

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13. Coroneos E, Martinez M, McKenna S et al. Differential regulation of sphingomyelinase and ceramidase activities by growth factors and cytokines. J Biol Chem 1995; 270:23305-23309. 14. Nikolva-Karakashian M, Morgan ET, Alexander C et al. Bimodal regulation of ceramidase by interleukin-1: Implications for the regulation of cytochrome P450 2C11 (CYP2C11) J Biol Chem 1997; 272:18718-18724. 15. Bernardo K, Hurwitz R, Zenk T et al. Purification, characterization, and biosynthesis of human acid ceramidase. J Biol Chem 1995; 270:11098-11102. 16. Koch J, Gartner S, Li CM et al. Molecular cloning and characterization of a full-length complementary DNA encoding human acid ceramidase. J Biol Chem 1996; 271:33110-33115. 17. Li C-M, Hong S-B, Kopal G et al. Cloning and characterization of the full-length cDNA and genomic sequences encoding murine acid ceramidase. Genomics 1998; 50:267-274. 18. Okino N, Ichinose S, Omori A et al. Molecular cloning, sequencing, and expression of the gene encoding alkaline ceramidase from Pseudomonas aeruginosa: Cloning of a ceramidase homologue from Mycobacterium tuberculosis. J Biol Chem 1999; 274:36616-36622. 19. Tani M, Okino N, Mori K et al. Molecular cloning of the full-length cDNA encoding mouse neutral ceramidase: A novel but highly conserved gene family of neutral/alkaline ceramidases. J Biol Chem 2000; 275:11229-11234. 20. Mitsutake S, Tani M, Okino N et al. Purification, characterization, molecular cloning, and subcellular distribution of neutral ceramidase of rat kidney. J Biol Chem 2001; 276:26249-26259. 21. Bawab SE, Roddy P, Qian T et al. Molecular cloning and characterization of a human mitochondrial ceramidase. J Biol Chem 2000; 275:21508-21513. 22. Mao C, Xu R, Bielawska A et al. Cloning of an alkaline ceramidase from Saccharomyces cerevisiae. J Biol Chem 2000; 275:6876-6884. 23. Mao C, Xu R, Szulc ZM et al. Cloning and characterization of a novel human alkaline ceramidase. J Biol Chem 2001; 276:26577-26588. 24. Romiti E, Meacci E, Tani M et al. Neutral/alkaline and acid ceramidase activities are actively released by murine endothelial cells. Biochem Biophys Res Commun 2000; 275:746-751. 25. Okino N, Tani T, Imayama S et al. Purification and characterization of a novel ceramidase from Pseudomonas aeruginosa. J Biol Chem 1998; 273:14368-14373. 26. Ito M, Kurita T, Kita K. A novel enzyme that cleaves the N-acyl linkage of ceramides in various glycosphingolipids as well as sphingomyelin to produce their lyso forms. J Biol Chem 1995; 270:24370-24374. 27. Imokawa G, Abe A, Jin K et al. Decreased level of ceramides in stratum corneum of atopic dermatitis: an etiologic factor in atopic dry skin? J Invest Dermatol 1991; 96:523-526. 28. Ohnishi Y, Okino N, Ito M et al. Ceramidase activity in bacterial skin flora as a possible cause of ceramide deficiency in atopic dermatitis. Clinic. Diagnostic Lab Immun 1999; 6:101-104. 29. Kita K, Sueyoshi N, Okino N et al. Activation of bacterial ceramidase by anionic glycerophospholipids: Possible involvement in ceramide hydrolysis on atopic skin by Pseudomonas ceramidase. Biochem J 2001; 362:619-626. 30. Leyden JJ, Marples RR, Kligman AM. Staphylococcus aureus in the lesions of atopic dermatitis. Brit J Dermatol 1974; 90:525-530. 31. Peters JE, Galloway DR. Purification and characterization of an active fragment of the LasA protein from Pseudomonas aeruginosa: Enhancement of elastase activity. J Bacteriol 1990; 172:2236-2240. 32. Anderson DS. The acid-base balance of the skin. Brit J Dermatol 1951; 63:283-296. 33. Norris FA, Wilson MP, Wallis TS et al. SopB, a protein required for virulence of Salmonella dublin, is an inositol phosphate phosphatase. Proc Natl Acad Sci USA 1998; 95:14057-14059. 34. Tani M, Okino N, Mitsutake M et al. Purification and characterization of a neutral ceramidase from mouse liver: A single protein catalyses the reversible reaction in which ceramide is both hydrolysed and synthesized. J Biol Chem 2000; 275:3462-3468. 35. Bawab SEI, Bielawska A, Hannun YA. Purification and characterization of a membrane-bound non-lysosomal ceramidase from rat brain. J Biol Chem 1999; 274:27948-27955. 36. Ito M, Mitsutake S, Tani M et al. Enzymatic synthesis of [14C]ceramide, [14C]glycosphingolipids, and ω-aminoceramide. Method in Enzymology, 1999; 311:682-689. 37. Tani M, Okino N, Mitsutake S et al. Specific and sensitive assay for alkaline and neutral cermidases involving C12-NBD-ceramide. J Biochem (Tokyo) 1999; 125:746-749. 38. Bawab SEI, Birbes H, Roddy P et al. Biochemical characterization of the reverse activity of rat brain ceramidase: A CoA-independent and fumonisin B1 insensitive ceramide synthase. J Biol Chem 2001; 276:16758-16766. 39. Bendall DS, de Duve C. Tissue-fractionation studies. The activation of latent dehydrogenases in mitochondria from rat liver. Biochem J 1960; 74:44-450.

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40. Romiti E, Meacci E, Tanzi G et al. Localization of neutral ceramidase in caveolin-enriched light membranes of murine endothelial cells. FEBS Lett 2001; 506:163-168. 41. Prinetti A, Chigorno V, Tettamanti G et al. Sphingolipid-enriched membrane domains from rat cerebellar granule cells differentiated in culture: A compositional study. J Biol Chem. 2000; 275:11658-11665. 42. Schissel SL, Schuchman EH, Williams KJ, et al. Zn2+-stimulated sphingomyelinase is secreted by many cell types and is a product of the acid sphingomyelinase gene. J Biol Chem. 1996; 271:18431-18436.

CHAPTER 6

The Role of Serine/Threonine Protein Phosphatases in Ceramide Signaling Charles E. Chalfant and Yusuf A. Hannun

Abstract

S

phingolipids serve as potential reservoirs for bioactive lipids and are now included with the well established mediators of signal transduction such as diacylglycerol, phosphatidylinositides, and eicosanoids, all derived from glycerolipids.1-6 Furthermore, sphingolipid metabolism has clearly been shown to be involved in the regulation of cell growth, differentiation, and programmed cell death.1-6 Indeed, there have been over 4000 studies conducted on sphingolipids during the past five years, and a majority have been directed towards understanding the emerging role of ceramide as a cellular bioregulator. Thus, with the demonstration that ceramide mediates various signaling cascades, there is now a necessity for defining direct targets of ceramide and specific mechanisms regulated by ceramide. The search for potential direct targets for ceramide action led to the identification of several candidate ceramideregulated enzymes such as ceramide-activated protein kinases (CAPK), cathepsin D, and a key set of targets, serine/threonine protein phosphatases.7-12 Two serine/threonine phosphatases have been shown to be ceramide responsive in vitro and in vivo, protein phosphatase-1 (PP1) and protein phosphatase 2A (PP2A).10,11,13-16 These protein phosphatases are now collectively termed ceramide-activated protein phosphatases (CAPPs). This Chapter will discuss the relevant mechanisms by which CAPP is activated by ceramide in vitro and its emerging role in regulating various cascades of signal transduction through modulation of specific phospho-targets.

Identification of a Ceramide-Activated Protein Phosphatase (CAPP) Based on biochemical parameters, Ser/Thr protein phosphatases were initially divided into two classes, type 1 protein phosphatases (PP1) and type 2 protein phosphatases (PP2A).17 The PP1 class is inhibited by two heat-stable proteins termed inhibitor 1 (I-1) and inhibitor 2 (I-2) whereas type 2 or PP2 class phosphatases are insensitive to these proteins, but inhibited by the small t-antigen of SV40.17-19 Type 2 phosphatases can be further subdivided into spontaneously active (PP2A), Ca+2-dependent (PP2B) and Mg+2-dependent (PP2C) classes.17,19 In the past few years, many novel serine/threonine phosphatases such as protein phosphatase-V and – X have been identified that do not fit into any of these classes.20 Both PP1 and PP2A are composed of a catalytic subunit bound by one or more regulatory subunits.17-19 These subunits regulate subcellular localization, substrate specificity, enzyme activity, and in some cases cofactor binding.17-19 The catalytic and regulatory subunits are also regulated by covalent modifications such as phosphorylation and methylation.18,19 Thus, regulation of these protein phosphatases is very complex requiring coordination of

Ceramide Signaling, edited by Anthony H. Futerman. ©2002 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Ceramide Signaling

phosphorylation, methylation, subunit expression, and as discussed in this Chapter, lipid second messengers. Originally, ceramide was shown to increase the activity of a serine/threonine protein phosphatase(s) in crude cytosolic extracts.10,11 This in vitro activation was achieved by Derythro-ceramide, but not by the biologically inactive dihydro-ceramide.10,11 From this initial observation, a predominant ceramide-activated protein phosphatase was shown to exist in rat brain,21 and this phosphatase was identified as a member of the 2A (PP2A) family of serine/ threonine phosphatases.10,11 This conclusion was based on studies demonstrating that pretreatment with okadaic acid inhibited the ability of ceramide to activate a serine/threonine phosphatase in T9 glioma cell extracts.10,11 To conclusively demonstrate that PP2A is activated by ceramide, a novel chromatographic approach was utilized to purify the major CAPP activity from rat brain to near homogeneity. 21 This approach used hydrophobic interaction chromatography on phenyl sepharose followed by anion-exchange on MonoQ.21 CAPP purified in this manner was found to be composed of three major bands on SDS-PAGE which comigrated with the three subunits of heterotrimeric PP2A.21 Immunological studies then identified CAPP as heterotrimeric (AB’C and ABαC) and heterodimeric (AC) PP2A species.21 Further demonstrating that PP2A was a form of CAPP, heterotrimeric PP2A (AB’C) and heterodimeric PP2A (AC) purified from rabbit skeletal muscle were shown to be activated by ceramide directly and in a specific manner.21 These results were further supported by the coelution of CAPP with PP2A on size-exclusion chromatography and by their immunologic identity.21 These studies clearly demonstrated that PP2A was indeed a form of CAPP and provided a convenient and efficient method for isolating the PP2A species of CAPP to allow for in-depth biochemical analysis.21 Another eluting species of ceramide-activated phosphatases was also noted, and further studies showed that this was PP1, a closely-related serine/threonine phosphatase.13,14 In vitro studies showed that purified PP1 responds to ceramide in vitro with similar specificities as PP2A,13,14 thus establishing PP1 as a second CAPP.

In Vitro Regulation of CAPP by Ceramide Several studies have now shown that short chain (cell permeable) ceramides activate PP2A and the catalytic subunit of PP1 (PP1c) in vitro.10,14,21,22 D-e-C2-ceramide activated heterotrimeric PP2A (AB’C) up to 3.5-fold with an EC50 of approximately 5 µM and to a lesser extent (up to 2.5-fold) the heterodimeric form (AC) of PP2A.10,21 Chain length studies on heterotrimeric PP2A demonstrated that ceramides possessing hexanoyl, decanoyl, and myristoyl, but not stearoyl acyl chains also activated the phosphatase.10,21 The longer chain ceramides failed to activate due to solubility problems in delivering those hydrophobic ceramides (see below). Activation of heterotrimeric PP2A was ceramide-specific, as closely related lipids had no effect on PP2A activity.10,21 Moreover, dihydro-C2-ceramide (which lacks the trans 4-5 double bond found in ceramide) did not activate PP2A in vitro, but actually inhibited PP2A activity.10,21 These findings were particularly important for several reasons. First, they demonstrated chain-length selectivity in the activation of PP2A. Second, dihydroceramide is a metabolic precursor of ceramide, thus establishing metabolic specificity of ceramide in activation of CAPP. Finally, dihydroceramides had been shown not to mimic the cellular activities of ceramide in inducing apoptosis, cell cycle arrest, or differentiation.23 Thus, these corresponding specificities in cellular action and in vitro activation of CAPP suggested CAPP as a relevant cellular target for ceramide. To further demonstrate that this activation was specific, the stereospecificity of activation of these phosphatases by ceramides was examined. Thus, the stereoisomers of the naturally occurring D-erytho-C2 ceramide (2S, 3R) were synthesized. These were its enantiomer, L-erytho-C2 ceramide (2R, 3S), and the two diastereoisomers in the threo conformation, D-threo-C2 ceramide and L-threo-C2 ceramide, which differ from erythro in that the C-2 configuration relative to C3 is in a cis-conformation unlike erythro in which the groups are in a trans-conformation. PP2A

The Role of Serine/Threonine Protein Phosphatases in Ceramide Signaling

51

demonstrated some stereospecificity with the short chain ceramides in that only the D- and Lerythro forms of C2-ceramide activated PP2A and not the D- and L-threo forms.10,21 PP1 was also activated by short chain ceramides in vitro. In these studies, D-e-C6- and De-C2-ceramide activated PP1c in a dose dependent manner (Kishikawa K, Chalfant CE, Lee JY, Hannun YA. 2001; unpublished findings). PP1 showed similar N-acyl chain length dependencies (as PP2A) since the N-acetyl, hexanoyl, and decanoyl, but not stearoyl ceramides activated PP1 in vitro (Kishikawa K, Chalfant CE, Lee JY, Hannun YA. 2001; unpublished findings). Similar to PP2A, the activation was specific as again closely related lipids as well as the inactive form, dihydro-C6-ceramide, failed to activate PP1 (Kishikawa K, Chalfant CE, Lee JY, Hannun YA. 2001; unpublished findings). PP1 also demonstrated stereospecificity with Derythro-C6-ceramide activating to the greatest extent compared to its stereoisomers (Kishikawa K, Chalfant CE, Lee JY, Hannun YA. 2001; unpublished findings). A major problem that hampered further evaluation of the biochemical regulation of CAPP in vitro had been the lack of an effect of long chain (natural) ceramides. Since long chain neutral lipids are insoluble in aqueous solutions, delivering long chain ceramides in vitro and in vivo is difficult and limited. To overcome this problem, long chain ceramides were solubilized with 2% dodecane at a final reaction concentration of 0.02%.13 Dodecane alone exerted an inhibitory effect on phosphatase activity, but solubilized D-e-C18 ceramide clearly activated the PP1α catalytic subunit (PP1αc), PP1γ catalytic subunit (PP1γc), PP2Ac, and PP2A trimer in a dose-dependent manner.13 This activation was examined for specificity by long chain ceramides using stereoisomers of D-e-C18 ceramide.13 Only D-e-C18 ceramide activated each phosphatase. These observations are significant for at least three reasons.13 First, activation of CAPP by natural ceramides was demonstrated. Second, specificity for ceramide was clearly demonstrated for the long chain ceramide, and the stereochemical specificity became better defined with the long chain isomers than with the short chain ones. Third, a specific ceramidebinding/interaction site was now suggested to be present on the catalytic subunit of at least PP1 and PP2A since they respond directly to ceramide. Since these observations, unsaturated natural ceramides dispersed in aqueous buffer by sonication have been shown to activate both PP1c and PP2Ac without the need for dodecane solubilization (Chalfant CE, Hannun YA. 2001; unpublished findings).

Selective CAPP Substrates in Cells Since initial studies showed that the specificity for CAPP activation in vitro closely resembled the specificity for various cellular activities of ceramide such as apoptosis,5,10,11,13,14,24,25 CAPP emerged as a key candidate for mediating cellular activities of ceramide through dephosphorylation of key substrates. Several criteria for establishing a CAPP substrate are proposed: 1) exogenous ceramides induce the dephosphorylation of the particular phospho-substrate; 2) inhibitors of serine/threonine protein phosphatase block this effect; 3) exogenous dihydroceramide should not affect the phosphorylation state of the particular phospho-substrate; and 4) the target phospho-protein should act as an in vitro substrate for CAPP. Using these criteria, several targets for CAPP have been identified by multiple groups (Fig. 1).

Protein Kinase Cα (PKCα) The first study demonstrating a CAPP substrate in cells came in 1996 from Obeid and coworkers.16 They first observed that in Molt-4 leukemia cells, ceramide inhibited phorbol 12myristate 13-acetate (PMA)-induced phosphorylation of many substrates, suggesting a role for ceramide in regulating the protein kinase C (PKC) pathway.16,26 PKCα is a key member of the PKC family that is involved in signal transduction, growth regulation, and metabolic regulation. Importantly, PKCα activity has been specifically implicated as an anti-apoptotic mechanism, and it has even been proposed that inactivation of PKCα is important in inducing apoptosis.26 Therefore, the mechanism by which ceramide regulated PKCα was pursued, and ceramide was found to inhibit basal and PMA-induced phosphorylation of PKCα (i.e.,

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Ceramide Signaling

Figure 1. Schematic of known in vivo substrates of ceramide-activated protein phosphatases.

autophosphorylation).16 These results suggested that the effects of ceramide were at the level of regulation of PKC rather than its substrates. Ceramide did not directly inhibit PKCα in vitro nor did ceramide modulate the levels of PKCα protein, or inhibit PMA-induced translocation of PKCα.16 Using an immunoprecipitation assay for PKCα activity, treatment of Molt-4 cells with ceramide resulted in a concentration- and time-dependent inhibition of immunoprecipitated PKCα activity.16 The inactive form of ceramide, dihydro-C6 ceramide had no effect on PKCα activity.16 Ceramide was found to block the phosphorylation of PKCα in cells.16 Together, these results suggested that ceramide inactivated PKC by removing critical phosphates (such as thr 500 in the activation loop). Indeed, using an antibody specific for this site, it was shown that ceramide induces specifically the removal of this phosphate.27 Mechanistically (from the ceramide point of view), okadaic acid at concentrations that inhibit the serine/threonine protein phosphatase PP2A, blocked ceramide-induced dephosphorylation of PKCα in Molt-4 cells.16 These results indicated a direct role for ceramide and ceramide-activated protein phosphatase in mediating the inactivation of PKCα in Molt-4 cells.

c-Jun

Also in 1996, Galarreta and co-workers identified c-Jun as a target for CAPP in cells.28 They demonstrated that treatment with exogenous ceramide or TNFα, a ceramide-generating agonist, led to time-dependent dephosphorylation of c-Jun in A431 cells.28 Furthermore and importantly, treatment of these cells with bacterial sphingomyelinase (B-SMase) also induced the dephosphorylation of c-Jun, demonstrating for the first time that endogenously generated ceramide (in this case, ceramide generated at the plasma membrane) induced the dephosphorylation of a CAPP target.28 Pre-treatment of these cells with okadaic acid completely blocked the effects of exogenous ceramide, TNFα, and bacterial SMase on c-Jun, suggesting a PP2A form of CAPP.28 Therefore, c-Jun was shown to be a substrate for CAPP in vitro fulfilling all of the criteria for the designation of a CAPP substrate.

Retinoblastoma Gene Product (Rb) Because of the essential involvement of Rb in the G0/G1 phase of the cell cycle, the effects of ceramide on Rb were investigated. Rb was dephosphorylated, but not proteolytically cleaved, following ceramide treatment in Molt-4 cells overexpressing Bcl-2.11,29-31 Using this model, it was found that D-e-C6 ceramide induced a rapid (within 2-6 hours) dephosphorylation of Rb

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in a dose-dependent manner.11,29-31 In addition, the inactive form of C6-ceramide, dihydroC6-ceramide, did not induce dephosphorylation of Rb demonstrating lipid specificity.11,29-31 Moreover, the PP1 and PP2A inhibitor, calyculin A, blocked ceramide-induced Rb dephosphorylation, suggesting a role for CAPP in regulating this mechanism.11,29-31 Of importance, okadaic acid at concentrations that specifically inhibit PP2A did not affect ceramide dephosphorylation of Rb, suggesting for the first time, a PP1-dependent mechanism.11,29-31 These results were further supported by the demonstration that Rb is a substrate for PP1 in vitro and ceramide increased the in vitro activity of PP1 toward Rb (Kishikawa K, Chalfant CE, Lee JY, Hannun YA. 2001; unpublished findings). Taken together, these results defined a mechanism by which ceramide regulates Rb dephosphorylation and suggested a different species of CAPP, PP1, responsible for imparting ceramide effects toward a specific in vivo target (Rb). Furthermore, these studies showed that okadaic acid and calyculin A inhibited distinct effects of ceramide, and implicated the action of different phosphatases.

Bcl-2 Recently, several key apoptotic factors have been shown to be regulated by phosphorylation/ dephosphorylation via serine/threonine protein phosphatases. For example, Bcl-2 phosphorylation on serine 70 has been demonstrated to be necessary for its protective/survival function, and dephosphorylation of Bcl-2 serine 70 leads to BAD/Bcl-2 heterodimerization, effectively inhibiting the function of Bcl-2.32,33 Interestingly, serine 70 is a direct target for phosphorylation by PKCα and dephosphorylation by PP2A. Recently, May and co-workers demonstrated increased mitochondrial PP2A activity and specific dephosphorylation of mitochondrial Bcl-2 in response to ceramide.32,33 Again, okadaic acid was able to inhibit this effect, further implicating the PP2A form of CAPP in this ceramide effect.32,33 Thus, PP2A has been demonstrated to dephosphorylate both a Bcl-2 kinase (PKCα) and Bcl-2 in response to ceramide, suggesting a possible signaling complex. Furthermore, this report validated the signal transduction sequence for ceramide since other studies had placed ceramide formation somewhere between the action of upstream and downstream caspases, with Bcl-2 acting downstream of ceramide in that same pathway.34,35 Thus, ceramide regulates the phosphorylation of Bcl-2 and its antiapoptotic function via CAPP, clearly placing ceramide generation upstream of Bcl-2 (Fig. 2).3336

Akt/PKB Multiple recent reports have implicated the protein kinase Akt (also known as PKB) as a target for ceramide action.37-41 Akt/PKB plays key roles in insulin action and in mitogenic and anti-apoptotic signaling.42,43 Akt/PKB is activated by phosphoinositides generated by PI-3 kinase and is also a substrate for PDK, an activating protein kinase, which is also activated by phosphoinositides.9,44,45 In turn, Akt/PKB has been shown to phosphorylate a number of key substrates including Bad, caspase 9, and others.9,43,44 Inactivation of Akt, a known PP2A substrate, has been shown to be necessary for activation of caspase 9 and Bad,9,44 and has been proposed as a necessary event in turning off a key anti-apopotic mechanism. Importantly, several studies have demonstrated the ability of ceramide to induce dephosphorylation of Akt/PKB with concomitant loss of function/activity.9,37-41,45 These studies can be summarized as follows: First, treatment of mouse and rat skeletal muscle cells with exogenous ceramide has been demonstrated to inhibit Akt/PKB activity, and this effect was blocked by pre-treatment with okadaic acid.38,40-42 This implicated PP2A in regulating the phosphostate of Akt/PKB and, thus, its activity. Second, impairment of Akt/PKB function and insulin sensitivity in adipocytes corresponds with increased levels of ceramide.46,47 Third, ceramide has been shown to block the translocation of Akt/PKB to the membrane in response to such agonists as insulin and PDGF.39 Lastly, Akt/PKB has been shown to rescue adipocytes from apoptosis in response to insulin, and ceramide blocked this effect.46 These data all suggest that ceramide, in most cases through a ceramide-activated protein phosphatase, inactivates Akt/PKB.

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Figure 2. Schematic of TNFα signaling in apoptosis. The schematic depicts a general scheme of TNFα signaling demonstrating the function of Bcl-2 and PKCα downstream of ceramide generation. PP2A is shown to dephosphorylate both Bcl-2 and PKCα in response to ceramide allowing for the eventual activation of downstream caspases.

SR Proteins Recently, exogenous ceramide was demonstrated to induce the dephosphorylation of SR proteins, a family of serine/arginine (RS) domain containing proteins that regulate constitutive and alternative pre-mRNA processing.15,48-56 It was demonstrated that treatment of Jurkat cells with D-e-C6 ceramide produced a time- and dose-dependent decrease in the phosphorylation of all detectable SR protein species, SRp70, SRp55, SRp40, and SRp30.15 Treatment of Jurkat cells with the biologically inactive D-e-dihydroC6 ceramide had no effect on the phosphorylation state of SR proteins.15 Caspase inhibitors had also no effect on the dephosphorylation of SR proteins in response to exogenous ceramide, demonstrating that this effect was independent or upstream of caspase activation.15 As with Rb dephosphorylation, calyculin A, but not okadaic acid blocked ceramide-induced dephosphorylation of SR proteins, demonstrating a role for protein phosphatase-1.15 These results, therefore, demonstrated a specific effect of D-e-C6 ceramide on the dephosphorylation of SR proteins via activation of CAPP. Taken together, all of these studies thus far are beginning to demonstrate the important effects of ceramide on the dephosphorylation of several important substrates linked to signaling cascades.

Role of Endogenous Ceramide in Regulating CAPP Activity One of the main difficulties in validating CAPP in cells had been the absence of studies demonstrating a dependence on the generation of endogenous ceramide for the dephosphorylation of a particular phospho-protein. As discussed previously in the Chapter, B-SMase treatment was shown to induce activation of PP2A leading to the dephosphorylation of c-JUN, thus demonstrating the ability of endogenous ceramide to exert specific functions.28 Although this study showed the ability of endogenous ceramide to activate CAPP in cells, a physiological role for endogenous ceramide in regulating CAPP in cells was not established. In order to establish a role for CAPP in signal transduction, studies are needed that utilize relevant agonists

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(e.g., TNFα) and demonstrate a necessary role for the generation of ceramide to activate CAPP. Indeed, two such models of CAPP activation have been defined recently. First, fumonisin B1, and inhibitor of Co-A dependent ceramide synthase and the generation of de novo ceramide, was shown to block dephosphorylation of PKCα by protein phosphatase 2A in response to TNFα in L929 cells (Fig. 3).27 Second, the same inhibitor of de novo ceramide generation also blocked the dephosphorylation of SR proteins in response to Fas activation (Fig. 3).15 These two reports were milestones validating the existence of ceramide-activated protein phosphatase in cells and demonstrating the necessity of generating endogenous ceramide in order to activate CAPP. Furthermore, these studies demonstrate that de novo ceramide can activate both PP1 and PP2A forms of CAPP. Now the question can be raised whether ceramide generated by other pathways can activate CAPP, only specific forms of CAPP, or even CAPPs localized in specific cellular compartments. Further studies are needed to answer these questions, but the identification of these ceramidedependent mechanisms now also allows for the investigation of mechanisms downstream of CAPP. Thus, the recent availability of specific inhibitors of pathways for generating ceramide in response to exogenous agonists and molecular tools (such as the overexpression of enzymes that clear ceramide, or anti-sense constructs for enzymes that generate ceramide), should now allow the modulation of ceramide levels, thereby, determining their effects on signaling events. Based on this, the criteria for defining a particular phospho-target as a substrate for CAPP should be expanded, and the designation as a CAPP substrate should now require the additional criterion of demonstrating that specific inhibition of ceramide generation leads to subsequent inhibition of the dephosphorylation of the phospho-substrate in question.

General Mechanisms Regulated by Ceramide-Activated Protein Phosphatases Studies using specific inhibitors of serine/threonine protein phosphatases have demonstrated a clear role for CAPPs in regulating key biological responses. These studies and observations are briefly discussed below along with their implications.

Apoptosis A role for serine-threonine protein phosphatases in regulating apoptosis has been established by many laboratories. Inhibitors of serine/threonine protein phosphatases have been demonstrated to block apoptosis induced by docosahexanoic acid, cytosine arabinoside, interleukin-3 withdrawal, heat stress, γ-radiation, and etoposide.40,46,57,58 Most of these apoptosis-inducers also cause an increase in ceramide levels, and several studies have specifically characterized a role for CAPPs in the apoptotic response. First, CAPP has been shown to be involved in c-Myc downregulation (a prelude to apoptosis in many cell models) in response to ceramide and TNFα.11 Second, CAPP has been shown to be involved in activation of caspases, and inhibition of CAPP blocks both apoptosis and caspase activation.59 Third, activation of CAPP has been shown to be involved in PC12 apoptosis and to block cell survival pathways mediated by nerve growth factor.40 Thus, many reports have shown both a correlation and a direct link between serine/threonine protein phosphatases, ceramide generation, and apoptosis in many cell models.

Differentiation Two studies demonstrate a role for serine/threonine protein phosphatases in regulating neural cell differentiation. The first study demonstrated that both okadaic acid and calyculin A blocked the differentiation of Neuro 2a cells induced by lactacystin.60 In another study, Tettamanti and co-workers implicated CAPP in regulating the differentiation of Neura 2a cells.61,62 They showed that okadaic acid blocked Neura 2a cell differentiation under conditions that increased ceramide levels.61,62 This study clearly implicated the PP2A subspecies of CAPP and demonstrated a role for CAPP in mediating neural cell differentiation.

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Figure 3. Schematic of serine/threonine protein phosphatases activated by de novo ceramide.

Cell Growth and Proliferation Probably the most impressive body of work implicating CAPP in regulating specific cellular mechanisms is in the field of cell growth and proliferation. Many laboratories have established a role for CAPP genetically, biochemically, and at the level of pharmacology. Genetically, Nickels and Broach demonstrated a necessity for yeast CAPP in the induction of ceramide-mediated cell cycle arrest.25 Briefly, they demonstrated the PP2A yeast homologue was necessary for arresting yeast cells in the G1 phase and mutation of any of the heterotrimeric subunits which composed yeast PP2A rendered the cells resistant to ceramide-induced cell cycle arrest.25 In mammalian cells, we have already discussed the CAPP target, Rb, and dephosphorylation of Rb by CAPP has been shown to induce cell cycle arrest.29 Recently, the hypo-phosphorylated form of Rb has been shown to be an efficient substrate for caspase 3 unlike the phosphorylated form.63 Furthermore, cyclin-dependent kinase II (CDK2) has been recently shown as a substrate for CAPP.64 This study demonstrated that inhibitors of PP1 and PP2A block the inactivation of CDK2 in response to ceramide.64 Since inactivation of CDK2 has been demonstrated to induce the dephosphorylation of Rb, this illustrates another example of a signaling complex where CAPP both inactivates a kinase and its substrate through dephosphorylation.64

Insulin Secretion and Action The role of CAPP in insulin signaling is becoming an emergent theme. First, ceramide and CAPP have been implicated in blocking insulin secretion and induction of apoptosis in isolated beta cells.65 This finding taken together with the knowledge that increased cytokines, specifically those that increase ceramide levels in cells (e.g., TNFα), were upregulated in diabetic patients led to a recent flood of reports on inhibition of insulin action in response to

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Figure 4. Pathways of signal transduction regulated by ceramide-activated protein phosphatases. The schematic depicts known pathways of signal transduction modulated by ceramide and ceramide-activated protein phosphatase. The diagram also depicts other candidate targets for ceramide including KSR, PKCα, and cathepsin D.

ceramide.66,67 In these reports, exogenous ceramide treatment was shown to block both insulin-stimulated glucose transport (ISGT) and insulin-stimulated glycogen synthesis (ISGS).38,39,41,42,45,46,68,69 Okadaic acid in both instances inhibited the effect of ceramide on ISGT and ISGS implicating the PP2A form of CAPP. Since okadaic acid has also been established as an inducer of ISGT, a role for PP2A inhibition (CAPP inhibition) has been hypothesized as a necessary mechanism in insulin responses to permit kinase cascades to proceed.70 As previously discussed, one role of CAPP, mechanistically, is the inhibition of Akt/PKB in response to ceramide.38,39,41,42,45,47,65,69 Thus, CAPP has been shown to play an integral role in regulating both insulin secretion and insulin responses, making it a possible target for the design of therapies to alleviate the diabetic condition.

Regulation of mRNA Processing as a Specific Pathway Regulated by CAPP Through examination of possible downstream mechanisms regulated by ceramide-responsive enzymes, a common theme of substrates for both CAPP and PKCζ was recognized in the alternative processing of pre-mRNA. Both PP1 and PKCζ have been demonstrated to regulate protein factors involved in the mechanism of alternative splicing.15 Specifically, in response to endogenous ceramide, PP1 dephosphorylated SR proteins, a family of factors that regulate constitutive and alternative processing of pre-mRNA.15,49,51-54,71-73 Dephosphorylation of SR proteins effectively inhibits many of their functions and has been reported to regulate alternative processing of pre-mRNA.49,51-54,71-73 On the other hand, ceramide has been shown to induce PKCζ to phosphorylate hnRNPA1, a known regulator of the alternative processing of pre-mRNA and antagonist of SR proteins.7,74 Phosphorylation of hnRNP A1 induces its nuclear translocation and increases its RNA binding ability, thereby enhancing its function as an antagonist of SR proteins.7,74,75 Therefore, ceramide, via the “cross-talk” of two direct targets, effectively diminishes the ability of SR proteins to regulate pre-mRNA processing by inhibiting

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activity directly (dephosphorylation) and indirectly (activation of an SR protein antagonist). Thus, based on these observations, we propose that ceramide may regulate the alternative splicing of genes that play an important role in cellular stress responses and apoptosis. Indeed, examination of the literature reveals that both Bcl-x and caspase 9 genes produced splicing variants with opposing functions.76,77 The Bcl-x splice variant, Bcl-x(L) and the caspase 9 splice variant, caspase 9b, inhibit apoptosis in contrast to the pro-apoptotic splice variants, Bcl-x(s) and caspase 9.76,77 Ceramide was found to regulate the alternative splicing of both Bclx and caspase 9, reducing the expression of the cell survival factors, caspase 9b and Bcl-x(L) while increasing the expression of the pro-apoptotic factors, Bcl-x(s) and caspase 9.78 Ceramideinduced alternative splicing was blocked by inhibitors of serine-threonine protein phosphatases and of the de novo ceramide pathway, demonstrating a role for CAPP and endogenous ceramide in regulating this mechanism.78 These observations were important in that a novel, direct, and specific mechanism mediated by a ceramide-activated protein phosphatase and endogenous ceramide has now been defined. It is now obvious that regulation of sphingolipid pathways plays an important and complex role in regulating many cellular responses. Through careful examination of ceramide signaling, the various cascades of ceramide action are now being delineated (Fig. 4). This conclusion increases the need for intense investigation of sphingolipid-mediated mechanisms and with the emergence of new molecular and biochemical tools for sphingolipid metabolic enzymes, new mechanisms and targets mediated by ceramide-activated protein phosphatases and other ceramide-responsive enzymes will soon be discovered.

Acknowledgements This work was supported by a grant (to Y.A.H.) from the National Institutes of Health (CA87584 & GM43825), a National Research Service Award (to C.E.C.) from the National Institutes of Health (GM19953-02), and a VA Merit Review (to C.E.C.) from the Veteran’s Administration.

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67. Cavallo MG, Pozzilli P, Bird C et al. Cytokines in sera from insulin-dependent diabetic patients at diagnosis. Clin Exp Immunol 1991; 86:256-259. 68. Begum N, Ragolia L. Effect of tumor necrosis factor-alpha on insulin action in cultured rat skeletal muscle cells. Endocrinology 1996; 137:2441-2446. 69. Hajduch E, Balendran A, Batty IH et al. Ceramide impairs the insulin-dependent membrane recruitment of protein kinase B leading to a loss in downstream signalling in L6 skeletal muscle cells. Diabetologia 2001; 44:173-183. 70. Standaert ML, Bandyopadhyay G, Sajan MP et al. Okadaic acid activates atypical protein kinase C (zeta/lambda) in rat and 3T3/L1 adipocytes. An apparent requirement for activation of Glut4 translocation and glucose transport. J Biol Chem 1999; 274:14074-14078. 71. Mermoud JE, Cohen PT, Lamond AI. Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism. Embo J 1994; 13:5679-5688. 72. Du C, McGuffin ME, Dauwalder B et al. Protein phosphorylation plays an essential role in the regulation of alternative splicing and sex determination in Drosophila. Mol Cell 1998; 2:741-750. 73. Misteli T, Spector DL. Serine/threonine phosphatase 1 modulates the subnuclear distribution of pre-mRNA splicing factors. Mol Biol Cell 1996; 7:1559-1572. 74. Municio MM, Lozano J, Sanchez P et al. Identification of heterogeneous ribonucleoprotein A1 as a novel substrate for protein kinase C zeta. J Biol Chem 1995; 270:15884-15891. 75. van der Houven van Oordt W, Diaz-Meco MT, Lozano J et al. The MKK(3/6)-p38-signaling cascade alters the subcellular distribution of hnRNP A1 and modulates alternative splicing regulation. J Cell Biol 2000; 149:307-316. 76. Minn AJ, Boise LH, Thompson CB. Bcl-x(S) antagonizes the protective effects of Bcl-x(L). J Biol Chem 1996; 271:6306-6312. 77. Srinivasula SM, Ahmad M, Guo Y et al. Identification of an endogenous dominant-negative short isoform of caspase-9 that can regulate apoptosis. Cancer Res 1999; 59:999-1002. 78. Chalfant CE, Rathman K, Pinkerman RL et al. De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. J Biol Chem 2002; in press.

CHAPTER 7

Kinase Suppressor of Ras as a CeramideActivated Protein Kinase D. Brent Polk, Jose Lozano and Richard N. Kolesnick

Abstract

C

eramide has received attention as a second messenger in a number of biological systems determining cellular proliferation, differentiation and apoptosis. Among the targets of ceramide activation, kinase suppressor of Ras (KSR) is a novel serine/threonine kinase, identified in genetic screens in Drosophila melanogaster and Caenorhabditis elegans, that regulates signaling through the Ras/Mitogen-activated protein kinase (MAPK) pathway. Inactivating mutations of KSR suppress the developmental effects orchestrated by activated Ras pathways in these organisms. Through biochemical assays, KSR was previously identified as a ceramide-activated membrane-associated proline-directed kinase stimulated by tumor necrosis factor to phosphorylate Raf-1, and initiate the MAPK pathway. Recent data show that KSR also functions as a scaffold protein coordinating proteins of the MAPK signaling module. Cellular biological evidence points to KSR activation of Raf-1 as a regulatory step for such diverse signaling functions as epidermal growth factor-mediated proliferation and TNF-mediated survival.

Introduction Kinase suppressor of Ras (KSR) was originally cloned in genetic screening assays designed to identify positive modifiers of the Ras pathway in Caenorhabditis elegans and Drosophila melanogaster.1-3 Isolation of human and murine KSR demonstrated highly conserved sequence homology, suggesting KSR signaling was also evolutionarily conserved.3,4 The ceramide-activated protein kinase (CAPK), identified by an in-gel kinase assay as a 97 kDa proline-directed Ser/Thr kinase, was subsequently recognized as KSR.5 This was based on the demonstration that the unique substrate recognition motif for CAPK6 was contained within Raf-1 and utilized by KSR for Raf-1 phosphorylation and activation.7 Nevertheless, the function of KSR as a kinase has remained controversial. Some groups have not been able to reproduce the kinase activity of KSR and have suggested KSR may not function as a kinase,8-10 while others have reported kinases co-immunoprecipitating with KSR may account for the observed kinase activity.11 These observations have led to the suggestion that KSR functions as a complex scaffolding protein coordinating a number of the kinases of the Ras/Raf/MEK-1/ERK MAPK signaling pathway (recently reviewed12). However, through use of a two-stage kinase assay where KSR never contacts any other kinase but Raf-1, both our laboratories have now independently reported Thr phosphorylation of Raf-1 by KSR following epidermal growth factor (EGF) or tumor necrosis factor (TNF) treatment of cells.13,14 Taken together, these findings suggest KSR has both a scaffolding role and requires utilization of its regulable kinase activity for the full complement of signaling and cellular functions. Recent evidence indicated that the Ceramide Signaling, edited by Anthony H. Futerman. ©2002 Eurekah.com and Kluwer Academic / Plenum Publishers.

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kinase activity of KSR is necessary for activation of anti-apoptotic pathways by TNF.15 This review will focus on KSR as a CAPK, which regulates signaling pathways that determine cellular proliferation, differentiation and apoptosis.

KSR Structure and Activity KSR is comprised of five distinct domains, termed CA1-CA5, the first four of which are within the N-terminus (Fig. 1).3 CA1 is unique to KSR and its function awaits characterization. CA2 is a Src homology 3 (SH3) recognition site and CA3 a Cys-rich domain similar to the lipid-binding domain of protein kinase C (PKC). CA4 is a Ser/Thr-rich region resembling the CR2 domain of Raf-1. The C-terminal region of KSR (CA5) contains the 11 conserved kinase subdomains found in all kinases. However, it is not clear whether KSR is a Tyr or Ser/ Thr kinase. The sequence YI(L)APE in subdomain VIII, which is conserved among Ser/Thr kinases, is present in all KSR genes cloned so far, yet C. elegans and D. melanogaster KSR also contain the sequence HKDLR indicative of Tyr kinases.3 Mouse and human KSR have an Arg in kinase subdomain II instead of the conserved Lys normally involved in ATP binding in mammalian kinases. Hence, some investigators have argued that mammalian KSR might not even act as a kinase.3,8,10,16 Nonetheless, Morrison and co-workers showed conversion of the Arg to a Met or the conserved DFG motif to VFG, kinase-inactivating mutations, prevented the catalytic unit of KSR from blocking Ha-RasV12-induced germinal vesicle breakdown (GVBD) in Xenopus oocytes. Similarly, Guan and co-workers demonstrated that the Arg to Met mutation prevented KSR inhibition of Elk-1 phosphorylation and activation in COS1 cells.17 These studies provide strong evidence that KSR indeed possesses kinase activity. Conversion of Arg to Lys, however, which should restore kinase activity to KSR if the Arg in subdomain II was naturally inactivating, failed to alter KSR inhibition of GVBD16 or Elk-1 activation.17 A potential explanation for this apparent discrepancy can be found in the recent publication of another Ser/Thr protein kinase, WNK1, also lacking a Lys in the catalytic subdomain II.18 Molecular modeling of WNK1 demonstrated an alternative Lys within this subdomain was oriented within the ATP binding cleft of WNK1, such that it might serve a similar ATP coordinating function, a postulate supported by mutagenesis at this position.18 Consistent with mammalian KSR acting as a kinase, Morrison and co-workers recently showed that the murine brain KSR isoform, a KSR splice variant, retains ATP binding capability.19 Determining if KSR directly phosphorylates and activates Raf-1 has been the most contentious issue regarding the role of KSR in signal transduction. The original studies of the kinase activity of KSR to signal Raf-1 activation in response to TNF or elevation of cellular ceramide levels5 used a one stage in vitro reconstitution assay comprised of recombinant Raf-1, MEK1 and ERK-2/MAPK.13 Based on the inability to reconstitute the kinase activity of KSR and the fact that numerous kinases co-immunoprecipitated with KSR, Morrison and co-workers proposed that the observed activity was not intrinsic to KSR.9 Their hypothesis is that the primary mode of KSR signaling is via protein-protein interaction (the scaffolding model). Consistent with this model, expression of the KSR N-terminus alone (∆C539) activated Raf-1 in Xenopus oocytes and cooperated with Ha-RasV12 to induce GVBD,16 and transgenic restoration of kinase defective C. elegans ksr-1 reverted the phenotype of a strong loss-of-function KSR mutant.10 Nevertheless, a two-stage in vitro activity assay for KSR in which KSR never comes in contact with any kinases other than Raf-1 would appear to show that KSR does indeed phosphorylate and activate Raf-1.13,14 During the first stage of this assay, highly purified KSR is incubated only with a homogenous preparation of Raf-1 in a reaction mixture containing ATP. In the second stage, phosphorylated/activated Raf-1 is separated from KSR and incubated with a reaction mixture containing unactivated GST-MEK, unactivated GST-MAPK and a human GST-Elk-1 fusion protein. Phosphorylation of Elk-1 serves as readout for reconstitution of MAPK signaling. KSR, washed to apparent homogeneity with high salt, retained its activity toward Raf-1 in this assay, indicating the kinase activity was intrinsic to KSR.13 A caveat to these studies is that the highly purified KSR might still be contaminated with low levels of an

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Figure 1. Schematic comparison of Raf-1 and KSR homology domains. Numbers indicate position of amino acid residues. The highly conserved regions are indicated as CR1-3 in Raf-1 and CA1-5 in KSR with the kinase domains indicated in green. Shown in blue are the reported phosphorylation sites for both proteins (see refs. 8, 11 and 29). The two Ras-binding domains on Raf are indicated by the tan and orange colored boxes partially overlapping the CR1 region (reviewed in ref. 33). To view color version of Figure please visit http://www.eurekah.com/chapter.php?chapid=630&bookid=13&catid=56.

unknown activity capable of activating Raf-1. Further, the Raf-1 must be partially pre-activated in order for it to serve as an effective KSR substrate. The question arises as to why it has been so difficult for some groups to detect the intrinsic kinase activity of KSR. KSR is virtually inactive in many resting cells, but its activity is markedly increased by EGF, TNF or ceramide treatment.13,14 Hence, KSR activity will go unrecognized in many systems if examined while unstimulated, perhaps contributing to the inability of some groups to detect substantive activity. Alternately, the recent studies of Morrison and coworkers may provide an answer to this dilemma, in some instances. Almost all studies to date have been performed using systems overexpressing recombinant KSR to levels far beyond the endogenous KSR concentration (which is usually quite small) and thus the discrepancies observed may reflect gene dosage. These investigators demonstrated that low KSR expression cooperated with activated Ras to increase MAPK signaling, while high dose KSR inhibited MAPK activation and Xenopus oocyte maturation.8 We suggest that the kinase activity of KSR may be similarly affected by dose, resulting in inconsistencies between laboratories. In agreement with our contention that KSR possesses kinase activity towards Raf-1, we isolated endogenous KSR from A431 cells, which contain high levels of activated EGFR, and demonstrated constitutively enhanced kinase activity.13 Thus, the activity we observed cannot be attributed to overexpression of the recombinant protein. Alternatively, differences between groups may reflect cell-type specific signaling as, for instance, KSR transduces apoptotic signals in response to ceramide in COS-7 cells only if they express BAD, a pro-apoptotic Bcl-2 family member.20 Further, in intestinal cells, TNF, but not EGF, stimulates KSR phosphorylation and kinase activity,14,15 whereas EGF stimulates KSR activity in COS-7 cells,13,21 indicating cell-type specific upstream activation. Additional investigation is required to resolve this dilemma.

Mechanisms of KSR Activation Cellular KSR exists in a multiprotein signaling complex of up to 1000 kDa consisting of components of the MAPK pathway (Raf-1/MEK/MAPK), heat-shock proteins (HSP90/HSP70/ HSP68/p50CDC37), Cdc25C-activated kinase (C-TAK1), 14-3-3 proteins, the βγ subunits of G-proteins and several other yet unidentified proteins (p33/p34/p36/p60).10 Raf-1 and MAPK

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interaction with KSR appear transient and are enhanced by growth factor stimulation or overexpression of activated Ras. Alternately, MEK, C-TAK1 and 14-3-3 proteins are constitutively bound to KSR. The role of heat-shock proteins in KSR regulation is still uncertain but they may stabilize KSR molecules or the whole complex, since treatment of cells with geldanamycin, a known inhibitor of HSP90, reduces the KSR half-life.10 Alternately, 14-3-3 proteins may help sequester KSR within the cytoplasm, in unstimulated cells (see below).22 It is also not clear whether all complexed proteins interact directly with KSR, although MEK, MAPK, 14-3-3 and the γ subunit of G-proteins do so in the yeast two-hybrid system. Thus, besides its kinase function, KSR should be considered as a scaffold protein that efficiently coordinates different components of a signaling module.9,10,23 The scaffolding and enzymatic (kinase) functions of KSR may be more or less relevant depending on cellular context, or both KSR functions may be necessary for full signaling. For example, a potential physiological role for KSR is to bring MEK to the plasma membrane10 where it can be activated by Raf-1 (scaffold function) while for Raf-1 to become active, KSR kinase activity may be required.5,13,15 Consistent with this hypothesis of KSR as a dual function signal transducer, all mutations that blocked MEK1 binding also prevented KSR-enhanced MAPK signaling and differentiation in PC-12 cells.19 In our laboratories, KSR kinase activity is increased following cellular treatment with EGF or TNF, or by increasing ceramide levels.5, 13, 15 Further, ceramide treatment of isolated KSR enhanced its kinase activity in vitro.5 In fact, ceramide may bind directly to KSR.5 A number of proteins have now been reported to bind ceramide including PKCζ,24 Raf-1,25 cathepsin D,26 and even antibodies have been isolated that specifically detect ceramide.27 PKCζ,24 Raf-1,25 and KSR5 all contain a Cys-rich domain (CRD) with high homology to the DAG/phorbol ester binding domain of conventional PKC isoforms [CxxCxnCxxC motifs (where x = any amino acid)]. The DAG/phorbol ester-binding motif of PKC also requires several surrounding hydrophobic amino acids to form the hydrophobic pocket that binds the lipid.28 Since ceramide and DAG exhibit high structural similarity,29 and since the CRDs of Raf-1 and KSR are highly homologous to that of PKC, the Raf-1 and KSR CRDs were suggested to represent ceramide binding sites.28 Recent investigations by Grassme et al30 addressed this issue directly, finding that [14C16]ceramide bound directly to fusion proteins comprised of the CA3 domain of KSR or the CR1 domain of Raf-1. In contrast, other radiolabeled lipids or dihydroceramide did not bind these domains. Further, binding was displaced by an excess of unlabelled C16 ceramide, but not by unlabeled SM, dihydroceramide or arachidonic acid, providing additional evidence for specificity.

Mechanisms of Raf-1 Activation via KSR The Raf-1 Ser/Thr kinase is a central component in many signaling pathways, functioning to connect upstream Tyr kinases and Ras to downstream Ser/Thr kinases.31-33 Upon activation, Raf-1 phosphorylates and activates MEK1, resulting in propagation of the signal to MAPK. MAPK phosphorylates several regulatory proteins in the cytoplasm and nucleus to alter transcription and translation.34,35 Raf-1 displays three distinctive domains shown in Fig. 1: a Cys-rich amino terminal domain (CR1), a Ser/Thr-rich domain (CR2), and a carboxyl-terminal kinase domain (CR3).36-39 Raf1 activation in vivo is initiated when cytoplasmic Raf-1, through the Ras binding domain in CR1, couples to active GTP-bound Ras and is recruited to the plasma membrane. After binding Ras, Raf-1 becomes activated by a complex and still incompletely understood mechanism involving phosphorylation and interaction with membrane lipids.40-45 Confirmation of an essential role for phosphorylation in regulating Raf-1 kinase function derives from the identification and mutation of critical in vitro and in vivo phosphorylation sites. In resting cells, Raf1 is phosphorylated exclusively on Ser (Ser43, Ser621, Ser624) and Thr residues (Thr268). Ser and Thr phosphorylation can be further increased on these and other sites (Ser259, Ser388/389, Ser497/499, Thr269) by mitogenic stimuli and cytokines, and is often accompanied by Tyr phosphorylation at

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positions 340 and 341.5,7,14,37,42,46-56 The relative contribution of these sites to Raf-1 activation is cell type- and stimulus-specific. Regarding the interaction with membranes, recent studies by Morrison and co-workers suggest that ceramide is the lipid involved, either directly or indirectly, in Raf-1 activation.57 Since its discovery as a component of the Ras pathway, much effort has been invested to elucidate the precise role of KSR/CAPK in regulating the Raf/MAPK cascade. Yao and colleagues first demonstrated reconstitution of Raf-1/MEK signaling by CAPK isolated from TNFtreated HL-60 cells using an in vitro kinase assay, and defined Thr269 as a potential direct phosphorylation site.7 Using the recently established two-stage in vitro assay for KSR kinase activity, in which KSR contacts only Raf-1, we defined EGF as a potent activator of KSR kinase activity.13 In vitro, KSR immunopurified from EGF-treated COS7 cells specifically phosphorylated Raf-1 on Thr residues during the first stage of the two-stage kinase assay, while KSR isolated from resting cells was inactive. Using purified wild type and mutant Raf-1 proteins, we defined Thr269 as the major Raf-1 site phosphorylated by KSR in vitro, and showed that phosphorylation of this site was essential for Raf-1 activation by KSR. KSR acted via trans-phosphorylation, not by increasing Raf-1 autophosphorylation, as kinase inactive Raf1(K375M) served as an equally effective KSR substrate. In vivo, low physiologic doses of EGF (0.001-0.1 ng/ml) stimulated KSR activation, and induced Thr269 phosphorylation and activation of Raf-1. However, low dose EGF did not induce Ser or Tyr phosphorylation of Raf-1. High dose EGF (10-100 ng/ml) induced no additional Thr269 phosphorylation but rather increased Raf-1 phosphorylation on Ser residues and Tyr340/341. A Raf-1 mutant with Val substituted for Thr269 was unresponsive to low-dose EGF, but was Ser and Tyr340/341 phosphorylated and partially activated at high EGF doses. Raf-1(T269A) could still be activated by phorbol ester stimulation indicating that substitution of this amino acid did not globally inactivate Raf kinase. These studies provided evidence that Thr269 was the major Raf-1 site phosphorylated by KSR. Further, phosphorylation of this site appeared essential for Raf-1 activation by KSR in vitro and for optimal Raf-1 activation in response to physiological EGF stimulation in vivo. These studies also emphasized that KSR was essentially inactive in some unstimulated cells, perhaps, as stated above, accounting for the inability of some investigators to observe activity in KSR kinase assays. Another issue regarding the role of KSR in Raf-1 activation focuses on the role of ceramide in the Raf-1 activation process. Several groups have shown that cell-permeable ceramide, SMasegenerated endogenous ceramide, or TNF increased MAPK activity through Raf-1 activation.7,14,25,28,58 However, others have shown TNF activation of MAPK is Raf-1-independent,59 or even that TNF attenuated MAPK activation.60 The original observation that ceramide induced activation of KSR in intact cells, was supported by the observation that short-chain and long-chain ceramides increased the activity of KSR in vitro using the original one stage kinase assay. Yan and Polk recently provided additional support for the concept that KSR is a CAPK using YAMC intestinal cells, in which TNF, short chain ceramide analogs or SMase induced rapid KSR activation.14 Further, long-chain ceramide directly activated KSR immunopurified from these cells (Yan and Polk, unpublished data). Although definitive information is not available as to the mechanism of ceramide activation, it likely occurs through the conserved KSR CRD, CA3.30 Additional investigation is necessary to determine in which cells this ceramide-dependent mechanism is operative. The Raf-1 CRD, CR1, may also be involved in ceramide signaling through this cascade, as Morrison and co-workers recently showed it was essential for ceramide-induced Raf-1 activation.57 The effect of EGF, however, to activate KSR, is likely to occur by an alternative mechanism, as EGF does not increase ceramide levels.

Subcellular Localization KSR is largely localized to the cytoplasmic compartment in unstimulated cells but upon serum stimulation61 or in the presence of activated Ras9 it translocates to the plasma membrane in a complex with Raf-1, and increases Raf-1 kinase activity. The Cys-rich CA3 domain

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appears essential for membrane localization of KSR and the mechanism might involve interaction with lipid.28 How KSR translocates to the plasma membrane remains to be clarified, although it may involve, in part, the de-phosphorylation of Ser392. It is unlikely that it will involve binding to activated Ras, as with Raf-1, since KSR does not directly interact with Ras in all experiments performed so far.61

Role of KSR Phosphorylation Sites KSR phosphorylation may be a mechanism for regulation of KSR function and/or subcellular localization. KSR is highly phosphorylated in vivo on at least 12 Ser/Thr residues.8,11 Three were found to be MAPK phosphorylation sites (Thr260, Thr274 and Ser443), and were Ras- and PDGF-inducible. The functional consequences of KSR phosphorylation by MAPK remains obscure since mutation of the corresponding residues has no affect on the ability of KSR to augment Ras signaling in the Xenopus oocyte meiotic maturation assay.8 Alternately, mutation of Ser297 and Ser392 to Ala in KSR abolishes 14-3-3 binding, confirming these are 143-3 binding sites. Recent studies provide evidence that C-TAK1 phosphorylates Ser297 and Ser392, resulting in 14-3-3 protein binding and KSR sequestration in the cytoplasm of resting cells.22 The complete characterization of the kinases responsible for KSR phosphorylation and their modulation will, undoubtedly, shed new light on regulation of KSR function.

Biological Effects of KSR In contrast to the emerging body of work relating to the signaling pathway activated by KSR, there is little information regarding its biologic function. The available information suggests that KSR, as a regulator of the Ras/Raf pathway, may have essential roles in such programs as proliferation, differentiation and apoptosis.4 KSR, via Raf, has been reported to be required for Xenopus laevis oocyte maturation, cellular transformation and D. melanogaster eye development.5, 9, 16, 61 For Xenopus oocyte maturation, KSR overexpression was ineffective alone in inducing GVBD, while enhancing the effect of activated Ras. Nevertheless, overexpression of KSR along with 14-3-3 proteins induced Xenopus oocyte maturation, which could be blocked by dominant-negative Raf-1.61 Alternately, isolated expression of the kinase domain of KSR blocked Xenopus oocyte maturation and eye development in Drosophila.16 Similarly, overexpression of full length KSR in D. melanogaster blocked the R7 photoreceptor formation.8 These studies suggest that all studies involving expression of KSR constructs must be evaluated with some reserve, as the biological outcome may largely dependent on the level of expression.8 Loss-of-function KSR mutants have been useful in the assessment of the biologic actions of KSR. In C. elegans, strong loss of KSR function blocked induction of the multiple vulvae phenotype characteristic of the activated Ras pathway.1,2 Loss-of-function of a C. elegans protein phosphatase 2A (sur-6) appeared to act in concert with loss of function KSR in regulating this event.62 Interestingly, these mutations did not affect normal vulval development in animals expressing wild-type Ras. Expression of a dominant-negative KSR, in which two conserved Asps normally involved in coordination of ATP within the binding cleft were mutated to Ala, has also been useful in evaluating the role of KSR in differentiation. Dominant negative KSR blocked TNF-induced growth arrest and differentiation in intestinal epithelial cells,63,64 an event signaled through the Raf-1/MEK-1/ERK MAP kinase pathway.65 The decision to undergo programmed cell death or survive may also be impacted by KSR signaling. A wealth of genetic, biologic, and pharmacological data support ceramide as a principal mediator of the apoptotic response.66 Evidence suggests that the responses to ceramide may be cell type- and stimulus-specific. Recent studies show that the availability of KSR is determinant in TNF-induced apoptotic death of mouse colon cells. In wild type colon cells, TNF, which stimulates ceramide elevation, and exogenous ceramide, induced a differentiation program. However, either downregulation of KSR protein by anti-sense cDNA expression, or the expression of a dominant-negative KSR construct,5 converted these cells to undergo

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apoptosis.15 This appeared to result from the absence of coordinated activation of the ERK and NFκB anti-apoptotic pathways through Raf-1, since Raf-1 activation was needed for stimulation of both pathways, and simultaneous pharmacological or molecular inhibition of both pathways was necessary to recapitulate the effect of inhibiting KSR.15 Cell type may play an important role in KSR-regulated anti-apoptosis. While dominant-negative KSR or anti-sense inactivation of KSR in YAMC and mouse small intestinal epithelial (MSIE) cells inhibited both NFκB and ERK/MAPK activation,15 reducing KSR kinase activity/expression had no effect on either TNF-activated pathway in HeLa or A431 cells.

Conclusions and Remaining Questions Although there is substantial disagreement as to the mechanism of KSR signaling, in our opinion the best fit for all the data is that KSR is a dual function signal transducer, acting both as a scaffolding protein and a signaling kinase initiating Raf-1/MEK-1/ERK MAPK activation by the EGF and TNF receptors. The results further indicate that the requirement of KSR kinase activity for initiation of these pathways is cell context dependent. Significant questions remain regarding the biological role of KSR, the mechanism of activation by ceramide, and the role of protein phosphorylation in KSR function. To date, only two of the kinases responsible for phosphorylation of five of the reported twelve phosphorylation sites have been identified. It is likely that the identification of these proteins and their regulation will lead to a greater understanding of the mechanisms regulating KSR function.

References 1. Kornfeld K, Hom DB, Horvitz HR. The ksr-1 gene encodes a novel protein kinase involved in Ras-mediated signaling in C. elegans. Cell 1995; 83:903-913. 2. Sundaram M, Han M. The C. elegans ksr-1 gene encodes a novel Raf-related kinase involved in Ras-mediated signal transduction. Cell 1995; 83:889-901. 3. Therrien M, Chang HC, Solomon NM et al. KSR, a novel protein kinase required for RAS signal transduction. Cell 1995; 83:879-888. 4. Downward J. KSR: A novel player in the Ras pathway. Cell 1995; 83:831-834. 5. Zhang Y, Yao B, Delikat S et al. Kinase supressor of Ras is ceramide-activated protein kinase. Cell 1997; 89:63-72. 6. Joseph CK, Byun H-S, Bittman R et al. Substrate recognition by ceramide-activated protein kinase: Evidence that kinase activity is proline-directed. J Biol Chem 1993; 268:20002-20006. 7. Yao B, Zhang Y, Delikat S et al. Phosphorylation of Raf by ceramide-activated protein kinase. Nature 1995; 378:307-310. 8. Cacace AM, Michaud NR, Therrien M et al. Identification of constitutive and Ras-inducible phosphorylation sites of KSR: Implications for 14-3-3 binding, mitogen-activated protein kinase binding, and KSR overexpression. Mol Cell Biol 1999; 19:229-240. 9. Michaud NR, Therrien M, Cacace A et al. KSR stimulates Raf-1 activity in a kinase-independent manner. Proc Natl Acad Sci USA 1997; 94:12792-12796. 10. Stewart S, Sundaram M, Zhang Y et al. Kinase suppressor of Ras forms multiprotein signaling complex and modulates MEK localization. Mol Cell Biol 1999; 19:5523-5534. 11. Volle DJ, Fulton JA, Chaika OV et al. Phosphorylation of the kinase suppressor of Ras by associated kinases. Biochemistry 1999; 38:5130-5137. 12. Morrison D. KSR: A MAPK scaffold of the Ras pathway? J Cell Sci 2001; 114:1609-1612. 13. Xing HR, Lozano J, Kolesnick R. Epidermal growth factor treatment enhances the kinase activity of kinase suppressor of Ras. J Biol Chem 2000; 275:17276-17280. 14. Yan F, Polk DB. Kinase suppressor of Ras is necessary for tumor necrosis factor α activation of extracellular signal-regulated kinase/mitogen-activated protein kinase in intestinal epithelial cells. Cancer Res 2001; 61:963-969. 15. Yan F, John SK, Polk DB. Kinase suppressor of Ras determines survival of intestinal cells exposed to tumor necrosis factor. Cancer Res 2001; 61:8668-8675. 16. Therrien M, Michaud NR, Rubin GM et al. KSR modulates signal propagation within the MAPK cascade. Genes Dev 1996; 10:2684-2695. 17. Sugimoto T, Stewart S, Han M et al. The kinase suppressor of Ras (KSR) modulates growth factor and Ras signaling by uncoupling Elk-1 phosphorylation from MAP kinase activation. Embo J 1998; 17(6):1717-27.

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18. Xu B-E, English JM, Wilsbacher JL et al. WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II. J Biol Chem 2000; 275(22):16795-16801. 19. Muller J, Cacace AM, Lyons WE et al. Identification of B-KSR1, a novel brain-specific isoform of KSR1 that functions in neuronal signaling. Mol Cell Biol 2000; 20(15):5529-5539. 20. Basu S, Bayoumy S, Zhang Y et al. BAD enables ceramide to signal apoptosis via Ras and Raf-1. J Biol Chem 1998; 273:30419-30426. 21. Xing HR, Kolesnick R. Kinase suppressor of Ras signals through Thr269 of c-Raf-1. J Biol Chem 2001; 276:9733-9741. 22. Muller J, Ory S, Copeland T et al. C-TAK1 regulates Ras signaling by phosphorylating the MAPK scaffold, KSR1. Mol Cell 2001; 8(5):983-993. 23. Yu W, Fantl WJ, Harrowe G et al. Regulation of the MAP kinase pathway by mammalian Ksr through direct interaction with MEK and ERK. Curr Biol 1997; 8:56-64. 24. Muller G, Ayoub M, Storz P et al. PKC ζ is a molecular switch in signal transduction of TNF-α, bifunctionally regulated by ceramide and arachidonic acid. EMBO J 1995; 14:1961-1969. 25. Huwiler A, Brunner J, Hummel R et al. Ceramide-binding and activation defines protein kinase cRaf as a ceramide-activated protein kinase. Proc Natl Acad Sci USA 1996; 93:6959-6953. 26. Heinrich M, Wickel M, Schneider-Brachert W et al. Cathepsin D targeted by acid sphingomyelinasederived ceramide. Embo J 1999; 18(19):5252-5263. 27. Vieira LM, Sampaio EP, Nery JA et al. Immunological status of ENL (erythema nodosum leprosum) patients: Its relationship to bacterial load and levels of circulating IL-2R. Rev Inst Med Trop Sao Paulo 1996; 38(2):103-111. 28. van Blitterswijk WJ. Hypothesis: Ceramide conditionally activates atypical protein kinase C, Raf-1 and KSR through binding to their cysteine-rich domains. Biochem J 1998; 331:679-680. 29. Pascher I, Lundmark M, Nyholm PG et al. Crystal structures of membrane lipids. Biochim Biophys Acta 1992; 1113(3-4):339-373. 30. Grassme H, Schwarz S, Gulbins E. Molecular mechanisms of ceramide-mediated CD95 clustering. Biochem Biophys Res Commun 2001; 284(4):1016-1030. 31. Marshall CJ. MAP kinase kinase kinase, MAP kinase kinase and MAP kinase. Curr Opin Genet Dev 1994; 4(1):82-89. 32. Moodie SA, Wolfman A. The 3Rs of life: Ras, Raf and growth regulation. Trends Genet 1994; 10(2):44-48. 33. Morrison DK, Cutler RE Jr. The complexity of Raf-1 regulation. Curr Opin Cell Biol 1997; 9:174-179. 34. Crews CM, Erikson RL. Extracellular signals and reversible protein phosphorylation: What to Mek of it all. Cell 1993; 74(2):215-217. 35. Seger R, Krebs EG. The MAPK signaling cascade. Faseb J 1995; 9(9):726-735. 36. Ghosh S, Xie WQ, Quest AF et al. The cysteine-rich region of raf-1 kinase contains zinc, translocates to liposomes, and is adjacent to a segment that binds GTP-ras. J Biol Chem 1994; 269(13):10000-10007. 37. Morrison DK, Heidecker G, Rapp UR et al. Identification of the major phosphorylation sites of the Raf-1 kinase. J Biol Chem 1993; 268:17309-17316. 38. Williams NG, Roberts TM. Signal transduction pathways involving the Raf proto-oncogene. Cancer Metastasis Rev 1994; 13(1):105-116. 39. Williams NG, Roberts TM, Li P. Both p21ras and pp60v-src are required, but neither alone is sufficient, to activate the Raf-1 kinase. Proc Natl Acad Sci USA 1992; 89(7):2922-2926. 40. Chuang E, Barnard D, Hettich L et al. Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues. Mol Cell Biol 1994; 14(8):5318-5325. 41. Leevers SJ, Paterson HF, Marshall CJ. Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature 1994; 369(6479):411-414. 42. Magnuson NS, Beck T, Vahidi H et al. The Raf-1 serine/threonine protein kinase. Semin Cancer Biol 1994; 5(4):247-253. 43. Marais R, Light Y, Mason C et al. Requirement of Ras-GTP-Raf complexes for activation of Raf1 by protein kinase C. Science 1998; 280(5360):109-112. 44. Marais R, Light Y, Paterson HF et al. Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation. EMBO J 1995; 14:3136-3145. 45. Winkler DG, Cutler RE Jr, Drugan JK et al. Identification of residues in the cysteine-rich domain of Raf-1 that control Ras binding and Raf-1 activity. J Biol Chem 1998; 273(34):21578-21584. 46. Carroll MP, May WS. Protein kinase C-mediated serine phosphorylation directly activates Raf-1 in murine hematopoietic cells. J Biol Chem 1994; 269(2):1249-1256.

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47. Cutler RE Jr, Stephens RM, Saracino MR et al. Autoregulation of the Raf-1 serine/threonine kinase. Proc Natl Acad Sci USA 1998; 95(16):9214-9219. 48. Dent P, Jelinek T, Morrison DK et al. Reversal of Raf-1 activation by purified and membraneassociated protein phosphatases. Science 1995; 268:1902-1906. 49. Dent P, Reardon DB, Morrison DK et al. Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro [published erratum appears in Mol Cell Biol 1995 Sep;15(9):5203]. Mol Cell Biol 1995; 15(8):4125-4135. 50. Diaz B, Barnard D, Filson A et al. Phosphorylation of Raf-1 serine 338-serine 339 is an essential regulatory event for Ras-depentdent activation and biological signaling. Mol Cell Biol 1997; 17:4509-4516. 51. Fabian JR, Daar IO, Morrison DK. Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase. Mol Cell Biol 1993; 13(11):7170-7179. 52. Izumi T, Tamemoto H, Nagao M et al. Insulin and platelet-derived growth factor stimulate phosphorylation of the c-raf product at serine and threonine residues in intact cells. J Biol Chem 1991; 266(12):7933-7939. 53. Kaplan DR, Morrison DK, Wong G et al. PDGF β-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex. Cell 1990; 61(1):125-33. 54. Kolch W, Heidecker G, Kochs G et al. Protein kinase C alpha activates RAF-1 by direct phosphorylation. Nature 1993; 364(6434):249-252. 55. Morrison DK, Kaplan DR, Rapp U et al. Signal transduction from membrane to cytoplasm: Growth factors and membrane-bound oncogene products increase Raf-1 phosphorylation and associated protein kinase activity. Proc Natl Acad Sci USA 1988; 85(23):8855-8859. 56. Zimmermann S, Moelling K. Phosphorylation and regulation of Raf by Akt (protein kinase B). Science 1999; 286(5445):1741-1744. 57. Zhou M, Horita DA, Waugh DS et al. Solution structure and functional analysis of the cysteinerich C1 domain of kinase suppressor of Ras (KSR). J Mol Biol 2002; 315(3):435-446. 58. Kalb A, Bluethmann H, Moore MW et al. Tumor necrosis factor receptors (Tnfr) in mouse fibroblasts deficient in Tnfr1 or Tnfr2 are signaling competent and activate the mitogen-activated protein kinase pathway with differential kinetics. J Biol Chem 1996; 271(45):28097-28104. 59. Winston BW, Lange-Carter CA, Gardner AM et al. Tumor necrosis factor a rapidly activates the mitogen-activated protein kinase (MAPK) cascade in a MAPK kinase kinase-dependent, c-Raf-1independent fashion in mouse macrophages. Proc Natl Acad Sci USA 1995; 92:1614-1618. 60. Muller G, Storz P, Bourteele S et al. Regulation of Raf-1 kinase by TNF via its second messenger ceramide and cross-talk with mitogenic signaling. EMBO J 1998; 17:732-742. 61. Xing H, Kornfeld K, Muslin AJ. The protein kinase KSR interacts with 14-3-3 protein and Raf. Curr Biol 1997; 7:294-300. 62. Sieburth DS, Sundaram M, Howard RM et al. A PP2A regulatory subunit positively regulates Ras-mediated signaling during Caenorhabditis elegans vulval induction. Genes Dev 1999; 13(19):2562-2569. 63. Kaiser GC, Polk DB. Tumor necrosis factor α regulates proliferation in a mouse intestinal cell line. Gastroenterology 1997; 112:1231-1240. 64. Ziambaras T, Rubin DC, Perlmutter DH. Regulation of sucrase-isomaltase gene expression in human intestinal epithelial cells by inflammatory cytokines. J Biol Chem 1996; 271:1237-1242. 65. Kaiser GC, Yan F, Polk DB. Conversion of TNFa from antiproliferative to proliferative ligand in mouse intestinal epithelial cells by regulating mitogen-activated protein kinase. Exp Cell Res 1999; 249:349-358. 66. Mathias S, Pena LA, Kolesnick RN. Signal transduction of stress via ceramide. Biochem J 1998; 335(Pt 3):465-480.

CHAPTER 8

Ceramide in Apoptosis: The FAN Thesis, Not a Fantasy Bruno Ségui, Olivier Cuvillier, Sophie Malagarie-Cazenave, Sophie Lévêque, Valérie Gouazé, Nathalie Andrieu-Abadie, and Thierry Levade

Abstract

T

he sphingolipid ceramide has recently been proposed as a new apoptotic cell death mediator. Here the role of the FAN (Factor Associated with Neutral SMase activation) protein in apoptosis signal transduction is discussed. FAN was initially described to associate with the TNF receptor mediating the activation of neutral sphingomyelinase (N-SMase), an enzyme releasing ceramide from the membrane phospholipid sphingomyelin (SM). We now suggest that FAN can mediate the apoptotic response of receptors such as TNF-R1 and CD40, and mediate other effects. As a regulator of ceramide formation, FAN thus appears as a new player in apoptosis signaling, highlighting the potential function of the sphingolipid second messengers in signal transduction.

Apoptosis, Ceramide and SMases Apoptosis is a sophisticated, tightly regulated process allowing metazoan organisms to eliminate unwanted or damaged cells.1 Excessive or insufficient apoptosis is likely to result in the development of either degenerative or malignant diseases. Attempts to decipher the signaling pathways that control apoptosis have led to the identification of two main routes, the death receptor pathway and the mitochondrial pathway.2 The former is launched by oligomerization of so-called death receptors (i.e., members of the TNF receptor superfamily), and involves autocatalytic processing of caspase-8, therefore leading to executioner caspase cleavage and cell dismantling. The latter implicates the release of apoptogenic factors from mitochondria including cytochrome c, which in turn can initiate a caspase cascade involving downstream executioner caspases. Certain sphingolipids such as ceramide, sphingosine and sphingosine 1-phosphate also appear to fit within these apoptosis signaling schemes.3-5 Although ceramide was proposed to act as a second messenger in TNF-induced apoptosis for the first time in 1993,6 little has been learned since then about the mechanism of generation, mode of action, and relative contribution to apoptosis of this sphingolipid. Direct molecular targets of ceramide and upstream pathways that govern and regulate ceramide production remain to be identified and/or characterized. These questions are illustrated by the current controversy regarding the SMase(s) implicated in the generation of the proapoptotic ceramide.7 Ceramide Signaling, edited by Anthony H. Futerman. ©2002 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Based on previous work from our laboratory and others (see ref. 7), we have proposed that a neutral pH optimum SMase is responsible for the production of a bioactive ceramide. We now suggest that under certain situations, the adapter protein FAN can modulate ceramide generation and subsequent apoptosis.

FAN: A Link Between TNF-R1 and Ceramide Production The pleiotropic effects of TNF are initiated by oligomerization (upon or prior to binding of the cytokine) of specific plasma membrane receptors, TNF-R1 (also named CD120a or p55 TNFR) and TNF-R2 (CD120b or p75 TNFR).8 Most of the TNF effects, including its antitumoral activity, are thought to be mediated by TNF-R1 and implicate different domains of its intracytoplasmic region.8,9 Elegant structure-function analyses by Krönke and co-workers have demonstrated that distinct regions of TNF-R1 can trigger the formation of ceramide from SM.10 First, an 80 amino acid C-terminal domain of the receptor, called the ‘death domain’, signals activation of an acidic pH optimum, possibly endosomal, SMase through the recruitment of the adapter proteins TRADD and FADD.11,12 Of note, this acid sphingomyelinase (A-SMase) activity does not seem to be influenced by the FADD-interacting protein, procaspase-8.11 Second, activation of a neutral, plasma membrane-associated, SMase is promoted by TNF via binding of the adapter protein FAN to a short TNF-R1 domain called NSD (Neutral Sphingomyelinase Domain) that is located N-terminally with respect to the ‘death domain’.13 FAN is a 917 amino acid protein belonging to the WD-40 family14 that is characterized by the presence of motifs facilitating protein-protein interactions and includes proteins serving regulatory functions in signal transduction, apoptosis and vesicle traffic (Fig. 1).15 Binding of FAN to TNF-R1 occurs via its C-terminal region that contains the WD repeats. Abrogation of FAN function by overexpression of an N-terminally-deleted FAN mutant (which exhibits a dominant-negative effect) or gene knockout has clearly shown that FAN is required for ceramide production by N-SMase.14,16

FAN and TNF-R1- and CD40-Mediated Apoptosis Because we have shown that TNF-stimulated SM breakdown was not altered in fibroblasts derived from Niemann-Pick disease,17 i.e., cells having a genetic deficiency of A-SMase activity, we investigated whether FAN, as a regulator of the putative proapoptotic messenger ceramide, could modulate TNF-induced apoptosis. Stable expression of the truncated, dominant-negative form of FAN in human transformed fibroblasts resulted in i) abrogation of TNFinduced SM hydrolysis (Fig. 2), ii) suppression of subsequent ceramide formation, and iii) inhibition of cell death.18 Further analysis of these effects indicated that in fibroblasts expressing the truncated FAN i) the release of cytochrome c into the cytosol was impaired, ii) the activity of proteases acting on the DEVD substrate motif was considerably decreased as compared to empty vector-transfected cells, iii) the proteolytic cleavage of caspase-3, -7, and -8 was inhibited. Evidence that FAN is implicated in the regulation of TNF-R1-mediated apoptosis was further provided by the observation that fibroblasts from FAN-deficient mice were also partially resistant to TNF-induced apoptosis.18 Of note, cytotoxicity of both human cells overexpressing the dominant-negative form of FAN and murine FAN-deficient cells was overcome by treatment with short-chain ceramide. This suggests that abrogation of FAN functions indeed impairs TNF-R1 cytotoxic signaling by blocking ceramide generation. In addition, hydrolysis of SM and induction of cell death by daunorubicin, an anticancer agent known to trigger apoptosis and to activate the SM-ceramide pathway,19 proceeded normally in FANmutated cells (Fig. 2), indicating that FAN is not a general regulator of N-SMase activation and apoptosis.

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Figure 1. Structure of FAN. Abbreviation : BEACH, BEige And Chediak-Higashi.

These findings strongly favor the idea that other domains of the TNF-R1, besides the well known ‘death domain’, are involved in TNF-induced apoptosis. So far, the only known region that was believed to be required for this activity was the ‘death domain’, extending from amino acid 326 to amino acid 413.20 Indeed, deletions of this region abolished the cytotoxic action of TNF on murine L929 cells. Nevertheless, ablation of the cytoplasmic sequence spanning from amino acid 212 to amino acid 326, but not a truncation of amino acid 212-308, led to a reduction of ~50% of the TNF-induced cell death, suggesting that a more membrane-proximal domain than the ‘death domain’ was also implicated in the cytotoxic effect of TNF.20 According to Goeddel’s studies, this domain covers the NSD region (amino acid 309-319), which binds to the FAN protein.20 Although our findings demonstrate that the apoptotic effect of TNF requires a signal emanating from a membrane-proximal region of the TNF-R1 transduced by FAN, they still raise a number of questions regarding the mechanism of action of FAN. Specifically, the mode of activation by FAN of a N-SMase remains to be elucidated. Likewise, how is the FANmediated pathway(s) in TNF-R1-induced apoptosis connected to the other well-established components of the programmed cell death machinery? Because CD40 is a member of the TNF receptor superfamily,21,22 which includes the p75 Nerve growth factor receptor and FasR/CD95, all known to trigger the production of ceramide,5 we examined whether CD40 engagement could promote ceramide formation and whether FAN could be involved in CD40 responses. These studies were carried out on cultured B lymphoid cells and fibroblasts, as CD40 is known to be expressed in these cell types. Whereas CD40 ligation promotes mature B cell proliferation, it induces apoptosis in transformed cells of both hematopoietic and non-hematopoietic origin.23 We have determined that cell treatment with an agonistic anti-CD40 antibody or with recombinant CD40 ligand triggers SM hydrolysis (Fig. 2) and ceramide production through the stimulation of a N-SMase.24 In addition, exposure of B cells to exogenous ceramides or treatments that lead to increased intracellular ceramide concentrations mimicked the inhibitory effect of anti-CD40 on thymidine incorporation, suggesting that at least some CD40 responses could be signaled by ceramide. On transformed fibroblasts, CD40 ligation induced apoptosis, and this effect was inhibited by overexpression of a dominant-negative form of FAN.24

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Figure 2. Effect of FAN on the SM content upon TNF, CD40 ligand, and daunorubicin treatments. Human fibroblasts stably transfected with the empty vector pcDNA3 or a vector encoding the truncated FAN (∆FAN) were labelled with [3H]choline and then treated for the indicated times with 50ng/ml recombinant human TNF (left), 5µg/ml of soluble CD40 ligand (center) or 1µM daunorubicin (right). The [3H]cholinelabelled SM content was then determined.

These data indicated that CD40-mediated apoptosis could involve FAN. As a matter of fact, expression of the truncated FAN inhibited CD40-induced SM degradation and ceramide generation. Furthermore, co-immunoprecipitation experiments showed that CD40, like the TNF-R1, interacts with FAN.24 Although the intracellular domain of CD40 shares only limited homology with that of TNF-R1, it harbors a polypeptide sequence (QETLH) quite homologous to that (EDSAH) of the NSD of TNF-R1, suggesting possible functional similarities between these two receptors.

FAN and other Effects Besides its involvement in apoptosis triggered by TNF-R1 and CD40 seen on cultured cells, FAN, which was originally described to bind TNF-R1, seems to be also involved in other biological effects of these two receptors and of other receptors as well (see Table 1). Regarding TNF effects, activation of c-Raf-1 kinase has recently been proposed to occur through two cooperative cytoplasmic domains of TNF-R1, one of which is the NSD.25 In addition, we have recently obtained evidence that IL-6 secretion stimulated by TNF (as well as by CD40 ligation) on fibroblasts is partially dependent on FAN (unpublished data). Finally, TNF-stimulated actin reorganization in macrophages is dependent on the FAN-binding region of TNF-R1.26 However, activation of ERK1/2 (p44/p42 MAPK), phosphorylation of cytosolic phospholipase A2 and subsequent arachidonic acid release, events previously ascribed to the FANmediated N-SMase signaling pathway,14,27 occur normally in the absence of FAN,16,18,28 consistent with the idea that these responses are mediated by the ‘death domain’.29 In addition, activation of p38 MAPK also seems to be independent of NSD and FAN (unpublished data). Finally, TNF-induced cell surface expression of the adhesion molecule CD54 appears unaffected

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Table 1. Biological responses regulated by FAN. Listed are the effects induced by TNF-R1, CD40 or CB1 receptor stimulation that are (+) or not (-) regulated by FAN Effect

N-SMase activation SM hydrolysis Ceramide generation Caspase activation ERK 1/2 activation NF-κB nuclear translocation p38 activation cPLA2 activation c-Raf-1 CD54 expression IL-6 secretion Actin polymerization Apoptosis

TNF-R1

Receptor CD40

CB1

+ + + + + + + +

+ + + ND ND ND ND ND ND ND + ND +

ND + + ND ND ND ND ND ND ND ND ND ND

Abbreviations: N-SMase, neutral sphingomyellinase; ERK, extracellular signal-regulated kinase; cPLA2, cytosolic phospholipase A2; IL-6, interleukin-6; ND, not determined (or not applicable).

by abrogation of FAN function.18 Thus, only a subset of TNF responses that are presumably not (exclusively) mediated by the ‘death domain’, implicate FAN and possibly ceramide. A G-protein coupled receptor, the CB1 receptor for cannabinoids, has recently been described to signal some of its effects, notably regulation of brain energy metabolism, via the early production of ceramide30 (and see Chapter 13). This generation, which indeed occurred within minutes and implied a SMase, does not involve Gi/0 proteins and seems to be regulated by FAN.31 The DCLHK stretch in the CB1 receptor could potentially be responsible for the binding of FAN to the receptor. In contrast to the CB1-elicited late and sustained ceramide production that is involved in cannabinoid-induced apoptosis of glioma cells, acute FANregulated ceramide generation appears to mediate the stimulation of glucose consumption and ketogenesis, important functions of astrocytes.30 The in vivo physiological significance of FAN (and of ceramide produced by a N-SMase) can be determined by examining the phenotype of FAN-deficient mice. These animals do not show gross abnormalities in general health and reproduction, but exhibit a defective repair process of the skin permeability barrier.16 This is an interesting observation because i) ceramide is known to be a quantitatively and qualitatively important constituent of the epidermis, where it contributes to the hydrophobic coat of the stratum corneum (preventing water loss);32 and ii) TNF-R1 promotes cutaneous barrier repair via stimulation of SMases.33 It is possible that an insufficient production of ceramide after disruption of the permeability barrier accounts for a delayed repair. In this scenario, FAN would represent, via N-SMase and ceramide, an important component in the signaling pathways that contribute to epidermal barrier homeostasis and possibly wound healing.

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Figure 3. Role of FAN in TNF-R1-mediated responses. Abbreviations : Cyt c, cytochrome c ; DD, Death Domain ; FADD, Fas-Associated Death Domain protein; FAN, Factor Associated with Neutral SMase activation ; NSD, Neutral Sphingomyelinase Domain; N-SMase, neutral sphingomyelinase; SM, sphingomyelin ; TRADD, TNF-R Associated Death Domain protein.

Conclusion: Is Ceramide in Apoptosis Still a FANtasy? Despite numerous observations on many different cell systems, the actual contribution of ceramide in the general signaling network that leads to apoptotic cell death has been debated.34-37 Recent experimental evidence has, however, been obtained that the adapter protein FAN fulfills an important role in some receptor-mediated biological effects, including apoptosis. As far as we know, the only definite action of FAN is to elicit N-SMase activation in response to TNF-R1 stimulation. It is therefore tempting to envision the product of SMase action, ceramide, as a player in apoptosis signaling (Fig.3). The above findings on FAN suggest that ceramide (or possibly its metabolites) acts by amplifying the classical apoptotic pathways, e.g., those initiated at the ‘death domain’ of death receptors. Rather than being ‘the’ lipid mediator of apoptosis, ceramide appears as one component that cooperates with others to ultimately result in a full apoptotic response (at least in fibroblasts). Such a hypothesis would not be inconsistent, given the redundancy of the various signaling pathways involved in apoptosis induction or regulation.1,2 These observations raise a number of questions. Among those: is the spectrum of FANmediated effects related to ceramide only? How does FAN regulate N-SMase and what are the (likely) protein intermediates? What is the nature of receptors that couple to FAN? Are other (e.g., immunological) defects than the skin abnormality associated to the FAN deficiency in mice? Is FAN or its homologues expressed in non-mammalian organisms? Future work is expected to

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clarify these issues, allowing elucidation of both FAN and ceramide functions, and presumably leading to the development of novel potential approaches for therapeutic intervention in pathological states.

Acknowledgments The authors thank Drs. S. Adam-Klages, M. Krönke, I. Galve-Roperh and M. Guzman for fruitful discussion and collaborative work. Financial support by the Inserm, the Fondation pour la Recherche Médicale, and Université Paul Sabatier is gratefully acknowledged.

References 1. Raff M. Cell suicide for beginners. Nature 1998; 396:119-122. 2. Hengartner MO. The biochemistry of apoptosis. Nature 2000; 407:770-776. 3. Spiegel S, Merrill Jr AH. Sphingolipid metabolism and cell growth regulation. FASEB J 1996; 10:1388-1397. 4. Kolesnick RN, Krönke M. Regulation of ceramide production and apoptosis. Annu Rev Physiol 1998; 60:643-665. 5. Hannun YA, Luberto C. Ceramide in the eukaryotic stress response. Trends Cell Biol 2000; 10:73-80. 6. Obeid LM, Linardic CM, Karolak LA et al. Programmed cell death induced by ceramide. Science 1993; 259:1769-1771. 7. Levade T, Jaffrézou JP. Signalling sphingomyelinases: which, where, how and why? Biochim Biophys Acta 1999; 1438:1-17. 8. Vandenabeele P, Declercq W, Beyaert R et al. Two tumour necrosis factor receptors: Structure and function. Trends Cell Biol 1995; 5:392-399. 9. Wallach D, Varfolomeev EE, Malinin NL et al. Tumor necrosis factor receptor and Fas signaling mechanisms. Annu Rev Immunol 1999; 17:331-367. 10. Adam-Klages S, Schwandner R, Adam D et al. Distinct adapter proteins mediate acid versus neutral sphingomyelinase activation through the p55 receptor for tumor necrosis factor. J Leukoc Biol 1998; 63:678-682. 11. Schwandner R, Wiegmann K, Bernardo K et al. TNF receptor death domain-associated proteins TRADD and FADD signal activation of acid sphingomyelinase. J Biol Chem 1998; 273:5916-5922. 12. Wiegmann K, Schwandner R, Krut O et al. Requirement of FADD for tumor necrosis factorinduced activation of acid sphingomyelinase. J Biol Chem 1999; 274:5267-5270. 13. Adam D, Wiegmann K, Adam-Klages S et al. A novel cytoplasmic domain of the p55 Tumor Necrosis Factor receptor initiates the neutral sphingomyelinase pathway. J Biol Chem 1996:14617-14622. 14. Adam-Klages S, Adam D, Wiegmann K et al. FAN, a novel WD-repeat protein, couples the p55 TNF-receptor to neutral sphingomyelinase. Cell 1996; 86:937-947. 15. Smith TF, Gaitatzes C, Saxena K et al. The WD repeat: a common architecture for diverse functions. Trends Biochem Sci 1999; 24:181-185. 16. Kreder D, Krut O, Adam-Klages S et al. Impaired neutral sphingomyelinase activation and cutaneous barrier repair in FAN-deficient mice. EMBO J 1999; 18:2472-2479. 17. Andrieu N, Salvayre R, Levade T. Evidence against involvement of the acid lysosomal sphingomyelinase in the tumour-necrosis-factor- and interleukin-1-induced sphingomyelin cycle and cell proliferation in human fibroblasts. Biochem J 1994; 303:341-345. 18. Ségui B, Cuvillier O, Adam-Klages S et al. Involvement of FAN in tumor necrosis factor-induced apoptosis. J Clin Invest 2001; 108:143-151. 19. Jaffrézou J, Levade T, Bettaieb A et al. Daunorubicin-induced apoptosis: triggering of ceramide generation through sphingomyelin hydrolysis. EMBO J 1996; 15:2417-2424. 20. Tartaglia LA, Ayres TM, Wong GHW et al. A novel domain within the 55 kd TNF receptor signals cell death. Cell 1993; 74:845-853. 21. van Kooten C, Banchereau J. Functions of CD40 on B cells, dendritic cells and other cells. Curr Opin Immunol 1997; 9:330-337.

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22. Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 2001; 104:487-501. 23. Hess S, Engelmann H. A novel function of CD40: induction of cell death in transformed cells. J Exp Med 1996; 183:159-167. 24. Ségui B, Andrieu-Abadie N, Adam-Klages S et al. CD40 signals apoptosis through FAN-regulated activation of the sphingomyelin-ceramide pathway. J Biol Chem 1999; 274:37251-37258. 25. Hildt E, Oess S. Identification of Grb2 as a novel binding partner of tumor necrosis factor (TNF) receptor I. J Exp Med 1999; 189:1707-1714. 26. Peppelenbosch M, Boone E, Jones GE et al. Multiple signal transduction pathways regulate TNFinduced actin reorganization in macrophages: Inhibition of Cdc42-mediated filopodium formation by TNF. J Immunol 1999; 162:837-845. 27. Wiegmann K, Schütze S, Machleidt T et al. Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 1994; 78:1005-1015. 28. Lüschen S, Adam D, Ussat S et al. Activation of ERK1/2 and cPLA2 by the p55 TNF receptor occurs independently of FAN. Biochem Biophys Res Commun 2000; 274:506-512. 29. Boone E, Vandevoorde V, De Wilde G et al. Activation of p42/p44 mitogen-activated protein kinases (MAPK) and p38 MAPK by tumor necrosis factor (TNF) is mediated through the death domain of the 55-kDa TNF receptor. FEBS Lett 1998; 441:275-280. 30. Guzman M, Galve-Roperh I, Sanchez C. Ceramide: A new second messenger of cannabinoid action. Trends Pharmacol Sci 2001; 22:19-22. 31. Sanchez C, Rueda D, Segui B et al. The CB1 cannabinoid receptor of astrocytes is coupled to sphingomyelin hydrolysis through the adaptor protein Fan. Mol Pharmacol 2001; 59:955-959. 32. Gray GM, White RJ. Glycosphingolipids and ceramides in human and pig epidermis. J Invest Dermatol 1978; 70:336-341. 33. Jensen JM, Schutze S, Forl M et al. Roles for tumor necrosis factor receptor p55 and sphingomyelinase in repairing the cutaneous permeability barrier. J Clin Invest 1999; 104:1761-1770. 34. Hofmann K, Dixit VM. Ceramide in apoptosis: Does it really matter? Trends Biochem Sci 1998; 23:374-377. 35. Kolesnick R, Hannun YA. Ceramide and apoptosis. Trends Biochem Sci 1999; 24:224-225. 36. Radin NS. Apoptotic death by ceramide: Will the real killer please stand up? Med Hypotheses 2001; 57:96-100. 37. Venkataraman K, Futerman AH. Ceramide as a second messenger: Sticky solutions to sticky problems. Trends Cell Biol 2000; 10:408-412.

CHAPTER 9

The Cross-Talk Between Nitric Oxide and Ceramide and Its Role in Apoptosis Regulation Rico Barsacchi, Clara Sciorati and Emilio Clementi

Abstract

N

itric oxide (NO), a short-lived pleiotropic messenger, is known to interact with signaling pathways operated by other messenger molecules, including ions, cyclic nucleotides, protein kinases and phosphatases. Through these interactions NO regulates a variety of biological functions. Recently, NO has been shown to interact with ceramide signaling by regulating enzymes in the metabolic pathway of sphingolipids. This leads to increases or decreases in ceramide cell content, depending on the concentration of NO and the time of exposure. Ceramide in turn may activate NO synthases to generate NO. The cross-talk between the two messenger molecules appears of biological relevance in the regulation of the signal transduction pathways triggered by death receptors and leads to modulation of the ability of these receptors to induce cell death via apoptosis.

Introduction Nitric oxide (NO) is a short lived diffusible messenger synthesized from L-arginine by three NO synthases (NOS). Of these enzymes, the inducible isoform is expressed after cell stimulation by a variety of cytokine and bacterial products, while the endothelial and neuronal NOS are constitutively expressed and regulated by intracellular messenger molecules.1 Nitric oxide exerts a variety of biological functions among which is the regulation of cell death. Long lasting generation of NO, as occurs following expression of inducible NOS, is proapoptotic in most cell types.2 In contrast, the low physiological concentrations produced by the constitutive isoforms protect from apoptosis triggered by a variety of stimuli, including death receptors belonging to the tumor necrosis factor-α receptor (TNF-RI)/CD95 superfamily.3 Apoptosis stimulated by these receptors has a key role in a number of physiological processes, e.g., the development and remodeling of tissues and organs, and also in the onset and progress of various pathological conditions.4 Death receptors initiate apoptosis through a defined series of signal transduction events, i.e., recruitment of adapter proteins with formation of the so-called DISC complex, release of mitochondrial cytochrome c and activation of the caspase cascade.5 Most of these steps are known targets for the protective action by NO, as extensively reviewed in ref. 3. In addition, Ceramide Signaling, edited by Anthony H. Futerman. ©2002 Eurekah.com and Kluwer Academic / Plenum Publishers.

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death receptors activate the sphingolipid metabolic pathway with ensuing generation of ceramide, which contributes to their apoptogenic effect.6 Recent papers have proposed that interference with ceramide signaling is an additional mechanism through which NO regulates apoptosis induced by death receptors. This chapter will review the molecular mechanism of interaction between NO and ceramide in the context of apoptosis regulation.

Nitric Oxide Regulates Ceramide Generation In human leukemia and rat mesangial cells, treatment with high, micromolar concentrations of NO increases ceramide levels and leads to apoptosis.7,8 Of importance, increases in ceramide content have been detected only after hours (2 to 24) of treatment with NO.7-9 This effect of NO appears to result from actions on various enzymes of the SM metabolic pathway, i.e., inhibition of ceramidases and activation of N-SMase(s) and A-SMase(s).7,8 By contrast, when the metabolic pathways leading to the generation of ceramide are activated by physiological agonists, such as ligands of death receptors, ceramide levels are reduced by NO. We have shown this in human γδ T lymphocytes and in the monocytic line U937, where ceramide generation is triggered within minutes after activation of CD95 or TNF-RI.10,11 This inhibitory effect of NO is fast in onset (minutes) and is observed at low concentrations of the gas (Fig. 1). Nitric oxide appears to act through activation of the soluble guanylyl cyclase with generation of cyclic GMP (cGMP), because its effect is mimicked by cGMP analogs and blocked by ODQ, a specific inhibitor of guanylyl cyclase (see Fig. 1 and refs. 10, 11). Preliminary evidence indicates that the reduction of ceramide levels by NO is due to cGMP-dependent inhibition of both A-SMase(s) and N-SMase(s) (Barsacchi and Clementi, unpublished data). The inhibitory effect of NO on ceramide generation is relevant to the regulation of apoptosis. As shown in Figure 2, administration of exogenous ceramide reverses the inhibition by NO of TNFα-triggered apoptosis. Of importance, regulation of apoptosis by NO/ceramide is exerted in the early phases of the apoptogenic signal transduction cascade initiated by TNFα. In particular, ceramide enhances the recruitment of the protein TRADD to TNF-RI and the activation of caspase 8 (Fig. 3), thereby increasing apoptosis triggered by the cytokine.11,12 Exogenous NO promotes an inhibition of these signaling events, and thus of apoptosis, through its ability to reduce ceramide generation (Fig.2). The early events described above account for only part of the effect of NO on ceramide signaling and apoptosis. In lymphocytes, eosinophils and neuroblastoma cells, NO has also been shown to inhibit apoptosis induced by the addition of exogenous ceramide 10,13,14, through mechanisms partially dependent on cGMP generation. Thus, the molecular levels at which NO exerts its action are multiple and are located both upstream and downstream of ceramide formation.

Ceramide Regulates Nitric Oxide Generation Relationships between NO and ceramide are not limited to a one-way direction, i.e., NO regulating ceramide generation, but occur also the other way round, because the lipid messenger can increase bioavailable NO. Addition of exogenous SMase and ceramide promotes expression of the inducible NOS gene some hours after cell treatment.15-17 The subsequent long lasting NO generation participates in the toxic effect of the above compounds.15-17 Ceramide may trigger NO generation also via activation of endothelial NOS (eNOS). This is a non genomic effect which occurs within minutes after administration of the lipid messenger and results in generation of low, physiological concentrations of NO.18 Ceramide-dependent activation of eNOS appears of biological relevance as it is the mechanism by which CD95 and TNF-RI stimulate the generation of NO (see Fig. 4 and Clementi, unpublished observation). Activation of eNOS by these receptors occurs within minutes and NO thus generated appears to act as a negative regulator of their apoptogenic signals.10,19 We

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Figure 1. Nitric oxide inhibits CD95 and TNFα-induced ceramide generation. Human γδ T lymphocytes (Panel A) or U937 cells (Panel B) were pretreated for 15 minutes with the NO donor SNAP (NO) or the cGMP analog 8 Br-cGMP (cGMP). Ceramide production was stimulated by activation of CD95 or TNF-R1. Ceramide content was measured by the diacylglycerol kinase assay followed by thin layer chromatography and densitometric analysis of the autoradiographies thus obtained. Ceramide concentrations are expressed as percentage above those measured in unstimulated cell samples run in parallel. Values + standard error of the means are from 4 experiments. Adapted from refs 10 and 11, in which details of the methods used are given.

have observed this in γδ T lymphocytes, where eNOS expression is stimulated after cell activation by antigens and decreases progressively.10 In these cells eNOS activity correlates negatively with susceptibility to CD95-induced apoptosis, and blockade of NO generation by the NOS inhibitor L-NAME confers sensitivity to CD95 to otherwise resistant cells (Panel A in Fig. 5). Lymphocytes, therefore, use eNOS to tune the level of their response to CD95 activation and

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Figure 2. Ceramide increases while NO/cGMP decrease TNFα-induced apoptosis in U937 cells. Cells were treated for 3 hours with TNFα in the presence of the protein synthesis inhibitor cycloheximide, with or without SNAP (NO), 8 Br-cGMP (cGMP), and the membrane permeant C2 ceramide (Cer) in various combinations, as indicated in the key. Apoptosis was measured by DNA fragmentation analysis as described in ref 11. Values + standard error of the means are from 4 experiments.

to regulate their life span after antigen stimulation.10 Similarly, in HeLa cell clones transfected with the eNOS under a tetracycline-responsive element, we found that induction of eNOS was sufficient to protect from TNFα-induced apoptosis.19 Again, this resistance to apoptosis was lost after treatment with L-NAME (Panel B in Fig. 5). The relevance of eNOS in controlling apoptosis has been suggested also by experiments in other cellular models.20-22 Taken together, the evidence summarized above suggests that a crosstalk between NO and ceramide constitutes an important built-in mechanism by which death receptors control their own apoptotic program.

The Cross-Talk Between Nitric Oxide and Ceramide in the Context of Apoptosis The model of Figure 6 shows a hypothetical scheme of the functional interactions among death receptors, nitric oxide and ceramide. The model is based on data about TNF-RI because information on this receptor is more complete. Activation results in the tightening of the bonds between the three TNF-RI subunits leading to recruitment of the DISC proteins TRADD and FADD, with ensuing activation of caspase 8.5,23 Concomitantly, SMase(s) are activated with generation of ceramide. Caspase 8 and ceramide activate the downstream cascade of events leading to apoptotic cell death.5,24 In

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Figure 3. Ceramide and NO/cGMP regulate TNFα-induced TRADD recruitment and caspase 8 activation in U937 cells. TRADD recruitment to TNF-RI (Panel A) was analyzed by Western blotting of immunoprecipitates obtained with an anti TNF-R1 antibody from cell lysates prepared 5 minutes after TNFα treatment in the absence or presence of C2 ceramide (Cer) or SNAP (NO). Lane marked with L was loaded with 50 µg whole cell homogenate as internal control. Caspase 8 activity (Panel B) was measured using a specific fluorescent substrate in lysates of cells treated for 5 minutes with TNFα in the presence or absence of the other compounds. Values + standard errors of the means are expressed as percentage of those observed in untreated controls run in parallel (n = 4).

addition, ceramide amplifies the initial signaling events at TNF-RI by increasing TRADD recruitment. This feed-forward mechanism, however, is counterbalanced by ceramide itself through activation of eNOS and generation of NO, inhibition of SMase(s) and eventual reduction in ceramide levels. In addition, NO may counterbalance the apoptogenic effect of ceramide also because of its action on additional events involved in TNFα-induced apoptosis, e.g., inhibition of cytochrome c release, inhibition of caspases, increased expression of anti

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Figure 4. TNF-R1 activates eNOS through ceramide. HeLa cell clones expressing eNOS under a tetracyclin-inducible promoter (“Tet-off”) were treated with or without doxycyclin in order to obtain cells either expressing or not eNOS (see Panel B in Fig. 5). The cells were then treated for 15 min with TNFα, in the presence or absence of each of the following: the NOS inhibitor L-NAME; the glucosylceramide synthase inhibitor PDMP; or the ceramide synthase inhibitor fumonisin B1 (Fum), which increase or decrease, respectively, the concentrations of ceramide generated by stimulation of TNF-RI.11 eNOS activity was evaluated by assessing the conversion of arginine into citrulline as described in ref. 19. Cells expressing eNOS respond to TNF-RI stimulation with increases in NOS activity, which are stronger if ceramide generation by the death receptor is increased by PDMP, and reduced if ceramide content is lowered by Fum. Values + standard errors of the means are from 4 experiments.

apoptotic proteins, as detailed in ref. 3. The functional coupling between eNOS and TNF-RI might be facilitated by the subcellular localization of the enzyme and the receptor, which are both concentrated at the caveolae of the plasma membrane.25-27 The picture described above might be further complicated when TNF-RI stimulation leads to induction of the expression of inducible NOS. This may be triggered by the death receptor through the activation of multiple transcription factors, including NF-κB, some of

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Figure 5. eNOS activity regulates CD95 and TNFα-induced apoptosis. Panel A: γδ T lymphocytes were activated by antigenic stimulation and analyzed after 14 and 28 days, i.e., when they were resistant or sensitive, respectively, to CD95-induced apoptosis.10 The picture shows Western blot analyses of eNOS expression in cell lysates. A positive control for eNOS expression (st, lysate from porcine aortic endothelial cells) was loaded in parallel. The graph shows apoptosis induction by CD95 either alone or in the presence of L-NAME. Apoptosis (expressed as percentage of apoptotic cells) was assessed by flow cytometry 6 hours after treatment measuring exposure of phosphatidylserine on the outer leaflet of the plasma membrane. Values + standard errors of the means are from 5 experiments. Details of the methods used are given in ref. 10, which is partial source for the results shown. Panel B: HeLa cell clones expressing eNOS under a Tet-off inducible promoter were treated with or without doxycyclin. Levels of expression of the protein in eNOS-expressing (+) or eNOS-deficient (-) cells were analyzed by Western blotting as shown in the picture. A positive control for eNOS expression (st, lysate from porcine aortic endothelial cells) was loaded in parallel. The graph shows apoptosis induction by TNFα, administered either alone or in the presence of L-NAME. Apoptosis was assessed by flow cytometry 12 hours after treatment as described in A. Values + standard errors of the means are from 5 experiments. Details of the methods used are given in ref. 19.

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Figure 6. Schematic representation of the cross-talk between NO and ceramide cross-talk in regulation of apoptotic signaling by TNF-RI. For details see text. Abbreviations in the figure not specified in the text are as follows: Casp 8 for caspase 8; iNOS for inducible NOS; SMase for SMase(s). The difference in character size used to represent NO indicates low and high concentrations of the gas.

which are activated by ceramide itself.15,16 The sustained generation of NO by this NOS isoform may contribute to cell death through a variety of mechanisms, e.g., alteration of energy metabolism, modifications in gene expression, generation of ceramide and highly reactive oxidant species.2 Apoptosis induced by death receptors plays a role in diseases such as atherosclerosis, vasculitis, graft rejection, gastric damage and various forms of hepatitis.10, 28-30 This consideration emphasizes the importance of the cross-talk between ceramide and nitric oxide since it may help defining new targets and strategies for therapeutic intervention.

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References 1. Stuehr DJ. Mammalian nitric oxide synthases. Biochim Biophys Acta 1999; 1411:217-230. 2. Brune B, von Knethen A, Sandau KB. Nitric oxide (NO): an effector of apoptosis. Cell Death Differ 1999; 6:969-975. 3. Liu L, Stamler JS. NO: an inhibitor of cell death. Cell Death Differ 1999; 6:937-942. 4. Aggarwal BB, Natarajan K. Tumor necrosis factors: developments during the last decade. Eur Cytokine Netw 1996; 7:93-124. 5. Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science. 1998; 281:1305-1308. 6. Hannun YA. Functions of ceramide in coordinating cellular responses to stress. Science 1996; 274:1855-1859. 7. Takeda Y, Tashima M, Takahashi A et al. Ceramide generation in nitric oxide-induced apoptosis. Activation of magnesium-dependent neutral sphingomyelinase via caspase-3. J Biol Chem 1999; 274:10654-10660. 8. Huwiler A, Pfeilschifter J, van den Bosch H. Nitric oxide donors induce stress signaling via ceramide formation in rat renal mesangial cells. J Biol Chem 1999; 274:7190-7195. 9. Nagano S, Takeda M, Ma L et al. Cytokine-induced cell death in immortalized Schwann cells: roles of nitric oxide and cyclic AMP. J Neurochem 2001; 77:1486-1495. 10. Sciorati C, Rovere P, Ferrarini M et al. Autocrine nitric oxide modulates CD95-induced apoptosis in γδ T lymphocytes. J Biol Chem 1997; 272:23211-23215. 11. De Nadai C, Sestili P, Cantoni O et al. Nitric oxide inhibits tumor necrosis factor-α-induced apoptosis by reducing the generation of ceramide. Proc Natl Acad Sci USA 2000; 97:5480-5485. 12. Higuchi M, Singh S, Jaffrezou JP et al. Acidic sphingomyelinase-generated ceramide is needed but not sufficient for TNF-induced apoptosis and nuclear factor-kappa B activation. J Immunol 1996; 157:297-304. 13. Lièvremont JP, Sciorati C, Morandi E et al. The p75(NTR)-induced apoptotic program develops through a ceramide-caspase pathway negatively regulated by nitric oxide. J Biol Chem 1999; 274:15466-15472. 14. Hebestreit H, Dibbert B, Balatti I et al. Disruption of fas receptor signaling by nitric oxide in eosinophils. J Exp Med 1998; 187:415-425. 15. Katsuyama K, Shichiri M, Marumo F et al. Role of nuclear factor-kappa B activation in cytokineand sphingomyelinase-stimulated inducible nitric oxide synthase gene expression in vascular smooth muscle cells. Endocrinol 1998; 139:4506-4512. 16. Vann LR, Twitty S, Spiegel S et al. Divergence in regulation of nitric oxide synthase and its cofactor tetrahydrobiopterin by tumor necrosis factor-α. J Biol Chem 2000; 275:13275-13281. 17. Zhou YT, Grayburn P, Karim A et al. Lipotoxic heart disease in obese rats: implications for human obesity. Proc Natl Acad Sci USA 2000; 97:1784-1789. 18. Igarashi J, Thatte HS, Prabhakar P et al. Calcium-independent activation of endothelial nitric oxide synthase by ceramide. Proc Natl Acad Sci USA 1999; 96:12583-12588. 19. Bulotta S, Barsacchi R, Rotiroti D et al. Activation of the endothelial nitric oxide synthase by tumor necrosis factor α. A novel feedback mechanism regulating cell death. J Biol Chem 2001; 276:6529-6536. 20. Dimmeler S, Zeiher AM. Nitric oxide: an endothelial cell survival factor. Cell Death Differ 1999; 6:964-968. 21. Furuke K, Burd PR, Horvath-Arcidiacono JA er al. Human NK cells express endothelial nitric oxide synthase and nitric oxide protects them from activation-induced cell death by regulating expression of TNF-α. J Immunol 1999; 163:1473-1480. 22. Murphy PR, Limoges M, Dodd F et al. Fibroblast growth factor-2 stimulates endothelial nitric oxide synthase expression and inhibits apoptosis by a nitric oxide-dependent pathway in Nb2 lymphoma cells. Endocrinol 2001; 142:81-88. 23. Chan FK, Chun HJ, Zheng L et al. A domain in TNF receptors that mediates ligand-independent receptor assembly and signaling. Science 2000; 288:2351-2354. 24. Hannun YA, Luberto C. Ceramide in the eukaryotic stress response.Trends Cell Biol 2000; 10:73-80. 25. Shaul PW, Smart EJ, Robinson Lj et al. Acylation targets endothelial nitric oxide synthase to plasmalemmal caveolae. J Biol Chem 1996; 271:6518-6522.

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26. Garcia-Cardena G, Oh P, Liu J et al. Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: implications for nitric oxide signaling. Proc Natl Acad Sci USA 1996; 93:6448-6453. 27. Ko YG, Lee JS, Kang YS et al. TNF-α-mediated apoptosis is initiated in caveolae-like domains. J Immunol 1999; 162:7217-7223. 28. Appleyard CB, McCafferty DM, Tigley A et al. Tumor necrosis factor mediation of NSAID-induced gastric damage: role of leukocyte adherence. Am J Physiol 1996; 270:G42-G48. 29. Beutler B, Bazzoni F. TNF, apoptosis and autoimmunity: a common thread? Blood Cells Mol Dis 1998; 24:216-230. 30. Bradham CA, Plumpe J, Manns MP et al. Mechanisms of hepatic toxicity. I. TNF-induced liver injury. Am J Physiol 1998; 275:G387-G392.

CHAPTER 10

Crosstalk of Ceramide with Cell Survival Signaling Toshiro Okazaki, Tadakazu Kondo, Mitsumasa Watanabe, Yoshimitsu Taguchi and Takeshi Yabu

Summary

I

n addition to the involvement of ceramide in pro-apoptotic signaling, the role of ceramide as an anti-apoptotic, or cell survival signal, has also been investigated. So far, many antiapoptotic molecules such as protein kinase C (PKC), mitogen-activated protein (MAP) kinase, phospholipase D (PLD), heat shock proteins (HSPs), p21 ras, phosphatidylinositol-3 kinase (PI-3k)/Akt kinase, NF-κB, reactive oxygen intermediate (ROI) scavengers, calcium signaling and cytokines (growth factors) have been recognized as anti-apoptotic or survival molecules. In this Chapter we discuss their crosstalk with ceramide-related pro-apoptotic actions and discuss the importance of the balance between pro- and anti-apoptotic signaling in ceramide-induced apoptosis for the decision of cell fate.

Introduction Since the demonstration that ceramide is a lipid mediator for cell differentiation, a diverse array of stresses were suggested to increase intracellular ceramide content and to activate its downstream signaling pathways so as to execute apoptosis.1-3 In terms of the mechanisms by which ceramide content is regulated, the ceramide-generating systems, such as sphingomyelin (SM) hydrolysis by SMase, and de novo synthesis by ceramide synthase and palmitoyl-CoA transferase, are known to increase apoptosis, while a ceramide-metabolizing system, such as conversion to glucosylceramide (GC) by GC synthase, deacylation to sphingosine by ceramidase, and re-synthesis to SM by SM synthase, decrease apoptosis. Recently, biochemical and molecular analyses have been undertaken to investigate the mechanism and regulation of ceramide generation and many mammalian genes of the enzymes involved in ceramide synthesis/metabolism have been cloned, with the exception of SM synthase and ceramide synthase.4-12 However, although ceramide-activated protein kinase and phosphatase, jun N-terminal kinases, protein kinase C ζ and many others were suggested to be candidates for direct downstream targets of ceramide action, the precise mechanism by which ceramide directly connects to these molecules remains to be determined. In addition to the role of ceramide in pro-apoptotic signaling, the role of anti-apoptotic or survival signaling has also been investigated in ceramide-induced apoptosis. For example, it was reported that (1) protein kinase C (PKC), (2) mitogen-activated protein (MAP) kinase, (3) phospholipase D (PLD), (4) heat shock proteins (HSPs), (5) p21 ras, (6) phosphatidylinositol3 kinase (PI-3k)/Akt kinase, (7) NF-κB, (8) reactive oxygen intermediate (ROI) scavengers such as glutathione, superoxide dismutase (SOD) and catalase, (9) calcium signaling, and (10) cytokines (growth factors), which have been recognized as anti-apoptotic or survival molecules, Ceramide Signaling, edited by Anthony H. Futerman. ©2002 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Figure 1. Key players in the regulation of ceramide-mediated apoptosis. Ceramide is related to many kinds of pro-apoptotic signaling molecules such as N-terminal jun kinase (JUNK), retinoblastoma protein (Rb), caspases ROS and p-53 in stress-induced apoptosis. In contrast, a diverse array of anti-apoptotic molecules, such as PI-3 kinase/Akt kinase, HSP-70, NF-κB and PKC are known to act as suppressors of ceramide-related apoptosis. The crosstalk between ceramide-related proapoptotic signaling and growth factor-induced anti-apoptotic signaling is complicated, but of great importance in order to understand the mechanism of stress-induced apoptosis.

were able to cross-talk with ceramides, as shown in Fig. 1. We now summarize how ceramide action relates to anti-apoptotic signals in the induction of apoptosis. (1) PKC; The ability of ceramide to bind to PKC α,δ and ζ was reported.13,14 Ceramide activated PKC ζ by inducing its translocation to the membrane in primary cultures of astrocytes,15 and induced cell cycle arrest through activation of the stress-activated protein kinase complex.13 Recently, ceramide was shown to induce the cleavage of PKC δ and ε in induction of apoptosis;16,17 after cytosolic translocation some part of PKC δ and ε may be degraded by caspase-3. More recently, tyrosine phosphorylation of PKC δ and its activation in the Golgi apparatus was found to be induced by ceramide in HeLa cells.18 In addition, cytosolic translocation of PKC δ and inhibition of PKC α was shown by ceramide,19,20 and antigen-induced translocation of α, β1 and β2 isozymes was specifically prevented by C2-ceramide.21 Therefore, ceramide may induce the inhibition of the function of PKC δ and ε by decreasing the active form of the enzyme in the membrane; we found that auto-phosphorylation of membranous PKC δ was decreased by ceramide in HL-60 cells, but it still remains to be seen whether cytosolic PKC is activated or not. In contrast, PKC activation by phorbol esters inhibited daunomycin-induced apoptosis through suppression of SMase activity and ceramide generation, while inhibition of PKC induced apoptosis and ceramide generation occurs by activating neutral N-SMase.22,23 (2) MAP kinase; Although MAP kinase was activated by treatment with bacterial SMase or C2-ceramide in HL-60 cell differentiation,24 recently it was shown that MAP kinase is not involved in ceramide-induced differentiation in monoblastic U-937 cells, because inhibition of MEK with PD98059 did not inhibit monocytic differentiation by ceramide.25 On the contrary, ceramide was reported to inhibit phosphorylation of MAP kinase p42 and p44 and to reduce IgG-dependent phagocytosis in neutrophils.26

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(3) PLD; In vitro, it seems to be consistent that ceramide inhibits PLD,27,28 but the situation in vivo is less conclusive. C2-ceramide inhibited Ag- or PMA-induced activation of phospholipase D (PLD), whereas the Ca2+ ionophore, A23187-induced PLD activation was not affected, probably because ceramide inhibited the Ca2+ increase through PKC.21 Thyroid-stimulating hormone-induced PLD activation was inhibited by ceramide in an early phase of thyroid cell apoptosis.29 However, recently it was shown that C6-ceramide, or ceramide generated by Fas cross-linking, does not affect PKC β-dependent PLD activity in A20 cells.30 (4) HSP; The levels of the small heat shock protein, αB-crystallin, in NIH-3T3 cells, as measured by Northern blot and immunoblotting analyses, were increased by the addition of C2-ceramide.31 In contrast, we reported that heat shock protein HSP-70, which binds to Apaf1 or apoptosis-inducing factor (AIF) to inhibit activation of caspase-3 and apoptosis,32,33 was inhibited by C2-ceramide at mRNA and protein levels in a dose- and time-dependent manner, but HSP-60 and -90 were not affected.34 The inhibition of HSP-70 by ceramide was shown to be dependent on transcriptional regulation as judged by run-on experiments, but not on HSinduced transcription factors, heat shock factor (HSF)-1 and 2.34 In HS-resistant cells HSinduced ceramide generation and an increase of HSP-70 without execution of apoptosis suggests that HSP-70 increased in response to stress-induced proapoptotic signals. (5) ras and (6) PI-3k/Akt kinase; Ras and raf-1-dependent activation of PI-3k was detected in ceramide-, TNF-α− or Fas-induced apoptosis,35,36 but ceramide inhibited PI-3k-dependent Akt kinase by dephosphorylating serine 473 or inactivating 3-phosphoinositide-dependent kinase (PDK)-1 in apoptosis induction.37,38 Thus, ceramide may inhibit an Akt-dependent pathway mediated by PI-3k to execute apoptosis, while in response to a pro-apoptotic signaling, the lipid mediators such as phosphatidylinositol bisphosphate (PIP2) seems to act through ras-dependent PI-3k to intensify survival signals. This notion led us to investigate the precise mechanism of mutual regulation between ceramide and the PI-3k/Akt kinase pathway in apoptosis in order to understand the cross talk between the proapoptotic action of ceramide and anti-apoptotic/survival signaling. (7) NF-κB; The relationship between ceramide and NF-κB activation in apoptosis is controversial. Some reports showed that TNF-α induced ceramide generation and activation of NF-κB,39 but others suggested the lack of NF-κB activation because of exogenous ceramide.40,41 We also found that TNF-α or daunomycin-induced NF-κB activation was not induced by Nacetylceramide or N-hexanoylceramide (C2- and C6-ceramide) in Jurkat cells (data not shown). Therefore, the evidence for NF-κB in ceramide-mediated apoptosis is inconsistent, although recently inhibition of NF-κB activation was suggested to be closely related to overcoming the resistance of the cells to induction of apoptosis.42 (8) ROI scavenger; It is well know that ceramide-induced apoptosis is involved in the generation of oxidative damage, especially in mitochondria.43 C2-ceramide affected the mitochondrial electron transport system and decreased glutathione content to generate ROI,44,45 and glutathione inhibited ceramide formation by TNF-α and hypoxia through inhibition of SMase.46,47 We also found that another ROI scavenger, catalase, and SOD rescued the cells from apoptosis induced by C2-ceramide. These results suggest that oxidative damage is located upstream and downstream of ceramide action and may be one of the indispensable signals in ceramide-induced apoptosis. (9) Calcium; Many growth factors utilize the second messenger, inositol triphosphate (InsP3), to set up prolonged calcium signals, which activate the immediate early genes responsible for inducing resting cells (G0) to re-enter the cell cycle and DNA synthesis.48 Ceramide inhibits sodium or L type-calcium channel,49,50 and increased calcium-dependent translocation of phospholipase A2 (PLA2) from the cytosol to membranes in platelets.51 Rhod-2 fluorescence in mitochondria increased to a maximum within 3 hours after C2-ceramide treatment or serum withdrawal.52 Decreased endoplasmic reticulum (ER) calcium occurs 4 h after low serum treatment and precedes apoptosis; significant elevations in ceramide are observed 16 h after reduced serum treatment, suggesting ceramide action is downstream of calcium signaling in apoptosis.53

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However, no evidence for the close relationship between ceramide action and calcium increase has yet been obtained in cell proliferation and apoptosis. (10) Cytokines (growth factors); It was reported that sub-micromolar concentrations of C2-ceramide induced IL-6 gene expression and protein production as effectively as interleukin (IL)-1 β in human fibroblasts.54 In contrast C2-ceramide suppressed IL-6 synthesis induced by prostaglandin E1, 12-O-tetradecanoylphorbol-13-acetate (TPA), cholera toxin, forskolin or dibutyryl cAMP in osteoblast-like MC3T3-E1 cells.55 We found that ceramide and sphingosine suppress TPA-induced IL-6 secretion from fibroblasts. In addition, transforming growth factor (TGF)-β1 rescued ceramide-induced cell death without affecting the cell cycle and decreasing the level of Bcl-2, thereby abolishing CPP32/Yama protease activation.56 Over-expression of cyclooxygenase-2 and hepatocyte growth factor (HGF)-induced COX-2 inhibited ceramide-induced apoptosis in RGM-1 gastric epithelial cells.57 When primary cultures of quiescent astrocytes from rat cerebellum were stimulated to proliferate by mitogenic doses of basic fibroblast growth factor (bFGF), ceramide formation was decreased.58 Therefore, many kinds of growth factors seem to modulate ceramide-related apoptosis downstream or upstream of ceramide action, but the precise mechanism by which ceramide signaling regulates growth factor-dependent cell proliferation still remains to be clarified These results strongly suggest that the severity of ceramide action on apoptosis is closely related to anti-apoptotic signaling, and as shown above, the regulation of the cross talk between ceramide and anti-apoptotic signals still needs to be fully established. Ceramide inhibits antiapoptotic signals in some conditions, but enhances them in other conditions probably due to the difference of time and extent of ceramide generation after induction of stress. Thus, antiapoptotic signals may be enhanced by the incomplete pro-apoptotic action mediated by ceramide, and be suppressed by executive apoptotic action by ceramide. It seems to be critical for deciding the fate of the cells, apoptosis or cell survival, to balance the forces between anti-apoptotic and pro-apoptotic signaling regulated by ceramide (Fig. 1).

Regulation of PI-3 Kinase-Dependent Signaling by Ceramide How does ceramide affect the PI-3k/Akt kinase pathway? In addition to a direct regulation of Akt kinase, such as dephosphorylation of serine 473 and inhibition of PDK-1 by ceramide, a mechanism by which ceramide affects the intracellular topology of Akt kinase or PI-3k has recently been proposed. Firstly, it was reported that ceramide-generating stresses such as UV, irradiation and hyper-osmolarity, down-regulated the activities of PI-3k and Akt kinase through acid SMase because acid SMase-deficient lymphoblasts did not show any increase of ceramide and down-regulation of PI-3k signaling.59 It was then found that without impairing the phosphorylation of insulin receptor substrate 1, or a loss in phosphoinositide 3-kinase activation, ceramide caused a failure to activate Akt kinase due to a defect of recruitment of Akt kinase to the plasma membrane.60 Similar inhibitory results by ceramide on Akt kinase translocation were shown in PDGF-stimulated NIH-3T3 fibroblasts.61 Moreover, upstream of Akt kinase, PI-3k-dependent signalling was reported to be abrogated by ceramide-regulated caveolin 1 without affecting its expression, association and autophosphorylation, suggesting that ceramide alters the localization of PI-3k through modulation of membrane rafts consisting of caveolae.62 In contrast to ceramide-mediated inhibition of PI-3k, PI-3k activation through acid SMase was suggested as Niemann-Pick disease-derived lymphoblasts, which are deficient in acid SMase, did not show an increase of PI-3k by insulin.63 Since the authors’ conclusion was based upon the use of a SMase-deficient cell system instead of the experimental condition where intracellular ceramide was directly decreased, the results suggesting activation of PI-3k by ceramide seems to be difficult to reconcile with other data showing an inhibitory action of ceramide on growth factor-induced activation of PI-3k/Akt kinase. Next, how does ceramide affect the growth factor-mediated PI-3k/Akt kinase pathway? We recently found that C2-ceramide inhibited insulin-like growth factor (IGF)-1-induced PI-3k activation and subsequent autophosphorylation of Akt kinase in human leukemia HL-60 cells.

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Ceramide was also reported to inhibit IGF-1-induced proliferation by arresting the cells in the S phase in human breast cancer MCF-7 and T47D cells.64 Downstream of the PI-3k/Akt pathway in the initiation and elongation of translation, it is thought important that the initiation factor 4E-binding protein 1 (4E-BP1) is phosphorylated and sequesters cap binding protein eIF-4E for its activation in insulin-induced proliferation.65 When H2O2 inhibited cell growth, H2O2 stimulated dephosphorylation of 4E-BP1 by increasing protein phosphatase (PP1/PP2A) activity in cardiac myocytes.66 When we treated HL-60 cells with IGF-1 in the presence or absence of C2-ceramide, an increase of phosphorylation of 4E-BP1 was inhibited, suggesting that ceramide utilizes the inhibitory regulation of protein synthesis via translational capping to induce apoptosis. Recently, a large body of evidence for nuclear localization of ceramide, PI-3k and Akt kinase has emerged. For example, increase of nuclear PI-3k in granulocytic differentiation of HL-60 cells or NGF-treated PC12 cells was detected,67,68 while after activation, Akt kinase was shown to translocate from the plasma membrane to the nucleus.69 Nuclear activation of SMase and increase of ceramide were reported to play a role in hepatocyte apoptosis.70 Moreover, we found that a considerably large amount of ceramide is located in the nuclei of HL-60 cells as judged by western blotting using an anti-ceramide antibody. Therefore, although it still remains to be clarified how and where in the cell ceramide interacts with PI-3k or Akt kinase, ceramide may crosstalk with the PI-3k/Akt kinase pathway not only in the plasma membrane but also in other intracellular sites such as the nucleus to regulate cell apoptosis and survival.

Regulation of Ceramide-Related Signaling by PI-3 Kinase

It is well known that the PI-3k pathway is deeply involved in the regulation of apoptosis.71 Over-expression of PI-3k or Akt kinase caused protection of cells against staurosporine-induced apoptosis without an increase in ceramide formation, and anti-inflammatory cytokines, IL-10 and IL-13 were suggested to inhibit TNF-α- induced ceramide formation through activation of PI-3k in rat primary astrocytes.72 As a mechanism by which PI-3k signaling inhibits ceramide generation, the inhibition of SMase was suggested because IGF-1/PI-3k inhibited bacterial SMase-induced ceramide generation.73 However, it has not been elucidated whether stress-induced ceramide generation is inhibited by IGF-1/PI-3k anti-apoptotic signaling. Therefore, we recently examined whether ceramide generation is inhibited by IGF-1 in heat-shock (HS)-induced apoptosis. We demonstrated that ceramide generation induced by HS at 43˚C for 30 min was not affected by 100 ng/ml IGF-1 in HL-60 cells, when IGF-1 restored HSinduced apoptosis by approximately 50%. However, HS-induced caspase-3 activation and oxidative damage judged by lipid peroxidation was inhibited by IGF-1, and the inhibition by IGF-1 of caspase-3 and oxidative damage was recovered in the presence of the PI-3k inhibitor, wortmannin. These results suggest that PI-3k-dependent anti-apoptotic signaling acts not only upstream of ceramide but also downstream of ceramide, including activation of caspase and oxidative damage. Since the ceramide content of mitochondria is increased by stress-induced apoptosis,43 IGF-1 may inhibit caspase-3 activation by abrogating the ceramide-induced increase of mitochondrial membrane potential to release oxidative damage. HS-increased ceramide signaling was inhibited by IGF-1/PI-3k at the step of caspase-3 activation probably through inhibition of caspase-9 and activation of Bcl-2 via Akt kinase action, and generation of oxidative damage was subsequently blocked. How does ceramide-induced caspase-3 increase oxidative damage? We investigated the relationship of the caspase and reactive oxygen intermediate (ROI) scavenger system. Although ceramide was reported to decrease glutathione,44 we found that C2-ceramide inhibited catalase activity in a dose- and time-dependent manner and that this inhibition of catalase by ceramide was restored by IGF-1 and overexpression of PI-3k in HL-60 cells. Since ceramide-induced apoptosis was suppressed by various kinds of ROI scavengers, glutathione, catalase and SOD, execution of ceramide-mediated apoptosis through oxidative damage may be regulated by the caspase-3-dependent ROI scavenger system. In summary, these results suggest that IGF-1/PI-3 kinase inhibited C2-ceramide-induced

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t

Figure 2. Interaction of ceramide-related pro-apoptotic signaling with PI-3 kinase/Akt kinase pathway. IGF-1 activates the PI-3 kinase/Akt kinase pathway to induce cell growth via activation of S6 kinase and other mechanisms. Ceramide induces apoptosis by increasing oxidative damage via direct generation of ROS and inhibition of ROS scavengers such as glutathione, catalase and superoxide dismutase (SOD). Ceramide-activated caspase-3 inhibits catalase function at the mRNA and protein level. In inhibition by IGF-1 of ceramide-induced apoptosis, PI-3 kinase suppressed ceramide-induced oxidative damage by inhibiting caspase-3 activation, while ceramide inhibits IGF-1-induced PI-3 kinase.

apoptosis due to relieving oxidative damage, which resulted from the inhibition of catalase by activated caspase-3 (Fig. 2). In the future, analysis of the topological mechanism by which ceramide connects to PI-3k signaling would be required to understand the precise physiological relationship of ceramide-mediated apoptosis and PI-3k/Akt kinase-dependent cell survival.

Concluding Remarks It has been investigated whether ceramide may play a role in apoptosis, secretion and differentiation. However, we have not yet succeeded in understanding the precise mechanisms by which ceramide regulates a diverse array of cell functions, since it is still unknown what molecule(s) is directly downstream of ceramide action, what kind of metabolic pathway is most involved in ceramide generation, and where in the cells ceramide signaling works. In this Chapter, we proposed that a possible role of ceramide as a inhibitory regulator of cell survival or growth related to the PI-3 kinase/Akt kinase pathway, coincidentally with reciprocal action of ceramide as pro-apoptotic signal, should be fully considered in attempts to understand the physiological role of ceramide signaling.

References 1. Okazaki T, Kondo T, Kitano T et al. Diversity and complexity of ceramide signalling in apoptosis. Cell Signal. 1998; 10:685-692. 2. Kolesnick RN, Kronke M. Regulation of ceramide production and apoptosis. Annu Rev Physiol 1998; 60:643-65. 3. Hannun YA, Obeid LM. Mechanisms of ceramide-mediated apoptosis. Adv Exp Med Biol 1997; 407:145-149.

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4. Hofmann K, Tomiuk S, Wolff G et al. Cloning and characterization of the mammalian brainspecific, Mg2+-dependent neutral sphingomyelinase. Proc Natl Acad Sci USA 2000; 97:5895-5900. 5. Chatterjee S, Han H, Rollins S et al. Molecular cloning, characterization, and expression of a novel human neutral sphingomyelinase. J Biol Chem 1999; 274:37407-37412. 6. Newrzella D, Stoffel W. Molecular cloning of the acid sphingomyelinase of the mouse and the organization and complete nucleotide sequence of the gene. Biol Chem Hoppe Seyler 1992; 373:1233-1238. 7. Mao C, Xu R, Szulc Z et al. Cloning and characterization of a novel human alkaline ceramidase. A mammalian enzyme that hydrolyzes phytoceramide. J Biol Chem 2001; 276:26577-26588. 8. Mitsutake S, Tani M, Okino N et al. Purification, characterization, molecular cloning, and subcellular distribution of neutral ceramidase of rat kidney. J Biol Chem 2001; 276:26249-26259. 9. Tani M, Okino N, Mori K et al. Molecular cloning of the full-length cDNA encoding mouse neutral ceramidase. A novel but highly conserved gene family of neutral/alkaline ceramidases. J Biol Chem 2000; 275:11229-11234. 10. Ichikawa S, Sakiyama H, Suzuki G et al. Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis. Proc Natl Acad Sci USA 1996; 93:12654. 11. Weiss B, Stoffel W. Human and murine serine-palmitoyl-CoA transferase—cloning, expression and characterization of the key enzyme in sphingolipid synthesis. Eur J Biochem 1997; 249:239-247. 12. Hanada K, Hara T, Nishijima M et al. A mammalian homolog of the yeast LCB1 encodes a component of serine palmitoyltransferase, the enzyme catalyzing the first step in sphingolipid synthesis. 1997; J Biol Chem 272:32108-32114. 13. Bourbon NA, Yun J, Kester M. Ceramide directly activates protein kinase C zeta to regulate a stress-activated protein kinase signaling complex. J Biol Chem 2000; 275:35617-35623. 14. Huwiler A, Fabbro D, Pfeilschifter J. Selective ceramide binding to protein kinase C-alpha and -delta isoenzymes in renal mesangial cells. Biochemistry 1998; 37:14556-14562. 15. Galve-Roperh I, Haro A, Diaz-Laviada I. Ceramide-induced translocation of protein kinase C zeta in primary cultures of astrocytes. FEBS Lett 1997; 415:271-274. 16. Koriyama H, Kouchi Z, Umeda T et al. Proteolytic activation of protein kinase C delta and epsilon by caspase-3 in U937 cells during chemotherapeutic agent-induced apoptosis. Cell Signal 1999; 11:831-838. 17. Datta R, Kojima H, Yoshida K et al. Caspase-3-mediated cleavage of protein kinase C theta in induction of apoptosis. J Biol Chem 1997; 272:20317-20320. 18. Kajimoto T, Ohmori S, Shirai Y et al. Subtype-specific translocation of the delta subtype of protein kinase C and its activation by tyrosine phosphorylation induced by ceramide in HeLa cells. Mol Cell Biol 2001; 21:1769-1783. 19. Sawai H, Hannun YA. Ceramide and sphingomyelinases in the regulation of stress responses. Chem Phys Lipids 1999; 102:141-147. 20. Jones MJ, Murray AW. Evidence that ceramide selectively inhibits protein kinase C-alpha translocation and modulates bradykinin activation of phospholipase D. J Biol Chem 1995; 270:5007-5013. 21. Nakamura Y, Nakashima S, Ojio K et al. Ceramide inhibits IgE-mediated activation of phospholipase D, but not of phospholipase C, in rat basophilic leukemia (RBL-2H3) cells. J Immunol 1996; 156:256-262. 22. Chmura SJ, Nodzenski E, Weichselbaum RR et al. Protein kinase C inhibition induces apoptosis and ceramide production through activation of a neutral sphingomyelinase. Cancer Res 1996; 56:2711-2714. 23. Mansat V, Laurent G, Levade T et al. The protein kinase C activators phorbol esters and phosphatidylserine inhibit neutral sphingomyelinase activation, ceramide generation, and apoptosis triggered by daunorubicin. Cancer Res 1997; 57:5300-5304. 24. Raines MA, Kolesnick RN, Golde DW. Sphingomyelinase and ceramide activate mitogen-activated protein kinase in myeloid HL-60 cells. J Biol Chem 1993; 268:14572-14575. 25. Ragg SJ, Kaga S, Berg KA et al. The mitogen-activated protein kinase pathway inhibits ceramideinduced terminal differentiation of a human monoblastic leukemia cell line, U937. J Immunol 1998; 161:1390-1398. 26. Suchard SJ, Mansfield PJ, Boxer LA et al. Mitogen-activated protein kinase activation during IgGdependent phagocytosis in human neutrophils: inhibition by ceramide. J Immunol 1997; 158:4961-4967. 27. Venable ME, Bielawska A, Obeid LM. Ceramide inhibits phospholipase D in a cell-free system. J Biol Chem 1996; 271:24800-24805.

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28. Singh IN, Stromberg LM, Bourgoin SG et al. Ceramide inhibition of mammalian phospholipase D1 and D2 activities is antagonized by phosphatidylinositol 4,5-bisphosphate. Biochemistry 2001; 40:11227-12233. 29. Park BJ, Kim JH, Han JS et al. Effect of ceramide on apoptosis and phospholipase D activity in FRTL-5 thyroid cells. Exp Mol Med 1999; 31:142-150. 30. Han J, Shin I. Ceramide does not inhibit protein kinase C beta-dependent phospholipase D activity stimulated by anti-Fas monoclonal antibody in A20 cells. Cell Signal 2000; 12:731-736. 31. Chang Y, Abe A, Shayman JA. Ceramide formation during heat shock: a potential mediator of alpha B-crystallin transcription. Proc Natl Acad Sci USA 1995; 92:12275-12279. 32. Ravagnan L, Gurbuxani S, Susin SA et al. Heat-shock protein 70 antagonizes apoptosis-inducing factor. Nat Cell Biol 2001; 3:839-843. 33. Beere HM, Wolf BB, Cain K et al. Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat Cell Biol 2000; 2:469-475. 34. Kondo T, Matsuda T, Tashima M et al. Suppression of heat shock protein-70 by ceramide in heat shock-induced HL-60 cell apoptosis. J Biol Chem 2000; 275:8872-8879. 35. Hanna AN, Chan EY, Xu J et al. A novel pathway for tumor necrosis factor-alpha and ceramide signaling involving sequential activation of tyrosine kinase, p21(ras), and phosphatidylinositol 3kinase. J Biol Chem 1999; 274:12722-12729. 36. Gulbins E, Brenner B, Koppenhoefer U et al. Fas or ceramide induce apoptosis by Ras-regulated phosphoinositide-3-kinase activation. J Leukoc Biol 1998; 63:253-263. 37. Schubert KM, Scheid MP, Duronio V. Ceramide inhibits protein kinase B/Akt by promoting dephosphorylation of serine 473. J Biol Chem 2000; 275:13330-13335. 38. Zhou H, Summers SA, Birnbaum MJ et al. Inhibition of Akt kinase by cell-permeable ceramide and its implications for ceramide-induced apoptosis. J Biol Chem 1998; 273:16568-16575. 39. Aggarwal BB, Higuchi M. Role of ceramide in tumour necrosis factor-mediated apoptosis and nuclear factor-kappa B activation. Biochem Soc Trans 1997; 25:1166-11671. 40. Gamard CJ, Dbaibo GS, Liu B et al. Selective involvement of ceramide in cytokine-induced apoptosis. Ceramide inhibits phorbol ester activation of nuclear factor kappaB. J Biol Chem 1997; 272:16474-16481. 41. Johns LD, Sarr T, Ranges GE. Inhibition of ceramide pathway does not affect ability of TNFalpha to activate nuclear factor-kappa B. J Immunol 1994; 152:5877-5882. 42. Aggarwal BB. Apoptosis and nuclear factor-kappa B: a tale of association and dissociation. Biochem Pharmacol 2000; 60:1033-1039. 43. Garcia-Ruiz C, Colell A, Mari M et al. Direct effect of ceramide on the mitochondrial electron transport chain leads to generation of reactive oxygen species. Role of mitochondrial glutathione. J Biol Chem 1997; 272:11369-11377. 44. Davis MA, Flaws JA, Young M. et al. Effect of ceramide on intracellular glutathione determines apoptotic or necrotic cell death of JB6 tumor cells. Toxicol Sci 2000; 53:48-55. 45. Fernandez-Ayala DJ, Martin SF, Barroso MP et al. Coenzyme Q protects cells against serum withdrawal-induced apoptosis by inhibition of ceramide release and caspase-3 activation. Antioxid Redox Signal 2000; 2:263-275. 46. Yoshimura S, Banno Y, Nakashima S et al. Inhibition of neutral sphingomyelinase activation and ceramide formation by glutathione in hypoxic PC12 cell death. J Neurochem 1999; 73:675-683. 47. Liu B, Andrieu-Abadie N, Levade T et al. Glutathione regulation of neutral sphingomyelinase in tumor necrosis factor-alpha-induced cell death. J Biol Chem 1998; 273:11313-11320. 48. Berridge MJ. Calcium signalling and cell proliferation. Bioessays 1995; 17:491-500. 49. Condrescu M, Reeves JP. Inhibition of sodium-calcium exchange by ceramide and sphingosine. J Biol Chem 2001; 276:4046-4054. 50. Chik CL, Li B, Negishi T et al. Ceramide inhibits L-type calcium channel currents in rat pinealocytes. Adv Exp Med Biol 1999; 460:51-59. 51. Kitatani K, Oka T, Murata T et al. Acceleration by ceramide of calcium-dependent translocation of phospholipase A2 from cytosol to membranes in platelets. Arch Biochem Biophys 2000; 382:296-302. 52. Muriel MP, Lambeng N, Darios F et al. Mitochondrial free calcium levels (Rhod-2 fluorescence) and ultrastructural alterations in neuronally differentiated PC12 cells during ceramide-dependent cell death. J Comp Neurol 2000; 426:297-315. 53. Jayadev S, Barrett JC, Murphy E. Elevated ceramide is downstream of altered calcium homeostasis in low serum-induced apoptosis. Am J Physiol Cell Physiol 2000; 279:C1640-1647. 54. Laulederkind SJ, Bielawska A, Raghow R et al. Ceramide induces interleukin 6 gene expression in human fibroblasts. J Exp Med 1995; 182:599-604.

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55. Shinoda J, Kozawa O, Tokuda H. et al. Effect of ceramide on interleukin-6 synthesis in osteoblastlike cells. Cell Signal 1999; 11:435-441. 56. Kuo ML, Chen CW, Jee SH et al. Transforming growth factor beta1 attenuates ceramide-induced CPP32/Yama activation and apoptosis in human leukaemic HL-60 cells. Biochem J 1997; 327:663-667. 57. Shimada T, Hiraishi H, Terano A. Hepatocyte growth factor protects gastric epithelial cells against ceramide-induced apoptosis through induction of cyclooxygenase-2. Life Sci 2000; 68:539-546. 58. Riboni L, Viani P, Bassi R et al. Biomodulatory role of ceramide in basic fibroblast growth factorinduced proliferation of cerebellar astrocytes in primary culture. Glia 2000; 32:137-145. 59. Zundel W, Giaccia A. Inhibition of the anti-apoptotic PI(3)K/Akt/Bad pathway by stress. Genes Dev 1998; 12:1941-1946. 60. Hajduch E, Balendran A, Batty IH et al. Ceramide impairs the insulin-dependent membrane recruitment of protein kinase B leading to a loss in downstream signalling in L6 skeletal muscle cells. Diabetologia 2001; 44:173-183. 61. Stratford S, DeWald DB, Summers SA. Ceramide dissociates 3'-phosphoinositide production from pleckstrin homology domain translocation. Biochem J 2001; 354:359-368. 62. Zundel W, Swiersz LM, Giaccia A. Caveolin 1-mediated regulation of receptor tyrosine kinaseassociated phosphatidylinositol 3-kinase activity by ceramide. Mol Cell Biol 2000; 20:1507-1514. 63. Huang C, Ma WY, Ding M et al. Involvement of sphingomyelinase in insulin-induced phosphatidylinositol 3-kinase activation. Faseb J 2001; 15:1113-1124. 64. Perks CM, Gill ZP, Newcomb PV et al. Activation of integrin and ceramide signalling pathways can inhibit the mitogenic effect of insulin-like growth factor I (IGF-I) in human breast cancer cell lines. Br J Cancer 1999; 79:701-706. 65. Grolleau A, Wietzerbin J, Beretta L. Defect in the regulation of 4E-BP1 and 2, two repressors of translation initiation, in the retinoid acid resistant cell lines. NB4-R1 and NB4-R2, Leukemia 2000; 14:1909-1914. 66. Pham FH, Sugden PH, Clerk A. Regulation of protein kinase B and 4E-BP1 by oxidative stress in cardiac myocytes. Circ Res 2000; 86:1252-1258. 67. Marchisio M, Bertagnolo V, Colamussi ML et al. Phosphatidylinositol 3-kinase in HL-60 nuclei is bound to the nuclear matrix and increases during granulocytic differentiation. Biochem Biophys Res Commun 1998; 253:346-351. 68. Neri LM, Martelli AM, Borgatti P et al. Increase in nuclear phosphatidylinositol 3-kinase activity and phosphatidylinositol (3,4,5) trisphosphate synthesis precede PKC-zeta translocation to the nucleus of NGF-treated PC12 cells. Faseb J 1999; 13:2299-2310. 69. Andjelkovic M, Alessi DR, Meier R et al. Role of translocation in the activation and function of protein kinase B. J Biol Chem 1997; 272:31515-31524 70. Tsugane K, Tamiya-Koizumi, K, Nagino M et al. A possible role of nuclear ceramide and sphingosine in hepatocyte apoptosis in rat liver. J Hepatol 1999; 31:8-17. 71. Webb PR, Wang KQ, Scheel-Toellner D et al. Regulation of neutrophil apoptosis: a role for protein kinase C and phosphatidylinositol-3-kinase. Apoptosis 2000; 5:451-458. 72. Goswami R, Kilkus J, Dawson SA et al. Overexpression of Akt (protein kinase B) confers protection against apoptosis and prevents formation of ceramide in response to pro-apoptotic stimuli. J Neurosci Res 1999; 57:884-893. 73. Burow ME, Weldon CB, Collins-Burow BM et al. Cross-talk between phosphatidylinositol 3-kinase and sphingomyelinase pathways as a mechanism for cell survival/death decisions. J Biol Chem 2000; 275:9628-9635.

CHAPTER 11

Ceramide in the Regulation of Neuronal Development: Two Faces of a Lipid Christian Riebeling and Anthony H. Futerman

Abstract

T

he notion proposed over a decade ago that ceramide is involved in cellular signaling events has stimulated a large number of studies that have attempted to define the precise function(s) of ceramide in signaling, and has revived interest in understanding the mechanisms of regulation of sphingolipid metabolism. Ceramide is now known to be involved in cellular events as diverse as proliferation, differentiation, senescence and apoptosis, and the ceramide that is involved in these processes can come from either SM breakdown or from de novo synthesis. Studies from our laboratory have helped define the role of ceramide derived from SM in the regulation of neuronal growth and development. Intriguingly, ceramide generated from SM upon binding of nerve growth factor to neurotrophin receptors can either induce neuronal cell death, or induce survival and outgrowth. Which of these two processes occurs is dictated by the developmental stage of the neuron, which is itself reflected in the level of expression of the neurotrophin receptor, p75NTR. The biochemical status of down-stream signaling molecules may be an additional determinant of how ceramide performs its disparate roles.

Ceramide and Neuronal Development and Death The development of the nervous system is accompanied by a considerable amount of programmed cell death. Neurons are produced in excess and compete with each other for limiting amounts of neurotrophins secreted by target cells. Indeed, the assumption that neurons die simply by passive starvation in the absence of trophic factors has been challenged by the finding that neurotrophins themselves can induce apoptotic cell death under certain conditions.1 Apoptotic cell death is characterized by removal of the dying cell without an inflammatory response. Several characteristic morphological changes occur during apoptosis, including shrinking of the cytoplasm, plasma membrane blebbing, nuclear chromatin condensation, and fragmentation of genomic DNA. In the early stages of apoptosis, phosphatidylserine is exposed on the cell surface, triggering cell engulfment by neighboring cells or phagocytes. The fundamental biochemical elements of the apoptotic pathway are conserved throughout the animal kingdom. Among the factors that regulate mammalian apoptosis are the Bcl-2 family of proteins, the adaptor protein ‘Apoptotic protease-activating factor 1’ (Apaf-1), and the cysteine protease family of caspases. Neurons, as might be expected, share the same basic apoptotic program as other cell types, but different types of neurons, and neurons at different developmental stages, express different combinations of Bcl-2 and caspase family members, which might be one way of controling the specificity of regulation.2 Ceramide has been implicated as a key player in the apoptosis of various cell types, including neurons. 3 Ceramide can be produced by sphingomyelin (SM) hydrolysis by Ceramide Signaling, edited by Anthony H. Futerman. ©2002 Eurekah.com and Kluwer Academic / Plenum Publishers.

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sphingomyelinases (SMase)4 with either an acidic (A-SMase) or neutral (N-SMase) pH optimum, or by de novo synthesis.5 In our laboratory, we have used neurons cultured from embryonic mice or rat hippocampus to study the role of ceramide (and other sphingolipids) in both neuronal development and in neuronal death. These neurons are cultured in such a way that axons and dendrites can be distinguished both morphologically and biochemically.6 Briefly, post-mitotic hippocampal neurons are plated on cover slips coated with poly-L-lysine. Cover slips can either be transferred to culture dishes containing a monolayer of astroglia,6 or transferred into a culture dish that contains B27 medium,7 allowing neuronal survival in the absence of a glial monolayer.8 Importantly, in both systems, cultures are maintained in serum-free medium. The growth of cultured hippocampal neurons has been classified into five distinct developmental stages.9 Stage 1 is characterized by a neuronal cell body exhibiting many lamellipodia. In stage 2, the lamellipodia are lost and a number of short processes, known as ‘minor processes’, are formed from the cell body. Within hours, one of the minor processes starts to grow rapidly and develops axonal characteristics; such neurons are ‘stage 3’. Branches are formed from the axon as collaterals, and as each new branch emerges, the growth cone of the original axon loses its lamellipodial appearance and elongation stops. Dendrites develop from minor processes in stage 4. Finally in stage 5, synaptic contacts between axons and dendrites are created. We originally demonstrated that ceramide-generation could regulate neuronal development, since it enhanced the formation of minor processes from lamellipodia.3 Moreover, in stage 3 neurons, the glycosylated metabolite of ceramide, glucosylceramide (GlcCer), is required for normal10 and accelerated11 axonal growth. Intriguingly, at both stages high concentrations of ceramide induce apoptosis3. We have gone on to demonstrate that ceramide produced endogenously (rather than exogenously-applied short-acyl chain ceramides, or ceramide produced by exogenously added bacterial or human SMase) in response to the binding of neurotrophins to the p75NTR receptor8 can regulate the rate of minor process formation, or of apoptosis in hippocampal neurons. The decision whether to enter these diametrically opposed pathways depends on the expression status of neurotrophin receptors and, as a consequence, intracellular ceramide levels.12

Neurotrophins as Modulators of Neuronal Survival and Death Neurotrophins were originally thought to be mainly involved in promoting neuronal differentiation and survival. Although this concept still remains true, recent work has demonstrated that neurotrophins can also directly induce neuronal apoptosis.2 In mammals, four neurotrophins are known, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Neurotrophins are produced as precursor proteins, which are cleaved at dibasic amino acids to form mature forms of 118-120 amino acids. In their mature form they generally function as noncovalently associated homodimers, but at least some neurotrophin subunits are able to form heterodimers with other neurotrophins.13 All four neurotrophins interact with two receptor types, the shared p75NTR and three distinct receptor tyrosine kinases of the trk receptor family, trkA, trkB, and trkC. Neurotrophins directly bind and dimerize trk receptors, resulting in activation of the tyrosine kinase present in the cytoplasmic domain, which results in subsequent tyrosine phosphorylation of selective cellular substrates, such as phospholipase Cγ14 and phosphatidylinositol-3-kinase.15 Tyrosine kinase-mediated signaling by endogenous trk receptors promotes survival and/or differentiation in all neuronal populations examined to date. The other neurotrophin receptor, p75NTR, is a distant member of the tumor necrosis factor (TNF) receptor family16 and was the first neurotrophin receptor identified. It comprises an extracellular region of four cysteine-rich repeats, all of which are required for ligand binding, a single transmembrane domain, and a cytoplasmic tail. No known catalytic motifs are present in the cytoplasmic tail but a region of approximately 80 amino acids near the C-terminus

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displays strong homology to the death domains of TNF receptors.17 Initially p75NTR was thought to be a low-affinity receptor specific for NGF but it was later shown to bind all neurotrophins with a very similar affinity. Recent studies have shown that unprocessed pro-neurotrophins represent a large amount of the secreted forms, and that pro-NGF binds to p75NTR with an affinity that is five-fold higher than the mature form; in contrast, trkA preferentially binds to the processed form of NGF.18 The first evidence (Chapter 12) for the involvement of ceramide in the down-stream signaling pathway elicited by NGF binding to p75NTR was obtained in T9 glioma cells, a cell line that does not express trkA. The latter is of particular importance since it has been suggested that the major function of p75NTR was to modify the activity of trk receptors; thus, ligand binding by p75NTR leads to an increase in high affinity trkA binding sites, enhanced trkA autophosphorylation in response to NGF, and increased selectivity of trkA for the neurotrophin.19 In T9 glioma cells, NGF binding led to an increase in intracellular ceramide levels,20 which resulted from binding of NGF to p75NTR since these cells do not express trkA receptors. In contrast, in PC12 cells that do express trkA and p75NTR, NGF did not induce ceramide generation,21 whereas BDNF, which binds p75NTR but not trkA, led to increased ceramide levels, suggesting that trkA negates the signaling properties of p75NTR with respect to ceramide generation (Fig. 1). Although various p75NTR-interacting proteins have been identified, including members of the TRAF family,22 NRIF,23 NADE24 and NRAGE,25 their precise roles in the apoptotic signaling cascade have not yet been defined, and little is known about how these interact with the ceramide signaling pathway. Our data, discussed below, has addressed some of these issues by analyzing a number of the down-stream interactors that are involved in NGF-induced ceramide generation via N-SMase, but not A-SMase, in hippocampal neurons that express p75NTR but not trkA.

SMases and Their Regulation The major form of SMase found in the brain is N-SMase, and we would like to speculate that N-SMase, rather than the A-SMase that is found at high levels in other tissues, is the SMase that is largely responsible for ceramide generation in the central nervous system during normal development, particularly that which is under control of trophic factors. This is based on our finding that A-SMase is not involved in signaling through the p75NTR in cultured hippocampal neurons,8 as neurons derived from wild type or A-SMase-deficient26 mice are equally susceptible to NGF-induced cell death.8 In contrast, excitotoxic cell death is partially abolished in A-SMase-deficient neurons,8,27 suggesting that different pathways of ceramide generation, via A-SMase or N-SMase, can exist in the same neuron, and are involved in ceramide generated from distinct ligand-receptor interactions. This would be consistent with many observations showing an important role for A-SMase in ceramide generation in response to apoptotic signals. For instance, in the case of the apoptotic pathway initiated by binding of TNF to the TNF receptor 1, A-SMase is activated by the TNF receptor death domain-associated proteins TRADD and FADD28, and in Kym-1 rhabdomyosarcoma cells, ceramide generated via A-SMase in response to TNF treatment occurs in the final stage of apoptosis.29 Unfortunately, progress in identifying and cloning N-SMase has lagged well behind that of A-SMase. Several putative N-SMase activities have been purified over the past few years, but a convincing case that any of them are the genuine N-SMase is still lacking. A neutral, membrane-associated, Mg2+-stimulated SMase sensitive to the inhibitor scyphostatin30 as well as to glutathione31 has been purified from bovine brain,32 but not cloned. Purification of N-SMase from rat liver plasma membrane has revealed a distinct enzyme not affected by glutathione,33 and six distinguishable activities were purified from heavy membrane fractions from bovine brain.34 The first cloned putative N-SMase35 was shown to reside in the endoplasmic reticulum and is most likely a phospholipase C specific for lyso-platelet activating factor.36,37 A second N-SMase was identified by expression cloning of a human kidney cDNA library38 and the product of a brain specific clone was shown to reside in the Golgi apparatus.39 Thus, to date identification of a genuine N-SMase remains elusive.

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Figure 1. The cross-talk between trkA and p75NTR. Upon binding of NGF, trkA abolishes the activation of N-SMase by p75NTR, whereas p75NTR enhances signaling through trkA by an unknown mechanism. PI-3K, phosphatidylinositol-3-kinase; PLCγ, phospholipase Cγ; TRAF, TNF receptor-associated factor.

However, despite the lack of a genuine cloned N-SMase, there is little doubt that this enzyme is involved in ceramide generation in signaling pathways. Support for this has been obtained, amongst others, by studies showing that activation of N-SMase by TNF occurs via a distinct protein-protein interaction, involving the cytoplasmic domain of the TNF receptor 1 called the NSD (neutral SMase activation domain), which binds to the adaptor protein FAN40 (factor associated with neutral SMase activation) (Chapter 8). FAN, a member of the family of WD-repeat proteins, activates N-SMase by an unknown mechanism. Other potential players involved in N-SMase activation have been identified. Activation of phospholipase A2 and arachidonic acid accumulation precedes SM hydrolysis in response to TNF in HL60 cells;41 moreover, cells lacking phospholipase A2 are resistant to TNF-induced apoptosis and susceptibility to TNF can be restored by ectopic expression of phospholipase A2.42 A further level of regulation of N-SMase occurs by intracellular oxidation through the inhibition of N-SMase by reduced glutathione.31 Levels of oxidized glutathione are elevated in response to TNF in rat primary astrocytes, oligodendrocytes, microglia, and C6 glial cells.43 Thus, some up- and down-stream players in the N-SMase pathway are beginning to emerge.

Downstream Players in the Ceramide Response Recent studies in our laboratory have identified two down-stream players in the pathway initiated by binding of NGF to p75NTR and subsequent activation of N-SMase, namely death-associated protein (DAP)-kinase44 and jun kinase,8 although it should be stressed that neither of these kinases are the initial proteins with which N-SMase interacts. DAP-kinase is a Ca2+/calmodulin-regulated serine/threonine kinase mediating the apoptotic responses of several intra- and extracellular stress signals. It is a multi-domain protein containing a conserved death domain.17 The pro-apoptotic function of DAP-kinase requires the joint action of several domains and depends on autophosphorylation.45 In the developing and adult rat central nervous system, DAP-kinase mRNA is widely expressed in proliferative and post mitotic regions within the cerebral cortex, hippocampus and cerebellar Purkinje cells, from embryonic day 13 onwards.46 Expression in the brain is markedly decreased post-natally, but remains high in a number of areas, particularly the hippocampus.

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In culture, hippocampal neurons derived from DAP-kinase-deficient mice are almost completely resistant to treatment with short-chain ceramide and to NGF.44 Interestingly, phosphorylation of serine 308, which is required for auto-inhibition of DAP-kinase, is rapidly reduced in response to ceramide.45 However, a C-terminal peptide of DAP-kinase distinct from the auto-phosphorylation site efficiently blocks apoptosis by either short-chain ceramide or treatment with bacterial SMase.44 Moreover, over-expression of a kinase-dead mutant of DAP-kinase in PC12 cells abolishes ceramide-induced apoptosis.47 In addition, protein levels of DAP-kinase increase early in response to ceramide in hippocampal neurons, suggesting a further level of regulation. Together, these data suggest that although DAP-kinase is a central player in ceramide-induced cell death, the pathway in which DAP-kinase is involved is not the only one via which ceramide can induce apoptosis in neurons, since ceramide can kill neurons after long times even in DAP-kinase-deficient mice,44 again indicating the variety of levels at which the response to ceramide can be regulated. Ceramide generation is also up-stream to jun kinase in the signaling pathway elicited by NGF binding to p75NTR in hippocampal neurons. Previous studies48 had implicated jun kinase in neuronal death induced by neurotrophins, and we recently demonstrated that incubation of neurons with scyphostatin prior to incubation with NGF, blocked both neuronal cell death, and importantly, jun kinase activation.8 Dual roles have been suggested for jun kinase, in development and stress responses, with different jun kinase pools serving different functions.49 Our data show that ceramide generation is necessary for both NGF-induced neuronal cell death and jun kinase activation, suggesting that ceramide somehow regulates or modulates one of the interactors or kinases up-stream of jun kinase. The current state of knowledge regarding activation pathways to jun kinase50 does not allow the delineation of a precise mechanism by which ceramide might activate jun kinase. An intriguing option might involve modulation of the accessibility of p75NTR and interactor proteins to jun kinase via scaffolding proteins such as the jun-interacting proteins.51 Mediators that could provide an initiating link from p75NTR to jun kinase have not yet been determined, although a growing list of p75NTR interactors has emerged in recent years, as discussed above. It will obviously be of interest to examine the effects of manipulating ceramide levels on the interaction of these diverse proteins with p75NTR, and on the down-stream cascades thus activated. A number of other putative down-stream targets of ceramide have been identified in non-neuronal cells. By affinity chromatography, cathepsin D was identified as a ceramide-binding protein,52 and a number of protein phosphatases (Chapter 6) and protein kinases have also been identified. However, the mode of their activation or inhibition by ceramide has not been fully characterized, and none of them has been shown to unambiguously directly bind to ceramide. A relatively well-studied example is protein phosphatase 2A, which is activated upon ceramide treatment.53 Protein phosphatase 2A inactivates protein kinase C (PKC)54 and protein kinase B/Akt55, as well as down-stream targets of these kinases involved in apoptosis, such as bcl-2.56 The dephosphorylation of bcl-2 results in loss of its anti-apoptotic potency. Protein phosphatase 1 is also involved in the ceramide dependent dephosphorylation of the transcriptional regulator retinoblastoma gene product (Rb)57. In addition, classical and novel PKC isoforms are inhibited by ceramide, most probably via protein phosphatase 2A, whereas the activity of the atypical isoform PKCζ is stimulated;58 interestingly, PKCζ is involved in activation of jun kinases.59 In contrast, kinase suppressor of Ras (KSR) has been shown to be required for ceramide-induced activation of the mitogen activated protein kinase (MAPK) p42/p4460 (Chapter 7). It is not possible to conclude a section on down-stream targets of ceramide without a brief discussion of the role of mitochondria in this process, particularly as there is emerging evidence suggesting a direct role for ceramide in the biochemical events that occur in mitochondria during apoptosis. By way of example, stimulation of the CD95 receptor upon binding of a CD95 ligand induces formation of a signaling complex,61 in which caspase-8 is activated,

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which subsequently cleaves procaspase-3 resulting in activation of the main executor caspase, caspase-3. In the majority of cell types, caspase-3, and thus apoptosis, is not directly activated but requires the activation of caspase-9 which involves the mitochondria;62 this is also the case in neurons.63 Jun kinase may provide a link between ceramide and mitochondria since active jun-kinase can induce cytochrome c release from mitochondria in a cell free assay.64 All mitochondrial activities in apoptosis can be blocked by over-expression of bcl-2 or bcl-xL.65 Thus, ceramide-induced cytochrome c release from isolated mitochondria can be prevented by pre-incubation with Bcl-2.66 Importantly, Bcl-2 expression is widespread in the developing central nervous system, remains high in the adult peripheral nervous system,67 and is up-regulated in response to ischemia. Moreover, bcl-2 overexpression can block apoptosis induced by neurotrophin withdrawal,68 suggesting that the mitochondria, as it does in other cells, will also play a key role in ceramide-induced apoptosis in the nervous system.

A Role for de novo Ceramide Synthesis To date, we have mainly discussed ceramide generated via the action of SMases. However, there is good evidence that ceramide synthesized de novo also plays an important role in apoptosis. Ceramide synthesis occurs on the cytosolic leaflet of the endoplasmic reticulum69 and therefore has a distinct topology from ceramide generated via SMases, which is probably produced at the inner leaflet of the plasma membrane although this issue has still not been completely clarified.70 Nevertheless, inhibition of ceramide synthesis by fumonisin B1 often blocks apoptosis,71 suggesting that de novo ceramide synthesis also participates in apoptotic and developmental processes. Ceramide production by this route is induced by DNA-damaging agents5 and also by TNF,72 and shows the same apoptotic features as ceramide generated from SM. Likewise, in hippocampal neurons, NGF-induced cell death can be abolished by long-term (24 h) treatment with fumonisin B1 (unpublished data), although it is not clear if this effect is due to altering cellular levels of SM, or directly due to de novo ceramide synthesis. In contrast, the development of hippocampal neurons is not altered by treatment with fumonisin B1 within the first 24 to 48 h in culture,10,73 suggesting that sphingolipid synthesis is not required for minor process formation at stage 2, or the initiation of axonal outgrowth in early stage 3. Later in stage 3, fumonisin B1 abolishes axonal elongation and this effect is due to a reduction in the rate of synthesis of GlcCer,3,74 which is required for axonal growth, rather than accumulation of ceramide. Thus, an integrated view of the role of ceramide in regulating neuronal growth or death must take into account the possibility, at least over long-time periods, that ceramide is rapidly metabolized, and the effect of the metabolites needs to be carefully distinguished from that of ceramide per se.

Signaling Through Ceramide Metabolites Notable amongst ceramide metabolites is sphingosine-1-phosphate, which is produced by the hydrolysis of ceramide to sphingosine and its subsequent phosphorylation by sphingosine kinase. Several ceramidases potentially responsible for the first step in this pathway have been identified, residing in lysosomes,75 plasma membrane76 and mitochondria77 (Chapters 4 and 5). Sphingosine is known to cause apoptosis in several cell types, including hippocampal neurons.78 In contrast, sphingosine-1-phosphate, which is a paracrine as well as an intracellular survival factor, often works in direct contrast to ceramide.79 However, it induces cell death in hippocampal neurons80 and can cause their developmental arrest in stage 1 (unpublished results). GlcCer can also directly affect neuronal development, and the development of other cell types, and its synthesis from ceramide can protect against the pro-apoptotic effects of ceramide. In cancer cells, ceramide glycosylation to GlcCer rescues them from ceramide-induced apoptosis and is associated with multidrug resistance81 (Chapter 14). In hippocampal neurons, GlcCer is required for normal axon growth during stage 33 and its synthesis is stimulated by laminin and basic fibroblast growth factor (bFGF).11 Treatment of neurons at this stage with either fumonisin

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Figure 2. The dual role of ceramide in the development of hippocampal neurons. At stage 1, low expression of p75NTR results in low levels of ceramide generation upon binding of NGF, and formation of minor processes is induced. Note that this can also be achieved by exogenous addition of low concentrations of ceramide. Later, in stage 3, p75NTR is expressed at higher levels and higher levels of ceramide are generated in response to NGF binding. Apoptosis is induced, and can be abolished by inhibition of N-SMase activity or of DAP-kinase.

B1 or D-erythro-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) blocks axonal outgrowth.10 In contrast, the glucocerebrosidase inhibitor, conduritol-B-epoxide, stimulates axonal growth by enhancing either the rate of formation or stabilization of axonal branches. Axonal outgrowth is accompanied by a tremendous increase of membrane surface, and in this respect it is of interest that the activity of the rate-limiting enzyme of phosphatidylcholine biosynthesis, CTP:phosphocholine cytidylyltransferase (CCT), in brain and hippocampal neurons is directly activated by GlcCer.82 Thus, GlcCer may ‘signal’ via direct activation of CCT, whereas ceramide essentially has no effect on this enzyme.82

Conclusions Ceramide is an important signaling molecule involved in the regulation of the development and the death of neurons. It can be generated via binding of NGF to p75NTR leading to activation of N-SMase. The level of ceramide generated can dictate whether development is stimulated or whether apoptosis is induced. The mechanism by which ceramide induces these disparate effects is not known (Fig. 2), but may involve activation of different down-stream signaling pathways, such as DAP kinase and jun kinase. Alternatively, the possibility that a specific concentration of ceramide must be reached to allow formation of membrane rafts,83,84 and thus sequestering and activation of receptors in this membrane domain, is an attractive new hypothesis that deserves wide-spread attention. Although this aspect of ceramide signaling has not been discussed in this review, renewed interest in the biophysics of ceramide (Chapter 2), and the effect of its generation on properties of the lipid bilayer,70 are sure to add an additional layer of excitement to the study of ceramide signaling in neurons and in other cells types.

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Acknowledgements Christian Riebeling is supported by a Research Training Network fellowship from the European Union (HPRN-CT-2000-00077). Work in the Futerman laboratory is currently supported by the Israel Science Foundation, the European Union (RTN1-1999-00382, and QLG3-CT-1999-573), the Buddy Taub Foundation, the National Niemann-Pick Foundation, and the Minerva Foundation, Munich, Germany.

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CHAPTER 12

Neurons, Neurotrophins and Ceramide Signaling: Do Domains and Pores Contribute to the Dichotomy? Rick T. Dobrowsky

Introduction

O

ver the last decade, ceramide has received considerable notoriety as a lipid second messenger that mediates a variety of cell stress responses induced by numerous agonists and environmental stimuli. Although ceramide signaling continues to garner the greatest interest due to its contribution to apoptosis, ceramide production may also stimulate mitogenic signals, promote the activity of growth arrest pathways and induce or inhibit cell differentiation. Not surprisingly, studies on the role of ceramide signaling in the central and peripheral nervous systems have revealed a similar myriad of complex responses. For example, ceramide may induce neuronal apoptosis or protect from apoptosis induced by excitotoxins or growth factor withdrawal,1 inhibit axonal growth or promote axonal or dendritic growth,2 inhibit the inwardly rectifying K+ current3 or enhance the outward delayed rectifier K+ current4 and in response to cannabinoids (Chapter 13), control metabolic processes or induce apoptosis of glial cells.5 The factors that influence how a cell may respond to ceramide production are slowly being identified. In general, these factors may be classified into specific protein targets,6,7 potential influence of ceramide production on lipid domain formation at the site of generation,8-11 and the integrated interaction between enzymes producing ceramide with those controlling its degradation.12 These categories are not mutually exclusive since activation of ceramide metabolizing ehnzymes may not only inactivate ceramide, but also activate a “sphingolipid biostat” by enhancing the production of sphingosine-1-phosphate, promoting cell growth and survival.13 Moreover, the site of ceramide production may affect its ability to interact with other lipid constituents and impart biophysical properties specific for particular ceramideprotein interactions. It is now more or less accepted that most cell types, including neurons and glia, contain specific lipid microdomains that are relatively depleted of phospholipids but enriched in SM, cholesterol, ceramide and glycosphingolipids.14 These domains have a lipid phase behavior, called the liquid-ordered phase, that is intermediate to the more fluid liquid-crystalline or more solid gel phases.15 Although similarities exist in the extension and packing of acyl chains between the gel and liquid-ordered phase, the latter has a greater degree of lateral mobility.16 Two biophysical properties of liquid-ordered domains are their insolubility in non-ionic Ceramide Signaling, edited by Anthony H. Futerman. ©2002 Eurekah.com and Kluwer Academic / Plenum Publishers.

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detergents at low temperature and low buoyant density. Accordingly, isolation of these domains following detergent extraction of cells and centrifugation through sucrose gradients has facilitated the characterization of lipid raft domains.17 Although numerous names have been given to liquid-ordered domains, lipid rafts is a general descriptor that has some consensus. However, specialized detergent-insoluble raft domains also exist. For example, caveolae share a similar lipid composition with lipid rafts but also incorporate the protein caveolin-1.18 In contrast, glycosphingolipid signaling domains have been described that are deficient in SM but still remain insoluble in detergent.19 Importantly, biophysical studies support that ceramide may become concentrated in lipid rafts and actually stabilize domain formation.9,11 These biophysical interactions may underlie the role of ceramide-rich lipid rafts in controlling some aspects of cell signaling.20,21 Further, ceramides can form pores in phospholipid-rich planar membranes.10 This raises the possibility that, in certain membranes in vivo, ceramide may form channels.10 The broad goal of this brief retrospective is to examine if knowledge of ceramide domain/ pore formation may help explain or predict the outcome of some aspects of neurotrophininduced ceramide signaling in axonal growth and cell death. To this end, we will present an overview of recent findings on how the compartmentalized production of ceramide may promote distinct ceramide-lipid, ceramide-protein interactions at the plasma membrane versus internal membranes. However, the reader is also directed to additional reviews on how the biophysical properties of ceramide in phospholipid bilayers may regulate signaling.22,23

Influence of Ceramide on Lipid Raft Domains The bulk of biological membranes are composed of phospholipids containing relatively unsaturated acyl chains with multiple cis double bonds. In general, phospholipids do not undergo tight packing within the membrane, do not undergo extensive intermolecular hydrogen bonding and are likely to be in the typical liquid-crystalline state.24 In contrast, sphingolipids primarily contain saturated acyl chains or long acyl chains (C22-C24) that contain one double bond. These saturated acyl chains promote tight packing and the presence of both the amide linkage and allylic hydroxyl group as components of the sphingoid backbone may facilitate extensive hydrogen bonding between sphingolipids.25 Although these properties of sphingolipids are critical for contributing to formation of liquid-ordered domains, they are not sufficient. Detergent resistant lipid rafts isolated from plasma membranes may contain about 15-33 mol% cholesterol depending upon the cell and developmental stage.11,17,26 Importantly, the detergent-insolubility of lipid rafts is attributed solely to the lipid composition of these domains and is independent of protein components.27 The presence of cholesterol is a critical component for formation of liquid-ordered domains since it selectively promotes tight packing with saturated lipids.9,27-29 It is also important to note that the liquid-ordered phase may form between cholesterol and disaturated molecular species of phosphatidylcholine.30 However, SM and glycosphingolipids are the primary source of these saturated acyl chains in plasma membranes. Interestingly, some sterols such as coprostanol, androstenol and cholesterol sulfate inhibit formation of liquid-ordered domains.29 Consistent with this observation, 4-cholesten3β-one, an oxidation product of cholesterol, can disrupt the formation of lipid rafts and decrease receptor-linked tyrosine kinase signaling in caveolae by interfering with the ability of the activated receptor to couple to downstream signaling partners.31 Ceramide is also a constitutive component of lipid raft domains. Several studies have reported that the basal amount of ceramide associated with lipid rafts may account for 50-60% of total cellular ceramide.26,32-35 Not surprisingly, agonists such as interleukin-1β and tumor necrosis factor-α (TNF) that increase cellular ceramide levels at the plasma membrane may also increase the ceramide content of raft domains.35,36 However, TNF can also induce the hydrolysis of cholesterol-poor pools of SM located outside of liquid-ordered (lipid raft)

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domains,32 leading to potential differences in the properties of ceramide produced in these distinct membrane regions. Biophysical studies strongly support that ceramide can form microdomains and/or alter the physical properties of lipid rafts (see Chapters 2 and 3). The effects of ceramide, however, are influenced by its acyl chain length, and the presence or absence of cholesterol and phospholipids in raft domains.25,37,38 In the absence of cholesterol, ceramide-SM complexes may readily form due to similarities in acyl chain lengths and formation of a hydrogen bond network.11 Indeed, the presence of SM augments the formation of ceramide-enriched microdomains.25 Given that naturally occurring long-chain ceramides have a phase transition temperature above that of SM, a localized change in the SM:cermide ratio may dramatically stabilize the gel state of the local region and induce partitioning from the more fluid and surrounding phospholipids.11 Although ceramide-SM interactions promote ordered phase formation, the properties of these domains are likely to be distinct form those formed in a liquid-ordered phase in the presence of cholesterol.9,29 A recent study of cerebellar granule cells differentiated in vitro for 2-17 days indicates that lipid rafts contain about 1-1.5 mol% ceramide relative to total lipid (phospholipid+sphingolipid+sterol).26,33 Typically, cellular levels of ceramide rise from 2-5 fold depending upon the type of stimulus.39-41 Thus, it would not be unreasonable to expect ceramide levels to range from at least 2-7.5 mol% relative to total lipid in this compartment (this is probably a conservative estimate). Interestingly, addition of 10 mol% bovine brain ceramides to SM-rich membranes containing 33 mol% cholesterol had no effect in increasing acyl chain order of the membrane, i.e., no formation of ceramide-SM complexes.11 In contrast, addition of as little as 3 mol% of non-hydroxylated ceramides significantly stabilized domain formation in SM-phospholipid rich membranes containing 15 mol% cholesterol.9 Thus, the presence of phospholipids within raft domains may increase the ability of ceramide to stabilize domain formation. Indeed, lipid rafts do contain some phospholipid,26,32,33 and are important sites for agonist-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate.42,43 Taken together, these data strongly support that 1) ceramide production in lipid rafts can affect the stability of liquid-ordered domains and 2) ceramide can produce its own microdomains (not a liquid-ordered phase) with distinct biophysical properties from the surrounding plasma membrane.

Effect of Ceramides on SM-Cholesterol Poor Membranes The de novo metabolic route for ceramide production begins with the formation of the sphingoid backbone via the enzyme serine-palmitoyl transferase to form 3-ketosphinganine. Following reduction, sphinganine may then serve as substrate for the enzyme dihydroceramide synthase (often called ceramide synthase).12 Although agonist-induced ceramide production may occur via SM hydrolysis, Kolesnick and colleagues were the first to show that chemotherapeutics may also produce apoptogenic pools of ceramide via activation of ceramide synthase.44 Subsequently, numerous reports have ascribed a role for the de novo pathway in ceramidemediated biologies. The enzyme ceramide synthase localizes to the endoplasmic reticulum and possibly mitochondria.45 These membranes are rather deficient in SM and cholesterol46 and do not form liquid-ordered domains.47 Depending on the species, purified mitochondrial membranes isolated from adult brain tissue contain approximately 1.9-3.7 mol% SM and about 7 mol% cholesterol48 (both relative to total lipid); plasma membrane values range from 10-20 mol% and 30-40 mol% for SM and cholesterol, respectively.47 The decreased cholesterol and increased phospholipid content of these membranes may have specific consequences on the formation of ceramide-rich domains. Indeed, addition of up to 25 mol% of bovine brain ceramides to model membranes composed of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) or 1,2-dipalmitoyl phosphatidylcholine (DPPC) did not result in microdomain formation.11 In contrast, addition of 8-12 mol % bovine brain ceramides to

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DPPC bilayers resulted in the greatest cluster size (number of molecules) in the ceramide-enriched phase which decreased upon addition of 25-40 mol % ceramide.49 Similarly, addition of 10 mol% bovine brain ceramides to 1,2-dimyristoyl phosphatidylcholine (DMPC) bilayers resulted in ceramide microdomain formation.50 This was attributed to the hydrophobic mismatch between the longer chain acyl species present in bovine brain ceramides and the relatively short myristoyl chain of the DMPC.50 However, this same group further reported that ceramide-enriched microdomains were also formed by SMase treatment of POPC:N-palmitoylSM (C16:0 SM) bilayers.25 Although acyl chain mismatch is not as apparent in this system, increased ceramide domain formation may arise from the increased tendency of the newly produced C16:0 ceramide to form a gel phase at 37oC.11 Further, a recent biophysical study using mixed lipid monolayer films containing DMPC with either N-palmitoyl-sphingosine (C16:0 ceramide) or N-nervonoyl-sphingosine (C24:1 ceramide) found that the different ceramides created very distinct “domain morphologies”.51 Although it is difficult to directly extrapolate these studies to interactions within more complex biological membranes, collectively, they support that the ability of ceramide to form a microdomain is strongly influenced by the mol% ceramide and the molecular species of the acyl chains in both ceramide and the surrounding phospholipids. Can we now relate any of the biophysical data to what may occur during de novo synthesis of ceramide in response to an agonist in cholesterol-poor membranes? B-cell receptor crosslinking by anti-IgM antiserum leads to activation-induced cell death via an apoptotic pathway.52 In Burkitt’s lymphoma Ramos cells, a critical component of this pathway is the initial de novo synthesis of C16:0 ceramide at 6 hr followed by a latter increase in the formation of a C24:1 ceramide species.53 Importantly, inhibition of de novo ceramide production with fumonisin B1 (a ceramide synthase inhibitor) blocked anti-IgM-induced ceramide production, loss of mitochondrial function and apoptosis.53 This result would suggest that de novo ceramide production within either mitochondrial or microsomal membranes is necessary to induce mitochondrial dysfunction. Based upon the biophysical data presented above, these results raise the possibility that the early production of C16:0 ceramide may initially promote more lateral phase separation leading to disruption of mitochondrial processes, i.e., changes in permeability or increased oxidation.54 However, no evidence exists to strongly support or negate a role of ceramide-domain formation in disrupting mitochondrial function. Alternatively, rapid production of C16:0 ceramide may lead to the formation of a stable channel which has been demonstrated in cholesterol poor-phospholipid rich lipid membranes.10 The size of pores composed of C16:0 ceramide is estimated to be large enough to accommodate the release of cytochrome c if they form in the outer mitochondrial membrane.10 In this regard, direct addition of C2-ceramide, which can also form a stable pore,10 to isolated mitochondria induced cytochrome c release. 55 A complicating point to this discussion, however, is that de novo ceramide synthesis occurs via the formation of dihydroceramide, a molecule that can not form channels.10 Formation of ceramide requires enzymatic desaturation of the dihydro precursor. Although increases in mitochondrial levels of ceramide have been demonstrated in vivo,56 it remains unclear whether ceramide formation occurs directly in mitochondria, via the action of the microsomal dihydroceramide desaturase57 upon mitochondrial pools of newly formed dihydroceramide, or as a result of transfer from the endoplasmic reticulum. A caveat to the latter possibility is that the half-time for the rate of ceramide transfer between lipid bilayers is in the orders of days.58 Finally, since acyl chain length may influence both ceramide microdomain morphology51 and microdomain formation in phospholipid-rich membranes,11 it would not be unreasonable to expect that increased de novo production of increasingly heterogeneous ceramide species53 may affect domain formation/function and influence ceramide signaling. It will be interesting to determine if the addition of more hydrophobic ceramides may affect channel formation by C16:0 ceramide.

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Are There any Relationships Between Neurotrophins, Ceramide, Lipid Rafts and Neuronal Biology? Neurotrophins comprise a family of growth factors that serve varied roles in the development, survival and differentiation of various neuronal populations.59 Neurotrophins mediate their affects via interaction with two receptors, the Trk family of receptor-linked tyrosine kinases and p75NTR, the common neurotrophin receptor.60 p75NTR activation may contribute prominently to cell stress responses and apoptosis61 via increased ceramide production41,62-66, activation of stress-activated protein kinases,67-69 and modulation of NF-κB activity.70-72 Not surprisingly, p75NTR-dependent ceramide generation has been associated with the induction of apoptosis,73 neuroprotection64,74 and increasing axonal growth.41 Indeed, ceramide can directly increase74,75 or decrease76,77 axonal outgrowth depending, at least in part, upon the concentration and developmental stage of the neuron.74,75 The effect of ceramide on axonal growth may provide an interesting model to explore how lipid rafts and ceramide domain formation may contribute to or affect neurotrophin signaling. For example, increasing ceramide within distal axons of sympathetic neurons inhibits axonal growth.76,77 Interestingly, addition of ceramide to the cell bodies of sympathetic neurons had no effect on axonal elongation since the ceramide was not efficiently transported anterogradely.77 Further, increases in the ceramide content of distal axons inhibited the uptake of NGF but enhanced the activation of Trk A by NGF. This agrees with previous reports that ceramide can inhibit endocytosis78 and enhance both NGF-dependent and NGF-independent activation of Trk A.79 These results raise the possibility that ceramide domain formation may affect neurotrophin activity by inducing Trk A clustering in lipid rafts80 or enhancing clustering of Trk A pools localized outside of lipid rafts.34 Another twist may be that ceramide production via p75NTR may enhance clustering of this receptor.* Although the role of p75NTR receptor clustering is unknown, this might be associated with the induction of apoptosis via p75NTR.73 It is intriguing to speculate that one role of ceramide production in p75NTR signaling may be to enhance clustering of the receptor and regulate coupling to other signal transduction pathways. It should also be noted that the effect of ceramide on neurotrophin activity may be influenced by the presence of other protein components of lipid rafts. For example, caveolin-1 can bind to Trk A and inhibit its activation.81 Further, ceramide generated through activation of a lipidraft-associated A-SMase may also inhibit downstream signaling through tyrosine kinase receptors.82 Interestingly, ceramide enrichment in caveolae was associated with inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) via an interaction with caveolin-1;82 it is also relevant to note that Trk A activation may lead to inhibition of a lipid-raft associated A-SMase via activation of PI 3-kinase.83 Although the above results implicate a role for ceramide domain formation in these cell membranes, the data is not conclusive. The use of sterols which promote or inhibit formation of liquid-ordered domains may help to further define the role of ceramide interactions with these lipid domains and the contribution of this interaction to neurotrophin signaling. In contrast to the effect of ceramide on inhibiting axonal growth of sympathetic neurons, exogenous ceramide stimulates the early transition of hippocampal neurons from a rounded cell lacking processes (stage 1) to one bearing several short processes of similar length (stage 2). Brann et al41 demonstrated that axonal elongation and maturation to stage 3 hippocampal neurons required nerve growth factor-induced ceramide production through p75NTR. Although the effect of ceramide on inducing axonal growth at stage 3 is attributed to its conversion to glucosylceramide,74,75,84 ceramide signaling was suggested to be critical to increasing the stage 1 to stage 2 transition since inhibitors of glucosylceramide production did not block this effect.75 Since stage 3, and presumably earlier stage neurons, lack lipid rafts due to their low SM * Dremina and Dobrowsky, unpublished observation

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content,84 these results raise the possibility that ceramide may form distinct and less ordered membrane phases in young neurons versus those possessing lipid rafts (stage 4-5).84 Indeed, low concentrations of ceramide do not promote survival but induce death of stage 4-5 neurons, a time when lipid raft domains are present.74,84 As discussed above, this would correlate with the potential to form more ordered ceramide domains in the membranes. However, if differences in the potential order/stability of a ceramide domain exist at different developmental stages, it remains unknown whether this is a critical factor in affecting how ceramide might differentially couple to differentiation or death signals. However, data does exist indicating that changes in the stability of liquid-ordered domains can influence protein-protein and lipid-protein interactions.18 Thus, it would seem that understanding how ceramide may interact with proteins would facilitate predicting if differences in the potential order/stability of a ceramide domain is relevant to directing the outcome of ceramide signaling. Several proteins, kinase suppressor of ras (KSR), raf-1 and PKCζ have been identified to bind ceramide with relatively high affinity. 85,86 It is interesting that at least two of these proteins have been localized to lipid raft domains.18 Moreover, since ras also localizes to lipid rafts,18 it will not be surprising to find KSR in these domains. One hypothesis is that high affinity ceramide:protein interactions may occur via a cysteine-rich domain present in raf-1 and PKCζ. Molecular modeling predicts that this cysteine-rich region may form a cleft for interaction, via hydrogen bonding, with the amide and allylic hydroxyl of ceramide.88 Increased stability of a lipid raft-associated ceramide domain may provide a more competent scaffold for the stereospecific interaction of ceramide with KSR, raf-1 or PKCζ or possibly protein phosphatases.87 However, no evidence exists that the plethora of ceramide responses in various cells is regulated solely through the interaction of ceramide with high affinity binding proteins. Perhaps an example may also be taken from proteins that interact with other membrane lipids. It is well recognized that proteins containing pleckstrin homology (PH) domains can interact with high affinity to phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate.89 Some PH-domain containing proteins, however, bind phosphatidylinositol polyphosphates weakly or with low specificity.90 The regulated avidity model for recruitment of low affinity PH-domain containing proteins to membrane regions enriched in phosphatidylinositol polyphosphates implicates the formation of an oligomeric complex of individual proteins whose collective binding energies would be greater than any one individual component.91,92 Although phospholipids exist primarily in a fluid phase, in principle, this general concept should also be applicable to ceramide signaling. If cytosolic proteins that are activated/inhibited by increased ceramide levels interact with ceramide-enriched domains via hydrogen bonding, then it is not hard to envision that formation of an oligomeric protein complex, via phosphorylation or through interaction between protein-protein interaction modules, would have more potential sites for stable interaction. Thus, even a more fluid ceramide-enriched membrane region may be a sufficiently stable scaffold for (or stabilized by) oligomerized/self-associated low affinity binding proteins that collectively provide an increased avidity for the lipid domain (Fig. 1). Since ceramide present in liquid-ordered domains undergoes tighter packing distinct from that of a non-raft SM-ceramide domain,9,11 ceramide generation within lipid rafts may provide some spatial specificity by increasing the stringency of low affinity interactions. If ceramide generation is prolonged, changes in the size or number of ceramide-enriched domains may directly affect the avidity of the complex. Thus, it is conceivable that differences in the avidity of an oligomeric complex for a given ceramide-enriched domain may vary the amplitude and duration of the ceramide signal, even through a common signaling molecule, leading to qualitatively different biological responses.93 This is not unprecedented since transient activation of stress-activated protein kinases may promote cell growth whereas prolonged activation may induce apoptosis.94,95 Further identification of protein structures that may form a “sphingolipid-binding motif” would obviously facilitate exploration of this model.

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Figure 1. Hypothetical model of low affinity protein interactions with SM/ceramide-enriched domains via regulated avidity. Low affinity interactions of protein monomers with ceramide-enriched domains may be enhanced either by (a) proximal oligomerization of ceramide-associated monomers or by interaction of a protein complex preformed in the cytoplasm. The kinetics for formation of a stable complex with the ceramide domain (b) may be regulated, in part, by the magnitude of co-operation of individual monomers in contributing to high avidity association of the protein complex with the membrane (indicated by arrow width). Further stabilization of high-avidity association may occur as individual ceramide-enriched domains coalesce (c). Black, grey and open membrane components represent SM, ceramide and phospholipid molecules, respectively. Black spotted ovals of and rectangular hatched region of hypothetical protein monomers represent putative low affinity ceramide-binding and protein:protein interaction domains, respectively.

Conclusions At this point, our current knowledge of ceramide domain/pore formation can not help explain or predict the outcome of neurotrophin-induced ceramide signaling in the nervous system. However, it is apparent that formation of ceramide-enriched domains seems critical to some aspects of ceramide-mediated apoptosis.20,21 Recent development of a ceramide antibody (15B4 from Alexis) may help facilitate localization of ceramide-enriched domains in neuronal membranes. Future development of approaches to modify formation of these various regions will also facilitate identifying their potential role in signaling. Despite the lack of answers, there is little doubt that ceramide production is a component of neurotrophin signaling and can influence neuronal responses to cytokines and nerve growth factors. Whether domains or pores, determining if differences in phase partitioning in the membrane may direct ceramide signaling will stimulate a lot of neurons.

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49. Carrer DC, Maggio B. Phase behavior and molecular interactions in mixtures of ceramide with dipalmitoylphosphatidylcholine. J Lipid Res 1999; 40:1978-1989. 50. Holopainen JM, Lehtonen JY, Kinnunen PK. Lipid microdomains in dimyristoylphosphatidylcholineceramide liposomes. Chem Phys Lipids 1997; 88:1-13. 51. Holopainen JM, Brockman HL, Brown RE et al. Interfacial interactions of ceramide with dimyristoylphosphatidylcholine: impact of the N-acyl chain. Biophys J 2001; 80:765-775. 52. Weisner DA, Kilkus JP, Gottschalk AR et al. Anti-immunoglobulin-induced apoptosis in WEHI 231 cells involves the slow formation of ceramide from sphingomyelin and is blocked by bcl-xL. J Biol Chem 1997; 272:9868-9846. 53. Kroesen BJ, Pettus B, Luberto C et al. Induction of apoptosis through B-cell receptor cross-linking occurs via de novo generated C16-ceramide and involves mitochondria. J Biol Chem 2001; 276: 13606-13614. 54. Gudz TI, Tserng K-Y, Hoppel CL. Direct inhibition of mitochondrial respiratory chain complex III by cell-permeable ceramide. J Biol Chem 1997; 272:24154-24158. 55. Ghafourifar P, Klein SD, Schucht O et al. Ceramide induces cytochrome c release from isolated mitochondria. Importance of mitochondrial redox state. J Biol Chem 1999; 274:6080-6084. 56. Garcia-Ruiz C, Colell A, Maris M et al. Direct effects of ceramide on the mitochondrial electron transport chain leads to generation of reactive oxygen species. J Biol Chem 1997; 272:11369-11377. 57. Michel C, van Echten-Deckert G, Rother J et al. Characterization of ceramide synthesis. A dihydroceramide desaturase introduces the 4,5-trans-double bond of sphingosine at the level of dihydroceramide. J Biol Chem 1997; 272:22432-22437. 58. Simon CGJ, Holloway PW, Gear AR. Exchange of C(16)-ceramide between phospholipid vesicles. Biochemistry 1999; 38:14676-14682. 59. Huang EJ, Reichardt LF. Neurotrophins: Roles in neuronal development and function. Annu Rev Neurosci 2001; 24:677-736. 60. Sofroniew MV, Howe CL, Mobley WC. Nerve growth factor signaling, neuroprotection, and neural repair. Annu Rev Neurosci 2001; 24:1217-1281. 61. Dobrowsky RT, Carter BD. p75 Neurotrophin receptor signaling: Mechanisms for neurotrophic modulation of cell stress? J Neurosci Res 2000; 61:237-243. 62. Dobrowsky RT, Werner MH, Castellino AM et al. Activation of the sphingomyelin cycle through the low-affinity neurotrophin receptor. Science 1994; 265:1596-1599. 63. Frago LM, Leon Y, de la Rosa EJ et al. Nerve growth factor and ceramides modulate cell death in the early developing inner ear. J Cell Sci 1998; 111:549-556. 64. Kume T, Nishikawa H, Tomioka H et al. p75-mediated neuroprotection by NGF against glutamate cytotoxicity in cortical cultures. Brain Res 2000; 852:279-289. 65. Lievremont JP, Sciorati C, Morandi E et al. The p75(NTR)-induced apoptotic program develops through a ceramide-caspase pathway negatively regulated by nitric oxide. J Biol Chem 1999; 274:15466-15472. 66. Blochl A, Sirrenberg C. Neurotrophins stimulate the release of dopamine from rat mesencephalic neurons via Trk and p75Lntr receptors. J Biol Chem 1996; 271:21100-21107. 67. Cassacia-Bonnefil P, Carter BD, Dobrowsky RT et al. Nerve growth factor-mediated death of oligodendrocytes by the p75 neurotrophin receptor. Nature 1996; 383:716-719. 68. Bamji SX, Majdan M, Pozniak CD et al. The p75 neurotrophin receptor mediates neuronal apoptosis and is essential for naturally occurring sympathetic neuron death. J Cell Biol 1998; 140:911-923. 69. Wang JJ, Tasinato A, Ethell DW et al. Phosphorylation of the common neurotrophin receptor p75 by p38beta2 kinase affects NF-kappaB and AP-1 activities. J Mol Neurosci 2000; 15:19-29. 70. Carter BD, Kaltschmidt C, Kaltschmidt B et al. Selective activation of NF-κB by nerve growth factor through the neurotrophin receptor p75. Science 1996; 272:542-545. 71. Gentry JJ, Casaccia-Bonnefil P, Carter BD. Nerve growth factor activation of NF-κB through its p75 receptor is an anti-apoptotic signal in RN22 schwannoma cells. J Biol Chem 2000; 275:7558-7565. 72. Bhakar AL, Roux PP, Lachance C et al. The p75 neurotrophin receptor (p75NTR) alters tumor necrosis factor-mediated NF-kappaB activity under physiological conditions, but direct p75NTRmediated NF-kappaB activation requires cell stress. J Biol Chem 1999; 274:21443-21449. 73. Barker PA. p75NTR: A study in contrasts. Cell Death Differen 1998; 5:346-356.

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74. Mitoma J, Ito M, Furuya S et al. Bipotential roles of ceramide in the growth of hippocampal neurons: promotion of cell survival and dendritic outgrowth in dose- and developmental stagedependent manners. J Neurosci Res 1998; 51:712-722. 75. Schwarz A, Futerman AH. Distinct roles for ceramide and glucosylceramide at different stages of neuronal growth. J Neurosci 1997; 17:2929-2938. 76. Posse de Chaves E, Bussiere M, Vance DE et al. Elevation of ceramide within distal neurites inhibits neurite growth in cultured rat sympathetic neurons. J Biol Chem 1997; 272:3028-3035. 77. Posse de Chaves E, Bussiere M, MacInnis B et al. Ceramide inhibits axonal growth and nerve growth factor uptake without compromising the viability of sympathetic neurons. J Biol Chem 2001; 276:36207-36214. 78. Chen CS, Rosenwald AG, Pagano RE. Ceramide as a modulator of endocytosis. J Biol Chem 1995; 270:13291-13297. 79. MacPhee I, Barker PA. Extended ceramide exposure activates the trkA receptor by increasing receptor homodimer formation. J Neurochem 1999; 72:1423-1430. 80. Huang C, Zhou J, Feng AK et al. Nerve growth factor signaling in caveolae-like domains at the plasma membrane. J Biol Chem 1999; 274:36707-36714. 81. Bilderback TR, Lisanti MP, Dobrowsky RT. Caveolin interacts with Trk A and p75NTR and regulates neurotrophin signaling pathways. J Biol Chem 1999; 274:257-263. 82. Zundel W, Swiersz LM, Giaccia A. Caveolin 1-mediated regulation of receptor tyrosine kinaseassociated phosphatidylinositol 3-kinase activity by ceramide. Mol Cell Biol 2000; 20:1507-1514. 83. Bilderback TR, Gazula V-R, Dobrowsky RT. Phosphoinositide 3-kinase regulates crosstalk between Trk A tyrosine kinase and p75NTR-dependent sphingolipid signaling pathways. J Neurochem 2001; 76:1540-1551. 84. Ledesma MD, Brugger B, Bunning C et al. Maturation of the axonal plasma membrane requires upregulation of sphingomyelin synthesis and formation of protein-lipid complexes. EMBO J 1999; 18:1761-1771. 85. Zhang Y, Yao B, Delikat S et al. Kinase suppressor of ras is ceramide-activated protein kinase. Cell 1997; 89:63-72. 86. Muller G, Ayoub M, Storz P et al. PKC ζ is a molecular switch in signal tranduction of TNF-α, bifunctionally regulated by ceramide and arachidonic acid. EMBO J 1995; 14:1961-1969. 87. Chalfant CE, Kishikawa K, Mumby MC et al. Long chain ceramides activate protein phosphatase1 and protein phosphatase-2A. Activation is stereospecific and regulated by phosphatidic acid. J Biol Chem 1999; 274:20313-20317. 88. van Blitterswijk WJ. Hypothesis: Ceramide conditionally activates atypical protein kinases C, raf-1 and KSR through binding to their cysteine-rich domains. Biochem J 1998; 331:679-680. 89. Franke TF, Kaplan DK, Cantley LC et al. Direct regulation of the Akt proto-oncogene product by phosphatidyl-3,4-bisphosphate. Science 1997; 275:665-668. 90. Kavran JM, Klein DE, Lee A et al. Specificity and promiscuity in phosphoinositide binding by pleckstrin homology domains. J Biol Chem 1998; 273:30497-30508. 91. Lemmon MA, Ferguson KM. Signal-dependent membrane targeting by pleckstrin homology (PH) domains. Biochem J 2000; 350:1-18. 92. Mao Y, Nickitenko A, Duan X et al. Crystal structure of the VHS and FYVE tandem domains of Hrs, a protein involved in membrane trafficking and signal transduction. Cell 2000; 100:447-456. 93. Pawson T, Nash P. Protein-protein interactions define specificity in signal transduction. Genes Dev 2000; 14:1027-1047. 94. Roulston A, Reinhard C, Amiri P et al. Early activation of c-Jun N-terminal kinase and p38 kinase regulate cell survival in response to tumor necrosis factor alpha. J Biol Chem 1998; 273:10232-10239. 95. Guo YL, Baysal K, Kang B et al. Correlation between sustained c-Jun N-terminal protein kinase activation and apoptosis induced by tumor necrosis factor-alpha in rat mesangial cells. J Biol Chem 1998; 273:4027-4034.

CHAPTER 13

Ceramide Signaling in Cannabinoid Action Ismael Galve-Roperh, Cristina Sánchez, Teresa Gómez del Pulgar, Guillermo Velasco, Daniel Rueda, Cristina Blázquez and Manuel Guzmán

Summary

C

annabinoids, the active components of Cannabis sativa (marijuana) and their endogenous counterparts, exert their effects by binding to specific Gi/o-protein-coupled receptors that modulate adenylyl cyclase, ion channels and extracellular signal-regulated kinase. Recent research has shown that the CB1 cannabinoid receptor is also coupled to the generation of the lipid second messenger ceramide via two different pathways: SM hydrolysis and ceramide synthhesis de novo. Ceramide in turn mediates different cannabinoid actions such as stimulation of metabolism in primary astrocytes and induction of apoptosis in glioma cells, depending on the origin and characteristics of the generated ceramide pool. Of importance, cannabinoid-induced apoptosis of glioma cells in vitro correlates with their ability to induce tumor regression of malignant gliomas in vivo. These findings provide a new conceptual view on how seven-transmembrane cannabinoid receptors signal and raise exciting therapeutic and physiological questions.

Introduction Cannabinoids, the active components of marijuana and their endogenous counterparts, exert a wide variety of central and peripheral effects, some of them with potential clinical applications (Box 1). Extensive molecular and pharmacological studies have demonstrated that cannabinoids act via seven-transmembrane receptors coupled to Gi/o-proteins that signal inhibition of adenylyl cyclase and activation of the extracellular signal-regulated kinase (ERK) cascade.1,2 The CB1 receptor also modulates ion channels, inducing, for example, inhibition of N- and P/Q-type voltage-sensitive Ca2+ channels and activation of G-protein-activated inwardly rectifying K+ channels1,2 (Fig. 1). Besides these well-established cannabinoid receptorcoupled signaling events, cannabinoid receptors have been shown to activate c-Jun N-terminal kinase, p38 mitogen-activated protein kinase and protein kinase B (PKB).3-5 Moreover, recent observations demonstrate that cannabinoid receptor activation triggers the generation of ceramide, that in turn may be important in the control of cell fate. Ceramide may induce apoptosis in different tissues including the central nervous system6,7 and also exerts important regulatory effects on neuronal growth and development.8,9 Moreover, intracellular ceramide accumulation occurs in neurodegenerative diseases, trauma, epilepsy and ischaemia/stroke6,10. The aim of this review is to examine this new aspect of cannabinoid-mediated signal transduction in which a seven-transmembrane receptor signals via ceramide accumulation, and to discuss its potential therapeutic and physiological implications. Ceramide Signaling, edited by Anthony H. Futerman. ©2002 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Box 1. The Endocannabinoid System Cannabinoids, the active components of marijuana (Cannabis sativa) and their endogenous counterparts, exert their actions through specific G-protein-coupled receptors1,2. The first cannabinoid receptor (CB1) was cloned from a cortical rat brain cDNA library3, and a second subtype of cannabinoid receptor (CB2) was subsequently cloned from human promyelocitic HL-60 cells4. Both CB1 and CB2 are members of the seven-transmembrane receptor superfamily. CB1 is mainly expressed in the central nervous system, although it is also expressed in peripheral nerve terminals and a variety of extra-neural tissues. By contrast, CB2 expression is mostly restricted to the immune system. Cannabinoid receptor agonists comprise several types of compounds. Classical cannabinoids include both the cannabinoids produced by marijuana and some synthetic analogs. The most relevant plantderived cannabinoids are ∆9-tetrahydrocannabinol (THC) (see Fig. in box), ∆8-THC (almost as active as ∆9-THC but much less abundant in the plant), cannabinol and cannabidiol (both produced in significant amounts but much less potent as cannabimimetic agents). Synthetic cannabinoids include (a) compounds with the classical cannabinoid structure such as HU210 (Fig. 1), and (b) non-classical cannabinoids such as various bi- and tricyclic analogs of THC, and aminoalkylindols. Of importance, a family of endogenous cannabinoids has been characterized5,6, which together with their receptors3,4 and specific processes of synthesis7, uptake8 and degradation9, point to the existence of an endocannabinoid system. The first endogenous cannabinoid was described by Mechoulam and coworkers10. It was named anandamide (arachidonoylethanolamide), from the sanscrit ananda, “internal bliss”, and making reference to its chemical structure (the amide of arachidonic acid and ethanolamine) (Fig. 1). Subsequently, other endogenous cannabinoid receptor ligands such as 2-arachidonoylglycerol have been described11,12. These compounds, although structurally not related to THC, bind to cannabinoid receptors and mimic the effects of plant-derived and synthetic cannabinoids. Research on signal transduction mechanisms by cannabinoids has greatly benefited from the existence of selective cannabinoid receptor antagonists such as SR141716 and SR144528 for CB1 and CB2, respectively1. The endocannabinoid system acts as a neuromodulatory system that generally inhibits the release of neurotransmitters such as glutamate, dopamine and GABA by acting as retrograde messengers and inhibiting Ca2+ influx5,6,13. Thus the endogenous cannabinoid system exerts regulatory functions in a wide variety of physiological processes with potential therapeutic implications. Two oral preparations of cannabinoids (Marinol® and Cesamet®) are clinically available and prescribed to stimulate appetite and suppress vomiting and nausea in chemotherapy-treated AIDS and cancer patients2. Likewise, a phase III clinical trial is in progress for the treatment of obesity with SR141716. Cannabinoids also participate in the control of pain responses via CB1 receptors, and in some cases by interactions with the vanilloid and opioid systems1,2,6. Other cannabinoid actions include movement control and suppression of tremor and spasticity in multiple sclerosis14, neuroprotection15, control of vascular tone16 and regulation of immune function17. Moreover, the ability of these compounds to regulate the cell survival/death decision constitutes an active area of current cannabinoid research18. 1. Pertwee RG. Cannabinoid receptor ligands: clinical and neuropharmacological considerations, relevant to future drug discovery and development. Exp Opin Invest Drugs 2000; 9:1-19. 2. Porter AC, Felder CC. The endocannabinoid nervous system: unique opportunities for therapeutic intervention. Pharmacol Ther 2001; 90:45-60. 3. Matsuda LA , Lolait SJ, Brownstein MJ et al. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 1990; 346:561-564. 4. Munro S, Thomas KL, Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature 1993; 365: 61-65. 5. Di Marzo V, Melck D, Bisogno T et al. Endocannabinoids: endogenous cannabinoid receptor ligands with neuromodulatory action. Trends Neurosci 1998; 21:521-528.

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6. Piomelli D, Giuffrida A, Calignano A et al. The endocannabinoid system as a target for therapeutic drugs. Trends Pharmacol Sci 2000; 21:218-224. 7. Di Marzo V, Fontana A, Cadas H et al. Formation and inactivation of endogenous cannabinoid anandamide. Nature 1994; 372:686-691. 8. Beltramo M, Stella N, Calignano A et al. Functional role of high-affinity anandamide transport, as revealed by selective inhibition. Science 1997; 277:1094-1097. 9. Cravatt BF, Giang DK, Mayfield SP et al. Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides. Nature 1996; 384:83-87. 10. Devane WA, Hanus L, Breuer A et al. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 1992; 258:1946-1949. 11. Mechoulam R, Ben Shabat S, Hanus L et al. Identification of an endogenous 2monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem Pharmacol 1995; 50:83-90. 12. Stella N, Schweitzer P, Piomelli D. A second endogenous cannabinoid that modulates long-term potentiation. Nature 1997; 388:773-778. 13. Wilson RI, Nicoll RA. Endogenous cannabinoids mediate retrograde signaling at hippocampal synapses. Nature 2001; 410:588-592. 14. Baker D, Pryce G, Croxford JL et al. Cannabinoids control spasticity and tremor in a multiple sclerosis model. Nature 2000; 404:84-87. 15. Nagayama T, Sinor AD, Simon RP et al. Cannabinoids and neuroprotection in global and focal cerebral ischemia and in neuronal cultures. J Neurosci 1999; 19: 2987-2995. 16. Wagner JA, Varga K, Ellis EF et al. Activation of peripheral CB1 cannabinoid receptors in haemorrhagic shock. Nature 1997; 390:518-521. 17. Klein TW, Newton C, Friedman H. Cannabinoid receptors and immunity. Immunol Today 1998; 19:373-381. 18. Guzmán M, Galve-Roperh I, Sánchez C. Ceramide, a new second messenger of cannabinoid action. Trends Pharmacol Sci 2001; 22:19-22.

Ceramide Generation Acute CB1 receptor activation by different cannabinoids induces SM hydrolysis in both primary astrocytes11,12 and C6 glioma cells,13,14 with a maximal effect at ~15 min. As expected, this stimulation is concomitant with an increase in ceramide levels (maximum twofold). The functional coupling of receptors to SMases may involve different adaptor proteins. One of these is the factor associated with neutral SMase activation (FAN) (see Chapter 8 ). A role for FAN in CB1 receptor-evoked SM hydrolysis is supported by coimmunoprecipitation experiments demonstrating the binding of FAN to the activated CB1 receptor and by the resistance of cells expressing dominant-negative FAN to cannabinoid-induced SM breakdown.15 Of interest,

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Figure 1. Fig. 1. Signaling pathways activated by the CB1 cannabinoid receptor. Plant derived and endogenous cannabinoids produce most of their effects by binding to the CB1 receptor. Cannabinoid receptor signals inhibition of the adenylyl cyclase (AC)-protein kinase A (PKA) pathway and activation of the extracellular signal-regulated kinase (ERK) cascade via Gi/o protein. The CB1 receptor also modulates ion channels, inducing, for example, inhibition of N- and P/Q-type voltage-sensitive Ca2+ channels (VSCC). All these events participate in the control of cell function by cannabinoids. In addition the cannabinoid receptor also mediates ceramide accumulation. Activation of the receptor can produce two peaks of ceramide. Short-term ceramide generation involves sphingomyelin (SM) hydrolysis via sphingomyelinase (SMase) activation possibly through the adaptor protein FAN. Long-term ceramide generation may occur via serine palmitoyltranferase (SPT) induction and enhanced ceramide synthesis de novo. FA: fatty acyl-CoA.

both G-protein β subunits and FAN are members of the WD-repeat protein family.16 The potential association of FAN to the CB1 receptor is supported by the homology between a sequence in the 55-kDa tumor necrosis factor (TNF) receptor FAN-binding domain (DSAHK) and a sequence in the cytoplasmic region of the CB1 receptor (DCLHK). This homology is higher than that shared by the 55-kDa TNF receptor FAN-binding domain (EDSAH) and the sequence recently proposed to bind FAN in the immunoregulatory transmembrane protein CD40 (QETLH).17 Recently, another G-protein-coupled-receptor—the receptor for the chemokine growth-related gene product β—has been reported to evoke SM hydrolysis through SMase activation.18 In addition to short-term ceramide generation through SM hydrolysis, long-term ceramide accumulation through enhanced synthesis de novo or impaired clearance and/or metabolism has been gaining appreciation as alternative means of generating a signaling pool of ceramide.19 In this context, cannabinoid receptor activation also evokes sustained ceramide accumulation. In glioma cells, cannabinoids induce a sustained peak of ceramide starting at day 2-3 of

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treatment and reaching a maximal four-fold increase at day 5 (Ref. 14). Serine palmitoyltransferase (SPT), the rate-limiting enzyme for ceramide synthesis de novo, might be a potential candidate to be involved in this effect. By using different subclones of glioma cells, we have observed that cells that respond to cannabinoid stimulation with sustained ceramide generation also show increased SPT activity. In contrast, cells that are refractory for cannabinoid-induced sustained ceramide accumulation are also negative for SPT induction. Moreover, pharmacological inhibition of ceramide synthesis de novo, but not of SMases, prevents cannabinoid action relying on sustained ceramide accumulation (Gómez del Pulgar T, Velasco G, Sánchez C, Haro A, Guzmán M, submitted). Another G-protein-coupled receptor, the angiotensin II type 2 receptor, also induces ceramide accumulation via enhanced synthesis de novo.20 In summary, besides previously recognized modulators of ceramide signaling such as stress stimuli and the death receptor family, cannabinoid receptors constitute an example of seven-transmembrane receptors coupled to ceramide production.

Ceramide Targets Different protein kinases involved in the control of cell survival, such as members of the mitogen- and stress-activated protein kinase families, are potential targets of ceramide action.6 Based on pharmacological data showing prevention of cannabinoid actions by the ERK kinase (MEK) inhibitor PD98059, the mechanism of ceramide-induced activation of ERK was investigated. In parallel to both acute and sustained cannabinoid-induced ceramide generation, Raf1 and ERK activation was detected in glial cells11,14 (Fig.1), whereas increased activity of kinase supressor of Ras, a different ceramide-activated protein kinase,21 could not be evidenced. These results are in line with the proposed ability of Raf-1 to directly bind ceramide,22 although the functional consequences of this process are not completely understood.23 Another potential target that may mediate ceramide actions is the phosphoinositide 3’-kinase/PKB pathway, which is inhibited by ceramide.24 In this context, recent data indicate that such ceramide-mediated inhibition of PKB might also contribute to some cannabinoid actions in the control of cell fate (Gómez del Pulgar T, Velasco G, Sánchez C, Haro A, Guzmán M, submitted).

Ceramide Function The early peak of ceramide induced by cannabinoids has been related to the regulation of metabolic functions. Thus, cannabinoids stimulate the utilization of glucose, the main source of brain energy metabolism, and the production of ketone bodies, an alternative source of energy when glucose deprivation ensues, in primary astrocytes.25 Both effects are prevented by cannabinoid receptor antagonism and are preceded by a rapid and transient increase in the levels of ceramide. Ceramide in turn seems to mediate the stimulation of glucose consumption via the Raf-1/MEK/ERK cascade,11 and the stimulation of ketogenesis via the outer-mitochondrial-membrane carnitine palmitoyltransferase, the rate-limiting enzyme for fatty acid oxidation.12 One of the most important functions of astrocytes is the regulation of brain energy metabolism by providing neurons with anaplerotic metabolites and substrates for generation of energy. It is therefore tempting to speculate that the endogenous cannabinoid system might regulate via ceramide the amount and type of nutrients supplied by astrocytes to neurons as source of carbon for neuronal biosynthetic processes (e.g., myelination) and oxidative metabolism (e.g., synaptic activity). Various cannabinoids have been shown to induce apoptosis of neural cells such as glioma, astrocytoma, neuroblastoma and hippocampal neurons.26 In addition, cannabinoids exert an antiproliferative effect on breast and prostate cancer cells.26 The important role of ceramide in the induction of apoptosis prompted us to explore the hypothesis that cannabinoid-induced ceramide accumulation may participate in the induction of apoptosis.14 Using subclones of

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glioma cells with different sensitivity to cannabinoids (see above) it was demonsrated that sustained but not acute ceramide generation and ERK activation is responsible for cannabinoid-induced apoptosis.14 Likewise, primary astrocytes, in which cannabinoids evoke acute but not sustained ceramide generation and ERK activation, are resistant to the apoptotic effect of the cannabinoids.11,14 Of importance, if sustained ceramide accumulation and ERK activation is induced by enhanced ceramide synthesis de novo through palmitate loading, primary astrocytes undergo apoptosis,27 showing therefore that the ceramide-regulated cell death machinery is functional in these cells. In addition, because the endogenous cannabinoid system has been suggested to play a specific role in brain development,28 ceramide generated following cannabinoid receptor activation might control neural cell fate during this process.

Therapeutic Implications Plant-derived and synthetic cannabinoids do not only induce apoptosis in vitro but also exert an anti-tumoral action in vivo without significant collateral effects.15,29 Thus cannabinoids induce tumor regression in rats with intracranial malignant gliomas. Moreover, cannabinoids inhibit the growth of tumors generated by subcutaneous injection to immune-deficient mice of rat glioma cells as well as grade IV human astrocytoma cells.15,29 It is worth noting that CB2 selective agonists, which are devoid of typical CB1-mediated psychoactive effects, are also effective inhibitors of glioma growth in vivo.29 The case of gliomas is of particular interest because they are one of the most malignant forms of cancer, and conventional therapies, including surgery, radiotherapy, chemotherapy and immunotherapy, are usually uneffective or just palliative.30 It is therefore essential to develop new therapeutic strategies that acting alone or in combination might allow better clinical results. Ceramide accumulation is a factor that enhances the effectivenes of chemotherapy31 and radiotherapy32 treatments in certain models. In addition, ceramide may have antitumoral properties either alone33 or in combined therapies.34.Thus, it is tempting to propose that cannabinoids might be therapeutic agents for the management of malignant brain tumours owing to their ability to generate ceramide.

Concluding Remarks Recent investigations have shown that cannabinoid receptor activation can induce two peaks of ceramide generation which have different kinetics (minute- versus day-range), magnitude (two versus fourfold), mechanistic origin (SM hydrolysis versus ceramide synthesis de novo) and function (metabolic regulation versus induction of apoptosis). These observations open a new conceptual view on the mechanism of cannabinoid action, and contribute to the novel idea that seven transmembrane receptors may signal via mechanisms alternative to the classical heterotrimeric- G-protein paradigm.35 Clearly further research is required to unravel the molecular mechanism responsible for the coupling of cannabinoid receptors to ceramide generation. Keeping in mind the important role of ceramide in the control of cell fate, together with the increasing array of cell functions modulated by cannabinoids, we anticipate that the “cannabinoid-ceramide connection” could lead to important therapeutic and physiological possibilities as exemplified by the case of glioma management.

Acknowledgements We are indebted to ML Ceballos, C Corbacho, M Cortés, JW Huffman, M Izquierdo and S Ramón y Cajal for their participation in our investigations discussed herein. Work in the authors’ laboratory is supported by grants from Comisión Interministerial de Ciencia y Tecnología (PM 98/0079) and Comunidad Autónoma de Madrid (CAM 08.5/0017/98) and Fundación Ramón Areces.

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25. Guzmán M, Sánchez C. Effects of cannabinoids on energy metabolism. Life Sci 1999; 65:657-664. 26. Guzmán M, Sánchez C, Galve-Roperh I. Control of the cell survival/death decision by cannabinoids. J Mol Med 2001; 78:613-625. 27. Blázquez C, Galve-Roperh I, Guzmán M. De novo-synthesized ceramide signals apoptosis in astrocytes via extracellular signal-regulated kinase. FASEB J 2000; 14:2315-2322. 28. Fernández Ruiz JJ, Berrendero F, Hernández ML et al. The endogenous cannabinoid system and brain development. Trends Neurosci 2000; 23:14-20. 29. Sánchez C, Ceballos ML, Gómez del Pulgar MT et al. Inhibition of glioma growth in vivo by selective activation of the CB2 cannabinoid receptor. Cancer Res 2001; 61:5784-5789. 30. Holland EC. Glioblastoma multiforme: The terminator. Proc Natl Acad Sci USA 2000; 97:6242-6244. 31. Maurer BJ, Melton L, Billups C et al. Synergistic cytotoxicity in solid tumor cell lines between N(4-Hydroxiphenyl)retinamide and modulators of ceramide metabolism. J Natl Cancer Inst 2000; 92:1897-1909. 32. Santana P, Pena LA, Haimovitz-Friedman A et al. Acid Smase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis. Cell 1996; 86:189-199. 33. Schmelz EM, Bushnev AS, Dillehay DL et al. Ceramide-β-D-glucuronide: synthesis, digestion, and suppression of early markers of colon carcinogenesis. Cancer Res 1999; 59:5768-5772. 34. Radin NS. Killing cancer cells by poly-drug elevation of ceramide levels. A hypothesis whose time has come? Eur J Biochem 2001; 268:193-204. 35. Brzostowski JA, Kimmel AR. Signaling at zero G: G-protein-independent functions for 7-TM receptors. Trends Biochem Sci 2001; 26:291-297.

CHAPTER 14

Ceramide Glycosylation and Chemotherapy Resistance Myles C. Cabot

M

ultidrug resistance, inherent or acquired, is a frequent characteristic of cancer cells and is difficult to predict and to manage. Multidrug resistance is caused by multiple mechanisms, including the dysfunctional metabolism of the lipid second messenger ceramide. The cytotoxic effect of various chemotherapeutics is decreased when the generation of ceramides is impaired, which results in the ineffectiveness of routine dosage and the need for higher, even more toxic drug levels. Needless to emphasize, this is a most undesirable situation; patients and oncologists would welcome its possible correction. Here we review ceramide metabolism with relationship to both blocking and potentiating the toxic response of cancer cells to chemotherapy. It is hoped that administering agents to target ceramide metabolism in combination with chemotherapy will improve response rates, especially in those patients with metastatic disease. The link between ceramide metabolism and chemotherapy response was alluded to by earlier studies with anthracyclines. Shortly thereafter, the association between anthracycline resistance and accelerated ceramide metabolism through glucosylceramide synthase (GCS) was revealed. The signaling pathways activated by daunorubicin, including a SMase-initiated ceramide pathway, have been the subject of a recent review.1 The enzyme ceramide synthase, by way of the de novo pathway, was shown to mediate daunorubicin-induced apoptosis in leukemia cells.2 Exposing cells to drug heightened ceramide synthase activity, promoting ceramide formation and increasing the percentage of apoptotic cells. Introducing Fumonisin B1 to inhibit ceramide synthase negated the cytotoxic impact of daunorubicin, a result which clearly affirmed ceramide’s role in anthracycline action. In a round of similar studies using human leukemia cell models, Jaffrezou et al3 found that daunorubicin likewise increased ceramide levels, albeit via SM hydrolysis and not ceramide synthase; nevertheless, apoptosis was the end result. Doxorubicin also promotes ceramide elevation, this work being conducted in MCF-7 breast cancer cells.4,5 Moving into the realm of multidrug resistance (MDR) brought recognition to the enzyme catalyzing ceramide glycosylation, GCS, as an important facet of cellular response to chemotherapy. Studies comparing chemotherapy-sensitive and chemotherapy-resistant cancer cells demonstrated a clear distinction in cerebroside content shown by an accumulation of a glycosylated form of ceramide, glucosylceramide, in the drug-resistant cells.6 This finding holds true for drug-resistant cancer cells derived from breast, ovary, melanoma,7 and prostate (author’s unpublished observations), and recent studies have confirmed this in a multidrug resistant cell line derived from HT29 human colon carcinoma.8 Investigations into the mechanism underlying the glucosylceramide surplus in chemotherapyresistant cells revealed that exposure of wild-type cells to doxorubicin prompted an increase in ceramide and cell death attributable to apoptosis, whereas in chemotherapy-resistant cells treated Ceramide Signaling, edited by Anthony H. Futerman. ©2002 Eurekah.com and Kluwer Academic / Plenum Publishers.

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in the same fashion, the ceramide generated was converted to glucosylceramide and the cytotoxic response was nil.4 This indicated that drug-resistant cells had an enhanced capacity for ceramide metabolism through the glycosylation pathway. An evaluation of ceramide toxicity showed wild-type cells susceptible to cell-permeable C6-ceramide, whereas drug-resistant cells were tolerant.9 Analysis of the metabolic fate of ceramide in these cells showed wild-type MCF-7 contained only free C6-ceramide whereas doxorubicin-resistant cells contained only high amounts of glucosyl-C6-ceramide. With this work, the importance of GCS in regulating cellular response to ceramide and perhaps to ceramide-generating drugs such as the anthracyclines, became apparent. To more clearly establish the influence of ceramide metabolism on drug resistance, the GCS gene was introduced into MCF-7 cells using a retroviral tetracycline-on expression system.10 The cell line that was developed, “MCF-7/GCS,” expressed an 11-fold higher level of GCS activity in the expression-on mode, compared to the parent cell line, and demonstrated strong resistance to doxorubicin and to ceramide analogs.11 Ceramide resistance displayed by MCF7/GCS cells paralleled the activity of the expressed GCS. In addition, resistance to TNF-α, which employs ceramide as a second messenger in the cell-killing response, was also a property of MCF-7/GCS cells.12 It was shown that TNF-α had little influence on the induction of apoptosis or on growth arrest in MCF-7/GCS cells, and lipid metabolism studies revealed that TNF-α promoted a ceramide increase in MCF-7 cells and a glucosylceramide increase in MCF7/GCS cells. Further, TNF-α-induced caspase activity was halted as a result of GCS transfection. Doxorubicin and TNF-α resistance in MCF-7/GCS cells was related to hyperglycosylation of ceramide and not to shifts in the levels of P-gp, Bcl-2, or TNF receptor 1 expression.11,12 The role of ceramide metabolism in regulating the cellular response to chemotherapy has also been demonstrated employing a reverse tactic, by introducing GCS-antisense into doxorubicin-resistant cells. These studies showed that decreasing cellular glycosylation potential through GCS antisense transfection heightened chemotherapy sensitivity in doxorubicin-resistant cells, effectively reversing resistance.13 The antisense cell line displayed by RT-PCR, Western blot, and in vitro assays, decreased GCS mRNA, GCS protein, and GCS enzymatic activity. These cells were 28 times more sensitive to doxorubicin compared to the parent MCF-7-AdrR cell line. Under doxorubicin stress, GCS-antisense transfected cells displayed time- and dosedependent increases in endogenous ceramide and dramatically higher levels of caspase activity, compared to control cells. GCS antisense transfection also heightened sensitivity to Vinca alkaloids and taxanes but had little impact on sensitivity to either 5-FU or cisplatin.14 Inasmuch as the data from gene transfection studies positions GCS as a force to overcome multidrug resistance in chemotherapy (Fig. 1), manipulation of ceramide metabolism by drug intervention can also provide a viable therapeutic avenue. Ceramide levels are readily enhanced by the introduction of ceramide generating agents alone or in conjunction with inhibitors of ceramide metabolism ( Fig. 2). A number of classical P-glycoprotein substrates influence ceramide metabolism by hindering its conversion to glucosylceramide. Tamoxifen, used in the Dartmouth regimen for treatment of melanoma,15 evaluated for treatment of pancreatic carcinoma and malignant gliomas,16,17 and employed in a biochemotherapy regimen for patients with metastatic melanoma,18 inhibits glucosylceramide synthesis in vinblastine-resistant carcinoma.19 In doxorubicin-resistant cells, clinically relevant concentrations of tamoxifen, verapamil, and cyclosporin A, markedly decrease glucosylceramide levels with IC50 values of 1.0, 0.8, and 2.3 mM, respectively.20 Toremifene, an anti-estrogen with chemistry and pharmacology similar to tamoxifen, is equally effective.9 These studies suggest that the P-glycoprotein-targeted agents act not only by blocking cellular drug efflux but also through enhancing ceramide levels. RU486 (mifepristone) inhibits growth and induces apoptosis in MCF-7 cells,21 and retards ceramide glycosylation in MCF-7-doxorubicin-resistant cells.9 Ketoconazole overcomes resistance to doxorubicin and vinblastine in cancer cells,22 displays activity in advanced prostate cancer patients,23 and it inhibits glucosylceramide synthesis (author’s unpublished observations). Studies with tamoxifen analogs corroborate the idea that MDR reversal and inhibition of cellular glucosylceramide

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Figure 1. Shifting cellular chemotherapy tolerance through GCS gene transfection.

synthesis are allied. For example, exposure of doxorubicin-resistant cells to triphenylethylene, the tamoxifen nucleus minus the dimethylethanolamine moiety, neither inhibits ceramide glycosylation nor sensitizes cells to doxorubicin, but inclusion of tamoxifen with doxorubicin decreases glucosylceramide production, enhances ceramide generation, and sensitizes cells.20,24 Similarly, cis-tamoxifen is devoid of chemosensitizing activity, indicating a stereochemical requirement. Studies using chemical inhibitors of GCS further highlight a relationship between ceramide metabolism and chemotherapy efficacy. PPMP (1-phenyl-2-palmitoylamino-3-morpholino1-propanol), a commercially available inhibitor of ceramide glycosylation,25 sensitizes breast cancer cells to doxorubicin,20 and neuroblastoma to Taxol and vincristine.26 These enzyme inhibitors are structural analogs of the natural GCS substrate. A chemical cousin, PPPP (1phenyl-2 palmitoylamino-3-pyrrolidino-1-propanol) completely blocks glucosylceramide synthesis in vincristine-resistant leukemia and in combination, enhances vincristine-induced cytotoxicity,27 and pretreatment of melanoma cells with PPPP markedly reduces tumor formation and metastatic potential in mice.28 In metastatic human colon cancer, raising the levels of ceramide by direct administration of ceramide analogs and ceramidase inhibitors induces apoptotic cell death, preventing tumor growth.29 Preferential killing of drug-resistant epidermoid carcinoma cells over drug-sensitive counterparts upon exposure to PDMP and PPPP has also been shown.30 A reduction in glucosylceramide levels accompanied PDMP-induced apoptosis, prompting the authors to suggest that manipulation of glucosylceramide levels is an avenue for preferential destruction of drug-resistant cancer cells. Spinedi et al31 showed that PDMP suppresses glucosylceramide synthesis and potentiates the apoptotic effect of C6-ceramide in neuroepithelioma, suggesting that glucosylceramide synthesis is a mechanism to escape ceramide-governed apoptosis. Similarly, by targeting ceramide metabolism, Sietsma et al26 showed that PDMP increases neuroblastoma sensitivity to chemotherapy, and that the increase involves a sustained elevation of ceramide. Work with SDZ PSC 833 ([3’-keto-bmt1]-[val-2]-cyclosporine), signaled an important step forward in demonstrating both the cytotoxic principles of ceramide and the impact of manipulating the ceramide pathway for therapeutic purposes. SDZ PSC 833 is a second generation MDR modulator that interferes with P-glycoprotein-mediated drug efflux.32 Our group first reported that SDZ PSC 833 alone strongly activates cellular ceramide formation, and that the increase in ceramide was mirrored by a progressive decline in cell survival.33 Cells having an enhanced capacity for ceramide glycosylation were more resistant to SDZ PSC 833, as opposed to drug-sensitive cells.6,24 Lipid metabolism studies revealed that SDZ PSC 833 resistance was allied with rapid conversion of ceramide to glucosylceramide.24 Studies with P-glycoprotein negative cells and with mdr-1-transfected cells showed that SDZ PSC 833 elicits ceramide generation independently of P-glycoprotein.34 In other studies it was revealed that drug sensitivity in leukemia could be enhanced by SDZ PSC 833 through perturbations in SM-ceramide pathways and not by modified drug efflux parameters.35

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Figure 2. Manipulation of Cellular Ceramide Levels Through Drug Intervention. The drugs shown exert a stimulatory influence (+) on SPT (serine palmitoyltransferase) and Cer Syn (ceramide synthase), or have an inhibitory impact (-) on GCS (glucosylceramide synthase). In combination, this enhances intracellular levels of ceramide.

Tilly and colleagues, using an inhibitor of ceramide-induced cell death, showed that cell destruction by chemotherapy was preventable if ceramide metabolism was manipulated.36 Numerous studies now clearly demonstrate that co-administration of agents that target ceramide metabolism enhances the cytotoxic impact of chemotherapy. For example, vinblastine toxicity is intensified by SDZ PSC 833 in P-glycoprotein-poor cells, and both drugs at low dose promote ceramide formation.37 Doxorubicin and tamoxifen synergize to increase ceramide formation, complementing cytotoxicity.5,20 Another example of enhanced drug efficacy through ceramide targeting is shown by the influence of RU486 on doxorubicin.5,9 A three-component regimen consisting of doxorubicin and two agents that enhance ceramide formation, tamoxifen and SDZ PSC 833, can bring cell viability to zero.24 Suramin, a polysulfonated naphthylurea, disrupts glycolipid metabolism and elicits apoptosis through ceramide pathways in a number of cancer cell lines.38 In Bcl2-transfected prostate cancer cells that are resistant to doxorubicin, a doxorubicin/suramin combination was shown effective in circumventing resistance.39 Suramin is currently in clinical trials for treatment of breast and prostate cancer.40-42 Aberrant ceramide generation is also involved in prostate cancer cell resistance to chemotherapy,43 and it was recently shown that ceramidase is overexpressed in prostate cancer.44 These findings justify exploring a ceramide-targeted approach for the treatment of prostate cancer. More evidence that the ceramide pathway can be manipulated for therapeutic purposes stems from work with the novel synthetic retinoid, N-(4-hydroxyphenyl) retinamide (4-HPR). 4-HPR has been shown to significantly increase the levels of ceramide and induce mixed apoptosis/necrosis in highly drug-resistant human neuroblastoma cell lines.45 The cytotoxicity of 4-HPR can be enhanced by adding modulators of ceramide metabolism,46 such as L-threodihydrosphingosine (safingol), a sphingosine analog and protein kinase-C inhibitor,47 and PPMP and tamoxifen. Safingol has been shown to be synergistic with 4-HPR and produces a 100- to 10,000-fold increase in cytotoxicity, compared to 4-HPR alone, in neuroblastoma, lung,

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melanoma, prostate, colon, and pancreatic cancer cell lines.46 The safingol/4-HPR combination is minimally toxic to fibroblasts and bone marrow myeloid progenitor cells. The involvement of ceramide metabolism in cancer cell response to chemotherapy is farreaching. Exogenous addition of ceramide enhances Taxol-induced apoptosis in leukemic T cells.48 Ceramide also enhances Taxol-induced apoptosis in head and neck squamous cell carcinoma, suggesting that a Taxol/ceramide combination therapy may be a promising alternative to conventional treatment of head and neck cancers.49 Research in breast cancer shows that Taxol elicits de novo ceramide generation and apoptosis; however, addition of ceramide synthesis inhibitors, such as L-cycloserine, checks Taxol-induced apoptosis.50 These data imply that ceramide is obligate for Taxol-elicited cytotoxicity. The theme on the interplay of ceramide with chemotherapy response continues to expand. Recent reviews by Norman Radin caption this idea.51,52 Many studies now indicate that, in addition to SMase, de novo enzymes of ceramide synthesis are also viable targets for chemotherapeutic agents. Etoposide regulates serine palmitoyltransferase, the rate-limiting enzyme in de novo ceramide synthesis.53 In human neuroblastoma, 4-HPR elevates ceramide via coordinate activation of serine palmitoyltransferase and ceramide synthase.54 Controlling ceramide metabolism is an attractive approach to cancer treatment. The majority of drugs that would be used are already available in the clinic, expediting the application of principle to practice. Ceramide metabolism can also be impacted by gene therapy. This has been illustrated through the application of antisense GCS, which is effective in the complete reversal of doxorubicin resistance in a breast cancer cell model.14

References 1. Laurent G, Jaffrézou J-P. Signaling pathways activated by daunorubicin. Blood 2001; 98:913-924. 2. Bose R, Verheij M, Haimovitz-Friedman A et al. Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals. Cell 1995; 82:405-414. 3. Jaffrezou JP, Levade T, Bettaieb A et al. Daunorubicin-induced apoptosis: triggering of ceramide generation through sphingomyelin hydrolysis. EMBO J 1996; 15:2417-2424. 4. Cabot MC, Giuliano AE. Apoptosis-a cell mechanism important for cytotoxic response to adriamycin and a lipid metabolic pathway that facilitates escape. Breast Cancer Res Treat 1997; 47:71. 5. Lucci A, Han TY, Liu YY et al. Modification of ceramide metabolism increases cancer cell sensitivity to cytotoxics. Int J Oncol 1999; 15:541-546. 6. Lavie Y, Cao H, Bursten SL et al. Accumulation of glucosylceramides in multidrug-resistant cancer cells. J Biol Chem 1996; 271:19530-19536. 7. Lucci A, Cho WI, Han TY et al. Glucosylceramide: a marker for multiple-drug resistant cancers. Anticancer Res 1998; 18:475-480. 8. Kok JW, Veldman RJ, Klappe K et al. Differential expression of sphingolipids in MRP1 overexpressing HT29 cells. Int J Cancer 2000; 87:172-178. 9. Lucci A, Giuliano AE, Han TY et al. Ceramide toxicity and metabolism differ in wild-type and multidrug-resistant cancer cells. Int J Oncol 1999; 15:535-540. 10. Liu YY, Cabot MC. Development of a mammalian tet-on expression cell line: glucosylceramide synthase regulates TNF-α-induced apoptosis. In: DeLey M, ed. Human Cytokine Protocols in Methods in Molecular Biology. Totowa: Humana Press, Inc., 2002. 11. Liu YY, Han TY, Giuliano AE et al. Expression of glucosylceramide synthase, converting ceramide to glucosylceramide, confers adriamycin resistance in human breast cancer cells. J Biol Chem 1999; 274:1140-1146. 12. Liu YY, Han TY, Giuliano AE et al. Glycosylation of ceramide potentiates cellular resistance to tumor necrosis factor-alpha-induced apoptosis. Exp Cell Res 1999; 252:464-470. 13. Liu YY, Han TY, Giuliano AE et al. Uncoupling ceramide glycosylation by transfection of glucosylceramide synthase antisense reverses adriamycin resistance. J Biol Chem 2000; 275:7138-7143. 14. Liu Y-Y, Han T-Y, Giuliano AE et al. Ceramide glycosylation potentiates cellular multidrug resistance. FASEB J 2001; 15:719-730. 15. Del Prete SA, Maurer LH, O’Donnell J et al. Combination chemotherapy with cisplatin, carmustine, dacarbazine, and tamoxifen in metastatic melanoma. Cancer Treat Rep 1984; 68:1403-1405.

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16. Gelmann EP. Tamoxifen for the treatment of malignancies other than breast and endometrial carcinoma. Semin Oncol 1997; 24:S1-65-S1-70. 17. Couldwell WT, Weiss MH, DeGiorgio CM et al. Clinical and radiographic response in a minority of patients with recurrent malignant gliomas treated with high-dose tamoxifen. Neurosurgery 1993; 32:485-489; discussion 489-490. 18. O’Day SJ, Boasberg PD, Kristejda TS et al. High-dose tamoxifen added to concurrent biochemotherapy with decrescendo interleukin-2 in patients with metastatic melanoma. Cancer 2001; 92:609-619. 19. Cabot MC, Giuliano AE, Volner A et al. Tamoxifen retards glycosphingolipid metabolism in human cancer cells. FEBS Lett 1996; 394:129-131. 20. Lavie Y, Cao HT, Volner A et al. Agents that reverse multidrug resistance, tamoxifen, verapamil, and cyclosporin A, block glycosphingolipid metabolism by inhibiting ceramide glycosylation in human cancer cells. J Biol Chem 1997; 272:1682-1687. 21. El Etreby MF, Liang Y, Wrenn RW et al. Additive effect of mifepristone and tamoxifen on apoptotic pathways in MCF-7 human breast cancer cells. Breast Cancer Res Treat 1998; 51:149-168. 22. Siegsmund MJ, Cardarelli C, Aksentijevich I et al. Ketoconazole effectively reverses multidrug resistance in highly resistant KB cells. J Urol 1994; 151:485-491. 23. Small EJ, Baron AD, Fippin L et al. Ketoconazole retains activity in advanced prostate cancer patients with progression despite flutamide withdrawal. J Urol 1997; 157:1204-1207. 24. Lucci A, Han TY, Liu YY et al. Multidrug resistance modulators and doxorubicin synergize to elevate ceramide levels and elicit apoptosis in drug-resistant cancer cells. Cancer 1999; 86:300-311. 25. Abe A, Inokuchi J, Jimbo M et al. Improved inhibitors of glucosylceramide synthase. J Biochem (Tokyo) 1992; 111:191-196. 26. Sietsma H, Veldman RJ, Kolk D et al. 1-phenyl-2-decanoylamino-3-morpholino-1-propanol chemosensitizes neuroblastoma cells for taxol and vincristine. Clin Cancer Res 2000; 6:942-948. 27. Olshefski RS, Ladisch S. Glucosylceramide synthase inhibition enhances vincristine-induced cytotoxicity. Int J Cancer 2001; 93:131-138. 28. Deng W, Li R, Ladisch S. Influence of cellular ganglioside depletion on tumor formation. J Natl Cancer Inst 2000; 92:912-917. 29. Selzner M, Bielawska A, Morse MA et al. Induction of apoptotic cell death and prevention of tumor growth by ceramide analogues in metastatic human colon cancer. Cancer Res 2001; 61:1233-1240. 30. Nicholson KM, Quinn DM, Kellett GL et al. Preferential killing of multidrug-resistant KB cells by inhibitors of glucosylceramide synthase. Br J Cancer 1999; 81:423-430. 31. Spinedi A, Bartolomeo SD, Piacentini M. Apoptosis induced by N-hexanoylsphingosine in CHP100 cells associates with accumulation of endogenous ceramide and is potentiated by inhibition of glucocerebroside synthesis. Cell Death Differ 1998; 5:785-791. 32. Archinal-Mattheis A, Rzepka RW, Watanabe T et al. Analysis of the interactions of SDZ PSC 833 ([3’-keto-Bmtl-Val2]-cyclosporine), a multidrug resistance modulator, with P-glycoprotein. Oncol Res 1995; 7:603-610. 33. Cabot MC, Giuliano AE, Volner A et al. Tamoxifen retards glycosphingolipid metabolism in human cancer cells. FEBS Lett 1996; 394:129-131. 34. Goulding CW, Giuliano AE, Cabot MC. SDZ PSC 833 the drug resistance modulator activates cellular ceramide formation by a pathway independent of P-glycoprotein. Cancer Lett 2000; 149:143-151. 35. Pallis M, Russell N. P-glycoprotein plays a drug-efflux-independent role in augmenting cell survival in acute myeloblastic leukemia and is associated with modulation of a sphingomyelin-ceramide apoptotic pathway. Blood 2000; 95:2897-2904. 36. Perez GI, Knudson CM, Leykin L et al. Apoptosis-associated signaling pathways are required for chemotherapy-mediated female germ cell destruction. Nat Med 1997; 3:1228-1232. 37. Cabot MC, Giuliano AE, Han T-Y et al. SDZ PSC 833, the cyclosporine A analogue and multidrug resistance modulator, activates ceramide synthesis and increases vinblastine sensitivity in drug-sensitive and drug-resistant cancer cells. Cancer Res 1999; 59:880-885. 38. Gill JS, Windebank AJ. Role of ceramide in suramin-induced cancer cell death. Cancer Lett 1997; 119:169-176. 39. Tu SM, McConnell K, Marin MC et al. Combination adriamycin and suramin induces apoptosis in bcl-2 expressing prostate carcinoma cells. Cancer Lett 1995; 93:147-155. Erratum in Cancer Lett 1996; 99:247. 40. Lawrence JB, Conover CA, Haddad TC et al. Evaluation of continuous infusion suramin in metastatic breast cancer: impact on plasma levels of insulin-line growth factors (IGFs) and IGF-binding proteins. Clin Cancer Res 1997; 3:1713-1720.

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41. Beedassy A, Cardi G. Chemotherapy in advanced prostate cancer. Semin Oncol 1999; 26:428-438. 42. Smith DC. Chemotherapy for hormone refractory prostate cancer. Urol Clin North Am 1999; 26:323-331. 43. Wang XZ, Beebe JR, Pwiti L et al. Aberrant sphingolipid signaling is involved in the resistance of prostate cancer cell lines to chemotherapy. Cancer Res 1999; 59:5842-5848. 44. Seelan RS, Qian C, Yokomizo A et al. Human acid ceramidase is overexpressed but not mutated in prostate cancer. Genes Chromosomes Cancer 2000; 29:137-146. 45. Maurer BJ, Metelitsa LS, Seeger RC et al. Increase of ceramide and induction of mixed apoptosis/ necrosis by N-(4-hydroxyphenyl)retinamide in neuroblastoma cell lines. J Natl Cancer Inst 1999; 91:1138-1146. 46. Maurer BJ, Melton L, Billups C et al. Synergistic cytotoxicity in solid tumor cell lines between N(4-hydroxyphenyl)retinamide and modulators of ceramide metabolism. J Natl Cancer Inst 2000; 92:1897-1909. 47. Schwartz GK, Ward D, Saltz L et al. A pilot clinical/pharmacological study of the protein kinase C-specific inhibitor safingol alone and in combination with doxorubicin. Clin Cancer Res 1997; 3:537-543. 48. Myrick D, Blackinton D, Klostergaard J et al. Paclitaxel-induced apoptosis in Jurkat, a leukemic T cell line, is enhanced by ceramide. Leuk Res 1999; 23:569-578. 49. Mehta S, Blackinton D, Omar I et al. Combined cytotoxic action of paclitaxel and ceramide against the human Tu138 head and neck squamous carcinoma cell line. Cancer Chemother Pharmacol 2000; 46:85-92. 50. Charles AG, Han T-Y, Liu YY et al. Taxol-induced ceramide generation and apoptosis in human breast cancer cells. Cancer Chemother Pharmacol 2001; 47:444-450. 51. Radin NS. Killing cancer cells by poly-drug elevation of ceramide levels—a hypothesis whose time has come? Eur J Biochem 2001; 268:193-204. 52. Radin NS. Apoptotic death by ceramide: will the real killer please stand up? Med Hypotheses 2001; 57:96-100. 53. Perry DK, Carton J, Shah AK et al. Serine palmitoyltransferase regulates de novo ceramide generation during etoposide-induced apoptosis. J Biol Chem 2000; 275:9078-9084. 54. Wang H, Maurer BJ, Reynolds CP et al. N-(4-hydroxyphenyl)retinamide elevates ceramide in neuroblastoma cell lines by coordinate activation of serine palmitoyltransferase and ceramide synthase. Cancer Res 2001; 61:5102-5105.

CHAPTER 15

Ceramide in Serum Lipoproteins: Function and Regulation of Metabolism Mariana N. Nikolova-Karakashian

Abstract

S

erum ceramide levels increase during the acute phase response to inflammation in animal models and in humans. Two major mechanisms appear to mediate these changes. The bacterial endotoxin, LPS, stimulates serine-palmitoyl transferase (SPT) mRNA levels and activity in liver, thus increasing de novo synthesis of ceramide. This is paralleled by an increase in ceramide levels in very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) fractions, but not in albumin and HDL fractions. Inflammatory mediators also stimulate the secretion of Zn2+-dependent sphingomyelinase (sSMase) by endothelial cells and macrophages, that acts on LDL particles trapped in the sub-endothelial space and hydrolyses LDL sphingomyelin (SM) to ceramide. The resulting ceramide-enriched LDL have highly atherogenic properties. They are taken up by the endothelial cells in a receptor-mediated fashion and can deliver excess ceramide to the cells. This is paralleled by increased incidence of cell death. In turn, sSMase-treated lipoproteins undergo spontaneous aggregation, they are retained in the sub-endothelial space and induce foam cell formation in macrophages. In vivo data, using knockout mouse models, confirm further that LDL enriched in ceramide are an alternative route to increase intracellular ceramide levels and to affect the pro-atherogenic properties of LDL.

Introduction Extensive studies over the last decade implicate generation of ceramide in the eukaryotic stress response and propose a role of ceramide as a “biostat” in the stress response.1 The experimental foundations for this concept are based on observations that multiple stress agents, such as inducers of apoptosis and inflammatory cytokines, generate ceramide intracellularly either by stimulating hydrolysis of SM at different subcellular compartments,2-5 or by activating de novo synthesis in the endoplasmic reticulum.6,7 In turn, enhancement of intracellular levels of ceramide is sufficient to induce many of the stress responses associated with these agonists. Moreover, activation of pathways for ceramide clearance, e.g., turnover to sphingosine and consequently to sphingosine phosphate, can prevent the detrimental effects of some stress agonists in many cell types, thus supporting a role of ceramide as a stress “biostat”. 8,9,10 In addition to being generated intracellularly, ceramide is a component of serum lipoproteins and its levels increase during experimental models of inflammation11,12 as well as during some inflammation-based disease conditions. The functional significance of these increases is not well understood, however a role in the onset of atherosclerosis has been proposed and supported by numerous experimental evidence. Two possible scenarios have been investigated. First, increases in ceramide concentrations in LDL, when paralleled by reciprocal decreases in SM mass, induce extensive aggregation13 and oxidation14 of LDL particles. Both of these Ceramide Signaling, edited by Anthony H. Futerman. ©2002 Eurekah.com and Kluwer Academic / Plenum Publishers.

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modifications are intimately involved in the induction of macrophage foam cell formation, regulation of endothelial functions and smooth muscle cell proliferation. In turn, the uptake of LDL with increased ceramide concentrations may lead to elevation of intracellular ceramide, thus providing an alternative route for regulation of intracellular ceramide levels. The goal of this review is to summarize the current information on ceramide present in serum, with a focus on the mechanisms that regulate its levels and its possible functional significance.

Ceramide is a Component of Serum Lipoproteins SM is a structural constituent of lipoproteins and the second major phospholipid after phosphatidylcholine. However, very little is known with respect to ceramide. As far as one can readily ascertain, the only published study15 to quantify the mass of ceramide in serum was done in rats, which differ greatly from humans in respect to their lipoprotein profiles. According to this study, ceramide is present in serum, and VLDL has the highest ceramide concentration (6.5 nmol/mg protein), followed by LDL with 0.5 nmol/mg protein, HDL with 0.2 nmol/mg protein, and the albumin fraction that has less than 0.1 nmol/mg protein. Our studies show that in humans, ceramide is distributed similarly and HDL and albumin fractions have low levels of ceramide. However, the levels in LDL are comparable, and even higher than those in VLDL (Table 1). Since the majority of LDL are derived from VLDL (with a small percent being secreted directly from the liver) the differences with rats may be due to the different rates of conversion of VLDL to LDL and/or different catabolic rates of LDL. These results demonstrate that the majority of serum ceramide in humans is in VLDL and LDL particles, suggesting that the regulation of serum ceramide levels is likely to occur via the enzymes that determine VLDL and LDL ceramide content. Below we will concentrate on the two major pathways implicated in that, namely de novo synthesis in liver, and degradation of LDL SM.

Secretion of Ceramide in the Form of VLDL by the Liver

In 1995, Merrill at all15 showed that isolated rat hepatocytes secrete up to 5% of the newly synthesized sphingolipids (the majority of which is ceramide) in the form of VLDL. A number of factors can affect the levels of ceramide in the newly secreted VLDL. For example, the addition of palmitic acid, but not stearic or oleic acid, enhances ceramide secretion due to stimulation of de novo synthesis of long-chain bases by serine-palmitoyl transferase (SPT). In turn, inhibition of de novo synthesis of sphingolipids with Fumonisin B1 (FB1), (Fig. 1),a mycotoxin inhibitor of sphinganine-N-acyltransferase (see Chapter 1), reduces overall sphingolipid synthesis and secretion by 90%. In both cases, the rate of secretion of ApoB, the protein component of VLDL, is not affected. These data thus suggest that ceramide concentration in VLDL and LDL may be regulated independently of the mechanisms that determine the overall rate of lipoprotein secretion by the liver. Evidence shows that the ceramide concentration in VLDL and LDL is also regulated in vivo. Induction of systemic inflammatory response in hamsters by administration of the bacterial endotoxin, lipopolisaccharide (LPS), induces an elevation of ceramide in VLDL and LDL after 16 to 24 hours .11 Increases in VLDL and LDL ceramide content (2 to 3 fold over basal levels) are also found in humans injected with LPS (data submitted for publication). For the VLDL particles, these increases are noticeable at 3 to 6 hours after LPS administration, while in LDL, the increases start at a later time point (6 to 24 hours). In contrast, the ceramide levels in HDL and lipoprotein-deficient serum are not affected. In hamsters, LPS administration up-regulates liver SPT mRNA levels, SPT activity and de novo synthesis of sphingolipids,11 thus suggesting that stimulation of de novo synthesis of ceramide mediates ceramide increases in VLDL and LDL. This is supported further by studies using a radiolabeled substrate for SPT, [3H]-serine. These studies show an increase in [3H]ceramide in VLDL and LDL particles after LPS administration. In addition, [3H]-SM and

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Table 1. Analyses of ceramide mass in human lipoproteins LPDS

VLDL

LDL

HDL

1.368±0.288 5.2±0.4

Ceramide nmol/mg.Protein 0.273±0.041 % 15.1±3.2

2.594±0.291 41.5±5.9

4.220±0.138 38.0±4.8

DHceramide pmol/mg.Protein 5.7±0.9 % 25.04±3.9

226.4±0.6 40.3±2.7

261.8±2.9 30.0±2.3

24.2±0.1 4.8±0.8

Lipoproteins were isolated from human plasma and the mass of ceramide and dihydroceramide was analyzed by TLC/HPLC. The percent value is calculated based on the protein recovery in each fraction and is a percent of the total plasma ceramide/dihydroceramide. Abbreviations: LPDS, lipoprotein deficient serum; VLDL, very low density lipoproteins; LDL, low-density lipoproteins; HDL, high density lipoproteins; DHCeramide, dihydroceramide.

[3H]-GlcCer levels also increase.12 In view of the role of ceramide as a rate-limiting precursor in the synthesis of these sphingolipids, this observation provides additional evidence which supports the role of de novo synthesis in the increases in VLDL and LDL ceramide levels. The mechanism for induction of SPT mRNA expression during inflammation is unclear. When hamsters are injected with TNFα and IL-1β instead of LPS, only the IL-1β treatment reproduces the effects of LPS.11,12 This suggests that LPS-induced secretion of IL-1β may be involved; however, direct evidence for its role is still lacking. Other conditions, such as atherogenic diet (0.15% cholesterol and 12.8% milk fat) and aging also increase serum ceramide levels (unpublished data). Because 26% of the milk fat present in the diet is palmitic acid, it is likely that increased levels of ceramide are caused by activation of SPT activity, similar to the aforementioned effects of palmitic acid in isolated hepatocytes.

Generation of Ceramide in LDL Particles In addition to incorporation by de novo synthesis, ceramide levels in LDL are modulated by degradation of SM present in the lipoproteins. Two different SMases have been described to act on LDL.

Zn2+-Dependent Secreted SMase While the changes in de novo synthesis in the liver affect the concentrations of ceramide in circulating lipoproteins, sSMase has been suggested to alter ceramide levels in LDL trapped in the sub-endothelial space. The role of sSMase in modifying LDL ceramide levels is relatively well understood and readers are referred to a recent review article16 for more information. The sSMase is the product of the ASMase gene and is the only known secreted SMase in mammals. The protein is secreted mainly by the endothelial cells, and, to a lesser extent by macrophages in an inflammation-dependent manner. The secretion of sSMase by macrophages is stimulated in vitro by IL-1β.17 The mechanism(s) for the effects of IL-1β is not well understood, however it is clear that cytokine treatment affects a step in the post-translational modification of the protein that determines whether it will be targeted to lysosomes (and act as an acid SMase) or will be secreted along the exocytotic pathway.18 Another puzzling feature of the sSMase is that while the lysosomal form of the enzyme has an acidic pH optimum and does not require Zn2+, the secreted form is Zn2+-dependent and is active at neutral pH.19

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Figure 1. Incorporation of ceramide and complex sphingolipids in VLDL particles in liver. The following enzymes of sphingolipid synthesis are shown: 1, serine-palmitoyl transferase (SPT); 2, sphinganine-N-acyl transferase; 3, dihydroceramide desaturase; 4, SM synthase; 5, glucosylceramide synthase.

Modifications of LDL such as oxidation or phospholipase A2 treatment increase the susceptibility of LDL to sSMase. Furthermore, sSMase is activated by the presence of a proteoglycan matrix that immobilizes the LDL in the sub-intimal space.20 Finally, atherosclerotic arterial lesions have increased sSMase protein levels (and higher ceramide levels) as compared to normal aorta.20 These observations support the idea that Zn2+-dependent SMase plays a role in modifying the ceramide concentration in LDL trapped in the sub-entothelium. At the same time however, it is still possible for sSMase-altered LDL particles to be released back into the circulation and thus to contribute to the regulation of overall ceramide levels in the serum. Furthermore, administration of LPS to C57/Bl6 mice induced an increase in the Zn2+ -dependent SMase activity in the serum, which also supports the possibility that sSMase may contribute to the increases in ceramide levels in circulating LDL21 (Fig. 2).

LDL-Associated Sphingomyelinase When giant negatively charged SM-containing liposomes have been used as a substrate, an intrinsic SMase activity of native LDL but not oxidized LDL, VLDL and HDL particles has been found.22 The biochemical characterization of this activity, however, is incomplete and details regarding its substrate specificity, pH and cationic dependence are lacking. Based on sequence comparison between bacterial SMases and apoB, it has been suggested that this enzymatic activity could be an intrinsic property of apoB. The functional significance of LDLassociated SMase activity is unclear; however one can envision that SM localized in the outer shell of neighboring LDL particles may be targeted.

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Figure 2. An excessive generation of ceramide in liver (Mechanism A) during acute inflammation leads to increased incorporation of ceramide in VLDL and LDL. Alternatively, inflammation can induce the secretion of Zn2+-dependent SMase (Mechanism B) by the endothelial cells that degrades LDL SM to ceramide. Increases in LDL ceramide levels cause LDL aggregation, retention in sub-intimal space and foam cell formation.

Biological Consequences of Elevation of Ceramide Concentrations in LDL Effects of Ceramide on LDL Properties Conversion of LDL SM to ceramide has been shown to increase the aggregation rate of LDL particles,13,18 to enhance arterial matrix binding20 and to induce foam cell formation.23 LDL isolated from ApoE deficient mice that spontaneously develop atherosclerotic lesions have high concentration of SM as compared to normal C57/Bl6 mice.23 When these lipoproteins are treated with sSMase in vitro, they aggregate much faster than SMase-treated control LDL. Furthermore, the resulting ceramide-rich lipoproteins are potent inducers of macrophage foam cell formation and stimulate cholesterol esterification by ~5-fold. When the ceramide content of lesional LDL was assayed it was found to be 10 to 15 fold higher than that of serum LDL. Furthermore, the excess ceramide was found exclusively in the aggregated lesional LDL, while those that were monomeric had low ceramide concentration.13 These studies provide convincing evidence that increased conversion of SM to ceramide in LDL particles stimulates aggregation and, at least, correlates with the development of atherosclerotic plaques. It is unclear however, if it is the lack of SM or the increase in ceramide content that is directly responsible for the aggregation. The sSMase is likely to act on SM molecules that are randomly distributed on the lipoprotein surface. This may not be sufficient to induce LDL aggregation because clearly, the formation of LDL aggregates requires the formation of large hydrophobic

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areas on the LDL surface. Biophysical studies however have shown that, in large liposomes, ceramide can self-segregate and can form domains.24 Therefore, it is likely that the excess generation of ceramide rather than the lack of SM is the immediate inducer of LDL aggregation. Experimental evidence also supports this notion, since SMase-induced aggregation can be observed even in the presence of excess SM.13 The increased conversion of LDL SM to ceramide, at least in vitro, may also increase the susceptibility of LDL for oxidation.15 LDL treated with bacterial SMase are oxidized to a much greater extent than non-treated LDL particles. It has been proposed that degradation of LDL SM increases the exposure of LDL interior to reactive oxygen species. The functional significance of these observations however is unclear because the current understanding of the oxidation processes in atherosclerosis is that LDL become oxidized in the circulation, and not in sub-endothelium space, where LDL particles are exposed to sSMase. The role of LDL ceramide for the development of atherosclerosis is also supported by in vivo studies. ApoE deficient mice that also lack sSMase develop less atherosclerotic plaques, only about 37% (based on the total plaque areas) of that in an ApoE knockout that has SMase activity.25 This remarkable protective effect has been found in both genders and in both early and advanced plaques. Importantly, cholesterol levels in the two mice were not affected. Thus, these experiments provide strong in vivo evidence that an increase in ceramide levels in LDL promotes atherosclerosis. The few epidemiological data that are available for humans also confirm this concept. For example, pre-menopausal women have significantly lower level of SM in plasma than age-matched male controls. However, during conditions characterized with increased risk for development of atherosclerosis such as menopause, diabetes and hypertriglyceremia, SM levels in plasma increase and reach those found in males.26,27 Together with the aforementioned observations in ApoE knockout mice, these data show that increases in SM LDL levels, and consequently increased potential to generate excessive ceramide correlates with the risk for development of atherosclerosis.

Effects of LDL-Derived Ceramide on Intracellular Ceramide Homeostasis Circulating LDL is taken-up by the cells of the vasculature via receptor mediated endocytosis. While the intracellular metabolic fate of cholesterol and ApoB components of LDL is relatively well elucidated, that of LDL-derived sphingolipids is unclear. It is presumed that once delivered to the lysosomes, sphingolipids are efficiently degrdaded by acidic SMase and ceramidase to sphingosine. The latter could either be degraded further to hexadecenal and ethanolamine phosphate via cytosolic sphingosine kinase and sphingosine-phosphate lyase, or re-acylated to form ceramide. In turn, increased supply of ceramide from exogenous sources may affect the de novo synthesis of ceramide by a feed-back mechanism. None of these possibilities however has been tested experimentally, and it is unclear whether the clearance pathway is efficient to turnover the excess ceramide derived from sphingolipid-rich LDL. Ongoing research in our laboratory has focused on understanding the metabolic fate of ceramide derived from ceramide-rich LDL particles. The major findings of these studies are currently being prepared for publication and can be summarized as follows: In vitro, LDL can be enriched with short-chain ceramide analogues, C2- and C6-ceramide, in a specific and controllable manner and can form stable complexes, referred to as ceramideenriched LDL (Cer-LDL). When LDL are enriched with ceramide at a level of 18 nmol/mg protein (corresponding to a 3-4 fold increase over the normal ceramide concentrations in LDL), and used to treat human microvascular endothelial cells, HMEC-1, accumulation of the respective ceramide analogue is detected within 4 hours of treatment and reaches up to 0.25 nmol/mg protein. Such accumulation is unlikely to be a property of the short-chain ceramide only, because both, C2- and C6-ceramide have been shown to be good substrates for ceramidase activities in liver28 and other cell types.29 Furthermore, the appearance of exogenous ceramide in the cells follows closely the kinetics of receptor-mediated uptake and can be efficiently competed by native LDL.

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Based on Annexin V and Hoechst staining, treatment with Cer-LDL (100µg/ml) is sufficient to induce apoptosis (reaching 10 to 15% of all cells), while control LDL is inactive. Remarkably, equimolar concentrations of C2-ceramide (1.8µM) when added as an ethanol vehicle, is insufficient to induce cell death despite the fact that a higher percent of the exogenous ceramide is internalized. These comparisons show that LDL-derived ceramide is more efficient in inducing cell death than ceramide delivered by the conventional experimental (and non-physiological) method. This higher efficiency is clearly not a result of higher intracellular mass accumulation, but rather of targeting to specific sub-cellular localizations and/or activation of additional signaling mechanisms that may promote apoptosis. Taken together, these data therefore suggest that LDL-derived ceramide may have bioactive properties similar to those described for ceramide generated endogenously. Moreover, in this case, the effects on cell functions are independent of agonist-induced activation of cellular SMases. Finally, they suggest that increases in serum ceramide levels during inflammation may contribute to the endothelial cell death observed in conditions related to septic shock or diseases characterized with chronic secretion of inflammatory cytokines.

Conclusions and Future Directions The observations summarized in this article provide initial evidence that ceramide can act as a bioactive molecule not only when generated intracellularly, but also outside the cells, and in some occasions in a tissue that is distant from the targeted cell. This implies that in vivo, in addition to ceramide generated intracellularly in response to different agonists, other sources of ceramide may contribute to the overall level of ceramide in the cells, and respectively to its biological effects. This scenario depends on the presence of specific pathways for internalization, such as, LDL receptors and scavenger receptors for the monomeric ceramide-rich LDL, or specific phagocytosis-like internalization processes for the aggregated LDL that may not be present in all cell types. It is likely however, that in cells that are actively involved in lipoprotein uptake, such as macrophages, vascular endothelial cells and smooth muscle cells, as well as cells of adipose tissue, LDL as a source of ceramide could be important in inflammatory-like pathological conditions. Since chronic, low amplitude inflammation underlies the onset of many diseases and pathophysiological processes, such as atherosclerosis and aging, to name a few, LDL as a source of ceramide could be important for understanding the role of ceramide as a stress mediator in vivo.

References 1. Hannun YA, Luberto C, Argraves KM. Enzymes of sphingolipid metabolism: from modular to integrative signaling. Biochemistry 2001; 40(16):4893-903. 2. Kronke M. Involvement of sphingomyelinases in TNF signaling pathways. Chem Phys Lipids 1999; 102(1-2):157-66. 3. Linardic CM, Hannun YA. Identification of a distinct pool of sphingomyelin involved in the sphingomyelin cycle. J Biol Chem 1994; 269(38):23530-7. 4. Santana P, Pena LA, Haimovitz-Friedman et al. Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis. Cell 1996; 86(2):189-99. 5. Birbes H, El Bawab S, Hannun YA et al. Selective hydrolysis of a mitochondrial pool of sphingomyelin induces apoptosis. FASEB J 2001; 15(14):2669-79. 6. Bose R, Verheij M, Haimovitz-Friedman A et al. Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals. Cell 1995; 82(3):405-14. 7. Perry DK, Carton J, Shah AK et al. Serine palmitoyltransferase regulates de novo ceramide generation during etoposide-induced apoptosis. J Biol Chem 2000; 275(12):9078-84. 8. Cuvillier O, Rosenthal DS, Smulson ME et al. Sphingosine 1-phosphate inhibits activation of caspases that cleave poly (ADP-ribose) polymerase and lamins during Fas- and ceramide-mediated apoptosis in Jurkat T lymphocytes. J Biol Chem 1998; 273(5):2910-6. 9. Dawson G, Goswami R, Kilkus J et al. The formation of ceramide from sphingomyelin is associated with cellular apoptosis. Acta Biochim Pol 1998; 45(2):287-97. 10. Xia P, Wang L, Gamble JR et al. Activation of sphingosine kinase by tumor necrosis factor-alpha inhibits apoptosis in human endothelial cells. J Biol Chem 1999; 274(48):34499-505.

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11. Memon RA, Holleran WM, Moser AH et al. Endotoxin and cytokines increase hepatic sphingolipid biosynthesis and produce lipoproteins enriched in ceramides and sphingomyelin. Arterioscler Thromb Vasc Biol 1999; 18(8):1257-65. 12. Memon RA, Holleran WM, Uchida Y et al. Regulation of glycosphingolipid metabolism in liver during the acute phase response. J Biol Chem 1999; 274(28):19707-13. 13. Schissel SL, Tweedie-Hardman J, Rapp JH et al. Rabbit aorta and human atherosclerotic lesions hydrolyse the sphingomyelin of retained low-density lipoprotein. Proposed role for arterial-wall sphingomyelinase in subendothelial retention and aggregation of atherogenic lipoproteins. J Clin Invest 1996; 98(6):1455-64. 14. Subbaiah PV, Subramanian VS, Wang K. Novel physiological function of sphingomyelin in plasma. Inhibition of lipid peroxidation in low density lipoproteins. J Biol Chem 1999; 274(51):36409-14. 15. Merrill AH Jr, Lingrell S, Wang E et al. Sphingolipid biosynthesis de novo by rat hepatocytes in culture. Ceramide and sphingomyelin are associated with, but not required for, very-low density lipoprotein (VLDL) secretion. J Biol Chem 1995; 270:13834-13841. 16. Tabas I. Secretory sphingomyelinase. Chem Phys Lipids 1999; 102(1-2):123-30. 17. Marathe S, Schissel SL, Yellin MJ et al. Human vascular endothelial cells are a rich and regulatable source of secretory sphingomyelinase. Implications for early atherogenesis and ceramide-mediated cell signaling. J Biol Chem 1998; 273(7):4081-8. 18. Schissel SL, Keesler GA., Schuchman E.H et al. The cellular trafficking and zinc dependence of secretory and lysosomal sphingomyelinase, two products of the acid sphingomyelinase gene. J Biol Chem 1998:273(29) 18250-9. 19. Schissel SL, Jiang X, Tweedie-Hardman J et al. Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral pH. Implications for atherosclerotic lesion development J Biol Chem 1998; 273(5):.2738-46. 20. Marathe S, Kuriakose G, Williams KJ et al. Sphingomyelinase, an enzyme implicated in atherogenesis, is present in atherosclerotic lesions and binds to specific components of the subendothelial extracellular matrix . Arterioscler Thromb Vasc Biol 1999; 19(11):2648-58. 21. Wong ML, Xie B, Beatini N et al. Acute systemic inflammation up-regulates secretory sphingomyelinase in vivo: a possible link between inflammatory cytokines and atherogenesis. Proc Natl Acad Sci USA 2000; 97(15):8681-6. 22. Holopainen JM, Medina OP, Metso AJ et al. Sphingomyelinase activity associated with human plasma low density lipoprotein. J Biol Chem 2000; 275(22):16484-9. 23. Marathe S, Choi Y, Leventhal AR et al. Sphingomyelinase converts lipoproteins from apolipoprotein E knockout mice into potent inducers of macrophage foam cell formation. Arterioscler Thromb Vasc Biol. 2000; 20(12):2607-13. 24. Holopainen JM, Angelova MI, Kinnunen PK. Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes. Biophys J 2000; 78(2):830-8. 25. Marathe S., Tribble D., Kuriakose G. et al. Sphingomyelinase transgenic and knockout mice: Direct evidence that SMase is atherogenic in vivo. Circulation (Abstr.) 1999; (100):I-695. 26. Bagdade JD, Buchanan WF, Pollare T et al. Abnormal lipoprotein phospholipid composition in patients with essential hypertension. Atherosclerosis 1995 117(2):209-15. 27. Lane JT, Subbaiah PV, Otto ME et al. Lipoprotein composition and HDL particle size distribution in women with non-insulin-dependent diabetes mellitus and the effects of probucol treatment. J Lab Clin Med 1991; 118(2):120-8. 28. Nikolova-Karakashian MN, Morgan ET, Alexander C et al. Bimodal regulation of ceramidase by interleukin-1ß: Implication for the regulation of cytochrome P450 2C11 (CYP2C11). J Biol Chem 1997; 272:18718-18724. 29. Ogretmen B, Pettus BJ, Rossi MJ et al. Biochemical mechanisms of the generation of endogenous long chain ceramide in response to exogenous short chain ceramide in the A549 human lung adenocarcinoma cell line: Role for endogenous ceramide in mediating the action of exogenous ceramide. J Biol Chem 2002; Jan 28 (e-publication). 30. Chen J, Nikolova-Karakashian MN, Merrill AH Jr et al. Regulation of cytochrome P4502C11 (CYP2C11) gene expression by interleukin-1, sphingomyelin hydrolysis and ceramides in rat hepatocytes. J Biol Chem 1995; 270:25233-25238.

CHAPTER 16

Therapeutic Implications of CeramideRegulated Signaling Cascades Mark Kester, Jong K. Yun, Tom Stover and Lakshman Sandirasegarane

Abstract

F

rom “Bench to Bedside” is the often-used phrase that alludes to the potential clinical or therapeutic benefits of innovative basic science research or technologies. However, the “Bench to Bedside” phrase is often not supported by translationally-based research that exploits basic science discoveries. Based upon major leaps in our understanding of the biochemistry, biophysics and molecular biology of lipid-derived second messengers, the sphingolipid field is finally in position to embrace the “Bench to Beside” concept. This chapter will focus on studies that have begun to define the therapeutic potential of strategies that alter endogenous levels of ceramide or ceramide analogues in cardiovascular and cancer models. Wherever possible, results from translationally-based studies will be used to clarify or re-evaluate controversies in the literature.

The Bench—Ceramides and Signaling Cascades The in vivo use of ceramide analogues, ceramide mimetics or agents that alter endogenous ceramide concentration can only be a reality after we fundamentally understand the mechanisms of action of ceramides on the multiplicity of redundant, inter-related signaling cascades that allow cells to communicate. It is now well established that ceramide interacts with multiple signaling cascades leading to cell growth arrest and/or apoptosis.1,2 Even though several candidate targets for ceramide have been suggested, including KSR,3 (see Chapter 7) PKCζ,4-8 MEKK1,7,9 PP2a,10 (see Chapter 6) VAV,11 PKR,12 cathepsin D13 and calpain,14 the ability of ceramide to directly bind to these intracellular signaling elements through a discrete ceramidebinding domain(s) has not been rigorously proven. Yet, a consensus of opinion is emerging, suggesting that ceramide interacts with multiple upstream elements in signaling cascades through generation and localization in discrete membrane microdomains.15-17 It can also be envisioned that ceramide ultimately acts as a cofactor, providing the specificity and selectivity necessary for multiple kinase interactions, leading to the formation of signaling complexes. Scaffold or anchoring proteins can regulate the interaction of multiple signaling elements to form these “signalosomes”.18-21 It is likely that lipid-derived second messengers, including ceramide, can augment these protein/protein interactions through specific lipid/ protein interactions at discrete lipid-binding domains. In this way, ceramide can regulate the formation of unique, tissue-specific, signalosomes linked to growth regulation and apoptosis. In an analogous fashion, TNF “receptosomes” have been implicated as signaling vesicles, transmitting acid-SMase-generated ceramide and co-localized cathepsin from the endosome into the cytosol where it can access substrates.13 Our laboratory has recently reported three synergistic mechanisms by which ceramide can regulate signalosome formation in smooth muscle cell pericytes, and human embryonic kidney Ceramide Signaling, edited by Anthony H. Futerman. ©2002 Eurekah.com and Kluwer Academic / Plenum Publishers.

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cells7,8,22 (Fig. 1). These signaling mechanisms are based upon earlier studies, from multiple investigators using various cell types, demonstrating that ceramide induces cell cycle arrest and/or apoptosis through simultaneous activation of stress-activating protein kinases (SAPK) and inhibition of extracellular signal regulated kinase (ERK) and /or AKT.23-32 However, the precise mechanism(s) by which ceramide concomitantly regulates these multiple signaling cascades have not been defined. Recent studies have suggested that interactions between ceramide and distinct PKC isotypes may be a unifying paradigm underscoring ceramide regulation of signalosomes. In the first mechanism, cell-permeable or physiological ceramides activate either immunoprecipitated or recombinant PKCζ, which induces formation of a MEKK1/SEK/SAPK signalosome.7 In a similar scenario, Wang et al have shown that PKCζ is a signaling intermediate for ceramide-activated SAPK in PC12 cells.26 In the second mechanism, ceramide competitively inhibits DAG-stimulated PKCε to disrupt the formation of a Raf/MEK/ERK signalosome, an event that is also associated with growth arrest.22 A third mechanism by which ceramide checks cell cycle progression is through a PKCζ-dependent, PI-3-K-independent, inactivation of the pro-survival kinase, AKT.8 This mechanism also suggests that ceramide regulates binding interactions between PKCζ and upstream elements in the AKT cascade. These interactions are specific to individual PKC isotypes as ceramide induces PKCζ, but not PKCε, interactions with AKT. It is also possible that the actions of PKCζ to function as either an anti-mitogenic or pro-mitogenic kinase can be a consequence of specific lipid-derived second messengers (ceramide/PI3-lipids/arachidonate) that differentially couple PKCζ to either anti- or pro-mitogenic signalosomes.4,8,33 As we begin to manipulate endogenous levels of ceramide in pre-clinical models, confirmation of these signaling mechanisms in vivo will further elucidate their overall biological relevance. The role of ceramide to interact with and differentially regulate PKC isotypes is still somewhat controversial. Multiple studies have shown that ceramide activates atypical PKCs, including PKCζ,4-8,26 while inactivating conventional or novel PKC isotypes.22,34-36 However, studies with photoaffinity-labeled ceramide were unable to reproduce these observations.37 Until we define the ceramide-binding domain(s) of individual PKC isotypes, we are left with the intriguing hypothesis of Van Blitterswijk,38 in which it was proposed that ceramide, but not diacylglycerol (DAG), binds to the unique single cysteine rich domain (CRD) of PKCζ. In contrast, ceramide competes with DAG for either of the two CRD sites on conventional and novel PKC to inhibit activation. This hypothesis provides a plausible explanation for the Yin/ Yang relationship observed between ceramide and DAG in terms of cell cycle progression.39 This may be analogous to the competitive interactions observed between ester- and etherlinked diglycerides40 as well as between arachidonate and ceramide4 for PKC isotypes. Understanding these specific lipid/protein interactions at both a molecular and biochemical level has the potential to allow future studies to translate these two-dimensional cell culture studies to three-dimensional in vivo models, in essence “kicking it up a notch”.

The Bedside—Ceramides and Cardiovascular Disease The role of ceramides in the physiological and pathophysiological regulation of vascular smooth muscle function has been previously reviewed.41-45 Yet controversies still remain as to the exact role of ceramide in remodeling of atherosclerotic lesions or in hypertension. Translationally-based studies have been able to clarify some of these discrepancies. The role of ceramide as an anti-proliferative lipid in vivo is particularly well illustrated in studies by Johns et al, in which neutral SMase-generated ceramide formation is reduced in spontaneously hypertensive rats (SHR), contributing to increased VSMC proliferation.46 Even though ceramide, by itself, is an anti-proliferative metabolite, studies suggest that ceramide, as a component of oxidized low-density lipoproteins, contributes to the development of atherosclerotic lesions.47 Moreover, it has been suggested that SM-generated ceramide correlates with aggregated LDL particles.48 However, it is now believed that ceramide metabolites, including lactosylceramide and sphingosine-1-phosphate, are the ultimate mediators of oxidized LDL-induced VSM proliferation and differentiation.43,49 Alternatively, ceramide may be beneficial in limiting

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Figure 1. Ceramide induces cell cycle arrest or apoptosis through multiple signal transduction cascades. For color version please visit http://www.eurekah.com/chapter.php?chapid=704&bookid=13&catid=56.

atherosclerosis through ceramide-dependent eNOS expression50 and inhibition of TNF-induced adhesion protein expression,51 subsequent to HDL binding to endothelial scavenger receptors. Ceramide also inhibits gene transcription of sterol regulatory element binding proteins to mediate a physiological feedback mechanism to lower cholesterol biosynthesis.52 Taken together, these studies directly illustrate some of the major difficulties in interpreting and rationalizing results between in vitro and in vivo models. Questions regarding multiple bioactive metabolites, subcellular localization, specific targets and intercalation into bilayers (bioavailability) may confound interpretations. For example, ambiguous results could be a result of divergent metabolic pathways leading to the formation of either pro-mitogenic (sphingosine-1-phosphate, lactosylceramide, galactosylceramide) or anti-mitogenic (sphingosine, sphinganine) metabolites.53-55 Furthermore, ceramide generation in the mitochondria56 as well as the nucleus,57 but not in the plasma membrane, may be critical events in apoptosis. Yet, two recent studies have documented the potential clinical utility of ceramides as anti-proliferative, vasodilatory or protective agents in arteries.58, 59 Both of these studies take advantage of local, direct and acute delivery systems for cell-permeable ceramide analogues that are less likely to be metabolized than corresponding physiological ceramides. In the first in vivo pre-clinical model, a cell-permeable ceramide analogue was delivered directly and acutely to the site of vascular injury. It was shown that C6-ceramide-coated balloon catheters prevent neointimal hyperplasia in rabbit carotid arteries58 (Fig. 2). The clinical correlate for such a model is restenosis after balloon angioplasty and/or stenting. Secondary occlusion of stented coronary arteries affects nearly 20% of the 1.5 million patients who undergo coronary angioplasty worldwide. The mechanism by which ceramide induces cell cycle arrest in stretch-injured vascular smooth muscle cells was mediated through inactivation of both ERK and AKT signaling cascades, validating in vitro findings.8,22,58 This study documents that

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Figure 2. Ceramide-coated balloon catheters limit neointimal hyperplasia after stretch injury in rabbit carotid arteries. Top panels, H/E-stained carotid arteries, two weeks post-angioplasty. Bottom panels, Proliferating cell nuclear antigen expression in rabbit carotid arteries after stretch injury. For color version please visit http://www.eurekah.com/chapter.php?chapid=704&bookid=13&catid=56.

intra-arterial drug delivery is technically feasible for cell-permeable lipids that target growth factor signaling cascades. Moreover, ceramide analogues or mimetics as adjuncts to polymer based coatings or as the coating themselves are uniquely suited to drug delivery platforms. These studies support future initiatives to locally and acutely deliver ceramide analogues as therapeutics to diminish proliferative pathologies. Innovative strategies to deliver ceramide analogues from various platforms, including stents or grafts, in proliferative smooth muscle pathologies such as restenosis after angioplasty, hemodialysis access failure, anastamoses or transjugular intrahepatic portosystemic shunting are currently under investigation. In the second in vivo pre-clinical model, C8-ceramide was shown to significantly reduce focal cerebral ischemia in SHR rats.59 This study supports in vitro findings that have implicated TNF-generated ceramide in induction of tolerance to ischemia.60 It is speculated that ceramide-induced preconditioning protects cultured astrocytes against the proinflammatory effects of TNFα.61 In addition, this study alludes to the therapeutic applicability of intravenous or intracisternal infusion of cell-permeable ceramide analogues. In an analogous fashion, the ceramide mimetic, trimethylsphingosine, serves a protective role for myocardium and endothelium after ischemic/reperfusion injury.62 In addition, ceramide-activated KSR has been implicated as a compensatory pathway minimizing the pro-apoptotic and pro-inflammatory actions of TNF in irritable bowel disease patients.63 Taken together, these in vivo studies may help redefine ceramides as potential therapeutics with anti-proliferative and protective properties. The putative clinical utility of ceramide analogues or related lipometics may rely on the differential effects ceramide has on multiple phenotypes in different tissues. We can speculate

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that ceramide functions as the ultimate “triple threat” to prevent restenosis or limit infarct size after ischemia by inducing vascular smooth muscle cell cycle arrest, maintaining wound-healing responses and diminishing the pro-inflammatory milieu. For example, C6-ceramide-coated balloon embolectomy catheters preferentially target dysfunctional, proliferative, vascular smooth muscle, inducing cell cycle arrest without inducing significant apoptosis or without diminishing the wound healing response.58 In fact, in unpublished data, we have shown that ceramidecoated catheters augment expression of endothelial cell-derived PDGF-ββ, supporting reendothelialization and the potential to minimize pro-thrombogenic side effects of coated-stents. In addition, ceramide does not increase the expression of adhesion molecules in human vascular endothelial cells.64 Yet, in contrast to vascular smooth muscle, ceramide has been shown to activate pro-mitogenic cascades such as ras/ERK in fibroblasts,65 events consistent with wound healing responses. It is also plausible that exogenously delivered cell-permeable ceramides diminish recruitment of macrophage precursors to atherosclerotic or infracted lesions or vulnerable plaques. Supporting this contention are studies demonstrating that Fas ligand, which signals via ceramide, could diminish vessel inflammation by targeting macrophage/mononuclear cell infiltrates and not vascular endothelial cells.66,67,68 The ability of exogenous ceramide to diminish macrophage recruitment through apoptosis may be a result of inactivation of AKT signaling.69 Tissue-specific signaling cascades may be responsible for the orchestrated in vivo phenotype observed after local delivery of ceramide analogues. In addition to ceramide’s well described role to inhibit cell cycle progression in airway smooth muscle,25 rat glomerular mesangial cells,24 A7r5 rat vascular smooth muscle cells8 as well as human coronary artery smooth muscle45, ceramide has also been implicated in vascular smooth muscle contractile responses. Ceramide has been shown to induce vasodilation of phenylephrine-contracted, endothelium denuded, rat thoracic aortic rings.70, 71 In contrast, ceramide has been reported to mediate contraction of rabbit rectosigmoid smooth muscle72,73 as well as inhibition of endothelium-dependent vasodilation in bovine coronary arteries.74 These discrepancies could be due to the role of an intact endothelial lining generating nitric oxide. Again data from local ceramide-delivery studies to limit infarct damage or stenosis58,59 may help clarify the discrepancies, arguing for a vasodilatory action of ceramide in vivo.

The Bedside—Ceramides and Cancer The potential benefit of ceramide-based chemotherapy in cancer is based on the ability of exogenous short-chain ceramide analogues to induce apoptosis in transformed/cancer cell lines. To date, the exact mechanism(s) of ceramide-mediated cell signaling leading to apoptosis has not been clearly defined. Yet, many clinically important cytotoxic agents appear to be effective by synergizing with ceramide-mediated apoptotic signaling pathway in cancer cells. The cytotoxic effect of taxol is linked to the de novo synthesis of ceramide in MDA-MB 468 human breast cancer cells, and taxol-dependent cytotoxicity is abolished when ceramide formation is blocked using L-cycloserine, an inhibitor of de novo ceramide synthesis.75 Moreover, exogenous ceramide synergistically augmented taxol-induction of apoptosis.76 Doxorubicin also promotes ceramide formation and apoptosis in breast cancer cells.77 Tamoxifen has been shown to increase cellular ceramide levels by blocking conversion of ceramide to glucosylceramide, which was independent of estrogen receptor status.78,79 Furthermore, the combination of tamoxifen with agents, such as doxorubicin or cyclosporin A analogue, has been shown to exert synergistic effects on ceramide formation.80 Recent provocative pre-clinical studies by Schmelz et al suggest supplementing milk glycosphingolipids and C16-ceramide into animal diets can suppress colonic neoplasia by as much as 50%.81,82 In fact, it has been suggested that ceramides, as functional components of dairy foods and soybeans, can potentially lower colon carcinogenesis as well as lower cholesterol and LDL with elevation of HDL levels.83 Yet, the clinical utility of systemic delivery of exogenous ceramide may be limited by ceramide metabolism, again arguing for local delivery strategies. Ceramide can serve as a precursor for the synthesis of sphingosine-1-phosphate (S-1-P)

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and glucosylceramide (GlcCer), which are implicated in cancer cell growth. This metabolic conversion of ceramide into S-1-P and/or GlcCer may switch cancer cells from an apoptotic state to a cell growth state. Supporting this, the production of GlcCer from ceramide has been shown to prevent the induction of apoptosis and stimulate cancer cell growth.84,85 Additionally, studies show that S-1-P stimulates cell growth and inhibits apoptotic cell death by serving as both an extracellular regulator and an intracellular second messenger,86-89 indicating that targeting ceramidase or sphingosine kinase, which metabolize ceramide into mitogenic S-1-P, may be an effective treatment strategy against cancer. To this end, Novogen Inc. has begun clinical studies with a putative sphingosine kinase inhibitor.90 Other potential drug targets include inhibitors of ceramide hydrolysis, ceramide glucosylation, ceramide phosphorylation, and sphingosine phosphorylation. Additionally, activators of SM hydrolysis, glucosylceramide hydrolysis, de novo ceramide synthesis, S-1-P phosphohydrolysis, and Cer-1-P phosphohydrolysis could contribute to multi-drug-induced elevation of intracellular ceramide. Interestingly, multi-drug resistant (MDR) cancers may be linked to augmented ceramide metabolism. Exposure to doxorubicin increases ceramide levels in drug-sensitive MCF-7 breast cancer cells, but not in the doxorubicin-resistant MCF-7-AdrR cells.77 Additionally, neither C6-Cer nor tamoxifen (a known inhibitor of GlcCer synthase) was cytotoxic alone, but the addition of tamoxifen to the C6-Cer treatment regimen decreased MCF-7-AdrR cell viability and elicited apoptosis. Further treatment of these cells with adriamycin stimulated an increase in endogenous ceramide levels only if co-administered with tamoxifen, in which case augmented ceramide levels correlated with a further decline in cell viability. Since MCF-7-AdrR cells have a high level of GlcCer synthase activity, these cells may display resistance to exogenous cell-permeable ceramide as well as chemotherapeutic agents (i.e., doxorubicin and adriamycin) through metabolism of ceramide into GlcCer. Studies in KB-V-1 MDR human epidermoid carcinoma cells also allude to the efficacy of augmenting endogenous ceramide levels in aggressive, MDR cancers. In these transformed cells, the multi-drug resistance modulator PSC 833 (a cyclosporin derivative) induced ceramide synthesis, which was blocked by fumonisin B1.91 Similar studies in KG1a cells, which are resistant to TNFα and do not produce ceramide upon cytokine stimulation, can be sensitized by PSC 833, to restore ceramide generation by activation of neutral, but not acid, SMase activity.92 Additional evidence for inhibition of GlcCer synthesis as a potential target to augment endogenous ceramide levels and induce apoptosis comes from studies in a human neuroepithelioma cell model.93 Although having no effect on cell viability when administered alone, GlcCer accumulation was fully suppressed by PDMP (D-threo-1-phenyl-2- decanoylamino-3morpholino-1-propanol), significantly potentiating the apoptotic effect of C6-Cer. Thus, it is not surprising that targeting the GlcCer synthase with antisense cDNA or oligonucleotides reversed adriamycin-resistance in breast cancer cells.94 Taken together, these studies strongly suggest that targeted inhibition of ceramide metabolism potentiates cellular sensitivity to exogenous ceramide as well as other chemotherapeutic agents. This potentially translates to the use of lower doses of common anti-neoplastic drugs in ceramide-enhancing drug cocktails, as to induce synergistically cancer cell death with reduced overall side effects. In addition to mediating some of the effects of cancer chemotherapy, ceramide has been implicated as the modulator of radiation therapy-induced cell death.95 Acid SMase-generated ceramide was shown to mediate radiation-induced apoptosis of micro-vascular and intestinal endothelial cells as well as lymphoid and haematopoietic cells.95,96 These events are critical for diminished tumor microvessel angiogenesis and subsequent diminished metastasis. These studies also suggest caution with brachytherapy (local irradiation) to inhibit restenosis, due to prothrombogenic complications of radiation-induced endothelial cell death. Supporting the “Yin/ Yang” relationship between signaling lipids, S-1-P prevented radiation-induced ovarian damage, providing a novel therapeutic approach to preserve ovarian function in vivo.97 In a similar fashion, S-1-P has been shown to induce vasculogenesis and angiogenesis through activation of the EDG-1-receptor, in vivo.98

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The Bedside—Other Potential Applications for Ceramide-Based Therapeutics Some of the initial in vivo applications of ceramide have been documented in cosmetology, with ceramide analogues and mimetics added to skin creams and shampoos.99,100 The rationales for these products are studies demonstrating that ceramide may induce keratinocyte cell death as well as augment barrier formation. 101, 102 Another novel in vivo application may be to target inositolphosphorylceramide formation in fungal infections. 103 Fungal pathogenesis in immuno-compromised patients is often life threatening. The formation of inositolphosphorylceramide analogues in fungi is essential for viability, suggesting inhibitors of IPC formation would make ideal antifungal drug candidates.103 It is even possible to contemplate ceramide-based therapeutic initiatives to limit viral infection, as fusion of HIV1 into the plasma membrane of CD4+ cells is dependent on the glycosphingolipids of membrane microdomains104 Targeting ceramide metabolism may also prove to be beneficial for diabetic patients. There is strong evidence that increased endogenous ceramide levels are associated with insulin resistance and the diabetic state. Studies, from as early as 1990, have shown that in the insulinresistant state, intracellular concentrations of ceramide are elevated in rat skeletal muscle. 105 Ceramide analogues have also been shown to diminish insulin-induced glucose transport in cultured adipocytes.106,107 To begin identifying the mechanisms underlying ceramide-induced insulin-resistance, ceramide has been shown to inhibit insulin-induced glucose transport by its inhibitory effect on AKT phosphorylation and GLUT4 translocation in 3T3-L1 adipocytes.108 This diminished AKT activity is a result of diminished AKT translocation and an augmented AKT phosphatase, independent of PI-3-kinase regulation.31 It is likely that ceramide metabolites also contribute to diabetic complications, as an increased accumulation of GlcCer has been observed in the kidneys of diabetic rats, which correlated with increased renal hypertrophy.109 Alternatively, glycosphingolipid formation may represent a significant pathway for glucose utilization in early diabetic nephropathy. In addition to diabetes, targeting ceramide metabolism to achieve fat reduction may be feasible, as ceramide blocks adipogenesis by decreasing the phosphorylation of C/EBP, a stress-activated transcriptional factor.110 In this model, inhibition of ceramide production reversed TNF-induced insulin resistance.110 In diseases of diminished leptin production, ceramide accumulation has been shown to induce apoptosis in lipid-laden pancreatic beta cells and skeletal muscle.111 Taken together, these studies suggest that dysfunctional ceramide metabolism may play a role in the diabetic state.

Conclusions—Back to the Bench The failure of promising therapeutics in clinical trials underscores the complexity and redundancy of signaling cascades regulating cell growth or apoptosis. Thus, to be successful, translational research must be dependent upon basic scientific studies that define novel signaling targets for in vivo investigation and validation. The identification of ceramide-binding domains on putative targets should allow the formulation of even more specific or potent ceramide analogues or mimetics for study. It is truly the complementation of in vitro and in vivo studies that allow for the optimization of therapeutics that exploit these defined targets. As a case in point, targeted and local ceramide-based delivery methodologies are presently being optimized. The potential to incorporate anti-mitogenic ceramide analogues into conventional or cationic liposomal delivery systems may not only improve efficiency of transfer but also serve to augment gene transfer or oligonucleotide targeting therapies. These types of initiatives are only possible in an atmosphere of collaboration between basic and clinical scientists with combined skills in biophysics, molecular biology, pharmacology and bioengineering.

Acknowledgements MK is supported by grants DK53715 and HL66371 from the National Institutes of Health and participates in a related project sponsored, in part, by MD3, Inc.

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106. Brindley DN, Wang CN, Mei J et al. Tumor necrosis factor-alpha and ceramides in insulin resistance. Lipids 1999; 34(Suppl):S85-88. 107. Wang CN, O’Brien L, Brindley DN. Effects of cell-permeable ceramides and tumor necrosis factor-alpha on insulin signaling and glucose uptake in 3T3-L1 adipocytes. Diabetes 1998; 47:24-31. 108. Summers SA, Garza LA, Zhou H et al. Regulation of insulin-stimulated glucose transporter GLUT4 translocation and Akt kinase activity by ceramide. Mol Cell Biol 1998; 18:5457-5464. 109. Zador IZ, Deshmukh GD, Kunkel R et al. A role for glycosphingolipid accumulation in the renal hypertrophy of streptozotocin-induced diabetes mellitus. J Clin Invest 1993; 91:797-803. 110. Grigsby RJ, Dobrowsky RT. Inhibition of ceramide production reverses TNF-induced insulin resistance. Biochem Biophys Res Commun 2001; 287:1121-1124. 111. Unger RH, Zhou YT. Lipotoxicity of beta-cells in obesity and in other causes of fatty acid spillover. Diabetes 2001; 50 Suppl 1:S118-121.

Index A

F

Acid ceramidase 30-32, 47, 111 Acyl chain 11-15, 32, 33, 51, 102, 115, 116 Akt kinase 91-96 Alkaline ceramidase 29, 30, 34-38 Aminopentol 1, 2, 5-7 Apoptosis 5, 9, 10, 12, 13, 15, 24, 29-32, 41, 47, 50, 51, 53-56, 58, 63, 64, 68, 69, 73-78, 81-85, 87, 88, 91-96, 101-111, 113, 116-119, 125, 129, 130, 133-137, 141, 147, 149-151, 153-155 Atherosclerosis 10, 88, 141, 146, 147, 151 Axon 102, 106, 117

FAN 73-79, 104, 109, 127, 128 Farber’s disease 30 Fas 31, 55, 79, 93, 110, 153 Fumonisin 1-6, 33, 35, 55, 86, 106, 116, 133, 142, 154

C Cancer 47, 95, 106, 110, 111, 126, 129, 130, 133-137, 149, 153, 154 Cannabinoid(s) 77, 113, 125-130 Caveolae 10, 36, 86, 94, 110, 114, 117 CD40 22-24, 31, 73, 75-77, 128 CD95 21-24, 31, 81-83, 87, 105, 110, 111 Cell cycle arrest 50, 56, 92, 150, 151, 153 Ceramidase 2, 10, 29-38, 41, 47, 91, 111, 135, 136, 146, 154 Ceramide(s) 1-6, 9-16, 21-25, 29-37, 41-47, 49-58, 63-69, 73-86, 88, 91-96, 101-111, 113-119, 125, 127-130, 133-137, 141-147, 149-155 Ceramide-activated protein phosphatase (CAPP) 14, 21, 49-58, 110 Chemotherapy 126, 130, 133-137, 153, 154 Cholesterol 4, 12, 13, 21, 22, 46, 113-116, 143, 145, 146, 151, 153 Clustering 21, 23, 24, 117

G G-protein(s) 21, 65, 66, 77, 125, 126, 128-130 Giant liposomes 12 Glia 113 Glioma 50, 77, 103, 125, 127-130 Glucosylceramide(s) 1- 5, 86, 91, 102, 108, 110, 111, 117, 133-136, 144, 153, 154

H Hippocampus 102, 104, 108

I Inflammation 29, 32, 141, 143, 145, 147, 153 Insulin 53, 56, 57, 94, 95, 155

J jun kinase 92, 104-107, 110

K

DAP kinase 107, 109

Kinase(s) 2, 9, 10, 14, 21, 23, 30, 35, 41, 45, 47, 49, 51, 53, 56, 57, 63-69, 76, 77, 81, 83, 91-96, 102, 104-110, 114, 117, 118, 125, 128, 129, 136, 146, 149, 150, 154, 155 KSR 21, 56, 63-69, 105, 118, 149, 152

E

L

Endothelial cell(s) 46, 87, 110, 141, 143, 145-147, 153, 154 Extracellular signal-regulated kinase 77, 110, 125, 128

Lateral organization 11, 12 Lipid domains 113, 117, 118 Lipid raft 22, 94, 107, 111, 114, 115, 117, 118

D

Ceramide Signaling

162 Lipoprotein(s) 10, 13, 29, 35, 141-143, 145, 147, 150 Liver 5, 6, 33, 35, 45, 46, 103, 109, 110, 141-145 Low density lipoprotein 10, 143

M MAP kinase 10, 68, 92 Membrane proteins 10, 14-16, 128 Membrane vesiculation 13 Metastasis 154 Microdomain 11, 12, 14-16, 23, 113, 115, 116, 149, 155 Mitochondria 10, 35, 36, 42, 44, 73, 93, 95, 105, 106, 110, 115, 116 Multidrug resistance 106, 133, 134, 154 Mycobacterium 24, 29, 33, 43, 45

N N-SMase (neutral-sphingomyelinase) 21, 73-79, 82, 92, 102-104, 107 Nerve growth factor 22, 55, 75, 101, 102, 108, 109, 117, 119 Neuron(s) 101-103, 105-111, 113, 117-119, 129 Neurotrophin(s) 101-103, 105, 106, 108-110, 114, 117, 119 Neutral ceramidase 32-35, 111 Nitric oxide 29, 81-84, 88, 153 NOS 81-88, 151

O Oxidative damage 93, 95, 96

P p75 22, 74, 75, 108-110 PI-3 kinase 53, 92, 95, 96 Pores 114, 116, 119 Protein kinase 2, 14, 21, 45, 47, 49, 51, 53, 63, 64, 81, 91, 92, 105, 109, 110, 117, 118, 125, 128, 129, 136, 150 Pseudomonas 29, 32, 33, 35, 42, 43, 45

R Ras 21, 63, 65, 66-68, 91, 93, 105, 110, 118, 129, 153

S Serine palmitoyltransferase (SPT) 10, 47, 128, 129, 136, 137, 141-143 Serine/threonine protein phosphatase(s) 49, 50-53, 55, 56 Serum 67, 93, 102, 108, 141-145, 147 Signaling 2-4, 9, 10, 11, 15, 21-25, 30, 31, 49, 53-56, 58, 63-69, 73, 74, 76-78, 81, 82, 85, 88, 91-96, 101-111, 113, 114, 116-119, 125, 127-129, 133, 147, 149-155 SM (sphingomyelin) 2, 4, 5, 9-13, 21, 22, 29, 42, 45, 46, 66, 73-77, 79, 82, 91, 101, 104, 106, 113-119, 125, 127, 128, 130, 133, 135, 141-146, 150, 154 SMase (sphingomyelinases) 10, 12, 13, 21-24, 35, 42, 46, 52, 54, 67, 73-79, 82, 84, 85, 88, 91-95, 102-107, 116, 117, 127-129, 133, 137, 141, 143-147, 149, 150, 154 Sphingoid base 1-6, 21, 22, 30, 35, 37, 110 Sphingolipid(s) 1-6, 12, 21-25, 31, 35-37, 41, 45, 49, 58, 73, 81, 82, 101, 102, 106, 108-110, 113-115, 118, 142-144, 146, 149 Sphingosine-1-phosphate 41, 106, 111, 113, 150, 151, 153 SR protein(s) 54, 55, 57, 58

T Tandem mass spectrometry 3, 5, 6 TNF 63-69, 73-79, 81-86, 88, 93, 95, 102-104, 106, 108, 109, 114, 128, 134, 149, 151, 152, 155 TNF receptor 69, 73, 75, 103, 104, 108, 109, 128, 134 TNF-R1 73-79, 83, 85, 86