Neuroendocrinology, Volume 181: THE NORMAL NEUROENDOCRINE SYSTEM (Progress in Brain Research)

PROGRESS IN BRAIN RESEARCH

VOLUME 181

NEUROENDOCRINOLOGY: THE NORMAL NEUROENDOCRINE SYSTEM EDITED BY

LUCIANO MARTINI Department of Endocrinology, University of Milano, Milano, Italy

GEORGE P. CHROUSOS First Department of Pediatrics Athens University Medical School, Athens, Greece

FERNAND LABRIE Molecular Endocrinology Laval University, Quebec City, Canada

KAREL PACAK Section on Medical Neuroendocrinology NICHD-NIH, Bethesda, MD, USA

DONALD W. PFAFF Laboratory of Neurobiology and Behavior, Rockefeller University, New York, NY, USA

AMSTERDAM – BOSTON – HEIDELBERG – LONDON – NEW YORK – OXFORD PARIS – SAN DIEGO – SAN FRANCISCO – SINGAPORE – SYDNEY – TOKYO

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands Linacre House, Jordan Hill, Oxford OX2 8DP, UK 360 Park Avenue South, New York, NY 10010-1710 First edition 2010 Copyright
2010 Elsevier B.V. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-444-53617-4 ISSN: 0079-6123 For information on all Elsevier publications visit our website at elsevierdirect.com

Printed and bound in Great Britain 10 11 12 13

10 9 8 7 6 5 4 3 2 1

Working together to grow libraries in developing countries www.elsevier.com | www.bookaid.org | www.sabre.org

List of Contributors E. Aguilar, Department of Cell Biology, Physiology and Immunology, University of Córdoba, Córdoba, Spain M.-A. Arévalo, Instituto Cajal, Consejo Superior de Investigaciones Científicas, Madrid, Spain I. Azcoitia, Departamento de Biología Celular, Facultad de Biología, Universidad Complutense, Madrid, Spain W.C. Boon, Florey Neuroscience Institutes; Centre of Neuroscience, Melbourne University, Parkville, Victoria, Australia; Prince Henry’s Institute; Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia J.D.Y. Chow, Prince Henry’s Institute; Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia X. Fan, Department of Histology and Embryology, Third Military Medical University, Chongqing, China L. Fuentes-Broto, Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA E.-H. Gan, Endocrine Research Group, Institute of Human Genetics, University of Newcastle-on-Tyne, Newcastle-upon-Tyne, UK L.M. Garcia-Segura, Instituto Cajal, Consejo Superior de Investigaciones Científicas, Madrid, Spain J.-Å. Gustafsson, Department of Biosciences and Nutrition, Karolinska Institute, Huddinge, Sweden; Center for Nuclear Receptors and Cell Signalling, Department of Biology and Biochemistry, University of Houston, Houston, TX, USA I. Huhtaniemi, Department of Surgery and Cancer, Imperial College London, Hammersmith Campus, London, UK P.S. Kalra, Department of Physiology and Functional Genomics, McKnight Brain Institute, College of Medicine, University of Florida, Gainesville, FL, USA S.P. Kalra, Department of Neuroscience, McKnight Brain Institute, College of Medicine, University of Florida, Gainesville, FL, USA D.M. Keenan, Department of Statistics, University of Virginia, Charlottesville, VA, USA F. Labrie, Oncology, Molecular Endocrinology and Human Genomics Research Center (CREMOGH), Department of Molecular Medicine, Laval University and Laval University Hospital Research Center (CRCHUL), Quebec, Canada S.A. Laporte, Departments of Medicine and Cell Biology, McGill University, Montreal, Quebec, Canada D.M. Lonard, Department of Molecular and Cellular Biology, Baylor College of Medicine, TX, USA V. Luu-The, Oncology, Molecular Endocrinology and Human Genomics Research Center (CREMOGH), Department of Molecular Medicine, Laval University and Laval University Hospital Research Center (CRCHUL), Quebec, Canada G. Murakami, Laboratory of Neurobiology and Behavior, The Rockefeller University, NY, USA B.W. O’Malley, Department of Molecular and Cellular Biology, Baylor College of Medicine, TX, USA G. Pelletier, Oncology, Molecular Endocrinology and Human Genomics Research Center (CHUL), Quebec, Canada D.W. Pfaff, Laboratory of Neurobiology and Behavior, The Rockefeller University, NY, USA S.M. Pincus, Independent Mathematician, Guilford, CT, USA v

vi

R. Pineda, Department of Cell Biology, Physiology and Immunology, University of Córdoba, Córdoba, Spain L. Pinilla, Department of Cell Biology, Physiology and Immunology, University of Córdoba, Córdoba, Spain B.I. Posner, Departments of Medicine and Cell Biology, McGill University, Montreal, Quebec, Canada R. Quinton, Endocrine Research Group, Institute of Human Genetics, University of Newcastle-on-Tyne, Newcastle-upon-Tyne, UK R.J. Reiter, Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA S. Rivest, Laboratory of Endocrinology and Genomics, CHUQ Research Center and Department of Molecular Medicine, Laval University, Quebec, Canada E.R. Simpson, Prince Henry’s Institute; Department of Biochemistry, Monash University, Clayton, Victoria, Australia V. Stanišic, Department of Molecular and Cellular Biology, Baylor College of Medicine, TX, USA D.-X. Tan, Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA M. Tena-Sempere, Department of Cell Biology, Physiology and Immunology, University of Córdoba, Córdoba, Spain J.D. Veldhuis, Department of Medicine, Endocrine Research Unit, Mayo School of Graduate Medical Education, Clinical Translational Science Center, Mayo Clinic, Rochester, MN, USA M. Warner, Department of Biosciences and Nutrition, Karolinska Institute, Huddinge, Sweden; Center for Nuclear Receptors and Cell Signalling, Department of Biology and Biochemistry, University of Houston, Houston, TX, USA Z.M. Weil, Laboratory of Neurobiology and Behavior, The Rockefeller University, NY, USA H. Xu, Center for Nuclear Receptors and Cell Signalling, Department of Biology and Biochemistry, University of Houston, Houston, TX, USA

“en guise d’introduction …’’

As presented to me by Luciano Martini, Neuroendocrinology: volume 181 the normal neuroendocrine system and volume 182 pathological situations and diseases will be two virtual volumes of over 500 virtual pages written by a group of distinguished colleagues all well qualified, many of these friends of old. It is not my intention to present or discuss in any shape or form the enormous sum that these few lines will precede and accompany. I would need proficiency in several foreign languages, foreign to me certainly. I am referring to the languages of molecular biology and the immensely intellectual country they come from and serve. Reading the list of titles of the many chapters, these two volumes will be made of, is in itself the description of what happened to that field of knowledge since the word neuro-endocrinology (with the hyphen) was coined and used for the first time in 1946 as the title of the also (already) enormous book (1106 pages) Traité de Neuro-endocrinologie by Gustave Roussy and Michel Mosinger. With the well-established knowledge of neurotransmitters as small molecules (acetylcholine, catecholamines, etc.) between nerve endings (synapses) (Sherrington, von Euler, etc.) and that of hormones (coining of the word by Starling in 1904 for secretin out of extracts of duodenal tissue), the concept of – and the word – neurosecretion appeared in the 1940s with the stunning images by the Scharrers (Ernst and Berta) of protein granules (Gomori stain) in neuronal cell bodies and moving along with axoplasmic flow. In vertebrates, that was essentially and originally dealing with observations in the hypothalamus and the posterior pituitary. But similar images were also found in invertebrates, leading to a major extension of the concept. And when the interest started about the mechanisms involved in the control of regulation of the anterior pituitary functions, involving both hypothalamic centres and unusual capillary connections, the very concept of specific molecules of neuronal origin travelling to the pituitary became the question of the day (see Geoffrey W. Harris’s Neural Control of the Pituitary Gland, 1955), which was eventually answered after over 17 years of research with the characterization of all the suspected releasing factors plus an unexpected inhibitory factor, somatostatin, all first characterized in extracts of hypothalamic tissues. So far, a rather linear way of thinking, so to speak. But all along and more and more intriguing were the observations generated by the molecular biology approach I mentioned above, that followed: syntheses of analogues of the original sequences with agonist or antagonist activities, recognition of multiple receptors for each and all of these peptides, cDNA cloning of all, etc. So much so that the conclusion was reached that each and all of these ligands and their receptors were actually quite ubiquitous and functional throughout the organism and not only located in the hypothalamus and other classical structures of the nervous system.

vii

viii

One remarkable example, out of a large library of the same: presence of the full CRH system – peptides, mRNAs, receptors, binding proteins … in adipose tissues (e.g. Seres, J., Bornstein, S. R., Seres, P., Willenberg, H. S., Schulte, K. M., Scherbaum, W.A., et al. (2004). Corticotropin-releasing hormone system in human adipose tissue. Journal of Clinical Endocrinology and Metabolism, 89, 965– 970). Also, that several of these peptides and their chemistry are involved in psychological events normal and abnormal is another of these now unquestionable conclusions as discussed in several chapters here; David DeWied (1926–2004) was a precursor. Let me close here these simple opening lines for what will be major reference volumes – if the word may apply to a virtual entity. We do indeed live in a world where even the virtual is real … Roger Guillemin, MD Nobel Laureate The Salk Institute for Biological Studies, La Jolla, California, United States of America

L. Martini (Eds.) Progress in Brain Research, Vol. 181 ISSN: 0079-6123 Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 1

Cellular signalling: peptide hormones and growth factors Barry I. Posner
and Stephane A. Laporte Departments of Medicine and Cell Biology, McGill University, Montreal, Quebec, Canada

Abstract: Peptide hormones and growth factors initiate signalling by binding to and activating their cell surface receptors. The activated receptors interact with and modulate the activity of cell surface enzymes and adaptor proteins which entrain a series of reactions leading to metabolic and proliferative signals. Rapid internalization of ligand–receptor complexes into the endosomal system both prolongs and augments events initiated at the cell surface. In addition endocytosis brings activated receptors into contact with a wider range of substrates giving rise to unique signalling events critical for modulating proliferation and apoptosis. Within the endosomal system, receptor function is regulated by lowering vacuolar pH, augmenting ligand proteolysis and promoting receptor kinase dephosphorylation. Ubiquitination–deubiquitination plays a key role in regulating receptor traffic through the endosomal system resulting in either recycling to the cell surface or degradation in multivesicular–lysosomal elements. From a clinical perspective there are several studies showing that manipulating endosomal processes may constitute a new therapeutic strategy. Keywords: growth factors; surface receptors; internalization of ligand–receptor complexes; endocytosis; ubiquitination–deubiquitination

proteins leading to the entrainment of the signalling process. For a number of years it was believed that hormone–growth factor signalling was exclusive to the cell surface. However the observation of intracellular receptors (Bergeron et al., 1978) and the appreciation of rapid receptor-dependent internalization and concentration of ligands into non-lysosomal intracellular structures (Bergeron et al., 1979; Josefsberg et al., 1979) led to the hypothesis that signalling was extended beyond the cell surface throughout the ‘exoplasmic’ space delineated by the endosomal system (Posner et al., 1980). This view was strengthened by the demonstration that Insulin Receptor Kinase and epidermal growth

Historical survey The action of polypeptide hormones and growth factors is initiated following their binding to cognate cell surface receptors defined as sites with high affinity and specificity for their respective ligands (Roth, 1973). This is followed by events at the cell surface involving oligomerization of ligand–receptor complexes (Burgess, 2008) and/or their interaction with cell surface




Corresponding author. Tel.: þ1-514-398-8094; Fax: þ1-514-398-3923; E-mail: [email protected]

DOI: 10.1016/S0079-6123(08)81001-1

1

2

factor receptor (IRK and EGFR) rapidly concentrated into well-defined endosomal structures, wherein they were more highly activated than at the cell surface (Kay et al., 1986; Khan et al., 1986). The observation that activated endosomal EGFR recruited key signalling modules to a much greater extent than the cell surface EGFR further strengthened the hypothesis that signalling occurred intracellularly (Di Guglielmo et al., 1994). Subsequent studies have greatly amplified this model of cell signalling for a large range of peptide hormones and growth factors (Murphy et al., 2009). Although it soon became clear that signalling initiated at the cell surface continues and is prolonged in the endosomal system (signalling by extension) (Bevan et al., 1996), there are now also clear examples of signalling events initiated within the endosomal apparatus (signalling by sequestration) (Sorkin and von Zastrow, 2009). In this chapter we address both the key cell surface and endosomal events involved in signalling by peptide hormones and growth factors focussing on illustrative examples while attempting to give a sense of the breadth of these phenomena.

Cell surface events: the initiation of signalling The receptor tyrosine kinases The mechanisms of signalling have been well studied for the receptor tyrosine kinases (RTKs), especially the IRK and the EGFR kinase. The IRK is a heterotetrameric molecule consisting of two a–b dimers wherein each a subunit is linked by disulphide bonds to a corresponding b subunit, a transmembrane protein containing on its intracellular domain the tyrosine kinase activity of the IRK. The a subunits are externally oriented peripheral proteins, also linked by disulphide bonds, which form a binding pocket for a single insulin molecule (Yip and Ottensmeyer, 2003). Activation of the IRK occurs on insulin binding and is necessary for the initiation of insulin signalling (Ellis et al., 1986). Studies with peroxovanadium

compounds (pVs), powerful phosphotyrosine phosphatase (PTP) inhibitors, have shown IRK activation and insulin signalling in the absence of insulin, thus establishing that kinase activation is not only necessary but also sufficient for the full insulin response (Posner et al., 1994). Activation of the IRK results from autophosphorylation on specific b subunit tyrosine residues, which is effected by transphosphorylation between adjacent a–b dimers in the heterotetrameric insulin molecule (Boni-Schnetzler et al., 1986) (Fig. 1a). In contrast to the IRK the EGFR is a monomer which, on binding a single molecule of EGF, dimerizes, thus permitting transphosphorylation on specific tyrosine kinases of the cytosolic domain of the EGFR and hence activation (Lemmon et al., 1997) (Fig. 1a). In both cases the activated kinase effects phosphorylation of downstream scaffolding proteins on tyrosine residues which function as binding motifs for the recruitment of Src Homology 2 domain (SH2)-containing proteins, necessary intermediates in the entrainment of the signalling response. In the case of the IRK the two principal signalling scaffolds are IRS-1 and IRS-2 (Kahn and White, 1988), and in the case of the EGFR, Gab-1 and Gab-2 (HolgadoMadruga et al., 1996). RTKs can also function as scaffolds as illustrated particularly for the EGFR. Other related RTKs such as those activated by neurotrophins (TrkA) (Miller and Kaplan, 2001), the platelet-derived growth factor (PDGF) (Claesson-Welsh, 1994) and vascular endothelial growth factor (VEGF) (Neufeld et al., 1994) display similar modes of activation as the EGFR and can stimulate many of the same downstream signalling modules including the Ras–Raf–MEK– Erk and PI3K–Akt pathways.

Receptor serine/threonine kinases The transforming growth factor b (TGF-b) is the prototype of a superfamily, composed of >40 related proteins [including activin, nodal, bone morphogenic proteins (BMPs), myostatin and

3 (a)

Insulin

EGF

IRK

α α

β

EGFR

β P P

Ras

IRS

P P

Raf

P P

Grb2

P P

P SHC P PI3k

P P

Gab

SOS

SOS

Grb2 P

MEK

P

IRS

Gab

P

PI3k

P

Erk

PI3k

P

P

Metabolic and proliferative signals (b) TGFβ GPCR γ βα

α E P P

β γ

II I I

P SAR SM A AD 2

II I I P

II II

II

GRKs

TGFβ receptor Second messengers - cAMP - Ins 1,4,5 P3 - Diacyl Glycerol - Ca2+

Fig. 1. Cell surface signalling events. (a) The insulin receptor kinase (IRK) and the EGF receptor (EGFR). One insulin molecule binds in a pocket made by the two extracellular a subunits of the IRK leading to tyrosine autophosphorylation and activation of the IRK. This evokes tyrosine phosphorylation of insulin receptor substrates (IRS), principally IRS-1 and IRS-2. The phosphotyrosine sites of IRS recruit SH2 domain-containing adaptors leading to activation of their associated enzyme functions. Binding of ligand to each EGFR monomer promotes and stabilizes the dimer configuration thus permitting tyrosine autophosphorylation. The tyrosinephosphorylated EGFR can phosphorylate substrates (viz. Gab-1 and Gab-2) or, via their SH2 domains, recruit adaptor molecules. Both processes lead to enzyme activation and signalling. (b) GPCR and TGF-b receptors. Binding of TGF-b to subunit Type II of the TGF-b receptor, a constitutively active serine/threonine kinase, promotes binding to and phosphorylation of subunit Type I in the final bidimeric complex. The phosphorylation site docks SARA and Smad2 which begins the signalling process for TGF-b receptor. Binding of ligand to the GPCR promotes its association with GTP-bound a subunit and cell surface enzymes like adenyl cyclase and phospholipase C. Their resultant activation generates second messenger molecules which promote downstream intracellular signalling.

others]. They play a prominent role in modulating cellular proliferation, differentiation, metabolism and apoptosis, and defects in their signal transduction have been implicated in human diseases including tissue fibrosis and cancer (Massague and Gomis, 2006). The TGF-b receptor consists of two

distinct subunits (Types I and II) both of which are transmembrane Ser/Thr kinases. The binding of TGF-b generates a bidimeric complex in which the constitutively active Type II subunit phosphorylates and activates the Type I subunit (Fig. 1b). This promotes the recruitment to the Type I

4

subunit of a family of adaptor proteins in particular SARA (Smad anchor for receptor activation) which facilitates the further recruitment and phosphorylation of Smad2, a transcription factor mediating a range of TGF-b action (Shi and Massague, 2003).

Signalling by G protein-regulated receptors G protein-coupled receptors (GPCRs), a subset of seven transmembrane-spanning receptor family, represent the largest class of hormone receptors, and account for up to 50% of marketed drug targets (Ma and Zemmel, 2002). Binding of hormone to receptor results in coupling of the complex to heterotrimeric G proteins (abg) resulting in the conversion of guanosine diphosphate (a-GDP) to Guanosine-50 -triphosphate bound alpha subunit (a-GTP), and its dissociation from the ßg subunits (Fig. 1b). The dissociated a-GTP and bg subunits interact with and modulate the function of enzymes [viz. adenyl cyclase (AC) and phospholipase C (PLC)] and channels (Dorsam and Gutkind, 2007; Lefkowitz, 2007; Rozengurt, 2007) (Fig. 1). For example, the vasopressin V2 receptor (V2R) and angiotensin type 1A receptor (AT1A) can couple to the Gs and the Gq heterotrimeric complexes, and engage AC and PLC, respectively, to generate cyclic adenosine monophosphate (cAMP), and diacylglycerol (DAG) and Ca2þ (Fig. 1b) which modulate cellular enzyme activities leading to changes in cell function and growth. It has also been found that GPCRs can transactivate other plasma membrane receptors as first observed in Rat-1 fibroblasts where stimulation of endothelin-1, lipopolysaccharide (LPS) and thrombin receptors promoted EGFR autophosphorylation and mitogen-activated protein kinase (MAPK) activation (Daub et al., 1996). This mechanism has been shown to apply to many GPCRs and different RTKs (viz. PDGFR, FGFR, TrkA and IGF-R) in various cell types (Asakura et al., 2002; Shah and Catt, 2004; Wetzker and Bohmer, 2003). The mechanisms for trans-activation of RTKs have been shown to involve the shedding of endogenous autocrine/paracrine ligands for the RTKs (Pierce et al., 2001; Wetzker and

Bohmer, 2003), and the phosphorylation of RTKs by non-receptor tyrosine kinases like Src that are activated downstream perhaps by recruited arrestin proteins, which have been shown to bind to Src (Luttrell et al., 1999), and could thus constitute a signalling scaffold for RTK trans-activation (Maudsley et al., 2000). Termination of GPCR signalling is mediated in part by the intrinsic GTPase activity of the a subunit resulting in the conversion of a-GTP to a-GDP and reassembly of the heterotrimeric (abg) complex. In addition, cell surface GPCRs are phosphorylated through the activation of downstream kinases (viz. cAMP-dependent protein kinase A (PKA) and Protein kinase C (PKC)) as well as by G protein-specific receptor kinases (GRKs) (Ferguson, 2001; Krupnick and Benovic, 1998; Lefkowitz, 1998; Luttrell, 2008). The latter promotes binding of the arrestin proteins (barr1 and barr2) to receptors, and their uncoupling from G protein-mediated signalling. Phosphorylation by GRKs of GPCRs (their only target) requires receptor occupancy and is often referred to as homologous desensitization. More recent work underscores the importance of arrestins as adaptors for the trafficking and intracellular signalling of GPCRs (see below).

The signalling endosome; patterns of intracellular signalling The receptor kinases The binding of insulin and EGF to their respective RTKs induces rapid internalization of ligand–receptor complexes into endosomes from which they recycle to the cell surface or are sorted to multivesicular–lysosomal entities for degradation (Beguinot et al., 1984). Internalization was originally viewed as a mechanism for the removal of ligand–receptor complexes from the plasma membrane, and for the downregulation of signalling, a view supported by studies on an internalization-defective EGFR mutant (c0 973) which displayed enhanced signalling capacity as compared to wild-type EGFR (Wells et al., 1990). However there is now abundant evidence that the intracellular concentration of RTKs and

5

demonstrated (Di Guglielmo et al., 1994), and it was shown that full activation of the Erk signalling pathway required clathrin-mediated EGFR endocytosis (Vieira et al., 1996). These observations suggest that spatial control of signalling involves compartmentalization of signalling components into signalling endosomes (Bevan et al., 1996) (Fig. 2). Further control appears to be effected by sequestration into sub-domains within a given

other receptors for peptide hormones and growth factors is important in the temporal and spatial regulation of cell signalling (Bevan et al., 1996; Miaczynska et al., 2004b; Sorkin and Von Zastrow, 2002). Thus, it was initially shown that activated RTKs were concentrated in endosomes (Kay et al., 1986; Khan et al., 1986) (Fig. 2). In the case of the activated endosomal EGFR augmented recruitment of downstream signalling components Grb2, SOS and Shc was

AP-2 P P

P P

βA rr

P P

rr

P SA RA SM AD 2

βA

II I I

P P

P

P

P

P

Src Recycling

Early endosome Endosome

PTP Erk

Endosome

P

Raf

P

4

r

βAr

Erk

Raf P

P P

P P

Grb2

Ras

IRS Grb2 P SOS

TF

SMAD 2

P

TF

P PT

P

Mek P SMAD

P

P

Late P18 P14 Mek endosome MP1

P

SMAD 4

SMAD 2

P

IRS

SARA SMAD 2

fts

P

Ra

II I I

Transcription

P P

PI3k

Akt

SMAD 4

Metabolic and proliferative signals

Fig. 2. Signalling endosomes. Receptors are partially activated at the cell surface and undergo further activation following internalization into endosomes. Endosomal IRK can tyrosine-phosphorylate IRS but its duration of action is curtailed by an associated PTP(s) which dephosphorylates and inactivates the receptor leading to its recycling back to the plasma membrane. EGFR is internalized and concentrated in late endosomal rafts where its proximity to the MEK–Erk complex leads to prolonged Erk activation. The complex of activated TGF-b receptor with SARA–Smad is concentrated in endosomes where Smad2 phosphorylation and binding to Smad4 generates a heterodimer which enters the nucleus and modulates transcription. The binding of barrestin to phosphoserine sites in the intracellular domains of the GPCR sequesters it away from the cell surface by facilitating its interaction with clathrin and AP2. barrestin acts as a scaffold for the recruitment of endosomal signalling complexes leading to the activation of Erk, other kinases and signalling events.

6

organelle such as membrane rafts which are dynamic, nano-scale structures rich in cholesterol and sphingolipids (Hancock, 2006). It has been proposed that the localization of EGFR and other receptors in rafts modulates their signalling properties (Balbis et al., 2007; Pike, 2005; Wang et al., 2009) (Fig. 2). Indeed, data suggest that clathrin-mediated endocytosis of TGF-b complexes are important for signal propagation, while caveolin-mediated internalization might serve only for receptor downregulation (Di Guglielmo et al., 2003).

Signalling can initiate in the endosomal system The above documented signalling by extension in which receptors activated at the cell surface continue to remain activated and competent for signalling during a substantial part of their intracellular itinerary. However it was subsequently shown that signalling could be initiated in the endosomal system. The pVs were discovered and characterized as phosphotyrosine phosphatase inhibitors in the early 1990s (Posner et al., 1994). Administration of pVs in the presence of colchicine (an inhibitor of recycling) was shown to exclusively activate intracellular IRKs and effect insulin signalling (Bevan et al., 1995a). Subsequent work by Wang et al. showed that selective activation of the EGFR restricted to endosomes also resulted in the full range of downstream signalling including cell proliferation and survival (Wang et al., 2002). Thus, intracellular signalling can be initiated from within the endosomal space. Internalization to late endosomes was for some time considered to reflect the termination of intracellular signalling. Recent evidence for EGF-induced late endosomal signalling comes from the work of Teis and colleagues who showed that the adaptor protein p14 is required to localize the MEK partner 1 protein (MP1)–MAPK signalling complex to rafts in late endosomes (Teis et al., 2002). Knocking down p14 inhibited the late prolonged (endosomal) but not the early brief (plasma membrane) phase of MAPK activation

(Teis and Huber, 2003; Teis et al., 2002). The demonstration that hyperactivated EGFR concentrates in late endosomal rafts (Balbis et al., 2007) suggests a mechanism for the activation of the MAPK cascade in this compartment (Balbis and Posner, 2010) (Fig. 2).

Neurotrophin and its receptor The neurotrophins, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), play an important role in cell survival and differentiation through the activation of the PI3K–Akt and Ras–Raf–MEK–Erk signalling pathways. Studies have shown that the addition of NGF to the distal ends of axons and dendrites triggers a retrograde signal that elicits a response in the cell body (Bhattacharyya et al., 1997). NGF binding to the TrkA receptor triggers the internalization and concentration of the activated TrkA in endosomes wherein it associates with downstream signalling proteins (Grimes et al., 1996; Riccio et al., 1997; Wu et al., 2009). The retrograde transport of activated NGF–TrkA complexes is dependent upon TrkA kinase activity and requires microtubules and the microtubule motor dynein (Watson et al., 1999, 2001) (Fig. 3). Initially, TrkA signalling endosomes are early endosomes based on their content of Rab5 GTPase and early endosome antigen 1 (EEA1) proteins but subsequently undergo transformation into late endosomal structures (Watson et al., 2001). In PC12 cells, NGF induces the assembly of an endosomal signalling complex containing TrkA, MAPK and Rap-1 (Wu et al., 2001). As with the EGFR, NGF signalling at the plasma membrane (PM) causes transient Erk activation whereas endosomal signalling produces sustained MAPK activation including that of Erk5. The activation of Erk5 by the retrograde route, but not by the application of NGF to the neuronal soma, activates transcription events in the nucleus needed for neuronal survival and dendrite extension (Heerssen et al., 2004; Watson et al., 2001) (Fig. 3).

7

ERK1/2

P

NGF P

Erk5

TrkA

Endosome

Nuclear gene regulation

Rap MAPK P

Erk5 Rap MAPK

Mek5

P

BRaf Axon terminal

Neuron cell body

Fig. 3. Neurotrophin signalling by retrograde axonal transport. The binding of NGF to its receptor (TrkA) at the axon terminal gives rise to an endosomal complex able to activate MAP kinase. The vesicles containing these complexes undergo microtubule-dependent retrograde transport to the neuronal cell body where it gives rise to Erk5 activation and nuclear transport yielding the phosphorylation of transcription factors and changes in signalling. Activation of TrkA at the cell body also activates Erk5 but there is no nuclear access or altered transcription. This highlights the importance of compartmentation in determining the specificity of signalling.

Receptor Ser/Thr kinase Although the TGF-b receptor appears to undergo initial activation at the cell surface it reaches full activation only after entering the endosomal compartment in which SARA is especially concentrated by virtue of its FYVE domain (Hayes et al., 2002). It has been observed that although complexes of SARA– Smad2 occur at the cell surface the activation of Smads is largely endosomal (Penheiter et al., 2002). Disruption of SARA–Smad complexes in endosomes was shown to inhibit the nuclear translocation of Smad2 and thus TGF-b signalling (Hayes et al., 2002). Impairing endocytosis of the receptor–SARA–Smad complex was found to markedly inhibit signalling (Hu et al., 2008). Thus, a key step in TGF-b signalling is the activation of Smad and/or the recruitment of co-Smads in the endosomal compartment (Fig. 2).

The GPCRs Regulators of GPCR desensitization, such as GRKs and arrestins, are important in regulating GPCR internalization. Thus, overexpression of GRK2 increases phosphorylation and

internalization of acetylcholine receptors (m2) whereas blocking GRK2 action has the opposite effect (Tsuga et al., 1994). The role of GRK and arrestin in receptor internalization has now been established for many GPCRs (Claing et al., 2002; Ferguson, 2001; Lefkowitz, 1998; Marchese et al., 2003). The molecular basis for the endocytic adaptor function of arrestins derives from their ability to directly bind clathrin and its adaptor AP2 (Goodman et al., 1996; Laporte et al., 1999), thus enabling the clustering of receptor into clathrin-coated (CC) pits, a prerequisite for endocytosis (Fig. 2). The recruitment of Src generates GPCR–barrestin– Src complexes in CC pits (Luttrell et al., 1999), and Src-dependent tyrosine phosphorylation of proteins of the CC pits appears to play a key role in furthering GPCR internalization (Ahn et al., 1999, 2002; Fessart et al., 2005, 2007; Huang et al., 2003; Zimmerman et al., 2009). Internalized GPCRs rapidly concentrate in endosomes from which they can be recycled to the cell surface or directed to multivesicular bodies/lysosomes for degradation. Some GPCRs [Class A viz. the b2-adrenergic receptor (b2AR)] recycle rapidly while others (Class B viz. the V2R) remain associated with barrestin in endosomes and hence recycle more slowly (Oakley et al., 1999, 2000).

8

There is now clear evidence that endosomal GPCRs and their effectors constitute a platform for intracellular signalling (Sorkin and Von Zastrow, 2002). Thus, in yeast, the Ga protein, Gpa1, interacts in endosomes with both the catalytic and regulatory subunits of yeast PI3K to increase kinase activity and generate PtdIns3P (Slessareva et al., 2006). Activated endosomal GPCRs continue second messenger production as in thyroid follicles where a fluorescent biosensor was used to detect ongoing cAMP production after agonist-induced internalization of the Thyroid-stimulating hormone (TSH) receptor in close association with Gas and AC (Calebiro et al., 2009). Following stimulation with PTH1-34 a similar approach detected sustained cAMP production, which coincided with co-localization of agonist receptor complexes, Gas and AC in endosomes (Ferrandon et al., 2009). Whereas activation of the sphingosine-1-phosphate (SP1) receptor by SP1 evoked transient internalization and Gai-dependent inhibition of cAMP production, the synthetic compound FTY720P led to both sustained internalization and inhibition of AC (Mullershausen et al., 2009). It is now clear that GPCR–barrestin complexes concentrate in endosomes and form complexes with components of the MAPK signalling pathway (Fig. 2). Thus, stimulation of the AT1R, V2R, substance P receptor (NK-1) and protease-activated receptor 2 (PAR-2) resulted in the endosomal accumulation of receptor–b arrestin–MAPK–Erk2 signalling scaffolds (DeFea et al., 2000a, 2000b; Luttrell et al., 2001; Tohgo et al., 2003) (Fig. 2). Endosomal complexes of activated AT1R– barrestin2– Ask1–MKK4 and Ask1-dependent phosphorylated c-Jun NH2-terminal kinase (JNK3) have been observed (McDonald et al., 2000). Agonist activation of NK1 receptors yielded internalized receptor–barrestin–c-Src-activated Erk1/2 while stimulation of PAR2 induced the assembly of internalized receptor–barrestin1–Raf-1-activated ERK1/2 complexes (DeFea et al., 2000a, 2000b). Whereas G protein-dependent Erk activation is transient, that deriving from endosomal receptor–barrestin complexes manifests a prolonged time course and is sensitive to barrestin depletion but insensitive to the inhibition of G

protein-dependent signalling (Ahn et al., 2004; Camina et al., 2007; Charest et al., 2007; Gesty-Palmer et al., 2006; Milasta et al., 2005; Sneddon and Friedman, 2007). This differentiation was strikingly demonstrated in studies on the parathyroid hormone (PTH) receptor. Whereas PTH1-34 activated both the G proteinand barrestin-dependent Erk activation, PTHNBR activated only Gs-dependent signalling, and PTH-IA activated only the barrestin-dependent signalling (Gesty-Palmer et al., 2006). Although barrestin-dependent Erk activation appears to be essentially endosomal, there are studies demonstrating the formation of barrestin signalling complexes and ERK1/2 activation at the plasma membrane (DeFea et al., 2000a; Luttrell et al., 1999; Scott et al., 2006; Terrillon and Bouvier, 2004).

Endosomal signalling: requirements and regulation in health and disease Signalling derived from endosomes As noted above there is overwhelming evidence that signalling in endosomes both sustains and amplifies signals initiated at the cell surface (signalling by extension). There is now substantial evidence that signals are uniquely generated from within the endosomal compartment in physiologic conditions. Thus, internalized EGFR triggers Adaptor protein, Phosphotyrosine interaction, PH domain and Leucine zipper containing 1 (APPL1) release from endosomes and its translocation to the nucleus where it interacts with components of the NuRD histone deacetylation complex and regulates cell proliferation (Miaczynska et al., 2004a). More recently, APPL1 was shown to regulate b-catenin-dependent gene transcription, a key step in the Wnt signalling pathway (Rashid et al., 2009). APPL1 has been found to interact with a diverse set of receptors as well as signalling proteins suggesting that it might serve as an endosomal signalling platform for multiple pathways, including the regulation of the signalling specificity of Akt on endosomes (Schenck et al., 2008).

9

Notch is a heterodimeric transmembrane protein whose signalling function is conserved throughout the animal kingdom, and is involved in various developmental processes including stem cell maintenance (Bray, 2006). Ligand binding effects extracellular cleavage of the transmembrane subunit (Tien et al., 2009). There is evidence, particularly from experiments in Drosophila, that further cleavage by an endosomal g-secretase complex generates a cytosolic fragment which enters the nucleus and regulates gene transcription (Vaccari et al., 2008; Yan et al., 2009). Thus, Notch trafficking through the endosomal apparatus appears to be critical for signalling and alterations therein may lead to developmental abnormalities. A further example of the same phenomenon was described for signalling by the RTK c-Met, the receptor for hepatocyte growth factor receptor whose activation induces cell proliferation, migration, morphogenesis and survival (McShane and Zerial, 2008). The accumulation of activated c-Met in perinuclear endosomes is necessary for the phosphorylation and nuclear accumulation of its downstream transcription factor STAT3 (signal transducer and activator of transcription 3) (Kermorgant and Parker, 2008). Indeed, it has been argued that many proteins involved in endosomal trafficking play a role in modulating nuclear factors and hence transcription (Pyrzynska et al., 2009). Tumour necrosis factor (TNF), the prototype of a superfamily of cytokines and their related receptors, binds to two cell surface receptors of which TNFR1 mediates apoptosis. Although the TNF–TNFR1 complex at the cell surface can activate neutral sphingomyelinase (nSMase) leading to pleiotropic responses including proliferation and differentiation, it is clear that its intracellular accumulation leads to very different consequences (Schutze et al., 2008). Recent work has shown that the endosomal accumulation of TNF–TNFR1–adaptor complexes results in the intracellular activation of caspase 3 and the entrainment of apoptosis (Schneider-Brachert et al., 2004). The stimulation of endosomal acidic sphingomyelinase (aSMase) by activated TNFR1 leads to ceramide

formation, the activation of cathepsin D and then caspase 9 with a further augmentation of apoptosis (Heinrich et al., 2004). Other members of this family (viz. CD95) must also undergo endocytosis in order to promote apoptosis in target cells (Lee et al., 2006). There are other examples in which ligand– receptor compartmentalization is critical for cellular responses. Noteworthy is the family of innate immune receptors, the toll receptor family (TLR), which respond to a range of microbial ligands and other biological products (Barton and Kagan, 2009). Finally, it is worth mentioning that there is now evidence that silencing by small RNAs is intimately linked to endosomal trafficking (Lee et al., 2009). Thus, the endosomal system stands at the centre of signal induction and hence the modulation of many biologic processes.

Regulation of endosomal signalling Several endosomal processes bring about the termination of intracellular signalling. 1. Acidification and proteolysis. As ligand–receptor complexes move through the endosomal system from early to late structures, vacuolar pH is lowered by the action of vacuolar ATPases. There is consequent dissociation of ligand from receptor thus facilitating the action of endosomal proteases to degrade internalized ligands (Posner, 2003). This has been well studied in the case of insulin where an endosomal acidic protease plays an important role in the clearance of internalized insulin thus contributing to limiting the duration of receptor activation and hence signalling (Authier et al., 1994). 2. Dephosphorylation of RTKs. RTK-associated PTP activity was noted for both endosomal insulin and EGF receptors (Faure et al., 1992). In subsequent studies using the pVs, it was shown that dephosphorylation was a dynamic process that played a critical role in abrogating the intracellular action of RTKs as well as promoting their recycling to the cell surface (Bevan et al., 1995b).

10

3. Binding of inhibitory adaptors. The binding of adaptors, which inhibit intracellular receptor function, has been observed as in the desensitization of G protein activation by the binding of barrestins to GPCRs (see above). The TGF-b receptor requires the recruitment of Smad2 for its action. Smad7 can bind in place of Smad2 leading to the abrogation of signalling (Shi and Massague, 2003). Depending on which adaptor protein binds to the cytosolic domain of the TNFR1 trimer the consequences can be either nSMase activation at the cell surface and anti-apoptotic signalling or the endosomal activation of aSMase and the entrainment of apoptosis (Schutze et al., 2008). 4. Ubiquitination and deubiquitination. The covalent linking of ubiquitin to lysine residues in target proteins is a key modulator of their distribution, function and fate. Thus, agonist-dependent ubiquitination of GPCRs and RTKs in endosomes plays an important role in their trafficking from early to late elements of the endosomal apparatus (Marchese and Benovic, 2001; Shenoy, 2007). Thus, EGFR internalization is unaffected by ubiquitination but efficient trafficking to late endosomes and multivesicular bodies (MVBs) requires ubiquitination (Huang et al., 2007). On the other hand deubiquitination is essential for the post-endocytic sorting in MVBs – lysosomes and subsequent degradation of EGFRs (Mizuno et al., 2005). Deubiquitination earlier in the internalization sequence can result in augmented recycling of receptors and hence reduced degradation as seen for the b2AR (Shenoy et al., 2001).

Pathophysiologic processes and endosomes The above considerations argue that endosomal signalling plays a central role in the functioning of multiple receptor systems, and thus has significant physiological consequences. It is therefore not surprising to find disturbances in endosomal function contributing importantly to disease states as in the role of endosomal

b-secretase to generate b-amyloid in Alzheimer’s disease (He et al., 2007). In recent times there have been several studies indicating the value of targeting endosomal processes for drug development. Of interest is the recent observation that a membrane-targeted inhibitor of endosomal b-secretase was much more efficient than free compound in inhibiting b-secretase activity and therefore of potential therapeutic value in Alzheimer’s disease (Rajendran et al., 2008). There is evidence suggesting that lithium action on behavior depends on its ability to disrupt endosomal components of GPCR signalling (Beaulieu et al., 2008), and that nicotinic acid’s flushing effect derives from its effect on endosomal modulation of prostaglandin synthesis (Walters et al., 2009). A final example is the therapeutic targeting of SP1 receptors which modulate the immune process in both lymphocytes and endothelial cells. Recent work has identified an immunomodulator whose efficacy derives from its ability to promote prolonged endosomal signalling in target cells (Mullershausen et al., 2009).

Acknowledgements We are especially grateful to Mr. Victor Dumas who prepared the figures for this manuscript. We also thank the Canadian Institutes of Health Research for their ongoing support of our laboratories.

Abbreviations AC AP2 APPL1

aSMase AT1A b2AR BDNF

adenyl cyclase clathrin adaptor protein 2 Adaptor protein, Phosphotyrosine interaction, PH domain and Leucine zipper containing 1 acidic sphingomyelinase angiotensin type 1A receptor b2-adrenergic receptor brain-derived neurotrophic factor

11

BMP cAMP CC DAG EEA1 EGFR Erk FGFR Gab-1 a-GDP GPCR GRK a-GTP IGF-R IRK IRS-1 and IRS-2 JNK3

LPS m2 MAPK MEK MP1 MVB NGF NK-1 nSMase NuRD PAR-2 PDGF PDGFR PI3K PKA

bone morphogenic protein cyclic adenosine monophosphate clathrin-coated diacylglycerol early endosome antigen 1 epidermal growth factor receptor extracellular signal-regulated kinase fibroblast growth factor receptor GRB2-associated-binding protein 1 guanosine diphosphate G protein-coupled receptor G protein-specific receptor kinase Guanosine-50 -triphosphate bound alpha subunit Insulin-like growth factor receptor insulin Receptor Kinase insulin receptor substrate 1 and 2 Ask1-dependent phosphorylated c-Jun NH2terminal kinase lipopolysaccharide acetylcholine receptor m2 mitogen-activated protein kinase mitogen-activated protein kinase kinase MEK partner 1 protein multivesicular body nerve growth factor substance P receptor 1 neutral sphingomyelinase nucleosome remodelling and histone deacetylase protease-activated receptor 2 platelet-derived growth factor platelet-derived growth factor receptor phosphoinositide 3-kinase cAMP-dependent protein kinase A

PKC PLC PM PTH PTP pV RTK SARA SH2 SP1 STAT TGF-b TLR TNF TNFR1 TrkA TSH V2R VEGF

Protein kinase C phospholipase C plasma membrane parathyroid hormone phosphotyrosine phosphatase peroxovanadium compound receptor tyrosine kinase Smad anchor for receptor activation Src Homology 2 domain sphingosine-1-phosphate signal transducer and activator of transcription transforming growth factor b toll receptor family tumour necrosis factor tumor necrosis factor receptor 1 neurotrophic tyrosine kinase receptor thyroid-stimulating hormone vasopressin V2 receptor vascular endothelial growth factor

References Ahn, S., Kim, J., Lucaveche, C. L., Reedy, M. C., Luttrell, L. M., Lefkowitz, R. J., et al. (2002). Src-dependent tyrosine phosphorylation regulates dynamin self-assembly and ligandinduced endocytosis of the epidermal growth factor receptor. The Journal of Biological Chemistry, 277, 26642–26651. Ahn, S., Maudsley, S., Luttrell, L. M., Lefkowitz, R. J., & Daaka, Y. (1999). Src-mediated tyrosine phosphorylation of dynamin is required for beta2-adrenergic receptor internalization and mitogen-activated protein kinase signaling. The Journal of Biological Chemistry, 274, 1185–1188. Ahn, S., Shenoy, S. K., Wei, H., & Lefkowitz, R. J. (2004). Differential kinetic and spatial patterns of beta-arrestin and G protein-mediated ERK activation by the angiotensin II receptor. The Journal of Biological Chemistry, 279, 35518– 35525. Asakura, M., Kitakaze, M., Takashima, S., Liao, Y., Ishikura, F., Yoshinaka, T., et al. (2002). Cardiac hypertrophy is inhibited by antagonism of ADAM12 processing of HB-EGF: Metalloproteinase inhibitors as a new therapy. Nature Medicine, 8, 35–40. Authier, F., Rachubinski, R. A., Posner, B. I., & Bergeron, J. J. (1994). Endosomal proteolysis of insulin by an acidic thiol

12 metalloprotease unrelated to insulin degrading enzyme. Journal of Biological Chemistry, 269, 3010–3016. Balbis, A., Parmar, A., Wang, Y., Baquiran, G., & Posner, B. I. (2007). Compartmentalization of signaling-competent epidermal growth factor receptors in endosomes. Endocrinology, 148, 2944–2954. Balbis, A., & Posner, B. I. (2010). Compartmentalization of EGFR in cellular membranes: Role of membrane rafts. Journal of Cellular Biochemistry, In Press. Barton, G. M., & Kagan, J. C. (2009). A cell biological view of toll-like receptor function: Regulation through compartmentalization. Nature Reviews Immunology, 9, 535–542. Beaulieu, J. M., Marion, S., Rodriguiz, R. M., Medvedev, I. O., Sotnikova, T. D., Ghisi, V., et al. (2008). A beta-arrestin 2 signaling complex mediates lithium action on behavior. Cell, 132, 125–136. Beguinot, L., Lyall, R. M., Willingham, M. C., & Pastan, I. (1984). Down-regulation of the epidermal growth factor receptor in KB cells is due to receptor internalization and subsequent degradation in lysosomes. Proceedings of the National Academy of Sciences of the United States of America, 81, 2384–2388. Bergeron, J. J., Posner, B. I., Josefsberg, Z., & Sikstrom, R. (1978). Intracellular polypeptide hormone receptors. The demonstration of specific binding sites for insulin and human growth hormone in Golgi fractions isolated from the liver of female rats. Journal of Biological Chemistry, 253, 4058–4066. Bergeron, J. J., Sikstrom, R., Hand, A. R., & Posner, B. I. (1979). Binding and uptake of 125i-insulin into rat liver hepatocytes and endothelium. An in vivo radioautographic study. Journal of Cell Biology, 80, 427–443. Bevan, A. P., Burgess, J. W., Drake, P. G., Shaver, A., Bergeron, J. J., & Posner, B. I. (1995a). Selective activation of the rat hepatic endosomal insulin receptor kinase. Role for the endosome in insulin signaling. Journal of Biological Chemistry, 270, 10784–10791. Bevan, A. P., Drake, P. G., Bergeron, J. J., & Posner, B. I. (1996). Intracellular signal transduction: The role of endosomes. Trends in Endocrinology and Metabolism, 7, 13–21. Bevan, A. P., Drake, P. G., Yale, J. F., Shaver, A., & Posner, B. I. (1995b). Peroxovanadium compounds: Biological actions and mechanism of insulin-mimesis. Molecular & Cellular Biochemistry, 153, 49–58. Bhattacharyya, A., Watson, F. L., Bradlee, T. A., Pomeroy, S. L., Stiles, C. D., & Segal, R. A. (1997). Trk receptors function as rapid retrograde signal carriers in the adult nervous system. Journal of Neuroscience, 17, 7007–7016. Boni-Schnetzler, M., Rubin, J. B., & Pilch, P. F. (1986). Structural requirements for the transmembrane activation of the insulin receptor kinase. The Journal of Biological Chemistry, 261, 15281–15287. Bray, S. J. (2006). Notch signalling: A simple pathway becomes complex. Nature Reviews Molecular Cell Biology, 7, 678–689. Burgess, A. W. (2008). EGFR family: Structure physiology signalling and therapeutic targets. Growth Factors, 26, 263–274.

Calebiro, D., Nikolaev, V. O., Gagliani, M. C., De Filippis, T., Dees, C., Tacchetti, C., et al. (2009). Persistent cAMPsignals triggered by internalized G-protein-coupled receptors. PLoS Biology, 7, e1000172. Camina, J. P., Lodeiro, M., Ischenko, O., Martini, A. C., & Casanueva, F. F. (2007). Stimulation by ghrelin of p42/p44 mitogen-activated protein kinase through the GHS-R1a receptor: Role of G-proteins and beta-arrestins. Journal of Cellular Physiology, 213, 187–200. Charest, P. G., Oligny-Longpre, G., Bonin, H., Azzi, M., & Bouvier, M. (2007). The V2 vasopressin receptor stimulates ERK1/2 activity independently of heterotrimeric G protein signalling. Cellular Signalling, 19, 32–41. Claesson-Welsh, L. (1994). Platelet-derived growth factor receptor signals. The Journal of Biological Chemistry, 269, 32023–32026. Claing, A., Laporte, S. A., Caron, M. G., & Lefkowitz, R. J. (2002). Endocytosis of G protein-coupled receptors: Roles of G protein-coupled receptor kinases and beta-arrestin proteins. Progress of Neurobiology, 66, 61–79. Daub, H., Weiss, F. U., Wallasch, C., & Ullrich, A. (1996). Role of transactivation of the EGF receptor in signalling by G-protein-coupled receptors. Nature, 379, 557–560. Defea, K. A., Vaughn, Z. D., O’Bryan, E. M., Nishijima, D., Dery, O., & Bunnett, N. W. (2000a). The proliferative and antiapoptotic effects of substance P are facilitated by formation of a beta-arrestin-dependent scaffolding complex. Proceedings of the National Academy of Sciences of the United States of America, 97, 11086–11091. Defea, K. A., Zalevsky, J., Thoma, M. S., Dery, O., Mullins, R. D., & Bunnett, N. W. (2000b). Beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2. The Journal of Cell Biology, 148, 1267–1281. Di Guglielmo, G. M., Baass, P. C., Ou, W. J., Posner, B. I., & Bergeron, J. J. (1994). Compartmentalization of SHC, GRB2 and mSOS, and hyperphosphorylation of Raf-1 by EGF but not insulin in liver parenchyma. EMBO Journal, 13, 4269–4277. Di Guglielmo, G. M., Le Roy, C., Goodfellow, A. F., & Wrana, J. L. (2003). Distinct endocytic pathways regulate TGF-beta receptor signalling and turnover. Nature Cell Biology, 5, 410–421. Dorsam, R. T., & Gutkind, J. S. (2007). G-protein-coupled receptors and cancer. Nature Reviews Cancer, 7, 79–94. Ellis, L., Clauser, E., Morgan, D. O., Edery, M., Roth, R. A., & Rutter, W. J. (1986). Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose. Cell, 45, 721–732. Faure, R., Baquiran, G., Bergeron, J. J., & Posner, B. I. (1992). The dephosphorylation of insulin and epidermal growth factor receptors. Role of endosome-associated phosphotyrosine phosphatase(s). Journal of Biological Chemistry, 267, 11215– 11221. Ferguson, S. S. (2001). Evolving concepts in G proteincoupled receptor endocytosis: The role in receptor

13 desensitization and signaling. Pharmacological Reviews, 53, 1–24. Ferrandon, S., Feinstein, T. N., Castro, M., Wang, B., Bouley, R., Potts, J. T., et al. (2009). Sustained cyclic AMP production by parathyroid hormone receptor endocytosis. Nature Chemical Biology, 5, 734–742. Fessart, D., Simaan, M., & Laporte, S. A. (2005). c-Src regulates clathrin adapter protein 2 interaction with beta-arrestin and the angiotensin II type 1 receptor during clathrinmediated internalization. Molecular Endocrinology, 19, 491–503. Fessart, D., Simaan, M., Zimmerman, B., Comeau, J., Hamdan, F. F., Wiseman, P. W., et al. (2007). Src-dependent phosphorylation of beta2-adaptin dissociates the beta-arrestinAP-2 complex. Journal of Cell Science, 120, 1723–1732. Gesty-Palmer, D., Chen, M., Reiter, E., Ahn, S., Nelson, C. D., Wang, S., et al. (2006). Distinct beta-arrestin- and G proteindependent pathways for parathyroid hormone receptorstimulated ERK1/2 activation. The Journal of Biological Chemistry, 281, 10856–10864. Goodman, O. B. Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., et al. (1996). Betaarrestin acts as a clathrin adaptor in endocytosis of the beta2-adrenergic receptor. Nature, 383, 447–450. Grimes, M. L., Zhou, J., Beattie, E. C., Yuen, E. C., Hall, D. E., Valletta, J. S., et al. (1996). Endocytosis of activated TrkA: Evidence that nerve growth factor induces formation of signaling endosomes. Journal of Neuroscience, 16, 7950–7964. Hancock, J. F. (2006). Lipid rafts: Contentious only from simplistic standpoints. Nature Reviews Molecular Cell Biology, 7, 456–462. Hayes, S., Chawla, A., & Corvera, S. (2002). TGF beta receptor internalization into EEA1-enriched early endosomes: Role in signaling to smad2. The Journal of Cell Biology, 158, 1239– 1249. He, X., Cooley, K., Chung, C. H., Dashti, N., & Tang, J. (2007). Apolipoprotein receptor 2 and X11 alpha/beta mediate apolipoprotein E-induced endocytosis of amyloid-beta precursor protein and beta-secretase, leading to amyloid-beta production. Journal of Neuroscience, 27, 4052–4060. Heerssen, H. M., Pazyra, M. F., & Segal, R. A. (2004). Dynein motors transport activated trks to promote survival of targetdependent neurons. Nature Neuroscience, 7, 596–604. Heinrich, M., Neumeyer, J., Jakob, M., Hallas, C., Tchikov, V., Winoto-Morbach, S., et al. (2004). Cathepsin D links TNFinduced acid sphingomyelinase to bid-mediated caspase-9 and -3 activation. Cell Death Differential, 11, 550–563. Holgado-Madruga, M., Emlet, D. R., Moscatello, D. K., Godwin, A. K., & Wong, A. J. (1996). A grb2-associated docking protein in EGF- and insulin-receptor signalling. Nature, 379, 560–564. Hu, H., Milstein, M., Bliss, J. M., Thai, M., Malhotra, G., Huynh, L. C., et al. (2008). Integration of transforming growth factor beta and RAS signaling silences a RAB5 guanine nucleotide exchange factor and enhances growth factor-directed cell migration. Molecular and Cellular Biology, 28, 1573–1583.

Huang, F., Goh, L. K., & Sorkin, A. (2007). EGF receptor ubiquitination is not necessary for its internalization. Proceedings of the National Academy of Sciences of the United States of America, 104, 16904–16909. Huang, F., Jiang, X., & Sorkin, A. (2003). Tyrosine phosphorylation of the beta2 subunit of clathrin adaptor complex AP2 reveals the role of a di-leucine motif in the epidermal growth factor receptor trafficking. The Journal of Biological Chemistry, 278, 43411–43417. Josefsberg, Z., Posner, B. I., Patel, B., & Bergeron, J. J. (1979). The uptake of prolactin into female rat liver. Concentration of intact hormone in the Golgi apparatus. Journal of Biological Chemistry, 254, 209–214. Kahn, C. R., & White, M. F. (1988). The insulin receptor and the molecular mechanism of insulin action. The Journal of Clinical Investigation, 82, 1151–1156. Kay, D. G., Lai, W. H., Uchihashi, M., Khan, M. N., Posner, B. I., & Bergeron, J. J. (1986). Epidermal growth factor receptor kinase translocation and activation in vivo. Journal of Biological Chemistry, 261, 8473–8480. Kermorgant, S., & Parker, P. J. (2008). Receptor trafficking controls weak signal delivery: A strategy used by c-Met for STAT3 nuclear accumulation. The Journal of Cell Biology, 182, 855–863. Khan, M. N., Savoie, S., Bergeron, J. J., & Posner, B. I. (1986). Characterization of rat liver endosomal fractions. In vivo activation of insulin-stimulable receptor kinase in these structures. Journal of Biological Chemistry, 261, 8462–8472. Krupnick, J. G., & Benovic, J. L. (1998). The role of receptor kinases and arrestins in G protein-coupled receptor regulation. Annual Review of Pharmacology and Toxicology, 38, 289–319. Laporte, S. A., Oakley, R. H., Zhang, J., Holt, J. A., Ferguson, S. S., Caron, M. G., et al. (1999). The beta2-adrenergic receptor/betaarrestin complex recruits the clathrin adaptor AP-2 during endocytosis. Proceedings of the National Academy of Sciences of the United States of America, 96, 3712–3717. Lee, K. H., Feig, C., Tchikov, V., Schickel, R., Hallas, C., Schutze, S., et al. (2006). The role of receptor internalization in CD95 signaling. The EMBO Journal, 25, 1009–1023. Lee, Y. S., Pressman, S., Andress, A. P., Kim, K., White, J. L., Cassidy, J. J., et al. (2009). Silencing by small RNAs is linked to endosomal trafficking. Nature Cell Biology, 11, 1150–1156. Lefkowitz, R. J. (1998). G protein-coupled receptors. III. New roles for receptor kinases and beta-arrestins in receptor signaling and desensitization. The Journal of Biological Chemistry, 273, 18677–18680. Lefkowitz, R. J. (2007). Seven transmembrane receptors: A brief personal retrospective. Biochimica et Biophysica Acta, 1768, 748–755. Lemmon, M. A., Bu, Z., Ladbury, J. E., Zhou, M., Pinchasi, D., Lax, I., et al. (1997). Two EGF molecules contribute additively to stabilization of the EGFR dimer. The EMBO Journal, 16, 281–294. Luttrell, L. M. (2008). Reviews in molecular biology and biotechnology: Transmembrane signaling by G protein-coupled receptors. Molecular Biotechnology, 39, 239–264.

14 Luttrell, L. M., Ferguson, S. S., Daaka, Y., Miller, W. E., Maudsley, S., Della Rocca, G. J., et al. (1999). Betaarrestin-dependent formation of beta2 adrenergic receptorsrc protein kinase complexes. Science, 283, 655–661. Luttrell, L. M., Roudabush, F. L., Choy, E. W., Miller, W. E., Field, M. E., Pierce, K. L., et al. (2001). Activation and targeting of extracellular signal-regulated kinases by betaarrestin scaffolds. Proceedings of the National Academy of Sciences of the United States of America, 98, 2449–2454. Ma, P., & Zemmel, R. (2002). Value of novelty? Nature Reviews Drug Discovery, 1, 571–572. Marchese, A., & Benovic, J. L. (2001). Agonist-promoted ubiquitination of the G protein-coupled receptor CXCR4 mediates lysosomal sorting. The Journal of Biological Chemistry, 276, 45509–45512. Marchese, A., Chen, C., Kim, Y. M., & Benovic, J. L. (2003). The ins and outs of G protein-coupled receptor trafficking. Trends in Biochemical Sciences, 28, 369–376. Massague, J., & Gomis, R. R. (2006). The logic of TGFbeta signaling. FEBS Letters, 580, 2811–2820. Maudsley, S., Pierce, K. L., Zamah, A. M., Miller, W. E., Ahn, S., Daaka, Y., et al. (2000). The beta(2)-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor. The Journal of Biological Chemistry, 275, 9572–9580. Mcdonald, P. H., Chow, C. W., Miller, W. E., Laporte, S. A., Field, M. E., Lin, F. T., et al. (2000). Beta-arrestin 2: A receptor-regulated MAPK scaffold for the activation of JNK3. Science, 290, 1574–1577. Mcshane, M. P., & Zerial, M. (2008). Survival of the weakest: Signaling aided by endosomes. The Journal of Cell Biology, 182, 823–825. Miaczynska, M., Christoforidis, S., Giner, A., Shevchenko, A., Uttenweiler-Joseph, S., Habermann, B., et al. (2004a). APPL proteins link Rab5 to nuclear signal transduction via an endosomal compartment. Cell, 116, 445–456. Miaczynska, M., Pelkmans, L., & Zerial, M. (2004b). Not just a sink: Endosomes in control of signal transduction. Current Opinion Cell Biology, 16, 400–406. Milasta, S., Evans, N. A., Ormiston, L., Wilson, S., Lefkowitz, R. J., & Milligan, G. (2005). The sustainability of interactions between the orexin-1 receptor and beta-arrestin-2 is defined by a single C-terminal cluster of hydroxy amino acids and modulates the kinetics of ERK MAPK regulation. The Biochemical Journal, 387, 573–584. Miller, F. D., & Kaplan, D. R. (2001). On trk for retrograde signaling. Neuron, 32, 767–770. Mizuno, E., Iura, T., Mukai, A., Yoshimori, T., Kitamura, N., & Komada, M. (2005). Regulation of epidermal growth factor receptor down-regulation by UBPY-mediated deubiquitination at endosomes. Molecular Biology Cell, 16, 5163–5174. Mullershausen, F., Zecri, F., Cetin, C., Billich, A., Guerini, D., & Seuwen, K. (2009). Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors. Nature Chemical Biology, 5, 428–434.

Murphy, J. E., Padilla, B. E., Hasdemir, B., Cottrell, G. S., & Bunnett, N. W. (2009). Endosomes: A legitimate platform for the signaling train. Proceedings of the National Academy of Sciences of the United States of America, 106, 17615–17622. Neufeld, G., Tessler, S., Gitay-Goren, H., Cohen, T., & Levi, B. Z. (1994). Vascular endothelial growth factor and its receptors. Progress of Growth Factor Research, 5, 89–97. Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S., & Caron, M. G. (1999). Association of beta-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization. The Journal of Biological Chemistry, 274, 32248–32257. Oakley, R. H., Laporte, S. A., Holt, J. A., Caron, M. G., & Barak, L. S. (2000). Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein-coupled receptors delineate two major classes of receptors. The Journal of Biological Chemistry, 275, 17201–17210. Penheiter, S. G., Mitchell, H., Garamszegi, N., Edens, M., Dore, J. J. Jr., & Leof, E. B. (2002). Internalizationdependent and -independent requirements for transforming growth factor beta receptor signaling via the smad pathway. Molecular and Cellular Biology, 22, 4750–4759. Pierce, K. L., Luttrell, L. M., & Lefkowitz, R. J. (2001). New mechanisms in heptahelical receptor signaling to mitogen activated protein kinase cascades. Oncogene, 20, 1532–1539. Pike, L. J. (2005). Growth factor receptors, lipid rafts and caveolae: An evolving story. Biochimica et Biophysica Acta, 1746, 260–273. Posner, B. I. (2003). Regulation of insulin receptor kinase activity by endosomal processes: Possible areas for therapeutic intervention. Current Opinion in Investigational Drugs, 4, 430–434. Posner, B. I., Faure, R., Burgess, J. W., Bevan, A. P., Lachance, D., Zhang-Sun, G., et al. (1994). Peroxovanadium compounds. A new class of potent phosphotyrosine phosphatase inhibitors which are insulin mimetics. Journal of Biological Chemistry, 269, 4596–4604. Posner, B. I., Patel, B., Verma, A. K., & Bergeron, J. J. (1980). Uptake of insulin by plasmalemma and Golgi subcellular fractions of rat liver. Journal of Biological Chemistry, 255, 735–741. Pyrzynska, B., Pilecka, I., & Miaczynska, M. (2009). Endocytic proteins in the regulation of nuclear signaling, transcription and tumorigenesis. Molecular Oncology, 3, 321–338. Rajendran, L., Schneider, A., Schlechtingen, G., Weidlich, S., Ries, J., Braxmeier, T., et al. (2008). Efficient inhibition of the Alzheimer’s disease beta-secretase by membrane targeting. Science, 320, 520–523. Rashid, S., Pilecka, I., Torun, A., Olchowik, M., Bielinska, B., & Miaczynska, M. (2009). Endosomal adaptor proteins APPL1 and APPL2 are novel activators of beta-catenin/ TCF-mediated transcription. The Journal of Biological Chemistry, 284, 18115–18128.

15 Riccio, A., Pierchala, B. A., Ciarallo, C. L., & Ginty, D. D. (1997). An NGF-TrkA-mediated retrograde signal to transcription factor CREB in sympathetic neurons. Science, 277, 1097–1100. Roth, J. (1973). Peptide hormone binding to receptors: A review of direct studies in vitro. Metabolism, 22, 1059–1073. Rozengurt, E. (2007). Mitogenic signaling pathways induced by G protein-coupled receptors. Journal of Cellular Physiology, 213, 589–602. Schenck, A., Goto-Silva, L., Collinet, C., Rhinn, M., Giner, A., Habermann, B., et al. (2008). The endosomal protein APPL1 mediates akt substrate specificity and cell survival in vertebrate development. Cell, 133, 486–497. Schneider-Brachert, W., Tchikov, V., Neumeyer, J., Jakob, M., Winoto-Morbach, S., Held-Feindt, J., et al. (2004). Compartmentalization of TNF receptor 1 signaling: Internalized TNF receptosomes as death signaling vesicles. Immunity, 21, 415–428. Schutze, S., Tchikov, V., & Schneider-Brachert, W. (2008). Regulation of TNFR1 and CD95 signalling by receptor compartmentalization. Nature Reviews Molecular Cell Biology, 9, 655–662. Scott, M. G., Pierotti, V., Storez, H., Lindberg, E., Thuret, A., Muntaner, O., et al. (2006). Cooperative regulation of extracellular signal-regulated kinase activation and cell shape change by filamin A and beta-arrestins. Molecular and Cellular Biology, 26, 3432–3445. Shah, B. H. & Catt, K. J. (2004). GPCR-mediated transactivation of RTKs in the CNS: Mechanisms and consequences. Trends Neuroscience, 27, 48–53. Shenoy, S. K. (2007). Seven-transmembrane receptors and ubiquitination. Circulation Research, 100, 1142–1154. Shenoy, S. K., Mcdonald, P. H., Kohout, T. A., & Lefkowitz, R. J. (2001). Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin. Science, 294, 1307–1313. Shi, Y., & Massague, J. (2003). Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell, 113, 685–700. Slessareva, J. E., Routt, S. M., Temple, B., Bankaitis, V. A., & Dohlman, H. G. (2006). Activation of the phosphatidylinositol 3-kinase vps34 by a G protein alpha subunit at the endosome. Cell, 126, 191–203. Sneddon, W. B., & Friedman, P. A. (2007). Beta-arrestin-dependent parathyroid hormone-stimulated extracellular signalregulated kinase activation and parathyroid hormone type 1 receptor internalization. Endocrinology, 148, 4073–4079. Sorkin, A., & Von Zastrow, M. (2002). Signal transduction and endocytosis: Close encounters of many kinds. Nature Reviews Molecular Cell Biology, 3, 600–614. Sorkin, A., & Von Zastrow, M. (2009). Endocytosis and signalling: Intertwining molecular networks. Nature Reviews Molecular Cell Biology, 10, 609–622. Teis, D., & Huber, L. A. (2003). The odd couple: Signal transduction and endocytosis. Cellular and Molecular Life Sciences, 60, 2020–2033. Teis, D., Wunderlich, W., & Huber, L. A. (2002). Localization of the MP1-MAPK scaffold complex to endosomes is

mediated by p14 and required for signal transduction. Developmental Cell, 3, 803–814. Terrillon, S., & Bouvier, M. (2004). Receptor activity-independent recruitment of betaarrestin2 reveals specific signalling modes. The EMBO Journal, 23, 3950–3961. Tien, A. C., Rajan, A., & Bellen, H. J. (2009). A notch updated. The Journal of Cell Biology, 184, 621–629. Tohgo, A., Choy, E. W., Gesty-Palmer, D., Pierce, K. L., Laporte, S., Oakley, R. H., et al. (2003). The stability of the G proteincoupled receptor-beta-arrestin interaction determines the mechanism and functional consequence of ERK activation. The Journal of Biological Chemistry, 278, 6258–6267. Tsuga, H., Kameyama, K., Haga, T., Kurose, H., & Nagao, T. (1994). Sequestration of muscarinic acetylcholine receptor m2 subtypes. Facilitation by G protein-coupled receptor kinase (GRK2) and attenuation by a dominant-negative mutant of GRK2. The Journal of Biological Chemistry, 269, 32522–32527. Vaccari, T., Lu, H., Kanwar, R., Fortini, M. E., & Bilder, D. (2008). Endosomal entry regulates notch receptor activation in Drosophila melanogaster. The Journal of Cell Biology, 180, 755–762. Vieira, A. V., Lamaze, C., & Schmid, S. L. (1996). Control of EGF receptor signaling by clathrin-mediated endocytosis. Science, 274, 2086–2089. Walters, R. W., Shukla, A. K., Kovacs, J. J., Violin, J. D., Dewire, S. M., Lam, C. M., et al. (2009). Beta-arrestin1 mediates nicotinic acid-induced flushing, but not its antilipolytic effect, in mice. The Journal of Clinical Investigation, 119, 1312–1321. Wang, Y., Pennock, S., Chen, X., & Wang, Z. (2002). Endosomal signaling of epidermal growth factor receptor stimulates signal transduction pathways leading to cell survival. Molecular and Cellular Biology, 22, 7279–7290. Wang, Y., Posner, B. I., & Balbis, A. (2009). Compartmentalization of epidermal growth factor receptor in liver plasma membrane. Journal of Cellular Biochemistry, 107, 96–103. Watson, F. L., Heerssen, H. M., Bhattacharyya, A., Klesse, L., Lin, M. Z., & Segal, R. A. (2001). Neurotrophins use the Erk5 pathway to mediate a retrograde survival response. Nature Neuroscience, 4, 981–988. Watson, F. L., Heerssen, H. M., Moheban, D. B., Lin, M. Z., Sauvageot, C. M., Bhattacharyya, A., et al. (1999). Rapid nuclear responses to target-derived neurotrophins require retrograde transport of ligand-receptor complex. Journal of Neuroscience, 19, 7889–7900. Wells, A., Welsh, J. B., Lazar, C. S., Wiley, H. S., Gill, G. N., & Rosenfeld, M. G. (1990). Ligand-induced transformation by a noninternalizing epidermal growth factor receptor. Science, 247, 962–964. Wetzker, R., & Bohmer, F. D. (2003). Transactivation joins multiple tracks to the ERK/MAPK cascade. Nature Reviews Molecular Cell Biology, 4, 651–657. Wu, C., Cui, B., He, L., Chen, L., & Mobley, W. C. (2009). The coming of age of axonal neurotrophin signaling endosomes. Journal of Proteomics, 72, 46–55.

16 Wu, C., Lai, C. F., & Mobley, W. C. (2001). Nerve growth factor activates persistent Rap1 signaling in endosomes. Journal of Neuroscience, 21, 5406–5416. Yan, Y., Denef, N., & Schupbach, T. (2009). The vacuolar proton pump, V-ATPase, is required for notch signaling and endosomal trafficking in Drosophila. Developmental Cell, 17, 387–402.

Yip, C. C., & Ottensmeyer, P. (2003). Three-dimensional structural interactions of insulin and its receptor. The Journal of Biological Chemistry, 278, 27329–27332. Zimmerman, B., Simaan, M., Lee, M. H., Luttrell, L. M., & Laporte, S. A. (2009). c-Src-mediated phosphorylation of AP-2 reveals a general mechanism for receptors internalizing through the clathrin pathway. Cellular Signalling, 21, 103–110.

L. Martini (Eds.) Progress in Brain Research, Vol. 181 ISSN: 0079-6123 Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 2

Neuroendocrine control of energy homeostasis: update on new insights Satya P. Kalra1,
and Pushpa S. Kalra2 1

Department of Neuroscience, McKnight Brain Institute, College of Medicine, University of Florida, Gainesville, Florida, United States 2 Department of Physiology and Functional Genomics, McKnight Brain Institute, College of Medicine, University of Florida, Gainesville, Florida, United States

Abstract: Recent upsurge in research has uncovered distinct circuitries that regulate appetite, energy expenditure and fat accrual under the supervision of hormonal feedback signalling of adipocyte leptin and gastric ghrelin in the hypothalamic integration of energy homeostasis. A host of messenger molecules of diverse chemical composition and origin mediate the crosstalk between the three circuitries. Leptin is now recognized as the mandatory afferent signal in maintenance of weight homeostasis. Leptin insufficiency in the hypothalamus due to diminished transport of leptin across the blood–brain barrier (BBB) imposed by environmental causes, such as consumption of energy-enriched diets and diminished energy expenditure, orchestrates unregulated fat accrual and the attendant disease cluster of metabolic syndrome. Bioavailability of leptin selectively in the hypothalamic targets with the aid of gene therapy successfully averted the environmentally induced metabolic afflictions and normalized lifespan. Thus, sustenance of optimal sufficiency in leptin signalling solely in the hypothalamus is a novel strategy to combat the worldwide epidemic of obesity and metabolic syndrome. Keywords: appetite; energy expenditure; fat accrual; leptin; hypothalamus

in some patients. Indeed, experimental evidence in the 1940s and 1950s that electrolytic lesions in the ventromedial hypothalamus (VMH) elicited relentless hyperphagia, weight gain and morbid obesity, whereas lesions in the lateral hypothalamus (LH) conferred immobility, aphagia and wasting, led to enunciation of a dual-centre hypothesis – the VMH as the satiety centre and the LH as the hunger centre (Bernardis and Bellinger, 1996; Bray, 1998; Kalra et al., 1999; King, 2006). The adipostat and the setpoint formulations were invoked to explain the dynamic response of the hypothalamic centres to feedback from adipocytes in order to maintain weight within a narrow range through the lifetime (Bray, 1998; Powley et al., 1980). During the

Introduction The cause and effect relationship between nutrition, energy imbalance and human health has been appreciated since time immemorial (Bray, 1998; Kalra and Kalra, 2005). Therapeutic strategies adapted in ancient civilizations to alleviate pain and suffering from metabolic diseases are still in vogue. Clinical observations at the turn of the 20th century suggested that abnormal architecture of the basal aspect of the brain was associated with obesity


Corresponding author. Tel.: þ1-352-373-4739; Fax: þ352-294-0191; E-mail: [email protected]

DOI: 10.1016/S0079-6123(08)81002-3

17

18

Synthesis Action and

Cerebral cortex

Regulation

Hippocampus

PC

Thalamus Midbrain

AC

Preoptic area

PVN

SCN

Pons

DMN LH VMN

OC

ARC

Pituitary

Fig. 1. A diagrammatic representation of hypothalamic sites associated with appetite-regulating signal pathways on a sagittal section near midline of the rat brain. AC, anterior commissure; OC, optic chiasm; PC, posterior commissure. Synthesis – hypothalamic sites involved in synthesis of orexigenic and anorexigenic signals. Action – hypothalamic areas where orexigenic and anorexigenic messengers act. Regulation – ypothalamic sites involved in regulation of synthesis, release, and action of orexigenic and anorexigenic signals. Modified, with permission, from Kalra et al. (1999).

following two decades of controversy over the validity of the dual-centre hypothesis, brain monoamines were identified as putative neurochemical signals in mediating the stimulation and termination of appetite (Kalra et al., 1999; Stricker, 1990). The modern era of the precise understanding of the neural control of energy homeostasis by brain peptidergic, and not monoaminergic, systems was ushered in the mid-1980s with the discovery of neuropeptide Y (NPY) as a physiological appetite transducer in the hypothalamic arcuate nucleus– paraventricular nucleus (ARC–PVN) axis (Fig. 1) (Clark et al., 1984; Kalra and Kalra, 2004a, 2006a; Kalra et al., 1991, 1999). Isolation of leptin from fat tissue to serve as a putative adipostat and of ghrelin from stomach as an orexigenic signal in the 1990s firmly established the existence of neuroendocrine regulation of energy homeostasis by a closed feedback loop between peripheral afferent hormonal signals and the hypothalamic NPYergic system (Asakawa et al., 2001; Kalra and Kalra, 2003, 2004b, 2006a; Kalra et al., 1991, 1999; Zhang et al., 1994).

The current explosion, shortly thereafter, in basic and clinical research to delineate physiological, cellular, molecular and genetic basis of the hypothalamic integration of energy homeostasis was fuelled by epidemiological surveys that repeatedly affirmed the worldwide pandemic of obesity, diabetes and escalation in the attendant comorbidities forecasted the imminent shortened lifespan (Flegal et al., 2005; Friedman, 2009; Grundy, 2004; Hill et al., 2003). The information to date since the publication of a comprehensive review by us on the subject of the neuroendocrine control of energy homeostasis (Kalra et al., 1999) is briefly collated here to highlight the three newly identified hypothalamic circuitries that regulate appetite, energy expenditure and storage of unexpended energy into fat under the direction of the ever-changing hormonal feedback milieu driven by environmental shifts in nutrition and lifestyle. In addition, loci in the hormonal feedback and neural signalling that dysregulate energy homeostasis to orchestrate an abnormal rate of fat deposition and pathophysiological sequelae underlying the disease cluster of metabolic syndrome

19

tissue (BAT), white adipose tissue (WAT), pancreas, liver and skeletal muscle, the obligatory participants in hypothalamic integration of metabolic and energy homeostasis (Fig. 3).

(Grundy, 2004; Hill et al., 2003; Kalra and Kalra, 2004b, 2006b) are critically analyzed.

Neuroanatomy of hypothalamic circuitries involved in energy homeostasis Appetite regulating network Integration of energy homeostasis on a daily basis by the hypothalamus involves crosstalk among three distinct circuitries – the appetite regulating network (ARN), the energy expending network (EEN) and the fat accrual network (FAN) extending caudally from the medial preoptic area (MPOA) to the PVN, ARC, VMH and LH in the diencephalon (Figs. 1 and 2) (Kalra et al., 1999; Wynne et al., 2005). The interconnected perikarya expressing diverse messenger molecules in these circuitries innervate various extrahypothalamic sites and hindbrain relay centres to eventually terminate in the periphery in the brown adipose

In rodents and primates, the propagation and termination of appetitive drive and the restraint on appetite during the intermeal interval on a daily basis is regulated by a hardcore wiring consisting of interconnected orexigenic and anorexigenic circuits within the ARN (Kalra et al., 1999). The orexigenic circuit of the ARN is comprised of a subpopulation of perikarya in the ARC that coexpress NPY, g-aminobutyric acid (GABA) and agouti-related peptide (AgRP) and project both locally in the ARC and dorsally into the PVN (Fig. 2) (Dietrich and Horvath, 2009; Kalra and Kalra,

Afferent hormonal feedback signaling and appetite regulating network Time Line Year Signal 1984 NPY 1994

APPETITE

PVN

(–)

LEPTIN

1999 GHRELIN

POMC CART

(+) NPY GABA AgRP

(+)

ARC NPY GABA AgRP

III V (–)

LEPTIN

(+) GHRELIN

(–) Fat Stomach

(+) INSULIN Pancreas

Fig. 2. Dynamics in the feedback circuitry involved in the integration of appetite. The time line for the discovery of the three signals in regulating appetite is shown on the left. The primary components of the appetite-regulating network (shown on the right), the orexigenic neuropeptide Y (NPY) and coexpressed GABA and agouti-related peptide (AgRP), in an interplay with the anorexigenic pathway emanating from neurons that coexpress proopiomelanocortin (POMC), cocaine- and amphetamine-regulating transcript (CART) and nesfatin (not shown) stimulate and terminate appetite, respectively. The synthesis and release of these peptides in the arcuate nucleus (ARC)–paraventricular nucleus (PVN) axis is regulated by two functionally opposed afferent hormonal signals: leptin from adipocytes (fat tissue) and ghrelin from the stomach. The feedback relationship in the fat–stomach–pancreas axis in the periphery, the dual action of leptin at the level of ARC and stomach to regulate the orexigenic effects of ghrelin and stimulation of adipogenesis by insulin are also shown. þ, stimulatory; –, inhibitory; III V, third cerebroventricle. Modified, with permission, from Kalra & Kalra (2005).

20

(b)

(a)

Leptin gene therapy

Leptin insufficiency 2 Leptin mutant Leptin R mutants Lipodystrophy Insulinopenia (Type 1 diabetes)

Leptin sufficiency Hypothalamic network Hormonal signal Obesity

1 Neural signal

LEPTIN

LEPTIN

WAT WAT INSULIN

INSULIN Pancreas

Liver

Muscle

BAT

Glucose disposal Hyperinsulinemia

Pancreas

Liver

Muscle

BAT

Hyperglycemia Glucose disposal 3

Diabetes

Fig. 3. Schematic depiction of the role of leptin insufficiency and sufficiency in the hypothalamus in sustaining normoglycemia and diabetes, respectively. (a) Leptin insufficiency in the hypothalamus conferred by either  1 restriction of leptin transport engendered by hyperleptinaemia due to obesity (see also Fig. 4) or that  2 manifests due to a deficiency in leptin signalling as in leptin mutants, leptin receptor mutants, lipodystrophy or insulinopenia (type 1 diabetes), cumulatively results  3 in removal of hypothalamic restraint on pancreatic insulin secretion and attenuation of glucose disposal, the antecedent sequelae, hyperinsulinaemia and hyperglycaemia of diabetes. (b) Leptin sufficiency in the hypothalamus is maintained by an active process of leptin transport across blood–brain barrier as dictated by circulating levels of leptin produced by WAT or it can be maintained experimentally by central leptin gene therapy. The independent course traversed by leptin-responsive pathways emanating from the hypothalamic EEN and FAN and descending caudally through the brainstem, innervate pancreas, liver, skeletal muscle and BAT in the periphery. Information relay along these neural tracts maintains glucose homeostasis and energy expenditure. For details see text. WAT, white adipose tissue; EEN, energy expending network; FAN, fat accrual network; BAT, brown adipose tissue. Modified, with permission, from Kalra (2009a).

2004b, 2006a; Kalra et al., 1999; Pu et al., 1999; Tong et al., 2008). Within the ARC, they synapse on the anorexigenic melanocortin circuit composed of proopiomelanocortin (POMC) neurons that coexpress a-melanocortin stimulating hormone (a-MSH), cocaine- and amphetamine-regulated transcript (CART) and nesfatin (Kalra et al., 1999; Wu et al., 2009; Wynne et al., 2005). Each of these neuropeptides inhibits feeding (Kalra et al., 1999; Lenz and Diamond, 2008; Shimizu et al., 2009; Wynne et al., 2005) and, seemingly, synergistically through MC3 and MC4 receptors in the PVN restrains the appetitive drive during intermeal intervals. Under the direction of afferent feedback signals from the periphery the temporally based periodic expression of the appetitive drive is propagated by a two-prong action of the orexigenic circuitry in the ARC–PVN axis. Co-release of NPY and

GABA in the PVN directly stimulates feeding by synergistically activating cognate NPY Y1 and Y5 and GABAA receptors and indirectly by disinhibiting the anorexigenic melanocortin MC4 signalling to repress the tonic restraint (Kalra and Kalra, 2004b; Kalra et al., 1991, 1999; Lenz and Diamond, 2008; Pu et al., 1999). This timely interplay of the hypothalamic hardcore wiring of the ARN is essential in sustenance of the daily pattern of ingestive behavior because rodents starve in the complete absence of orexigenic NPY, AgRP and GABA and anorexigenic MC4 receptor signalling confers hyperphagia and obesity (Coll et al., 2007; Farooqi and O’Rahilly, 2005; Friedman, 2009; Luquet et al., 2005; Tong et al., 2008). The role of additional signalling molecules produced in the hypothalamus that stimulate or inhibit appetitive drive, either independently or via

21

modulation of the ARN, has also been examined extensively. The endogenous endocannabinoids anandamide and 2-arachidonoylglycerol may modulate the daily food intake pattern through their cognate receptor CB1 in the VMH (Richard et al., 2009). However, their relevance in the genesis of the daily periodic appetitive drive by the ARN is unclear. A subpopulation of neurons in the LH express the neuropeptide orexin which seemingly stimulates feeding by evoking NPY release in the PVN through synaptic links with the NPY expressing perikarya in the ARC (Horvath et al., 1999; Kalra et al., 1999; Lenz and Diamond, 2008; Wynne et al., 2005).

Energy expending network In comparison to that of the ARN, much less has been revealed on the precise working of the hypothalamic EEN at physiological, cellular and molecular levels. The basic framework of the EEN consists of neuronal links between BATs, as revealed by retrograde mapping studies (Bamshad et al., 1999; Bartness et al., 2005), and the dynamic shifts in uncoupling protein 1 (UCP1) mRNA levels in the BAT, a highly reliable marker of the rate of non-shivering thermogenic energy expenditure (Fig. 3) (Dhillon et al., 2001a; Kalra, 2001, 2008a; Kalra and Kalra, 2005; Keen-Rhinehart et al., 2005). It is recognized now that leptin and cold-induced increase in information relay along the efferents from the MPOA, ARC and VMH to BAT stimulate thermogenic energy expenditure not only in rodents but also in humans (Bagnasco et al., 2002a; Bamshad et al., 1999; Chen et al., 1998; Luiten et al., 1987; Sved et al., 2001; Virtanen et al., 2009). Interruption of these relays either surgically, age-related or due to nutritional environments reduces energy expenditure and promotes deposition of unexpended energy into body fat (Bagnasco et al., 2002a, 2002b; Bagnasco et al., 2003; Dube et al., 2002; Dube et al., 2007; Pu et al., 2003; Virtanen et al., 2009). Intriguingly, recent identification of new messenger molecules has resurrected a significant participation of VMH and LH in energy homeostasis. Steroidogenic factor-1 (SF-1) expressing

neurons and the brain-derived neurotrophic factor (BDNF) and its receptor TrkB expressing neurons in the VMH are apparently strong candidates for a role in the regulation of energy expenditure by leptin, independent of effects on phagia, if any (Dhillon et al., 2006; Gray et al., 2006; Rios et al., 2001; Xu et al., 2003). In fact, germline and conditional knockout of these signalling molecules in various paradigms have uncovered a divergent operation of the EEN and ARN (Dhillon et al., 2006; Gray et al., 2006; Rios et al., 2001; Xu et al., 2003; Yeo et al., 2004). Consequently, an insight into the anatomical relationship of SF-1 and BDNF expressing neuronal subpopulations within the ARN and EEN should shed new light on their precise interplay in the hypothalamic control of energy homeostasis. Although, like orexin, melanin concentrating hormone (MCH) produced by a subpopulation of neurons in LH also stimulates feeding, new emerging evidence suggested that this neuropeptide may primarily be involved in the control of general activity and metabolism (Balthasar et al., 2005; Bochukova et al., 2009; Farooqi and O’Rahilly, 2005; Kalra et al., 1999). Despite mild hyperphagia, MCH-R knockout mice were lean, primarily due to hyperactivity and hypermetabolism (Alon and Friedman, 2006; Kublaoui et al., 2006). Genetic mutations in the preproMCH gene also produced lean phenotype in wild-type (WT) and leptin-deficient obese ob/ob mice. The late onset of hypophagia and lean phenotype were apparently a result of increased energy expenditure and hypermetabolism (Alon and Friedman, 2006; Kublaoui et al., 2006). It is highly likely that BAT-mediated thermogenic energy expenditure markedly augmented energy expenditure in MCH-deficient mice (Alon and Friedman, 2006; Kublaoui et al., 2006), and that the leptin-induced enhanced relays from the MPOA, ARC and VMH en route to BAT mobilize linkages with the inhibitory MCH signalling and thereby serve as a mandatory efferent pathway within the EEN in regulation of energy disposal (Bagnasco et al., 2002a; Balthasar et al., 2005; Bamshad et al., 1999; Chen et al., 1998; Sved et al., 2001; Virtanen et al., 2009).

22

In this respect, the reports that the FTO (fat mass and obesity-associated) gene, expressed in hypothalamic ARC, PVN and VMH and in the hindbrain, enhanced fat accrual in humans and rodents reveal a new mode of the operation of the EEN (Fischer et al., 2009; Fredriksson et al., 2008). Hypoleptinemia evoked by food deprivation or restriction increased FTO gene expression in parallel with the well-documented rises in hypothalamic NPY and decreases in POMC gene expression. FTO deficiency (fto–/–) induced lifetime growth retardation and lean phenotype, characterized by reduced fat and a lean body mass, which was also attributable solely to enhanced energy expenditure since the locomotor activity and energy intake were unchanged (Fischer et al., 2009; Fredriksson et al., 2008). Further, reduced FTO expression protected rodents from the high-fat diet (HFD)-induced obesity, and a single nuclear polymorphism (SNP) in FTO gene was associated with childhood and adult obesity (Fischer et al., 2009; Fredriksson et al., 2008). These findings clearly implicated FTO–ARN link as a novel line of communication engaged specifically in the control of energy expenditure. Consequently, a precise morphological and physiological linkage of FTO gene expressing neurons with various components of the ARN in the ARC–PVN axis, and the neighbouring modulatory network of SF-1, MCH and orexin signalling in the VMH–LH axis, should be undertaken for a global understanding of the diverse ways the hypothalamus integrates appetite and energy expenditure.

Fat accrual network Disruptions in hypothalamic signalling either due to ablation of hypothalamic nuclei with electrolytic lesions, application of neurotoxin in discrete hypothalamic sites or surgical transection of efferent outflow promptly increase pancreatic insulin secretion contemporaneous with decreased rates of glucose disposal and hyperglycaemia, leading to increased rate of fat deposition (Bamshad et al., 1999; Bray, 1998; Dube et al., 2007; Dube et al., 1999; Kalra and Kalra, 2004b, 2007; Pu et al., 2003). Contrary to the long-held assumption that

the attending hyperphagia alone promotes increased fat accrual in these paradigms, it is now amply clear that it is the antecedent hyperinsulinaemia resulting from a loss of hypothalamic restraint on the obesogenic pancreatic insulin secretion, accompanied by reduced rates of glucose disposal by the liver, skeletal muscles and BAT, that is responsible for the increased phagia and fat deposition observed (Fig. 3) (Beretta et al., 2002; Boghossian et al., 2006a, 2006b). A wealth of experimental evidence, including retrograde mapping studies, is consistent with the new insight that neural efferents to pancreas, skeletal muscles, BAT and WAT constitute the FAN, a distinct circuitry but operating along with ARN and EEN in the hypothalamic integration of energy homeostasis (Bamshad et al., 1999; Bamshad et al., 1998; Bartness and Bamshad, 1998; Bartness et al., 2005; Buijs et al., 2001; Kreier et al., 2006; Uyama et al., 2004). As expounded in the following sections, it is safe to envision that afferent hormonal feedback signalling engages the FAN to neurally link the peripheral organs to regulate fat accrual in the body (Fig. 3).

Peripheral feedback signals in the hypothalamic integration of energy homeostasis Adipostat signals It was recognized early on that in order to maintain weight within a narrow range on a daily basis peripheral signals not only relay information on energy status in the body to the hypothalamus, but they also propagate efferent neural signals within the hypothalamus that, in turn, feed back to quantitatively and qualitatively monitor afferent hormonal signalling from the periphery. Insulin was considered as one such adipostat signal because central injections of insulin suppressed food intake, possibly through repression of NPYergic signalling in the ARC–PVN, insulin was detected in the cerebrospinal fluid and insulin receptors were visualized in the hypothalamus and various extrahypothalamic regions (Coll et al., 2007; Kalra et al., 1999; Plum et al., 2006; Schwartz et al., 1992; Wynne et al., 2005). However, the evidence

23

accumulated over the ensuing two decades showing that hyperinsulinemia coexists with obesity in rodents and humans, central administration of insulin was ineffective in suppressing food intake or body weight in various models of obesity, equivocal evidence of expression of insulin receptors by neurons of the ARN, and the demonstration that insulin receptor knockout mice not only failed to display the expected obesity, but a small weight gain was limited to female phenotype, were not consistent with the hypothesis that insulin serves as a primary physiological adipostat signal to the hypothalamus (Friedman, 2009; Lenz and Diamond, 2008; Plum et al., 2006). On the other hand, the new emerging evidence has assigned a major role for insulin in fat accrual insulin a major participant in fat accrual in the body by directly promoting adipogenesis and in the secretion of various adipokines that, either independently or in concert, play distinct roles in the regulation of energy homeostasis (Fig. 2) (Kalra, 2007, 2008a).

systemic administration of each was found to inhibit food intake to varying degrees in rodents and humans (Coll et al., 2007; Kalra, 2008a; Neary and Batterham, 2009; Wynne et al., 2005), their relevance in terms of where and how they participate to confer periodic feeding pattern in rodents and man remains unclear in view of the following observations: (1) PP, PYY1–36 and PYY3–36 stimulate and not inhibit feeding when administered centrally, (2) although blood levels of GLP-1 and GLP-2, CCK and PP correlate temporally with feeding episodes, it is unknown whether similar correlations exist with their levels in the brain, (3) a few of these peptides fail to cross BBB, (4) exact neuronal targets of these anorexigenic peptides within ARN have not been visualized and finally (5) suppression of appetite by these peptides may be an unpleasant pharmacologic response resulting from their pleiotropic effects either alone or together (Coll et al., 2007; Kalra, 2008a; Neary and Batterham, 2009; Wynne et al., 2005).

Anorexigenic signals Steroids A spectrum of signal molecules of diverse chemical composition and origins has been proposed as satiety or anorexigenic signals operating through the hypothalamic ARN (Coll et al., 2007; Kalra, 2008a; Neary and Batterham, 2009; Wynne et al., 2005). In general, these hormones reach the hypothalamic ARN by passive diffusion, by active transport by a saturable receptor-mediated process across the blood–brain barrier (BBB) or, indirectly, by neural relays via a circuitous route along the vagal fibres to the nucleus solitarius tract for ultimate transmission to the hypothalamus (Coll et al., 2007; Kalra, 2008a; Kalra et al., 1999; Neary and Batterham, 2009; Wynne et al., 2005). The anorexigenic afferent peptidergic signals so far evaluated are cholecystokinin (CCK), glucagon-like peptides 1 and 2 (GLP-1 and GLP-2) and oxymodulin from the gastrointestinal tract, and the members of the pancreatic polypeptide family, pancreatic polypeptide (PP), PYY1–36 and PYY3–36 from the pancreatic axocrine cells (Clark et al., 1984; Kalra and Kalra, 2003, 2004b, 2006a; Kalra et al., 1998b, 1999). Although

Gonadal and adrenal steroids impact hypothalamic integration of energy homeostasis in varied ways in rodents and humans (Kalra et al., 1999). Adrenocorticoids – corticosterone and cortisol – promote weight gain by stimulating food intake through upregulation of NPY efflux in the ARC–PVN axis (Kalra et al., 1999). It is, therefore, possible that chronic activation of the hypothalamic–pituitary– adrenal axis adversely impacts energy balance. Gonadal steroids also readily affect weight homeostasis as gonadectomy elicits hyperphagia and fat accrual and steroid replacement normalizes these responses by restraining NPY signalling in the ARC–PVN axis (Bagnasco et al., 2002b; Bonavera et al., 1994; Torto et al., 2006).

Ghrelin Ghrelin secreted by oxyntic cells of the stomach is the only peripheral hormonal signal known to stimulate appetite and adiposity by selectively

24

activating the ARN (Asakawa et al., 2001; Kalra and Kalra, 2003; Kalra et al., 2003; Kalra et al., 2005). Daily administration of ghrelin promotes overeating and obesity. Ghrelin is secreted in a tonic pulsatile manner during the intermeal interval and high-amplitude pulse discharge before the meal time propagates appetitive drive by selectively stimulating the release of NPY in the ARC–PVN axis (Fig. 2) (Kalra et al., 2005). Expression of hunger in response to food deprivation or restriction is associated with ghrelin hypersecretion. Seemingly, ghrelin is an important physiological signal in the genesis of the daily periodic ingestive behavior (Kalra and Kalra, 2003; Kalra et al., 2003, 2005). However, a role of ghrelin in promoting obesity is less certain because ghrelin secretion is markedly reduced concomitant with hyperleptinaemia in obese rodents and humans and leptin administration suppresses ghrelin secretion (Fig. 2) (Dube et al., 2002; Kalra and Kalra, 2003; Kalra et al., 2003, 2005; Otukonyong et al., 2005a, 2005b; Ueno et al., 2004).

Leptin There is a general consensus that leptin secreted primarily by adipocytes and stomach is an obligatory afferent hormonal signal that confers the daily periodic appetitive drive by restraining food intake through a two-prong receptor-mediated action on the core ARN in the ARC–PVN axis and the modulating circuitry in the MPOA, VMH and LH complex (Fig. 2) (Friedman and Halaas, 1998; Kalra and Kalra, 1996, 2003; Kalra et al., 1999, 2003). High-amplitude episodic leptin discharge in the intermeal interval tonically restrains the appetitive drive by activating the anorexigenic melanocortin signalling and, simultaneously, repressing the orexigenic NPY signalling in the PVN–ARC axis. As leptin hypersecretion diminishes gradually prior to the mealtime, the restraint on the ARN is curtailed leading to activation of NPY, GABA and AgRP release in the ARC–PVN axis, a response assisted by the rising preprandial titres of ghrelin, to cumulatively propagate expression of appetite

(Boghossian et al., 2006a; Kalra and Kalra, 1996, 2003; Kalra et al., 1999, 2003; Kalra, 2008a; Xu et al., 1999; Yokosuka et al., 1998). Post-prandial leptin hypersecretion is believed to reassert the restraint on feeding by countering the orexigenic action of ghrelin directly at the level of NPYergic signalling and by suppressing ghrelin secretion from oxyntic cells in the stomach and upregulating anorexigenic melanocortin signalling directly (Fig. 2) (Boghossian et al., 2006a; Kalra and Kalra, 1996, 2003; Kalra et al., 1999, 2003, 2005; Yokosuka et al., 1998). Independent of its causal role in the regulation of periodic appetite drive through the ARN, leptin exerts a regulatory control on energy expenditure and storage of unexpended energy. Within the EEN, leptin is documented to stimulate the BAT-mediated thermogenic energy expenditure by activating the leptin receptor expressing neurons, SF-1 and BDNF in the VMH and orexin and MCH in the LH (Alon and Friedman, 2006; Coll et al., 2007; Dhillon et al., 2006; Kalra, 2008a; Kalra and Kalra, 2003; Kublaoui et al., 2006; Luquet et al., 2005; Rios et al., 2001). The regulatory role of leptin on the FAN by a tonic restraint on the secretion of obesogenic pancreatic insulin, augmentation of glucose metabolism and disposal in the periphery through the NPY and melanocortin pathways from the ARC has recently been uncovered (Fig. 3) (Bagnasco et al., 2002a, 2002b; Beretta et al., 2002; Dhillon et al., 2000, 2001a; Dhillon et al., 2001b; Kalra and Kalra, 2006a, 2007; Ren et al., 2007; Rios et al., 2001; Shimizu et al., 2009). Whether these three hypothalamic regulatory circuits crosstalk or operate independently under the influence of the environmentally induced dynamic fluctuations in leptin signalling remains to be clarified.

Mechanisms underlying dysregulation of energy homeostasis and obesity in rodents and humans Hyperphagia The comprehensive new understanding of the hypothalamic control of energy homeostasis in the preceding sections has, quite unexpectedly,

25

uncovered diverse loci capable of dysregulating energy homeostasis by enforcing hyperphagia, diminution in energy expenditure, or unleashing neural and endocrine factors that impel incessant fat accrual and morbid obesity (Friedman, 2009; Grundy, 2004; Hill et al., 2003; Kalra, 2001; Kalra et al., 1999). These loci are vulnerable to shifts in internal and external environments. In rodents, experimentally or genetically induced hyperphagia and obesity manifest readily in response to either upregulation of orexigenic or dampening of anorexigenic circuits of the ARN, orchestrated by imbalances in synthesis, release or action of the neurotransmitters and decrease of their target receptors (Dube et al., 1999, 2007; Kalra, 2001; Kalra et al., 1998a; Kalra et al., 1999). Paradoxically, suboptimal release of orexigenic signals, such as NPY within the ARN, provokes hyperphagia instead of decreased daily intake, presumably due to post-receptor supersensitivity (Dube et al., 2007; Kalra et al., 1997, 1998a). On the other hand, nutritional environments that enforce consumption of calorie-enriched diets reduce gram/ day intake (Boghossian et al., 2006a; Dube et al., 2002; Kalra, 2008a; Lindqvist et al., 2005). The attendant obesity in these rodents has been attributed to the increased kilocalories/gram in the diets (Kalra, 2008a; Lindqvist et al., 2005; Otukonyong et al., 2005a, 2005b). In humans, relentless hyperphagia is responsible for morbid obesity in genetic disorders of Prader–Willi Syndrome, Alstrom Syndrome, WAGR Syndrome, Fragile X Syndrome and Bardet–Biedl Syndrome (Goldstone, 2006). Whether defective signalling in the hypothalamic ARN is the underlying cause of compulsive eating in these syndromes is unknown. With the exception of eating disorders of unknown aetiology, hyperphagia with or without changes in energy expenditure in the genetically based disruptions in the three hypothalamic circuitries accounts for only 1–6% of adult and childhood obesity (Bochukova et al., 2009; Farooqi and O’Rahilly, 2005; Grundy, 2004; Hill et al., 2003). Epidemiological and clinical evidence uniformly endorse the view that environmental factors, such as consumption of energy-enriched diets and sedentary lifestyle, promote overeating and disrupt FAN to jointly

confer the increase in the incidence of obesity worldwide (Grundy, 2004; Hill et al., 2003; Kalra, 2008a; Kalra and Kalra, 2005).

Disruptions in afferent leptin signalling Dysregulation in central leptin feedback due to drastic environmental modifications has emerged as a leading cause of obesity, diabetes and various life-threatening comorbidities (Kalra, 2008a). In the 1990s development of leptin resistance by hypothalamic targets was proposed to explain the environmentally induced obesity after it was noted that fat accrual remained unabated despite marked increases in circulating leptin levels (Coll et al., 2007; Friedman, 2009; Kalra, 2001; Kalra et al., 1999; Wynne et al., 2005). Evidence accumulated over a decade from clinical and animal experiments that closely evaluated the cause–effect temporal relationship between hyperleptinaemia and central leptin resistance at cellular and molecular levels not only failed to endorse the central leptin resistance formulation, but it is now consistent with an alternate proposal that environmentally based metabolic derangements provoke defective patterns in leptin secretion that confer quantitatively insufficient leptin signalling at targets within the ARN, EEN and FAN circuitries resulting in increased appetitive drive, diminished rate of energy expenditure and enhanced fat accrual (Figs. 3 and 4) (Chen and Heiman, 2000; Kalra, 2001, 2008a; Kastin and Pan, 2006; Montague et al., 1997; Sahu, 2002; Ziylan et al., 2009). This central leptin insufficiency hypothesis is supported by observations that leptin transport across the BBB to the hypothalamus is impaired in obese humans and rodents and the existing suboptimal leptin levels available at hypothalamic targets were, indeed, associated with a slow and steady increase in adiposity with age and that induced by HFD consumption (Fig. 4) (Kalra, 2008a; Kastin and Pan, 2006). Further, it was also evident that a rise in blood and diminution in brain leptin levels preceded the weight gain, and this inverse relationship prevailed with consumption of energy-enriched diets. Furthermore, in marked contrast to a clinical report that only a very small reduction in weight was seen

26

Leptin insufficiency syndrome in causation of metabolic diseases Decreased leptin transport across BBB

Leptin insufficiency

Derangements in hypothalamic regulation of energy homeostasis

Decreased restraint on pancreatic insulin secretion

Hyperinsulinaemia

Decreased glucose metabolism in peripheral organs

Decreased energy expenditure

Increased blood glucose and energy fuels

Hyperleptinaemia

Adipogenesis and obesity Consumption of energyenriched diets

Metabolic syndrome (e.g. type 2 diabetes)

Fig. 4. A flow chart of sequential metabolic and neural events initiated by consumption of energy-enriched diets leading to leptin insufficiency in the brain and derangements in the hypothalamic regulation of insulin secretion, glucose metabolism and energy expenditure that together promote fat accrual and metabolic disorders including type 2 diabetes. For details see text. Reproduced, with permission, from Kalra (2008a).

after boosting blood levels in severely hyperleptinemic obese human subjects (Heymsfield et al., 1999), a minute increase in leptin levels centrally to overcome leptin insufficiency or leptin receptor signalling suppressed food intake and weight in obese rodents (Bagnasco et al., 2002a, 2003; Boghossian et al., 2005; Boghossian et al., 2006a; Dube et al., 2002; Kalra, 2008a, 2009b; KeenRhinehart et al., 2005; Van Heek et al., 1997).

Sustenance of leptin sufficiency in the hypothalamus by leptin gene transfer confers global health benefits Recognition of this evidence-based inference that central leptin insufficiency may underlie the pandemic of environmentally induced metabolic inflictions sparked exploration of new

strategies to address the question of whether prevention of central leptin insufficiency can curtail this modern scourge? Although the supply of leptin to the hypothalamus by either single or multiple injections or continuous intracerebroventricular infusion can suppress food intake and/or body weight gain through the duration of the treatment, these delivery procedures were not only cumbersome, but also fraught with the confounding pleiotropic effects of leptin leaking into the general circulation (Kalra, 2008a). Increasing leptin concentrations peripherally by the systemic administration of adenovirus encoding the leptin gene (Ad-lep) markedly suppressed food intake and depleted fat in the body for extended periods. However, these fat-depleting benefits were found to be mediated centrally because the effectiveness of hyperleptinaemia after a single systemic Ad-lep

27

injection was obliterated in VMH-ablated rats (Wang et al., 1999). On the other hand, leptin supply selectively in the hypothalamus with the aid of ectopic leptin gene expression by the safe, non-pathogenic and non-immunogenic recombinant adeno-associated virus vector encoding leptin (rAAV-lep) was remarkably successful in imposing energy homeostasis in rodents (Fig. 3) (Carter and Burstein, 2004; Dhillon et al., 2000, 2001a, 2001b; Kalra and Kalra, 2001; Taymans et al., 2007; Wilson, 2009; Wright, 2009). Indeed, normal operation of ARN prevailed after a single rAAV-lep injection intracerebroventricularly or into hypothalamic sites – MPOA, ARC, PVN and VMH, as shown by suppression of fat accumulation, circulating levels of adipokines, including leptin, tumour necrosis factor a, adiponectin, triglycerides and free fatty acids, with or without accompanying decreases in food intake in various rodent models (Figs. 3 and 4) (Bagnasco et al., 2002a, 2003; Beretta et al., 2002; Boghossian et al., 2005, 2006a, 2006b; Dhillon et al., 2001a, 2001b; Dube et al., 2002; Kalra and Kalra, 2005; Lecklin et al., 2005). These longlasting global benefits of a single central injection of rAAV-lep were attributable both to suppression of orexigenic and upregulation of

anorexigenic signalling in the hypothalamic ARN along with upregulation of EEN, as shown by enhanced thermogenic energy expenditure. Maintenance of leptin sufficiency in the hypothalamus by gene therapy also curbed the age-related and HFD-induced derangements in the operation of FAN by preventing hyperinsulinaemia, insulin resistance and hyperglycaemia through a neurally mediated restraint on pancreatic insulin secretion, and simultaneous increase in glucose disposal and metabolism in BAT, liver and skeletal muscles (Figs. 3 and 5) (Bagnasco et al., 2002a, 2003; Beretta et al., 2002; Boghossian et al., 2005, 2006a, 2006b; Dhillon et al., 2000, 2001a, 2001b; Kalra, 2009a, 2009b; Kojima et al., 2009; Lecklin et al., 2005). Observations that leptin repletion in rodents and patients suffering from severe leptinopenia of lipodystrophy abrogated hyperinsulinaemia and diabetes, presumably by a central action, support the central leptin insufficiency hypothesis in the genesis of diabetes (Ebihara et al., 2007; Park et al., 2008; Shimomura et al., 1999). Various additional health benefits of central leptin replenishment including normalized neuroendocrine control of reproduction, brain growth, skeletal architecture, suppression of inflammatory

Energy homeostasis modifiers Genetic, Lifestyle, Nutritional

Hypothalamic leptin insufficiency

Central leptin gene therapy

Hypothalamic leptin sufficiency

Consequences

Consequences Obesity Energy expenditure Energy intake Metabolic syndrome: Dyslipidemia Diabetes Cardiovascular risks Skeletal abnormalities Life span symbols:

, stimulation;

, suppression

Fig. 5. Global health benefits of central leptin gene therapy to overcome hypothalamic leptin insufficiency due to energy homeostasis modifiers. Modified, with permission, from Kalra (2009b).

28

cytokines, cardiomyopathy and stimulation of ghrelin and decrease in insulin-like growth factor 1 in circulation were observed (Fig. 5) (Beretta et al., 2002; Boghossian et al., 2007; Dube et al., 2008; Iwaniec et al., 2007; Kalra, 2007, 2009a, 2009b; Kalra et al. 2009; Lecklin et al., 2005; Malhotra et al., 2009). Finally, amelioration of life-threatening metabolic comorbidities along with neuroendocrine benefits, in toto, prevented early mortality and restored normal lifespan in leptin-deficient mice (Boghossian et al., 2007).

Abbreviations Ad-lep AgRP ARC ARN a-MSH BAT BBB BDNF

Conclusion

CART

In summary, multidisciplinary research has unravelled the framework that encompasses the operation of ARN, EEN and FAN and the crosstalk among them via a multitude of neurotransmitters under the supervision of afferent adipocyte leptin and gastric ghrelin in the hypothalamic integration of energy balance. Hyperleptinaemia due to environmental causes, consumption of energy-enriched diet and lifestyle of reduced energy expenditure renders the BBB impervious to leptin entry into the brain. The resultant leptin insufficiency in the hypothalamus orchestrates energy imbalance in favour of unregulated fat accrual (Fig. 4). Leptin gene therapy to overcome leptin insufficiency, solely in the hypothalamus in varied rodent paradigms, was found to be safe and durable in reinstating energy homeostasis for extended duration of experiments (Fig. 5). Collectively, this new insight into the neuroendocrinology of energy homeostasis advocates clinical evaluation of the potential of interventional neurotherapy aimed at stabilizing leptin signalling solely in the hypothalamus to combat the modern epidemic of obesity and attendant disease cluster of metabolic syndrome.

CCK EEN FAN GABA GLP-1 GLP-2 HFD LH MCH

Acknowledgement Research embodied in this review is supported by a grant from the National Institutes of Health (DK37273).

MPOA NPY POMC PP PVN rAAV-lep

SF1 SNP UCP1 VMH WAT WT

adenovirus encoding leptin gene agouti-related peptide arcuate nucleus appetite regulating network a-melanocortin stimulating hormone brown adipose tissue blood brain barrier brain-derived neurotrophic factor cocaine- and amphetamineregulated transcript cholecystokinin energy expending network fat accrual network g-aminobutyric acid glucagon-like peptide 1 glucagon-like peptide 2 high fat diet lateral hypothalamus melanin concentrating hormone medial preoptic area neuropeptide Y proopiomelanocortin pancreatic polypeptide paraventricular nucleus recombinant adeno-associated virus vector encoding leptin gene steroidogenic factor 1 single nucleotide polymorphism uncoupling protein 1 ventromedial hypothalamus white adipose tissue wild type

References Alon, T. & Friedman, J. M. (2006). Late-onset leanness in mice with targeted ablation of melanin concentrating hormone neurons. Journal of Neuroscience, 26, 389–397.

29 Asakawa, A., Inui, A., Kaga, T., Yuzuriha, H., Nagata, T., Ueno, N., et al. (2001). Ghrelin is an appetite-stimulatory signal from stomach with structural resemblance to motilin. Gastroenterology, 120, 337–345. Bagnasco, M., Dube, M. G., Kalra, P. S., & Kalra, S. P. (2002a). Evidence for the existence of distinct central appetite and energy expenditure pathways and stimulation of ghrelin as revealed by hypothalamic site-specific leptin gene therapy. Endocrinology, 143, 4409–4421. Bagnasco, M., Dube, M. G., Katz, A., Kalra, P. S., & Kalra, S. P. (2003). Leptin expression in hypothalamic PVN reverses dietary obesity and hyperinsulinemia but stimulates ghrelin. Obesity Research, 11, 1463–1470. Bagnasco, M., Kalra, P. S., & Kalra, S. P. (2002b). Plasma leptin levels are pulsatile in adult rats: Effects of gonadectomy. Neuroendocrinology, 75, 257–263. Balthasar, N., Dalgaard, L. T., Lee, C. E., Yu, J., Funahashi, H., Williams, T., et al. (2005). Divergence of melanocortin pathways in the control of food intake and energy expenditure. Cell, 123, 493–505. Bamshad, M., Aoki, V. T., Adkison, M. G., Warren, W. S., & Bartness, T. J. (1998). Central nervous system origins of the sympathetic nervous system outflow to white adipose tissue. American Journal of Physiology, 275, R291–R299. Bamshad, M., Song, C. K., & Bartness, T. J. (1999). CNS origins of the sympathetic nervous system outflow to brown adipose tissue. American Journal of Physiology, 276, R1569–R1578. Bartness, T. J. & Bamshad, M. (1998). Innervation of mammalian white adipose tissue: Implications for the regulation of total body fat. American Journal of Physiology, 275, R1399–R1411. Bartness, T. J., Kay Song, C., Shi, H., Bowers, R. R., & Foster, M. T. (2005). Brain-adipose tissue cross talk. Proceedings of the Nutrition Society, 64, 53–64. Beretta, E., Dube, M. G., Kalra, P. S., & Kalra, S. P. (2002). Long-term suppression of weight gain, adiposity, and serum insulin by central leptin gene therapy in prepubertal rats: Effects on serum ghrelin and appetite-regulating genes. Pediatric Research, 52, 189–198. Bernardis, L. L. & Bellinger, L. L. (1996). The lateral hypothalamic area revisited: Ingestive behavior. Neuroscience and Biobehavioral Reviews, 20, 189–287. Bochukova, E. G., Huang, N., Keogh, J., Henning, E., Purmann, C., Blaszczyk, K., et al. (2009). Large, rare chromosomal deletions associated with severe early-onset obesity. Nature, 463, 666–670. Boghossian, S., Dube, M. G., Torto, R., Kalra, P. S., & Kalra, S. P. (2006a). Hypothalamic clamp on insulin release by leptin-transgene expression. Peptides, 27, 3245–3254. Boghossian, S., Lecklin, A., Dube, M. G., Kalra, P. S., & Kalra, S. P. (2006b). Increased leptin expression in the dorsal vagal complex suppresses adiposity without affecting energy intake and metabolic hormones. Obesity, 14, 1003–1009. Boghossian, S., Lecklin, A. H., Torto, R., Kalra, P. S., & Kalra, S. P. (2005). Suppression of fat deposition for the life time of rodents with gene therapy. Peptides, 26, 1512–1519.

Boghossian, S., Ueno, N., Dube, M. G., Kalra, P., & Kalra, S. (2007). Leptin gene transfer in the hypothalamus enhances longevity in adult monogenic mutant mice in the absence of circulating leptin. Neurobiology of Aging, 28, 1594–1604. Bonavera, J. J., Dube, M. G., Kalra, P. S., & Kalra, S. P. (1994). Anorectic effects of estrogen May be mediated by decreased neuropeptide- Y release in the hypothalamic paraventricular nucleus. Endocrinology, 134, 2367–2370. Bray, G. A. (1998). Historical framework for the development of ideas about obesity. In George A. Bray, Claude Bouchard, & W. P. T. James (Eds.), Handbook of obesity Vol. 1 (pp. 1–29). New York: Marcel Dekker Inc. Buijs, R. M., Chun, S. J., Niijima, A., Romijn, H. J., & Nagai, K. (2001). Parasympathetic and sympathetic control of the pancreas: A role for the suprachiasmatic nucleus and other hypothalamic centers that are involved in the regulation of food intake. The Journal of Comparative Neurology, 431, 405–423. Carter, B. J. & Burstein, H. (2004). Adeno-associated virus and AAV vectors for gene delivery. In N. Templeton-Smith (Ed.), Theruapeutics mechanisms and strategies (pp. 71– 1011). New York: Marcel Dekker. Chen, Y. & Heiman, M. L. (2000). Chronic leptin administration promotes lipid utilization until fat mass is greatly reduced and preserves lean mass of normal female rats. Regulatory Peptides, 92, 113–119. Chen, X. M., Hosono, T., Yoda, T., Fukuda, Y., & Kanosue, K. (1998). Efferent projection from the preoptic area for the control of non- shivering thermogenesis in rats. Journal of Physiology (Paris), 512, 883–892. Clark, J. T., Kalra, P. S., Crowley, W. R., & Kalra, S. P. (1984). Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology, 115, 427–429. Coll, A. P., Farooqi, I. S., & O’Rahilly, S. (2007). The hormonal control of food intake. Cell, 129, 251–262. Dhillon, H., Ge, Y., Minter, R. M., Prima, V., Moldawer, L. L., Muzyczka, N., et al. (2000). Long-term differential modulation of genes encoding orexigenic and anorexigenic peptides by leptin delivered by rAAV vector in ob/ob mice. Relationship with body weight change. Regulatory Peptides, 92, 97–105. Dhillon, H., Kalra, S. P., & Kalra, P. S. (2001a). Dose-dependent effects of central leptin gene therapy on genes that regulate body weight and appetite in the hypothalamus. Molecular Therapy, 4, 139–145. Dhillon, H., Kalra, S. P., Prima, V., Zolotukhin, S., Scarpace, P. J., Moldawer, L. L., et al. (2001b). Central leptin gene therapy suppresses body weight gain, adiposity and serum insulin without affecting food consumption in normal rats: A long-term study. Regulatory Peptides, 99, 69–77. Dhillon, H., Zigman, J. M., Ye, C., Lee, C. E., McGovern, R. A., Tang, V., et al. (2006). Leptin directly activates SF1 neurons in the VMH, and this action by leptin is required for normal body-weight homeostasis. Neuron, 49, 191–203. Dietrich, M. O. & Horvath, T. L. (2009). GABA keeps up an appetite for life. Cell, 137, 1177–1179.

30 Dube, M. G., Beretta, E., Dhillon, H., Ueno, N., Kalra, P. S., & Kalra, S. P. (2002). Central leptin gene therapy blocks high fat diet-induced weight gain, hyperleptinemia and hyperinsulinemia: Effects on serum ghrelin levels. Current Medical Literature – Diabetes, 51, 1729–1736. Dube, M. G., Kalra, S. P., & Kalra, P. S. (2007). Low abundance of NPY in the hypothalamus can produce hyperphagia and obesity. Peptides, 28, 475–479. Dube, M. G., Torto, R., & Kalra, S. P. (2008). Increased leptin expression selectively in the hypothalamus suppresses inflammatory markers CRP and IL-6 in leptin-deficient diabetic obese mice. Peptides, 29, 593–598. Dube, M. G., Xu, B., Kalra, P. S., Sninsky, C. A., & Kalra, S. P. (1999). Disruption in neuropeptide Y and leptin signaling in obese ventromedial hypothalamic-lesioned rats. Brain Research, 816, 38–46. Ebihara, K., Kusakabe, T., Hirata, M., Masuzaki, H., Miyanaga, F., Kobayashi, N., et al. (2007). Efficacy and safety of leptin-replacement therapy and possible mechanisms of leptin actions in patients with generalized lipodystrophy. Journal of Clinical Endocrinology Metabolism, 92, 532–541. Farooqi, I. S. & O’Rahilly, S. (2005). Monogenic obesity in humans. Annual Review of Medicine, 56, 443–458. Fischer, J., Koch, L., Emmerling, C., Vierkotten, J., Peters, T., Bruning, J. C., et al. (2009). Inactivation of the fto gene protects from obesity. Nature, 458, 894–898. Flegal, K. M., Graubard, B. I., Williamson, D. F., & Gail, M. H. (2005). Excess deaths associated with underweight, overweight, and obesity. JAMA, 293, 1861–1867. Fredriksson, R., Hagglund, M., Olszewski, P. K., Stephansson, O., Jacobsson, J. A., Olszewska, A. M., et al. (2008). The obesity gene, FTO, is of ancient origin, up-regulated during food deprivation and expressed in neurons of feeding-related nuclei of the brain. Endocrinology, 149, 2062–2071. Friedman, J. M. (2009). Obesity: Causes and control of excess body fat. Nature, 459, 340–342. Friedman, J. M. & Halaas, J. L. (1998). Leptin and the regulation of body weight in mammals. Nature, 395, 763–770. Goldstone, A. P. (2006). The hypothalamus, hormones, and hunger: Alterations in human obesity and illness. Progress in Brain Research, 153, 57–73. Gray, J., Yeo, G. S., Cox, J. J., Morton, J., Adlam, A. L., Keogh, J. M., et al. (2006). Hyperphagia, severe obesity, impaired cognitive function, and hyperactivity associated with functional loss of one copy of the brain-derived neurotrophic factor (BDNF) gene. Current Medical Literature – Diabetes, 55, 3366–3371. Grundy, S. M. (2004). Obesity, metabolic syndrome, and cardiovascular disease. Journal of Clinical Endocrinology Metabolism, 89, 2595–2600. Heymsfield, S. B., Greenberg, A. S., Fujioka, K., Dixon, R. M., Kushner, R., Hunt, T., et al. (1999). Recombinant leptin for weight loss in obese and lean adults: A randomized, controlled, dose-escalation trial. JAMA, 282, 1568–1575. Hill, J. O., Wyatt, H. R., Reed, G. W., & Peters, J. C. (2003). Obesity and the environment: Where do we go from here? Science, 299, 853–855.

Horvath, T. L., Diano, S., & van den Pol, A. N. (1999). Synaptic interaction between hypocretin (orexin) and neuropeptide Y cells in the rodent and primate hypothalamus: A novel circuit implicated in metabolic and endocrine regulations. Journal of Neuroscience, 19, 1072–1087. Iwaniec, U. T., Boghossian, S., Lapke, P. D., Turner, R. T., & Kalra, S. P. (2007). Central leptin gene therapy corrects skeletal abnormalities in leptin-deficient ob/ob mice. Peptides, 28, 1012–1019. Kalra, S. P. (2001). Circumventing leptin resistance for weight control. Proceedings of the National Academy of Sciences of the United States of America, 98, 4279–4281. Kalra, S. P. (2007). Global life-long health benefits of repression of hypothalamic NPY system by central leptin gene therapy. Current Topics Medicinal Chemistry, 7, 1675–1681. Kalra, S. P. (2008a). Central leptin insufficiency syndrome: An interactive etiology for obesity, metabolic and neural diseases and for designing new therapeutic interventions. Peptides, 29, 127–138. Kalra, S. P. (2008b). Disruption in the leptin-NPY link underlies the pandemic of diabetes and metabolic syndrome: New therapeutic approaches. Nutrition, 24, 820–826. Kalra, S. P. (2009a). Central leptin gene therapy ameliorates diabetes type 1 and 2 through two independent hypothalamic relays; a benefit beyond weight and appetite regulation. Peptides, 30, 1957–1963. Kalra, S. P. (2009b). Increased leptin supply to hypothalamus by gene therapy confers life-long benefits on energy homeostasis, disease cluster of metabolic syndrome-diabetes type 1 and 2, dyslipidemia and cardiovascular ailmentsand bone remodeling. In S. Shioda, , I. Homma, & Kato, N. (Eds.), Transmitters and modulators in health and disease (pp. 19–30). Tokyo: Springer. Kalra, S. P., Bagnasco, M., Otukonyong, E. E., Dube, M. G., & Kalra, P. S. (2003). Rhythmic, reciprocal ghrelin and leptin signaling: New insight in the development of obesity. Regulatory Peptides, 111, 1–11. Kalra, S. P., Dube, M. G., & Iwaniec, U. T. (2009). Leptin increases osteoblast-specific osteocalcin release through a hypothalamic relay. Peptides, 30, 967–973. Kalra, S. P., Dube, M. G., Pu, S., Xu, B., Horvath, T. L., & Kalra, P. S. (1999). Interacting appetite-regulating pathways in the hypothalamic regulation of body weight. Endocrine Reviews, 20, 68–100. Kalra, S. P., Dube, M. G., Sahu, A., Phelps, C. P., & Kalra, P. S. (1991). Neuropeptide Y secretion increases in the paraventricular nucleus in association with increased appetite for food. Proceedings of the National Academy of Sciences of the United States of America, 88, 10931–10935. Kalra, P. S., Dube, M. G., Xu, B., Farmerie, W. G., & Kalra, S. P. (1998a). Neuropeptide Y (NPY) Y1 receptor mRNA is upregulated in association with transient hyperphagia and body weight gain: Evidence for a hypothalamic site for concurrent development of leptin resistance. Journal of Neuroendocrinology, 10, 43–49. Kalra, P. S., Dube, M. G., Xu, B., & Kalra, S. P. (1997). Increased receptor sensitivity to neuropeptide Y in the

31 hypothalamus May underlie transient hyperphagia and body weight gain. Regulatory Peptides, 72, 121–130. Kalra, S. P. & Kalra, P. S. (1996). Nutritional infertility: The role of the interconnected hypothalamic neuropeptide Y-galanin-opioid network. Frontiers in Neuroendocrinology, 17, 371–401. Kalra, S. P. & Kalra, P. S. (2001). Viral vectors as probes to decipher brain circuitry for weight control. Trends in Endocrinology and Metabolism, 12, 377–378. Kalra, S. P. & Kalra, P. S. (2003). Neuropeptide Y: A physiological orexigen modulated by the feedback action of ghrelin and leptin. Endocrine, 22, 49–55. Kalra, S. P. & Kalra, P. S. (2004a). NPY – an endearing journey in search of a neurochemcial on/off switch for appetite, sex and reproduction. Peptides, 25, 465–471. Kalra, S. P. & Kalra, P. S. (2004b). NPY and cohorts in regulating appetite, obesity and metabolic syndrome: Beneficial effects of gene therapy. Neuropeptides, 38, 201–211. Kalra, S. P. & Kalra, P. S. (2005). Gene transfer technology: A preventive neurotherapy to curb obesity, ameliorate metabolic syndrome and extend life-expectancy. Trends in Pharmacological Sciences, 26, 488–495. Kalra, S. P. & Kalra, P. S. (2006a). Neuropeptide Y: A conductor of the appetite-regulating orchestra in the hypothalamus. In A. J. Kastin (Ed.), The handbook of biologically active peptides (pp. 889–894). Burlington, MA: Academic Press. Kalra, S. P. & Kalra, P. S. (2006b). Subjugation of hypothalamic NPY and cohorts with central leptin gene therapy alleviates dyslipidemia, insulin resistance, and obesity for lifetime. In Z. Zukowska, & G. Feuerstein (Eds.), NPY family of peptides in neurobiology, cardiovascular and metabolic disorders: Genes, functions and therapeutics (pp. 157–169). Switzerland: Birhauser Verlag Publishing House. Kalra, S. P. & Kalra, P. S. (2007). To subjugate NPY is to improve the quality of life and live longer. Peptides, 28, 413–418. Kalra, S. P., Ueno, N., & Kalra, P. S. (2005). Stimulation of appetite by ghrelin is regulated by leptin restraint: Peripheral and central sites of action. The Journal of Nutrition, 135, 1331–1335. Kalra, S. P., Xu, B., Dube, M. G., & Kalra, P. S. (1998b). Nutritional infertility, mismanagement in central neuropeptidergic and peripheral insulin – leptin signaling. In W. Hansel, G. Bray, & Ryan, D. H. (Eds.), Nutrition and reproduction (pp. 25–70). Baton Rouge, LA: State University Press. Kastin, A. J. & Pan, W. (2006). Intranasal leptin: Blood-brain barrier bypass (BBBB) for obesity? Endocrinology, 147, 2086–2087. Keen-Rhinehart, E., Kalra, S. P., & Kalra, P. S. (2005). AAV mediated leptin receptor installation improves energy balance and the reproductive status of obese female koletsky rats. Peptides, 26, 2567–2578. King, B. M. (2006). The rise, fall, and resurrection of the ventromedial hypothalamus in the regulation of feeding behavior and body weight. Physiology & Behavior, 87, 221–244.

Kojima, S., Asakawa, A., Amitani, H., Sakoguchi, T., Ueno, N., Inui, A., et al. (2009). Central leptin gene therapy, a substitute for insulin therapy to ameliorate hyperglycemia and hyperphagia, and promote survival in insulin-deficient diabetic mice. Peptides, 30, 962–966. Kreier, F., Kap, Y. S., Mettenleiter, T. C., van Heijningen, C., van der Vliet, J., Kalsbeek, A., et al. (2006). Tracing from fat tissue, liver, and pancreas: A neuroanatomical framework for the role of the brain in type 2 diabetes. Endocrinology, 147, 1140–1147. Kublaoui, B. M., Holder, J. L. Jr., Gemelli, T., & Zinn, A. R. (2006). Sim1 haploinsufficiency impairs melanocortinmediated anorexia and activation of paraventricular nucleus neurons. Molecular Endocrinology, 20, 2483–2492. Lecklin, A. H., Dube, M. G., Torto, R., Kalra, P. S., & Kalra, S. P. (2005). Perigestational suppression of weight gain with central leptin gene therapy results in lower weight F1 generation. Peptides, 26, 1176–1187. Lenz, A. & Diamond, F. B. Jr. (2008). Obesity: The hormonal milieu. Current Opinion Endocrinology Diabetes Obesity, 15, 9–20. Lindqvist, A., de la Cour, C. D., Stegmark, A., Hakanson, R., & Erlanson-Albertsson, C. (2005). Overeating of palatable food is associated with blunted leptin and ghrelin responses. Regulatory Peptides, 130, 123–132. Luiten, P. G., ter Horst, G. J., & Steffens, A. B. (1987). The hypothalamus, intrinsic connections and outflow pathways to the endocrine system in relation to the control of feeding and metabolism. Progress of Neurobiology, 28, 1–54. Luquet, S., Perez, F. A., Hnasko, T. S., & Palmiter, R. D. (2005). NPY/AgRP neurons are essential for feeding in adult mice but can be ablated in neonates. Science, 310, 683–685. Malhotra, A., Vashistha, H., Yadav, V. S., Dube, M. G., Kalra, S. P., Abdellatif, M., et al. (2009). Inhibition of p66ShcA redox activity in cardiac muscle cells attenuates hyperglycemia-induced oxidative stress and apoptosis. American Journal of Physiology. Heart and Circulatory Physiology, 296, H380–H388. Montague, C. T., Farooqi, I. S., Whitehead, J. P., Soos, M. A., Rau, H., Wareham, N. J., et al. (1997). Congenital leptin deficiency is associated with severe early-onset obesity in humans. Nature, 387, 903–908. Neary, M. T. & Batterham, R. L. (2009). Peptide YY: Food for thought. Physiology & Behavior, 97, 616–619. Otukonyong, E. E., Dube, M. G., Torto, R., Kalra, P. S., & Kalra, S. P. (2005a). Central leptin differentially modulates ultradian secretory patterns of insulin, leptin and ghrelin independent of effects on food intake and body weight. Peptides, 26, 2559–2566. Otukonyong, E. E., Dube, M. G., Torto, R., Kalra, P. S., & Kalra, S. P. (2005b). High fat diet-induced ultradian leptin and insulin hypersecretion and ghrelin is absent in obesityresistant rats. Obesity Research, I13, 991–999. Park, J. Y., Chong, A. Y., Cochran, E. K., Kleiner, D. E., Haller, M. J., Schatz, D. A., et al. (2008). Type 1 diabetes associated with acquired generalized lipodystrophy

32 and insulin resistance: The effect of long-term leptin therapy. Journal of Clinical Endocrinology Metabolism, 93, 26–31. Plum, L., Belgardt, B. F., & Bruning, J. C. (2006). Central insulin action in energy and glucose homeostasis. The Journal of Clinical Investigation, 116, 1761–1766. Powley, T. L., Opsahl, C. J., Cox, J. E., & Weingarten, H. P. (1980). The role of the hypothalmus in energy homeostasis. In P. J. Morgane & J. Planksepp (Eds.), Handbook of the hypothalamus. Part A: Behavioral studies of the hypothalamus Vol. 3 (pp. 211–298). New York: Marcel Dekker, Inc. Pu, S., Dube, M. G., Kalra, S. P., & Kalra, P. S. (2003). 33rd annual society for neuroscience meeting (pp. W-33, Abs #831.7). New Orleans, LA: Society for Neuroscience. Pu, S., Jain, M. R., Horvath, T. L., Diano, S., Kalra, P. S., & Kalra, S. P. (1999). Interactions between neuropeptide Y and gamma-aminobutyric acid in stimulation of feeding: A morphological and pharmacological analysis. Endocrinology, 140, 933–940. Ren, D., Zhou, Y., Morris, D., Li, M., Li, Z., & Rui, L. (2007). Neuronal SH2B1 is essential for controlling energy and glucose homeostasis. The Journal of Clinical Investigation, 117, 397–406. Richard, D., Guesdon, B., & Timofeeva, E. (2009). The brain endocannabinoid system in the regulation of energy balance. Best Practice and Research Clinical Endocrinology Metabolism, 23, 17–32. Rios, M., Fan, G., Fekete, C., Kelly, J., Bates, B., Kuehn, R., et al. (2001). Conditional deletion of brain-derived neurotrophic factor in the postnatal brain leads to obesity and hyperactivity. Molecular Endocrinology, 15, 1748–1757. Sahu, A. (2002). Resistance to the satiety action of leptin following chronic central leptin infusion is associated with the development of leptin resistance in neuropeptide Y neurones. Journal of Neuroendocrinology, 14, 796–804. Schwartz, M. W., Figlewicz, D. P., Baskin, D. G., Woods, S. C., & Porte, D. Jr. (1992). Insulin in the brain: A hormonal regulator of energy balance. Endocrine Reviews, 13, 387–414. Shimizu, H., Oh, I. S., Okada, S., & Mori, M. (2009). Nesfatin1: An overview and future clinical application. Endocrine Journal, 56, 537–543. Shimomura, I., Hammer, R. E., Ikemoto, S., Brown, M. S., & Goldstein, J. L. (1999). Leptin reverses insulin resistance and diabetes mellitus in mice with congenital lipodystrophy. Nature, 401, 73–76. Stricker, E. M. (Ed.) (1990). Handbook of behavioral neurobiology. In Neurobiology of food and fluid intake Vol. 10 (pp. 1–553). New York: Plenum Press. Sved, A. F., Cano, G., & Card, J. P. (2001). Neuroanatomical specificity of the circuits controlling sympathetic outflow to different targets. Clinical and Experimental Pharmacology and Physiology, 28, 115–119. Taymans, J. M., Vandenberghe, L. H., Haute, C. V., Thiry, I., Deroose, C. M., Mortelmans, L., et al. (2007).

Comparative analysis of adeno-associated viral vector serotypes 1, 2, 5, 7, and 8 in mouse brain. Human Gene Therapy, 18, 195–206. Tong, Q., Ye, C. P., Jones, J. E., Elmquist, J. K., & Lowell, B. B. (2008). Synaptic release of GABA by AgRP neurons is required for normal regulation of energy balance. Nature Neuroscience, 11, 998–1000. Torto, R., Boghossian, S., Dube, M. G., Kalra, P. S., & Kalra, S. P. (2006). Central leptin gene therapy blocks ovariectomy-induced adiposity. Obesity (Silver Spring), 14, 1312–1319. Ueno, N., Dube, M. G., Inui, A., Kalra, P. S., & Kalra, S. P. (2004). Leptin modulates orexigenic effects of ghrelin and attenuates adiponectin and insulin levels and selectively the dark-phase feeding as revealed by central leptin gene therapy. Endocrinology, 145, 4176–4184. Uyama, N., Geerts, A., & Reynaert, H. (2004). Neural connections between the hypothalamus and the liver. The Anatomical Record Part A, Discoveries Molecular Cellular, and Evolutionary Biology, 280, 808–820. Van Heek, M., Compton, D. S., France, C. F., Tedesco, R. P., Fawzi, A. B., Graziano, M. P., et al. (1997). Dietinduced obese mice develop peripheral, but not central, resistance to leptin. The Journal of Clinical Investigation, 99, 385–390. Virtanen, K. A., Lidell, M. E., Orava, J., Heglind, M., Westergren, R., Niemi, T., et al. (2009). Functional brown adipose tissue in healthy adults. The New England Journal of Medicine, 360, 1518–1525. Wang, Z. W., Zhou, Y. T., Kakuma, T., Lee, Y., Higa, M., Kalra, S. P., et al. (1999). Comparing the hypothalamic and extrahypothalamic actions of endogenous hyperleptinemia. Proceedings of the National Academy of Sciences of the United States of America, 96, 10373–10378. Wilson, J. M. (2009). Progress in the commercial-scale production of adeno-associated viral vectors. Human Gene Therapy, 20, 695. Wright, J. F. (2009). Transient transfection methods for clinical adeno-associated viral vector production. Human Gene Therapy, 20, 698–706. Wu, Q., Boyle, M. P., & Palmiter, R. D. (2009). Loss of GABAergic signaling by AgRP neurons to the parabrachial nucleus leads to starvation. Cell, 137, 1225–1234. Wynne, K., Stanley, S., McGowan, B., & Bloom, S. (2005). Appetite control. Journal of Endocrinology, 184, 291–318. Xu, B., Goulding, E. H., Zang, K., Cepoi, D., Cone, R. D., Jones, K. R., et al. (2003). Brain-derived neurotrophic factor regulates energy balance downstream of melanocortin-4 receptor. Nature Neuroscience, 6, 736–742. Xu, B., Kalra, P. S., Farmerie, W. G., & Kalra, S. P. (1999). Daily changes in hypothalamic gene expression of neuropeptide Y, galanin, proopiomelanocortin, and adipocyte leptin gene expression and secretion: Effects of food restriction. Endocrinology, 140, 2868–2875. Yeo, G. S., Connie Hung, C. C., Rochford, J., Keogh, J., Gray, J., Sivaramakrishnan, S., et al. (2004). A de novo mutation affecting human TrkB associated with severe

33 obesity and developmental delay. Nature Neuroscience, 7, 1187–1189. Yokosuka, M., Xu, B., Pu, S., Kalra, P. S., & Kalra, S. P. (1998). Neural substrates for leptin and neuropeptide Y (NPY) interaction: Hypothalamic sites associated with inhibition of NPY-induced food intake. Physiology & Behavior, 64, 331–338.

Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., & Friedman, J. M. (1994). Positional cloning of the mouse obese gene and its human homologue. Nature, 372, 425–432. Ziylan, Y. Z., Baltaci, A. K., & Mogulkoc, R. (2009). Leptin transport in the central nervous system. Cell Biochemistry and Function, 27, 63–70.

L. Martini (Eds.) Progress in Brain Research, Vol. 181 ISSN: 0079-6123 Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 3

Reproductive behaviors: new developments in concepts and in molecular mechanisms Zachary M. Weil, Gen Murakami and Donald W. Pfaff Laboratory of Neurobiology and Behavior, The Rockefeller University, New York, United States of America

Abstract: New developments in the analysis of mechanisms for reproductive behaviors are reviewed. Conceptually, the concept of generalized arousal (GA) of the central nervous system (CNS) is considered. Breeding for high and low GA, we show an impact of GA on sexual arousal of male mice, and also find that the structure of GA in the CNS of males and females is not the same. Further, we propose, theoretically, that among epithelial tissues in humans, there are correlations among their innervation densities and their ability to trigger arousal. In new technical developments, we analyze transcriptional effects of estrogens in the hypothalamic neurons that regulate lordosis behavior. The rapid effect of estradiol to increase acetylation of histones in ventromedial hypothalamic neurons could be tied into transcriptional activation, but the effect of estradiol to increase methylation of histone 3, lysine 9 (H3K9) is puzzling. This work seeks to discover the coactivator dynamics underlying transcriptional effects of estrogens on sex behavior. Keywords: arousal; sex difference; lordosis; histones; epigenetics

approach: an introduction to methods for studying changes in the chromatin overlying genes crucial for the transcriptional changes in hypothalamic and preoptic neurons, which in turn are crucial for reproductive behaviors.

Introduction Since the purpose of this volume of Progress in Brain Research is to review new developments in neuroendocrinology, we shall not go back over the huge bodies of fact that are covered for female sexual behaviors (Lee et al., 2009) and male sexual behaviors (Hull et al., 2009) in Hormones, Brain and Behavior (Academic Press/Elsevier, 2009). Instead, we’ll open up a discussion about a new emphasis on concepts of the brain’s regulation of hormone-dependent sexual behaviors in the female and male. This will be followed by a novel chemical

Generalized arousal and its impact on sex behaviors The most powerful and essential force in the vertebrate central nervous system (CNS) has been proposed (Pfaff, 2006) as originating from primitive reticular neurons in the lower brainstem. We have gathered evidence for its existence using three approaches: (1) by principal components analysis, showing that about one-third of the variance of the



Corresponding author. Tel.: +212-327-8667; Fax: +212-327-8643; E-mail: [email protected]

DOI: 10.1016/S0079-6123(08)81003-5

35

36

data in arousal-related assays (of mouse behavior) is due to generalized arousal (GA) force (Garey et al., 2003); (2) by acknowledging the large amount of neuroanatomical, neurophysiological and genomic evidence for the detailed mechanisms supporting GA (reviewed in Pfaff, 2006); and (3) by breeding mice for high and low GA (Weil et al., 2010). Given that generalized CNS arousal actually exists, what are its impacts on female and male sexual behaviors? In the female, new neuropharmacological results prove that arousal-enhancing drugs, particularly amphetamines, increase the expression of lordosis behavior (Holder et al., 2009). As reported in Weil et al. (2010), we have new evidence that the mechanistic structure of generalized CNS arousal mechanisms are not the same in female mice as in male mice. Mice are being bred for high GA and low GA according to the operational definition stated in Pfaff (2006). Male and female mice derived from ‘high’ parents are referred to as HM and HF, respectively, while the offspring of ‘low’ parents are LM and LF. Males from the high line and those offspring who exhibited high levels of GA exhibited a

Mounts before first intromission

10 0 High

10 0 High

40 30 20 10 0 Low

Offspring arousal

40 30 20 10 0 High

(d) 4 3 2 1 0

Low

Parental arousal

(f) Number of intromissions before ejaculation

50

High

20

Low

Parental arousal

(e)

30

Low

Offspring arousal

Hit ratio

Intromission: mount ratio

20

(c) 40

Latency to ejaculate after first intromission (sec)

Number of intromissions before ejaculation

Mounts before first intromission

30

Latency to ejaculate after 1st intromission

High

Parental arousal

(g) 4 3 2 1 0

High

1.0 0.8 0.6 0.4 0.2 0.0 High

Low

Low

Offspring arousal

Low

Parental arousal

(h) Intromission: mount ratio

(b) 40

Mounts before first intromission

Intromissions before ejaculation

Latency to ejaculate after first intromission (Log10 sec)

(a)

specific pattern of sexual behavior associated with a higher level of excitability and sexual arousal (Fig. 1). High-arousal males exhibited more mounts before intromission and, then, fewer intromissions before ejaculating and rapid ejaculation after the first intromission. Additionally, the percentage of mount attempts that were successful in leading to intromission was significantly lower among male mice from the higharousal line. The pattern of sexual behavior indicates that high-arousal males were excitable in an inappropriate manner, as indicated by the large numbers of early mounts without intromission and the very low intromission: total mount ratio. Finally, to extract information about the most prominent feature of the data gathered from our GA assay, we used a mathematical method called principal components analysis. This method is used here to analyze the relative contributions of motor, sensory and emotional (fear) measures as they influence the largest, most elementary dimension of arousal. That is, the most generalized, elementary force operating in our arousal assay is revealed by a forced one-component

1.0 0.8 0.6 0.4 0.2 0.0 High

Low

Offspring arousal

Fig. 1. Male mice bred for high levels of generalized CNS arousal were excitable, made many quick and unsuccessful mounts of the female and, once having achieved an intromission, quickly achieved ejaculation. In this figure their male sex behavior is compared to male mice bred for low levels of generalized CNS arousal (from Weil et al., 2010). Thus illustrated is a contribution of generalized CNS arousal to one specific type of arousal, sexual arousal.

37

solution of our data set. The most interesting comparisons to come out of the principal components analysis are illustrated in Fig. 2. It demonstrates the separate contributions of motor activity, olfactory responsivity and fear to Principal Component #1, the component that quantifies the most generalized, powerful force generating behavior in these arousal assays. In Fig. 2, when a measure has a (–) sign, it means that it was, indeed, grouped with the forces on Principal Component #1, but in the reverse direction (low values of that behavior are strongly associated with Principal Component #1’s contribution to the production of arousal-related behaviors). Principal Component #1 reflects a high degree of motor activity. Our analysis raises the question of whether the structures of arousal functions are the same in males and females. Fig. 2 shows that the major differences between HM and LM come from the large contribution of motor activity of HM to Principal Component #1, as well as a difference in the contribution of fear.

In fact, it is the failure of motor activity driven by Principal Component #1 that makes those males LM rather than HM. HM have high movement rates and are skittish. Females are different. The major difference between HF and LF comes from the strong reactivity of the HF to olfactory input. With respect to sex differences between HM and HF, there are large differences in the contributions of olfactory responsiveness to Principal Component #1, as well as fear. We speculate that a HF female ready to mate, having spent much time in her burrow, will emerge from her burrow just before ovulation. She must lack fear and locomote extensively, spreading the odour of vaginal secretions, a form of courtship behavior that encourages males to mate just as she is ovulating. In turn, her powerful olfactory response will help her choose healthy vigorous males as potential fathers of her litter. Between LM and LF, the major difference is due to the fact that the LF had a large motor contribution to Principal Component #1, as well as a smaller difference between

Males

Females

Motor +

Motor

+

+ Fear

.589

.587

.481

High –

.09

+

Olfactory

.518

–

+ Olfactory

–.234

Fear –

–

– + .699

+

+ Fear

.262

Low –.211

–

–.388

Fear –

+

Olfactory

–

+

–.147

Olfactory

–.167

–

–

Fig. 2. The structure of generalized CNS arousal is not the same in female mice as compared to males (from Weil et al., 2010). Here the contributions of motor, sensory (olfactory) and emotional (fear) measurements to the primary component of CNS arousal are quantified. See text for explanation.

38

LM and LF in fear. From this application of principal components analysis, we infer that the structure of the primary arousal component is not the same in males as in females.

Mechanisms initiating generalized and sexual arousal in humans We have summarized sources of sexual arousal from various levels of the neuraxis (MartinAlguacil et al., 2006). Although a large number of studies with animal brain emphasize the importance of the hypothalamus and preoptic area in the regulation of sexual behavior, we clearly recognize the power of genital stimulation, in humans at least, for arousal sexual arousal. Therefore, we have begun the histochemical characterization of epithelial cells in the critical genital areas. For example, the expression of immunoreactive estrogen receptor (ER)-alpha differs significantly from that of ER-beta (Martin-Alguacil et al., 2008b; Schober et al., 2008) and has an interesting relation to the expression of the neuronal isoform of nitric oxide synthase (nNOS) (Martin-Alguacil et al., 2008a). Extrapolating from our studies with ER expression in human genital tissues, we propose (Schober et al., 2010, in preparation) the following new concept – across the entire range of human epithelia, three different functions are strictly correlated with one another: (1) a higher density of innervation and vascularization is correlated with (2) greater speed and efficiency of wound healing and (3) a more powerful ability to stimulate CNS arousal upon stimulation of the epithelial surface. New studies will (1) systematically test this theory using human tissue and (2) analyze the mechanisms by which this convergence of functions is produced.

Chromatin: steroid hormone-induced covalent modifications of histone tails New mRNA and protein synthesis in hypothalamic neurons are required for estrogenic facilitation of lordosis behavior (reviewed in Pfaff,

1980). This discovery led to a long series of experiments in which genes that had two properties were identified: (1) their mRNA levels were increased in hypothalamic neurons after E administration and (2) their gene products would foster female reproductive behaviors (summarized in Pfaff, 1999). In turn, the emphasis on gene–behavior relationships sprung two very surprising observations: the loss of one gene (ERalpha) could cause females to be treated like males and to respond like males (Ogawa et al., 1996), and secondly, an individual gene could have opposite behavioral effects in male brains as compared to female brains (summarized in Ogawa et al., 2004). All of these and other observations (e.g. Ogawa et al., 1998a, 1998b) caused us to focus on hormonefacilitated gene transcription. The initiation of transcription is widely understood to involve changes in chromatin, the set of proteins that guard access to DNA by transcription factors such as ERs (illustrated in Figs. 3–5). Chromatin, the principal structural protein associated with DNA molecules, has a potential regulatory role in gene expression. Chromatin is the principal structural protein around which DNA is wrapped and it is made up of four canonical proteins termed H2A, H2B, H3 and H4, the structure of which have been largely conserved over evolutionary time. Broadly, DNA exists in two states, (1) euchromatin, wherein DNA is relatively loosely coiled around chromatin and which is associated with transcriptionally active genes. This is in contrast to (2) heterochromatin where DNA is tightly wrapped around histone peptides and thus the underlying DNA is inaccessible to the transcriptional machinery. These states were long considered developmentally programmed and static. However, these broad categories are significantly complicated by dozens of covalent modifications of histone tail regions which can more subtly regulate gene expression. Modification of histone tails with the additions of acetyl, phosphate, methyl, sumo and ADP ribosyl groups at specific amino acid residues alone and in combination has specific regulatory effects on the genes associated with modified chromatin. The complexity of this system has lead Professor

39 Nucleosome remodelling permits repressed derepressed transcription machinery Transcription factors and coactivators

(simplest view) Fig. 3. Inactive DNA is tightly wound, whereas DNA accessible to transcription factors is no longer tied up in nucleosomes.

Histone chemical status regulates access to DNA by transcription factors Various modifications applied to histone tails (methylation, acetylation, imination, phosphorylation)

H4 H3 H2A H2B

Nucleosome

H3 H4 Inactive DNA Fig. 4. Nucleosomes in which inactive DNA is wrapped are characterized by tails of histone proteins emerging in a manner that leaves them susceptible to chemical modification. In the usual case for mammalian cells, acetylation of amino acids on these tails is associated with increased rates of transcription and methylation is associated with repression of transcription. We have tied increased acetylation to estrogenic effects in the region of the ventromedial hypothalamus important for regulating lordosis behavior (Weil et al., 2009).

C. David Allis and colleagues here at Rockefeller to suggest that these covalent modifications of histone peptides taken together form a ‘histone code’ which provide important epigenetic regulation of gene expression. Therefore, with respect to female sex behavior, it was natural for us to try and draw histone modification into E-caused transcriptional changes in the VMH neurons essential for E-dependent lordosis behavior (Weil et al., 2009). Results showed that treatment with 17b-estradiol rapidly methylates H3K9 in the VMH, and also that treatment with

17b-estradiol rapidly increased the acetylation status of histone H4 proteins in the VMH. These changes are highly likely to be relevant to lordosis behavior because double-identified ER-alpha positive cells in the ventromedial hypothalamus are shown by our new results to exhibit H4 acetylation. We have further reason to believe that histone acetylation within VMH neurons is causally linked to female reproductive behavior, because microinjections of the histone deacetylase inhibitor into the VMH potentiates estrogen-induced lordosis

40

ER

ER

D B D

DB D TCAnnnTGACC G T AG

GR

AnnnTGTT AAC CT AG

D

B

D

D B D

GR

Transcribing NOT Transcribing DBD DNA binding domain ER Estrogen receptors GR Glucocorticoid receptors

Fig. 5. The net result of loosening DNA tight connections with nucleosomes is thought to lie in freeing up transcription factor recognition elements, illustrated here by consensus nucleotide base sequences for estrogen receptors (ERs) and glucocorticoid receptors (GRs).

behavior and reduces rejection behaviors (Weil et al., 2009). Similar considerations apply to maternal behavior. The optimal pattern of hormone exposure for the onset of maternal behaviors in female laboratory mammals comprises a sudden drop of progesterone levels in the blood coupled with high levels of estrogens in the blood. Surprisingly, however, it had not been determined exactly what duration of estrogen exposure would be minimally sufficient for the facilitation of maternal behaviors. We have just finished the appropriate experiments by ovariectomizing female mice 11 days before tests of maternal behaviors and, at the same time, implanting progesterone capsules subcutaneously, which will subsequently be removed two days before behavioral testing. The question is: how long must estrogens circulate in progesterone-withdrawn mice, for maternal behaviors to occur? The answer was surprising: estrogens administered as little as two hours before testing could enhance maternal behaviors in progesterone-withdrawn female mice (Murakami et al., unpublished data, 2010). The advantage of this short requirement of estrogen treatment is that it permits us to concentrate

on a short time window during estrogen-triggered behaviorally important transcriptional changes. In turn, this knowledge tells us exactly when to look for histone chemical modifications in preoptic area neurons, a project that we are finishing now (Murakami et al., unpublished data, 2010). From unpublished experiments, we know that the schedule of E administration consistent with the initiation of maternal behaviors is associated with global changes of histone chemistry in the preoptic neurons essential for maternal behavior, and that these changes include both methylation and acetylation (Murakami et al., unpublished data, 2010), but we neither have tried to draw these histone modifications into transcriptional arguments, nor have we proven that these histone modifications are essential for the initiation of maternal behavior. Those experiments will be initiated soon in this laboratory. Although the data and molecular concepts of histone modifications fit very clearly with access to estrogen-sensitive genes by ligand-activated transcription factors, ERs, phenomena in neuroendocrine neurons are not always so simple. The changes in methylation status of specific histones caused by acute stress in hippocampal

41

neurons (Hunter et al., 2009) have not, so far, been mimicked by corticosterone injections as they might have been expected to be. Alternate routes of stress effects, therefore, are being explored currently. Our current emphasis on the chemical modifications of histone tails in hypothalamic and preoptic neurons brings us through the next mechanistic step in demonstrating how gene–behavior causal relations occur. After all, Ogawa et al. proposed that deletion of a single gene could cause a female mouse to be treated like a male and to behave like a male (Ogawa et al., 1996). Altogether, knocking out the gene for ER-alpha caused a panoply of changes in sexual, aggressive and maternal behaviors (Ogawaet al., 1997, 1998a, 1998b). Taken together, the Ogawa et al. (2004) findings proved that the same gene could have opposite behavioral effects (on aggressive behavior) in females as that same gene has in males. In sum, because of the clear relationships between patterns of gene expression and behavior in neuroendocrine systems, we predict that sex steroid effects on reproductive behaviors – both sexual and maternal – will provide some of the most chemically detailed and functionally important examples of histone modifications in nerve cells. References Garey, J., Goodwillie, A., Frohlich, J., Morgan, M., Gustafsson, J.-A., Smithies, O., et al. (2003). Genetic contributions to generalized arousal of brain and behaviour. Proceedings of the National Academy of Sciences, U. S. A., 100(19), 11019–11022. Holder, M., Hadjimarkou, M. M., Zup, S. L., Blutstein, T., Benham, R. S., McCarthy, M. M., Mong, J. A. (2009). Methamphetamine facilitates female sexual behavior and enhances neuronal activation in the medial amygdale and the ventromedial nucleus of the hypothalamus. Psychoneuroendocrinology, 35(2), 197–208. Hull, E., Rodriguez-Manzo, G. (2009). Males sex behavior. In D. Pfaff, A. Arnold, A. Etgen, S. Fahrbach, R. Rubin, & E. Hormones, (Eds.), Brain and behavior (2nd ed.). San Diego, CA: Academic Press/Elsevier. Hunter, R., McCarthy, K., Milne, T., Pfaff, D., & McEwen, B. (2009). Regulation of hippocampal H3 histone methylation by acute and chronic stress. Proceedings of National Academy of Sciences, 106(49), 20912–20917.

Lee, A. W., Kow, L.-M., Devidze, N., Ribeiro, A., MartinAlguacil, N., Schober, J., et al. (2009). Genetic mechanisms in neural and hormonal controls over female reproductive behaviors. Hormones, Brain and Behavior, 2, 1163–1186. Martin-Alguacil, N., Schober, J., Kow, L. M., & Pfaff, D. W. (2006). Arousing properties of the vulvar epithelium. Journal of Urology, 176(2), 456–462, Review. Martin-Alguacil, N., Schober, J., Kow, L.-M., & Pfaff, D. (2008a). Oestrogen receptor expression and neuronal nitric oxide synthase in the clitoris and preputial gland structures of mice. British Journal of Urology International, 102, 1719–1723. Martin-Alguacil, N., Schober, J. M., Sengelaub, D., Pfaff, D. W., & Shelley, D. N. (2008b). Clitoral sexual arousal: Neuronal tracing study from the clitoris through the spinal tracts. Journal of Urology, 180, 1241–1248. Ogawa, S., Choleris, E., & Pfaff, D. W. (2004). Genetic influences on aggressive behaviors and arousability in animals. Annals of the New York Academy of Sciences, 1036, 257–266. Ogawa, S., Eng, V., Taylor, J., Lubahn, D., Korach, K., & Pfaff, D. (1998a). Roles of estrogen receptor-alpha gene expression in reproduction-related behaviors in female mice. Endocrinology, 139, 5070–5081. Ogawa, S., Lubahn, D. B., Korach, K. S., & Pfaff, D. W. (1997). Behavioral effects of estrogen receptor gene disruption in male mice. Proceedings of National Academic Science U.S.A., 94, 1476–1481. Ogawa, S., Taylor, J., Lubahn, D. B., Korach, K. S., & Pfaff, D. W. (1996). Reversal of sex roles in genetic female mice by disruption of estrogen receptor gene. Neuroendocrinology, 64, 467–470. Ogawa, S., Washburn, T., Taylor, J., Lubahn, D., Korach, K., & Pfaff, D. (1998b). Modifications of testosterone-dependent behaviors by estrogen receptor-alpha gene disruption in male mice. Endocrinology, 139, 5058–5069. Pfaff, D. W. (1980). Estrogens and brain function: Neural analysis of a hormone-controlled mammalian reproductive behavior. New York: Springer-Verlag. Pfaff, D. W. (1999). Drive: Neurobiological and molecular mechanisms of sexual motivation. Cambridge: The M.I.T. Press. Pfaff, D. W. (2006). Brain arousal and information theory: Neural and genetic mechanisms. Cambridge, Mass: Harvard University Press. Schober, J. M., Martin-Alguacil, N., Kow, L.-M., & Pfaff, D. W. (2008). Estrogen receptors and their relation to neural receptive tissue of the labia minora. British Journal of Urology International, 101(11), 1401–1406. Weil, Z. M., Hunter, R. G., McEwen, B. S., Allis, C., & Pfaff, D. W. (2009). Estrogenic regulation of histone acetylation in ventromedial hypothalamic neurons. Chicago, IL: Society for Neuroscience. Weil, Z. M., Zhang, Q., Hornung, A., Blizard, D., & Pfaff, D. W. (2010). Impact of generalized brain arousal on sexual behavior. Proceedings of the National Academy of Sciences, 107(5), 2265–2270.

L. Martini (Eds.) Progress in Brain Research, Vol. 181 ISSN: 0079-6123 Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 4

Interactions between the immune and neuroendocrine systems Serge Rivest
Laboratory of Endocrinology and Genomics, CHUQ Research Center and Department of Molecular Medicine, Laval University, Quebec, Canada

Abstract: The growing spark of interest in research concerning the molecular links between the nervous, endocrine and immune systems has caused an explosion of new knowledge concerning the fine mechanisms that orchestrate the integrated response to an immune challenge. For instance, elevation in plasma glucocorticoid (GC) levels is one of the most powerful and well-controlled feedback mechanisms on the proinflammatory signal transduction machinery taking place across the organism. Circulating inflammatory molecules have the ability to target their cognate receptors at the levels of blood–brain barrier, the latter in return produces specific prostaglandins (PGs). This chapter presents the brain circuits involved in the activation of the hypothalamic–pituitary–adrenal (HPA) axis by endogenously produced prostaglandin E2 (PGE2) during systemic innate immune insults. Keywords: blood–brain barrier; brain circuits; glucocorticoids; hypothalamic–pituitary–adrenal axis; NF-kB, paraventricular nucleus of the hypothalamus; proinflammatory cytokines; prostaglandin E2

circulating neutrophils and monocytes and locally resident macrophages, together mount rapidly an inflammatory response that is characterized, among other features, by the secretion of cytokines (Fig. 1). Their secretion into the bloodstream is a key step in triggering the neuronal activity and subsequent neurophysiological responses that take place during systemic and localized tissue insults. Cytokines influence many neuroendocrine systems, the most prominent of which is the activation of the hypothalamic–pituitary–adrenal (HPA) axis, resulting in the release of adrenocorticotrophic hormone (ACTH) and glucocorticoids (Rivest, 2009). Once present in the bloodstream, these cytokines have also the ability to increase body temperature (fever) and

Introduction Inflammation is a general name for reactions occurring after most kinds of tissue injuries or infections or immunologic stimulations as a host defense against foreign or altered endogenous substances (Akira et al., 2006). The local inflammatory reaction is characterized by an initial increase of blood flow to site of injury, enhanced vascular permeability and selective accumulation of different effector cells from the peripheral blood to injured regions. These cells, mostly


Corresponding author. Tel.: þ418-654-2296; Fax: þ418-654-2761; E-mail: [email protected]

DOI: 10.1016/S0079-6123(08)81004-7

43

44

EP4 Microglial cell

PGE2 ? IL-6R

COX-2 IL-1R

TNFR

Neutrophil LPS LBP/Septin

CD14/TLR4

sCD14

IL-1

CD14/TLR4

TNF-α

Monocyte

CD14/TLR4

CD14/ TLR4

IL-1 TNF-α

Macrophage

IL-6

TNFR IL-1R COX IL-6R -2 ?

IL-6

PGE2

Fig. 1. Innate immune response evoked by endotoxic lipopolysaccharide (LPS). LPS is a major component of the outer membranes of Gram-negative bacteria, which is the best-characterized example of innate recognition associated with a robust inflammatory response by phagocytic cells. Secretion of cytokines by circulating monocytes/neutrophils and tissue macrophages by LPS requires a series of mechanisms in cascade, the first step being the binding of the LPS with the serum proteins LPS-binding protein (LBP) or septins. The newly formed complex may then activate different populations of cells by binding to its CD14 receptor and toll-like receptor 4 (TLR4). The latter is the actual signalling receptor for LPS. Two forms of CD14 receptors can be found. The first is present on the surface of myeloid cells (mCD14) and acts as a glycosyl-phosphatidylinositol (GPI)-anchored membrane glycoprotein; the other form is soluble in the serum (sCD14) and lacks the GPI properties, though it can bind LPS to activate cells devoid of mCD14, such as endothelial cells. LBP is not essential for LPS signalling, but the LPS–LBP complex is particularly powerful in activating cells of myeloid origin, that is neutrophils, monocytes, macrophages and microglia. One of the well-known consequences of such activation is the production of proinflammatory cytokines, such as interleukin-1b (IL-1b), tumour necrosis factor alpha (TNF-a) and IL-6. These cytokines may, in turn, bind to their cognate receptors expressed on the surface of cells forming the blood–brain barrier (BBB); but they are not essential for mediating the effects of LPS in the CNS. LPS is an exogenous ligand for cerebral tissue that expresses both LPS receptors. Such an innate immune/inflammatory response also takes place during tissue injuries and in presence of more complex pathogens (bacteria, viruses, etc.). See text for details.

cause sickness behaviors (Dantzer et al., 2008). The proinflammatory cytokine interleukin-1, especially its b form (IL-1b), is probably the most important molecule capable of modulating

cerebral functions during systemic and localized inflammation. Systemic IL-b injection activates the neurons involved in the control of autonomic functions, and neutralizing antibodies or IL-1

45

receptor antagonist are capable of preventing numerous responses during inflammatory stimuli. Studies in IL-1b-deficient mice have provided solid and compelling evidence supporting the critical role played by this cytokine in neural– immune interaction (Laflamme et al., 1999). Other cytokines implicated in neuroendocrine and febrile responses include tumour necrosis factor-a (TNF-a) and IL-6. Like IL-1b, intravenous (i.v.) TNF-a injection has a profound influence on the brain, fever and hormones of the HPA axis (Nadeau and Rivest, 1999). In contrast, the role of IL-6 in neuroendocrine and febrile responses is quite controversial (Vallières and Rivest, 1997, 1999; Vallières et al., 1997). IL-6 has been defined as one of the principal endogenous pyrogens from the observation that IL-6- deficient mice were unable to develop normal fever in response to lipopolysaccharide (LPS) and IL-1b. It has also been demonstrated that prostaglandins (PGs) mediate IL-6-induced fever and activate the HPA axis, but IL-6 is unable to stimulate PG formation in the brain. However, a specific antiserum to IL-6 (IL-6AS) was able to prevent LPSinduced febrile response and cyclooxygenase 2 (COX-2)-deficient mice do not develop fever in response to intracerebroventricular (i.c.v.) IL-6 injection. The mechanisms by which IL-1b and IL-6 activate the autonomic functions are likely to be quite different and IL-1b is clearly a more potent cytokine than IL-6 to trigger prostaglandin E2 (PGE2) synthesis in the central nervous system (CNS) (Rivest et al., 2000).

Possible pathways by which circulating IL-1b signals the brain The transport of soluble substances across vascular compartments occurs via either paracellular or transcellular mechanisms. Within the cerebrovasculature, the paracellular route is particularly impermeable due to the presence of the blood– brain barrier (BBB). The BBB consists primarily of non-fenestrated endothelial cells that are connected by tight junctions. Furthermore, the large molecular sizes (13–15 kDa) and hydrophilic nature of cytokines preclude their transcellular

movement by simple diffusion to any appreciable extent, and early studies concluded that the BBB was impermeable to IL-1b. It is nevertheless possible that endogenous cytokines diffuse across the BBB during extreme periods of fever or in presence of high levels in plasma during a long period of time. Regions relatively devoid of BBB permit cytokine interaction with the neuronal elements. The circumventricular organs (CVOs) contain capillaries with rather greater permeability than the rest of the CNS and the vascular density in these regions is extraordinarily high. Therefore, the CVOs have been proposed as potential sites of action, in particular the structure lining the anteroventral third ventricle (AV3V) region, namely the organum vasculosum of the lamina terminalis (OVLT). The subfornical organ (SFO), median eminence (ME) and area postrema (AP) are the other potential target CVOs (Rivest, 2009). On the other hand, medullary group of cells may play an important role in processing visceral sensory information carried out by the vagus and glossopharyngeal nerves (Dantzer et al., 2008). The vagus nerve has indeed been proposed to provide a rapid communication pathway for cytokine signalling between the periphery and brain, but this depends on the doses and route of administration. On the other hand, a strong body of evidence now supports the concept that cytokines, particularly IL-1, act on the microvasculature with consequent release of local signalling molecules, namely PGs (Zhang and Rivest, 2003). Indeed, brain microvessels exhibit constitutive expression levels of IL-1 type 1 receptor (IL-1R1) transcript and systemic IL-1b injection causes a rapid and profound transcriptional activation of the gene encoding COX-2 in cells lining the CNS blood vessels (Ericsson et al., 1995, Lacroix and Rivest, 1996, 1998). Circulating IL-1 also induces microsomal prostaglandin E synthase (mPGES-1), which colocalizes with COX-2 in the perinuclear region of the cerebral endothelium (Blais et al., 2005). Of great interest are the data that mPGES-1-deficient mice failed to develop fever in response to both systemic LPS

46

and IL-1, but not to centrally administered PGE2 (Engblom et al., 2003; Saha et al., 2005). These data clearly underline the critical role of the PGE2 machinery in the endothelium of the brain blood vessels to activate the autonomic functions during systemic inflammation.

Biosynthesis of PGs Prostanoids are a group of 20-carbon unsaturated fatty acid derivates that are produced via a complex enzymatic cascade. First, cyclooxygenase activity adds molecular oxygen to the unsaturated fatty acid arachidonic acid, generating prostaglandin G2 (PGG2). PGG2 is then converted to PGH2 by the peroxidase activity of the enzyme. Once generated, PGH2 is rapidly converted to prostaglandins (PGD2, PGE2, PGF2a), prostacylin (PGI2) and thromboxane A2 (TxA2) by tissuespecific synthases. Prostaglandin E synthase has recently been identified and designated as microsomal PGES (mPGES) and cytosolic PGES (cPGES). These molecules or their derivatives interact with specific receptors to modulate cell function (Zhang and Rivest, 2003). The diversity of the tissue-specific synthases and receptors gives rise to a wide range of potential biologic functions for the prostanoids. Prostaglandin G, PGH, PGI, and TxA are chemically unstable and are degraded into inactive products under physiological conditions, with a half-life of 30 seconds to a few minutes. Other PGs, although chemically stable, are metabolized quickly. It is therefore believed that prostanoids work locally, acting only in the vicinity of the site of production to serve as potent autocrine and paracrine mediators in a wide variety of physiological processes. Although PGE2 rises in blood promptly after the entry of microorganisms or in response to the endotoxin LPS and cytokines, it is now generally accepted that the PGE2 detected in the brain is not derived from the blood, but is rather produced directly within the CNS. It is therefore proposed here that circulating LPS and cytokines bind to their cognate receptors onto endothelial and/or monocytic cells lining the BBB, which leads to proinflammatory signalling

and transcription of the enzymes responsible for the PGE2 formation in the cerebral tissue (Ek et al., 2001). It is interesting to note that systemic inflammatory insults induce COX-2 and mPGES in a rather unspecific manner across the cerebral blood vessels and small capillaries, while the neuronal activity is limited to selective nuclei, including the endocrine hypothalamus. It is thus possible that expression of specific PGE2 receptors within parenchymal cells adjacent to the site of production determines the action of the PG in the brain.

PGE2 sites of action Classic prostanoid receptors comprise a family of eight encoding transmembrane G-proteincoupled receptors (Narumiya et al., 1999). These receptors are classified on the basis of selective affinities for naturally occurring prostanoids. There are distinct receptors for TxA2, PGI2, PGF2a and PGD2 (namely, TP, IP, FP and DP, respectively) and four different receptors for PGE2 (EP1–4). Multiple alternatively spliced isoforms exist for the PGE2 EP3 receptor (EP3a,3b,3g ). They share common extracellular and membrane-spanning regions, but differ in intracellular and carboxy-terminal domains. Each receptor is associated with a unique G-protein and consequently a unique second messenger system, namely elevation of intracellular Caþþ (EP1) and stimulation (EP2, EP4) or inhibition (EP3) of adenylate cyclase. Despite the presence of some conserved sequences, overall homology among the prostanoid receptors is quite limited, ranging from 20% to 30%. On the other hand, the homology of a given type or subtype of receptor among various species is considerably higher. Each of the eight types and subtypes of receptors shows selective ligand-binding specificity that distinguishes it from the others. In addition to transmembrane receptors, the peroxisome proliferator-activated receptor-g (PPAR-g) is a member of the nuclear receptor family of transcription factors that can be activated by binding to PGD derivates, such as 15-deoxy-D12,14 prostaglandin J2 (15d-PGJ2).

47

In 1988, the distribution of [3H] PGE2-binding sites, presumably PGE2 receptors, was first demonstrated in the monkey diencephalon, which was followed by more detailed analysis of [3H] PGE2-binding sites in rat brain. PGE2binding sites were located in a number of discrete brain regions, including thalamic and hypothalamic nuclei, ventral hippocampus, central grey, superior colliculus, parabrachial nucleus (PB), locus coeruleus (LC), raphe nuclei, spinal trigeminal nuclei and nucleus of the solitary tract (NTS). In situ hybridization was thereafter used to determine the exact distribution of each PGE2 receptor subtype. The hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei exhibited EP1-expressing cells, but a wide distribution was found for the gene encoding the EP3 subtype. We and others have reported a very distinct pattern of EP2- and EP4-expressing neurons throughout rat brain, namely EP2 receptor mRNA was detected in the bed nucleus of the stria terminalis (BnST), lateral septum (LS), SFO, ventromedial hypothalamic nucleus (VMH), central nucleus of the amygdala (CeA), LC and AP, while EP4 receptor transcript was located mainly in regions involved in the control of neuroendocrine and autonomic activities (Zhang and Rivest, 1999, 2000, 2003). Moderate doses of LPS or IL-1b activate EP4 neurons and this activation is prevented when animals are pre-treated with COX inhibitors (Zhang and Rivest, 2000). All four PGE2 EP receptor mRNAs are expressed in the anteromedial preoptic region that plays a crucial role in the febrile response (Lazarus et al., 2007; Oka et al., 2000; Scammell et al., 1996). Among these, only EP4 receptor mRNA is strongly expressed throughout the PGE2-sensitive regions, including the OVLT, ventromedial preoptic nucleus (VmPO) and median preoptic nucleus (MnPO). These EP4-expressing neurons are also activated by systemic inflammation, whereas EP2 and EP3-positive neurons do not respond. EP1 receptor mRNA is present in PGE2-sensitive regions, but its expression level is weak. This led us to believe that EP4 may be the key binding and functional receptor for PGE2 in the brain to activate the circuits involved in the autonomic control.

Nevertheless, pharmacological and genetic mutation experiments suggest otherwise. Drugs with agonist and antagonist properties for each EP receptor were used in rats (Oka and Hori, 1994). An i.c.v. injection of 17-phenyl-o-trinorPGE2 (an EP1 and EP3a receptor agonist), but not butaprost, M&B28767, or 11-deoxy-PGE1 (EP2, EP3a and EP4 receptor agonists, respectively), induced fever, and SC19220 (an EP1 receptor antagonist) prevented the febrile response to PGE2. Another pharmacological study indicated that EP2 or EP3 receptor might be the receptors necessary to produce fever (Parrott and Vellucci, 1996). In mice bearing genetic deletions of the EP1–4 receptors, only EP3-knockout animals failed to show the early phase of fever (up to one hour) after LPS i.v. or PGE2 i.c.v. injection (Ushikubi et al., 1998). It is important to note, however, that EP4-deficient mice do not survive and have to be intercrossed in a different background. It is therefore quite difficult to compare them with the other genedeficient mice, and inducible EP4 knockout mice will be essential to clearly define the role of this receptor in the brain of mature animals. The relative lack of specificity of EP4 antagonists and agonists in the species studied may also explain the pharmacological data. It is nevertheless possible that the anatomy and circuits unravelled by functional indices of neuronal activity have little to do with the physiological outcomes, namely fever and increased activity of the HPA axis.

Functional circuits by intraparenchymal PGE2 Without such anatomical work, however, it is not possible to clarify the exact pathways and groups of neurons involved in neural–immune interaction. Indeed, the final picture is far from complete at this time. The following cascade of events is based on the data described above, but few steps still remain to be fully validated and are proposed as working hypotheses: 1. Endotoxin LPS reaches the bloodstream and binds to the serum protein LPS-binding protein (LBP). The newly formed complex then binds to

48

membrane CD14 and TLR4 receptors located on the surface of myeloid cells, triggering NF-kB signalling, nuclear translocation and cytokine gene transcription (Fig. 1). Pro-IL-1b and -TNF-a have to be cleaved by specific cytosolic enzymes before being transported into the extracellular environment. Recent data suggest a critical role of extracellular ATP and the P2X7 receptor in this final secretion process. Once in the bloodstream, both IL-1b and TNF-a can act on distant organs to trigger different physiological responses by binding to their respective type I receptors. 2. The endothelia and perivascular cells are the most obvious target cells in the CNS, because they constitutively express numerous cytokine receptors and they are highly sensitive to low circulating levels of proinflammatory molecule. Please note that circulating LPS is also a direct ligand for these cells and the release of circulating cytokines is not a prerequisite for LPS activation of the endothelial and perivascular cells lining the BBB. 3. Circulating LPS/LBP, IL-1 and TNF initiate COX-2 and mPGES transcriptions through an NF-kB and MAPK pathway, which is activated within seconds upon ligand–receptor interaction. This leads to PGE2 synthesis and release by the endothelium and pericytes of the brain capillaries. The PG may therefore diffuse across the brain parenchymal elements and bind to specific EP receptors at the surface of the neurons controlling the autonomic functions. 4. Whether one or more EP receptors participate in the neuronal response to PGE2 is still a matter of great debate, but the neuroanatomy weighs in favour of the EP4 receptor subtype. This receptor is found in key regions that control fever, HPA axis activity and other autonomic functions. EP4-expressing neurons are activated (as revealed by spontaneous induction of the immediate-early gene (IEG) c-fos) in response to centrally injected PGE2 and systemically administered IL-1 and LPS. Selective and non-selective COX inhibitors have the ability to prevent such functional activation of these neurons. EP4-expressing neurons display an integrated circuit

associated with the control of autonomic functions. The EP4 receptor is associated with Gs-protein and stimulation of cyclicAMP (cAMP), which is the most potent transduction pathway involved in the release of corticotropin-releasing factor (CRF) from the PVN and increase in HPA axis activity. There is also evidence in support for cAMP signalling in thermo-sensitive neurons of the VmPO. 5. On the other hand, PGE2 may inhibit major inhibitory neurons (e.g. g-aminobutyric acid (GABA)) projecting to the nuclei that control the autonomic circuits. This would explain the data that only EP3-deficient mice fail to exhibit a febrile response to inflammatory stimuli. This particular receptor is associated with a Gi-coupled protein inhibiting cAMP upon its activation, which may take place in GABA neurons that innervate the VmPO and PVN. Obviously, these neurons would not express c-fos or other indices of neuronal activation in response to PGE2. Functional anatomical data supporting this concept are nevertheless lacking, because we have as yet no reliable tools to assess inhibition rather than stimulation in vivo. If interaction of PGE2 with its EP3 receptor blocks the activity of GABA neurons innervating the VmPO and PVN, then the spontaneous induction of IEGs in these regions may be indirectly attributable to GABA inhibition rather than to a direct interaction of PGE2 with its EP4 receptor. This is a chickenand-egg problem that will have to be resolved with appropriate tools and animal models.

Alternative pathways The PGE2 production in the CNS may rely on two different pathways: (1) a fast transient production in endothelial cells and CVOs dependent on activation (possibly through MAPK p38 phosphorylation) of available constitutive PGE2 synthesis enzymes (cPLA2, COX-1, cPGES, mPGES-2), independent of transcription and NF-kB activation (Fig. 2), and (2) a slow sustained PGE2 synthesis in the endothelial cells and perivascular microglia (that are better

49

NTS

X

C2 A2

POA

X

X

GABA

cVLM GABA

A1

X X

X

GABA

C1 ACTH

rVLM

CRF

CORT

PGE2

cPGES COX-1 cPLA2-α

Available AA stores MAPK (p38)

TLR4 TNFR1

IL-1RI

Fig. 2. Hypothetical model for the fast EP3-dependent PGE2 response in the brain. Circulating inflammatory factors (IL-1, TNF, LPS) bind to their receptors expressed on the surface of endothelial cells of the brain microvasculature. This leads to a rapid use of available constitutive enzymes (cyclooxygenase (COX)-1, cytosolic phospholipase A2 (cPLA2), cytosolic prostaglandin E synthase (cPGES)) and arachidonic acid (AA) stores to transiently produce and release PGE2 (possibly through MAPK p38 mechanisms). This PGE2 acts on its EP3 receptor on GABAergic afferences to the PVN, which rapidly turns off the inhibition of the corticotropinreleasing factor (CRF)-releasing neurons. A similar scenario may happen at the medial preoptic area (POA), C1 catecholaminergic neurons of the rostral ventrolateral medulla (rVLM) and C2 catecholaminergic neurons of the solitary tract nucleus (NTS), where the binding of PGE2 to the EP3 receptor inhibits excitation of these GABAergic projections of PVN neurons.

equipped to respond to cytokines and pathogens) involving the robust induction of the principal enzymes involved in PGE2 synthesis (especially sPLA2-IIA, COX-2 and mPGES-1) that are NF-k B- and MAPK-mediated gene products. The slow transcription-mediated response would be massive but could offer a way through which GCs would then exert their negative feedback on NF-kB

activity to shutdown the CNS production of PGE2. The fast response could be involved in priming the later delayed response and preventing drastic changes to homeostatic processes that the delayed response would have problems overcoming. A study looking at the effects of specific COX-1 and COX-2 inhibitors supports such a role for the COX1-dependent early response, since the ablation of

50

COX-1 activity following LPS administration led to a fast hypothermic response, which could not be overcome by the later COX-2-dependent fever response. This study emphasizes the importance of the coordination between the fast and delayed responses. The population of cells containing the enzymes responsible for PGE2 synthesis during systemic inflammation is still debated. Numerous papers have provided clear evidence that both COX-2 and mPGES-1 transcripts and proteins are expressed essentially within the endothelium of the brain blood vessels. On the other hand, few studies have shown that perivascular myeloid cells are responsible to deliver PGE2 in response to systemic inflammatory stimuli. The action of this prostaglandin may therefore be dependent on its cellular source, the endogenous pool of PGE2 in endothelial cells and the receptor subtypes expressed on their cellular targets. It has been shown that EP4 is present on the post-synaptic sites of hypothalamic stimulatory neurons, whereas GABA neurons contain presynaptic EP3 receptors in their terminals. Activation of both EP3 and EP4 receptors is actually involved for the excitatory regulation of supraoptic neurons by PGE2. Taking these data into consideration, we propose that both receptors contribute to the activation of PVN neurons during systemic inflammation, but at different times. Stimulation of the cPLA2–COX-1–cPGES pathway would lead to a rapid secretion of PGE2 from endothelial cells and then cause an immediate activation of the CRF neurons through inhibition of GABA afferents that contain EP3 receptors (Fig. 2). The hypothalamic PVN receives robust innervation from GABA neurons that play a critical role in the control of the HPA axis during stress. Inhibition of GABAergic neurons by EP3 may take place locally at the level of the nerve terminals within the PVN or through distal inhibition of GABA projections originating from different areas, such as the preoptic area (POA), PB, rostral ventrolateral medulla (rVLM) and/or NTS. PGE2 synthesis under the control of the sPLA2IIA–COX-2–mPGES-1 pathway would take the relay and trigger different populations of neurons

(Fig. 3). Enzymes of this pathway are not constitutively present and have to be induced by the proinflammatory signal transduction pathways. It is also possible that both endothelial and microglial cells contribute to the PGE2 production in the delayed phase. Once secreted, PGE2 would maintain CRF activity via EP4 stimulation either directly or through activation of the A1 and A2 catecholaminergic circuits from the caudal ventrolateral medulla (cVLM) and NTS, respectively. These mechanisms fit nicely with the hypothesis of a fast and delayed PGE2 response, where the fast phase could be associated with the disinhibition of CRF-releasing neurons of the PVN, without the need for time- and energy-hungry transcription. This response could also prime the PVN to become more receptive to the massive release of PGE2 by endothelial and perivascular microglial cells with their newly transcribed PGE2-synthesizing enzymes. In support of this hypothesis is the specific increase in EP4 mRNA levels in CRF neurons and catecholaminergic afferences to PVN following a systemic immune challenge. The sustained GC release provides direct inhibitory feedback on these events at the level of transcriptional machinery, which is the most powerful endogenous immunosuppressive mechanism. This innovative dual regulation of PGE2 production and action in the brain could have important implications for an appropriate control of fever, HPA axis and other autonomic functions.

Concluding remarks More work is clearly needed to unravel the finetuning of the circuits and signal transduction pathways that participate in the neural–immune interface. There is, however, no doubt that PGE2 plays a determinant role in activating or inhibiting the key populations of neurons that are all together involved in engaging the physiological responses necessary for the organism to restore health during illness. An appropriate febrile response and a time-dependent release of glucocorticoids are major phenomena in which the brain has a direct impact on the systemic innate immune system.

51

NTS C2 A2

POA GABA

cVLM GABA

A1

GABA C1 ACTH

rVLM

CRF AVP

PGE2

CORT

Gene induction

mPGES-1 COX-2 sPLA2-IIA

NF-κB TLR4 TNFR1 IL-1RI

Fig. 3. Delayed EP4-dependent PGE2 response in the brain during systemic immune insults. The delayed PGE2 response requires transcription of the inducible enzymes (secretory phospholipase A2 (sPLA2-IIA), cyclooxygenase (COX)-2, microsomal prostaglandin E synthase (mPGES)-1)) through NF-kB and AP-1 mediated transcriptional mechanisms. These enzymes permit the massive production of PGE2 that acts on the EP4 receptors in the paraventricular nucleus (PVN) and at the adrenergic A1 region of the caudal ventrolateral medulla (cVLM) and A2 region of the nucleus of the solitary tract (NTS) to enhance corticotropin-releasing factor (CRF) and vasopressin (AVP) release into the infundibular system. This leads to the release of corticotroph adrenocorticotrophic hormone (ACTH) and subsequently biosynthesis and secretion of corticosterone (CORT) from the adrenal medulla. The latter hormone acts as negative feedback on the transcription of the enzymes responsible for the production of PGE2 and prevents overproduction of inflammatory molecules during innate immunity.

Imbalances in these two regulatory systems are actually becoming hallmarks of autoimmune diseases and neurodegenerative disorders. Clarifying the exact mechanisms and circuits that allow such fine neurophysiological outcomes will obviously be essential for designing appropriate therapeutic strategies when dysfunction occurs during the

acute-phase response. The best guess at this time leads to the key kinases controlling the proinflammatory signal transduction pathways in cells of the BBB, PGE2 biosynthetic machinery and release and specific EP receptor subtypes expressed on the surface of the neuronal populations that control endocrine and autonomic functions.

52

Acknowledgments Our work is supported by the Canadian Institutes of Health Research (CIHR) and the author holds a Canadian Research Chair (CRC) in Neuroimmunology. References Akira, S., Uematsu, S., & Takeuchi, O. (2006). Pathogen recognition and innate immunity. Cell, 124, 783–801. Blais, V., Turrin, N. P., & Rivest, S. (2005). Cyclooxygenase 2 (COX-2) inhibition increases the inflammatory response in the brain during systemic immune stimuli. Journal of Neurochemistry, 95, 1563–1574. Dantzer, R., O’Connor, J. C., Freund, G. G., Johnson, R. W., & Kelley, K. W. (2008). From inflammation to sickness and depression: When the immune system subjugates the brain. Nature Reviews Neuroscience, 9, 46–56. Ek, M., Engblom, D., Saha, S., Blomqvist, A., Jakobsson, P. J., & Ericsson-Dahlstrand, A. (2001). Inflammatory response: Pathway across the blood–brain barrier. Nature, 410, 430– 431. Engblom, D., Saha, S., Engstrom, L., Westman, M., Audoly, L. P., Jakobsson, P. J., et al. (2003). Microsomal prostaglandin E synthase-1 is the central switch during immuneinduced pyresis. Nature Neuroscience, 6, 1137–1138. Ericsson, A., Liu, C., Hart, R. P., & Sawchenko, P. E. (1995). Type-1 interleukin-1 receptor in the rat brain: Distribution, regulation, and relationship to sites of IL-1-induced cellular activation. The Journal of Comparative Neurology, 361, 681– 698. Lacroix, S. & Rivest, S. (1996). Role of cyclo-oxygenase pathways in the stimulatory influence of immune challenge in the transcription of a specific CRF receptor subtype in the rat brain. Journal of Chemical Neuroanatomy, 10, 53–71. Lacroix, S. & Rivest, S. (1998). Effect of acute systemic inflammatory response and cytokines on the transcription of the genes encoding cyclooxygenase enzymes (COX-1 and COX-2) in the rat brain. Journal of Neurochemistry, 70, 452–466. Laflamme, N., Lacroix, S., & Rivest, S. (1999). An essential role of interleukin-1b in mediating NF-kB activity and COX2 transcription in cells of the blood–brain barrier in response to systemic and localized inflammation, but not during endotoxemia. Journal of Neuroscience, 19, 10923–10930. Lazarus, M., Yoshida, K., Coppari, R., Bass, C. E., Mochizuki, T., Lowell, B. B., et al. (2007). EP3 prostaglandin receptors in the median preoptic nucleus are critical for fever responses. Nature Neuroscience, 10, 1131–1133. Nadeau, S. & Rivest, S. (1999). Effects of circulating tumor necrosis factor (TNF) on the neuronal activity and expression of the genes encoding the TNF receptors (p55 and p75) in the rat brain: A view from the blood–brain barrier. Neuroscience, 93, 1449–1464.

Narumiya, S., Sugimoto, Y., & Ushikubi, F. (1999). Prostanoid receptors: Structures, properties, and functions. Physiology Review, 79, 1193–1226. Oka, T. & Hori, T. (1994). EP1-receptor mediation of prostaglandin E2-induced hyperthermia in rats. American Journal of Physiology, 267, R289–R294. Oka, T., Oka, K., Scammell, T. E., Lee, C., Kelly, J. F., Nantel, F., et al. (2000). Relationship of EP(1-4) prostaglandin receptors with rat hypothalamic cell groups involved in lipopolysaccharide fever responses. The Journal of Comparative Neurology, 428, 20–32. Parrott, R. F. & Vellucci, S. V. (1996). Effects of centrally administered prostaglandin EP receptor agonists on febrile and adrenocortical responses in the prepubertal pig. Brain Research Bulletin, 41, 97–103. Rivest, S. (2009). Regulation of innate immune responses in the brain. Nature Reviews Immunology, 9, 429–439. Rivest, S., Lacroix, S., Vallières, L., Nadeau, S., Zhang, J., & Laflamme, N. (2000). How the blood talks to the brain parenchyma and the paraventricular nucleus of the hypothalamus during systemic inflammatory and infectious stimuli. Proceedings of Society Experimental Biology Medicine, 223, 22–38. Saha, S., Engstrom, L., Mackerlova, L., Jakobsson, P. J., & Blomqvist, A. (2005). Impaired febrile responses to immune challenge in mice deficient in microsomal prostaglandin e synthase-1. American Journal of Physiology Regulatory Integrative and Comparative Physiology, 288, R1100–R1107. Scammell, T. E., Elmquist, J. K., Griffin, J. D., & Saper, C. B. (1996). Ventromedial preoptic prostaglandin E2 activates fever-producing autonomic pathways. Journal of Neuroscience, 16, 6246–6254. Ushikubi, F., Segi, E., Sugimoto, Y., Murata, T., Matsuoka, T., Kobayashi, T., et al. (1998). Impaired febrile response in mice lacking the prostaglandin E receptor subtype EP3. Nature, 395, 281–284. Vallières, L., Lacroix, S., & Rivest, S. (1997). Influence of interleukin-6 on neural activity and transcription of the gene encoding corticotropin-releasing factor in the rat brain: An effect depending upon the route of administration. European Journal of Neuroscience, 9, 1461–1472. Vallières, L. & Rivest, S. (1997). Regulation of the genes encoding interleukin-6, its receptor, and gp130 in the rat brain in response to the immune activator lipopolysaccharide and the proinflammatory cytokine interleukin-1b. Journal of Neurochemistry, 69, 1668–1683. Vallières, L. & Rivest, S. (1999). Interleukin-6 is a needed proinflammatory cytokine in the prolonged neural activity and transcriptional activation of corticotropin-releasing factor during endotoxemia. Endocrinology, 140, 3890– 3903. Zhang, Y. H., Lu, J., Elmquist, J. K., & Saper, C. B. (2000). Lipopolysaccharide activates specific populations of hypothalamic and brainstem neurons that project to the spinal cord. Journal of Neuroscience, 20, 6578–6586. Zhang, Y. H., Lu, J., Elmquist, J. K., & Saper, C. B. (2003). Specific roles of cyclooxygenase-1 and cyclooxygenase-2

53 in lipopolysaccharide-induced fever and fos expression in rat brain. The Journal of Comparative Neurology, 463, 3–12. Zhang, J. & Rivest, S. (1999). Distribution, regulation and colocalization of the genes encoding the EP2- and EP4PGE2 receptors in the rat brain and neuronal reponses to systemic inflammation. European Journal of Neuroscience, 11, 2651–2668.

Zhang, J. & Rivest, S. (2000). A functional analysis of EP4 receptor-expressing neurons in mediating the action of PGE2 within specific nuclei of the brain in response to circulating interleukin-1b. Journal of Neurochemistry, 74, 2134–2145. Zhang, J. & Rivest, S. (2003). Is survival possible without arachidonate metabolites in the brain during systemic infection? News Physiology Science, 18, 137–142.

L. Martini (Eds.) Progress in Brain Research, Vol. 181 ISSN: 0079-6123 Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 5

Physiological roles of the kisspeptin/GPR54 system in the neuroendocrine control of reproduction Rafael Pineda, Enrique Aguilar, Leonor Pinilla and Manuel Tena-Sempere
Department of Cell Biology, Physiology and Immunology, University of Córdoba, Córdoba, Spain

Abstract: Reproductive maturation and function are maintained by a complex neurohormonal network that integrates at the so-called hypothalamic–pituitary–gonadal (HPG) axis. This system is hierarchically controlled by the decapeptide, GnRH, which in turn is under the dynamic regulation of multiple stimulatory and inhibitory pathways, including peripheral signals (prominently, sex steroids) and different central modulators. Among the latter, considerable interest has been raised recently by the identification of the major roles and mechanisms of action of kisspeptins, a family of neuropeptides encoded by the Kiss1 gene, which acting via the G protein-coupled receptor, GPR54, have been shown to play essential functions as potent activators and major gatekeepers of the HPG axis. Indeed, kisspeptin neurons, whose mere existence and neuroendocrine dimension had escaped from general attention up to five years ago, have been now universally recognized as key players in the control of critical aspects of reproductive development and function, from sexual differentiation to regulation of GnRH/gonadotropin secretion and the metabolic gating of fertility. In this chapter, we will provide a concise summary of the state of the art in this rapidly evolving area of neuroendocrinology, with special emphasis on recent developments and contentious issues that are likely to attract considerable attention in the coming years. Keywords: kisspeptins; Kiss1; GPR54; Kiss1R; GnRH; gonadotropins; puberty; ovulation; feedback; sex steroids

regulatory cascade, the major hierarchical element is a neuronal population, mostly located at the preoptic area of the hypothalamus, that secretes the decapeptide, gonadotropin-releasing hormone (GnRH) (Herbison, 2006; Schwartz, 2000), which in turn activates the secretion of pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and thereby stimulates gonadal maturation and functions. As such, GnRH neurons are considered as major integrators and common output pathway for a wide diversity of

Introduction The neuroendocrine control of reproduction relies on the concerted action of different neural and hormonal signals that originate and/or impinge at different levels of the so-called hypothalamic– pituitary–gonadal (HPG) axis. Within this


Corresponding author. Tel.: þ34-957-218281; Fax: þ34-957-218288; E-mail: [email protected]

DOI: 10.1016/S0079-6123(08)81005-9

55

56

regulators of reproductive function, which influence downstream elements of the HPG axis by virtue of their ability to modulate the secretion of GnRH (Herbison, 2006). Indeed, many neuropeptides, originating either from other hypothalamic nuclei or brain areas, are involved in the maturation and fine-tuning of GnRH neurons (Herbison, 2006). Similarly, different peripheral signals participate in the dynamic regulation of GnRH release. Among these, gonadal sex steroids (and peptides) feedback in negative (or positive) fashion to the hypothalamic–pituitary levels to keep the function of the HPG axis in balance (Schwartz, 2000; Tena-Sempere and Huhtaniemi, 2003). While the major neurohormonal elements of the HPG system have been well settled for decades, studies conducted mostly during the past years have allowed to expose a considerable degree of complexity of the hypothalamic (brain) pathways responsible for the central control of reproductive function. Such a complexity is reflected by the existence of multiple neuronal afferents to GnRH neurons, with either stimulatory (e.g. glutamate, norepinephrine) or inhibitory [e.g. g-aminobutyric acid (GABA), endogenous opioids] actions, although some of the latter (e.g. GABA) can also stimulate GnRH neurons under some conditions. Similarly, it has been recognized that GnRH neurons are also under the control of signals originating from glial cells, such as neurotrophic factors and glutamate. Altogether, these central neuronal and glial pathways are fundamental for the dynamic regulation of pulsatile GnRH secretion (Herbison, 2006; Ojeda et al., 2008), thereby providing the central drive for the function of the reproductive axis in different physiological states. As yet, the nature and mechanisms of action of such a network of regulatory signals remain to be fully characterized. In this context, we have witnessed in the past five years the emergence of a novel neuronal circuitry, involving a family of peptides termed kisspeptins – as products of the Kiss1 gene – whose recognition as indispensable elements in the central control of the HPG axis has forced us to redefine our current understanding of reproductive neuroendocrinology (Oakley et al., 2009; Roa et al., 2008a). The most salient

features of this novel system are comprehensively summarized in the following sections. Yet, due to the aims and scope of this chapter, especial emphasis will be made to identify and eventually address hot areas and contentious issues in the field, as they are likely to become the subject of active investigation from year 2010 onwards.

Kisspeptins and GPR54: discovery and proposed nomenclature Kisspeptins are a family of structurally related peptides, encoded by the KISS1/Kiss1 gene, that operate via binging and subsequent activation of the G protein-coupled receptor, GPR54 (Oakley et al., 2009). The elements (genes and peptides) of this ligand–receptor system were sequentially identified between 1996 and 2001, and their identification took place in the field of cancer biology (an area apparently unrelated to neuroendocrinology) (Oakley et al., 2009; Roa et al., 2008a). The first element of the system to be characterized was the KISS1 gene, whose transcript was found (in 1996) to be selectively repressed in melanoma cell lines with high metastatic capacity (Lee et al., 1996). Three years later, GPR54 was cloned in rat brain, originally as an orphan receptor (therefore, not related to Kiss1), by virtue of its partial homology with the transmembrane regions of galanin receptors (Lee et al., 1999). However, it was not until 2001, when the major peptide products of the Kiss1 gene were characterized as a family of structurally related peptides, globally termed kisspeptins, arising from the differential proteolytic processing of a common precursor (see Fig. 1). In fact, the major peptide product of the family, of 54-amino-acid length in humans (i.e. kisspeptin-54), was termed metastin due to its ability to suppress tumour invasion (Ohtaki et al., 2001). In the same year, it was demonstrated that the 10-amino-acid stretch at C terminus (also termed kisspeptin-10 or Kp-10) was sufficient for receptor activation, and that all known kisspeptins can bind to GPR54, which was then defined as the cognate receptor of Kiss1-derived peptides (Kotani et al., 2001). At that time, however, the

57 Prepro-Kisspeptin 1

NH2-

19 20

65 68

121 124

Kisspeptin-54

signal

145

- COOH

Kp-54 Kp-14

Kisspeptins Kp-13 Kp-10

Fig. 1. Structure of kisspeptins – the peptide products of the KISS1 gene. Different kisspeptins are generated by proteolytic cleavage from a common precursor of 145 amino acids, prepro-kisspeptin, which contains a 19-amino-acid signal peptide and a central 54-amino-acid region, kisspeptin-54 (Kp-54; formerly termed metastin). Lower-molecular-weight forms of kisspeptins include Kp-14, Kp-13 and Kp-10; the latter corresponds to the common C-terminal 10-amino-acid stretch that contains the RFamide motif and is sufficient to activate GPR54. Adapted from Roa et al. (2008a), with substantial modifications.

known biological functions of kisspeptins were virtually restricted to their ability to constrain tumour spread, thus being catalogued as metastasis-suppressor factors. Although the elements of the Kiss1 system were all well characterized by 2001, it was not until the end of 2003 when the reproductive dimension of the system was disclosed. This unsuspected facet of kisspeptins was exposed by the striking observation that some forms of idiopathic hypogonadotropic hypogonadism were associated with inactivating mutations of GPR54 gene (de Roux et al., 2003; Seminara et al., 2003). This hypogonadal phenotype of central origin was also observed in mouse models where the GPR54 gene had been knocked out (Funes et al., 2003; Seminara et al., 2003), findings that have been later replicated in Kiss1 null mice (d’Anglemont de Tassigny et al., 2007; Lapatto et al., 2007). Altogether, these clinical and experimental observations set the ground for specific analyses on the physiological roles, major regulatory mechanisms and modes of action of kisspeptins in the neuroendocrine control of the HPG axis. Indeed, research activities along these lines have increase exponentially over the past five years, and an ever-growing number of publications have been produced recently in the field (e.g. >160 papers added to PubMed during 2009). This considerable research effort has allowed a very rapid expansion of our knowledge of the neuroanatomy, physiology, pharmacology and pathophysiology of this ‘novel’ neuroendocrine system.

Although placental mammals have been shown to have a single Kiss1 gene in their genomes, recent studies in fish and other non-mammalian species have allowed the identification of a second Kiss-like gene, named Kiss2 (Felip et al., 2009). This gene encodes a decapeptide with substantial divergence from mammalian Kp-10 (encoded by Kiss1). Yet, in species such as the teleost sea bass, this Kiss2-derived decapeptide was found to be more potent than Kp-10 in eliciting gonadotropin secretion (Felip et al., 2009). In addition, in contrast to mammals, molecular variability at the GPR54 (due to alternative splicing or gene duplication) has been described in non-mammalian species (Mechaly et al., 2010; Mechaly et al., 2009b), phenomenon whose functional relevance is yet to be fully characterized. A comprehensive overview of some evolutionary aspects of the kisspeptin/GPR54 system has been recently provided and can be found elsewhere (Lee et al., 2009). Given the focus of the present chapter, we will concentrate our attention on Kiss1 (and their peptide products), as representative of the mammalian system. As it is usually the case with rapidly evolving areas of biology, some differences/inconsistencies in the nomenclature of the elements of the kisspeptin system have been detected over the past years. For this chapter, we will partially adopt the recent proposal for nomenclature by Gottsch and colleagues (Gottsch et al., 2009a), where KISS1 and Kiss1 are used to name the human and nonhuman genes, respectively. In addition, the peptide

58

products of KISS1/Kiss1 will be globally termed kisspeptins (Kp), with a numeric extension to indicate the amino acid length (e.g. Kp-10), if relevant. However, in contrast to the above proposal, to keep the homogeneity with previous literature, the kisspeptin receptor will be termed GPR54, although we acknowledge that the use of Kiss1 receptor (Kiss1R) is fully valid as well. Finally, Kiss1 or kisspeptin will be used for global reference to the ligand–receptor system, when no given species is alluded to.

Neuroanatomy of kisspeptin neurons in mammals Before the reproductive dimension of this system was disclosed, characterization of the neuroanatomical distribution of Kiss1/kisspeptin and GPR54 expression had been initiated. This, however, was mostly based on reverse transcriptase–polymerase chain reaction (RT-PCR) assays upon fragments of different brain regions or immunohistochemical analyses with antibodies whose specificity had not been thoroughly characterized. Yet, such studies allowed to set the contention that the elements of this system (as mRNA and/or protein) are expressed in different areas of the central nervous system (CNS). Thus, KISS1 mRNA was detected in the human brain with a scattered distribution, including prominent signals at the basal ganglia and the hypothalamus. Similarly, GPR54 gene expression was also found in the spinal cord and different brain areas, such as basal ganglia, amygdala, substantia nigra, hippocampus and hypothalamus (Muir et al., 2001; Ohtaki et al., 2001). In addition, one study documented the presence of kisspeptin-like immunoreactivity (IR) in a wide diversity of brain regions, with the strongest signals at the hypothalamic dorsomedial (DMN), ventromedial (VMN) and arcuate (ARC) nuclei, as well as at the nucleus of the solitary tract and caudal ventrolateral medulla (Brailoiu et al., 2005). Finally, fibers with clear-cut kisspeptin-IR were described in different areas of the telencephalon, diencephalon, mesencephalon, metencephalon and myelencephalon (Brailoiu et al., 2005). As general

call of caution, however, results from this study would need replication, given the uncertainties about the specificity of the kisspeptin antibody used, since it may react with other RFamide peptides, such as RFRP1 and RFRP3 (Mikkelsen and Simonneaux, 2009). The advent of the ‘reproductive’ era of kisspeptins raised considerable interest on the detailed characterization of their patterns of distribution within the hypothalamus. Indeed, in mammals, the presence of a population of kisspeptin neurons at the ARC (or the equivalent infundibular region) has been unambiguously documented in the mouse, rat, sheep, horse, pig, monkey and human, as defined by expression of Kiss1 mRNA and/or kisspeptin-IR (Mikkelsen and Simonneaux, 2009). In addition, detailed analyses conducted in rodents have allowed identifying Kiss1/kisspeptinþve neurons at other hypothalamic sites such as the anteroventral periventricular nucleus (AVPV) and, more faintly, at the DMN (Clarkson et al., 2009b; Mikkelsen and Simonneaux, 2009). On the former, elegant neuroanatomical studies in mice have suggested that rather than being restricted to the AVPV, kisspeptin neurons at this site constitute a continuum also involving the contiguous periventricular preoptic area, the so-called rostral periventricular area of the third ventricle (RP3V) (Clarkson et al., 2009b). However, the ARC and AVPV populations of kisspeptin neurons appear to display important anatomical differences in terms of projections to GnRH neurons, as direct appositions have been reported only from the AVPV (Clarkson and Herbison, 2006). Yet, it is plausible that Kiss1 neurons from the ARC may interact with elements of the pre-synaptic network controlling GnRH neurons, if not with GnRH neurons themselves. In addition, it has to be noted that the magnitude (and even the existence) of this AVPV population is highly dependent on the species, as it seems to be absent in the sheep, horse and primates, but present in pig (Decourt et al., 2008; Mikkelsen and Simonneaux, 2009; Tomikawa et al., 2010). On the other hand, the presence of a population of kisspeptin neurons at the DMN remains contentious, as Kiss1 RNA expression has not been detected at this area, and the abundance of RFRPs in this nucleus might have yielded

59

false-positives in some immunohistochemical approaches (Mikkelsen and Simonneaux, 2009). The functional significance of these different populations of Kiss1 neurons will be discussed in the section ‘Kisspeptin neurons: roles in feedback control of gonadotropins’. In contrast to the detailed characterization of the patterns of Kiss1/kisspeptin expression at the hypothalamus, the actual distribution of GPR54þve cells in the brain has remained poorly defined for years. This might be due, at least partially, to the lack of reliable antisera against GPR54. Anyhow, initial in situ hybridization data demonstrated expression of this transcript in the hypothalamus, and specifically in GnRH neurons (Irwig et al., 2004), thereby providing a discernible pathway for the direct actions of kisspeptins on GnRH secretion (see the section ‘Kisspeptins and the control of gonadotropin secretion: roles and mechanisms’). Notably, in the final stage of preparation of this chapter, a study using a transgenic GPR54 LacZ knock-in mouse line has been published which confirms the expression of kisspeptin receptors in GnRH neurons at the rostral preoptic area (rPOA), and provides the first reliable map of GPR54þve neurons in different brain areas, with the highest density at the dentate gyrus of the hippocampus (Herbison et al., 2010). It remains intriguing what would be the functional relevance of GPR54 expression at brain areas other than the hypothalamus, such as the hippocampus, where kisspeptins have been shown to modulate synaptic transmission in experimental settings (Arai et al., 2005).

Kisspeptins and the control of gonadotropin secretion: roles and mechanisms Upon the disclosure of the reproductive facet of kisspeptins and GPR54, an explosive research activity commenced worldwide aiming to define their actual physiological roles and mechanisms of action in the neuroendocrine control of the HPG axis. Indeed, initial pharmacological studies conducted between 2004 and 2005 demonstrated that kisspeptins are very potent stimulators of LH

and, to a lesser extent, FSH release in different species, such as the rat, mouse, sheep, monkey and human (Dhillo et al., 2005; Gottsch et al., 2004; Messager et al., 2005; Navarro et al., 2004a, 2005a, 2005b; Shahab et al., 2005). These stimulatory effects have been later confirmed in other mammalian and non-mammalian species (Roa et al., 2008a). Of note, detailed pharmacological studies in rodents have evidenced some differences between the patterns of LH and FSH responses to kisspeptins, such as their relative magnitude (higher for LH) and time course (faster for LH), as well as in the threshold and mean effective doses (two orders of magnitude lower for LH) (Navarro et al., 2005a, 2005b; Tovar et al., 2006); for illustration, see Fig. 2. Notwithstanding, based on comparative analyses with other well-known stimulators of the HPG axis, it is now universally accepted that kisspeptins are among the most potent elicitors of the GnRH/gonadotropin system (Roa et al., 2008a). There is general consensus that the primary site of action of kisspeptins for the above effects on gonadotropin secretion is the hypothalamus, where they ultimately lead to the stimulation of GnRH secretion (Oakley et al., 2009; Roa et al., 2008a). Evidence for the expression of GPR54 in GnRH neurons has been obtained by in situ hybridization analyses in rodents (Irwig et al., 2004) and more recently from expression and functional studies using human and murine GnRH cell lines (Jacobi et al., 2007; Novaira et al., 2009; Quaynor et al., 2007), thus providing the basis for direct effects of kisspeptins on this neuronal population. Yet, their tumourogenic nature and the lack of replication in some studies force us to consider data from GnRH cell lines (such as GT1-7) with caution. Notwithstanding, the obligatory role of GnRH for the stimulatory effects of kisspeptins has been demonstrated in models of genetic inactivation (i.e. hpg mouse) or pharmacological blockade of the decapeptide (Roa et al., 2009a). In good agreement, kisspeptins have been shown to elicit, in a dosedependent manner, GnRH secretion in different species, both in vivo and by hypothalamic explants in vitro (Castellano et al., 2005; Roa et al., 2009a). The signalling cascade activated by kisspeptin to stimulate GnRH secretion appears to involve the

60 (a) LH 14 (ng/mL) 12

FSH 24 (ng/mL) 22

10

20

8

18

6

16

4

14

2

12

Vehicle

100 fmol

1 pmol

10 pmol

100 pmol

1 nmol

10 nmol

0-min

30-min

60-min

90 min

120-min

1 50.-min

180-min

10 nmol

1 nmol

100 pmol

10 pmol

1 pmol

100 fmol

Vehicle

Dose-Response (Kp-10) (b) LH 14 (ng/mL) 12

FSH 24 (ng/mL) 22

10

20 18 16 14 12

8 6 4 2

180-min

1 50.-min

120-min

90 min

60-min

30-min

0-min

Time-Course (Kp-10; 1 nmol) Fig. 2. Prototypical gonadotropin responses to kisspeptin stimulation in rats. Schematic illustrations are presented of the patterns of LH and FSH responses to intra-cerebral administration of Kp-10. Both dose-dependent (panel A) and time-dependent (panel B) gonadotropic responses are shown. Hormonal values are adapted from original data from Navarro et al. (2005a, 2005b) and our unpublished observations.

activation of phospholipase-C (PLC), but not of adenylate cyclase, the mobilization of intra-cellular Ca2þ stores and the recruitment of ERK1/2 and p38 kinases, as evidenced by secretory analyses using hypothalamic explants ex vivo (Castano et al., 2009; Castellano et al., 2006b; Roa et al., 2009a). This has been recently confirmed by electrophysiological recordings and calcium imaging in GnRH neurons, which documented that kisspeptin excitation of GnRH neurons is conveyed through a PLC-/ calcium-dependent pathway regulating multiple ion channels, including potassium and transient receptor potential (TRP) channels (Liu et al., 2008; Zhang et al., 2008). Although the capacity of kisspeptins to ultimately activate GnRH neurons, as evidenced by c-fos induction and action potential firing, is undisputed (Oakley et al., 2009), some debate persists

on how and where specifically this action is conducted. Thus, direct actions of kisspeptins are likely to take place not only at neuronal perikarya, but also at GnRH nerve terminals located at the median eminence, as indirectly suggested by the rapid stimulatory actions of systemically delivered kisspeptins (Tovar et al., 2006), and recently demonstrated by the use of mediobasal hypothalamic fragments from wt and GPR54 KO mice (d’Anglemont de Tassigny et al., 2008). In addition, electrophysiological studies have evidenced that, besides these direct effects, some of the actions of kisspeptins may be conducted transsynaptically, that is, via modulation of other prominent afferents to GnRH neurons, such as glutamate and GABA pathways (PieleckaFortuna and Moenter, 2010). The relative physiological relevance of the different modes/sites

61

of action described above (indirect vs. direct; cell bodies vs. nerve terminals) is yet to be characterized. Finally, whereas there is general agreement that the hypothalamus is the primary site for their stimulatory effects on gonadotropin secretion, the possibility of additional actions of kisspeptins directly at the pituitary level has been put forward, although results in this front remain contentious (Roa et al., 2008a). In this sense, several studies have documented the ability of kisspeptins to elicit LH release by pituitary tissue in vitro, at least in the rat, bovine and ovine species (Gutierrez-Pascual et al., 2007; Navarro et al., 2005b; Smith et al., 2007; Suzuki et al., 2008), whereas kisspeptins have been detected in hypophysial portal blood in the sheep (Smith et al., 2007), and immunohistochemical evidence suggestive of potential direct pituitary effects has been recently produced in the monkey (Ramaswamy et al., 2009). In addition, expression of Kiss1 and GPR54 mRNA has been documented in the rat and sheep pituitary, under the control of GnRH and/or sex steroids (Bellingham et al., 2009; Richard et al., 2008), features that are indicative of a functional role at the pituitary level. Admittedly, however, few other studies have been unable to detect direct pituitary actions of kisspeptins in rats (Matsui et al., 2004; Thompson et al., 2004), and the physiological relevance of such direct effects, as per induction of the pre-ovulatory surge of gonadotropins, has been questioned in sheep (Smith et al., 2007). In this scenario, further investigation is warranted as to elucidate the actual roles and eventual mechanisms of action of kisspeptins at the pituitary level. Of note, fragmentary evidence suggests that such pituitary actions of kisspeptins might also involve the modulation of other neuroendocrine axes, such as the somathotrope system (GutierrezPascual et al., 2007). Kisspeptin neurons: roles in sexual differentiation and puberty onset Considering the prominent effects of kisspeptins on GnRH/gonadotropin secretion, and the discernible neuroendocrine pathways involving

Kiss1 neurons, specific analyses addressing their potential implication in some key aspects of sexual maturation have been undertaken in recent years. These have targeted both the process of brain sexual differentiation as well as puberty onset. Concerning sex differentiation, studies in adult rodents have demonstrated that the AVPV population of Kiss1 neurons is sexually dimorphic (i.e. larger in females at adulthood) (Kauffman et al., 2007). Such dimorphism is likely due to the organizing effects of sex steroids during the critical (neonatal) period of sexual maturation of the brain (Kauffman et al., 2007). This is evidenced by studies involving neonatal exposure to high doses of androgen in genetic female rats, which resulted in a dramatic decrease of Kiss1 mRNA expression at AVPV in adulthood, that is, ‘masculinized’ pattern of expression (Kauffman et al., 2007). Similarly, analyses in genetic male rats submitted to neonatal gonadectomy (GNX) demonstrated increased expression of Kiss1 at the AVPV in adulthood, that is, ‘feminized’ pattern of expression (Homma et al., 2009). Importantly, these changes in AVPV Kiss1 neurons are tightly correlated with functional modifications in the capacity to display an essential sexually differentiated trait, that is, the ability of estrogen to elicit positive feedback in terms of gonadotropin secretion (see the section ‘Kisspeptin neurons: roles in feedback control of gonadotropins’), which is detectable in adult genetic females and neonatally GNX males, but absent in genetic males and neonatally androgenized female rats (Homma et al., 2009). The sensitivity of the developing Kiss1 system to the organizing effects of sex steroids is further documented by observations in afetoprotein (AFP) KO mice, where the congenital lack of this scavenger protein of circulating estrogens results in excessive estrogenic input during early development. In this model, sexual differentiation and adult function of Kiss1 system is severely disrupted (Gonzalez-Martinez et al., 2008). In the same vein, we have documented that neonatal exposures to synthetic estrogens, known to disturb proper activation and function of the gonadotropic axis (Tena-Sempere et al., 2000), persistently suppressed the hypothalamic

62

expression of Kiss1 gene at the expected time of puberty and adulthood in the rat (Navarro et al. 2004a, 2009b). In addition to their role in brain sexual differentiation, the initial findings that congenital absence of functional GPR54 or kisspeptins in humans and/or mice is associated with lack of puberty onset and variable degrees of sexual immaturity (d’Anglemont de Tassigny et al., 2007; de Roux et al., 2003; Lapatto et al., 2007; Seminara et al., 2003) fueled specific analyses on the putative involvement of this system in the timing of puberty, as these original observations did not allow the dissection of the underlying mechanisms. Studies conducted mostly in laboratory rodents over the past few years have exposed some key aspects of the complex pattern of developmental activation of Kiss1 neurons along pubertal maturation, which is likely to serve as essential element in the central networks controlling the pubertal activation of the HPG axis. This phenomenon appears to include not only an increase in the endogenous kisspeptin tone, which appears to be sufficient to drive the gonadotropic axis to a state of full (pubertal) activity (Navarro et al., 2004a, 2004b; Plant et al., 2006; Shahab et al., 2005), but also a rise in the efficiency of GPR54 signalling and in the sensitivity to the stimulatory effects of kisspeptin in terms of GnRH/LH responses (Castellano et al., 2006b; Han et al., 2005), that seems to be coupled to state of resistance to desensitization to kisspeptin stimulation. Finally, pubertal changes of the hypothalamic Kiss1 system also involve dramatic plastic modifications in the number of kisspeptin neurons (i.e. at the ARC and AVPV) and their projections to GnRH neurons from specific hypothalamic areas (i.e. AVPV), both of which seem to significantly increase along the pubertal transition (Clarkson and Herbison, 2006). Indirect evidence strongly suggests that such a concerted and multi-faceted pattern of maturation of the kisspeptin system is functionally relevant for the precise timing of puberty, as the gonadotropic axis acquires maximal responsiveness to kisspeptin activation at much earlier stages of postnatal development (e.g. neonatal/ infantile period) (Castellano et al., 2006b;

Nazian, 2006). This would imply that inappropriate (precocious) activation of this system may lead to earlier puberty, as suggested by pharmacological studies in rats (Castellano et al., 2006b; Navarro et al., 2004b). In the same vein, recent studies using a potent antagonist have documented that blockade of brain kisspeptin signalling results in disruption of puberty onset in female rats (Pineda et al., 2010). Despite the general consensus that kisspeptins are essential gatekeepers of puberty onset, there are key aspects of this phenomenon that are yet to be elucidated. First, assuming the consistent activation of kisspeptin expression and signalling along puberty, the factors responsible for such a developmental switch remain ill defined. In addition, the actual nature of kisspeptin function, either as trigger or positive amplifier of the rise in the neurosecretory activity of GnRH neurons at the time of puberty, awaits to be defined. In this context, recent experimental work in female mice evidenced that the increase in kisspeptin expression at the hypothalamus at the time of puberty requires some degree of estrogenic input from the ovary (Clarkson et al., 2009a). Thus, conditions of low or null estrogen levels, such as GNX or inactivation of aromatase gene, resulted in a dramatic decrease in kisspeptin content at the AVPV/RP3V in pubertal females (Clarkson et al., 2009a). These observations led to the appealing hypothesis that a positive feedback loop between ovarian estrogen and the hypothalamus, involving Kiss1 neurons at the AVPV, may exist before puberty, as it has been thoroughly documented in adult females (see the section ‘Kisspeptin neurons: roles in feedback control of gonadotropins’). Given that ovarian secretion of sex steroid is dependent on circulating gonadotropins, this hypothesis would imply that, rather than genuine triggers, kisspeptin neurons would operate as estrogen-dependent amplifiers of GnRH neuronal activity in the pre-pubertal period (Clarkson et al., 2009a), a model tentatively depicted in Fig. 3. Yet, it remains to be defined which level of estrogenic input is required for the pubertal activation of the Kiss1 system at the AVPV, and whether kisspeptin neurons at other hypothalamic nuclei, such as the ARC, do play

63

AVPV

+ Kiss-independent inputs

↑↑↑ Kiss1-GnRH Appositions ↑ GPR54 Expression

Kiss1

GnRH

↑ Kiss1 tone

Kiss1

+

AVPV

GnRH ↑↑Responsiveness

Hypothalamus

+

+

+

+

Pituitary

GnRH

GnRH

Pubertal transition

+

LH FSH

+ LH E2

FSH

E2

Ovary

Fig. 3. Kiss1 system and the neuroendocrine control of puberty onset. A tentative model for the maturational and functional changes of the Kiss1 system putatively involved in the onset of puberty is presented. Enhancement of kisspeptin tone and Kiss1 neuron projections to GnRH neurons is observed along puberty, a phenomenon that appears to require the permissive/driving action of ovarian-derived estrogens, whose secretion should be initially triggered in a kisspeptin-independent manner. In addition, an increase in the sensitivity to and signalling efficiency of kisspeptins takes place at the pubertal transition. The potential roles of Kiss1 neurons at the ARC in the timing/control of male and female puberty are less well characterized, and have not been schematized in the figure. For further details, see the section ‘Kisspeptin neurons: roles in sexual differentiation and puberty onset’ and references therein.

any discernible role in the timing of puberty. On the latter, it is remarkable that the ARC population of Kiss1 neurons is clearly predominant in male rodents, as well as in other species, such as sheep and primates, regardless of sex. Finally, in addition to sex steroids, other regulators of the hypothalamic Kiss1 system at puberty are likely to exist. These may involve peripheral hormones (e.g. leptin, ghrelin, insulin, insulin-like growth factor (IGF)) and central neuropeptides (e.g. Neuropeptide Y (NPY), melanocortins), as described in the sections ‘Metabolic regulation of the kisspeptin system’ and ‘Other regulators of the kisspeptin system: neuropeptides, hormones and environmental

cues’, although their specific roles, if any, during the pubertal transition remain virtually unexplored. Kisspeptin neurons: roles in feedback control of gonadotropins Upon disclosure of its reproductive dimension, one of the features of the Kiss1 system that was first recognized was the ability of sex steroids to regulate Kiss1 gene expression at the hypothalamus. Thus, expression analyses in hypothalamic fragments from GNX rats demonstrated a consistent elevation of Kiss1 mRNA levels in both males and females, which were tightly paralleled by

64

concomitant changes in circulating gonadotropins due to the removal of the negative feedback of gonadal steroids (Navarro et al., 2004a). Indeed, sex steroid replacement fully reversed these responses, as testosterone supplementation in males and estrogen administration in females totally prevented the increase in Kiss1 mRNA and serum gonadotropin levels following GNX (Navarro et al., 2004a). These initial observations were later refined by in situ hybridization analyses in rodents, which documented a significant increase in Kiss1 mRNA expression after GNX at the ARC (Smith et al., 2005a, 2005b), an area classically recognized as central for the negative feedback of sex steroids. Moreover, the rise in Kiss1 mRNA levels at the ARC following GNX was prevented by sex steroid replacement (Smith et al., 2005a, 2005b). Analogous observations have been made in other species, such as the sheep, monkey and human (Oakley et al., 2009; Roa et al., 2008a), for the latter, in a condition of gonadal insufficiency, such as the menopause (Rometo et al., 2007). Altogether, these findings suggested the involvement of Kiss1 neurons at the ARC in mediating the negative feedback effects of sex steroids on gonadotropin secretion. This contention is supported by the observation that such a negative feedback regulation is apparently absent in mice lacking functional GPR54 signalling (Dungan et al., 2007). Neuroanatomical analyses also demonstrated that, in contrast to the ARC, Kiss1 mRNA levels at the AVPV decreased after GNX and increased after sex steroid replacement (Smith et al., 2005a, 2005b). These seminal observations revealed a diametrically opposite behavior of kisspeptin neurons at this site, and immediately raised the possibility of their involvement in mediating the positive feedback actions of estrogen, as driving force for the generation of the pre-ovulatory surge of gonadotropins in the female. This contention has been further supported by functional studies showing activation of AVPV Kiss1 neurons at the time of the surges and following estrogen priming (Smith et al., 2006b), as well as by data from GPR54 KO mice, where the ability of estrogen to elicit positive feedback and GnRH neuronal activation is abrogated

(Clarkson et al., 2008). Admittedly however, one study has documented that even in the congenital absence of GPR54 signalling some residual positive feedback actions of estrogen can be detected (Dungan et al., 2007); the reasons for the discrepancy between these studies remain obscure. In any event, the fundamental role of kisspeptins in the generation of the pre-ovulatory surges of gonadotropins is further demonstrated by studies of immunoneutralization of central kisspeptins, which resulted in blockade of the ovulatory surge of LH (Kinoshita et al., 2005). Similarly, very recent studies in cyclic female rats have demonstrated that central infusion of a potent kisspeptin antagonist is sufficient to inhibit the pre-ovulatory peaks of LH and FSH (Pineda et al., 2010). Taken as a whole, these data suggest that the pre-ovulatory peak of estrogen activates the transcription of Kiss1 gene, thereby inducing an increase in kisspeptin tone/secretion, which in turn heightens the secretory activity of GnRH neurons, and thus triggers the surge of gonadotropins preceding ovulation. One of the most intriguing aspects of sex steroid regulation of the hypothalamic Kiss1 system is how the very same regulator (estrogen) is able to increase Kiss1 mRNA levels in AVPV neurons, but lowers its expression at the ARC. This opposite behavior is not apparently related with a differential pattern of expression of a and b forms of estrogen receptors (ERs) between these two hypothalamic sites (Smith et al., 2005a). In fact, pharmacological and functional genomic studies in rats and mice have demonstrated that the regulatory actions of estrogen on Kiss1 expression are mediated via ERa (Navarro et al., 2004a; Smith et al., 2005a). Interestingly, by the use of a mouse model engineered to lack classical ER signalling through direct biding to DNA, it has been recently demonstrated that the acute actions of estrogens on Kiss1 mRNA expression at the ARC and AVPV are differentially mediated: the stimulatory actions of estradiol on Kiss1 mRNA levels at the AVPV are conducted through classical ER–DNA interactions, whereas the inhibitory effects of estrogen on Kiss1 expression at the ARC do not require this classical

65

pathway, suggesting alternative regulatory mechanisms (either ERE-independent genomic actions or non-nuclear receptor-mediated events) (Gottsch et al., 2009b). Such a differential mechanism of action may justify the divergent actions of estrogen on Kiss1 expression at these two hypothalamic nuclei. Other interesting facet of estrogen regulation of kisspeptin signalling that has arisen recently is the possibility of post-transcriptional modulatory actions, that is, that estrogen could not only influence the expression of Kiss1 gene, but also kisspeptin responsiveness and/or neurosecretion. Thus, selective blockade of ERa, which prevented the generation of the ovulatory surges of gonadotropins, also markedly decreased net LH and FSH responses to Kp-10 in cyclic female rats at the pre-ovulatory stage (Roa et al., 2008b, 2008c). In good agreement, net gonadotropin responses to Kp-10 were significantly reduced in GNX females (Roa et al., 2006). These observations are in keeping with electrophysiological studies in GNX mice that demonstrated a significant decrease in kisspeptin-stimulated GnRH neuronal activity that could be rescued by estradiol replacement in vivo (Pielecka-Fortuna et al., 2008). Given the conspicuous lack of ERa in GnRH neurons (Herbison, 2006), such modulatory actions may take place at upstream afferents. In fact, recent studies have suggested that kisspeptin increases GABA and glutamate neurotransmission to GnRH neurons in an estradioldependent manner (Pielecka-Fortuna and Moenter, 2010). Alternatively, part of these post-transcriptional regulatory effects might be mediated via ERb, which is expressed in GnRH neurons and whose blockade also results in subtle, but discernible alterations of kisspeptin responsiveness (Roa et al., 2008b, 2008c). As for the ovulatory surge of gonadotropins, the above observations strongly suggest that, in addition to transcriptional effects on Kiss1 gene, estrogen is able to increase the responsiveness of GnRH neurons to kisspeptin stimulation, thereby allowing the maximal activation of gonadotropin secretion at the pre-ovulatory period. An integral model for the transcriptional and posttranscriptional mechanisms whereby estrogen

positively regulates kisspeptin signalling for the generation of the pre-ovulatory surge of gonadotropins is depicted in Fig. 4.

Metabolic regulation of the kisspeptin system Besides regulation by sex steroids, other essential modifiers of reproductive maturation and function have been shown to participate in the control of the hypothalamic Kiss1 system. In this context, compelling evidence has demonstrated that Kiss1 expression at the hypothalamus is under the regulation of metabolic cues, as potential mechanism for the modulation of puberty onset and fertility by the body energy status and nutritional factors (Castellano et al., 2009b; Roa et al., 2008a). Thus, conditions of negative energy balance and metabolic stress have been shown to induce variable degrees of inhibition of Kiss1 mRNA levels at the hypothalamus in pubertal and adult rodents (Castellano et al., 2005, 2006c; Castellano et al., 2009a; Luque et al., 2007). Moreover, the state of hypogonadotropism and lack of puberty induced by chronic sub-nutrition in female rats could be rescued (at least partially) by administration of exogenous kisspeptin (Castellano et al., 2005). Similarly, defective gonadotropin secretion in conditions of severe metabolic stress, such as uncontrolled diabetes, could be normalized by kisspeptin administration (Castellano et al., 2006c, 2009a). Altogether, this experimental evidence supports the hypothesis that kisspeptin neurons at the hypothalamus operate as sensors and neuroendocrine conduits for conveying metabolic information onto reproductive centres, presumably GnRH neurons. The neuroendocrine pathways whereby Kiss1 expression is modulated by nutritional and metabolic factors is yet to be fully characterized, but solid evidence has pointed out the involvement of the adipose hormone, leptin, in this phenomenon. Thus, studies in GNX ob/ob male mice revealed a marked reduction of Kiss1 mRNA levels at the ARC in the absence of leptin that could be rescued by central leptin administration (Smith et al., 2006a), response that has also been observed in gonadal-intact

66

Negative feedback (ARC)

Positive feedback (AVPV)

Kiss1

+

-

+

GnRH

+ +

Hypothalamus +

GnRH

Pituitary

+ Ovary

E2

LH

LH FSH

AM

PM

Pre-ovulatory Surge

E2 P

Fig. 4. Kiss1 system and the generation of the pre-ovulatory surges of gonadotropins. The proposed roles of kisspeptin neurons at AVPV in mediating the positive feedback effects of ovarian sex steroids on GnRH and gonadotropin secretion, as suggested on the basis of rodent data, are presented. The rise of estradiol (E2) levels at the pre-ovulatory period stimulates Kiss1 gene expression at the AVPV that, in the presence of activated receptors for progesterone (P), contributes to the induction of pre-ovulatory surge of LH (positive feedback) during the afternoon/evening preceding ovulation. In addition to transcriptional effects, E2 seems to elicit a state of enhanced responsiveness to kisspeptin, likely at the level of GnRH neurons, during the peri-ovulatory period (dotted line). For comparative purposes, the tentative model for negative feedback control of gonadotropin secretion by E2, involving Kiss1 neurons at the ARC, is also depicted. For further details, see the section ‘Kisspeptin neurons: roles in feedback control of gonadotropins’ and references therein.

ob/ob mice (Luque et al., 2007). Similarly, suppressed expression of Kiss1 was also documented at the hypothalamus of male and female rats under severe metabolic stress, due to uncontrolled diabetes (Castellano et al., 2006c), suppression that was reversed by central infusion of leptin. Finally, as indirect evidence, leptin has been shown to increase Kiss1 mRNA levels in the murine hypothalamic cell line, N6 (Luque et al., 2007). In fact, expression of leptin receptor gene has been demonstrated in Kiss1þve neurons in the ARC (Smith et al., 2006a), thereby providing the molecular basis for such direct actions of leptin on Kiss1 expression. Importantly, such a leptin–kisspeptin pathway provides a tenable explanation for the apparent conundrum that leptin is capable of influencing GnRH secretory function, but its receptors are not expressed in GnRH neurons, as elegantly demonstrated recently (Quennell et al., 2009).

Overall, the above data strongly suggest that leptin, as key signal of energy sufficiency, functions as regulator of the hypothalamic Kiss1 system, thereby participating in the metabolic control of the GnRH/gonadotropic axis (Castellano et al., 2009b). The molecular mechanisms whereby leptin regulates Kiss1 expression (and function) at the hypothalamus have begun to be deciphered recently. In an elegant series of studies, Altarejos et al. (2008) have demonstrated that the Creb1regulated transcription coactivator-1 (Crct1) is involved in mediating at least part of leptin effects on the hypothalamic Kiss1 system. Thus, Crtc1 KO mice were shown to display not only obese and hyperphagic phenotypes, but also to be infertile. The physiological substrate for such a phenotype appears to involve the ability of leptin to dephosphorylate (and activate) Crtc1, which in

67

turn stimulates the recruitment of Crct1 to Kiss1 gene promoter and the expression of Kiss1 mRNA at the hypothalamus (Altarejos et al., 2008). As call of caution, however, a very recent report has been unable to replicate the consequences of functional inactivation of Crtc1 on mouse fertility (Breuillaud et al., 2009); the reasons for such discrepancy remain obscure. This tentative leptin–Crtc1–Kiss1 pathway might interplay with (or be parallel to) another metabolic signalling system, involving the mammalian target of rapamycin (mTOR), which appears to participate also in the central regulation of Kiss1 expression and the HPG axis (Roa et al., 2009b). Notably, mTOR signalling at the ARC had been proposed as transducer for leptin effects on energy homeostasis and food intake (Cota et al., 2006); yet, its potential involvement in the metabolic regulation of puberty onset and fertility remained unexplored. Recent data demonstrated that blockade of central mTOR disrupts the normal timing of puberty in female rats. More importantly, inhibition of mTOR signalling prevented the permissive effects of leptin in terms of puberty onset, and resulted in the suppression of Kiss1 mRNA levels, mainly at the ARC (Roa et al., 2009b). Altogether, these findings provide evidence for the existence of a leptin–mTOR–kisspeptin pathway in the metabolic control of puberty, whose neuroanatomical basis is yet to be defined. Whether other central fuel-sensing mechanisms are also involved in the metabolic regulation of Kiss1 expression at the hypothalamus remains unexplored.

Other regulators of the kisspeptin system: neuropeptides, hormones and environmental cues While the involvement of sex steroids and leptin in the control of Kiss1 expression at the hypothalamus has been well characterized, the roles of other putative regulators of this system, either of central or peripheral origin, has received to date less attention. Notwithstanding, some recent studies have begun to elucidate the participation of additional hormones and

neuropeptides in the control of the hypothalamic expression of Kiss1. Likewise, compelling evidence gathered in the past three years strongly suggest that kisspeptin neurons play a central role in the transmission of environmental (e.g. photoperiodic) influences onto the HPG axis, and are also the target for the dysregulation of reproductive function in ageing and stress conditions. In the metabolic front, other neuroendocrine regulators of the Kiss1 system are likely to include NPY, IGF1 and ghrelin. In contrast, no evidence for a role of insulin in the direct control of hypothalamic expression of Kiss1 has been obtained using in vivo and in vitro settings (Castellano et al., 2006c; Luque et al., 2007). Experimental evidence supporting a role of NPY in the regulation of Kiss1 expression comes from in vivo studies using NPY KO mice, which displayed significantly lower levels of Kiss1 mRNA at the hypothalamus, and in vitro studies using the hypothalamic cell line, N6, where Kp-10 was capable of inducing a significant increase in Kiss1 gene expression (Luque et al., 2007). This apparent stimulatory effect of NPY seems to be at odds with the reported ability of leptin to inhibit NPY expression at ARC (Schwartz et al., 1996), thus making it unlikely that NPY is the mediator for the stimulatory effects of leptin on Kiss1 gene expression. In addition, IGF1 has been reported to activate the expression of Kiss1 mRNA at the AVPV in pre-pubertal female rats (Hiney et al., 2009), although blockade of its receptor failed to alter Kiss1 mRNA levels at periventricular hypothalamic areas in adult female rats (Todd et al., 2007); neither did IGF1 increase Kiss1 mRNA expression in N6 cells (Luque et al., 2007). Opposite to these stimulatory actions, the gutderived hormone, ghrelin, has been shown to inhibit hypothalamic Kiss1 mRNA levels in female rats (Forbes et al., 2009), an action that is fully compatible with (and likely mechanistically relevant for) the reported ability of ghrelin, as signal of energy insufficiency, to suppress gonadotropin secretion and puberty onset (Tena-Sempere, 2008). Also, in the metabolic arena, a recent report has demonstrated that melanocortins can

68

activate Kiss1 gene expression at the hypothalamic preoptic area, as potential mechanism for its stimulatory actions of the HPG axis in sheep (Backholer et al., 2009). In addition, the orexigenic signal melanin-concentrating hormone (MCH) has been very recently shown to suppress kisspeptin-induced stimulation of GnRH neurons (Wu et al., 2009), thereby contributing to the neuroendocrine mechanisms for linking energy homeostasis and the function of the gonadotropic axis. Recent exciting findings in the field strongly suggest that neurokinin B (NKB), dynorphin (Dyn) and the gonadotropin-inhibitory hormone, GnIH, may also be involved in the neuroendocrine control of the hypothalamic Kiss1 system. While the evidence relating NKB, Dyn and Kiss1 will be summarized in the section ‘Kisspeptins in year 2010: future challenges and expected developments’, we stress here that a reciprocal interplay between kisspeptins and the mammalian homologue of GnIH, namely, RFRP, has been described in sheep as important circuitry for the central control of GnRH neurons (Smith et al., 2008). According to the proposed model, a concomitant activation of kisspeptin neurons at the ARC and inhibition of RFRP neurons at the dorsomedial hypothalamus take place at states of maximal activation of the HPG axis, such as the breeding season in sheep (Smith et al., 2008). Whether such a reciprocal regulatory network between Kiss1 and GnIH/RFRP neurons exits in other mammalian (or non-mammalian) species is yet to be defined. If so, it would be reasonable to predict a prominent role for such a network in the dynamic regulation of reproductive function, through the balance between stimulatory (kisspeptin) and inhibitory (RFRP) signals, both belonging to the family of RFamide neuropeptides (Kriegsfeld, 2006). Analyses in seasonal breeders strongly suggest that hypothalamic expression of Kiss1 is also under the influence of photic cues, as means to provide the neuroendocrine basis for the well-known link between photoperiod/season and reproductive capacity in these species (Clarke et al., 2009; Greives et al., 2007; Revel

et al., 2007; Simonneaux et al., 2009). Although some differences on the hypothalamic Kiss1 populations affected by changes in day length have been detected among species (Syrian hamster, Siberian hamster and sheep), the consensus exists that kisspeptin neurons do play a nodal role for conveying such environmental information onto GnRH neurons. Studies in Syrian hamsters have pointed out an important role of the pineal-derived neurotransmitter, melatonin, in this regulatory pathway (Revel et al., 2009; Simonneaux et al., 2009). Thus, in hamsters subjected to partial light deprivation (short-days, eight-hour light/day), as signal for inhibition of HPG axis, a significant reduction of Kiss1 mRNA levels at ARC was detected, which was prevented by pinealectomy (Revel et al., 2006). In contrast, in hamsters under long days (14-hour light/day), with full HPG activity, melatonin lowered hypothalamic Kiss1 mRNA expression (Simonneaux et al., 2009). Finally, the fact that exogenous kisspeptin administration was sufficient to rescue gonadal regression in short-day hamsters further supports the important role of Kiss1 neurons in such environmental regulation of the reproductive system (Revel et al., 2006). Interestingly, Kiss1 and GnIH/RFRP neurons have been suggested to be oppositely regulated by photic cues in sheep and hamsters (Paul et al., 2009; Smith et al., 2008), thus further supporting the reciprocal interaction between these neuronal pathways in the dynamic control of GnRH function.

Kisspeptins in year 2010: future challenges and expected developments Disclosure of the reproductive dimension of kisspeptins and GPR54 in late 2003 opened up a new era in our understanding of the neuroendocrine networks responsible for the sophisticated control of the HPG axis along the life span. Indeed, functional characterization of the roles and regulatory mechanisms of kisspeptin neurons, located at specific hypothalamic nuclei, has attracted considerable attention among neuroendocrinologists in

69

recent years, efforts that have led to the identification of discernible pathways involving Kiss1 neurons with essential roles in the regulation of key aspects of the maturation and function of the reproductive axis (Roa et al., 2008a). Moreover, recognition of the essential roles of such kisspeptin circuitries has prompted reproductive physiologists to reassess the functional roles and mechanisms of action of many classical neurotransmitters and neuropeptides, already known to modulate GnRH function, within this ‘novel’ neuroendocrine framework (Roa et al., 2008a). However, despite the astonishing progress in the field, several facets of the physiology of the Kiss1 system remain ill defined and are likely to concentrate substantial research efforts in the coming years. Some of these will be briefly delineated in this section, as a means to provide a tenable forecast for the progress of the field in year 2010 and beyond. As extensively discussed in the previous sections, the neuroanatomy of Kiss1 neurons, especially within the hypothalamus, has been extensively characterized in different mammalian and nonmammalian species in the past few years. However, some overt divergences across species have been identified (e.g. the existence of a prominent Kiss1 neuronal population at the AVPV in rodents, but not in sheep and primates), whose functional relevance is yet to be fully defined. More importantly, the mapping of GPR54 expression in different hypothalamic and extra-hypothalamic areas remains less well defined, in part due to the lack of reliable antibodies for immunohistochemical analysis. In the same line, there is paucity of data on the actual distribution of afferents to and projections of Kiss1 neurons, an area that requires further refinement. Indeed, of the two prominent neuronal populations expressing Kiss1/ kisspeptins in mouse hypothalamus, direct synaptic contacts with GnRH neurons have been only documented for that located at the AVPV (Clarkson and Herbison, 2006). In contrast, the neuroendocrine pathways whereby Kiss1 neurons at the ARC (i.e. the most abundant in male rodents and in other mammals regardless of sex) regulate GnRH functions remain unsolved. Similarly, despite the functional/pharmacological identification of an array of potential neuropeptide and hormonal regulators of

Kiss1 expression at the hypothalamus (as summarized in the section ‘Other regulators of the kisspeptin system: neuropeptides, hormones and environmental cues’), the neuronal, and eventual glial, inputs received by Kiss1 neurons at different hypothalamic nuclei are yet to be characterized. Overall, it is anticipated that considerable efforts will be paid to complete the characterization of the neuroanatomical features of Kiss1 pathways in different species, including humans, as a means to help refine of our knowledge of the physiology of this system. In the functional front, several facets of the Kiss1 system remain poorly characterized and will require further attention in the near future. Among these, there is as yet limited information about the different intra-cellular signalling pathways and functional domains of GPR54, although significant developments in this front have taken place recently, such as the characterization of the important roles of the intra-cellular loop 2 of GPR54 in the catalytic activation of Ga subunit (Wacker et al., 2008), and the ability of the GPR serine/threonine kinase 2, GRK2, and b-arrestin to modulate GPR54 signalling (Pampillo et al., 2009), information that will help to define the basis for a better pharmacological manipulation of this system. In the same line, the recent characterization of the intra-cellular and electrophysiological mechanisms responsible for the activation of GnRH neurons in response to kisspeptin (Liu et al., 2008; Zhang et al., 2008) will improve our understanding of the mechanism of action and auto-regulation (e.g. desensitization) of kisspeptin effects in key neuronal targets. Other functional aspect that is likely to draw considerable attention in the coming years is that related to the neuroendocrine signals and molecular mechanisms responsible for the regulation of Kiss1 expression at the hypothalamus. Specific comments on this point can be found in the sections ‘Kisspeptin neurons: roles in sexual differentiation and puberty onset’, ‘Kisspeptin neurons: roles in feedback control of gonadotropins’ and ‘Metabolic regulation of the kisspeptin system’. As additional exciting development in this front, compelling evidence is mounting that Kiss1 neurons also express other neuropeptides with discernible roles in the control of the GnRH/

70

gonadotropin axis, such as NKB and Dyn, which may play important roles in the (auto)regulation of kisspeptinergic pathways. This contention was first recognized in the sheep (Goodman et al., 2007), but has been recently confirmed (and refined) in the mouse, where hypothalamic Kiss1, NKB and/or Dyn expression has been shown to be co-regulated during puberty and by gonadal inputs (Gottsch et al., 2009b; Kauffman et al., 2010; Navarro et al., 2009a). Accordingly, it has been recently proposed that Kiss1 neurons should be considered as kisspeptin/ NKB/Dyn neurons (thus renamed as KNDy neurons) (Cheng et al., 2010), where NKB and Dyn are likely to operate as auto-regulators of kisspeptin output onto GnRH neurons (Navarro et al., 2009a). While the existence and functional relevance of such neuronal population need to be confirmed in different mammalian species, the importance of NKB signalling in the control of the HPG axis is further stressed by the recent observations that humans with inactivating mutations in TAC3 and TAC3R genes, which encode NKB and its receptor, respectively, suffer from hypogonadotropic hypogonadism (Topaloglu et al., 2009). In this context, it is predicted that considerable research efforts will be devoted to characterize the roles of NKB, and possibly Dyn, in the dynamic control of key facets of the HPG axis, from sexual differentiation to puberty onset and gonadotropin secretion, in close parallelism with recent similar studies on the Kiss1 system. Other intriguing aspect of the Kiss1 system that awaits further characterization is the presence and functional relevance of kisspeptins and GPR54 in different peripheral tissues. Thus, expression of Kiss1/kisspeptin and/or GPR54 has been documented in several reproductive (e.g. placenta, ovary) and non-reproductive (e.g. pancreas, adipose, adrenal glands, blood vessels) organs (Brown et al., 2008; Castellano et al., 2006a; Gaytan et al., 2009; Mead et al., 2007; Roa et al., 2008a; Takahashi et al., 2010). Accordingly, kisspeptins have been suggested to play discernible regulatory roles in the control of a wide spectrum of biological functions including trophoblast invasion, ovulation, insulin secretion, aldosterone production and cardiovascular function. However, the physiological relevance of such peripheral functions has been questioned by the apparent

lack of additional phenotypes in GPR54 or Kiss1 KO mice. Indeed, in the reproductive front, it is unquestioned that the primary site of action of kisspeptins in the control of the HPG axis is the hypothalamus, as extensively revised in the section ‘Kisspeptins and the control of gonadotropin secretion: roles and mechanisms’. Notwithstanding, such a predominant central action does not preclude the possibility of subtle modulatory roles of kisspeptin signalling at other peripheral sites, biological effects whose nature and relative importance warrants specific investigation in the coming years. In terms of ‘translational’ medicine, the features of the Kiss1 system described in the previous sections make it tempting to propose that alterations of this system might result in different types of reproductive disorders, ranging from precocious puberty to infertility. This is documented by the striking phenotypes of human and murine models of inactivation of GPR54 and/or Kiss1 genes. Similarly, a recent report has documented the potential association between activating mutations of GPR54 and precocious puberty (Teles et al., 2008). Admittedly, these cases appear to be very rare in human populations, but it is plausible that polymorphic variations in these genes, either alone or in combination with other allelic variants, might contribute to define changes in the timing of puberty and/or fertility, a possibility that will require specific analysis, as recently initiated in the context of puberty (Luan et al., 2007a, 2007b). Also, in the translational front, studies in experimental animal models have allowed to hypothesize that reproductive dysfunction associated with severe metabolic alterations, stress or ageing are coupled to changes in the expression and/or function of Kiss1 system at the hypothalamus (Kinsey-Jones et al., 2009; Lederman et al., 2009). The relevance of such mechanism in stress-associated reproductive failure and reproductive senescence in humans is yet to be determined. Finally, extensive pharmacological characterization of kisspeptins, as extraordinarily potent elicitors of gonadotropin secretion in different mammalian species, including male and female humans (Dhillo et al., 2005, 2007), has set the

71

(Curtis et al., 2010) may help to improve available protocols of pharmacological/hormonal activation of the gonadotropic axis in different species, including humans. In conclusion, in this chapter we have attempted to provide a comprehensive overview of the major developments that have taken place in recent years in our knowledge of the physiology of the Kiss1 system, with specific attention to the disclosure and in-depth characterization of its essential roles in the neuroendocrine control of the HPG axis. Strikingly, although Kiss1 and GPR54 were first recognized as metastasis-suppressor factors, and their neuroendocrine dimension had remained unsuspected up to six years ago, it is now universally accepted that Kiss1 neurons at the hypothalamus do have a nodal position in the hierarchy of afferent pathways controlling GnRH neurons, thereby playing a key role in the regulation of fundamental aspects of the maturation and function of the gonadotropic axis. This major hierarchical position is schematically depicted in Fig. 5. Overall, identification of kisspeptins is now regarded as one of the major breakthroughs

ground for the definition of protocols of therapeutic intervention of the HPG axis using kisspeptin analogues. In this context, different studies have addressed the major structure–function relationships of kisspeptin molecules, and a number of analogues with either agonistic or antagonistic activities have been very recently reported (Curtis et al., 2010; Gutierrez-Pascual et al., 2009; Roseweir et al., 2009). Generation of such compounds will not only provide better tools for the dissection of the physiological roles of kisspeptins in the control of the HPG axis, but will likely contribute to enlarge the available options for the pharmacological intervention on this hormonal system. For instance, initial studies using kisspeptin antagonists have documented that while blockade of kisspeptin signalling can prevent the normal occurrence of the pre-ovulatory surge of gonadotropins, it does not apparently suppress basal gonadotropin levels (Pineda et al., 2010), a phenomenon that is in contrast to that observed after administration of GnRH analogues and may have potential pharmacological interest. Similarly, the very recent development of long-acting kisspeptin agonists Σ Inputs

Gonadal hormones • Negative Feedback (ARC)

Glutamate GABA Others

• Positive Feedback (AVPV)

+/-?

GPR54

Kiss1 Neuron

NKB

Developmental cues

Dyn → Integrator

+

• Sex Differentiation • Puberty Onset

Metabolic factors • Nutritional Status

GnRH Neuron

+

• Leptin (mTOR/Crtc1) • NPY,IGF1, Melanocortins • Ghrelin, MCH

Rhythms/photoperiod • Seasonal breeding

GnRH → Effector

• Melatonin

Fig. 5. Kiss1 neurons as nodal centre for the integration of regulatory signals of the HPG axis. GnRH neurons, as final output pathway for the downstream regulation of the elements of the reproductive axis, are under the dynamic control of discrete populations of Kiss1 neurons, located at specific hypothalamic nuclei, which operate directly at different levels (perikarya and nerve terminals) of GnRH neurons, and eventually also via indirect actions upon other afferent pathways. Overall, Kiss neurons are proposed to function as essential node for the integration of a wide diversity of regulatory signals, from sex steroids and developmental cues to metabolic factors and environmental signals, affecting the gonadotropic axis. The complexity of this neuronal population is illustrated by recent findings on the co-expression of other neuropeptides (such as NKB and Dyn), putatively involved in the auto-regulation of these neurons and the fine-tuning of HPG axis. Adapted from Roa et al. (2008a), with substantial modifications.

72

in contemporary neuroendocrinology, and considered as (one of) the most important finding in reproductive physiology since the isolation and characterization of GnRH in early 1970s. In any event, although the progress in this field has been astonishingly rapid, many aspects of the Kiss1 system remain to be fully disclosed, thus making it predictable that kisspeptins and their receptor will continue to fuel (and inspire) the work of a large number of neuroendocrinologists in the next decade.

Acknowledgements The authors are indebted to the members of the research team at the Physiology Section of the University of Cordoba, who actively participated in the generation of experimental data discussed herein. The work from the authors’ laboratory reviewed in this chapter was supported by grants BFU 2005-07446 and BFU 2008-00984 (Ministerio de Ciencia e Innovación, Spain), funds from Instituto de Salud Carlos III (Red de Centros RCMN C03/08 and Project PI042082; Ministerio de Sanidad, Spain), and EU research contracts EDEN QLK4-CT-2002-00603 and DEER FP7ENV-2007-1. CIBER is an initiative of Instituto de Salud Carlos III (Ministerio de Sanidad, Spain).

Abbreviations AFP ARC AVPV CNS DMN FSH GABA GnRH GNX GPR54 HPG IGF

a-fetoprotein arcuate nucleus anteroventral periventricular nucleus central nervous system dorsomedial nucleus follicle-stimulating hormone g-aminobutyric acid gonadotropin-releasing hormone gonadectomy G protein-coupled receptor 54 hypothalamic–pituitary– gonadal insulin-like growth factor

IR LH MCH mTOR NKB NPY PLC RFRP1 and RFRP3 RP3V rPOA RT-PCR TRP VMN

immunoreactivity luteinizing hormone melanin-concentrating hormone mammalian target of rapamycin neurokinin B Neuropeptide Y phospholipase-C Arg-Phe-related peptide-1 and -3 rostral periventricular area of the third ventricle rostral preoptic area reverse transcriptase– polymerase chain reaction transient receptor potential ventromedial nucleus

References Altarejos, J. Y., Goebel, N., Conkright, M. D., Inoue, H., Xie, J., Arias, C. M., et al. (2008). The Creb1 coactivator Crtc1 is required for energy balance and fertility. Nature Medicine, 14, 1112–1117. Arai, A. C., Xia, Y. F., Suzuki, E., Kessler, M., Civelli, O., & Nothacker, H. P. (2005). Cancer metastasis-suppressing peptide metastin upregulates excitatory synaptic transmission in hippocampal dentate granule cells. Journal of Neurophysiology, 94, 3648–3652. Backholer, K., Smith, J., & Clarke, I. J. (2009). Melanocortins may stimulate reproduction by activating orexin neurons in the dorsomedial hypothalamus and kisspeptin neurons in the preoptic area of the ewe. Endocrinology, 150, 5488–5497. Bellingham, M., Fowler, P., Amezaga, M., Rhind, A., Cotinot, C., Madon-Pepin, B., et al. (2009). Exposure to a complex cocktail of environmental endocrine disrupting compounds disturbs the kiss1/GPR54 system in ovine hypothalamus and pituitary gland. Environmental Health Perspectives, 117, 1556–1562. doi: 10.1289/eph. 0900699. Brailoiu, G. C., Dun, S. L., Ohsawa, M., Yin, D., Yang, J., Chang, J. K., et al. (2005). KiSS-1 expression and metastinlike immunoreactivity in the rat brain. The Journal of Comparative Neurology, 481, 314–329. Breuillaud, L., Halfon, O., Magistretti, P. J., Pralong, F. P., & Cardinaux, J. R. (2009). Mouse fertility is not dependent on the CREB coactivator Crtc1. Nature Medicine, 15, 989–990, author reply 991. Brown, R. E., Imran, S. A., Ur, E., & Wilkinson, M. (2008). KiSS-1 mRNA in adipose tissue is regulated by sex

73 hormones and food intake. Molecular and Cellular Endocrinology, 281, 64–72. Castano, J. P., Martinez-Fuentes, A. J., Gutierrez-Pascual, E., Vaudry, H., Tena-Sempere, M., & Malagon, M. M. (2009). Intracellular signaling pathways activated by kisspeptins through GPR54: Do multiple signals underlie function diversity? Peptides, 30, 10–15. Castellano, J. M., Gaytan, M., Roa, J., Vigo, E., Navarro, V. M., Bellido, C., et al. (2006a). Expression of KiSS-1 in rat ovary: Putative local regulator of ovulation? Endocrinology, 147, 4852–4862. Castellano, J. M., Navarro, V. M., Fernandez-Fernandez, R., Castano, J. P., Malagon, M. M., Aguilar, E., et al. (2006b). Ontogeny and mechanisms of action for the stimulatory effect of kisspeptin on gonadotropin-releasing hormone system of the rat. Molecular and Cellular Endocrinology, 257–258, 75–83. Castellano, J. M., Navarro, V. M., Fernandez-Fernandez, R., Nogueiras, R., Tovar, S., Roa, J., et al. (2005). Changes in hypothalamic KiSS-1 system and restoration of pubertal activation of the reproductive axis by kisspeptin in undernutrition. Endocrinology, 146, 3917–3925. Castellano, J. M., Navarro, V. M., Fernandez-Fernandez, R., Roa, J., Vigo, E., Pineda, R., et al. (2006c). Expression of hypothalamic KiSS-1 system and rescue of defective gonadotropic responses by kisspeptin in streptozotocin-induced diabetic male rats. Current Medical Literature – Diabetes, 55, 2602–2610. Castellano, J. M., Navarro, V. M., Roa, J., Pineda, R., SanchezGarrido, M. A., Garcia-Galiano, D., et al. (2009a). Alterations in hypothalamic KiSS-1 system in experimental diabetes: Early changes and functional consequences. Endocrinology, 150, 784–794. Castellano, J. M., Roa, J., Luque, R. M., Dieguez, C., Aguilar, E., Pinilla, L., et al. (2009b). KiSS-1/kisspeptins and the metabolic control of reproduction: Physiologic roles and putative physiopathological implications. Peptides, 30, 139–145. Cheng, G., Coolen, L. M., Padmanabhan, V., Goodman, R. L., & Lehman, M. N. (2010). The kisspeptin/neurokinin B/ dynorphin (KNDy) cell population of the arcuate nucleus: Sex differences and effects of prenatal testosterone in the sheep. Endocrinology, 151, 301–311. Clarke, I. J., Smith, J. T., Caraty, A., Goodman, R. L., & Lehman, M. N. (2009). Kisspeptin and seasonality in sheep. Peptides, 30, 154–163. Clarkson, J., Boon, W. C., Simpson, E. R., & Herbison, A. E. (2009a). Postnatal development of an estradiol-kisspeptin positive feedback mechanism implicated in puberty onset. Endocrinology, 150, 3214–3220. Clarkson, J., d’Anglemont de Tassigny, X., Moreno, A. S., Colledge, W. H., & Herbison, A. E. (2008). KisspeptinGPR54 signaling is essential for preovulatory gonadotropin-releasing hormone neuron activation and the luteinizing hormone surge. Journal of Neuroscience, 28, 8691–8697. Clarkson, J., d’Anglemont de Tassigny, X., Colledge, W. H., Caraty, A., & Herbison, A. E. (2009b). Distribution of kisspeptin neurons in the adult female mouse brain. Journal of Neuroendocrinology, 21, 673–682.

Clarkson, J. & Herbison, A. E. (2006). Postnatal development of kisspeptin neurons in mouse hypothalamus; sexual dimorphism and projections to gonadotropin-releasing hormone neurons. Endocrinology, 147, 5817–5825. Cota, D., Proulx, K., Smith, K. A., Kozma, S. C., Thomas, G., Woods, S. C., et al. (2006). Hypothalamic mTOR signaling regulates food intake. Science, 312, 927–930. Curtis, A. E., Cooke, J. H., Baxter, J. E., Parkinson, J. R., Bataveljic, A. S., Ghatei, M. A., et al. (2010). A kisspeptin10 analogue with greater in vivo bioactivity than kisspeptin10. American Journal of Physiology Endocrinology Metabolism, 298, E296–E303. d’Anglemont de Tassigny, X., Fagg, L. A., Carlton, M. B., & Colledge, W. H. (2008). Kisspeptin can stimulate gonadotropin-releasing hormone (GnRH) release by a direct action at GnRH nerve terminals. Endocrinology, 149, 3926–3932. d’Anglemont de Tassigny, X., Fagg, L. A., Dixon, J. P., Day, K., Leitch, H. G., Hendrick, A. G., et al. (2007). Hypogonadotropic hypogonadism in mice lacking a functional kiss1 gene. Proceedings of the National Academy of Sciences of the United States of America, 104, 10714–10719. de Roux, N., Genin, E., Carel, J. C., Matsuda, F., Chaussain, J. L., & Milgrom, E. (2003). Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proceedings of the National Academy of Sciences of the United States of America, 100, 10972–10976. Decourt, C., Tillet, Y., Caraty, A., Franceschini, I., & Briant, C. (2008). Kisspeptin immunoreactive neurons in the equine hypothalamus interactions with GnRH neuronal system. Journal of Chemical Neuroanatomy, 36, 131–137. Dhillo, W. S., Chaudhri, O. B., Patterson, M., Thompson, E. L., Murphy, K. G., Badman, M. K., et al. (2005). Kisspeptin-54 stimulates the hypothalamic-pituitary gonadal axis in human males. Journal of Clinical Endocrinology Metabolism, 90, 6609–6615. Dhillo, W. S., Chaudhri, O. B., Thompson, E. L., Murphy, K. G., Patterson, M., Ramachandran, R., et al. (2007). Kisspeptin-54 stimulates gonadotropin release most potently during the preovulatory phase of the menstrual cycle in women. Journal of Clinical Endocrinology Metabolism, 92, 3958–3966. Dungan, H. M., Gottsch, M. L., Zeng, H., Gragerov, A., Bergmann, J. E., Vassilatis, D. K., et al. (2007). The role of kisspeptin-GPR54 signaling in the tonic regulation and surge release of gonadotropin-releasing hormone/luteinizing hormone. Journal of Neuroscience, 27, 12088–12095. Felip, A., Zanuy, S., Pineda, R., Pinilla, L., Carrillo, M., TenaSempere, M., et al. (2009). Evidence for two distinct KiSS genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals. Molecular and Cellular Endocrinology, 312, 61– 71. Forbes, S., Li, X. F., Kinsey-Jones, J., & O’byrne, K. (2009). Effects of ghrelin on kisspeptin mRNA expression in the hypothalamic medial preoptic area and pulsatile luteinising hormone secretion in the female rat. Neuroscience Letters, 460, 143–147.

74 Funes, S., Hedrick, J. A., Vassileva, G., Markowitz, L., Abbondanzo, S., Golovko, A., et al. (2003). The KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system. Biochemical and Biophysical Research Communications, 312, 1357–1363. Gaytan, F., Gaytan, M., Castellano, J. M., Romero, M., Roa, J., Aparicio, B., et al. (2009). KiSS-1 in the mammalian ovary: Distribution of kisspeptin in human and marmoset and alterations in KiSS-1 mRNA levels in a rat model of ovulatory dysfunction. American Journal of Physiology Endocrinology Metabolism, 296, E520–E531. Gonzalez-Martinez, D., de Mees, C., Douhard, Q., Szpirer, C., & Bakker, J. (2008). Absence of gonadotropin-releasing hormone 1 and kiss1 activation in alpha-fetoprotein knockout mice: Prenatal estrogens defeminize the potential to show preovulatory luteinizing hormone surges. Endocrinology, 149, 2333–2340. Goodman, R. L., Lehman, M. N., Smith, J. T., Coolen, L. M., de Oliveira, C. V., Jafarzadehshirazi, M. R., et al. (2007). Kisspeptin neurons in the arcuate nucleus of the ewe express both dynorphin A and neurokinin B. Endocrinology, 148, 5752–5760. Gottsch, M. L., Clifton, D. K., & Steiner, R. A. (2009a). From KISS1 to kisspeptins: An historical perspective and suggested nomenclature. Peptides, 30, 4–9. Gottsch, M. L., Cunningham, M. J., Smith, J. T., Popa, S. M., Acohido, B. V., Crowley, W. F., et al. (2004). A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. Endocrinology, 145, 4073–4077. Gottsch, M. L., Navarro, V. M., Zhao, Z., Glidewell-Kenney, C., Weiss, J., Jameson, J. L., et al. (2009b). Regulation of kiss1 and dynorphin gene expression in the murine brain by classical and nonclassical estrogen receptor pathways. Journal of Neuroscience, 29, 9390–9395. Greives, T. J., Mason, A. O., Scotti, M. A., Levine, J., Ketterson, E. D., Kriegsfeld, L. J., et al. (2007). Environmental control of kisspeptin: Implications for seasonal reproduction. Endocrinology, 148, 1158–1166. Gutierrez-Pascual, E., Leprince, J., Martinez-Fuentes, A. J., Segalas-Milazzo, I., Pineda, R., Roa, J., et al. (2009). In vivo and in vitro structure-activity relationships and structural conformation of kisspeptin-10-related peptides. Molecular Pharmacology, 76, 58–67. Gutierrez-Pascual, E., Martinez-Fuentes, A. J., Pinilla, L., Tena-Sempere, M., Malagon, M. M., & Castano, J. P. (2007). Direct pituitary effects of kisspeptin: Activation of gonadotrophs and somatotrophs and stimulation of luteinising hormone and growth hormone secretion. Journal of Neuroendocrinology, 19, 521–530. Han, S. K., Gottsch, M. L., Lee, K. J., Popa, S. M., Smith, J. T., Jakawich, S. K., et al. (2005). Activation of gonadotropinreleasing hormone neurons by kisspeptin as a neuroendocrine switch for the onset of puberty. Journal of Neuroscience, 25, 11349–11356. Herbison, A. E. (2006). Physiology of the gonadotropin-releasing hormone neuronal network. In J. D. Neill (Ed.), The Physiology of Reproduction. Elsevier/Academic Press, San Diego (USA).

Herbison, A. E., d’Anglemont de Tassigny, X., Doran, J., & Colledge, W. H. (2010). Distribution and postnatal development of GPR54 gene expression in mouse brain and gonadotropin-releasing hormone neurons. Endocrinology, 151, 312–321. Hiney, J. K., Srivastava, V. K., Pine, M. D., & Les Dees, W. (2009). Insulin-like growth factor-I activates KiSS-1 gene expression in the brain of the prepubertal female rat. Endocrinology, 150, 376–384. Homma, T., Sakakibara, M., Yamada, S., Kinoshita, M., Iwata, K., Tomikawa, J., et al. (2009). Significance of neonatal testicular sex steroids to defeminize anteroventral periventricular kisspeptin neurons and the GnRH/LH surge system in male rats. Biology of Reproduction, 81, 1216–1225. Irwig, M. S., Fraley, G. S., Smith, J. T., Acohido, B. V., Popa, S. M., Cunningham, M. J., et al. (2004). Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat. Neuroendocrinology, 80, 264–272. Jacobi, J. S., Martin, C., Nava, G., Jeziorski, M. C., Clapp, C., & MartinezDeLaEscalera, G. (2007). 17-Beta-estradiol directly regulates the expression of adrenergic receptors and kisspeptin/GPR54 system in GT1-7 GnRH neurons. Neuroendocrinology, 86, 260–269. Kauffman, A. S., Gottsch, M. L., Roa, J., Byquist, A. C., Crown, A., Clifton, D. K., et al. (2007). Sexual differentiation of kiss1 gene expression in the brain of the rat. Endocrinology, 148, 1774–1783. Kauffman, A. S., Navarro, V. M., Kim, J., Clifton, D., & Steiner, R. A. (2010). Sex differences in the regulation of kiss1/NKB neurons in juvenile mice: Implications for the timing of puberty. American Journal of Physiology Endocrinology Metabolism. (in press). Kinoshita, M., Tsukamura, H., Adachi, S., Matsui, H., Uenoyama, Y., Iwata, K., et al. (2005). Involvement of central metastin in the regulation of preovulatory luteinizing hormone surge and estrous cyclicity in female rats. Endocrinology, 146, 4431–4436. Kinsey-Jones, J. S., Li, X. F., Knox, A. M., Wilkinson, E. S., Zhu, X. L., Chaudhary, A. A., et al. (2009). Down-regulation of hypothalamic kisspeptin and its receptor, kiss1r, mRNA expression is associated with stress-induced suppression of luteinising hormone secretion in the female rat. Journal of Neuroendocrinology, 21, 20–29. Kotani, M., Detheux, M., Vandenbogaerde, A., Communi, D., Vanderwinden, J. M., Le Poul, E., et al. (2001). The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. The Journal of Biological Chemistry, 276, 34631–34636. Kriegsfeld, L. J. (2006). Driving reproduction: RFamide peptides behind the wheel. Hormones and Behavior, 50, 655–666. Lapatto, R., Pallais, J. C., Zhang, D., Chan, Y. M., Mahan, A., Cerrato, F., et al. (2007). Kiss1/mice exhibit more variable hypogonadism than GPR54/mice. Endocrinology, 148, 4927– 4936. Lederman, M. A., Lebesgue, D., Gonzalez, V. V., Shu, J., Merhi, Z. O., Etgen, A. M., et al. (2009). Age-related LH

75 surge dysfunction correlates with reduced responsiveness of hypothalamic anteroventral periventricular nucleus kisspeptin neurons to estradiol positive feedback in middle-aged rats. Neuropharmacology, 58, 314–320. Lee, J. H., Miele, M. E., Hicks, D. J., Phillips, K. K., Trent, J. M., Weissman, B. E., et al. (1996). KiSS-1, a novel human malignant melanoma metastasis-suppressor gene. Journal of the National Cancer Institute, 88, 1731–1737. Lee, D. K., Nguyen, T., O’Neill, G. P., Cheng, R., Liu, Y., Howard, A. D., et al. (1999). Discovery of a receptor related to the galanin receptors. FEBS Letters, 446, 103–107. Lee, Y. R., Tsunekawa, K., Moon, M. J., Um, H. N., Hwang, J. I., Osugi, T., et al. (2009). Molecular evolution of multiple forms of kisspeptins and GPR54 receptors in vertebrates. Endocrinology, 150, 2837–2846. Liu, X., Lee, K., & Herbison, A. E. (2008). Kisspeptin excites gonadotropin-releasing hormone neurons through a phospholipase C/calcium-dependent pathway regulating multiple ion channels. Endocrinology, 149, 4605–4614. Luan, X., Yu, H., Wei, X., Zhou, Y., Wang, W., Li, P., et al. (2007a). GPR54 polymorphisms in Chinese girls with central precocious puberty. Neuroendocrinology, 86, 77–83. Luan, X., Zhou, Y., Wang, W., Yu, H., Li, P., Gan, X., et al. (2007b). Association study of the polymorphisms in the KISS1 gene with central precocious puberty in Chinese girls. European Journal of Endocrinology, 157, 113–118. Luque, R. M., Kineman, R. D., & Tena-Sempere, M. (2007). Regulation of hypothalamic expression of KiSS-1 and GPR54 genes by metabolic factors: Analyses using mouse models and a cell line. Endocrinology, 148, 4601–4611. Matsui, H., Takatsu, Y., Kumano, S., Matsumoto, H., & Ohtaki, T. (2004). Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat. Biochemical and Biophysical Research Communications, 320, 383–388. Mead, E. J., Maguire, J. J., Kuc, R. E., & Davenport, A. P. (2007). Kisspeptins are novel potent vasoconstrictors in humans, with a discrete localization of their receptor, G protein-coupled receptor 54, to atherosclerosis-prone vessels. Endocrinology, 148, 140–147. Mechaly, A. S., Vinas, J., & Piferrer, F. (2009b). Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese sole (Solea senegalensis). Biology of Reproduction, 80, 60–69. Mechaly, A. S., Vinas, J., Murphy, C., Reith, M., & Piferrer, F. (2010). Gene structure of the kiss1 receptor-2 (kiss1r-2) in the Atlantic halibut: Insights into the evolution and regulation of kiss1r genes. Molecular and Cellular Endocrinology, 317, 78–89. Messager, S., Chatzidaki, E. E., Ma, D., Hendrick, A. G., Zahn, D., Dixon, J., et al. (2005). Kisspeptin directly stimulates gonadotropin-releasing hormone release via G proteincoupled receptor 54. Proceedings of the National Academy of Sciences of the United States of America, 102, 1761–1766. Mikkelsen, J. D. & Simonneaux, V. (2009). The neuroanatomy of the kisspeptin system in the mammalian brain. Peptides, 30, 26–33.

Muir, A. I., Chamberlain, L., Elshourbagy, N. A., Michalovich, D., Moore, D. J., Calamari, A., et al. (2001). AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1. The Journal of Biological Chemistry, 276, 28969–28975. Navarro, V. M., Castellano, J. M., Fernandez-Fernandez, R., Barreiro, M. L., Roa, J., Sanchez-Criado, J. E., et al. (2004a). Developmental and hormonally regulated messenger ribonucleic acid expression of KiSS-1 and its putative receptor, GPR54, in rat hypothalamus and potent luteinizing hormone-releasing activity of KiSS-1 peptide. Endocrinology, 145, 4565–4574. Navarro, V. M., Castellano, J. M., Fernandez-Fernandez, R., Tovar, S., Roa, J., Mayen, A., et al. (2005a). Effects of KiSS1 peptide, the natural ligand of GPR54, on follicle-stimulating hormone secretion in the rat. Endocrinology, 146, 1689–1697. Navarro, V. M., Castellano, J. M., Fernandez-Fernandez, R., Tovar, S., Roa, J., Mayen, A., et al. (2005b). Characterization of the potent luteinizing hormone-releasing activity of KiSS-1 peptide, the natural ligand of GPR54. Endocrinology, 146, 156–163. Navarro, V. M., Fernandez-Fernandez, R., Castellano, J. M., Roa, J., Mayen, A., Barreiro, M. L., et al. (2004b). Advanced vaginal opening and precocious activation of the reproductive axis by KiSS-1 peptide, the endogenous ligand of GPR54. Journal of Physiology (Paris), 561, 379–386. Navarro, V. M., Gottsch, M. L., Chavkin, C., Okamura, H., Clifton, D. K., & Steiner, R. A. (2009a). Regulation of gonadotropin-releasing hormone secretion by kisspeptin/dynorphin/neurokinin B neurons in the arcuate nucleus of the mouse. Journal of Neuroscience, 29, 11859–11866. Navarro, V. M., Sanchez-Garrido, M. A., Castellano, J. M., Roa, J., Garcia-Galiano, D., Pineda, R., et al. (2009b). Persistent impairment of hypothalamic KiSS-1 system after exposures to estrogenic compounds at critical periods of brain sex differentiation. Endocrinology, 150, 2359–2367. Nazian, S. J. (2006). Role of metastin in the release of gonadotropin-releasing hormone from the hypothalamus of the male rat. Journal of Andrology, 27, 444–449. Novaira, H. J., Ng, Y., Wolfe, A., & Radovick, S. (2009). Kisspeptin increases GnRH mRNA expression and secretion in GnRH secreting neuronal cell lines. Molecular and Cellular Endocrinology, 311, 126–134. Oakley, A. E., Clifton, D. K., & Steiner, R. A. (2009). Kisspeptin signaling in the brain. Endocrine Reviews, 30, 713–743. Ohtaki, T., Shintani, Y., Honda, S., Matsumoto, H., Hori, A., Kanehashi, K., et al. (2001). Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature, 411, 613–617. Ojeda, S. R., Lomniczi, A., & Sandau, U. S. (2008). Glialgonadotrophin hormone (GnRH) neuron interactions in the median eminence and the control of GnRH secretion. Journal of Neuroendocrinology, 20, 732–742. Pampillo, M., Camuso, N., Taylor, J. E., Szereszewski, J. M., Ahow, M. R., Zajac, M., et al. (2009). Regulation of GPR54 signaling by GRK2 and {beta}-arrestin. Molecular Endocrinology, 23, 2060–2074.

76 Paul, M. J., Pyter, L. M., Freeman, D. A., Galang, J., & Prendergast, B. J. (2009). Photic and nonphotic seasonal cues differentially engage hypothalamic kisspeptin and RFamide-related peptide mRNA expression in Siberian hamsters. Journal of Neuroendocrinology, 21, 1007–1014. Pielecka-Fortuna, J., Chu, Z., & Moenter, S. M. (2008). Kisspeptin acts directly and indirectly to increase GnRH neuron activity and its effects are modulated by estradiol. Endocrinology, 149, 1979–1986. Pielecka-Fortuna, J. & Moenter, S. M. (2010). Kisspeptin increases {gamma}-aminobutyric acidergic and glutamatergic transmission directly to gonadotropin-releasing hormone neurons in an estradiol-dependent manner. Endocrinology, 151, 291–300. Pineda, R., Garcia-Galiano, D., Roseweir, A., Romero, M., Sanchez-Garrido, M. A., Ruiz-Pino, F., et al. (2010). Critical roles of kisspeptins in female puberty and preovulatory gonadotropin surges as revealed by a novel antagonist. Endocrinology, 151, 722–730. Plant, T. M., Ramaswamy, S., & Dipietro, M. J. (2006). Repetitive activation of hypothalamic G protein-coupled receptor 54 with intravenous pulses of kisspeptin in the juvenile monkey (Macaca mulatta) elicits a sustained train of gonadotropin-releasing hormone discharges. Endocrinology, 147, 1007–1013. Quaynor, S., Hu, L., Leung, P. K., Feng, H., Mores, N., Krsmanovic, L. Z., et al. (2007). Expression of a functional G protein-coupled receptor 54-kisspeptin autoregulatory system in hypothalamic gonadotropin-releasing hormone neurons. Molecular Endocrinology, 21, 3062–3070. Quennell, J. H., Mulligan, A. C., Tups, A., Liu, X., Phipps, S. J., Kemp, C. J., et al. (2009). Leptin indirectly regulates gonadotropin-releasing hormone neuronal function. Endocrinology, 150, 2805–2812. Ramaswamy, S., Gibbs, R. B., & Plant, T. M. (2009). Studies of the localisation of kisspeptin within the pituitary of the rhesus monkey (Macaca mulatta) and the effect of kisspeptin on the release of non-gonadotropic pituitary hormones. Journal of Neuroendocrinology, 21, 795–804. Revel, F. G., Ansel, L., Klosen, P., Saboureau, M., Pevet, P., Mikkelsen, J. D., et al. (2007). Kisspeptin: A key link to seasonal breeding. Reviews in Endocrine & Metabolic Disorders, 8, 57–65. Revel, F. G., Masson-Pevet, M., Pevet, P., Mikkelsen, J. D., & Simonneaux, V. (2009). Melatonin controls seasonal breeding by a network of hypothalamic targets. Neuroendocrinology, 90, 1–14. Revel, F. G., Saboureau, M., Masson-Pevet, M., Pevet, P., Mikkelsen, J. D., & Simonneaux, V. (2006). Kisspeptin mediates the photoperiodic control of reproduction in hamsters. Current Biology, 16, 1730–1735. Richard, N., Galmiche, G., Corvaisier, S., Caraty, A., & Kottler, M. L. (2008). KiSS-1 and GPR54 genes are co-expressed in rat gonadotrophs and differentially regulated in vivo by oestradiol and gonadotrophinreleasing hormone. Journal of Neuroendocrinology, 20, 381–393.

Roa, J., Aguilar, E., Dieguez, C., Pinilla, L., & Tena-Sempere, M. (2008a). New frontiers in kisspeptin/GPR54 physiology as fundamental gatekeepers of reproductive function. Frontiers in Neuroendocrinology, 29, 48–69. Roa, J., Castellano, J. M., Navarro, V. M., Handelsman, D. J., Pinilla, L., & Tena-Sempere, M. (2009a). Kisspeptins and the control of gonadotropin secretion in male and female rodents. Peptides, 30, 57–66. Roa, J., Garcia-Galiano, D., Varela, L., Sanchez-Garrido, M. A., Pineda, R., Castellano, J. M., et al. (2009b). The mammalian target of rapamycin as novel central regulator of puberty onset via modulation of hypothalamic kiss1 system. Endocrinology, 150, 5016–5026. Roa, J., Vigo, E., Castellano, J. M., Gaytan, F., Garcia-Galiano, D., Navarro, V. M., et al. (2008b). Follicle-stimulating hormone responses to kisspeptin in the female rat at the preovulatory period: Modulation by estrogen and progesterone receptors. Endocrinology, 149, 5783–5790. Roa, J., Vigo, E., Castellano, J. M., Gaytan, F., Navarro, V. M., Aguilar, E., et al. (2008c). Opposite roles of estrogen receptor (ER)-alpha and ERbeta in the modulation of luteinizing hormone responses to kisspeptin in the female rat: Implications for the generation of the preovulatory surge. Endocrinology, 149, 1627–1637. Roa, J., Vigo, E., Castellano, J. M., Navarro, V. M., FernandezFernandez, R., Casanueva, F. F., et al. (2006). Hypothalamic expression of KiSS-1 system and gonadotropin-releasing effects of kisspeptin in different reproductive states of the female rat. Endocrinology, 147, 2864–2878. Rometo, A. M., Krajewski, S. J., Voytko, M. L., & Rance, N. E. (2007). Hypertrophy and increased kisspeptin gene expression in the hypothalamic infundibular nucleus of postmenopausal women and ovariectomized monkeys. Journal of Clinical Endocrinology Metabolism, 92, 2744–2750. Roseweir, A. K., Kauffman, A. S., Smith, J. T., Guerriero, K. A., Morgan, K., Pielecka-Fortuna, J., et al. (2009). Discovery of potent kisspeptin antagonists delineate physiological mechanisms of gonadotropin regulation. Journal of Neuroscience, 29, 3920–3929. Schwartz, N. B. (2000). Neuroendocrine regulation of reproductive cyclicity. In P. M. Conn & M. E. Freeman (Eds.), Neuroendocrinology in Physiology and Medicine. Totowa, NJ: Humana Press. Schwartz, M. W., Seeley, R. J., Campfield, L. A., Burn, P., & Baskin, D. G. (1996). Identification of targets of leptin action in rat hypothalamus. The Journal of Clinical Investigation, 98, 1101–1106. Seminara, S. B., Messager, S., Chatzidaki, E. E., Thresher, R. R., Acierno, J. S. Jr., Shagoury, J. K., et al. (2003). The GPR54 gene as a regulator of puberty. The New England Journal of Medicine, 349, 1614–1627. Shahab, M., Mastronardi, C., Seminara, S. B., Crowley, W. F., Ojeda, S. R., & Plant, T. M. (2005). Increased hypothalamic GPR54 signaling: A potential mechanism for initiation of puberty in primates. Proceedings of the National Academy of Sciences of the United States of America, 102, 2129–2134.

77 Simonneaux, V., Ansel, L., Revel, F. G., Klosen, P., Pevet, P., & Mikkelsen, J. D. (2009). Kisspeptin and the seasonal control of reproduction in hamsters. Peptides, 30, 146–153. Smith, J. T., Acohido, B. V., Clifton, D. K., & Steiner, R. A. (2006a). KiSS-1 neurons are direct targets for leptin in the ob/ob mouse. Journal of Neuroendocrinology, 18, 298–303. Smith, J. T., Coolen, L. M., Kriegsfeld, L. J., Sari, I. P., Jaafarzadehshirazi, M. R., Maltby, M., et al. (2008). Variation in kisspeptin and RFamide-related peptide (RFRP) expression and terminal connections to gonadotropinreleasing hormone neurons in the brain: A novel medium for seasonal breeding in the sheep. Endocrinology, 149, 5770–5782. Smith, J. T., Cunningham, M. J., Rissman, E. F., Clifton, D. K., & Steiner, R. A. (2005a). Regulation of kiss1 gene expression in the brain of the female mouse. Endocrinology, 146, 3686–3692. Smith, J. T., Dungan, H. M., Stoll, E. A., Gottsch, M. L., Braun, R. E., Eacker, S. M., et al. (2005b). Differential regulation of KiSS-1 mRNA expression by sex steroids in the brain of the male mouse. Endocrinology, 146, 2976–2984. Smith, J. T., Popa, S. M., Clifton, D. K., Hoffman, G. E., & Steiner, R. A. (2006b). Kiss1 neurons in the forebrain as central processors for generating the preovulatory luteinizing hormone surge. Journal of Neuroscience, 26, 6687–6694. Smith, J. T., Rao, A., Pereira, A., Caraty, A., Millar, R. P., & Clarke, I. J. (2007). Kisspeptin is present in ovine hypophysial portal blood, but does not increase during the preovulatory luteinizing hormone surge: Evidence that gonadotropes are not direct targets of kisspeptin in vivo. Endocrinology, 149, 1951–1955. Suzuki, S., Kadokawa, H., & Hashizume, T. (2008). Direct kisspeptin-10 stimulation on luteinizing hormone secretion from bovine and porcine anterior pituitary cells. Animal Reproduction Science, 103, 360–365. Takahashi, K., Shoji, I., Shibasaki, A., Kato, I., Hiraishi, K., Yamamoto, H., et al. (2010). Presence of kisspeptin-like immunoreactivity in human adrenal glands and adrenal tumors. Journal of Molecular Neuroscience, (in press). Teles, M. G., Bianco, S. D., Brito, V. N., Trarbach, E. B., Kuohung, W., Xu, S., et al. (2008). A GPR54-activating mutation in a patient with central precocious puberty. The New England Journal of Medicine, 358, 709–715. Tena-Sempere, M. (2008). Ghrelin as a pleotrophic modulator of gonadal function and reproduction. Nature Clinical Practice Endocrinology and Metabolism, 4, 666–674. Tena-Sempere, M. & Huhtaniemi, I. (2003). Gonadotropins and gonadotropin receptors. In B. C.J.M. Fauser (Ed.),

Reproductive Medicine – Molecular, Cellular and Genetic Fundamentals. New York: Parthenon Publishing. Tena-Sempere, M., Pinilla, L., Gonzalez, L. C., & Aguilar, E. (2000). Reproductive disruption by exposure to exogenous estrogenic compounds during sex differentiation: Lessons from the neonatally estrogenized male rat. Current Topics Steroid Research, 3, 23–37. Thompson, E. L., Patterson, M., Murphy, K. G., Smith, K. L., Dhillo, W. S., Todd, J. F., et al. (2004). Central and peripheral administration of kisspeptin-10 stimulates the hypothalamic-pituitary-gonadal axis. Journal of Neuroendocrinology, 16, 850–858. Todd, B. J., Fraley, G. S., Peck, A. C., Schwartz, G. J., & Etgen, A. M. (2007). Central insulin-like growth factor 1 receptors play distinct roles in the control of reproduction, food intake, and body weight in female rats. Biology of Reproduction, 77, 492–503. Tomikawa, J., Homma, T., Tajima, S., Shibata, T., Inamoto, Y., Takase, K., et al. (2010). Molecular characterization and estrogen regulation of hypothalamic KISS1 gene in the pig. Biology of Reproduction, 82, 313–319. Topaloglu, A. K., Reimann, F., Guclu, M., Yalin, A. S., Kotan, L. D., Porter, K. M., et al. (2009). TAC3 and TACR3 mutations in familial hypogonadotropic hypogonadism reveal a key role for neurokinin B in the central control of reproduction. Nature Genetics, 41, 354–358. Tovar, S., Vazquez, M. J., Navarro, V. M., Fernandez-Fernandez, R., Castellano, J. M., Vigo, E., et al. (2006). Effects of single or repeated intravenous administration of kisspeptin upon dynamic LH secretion in conscious male rats. Endocrinology, 147, 2696–2704. Wacker, J. L., Feller, D. B., Tang, X. B., Defino, M. C., Namkung, Y., Lyssand, J. S., et al. (2008). Disease-causing mutation in GPR54 reveals the importance of the second intracellular loop for class A G-protein-coupled receptor function. The Journal of Biological Chemistry, 283, 31068– 31078. Wu, M., Dumalska, I., Morozova, E., Van Den Pol, A., & Alreja, M. (2009). Melanin-concentrating hormone directly inhibits GnRH neurons and blocks kisspeptin activation, linking energy balance to reproduction. Proceedings of the National Academy of Sciences of the United States of America, 106, 17217–17222. Zhang, C., Roepke, T. A., Kelly, M. J., & Ronnekleiv, O. K. (2008). Kisspeptin depolarizes gonadotropin-releasing hormone neurons through activation of TRPC-like cationic channels. Journal of Neuroscience, 28, 4423–4434.

L. Martini (Eds.) Progress in Brain Research, Vol. 181 ISSN: 0079-6123 Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 6

Regulation of complex pulsatile and rhythmic neuroendocrine systems: the male gonadal axis as a prototype Johannes D. Veldhuis1,
, Daniel M. Keenan2 and Steven M. Pincus3 1

Department of Medicine, Endocrine Research Unit, Mayo School of Graduate Medical Education, Clinical Translational Science Center, Mayo Clinic, Rochester, Minnesota, United States of America 2 Department of Statistics, University of Virginia, Charlottesville, Virginia, Unites States of America 3 Independent Mathematician, Guilford, Connecticut, United States of America

Abstract: Hormone-secreting glands communicate via intermittent (pulsatile or rhythmic) signal exchange. Signals act upon target glands via implicit (not directly observable) stimulatory and inhibitory dose–response functions. Time delays operate, since secreted hormones do not arrive at or act on responsive cells instantaneously. Neuroendocrine systems are unique examples, therefore, of intermittent time-delayed dose-dependent homeostatic ensembles. Investigating such ensembles thus requires estimating secretion from plasma concentrations, recognizing biological time-delays and reconstructing unobserved feedforward (agonist) and feedback (antagonist) dose–response interfaces as illustrated primarily for the GnRH–LH–T–axis, and secondarily for the corticotropic and somatotropic axes. In this manner, each neuroendocrine system is viewed as a whole, rather than the sum of individual parts. Keywords: cortisol; rhythms; secretion; pulses; GH; LH; testosterone; ACTH

system, are uniformly composite in neuroanatomic structure. Hormonal systems exhibit intricate elements, such as peptidyl, steroidal, gaseous (nitric oxide), fatty acyl (ghrelin), prostanoid (PGE2, PGF2-alpha) and neurotransmitter signals. Moreover, time-dependent specification of signal synthesis, release, action, dissipation, modulation and exchange endows an involved (non-linear, stochastic and time-delayed) nature to all endocrine axes. The conjoint effects of composite structure, intricate elements and time-varying interactions confer adaptive capabilities in physiological

Introduction Complexity of endocrine systems arises at several levels. Here, complexity is taken to mean, ‘the condition or state of having a composite structure or an intricate or involved nature’ (Oxford English Dictionary, O.E.D.). Endocrine axes, such as the limbic–hypothalamic–pituitary–adrenal–autonomic


Corresponding author. Tel.: þ507-255-0902; Fax: þ507-255-0901; E-mail: [email protected]

DOI: 10.1016/S0079-6123(08)81006-0

79

80

contexts and homeostatic resiliency to pathogenetic stresses. Rhythmic refers to processes marked by ‘a (more or less) regulated succession of strong and weak or otherwise differing influences’ (O.E.D.). Successive influences in hormonal systems include trains of alternating low- and high-signal activity, designated nadirs and pulses (Evans et al. 1992; Giustina and Veldhuis, 1998; Urban et al. 1988; Veldhuis et al., 2006, 2008). In most biological systems, intermittent signal exchange is both circadian (approximately daily) and ultradian (characterized by multiple events distributed over a single day). Ultradian signal exchange confers several theoretical benefits to biological systems (Table 1). Precisely rhythmic behavior is unusual in living organisms; that is, recurring events are not strictly regular with near-zero intraindividual and interindividual variabilities. Among more consistent rhythms are circadian temperature, cortisol and melatonin excursions and Table 1. Implications signalling

of

episodic

(ultradian)

endocrine

• Pulses are outcome of phasic neural signalling to endocrine glands (Veldhuis et al., 1989) • Obviate downregulation of receptor–effector system (Giustina & Veldhuis, 1998, Homme, Schaefer, Mehls, & Schmitt, 2009) • Enhance economy of glandular secretory activity (Veldhuis et al., 1987a, 1989) • Amplitude and frequency modulation can generate circadian rhythms (Veldhuis et al., 1990a) • Selective signalling and gene induction (Bedecarrats & Kaiser, 2003; Lotinun, Sibonga, & Turner, 2002; Zhou, Wang, Hadley, Corey, & Vasilatos-Younken, 2005) • Tissue-specific responsesa (Chow, Sharma, & Jusko, 1999) • Species-related differences (Cravener, Vasilatos-Younken, & Andersen, 1990) • Mode of hormone processing (e.g. LH and FSH) (McNeilly, Crawford, Taragnat, Nicol, & McNeilly, 2003) • Augment or mute dose–response function (Tolic, Mosekilde, & Sturis, 2000) • Modulate potentiation by other effectors (Bucholtz et al. 2000) • Regulate feedback efficacy (Zwart, Iranmanesh, & Veldhuis, 1997) a

GH pulses activate lipolysis in fat and IGF-I expression in skeletal muscle and bone (Isgaard, Carlsson, Isaksson, & Jansson, 1988; Laursen et al. 2001; Sun, Lee, Almon, & Jusko, 1999).

ultradian insulin and glucagon pulses, which exhibit intraindividual coefficients of variation as low as 8–20% under physiological conditions (Veldhuis 2002, 2008). The least reproducible interevent (interpulse) times are associated with ultradian arginine vasopressin [AVP; antidiuretic hormone(ADH)], growth hormone (GH), luteinizing hormone (LH), adrenocorticotrophic hormone (ACTH), prolactin, folliclestimulating hormone (FSH), thyroid-stimulating hormone (TSH), beta-endorphin, parathormone, cortisol, sex-steroid and inhibin secretory episodes, which manifest highly irregular timing closely resembling a renewal process (Evans et al. 1992; Giustina and Veldhuis, 1998; Urban et al. 1988; Veldhuis et al., 2006, 2008). Renewal processes act like random pulse generators, wherein the timing of prior events does not predict the exact timing of the next event (stated technically, successive interevent intervals are not necessarily or typically correlated). Endocrinologists and physiologists term apparently random pulse sequences episodic, to distinguish them from expressly rhythmic. The distinction is important, since episodic pulses are not so amenable to classical methods of timeseries analyses (e.g. spectral density, autocorrelation, Fourier expansion), which require more uniform rhythmicity. Table 2 summarizes several of the challenges inherent in analyzing episodic ultradian signals. Consequently, a specialized armamentarium of analytical tools is necessary to quantify the number, size and shape of episodic endocrine signals (Keenan and Veldhuis, 2003; Veldhuis et al., 2008). Non-classical methodologies are especially suited to analyses of secretion

Table 2. Challenges of ultradian pulse analysis • The size, shape, number and location of bursts are unknown • Interpulse time delays are random • Data sets are sparse (short) and noisy (confounded by sampling and measurement error) • Elimination processes are controlled by protein binding, biotransformation and removal • Basal secretion confounds detection of confluent pulses • Kinetics differ by hormone, among individuals and across species

81

of anterior and posterior pituitary hormones, parathormone, downstream targets of LH and FSH [estradiol, progesterone, inhibin B and testosterone (T)] and ACTH (cortisol and aldosterone), and possibly juxtaglomerular macula-densa renin and gastric ghrelin. The greater regularity of the sequential timing of insulin and glucagon pulses makes analysis of these pancreatic-islet hormones tractable to both classical and novel timeseries techniques.

Hypothalamo-pituitary-target organ systems Hypothalamus Hormonal axes that include the pituitary gland are uniformly supervised by (anterior pituitary) or comprise (posterior pituitary) hypothalamic

neurons. Pituitary secretion is further modulated by local non-homotypic factors and regulated by systemic and target tissue-derived feedback signals. Fig. 1a illustrates this concept. Foremost among such axes are diads, triads, tetrads and pentads comprising dopamine-prolactin, oxytocinuterine contractions, AVP–osmolality–baroreceptors, TRH–TSH–thyroxine, GnRH–LH–sex steroids, CRH/AVP–ACTH–cortisol, GnRH/activin/ inhibin–FSH–estradiol and GHRH–somatostatin– ghrelin–GH–IGF-I. Ensemble (complete-axis) regulation arises from time-delayed, non-linear (saturable and asymptotic), combined stochastic (random) and deterministic (rule-based) communications within an axis and (to a less well understood extent) among different axes. For example, sex steroids modify the direction (inhibition or stimulation) and/or strength (potency, efficacy or sensitivity) of homeostatic signalling between

(a) Neuroanatomic bases of hormone pulsatility {cortex, brain stem}

(b) Stochastic elements in a neuroendocrine feedback system

Periventricular & Arcuate Nuclei

Portal microvascular system

1. Procedural/experimental uncertainties

Efectors

Assay

Hypothalamus

Neuroglial cells

Samples 2. Cellular nonuniformity; blood admixture

ACTH GH Prolactin TSH LH FSH

Folliculostellate cells

ADH Oxytocin

Distance

3. Pulse–regeneration properties

Secretion

Pituitary gland

Release

Pituitary stalk

4. Dose–response linkages

Response

Time Systemic circulation {ffa, glucocorticoids, aldosterone, Oestrogen, androgen, thyroxine, IGF-I, insulin, IL-6, TNFα, adiponectin} Target tissues {hepatic IGF-I, adrenal corticosteroids, gonadal sex steroids, thyroid T4/T3, pancreatic beta-cell insulin, macrophage cytokines, fat-cell adipokines}

Agonist

Fig. 1. Panel A. Schematic representation of fundamental neuroendocrine axis. A neuroendocrine ensemble comprises CNS inputs (cerebral cortex, brainstem and hypothalamus); delivery of hypothalamic effectors to the anterior pituitary gland via the portal microvascular system and to the posterior pituitary via by direct neural extension of ADH (AVP) and oxytocin axons; pituitary hormone-selective secretory cells; local intrapituitary regulators (e.g. membrane contact-mediated and paracrine effects of folliculostellate cells); remote target tissues and their secreted feedback products; and systemic factors, such as free fatty acids (ffa), which secondarily modify the CNS-hypothalamo-pituitary unit. Neuroendocrine rhythmicity requires dynamic interactions within the collective system (ensemble). Panel B. Random (stochastic) effects that enter into neuroendocrine rhythmicity and its analytical estimation.

82

dopamine-mediated hypothalamic (Evans et al. 1992; Urban et al. 1988).

GnRH–LH, GnRH/FSH, CRH–ACTH, ACTH– cortisol, cortisol/LH, GHRH/GH, ghrelin/GH, somatostatin/GH, GH/IGF-I and among baroreceptors/osmolality/AVP. Major technical and experimental needs in the field include further evolution of well-articulated analytical methods to evaluate, and empirical knowledge to define, linkages within and among neuroendocrine ensembles.

feedback

Systemic effectors A third repertoire of signals impinging upon pituitary secretion comprises blood-borne effectors (Evans et al. 1992; Giustina and Veldhuis, 1998; Urban et al. 1988; Veldhuis et al., 2006). Prominent among such regulators are free fatty acids, which suppress GH secretion; cortisol, which represses ACTH secretion; thyroxine, which directly inhibits TSH secretion; IGF-I, which decreases GH secretion in some species and estrogen, which augments prolactin and GH synthesis in certain mammals (Giustina and Veldhuis, 1998).

Pituitary locus Intrapituitary regulation increases the complexity of hypothalamo-pituitary-target systems by modulating primary secretory cells and their lineages. Table 3 presents several putative intrapituitary regulatory interactions. For example, the presence of folliculostellate (macrophage-like immune) cells in the pars distalis allows for cytokine-dependent regulation of somatotropes (suppressed) and corticotropes (stimulated) by factors like interleukin-6 and tumour necrosis-factor alpha (Veldhuis et al., 2006). Annexin (earlier known as lipocortin1), produced by folliculostellate cells under glucocorticoid drive, may partially transduce negative feedback onto corticotropes, thus opposing cytokine activation. Heterologous hypothalamopituitary signalling bolsters pituitary regulation further. An instance is hypothalamic GnRH drive of lactotrope prolactin secretion, which in turn suppresses GnRH/LH secretion via

Feedback signals Target organs such as the testis, ovary, adrenal gland, liver and calcified bone matrix (under the control of effectors like LH, FSH, ACTH, insulin and parathormone) exert feedback on the hypothalamo-pituitary unit, pancreatic islets and parathyroid glands via autonomic neural (testis, ovary and adrenal gland) and soluble endocrine signals (testosterone, inhibin B, cortisol, glucose and calcium). Products of target glands typically inhibit secretion of the cognate agonist, except during the preovulatory LH surge when ovarian

Table 3. Putative intrapituitary regulatory interactions Systemically secreting cell

Interacting cell

Corticotrope (ACTH)

Folliculostellate

Gonadotrope (LH/ FSH)

Folliculostellate

Thyrotrope (TSH) Lactotrope (prolactin)

Folliculostellate Gonadotrope

Somatotrope (GH)

Neuroglia

Interaction

Annexin-1 TNF-a follistatin PACAP T4 ! T3 ? adenosine ? nitric oxide IGF-I

Plus and minus signs denote stimulation and inhibition, respectively. ? denotes proposed but not proven.

References –

Omer, Meredith, Morris, & Christian (2006)

þ –

Ooi, Tawadros, & Escalona, (2004) Winters & Moore (2004)

þ – þ

Winters & Moore (2007) Fliers, Unmehopa, & Alkemade (2006) Giustina & Veldhuis, (1998); Yu, Kimura, Walczewska, Porter, & McCann (1998)

– –

Veldhuis et al. (2006)

83

sex steroids potentiate GnRH-induced secretion (Evans et al. 1992).

LH

Random (stochastic) elements Understanding endocrine systems requires visualizing the composite ensemble of agonists (feedforward signals) and antagonists (feedback signals), regulatory interfaces (implicit dose– response functions) and associated time delays (reflecting neural or circulatory transfer of signals and response delays between secretory glands and target cells). Beyond deterministic (causally linked) biological inputs into the hypothalamo-pituitary unit, Fig. 1b emphasizes the recent recognition that random (stochastic) variations arise from both experimental methodology (sample collection and processing) and biological processes (e.g. non-uniformity of consecutive pulse sizes or interpulse-interval lengths) (Bergendahl et al., 1996; Keenan et al., 2000). Valid analysis must correctly allow for such seemingly random effects or uncertainty in the data (Keenan et al., 1998, 2001, 2004a; Veldhuis et al., 2008).

Integrating pulsatile and 24-hour rhythmic secretion patterns Experimental data in multiple species (viz. the rat, mouse, pig, cow, dog, guinea pig, hamster and primate) strongly indicate that the major components of neuroendocrine axes (hypothalamus, pituitary, systemic blood and target organs) regulate hormone secretion by selectively affecting secretory-burst number (frequency), size (mass or amplitude), shape (waveform) and interpulse-nadir concentrations (partially reflecting basal secretion). In particular, nutritional, agerelated, circadian (24-hour rhythmic), sleep- and activity-associated variations in mean hormone concentrations are mediated via differences in secretory-burst amplitude, frequency and possibly basal secretion. However, basal-secretion models are less consistent, making definitive statements difficult.

The time scales of pulsatile and circadian hormone secretion differ by about one order of magnitude for pituitary hormones, sex steroids, cortisol, aldosterone and renin (nominal interpulse intervals 45–180 minute) and by two orders of magnitude for insulin, glucagon and parathormone secretion (interpulse intervals 4–12 minute). Table 4 summarizes inferred primary pulsedefined mechanisms in humans that yield 24-hour rhythmicity of anterior pituitary-hormone concentrations (Veldhuis et al., 1990a). Notable are ACTH and insulin, which exhibit principally meal-related and 24-hour oscillations in secretoryburst mass (proportionate to pulse size). No anterior-pituitary hormone maintains exclusively burst frequency-dependent rhythmicity of 24-hour concentrations (Evans et al. 1992; Giustina and Veldhuis, 1998; Urban et al. 1988; Veldhuis et al., 2006). LH, FSH, TSH, prolactin and GH manifest combined variations in secretory-burst mass (amplitudes somewhat larger at night) and frequency (slower for LH at night in follicularphase women and more rapid for TSH, prolactin and GH at night in men and women). Prolactin and TSH secretory bursts get larger just before sleep onset, and GH during deep sleep (Veldhuis and Johnson, 1988; Veldhuis et al., 1990a, 1990b). Oxytocin and AVP (ADH) have been less well studied in this manner (Veldhuis et al., 2008). Cortisol, testosterone, progesterone and estradiol Table 4. Primary contributions to diurnally rhythmic hormone concentrations Hormone

Pulse size

Pulse number

LH FSH Prolactin TSH ACTH Renin Insulin Glucagon PTH Cortisol Sex steroids Aldosterone

þ þ þ þ þ þ þ þ þ þ þ þ

þ þ þ þ – þ – – – þ þ ?

þ denotes 24-hour rhythm. – no 24-hour rhythm.

84

pulses evince both amplitude (the primary contributor) and frequency variations, which determine 24-hour concentration rhythms (Iranmanesh et al., 1989; Rossmanith et al., 1990; Veldhuis et al. 1987b, 1988, 1989). The frequency of anterior-pituitary hormone pulses is endowed by cognate hypothalamic signals (TRH for TSH, GnRH for LH and FSH, CRH/AVP for ACTH, etc.), but amplitude modulation is far more complex. In fact, amplitude is also controlled by physical factors involved in hormone disappearance (Fig. 2). Several major factors determine pituitaryhormone secretory-burst amplitude (maximal secretion rate attained within a burst) and more specifically mass (amount of hormone secreted per burst per unit distribution volume). Key factors include (1) hypothalamic signals, for example GHRH and somatostatin for GH bursts; (2) pituitary cell-specific responsiveness, for example gonadotrope responsiveness to GnRH; (3) non-homotypic intrapituitary mechanisms, for example follistatin derived from folliculostellate cells, repressing activin’s drive of FSH-secreting gonadotropes and (4) extrapituitary systemic factors, for example systemic cortisol and interleukin-6, which regulate corticotrope responsivity to corticotropinreleasing hormone (CRH) and AVP. Core mechanisms of amplitude and frequency modulation allow Threefold control of hormone disappearance Endocrine cells

Capillary

Force

Model

Diffusion

Partial derivative on space and time

Advection

First derivative on time

Elimination

Finite probability

finely graded adaptations to physiological state, gender, age, sleep, physical activity and nutrition. This theme is readily illustrated in the gonadal axis, in which GnRH neurons are innervated by estrogenresponsive excitatory (kisspeptin, glutamate) and inhibitory (GABAergic) CNS neurons; gonadotrope cells are regulated by intrapituitary cytokines, inhibin, follistatin and activin and extrapituitary inhibin and sex steroids are produced gonadally under stimulatory (LH, FSH, IGF-I) and inhibitory (cytokine, CRH, NPY) control, permitting both negative and positive regulation in the female, and negative feedback in the male (Evans et al. 1992; Urban et al. 1988) (Fig. 1).

Quantification of ensemble control An outstanding challenge is the creation of increasingly complex yet analytically tractable models of neuroendocrine signalling ensembles. Tractable analytical models are important in order to (1) make quantitative comparisons among physiological states during development, adulthood and ageing, between genders, under nutritionally controlled conditions, during sleep and wakefulness, in response to stress, exercise and rest and in relation to pathologic aberrations; (2) test the coherence and validity of current neuroendocrine-axis concepts by formalizing the dynamics of proposed pathway connections; (3) assess deficits in the field, which if addressed would clarify key regulatory mechanisms and (4) assist in formulating relevant experiments to test new hypotheses. With this motivation, Fig. 3 presents simplified core regulatory models of the gonadal axis, somatotropic axis and corticotropic axis (Farhy and Veldhuis, 2005; Farhy et al., 2007; Keenan et al., 2001, 2004a).

Hormone elimination Fig. 2. Concepts of diffusion (random molecular motion in solution), advection (linear transport by circulatory flow) and metabolism (irreversible loss by degradation, removal or transformation). Adapted with permission from Veldhuis et al. (2008; Fig. 7b).

The pathways shown schematically in Fig. 3a illustrate essential regulatory connections among core gonadal-axis signals: GnRH, LH and testosterone (in the female, estradiol and progesterone). These signals act upon downstream target glands,

85

(a)

Dynamic structure of gonadal axis

Hypothalamus

(b)

GnRH

Dynamic linkages in somatotropic axis GHRH

(–)

(+) (–)

(+)

(+)

(±) (–)

Pituitary

LH

(–)

(+)

Ghrelin

(–)

(+)

(+)

(+)

FSH (±)

GH (+)

(+)

Somatostatin

(–)

(±) (+)

(–)

(+) Target gland

T, Prog

E2

(c)

IGF-I

Key elements in corticotropic axis CRH

(–)

AVP

(–)

(+)

(+) (+) ACTH (+)

(–)

Cortisol

Fig. 3. Panels A–C depict minimal pathways that supervise dynamics of the gonadal axis (T and E2 in the male, and T, E2 and progesterone in the female) (a), the somatotropic axis (b), and the corticotropic (stress-adaptive) axis (c). Plus and minus signs denote stimulation and inhibition, respectively. CRH, corticotropin (ACTH)-releasing hormone; AVP, arginine vasopressin (ADH: antidiuretic hormone). Unpublished drawings.

GnRH ! pituitary, LH ! Leydig cells, LH ! granulosa and theca cells, E2 and T ! hypothalamic GnRH, T ! E2 ! pituitary LH and progesterone ! GnRH. This rudimentary structure omits GnRH ! prolactin, prolactin ! GnRH (inhibition) and FSH/inhibin B/activin A/follistatin. Whatever the (parsimonious) model, incorporation of dynamic signal exchange requires valid representation of intermittent and continuous signal secretion, transfer and elimination. Elimination is jointly determined by an admixture of diffusion (random molecular motion) and advection (forward motion) in the bloodstream, and metabolism (finite irreversible loss from the body). In most endocrine systems, diffusion, advection and metabolism can be accounted for collectively by a biexponential decay model, as

argued mathematically from first principles (Keenan and Veldhuis, 2004; Keenan et al., 2000). Fig. 4 illustrates the processes of diffusion, advection and metabolism that give rise to an exponential formulation. The decay of bloodhormone concentrations, X(t), in the presence of only basal (non-pulsatile) secretion (ß0) given a starting concentration, X(0), can be written simply as XðtÞ ¼ ðAe  a1 t þ ð1  AÞe  a2 t ÞXð0Þ
 A ð1  AÞ  a1 t  a2 t ð1  e Þþ ð1  e Þ þ 0  a1 a2 þ Error

ð1Þ

86

Stepwise deconvolution estimation of secretion and elimination

Hormone concentration

8

* * Observed data Predicted (reconvolution) curve

6

4

2

0

0

500

1000

1500

Time (min)

Fig. 4. Hormone concentrations (traced by asterisks, observed data) rendered analytically as the conjoint result of secretory bursts and biexponential decay (continuous curvilinear lines). Plot was obtained by beginning (but not finalizing) secretion and elimination estimates by deconvolution analysis. Adapted with permission from Veldhuis et al. (2008; Fig. 5b).

Dose–response concept In biological systems, molecules reaching their target receptors generate cellular responses via monotonic asymptotic dose–response functions (Keenan and Veldhuis, 2003). In embodying this concept, a four-parameter logistic structure arises from expected conservation of mass, cooperative sigmoidal-like activation (or inhibition) of the receptor–effector response pathway and maximal (or minimal) target-cell response boundaries due to rate-limiting steps in the responding cell (Kirkland and Goldberg). The general structure of a sigmoidally increasing agonist-driven effectorresponse function is illustrated for LH’s concentration-dependent drive of T secretion, as follows:

Tsec ¼ Basal sec þ

Efficacy 1þ

e  ½ED50 þ Sens  LHcon 

ð2Þ

þ Error where basal denotes non-pulsatile T secretion (T secretion asymptotically estimated at zero pulsatile LH drive); LH efficacy defines maximal T secretion at asymptotically infinite LH

concentrations; ED50 signifies the LH concentration inducing one-half maximal T secretion and target-cell (testicular Leydig-cell) sensitivity is the maximal absolute slope of the dose– response relationship (the incremental T-secretory response to a unit increase in LH concentrations). A suggested formalism for neuroendocrine dose–response interfaces (defined by functions) is to relate the agonist (or inhibitor) concentration to the response secretion rate (rather than response concentration) (Keenan and Veldhuis, 2003, 2004; Keenan et al., 2009a; Keenan et al., 2004b, 2006; Liu et al., 2005a; Veldhuis et al., 2008). This is because agonist/inhibitor input to a receptor is proportionate to the hormone concentration, whereas the glandular response is fundamentally a secretion-rate change. The question thus emerges, How is secretion estimated from a series of concentration measurements? This query is significant in neuroendocrinology, inasmuch as neuronal or glandular secretion rates are not usually directly measurable without invasive procedures to quantify both time-varying blood flow (e.g. at the pituitary, testis or hypothalamus)

87

concentration profiles into predicted (rather than observed) secretory bursts and basal (non-pulsatile) secretion. The predicted secretion profiles are depicted below each measured concentration profile.

and sample-by-sample neurohormone concentrations simultaneously. Both measurements must be accomplished at sufficient frequency to avoid censoring (partially or entirely overlooking) secretory bursts. In large animals like the horse, sheep and pig, highly frequent blood samples can be obtained over more prolonged periods. Fig. 5 depicts an exceptional record of GnRH, LH and FSH concentrations (top) measured simultaneously every 0.5 minute (30 seconds) in the conscious, procedurally adapted, unanaesthetized female horse during the luteal phase. Directly measured data are here termed observed. By an indirect process referred to in mathematics as deconvolution analysis, one can deconvolve (decompose, unravel) the three hormone

Estimating hormone secretion from concentration data Concept of deconvolution Deconvolution analysis is a computer-assisted mathematical technique to estimate underlying augmentative and dissipative functions, which gave rise to fluctuating data (Keenan et al., 2001,

Validating LH pulse trains: three types

(a)

(IU/l)

12

TN *

8

LH infused (N = 5 pulses) FP *

4 0 0

100

200

300

400

500

600

(b)

Illustrative in vivo validation of LH-pulse detection 10-min data

(IU/l)

8

1.0

6

FP *

FN *

4 2

0.8

0 0

200

400

40

800

1000 1200 1400

LH Sheep (N = 7 pulses)

(ng/l)

60

600

FN *

FP *

20

Sensitivity

LH concentration

LH simulated (N = 16 pulses)

*

0.6 0.4

N=6 Sheep

0.2 Mean ± SD 0.0 0.95 1.0

0 0

50

100

150 200 250 Time (min)

300

350

1.0 1.0 Specificity

Variable-Gamma waveform

AutoDecon

1.0

* Cluste8

PULSE4

Fig. 5. Panel A. Three types of validating paradigms comprising 10-minute measured (top and bottom) and model-simulated (middle) LH concentrations. Arrows mark pulses that were estimated by deconvolution analysis. Top. Profiles of LH concentrations created by pharmacological blockade of GnRH action using ganirelix and intravenous infusion of five consecutive pulses of recombinant human LH in a young man. Middle – Computer-simulated LH profile-comprising 16 true pulses. Bottom – Jugular-venous LH pulses (N = 7) corroborated in an ovariectomized ewe by simultaneous portal sampling of GnRH. The profiles are chosen to illustrate true-negative (TN), true-positive (TP), false-negative (FN), and false-positive (FP) pulses. Asterisks above the data indicate the locations of TN and TP pulses as well as FN or FP errors. Panel B. Sensitivity (y axis) and specificity (x axis) of LHpulse detection illustrated for three publically available deconvolution models: variable-waveform (Gamma) model (Chattopadhyay et al., 2008; Keenan et al., 2003b), AutoDecon and PULSE 4 (Veldhuis et al., 2008), and one discrete peak-detection method (Cluster8) (Veldhuis & Johnson, 1986). TP pulses were based upon simultaneous GnRH and LH sampling in the portal and pituitary blood of six ovariectomized sheep, as described in (Liu et al. 2009).

88

(c)

Validation of GH-pulse detection by pulsatile GHRH injections

N = 304 GH pulses (N = 19 men) 100

92

90

91

91

Sensitivity

Specificity

Positive

Negative

Percentage (%)

80

60

40

20

0

Predictive accuracy 1.8 Observed Predicted

1.6

GH concentration (µg/L)

1.4 FP

1.2 1.0

FN

0.8 0.6 0.4 0.2 0 0800

1400

2000 Clock time (hour)

0200

0800

Fig. 5. (Continued) Panel C. Discriminative indices (top) of variable-waveform (Gamma) deconvolution analysis using a total of 304 TP GH pulses induced by repetitive bolus injection of GHRH in 19 men (Iranmanesh et al. 1998). Pulsatile GH profile illustrated in one man (bottom). Sensitivity, specificity and (positive vs. negative) predictive accuracy are defined in Veldhuis et al. (2008). Unpublished line drawings.

2004a; Liu et al. 2009; Veldhuis et al., 1987a). For example, in seismology, a recorded earth tremor (defined by episodic excursion of a pen writing on

a revolving drum) might be deconvolved to estimate the primary shock wave (an augmentation process), and the associated attenuation

89

(dissipation) process acting on the propagated shock wave en route to the seismograph. The seismic deconvolution problem is solved using logical choices of mathematical functions for augmentation and attenuation. In relation to pulsatile hormone-concentration profiles, the goal is to reconstruct unobserved basal and pulsatile secretory contributions to the profiles and (biexponential) elimination kinetics of secreted hormone, while allowing for random measurement error. Deconvolution analysis is concerned with recovering (estimating) secretion and elimination functions from serial concentration data. When the elimination function is known a priori, the deconvolution problem entails only estimating secretion, and thus is greatly simplified (Evans et al. 1992; Giustina and Veldhuis, 1998; Urban et al. 1988; Veldhuis et al., 2006, 2008). Usually one assumes a general form for random (stochastic) error affecting the measurements (e.g. Gaussian variations). In some circumstances, neither the secretion nor the elimination function is known under the particular conditions being studied. Deconvolution analysis is still possible if one assumes some plausible, empirically relevant, shape for underlying secretory bursts. An early methodology of this kind assumed a Gaussianlike symmetric secretory-event waveform (time course of secretion rates within a burst) (Veldhuis et al., 1987a). Direct sampling of pituitary venous blood and in vitro perifusion of endocrine cells revealed that the Gaussian assumption was often violated, as shown in Fig. 5 (top) (Clarke et al., 2002; Keenan et al. 2004a, 2009a; Veldhuis et al., 2002, 2006; Veldhuis et al., 2007). To resolve this obstacle, one can employ variablewaveform deconvolution analysis (Keenan and Veldhuis, 2004; Keenan et al., 2001, 2003b, 2004a, 2005; Liu et al. 2009). In one such technique, a so-called generalized Gamma density (probability function) replaces the Gaussian distribution of secretion rates constituting bursts. The variable-waveform (Gamma) methodology includes one more parameter than the two-parameter Gaussian (defined by mean and standard deviation (SD)). The three parameters introduce flexibility by allowing variable (1) steepness of

burst onset (ascent), (2) peakedness of the burst and (3) steepness of burst offset (descent): Fig. 5 (bottom). The variable-waveform concept thus embodies a continuous spectrum of possible secretory-burst shapes, as well as nearly symmetric Gaussian shapes as a subset. If the release process is relatively uniform in any given hormone time series, a normalized secretory waveform can be estimated as an average of all bursts. In this simplification, bursts differ from each other primarily in size, rather than in both shape and size. One may then formulate pulsatile secretion as the sum of homogeneously shaped bursts (normalized waveforms), each burst having a mean mass modified by a random effect to incorporate pulse-by-pulse biological variability in burst size. Accordingly, hormone-concentration profiles can be represented by the convolution integral (integrated product) of the pulsatile secretion function and the elimination function plus measurement error, as follows:

Zt XðtÞ ¼

ðAe  1 ðt  r Þ þ ð1  AÞe  2 ðt  r Þ Þ

0

ð3Þ

 PðrÞdr þ Error where P(r) is the pulsatile production (secretion) rate function comprising the summed products of secretory-burst shape (flexible Gamma density) and individual secretory burst mass with a random effect (Fig. 6). The resultant model of combined basal (b0) and pulsatile hormone secretion with biexponential elimination is then simply:


X ðt Þ ¼ 0 

 A 1A  1 t  2 t ð1  e Þþ ð1  e Þ 1 2

Zt   þ Ae  1 ðtr Þ þ ð1  AÞe  2 ðtr Þ 0

P ðr Þdr þ Error Conc ¼ Basal sec þ Elimination þ Pulsatile sec þ Error

ð4Þ

90

Properties of secretory-burst waveform Generalized gamma density Mode = β2[β1–

Normalized secretion rate (probability)

Peakedness β3

Upstroke β1β3

1 β3

1/β3

Downstroke β3/β2

Time (min) Fig. 6. Simplified schema of a variable-waveform (generalized Gamma probability density) secretory-burst model defined by three (beta) parameters, which together control upstroke steepness, peakedness, the mathematical mode (time-delay from onset to maximal secretion rate) and downstroke steepness. A Gaussian model would have two parameters, enforcing strict symmetry. Unpublished line drawing.

This formalism applies when the pulse locations are known or estimable in the hormone time series.

Detecting putative pulses A major difficulty had been valid estimation of pulse times (burst onsets), which in earlier approaches were nominally identified by thresholds [e.g. a 20% increase in hormone concentration or three times the intraassay coefficient of variation (CV)]; cluster analysis (an unpaired t-test); a consecutive string of three or more positive residuals and by other plausible but empirical methods (Veldhuis et al., 2008). One recent alternative is incremental smoothing using a non-linear modification of the heat equation, wherein the hormone-concentration profile is repeatedly scanned by successively dropping the weakest (most-shallow) potential nadir before pulse onset. The idea is to create an exhaustive family of possible pulse-time sets

(Chattopadhyay et al., 2008; Keenan et al., 2005; Liu et al. 2009), as illustrated in Fig. 7. Candidate sets include the maximal possible number of pulse onsets [‘dips’, defined by a change in first derivative from negative (downslope) to positive (upstroke of pulse)], as well as successive sets each containing one less candidate pulse time. The ultimate result of infinite smoothing would be a straight line with no pulses, and the penultimate set would contain the single most dominant pulse, and so on. This reductio ad absurdum exercise creates a compendium of potential pulse-time sets, such as sets containing 21, 20, 19,…1 pulses. Deconvolution analysis (Equation 4) is applied to each candidate pulse-time set in the family to estimate secretion, elimination and random effects. Thereafter, a statistical comparison is made among the candidate fits by formal criteria, such as the Akaike information criterion (AIC) or Bayesian information criterion (BIC), as discussed in Chattopadhyay et al.(2008). By this means, possible pulsetime sets are first obtained independently;

91

(a)

Maximum-likelihood deconvolution construct

Concentration

Secretion rate

Concentration

Gamma density

Stochastic error

Biexponential decay +

*

Time

Time

Time

Time

Integrate

(b)

LH Peak identification by nonlinear diffusion

LH Con (IU/L)

5.0

Beginning putative pulse times No of pulses: 36

2.5 0 0 40

200

400

600

800

1000

Putative pulse times, as the algorithm runs 2

N = 36

1 3

4

5

30 Pulse time sets

1200

20

10 N=6 0

0

200

LH Con (IU/L)

5.0

400

600

800

1000

1200

No of pulses at 3000 steps: 6

2.5 0 0

200

400

600

800

1000

1200

Time (min)

Fig. 7. Panel A. General concept of deconvolution analysis. A pulsatile hormone-concentration profile (here illustrated by one peak, far left) is deconvolved (decomposed) into a succession of secretory bursts of individual sizes and of common shape (flexible Gamma probability waveform) (left middle). Each secreted molecule is subject to diffusion, advection and metabolism (described by biexponential kinetics, right middle). Measured concentrations are confounded by random (stochastic) procedural and assay errors (far right). Panel B. Example of (non-linear heat equation-based) incremental smoothing applied to detect possible pulse-onset times (asterisks) in an LH profile (top). All nadirs (N = 36) are marked initially by first-derivative changes (vertical green bars on x axis at top and columns of blue asterisks in middle). Algorithmic iterations then successively eliminate the mathematically most diminutive nadir one at a time, yielding multiple decremental sets of potential pulse times (successive rows of asterisks). Circled numbers show the time-locations of successively deleted pulse onsets. Deconvolution analysis is performed on each candidate pulse-time set using a non-linear mixed-effects model conditioned on pulse times. The Akaike information criterion is applied to distinguish the optimal pulse-time set (bottom), which in this instance comprised 10 pulses (red plus signs on x axis, bottom).

92

(c)

LH-pulse detection surface

1.0

Normalized LH con (IU/L)

0.8

0.6

0.4

0.2

0 3000 1500 2000 1000 Algorithmic iterations

1000

500 0

0

Observational time (min)

Fig. 7. (Continued) Panel C. Three-dimensional rendering of 3000 successive algorithmic pulse-set searches applied to (unitnormalized) time-varying LH concentrations (z axis). Candidate LH pulse-time sets (blue asterisks) are defined in observational time (x axis) as a function of algorithmic iterations (y axis). Infinite iterations would ultimately smooth away all LH fluctuations yielding a zero-slope plane of z-intercept 0.5. Unpublished drawings.

deconvolution-parameter estimates are mathematically conditioned on a priori candidate pulse-time sets; and, the final model is selected objectively with a penalized AIC or BIC, which penalty includes the number of pulse times (Veldhuis et al., 2008). A statistical penalty is necessary for adding pulses, because an arbitrary model could fit any data set if a high number (e.g. N–1 for N data points) of pulses or parameters were allowed. That type of fit would provide little biological information.

Validation required for deconvolution analysis Any deconvolution model should be validated empirically and demonstrated to be realizable

(solvable) mathematically (Veldhuis et al., 2008). Fig. 8a illustrates three empirical validation paradigms,namely infused pulses of recombinant LH in a man after pharmacological inhibition of LH secretion; computersimulated LH-pulse profiles and LH pulses in an ovariectomized ewe, wherein true LH pulses are identified by matching GnRH peaks assayed in pituitary-venous blood. For these three particular validation models, one recent methodology achieved 93% sensitivity and 94% specificity (Liu et al. 2009). Mathematical verification of realizability requires direct proof of parameter asymptotics (Chattopadhyay et al., 2008; Keenan et al., 2005).

93

Analytical reconstruction of LH pulse-renewal process Late follicular

Post-menopausal 60

LH concentration (IU/L)

8

6 40 4 20 2

0

0 0

500

1000

0

1500

20

20

Weibull density –3 (x 10 )

1500

Time (min)

Time (min)

Probability of time delay

15

15 10

10

lambda = 20.4 gamma = 2.0

5 0

1000

500

lambda = 18.4 gamma = 4.2

5 0

0

50

100

150

200

250

Interpulse interval (min)

300

0

50

100

150

200

250

300

Interpulse interval (min)

Fig. 8. Variable-waveform (Gamma density) deconvolution analysis applied to LH time series in two women, one of pre- (left) and one of post- (right) menopausal age. Vertical bars on the x axes denote significant pulse-onset locations over 24 hour. Interrupted curves give deconvolution-estimated LH concentrations (top). Interpulse-interval lengths (times in minutes) are plotted below, according to a flexible two-parameter Weibull probability distribution (as distinct from a 1-parameter Poisson process where SD = mean). Lambda, number of pulses/24 hour; gamma, measure of variability, wherein higher values denote less variability (maximal variability occurs when gamma =v1, defining an exponential Poisson random-renewal process). Adapted from Veldhuis et al. (2008; Fig 6B).

Physiological information gained by deconvolution analysis The purposes of deconvolution analysis are to gain greater insights into the regulation of otherwise unobserved pulsatile secretion; account for variations in hormone elimination half-times among conditions and subjects and quantify the apparently basal (non-pulsatile) component of hormone release, which putatively represents a constitutive or sparingly regulated (short-term

stable) mode of secretion. A flexible-waveform deconvolution methodology also allows quantification of secretory-burst shape, namely relative contributions of initial and delayed secretion (burst ascent before and descent after maximal secretion). Estradiol, GHRH, ghrelin and somatostatin appear to determine the waveform of GH secretory bursts, doubling (agonists) or halving (somatostatin) the mode (time delay from burst onset to maximal secretion) (Veldhuis and Keenan, 2008; Veldhuis et al., 2007).

94

Deconvolution also permits post hoc estimation of the probability distribution of interpulse intervals (waiting times between successive secretory bursts), after optimal pulse-time selection via AIC or BIC. Fig. 8b illustrates interpulse waiting-time distributions for LH in a young woman studied in the latefollicular phase of the menstrual cycle (left) and a post-menopausal woman (right) (Keenan et al., 2003a). Interpulse-interval times conform well to a two-parameter Weibull renewal process (type of random distribution). The two Weibull parameters, lambda and gamma, define mean pulsing rate (frequency) and variability of interpulse-interval lengths (unitless). This model is much more general than the classic one-parameter Poisson process, in which the mean pulsing frequency definitionally equals the pulsing SD (Keenan et al., 1998, 2000). Therefore, the variability (coefficient of variation = SD/mean  100%) of interpulse-interval times in a Poisson process is always 100% (Keenan et al., 1998). Most critically, empirical data in most hormonal time series strongly violate the CV = 100% mandate, as noted below, necessitating an alternate and more general model framework than the Poisson process. The more flexible Weibull process includes the strongly exponentially right-tailed Poisson as a subset (when gamma = 1), and a nearly symmetric Gaussian (when gamma ! infinity) as extreme subsets, while allowing for continuous interpulse variabilities (CVs) between 0 and 100% (Keenan and Veldhuis, 2000). Flexibility is important, inasmuch as interpulse-interval CVs for hormones are as low as 8–15% for relatively regularly recurring parathormone and insulin pulses and 30–65% for irregular pituitary, adrenal and gonadal-hormone pulses. Illustrative Weibull estimates for LH pulsatility of gamma = 2.0 in a premenopausal and gamma = 4.2 in a post-menopausal woman (Fig. 8b) quantify much more consistent (less variable) interpulse-interval lengths in the menopausal context, when ovarian negative feedback is largely silenced (Keenan and Veldhuis, 2000; Keenan et al., 2003a). Analogously, older men compared with young men exhibit greater uniformity (less variability) in LH interpulse-interval lengths (higher gamma), and also manifest increased mean LH pulse frequency (higher lambda) (Keenan &

Veldhuis, 2001). The extent to which altered GnRH/LH pulse-generation properties in older women and men reflect reduced gonadally derived negative feedback vis-à-vis attenuated intrahypothalamic restraint of GnRH pulsing is not yet known.

Control of a neuroendocrine ensemble Concept of deterministic dynamics Neuroendocrine dynamics extend beyond regulation of basal and pulsatile hormone secretion and elimination. A hallmark of hormonal ensembles is homeostatic control. Homeostasis is transduced via pathway-specific time-delayed dose–response interfaces, which link hormone concentrations to target-gland secretion rates. Ensemble is defined here as a collection of interacting elements, such as GnRH, LH and T, which exert and are subject to mutual control, thereby conferring dynamic connectivity and adaptive homeostasis. Fig. 9a schematizes basic connectivity within the malegonadal ensemble, wherein (1) GnRH pulses drive LH pulses; (2) LH pulses stimulate T pulses, and maintain basal T secretion; (3) T upon entering the blood associates with and dissociates from sex hormone-binding globulin (SHBG) or albumin or remains free (unbound); (4) systemic T concentrations repress hypothalamic GnRH burst size and number by negative feedback; and (5) aromatization of T to E2 inhibits GnRH-driven pituitary LH secretion non-competitively (not shown). According to this simplified model, quantifying homeostatic behavior of the male GnRH– LH–T axis would require at a minimum estimating four implicit dose–response functions that subserve how (1) GnRH concentrations stimulate LH secretion; (2) LH concentrations drive T secretion (abbreviated LH ! T); (3) T concentrations inhibit GnRH pulse frequency and (4) T concentrations suppress the amount (mass) of GnRH released per burst (Keenan and Veldhuis, 2009; Keenan et al. 2006). Other dose–response functions not yet incorporated into existing analytical constructs would entail E2 (derived from T) acting on kisspeptin neurons to repress drive of

95

(–)

[GnRH]

Hypothalamus Circulation

(+)

Time

[GnRH]

(a) Frequency Mass

[T]

Pituitary gland

[LH] (+)

[LH]

LH Feedforward on T

T sec

Potency/Sensitivity/Efficacy Slope

ED50

(–)

(–)

Time

Maximum

[Estradiol]

Construct of GnRH–LH–testosterone ensemble

(–) T Feedback on GnRH and LH

[LH] (+) Total [T]

(+) Time

Free T SHBG-T Albumin-T

(–)

(b)

Fate of secreted testosterone Gonadotropes

Leydig cells Te Secr Rate

LH

LH

Spermatic vein Te Secr Rate Time

Time Exchangeable plasma space k1

Te

SHBG-Te + SHBG k2

k3

k4

Albumin-Te + Albumin k5 Elimination

Diffusion Advection Binding Metabolism

Fig. 9. Panel A. Construct (model) of male GnRH–LH–T ensemble, defined by specific signals, time delays, and dose–response interfaces. Hypothalamic GnRH neurons (top center) discharge the homonymous peptide into portal microvascular blood (circulation). GnRH pulses drive LH secretory bursts at the pituitary level (middle center). LH concentrations feed forward onto (stimulate) testicular Leydig cells via implicit (estimable) LH ! T dose–response functions, defined by potency (inversely related to ED50), sensitivity (slope) and efficacy (maximal T secretion rate). Blood-borne T may remain free (unbound) or bind to SHBG (high affinity, low capacity) or albumin (low affinity, high capacity) (bottom row). T feeds back onto (inhibits) GnRH-stimulated LH secretion at the pituitary level after aromatization to estradiol (right middle), and onto GnRH secretory-burst size (mass) and GnRH secretory-burst frequency (right top). T may act via native T, 5 alpha-reduced T and estradiol via distinguishable dose–response functions. The simplified ensemble is amenable to model-based analysis (Chattopadhyay et al., 2008; Keenan & Veldhuis, 2009; Veldhuis et al., 2008). Neuroendocrine pulsatility and rhythmicity are endowed by the interactions, rather than by any single locus operating in isolation. Unpublished line drawing. Panel B. Basic fate of T secreted into the bloodstream under pulsatile LH drive, yielding spermatic-vein T pulses superimposed upon basal T secretion (top). T in plasma is available to diffusion (random molecular motion), advection (linear motion due to blood flow), binding to plasma proteins, and irreversible loss (metabolism, bottom). Adapted from Veldhuis et al. (2008; Fig. 4).

96

GnRH and on the pituitary to non-competitively block GnRH’s stimulation of LH (and FSH) secretion (Schnorr et al., 2001). In addition to creating dose–response functions, one must correctly partition T’s binding to and release from carrier proteins in the circulation, as illustrated in Fig. 9b. In one analytical construct, calculations of free, SHBG- and albumin-bound T concentrations proceed simultaneously with estimation of T’s secretion and elimination rates as well as the LH ! T dose–response function (Keenan and Veldhuis, 2004, 2009; Keenan et al. 2006). Necessary data for such analyses comprise serial concentrations of LH and total T, a pooled estimate of SHBG and albumin concentrations for the time series, and published rate constants (k values) for T’s binding to and dissociation from the two main carrier proteins, SHBG and albumin.

Random (stochastic) facets The ensemble (network-like) properties of GnRH–LH–T interactions are paradigmatic of other neuroendocrine systems, in which homeostasis is maintained via time-delayed feedforward (stimulatory) and feedback (inhibitory) connections. The connections (pathways) comprise plasma hormone concentrations (determined fivefold by distribution volume, diffusion, advection, metabolism and secretion rates), which stimulate or inhibit a target gland via unobserved but estimable dose–response functions (Keenan and Veldhuis, 2009, Keenan et al., 2001, 2004a, 2006, 2009a). In the human male gonadal axis, concentrations of LH and T are measured, whereas those of GnRH must be estimated simultaneously with all GnRH– LH–T elimination, secretion and dose–response features by analytical means. In addition to deterministic (rule-based) facets of a closed-loop (fully interconnected) neuroendocrine system, randomness (stochastic variability) enters into overall dynamics (Fig. 1b). Sources of random variability are both technical (e.g. unwitting inconsistencies in the precise timing of blood samples, sample aliquoting, freezing, thawing, pipetting and assay, including data reduction)

and biological (e.g. apparent randomness of interpulse-waiting times, successive pulse sizes, hormone diffusion). An important aspect of model formulation is judicious placement of delimited allowable random effects to reflect essential de facto variability in the system. Imposition of excessive random effects would erode determinability of key parameters, whereas omission of true random effects could bias model estimates (Veldhuis et al., 2008). Several recent ensemble models allow random effects (stochastic variability) principally in hormone measurements, consecutive burst-mass values, sequential interpulse-interval lengths and pulse-by-pulse dose–response efficacy (or potency or sensitivity, separately). The mathematical determinability of key parameters in such constructs has been verified (Chattopadhyay et al., 2008; Keenan et al., 2000, 2001, 2004a, 2005, 2009a). Allowable pulse-by-pulse dose–response variability is consonant with a myriad of empirical observations in vitro and in vivo, showing non-uniformity of sequential secretory responses to fixed or similar recurrent stimuli or inhibitors (Evans et al. 1992; Giustina and Veldhuis, 1998; Urban et al. 1988; Veldhuis et al., 2006, 2008). Non-uniformity of endocrine responses to successive signal inputs may represent fluctuating potentiation and attenuation of target-cell response mechanisms, originating at the level of receptor–effector coupling and later steps in the signalling response. Fig. 10 depicts the analytical outcome of incorporating statistical allowance for possible random (stochastic) pulse-by-pulse variability in LH ! T feedforward dose–response (1) potency (left), (2) efficacy (middle) and (3) sensitivity (right) in an intact ram. The random-effects models are depicted separately, inasmuch as methods do not yet exist for reliably partitioning random effects among the three stochastic models simultaneously. Helpful experiments to address this issue might include direct in vivo monitoring of Leydig-cell T-secretory responses to an experimentally controlled train of LH pulses, and analogously direct sampling of adrenal zona-fasciculata cortisol secretory responses to a prespecified pulsatile ACTH clamp.

97

Reconstruction of GnRH, LH and testosterone interactions GnRH

30 20

Conc (ng/L)

40

0.8 0.6 0.4 0.2

10 0 200

400

600

0

800

0

0.8

Potency shift

0.6 0.4 0.2

0

0.5

200

400

600

4 2 0

800

1.0

0.8

0

Efficacy shift

0.4 0.2

LH signal (µg/L)

0

0.5

1.0

LH signal (µg/L)

200

400

600

800

Time (min)

0.6

0

----

6

Time (min)

T sec rate (µg/L/min)

T sec rate (µg/L/min)

Time (min)

0

Observed 8 Predicted

----

T sec rate (µg/L/min)

Conc (ng/L)

Observed 1.0 Predicted

Ram ----

50

0

10

1.2

Conc (ng/L)

Observed Predicted

60

Testosterone

LH

70

0.8

Sensitivity shift

0.6 0.4 0.2 0

0

0.5

1.0

LH signal (µg/L)

Fig. 10. GnRH–LH–T system sampled invasively every 10-minutes in a conscious ram. Top row shows measured (observed, continuous lines) and estimated (deconvolution-predicted, interrupted lines) hormone-concentration profiles. The bottom row (left-to-right) depicts analytically reconstructed LH ! T dose–response curves allowing separately for possible random effects on potency, efficacy or sensitivity. Different curves in each panel arise from allowable pulse-by-pulse random effects on the indicated LH-Te dose–response parameter. The concept is discussed further in Keenan et al. (2004a). Unpublished plots.

System-level adaptations in pathophysiology The neuroendocrinologist’s goal is often to discern development-, gender-, age-, nutrition-, sleepwake- and activity-related as well as pathophysiological mechanisms that supervise a complex hormonal system. Models exist at all levels of organization a shown in Table 5. For example, using the human GnRH–LH–T system as an example of multipathway control, one could ask how age determines the implicit (unobserved but estimable) dose–response properties of GnRH ! LH stimulation, LH ! T feedforward and T ! GnRH feedback (Keenan et al. 2006). The general ensemble concept in this case may be amplified by incorporating the potential effect of age as a regression parameter (Keenan and Veldhuis, 2009). Fig. 11 illustrates the outcome of such analyses in 24 healthy men aged 20–72 years. Age

decreases each of LH ! T dose–response potency (increases ED50, Panel A), efficacy (maximal T response, Panel B) and sensitivity (maximal positive slope, Panel C) by 49–78%. The power of the cohort-regression strategy derives from the use of all data sets simultaneously in making a global estimate of the impact of a physiological variable like age. The same could be done for other covariates like body-mass index, abdominal visceral fat, insulin concentrations inter alia.

Appraisal of negative feedback in a neuroendocrine system Several approaches exist to quantify changes in negative feedback. One method is to estimate analytically the inhibitory action of an observed signal (e.g. measured T concentrations) on an

98 Table 5. Illustrative model forms Primary model

Structure

References

(a) Subcellular Receptor activation GnRH signalling

Pulse vs. constant PTH Post-receptor events Beta-cell insulin secretion Pancreatic beta cell

Potter et al. (2005) Scullion, Brown, & Leng, (2004), Washington, Blum, Reed, & Conn (2004) Kennedy, Kauri, Dahlgren, & Jung (2002) Bergsten (2002)

Autofeedback

Li & Khadra (2008)

Feedback and delay Ensemble construct Non-linear dynamics

Farhy & Veldhuis (2003, 2004, 2005) Keenan et al. (2004a, 2006) Clark, Schlosser, & Selgrade (2003), Rasgon et al. (2003) Keenan et al. (2001, 2009a), Lenbury & Pornsawad (2005) Fricke, Lehmkuhl, & Pfaff (2006)

Metabolic oscillations Calcium oscillations (b) Cellular GnRH firing synchrony (c) Pulsatile Ensembles GH pulsatility LH pulsatility Menstrual cycle ACTH pulsatility

Ensemble model

Feeding/satiety signals (d) Target Gland Feedforward drive

Iinterconnectivity

Toxic glucocorticoid damage Receptor desensitization (e) Circadian Circadian clocks (f) Circannual Circannual

ACTH ! cortisol, GH ! IGF-I, LH ! T Systems biology

Giovannini, Marzetti, Borst, & Leeuwenburgh (2008)

Continuous PTH signal

Homme et al. (2009)

Phosphorylation and feedback

Leloup (2009), Miller et al. (2004)

Melatonin-lactotrope

Lincoln, Clarke, Hut, & Hazlerigg (2006)

observed or an unobserved signal (e.g. LH concentrations or hypothalamic GnRH outflow) (Keenan et al. 2006). This requires assuming an ensemble-like dynamic structure for the GnRH– LH–T or other axis. Ensemble-based feedback analyses have been performed using paired LH– T time series in men aged 20–72 years, yielding the inference that age reduces the efficacy of T’s feedback onto endogenous GnRH-dependent LH secretion by about 42% (Keenan and Veldhuis, 2009). In the same model, age did not affect T’s concentration-dependent inhibition of GnRH–LH pulse frequency. Estimated outflow (combined secretion and action) of the unobserved but analytically estimable (virtual) GnRH signal declined by 71% with age. The model also reconstructs pulsatile LH secretion rates as a joint function of virtual GnRH concentrations (via a stimulatory

Keenan et al. (2004a, 2004b), Sun et al. (1999)

three-parameter logistic function) and free T concentrations (via an inhibitory one-parameter exponential function) (Fig. 12). The resulting estimates are interrelated by a three-dimensional response surface. In other analyses, statistical comparisons by age stratum confirmed that responses of LH secretion to virtual GnRH feedforward and free T feedback are markedly diminished in eight older men (aged 50–72 years) compared with 10 young men (aged 20–35 years) (Keenan et al. 2006). The conceptual framework illustrated for the male GnRH–LH–T axis is expressly relevant to other neuroendocrine ensembles. Examples comprise the female GnRH–LH–progesterone– estradiol axis by inclusion of two sex-steroid feedback functions (Evans et al. 1992); the CRH–AVP–ACTH-cortisol axis via allowance

99 Potency of LH decreases (ED50 increases) with age 250 200 150 100 50 0 80 70

N = 24

60

Feedforward efficacy of LH declines with age

(b) T secretion (ng/dL/8 hour)

T secretion (ng/dL/8 hour)

(a)

50

40

Age (years)

30

20

2

0

N

200

= 24

100

8

6

4

300

0 80 70

60

50

40

30

LH conc (IU/L)

LH conc (IU/L)

300

80 years

2.2

1.8

50

Efficacy parameter

P = 0.023

150 100

T secretion (ng/dL/8 hour)

20 years

200

20 year 200

100

80 year

50% 0

0

280

2.6

ED50(IU/L)

T secretion (ng/dL/8 hour)

250

8

6

4

2

20 0

Age (years)

0

2

4

6

1.4 20

8

30

40

50

60

70

0

2

(c)

6

8

200 160

49%

120 20

30

40

50

60

70

80

80 Age (years)

LH conc (IU/L)

Age (years)

LH conc (IU/L)

4

P < 0.005

240

T secretion (ng/dL/8 hour)

Leydig-cell sensitivity to LH decreases with age 300

N = 24

200 100 0

80

70

60

50

40

30

20

Age (years)

20 years

200

100 80 years 0

LH conc (IU/L)

1.4

Sensitivity parameter

T secretion (ng/dL/8 hour)

300

8

6

4

2

0

0

2

4

6

LH Conc (IU/L)

8

P < 0.01 1.0

0.6

78% 0.2 20

30

40

50

60

70

80

Age (years)

Fig. 11. Populational (full-cohort) non-invasive analysis of impact of age in 24 men on analytically estimated LH ! T feedforward dose-responsiveness using serial measurements of both hormones. Separate figures present the LH ED50 (an inverse measure of potency, Panel A), LH efficacy (maximal T secretion rate, Panel B), and testicular Leydig-cell sensitivity to LH (maximal positive slope of the dose–response, Panel C). Top. Regression of LH ! T logistic dose–response function linearly on age. Bottom left. Plot of LH ! T dose–response parameter isobars at extrapolated ages of 20 and 80 years. Bottom right. Regression of individual dose– response parameter on age. Adapted from Keenan & Veldhuis (2009; Fig. 4).

for two hypothalamic feedforward functions in the horse and a combined CRH–AVP feedforward signal in the human (Keenan et al., 2001, 2004b, 2009a, 2009b); the GHRH–ghrelin–somatostatin–GH system, as foreshadowed in a recent simulation model that still requires an analytical component (Farhy and Veldhuis, 2005); the TRH–TSH–thyroxine/triiodothyronine ensemble; and the osmolality–baroreceptor–AVP (ADH) system, among others. These neuroendocrine concepts have thematic analogy to some pharmacodynamic models, which quantify drug kinetics and tissue responses concomitantly (Conolly and Andersen, 1991; Dahl et al. 2010).

Ensemble estimates of neuroendocrine dynamics One might ask whether reliable integrative measures of ensemble performance exist, which may be applied without prior knowledge of underlying structure of the system. Two main tools allow model-free estimates of altered neuroendocrine dynamics, even when system connectivity, time delays, dose– response functions and kinetics are not known, namely neural networks and approximate entropy (Veldhuis et al., 2008). These methods have different discriminative bases and relative limitations.

100

Impact of age on T’s repression of GnRH Fdfwd 1.5

Young men (20–35 year)

LH secretion rate (IU/L/min)

1.0 0.5 0 10

10

5 00 1.5

5

Older men (50–72 year)

Approximate entropy

1.0 0.5 0 10 5 Virtual GnRH signal

10 0 0

need to specify any distinct model structure. The method has been applied to discriminate pituitary tumoural (acromegaly associated) from normal GH secretory patterns and pathological from physiological glucose profiles (Prank et al., 1996, 1998). Further work is needed in this area to enhance replicability.

5 Free T conc (ng/dL)

Fig. 12. Three-dimensional response surface depicting the joint dependence of LH secretion rates (z axis) on (i) nonlinear (3-parameter logistic) feedforward by estimated (unobserved) GnRH concentrations (y axis) and (ii) exponential feedback (inhibition) by calculated free (unbound) T concentrations (x axis). Data are from 10 young (ages 20–35 year) and 8 older (ages 50–72 year) men. Age is associated with decreased GnRH outflow and attenuated T negative feedback. Adapted from Keenan et al. (2006; Fig. 3a).

Neural networks Advances in artificial intelligence and oncologic prediction strategies have introduced and illustrated the use of empirical neural-like networks, structured mathematically much like multiple-regression or polynomial-based equation systems (Brabant and Prank, 2000). Adjustable coefficients are employed to represent variable strengths of multiple potentially relevant factors acting via one or more tiers of pathways. The pathways, in the broadest sense, emulate interneuronal connections. The neural network must be trained on representative data sets to tune the linking coefficients, and then tested on a second independent set of data before application to unknown data. The purpose is to discriminate aggregate features in the data that are not readily defined by usual model parameters. A theoretical limitation of the artificial neural-network approach is reproducibility across data sets in the same and different laboratories. An advantage is the lack of

Approximate entropy (ApEn) is an ensemble measure of the relative randomness vis-à-vis regularity of time series (Pincus 1991, 2000b). The motivation is to distinguish between (hormone) time series that appear quite different in subpattern reproducibility, yet may have similar means and variances. ApEn provides a sensitive (>90%) and specific (>90%) statistic to discriminate degree of pattern consistency, orderliness or regularity (Pincus et al., 1999, 2000; Pincus et al. 1996a, 1997). ApEn is a single number between 0 (perfectly ordered, like a theoretic sine wave) and 2.30 (ln 10, maximally random). Technically, ApEn sums the marginal probabilities that subpatterns of any data length (e.g. m) recur within a tolerance (range) r upon next incremental comparison (at m þ 1). ApEn is the average value of the (negative) logarithms of conditional probabilities (all between 0 and 1.0). Thus, smaller ApEn signifies greater pattern reproducibility, and larger ApEn greater randomness (Pincus 2000a). The sensitivity of this metric is such that it can distinguish between the relative randomness of pi (ratio of circumference to diameter), a transcendental constant, and square root of two, expressed in either base 2 or base 10 to 100,000 decimal places (Pincus and Kalman, 1997). The utility of ApEn lies in its capability to discriminate between processes driven by different admixtures of deterministic (strictly repeatable) and random (causally unassignable) effects, as sketched in Fig. 13a. ApEn can be applied reliably to finite time series of length, N, obtained in lifescience experiments (Pincus and Singer, 1996), in contrast to usual entropy measures like the Kolmogorov–Sinai statistic that require nearly

(b)

(a)

Disorderliness and asynchrony in agling 2.0

(nearly random)

Intermediate entropy (measurable orderliness)

LH ApEn

High entropy

(Relative randomness)

Approximate entropy concept of regularity

LH concentration P < 0.001

1.5

+

1.0

+ (N = 13) 0.5

(highly ordered)

2.5 Cross-ApEn (Relative asynchrony)

Low entropy

(N = 13)

LH concentration vs NPT P < 0.001

2.0

1.5

1.0 (N = 10)

(N = 8)

Young

Older

0.5

Fig. 13. Panel A. Schematic depiction of approximate entropy (ApEn) concept. ApEn is a measure of the relative admixture of randomness in deterministic processes. Higher ApEn (top) denotes greater process randomness (less orderly or less reproducible subpatterns in the data) compared with the middle and bottom curves. Unpublished. Panel B. Individual ApEn and cross-ApEn as measures of single time-series randomness and paired time-series joint asynchrony, respectively. Top. LH ApEn in 14 young and 13 older men. Bottom. Cross-ApEn of overnight LH and nocturnal penile tumescence (NPT) measurements in 10 young and 8 older men. Higher ApEn and cross-ApEn values in older than young men indicate that age erodes the orderliness of LH secretion, and reduces the joint synchrony of LH and NPT oscillations. Adapted with permission from Veldman, Frolich, Pincus, Veldhuis, & Roelfsema (2000b; Fig. 3).

101

102

infinite and noise-free time series (Pincus 1991; Pincus et al., 1999). Moreover, ApEn confers both interpretability and reproducibility, namely higher ApEn (more irregular series) implies less strictly deterministic (rule-based, predictable) control, often due to loss of feedback regulation or erosion of pathway connectivity within the ensemble (Pincus 1994; Veldhuis et al., 2001a, 2001c). Reproducibility is achieved by defining ApEn as a family of statistics, each designated by (m, r, N), where m is the template size used in evaluating subpattern length; r is a normalized threshold range for testing subpattern similarity and N is the number of sequential observations in the time series. The threshold, r, is typically normalized to the overall variability of the time series by expressing it as a fraction of the series SD, for example 0.2 of the individual series SD for data series of length N > 60. The template size, m, is usually 1 for N < 300. Thus, typically ApEn (m, r, N) is defined by m = 1, r = 0.2  SD and N = 145 (10-minute samples collected for 24 hour). For neuroendocrine time series of length 17 < N < 60, suitable choices of r (at m = 1) have been published (Pincus et al., 1999). Fig. 13b (top) compares ApEn of LH concentration-time series in young and older men (Pincus et al. 1996b). Age remarkably elevates LH ApEn, denoting greater relative randomness (reduced network inputs or decreased negative feedback) in the LH-secretion process. Lesser reproducibility (greater disorderliness) of hormone patterns in ageing individuals is not restricted to LH secretion, but is also evident for T, FSH, GH, ACTH, cortisol and insulin (Friend et al., 1996; Meneilly et al., 1997; Pincus et al., 1997; Veldhuis et al. 2009; Veldman et al., 2000a). Prolactin, TSH, AVP (ADH), melatonin, glucagon, parathormone, estradiol and progesterone have not been evaluated in relation to age. Most endocrine tumours are marked by elevated ApEn of the cognate hormone (Hartman et al. 1994; Roelfsema et al., 2008; Siragy et al., 1995; Van den Berg et al., 1997; Veldman et al. 1999). ApEn also identifies a striking gender difference in young-adult GH secretion profiles, with greater irregularity in women than men and

in the female than male rat (Pincus et al. 1996a). ApEn of GH increases in midpuberty (compared with prepuberty and adulthood) in boys, and in subjects administered T or estrogen (Pincus et al., 2000; Veldhuis et al. 1997). Quantifiable differences in LH and T secretory regularity also emerge and recede across normal puberty in boys (Veldhuis et al. 2001b). The consistency of age-, gender-, nutrition- and development-related effects on the relative orderliness of GH and LH secretory patterns provides strong evidence of altered GHRH–ghrelin–GH–IGF-I and GnRH– LH–T feedback/feedforward ensemble control in different physiological settings. Relative feedback/ feedforward strength is an important determinant of ApEn, since experimental modification of negative feedback by T, cortisol, IGF-I and T4 onto LH, ACTH, GH and TSH secretion, respectively, significantly alters secretory orderliness (Veldhuis et al. 2001c). Moreover, fixed feedforward (e.g. by GHRH injections) elevates ApEn of stimulated hormone (GH) release. Analogously, in reductionistic networks (e.g. logistic, autoregressive moving-average, stochastic differentialequation and coupled-linear systems), attenuation or deletion of connectivity increases ApEn (Pincus 1991; Pincus and Goldberger, 1994; Pincus and Kalman, 1997). Because the ApEn calculation is independent of knowledge of underlying model structure and absolute scale, biological applicability is broad. For example, the model-invariance of ApEn has allowed analysis of complex EEG records with good discrimination between pathological and normal states (Srinivasan et al., 2007). In neuroendocrine time series, ApEn estimates have SDs of 0.06–0.08 (unitless), demonstrating high consistency (Pincus 2000a). Intraindividual test-retest reliability of ApEn is 8–12% based upon consecutive 24-hour GH time series in healthy adults (Friend et al., 1996), defining high biological reproducibility. The scale-independence of ApEn means that a uniform change in the absolute value of the data (even if multiplied by an arbitrary constant) does not alter the estimate. Analogously, ApEn is translation-independent, signifying that a constant could be added to or subtracted from each

103

measurement without changing ApEn. The foregoing characteristics ensure good discrimination between complex ensemble (network-like) dynamics. A quantitative inference of altered systems dynamics, in turn, invites the design of more probing mechanistic experiments to elucidate the nature (specific pathway, time delay or dose– response properties) of the alteration (Veldhuis et al., 2008).

Cross-approximate entropy A significant limitation of earlier analytical approaches to synchrony analysis (e.g. between GnRH and LH, or ACTH and cortisol) was reliance on linear methods requiring a fixed lag (time delay between samples in the two series being tested for synchrony) (Iranmanesh et al., 1989; Veldhuis and Johnson, 1988). Cross-correlation and cross-spectral analysis are classical linear methods (Davis 1988; Wei 1990). However, effector-response coupling lag times are not precisely uniform, even in the same timeseries pair (Veldhuis et al., 1994). Complementary to linear techniques is cross-approximate entropy (cross-ApEn), designed to appraise the degree of joint synchrony between parallel time series independently of time lag (Pincus 2000a, 2000b; Pincus et al. 1996b). Cross-ApEn is a bivariate extension of univariate ApEn (Pincus and Kalman, 1997). For good reproducibility, the paired time series are first normalized by zscore transformation. Analogous to ApEn, cross-ApEn takes the mean (negative) logarithm of the conditional probabilities that subpatterns of length m = 1 (in the first series) are matched by similar patterns of length m þ 1 (in the second series) within a threshold range r. Thus, cross-ApEn allows pathway-specific (pairwise) quantification of pattern synchrony. Examples are cross-ApEn of paired series like insulin–glucose, ACTH–cortisol, GH–cortisol, LH–T or LH–NPT (nocturnal penile tumescence, a marker of brainstem adrenergic outflow) (Charmandari et al. 2001; Pincus et al. 1996b; Roelfsema et al., 1998; Veldhuis et al., 1999, 2000). Higher

cross-ApEn quantifies greater asynchrony (less joint synchrony). This is illustrated by higher cross-ApEn of the LH–NPT pathway in healthy older than young men (Fig. 13 (bottom), thus delineating disruption of gonadal-axis synchrony in ageing (Veldhuis et al., 1999). Age-related erosion of joint hormone synchrony is also evident for LH–T, LH–FSH and LH–prolactin pairs in men (Liu et al. 2006; Veldhuis et al., 2000). Comparable neuroendocrine analyses are not yet available in women. The concept of directionally selective crossApEn was introduced recently (Liu et al., 2005a, 2005b). The computational difference is simply which member of the paired series serves as the template for pattern comparisons. The notion is illustrated by calculating ACTH–cortisol feedforward cross-ApEn and separately cortisol–ACTH feedback cross-ApEn (Fig. 14a). Both feedforward and feedback cross-ApEn values in the corticotropic axis increase with age in men (Veldhuis et al. 2009), signifying erosion of bidirectional corticotrope-adrenal secretory synchrony. Cortisol– ACTH feedback synchrony deteriorates (higher ApEn) with age in women also. Analogously, both forward and reverse cross-ApEn values in the male gonadal axis rise with age, denoting combined deterioration of LH ! T feedforward and T ! LH feedback synchrony (Liu et al., 2005a, 2005b) (Fig. 14b). Higher cross-ApEn with age provides non-invasive evidence of pathway-selective impairment in signalling within the corticotropic and gonadotropic axes. Recent refinements of ApEn and cross-ApEn computations allow estimation of individual SDs of ApEn and cross-ApEn for any given time series, wherein template matches (m and m þ 1) are randomly resampled (jackknife procedure). Resampled ApEn can be shown to reduce bias in time series of smaller N. Fig. 14c displays individual-subject probability distributions and joint (combined-cohort) distributions for LH ApEn in older and young men. Unpaired parametric and non-parametric comparisons of the distributions document higher LH ApEn in older than young men, signifying prominent curtailment of secretory regularity.

104

Reverse (inhibitor) template

Feedforward

Feedback

O

2.2

Reverse cross-ApEn

ACTH sec

Cortisol sec

O

2.0 1.8 1.6 1.4

Time

2.0 1.8 1.6 1.4

1.2

Time

(c)

P = 0.009

2.2

Time

O O

2.4 P = 0.02

O

Time

O

T→ LH inhibition

LH→T stimulation 2.4

Forward cross-ApEn

O

Loss of LH-T-LH synchrony in older men

(b)

Schema of bidirectional cross-ApEn analysis Forward (stimulus) template Cortisol con

ACTH con

(a)

1.2 Y

Y

O

O

Impact of age on ACTH-cort and cort-ACTH synchrony 2.0

ACTH-cortisol feedforward asynchrony

1.5

Asynchrony (synchrony loss)

1.0 0.5

P = 0.018 R 2 = 0.22

N = 26 men 0.0 20 2.0

30

40

50

60

70

80

Cortisol-ACTH feedback asynchrony

1.5 1.0 0.5 0.0 20

P = 0.021 R 2 = 0.20 30

40

50 60 Age (year)

70

80

Fig. 14. Panel A. Concept of directional (forward or reverse) cross-ApEn. ACTH-cortisol feedforward (left) and cortisol-ACTH feedback (right) cross-ApEn comparisons are illustrated. Adapted from Liu et al. (2005a; Fig. 2). Panel B. Increased forward (left) and reverse (right) cross-ApEn for LH ! T and T ! LH, respectively, in older men, defining erosion of stimulatory and inhibitory regulation within the gonadal axis. Adapted from Liu et al. (2005b; Fig. 3). Panel C. Probability distributions of LH ApEn estimates in individual young and older men (solid lines), and the joint (combined-cohort) ApEn distributions in the separate age strata (interrupted curves). Probability distributions of ApEn estimates were obtained by a within-series resampling (jackknife) procedure. University of Virginia Ph.D. thesis of Xin Wang, Department of Statistics, June 2009.

105

Discrete pulse-coincidence estimates If pulse times are marked reliably in paired hormone time series, one can test whether pulse coincidences (either exact peak concordance or concordance within a finite time window) occur more or less often than expected on the basis of chance associations alone (Veldhuis et al., 1991, 1992). One strategy is to assume that a pulse does or does not occur at each sampling time (or within a delimited time window of several consecutive samples). Pulse times in the two hormone time series are then viewed as arising from binary processes. The null hypothesis is that pulse concordance between the paired series reflects solely random associations. The mathematical properties of the joint distribution of two putatively independent binary series is a hypergeometric distribution (Veldhuis et al., 1991), from which P values may be calculated for rejecting the null hypothesis of random concordance. This type of analysis discloses highly non-random (increased) associations between paired GnRH and LH, LH and FSH, LH and T, LH and prolactin, beta-endorphin and ACTH and ACTH and cortisol pulses in healthy men (Veldhuis et al., 1991, 1992, 1994). Similar analyses would be applicable to paired pulses GH and free IGF-I, TSH and free thyroxine, renin and aldosterone inter alia. The hypergeometric calculation has the weakness of underestimating P values, since actual pulse times rarely occur in consecutive samples due to physiological refraction. In addition, pulse-time estimates have methodological uncertainty, since they depend in part on sampling frequency, pulse size and shape, hormone half-life, assay variability and technique of pulse detection.

estimate unobserved signals within an ensemble analytically, for example to reconstruct GnRH outflow from knowledge of LH and T fluctuations; (3) more comprehensive ensemble mathematical constructs that meld macroscopic (blood-borne), microscopic (cellular) and molecular (genomic, proteomic) components of a neuroendocrine system; (4) appropriate measures of multiseries synchrony (e.g. GnRH–LH–FSH–prolactin), as distinguished from the traditional pairwise perspective; (5) greater insights into specific linkages among circadian, sleep-related and neurohormone secretion; and (6) specialized strategies to quantify mechanisms mediating queuing-like time delays in signal exchange and refractory periods. Such advancements are necessary to diagnose the type and quantify the degree of physiological adaptations and pathological alterations of system-level neuroendocrine behavior more aptly. From a medical perspective, the objective is to ascertain regulatory changes well before the emergence of catastrophic or irreversible system failure, and to evaluate the efficacy of therapies and interventions.

Acknowledgments We thank Donna Scott for support of manuscript preparation and Ashley Bryant for data analysis and graphics. Supported in part via the Clinical Translational Research Center Grant MO1 RR00585 to the Mayo Clinic and Foundation from the National Center for Research Resources (Rockville, MD) and R01 NIA AG019695, AG23133, AG29362, DK73148 and R21 AG29215 and AG23777 from the National Institutes of Health (Bethesda, MD).

Innovative prospects

Abbreviations

Preeminent issues that need to be addressed to enhance the study of neuroendocrine systems include (at least) the development of (1) novel non-invasive measures of negative feedback to complement cross-correlation, cross-spectrum, dose–response estimation, cross-ApEn and hypergeometric models; (2) improved methods to

ADH AIC ApEn AVP BIC CRH

antidiuretic hormone Akaike information criterion approximate entropy arginine vasopressin Bayesian information criterion corticotropin-releasing hormone

106

cross-ApEn CV FSH GH LH NPT SD SD T TSH

cross-approximate entropy coefficient of variation follicle stimulating hormone growth hormone luteinizing hormone nocturnal penile tumescence standard deviation standard deviation testosterone thyroid-stimulating hormone

References Bedecarrats, G. Y. & Kaiser, U. B. (2003). Differential regulation of gonadotropin subunit gene promoter activity by pulsatile gonadotropin-releasing hormone (GnRH) in perifused LbetaT2 cells: Role of GnRH receptor concentration. Endocrinology 144, 1802–1811. Bergendahl, M., Vance, M. L., Iranmanesh, A., Thorner, M. O., & Veldhuis, J. D. (1996). Fasting as a metabolic stress paradigm selectively amplifies cortisol secretory burst mass and delays the time of maximal nyctohemeral cortisol concentrations in healthy men. Journal of Clinical Endocrinology and Metabolism 81, 692–699. Bergsten, P. (2002). Role of oscillations in membrane potential, cytoplasmic Ca2þ, and metabolism for plasma insulin oscillations. Current Medical Literature – Diabetes 51 (Suppl. 1), S171–S176. Brabant, G. & Prank, K. (2000). Prediction and significance of the temporal pattern of hormone secretion in disease states. Novartis Foundation Symposium 227, 105–114. Bucholtz, D. C., Chiesa, A., Pappano, W. N., Nagatani, S., Tsukamura, H., Maeda, K. I., et al. (2000). Regulation of pulsatile luteinizing hormone secretion by insulin in the diabetic male lamb. Biology of Reproduction 62, 1248–1255. Charmandari, E., Pincus, S. M., Matthews, D. R., Dennison, E., Fall, C. H., & Hindmarsh, P. C. (2001). Joint growth hormone and cortisol spontaneous secretion is more asynchronous in older females than in their male counterparts. Journal of Clinical Endocrinology and Metabolism 86, 3393–3399. Chattopadhyay, S., Veldhuis, J. D., & Keenan, D. M. (2008). Probabilistic recovery of pulsatile, secretory and kinetic structure: An alternating discrete and continuous schema. Quarterly of Applied Mathematics 66, 401–421. Chow, F. S., Sharma, A., & Jusko, W. J. (1999). Modeling interactions between adrenal suppression and T-helper lymphocyte trafficking during multiple dosing of methylprednisolone. Journal of Pharmacokinetics and Biopharmaceutics 27, 559–575.

Clark, L. H., Schlosser, P. M., & Selgrade, J. F. (2003). Multiple stable periodic solutions in a model for hormonal control of the menstrual cycle. Bulletin of Mathematical Biology 65, 157–173. Clarke, I., Moore, L., & Veldhuis, J. D. (2002). Intensive direct cavernous sinus sampling identifies high-frequency nearly random patterns of FSH secretion in ovariectomized ewes: Combined appraisal by radioimmunoassay and bioassay. Endocrinology 143, 117–129. Conolly, R. B. & Andersen, M. E. (1991). Biologically based pharmacodynamic models: Tools for toxicological research and risk assessment. Annual Review of Pharmacology and Toxicology 31, 503–523. Cravener, T. L., Vasilatos-Younken, R., & Andersen, B. J. (1990). Hepatomegaly induced by the pulsatile, but not continuous, intravenous administration of purified chicken growth hormone in broiler pullets: Liver composition and nucleic-acid content. Poultry Science 69, 845–848. Dahl, S. G., Aarons, L., Gundert-Remy, U., Karlsson, M. O., Schneider, Y. J., Steimer, J. L., et al. (2010). Incorporating physiological and biochemical mechanisms into pharmacokinetic-pharmacodynamic models: A conceptual framework. Basic and Clinical Pharmacology Toxicology 106(1), 2–12. Davis, W. W. (1988). Use of image data in a control problem. In J. C. Spall (Ed.), Bayesian analysis of time series and dynamic models (pp. 285–311). Dekker: New York. Evans, W. S., Sollenberger, M. J., Booth, R. A., Jr., Rogol, A. D., Urban, R. J., Carlsen, E. C., et al. (1992). Contemporary aspects of discrete peak detection algorithms. II. The paradigm of the luteinizing hormone pulse signal in women. Endocrine Reviews 13(1), 81–104. Farhy, L. S., Bowers, C. Y., & Veldhuis, J. D. (2007). Modelprojected mechanistic bases for sex differences in growthhormone regulation in humans. American Journal of Physiology Regulatory, Integrative and Comparative Physiology 292, R1577–R1593. Farhy, L. S. & Veldhuis, J. D. (2003). Joint pituitary-hypothalamic and intrahypothalamic autofeedback construct of pulsatile growth hormone secretion. American Journal of Physiology Regulatory, Integrative and Comparative Physiology 285, R1240–R1249. Farhy, L. S. & Veldhuis, J. D. (2004). Putative GH pulse renewal: Periventricular somatostatinergic control of an arcuate-nuclear somatostatin and GH-releasing hormone oscillator. American Journal of Physiology 286, R1030–R1042. Farhy, L. S. & Veldhuis, J. D. (2005). Deterministic construct of amplifying actions of ghrelin on pulsatile GH secretion. American Journal of Physiology Regulatory, Integrative and Comparative Physiology 288, R1649–R1663. Fliers, E., Unmehopa, U. A., & Alkemade, A. (2006). Functional neuroanatomy of thyroid hormone feedback in the human hypothalamus and pituitary gland. Molecular and Cellular Endocrinology 251, 1–8. Fricke, O., Lehmkuhl, G., & Pfaff, D. W. (2006). Cybernetic principles in the systematic concept of hypothalamic feeding control. European Journal of Endocrinology 154, 167–173. Friend, K., Iranmanesh, A., & Veldhuis, J. D. (1996). The orderliness of the growth hormone (GH) release process

107 and the mean mass of GH secreted per burst are highly conserved in individual men on successive days. Journal of Clinical Endocrinology and Metabolism 81, 3746–3753. Giovannini, S., Marzetti, E., Borst, S. E., & Leeuwenburgh, C. (2008). Modulation of GH/IGF-1 axis: Potential strategies to counteract sarcopenia in older adults. Mechanism of Ageing and Development 129, 593–601. Giustina, A. & Veldhuis, J. D. (1998). Pathophysiology of the neuroregulation of growth hormone secretion in experimental animals and the human. Endocrine Reviews 19, 717–797. Hartman, M. L., Pincus, S. M., Johnson, M. L., Matthews, D. H., Faunt, L. M., Vance, M. L., et al. (1994). Enhanced basal and disorderly growth hormone secretion distinguish acromegalic from normal pulsatile growth hormone release. The Journal of Clinical Investigation 94, 1277–1288. Homme, M., Schaefer, F., Mehls, O., & Schmitt, C. P. (2009). Differential regulation of RGS-2 by constant and oscillating PTH concentrations. Calcified Tissue International 84, 305–312. Iranmanesh, A., Lizarralde, G., Johnson, M. L., & Veldhuis, J. D. (1989). Circadian, ultradian and episodic release of beta endorphin in men, and its temporal coupling with cortisol. Journal of Clinical Endocrinology and Metabolism 68, 1019–1026. Iranmanesh, A., South, S., Liem, A. Y., Clemmons, D., Thorner, M. O., Weltman, A., et al. (1998). Unequal impact of age, percentage body fat, and serum testosterone concentrations on the somatotrophic, IGF-I, and IGF-binding protein responses to a three-day intravenous growth hormonereleasing hormone pulsatile infusion in men. European Journal of Endocrinology 139, 59–71. Isgaard, J., Carlsson, L., Isaksson, O. G.P., & Jansson, J. O. (1988). Pulsatile intravenous growth hormone (GH) infusion to hypophysectomized rats increases serum-like growth factor I messenger ribonucleic acid in skeletal tissues more effectively than continuous infusion. Endocrinology 123, 2605–2610. Keenan, D. M., Alexander, S. L., Irvine, C. H.G., Clarke, I. J., Canny, B. J., Scott, C. J., et al. (2004a). Reconstruction of in vivo time-evolving neuroendocrine dose-response properties unveils admixed deterministic and stochastic elements. Proceedings of the National Academy of Science United States of Ameriaca 101, 6740–6745. Keenan, D. M., Alexander, S., Irvine, C. H., & Veldhuis, J. D. (2009a). Quantifying nonlinear interactions within the hypothalamo-pituitary-adrenal axis in the conscious horse. Endocrinology 150, 1941–1951. Keenan, D. M., Chattopadhyay, S., & Veldhuis, J. D. (2005). Composite model of time-varying appearance and disappearance of neurohormone pulse signals in blood. Journal of Theoretical Biology 236, 242–255. Keenan, D. M., Evans, W. S., & Veldhuis, J. D. (2003a). Control of LH secretory-burst frequency and interpulse-interval regularity in women. American Journal of Physiology 285, E938–E948. Keenan, D. M., Licinio, J., & Veldhuis, J. D. (2001). A feedback-controlled ensemble model of the stress-responsive hypothalamo-pituitary-adrenal axis. Proceedings of the National Academy of Science United States of Ameriaca 98, 4028–4033.

Keenan, D. M., Roelfsema, F., Biermasz, N., & Veldhuis, J. D. (2003b). Physiological control of pituitary hormone secretoryburst mass, frequency and waveform: A statistical formulation and analysis. American Journal of Physiology 285, R664–R673. Keenan, D. M., Roelfsema, F., Carroll, B. J., Iranmanesh, A., & Veldhuis, J. D. (2009b). Sex defines the age dependence of endogenous ACTH-cortisol dose responsiveness. American Journal of Physiology Regulatory, Integrative and Comparative Physiology 297, R515–R523. Keenan, D. M., Roelfsema, F., & Veldhuis, J. D. (2004b). Endogenous ACTH concentration-dependent drive of pulsatile cortisol secretion in the human. American Journal of Physiology Endocrinology and Metabolism 287, E652–E661. Keenan, D. M., Sun, W., & Veldhuis, J. D. (2000). A stochastic biomathematical model of the male reproductive hormone system. SIAM Journal on Applied Mathematics 61, 934–965. Keenan, D. M., Takahashi, P. Y., Liu, P. Y., Roebuck, P. D., Nehra, A. X., Iranmanesh, A., et al. (2006). An ensemble model of the male gonadal axis: Illustrative application in aging men. Endocrinology 147, 2817–2828. Keenan, D. M. & Veldhuis, J. D. (2000). Explicating hypergonadotropism in postmenopausal women: A statistical model. American Journal of Physiology 278, R1247–R1257. Keenan, D. M. & Veldhuis, J. D. (2001). Disruption of the hypothalamic luteinizing-hormone pulsing mechanism in aging men. American Journal of Physiology 281, R1917–R1924. Keenan, D. M. & Veldhuis, J. D. (2003). Mathematical modeling of receptor-mediated interlinked systems. In Helen L. Henry & Anthony W. Norman (Eds.), Encyclopedia of hormones (pp. 286–294). San Diego, CA: Academic Press. Keenan, D. M. & Veldhuis, J. D. (2004). Divergent gonadotropin-gonadal dose-responsive coupling in healthy young and aging men. American Journal of Physiology 286, R381– R389. Keenan, D. M. & Veldhuis, J. D. (2009). Age-dependent regression analysis of male gonadal axis. American Journal of Physiology Regulatory, Integrative and Comparative Physiology 297 (5), R1215–1227. Epub July 8, 2009. Keenan, D. M., Veldhuis, J. D., & Yang, R. (1998). Joint recovery of pulsatile and basal hormone secretion by stochastic nonlinear random-effects analysis. American Journal of Physiology 275, R1939–R1949. Kennedy, R. T., Kauri, L. M., Dahlgren, G. M., & Jung, S. K. (2002). Metabolic oscillations in beta-cells. Current Medical Literature – Diabetes 51 (Suppl. 1), S152–S161. Laursen, T., Gravholt, C. H., Heickendorff, L., Drustrup, J., Kappelgaard, A. M., Jorgensen, J. O., et al. (2001). Longterm effects of continuous subcutaneous infusion versus daily subcutaneous injections of growth hormone (GH) on the insulin-like growth factor system, insulin sensitivity, body composition, and bone and lipoprotein metabolism in GHdeficient adults. Journal of Clinical Endocrinology and Metabolism 86, 1222–1228. Leloup, J. C. (2009). Circadian clocks and phosphorylation: Insights from computational modeling. Central European Journal of Biology 4, 290–303.

108 Lenbury, Y. & Pornsawad, P. (2005). A delay-differential equation model of the feedback-controlled hypothalamuspituitary-adrenal axis in humans. Mathematical Medicine and Biology 22, 15–33. Li, Y. X. & Khadra, A. (2008). Robust synchrony and rhythmogenesis in endocrine neurons via autocrine regulations in vitro and in vivo. Bulletin of Mathematical Biology 70, 2103–2125. Lincoln, G. A., Clarke, I. J., Hut, R. A., & Hazlerigg, D. G. (2006). Characterizing a mammalian circannual pacemaker. Science 314, 1941–1944. Liu, P. Y., Keenan, D. M., Kok, P., Padmanabhan, V., O’Byrne, K. T., & Veldhuis, J. D. (2009). Sensitivity and specificity of pulse detection using a new deconvolution method. American Journal of Physiology Endocrinology and Metabolism 297, E538–E544. Liu, P. Y., Pincus, S. M., Keenan, D. M., Roelfsema, F., & Veldhuis, J. D. (2005a). Analysis of bidirectional pattern synchrony of concentration-secretion pairs: Implementation in the human testicular and adrenal axes. American Journal of Physiology 288, R440–R446. Liu, P. Y., Pincus, S. M., Keenan, D. M., Roelfsema, F., & Veldhuis, J. D. (2005b). Joint synchrony of reciprocal hormonal signaling in human paradigms of both ACTH excess and cortisol depletion. American Journal of Physiology Endocrinology and Metabolism 289, 160–165. Liu, P. Y., Pincus, S. M., Takahashi, P. Y., Roebuck, P. D., Iranmanesh, A., Keenan, D. M., et al. (2006). Aging attenuates both the regularity and joint synchrony of LH and testosterone secretion in normal men: Analyses via a model of graded GnRH receptor blockade. American Journal of Physiology 290, E34–E41. Lotinun, S., Sibonga, J. D., & Turner, R. T. (2002). Differential effects of intermittent and continuous administration of parathyroid hormone on bone histomorphometry and gene expression. Endocrine 17, 29–36. McNeilly, A. S., Crawford, J. L., Taragnat, C., Nicol, L., & McNeilly, J. R. (2003). The differential secretion of FSH and LH: Regulation through genes, feedback and packaging. Reproduction Supplement 61, 463–476. Meneilly, G. S., Ryan, A. S., Veldhuis, J. D., & Elahi, D. (1997). Increased disorderliness of basal insulin release, attenuated insulin secretory-burst mass, and reduced ultradian rhythmicity of insulin secretion in older individuals. Journal of Clinical Endocrinology and Metabolism 82, 4088–4093. Miller, B. H., Olson, S. L., Turek, F. W., Levine, J. E., Horton, T. H., & Takahashi, J. S. (2004). Circadian clock mutation disrupts estrous cyclicity and maintenance of pregnancy. Current Biology 14, 1367–1373. Omer, S., Meredith, D., Morris, J. F., & Christian, H. C. (2006). Evidence for the role of adenosine 50 -triphosphate-binding cassette (ABC)-A1 in the externalization of annexin 1 from pituitary folliculostellate cells and ABCA1-transfected cell models. Endocrinology 147, 3219–3227. Ooi, G. T., Tawadros, N., & Escalona, R. M. (2004). Pituitary cell lines and their endocrine applications. Molecular and Cellular Endocrinology 228, 1–21.

Pincus, S. M. (1991). Approximate entropy as a measure of system complexity. Proceedings of the National Academy of Science United States of Ameriaca 88, 2297–2301. Pincus, S. M. (1994). Greater signal regularity May indicate increased system isolation. Mathematical Biosciences 122, 161–181. Pincus, S. M. (2000a). Irregularity and asynchrony in biologic network signals. Methods in Enzymology 321, 149–182. Pincus, S. M. (2000b). Orderliness of hormone release. In D. J. Chadwick, & J. A. Goode (Eds.), Mechanisms and biological significance of pulsatile hormone secretion (pp. 82–104). New York: John Wiley and Sons, Ltd. Pincus, S. M., Gevers, E., Robinson, I. C.A.F., van den Berg, G., Roelfsema, F., Hartman, M. L., et al. (1996a). Females secrete growth hormone with more process irregularity than males in both human and rat. American Journal of Physiology 270, E107–E115. Pincus, S. M. & Goldberger, A. L. (1994). Physiological timeseries analysis: What does regularity quantify? American Journal of Physiology 266, H1643–H1656. Pincus, S. M., Hartman, M. L., Roelfsema, F., Thorner, M. O., & Veldhuis, J. D. (1999). Hormone pulsatility discrimination via coarse and short time sampling. American Journal of Physiology 277, E948–E957. Pincus, S. M. & Kalman, R. E. (1997). Not all (possibly) “random” sequences are created equal. Proceedings of the National Academy of Science United States of Ameriaca 94, 3513–3518. Pincus, S. M., Mulligan, T., Iranmanesh, A., Gheorghiu, S., Godschalk, M., & Veldhuis, J. D. (1996b). Older males secrete luteinizing hormone and testosterone more irregularly, and jointly more asynchronously, than younger males. Proceedings of the National Academy of Science United States of Ameriaca 93, 14100–14105. Pincus, S. M. & Singer, B. H. (1996). Randomness and degrees of irregularity. Proceedings of the National Academy of Science United States of Ameriaca 93, 2083–2088. Pincus, S. M., Veldhuis, J. D., Mulligan, T., Iranmanesh, A., & Evans, W. S. (1997). Effects of age on the irregularity of LH and FSH serum concentrations in women and men. American Journal of Physiology 273, E989–E995. Pincus, S. M., Veldhuis, J. D., & Rogol, A. D. (2000). Longitudinal changes in growth hormone secretory process irregularity assessed transpubertally in healthy boys. American Journal of Physiology Endocrinology and Metabolism 279, E417–E424. Potter, L. K., Greller, L. D., Cho, C. R., Nuttall, M. E., Stroup, G. B., Suva, L. J., et al. (2005). Response to continuous and pulsatile PTH dosing: A mathematical model for parathyroid hormone receptor kinetics. Bone 37, 159–169. Prank, K., Jurgens, C., von zur Muhlen, A., & Brabant, G. (1998). Predictive neural networks for learning the time course of blood glucose levels from the complex interaction of counterregulatory hormones. Neural Computation 10, 941–953. Prank, K., Kloppstech, M., Nowlan, S. J., Sejnowski, T. J., & Brabant, G. (1996). Self-organized segmentation of time

109 series: Separating growth hormone secretion in acromegaly from normal controls. Biophysical Journal 70, 2540–2547. Rasgon, N. L., Pumphrey, L., Prolo, P., Elman, S., Negrao, A. B., Licinio, J., et al. (2003). Emergent oscillations in a mathematical model of the human menstrual cycle. CNS Spectrums 8, 805–814. Roelfsema, F., Pereira, A. M., Keenan, D. M., Veldhuis, J. D., & Romijn, J. A. (2008). Thyrotropin secretion by thyrotropinomas is characterized by increased pulse frequency, delayed diurnal rhythm, enhanced basal secretion, spikiness, and disorderliness. Journal of Clinical Endocrinology and Metabolism 93, 4052–4057. Roelfsema, F., Pincus, S. M., & Veldhuis, J. D. (1998). Patients with cushing’s disease secrete adrenocorticotropin and cortisol jointly more asynchronously than healthy subjects. Journal of Clinical Endocrinology and Metabolism 83, 688–692. Rossmanith, W. G., Laughlin, G. A., Mortola, J. F., & Yen, S. S. (1990). Secretory dynamics of oestradiol (E2) and progesterone (P4) during periods of relative pituitary LH quiescence in the midluteal phase of the menstrual cycle. Clinical Endocrinology (Oxford) 32, 13–23. Schnorr, J. A., Bray, M. J., & Veldhuis, J. D. (2001). Aromatization mediates testosterone’s short-term feedback restraint of 24-hour endogenously driven and acute exogenous GnRHstimulated LH and FSH secretion in young men. Journal of Clinical Endocrinology and Metabolism 86, 2600–2606. Scullion, S., Brown, D., & Leng, G. (2004). Modelling the pituitary response to luteinizing hormone-releasing hormone. Journal of Neuroendocrinology 16, 265–271. Siragy, H. M., Vieweg, W. V.R., Pincus, S. M., & Veldhuis, J. D. (1995). Increased disorderliness and amplified basal and pulsatile aldosterone secretion in patients with primary aldosteronism. Journal of Clinical Endocrinology and Metabolism 80, 28–33. Srinivasan, V., Eswaran, C., & Sriraam, N. (2007). Approximate entropy-based epileptic EEG detection using artificial neural networks. IEEE Transactions on Information Technology in Biomedicine 11, 288–295. Sun, Y. N., Lee, H. J., Almon, R. R., & Jusko, W. J. (1999). A pharmacokinetic/pharmacodynamic model for recombinant human growth hormone effects on induction of insulin-like growth factor I in monkeys. The Journal of Pharmacology and Experimental Therapeutics 289, 1523–1532. Tolic, I. M., Mosekilde, E., & Sturis, J. (2000). Modeling the insulin-glucose feedback system: The significance of pulsatile insulin secretion. Journal of Theoretical Biology 207, 361–375. Urban, R. J., Evans, W. S., Rogol, A. D., Kaiser, D. L., Johnson, M. L., & Veldhuis, J. D. (1988). Contemporary aspects of discrete peak detection algorithms. I. The paradigm of the luteinizing hormone pulse signal in men. Endocrine Reviews 9, 3–37. Van den Berg, G., Pincus, S. M., Veldhuis, J. D., Frolich, M., & Roelfsema, F. (1997). Greater disorderliness of ACTH and cortisol release accompanies pituitary-dependent cushing’s disease. European Journal of Endocrinology 136, 394–400. Veldhuis, J. D. (2002). Biorhythms, pulsatility and hormone resistance. In G. P. Chrousos, J. M. Olefsky, & E. Samols (Eds.), Hormone resistance and hypersensitivity states (pp. 11–44). Philadelphia, PA: Lippincott Williams & Wilkins.

Veldhuis, J. D. (2008). Pulsatile hormone secretion: Mechanisms, significance and evaluation. In D. Lloyd, & E. L. Rossi (Eds.), Ultradian rhythms from molecules to mind: A new vision of life (pp. 229–248). New York: Springer. Veldhuis, J. D., Carlson, M. L., & Johnson, M. L. (1987a). The pituitary gland secretes in bursts: Appraising the nature of glandular secretory impulses by simultaneous multipleparameter deconvolution of plasma hormone concentrations. Proceedings of the National Academy of Science United States of Ameriaca 84, 7686–7690. Veldhuis, J. D., Evans, W. S., Kolp, L. A., Rogol, A. D., & Johnson, M. L. (1988). Physiological profiles of episodic progesterone release during the mid-luteal phase of the human menstrual cycle: Analysis of circadian and ultradian rhythms, discrete pulse properties, and correlations with simultaneous LH release. Journal of Clinical Endocrinology and Metabolism 66, 414–421. Veldhuis, J. D., Fletcher, T. P., Gatford, K. L., Egan, A. R., & Clarke, I. J. (2002). Hypophyseal-portal somatostatin (SIRF) and jugular venous growth hormone secretion in the conscious unrestrained ewe. Neuroendocrinology 75, 83–91. Veldhuis, J. D., Iranmanesh, A., Godschalk, M., & Mulligan, T. (2000). Older men manifest multifold synchrony disruption of reproductive neurohormone outflow. Journal of Clinical Endocrinology and Metabolism 85, 1477–1486. Veldhuis, J. D., Iranmanesh, A., Johnson, M. L., & Lizarralde, G. (1990a). Twenty-four hour rhythms in plasma concentrations of adenohypophyseal hormones are generated by distinct amplitude and/or frequency modulation of underlying pituitary secretory bursts. Journal of Clinical Endocrinology and Metabolism 71, 1616–1623. Veldhuis, J. D., Iranmanesh, A., Johnson, M. L., & Lizarralde, G. (1990b). Amplitude, but not frequency, modulation of ACTH secretory bursts gives rise to the nyctohemeral rhythm of the corticotropic axis in man. Journal of Clinical Endocrinology and Metabolism 71, 452–463. Veldhuis, J. D., Iranmanesh, A., Lizarralde, G., & Johnson, M. L. (1989). Amplitude modulation of a burst-like mode of cortisol secretion subserves the circadian glucocorticoid rhythm in man. American Journal of Physiology 257, E6–E14. Veldhuis, J. D., Iranmanesh, A., Mulligan, T., & Pincus, S. M. (1999). Disruption of the young-adult synchrony between luteinizing hormone release and oscillations in follicle-stimulating hormone, prolactin, and nocturnal penile tumescence (NPT) in healthy older men. Journal of Clinical Endocrinology and Metabolism 84, 3498–3505. Veldhuis, J. D. & Johnson, M. L. (1986). Cluster analysis: A simple, versatile and robust algorithm for endocrine pulse detection. American Journal of Physiology 250, E486–E493. Veldhuis, J. D. & Johnson, M. L. (1988). Operating characteristics of the human male hypothalamo-pituitary-gonadal axis in men: Circadian, ultradian, and pulsatile release of prolactin and its temporal coupling with luteinizing hormone. Journal of Clinical Endocrinology and Metabolism 67, 116–123.

110 Veldhuis, J. D., Johnson, M. L., Faunt, L. M., & Seneta, E. (1994). Assessing temporal coupling between two, or among three or more, neuroendocrine pulse trains: Cross-correlation analysis, simulation methods, and conditional probability testing. Methods in Neuroscience 20, 336–376. Veldhuis, J. D., Johnson, M. L., & Seneta, E. (1991). Analysis of the co-pulsatility of anterior pituitary hormones. Journal of Clinical Endocrinology and Metabolism 73, 569–576. Veldhuis, J. D., Johnson, M. L., Seneta, E., & Iranmanesh, A. (1992). Temporal coupling among luteinizing hormone, follicle stimulating hormone, beta-endorphin, and cortisol pulse episodes in vivo. Acta Endocrinologica (Copenh) 126, 193–200. Veldhuis, J. D., Johnson, M. L., Veldhuis, O. L., Straume, M., & Pincus, S. (2001a). Impact of pulsatility on the ensemble orderliness (approximate entropy) of neurohormone secretion. American Journal of Physiology 281, R1975–R1985. Veldhuis, J. D. & Keenan, D. M. (2008). Secretagogues govern growth-hormone secretory-burst waveform and mass in healthy eugonadal and short-term hypogonadal men. European Journal of Endocrinology 159, 547–554. Veldhuis, J. D., Keenan, D. M., & Bowers, C. Y. (2007). Estimation of the size and shape of GH secretory bursts in healthy women using a physiological estradiol clamp and variable-waveform deconvolution model. American Journal of Physiology Regulatory, Integrative and Comparative Physiology 293, R1013–R1021. Veldhuis, J. D., Keenan, D. M., & Pincus, S. M. (2008). Motivations and methods for analyzing pulsatile hormone secretion. Endocrine Reviews 29, 823–864. Veldhuis, J. D., King, J. C., Urban, R. J., Rogol, A. D., Evans, W. S., Kolp, L. A., et al. (1987b). Operating characteristics of the male hypothalamo-pituitary-gonadal axis: Pulsatile release of testosterone and follicle-stimulating hormone and their temporal coupling with luteinizing hormone. Journal of Clinical Endocrinology and Metabolism 65, 929–941. Veldhuis, J. D., Metzger, D. L., Martha, Jr., Mauras, P. M., Kerrigan, N., Keenan, J. R., et al., S. M. (1997). Estrogen and testosterone, but not a non-aromatizable androgen, direct network integration of the hypothalamo-somatotrope (growth hormone)-insulin-like growth factor I axis in the human: Evidence from pubertal pathophysiology and sexsteroid hormone replacement. Journal of Clinical Endocrinology and Metabolism 82, 3414–3420. Veldhuis, J. D., Pincus, S. M., Mitamura, R., Yano, K., Suzuki, N., Ito, Y., et al. (2001b). Developmentally delimited emergence of more orderly luteinizing hormone and testosterone secretion in late prepuberty in boys. Journal of Clinical Endocrinology and Metabolism 86, 80–89. Veldhuis, J. D., Roelfsema, F., Iranmanesh, A., Carroll, B. J., Keenan, D. M., & Pincus, S. M. (2009). Basal, pulsatile, entropic (patterned) and spiky (staccato-like) properties of ACTH secretion: Impact of age, gender and body mass index. Journal of Clinical Endocrinology and Metabolism 94(10), 4045–4052. Ref Type: Abstract.

Veldhuis, J. D., Roemmich, J. N., Richmond, E. J., & Bowers, C. Y. (2006). Somatotropic and gonadotropic axes linkages in infancy, childhood, and the puberty-adult transition. Endocrine Reviews 27, 101–140. Veldhuis, J. D., Straume, M., Iranmanesh, A., Mulligan, T., Jaffe, C. A., Barkan, A., et al. (2001c). Secretory process regularity monitors neuroendocrine feedback and feedforward signaling strength in humans. American Journal of Physiology 280, R721–R729. Veldman, R. G., Frolich, M., Pincus, S. M., Veldhuis, J. D., & Roelfsema, F. (2000a). Apparently complete restoration of normal daily adrenocorticotropin, cortisol, growth hormone, and prolactin secretory dynamics in adults with cushing’s disease after clinically successful transsphenoidal adenomectomy. Journal of Clinical Endocrinology and Metabolism 85, 4039–4046. Veldman, R. G., Frolich, M., Pincus, S. M., Veldhuis, J. D., & Roelfsema, F. (2000b). Hyperleptinemia in women with cushing’s disease is driven by high-amplitude pulsatile but orderly and rhythmic leptin secretion. European Journal of Endocrinology 144, 21–27. Veldman, R. G., van den Berg, G., Pincus, S. M., Frolich, M., Veldhuis, J. D., & Roelfsema, F. (1999). Increased episodic release and disorderliness of prolactin secretion in both micro- and macroprolactinomas. European Journal of Endocrionology 140, 192–200. Washington, T. M., Blum, J. J., Reed, M. C., & Conn, P. M. (2004). A mathematical model for LH release in response to continuous and pulsatile exposure of gonadotrophs to GnRH. Theoretical Biology and Medical Modelling 1, 9–26. Wei, W. W.S. (1990). Vector AR(1) models. Time-series analysis (pp. 332–342). New York: Addison-Wesley Publishing Company. Winters, S. J. & Moore, J. P. (2004). Intra-pituitary regulation of gonadotrophs in male rodents and primates. Reproduction 128, 13–23. Winters, S. J. & Moore, J. P. (2007). Paracrine control of gonadotrophs. Seminars in Reproductive Medicine 25, 379–387. Yu, W. H., Kimura, M., Walczewska, A., Porter, J. C., & McCann, S. M. (1998). Adenosine acts by A1 receptors to stimulate release of prolactin from anterior-pituitaries in vitro. Proceedings of the National Academy of Sciences of the United States of America 95, 7795–7798. Zhou, Y., Wang, X., Hadley, J., Corey, S. J., & VasilatosYounken, R. (2005). Regulation of JAK2 protein expression by chronic, pulsatile GH administration in vivo: A possible mechanism for ligand enhancement of signal transduction. General and Comparative Endocrinology 144, 128–139. Zwart, A. D., Iranmanesh, A., & Veldhuis, J. D. (1997). Disparate serum free testosterone concentrations and degrees of hypothalamo-pituitary-LH suppression are achieved by continuous versus pulsatile intravenous androgen replacement in men: A clinical experimental model of ketoconazoleinduced reversible hypoandrogenemia with controlled testosterone add-back. Journal of Clinical Endocrinology and Metabolism 82, 2062–2069.

L. Martini (Eds.) Progress in Brain Research, Vol. 181 ISSN: 0079-6123 Copyright Ó 2010 Elsevier B.V. All rights reserved.

CHAPTER 7

Physiological significance of the rhythmic secretion of hypothalamic and pituitary hormones Earn-Hui Gan and Richard Quinton
Endocrine Research Group, Institute of Human Genetics, University of Newcastle-on-Tyne, Newcastle-upon-Tyne, United Kingdom

Abstract: The various hypothalamic–pituitary–end-organ/gland axes are central to the regulation of mammalian homeostasis. These have a core role in integrating the response of both endocrine and nervous systems to external and internal stimuli, by means of multi-level signalling through negative and positive feedback loops. The content of these hormonal signals is overwhelmingly conveyed in a rhythmic secretory pattern (frequency modulation of signal) that is energetically more efficient in transmitting neuroendocrine signals than the alternatives (modulation of signal by amplitude or by total area-under-curve). These rhythmic neuroendocrine secretions are individually distinct but the majority display a common feature of low-level basal secretion with superimposed pulsatile rhythms. The underlying mechanisms contributing to this unique rhythmic secretion are complicated and incompletely understood, but are beginning to be better defined as a result of several elegant studies performed in recent years. In some cases, signal transduction in the target tissue is critically dependent upon a pulsatile input, but in others the observed pulsatility is a downstream echo of obligate pulsatility exhibited by a higher-level control hormone. Thus, the gonads are presented with a pulsatile gonadotrophin signal, not because this is essential to gonadotrophin action (the same level of stimulation can be elicited by a continuous input), but as a downstream consequence of pulsatile GnRH-mediated stimulation of pituitary gonadotrophs. By contrast, rhythmicity of signal is embedded at all levels of the hypothalamo–pituitary–adrenal axis. Hypothalamic–pituitary rhythmic secretions are influenced by various internal and external inputs such as age, gender, sleep and wakefulness, food intake, light (photoperiod) or exposure to stress. Understanding the physiological significance of the rhythmic secretion of hypothalamic and pituitary hormones has the potential to provide insights into disease mechanisms, to validate diagnostic tests and, ultimately, to help develop novel therapeutic interventions. This chapter will overview the physiological basis of rhythmic secretion of hypothalamic and pituitary hormones, principally in humans and, by reference to specific examples, describe the various feedback loops and internal and external stimuli that precisely determine these neuroendocrine secretory patterns. Keywords: rhythmic secretory patterns GH TSH GnRH photoperiod




Corresponding author. Tel.: þ44-191-2824635; Fax: þ44-191-2820129 E-mail: [email protected]

DOI: 10.1016/S0079-6123(08)81007-2

111

112

Introduction Oscillating and pulsatile signals in neuroendocrine axes have been demonstrated in virtually all mammalian species studied, from rodents to human (Gudmundsson and Carnes, 1997). In mammals, this observable rhythm is generated by an endogenous circadian clock located within the suprachiasmatic nucleus (SCN) of the hypothalamus (Cermakian and Boivin, 2009; Klein et al., 1991; Moore and Silver, 1998). This time-controlling mechanism is shown to be an intrinsic property at the cellular level, with circadian rhythmicity being constitutively exhibited in vitro by SCN neurons (Cermakian and Boivin, 2003, 2009; Ko and Takahashi, 2006). This property results from the differential expression of various clock genes (e.g. Per1-3, Cry1-2) that have been isolated based on sequence homology with known clock genes in Drosophila melanogaster. Their associated protein products, including neuropeptides, transcription factors and metabolic enzymes, contribute to both negative and positive feedback loops in neuroendocrine axes (Cermakian and Boivin, 2009). In this chapter, we will focus on patterns and control mechanisms of rhythmic hormone secretion, particularly in humans, with specific reference to growth hormone (GH), adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), prolactin (PRL) and oxytocin. The gonadotropic axis and its hypothalamic regulator are discussed elsewhere in this volume. However, before beginning our description, it is necessary to define a number of key terms and definitions. The ‘diurnal’ (or perhaps more accurately, ‘ultradian’) rhythm is a 24-hour periodicity that reflects the superimposition of external and internal stimuli on the endogenous (circadian) signal. Endogenous neurosecretory rhythms are defined as ‘circadian’, because they typically cycle over a period of around (but never exactly) 24 hours. The two principal environmental cues that act to resynchronise the endogenous circadian rhythms are the light–dark and rest–activity cycles. A rhythmic signal can encode information in three principal ways: through the pulse frequency (frequency modulation – as in the

‘FM radio’ signal), the integrated pulse amplitude (amplitude modulation – as in the ‘AM radio’ signal) or through the total area-under-curve. Of these, the former is typically most energetically efficient (Goldbeter et al., 2000).

Neurohumoral regulation of growth hormone secretion GH is essential for promoting somatic growth from the end of the second year of life until puberty (in man), by regulating protein synthesis, lipolysis and skeletal growth. It also has a homeostatic role in adult life (Davidson, 1987; Furuhata et al., 2002). GH secretion is predominantly pulsatile in healthy young adults and carries significant physiological signals to target tissues by determining the GH-receptor turnover, mode of second-messenger signalling, cell-specific gene expression and distinct metabolic responses (Achermann et al., 1999; Giustina and Veldhuis, 1998; Veldhuis and Bowers, 2009; Veldhuis et al., 2009a).

Relationship between pulsatile GH secretion and IGF-1 level In humans, 85% of GH secretion is pulsatile, with up to 10 pulses of GH secretion in a 24hour period (Ionescu and Frohman, 2006; Stolar and Baumann, 1986). Many of the physiological effects of GH are mediated indirectly through stimulation of insulin-like growth factor 1 (IGF-1) secretion by the liver and other target tissues. The circulating IGF-1 level strongly correlates with the pattern of rhythmic GH secretion (Veldhuis and Bowers, 2009). The attenuation of GH pulse-dependent STAT5b (signal transducer and activator of transcription 5B) signalling is shown to associate with the diminution of IGF-1 concentration, which in turn leads to growth failure (Davey et al., 1999; Veldhuis and Bowers, 2009). Most studies have demonstrated that mean GH concentration, as reflected by pulse amplitude, best correlates with serum IGF-1 concentration (i.e. amplitude

113 25

20

GH(mU/L)

15

10

08:00

08:40

07:20

06:00

06:40

04:40

05:20

04:00

03:20

02:00

02:40

01:20

00:40

23:20

00:00

22:40

21:20

22:00

20:40

20:00

19:20

18:40

18:00

17:20

16:40

16:00

15:20

14:00

14:40

13:20

12:40

0

12:00

5

Time Fig. 1. Twenty-hour GH profile in a normal 18-year-old girl.

modulation of signal), though a raised basal GH secretion (reflecting ‘area-under-curve’), as observed for instance in patients with acromegaly, clearly also has a role (Ionescu and Frohman, 2006; Veldhuis et al., 1995). In man, pulsatile GH secretion peaks in the evening and displays a distinctive composite of low-frequency volleys and high-frequency secretory bursts (Fig. 1) (Farhy and Veldhuis, 2003). Multi-burst volleys recur every 1.5–2.5 hours in humans, with rapid discrete GH pulses arising every 30–60 minutes within an individual volley (Farhy and Veldhuis, 2003; Gevers et al., 1998; Giustina and Veldhuis, 1998; Hartman et al., 1991, 1992).

Neuropeptide interactions in the generation of GH pulses GH pulses are instituted by a multi-signal interaction between various neuropeptides, including the

amplifying signals, GHRH (growth-hormone-releasing hormone) and GHS (growth hormone secretagogue or ghrelin), the inhibitory factor somatostatin (SRIF), and feedback-inhibition from circulating IGF-1 (Bluet-Pajot et al., 1998; Frohman, 1996; Furuhata et al., 2002; Shuto et al., 2002; Veldhuis and Bowers, 2009). The interplay between episodic facilitative drive by GHRH, intermittent suppression by central neural SRIF and reversible negative feedback by both systemic and local central nervous system GH secretion and peripherally derived IGF-1 result in discrete GH pulses (Farhy and Veldhuis, 2004; Giustina and Veldhuis, 1998; Mueller et al., 1999). Several other potent neuromodulators probably also influence the GH axis, including endogenous opioids, neuropeptide Y, galanin, cholinergic and adrenergic neurotransmitters, gammaaminobutyric acid and sex steroids (Veldhuis, 1998). Ghrelin, the endogenous ligand of the GHS receptor, is secreted dominantly by the gastrointestinal tract and is the only known circulating orexigen.

114

Although not essential for GH secretion, it does seem to amplify GHRH-mediated GH pulses (Farhy and Veldhuis, 2005). Differential timing of the release of these neuropeptides, along with superimposed variation reflecting age and sexual dimorphism, contributes to the observed pulsatility of GH secretion (Farhy et al., 2002; Haus, 2007).

Control of GH secretion: lessons from animal studies Peaks and troughs of GH secretion in rodents have been shown to result from a complex interactive network. This comprises (1) time-delayed negative feedback to stimulate SRIF secretion from the periventricular nucleus (PEV) of the hypothalamus into the hypophysial portal circulation, which in turn antagonises GH release from the anterior pituitary (Farhy and Veldhuis, 2004; Robinson, 1991), and (2) a GHRH–SRIF oscillator in the arcuate nucleus (ARC) of the hypothalamus (Fig. 2) (Farhy and Veldhuis, 2004; Giustina and Veldhuis, 1998; Mueller et al., 1999). SRIF receptor is expressed by both (ARC) GHRHpositive neurons and (PEV and ARC) SRIF-

positive neurons (Bertherat et al., 1992; Farhy and Veldhuis, 2004). There is presumed to be a time-delayed, short-latency, reciprocal interaction between the actions of GHRH and SRIF to create a damped intra-arcuate oscillator (Farhy and Veldhuis, 2004). An unequal feedback latency exists between the systemic GH input to (PEV) SRIF secretion (long loop feedback) and (ARC) GHRH secretion to drive SRIF-mediated inhibition of GH secretion (short loop feedback), leading to prolonged intervolley duration and short intra-volley intervals (Farhy and Veldhuis, 2004). It is postulated that the systemic GH pulses stimulate (PEV) SRIFdependent inhibition of somatotrope GH release, by reducing (1) GHRH secretion into portal blood and (2) intra-hypothalamic GHRH feed-forward on (ARC) SRIF secretion, leading to an intervolley interval of reduced GH secretion (Farhy and Veldhuis, 2004).

Sexual dimorphism in the pattern of pulsatile GH secretion The reduction in circulating GH and hypothalamic (PEV) SRIF concentration during an inter-pulse

Arcuate Nucleus

Periventricular Nucleus

SRIF

SRIF

GHRH

Pituitary Peripheral

GH

IGH-1

Stimulatory effects Inhibitory effects Fig. 2. Modulation of pulsatile GH secretion: schema of interaction between hypothalamus (arcuate and periventricular nuclei), pituitary and peripheral tissues.

115

trough disinhibits the putative GHRH–SRIF oscillator, thereby triggering the high-frequency, amplitude-damped GHRH and GH secretory bursts demonstrated in male rats. In females, a decline in GH-induced (PEV) SRIF release will attenuate (PEV) SRIF-enforced suppression and disinhibition of coupled (ARC) GHRH–SRIF interactions, leading to discrete, high-frequency and low-amplitude GH pulses with only occasional volley-like complexes (Clark et al., 1986, 1987; Farhy and Veldhuis, 2004; Pincus et al., 1996). This is in keeping with a study in young human adults showing that, in the fasting state, women secrete several-fold more GH in bursts than men, and that the somatosergic outflow in women is opposed by greater feedforward by both GHRH and GHS (SoaresWelch et al., 2005). Sexual dismorphism of the human GH secretory pattern is also demonstrated during sleep. In men, the highest pulse occurs after sleep onset within the first phase of slow-wave sleep and accounts for the highest secretory output per 24 hours – even up to 70% (Haus, 2007; Ho et al., 1987). By contrast, females exhibit a wider distribution of GH pulses throughout the day, with the sleep-onset-entrained GH surge accounting for a smaller fraction of the total 24-hour secretion (Haus, 2007; Ho et al., 1987). These observations help explain the alterations in GH secretion observed with sleep disorders, among shift workers or in trans-meridian travellers (Cauter et al., 1998; Haus, 2007). Effects of ageing on pulsatile GH secretion GH secretion is reduced in older men and women (Veldhuis, 2008). In comparison with the volley-like clusters of prominent pulses observed in pubertal children, the elderly display frequent, isolated and low-amplitude GH bursts. This results in a reduced circadian mean GH concentration by selectively reducing pulsatile GH secretion (Farhy and Veldhuis, 2004; Hartman et al., 1991; Martha et al., 1992; Veldhuis et al., 2000). Young adults also exhibit a notably higher GH secretory response to

lowering of IGF-1 levels, compared with elderly subjects (Veldhuis, 2008; Veldhuis et al., 2006), which implies an impaired hypothalamic–pituitary drive to GH secretion with ageing, possibly via diminished responsiveness to GHRH and increased responsiveness to SRIF (Haus, 2007; Martin et al., 1997). Potential estrogen-independent factors contributing to the depletion of GH in older woman includes catecholamines, thyroxines, cortisol, free fatty acids, inflammatory cytokines and regulatory peptides (Veldhuis 2008; Veldhuis et al., 2005). In men, testosterone increases pulsatile GH secretion by augmentation of pulse amplitude (Gentili et al., 2002; Iranmanesh et al., 1998; Veldhuis, 2008). However, GH pulse frequency, GH elimination kinetic or hepatic action of GH to stimulate IGF-1 production is not affected by age or testosterone (Gentili et al., 2002; Muniyappa et al., 2007; Veldhuis et al., 2006). Hence, the full extent of the interplay between testosterone and age in affecting GH secretory burst size is still not entirely clear.

Effects of developmental stage and pubertal status on GH pulsatile secretion Developmental stage and the physiological changes in response to adiposity, aerobic capacity and ageing have significant effects on GH secretion. Smaller GH pulses, as quantified by less GH secreted per burst (which diminishes the mean GH concentration), are observed in normal midchildhood, in association with low aerobic capacity, in young adults with hypogonadism and in obesity (Lang et al., 1987; Lieman et al., 2001; Veldhuis et al., 2009; Weltman et al., 1994). The first day of infant life and the period corresponding to Tanner stages IV–V of puberty represent periods of dominant amplitude modulation of signal, being associated with significant augmentation of the GH pulses with no change in GH pulse frequency, GH half life or GH basal release (Gentili et al., 2002; Shah et al., 1999; Veldhuis et al., 2006).

116

Pulsatile GH secretion can be stimulated by fasting or by GHRH/GHS or estradiol, but is diminished by factors associated with food intake, abdominal visceral fat and ageing (Lang et al., 1987; Vahl et al., 1997; Veldhuis and Bowers, 2003; Veldhuis et al., 2009b). Veldhuis found a strong negative impact of computed tomographyestimated abdominal visceral fat on fasting and GHRH/GHS-stimulated pulsatile GH secretion in an estrogen-enriched milieu (Veldhuis et al., 2009).

Unresolved or poorly understood issues Recent human experiments have demonstrated interesting and distinct findings in response to different patterns of GH or GHRH infusion. For instance, the observed pulsatility of GH secretion in post-menopausal women is maintained under continuous stimulation with GHRH and growth hormone-releasing peptide 2 (GHRP-2) (Haus, 2007; Veldhuis et al., 2002, 2006). Likewise, continuous GHS infusion in adults for 1–30 days resulted in augmented GH secretory burst activity, but with unchanged GH pulse frequency and circadian rhythmicity (Haus, 2007; Shah et al., 1999). Whereas bolus GH infusion enhanced lipolysis to a greater extent than continuous delivery, it was less effective at increasing the IGF-1 concentration (Haus, 2007; Jorgensen et al., 1991). Little is known about the physiological significance of tonic basal GH secretion, but an increase is observed experimentally in mice with knockout of the type 1 SRIF receptor and clinically in humans with acromegaly (Hartman et al., 1994; Veldhuis et al., 2009). It is impaired with advancing age and by exogenous estrogen administration (Veldhuis et al., 2009).

Regulation of corticotrophic function and action Activation of the hypothalamus–pituitary–adrenal (HPA) axis involves the pulsatile secretion of corticotropin-releasing hormone (CRH) and of arginine vasopressin (AVP) from the PEV. CRH triggers the release of ACTH from pituitary

corticotrophs; ACTH in turn upregulates adrenal cortical enzymatic activity, principally conversion of cholesterol to pregnenolone (the first and rate-limiting step in the steroid biosynthetic pathway), leading to increased cortisol secretion (Crofford et al., 2004). Cortisol is a key mediator of vascular integrity, immune response, energy balance and metabolism (Baigent, 2001; Richard et al., 2000). Diurnal variation in (PEV) CRH and ACTH secretion is regulated by the endogenous circadian clock located in the SCN, but is also modulated by direct inhibitory feedback from circulating glucocorticoids, and by a few inhibitory and excitatory pathways from the hippocampus, amygdale, stria terminalis, brain stem nucleus and other brain centres that allow HPA activity to vary according to the prevailing psychological, physical and immunological stressors (Lightman et al., 2002).

Patterns of pulsatile hormone release within the hypothalamo–pituitary–adrenal axis The HPA axis exhibits a mixture of basal and pulsatile hormone release. The ultradian pulse pattern (spikes within 24 hours) is superimposed upon the underlying circadian rhythms and is entrained to the light–dark and sleep–wake cycles (Czeisler, 1995; Veldhuis et al., 1990, 2009). The corticotropic axis receives time information via oscillator neurons in the SCN that input to (ARC) CRH-ergic neurons, which then release CRH into the portal system in a periodic and pulsatile manner (Haus, 2007). ACTH is then secreted in a pulsatile pattern superimposed on a circadian variation in response to the pulsatile CRH secretion, followed by corresponding pulses of cortisol secretion into the adrenal veins (Haus, 2007). There is also some evidence for intrinsic pulsatility of ACTH secretion by pituitary corticotrophs, independent of hypothalamic inputs (Gambacciani et al., 1987; Gudmundsson and Carnes, 1997). In vitro studies suggest that CRH may predominantly regulate sustained HPA axis activation, whereas AVP is mainly responsible for acute, short-term stimulation (Gudmundsson and

117

Carnes, 1997; Watanabe et al., 1989). HPA axis activity varies according to stress and mealtimes, with specific alterations in the timing, intensity and duration of any stress stimulus resulting in widely varying patterns of ACTH pulsatile activity across the day (Crofford et al., 2004; Dallman et al., 1992). Studies also demonstrate acute inhibition of ACTH-mediated cortisol secretion with the onset of sleep (Haus, 2007; Weibel et al., 1995).

Periodicity of ACTH secretion About 12–18 pulses per 24-hour span of ACTH have been documented and a diurnal variation is seen, with trough ACTH secretion in the evening and the peak just before awakening in the early morning (Crofford et al., 2004; Haus, 2007). The corresponding cortisol peak in the early morning (around 07.00–08.00 hour) is followed by a gradual decline during the daytime and a ‘quiet period’ of lower adrenal responsiveness, with continuous, small-amplitude pulses throughout the evening and early night hours (Haus, 2007). The existence of this ‘quiet period’ when the cortisol secretory response to sustained ACTH pulses is relatively blunted indicates, perhaps, an intrinsic circadian propertyvariable ACTH responsiveness within the adrenal cortex (Haus, 2007; Haus and Touitou, 1994). Overall, the percentage pulsatile ACTH secretion predicts the mean daily cortisol concentration, suggesting that pulsatility of pituitary ACTH secretion is functionally important to (adrenal) target tissue responsiveness, rather than merely echoing pulsatility of upstream hypothalamic hormones (Leng and Brown, 1997).

Effects of ageing, gender and body adiposity on ACTH secretion A recent study on the impact of age, gender and body mass upon ACTH dynamics showed that basal, pulsatile and total 24-hour secretory rates were higher in men than women (Veldhuis

et al., 2009). This is in keeping with previous studies showing higher concentrations of ACTH in men than in women, both before and after psychological stress exposure, and consistent with the postmortem observation that CRHimmunoreactive neurons are more abundant in older men than older women (Bao and Swaab, 2007; Traustadottir et al., 2003; Uhart et al., 2006; Veldhuis et al., 2009). Cortisol–ACTH feedback-synchrony deteriorates with age in both men and women, and cortisol secretory patterns are more irregular with advancing age. Nevertheless, age does not seem to be a significant influence on the basal, pulsatile or total concentration of ACTH secretion (Leng and Brown, 1997). The possible mechanisms of age-related decline in the inhibition of the hypothalamo–corticotroph unit remain speculative. Increased body mass index (BMI) is also shown to be associated with enhanced pulsatile, basal and total 24-hour ACTH (Leng and Brown, 1997). The explanation for this positive relationship between ACTH secretion and BMI is unclear, but might be due to obesity-modulated amplification of ACTH responsiveness (Katz et al., 2000; Kopelman et al., 1988, Veldhuis et al., 2009). The possible effects of hyperinsulinemia, increased adrenergic outflow and circulating cytokines or adipokines require further study (Leng and Brown, 1997; Solano et al., 2001; Vicennati et al., 2006).

The glucocorticoid ultradian rhythm is maintained across the blood–brain barrier Using rapid-sampling in vivo microdialysis, Droste et al. demonstrated that the basal ultradian rhythm for corticosterone in male and female rats (around 1.2 pulses/hour) is maintained across the blood–brain barrier, allowing brain exposure to glucocorticoids to modulate emotion, motivation and cognition. However, peak levels of corticosterone secreted in response to stress are delayed by 20 minutes in the brain extracellular fluid compared to plasma, even though clearance occurs concurrently. This results in a

118

smaller exposure of the brain to stress-induced corticosterone than would be predicted from plasma hormone concentrations (Droste et al., 2008, 2009).

Regulation of thyrotroph function Thyroid hormone secretion is necessarily very tightly controlled, being essential for energy homeostasis and basal heat production (Morley, 1981). Thyrotrophin-releasing hormone (TRH) stimulates the release of both TSH and PRL from the anterior pituitary and demonstrates a light-dark-dependent circadian rhythm (Covarrubias et al., 1988; Haus, 2007; Martino et al., 1985). It also interacts with other circadian periodic neurotransmitters such as 5-HT (5-hydroxytryptamine), dopamine and noradrenaline (Bennett et al., 1989; Haus, 2007). This pulsatile pattern of TRH secretion is biologically important, because pituitary thyrotrophs are more responsive to intermittent than continuous TRH stimulation, in terms of TSH secretion (Haus, 2007; Spencer et al., 1980). Pulsatility of TSH secretion is not correlated with serum thyroid hormone levels, implying that TSH pulses are predominantly TRH driven (Brabant et al., 1991).

Ultradian pattern and regulation of TSH secretion TSH secretion is characterised by a diurnal variation with superimposed bursts (spiky secretion) (Roelfsema et al., 2009b). It demonstrates a series of discrete pulses with an average frequency of 0.4 pulses/hour in normal men and women (Brabant et al., 1990; Haus, 2007; Nicolau and Haus, 1994). These pulses show an unequal distribution, with clustering of pulses during the evening and night hours and consequent fusion of pulses resulting in a nocturnal rise in pulse amplitude. Hence, peak TSH concentration is between 2300 and 0400 hours, with subsequent decline to a nadir between 1200 and 1400 (Brabant et al., 1990; Greenspan et al., 1986;

Haus, 2007; Mantzoros et al., 2001). TSH pulsatile secretion is regulated through interplay between an apparent hypothalamic oscillator (modulated by inhibitory influences from dopamine, SRIF and other inhibitory neurotransmitters), direct stimulation from hypothalamic TRH and the negative feedback effect of circulating thyroid hormones (T3 and T4) (Mariotti, 2006; Roelfsema et al., 2009b). Sleep deprivation augments the circadian variation in TSH secretion attributed to changes in pulse amplitude, but not pulse frequency. Similarly, resumption of sleep (or fasting) suppresses TSH secretion by reducing pulse amplitude (Brabant et al., 1990; Romijin et al., 1990).

Effects of ageing and adiposity on ultradian TSH secretion Studies to date have shown no evidence for sexual dimorphism of TSH concentration, TSH pulsatile or basal secretion, TSH pattern regularity, or TSH diurnal release. The relationship between diurnal TSH secretion and body mass index has not been studied extensively, but some studies have shown increased TSH secretion in obese women compared with normal-weight, age-matched controls. This difference was reduced by weight reduction or administration of bromocriptin (Kok et al., 2005; Roelfsema et al., 2009a). The adipocyte-derived hormone leptin demonstrates a pattern of ultradian fluctuation that is synchronous with TSH, and it may turn out to be an important modulator of the thyrotroph axis via a stimulating effect on TRH synthesis and release (Lechan and Fekete, 2006; Roelfsema et al., 2009b). TSH secretion and the circadian rhythm remain intact in the elderly but a blunted nocturnal peak has been found. This is mainly due to a decrease in TSH pulse amplitude, as higher daytime trough levels result in higher mean concentration (Barreca et al., 1985; Greenspan et al., 1991; Haus, 2007; van Coevorden et al., 1989). In comparison with children or young adults, elderly subjects showed a slight phase delay in the circadian peak time of TSH and more marked phase

119

delay in T3, T4 and rT3, suggesting a delayed response of the thyroid gland to TSH stimulation in the elderly (Haus, 2007; Nicolau and Haus, 1994).

Regulation of lactotroph function The regulation of PRL secretion is complex and not fully understood. Release is regulated by various stimulatory and inhibitory secretagogues involving the hypothalamus, peripheral peptide modulators and sex hormones. Factors identified include TRH, dopamine, oxytocin, vasoactive intestinal peptides (VIPs), estrogen and growth factor, such as EGF (epidermal growth factor) and FGF-2 (fibroblast growth factor-2) (Haus, 2007; Iranmanesh et al., 1999; Tolis and Franks, 1979). In addition, external stimuli such as nipple stimulation, emotional stress and physical exercise are contributory factors in PRL secretion (Hauffa, 2001). PRL is primarily under inhibitory neural control of the hypothalamus via tonic suppression mediated by dopamine, which then exerts negative feedback on the ARC (Hauffa, 2001). PRL concentration rises during pregnancy and postpartum period (high estrogen states), but the 24-hour secretory pattern is maintained (Tay et al., 1996). Nursing reflexes dominate the pulsatile secretion during postpartum, with PRL pulses follow suckling episodes and usual nocturnal rise only apparent after cessation of breast-feeding (Haus, 2007; Tay et al., 1996).

Effect of light–dark and sleep–wake cycles on prolactin secretion PRL release exhibits basal, episodic (ultradian and pulsatile) and circadian rhythm in human and animal studies (Iranmanesh et al., 1999; Veldhuis et al.,1990). The normal secretory pattern of PRL comprises intermittent pulses, occurring every two to three hours with variable amplitude (Haus, 2007; Veldhuis et al., 1992). The highest plasma PRL concentration occurring late at night coincides with rapid eye movement (REM) sleep in both men and non-

lactating, non-pregnant women (Haus, 2007; Obal et al., 1994; Roky et al., 1995). This night-time elevation results from an increase in basal PRL secretion and pulse amplitude, but not in pulse frequency (Veldhuis et al., 1990). However, during nursing, nipple stimulation becomes the main regulator of circulating plasma PRL, with reflex elevation of both PRL and oxytoxin levels in lactating women (Haus, 2007; Leng and Brown, 1997). PRL pulse amplitude is also enhanced during sleep periods, irrespective of the time of the day, with immediate offset of active PRL secretion upon awakening (Haus, 2007; Reppert and Weaver, 2001). On the other hand, there is modest elevation in PRL concentration around the time of the usual sleep onset, even when subjects remain awake (Haus, 2007). These findings suggest that PRL is mainly governed by a circadian rhythm with superimposed pulsatile secretion modulated by the sleep–wakefulness pattern; peak PRL concentration occurs when sleep onset coincides with circadian rhythmicity (Haus, 2007; Spiegel et al., 1999; Waldstreicher et al., 1996).

Effect of ageing on prolactin secretion Ageing is associated with a significant decrement in circulating PRL concentrations, with reduced secretory burst mass, pulse amplitude and decline in the maximal PRL secretory rate achieved within each pulse, without alteration of pulse duration (Iranmanesh et al., 1999). This has been shown in studies involving older men and estrogen-unreplaced post-menopausal women (Iranmanesh et al., 1999; Maddox et al., 1991). These phenomena could be explained by the accumulation of PRL-derived amyloid in the human ageing pituitary gland and reduction in PRL-binding sites in the choroid plexus, hippocampus and hypothalamus in elderly individuals, contributing to a decline in lactotroph cell responsiveness to available stimulating hormones and/or a reduction in lactotroph cell mass (Di Carlo et al., 1992; Goya et al., 1991; Iranmanesh et al., 1999; Westermark et al.,

120

1997). The bioactivity of circulating PRL may also fall in elderly men and women (Iranmanesh et al., 1999; Maddox et al., 1991). In addition there was also greater ‘disorderliness’ with higher approximate entropy of PRL release seen in the older men suggesting lack of robustness in the synchrony of the regulation of PRL secretion (Iranmanesh et al., 1999).

Interaction between pulsatile secretion of prolactin and LH Many studies have showed synchrony between spontaneous pulsatile luteinizing hormone (LH) and PRL secretion, mediated through gonadotropin-releasing hormone (GnRH), in both normal or hypogonal women, with a strong positive relationship in pulse frequency and amplitude (Casper and Yen, 1981; Mais and Yen, 1986; Masaoka et al., 1988). In menstruating women, the pulse frequency of both LH and PRL is markedly lower and the pulse amplitude notably higher during luteal phase, compared with the follicular phase (Filicori et al., 1986; Masaoka et al., 1988). PRL levels increase following infusion of GnRH or agonist, and intermittent administration of GnRH has elicited pulsatile release of PRL in the luteal phase (Casper and Yen, 1981; Masaoka et al., 1988), confirming that, under some conditions, GnRH can act as a PRL-releasing factor, either via direct action on pituitary lactotrophs, or at a hypothalamic level, whereby cyclic attenuation of dopamine activity by episodic discharge of GnRH results in pulsatile release of both PRL and LH (Masaoka et al., 1988).

Physiological significance of pulsatile oxytocin secretion Along with AVP (which does not exhibit pulsatile secretion), oxytoxin is synthesised in the magnocellular neuroendocrine cells of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus and stored in secretory granules in the axon terminals that constitute

the posterior pituitary (Rubin et al., 1978). Oxytocin has an important role in lactation and normal progression of labour in human (Leng and Brown, 1997). Oxytocin neurons discharge action potential bursts in response to the suckling of hungry rat pups, at an interval of 5–10 minutes, resulting in synchronised firing and, hence, pulsatile oxytocin secretion (Wakerley and Ingram, 1993). The pulse of oxytocin then enters the blood as a bolus in high concentration to induce myoepithelial contraction and milk let-down (Bicknell, 1988; Leng and Brown, 1997). Gradual increases in oxytocin levels or continuous infusion of oxytocin elicit progressive desensitisation of the mammary receptors (Bicknell, 1988; Leng and Brown, 1997). Hence, pulses of oxytocin are optimally efficient in triggering lactation with brief, synchronised and high-frequency discharge from oxytocin cells (Leng and Brown, 1997). Oxytocin also has a facilitatory role in parturition; experimental inhibition of oxytocin action interrupts parturition and full restoration action requires restoration of normal oxytocin secretory pattern (Leng and Brown, 1997). However, both oxytocin knock-out mice and oxytocin-deficient women can have normal labour.

Discussion The phenomenon of oscillatory secretion is not exclusive to neuroendocrine axes and is widely found in various biological systems such as peripheral inter-cellular signalling (e.g. insulin secretion from beta-islet cells) and intra-cellular messengers [e.g. cyclic adenosine monophosphate (AMP) secretion] (Li and Goldbetet, 1992). Pulsatility appears to be a common and fundamental means of biological communication and organisation that is even present in a primitive organism such as slime mould (Dictyostelium discoideum). Slime mould is noted to respond to an external pulse of cyclic AMP every five minutes, but not to a continuous stimulus or to a shorter pulse interval (Martiel and Goldbeter, 1987). Biological oscillations were once considered to be the results of breakdown of effective self-regulation (Savageau, 1976), but subsequent work

121

has clearly evidenced their physiological advantages. Biological oscillation potentially confer five functional benefits in respect of (1) temporal organisation, entrainment and synchronisation, (2) spatial organisation, (3) prediction of repetitive events, (4) energy efficiency of frequency encoding of information and (5) precision of control (Rapp, 1987). Energy efficiency was first demonstrated in oscillatory insulin-driven glycolysis, with a reduction in 5–10% of free energy requirements (Rehmus et al., 1981) and other studies have demonstrated a close relationship between a specific pattern of periodic stimulation and the optimal responsiveness of the target cells in various cellular systems. Periodic stimuli were found to be more efficient than random or chaotic signals in a study based on a model on receptor desensitisation, with cellular responsiveness being more sensitive to variations in the time interval between pulses than to variations in the duration of each pulse (Li and Goldbetet, 1992). The key to maintaining high responsiveness is to ensure adequate recovery time, so not surprisingly, continuous stimulation induces a decrease in the responsiveness of target cells – due to receptor desensitisation – that in turn protects target cells from the adverse consequences of over-stimulation. Hence, pulsatile signals appears to be the optimal mode of communication with downstream targets by achieving a balance between two distinct demands: (1) a reasonable ligand-free time to elapse for the receptor to resensitise up to a sufficient level and (2) time elapsed before the next stimulus arrives should not be so long as to overly constrain the number of significant responses that the receptor can generate in a given period (Li and Goldbetet, 1992). However, because of differences in receptor kinetics and on/off times, not all receptor systems demonstrate desensitisation. Biological oscillations enable the frequency encoding of physiological information. A constant hormone concentration in the fluid surrounding a specialised secretory cell can result in sustained periodic oscillation of that cell’s membrane potential. Moreover, the frequency of the oscillation can be tuned according to the extracellular hormone concentration, further enhancing the precision of

control (Berridge and Rapp, 1979). Hence, alterations in hormone concentration arising from pulsatility elicit corresponding change in membrane oscillator frequency that tends to optimise the physiological consequences (Rapp, 1987). In view of various physiological advantages in biological oscillations, it is not surprising to find that ultradian and circadian rhythms, which have been known about in hypothalamic–pituitary– end-organ axes for decades, appear to be a common phenomenon in mammalian species. This physiological rhythm of hormonal secretion is demonstrated throughout human life span from the newborn period, through childhood, puberty, pregnancy and lactation, to ageing, and may exhibit sexual dimorphism (Hauffa, 2001). Pulsatility in the neuroendocrine axes involves a dynamic interaction via non-linear, timedelayed, dose-responsive feedback and feedforward activities (Veldhuis et al., 2001). Each neuroendocrine axis exhibits unique time-dependent secretory patterns to presumptively achieve homeostasis. Hence, neuroendocrine disease could be attributed to the disruption of multilevel, inter-nodal communication or within a control locus itself (Pincus et al., 1999; Veldhuis et al., 2001). The use of frequent hormone sampling techniques in conjunction with sophisticated new mathematical tools for the analysis of the behavior of these integrated biological networks has enabled us to gain some insight into the pathophysiology of human disease associated with altered pulsatility (Veldhuis et al., 2001). In addition, novel parameters describing the overall orderliness of neurohormone secretion, such as approximate entropy (ApEn) may facilitate the identification of disease-specific alterations of hormone pulsatile patterns, potentially leading to targeted biopharmaceutical modification of pulsatile hormonal secretion as a form of disease therapy in the future.

Abbreviations ACTH AMP ARC

adrenocorticotropic hormone adenosine monophosphate arcuate nucleus

122

AVP BMI CRH EGF FGF-2 GH GHRH GHRP-2 GHS GnRH HPA 5-HT IGF-1 LH PEV PRL PVN REM SCN SON SRIF STAT5b TRH TSH VIP

arginine vasopressin body mass index corticotropin-releasing hormone epidermal growth factor fibroblast growth factor-2 growth hormone growth-hormone-releasing hormone growth hormone-releasing peptide 2 growth hormone secretagogue gonadotropin-releasing hormone hypothalamus–pituitary– adrenal 5-hydroxytryptamine insulin-like growth factor 1 luteinizing hormone periventricular nucleus prolactin paraventricular nucleus rapid eye movement suprachiasmatic nucleus supraoptic nucleus somatostatin signal transducer and activator of transcription 5B thyrotrophin-releasing hormone thyroid-stimulating hormone vasoactive intestinal peptide

References Achermann, J. C., Brook, C. G., Robinson, I. C., Matthews, D. R., & Hindmarsh, P. C. (1999). Peak and trough growth hormone concentration influence growth hormone and serum insulin like growth factor-1 concentrations in short children. Clinical Endocrinology (Oxford), 50, 301–308. Baigent, S. M. (2001). Peripheral corticotrophin-releasing hormone and urocortin in the control of the immune response. Peptides, 22(5), 809–820. Bao, A. M. & Swaab, D. F. (2007). Gender difference in agerelated number of corticotrophin-releasing hormone-expressing neurons in the human hypothalamic paraventricular

nucleus and the role of sex hormones. Neuroendocrinology, 85, 27–36. Barreca, T., Franceschini, R., Messina, V., Bottaro, L., & Rolandi, E. (1985). 24-Hour thyroid-stimulating hormone secretory pattern in elderly men. Gerontology, 31(2), 119–123. Bennett, G. W., Marsden, C. A., Fone, K. C.F., Johnson, J. V., & Heal, D. J. (1989). TRH-catecholamine interactions in brain and spinal cord. Annals of the New York Academy of Science, 553, 106–120. Berridge, M. J. & Rapp, P. E. (1979). A comparative survery of the function, mechanism and control of cellular oscillators. Journals of Experimental Biology, 81, 217–279. Bertherat, J., Dournaud, P., Berod, A., Normand, E., Block, B., Rostene, W., et al. (1992). Growth hormone-releasing hormone-synthesizing neurons are a subpopulation of somatostatin receptor-labeled cells in the rat arcuate nucleus: A combined in situ hybridization and receptor light-microscopic radioautographic study. Neuroendocrinology, 25, 25–33. Bicknell, R. J. (1988). Optimizing release from peptide hormone secretory nerve terminals. The Journal of Experimental Biology, 139, 51–65. Bluet-Pajot, M. T., Epelbaum, J., Gourdji, D., Hammond, C., & Kordon, C. (1998). Hypothalamic and hypophyseal regulation of growth hormone secretion. Cellular and Molecular Neurobiology, 18, 101–123. Brabant, G., Prank, K., Hoang-Vu, C., Hesch, R. D., & von zur Muhlen, A. (1991). Hypothalamic regulation of pulsatile thyrotropin secretion. Journal of Clinical Endocrinology & Metabolism, 72, 145–150. Brabant, G., Prank, K., Ranft, U., Schuermeyer, T., Wagner, T. O., Hauser, H., et al. (1990). Physiological regulation of circadian and pulsatile thyrotropin secretion in normal man and woman. Journal of Clinical Endocrinology & Metabolism, 70(2), 403–409. Casper, R. F. & Yen, S. S.C. (1981). Simultaneous pulsatile release of prolactin and luteinizing hormone induced by luteinizing hormone-releasing factor agonist. Journal of Clinical Endocrinology & Metabolism, 52, 934. Cauter, E. V., Plat, L., & Copinschi, G. (1998). Interrelations between sleep and the somatotropic axis. Sleep, 21(6), 553–566. Cermakian, N. & Boivin, D. B. (2003). A molecular perspective of human circadian rhythm disorders. Brain Research Reviews, 42, 204–220. Cermakian, N. & Boivin, D. B. (2009). The regulation of central and peripheral circadian clocks in humans. Obesity, 10(Suppl. 2), 25–36. Clark, R. G., Carlsson, L. M.S., Rafferty, B., & Robinson, I. C.A.F. (1987). Growth hormone secretory profiles in conscious female rats. Journal of Endocrinology, 114, 399–407. Clark, R. G., Chamber, G., Lewin, J., & Robinson, I. C.A.F. (1986). Automated repetitive microsampling of blood: Growth hormone profiles in conscious male rats. Journal of Endocrinology, 111, 27–35. Covarrubias, L., Uribe, R. M., Mendes, M., Charli, J. L., & Joseph-Bravo, P. (1988). Neuronal TRH synthesis: Developmental and circadian TRH mRNA levels. Biochemical and Biophysical Research Communications, 151(1), 615–622.

123 Crofford, L. J., Young, E. A., Engleberg, N. C., Korszun, A., Brucksch, C. B., McClure, L. A., et al. (2004). Basal circadian and pulsatile ACTH and cortisol secretion in patients with fibromyalgia and/or chronic fatigue syndrome. Brain, Behavious and Immunity, 18, 314–325. Czeisler, C. A. (1995). The effect of light on the human circadian pacemaker. In D. J. Chadwick, , K. Ackrill (Eds.), Circadian clocks and their adjustments. Ciba Foundation Symposium (Vol. 183, pp. 254–302). New York: John Wiley & Sons. Dallman, M. F., Akana, S. F., Scribner, K. A., Bradbury, M. J., Walker, C. D., Walker, A., et al. (1992). Stress, feedback and facilitation in the hypothalamo-pituitary-adrenal axis. Journal of Neuroendocrinology, 5, 517–526. Davey, H. W., Park, S. H., Grattan, D. R., McLachlan, M. J., & Waxman, D. J. (1999). STAT5b-deficient mice are growth hormone pulse-resistent. Role of STAT5b in sex-specific liver p450 expression. The Journal of Biological Chemistry, 274, 35331–35336. Davidson, M. B. (1987). Effects of growth hormone on carbohydrate and lipid metabolism. Endocrine Reviews, 8, 115–131. Di Carlo, R., Muccioli, G., Papotti, M., & Bussolati, G. (1992). Characterization of prolactin receptor in human brain and choroid plexus. Brain Research, 570, 341–346. Droste, S. K., de Groote, L., Atkinson, H. C., Lightman, S. L., Reul, J. M., & Linthorst, A. C. (2008). Corticosterone levels in the brain show a distinct ultradian rhythm but a delayed response to a forced swim stress. Endocrinology, 149, 3244–3253. Droste, S. K., de Groote, L., Lightman, S. L., Reul, J. M., & Linthorst, A. C. (2009). The ultradian and circadian rhythms of free corticosterone in the brain are not affected by gender: An in vivo microdialysis study in Wistar rats. Journal of Neuroendocrinology, 21, 32–40. Farhy, L. S., Straume, M., Johnson, M. L., Kovatchev, B., & Veldhuis, J. D. (2002). Unequal autonegative feedback by GH models the sexual dimorphism in GH secretory dynamics. American Journal of Physiology Regulatory, Integrative and Comparative Physiology, 282, R753–R764. Farhy, L. S. & Veldhuis, J. D. (2003). Joint pituitary-hypothalamic and intrahypothalamic autofeedback construct of pulsatile growth hormone secretion. American Journal of Physiology Regulatory, Integrative and Comparative Physiology, 285, R1240–R1249. Farhy, L. S. & Veldhuis, J. D. (2004). Putative GH pulse renewal: Periventricular somatostatinergic control of an arcuate-nuclear somatostatin and GH-releasing hormone oscillator. American Journal of Physiology Regulatory, Integrative and Comparative Physiology, 286, R1030–R1042. Farhy, L. S. & Veldhuis, J. D. (2005). Deterministic construct of amplifying actions of ghrelin on pulsatile growth hormone secretion. American Journal of Physiology Regulatory, Integrative and Comparative Physiology, 288, R1649–R1663. Filicori, M., Santoro, N., Merriam, G. R., & Cowley, W. F. Jr. (1986). Characterization of the physiological pattern of episodic gonadotropin secretion throughout the human menstrual cycle. Journal of Clinical Endocrinology & Metabolism, 62, 1136.

Frohman, L. A. (1996). New insight into the regulation of somatotrope function using genetic and transgenic models. Metabolism, 45, 1–3. Furuhata, Y., Nishihara, M., & Takahashi, M. (2002). Effects of pulsatile secretion of growth hormone on fat deposition in human GH transgenic rats. Nutrition Research Reviews, 15, 231–244. Gambacciani, M., Liu, J. H., Swartz, L. H., Tuaros, V. S., Rasmussen, D. D., & Yen, S. S.C. (1987). Intrinsic pulsatility of ACTH release from the human pituitary in vitro. Clinical Endocrionology (Oxford), 26, 557–563. Gentili, A., Mulligan, T., Godschalk, M., Clore, J., Patrie, J., Iranmanesh, A., et al. (2002). Unequal impact of short-term testosterone repletion on the somatotropic axis of young and older men. Journal of Clinical Endocrinology & Metabolism, 87, 825–834. Gevers, E., Pincus, S. M., Robinson, I. C.A.F., & Veldhuis, J. D. (1998). Differential orderliness of the GH release process in castrate male and female rats. American Journal of Physiology Regulatory, Intergrative and Comparative Physiology, 274, R437–R444. Giustina, A. & Veldhuis, J. D. (1998). Pathophysiology of the neuroregulation of growth hormone secretion in experimental animals and human. Endocrine Reviews, 19, 717–797. Goldbeter, A., Dupont, G., & Halloy, J. (2000). The frequency encoding of pulsatility. In P. J. Chadwick & J. A. Gode (Eds.), Mechanism and biological significance of pulsatile hormone secretion. Novartis Foundation Symposium (Vol. 221, pp. 19–45). Chichester: John Wiley & Sons. Goya, R. G., Castro, M. G., & Meites, J. (1991). Differential effect of aging on serum levels of prolactin and alphamelanotropin in rats. Proceedings of the Society for Experimental Biology and Medicine, 196, 218–221. Greenspan, S. L., Klibanski, A., Rowe, J. W., & Elahi, D. (1991). Age-related alterations in pulsatile secretion of TSH: Role of dopaminergic regulation. American Journal of Physiology, 260(3), E486–E491. Greenspan, S. L., Klibanski, A., Schoenfeld, D., & Ridgway, E. C. (1986). Pulsatile secretion of thyrotropin in man. Journal of Clinical Endocrinology & Metabolism, 63, 661–668. Gudmundsson, A. & Carnes, M. (1997). Pulsatile adrenocoticotropic hormone: An overview. Biological Psychiatry, 41, 342–365. Hartman, M. L., Faria, A. C., Vance, M. L., Johnson, M. L., Thorner, M. O., & Veldhuis, J. D. (1991). Temporal structure of in vivo growth hormone secretory events in man. American Journal of Physiology Endocrinology and Metabolism, 260, E101–E110. Hartman, M. L., Pincus, S. M., Johnson, M. L., Matthews, D. H., Faunt, L. M., Vance, M. L., et al. (1994). Enhanced basal and disorderly growth hormone secretion distinguish acromegalic from normal pulsatile growth hormone release. The Journal of Clinical Investigation, 94, 1277–1288. Hartman, M. L., Veldhuis, J. D., Johnson, M. L., Lee, M. M., Alberti, K. G., Samojlik, E., et al. (1992). Augmented growth hormone secretory burst frequency and amplitude mediate enhanced GH secretion during a

124 two-day fast in normal men. Journal of Clinical Endocrinology & Metabolism, 74, 757–765. Hauffa, B. P. (2001). Clinical implications of pulsatile hormone signals. Growth Hormone & IGF Research, 11(Suppl. A), S1–S8. Haus, E. (2007). Chronobiology in the endocrine system. Advanced Drug Delivery Reviews, 59, 985–1014. Haus, E. & Touitou, Y. (1994). Principles of clinical chronobiology. In Y. Touituo & E. Haus (Eds.), Biologic rhythms in clinical and laboratory medicine (2nd ed., pp. 6–34). New York: Springer-Verlag. Ho, K. Y., Evans, W. S., Blizzard, R. M., Veldhuis, J. D., Merriam, G. R., Samojlik, E., et al. (1987). Effects of sex and age on the 24-hour profile of growth hormone secretion in man: Importance of endogenous estradiol concentrations. Journal of Clinical Endocrinology & Metabolism, 64(1), 51–58. Ionescu, Ma. nd & Frohman, L. A. (2006). Pulsatile secretion of growth hormone persists during continuous stimulation by CJC-1295, a long acting GH-releasing hormone analog. Journal of Clinical Endocrinology & Metabolism, 91, 4792–4797. Iranmanesh, A., Mulligan, T., & Veldhuis, J. D. (1999). Mechanisms subserving the physiological nocturnal relative hypoprolactinemia of healthy older men: Dual decline in the prolactin secretory burst mass and basal release with preservation of pulse duration, frequency and interpulse interval – a general clinical research center study. Journal of Clinical Endocrinology & Metabolism, 84, 1083–1090. Iranmanesh, A., South, S., Liem, A. Y., Clemmons, D., Thorner, M. O., Weltman, A., et al. (1998). Unequal impact of age, percentage body fat and testosterone concentrations on the somatotrophic, IGF-1 and IGF-binding protein responses to a three-day intravenous growth hormonereleasing hormone pulsatile infusion in men. European Journal of Endocrinology, 139, 59–71. Jorgensen, J. O., Blum, W. F., Moller, N., Christiansen, J., Alberti, K. G., & Orskov, H. (1991). Short term changes in serum insulin-like growth factor and IGF binding protein 3 after different modes of intravenous growth hormone exposure in GH-deficient patients. Journal of Clinical Endocrinology and Metabolism, 72, 582–587. Katz, J. R., Taylor, N. G., Perry, L., Yudkin, J. S., & Coppack, S. W. (2000). Increased response of cortisol and ACTH to corticotrophin releasing hormone in centrally obese men, but not in post-menopausal women. International Journal of Obesity and Related Metabolic Disorder, 24(Suppl. 2), S138–S139. Klein, D., Moore, R. Y., & Reppert, S. M. (1991). Suprachiasmatic nucleus: The mind’s clock. New York: Oxford University Press. Ko, C. H. & Takahashi, J. S. (2006). Molecular components of the mammalian circadian clock. Human Molecular Genetics, 15, R271–R277. Kok, P., Roelfsema, F., Langendonk, J. G., Frolich, M., Burggraaf, J., Meinders, A. E., et al. (2005). High circulating thyrotropin levels in obese women are reduced after body weight loss induced by caloric restriction. Journal of Clinical Endocrinology and Metabolism, 90, 4659–4663. Kopelman, P. G., Grossman, A., Lavender, P., Besser, G. M., Rees, L. H., & Coy, D. (1988). The cortisol response to

corticotrophin-releasing factor is blunted in obesity. Clinical Endocrinology (Oxford), 28, 15–18. Lang, I., Schernthaner, G., Pietschmann, P., Kurz, R., Stephenson, J. M., & Templ, H. (1987). Effects of age and sex on growth hormone response to growth hormone-releasing hormone in healthy individuals. Journal of Clinical Endocrinology & Metabolism, 65, 535–540. Lechan, R. M. & Fekete, C. (2006). The TRH neuron: A hypothalamic integrator of energy metabolism. Progress in Brain Research, 153, 209–235. Leng, G. & Brown, D. (1997). The origins and significance of pulsatility in hormone secretion from the pituitary. Journal of Neuroendocrinology, 9, 493–513. Li, Y. X. & Goldbetet, A. (1992). Pulsatile signalling in intercellular communication. Biophysical Journal, 61, 161–171. Lieman, H. J., Adel, T. E., Forst, C., von Hagen, S., & Santoro, N. (2001). Effects of aging and estradiol supplementation on GH axis dynamics in women. Journal of Clinical Endocrinology & Metabolism, 86, 3918–3923. Lightman, S. L., Windle, R. J., Ma, X. M., Harbuz, M. S., Shanks, N. M., Julian, M. D., et al. (2002). Hypothalamicpituitary-adrenal function. Archives of Physiology and Biochemistry, 110(1–2), 90–93. Maddox, P. R., Jones, D. L., & Mansel, R. E. (1991). Basal prolactin and total lactogenic hormone levels by microassay and immunoassay in normal human sera. Acta Endocrinologica (Copenh), 12, 621–627. Mais, M. & Yen, S. S.C. (1986). Prolactin-releasing action of gonadotropin-releasing hormone in hypogonal women. Journal of Clinical Endocrinology & Metabolism, 62, 1089. Mantzoros, C. S., Ozata, M., Negrao, A. B., Suchard, M. A., Ziotopoulou, M., Caglayan, S., et al. (2001). Synchonicity of frequently sampled thyrotropin (TSH) and leptin concentrations in healthy adults and leptin-deficient subjects: Evidence for possible partial TSH regulation by leptin in humans. Journal of Clinical Endocrinology & Metabolism, 86, 3284–3291. Mariotti, S. (2006). Normal physiology of the hypothalamicpituitary-thyroidal system and relation to the neural system and other endocrine gland. Chapter 4. Thyroid disease manager. www.thyroidmanager.org/chapter4 Martha, P. M. Jr., Goorman, K. M., Blizzard, R. M., Rogol, A. D., & Veldhius, J. D. (1992). Endogenous growth hormone secretion and clearance rates in normal boy as determined by deconvolution analysis: Relationship to age, puberty status and body mass. Journal of Clinical Endocrinology & Metabolism, 73, 336–344. Martiel, J.-L. & Goldbeter, A. (1987). A model based on receptor desensitization for cyclic AMP signalling in Dictyostelium cells. Biophysical Journal, 52, 807–828. Martin, F. C., Yeo, A. L., & Sonksen, P. H. (1997). Growth hormone secretion in the elderly: Aging and the somatopause. Bailliere’s Clinical Endocrinology and Metabolism, 11, 223–250. Martino, E., Bambini, G., Vaudagna, G., Breccia, M., & Baschierim, L. (1985). Effects of continuous light and dark exposure on hypothalamic thyrotropin-releasing hormone in rats. Journal of Endocrinological Investigation, 8, 31–33.

125 Masaoka, K., Kitazawa, M., & Kumasaka, T. (1988). Pulsatile secretion of prolactin and luteinizing hormone and their synchronous relationship during the human menstrual cycle. Gynecological Endocrinology, 2, 293–303. Moore, R. Y. & Silver, R. (1998). Suprachiasmatic nucleus organisation. Chronobiology International, 15, 475–487. Morley, J. E. (1981). Neuroendocrine control of thyrotropin secretion. Endocrine Reviews, 12, 396–436. Mueller, E. E., Locatelli, V., & Cocchi, D. (1999). Neuroendocrine control of growth hormone secretion. Physiological Reviews, 79, 511–607. Muniyappa, R., Sorkin, J. D., Veldhuis, J. D., Harman, S. M., Munzer, T., Bhasin, S., & Blackman, M. R., (2007). Long term testosterone supplementation augments overnight growth hormone secretion in healthy older man. American Journal of Physiology Endocrinology and Metabolism, 293, E769–E775. Nicolau, G. Y. & Haus, E. (1994). Chronobiology of the hypothalamic-pituitary-thyroid axis. In Y. Touitou & E. Haus (Eds.), Biologic rhythms in clinical and laboratory medicines (2nd ed., pp. 330–347). New York: SpringerVerlag. Obal, Jr., Payne, F., Kacsoh, L., Opp, B., Kapas, M., Grosvenor, L., et al. (1994). Involvement of prolactin in the REM sleep-promoting activity of systemic vasoactive intestinal peptide (VIP). Brain Research, 645, 143–149. Pincus, S. M., Gevers, E., Robinson, I. C.A.F., Van de Berg, G., Roelfsema, F., Hartman, M. L., et al. (1996). Females secrete growth hormone with more process irregularity than males in both human and rat. American Journal of Physiological Endocrinology and Metabolism, 270, E107–E115. Pincus, S. M., Hartman, M. L., Roelfsema, F., Thorner, M. O., & Veldhuis, J. D. (1999). Hormone pulsatility discrimination via coarse and short time sampling. American Journal of Physiological Endocrinology and Metabolism, 277, E948–E957. Rapp, P. E. (1987). Why are so many biological systems periodic? Progress in Neurobiology, 29, 261–273. Rehmus, P., Termonia, Y., & Ross, J. (1981). Dissipation in driven oscillatory chemical systems with application to glycolysis. Kinam, 3, 123–155. Reppert, S. M. & Weaver, D. R. (2001). Molecular analysis of mammalian circadian rhythm. Annual Review of Physiology, 63, 647–676. Richard, D., Huang, Q., & Timofeeva, E. (2000). The corticotrophin-releasing hormone system in the regulation of energy balance in obesity. International Journal of Obesity and Related Metabolic Disorder, 24(2), S36–S39. Robinson, I. C.A.F. (1991). The growth hormone secretory pattern: A response to neuroendocrine signals. Acta Paediatrica Scandinavica Supplement, 372, 70–78. Roelfsema, F., Biermasz, N. R., Frolich, M., Keenan, D. M., Veldhuis, J. D., & Romijin, J. A. (2009a). Diminished and irregular thyrotropin secretion with preserved diurnal rhythm in patients with active acromegaly. Journal of Clinical Endocrinology & Metabolism, 94, 1945–1950. Roelfsema, F., Pereira, A. M., Veldhuis, J. D., Adriaanse, R., Endert, E., Fliers, E., et al. (2009b). Thyrotropin secretion

profiles are not different in men and women. Journal of Clinical Endocrinology & Metabolism, 94, 3963–3967. Roky, R., Obal, Jr., Valatx, F., Bredow, J. L., Fang, S., Pagano, J., et al., (1995) Prolactin and rapid eye movement sleep regulation. Sleep, 18(7), 536–542. Romijin, J. A., Adriaanse, R., Brabant, G., Prank, K., Endert, E., & Wiersinga, W. M. (1990). Pulsatile secretion of thyrotropin during fasting: A decrease of thyrotropin pulse amplitude. Journal of Clinical Endocrinology & Metabolism, 70, 1631–1636. Rubin, R. T., Poland, R. E., Gouin, P. R., & Tower, B. B. (1978). Secretion of hormones influencing water and electrolyte balance (antidiuretic hormone, aldosterone, prolactin) during sleep in normal adult men. Psychosomatic Medicine, 40, 44–59. Savageau, M. A. (1976). Biochemical system analysis. A study of function and design in molecular biology (p. 121). Reading, MA: Addison-Wesley. Shah, N., Evans, W. S., Bowers, C. Y., & Veldhuis, J. D. (1999). Tripartite neuroendocrine activation of the human growth hormone axis in women by continuous 24-hour GH-releasing peptide (GHRP-2) infusion: Pulsatile, entropic and nystohemeral mechanisms. Journal of Clinical Endocrinology and Metabolism, 84, 2140–2150. Shuto, Y., Shibasaki, T., Otagiri, A., Kuriyama, H., Ohata, H., Tamura, H., et al. (2002). Hypothalamic growth hormone secretagogue receptor regulates growth hormone secretion, feeding and adiposity. The Journal of Clinical Investigation, 109, 1429–1436. Soares-Welch, C., Farhy, L., Mielke, K. L., Mahmud, F. H., Miles, J. M., Bowers, C. Y., et al. (2005). Complementary secretagogue pairs unmask prominent gender-related contrast in mechanism of growth hormone pulse renewal in young adults. Journal of Clinical Endocrinology & Metabolism, 90, 2225–2232. Solano, M. P., Kumar, M. M., Fernandez, B., Jones, L., & Goldberg, R. B. (2001). The pituitary response to ovine corticotrophin-releasing hormone is enhanced in obesed men and correlates with insulin resistance. Hormone and Metabolic Research, 33, 39–43. Spencer, C. A., Greenstadr, M. A., Wheeler, W. S., Kletzky, O. A., & Nicoloff, J. T. (1980). The influence of a long-term low dose thyrotropin-releasing hormone infusions on serum thyrotropin and prolactin concentrations in man. Journal of Clinical Endocrinology and Metabolism, 51(4), 771–775. Spiegel, K., Leproult, R., & Van Cauter, E. (1999). Impact of sleep debt on metabolic and endocrine function. Lancet, 354, 1435–1439. Stolar, M. W. & Baumann, G. (1986). Secretory patterns of growth hormone during basal periods in man. Metabolism, 25, 883–888. Tay, C. C., Glasier, A. F., & McNeilly, A. S. (1996). Twenty-four hour patterns of prolactin secretion during lactation and the relationship to suckling and the resumption of fertility in breast-feeding women. Human Reproduction, 11(5), 950–955. Tolis, G. & Franks, S. (1979). Physiology and pathology of prolactin secretion. In G. Tolis, F. Labrie, & J. B. Martin (Eds.), Clinical endocrinology: A pathophysiological approach (pp. 291–317). New York: Raven Press.

126 Traustadottir, T., Bosch, P. R., & Matt, K. S. (2003). Gender differences in cardiovascular and hypothalamic-pituitaryadrenal axis responses to psychological stress in healthy older adult men and women. Stress, 6, 133–140. Uhart, M., Chong, R. Y., Oswald, L., Lin, P. I., & Wand, G. S. (2006). Gender differences in hypothalamic-pituitary-adrenal axis reactivity. Psychoneuroendocrinology, 31, 642–652. Vahl, N., Jorgensen, J. O., Skjaerback, C., Veldhuis, J. D., Orskov, H., & Christiansen, J. (1997). Abdominal adiposity rather than age and sex predicts the mass and patterned regularity of growth hormone secretion in mid-life healthy adults. American Journal of Physiology Endocrinology and Metabolism, 272, E1108–E1116. Van Coevorden, A., Laurent, E., Decoster, C., Kerkhofs, M., Neve, P., van Cauter, E., et al. (1989). Decreased basal and stimulated thyrotropin secretion in healthy elderly men. Journal of Clinical Endocrinology & Metabolism, 69(1), 177–185. Veldhuis, J. D. (1998). Review: Neuroendocrine control of pulsatile growth hormone release in the human: Relationship with gender. Growth Hormone & IGF Reseach, 8, 49–59. Veldhuis, J. D. (2008). Aging and hormones of the hypothalo-pituitary axis: Gonadortopic axis in men and somatotropic axes in men and women. Ageing Research Reviews, 7, 189–208. Veldhuis, J. D. & Bowers, C. Y. (2003). Three-peptide control of pulsatile and entropic feedback-sensitive modes of growth hormone secretion: Modulation by estrogen and aromatizable androgen. Journal of Pediatric Endocrinology and Metabolism, 16, 587–605. Veldhuis, J. D. & Bowers, C. Y. (2009a). Determinants of GHreleasing hormone and GH-releasing peptide synergy in men. American Journal of Physiology Endocrinology and Metabolism, 296, E1085–E1092. Veldhuis, J. D., Erickson, D., Mielke, K., Fahry, L. S., Keenan, D. M., & Bowers., C. Y. (2005). Dinstinctive inhibitory mechanisms of age and relative visceral adiposity on GH secretion in pre- and postmenopausal women studied under a hypogonal clamp. Journal of Clinical Endocrinology and Metabolism, 90, 6006–6013. Veldhuis, J. D., Evans, W. S., & Bowers., C. Y. (2002). Impact of estradiol supplementation on dual peptidyl drive of growth hormone secretion in postmenopausal women. Journal of Clinical Endocrinology and Metabolism, 87(2), 859–866. Veldhuis, J. D., Hudson, S. B., Erickson, D., Bailey, J. N., Reynolds, G. A., & Bowers, C. Y. (2009b). Relative effects of estrogen, age and visceral fat on pulsatile growth hormone secretion in healthy women. American Journal of Physiology Endocrinology and Metabolism, 297, E367–E374. Veldhuis, J. D., Iranmanesh, A., Naftolowitz, D., Tatham, N., Cassidy, F., & Carroll, B. J. (1990). Twenty-four rhythms in plasma concentration of adenohypophyseal hormones are generated by distinct amplitude and/or frequency modulation of underlying pituitary secretory bursts. Journal of Clinical Endocrinology & Metabolism, 71, 1616–1623. Veldhuis, J. D., Johnson, M. L., Lizarralde, G., & Iranmanesh, A. (1992). Rhythmic and nonrhythmic modes of anterior pituitary gland secretion. Chronobiology International, 9(5), 371–379.

Veldhuis, J. D., Liem, A. Y., South, S., Weltman, A., Weltman, J., Clemmons, D. A., et al. (1995). Differential impact of age, sex hormones and obesity on basal versus pulsatile growth hormone secretion in men as assessed in an ultrasensitive chemiluminescence assay. Journal of Clinical Endocrinology and Metabolism, 80, 3209–3222. Veldhuis, J. D., Roelfsema, F., Iranmanesh, A., Carroll, B. J., Keenan, D. M., & Pincus, S. M. (2009). Basal, pulsatile, entropic (patterned) and spiky(staccato-like) properties of ACTH secretion: Impact of age, gender and body mass index. Journal of Clinical Endocrinology and Metabolism, 94(10), 4045–4052. Veldhuis, J. D., Roemmich, J. N., Richmond, E.Ja. nd, & Bowers, C. Y. (2006). Somatotropic and gonadotropic axes linkage in infancy, childhood and the puberty-adult transition. Endocrine Reviews, 27, 101–140. Veldhuis, J. D., Roemmich, J. N., & Rogol, A. D. (2000). Gender and sexual maturation-dependant contrasts in the neuroregulation of growth hormone secretion in prepubertal and the late adolescent males and females–a general clinical research center-based study. Journal of Clinical Endocrinology and Metabolism, 85, 2385–2394. Veldhuis, J. D., Straume, M., Iranmanesh, A., Mulligan, T., Jaffe, C., Barkan, A., et al. (2001). Secretory process regularity monitors neuroendocrine feedback and feedforward signalling strength in humans. American Journal of Physiology Regulatory, Intergrative and Comparative Physiology, 280, R721–R729. Vicennati, V., Ceroni, L., Genghini, S., Patton, L., Pagotto, U., & Pasquali, R. (2006). Sex difference in the relationship between the hypothalamic-pituitary-adrenal axis and sex hormones in obesity. Obesity (Silver Spring), 12, 235–243. Wakerley, J. B. & Ingram, C. D. (1993). Synchronisation of bursting in hypothalamic oxytocin neurones: Possible coordinating mechanisms. News in Physiological Sciences, 8, 129–144. Waldstreicher, J., Duffy, J. F., Brown, E. N., Rogacz, S., Allan, J. S., & Czeisler, C. A. (1996). Gender differences in the temporal organisation of prolactin secretion: Evidence for a sleep-independent circadian rhythm of circulating PRL levels – a clinical research center study. Journal of Clinical Endocrinology & Metabolism, 81(4), 1483–1487. Watanabe, T., Oki, Y., & Orth, D. N. (1989). Kinetic actions and interactions of arginine vasopressin, angiotensin-II and oxytocin on adrenocorticotropin secretion by rat anterior pituitary cells in the microperfusion system. Endocrinology, 125, 1921–1931. Weibel, L., Follenius, M., Spiegel, K., Ehrhart, J., & Brandenberger, G. (1995). Comparative effect of night and daytime sleep on the 24-hour cortisol secretory profile. Sleep, 18(7), 549–556. Weltman, A., Weltman, J. Y., Hartman, M. L., Abbott, R. D., Rogol, A. D., Evans, W. S., et al. (1994). Relationship between age, percentage body fat, fitness and 24-hour growth hormone release in healthy young adults: Effects of gender. Journal of Clinical Endocrinology and Metabolism, 78, 543–548. Westermark, P., Eriksson, L., Engstrom, U., Enestrom, S., & Sletten, K. (1997). Prolactin-derived amyloid in the aging pituitary gland. American Journal of Physiology, 150, 67–73.

L. Martini (Eds.) Progress in Brain Research, Vol. 181 ISSN: 0079-6123 Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 8

Melatonin: a multitasking molecule Russel J. Reiter
, Dun-Xian Tan and Lorena Fuentes-Broto Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America

Abstract: Melatonin (N-acetyl-5-methoxytryptamine) has revealed itself as an ubiquitously distributed and functionally diverse molecule. The mechanisms that control its synthesis within the pineal gland have been well characterized and the retinal and biological clock processes that modulate the circadian production of melatonin in the pineal gland are rapidly being unravelled. A feature that characterizes melatonin is the variety of mechanisms it employs to modulate the physiology and molecular biology of cells. While many of these actions are mediated by well-characterized, G-protein coupled melatonin receptors in cellular membranes, other actions of the indole seem to involve its interaction with orphan nuclear receptors and with molecules, for example calmodulin, in the cytosol. Additionally, by virtue of its ability to detoxify free radicals and related oxygen derivatives, melatonin influences the molecular physiology of cells via receptor-independent means. These uncommonly complex processes often make it difficult to determine specifically how melatonin functions to exert its obvious actions. What is apparent, however, is that the actions of melatonin contribute to improved cellular and organismal physiology. In view of this and its virtual absence of toxicity, melatonin may well find applications in both human and veterinary medicine. Keywords: melatonin; melatonin receptors; seasonal reproduction; circadian rhythms; sleep; cancer; free radicals; antioxidant

aggregation in the melanocytes of amphibians and its derivation from serotonin (‘-tonin’). Soon after its discovery, its synthetic pathway in the pineal gland was identified (Axelrod and Weissbach, 1960). One of the earliest findings regarding the production of melatonin in the pineal is that it is primarily synthesized and secreted at night and that the circadian rhythm of melatonin production is determined by the prevailing light– dark cycle (Axelrod et al., 1965). As a consequence, nocturnal circulating melatonin levels are higher at night than during the day in all species examined.

Introduction Identification of the pineal gland as a genuine organ of internal secretion was established by the isolation and identification of the methoxy derivative of serotonin, that is N-acetyl-5methoxytryptamine, in bovine pineal tissue by Lerner et al., (1958, 1959) in the late 1950s. The newly discovered molecule was named melatonin because of its effects on melanin (‘mela-’)


Corresponding author. Tel.: þ210-567-3859; Fax: þ210-567-6948; E-mail: [email protected]

DOI: 10.1016/S0079-6123(08)81008-4

127

128

Besides the pineal gland, many organs may have the capability of producing melatonin given the high levels they contain (Hardeland, 2009); however, the diurnal rhythm of blood melatonin is exclusively a function of its secretion by the pineal gland. Of interest is that, in addition to the blood, melatonin is detectable in other fluids as well, for example fluid of the anterior chamber of the eye (Yu et al., 1990), cerebrospinal fluid (CSF) (Skinner and Malpaux, 1999), bile (Tan et al., 1999), ovarian follicular fluid (Nakamura et al., 2003), and so on. Moreover, these fluids often have concentrations much higher than in the serum suggesting that melatonin is either concentrated against a gradient or that melatonin in these fluids is not derived from the blood vascular system. The unexpectedly wide distribution of melatonin in the organs and fluids of mammals also portends equally diverse actions, some of which are summarized in this brief review.

Melatonin synthesis, release and catabolism The metabolic pathway of tryptophan that culminates in melatonin generation is well known (Fig. 1). The quantity of serotonin in the gland is determined by the activity of tryptophan hydroxylase (TPH). The expression of TPH mRNA as well as the activity of the enzyme vary over a light–dark cycle and are clock-driven rhythms; peak levels of both occur at night (Sugden, 2003) coincident with maximal melatonin generation. The amount of serotonin in the pineal gland is reportedly exceptionally high, so low levels of this precursor are never rate limiting in melatonin synthesis (Quay, 1963). Whereas the rate of melatonin synthesis from serotonin is generally considered to be coupled to the activity of arylalkylamine N-acetyltransferase (AANAT) (Iuvone et al., 2005), there are times when this may not be the case (Liu and Borjigin, 2005). It is usual that the rhythm of pineal AANAT runs in parallel with that of pineal and blood melatonin levels with all three parameters exhibiting peak levels at night (Reiter, 1991a, 1991b). Thus, it is generally accepted that the

concentrations of melatonin in the peripheral circulation can be used as an index of the metabolic activity of the pineal gland at virtually the same time. The 24-hour oscillations in AANAT activity and melatonin production are driven by the master circadian clock in the anterior hypothalamus, that is the suprachiasmatic nucleus (SCN) (Hastings et al., 2008). At night during darkness, the SCN sends an electrical signal to the pineal gland which causes the release of norepinephrine (NE) from post-ganglionic sympathetic nerve endings onto the pinealocytes which initiates and sustains, primarily via b1-adrenergic receptors, elevated melatonin production (Maronde and Stehle, 2007). The neural circuitry that connects the SCN to the mammalian pineal gland is remarkably complex and involves projections from the master clock to the paraventricular nucleus (PVN). Axons from neurons in the PVN descend through the brain stem to the intermediolateral cell column (ILCC) of the upper thoracic cord where they synapse on preganglionic sympathetic neural parikarya. Along with other axons that control sympathetic responses in the head, ILCC axons carrying information destined for the pineal gland exit the spinal cord and pass up the sympathetic trunk to the superior cervical ganglia where the final synapse in the pathway is made. Axons of post-ganglionic sympathetic neurons then follow blood vessels to the pineal gland where, in the tentorium cerebelli, they form discrete bundles of nerves, the nervi conarii. These fibres enter the pineal (Fig. 1) (Kappers, 1965; Moller, 1976; Moore, 1996) and terminate on the functional units of the pineal, the pinealocytes. A parasympathetic innervation of the pineal gland has also been described, but its importance in determining melatonin production is negligible (Kappers, 1969). Since NE released from sympathetic nerve endings in the pineal gland provides the primary stimulus for nocturnal melatonin synthesis, it might be expected that stressful stimuli, which discharge NE from other sites and elevate circulating levels of the catecholamine, would arrive at the

129 Paraventricular nucleus Biological clock (suprachiasmatic nucleus)

Pineal gland

Nervi cornarii (in tentorium cerebelli) Pinealocyte TRP TPH 5-TRP AADD

5-HT AA-NAT

NAS HIOMT

MEL

Retinohypothalamic tract (from melanopsin-containing retinal neurons)

Long days

Short days

d

d

Melatonin rhythm Upper thoracic cord

Intermediolateral cell column (sympathetic neurons)

Post-ganglionic sympathetic axon Superior cervical ganglion Preganglionic sympathetic axon

Fig. 1. Neural pathway connecting the eyes (the melanopsin-containing ganglion cells of the retina) with the pineal gland and the synthesis of melatonin (MEL) from tryptophan (TRP) in a pinealocyte. Just prior to their entrance into the pineal gland the postganglionic fibres form discrete nerve bundles in the tentorium cerebelli, the meninges that lie between the cerebrum and cerebellum. TPH, tryptophan hydroxylase; 5-TRP, 5-hydroxytryptophan; AADD, L-amino acid decarboxylase; 5-HT, serotonin, AANAT, arylalkylamine N-acetyltransferase; NAS, N-acetylserotonin; HlOMT, hydroxyindole-O-methyltransferase. The duration of the nocturnal elevation of melatonin varies with the season of the year.

pinealocytes and augment daytime melatonin synthesis. This does not occur, however, since the sympathetic nerve endings in the pineal gland function as an effective ‘sink’ and sequester excess circulating NE (Parfitt and Klein, 1976). This is important because it maintains the integrity of the melatonin cycle which provides key circadian information to other organs. Many other neurotransmitters are found in the pineal gland, but their functional significance remains unknown. The circadian production of melatonin with peak nocturnal levels is characteristic of all

vertebrates examined regardless of their specific activity pattern, that is diurnal or nocturnal. In humans, a melatonin rhythm is absent at birth but develops within six months after delivery. Thereafter, a robust melatonin cycle persists until about middle age after which the nighttime peak of melatonin dwindles such that in elderly humans (Sack et al., 1986), as in other aged mammals (Reiter et al., 1980, 1981), the cyclic production of melatonin in the pineal and its rhythm in the blood is markedly dampened. Considering its importance to the normal physiology of the organism, the age-associated drop in

130

melatonin may have a number of negative consequences (see below). Although the pineal is classified as an endocrine gland, it is atypical for several reasons: (1) its synthetic and secretory activities are regulated exclusively by the neural input; it is not routinely influenced by hormones from other endocrine organs; (2) the pineal does not store residual amounts of its secretory product, that is melatonin. It is a feature of many classic endocrine cells to hoard large amounts of the hormones they produce; (3) melatonin has both receptor-mediated and receptor-independent actions, which are uncommonly widespread. Hormones from the classic endocrine organs typically have rather specific actions on a determined group of cells. Circulating melatonin is taken up into hepatocytes where it is primarily 6-hydroxylated by hepatic P450 mono-oxygenases. The product is then conjugated to sulphate, released from liver cells and excreted in the urine as 6-sulfatoxymelatonin (Skene et al., 2001). CYP1A1, CYP1A2 and CYP1B1 are involved in the 6-hydroxylation of melatonin (Ma et al., 2005). Polycyclic hydrocarbons in cigarette smoke induce CYP1A2; hence, habitual smokers have lower circulating levels of melatonin relative to intervals when they refrain from smoking (Ursing et al., 2005). Besides its receptor-mediated actions, melatonin also has direct free radical scavenging, receptor-independent effects. During these processes other metabolites are produced including cyclic 3-hydroxymelatonin, N1-acetyl-N2-formyl5-methoxykynuramine (AFMK) and N1-acetyl5-methoxykynuramine (AMK) (Tan et al., 2002). These products are also excreted in the urine, albeit in low quantities under usual conditions (Ma et al., 2006).

Melatonin synthesis: light regulation Substantial progress has been made in the last decade in our understanding as to how environmental light relates to the biological clock, the SCN, and pineal melatonin synthesis. Of great interest is that the conventional retinal

photoreceptors, that is the rods and cones, have only a minor role in the light-mediated inhibition of pineal melatonin production. Rather, a highly specialized group of retinal ganglion cells which constitute 1–2% of the total number of neurons in the ganglion cell layer is responsible for detecting and transducing the critical wavelengths of light that result in pineal melatonin synthesis inhibition (Brainard et al., 2008; Jasser et al., 2006). This alternative set of photoreceptors is endowed with a unique photopigment, melanopsin, which only responds to a relatively narrow band of visible blue wavelengths (roughly 460–480 nm) that regulates the SCN and suppresses pineal melatonin synthesis (Kumbalasiri and Provencio, 2005). Thus, the mammalian retina has essentially two visual systems, one of which subserves vision (visual vision) and one of which influences the biological clock (circadian vision) (Foster and Hankins, 2007). As a result, individuals who are profoundly blind due to degeneration of the outer retinal layer (the rods and cones) still have circadian vision which is capable of regulating the SCN and the diurnal melatonin cycle. Conversely, surgical removal of the orbital globes eliminates both visual and circadian vision. Rather than sending an electrical message to the visual cortex, axons of the melanopsin-containing ganglion cells project prominently to the SCN (Hankins and Lucas, 2002). Via this route, light activation of melanopsin modulates clock gene expression in the SCN (Foster and Hankins, 2007) and likewise suppresses nocturnal melatonin synthesis (Fig. 2). One potential problem facing modern societies is the widespread use/misuse of artificial light at night. This light, if of the proper wavelength and sufficient irradiance, alters the function of the biological clock and restricts pineal melatonin production. Considering that artificial light is commonplace in modern societies and in view of the large number of individuals who work at night (an estimated one-fourth of the world’s workforce), humans are drastically reducing the amount of melatonin they are capable of producing and severely disturbing their biological clock which, during evolution, was exclusively synchronized by the rising and setting of the sun (Erren and

131

Capillary

Pinealocyte

Tryptophan

MT1

MT2

Tryptophan 5-hydroxilase

SCN

5-Hydroxitryptophan

Clock proteins

L-tryptophan descarboxylase

PVN

Serotonin P CREB

(Inhibitory)

Clock genes

P CREB

AANAT

GABA

N-acetylserotonin HIOMT HC

PKA

O

CH

H N

CaMK

PKA

O N H

cAMP

Ca2+

NO

cAMP

Melatonin

PAC2

β1

Capillary

NMDA

Gs

AC

NE

PACAP

L-glutamate 3rdV

SCN RHT Retinal ganglion cell

ILCC

Light melanopsin 460–480nm wavelengths

AP

PP

SCG

Fig. 2. Specific wavelengths of blue light (roughly 460–480 nm) are detected by the new-discovered photopigment, melanopsin, in a small percentage (1–2%) of the retinal ganglion cells. The axons of the melanopsin-containing ganglion cells project to the suprachiasmatic nuclei (SCN) via the retinohypothalamic tract (RHT) in the optic nerve. These neurons release glutamate and pituitary adenylate cyclase activating polypeptide (PCACP) which cause entrainment of clock gene expression in the SCN. The circadian activity of the cells within the SCN is a function of autoregulatory feedback loops. To modulate pineal melatonin production, axons from SCN neurons project to the PVN of the hypothalamus where they release g-aminobutyric acid (GABA). Nerve cell bodies in the PVN have axons that descend through the brain stem and eventually synapse of neurons in the intermediolateral cell column (ILCC), which are preganglionic sympathetic neurons. Axons of these neurons eventually terminate on pinealocytes after an additional synapse in the superior cervical ganglion (SCG). The release of norepinephrine, which occurs during the night, stimulates the synthesis and release of melatonin. Melatonin is discharged into the blood vascular system and possibly also into the cerebrospinal fluid (CSF) of the third ventricle (3rd V). Both blood and CSF melatonin have ready access to the SCN neurons where it acts on MT1 and MT2 receptors. This means, melatonin influences the firing rate of the SCN neurons thereby resetting the circadian pacemaker and regulating circadian processes such as sleep. AANAT = arylalkylamine N-acetyltransferace; AC = adenylate cyclase; AP = anterior pituitary gland; b1 = beta-adrenergic receptor; CaMK = calmodulin kinase; cAMP = cyclic adenosine monophosphate; CREB = cAMP response-element-binding protein; GS = G stimulatory protein; HIOMT = hydroxyindole-O-methyltransferase (also known as acetyl serotonin-O-methyltransferase); NO = nitric oxide; PKA = protein kinase A; PP = posterior pituitary.

Reiter, 2009a). Also, the excessive use of artificial light at night, sometimes referred to as light pollution, may be rendering humans relatively melatonin deficient. Currently, there is concern that

excessive light exposure after darkness onset, because it disturbs the function of the biological clock as well as truncates melatonin production, may result in a myriad of pathophysiologies

132

(Erren and Reiter, 2009b; Turek et al., 2005). As a result, suggestions have been made regarding proper light hygiene to ensure optimal circadian regulation and melatonin production (Erren and Reiter, 2009c).

Melatonin: receptors and signal transduction mechanisms Melatonin influences cellular physiology via membrane receptors, nuclear binding sites and after its interaction with cytosolic molecules. Additionally, melatonin has receptor-independent actions by virtue of its ability to directly scavenge menacing free radicals and related reactants. Much of what is known concerning the regulatory actions of melatonin stems from its binding to receptors in the cell membrane. Although originally thought to have a rather limited distribution, with more advanced methodologies, these receptors have now been uncovered in virtually every organ and on many cells in all organisms (Dubocovich and Markowska, 2005). The membrane receptors that were originally cloned from Xenopus melanocytes were subsequently found to have homologs in vertebrates (Reppert et al., 1994, 1995). The two major membrane receptors are currently identified as MT1 and MT2; they are members of the G proteincoupled receptor family with seven transmembrane domains. These receptors differ somewhat in terms of their affinities for the natural ligand, melatonin. In the case of the human MT1 and MT2 receptors, the respective Ki values are 80.7 and 383 pM (Kato et al., 2005). These values are roughly similar to those measured in other species. A third melatonin receptor, MT3, is seemingly identical to the cystosolic enzyme, quinone reductase 2 (QR2) (Boutin et al., 2008). Melatonin, depending on the specific cell and on the species, activates a variety of different second messenger cascades after its binding to membrane receptors. A major means by which the MT1 and MT2 receptors regulate intracellular processes is via an inhibition of adenylate cyclase, a reduction in cAMP and modulation of protein kinase A (PKA) activity; this action involves a

pertussis toxin sensitive Gi protein (Dubocovich et al., 2003). Activated MT1 receptors also inhibit cAMP response-element-binding protein (CREB) phosphorylation and the formation of immediate early gene products, c-fos and jun B (Ross et al., 1996; von Gall et al., 2002). Stimulation of both the MT1 and MT2 receptor activates phospholipase C-b with a commensurate elevation of inositol-(1,4,5)-triphosphate (IP3)/Ca2þ and 1,2-diacylglycerol (Alarma-Estrany and Pintor, 2007). Stimulation of MT1 is also accompanied by an elevation of the phosphorylation of mitogen-activated protein kinase MEK1/2 as well as extracellular signal-regulated kinase (Witt-Enderby et al., 2000). Activation of the MT1 receptor also increases potassium conductance by activating Kir3 (GIRK) inward rectifier potassium channels (Nelson et al., 1996) and potentiates prostaglandin F2a- and ATPmediated stimulation of the activity of protein kinase C (PKC) (Roka et al., 1999). In some cells, membrane melatonin receptors are linked with an inhibition of cGMP and DNA synthesis (Jockers et al., 1997). The signalling mechanisms of melatonin via its membrane receptors are obviously highly complex and vary with the cell type and possibly also with the species. The interested reader should consult the individual publications in this area as well as recent comprehensive reviews related to this subject (Hardeland, 2009; Witt-Enderby et al., 2006). Although nuclear binding sites for melatonin have frequently been disputed, currently they are accepted as being real by many, albeit perhaps not all, scientists working in the field. These binding sites are members of the retinoic acid receptor superfamily and include RORa1, RORa2, RZRa and RZRb (Carlberg, 2000). Among other possible functions, these binding sites are involved in melatonin’s inhibition of inflammation (Carrillo-Vico et al., 2005) and possibly also in the stimulation of antioxidative enzymes (TomasZapico and Coto-Montes, 2005). Evidence suggests there is cooperation between melatonin membrane receptors and the nuclear binding sites in terms of mediating some actions of the indoleamine.

133

Considering the numerous actions of calmodulin (CaM) in cell physiology, for example as a regulator of several protein kinases and other enzymes, the fact that melatonin reportedly binds to this cytosolic protein could be of considerable significance (Benitez-King and Anton-Tay, 1993; Pozo et al., 1997). Actions of melatonin attributed to its effects via CaM include its regulation of the cytoskeleton (Benitez-King and Anton-Tay, 1993) and neuronal nitric oxide synthase (Pozo et al., 1997). The modulation of the cytoskeleton by melatonin also involves membrane receptors as recently demonstrated by Benitez-King and colleagues (2009). Intracellular actions of melatonin, that is in the cytosol and nucleus, are supported by the fact that this indoleamine readily passes through cell membranes and seemingly has access to all subcellular organelles (Hevia et al., 2008). Notwithstanding this, there is still a great deal that is unknown about the specific intracellular signalling pathways of melatonin. For the time being, it seems safe to conclude that melatonin’s ability to alter molecular processes within cells is unimaginably complex and still requires substantial definition. The non-receptor-mediated actions of melatonin relate to its direct free radical scavenging activity. This only requires that melatonin be in the vicinity, that is within the reaction cage, of the radical when it is formed (Fig. 2). Given that melatonin protects lipids, proteins and nuclear DNA from oxidative damage suggests that its intracellular distribution is wide. The mechanism by which melatonin detoxifies radicals is via electron donation (Tan et al., 2002). Melatonin has been found to neutralize the most toxic oxidizing agents generated within cells, that is the hydroxyl radical (•OH) and the peroxynitrite anion (ONOO–). Additionally, however, melatonin reportedly scavenges singlet oxygen (1O2), superoxide anion radical (O•– 2 ), hydrogen peroxide (H2O2), nitric oxide (NO•) and hypochlorous acid (HClO). Besides melatonin, several metabolites that are formed when melatonin interacts with a radical are also highly effective scavengers. These products include cyclic 3-hydroxymelatonin, AFMK and AMK. For additional details on

the antioxidant activity of the melatonin, the reader is invited to consult several recent reviews (Allegra et al., 2003; Hardeland et al., 2009; Reiter et al., 2009a).

Melatonin and seasonal reproduction In animals living under natural conditions, conveying information about the changing ambient photoperiod duration is one of the essential functions of the melatonin rhythm. Thus, the cyclic production of melatonin not only functions as a ‘clock’but also as a ‘calendar’ (Reiter, 1993). This latter function is achieved due to the fact that as night length changes, the duration of the nocturnal elevation of melatonin likewise increases or decreases in length accordingly (Brainard et al., 1982). Thus, the changing melatonin cycle provides the internal organs time of year information. This allows the melatonin rhythm to regulate seasonal reproductive collapse and recrudescence as well as pelage changes (both colour and growth) and body weight (Reiter, 1980). Even before the discovery of melatonin there was a modicum of evidence that the pineal had some regulatory influence on reproductive physiology. This was not conclusively documented, however, until it was shown that the short photoperiod-induced involution of the gonads of both male and female Syrian hamsters was prevented either by surgical removal of the pineal gland or by the sympathetic denervation of the organ (Hoffman and Reiter, 1965, 1966; Reiter and Hester, 1966). On the basis of the outcomes of these studies, the authors speculated that the annual changes in reproductive capability in seasonally breeding animals were determined by altered pineal function induced by changes in day and night length. This was proven several years later when it was found that the winter collapse of the reproductive system typical of hamsters maintained under natural photoperiod and temperature conditions during the winter months maintained morphologically and physiologically active testes and accessory sex organs if they had been pinealectomized (Reiter, 1973). That melatonin was responsible for the seasonal involution

134

of the reproductive organs was shown shortly thereafter (Reiter et al., 1976; Tamarkin et al., 1976); additionally it was found that long daily duration of elevated melatonin (which occurs during the winter months) as opposed to the amplitude of the nocturnal melatonin peak was associated with reproductive involution in the Syrian hamster (Carter and Goldman, 1983). These studies serve as the basis for the now well-known association between the ambient photoperiod and reproductive fluctuations in both long-day and short-day seasonal breeders (Fig. 3). When prolonged daily melatonin elevations were shown to inhibit the neuroendocrinereproductive system, the indole was classified as an antigonadal or antigonadotropic molecule. This classification turned out to be erroneous when it was realized that many species are sexually active and breed during the winter when daily night-time elevated melatonin levels are prolonged. From this it was obvious that the seasonally changing melatonin message is used by both long-day (e.g. hamsters) and short-day (e.g. sheep) breeders to synchronize their reproductive cycle with the appropriate season.

What this means is that melatonin is neither antigonadotropic nor progonadotropic. Rather the melatonin message is a passive signal which provides all species with seasonal information; how this message is used, however, is species specific. The melatonin signal allows seasonal breeders to anticipate the changing seasons and make the necessary adjustments in advance of the actual sexually quiescent interval or breeding period. Within the past two decades the cellular and molecular mechanisms by which melatonin regulates the annual reproductive cycle of seasonal breeders has been at least partially elucidated. Melatonin receptors, particularly MT1, have been identified in various hypothalamic nuclei, in the pars tuberalis (PT) of the anterior pituitary gland, and in the gonads themselves (Dubocovich and Markowska, 2005; Morgan et al., 1994, 1999; Soares et al., 2003). Any or all of these may be operative in the general control of reproductive physiology by melatonin, but those in the neuroendocrine axis seem to relate specifically to the regulation of seasonal changes in reproductive collapse and recrudescence as well as behaviors such as

Recrudescent

Vernal equinox

Long-day breeders

D

Atrophic

Functional status of neuroendocrin–reproductive axis

Autumnal equinox

N

Short-day breeders

D

Duration of nocturnal melatonin is critical in determining reproductive status

J

J

A

D

N

Delivery of offspring

D

Changing duration of nocturnal melatonin levels provides important anticipatory information

S O N D J Month (Northern hemisphere)

F

M

A

M

Fig. 3. Seasonal changes in reproductive competence in photoperiod-dependent long-day and short-day breeding mammals. Longday breeders mate during the summer when nocturnally elevated melatonin is of short duration; conversely, short-day breeders mate during the winter months when elevated night melatonin levels are prolonged. Regardless of when animals mate, delivery of the young occurs in the late spring and early summer, a time that maximizes survival of the offspring. The changing photoperiod (and melatonin rhythm) allows animals to anticipate upcoming seasons and adjust their reproductive status accordingly.

135

hibernation, migration and pelage growth and colour changes (Lincoln et al., 2003, 2006). The majority of the studies designed to uncover the processes by which the circannual clock and the melatonin rhythm modulate the annual cycle of reproduction have employed a limited number of species. The short-day breeders that have been most frequently used are various breeds of sheep (Hazlerigg et al., 1996), while long-day breeding hamsters often represent this group of mammals (Stirland et al., 2001). Both have contributed significantly to an understanding of the molecular mechanisms by which the photoperiod, via the melatonin signal, regulates seasonal hormonal changes, especially in prolactin. The scientists working in this difficult area have shown extreme diligence considering the very small size of the pituitary gland region (PT) that is intimately involved with some of these processes. An early study revealed that the regulation of gonadotropins, that is luteinizing hormone (LH) and follicle-stimulating hormone (FSH), by melatonin involved neurons in the hypothalamus while prolactin regulation by melatonin was in the pituitary gland itself (Lincoln and Clarke, 1994). This conclusion was drawn after the hypothalamus was surgically disconnected from the pituitary; this procedure eliminated melatonin regulation of gonadotropin secretion, but spared the ability of the photoperiod and melatonin to modulate the circannual rhythm of prolactin secretion (Fig. 4). The pituitary region in which melatonin acts is a PT. This small group of cells is associated with the pars distalis (PD) but it is adherent to the ventral surface of the median eminence and in close proximity to the primary portal plexus, the blood of which drains, via long portal veins, into the secondary portal plexus in the PD. According to currently available information, the circannual regulation of prolactin secretion is influenced by the changing duration of the nocturnal melatonin message. The PT contains, among other cells, a PT-specific cell type which is morphological distinct from those present in the PD (Wittkowski et al., 1999) and whose physiology is altered in response to melatonin application (Bockers et al., 1995). These specialized lactotrophs which

contain membrane melatonin receptors produce a PT-derived prolactin peptidic secretagogue, termed tuberalin (Hazlerigg et al., 1996; Morgan et al., 1996). After its release from PT cells, tuberalin possibly diffuses into the primary portal plexus of the median eminence, thereby gaining access to prolactin-containing cells of the PD. Tuberalin is classified as a prolactin releasing factor because it stimulates the secretion of prolactin from primary cultures of PD cells. These proposed relations are depicted in Fig. 3. Additionally, thyroid-stimulating hormone (TSH) is also involved in melatonin-mediated waxing and waning of the reproductive system of mammals. TSH, released from PT cells, regulates the conversion of inactive thyroxine (T4) to active tri-iodothyronine (T3) in nearby tanycytes which line the infundibular recess of the third ventricle (Hanon et al., 2010). Specifically, how T3 then influences the key genes involved in driving the physiology of the neuroendocrine-reproductive axis has yet to be definitively determined. One likely possibility, however, is that T3, generated in the tanycytes, is released and acts on kisspeptin neurons in the adjacent arcuate nuclei (ARC) of the hypothalamus. Kisspeptin induces GnRH expression and release from nerve terminals ending near capillaries of the primary portal plexus (Colledge, 2009; Revel et al., 2006). After its discharge, GnRH flows into the capillaries of the PD to mediate the release of gonadotropins. As a consequence, T3, which is profoundly regulated by the photoperiod and by melatonin, as well as the tanycytes are essential components of the mechanisms gating seasonal reproduction (Barrett et al., 2007). Melatonin receptors have also been identified in the gonads of both genders as well as at some accessory organ sites; moreover, melatonin may be synthesized in peripheral reproductive tissues (Tamura et al., 2009). Additionally, melatonin may influence mate selection in some species and protect the reproductive system from oxidative stress due to its ability to annihilate free radicals. Three recent review articles should be consulted for information on the peripheral reproductive actions of melatonin (Reiter et al., 2009b; Tamura et al., 2008, 2009).

136

Photoperiod-driven melatonin rhythm day

night

day

Hypothalamic neuroendocrine cells Median eminence

Hypothalamus

Hypothalamo-pituitary disconnection

Melatonin

Primary portal plexus Pars distalis

Pars tuberalis

Long portal vessels

MT1

Thyrotroph

Tuberalin

Secondary portal plexus

Lactotroph

Prolactin Fig. 4. The presumptive mechanisms whereby the seasonally changing melatonin message influences the secretion of prolactin. In the pars tuberalis (PT) of the adenohypophysis, melatonin interacts with MT1 receptors on PT-specific cells where it influences clock gene expression (Morgan et al., 1999) and decodes the melatonin signal. This culminates in the regulation of a peptide called tuberalin (a prolactin stimulating factor), which is released from cells of the PT and presumably gains access to the primary portal plexus (PPP) in the median eminence. Tuberalin then passes to the pars distalis (PD) via the long portal veins (LPV) into the secondary portal plexus (SPP). From here, it diffuses into the PD and acts as a subpopulation of lactotrophs to promote prolactin secretion. The melatonin message in seasonally breeding species also influences gonadotrophin (LH and FSH) release from the PD after it interacts with cells in the mediobasal hypothalamus. This is surmised from a study in which the hypothalamus and pituitary gland were surgically disconnected (HPD); in this case, the ability of melatonin to influence gonadotrophin secretion from the PD was lost.

Melatonin, circadian rhythms and sleep The SCN was one of the earliest sites in which melatonin receptors were identified (Dubocovich and Markowska, 2005). The mammalian SCN as well as immortalized SCN2.2 neurons express mRNA and protein for both the MT1 and MT2 membrane receptors (Rivera-Bermudez et al., 2004). As in other tissues, the actions of melatonin on its receptors in the SCN involve multiple signalling pathways (Liu et al., 1997).

In SCN2.2 cells, melatonin inhibits forskolinstimulated cAMP accumulation with this response being blocked by the membrane receptor blocker, luzindole. In slices collected from the mouse brain, melatonin interferes with pituitary adenylate cyclase-activating polypeptide (PACAP) stimulation of CREB phosphorylation by acting specific on the MT1 receptor (von Gall et al., 2000). In contrast, in SCN2.2 cells and in the rat SCN activation of the MT2 membrane receptor mediates a rise in neuronal PKC activity (Hunt et al., 2001).

137

Whether nuclear receptors for melatonin have any functional importance for neurons in the SCN remains undetermined. One of the initial uses of melatonin in the human was to minimize the circadian disruption that occurred during jet lag (Arendt et al., 1988). Because of results such as these, melatonin has been classified as a chronobiotic, a moniker that is certainly deserved considering its actions on circannual and circadian rhythmicity (Arendt, 2006). The endogenous nocturnal melatonin peak in humans is accompanied by a drop in core body temperature (Cagnacci et al., 1992) and a reduction in both systolic and diastolic blood pressure (Reiter et al., 2009c; Simko and Pechanova, 2009). The night-time melatonin rise is likewise related to maximal tiredness/fatigue, as well as minimal degree of alertness and physical and mental performance. Bright-light exposure at night, which inhibits the nocturnal rise in melatonin also leads to an elevation in body temperature, a higher level of performance, reduced sleepiness and an absence of reduction in blood pressure. Exogenous melatonin administration during the day reportedly promotes sleepiness and in some cases reduces core body temperature (Kräuchi et al., 2006). Similarly, giving melatonin to humans at night lowers blood pressure; this action is particularly important given that individuals who drop their blood pressure at night reportedly survive longer (Ohkubo et al., 1997). The action of melatonin on core body temperature is presumably mediated via central temperature regulating processes in the hypothalamus, although this has never been proven. The depressive effect of melatonin on blood pressure may also involve central blood pressure regulating mechanisms at the level of the hypothalamus or in the area postrema. Moreover, melatonin has endothelium-relaxing actions and it scavenges free radicals which would reduce blood pressure; any or all of these actions may contribute to the ability of melatonin to modulate nocturnal blood pressure in humans (Reiter et al., 2009c). The ability of melatonin to modulate sleep has been a field of active investigation for several

decades. Under well-controlled conditions, the evening rise in melatonin in humans coincides closely with the opening of the ‘sleep gate’ (Lavie, 1997) and it follows a period of wakefulness referred to as the ‘forbidden zone for sleep’ (Shochat et al., 1997). Despite the apparent association of elevated levels of melatonin with sleep, an association of the indole with any specific sleep stage has not been forthcoming. Some of the most compelling evidence supporting a relationship between high endogenous melatonin levels and sleep is provided by free-running, profoundly blind subjects where both sleepiness and reduced body temperature accompany maximal melatonin levels even when they occur during the day (Dijk et al., 1997). A large number of scientific publications have shown that oral doses of melatonin, usually ranging from 0.25 to 10 mg (and possibly higher), promote sleepiness and/or advance sleep onset when administered in the evening (Zawilska et al., 2009). Likewise, melatonin consumed during the day by humans reportedly causes an elevated sleep propensity and alters the waking EEG two to four hours after the indole is taken. The phase-shifting effects of melatonin on the timing of sleep are supported by a large body of evidence, although total sleep time usually is not increased when melatonin is taken (Rajaratnam et al., 2004). Actually, melatonin taken in the evening typically phase advances sleep onset while when injected in the morning it phase delays sleep the subsequent night; these differences are explainable in terms of the phase–response curve (PRC) for melatonin (Lewy et al., 1997). A conspicuous sign of circadian rhythm disruption is poor quality sleep. These disruptions are obvious in jet lagged and night-shift workers and in individuals with delayed sleep phase syndrome (DSPS) or advanced sleep phase syndrome (ASPS). Likewise, circadian rhythm disorders and reduced melatonin levels may account for poor sleep quantity in elderly humans. While there have been numerous studies in terms of melatonin’s ability to correct circadian disorders in individuals during jet lag or in night-shift

138

workers, the findings are inconsistent with both positive effects being reported and publications claiming that melatonin is ineffectual in promoting improved circadian adaption (Zawilska et al., 2009). The variability of the outcomes of these studies may relate to the timing of the administration of melatonin (based on its PRC) and the confounding factor of light exposure after darkness onset. Despite the inconsistency of some findings, the American Academy of Sleep Medicine supports the use of melatonin in jet lag, non-24-hour sleep/wake disorders, and some other situations where sleep is compromised due to circadian disturbances (Morgenthaler et al., 2007). Melatonin has also been successfully used to treat sleep disorders in children with neurodevelopment disabilities (Wasdell et al., 2008). It has generally been tacitly assumed that the efficacy of melatonin in modulating circadian rhythms and sleep propensity are mediated by receptors on neurons in the SCN. While this assumption seems intuitively accurate, few details of specific mechanisms involved have revealed themselves. The pharmaceutical industry has embarked on an investigative path to identify melatonin analogues which are effective in adjusting circadian rhythms, promoting sleep and possibly for eventual use in other conditions.

Melatonin and cancer inhibition While the majority of published reports have been concerned with the ability of melatonin to inhibit the progression of already established tumours, there is also evidence that melatonin may reduce events that lead to the initiation of cancer. Thus, many tumours develop following damage to nuclear DNA when it goes unrepaired. Injury to DNA is frequently a result of free radicals (Cerutti et al., 1994). Since melatonin readily neutralizes free radicals, it protects DNA from the oxidative damage they inflict (Assayed and Abd El-Aty, 2009, Karbownik and Reiter, 2000). For any antioxidant to protect the genome from destruction by the highly toxic •OH, the protective agent must essentially

straddle the DNA since the most toxic and reactive radicals are estimated to travel only a few angstroms and have a half-life of only a few nanoseconds before interacting with a bystander molecule such as DNA. The high efficiency of melatonin in reducing free radical-mediated mangling of DNA indicates that the indoleamine must be in the nucleus in sufficient concentrations to counteract the damaging effects of any radicals produced in the vicinity (MenendezPelaez and Reiter, 1993). Besides preventing destruction of the genome, melatonin may also aid in its repair once it has been mutilated (Sliwinski et al., 2007). Due to these two actions, melatonin may limit tumour cell initiation and thereby cancer frequency. As mentioned above, the vast majority of studies that have tested melatonin as an oncostatic agent have been concerned with its ability to inhibit tumour progression (Blask et al., 2005a; Korkmaz et al., 2009a; Vijayalaxmi et al., 2002). In addition to suppressing the proliferative activity of tumour cells (Cos and Sanchez-Barcelo, 2000; Panzer and Viljoen, 1997; Shiu, 2007), melatonin often enhances apoptosis in many cancer cells (Sainz et al., 2003). Moreover, the indole possess anti-metastatic actions, likely due to its ability to enhance the production of cell surface adhesion molecules (Cos and Sanchez-Barcelo, 2000) and by preventing the breakdown of the intracellular matrix (Ortiz-Lopez et al., 2009). In addition to these actions, melatonin causes the transformation of cell division-prone cancer cells into more differentiated mitotically resistant cells (Sainz et al., 2005). Thus, melatonin has at least four means of limiting tumour progression, that is anti-proliferation, anti-metatasis, proapoptotic and prodifferentiation. Given that melatonin employs each of these actions to restrict cancer, it is an attractive candidate for supplemental use in individuals at risk for tumour development and as a treatment agent in combination with conventional therapies. What is somewhat perplexing, however, are the multiple mechanisms that have been described to explain melatonin’s activity as an anti-tumour agent (Jung and Ahmad, 2006; Reiter, 2004). One proposed mechanism to explain the inhibition

139

of cancer cell proliferation by melatonin involves its action in reducing the uptake of linoleic acid (LA) by breast cancer cells (Blask et al., 2005b). LA is an essential and major fatty acid that is taken into cells through a specific fatty acid transporter. Melatonin restricts LA transport into MCF-7 breast cancer cells after it interacts with cell membrane receptors (MT1 and MT2); this results in an inhibition of adenylate cyclase and cAMP and the closure of the fatty acid transporter. When circulating melatonin levels are low,that is day-time values, LA readily enters breast cancer cells where it is oxidized to 13-hydroxyoctadecadienoic acid (13-HODE), a mitogenic signalling molecule, by 15-lipoxygenase (15-LOX-1). This promotes the activation of the MEK–ERK1/2 pathway that serves to promote cell proliferation and tumour growth (Blask et al., 2005b). When blood melatonin levels are high, that is at night-time physiological concentrations (low nM range), the indole acts via the membrane receptors to close the fatty acid transporter, limit LA uptake and shut down cell proliferation (Blask et al., 2005b). The implication of these findings are severalfold: (1) breast cancer cell proliferation may be greater during the day (low melatonin) than at night (high melatonin); (2) any factor that reduces night-time melatonin concentrations, for example light exposure during darkness, drugs, increased age, and so on, may promote breast cancer cell growth and (3) supplemental melatonin may inhibit cancer cell proliferation. While many of these studies have used MCF-7 human breast cancer cells, similar results are obtained with human leiomyosarcoma (Dauchy et al., 2009) and rat hepatoma cells (Blask et al., 2005b). An alternative mechanism that may be involved in cancer inhibition by melatonin is the regulation of telomerase. Telomerase is a specialized ribonucleoprotein DNA polymerase that synthesizes the telomeric extensions of linear eukaryotic chromosomes. These extensions on the end of each chromosome are essential for maintaining a sturdy chromosomal structure. In most normal cells telomerase activity is negligible, so telomeres become shorter with each cell division, making the chromosomes unstable and susceptible to damage and the cell more likely to undergo apoptosis.

In many cancer cells, telomerase activity is upregulated. Thus, even though cancer cells divide frequently, the highly active telomerase generates new telomeric extensions which enhance the stability of their chromosomes. Because of this, inhibiting telomerase activity in tumour cells is a viable target of pharmaceutical anti-cancer drugs. When immune-compromised mice harbouring growing xenografts of MCF-7 human breast cancer cells are given supplemental melatonin for five weeks in their drinking water, tumour size and number of metastases were highly significantly reduced compared to these parameters in nonmelatonin treated animals (Leon-Blanco et al., 2003). Telomerase activity, estimated using the TRAP assay, revealed that it was likewise significantly reduced in the breast tumours of the mice that received melatonin. In a series of accompanying in vitro experiments, melatonin was tested for its ability to alter different catalytic subunits of telomerase including TERT and TR using RT-PCR and Southern blot. At both physiological (1 nM) and pharmacological (10 mm) concentrations, melatonin reduced the mRNA for the catalytic subunit of telomerase, that is TERT. Similarly, the TR subunit mRNA of telomerase was likewise inhibited. Since these two subunits constitute the core of the enzyme and TERT is necessary for the activity of telomerase, these findings indicate a viable epigenetic mechanism by which melatonin may inhibit human breast cancer growth. As noted above, telomerase activity is an important therapeutic target of the pharmaceutical industry for cancer inhibition and the use of melatonin may well aid in curtailing breast tumour growth (Leon-Blanco et al., 2003). Given that telomerase activity is upregulated in many cancer cells, melatonin may be a useful agent to inhibit the activity of this enzyme and to limit the growth of several tumour types. Other major mechanisms whereby melatonin retards the growth of hormone-dependent cancers include its ability to inhibit aromatase, an enzyme which metabolizes androgen precursors to estrogens, as well as its action at the level of the estrogen receptor (ER), specifically ERa; this latter action reduces the stimulatory effects of

140

proliferation of hormone-dependent mammary cancer (Kiefer et al., 2005; Sanchez-Barcelo et al., 2005). The results of these and related studies are summarized in Fig. 5. Most recently, another perspective has been presented to explain the oncostatic activity of melatonin. The sirtuin family of class III histone deacetylases (HDACs) is related to longevity of normal cells and they are often upregulated in

endogenous estrogens on mammary cancer cell proliferation. Agents which inhibit aromatase are referred to as selective estrogen enzyme modulators (SEEM) while the latter are identified as selective estrogen receptor modulators (SERM). These anti-estrogen effects of melatonin have been thoroughly exploited by two research groups who have presented compelling data which define a mechanism by which melatonin likely limits cell

Melatonin

Down regulates E2 synthesis

SEEM actions

E2

Ovaries

Adernal cortex (z. reticularis)

aromatase

Androgens

NH3 MT1 receptor

Cell membrane COOH

E2

aromatase

Androgens

(epithelial cells) SEEM actions

SERM actions

E2

Melatonin

ERα

ERE DNA Nucleus Transcription

Fig. 5. Some of the proposed mechanisms by which melatonin interferes with the growth of estrogen-mediated mammary cancer cell growth. In this scheme, melatonin exhibits both SEEM (selective estrogen enzyme modulation) and SERM (selective estrogen receptor modulation) actions. Melatonin inhibits the activity of the estrogen (E2)-synthesizing enzyme, aromatase. Melatonin also may curtail E2 produced by the ovary. Via these two means, melatonin reduces the amount of E2 available to stimulate cancer cell proliferation. The SERM actions involve melatonin’s ability to limit the expression of the estrogen receptor (ERa) and inhibit the binding of the E2–ER complex to the estrogen response element (ERE) in the DNA. These latter actions occur after melatonin interacts with a receptor (MT1) in the cell membrane of a mammary epithelial cell. This figure is primarily based on the findings of Sanchez-Barcelo et al. (2005) and Kiefer and colleagues (2005).

141

cancer cells. Especially, SIRT1, a nicotinamide adenine dinucleotide (NADþ)-dependent sirtuin, promotes cell survival by limiting apoptosis. It is also now realized that the metabolic function of SIRT1 is influenced by circadian processes which, when they become deregulated, may contribute to an increased cancer risk. In a provocative and imaginative review, Jung-Hynes and Ahmad (2009) present a strong argument for the involvement of melatonin in the regulation of clock genes, SIRT1 expression and cancer. Especially in the elderly where melatonin levels wane, circadian rhythms deteriorate and cancer risk increases, supplemental melatonin may be of benefit because it inhibits SIRT1 activity in tumour cells which would increase the likelihood of them undergoing apoptosis (JungHynes et al., 2010). The ability of melatonin to promote apoptosis of cancer cells has been reported by a number of investigators (Sainz et al., 2005), but this is the first time it has been linked to the inhibition of SIRT1. Both the peroxisome proliferator-activated receptors (PPARs) as well as the nuclear receptors (RXRs), for which melatonin is an endogenous ligand, play crucial roles in inducing apoptosis in a number of cancer cell types (James et al., 2003). When cultured MDA-MB-231 human breast cancer cells were treated with the PPAR agonist, troglitazone, and melatonin, 84% of the cells underwent apoptosis within 72 hours; this was much greater than the apoptotic response induced by either troglitazone or melatonin alone (Korkmaz et al., 2009b). These findings are consistent with the idea that melatonin’s oncostatic activity may work via epigenetic mechanisms to limit tumour growth (Korkmaz et al., 2009a). Currently, light at night is being examined as a causative factor for the elevated breast cancer risk in women and prostate cancer in men. As noted above, light exposure after darkness onset alters biological rhythmicity and suppresses melatonin; both these changes have been speculated to contribute to the increased incidence of breast and prostate cancer in the human population (Reiter et al., 2007; Stevens, 2009). Moreover, these alterations may contribute to an elevated cancer risk of all types (Erren and Reiter, 2009b).

Another issue that should be considered in reference to melatonin and cancer is the fact that, in most cases, cancer is an age-related disease. As individuals age, the integrity of the circadian system deteriorates and endogenous melatonin levels fall (Jung-Hynes and Ahmad, 2009; Jung-Hynes et al., 2010). Based on currently available evidence, it is possible that these two events may conspire to elevate cancer risk in the elderly.

Melatonin as a free radical scavenger and antioxidant The ability of melatonin to quench the devastatingly reactive and toxic •OH was initially uncovered in 1993 (Tan et al., 1993). Since then, this action of melatonin has been repeatedly confirmed (Tan et al., 2002). Moreover, in addition to neutralizing the •OH, melatonin has been found to detoxify other damaging oxidizing 1 agents including the ONOO–, O•– 2 , H 2O 2, O 2, NO•, LOO• and HClO (Reiter et al., 2009d). Of significant interest and importance are the observations that not only is melatonin a highly effective scavenger of a variety of toxic radicals, but the metabolites that result from the interaction of melatonin with free radicals are equally proficient at neutralizing damaging reactants. The first of these to be discovered was cyclic 3-hydroxymelatonin (Tan et al., 1998). Like its parent molecule, melatonin, cyclic 3-hyroxymelatonin functions as a radical scavenger and in the process is converted to AFMK (Hardeland et al., 2009). AFMK is also formed directly when melatonin interacts with H2O2 (Tan et al., 2000). AFMK is also highly effective in scavenging toxic radicals resulting in the generation of AMK. From this description, it is apparent that melatonin and several of its metabolites are able to protect cells from oxidative damage due to their combined actions in detoxifying radicals. These sequential reactions make melatonin highly efficient in reducing oxidative damage to subcellular organelles and cells with this series of reactions being referred to as melatonin’s antioxidative cascade (Fig. 6).

Fig. 6. Melatonin and its metabolites are radical scavengers. This sequence of reactions is referred to as the antioxidant cascade of melatonin. In this sequence melatonin detoxifies the •OH to generate cyclic 3-hydroxymelatonin, which also functions as a scavenger with the resulting product being AFMK. Melatonin may also directly neutralize H2O2 to produce AFMK. AFMK, like its precursors, is also a scavenger and in doing so it generates AMK. AMK may also detoxify radicals with several metabolites, some of which have not been structurally identified, being produced.

143

Oxygen (O2) is the obligatory precursor of a variety of free radicals. Its chemical reduction gives rise to the majority of damaging radicals and related reactants. While radicals are generated throughout all cells, mitochondria are important contributors to free radical production. This results from the fact that as electrons are shunted between the complexes of the electron transport chain (ETC), some escape and reduce nearby O2 molecules causing the formation of the O•– 2 which, as described in Fig. 7, goes on to form more toxic and damaging agents. Given that mitochondria are a major site of free radical generation, for melatonin to be successful in reducing cellular damage and death, it

would be essential that it gains access to the mitochondria where it would have the opportunity to interrupt O•– 2 formation and, therefore, the generation of all subsequent radical products as well. Using a combination of time-lapse conventional, confocal and multiphoton fluorescent imaging microscopy coupled with non-invasive mitochondria-targeted probes, Jou et al (2004, 2007) examined melatonin’s ability to quench radicals at the mitochondrial level in cultured rat brain astrocytes. When astrocytes were challenged with an oxidizing agent, H2O2, mitochondrial reactive oxygen species generation increased significantly as visualized using dichlorofluorescein and dihydrorhodamine-123; these changes

Citrulline 1O 2

NO

Arginine NOS (

ONOO–

)

Photo excitation

Highly destructive to lipids, DNA and proteins

•OH

& ONOO– cannot be enzymatically removed

e–

O2

O2• –

O2• –

formed at mt Complexes I & III and to lesser extent through out cell

OH

e– CuZnSOD( ) MnSOD(

Transition metals

e–

)

H2O + O2 MPO(

)

GPx( )

H2O + HOCl

GSH H2O + O2 Glutamate Gysteine

CAT

GCL(

GSSG

H2O2

)

Glycine Glutamylcysteine

GRd( )

Fig. 7. The chemical reduction of molecular oxygen (O2) gives rise to a number of damaging radicals and related products. The most damaging of these is the hydroxyl radical (•OH) and the peroxynitrite anion (ONOO–), both of which are scavenged by melatonin and/or its metabolites. Besides functioning as an effective radical scavenger, melatonin stimulates (as indicated by ") a number of antioxidative enzymes while inhibiting (as indicated by #) a pro-oxidative enzyme, nitric oxide synthase (NOS). CAT, catalase; CuZnSOD, copper/zinc superoxide dismutase; MnSOD, magnesium superoxide dismutase; GCL, glutamylcysteine ligase; GPx, glutathione peroxidase; GRd, glutathione reductase; MPO, myeloperoxidase; 1O2, singlet oxygen; O•– 2 , superoxide anion radical; H2O2, hydrogen peroxide, HOCl, hypochlorous acid; GSH, reduced glutathione; GSSG, oxidized glutathione; e–, electron.

144

were virtually totally prevented when H2O2-treated cells were incubated with melatonin. Additionally, melatonin reduced the typical morphological changes (cell membrane blebbing, condensation of the nucleus, rounding up the cell and mitochondrial swelling) associated with elevated oxidative stress. Each of the detrimental morphological changes observed in H2O2-challenged cells is a prelude to apoptosis, which was also abundantly observed in the astrocytes. Since melatonin clearly prevented mitochondrial free radical generation and the downstream events that are consequential in cell death, the indole also reduced apoptosis. Jou and colleagues (2004) confirmed this by showing that melatonin also limited opening of mitochondrial transition pore, cytochrome c release, caspase 3 activation and DNA laddering. In their second well-illustrated study, Jou and co-workers (2007) used two special cells that have a defective mitochondrial respiratory chain due to a large-scale deletion (4977 base pairs) of the mitochondrial DNA (referred to as the common deletion, or CD). In addition to causing a severe ATP deficiency, these cells generate free radicals at an inordinately high rate. In these cells, the addition of melatonin dramatically reduced free radical generation where the mitochondria were suffering with the CD. Moreover, when these cells are challenged with secondary oxidative stress, in this case the addition H2O2, melatonin again was highly effective in limiting free radical generation and cellular changes typical of cells undergoing apoptosis. Of special interest is that respiratory chain defects, as exhibited in the CD cells, are centrally involved in a variety of pathological conditions and diseases suggesting that melatonin may have significant utility in treating the socalled mitochondrial diseases (Acuna-Castroviejo et al., 2010). The findings related to the anti-apoptotic activity of melatonin has special importance in the central nervous system given that in all the major neurodegenerative diseases neuronal death via apoptosis is a major feature of the disease. The high level of vulnerability of the brain to oxidative stress stems from reasons unique to the central nervous system. This includes the fact

that, (1), the brain, although on average only 2% of the body weight uses at rest 20% of the inhaled O2; this percentage rises when the brain is mentally active. Obviously, since O2 is the precursor of many toxic reactants, the high utilization of O2 by the CNS makes it highly susceptible to free radical abuse. (2), The brain is rich in easily oxidized fatty acids. In addition to lipid-rich cell membranes which are very extensive in neurons due to their long processes, the brain contains large quantities of fats in the form of myelin. (3), Some regions of the brain contain high concentrations of the antioxidant, ascorbic acid (AA); however, in the presence of free iron, which occurs as a result of haemorrhage within the CNS, AA generates large quantities of the most damaging radical, the •OH. Finally, (4), the brain is unexpectedly poorly equipped with antioxidative enzymes which play a major role in metabolizing highly toxic radicals to innocuous products. Apoptosis, so-called programmed cell death, is a feature characteristic of acute and chronic, frequently age-related, neurodegenerative diseases. Acutely, melatonin has been highly successfully used to reduce infarct volume, oxidative damage and cell death in the brain and spinal cord following induced ischemia/reperfusion (Cervantes et al., 2008; Reiter et al., 2005) or trauma (Esposito et al., 2009; Samantaray et al., 2009). The chronic application of melatonin has been found to be beneficial in more slowly developing neurodegenerative conditions as well (Pappolla et al., 2000; Reiter et al., 2004; Wang, 2009) and in brain ageing (Carretero et al., 2009; Reiter et al., 1999). The initiation and continuation of neuronal loss as a consequence of normal ageing and during the development of various forms of dementia is often a result of cell death via apoptosis. Apoptosis involves the activation of caspases, which induce cellular implosion and loss. There are two major apoptotic signalling pathways referred to the extrinsic and intrinsic paths. The extrinsic apoptotic pathway, also called the death receptor pathway, is initiated by death receptors, for example CD95/APO-1/Fas, on the cell membrane and involves caspase-8/Bid and caspase 10 activation

145

(Ashkenazi and Dixit, 1998). The intrinsic apoptotic pathway (the mitochondrial pathway) involves the release of proapoptotic molecules from the mitochondria (Friedlander, 2003). Obviously, as documented in the publications of Jou et al (2004, 2007), melatonin has ready access to the mitochondria where it scavenges free radicals. This action likely contributes to the ability of melatonin to inhibit the intrinsic pathway of apoptosis and reduce neuronal loss in experimental models of Alzheimer’s disease (AD) (Olcese et al., 2009; Pappolla et al., 1997; Zhou et al., 2008) and Parkinson’s disease (PD) (Chetsawang et al., 2009; Lin et al., 2008; Saravanan et al., 2008). The vast majority of published reports confirm that melatonin suppresses the intrinsic apoptotic pathway. Wang and co-workers (2008) screened a library of more than 1000 FDA-approved drugs as part of the Neurodegeneration Drug Screening Consortium of the NINDS for their ability to inhibit the release of cytochrome c from Ca2þ-stimulated mitochondria. In this test, melatonin was one of the best in reducing cytochrome c discharge from mitochondria and was listed at position 14 out of the total number of drugs tested. This, along with its ability to easily cross the blood–brain barrier and its virtual absence of toxicity, makes it a high-priority molecule to examine in reference to possibly forestalling neurodegenerative diseases of the aged. The findings briefly summarized here also provoke the question as to whether the drop in endogenous melatonin levels during ageing contributes to free radical-mediated brain ageing (Reiter et al., 2000).

Concluding remarks Melatonin is a remarkably functionally pleiotropic neuroendocrine molecule with actions mediated by membrane receptors, cytosolic and nuclear binding sites and via its receptor-independent functions. In some cases it is difficult to determine by which of these processes melatonin mediates its effects. The fact that melatonin is produced in all species of the animal kingdom from unicells to

humans suggests its early evolution and functional importance. While in humans it is widely known that melatonin is synthesized in the pineal gland, this may be only one site of melatonin production. The essential nature of melatonin synthesis in the pineal gland is that its rhythm provides all species with a circadian melatonin signal which is essential for a number of the physiological effects of this functionally diverse molecule. This brief survey clearly did not summarize all of the proposed functions of melatonin. For example, its actions on immunoregulation, skin physiology, retinal function, bone physiology, ageing, oral health,and so on can be found in other comprehensive reviews. Obviously, while tremendous advances have been made in establishing melatonin as a beneficial component of optimal health, a great deal still remains to be learned about the mechanisms involved. References Acuna-Castroviejo, D., Escames, G., Lopez, L. C., & Reiter, R. J. (2010). Melatonin, mitochondrial homeostasis and mitochondrial-related diseases: An update. Current Topics in Medicinal Chemistry. Alarma-Estrany, P. & Pintor, J. (2007). Melatonin receptors in the eye: Location, second messengers and role in ocular physiology. Pharmacology Therapeutics, 113(3), 507–522. Allegra, M., Reiter, R. J., Tan, D. X., Gentile, C., Tesoriere, L., & Livrea, M. A. (2003). The chemistry of melatonin’s interaction with reactive species.. Journal of Pineal Research, 34(1), 1–10. Arendt, J. (2006). Melatonin and human rhythms. Chronobiology International, 23(1–2), 21–37. Arendt, J., Aldhous, M., & Wright, J. (1988). Alleviation of jetlag by melatonin: Preliminary results of controlled double blind trial. British Medical Journal (Clinical Research Edition), 292(7), 1170. Ashkenazi, A. & Dixit, V. M. (1998). Death receptors: Signaling and modulation. Science, 281(5381), 1305–1308. Assayed, M. E. & Abd El-Aty, A. M. (2009). Protection of rat chromosomes by melatonin against gamma irradiationinduced damage. Mutation Research, 677(1–2), 14–20. Axelrod, J. & Weissbach, H. (1960). Enzymatic O-methylation of N-acetylserotonin to melatonin. Science, 134(131), 1312. Axelrod, J., Wurtman, R. J., & Snyder, S. H. (1965). Control of hydroxyindole-O-methyltransferase activity in the rat pineal gland by environmental light. The Journal of Biological Chemistry, 240(1), 949–954. Barrett, P., Ebling, F. J.P., Schuhler, S., Wilson, D., Ross, A. W., Warner, A., et al. (2007). Hypothalamic thyroid

146 hormone catabolism acts as a gatekeeper for the seasonal control of body weight and reproduction. Endocrinology, 148(8), 3608–3617. Benitez-King, G. & Anton-Tay, F. (1993). Calimodulin mediates melatonin cytoskeletal effects. Experientia, 49(8), 635–641. Benitez-King, G., Soto-Vego, E., & Ramirez-Rodriguez, G. (2009). Melatonin modulates microfilament phenotypes in epithelial cells: Implications for adhesion and inhibition of cancer cell migration. Histology and Histopathology, 24(6), 789–799. Blask, D. E., Brainard, G. C., Dauchy, R. T., Hanifin, J. P., Davidson, L. K., Krause, J. A., et al. (2005b). Melatonindepleted blood from premenopausal women exposed to light at night stimulates growth of breast cancer xenografts in nude rats. Cancer Research, 65(23), 11174–11184. Blask, D. E., Dauchy, R. T., & Sauer, L. A. (2005a). Putting cancer to sleep at night: The neuroendocrine/circadian melatonin signal. Endocrine, 27(2), 179–188. Bockers, T. M., Niklowitz, P., Bockmann, J., Fauteck, J. D., Wittkowski, W., & Kreutz, M. R. (1995). Daily melatonin injections induce cytological changes in pars tuberalis-specific cells Similar To short photoperiod. Journal of Neuroendocrinology, 7(8), 607–613. Boutin, J. A., Marcheteau, E., Hennig, P., Moulharat, N., Berger, S., Delagrange, P., et al. (2008). MT3/QR2 melatonin binding site does not use melatonin as a substrate or a cosubstrate. Journal of Pineal Research, 45(4), 524–531. Brainard, G. C., Petterborg, L. J., Richardson, B. A., & Reiter, R. J. (1982). Pineal melatonin in syrian hamsters: Circadian and seasonal rhythms in animals maintained under laboratory and natural conditions. Neuroendocrinology, 35(5), 342–348. Brainard, G. C., Sliney, D., Hanifin, J. P., Glickman, C., Byrne, B., Greeson, J. M., et al. (2008). Sensitivity of the human circadian system to short-wavelength (420 nm) light. Journal of Biological Rhythms, 23(5), 379–386. Cagnacci, A., Elliott, J. A., & Yen, S. S. (1992). Melatonin: A major regulator of the circadian rhythm of core temperature in humans. Journal of Clinical Endocrinology and Metabolism, 75(2), 447–452. Carlberg, C. (2000). Gene regulation by melatonin. Annals of the New York Academy of Sciences, 917, 387–396. Carretero, M., Escames, G., Lopez, L. C., Venegas, C., Dayoub, J. C., Garcia, L., et al. (2009). Long-term melatonin administration protects brain mitochondria from aging. Journal of Pineal Research, 47, 192–200. Carrillo-Vico, A., Guerrero, J. M., Lardone, P. J., & Reiter., R. J. (2005). A review of the multiple actions of melatonin on the immune system. Endocrine, 27(2), 189–200. Carter, D. S. & Goldman, B. D. (1983). Antigonadal effects of timed melatonin infusion in pinealectomized male djungarian hamsters (phodopus sungorus sungorus): Duration is the critical parameter. Endocrinology, 113(4), 1261– 1267. Cerutti, P., Ghosh, R., Oya, Y., & Amstad, P. (1994). The role of cellular antioxidant defense in oxidant carcinogenesis. Environmental Health Perspectives, 102(Suppl. 10), 123–129.

Cervantes, M., Morali, G., & Letechipia-Vallejo, G. (2008). Melatonin and ischemia-reperfusion injury to the brain. Journal of Pineal Research, 145, 1–7. Chetsawang, B., Chetsawang, J., & Govitrapong, P. (2009). Protection against cell death and sustained tyrosine hydroxylase phosphorylation in hydrogen peroxide- and MPTPtreated human neuroblastoma cells with melatonin. Journal of Pineal Research, 46(1), 36–42. Colledge, W. H. (2009). Kisspeptins and GnRH neuronal signaling. Trends in Endocrinology and Metabolism, 20(3), 115–121. Cos, S. & Sanchez-Barcelo, E. J. (2000). Melatonin and mammary pathological growth. Frontiers in Neuroendocrinology, 21(2), 133–170. Dauchy, R. T., Blask, D. E., Dauchy, E. M., Davidson, L. K., Tirrell, P. C., Greene, M. W., et al. (2009). Antineoplastic effects of melatonin on a rare malignancy of mesenchymal origin: Melatonin receptor-mediated inhibition of signal transduction, linoleic acid metabolism and growth in tissue isolated human leiomyosarcoma xenografts. Journal of Pineal Research, 47(1), 32–42. Dijk, D. J., Shanahan, T. L., Duffy, J. F., Rouda, J. M., & Czeisler, C. A. (1997). Variation of electroencephalographic activity during non-rapid eye movement and rapid eye movement sleep with phase of circadian melatonin rhythm in humans. Journal of Physiology (Paris), 505(Pt. 3), 851–858. Dubocovich, M. L. & Markowska, M. (2005). Functional MT1 an MT2 melatonin receptors in mammals. Endocrine, 27(2), 101–110. Dubocovich, M. L., Rivera-Bermudez, M. A., Gerdin, M. J., & Masana, M. I. (2003). Molecular pharmacology, regulation and function of mammalian melatonin receptors. Frontiers in Bioscience, 8, d1093–d1098. Erren, T. C. & Reiter, R. J. (2009a). Defining chronodisruption. Journal of Pineal Research, 46(3), 245–247. Erren, T. C. & Reiter, R. J. (2009b). A generalized theory of carcinogenesis Due To chronodisruption. Neuroendocrinology Letters, 29(6), 815–821. Erren, T. C. & Reiter, R. J. (2009c). Light hygiene: Time to make preventive use of insights – old and new – in the nexus of the drug light, melatonin, clocks, chronodisruption and public health. Medical Hypotheses, 73(4), 537–541. Esposito, E., Genovese, T., Caminiti, R., Bramanti, P., Meil, R., & Cuzzocrea, S. (2009). Melatonin reduces stress activated/ mitogen-activated protein kinases in spinal cord injury. Journal of Pineal Research, 46(1), 79–86. Foster, R. G. & Hankins, M. W. (2007). Circadian vision. Current Biology, 17(17), R746–R751. Friedlander, R. M. (2003). Apoptosis and caspases in neurodegenerative diseases. The New England Journal of Medicine, 348(14), 1365–1375. Hankins, M. W. & Lucas, R. J. (2002). The primary visual pathway in humans is regulated According To long-term light exposure through the action of a non-classic photopigment. Current Biology, 12(3), 191–198. Hanon, E. A., Routledge, K., Dardente, H., Masson-Pevet, M., Morgan, P. J., & Hazlerigg, D. G. (2010). Effect of photoperiod on the TSH neuroendocrine system of the european

147 hamster (Cricetus cricetus). Journal of Neuroendocrinology, 22(1), 51–55. Hardeland, R. (2009). Melatonin signaling mechanisms of a pleiotropic agent. Biofactors, 35(2), 183–192. Hardeland, R., Tan, D. X., & Reiter, R. J. (2009). Kynuramines, metabolites of melatonin and other indoles: The resurrection of an almost forgotten class of biogenic amines. Journal of Pineal Research, 47(2), 109–126. Hastings, M. H., Maywood, E. S., & Reddy, A. B. (2008). Two decades of circadian time. Journal of Neuroendocrinology, 28(5), 812–819. Hazlerigg, D. G., Hastings, M. H., & Morgan, P. J. (1996). Production of prolactin releasing factor by the ovine pars tuberalis. Journal of Neuroendocrinology, 8(7), 489–492. Hevia, D., Sainz, R. M., Blanca, D., Quiros, I., Tan, D. X., Rodriguez, C., et al. (2008). Melatonin uptake in prostate cancer cells: Intracellular transport versus simple passive diffusion. Journal of Pineal Research, 45(3), 247–257. Hoffman, R. A. & Reiter, R. J. (1965). Pineal gland: Influence on gonads of male hamsters. Science, 148(3677), 1609–1611. Hoffman, R. A. & Reiter, R. J. (1966). Responses of some endocrine organs of female hamsters to pinealectomy and light. Life Sciences, 5(12), 1147–1151. Hunt, A. E., Al-Ghoul, W. M., Gillette, M. V., & Dubocovich, M. L. (2001). Activation of MT(2) melatonin receptors in rat suprachiasmatic nucleus phase advances the circadian clock. American Journal of Physiology-Cell Physiology, 280(1), C110-C118. Iuvone, P. M., Tosini, G., Pozdeyev, N., Hague, R., Klein, D. C., & Chaurasia, S. S. (2005). Circadian clocks, clock networks, arylalkylamine N-acetyltransferase, and melatonin in the retina. Progress in Retinal and Eye Research, 24(4), 433–436. James, S. Y., Lin, F., Kolluri, S. K., Dawson, M. I., & Zhang, X. K. (2003). Regulation of retinoic acid receptor beta expression by peroxisome proliferator-activated receptor gamma ligands in cancer cells. Cancer Research, 63(13), 3531–3538. Jasser, S. A., Blask, D. E., & Brainard, G. C. (2006). Light during darkness and cancer: Relationships in circadian photoreception and tumor biology. Cancer Causes Control, 17(4), 515–523. Jockers, R., Petit, I., Lacroix, I., de Coppet, P., Barrett, P., Morgan, P. J., et al. (1997). Novel isoforms of Mel1c melatonin receptors modulating intracellular cyclic guanosine 31,51monophosphate levels. Molecular Pharmacology, 11(8), 1070–1081. Jou, M. J., Peng, T. I., Reiter, R. J., Jou, S. B., Wu, H. Y., & Wen, S. T. (2004). Visualization of the antioxidative effects of melatonin at the mitochondrial level during oxidative stress-induced apoptosis of rat brain astrocytes. Journal of Pineal Research, 37(1), 55–70. Jou, M. J., Peng, T. I., Yu, P. Z., Jou, S. B., Reiter, R. J., Chen, J. Y., et al. (2007). Melatonin protects against common deletion of mitochondrial DNA-augmented mitochondrial oxidative stress and apoptosis. Journal of Pineal Research, 43(4), 389–403.

Jung, B. & Ahmad, W. (2006). Melatonin in cancer management: Progress and promise. Cancer Research, 66(20), 9789– 9793. Jung-Hynes, B. & Ahmad, N. (2009). SIRT1 controls circadian clock circuitry and promotes cell survival: A connection with age-related neoplasms. The FASEB Journal, 23(9), 2803– 2809. Jung-Hynes, B., Reiter, R. J., & Ahmad, N. (2010). Sirtuins, melatonin and circadian rhythms: A bridge between cancer and aging. Journal of Pineal Research, 48(1), 9–19. Kappers, J. A. (1965). Survey of the innervation of the epiphysis cerebri and accessory pineal organs of vertebrates. Progress in Brain Research, 10, 87–153. Kappers, J. A. (1969). The mammalian pineal organ. Journal of Neuro-Visceral Relations, 31(Suppl. 9), 140–184. Karbownik, M. & Reiter, R. J. (2000). Antioxidative effects of melatonin in protection against cellular damage caused by ionizing radiation. Proceedings of Society Experimental Biology Medicine, 225(1), 9–22. Kato, K., Hirai, K., Nishiyama, K., Uchikawa, O., Fukatsu, K., Ohkawa, S., et al. (2005). Neurochemical properties of ramelteon (TAK-375), a selective MT1/MT2 receptor agonist. Neuropharmacology, 48(2), 301–310. Kiefer, T. L., Lai, L., Yuan, L., Dong, C., Burow, M. E., & Hill, S. M. (2005). Differential regulation of estrogen receptor-alpha, glucocorticoid receptor and retinoic acid receptor-alpha transcriptional activity by melatonin is mediated via different G proteins. Journal of Pineal Research, 38(4), 231–239. Korkmaz, A., Sanchez-Barcelo, E., Tan, D. X., & Reiter, R. J. (2009a). Role of melatonin in the epigenetic regulation of breast cancer. Breast Cancer Research and Treatment, 115(1), 13–27. Korkmaz, A., Tamura, H., Manchester, L. C., Ogden, G. B., Tan, D. X., & Reiter, R. J. (2009b). Combination of melatonin and peroxisome proliferator-activated receptor-g agonist induces apoptosis in breast cancer cell line. Journal of Pineal Research, 46(1), 115–116. Kräuchi, K., Cajochen, C., Pache, M., Flammer, J., & WirzJustice, A. (2006). Thermoregulatory effects of melatonin in relation to sleepiness. Chronobiology International, 23(1–2), 475–484. Kumbalasiri, T. & Provencio, I. (2005). Melanopsin and other novel mammalian opsins. Experimental Eye Research, 81(4), 368–375. Lavie, P. (1997). Melatonin: Role in gating nocturnal rise in sleep propensity. Journal of Biological Rhythms, 12(6), 657–665. Leon-Blanco, M. M., Guerrero, J. M., Reiter, R. J., Calvo, J. R., & Pozo, D. (2003). Melatonin inhibits telomerase activity in the MCF-7 tumor cell line both in vivo and in vitro. Journal of Pineal Research, 35(3), 204–211. Lerner, A. B., Case, J. D., & Heinzelmann, R. V. (1959). Structure of melatonin. Journal of the American Chemical Society, 81(4), 6084–6085. Lerner, A. B., Case, J. D., Takahashi, Y., Lee, Y., & Mori, W. (1958). Isolation of melatonin, the pineal factor that lightens melanocytes. Journal of the American Chemical Society, 80(2), 2587.

148 Lewy, A. J., Bauer, V. K., Ahmed, S., Thomas, K. H., Cutter, N. L., Singer, C. M., et al. (1997). The human phase response curve (PRC) to melatonin is about 12 hours Out Of phase with the PRC to light. Chronobiology International, 15(1), 71–83. Lin, C. H., Huang, J. Y., Ching, C. H., & Chuang, J. I. (2008). Melatonin reduces neuronal loss, downregulation of dopamine transporter, and upregulation of D2 receptor in rotenone-induced parkinsonian rats. Journal of Pineal Research, 44(2), 205–213. Lincoln, G. A., Andersson, H., & Laudon, A. (2003). Clock genes in calendar cells as the basis of annual timekeeping in mammals – a unifying hypothesis. Journal of Endocrinology, 179(1), 1–13. Lincoln, G. A. & Clarke, I. J. (1994). Photoperiodicallyinduced cycles in the secretion of prolactin in hypothalamopituitary disconnected rams: Evidence for translation of the melatonin signal in the pituitary gland. Journal of Neuroendocrinology, 6(3), 251–260. Lincoln, G. A., Clarke, I. L., Hut, R. A., & Halerigg, D. G. (2006). Characterizing a mammalian circannual pacemaker. Science, 314(5807), 1941–1944. Liu, T. & Borjigin, J. (2005). N-acetyltransferase is not the rate limiting enzyme in melatonin synthesis at night. Journal of Pineal Research, 39(1), 91–96. Liu, C., Weaver, D. R., Jin, X., Shearman, L. P., Pieschi, R. L., Gribkoff, V. K., et al. (1997). Molecular dissection of two distinct actions of melatonin on the suprachiasmatic circadian clock. Neuron, 19(6), 91–102. Ma, X., Idle, J. R., Krausz, K. W., & Gonzalez, F. J. (2005). Melabolism of melatonin by human cytochromes P450. Drug Metabolism and Disposition, 33(4), 489–494. Ma, X., Idle, J. R., Krausz, K. W., Tan, D. X., Ceraulo, L., & Gonzalez, F. J. (2006). Urinary metabolites and antioxidant products of exogenous melatonin in the mouse. Journal of Pineal Research, 40(4), 343–349. Maronde, E. & Stehle, J. (2007). The mammalian pineal gland: Known facts, unknown facets. Trends in Endocrinology and Metabolism, 18(4), 142–149. Menendez-Pelaez, A. & Reiter, R. J. (1993). Distribution of melatonin in mammalian tissues: The relative importance of nuclear versus cytosolic localization. Journal of Pineal Research, 15(2), 59–69. Moller, M. (1976). The ultrastructure of the human fetal pineal gland. II. Innervation and cell junctions. Cell and Tissue Research, 169(1), 7–21. Moore, R. Y. (1996). Neural control of the pineal gland. Behavioural Brain Research, 73(1–2), 125–130. Morgan, P. J., Barrett, P., Howell, H. E., & Helliwell, R. (1994). Melatonin receptors, molecular pharmacology and physiological significance. Neurochemistry International, 24(2), 101–146. Morgan, P. J., Messager, S., Webster, C., Barrett, P., & Ross, A. (1999). How does the melatonin receptor decode a photoperiodic signal in the pars tuberalis? Advances in Experimental Medicine and Biology, 460, 165–174. Morgan, P. J., Webster, C. A., Mercer, J. G., Ross, A. W., Hazlerigg, D. G., MacLean, A., et al. (1996). The ovine pars tuberalis secretes a factor(s) that regulates gene

expression in both lactotropic and non-lactotropic pituitary cells. Endocrinology, 137(9), 4018–4026. Morgenthaler, T. I., Lee-Chiong, T., Alessi, C., Friedman, L., Aurora, R. N., Boehlecke, B., et al. (2007). Standards of practice committee of the american academy of sleep medicine: Practice parameters for the clinical evaluation and treatment of circadian rhythm sleep disorders. Sleep, 30(12), 1445–1459. Nakamura, Y., Tamura, H., Takayama, H., & Kato, H. (2003). Increased endogenous level of melatonin in human preovulatory follicles does not directly influence progesterone production. Fertility and Sterility, 80(4), 1012–1016. Nelson, C. S., Marino, J. L., & Allen, C. N. (1996). Melatonin receptors activate heteromeric G-protein coupled kir3 channels. NeuroReport, 7(3), 717–720. Ohkubo, T., Imai, Y., Tsuji, I., Nagai, K., Watanabe, N., Minami, N., et al. (1997). Relation between nocturnal decline in blood pressure and mortality: The ohasama study. American Journal of Hypertension, 10(11), 1201–1207. Olcese, J. M., Cao, C., Mori, T., Mamcarz, M. D., Maxwell, A., Runfeldt, M. J., et al. (2009). Protection against cognitive deficits and markers of neurodegeneration by long-term oral administration of melatonin in a transgenic model of alzheimer disease. Journal of Pineal Research, 47(1), 82–96. Ortiz-Lopez, L., Morales-Mulia, S., Ramirez-Rodriguez, G., & Benitez-King, G. (2009). ROCK-regulated cytoskeletal dynamics participate in the inhibitory effect of melatonin on cancer cell migration. Journal of Pineal Research, 46(1), 15–21. Panzer, A. & Viljoen, M. (1997). The validity of melatonin as an oncostatic agent. Journal of Pineal Research, 22(4), 184–202. Pappolla, M. A., Chyan, Y. J., Poeggeler, B., Frangione, B., Wilson, G., Ghiso, J., et al. (2000). An assessment of the antioxidant and the antiamyloidogenic properties of melatonin: Implications for alzheimer’s disease. Journal of Neural Transmission, 107(2), 203–231. Pappolla, M. A., Sos, M., Omar, R. A., Bick, R. J., HicksonBick, D. L., Reiter, R. J., et al. (1997). Melatonin prevents death of neuroblastoma cells exposed to alzheimer amyloid peptide. Journal of Neuroscience, 17(5), 1683–1690. Parfitt, A. G. & Klein, D. C. (1976). Sympathetic nerve endings in the pineal gland protect against acute stress-induced increase in N-acetyl-transferase (EC 2.3.1.5) activity. Endocrinology, 99(3), 840–851. Pozo, D., Reiter, R. J., Calvo, J. R., & Guerrero, J. M. (1997). Inhibition of cerebellar nitric oxide synthase and cyclic GMP production by melatonin via complex formation with calimodulin. Journal of Cellular Biochemistry, 6(3), 430–442. Quay, W. B. (1963). Circadian rhythm of rat pineal serotonin and its modification by estrous cycle and photoperiod. General and Comparative Endocrinology, 3(5), 473–479. Rajaratnam, S. M., Middleton, B., Stone, B. N., Arendt, J., & Dijk, D. J. (2004). Melatonin advances the circadian timing of EEG sleep and directly facilitates sleep without altering its duration in extended sleep opportunities in humans. Journal of Physiology (Paris), 561(1, Pt. 1), 339–351.

149 Reiter, R. J. (1973). Pineal control of a seasonal reproductive rhythm in male golden hamsters exposed to natural daylight and temperature. Endocrinology, 92(2), 423–430. Reiter, R. J. (1980). The pineal and its hormones in the control of reproduction in mammals. Endocrine Reviews, 1(2), 109– 131. Reiter, R. J. (1991a). Pineal melatonin: Cell biology of its synthesis and of its physiological interactions. Endocrine Reviews, 12(1), 151–180. Reiter, R. J. (1991b). Melatonin: The chemical expression of darkness. Molecular and Cellular Endocrinology, 79(1–3), C153–C158. Reiter, R. J. (1993). The melatonin rhythm: Both a clock and a calendar. Experentia, 49(8), 654–664. Reiter, R. J. (2004). Mechanisms of cancer inhibition by melatonin. Journal of Pineal Research, 37, 213–214. Reiter, R. J., Blask, D. E., Johnson, L. Y., Rudeen, P. K., Vaughan, M. K., & Waring, P. J. (1976). Melatonin inhibition of reproduction in the male hamster: Its dependency on time of day of administration and on an intact and sympathetically innervated pineal gland. Neuroendocrinology, 22(2), 107– 116. Reiter, R. J., Cabrera, J., Sainz, R. M., Mayo, J. C., Manchester, L. C., & Tan, D. X. (2000). Melatonin as a pharmacological agent against neuronal loss in experimental models of huntington’s disease, alzheimer’s disease and parkinsonism. Annals of the New York Academy of Sciences, 890, 471–485. Reiter, R. J., Craft, C. M., Johnson, J. E., Jr., King, T. S., Richardson, B. A., Vaughn, G. M., et al. (1981). Age-associated reduction in nocturnal melatonin levels in female rats. Endocrinology, 109(4), 1295–1297. Reiter, R. J. & Hester, R. J. (1966). Interrelationships of the pineal gland, the superior cervical ganglia and the photoperiod in the regulation of the endocrine systems of hamsters. Endocrinology, 79(6), 1168–1170. Reiter, R. J., Paredes, S. D., Manchester, L. C., & Tan, D. X. (2009a). Reducing oxidative/nitrosative stress: A newly-discovered genre for melatonin. Critical Reviews in Biochemistry and Molecular Biology, 44(4), 175–200. Reiter, R. J., Paredes, S. D., Manchester, L. C., & Tan, D. X. (2009d). Reducing oxidative/nitrosative stress: A newly-discovered genre for melatonin. Critical Reviews in Biochemistry and Molecular Biology, 44(4), 175–200. Reiter, R. J., Richardson, B. A., Johnson, L. Y., Ferguson, B. N., & Dinh, D. T. (1980). Pineal melatonin rhythm: Reduction in aging syrian hamsters. Science, 210(4476), 1372–1373. Reiter, R. J., Tan, D. X., Kim, S. J., Manchester, L. C., Qi, W., Garcia, J. J., et al. (1999). Augmentation of indices of oxidative damage in life-long melatonin-deficient rats. Mechanisms of Ageing and Development, 110(2), 157–173. Reiter, R. J., Tan, D. X., & Korkmaz, A. (2009c). The circadian melatonin rhythm and its modulation: Possible impact on hypertension. Journal of Hypertension, 27(Suppl. 6), S17– S20. Reiter, R. J., Tan, D. X., Korkmaz, A., Erren, T. C., Peikarski, C., Tamura, H., et al. (2007). Light at night,

chronodisruption, melatonin suppression, and cancer risk: A review. Critical Reviews in Oncogenesis, 13(4), 303–328. Reiter, R. J., Tan, D. X., Leon, J., Kilic, U., & Kilic, E. (2005). When melatonin gets on your nerves: Its beneficial actions in experimental models of stroke. Experimental Biology and Medicine, 230(1), 104–117. Reiter, R. J., Tan, D. X., Manchester, L. C., Paredes, S. D., Mayo, J. C., & Sainz, R. M. (2009b). Melatonin and reproduction revisited. Biology of Reproduction, 81(3), 445–456. Reiter, R. J., Tan, D. X., & Pappolla, M. A. (2004). Melatonin relieves the neural oxidative burden that contributes to dementias. Annals of the New York Academy of Sciences, 1035, 179–196. Reppert, S. M., Godson, C., Mahle, C. D., Weaver, D. R., Slaugenhaupt, S. A., & Gusella, J. F. (1995). Molecular characterization of a second melatonin receptor expressed in the human retina and brain: The Mel1b melatonin receptor. Proceedings of the National Academy of Sciences United States of America, 92(19), 8734–8738. Reppert, S. M., Weaver, D. R., & Ebisawa, T. (1994). Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses. Neuron, 13(5), 1177–1185. Revel, F. G., Saboureau, M., Masson-Pevet, M., Pevet, P., Mikkelsen, J. D., & Simonneaux, V. (2006). Kisspeptin mediates the photoperiodic control of reproduction in hamsters. Current Biology, 16(17), 1730–1735. Rivera-Bermudez, M. A., Massana, M. L., Brown, G. M., Earnst, D. J., & Dubocovich, M. L. (2004). Immortalized cells from the rat suprachiasmatic nucleus express functional melatonin receptors. Brain Research, 1002(1–2), 21–27. Roka, F., Brydon, L., Waldhoer, M., Strosberg, A. D., Freissmuth, M., Jockers, R., et al. (1999). Tight association of the human Mel1a-melatonin receptor and Gi: Precoupling and constitutive activity. Molecular Pharmacology, 56(5), 1014– 1024. Ross, A. W., Barrett, P., Mercer, J. G., & Morgan, P. J. (1996). Melatonin suppresses the induction of AP-1 transcription factor components in the pars tuberalis of the pituitary. Molecular and Cellular Endocrinology, 123(1), 71–80. Sack, R. I., Lewy, A. J., Erb, D. L., Vollmer, W. M., & Singer, C. M. (1986). Human melatonin production decreases with age. Journal of Pineal Research, 3(4), 379–388. Sainz, R. M., Mayo, J. C., Rodriguez, C., Tan, D. X., LopezBurillo, S., & Reiter, R. J. (2003). Melatonin and cell death: Differential actions on apoptosis in normal and cancer cells. Cellular and Molecular Life Sciences, 60(7), 1407–1426. Sainz, R. M., Mayo, J. C., Tan, D. X., Leon, J., Manchester, L. C., & Reiter, R. J. (2005). Melatonin reduces prostate cancer cell growth leading to neurodifferentiation via a receptor and PKA mechanism. The Prostate, 63(1), 29–43. Samantaray, S., Das, A., Thakore, N. P., Matzelle, D. D., Reiter, R. J., Ray, S. K., et al. (2009). Therapeutic potential of melatonin in traumatic central nervous system injury. Journal of Pineal Research, 47(2), 134–142. Sanchez-Barcelo, E. J., Cos, S., Mediavilla, M. D., MartinezCampa, C., Gonzalez, A., & Alonso-Gonzalez, C. (2005).

150 Melatonin-estrogen interactions in breast cancer. Journal of Pineal Research, 38(4), 217–222. Saravanan, K. S., Sindhu, K. M., & Mohanakumar, K. P. (2008). Melatonin protects against rotenone-induced oxidative stress in a hemiparkinsonian rat mode. Journal of Pineal Research, 42(3), 247–253. Shiu, S. Y. (2007). Toward rational and evidence-based use of melatonin in prostate cancer prevention and treatment. Journal of Pineal Research, 43(1), 1–9. Shochat, T., Luboshisky, R., & Lamie, P. (1997). Nocturnal melatonin onset is phase locked to the primary sleep gate. American Journal of Physiology, 273(1, Pt. 2), R364–R370. Simko, F. & Pechanova, O. (2009). Potential roles of melatonin and chronotherapy among the new trends in hypertension treatment. Journal of Pineal Research, 47(2), 127–133. Skene, D. J., Papagiannidou, E., Hashemi, E., Snelling, J., Lewis, D. F., Fernandez, M., et al. (2001). Contribution of CYP1A2 in the hepatic metabolism of melatonin: Studies with isolated microsomal preparations and liver slices. Journal of Pineal Research, 31(4), 333–342. Skinner, D. C. & Malpaux, B. (1999). High melatonin concentrations in third ventricular cerebrospinal fluid are not Due To galen vein blood recirculating through the choroid plexus. Endocrinology, 140(10), 4399–4405. Sliwinski, T., Rosej, W., Morawiec-Baida, A., Morawiec, Z., Reiter, R. J., & Blasiak, J. (2007). Protective action of melatonin against oxidative damage: Chemical inactivation versus base-excision repair. Mutation Research, 634(1–2), 220–227. Soares, J. M. Jr., Masana, M. I., Ersakin, C., & Dubocovich, M. L. (2003). Functional melatonin receptors in rat ovaries at various stages of the estrous cycle. The Journal of Pharmacology and Experimental Therapeutics, 306(2), 694–702. Stevens, R. J. (2009). Working against our endogenous circadian clock: Breast cancer and electric lighting in the modern world. Mutation Research, 679(1–2), 6–8. Stirland, J. A., Johnston, J. D., Cagampang, F. R.A., Morgan, P. J., Castro, M. C., White, M. R.H., et al. (2001). Photoperiodic regulation of prolactin gene expression in the syrian hamster by a pars tuberalis-derived factor. Journal of Neuroendocrinology, 13(2), 147–157. Sugden, D. (2003). Comparison of circadian expression of tryptophan hydroxylase isoforms mRNAs in the rat pineal gland using real-time PCR. Journal of Neurochemistry, 86(5), 1308–1311. Tamarkin, L., Westrom, W. K., Hamill, A. I., & Goldman, B. C. (1976). Effect of melatonin on the reproductive systems of male and female syrian hamsters: A diurnal rhythm in sensitivity to melatonin. Endocrinology, 99(6), 1534–1541. Tamura, H., Nakamura, Y., Korkmaz, A., Manchester, L. C., Tan, D. X., Sugino, N., et al. (2009). Melatonin and the ovary: Physiological and pathophysiological implications. Fertility and Sterility, 92(1), 328–343. Tamura, H., Nakamura, Y., Terron, M. P., Flores, L. J., Manchester, L. C., Tan, D. X., et al. (2008). Melatonin and pregnancy in the human. Reproductive Toxicology, 25(3), 291–303.

Tan, D. X., Chen, L. D., Poeggeler, B., Manchester, L. C., & Reiter, R. J. (1993). Melatonin: A potent, endogenous hydroxyl radical scavenger. Endocrine Journal, 1(1), 57–60. Tan, D. X., Manchester, L. C., Reiter, R. J., Plummer, B. F., Hardies, L. J., Weintraub, S. T., et al. (1998). A novel melatonin metabolic, cyclic 3-hydroxy-melatonin, and biomarker of in vivo hydroxyl radical generation. Biochemical and Biophysical Research Communications, 253(3), 614–620. Tan, D. X., Manchester, L. C., Reiter, R. J., Plummer, B. F., Limson, J., Weintraub, S. T., et al. (2000). Melatonin directly scavenges hydrogen peroxide: A potentially new metabolic pathway of melatonin biotransformation. Free Radical Biology & Medicine, 29, 1177–1185. Tan, D. X., Manchester, L. C., Reiter, R. J., Qi, W., Hanes, M. A., & Farley, N. J. (1999). High physiological levels of melatonin in the bile of mammals. Life Sciences, 65(23), 2523–2529. Tan, D. X., Reiter, R. J., Manchester, L. C., Yan, M. T., ElSawi, M., Sainz, R. M., et al. (2002). Chemical and physical properties and potential mechanisms: Melatonin as a broad spectrum antioxidant and free radical scavenger. Current Topics in Medicinal Chemistry, 2(2), 181–198. Tomas-Zapico, C. & Coto-Montes, A. (2005). A proposed mechanism to explain the stimulatory effect of melatonin on antioxidative enzymes. Journal of Pineal Research, 39 (2), 99–104. Turek, F. W., Josh, U. C., Kohsaka, A., Lin, E., Ivanova, G., McDearmon, E., et al. (2005). Obesity and metabolic syndrome in circadian clock mutant mice. Science, 308(5724), 1043–1045. Ursing, C., von Bohr, C., Brismar, K., & Rojdmark, S. (2005). Influence of cigarette smoking on melatonin levels in man. European Journal of Clinical Pharmacology, 61(3), 197–201. Vijayalaxmi, T., Jr., C. R., Reiter, R. J., & Herman, T. S. (2002). Melatonin: From basic research to cancer treatment clinics. Journal of Clinical Oncology, 20(10), 2575–2601. von Gall, C., Garabette, M. L., Kell, C. A., Frenzel, S., Dehghani, F., Schumm-Draeyer, P. M., et al. (2002). Rhythmic gene expression in pituitary depends on heterologous sensitization by the neurohormone melatonin. Nature Neuroscience, 5(3), 234–238. von Gall, C., Weaver, D. R., Kock, M., Korf, H. W., & Stehle, J. H. (2000). Melatonin limits transcriptional impact of phosphoCREB in mouse SCN via the Mel1a receptor. NeuroReport, 11(9), 1803–1807. Wang, X. (2009). The antiapoptatic activity of melatonin in neurodegenerative diseases. CNS Neuroscience & Therapeutics, 15(4), 345–357. Wang, X., Zhu, S., Pei, Z., Drozda, M., Stavroskoya, J. G., Del Signore, S. J., et al. (2008). Inhibitors of cytochrome c release with therapeutic potential for huntington’s disease. Journal of Neuroscience, 28(38), 9473–9485. Wasdell, M. B., Jan, J. E., Bomben, M. M., Freeman, R. D., Reitveld, W. J., Tai, J., et al. (2008). A randomized placebocontrolled trial of controlled release melatonin treatment of delayed sleep phase syndrome and impaired sleep

151 maintenance in children with neurodevelopmental disabilities. Journal of Pineal Research, 44(1), 57–64. Witt-Enderby, P., MacKenzie, R. S., McKeon, R. M., Carroll, E. A., Bordt, S. L., & Melan, M. A. (2000). Melatonin induction of filamentous structures in non-neuronal cells that is dependent on expression of the human mt1 melatonin receptor. Cell Motility and the Cytoskeleton, 46(1), 28–42. Witt-Enderby, P. A., Radio, N. M., Doctor, J. S., & Davis, V. L. (2006). Therapeutic treatments potentially mediated by melatonin receptors: Potential clinical uses in the prevention of osteoporosis, cancer and as an adjuvant therapy. Journal of Pineal Research, 41(4), 297–305. Wittkowski, W., Bockmann, J., Kreutz, M. R., & Bockers, T. M. (1999). Cell and molecular biology of the pars tuberalis

of the pituitary. International Review of Cytology, 185, 157–194. Yu, H. S., Yee, R. W., Hawes, K. A., & Reiter, R. J. (1990). Diurnal rhythms of immunoreactive melatonin in the aqueous humor and serum of male pigmented rabbits. Neuroscience Letters, 116(3), 309–314. Zawilska, J. B., Skene, D. J., & Arendt, J. (2009). Physiology and pharmacology of melatonin in relation to biological rhythms. Pharmacological Reports, 61(3), 363–410. Zhou, J., Zhang, S., Zhao, X., & Wei, T. (2008). Melatonin impairs NADPH oxidase assembly and decreases superoxide anion production in microglia exposed to amyloid- b1-42. Journal of Pineal Research, 45(2), 157–165.

L. Martini (Eds.) Progress in Brain Research, Vol. 181 ISSN: 0079-6123 Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 9

Modulation of steroid hormone receptor activity Vladimir Staniši´c, David M. Lonard and Bert W. O’Malley
Department of Molecular and Cellular Biology, Baylor College of Medicine, Texas, United States of America

Abstract: Classical steroid hormones (SHs) – estrogens, androgens, progestins, glucocorticoids and

mineralocorticoids – play critical roles in the regulation of reproduction, metabolism and cancer. SHs act via their cognate steroid hormone receptors (SHRs) in multiple target tissues throughout the body, exerting their physiological effects through nuclear receptor (NR)-mediated gene transcription. Since SHRs are the mediators of steroid hormone signalling in cells, regulation of their expression and function is critical for appropriate physiological responses to SHs. Cells regulate SHRs by determining the cellular concentration of SHR proteins in the cell and by tightly regulating their activity through post-translational modifications and interactions with coactivator protein complexes. In this chapter we will examine each of these regulatory mechanisms and assess their functional impact on the activity of SHRs. Keywords: steroid hormone; steroid hormone receptor; coactivator; hormone-mediated transcription; steroid receptor degradation; post-translational modifications

gonads. Adrenocortical steroids include glucocorticoids such as cortisol and mineralocorticoids such as aldosterone. In general, glucocorticoids regulate carbohydrate, lipid and protein metabolism and the responses to stress and inflammation. Mineralocorticoids are involved in the regulation of electrolyte homeostasis and extracellular and intracellular fluid volume. Gonadal sex steroids are produced in the testes in males and in ovaries in females. Both testes and ovaries produce androgens (testosterone) and estrogens (17b-estradiol). Progestins (progesterone) are produced in the ovaries and in the placenta. Like adrenal steroids, gonadal SHs affect a broad array of molecular and physiological processes in cells and tissues throughout the body. Gonadal hormones are involved in the regulation of an organism’s development, sexual maturation and differentiation, behavior and the homeostasis of both reproductive and non-reproductive tissues.

Introduction Steroid hormones (SHs) are derivatives of cholesterol that are biosynthesized through a number of biochemical reactions; a flow chart describing their biosynthesis is presented in Fig. 1. In essence, synthesis of SH involves the conversion of cholesterol to pregnolone that is subsequently converted to 17-hydroxypregnolone, 17-hydroxyprogesterone and progesterone. 17-Hydroxypregnolone can be converted into testosterone, a precursor for estrogen, while 17-hydroxyprogesterone can be converted into cortisol. Progesterone is itself a precursor for aldosterone. The biosynthesis of SHs primarily takes place in the adrenal cortex and in the


Corresponding author. Tel.: þ713-798-6205; Fax: 713-798-5599; E-mails: [email protected]

DOI: 10.1016/S0079-6123(08)81009-6

153

154

HO Cholesterol CH3 C=0 CH3 C=0

CH3 C=0 HO

OH

Pregnenolone O Progesterone (progestin)

HO 17-Hydroxypregnenolone

CH2OH

CH2OH OH

O Testosterone (androgen)

OH

HO

C=0

O

HO Estradiol (estrogen)

Cortisol (glucocorticoid)

HO

OH

OHC C = 0

O Aldosterone (mineralocorticoid)

Fig. 1. Synthesis of steroid hormones. All steroid hormones are derived from cholesterol. Represented is a simplified schematic of biochemical pathways that lead to conversion of cholesterol to major steroid hormones.

Finally, the consequences of reduced levels after menopause or andropause or their misregulation can lead to numerous pathological conditions such as osteoporosis and depression (Guyton and Hall, 2006; Voet and Voet, 2004). As derivatives of cholesterol, SHs are hydrophobic and are transported in the blood via carrier proteins such as transcortin and albumin. They freely diffuse through the plasma membrane and interact with their cognate receptors in target tissues. The general mechanism of SH action was elucidated in 1972 by O’Malley and colleagues (Means et al., 1972; O’Malley and Means, 1974; Tsai et al., 1975), who were studying the function of estrogen and progesterone and their cognate SHRs in the chicken oviduct and in vitro. Their work laid a foundation for further research on SH function, and eventually to the discovery of the

coactivators. These pioneering studies culminated in the present model of molecular events that define the action of the steroid hormones at the cellular level (Fig. 2): (1) steroid hormones bind to steroid hormone receptors located in the cytoplasm or nucleus of the cell; (2) binding of hormone to receptor causes receptor dissociation from chaperone proteins and receptor dimerization; (3) hormone-bound receptors translocate to the nucleus and interact with DNA; (4) in the DNA, hormone-bound receptors cause dissociation of inhibitory repressor molecules and the recruitment of steroid receptor coactivators; (5) receptor–coactivator complexes decondense and open the chromatin by histone modification and displacement; (6) receptor–coactivator complexes recruit general transcription factors (GTFs) and RNA polymerase II and (7)

155

Albumin

HSP90

NR HSP90

HSP90 NR

NR

NR

Coactivators

Co

re

pr

es

or

s

NR

NR

NR

GTF POL II GTF GTF

Fig. 2. Mechanism of action of steroid hormones. Steroid hormones are transported in the blood bound to carrier protein (albumin). Steroid hormones are liposoluble molecules that freely translocate through cell membrane. Once inside the cell, steroid hormone binds to nuclear receptors (NR) that are located in the cytosol and is bound to heat shock protein 90 (HSP90). Upon binding of steroid hormone to the receptor, the receptor is released from HSP90, dimerizes and translocates to the nucleus. In the nucleus, NR binds to DNA and initiates transcription by recruiting coactivators, general transcription factors (GTF) and RNA polymerase II (RNA pol II).

receptor–coactivator complexes maintain an open chromatin state and promote transcriptional elongation, mRNA splicing and eventually degradation of the activated transcription factors at the gene regulation site. The existence of protein receptors for SHs was first elucidated in the early 1960s by the pioneering work of Jensen and Gorski (Gorski and Nicolette, 1963; Jacobson and Jensen, 1962; Jensen, 1962; Jensen and DeSombre, 1973; Toft and Gorski, 1966), who used tritium-labelled estradiol to isolate and characterize the subcellular and molecular constituents that bind estrogen in the rat uterus. The development of recombinant DNA technologies in the 1970s and 1980s led to the cloning of SHRs and determination of their primary amino acid sequences. The first nuclear hormone receptor to be cloned was the glucocorticoid

receptor (GR) in 1985 (Evans, 2005; Hollenberg et al., 1985; Miesfeld et al., 1986). This was followed by cloning of estrogen receptor-a (ERa) (Greene et al., 1986; Green et al., 1986; Miesfeld et al., 1984) and other steroid receptors soon followed, leading to the establishment of the nuclear receptor (NR) superfamily of transcription factors and the realization that they share several major structural and functional features (Evans, 1988a, 1988b; Green and Chambon, 1988; Gustafsson et al., 1986; Miesfeld et al., 1986). Like other members of the nuclear hormone receptor superfamily, SHRs are functionally composed of three critical modular domains: a hormone-independent activation function 1 (AF1) domain, a DNA-binding domain (DBD) and a hormone-dependent activation function 2 (AF2) domain that is activated allosterically

156 Domains: A/B NH3

AF1

C

D

E

DBD

H

AF2

F COOH

Function: AF1 – Activation function 1 – hormone independent activation of the receptor DBD – DNA binding domain H – Hinge region – important for protein–protein interactions of the receptor and receptors post-translational modifications AF2 – Activation function 2 – contains ligand-binding domain, and ligand-dependent transcriptional function, protein-protein interactions Fig. 3. General outline of functional and structural domains of steroid hormone receptors. Structural domains (A–F) and corresponding functional modalities of steroid hormone receptors are depicted.

upon ligand binding (Mangelsdorf et al., 1995). Here, we will focus on ERa as a prototypical model for SHR structure and discuss the relationship between its structural features and its biological functions (Fig. 3). ERa consists of 595 amino acid residues with a molecular mass of 66 kDa (Bourguet et al., 2000; Evans, 1988b; Klinge, 2000; Nagy and Schwabe, 2004; Pike, 2006). It is composed of six domains that comprise the AF1, DBD, AF2 and other portions of the receptor (Fig. 3) (Bourguet et al., 2000; Evans, 1988b; Klinge, 2000; Nagy and Schwabe, 2004). The A and B domains are contained within the receptor’s AF1 and are implicated in hormone-independent transcriptional activation of the receptor. The C domain represents the DBD of the molecule and is composed of two zinc-finger motifs that are responsible for DNA sequence-specific binding to estrogen response elements (EREs). The D domain or the hinge region is a 39 amino acid long linker between the DNA and ligand-binding regions (LBDs) of ERa. Functionally, it contains a nuclear localization signal (NLS) and is implicated in interactions with some coregulator molecules. The E domain of ERa is responsible for ligand binding and doubles as the ligand-activated AF2 domain. The LBD is composed of 12 alpha helixes, five of which (helixes 3, 6, 8, 11 and 12) form a hydrophobic ligand-binding cleft (Bourguet

et al., 2000; Evans, 1988b; Klinge, 2000; Nagy and Schwabe, 2004; Pike, 2006). Upon binding to E2, this region undergoes a conformational change such that helix 12 is displaced over the opening of the ligand-binding pocket. This change in the position of helix 12 creates a coactivator-binding surface that forms specific interactions with LXXLL helical motifs present in many coactivators (Klinge, 2000; Lonard and O’Malley, 2007, 2008a, 2008b). Finally, there is the C-terminal F domain whose role in the receptor function is less clear but has been shown to be involved in receptor dimerization (McKenna et al., 1999). The functional domains contained in ERa also are found with minor variations in all other steroid hormone receptors. The structural and functional conservation between receptors points not only to the similar mechanism of action and shared evolutionary origin, but also to similar modes of receptor regulation. Given the significance of SHs and SHRs in an organism’s basic physiological and reproductive processes, regulation of their function depends on numerous regulatory systems. It is not surprising then to learn that SHs regulate hundreds of genes in all tissues of the body. SHR activity is regulated on several different levels: through regulation of SHR expression, protein concentration and stability,

157

through SHR post-translational modifications (PTMs) and through interactions of the receptors with coregulators.

Regulation of steroid hormone receptor availability by control of steroid receptor promoter activity Cells regulate the basal expression of SHRs by regulating the transcription of receptor mRNA, its translation and its protein stability. Given that steroid receptors are important and potent transcription factors, the level and the complexity of regulation of their mRNA expression is not surprising. More than seven different promoters have been identified and characterized that are upstream of the ERa gene. The ERa gene (ESR1) is located on chromosome 6, spans 300 kb and is encoded by eight exons (Ponglikitmongkol and Chambon 1988). ERa promoters are designated as A–F, T1 and T2, and span more than 150 kb of genomic DNA (Fig. 4) (Walter et al., 1985). These promoters

produce mRNA with different 50 untranslated regions that are spliced to a common acceptor site located at the þ163 position in the transcript. The location and the amount of endocrine hormone receptor expression in the organism are primary determinants of the endocrine hormone tissue specificity and physiological effect of individual SHs. ERa is primarily expressed in the breast, uterus, ovary, prostate, testes, epididymis, bones and brain, and this expression is controlled by the complex structure of the different ERa promoters. For example, the A promoter is utilized primarily in normal breast and in some breast and uterine cancer cell lines. On the other hand, promoter C is active mostly in breast cancerderived cell lines, but its activity is less pronounced in normal breast tissue (Grandien et al., 1993, 1995). Promoter B function is linked to ERa expression in endometrial cancer-derived cells but not in breast and uterine cancer cell lines (Grandien et al., 1995). Promoter E is liver specific while promoter F regulates ERa gene transcription in osteoblasts, and T1 and T2 promoters are associated with ERa expression in the testes (Flouriot

miR206

miR22 AUFp45

mRNA miR221/222

miR18a Coding region

3‘UTR region

ATG

DNA ENH.

F

E

T1

T2

D

C

B

A

Promoter region +1

+233

Fig. 4. Regulation of ERa at the level of gene expression and mRNA stability and translation. Lower panel. ERa gene expression is regulated by at least 8 promoters (labelled A–T and enhancer – ENH). Different promoters determine tissue specificity, timing and magnitude of ERa gene expression. Depiction of ERa promoters is based on the nomenclature by Kos et al. (2001) Upper panel. ERa mRNA is regulated by four micro-RNAs and mRNA-binding protein AUFp45. Binding of these molecules to ERa mRNA affects its stability and translation.

158

et al., 1998; Kos et al., 2001; Pinzone et al., 2004). In tissues and cell lines that do not express the receptor, these promoters are methylated and silenced by the action of the DNMT1 DNA methylase (Pinzone et al., 2004). Despite the number of ERa promoters present, their individual intrinsic transcriptional potential is low. Containing no classical TATA, CCAAT or GC boxes, they provide robust ERa mRNA expression with spatial-temporal precision through the combined action of binding of transcription factors interacting at the different sites (Kos et al., 2001). Estrogen and selective estrogen receptor modulators (SERMs) are among the major modulators of ERa promoter activity (Castles et al., 1997; Donaghue et al., 1999; Griffin et al., 1998; Treilleux et al., 1997). Other nuclear receptors such as the progesterone receptor (PR), androgen receptor (AR), vitamin D receptor (VDR) and retinoid X receptor (RXR) also can regulate ERa gene promoter activity. Peptide hormones and growth factors regulate ERa promoter either by direct interaction with the DNA or through the activation of different signalling cascades (involving PKA, PKB, PKC and other signalling kinases). The fact that many growth regulatory pathways control the ERa promoter indicates that ERa is a potent mitogen whose expression must be controlled by a variety of signalling growth factors (Pinzone et al., 2004). From the transcriptional complexity seen at ERa promoter, a regulatory mechanism emerges that is sensitive to the immediate and long-term needs of the organism, a mechanism capable of converting ERa protein levels from a low level of basal constitutive expression to acute or chronic elevations depending upon acute extracellular events and intracellular signalling cascades. This mechanism provides for dynamic control of ERa expression in response to changes in the environment such as stress or the administration of estrogen analogues in anti-cancer therapy and the consumption of phyto-estrogens in the diet. Estrogen receptor b (ERb) is located on chromosome 14 and contains eight exons that are transcribed from at least two different promoters

termed ON and OK. Similar to that seen for ERa, the two ERb promoters are responsible for differential tissue expression of the receptor. ERb expression also is regulated in a developmentally dependent manner. Unlike ERa, the expression of ERb is regulated by circadian rhythm. Negative circadian regulators, PER and CRY bind an E-box motif in the ERb promoter region and regulate circadian ERb mRNA both in synchronized cell culture lines and in mouse tissues (Zhao et al., 2008a). This finding is of physiological relevance considering the tissue distribution of ERb, which links it to mood disorders and reproductive problems in female shift workers and in the development of some tumours. The ERb promoter is methylated and aberrant methylation patterns are correlated with a number of disease states including prostate, breast, endometrial and ovarian cancers and atherosclerosis. Unlike ERa and ERb that are coded by two different genes on different chromosomes, progesterone receptors A and B isoforms are produced from two distinct promoters of a single PR gene (Kastner et al., 1990) located on chromosome 11q22. PR-A and PR-B are expressed in an estrogen-dependent manner although no identifiable consensus ERE is present in their 50 upstream region. Furthermore, both isoforms are expressed in a similar temporal and spatial manner and to a similar extent. Methylation of PR-B can lead to an imbalance between the two isoforms, and it is potentially seen as a causative agent in endometriosis and breast cancer. The androgen receptor is located on the X chromosome at Xq11-1. Its core promoter does not contain classical TATA or CAAT boxes, but it does contain an SP1-binding site at –46 bp and a cAMP response element (CRE) at –508 bp. Consequently, AR is upregulated by cAMP analogues. In addition, NF-kB and TNFa-binding sites are located in a distal part of the AR core promoter. The expression of AR is developmentally regulated and decreases with age. It is also regulated by its ligand; prolonged treatment with dihydroxytestosterone (DHT) decreases the expression level of AR. However, this effect is cell type

159

specific and occurs in LNCaP and T47-D cell lines but not in human hepatocellular carcinoma or osteoblastic cell lines. Finally, methylation of the proximal promoter and first exon has been observed in some instances and it is correlated with the development of androgenindependent prostate cancers (Waltering et al., 2006a). The human GR gene is located on chromosome 5 q11–q13 and is composed of nine exons. Like AR, the GR promoter does not contain TATA and CCAAT boxes, but does possess an SP1-binding site. The GR promoter region also contains AP1-, YY1-, NF-kB- and GR-binding sites (Breslin et al., 2001; Nobukuni et al., 2002; Yudt and Cidlowski, 2002). Three distinct promoters (A–C) produce mRNAs that differ in their 50 UTR regions. However, these mRNAs are subsequently spliced to a common translational start site in the second exon. It is speculated that the existence of multiple promoters accommodates cell-type specificity of GR expression and the complex regulation of the GR promoter by a number of transcription factors. In addition, a number of GR isoforms have been identified. These isoforms arise from a single gene through different splicing mechanisms and alternate translational initiations; GR isoforms are regulated in a tissuespecific manner and influence cell-specific response to glucocorticoids (Duma et al., 2006; Lu and Cidlowski, 2006). The mineralocorticoid receptor (MR) gene is located on chromosome 4q31.1-31.2 and is expressed in many tissues, such as kidney, colon, heart, hippocampus, brown adipose tissue and sweat glands. Its 50 upstream region contains two promoters (a and b), and it has three CAAT and TATA elements distributed from the 50 untranslated region to the first intron (Listwak et al., 1996). The MR promoter also contains SP1 and CREB sites (Listwak et al., 1996). The level of MR in the brain is controlled at the mRNA level and is regulated by prolonged administration of antidepressants such as imipramine (Brady et al., 1991) that increase its levels in the hippocampus.

Regulation of steroid hormone receptor availability by modulation of steroid receptor mRNA stability and translation Regulation of steroid receptor mRNA stability and translation occurs primarily through binding of specific mRNA-binding proteins or microRNAs (miRNA) to 30 UTR regulatory elements. Two regulatory elements are most prominent, AU-rich elements (AREs) and C-rich elements. AREs containing multiple copies of a 50 -AUUUA-30 motif destabilize mRNAs, while C-rich elements are recognized and differentially regulated by poly(C)-binding proteins. The miRNAs are a class of regulatory modalities that interact with specific complementary sequences present in the 30 UTR region of the receptor’s mRNA. All steroid hormone receptors contain an unusually long 30 UTR and a large number of ARE sequences in the 30 UTR, and allow steroid hormones to auto-regulate the mRNA expression levels of their cognate receptors (Ing, 2005). The ERa mRNA is 4.3 kb long and has a steady-state half-life of approximately five hours (Fig. 4). The mRNA contains an extensive 30 UTR which is three times as long as its open reading frame. The ERa 30 UTR is known to contain several regulatory elements including 14 putative class I AREs (Green et al., 1986; Keaveney et al., 1993; Kenealy et al., 2000), but its stability can be altered in response to different stimuli (Kenealy et al., 2000). Proteins such as AUFp45 bind ERa mRNA and increase its stability by protecting it from RNases (Ing et al., 2008). Recently, several miRNAs – miR18a, miR22, miR206 and miR221/222 – have been shown to bind and negatively affect ERa mRNA stability and/or translation (Adams et al., 2007; Liu et al., 2009; Pandey and Picard, 2009; Zhao et al., 2008b). Functionally, the expression of miRNAs leads to a decrease in the cellular receptor pool and subsequently to attenuated cellular responses to estrogen stimulation. Androgen receptor mRNA spans 7 kbs and is extensively regulated post-transcriptionally (Waltering et al., 2006b). Towards the 50 end of the

160

AR 30 UTR, the RNA-binding protein HuR binds and stabilizes mRNA by interacting with AREs. HuR belongs to the Elav/Hu family of RNA-binding proteins, which in addition to being a regulator of mRNA stability also has a function in shuttling AR mRNA from the nucleus to the cytoplasm. Two proteins bind the C-rich elements in the AR mRNA-CP1 and -CP2; both affect AR mRNA stability and the rate of translation (Wilce et al., 2002). Recently, a new regulator of AR mRNA translation, hnRNP-K, has been identified in prostate cancer cells. The hnRNP-K can bind to both the 50 and 30 UTR regions as well as the coding region of the AR mRNA to inhibit its translation (Mukhopadhyay et al., 2009). Overall, androgens and progestins generally increase the stability of AR and PR mRNA in prostate and endometrial cancers, respectively (Ing, 2005). Human GR mRNA contains numerous ARE elements and is subject to post-transcriptional regulation (Schaaf and Cidlowski, 2002). In addition to its regulation by a protein binding to an ARE element in the GR 30 UTR, GR mRNA is also negatively regulated by two miRNAs – miR18 and miR124a. Interestingly, while miR18 is broadly expressed in many tissues, miR124a is exclusively expressed in the brain, suggesting a probable tissue-specific regulatory requirement for controlling GR mRNA expression in the nervous system (Vreugdenhil et al., 2009). Generally, glucocorticoids tend to decrease the stability of GR mRNA in the kidney, liver and colon cells (Ing, 2005).

Regulation of steroid hormone receptors at the level of protein stability The ubiquitin proteasome system (UPS) regulates SHR protein stability. The UPS is a complex biochemical system that regulates the stability of cellular proteins by virtue of a number of enzymes responsible for protein degradation (Hershko, 1996; Hershko and Ciechanover, 1998; Nandi et al., 2006). In the initial reaction of the UPS, the 76 amino acid protein ubiquitin is covalently attached to protein substrates destined for degradation. Ubiquitin attachment

is mediated by three classes of enzymes: a sole ubiquitin-activating enzyme (E1) first charges an ubiquitin molecule and transfers it to an E2-ubiquitin conjugating enzyme; ubiquitin is subsequently transferred to the target protein from E2 by the activity of an E3-ubiquitin ligase. The temporal, spatial, contextual and protein specificities of ubiquitin-protein attachments are regulated by several hundred different ubiquitin E3 ligases. Once a protein substrate is polyubiquitinated, it can become a target for degradation by the 26S proteasome complex. After the initial attachment of the first ubiquitin to a target protein, subsequent ubiquitins can be added to this ubiquitin, forming a polyubiquitin chain. Depending on the lysine linkage of branching ubiquitin molecules (K6, K11, K27, K33, K48 and K63), a polyubiquitinated protein can be fated for degradation or other cellular processes. K48-based polyubiquitin linkages lead to proteasome-mediated protein degradation. The 26S proteasome is composed of the 20S proteasome proteolytic core and the 19S cap that serves to recruit and channel substrate proteins into the 20S core. The reversal of protein ubiquitination can be effected through a class of proteases termed deubiquitinating enzymes (DUBs). Although ubiquitination is primarily thought of as a PTM event associated with protein degradation, monoubiquitin or polyubiquitin linkages other than K48 can affect protein localization, protein trafficking, secretion, nuclear export, ER processing, transcriptional activity and DNA binding (Nandi et al., 2006). ERa protein turnover is extensively regulated by the UPS. ERa activity and stability are intimately linked so that inhibition of ERa degradation leads to its stabilization, but also to its loss of transcriptional activity. UPS-dependent turnover of the receptor is required for its transcriptional function (Alarid et al., 1999; Khissiin and Leclercq, 1999; Lonard et al., 2000; Metivier et al., 2003; Nawaz et al., 1999a; Reid et al., 2003; Stenoien et al., 2001; Wijayaratne and McDonnell, 2001). Ubiquitin E3 ligases that regulate unliganded ERa protein stability and activity include CHIP (carboxyl terminus of Hsc70-interacting protein)

161

and breast cancer 1, early onset (BRCA1) (Ballinger et al., 1999; Fan et al., 2005; Tateishi et al., 2004; Zheng et al., 2001). CHIP is part of a molecular chaperone complex that contains Hsp90, Hsp70, Hsp40 and BAG-1 proteins. In accordance with its established chaperone activity, experimental evidence indicates that CHIP functions to recognize and remove misfolded ERa. CHIP interaction with the ERa ligandbinding domain leads to increased ERa ubiquitination and increased ERa turnover. BRCA1 also is shown to interact and mono-ubiquitinate ERa at lysine 302 in the hinge region of the receptor (Zheng et al., 2001). Although this modification has not been conclusively linked to receptor degradation, it is hypothesized to have a functional effect on ERa transcriptional activity. In its liganded state ERa is modified by E6-associated protein (E6-AP/UBE3A) and MDM2. ERa association with E6-AP is reciprocal to Ca2þ-dependent calmodulin binding to ERa. E6-AP destabilizes the receptor and leads to increased receptor activity (Li et al., 2006; Nawaz et al., 1999b). MDM2 interaction with ERa also coactivates the receptor (Liu et al., 2000; Saji et al., 2001). Furthermore, the COP9 signalosome (CSN) is involved in the regulation of ERa degradation by regulating the activity of these E3 ligases (Fan et al., 2003). Recently, deubiquitinating enzymes have been reported to regulate ERa protein stability and activity. The deubiquitinating enzyme OTUB1 has been found to directly bind and deubiquitinate ERa in cells and in vitro. ERa deubiquitination stabilizes the receptor in the chromatin fraction of Ishikawa endometrial cancer cells and represses ERa transcriptional activity (Stanisic et al., 2009). The presence of both ubiquitinating and deubiquitinating enzymes on ERa suggests that there is a dynamic regulation of ERa ubiquitin status during transcription. Stability of the receptor is coupled with the receptor’s cycling on the promoter of ERa target genes and disruption of either receptor’s stability or its cycling leads to the abolishment of transcription (Reid et al., 2003). As ERa binds to the promoter of target genes, it recruits coactivator complexes and components

of the UPS system (ubiquitin E3 ligases, proteasome, deubiquitinating enzymes). In this situation the UPS is hypothesized to ensure that the ordered procession of recruited cofactors proceeds uninterrupted by sequentially removing ‘used up’ coactivator factors, and by eliminating the receptor itself after each successful round of transcription. This process forms a basis for a ‘ubiquitin clock’ present both for the receptor and coactivators, which ensures maximum efficacy of transcription while tightly controlling the amount and usage of available transcriptional constituents (Wu et al., 2007). Experimental data show that if this process is interrupted by the addition of proteasome inhibitors or the removal of cellular ubiquitin (Lonard et al., 2000), transcription is irrevocably stopped by the stabilization and immobilization of the receptor in the nuclear matrix. Unlike ERa, the degradation of ERb is not coupled with ERb activation, but is instead required for the down-regulation of ERb transcriptional activity. E3 ligase CHIP is charged with the clearing of ERb and shutting down its signalling in MDA-MB231 breast cancer cells (Tateishi et al., 2006). Similar to ERa, PR protein is preferentially degraded by the UPS in its liganded state. Within six hours of progesterone treatment, 95% of liganded PR is degraded; in contrast, the half-life of unliganded PR is 21 hours (Ismail and Nawaz, 2005; Nardulli and Katzellenbogen, 1988). Proteasomal degradation of PR is preceded by PR phosphorylation at serine-294 by mitogen-activated protein kinase (Lange et al., 2000). Also, the yeast E3-ubiquitin ligase RSP5 and its human orthologue hRPF1 positively affect PR-mediated transcription (Imhof and McDonnell, 1996). UbcH7, an E2-conjugating enzyme, and E6-AP E3 ligase stimulate PR transcription in a coordinated manner (Faus and Haendler, 2006b; Imhof and McDonnell, 1996; Verma et al., 2004). The ubiquitin E3 ligase BRCA-1 also binds PR; however, the BRCA-1 interaction with PR negatively regulates PR transcriptional activity (Eakin et al., 2007; Ma et al., 2006). A CUE domain containing 2 (CUEDC2) binds PR and enhances ubiquitination of PR on lysine 388 and causes hormone-dependent

162

ERa is phosphorylated in response to secondary messenger pathways. ERa is phosphorylated on both serine and tyrosine residues, but there is only one tyrosine phosphorylation site recognized in ERa and phosphorylation on this site is not hormone regulated (Lannigan, 2003). In general, phosphorylation of ERa by different kinases changes ERa affinity for E2, 4-hydroxytamoxifen (4HT), EREs in the DNA, and for its interactions with coactivators (Lannigan, 2003; Likhite et al., 2006). ERa is phosphorylated in a hormonedependent manner on serines S104, S106, S118 and 167 (Likhite et al., 2006). Phosphorylation of ERa on serine S118 is the most prominent phospho-modification of ERa both in vitro and in vivo (Chen et al., 2002; Lannigan, 2003; Murphy et al., 2006). Phosphorylation of S118 is effected by two different independent cellular pathways, and in both cases it conveys activity to the receptor. Hormone binding to the ERa induces rapid S118 phosphorylation by cyclin-dependent protein kinase Cdk7. Also, growth factor signals activate the MAPK signalling pathway and induce hormone-independent phosphorylation of S118 by ERK1/2 kinase. This hormone-independent phosphorylation of S118 mechanistically underscores the hormone-independent activation of ERa via its AF1, and it is implicated as the possible mechanism for the agonistic action of 4HT. In addition to S118, two neighbouring serines, S104 and S106 also have been shown to be phosphorylated in vitro by the MAPK pathway in an E2-dependent manner by the cyclin A/CDK2dependent kinase (Lannigan, 2003; Thomas

degradation of PR. Consequently, loss of CUEDC2 leads to a decrease in PR transcriptional activity in breast cancer cells and progesterone-dependent proliferation in cancer cells (Zhang et al., 2007). AR transcriptional activity is negatively regulated by UPS-mediated receptor turnover. The E3 ligase MDM2 ubiquitinates and destabilizes AR following AR phosphorylation by Akt. These events lead to a decrease in AR activity. The CHIP E3 ligase interacts with AR through its N-terminal domain and limits its function (He et al., 2004). Proteins that stabilize AR, such as tumour susceptibility gene product TSG101, enhance AR monoubiquitination and increase AR transcriptional activity (Burgdorf et al., 2004). Both GR and MR also are subject to proteasomal degradation. In the case of GR, the E3 ligase hRPF1 binds and coactivates GR transcription (Imhof and McDonnell, 1996). However, a mutation in the GR degron region stabilizes the receptor and causes its transcriptional enhancement. In addition, treatment of cells with the proteasomal inhibitor MG132 both stabilizes GR and leads to increased GR-mediated transcription (Wallace and Cidlowski, 2001). Although MR contains degrons in its protein sequence and is degraded by UPS, the relationship between MR and the proteasome has not been established.

Receptor post-translational modifications ERa is phosphorylated both in the presence and absence of ligand (Fig. 5). In the unliganded state,

CyclinA/CDK2

Cdk7 ERK 1/2

S104 S106 S118 NH3

AF1

RSK AKT Casein kinase B p300 SET7

S167

PIAS1, PIAS3

K266 K268 K299 K302 K303

DBD

H

AF2

COOH

Fig. 5. Post-translational regulation of ERa. Steroid hormone receptors are regulated by a plethora of PTMs. In the example of ERa, signalling cascades converge on ERa protein and regulate its function by phosphorylation, acetylation, methylation and SUMOylation.

163

et al., 2008). S167 is phosphorylated in a ligandindependent manner by p90 ribosomal S6 kinase RSK, and AKT, in response to the activation of the PI3 kinase pathway (Lannigan, 2003). In vitro, S167 can be phosphorylated by casein kinase B (Lannigan, 2003). In a study of ERa positive breast cancer patients, phosphorylation of ERa at Ser167 was identified as a marker of longer disease-free status and overall survival (Jiang et al., 2007). The ERa hinge region is extensively modified by several PTMs, and is the site of dynamic interplay between acetylation, methylation, SUMOylation and ubiquitination (Fig. 5). This centre of post-translational processing presumably reflects the functional importance of this region for finetuning the interactions of ERa with DNA and coactivators. ERa is acetylated by p300 in a hormone-dependent manner on lysines K266 and K268 (Kim et al., 2006). This acetylation increases ERa estrogen-dependent interaction with DNA and coactivates ERa-mediated transcription in transient reporter assays (Kim et al., 2006; Wang et al., 2001a). Lysines K302 and K303 in the hinge region of ERa also have been recognized as potential p300 acetylation sites since experimentally created mutations of these sites reduce ERa transactivation while at the same time lowering the acetylation status of the receptor (Cui et al., 2004; Wang et al., 2001a). It has been reported that phosphorylation of serine S305 by PKA prevents acetylation of lysine K303 and attenuates ERa transcriptional activity (Cui et al., 2004). Although the receptor recruits methyltransferases for the purpose of chromatin modification, it has been shown that methyltransferases also can methylate the receptor. Lysine 302 is reported to be methylated in vivo and in vitro by the SET7 lysine methyltransferase. SET7 is recruited by receptors to methylate histone H3K4, but it can act to methylate transcription factors as well. Methylation of ERa K302 stabilizes ERa, indicating that K302 is a potential site of ERa ubiquitination. In addition, loss of SET7 attenuates E2-dependent activation of ERa-regulated genes in MCF-7 cells (Subramanian et al., 2008). Therefore, it is possible that the methylation of the hinge region of ERa stabilizes the receptor and increases its transcriptional activity.

Although ERa does not contain a consensus SUMO conjugation site, ERa has been shown to be SUMOylated in cells and in vitro (Sentis et al., 2005). SUMOylation of ERa appears to be strictly hormone dependent and it occurs in the hinge region of the molecule, affecting K299, K302, K303 lysine triad. SUMOylation of the hinge region positively affects estrogen-dependent ERa transcriptional activity in transient reporter assays. Attachment of the SUMO-1 modifier to the ERa hinge region is mediated by protein inhibitor of activated signal transducer and activator of transcription PIAS1 and PIAS3 E3 SUMO ligases. However, the same report shows that PIAS1 and PIAS3, as well as Ubc9, can coactivate ERa transcription independently from their SUMO-1 conjugation activity (Sentis et al., 2005). AR is phosphorylated on at least nine serine residues throughout the molecule. Serine S213 was shown to be activated by PI3K/Akt in response to treatment with the synthetic androgen R1881. Another phosphorylation site, Ser 650, is phosphorylated by MAPK kinase (MKK), c-Jun N-terminal kinase (JNK) or MKK6–p38 signalling pathways. Phosphorylation of Ser 650, which is located adjacent to the nuclear export signal, prevents the export of AR from the nucleus and thereby regulates AR activity (Gioeli et al., 2006). AR activity is substantially linked with its acetylation status. A number of studies have shown that acetylation of AR causes coactivator recruitment and receptor coactivation. AR is acetylated in vitro and in vivo by p300 and cAMP-response element-binding protein (CBP) on lysines K632 and K633 in the vicinity of its DBD. This modification is required for the receptor’s coactivation since the mutation in the conserved acetylation site diminished the receptor’s response to hormone (Fu et al., 2000). Acetylation of the same conserved site was also observed in the study of bombesin-induced AR transactivation. Bombesin and DHT act via Src and PKCd signalling pathways to activate p300 acetyltransferase, leading to the activation of AR activity in prostate cancer cells (Gong et al., 2006). Furthermore, acetylation of AR leads to the recruitment of coactivator complexes and the dismissal of corepressors,

164

leading to increased residency of the receptor on promoters and enhanced proliferation of prostate cancer cells (Fu et al., 2003). In addition, other studies have shown that AR is also acetylated by coactivator protein Tip60 which plays a key role in prostate cancer development, and that loss of acetylation of the receptor is linked with defects in trafficking, misfolding and aggregation similar to those seen in polyglutamine aggregates (Gaughan et al., 2002; Halkidou et al.; Korkmaz et al., 2004; Thomas et al., 2004). AR SUMOylation is hormone dependent, and it is generally repressive with respect to the transcriptional function of the receptor. AR is SUMOylated on lysines K386 and K520 by SUMO E3 ligases PIAS1 and PIASxa (Nishida and Yasuda, 2002; Poukka et al., 2000). These events cause transcriptional repression of AR. On the other hand PIASlike SUMO ligase hZimp10 promotes SUMOdependent activation of the receptor in an androgen-specific manner (Poukka et al., 1999; Sharma et al., 2003). PR activity is regulated by several protein kinases including MAPKs, CDK2 and casein kinase II. PR phosphorylation on Ser 294 by MAPK is an activating event that is linked to proteasome-mediated degradation of the receptor protein (Lange et al., 2000; Qiu & Lange, 2003). In addition, PR phosphorylation was found to be important for controlling the receptor’s subcellular localization. Although PR is likely regulated by protein acetylation, no acetyl transferases have been identified to date to interact with and acetylate the receptor. A SUMOylation site has been mapped in the PR molecule on lysine K388 in the N-terminus. Ligand binding is required for the SUMOylation of this site, and it leads to the auto-inhibition and transrepression of PR transcriptional activity (Abdel-Hafiz et al., 2002). Further studies have shown that SUMOylation also leads to decreased ligand affinity for the receptor and slowed receptor down-regulation. However over-expression of SUMO-1 leads to increased PR activity, presumably through SUMO-dependent enhancement of activity of

coactivators such as steroid receptor coactivator-1 (SRC-1) (Abdel-Hafiz et al., 2009). GR is subject to complex combinatorial phosphorylation interplay by MAPKs, CDKs and casein kinase II. Phosphorylation of the serine residues (S113, S141, S203, S211 and S226) in the N-terminal domain of the molecule has been linked to the receptor’s function and disruption of these phosphorylations led to a decrease in transcriptional activity in some experimental systems (Almlof et al., 1995; Krstic et al., 1997). Furthermore, GR subcellular localization has been shown to be dependent on phosphorylation of S203 and S211 (Ismaili and Garabedian, 2004a). Recently, interplay between phosphorylation of S211 and S226 has been implicated in the regulation of GR transcriptional coactivation. It has been determined that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226 (Chen et al., 2008). Also, GSK-3b has been shown to phosphorylate GR on serine 404. This modification caused inhibition of glucocorticoid-dependent NF-kB transrepression and decreased glucocorticoid-dependent cell death of osteoblasts (Galliher-Beckley et al., 2008). In the rat brain, GR is phosphorylated in the nucleus on residue S232 by Cdk5 following chronic stress. In the same system, phosphorylation of serine S246 by JNK is decreased upon phosphorylation of S246 (Adzic et al., 2009). In addition to kinases, a number of phosphatases also have been implicated in the regulation of GR activity. In MCF-7 and T47-D breast cancer cells, estradiol inhibits GR activity by downregulating active S211 phosphorylated form of the receptor by inducing protein phosphatase 5 (PP5) (Zhang et al., 2009). Other phosphatases such as PP1 and PP2 also play roles in reversing GR phosphorylation and inhibit its function (Faus and Haendler, 2006a; Ismaili and Garabedian, 2004b). GR is acetylated in a hormone-dependent manner on lysines K494 and K495. Inhibition of this modification as a result of impaired histone deacetylase 2 (HDAC2) activity causes down-regulation of glucocorticoid-dependent NF-kB-mediated gene expression (Ito et al., 2006). GR is also a target for

165

acetylation by the histone acetyl transferase CLOCK and its heterodimer partner BMAL1. These proteins are self-oscillating transcription factors involved in the regulation of circadian rhythms in the central and peripheral nervous system. CLOCK/BMAL1 has been shown to negatively affect GR-mediated transcription (Nader et al., 2009). GR is SUMOylated on lysines K277, K293 and K703, and this modification is positively correlated with its transcriptional activity (Le Drean et al., 2002). GR has been found to interact with the SUMO conjugating enzyme Ubc9 through its ligand-binding domain (Cho et al., 2005; Gottlicher et al., 1996). MR is preferentially phosphorylated on its N-terminal domain in the presence of aldosterone (Faus and Haendler, 2006a). In addition, the activation of PKA has been correlated with increased MR affinity for DNA. MR is SUMOylated on lysines K89, K399, K428, K494 and K953, and the elimination of these sites leads to increased MR transcriptional activity, indicating that SUMOylation has a negative effect on the receptor’s transcription activity. Acetylation of MR has not been reported, despite the presence of a putative acetylation site (Faus and Haendler, 2006a).

Regulation of steroid hormone receptor activity by coregulator molecules Although genome-wide ERa–DNA binding studies have identified several thousand widely dispersed ERa–DNA binding sites, only a handful of these sites have been experimentally shown to regulate the transcription of ERa target genes (Green and Carroll, 2007). These findings together with the finding that forkhead protein binding motifs are enriched in a genome-wide screen of ERa-binding sites suggest the existence of pioneer factors that function to define and license gene promoters for subsequent transcription initiation steps (Cirillo et al., 2002; Green and Carroll, 2007). FoxA1 is thought to initially contact compact chromatin and act to disrupt internucleosomal interactions

mediated by H3/H4 histone tetramers (Cirillo et al., 2002). FoxA1 presumably achieves this by mimicking histone molecules (Green and Carroll, 2007). Studies of the cyclin D1 (CCDN1) promoter (Eeckhoute et al., 2006) indicate that FoxA1 relaxes and opens chromatin in the enhancer 2 (enh2) region of the CCDN1 promoter; this event leads to the E2dependent recruitment of Sp1 and ERa. FoxA1 expression is well correlated with the expression pattern of ERa in luminal breast cancer samples (Green and Carroll, 2007; Thorat et al., 2008). In addition to FoxA1, GATA3 has been shown to play a role as a pioneer factor for ERa (Eeckhoute et al., 2007; Kouros-Mehr et al., 2006). GATA3 expression is recognized as a marker for ERa positive tumours, and its disruption in the GATA3 knockout mouse leads to a phenotype that mimics that of the ERa knockout mouse (Eeckhoute et al., 2007; Mallepell et al., 2006). Importantly, ERa regulates the expression of both FoxA1 and GATA3, while at the same time, these pioneer transcription factors also regulate the expression of ERa (Eeckhoute et al., 2007; Green and Carroll, 2007) forming a feedback regulatory loop. Pioneer factor-mediated chromatin relaxation and opening is thought to lead to hormonedependent recruitment of ERa to cis regulatory elements in promoter regions. In subsequent steps, chromatin remodelling coregulator complexes are recruited to the site of impending transcription. There are two major groups of chromatin remodelling coactivator complexes: (1) ATP-dependent chromatin remodelling complexes and (2) enzymes that covalently modify histones by acetylation, methylation, phosphorylation and ubiquitination. ATP-dependent chromatin remodelling complexes such as hSWI–SNF use the energy of ATP hydrolysis to displace nucleosomes along the DNA in a process termed nucleosome sliding, thereby increasing nucleosome accessibility to transcription factors, to change rotational phasing of DNA on the nucleosome and to reduce negative supercoiling of circular chromatin templates (Green and Carroll, 2007; Kassabov et al., 2003). ATP-dependent chromatin remodelling

166

complexes are multi-subunit protein complexes, and ERa is shown to interact in an E2-dependent manner with many of its subunits (Chiba et al., 1994; DiRenzo et al., 2000; Garcia-Pedrero et al., 2006; Green and Carroll, 2007; Ichinose et al., 1997; Kassabov et al., 2003). BRG1 is a component of the hSWI–SNF coactivator complex that interacts with ERa AF2 in an ATP- and E2-dependent manner (Chiba et al., 1994; DiRenzo et al., 2000; Ichinose et al., 1997). BAF57 is another hSWI–SNF subunit that is shown to participate in ERa-mediated gene expression. In cell culture, BAF57 coactivates ERa-mediated transcription.

It binds to the ERa hinge region and has been shown to help recruit p160 coactivators (GarciaPedrero et al., 2006; Green and Carroll, 2007). A major addition to the basic model of steroid receptor-mediated transcription came with the observation made using in vitro transcription systems that NR and basic transcription machinery alone are insufficient to induce a strong transcriptional output (Fig. 6) (Lonard and O’Malley, 2007, 2008a, 2008b; Spelsberg et al., 1972). This realization was followed with the identification of the first steroid receptorassociated protein 160 (ERAP160) (Halachmi

(2)

(1) NR

NR

FOXA1 SRC1

SRC3 Corepressor

Corepressor NR

Corepressor

p300

Corepressor

(3)

NR BAF57

(4) S

HAT

HM

Coactivators

NR

LTD

26

HAT

HM Coactivators

NR

NR

UB.

LTD

GTF POL II NR GTF GTF UB.

Fig. 6. Nuclear receptors (NR) initiate transcription of steroid hormone responsive genes. (1) In an inactive transcriptional state, gene promoters are tightly wrapped in the nucleosomes and bound with the repressor molecules. Upon hormone stimulation, FOXA1 pioneering factor binds to the gene promoter and navigates the binding of the nuclear receptor such as ERa. (2) NR binds DNA and recruits primary coactivators such as SRC1, SRC3, p300, BAF57, etc. (3) Protein–protein interactions of primary coactivators lead to the recruitment of secondary coactivators with enzymatic activity: HAT, histone acetyl transferases; HM, histone methylases; LTD, demethylases; UB,ubiquitination enzymes (E3 ligases, deubiquitinating enzymes) and others. (4) Finally, general transcription factors (GTF) and RNA polymerase II (RNA pol II) are recruited and transcription initiated and maintained. The recruitment of the components of the ubiquitin proteasome system and proteasome (26S) for the regulated, activity-dependent degradation of coactivators and the receptor is critical for the successful continuation of the transcription.

167

et al., 1994). Also, the observation that the addition of two different, non-overlapping steroid receptors into a cell leads to transcriptional interference and squelching suggested that there is a common limiting pool of molecules utilized by different transcription factors during transcription. These observations led to the hypothesis that a group of yet undiscovered transcriptional players is required for robust steroid receptor transcriptional activity. Soon after, Oñate et al. (1995) cloned and identified SRC-1 in a yeast two-hybrid screen using the progesterone receptor LBD as a bait protein; SRC-1 was shown to be a potent activator of transcription for most steroid receptors. In subsequent years many more coactivators and corepressors (>350) were identified with diverse structural and enzymatic capabilities that collectively orchestrate and execute a highly complex sequence of molecular events at the gene promoter that leads to productive transcription (Cavailles et al., 1995; Halachmi et al., 1994; Kamei et al., 1996; Le Douarin et al., 1995; Lee et al., 1995; Lonard and O’Malley, 2007, 2008a, 2008b; Oñate et al., 1995). There are two types of coactivators – primary coactivators and secondary coactivators. Primary coactivators directly bind to SHRs and usually serve as scaffolds for the recruitment and the exchange of the secondary coactivators. Secondary coactivators do not bind directly to the SHRs but interact indirectly through the primary coactivators, but their enzymatic functions are indispensible for the receptor’s activity. Neither primary nor secondary coactivators bind ligands or DNA but are recruited by the receptors to the chromatin, usually in a hormone-dependent manner. The p160 coactivators or the steroid receptor coactivators SRC-1, SRC-2 and SRC-3 are the most studied of the primary coactivators; they play critical roles in chromatin remodelling and the assembly of transcription initiation complexes upon the recruitment of ligand-activated receptors to the promoter (Fig. 6). Although they were discovered in the contexts of different receptors, the SRC-family coactivators enhance the transcriptional activity of most steroid receptors and many other transcription factors as well. Although SRC-1

and SRC-3 have intrinsic histone acetyltransferase (HAT) activity, their major function (as well as the function of SRC-2) is to provide scaffolding and to direct the recruitment of other enzymatically active molecules. This function of SRCs is mediated by protein–protein interactions with secondary coactivators via distinct domains in the SRC coactivators. The molecular weight of p160/SRC proteins is 160 kDa, and they contain several conserved domains. A key motif contained in SRC proteins structure responsible for their interaction with SHRs is the LXXLL NR box motif. This motif interacts with the hydrophobic cleft formed in the LBD of the receptor upon ligand binding. An important feature of the LXXLL motif is that it binds with different affinities to different SHRs depending upon the amino acid context in which the LXXLL is found. In addition, SRCs can interact with the AF-1 domains of the receptors through their C-terminal regions. SRC interactions with secondary coactivators are mediated by three transcriptional activation domains (ADs). These domains contain intrinsic activating capacity as assessed by the transcriptional ability of isolated AD fused to the Gal4 DBD. AD1 interacts with and recruits acetyltransferases such as p300, CBP and PCAF. AD2 recruits histone methyltransferases CARM1 and PRMT1. Unlike AD1 and AD2 that are located in the C-terminal domain of the SRC molecule, AD3 is located on the N-terminus and contains basic helix-loop-helix PAS domain (bHLH-PAS). This domain interacts with a plethora of coactivators such as ANCO1, BAF57, CoCoA, Flil, GAC63 and others. The secondary coactivators associate with the steroid receptors mainly through interactions with the p160/SRCs; the HATs p300 and CBP serve as prime examples of secondary coactivators (Fig. 6). Although they can directly interact with the receptors, they primarily function as secondary coactivators in partnership with SRC family coactivators. Once recruited to the promoter, p300 and CBP HAT activities enable chromatin decondensation and the opening of the chromatin. This function results in the neutralization of the positively charged histone tails with negatively charged acetyl groups. Neutralized histone tails lose their affinity for negatively charged DNA, resulting in greater

168

access for other factors to DNA. By acetylating histones, p300 and CBP promote the recruitment of RNA polymerase II and the basic transcription factors TBP and TFIIB. Protein arginine methyltransferases CARM1 and PRMT1 also are secondary coactivators that impact SHR transcription by methylating histone tails. CARM1 methylates histone H3 on arginine residues R2, R17 and R26 in response to hormone binding to the steroid receptor. In addition to its histone-methylating function, CARM1 is also responsible for methylating coactivators such as SRC-3, p300 and CBP. PRMT1 methylates R3 on histone H4 in response to hormone stimulation of steroid receptors. Methylation of R3 is just one step in a string of sequential methylation, acetylation and ubiquitination events that take place on histone tails, ultimately leading to the recruitment of RNA PolII. Hormone-dependent binding of CoCoA to SRC coactivator is mediated by the AD3 domain of the SRC coactivators. CoCoA is a secondary coactivator that exerts its stimulatory function through protein–protein interactions with mediator complex and basal transcription factors such as TBP and TAF9. CoCoA acts synergistically with p300 and CARM1 to promote hormone-dependent, steroid receptor-mediated transcription. When steroid hormone receptors recruit coactivator complexes to chromatin, they orchestrate a highly complex and ordered sequence of events that enable transcription to progress from the initial chromatin opening to recruitment of RNA polymerase, transcription initiation, transcription elongation and transcription termination (Fig. 6). In the cell, coactivators serve as ‘master regulators’ that act to integrate cellular and extracellular events with the process of gene transcription with the goal of producing a coherent physiologic transcriptional response (Lonard and O’Malley, 2007, 2008a, 2008b). In this way, coregulators act as integrators and processors of cellular signals and relay those signals to the chromatin to generate the transcriptional responses appropriate to upstream signals. It has been shown recently that coregulators also participate in processes outside of transcription. Their expanded roles include regulation of mRNA processing,

export and mRNA translation into protein and other cellular events (Lonard and O’Malley, 2007, 2008a, 2008b). Therefore, steroid hormone-dependent transcription is absolutely contingent on the recruitment and interaction of these cofactors with SHRs. For example, dependent upon cell and gene context, ERa recruits all three SRC coactivators (Anzick et al., 1997; Demarest et al., 2002; Green and Carroll, 2007; Hong et al., 1997; Kamei et al., 1996; Lonard and O’Malley, 2007, 2008a, 2008b; Torchia et al., 1998). Once recruited to chromatin by ERa, SRC coactivators engage in an elaborate process of recruitment and exchange of enzymatically active molecules that maintain the open state of chromatin, and temporally and spatially direct the assembly of the basal transcription machinery. ERa recruitment of SRC coactivators causes their interaction with CBP and p300. CBP and p300 HATs acetylate histone H3 at lysine K14 and histone H4 at lysines K5 and K8. Furthermore, they acetylate histone H2A, and H2B lysines (Chen et al., 1997; Demarest et al., 2002; Kamei et al., 1996; Kim et al., 2001; Kobayashi et al., 2000; MartinezBalbas et al., 1998; Schiltz et al., 1999; Spencer et al., 1997; Webb et al., 1998). CBP and p300 are required for the initial decondensation of the chromatin and their functions are often thought to be redundant; however, they are both recruited to the promoter and show different cycling dynamics at the same promoter. In addition to its interaction with coactivators, p300 interacts with the ERa AF2 domain where it promotes functional synergism between the AF1 and AF2 domains (Green and Carroll, 2007; Kobayashi et al., 2000). p300/CBP-associated factor (PCAF) is a homologue of the yeast GCN5 and contains intrinsic HAT activity. PCAF is not directly recruited to ERa, but interacts with ERa via SRC-1 (Santos-Rosa et al., 2003). PCAF has been shown to be a transcriptional coactivator by increasing E2-dependent transcription of ERa by acetylating H3K9 and H3K14 (Santos-Rosa et al., 2003; Xu et al., 1998). Another class of enzymes that are recruited to the site of ERa transcription by the p160 coactivators includes the methyltransferases CARM1

169

and PRMT1 (Bedford and Richard, 2005; Metivier et al., 2003; Qi et al., 2002; Strahl et al., 2001; Wang et al., 2001b). PRMT1 adds methyl groups to histone H4 arginine R3 (Strahl et al., 2001; Wang et al., 2001b). This methylation is essential for ERa transcription as it is one of the earliest histone modification marks that ensues after hormone treatment. Histone H4R3 methylation is thought to precede histone acetylation and is considered to be a licensing event on the nucleosome (Strahl et al., 2001; Wang et al., 2001b). CARM1 functions both as a methyltransferases and as an adaptor protein. As a methyl transferase CARM1 methylates histone H3. It does not interact directly with ERa but it is recruited by an SRC coactivator simultaneously with p300. As an adaptor protein, CARM1 acts to recruit BRG1, a member of the ATP-dependent chromatin remodelling complex (Bauer et al., 2002; Bedford and Richard, 2005; Ma et al., 2001; Metivier et al., 2003; Qi et al., 2002; Schurter et al., 2001). The action of methylases is reversed by the action of demethylases such as lysine-specific demethylase LSD1 that removes methylation marks from the histone. The activity of coactivator proteins at the site of impending ERa-mediated transcription creates a pathway for the recruitment of basal transcription factors. RNA polymerase II (RNAPII) binding and initiation of transcription requires the assembly of a complement of basal transcription factors that include TFII A, B, D, E, F and H.

Conclusion Given the important and fundamental roles that SHs have in physiology (and pathology), precise and dynamic regulation of their activity is of paramount importance. Organisms achieve this regulation by employing a myriad of molecular regulatory mechanisms. By regulating the amount and the activity of receptors present at any one time, cells regulate the magnitude and duration of hormonal signalling. Cells regulate the amount and activity of receptors at five distinct levels: (1) by regulating the promoters of receptors; (2) by regulating the stability and translation of receptor

mRNAs; (3) by regulating the stability and degradation of receptor proteins; (4) by modulating SHR activity through post-translational modifications and finally (5) by modulating steroid receptor activity via protein–protein interactions and through the key activities of coactivator molecules. The complexity of SHR regulation and the sheer number of the molecular constituents involved in its regulation serve as a testimony to the importance of SHRs for the survival of an organism. Given their widespread roles in regulating physiology, metabolism and reproduction, it should not be surprising that their misregulation often leads to a number of disease states. Knowledge of the mechanisms that underlie the regulation of SHR action is essential for understanding their roles in homeostasis, in human disease states and in the development of potential diagnostics and therapeutics to modulate their biological functions.

Abbreviations AD AF1 AR AREs CBP CRE CSN CUEDC2 DBD DHT DUB ERa ERE GR GTF HAT HDAC2 JNK LBD miRNA MKK MR NLS

activation domains activation function 1 androgen receptor AU-rich elements cAMP-response elementbinding protein cAMP response element COP9 signalosome CUE domain containing 2 DNA-binding domain dihydroxytestosterone deubiquitinating enzymes estrogen receptor-a estrogen response element glucocorticoid receptor general transcription factor histone acetyltransferase histone deacetylase 2 c-Jun N-terminal kinase ligand-binding region micro-RNAs MAPK kinase mineralocorticoid receptor nuclear localization signal

170

NR PCAF PR PTM RXR SERM SH SHR SRC-1 UPS VDR

nuclear receptor p300/CBP-associated factor progesterone receptor post-translational modification retinoid X receptor selective estrogen receptor modulator steroid hormone steroid hormone receptor steroid receptor coactivator-1 ubiquitin proteasome system vitamin D receptor

References Abdel-Hafiz, H., Dudevoir, M. L., & Horwitz, K. B. (2009). Mechanisms underlying the control of progesterone receptor transcriptional activity by SUMOylation. Journal of Biological Chemistry, 284, 9099–9108. Abdel-Hafiz, H., Takimoto, G. S., Tung, L., & Horwitz, K. B. (2002). The inhibitory function in human progesterone receptor n termini binds SUMO-1 protein to regulate autoinhibition and transrepression. Journal of Biological Chemistry, 277, 33950–33956. Adams, B. D., Furneaux, H., & White, B. A. (2007). The microribonucleic acid (miRNA) miR-206 targets the human estrogen receptor-{alpha} (ER{alpha}) and represses ER{alpha} messenger RNA and protein expression in breast cancer cell lines. Molecular Endocrinology, 21, 1132–1147. Adzic, M., Djordjevic, J., Djordjevic, A., Niciforovic, A., Demonacos, C., Radojcic, M., et al. (2009). Acute or chronic stress induce cell compartment-specific phosphorylation of glucocorticoid receptor and alter its transcriptional activity in wistar rat brain. Journal of Endocrinology, 202, 87–97. Alarid, E. T., Bakopoulos, N., & Solodin, N. (1999). Proteasome-mediated proteolysis of estrogen receptor: A novel component in autologous down-regulation. Molecular Endocrinology, 13, 1522–1534. Almlof, T., Wright, A. P., & Gustafsson, J. A. (1995). Role of acidic and phosphorylated residues in gene activation by the glucocorticoid receptor. The Journal of Biological Chemistry, 270, 17535–17540. Anzick, S. L., Kononen, J., Walker, R. L., Azorsa, D. O., Tanner, M. M., Guan, X.-Y., et al. (1997). AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science, 277, 965–968. Ballinger, C. A., Connell, P., Wu, Y., Hu, Z., Thompson, L. J., Yin, L.-Y., et al. (1999). Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions. Molecular and Cellular Biology, 19, 4535–4545.

Bauer, U. M., Daujat, S., Nielsen, S. J., Nightingale, K., & Kouzarides, T. (2002). Methylation at arginine 17 of histone H3 is linked to gene activation. EMBO Reports, 3, 39–44. Bedford, M. T. & Richard, S. (2005). Arginine methylation: An emerging regulator of protein function. Molecular Cell, 18, 263–272. Bourguet, W., Germain, P., & Gronemeyer, H. (2000). Nuclear receptor ligand-binding domains: Three-dimensional structures, molecular interactions and pharmacological implications. Trends in Pharmacological Sciences, 21, 381–388. Brady, L. S., Whitfield, Jr., Fox, H. J., Gold, R. J., & Herkenham, P. W. M. (1991) Long-term antidepressant administration alters corticotropin-releasing hormone, tyrosine hydroxylase, and mineralocorticoid receptor gene expression in rat brain. Therapeutic implications. Journal of Clinical Investigation 87, 831–837. Breslin, M. B., Geng, C.-D., & Vedeckis, W. V. (2001). Multiple promoters exist in the human GR gene, one of which is activated by glucocorticoids. Molecular Endocrinology, 15, 1381–1395. Burgdorf, S., Leister, P., & Scheidtmann, K. H. (2004). TSG101 interacts with apoptosis-antagonizing transcription factor and enhances androgen receptor-mediated transcription by promoting its monoubiquitination. Journal of Biological Chemistry, 279, 17524–17534. Castles, C. G., Oesterreich, S., Hansen, R., & Fuqua, S. A.W. (1997). Auto-regulation of the estrogen receptor promoter. The Journal of Steroid Biochemistry and Molecular Biology, 62, 155–163. Cavailles, V., Dauvois, S., L’Horset, F., Lopez, G., Hoare, S., Kushner, P. J., et al. (1995). Nuclear factor RIP140 modulates transcriptional activation by the estrogen receptor. EMBO Journal, 14, 3741–3751. Chen, W., Dang, T., Blind, R. D., Wang, Z., Cavasotto, C. N., Hittelman, A. B., et al. (2008). Glucocorticoid receptor phosphorylation differentially affects target gene expression. Molecular Endocrinology, 22, 1754–1766. Chen, H., Lin, R. J., Schiltz, R. L., Chakravarti, D., Nash, A., Nagy, L., et al. (1997). Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300. Cell, 90, 569–580. Chen, D. S., Washbrook, E., Sarwar, N., Bates, G. J., Pace, P. E., Thirunuvakkarasu, V., et al. (2002). Phosphorylation of human estrogen receptor alpha at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera. Oncogene, 21, 4921–4931. Chiba, H., Muramatsu, M., Nomoto, A., & Kato, H. (1994). Two human homologues of saccharomyces cerevisiae SWI2/ SNF2 and drosophila brahma are transcriptional coactivators cooperating with the estrogen receptor and the retinoic acid receptor. Nucleic Acids Research, 22, 1815–1820. Cho, S., Kagan, B. L., Blackford, J. A., Jr., Szapary, D., & Simons, S. S., Jr. (2005). Glucocorticoid receptor ligand binding domain is sufficient for the modulation of glucocorticoid induction properties by homologous receptors, coactivator transcription intermediary factor 2, and ubc9. Molecular Endocrinology, 19, 290–311.

171 Cirillo, L. A., Lin, F. R., Cuesta, I., Friedman, D., Jarnik, M., & Zaret, K. S. (2002). Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4. Molecular Cell, 9, 279–289. Cui, Y., Zhang, M., Pestell, R., Curran, E. M., Welshons, W. V., & Fuqua, S. A.W. (2004). Phosphorylation of estrogen receptor {alpha} blocks its acetylation and regulates estrogen sensitivity. Cancer Research, 64, 9199–9208. Demarest, S. J., Martinez-Yamout, M., Chung, J., Chen, H., Xu, W., Dyson, H. J., et al. (2002). Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators. Nature, 415, 549–553. DiRenzo, J., Shang, Y., Phelan, M., Sif, S., Myers, M., Kingston, R., et al. (2000). BRG-1 is recruited to estrogen-responsive promoters and cooperates with factors involved in histone acetylation. Molecular and Cellular Biology, 20, 7541–7549. Donaghue, C., Westley, B. R., & May, F. E.B. (1999). Selective promoter usage of the human estrogen receptor-{alpha} gene and its regulation by estrogen. Molecular Endocrinology, 13, 1934–1950. Duma, D., Jewell, C. M., & Cidlowski, J. A. (2006). Multiple glucocorticoid receptor isoforms and mechanisms of posttranslational modification. The Journal of Steroid Biochemistry and Molecular Biology, 102, 11–21. Eakin, C. M., MacCoss, M. J., Finney, G. L., & Klevit, R. E. (2007). Estrogen receptor Î+ is a putative substrate for the BRCA1 ubiquitin ligase. Proceedings of the National Academy of Sciences, 104, 5794–5799. Eeckhoute, J. R.M., Carroll, J. S., Geistlinger, T. R., TorresArzayus, M. I., & Brown, M. (2006). A cell-type-specific transcriptional network required for estrogen regulation of cyclin D1 and cell cycle progression in breast cancer. Genes and Development, 20, 2513–2526. Eeckhoute, J., Keeton, E. K., Lupien, M., Krum, S. A., Carroll, J. S., & Brown, M. (2007). Positive cross-regulatory loop ties GATA-3 to estrogen receptor {alpha} expression in breast cancer. Cancer Research, 67, 6477–6483. Evans, R. (1988a). The steroid and thyroid hormone receptor superfamily. Science, 240, 889–895. Evans, R. M. (1988b). The steroid and thyroid hormone receptor superfamily. Science, 240, 889–895. Evans, R. M. (2005). The nuclear receptor superfamily: A rosetta stone for physiology. Molecular Endocrinology, 19, 1429–1438. Fan, M., Bigsby, R. M., & Nephew, K. P. (2003). The NEDD8 pathway is required for proteasome-mediated degradation of human estrogen receptor (ER)-{alpha} and essential for the antiproliferative activity of ICI 182,780 in ER{alpha}-positive breast cancer cells. Molecular Endocrinology, 17, 356–365. Fan, M., Park, A., & Nephew, K. P. (2005). CHIP (carboxyl terminus of hsc70-interacting protein) promotes basal and geldanamycin-induced degradation of estrogen receptor{alpha}. Molecular Endocrinology, 19, 2901–2914. Faus, H. & Haendler, B. (2006a). Post-translational modifications of steroid receptors. Biomedicine and Pharmacotherapy, 60, 520–528.

Faus, H. & Haendler, B. (2006b). Post-translational modifications of steroid receptors. Biomedecine and Pharmacotherapy, 60, 520–528. Flouriot, G., Griffin, C., Kenealy, M., Sonntag-Buck, V., & Gannon, F. (1998). Differentially expressed messenger RNA isoforms of the human estrogen receptor-{alpha} gene are generated by alternative splicing and promoter usage. Molecular Endocrinology, 12, 1939–1954. Fu, M., Rao, M., Wang, C., Sakamaki, T., Wang, J., Di Vizio, D., et al. (2003). Acetylation of androgen receptor enhances coactivator binding and promotes prostate cancer cell growth. Molecular and Cellular Biology, 23, 8563–8575. Fu, M., Wang, C., Reutens, A. T., Wang, J., Angeletti, R. H., Siconolfi-Baez, L., et al. (2000). P300 and p300/cAMP-response element-binding protein-associated factor acetylate the androgen receptor at sites governing hormone-dependent transactivation. Journal of Biological Chemistry, 275, 20853–20860. Galliher-Beckley, A. J., Williams, J. G., Collins, J. B., & Cidlowski, J. A. (2008). Glycogen synthase kinase 3{beta}mediated serine phosphorylation of the human glucocorticoid receptor redirects gene expression profiles. Molecular and Cellular Biology, 28, 7309–7322. Garcia-Pedrero, J. M., Kiskinis, E., Parker, M. G., & Belandia, B. (2006). The SWI/SNF chromatin remodeling subunit BAF57 is a critical regulator of estrogen receptor function in breast cancer cells. The Journal of Biological Chemistry, 281, 22656–22664. Gaughan, L., Logan, I. R., Cook, S., Neal, D. E., & Robson, C. N. (2002). Tip60 and histone deacetylase 1 regulate androgen receptor activity through changes to the acetylation status of the receptor. Journal of Biological Chemistry, 277, 25904–25913. Gioeli, D., Black, B. E., Gordon, V., Spencer, A., Kesler, C. T., Eblen, S. T., et al. (2006). Stress kinase signaling regulates androgen receptor phosphorylation, transcription, and localization. Molecular Endocrinology, 20, 503–515. Gong, J., Zhu, J., Goodman, O. B., Pestell, R. G., Schlegel, P. N., Nanus, D. M., & Shen, R. (2006). Activation of p300 histone acetyltransferase activity and acetylation of the androgen receptor by bombesin in prostate cancer cells. Oncogene. 25, 2011–2021. Gorski, J. & Nicolette, J. A. (1963). Early estrogen effects on newly synthesized RNA and phospholipid in subcellular fractions of rat uteri. Archives of Biochemistry and Biophysics, 103, 418–423. Gottlicher, M., Heck, S., Doucas, V., Wade, E., Kullmann, M., Cato, A. C., et al. (1996). Interaction of the ubc9 human homologue with c-Jun and with the glucocorticoid receptor. Steroids, 61, 257–262. Grandien, K., Backdahl, M., Ljunggren, O., Gustafsson, J., & Berkenstam, A. (1995). Estrogen target tissue determines alternative promoter utilization of the human estrogen receptor gene in osteoblasts and tumor cell lines. Endocrinology, 136, 2223–2229. Grandien, K. F.H., Berkenstam, A., Nilsson, S., & Gustafsson, J.-A. (1993). Localization of DNase i hypersensitive sites in the human oestrogen receptor gene correlates with the

172 transcriptional activity of two differentially used promoters. Journal of Molecular Endocrinology, 10, 269–277. Green, K. A. & Carroll, J. S. (2007). Oestrogen-receptormediated transcription and the influence of co-factors and chromatin state. Nature Reviews Cancer, 7, 713–722. Green, S. & Chambon, P. (1988). Nuclear receptors enhance our understanding of transcription regulation. Trends in Genetics, 4, 309–314. Green, S., Walter, P., Kumar, V., Krust, A., Bornert, J.-M., Argos, P. & Chambon, P. (1986). Human oestrogen receptor cDNA: Sequence, expression and homology to v-erb-A. Nature, 320, 134–139. Greene, G. L., Waterfield, G. P., Baker, M., Hort, A., & Shine, Y. (1986). Sequence and expression of human estrogen receptor complementary DNA. Science, 231, 1150–1154. Griffin, C., Flouriot, G., Sonntag-Buck, V., Nestor, P., & Gannon, F. (1998). Identification of novel chicken estrogen receptor-{alpha} messenger ribonucleic acid isoforms generated by alternative splicing and promoter usage. Endocrinology, 139, 4614–4625. Gustafsson, J. A., Carlstedt-Duke, J., Wrange, O., Okret, S., & Wikstrom, ¨ A. C. (1986). Functional analysis of the purified glucocorticoid receptor. The Journal of Steroid Biochemistry, 24, 63–68. Guyton, A. C. & Hall, J. E. (2006). Textbook of medical physiology. Philadelphia: Elsevier Saunders. Halachmi, S., Marden, E., Martin, G., MacKay, H., Abbondanza, C., & Brown, M. (1994). Estrogen receptor-associated proteins: Possible mediators of hormone-induced transcription. Science, 264, 1455–1458. Halkidou, K., Gnanapragasam, V.J., Mehta, P.B., Logan, I.R., Brady, M.E., Cook, S., et al. (2003). Expression of tip60, an androgen receptor coactivator, and its role in prostate cancer development. Oncogene, 22, 2466-2477. He, B., Bai, S., Hnat, A. T., Kalman, R. I., Minges, J. T., Patterson, C., et al. (2004). An androgen receptor NH2terminal conserved motif interacts with the COOH terminus of the hsp70-interacting protein (CHIP). Journal of Biological Chemistry, 279, 30643–30653. Hershko, A. (1996). Lessons from the discovery ofthe ubiquitin system. Trends in Biochemical Sciences, 21, 445–449. Hershko, A. & Ciechanover, A. (1998). The ubiquitin system. Annual Review of Biochemistry, 67, 425–479. Hollenberg, S. M., Ong, W. C., Cerelli, E. S., Oro, G., Lebo, A., Thompson, R., et al. (1985). Primary structure and expression of a functional human glucocorticoid receptor cDNA. Nature 318, 635–641. Hong, H., Kohli, K., Garabedian, M., & Stallcup, M. (1997). GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin d receptors. Molecular and Cellular Biology, 17, 2735–2744. Ichinose, H., Garnier, J.-M., Chambon, P., & Losson, R. (1997). Ligand-dependent interaction between the estrogen receptor and the human homologues of SWI2/SNF2. Gene, 188, 95–100. Imhof, M. & McDonnell, D. (1996). Yeast RSP5 and its human homolog hRPF1 potentiate hormone-dependent activation

of transcription by human progesterone and glucocorticoid receptors. Molecular and Cellular Biology, 16, 2594–2605. Ing, N. H. (2005). Steroid hormones regulate gene expression posttranscriptionally by altering the stabilities of messenger RNAs. Biology of Reproduction, 72, 1290–1296. Ing, N. H., Massuto, D. A., & Jaeger, L. A. (2008). Estradiol up-regulates AUF1p45 binding to stabilizing regions within the 3’-untranslated region of estrogen receptor {alpha} mRNA. The Journal of Biological Chemistry, 283, 1764–1772. Ismail, A. & Nawaz, Z. (2005). Nuclear hormone receptor degradation and gene transcription: An update. IUBMB Life, 57, 483–490. Ismaili, N. & Garabedian, M. J. (2004a). Modulation of glucocorticoid receptor function via phosphorylation. Annals of the New York Academy of Sciences, 1024, 86–101. Ismaili, N. & Garabedian, M. J. (2004b). Modulation of glucocorticoid receptor function via phosphorylation. Annals of the New York Academy of Sciences, 1024, 86–101. Ito, K., Yamamura, S., Essilfie-Quaye, S., Cosio, B., Ito, M., Barnes, P. J., et al. (2006). Histone deacetylase 2-mediated deacetylation of the glucocorticoid receptor enables NF{kappa}b suppression. The Journal of Experimental Medicine, 203, 7–13. Jacobson, H. I. & Jensen, E. V. (1962). Basic guides to the mechanism of estrogen action. Recent Progress in Hormone Research, 18, 387–414. Jensen, E. V. (1962). On the mechanism of estrogen action. Perspectives in Biology and Medicine, 6, 47–59. Jensen, E. V. & DeSombre, E. R. (1973). Estrogen-receptor interaction: Estrogenic hormones effect transformation of specific receptor proteins to a biochemically functional form. Science, 182(4108), 126–134. Jiang, J., Sarwar, N., Peston, D., Kulinskaya, E., Shousha, S., Coombes, R. C., et al. (2007). Phosphorylation of estrogen receptor-alpha at ser(167) is indicative of longer disease-free and overall survival in breast cancer patients. Clinical Cancer Research, 13, 5769–5776. Kamei, Y., Xu, L., Heinzel, T., Torchia, J., Kurokawa, R., Gloss, B., et al. (1996). A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell, 85, 403–414. Kassabov, S. R., Zhang, B., Persinger, J., & Bartholomew, B. (2003). SWI/SNF unwraps, slides, and rewraps the nucleosome. Molecular Cell, 11, 391–403. Kastner, P., Krust, A., Turcotte, B., Stropp, U., Tora, L., Gronemeyer, H., et al. (1990). Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms a and B. The EMBO Journal, 9, 1603–1614. Keaveney, M., Parker, M. G., & Gannon, F. (1993). Identification of a functional role for the 3’ region of the human oestrogen receptor gene. Journal of Molecular Endocrinology, 10, 143–152. Kenealy, M.-R., Flouriot, G., Sonntag-Buck, V., Dandekar, T., Brand, H., & Gannon, F. (2000). The 30 -untranslated region of the human estrogen receptor {alpha} gene mediates rapid

173 messenger ribonucleic acid turnover. Endocrinology, 141, 2805–2813. Khissiin, A. E. & Leclercq, G. (1999). Implication of proteasome in estrogen receptor degradation. FEBS Letters, 448, 160–166. Kim, M. Y., Hsiao, S. J., & Kraus, W. L. (2001). A role for coactivators and histone acetylation in estrogen receptor alpha-mediated transcription initiation. EMBO Journal, 20, 6084–6094. Kim, M. Y., Woo, E. M., Chong, Y. T.E., Homenko, D. R., & Kraus, W. L. (2006). Acetylation of estrogen receptor {alpha} by p300 at lysines 266 and 268 enhances the deoxyribonucleic acid binding and transactivation activities of the receptor. Molecular Endocrinology, 20, 1479–1493. Klinge, C. M. (2000). Estrogen receptor interaction with coactivators and co-repressors[small star, filled]. Steroids, 65, 227–251. Kobayashi, Y., Kitamoto, T., Masuhiro, Y., Watanabe, M., Kase, T., Metzger, D., et al. (2000). P300 mediates functional synergism between AF-1 and AF-2 of estrogen receptor alpha and beta by interacting directly with the N-terminal A/B domains. The Journal of Biological Chemistry, 275, 15645–15651. Korkmaz, C., Fronsdal, K., Zhang, Y., Lorenzo, P., & Saatcioglu, F. (2004). Potentiation of androgen receptor transcriptional activity by inhibition of histone deacetylation–rescue of transcriptionally compromised mutants. Journal of Endocrinology, 182, 377–389. Kos, M., Reid, G., Denger, S., & Gannon, F. (2001). Minireview: Genomic organization of the human ERalpha gene promoter region. Molecular Endocrinology, 15, 2057–2063. Kouros-Mehr, H., Slorach, E. M., Sternlicht, M. D., & Werb, Z. (2006). GATA-3 maintains the differentiation of the luminal cell fate in the mammary gland. Cell, 127, 1041–1055. Krstic, M. D., Rogatsky, I., Yamamoto, K. R., & Garabedian, M. J. (1997). Mitogen-activated and cyclin-dependent protein kinases selectively and differentially modulate transcriptional enhancement by the glucocorticoid receptor. Molecular and Cellular Biology, 17, 3947–3954. Lange, C. A., Shen, T., & Horwitz, K. B. (2000). Phosphorylation of human progesterone receptors at serine-294 by mitogen-activated protein kinase signals their degradation by the 26s proteasome. Proceedings of the National Academy of Sciences of the United States of America, 97, 1032–1037. Lannigan, D. A. (2003). Estrogen receptor phosphorylation. Steroids, 68, 1–9. Le Douarin, B., Zechel, C., Garnier, J. M., Lutz, Y., Tora, L., Pierrat, B., et al. (1995). The N-terminal part of TIF1, a putative mediator of the ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T18. EMBO Journal, 14, 2020–2033. Le Drean, Y., Mincheneau, N., Le Goff, P., & Michel, D. (2002). Potentiation of glucocorticoid receptor transcriptional activity by sumoylation. Endocrinology, 143, 3482–3489. Lee, J. W., Ryan, F., Swaffield, J. C., Johnston, S. A., & Moore, D. D. (1995). Interaction of thyroid-hormone receptor with a conserved transcriptional mediator. Nature, 374, 91–94.

Li, L., Li, Z., Howley, P. M., & Sacks, D. B. (2006). E6AP and calmodulin reciprocally regulate estrogen receptor stability. The Journal of Biological Chemistry, 281, 1978–1985. Likhite, V. S., Stossi, F., Kim, K., Katzenellenbogen, B. S., & Katzenellenbogen, J. A. (2006). Kinase-specific phosphorylation of the estrogen receptor changes receptor interactions with ligand, deoxyribonucleic acid, and coregulators associated with alterations in estrogen and tamoxifen activity. Molecular Endocrinology, 20, 3120–3132. Listwak, S. J., Gold, P. W., & Whitfield, H. J. (1996). The human mineralocorticoid receptor gene promoter: Its structure and expression. The Journal of Steroid Biochemistry and Molecular Biology, 58, 495–506. Liu, G., Schwartz, J. A., & Brooks, S. C. (2000). Estrogen receptor protects p53 from deactivation by human double minute-2. Cancer Research, 60, 1810–1814. Liu, W.-H., Yeh, S.-H., Lu, C.-C., Yu, S.-L., Chen, H.-Y., Lin, C.-Y., et al. (2009). MicroRNA-18a prevents estrogen receptor-[alpha] expression, promoting proliferation of hepatocellular carcinoma cells. Gastroenterology, 136, 683– 693. Lonard, D. M., Nawaz, Z., Smith, C. L., & O’Malley, B. W. (2000). The 26s proteasome is required for estrogen receptoralpha and coactivator turnover and for efficient estrogen receptor-alpha transactivation. Molecular Cell, 5, 939–948. Lonard, D. M. & O’Malley, B. W. (2007). Nuclear receptor coregulators: Judges, juries, and executioners of cellular regulation. Molecular Cell 27, 691–700. Lonard, D. M. & O’Malley, B. W. (2008a). Gene transcription: Two worlds merged. Nature, 452, 946–947. Lonard, D. M. & O’Malley, B. W. (2008b). SRC-3 transcription-coupled activation, degradation, and the ubiquitin clock: Is there enough coactivator to go around in cells? Science Signaling, 1, pe16. Lu, N. Z. & Cidlowski, J. A. (2006). Glucocorticoid receptor isoforms generate transcription specificity. Trends in Cell Biology, 16, 301–307. Ma, H., Baumann, C. T., Li, H. W., Strahl, B. D., Rice, R., Jelinek, M. A., et al. (2001). Hormone-dependent, CARM1-directed, arginine-specific methylation of histone H3 on a steroid-regulated promoter. Current Biology, 11, 1981–1985. Ma, Y., Katiyar, P., Jones, L. P., Fan, S., Zhang, Y., Furth, P. A., et al. (2006). The breast cancer susceptibility gene BRCA1 regulates progesterone receptor signaling in mammary epithelial cells. Molecular Endocrinology, 20, 14–34. Mallepell, S., Krust, A. E., Chambon, P., & Brisken, C. (2006). Paracrine signaling through the epithelial estrogen receptor Î+ is required for proliferation and morphogenesis in the mammary gland. Proceedings of the National Academy of Sciences of the United States of America, 103, 2196–2201. Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K., et al. (1995). The nuclear receptor superfamily: the second decade. Cell, 83, 835–839. Martinez-Balbas, M. A., Bannister, A. J., Martin, K., HausSeuffert, P., Meisterernst, M., & Kouzarides, T. (1998). The acetyltransferase activity of CBP stimulates transcription. EMBO Journal, 17, 2886–2893.

174 McKenna, N. J., Xu, J., Nawaz, Z., Tsai, S. Y., Tsai, M. J., & O’Malley, B. W. (1999). Nuclear receptor coactivators: Multiple enzymes, multiple complexes, multiple functions. The Journal of Steroid Biochemistry and Molecular Biology, 69, 3–12. Means, A. R., Comstock, J. P., Rosenfeld, G. C., & O’Malley, B. W. (1972). Ovalbumin messenger RNA of chick oviduct: Partial characterization, estrogen dependence, and translation in vitro. Proceedings of the National Academy of Sciences of the United States of America, 69, 1146–1150. Metivier, R., Penot, G., Hubner, M. R., Reid, G., Brand, H., Kos, M., et al. (2003). Estrogen receptor-alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell, 115, 751–763. Miesfeld, R., Okret, S., Wikstrom, A. C., Wrange, O., Gustafsson, J. A., & Yamamoto, K. R. (1984). Characterization of a steroid hormone receptor gene and mRNA in wild-type and mutant cells. Nature, 312, 779–781. Miesfeld, R., Rusconi, S., Godowski, P. J., Maler, B. A., Sample, O., Wikstrom, ¨ A.-C., et al. (1986). Genetic complementation of a glucocorticoid receptor deficiency by expression of cloned receptor cDNA. Cell, 46, 389–399. Mukhopadhyay, N. K., Kim, J., Cinar, B., Ramachandran, A., Hager, M. H., Di Vizio, D., et al. (2009). Heterogeneous nuclear ribonucleoprotein k is a novel regulator of androgen receptor translation. Cancer Research, 69, 2210–2218. Murphy, L. C., Weitsman, G. E., Skliris, G. P., Teh, E. M., Li, L., Peng, B., et al. (2006). Potential role of estrogen receptor ot (ER alpha) phosphorylated at Serine(118) in human breast cancer in vivo. The Journal of Steroid Biochemistry and Molecular Biology, 102, Issues 1-5, pp. 139–146. Nader, N., Chrousos, G. P., & Kino, T. (2009). Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: Potential physiological implications. The FASEB Journal, 23, 1572–1583. Nagy, L. & Schwabe, J. W.R. (2004). Mechanism of the nuclear receptor molecular switch. Trends in Biochemical Sciences, 29, 317–324. Nandi, D., Tahiliani, P., Kumar, A., & Chandu, D. (2006). The ubiquitin-proteasome system. Journal of Biosciences, 31, 137–155. Nardulli, A. M. & Katzellenbogen, B. S. (1988). Progesterone receptor regulation in T47D human breast cancer cells: Analysis by density labeling of progesterone receptor synthesis and degradation and their modulation by progestin. Endocrinology, 122, 1532–1540. Nawaz, Z., Lonard, D. M., Dennis, A. P., Smith, C. L., & O’Malley, B. W. (1999a). Proteasome-dependent degradation of the human estrogen receptor. Proceedings of the National Academy of Sciences of the United States of America, 96, 1858–1862. Nawaz, Z., Lonard, D. M., Smith, C. L., Lev-Lehman, E., Tsai, S. Y., Tsai, M.-J., et al. (1999b). The angelman syndromeassociated protein, E6-AP, is a coactivator for the nuclear

hormone receptor superfamily. Molecular and Cellular Biology, 19, 1182–1189. Nishida, T. & Yasuda, H. (2002). PIAS1 and PIASxÎ+ function as SUMO-E3 ligases toward androgen receptor and repress androgen receptor-dependent transcription. Journal of Biological Chemistry, 277, 41311–41317. Nobukuni, Y., Smith, C. L., Hager, G. L., & Detera-Wadleigh, S. D. (2002). Characterization of the human glucocorticoid receptor promoter. Biochemistry, 34, 8207–8214. Oñate, S. A., Tsai, S. Y., Tsai, M.-J., & O’Malley, B. W. (1995). Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science, 270, 1354–1357. O’Malley, B. W. & Means, A. R. (1974). Female steroid hormones and target cell nuclei: The effects of steroid hormones on target cell nuclei are of major importance in the induction of new cell functions. Science, 183, 610–620. Pandey, D. P. & Picard, D. (2009). MiR-22 inhibits estrogen signaling by directly targeting the estrogen receptor {alpha} mRNA. Molecular and Cellular Biology, 29(13), 3783–3790. Pike, A. C.W. (2006). Lessons learnt from structural studies of the oestrogen receptor. Best Practice and Research Clinical Endocrinology and Metabolism, 20, 1–14. Pinzone, J. J., Stevenson, H., Strobl, J. S., & Berg, P. E. (2004). Molecular and cellular determinants of estrogen receptor alpha expression. Molecular and Cellular Biology, 24, 4605–4612. Ponglikitmongkol, S. G. & Chambon, P. (1988). Genomic organization of the human oestrogen receptor gene. The EMBO Journal, 7, 3385–3388. Poukka, H., Aarnisalo, P., Karvonen, U., & Palvimo, J. (1999). Ubc9 interacts with the androgen receptor and activates receptor-dependent transcription. Journal of Biological Chemistry, 274, 19441–19446. Poukka, H., Karvonen, U., Janne, O. A., & Palvimo, J. J. (2000). Covalent modification of the androgen receptor by small ubiquitin-like modifier 1 (SUMO-1). Proceedings of the National Academy of Sciences of the United States of America, 97, 14145–14150. Qi, C., Chang, J., Zhu, Y. W., Yeldandi, A. V., Rao, S. M., & Zhu, Y. J. (2002). Identification of protein arginine methyltransferase 2 as a coactivator for estrogen receptor alpha. Journal of Biological Chemistry, 277, 28624–28630. Qiu, M. & Lange, C. A. (2003). MAP kinases couple multiple functions of human progesterone receptors: Degradation, transcriptional synergy, and nuclear association. The Journal of Steroid Biochemistry and Molecular Biology, 85, 147–157. Reid, G., Hubner, M. R., Metivier, R., Brand, H., Denger, S., Manu, D., et al. (2003). Cyclic, proteasome-mediated turnover of unliganded and liganded ERalpha on responsive promoters is an integral feature of estrogen signaling. Molecular Cell, 11, 695–707. Saji, S., Okumura, N., Eguchi, H., Nakashima, S., Suzuki, A., Toi, M., et al. (2001). MDM2 enhances the function of estrogen receptor alpha in human breast cancer cells. Biochemical and Biophysical Research Communications, 281, 259–265.

175 Santos-Rosa, H., Valls, E., Kouzarides, T., & Martinez-Balbas, M. (2003). Mechanisms of P/CAF auto-acetylation. Nucleic Acids Research, 31, 4285–4292. Schaaf, M. J.M. & Cidlowski, J. A. (2002). AUUUA motifs in the 3’UTR of human glucocorticoid receptor [alpha] and [beta] mRNA destabilize mRNA and decrease receptor protein expression. Steroids, 67, 627–636. Schiltz, R. L., Mizzen, C. A., Vassilev, A., Cook, R. G., Allis, C. D., & Nakatani, Y. (1999). Overlapping but distinct patterns of histone acetylation by the human coactivators p300 and PCAF within nucleosomal substrates. The Journal of Biological Chemistry, 274, 1189–1192. Schurter, B. T., Koh, S. S., Chen, D., Bunick, G. J., Harp, J. M., Hanson, B. L., et al. (2001). Methylation of histone H3 by coactivator-associated arginine methyltransferase 1. Biochemistry, 40, 5747–5756. Sentis, S., Le Romancer, M., Bianchin, C., Rostan, M.-C., & Corbo, L. (2005). Sumoylation of the estrogen receptor {alpha} hinge region regulates its transcriptional activity. Molecular Endocrinology, 19, 2671–2684. Sharma, M., Li, X., Wang, Y., Zarnegar, M., Huang, C.-Y., Palvimo, J. J., et al. (2003). HZimp10 is an androgen receptor co-activator and forms a complex with SUMO-1 at replication foci. The EMBO Journal, 22, 6101–6114. Spelsberg, T. C., Steggles, A. W., Chytil, F., & O’Malley, B. W. (1972). Progesterone-binding components of chick oviduct. V. Exchange of progesterone-binding capacity from target to nontarget tissue chromatins. The Journal of Biological Chemistry, 247, 1368–1374. Spencer, T. E., Jenster, G., Burcin, M. M., Allis, C. D., Zhou, J., Mizzen, C. A., et al. (1997). Steroid receptor coactivator-1 is a histone acetyltransferase. Nature, 389, 194–198. Stanisic, V., Malovannaya, A., Qin, J., Lonard, D. M., & O’Malley, B. W. (2009). OTU-domain containing ubiquitin aldehyde binding protein 1 (OTUB1) deubiquitinates estrogen receptor - alpha (ERalpha) and affects ERalpha transcriptional activity. The Journal of Biological Chemistry, 284, 16135–16145. M109.007484. Stenoien, D. L., Patel, K., Mancini, M. G., Dutertre, M., Smith, C. L., O’Malley, B. W., et al. (2001). FRAP reveals that mobility of oestrogen receptor-[alpha] is ligand- and proteasome-dependent. Nature Cell Biology, 3, 15–23. Strahl, B. D., Briggs, S. D., Brame, C. J., Caldwell, J. A., Koh, S. S., Ma, H., et al. (2001). Methylation of histone H4 at arginine 3 occurs in vivo and is mediated by the nuclear receptor coactivator PRMT1. Current Biology, 11, 996–1000. Subramanian, K., Jia, D., Kapoor-Vazirani, P., Powell, D. R., Collins, R. E., Sharma, D., et al. (2008). Regulation of estrogen receptor [alpha] by the SET7 lysine methyltransferase. Molecular Cell, 30, 336–347. Tateishi, Y., Kawabe, Y., Chiba, T., Murata, S., Ichikawa, K., Murayama, A., et al. (2004). Ligand-dependent switching of ubiquitin-proteasome pathways for estrogen receptor. The EMBO Journal, 23, 4813–4823. Tateishi, Y., Sonoo, R., Sekiya, Y.-I., Sunahara, N., Kawano, M., Wayama, M., et al. (2006). Turning off estrogen receptor {beta}-

mediated transcription requires estrogen-dependent receptor proteolysis. Molecular and Cellular Biology, 26, 7966–7976. Thomas, M., Dadgar, N., Aphale, A., Harrell, J. M., Kunkel, R., Pratt, W. B., et al. (2004). Androgen receptor acetylation site mutations cause trafficking defects, misfolding, and aggregation Similar To expanded glutamine tracts. Journal of Biological Chemistry, 279, 8389–8395. Thomas, R. S., Sarwar, N., Phoenix, F., Coombes, R. C., & Ali, S. (2008). Phosphorylation at serines 104 and 106 by erk1/2 MAPK is important for estrogen receptor-alpha activity. Journal of Molecular Endocrinology, 40, 173–184. Thorat, M. A., Marchio, C., Morimiya, A., Savage, K., Nakshatri, H., Reis-Filho, J. S., et al. (2008). Forkhead box A1 expression in breast cancer is associated with luminal subtype and good prognosis. Journal of Clinical Pathology, 61, 327–332. Toft, D. & Gorski, J. (1966). A receptor molecule for estrogens: Isolation from the rat uterus and preliminary characterization. Proceedings of the National Academy of Sciences of the United States of America, 55, 1574–1581. Torchia, J., Glass, C., & Rosenfeld, M. G. (1998). Co-activators and co-repressors in the integration of transcriptional responses. Current Opinion in Cell Biology, 10, 373–383. Treilleux, I., Peloux, N., Brown, M., & Sergeant, A. (1997). Human estrogen receptor (ER) gene promoter-P1: Estradiol-independent activity and estradiol inducibility in ERþ and ER- cells. Molecular Endocrinology, 11, 1319–1331. Tsai, S.-Y., Tsai, M.-J., Schwartz, R., Kalimi, M., Clark, J. H., & O’Malley, B. W. (1975). Effects of estrogen on gene expression in chick oviduct: Nuclear receptor levels and initiation of transcription. Proceedings of the National Academy of Sciences of the United States of America, 72, 4228–4232. Verma, S., Ismail, A., Gao, X., Fu, G., Li, X., O’Malley, B. W., et al. (2004). The ubiquitin-conjugating enzyme UBCH7 acts as a coactivator for steroid hormone receptors. Molecular and Cellular Biology, 24, 8716–8726. Voet, D. & Voet, J. G. (2004). Biochemistry. New York: J. Wiley & Sons. Vreugdenhil, E., Verissimo, C. S.L., Mariman, R., Kamphorst, J. T., Barbosa, J. S., Zweers, T., et al. (2009). MicroRNA 18 and 124a down-regulate the glucocorticoid receptor: Implications for glucocorticoid responsiveness in the brain. Endocrinology, 150, 2220–2228. Wallace, A. D. & Cidlowski, J. A. (2001). Proteasomemediated glucocorticoid receptor degradation restricts transcriptional signaling by glucocorticoids. Journal of Biological Chemistry, 276, 42714–42721. Walter, P., Green, S., Greene, G., Krust, A., Bornert, J. M., Jeltsch, J. M., et al. (1985). Cloning of the human estrogen receptor cDNA. Proceedings of the National Academy of Sciences of the United States of America, 82, 7889–7893. Waltering, K. K., Wallen, M. J., Tammela, T. L., Vessella, R. L., & Visakorpi, T. (2006a). Mutation screening of the androgen receptor promoter and untranslated regions in prostate cancer. The Prostate, 66, 1585–1591. Waltering, K. K., Wallén, M. J., Tammela, T. L.J., Vessella, R. L., & Visakorpi, T. (2006b). Mutation screening of the

176 androgen receptor promoter and untranslated regions in prostate cancer. The Prostate, 66, 1585–1591. Wang, C., Fu, M., Angeletti, R. H., Siconolfi-Baez, L., Reutens, A. T., Albanese, C., et al. (2001a). Direct acetylation of the estrogen receptor alpha hinge region by p300 regulates transactivation and hormone sensitivity. The Journal of Biological Chemistry, 276, 18375–18383. Wang, H. B., Huang, Z. Q., Xia, L., Feng, Q., ErdjumentBromage, H., Strahl, B. D., et al. (2001b). Methylation of histone H4 at arginine 3 facilitating transcriptional activation by nuclear hormone receptor. Science, 293, 853–857. Webb, P., Nguyen, P., Shinsako, J., Anderson, C., Feng, W., Nguyen, M. P., et al. (1998). Estrogen receptor activation function 1 works by binding p160 coactivator proteins. Molecular Endocrinology, 12, 1605–1618. Wijayaratne, A. L. & McDonnell, D. P. (2001). The human estrogen receptor-alpha is a ubiquitinated protein whose stability is affected differentially by agonists, antagonists, and selective estrogen receptor modulators. The Journal of Biological Chemistry, 276, 35684–35692. Wilce, J. A., Leedman, P. J., & Wilce, M. C.J. (2002). RNAbinding proteins that target the androgen receptor mRNA. IUBMB Life, 54, 345–349. Wu, R. C., Feng, Q., Lonard, D. M., & O’Malley, B. W. (2007). SRC-3 coactivator functional lifetime is regulated by a phospho-dependent ubiquitin time clock. Cell, 129, 1125–1140. Xu, W. T., Edmondson, D. G., & Roth, S. Y. (1998). Mammalian GCN5 and P/CAF acetyltransferases have homologous

amino-terminal domains important for recognition of nucleosomal substrates. Molecular and Cellular Biology, 18, 5659–5669. Yudt, M. R. & Cidlowski, J. A. (2002). The glucocorticoid receptor: Coding a diversity of proteins and responses through a single gene. Molecular Endocrinology, 16, 1719– 1726. Zhang, Y., Leung, D. Y., Nordeen, S. K., & Goleva, E. (2009). Estrogen inhibits glucocorticoid action via protein phosphatase 5 (PP5)-mediated glucocorticoid receptor dephosphorylation. The Journal of Biological Chemistry, 284, 24542– 24552. Zhang, P.-J., Zhao, J., Li, H.-Y., Man, J.-H., He, K., Zhou, T., et al. (2007). CUE domain containing 2 regulates degradation of progesterone receptor by ubiquitin-proteasome. The EMBO Journal, 26, 1831–1842. Zhao, C., Dahlman-Wright, K., & Gustafsson, J. A. (2008a). Estrogen receptor beta: An overview and update. Nuclear Receptor Signaling, 6, e003. Zhao, J.-J., Lin, J., Yang, H., Kong, W., He, L., Ma, X., et al. (2008b). MicroRNA-221/222 negatively regulates estrogen receptor{alpha} and is associated with tamoxifen resistance in breast cancer. The Journal of Biological Chemistry, 283, 31079–31086. Zheng, L., Annab, L. A., Afshari, C. A., Lee, W. H., & Boyer, T. G. (2001). BRCA1 mediates ligand-independent transcriptional repression of the estrogen receptor. Proceedings of the National Academy of Sciences of the United States of America, 98, 9587–9592.

L. Martini (Eds.) Progress in Brain Research, Vol. 181 ISSN: 0079-6123 Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 10

The intracrine sex steroid biosynthesis pathways Van Luu-The
and Fernand Labrie Oncology, Molecular Endocrinology and Human Genomics Research Center (CREMOGH), Department of Molecular Medicine, Laval University and Laval University Hospital Research Center (CRCHUL), Quebec, Canada

Abstract: There is an increasing number of differences reported between the steroidogenesis pathways described in the traditional literature related to gonadal steroidogenesis and the more recent observations achieved using new technologies, especially molecular cloning, pangenomic expression studies, precise quantification of mRNA expression using real-time PCR, use of steroidogenic enzymes stably transfected in cells, detailed enzymatic activity analysis in cultured cell lines and mass spectrometry analysis of steroids. The objective of this chapter is to present steroidogenesis in the light of new findings that demonstrate pathways of biosynthesis of estradiol (E2) and dihydrotestosterone (DHT) from adrenal dehydroepiandrosterone (DHEA) in peripheral intracrine tissues which do not involve testosterone as intermediate as classically found in the testis and ovary. Steroidogenic enzymes different from those of the ovary and testis act in a tissue-specific manner to catalyze the transformation of DHEA into active sex steroids. These new pathways are especially important in post-menopausal women where all estrogens and practically all androgens are made at their site of action in peripheral tissues from DHEA, the precursor of adrenal origin. In men, on the other hand, from 40 to 50% of androgens are made in peripheral tissues from adrenal DHEA, thus indicating the major importance of the intracrine pathways in both men and women. We also examine the molecular evolution of steroidogenic enzymes which explains the major differences in steroid metabolism observed between laboratory animals and humans.

Keywords: steroidogenesis; menopause; dehydroepiandrosterone; estrogens; androgens

now well established that an additional source of sex steroids plays a major role in humans of both sexes (Fig. 1, right). For example, in men who have their testicles surgically removed or who are treated with gonadotropin-releasing hormone (GnRH) agonists that completely block testicular androgen secretion (Labrie et al., 1980), it is observed that while the blood levels of testosterone are reduced by 95–97%, the concentrations of intra-prostatic dihydrotestosterone (DHT) is only decreased

Introduction In addition to the sex steroids of gonadal origin that are synthesized in the testicles and ovaries before being released in the blood in order to reach all tissues to exert their action in the classical endocrine manner (Fig. 1, left), it is


Corresponding author. Tel.: +418-654-2296; Fax: +418-654-2761; E-mail: [email protected]

DOI: 10.1016/S0079-6123(08)81010-2

177

178

GnRH CRH

LH ACTH

Testis

Testo

Adrenal gland Circulation DHEA

Testo

4-dione

Circulation

E2 E1

A-dione

E2

DHT

E1S

ADT-G + 3α-diol-G

Circulation

Ovary

Peripheral target tissues Circulation Fig. 1. Schematic representation of the ovarian, testicular and adrenal sources of sex steroids or global sex steroid availability in women and men. After menopause, the secretion of E2 by the ovaries ceases. Consequently, in post-menopausal women, estrogens and nearly all androgens are made locally in peripheral target intracrine tissues. The pre- and post-menopausal ovary secretes small amounts of testo directly into the circulation, where it has an inhibitory effect (–) on GnRH secretion in the brain. Much larger amounts are secreted by the testis. Conversely, the adrenal glands – as well as secreting cortisol that decreases CRH secretion which stimulates ACTH levels – secrete large amounts of DHEA. This inactive precursor is converted in specific target tissues into androgens and/or estrogens via the process of intracrinology. Only small amounts of these peripherally made sex steroids diffuse into the circulation, thus avoiding the possibility of gaining information about the intra-cellular made and active sex steroids. The active androgens and estrogens are inactivated locally before being released in the circulation. The intra-cellular androgens are metabolized into the metabolites ADT and 3a-diol which are then further transformed into the more water-soluble glucuronide derivatives and released into the blood where they can be measured as parameter of total androgenic activity. Abbreviations: ACTH, adrenocorticotropin; CRH, corticotrophin-releasing hormone; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; E1, estrone; E1S, estrone sulphate; E2, estradiol; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; testo, testosterone; ADT-G, androsterone glucuronide; 3a-diol-G, 3a-diol-3 or 17-glucuronide.

by an average of 40% as shown by the various data available in the literature (Labrie, 2007; Labrie et al., 1985). Such findings clearly indicate the existence of an important local

biosynthesis of androgens in the prostate (Fig. 1). Most of the other peripheral tissues such as skin, liver and adipose tissue also show local biosynthesis of sex steroids that exert their

179

action locally in an intracrine manner (Labrie, 1991; Labrie et al., 2005). In other words, the action of the sex steroids made from dehydroepiandrosterone (DHEA) is exerted in the same cells where the steroids are synthesized and inactivated (Labrie, 1991). The role of intracrinology or local biosynthesis of sex steroids from adrenal DHEA is even more dramatic in women where, after menopause, all estrogens and practically all androgens are made from DHEA in a tissue-specific manner by the process of intracrinology (Labrie et al., 2005). The importance of the peripheral formation of estrogens and androgens from DHEA has recently been clearly demonstrated in post-menopausal women where a rapid correction of all their symptoms and signs of vaginal atrophy accompanied by improved sexual function was achieved rapidly by intravaginal DHEA administration with no change in the blood levels of estrogens and androgens (Labrie et al., 2008, 2009a, 2009b, 2009c). There are, moreover, a series of data that indicate low DHEA as responsible for the other problems of

menopause related to hormonal deficiency (Labrie, 2007). The local steroid biosynthesis of sex steroids by the process of intracrinology in peripheral tissues in humans is supported by the cloning and characterization of all the enzymes required which include types 1 (Dumont et al., 1992b; Lachance et al., 1990; Lorence et al., 1990; Luu-The et al., 1989) and 2 (Lachance et al., 1991; Rheaume et al., 1991) 3b-hydroxysteroid dehydrogenases (3b-HSDs), types 1, 2, 3, 5, 7, 8, 12, 14 and 15 17b-HSDs (Bellemare et al., 2009; Dufort et al., 1999; Geissler et al., 1994; Krazeisen et al., 1999; Labrie et al., 1995; Luu-The, 2001; Luu-The et al., 2006; Luu The et al., 1989, 1990a; Peltoketo et al., 1988; Wu et al., 1993) as well as types 1, 2 and 3 5a-reductases (Andersson and Russell, 1990; Andersson et al., 1991; Labrie et al., 1992; Uemura et al., 2008; Yamana et al., 2009) that are expressed in a tissue-specific manner (Fig. 2). The virilization of boys having a deficit in type 2 3b-HSD and data in men with aromatase deficiency (Bilezikian et al., 1998; Carani et al., 1997; Morishima et al., 1995) also

DHEA-S Sulf

DHEA

17β-HSD 1

5-diol

17β-HSD 2, 4

3β-HSD

3β HSD

17β-HSD 3, 5

4-dione 5α-red 1, 2

Testo 17β-HSD 2, 10 5α-red 1, 2 17β-HSD-3, 5, 15

A-dione

DHT 17β-HSD 2, 9, 10

3α-HSD

Aromatase

3α-HSD 17β-HSD 3, 5, 15

ADT

3α-diol

Aromatase

17β-HSD 2, 9, 10, 14

17β-HSD 1, 7, 12

E1

E2

17β-HSD 2, 4, 8, 14

Fig. 2. Schematic representation of the complete steroidogenic endocrine and intracrine pathways in human gonadal and peripheral tissues, respectively.

180

and 12 17b-HSDs (Figs. 2 and 3). These genes share a percentage of amino acid sequence homology of less than 70% and possess the same genomic structure. The function and activity of these genes are somewhat conserved, although the amino acid sequences show large differences. It is worth noting that, in general, the rodent and human genes share a percentage of homology between 70 and 80%. In addition, it should be mentioned that some gene families possess different copy numbers and tissue distribution. Consequently, different steroid metabolic profiles are expected between laboratory animals and humans, in addition to differences in the level of activity of the enzymes present. 2. The ‘conserved structures–different activities’ class is composed of genes that are more recently duplicated, namely, after the separation between rodent and human, such as the types 1 and 2 3b-HSD that share 94% amino acid identity, many of the enzymes of the aldo-keto reductase family, such as type 5 17bHSD, types 1 and 3 3a-HSDs, 20a-HSD and the UDP-glucuronosyl transferase (UGT1A family). These genes share a percentage of homology of more than 80% but encode proteins having different activities. For example, human type 5 17b-HSD and types 1 and 3 3a-HSD share 88% amino acid identity

confirm the importance of local and tissue-specific biosynthesis of sex steroids.

Molecular evolution and different classes of steroidogenic enzymes Structural studies of the enzymes involved in the formation and degradation of sex steroids indicate that many steroidogenic enzyme families are generated by duplication and divergence such as observed for the 3b-HSDs, 5areductases, 3a-HSDs and uridine diphosphate (UDP)-glucuronosyl transferases (UGTs) (Baker, 2001b; Baker, Luu-The, Simard, & Labrie, 1990), while others such as some 17bHSDs are generated by convergent evolution of activity (Baker, 2001a). In addition, it can be noticed that the enzymes responsible for the formation of estrogens and androgens in peripheral tissues differ markedly between species (Luu-The, 2001). The steroidogenic enzymes can be divided into three molecular evolutionary classes (Dufort et al., 2001; Luu-The, 2001; Luu-The et al., 2001): 1. The class of ‘conserved structures–conserved activities’ is composed of genes that are duplicated before divergence between rodent and human. This class includes the genes encoding types 1 and 2 5a-reductases, types 3

Molecular evolution of steroidogenic enzymes

Species Enzymes (amino acid identity %)

800

Rat and mouse Monkey Human

Vertebrates 17β-HSDs

Types 3 & 12 17β-HSD 5α-reductases

20

600

Aldo-keto reductases + 3β-HSD

45

400

60

200

70–80 94 100

100

Molecular evolution scale (millions years) Fig. 3. Schematic representation of the molecular evolution of steroidogenic enzymes.

181

but catalyze different activities. Similarly, although type 3 3a-HSD and 20a-HSD share 97% amino acid identity, they possess different substrate specificities. Similar structure–activity relationships are also observed with genes of the UGT1A family. The genes belonging to this evolutionary class encode the main enzymes responsible for the different profiles of steroid metabolism between humans and rodents. In fact, many enzymes existing in humans do not even have their equivalent in rodents. 3. The ‘different structures–conserved activity’ class represents enzymes derived from genes having convergent evolution of activity such as those encoding 17b-HSDs (Baker, 2001a), with the exception of types 3 and 12 17b-HSD that are duplicated genes (Luu-The et al., 2006). These genes share very low homology (less than 25%) and are derived from ancestral genes having different structures and functions. However, all of the enzymes encoded by these genes catalyze the oxidation or reduction of the keto and hydroxyl groups, respectively, at position 17 of the steroid nucleus. These enzymes play a crucial role in the activation and inactivation of androgens and estrogens. It is noteworthy that some genes encode enzymes specific for C19-substrates such as types 3, 5, 10, 11, 14 and 15 17b-HSDs, while others are specific for C18-substrates such as types 1, 7, 8 and 12 17b-HSDs. On the other hand, type 2 17b-HSD is able to catalyze enzymatic reactions of both C18- and C19-substrates. Many of these enzymes are also capable of transforming substrates other than androgens and estrogens. This ability seems to be acquired from ancestral functions existing in unicellular organisms (yeast) and invertebrates (Caenorhabditis elegans) where androgens and estrogens are not functional because of the lack of androgen and estrogen receptors. Indeed, androgen and estrogen receptors and their ligands only appeared just before or during the pre-cambrian period (Baker, 1997; Bertrand et al., 2004; Laudet, 1997).

Does convergent evolution result from modification of an old enzymatic structure or an adaptation of existing activities of a multisubstrate enzyme to a new substrate? Example of type 12 17b-HSD We have recently isolated and characterized orthologs of type 12 17b-HSD in many species including the nematode C. elegans (Desnoyers et al., 2007), mouse (Blanchard and Luu-The, 2007), monkey (Liu and Zheng et al., 2007) and human (Luu-The et al., 2006). The data obtained show that the gene encoding type 12 17b-HSD is highly conserved with a common ancestor found in C. elegans, namely, LET-767. This ancestral gene leads, in addition to type 12 17b-HSD, to type 3 17b-HSD, a key enzyme in steroidogenesis in the testis. The deficiency of type 3 17b-HSD impairs testosterone biosynthesis in the human testis and is the cause of male pseudohermaphroditism in boys having the mutated gene (Geissler et al., 1994). LET-767 in C. elegans possesses 42 and 40% amino acid identity with human types 3 and 12 17b-HSDs, respectively. The expression of LET-767 in HEK-293 cells shows that the enzyme is able to catalyze the conversion of estrone into estradiol (activity of type 12 17b-HSD) as well as androstenedione into testosterone (activity of type 3 17b-HSD). It is worth noting that estradiol and testosterone are not functional and most probably do not exist in C. elegans which does not possess androgen and estrogen receptors (Baker, 1997; Laudet, 1997). Mutations that inactivate LET-767 lead to a growth inhibition and a reduction of the size of the nematode similar to the observations made in wild-type C. elegans cultured in the absence of cholesterol, thus suggesting that LET-767 could be involved in cholesterol metabolism (Kuervers et al., 2003). Others have shown that C. elegans having inactivated LET-767 have impaired longchain fatty acid biosynthesis (Entchev et al., 2008). Characterization and comparison of the mouse (Blanchard and Luu-The, 2007) and monkey (Liu and Zheng et al., 2007) type 12 17b-HSDs with the human enzyme (Luu-The et al., 2006) show that while the monkey enzyme is estrogen-specific

182

similar to the human enzyme, the mouse enzyme is able to catalyze both androgen and estrogen substrates, as observed with LET-767. Using molecular modelization confirmed by site-directed mutagenesis, we have identified the involvement of amino acid 234 in the steric hindrance of the entrance of C18- and C19-steroids (Luu-The et al., 2006). Indeed, in the monkey and human enzymes, this amino acid is a phenylalanine, a voluminous amino acid that prevents the entrance of C19-steroids, thus conferring type 12 17b-HSD selectivity for estrogens. On the other hand, in the C. elegans and mouse, the corresponding amino acids are methionine and leucine, respectively. These amino acids are less voluminous than phenylalanine and allow the access of androgens, as well as estrogens, to the binding site. In a study performed in pre-adipocytes and differentiated adipocytes, Bellemare et al. (Bellemare et al., 2009) have shown that type 12 17b-HSD expression is significantly increased in differentiated adipocytes. This increase in expression levels of type 12 17b-HSD corresponds to the increase in the ability to catalyze the transformation of estrone into estradiol in differentiated adipocytes. Since the expression of the two other estrogenic 17b-HSDs, namely, types 1 and 7 17b-HSD, are not increased under those circumstances, the data strongly suggest that type 12 17b-HSD is responsible for the increased conversion of estrone into estradiol in differentiated adipocytes. The ability of LET-767 to catalyze the formation of estradiol and testosterone, although these compounds are not functional in C. elegans, suggests that the convergent evolution of 17b-HSD activities is due to an adaptation of an existing activity of a multi-substrate enzyme to a newly appearing substrate rather than the evolution of an old enzymatic structure to accommodate a new substrate. It also suggests that if two or more substrates are present in a multi-cellular organism, the enzyme could exert multiple functions, depending upon the intra-cellular environment. This is most probably the case of the vertebrate type 12 17b-HSD that possesses dual activities, namely, an ancestral 3-ketoacyl-CoA reductase

activity involved in the elongation of long-chain fatty acids, and a more recent type 12 17b-HSD activity involved in the formation of estradiol and testosterone in the mouse and estradiol in the monkey and human. The bifunctional activity of type 12 17b-HSD is in agreement with data obtained by our group showing embryonic lethality of the mouse having homozygous deletion of the type 12 17b-HSD gene (HSD17B12/), while the heterozygous deletion leads to a reduction of androgen and estrogen levels (Bellemare et al., 2009). The cause of embryonic lethality is possibly due to impairment of the formation of long-chain fatty acids, since mice having aromatase (Fisher et al., 1998) or estrogen receptor (Korach, 1994) genes deleted do not show embryonic lethality. It is noteworthy that while the mouse type 12 17b-HSD is able to efficiently catalyze the transformation of both androgen and estrogen substrates (Blanchard and Luu-The, 2007), the human enzyme is more selective for estrogen (Luu-The et al., 2006). This difference in type 12 17b-HSD activity contributes to the important difference of steroid metabolism between laboratory animals and humans. Another example of convergent evolution in steroidogenic enzymes, in which an ancestor gene is able to catalyze the transformation of a more recent substrate, is the gene det2 (deetiolated 2) of Arabidopsis thaliana and type 2 5areductase. DET2 is a gene that encodes a protein that transforms campesterol (a brassinoid) into campestanol (Noguchi et al., 1999) and also plays an important role in light-regulated development in arabidopsis as well as in cotton fiber cell initiation and elongation (Luo et al., 2007). When expressed in HEK-293 cells, this enzyme is also able to catalyze the transformation of testosterone into dihydrotestosterone (Li et al., 1997), a reaction catalyzed by vertebrate 5a-reductase. On the other hand, human type 2 5a-reductase expressed in det2 mutant plants can substitute for DET2 in brassinosteroid biosynthesis (Li et al., 1997). Other studies based upon sequence homology suggest that types 3 and 12 17b-HSD are orthologs of the gene YBR159w in yeast Saccharomyces cerevisiae described by Moon and Horton (Moon

183

and Horton, 2003). The enzyme encoded by this gene in yeast possesses 3-ketoacyl-CoA reductase activity involved in the elongation of long-chain fatty acids. The same authors have shown that the human type 12 17b-HSD expressed in HEK-293 cells is able to catalyze 3-ketoacyl-CoA reductase activity involved in the transformation of palmitic acid into stearic acid.

Marked differences of steroidogenic enzymes between humans, monkeys and rodents Since sex steroids characterized by the presence of a 17b-hydroxy group and their corresponding androgen and estrogen receptors appeared with the vertebrates some 400,000 million years ago (Colbert and Morales, 1991) (Fig. 3), the common ancestor genes found in yeast, plants, worms and insects from which members of 17b-HSD family are derived should be very different and, most probably, are unlikely to exert the same activities and roles. Taking into account the molecular evolutionary aspects mentioned above that create differences in activity (amino acid changes in the coding region) or tissue distribution (especially nucleotide change (s) in the promoter region), it seems of interest to compare some human, monkey and rodent steroidogenic enzymes (Table 1). It can be seen that marked differences are observed, not only in activity but also in the tissue distribution of the enzymes. It can be seen in Table 1 that types 1 and 12 17b-HSD selectively catalyze the transformation of E1 into E2 in human (Dumont et al., 1992a; Luu-The et al., 1990b, 2006), while, in the rodent, these two enzymes catalyze, in addition, the transformation of 4-dione into testo (Blanchard and Luu-The, 2007; Nokelainen et al., 1996). On the other hand, human type 5 17b-HSD very poorly catalyzes the transformation of E1 into E2 (Dufort et al., 1999) while this activity is high with the mouse enzyme (Deyashiki et al., 1995; Liu et al., 2007). There are also major differences between rodent, sub-human primate and human 3b-HSDs. In humans, the 3b-HSD gene family has two genes

and five pseudogenes grouped in a cluster on chromosome 1 (band 1p13.1) (Berube et al., 1989; Luu The et al., 1992; McBride et al., 1999). In humans, type 1 3b-HSD is expressed essentially in peripheral tissues, namely, the placenta, skin, mammary gland and prostate (Rheaume et al., 1991), while type 2 3b-HSD is exclusively expressed in the adrenal gland, testis and ovary. The deficiency in human type 3 3b-HSD is responsible for congenital adrenal hyperplasia associated with male pseudohermaphroditism (Russell et al., 1994; Simard et al., 2005). In the mouse, on the other hand, there are six functional types of 3b-HSD, while in the Macaca fascicularis, there is only one functional 3b-HSD (Liu, 2007). The above-mentioned data strongly suggest that types 1 and 2 3b-HSD are duplicated after the divergence from the Macaca fascicularis 3b-HSD gene. Accordingly, the two human enzymes share 94% amino acid sequence identity, confirming that they are duplicated after the divergence with the rodent and most probably from duplication of the single Macaca fascicularis gene. It is noteworthy that only humans possess two 3b-HSDs which possess a 10-fold difference in activity and affinity. Interestingly, the enzyme that possesses higher activity and affinity is one permitting the intracrine transformation of DHEA into 4-dione and 5-diol into testosterone in peripheral intracrine tissues (Labrie, 1991). Type 2 3b-HSD, on the other hand, has lower affinity and activity, and is responsible for adrenal congenital hyperplasia (Rheaume et al., 1991, 1992). In the mouse adrenal gland and testis, there are two types of 3b-HSDs (Payne and Hales, 2004). Accordingly, the probability of having 3b-HSD deficiency in the mouse is much lower than in humans. It is noteworthy that in the rodent placenta, there is an absence of enzymes involved in the biosynthesis of E2, namely, 3b-HSD, aromatase and type 1 17b-HSD (Payne and Hales, 2004), while these enzymes are expressed at very high levels in humans. In addition, the enzyme 17ahydroxylase/17, 20-lyase is absent in the mouse adrenals (Brock and Waterman, 1999; Luu-The

Table 1. Comparison of the activity and tissue distribution of some steroidogenic enzymes between human, monkey and rodents

Enzyme (human) 3b-HSD1

Percentage homology of amino acid sequence 70% homology with rodent 3b-HSD

5a-reductase type 2

94% homology with human type 1 70% homology with rodent 3b-HSD 62% homology with rodent 48.4% identity with human type 2 75% homology with rodent

Aromatase

48.4% identity with human type 1 81% homology with rodent

3b-HSD2

5a-reductase type 1

Enzymatic activity

Site (tissue) of expression

DHEA ! 4-dione

Human: placenta, skin, brain

Preg ! prog

– Could be equivalent to rat type 4 and mouse type 6 that are expressed in the skin, however, mouse type 6 is also expressed in the testis

High activity and affinity, similar to rodent enzymes Low activity

Human: adrenals and gonads

– does not have equivalent in rodent, in term of activity 4-Dione ! 5a-dione (high activity) Testo ! DHT (low activity)

– could be equivalent with mouse type 1 that is expressed in the adrenals and gonads Human: liver, skin Rodent: ventral prostate (epithelial cells), epididymis, liver

4-Dione ! 5a-dione

Human: prostate (stromal cells), epididymis, seminal vesicle, genital skin (fibroblasts) Rodent: prostate (stromal cells), epididymis

Testo ! DHT 4-Dione ! E1 (high activity) Testo ! E2 (weak activity)

Type 1 17b-HSD

63% homology with rodent

E1 ! E2 (also 4-dione ! testo in rodent)

Type 3 17b-HSD

72.5% homology with rodent

4-Dione ! testo

Human: placenta, pre-ovulatory follicle, corpus luteum, testis, adipose tissue Rodent: pre-ovulatory follicle, corpus luteum, testis, adipose tissue, absent in placenta Human: placenta and granulosa cells of human ovary Rodent: granulosa cells of developing follicles, absent in placenta Human: testis Rodent: testis

Type 5 17b-HSD

76% homology

Type 7 17b-HSD

85% homology with rodent

Type 12 17b-HSD

81% with rodent

Human: 4-dione ! testo 5a-dione ! DHT Prog ! 20a-OHProg Rodent: 4-dione ! testo 5adione ! DHT E1 ! E2 E1 ! E2 DHT ! 3b-diol Human: E1 ! E2 Mouse: E1 ! E2

Type 15 17b-HSD

85% homology with rodent

4-Dione ! testo DHEA ! 5-diol 5a-Dione ! DHT ADT ! 3a-diol 4-Dione ! testo (weak)

Human: liver, muscle, small intestine, kidney, skin and mammary gland Rodent: liver

Human: liver, ovary Rodent: liver, heart, kidney and epididymis Human: liver, mammary gland, uterus, skin, adipose tissue, heart and brain Rodent: adrenals, testis, liver, kidney, epididymis, adipose tissue and brain

Human: prostate, liver, kidney, brain, heart, pancreas and skin, (very low in testis) Rodent: testis, liver, kidney, (absence in prostate)

The abbreviations and symbols used are: DHEA: dehydroepiandrosterone; 5-diol: 5-androstene-3b,17b-diol; 4-dione: 4-androstenedione; 5a-dione: 5a-androstane-3,17-dione; testo: testosterone; DHT: dihydrotestosterone; E1: estrone; E2: estradiol; 3a-diol: 5a-androstane-3a,17b-diol; 3b-diol: 5a-androstane-3b,17b-diol; preg: pregnenolone; prog: progesterone; HSD: hydroxysteroid dehydrogenase.

186

(3a-HSDs and UGTs) belong to the class ‘conserved structures–different activities’ described above. These genes diverged after the separation between rodents and primates and also between primates and humans, thus implying that they could not have orthologs in other species. In addition, although showing homology, ortholog genes can encode for enzymes having different substrate specificities. The major pathway of final androgen and estrogen inactivation in humans is glucuronidation that occurs by the addition of a polar glycosyl group to small hydrophilic molecules, thus facilitating their excretion (Belanger et al., 2003) (Figs. 1 and 4). The enzymes responsible for this transformation are members of the UGT family (Mackenzie et al., 2005, 1997). In humans, UGT enzymes are expressed in the liver and a long series of extra-hepatic tissues, including the kidney, brain, skin, adipose and reproductive tissues (Guillemette et al., 2004) and as expected, androgen glucuronides are present

et al., 2005), while it is highly expressed in humans (Luu-The et al., 2005). These observations can explain the low levels of C19-steroids in the circulation in rodents, while the concentration of circulating C19-steroids, especially DHEA and its sulphate DHEA-S, is very high in humans.

Species differences in sex steroid-inactivating enzymes In addition to the marked species difference in estrogen and androgen formation between species, there are also major differences between species in tissue distribution and activity of the steroid-inactivating enzymes in peripheral tissues ranging from the absence of glucuronidation enzymes in rodents to very high levels in primates and intermediate concentrations in humans. This observation is most probably due to the fact that most of the inactivating enzymes

Pathways of sex steroid biosysthesis Endocrine

(a)

Intracrine Peripheral tissues

(b)

Ovary and testis

Intra-gonadal

Cholesterol

Adrenal gland

DHEA 3β-HSD-2 Androstenedione (4-dione)

DHEA

DHEA

17βHSD3, 5

Testo

DHEA

DHEA 3β-HSD-1

Aromatase Androstenedione (4-dione) Aromatase

E2

5α-reductase 17βHSD5, 15

DHEA

Testo

E2

Circulation Testo

5α−Androstenedione (4-dione)

DHT 17βHSD2

DHEA

E2

17βHSD1, 7, 12

Testo 5α-reductase

Estrone

Estradiol 17βHSD2

DHT

Intracrine tissues Target tissues E2

Circulation

DHEA

Circulation

DHEA

Testo

Fig. 4. Schematic representation of pathways requiring (a) and not requiring (b) testosterone as intermediate.

187

in the circulation which is their route of elimination (Belanger et al., 1991). The extra-hepatic expression and activity of the androgenconjugating UGT enzymes are major determinants of the local inactivation of androgens and estrogens, thus playing an important role in the regulation of local androgen and estrogen concentration and action or intracrinology (Belanger et al., 2003). It is important to indicate that the concentrations of the active steroids testosterone and estradiol in the circulation represent the sum of the steroids secreted by the gonads and the concentration of the steroids leaking from the peripheral tissues following DHEA transformation by the intracrine enzymes (Figs. 2 and 4). It is thus important to indicate that the low circulating levels of E2 or testo observed after castration, in men or after menopause or ovariectomy in women, does not mean that these steroids do not play an important intra-cellular role. Consequently, the conjugated steroids are a better representative of the global exposure to each class of steroids (Labrie et al., 2006).

Does the 17b-HSD step precede or follow aromatase or 5a-reductase action in the steroidogenic pathways in peripheral tissues? The traditional understanding is that estradiol and DHT are always synthetized from the aromatization and 5a-reduction of testosterone, respectively (Fig. 4a). This pathway indicates that the steps of aromatization and 5a-reduction follow the step catalyzed by type 3 17b-HSD and that estrogenic 17b-HSD is not involved. Contrary to this belief, the cloning of estrogenspecific 17b-HSD and observation of the higher affinity of aromatase (Kellis and Vickery, 1987; Reed and Ohno, 1976) and 5a-reductase (Andersson and Russell, 1990; Russell and Wilson, 1994; Sugimoto et al., 1995) for 4-androstenedione than for testosterone are strongly in favour of the biosynthetic pathway in which the step catalyzed by 17b-HSDs follows the step catalyzed by aromatase (Fig. 4b). On the other hand, in line with this

pharmacokinetic information, DHT is made from A-dione following 5a-reduction of 4-dione (Fig. 4b). These pathways do not require testosterone as an intermediate as traditionally described. Using [14C]-labelled DHEA and 4-androstenedione and inhibitors of aromatase and 5a-reductase, we have clearly demonstrated that these pathways are the major pathways responsible for the formation of estradiol in JEG-3 cells (Samson et al., 2009b) and DHT in DU-145 (Samson et al., 2009a) and the SZ95 sebaceous gland cell line (Samson et al., 2009c). These pathways are thus specific to intracrinology while, in the testis and ovary, the pathways illustrated in Fig. 4a are operative. Although testosterone can be aromatized into estradiol or 5a-reduced into DHT in vitro with the appropriate enzymes, we believe, in agreement with the recently observed thermodynamics of the reaction, that the main pathways for the biosynthesis of estradiol and DHT in cultured cells and most probably also in vivo in humans, in the presence of 4-androstenedione, do not require testosterone as an intermediate. The reason for the novelty of this observation is most probably due, as mentioned above, to the general belief that steroid concentrations in the circulation represent the concentrations available for transformation in the cells or tissues while, as the more recent information on intracrinology indicates, the circulating concentrations of sex steroids do not represent the intra-cellular concentrations of the same steroids (Labrie et al., 2005). These data are also in agreement with the presence of two androgens, namely, testosterone and DHT, that are synthesized by two different pathways. The higher affinity of testosterone for the androgen receptor (AR) (Km  108, 109 M) than 5a-reductases 6 (Km  10 M) is also in favour of the role of testosterone as ligand for AR without requiring its transformation into DHT, since in tissues that express both AR and 5a-reductases, testosterone will preferentially bind to AR and exert its androgenic activity before binding to 5a-reductases.

188

Biosynthesis of testosterone Testosterone is produced from the transformation of 4-androstenedione by types 3 or 5 17bHSDs in tissues or cells in which the expression level of 5a-reductases is low or absent, such as in the testicles and muscle. Type 3 17b-HSD is mainly expressed in the testis and is responsible for the formation of testosterone necessary for the formation of the internal male reproductive structures. Inactivation of type 3 17b-HSD in patients who have a mutated gene is responsible for male pseudohermaphroditism (Geissler et al., 1994). The typical features of patients who have a mutated type 3 17b-HSD gene are ambiguous female external genitalia and virilization at puberty (Wilson, 1978). At surgery, testes and epididymes are found in the inguinal canals, whereas lower Wolffian duct structures are male in character, including the seminal vesicles and ejaculatory ducts. Type 3 17b-HSD is the main enzyme that produces testicular testosterone that acts as androgen in an endocrine manner in all peripheral target tissues. A defect in type 2 5a-reductase also causes male pseudohermaphroditism (Andersson et al., 1991) with ambiguous external genitalia. However, in contrast with type 3 17b-HSD gene deficiency, the Wolffian structures are normally differentiated (Peterson et al., 1977), suggesting that testo and DHT exert their androgenic activity in a different manner via the same AR. It is possible that the co-activators/co-repressors recruited in the presence of testosterone and DHT are different in identity and concentration. Mutations that inactivate the genes encoding AR cause the X-linked androgen-insensitive syndrome (AIS) (ImperatoMcGinley et al., 1990; Xu et al., 2003). In these patients, the development of both the internal and external male reproductive structures are altered suggesting that both testosterone and DHT are acting through the same AR. Another tissue that possesses low levels of 5a-reductase and high levels of testosteroneproducing enzyme is the muscle that expresses high levels of type 5 17b-HSD. However, type 5 17b-HSD is a multi-substrate enzyme having the additional ability to convert 5a-androstane-

3,17-dione into DHT and androsterone (ADT) into 5a-androstane-3a,17b-diol. Thus, in tissues that also possess 5a-reductases, type 5 17b-HSD is involved in the formation of DHT and 5aandrostane-3a,17b-diol in the pathway that does not require testosterone as an intermediate. In women, the biosynthesis of testosterone in tissues that possess low levels of 5a-reductase most probably involves type 5 17b-HSD, as previously described (Luu-The et al., 2001).

Biosynthesis of DHT In tissues and cells that express 5a-reductases such as the prostate, skin and liver, DHT, the most potent natural androgen, is most likely to be synthesized, as discussed above, by a pathway that does not require testosterone as an intermediate (Fig. 4b). These tissues are well known as androgen-sensitive tissues and the imbalance of DHT levels could be associated with diseases of the prostate gland, such as benign prostatic hyperplasia (BPH) and prostate cancer, or skin associated diseases such as alopecia, hirsutism and acne seborrea. The above-mentioned data strongly suggest that depending upon the steroid precursors and enzymes present in a specific cell, AR can be modulated by testosterone or DHT. In addition to 5a-reductases that catalyze the conversion of 4-androstenedione into 5a-androstanedione, the conversion of 5a-androstanedione into DHT requires the presence of 17b-HSDs. At present, the two most likely candidates among 15 17b-HSDs identified (Fig. 4b) are types 5 and 15 17b-HSD (Luu-The et al., 2008).

Biosynthesis of estradiol Estradiol, the most potent natural estrogen, plays a crucial role in the proliferation of normal and cancerous breast and uterine cells. The role of local E2 synthesis is increasingly recognized and prevails over the traditional belief that E2 is exclusively synthesized in the gonads and delivered to peripheral tissues through the circulation. Although the biosynthetic pathway of E2 was

189

proposed a few decades ago, there is still, as mentioned above, some controversy about the sequential intervention of aromatase and 17b-HSD. In fact, traditional literature and textbooks indicate that E2 is produced by the transformation of 4-androstenedione (4-dione) into testosterone (T) by 17b-HSD and then by aromati17 -HSD Arom testo ! E2) zation of T into E2 (4-dione (Fig. 4b). However, the four-fold higher affinity of aromatase for 4-androstenedione than for testosterone using microsomes (Reed and Ohno, 1976) and the purified enzyme (Kellis and Vickery, 1987), respectively, are in favour of 4-androstenedione being the substrate for aromatase (Fig. 4b). Accordingly, the aromatase step should naturally precede the action of 17b-HSD. The role of the pathway is supported by the cloning of estrogen-specific 17b-HSDs (Krazeisen et al., 1999; Luu The et al., 1989, 1990a, 2006; Peltoketo et al., 1988). Aromatase represents an interesting case in which the expression of multiple tissue-specific transcripts is driven by different alternative promoters of the same gene (Simpson et al., 1994, 1997). These transcripts, however, possess the same coding sequence and thus encode the same protein structure in all tissues. It is worth noting that, after menopause, when the ovary is no longer functional, all the active estrogens in women are made in target tissues from inactive adrenal precursors (Labrie et al., 2001, 2005). Ironically, it is in the post-menopausal period that breast cancers are more frequently observed and are, in 75% of cases, efficiently treated by estrogen blockade, thus demonstrating the very important role of intracrine formation of estrogens (Labrie et al., 2005, Mouridsen et al., 2003).

!

References Andersson, S., Berman, D. M., Jenkins, E. P., & Russell, D. W. (1991). Deletion of steroid 5 alpha-reductase 2 gene in male pseudohermaphroditism. Nature, 354(6349), 159–161. Andersson, S. & Russell, D. W. (1990). Structural and biochemical properties of cloned and expressed human and rat steroid 5 alpha-reductases. Proceedings of the National

Academy of Sciences of the United States of America, 87 (10), 3640–3644. Baker, M. E. (1997). Steroid receptor phylogeny and vertebrate origins. Molecular and Cellular Endocrinology, 135 (2), 101–107. Baker, M. E. (2001a). Evolution of 17beta-hydroxysteroid dehydrogenases and their role in androgen, estrogen and retinoid action. Molecular and Cellular Endocrinology, 171 (1–2), 211–215. Baker, M. E. (2001b). Hydroxysteroid dehydrogenases: Ancient and modern regulators of adrenal and sex steroid action. Molecular and Cellular Endocrinology, 175(1–2), 1–4. Baker, M. E., Luu-The, Y., Simard, J., & Labrie, F. (1990). A common ancestor for mammalian 3 beta-hydroxysteroid dehydrogenase and plant dihydroflavonol reductase. The Biochemical Journal, 269(2), 558–559. Belanger, A., Brochu, M., Lacoste, D., Noel, C., Labrie, F., Dupont, A., et al. (1991). Steroid glucuronides: Human circulatory levels and formation by LNCaP cells. The Journal of Steroid Biochemistry and Molecular Biology, 40(4–6), 593–598. Belanger, A., Pelletier, G., Labrie, F., Barbier, O., & Chouinard, S. (2003). Inactivation of androgens by UDP-glucuronosyltransferase enzymes in humans. Trends in Endocrinology and Metabolism, 14(10), 473–479. Bellemare, V., Laberge, P., Noel, S., Tchernof, A., & Luu-The, V. (2009). Differential estrogenic 17beta-hydroxysteroid dehydrogenase activity and type 12 17beta-hydroxysteroid dehydrogenase expression levels in preadipocytes and differentiated adipocytes. Journal of Steroid Biochemistry and Molecular Biology, 114(3–5), 129–134. Bellemare, V., Phaneuf, D., & Luu-The, V. (2009). Type 12 17beta-hydroxysteroid dehydrogenase knock-out mouse: Reduction of androgen and estrogen levels in heterozygotes and embryonic lethality in homozygotes. 13th International Congress on Hormonal Steroids and Hormones & Cancer (p.P–119). Quebec. Bertrand, S., Brunet, F. G., Escriva, H., Parmentier, G., Laudet, V., & Robinson-Rechavi, M. (2004). Evolutionary genomics of nuclear receptors: From twenty-five ancestral genes to derived endocrine systems. Molecular Biology and Evolution, 21(10), 1923–1937. Berube, D., Luu The, V., Lachance, Y., Gagne, R., & Labrie, F. (1989). Assignment of the human 3 beta-hydroxysteroid dehydrogenase gene (HSDB3) to the p13 band of chromosome 1. Cytogenetics and Cell Genetics, 52(3–4), 199–200. Bilezikian, J. P., Morishima, A., Bell, J., & Grumbach, M. M. (1998). Increased bone mass as a result of estrogen therapy in a man with aromatase deficiency. The New England Journal of Medicine, 339(9), 599–603. Blanchard, P. G. & Luu-The, V. (2007). Differential androgen and estrogen substrates specificity in the mouse and primates type 12 17beta-hydroxysteroid dehydrogenase. Journal of Endocrinology, 194(2), 449–455. Brock, B. J. & Waterman, M. R. (1999). Biochemical differences between rat and human cytochrome P450c17 support

190 the different steroidogenic needs of these two species. Biochemistry, 38(5), 1598–1606. Carani, C., Qin, K., Simoni, M., Faustini-Fustini, M., Serpente, S., Boyd, J., et al. (1997). Effect of testosterone and estradiol in a man with aromatase deficiency. The New England Journal of Medicine, 337(2), 91–95. Colbert, E. H. & Morales, M. (1991). Evolution of vertebrates: A history of the backboned animals through time. New York: Wiley-Liss. Desnoyers, S., Blanchard, P.-G., St-Laurent, J.-F., Gagnon, S., Baillie, D. L., & Luu-The, V. (2007). C. elegans LET-767 is able to metabolize androgens and estrogens and likely shares common ancestor with human types 3 and 12 17bhydroxysteroid dehydrogenases. Journal of Endocrinology, 195, 271–279. Deyashiki, Y., Ohshima, K., Nakanishi, M., Sato, K., Matsuura, K., & Hara, A. (1995). Molecular cloning and characterization of mouse estradiol 17 beta-dehydrogenase (A-specific), a member of the aldoketoreductase family. The Journal of Biological Chemistry, 270(18), 10461–10467. Dufort, I., Labrie, F., & Luu-The, V. (2001). Human types 1 and 3 3alpha-hydroxysteroid dehydrogenases: Differential lability and tissue distribution. Journal of Clinical Endocrinology and Metabolism, 86(2), 841–846. Dufort, I., Rheault, P., Huang, X.-F., Soucy, P., & Luu-The, V. (1999). Characteristics of a highly labile human type 5 17beta-hydroxysteroid dehydrogenase. Endocrinology, 140(2), 568–574. Dumont, M., Luu-The, V., Dupont, E., Pelletier, G., & Labrie, F. (1992b). Characterization, expression, and immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase in human skin. The Journal of Investigative Dermatology, 99(4), 415–421. Dumont, M., Luu-The, V., de Launoit, Y., & Labrie, F. (1992a). Expression of human 17 beta-hydroxysteroid dehydrogenase in mammalian cells. The Journal of Steroid Biochemistry and Molecular Biology, 41(3–8), 605–608. Entchev, E. V., Schwudke, D., Zagoriy, V., Matyash, V., Bogdanova, A., Habermann, B., et al. (2008). LET-767 is required for the production of branched chain and long chain fatty acids in Caenorhabditis elegans. The Journal of Biological Chemistry, 283(25), 17550–17560. Fisher, C. R., Graves, K. H., Parlow, A. F., & Simpson, E. R. (1998). Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene. Proceedings of the National Academy of Sciences of the United States of America, 95(12), 6965–6970. Geissler, W. M., Davis, D. L., Wu, L., Bradshaw, K. D., Patel, S., Mendonca, B. B., et al. (1994). Male pseudohermaphroditism caused by mutations of testicular 17 beta-hydroxysteroid dehydrogenase 3. Nature Genetics, 7(1), 34–39. Guillemette, C., Belanger, A., & Lepine, J. (2004). Metabolic inactivation of estrogens in breast tissue by UDP-glucuronosyltransferase enzymes: An overview. Breast Cancer Research, 6(6), 246–254. Imperato-McGinley, J., Ip, N. Y., Gautier, T., Neuweiler, J., Gruenspan, H., Liao, S., et al. (1990). DNA linkage analysis

and studies of the androgen receptor gene in a large kindred with complete androgen insensitivity. American Journal of Medical Genetics, 36(1), 104–108. Kellis, J. T. Jr. & Vickery, L. E. (1987). Purification and characterization of human placental aromatase cytochrome P-450. The Journal of Biological Chemistry, 262(9), 4413–4420. Korach, K. S. (1994). Insights from the study of animals lacking functional estrogen receptor. Science, 266(5190), 1524–1527. Krazeisen, A., Breitling, R., Imai, K., Fritz, S., Moller, G., & Adamski, J. (1999). Determination of cDNA, gene structure and chromosomal localization of the novel human 17betahydroxysteroid dehydrogenase type 7(1). FEBS Letters, 460(2), 373–379. Kuervers, L. M., Jones, C. L., O’Neil, N. J., & Baillie, D. L. (2003). The sterol modifying enzyme LET-767 is essential for growth, reproduction and development in Caenorhabditis elegans. Molecular Genetics and Genomics, 270(2), 121–131. Labrie, F. (1991). Intracrinology. Molecular and Cellular Endocrinology, 78(3), C113–C118. Labrie, F. (2007). Drug insight: Breast cancer prevention and tissue-targeted hormone replacement therapy. Nature Clinical Practice Endocrinology and Metabolism, 3(8), 584–593. Labrie, F., Archer, D., Bouchard, C., Fortier, M., Cusan, L., Gomez, J. L., et al. (2009a). Intravaginal dehydroepiandrosterone (prasterone), a physiological and highly efficient treatment of vaginal atrophy. Menopause, 16(5), 907–922. Labrie, F., Archer, D., Bouchard, C., Fortier, M., Cusan, L., Gomez, J. L., et al. (2009b). Effect of intravaginal dehydroepiandrosterone (prasterone) on libido and sexual dysfunction in postmenopausal women. Menopause, 16(5), 923–931. Labrie, F., Archer, D., Bouchard, C., Fortier, M., Cusan, L., Gomez, J. L., et al. (2009c). Serum steroid levels during 12week intravaginal dehydroepiandrosterone administration. Menopause, 16(5), 897–906. Labrie, F., Belanger, A., Belanger, P., Berube, R., Martel, C., Cusan, L., et al. (2006). Androgen glucuronides, instead of testosterone, as the new markers of androgenic activity in women. The Journal of Steroid Biochemistry and Molecular Biology, 99(4–5), 182–188. Labrie, F., Bélanger, A., Cusan, L., Seguin, C., Pelletier, G., Kelly, P. A., et al. (1980). Antifertility effects of LHRH agonists in the male. Journal of Andrology, 1, 209–228. Labrie, F., Cusan, L., Gomez, J. L., Cote, I., Berube, R., Belanger, P., et al. (2008). Effect of intravaginal DHEA on serum DHEA and eleven of its metabolites in postmenopausal women. The Journal of Steroid Biochemistry and Molecular Biology, 111(3–5), 178–194. Labrie, F., Dupont, A., & Belanger, A. (1985). Complete androgen blockade for the treatment of prostate cancer. Important Advances in Oncology, 193–217. Labrie, Y., Durocher, F., Lachance, Y., Turgeon, C., Simard, J., Labrie, C., et al. (1995). The human type II 17 beta-hydroxysteroid dehydrogenase gene encodes two alternatively spliced mRNA species. DNA and Cell Biology, 14(10), 849–861.

191 Labrie, F., Luu-The, V., Belanger, A., Lin, S. X., Simard, J., Pelletier, G., et al. (2005). Is dehydroepiandrosterone a hormone? Journal of Endocrinology, 187(2), 169–196. Labrie, F., Luu-The, V., Labrie, C., & Simard, J. (2001). DHEA and its transformation into androgens and estrogens in peripheral target tissues: Intracrinology. Frontiers in Neuroendocrinology, 22(3), 185–212. Labrie, F., Sugimoto, Y., Luu-The, V., Simard, J., Lachance, Y., Bachvarov, D., et al. (1992). Structure of human type II 5 alpha-reductase gene. Endocrinology, 131(3), 1571–1573. Lachance, Y., Luu-The, V., Labrie, C., Simard, J., Dumont, M., de Launoit, Y., et al. (1990). Characterization of human 3bhydroxysteroid dehydrogenase/D5–D4 isomerase gene and its expression in mammalian cells. Journal of Biological Chemistry, 265(33), 20469–20475. Lachance, Y., Luu-The, V., Verreault, H., Dumont, M., Rheaume, É., Leblanc , G., et al. (1991). Structure of the human type II 3b-hydroxysteroid dehydrogenase/D5–D4 isomerase (3b-HSD) gene: Adrenal and gonadal specificity. DNA and Cell Biology, 10(10), 701–711. Laudet, V. (1997). Evolution of the nuclear receptor superfamily: Early diversification from an ancestral orphan receptor. Journal of Molecular Endocrinology, 19(3), 207–226. Li, J., Biswas, M. G., Chao, A., Russell, D. W., & Chory, J. (1997). Conservation of function between mammalian and plant steroid 5alpha-reductases. Proceedings of the National Academy of Sciences of the United States of America, 94(8), 3554–3559. Liu, H. (2007). Molecular cloning and characterization of enzymes involved in sex hormone formation and inactivation in cynomolgus monkey. Ph. D., Endocrinology and Physiology Program, Department of Physiology and Anatomy, Laval University, Quebec. Liu, H., Bellemare, V., Labrie, F., & Luu-The, V. (2007). Molecular characterization of the cynomolgus monkey Macaca fascicularis steroidogenic enzymes belonging to the aldo-keto reductase family. The Journal of Steroid Biochemistry and Molecular Biology, 104(1–2), 75–80. Liu, H., Zheng, S., Bellemare, V., Pelletier, G., Labrie, F., & Luu-The, V. (2007). Expression and localization of estrogenic type 12 17beta-hydroxysteroid dehydrogenase in the cynomolgus monkey. BMC Biochemistry, 8, 2. Lorence, M. C., Corbin, C. J., Kamimura, N., Mahendroo, M. S., & Mason, J. I. (1990). Structural analysis of the gene encoding human 3 beta-hydroxysteroid dehydrogenase/ delta 5 – 4-isomerase. Molecular Endocrinology, 4(12), 1850–1855. Luo, M., Xiao, Y., Li, X., Lu, X., Deng, W., Li, D., et al. (2007). GhDET2, a steroid 5alpha-reductase, plays an important role in cotton fiber cell initiation and elongation. The Plant Journal, 51(3), 419–430. Luu The, V., Labrie, C., Zhao, H. F., Couet, J., Lachance, Y., Simard, J., et al. (1989). Characterization of cDNAs for human estradiol 17 beta-dehydrogenase and assignment of the gene to chromosome 17: Evidence of two mRNA species with distinct 5’-termini in human placenta. Molecular Endocrinology, 3(8), 1301–1309.

Luu The, V., Lachance, Y., Leblanc, G., & Labrie, F. (1992). Human 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase: Characterization of the three additional related genes. 74th Annual Meeting of the Endocrine Society, San Antonio, Texas. Luu-The, V. (2001). Analysis and characteristics of multiple types of human 17b-hydroxysteroid dehydrogenases. Journal of Steroid Biochemistry and Molecular Biology, 76(1–5), 143–151. Luu-The, V., Belanger, A., & Labrie, F. (2008). Androgen biosynthetic pathways in the human prostate. Best Practice and Research Clinical Endocrinology and Metabolism, 22(2), 207–221. Luu-The, V., Dufort, I., Pelletier, G., & Labrie, F. (2001). Type 5 17b-hydroxysteroid dehydrogenase: Its role in the formation of androgens in women. Molecular and Cellular Endocrinology, 171(1–2), 77–82. Luu-The, V., Labrie, C., Simard, J., Lachance, Y., Zhao, H.-F., Couët, J., et al. (1990a). Structure of two in tandem human 17b-hydroxysteroid dehydrogenase genes. Molecular Endocrinology, 4(2), 268–275. Luu-The, V., Labrie, C., Zhao, H. F., Couet, J., Lachance, Y., Simard, J., et al. (1990b). Purification, cloning, complementary DNA structure, and predicted amino acid sequence of human estradiol 17 beta-dehydrogenase. Annals of the New York Academy of Sciences, 595, 40–52. Luu-The, V., Lachance, Y., Labrie, C., Leblanc, G., Thomas, J. L., Strickler, R. C., et al. (1989). Full length cDNA structure and deduced amino acid sequence of human 3bhydroxy-5-ene steroid dehydrogenase. Molecular Endocrinology, 3(8), 1310–1312. Luu-The, V., Pelletier, G., & Labrie, F. (2005). Quantitative appreciation of steroidogenic gene expression in mouse tissues: New roles for type 2 5alpha-reductase, 20alpha-hydroxysteroid dehydrogenase and estrogen sulfotransferase. The Journal of Steroid Biochemistry and Molecular Biology, 93 (2–5), 269–276. Luu-The, V., Tremblay, P., & Labrie, F. (2006). Characterization of type 12 17beta-hydroxysteroid dehydrogenase, an isoform of type 3 17beta-hydroxysteroid dehydrogenase responsible for estradiol formation in women. Molecular Endocrinology, 20(2), 437–443. Mackenzie, P. I., Bock, K. W., Burchell, B., Guillemette, C., Ikushiro, S., Iyanagi, T., et al. (2005). Nomenclature update for the mammalian UDP glycosyltransferase (UGT) gene superfamily. Pharmacogenetics and Genomics, 15(10), 677–685. Mackenzie, P. I., Owens, I. S., Burchell, B., Bock, K. W., Bairoch, A., Belanger, A., et al. (1997). The UDP glycosyltransferase gene superfamily: Recommended nomenclature update based on evolutionary divergence. Pharmacogenetics, 7(4), 255–269. McBride, M. W., McVie, A. J., Burridge, S. M., Brintnell, B., Craig, N., Wallace, A. M., et al. (1999). Cloning, expression, and physical mapping of the 3beta-hydroxysteroid dehydrogenase gene cluster (HSD3BP1-HSD3BP5) in human. Genomics, 61(3), 277–284.

192 Moon, Y. A. & Horton, J. D. (2003). Identification of two mammalian reductases involved in the two-carbon fatty acyl elongation cascade. The Journal of Biological Chemistry, 278(9), 7335–7343. Morishima, A., Grumbach, M. M., Simpson, E. R., Fisher, C., & Qin, K. (1995). Aromatase deficiency in male and female siblings caused by a novel mutation and the physiological role of estrogens. Journal of Clinical Endocrinology and Metabolism, 80(12), 3689–3698. Mouridsen, H., Gershanovich, M., Sun, Y., Perez-Carrion, R., Boni, C., Monnier, A., et al. (2003). Phase III study of letrozole versus tamoxifen as first-line therapy of advanced breast cancer in postmenopausal women: Analysis of survival and update of efficacy from the international letrozole breast cancer group. Journal of Clinical Oncology, 21(11), 2101–2109. Noguchi, T., Fujioka, S., Takatsuto, S., Sakurai, A., Yoshida, S., Li, J., et al. (1999). Arabidopsis det2 is defective in the conversion of (24r)-24-methylcholest-4-en-3-one to (24r)24-methyl-5alpha-cholestan-3-one in brassinosteroid biosynthesis. Plant Physiology, 120(3), 833–840. Nokelainen, P., Puranen, T., Peltoketo, H., Orava, M., Vihko, P., & Vihko, R. (1996). Molecular cloning of mouse 17 beta-hydroxysteroid dehydrogenase type 1 and characterization of enzyme activity. European Journal of Biochemistry, 236(2), 482–490. Payne, A. H. & Hales, D. B. (2004). Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocrine Reviews, 25(6), 947–970. Peltoketo, H., Isomaa, V., Maentausta, O., & Vihko, R. (1988). Complete amino acid sequence of human placental 17 betahydroxysteroid dehydrogenase deduced from cDNA. FEBS Letters, 239(1), 73–77. Peterson, R. E., Imperato-McGinley, J., Gautier, T., & Sturla, E. (1977). Male pseudohermaphroditism due to steroid 5alpha-reductase deficiency. American Journal of Medicine, 62(2), 170–191. Reed, K. C. & Ohno, S. (1976). Kinetic properties of human placental aromatase. Application of an assay measuring 3H2O release from 1beta,2beta-3h-androgens. The Journal of Biological Chemistry, 251(6), 1625–1631. Rheaume, É., Lachance, Y., Zhao, H.-F., Breton, N., Dumont, M., de Launoit, Y., et al. (1991). Structure and expression of a new complementary DNA encoding the almost exclusive 3b-hydroxysteroid dehydrogenase/D5–D4-isomerase in human adrenals and gonads. Molecular Endocrinology, 5(8), 1147–1157. Rheaume, E., Simard, J., Morel, Y., Mebarki, F., Zachmann, M., Forest, M. G., et al. (1992). Congenital adrenal hyperplasia due to point mutations in the type II 3 beta-hydroxysteroid dehydrogenase gene. Nature Genetics, 1(4), 239–245. Russell, A. J., Wallace, A. M., Forest, M. G., Donaldson, M. D., Edwards, C. R., & Sutcliffe, R. G. (1994). Mutation in the human gene for 3 beta-hydroxysteroid dehydrogenase type II leading to male pseudohermaphroditism without salt loss. Journal of Molecular Endocrinology, 12(2), 225–237. Russell, D. W. & Wilson, J. D. (1994). Steroid 5 alpha-reductase: Two genes/two enzymes. Annual Review of Biochemistry, 63, 25–61.

Samson, M., Labrie, F., & Luu-The, V. (2009a). Sequential transformation of 4-androstenedione into dihydrotestosterone in prostate carcinoma (DU-145) cells indicates that 4androstenedione and not testosterone is the substrate of 5alpha-reductase. Hormone Molecular Biology Clinical Investigation, 1, 63–72. Samson, M., Labrie, F., & Luu-The, V. (2009b). Specific estradiol biosynthetic pathway in choriocarcinoma (JEG-3) cell line. The Journal of Steroid Biochemistry and Molecular Biology, 116(3–5), 154–159. Samson, M., Labrie, F., Zouboulis, C. C., & Luu-The, V. (2009c). Biosynthesis of dihydrotestosterone by a pathway that does not require testosterone as intermediate in the SZ95 sebaceous gland cell line. Journal of Investigative Dermatology, 130(2), 602–604. Simard, J., Ricketts, M. L., Gingras, S., Soucy, P., Feltus, F. A., & Melner, M. H. (2005). Molecular biology of the 3betahydroxysteroid dehydrogenase/delta5-delta4 isomerase gene family. Endocrine Reviews, 26(4), 525–582. Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Hinshelwood, M. M., Graham-Lorence, S., et al. (1994). Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrine Reviews, 15(3), 342–355. Simpson, E. R., Michael, M. D., Agarwal, V. R., Hinshelwood, M. M., Bulun, S. E., & Zhao, Y. (1997). Cytochromes P450 11: Expression of the CYP19 (aromatase) gene: An unusual case of alternative promoter usage. The FASEB Journal, 11 (1), 29–36. Sugimoto, Y., Lopez-Solache, I., Labrie, F., & Luu-The, V. (1995). Cations inhibit specifically type I 5 alpha-reductase found in human skin. The Journal of Investigative Dermatology, 104(5), 775–778. Uemura, M., Tamura, K., Chung, S., Honma, S., Okuyama, A., Nakamura, Y., et al. (2008). Novel 5 alpha-steroid reductase (SRD5A3, type-3) is overexpressed in hormone-refractory prostate cancer. Cancer Science, 99(1), 81–86. Wilson, J. D. (1978). Sexual differentiation. Annual Review of Physiology, 40, 279–306. Wu, L., Einstein, M., Geissler, W. M., Chan, H. K., Elliston, K. O., & Andersson, S. (1993). Expression cloning and characterization of human 17 beta-hydroxysteroid dehydrogenase type 2, a microsomal enzyme possessing 20 alphahydroxysteroid dehydrogenase activity. The Journal of Biological Chemistry, 268(17), 12964–12969. Xu, W., Robert, C., Thornton, P. S., & Spinner, N. B. (2003). Complete androgen insensitivity syndrome due to X chromosome inversion: A clinical report. American Journal of Medical Genetics Part A, 120(3), 434–436. Yamana, K., Labrie, F., & Luu-The, V. (2009). Characterization of a new type 3 5alpha-reductase, an enzyme involves in the biosynthesis of dihydrotestosterone, highly expressed in skin, mammary glands, breast cancer cell lines and adipose tissue, and inhibited by Finasteride. Endocrine Society Meeting. 91th Annual Meeting (p. P2–P4), Washington, DC, U.S.A.

L. Martini (Eds.) Progress in Brain Research, Vol. 181 ISSN: 0079-6123 Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 11

Steroidogenic enzymes in the brain: morphological aspects Georges Pelletier
Oncology, Molecular Endocrinology and Human Genomics Research Center (CHUL), Quebec, Canada

Abstract: It is now well documented that brain tissue is capable of synthesizing de novo bioactive steroids, named neurosteroids, which are involved in the regulation of various functions in the brain, including behavioral, neuroendocrine and metabolic processes. In this chapter, we have summarized the current knowledge about the expression of enzymes involved in the biosynthesis and metabolism of steroids in the brain with special emphasis on the morphological localization of those enzymes. The results obtained following use of immunocytochemistry and/or in situ hybridization indicate that the enzymes are expressed in both neurons and glial cells distributed throughout the brain with some species-related variations. As observed at the electron microscopic level, the enzymes localized in the neurons or glial cells are not associated with any specific organelles, being distributed throughout the cytoplasm. Since usually one nerve cell expresses only one enzyme, it might be suggested that the neuroactive steroid synthesis that requires the action of several enzymes involves the intervention of several cells (neurons and/or glial cells). More work is required to fully establish the migration of precursors and active steroids in the brain. Keywords: steroidogenic enzymes; neurosteroids; brain; immunocytochemistry; in situ hybridization

plasma. In the early eighties, it was shown that the brain is capable of de novo biosynthesis of steroids. The first clue of neurosteroidogenesis came from the observations that pregnenolone, dehydroepiandrosterone (DHEA), and their sulphate derivatives accumulate in the brain of castrated and adrenalectomized rats (Corpéchot et al., 1981). Moreover, Robel et al., (1986) showed that circadian variations of steroid concentrations in brain tissue are not synchronized with those of steroids measured in peripheral blood. As a result, the term neurosteroids was proposed to designate the steroids synthesized in the brain, either de novo from cholesterol or

Introduction The steroid hormones exert a determinant role in brain development while regulating various physiological functions such as locomotion, feeding, sexual behaviors, learning and memory (for a review, see McEwen, 1994). It has been long believed that the steroids acting on the brain were originating from steroidogenic glands, including gonads, adrenal cortex and


Corresponding author. Tel.: þ418-525-4444; Fax: þ418-654-2761; E-mail: [email protected]

DOI: 10.1016/S0079-6123(08)81011-4

193

194

by in situ metabolism of blood-borne precursors (Baulieu, 1997; Robel & Baulieu, 1985). The concept of neurosteroidogenesis has been subsequently supported by morphological data showing the occurrence of steroidogenic enzymes or their mRNAs by immunohistochemistry and in situ hybridization, respectively (Do Rego et al., 2007; Dupont et al., 1994; Le Goascogne et al., 1987; Mellon and Vaudry, 2001; Mensah-Nyagan et al., 1994, 1996, 1999; Pelletier et al., 2003, 2007; Robel and Baulieu, 1985). The morphological approach has largely contributed to accurately determine the brain areas involved in the biosynthesis and metabolism of active neurosteroids as well as to identify the cell type (neurons/glial cells) expressing the different enzymes. The aim of this chapter is to summarize the current research about the expression of steroidogenic enzymes in the brain with special emphasis on their cellular localization.

Cytochrome P450 side-chain cleavage The first step in the synthesis of all steroid hormones is the conversion of cholesterol to pregnenolone by a mitochondrial enzyme, cytochrome P450 side-chain cleavage (scc) (Lieberman et al., 1984). This enzyme was first observed by Le Goascogne et al. (1987) who detected by immunocytochemistry the presence of immunoactive glial cells in the white matter throughout the rat brain. P450 scc protein has also been detected by Western blot analysis in the cerebellum and by immunochemistry in Purkinje cells (Tsutui, 1999; Ukena et al., 1998) as well as in pyramidal and granule neurons in the hippocampus of neonatal and adult rats (Kimoto et al., 2001). Moreover, the occurrence of mRNA encoding P450 scc has been demonstrated in mammalian brain by various approaches including reverse transcription polymerase chain reaction (RT-PCR), ribonuclease protection assay and in situ hybridization histochemistry (Compagnone et al., 1995; Mellon and Deschepper, 1993). In rat and mouse, the highest P450 scc gene

expression was found in the cerebral cortex. In the human brain, the highest concentrations of P450 scc mRNA were observed in the olfactory bulb, caudate nucleus, thalamus, corpus collosum, amygdala, hippocampus, cerebral cortex and cerebellum. The P450 scc protein was also identified in the human brain (Le Goascogne et al., 1989). Altogether, these results indicate that P450 scc is expressed in both neurons and glial cells in several brain areas.

3b-Hydroxysteroid dehydrogenase 3b-Hydroxysteroid dehydrogenase (3b-HSD) is a membrane-bound mitochondrial enzyme that catalyzes the conversion of D5-3b-hydroxysteroid into D4-3b-ketosteroids, leading to the formation of progesterone from pregnenolone and androstenedione from DHEA. In human, two isoforms of 3b-HSD have been characterized: type 1 is mainly expressed in the placenta, but also found in the skin and mammary gland (Luu-The et al., 1989; Rhéaume et al., 1991) while type 2 is predominantly expressed in the adrenal cortex and gonads (Rhéaume et al., 1991). Four isoforms of 3b-HSD have been described in rat (Zhao et al., 1990) and six isoforms in mouse (Simard et al., 1996). The first data on the presence of 3b-HSD in the brain have been provided by Weidenfeld et al., (1980), who showed that homogenates of rat amygdala and septum could convert pregnenolone into progesterone. Subsequent studies have confirmed the existence of bioactive 3b-HSD in brain tissues and primary cultures of oligodendrocytes and neurons (Bauer and Bauer, 1989; Robel et al., 1986; Ukena et al., 1999). Type 1 3b-HSD mRNA has been detected in several regions of the rat brain including the olfactory bulb, the olfactory tubercle, the caudate putamen, the nucleus accumbens, the cerebral cortex, the thalamus, the hypothalamus, the hippocampus, the septum, the medial habenular nucleus, the nucleus vestibularis lateralis, the nucleus vestibularis medialis, the nucleus vestibularis spinalis and the cerebellum (Dupont et al., 1994; Furukawa et al., 1998; Guennoun et al.,

195

1995; Kohchi et al., 1998; Meffre et al., 2007; Sanne and Krueger, 1995; Ukena et al., 1999). In the cerebellar cortex, 3b-HSD mRNA is expressed in Purkinje cells, granule cells and basket/stellate cells (Furukawa et al., 1998; Ukena et al., 1999). In other areas of the rat brain, the hybridization signal was only detected in neurons (Fig. 1) (Dupont et al., 1994; Furukawa et al., 1998; Guennoun et al., 1995; Kohchi et al., 1998; Meffre et al., 2007; Sanne and Krueger, 1995; Ukena et al., 1999). The type 1 3b-HSD gene is also expressed in the peripheral nervous system of rodents, particularly in dorsal root ganglion neurons and in Schwann cells of the sciatic nerve (Guennoun et al., 1997; Koenig et al., 1995; Robert et al., 2001). In the human brain, the presence of type 2 3b-HSD mRNA has been

demonstrated by real-time RT-PCR. The highest concentrations are found in the amygdala, the caudate nucleus, the corpus callosum, the hippocampus, the thalamus, the cerebellum and the spinal cord (Yu et al., 2002). Cultured human oligodendroglial, astroglial and neuronal cell lines all express 3b-HSD, suggesting that various types of glial cells have the capacity of synthesizing progesterone (Brown et al., 2000). The occurrence of 3b-HSD mRNA and/or protein has also been described in sub-mammalian vertebrates (Do Rego et al., 2009). In amphibians, the distribution of 3b-HSD has been determined by immunofluorescence in the brain of the frog Rana esculenta (Mensah-Nyagan et al., 1994) using an antiserum against the human placental type 1 3b-HSD in the brain of any vertebrates. In the frog brain, 3b-positive neurons are only found in diencephalic nuclei, notably in the anterior preoptic area, the posterior tuberculum, the nucleus of the periventricular organ, the suprachiasmatic nucleus, and the dorsal and ventral hypothalamic nuclei. A dense network of 3b-HSD-positive fibres is observed throughout the frog telencephalon, diencephalon and mesencephalon (Bruzzone et al., 2010; MensahNyagan et al., 1994). A similar distribution of 3b-HSD has been subsequently reported in hypothalamic nuclei of the newt Cynops pyrrhogaster (Inai et al., 2003).

Cytochrome P450C17

Fig. 1. Localization of 3b-hydroxysteroid dehydrogenase mRNA in the rat nucleus propositus hypoglossis. In situ hybridization was performed with a 35S-labelled cRNA probe. The silver grains (arrows) representing the hybridization signal are over the rough endoplasmic reticulum (RER) and nucleus (N) of a neuron. A synaptic contact (arrowhead) can be seen. × 15,000.

Cytochrome P450C17, also termed 17a-hydroxylase/C17.20 lyase, catalyzes the hydroxylation of C21 steroids (pregnenolone, progesterone) into C19 steroids (DHEA and androstenedione, respectively). Although the presence of DHEA and androstenedione in the brain is well documented (Akwa et al., 1991; Corpéchot et al., 1981, 1983; Jo et al., 1989; Lanthier and Patwardhan, 1986; Matsunaga et al., 2001; Mensah-Nyagan et al., 1996; Robel et al., 1986; Soma et al., 2004), the biosynthetic pathways leading to the formation of 17-hydroxylated steroids remain a matter of debate (Baulieu, 1998; Baulieu and Robel, 1998). Highly sensitive RT-PCR analyses failed to detect

196

the presence of P450C17 mRNA in any region of the adult rat brain (Mellon and Deschepper, 1993) while other studies revealed mRNA expression in the cerebellum and brainstem (Kohchi et al., 1998; Stromstedt and Waterman, 1995). Similarly, several attempts to demonstrate P450C17 in the brain using immunocytochemistry have been unsuccessful (Akwa et al., 1991; Le Goascogne et al., 1991) while other reports have described the presence of the immunoreactive protein in mesencephalic nuclei in the mouse embryo (Compagnone et al., 1995) and in the hippocampal formation in the adult rat (Hojo et al., 2004). In the hippocampus, P450C17 is localized to pyramidal neurons in the CA1–CA3 region and in granule cells of the dentate gyrus. The presence of P450C17 immunoreactivity has also been observed in hypothalamic periventricular neurons and in Purkinje cells of the cerebellar cortex (Yamada et al., 1997). In amphibians, the P450C17 enzyme has been localized in the brain by immunohistochemistry, using an antiserum against purified bovine testicular P450C17 (Do Rego et al., 2007). In the frog Rana esculenta, P450C17-immunoreactive cells are widely distributed in the telencephalon, diencephalon, mesencephalon and metencephalon (Do Rego et al., 2007). In particular, a dense accumulation of P450C17-positive neurons is observed in the periventricular regions of the diencephalon. Do Rego et al. (2007) has found a good correlation between the distribution of P450C17-positive cells and the localization of P450C17 bioactivity, indicating that the immunoreactivity detected in the frog brain corresponds to an active form of the enzyme.

(Moeller and Adamski, 2009). With the exception of 17b-HSD5, which is a member of the aldoketoreductase (AKR) family, all 17b-HSDs belong to the short-chain dehydrogenase/reductase (SDR) superfamily. In the mammalian brain, the localization of cells expressing different types of 17b-HSD has been investigated by immunocytochemistry, in situ hybridization and RT-PCR (Hojo et al., 2004, 2008; Mukai et al., 2006; Pelletier et al., 1995). In rodents, type 1 17b-HSD-like immunoreactivity is found in ependymal cells and astrocytes in the hippocampus, cerebral cortex, thalamus and hypothalamus (Hojo et al., 2008; Pelletier et al., 1995). As observed at the electron microscopic level, the enzyme immunoreactivity was observed throughout the cytoplasm without any association with organelles (Fig. 2). Expression of mRNAs encoding several isoforms of 17b-HSD has also been detected in the neonatal (Zwain and Yen, 1999) and adult rat brain (Hojo et al., 2004, 2008; Mukai et al., 2006). Very recently, using in situ hybridization, we have found both type 8 17b-HSD and type 10 17b-HSD mRNAs in the mouse brain. The hybridization signal could be detected in several hypothalamic nuclei including the arcuate, paraventricular, supraoptic and suprachiasmatic nuclei, in the hippocampus, the piriform cortex, the medial labenular nucleus, the facial nucleus,

17b-Hydroxysteroid dehydrogenase The enzyme 17b-hydroxysteroid dehydrogenase (17b-HSD) catalyzes the interconversion between the active and inactive forms of specific steroidal hormones in the final steps of their biosynthesis (Labrie et al., 2000). They are named according to their ability to catalyze oxidation or reduction of the 17-hydroxy or 17-keto functions of specific physiologically relevant steroids. Up to now, 15 types of 17b-HSDs are reported in vertebrates

Fig. 2. Immunoelectron microscopic localization of 17b-HSD type 1 in a glial cell in the rat median eminence. The gold particles (arrows) are observed over the cytoplasm without any specific association with organelles. No significant nuclear staining can be observed. N: nucleus. × 52,000.

197

(a)

(a)

(b)

(b)

Fig. 3. (a) Light microscope micrograph illustrating 17bhydroxysteroid dehydrogenase type 8 mRNA expression as studied by in situ hybridization using an antisense cRNA probe in the mouse cerebellar cortex. Accumulation of silver grains representing the in situ hybridization signal can be seen over the Purkinje cells (arrows). × 600. G: granular cells. (b) Section consecutive to that shown in (a) hybridized with the sense probe (negative control). Few dispersed grains can be observed.

the area postrema, the choroid plexus and cerebellar cortex (Purkinje cells) (Figs. 3 and 4). The occurrence of 17b-HSD mRNA and/or protein has also been investigated in the brain of sub-mammalian vertebrates. In amphibians, the distribution of 17b-HSD has been studied by immunohistochemistry using an antibody against type 1 human placental 17b-HSD (Dupont et al., 1991; Labrie et al., 1989; Mensah-Nyagan et al., 1996). In the brain of the frog Rana ridibunda, 17b-HSD-like immunoreactivity is contained in a sub-population of ependymal gliocytes bordering the lateral ventricles of the telencephalon in the medial pallium and the lateral septum. Networks of 17b-HSD-positive processes are seen in the anterior preoptic area, the corpus geniculatus

Fig. 4. (a) Light microscope micrograph showing 17bhydroxysteroid dehydrogenase type 10 mRNA expression as studied by in situ hybridization (radiolabelled antisense cRNA probe) in the mouse facial nucleus. Large motoneurons (arrows) are radiolabelled. (b) Section consecutive to that shown in (a) hybridized with the sense probe (negative control). Only a few grains can be detected. × 600.

lateralis and the thalamus (Mensah-Nyagan et al., 1996). 17b-HSD activity in the brain of mammals has been long suspected on the basis of occurrence of androgens and estrogens in cerebral tissue (Jaffe, 1969; Resko et al., 1979). Subsequently, it has been shown that monkey and human brain slices can convert D4 into T (Martel et al., 1992; Steckelbroeck et al., 1999) and DHEA into testosterone (T) and estradiol (Hojo et al., 2004; Kawato et al., 2002), thus confirming that the mammalian brain possesses 17b-HSD activity. In the frog brain, a good correlation is observed between the distribution of 17b-HSDimmunoreactive structures and the regional concentrations of T and 5a-dihydrotestosterone (5a-DHT) (Mensah-Nyagan et al., 1996). Moreover,

198

pulse-chase experiments with [3H]D5 progesterone as a precursor combined with high-performance liquid chromatography (HPLC) analysis have shown that frog brain explants are capable of synthesizing T and 5a-DHT (Do Rego et al., 2009), clearly indicating that the biosynthesis of androgens occurs in the brain of vertebrates.

5a-Reductase The enzyme 5a-reductase catalyzes the transformation of progesterone, T and 11-deoxycorticosterone into 5a-dihydroprogesterone, 5a-dihydrotestosterone and 5a-dihydrodeoxycorticosterone, respectively. Two 5a-reductase isozymes encoded by two distinct genes, designated type 1 and type 2, have been identified in rodents (Berman and Russell, 1993; Mahendroo et al., 1996; Normington and Russell, 1992), monkey (Levy et al., 1995) and human (Andersson et al., 1991; Labrie et al., 1992; Russell and Wilson, 1994). Both 5a-reductase isoforms are able to 5a-reduce all D4-3-ketosteroids, but they possess different optimal pH and substrate specificity, and thus distinct functional properties (Celotti et al., 1997; Normington and Russell, 1992; Wilson et al., 1993). 5a-Reductase type 1 is by far the most abundant molecular form in the brain of rat, mouse and human (Melcangi et al., 1998; Russel and Wilson, 1994; Steckelbroeck et al., 2001; Stoffel-Wagner, 2003; Thigpen et al., 1993; Torres and Ortega, 2003). In particular, in the rat brain, Northern blot analysis has shown the occurrence of high concentrations of 5a-reductase type 1 mRNA and much lower amounts of 5a-reductase type 2 mRNA (Lephart, 1993; Normington and Russell, 1992). In the rat brain, 5a-reductase immunoreactivity has been observed in glial and ependymal cells, including tanycytes (Pelletier et al., 1994). Immunolabelled cells were found in high concentration in the hypothalamus, thalamus, central cortex, cerebellar cortex (Fig. 5) and circumventricular organs. The 5a-reductase gene has been found to be primarily expressed in glial cells. At the ultrastructural level, the immunoreactive material appears to be distributed throughout the cytoplasm of glial cells without any particular

Fig. 5. Immunocytochemical localization of 5a-reductase type 1 in the rat cerebellar cortex. Bergmann cells (arrows) are immunolabelled while the Purkinje cells (P) do not contain any immunoreactive material. × 800.

association with mitochondria or other organelles (Kiyokage et al., 2005; Pelletier et al., 1994). On the other hand, in the mouse brain, 5a-reductase type 1 mRNA and immunoreactivity were detected in neurons, but not in glial cells (Agis-Balboa et al., 2006). 5a-Reductase type 1positive cells are mainly found in the mitral cell layer of the olfactory bulb, in layers 2, 3 and 5 of the neocortex, in the CA1–CA3 and dentate gyrus of the hippocampus, in the amygdala, and in granule and Purkinje cells of the cerebellum (Agis-Balboa et al., 2006). In the human brain, 5a-reductase type 1 mRNA has been detected in the temporal cortex, subcortical white matter and hippocampal tissue obtained from patients with chronic temporal lobe epilepsy (Stoffel-Wagner et al., 2000) as well as in the cerebellum, hypothalamus and pons from post-partum brains (Thigpen et al., 1993). In non-mammalian vertebrates, the distribution of 5a-reductase has been investigated in the brain of amphibians during development (Bruzzone et al., 2010; Vallarino et al., 2005) and in adult fish (Mathieu et al., 2001). In frog Rana esculenta tadpoles, 5a-reductase immunoreactivity was detected in both neurons and glial cells in the telencephalon, diencephalon, mesencephalon and rhombencephalon. It is noteworthy that the existence of 5a-reductase activity in the brain of mammals is well established

199

(Martini et al., 1996; Melcangi et al., 1993; NegriCesi et al., 1996; Steckelbroeck et al., 2001; Thigpen et al., 1993). In particular, rat brain homogenates are capable of converting progesterone into the 5a-reductase metabolite 5a-dehydroprogesterone (Celotti et al., 1992). Similarly, 5a-reductase activity has been shown in the human brain (Celotti et al., 1986; Steckelbroeck et al., 2001; StoffelWagner et al., 2000). These biochemical results are in agreement with the methodological findings showing the expression of 5a-reductase mRNA and 5a-reductase immunoreactivity.

3a-Hydroxysteroid dehydrogenase 3a-Hydroxysteroid dehydrogenase (3a-HSD) is a bifunctional enzyme that interconverts, in a reversible manner, the 5a-reduced steroids (5a-DHT and 5a-dihydroprogesterone (5a-DHP) into 3aandrostanediol and 3a,5a-tetrahydroprogesterone, respectively). 3a-HSD also catalyzes the reversible conversion of dihydrodeoxycorticosterone into tetrahydrodeoxycorticosterone. In humans, multiple cDNAs encoding various proteins structurally related to 3a-HSD have been reported (Qin et al., 1993). However, to date, only four functional isoforms of 3a-HSD have been characterized on the basis of their affinity for 5a-DHT (Khanna et al., 1995a, 1995b; Pawlowski et al., 1991; Penning et al., 2000, 2003). In contrast, rodents possess a single 3a-HSD isozyme that mediates all the oxidative and reductive reactions (Hara et al., 1988; Hoog et al., 1994; Pawlowski et al., 1991; Penning et al., 2003). In mammals, the presence of 3a-HSD mRNA and/or protein has been described in several brain regions. In rat, the highest concentration of 3a-HSD protein occurs in the olfactory bulb, while lower levels of the enzyme are found in the cerebral cortex, hypothalamus, cerebellum and pituitary (Compagnone and Mellon, 2000; Khanna et al., 1995b). In vitro and in vivo studies have demonstrated the presence of 3a-HSD bioactivity in the brain of rodents (Eechaute et al., 1999; Khanna et al., 1995a; Krieger and Scott, 1984; Martini et al., 1996; Penning, 1996) and primates (Bonsall et al., 1989, 1990). In rat, the highest activity is found

in the olfactory bulb and olfactory tubercle (Khanna et al., 1995a; Krieger and Scott, 1989; Penning 1996) indicating that the 3a-HSD protein detected in this region (Compagnone and Mellon, 2000; Khanna et al., 1995a) corresponds to a biologically active form of the enzyme. In the mouse brain, in situ hybridization studies have shown intense expression of 3a-HSD mRNA in the mitral and periglomerular cells of the olfactory bulb, in pyramidal neurons of the cerebral cortex, in glutamatergic neurons of the hypothalamic dorsomedial nucleus and in Purkinje cells of the cerebellum, and have revealed the existence of additional populations of 3a-HSD neurons in the CA1–CA3 region of the hippocampus and granule cells of the dentate gyrus (Agis-Balboa et al., 2006). In the human brain, both 3a-HSD type 2 and 3 mRNAs are widely expressed (Griffin and Mellon, 1999). In particular, 3a-HSD type 2 mRNA is found in the fontotemporal lobes, putamen, medulla and spinal cord (Griffin and Mellon, 1999) as well as in the subcortical white matter (Steckelbroeck et al., 2001), while 3a-HSD type 3 mRNA is mainly expressed in the putamen, cerebellum, medulla and spinal cord (Griffin and Mellon, 1999).

20a-Hydroxysteroid dehydrogenase The enzyme 20a-hydroxysteroid dehydrogenase (20a-HSD) catalyzes the conversion of progesterone into its inactive form, 20a-hydroxyprogesterone (Zhang et al., 2000). In the brain of several species, conversion of progesterone into 20a-reduced metabolites has been reported, indicating that some brain areas have 20a-HSD activity (Karavolas and Hodges, 1990). Recently, with a cRNA probe that has been successfully used to localize 20a-HSD by in situ hybridization in a variety of mouse tissues (Pelletier et al., 2003), we performed the localization of 20a-HSD mRNA in mouse brains. We demonstrated for the first time that 20a-HSD mRNA is expressed in neurons in the frontal, parietal, temporal and visual cortex as well as in the hippocampus (Pelletier et al., 2004). In the cortex, they are mostly concentrated in the external granular

200

(a)

of locally produced progesterone for progesterone receptors, and thus control the influence of progesterone on neuronal activity.

Cytochrome P450 aromatase

(b)

Fig. 6. (a) Light microscope micrograph illustrating the localization of 20a-hydroxysteroid dehydrogenase mRNA in the CAI layer of the mouse hippocampus as studied by in situ hybridization (radiolabelled antisense cRNA probe). A few pyramidal neurons (arrows) exhibit radiolabelling × 600. (b) Consecutive section hybridized with the sense probe (negative control). Only diffuse labelling can be observed. × 600.

layer, the external pyramidal layer and the inner granular layer. The molecular layer is generally devoid of any specific reaction. In the hippocampus, the labelling is mostly detected over pyramidal cells of the CAI layer (Fig. 6). Occasionally, labelled neurons were detected in the layer dorsal to the CAI layer. These findings suggest that neurons in cortical and hippocampal areas can convert progesterone into its inactive form, 20a-progesterone. Interestingly, progesterone receptors have been localized in neurons of the external granular layer, external pyramidal layer, inner granular layer and inner pyramidal layer of the cortex as well as pyramidal cells of the CA1 layer of the hippocampus, which also express 20a-HSD mRNA (Kato et al., 1994). 20a-HSD might therefore regulate the availability

Cytochrome P450 aromatase (P450arom) catalyzes the conversion of C19 androgens (androstenedione and testosterone) into C18 estrogens (estrone and estradiol, respectively). The presence of P450arom mRNA in the brain of mammals has been demonstrated by RT-PCR and in situ hybridization histochemistry (Harada and Yamada, 1992; Hojo et al., 2004; Lephart et al., 1992). In rat and mouse, intense expression of the P450arom gene occurs in the cerebral cortex, medial preoptic nucleus, bed nucleus of the stria terminalis, medial amygdala and hippocampus. The expression of the P450arom protein has been investigated in the brain of rodents by using antibodies against human placental P450arom. In rat, numerous P450arom-immunoreactive cells are observed in amygdaloid structures and in the supraoptic nucleus. A moderate density of positive cells is detected in the paraventricular and arcuate nuclei, the CA1–CA3 regions and dentate gyrus of the hippocampus, the subincerta nucleus, the paraventricular, lateral and dorsomedial hypothalamic nuclei (Hojo et al., 2004; Jakab et al., 1993, 1994; Sanghera et al., 1991). Double immunohistochemical labelling using specific markers have shown that P450arom is expressed only in neurons and not in glial cells (Beyer et al., 1994; Jakab et al., 1994; Lephart, 1996). In human brain, P450arom mRNA has been found to be widely expressed in the frontal and temporal cortex, subcortical white matter, hippocampus, thalamus, hypothalamus and pons (Sasano et al., 1998; Stoffel-Wagner et al., 1998; Yague et al., 2006). The presence of the P450arom protein has been confirmed by immunohistochemistry in the cerebral cortex, limbic system and hypothalamus (Ishunina et al., 2005; Naftolin et al., 1996; Yague et al., 2006). In human, immunoreactive P450arom is found not only in neurons but also in a sub-population of astroglial cells (Ishunina

201

et al., 2005; Naftolin et al., 1996; Yague et al., 2006). So far there have been several reports indicating that brain tissue homogenates from mammals, including primates, are able to catalyze the conversion of androstenedione into estrone and estradiol (Naftolin et al., 1975; Roselli et al., 1985, 1996; Steckelbroek et al., 1999). Moreover, localization of P450arom mRNA and immunoreactive neurons matches fairly well with the regional distribution of the enzymatic activity in the brain (Abdelgadir et al., 1997; George and Ojeda, 1982; MacLusky et al., 1986; Roselli et al., 1985; Steckelbroeck et al., 1999).

11b-Hydroxysteroid dehydrogenase The interconversion of glucocorticoids from their inactive (11-dehydrocorticosterone, cortisone) to their active forms (corticosterone, cortisol) is catalyzed by the enzyme 11b-hydroxysteroid dehydrogenase (11b-HSD) in specific tissues. Molecular cloning of the cDNAs encoding 11b-HSD revealed the existence of two isoforms of the enzyme, 11b-HSD type 1 and type 2, in humans and rodents (Tannin et al., 1991; Zhou et al., 1995). 11b-HSD type 1 is a bidirectional enzyme, predominantly displaying oxo-reductase activity, while 11b-HSD type 2 has potent dehydrogenase activity, inactivating glucocorticoids (Holmes and Seckl, 2006; Sandeep and Walker, 2001; Stewart and Krozowski, 1999). Using stereotaxic injection of radiolabelled corticosterone and dehydrocorticosterone into the rat hippocampus, Jellinck et al., (1999) demonstrated equal conversion of each steroid to the other one. In contrast, in cultured hippocampal cells, Rajan et al., (1996) showed that 11b-HSD type 1 acts primarily as a reductase by catalyzing the conversion of 11-dehydrocorticosterone to corticosterone. In the rat forebrain, Moisan et al., (1990) reported that 11b-HSD mRNA expression, as studied by in situ hybridization, could be found in the cerebral cortex, hippocampus and some hypothalamic areas such as medial preoptic area and arcuate nucleus. Although these authors did

not identify what 11b-HSD type was detected, it is likely that it was the type 1. In 11b-HSD type 1 knockout mice, hippocampal corticosterone levels are lower than those observed in wild-type controls (Yau and Seckl, 2001). 11b-HSD type 2 is expressed in the developing rat brain, but is switched off after brain has reached its full development (Diaz et al., 1998; Holmes and Seckl, 2006). Recently, we proceeded to the localization of 11b-HSD type 1 and type 2 mRNA using in situ hybridization in the male mouse brain (Pelletier et al., 2007). 11b type 1 was mostly localized in the cerebral cortex, hippocampus, amygdale, medial preoptic area and caudate-putamen nucleus, with the highest reaction in the cerebral cortex and hippocampus. As observed at the light microscopic level, in the hippocampus, a large number of neurons were labelled. They were particularly abundant in the stratum lucidum; CA1, CA2 and CA3 areas; molecular layer and gyrus dentatus (polymorph layer) (Fig. 7). In the cerebral cortex, radiolabelled cells were mostly found in layers 2 and 3 in the parietal cortex, with the highest concentration in the retrosplenial granular area. In

Fig. 7. (a) Light microscope micrograph showing the localization of 11b-hydroxysteroid dehydrogenase mRNA in the gyrus dentatus of the mouse hippocampus, as studied by in situ hybridization (radiolabelled antisense cRNA probe). Labelled neurons (arrows) can be seen in the granular and polymorph layers (arrows). MO: molecular layer. × 600. (b) Consecutive section hybridized with the sense probe (negative control). Only weak diffuse labelling can be detected. × 600.

202

the frontal and cingulated cortex, modest expression of 11b-HSD1 mRNA was also observed. Cells expressing the enzyme mRNA were also detected in the anterolateral area of the amygdale, medial preoptic area and caudate-putamen nucleus. High hybridization signal was also observed in the vasculature of choroid plexus and meninges. No 11b-HSD type 2 mRNA could be detected in the brain, while high expression of the enzyme mRNA could be observed in the kidney. These results suggest that in some brain areas, there is a local regulation of the levels of active glucocorticoids (cortisol, corticosterone).

Conclusion Since the first observation by Beaulieu and his collaborators (Corpéchot et al., 1981) that some hormonal steroids were in higher concentrations in the brain than in blood, there have been a large number of reports on the expression of steroidogenic enzymes in the brain. So far, most of the enzymes involved in the biosynthesis and metabolism of all the categories of steroid hormones (sex steroids and glucocorticoids) have been found to be expressed in the brain of many representative species of vertebrates. The concept of neurosteroidogenesis is now strongly supported by the occurrence of steroidogenic enzymes in nerve cells. The application of immunocytochemical and in situ hybridization techniques has contributed to determine the exact neuroanatomical distribution of key enzymes such as cytochrome P450 scc, 3b-HSD, cytochrome P450C17, 17b-HSD, P450 aromatase, 5a-reductase and 11b-HSD. The enzymes have been clearly shown to be expressed in both neurons and glial cells often largely distributed throughout the brain with some species-related variations. Interestingly, the presence of enzymatic activities for steroid biosynthesis and metabolism has also been demonstrated in neurons and/or glial cells. Contrary to classical steroid-secreting cells that contain several steroidogenic enzymes involved in the biosynthesis of active steroids from cholesterol, the nerve cells often possess only one enzyme. The synthesis of neuroactive

steroids such as dihydrotestosterone or allopregnenolone that requires the action of several enzymes involves movement of precursor molecules from one cell type to the other one, often from neurons to glial cells or the reverse. Clearly, more work is required to fully establish the migration of precursors and active steroids in the brain. References Abdelgadir, S. E., Roselli, C. E., Choate, J. V., & Resko, J. A. (1997). Distribution of aromatase cytochrome P450 messenger ribonucleic acid in adult rhesus monkey brains. Biology of Reproduction, 57, 772–777. Agis-Balboa, R. C., Pinna, G., Zhubi, A., Maloku, E., Veldic, M., Costa, E., et al. (2006). Characterization of brain neurons that express enzymes mediating neurosteroid biosynthesis. Proceedings of the National Academy of Sciences of the United States of America, 103, 14602–14607. Akwa, Y., Young, J., Kabbadj, K., Sancho, M. J., Zucman, D., Vourc’h, C., et al. (1991). Neurosteroids: biosynthesis, metabolism and function of pregnenolone and dehydroepiandrosterone in the brain. The Journal of Steroid Biochemistry and Molecular Biology, 40, 71–81. Andersson, S., Berman, D. M., Jenkins, E. P., & Russell, D. W. (1991). Deletion of steroid 5 alpha-reductase 2 gene in male pseudohermaphroditism. Nature, 354, 159–161. Bauer, H. C. & Bauer, H. (1989). Micromethod for the determination of 3-beta-HSD activity in cultured cells. The Journal of Steroid Biochemistry, 33, 643–646. Baulieu, E. E. (1997). Neurosteroids: of the nervous system, by the nervous system, for the nervous system. Recent Progress in Hormone Research, 52, 1–32. Baulieu, E. E. (1998). Neurosteroids: a novel function of the brain. Psychoneuroendocrinology, 23, 963–987. Baulieu, E. E. & Robel, P. (1998). Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) as neuroactive neurosteroids. Proceedings of the National Academy of Sciences of the United States of America, 95, 4089–4091. Berman, D. M. & Russell, D. W. (1993). Cell-type-specific expression of rat steroid 5 alpha-reductase isozymes. Proceedings of the National Academy of Sciences of the United States of America, 90, 9359–9363. Beyer, C., Green, S. J., Barker, P. J., Huskisson, N. S., & Hutchison, J. B. (1994). Aromatase-immunoreactivity is localised specifically in neurones in the developing mouse hypothalamus and cortex. Brain Research, 638, 203–210. Bonsall, R. W., Rees, H. D., & Michael, R. P. (1989). Identification of radioactivity in cell nuclei from brain, pituitary gland and genital tract of male rhesus monkeys after the administration of [3h]testosterone. The Journal of Steroid Biochemistry, 32, 599–608. Bonsall, R. W., Zumpe, D., & Michael, R. P. (1990). Comparisons of the nuclear uptake of [3h]-testosterone and its

203 metabolites by the brains of male and female macaque fetuses at 122 days of gestation. Neuroendocrinology, 51, 474–480. Brown, R. C., Cascio, C., & Papadopoulos, V. (2000). Pathways of neurosteroid biosynthesis in cell lines from human brain: regulation of dehydroepiandrosterone formation by oxidative stress and beta-amyloid peptide. Journal of Neurochemistry, 74, 847–859. Bruzzone, F., Do Rego, J. L., Luu-The, V., Pelletier, G., Vallarino, M., & Vaudry, H. (2010). Immunohistochemical localization and biological activity of 3beta-hydroxysteroid dehydrogenase and 5alpha-reductase in the brain of the frog, Rana esculenta, during development. Journal of Chemical Neuroanatomy, 50, 35–50. Celotti, F., Melcangi, R. C., & Negri-Cesi, P. (1986). A comparative study of the metabolism of testosterone in the neuroendocrine structures of several animal species. Neuroendocrinology Letters, 8, 227–236. Celotti F., Melcangi R., & Martini L. (1992). The 5a-reductase in the brain: molecular aspects and relation to the brain function. Front Neuroendocrinol, 13, 163–215. Celotti, F., Negri-Cesi, P., & Poletti, A. (1997). Steroid metabolism in the mammalian brain: 5alpha-reduction and aromatization. Biology of Reproduction, 44, 365–375. Compagnone, N. A., Bulfone, A., Rubenstein, J. L., & Mellon, S. H. (1995). Steroidogenic enzyme P450c17 is expressed in the embryonic central nervous system. Endocrinology, 136, 5212–5223. Compagnone, N. A. & Mellon, S. H. (2000). Neurosteroids: biosynthesis and function of these novel neuromodulators. Frontiers in Neuroendocrinology, 21, 1–56. Corpechot, C., Robel, P., Axelson, M., Sjovall, J., & Baulieu, E. E. (1981). Characterization and measurement of dehydroepiandrosterone sulfate in rat brain. Proceedings of the National Academy of Sciences of the United States of America, 78, 4704–4707. Diaz, R., Brown, R. W., & Seckl, J. R. (1998). Distinct ontogeny of glucocorticoid and mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase types I and II mRNAs in the fetal rat brain suggest a complex control of glucocorticoid actions. Journal of Neuroscience, 18, 2570–2580. Do Rego, J. L., Seong, J. Y., Burel, D., Leprince, J., Luu-The, V., Tsutsui, K., et al. (2009). Neurosteroid biosynthesis: enzymatic pathways and neuroendocrine regulation by neurotransmitters and neuropeptides. Frontiers in Neuroendocrinology, 30, 259–301. Do Rego, J. L., Tremblay, Y., Luu-The, V., Repetto, E., Castel, H., Vallarino, M., et al. (2007). Immunohistochemical localization and biological activity of the steroidogenic enzyme cytochrome P450 17alpha-hydroxylase/C17, 20-lyase (P450C17) in the frog brain and pituitary. Journal of Neurochemistry, 100, 251–268. Dupont, E., Labrie, F., Luu-The, V., & Pelletier, G. (1991). Localization of 17 beta-hydroxysteroid dehydrogenase throughout gestation in human placenta. The Journal of Histochemistry and Cytochemistry, 39, 1403–1407.

Dupont, E., Simard, J., Luu-The, V., Labrie, F., & Pelletier, G. (1994). Localization of 3 beta-hydroxysteroid dehydrogenase in rat brain as studied by in situ hybridization. Molecular and Cellular Neuroscience, 5, 119–123. Eechaute, W. P., Dhooge, W. S., Gao, C. Q., Calders, P., Rubens, R., Weyne, J., et al. (1999). Progesterone-transforming enzyme activity in the hypothalamus of the male rat. The Journal of Steroid Biochemistry and Molecular Biology, 70, 159–167. Furukawa, A., Miyatake, A., Ohnishi, T., & Ichikawa, Y. (1998). Steroidogenic acute regulatory protein (StAR) transcripts constitutively expressed in the adult rat central nervous system: colocalization of StAR, cytochrome P-450SCC (CYP XIA1), and 3beta-hydroxysteroid dehydrogenase in the rat brain. Journal of Neurochemistry, 71, 2231–2238. George, F. W. & Ojeda, S. R. (1982). Changes in aromatase activity in the rat brain during embryonic, neonatal, and infantile development. Endocrinology, 111, 522–529. Goascogne, C., Gouezou, M., Robel, P., Defaye, G., Chambaz, E., Waterman, M.R., et al. (1989). The cholesterol side-chain cleavage complex in human brain white matter. Journal of Neuroendocrinology, 1, 153–156. Griffin, L. D. & Mellon, S. H. (1999). Selective serotonin reuptake inhibitors directly alter activity of neurosteroidogenic enzymes. Proceedings of the National Academy of Sciences of the United States of America, 96, 13512–13517. Guennoun, R., Fiddes, R. J., Gouezou, M., Lombes, M., & Baulieu, E. E. (1995). A key enzyme in the biosynthesis of neurosteroids, 3 beta-hydroxysteroid dehydrogenase/delta 5delta 4-isomerase (3 beta-HSD), is expressed in rat brain. Brain Research. Molecular Brain Research, 30, 287–300. Guennoun, R., Schumacher, M., Robert, F., Delespierre, B., Gouezou, M., Eychenne, B., et al. (1997). Neurosteroids: expression of functional 3beta-hydroxysteroid dehydrogenase by rat sensory neurons and schwann cells. European Journal of Neuroscience, 9, 2236–2247. Hara, A., Inoue, Y., Nakagawa, M., Naganeo, F., & Sawada, H. (1988). Purification and characterization of NADP+-dependent 3 alpha-hydroxysteroid dehydrogenase from mouse liver cytosol. Journal of Biochemistry, 103, 1027–1034. Harada, N. & Yamada, K. (1992). Ontogeny of aromatase messenger ribonucleic acid in mouse brain: fluorometrical quantitation by polymerase chain reaction. Endocrinology, 131, 2306–2312. Hojo, Y., Hattori, T. A., Enami, T., Furukawa, A., Suzuki, K., Ishii, H. T., et al. (2004). Adult male rat hippocampus synthesizes estradiol from pregnenolone by cytochromes P45017alpha and P450 aromatase localized in neurons. Proceedings of the National Academy of Sciences of the United States of America, 101, 865–870. Hojo, Y., Murakami, G., Mukai, H., Higo, S., Hatanaka, Y., Ogiue-Ikeda, M., et al. (2008). Estrogen synthesis in the brain–role in synaptic plasticity and memory. Molecular and Cellular Endocrinology, 290, 31–43. Holmes, M. C. & Seckl, J. R. (2006). The role of 11betahydroxysteroid dehydrogenases in the brain. Molecular and Cellular Endocrinology, 248, 9–14.

204 Hoog, S. S., Pawlowski, J. E., Alzari, P. M., Penning, T. M., & Lewis, M. (1994). Three-dimensional structure of rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase: a member of the aldo-keto reductase superfamily. Proceedings of the National Academy of Sciences of the United States of America, 91, 2517–2521. Inai, Y., Nagai, K., Ukena, K., Oishi, T., & Tsutsui, K. (2003). Seasonal changes in neurosteroid concentrations in the amphibian brain and environmental factors regulating their changes. Brain Research, 959, 214–225. Ishunina, T. A., van Beurden, D., van der Meulen, G., Unmehopa, U. A., Hol, E. M., Huitinga, I., et al. (2005). Diminished aromatase immunoreactivity in the hypothalamus, but not in the basal forebrain nuclei in alzheimer’s disease. Neurobiology of Aging, 26, 173–194. Jaffe, R. B. (1969). Testosterone metabolism in target tissues. Hypothalamic and pituitary tissues of the adult rat and human fetus, and the immature rat epiphysis. Steroids, 14, 483–498. Jakab, R. L., Harada, N., & Naftolin, F. (1994). Aromatase(estrogen synthetase) immunoreactive neurons in the rat septal area. A light and electron microscopic study. Brain Research, 664, 85–93. Jakab, R. L., Horvath, T. L., Leranth, C., Harada, N., & Naftolin, F. (1993). Aromatase immunoreactivity in the rat brain: gonadectomy-sensitive hypothalamic neurons and an unresponsive “limbic ring” of the lateral septum-bed nucleusamygdala complex. The Journal of Steroid Biochemistry and Molecular Biology, 44, 481–498. Jellinck, P. H., Pavlides, C., Sakai, R. R., & McEwen, B. S. (1999). 11beta-hydroxysteroid dehydrogenase functions reversibly as an oxidoreductase in the rat hippocampus in vivo. The Journal of Steroid Biochemistry and Molecular Biology, 71, 139–144. Jo, D. H., Abdallah, M. A., Young, J., Baulieu, E. E., & Robel, P. (1989). Pregnenolone, dehydroepiandrosterone, and their sulfate and fatty acid esters in the rat brain. Steroids, 54, 287–297. Karavolas, H. J. & Hodges, D. R. (1990). Neuroendocrine metabolism of progesterone and related progestins. Ciba Foundation Symposium, 153(22–44), discussion 44–55. Kato, J., Hirata, S., Nozawa, A., & Yamada-Mouri, N. (1994). Gene expression of progesterone receptor isoforms in the rat brain. Hormones and Behavior, 28, 454–463. Kawato, S., Hojo, Y., & Kimoto, T. (2002). Histological and metabolism analysis of P450 expression in the brain. Methods in Enzymology, 357, 241–249. Khanna, M., Qin, K. N., & Cheng, K. C. (1995). Distribution of 3 alpha-hydroxysteroid dehydrogenase in rat brain and molecular cloning of multiple cDNAs encoding structurally related proteins in humans. The Journal of Steroid Biochemistry and Molecular Biology, 53, 41–46. Khanna, M., Qin, K. N., Wang, R. W., & Cheng, K. C. (1995). Substrate specificity, gene structure, and tissue-specific distribution of multiple human 3 alpha-hydroxysteroid dehydrogenases. The Journal of Biological Chemistry, 270, 20162–20168.

Kimoto, T., Tsurugizawa, T., Ohta, Y., Makino, J., Tamura, H., Hojo, Y., et al. (2001). Neurosteroid synthesis by cytochrome p450-containing systems localized in the rat brain hippocampal neurons: N-methyl-D-aspartate and calcium-dependent synthesis. Endocrinology, 142, 3578–3589. Kiyokage, E., Toida, K., Suzuki-Yamamoto, T., & Ishimura, K. (2005). Localization of 5alpha-reductase in the rat main olfactory bulb. The Journal of Comparative Neurology, 493, 381–395. Koenig, H. L., Schumacher, M., Ferzaz, B., Thi, A. N., Ressouches, A., Guennoun, R., et al. (1995). Progesterone synthesis and myelin formation by schwann cells. Science, 268, 1500–1503. Kohchi, C., Ukena, K., & Tsutsui, K. (1998). Age- and regionspecific expressions of the messenger RNAs encoding for steroidogenic enzymes p450scc, P450c17 and 3beta-HSD in the postnatal rat brain. Brain Research, 801, 233–238. Krieger, N. R. & Scott, R. G. (1984). 3 Alpha-hydroxysteroid oxidoreductase in rat brain. Journal of Neurochemistry, 42, 887–890. Krieger, N. R. & Scott, R. G. (1989). Nonneuronal localization for steroid converting enzyme: 3 alpha-hydroxysteroid oxidoreductase in olfactory tubercle of rat brain. Journal of Neurochemistry, 52, 1866–1870. Labrie, F., Luu-The, V., Labrie, C., Berube, D., Couet, J., Zhao, H. F., et al. (1989). Characterization of two mRNA species encoding human estradiol 17 beta-dehydrogenase and assignment of the gene to chromosome 17. The Journal of Steroid Biochemistry, 34, 189–197. Labrie, F., Luu-The, V., Lin, S. X., Simard, J., & Labrie, C. (2000). Role of 17 beta-hydroxysteroid dehydrogenases in sex steroid formation in peripheral intracrine tissues. Trends in Endocrinology and Metabolism, 11, 421–427. Labrie, F., Sugimoto, Y., Luu-The, V., Simard, J., Lachance, Y., Bachvarov, D., et al. (1992). Structure of human type II 5 alpha-reductase gene. Endocrinology, 131, 1571–1573. Lanthier, A. & Patwardhan, V. V. (1986). Sex steroids and 5en-3 beta-hydroxysteroids in specific regions of the human brain and cranial nerves. The Journal of Steroid Biochemistry, 25, 445–449. Le Goascogne, C., Robel, P., Gouezou, M., Sananes, N., Baulieu, E. E., & Waterman, M. (1987). Neurosteroids: cytochrome P-450scc in rat brain. Science, 237, 1212–1215. Le Goascogne, C., Sananes, N., Gouezou, M., Takemori, S., Kominami, S., Baulieu, E. E., et al. (1991). Immunoreactive cytochrome P-450(17 alpha) in rat and guinea-pig gonads, adrenal glands and brain. Journal of Reproduction and Fertility, 93, 609–622. Lephart, E. D. (1993). Brain 5alpha-reductase: cellular, enzymatic, and molecular perspectives and implications for biological function. Molecular and Cellular Neuroscience, 4, 473–484. Lephart, E. D. (1996). A review of brain aromatase cytochrome P450. Brain Research Brain Research Reviews, 22, 1–26. Lephart, E. D., Simpson, E. R., McPhaul, M. J., Kilgore, M. W., Wilson, J. D., & Ojeda, S.R. (1992). Brain aromatase

205 cytochrome P-450 messenger RNA levels and enzyme activity during prenatal and perinatal development in the rat. Brain Research Molecular Brain Research, 16, 187–192. Levy, M. A., Brandt, M., Sheedy, K. M., Holt, D. A., Heaslip, J. I., Trill, J. J., et al. (1995). Cloning, expression and functional characterization of type 1 and type 2 steroid 5 alpha-reductases from cynomolgus monkey: comparisons with human and rat isoenzymes. The Journal of Steroid Biochemistry and Molecular Biology, 52, 307–319. Lieberman, S., Greenfield, N. J., & Wolfson, A. (1984). A heuristic proposal for understanding steroidogenic processes. Endocrine Reviews, 5, 128–148. Luu The, V., Lachance, Y., Labrie, C., Leblanc, G., Thomas, J. L., Strickler, R.C., et al. (1989). Full length cDNA structure and deduced amino acid sequence of human 3 beta-hydroxy-5-ene steroid dehydrogenase. Molecular Endocrinology, 3, 1310–1312. MacLusky, N. J., Naftolin, F., & Goldman-Rakic, P. S. (1986). Estrogen formation and binding in the cerebral cortex of the developing rhesus monkey. Proceedings of the National Academy of Sciences of the United States of America, 83, 513–516. Mahendroo, M. S., Cala, K. M., & Russell, D.W. (1996). 5 Alpha-reduced androgens play a key role in murine parturition. Molecular Endocrinology, 10, 380–392. Martel, C., Rheaume, E., Takahashi, M., Trudel, C., Couet, J., Luu-The, V., et al. (1992). Distribution of 17 beta-hydroxysteroid dehydrogenase gene expression and activity in rat and human tissues. The Journal of Steroid Biochemistry and Molecular Biology, 41, 597–603. Martini, L., Celotti, F., & Melcangi, R. C. (1996). Testosterone and progesterone metabolism in the central nervous system: cellular localization and mechanism of control of the enzymes involved. Cellular and Molecular Neurobiology, 16, 271–282. Mathieu, M., Mensah-Nyagan, A. G., Vallarino, M., Do-Rego, J. L., Beaujean, D., Vaudry, D., et al. (2001). Immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase in the brain of the african lungfish protopterus annectens. The Journal of Comparative Neurology, 438, 123–135. Matsunaga M, Ukena K, Tsutsui K (2001). Expression and localization of cytochrome P450 17a-hydroxylase/c17, 20lyase in the avian brain, Brain Res 899, 112–122. Meffre, D., Delespierre, B., Gouezou, M., Schumacher, M., Stein, D. G., & Guennoun, R. (2007). 3Beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase mRNA expression in rat brain: effect of pseudopregnancy and traumatic brain injury. The Journal of Steroid Biochemistry and Molecular Biology, 104, 293–300. Melcangi, R. C., Celotti, F., Castano, P., & Martini, L. (1993). Differential localization of the 5 alpha-reductase and the 3 alpha-hydroxysteroid dehydrogenase in neuronal and glial cultures. Endocrinology, 132, 1252–1259. Melcangi, R. C., Poletti, A., Cavarretta, I., Celotti, F., Colciago, A., Magnaghi, V., et al. (1998). The 5alpha-reductase in the central nervous system: expression and modes of control. The Journal of Steroid Biochemistry and Molecular Biology, 65, 295–299.

Mellon, S. H. & Deschepper, C. F. (1993). Neurosteroid biosynthesis: genes for adrenal steroidogenic enzymes are expressed in the brain. Brain Research, 629, 283–292. Mellon, S. H. & Vaudry, H. (2001). Biosynthesis of neurosteroids and regulation of their synthesis. International Review of Neurobiology, 46, 33–78. Mensah-Nyagan, A. G., Do-Rego, J. L., Beaujean, D., LuuThe, V., Pelletier, G., & Vaudry, H. (1999). Neurosteroids: expression of steroidogenic enzymes and regulation of steroid biosynthesis in the central nervous system. Pharmacological Reviews, 51, 63–81. Mensah-Nyagan, A. G., Feuilloley, M., Dupont, E., DoRego, J. L., Leboulenger, F., Pelletier, G., et al. (1994). Immunocytochemical localization and biological activity of 3 beta-hydroxysteroid dehydrogenase in the central nervous system of the frog. Journal of Neuroscience, 14, 7306–7318. Mensah-Nyagan, A. M., Feuilloley, M., Do-Rego, J.L., Marcual, A., Lange, C., Tonon, M.C., et al. (1996). Localization of 17beta-hydroxysteroid dehydrogenase and characterization of testosterone in the brain of the male frog. Proceedings of the National Academy of Sciences of the United States of America, 93, 1423–1428. Moeller, G. & Adamski, J. (2009). Integrated view on 17betahydroxysteroid dehydrogenases. Molecular and Cellular Endocrinology, 301, 7–19. Moisan, M.P., Seckl, J. R., & Edwards, C. R. (1990). 11 Beta-hydroxysteroid dehydrogenase bioactivity and messenger RNA expression in rat forebrain: localization in hypothalamus, hippocampus, and cortex. Endocrinology, 127, 1450–1455. Mukai, H., Tsurugizawa, T., Ogiue-Ikeda, M., Murakami, G., Hojo, Y., Ishii, H., et al. (2006). Local neurosteroid production in the hippocampus: influence on synaptic plasticity of memory. Neuroendocrinology, 84, 255–263. Naftolin, F., Horvath, T. L., Jakab, R. L., Leranth, C., Harada, N., & Balthazart, J. (1996). Aromatase immunoreactivity in axon terminals of the vertebrate brain. An immunocytochemical study on quail, rat, monkey and human tissues. Neuroendocrinology, 63, 149–155. Naftolin, F., Ryan, K. J., Davies, I. J., Petro, Z., & Kuhn, M. (1975). The formation and metabolism of estrogens in brain tissues. Advanced Bioscience, 15, 105–121. Negri-Cesi, P., Poletti, A., & Celotti, F. (1996). Metabolism of steroids in the brain: a new insight into the role of 5alphareductase and aromatase in brain differentiation and functions. The Journal of Steroid Biochemistry and Molecular Biology, 58, 455–466. Normington, K. & Russell, D. W. (1992). Tissue distribution and kinetic characteristics of rat steroid 5 alpha-reductase isozymes. Evidence for distinct physiological functions. The Journal of Biological Chemistry, 267, 19548–19554. Pawlowski, J., Huizinga, M., & Penning, T. M. (1991). Isolation and partial characterization of a full-length cDNA clone for 3 alpha-hydroxysteroid dehydrogenase: a potential target enzyme for nonsteroidal anti-inflammatory drugs. Agents Actions, 34, 289–293.

206 Pelletier, G., Luu-The, V., & Labrie, F. (1994). Immunocytochemical localization of 5 alpha-reductase in rat brain. Molecular and Cellular Neuroscience, 5, 394–399. Pelletier, G., Luu-The, V., & Labrie, F. (1995). Immunocytochemical localization of type I 17 beta-hydroxysteroid dehydrogenase in the rat brain. Brain Research, 704, 233–239. Pelletier, G., Luu-The, V., Li, S., Bujold, G., & Labrie, F. (2007). Localization and glucocorticoid regulation of 11beta-hydroxysteroid dehydrogenase type 1 mRNA in the male mouse forebrain. Neuroscience, 145, 110–115. Pelletier, G., Luu-The, V., Li, S., & Labrie, F. (2004). Localization of 20alpha-hydroxysteroid dehydrogenase mRNA in mouse brain by in situ hybridization. Brain Research Molecular Brain Research, 125, 143–146. Pelletier, G., Luu-The, V., Li, S., Ren, L., & Labrie, F. (2003). Sex-related expression of 20alpha-hydroxysteroid dehydrogenase mRNA in the adult mouse. The Journal of Histochemistry and Cytochemistry, 51, 1425–1436. Penning, T.M. (1996). 3 Alpha-hydroxysteroid dehydrogenase: three dimensional structure and gene regulation. Journal of Endocrinology, 150, S175–S187. Penning, T. M., Burczynski, M. E., Jez, J. M., Hung, C. F., Lin, H. K., Ma, H., et al. (2000). Human 3alpha-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones. The Biochemical Journal, 351, 67–77. Penning, T. M., Jin, Y., Heredia, V. V., & Lewis, M. (2003). Structure-function relationships in 3alpha-hydroxysteroid dehydrogenases: a comparison of the rat and human isoforms. The Journal of Steroid Biochemistry and Molecular Biology, 85, 247–255. Qin, X., Klandorf, H., Porter, D. W., Holt, S. B., & Martin, W. G. (1993). Estrogen enhancement of ca-, mg-, and ca-mgstimulated adenosine triphosphatase activity in the chick shell gland. General and Comparative Endocrinology, 89, 4–10. Rajan, V., Edwards, C. R., & Seckl, J. R. (1996). 11 Betahydroxysteroid dehydrogenase in cultured hippocampal cells reactivates inert 11-dehydrocorticosterone, potentiating neurotoxicity. Journal of Neuroscience, 16, 65–70. Resko, J. A., Stadelman, H. L., & Norman, R. L. (1979). 17 Beta-hydroxysteroid dehydrogenase activity in the pituitary gland and neural tissue of rhesus monkeys. The Journal of Steroid Biochemistry, 11, 1429–1434. Rheaume, E., Lachance, Y., Zhao, H. F., Breton, N., Dumont, M., de Launoit, Y., et al. (1991). Structure and expression of a new complementary DNA encoding the almost exclusive 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase in human adrenals and gonads. Molecular Endocrinology, 5, 1147–1157. Robel, P. & Baulieu, E. E. (1985). Neuro-steroids-"-3-betahydroxy-delta-5-derivatives in the rodent brain. Neurochemistry International, 7, 953–958. Robel, P., Synguelakis, M., Halberg, F., & Baulieu, E. E. (1986). Persistence of the circadian rhythm of

dehydroepiandrosterone in the brain, but not in the plasma, of castrated and adrenalectomized rats. Comptes rendus de l'Académie des sciences. Série III, 303, 235–238. Robert, F., Guennoun, R., Desarnaud, F., Do-Thi, A., Benmessahel, Y., Baulieu, E.E., et al. (2001). Synthesis of progesterone in schwann cells: regulation by sensory neurons. European Journal of Neuroscience, 13, 916–924. Roselli, C. E., Horton, L. E., & Resko, J. A. (1985). Distribution and regulation of aromatase activity in the rat hypothalamus and limbic system. Endocrinology, 117, 2471–2477. Roselli, C. E., Klosterman, S. A., & Fasasi, T. A. (1996). Sex differences in androgen responsiveness in the rat brain: regional differences in the induction of aromatase activity. Neuroendocrinology, 64, 139–145. Russell, D. W. & Wilson, J. D. (1994). Steroid 5 alpha-reductase: two genes/two enzymes. Annual Review of Biochemistry, 63, 25–61. Sandeep, T. C. & Walker, B. R. (2001). Pathophysiology of modulation of local glucocorticoid levels by 11beta-hydroxysteroid dehydrogenases. Trends in Endocrinology and Metabolism, 12, 446–453. Sanghera, M. K., Simpson, E. R., McPhaul, M. J., Kozlowski, G., Conley, A. J., & Lephart, E. D. (1991). Immunocytochemical distribution of aromatase cytochrome P450 in the rat brain using peptide-generated polyclonal antibodies. Endocrinology, 129, 2834–2844. Sanne, J. L. & Krueger, K. E. (1995). Expression of cytochrome P450 side-chain cleavage enzyme and 3 beta-hydroxysteroid dehydrogenase in the rat central nervous system: a study by polymerase chain reaction and in situ hybridization. Journal of Neurochemistry, 65, 528–536. Sasano, H., Takashashi, K., Satoh, F., Nagura, H., & Harada, N. (1998). Aromatase in the human central nervous system. Clinical Endocrinology (Oxford), 48, 325–329. Simard J, Durocher F, Mébarki F, Turgeon C, Sanchez R, Labrie Y, Couet J, Trudel C, Rhéaume E, Morel Y, LuuThe V, Labrie F (1996). Molecular biology and genetics of the 3b-hydroxysteroid dehydrogenase/D5-D4 isomerase gene family. Journal of Endocrinol, 150, S189–S207. Soma, K. K., Alday, N. A., Hau, M., & Schlinger, B. A. (2004). Dehydroepiandrosterone metabolism by 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase in adult zebra finch brain: sex difference and rapid effect of stress. Endocrinology, 145, 1668–1677. Steckelbroeck, S., Stoffel-Wagner, B., Reichelt, R., Schramm, J., Bidlingmaier, F., Siekmann, L., et al. (1999). Characterization of 17beta-hydroxysteroid dehydrogenase activity in brain tissue: testosterone formation in the human temporal lobe. Journal of Neuroendocrinology, 11, 457–464. Steckelbroeck, S., Watzka, M., Stoffel-Wagner, B., Hans, V. H., Redel, L., Clusmann, H., et al. (2001). Expression of the 17beta-hydroxysteroid dehydrogenase type 5 mRNA in the human brain. Molecular and Cellular Endocrinology, 171, 165–168. Stewart, P. M. & Krozowski, Z. S. (1999). 11 Beta-hydroxysteroid dehydrogenase. Vitamins and Hormones, 57, 249–324.

207 Stoffel-Wagner, B. (2003). Neurosteroid biosynthesis in the human brain and its clinical implications. Annals of the New York Academy of Sciences, 1007, 64–78. Stoffel-Wagner, B., Beyenburg, S., Watzka, M., Blumcke, I., Bauer, J., Schramm, J., et al. (2000). Expression of 5alphareductase and 3alpha-hydroxisteroid oxidoreductase in the hippocampus of patients with chronic temporal lobe epilepsy. Epilepsia, 41, 140–147. Stoffel-Wagner, B., Watzka, M., Steckelbroeck, S., Schwaab, R., Schramm, J., Bidlingmaier, F., et al. (1998). Expression of CYP19 (aromatase) mRNA in the human temporal lobe. Biochemical and Biophysical Research Communications, 244, 768–771. Stromstedt, M. & Waterman, M. R. (1995). Messenger RNAs encoding steroidogenic enzymes are expressed in rodent brain. Brain Research Molecular Brain Research, 34, 75–88. Tannin, G. M., Agarwal, A. K., Monder, C., New, M. I., & White, P. C. (1991). The human gene for 11 beta-hydroxysteroid dehydrogenase. Structure, tissue distribution, and chromosomal localization. The Journal of Biological Chemistry, 266, 16653–16658. Thigpen, A. E., Silver, R. I., Guileyardo, J. M., Casey, M. L., McConnell, J. D., & Russell, D. W. (1993). Tissue distribution and ontogeny of steroid 5 alpha-reductase isozyme expression. The Journal of Clinical Investigation, 92, 903– 910. Torres, J. M. & Ortega, E. (2003). Differential regulation of steroid 5alpha-reductase isozymes expression by androgens in the adult rat brain. The FASEB Journal, 17, 1428–1433. Tsutsui, K. & Ukena, K. (1999). Neurosteroids in the cerebellar purkinje neuron and their actions (review). International Journal of Molecular Medicine, 4, 49–56. Ukena, K., Kohchi, C., & Tsutsui, K. (1999). Expression and activity of 3beta-hydroxysteroid dehydrogenase/delta5delta4-isomerase in the rat purkinje neuron during neonatal life. Endocrinology, 140, 805–813. Ukena, K., Usui, M., Kohchi, C., Tsutsui, K., Abdelgadir, S. E., Roselli, C. E., et al. (1998). Cytochrome P450 side-chain cleavage enzyme in the cerebellar purkinje neuron and its neonatal change in rats distribution of aromatase cytochrome P450 messenger ribonucleic acid in adult rhesus monkey brains. Endocrinology, 139, 137–147.

Vallarino, M., Mathieu, M., do-Rego, J. L., Bruzzone, F., Chartrel, N., Luu-The, V., et al. (2005). Ontogeny of 3 betahydroxysteroid dehydrogenase and 5 alpha-reductase in the frog brain. Annals of the New York Academy of Sciences, 1040, 490–493. Weidenfeld, J., Siegel, R. A., & Chowers, I. (1980). In vitro conversion of pregnenolone to progesterone by discrete brain areas of the male rat. The Journal of Steroid Biochemistry, 13, 961–963. Wilson, J. D., Griffin, J. E., & Russell, D. W. (1993). Steroid 5 alpha-reductase 2 deficiency. Endocrine Reviews, 14, 577–593. Yague, J. G., Munoz, A., de Monasterio-Schrader, P., Defelipe, J., Garcia-Segura, L. M., & Azcoitia, I. (2006). Aromatase expression in the human temporal cortex. Neuroscience, 138, 389–401. Yamada, H., Kominami, S., Takemori, S., Kitawaki, J., & Kataoka, Y. (1997). Immunohistochemical localization of cytochrome P450 enzymes in the rat brain, considering the steroid-synthesis in the neurons. Acta Histochemica et Cytochemica, 30, 609–616. Yau, J. L. & Seckl, J. R. (2001). 11beta-hydroxysteroid dehydrogenase type I in the brain; thickening the glucocorticoid soup. Molecular Psychiatry, 6, 611–614. Yu, L., Romero, D. G., Gomez-Sanchez, C. E., & GomezSanchez, E. P. (2002). Steroidogenic enzyme gene expression in the human brain. Molecular and Cellular Endocrinology, 190, 9–17. Zhang, Y., Dufort, I., Rheault, P., & Luu-The, V. (2000). Characterization of a human 20alpha-hydroxysteroid dehydrogenase. Journal of Molecular Endocrinology, 25, 221–228. Zhao, H. F., Rheaume, E., Trudel, C., Couet, J., Labrie, F., & Simard, J. (1990). Structure and sexual dimorphic expression of a liver-specific rat 3 beta-hydroxysteroid dehydrogenase/ isomerase. Endocrinology, 127, 3237–3239. Zhou, M. Y., Gomez-Sanchez, E. P., Cosby, D., & GomezSanchez, C. E. (1995). Cloning of a NAD+ -dependent 11 hydroxysteroid dehydrogenase from rat kidney. The Endocrine Society, 77th Annual Meeting. Washington, DC, June 14-17. Program & Abstracts: 413 (poster P2–489). Zwain, I. H. & Yen, S. S. (1999). Dehydroepiandrosterone: biosynthesis and metabolism in the brain. Endocrinology, 140, 880–887.

L. Martini (Eds.) Progress in Brain Research, Vol. 181 ISSN: 0079-6123 Copyright
2010 Elsevier B.V.

CHAPTER 12

The multiple roles of estrogens and the enzyme aromatase Wah Chin Boon1,2,3,4,
, Jenny D. Y. Chow3,4 and Evan R Simpson3,5 1 Florey Neuroscience Institutes, Parkville, Victoria, Australia Centre of Neuroscience, Melbourne University, Parkville, Victoria, Australia 3 Prince Henry’s Institute, Clayton, Victoria, Australia 4 Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia 5 Department of Biochemistry, Monash University, Clayton, Victoria, Australia 2

Abstract: Aromatase is the enzyme that catalyzes the last step of estrogen biosynthesis. It is expressed in many tissues such as the gonads, brain and adipose tissue. The regulation of the level and activity of aromatase determines the levels of estrogens that have endocrine, paracrine and autocrine effects on tissues. Estrogens play many roles in the body, regulating reproduction, metabolism and behavior. In the brain, cell survival and the activity of neurons are affected by estrogens and hence aromatase. Keywords: aromatase; CYP19; estrogens; brain; neuroprotection

(Allen and Doisy, 1983), it was not until the 1980s that the human aromatase cytochrome P450 protein was extracted from placental microsomes, which demonstrated that indeed a single enzyme is responsible for the multiple reaction steps in the aromatization process (reviewed by Santen et al., 2009). Even then, the structure of aromatase remained unknown for another two decades. In 2009, the aromatase crystal structure was finally revealed (Ghosh et al., 2009), and was the first natural mammalian full-length cytochrome P450 protein to be crystallized.

Aromatase Aromatase cytochrome P450 is the enzyme that catalyzes the last step of estrogen biosynthesis (Fig. 1), that is, the rate-limiting irreversible aromatization of androgens to estrogens. Although actions of estrogens (uterine and vaginal tissue changes during menstrual cycles in guinea pigs) were first described in 1917 (Stockard and Papanicolaou, 1917) and the responsible steroids estrone and 17b-estradiol were purified from urine of pregnant women in the next decades




Corresponding author. Tel.: þ61-3-8344-1888; Fax: þ61-3-9348-1707; E-mail: [email protected]

DOI: 10.1016/S0079-6123(08)81012-6

209

210

CH3 CH3

Cholesterol

HO

Side-chain cleavage (CYP11A) CH3 O CH3

HO

Pregnenolone

Progesterone

3β-HSD2

CYP17

CYP17

17α-OH-pregnenolone

17α-OH-progesterone

3β-HSD2

CYP17

Dehydroepiandrosterone (DHEA)

CYP17

16α-hydroxylase 3β-HSD2

CH3 CH3

CH3

O

O

CH3

OH

CH3 17β-HSD3

16α-OH-DHEA

O Androstenedione

Aromatase (CYP19)

Testosterone

Aromatase (CYP19) CH3

CH3

17β-HSD4

O

HO

Aromatase (CYP19)

CH3

OH OH

OH

CH3

O

OH

17β-HSD1

?

17β-HSD2

HO

HO

Estriol

HO

Estrone

17β-estradiol

Fig. 1. The biosynthesis of estrogens and participating steroidogenic enzymes. CYP11A, cholesterol side-chain cleavage; CYP17, 17a-hydroxylase; 3 -HSD2, 3b-hydroxysteroid dehydrogenase type 2; 17 -HSD1, 17b-hydroxysteroid dehydrogenase type 1; 17 -HSD2, 17b-hydroxysteroid dehydrogenase type 2; 17 -HSD3, 17b-hydroxysteroid dehydrogenase type 3; 17 -HSD4, 17b-hydroxysteroid dehydrogenase type 4; CYP19, aromatase.

211

Aromatization Aromatase belongs to the cytochrome P450 superfamily and thus it is a haem-binding protein (see review by Conley and Hinshelwood, 2001). It is localized at microsomal organelles of estrogen-producing cells. It has a high substrate (androgens) specificity that is conferred by the hydrophobic and polar residues lining the androstenedione cleft, which complements the steroid backbone. To catalyze the conversion of androgens to estrogens, aromatase forms a complex with NADPH-cytochrome P450 reductase (a ubiquitous flavoprotein). Aromatization occurs when the aromatase complex converts C19 androgen substrates to C18 estrogens in three consecutive reactions: (1) hydroxylation (2) oxidation and (3) demethylation. This results in the C19 A-ring being converted into a phenolic ring characteristic of estrogens. Demethylation removes the C19 angular methyl group releasing it as formic acid. These reactions require NADPH cytochrome P450 reductase, which transfers reducing equivalents from NADPH to P450arom during oxidation of the C19 angular methyl group to formic acid. There are three main C19 steroids: dehydroepiandrosterone testosterone and androstenedione. The production of certain estrogens appears to be tissue-specific due to substrate availability: the placenta converts 16a-hydroxydehydroepiandrosterone to estriol; the ovary aromatizes testosterone to estradiol and in adipose tissue androstenedione is aromatized to estrone (Osawa et al., 1987).

Aromatase expression Peripheral tissues Aromatase expression in pre-menopausal women is found in granulosa cells (the major site of expression) and the corpus luteum of the ovary (McNatty et al., 1976). It is also detected in human testicular Leydig and Sertoli cells (Brodie and Inkster, 1993), the epididymis (Carpino et al., 2004), germ cells (Lambard et al., 2004),

syncytotrophoblasts of the placenta (Kilgore et al., 1992) and numerous foetal tissues (Price et al., 1992; Toda et al., 1994). Extragonadal tissues expressing aromatase include adipose mesenchymal tissue (Mahendroo et al., 1993), skin fibroblasts (Harada, 1992), bone osteoblasts and osteoclasts (Nawata et al., 1995), skeletal and smooth muscle (Larionov et al., 2003) and vascular endothelium (Sasano et al., 1999). Estrogen receptors are expressed in the same tissues that express aromatase (Table 1).

Central nervous system Aromatase transcript expression has been detected in rodent and avian brains (as reviewed by Lephart et al., 2001b; Naftolin et al., 2001), markedly in the limbic systems, hypothalamus, preoptic nucleus, sexually dimorphic nucleus, bed nucleus of the striata terminalis, hippocampus (Hojo et al., 2004) and cerebellum (Sakamoto et al., 2003). It has been demonstrated that the male rat foetal hypothalamus expresses higher levels of aromatase than that of female rat (Hutchison et al., 1997). Male porcine hypothalamus has aromatase transcript levels four times that of female counterparts (Corbin et al., 2009) assayed by reverse transcriptase-polymerase chain reaction (RT-PCR). Aromatase expression was also detected in rat pituitary (Galmiche et al., 2006) by RT-PCR. High levels of aromatase activity were detected in the male rat periventricular preoptic nucleus and medial preoptic nucleus; intermediate levels in the suprachiasmatic preoptic nucleus, anterior hypothalamus, periventricular anterior hypothalamus and ventromedial nucleus; and low levels in the arcuate nucleusmedian eminence, lateral preoptic nucleus, supraoptic nucleus (SON), dorsomedial nucleus and lateral hypothalamus (Roselli et al., 1985). Using polyclonal antibody raised against peptide corresponding to rat aromatase sequence (Sanghera et al., 1991), intense immunostaining was observed in neurons of adult rat amygdaloid structures and SON as well as reticular

Table 1. The expression of estrogen receptors and aromatase in normal human tissues

Human tissue

ERa

ERb

Brain

Hypothalamus and forebrain (Osterlund et al., 2000)

Cortex and hippocampus (Gonzalez et al., 2007)

Skin

Fibroblasts (Haczynski et al., 2002)

Fibroblasts (Haczynski et al., 2002)

Cardiovascular

Cardiomyocytes (Mahmoodzadeh et al., 2009); vascular endothelium (Cruz et al., 2008)

Arteries and vascular stroma (Savolainen et al., 2001); smooth muscle cells (Hodges et al., 2000)

Bone Adipose

Osteoblasts, osteoclasts and osteocytes (Hoyland et al., 1997) Adipocytes and stromal cells (Price & O’Brien, 1993)

Osteoblasts, osteoclasts and osteocytes (Vidal et al., 1999) (Pedersen et al., 2001)

Liver

(Grandien, 1996)

Not reported

Placenta

Proliferating trophoblasts (Bukovsky et al., 2003)

Differentiating trophoblasts (Bukovsky et al., 2003)

Granulosa, theca and epithelium (Saunders et al., 2000)

Testis

Granulosa, theca and epithelium (Saunders et al., 2000) Not detected (Makinen et al., 2001)

Urogenital tract Adrenal glands

Not detected (Baquedano et al., 2007)

Zona reticularis (Baquedano et al., 2007)

Uterus

Proliferative glandular and stromal cells (major) (Mylonas et al., 2004)

Prostate

Stromal nuclei only (Leav et al., 2001)

Proliferative glandular and stromal cells (minor), vascular endothelium (Lecce et al., 2001; Mylonas et al., 2004) Multiple cell types (Pasquali et al., 2001)

Foetal tissues

Uterine mesenchyme (Glatstein & Yeh, 1995); Leydig cells (Shapiro et al., 2005b); prostate (Shapiro et al., 2005a); neurons (Gonzalez et al., 2007)

Gonadal cells (Shapiro et al., 2005b); umbilical vein endothelial cells (Toth et al., 2008); prostate (Shapiro et al., 2005a); neurons (Fried et al., 2004)

Gonads Ovary

Sertoli, Leydig and germ cells (Makinen et al., 2001, Moore et al., 1998)

Aromatase gene promoter Promoter 1f (Honda et al., 1994) Promoter 1.4 (Harada, 1992) Promoter 1.7 (endothelial cells) (Sebastian et al., 2002) Promoter 1.6 (Shozu et al., 1998) Promoter 1.4/1.3/II (Mahendroo et al., 1991) N/A (adult) Promoter 1.4 (foetal hepatocytes) (Zhao et al., 1995) Promoter 1a/2a/1.2 Promoter 1.8 (Demura et al., 2008) Promoter II (Means et al., 1991) Promoter II (Bulun et al., 1993) Promoter 1.3/II (Baquedano et al., 2007) Not expressed (Bulun et al., 2005) Promoter II (Ellem et al., 2004) Promoter 1.5 (Toda et al., 1994)

213

thalamic nucleus, olfactory tract and piriform cortex, whereas moderate to light immunoreactivity was observed in the paraventricular and arcuate nuclei and hippocampus. However, contradicting to previous reports, neurons in the bed nucleus stria terminalis, medial basal hypothalamic, and preoptic areas displayed little aromatase immunoreactivity. In contrast, robust aromatase immunoreactivity was detected in the medial preoptic area and hypothalamus of postnatal day 5 rat (Horvath et al., 1997), including periventricular regions, ventromedial and arcuate nuclei, as well as the limbic structures (the central and medial nuclei of the amygdala, stria terminalis, bed nucleus of the stria terminalis, lateral septum, medial septum, diagonal band of Broca, lateral habenula and all areas of the cingulate cortex). Despite the fact that there are various studies that have determined aromatase activity and expression in brain regions using animal models (Naftolin and MacLusky, 1982, Naftolin et al., 2001), studies in human postnatal brain tissue are limited due to the difficulty in obtaining fresh human brain tissue samples. Sasano et al., (1998) examined aromatase expression in various postmortem human brain regions using RT-PCR and demonstrated that aromatase is expressed widely in human brain regions such as pons, thalamus, hypothalamus and hippocampus. The expression of aromatase mRNA in human temporal lobe tissues was investigated by Stoffel-Wagner et al. (1998) and reported that aromatase expression levels did not differ significantly between men and women, but aromatase mRNA levels were significantly higher in adults than in children, accounting for differences of expression levels between children and adults. As an extension to the previous findings, Stoffel-Wagner et al., (1999) examined the aromatase expression in biopsy samples from 45 women and 54 men with epilepsy using nested competitive RT-PCR. They detected aromatase expression in hippocampus, temporal and frontal neocortex, with the temporal expressing significantly higher levels than frontal neocortex. No expression differences between sexes were observed in any of the brain regions

investigated. Recent studies have determined that in the human temporal cortex, aromatase is expressed in a large population of pyramidal neurons and in certain interneurons and astrocytes, suggesting that aromatase serves a significant role in human cerebral cortex (Yague et al., 2006). Aromatase expression was detected in normal human pituitary obtained from autopsy (13 males, 6 females, median age: 30 years, interquartile ranges 23–63) via quantitative RT-PCR and aromatase protein with immunohistochemical staining (Kadioglu et al., 2008). Although median relative expression level of aromatase mRNA of men (median DeltaCt = 42.6; interquartile ranges: 7.6–93.9) was higher than women (median DeltaCt = 3.9, interquartile ranges: 0–44.8), the difference is not statistically significant (p = 0.2) due to small sample size and large variations within groups. The aromatase levels were also not correlated with the age of the study subjects (p = 0.42; r = –0.21). As both estrogen receptor a and b are expressed in numerous sites of the brain (Azcoitia et al., 1999; Gonzalez et al., 2007; Mitra et al., 2003), estrogens produced locally in the brain could act in paracrine, intracrine or autocrine manner.

Regulation of aromatase expression Cyp19 (aromatase) gene expression regulation Aromatase is encoded by the Cyp19 gene. More than one copy of the Cyp19 gene has been isolated in fish (gonadal cyp19a1 and brain cyp19a2; Kazeto et al., 2001) and boar (cyp19a1, cyp19a2, cyp19a3; Corbin et al., 2009). It had been hypothesized that multiple isoforms of aromatase may exist in the human body (Osawa et al., 1987). However, from restriction mapping and genomic Southern analyses, Means et al. (1989) demonstrated that there is no evidence for more than one isoform of aromatase existing in the human genome. This was supported by surveying the human genome database after its complete elucidation (Bulun et al., 2003).

214

pathways and transcription factors that exist in different tissues, to which different promoters are responsive (Simpson et al., 1993). Activation of tissue-specific promoters triggers the transcriptional splicing of their associated exons I to the coding exons at a common splice junction (CSJ) 38 bp upstream of the transcription start site, resulting in alternative aromatase transcripts with unique 50 -ends. However, the mechanisms regulating the use of tissue-specific promoters and the resulting tissue-specific alternative splicing are not completely understood. Since 50 -ends are not translated, the resulting aromatase coding sequence and protein sequence are identical regardless of the promoter being used. Although alternative exons I are tissue-specific, they are by no means the only transcript variant present in one particular tissue. For instance, exons I.1, 2a and I.2 are all expressed in the placenta (although the latter two at very low levels), and adipose tissue contains transcript variants of exon I.4, I.3 and PII (Mahendroo et al., 1993). The 50 -UTR of human CYP19A1 and that of some other species have been studied extensively. Of the aromatase species sequenced so

The human CYP19A1 gene is located at chromosome 15q21.1, which consists of nine coding exons (II to X) and a 50 -untranslated region (50 UTR), altogether spanning approximately 123 kb in length (Bulun et al., 2003) (Fig. 2). The coding exons occupy 30 kb and the remaining 93 kb contains alternative promoter and untranslated first exons. To date, 11 tissue-specific alternative promoters/first exons have been characterized: promoter/exon I.1 (placenta major), 2a (placenta minor), I.4 (skin), I.5 (foetal tissues), I.7 (endothelium), 1f (brain), I.2 (placenta minor), I.6 (bone), I.3 (adipose and breast cancer), promoter II/exon PII (gonads) and the newly discovered I.8 (Demura et al., 2008). These promoters allow the regulation of CYP19A1 expression in a tissue-specific manner. For example, promoter II drives aromatase expression in the ovary (Adams et al., 2001; Michael et al., 1995) while promoter 1f is used to direct aromatase expression mainly in the brain (Honda et al., 1994), promoter I.4 to direct expression in adipose tissues (Mahendroo et al., 1993) and promoter I.1 to direct expression in the placenta (Kilgore et al., 1992). This is due to the presence of unique cell signalling

Promoters and untranslated Exons I (∼93 kb)

Coding exons II-X (–30 kb)

3’

5’

Simpson Harada

I.1 1a

2a

I.8 I.4 Ib

Placenta Placenta Minor 2 Major

I.5

Adipose Fetal stomal; tissues skin fibroblast; Foetal tissues Breast cancer Placenta; Multiple tissue s

I.7 If Vascular Brain endothelium Breast cancer

I.2 1e Placenta Minor 1

I.6

I.3 1c

PII II 1d 2

X 10

Bone Ovary; Ovary; Breast Prostate; cancer Testis; Breast cancer

Fig. 2. Human aromatase (CYP19A1) gene. A schematic representation of the CYP19A1 tissue-specific promoters and 50 -untranslated exons 1. Note that exons I.1, 1.4, 1.3, PII and 1.2 from the Simpson system of nomenclature are also known as 1a, 1b, 1c, 1d and 1e, respectively, in the Harada system of nomenclature according to chronological order of discovery.

215

far, that of mouse Cyp19A1 (chromosome 9) has the highest homology of 81% with human CYP19A1, followed by the equine, rat and chicken at 78, 77 and 73%, respectively (Seralini et al., 2003; Simpson et al., 1993). There is also only one copy of Cyp19a1 in the murine genome. So far, five tissue-specific promoters/ exons I have been described in the mouse gene, namely, the brain, ovary- (Honda et al., 1996), testis-specific exon I (Golovine et al., 2003) and the more recently described gonadal adiposespecific exon I (Chow et al., 2009), spanning approximately 75 kb upstream of the mouse Cyp19A1 transcription start site in the order: exon I.4 (gonadal fat; –75 kb), 1f (brain; – 36 kb), Etes (testis; –36 kb), exon I.3 (ovary and testis; –200 bp) and proximal exon PII (ovary). Thus, the regulation of aromatase in the human or rodent body is quite complex with the regulators acting at tissue-specific promoters of one CYP19A1 gene. Regulators of peripheral aromatase expression include hormones (e.g. androgens, estrogens and follicle-stimulating hormones), cytokines and growth factors (e.g. insulin-like growth factor-I) (refer to review by Simpson, 2004). Although no estrogen response elements have been reported in the promoter regions, expression of aromatase can be regulated by estrogens, for example, in the human placenta, estrogens induced estrogen receptor a (ERa) recruitment to the –255- to –155-bp region leading to histone modifications resulting in increased aromatase transcription (Kumar et al., 2009). Expression of aromatase in the avian (Steimer and Hutchison, 1981) and mammalian (Abdelgadir et al., 1994; Lephart, 1996) brain regions such as the hypothalamus can be enhanced by testosterone and estradiol (Balthazart et al., 2001b). However, the steroidal control of aromatase expression is neuroanatomically specific. It had been reported that castration of male rats had no effects on the aromatase activity in the amygdala but significantly reduced aromatase activity levels in the preoptic area to that of female rats (Roselli et al., 1985), and testosterone administration restored the aromatase activity.

Neurotransmitters acting through protein kinase C or G (Abe-Dohmae et al., 1996) at promoter 1f could also control the expression of aromatase. By contrast, expression of aromatase in the fish gonads and brain is regulated by the substantially different 50 -flanking promoters of cyp19a1 and cyp19a2 genes, through the involvement of different regulators/transcription factors. Gonadal-specific aromatase gene cyp19a1 contains three cAMP-responsive elements (CREs), an aryl hydrocarbon-responsive element (AhR/ Arnt), a steroidogenic factor 1 (SF-1) site and a TATA box whereas the brain-specific aromatase gene contains a single CRE, an estrogen-responsive element (ERE), a peroxisome proliferator-activated receptor a/retinoid X receptor a heterodimer-responsive element (PPARa/RXRa) and a TATA box. The predominant transcription initiation sites for cyp19a1 and cyp19a2 transcripts were 28 and 91 bp upstream from the putative translation initiation codon, respectively (Kazeto et al., 2001).

Regulation of aromatase activity The activity of aromatase activity has been shown to be regulated by phosphorylation. The first evidence came from the quail preoptic–hypothalamic homogenate – 15 minute preincubation with 1 mM ATP and 5 mM MgCl2 (a limiting factor for kinases) reduced the aromatase activity significantly down to 16.9% of that of controls (Balthazart, Baillien, & Ball, 2001c). Incidently, the inhibition by ATP/Mg2þ could be blocked by 10 mM staurosporine (a general serine/threonine kinase inhibitor) or 10 mM bisindolymaleimide (a Protein Kinase C (PKC) inhibitor) or 50 mM genistein (a general tyrosine kinase inhibtor) without affecting the basal aromatase activity. Although the exact sites of phosphorylation were not determined emperically in these studies, the quail aromatase sequence has consensus phoshorylation sites for Protein Kinase A (PKA) (T389), PKC (S363) and tyrosine kinase (Y354), which are also conserved in the human aromatase sequence

216

(Balthazart et al., 2001a). Protein kinase A (PKA) and PKC phosphorylate serine and/or threonine amino residues. The above studies are supported by the independent report that phosphorylation of murine aromatase inhibited its activity in COS7 cells (Miller et al., 2008). The mutation S118A blocked phosphorylation of the aromatase and showed increased specific enzymatic activity as compared to the wild-type controls, whereas the mutation S118D (mimics phosphorylation effects) exhibited decreased activity versus wild-type control. The structural importance of the S118 residue is also illustrated by the decreased protein stability of the mutants. In contrast, it has been demonstrated that phosphorylation of human aromatase at Y361 resulted in increased activity in MCF-7 (breast cancer cell line) after estradiol incubation through activation of tyrosine kinase c-Src by ligand-bound ERa (Catalano et al., 2009). This rapid regulation of aromatase would have application in the rapid effects of estrogens on neuronal functions, which will be discussed in the section below.

Multiple roles of aromatase The multiple roles of aromatase were uncovered by studying models of aromatase deficiency – both human and mice.

Human aromatase deficiency Natural mutations of the aromatase gene causing aromatase and estrogen deficiency are very rare in humans and often result from parents of consanguineous marriages. To date, there are only 15 known cases of patients (eight men and seven women) diagnosed with aromatase deficiency (Jones et al., 2007; Lanfranco et al., 2008). Reproductive abnormalities are the primary observations in patients diagnosed with aromatase deficiency. Female patients of aromatase deficiency are usually diagnosed and treated early in

life due to signs of pseudohermaphroditism at birth, pubertal abnormalities (mild virilization, cystic ovaries, hypergonadotrophins, elevated testosterone, low estrogen, enlarged clitoris), amenorrhea at the time of puberty and delayed maturation of bone development. Estrogen replacement therapy resulted in a growth spurt, breast development, menarche, suppression of gonadotropin levels, and resolution of the cysts (Harada et al., 1992; Jones et al., 2007). Aromatase deficiency causes abnormalities that are sexually dimorphic, and hence shedding light on the importance of estrogen unique to each of the genders. Male patients are usually diagnosed and treated much later in life, as pubertal development is normal, but signs of infertility and persistent bone growth become apparent (Jones et al., 2007; Lanfranco et al., 2008). The first male patient with aromatase deficiency was described in 1995 (Morishima et al., 1995). Aromatase deficiency in males does not affect the reproductive development to the same degree as that in females. Aromatase-deficient men develop progressive infertility in adulthood, with decreased sperm motility and/or low sperm counts as the common causes (Carani et al., 1997; Morishima et al., 1995), but sexual activity may also be affected (Carani et al., 1999). Metabolic abnormalities [such as slight truncal obesity, hyperinsulinemia, elevated serum triglyceride and low-density lipoprotein (LDL) cholesterol and high-density lipoprotein (HDL) cholesterol] as well as some liver dysfunction are present in male aromatase-deficient patients (reviewed by Jones et al., 2007). It is still uncertain whether untreated female aromatase-deficient patients will also develop fatty liver disease as observed in male patients. Neither cognitive function nor psychological profiles of these patients were reported.

Aromatase knockout mice The generation of the aromatase knockout (ArKO) mice (Fisher et al., 1998) revealed very similar phenotypes as those observed in aromatase-deficient humans, and has since

217

enabled scientists to study the physiological importance of estrogen in the laboratory. The ArKO mouse model was generated by deleting exon IX of the Cyp19A1 (replaced by a neomycin cassette) resulting in the expression of a nonfunctional aromatase enzyme, thus becoming estrogen-deficient and hyperandrogenic. Both genders presented with disrupted reproductive functions (Fisher et al., 1998; McPherson et al., 2001; Robertson et al., 1999), bone undermineralization (Oz et al., 2001), reduced blood pressure and baroreflex sensitivity (Head et al., 2004), autoimmunity (Shim et al., 2004) as well as an array of metabolic phenotypes such as increased adiposity (Jones et al., 2000; Misso et al., 2003) and hepatic steatosis (Hewitt et al., 2004) (Table 2). These metabolic phenotypes were also reported in the ArKO generated by Toda et al. (2001b). Interestingly, the metabolic phenotypes of the ArKO mice are sexually dimorphic. Female ArKO mice do not develop hepatic steatosis like the male ArKO; however, serum triglyceride was significantly elevated, which was likewise not observed in male ArKO mice. Female ArKO mice also have increased serum cholesterol and HDL levels, and both were reversible upon estrogen replacement. The pituitary of the female ArKO mice is smaller and the plasma growth hormone (GH) levels were decreased (Yan et al., 2005). This is accompanied by decreased transcript levels of GH-secretagogue receptor, GH-releasing hormone receptor in the pituitary that could be corrected by estrogen treatment.

ArKO brain and behavioral phenotypes Interestingly, the ArKO mouse model presents brain and behavioral phenotypes that were not previously observed in gonadectomized animals (refer to review by Hill and Boon, 2009). We have reported that neuronal apoptosis occurred in the frontal cortex of aged female ArKO mice (Hill et al., 2009) and in the hypothalamus of aged male ArKO mice (Hill et al., 2004) in the absence of external assault. This could be a consequence of decreased levels of anti-apoptotic

gene expressions and increased levels of proapoptotic gene expressions (Hill et al., 2007a, 2009). The ArKO mouse model developed in Harada’s laboratory (Honda et al., 1998), by targeted disruption of exon 1 and 2, had been reported to be more susceptible to neuronal toxins domoic acid (Azcoitia et al., 2001) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Morale et al., 2006). These male ArKO mice showed significant hilar neuronal loss in the hippocampus after injection of a low dose of domoic acid, which had no effect on the control littermates, indicating that aromatase deficiency increases the vulnerability of neurons to neurotoxic degeneration. These neonatal ArKO animals have decreased Purkinje dendritic growth, spinogenesis and synaptogenesis (Sasahara et al., 2007) as compared to the wild type (WT) animals during the same developmental period demonstrating that estrogens are important in promoting synapse formation. Our laboratories have demonstrated that both male and female ArKO mice had impaired spatial reference memory (Martin et al., 2003) although the female ArKO mice performed as well as the WT in the watermaze test (Boon et al., 2005). Our male ArKO mice presented loss of sexual behavior (Robertson et al., 2001) but developed compulsive behaviors such as excessive grooming and wheel-running activities (Hill et al., 2007b). The levels of catechol-Omethyltransferase (COMT) enzyme that metabolize dopamine were lowered in the hypothalamus of the male ArKO mice when compared to the WT mice. The compulsive behavior were ameliorated after estradiol administration (Hill et al., 2007b) with concomitant increase of COMT levels to WT levels. Depressive-like behavior was observed in the female ArKO model developed in Harada’s laboratory (Dalla et al., 2004) whereas the male ArKO did not display such behavior. The sexual and aggressive behaviors were disrupted in the male ArKO mice and they developed infanticide behavior (reviewed by Harada et al., 2009). Toda et al., (2001a) also reported the loss of agressive behavior in their male ArKO mice (Nemoto et al., 2000). Estrogen

Table 2. Key phenotypes of aromatase knockout (ArKO) mice ArKO (Fisher et al., 1998) Cyp19A1 exon 9 deletion

ArKO (Honda et al., 1998) Cyp19A1 exon 1 deletion

ArKO (Nemoto et al., 2000) Cyp19A1 exon 9 deletion

Phenotypes

Male

Female

Male

Female

Male

Female

Reproductive

Age-progressive infertility; impaired spermatogenesis (Robertson et al., 1999); prostate gland hyperplasia (McPherson et al., 2001) " adiposity " liver cholesterol, " serum leptin and insulin Hepatic steatosis

Infertile; disrupted folliculogenesis; haemorrhagic cystic follicles (Britt et al., 2001); underdeveloped uteri

Infertile; no significant external abnormalities

Infertile; underdeveloped uteri; No other significant external abnormalities

Infertile

" adiposity " serum leptin and insulin " serum TG, cholesterol and HDL (Misso et al., 2003)

No info

No info

Hepatic steatosis; impaired gene expression and hepatic enzyme activities of fatty acid b-oxidation (Nemoto et al., 2000) " HDL-cholesterol (Toda et al., 2001c) Insulin resistance (Takeda et al., 2003)

Infertile; anovulation, depletion of follicles; disorganized interstitial cells; haemorrhages in the ovaries (Toda et al., 2001d) Normal hepatic fatty acid b-oxidation (Toda et al. 2001c)

Decreased bone length and density; increased bone formation rate; increase B-cell lymphopioesis (Oz et al., 2001)

No info

No info

" bone resorption similar to females, but at 32 weeks bone loss is less compared to females (Miyaura et al., 2001)

Metabolic

# body lean mass, physical activity, calorie intake (Jones et al., 2000; Hewitt et al., 2003) Bone

Decreased bone formation rate (Oz et al., 2001)

Loss of cancellous bone, " bone resorption from 9 weeks old; more severe at 32 weeks. (Miyaura et al., 2001)

CNS/ behavioral

apoptosis in AN and MPO (Hill et al., 2004, 2007a); obsessivecompulsive behavior (Hill et al., 2007b); # sexual activity (Robertson et al., 2001), reduced spatial reference memory (Martin et al., 2003)

apoptosis in frontal cortex (Hill et al., 2009); reversible middle cerebral artery occlusion resulted in greater total and regional damage in female ArKO mice than ovarectomized WT controls (McCullough et al., 2003). Reduced spatial reference memory (Martin et al., 2003)

Others

Severe autoimmune exocrinopathy (Shim et al., 2004); reduced proliferation and enhances apoptosisrelated death in VSMCs (Ling et al., 2004); reduced acetylcholineinduced release of nitric oxide in aorta (Kimura et al., 2003)

Reduced blood pressure and baroreflex sensitivity (Head et al., 2004); severe autoimmune exocrinopathy (Shim et al., 2004); reduced proliferation and enhances apoptosisrelated death in VSMCs (Ling et al., 2004)

Increase vulnerability to neurotoxin (Azcoitia et al., 2001; Morale et al., 2006); prolonged latencies to mount and decreased numbers of mounts in response to receptive stimulus females; deficits in olfactory and visual cues for sexual partner preference (Bakker et al., 2002); severe deficits in social recognition (Pierman et al., 2008); anxiety and depressive-like symptoms (Dalla et al., 2005) –

Enhanced response to odour cues (Wesson et al., 2006)

Lack of aggressive behavior (Toda et al., 2001b); impairment in mounting behavior (Toda et al., 2001a)

No info

–

Thymic regression and reduced cellularity

–

(Li et al., 2002)

220

replacement could restore the sexual behavior deficit in male (Bakker et al., 2004) but not female ArKO mice (Bakker et al., 2002). In summary, the ArKO mouse models illustrated that aromatase plays important roles in regulating neuronal survival and functions as well as behaviors.

Physiological effects of estrogen Estrogen is considered to be the female steroid also because of its traditional role in the female reproductive system. But today, estrogen is implicated in a far wider range of actions including metabolic regulation, and neurological and behavioral effects in both male and female.

Reproductive effects

and the pattern of fat deposition, and the appearance of axillary and pubic hair also depend on estrogens.

Male Testosterone is the principal hormone for the development of male sexual characteristics. However, current research has shed light on the effects of estrogen in the male reproductive system and some of the effects of testosterone are mediated through its conversion to estrogen. Estrogen is believed to be critical in the normal development of male gonadal functions and spermatogenesis by controlling stem cell number and spermatid maturation in the seminiferous tubules (reviewed by Hess, 2003). The male ArKO testicular phenotype illustrated this role of estrogens (Robertson et al., 1999).

Female The ovary is the principal source of estrogens in the female circulation. Estrogens are responsible for the development of primary reproductive characteristics including growth and maturation of reproductive organs and breasts at puberty and maintaining their adult size and function through the reproductive age. The data gathered from studying the female reproductive phenotype of the ArKO mice confirmed that estrogens are responsible for the estrous cycle, folliculogenesis and ovulation. Female ArKO mice had atrophic uterus (Fisher et al., 1998) and reduced numbers of primordial and primary follicles (Britt et al., 2004) compared with WT mice and did not ovulate. However, oocytes retrieved from the ArKO antral follicles could be fertilized in vitro and developed to the blastocyst stage at the same rate as wild-type and heterozygote littermates (Huynh et al., 2004). Studies on female aromatase-deficient patient also supported these animal data (MacGillivray et al., 1998) and also indicated that secondary characteristics such as feminization of skeleton

Non-reproductive effects in peripheral tissues In peripheral tissues, estrogen generally promotes cellular division, regrowth and anabolism. Estrogen in the cardiovascular system promotes angiogenesis, vasodilation and vasoprotection against atherosclerosis and endothelial cells from apoptosis (reviewed by Mendelsohn, 2009). Nevertheless, estrogen is also known to promote thrombogenesis and therefore blood clotting. Estrogen replacement therapy and/or estrogen-based oral contraceptives are associated with increased risk of venous thrombosis (Canonico et al., 2008). Relevantly, estrogen reduces serum LDLs and hence increases HDLs in the circulation (reviewed by Knopp et al., 2006). In bone, estrogen has pro-formation effects and inhibits resorption to maintain normal bone mass, as well as stimulating epiphyseal closure (Lanfranco et al., 2008). Youthful skin possesses turgor and is free of wrinkles due to the estrogenic effect of hydration and collagen synthesis (Youn et al., 2003).

221

Central nervous system In brief, estrogen in the central nervous system (CNS) exhibits neurotrophic properties (Carrer et al., 2003), promotes synaptogenesis (Frankfurt et al., 1990) and differentiation from as early as foetal development through adulthood (Lephart et al., 2001a; Wang et al., 2003). It plays an important role in neuroprotection. Estrogens also act on hypothalamus and pituitary to inhibit gonadotropin release in men (Raven et al., 2006). Estrogen replacement to aromatase-deficient men decreased basal and gonadotropin-releasing hormone (GnRH)-stimulated secretion of luteinizing hormone (LH), LH-pulsed amplitude as well as lowered frequency of LH pulses (Rochira et al., 2006). Thus, this demonstrated that circulating estrogens exerted effects both at the pituitary level and at the hypothalamic level. However, the discrepancy among testosterone levels, the arrest of spermatogenesis and a slightly inappropriate respective increase of serum follicle-stimulating hormone (FSH) (lower than expected) suggest a possible role of estrogens in the priming and the maturation of hypothalamus–pituitary– gonadal axis in men, an event that has never occurred in these two subjects as a consequence of chronic estrogen deprivation. Estrogens affect all of the hypothalamic nuclei (such as the paraventricular nucleus or arcuate/ventromedial nucleus) that control energy homeostasis and appetite. The activities of hypothalamic neurones are influenced by estrogens through gene regulation and neuronal excitability (reviewed by Roepke, 2009), which are mediated by the classic estrogen receptors present in these nuclei as well as membrane-associated estrogen receptors.

Effects of aromatase in the brain In the 1970s, it was first demonstrated that estrogens could be produced in the brain by the local aromatization of testosterone (Naftolin et al., 1975). Research in this area has been gaining momentum and evidence is mounting to show that locally synthesized estrogens from

pregnenolone (Hojo et al., 2004) could modulate neuronal functions in addition to their neuroprotective effects.

Neuroprotective effects Estrogens have neuroprotective functions in the brain, especially in the elderly (Mulnard et al., 2000). As the plasma levels of estrogens in men are low, local production of estrogens in the brain will have major contributions to the neuroprotection. This holds true for post-menopausal women since their circulating levels of estrogens have plummeted after menopause. This highlights the importance of brain aromatase. Indeed, an increase in aromatase immunoreactivity (aromir) was found in the nucleus basalis of Meynert (NBM) and SON during normal ageing but a decreased arom-ir was found in the SON, infundibular nucleus (INF) and the medial mammillary nucleus (MMN) of Alzheimer’s disease (AD) patients as compared to non-sufferers (Ishunina et al., 2005). The significance of this localized production of estrogens is highlighted by the presence of estrogen receptor a in these brain regions and the expression levels are elevated in AD patients (Ishunina and Swaab, 2003). Another independent study (Yue et al., 2005) reported that female AD brains contained greatly reduced estrogen levels compared with those from age- and gender-matched normal control subjects although both AD and control subjects had comparably low levels of serum estrogen, again confirming that locally synthesized estrogens have neuroprotective effects. Further evidence that local estrogen production is neuroprotective comes from ArKO mouse. Reversible middle cerebral artery occlusion (90 minute; 22 hour reperfusion) resulted in greater total and regional damage in female ArKO mice than ovarectomized WT controls (McCullough et al., 2003). This infers that extragonadal estrogens play a critical role in neuroprotection. In addition, studies using amyloid precursor protein transgenic mice have reported no effect of ovariectomy or estrogen replacement on

222

b-amyloid deposition in the hippocampus and neocortex (Green et al., 2005). However, the estrogen-deficient APP23 mice [generated by cross-breeding the APP23 transgenic mouse (AD model) with the ArKO mouse] presented amyloid plaque formation at a younger age, accompanied by an increased b-amyloid peptide deposition and increased b-amyloid production (Yue et al., 2005). It is noteworthy that increased risk for AD is associated with polymorphisms of the aromatase (CYP19) gene (Huang and Poduslo, 2006; Iivonen et al., 2004). However, the mechanism(s) underlying the neuroprotective effects of brain estrogens has yet to be elucidated completely. One clue would be that AD brains are reportedly under severe oxidative stress, as a result of either amyloid b-mediated generated oxyradicals or perturbed ionic calcium balances within neurons and their mitochondria (Emilien et al., 2000; Xu et al., 2006). Estrogen has strong antioxidant actions (Bhavnani, 2003), and modifies inflammatory responses and may directly reduce amyloid b generation (van Groen and Kadish, 2005; Xu et al., 1998). Estradiol has also been reported to influence the expression of many genes in the brain that are relevant to estradiol’s ability to protect. These include genes involved in the balance of apoptosis and cell survival (reviewed by Wise et al., 2001; Bhavnani, 2003). In the ArKO mice, we have reported neuronal loss in the frontal cortex of aged female ArKO mice (Hill et al., 2009) and also dopaminergic cell loss in the hypothalamus of aged male ArKO mice (Hill et al., 2004, 2007a) in the absence of external assault such as neurotoxin or pathological agents. Increased levels of pro-apoptotic gene expression and decreased levels of antiapoptotic gene expression were detected in the brain regions affected. Hence, aromatase is essential for the survival of neurons. Another suggestion on the neuroprotective mechanism of estrogens is that estrogens upregulate cerebral apolipoprotein E (apoE), which is believed to be involved in neuronal protection and repair (Levin-Allerhand et al., 2001). During this study, ovariectomized mice were treated with

pharmacological levels of 17b-estradiol or placebo for five days. Results indicated an upregulation of apoE but not glial fibrillary acidic protein (GFAP) in the cortex and diencephalons of estradiol-treated mice brains, while both apoE and GFAP were equally upregulated in the hippocampus (LevinAllerhand et al., 2001). It was concluded that estradiol upregulates apoE, a neuroprotective and repair mechanism, in the cortex and diencephalons, while no upregulation of GFAP, a neuronal destructive mechanism, was seen in these areas of the brain. Therefore, estradiol may have neuroprotective effects on the brain by upregulation of apoE in the cortex and diencephalon. Ovariectomized rats with entorhinal cortex lesions showed an inhibition in the increase in GFAP and enhanced neurite outgrowth after estradiol replacement as compared to placebo-treated animals (Rozovsky et al., 2002). Estradiol also seemed to reorganize astrocytic laminin into extracellular fibrillar arrays, which have shown to support neurite outgrowth. Therefore, not only did estradiol inhibit increases of GFAP, but it also actually aids in the growth of neurites (Rozovsky et al., 2002). Similar estradiol repression of astrocyte GFAP neuroinflammatory response was observed in mesencephalic dopaminergic neurons after MPTP lesion to mouse substantia nigra pars compacta (Morale et al., 2006). Brain aromatase is normally expressed in the neurons but after injury such as neurotoxin kainic acid lesion, novel aromatase in astrocytes was detected by immunostaining and was co-localized with GFAP (Garcia-Segura et al., 1999). No aromatase immunostaining was detectable in astrocytes of control animals.

Sustaining brain glucose metabolism Estrogens may play an important role of sustaining glucose as the primary fuel source in the brain. The evidence presented so far includes the observation that estrogens increased the expression of neuronal glucose transporter subunits, for example, Glut1, Glut3 and Glut4 and glucose transport across the blood–brain barrier endothelium (Cheng et al., 2001). Simultaneously,

223

estrogens increased glycolytic enzyme activity of hexokinase, phosphofructokinase and pyruvate kinase (Kostanyan and Nazaryan, 1992). In addition, estrogens increase the levels of pyruvate dehydrogenase (Nilsen et al., 2007), which converts pyruvate, the product of glycolytic metabolism, to acetyl-CoA, which feeds into the tricarboxylic acid cycle for production of ATP. It may not be a coincidence that estrogens also increase the levels of ATP synthase and proteins involved in mitochondrial oxidative phosphorylation electron transfer (Nilsen et al., 2007). Thus, estrogen treatment increases glucose metabolism of the brain and this may be an important mechanism by which estrogens protect the brain from ageassociated metabolic decline. Significant decrease in the metabolism of the posterior cingulate cortex was observed during two-year follow-up examination in post-menopausal women not on estrogen replacement therapy whereas age-matched estrogen-treated women did not present such a decline (Rasgon et al., 2005).

Modulating effects on neurons

neurons substantia nigra pars compacta and dopamine transporter innervation of the caudate-putamen in adulthood (Morale et al., 2008).

Levels of neurotransmitter–receptor subunits Estrogens may influence the expression pattern and/or levels of neurotransmitter–receptor subunits mediated via the classic estrogen receptor pathway. For example, in the hippocampus, both ERa and ERb are present. Using the estrogen receptor-specific agonists, ERa selective agonist propylpyrazole triol (PPT) [1,3,5-tris (4-hydroxyphenyl)-4-propyl-1H-pyrazoletriol] and the ERb selective agonist DPN [2,3-bis (4-hydroxyphenyl) propionitrile] it was demonstrated that only diarylpropiolnitrile (DPN) administration had effects on the expression of alpha-amino-3-hydroxyl-5methyl-4-isoxazolepropionate (AMPA) receptor subunits GluR2 and GluR3, increasing and decreasing levels respectively, whereas estradiol, DPN and PPT increased AMPA-type glutamate receptor subunit GluR1 in the female rat stratum radiatum of dorsal hippocampus (Waters et al., 2009a).

Levels of neurotransmitters Physiological levels of estradiol have acute stimulatory effects on the dopaminergic activity in the ovarectomized rat striatum (Pasqualini et al., 1995). Subcutaneous injection of 17b- (but not 17a-) estradiol stimulated in situ tyrosine hydroxylase (TH) activity in a rapid dose-dependent manner, releasing newly synthesized dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC). This stimulation of DA synthesis was a consequence of an increase in TH levels. Incubation of striatal slices in the presence of 10(-9) M 17b- (but not 17a-) estradiol indeed evoked an approximate twofold increase in the Ki(DA) of one form of the enzyme. We have reported that less TH-positive neurons are present in the aged male hypothalamus of the ArKO mouse (Hill et al., 2004). Others have reported that aromatase deficiency from early embryonic life in the ArKO mice also significantly impaired the functional integrity of TH-positive

Dendritic spine formation Aromatase has been detected in axons and axon terminals in rat brains by immunostaining (Horvath et al., 1997). Previously, it has been shown that estrogens increase dendritic spine density and synaptogenesis in female (Frankfurt et al., 1990) and male rat (de Castilhos et al., 2008). Furthermore, ERa has also been detected by immunostaining in several extranuclear sites including dendrites, spines, terminals and axons in the hippocampus (McEwen et al., 2001; Romeo et al., 2005) with pro-estrous female rats having significantly more ERa in the dendrites than diestrous or male rats (Romeo et al., 2005). Estradiol and the ERa selective agonist PPT could induce significant increase in dendritic spines in vitro (Jelks et al., 2007); estrogen receptors have been localized along dendrites of cultured hippocampal neurons expressing N-Methyl-D-Aspartate

224

(NMDA) receptor subunit 1 through immunocytochemistry. The presence of aromatase in the axon terminals may infer that locally produced estrogens are acting directly on the localized estrogen receptors at synapses. One of the proposed mechanisms is that estrogens increase scaffolding proteins at the synpase such as post-synaptic density-95 (PSD-95) protein. Strong evidence has implicated that estrogens could increase the protein levels by upregulating the translation rate without increasing the transcription. It has been demonstrated in the NG108-15 neuroblastoma culture that estrogens rapidly stimulate an increase in PSD-95 protein levels without a concomittant rapid increase in PSD-95 mRNA levels. This stimulation could be inhibited by ERa antagonist ICI 182,780 or the PI3K inhibitor LY294002 (Akama and McEwen, 2003). The mechanism is mediated by Akt (protein kinase B) and 4E-BP1 (eukaryotic initiation factor-4E binding protein 1) phosphorylation. The translation of the PDS-95 transcripts at the dendrites is arrested by the binding of a protein complex containing 4E-BP1. Estrogens activate the phosphorylation of Akt (mediated through action of ERa), which in turn phosphorylates 4E-BP1 and leads to the dissociation of the inhibiting complex resulting in the translation of new PSD-95 immediately at the spine to increase spine maturation and synaptic formation. All this occurs in the dendrite, without involving the genomic action of estrogens (Waters et al., 2009b). Later, it is demonstrated in vitro that estrogen-induced phosphorylation of Akt is first apparent at 10 minute and maximal at 30 minute and could be blocked by an inhibitor of phosphatidylinositol-3kinase (PI3K) (Mannella and Brinton, 2006). The same study also showed that there is protein–protein interaction between ER and the PI3K regulatory subunit p85 in cultured cortical neurons. These in vitro data are supported by studies using cortical synaptoneurosome preparations (Dominguez et al., 2007), further illustrating that estrogens act at the synapses to modify dendritic spine formation and plasticity. Further evidence supporting this mechanism is the localization of phosphorylated Akt by immunostaining at the dendritic spines of rat

hippocampus. With the use of electron microscopy (Znamensky et al., 2003), the phosphorylated Akt could be detected at (i) dendritic spines (both cytoplasm and plasmalemma); (ii) spine apparati located within 0.1 micron of dendritic spine bases; (iii) endoplasmic reticula and polyribosomes in the cytoplasm of dendritic shafts and (iv) the plasmalemma of dendritic shafts. The localization density is correlated to the natural fluctuations of estrogen levels across the estrous cycle (Znamensky et al., 2003), which have previously been shown to cause cyclic changes in dendritic spine density and synaptogenesis in the rat hippocampus (Woolley and McEwen, 1992) levels. Estrogens also induce PSD-95 to form a ternary complex with calcium-activated neuronal NO synthase (nNOS) and glutamate NMDA receptor channels harbouring NR2B subunits as demonstrated in primary rat preoptic neuronal culture. Coupling of nNOS to NMDA receptor is accompanied by nitric oxide (NO) production. Again, this is an ERa-mediated event as the protein– protein interaction as well as NO production can be abolished by an ERa antagonist, ICI 182,780 (d’Anglemont de Tassigny et al., 2009). Dendritic spines are composed from actin microfilaments and recently, there are reports that estrogens have non-genomic effects on the re-modelling of actin cytoskeleton (reviewed by Sanchez and Simoncini, 2009). 17b-Estradiol addition to primary rat cortical cultures leads to phosphorylation and activation of WAVE1 (Wiskott-Aldrich syndrome protein (WASP)family verprolin homologous), which controls actin polymerization through the actin-related protein (Arp)-2/3 complex (Sanchez et al., 2009) in the neurons. This is mediated by ERa through the G protein/c-Src signalling cascade. Another non-genomic estrogen mechanism proposed is the activation of PI3 kinase that results in enhanced glutamate release from pre-;psynaptic neurons and leads to the activation of ionotropic glutamate receptors, which then activate mitogen-activated protein (MAP) kinases, thereby inducing dendritic spine formation during estradiol-induced organization of the hypothalamic synaptic patterning (Schwarz et al., 2008).

225

Allosteric effects

Conclusion

Aromatase has been detected by immunocytochemistry in axon terminals of vertebrate brains including quail, rat, monkey and human tissues (Naftolin et al., 1996) or at the surface of presynaptic vesicles (Hojo et al., 2004). Locally synthesized estrogens could serve as allosteric modulators of neurotransmitter receptors or ionotropic receptors. It has been reported that 17b-estradiol acts as a non-serotonin-competitive allosteric antagonist for the 5-hydroxytryptamine type 3 (5-HT3) receptor at the membrane surface of HEK-293 cells (Wetzel et al., 1998). 17b-Estradiol has also been reported to directly bind to the b subunit of the maxi-K channels (hSlo) and rapidly activates the channels (Valverde et al., 1999). As a consequence, estrogens can modulate the electrical activity of neurons within seconds. It has been demonstrated by electrophysiological studies that extremely low concentrations of estrogens acutely potentiate L-type voltage-gated Ca(2þ) channels (VGCCs) in hippocampal neurons, hippocampal slices, and HEK-293 cells transfected with neuronal L-type VGCC, via an ERindependent mechanism (Sarkar et al., 2008). Dihydropyridine site-specific L-type VGCC antagonist could displace membrane-bound estrogens, inferring that estrogens are interacting directly with the channels to rapidly induce Ca2þ influx (Sarkar et al., 2008). Remarkably, 17bestradiol pre-treatment of aged hippocampal neurons could normalize age-dysregulated intracellular Ca concentration responses after glutamate stimulation to that of middle-aged neurons in vitro (Brewer et al., 2006). In vitro study using neonatal hypothalamic neurons has also shown that estradiol pre-treatment could extend the duration of depolarization actions of gammaaminobutyric acid (GABA) as well as doubling the amplitude of Ca2þ transient induction (PerrotSinal et al., 2001) but the underlying mechanism remains to be elucidated. In summary, brain estrogens produced by brain aromatase are ‘neuroactive steroids’ that alter neuronal excitability by interacting with specific neurotransmitter receptors at the cell surface (Balthazart and Ball, 2006; Rupprecht and Holsboer, 1999).

Estrogens play multiple roles in regulating physiological functions. Mounting evidence has been gathered to support that estrogens act in paracrine or autocrine manners in the brain to regulate neuronal survival and brain functions. This is possible due to the expression of aromatase in specific brain regions/nuclei and cell populations. Since aromatase expression levels are highly regulated at the genomic levels and the aromatase activity are regulated by phosphorylations, the levels of brain estrogens could be tightly controlled.

References Abdelgadir, S. E., Resko, J. A., Ojeda, S. R., Lephart, E. D., McPhaul, M. J., & Roselli, C. E. (1994). Androgens regulate aromatase cytochrome P450 messenger ribonucleic acid in rat brain. Endocrinology, 135, 395–401. Abe-Dohmae, S., Takagi, Y., & Harada, N. (1996). Neurotransmitter-mediated regulation of brain aromatase: Protein kinase C- and G-dependent induction. Journal of Neurochemistry, 67, 2087–2095. Adams, M. M., Oung, T., Morrison, J. H., & Gore, A. C. (2001). Length of postovariectomy interval and age, but not estrogen replacement, regulate N-methyl-D-aspartate receptor mRNA levels in the hippocampus of female rats. Experimental Neurology, 170, 345–356. Akama, K. T. & McEwen, B. S. (2003). Estrogen stimulates postsynaptic density-95 rapid protein synthesis via the akt/ protein kinase B pathway. Journal of Neuroscience, 23, 2333–2339. Allen, E. & Doisy, E. A. (1983). Landmark article Sept 8, 1923. An ovarian hormone. Preliminary report on its localization, extraction and partial purification, and action in test animals. By Edgar Allen and Edward A. Doisy. JAMA, 250, 2681–2683. Azcoitia, I., Sierra, A., & Garcia-Segura, L. M. (1999). Localization of estrogen receptor beta-immunoreactivity in astrocytes of the adult rat brain. Glia, 26, 260–267. Azcoitia, I., Sierra, A., Veiga, S., Honda, S., Harada, N., & Garcia-Segura, L. M. (2001). Brain aromatase is neuroprotective. Journal of Neurobiology, 47, 318–329. Bakker, J., Honda, S. I., Harada, N., & Balthazart, J. (2002). The aromatase knock-out mouse provides new evidence that estradiol is required during development in the female for the expression of sociosexual behaviors in adulthood. Journal of Neuroscience, 22, 9104–9112. Bakker, J., Honda, S., Harada, N., & Balthazart, J. (2004). Restoration of male sexual behavior by adult exogenous

226 estrogens in male aromatase knockout mice. Hormones and Behavior, 46, 1–10. Balthazart, J., Baillien, M., & Ball, G. F. (2001a). Phosphorylation processes mediate rapid changes of brain aromatase activity. The Journal of Steroid Biochemistry and Molecular Biology, 79, 261–277. Balthazart, J., Baillien, M., & Ball, G. F. (2001b). Phosphorylation processes mediate rapid changes of brain aromatase activity. The Journal of Steroid Biochemistry and Molecular Biology, 79, 261–277. Balthazart, J., Baillien, M., & Ball, G. F. (2001c). Rapid and reversible inhibition of brain aromatase activity. Journal of Neuroendocrinology, 13, 63–73. Balthazart, J. & Ball, G. F. (2006). Is brain estradiol a hormone or a neurotransmitter? Trends in Neurosciences, 29, 241–249. Baquedano, M. S., Saraco, N., et al. (2007). Identification and developmental changes of aromatase and estrogen receptor expression in prepubertal and pubertal human adrenal tissues. Journal of Clinical Endocrinology and Metabolism, 92 (6), 2215–2222. Bhavnani, B. R. (2003). Estrogens and menopause: Pharmacology of conjugated equine estrogens and their potential role in the prevention of neurodegenerative diseases such as Alzheimer’s. The Journal of Steroid Biochemistry and Molecular Biology, 85, 473–482. Boon, W. C., Diepstraten, J., van der Burg, J., Jones, M. E., Simpson, E. R., & van den Buuse, M. (2005). Hippocampal NMDA receptor subunit expression and watermaze learning in estrogen deficient female mice. Brain Research. Molecular Brain Research, 140, 127–132. Brewer, G. J., Reichensperger, J. D., & Brinton, R. D. (2006). Prevention of age-related dysregulation of calcium dynamics by estrogen in neurons. Neurobiology of Aging, 27, 306–317. Britt, K.L., Drummond, A.E., et al. (2001). The ovarian phenotype of the aromatase knockout (ArKO) mouse. The Journal of Steroid Biochemistry and Molecular Biology, 79 (1–5), 181–185. Britt, K. L., Saunders, P. K., McPherson, S. J., Misso, M. L., Simpson, E. R., & Findlay, J. K. (2004). Estrogen actions on follicle formation and early follicle development. Biology of Reproduction, 71, 1712–1723. Brodie, A. & Inkster, S. (1993). Aromatase in the human testis. The Journal of Steroid Biochemistry and Molecular Biology, 44, 549–555. Bukovsky, A., Caudle, M.R., et al. (2003). Placental expression of estrogen receptor beta and its hormone binding variant – comparison with estrogen receptor alpha and a role for estrogen receptors in asymmetric division and differentiation of estrogen-dependent cells. Reproductive Biology and Endocrinology, 1, 36. Bulun, S.E., Imir, G., et al. (2005). Aromatase in endometriosis and uterine leiomyomata. The Journal of Steroid Biochemistry and Molecular Biology, 95(1–5), 57–62. Bulun, S. E., Sebastian, S., Takayama, K., Suzuki, T., Sasano, H., & Shozu, M. (2003). The human CYP19 (aromatase P450) gene: Update on physiologic roles and genomic

organization of promoters. The Journal of Steroid Biochemistry and Molecular Biology, 86, 219–224. Canonico, M., Plu-Bureau, L. G.D., & Scarabin, P. Y. (2008). Hormone replacement therapy and risk of venous thromboembolism in postmenopausal women: Systematic review and meta-analysis. BMJ, 336, 1227–1231. Carani, C., Qin, K., Simoni, M., Faustini-Fustini, M., Serpente, S., Boyd, J., et al. (1997). Effect of testosterone and estradiol in a man with aromatase deficiency. The New England Journal of Medicine, 337, 91–95. Carani, C., Rochira, V., Faustini-Fustini, M., Balestrieri, A., & Granata, A. R. (1999). Role of oestrogen in male sexual behaviour: Insights from the natural model of aromatase deficiency. Clinical Endocrinology (Oxford), 51, 517–524. Carpino, A., Romeo, F., & Rago, V. (2004). Aromatase immunolocalization in human ductuli efferentes and proximal ductus epididymis. Journal of Anatomy, 204, 217–220. Carrer, H. F., Cambiasso, M. J., Brito, V., & Gorosito, S. (2003). Neurotrophic factors and estradiol interact to control axogenic growth in hypothalamic neurons. Annals of the New York Academy of Sciences, 1007, 306–316. Catalano, S., Barone, I., Giordano, C., Rizza, P., Qi, H., Gu, G., et al. (2009). Rapid estradiol/ERalpha signaling enhances aromatase enzymatic activity in breast cancer cells. Molecular Endocrinology, 23, 1634–1645. Cheng, C. M., Cohen, M., Wang, J., & Bondy, C. A. (2001). Estrogen augments glucose transporter and IGF1 expression in primate cerebral cortex. The FASEB Journal, 15, 907–915. Chow, J. D., Simpson, E. R., & Boon, W. C. (2009). Alternative 50 -untranslated first exons of the mouse Cyp19A1 (aromatase) gene. The Journal of Steroid Biochemistry and Molecular Biology, 115, 115–125. Conley, A. & Hinshelwood, M. (2001). Mammalian aromatases. Reproduction, 121, 685–695. Corbin, C. J., Berger, T., Ford, J. J., Roselli, C. E., Sienkiewicz, W., Trainor, B. C., et al. (2009). Porcine hypothalamic aromatase cytochrome P450: Isoform characterization, sexdependent activity, regional expression, and regulation by enzyme inhibition in neonatal boars. Biology of Reproduction, 81, 388–395. Cruz, M.N., Agewall, S., et al. (2008). Acute dilatation to phytoestrogens and estrogen receptor subtypes expression in small arteries from women with coronary heart disease. Atherosclerosis, 196(1), 49–58. Dalla, C., Antoniou, K., Papadopoulou-Daifoti, Z., Balthazart, J., & Bakker, J. (2004). Oestrogen-deficient female aromatase knockout (ArKO) mice exhibit depressive-like symptomatology. European Journal of Neuroscience, 20, 217–228. Dalla, C., Antoniou, K., et al. (2005). Male aromatase-knockout mice exhibit normal levels of activity, anxiety and depressive-like symptomatology. Behavioural Brain Research, 163(2), 186–193. d’Anglemont de Tassigny, X., Campagne, C., Steculorum, S., & Prevot, V. (2009). Estradiol induces physical association of neuronal nitric oxide synthase with NMDA receptor and promotes nitric oxide formation via estrogen receptor activation in primary neuronal cultures. Journal of Neurochemistry, 109, 214–224.

227 de Castilhos, J., Forti, C. D., Achaval, M., & Rasia-Filho, A. A. (2008). Dendritic spine density of posterodorsal medial amygdala neurons can be affected by gonadectomy and sex steroid manipulations in adult rats: A Golgi study. Brain Research, 1240, 73–81. Demura, M., Reierstad, S., Innes, J. E., & Bulun, S. E. (2008). Novel promoter I.8 and promoter usage in the CYP19 (aromatase) gene. Reproduction Science, 15, 1044–1053. Dominguez, R., Liu, R., & Baudry, M. (2007). 17-Beta-estradiol-mediated activation of extracellular-signal regulated kinase, phosphatidylinositol 3-kinase/protein kinase B-Akt and N-methyl-D-aspartate receptor phosphorylation in cortical synaptoneurosomes. Journal of Neurochemistry, 101, 232–240. Ellem, S.J., Schmitt, J.F., et al. (2004). Local aromatase expression in human prostate is altered in malignancy. Journal of Clinical Endocrinology and Metabolism, 89(5), 2434–2441. Emilien, G., Beyreuther, K., Masters, C. L., & Maloteaux, J. M. (2000). Prospects for pharmacological intervention in Alzheimer disease. Archives of Neurology, 57, 454–459. Fisher, C. R., Graves, K. H., Parlow, A. F., & Simpson, E. R. (1998). Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene. Proceedings of the National Academy of Sciences of the United States of America, 95, 6965–6970. Frankfurt, M., Gould, E., Woolley, C. S., & McEwen, B. S. (1990). Gonadal steroids modify dendritic spine density in ventromedial hypothalamic neurons: A Golgi study in the adult rat. Neuroendocrinology, 51, 530–535. Fried, G., Andersson, E., et al. (2004). Estrogen receptor beta is expressed in human embryonic brain cells and is regulated by 17beta-estradiol. European Journal of Neuroscience, 20(9), 2345–2354. Galmiche, G., Corvaisier, S., & Kottler, M. L. (2006). Aromatase gene expression and regulation in the female rat pituitary. Annals of the New York Academy of Sciences, 1070, 286–292. Garcia-Segura, L. M., Wozniak, A., Azcoitia, I., Rodriguez, J. R., Hutchison, R. E., & Hutchison, J. B. (1999). Aromatase expression by astrocytes after brain injury: Implications for local estrogen formation in brain repair. Neuroscience, 89, 567–578. Ghosh, D., Griswold, J., Erman, M., & Pangborn, W. (2009). Structural basis for androgen specificity and oestrogen synthesis in human aromatase. Nature, 457, 219–223. Glatstein, I.Z. & Yeh, J. (1995). Ontogeny of the estrogen receptor in the human fetal uterus. Journal of Clinical Endocrinology and Metabolism, 80(3), 958–964. Golovine, K., Schwerin, M., & Vanselow, J. (2003). Three different promoters control expression of the aromatase cytochrome p450 gene (cyp19) in mouse gonads and brain. Biology of Reproduction, 68, 978–984. Gonzalez, M., Cabrera-Socorro, A., Perez-Garcia, C. G., Fraser, J. D., Lopez, F. J., Alonso, R., et al. (2007). Distribution patterns of estrogen receptor alpha and beta in the human cortex and hippocampus during development and adulthood. The Journal of Comparative Neurology, 503, 790–802. Grandien, K. (1996). Determination of transcription start sites in the human estrogen receptor gene and identification of a

novel, tissue-specific, estrogen receptor-mRNA isoform. Molecular and Cellular Endocrinology, 116(2), 207–212. Grandien, K. (1996). Determination of transcription start sites in the human estrogen receptor gene and identification of a novel, tissue-specific, estrogen receptor-mRNA isoform. Mol Cell Endocrinol, 116(2): 207–212. Green, P. S., Bales, K., Paul, S., & Bu, G. (2005). Estrogen therapy fails to alter amyloid deposition in the PDAPP model of Alzheimer’s disease. Endocrinology, 146, 2774– 2781. Harada, N. (1992). A unique aromatase (P-450AROM) mRNA formed by alternative use of tissue-specific exons 1 in human skin fibroblasts. Biochemical and Biophysical Research Communications, 189, 1001–1007. Harada, N., Ogawa, H., Shozu, M., & Yamada, K. (1992). Genetic studies to characterize the origin of the mutation in placental aromatase deficiency. The American Journal of Human Genetics, 51, 666–672. Harada, N., Wakatsuki, T., Aste, N., Yoshimura, N., & Honda, S. I. (2009). Functional analysis of neurosteroidal oestrogen using gene-disrupted and transgenic mice. Journal of Neuroendocrinology, 21, 365–369. Haczynski, J., Tarkowski, R., et al. (2002). Human cultured skin fibroblasts express estrogen receptor alpha and beta. International Journal of Molecular Medicine, 10(2), 149–153. Head, G. A., Obeyesekere, V. R., Jones, M. E., Simpson, E. R., & Krozowski, Z. S. (2004). Aromatase-deficient (ArKO) mice have reduced blood pressure and baroreflex sensitivity. Endocrinology, 145, 4286–4291. Hess, R. A. (2003). Estrogen in the adult male reproductive tract: A review. Reproductive Biology and Endocrinology, 1, 52. Hewitt, K. N., Pratis, K., Jones, M. E., & Simpson, E. R. (2004). Estrogen replacement reverses the hepatic steatosis phenotype in the male aromatase knockout mouse. Endocrinology, 145, 1842–1848. Hewitt, K. N., Boon, W. C. et al. (2003). The aromatase knockout mouse presents with a sexually dimorphic disruption to cholesterol homeostasis. Endocrinology, 144 (9), 3895–3903. Hill, R. A. & Boon, W. C. (2009). Estrogens, brain, and behavior: Lessons from knockout mouse models. Seminars in Reproductive Medicine, 27, 218–228. Hill, R. A., Chow, J., Fritzemeier, K., Simpson, E. R., & Boon, W. C. (2007a). Fas/FasL-mediated apoptosis in the arcuate nucleus and medial preoptic area of male ArKO mice is ameliorated by selective estrogen receptor alpha and estrogen receptor beta agonist treatment, respectively. Molecular and Cellular Neuroscience, 36, 146–157. Hill, R. A., Chua, H. K. et al. (2009). Estrogen deficiency results in apoptosis in the frontal cortex of adult female aromatase knockout mice. Mol Cellular Neurosci, 41(1), 1–7. Hill, R. A., McInnes, K. J., Gong, E. C., Jones, M. E., Simpson, E. R., & Boon, W. C. (2007b). Estrogen deficient male mice develop compulsive behavior. Biological Psychiatry, 61, 359–366. Hill, R. A., Pompolo, S., Jones, M. E.E., Simpson, E. R., & Boon, W. C. (2004). Estrogen deficiency leads to apoptosis in dopaminergic neurons in the medial pre-optic area and

228 arcuate nucleus of male mice. Molecular and Cellular Neuroscience, 27, 466–476. Hill, R. A., Pompolo, S., Jones, M. E., Simpson, E. R., & Boon, W. C. (2004). Estrogen deficiency leads to apoptosis in dopaminergic neurons in the medial preoptic area and arcuate nucleus of male mice. Molecular and Cellular Neuroscience, 27, 466–476. Hodges, Y.K., Tung, L., et al. (2000). Estrogen receptors alpha and beta: prevalence of estrogen receptor beta mRNA in human vascular smooth muscle and transcriptional effects. Circulation, 101(15), 1792–1798. Hojo, Y., Hattori, T. A., Enami, T., Furukawa, A., Suzuki, K., Ishii, H. T., et al. (2004). Adult male rat hippocampus synthesizes estradiol from pregnenolone by cytochromes P45017alpha and P450 aromatase localized in neurons. Proceedings of the National Academy of Sciences of the United States of America, 101, 865–870. Honda, S., Harada, N., Ito, S., Takagi, Y., & Maeda, S. (1998). Disruption of sexual behavior in male aromatasedeficient mice lacking exons 1 and 2 of the cyp19 gene. Biochemical and Biophysical Research Communications, 252, 445–449. Honda, S., Harada, N., & Takagi, Y. (1994). Novel exon 1 of the aromatase gene specific for aromatase transcripts in human brain. Biochemical and Biophysical Research Communications, 198, 1153–1160. Honda, S., Harada, N., & Takagi, Y. (1996). The alternative exons 1 of the mouse aromatase cytochrome P-450 gene. Biochimica et Biophysica Acta, 1305, 145–150. Horvath, T. L., Roa-Pena, L., Jakab, R. L., Simpson, E. R., & Naftolin, F. (1997). Aromatase in axonal processes of early postnatal hypothalamic and limbic areas including the cingulate cortex. The Journal of Steroid Biochemistry and Molecular Biology, 61, 349–357. Hoyland, J.A., Mee, A.P., et al. (1997). Demonstration of estrogen receptor mRNA in bone using in situ reverse-transcriptase polymerase chain reaction. Bone, 20(2), 87–92. Huang, R. & Poduslo, S. E. (2006). CYP19 haplotypes increase risk for Alzheimer’s disease. Journal of Medical Genetics, 43, e42. Hutchison, J. B., Beyer, C., Hutchison, R. E., & Wozniak, A. (1997). Sex differences in the regulation of embryonic brain aromatase. The Journal of Steroid Biochemistry and Molecular Biology, 61, 315–322. Huynh, K., Jones, G., Thouas, G., Britt, K. L., Simpson, E. R., & Jones, M. E. (2004). Estrogen is not directly required for oocyte developmental competence. Biology of Reproduction, 70, 1263–1269. Iivonen, S., Corder, E., Lehtovirta, M., Helisalmi, S., Mannermaa, A., Vepsalainen, S., et al. (2004). Polymorphisms in the CYP19 gene confer increased risk for Alzheimer disease. Neurology, 62, 1170–1176. Ishunina, T. A. & Swaab, D. F. (2003). Increased neuronal metabolic activity and estrogen receptors in the vertical limb of the diagonal band of Broca in Alzheimer’s disease: Relation to sex and aging. Experimental Neurology, 183, 159–172.

Ishunina, T. A., van Beurden, D., van der, M. G., Unmehopa, U. A., Hol, E. M., Huitinga, I., et al. (2005). Diminished aromatase immunoreactivity in the hypothalamus, but not in the basal forebrain nuclei in Alzheimer’s disease. Neurobiology of Aging, 26, 173–194. Jelks, K. B., Wylie, R., Floyd, C. L., McAllister, A. K., & Wise, P. (2007). Estradiol targets synaptic proteins to induce glutamatergic synapse formation in cultured hippocampal neurons: Critical role of estrogen receptor-alpha. Journal of Neuroscience, 27, 6903–6913. Jones, M. E., Boon, W. C., McInnes, K., Maffei, L., Carani, C., & Simpson, E. R. (2007). Recognizing rare disorders: Aromatase deficiency. Nature Clinical Practice Endocrinology and Metabolism, 3, 414–421. Jones, M. E., Thorburn, A. W., Britt, K. L., Hewitt, K. N., Wreford, N. G., Proietto, J., et al. (2000). Aromatase-deficient (ArKO) mice have a phenotype of increased adiposity. Proceedings of the National Academy of Sciences of the United States of America, 97, 12735–12740. Kadioglu, P., Oral, G., Sayitoglu, M., Erensoy, N., Senel, B., Gazioglu, N., et al. (2008). Aromatase cytochrome P450 enzyme expression in human pituitary. Pituitary, 11, 29–35. Kazeto, Y., Ijiri, S., Place, A. R., Zohar, Y., & Trant, J. M. (2001). The 50 -flanking regions of CYP19A1 and CYP19A2 in zebrafish. Biochemical and Biophysical Research Communications, 288, 503–508. Kilgore, M. W., Means, G. D., Mendelson, C. R., & Simpson, E. R. (1992). Alternative promotion of aromatase P-450 expression in the human placenta. Molecular and Cellular Endocrinology, 83, R9–R16. Kimura, M., Sudhir, K., et al. (2003). Impaired acetylcholineinduced release of nitric oxide in the aorta of male aromatase-knockout mice: regulation of nitric oxide production by endogenous sex hormones in males. Circulation Research, 93(12), 1267–1271. Knopp, R. H., Paramsothy, P., Retzlaff, B. M., Fish, B., Walden, C., Dowdy, A., et al. (2006). Sex differences in lipoprotein metabolism and dietary response: Basis in hormonal differences and implications for cardiovascular disease. Current Cardiology Reports, 8, 452–459. Kostanyan, A. & Nazaryan, K. (1992). Rat brain glycolysis regulation by estradiol-17 beta. Biochimica et Biophysica Acta, 1133, 301–306. Kumar, P., Kamat, A., & Mendelson, C. R. (2009). Estrogen receptor alpha (ERalpha) mediates stimulatory effects of estrogen on aromatase (CYP19) gene expression in human placenta. Molecular Endocrinology, 23, 784–793. Lambard, S., Galeraud-Denis, I., Saunders, P. T., & Carreau, S. (2004). Human immature germ cells and ejaculated spermatozoa contain aromatase and oestrogen receptors. Journal of Molecular Endocrinology, 32, 279–289. Lanfranco, F., Zirilli, L., Baldi, M., Pignatti, E., Corneli, G., Ghigo, E., et al. (2008). A novel mutation in the human aromatase gene: Insights on the relationship among serum estradiol, longitudinal growth and bone mineral density in an adult man under estrogen replacement treatment. Bone, 43, 628–635.

229 Larionov, A. A., Vasyliev, D. A., Mason, J. I., Howie, A. F., Berstein, L. M., & Miller, W. R. (2003). Aromatase in skeletal muscle. The Journal of Steroid Biochemistry and Molecular Biology, 84, 485–492. Leav, I., Lau, K.M., et al. (2001). Comparative studies of the estrogen receptors beta and alpha and the androgen receptor in normal human prostate glands, dysplasia, and in primary and metastatic carcinoma. The American Journal of Pathology, 159(1), 79–92. Lecce, G., Meduri, G., et al. (2001). Presence of estrogen receptor beta in the human endometrium through the cycle: expression in glandular, stromal, and vascular cells. Journal of Clinical Endocrinology and Metabolism, 86(3), 1379–1386. Lephart, E. D. (1996). A review of brain aromatase cytochrome P450. Brain Research. Brain Research Reviews, 22, 1–26. Lephart, E. D., Call, S. B., Rhees, R. W., Jacobson, N. A., Weber, K. S., Bledsoe, J., et al. (2001a). Neuroendocrine regulation of sexually dimorphic brain structure and associated sexual behavior in male rats is genetically controlled. Biology of Reproduction, 64, 571–578. Lephart, E. D., Lund, T. D., & Horvath, T. L. (2001b). Brain androgen and progesterone metabolizing enzymes: Biosynthesis, distribution and function. Brain Research. Brain Research Reviews, 37, 25–37. Levin-Allerhand, J., McEwen, B. S., Lominska, C. E., Lubahn, D. B., Korach, K. S., & Smith, J. D. (2001). Brain region-specific up-regulation of mouse apolipoprotein E by pharmacological estrogen treatments. Journal of Neurochemistry, 79, 796–803. Li, C. L., Toda, K., et al. (2002). Estrogen deficiency results in enhanced expression of smoothened of the hedgehog signaling in the thymus and affects thymocyte development. International Immunopharmacology, 2(6), 823–833. Ling, S., Dai, A., et al. (2004). Endogenous estrogen deficiency reduces proliferation and enhances apoptosis-related death in vascular smooth muscle cells: insights from the aromataseknockout mouse. Circulation, 109(4), 537–543. MacGillivray, M. H., Morishima, A., Conte, F., Grumbach, M., & Smith, E. P. (1998). Pediatric endocrinology update: An overview. The essential roles of estrogens in pubertal growth, epiphyseal fusion and bone turnover: Lessons from mutations in the genes for aromatase and the estrogen receptor. Hormone Research, 49(Suppl. 1), 2–8. Mahendroo, M.S., Means, G.D., et al. (1991). Tissue-specific expression of human P-450AROM. The promoter responsible for expression in adipose tissue is different from that utilized in placenta. The Journal of Biological Chemistry, 266(17), 11276–11281. Mahendroo, M. S., Mendelson, C. R., & Simpson, E. R. (1993). Tissue-specific and hormonally controlled alternative promoters regulate aromatase cytochrome P450 gene expression in human adipose tissue. The Journal of Biological Chemistry, 268, 19463–19470. Mahmoodzadeh, S., Fritschka, S., et al. (2009). Nuclear factorkappaB regulates estrogen receptor-alpha transcription in the human heart. The Journal of Biological Chemistry, 284(37), 24705–24714.

Makinen, S., Makela, S., et al. (2001). Localization of oestrogen receptors alpha and beta in human testis. Molecular Reproduction and Development, 7(6), 497–503. Mannella, P. & Brinton, R. D. (2006). Estrogen receptor protein interaction with phosphatidylinositol 3-kinase leads to activation of phosphorylated Akt and extracellular signalregulated kinase 1/2 in the same population of cortical neurons: A unified mechanism of estrogen action. Journal of Neuroscience, 26, 9439–9447. Martin, S., Jones, M., Simpson, E., & van den Buuse, M. (2003). Impaired spatial reference memory in aromatasedeficient (ArKO) mice. NeuroReport, 14, 1979–1982. McCullough, L. D., Blizzard, K., Simpson, E. R., Oz, O. K., & Hurn, P. D. (2003). Aromatase cytochrome P450 and extragonadal estrogen play a role in ischemic neuroprotection. Journal of Neuroscience, 23, 8701–8705. McEwen, B., Akama, K., Alves, S., Brake, W. G., Bulloch, K., Lee, S., et al. (2001). Tracking the estrogen receptor in neurons: Implications for estrogen-induced synapse formation. Proceedings of the National Academy of Sciences of the United States of America, 98, 7093–7100. McNatty, K. P., Baird, D. T., Bolton, A., Chambers, P., Corker, C. S., & McLean, H. (1976). Concentration of oestrogens and androgens in human ovarian venous plasma and follicular fluid throughout the menstrual cycle. Journal of Endocrinology, 71, 77–85. McPherson, S. J., Wang, H., Jones, M. E., Pedersen, J., Iismaa, T. P., Wreford, N., et al. (2001). Elevated androgens and prolactin in aromatase-deficient mice cause enlargement, but not malignancy, of the prostate gland. Endocrinology, 142, 2458–2467. Means, G. D., Mahendroo, M. S., Corbin, C. J., Mathis, J. M., Powell, F. E., Mendelson, C. R., et al. (1989). Structural analysis of the gene encoding human aromatase cytochrome P-450, the enzyme responsible for estrogen biosynthesis. The Journal of Biological Chemistry, 264, 19385–19391. Means, G.D., Kilgore, M.W., et al. (1991). Tissue-specific promoters regulate aromatase cytochrome P450 gene expression in human ovary and fetal tissues. Molecular Endocrinology, 5(12), 2005–2013. Mendelsohn, M. E. (2009). Estrogen actions in the cardiovascular system. Climacteric, 12(Suppl. 1), 18–21. Miyaura, C., Toda, K., et al. (2001). Sex- and age-related response to aromatase deficiency in bone. Biochemical and Biophysical Research Communications, 280(4), 1062–1068. Michael, M. D., Kilgore, M. W., Morohashi, K., & Simpson, E. R. (1995). Ad4BP/SF-1 regulates cyclic AMP-induced transcription from the proximal promoter (PII) of the human aromatase P450 (CYP19) gene in the ovary. The Journal of Biological Chemistry, 270, 13561–13566. Miller, T. W., Shin, I., Kagawa, N., Evans, D. B., Waterman, M. R., & Arteaga, C. L. (2008). Aromatase is phosphorylated in situ at serine-118. The Journal of Steroid Biochemistry and Molecular Biology, 112, 95–101. Misso, M. L., Murata, Y., Boon, W. C., Jones, M. E., Britt, K. L., & Simpson, E. R. (2003). Cellular and molecular

230 characterization of the adipose phenotype of the aromatasedeficient mouse. Endocrinology, 144, 1474–1480. Mitra, S. W., Hoskin, E., Yudkovitz, J., Pear, L., Wilkinson, H. A., Hayashi, S., et al. (2003). Immunolocalization of estrogen receptor beta in the mouse brain: Comparison with estrogen receptor alpha. Endocrinology, 144, 2055–2067. Moore, J.T., McKee, D.D., et al. (1998). Cloning and characterization of human estrogen receptor beta isoforms. Biochemical and Biophysical Research Communications, 247(1), 75–78. Morale, M. C., L’episcopo, F., Tirolo, C., Giaquinta, G., Caniglia, S., Testa, N., et al. (2008). Loss of aromatase cytochrome P450 function as a risk factor for Parkinson’s disease? Brain Research Reviews, 57, 431–443. Morale, M. C., Serra, P. A., L’episcopo, F., Tirolo, C., Caniglia, S., Testa, N., et al. (2006). Estrogen, neuroinflammation and neuroprotection in Parkinson’s disease: Glia dictates resistance versus vulnerability to neurodegeneration. Neuroscience, 138, 869–878. Morishima, A., Grumbach, M. M., Simpson, E. R., Fisher, C., & Qin, K. (1995). Aromatase deficiency in male and female siblings caused by a novel mutation and the physiological role of estrogens. Journal of Clinical Endocrinology and Metabolism, 80, 3689–3698. Miyaura, C., K. Toda, et al. (2001). Sex- and age-related response to aromatase deficiency in bone. Biochem Biophys Res Commun, 280(4): 1062–1068. Mulnard, R. A., Cotman, C. W., Kawas, C., van Dyck, C. H., Sano, M., Doody, R., et al. (2000). Estrogen replacement therapy for treatment of mild to moderate alzheimer disease: a randomized controlled trial. Alzheimer’s disease cooperative study. JAMA, 283(8), 1007–1015. Mylonas, I., Jeschke, U., et al. (2004). Immunohistochemical analysis of estrogen receptor alpha, estrogen receptor beta and progesterone receptor in normal human endometrium. Acta Histochemica, 106(3), 245–252. Naftolin, F., Horvath, T. L., & Balthazart, J. (2001). Estrogen synthetase (aromatase) immunohistochemistry reveals concordance between avian and rodent limbic systems and hypothalami. Experimental Biology and Medicine (Maywood), 226, 717–725. Naftolin, F., Horvath, T. L., Jakab, R. L., Leranth, C., Harada, N., & Balthazart, J. (1996). Aromatase immunoreactivity in axon terminals of the vertebrate brain. An immunocytochemical study on quail, rat, monkey and human tissues. Neuroendocrinology, 63, 149–155. Naftolin, F. & MacLusky, N. J. (1982). Aromatase in the central nervous system. Cancer Research, 42, 3274s–3276s. Naftolin, F., Ryan, K. J., Davies, I. J., Reddy, V. V., Flores, F., Petro, Z., et al. (1975). The formation of estrogens by central neuroendocrine tissues. Recent Progress in Hormone Research, 31, 295–319. Nawata, H., Tanaka, S., Tanaka, S., Takayanagi, R., Sakai, Y., Yanase, T., et al. (1995). Aromatase in bone cell: Association with osteoporosis in postmenopausal women. The Journal of Steroid Biochemistry and Molecular Biology, 53, 165–174. Nemoto, Y., Toda, K., Ono, M., Fujikawa-Adachi, K., Saibara, T., Onishi, S., et al. (2000). Altered expression of fatty acid-

metabolizing enzymes in aromatase-deficient mice. The Journal of Clinical Investigation, 105, 1819–1825. Nilsen, J., Irwin, R. W., Gallaher, T. K., & Brinton, R. D. (2007). Estradiol in vivo regulation of brain mitochondrial proteome. Journal of Neuroscience, 27, 14069–14077. Osawa, Y., Higashiyama, T., Fronckowiak, M., Yoshida, N., & Yarborough, C. (1987). Aromatase. The Journal of Steroid Biochemistry, 27, 781–789. Osterlund, M.K., Grandien, K., et al. (2000). The human brain has distinct regional expression patterns of estrogen receptor alpha mRNA isoforms derived from alternative promoters. Journal of Neurochemistry, 75(4), 1390–1397. Oz, O. K., Hirasawa, G., Lawson, J., Nanu, L., Constantinescu, A., Antich, P. P., et al. (2001). Bone phenotype of the aromatase deficient mouse. The Journal of Steroid Biochemistry and Molecular Biology, 79, 49–59. Pasqualini, C., Olivier, V., Guibert, B., Frain, O., & Leviel, V. (1995). Acute stimulatory effect of estradiol on striatal dopamine synthesis. Journal of Neurochemistry, 65, 1651–1657. Pasquali, D., Staibano, S., et al. (2001). Estrogen receptor beta expression in human prostate tissue. Molecular and Cellular Endocrinology, 178(1–2), 47–50. Pedersen, S.B., Bruun, J.M., et al. (2001). Demonstration of estrogen receptor subtypes alpha and beta in human adipose tissue: influences of adipose cell differentiation and fat depot localization. Molecular and Cellular Endocrinology, 182(1), 27–37. Perrot-Sinal, T. S., Davis, A. M., Gregerson, K. A., Kao, J. P., & McCarthy, M. M. (2001). Estradiol enhances excitatory gamma-aminobutyric [corrected] acid-mediated calcium signaling in neonatal hypothalamic neurons. Endocrinology, 142, 2238–2243. Pierman, S., Sica, M., et al. (2008). Activational effects of estradiol and dihydrotestosterone on social recognition and the arginine-vasopressin immunoreactive system in male mice lacking a functional aromatase gene. Hormones and Behavior, 54(1), 98–106. Price, T., Aitken, J., & Simpson, E. R. (1992). Relative expression of aromatase cytochrome P450 in human fetal tissues as determined by competitive polymerase chain reaction amplification. Journal of Clinical Endocrinology and Metabolism, 74, 879–883. Price, T.M. & O‘Brien, S.N. (1993). Determination of estrogen receptor messenger ribonucleic acid (mRNA) and cytochrome P450 aromatase mRNA levels in adipocytes and adipose stromal cells by competitive polymerase chain reaction amplification. Journal of Clinical Endocrinology and Metabolism, 77(4), 1041–1045. Rasgon, N. L., Silverman, D., Siddarth, P., Miller, K., Ercoli, L. M., Elman, S., et al. (2005). Estrogen use and brain metabolic change in postmenopausal women. Neurobiology of Aging, 26, 229–235. Raven, G., de Jong, F. H., Kaufman, J. M., & de Ronde, W. (2006). In men, peripheral estradiol levels directly reflect the action of estrogens at the hypothalamo-pituitary level to inhibit gonadotropin secretion. Journal of Clinical Endocrinology and Metabolism, 91, 3324–3328.

231 Robertson, K. M., O’Donnell, L., Jones, M. E., Meachem, S. J., Boon, W. C., Fisher, C. R., et al. (1999). Impairment of spermatogenesis in mice lacking a functional aromatase (cyp 19) gene. Proceedings of the National Academy of Sciences of the United States of America, 96, 7986–7991. Robertson, K. M., Simpson, E. R., Lacham-Kaplan, O., & Jones, M. E. (2001). Characterization of the fertility of male aromatase knockout mice. Journal of Andrology, 22, 825–830. Rochira, V., Zirilli, L., Genazzani, A. D., Balestrieri, A., Aranda, C., Fabre, B., et al. (2006). Hypothalamic-pituitary-gonadal axis in two men with aromatase deficiency: Evidence that circulating estrogens are required at the hypothalamic level for the integrity of gonadotropin negative feedback. European Journal of Endocrinology, 155, 513–522. Roepke, T. A. (2009). Oestrogen modulates hypothalamic control of energy homeostasis through multiple mechanisms. Journal of Neuroendocrinology, 21, 141–150. Romeo, R. D., McCarthy, J. B., Wang, A., Milner, T. A., & McEwen, B. S. (2005). Sex differences in hippocampal estradiol-induced N-methyl-D-aspartic acid binding and ultrastructural localization of estrogen receptor-alpha. Neuroendocrinology, 81, 391–399. Roselli, C. E., Horton, L. E., & Resko, J. A. (1985). Distribution and regulation of aromatase activity in the rat hypothalamus and limbic system. Endocrinology, 117, 2471–2477. Rozovsky, I., Wei, M., Stone, D. J., Zanjani, H., Anderson, C. P., Morgan, T. E., et al. (2002). Estradiol (E2) enhances neurite outgrowth by repressing glial fibrillary acidic protein expression and reorganizing laminin. Endocrinology, 143, 636–646. Rupprecht, R. & Holsboer, F. (1999). Neuroactive steroids: Mechanisms of action and neuropsychopharmacological perspectives. Trends in Neurosciences, 22, 410–416. Sakamoto, H., Mezaki, Y., Shikimi, H., Ukena, K., & Tsutsui, K. (2003). Dendritic growth and spine formation in response to estrogen in the developing Purkinje cell. Endocrinology, 144, 4466–4477. Sanchez, A. M., Flamini, M. I., Fu, X. D., Mannella, P., Giretti, M. S., Goglia, L., et al. (2009). Rapid signaling of estrogen to WAVE1 and moesin controls neuronal spine formation via the actin cytoskeleton. Molecular Endocrinology, 23, 1193– 1202. Sanchez, A. M. & Simoncini, T. (2009). Extra-nuclear signaling of ERalpha to the actin cytoskeleton in the central nervous system. Steroids. (in press). Sanghera, M. K., Simpson, E. R., McPhaul, M. J., Kozlowski, G., Conley, A. J., & Lephart, E. D. (1991). Immunocytochemical distribution of aromatase cytochrome P450 in the rat brain using peptide-generated polyclonal antibodies. Endocrinology, 129, 2834–2844. Santen, R. J., Brodie, H., Simpson, E. R., Siiteri, P. K., & Brodie, A. (2009). History of aromatase: Saga of an important biological mediator and therapeutic target. Endocrine Reviews, 30, 343–375. Sarkar, S. N., Huang, R. Q., Logan, S. M., Yi, K. D., Dillon, G. H., & Simpkins, J. W. (2008). Estrogens directly potentiate neuronal L-type Ca2þ channels. Proceedings of the National Academy of Sciences of the United States of America, 105, 15148–15153.

Sasahara, K., Shikimi, H., Haraguchi, S., Sakamoto, H., Honda, S., Harada, N., et al. (2007). Mode of action and functional significance of estrogen-inducing dendritic growth, spinogenesis, and synaptogenesis in the developing Purkinje cell. Journal of Neuroscience, 27, 7408–7417. Sasano, H., Murakami, H., Shizawa, S., Satomi, S., Nagura, H., & Harada, N. (1999). Aromatase and sex steroid receptors in human vena cava. Endocrine Journal, 46, 233–242. Sasano, H., Takashashi, K., Satoh, F., Nagura, H., & Harada, N. (1998). Aromatase in the human central nervous system. Clinical Endocrinology (Oxford), 48, 325–329. Saunders, P.T., Millar, M.R., et al. (2000). Differential expression of estrogen receptor-alpha and -beta and androgen receptor in the ovaries of marmosets and humans. Biology of Reproduction, 63(4), 1098–1105. Savolainen, H., Frosen, J., et al. (2001). Expression of the vasculoprotective estrogen receptor subtype beta in rat and human cardiac allograft vasculopathy. Transplantation Proceedings, 33(1–2), 1605. Schwarz, J. M., Liang, S. L., Thompson, S. M., & McCarthy, M. M. (2008). Estradiol induces hypothalamic dendritic spines by enhancing glutamate release: A mechanism for organizational sex differences. Neuron, 58, 584–598. Sebastian, S., Takayama, K., et al. (2002). Cloning and characterization of a novel endothelial promoter of the human CYP19 (aromatase P450) gene that is up-regulated in breast cancer tissue. Molecular Endocrinology, 16(10), 2243–2254. Seralini, G. E., Tomilin, A., Auvray, P., Nativelle-Serpentini, C., Sourdaine, P., & Moslemi, S. (2003). Molecular characterization and expression of equine testicular cytochrome P450 aromatase. Biochimica et Biophysica Acta, 1625, 229–238. Shapiro, E., Huang, H., et al. (2005a). Immunolocalization of estrogen receptor alpha and beta in human fetal prostate. The Journal of Urology, 174(5), 2051–2053. Shapiro, E., Huang, H., et al. (2005b). Immunolocalization of androgen receptor and estrogen receptors alpha and beta in human fetal testis and epididymis. The Journal of Urology, 174(4 Pt 2), 1695–1698, discussion 1698. Shim, G. J., Warner, M., Kim, H. J., Andersson, S., Liu, L., Ekman, J., et al. (2004). Aromatase-deficient mice spontaneously develop a lymphoproliferative autoimmune disease resembling Sjogren’s syndrome. Proceedings of the National Academy of Sciences of the United States of America, 101, 12628–12633. Shozu, M., Zhao, Y., et al. (1998). Multiple splicing events involved in regulation of human aromatase expression by a novel promoter, I.6. Endocrinology, 139(4), 1610–1617. Simpson, E. R. (2004). Aromatase: Biologic relevance of tissuespecific expression. Seminars in Reproductive Medicine, 22, 11–23. Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Corbin, C. J., & Mendelson, C. R. (1993). Tissuespecific promoters regulate aromatase cytochrome P450 expression. The Journal of Steroid Biochemistry and Molecular Biology, 44, 321–330. Steimer, T. & Hutchison, J. B. (1981). Androgen increases formation of behaviourally effective oestrogen in dove brain. Nature, 292, 345–347.

232 Stockard, C. R. & Papanicolaou, G. N. (1917). A rhythmical “heat period” in the guinea-pig. Science, 46, 42–44. Stoffel-Wagner, B., Watzka, M., Schramm, J., Bidlingmaier, F., & Klingmuller, D. (1999). Expression of CYP19 (aromatase) mRNA in different areas of the human brain. The Journal of Steroid Biochemistry and Molecular Biology, 70, 237–241. Stoffel-Wagner, B., Watzka, M., Steckelbroeck, S., Schwaab, R., Schramm, J., Bidlingmaier, F., et al. (1998). Expression of CYP19 (aromatase) mRNA in the human temporal lobe. Biochemical and Biophysical Research Communications, 244, 768–771. Takeda, K., Toda, K., et al. (2003). Progressive development of insulin resistance phenotype in male mice with complete aromatase (CYP19) deficiency. Journal of Endocrinology, 176(2), 237–246. Toda, K., Saibara, T., Okada, T., Onishi, S., & Shizuta, Y. (2001a). A loss of aggressive behaviour and its reinstatement by oestrogen in mice lacking the aromatase gene (Cyp19). Journal of Endocrinology, 168, 217–220. Toda, K., Simpson, E. R., Mendelson, C. R., Shizuta, Y., & Kilgore, M. W. (1994). Expression of the gene encoding aromatase cytochrome P450 (CYP19) in fetal tissues. Molecular Endocrinology, 8, 210–217. Toda, K., Takeda, K., Akira, S., Saibara, T., Okada, T., Onishi, S., et al. (2001b). Alternations in hepatic expression of fattyacid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17beta-estradiol or a peroxisome proliferator. The Journal of Steroid Biochemistry and Molecular Biology, 79, 11–17. Toda, K., Takeda, K., et al. (2001c). Alternations in hepatic expression of fatty-acid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17beta-estradiol or a peroxisome proliferator. The Journal of Steroid Biochemistry and Molecular Biology, 79(1–5), 11–17. Toda, K., Takeda, K. et al. (2001). Targeted disruption of the aromatase P450 gene (Cyp19) in mice and their ovarian and uterine responses to 17beta-oestradiol. J Endocrinol, 170 (1), 99–111. Toth, B., Saadat, G., et al. (2008). Human umbilical vascular endothelial cells express estrogen receptor beta (ERbeta) and progesterone receptor a (PR-A), but not ERalpha and PR-B. Histochemistry and Cell Biology, 130(2), 399–405. Valverde, M. A., Rojas, P., Amigo, J., Cosmelli, D., Orio, P., Bahamonde, M. I., et al. (1999). Acute activation of Maxi-K channels (hSlo) by estradiol binding to the beta subunit. Science, 285, 1929–1931. van Groen, T. & Kadish, I. (2005). Transgenic AD model mice, effects of potential anti-AD treatments on inflammation and pathology. Brain Research. Brain Research Reviews, 48, 370–378. Vidal, O., Kindblom, L.G., et al. (1999). Expression and localization of estrogen receptor-beta in murine and human bone. Journal of Bone and Mineral Research, 14(6), 923–929. Wang, L., Andersson, S., & Gustafsson, J. A. (2003). Estrogen receptor (ER)beta knockout mice reveal a role for ERbeta in migration of cortical neurons in the developing brain. Proceedings of the National Academy of Sciences of the United States of America, 100, 703–708.

Waters, E. M., Mitterling, K., Spencer, J. L., Mazid, S., McEwen, B. S., & Milner, T. A. (2009a). Estrogen receptor alpha and beta specific agonists regulate expression of synaptic proteins in rat hippocampus. Brain Research, 1290, 1–11. Waters, E. M., Mitterling, K., Spencer, J. L., Mazid, S., McEwen, B. S., & Milner, T. A. (2009b). Estrogen receptor alpha and beta specific agonists regulate expression of synaptic proteins in rat hippocampus. Brain Research, 1290, 1–11. Wetzel, C. H., Hermann, B., Behl, C., Pestel, E., Rammes, G., Zieglgansberger, W., et al. (1998). Functional antagonism of gonadal steroids at the 5-hydroxytryptamine type 3 receptor. Molecular Endocrinology, 12, 1441–1451. Wise, P. M., Dubal, D. B., Wilson, M. E., Rau, S. W., Bottner, M., & Rosewell, K. L. (2001). Estradiol is a protective factor in the adult and aging brain: Understanding of mechanisms derived from in vivo and in vitro studies. Brain Research. Brain Research Reviews, 37, 313–319. Wesson, D.W., Keller, M., et al. (2006). Enhanced urinary odor discrimination in female aromatase knockout (ArKO) mice. Hormones and Behavior, 49(5), 580–586. Woolley, C. S. & McEwen, B. S. (1992). Estradiol mediates fluctuation in hippocampal synapse density during the estrous cycle in the adult rat. Journal of Neuroscience, 12, 2549–2554. Xu, H., Gouras, G. K., Greenfield, J. P., Vincent, B., Naslund, J., Mazzarelli, L., et al. (1998). Estrogen reduces neuronal generation of Alzheimer beta-amyloid peptides. Nature Medicine, 4, 447–451. Xu, H., Wang, R., Zhang, Y. W., & Zhang, X. (2006). Estrogen, beta-amyloid metabolism/trafficking, and Alzheimer’s disease. Annals of the New York Academy of Sciences, 1089, 324–342. Yague, J. G., Munoz, A., Monasterio-Schrader, P., Defelipe, J., Garcia-Segura, L. M., & Azcoitia, I. (2006). Aromatase expression in the human temporal cortex. Neuroscience, 138, 389–401. Yan, M., Jones, M. E., Hernandez, M., Liu, D., Simpson, E. R., & Chen, C. (2005). Oestrogen replacement in vivo rescues the dysfunction of pituitary somatotropes in ovariectomised aromatase knockout mice. Neuroendocrinology, 81, 158–166. Youn, C. S., Kwon, O. S., Won, C. H., Hwang, E. J., Park, B. J., Eun, H. C., et al. (2003). Effect of pregnancy and menopause on facial wrinkling in women. Acta Dermato-Venereologica, 83, 419–424. Yue, X., Lu, M., Lancaster, T., Cao, P., Honda, S., Staufenbiel, M., et al. (2005). Brain estrogen deficiency accelerates Ab plaque formation in an Alzheimer’s disease animal model. Proceedings of the National Academy of Sciences of the United States of America, 102, 19198–19203. Znamensky, V., Akama, K. T., McEwen, B. S., & Milner, T. A. (2003). Estrogen levels regulate the subcellular distribution of phosphorylated Akt in hippocampal CA1 dendrites. Journal of Neuroscience, 23, 2340–2347. Zhao, Y., Mendelson, C. R. et al. (1995). Characterization of the sequences of the human CYP19 (aromatase) gene that mediate regulation by glucocorticoids in adipose stromal cells and fetal hepatocytes. Molecular Endocrinology, 9(3), 340–349.

L. Martini (Eds.) Progress in Brain Research, Vol. 181 ISSN: 0079-6123 Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 13

ERb in CNS: new roles in development and function Xiaotang Fan1, Haiwei Xu3, Margaret Warner2,3 and Jan-Åke Gustafsson2,3
1

Department of Histology and Embryology, Third Military Medical University, Chongqing, China 2 Department of Biosciences and Nutrition, Karolinska Institute, Huddinge, Sweden 3 Center for Nuclear Receptors and Cell Signalling, Department of Biology and Biochemistry, University of Houston, Houston, Texas, United States of America

Abstract: Estrogen acting through two estrogen receptors (ERs), ERa and ERb, regulates multiple functions in the central nervous system. Studies in rodent brains have revealed that ERa is the predominant ER in the hypothalamus and controls reproduction. ERb influences on non-reproductive processes and appears to be the main ER subtype expressed in the cerebral cortex, the hippocampus, the cerebellum and the dorsal raphe. During embryogenesis, estrogen plays an important role in brain development regulating maturation of distinct brain structures, thereby contributing to modulation of the function of these structures. Studies on the brains of ERb knockout mice revealed that, during embryonic brain development, ERb affects cortical layering and interneuron migration, thus playing a key role in brain morphogenesis. This chapter will focus on the roles of ERb in several aspects of the development and function of the mammalian central nervous system. Keywords: ERb; development; cerebral cortex; hippocampus; cerebellum; spinal cord

thought to mediate all of the actions of E2 in animal tissues. In 1996 a second ER subtype (ERb) was discovered (Kuiper et al., 1996). The two ERs are the products of two independent genes, located on different chromosomes. The ERb gene is located on human chromosome 14 (Enmark et al., 1997), whereas ERa is located on human chromosome 6 (Green et al., 1986). Characterization of ERs revealed that although they share 97% similarity in the DNA-binding domain (DBD), there is only a 55% similarity in the ligand-binding domain LBD (Fig. 1) and there is little homology in the N-terminal transactivation domain and carboxy-terminal F domain. These structural features account for the fact that both ERs bind to similar response elements on DNA

Introduction Estrogen receptors (ERs) are members of the nuclear receptor (NR) superfamily of transcription factors. In 1958, Elwood Jensen, using H3-labelled 17b-estradiol (E2), first demonstrated the existence of an intracellular receptor mediating actions of E2 in the female reproductive tract. Subsequently, Greene cloned the cDNA from uterus, with the help of antibodies raised in the Jensen laboratory (Greene et al., 1986). Until 1996 this remained the only known ER and was


Corresponding author. Tel.: +832-842-8803; Fax: +713-743-0634; E-mail: [email protected]

DOI: 10.1016/S0079-6123(08)81013-8

233

234 hERα hERβ

A/B A/B 18%

D

C C

D

97% 30%

E E 59%

F

595aa

F 530aa 18% Homology

Fig. 1. Comparison of human estrogen receptors (ERa and ERb). Full-length human ERa is 595 amino acids long; and full-length human ERb is 530 amino acids long. Both ERb and ERa have five domains. (A/B) The N-terminal domain; (C) The DNA-binding domain; (D) The hinge; (E): The ligand-binding domain; (F) The F region.

but exhibit ligand selectivity, which has permitted pharmaceutical companies to develop subtype-specific ligands (Chadwick et al. 2005; Leventhal et al. 2006; Neubauer et al. 2003; Stovall and Pinkerton 2009). In addition, chemicals in the environment, such as natural plant compounds (phytoestrogens) and industrial byproducts (xenoestrogens), differ in their affinity for ERa and ERb (Kuiper et al., 1997). E2 binds equally well to both receptors. Genistein has an approximately 10-fold higher affinity for ERb (0.3 nM) than for ERa and raloxifene binds preferentially to ERb (Barkhem et al., 1998; Bolger et al., 1998; Jordan et al., 1985; Oostenbrink et al., 2000). The two estrogen metabolites estrone and estriol, thought to be inactive metabolites of E2, actually bind to ERa and ERb with significant affinity (in the low nanomolar range) and, because their levels can be quite high, for example during pregnancy, it should be considered that they may be modulators of estrogen signalling in vivo. At present there are two commercially available non-steroidal compounds which are selective agonists or antagonists for ERa or ERb. Propylpyrazoletriol (PPT) is approximately 1000fold more potent as an agonist on ERa than on ERb, whereas diarylpropionitrile (DPN) is an ERb-selective agonist (Harrington et al., 2003). These subtype-selective agonists are widely used in vivo and in vitro to define the actions of ERa and ERb. Differences in the expression patterns of ERa and ERb in adult brains indicate distinct functions of the two receptors. ERa expression is high in the areas of the rodent brain associated with reproduction, such as the

hypothalamus, bed nucleus of the stria terminalis (BNST), the preoptic area/anterior hypothalamus and hypothalamic arcuate (ARC) and ventromedial (VMH) nuclei which can be regulated by circulating levels of estrogens in a region-dependent manner (Kruijver et al., 2002; Laflamme et al., 1998; Miranda and ToranAllerand, 1992; Wilson et al., 2008). In the human brain, ERb appears to be the predominant receptor in areas such as the cerebral cortex, the hippocampus, the anterior olfactory nucleus, the cerebellum, the dorsal raphe, the substantia nigra and ventral tegmental area of the midbrain and several brain stem nuclei (Osterlund and Hurd, 2001; Osterlund et al., 2000; Pérez et al., 2003). In vitro studies have shown that ERa and ERb have the ability to heterodimerize when they coexist in the same nucleus and that hetero- and homodimers display different transcriptional activities (Cowley et al., 1997; Matthews and Gustafsson, 2003; Pettersson et al., 1997). Both ERa and ERb are expressed in certain brain regions such as the preoptic area, BNST, ventromedial nucleus (VMN), medial amygdala and spinal cord (Fan et al., 2007), but their co-localization in cell nuclei of these regions remains to be assessed.

ERs in the embryonic brain There are many published studies on expression of ERa and ERb during embryonic brain development in rodent and human foetuses. There is a consensus that both ERs are expressed in the embryonic brain but the picture is less clear on the details of timing, protein expression, size of

235

the protein, type of neurons and brain areas where expression occurs. This is not surprising because the embryonic brain is difficult to work with even when the proteins under study are abundantly expressed and steroid receptors are not abundant in cellular proteins. The method of fixation of the brain; the specificity of the antibodies; identification of DNA fragments after RT-PCR and identification of the protein bands seen on western blots are all extremely important and vary from lab to lab (Al-Bader et al., 2008; Fan et al., 2006; Guo et al., 2001; Kritzer, 2006; Miranda and Toran-Allerand, 1992; Prewitt and Wilson, 2007; Zsaenovszky and Belcher, 2001). The human foetal brain has been examined by Fried et al. (2004), Gonzalez et al. (2007) and Takeyama et al. (2001). These are rare and unique investigations because of the difficulty of obtaining samples under conditions which permit good studies.

ERb affects neuronal migration during later stages of corticogenesis Cortical development begins with the formation of the preplate followed by the appearance of the cortical plate (CP), which is the precursor of most of the cortex. Layering of neurons occurs in an ‘inside-out’ manner, with the earliest-generated neurons positioned in the deepest layers and later-generated neurons migrating along processes of the radial glia beyond previously established layers to settle at progressively more superficial levels (Angevine and Sidman, 1961; Neubauer et al., 2003; Gupta et al., 2002). This inside-out gradient of migration is critical not only for cortical structure but also for establishment of correct neural connections. Defects in neuronal migration always induce retardation of cerebral cortex morphogenesis and mental development (Gleeson and Walsh, 2000; McManus and Golden, 2005). In the cortex of mice, the period of neurogenesis is between E11 and E17, and during this period the majority of CP neurons are generated from the ventricular zone (VZ). Studies on ERb

knockout (ERbKO) mice have revealed that early cortical development is not affected by lack of ERb; at E14.5, there were no discernable differences between the brains of ERbKO mice and their wild-type (WT) littermates in either size or gross morphology (Wang et al., 2003). Localization of ERa in the embryonic VZ cells indicates that ERa may affect early cortical neurogenesis, although no obvious brain abnormalities have been observed in the ERa knockout (ERaKO) mouse (Wang et al., 2001). Thus, the importance of ERa in development of the cerebral cortex remains to be investigated. However, at E18.5 the brains of ERbKO mice were clearly smaller than those of littermate controls, especially in the cerebral cortex. Nissl staining showed unequivocal evidence of a thinner cortex in the ERbKO brains of both male and female mice. No morphological differences were evident in the hippocampus, thalamus or hypothalamus when ERbKO and WT mice were compared (Wang et al., 2003). Thus, ERb affects cerebral cortex development at later stages of corticogenesis resulting in severe neuronal deficit in the somatosensory cortex, especially layers II, III, IV and V, in adult ERbKO mice (Wang et al., 2001). Neuronal labelling with BrdU pinpointed the defect to neuronal migration during the late stage of corticogenesis. Neurons generated later and destined to layers II–IV were delayed in their migration in mice lacking ERb. As the mice aged, the neurons labelled at E15.5 and E16.5 eventually did migrate to the correct position in the cortex, but there were far fewer of these neurons than in control mice (Wang et al., 2003). These observations were made in both male and female mice and strongly suggest that ERb plays an important role in the regulation of migration of cortical neurons in the upper laminae during perinatal life. Radial glial cells have a function in neuronal migration and laminar patterning of the cortex (Weissman et al., 2003). The radial glial cells of cerebral cortex in ERbKO mice are morphologically different from those in WT mice. When stained for nestin, an intermediate filament shared by radial glial cells and

236

neuronal precursors, the processes of the radial glial cells appear to be truncated or less organized into radial formations. Such abnormal processes might not be able to provide guidance for migrating neurons (Wang et al., 2003). Therefore, loss of ERb-induced effects in the radial glial cells might partly contribute to retarded neuronal migration. Epidermal growth factor (EGF) receptor and its ligands, including heparin-binding EGF and TGFa, are highly expressed in the germinal layers of the telencephalon and are important in the control of migration in the developing telencephalon (Caric et al., 2001; Lopez-Bendito et al., 2006). Loss of EGF receptor (EGFR) in mice leads to accumulation of neuronal precursors in telencephalic proliferative zones and defects in migration, while over-expression of EGFR leads to increased radial migration in the cortex and olfactory bulb (Sobeih and Corfas, 2002). In the brains of WT mice, EGFR expression is localized in two stripes, corresponding to subplate/white matter and the superficial marginal zone; but in ERbKO mice, EGFR expression was significantly decreased and especially lost in the superficial marginal zone (Fan et al., 2006). These results can mean either that, in ERbKO mice, EGFR-positive neurons failed to reach the superficial marginal zone at later stages of embryonic development or that ERb, through modulation of EGFR expression, affects migration of post-mitotic neurons to superficial cortical layers.

3b-Adiol as a ligand for ERb function in corticogenesis Estradiol synthesis occurs in the developing brain at E18.5 and this time when sexual imprinting of the brain occurs (Colciago et al., 2005). ERb is expressed in the brain from E12.5, a time when E2 is not synthesized in the CNS and when the brain is protected from maternal E2 by its sequestration on alpha-foetoprotein (AFP). Although ERb is essential for neuronal migration and cortical layering, E2 itself does not appear to be necessary as the brain of aromatase knockout (ArKO) mice, which cannot make E2, has not been reported to

show major neuronal abnormalities (Dalla et al., 2005). If ERb is essential for correct layering of the cortex at a time when E2 is not present in the brain, ERb may be acting independently of ligand or there might be an alternative ligand for ERb in the developing brain. The dihydrotestosterone metabolite, 5aandrostane-3b, 17b-diol (3b-Adiol) is secreted by the immature ovaries and testes and seems to be a promising alternative ligand for ERb during development (Weihua et al., 2002). As 3b-Adiol is synthesized in situ in cells expressing 5a-reductase and 17b-hydroxysteroid dehydrogenase type 7 (17bHSD type 7), its level in the brain is not affected by AFP. Mice in which the 17bHSD type 7 has been knocked out do not survive embryonic day 10.5 and exhibit major brain and heart defects (Shehu et al., 2008). CYP7B1 is the enzyme responsible for hydroxylation and inactivation of the estrogenicity of 3bAdiol. CYP7B1 knockout (CYP7B1KO) mice cannot hydroxylate 3b-Adiol and there are high levels of this estrogenic steroid in the brain of these mice. One consequence of the accumulation of 3b-Adiol in the brain is that the size of the forebrain area is larger than that in normal mice and this results in compression of the lateral ventricles. The enlarged cortex and the compression of the lateral ventricles remain in postnatal mice until puberty when the brain size normalizes (Sugiyama et al., 2009). The abnormal brain size appears to be due to a reduction in the number of cells undergoing apoptosis between E13.5 and E15.5, in the ventricular/subventricular zone (Sugiyama et al., 2009). Taken together, these results suggest that 3b-Adiol is the functional ligand for ERb in the developing brain.

ERb expression in the neonatal brain In the frontal cortex, there is a rapid down-regulation of ERs between P7 and P9. As judged from the undetectable levels of ERs, the peripubertal period from P14 to P35 appears to be a window in time when the brain is insensitive to estrogen. Four months after birth, there was a gradual

237

increase in the number of ERa- and ERb-positive cells (Sugiyama et al., 2009).

ERb expression in the hippocampus and its roles in hippocampal-dependent behavior ERb is the predominant ER in the hippocampus The hippocampus is a key brain region regulating complex cognitive and emotional responses, and is implicated in the aetiology of depressive and anxiety disorders, many of which exhibit some degree of sex difference. Both clinical data and animal experiments indicate that E2 acting on ERs affects hippocampal development and function. ERs are expressed in the rodent hippocampus (Levin, 2001; Li et al., 1997). In adult rat hippocampus, ERa immunoreactivity (IR) is localized in select interneuron nuclei and several extranuclear locations, including dendritic spines and axon terminals, while ERb-IR is primarily in the perikarya and proximal dendrites of pyramidal and granule cells. ERb-IR was also seen in a few non-principal cells and scattered nuclei in the ventral subiculum and CA3 region (Milner et al., 2005). RT-PCR analysis revealed that expression of aromatase as well as ERs (a/b) is developmentally regulated in the mouse hippocampus. Aromatase expression increased during the first two post-natal weeks and decreased to lower levels in adults. ERa/ERb mRNAs did not fluctuate significantly throughout pre- and post-natal development but revealed a distinct sex-specific pattern at the end of the first post-natal week (Prewitt and Wilson, 2007). With home-made antibodies that selectively recognize the C-terminal part of ERb, we have shown that ERb protein appeared in the nuclei of hippocampal neurons at E15.5, and increased to moderate level at E18.5 (Fan et al., 2006). In our hands there was no cytoplasmic ERb staining. The presence of both ER subtypes in the developing hippocampus has also been observed in other mammals including sheep and human (Schaub et al., 2008). Abundant ERb mRNA was localized in the hippocampal formation of human (primarily the

subiculum) (Osterlund et al., 2000). At the protein level, the expression of ERa and ERb displayed different spatial-temporal patterns. Western blotting and immunohistochemistry of the adult hippocampus revealed lower protein levels of ERa than ERb (Gonzalea et al., 2007). In the macaque monkey brain, ERb is widely expressed in the adult hippocampus, whereas ERa immunoreactivity is either absent or very rare (Takahashi et al., 2004). Interestingly, ultrastructural analysis of the brains of rats at diestrus revealed that the cellular and subcellular localization of ERb-IR was generally similar to that of ERa, except that ERb was more extensively found at extranuclear sites. ERb-IR was found in plasma membranes primarily of principal cell perikarya and proximal dendrites, spines arising from pyramidal and granule cell dendrites and pre-terminal axons and axon terminals (Milner et al., 2005). ERb presence in dendrites and axons may suggest its roles in important hippocampus-related functions such as learning and memory.

ERb affects hippocampal-dependent spatial learning Several lines of evidence suggest that hormonal changes play an important role in the incidence of cognitive decline after menopause and also in the development of Alzheimer’s disease (Foy et al., 2008). Animal studies have shown that E2 can influence cognitive processes of female rodents. Performance in the eye-blink conditioning, passive avoidance and object recognition tasks is related to E2 levels during the estrous cycle, being better in high E2 phases (Walf et al., 2006). When E2 is administered to ovariectomized (OVX) rats, performance in hippocampus-mediated tasks, such as the water maze, radial arm maze, four-arm plus maze and passive avoidance, is improved over that produced by vehicle administration (Cheryl et al., 2007). E2 exerts some of its effects on the hippocampus via classical genomic actions of steroid receptors. Studies of ERbKO and ERaKO mice have

238

revealed that the two ERs affect different types of learning. ERb specifically influences hippocampus-dependent spatial learning, while ERa mainly affects emotional learning that is likely to rely on the amygdala (Amin et al., 2005). OVX ERbKO females were slower than WT females to learn in the Morris water maze. Surprisingly, this learning deficit in the knockout females was enhanced by administration of exogenous E2 in a dose-dependent manner. In ERbKOs given a low dose of E2, learning was slow but ERbKOs who were administered a higher dose of E2 failed to learn the task. In contrast, all WT females displayed significant learning (Rissman et al., 2002). One recent study has further confirmed the roles of ERb in hippocampus-dependent spatial learning using ERbKO, ERaKO and WT mice (Liu et al., 2008). In this study, E2 did not improve cognitive functions in hippocampus-dependent spatial tasks in ERbKO mice, while both ERaKO and WT mice showed improvement in both the radial arm maze and Morris water maze. Furthermore, a selective ERb agonist (WAY-200070) was able to mimic the effects of E2 in a similar hippocampus-dependent task, whereas an ERa selective agonist (PPT) was not. ERb activation also increased long-term potentiation (LTP) in hippocampal slices. This effect was not seen in slices from ERbKO mice (Liu et al., 2008). In men, chronic isoflavone supplementation can improve spatial working memory, but does not affect auditory and episodic memory (Thorp et al., 2009). However, cognition was not influenced by estradiol benzoate (EB), the selective ERb agonist DPN or the ER modulator tamoxifen (Lacreuse et al., 2009) in young adult female macaques. Additional studies are needed to determine whether the cognitive effects of estrogens in monkeys of more advanced age are mediated by ERb, ERa or complex interactions between the two receptors. ERb-selective ER modulators (SERMS) or dietary phytoestrogens, which have selective actions on ERb, enhance performance in hippocampal tasks, such as the water maze, radial arm maze and inhibitory avoidance tasks (Madeline and Cheryl, 2006). OVX WT mice, but not ERbKO, administered E2 or DPN, spent a

greater percentage of time exploring a novel object in the object recognition task and a displaced object in the object placement task (Walf et al., 2008b). Thus, actions on ERb may be important for E2 or SERMs to enhance cognitive performance of female mice. Together, these findings suggest that ERb activation enhances learning and memory.

Mechanisms of ERb modulating hippocampaldependent spatial learning E2 has been reported to promote the formation of new dendritic spines and excitatory synapses in the hippocampus (Gazzaley et al., 2002). OVX adult rats pre-treated with E2 (60 mg/kg) showed increased spine-synapse density as rapidly as 30 minutes after E2 injection. Furthermore, administration of E2 (1–4 days) to OVX adult female rats increased the density of spines in the stratum radiatum of CA1 pyramidal neurons to the level seen in intact rats (Parducz et al., 2006). In vitro investigations have also shown that the spine density in CA1 area of cultured hippocampal slices was increased following several days’ treatment of E2. (Murphy and Segal, 1996; Pozzo-Miller et al., 1999). Prange-Kiel and Rune (2006) have reported that the hippocampus can synthesize E2 and that this is of biological significance. Using cultured slices of the stratum radiatum of the CA1 region, this group showed that the suppression of endogenous E2 synthesis by letrozole treatment for 4 days significantly decreased the density of spines, spine-synapses, spinophilin (spine marker) and synaptophysin (presynaptic marker). A marked decrease in the density of hippocampal synapses was observed between proestrus and estrus in rat (Woolley and McEwen, 1992). Expression of ERb mRNA and protein was high at estrus and low at proestrus. Thus the level of ERb expression seems to be related to E2 action on synapse structure and synaptic plasticity. Several studies (Bodo and Rissman, 2006; Choleris et al., 2008) have shown that when OVX mice are treated with E2, there are

239

no effects on total spine number but an increase in mushroom spines on CA1 pyramidal neurons. Furthermore, ERb-agonist-treated mice showed increased dendritic branching in CA1 pyramidal neurons, significantly increasing the ‘mushroom’ type of dendritic spines on the granule cells in the dentate gyrus without effects on total spine number. In in vitro studies, ERb appeared to have the opposite effect: Decreases in ERb expression correlated with an increase in synapse formation in hippocampal cultures. In these studies, over-expression of ERb resulted in lowering of the dendritic spine density that was elevated by E2 (Szymczak et al., 2006). Much more work is needed to resolve the discrepancies between in vivo and in vitro studies of the role of ERb on synapse formation in hippocampal neurons. E2-induced synaptogenesis is regulated by synaptic proteins. AMPARs are hetero-oligomeric proteins made of the subunits metabotropic glutamate receptors, GluR1–GluR4 (also known as GluRA–D). Malinow and Malenka (2002) have demonstrated that recruitment of AMPARs during LTP is from lateral diffusion of spine surface receptors containing GluR1. The activation of synapses caused by this trafficking is one mechanism for the long-term maintenance of increased synaptic strength and the formation of memories. Man et al. (2007) have demonstrated that ERb activation increased surface levels of GluR1 phosphorylated at Ser845, thereby regulating the subcellular trafficking of GluR1. Over-expression of post-synaptic density protein (PSD-95) has been shown to increase AMPAR currents by selectively delivering GluR1-containing receptors to synapses (Ehrlich et al., 2007). The ERb agonist WAY-200070 induced GluR1 while activation of ERb with DPN increased GluR2 and decreased GluR3. DPN also had more widespread effects in the CA1 region as it also increased GluR2 in the stratum oriens and stratum pyramidal. In ERbKO mice there is deficient LTP in the CA1 region as well as compromised hippocampusdependent memory. ER2 can potentiate CA3CA1 LTP in WT but not in ERbKO mice (Liu et al., 2008). These findings suggest that ERb is a modulator of hippocampal synaptic plasticity.

E2-induced synaptogenesis clearly depends on post-synaptic N-methyl-D-aspartate receptor (NMDAR) transmission because antagonists of NMDARs block E2-induced synaptogenesis in vivo and in vitro (Jelks et al., 2007; Smith et al., 2009). E2 is also involved in modulating NMDA receptors composed of the NR1/2B subunits and synapse formation. The NMDAR2B gene promoter contains an SP1 sequence by which ERa could activate its transcription. Hence, E2 could increase NMDA receptor density via ERa. This conclusion was confirmed by use of OVX rats in which decreased NMDA/NR2B level in the hippocampal CA1 oriens and CA1 radiatum could be reversed by E2 or PPT but not by DPN treatment (Jelks et al., 2007). Thus ERa and ERb affect synapse formation through different synaptic proteins. Adult hippocampal neurogenesis has been observed in birds, reptiles, rodents and primates, including humans (El-Bakri et al., 2004; Eriksson et al., 1998). Neurons are born in the underlying subgranular layer and move into the granule cell layer (GCL) to become mature granule neurons. Neurogenesis in adult hippocampus contributes to improving learning and memory as has been widely demonstrated in animal experiments (Kempermann et al., 1997; Lipkind et al., 2002; Shors et al., 2002). Extensive co-localization of immunoreactive markers for cell proliferation and differentiation with mRNAs for ERa and ERb points to a direct modulation of dentate cell proliferation, differentiation and survival by E2 (Isgor and Watson, 2005). Moreover, recent studies have revealed that activation of both ERs increases cell proliferation within the dentate gyrus but ERb appears to be a more effective regulator of this proliferation (Mazzucco et al., 2006). ERb promotion of adult hippocampal neurogenesis may be a mechanism positively affecting learning and memory.

ERb activation affects anxiety and/or depressive behavior In women, the incidence of anxiety and/or depression is increased when E2 levels are on the decline. Both E2 withdrawal after pregnancy and

240

treatment with tamoxifen for breast cancer increase the risk of depression. In contrast, when E2 levels are higher, either naturally or via hormone therapy (HT), anxiety is decreased. Animal experiments have revealed that male rats exhibit significantly higher levels of contextual fear memory than female rats. Male rats also showed higher levels of freezing than cycling female rats after contextual fear conditioning. Furthermore, female rats subjected to conditioning in proestrus and estrus displayed higher enhancement of fear extinction than male rats. OVX rats or mice given systemic or intrahippocampal E2 spend more time in the open arms of the elevated plus maze than their vehicleadministered counterparts, indicating that E2 is anxiolytic (Walf and Frye, 2007a; Walf and Frye, 2007b; Walf et al., 2009c). Comparison of the neurochemical profiles of ArKO and WT females has shown that estrogens modulate dopaminergic, serotonergic and noradrenergic brain activities. There is no difference in spontaneous motor activity, exploration or anxiety between ArKO and WT female mice, but ArKO females display ‘depressive-like’ symptoms, for example increased passive behaviors, such as floating, in repeated sessions of the forced swim test. This depressivelike behavior could not be reversed by treatment with E2, suggesting that E2 affects hippocampus function in a developmentally regulated manner (Dalla et al., 2004). There is evidence that the anti-anxiety and anti-depressive effects of E2 may be mediated by ERb in the hippocampus (Walf et al., 2006). E2 or ERb-selective SERMs administered into the OVX mice caused a decrease in anxiety and depressive behavior, that is the mice spent more time in the open arms of the plus maze, and spent less time immobile than did vehicle-treated mice (Walf et al., 2008b; Walf et al., 2009a). In contrast, administration of ERa-specific SERMs failed to decrease anxiety and depressive behavior. Comparison of behavior of ER knockout and WT mice has revealed that intact female ERbKO mice spent less time in the open arms of the elevated plus maze compared to WT and ERaKO mice (Lund et al., 2005). In addition, E2-treated OVX ERbKO mice

demonstrated greater anxiety behavior than did their WT counterparts in the plus maze (Walf et al., 2009b). This is consistent with another study in which antisense oligonucleotide (AS-ODN) targeted to ERa or ERb was used to explore ER function in anxiety and depression. OVX rats infused with ERb AS-ODNs had decreased ERb immunoreactivity in the brain and had significantly decreased open field central entries, decreased plus maze open arm time and entries, increased time spent immobile and decreased time spent swimming in the forced swim test than did rats administered ERa AS-ODNs, vehicle or scrambled AS-ODNs (Walf et al., 2008a). Furthermore, administration of the ERb agonist, DPN, but not the ERa agonist, PPT, enhanced extinction of contextual fear in OVX female rats. Furthermore, intrahippocampal injection of E2 or DPN before extinction training in OVX female rats remarkably reduced the levels of freezing response during extinction trials (Hughes et al., 2008). These results reveal that E2-mediated enhancement of fear extinction involves the activation of ERb. The mechanisms underlying the effects of ERb are related to its function in modulating the activity of 5-HT neurons in the dorsal raphe nuclei. When OVX female rats are given DPN, there is an increase in tryptophan hydroxylase (TPH)-2 mRNA in the caudal and mid-dorsal DRN and this is accompanied by a decrease in despair-like behavior. There was no affect on anxiety (Donner and Handa, 2009).

Role of ERb in protection of hippocampal neurons E2 at physiological concentrations modulates apoptotic cell death cascades and prevents neuronal death in experimental models of focal and global ischaemia (Carswell et al., 2004). Several lines of evidence indicate that E2 can exert protection against ischaemia-induced changes: (1) E2 prevented cell death in hippocampal CA1 of intact male gerbils and OVX female rats and gerbils (Merchenthaler et al., 2003); (2) the size of

241

lesions following ischaemic brain injury was smaller in either intact or OVX WT animals than in ArKO mice (McCullough et al., 2003); (3) in organotypic hippocampal slice culture system, E2 preconditioning significantly protected the hippocampal CA1 region against ischaemia (Raval et al., 2006); (4) in organotypic hippocampal slice cultures subjected to oxygen–glucose deprivation for 60 minutes, pre-treatment with E2 resulted in reduction in cellular injury in CA1 region by 46% compared to the vehicle control group (Cimarosti et al., 2006). The molecular basis of E2-mediated neuroprotection against brain ischaemia remains obscure but ERs are clearly involved as the broad-spectrum ER antagonist ICI 182,780 (intracerebroventricularly, 0 and 12 hours after ischaemia) abolished E2 protection (Miller et al., 2005). In terms of the receptor subtype involved, it seems that E2 can act via both ERa and ERb to protect CA1 neurons in rodents from global ischaemia-induced death. Furthermore, ERb plays a role in protection of hippocampal neurons from excitotoxic injury. Both PPT and DPN protected hippocampal neurons against glutamate-induced cell death in a dosedependent manner (Zhao et al., 2004).

Role of ERb in development of the cerebellum NRs control gene regulation throughout the development of the CNS, including the cerebellum (Gofflot et al., 2007). E2 acts on Purkinje cells through intranuclear receptor-mediated mechanisms during cerebellar development (Tsutsui, 2006; Tsutsui et al., 2004). In the cerebellum of adult rats or mice, in situ hybridization and immunohistochemistry studies have detected ERb mRNA and protein in Purkinje cells and scattered cells within the granular layer. In contrast, ERa expression in the adult cerebellum was undetectable by in situ hybridization and immunohistochemistry, although RTPCR and western blot analyses detected very low levels of ERa mRNA and protein. Furthermore, E2 treatment reduced the number of ERb mRNA-expressing Purkinje cells but had no significant effects on the cerebellar ERa mRNA

levels. Thus, it appears that the physiological effects of E2 on the adult cerebellum are mainly exerted through ERb. Both ERa and ERb contribute to the development of the cerebellum (Jakab et al., 2001). During mouse embryogenesis, ERb appeared at a high level in the cerebellar primordium as early as E12.5, although the function of ERb in the early cerebellum is not clear. At P0, there was strong ERb expression in the external germinal layer (EGL) and some scattered ERb-positive cells were found in the deeper layers of the cortex. At P7, ERb was expressed in the proliferating and differentiating granule cell precursors of EGL. By P21, ERb was expressed in granule and basket/stellate cells, but not in Purkinje cells (Qin et al., 2007). In the developing rat cerebellum, ERb appeared in Purkinje cells at P6, with peak intensities of immunostaining coinciding with the initiation of axonal and dendritic growth occuring between P7 and P8, when dendritic arbour growth is initiated. As was observed in granule cells, the most intense immunolabelling of Purkinje cells occurred during growth of processes, with particularly intense immunolabelling localized to the apical cytoplasmic swellings where dendrite growth occurs. Because the genesis and migration of Purkinje cells is primarily completed by birth, the observed pattern of ERb expression in Purkinje cells is also most consistent with ERb playing a role in the growth of cellular processes rather than functions related to proliferation. From the third post-natal week, ERb-IR was also detected in the later developing Golgi, stellate and basket neurons. Once induced, expression of ERb remained high during maturation of Purkinje cell dendrites and was maintained in the adult (Ikeda and Nagai, 2006). Based on the observed temporal and cell-type-specific changes in ERb expression and the correlation of these changes with known events during cerebellar development, it has been suggested that ERb is involved in post-mitotic developmental events related to migration, extension of cellular processes of cerebellar neurons and glia and the regulation of synaptic dynamics. In another study, quantitative real-time RT-PCR demonstrated that expression levels of cerebellar

242

ERb mRNA remained essentially unchanged during post-natal development (Mitra et al., 2003). In contrast, levels of cerebellar ERa mRNA in neonatal pups were significantly higher than in adults. In situ hybridization and immunohistochemistry demonstrated that ERb expression occurred in Golgi-type neurons in the granular layer at P7, in Purkinje cells at P14 and in basket cells in the molecular layer at P21 and was detected in all cell types in the adult cerebellum, suggesting a role for ERb in neuronal differentiation and maintenance. ERa mRNA and protein were expressed by Purkinje cells at all ages examined. ERa-expressing Purkinje cells were confined to the anterior lobes at day P7 but distributed in most lobes at P14 and P21. In the adult cerebellum, however, only a few ERa-IR Purkinje cells were observed. Thus, ERa expression was transiently increased during the time when Purkinje cell dendritic growth and synapse formation occurred, suggesting a role for ERa in Purkinje cell differentiation (Guo et al., 2001). The discrete expression profiles of ERa and ERb in the developing cerebellum suggest that the two ERs play distinct roles in cerebellar development. In vivo experiments have shown that administration of EB to newborn rats induced dendritic outgrowth of Purkinje cells. Furthermore, in vitro treatment with estradiol using cerebellar slice cultures from newborn rats resulted in the promotion of dendritic outgrowth of Purkinje cells. Thus, both in vitro and in vivo studies suggest that neonatal estradiol is essential for the promotion of dendritic growth of the Purkinje cells (Sakamoto et al., 2003). In addition to promoting dendritic growth, in vitro treatment of cerebellar slice cultures from newborn rats with E2 also increased the density of dendritic spines on Purkinje cells.

Role of ERb in dorsal horn morphogenesis of spinal cord and pain modulation ERb expression in the spinal cord is related to dorsal horn morphogenesis The dorsal spinal cord plays a critical role in organizing responses to sensory input, as it contains the neurons that integrate and relay

somatosensory information entering the spinal cord from sensory neurons in the periphery to central targets. The different types of dorsal spinal cord developed from the embryonic neural tube are essential for perception relay in the spinal cord. It has been proposed that the lamina II interneurons in the spinal cord that express calretinin (CR) and calbindin (CB) may relate to sensory pathways. In the dorsal horn, dorsal root ganglion (DRG) afferents innervate not only the secondary sensory neurons but also the lamina II interneurons, which can have either excitatory or inhibitory effects on the secondary sensory neurons such that pain signals can be modulated. Nociceptive neurons are specified early during development and precede the formation of synaptic contacts with their future peripheral or central targets. The establishment of lamina I and II is essential in the development and functional maturation of nociceptive circuits and subsequent processing of noxious and thermal sensitivity in mammals. Therefore, CRand CB-positive interneurons may contribute to establishing proper connections with the corresponding primary afferents. We have demonstrated that ERb is the predominant ER involved in the development of the dorsal horn (Fan et al., 2007). During embryogenesis, ERb is expressed in the spinal cord as early as E13.5; at middle and later embryonic ages, ERb was strongly expressed in the dorsal horn, mainly in the lamina I and II. In contrast, ERa-positive neurons were not detected before E15.5, and the level of expression of ERa was lower than that of ERb. In the neonatal spinal cord, both ERb and ERa were mainly localized in the superficial layers of the dorsal horn. ERb-positive neurons were less numerous than ERa-containing neurons in the outer layer but were more abundant in the deeper layers of the spinal cord (Fig. 2). In ERbKO mice, there is retarded neuronal development in the dorsal horns and loss of CRpositive neurons in the superficial layers. Double staining for ERb and CR showed that, in the dorsal horn of WT neonates (P0), most of the CR-positive neurons in the superficial dorsal horn also expressed ERb. In ERbKO mice at P0, there were few CR-positive cells, indicating that the developmental neuronal deficit remained. Furthermore, ERb is

243

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)

(l)

Fig. 2. ERb and ERa expression in the spinal cord during embryogenesis. (a) At E13.5, ERb is mainly localized in the superficial layers of the dorsal horn in the lumbar region of the spinal cord. (b and c) In E14.5 lumbar (b) and thoracic (c) spinal cords, ERb is strongly expressed in the laminae I–II. There is also distinct nuclear staining in the lateral region of lamina V and near the central canal. (d and e) In E15.5 thoracic (d) and cervical (e) spinal cords, ERb is found in laminae I–II; some ERb-positive cells are also localized in laminae III–V. (f) In the sagittal section of lumbo–sacral spinal cords at E15.5, dense and deep ERb staining is seen in the superficial layers. (g and h) In sagittal sections of thoracic spinal cords at E15.5, there are some ERa-positive cells in the dorsal horn (h), and the number of ERb-positive cells far exceeds that of ERa-positive cells (g). (i) At E17.5 in the lumbar spinal cord, more ERb-labelled cells appear in lamina II. (j–l) At E16.5 in the dorsal horn of the lumbar spinal cord, most of the ERa-positive cells also express ERb. CC, central canal. (Scale bar: 20 mm.) (From Fan et al. 2007). Copyright PNAS)

essential for interneuron survival throughout life because, in ERbKO mice, there were abnormalities in distribution and number of interneurons in the adult spinal cord. The number of CR-labelled cells in lamina II in 3- and 18-month-old mice was much lower in ERbKO than in WT mice. There was also a decrease in CB-positive cells in the outer layer II (IIo) in ERbKO mice at three months of age. The worsening of the neuronal losses with age suggests that ERb is essential throughout life for maintenance of the integrity of sensory pathways in the spinal cord. All of these data suggest that ERb can affect dorsal horn morphogenesis through modulating interneuron development of the superficial lamina of the dorsal horn.

Role of ERb in pain modulation Clinical data have indicated that females appear more sensitive to pain than males and experience

a greater prevalence of pain disorders. In various nociceptive assays of inflammation, female rats show higher sensitivity to pain than do males. This sexual dimorphism in pain perception and painful disorders indicates that gonadal hormones are able to modulate noxious signal processing (Amandusson et al., 1999; Dao and LeREsche, 2000; Liu and Gintzler, 2000). Using the oro-facial formalin model in ArKO mice and focusing on behavior and neurotransmitter changes in trigeminal nucleus caudalis (TNC) during the interphase of pain inhibition, Multon et al. (2005) suggested that permanent and total lack of estrogens attenuates anti-nociceptive and enhances pro-nociceptive CNS mechanisms. The relation between estrogens and pain seems complex. Experimental studies on the role of estrogens in pain processing indicate pro- as well as anti-nociceptive effects (Craft, 2007; KalbasiAnaraki et al., 2008; Sanoja and Cervero, 2008). These contradictions mainly seem to be due to

244

differences in experimental models used and the levels studied along the pain processing pathways. Both ERa and ERb appear to play key roles in pain modulation. Neuroanatomical studies in male and female mice reveal that both types of ERs are widely expressed in brain regions involved in pain perception (hypothalamus, limbic system and midbrain) and pain modulation (periaqueductal grey and locus ceruleus) (Vanderhorst et al., 2005). ERa and ERb may have distinct functional roles consistent with their non-overlapping expression patterns (Papka et al., 2001). It has been reported that ERa and ERb are expressed in rat DRG neurons, and that estrogen may effectively protect DRG neurons during early post-natal development (Patrone et al., 1999). Moreover, in the rat and mouse, both ERa and ERb have been shown to be expressed in the dorsal horn of adult spinal cords, in laminae I and II, an area involved in receiving and processing nociceptive information (Burke et al., 2000). In an animal model of inflammatory pain, E2induced analgesia can be reversed by tamoxifen. In another study, estradiol replacement in OVX rats attenuated the chronic phase of the formalin response at high formalin concentrations (Mannino et al., 2007). In contrast, 17a-estradiol as an inactive isomer of E2 failed to give the same attenuation, suggesting that, in this experimental system, E2 actions are mediated through genomic ER-mediated mechanisms (Teoh et al., 2000). We investigated the distribution of the primary afferents that target dorsal horn neurons. In three-month-old WT mice, there were more CGRP- and SP-positive fibres in the dorsal horn of ERbKO than in that of WT mice. In contrast, there was no difference in IB4-positive fibres between ERbKO and WT mice. Peptidergic fibre terminals expressing SP and/or CGRP have been observed in lamina I–II at E18–19, whereas the IB4þ subset of C fibre synaptic terminals appeared at P5 (Fitzgerald and Swett, 1983; Marti et al., 1987). Loss of ERb had little effect on CGRP or SP in DRG neurons, but induced higher IB4 expression. This suggests that peptidergic fibres do carry peptides from the DRG to the dorsal horn but that, in ERbKO mice, there are fewer interneurons in

the dorsal horn to interact with the peptides. This leads to accumulation of SP and CGRP in the afferent fibres. A recent study has reported that ERb 041, a selective ERb agonist, is antihyperalgesic in pre-clinical models of chemicalinduced and acute inflammatory pain (Leventhal et al., 2006). Combining this information with our results, we can infer that CR and CB neurons in the spinal cord may be the activation site through which ERb can modulate the pain sensitivity. As mentioned above, genistein binds to both ERs with a higher affinity for ERb than ERa. In a neuropathic mouse model induced by means of chronic sciatic nerve constriction, genistein reversed pain hypersensitivity in a timeand dose-dependent manner, suggesting involvement of ERb in painful neuropathy amelioration (Valsecchi et al., 2008). Furthermore, ERb-131, a potent and selective agonist for ERb, alleviated the nociception in the chronic complete Freund’s adjuvant model, but was without effect on nociception induced by either carrageen or formalin. In another study, ERb-131 was observed to alleviate tactile hyperalgesia induced by capsaicin, and reversed tactile allodynia caused by spinal nerve ligation and various chemical insults (Piu et al., 2007). Moreover, ERb131 did not influence the pain threshold of normal healthy animals. Thus, ERb agonist is a critical effector in attenuating a broad range of anti-nociceptive states. ERa and ERb have different effects on pain modulation. When formalin was injected into the lower lip of female rats, the nociceptive-responsive neurons in the medullar dorsal horn in which c-fos was increased were ERa-positive, thus providing a possible morphological basis for E2–ERa action to directly regulate pain transmission in the spinal cord (Amandusson and Blomqvist, 2010). Using DRG neurons from WT, ERaKO and ERbKO mice in vitro, ATP, an analgesic agent, induced [Ca2þ]i transients in DRG neurons. This increase in [Ca2þ]i transients could be inhibited by E2 in DRG neurons from ERbKO mice and WT mice, but not in DRG neurons from ERaKO mice. These results show that mouse DRG neurons express ERs and that the rapid attenuation of ATP-induced [Ca2þ]i signalling is mediated by

245

membrane-associated ERa (Cornil et al., 2006). ERa and ERb seem to mediate pain modulation through different mechanisms; more work is needed to better define these mechanisms.

Conclusions In summary, a variety of studies discussed in this review have described ERb expression and activity in patterning the developing CNS and have furthered our understanding of the roles of ERbmediated estrogen action in CNS development and function. ERb-mediated estrogen actions are important in CNS structural morphogenesis and higher brain functions such as cognition and mood, and may be implicated in mental disorders, depression, Alzheimer’s disease, motor dysfunction and pain. Future studies should include validation of ERb as a target for diseases affecting the CNS.

Acknowledgements This work was supported by a grant from the Swedish Cancer Fund. References Al-Bader, M. D., El-Abdallah, A. A., & Redzic, Z. B. (2008). Ontogenic profile of estrogen receptor alpha and beta mRNA and protein expression in fetal rat brain. Neuroscience Letters, 440(3), 222–226. Amandusson, S. & Blomqvist, A. (2010). Estrogen receptor-a expression in nociceptive-responsive neurons in the medullary dorsal horn of the female rat. European Journal of Pain, 14(3), 245–248. Amandusson, A., Hallbeck, M., Hallbeck, A. L., Hermanson, O., & Blomqvist, A. (1999). Estrogen-induced alterations of spinal cord enkephalin gene expression. Pain, 83(2), 243– 248. Amin, Z., Canli, T., & Epperson, C. N. (2005). Effect of estrogen-serotonin interactions on mood and cognition. Behavioral and Cognitive Neuroscience Reviews, 4(1), 43–58. Angevine Jun, J. B. & Sidman, R. L. (1961). Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse. Nature, 192, 766–768. Barkhem, T., Carlsson, B., Nilsson, Y., Enmark, E., Gustafsson, J.-A., & Nilsson, S. (1998). Differential response of estrogen receptor alpha and estrogen receptor beta to partial

estrogen agonists/antagonists. Molecular Pharmacology, 54 (1), 105–112. Bodo, C. & Rissman, E. F. (2006). New roles for estrogen receptor beta in behavior and neuroendocrinology. Frontiers in Neuroendocrinology, 27(2), 217–232. Bolger, R., Wiese, T. E., Ervin, K., Nestich, S., & Checovich, W. (1998). Rapid screening of environmental chemicals for estrogen receptor binding capacity. Environmental Health Perspectives, 106(9), 551–557. Burke, K. A., Schroeder, D. M., Abel, R. A., Richardson, S. C., Bigsby, R. M., & Nephew, K. P. (2000). Immunohistochemical detection of estrogen receptor alpha in male rat spinal cord during development. Journal of Neuroscience Research, 61(3), 329–337. Caric, D., Raphael, H., Viti, J., Feathers, A., Wancio, D., & Lillien, L. (2001). EGFRs mediate chemotactic migration in the developing telencephalon. Development, 128(21), 4203– 4216. Carswell, H. V., Macrae, I. M., Gallagher, L., Harrop, E., & Horsburgh, K. J. (2004). Neuroprotection by a selective estrogen receptor beta agonist in a mouse model of global ischemia. American Journal of Physiology. Heart and Circulatory Physiology, 287(4), 1501–1504. Chadwick, C. C., Chippari, S., Matelan, E., Borges-Marcucci, L., Eckert, A. M., Keith, J. C., Jr, et al. (2005). Identification of pathway-selective estrogen receptor ligands that inhibit NF-kappaB transcriptional activity. Proceedings of the National Academy of Sciences of the United States of America, 102(7), 2543–2548. Cheryl, A. F., Caryn, K. D., & Alicia, A. W. (2007). Estrogens and progestins enhance spatial learning of intact and ovariectomized rats in the object placement task. Neurobiology of Learning and Memory, 88(2), 208–216. Choleris, E., Clipperton, A. E., Phan, A., & Kavaliers, M. (2008). Estrogen receptor beta agonists in neurobehavioral investigations. Current Opinion in Investigational Drugs, 9 (7), 760–773. Cimarosti, H., O’Shea, R. D., Jones, N. M., Horn, A. P., Simão, F., Zamin, L. L., et al. (2006). The effects of estradiol on estrogen receptor and glutamate transporter expression in organotypic hippocampal cultures exposed to oxygen–glucose deprivation. Neurochemical Research, 31(4), 483–490. Colciago, A., Celotti, F., Pravettoni, A., Mornati, O., Martini, L., & Negri-Cesi, P. (2005). Dimorphic expression of testosterone metabolizing enzymes in the hypothalamic area of developing rats. Brain Research. Developmental Brain Research, 155(2), 107–116. Cornil, C. A., Ball, G. F., & Balthazart, J. (2006). Functional significance of the rapid regulation of brain estrogen action: where do the estrogens come from? Brain Research, 1126(1), 2–26. Cowley, S. M., Hoare, S., Mosselman, S., & Parker, M. G. (1997). Estrogen receptors alpha and beta form heterodimers on DNA. Journal of Biological Chemistry, 272(32), 19858–19862. Craft, R. M. (2007). Modulation of pain by estrogens. Pain, 132 (Suppl. 1), s3–s12.

246 Dalla, C., Antoniou, K., Papadopoulou-Daifoti, Z., Balthazart, J., & Bakker, J. (2004). Oestrogen -deficient female aromatase knockout (ArKO) mice exhibit depressive-like symptomatology. European Journal of Neuroscience, 20(1), 217–228. Dalla, C., Antoniou, K., Papadopoulou-Daifoti, Z., Balthazart, J., & Bakker, J. (2005). Male aromatase-knockout mice exhibit normal levels of activity, anxiety and “depressivelike” symptomatology. Behavioural Brain Research, 163(2), 186–193. Dao, T. T. & LeREsche, L. (2000). Gender differences in pain. Journal of Orofacial Pain, 14(3), 169–184. Donner, N. & Handa, R. J. (2009). Estrogen receptor beta regulates the expression of tryptophan- hydroxylase 2 mRNA within serotonergic neurons of the rat dorsal raphe nuclei. Neuroscience, 163(2), 705–718. Ehrlich, I., Klein, M., Rumpel, S., & Malinow, R. (2007). PSD95 is required for activity-driven synapse stabilization. Proceedings of the National Academy of Sciences United States of America, 104(10), 4176–4181. El-Bakri, N. K., Islam, A., Zhu, S., Elhassan, A., Mohammed, A., Winblad, B., et al. (2004). Effects of estrogen and progesterone treatment on rat hippocampal NMDA receptors: Relationship to Morris water maze performance. Journal of Cellular and Molecular Medicine, 8(4), 537–544. Enmark, E., Pelto-Huikko, M., Grandien, K., Lagercrantz, S., Lagercrantz, J., Fried, G., et al. (1997). Human estrogen receptor beta-gene structure, chromosomal localization, and expression pattern. Journal of Clinical Endocrinology and Metabolism, 82(12), 4258–4265. Eriksson, P. S., Perfilieva, E., Bjork-Eriksson, T., Alborn, A. M., Nordborg, C., Peterson, D. A., et al. (1998). Neurogenesis in the adult human hippocampus. Nature Medicine, 4 (11), 1313–1317. Fan, X., Kim, H. J., Warner, M., & Gustafsson, J.-A. (2007). Estrogen receptor beta is essential for sprouting of nociceptive primary afferents and for morphogenesis and maintenance of the dorsal horn interneurons. Proceedings of the National Academy of Sciences of the United States of America, 104(34), 13696–13701. Fan, X., Warner, M., & Gustafsson, J.-A. (2006). Estrogen receptor beta expression in the embryonic brain regulates development of calretinin-immunoreactive GABAergic interneurons. Proceedings of the National Academy of Sciences United States of America, 103(51), 19338–19343. Fitzgerald, M. & Swett, J. (1983). The termination pattern of sciatic nerve afferents in the substantia gelatinosa of neonatal rats. Neuroscience Letters, 43(2–3), 149–154. Foy, M. R., Baudry, M., Diaz Brinton, R., & Thompson, R. F. (2008). Estrogen and hippocampal plasticity in rodent models. Journal of Alzheimer’s Disease, 15(4), 589–603. Fried, G., Andersson, E., Csoregh, L., Enmark, E., Gustafsson, J.-A., Aanesen, A., et al. (2004). Estrogen receptor beta is expressed in human embryonic brain cells and is regulated by 17beta-estradiol. European Journal of Neuroscience, 20 (9), 2345–2354. Gazzaley, A., Kay, S., & Benson, D. L. (2002). Dendritic spine plasticity in hippocampus. Neuroscience, 111(4), 853–862.

Gleeson, J. G. & Walsh, C. A. (2000). Neuronal migration disorders: From genetic diseases to developmentalmechanisms. Trends in Neurosciences, 23(8), 352–359. Gofflot, F., Chartoire, N., Vasseur, L., Heikkinen, S., Dembele, D., Le Merrer, J., et al. (2007). Systematic gene expression mapping clusters nuclear receptors according to their function in the brain. Cell, 131(2), 405–418. González, M., Cabrera-Socorro, A., Pérez-Garc´ıa, C. G., Fraser, J. D., López, F. J., Alonso, R., et al. (2007). Distribution patterns of estrogen receptor alpha and beta in the human cortex and hippocampus during development and adulthood. The Journal of Comparative Neurology, 503(6), 790–802. Green, S., Walter, P., Kumar, V., Krust, A., Bornert, J. M., Argos, P., et al. (1986). Human oestrogen receptor cDNA: Sequence, expression and homology to v-erb-A. Nature, 320 (6058), 134–139. Greene, G. L., Gilna, P., Waterfield, M., Baker, A., Hort, Y., & Shine, J. (1986). Sequence and expression of human estrogen receptor complementary DNA. Science, 231(4742), 1150– 1154. Guo, X. Z., Su, J. D., Sun, Q. W., & Jun, D. S. (2001). Expression of estrogen receptor (ER)-alpha and -beta transcripts in the neonatal and adult rat cerebral cortex, cerebellum, and olfactory bulb. Cell Research, 11(4), 321–324. Gupta, A., Tsai, L. H., & Wynshaw-Boris, A. (2002). Life is a journey: A genetic look at neocortical development. Nature Reviews Genetics, 3(5), 342–355. Harrington, W. R., Sheng, S., Barnett, D. H., Petz, L. N., Katzenellenbogen, J. A., & Katzenellenbogen, B. S. (2003). Activities of estrogen receptor alpha- and beta-selective ligands at diverse estrogen responsive gene sites mediating transactivation or transrepression. Molecular and Cellular Endocrinology, 206(1–2), 13–22. Hughes, Z. A., Liu, F., Platt, B. J., Dwyer, J. M., Pulicicchio, C. M., Zhang, G., et al. (2008). WAY-200070, a selective agonist of estrogen receptor beta as a potential novel anxiolytic/antidepressant agent. Neuropharmacology, 54(7), 1136–1142. Ikeda, Y. & Nagai, A. (2006). Differential expression of the estrogen receptors alpha and beta during postnatal development of the rat cerebellum. Brain Research, 1083 (1), 39–49. Isgor, C. & Watson, S. J. (2005). Estrogen receptor alpha and beta mRNA expressions by proliferating and differentiating cells in the adult rat dentate gyrus and subventricular zone. Neuroscience, 134(3), 847–856. Jakab, R. L., Wong, J. K., & Belcher, S. M. (2001). Estrogen receptor beta immunoreactivity in differentiating cells of the developing rat cerebellum. The Journal of Comparative Neurology, 430(3), 396–409. Jelks, K. B., Wylie, R., Floyd, C. L., Johnson, M. L., Lai, D., Hui, S. L., et al. (2007). Estradiol targets synaptic proteins to induce glutamatergic synapse formation in cultured hippocampal neurons: Critical role of estrogen receptor-alpha. Journal of Neuroscience, 27(26), 6903–6913. Jordan, V. C., Mittal, S., Gosden, B., Koch, R., & Lieberman, M. E. (1985). Structure-activity relationships of estrogens. Environmental Health Perspectives, 61, 97–110.

247 Kalbasi-Anaraki, D., Sianati, S., Sadeghi, M., Ghasemi, M., Paydar, M. J., Ejtemaei Mehr, S., et al. (2008). Modulation by female sex hormones of the cannabinoid-induced catalepsy and analgesia in ovariectomized mice. European Journal of Pharmacology, 586(1–3), 189–196. Kempermann, G., Kuhn, H. G., & Gage, F. H. (1997). More hippocampal neurons in adult mice living in an enriched environment. Nature, 386(6624), 493–495. Kritzer, M. F. (2006). Regional, laminar and cellular distribution of immunoreactivity for ERb in the cerebral cortex of hormonally intact, postnatally developing male and female rats. Cerebral Cortex, 16(8), 1181–1192. Kruijver, F. P., Balesar, R., Espila, A. M., Unmehopa, U. A., & Swaab, D. F. (2002). Estrogen receptor-alpha distribution in the human hypothalamus in relation to sex and endocrine status. The Journal of Comparative Neurology, 454(2), 115– 139. Kuiper, G. G., Carlsson, B., Grandien, K., Enmark, E., Häggblad, J., Nilsson, S., et al. (1997). Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology, 138(3), 863–870. Kuiper, G. C., Enmark, E., Pelto-Huicco, M., Nilsson, S., & Gustafsson, J.-A. (1996). Cloning of a novel receptor expressed in rat prostate and ovary. Proceedings of the National Academy of Sciences United States of America, 93 (12), 5925–5930. Lacreuse, A., Wilson, M. E., & Herndon, J. G. (2009). No effect of different estrogen receptor ligands on cognition in adult female monkeys. Physiology & Behavior, 96(3), 448– 456. Laflamme, N., Nappi, R. E., Drolet, G., Labrie, C., & Rivest, S. (1998). Expression and neuropeptidergic characterization of estrogen receptors (ERalpha and ERbeta) throughout the rat brain: Anatomical evidence of distinct roles of each subtype. Journal of Neurobiology, 36(3), 357–378. Leventhal, L., Brandt, M. R., Cummons, T. A., Piesla, M. J., Rogers, K. E., & Harris, H. A. (2006). An estrogen receptorbeta agonist is active in models of inflammatory and chemical-induced pain. European Journal of Pharmacology, 553 (1–3), 146–148. Levin, E. R. (2001). Cell localization, physiology, and nongenomic actions of estrogen receptors. Journal of Applied Physiology, 91(4), 1860–1867. Li, X., Schwartz, P. E., & Rissman, E. F. (1997). Distribution of estrogen receptor-b-like immunoreactivity in rat forebrain. Neuroendocrinology, 66(2), 63–67. Lipkind, D., Nottebohm, F., Rado, R., & Barnea, A. (2002). Social change affects the survival of new neurons in the forebrain of adult songbirds. Behavioural Brain Research, 133(1), 31–43. Liu, F., Day, M., Muñiz, L. C., Bitran, D., Arias, R., RevillaSanchez, R., et al. (2008). Activation of estrogen receptorbeta regulates hippocampal synaptic plasticity and improves memory. Nature Neuroscience, 11(3), 334–343. Liu, N. J. & Gintzler, A. R. (2000). Prolonged ovarian sex steroid treatment of male rats produces antinociception:

Identification of sex-based divergent analgesic mechanisms. Pain, 85(1–2), 273–281. Lopez-Bendito, G., Cautinat, A., Sanchez, J. A., Bielle, F., Flames, N., Garratt, A. N., et al. (2006). Tangential neuronal migration controls axon guidance: A role for neuregulin-1 in thalamocortical axon navigation. Cell, 125(1), 127–142. Lund, T. D., Rovis, T., Chung, W. C., & Handa, R. J. (2005). Novel actions of estrogen receptor-beta on anxiety-related behaviors. Endocrinology, 146(2), 797–807. Madeline, E. R. & Cheryl, A. F. (2006). ERb-selective SERMs produce mnemonic-enhancing effects in the inhibitory avoidance and water maze tasks. Neurobiology of Learning and Memory, 85(2), 183–191. Malinow, R. & Malenka, R. C. (2002). AMPA receptor trafficking and synaptic plasticity. Annual Review of Neuroscience, 25, 103–126. Man, H. Y., Sekine-Aizawa, Y., & Huganir, R. L. (2007). Regulation of a-amino-3-hydroxy- 5-methyl- 4-isoxazolepropionic acid receptor trafficking through PKA phosphorylation of the Glu receptor 1 subunit. Proceedings of the National Academy of Sciences United States of America, 104 (9), 3579–3584. Mannino, C. A., South, S. M., Quinones-Jenab, V., & Inturrisi, C. E. (2007). Estradiol replacement in ovariectomized rats is antihyperalgesic in the formalin test. Journal of Pain, 8(4), 334–342. Marti, E., Gibson, S. J., Polak, J. M., Facer, P., Springall, D. R., Van Aswegen, G., et al. (1987). Ontogeny of peptide- and amine-containing neurones in motor, sensory, and autonomic regions of rat and human spinal cord, dorsal root ganglia, and rat skin. The Journal of Comparative Neurology, 266(3), 332–359. Matthews, J. & Gustafsson, J.-A. (2003). Estrogen Signaling: A Subtle Balance Between ERa and ERß. Molecular Interventions, 3(5), 281–292. Mazzucco, C. A., Lieblich, S. E., Bingham, B. I., Williamson, M. A., Viau, V., & Galea, L. A. (2006). Both estrogen receptor alpha and estrogen receptor beta agonists enhance cell proliferation in the dentate gyrus of adult female rats. Neuroscience, 141(4), 1793–1800. McCullough, L. D., Blizzard, K., Simpson, E. R., Oz, O. K., & Hurn, P. D. (2003). Aromatase cytochrome P450 and extragonadal estrogen play a role in ischemic neuroprotection. Journal of Neuroscience, 23(25), 8701–8705. McManus, M. F. & Golden, J. A. (2005). Neuronal migration in developmental disorders. Journal of Child Neurology, 20(4), 280–286. Merchenthaler, I., Dellovade, T. L., & Shughrue, P. J. (2003). Neuroprotection by estrogen in animal models of global and focal ischemia. Annals of the New York Academy of Sciences, 1007, 89–100. Miller, N. R., Jover, T., & Cohen, H. W. (2005). Estrogen can act via estrogen receptor a and b to protect hippocampal neurons against global ischemia-induced cell death. Endocrinology, 146(7), 3070–3079. Milner, T. A., Ayoola, K., Drake, C. T., Herrick, S. P., Tabori, N. E., McEwen, B. S., et al. (2005). Ultrastructural

248 localization of estrogen receptor beta immunoreactivity in the rat hippocampal formation. The Journal of Comparative Neurology, 491(2), 81–95. Miranda, R. C. & Toran-Allerand, C. D. (1992). Developmental expression of estrogen receptor mRNA in the rat cerebral cortex: A nonisotopic in situ hybridization histochemistry study. Cerebral Cortex, 2(1), 1–15. Mitra, S. W., Hoskin, E., Yudkovitz, J., Pear, L., Wilkinson, H. A., Hayashi, S., et al. (2003). Immunolocalization of estrogen receptor beta in the mouse brain: Comparison with estrogen receptor alpha. Endocrinology, 144(5), 2055– 2067. Multon, S., Pardutz, A., Mosen, J., Hua, M. T., Defays, C., Honda, S., et al. (2005). Lack of estrogen increases pain in the trigeminal formalin model: A behavioural and immunocytochemical study of transgenic ArKO mice. Pain, 114(1–2), 257–265. Murphy, D. D. & Segal, M. (1996). Regulation of dendritic spine density in cultured rat hippocampal neurons by steroid hormones. Journal of Neuroscience, 16(13), 4059–4068. Nadarajah, B. (2003). Radial glia and somaltranslocation of neurons in the developing cerebral cortex. Glia, 43(1), 33–36. Neubauer, B. L., McNulty, A. M., Chedid, M., Chen, K., Goode, R. L., Johnson, M. A., et al. (2003). The selective estrogen receptor modulator trioxifene (LY133314) inhibits metastasis and extends survival in the PAIII rat prostatic carcinoma model. Cancer Research, 63(18), 6056–6062. Oostenbrink, B. C., Pitera, J. W., van Lipzig, M. M., Meerman, J. H., & van Gunsteren, W. F. (2000). Simulations of the estrogen receptor ligand-binding domain: Affinity of natural ligands and xenoestrogens. Journal of Medicinal Chemistry, 43(24), 4594–4605. Osterlund, M. K., Gustafsson, J.-A., Keller, E., & Hurd, Y. L. (2000). Estrogen receptor beta (ERbeta) messenger ribonucleic acid (mRNA) expression within the human forebrain: Distinct distribution pattern to ERalpha mRNA. Journal of Clinical Endocrinology and Metabolism, 85(10), 3840–3846. Osterlund, M. K. & Hurd, Y. L. (2001). Estrogen receptors in the human forebrain and the relation to neuropsychiatric disorders. Progress in Neurobiology, 64(3), 251–267. Papka, R. E., Storey-Workley, M., Shughrue, P. J., Merchenthaler, I., Collins, J. J., Usip, S., et al. (2001). Estrogen receptor-alpha and beta-immunoreactivity and mRNA in neurons of sensory and autonomic ganglia and spinal cord. Cell and Tissue Research, 304(2), 193–214. Parducz, A., Hajszan, T., Maclusky, N. J., Hoyk, Z., Csakvari, E., Kurunczi, A., et al. (2006). Synaptic remodeling induced by gonadal hormones: Neuronal plasticity as a mediator of neuroendocrine and behavioral responses to steroids. Neuroscience, 138(3), 977–985. Patrone, C., Andersson, S., Korhonen, L., & Lindholm, D. (1999). Estrogen receptor-dependent regulation of sensory neuron survival in developing dorsal root ganglion. Proceedings of the National Academy of Sciences United States of America, 96(10), 10905–10910.

Pérez, S. E., Chen, E. Y., & Mufson, E. J. (2003). Distribution of estrogen receptor alpha and beta immunoreactive profiles in the postnatal rat brain. Brain Research. Developmental Brain Research, 145(1), 117–139. Pettersson, K., Grandien, K., Kuiper, G. G., & Gustafsson, J.-A. (1997). Mouse estrogen receptor beta forms estrogen response element-binding heterodimers with estrogen receptor alpha. Molecular Endocrinology, 11(10), 1486–1496. Piu, F., Hyldtoft, L., Gardell, L., Cheevers, C., DelTredici, A., Andersen, C., et al. (2007). A selective estrogen receptor beta agonist modulates neuropathic and inflammatory pain states. European Journal of Pain, 11S1, 174. Pozzo-Miller, L. D., Inoue, T., & Murphy, D. D. (1999). Estradiol increases spine density and NMDA dependent Ca2þ transients in spines of CA1 pyramidal neurons from hippocampal slices. Journal of Neurophysiology, 81(3), 1404–1411. Prange-Kiel, J. & Rune, G. M. (2006). Direct and indirect effects of estrogen on rat hippocampus. Neuroscience, 138(3), 765–772. Prewitt, A. K. & Wilson, M. E. (2007). Changes in estrogen receptor-alpha mRNA in the mouse cortex during development. Brain research, 1134(1), 62–69. Qin, J., Suh, J. M., Kim, B. J., Yu, C. T., Tanaka, T., Kodama, T., et al. (2007). The expression pattern of nuclear receptors during cerebellar development. Developmental Dynamics, 236(3), 810–820. Raval, A. P., Bramlett, H., & Perez-Pinzon, M. A. (2006). Estrogen preconditioning protects the hippocampal CA1 against ischemia. Neuroscience, 141(4), 1721–1730. Rissman, E. F., Heck, A. L., Leonard, J. E., Shupnik, M. A., & Gustafsson, J.-A. (2002). Disruption of estrogen receptor beta gene impairs spatial learning in female mice. Proceedings of the National Academy of Sciences of the United States of America, 99(6), 3996–4001. Sakamoto, H., Mezaki, Y., & Shikimi, H. (2003). Dendritic growth and spine formation in response to estrogen in the developing Purkinje cell. Endocrinology, 144(10), 4466–4477. Sanoja, R. & Cervero, F. (2008). Estrogen modulation of ovariectomy-induced hyperalgesia in adult mice. European Journal of Pain, 12(5), 573–581. Schaub, C. E., Gersting, J. A., Keller-Wood, M., & Wood, C. E. (2008). Development of ER-a and ER-b expression in the developing ovine brain and pituitary. Gene Expression Patterns, 8, 457–463. Shehu, A., Mao, J., Gibori, G. B., Halperin, J., Le, J., Devi, Y. S., et al. (2008). Prolactin receptor-associated protein/ 17beta-hydroxysteroid dehydrogenase type 7 gene (Hsd17b7) plays a crucial role in embryonic development and fetal survival. Molecular Endocrinology, 22(10), 2268– 2277. Shors, T. J., Townsend, D. A., Zhao, M., Kozorovitskiy, Y., Gould, E. (2002). Neurogenesis may relate to some but not all types of hippocampal dependent learning. Hippocampus, 12, 578–584. Smith, C. C., Veddler, L. C., & McMahon, L. L. (2009). Estradiol and the relationship between dendritic spines, NR2B containing NMDA receptors, and the magnitude of

249 long-term potentiation at hippocampal CA3-CA1 synapses. Psychoneuroendocrinology, Suppl. 1, S130–S142. Sobeih, M. & Corfas, G. (2002). Extracellular factors that regulate neuronal migration in the central nervous system. International Journal of Developmental Neuroscience, 20(3– 5), 349–357. Stovall, D. W. & Pinkerton, J. V. (2009). MF-101, an estrogen receptor beta agonist for the treatment of vasomotor symptoms in peri- and postmenopausal women. Current Opinion in Investigational Drugs, 10(4), 365–371. Sugiyama, N., Andersson, S., Lathe, R., Fan, X., AlonsoMagdalena, P., Schwend, T., et al. (2009). Spatiotemporal dynamics of the expression of estrogen receptors in the postnatal mouse brain. Molecular Psychiatry, 14(2), 223–232. Szymczak, S., Kalita, K., Jaworski, J., Mioduszewska, B., Savonenko, A., Markowska, A., et al. (2006). Increased estrogen receptor beta expression correlates with decreased spine formation in the rat hippocampus. Hippocampus, 16(5), 453–463. Takahashi, N., Tonchev, A. B., Koike, K., Murakami, K., Yamada, K., Yamashima, T., et al. (2004). Expression of estrogen receptor-beta in the postischemic monkey hippocampus. Neuroscience Letters, 369(1), 9–13. Takeyama, J., Suzuki, T., Inoue, S., Kaneko, C., Nagura, H., Harada, N., et al. (2001). Expression and cellular localization of estrogen receptors alpha and beta in the human fetus. Journal of Clinical Endocrinology and Metabolism, 86(5), 2258–2262. Taylor, A. H. & Al-Azzawi, F. (2000). Immunolocalisation of oestrogen receptor beta in human tissues. Journal of Molecular Endocrinology, 24(1), 145–155. Teoh, H., Quan, A., Leung, S. W., & Man, R. Y. (2000). Differential effects of 17b-estradiol and testosterone on the contractile responses of porcine coronary arteries. British Journal of Pharmacology, 129(7), 1301–1308. Thorp, A. A., Sinn, N., Buckley, J. D., Coates, A. M., & Howe, P. R. (2009). Soya isoflavone supplementation enhances spatial working memory in men. British Journal of Nutrition, 102 (9), 1348–1354. Tsutsui, K. (2006). Biosynthesis and organizing action of neurosteroids in the developing Purkinje cell. The Cerebellum, 5 (2), 89–96. Tsutsui, K., Sakamoto, H., Shikimi, H., & Ukena, K. (2004). Organizing actions of neurosteroids in the Purkinje neuron. Neuroscience Research, 49(3), 273–279. Valsecchi, A. E., Franchi, S., Panerai, A. E., Sacerdote, P., Trovato, A. E., & Colleoni, M. (2008). Genistein, a natural phytoestrogen from soy, relieves neuropathic pain following chronic constriction sciatic nerve injury in mice: Anti-inflammatory and antioxidant activity. Journal of Neurochemistry, 107(1), 230–240. Vanderhorst, V. G., Gustafsson, J.-A., & Ulfhake, B. (2005). Estrogen receptor-alpha and -beta immunoreactive neurons in the brainstem and spinal cord of male and female mice: Relationships to monoaminergic, cholinergic, and spinal projection systems. The Journal of Comparative Neurology, 488 (2), 152–179.

Walf, A. A., Ciriza, I., Garcia-Segura, L. M., & Frye, C. A. (2008a). Antisense oligodeoxynucleotides for estrogen receptor-beta and alpha attenuate estradiol’s modulation of affective and sexual behavior, respectively. Neuropsychopharmacology, 33(2), 431–440. Walf, A. A. & Frye, C. A. (2007a). Estradiol decreases anxiety behavior and enhances inhibitory avoidance and gestational stress produces opposite effects. Stress, 10(3), 251–260. Walf, A. A. & Frye, C. A. (2007b). Administration of estrogen receptor beta-specific selective estrogen receptor modulators to the hippocampus decrease anxiety and depressive behavior of ovariectomized rats. Pharmacology Biochemistry and Behavior, 86(2), 407–414. Walf, A. A., Koonce, C. J., & Frye, C. A. (2008b). Estradiol or diarylpropionitrile administration to wild type, but not estrogen receptor beta knockout, mice enhances performance in the object recognition and object placement tasks. Neurobiology of Learning and Memory, 89(4), 513–521. Walf, A. A., Koonce, C. J., & Frye, C. A. (2009a). Adult female wildtype, but not oestrogen receptor beta knockout, mice have decreased depression-like behaviour during pro-oestrus and following administration of oestradiol or diarylpropionitrile. Journal of Psychopharmacology, 23(4), 442–450. Walf, A. A., Koonce, C., Manleye, K., & Frye, C. A. (2009b). Proestrous compared to diestrous wildtype, but not estrogen receptor beta knockout, mice have better performance in the spontaneous alternation and object recognition tasks and reduced anxiety-like behavior in the elevated plus and mirror maze. Behavioural Brain Research, 196(2), 254–260. Walf, A. A., Paris, J. J., & Frye, C. A. (2009c). Chronic estradiol replacement to aged female rats reduces anxiety-like and depression-like behavior and enhances cognitive performance. Psychoneuroendocrinology, 34(6), 909–916. Walf, A. A., Rhodes, M. E., & Frye, C. A. (2006). Ovarian steroids enhance object recognition in naturally cycling and ovariectomized, hormone-primed rats. Neurobiology of Learning and Memory, 86(1), 35–46. Wang, L., Andersson, S., Warner, M., & Gustafsson, J. A. (2001) Morphological abnormalities in the brains of estrogen receptor b knockout mice. Proceedings of the National Academy of Sciences of the United States of America, 98, 2792–2796. Wang, L., Andersson, S., Warner, M., & Gustafsson, J.-A. (2003). Estrogen receptor (ER) beta knockout mice reveal a role for ERbeta in migration of cortical neurons in the developing brain. Proceedings of the National Academy of Sciences United States of America, 100(2), 703–708. Weihua, Z., Lathe, R., Warner, M., & Gustafsson, J.-A. (2002). An endocrine pathway in the prostate, ERbeta, AR, 5alpha-androstane-3beta, 17beta-diol, and CYP7B1, regulates prostate growth. Proceedings of the National Academy of Sciences of the United States of America, 99 (21), 13589–13594. Weissman, T., Noctor, S. C., Clinton, B. K., Honig, L. S., & Kriegstein, A. R. (2003). Neurogenic radial glial cells in

250 reptile, rodent and human: From mitosis to migration. Cerebral Cortex, 13(6), 550–559. Wilson, M. E., Westberry, J. M., & Prewitt, A. K. (2008). Dynamic regulation of estrogen receptor-alpha gene expression in the brain: a role for promoter methylation? Frontiers in Neuroendocrinology, 29(3), 375–385. Woolley, C. S. & McEwen, B. S. (1992). Estradiol mediates fluctuation in hippocampal synapse density during the estrous cycle in the adult rat. Journal of Neuroscience, 12(7), 2549–2554.

Zhao, L., Wu, T., & Brinton, R. D. (2004). Estrogen receptor subtypes alpha and beta contribute to neuroprotection and increased Bcl-2 expression in primary hippocampal neurons. Brain Research, 1010(1–2), 22–34. Zsaenovszky, A. & Belcher, S. M. (2001). Identification of a developmental gradient of estrogen receptor expression and cellular localization in the developing and adult female rat primary somatosensory cortex. Brain Research. Developmental Brain Research, 129(1), 39–46.

L. Martini (Eds.) Progress in Brain Research, Vol. 181 ISSN: 0079-6123 Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 14

Interactions of estradiol and insulin-like growth factor-I signalling in the nervous system: New advances Luis M. Garcia-Segura1,
, Mar´ıa-Angeles Ar evalo1 and Iñigo Azcoitia2 Instituto Cajal, Consejo Superior de Investigaciones Cient´ıficas, Madrid, Spain Departamento de Biolog´ıa Celular, Facultad de Biolog´ıa, Universidad Complutense, Madrid, Spain 1

2

Abstract: Estradiol and insulin-like growth factor-I (IGF-I) interact in the brain to regulate a variety of developmental and neuroplastic events. Some of these interactions are involved in the control of hormonal homeostasis and reproduction. However, the interactions may also potentially impact on affection and cognition by the regulation of adult neurogenesis in the hippocampus and by promoting neuroprotection under neurodegenerative conditions. Recent studies suggest that the interaction of estradiol and IGF-I is also relevant for the control of cholesterol homeostasis in neural cells. The molecular mechanisms involved in the interaction of estradiol and IGF-I include the cross-regulation of the expression of estrogen and IGF-I receptors, the regulation of estrogen receptor-mediated transcription by IGF-I and the regulation of IGF-I receptor signalling by estradiol. Current investigations are evidencing the role exerted by key signalling molecules, such as glycogen synthase kinase 3 and b-catenin, in the cross-talk of estrogen receptors and IGF-I receptors in neural cells. Keywords: Akt; b-catenin; estrogen receptors; glia; glycogen synthase kinase 3; IGF-I receptors; mitogenactivated protein kinase; neuronal development; neuroprotection; phosphatidylinositol 3-kinase; synaptic plasticity; Tau

actions in the brain and influences neuronal development, synaptic plasticity, neuroendocrine regulation, adult neurogenesis and cognition (Åberg et al., 2006; Aleman and Torres-Alemán, 2009; Fernandez et al., 2007; Torres-Aleman, 1999). IGF-I is also a potent neuroprotective molecule (Åberg et al., 2006; Carro et al., 2001, 2003). IGF-I is actively transported across the blood– brain barrier (Carro et al., 2006; Reinhardt and Bondy, 1994). Therefore, brain function is

Introduction Insulin-like growth factor-I (IGF-I) is a hormone of the somatotrophic axis and a local factor produced in many tissues, including the nervous system (LeRoith, 2008). IGF-I has pleiotropic


Corresponding author. Tel.: þ34-915854729; Fax: þ34-915854754; E-mail: [email protected]

DOI: 10.1016/S0079-6123(08)81014-X

251

252

affected by both locally produced and circulating IGF-I. The mechanism of action of IGF-I in the brain involves its interaction with other growth factors, such as brain-derived neurotrophic factor (BDNF) (Carro et al., 2000; Ding et al., 2006) and vascular endothelial growth factor (VEGF) (Lopez-Lopez et al., 2004, 2007). In addition, IGF-I interacts with the ovarian hormone estradiol in the regulation of multiple events in the nervous system. The first evidence of an interaction of estradiol and IGF-I in the nervous system was obtained by Toran-Allerand et al., (1988), showing a synergic effect of estradiol and insulin on neuritic growth in foetal rat and mouse brain explants. Although IGF-I was not directly tested in this study, it was proposed that the effect of insulin could be mediated by the activation of IGF-I receptors. Several years later, Ma et al. (1994) reported that insulin and IGFs promote the elongation of neurites and growth arrest of a neuroblastoma cell line, but only when these cells were transfected with estrogen receptor (ER) a. Since then, numerous studies have examined the functional consequences of the interaction of estradiol and IGF-I in the nervous system and have analyzed the mechanisms involved in such an interaction. Immunohistochemical analyses have revealed that many neurons and glial cells co-express ERs and the IGF-I receptors in the central nervous system (Cardona-Gómez et al., 2000; Garcia-Segura et al., 2000; Quesada et al., 2007). In addition, biochemical studies have detected that, in brain cells, ERa forms part of a macromolecular complex with IGF-I receptor and with several components of IGF-I receptor signalling cascade, such as p85, insulin receptor substrate 1 (IRS1), glycogen synthase kinase 3b (GSK3b) and b-catenin (CardonaGomez et al., 2004; Mendez et al., 2003). Here we will review the functional consequences of these molecular interactions for neuronal development, synaptic plasticity, neuroendocrine regulation, adult neurogenesis, brain cholesterol homeostasis and neuroprotection. Furthermore, we will consider recent advances in the

investigation of the molecular mechanisms involved in the cross-talk of ERs and IGF-I receptors in neural cells.

Functional intreactions of estradiol and IGF-I in the nervous system Development Insulin/IGF-I signalling is essential for neural development in vertebrates (Bateman and McNeill, 2006; Hernandez-Sanchez et al., 2006; Varela-Nieto et al., 2004). IGF-I has many developmental effects in the central nervous system, regulating the proliferation, maturation and differentiation of neural stem cells (Otaegi et al., 2006; Ye and D’Ercole, 2006) and promoting the production of neurons (Camarero et al., 2003; Russo et al., 2005; Vicario-Abejon et al., 2003) and oligodendrocytes (Hsieh et al., 2004; Vicario- Abejon et al., 2003; Zeger et al., 2007). IGF-I also promotes the membrane expansion of nerve growth cones (Laurino et al., 2005) and axon outgrowth (Ozdinler and Macklis, 2006), and regulates neuronal migration (Jiang et al., 1998), synaptogenesis (O’Kusky et al., 2003) and other aspects of the post-natal development of the central nervous system, including the process of myelination by preventing the apoptotic death of oligodendrocytes during post-natal life (Popken et al., 2004). IGF-I interacts with estradiol in the regulation of developmental events in the nervous system. An interaction between these two factors may contribute to the generation of structural sex differences in the brain by the regulation of the survival and differentiation of developing neurons in brain areas involved in the regulation of neuroendocrine events and reproduction (Carrer and Cambiasso, 2002). Several studies have demonstrated an interdependence of ERs and IGF-I receptor in the promotion of the survival and differentiation in primary cultures of developing hypothalamic neurons (Cambiasso et al., 2000; Carrer and Cambiasso, 2002; Dueñas et al., 1996). Both

253

estradiol and IGF-I promote neuronal survival and differentiation in primary neuronal cultures grown in a defined medium deprived of serum and hormones. In these cultures, the induction of neuronal survival and differentiation by IGF-I was prevented by inhibiting the synthesis of ERa, using a specific antisense oligonucleotide (Dueñas et al., 1996). The effect of IGF-I was also prevented by the ER antagonist ICI 182780 (Dueñas et al., 1996). In turn, the promotion of neuronal survival and differentiation by estradiol was prevented by blocking the synthesis of IGF-I in the cultures using a specific IGF-I antisense oligonucleotide (Dueñas et al., 1996), as well as by the pharmacological blockade of the mitogenactivated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K) signalling pathways, both activated by the IGF-I receptor (Garcia-Segura et al., 2000). Therefore, ERa mediates effects of IGF-I and IGF-I receptor mediates effects of estradiol on the survival and differentiation of hypothalamic neurons (Fig. 1). Similar results have been obtained in PC12 cells transfected with ERa. Both estradiol and IGF-I increased neuritic growth and antagonists of ERs and IGF-I receptor blocked the effect of both factors, indicating that ERa mediates effects of IGF-I and IGF-I receptor mediates effects of estradiol on neuritic growth in PC12 cells (Topalli and Etgen, 2004). An interaction of estradiol with IGF-I receptors has also been detected in the development of ventromedial hypothalamic neurons (Cambiasso et al., 2000; Carrer and Cambiasso, 2002). Estradiol induces an increase in axonal growth and in the expression of TrkB and IGFI receptors in ventromedial hypothalamic neurons derived from male rats. However, this effect is detected only when neurons are cultured in the presence of conditioned media from glial cells removed from target regions. This suggests that growth factors released by glia are involved in the neuritogenic effect of estradiol, and that IGF-I receptors are involved in this effect. Therefore, estradiol, IGF-I and its respective receptors may interact via diverse mechanisms in the regulation of neural development.

(a)

E2

ER IGF-IR IGF-I (b)

E2

x ER

IGF-IR IGF-I (c)

E2

ER

x

IGF-IR IGF-I Fig. 1. Estradiol and IGF-I interact in the promotion of survival and differentiation of developing hypothalamic neurons. (a) Estradiol (E2) and IGF-I prevent neuronal death induced by serum deprivation and promote neuronal differentiation in primary hypothalamic cultures. (b) The effect of IGF-I is prevented by the incubation with an ER antagonist (ICI 182780) or by inhibiting the synthesis of ERa, using a specific antisense oligonucleotide. (c) The effect of estradiol is prevented by blocking the synthesis of IGF-I in the cultures using a specific IGF-I antisense oligonucleotide, as well as by the pharmacological blockade of IGF-I receptor (IGF-IR) associated signalling.

Neuroendocrine regulation and reproduction As an endocrine signal, IGF-I represents a link between the growth and reproductive axes and the interaction between IGF-I and estradiol in the brain is of particular physiological relevance for the regulation of growth, sexual maturation and adult neuroendocrine function. IGF-I from a peripheral origin appears to be one of the signals related to the initiation of puberty (Hiney et al., 1996, 2009; Wilson, 1998). The central administration of IGF-I stimulates prepubertal luteinizing hormone secretion and this effect is dependent on the presence of estradiol (Hiney et al., 2004). Functional interactions between estradiol and IGF-I receptor have

254

been reported on the expression of a1B-adrenoceptor in the hypothalamus and preoptic area, on luteinizing hormone release and on estradioldependent reproductive behavior (Etgen et al., 2006). The catecholamine neurotransmitter norepinephrine and a1B-adrenoceptors in the preoptic area and hypothalamus are involved in the temporal coordination of lordosis behavior and the luteinizing hormone surge (Etgen et al., 1992; Herbison, 1997). Estradiol increases the expression of a1b-adrenoceptors in the preoptic area and hypothalamus (Karkanias et al., 1996; Petitti et al., 1992). This effect is blocked by the antagonism of brain IGF-I receptors (Quesada and Etgen, 2001, 2002). In addition, the pharmacological blockade of brain IGF-I receptors during estrogen priming prevents the induction of the luteinizing hormone surge in response to estradiol and progesterone (Quesada and Etgen, 2002) and attenuates lordosis behavior (Etgen, 2003; Quesada and Etgen, 2002). Furthermore, chronic inhibition of brain IGF-I receptor abolishes estrous cycles (Todd et al., 2007). IGF-I may affect gonadotropin levels by actions in the arcuate nucleus and the median eminence. IGF-I levels increase dramatically in the rat arcuate nucleus with the onset of female puberty, and fluctuate during the estrus cycle in parallel to the release of gonadotropins (Dueñas et al., 1994). The fluctuation of IGF-I levels in the arcuate nucleus and median eminence may be in part mediated by tanycytes, specialized glial cells located in the arcuate nucleus and the median eminence (Rodriguez et al., 2005). Tanycytes express IGF-I receptors and accumulate IGF-I from the cerebrospinal fluid (Fernandez-Galaz et al., 1996). This accumulation of IGF-I by tanycytes is mediated by IGF-I receptors, and shows marked differences during the estrus cycle. High levels of IGF-I immunoreactivity in tanycytes and astrocytes are detected in the afternoon of proestrus and the morning of estrus in the arcuate nucleus of cycling female rats, then IGF-I immunoreactivity declines in the morning of metoestrus. Therefore, IGF-I immunoreactivity follows the changes in circulating levels of estradiol and progesterone

during the estrus cycle. Indeed, estradiol and progesterone regulate IGF-I immunoreactivity in arcuate glial cells. IGF-I immunoreactivity is low in glial cells of ovariectomized rats and increases in a dose-dependent manner when ovariectomized rats are injected with estradiol, an effect blocked by the simultaneous administration of progesterone (Dueñas et al., 1994; Fernandez-Galaz et al., 1996, 1997). The effect of estradiol may be mediated by the overexpression of IGF-binding protein-2 by tanycytes (Cardona-Gomez et al., 2000). Therefore, estradiol, acting on tanycytes, may regulate IGFI incorporation in the arcuate nucleus. Changes in the levels of IGF-I in the arcuate nucleus may affect synaptic plasticity in this brain region. The arcuate nucleus shows estradiol-induced and estrus-cycle-related plastic changes in synaptic connectivity and glial–neuronal interactions in female rodents. During the pre-ovulatory and ovulatory phases of the estrus cycle, there is a transient disconnection of axosomatic GABAergic synapses on the arcuate neuronal perikarya of adult female rats and a transient increase in excitatory synaptic inputs on dendritic spines. The synaptic remodelling is induced by estradiol and is linked to plastic modifications in astroglial processes and to modifications in the frequency of neuronal firing of arcuate neurons (Csakvari et al., 2007; Garcia-Segura et al., 1994; Naftolin et al., 2007; Olmos et al., 1989; Parducz et al., 2002). Results from in vivo experiments using intracerebroventricular infusion of specific receptor antagonists have shown that both ERs and IGF-I receptors are involved in the induction of synaptic and glial plastic modifications in the arcuate nucleus during the estrus cycle (Cardona-Gomez et al., 2000; Fernandez-Galaz et al., 1997, 1999). Under control conditions, the number of synaptic inputs on arcuate neuronal perikarya of cycling female rats decreases between the morning and afternoon of proestrus, remains low in estrus and recovers the following day; these changes are accompanied by the remodelling of astroglial processes. To explore whether IGF-I may affect this plasticity, an IGF-I receptor antagonist (JB1) was infused

255

into the lateral cerebral ventricle of cycling female rats to neutralize the local action of IGF-I in the brain. The intracerebroventricular administration of the IGF-I receptor antagonist JB1 resulted in the blockage of the phasic remodelling of synapses and glial processes observed in the arcuate nucleus during the estrus cycle (Fernandez-Galaz et al., 1999). In contrast, JB1 did not affect the number of synapses in proestrus rats, suggesting that IGF-I receptor activation is necessary for the estrus-cycle-associated synaptic plastic changes but not for the normal maintenance of synaptic inputs. The decrease in the number of synapses on the day of proestrus can be mimicked by the injection of estradiol to ovariectomized rats. The intracerebroventricular administration of ICI 182780, an antagonist of ERs, or the antagonist of IGF-I receptors JB1, or even the simultaneous administration of both receptor antagonists, did not affect the basal number of axosomatic synapses. This indicates that the activation of ERs and IGF-I receptors is not necessary for the maintenance of the synaptic inputs on arcuate neuronal perikarya. In contrast, the infusion in the lateral cerebral ventricle of the ER antagonist prevented the decrease in axosomatic synapses after the administration of estradiol to ovariectomized rats. This finding indicates that estradiolinduced synaptic plasticity in the arcuate nucleus is mediated by the activation of ERs. In addition, the estradiol-induced decrease in the number of axosomatic synapses is also prevented by the administration of the IGF-I receptor antagonist, either alone or in combination with the ER antagonist, indicating that the estradiol-induced decrease in the number of axosomatic synapses in the arcuate nucleus of ovariectomized rats is dependent on IGF-I receptors (Cardona-Gomez et al., 2000). Therefore, it seems that both ERs and IGF-I receptors are involved in the induction of synaptic plasticity in the hypothalamus of intact rats during the estrus cycle. Arcuate neurons express ERs and IGF-I receptors (Garcia-Segura et al., 1997; Shughrue

et al., 1997) and may therefore be a direct target for both estradiol and IGF-I. IGF-I may affect pre- and/or post-synaptic mechanisms, since ultrastructural studies have shown that the IGF-I receptor is present both in axosomatic pre-synaptic terminals as well as in neuronal perikarya of the rat arcuate nucleus (GarciaSegura et al., 1997). In addition, arcuate astrocytes are also a target for IGF-I, since they also express IGF-I receptors (Garcia-Segura et al., 1997). Arcuate astrocytes appear to be directly involved in the regulation of synaptic inputs to arcuate neurons, since synaptic disconnection of arcuate neurons in estrus females is accompanied by an increased extension of glial fibrillary acidic protein (GFAP) immunoreactive astrocytic processes (detected by light microscopy) and by an increase in the ensheathment of neuronal surfaces by astrocytic processes (detected by electron microscopic analysis). Interestingly, studies on hypothalamic tissue fragments from ovariectomized rats have shown that IGF-I receptor activation is needed for the induction of GFAP changes by estrogen in the arcuate nucleus (Fernandez-Galaz et al., 1997). In addition, tanycytes may play an important role in the interaction of estradiol and IGF-I for the regulation of synaptic plasticity, regulating the levels of IGF-I in the arcuate nucleus (Dueñas et al., 1994; Fernandez-Galaz et al., 1996, 1997). Indeed, the administration of the IGF-I receptor antagonist JB1 in the rat lateral cerebral ventricle is able to block both the accumulation of IGF-I by arcuate nucleus tanycytes and estrogen-induced synaptic plasticity (FernandezGalaz et al., 1996, 1997; Garcia-Segura et al., 1994, 1999). The interaction of estradiol and IGF-I in the regulation of synaptic plasticity in the arcuate nucleus may be involved in the effects of these two factors on the regulation of gonadotropins and estrous cycles. In addition, since the plastic reorganization of synapses in the arcuate nucleus is also involved in the hormonal control of feeding (Gao et al., 2007; Horvath, 2005, 2006), the interaction of estradiol and IGF-I on synaptic plasticity may also affect energy balance.

256

Adult neurogenesis Neural precursors located in the subgranular zone of the dentate gyrus of the hippocampus proliferate in adult rodents. The newly generated neurons are functional, integrate in hippocampal circuits and may be involved in certain forms of hippocampal-dependent learning and in the control of affection. IGF-I has been identified as one of the modulators of adult neurogenesis (Åberg et al., 2000). This effect of IGF-I may have a strong impact on hippocampal function and cognition. Neurogenesis in the subgranular layer of the hippocampus progressively and significantly decreases with ageing. This decrease affects both the generation of newly generated cells and their differentiation. Some evidence suggests that there is a link between decreased neurogenesis and cognitive impairment in aged rats. Interestingly, the intracerebroventricular infusion of IGF-I to senescent rats significantly restored neurogenesis through an approximately threefold increase in the production of new neurons (Lichtenwalner et al., 2001). IGF-I also partially restores neurogenesis and learning performance in the water maze task in aged rats submitted to stress during the pre-natal period (Darnaudéry et al., 2006). Furthermore, peripheral IGF-I mediates the effects of physical exercise on hippocampal neurogenesis. Voluntary running in rodents increases cell proliferation and neurogenesis in the adult dentate gyrus (van Praag et al., 1999). This is associated with an increase in the uptake of circulating IGF-I by cells of different brain regions, including the hippocampus (Carro et al., 2001). In addition, the prevention of the entrance of circulating IGF-I into the brain of rats undergoing exercise, by the subcutaneous infusion of a blocking IGF-I antiserum, results in a complete inhibition of exercise-induced increases in the number of new neurons in the hippocampus (Trejo et al., 2001). Estradiol has also been shown to modulate neurogenesis in the adult hippocampus (Banasr et al., 2001; Ormerod and Galea, 2001; Tanapat et al., 1999). Administration of estradiol has a biphasic effect on cell proliferation: it first enhances proliferation in the dentate gyrus

within four hours of the hormonal administration (Banasr et al., 2001, Ormerod and Galea, 2001; Tanapat et al., 1999) and then, after 24–48 hours, it suppresses cell proliferation (Ormerod and Galea, 2001; Ormerod et al., 2003). The interaction of estradiol and IGF-I on the regulation of adult hippocampal neurogenesis has been studied in ovariectomized rats to which a silastic capsule filled with a mixture of cholesterol and estradiol, or estradiol alone, was inserted subcutaneously (Perez-Martin et al., 2003). In addition, an osmotic minipump filled with saline, IGF-I, the ER antagonist ICI 182780 or a mixture of IGF-I and the ER antagonist was subcutaneously implanted. Minipumps were attached to a brain infusion cannula that was implanted into the right lateral cerebral ventricle. All animals received six daily intraperitoneal injections of 5-bromo-2-deoxyuridine (BrdU) to label proliferating cells. Cells incorporating BrdU were found in the hilus of the dentate gyrus of the hippocampus, in the subgranular zone and in the inner part of the granule cell layer. Treatment with IGF-I increased the number of total BrdUpositive cells. Interestingly, the ER antagonist ICI 182780 blocked the effect of IGF-I, independent of whether the animals were treated or not with estradiol. ICI 182780 by itself did not affect the number of BrdU-positive cells. A study using confocal microscopy to determine the number of BrdU cells labelled with neuronal marker bIII-tubulin revealed that the total number of BrdU-labelled neurons was significantly higher in animals treated either with IGF-I alone or with IGF-I and estradiol, compared to control rats. Interestingly, rats treated with IGF-I and estradiol showed a higher number of BrdU-positive neurons than rats treated with IGF-I alone, and the anti-estrogen ICI 182780 blocked the effect of IGF-I on the number of BrdU neurons. Therefore, it seems that ERs are involved in the effect of IGF-I on hippocampal neurogenesis. It is important to note that the blockade of IGF-Iinduced neurogenesis by the ER antagonist ICI 182780 was observed in ovariectomized rats in absence of estradiol replacement, indicating that the implication of ERs in the neurogenic effects of IGF-I is independent of ovarian estradiol. The

257

effects of IGF-I on adult hippocampal neurogenesis may be, in part, mediated by the regulation by IGF-I of the activity of ERs (see below). ERs may then affect adult hippocampal IGF-I-induced neurogenesis by participating as a component of IGF-I receptor signalling. This interaction of IGF-I with ERs may occur directly in the proliferating cells, since they express receptors for both IGF-I and estrogen (Isgor and Watson, 2005; Perez-Martin et al., 2003). In addition, the activity of ERs may be necessary to maintain an adequate expression of IGF-I receptors in the hippocampus in order to sustain the effects of IGF-I on neurogenesis (Cardona-Gomez et al., 2001).

Neuroprotection Different neurodegenerative conditions are associated with modifications in serum IGF-I levels (Busiguina et al., 2000; Carro et al., 2000). Furthermore, mice with low levels of serum IGF-I, as a consequence of specific targeted disruption of the IGF-I gene in the liver, had reduced neurogenesis in the hippocampus together with impaired spatial learning (Trejo et al., 2008). Moreover, the disruption of IGF-I input to the brain promotes amyloidosis, cognitive disturbance, hyperphosphorylated Tau deposits, gliosis and synaptic protein loss (Carro et al., 2006). This finding supports the hypothesis that disrupted IGF-I signalling may be involved in the pathology of Alzheimer’s disease. Indeed, systemic IGF-I promotes brain b-amyloid clearance, stimulating the neuronal release of the molecule and the transport into the brain of bamyloid carrier proteins that will take the molecule out of the brain (Carro and Torres-Aleman, 2004; Carro et al., 2002). Therefore, decreased systemic IGF-I levels may result in an impaired b-amyloid clearance. In addition to the neuroprotective effects of peripheral IGF-I, local IGF-I produced in the nervous system may also play a role in neuroprotection. Brain injury induces the synthesis of IGF-I and estradiol by reactive astrocytes (GarciaEstrada et al., 1992; Garcia-Segura, 2008; Hwang et al., 2004; Saldanha et al., 2009) and up-regulates ERs, IGF-I receptors, and IGF-binding proteins in

reactive glia (Beilharz et al., 1998; Blurton-Jones and Tuszynski, 2001; Chung et al., 2003; GarciaOvejero et al., 2002). Therefore, estradiol and IGF-I released by reactive glia may act directly on these cells or on neighbouring neurons, regulating reactive gliosis, neuronal survival and the reorganization of neural tissue after injury. Indeed, IGF-I and estradiol interact to regulate the plastic response of the brain after injury and during neurodegenerative conditions. IGF-I promotes neuronal survival and inhibits neuronal apoptosis in vitro and in vivo in a variety of experimental models of neurodegeneration (Åberg et al., 2006; Carro et al., 2003; Trejo et al., 2004). In vivo, IGF-I has also been shown to be a neuroprotective factor against a variety of neurodegenerative conditions, including hypoxic-ischaemic brain injury (Guan et al., 1993, 2003), excitotoxicity (Azcoitia et al., 1999; Carro et al., 2001) and cerebellar ataxia (Fernandez et al., 1998; Fernandez, Carro et al., 1999, 2005). IGF-I neuroprotective effects are exerted by the activation of the main intracellular signalling pathways associated with IGF-I receptors, the MAPK and the PI3K/Akt pathways (Guan et al., 2003). In particular, the inhibition of GSK3b activity, which is downstream of the PI3K/Akt pathway, seems to be an essential step in the neuroprotective mechanism (Brywe et al., 2005). The interaction of IGF-I and estradiol in neuroprotection has been assessed in ovariectomized rats in vivo, using systemic administration of kainic acid to induce degeneration of hippocampal hilar neurons, an experimental model of excitotoxic cell death (Fig. 2). Both the systemic administration of estradiol and the intracerebroventricular infusion of IGF-I prevent hilar neuronal loss induced by kainic acid. The neuroprotective effect of estradiol is blocked by the intracerebroventricular infusion of an IGF-I receptor antagonist, while the neuroprotective effect of IGF-I is blocked by the intracerebroventricular infusion of the ER antagonist ICI 182780 (Azcoitia et al., 1999). Similar results have been obtained in ovariectomized rats after the unilateral infusion of 6-hydroxdopamine into the medial forebrain bundle to lesion the nigrostriatal dopaminergic pathway. Pre-treatment with estradiol

258

(a)

E2

ER KA IGF-IR IGF-I (b)

ischaemia, in which both estradiol and IGF-I prevent neuronal loss in hippocampal CA1, simultaneous treatment with both factors do not have an additive effect (Traub et al., 2009). This suggests that both factors may use the same signalling mechanisms to exert neuroprotection.

E2

x ER

Estradiol, IGF-I and brain cholesterol

KA

IGF-IR IGF-I (c)

E2

ER KA

x

IGF-IR IGF-I Fig. 2. IGF-I and estradiol interact in the neuroprotection of hippocampal hilar neurons in vivo against the systemic administration of kainic acid (KA). (a) Both the systemic administration of estradiol (E2) and the intracerebroventricular infusion of IGF-I prevent hilar neuronal loss induced by kainic acid. (b) the neuroprotective effect of IGF-I is blocked by the intracerebroventricular infusion of an ER antagonist (ICI 182780). (c) The neuroprotective effect of estradiol is blocked by the intracerebroventricular infusion of an IGF-I receptor (IGFIR) antagonist (JB1).

or IGF-I prevents the loss of substantia nigra compacta neurons, the decrease in dopaminergic innervation of the striatum and the related motor disturbances. Blockage of IGF-I receptor by the intracerebroventricular administration of the IGF-I receptor antagonist JB1 attenuates the neuroprotective effects of both estrogen and IGF-I (Quesada and Micevych, 2004). In addition, the neuroprotective action of estradiol against 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) toxicity in the nigro-striatal system of male mice is associated to the regulation of IGF-I receptor signalling (D’Astous et al., 2006). These findings suggest that the neuroprotective actions of estradiol and IGF-I after brain injury depend on the co-activation of both ERs and IGF-I receptor in neural cells. Furthermore, in a model of global cerebral

Another interesting interaction between estradiol and IGF-I in the brain has been recently discovered by studies that have assessed the neuroprotective role of the selective Alzheimer’s disease indicator 1 gene (Seladin-1/Dhcr24). This gene encodes for the enzyme 3-beta-hydroxysterol delta-24-reductase (DHCR24), which converts desmosterol into cholesterol (Peri et al., 2009). Seladin-1/DHCR24 confers to neuronal cells resistance against b-amyloid and oxidative stressinduced apoptosis and inhibits the activation of caspase-3 (Cecchi et al., 2008; Greeve et al., 2000; Peri and Serio, 2008a, 2008b; Peri et al., 2009). In this context, it is of interest that the expression of seladin-1/DHCR24 is reduced in the brain of Alzheimer’s disease patients. The down-regulation of seladin-1/DHCR24 expression is paralleled by an increase in the amount of hyperphosphorylated Tau (Iivonen et al., 2002). Seladin-1 has been shown to mediate neuroprotective effects of estradiol. The silencing of seladin-1/DHCR24 expression by small interfering RNA methodology prevents the neuroprotective effect of estradiol against b-amyloid and oxidative stress toxicity (Luciani et al., 2008; Peri and Serio, 2008a, 2008b; Peri et al., 2009). Estradiol increases the expression of seladin-1/ DHCR24 in human derived neuronal cells (Benvenuti et al., 2005; Peri and Serio, 2008a, 2008b; Peri et al., 2009). This effect has also been observed with the selective ERa agonist propylpyrazole-triol, whereas the selective ERb agonist diarylpropionitrile was less potent in increasing seladin-1/DHCR24 levels (Benvenuti et al., 2005; Peri et al., 2009). These findings suggest that estradiol regulates seladin-1/DHCR24 expression via an ERa-mediated mechanism. This mechanism involves a direct binding of

259

ERa to functionally active half-palindromic estrogen responsive elements (EREs) in the seladin-1/DHCR24 promoter (Luciani et al., 2008). Although local cholesterol synthesis in the brain is neuroprotective, it has been suggested that high cholesterol levels in plasma increase the risk of Alzheimer’s disease (Jick et al., 2000; Kivipelto et al., 2001; Pappolla et al., 2003; Wolozin, 2004). There is therefore an apparent contradiction between the neuroprotective effects of local cholesterol synthesis and the damaging effects of circulating cholesterol. The importance of membrane cholesterol levels to maintain proper cellular function may be one of the factors that explains the deleterious effect of decreased cholesterol synthesis in neural cells (Etgen, 2008; Peri et al., 2009). It is also important to consider that there is little transfer of cholesterol from the peripheral circulation to the brain. However, it has been recently proposed that 27-hydroxycholesterol, a product of cholesterol oxidation that crosses the blood–brain barrier (Heverin et al., 2005), may be involved in the negative effects of hypercholesterolaemia in the brain (Sharma et al., 2008). Interestingly, 27-hydroxycholesterol reduces IGF-I levels and alters IGF-I receptor signalling in organotypic hippocampal slices and this is associated with increased levels of aggregated amyloid b (Sharma et al., 2008). IGF-I, which is neuroprotective in experimental models of Alzheimer’s disease (Torres-Aleman, 2008), increases the expression of seladin-1/DHCR24 in human derived neuroblastoma cells (Giannini et al., 2008; Peri and Serio, 2008a; Peri et al., 2009). Therefore, it is conceivable that hypercholesterolaemia may decrease local cholesterol synthesis in the brain via 27-hydroxycholesterol and IGF-I. In addition, estradiol increases the release of IGF-I by human derived neuronal cells (Giannini et al., 2008). Thus, estradiol may contribute to increase brain cholesterol synthesis by direct effects on the seladin-1/DHCR24 promoter and by increasing local IGF-I levels (Fig. 3). Cholesterol synthesis may therefore represent another point of interaction of estradiol and IGF-I in the brain to promote neuroprotection.

Desmosterol

+

+

Cholesterol Membranes IGF-I

Steroidogenesis

Estradiol Fig. 3. Interactions of estradiol and IGF-I on human-derived neuronal cells in the regulation of seladin-1/DHCR24, the enzyme that transforms desmosterol into cholesterol. Estradiol upregulates seladin-1/DHCR24 expression by direct effects on the seladin-1/DHCR24 gene promoter. In addition, estradiol upregulates IGF-I levels in neurons. IGF-I, in turn, also promotes seladin-1/DHCR24 expression. Therefore, estradiol and IGF-I may interact in the regulation of cholesterol levels in neurons. In addition to its role in membranes, cholesterol enters in the steroidogenic pathway and may be metabolized to estradiol within the brain.

Molecular mechanisms involved in the interaction of estradiol and IGF-I in the nervous system Cross-regulation of the expression of ERs and IGF-I receptor in the brain Part of the interactions between estradiol and IGF-I in the nervous system may be mediated by a cross-regulation of ERs and IGF-I receptor. The activity of IGF-I receptors regulates the expression of estrogen receptors in the rat brain (Cardona-Gomez et al., 2001). Intracerebroventricular infusion of IGF-I in ovariectomized rats results in the down-regulation of ERa protein levels in the hypothalamus and the up-regulation of ERa levels in the hippocampus, compared with control rats infused with vehicle. No significant effect is observed in the cerebral cortex. The infusion of the IGF-I receptor antagonist JB1 has effects opposite to those of IGF-I: ERa levels were increased in the hypothalamus and decreased in the hippocampus, compared with control rats infused with vehicle. The IGF-I receptor antagonist is also able to induce a

260

significant decrease in ERa levels in the cerebral cortex. The intracerebroventricular infusion of IGF-I, or of the IGF-I receptor antagonist JB1, does not significantly affect ERb expression in the hypothalamus, the hippocampus or the cerebral cortex. However, IGF-I increases and JB1 decreases the expression of ERb in the cerebellum (Cardona-Gomez et al., 2001). These findings suggest that IGF-I receptor (IGF-IR) regulates the expression of ERs in the female rat central nervous system, that this effect is regionally specific and that it is selective for the two ER subtypes. Reciprocally, estradiol and the activity of ERs regulate the expression of IGF-I receptors in the nervous system. Estradiol increases the expression of IGF-I receptor (Cambiasso et al., 2000; Pons and Torres-Aleman, 1993) and IGF-binding proteins (Pons and Torres-Aleman, 1993) in monolayer hypothalamic cultures. Estradiol also increases the levels of IGF-I and IGF-binding protein-2 in human derived neuronal cells (Giannini et al., 2008) and in the rat hypothalamus in vivo (Cardona-Gomez et al., 2000; Dueñas et al., 1994). In addition, estradiol decreases the release of IGF-binding protein-4 by human derived neuronal cells (Giannini et al., 2008). Furthermore, the inhibition of ERs in the brain of ovariectomized rats by the chronic intracerebroventricular infusion of ICI 182780 results in the down-regulation of IGF-I receptor mRNA and protein levels in the hippocampus and the cerebral cortex compared with the levels observed in control animals given vehicle (Cardona-Gomez et al., 2001). A similar decrease is observed in IGF-I receptor mRNA levels in the hypothalamus of the rats treated with the ER antagonist. However, the inhibition of ERs does not affect IGF-I receptor protein levels in this brain region (Cardona-Gomez et al., 2001). Interestingly, the effect of the inhibition of ERs in the cerebellum is different from other brain regions. In the cerebellum, the ER antagonist results in an increase in IGF-I receptor mRNA and protein levels (Cardona-Gomez et al., 2001). The regional differences in the regulation of IGF-I receptors by the activity of ERs may be due to the different levels of expression of the a and b forms of ERs, since ERb is the predominant, if not the exclusive, form

in the cerebellum (Shughrue et al., 1997), while in the other brain areas studied both ER subtypes are expressed.

Interaction of ERs with the IGF-I receptor As in other cell types, the actions of estradiol mediated by classical ERs in neurons include nuclear-initiated ER signalling and membrane/ cytoplasm-initiated ER signalling. The nuclearinitiated ER signalling by classical ERs (ERa and ERb) is the best characterized mode of action of estradiol and involves transcriptional actions at ERE sequences in the genome (Gruber et al., 2004). In addition, classical ERs may act as transcriptional cofactors interacting with other DNA-binding elements (Cerciat et al., 2010; Ghisletti et al., 2005; Kushner et al., 2000). Membrane/cytoplasm-initiated ER signalling involves the interaction of classical ERs in the membrane or the cytoplasm with G-proteins, cAMP, glutamate receptors, signalling kinases and phosphatases (Kelly and Rønnekleiv, 2008; Marin et al., 2009; Micevych and Dominguez, 2009; Micevych and Mermelstein, 2008; Simpkins et al., 2009; Vasudevan and Pfaff, 2007) as well as actions on the mitochondria (Simpkins et al., 2009). The membrane/ cytoplasm-initiated ER signalling may finally regulate transcription at ERE-independent sites, such as the regulation of b-cateninmediated transcription (Varea et al., 2009a, 2009b). Experimental evidence accumulated over the years in different cell types strongly supports the idea that the membrane and nuclear ERa share a common transcriptional origin (Micevych and Mermelstein, 2008; Pedram et al., 2006; Razandi et al., 2003). Classical ERs associate to the plasma membrane at specific membrane domains, the caveolae, specialized microdomains that have a particular lipid composition and a high density of lipid-anchored proteins (Anderson, 1998), allowing the interaction of receptors and signalling proteins (Smart et al., 1995). Caveolins, structural proteins of caveolae, act as anchoring factors for ERs in the membrane (Sotgia et al., 2006; Zhang et al., 2006) and allow the interaction of ERs with

261

other molecules such as glutamate receptors (Mermelstein 2009; Micevych and Mermelstein, 2008), voltage-dependent anion channel (VDAC) (Ram´ırez et al., 2009) and the IGF-I receptor (Marin et al., 2009). The interaction of ERs and IGF-I receptor in the brain is hormonally regulated. Estradiol administration to adult ovariectomized rats results in a transient increase in the association between IGF-I receptor and ERa in the brain (Mendez et al., 2003). The interaction is coincident in time with an increase in tyrosine phosphorylation of IGF-I receptor. Estradiol also increases the interaction between the p85 subunit of the PI3K and IRS-1 (Mendez et al., 2003), one of the first events in the signal transduction of the IGF-I receptor, suggesting that the increase in IGF-I receptor phosphorylation induced by estradiol reflects functional activation of this receptor. In addition ERa interacts with other components of the IGF-I receptor signalling pathway, such as p85 (Mendez et al., 2003). This interaction is present in control ovariectomized animals and is increased after estradiol treatment. A similar estradiol-induced association of ERa and p85 occurs in the mammary cancer cell line MCF-7 (Castoria et al., 2001) and in human vascular endothelial cells (Simoncini et al., 2000). In addition, ERa interacts with GSK3b and b-catenin in the hippocampus and the presence of estradiol releases b-catenin from this complex (Cardona-Gomez et al., 2004). Interestingly, the interaction between ERa and the IGF-I receptor is also increased by the intracerebroventricular administration of IGF-I (Mendez et al., 2003). These findings suggest that the interaction of ERa with IGF-I receptor is part of the mechanisms involved in the signalling of both IGF-I and estradiol in the brain (Fig. 4).

Regulation of ER transcriptional activity by IGF-I in neural cells In addition to classical activation of ER by estradiol binding, ER transcriptional activity can be regulated by ligand-independent mechanisms. Intracellular kinase signalling pathways, activated

Oestradiol

IGF-I

IGF-IR ERα

IRS-1

PI3K Akt β-Cat

β-Cat ERα

GSK3β

β-Cat

β-Cat LEF-1

Oestradiol Fig. 4. Estradiol and IGF-I interact in the regulation of IGF-I signalling in the nervous system. Estradiol and IGF-I regulate the interaction of ERa with IGF-I receptor (IGF-IR) and with components of IGF-IR-associated signalling, such as insulin receptor substrate-1 (ISR-1), phosphatidylinositol 3-kinase (PI3K), Akt, glycogen synthase kinase 3b (GSK3b) and b-catenin. Through this signalling pathway, estradiol and IGF-I may interact in the regulation of neuronal development, synaptic plasticity, neuronal survival, the expression of antiapoptotic molecules and the phosphorylation of Tau. By the inhibition of GSK3b, estradiol induces the stabilization of b-catenin and its translocation to the cell nucleus. In the cell nucleus, b-catenin interacts with ERa to regulate ER-mediated transcription and with lymphoid enhancer binding factor-1 (LEF-1) to regulate LEF-1-mediated transcription.

by extracellular growth or trophic factors, regulate the ability of ERs to promote changes in gene expression. IGF-I is one of the extracellular regulators of these kinase pathways that have been shown to promote ER-dependent transcription. In different cell lines, including neuroblastoma cells, insulin and IGF-I activate ERs in the absence of estradiol and regulate ER-mediated gene expression (Agrati et al., 1997; Font de Mora and Brown, 2000; Klotz et al., 2002; Ma et al., 1994; Martin et al., 2000; Patrone et al., 1996). In neuroblastoma cells, IGF-I may have a different

262

regulation of the activity of ERa depending on whether the ER ligand is present or not. In absence of estradiol, IGF-I increases ERa activity using the Ras–MAPK signalling pathway (Patrone et al., 1998). In contrast, IGF-I negatively regulates ERa transcriptional activity in presence of estradiol (Mendez and Garcia-Segura, 2006). This effect of IGF-I is mediated by the PI3K–Akt– GSK3b pathway, which induces the translocation of b-catenin to the cell nucleus (Fig. 4). In turn, bcatenin binds to ERa in the nucleus and inhibits its transcriptional activity (Mendez and GarciaSegura, 2006). This has been demonstrated in N2a neuroblastoma cells, where IGF-I induces a significant activation of ER-mediated transcription by a PI3K independent mechanism. In contrast, the addition of estradiol and IGF-I produces a smaller response than that observed in the presence of estradiol alone. This inhibitory effect of IGF-I is no longer detected in the presence of the PI3K inhibitor wortmannin or when a kinasedead form of the IGF-I receptor is transfected in N2a cells (Mendez and Garcia-Segura, 2006). Wortmannin, per se, produces a decrease in the serine 9 phosphorylation of GSK3b and induces a dose-dependent increase in ER-mediated transcription. Interestingly, GSK3 inhibitors block the effect of wortmannin on ER activity, suggesting that GSK3 mediates the effect of PI3K on ER-mediated transcription (Mendez and GarciaSegura, 2006). Furthermore, the blockage of the PI3K pathway by the over-expression of a truncated form of p85 that lacks a small region implicated in the interaction with p110, the catalytic subunit of PI3K, results in a significant decrease in the serine 9 phosphorylation of GSK3b and a significant increase in ERmediated transcription compared with cells over-expressing the wild-type form of p85. The increase in ER-mediated transcription in N2a cells caused by the truncated form of p85 is no longer present in the presence of the GSK3 inhibitors (Mendez and Garcia-Segura, 2006). These findings are compatible with the possibility of a regulation of ER transcriptional activity by GSK3 driven by PI3K. Akt, which is downstream of PI3K, seems to be involved in this

action since the pharmacological inhibition of Akt produces a decrease in the serine 9 inhibitory phosphorylation of GSK3b and a dosedependent increase in ER-mediated transcription. GSK3 inhibitors block the effect of Akt inhibition on ER activity, suggesting that GSK3 mediates the effect of Akt on ER-mediated transcription (Mendez and Garcia-Segura, 2006). Furthermore, the regulation of nuclear ERa activity by GSK3 can be elicited by extracellular treatment with IGF-I or by the over-expression of IGF-I receptor in N2a cells (Mendez and Garcia-Segura, 2006). The role of GSK3 in the regulation of ER-mediated transcription in N2a cells has been investigated by assessing the effects of different pharmacological inhibitors of GSK3 in an estrogen-responsive gene reporter assay. All the GSK3 inhibitors tested result in a decrease in estradiol-induced ER-mediated transcription in a dose-dependent manner. The inhibitory effects are also observed when cells are stimulated with PPT, the ERa-specific agonist. In addition, the over-expression of GSK3b results in a significant increase in the response of the reporter construct to estradiol. In contrast, the enhancement of ERmediated transcription is not observed when a mutated, kinase-inactive form of GSK3 is transfected (Mendez and Garcia-Segura, 2006). b-Catenin seems to be involved in the effect of GSK3 on ER-mediated transcription in N2a cells. Co-localization of ERa and b-catenin has been detected in the cell nucleus of N2a cells and this co-localization is strongly increased by the pharmacological inhibition of GSK3 (Mendez and Garcia-Segura, 2006). In addition, an interaction between ERa and b-catenin in N2a cells has been detected in immunoprecipitation experiments (Mendez and Garcia-Segura, 2006). Furthermore, estradiol, PI3K and GSK3 inhibitors regulate this interaction. The interaction between ERa and bcatenin is lowered to minimal levels after 45 minutes of estradiol treatment, returned to control values one hour after addition of the hormone and remained at basal levels 24 hours after hormone treatment. The inhibition of PI3K with wortmannin also induces a decrease in the interaction between ERa and b-catenin that is

263

detectable 1 and 10 hours after the beginning of treatment. In contrast, the pharmacological inhibition of GSK3 induces a rapid increase in the levels of b-catenin associated with ERa. Further evidence of the involvement of GSK3 and b-catenin in the regulation that the PI3K pathway exerts on ER-mediated transcription has been obtained by using a mutant, non-degradable form of b-catenin in combination with reporter gene experiments. The mutation of serine 33 to a tyrosine (b-catenin S33Y) blocks GSK3 phosphorylation of this residue and thus prevents proteasome-dependent b-catenin degradation and results in higher expression levels than the wild-type form. Probably as a consequence of this, the levels of ERa associated with the nondegradable mutant of b-catenin are also higher (Mendez and Garcia-Segura, 2006). Overexpression of the mutated form of b-catenin results in a similar induction of ER-mediated transcription as over-expression of the wild-type form. However, wortmannin does not enhance ER transcriptional activity in cells over-expressing mutant b-catenin, an effect that is evident in cells expressing wild-type b-catenin (Mendez and Garcia-Segura, 2006).

Regulation of IGF-I receptor signalling by estradiol in the brain In vitro and in vivo studies have shown that estradiol rapidly activates in the brain the MAPK signalling pathway (Cardona-Gomez et al., 2001, 2002; Carrer et al., 2003; Dhandapani and Brann, 2007; Toran-Allerand et al., 1999). The MAPK signalling pathway mediates effects of estradiol on axonal growth (Carrer et al., 2003), synaptic plasticity (Ogiue-Ikeda et al., 2008), behavior (Etgen and Acosta-Martinez, 2003; Walf and Frye, 2008) and neuroprotection (Bryant et al., 2006; Guerra et al., 2004; JoverMengual et al., 2007; Kuroki et al., 2001; Lebesgue et al., 2009; Marin et al., 2005). Estradiol also activates the PI3K–Akt signalling pathway in the brain (Bourque et al., 2009; Cardona-Gomez et al., 2002; Garcia-Segura et al., 2006; Mendez et al., 2005; Toran-Allerand et al., 1999). The

activation of Akt by estradiol mediates hormonal effects on synaptic plasticity (Znamensky et al., 2003), lordosis behavior (Etgen and Acosta-Martinez, 2003) and neuronal survival under neurodegenerative conditions (Honda et al., 2000; Marin et al., 2005; Quesada et al., 2008; Wang et al., 2006; Yu et al., 2004; Zhang et al., 2001). Via the activation of Akt, estradiol may also activate the MAPK pathway in neurons (Mannella and Brinton, 2006). In addition, Akt regulates several transcription factors that control neuronal survival, such as nuclear factor (NF)-kB (Kane et al., 1999), cAMP response element binding (CREB) protein (Pugazhenthi et al., 2000) and several members of the Forkhead family (Brunet et al., 1999; Kops et al., 1999; Tang et al., 1999). In addition, activation of Akt may promote neuronal survival by the suppression of Bad-induced cell death (Datta et al., 1997; del Peso et al., 1997) and by the enhancement of the expression of the antiapoptotic Bcl-2 (Pugazhenthi et al., 2000). Estradiol is known to up-regulate Bcl-2 and other antiapoptotic Bcl-2 family members in brain neurons (Dubal et al., 1999; Garcia-Segura et al., 1998; Nilsen and Diaz Brinton, 2003; Yao et al., 2007; Zhao et al., 2004) and Akt may be involved in this hormonal effect (D’Astous et al., 2006). Regulating the activity of Akt in the brain, estradiol also affects the activity of GSK3b (Cardona-Gomez et al., 2004). This kinase regulates the phosphorylation of microtubuleassociated proteins and, therefore, microtubule dynamics, growth and retraction of neuronal processes and synapse formation and plasticity (Arevalo and Chao, 2005; Garrido et al., 2007; Jones et al., 2003; Peineau et al., 2007; Zhou et al., 2004). Under pathological conditions, GSK3b may be responsible for the hyperphosphorylation of Tau in Alzheimer’s disease (Lovestone et al., 1994) and after cerebral ischaemia (Cardona-Gomez et al., 2006). Interestingly, estradiol increases the amount of inactive GSK3b (ser-9 phosphorylated) and decreases the phosphorylation of Tau in the hippocampus in ovariectomized rats (Cardona-Gomez et al., 2004). After cerebral ischaemia, estradiol decreases hippocampal injury, inhibits GSK3b, decreases

264

the hyperphosphorylation of Tau and decreases the interaction of Tau with GSK3b and AMPA glutamate receptors (Cardona-Gomez et al., 2006). In addition, the phosphorylation and inhibition of GSK3b is associated to an enhanced neuronal survival (Cross, Alessi, Cohen, Andjelkovich, & Hemmings, 1995) and to the promotion of neuronal survival by estradiol (D’Astous et al., 2006; Goodenough et al., 2005). In addition, the inhibition of GSK3b by estradiol in neuroblastoma cells and in primary cortical neurons is associated with the stabilization of b-catenin and its translocation to the cell nucleus where it acts as a co-transcriptional modulator using canonical T cell factor (TCF)/lymphoid enhancer binding factor-1 (LEF-1)-mediated transcription, in a similar fashion to that produced by Wnt3a (Fig. 4) (Varea et al., 2009a, 2009b).

Future directions As we have seen in this chapter, new information on the functional consequences of the interaction of estradiol and IGF-I for neural development and function has accumulated in recent years. However, many important aspects remain to be addressed. For instance, while estradiol and IGF-I have been shown to interact in the regulation of synaptic plasticity in the hypothalamus, it is still necessary to explore whether these two factors interact in the regulation of synaptic plasticity in cognitive areas, such as the prefrontal cortex and the hippocampus. In addition, although it is known that estradiol and IGF-I have effects on cognition and affection, their interaction on the regulation of these parameters has not been assessed. There has been also a significant advance in the understanding of the molecular mechanisms of interaction of estradiol and IGF-I in the nervous system. We now know that IGF-I regulates ER transcriptional activity and estradiol regulates IGF-I receptor signalling in neural cells and we know details on the molecular mechanisms involved in this cross-talk. Future directions may include the assessment of the interaction of ERs and IGF-I receptors with other signalling systems

in the nervous system, such as Wnt signalling (Varea et al., 2009a, 2009b), and the study of the functional consequences of such interactions. Another open question is the possible modification in the cross-talk of ERs and IGF-I receptors in the brain during the different life stages, since plasma levels of estradiol and IGF-I change during development, puberty, reproductive cycles and ageing. Recent studies have shown that there is a maturation in the interaction of estradiol and IGF-I receptor signalling in the prefrontal cortex of female rats during puberty, which is dependent on ovarian hormones (Sanz et al., 2008). We do not know if similar changes occur in other brain regions or during other life stages. In particular, it is important to determine whether the cross-talk between IGF and ERs in the brain is affected by ageing and menopause, when circulating levels of both factors decrease. Brain pathology may also potentially change the interaction of estradiol and IGF-I in the brain, since ERs (Blurton-Jones and Tuszynski, 2001; Dubal et al., 2006; Garcia-Ovejero et al., 2002), IGF-I receptors (Chung et al., 2003), estradiol synthesis (Garcia-Segura et al., 1999; Garcia-Segura, 2008; Saldanha et al., 2009), IGFI levels (Beilharz et al., 1998; Garcia-Estrada et al., 1992; Hwang et al., 2004) and the expression of IGF-binding proteins (Beilharz et al., 1998) are increased in brain cells after some forms of acute injury (Fig. 5). In addition, decreased circulating

ERs

Neurons

Aromatase ERs IGF-I IGF-IR Astrocytes Injury

Fig. 5. Brain pathology may potentially change the interaction of estradiol and IGF-I in the brain, since the expression of aromatase (estradiol synthesis), ERs, IGF-I and IGF-I receptor (IGF-IR) are increased in neural cells after acute brain injuries. However, chronic neurodegeneration, such as Alzheimer’s disease, may be associated with a downregulation of estradiol brain synthesis and of ER and IGF-I receptor signalling.

265

levels of estradiol and IGF-I are also detected in a proportion of patients after traumatic brain injury (Agha et al., 2004). On the other hand, estrogen receptors and aromatase are down-regulated in the brain of Alzheimer’s disease patients (Ishunina et al., 2007), where IGF-I resistance also appears to occur. Finally, the ability of estradiol and of synthetic estrogenic compounds to increase IGF-I receptor signalling in the brain (D’Astous et al., 2006) may allow the development of new selective ER modulators with neuroprotective and antiinflammatory properties based on the activation of such signalling. Acknowledgements Support from Ministerio de Ciencia e Innovación, Spain (BFU2008-02950-C03-01 and BFU200802950-C03-02) is acknowledged. References Åberg, M. A., Aberg, N. D., Hedbacker, H., Oscarsson, J., & Eriksson, P. S. (2000). Peripheral infusion of IGF-I selectively induces neurogenesis in the adult rat hippocampus. Journal of Neuroscience, 20, 2896–2903. Åberg, N. D., Brywe, K. G., & Isgaard, J. (2006). Aspects of growth hormone and insulin-like growth factor-I related to neuroprotection, regeneration, and functional plasticity in the adult brain. The Scientific World Journal, 6, 53–80. Agha, A., Rogers, B., Sherlock, M., O’Kelly, P., Tormey, W., Phillips, J., et al. (2004). Anterior pituitary dysfunction in survivors of traumatic brain injury. Journal of Clinical Endocrinology and Metabolism, 89(10), 4929–4936. Agrati, P., Garnier, M., Patrone, C., Pollio, G., Santagati, S., Vegeto, E., et al. (1997). SK-ER3 neuroblastoma cells as a model for the study of estrogen influence on neural cells. Brain Research Bulletin, 44, 519–523. Aleman, A. & Torres-Alemán, I. (2009). Circulating insulin-like growth factor I and cognitive function: Neuromodulation throughout the lifespan. Progress in Neurobiology, 89, 256–265. Anderson, R. G. (1998). The caveolae membrane system. Annual Review of Biochemistry, 67, 199–225. Arevalo, J. C. & Chao, M. V. (2005). Axonal growth: Where neurotrophins meet wnts. Current Opinion in Cell Biology, 17, 112–115. Azcoitia, I., Sierra, A., & Garcia-Segura, L. M. (1999). Neuroprotective effects of estradiol in the adult rat hippocampus: Interaction with insulin-like growth factor-I signalling. Journal of Neuroscience Research, 58, 815–822.

Banasr, M., Hery, M., Brezun, J. M., & Daszuta, A. (2001). Serotonin mediates oestrogen stimulation of cell proliferation in the adult dentate gyrus. European Journal of Neuroscience, 14, 1417–1424. Bateman, J. M. & McNeill, H. (2006). Insulin/IGF signaling in neurogenesis. Cellular and Molecular Life Sciences, 63, 1701–1705. Beilharz, E. J., Russo, V. C., Butler, G., Baker, N. L., Connor, B., Sirimanne, E. S., et al. (1998). Co-ordinated and cellular specific induction of the components of the IGF/IGFBP axis in the rat brain following hypoxic-ischemic injury. Brain Research. Molecular Brain Research, 59, 119–134. Benvenuti, S., Luciani, P., Vannelli, G. B., Gelmini, S., Franceschi, E., Serio, M., et al. (2005). Estrogen and selective estrogen receptor modulators exert neuroprotective effects and stimulate the expression of selective alzheimer’s disease indicator-1, a recently discovered antiapoptotic gene, in human neuroblast long-term cell cultures. Journal of Clinical Endocrinology and Metabolism, 90, 1775–1782. Blurton-Jones, M. & Tuszynski, M. H. (2001). Reactive astrocytes express estrogen receptors in the injured primate brain. The Journal of Comparative Neurology, 433, 115–123. Bourque, M., Dluzen, D. E., & Di Paolo, T. (2009). Neuroprotective actions of sex steroids in parkinson’s disease. Frontiers in Neuroendocrinology, 30, 142–157. Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., et al. (1999). Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor. Cell, 96, 857–868. Bryant, D. N., Sheldahl, L. C., Marriott, L. K., Shapiro, R. A., & Dorsa, D. M. (2006). Multiple pathways transmit neuroprotective effects of gonadal steroids. Endocrine, 29(2), 199–207. Brywe, K. G., Mallard, C., Gustavsson, M., Hedtjärn, M., Leverin, A. L., Wang, X., et al. (2005). IGF-1 neuroprotection in the immature brain after hypoxia-ischemia, involvement of akt and GSK3b. European Journal of Neuroscience, 21, 1489–1502. Busiguina, S., Fernandez, A. M., Barrios, V., Clark, R., Tolbert, D. L., Berciano, J., et al. (2000). Neurodegeneration is associated to changes in serum insulin-like growth factors. Neurobiology of Disease, 7, 657–665. Camarero, G., Leon, Y., Gorospe, I., De Pablo, F., Alsina, B., Giraldez, F., et al. (2003). Insulin-like growth factor 1 is required for survival of transit-amplifying neuroblasts and differentiation of otic neurons. Developmental Biology, 262, 242–253. Cambiasso, M. J., Colombo, J. A., & Carrer, H. F. (2000). Differential effect of oestradiol and astroglia-conditioned media on the growth of hypothalamic neurons from male and female rat brains. European Journal of Neuroscience, 12, 2291–2298. Cardona-Gomez, G. P., Chowen, J. A., & Garcia-Segura, L. M. (2000). Estradiol and progesterone regulate the expression of insulin-like growth factor-I receptor and insulin-like growth factor binding protein-2 in the hypothalamus of adult female rats. Journal of Neurobiology, 43, 269–281.

266 Cardona-Gomez, G. P., Mendez, P., DonCarlos, L. L., Azcoitia, I., & Garcia-Segura, L. M. (2001). Interactions of estrogens and insulin-like growth factor-I in the brain: Implications for neuroprotection. Brain Research Reviews, 37, 320–334. Cardona-Gomez, G. P., Mendez, P., & Garcia-Segura, L. M. (2002). Synergistic interaction of estradiol and insulin-like growth factor-I in the activation of PI3K/akt signaling in the adult rat hypothalamus. Brain Research. Molecular Brain Research, 107, 80–88. Cardona-Gomez, G. P., Perez, M., Avila, J., Garcia-Segura, L. M., & Wandosell, F. (2004). Estradiol inhibits GSK3 and regulates interaction of estrogen receptors, GSK3 and betacatenin, in the hippocampus. Molecular and Cellular Neuroscience, 25, 363–373. Cardona-Gomez, G. P., Trejo, J. L., Fernandez, A. M., & Garcia-Segura, L. M. (2000). Estrogen receptors and insulin-like growth factor-I receptors mediate estrogen-dependent synaptic plasticity. NeuroReport, 11, 1735–1738. Cardona-Gómez, G. P., Arango-Davila, C., Gallego-Gómez, J. C., Barrera-Ocampo, A., Pimienta, H., & Garcia-Segura, L. M. (2006). Estrogen dissociates tau and alpha-amino-3hydroxy-5-methylisoxazole-4-propionic acid receptor subunit in postischemic hippocampus. NeuroReport, 17(12), 1337–1341. Cardona-Gómez, G. P., DonCarlos, L., & Garcia-Segura, L. M. (2000). Insulin-like growth factor I receptors and estrogen receptors colocalize in female rat brain. Neuroscience, 99(4), 751–760. Cardona-Gómez, G. P., Mendez, P., DonCarlos, L. L., Azcoitia, I., & Garcia-Segura, L. M. (2002). Interactions of estrogen and insulin-like growth factor-I in the brain: Molecular mechanisms and functional implications. The Journal of Steroid Biochemistry and Molecular Biology, 83(1-5), 211–217. Carrer, H. F. & Cambiasso, M. J. (2002). Sexual differentiation of the brain: Genes, estrogen, and neurotrophic factors. Cellular and Molecular Neurobiology, 22, 479–500. Carrer, H. F., Cambiasso, M. J., Brito, V., & Gorosito, S. (2003). Neurotrophic factors and estradiol interact to control axogenic growth in hypothalamic neurons. Annals of the New York Academy of Sciences, 1007, 306–316. Carro, E., Nunez, A., Busiguina, S., & Torres-Aleman, I. (2000). Circulating insulin-like growth factor I mediates effects of exercise on the brain. Journal of Neuroscience, 20, 2926–2933. Carro, E. & Torres-Aleman, I. (2004). The role of insulin and insulin-like growth factor I in the molecular and cellular mechanisms underlying the pathology of alzheimer’s disease. European Journal of Pharmacology, 490, 127–133. Carro, E., Trejo, J. L., Busiguina, S., & Torres-Alemán, I. (2001). Circulating insulin-like growth factor I mediates the protective effects of physical exercise against brain insults of different etiology and anatomy. Journal of Neuroscience, 21, 5678–5684. Carro, E., Trejo, J. L., Gomez-Isla, T., LeRoith, D., & TorresAleman, I. (2002). Serum insulin-like growth factor I

regulates brain amyloid-beta levels. Nature Medicine, 8, 1390–1397. Carro, E., Trejo, J. L., Nunez, A., & Torres-Aleman, I. (2003). Brain repair and neuroprotection by serum insulin-like growth factor I. Molecular Neurobiology, 27, 153–162. Carro, E., Trejo, J. L., Spuch, C., Bohl, D., Heard, J. M., & Torres-Aleman, I. (2006). Blockade of the insulin-like growth factor I receptor in the choroid plexus originates alzheimer’s-like neuropathology in rodents: New cues into the human disease? Neurobiology of Aging, 27, 1618–1631. Castoria, G., Migliaccio, A., Bilancio, A., Di Domenico, M., de Falco, A., Lombardi, M., et al. (2001). PI3-kinase in concert with src promotes the S-phase entry of oestradiol-stimulated MCF-7 cells. The EMBO Journal, 20, 6050–6059. Cecchi, C., Rosati, F., Pensalfini, A., Formigli, L., Nosi, D., Liguri, G., et al. (2008). Seladin-1/DHCR24 protects neuroblastoma cells against abeta toxicity by increasing membrane cholesterol content. Journal of Cellular and Molecular Medicine, 12(5B), 1990–2002. Cerciat, M., Unkila, M., Garcia-Segura, L. M., & Arevalo, M. A. (2010). Selective estrogen receptor modulators decrease the production of interleukin-6 and interferongamma-inducible protein-10 by astrocytes exposed to inflammatory challenge in vitro. Glia, 58(1), 93–102. Chung, Y. H., Joo, K. M., Shin, C. M., Lee, Y. J., Shin, D. H., Lee, K. H., et al. (2003). Immunohistochemical study on the distribution of insulin-like growth factor I (IGF-I) receptor in the central nervous system of SOD1(G93A) mutant transgenic mice. Brain Research, 994, 253–259. Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M., & Hemmings, B. A. (1995). Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature, 378, 785–789. Csakvari, E., Hoyk, Z., Gyenes, A., Garcia-Ovejero, D., Garcia-Segura, L. M., & Párducz, A. (2007). Fluctuation of synapse density in the arcuate nucleus during the estrous cycle. Neuroscience, 144(4), 1288–1292. Darnaudéry, M., Perez-Martin, M., Belizaire, G., Maccari, S., & Garcia-Segura, L. M. (2006). Insulin-like growth factor 1 reduces age-related disorders induced by prenatal stress in female rats. Neurobiology of Aging, 27, 119–127. D’Astous, M., Mendez, P., Morissette, M., Garcia-Segura, L. M., & Di Paolo, T. (2006). Implication of the phosphatidylinositol-3 kinase/protein kinase B signaling pathway in the neuroprotective effect of estradiol in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mice. Molecular Pharmacology, 69, 1492–1498. Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., et al. (1997). Akt phosphorylation of BAD couples survival signals to cell-intrinsic machinery. Cell, 91, 231–241. del Peso, L., Gonzalez-Garcia, M., Page, C., Herrera, R., & Nunez, G. (1997). Interleukin-3-induced phosphorylation of BAD through the protein kinase akt. Science, 278, 687–689. Dhandapani, K. M. & Brann, D. W. (2007). Role of astrocytes in estrogen-mediated neuroprotection. Experimental Gerontology, 42(1-2), 70–75.

267 Ding, Q., Vaynman, S., Akhavan, M., Ying, Z., & GomezPinilla, F. (2006). Insulin-like growth factor I interfaces with brain-derived neurotrophic factor-mediated synaptic plasticity to modulate aspects of exercise-induced cognitive function. Neuroscience, 140, 823–833. Dubal, D. B., Rau, S. W., Shughrue, P. J., Zhu, H., Yu, J., Cashion, A. B., et al. (2006). Differential modulation of estrogen receptors (ERs) in ischemic brain injury: A role for ERalpha in estradiol-mediated protection against delayed cell death. Endocrinology, 147(6), 3076–3084. Dubal, D. B., Shughrue, P. J., Wilson, M. E., Merchenthaler, I., & Wise, P. M. (1999). Estradiol modulates bcl-2 in cerebral ischemia: A potential role for estrogen receptors. Journal of Neuroscience, 19, 6385–6393. Dueñas, M., Luqu´ın, S., Chowen, J. A., Torres-Alemán, I., Naftolin, F., & Garcia-Segura, L. M. (1994). Gonadal hormone regulation of insulin-like growth factor-I-like immunoreactivity in hypothalamic astroglia of developing and adult rats. Neuroendocrinology, 59, 528–538. Dueñas, M., Torres-Aleman, I., Naftolin, F., & Garcia-Segura, L. M. (1996). Interaction of insulin-like growth factor-1 and estradiol signaling pathways on hypothalamic neuronal differentiation. Neuroscience, 74, 531–539. Etgen, A. M. (2003). Ovarian steroid and growth factor regulation of female reproductive function involves modification of hypothalamic alpha 1-adrenoceptor signaling. Annals of the New York Academy of Sciences, 1007, 153–161. Etgen, A. M. (2008). Estrogens and alzheimer’s disease: Is cholesterol a link? Endocrinology, 149(9), 4253–4255. Etgen, A. M. & Acosta-Martinez, M. (2003). Participation of growth factor signal transduction pathways in estradiol facilitation of female reproductive behavior. Endocrinology, 144, 3828–3835. Etgen, A. M., González-Flores, O., & Todd, B. J. (2006). The role of insulin-like growth factor-I and growth factorassociated signal transduction pathways in estradiol and progesterone facilitation of female reproductive behaviors. Front. Neuroendocrinology, 27(4), 363–375. Etgen, A. M., Ungar, S., & Petitti, N. (1992). Estradiol and progesterone modulation of norepinephrine neurotransmission: Implications for the regulation of female reproductive behavior. Journal of Neuroendocrinology, 31, 799–807. Fernandez, A. M., Carro, E. M., Lopez-Lopez, C., & TorresAleman, I. (2005). Insulin-like growth factor I treatment for cerebellar ataxia: Addressing a common pathway in the pathological cascade? Brain Research. Brain Research Reviews, 50, 134–141. Fernandez, S., Fernandez, A. M., Lopez-Lopez, C., & TorresAleman, I. (2007). Emerging roles of insulin-like growth factor-I in the adult brain. Growth Hormone & IGF Research, 17, 89–95. Fernandez, A. M., de la Vega, A. G., Planas, B., & TorresAleman, I. (1999). Neuroprotective actions of peripherally administered insulin-like growth factor I in the injured olivocerebellar pathway. European Journal of Neuroscience, 11, 2019–2030.

Fernandez, A. M., de la Vega, A. G., & Torres-Aleman, I. (1998). Insulin-like growth factor I restores motor coordination in a rat model of cerebellar ataxia. Proceedings of the National Academy of Sciences United States of America, 95, 1253–1258. Fernandez-Galaz, M. C., Morschl, E., Chowen, J. A., TorresAleman, I., Naftolin, F., & Garcia-Segura, L. M. (1997). Role of astroglia and insulin-like growth factor-I in gonadal hormone-dependent synaptic plasticity. Brain Research Bulletin, 44, 525–531. Fernandez-Galaz, M. C., Naftolin, F., & Garcia-Segura, L. M. (1999). Phasic synaptic remodelling of the arcuate nucelus during the estrous cycle depends on insulin-like growth factor-I receptor activation. Journal of Neuroscience Research, 55, 286–292. Fernandez-Galaz, M. C., Torres-Alemán, I., & Garcia-Segura, L. M. (1996). Endocrine-dependent accumulation of IGF-I by hypothalamic glia. NeuroReport, 8, 373–377. Font de Mora, J. & Brown, M. (2000). AIB1 is a conduit for kinase-mediated growth factor signaling to the estrogen receptor. Molecular and Cellular Biology, 20, 5041–5047. Gao, Q., Mezei, G., Nie, Y., Rao, Y., Choi, C. S., Bechmann, I., et al. (2007). Anorectic estrogen mimics leptin’s effect on the rewiring of melanocortin cells and Stat3 signaling in obese animals. Nature Medicine, 13, 89–94. Garcia-Estrada, J., Garcia-Segura, L. M., & Torres-Aleman, I. (1992). Expression of insulin-like growth factor I by astrocytes in response to injury. Brain Research, 592, 343–347. Garcia-Ovejero, D., Veiga, S., Garcia-Segura, L. M., & DonCarlos, L. L. (2002). Glial expression of estrogen and androgen receptors after rat brain injury. The Journal of Comparative Neurology, 450, 256–271. Garcia-Segura, L. M. (2008). Aromatase in the brain: Not just for reproduction anymore. Journal of Neuroendocrinology, 20(6), 705–712. Garcia-Segura, L. M., Cardona-Gomez, G. P., Chowen, J. A., & Azcoitia, I. (2000). Insulin-like growth factor-I receptors and estrogen receptors interact in the promotion of neuronal survival and neuroprotection. Journal of Neurocytology, 29, 425–437. Garcia-Segura, L. M., Cardona-Gomez, P., Naftolin, F., & Chowen, J. A. (1998). Estradiol upregulates Bcl-2 expression in adult brain neurons. NeuroReport, 9, 593–597. Garcia-Segura, L. M., Chowen, J. A., Párducz, A., & Naftolin, F. (1994). Gonadal hormones as promoters of structural synaptic plasticity: Cellular mechanisms. Progress in Neurobiology, 44, 279–307. Garcia-Segura, L. M., Naftolin, F., Hutchison, J. B., Azcoitia, I., & Chowen, J. A. (1999). Role of astroglia in estrogen regulation of synaptic plasticity and brain repair. Journal of Neurobiology, 40, 574–584. Garcia-Segura, L. M., Rodriguez, J. R., & Torres-Alemán, I. (1997). Localization of the insulin-like growth factor I receptor in the cerebellum and hypothalamus of adult rats: An electron microscopic study. Journal of Neurocytology, 26, 479–490.

268 Garcia-Segura, L. M., Sanz, A., & Mendez, P. (2006). Crosstalk between IGF-I and estradiol in the brain: Focus on neuroprotection. Neuroendocrinology, 84, 275–279. Garrido, J. J., Simon, D., Varea, O., & Wandosell, F. (2007). GSK3 alpha and GSK3 beta are necessary for axon formation. FEBS Letters, 581, 1579–1586. Ghisletti, S., Meda, C., Maggi, A., & Vegeto, E. (2005). 17bEstradiol inhibits inflammatory gene expression by controlling NF-kB intracellular localization. Molecular and Cellular Biology, 25, 2957–2968. Giannini, S., Benvenuti, S., Luciani, P., Manuelli, C., Cellai, I., Deledda, C., et al. (2008). Intermittent high glucose concentrations reduce neuronal precursor survival by altering the IGF system: The involvement of the neuroprotective factor DHCR24 (Seladin-1). Journal of Endocrinology, 198(3), 523–532. Goodenough, S., Schleusner, D., Pietrzik, C., Skutella, T., & Behl, C. (2005). Glycogen synthase kinase 3beta links neuroprotection by 17beta-estradiol to key Alzheimer processes. Neuroscience, 132, 581–589. Greeve, I., Hermans-Borgmeyer, I., Brellinger, C., Kasper, D., Gomez-Isla, T., Behl, C., et al. (2000). The human DIMINUTO/DWARF1 homolog seladin-1 confers resistance to Alzheimer’s disease-associated neurodegeneration and oxidative stress. Journal of Neuroscience, 20(19), 7345–7352. Gruber, C. J., Gruber, D. M., Gruber, I. M., Wieser, F., & Huber, J. C. (2004). Anatomy of the estrogen response element. Trends in Endocrinology and Metabolism, 15, 73–78. Guan, J., Bennet, L., Gluckman, P. D., & Gunn, A. J. (2003). Insulin-like growth factor-1 and post-ischemic brain injury. Progress in Neurobiology, 70, 443–462. Guan, J., Williams, C., Gunning, M., Mallard, C., & Gluckman, P. (1993). The effects of IGF-1 treatment after hypoxicischemic brain injury in adult rats. Journal of Cerebral Blood Flow and Metabolism, 13, 609–616. Guerra, B., Diaz, M., Alonso, R., & Marin, R. (2004). Plasma membrane oestrogen receptor mediates neuroprotection against beta-amyloid toxicity through activation of Raf-1/ MEK/ERK cascade in septal-derived cholinergic SN56 cells. Journal of Neurochemistry, 91, 99–109. Herbison, A. E. (1997). Noradrenergic regulation of cyclic GnRH secretion. Reviews of Reproduction, 2, 1–6. Hernandez-Sanchez, C., Mansilla, A., de la Rosa, E. J., & de Pablo, F. (2006). Proinsulin in development: New roles for an ancient prohormone. Diabetologia, 49, 1142–1150. Heverin, M., Meaney, S., Lutjohann, D., Diczfalusy, U., Wahren, J., & Bjorkhem, I. (2005). Crossing the barrier: Net flux of 27-hydroxycholesterol into the human brain. The Journal of Lipid Research, 46, 1047–1052. Hiney, J. K., Srivastava, V. K., Dearth, R. K., & Dees, W. L. (2004). Influence of estradiol on insulin-like growth factor-1induced luteinizing hormone secretion. Brain Research, 1012, 91–97. Hiney, J. K., Srivastava, V., Nyberg, C. L., Ojeda, S. R., & Dees, W. L. (1996). Insulin-like growth factor-I of peripheral

origin acts centrally to accelerate the initiation of female puberty. Endocrinology, 137, 3717–3727. Hiney, J. K., Srivastava, V. K., Pine, M. D., & Les Dees, W. (2009). Insulin-like growth factor-I activates KiSS-1 gene expression in the brain of the prepubertal female rat. Endocrinology, 150(1), 376–384. Honda, K., Sawada, H., Kihara, T., Urushitani, M., Nakamizo, T., Akaike, A., et al. (2000). Phosphatidylinositol 3-kinase mediates neuroprotection by estrogen in cultured cortical neurons. Journal of Neuroscience Research, 60, 321–327. Horvath, T. L. (2005). The hardship of obesity: A soft-wired hypothalamus. Nature Neuroscience, 8, 561–565. Horvath, T. L. (2006). Synaptic plasticity in energy balance regulation. Obesity (Silver Spring), 14(Suppl. 5), 228S–233S. Hsieh, J., Aimone, J. B., Kaspar, B. K., Kuwabara, T., Nakashima, K., & Gage, F. H. (2004). IGF-1 instructs multipotent adult neural progenitor cells to become oligodendrocytes. The Journal of Cell Biology, 164, 111–122. Hwang, I. K., Yoo, K. Y., Park, S. K., An, S. J., Lee, Y. L., Choi, S. Y., et al. (2004). Expression and changes of endogenous insulin-like growth factor-1 in neurons and glia in the gerbil hippocampus and dentate gyrus after ischemic insult. Neurochemistry International, 45, 149–156. Iivonen, S., Hiltunen, M., Alafuzoff, I., Mannermaa, A., Kerokoski, P., Puoliväli, J., et al. (2002). Seladin-1 transcription is linked to neuronal degeneration in Alzheimer’s disease. Neuroscience, 113, 301–310. Isgor, C. & Watson, S. J. (2005). Estrogen receptor alpha and beta mRNA expressions by proliferating and differentiating cells in the adult rat dentate gyrus and subventricular zone. Neuroscience, 134, 847–856. Ishunina, T. A., Fischer, D. F., & Swaab, D. F. (2007). Estrogen receptor alpha and its splice variants in the hippocampus in aging and Alzheimer’s disease. Neurobiology of Aging, 28 (11), 1670–1681. Jiang, J., McMurtry, J., Niedzwiecki, D., & Goldman, S. A. (1998). Insulin-like growth factor-1 is a radial cell-associated neurotrophin that promotes neuronal recruitment from the adult songbird edpendyma/subependyma. Journal of Neurobiology, 36, 1–15. Jick, H., Zornberg, G. L., Jick, S. S., Seshadri, S., & Drachman, D. A. (2000). Statins and the risk of dementia. Lancet, 356, 1627–1631. Jones, D. M., Tucker, B. A., Rahimtula, M., & Mearow, K. M. (2003). The synergistic effects of NGF and IGF-1 on neurite growth in adult sensory neurons: Convergence on the PI 3kinase signaling pathway. Journal of Neurochemistry, 86, 1116–1128. Jover-Mengual, T., Zukin, R. S., & Etgen, A. M. (2007). MAPK signaling is critical to estradiol protection of CA1 neurons in global ischemia. Endocrinology, 148(3), 1131–1143. Kane, L. P., Shapiro, V. S., Stokoe, D., & Weiss, A. (1999). Induction of NF-kappaB by the Akt/PKB kinase. Current Biology, 9, 601–604. Karkanias, G. B., Ansonoff, M. A., & Etgen, A. M. (1996). Estradiol regulation of alpha 1b-adrenoceptor mRNA in

269 female rat hypothalamus-preoptic area. Journal of Neuroendocrinology, 8, 449–455. Kelly, M. J. & Rønnekleiv, O. K. (2008). Membrane-initiated estrogen signaling in hypothalamic neurons. Molecular and Cellular Endocrinology, 290, 14–23. Kivipelto, M., Helkala, E. L., Hanninen, T., Laakso, M. P., Hallikainen, M., Alhainen, K., et al. (2001). Midlife vascular risk factors and late-life mild cognitive impairment: A population-based study. Neurology, 56, 1683–1689. Klotz, D. M., Hewitt, S. C., Ciana, P., Raviscioni, M., Lindzey, S. C., Foley, J., et al. (2002). Requirement of estrogen receptor-alpha in insulin-like growth factor-1 (IGF-1)-induced uterine responses and in vivo evidence for IGF-1/estrogen receptor cross-talk. The Journal of Biological Chemistry, 277, 8531–8537. Kops, G. J., de Ruiter, N. D., De Vries-Smits, A. M., Powell, D. R., Bos, J. L., & Burgering, B. M. (1999). Direct control of the Forkhead transcription factor AFX by protein kinase B. Nature, 398, 630–634. Kuroki, Y., Fukushima, K., Kanda, Y., Mizuno, K., & Watanabe, Y. (2001). Neuroprotection by estrogen via extracellular signal-regulated kinase against quinolinic acid-induced cell death in the rat hippocampus. European Journal of Neuroscience, 13, 472–476. Kushner, P. J., Agard, D. A., Greene, G. L., Scanlan, T. S., Shiau, A. K., Uht, R. M., et al. (2000). Estrogen receptor pathways to AP-1. The Journal of Steroid Biochemistry and Molecular Biology, 74(5), 311–317. Laurino, L., Wang, X. X., de la Houssaye, B. A., Sosa, L., Dupraz, S., Caceres, A., et al. (2005). PI3K activation by IGF-1 is essential for the regulation of membrane expansion at the nerve growth cone. Journal of Cell Science, 118, 3653– 3662. LeRoith, D. (2008). Clinical relevance of systemic and local IGF-I: Lessons from animal models. Pediatric Endocrinology Reviews, 5(Suppl. 2), 739–743. Lebesgue, D., Chevaleyre, V., Zukin, R. S., & Etgen, A. M. (2009). Estradiol rescues neurons from global ischemiainduced cell death: Multiple cellular pathways of neuroprotection. Steroids, 74(7), 555–561. Lichtenwalner, R. J., Forbes, M. E., Bennett, S. A., Lynch, C. D., Sonntag, W. E., & Riddle, D. R. (2001). Intracerebroventricular infusion of insulin-like growth factor-I ameliorates the age-related decline in hippocampal neurogenesis. Neuroscience, 107, 603–613. Lopez-Lopez, C., Dietrich, M. O., Metzger, F., Loetscher, H., & Torres-Aleman, I. (2007). Disturbed cross talk between insulin-like growth factor I and AMP-activated protein kinase as a possible cause of vascular dysfunction in the amyloid precursor protein/presenilin 2 mouse model of Alzheimer’s disease. Journal of Neuroscience, 27(4), 824–831. Lopez-Lopez, C., LeRoith, D., & Torres-Aleman, I. (2004). Insulin-like growth factor I is required for vessel remodeling in the adult brain. Proceedings of the National Academy of Sciences of the United States of America, 101(26), 9833–9838. Lovestone, S., Reynolds, C. H., Latimer, D., Davis, D. R., Anderton, B. H., Gallo, J. M., et al. (1994). Alzheimer’s

disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells. Current Biology, 4, 1077–1086. Luciani, P., Deledda, C., Rosati, F., Benvenuti, S., Cellai, S., Dichiara, F., et al. (2008). Seladin-1 is a fundamental mediator of the neuroprotective effects of estrogen in human neuroblast long-term cell cultures. Endocrinology, 149, 4256–4266. Ma, Z. Q., Santagati, S., Patrone, C., Pollio, G., Vegeto, E., & Maggi, A. (1994). Insulin-like growth factors activate estrogen receptor to control the growth and differentiation of the human neuroblastoma cell line SK-ER3. Molecular Endocrinology, 8, 910–918. Mannella, P. & Brinton, R. D. (2006). Estrogen receptor protein interaction with phosphatidylinositol 3-kinase leads to activation of phosphorylated Akt and extracellular signalregulated kinase 1/2 in the same population of cortical neurons: A unified mechanism of estrogen action. Journal of Neuroscience, 26, 9439–9447. Marin, R., Diaz, M., Alonso, R., Sanz, A., Arévalo, M. A., & Garcia-Segura, L. M. (2009). Role of estrogen receptor alpha in membrane-initiated signaling in neural cells: Interaction with IGF-1 receptor. The Journal of Steroid Biochemistry and Molecular Biology, 114, 2–7. Marin, R., Guerra, B., Alonso, R., Ramirez, C. M., & Diaz, M. (2005). Estrogen activates classical and alternative mechanisms to orchestrate neuroprotection. Current Neurovascular Research, 2, 287–301. Martin, M. B., Franke, T. F., Stoica, G. E., Chambon, P., Katzenellenbogen, B. S., Stoica, B. A., et al. (2000). A role for Akt in mediating the estrogenic functions of epidermal growth factor and insulin-like growth factor I. Endocrinology, 141, 4503–4511. Mendez, P., Azcoitia, I., & Garcia-Segura, L. M. (2003). Estrogen receptor alpha forms estrogen-dependent multimolecular complexes with insulin-like growth factor receptor and phosphatidylinositol 3-kinase in the adult rat brain. Brain Research. Molecular Brain Research, 112, 170–176. Mendez, P., Azcoitia, I., & Garcia-Segura, L. M. (2005). Interdependence of oestrogen and insulin-like growth factor-I in the brain: Potential for analysing neuroprotective mechanisms. Journal of Endocrinology, 185, 11–17. Mendez, P. & Garcia-Segura, L. M. (2006). Phosphatidyl inositol 3 kinase (PI3K) and glycogen synthase kinase 3 (GSK3) regulate estrogen receptor mediated transcription in neuronal cells. Endocrinology, 147, 3027–3039. Mermelstein, P. G. (2009). Membrane-localised oestrogen receptor alpha and beta influence neuronal activity through activation of metabotropic glutamate receptors. Journal of Neuroendocrinology, 21(4), 257–262. Micevych, P. & Dominguez, R. (2009). Membrane estradiol signaling in the brain. Frontiers in Neuroendocrinology, 30, 315–327. Micevych, P. E. & Mermelstein, P. G. (2008). Membrane estrogen receptors acting through metabotropic glutamate receptors: An emerging mechanism of estrogen action in brain. Molecular Neurobiology, 38, 66–77.

270 Naftolin, F., Garcia-Segura, L. M., Horvath, T. L., Zsarnovszky, A., Demir, N., Fadiel, A., et al. (2007). Estrogeninduced hypothalamic synaptic plasticity and pituitary sensitization in the control of the estrogen-induced gonadotrophin surge. Reproduction Science, 14, 101–116. Nilsen, J. & Diaz Brinton, R. (2003). Mechanism of estrogen-mediated neuroprotection: Regulation of mitochondrial calcium and Bcl-2 expression. Proceedings of the National Academy of Sciences United States of America, 100, 2842–2847. Ogiue-Ikeda, M., Tanabe, N., Mukai, H., Hojo, Y., Murakami, G., Tsurugizawa, T., et al. (2008). Rapid modulation of synaptic plasticity by estrogens as well as endocrine disrupters in hippocampal neurons. Brain Research Reviews, 57(2), 363–375. O’Kusky, J. R., Ye, P., & D’Ercole, A. J. (2003). Increased expression of insulin-like growth factor I augments the progressive phase of synaptogenesis without preventing synapse elimination in the hypoglossal nucleus. The Journal of Comparative Neurology, 464, 382–391. Olmos, G., Naftolin, F., Perez, J., Tranque, P. A., & GarciaSegura, L. M. (1989). Synaptic remodeling in the rat arcuate nucleus during the estrous cycle. Neuroscience, 32, 663–667. Ormerod, B. K. & Galea, L. A. (2001). Reproductive status influences cell proliferation and cell survival in the dentate gyrus of adult female meadow voles: A possible regulatory role for estradiol. Neuroscience, 102, 369–379. Ormerod, B. K., Lee, T. T.-Y., & Galea, L. A.M. (2003). Estradiol initially enhances but subsequently suppresses (via adrenal steroids) granula cell proliferation in the dentate gyrus of adult female rats. Journal of Neurobiology, 55, 247–260. Otaegi, G., Yusta-Boyo, M. J., Vergano-Vera, E., MendezGomez, H. R., Carrera, A. C., Abad, J. L., et al. (2006). Modulation of the PI 3-kinase-Akt signaling pathway by IGF-I and PTEN regulates the differentiation of neural stem/precursor cells. Journal of Cell Science, 119, 2739–2748. Ozdinler, P. H. & Macklis, J. D. (2006). IGF-I specifically enhances axon outgrowth of corticospinal motor neurons. Nature Neuroscience, 9, 1371–1381. Pappolla, M. A., Bryant-Thomas, T. K., Herbert, D., Pacheco, J., Fabra, G. M., Manjon, M., et al. (2003). Mild hypercholesterolemia is an early risk factor for the development of Alzheimer amyloid pathology. Neurology, 61, 199–205. Parducz, A., Hoyk, Z., Kis, Z., & Garcia-Segura, L. M. (2002). Hormonal enhancement of neuronal firing is linked to structural remodelling of excitatory and inhibitory synapses. European Journal of Neuroscience, 16(4), 665–670. Patrone, C., Gianazza, E., Santagati, S., Agrati, P., & Maggi, A. (1998). Divergent pathways regulate ligand-independent activation of ERa in SK-N-BE neuroblastoma and COS-1 renal carcinoma cells. Molecular Endocrinology, 12, 835–841. Patrone, C., Ma, Z. Q., Pollio, G., Agrati, P., Parker, M. G., & Maggi, A. (1996). Cross-coupling between insulin and

estrogen receptor in human neuroblastoma cells. Molecular Endocrinology, 10, 499–507. Pedram, A., Razandi, M., & Levin, E. R. (2006). Nature of functional estrogen receptors at the plasma membrane. Molecular Endocrinology, 20(9), 1996–2009. Peineau, S., Taghibiglou, C., Bradley, C., Wong, T. P., Liu, L., Lu, J., et al. (2007). LTP inhibits LTD in the hippocampus via regulation of GSK3beta. Neuron, 53, 703–717. Perez-Martin, M., Azcoitia, I., Trejo, J. L., Sierra, A., & Garcia-Segura, L. M. (2003). An antagonist of estrogen receptors blocks the induction of adult neurogenesis by insulin-like growth factor-I in the dentate gyrus of adult female rat. European Journal of Neuroscience, 18, 923–930. Peri, A., Danza, G., Benvenuti, S., Luciani, P., Deledda, C., Rosati, F., et al. (2009). New insights on the neuroprotective role of sterols and sex steroids: The seladin-1/ DHCR24 paradigm. Frontiers in Neuroendocrinology, 30 (2), 119–129. Peri, A. & Serio, M. (2008a). Neuroprotective effects of the Alzheimer’s disease-related gene seladin-1. Journal of Molecular Endocrinology, 41(5), 251–261. Peri, A. & Serio, M. (2008b). Estrogen receptor-mediated neuroprotection: The role of the Alzheimer’s disease-related gene seladin-1. Neuropsychiatric Disease and Treatment, 4 (4), 817–824. Petitti, N., Karkanias, G. B., & Etgen, A. M. (1992). Estradiol selectively regulates alpha 1B-noradrenergic receptors in the hypothalamus and preoptic area. Journal of Neuroscience, 12, 3869–3876. Pons, S. & Torres-Aleman, I. (1993). Estradiol modulates insulin-like growth factor I receptors and binding proteins in neurons from the hypothalamus. Neuroendocrinology, 5, 267–271. Popken, G. J., Hodge, R. D., Ye, P., Zhang, J., Ng, W., O’Kusky, J. R., et al. (2004). In vivo effects of insulin-like growth factor-I (IGF-I) on prenatal and early postnatal development of the central nervous system. European Journal of Neuroscience, 19, 2056–2068. Pugazhenthi, S., Nesterova, A., Sable, C., Heidenreich, K. A., Boxer, L. M., Heasley, L. E., et al. (2000). Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element-binding protein. The Journal of Biological Chemistry, 275, 10761–10766. Quesada, A. & Etgen, A. M. (2001). Insulin-like growth factor1 regulation of alpha(1)-adrenergic receptor signaling is estradiol dependent in the preoptic area and hypothalamus of female rats. Endocrinology, 142, 599–607. Quesada, A. & Etgen, A. M. (2002). Functional interactions between estrogen and insulin-like growth factor-I in the regulation of alpha 1B-adrenoceptors and female reproductive function. Journal of Neuroscience, 22, 2048–2401. Quesada, A., Lee, B. Y., & Micevych, P. E. (2008). PI3 kinase/ Akt activation mediates estrogen and IGF-1 nigral DA neuronal neuroprotection against a unilateral rat model of Parkinson’s disease. Developmental Neurobiology, 68(5), 632–644.

271 Quesada, A. & Micevych, P. E. (2004). Estrogen interacts with the IGF-1 system to protect nigrostriatal dopamine and maintain motoric behavior after 6-hydroxdopamine lesions. Journal of Neuroscience Research, 75, 107–116. Quesada, A., Romeo, H. E., & Micevych, P. (2007). Distribution and localization patterns of estrogen receptor-beta and insulin-like growth factor-1 receptors in neurons and glial cells of the female rat substantia nigra: Localization of ERbeta and IGF-1R in substantia nigra. The Journal of Comparative Neurology, 503(1), 198–208. Ram´ırez, C. M., González, M., D´ıaz, M., Alonso, R., Ferrer, I., Santpere, G., et al. (2009). VDAC and ERalpha interaction in caveolae from human cortex is altered in Alzheimer’s disease. Molecular and Cellular Neuroscience, 42(3), 172–183. Razandi, M., Alton, G., Pedram, A., Ghonshani, S., Webb, P., & Levin, E. R. (2003). Identification of a structural determinant necessary for the localization and function of estrogen receptor alpha at the plasma membrane. Molecular and Cellular Biology, 23(5), 1633–1646. Reinhardt, R. R. & Bondy, C. A. (1994). Insulin-like growth factors cross the blood-brain barrier. Endocrinology, 135, 1753–1761. Rodriguez, E. M., Blazquez, J. L., Pastor, F. E., Pelaez, B., Pena, P., Peruzzo, B., et al. (2005). Hypothalamic tanycytes: A key component of brain-endocrine interaction. International Review of Cytology, 247, 89–164. Russo, V. C., Gluckman, P. D., Feldman, E. L., & Werther, G. A. (2005). The insulin-like growth factor system and its pleiotropic functions in brain. Endocrine Reviews, 26, 916–943. Saldanha, C. J., Duncan, K. A., & Walters, B. J. (2009). Neuroprotective actions of brain aromatase. Frontiers in Neuroendocrinology, 30(2), 106–118. Sanz, A., Carrero, P., Pern´ıa, O., & Garcia-Segura, L. M. (2008). Pubertal maturation modifies the regulation of insulin-like growth factor-I receptor signaling by estradiol in the rat prefrontal cortex. Developmental Neurobiology, 68(8), 1018–1028. Sharma, S., Prasanthi, R. P.J., Schommer, E., Feist, G., & Ghribi, O. (2008). Hypercholesterolemia-induced Abeta accumulation in rabbit brain is associated with alteration in IGF-1 signaling. Neurobiology of Disease, 32(3), 426–432. Shughrue, P. J., Lane, M. V., & Merchenthaler, I. (1997). Comparative distribution of estrogen receptor-alpha and beta mRNA in the rat central nervous system. The Journal of Comparative Neurology, 388, 507–525. Simoncini, T., Hafezi-Moghadam, A., Brazil, D. P., Ley, K., Chin, W. W., & Liao, J. K. (2000). Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol3-OH kinase. Nature, 407, 538–541. Simpkins, J. W., Yi, K. D., & Yang, S. H. (2009). Role of protein phosphatases and mitochondria in the neuroprotective effects of estrogens. Frontiers in Neuroendocrinology, 30, 93–105. Smart, E. J., Ying, Y. S., Anderson, R. G., & Bloom, G. S. (1995). Hormonal regulation of caveolae internalization. The Journal of Cell Biology, 131, 1421–1433.

Sotgia, F., Rui, H., Bonuccelli, G., Mercier, I., Pestell, R. G., & Lisanti, M. P. (2006). Caveolin-1, mammary stem cells, and estrogen-dependent breast cancers. Cancer Research, 66(22), 10647–10651. Tanapat, P., Hastings, N. B., Reeves, A. J., & Gould, E. (1999). Estrogen stimulates a transient increase in the number of new neurons in the dentate gyrus of the adult female rat. Journal of Neuroscience, 19, 5792–5801. Tang, E. D., Nunez, G., Barr, F. G., & Guan, K. L. (1999). Negative regulation of the forkhead transcription factor FKHR by Akt. The Journal of Biological Chemistry, 274, 16741–16746. Todd, B. J., Fraley, G. S., Peck, A. C., Schwartz, G. J., & Etgen, A. M. (2007). Central insulin-like growth factor 1 receptors play distinct roles in the control of reproduction, food intake, and body weight in female rats. Biology of Reproduction, 77 (3), 492–503. Topalli, I. & Etgen, A. M. (2004). Insulin-like growth factor-I receptor and estrogen receptor crosstalk mediates hormoneinduced neurite outgrowth in PC12 cells. Brain Research, 1030, 116–124. Toran-Allerand, C. D., Ellis, L., & Pfenninger, K. H. (1988). Estrogen and insulin synergism in neurite growth enhancement in vitro: Mediation of steroid effects by interactions with growth factors? Brain Research, 469, 87–100. Toran-Allerand, C. D., Singh, M., & Sétáló, G. (1999). Novel mechanisms of estrogen action in the brain: New players in an old story. Frontiers in Neuroendocrinology, 20, 97–121. Torres-Aleman, I. (1999). Insulin-like growth factors as mediators of functional plasticity in the adult brain. Hormone and Metabolic Research, 31, 114–119. Torres-Aleman, I. (2008). Mouse models of Alzheimer’s dementia: Current concepts and new trends. Endocrinology, 149(12), 5952–5957. Traub, M. L., De Butte-Smith, M., Zukin, R. S., & Etgen, A. M. (2009). Oestradiol and IGF1 reduce cell loss after global ischemia in middle-aged female rats. Journal of Neuroendocrinology, 21(12), 1038–1044. Trejo, J. L., Carro, E., Garcia-Galloway, E., & Torres-Aleman, I. (2004). Role of insulin-like growth factor I signaling in neurodegenerative diseases. Journal of Molecular Medicine, 82, 156–162. Trejo, J. L., Carro, E., & Torres-Alemán, I. (2001). Circulating insulin-like growth factor I mediates exercise-induced increases in the number of new neurons in the adult hippocampus. Journal of Neuroscience, 21, 1628–1634. Trejo, J. L., Llorens-Mart´ın, M. V., & Torres-Alemán, I. (2008). The effects of exercise on spatial learning and anxiety-like behavior are mediated by an IGF-I-dependent mechanism related to hippocampal neurogenesis. Molecular and Cellular Neuroscience, 37, 402–411. van Praag, H., Kempermann, G., & Gage, F. H. (1999). Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nature Neuroscience, 2, 266–270.

272 Varea, O., Arevalo, M. A., Garrido, J. J., Garcia-Segura, L. M., Wandosell, F., & Mendez, P. (2009). Interaction of estrogen receptors with insulin-like growth factor-I and Wnt signaling in the nervous system. Steroids. Varea, O., Garrido, J. J., Dopazo, A., Mendez, P., GarciaSegura, L. M., & Wandosell, F. (2009). Estradiol activates beta-catenin dependent transcription in neurons. PLoS One, 4(4), e5153. Varela-Nieto, I., Morales-Garcia, J. A., Vigil, P., Diaz-Casares, A., Gorospe, I., Sanchez-Galiano, S., et al. (2004). Trophic effects of insulin-like growth factor-I (IGF-I) in the inner ear. Hearing Research, 196, 19–25. Vasudevan, N. & Pfaff, D. W. (2007). Membrane-initiated actions of estrogens in neuroendocrinology: Emerging principles. Endocrine Reviews, 28, 1–19. Vicario-Abejon, C., Yusta-Boyo, M. J., Fernandez-Moreno, C., & de Pablo, F. (2003). Locally born olfactory bulb stem cells proliferate in response to insulin-related factors and require endogenous insulin-like growth factor-I for differentiation into neurons and glia. Journal of Neuroscience, 23, 895–906. Walf, A. A. & Frye, C. A. (2008). Rapid and estrogen receptor beta mediated actions in the hippocampus mediate some functional effects of estrogen. Steroids, 73(9-10), 997–1007. Wang, R., Zhang, Q. G., Han, D., Xu, J., Lu, Q., & Zhang, G. Y. (2006). Inhibition of MLK3-MKK4/7-JNK1/2 pathway by Akt1 in exogenous estrogen-induced neuroprotection against transient global cerebral ischemia by a non-genomic mechanism in male rats. Journal of Neurochemistry, 99, 1543–1554. Wilson, M. E. (1998). Premature elevation in serum insulin-like growth factor-1 advances first ovulation in rhesus monkeys. Journal of Endocrinology, 158, 247–257. Wolozin, B. (2004). Cholesterol and the biology of Alzheimer’s disease. Neuron, 41, 7–10. Yao, M., Nguyen, T. V., & Pike, C. J. (2007). Estrogen regulates Bcl-w and Bim expression: Role in protection against

beta-amyloid peptide-induced neuronal death. Journal of Neuroscience, 27, 1422–1433. Ye, P. & D’Ercole, A. J. (2006). Insulin-like growth factor actions during development of neural stem cells and progenitors in the central nervous system. Journal of Neuroscience Research, 83, 1–6. Yu, X., Rajala, R. V., McGinnis, J. F., Li, F., Anderson, R. E., Yan, X., et al. (2004). Involvement of insulin/phosphoinositide 3-kinase/Akt signal pathway in 17 beta-estradiolmediated neuroprotection. The Journal of Biological Chemistry, 279, 13086–13094. Zeger, M., Popken, G., Zhang, J., Xuan, S., Lu, Q. R., Schwab, M. H., et al. (2007). Insulin-like growth factor type 1 receptor signaling in the cells of oligodendrocyte lineage is required for normal in vivo oligodendrocyte development and myelination. Glia, 55, 400–411. Zhang, L., Rubinow, D. R., Xaing, G., Li, B. S., Chang, Y. H., Maric, D., et al. (2001). Estrogen protects against beta-amyloid-induced neurotoxicity in rat hippocampal neurons by activation of Akt. NeuroReport, 12, 1919–1923. Zhang, X., Shen, P., Coleman, M., Zou, W., Boggie, B. W., Smith, L. M., et al. (2006). Caveolin-1 down-regulation activates estrogen receptor alpha expression and leads to 17beta-estradiol-stimulated mammary tumorigenesis. Anticancer Research, 25(1A), 369–375. Zhao, L., Wu, T. W., & Brinton, R. D. (2004). Estrogen receptor subtypes alpha and beta contribute to neuroprotection and increased Bcl-2 expression in primary hippocampal neurons. Brain Research, 1010, 22–34. Zhou, F. Q., Zhou, J., Dedhar, S., Wu, Y. H., & Snider, W. D. (2004). NGF-induced axon growth is mediated by localized inactivation of GSK-3beta and functions of the microtubule plus end binding protein APC. Neuron, 42, 897–912. Znamensky, V., Akama, K. T., McEwen, B. S., & Milner, T. A. (2003). Estrogen levels regulate the subcellular distribution of phosphorylated Akt in hippocampal CA1 dendrites. Journal of Neuroscience, 23, 2340–2347.

L. Martini (Eds.) Progress in Brain Research, Vol. 181 ISSN: 0079-6123 Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 15

A hormonal contraceptive for men: how close are we? Ilpo Huhtaniemi
Department of Surgery and Cancer, Imperial College London, Hammersmith Campus, London, United Kingdom

Abstract: Novel contraceptive methods for men are still not available, and the opinions about their need among experts and lay public are polarized between enthusiasm and scepticism. Of the different strategies, hormonal methods aimed at suppression of spermatogenesis have been most extensively studies, are most promising, and are the only approach with the potential of breakthrough in the near future. Their principle is to block pituitary gonadotropin secretion, which will eliminate the endocrine stimulus for testicular androgen production, thereby eliminating its support for spermatogenesis. Testosterone alone or in combination with progestin is the most promising lead. However, many obstacles still have to be overcome before a practical and acceptable method is available. The reasons for the slow progress are partly biological and partly practical and economical. It is difficult to design a method that would be effective in most men, have no side effects and be reversible, economical, and acceptable by all cultures. Unfortunately, the pharmaceutical industry is currently not participating in the development work, and the research in the field is suffering from lack of political and financial support. Ironically, with relative modest additional effort a hormonal contraceptive method for men would be available. We review in this chapter the main principles of hormonal male contraception, the results of the latest clinical trials and shed light on some future perspectives in the field. Keywords: male contraception; spermatogenesis; gonadotropins; sex steroids; testosterone; progestins

vasectomy, still no modern reversible contraceptive methods are available for men. Although widely used they are not optimal due to the condom’s limited user efficacy and vasectomy’s lack of reversibility. The current situation is a real missed opportunity in the quest for controlling the world population explosion, because now half of potential contraception users are left out. Although population overgrowth is not a concern of the developed world, the opportunity for men to participate more actively in family planning is an important gender equality issue. A novel male method would also provide contraception for couples who cannot use any of the currently available female methods, for example, during post-partum contraception.

Introduction World overpopulation remains one of the main challenges of mankind, and it even contributes to global warming by directly impinging on the level of the human carbon print. Besides sociopolitical measures, such as limiting family size by law and increasing the literacy rate of women, attempts to improve the quality and prevalence of usage of contraceptive methods are of major importance. Conspicuously, apart from condoms and


Corresponding author. Tel.: þ44-(0)20-75942104; Fax: þ44-(0)20-75942184; E-mail: [email protected]

DOI: 10.1016/S0079-6123(08)81015-1

273

274

than 60 years ago. Today, it is much more difficult to launch a new drug, and this in particular applies to contraceptives to be used by healthy individuals for many years.

There has always existed the popular cynicism and scepticism that if the ‘male pill’ were available, men would not use it and women would not trust the men. However, investigations have shown that if novel male methods were available they would be welcomed by the majority of men and women in all nationalities and religious groups (Anderson and Baird, 2002; Heinemann et al., 2005; Zhang et al., 2006). Moreover, only 2% of women in a stable relationship would not trust their partner to use the contraceptive (Glasier et al., 2000). The task to produce a new contraceptive for either sex is formidable, because it has to be highly effective, safe, reversible, easily accessible, inexpensive and culturally acceptable; it should not affect potency and libido; and it should be free from side effects. Biologically, the task with the male contraceptive is more challenging than it was for women. It was relatively easy to develop a hormone preparation that inhibited ovulation of a single oocyte once a month, whereas to stop the production or to inactivate sperm produced at a rate of 1000 per every heartbeat is much more difficult. Development of a male method has lagged behind for multiple reasons, which include the availability of safe and effective female methods and the purported reluctance of men to use contraceptive methods. Furthermore, the standards for safety and efficacy for new medications are now much stricter

Principles of hormonal male contraception One of the most widely tested principles of male contraception is the hormonal approach. It was discovered 70 years ago that treatment of men with testosterone (T) effectively suppresses their spermatogenesis (Heckel, 1939; McCullagh and McGurl, 1939). The explanation for this seemingly paradoxical response is in fact simple: when the circulating level of T increases by administration of exogenous hormone, enhanced negative feedback suppresses the secretion of the two pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (Figs. 1 and 2). LH is necessary for the stimulation of T production in testicular Leydig cells and for the maintenance of high intra-testicular level of this hormone, which is the most important hormone stimulating spermatogenesis (Fig. 3). FSH respectively enhances T action by maintaining the supporting function of Sertoli cells for spermatogenesis. The action of both gonadotropins is essential for qualitatively and quantitatively normal spermatogenesis. The anti-gonadotropic action of T can be boosted by

Hyp GnRH Analogs Testosterone Estradiol Progesterone

GnRH

Testosterone

GnRHR

Progestins

Pit

LH

FSH

LHR FSHR

Testes Testes

Inhibin

Fig. 1. The hypothalamic–pituitary–testicular axis, feedback loops of endogenous sex steroids (on the left) and sites of inhibitory actions of contraceptive hormones (on the right). Hyp = hypothalamus, Pit = pituitary gland.

275 4.0

4.0

LH (IU/L)

3.5

FSH (IU/L)

3.5

3.0

3.0

2.5

2.5

2.0

2.0

1.5

1.5

1.0

1.0

0.5

0.5

0.0

0.0 0

1

4

6

14

18

24

42 44 Week

FL 4 FL 8 FL 12 FL 16 FL 20 FL 24

0

1

4

6

14

18

24

42 44 Week

FL 4 FL 8 FL 12 FL 16 FL 20 FL 24

Fig. 2. The suppressive effect of treatment with T undecanoate/etonogestrel combination for 44 weeks on serum levels of LH and FSH. The two lines depict effects of two slightly different doses of the hormones. The suppression of gonadotropins is almost instantaneous, but their recovery following cessation of treatment occurs with some delay. The spermatogenic suppression in these subjects is depicted in Fig. 4. From Mommers et al. (2008) with permission.

Testosterone concentration

combining it with other anti-gonadotropic agents, such as gonadotropin-releasing hormone (GnRH) analogues and progestins (Fig. 1). Inclusion of androgen in the contraceptive regimen is necessary in order to maintain the normal extragonadal androgenic (potency and libido) and anabolic (muscle strength, bone density) effects of testicular

androgens. Suppression of spermatogenesis is reached with this type of contraceptive treatment in three to four months (Fig. 4), and it takes roughly the same time for sperm counts to return to the starting level after cessation of treatment. The slow onset of contraceptive efficacy is due to the biology of spermatogenesis with the 70-day

Steroid therapy Without androgenic effect

With androgenic effect

100 x Threshold testicular T level to maintain spermatogenesis

Add-back androgen

Testis Serum

Testis Serum

Testis Serum

Fig. 3. Schematic presentation of the 100-fold higher level of T in the testis tissue compared with peripheral serum (left) and the effect of anti-gonadotropic steroid treatment on these levels when the agent has no androgenic effect (middle) and when the agent is T (right). For the contraceptive efficacy it is important that the intra-testicular levels of T decrease below the threshold needed to maintain spermatogenesis. If the anti-gonadotropic treatment does not contain androgen, an addback dose of androgen (e.g. T) is needed to provide sufficient circulating androgen levels to maintain extragonadal androgen effects.

276 (a) 100

Percentage of men with a sperm concentration < cut off

90 80 70 60

Cut off:

50 40

< detection limit < =0.1 million per ml < =1 million per ml < =3 million per ml

30 20 10 0 0

6

12

16

20

24

30/34

42/44 Week

Fig. 4. An example of response of spermatogenesis to contraceptive treatment with androgen (T undecanoate i.m.) and progestin (etonogestrel implant) combination treatment. Spermatogenic suppression with sufficient contraceptive efficacy (sperm count <1 million/mL) is reached by about 90% in the men in 12 weeks. From Mommers et al. (2008) with permission.

spermatogenic cycle. The immature germ cells have to be washed off from the testis before the seminal fluid is free from residual sperm. Androgen treatment suppresses both gonadotropins, but apparently the suppression of LH, and LH-dependent T production, is crucial for the contraceptive effect. T can be considered the master switch of spermatogenesis, while FSH contributes to the quality and quantity of sperm. With the unravelling of structure and FSH-suppressing functions of inhibin (Fig. 1), there were high hopes that the principle of FSH suppression could be developed into a male contraceptive. However, neither men with inactivating FSH receptor mutation (Tapanainen et al., 1997) nor knockout mice for FSHb (Kumar et al., 1997) or FSH receptor (Abel et al., 2000) are azoospermic, and the inhibin/FSH approach has been largely abandoned. Although the hormonal approach to male contraception is promising, there are still multiple conceptual and practical obstacles before the final product is available for general use. A recent Cochrane Review on steroid hormones for contraception in men (Grimes et al., 2007) concluded: ‘No hormonal

birth control for men is ready for general use. Most trials were small pilot studies trying to differentiate hormone treatments. Larger trials with better methods are needed to test good leads in this area.’ Several comprehensive reviews on male hormonal contraception have recently summarized the older literature (Anderson and Baird, 2002; Grimes et al., 2007; Matthiesson and McLachlan, 2006; Page et al., 2008; Wenk and Nieschlag, 2006). We therefore concentrate below on the latest developments in the field and provide some predictions for future directions.

The various hormonal regimens tested for male contraception Testosterone alone Although the concept of T-based male contraception is old, the first seminal proof-of-concept trials on the use of T were carried out only about 20 years ago by the World Health Organization (Table 1) (WHO, 1990, 1996). It was established in these studies, using a treatment of 200 mg of T

Table 1. Male 'real-life' hormonal contraceptive efficacy trials without concomitant usage of a female method

Study

Number of couples

Failure to suppress þ dropouts

Became azoospermic N (%)

Completed efficacy phase N (%)

Pregnancies in efficacy phase N (%/person-year)

WHO (1990)

225

114

157 (57)

119 (53)

1 (0.8)

WHO (1996)

357

75

268 (75) 81 (23)b

280 (78)

4 (1.4)a

Gu et al. (2003) Turner et al. (2003)

305

9

296 (97)

280 (92)

1 (0.18)

55

4

49 (89) 2 (3.6)c

51 (93)

0 (0)

1045

43

1002 (96)d

733 (70)

9 (0.6)

Gu et al. (2009)

TE, testosterone enanthate; TU, testosterone undecanoate; DMPE, dimedroxyprogesterone acetate. a All pregnancies in oligozoospermic men. b Oligozoospermia 0.1–3 × 106 per mL in efficacy phase. c Oligozoospermia 0.1–1 × 106 per mL in efficacy phase. d Including severe oligozoospermia <1 × 106 per mL.

Treatment þ length of efficacy phase

Ethnicity

TE (200 mg/week) for 12 months TE (200 mg/week) for 12 months

Mixed

TU i.m. (500 mg/month) for six months DMPA (300 mg/3 month) þ 200 mg T implant for 12 months TU i.m. (500 mg/month) for 30 months

Chinese

Mixed

Caucasian

Chinese

278 Testosterone (T)

OH

O

OCO(CH2)5CH3

O

T enanthate

OH

OCO(CH2)9CH3

T undecanoate O

O

CH3

7α-methyl19-nor-T (MENT)

Fig. 5. The structure of testosterone (T), the two most widely used T esters (enanthate and undecanoate) used in the contraceptive trials, as well as a promising selective androgen receptor modulator (SARM), 7a-methyl-19-nor-T (MENT).

enanthate intra-muscular (i.m.) once weekly for 12 months (Fig. 5), that hormonal regimens to induce azoospermia can provide highly effective, sustained, and reversible male contraception with minimum side effects. The suppression of sperm counts to £3 × 106 per mL was originally considered sufficient for contraceptive efficacy, but the cutoff level was later reduced to £1 × 106 per mL (Nieschlag, 2009). Of the men completing the treatment, 89–100% became azoospermic in Chinese centres, but only 45–70% in those on Caucasian men. The better contraceptive efficacy of T treatment in Asian men has been documented in several subsequent studies, but its reasons remain unclear (see below). The insufficient contraceptive efficacy of T alone in Caucasian men prompted investigators to attempt combination of T with other antigonadotropic agents in Europe and North America (see below). The Chinese investigators have continued testing the T-alone regimen. A large phase III trial was recently published (Gu et al., 2009). In this study (Table 1), 500 mg of T undecanoate (U) i.m. (Fig. 5) was administered monthly for 30 months to 1045 men, of whom 733 (86%) completed the efficacy and follow-up phases. A total of 4.8% of the men failed to reach azoospermia

or severe oligozoospermia (<1 × 106 per mL) within six months. Post-suppression sperm rebound occurred in 1.3% of men, and nine pregnancies occurred in 1551 person-years of exposure during the 24-month efficacy phase. This resulted in a cumulative contraceptive failure rate of 1.1 per 100 men. The suppression of spermatogenesis was reversible in all but two subjects, and no serious side effects were reported. The excellent results of the Chinese trial may partly be due to the ethnicity of the study population, since good contraceptive efficacy of male hormonal regimens in South-East Asian populations has been a constant finding (Wenk and Nieschlag, 2006). Hence, the treatment was far superior to the 14% first-year failure rate of ‘typical use’ of condoms (Fu et al., 1999) and of 8% of female contraceptive pills (Kost et al., 2008; Trussell, 2004). It seems that T alone provides an efficacious method for male contraception in China, but in Caucasian populations, more effective means to suppress spermatogenesis are needed. Another key to the success of the Chinese study was apparently due to the T preparation used, i.m. TU dissolved in tea seed oil, which provided sustained release of hormone at injection intervals of 4

279

i.m. Testosterone enanthate

i.m. Testosterone undecanoate

Reference range

Serum testosterone

Peroral testosterone undecanoate

0

12 Hours

24

0

2 Weeks

4

0

2 Months

4

Fig. 6. Schematic presentation of pharmacokinetic profiles of the different modes of T administration. Peroral T undecanoate has to be administered several times a day to provide sufficient concentration. T enanthate has to be administered once a week for contraceptive purpose. T undecanoate in oil administered i.m. maintains constant androgen levels up to 12 weeks. Many side effects of T treatment have been ascribed to the unphysiologically high T levels immediately following administration of the short-acting regimens.

weeks. The earlier studies were carried out using T enanthate at one-week intervals (WHO, 1990, 1996). The 4-week interval of TU injections can be even extended up to 12 weeks if the hormone is dissolved in castor oil (Fig. 6), and an 8-week interval will be used in an ongoing WHO/CONRAD trial on TU and progestin.

Testosterone plus progestin Because of the sub-optimal efficacy of T alone in Caucasian men, several combination treatments have been tested to improve the level of suppression of gonadotropins and spermatogenesis. The most promising of them have been the combinations of T with progestins, in which case the T dose can be reduced and enhanced by antigonadotropic effect of progestins and possibly also through their direct testicular effects (El-Hefnawy et al., 2000). Multiple T and progestin combinations have been tested, and it can be concluded that they do provide better suppression of spermatogenesis than T alone (Page et al., 2008). The progestins tested have included oral and implant forms of levonorgestrel, oral

desogesterel, oral and implant etonogestrel (ENG), norethisterone enanthate, and medroxyprogesterone acetate (Fig. 7). The anti-androgenic effect of some progestins (cyproterone acetate, denogest) may further improve their anti-spermatogenic efficacy by suppressing action of the residual testicular levels of T (Meriggiola et al., 1996). The contraceptive effect of all progestins seems to be fairly similar, although the extent and spectrum of their side effects show some variance. The best androgen/progestin combinations reach about 90% contraceptive efficacy, and at the moment they seem as the most likely hormonal contraceptive applicable for Caucasian men. The seminal study on the T/progestin combination treatment was a pharmaceutical industry-driven male hormonal contraceptive trial conducted in collaboration with Organon-Schering-Plough and Merck-Schering (Mommers et al., 2008). A remarkable feature of this study was its doubleblind nature, which provided for the first time evidence-based information about the real nature and extent of side effects of hormonal contraceptives in men. The treatment entailed 354 Caucasian men who were treated with ENG implants combined with TU injections. Either low- or

280 CH3

CH3

C=O

O

HCOH H2C

O C CH3

Medroxprogesterone acetate

O

O CH

Levonorgestrel O

CH3

H3C H2C

H2C

CH3 OH

C=O C CH

OAc H2C

Desogestrel O

Cyproterone acetate

O Cl

Fig. 7. Examples of structures of progestins that have been used in male contraceptive trials.

high-release implants and two doses of TU were tested, including placebo implants and injections. Treatment duration was 42 or 44 weeks, with a 24-week post-treatment follow-up. Overall, gonadotropins (Fig. 2) and spermatogenesis (Fig. 4) were equally suppressed in all treatment groups, the latter reaching a sperm count £1 × 106 per mL at week 16 in 89% of men and in 94% of the high-release ENG groups. Only 3% of the participants never reached the levels of £1 × 106 per mL considered to provide effective contraception. Median recovery time of sperm to the levels of  20 × 106 per mL was 15 weeks. The treatment was well tolerated and the adverse effects included weight gain, mood changes, acne, sweating and libido change (see below). The findings of the Mommers et al. (2008) study were very important, because they were the first male hormonal contraceptive study where side effects were systematically monitored in a placebo-controlled fashion. It was concluded that the ENG implant/TU injection treatment was well tolerated and provided effective and reversible suppression of spermatogenesis. Admittedly, the implant/injection treatment regimen is cumbersome and unsuitable for wider use, but the

proof of principle was very important, that is, that sufficient contraceptive efficacy with a welltolerated hormonal treatment can be achieved. This study would have provided the stepping stone for further pharmaceutical development to achieve a more practical treatment mode with similar contraceptive efficacy. Unfortunately, both the pharmaceutical companies participating in the study were bought during the course of the study, and the new owners were not interested and/or motivated to develop the regimen further. It is at the moment uncertain how the promising concept can be developed into a final product in the absence of support from the industry. The remaining public sector agencies and committed academic researchers may not have the resources to complete the task.

Testosterone plus GnRH analogues GnRH agonists and antagonists have potent antigonadotropic action and are therefore logical agents to be tested for hormonal male contraception in combination with T. Agonists have not shown contraceptive efficacy, most likely because

281

of the rebound of FSH levels after initial suppression (Behre et al., 1992; Huhtaniemi et al., 1987). Although these compounds suppress circulating T to the castrate range, it is possible that the ‘addback’ level of T along with the residual constitutive T production and sustained FSH action provide an endocrine milieu able to support spermatogenesis. In contrast, GnRH antagonists achieving immediate and sustained suppression of both gonadotropins have proven efficacious in some (Pavlou et al., 1991) though not all (Bagatell et al., 1993) of the small trials so far conducted. A recent study with a newer GnRH antagonist, acyline, proved effective in providing prolonged and profound suppression of gonadotropins and T, and indicated that an injection twice a month may provide sufficient gonadotropin suppression for contraception (Herbst et al., 2004). Even longer-acting depot preparations of GnRH antagonists are now available for the treatment of prostate cancer (Crawford and Hou, 2009), and they might also be applicable for male contraception. The latest antagonist molecules have much reduced effect on histamine release and are therefore suitable for long-term treatments (Doehn et al., 2009). These compounds are potentially interesting alternatives for the progestin/T combination, but at least for the time being, their high price is prohibitive for their development into a widely accepted and affordable male contraceptive.

Other androgens The key questions in the selection of the androgen component to the contraceptive regimen are the intrinsic activity of the compound, its spectrum of effects, the duration of action and the route of administration. T is in principle the optimal molecule because it is the natural androgen and therefore the most logical ‘addback’ androgen to maintain the extragondal androgen effects. The drawback of T is its relatively low intrinsic bioactivity. T is produced by the testes at a rate of about 6 mg per day, and therefore rather high amounts of T have to be administered to maintain the normal circulating levels. This poses practical problems, because the solubility of T into the

injectable oil vehicles is limited, and for instance the TU injections in the hormonal contraception treatments have to be 4 mL (250 mg/mL castor oil), which makes them rather painful. It would be advantageous if the androgen treatment could be provided in a smaller volume of a more potent compound. T is not bioavailable through the peroral route because of its rapid metabolism in the alimentary tract and during the first passage through the liver. The 17a-alkylated T derivatives (old anabolic steroids) are orally active, but their hepatotoxicity excludes them from contraceptive use. The short duration of action of peroral TU (Fig. 6) makes it an inefficient and impractical alternative. New orally active steroidal and nonsteroidal androgens are being currently developed, and they may offer alternatives for the selection of contraceptive androgens (Jones et al., 2009). Selective androgen receptor modulators (SARMs), besides potentially having higher intrinsic biopotency, could provide the benefit of a desirable spectrum of tissue-specific actions. Because of the potential of long-term side effects of T especially on the prostate, prostate-sparing androgens with no 5a-reduction would be desirable. While maintaining their inhibitory action on gonadotropins and spermatogenesis, and supporting effects on potency, libido, and muscle and bone health, they would not have the potential side effect of promoting prostate growth. In contrast, the possibility of aromatization is considered an advantage because of its positive action on the bone. One potentially useful SARM is 7a-methyl-19-nortestosterone (MENT) (Fig. 5), which is aromatized but not 5a-reduced (LaMorte et al., 1994; von Eckardstein et al., 2003). Moreover, its biological activity exceeds that of T by about 10-fold, meaning that doses about 10% of the dose of T would be sufficient for the maintenance of androgenic activity in a contraceptive regimen. This would be a real advantage in the development of long-acting formulations. Promising preliminary findings on the possibility to replace T with MENT in contraceptive regimens have been obtained in two studies (von Eckardstein et al., 2003; Walton et al., 2007).

282

Prostate-sparing effects in animal experiments have been documented for some other SARMs, such as desoxymethyltestosterone and tetrahydrogestinone, and for some recently developed orally active steroidal (dimethandrolone) and non-steroidal SARMs (reviewed by Bhasin & Jasuja, 2009; Blithe, 2008; Page et al., 2008). An extra advantage of these compounds could be their partial progestin agonism, which might potentiate their anti-gonadotropic actions. Besides increased potency and/or favourable functional profile of the androgen, its duration of action is another important factor. The early hormonal contraceptive studies were carried out using T enanthate (Figs. 5 and 6), which had to be administered once a week to achieve sufficient concentrations for the contraceptive efficacy (WHO, 1990, 1996). T decanoate increases the injection intervals to 4–6 weeks (Brady et al., 2006). Sub-dermally implanted T pellets can be inserted every four to six months (Turner et al., 2003). Another treatment option is T gels, possibly combined with progestin. They seem to be acceptable to men despite the need of daily application (Page et al., 2006). A clear improvement was the advent of TU in injectable form (Figs. 5 and 6), which was used in the recent Chinese study at four-week intervals, but when dissolved in suitable oil it may be possible to prolong the injection intervals to 8–12 weeks (Zitzmann and Nieschlag, 2007). WHO has been developing another T ester, T buciclate, which showed excellent long-term depot effect suitable for male contraception (Rajalakshmi and Ramakrishnan, 1989). Unfortunately, its development is currently at halt because of lack of interest of industry to develop it further.

Remaining questions and problems Why do not all men suppress to azoospermia Effective suppression of intra-testicular (IT) T is the key to the anti-spermatogenic efficacy of a hormonal male contraceptive. ITT concentration is normally 2–2.5 mmol/L (Huhtaniemi et al., 1985; Matthiesson et al., 2005), which is 100-fold higher

than in peripheral circulation (Fig. 2). When gonadotropin secretion is blocked, ITT is suppressed by about 98%. The remaining 2%, about 50 nmol/ L, is still twice the normal peripheral concentration. If the normal ITT level is necessary for spermatogenesis, it is paradoxical why its suppression by 98% does not block spermatogenesis in more than two-thirds of Caucasian men. A logical explanation is that much lower testicular T level than normal is sufficient for spermatogenesis. The ITT level may be so high only because of the mundane reason that the testis is the site of T synthesis in the body. If much lower than normal levels of ITT can support spermatogenesis, then it is possible that the suppression of gonadotropins alone does not reduce T production enough to bring about azoospermia. There is in fact experimental evidence to support this contention. In LH receptor knockout mice, totally devoid of LH-stimulated T production, the constitutive gonadotropin-independent ITT level can maintain qualitatively full spermatogenesis (Zhang et al., 2003). These mice have very low, albeit measurable level of ITT, about 10 nmol/L, which is about 3% of that of control animals. As young adults the knockout mice are azoospermic, with spermatogenesis stopping at the round spermatid stage and failing to enter the androgen-dependent transit to elongating spermatids (Plant and Marshall, 2001). When reexamined at 12 months of age, surprisingly, qualitatively full spermatogenesis up to elongating spermatids could be observed in the knockout testes. However, if the mice were treated with anti-androgen between 6 and 12 months, to block action of the residual androgen, the progression of spermatogenesis beyond round spermatids was totally blocked. Hence, the gonadotropinindependent low T production can maintain spermatogenesis. The finding prompts the question whether more profound T suppression than achieved with gonadotropin blockage would be the key to consistent azoospermia in contraceptive treatments. In fact, we do not exactly know how low the human ITT has to be before spermatogenesis stops completely. Rodent data suggest that there may not be a threshold concentration at all, but a

283

linear correlation prevails between ITT and sperm production (Singh et al., 1995; Zirkin et al., 1989). The 50 nmol/L ITT level in gonadotropin-suppressed men (GnRH agonist for prostatic carcinoma and T for contraception) (Huhtaniemi et al., 1985; Matthiesson et al., 2005) is about twice the normal serum concentration. A similar level in rats in proportion to normal testicular T (20 nmol/L) clearly stimulates spermatogenesis (Zirkin et al., 1989), and in gonadotropin-deficient hpg mice, T treatment can induce qualitatively complete spermatogenesis at doses that do not produce measurable elevation in ITT (Singh et al., 1995). The crucial question whether simultaneous inhibition of spermatogenesis and maintenance of peripheral androgen actions is possible has never been addressed. Elimination of the residual ITT could be the key to consistent and complete suppression of spermatogenesis. One hypothesis is that non-suppressor men might more effectively convert their residual ITT to biologically more potent 5a-dihydrotestosterone (DHT), which would then maintain spermatogenesis even at low concentrations (Anderson et al., 1996). However, combination of 5a-reductase inhibitor to the T treatment was found ineffective in non-suppressor men (Kinniburgh et al., 2001; Matthiesson et al., 2005). Another possibility is that the residual gonadotropin-independent T production remains higher in non-suppressors, but measurements of ITT levels have not proven this (Page et al., 2007). Incomplete suppression of gonadotropins in nonresponders would be a logical explanation, but no proof for this has been obtained (Handelsman et al., 1995; Wallace et al., 1993). Differential dependence of spermatogenesis on hormonal stimulation (Handelsman et al., 1995) and pharmacogenetic differences in the rate of gonadotropin suppression (McLachlan et al., 2004) have also been suggested. In an integrated analysis of all clinical trials so far conducted, only two factors appeared clinically significant, that is, the use of progestins and ethnicity affected the treatment response (Liu et al., 2008). The latter study also concluded that conventional laboratory analyses are unlikely to identify the men who do not

suppress during contraceptive treatments. Progestin co-administration allows the use of smaller doses of T, which may reduce the ITT level below a threshold level needed in some men to maintain spermatogenesis. Of the different treatment regimens tested, the combination of T with the antiandrogen cyproterone acetate has been one of the most effective ones (Meriggiola et al., 2003), which suggests that elimination of the residual androgen action in the testis may really be critical. However, this particular regimen is not feasible because it may make the men ‘peripherally’ hypogonadal. A very recent study suggests that the higher fat mass in Caucasian men in comparison to Chinese men may after all contribute to their poorer gonadotropin suppression (Kornmann et al., 2009). Studies on genetic differences between suppressors and non-suppressors, for example, in the CAG repeat length of the androgen receptor exon 1 and CYP3A4, have yielded no reproducible findings (Eckardstein et al., 2002; Yu and Handelsman, 2001). One preliminary observation reported higher insulin-like factor 3 levels in nonsuppressor men (Amory et al., 2007). All in all, the reason for the variable suppression of spermatogenesis in the hormonal contraceptive trials remains enigmatic.

What are the short-term and long-term side effects The commonest short-term side effects of the contraceptive treatments include acne, weight gain, reduced testis size, increased haematocrite, adverse changes in blood lipids and changes in mood and sexual drive (Page et al., 2008). The anecdotal effects of androgens on aggression have not been observed. Potential long-term effects include atherogenic effects and increased risk of prostate cancer. However, no evidence for them exists, albeit the observation time in the treatment trials, usually up to 1–1.5 years, is too short. The longterm effects of progestins in men are even less well delineated, but effects on weight gain, suppression of high-density lipoprotein (HDL)-cholesterol, and increases in pro-inflammatory cytokines with increased cardiovascular risk may occur (Page et al., 2008). Altogether, the risks of androgen

284

and progestin treatment of men remain at the moment hypothetical, and the real ones can only be verified by long-term follow-up studies. Besides the hormones used, the route of their administration (peroral vs. injection/ transdermal) makes a difference in the side effect profile. The real drug-related side effects are difficult to decipher from most of the studies, because placebo controls have rarely been used. An exception is the recently conducted pharmaceutical industry-driven male contraceptive trial using T and progestin in a double-blind placebo-controlled fashion (Mommers et al., 2008). The drug-related, statistically significant adverse effects included acne (26 vs. 10%, active treatment vs. placebo), night sweating (27 vs. 8%), libido changes (usually increased; 13 vs. 0%) and weight gain (24 vs. 10%). No clinically relevant differences were found between active and placebo treatments in haematology, biochemistry, lipid parameters and prostate-specific antigen (PSA). These may reflect the route of administration of the hormones (sub-cutaneous and intra-muscular), which does not burden the liver. The study was not designed to monitor long-term safety, where potential adverse effects on the cardiovascular and prostate health are the main concerns. Whether the above, apparently objective side effects of male hormonal contraception are acceptable and/or possible to reduce by different hormone combinations remains to be studied. Conversely, we can also envision potential beneficial side effects of androgens in young men, which include increased lean body mass and decreased fat mass (Bhasin et al., 2001). They could in the long term prevent obesity, metabolic syndrome, osteoporosis and cardiovascular diseases. Because the contraceptive treatments will be given to young men for limited periods of time, it is not certain whether the temporary adverse lipid changes would have long-term harmful effects on diseases that normally appear tens of years later. However, this is a concern that needs due attention because in general very little is known about effects of progestin on men.

A small study confirmed the adverse effect of progestin on blood lipids in men (Herbst et al., 2003). They are in particular related to oral administration of progestins, due to their firstpass metabolism in the liver, which information may be important when developing the final contraceptive product. Concerning the effects of contraceptive steroids on inflammatory markers in men, associated with coronary heart disease, one study has reported that T þ progestin combination increased the level of the pro-inflammatory interleukin-6 (IL-6) and T alone decreased it (Zitzmann et al., 2005). A potentially important serious adverse effect in the long term is the increased incidence of prostate cancer. Although the growth of existing prostate cancer is dependent on androgens, the opinion whether the endogenous level of T or exogenously administered T correlates with the appearance of prostate cancer is controversial; no solid evidence for such a connection exists. Administration of even high levels of T to young men has not been found to increase significantly PSA levels (Cooper et al., 1996). Likewise, no consistent effects on prostate volume or PSA levels have been observed in the male contraceptive trials (Meriggiola et al., 2005). The information is still limited, and only long-term studies will reveal the real side effects of the hormonal contraceptive treatments.

What alternatives are there to improve the efficacy of hormonal contraception Increasing the androgen dose is clearly not the solution to improve the contraceptive efficacy because it would lead to increased side effects, and the concomitant increase in intra-testicular androgen levels could in fact reduce the effect. Therefore, combination of another anti-gonadotropic compound with minimal dose of T to maintain the extragonadal androgen effects is more likely to be successful, as the T þ progestin combinations demonstrate. But other alternatives should also be tested. As discussed above, a promising one is GnRH antagonist, but inhibiting the metabolism of T to the more active androgen,

285

DHT, did not bring improvement. Neither did combination of estrogen to T increase the azoospermia rate (Handelsman et al., 2000). A third alternative, not yet tested, is to block the residual testicular T production using an inhibitor of the 17b-hydroxysteroid dehydrogenase (17b-HSD) type III enzyme. This enzyme catalyzes the final distal step of T biosynthesis, that is, the conversion of androstenedione to T. It is envisioned that inhibition of this step would bring testicular T production to a complete halt, and would then have a more profound inhibiting effect on spermatogenesis than gonadotropin suppression alone. The author’s laboratory is currently testing this principle experimentally.

Conclusions and future perspectives Further innovations are needed to release the current stagnation in the field where androgen alone or androgen/progestin combinations are practically the only approaches being pursued in the development of a male contraceptive. One possibility is to test whether more effective suppression of ITT levels by enzyme inhibitors (see above) improves the contraceptive efficacy and reduces side effects. The availability of new targets through advances in genomic, proteomic and bioinformatics fields may reveal totally new and unexpected concepts, and finally a non-hormonal method may turn out to offer the best solution (Kopf, 2008). The number of genes with a crucial role in male fertility is vast, offering unlimited possibilities for intervention; 4% of the mouse genome are expressed in post-meiotic germ cells (Schultz et al., 2003). A very recent review identified 57 gene knockouts affecting spermatogenesis and 10 affecting fertilization (Naz et al., 2009). Knockout and transgenic RNAi mouse models are useful methods for target validation. The epididymis is a promising target, expressing numerous specific genes with crucial roles in sperm maturation (Sipila et al., 2009). The potential advantages of epididymal contraception are that it does not affect testicular endocrine or gametogenic function and its initiation and reversibility may be

much faster than those of the hormonal methods. If an epididymal gene product is ‘druggable’, that is, it is an ion channel, receptor, exchanger or enzyme, it may be possible to inhibit its function with small pharmacological molecules. Such a concept would lead to a very specific post-testicular contraceptive without the need to influence testicular function, either its hormone production or gametogenesis. Identification of epididymis-specific genes and unravelling of their significance in sperm maturation using knockout mouse models are likely to be important in this research (Naz et al., 2009; Sipila et al., 2009). Although biological therapeutics of larger molecular size, such as vaccines, antibodies, most peptides and proteins, might be effective, their high development and manufacturing cost would preclude them from becoming a widely available option. Despite the recognized need of novel contraceptives, also for men, as a global health issue, many practical hurdles besides the scientific ones still exist. From the industrial perspective, the current market of contraceptives is highly fragmented with several low-cost products easily available. With the development of a new product, patent protection, marketing issues, product liability and pricing have to be weighed against the researchand-development cost. They form the key practical equation that has to be solved between industry, academia and government in order that tangible progress can be made, and we would finally have the long sought-after ‘male pill’.

References Abel, M. H., Wootton, A. N., Wilkins, V., Huhtaniemi, I., Knight, P. G., & Charlton, H. M. (2000). The effect of a null mutation in the follicle-stimulating hormone receptor gene on mouse reproduction. Endocrinology, 141, 1795–1803. Amory, J. K., Page, S. T., Anawalt, B. D., Coviello, A. D., Matsumoto, A. M., & Bremner, W. J. (2007). Elevated end-of-treatment serum INSL3 is associated with failure to completely suppress spermatogenesis in men receiving male hormonal contraception. Journal of Andrology, 28, 548–554. Anderson, R. A. & Baird, D. T. (2002). Male contraception. Endocrine Reviews, 23, 735–762. Anderson, R. A., Wallace, A. M., & Wu, F. C. (1996). Comparison between testosterone enanthate-induced azoospermia

286 and oligozoospermia in a male contraceptive study. III. Higher 5 alpha-reductase activity in oligozoospermic men administered supraphysiological doses of testosterone. Journal of Clinical Endocrinology and Metabolism, 81, 902–908. Bagatell, C. J., Matsumoto, A. M., Christensen, R. B., Rivier, J. E., & Bremner, W. J. (1993). Comparison of a gonadotropin releasing-hormone antagonist plus testosterone (T) versus T alone as potential male contraceptive regimens. Journal of Clinical Endocrinology and Metabolism, 77, 427–432. Behre, H. M., Nashan, D., Hubert, W., & Nieschlag, E. (1992). Depot gonadotropin-releasing hormone agonist blunts the androgen-induced suppression of spermatogenesis in a clinical trial of male contraception. Journal of Clinical Endocrinology and Metabolism, 74, 84–90. Bhasin, S. & Jasuja, R. (2009). Selective androgen receptor modulators as function promoting therapies. Current Opinion in Clinical Nutrition and Metabolic Care, 12, 232–240. Bhasin, S., Woodhouse, L., & Storer, T. W. (2001). Proof of the effect of testosterone on skeletal muscle. The Journal of Endocrinology, 170, 27–38. Blithe, D. (2008). Male contraception: What is on the horizon? Contraception, 78, S23–S27. Brady, B. M., Amory, J. K., Perheentupa, A., Zitzmann, M., Hay, C. J., Apter, D., et al. (2006). A multicentre study investigating subcutaneous etonogestrel implants with injectable testosterone decanoate as a potential long-acting male contraceptive. Human Reproduction, 21, 285–294. Cooper, C. S., MacIndoe, J. H., Perry, P. J., Yates, W. R., & Williams, R. D. (1996). The effect of exogenous testosterone on total and free prostate specific antigen levels in healthy young men. The Journal of Urology, 156, 438–441, discussion 441–432. Crawford, E. D. & Hou, A. H. (2009). The role of LHRH antagonists in the treatment of prostate cancer. Oncology (Williston Park, New York), 23, 626–630. Doehn, C., Sommerauer, M., & Jocham, D. (2009). Degarelix for prostate cancer. Expert Opinion on Investigational Drugs, 18, 851–860. Eckardstein, S. V., Schmidt, A., Kamischke, A., Simoni, M., Gromoll, J., & Nieschlag, E. (2002). CAG repeat length in the androgen receptor gene and gonadotropin suppression influence the effectiveness of hormonal male contraception. Clinical Endocrinology, 57, 647–655. El-Hefnawy, T., Manna, P. R., Luconi, M., Baldi, E., Slotte, J. P., & Huhtaniemi, I. (2000). Progesterone action in a murine Leydig tumor cell line (mLTC-1), possibly through a nonclassical receptor type. Endocrinology, 141, 247–255. Fu, H., Darroch, J. E., Haas, T., & Ranjit, N. (1999). Contraceptive failure rates: New estimates from the 1995 national survey of family growth. Family Planning Perspectives, 31, 56–63. Glasier, A. F., Anakwe, R., Everington, D., Martin, C. W., van der Spuy, Z., Cheng, L., et al. (2000). Would women trust their partners to use a male pill? Human Reproduction, 15, 646–649. Grimes, D. A., Lopez, L. M., Gallo, M. F., Halpern, V., Nanda, K., & Schulz, K. F. (2007). Steroid hormones for

contraception in men. Cochrane Database of Systematic Reviews (Online), Apr 18; (2): CD004316. Gu, Y., Liang, X., Wu, W., Liu, M., Song, S., Cheng, L., et al. (2009). Multicenter contraceptive efficacy trial of injectable testosterone undecanoate in Chinese men. Journal of Clinical Endocrinology and Metabolism, 94, 1910–1915. Gu, Y.Q., Wang, X.H., Peng, L., Cheng, L.F., Huang. M.K., Huang, Z.J., & Zhang, G.Y. (2003). A munticenter contraceptive efficacy study of injectable testosterone undecanoate in healthy Chinese men. Journal of Clinical Endocrinology and Metabolism, 88, 562–568. Handelsman, D. J., Farley, T. M., Peregoudov, A., & Waites, G. M. (1995). Factors in nonuniform induction of azoospermia by testosterone enanthate in normal men. World Health Organization Task Force on Methods for the Regulation of Male Fertility. Fertility and Sterility, 63, 125–133. Handelsman, D. J., Wishart, S., & Conway, A. J. (2000). Oestradiol enhances testosterone-induced suppression of human spermatogenesis. Human Reproduction, 15, 672–679. Heckel, N. (1939). Production of oligospermia in a man by the use of testosterone propionate. Proceedings of the Society for Experimental Biology and Medicine, 40, 658–659. Heinemann, K., Saad, F., Wiesemes, M., White, S., & Heinemann, L. (2005). Attitudes toward male fertility control: Results of a multinational survey on four continents. Human Reproduction, 20, 549–556. Herbst, K. L., Anawalt, B. D., Amory, J. K., Matsumoto, A. M., & Bremner, W. J. (2003). The male contraceptive regimen of testosterone and levonorgestrel significantly increases lean mass in healthy young men in 4 weeks, but attenuates a decrease in fat mass induced by testosterone alone. Journal of Clinical Endocrinology and Metabolism, 88, 1167–1173. Herbst, K. L., Coviello, A. D., Page, S., Amory, J. K., Anawalt, B. D., & Bremner, W. J. (2004). A single dose of the potent gonadotropin-releasing hormone antagonist acyline suppresses gonadotropins and testosterone for 2 weeks in healthy young men. Journal of Clinical Endocrinology and Metabolism, 89, 5959–5965. Huhtaniemi, I., Nikula, H., & Rannikko, S. (1985). Treatment of prostatic cancer with a gonadotropin-releasing hormone agonist analog: Acute and long term effects on endocrine functions of testis tissue. Journal of Clinical Endocrinology and Metabolism, 61, 698–704. Huhtaniemi, I., Nikula, H., & Rannikko, S. (1987). Pituitarytesticular function of prostatic cancer patients during treatment with a gonadotropin-releasing hormone agonist analog. I. Circulating hormone levels. Journal of Andrology, 8, 355–362. Jones, A., Chen, J., Hwang, D. J., Miller, D. D., & Dalton, J. T. (2009). Preclinical characterization of a (S)-N-(4-cyano-3-trifluoromethyl-phenyl)-3-(3-fluoro,4-chlorophenoxy)-2hydroxy-2-methyl-propanamide: A selective androgen receptor modulator for hormonal male contraception. Endocrinology, 150, 385–395. Kinniburgh, D., Anderson, R. A., & Baird, D. T. (2001). Suppression of spermatogenesis with desogestrel and

287 testosterone pellets is not enhanced by addition of finasteride. Journal of Andrology, 22, 88–95. Kopf, G. S. (2008). Approaches to the identification of new nonhormonal targets for male contraception. Contraception, 78, S18–S22. Kornmann, B., Nieschlag, E., Zitzmann, M., Gromoll, J., Simoni, M., & von Eckardstein, S. (2009). Body fat content and testosterone pharmacokinetics determine gonadotropin suppression after intramuscular injections of testosterone preparations in normal men. Journal of Andrology, 30, 602–613. Kost, K., Singh, S., Vaughan, B., Trussell, J., & Bankole, A. (2008). Estimates of contraceptive failure from the 2002 national survey of family growth. Contraception, 77, 10–21. Kumar, T. R., Wang, Y., Lu, N., & Matzuk, M. M. (1997). Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nature Genetics, 15, 201–204. LaMorte, A., Kumar, N., Bardin, C. W., & Sundaram, K. (1994). Aromatization of 7 alpha-methyl-19-nortestosterone by human placental microsomes in vitro. The Journal of Steroid Biochemistry and Molecular Biology, 48, 297–304. Liu, P. Y., Swerdloff, R. S., Anawalt, B. D., Anderson, R. A., Bremner, W. J., Elliesen, J., et al. (2008). Determinants of the rate and extent of spermatogenic suppression during hormonal male contraception: An integrated analysis. Journal of Clinical Endocrinology and Metabolism, 93, 1774–1783. Matthiesson, K. L. & McLachlan, R. I. (2006). Male hormonal contraception: Concept proven, product in sight? Human Reproduction Update, 12, 463–482. Matthiesson, K. L., Stanton, P. G., O’Donnell, L., Meachem, S. J., Amory, J. K., Berger, R., et al. (2005). Effects of testosterone and levonorgestrel combined with a 5alphareductase inhibitor or gonadotropin-releasing hormone antagonist on spermatogenesis and intratesticular steroid levels in normal men. Journal of Clinical Endocrinology and Metabolism, 90, 5647–5655. McCullagh, E. & McGurl, F. (1939). Further observations on the clinical use of testosterone propionate. The Journal of Urology, 42, 1265–1267. McLachlan, R. I., Robertson, D. M., Pruysers, E., Ugoni, A., Matsumoto, A. M., Anawalt, B. D., et al. (2004). Relationship between serum gonadotropins and spermatogenic suppression in men undergoing steroidal contraceptive treatment. Journal of Clinical Endocrinology and Metabolism, 89, 142–149. Meriggiola, M. C., Bremner, W. J., Paulsen, C. A., Valdiserri, A., Incorvaia, L., Motta, R., et al. (1996). A combined regimen of cyproterone acetate and testosterone enanthate as a potentially highly effective male contraceptive. Journal of Clinical Endocrinology and Metabolism, 81, 3018–3023. Meriggiola, M. C., Costantino, A., Cerpolini, S., Bremner, W. J., Huebler, D., Morselli-Labate, A. M., et al. (2003). Testosterone undecanoate maintains spermatogenic suppression induced by cyproterone acetate plus testosterone undecanoate in normal men. Journal of Clinical Endocrinology and Metabolism, 88, 5818–5826.

Meriggiola, M. C., Costantino, A., Saad, F., D’Emidio, L., Morselli Labate, A. M., Bertaccini, A., et al. (2005). Norethisterone enanthate plus testosterone undecanoate for male contraception: Effects of various injection intervals on spermatogenesis, reproductive hormones, testis, and prostate. Journal of Clinical Endocrinology and Metabolism, 90, 2005–2014. Mommers, E., Kersemaekers, W. M., Elliesen, J., Kepers, M., Apter, D., Behre, H. M., et al. (2008). Male hormonal contraception: A double-blind, placebo-controlled study. Journal of Clinical Endocrinology and Metabolism, 93, 2572–2580. Naz, R. K., Engle, A., & None, R. (2009). Gene knockouts that affect male fertility: Novel targets for contraception. Frontiers in Bioscience, 14, 3994–4007. Nieschlag, E. (2009). Male hormonal contraception: Love’s labour’s lost? Journal of Clinical Endocrinology and Metabolism, 94, 1890–1892. Page, S. T., Amory, J. K., Anawalt, B. D., Irwig, M. S., Brockenbrough, A. T., Matsumoto, A. M., et al. (2006). Testosterone gel combined with depomedroxyprogesterone acetate is an effective male hormonal contraceptive regimen and is not enhanced by the addition of a GnRH antagonist. Journal of Clinical Endocrinology and Metabolism, 91, 4374–4380. Page, S. T., Amory, J. K., & Bremner, W. J. (2008). Advances in male contraception. Endocrine Reviews, 29, 465–493. Page, S. T., Kalhorn, T. F., Bremner, W. J., Anawalt, B. D., Matsumoto, A. M., & Amory, J. K. (2007). Intratesticular androgens and spermatogenesis during severe gonadotropin suppression induced by male hormonal contraceptive treatment. Journal of Andrology, 28, 734–741. Pavlou, S. N., Brewer, K., Farley, M. G., Lindner, J., Bastias, M. C., Rogers, B. J., et al. (1991). Combined administration of a gonadotropin-releasing hormone antagonist and testosterone in men induces reversible azoospermia without loss of libido. Journal of Clinical Endocrinology and Metabolism, 73, 1360–1369. Plant, T. M. & Marshall, G. R. (2001). The functional significance of FSH in spermatogenesis and the control of its secretion in male primates. Endocrine Reviews, 22, 764–786. Rajalakshmi, M. & Ramakrishnan, P. R. (1989). Pharmacokinetics and pharmacodynamics of a new long-acting androgen ester: Maintenance of physiological androgen levels for 4 months after a single injection. Contraception, 40, 399–412. Schultz, N., Hamra, F. K., & Garbers, D. L. (2003). A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets. Proceedings of the National Academy of Sciences of the United States of America, 100, 12201–12206. Singh, J., O’Neill, C., & Handelsman, D. J. (1995). Induction of spermatogenesis by androgens in gonadotropin-deficient (hpg) mice. Endocrinology, 136, 5311–5321. Sipila, P., Jalkanen, J., Huhtaniemi, I. T., & Poutanen, M. (2009). Novel epididymal proteins as targets for the development of post-testicular male contraception. Reproduction, 137, 379–389. Tapanainen, J. S., Aittomaki, K., Min, J., Vaskivuo, T., & Huhtaniemi, I. T. (1997). Men homozygous for an inactivating mutation of the follicle-stimulating hormone (FSH)

288 receptor gene present variable suppression of spermatogenesis and fertility. Nature Genetics, 15, 205–206. Trussell, J. (2004). Contraceptive failure in the United States. Contraception, 70, 89–96. Turner, L., Conway, A. J., Jimenez, M., Liu, P. Y., Forbes, E., McLachlan, R. I., et al. (2003). Contraceptive efficacy of a depot progestin and androgen combination in men. Journal of Clinical Endocrinology and Metabolism, 88, 4659–4667. von Eckardstein, S., Noe, G., Brache, V., Nieschlag, E., Croxatto, H., Alvarez, F., et al. (2003). A clinical trial of 7 alphamethyl-19-nortestosterone implants for possible use as a long-acting contraceptive for men. Journal of Clinical Endocrinology and Metabolism, 88, 5232–5239. Wallace, E. M., Gow, S. M., & Wu, F. C. (1993). Comparison between testosterone enanthate-induced azoospermia and oligozoospermia in a male contraceptive study. I: Plasma luteinizing hormone, follicle stimulating hormone, testosterone, estradiol, and inhibin concentrations. Journal of Clinical Endocrinology and Metabolism, 77, 290–293. Walton, M. J., Kumar, N., Baird, D. T., Ludlow, H., & Anderson, R. A. (2007). 7Alpha-methyl-19-nortestosterone (MENT) vs testosterone in combination with etonogestrel implants for spermatogenic suppression in healthy men. Journal of Andrology, 28, 679–688. Wenk, M. & Nieschlag, E. (2006). Male contraception: A realistic option? The European Journal of Contraception and Reproductive Health Care, 11, 69–80. World Health Organization (WHO) (1990). Contraceptive efficacy of testosterone-induced azoospermia in normal men. Lancet, 336, 955–959.

World Health Organization (WHO) (1996). Contraceptive efficacy of testosterone-induced azoospermia and oligozoospermia in normal men. Fertility and Sterility, 65, 821–829. Yu, B. & Handelsman, D. J. (2001). Pharmacogenetic polymorphisms of the AR and metabolism and susceptibility to hormone-induced azoospermia. Journal of Clinical Endocrinology and Metabolism, 86, 4406–4411. Zhang, F. P., Pakarainen, T., Poutanen, M., Toppari, J., & Huhtaniemi, I. (2003). The low gonadotropin-independent constitutive production of testicular testosterone is sufficient to maintain spermatogenesis. Proceedings of the National Academy of Sciences of the United States of America, 100, 13692–13697. Zhang, L., Shah, I. H., Liu, Y., Vogelsong, K. M., & Zhang, L. (2006). The acceptability of an injectable, once-a-month male contraceptive in China. Contraception, 73, 548–553. Zirkin, B. R., Santulli, R., Awoniyi, C. A., & Ewing, L. L. (1989). Maintenance of advanced spermatogenic cells in the adult rat testis: Quantitative relationship to testosterone concentration within the testis. Endocrinology, 124, 3043–3049. Zitzmann, M., Erren, M., Kamischke, A., Simoni, M., & Nieschlag, E. (2005). Endogenous progesterone and the exogenous progestin norethisterone enanthate are associated with a proinflammatory profile in healthy men. Journal of Clinical Endocrinology and Metabolism, 90, 6603–6608. Zitzmann, M. & Nieschlag, E. (2007). Androgen receptor gene CAG repeat length and body mass index modulate the safety of long-term intramuscular testosterone undecanoate therapy in hypogonadal men. Journal of Clinical Endocrinology and Metabolism, 92, 3844–3853.

Subject Index

acetylcholine receptors, 7 acute stress in hippocampal neurons, 40–41 adaptor protein p14 and MP1–MAPK signalling complex, 6 admixture of diffusion, 84–85 adrenocorticoids–corticosterone and weight gain, 23 adrenocorticotrophic hormone (ACTH), 43, 51, 80 24-hour span of, 117 secretion effects of ageing, gender and body adiposity on, 117 glucocorticoid ultradian rhythm, 117–118 secretion periodicity of, 117 advection in bloodstream, 84–85 agonist-driven effector response function, 86 agouti-related peptide (AgRP), 19 3a-hydroxysteroid dehydrogenase (3a-HSD) in human brain, 199–200 isoforms of, 199 in mammals, 199 20a-hydroxysteroid dehydrogenase (20a-HSD), 199–200 Akaike information criterion for optimal pulse-time set, 90–91 Alstrom syndrome, 25 Alzheimer’s disease development of, 237 seladin-1/DHCR24 expression in, 258–259 AMPARs protein, 239 androgeninsensitive syndrome (AIS), 188 angiotensin type 1A receptor (AT1A), 4 annexin feedback onto corticotropes, 82 anorexigenic pathway, 19 afferent peptidergic signals, 23 anterior pituitary-hormone concentrations yield 24-hour rhythmicity of, 83 anteroventral periventricular nucleus (AVPV), 58 anteroventral third ventricle (AV3V) region, 45

antidiuretic hormone(ADH), 80 apolipoprotein E (apoE), 222 apoptotic signalling pathways intrinsic and extrinsic apoptotic pathway, 144 appetite regulating network (ARN) appetite regulating signal pathways representation of hypothalamic sites associated with, 18 dynamics in feedback circuitry involved in, 19 approximate entropy (ApEn) calculation, 102–103 degree of pattern, 100 depiction of, 101 interpretability and reproducibility, 102 Kolmogorov–Sinai statistic, 100, 102 randomness, 100 utility of, 100 arachidonic acid (AA) and PGE2 release, 49 2-arachidonoylglycerol, 21 arcuate nucleus (ARC) of hypothalamus, 114 paraventricular nucleus (PVN) axis, 19 area postrema (AP), 45 aromatase knockout (ArKO) mouse model, 216 behavioral phenotypes in, 217–220 generation of, 216–217 aromatase, 209–210 aromatization process, 211 C19 androgen, conversion of, 211 NADPH-cytochrome P450 reductase, complex with, 211 deficiency in females and males, 216 estrogen biosynthesis, role in, 209–210 expression in central nervous system, 211, 213 in normal human tissues, 212 in peripheral tissues, 211 human aromatase (CYP19A1) gene, 214–215 multiple roles of 289

290

aromatase, 209–210 (Continued) ArKO brain and behavioral phenotypes, 217–220 aromatase knockout mice, 216–217 human aromatase deficiency, 216 regulation of aromatase activity, 215–216 Cyp19 gene expression regulation, 213–215 arrestin proteins barr1 and barr2, 4 molecular basis for endocytic adaptor function, 7 artificial light and melatonin production, 130–132 artificial neural-network approach, 100 arylalkylamine N-acetyltransferase (AANAT), 128 ascorbic acid (AA), 144 Ask1-dependent phosphorylated c-Jun NH2-terminal kinase (JNK3), 8 ATP-dependent chromatin remodelling complexes, 165–166 b2-adrenergic receptor (b2AR), 7 Bardet–Biedl Syndrome, 25 basal-secretion models, 83 b-catenin, 262–263 bed nucleus of stria terminalis (BnST), 47 betaendorphin, 80 3-beta-hydroxysterol delta-24-reductase (DHCR24), 258 3b-hydroxysteroid dehydrogenase (3b-HSD), 183, 194–195 3b-HSD mRNA in rat brain, 194–195 brain detection in, 194 in frog, 195 human brain areas, concentrations in, 195 isoforms in humans, 194 in peripheral nervous system of rodents, 195 17b-hydroxysteroid dehydrogenase (17b-HSD) in amphibians, 197 in mammalian brain, 196, 197 in mouse brain, 196 biological oscillations, 120–121 blood–brain barrier (BBB) cytokine interaction with neuronal elements and, 45

and IL-1b, 45 blood hormone concentrations decay, 85 bone morphogenic proteins (BMPs), 2–3 brain delayed EP4-dependent PGE2 response in, 51 EP3-dependent PGE2 response in, 49 expression of steroidogenic enzymes in, 194 3a-hydroxysteroid dehydrogenase, 199 20a-hydroxysteroid dehydrogenase, 199–200 5a-reductase, 198–199 3b-HSD, 194–195 11b-hydroxysteroid dehydrogenase, 201–202 17b-hydroxysteroid dehydrogenase, 196–198 cytochrome P450 aromatase, 200–201 cytochrome P450C17, 195–196 cytochrome P450 side-chain cleavage, 194 GPR54 expression at, 59 sexual differentiation and, 61 brain-derived neurotrophic factor (BDNF), 21, 252 cell survival and differentiation, role in, 6 breast cancers, in post-menopausal period, 189 5-bromo-2-deoxyuridine (BrdU), 256 brown adipose tissue (BAT), 19 mediated thermogenic energy expenditure, 21, 24 mRNA levels in, 21 caudal ventrolateral medulla (cVLM), 51 cell surface events initiation of signalling by G protein-regulated receptors, 4 receptor serine/threonine kinases, 2–4 receptor tyrosine kinases, 2 temporal and spatial regulation, 5 central nervous system (CNS), 35 PGE2 production in, 48–49 central nucleus of amygdala (CeA), 47 ceramide formation, 9 cerebral cortex development, role of ERs in, 235–236 cholecystokinin (CCK), 23 cholesterol SH synthesis from (see steroid hormones (SHs)) synthesis in brain, role of estradiol in, 258–259 chromatin, steroid hormone-induced covalent modifications DNA exists in euchromatin and heterochromatin, 38 loosening, 40

291

nucleosomes in, 39 initiation of transcription, 38 lordosis behavior, 38 mRNA and protein synthesis, 38 cigarette smoker, melatonin levels in, 130 circadian hormone secretion time scale, 83 circulating orexigen, 113 circumventricular organs (CVOs), 45 clathrin-coated (CC) pits GPCR–barrestin–Src complexes in, 7 clathrin-mediated EGFR endocytosis activation of Erk signalling pathway, 5 cocaine-and amphetamine-regulating transcript (CART), 19 corticosterone (CORT) from adrenal medulla, 51 corticotropin-releasing factor (CRF), 48 CRF-releasing neurons, inhibition, 49 cortisol, 80 and weight gain, 23 Creb1-regulated transcription coactivator-1 (Crct1), 66 cross-approximate entropy, 103 See also approximate entropy (ApEn) C19 steroids, 211 cyclic adenosine monophosphate (cAMP), 4 EP4 receptor, Gs-protein and stimulation, 48 production, 8 cyclic 3-hydroxymelatonin, 141–142 cyclooxygenase 2 (COX-2), 45 Cynops pyrrhogaster, 3b-HSD distribution in, 195 cytochrome P450 cytochrome P450 aromatase (P450arom), 200–201 cytochrome P450C17, 195–196 side-chain cleavage, 194 cytokines influence on neuroendocrine systems, 43 in neural–immune interaction, 45 in neuroendocrine and febrile responses, 45 cytosolic PGES (cPGES), 46 cytosolic phospholipase A2 (cPLA2), 49 deconvolution analysis, 87 concept of, 91 discriminative indices of variable-waveform, 88 elimination function, 89

Gaussian like symmetric secretory-event waveform, 89 generalized gamma density, 89 measurement error, 89 physiological information, 93–94 probability distribution of interpulse intervals, 94 putative pulses estimation, 90 recovering/estimating secretion and elimination, 89 statistical comparison, 90 validation, 92 dehydroepiandrosterone (DHEA), 179 dendritic spine, effect of estrogens on, 223–224 15-deoxy-D12,14 prostaglandin J2 (15d-PGJ2), 46 deubiquitination, 10 deubiquitinating enzymes (DUBs), 160 See also endosome signalling diacylglycerol (DAG), 4 diarylpropionitrile (DPN), 234 dihydrotestosterone (DHT), 177 biosynthesis of, 188 diurnally rhythmic hormone concentrations, 83 dopamine, 118 mediated hypothalamic feedback, 82 dose–response concept, 86–87 dual-centre hypothesis, 17 dynorphin (Dyn), 68 expression, 70 early endosome antigen 1 (EEA1) proteins, 6 EGF induced late endosomal signalling, 6 E2-induced synaptogenesis, 239 endocrine systems agonists and antagonists, 83 analytical models for quantification, 84 endocrine axes, 79 Gaussian assumption, 89 endogenous endocannabinoids anandamide, 21 endogenous neurosecretory rhythms, 112 endosome signalling GPCR desensitization, regulators of, 7 neurotrophin and receptor, 6 pathophysiologic processes, 10 patterns of, 4 receptor kinases, 4–5 receptor Ser/Thr kinase, 7 regulation

292

endosome signalling (Continued) acidification and proteolysis, 9 dephosphorylation of RTKs, 9–10 inhibitory adaptors binding, 10 ubiquitination and deubiquitination, 10 requirements and regulation in health and disease, 8 endotoxic lipopolysaccharide (LPS) LPS-binding protein (LBP) or septins, 44 energy expending network (EEN), 19 ARN and EEN, 21 FTO gene, 22 VMH and LH, 21 energy homeostasis gonadal and adrenal steroids, hypothalamic integration of, 23 neural control of, 18 neuroanatomy of hypothalamic circuitries involved in, 19 and obesity in rodents and humans, dysregulation disruptions in afferent leptin signalling, 25–26 hyperphagia, 25 leptin gene transfer, 26–28 ensembles based feedback analysis, 98–99 connectivity within, 94 construct of male GnRH–LH–T ensemble, 95 defined, 94 neuroendocrine dynamics for, 99 approximate entropy, 100–103 cross-approximate entropy, 103 discrete pulse-coincidence estimates, 105 neural networks, 100 TRH–TSH–thyroxine/triiodothyronine ensemble, 99 epidermal growth factor receptors (EGFR), 1, 236 autophosphorylation, 4 cell surface signalling events and, 2–3 NGF signalling and, 6 tyrosine kinases of cytosolic domain of, 2 episodic endocrine signalling implications, 80 ERb-selective ER modulators (SERMS), 238 estradiol biosynthesis, 188–189 estrogen, 220, 233 biosynthesis, role of aromatase in, 209–210 brain, effects in, 221 neuroprotective functions, 221–222

sustaining brain glucose metabolism, role in, 222–223 independent factors, 115 induced lordosis behavior, 39–40 neurons, modulating effects on allosteric effects, 225 dendritic spine formation, 223–224 neurotransmitter–receptor subunits levels, 223 non-reproductive effect in central nervous system, 221 in peripheral tissues, 220 replacement therapy, 216–217, 220 reproductive effects of female and male, 220 triggered, transcriptional changes in, 40 estrogen receptors (ERs) and aromatase in normal human tissues, expression of, 212 in embryonic brain, 234–235 estrogen receptor-b (ERb) anxiety and depressive behavior, affect on, 239–240 3b-Adiol as functional ligand for, 236 discovery of, 233 dorsal horn morphogenesis of spinal cord, role in, 242–243 hippocampus, expression in, 237 hippocampus-dependent spatial learning, influence on, 237–238 human brain, expression in, 234 neonatal brain, expression in, 236–237 neuronal migration during later stages of corticogenesis, role in, 235–236 pain modulation, effect on, 244–245 protection of hippocampal neurons, role in, 240–241 role in adult hippocampal neurogenesis, 238–239 role in cerebellar development, 241–242 estrogen receptor-a (ERa), functional and structural domains of, 156 nucleotide base sequences in, 40 roles in pain modulation, 243–245 subtypes of discovery, 233 and expression patterns in brain, 234 structural features, 233–234

293

subtype-selective agonists, 234 extracellular ATP and P2X7 receptor role, 48 fat accrual network (FAN), 19, 22 fat mass and obesity-associated (FTO) gene, 22 febrile response, PGE2 EP receptor mRNAs expression, 47 follicle-stimulating hormone (FSH), 55, 80, 276 Fragile X Syndrome, 25 g-aminobutyric acid (GABA), 19 PGE2 and, 48 Gaussian model, 90 generalized arousal (GA) force arousal-enhancing drugs, 36 and impact on sex behaviors, 35 levels of, 36 mechanisms initiating generalized and sexual arousal in humans, 38 structure of generalized CNS arousal, 37 ghrelin from stomach as orexigenic signal, 18 GHRH–ghrelin–somatostatin–GH system, 99 glial fibrillary acidic protein (GFAP), 222, 255 glucagon-like peptides 1 and 2 (GLP-1 and GLP-2), 23 glucocorticoid receptors (GRs), nucleotide base sequences in, 40 glucose metabolism of brain and estrogen treatment, 222–223 glycosyl-phosphatidylinositol (GPI)-anchored membrane glycoprotein, 44 gonadal axis dynamics, 85 gonadotropin-inhibitory hormone (GnIH), 68 gonadotropin-releasing hormone (GnRH), 55, 177, 288 activity and puberty, 62 Kiss1 system and generation of pre-ovulatory surges, 66 neuronal activity and estradiol replacement in vivo, 65 neurons, PLC-/calcium-dependent pathway, 60 peripheral signals and dynamic regulation, 56 G protein-coupled receptors (GPCRs) regulators of desensitization, 7 signalling termination, 4 G protein-specific receptor kinases (GRKs), 4 growth factors action, 1

growth hormone (GH), 80 control of secretion, 114 growth-hormone-releasing hormone (GHRH), 113 growth hormone secretagogue (GHS) receptor, 113 neuropeptide interactions and, 113–114 patterns of GH/GHRH infusion, 116 pulsatile GH secretion effect ageing on, 115 developmental stage and pubertal status, 115–116 patterns within hypothalamo–pituitary– adrenal axis, 116–117 secretion, neurohumoral regulation pulsatile GH secretion and IGF-1 level, 112–113 sexual dimorphism in, 114 twenty-hour GH profile, 113 heterologous hypothalamo-pituitary signalling, 82 high-fat diet (HFD)-induced obesity, 22 histone chemical modifications molecular concepts of, 40 in preoptic area neurons, 40 histone deacetylases (HDACs), 140 homeostatic signalling, 81 hormonal axes, 81 hormonal male contraception efficacy, alternatives to improve, 284–285 hormonal regimens tested other androgens, 281–282 testosterone alone, 276, 278–279 testosterone plus GnRH analogues, 280–281 testosterone plus progestin, 279–280 male real-life hormonal contraceptive efficacy trials, results of, 279 principles of, 274–276 short-term and long-term side effects of, 283–284 variable suppression of spermatogenesis in trials, 282–283 hormone concentrations hormonal ensembles, 94–96 secretion from concentration data deconvolution analysis, 87–94 secretory bursts and biexponential decay, 86 hormone–growth factor signalling, 1

294

27-hydroxycholesterol, 259 5-hydroxytryptamine (5-HT), 118 hyperinsulinemia with obesity, 23 hyperleptinaemia due to obesity and deficiency in leptin signalling, 20 hyperphagia, 24–25 hypoleptinemia and FTO gene expression, 22 hypothalamic–pituitary–adrenal (HPA) axis, 43 hypothalamic–pituitary–gonadal (HPG) axis, 55 gonadal sex steroids and feedback in negative/positive, 56 kisspeptins, 56 neurohormonal elements, 56 pubertal activation control, 62 stimulators of, 59 hypothalamo-pituitary-target organ systems feedback signals, 82 hypothalamus, 81–82 pituitary locus, 82 random/stochastic elements, 83 systemic effectors, 82 IGF-I and estradiol interaction in nervous system, 252 adult neurogenesis, 256–257 and cholesterol synthesis in brain, 258–259 for development, 252–253 future research, directions for, 264–265 molecular mechanisms involved in cross-regulation of ERs and IGF-I receptor, 259–260 ER transcriptional activity regulation, 261–263 interaction of ERs with IGF-I receptor, 260–261 regulation of IGF-I receptor signalling by estradiol, 263–264 neuroendocrine regulation and reproduction, 253–255 neuroprotection, 257–258 imipramine, 159 immunoreactive estrogen receptor (ER)-alpha expression in human genital tissues, 38 panoply of changes in sexual behaviors, 41 inflammation, 43 EP4-expressing neurons in, 47 PGE2 synthesis and

sPLA2-IIA–COX-2–mPGES-1 pathway, 50 PVN neurons activation, 50 inhibin secretory episodes, 80 innate immune response by endotoxic lipopolysaccharide (LPS), 44 insulin growth factor receptors (IRK), 1 cell surface signalling events and, 2–3 IRS-1 and IRS-2, signalling scaffolds, 2 tyrosine kinase activity of, 2 insulin/IGF-I signalling, 252 insulin-like growth factor 1 (IGF-1), 251–252 interaction with estradiol, 252–253 secretion and growth hormone, 112 See also IGF-I and estradiol interaction in nervous system insulin receptor substrates (IRS), 3 interlukin IL-1 microvasculature act on, 45 mPGES-1 and, 45 IL-1b signals in brain, 45–46 IL-6 injection prostaglandin E2 (PGE2) synthesis in CNS, 45 transcriptional activation and IL-1b injection, 45 IL-1 type 1 receptor (IL-1R1) transcript levels, 45 internalization-defective EGFR mutant (c0 973), 4 intrapituitary regulation, 82 kisspeptins action site, 59 biological functions as metastasis-suppressor factors, 57 future challenges and expected developments, 68–72 and gonadotropin secretion control GnRH secretion, 59 prototypical responses, 60 HPG axis, regulatory signals, 71 Kiss2 gene identification in fish and non-mammalian species, 57 KISS1/Kiss1 gene GPR54 mutations, 56–57 LH and FSH responses, 59 metabolic regulation control of hypothalamic Kiss1 system, 65

295

neuroendocrine pathways, 65 reproductive maturation and function, 65 uncontrolled diabetes, 65 neuroanatomy of neurons in mammals in brain regions, 58 hypothalamus, distribution within, 58–59 kisspeptin-like immunoreactivity (IR), 58 population at ARC, 58 regulators central role in transmission of environmental, 67 gene expression, 67 neuroendocrine regulators, 67 roles in feedback control of gonadotropins, 63–65 sexual differentiation and puberty onset, roles in, 61–63 structure, 57 Kolmogorov–Sinai statistic, 100, 102 lactotroph function regulation PRL secretion effect of ageing, 119–120 and LH, interaction between, 120 light–dark and sleep–wake cycles, 119 lateral hypothalamus (LH), lesions in, 17 leptin from fat tissue gene therapy global health benefits, 27 insufficiency and sufficiency in hypothalamus normoglycemia and diabetes, 20 leptin–Crtc1–Kiss1 pathway, 67 metabolic and neural events, hypothalamic regulation of, 26 as putative adipostat, 18 regulatory role on FAN, 24 LET-767, in C. elegans, 181 ligand–receptor complexes, 1 light–dark and sleep–wake cycles, prolactin secretion, 119 linoleic acid (LA), 139 lipopolysaccharide (LPS), 4 lordosis behavior, 38 luteinizing hormone (LH), 55, 80, 274 algorithmic pulse-set searches, 92 ApEn of, 102 concentrations, validating paradigms types, 87 interpulse waiting-time distributions for, 94 Leydig-cell sensitivity to, 99

non-invasive analysis, 99 pulse frequency, 94 three-dimensional response surface, 100 variable-waveform/Gamma density deconvolution analysis, 93 Weibull estimates for pulsatility of gamma, 94 male contraceptive methods development of new, standards for, 274 drawback of current methods, 273 hormonal approach to (see hormonal male contraception) population overgrowth and, 273 male pseudohermaphroditism, 181, 183, 188 mammalian target of rapamycin (mTOR), 67 MCF-7 human breast cancer cells, studies on, 139 medial preoptic area (MPOA), 19 median eminence (ME), 45 median preoptic nucleus (MnPO), 47 MEK–ERK1/2 pathway, in breast cancer cells, 139 melanin-concentrating hormone (MCH), 68 melanocortin pathways from ARC, 24 a-melanocortin stimulating hormone (a-MSH), 20 melatonin anti-estrogen effects of, 140 binding to membrane receptors, 132 and modulation of cytoskeleton, 133 MT1 and MT2 receptors, 132 and signalling mechanisms, 132 and cancer inhibition aromatase activity, inhibition of, 139–140 elderly, cancer risk in, 141 LA, reducing uptake of, 139 light exposure after darkness, consideration of, 141 SIRT1 expression, regulation of, 141 telomerase activity, regulation of, 139 tumour cell initiation, prevention of, 138 tumour progression, suppression of, 138 circadian rhythms and sleep, modulation of, 136–138 action on receptors on SCN, 136 circadian rhythm, disruption in, 137 core body temperature, action on, 137 depressive effect on blood pressure, 137 jet lag and sleep disorders, use in, 138 melatonin levels and sleep, relationship between, 137

296

melatonin (Continued) discovery of, 127 in fluids of mammals, 128 as free radical scavenger and antioxidant, 133 antioxidative cascade, 141–142 free radical production by chemical reduction of oxygen, 143 intrinsic apoptotic pathway, inhibition of, 145 mitochondrial free radical generation, prevention of, 144 neurodegenerative conditions and melatotin application, 144–145 oxidative damage and cell death in brain, reduction in, 144 6-hydroxylation of, 130 light regulation of synthesis, 130–132 nuclear binding sites for, 132 production in pineal gland, 127, 130 receptor-independent effects, 130 and seasonal reproduction gonadotropins, regulation of, 135 prolactin regulation, 135–136 Syrian hamsters, studies on, 133–134 TSH, role of, 135 tuberalin, role of, 135 synthesis from serotonin AANAT activity and, 128 circadian production with peak nocturnal levels, 129 cycle in humans, 129–130 master circadian clock in suprachiasmatic nuclei (SCN), 128 norepinephrine (NE), release of, 128–129 membrane rafts, 6 metabolism, 84–85 7a-methyl-19-nortestosterone (MENT), 281 microsomal prostaglandin (mPGES) E synthase, 45–46, 51 mitogen-activated protein kinase (MAPK), 253 activation, 4 GPCR–barrestin complexes and signalling pathway, 8 MAPK signalling pathway, 263 multivesicular–lysosomal entities for degradation, 4 N-acetyl-5-methoxytryptamine, see melatonin NADPH-cytochrome P450 reductase, 211

N2a neuroblastoma cells, ER-mediated transcription in, 262 nerve growth factor (NGF) NGF–TrkA complexes, retrograde transport, 6 nesfatin, 19 neuroendocrine axis pulsatility and rhythmicity, 95 representation, 81 neuroendocrine ensemble control deterministic dynamics, 94–96 GnRH–LH–T system, 97 random/stochastic facets, 96 neuroendocrine system appraisal of negative feedback in, 97–99 ensembles for dynamics, 99 approximate entropy, 100–103 cross-approximate entropy, 103 discrete pulse-coincidence estimates, 105 neural networks, 100 model forms, 98 neurogenesis in adult hippocampal, 239 neurokinin B (NKB), 68 neuropeptide Y (NPY) as physiological appetite transducer, 18 neurosteroidogenesis, 193–194 See also brain, expression of steroidogenic enzymes in neurotrophins (TrkA), 2 signalling by retrograde axonal transport, 7 neutral sphingomyelinase (nSMase) TNF–TNFR1 complex and, 9 nitric oxide synthase (nNOS) expression, 38 N-methyl-D-aspartate receptor (NMDAR), 239 non-linear mixed-effects model, 91 non-shivering thermogenic energy expenditure rate, 21 noradrenaline, 118 Notch, heterodimeric transmembrane protein signalling function, 9 trafficking through endosomal apparatus, 9 NPYergic signalling in ARC–PVN, 22–23 nucleosome sliding, 165 nucleus of solitary tract (NTS), 51 NuRD histone deacetylation complex, 8 orexin signalling and hyperphagia, 24–25 in VMH–LH axis, 22

297

organum vasculosum of lamina terminalis (OVLT), 45 osmolality–baroreceptor–AVP (ADH) system, 99 oxymodulin from gastrointestinal tract, 23 oxytocin, 120 See also pulsatile oxytocin secretion physiological significance pancreatic polypeptide (PP) administration and food intake, 23 parathormone, 80 parathyroid hormone (PTH) receptor studies on, 8 paraventricular (PVN) nuclei, 47 p300/CBP-associated factor (PCAF), 168 periodic appetite drive through ARN, 24 periodic stimuli, 121 peripheral feedback signals in hypothalamic integration of energy homeostasis adipostat signals, 22–23 anorexigenic signals, 23 ghrelin, 23–24 leptin, 24 steroids, 23 periventricular nucleus (PEV), 114 peroxisome proliferator-activated receptor-g (PPAR-g), 46 peroxovanadium compounds (pVs), 2 phosphatidylinositol 3-kinase (PI3K), 253 PI3K–Akt–GSK3b pathway, 262 PI3K–Akt signalling pathway, 263 phospholipase C (PLC), 4 activation, 60 phosphotyrosine phosphatase (PTP) inhibitors, 2, 6 pineal-derived neurotransmitter, 68 pituitary hormone secretory-burst amplitude and mass, 84 platelet-derived growth factor (PDGF), 2 Poisson process, 94 polypeptide hormones action, 1 post-prandial leptin hypersecretion restraint on feeding, 24 Prader–Willi Syndrome, 25 progesterone levels coupled with levels of estrogens in blood, 40 progestins, use in male contraceptive trials, 279–280

proinflammatory signal transduction pathways, 50 prolactin, 80 sleep–wakefulness pattern, 119 proopiomelanocortin (POMC), 19 propylpyrazoletriol (PPT), 234 prostacylin (PGI2), 46 prostaglandins (PGs) biosynthesis of, 46 functional circuits by intraparenchymal PGE2 EP3-dependent response in brain, 49 working hypotheses, 47–48 and IL-6-induced fever, 45 prostaglandin G2 (PGG2), 46 sites of action affinities for prostanoids, 46 febrile response, PGE2 EP receptor mRNAs expression, 47 [3H] PGE2-binding sites study, 47 prostanoid receptors, 46 protease-activated receptors 2 (PAR-2) receptor, 8 puberty onset, kisspeptins roles in, 61–63 pulsatile hormone secretion biexponential elimination, 89 concentration profile, 91 modulation, 114 rate function, 89 resultant model, 89 rhythmic pattern, 83 time scale, 83 variable-waveform, 90 pulsatile oxytocin secretion physiological significance, 120 pulsatility, 120–121 putative intrapituitary regulatory interactions, 82 radial glial cells, and neuronal migration, 235–236 Rana esculenta 3b-HSD distribution in, 195 17b-HSD in, 197 Ras–MAPK signalling pathway, 262 Rat-1 fibroblasts, plasma membrane receptors and, 4 receptor–effector response pathway, 86 receptor internalization GRK and arrestin, role of, 7 receptor serine/threonine kinases, 2–4

298

receptor tyrosine kinases (RTKs), 2 intracellular concentration, 4–5 phosphorylation by non-receptor tyrosine kinases, 4 trans-activation, 4 recombinant adeno-associated virus vector encoding leptin (rAAV-lep) injection intracerebroventricularly, 27 RNA polymerase II (RNAPII), 169 rostral periventricular area of third ventricle (RP3V), 58 secretory-burst model, 90 seladin-1/DHCR24, 258–259 selective androgen receptor modulators (SARMs), 281 selective estrogen enzyme modulators (SEEM), 140 sex steroid, 80 biosynthesis pathways and testosterone, 186–187 local and tissue-specific biosynthesis of, 178–180 sexual dismorphism of human GH secretory pattern, 114–115 SH2 domain-containing adaptors, 3 signal transducer and activator of transcription 5B (STAT5b) signalling, 112 signal transducer and activator of transcription 3 (STAT3), 9 single nuclear polymorphism (SNP) in FTO gene, 22 sleep-onset-entrained GH surge, 115 Smad anchor for receptor activation (SARA), 4 somatostatin (SRIF), 113 spermatogenesis, and action of gonadotropins, 276 sphingosine-1-phosphate (SP1), 8 steroid hormone receptors (SHRs) regulation by control of steroid receptor promoter activity, 155 AR expression, regulation of, 158–159 ERa promoter, role, 157 ERb promoter activity, regulation of, 158 GR promoter expression, regulation of, 159 mineralocorticoid receptor (MR) promoter, regulation of, 159 progesterone receptors A and B isoforms, expression, 158 regulation of ERa promoter activity, 158

by coregulator molecules, 165 ATP-dependent chromatin remodelling complexes, and ERa, 166–167 chromatin remodelling coactivator complexes, 165 coactivators in steroid receptor transcriptional activity, 166–167 ERa–DNA binding studies on, 165 ERa recruitment of SRC coactivators, 168–169 pioneer factors, role of, 165 primary coactivators, 167 role of coactivators, 168 secondary coactivators, 167–168 at level of protein stability AR activity, regulation of, 162 ERa protein, regulation of, 160–161 GR and MR, stabilization of, 162 PR protein, regulation of, 161–162 ubiquitin proteasome system (UPS) and, 160 post-translational regulation of AR activity, 163–164 of ERa, 162–163 of GR activity, 164–165 of MR activity, 165 of PR activity, 164 steroid receptor mRNA stability and translation, modulation of, 159–160 AR mRNA, regulation, 159–160 ERa mRNA, regulation, 159 GR mRNA, regulation, 160 regulatory elements, 159 steroid hormones (SHs), 153 action mechanism of, 154–155 binding to steroid hormone receptors (SHRs), 155 estrogen receptor-a (ERa), structure of, 156 protein receptors, existence of, 155 SHRs, functional and structural domains of, 155–156 synthesis of, 153–154 steroidogenic enzymes classes of conserved structures–conserved activities class, 180 conserved structures–different activities class, 180–181 different structuresconserved activities class, 181

299

convergent evolution in det2 of Arabidopsis thaliana and type 2 5a-reductase, 182 LET-767 of C. elegans and type 12 17b-HSD, 181–182 differences between humans, monkeys and rodents, 183–186 molecular evolution of, 180–181 species differences in sex steroid-inactivating enzymes, 186–187 steroidogenic factor-1 (SF-1), 21 subfornical organ (SFO), 45 6-sulfatoxymelatonin, 130 supraoptic (SON) nuclei, 47 system-level adaptations in pathophysiology, 97 telomerase activity, in tumour cells, 139 testosterone, biosynthesis of, 188 type 3 17b-HSD, role of, 188 type 5 17b-HSD, role of, 188 thromboxane A2 (TxA2), 46 thyroid-stimulating hormone (TSH), 80, 135 thyrotroph function regulation TSH secretion effects of ageing and adiposity, 118–119 ultradian pattern and regulation, 118 thyrotrophin-releasing hormone (TRH), 118 toll receptor family (TLR), 9 transforming growth factor b (TGF-b), 2 activation at cell surface, 7 SARA–Smad complexes and signalling, 7 Smad2 and action, 4 types I and II subunits, 3

transient receptor potential (TRP) channels, 60 transmembrane-spanning receptor family, 4 TrkA signalling endosomes Rab5 GTPase and early endosome antigen 1 (EEA1) proteins, 6 tryptophan hydroxylase (TPH), 128 tuberalin, 135 tumour necrosis factor (TNF) apoptosis and, 9 TNF-a injection, 45 TNFTNFR1adaptor complexes and intracellular activation of caspase 3, 9 tyrosine hydroxylase (TH), 223 ubiquitination, 10, see endosome signalling ubiquitin proteasome system (UPS), 160 ultradian signal exchange, challenges, 80 uncoupling protein 1 (UCP1), dynamic shifts in, 21 vascular endothelial growth factor (VEGF), 2, 252 vasoactive intestinal peptides (VIPs), 119 vasopressin V2 receptor (V2R), 4 ventromedial hypothalamus (VMH) cognate receptor CB1 in, 21 dual-centre hypothesis, 17 receptor TrkB expressing neurons in, 21 ventromedial preoptic nucleus (VmPO), 47 WAGR Syndrome, 25 Weibull renewal process, 94 white adipose tissue (WAT), 19 Wnt signalling pathway, 8